CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Guest Editors Sandro C. Esteves ANDROFERT, Clı´nica de Andrologia e Reproduc¸a˜o Humana e Centro de Refereˆncia para Reproduc¸a˜o Masculina. Campinas, Sa˜o Paulo, Brazil.
Ashok Agarwal Center for Reproductive Medicine, Cleveland Clinic Foundation, Cleveland. Ohio, USA Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Antonio Egidio Nardi Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonc¸a Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Gustavo Franco Carvalhal Faculdade de Medicina da Pontifı´cia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ivete Bedin Prado Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ludhmila Abrahao Hajjar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Fulvio Alexandre Scorza Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mauricio Etchebehere Universidade Estadual de Campinas Campinas, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Oswaldo Keith Okamoto Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Geraldo Busatto Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Patricia Rieken Macedo Rocco Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil
Carlos Serrano Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Emilia Inoue Sato Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Paulo Hoff Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sandro Esteves ANDROFERT - Andrology & Human Reproduction Clinic Campinas, SP, Brazil Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Silvia Vanessa Lourenc¸o Faculdade de Odontologia da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Thelma Suely Okay Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Editorial Board Universite´ de Sherbrooke Que´bec, Canada´
Witten/Herdecke University Witten, North Rhine - Westphalia, Germany
Abhijit Chandra King George’s Medical College Lucknow, India
Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil
Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Adauto Castelo Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Andrea Schmitt University of Goettingen Goettingen, Germany
Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil
Ademar Lopes Fundac¸a˜o Antoˆnio Prudente, Hospital do Caˆncer Sa˜o Paulo, SP, Brazil
Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Daniel Romero Mun˜oz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Euclides Ayres Castilho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Alberto Azoubel Antunes
Artur Brum-Fernandes
Edmund Neugebauer
Fa´bio Biscegli Jatene
Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Ernest Eugene Moore University of Colorado Denver Denver, CO, USA
Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Francisco Laurindo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ke-Seng Zhao Southern Medical University Guangzhou, China Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine University of London London, UK Marcelo Zugaib Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Sa˜o Paulo
Sa˜o Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mauro Perretti William Harvey Research Institute London, UK
Philip Cohen University of Houston Health Center Houston, Texas, USA Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´
Michael Gregory Sarr Mayo Clinic Rochester, MN, USA Milton de Arruda Martins Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA
Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica
Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA
Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Pedro Puech-Lea˜o Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Peter Libby
Brigham and Women’s Hospital Boston, Boston, MA, USA
Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Board of Governors Alberto Jose´ da Silva Duarte Aluisio Augusto Cotrim Segurado Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Carlos Roberto Ribeiro de Carvalho Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Neto Dalton de Alencar Fischer Chamone Daniel Romero Mun˜oz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euripedes Constantino Miguel Fa´bio Biscegli Jatene Flair Jose´ Carrilho Gerson Chadi Gilberto Luis Camanho Irene de Lourdes Noronha Irineu Tadeu Velasco Ivan Cecconello Jorge Elias Kalil
Editorial Director Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
Jose´ Antonio Franchini Ramires Jose´ Antonio Sanches Jose´ Eduardo Krieger Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Lenine Garcia Branda˜o Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Srougi Milton de Arruda Martins Nelson de Luccia Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff
Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini Roberto Kalil Roberto Zatz Roger Chammas Samir Rasslan Sandra Josefina Ferraz Ellero Grisi Selma Lancman Tarcı´sio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Venaˆncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob
Editorial Assistants Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ariane Maris Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil
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Clinics. Sa˜o Paulo: Scientific Journal of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clı´nicas da FMUSP, 1946–2004. 1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. CDD 610
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ISSN-1807-5932
CLINICS CONTENTS Clinics 2013 68(S1):1–167
EDITORIAL
The azoospermic male: current knowledge and future perspectives Sandro C. Esteves, Ashok Agarwal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
REVIEWS
Novel insights into the genetic and epigenetic paternal contribution to the human embryo Manoj Kumar, Kishlay Kumar, Shalu Jain, Tarannum Hassan, Rima Dada. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
The epidemiology and etiology of azoospermia Marcello Cocuzza, Conrado Alvarenga, Rodrigo Pagani. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Update in the evaluation of the azoospermic male Ahmet Gudeloglu, Sijo J. Parekattil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
The importance of semen analysis in the context of azoospermia Nabil Aziz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
A comprehensive review of genetics and genetic testing in azoospermia Alaa J. Hamada, Sandro C. Esteves, Ashok Agarwal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Obstructive azoospermia: reconstructive techniques and results Karen Baker, Edmund Sabanegh Jr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Medical management of non-obstructive azoospermia Rajeev Kumar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Hypogonadotropic Hypogonadism Revisited Renato Fraietta, Daniel Suslik Zylberstejn, Sandro C. Esteves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
The role of varicocele treatment in the management of non-obstructive azoospermia Kubilay Inci, Levent Mert Gunay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
An update on sperm retrieval techniques for azoospermic males Sandro C. Esteves, Ricardo Miyaoka, Jose´ Eduardo Orosz, Ashok Agarwal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Predictive factors for sperm retrieval and sperm injection outcomes in obstructive azoospermia: Do etiology, retrieval techniques and gamete source play a role? Ricardo Miyaoka, Sandro C. Esteves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Prognostic factors for sperm retrieval in non-obstructive azoospermia Sidney Glina, Marcelo Vieira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Laboratory processing and intracytoplasmic sperm injection using epididymal and testicular spermatozoa: what can be done to improve outcomes? Wana Popal, Zsolt P. Nagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception Bhushan K. Gangrade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Reproductive outcomes, including neonatal data, following sperm injection in men with obstructive and nonobstructive azoospermia: case series and systematic review Sandro C. Esteves, Ashok Agarwal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction? Veerle Vloeberghs, Greta Verheyen, Herman Tournaye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Biotechnological approaches to the treatment of aspermatogenic men Pedro Manuel Aponte, Stefan Schlatt, Luiz Renato de Franca. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
EDITORIAL
The azoospermic male: current knowledge and future perspectives Sandro C. Esteves,I* Ashok AgarwalII* I
ANDROFERT, Clı´nica de Andrologia e Reproduc¸a˜o Humana e Centro de Refereˆncia para Reproduc¸a˜o Masculina, Campinas, Sa˜o Paulo, Brazil. II Center for Reproductive Medicine, Cleveland Clinic Foundation, Cleveland, Ohio, USA. * Guest Editors.
This special issue is fully dedicated to the topic of azoospermia and contains the seminal work of renowned scientists and clinicians from seven countries on three continents. In seventeen chapters, a comprehensive review of the epidemiology, genetics, physiopathology, diagnosis, and management of azoospermia addresses our current knowledge on the topic. The clinical results of assisted reproductive techniques applied to this category of male infertility and the health of offspring originating from such fathers are critically analyzed. In addition, the challenges and the future biotechnological perspectives for the treatment of azoospermic males seeking fertility are discussed. Esteves SC, Agarwal A. The azoospermic male: current knowledge and future perspectives. Clinics. 2013;68(S1):1-4. E-mail: s.esteves@androfert.com.br Tel.: +55 19 3295-8877
Two major breakthroughs revolutionized the field of male infertility in the last three decades. The first was the development of intracytoplasmic sperm injection (ICSI) for the treatment of male factor infertility, and the second was application of ICSI to azoospermic males, with the demonstration that spermatozoa derived from either the epididymis or the testis were capable of normal fertilization and pregnancy. Azoospermia, defined as the complete absence of spermatozoa in the ejaculate, invariably results in infertility but does not necessarily imply sterility. In fact, azoospermia has been recognized as one of the most intriguing topics in male infertility. Due to the true nature of research involving the classical disciplines of physiology, biochemistry and molecular biology, a rapid rise in the volume of scientific knowledge regarding azoospermia has been obtained. Invariably, this has led to a better understanding of the multi-faceted aspects of azoospermia. However, there is still relatively little data within the literature supporting common clinical practices. The reproductive potential of azoospermic males with different etiologies is unclear; furthermore, the association of an increased risk of birth defects and potential iatrogenic transmission of genetic abnormalities with ICSI using sperm retrieved from these patients is still under debate. The scope of azoospermia related-infertility now covers a wide spectrum, including genetic studies, hormonal control, microsurgical and medical therapy, assisted reproduction techniques, and innovative stem cell research that aims to create artificial gametes. In this special issue of Clinics, we
invited leading, internationally recognized scientists and clinicians from the various sub-specialties to compile a collection of high-quality and comprehensive reviews highlighting the most current advances and contentious issues in azoospermia. Our aim is to provide readers with a thoughtful and wide-ranging review of the epidemiology, genetics, physiopathology, diagnosis, and management of azoospermia. The text is the first of its type and represents an invaluable tool for both basic scientists with an interest in sperm biology and clinicians (urologists, gynecologists, reproductive endocrinologists, and embryologists) working in the field of infertility. The selection of topics demonstrates the exciting breadth of this category of male infertility and the opportunity that research in this area holds for both understanding and improving the reproductive health of azoospermic males. This Special Issue commences with provocative insights into the genetic and epigenetic paternal contribution to the human embryo (1). Dada and colleagues from New Delhi present the current knowledge on the role of spermatozoa as highly specialized cells with the purpose of not only delivering competent paternal DNA to the oocyte but also providing a robust epigenetic contribution to embryogenesis. The paternal epigenetic contribution to embryogenesis requires that both the sperm DNA and the chromatin structure as a whole contain layers of regulatory elements that are sufficient to drive genes towards activation or silencing upon delivery to the egg. Changes in the epigenome are now known to affect gene expression, and several genes participating in spermatogenesis have been demonstrated to be epigenetically regulated. The second article by Marcello Cocuzza and colleagues from the University of Sa˜o Paulo is a comprehensive review of the epidemiology and etiology of azoospermia. According to these authors, azoospermia is identified in approximately 1% of all men and 10% to 15% of infertile males (2). With a population of approximately 3 billion
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.6061/clinics/2013(Sup01)01
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Editorial for the special issue on azoospermia Esteves S and Agarwal A
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people at reproductive age, a gross estimate indicates that approximately 10 million men worldwide are azoospermic. The authors explore several conditions that may lead to azoospermia and didactically divide them into pre-testicular, post-testicular, and testicular causes. Despite the advances in the diagnostic tools that identify men with genetic-origin azoospermia, many men are still classified as having idiopathic azoospermia because the specific etiological factor remains unidentifiable. Therefore, determining the etiology of azoospermia remains one of the main challenges in this field. Drs. Gudeloglu and Parekattil from the United States follow the theme by highlighting the importance of the clinical exam in the investigation of the azoospermic male. The authors detailed review defines why and how the clinical evaluation should be undertaken. They also discuss the usefulness and limitations of testis biopsies and imaging studies in the context of azoospermia and the importance of genetic counseling before using the spermatozoa from men with nonobstructive azoospermia (NOA) for assisted reproductive techniques (3). Dr. Aziz provides a laboratory perspective from the United Kingdom by cleverly defining azoospermia as a descriptive term for ejaculates that lack spermatozoa without implying a specific underlying cause (4). The author stresses that proper techniques are needed to reduce the amount of analytical error and enhance sperm count precision when evaluating semen specimens. The correct assessment of an initially azoospermic semen specimen should be followed by an examination of the pelleted semen to exclude cryptozoospermia, which is defined by the presence of a very small number of live sperm in a centrifuged pellet. An accurate assessment of very low sperm counts aims to avoid labeling severely oligozoospermic men as azoospermic, which is particularly important in the current era of assisted reproduction technology. Dr. Azizs chapter provides insightful information on the seminal plasma biomarkers that may aid in determining the causes of azoospermia. Laboratory seminology is clearly moving from the assessment of conventional semen profiles into the assessment of sperm function. This strategy is likely to aid in the understanding of the underlying pathophysiology of male infertility, and noninvasive biomarkers may be useful in discriminating NOA cases from obstructive azoospermia (OA). This Special Issue contains an authoritative article that provide the current knowledge on the genetic aspects of azoospermia and the testing available for clinical use. Dr. Hamada and his co-authors from the Cleveland Clinic provide an immense summary of results on the genetic aspects of male infertility and implication of these results on the diagnosis and treatment of azoospermic males (5). Molecular biology genetic testing involving the Y-chromosome can now correctly identify azoospermic men misdiagnosed as having idiopathic infertility. Moreover, Ychromosome testing is of prognostic value for sperm retrieval in NOA. The authors also present practical recommendations for testing, and they discuss the possible implications of using spermatozoa from men with genetic abnormalities for assisted conception. In the clinical setting, azoospermic patients are diagnosed as having obstructive or nonobstructive azoospermia. Obstructive azoospermia has been attributed to a mechanical blockage that can occur anywhere along the reproductive tract, including the vas deferens, epididymis, and ejaculatory
duct. OA is considered to be one of the most favorable prognostic conditions for male infertility because spermatogenesis is not disrupted, unlike in NOA. Drs. Baker and Sabanegh from the Cleveland Clinic discuss the current indications, techniques and results of reconstructive procedures in OA (6). These authors highlight the refinements in microsurgery that have optimized the success of reconstructive procedures; additionally, the authors indicate that the use of optical magnification is now the gold standard for vasal reconstruction. While the results of reconstructive procedures are excellent following vasectomies, other complex repairs may be required, especially in other etiological categories of OA. Despite being highly successful, microsurgical reconstruction may not be indicated in all men with OA, such as in patients with congenital bilateral absence of vas deferens (CBAVD) and certain cases of post-infectious obstructions or failed vasectomy reversals. In such cases, sperm retrieval can be performed for use with ICSI. Nonobstructive azoospermia, on the other hand, poses a different challenge. From the management standpoint, men with NOA are the most difficult to treat, and extensive debate exists on the benefit of intervention for this category of male infertility. Various conditions may cause NOA, including genetic and congenital abnormalities, post-infectious issues, exposure to gonadotoxins, medications, varicocele, trauma, endocrine disorders, and idiopathic causes. This Special Issue contains three articles that critically explore the role of medical and surgical therapy in nonobstructive azoospermia. Renato Fraietta and colleagues from the Federal University of SaËœo Paulo provide a timely review on hypogonadotropic hypogonadism (HH), which is a failure of spermatogenesis due to a lack of appropriate stimulation by gonadotropins (7). This category of patients includes not only congenital forms of HH but also a subset of men whose spermatogenic potential has been suppressed by excess androgens or steroids. These patients benefit from specific hormonal therapy and often show remarkable recovery of spermatogenic function with exogenously administered gonadotropins or gonadotropin releasing hormone. Unfortunately, not all men with NOA have HH. In fact, the larger category of NOA consists of men with intrinsic testicular impairment in which empirical medical therapy shows little benefit, as noted by Dr. Rajeev Kumar from the All India Institute of Medical Sciences in New Delhi (8). However, due to the developments in ART, a renewed interest in the role of interventions in this subset of NOA patients has developed. The author expertly discusses the role of medical therapy in these men to improve the quantity and quality of sperm that can be eventually retrieved from their ejaculates or from their testes for use in ICSI. In this sense, gonadotropins, aromatase inhibitors and non-steroidal antiestrogens show promise in achieving this endpoint. Lastly, Dr. Kubilay Inci from Turkey provides a critical appraisal on the role of varicocele repair for men with NOA (9). His authoritative review discusses the pathophysiology of varicocele-related infertility, and the discussion is supported by his own experience on the impact of varicocele repair in azoospermic patients. From the limited published data, it has been suggested that varicocele repair may not only allow small quantities of sperm to appear in the ejaculate but also may enhance the chances of retrieving sperm from the testis of these patients.
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Editorial for the special issue on azoospermia Esteves S and Agarwal A
Even a minimal restoration of sperm production facilitates sperm injection procedures. Currently, ART is the only option for most men with azoospermia-related infertility to have their biological offspring. Success has been achieved with ICSI in both obstructive and nonobstructive azoospermia, and the use of non-ejaculated sperm coupled with ICSI has become a worldwide established procedure. Surgical methods have been developed to retrieve spermatozoa from the epididymides and testes. After sperm retrieval, ICSI is used rather than standard IVF because ICSI has been shown to result in a significantly higher fertilization rate. A section of this Special Issue comprised of seven articles is fully dedicated to the use of assisted conception in azoospermia-related infertility. The authors of these selected titles have extensive publication records and more than a decade of clinical and/or laboratory experience in the management of azoospermic males using assisted conception. In the first article of this section, the authors prepared a comprehensive summary of the current methods for sperm retrieval and critically analyzed the advantages and disadvantages of each method (10). The authors note that the sperm retrieval (SR) method of choice is often based on the type of azoospermia and the attending surgeon’s preferences. However, SR should aim to both minimize damage to the reproductive tract, thereby preserving the chance of repeated retrieval attempts, and to obtain an adequate number of good quality sperm that can be immediately used for ICSI or alternatively cryopreserved for future ICSI attempts. Following this theme, two articles from Brazilian groups discuss the key elements for the success of sperm retrieval in obstructive and nonobstructive azoospermia (11,12). Dr. Miyaoka and colleagues summarize the current knowledge on the impact of several factors on sperm injection outcomes using surgically retrieved sperm from men with OA. The authors conclude that SR in OA is highly successful and that causes of obstruction and retrieval methods have little impact on SR success rates. Moreover, current evidence suggests that similar pregnancy outcomes are achieved by ICSI in OA using either fresh or frozen-thawed epididymal or testicular sperm (11). While a successful retrieval attempt is obtained in virtually all cases of OA, Drs. Glina and Vieira highlight the uncertainty of sperm acquisition in cases of NOA, thus making it desirable to determine the prognostic factors. The authors review several clinical and laboratory prognostic markers, such as the etiology of NOA, paternal age, testicular volume, serum levels of pituitary gonadotropins, genetic testing results, method of collection, testicular histopathology results and the impact of the laboratory tissue processing method; the authors concluded that the only unfavorable indicator for SR is the presence of microdeletions in the AZFa and/or AZFb regions of the Y chromosome long arm (12). Another key message from this review is that men with NOA are no longer considered sterile, even with elevated folliclestimulating hormone levels and small testes, because modern retrieval techniques can be used to collect testicular sperm and produce a healthy biological offspring via assisted conception. The laboratory management of surgically retrieved gametes requires special attention because spermatozoa collected from azoospermic men are often compromised in quality and more fragile. Drs. Popal and Nagy from Atlanta provide strategies for handling such gametes inside the laboratory and discuss potential dangers (13). Adherence to state of the art laboratory techniques and quality control are recommended to avoid jeopardizing the fertilizing potential of the sperm and chances of achieving a live birth. Several
techniques are described for optimizing the chances of harvesting spermatozoa from epididymal fluid and testicular tissue of azoospermic men. The concept of cryopreservation may also be used in association with sperm retrieval procedures. Some centers prefer to retrieve and intentionally cryopreserve sperm for future use. This strategy offers the advantage of avoiding ovarian stimulation when no sperm is obtained from testicular specimens. If sperm is retrieved and frozen, it can be thawed at any time, thereby avoiding the need to organize two operations (oocyte and sperm retrieval) on the same day. Additionally, cryopreservation may spare unused specimens that would be discharged after ICSI, which may be useful if the treatment cycle does not result in a pregnancy. Therefore, future ICSI attempts could be conducted without repeated surgical retrievals. In most cases of epididymal retrievals, motile sperm will be available after thawing, and ICSI outcomes using fresh motile or frozen-thawed epididymal sperm do not seem to differ. Cryopreservation of testicular sperm is also advisable, especially for men with NOA who often require multiple ICSI attempts to conceive but may not have an adequate number of sperm available for repeated retrieval attempts. These important aspects are discussed by Dr. Gangrade from Orlando, who also presents laboratory protocols for the cryopreservation of epididymal and testicular sperm and discusses the reproductive outcomes of using frozen-thawed gametes for sperm injections (14). The closing articles of the section dedicated to assisted reproduction are authored by the guest editors and Drs. Veerle and colleagues from the Centre of Reproductive Medicine in Brussels, who pioneered the introduction of ICSI and revolutionized the treatment of male infertility in the 90s. In our article, we summarized the data that have been generated on the reproductive potential of azoospermic men undergoing assisted conception (15). We performed a systematic review of the literature focusing on studies that directly compared pregnancy outcomes after sperm injections between couples whose male partner had OA or NOA. We also analyzed a personal database (SCE) of 370 couples who underwent ICSI according to the abovecited categories and compared the outcomes with a group of 465 non-azoospermic infertile males. In our series of 1,092 ICSI cycles performed in 835 male infertility patients, live birth rates were lowest in the NOA group. Miscarriage, ectopic pregnancy, and multiple pregnancy rates did not differ between clinical pregnancies achieved using ejaculated or non-ejaculated sperm from men with OA or NOA. In our series of 427 babies born with ICSI using sperm from non-azoospermic infertile fathers and azoospermic fathers with OA and NOA, the short-term neonatal outcomes were similar among groups, despite a tendency towards higher preterm birth in both azoospermia categories and lower gestational age for twins in OA. The overall perinatal death and malformation rates were 2.8% and 1.6%, respectively, and our results did not differ between deliveries that resulted from ICSI using ejaculated or non-ejaculated sperm from men with OA or NOA. In our review, we critically compare our results with other publications. We note that most published studies, including our data, suffer from methodological shortcomings. For instance, these studies were not designed to detect differences in live birth rates and not powered to detect differences in less frequent outcomes, such as malformations and other complications. Moreover, no follow-up study has yet compared the long-term
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Editorial for the special issue on azoospermia Esteves S and Agarwal A
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physical, neurological and developmental outcomes of children born with ICSI using sperm from azoospermic men with OA and NOA. We conclude that for now, the limited evidence on pregnancy and postnatal outcomes of ICSI using surgically derived sperm from azoospermic men is reassuring; however, a call for continuous monitoring is of utmost importance to support the recommendation of sperm retrieval and ICSI in azoospermia-related male infertility. Following this theme, an authoritative review by Dr. Veerle and co-authors provides an analysis of the results of immature germ cells used for ICSI (16). This strategy has been proposed in cases of NOA in which no spermatozoa can be retrieved. After the initial disappointing results, the in vitro culture of immature germ cells to more mature stages has been proposed as an approach to improve this poor outcome. More than a decade has passed since the introduction of ICSI with elongating and round spermatids; there is still a lot of uncertainty regarding the safety of this treatment option. The authors outline the clinical and scientific evidence for ICSI using immature germ cells and in vitro matured germ cells and describe the physiopathological mechanisms involved in fertilization. In addition, these authors suggest that despite reports of deliveries of healthy offspring, the method has very low efficiency; furthermore, most IVF programs worldwide have stopped spermatid injection. Several ethical and safety concerns related to the potential transmission of genomically imprinted disorders have been raised, leading to the ban of spermatid injection in countries such as the United Kingdom. Research toward the development of artificial gametes is timely due to the prevalence of NOA and inability of harvesting mature sperm from the testes in approximately half of patients. In addition, the overall efficiency of spermatid injection is disappointing, and the reproductive potential after ICSI using testicular sperm retrieved from azoospermic men with dysfunctional spermatogenesis is only fair. A recent breakthrough report by Japanese scientists at Kyoto University used stem cells from mouse embryos to create primordial germ cells, which were then able to differentiate in spermatozoa after testis transplantation in mice. This topic is the theme of the closing article of this Special Issue, namely the challenges and perspectives of biotechnology and stem cell research to treat the most severe cases of azoospermia and potentially ‘cure’ male sterility (17). This article is authored by the group led by Dr. Franca from the Federal University of Minas Gerais, Brazil, in collaboration with Dr. Schlatt from Munster, Germany. Dr. Franca is a leading authority in this field, and his provocative insights on cell biology call for a profound reflection. In their article, Dr. Franc¸a’s group proposes that men with incomplete spermatogenesis are collectively classified as aspermatogenic to indicate a highly severe testicular pathology with complete absence of spermatids and spermatozoa. The authors explore the novel biotechnological methods to rescue fertility while maintaining biological fatherhood. Human haploid-like cells have already been obtained from pluripotent stem cells of somatic origin using the novel technique of in vitro sperm derivation. Germ cell transplantation as a form of grafting is a promising method that may restore the fertility of prepubertal boys who previously received cancer treatments. Haploidization is being investigated as an option to create gametes based on biological cloning technology. Although promising, these methodologies are experimental,
and the production of human gametes in the laboratory is a highly complex process that has yet to be translated to reproductive medicine. This Special Issue of Clinics aims to be a landmark treatise on azoospermia. Scientists and clinicians from seven countries on three continents have contributed generously to the current scientific knowledge involving human azoospermia and its role in male reproductive health. We recommend its contents not only to students and researchers in the biological, veterinary and medical sciences but also to clinicians involved in the management of infertile couples and urologists, andrologists, gynecologists, embryologists and reproductive specialists interested in following the exponential growth in the knowledge of azoospermia. Due to the multidisciplinary nature of this category of male infertility, unsolved problems present themselves, and the opportunities for advancement continue to expand. We hope that readers will appreciate this Special Issue of Clinics and share our excitement in the study of azoospermia.
& REFERENCES 1. Kumar M, Kumar K, Jain S, Hassan T, Dada R. Novel insights into the genetic and epigenetic paternal contribution to the human embryo. Clinics. 2013;68(S1):5-14, http://dx.doi.org/10.6061/clinics/2013(Sup01)02. 2. Cocuzza M, Alvarenga C, Pagani R. The epidemiology and etiology of azoospermia. Clinics. 2013;68(S1):15-26, http://dx.doi.org/10.6061/ clinics/2013(Sup01)03. 3. Gudeloglu A, Parekattil SJ. Update in the evaluation of the azoospermic male. Clinics. 2013;68(S1):27-34, http://dx.doi.org/10.6061/clinics/2013 (Sup01)04. 4. Aziz N. The importance of semen analysis in the context of azoospermia. Clinics. 2013;68(S1):35-8, http://dx.doi.org/10.6061/clinics/2013(Sup01)05. 5. Hamada AJ, Esteves SC, Agarwal A. A comprehensive review of genetics and genetic testing in azoospermia. Clinics. 2013;68(S1):39-60, http://dx. doi.org/10.6061/clinics/2013(Sup01)06. 6. Baker K, Sabanegh Jr E. Obstructive azoospermia: reconstructive techniques and results. Clinics. 2013;68(S1):61-73, http://dx.doi.org/10. 6061/clinics/2013(Sup01)07. 7. Kumar R. Medical management of non-obstructive azoospermia. Clinics. 2013;68(S1):75-9, http://dx.doi.org/10.6061/clinics/2013(Sup01)08. 8. Fraietta R, Zylberstejn DS, Esteves SC. Hypogonadotropic Hypogonadism Revisited. Clinics. 2013;68(S1):81-8, http://dx.doi.org/10. 6061/clinics/2013(Sup01)09. 9. Inci K, Gunay LM. The role of varicocele treatment in the management of non-obstructive azoospermia. Clinics. 2013;68(S1):89-98, http://dx.doi. org/10.6061/clinics/2013(Sup01)10. 10. Esteves SC, Miyaoka R, Orosz JE, Agarwal A. An update on sperm retrieval techniques for azoospermic male. Clinics. 2013;68(S1):99-110, http://dx.doi.org/10.6061/clinics/2013(Sup01)11. 11. Miyaoka R, Esteves SC. Predictive factors for sperm retrieval and sperm injection outcomes in obstructive azoospermia: Do etiology, retrieval techniques and gamete source play a role? Clinics. 2013;68(S1):111-9, http://dx.doi.org/10.6061/clinics/2013(Sup01)12. 12. Glina S, Vieira M. Prognostic factors for sperm retrieval in nonobstructive azoospermia. Clinics. 2013;68(S1):121-4, http://dx.doi.org/ 10.6061/clinics/2013(Sup01)13. 13. Popal W, Nagy ZP. Laboratory processing and intracytoplasmic sperm injection using epididymal and testicular spermatozoa: what can be done to improve outcomes? Clinics. 2013;68(S1):125-30, http://dx.doi.org/10. 6061/clinics/2013(Sup01)14. 14. Gangrade BK. Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes in assisted conception. Clinics. 2013;68(S1):131-40, http://dx.doi.org/10.6061/clinics/2013(Sup01)15. 15. Esteves SC, Agarwal A. Reproductive outcomes, including neonatal data, following sperm injection in men with obstructive and nonobstructive azoospermia: case series and systematic review. Clinics. 2013;68(S1):141-9, http://dx.doi.org/10.6061/clinics/2013(Sup01)16. 16. Vloeberghs V, Verheyen G, Tournaye H. Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction? Clinics. 2013;68(S1):1516, http://dx.doi.org/10.6061/clinics/2013(Sup01)17. 17. Aponte PM, Schlatt S, Franca LR. Biotechnological approaches to the treatment of aspermatogenic men. Clinics. 2013;68(S1):157-67, http://dx. doi.org/10.6061/clinics/2013(Sup01)18.
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REVIEW
Novel insights into the genetic and epigenetic paternal contribution to the human embryo Manoj Kumar, Kishlay Kumar, Shalu Jain, Tarannum Hassan, Rima Dada Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences.
The integrity of the sperm genome and epigenome are critical for normal embryonic development. The advent of assisted reproductive technology has led to an increased understanding of the role of sperm in fertilization and embryogenesis. During fertilization, the sperm transmits not only nuclear DNA to the oocyte but also activation factor, centrosomes, and a host of messenger RNA and microRNAs. This complex complement of microRNAs and other non-coding RNAs is believed to modify important post-fertilization events. Thus, the health of the sperm genome and epigenome is critical for improving assisted conception rates and the birth of healthy offspring. KEYWORDS: Messenger RNA (mRNA); DNA damage; epigenome; DNA integrity; telomere. Kumar M, Kumar K, Jain S, Hassan T, Dada R. Novel insights into the genetic and epigenetic paternal contribution to the human embryo. Clinics. 2013;68(S1):5-14. Received for publication on August 18, 2012; Accepted for publication on August 20, 2012 E-mail: rima_dada@yahoo.co.in Tel.: 91 11 2658-8500 ext. 3517
all of which have a significant influence on the developing sperm cell. Sperm require these changes not only to shield the DNA throughout spermatogenesis but apparently, they also require these changes to contribute to the developmental program of the future embryo. Damage to genetic constituents and perturbations in the maintenance of these epigenetic changes have been demonstrated to affect fertilization potential and the early development of the embryo (7). This chapter will focus on the possible genetic and epigenetic contributions of sperm to the development of the human embryo.
& INTRODUCTION The paternal contribution to an embryo plays an important role in understanding early developmental processes and their effect on the health of a child. Sperm cells are highly differentiated, polarized and specialized, containing only the constituents required during and after fertilization (early embryonic development). However, the contribution of the male gamete to embryogenesis has not been well investigated. For quite some time, sperm have been considered mere vectors that carry the paternal genetic component to the oocyte. However, the contribution of sperm to the embryo has recently been better elucidated, with accumulating evidence suggesting that various spermatozoal components actively participate in early human development (1-4). During fertilization, the sperm transmits not only nuclear DNA but also oocyte activation factor (OAF) (critical for fertilization), centrosomes (critical for cell division) (5), and a population of messenger RNA (mRNA) that are of critical developmental importance (1,6). Studies investigating the epigenetic modifications in the developing sperm cell have provided new insights that may establish a more critical role for the sperm epigenome in the developing embryo. These non-genetic modifications include DNA methylation, histone tail modifications, targeted histone retention and protamine incorporation into the chromatin,
& SPERM GENETIC FACTORS Spermatozoal chromatin: effects on the developing embryo The maturation of spermatozoa consists primarily of three important phases: the initial proliferative phase, the meiotic division of the chromosomes, and the final maturation step (spermiogenesis). Of the 2806106 human sperm normally ejaculated into the vagina, only 200 reach the ampullary region of the oviduct where fertilization takes place (8). Fewer than 1 in 10,000 sperm get close enough to the egg to complete the process of fertilization. However, even these highly competent sperm are frequently not sufficient for sustaining the later development of the embryo. Sperm cells are highly specialized vehicles for transporting chromatin cargo, which consists of DNA and its associated proteins. The chromatin in mammalian sperm can broadly be divided into three major structural domains: (1) the vast majority of sperm DNA is coiled into toroids by protamines, (2) a much smaller percent (5-15%) remains bound to histones and thus retains its nucleosomal structure, and (3) DNA that is attached to the sperm nuclear matrix at MARs (matrix
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)02
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Sperm genome and epigenome in embryogenesis Kumar M et al.
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attachment regions) at intermediate intervals of approximately 50 kb throughout the genome (9). Sperm chromatin is essential for sperm function and subsequent embryonic development; defects in sperm chromatin have been linked to natural reproductive malfunctions, such as spontaneous abortion and assisted reproductive failure (10-12). These defects can include disrupted DNA integrity, which may affect fecundity and embryo growth, leading to embryo loss (13-15). Furthermore, the alteration of chromatin-associated proteins contributes to decreased fertility and poor embryonic growth (16). Most of the techniques used to detect sperm chromatin defects only detect gross defects in DNA integrity (17), while the roles of the associated proteins remain a mystery. Here, we will first discuss the role of sperm DNA and its associated proteins during embryonic growth. DNA damage (also referred to as DNA denaturation or fragmentation) is a common feature of human spermatozoa that affects DNA quality. Sperm DNA damage is thought to be induced by several mechanisms: (1) apoptosis during the process of spermatogenesis; (2) DNA breaks generated during the remodeling of sperm chromatin during the process of spermiogenesis; (3) post-testicular DNA fragmentation, which is induced by oxygen radicals, including the hydroxyl radical and nitric oxide, during sperm transport through the seminiferous tubules and the epididymis; (4) DNA fragmentation induced by endogenous caspases and endonucleases; (5) radiotherapy and chemotherapy; and (6) environmental toxicants and xenobiotics (18,19). Much concern has been expressed regarding the influence of sperm DNA integrity on abnormal reproductive outcomes (20,21). However, one of the well-established and extensively studied causes of DNA damage is oxidative stress, which causes the oxidation of the DNA bases and leads to the formation of various types of DNA adducts. These DNA adducts alter the function of the sperm genome, ultimately influencing in vivo or in vitro conception and the subsequent events that occur during early development (22). DNA fragmentation is more frequent in caudal epididymal and ejaculated sperm than testicular sperm (23-25). The persistence of high rates of DNA damage in ejaculated sperm is obvious, as mature spermatozoa possess a limited capacity to repair oxidative DNA lesions (26). It has been suggested that mammalian spermatozoa contain a mechanism by which they can digest their own DNA upon exposure to a stressful environment (27); however, the question of whether this mechanism is sufficient to cope with extensive damage arises. The answer lies in several studies postulating that the presence of a critical level of unrepaired DNA damage in embryos generated in vivo/in vitro explains the block in embryo development (28). The authors of these studies have arrived at the conclusion that a ‘‘late paternal effect’’ is responsible for blocking embryo development, which is evident from the outcomes of various assisted reproductive techniques (ARTs). The fragmentation of sperm DNA affects post-implantation embryonic development in ICSI procedures; specifically, high levels of sperm DNA fragmentation can compromise the viability of an embryo, resulting in pregnancy loss (21,29). Sperm carrying damaged DNA can complete the initial process of fertilization; however, the developmentally necessary genes in the damaged sperm DNA may hinder embryonic development upon activation of the embryonic genome at the 3-cell stage. Furthermore, sperm DNA damage has been linked to delayed chromosomal instability
in blastocysts and post-implantation developmental abnormalities (30,31). However, very little is currently known regarding the nature of germline DNA damage and the extent to which germ cells are capable of eliminating damaged DNA and completing the process of DNA repair. Oocytes have been shown to possess different repair pathways to handle a certain level of DNA damage in sperm (32). There is little information regarding the fidelity and nature of this oocyte repair mechanism, but these mechanisms are negatively impacted by age. The majority of couples with reduced reproductive fitness may be of advanced age, and women above 35-40 years of age may have a defective DNA repair system that may not be able to repair extensive DNA damage. Oocyte repair mechanisms may also be inhibited by the accumulation of oxidized bases, such as ethenonucleosides, in the sperm genome. The accumulation of such DNA adducts is mutagenic and, if not corrected, may increase the mutagenic load in early embryos. The analysis of sperm DNA fragmentation is a potentially valuable method for explaining the paternal origin of some unexplained and repeated ICSI fertilization and implantation failures (29). Such analysis may even help to determine the most efficient ART procedure (33) to reduce the probability of negative paternal contribution. A recent systematic review and meta-analysis of 2,969 couples showed a significant association between the level of DNA damage and pregnancy loss in both ART and spontaneous conceptions (34). These data affirm the clinical indication for the evaluation of sperm DNA damage prior to infertility treatment and the need to investigate the association between sperm DNA damage and recurrent pregnancy loss (10,35). The intact structural framework of chromatin, which consists of molecular regulatory factors, is also required for proper embryonic development (9). Among the three types of sperm chromatin structures discussed earlier, histonebound sperm DNA and MARs are inherited by the embryo and are most likely required for proper development. At least 2-15% of mammalian sperm chromatin is bound to histones rather than protamines (36-38). Histones are interspersed throughout the genome, primarily located at gene promoters (39). Another independent study also concluded that entire gene families important for embryo development are preferentially associated with histones in human spermatozoa (38). However, it will be interesting to know whether sperm histones are transmitted to the developing embryo. van der Heijden et al. (40,41) demonstrated that histones with specific modifications in the sperm cell are present in the paternal pronucleus, suggesting that these histones were never replaced. While the protamines in sperm chromatin are replaced with histones supplied by the oocyte after fertilization (42,43), this may not be necessary in regions where histones are already present in the sperm DNA. In the sperm nucleus, the chromatin is organized into loop domains that are attached to a proteinaceous structure, termed the nuclear matrix, every 20-120 kb. This organizes the chromatin into functional loops of DNA that help regulate DNA replication and gene transcription (9). Several studies have demonstrated a functional role for the sperm nuclear matrix during early embryogenesis. These data suggest two roles for the sperm nuclear matrix (44-47): to facilitate the proper association of DNA with the nuclear matrix (required for paternal pronuclear DNA replication in
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the one-cell embryo) and to serve as a checkpoint for sperm DNA integrity after fertilization.
expressed by the oocyte after fertilization. These nontesticular RNA-containing sperm can take up DNA and RNA in vitro; these molecules are not only delivered to but also expressed in the fertilized oocyte (72,73). This in vitro experiment showed that, in theory, foreign sperm RNA could play a role in early embryogenesis. Furthermore, the fact that some of these spermatozoal mRNAs are also found in zygotes indicates that these transcripts may be functionally important (74). Rassoulzadegan et al. (75) were the first to prove that sperm RNA can influence embryo development by studying the Kit gene-derived heritable effect, which appeared to be affected in heterozygous mice carrying a wild-type Kit allele and a silent Kit allele. This work was followed by a study showing that the pathological overexpression of Cdk9, a key regulator of cardiac growth in the mouse, could be induced and heritably transmitted following the intraooplasmic injection of RNAs targeting the gene, including the sequence-related expression of miR-1 miRNA. miR-1 is among a number of small inhibitory RNAs that are present in sperm (76). A recent report also demonstrated that some mRNAs can be translated de novo, supporting the hypothesis that a population of mRNAs may have a function during or beyond the process of fertilization (77). As there is no evidence that the proteins encoded by the majority of mRNAs found in mature spermatozoa are also present in sperm (67), these mRNAs may be viewed as potential contributors to early embryogenesis. In addition to mRNA, spermatozoa are enriched in antisense RNAs and microRNAs (miRNAs). These miRNAs have been detected in human spermatozoa, raising the possibility that these molecules may play a role in early fertilization events (6) and embryo development by regulating the expression of various genes. For example, sperm-borne microRNA-34c is required for the first cleavage division in mice (78). A recent survey of small RNAs in sperm also revealed a complex population of male-derived sncRNAs (small, non-coding RNAs) that are available for delivery upon fertilization (79). In addition to finding the miRNA class previously detected in mature mouse, porcine, and human spermatozoa (6,80,81), this survey also identified piRNAs for the first time (79). Any alteration in the amount or composition of sperm mRNAs may indicate abnormalities in spermatogenesis, which may later affect embryo development. The mRNA fingerprints of normozoospermic and teratozoospermic men have been shown to differ (82). Additionally, variations in the expression of two sperm RNAs coding for LDHC transcript variant 1 and TPX1 have been reported in men with poor sperm motility (83). Ostermeier et al. detected over 3,000 mRNA species in ejaculated spermatozoa through microarray analysis (84). The expression profiling of human spermatozoa by serial analysis of gene expression (SAGE) revealed 389 clustered genes, with a highly selective grouping among the most abundant SAGE tags (85). Approximately 25% of these tags (96) were related to DNA-dependent transcription or transcriptional regulation. In addition, a comparison of the spermatozoa used in homologous intrauterine insemination showed a difference in the transcripts of the two groups (patients achieving pregnancy versus those who did not; both fresh and frozen spermatozoa were used) (86). The authors found 741 exclusive transcripts that were expressed only in the pregnant group and 976 transcripts that were expressed only in the non-pregnant group. Several studies have reported that certain sperm transcripts do have an important role in
& SPERMATOZOAL TRANSCRIPTS: HIDDEN MESSENGERS In the previous section, the importance and contributions of sperm chromatin were discussed. In this section, we will further elaborate the role of sperm RNAs and their indispensability to the spermatozoa and embryo development. In the late 1950s and early 1960s, controversy regarding the role of sperm RNAs arose, and the presence of these molecules was questioned. Various landmark studies in the 1970s concluded that bovine spermatozoa were transcriptionally active but this activity was localized to the mitochondria (48-50). Contributing to these controversies, Pessot et al. demonstrated the presence of nuclear RNA in rat and human sperm. The RNA was extracted from sperm and analyzed by electrophoresis on a 10% polyacrylamide gel and 7 M urea. The electrophoretic profile revealed a complex set of bands ranging in size from tRNA to high-molecular-weight components. On average, an RNA content of approximately 0.1 pg per rat or human sperm was found (51). The repackaging of DNA into a nucleotorroidal conformation, which is approximately 20 times more condensed, enables the complete shutdown of the spermatid nucleus (52,53). The gradual shutdown of RNA transcription begins during meiosis, when the paired sex chromosomes are accommodated in the male germ cell, leading to the repression of gene expression on the X and Y chromosomes (54,55). The shutdown of transcriptional activity in human spermatozoa has been confirmed (56), strengthening the previous observations. The retention of mRNAs in spermatozoa begins to occur during the early stages of spermatogenesis. In the late 1990s, various studies documented the presence of different types of RNAs in human spermatozoa. Kumar et al. first documented the presence of c-MYC mRNA in the mid-piece and tail region of human spermatozoa (57). mRNAs coding for the following molecules have been found in human ejaculate spermatozoa: HLA (58), integrins (59), cyclic nucleotide phosphodiesterases (60), the L-type calcium channel and Ncadherin (61,62), estrogen and progestin-like receptors (63,64), nitric oxide synthase (NOS) (65), and, surprisingly, insulin (66), among others (67). Complex RNA populations have also been reported in the sperm of cows (68,69) and human spermatozoa (1,70,71). These mRNAs were assumed to be residues left over from spermatogenesis, but there is evidence that the spermatozoa deliver a unique set of mRNAs to the oocyte. Three different types of sperm mRNA have been discovered (70). The first group of mRNAs has a specific function during spermatogenesis but does not exhibit an obvious function post-fertilization. The presence of this set of remnant mRNAs could serve as a diagnostic tool with which to follow the fidelity of the later phases of spermatogenesis (71). A second group of RNAs (e.g., mRNA coding for PLC-z) also originates from the testicular germ cells and may have an additional role in the fertilized oocyte. A third not yet extensively studied group of sperm mRNAs (e.g., mRNA coding for clusterin) may originate from a non-testicular source and, after incorporation into the sperm, could be introduced into the oocyte during fertilization. It is of considerable interest that foreign RNA constructs can be introduced into the sperm cell to be
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early embryogenesis. The presence of mRNA transcripts encoding PSG-1 and HLA-E in human spermatozoa has previously been confirmed (84,85). Conversely, microarray analysis of the transcriptome of the human oocyte did not demonstrate the presence of PSG-1 mRNA and showed that HLA-E mRNA was down-regulated 87). An investigation of these transcripts showed significantly higher levels of PSG1 and HLA-E mRNA in the fertile group than the infertile group (88). In accordance with this study, a preliminary examination of the PSG1 gene also showed a lower level of PSG1 expression in the male partners of couples experiencing recurrent pregnancy loss than in fertile men (in press). It has been speculated that the PSG1 protein may play a crucial role in supporting early gestation and protecting the fetus from the maternal immune system. This hypothesis is supported by the fact that PSG1 is able to modulate monocyte/ macrophage metabolism to regulate T-cell activation and proliferation. In addition, PSG1 induces the secretion of antiinflammatory cytokines by monocytes (89). We recently investigated the expression of a few important genes (WNT5A, HSP90, and PRM2) that have been postulated to have a critical role in early development (unpublished). The PRM2 and HSP90 expression patterns were significantly altered in the male partners of couples with idiopathic recurrent pregnancy loss, while no association with recurrent pregnancy loss was found for WNT5A. Scientists investigating the heat-shock response (HSR) have focused on developmental processes because of the remarkably unusual characteristics of heat shock protein (Hsp) expression in pre-implantation embryos and gametogenesis. A striking Hsp expression pattern is exhibited in embryos during gametogenesis and in stem cell and differentiation models, and the expression of these proteins was shown to be stagespecific in both tissue models and male germ cells, the latter of which exhibited impaired abilities to mount a classical HSR. In addition, spermatogenesis and pre-implantation embryos showed extreme sensitivity to heat stress. The basal levels of HSFs and, even more interestingly, the ratios between different HSFs, which could vary from one individual to another, could contribute to reproductive success versus infertility or developmental success versus failure in humans. Our study revealed significantly higher levels of HSP90 and significantly lower PRM2 levels in the male partners of couples with idiopathic recurrent pregnancy loss. This result may be due to higher levels of free radicals causing oxidative stress, which is compensated for by increased HSP90 expression. High free radical levels in spermatozoa may cause a pronuclear block, impair cleavage and lead to blastomere fragmentation and poor-quality blastocysts. The glutathione system (GS) is an oxidative stress defense system in sperm that is specifically controlled by GPX family members and has been correlated with embryo morphology on day 3. The results of this study indicated that sperm-derived mRNA may condition the human embryo and persist to the cleavage stage (90). As explained earlier, protamines play a crucial role in the condensation of sperm chromatin and the protection of the paternal genome from internal and external environmental insults. Altered PRM2 expression is reported among men with poor fertilizing capacity (91), and a lack of PRM2 leads to sperm DNA damage and embryo death in mice (92). While high-throughput technologies have provided a glance at the mRNA population contained in spermatozoa, future studies should focus on the functional aspects of
these RNAs in the growing embryo. The results from such studies will further strengthen the correlation between the mRNA fingerprint of sperm and embryogenesis. Telomeres are evolutionarily conserved tandem hexameric repeats at the ends of chromosomes that maintain genomic integrity and chromosome stability. Telomeres serve as a biological clock and undergo attrition at a rate of 50-200 bp per cell division. The telomeres in germ cells are 10-20 kb in length, compared with 5-10 kb in somatic cells. The inheritance of telomere length in the embryo is a complex trait codetermined by the length of the telomeres in the sperm and ovum, the age of the father, free radical levels and gender. Tandem telomere repeats are rich in guanine, the nucleotide with the lowest oxidative potential and, thus, the most susceptibility to oxidative damage. We have previously shown high levels of free radicals to be associated with DNA damage in the semen and sperm, as well as shorter telomeres in the male partner of infertile couples and couples experiencing idiopathic recurrent pregnancy loss. As telomeres are histone-bound and located in the periphery of the sperm nucleus, telomeres are highly susceptible to oxidative damage. This induces GC to TA transitions, single- and double-strand breaks and accelerated telomere shortening. Inheriting shortened telomeres from the father may thus result in impaired cleavage and embryonic development. Zalenskaya et al. reported that sperm telomeres are the first structures to respond to the oocyte signal for pronucleus formation (93), and Rodriguez and colleagues (94) later reported that shortened sperm telomeres are associated with sperm DNA fragmentation and abnormal embryonic development. Concordance in telomere length is required for synapsis, homologous recombination, and normal chromosome segregation. Sperm with shortened telomeres show segregation abnormalities and nondisjunction, thus giving rise to aneuploid sperm after meiosis. Shortened telomere can increase the incidence of offspring with major or minor congenital malformations, childhood cancers, perinatal morbidity, developmental delay, and failure to thrive. In an ongoing study in our laboratory, we have found significantly shortened telomeres in the male partners of couples experiencing idiopathic recurrent pregnancy loss. We did not find an association with high levels of reactive oxygen species in this pilot study; however, this was a very clinically significant finding, and studies are still ongoing to further validate this result.
& THE SPERM EPIGENOME Genomic imprinting is a parental, origin-specific, genemarking phenomenon that is crucial for normal mammalian development. Imprinted genes are characterized by epigenetic modifications (95,96), including DNA methylation, and are associated with differentially methylated regions (DMRs) that are methylated on either the paternal or maternal allele. Epigenetics refers to phenotypic changes that are caused by mechanisms other than changes in the DNA sequence (thus the name epi- (above or over) genetics). The methylation of primary DMRs is presumed to be maintained throughout embryonic development, including the pre-implantation stages, during which extensive demethylation of the genome takes place. Recent studies have demonstrated that sperm have unique and potentially important epigenetic modifications.
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Male germ cells undergo unique and extensive chromatin and epigenetic remodeling soon after their specification (determination to become a spermatocyte) and during the differentiation process to become a mature spermatozoon (97). The epigenetic program of sperm is unique and tailored to meet the needs of this highly specialized cell. The unique nuclear protein landscape in sperm creates a chromatin structure that is between six and 20 times more dense than nucleosome-bound DNA, ultimately resulting in a tightly condensed nucleus (53,98). Although the mechanisms regulating and orchestrating specification and spermiogenesis remain poorly understood, some progress has been made in elucidating the molecular changes associated with these complex cellular changes. Recent studies analyzing sperm DNA methylation, histone modifications and the RNA transcripts in spermatozoa have further established the role of the sperm epigenetic program in the developing embryo. The importance of DNA methylation has been demonstrated globally, regionally, and at the single locus level in both humans and animal models. Multiple targeted studies have been performed in animal models to establish a clear role of DNA methylation in sperm and embryos. El Hajj et al. (99) suggested that the improper methylation of repetitive elements may be linked to recurrent pregnancy loss. Methylation abnormalities in the CREM promoter were observed in a subset of patients with protamine ratio abnormalities, as well as in patients presenting with various forms of male factor infertility (100). Recent data demonstrate that the aberrant methylation of promoters for specific genes (e.g., DAZL and MTHFR) and general gene classes, such as imprinted loci, is strongly associated with various forms of infertility and sperm defects in men (101-103). Twin studies have played an essential role in enabling the estimation of phenotypic heritability, and these studies now offer an opportunity to study epigenetic variation as a dynamic quantitative trait. Monozygotic (MZ) twin studies have proven to be very effective in answering key questions ranging from the genetics of social behavior and the nature versus nurture question to the heritability of phenotypic variation and disorders (104,105). Several studies have examined DNA methylation patterns in twins, and a recent study found that MZ twins exhibit a considerable degree of variability in DNA methylation patterns, which may impact the variability of gene expression and possible differences in disease susceptibility. Along with the current interest in CpG methylation, recent data have suggested that the intermediates formed during DNA demethylation may be important epigenetic regulators. Most prominent among these intermediates is 5hydroxymethylcytosine (5-hmC). A recent study by Pastor et al. revealed a pattern of 5-hmC enrichment in transcriptionally poised genes in stem cells (106). This unique localization suggests that 5-hmC has a role in embryonic stem cells and possibly the epigenome of multiple other cell types. The timing of the establishment and removal of methylation is critical to normal spermatogenesis. During cell division, the DNA in male germ cells is packaged in nucleosomes comprised of histone 2A (H2A), histone 2B (H2B), histone 3 (H3) and histone 4 (H4), all of which are susceptible to covalent modifications. Histones are basic proteins in eukaryotic nuclei that package DNA into nucleosomes. The H2A, H2B, H3 and H4 histones are
integral components of nucleosomes. Histone modifications, such as acetylation, methylation, ubiquitylation and phosphorylation, have emerged as the main players regulating epigenetic modifications. Each of these chemical modifications to histones can influence gene repression and/or activation. In post-implantation mammalian embryos, pluripotent cells in the epiblast give rise to primordial germ cells (PGCs). Germ cells undergo several changes in their epigenetic profile during the different stages of meiosis. PGCs enter mitotic arrest in males, whereas PGCs are arrested in prophase of meiosis I in females. Global nuclear remodeling occurs in haploid round spermatids, although some histone marks, such as H3K9me2, on the inactive X chromosome are retained (107,108). The testis-specific linker histone variant H1T2 appears at this stage and plays a crucial role in chromatin condensation during spermiogenesis (108). Later, the linker histone variant HIls1 (histone-1like protein in spermatids 1) is expressed in elongated spermatids. In the histone-protamine exchange process, nuclear histones become hyperacetylated during spermiogenesis and disassemble shortly thereafter, replaced by transition proteins (TP1 and TP2). In the final stage of spermiogenesis, the transition proteins are removed and replaced by protamines (109). The incorporation of protamines into sperm chromatin induces DNA compaction, which is important for the formation of spermatozoa and for providing a safe environment for the genome. The presence of somatic-like chromatin in the sperm nucleus could transmit different epigenetic information to offspring. Oakes et al. (110) suggested that the genome-wide DNA methylation pattern observed during spermiogenesis changes little after the pachytene spermatocyte stage. Many epigenetic modifiers, including DNA methyltransferases, histone modification enzymes and their regulatory proteins, play essential roles in germ cell development. Some of these modifiers are specifically expressed in germ cells, whereas others are more widely expressed. The crucial roles of germ-cell-specific genes, such as Dnmt3L and Prdm9, were revealed by conventional knockout studies (111-113). A recent report showed numerous intra- and inter-individual differences in the DNA methylation of human sperm samples, which could contribute to phenotypic differences in offspring. Furthermore, it has been reported that sperm samples from oligospermic patients often contain DNA methylation defects at imprinted loci (114,115). Kobayashi and his associates reported methylation defects in 17 of 78 embryos conceived by ART. He found that seven of these 17 embryos had inherited these altered methylation patterns paternally, while the rest were believed to have resulted from the ART process. The altered hormonal milieu associated with ART and the retrieval of epigenetically immature oocytes may result in an increase in epigenetic/imprinting defects in children conceived through ART. Imprinting errors in the developing fetus have been identified and shown to cause severe pathologies. Evidence suggests that Prader-Willi syndrome (PWS) and Angelman syndrome (AS) arise from the functional loss of several paternally expressed genes. The Prader-Willi/Angelman imprinted domain on human chromosome 15q11-q13 is regulated by an imprinting control regions (ICRs). Imprinting defects affecting the PWS/AS region can arise from the failure to demethylate the PWS-SRO in the male germ line, the failure to methylate the maternal PWS-SRO,
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embryogenesis or that are representative of the genome at large and are predictive of abnormal epigenetic modifications. Future studies will likely focus on the epigenetics of both gametes, as well as on the changes observed throughout embryogenesis. Thus, investigating the effects of genetic and epigenetic alterations in sperm and how these modifications arise and affect embryonic genes will be important in the prevention, diagnosis and treatment of disease.
or the failure to maintain PWS-SRO methylation after fertilization (116). Some studies have also suggested that the use of ART increases the risk of imprinting diseases, as the germ cells of infertile couples are prone to epigenetic instability. A pattern of decreased genome-wide methylation in sperm has also been associated with poor embryo quality in rats and decreased IVF pregnancy rates in humans (117). Benchaib et al. (117) used 5-methyl-cytosine immunostaining as an indicator of the genome-wide methylation pattern in sperm. He showed that decreased global methylation in semen samples from normozoospermic men was related to a poor pregnancy outcome from IVF (118), suggesting that global methylation status independently affects embryogenesis. Both the complex path of sperm production and the delicate balance of epigenetic and genetic factors during sperm maturation contribute to the formation of a mature sperm with the ability to fertilize an oocyte and contribute to the developing embryo. It has been proposed that the level of DNA methylation in human sperm could be linked to their ability to initiate a pregnancy in an assisted reproduction procedure (118). A defect at any one step may manifest as a syndrome of male infertility. The epigenome cycles through a series of precisely timed methylation changes during development, making the epigenome vulnerable to interference from environmental exposure (119). As the embryo grows, these parent-of-origin imprints are maintained in somatic tissues but erased in primordial germ cells so that imprints can be re-established in a sex-specific manner during gametogenesis. Histone modifications are thought to play a role in this sex-specific mark establishment, as the extensive loss of histone methylation and acetylation occurs along with the loss of DNA methylation (120). The methylation marks are then sustained throughout the individual’s lifetime until methylation marks are erased and re-established following fertilization of the next generation (120). Epigenetic programming plays an important role in an organism’s response to environmental stress during critical developmental periods (121). Epigenetic changes are not only heritable in somatic cells but can also be maintained during meiosis. Transgenerational epigenetic inheritance is governed by chromatin remodeling in Drosophila melanogaster (122), and the inheritance of coat color in successive generations of the Agouti viable yellow mouse is controlled by epigenetic mechanisms associated with the Agouti allele (123). The Agouti viable yellow (Avy) and Axin-Fused (AxinFu) mice are unique animal models that carry the Avy and AxinFu metastable epialleles, respectively. Researchers have used these mice to show that several nutritional and environmental exposures can alter epigenetic programming during gestation. For instance, exposure to methyl donors, such as folic acid, can hypermethylate the Avy and AxinFu alleles, leading to offspring mice that are brown (pseudoagouti) and to mice with straightened tails, respectively (124,125). The sperm cell has a highly differentiated and specialized morphology, and the epigenome of human sperm is unique, elegant and essential to embryogenesis. Epigenetic factors suggest that sperm play diverse and critical roles in regulating embryogenesis. Understanding the epigenetics of sperm and spermatogonia may be key in understanding the mechanisms of pluripotency, which has broad implications for potential therapies. Efforts should be aimed at identifying select candidate alleles that are key factors in
& EXPERT COMMENTARY Sperm are highly polarized cells that are both transcriptionally and translationally silent. At the time of fertilization, sperm transfer not only nuclear DNA but also oocyte activation factor, centrosomes, long-lived mRNAs, and small non-coding RNAs. It is believed that these mRNAs are transcripts for key developmental genes and that the small non-coding RNAs modify post-fertilization events. Due to its high polyunsaturated fatty acid content and the loss of the majority of cytosolic antioxidants, the mitochondrial and nuclear DNA of sperm are highly susceptible to oxidative damage. Mitochondria are the first site of free radical production and free radical-induced DNA damage. Thus, the sperm mtDNA is reduced to disposable elements at the time of fertilization. However, mtDNA accumulates sequence variations that produce high levels of free radicals, which damage both the mitochondrial and nuclear genomes. The mtDNA copy number in healthy sperm is 1-1.4, compared with approximately 5-10-fold more in sperm with morphological abnormalities and impaired motility. This high copy number of mtDNA when aging oocytes with a defective genetic filter are fertilized. The mtDNA copy number has a profound impact on the methylation pattern of nuclear genes. In a previous study from our laboratory, we established sperm DNA fragmentation indices of 30 and 26% in infertile males and the male partners of couple experiencing idiopathic recurrent pregnancy loss, respectively. The sperm genome is partitioned into a peripheral compartment that has histone-bound DNA, retains the nucleosomal structure and thus maintains imprints. This region of the genome contains promoters for developmentally important genes, transcription factors, signaling factors and microRNAs. The rest of the genome is packaged into a crystalline toroid by protamines. The retention of histones is not random but is significantly enriched in many developmentally important loci. Recent studies have also shown that the mtDNA copy number has a significant impact on the nuclear methylation pattern. Thus, one cannot study the nuclear genome in isolation because of the cross-talk between the mitochondrial and nuclear genomes of sperm. Telomeres are tandemly repeating hexameric units that cap chromosomal ends and are vital for genomic integrity and chromosomal stability. Concordance in telomere length in germ cells is necessary for synapsis, recombination and chromosome segregation. Rapid telomere attrition due to oxidative stress may result in meiotic recombination defects and segregation errors. This results in meiotic arrest and the generation of gametes with increased disjunction errors and aneuploidy. The inheritance of short telomeres from sperm may also impair cleavage, as optimal telomere length is a prerequisite for cell division. Short telomeres can also result in blastocysts with poor morphology. This may be one of the
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Sperm genome and epigenome in embryogenesis Kumar M et al. experimental work and helped in the manuscript drafting. Hassan T helped in the manuscript drafting.
key factors affecting pre- and post-implantation losses and the birth of offspring with major and minor congenital malformations and childhood cancers. Thus, the sperm genome is highly vulnerable to oxidative stress-induced DNA damage. Various lifestyle modifications (such as the increased intake of fruits and vegetables, exercise, meditation, yoga, cessation of smoking, and reduction in alcohol intake) can improve the health of the sperm genome and result in normal embryonic development and the birth of healthy offspring.
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& KEY ISSUES
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The integrity of the sperm genome and epigenome is critical for the birth of healthy offspring. The sperm nuclear genome is uniquely partitioned into compartments. These compartments include a central compact toroid, in which DNA is protamine-bound. This portion of the genome is transcriptionally and translationally inert. The peripheral compartment contains histone-bound DNA (5-15%) that retains the nucleosomal structure. This region contains promoters for developmentally important genes, microRNAs, and signaling factors. The histone-bound DNA is highly susceptible to environmental insults, especially oxidative damage. Telomeres, the nucleoprotein structures that constitute the biological molecular clock, cap the chromosomal ends and maintain chromosomal and genomic integrity. These guanine-rich repeats are highly susceptible to free radical-induced DNA damage. We believe that the rapid alteration of telomeres in the sperm genome underlies the etiology of infertility, which occurs as a result of accelerated telomere aging. Because the inheritance of telomere length is a complex trait, a short telomere length adversely affects cleavage and results in the generation of blastocysts with poor morphology. Sperm not only transfer the nuclear genome to oocytes but also transfer a stable population of developmentally important mRNAs. The sperm epigenome is maintained through the retention of histones, the compaction of major portions of the genome by protamines, DNA methylation, and covalent histone modifications. Because sperm lose the majority of cytosolic antioxidants at the time of spermiogenesis, sperm cells are highly vulnerable to free radical-induced DNA damage. Lower levels of key DNA repair enzymes have also been found in sperm (unpublished study from our laboratory). This finding may explain the persistence of DNA damage in sperm. The fertilization of oocytes by such sperm, either spontaneously or through assisted reproductive techniques, may help us to understand the pathogenesis of congenital malformations, childhood cancers and perinatal morbidity.
& AUTHOR CONTRIBUTIONS Dada R designed the study and the data acquisition procedure for determining telomere length, performed the DFI analysis, and drafted and revised the manuscript. Kumar K contributed to the experimental work and the drafting and revision of the manuscript. Kumar M contributed to the drafting and revision of the manuscript. Jain S performed the
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Abnormal methylation of the promoter of CREM is broadly associated with male factor infertility and poor sperm quality but is improved in sperm selected by density gradient centrifugation. Fertil Steril. 2011;95(7):2310-4, http://dx.doi.org/10. 1016/j.fertnstert.2011.03.096. 101. Navarro-Costa P, Nogueira P, Carvalho M, Leal F, Cordeiro I, CalhazJorge C, et al. Incorrect DNA methylation of the DAZL promoter CpG island associates with defective human sperm. Hum Reprod. 2010;25(10):2647-54, http://dx.doi.org/10.1093/humrep/deq200. 102. Pacheco SE, Houseman EA, Christensen BC, Marsit CJ, Kelsey KT, Sigman M, et al. Integrative DNA methylation and gene expression analyses identify DNA packaging and epigenetic regulatory genes associated with low motility sperm. PLoS One. 2011;6(6):e20280, http:// dx.doi.org/10.1371/journal.pone.0020280. 103. Wu H, D’Alessio AC, Ito S, Wang Z, Cui K, Zhao K, et al. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 2011;25(7):679-84, http://dx.doi.org/10.1101/gad.2036011. 104. Ebstein RP, Israel S, Chew SH, Zhong S, Knafo A. Genetics of human social behavior. Neuron. 2010;65(6):831-44, http://dx.doi.org/10.1016/ j.neuron.2010.02.020. 105. Torgersen S. The nature (and nurture) of personality disorders. Scand J Psychol. 2009;50(6):624-32. 106. Pastor WA, Pape UJ, Huang Y, Henderson HR, Lister R, Ko M, et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature. 2011;473(7347):394-7, http://dx.doi.org/10.1038/ nature10102. 107. Turner JM, Mahadevaiah SK, Ellis PJ, Mitchell MJ, Burgoyne PS. Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev Cell. 2006;10(4):521-9, http://dx.doi.org/10.1016/j.devcel.2006.02.009. 108. Namekawa SH, Park PJ, Zhang LF, Shima JE, McCarrey JR, Griswold MD, et al. Postmeiotic sex chromatin in the male germline of mice. Curr Biol. 2006;16(7):660-7, http://dx.doi.org/10.1016/j.cub.2006.01.066. 109. Rousseaux S, Caron C, Govin J, Lestrat C, Faure AK, Khochbin S. Establishment of male-specific epigenetic information. Gene. 2005;345(2):139-53, http://dx.doi.org/10.1016/j.gene.2004.12.004. 110. Oakes CC, La Salle S, Smiraglia DJ, Robaire B, Trasler JM. A unique configuration of genome-wide DNA methylation patterns in the testis. Proc Natl Acad Sci U S A. 2007;104(1):228-33, http://dx.doi.org/10. 1073/pnas.0607521104. 111. Hayashi K, Yoshida K, Matsui Y. A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature. 2005;438(7066):374-8, http://dx.doi.org/10.1038/nature04112. 112. Hata K, Okano M, Lei H, Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. 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121. Mathers JC, McKay JA. Epigenetics - potential contribution to fetal programming. Adv Exp Med Biol. 2009;646:119-23, http://dx.doi.org/ 10.1007/978-1-4020-9173-5_13. 122. Ruden DM, Lu X. Hsp90 affecting chromatin remodeling might explain transgenerational epigenetic inheritance in Drosophila. Curr Genomics. 2008;9(7):500-8, http://dx.doi.org/10.2174/138920208786241207. 123. Blewitt ME, Vickaryous NK, Paldi A, Koseki H, Whitelaw E. Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice. PLoS Genet. 2006;2(4):e49, http://dx.doi.org/10.1371/ journal.pgen.0020049. 124. Waterland RA, Travisano M, Tahiliani KG. Diet-induced hypermethylation at agouti viable yellow is not inherited transgenerationally through the female. FASEB J. 2007;21(12):3380-5, http://dx.doi.org/10. 1096/fj.07-8229com. 125. Cooney CA, Dave AA, Wolff GL. Maternal methyl supplements in mice affect epigenetic variation and DNA methylation of offspring. J Nutr. 2002;132(8 Suppl):2393S-400S. 126. Martin LA, Davies GL, Weigel MT, Betambeau N, Hills MJ, Salter J, et al. Pre-surgical study of the biological effects of the selective cyclooxygenase-2 inhibitor celecoxib in patients with primary breast cancer. Breast Cancer Res Treat. 2010;123(3):829-36, http://dx.doi.org/10.1007/ s10549-010-1100-z.
114. Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet. 2004;363(9422):1700-2, http://dx. doi.org/10.1016/S0140-6736(04)16256-9. 115. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet. 2007;16(21):2542-51, http://dx.doi. org/10.1093/hmg/ddm187. 116. Horsthemke B, Wagstaff J. Mechanisms of imprinting of the PraderWilli/Angelman region. Am J Med Genet A. 2008;146A(16):2041-52. 117. Benchaib M, Ajina M, Lornage J, Niveleau A, Durand P, Guerin JF. Quantitation by image analysis of global DNA methylation in human spermatozoa and its prognostic value in in vitro fertilization: a preliminary study. Fertil Steril. 2003;80(4):947-53, http://dx.doi.org/ 10.1016/S0015-0282(03)01151-8. 118. Benchaib M, Braun V, Ressnikof D, Lornage J, Durand P, Niveleau A, et al. Influence of global sperm DNA methylation on IVF results. Hum Reprod. 2005;20(3):768-73, http://dx.doi.org/10.1093/humrep/deh684. 119. Murphy SK, Jirtle RL. Imprinting evolution and the price of silence. Bioessays. 2003;25(6):577-88, http://dx.doi.org/10.1002/bies.10277. 120. Weaver JR, Susiarjo M, Bartolomei MS. Imprinting and epigenetic changes in the early embryo. Mamm Genome. 2009;20(9-10):532-43, http://dx.doi.org/10.1007/s00335-009-9225-2.
14
REVIEW
The epidemiology and etiology of azoospermia Marcello Cocuzza, Conrado Alvarenga, Rodrigo Pagani Universidade de Sa˜o Paulo, Faculdade de Medicina, Department of Urology, Sa˜o Paulo/SP, Brazil.
The misconception that infertility is typically associated with the female is commonly faced in the management of infertile men. It is uncommon for a patient to present for an infertility evaluation with an abnormal semen analysis report before an extensive female partner workup has been performed. Additionally, a man is usually considered fertile based only on seminal parameters without a physical exam. This behavior may lead to a delay in both the exact diagnosis and in possible specific infertility treatment. Moreover, male factor infertility can result from an underlying medical condition that is often treatable but could possibly be life-threatening. The responsibility of male factor in couple’s infertility has been exponentially rising in recent years due to a comprehensive evaluation of reproductive male function and improved diagnostic tools. Despite this improvement in diagnosis, azoospermia is always the most challenging topic associated with infertility treatment. Several conditions that interfere with spermatogenesis and reduce sperm production and quality can lead to azoospermia. Azoospermia may also occur because of a reproductive tract obstruction. Optimal management of patients with azoospermia requires a full understanding of the disease etiology. This review will discuss in detail the epidemiology and etiology of azoospermia. A thorough literature survey was performed using the Medline, EMBASE, BIOSIS, and Cochrane databases. We restricted the survey to clinical publications that were relevant to male infertility and azoospermia. Many of the recommendations included are not based on controlled studies. KEYWORDS: Male Infertility; Azoospermia; Semen Analysis. Cocuzza M, Alvarenga C, Pagani R. The epidemiology and etiology of azoospermia. Clinics. 2013;68(S1):15-26. Received for publication on March 5, 2012; Accepted for publication on March 29, 2012 E-mail: mcocuzza@uol.com.br Tel.: 55 11 2661-8080
cost benefits, risks and prognosis for treatment success. Clinicians should also provide adequate counseling for the couple and generous support for patients with severe male factor infertility. In the past, men with azoospermia were classified as infertile, and a sperm donor was initially considered one of the best options for conceiving. Currently, the knowledge that many causes of azoospermia can be reversed is widespread in the medical literature and practice. Thus, any trusted specialized assisted reproductive center will request a urologist/andrologist to provide sperm for an ART procedure. As a result, the urologist, even if he/she is not a specialist in the infertility field, is responsible for the adequate evaluation, diagnosis and treatment of the underlying condition whenever possible instead of only providing sperm extracted from the testicle or epididymis. This review discusses the most common causes of azoospermia that should be considered during the management of azoospermic men. We also included algorithms that will help to clarify and organize the etiologies and mechanisms of azoospermia.
& INTRODUCTION The development of intracytoplasmic sperm injection (ICSI) as an efficient therapy for severe male factor infertility has become an appropriate treatment for the majority of male reproductive tract deficiencies (1). Usually, even men with potentially treatable causes of infertility are treated with assisted reproductive techniques (ARTs) instead of specific therapy. However, once the diagnosis of azoospermia is established, no sperm can be found in the ejaculate; as a consequence, assisted reproduction cannot be applied due to the absence of sperm. Therefore, an understanding of azoospermia is very important for urologists. Azoospermia, defined as the absence of sperm in the ejaculate, is identified in approximately 1% of all men and in 10 to 15% of infertile males (2). A precise diagnosis of azoospermia and systematic evaluation of the patient to establish the disease etiology are needed to guide appropriate management options and to determine the associated
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
& DIAGNOSIS OF AZOOSPERMIA Azoospermia is defined as the complete absence of sperm from the ejaculate. This diagnosis must be confirmed by centrifugation of a semen specimen for 15 min at room
No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)03
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Palpation of the testes and measurement of their size is mandatory. Normal adult testicular measurements have been established to be at least 4.6 cm in length and 2.6 cm in width, resulting in a volume ranging from 18 to 20 cm3. Because 85% of the testicular volume is associated with sperm production, a decreased testicular size indicates impaired spermatogenic potential (15). The testes of patients with nonobstructive azoospermia will typically measure less than 15 cm3 in volume, and the epididymis will be flat. In the vast majority of patients, obstructive azoospermia may be easily distinguished from nonobstructive azoospermia through a thorough analysis of clinical diagnostic parameters. Ninety-six percent of men with obstructive azoospermia had follicle-stimulating hormone (FSH) levels of 7.6 mIU/ml or less or a testicular long axis greater than 4.6 cm. Conversely, 89% of men with nonobstructive azoospermia had FSH levels greater than 7.6 mIU/ml or a testicular long axis of 4.6 cm or less. Based on these results, we believe that an isolated diagnostic testicular biopsy is rarely indicated (16). The presence and consistency of both the vasa and epididymides should be evaluated. Palpation can complete the diagnosis of a bilateral congenital absence of the vas deferens, and scrotal exploration is not needed to make the diagnosis (Figure 1) (17). A digital rectal exam is necessary to look for masses and to examine the size and consistency of the prostate. Under normal conditions, the seminal vesicles may not be palpable but may be prominent in the setting of ejaculatory duct obstruction. Endocrine evaluation. An endocrinologic evaluation of patients who have severe male factor infertility leads to specific diagnoses and treatment strategies in a large population of infertile men (18). Although some authors recommend routine screening of the male hypothalamicpituitary-gonadal axis in all patients, endocrine screening of men with sperm counts of less than 10 million/mL based on serum testosterone and FSH levels alone will detect the vast majority of clinically significant endocrinopathies (19). In addition to cases of seminal parameter abnormalities, an investigation of those patients presenting with impaired sexual function or other clinical findings suggestive of endocrinopathy, such as a marked reduction in testicular size or gynecomastia, is recommended (20). If the testosterone level is low, a more complete evaluation will be necessary to analyze total and free testosterone, luteinizing hormone (LH), prolactin and estradiol levels. The information obtained from a complete endocrine profile may help to elucidate the etiology (21). Semen analysis. Azoospermic patients with a normal ejaculate volume may have either obstruction of the reproductive system or abnormalities in spermatogenesis. Azoospermic men with a low semen volume and normalsized testes may have ejaculatory dysfunction or ejaculatory duct obstruction. All patients presenting with absent ejaculation or low-volume ejaculation (,1.5 ml) should be asked to repeat the semen analysis and provide a postejaculation urine specimen. It is important to keep in mind that the majority of seminal fluid is contributed by the seminal vesicle. The ejaculated volume is an essential tool in the evaluation of an azoospermic patient, and the use of a diagnostic algorithm may prevent mistakes (Figure 1). Diagnostic testis biopsy. Testicular histology is the only definitive way to diagnose azoospermia. However, the
temperature with high-powered microscopic examination of the pellet and a centrifugation speed of at least 3,000 g (3). The semen analysis should be performed according to the 2010 World Health Organization guidelines, and at least two semen samples obtained more than two weeks apart should be examined (3,4). The finding of even small quantities of sperm in the centrifuged specimen excludes complete ductal obstruction and offers the potential for immediate sperm cryopreservation for ICSI cycles. Ron-El et al. showed that it is possible to detect sperm in 35% of the men who were thought to have nonobstructive azoospermia when a meticulous routine analysis of a centrifuged semen specimen was performed (5). These findings are interesting because patients routinely present with one or more semen analyses that were previously performed without using standardized centrifugation procedures. Urologists must always consider the need to repeat the seminal analysis to consistently confirm the diagnosis of azoospermia.
& EVALUATION OF AZOOSPERMIC PATIENTS Medical history. A complete evaluation should include a complete medical and surgical history, history of childhood illnesses (such as viral orchitis or cryptorchidism) and genital trauma, medications and allergies, and an inspection of past infections, such as sexually transmitted diseases (6). It is important to assess gonadotoxin exposures and prior radiation therapy or chemotherapy. In addition, approximately 1% of male infertility cases may be a consequence of a serious or potentially fatal disorder (7). Thus, it is always important to recognize that infertility may be the initial manifestation of a severe medical condition (8). Physical examination. A general physical examination is an essential part of the evaluation of an azoospermic man, and the patient should be examined in the supine and standing position in a warm room. A cold room causes contraction of the dartos and makes the examination difficult. The presence of clinical varicocele should be investigated and correctly classification, as some recent studies have shown that varicocele grade is related to the prognosis of treatment (9-12). These findings suggest that high-grade varicocele could be more frequently related to azoospermia, whereas varicocele grade I is not sufficient to explain the entire disease etiology. Appropriate sexual development must be assessed. Androgen deficiency should be suspected in the presence of diminished body hair distribution, gynecomastia or eunuchoid proportions (13). During the physical examination, men who are incompletely masculinized can be identified by excessively long extremities that occurred due to the absence of adequate epiphyseal closure at the time of puberty; this characteristic is observed in men with Kallmann’s or Klinefelter’s syndrome (14). The thyroid must be palpated, and the heart and lungs should be auscultated. The breasts should be observed and palpated for gynecomastia, which can be related to estrogen-secreting testicular tumors or adrenal tumors. The abdomen must be cautiously palpated. In addition to the general physical examination, a particular focus must be given to the genitalia, as described below.
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Figure 1 - Algorithm for the evaluation of patients with azoospermia.
pattern of the testis tissue is heterogeneous, and spermatogenesis most often occurs only in focal areas; therefore, a biopsy is rarely used as a diagnostic tool (22). Usually, testicular characteristics and laboratory findings are suggestive of nonobstructive azoospermia. Thus, testicular sperm extraction (TESE) can be performed at the same time as ART in a specialized assisted reproduction center, which allows for sperm cryopreservation during the procedure and avoids a testicular biopsy. In select patients with azoospermia, a normal testicular size, a palpable vas deferens and normal serum FSH levels, testis biopsies may be required to differentiate obstruction from disorders of spermatogenesis (Figure 1) (4). A normal testicular biopsy is pathognomonic for obstruction, and a vasography should be indicated to identify the site of the obstruction. Furthermore, scrotal exploration or endoscopic intervention may be required (Figure 1).
facilitate the restoration of fertility potential. Conversely, testicular disorders are generally irreversible, and the success rates for interventions associated with intrinsic testicular abnormalities are significantly lower. The etiologies, mechanisms and prognoses of azoospermia are summarized in Figure 2.
Pretesticular causes Pretesticular causes, also called secondary testicular failure, usually result from pathological endocrine conditions. Although an uncommon cause of male subfertility, up to 3% of infertile men will have an underlying endocrinopathy (19). Hypogonadotropic hypogonadism. Typical causes of hypogonadotropic hypogonadism (HGH) include Kallmann’s syndrome, pituitary trauma, pituitary tumors and anabolic steroid use. HGH is a rare cause of male infertility, and the disorder can be classified as congenital or acquired (23). Kallmann’s syndrome is a congenital cause of HGH and is associated with a malformation of the midline cranial structures (24). In this syndrome, the pathophysiology is a defect at the level of the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) due to the failure of the GnRH-releasing neurons to migrate to the olfactory lobe during development. Kallman syndrome is the most frequently reported form of congenital HGH, occurring in between 1:10,000 and 1:60,000 births (21). Segregation analysis in familial cases has demonstrated diverse inheritance patterns, suggesting the existence of several genes that regulate GnRH secretion. Genetic defects have been demonstrated in the KAL gene, located in the Xp22.3 region, that explain the X-linked form of the disease (25).
& ETIOLOGIES OF AZOOSPERMIA Although there are many causes of azoospermia, the etiologies of this disorder fall into three general categories: pretesticular, testicular and post-testicular. Pretesticular causes of azoospermia are endocrine abnormalities that adversely affect spermatogenesis. Testicular etiologies involve intrinsic disorders of spermatogenesis inside the testes. The post-testicular causes of azoospermia include obstruction of the ductal system at any location of the male reproductive tract. Every etiology of azoospermia is associated with a different prognosis, ranging from returns of production to simply finding sperm in the reproductive tract. The pretesticular and post-testicular abnormalities that cause azoospermia are commonly treatable, which may
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Figure 2 - Etiologies, mechanisms and prognoses of azoospermia.
hyperprolactinemia has not been shown to be useful (29). Prolactin has negligible physiological effects in men and is produced in the anterior pituitary. Hyperprolactinemia suppresses both FSH and LH and may be caused by medications, concurrent medical illnesses, tricyclic antidepressants, some antihypertensives, stress, or pituitary tumors (macroadenoma or microadenoma); the cause may also be idiopathic (30). The most common medications that induce hyperprolactinemia are phenothiazines, imipramine, methyldopa, and reserpine (21). The most common causes of hyperprolactinemia are prolactin-secreting microadenomas (,10 mm) and prolactin-secreting macroadenomas (.10 mm) (31). Symptoms of prolactinomas include infertility, depressed libido, galactorrhea, headache, fatigue, and erectile dysfunction. In patients with prolactin-secreting pituitary adenomas, gonadotropin and testosterone levels are commonly suppressed, whereas prolactin levels are elevated. The level of prolactin elevation provides insight into the type of pathology. Prolactin levels greater than 250 ng/ml, between 100 and 250 ng/ml, between 25 and 100 ng/ml, and between 0 and 25 ng/ml most commonly correspond to macroadenoma, microadenoma, pituitary stalk compression and normal levels, respectively (6). Androgen resistance. Androgen resistance occurs in approximately 1:60,000 births. More than 300 mutations have been found in the androgen receptor gene located on the X chromosome (Xq11-q12) (32). In addition to wellrecognized mutations localized in its 8 exons, mutations in the gene promoter region have also been reported (33). Because many mutations exist, the syndrome is clinically variable and ranges from phenotypic females (complete
The clinical presentation of Kallmann’s syndrome depends on the degree of hypogonadism. Most patients undergo delayed puberty, although those with less severe defects may present with a normal phenotype and mild subfertility. Other findings include anosmia, cleft palate and small testes. In more severe cases, congenital deafness, asymmetry of the cranium and the face, cerebellar dysfunction, cryptorchidism, and renal abnormalities may be present. Acquired causes of HGH include pituitary tumors, pituitary trauma, panhypopituitarism and anabolic steroid use. Exogenous testosterone inhibits the hypothalamicpituitary gonadal (HPG) axis and results in azoospermia by inhibiting gonadotropins via the feedback loop of the HPG axis, resulting in hypogonadism and infertility (18). Excess exogenous androgens from anabolic steroid use impair spermatogenesis by suppressing FSH levels and by depressing intratesticular testosterone levels (26). The initial evaluation of patients with suspected HGH may include a pituitary MRI to rule out a pituitary tumor (27). Pituitary tumors can cause local destruction of the anterior pituitary. The serum prolactin level should be measured, and hyperprolactinemia must be ruled out or treated before initiating gonadotropin replacement therapy. In patients with acquired HGH, normal spermatogenesis can usually be restored by treatment with exogenous gonadotropins or GnRH (28). Hyperprolactinemia. Hyperprolactinemia is a form of HGH caused by excessive prolactin secretion (6). An excess of prolactin inhibits the hypothalamic secretion of GnRH and has been implicated as a cause of reproductive and sexual dysfunction. Routine screening of infertile men for
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TESE, by providing sperm via ejaculation for ICSI (51). Moreover, the fertilizing ability and ICSI success rates have been described as superior when fresh motile ejaculated sperm are used compared with sperm provided by testicular biopsy or microsurgical TESE (4,52). Azoospermic patients may experience only intermittent sperm production that results in a temporary induction of spermatogenesis. Thus, semen cryopreservation is strongly recommended after the initial improvement following surgery (53). Azoospermic men with varicocele must decide whether to undergo varicocelectomy or microsurgical testicular sperm extraction with ICSI. Many of these patients will ultimately require ICSI, especially those with Sertoli cell-only patterns or maturation arrest at the spermatocyte stage (45,49,50,54). However, more than half of these men with maturation arrest at the spermatid stage or hypospermatogenesis can provide postoperative motile sperm via ejaculation (55,56). Therefore, a testicular biopsy during varicocele repair in azoospermic men provides histological data that can be used as a predictor of sperm appearance in the ejaculate after surgery and of success in sperm retrieval (45,50,57). Additionally, this information may assist couples in deciding how long to wait following varicocelectomy prior to proceeding with ART. The finding of a genetic etiology in infertile men with varicocele suggests that in such patients, Yq microdeletion screening should be performed to provide a proper diagnosis and to avoid unnecessary procedures that will most likely fail to improve testicular function due to a molecular/genetic basis (58). Undescended testes. Undescended testes are the most common genital malformation in boys and are noted in 2.7% of newborns and up to 0.8% of 1-year-olds (59). It is important to differentiate cryptorchid testes from retractile testes, a circumstance involving hyperactive cremasteric muscles that cause the testes to periodically reside in the inguinal canal or high scrotum. Suggested mechanisms for cryptorchidism-induced subfertility include testicular dysgenesis, an impaired endocrine axis, immunologic damage, and obstruction (60). Early treatment can potentially minimize the risk of infertility, and the success depends on the initial position of the testicle (61). This condition should be treated hormonally and/or surgically before the child’s first birthday; furthermore, the parents must be well informed about the risk, mainly because treatment before the age of 13 years does not seem to reduce the risk of malignancy (62). Although the majority of men with a history of unilateral undescended testes are capable of paternity, testicular volume and age at orchiopexy are independent predictors of fertility potential and sperm retrieval in men with a history of cryptorchidism (60,63). The incidence of azoospermia after treatment for undescended testes is approximately 13 and 34% in unilateral and bilateral cryptorchidism, respectively (60). However, a 30 and 80% incidence of azoospermia results from untreated unilateral and bilateral undescended testes, respectively (60). Testicular torsion. Testicular torsion occurs in approximately 1:4,000 males before the age of 25 years (64). This disorder demands immediate surgical exploration, and the risks of nonoperative management are well documented (65). Testicular preservation is usually achieved if surgical exploration is performed within 6 hours
androgen insensitivity) to normally virilized but infertile males (partial and minimal androgen insensitivity). Depending on the intensity of the defect, serum testosterone levels can be low, normal or high. It has been reported that as many as 40% of men with low or no sperm counts may have subtle androgen receptor abnormalities as the primary cause (34). Modern genetic research on the androgen receptor gene has also led to interesting new clinical correlations with male infertility. The androgen receptor gene has 8 exons, and it is known that a critical region of CAG nucleotide repeats, usually 15-30 in number, can be found in exon 1 (35). Extension of this repeat region results in spinal and bulbar muscular atrophy (Kennedy disease), a neurodegenerative disease that begins around age 30; this disorder involves muscle cramping and atrophy, including infertility from testicular atrophy. There is now enough evidence to propose that subtle alterations in this CAG repeat region may also be the cause of some cases of idiopathic infertility. Yoshida et al. recently detected longer than normal CAG nucleotide repeats in normally virilized men with idiopathic azoospermia (36). Furthermore, Casella et al. found that the testicular histology in azoospermic patients is associated with the polyglutamine length of the androgen receptor gene (37). Testicular etiologies. Testicular etiologies, broadly termed as primary testicular failure, are intrinsic disorders of spermatogenesis. Direct testicular pathology may derive from varicocele-induced testicular damage, undescended testes, testicular torsion, mumps orchitis, gonadotoxic effects from medications, genetic abnormalities and idiopathic causes. Primary testicular failure in conjunction with azoospermia, commonly termed nonobstructive azoospermia, is best managed by harvesting testicular sperm for eventual ICSI. However, the exact etiology should be determined whenever possible, and treatment may improve the success rates of sperm retrieval. Varicocele. Currently, there is convincing evidence in the literature that varicoceles produce a progressive harmful effect on the testis, and varicocelectomy has been shown to prevent the progressive decline in testicular function and reverse the damage (38-40). Additionally, varicocele repairs have been documented to improve pregnancy rates and ART outcomes (41-43). However, identifying the individuals with varicocele who will benefit from varicocele treatment remains a challenge for andrologists. Azoospermia in association with a varicocele occurs in between 5 and 10% of men (44-46). Tulloch, in 1955, was the first to report the recovery of sperm in the ejaculate and also subsequent pregnancy after varicocelectomy in a primarily azoospermic patient (47). At that time, these findings elicited renewed attention to varicocele treatment. However, it is still unknown why varicocele can have a devastating effect, leading to azoospermia, in some patients while 75% of men presenting with varicocele have normal semen findings (48,49). The large range of influence that varicocele exerts on testicular function suggests that there is currently no adequate diagnostic method with which to evaluate men presenting with clinical varicocele. Although sperm can be found in the ejaculate of azoospermic men following varicocele repair in 21 to 55% of cases, spontaneous pregnancies are extremely rare (45,46,49,50). However, varicocelectomy in this population may avoid the need for more invasive procedures, such as
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male infertility, occurring in between 1:500 and 1:1000 males (82). KS is characterized by X chromosome polysomy, with X disomy being the most common variant (47,XXY). Ninety percent of men with KS have non-mosaic X chromosome polysomes (83). Advanced maternal and paternal age have been associated with an increased risk of KS, and the number of sex-chromosome disomic sperm (24,XY) in the fathers of boys with KS increases markedly as paternal age increases (84,85). It is well known that men with KS present with a wide range of phenotypes and socioeconomic backgrounds, and these differences are the main reason for delayed diagnoses. It is estimated that only 10% of adolescents with KS are diagnosed before puberty (86). The classical phenotype of men with KS is characterized by tall eunuchoid body proportions; low testosterone levels; thin facial and pubic hair; small, hard testicles; a micropenis; sterility; and mild to moderate cognitive deficits (87). Affected men are also predisposed to diabetes mellitus, varicose veins, and chronic bronchitis, and they have a higher mortality rate due to breast cancer and non-Hodgkin lymphoma (88). Patients are commonly azoospermic, but testicular sperm extraction (TESE) may reveal spermatozoa in approximately 69% of KS men (89). Live births of children with normal karyotypes have been reported (90,91). The mosaic variant is less severe, and patients with this variant can present with normal testicular size, complete spermatogenesis and the presence of ejaculated sperm. b- 47,XYY Syndrome. This syndrome is caused by paternal nondisjunction during meiosis that results in YY sperm, occurring in approximately 1:1000 (92). These patients typically have a tall stature, demonstrate decreased intelligence and exhibit antisocial behavioral characteristics. Most men with this syndrome are azoospermic or severely oligospermic, and testis biopsy findings range from Sertoli cell-only to maturation arrest patterns (93). Unlike patients with KS, serum testosterone levels are normal in these patients. c- XX Male Syndrome. Etiologically, 90% of these patients present with a translocation of the SRY (testisdetermining region) from the Y to the X chromosome, occurring in approximately 1:20,000 (94). In the other 10%, either the translocation occurs to an autosomal chromosome, or the individual is SRY-negative (94). Typically, these patients have normal male external and internal genitalia and a normal hormonal profile. Because there is no translocation of the entire AZF region, these patients are azoospermic and therefore not candidates for testicular sperm extraction; these individuals should be provided with the option of donor sperm-assisted reproduction. d- Mixed Gonadal Dysgenesis. Patients with mixed gonadal dysgenesis normally have a mosaic 45,X0/46,XY genotype and anatomically have a testis on one side and a streak gonad on the other (92). Although this is a rare syndrome, the normally formed testis is often cryptorchidic and devoid of viable germ cells. The streak gonad is at risk of developing gonadoblastoma or seminoma and should be surgically removed (95). There are varying degrees of ambiguity of the external genitalia. e- Y-chromosome microdeletions. Tiepolo and Zuffardi were the first to publish a report on six azoospermic patients carrying deletions of the Y chromosome and postulated that factors controlling human spermatogenesis (azoospermia
after the onset of symptoms. In addition to duration, other factors, such as the degree of rotation, have been related to testicular salvage (66,67). The most significant complication of testicular torsion is a loss of the testis, which may lead to impaired fertility (68). Severe oligospermia or azoospermia is rare after unilateral testicular torsion; however, these conditions are possible when the contralateral testis has experienced any previous abnormality, such as orchiopexy for an undescended testis (69). Endocrine testicular function is expected to be normal in the event of a lost gonad. Conversely, the exocrine testicular function (spermatogenesis) is commonly affected (70,71). Patients with testicular torsion seem to have bilateral abnormalities that result in decreased spermatogenesis (72). It is unclear whether these abnormalities are due to an autoimmune process occurring after the rupture of the hemato-testicular barrier, leading to the formation of antisperm antibodies, or as a result of reperfusion-induced injury to the testis (71,73,74). Contralateral testicular biopsies are abnormal in up to 88% of cases at the time of torsion; therefore, some abnormalities are believed to be present even before the onset of torsion (68). Mumps orchitis. Since the introduction of a vaccine against the mumps virus, there has been a reduced risk of mumps and its complications. On the other hand, mumps orchitis should still be suspected in cases of scrotal swelling, as there has been a recent increase in mumps orchitis among pubertal and postpubertal males (75). Pubertal mumps orchitis occurs unilaterally in 67% of patients and bilaterally in 33% (76). Testicular atrophy occurs in 36% of those affected bilaterally, whereas infertility occurs in just 13% (77). However, prepubertal mumps orchitis has little effect on future fertility (76). Thus, it is important that urologists/andrologists are familiar with the diagnosis, treatment and complications of this condition. Gonadotoxins and medications. Drugs and medications may harm male fertility through four distinct mechanisms: 1) direct gonadotoxic effects, 2) alteration of the hypothalamic-pituitary-gonadal (HPG) axis, 3) ejaculation dysfunction, and 4) reduction in libido. Meanwhile, gonadotoxins affect spermatogenesis by direct injury to germ cells in the testis or by interfering with the function of the Sertoli cells (78). For this reason, a detailed history of medications, including prescribed, over-the-counter, illicit, and nutraceutical drugs, should be obtained. The vast majority of these medications are not able to cause azoospermia, but special attention should be given to patients using or being administered exogenous androgens, antiandrogens, chemotherapy agents or radiation therapy, as well as to patients exposed to environmental toxins, such as pesticides, fumigants, insecticides and solvents. Genetic. Chromosomal disorders are encountered at a higher frequency in the infertile compared with the fertile population (79). These chromosome alterations can currently be diagnosed in 15% of azoospermic and 5% of oligospermic men and represent one of the most common genetic defects in infertile men (80,81). Therefore, it is important that these men undergo genetic testing prior to the use of their sperm for ART. a- Klinefelter Syndrome. Klinefelter Syndrome (KS) is 45 times more common in men seeking infertility treatment than in the general male population and is the most common numerical chromosome anomaly observed in
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azoospermia with a testicular histology of Sertoli cell-only syndrome (107).
factor; AZF) may be located on the distal portion of the euchromatin segment of the long arm of the Y chromosome (Yq11) (96). Twenty years later, with the development of molecular biology technologies, the first spermatogenesis gene was identified, and the following three distinct intervals within the AZF region were mapped (from proximal to distal): Yq (AZFa, AZFb and AZFc) (97,98). Closest to the centromere, the AZFa region contains two genes that are important in the spermatogenesis process: DDX3Y (also known as DBY) and USP9Y (99). Microdeletion in the AZFa region occurs in almost 1% of men with nonobstructive azoospermia. Spermatogenic failure is the critical clinical consequence, and to date, there are no data in the literature to support the finding of sperm TESE (100). At first, the AZFb and AZFc regions seemed to be distinct and nonoverlapping; however, the precise characterization of the P5 to P1 interval has revealed that these regions are actually overlapping and correspond to different sites of ectopic homologous recombination within this region (101). Detected in approximately 1:4000 men and found in 13% of azoospermic and 6% of severely oligospermic men, AZFc is the most common microdeletion region (102). An AZFc microdeletion removes several genes that occupy this region, with the four copies of DAZ (Deleted in AZoospermia) being more clinically significant than with only one copy deleted (103,104). Oates et al. have offered the most pertinent clinical correlations to date regarding AZFc microdeletions. In their sample of 42 men, 38% were severely oligospermic and 62% were azoospermic. Of the azoospermic men, 67% had some level of spermatogenesis as detected from the testis biopsy; however, 19% of the overall group did not have sperm available from either the ejaculate or testis tissue (105). As previously described, the AZFb region is not distinct from the AZFc region but corresponds to another possible position of ectopic homologous recombination within that region that extends from the P5 palindrome to the P1 palindrome. The AZFb microdeletion is 6.2 Mb long, starting in the P5 palindrome and ending in the proximal portion of the P1 palindrome. The AZFb/AZFc microdeletion is longer (7.7 Mb), with its origin in P5 (as for AZFb) and its termination at the distal end of P1 (101). The greatest clinical meaning of these microdeletions associated is that there is a small likelihood of sperm retrieval by TESE (100). Combined, these two microdeletions may be found in approximately 1 to 3% of the NOA population (106). In a very elegant review of the literature, Foresta et al. found that almost 5,000 infertile men had been analyzed for the presence of Y microdeletions from 1992 to 2001. They found that the prevalence of deletions increases with more strict patient selection criteria. For instance, in unselected oligozoospermic men, the prevalence is 2.9%. The prevalence increases to 11.6% if idiopathic oligozoospermic patients are selected and to 14.3% if idiopathic severe oligozoospermia patients are included. In the same manner, unselected azoospermic patients show a deletion rate of 7.3%, but the exclusion of patients with obstructive azoospermia causes the prevalence to rise to 10.5% and to 18% if only idiopathic forms are considered. Furthermore, if patients are selected on the basis of their testicular structure, the prevalence is 24.7% in cases of idiopathic severe oligozoospermia with a testicular picture of severe hypospermatogenesis and 34.5% in cases of idiopathic
Post-testicular causes of azoospermia Post-testicular causes of azoospermia are due to either the obstruction of sperm delivery or ejaculatory dysfunction. The clinical management of obstructive azoospermia depends on its cause and also must take into account any coexisting infertility factors in the female partner (108). Therefore, both partners should be carefully evaluated before making any treatment recommendations. Men presenting with obstructive azoospermia may father children in one of two ways: surgical correction of the obstruction, which may allow the couple to conceive naturally; or retrieval of sperm directly from the epididymis or testis, followed by the use of ART (108). The surgical management of obstructive azoospermia varies with the site of obstruction and depends on the presence of pathological conditions, such as the absence of the vasa deferentia, vasal obstruction and ejaculatory duct obstruction. These conditions are described separately below. Absence of the vasa deferentia. Congenital bilateral absence of the vas deferens (CBAVD) is found in 1% of infertile men and in up to 6% of those with obstructive azoospermia (109). There are two possible mechanisms responsible for this condition: 1) mutations of the cystic fibrosis transmembrane regulator gene (CFTR) and 2) abnormalities in the differentiation of the mesonephric duct (110). Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians, with an incidence of 1:2500 births and a carrier frequency of 1:20 (33). The CFTR gene (7q31.2) contains 27 exons and is 250 base pairs in length. A three-base-pair deletion in exon 10 (delta F508) is the most common mutation found in the Caucasian population (111), but there are more than 800 different mutations that have been described. Another common mutation consists of an intron 8 anomaly, called 5T (112). Normally, seven to nine thymidines are present in this region; a reduction to the five-thymidine variant decreases the efficiency of the splicing of exon 9 and eventually leads to a 10-50% reduction in CFTR mRNA (113). Eighty percent of men with CBAVD and 43% of men with a congenital unilateral absence of the vas deferens have detectable CFTR gene mutations (17,113). The clinical features of CBAVD include normal testis size and preservation of spermatogenesis. The caput epididymis is always present, but the corpus and the cauda are found only occasionally. Seminal vesicles are often absent or atrophic but may also be enlarged or cystic. The ejaculate is acidic and low in volume [,1 ml]. Any insult to the Wolffian duct before week seven of gestation may impair urinary and reproductive tract formation including partial epididymal aplasia, seminal vesicle aplasia or hypoplasia, which may lead to a low ejaculate volume. Secondary findings include ipsilateral renal agenesis in 11% of patients with CBAVD and in 26% of patients with unilateral vasal absence (110). Imaging confirmation of renal agenesis is imperative in patients with unilateral absence of the vas deferens or in those with CBAVD lacking detectable CFTR gene mutations. Spermatozoa can be easily retrieved from the caput epididymis for use in conjunction with ICSI by using either
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microsurgical or percutaneous procedures (114). If the CFTR mutation is also present in the female, a preimplantation diagnosis can be performed to avoid the birth of a CF child or a CBAVD male. Vasal obstruction. The most frequent cause of nonpurposeful vasal obstruction is inadvertent injury during the performance of a hernia repair. This complication more frequently occurs when performed in infancy but can occur after any inguinal procedure where the vas and cord are manipulated (115). The diagnosis is suspected when examination reveals normal testicular size and the epididymis is full and firm. Instead of a direct vasal injury, the postoperative inflammatory response caused by the mesh may entrap and obstruct the inguinal vas deferens. The exact frequency of this problem is presently unknown. An estimated 80% of inguinal hernia operations involve the placement of a knitted polypropylene mesh to form a ‘tension-free’ herniorrhaphy. The prosthetic mesh induces a chronic foreign-body fibroblastic response, creating scar tissue that imparts strength to the floor and leads to fewer recurrences. However, little is known regarding the longterm effects of the polypropylene mesh on the vas deferens, especially with regard to fertility (116). The most common cause of obstruction of the vas deferens is vasectomy performed for elective sterilization (117). Approximately 500,000 vasectomies are performed in the United States each year (118). It is not surprising, therefore, that the most common indication to perform a vasovasostomy is to reverse a prior vasectomy to restore a man’s fertility due to a new marriage. Epididymal obstruction. Young’s syndrome is a triad of disorders that encompasses chronic sinusitis, bronchiectasis and obstructive azoospermia (119). Patients with this syndrome have only mildly impaired respiratory function and normal spermatogenesis. The pathophysiology of the condition is unclear but may involve abnormal ciliary function or abnormal mucus quality. The exact cause of azoospermia is not completely elucidated but is most likely due to obstruction of the epididymis by inspissated secretions. The diagnosis is based on the occurrence of chronic sinopulmonary infections and persistent azoospermia with normal spermatogenesis, after excluding cystic fibrosis and immotile-cilia syndrome. The sperm appear to be normal in patients with Young’s syndrome, and paternity has been documented in these patients (119). Ejaculatory duct obstruction. Ejaculatory duct obstruction is a pathological condition characterized by the obstruction of one or both ejaculatory ducts and may be either congenital or acquired (120,121). In 1973, Farley and Barnes initially described ejaculatory duct obstruction, which is responsible for 1 to 5% of male infertility cases (122,123). Although first described in azoospermic men showing complete blockage, it is now clear that obstruction may manifest in several ways, including azoospermia and oligoasthenospermia (124). Congenital causes include extrinsic compression of the ejaculatory ducts by Mu¨llerian (utricular) or Wolffian (diverticular) cysts. Acquired causes may be secondary to iatrogenic trauma (postsurgical), prostatic calcification, seminal vesicle calculi or infected-related scar tissue. Although there are no pathognomonic findings associated with ejaculatory duct obstruction, quite a few clinical findings are highly suggestive of this condition. Men with this condition present with low-volume azoospermia;
dilated seminal vesicles; and normal secondary sex characteristics, testes size, and hormonal profiles. Based on a suspicious semen analysis results, a transrectal ultrasound may be performed to confirm the diagnosis. Ejaculatory duct obstruction must not be confused with an obstruction of the vas deferens; approximately 80% of the volume of the semen is the gel-like fluid originating from the seminal vesicles, whereas the fraction from the testicles and epididymis, which contain the spermatozoa, accounts for only 5 to 10% of the volume of the semen. Thus, vasal obstruction usually does not influence the ejaculate volume (125). Disorders of Ejaculation. Although this is a relatively unusual cause of male infertility, disorders of ejaculation present an interesting challenge to the treating physician (126). Ejaculatory dysfunction includes a variety of disorders with individualized treatments. Ejaculatory dysfunction should be suspected in any patient with a low volume (,1.0 ml) of or absent ejaculate and should be distinguished from anorgasmia. Retrograde ejaculation can be defined as the abnormal backward flow of semen into the bladder with ejaculation; the etiology may be anatomic, neurogenic, pharmacologic or idiopathic. Pharmacological agents implicated in retrograde ejaculation include neuroleptics, tricyclic antidepressants, alpha-blockers used in the treatment of prostatism and certain antihypertensives (126-128). The diagnosis of retrograde ejaculation is made by examining the post-ejaculate urine for sperm. Although specific criteria have not been established for a positive post-ejaculate urinalysis, the finding of greater than 10 to 15 sperm per high-power field confirms the presence of retrograde ejaculation. In contrast, sperm will not be present in the urine of a patient with failure of emission, which must be diagnosed clinically.
& EXPERT COMMENTARY Azoospermia may be due to inadequate hormonal stimulation, impaired spermatogenesis or an obstruction. In the majority of cases, the assessment of both physical and laboratory information, including semen volume, testicular volume, the presence of bilateral vas deferens and serum FSH level, will aid in the differentiation between the three categories. Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system, including male infertility. In the future, the identification and characterization of different proteins expressed in the seminal plasma of men with normal and impaired spermatogenesis may aid in the elucidation of the molecular basis of male infertility and possibly azoospermia (129). Over the past three decades, revolutionary treatments for infertility using assisted reproductive technologies have been developed. The first major development occurred in 1978 with the birth of baby Louise Brown, who was conceived by in vitro fertilization. The next major development involved gamete micromanipulation reproduction techniques, such as intracytoplasmic sperm injection (1,130). These highly complex technologies have been applied with increasing frequency in the treatment of couples around the world. Over a million children have been conceived from assisted conception worldwide as of 2005 (131). All of these newer techniques appear to be increasingly less ’natural’, and there is a dearth of information on
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with reproductive specialists because the timing and coordination of care may help achieve the ultimate goals, as well as the better management of azoospermia to maximize sperm quality, in these patients. In an ideal world, cause-specific therapy of male-factor infertility would decrease the use of ART, thereby avoiding the costs, risks, complications, and treatment of the unaffected female partner.
these children beyond the neonatal period, especially with regard to fertile potential. Additionally, unlike most therapeutic procedures used in medicine, ARTs did not undergo meticulous safety testing prior to clinical use. There is no doubt that ICSI overcomes natural barriers and results in the transmission of possible genetic abnormalities to offspring. Infertility could be, in some way, a natural mechanism to block the transmission of these undesirable genetic traits to offspring. Although researchers believe that approximately 75% or more cases of infertility have a genetic basis, our current ability to diagnose these defects remains limited (132). However, before the development of ART, these men could not have reproduced by any means. Today, approximately 28% of azoospermic men presenting with genetic alterations can be diagnosed, allowing for adequate counseling before proceeding to ART (133). However, many patients with ‘idiopathic’ azoospermia are thought to have a contributing but as yet unidentifiable genetic cause. As such, there exists a risk of transmission of these abnormalities to offspring. As urologists, we are increasingly expected to be experts on the potential genetic basis of azoospermia. Genetic testing and counseling should always be considered in the management of these couples prior to treatment to allow the couples to make an informed decision as to whether to use the husband’s sperm. Karyotyping and Y-chromosome microdeletion analysis should be offered to all men with azoospermia due to primary testicular failure prior to the use of ART with their sperm. Because Y microdeletions are the most common molecularly defined causes of spermatogenic failure, significant numbers of Y-deleted boys could be expected to be fathered through ICSI. It has been demonstrated that spermatozoa from an oligozoospermic subject carrying a Yq deletion are able to fertilize oocytes in vitro, suggesting that sperm carrying a deletion possess all the characteristics required to regulate capacitation, the acrosome reaction, and the ability to penetrate and fertilize the oocyte (134). Although no other health problems are associated with microdeletion of the Y chromosome, few data exist on the phenotypes of the sons of fathers with these genetic abnormalities. Therefore, Y chromosome analysis should be offered to men who have nonobstructive azoospermia or severe oligospermia prior to performing an ICSI with their sperm. The simple palpation of the vas deferens could bring attention to a wide range of lethal syndromes that include the congenital absence of the vas deferens. If the doctor suspects any such syndrome, genetic testing for CFTR mutations in the female partner should be offered before proceeding with treatments that could utilize the sperm of a man with CBAVD. Currently, the recommendation is that if the female partner tests positive for a CFTR mutation, the male should be tested as well. However, if the female partner has a negative test for CFTR mutations, testing of the male partner is optional. Interest has been renewed in genetic studies to determine the underlying causes of idiopathic male infertility, but the overwhelming trend has been to sidestep improvements in diagnostic evaluation in favor of a more expensive, although more efficacious, option, namely ART (18). Wherever possible, an accurate diagnosis of the etiology of azoospermia is important prior to the initiation of the appropriate treatment. It is also imperative that urologists work intimately
& AUTHOR CONTRIBUTIONS Cocuzza M, Alvarenga A and Pagani R were responsible for writing the manuscript.
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Hinney B, Guttenbach M, Schmid M, Engel W, Michelmann HW. Pregnancy after intracytoplasmic sperm injection with sperm from a man with a 47,XXY Klinefelter’s karyotype. Fertil Steril. 1997;68(4):71820, http://dx.doi.org/10.1016/S0015-0282(97)00280-X. 92. Turek PJ, Pera RA. Current and future genetic screening for male infertility. Urol Clin North Am. 2002;29(4):767-92, http://dx.doi.org/10. 1016/S0094-0143(02)00090-3. 93. Wong EC, Ferguson KA, Chow V, Ma S. Sperm aneuploidy and meiotic sex chromosome configurations in an infertile XYY male. Hum Reprod. 2008;23(2):374-8. 94. Van der Auwera B, Van Roy N, De Paepe A, Hawkins JR, Liebaers I, Castedo S, et al. Molecular cytogenetic analysis of XX males using Y-specific DNA sequences, including SRY. Hum Genet. 1992;89(1):23-8, http://dx.doi.org/10.1007/BF00207036. 95. Denes FT, Cocuzza MA, Schneider-Monteiro ED, Silva FA, Costa EM, Mendonca BB, et al. The laparoscopic management of intersex patients: the preferred approach. 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101. Repping S, Skaletsky H, Lange J, Silber S, Van Der Veen F, Oates RD, et al. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet. 2002;71(4):906-22. 102. Reijo R, Alagappan RK, Patrizio P, Page DC. Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet. 1996;347(9011):1290-3, http://dx.doi.org/10.1016/S01406736(96)90938-1. 103. Lepretre AC, Patrat C, Mitchell M, Jouannet P, Bienvenu T. No partial DAZ deletions but frequent gene conversion events on the Y chromosome of fertile men. J Assist Reprod Genet. 2005;22(4):141-8, http://dx.doi.org/10.1007/s10815-005-4910-x. 104. Saxena R, de Vries JW, Repping S, Alagappan RK, Skaletsky H, Brown LG, et al. Four DAZ genes in two clusters found in the AZFc region of the human Y chromosome. Genomics. 2000;67(3):256-67, http://dx.doi. org/10.1006/geno.2000.6260. 105. Oates RD, Silber S, Brown LG, Page DC. Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod. 2002;17(11):2813-24, http://dx.doi.org/10.1093/ humrep/17.11.2813. 106. Oates RD. The genetic basis of male reproductive failure. Urol Clin North Am. 2008;35(2):257-70, ix, http://dx.doi.org/10.1016/j.ucl.2008. 01.015. 107. Foresta C, Moro E, Ferlin A. Y chromosome microdeletions and alterations of spermatogenesis. Endocr Rev. 2001;22(2):226-39, http:// dx.doi.org/10.1210/er.22.2.226. 108. The management of infertility due to obstructive azoospermia. Fertil Steril. 2008;90(5 Suppl):S121-4. 109. Ferlin A, Raicu F, Gatta V, Zuccarello D, Palka G, Foresta C. Male infertility: role of genetic background. Reprod Biomed Online. 2007;14(6):734-45, http://dx.doi.org/10.1016/S1472-6483(10)60677-3. 110. McCallum T, Milunsky J, Munarriz R, Carson R, Sadeghi-Nejad H, Oates R. Unilateral renal agenesis associated with congenital bilateral absence of the vas deferens: phenotypic findings and genetic considerations. Hum Reprod. 2001;16(2):282-8, http://dx.doi.org/10. 1093/humrep/16.2.282. 111. Uzun S, Gokce S, Wagner K. Cystic fibrosis transmembrane conductance regulator gene mutations in infertile males with congenital bilateral absence of the vas deferens. Tohoku J Exp Med. 2005;207(4):279-85. 112. Lebo RV, Grody WW. Variable penetrance and expressivity of the splice altering 5T sequence in the cystic fibrosis gene. Genetic testing. 2007;11(1):32-44, http://dx.doi.org/10.1089/gte.2006.9997. 113. Claustres M. Molecular pathology of the CFTR locus in male infertility. Reprod Biomed Online. 2005;10(1):14-41, http://dx.doi.org/10.1016/ S1472-6483(10)60801-2. 114. Tournaye H. Surgical sperm recovery for intracytoplasmic sperm injection: which method is to be preferred? Hum Reprod. 1999;14 Suppl 1:71-81, http://dx.doi.org/10.1093/humrep/14.suppl_1.71. 115. Matsuda T, Horii Y, Yoshida O. Unilateral obstruction of the vas deferens caused by childhood inguinal herniorrhaphy in male infertility patients. Fertil Steril. 1992;58(3):609-13. 116. Shin D, Lipshultz LI, Goldstein M, Barme GA, Fuchs EF, Nagler HM, et al. Herniorrhaphy with polypropylene mesh causing inguinal vasal obstruction: a preventable cause of obstructive azoospermia. Ann Surg. 2005;241(4):553-8, http://dx.doi.org/10.1097/01.sla.0000157318.13975. 2a. 117. Costabile RA, Spevak M. Characterization of patients presenting with male factor infertility in an equal access, no cost medical system. Urology. 2001;58(6):1021-4, http://dx.doi.org/10.1016/S0090-4295(01) 01400-5. 118. Marquette CM, Koonin LM, Antarsh L, Gargiullo PM, Smith JC. Vasectomy in the United States, 1991. Am J Public Health. 1995;85(5):644-9. 119. Handelsman DJ, Conway AJ, Boylan LM, Turtle JR. Young’s syndrome. Obstructive azoospermia and chronic sinopulmonary infections. N Engl J Med. 1984;310(1):3-9, http://dx.doi.org/10.1056/ NEJM198401053100102. 120. Pryor JP, Hendry WF. Ejaculatory duct obstruction in subfertile males: analysis of 87 patients. Fertil Steril. 1991;56(4):725-30. 121. Goldwasser BZ, Weinerth JL, Carson CC, 3rd. Ejaculatory duct obstruction: the case for aggressive diagnosis and treatment. J Urol. 1985;134(5):964-6. 122. Porch PP, Jr. Aspermia owing to obstruction of distal ejaculatory duct and treatment by transurethral resection. J Urol. 1978;119(1):141-2. 123. Farley S, Barnes R. Stenosis of ejaculatory ducts treated by endoscopic resection. J Urol. 1973;109(4):664-6. 124. Smith JF, Walsh TJ, Turek PJ. Ejaculatory duct obstruction. Urol Clin North Am. 2008;35(2):221-7, viii, http://dx.doi.org/10.1016/j.ucl.2008. 01.011. 125. Hopps CV, Goldstein M, Schlegel PN. The diagnosis and treatment of the azoospermic patient in the age of intracytoplasmic sperm injection.
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REVIEW
Update in the evaluation of the azoospermic male Ahmet Gudeloglu, Sijo J. Parekattil University of Florida, Winter Haven Hospital, Department of Urology, Winter Haven, FL, United States
Approximately 1% of all men in the general population suffer from azoospermia, and azoospermic men constitute approximately 10 to 15% of all infertile men. Thus, this group of patients represents a significant population in the field of male infertility. A thorough medical history, physical examination and hormonal profile are essential in the evaluation of azoospermic males. Imaging studies, a genetic workup and a testicular biopsy (with cryopreservation) may augment the workup and evaluation. Men with nonobstructive azoospermia should be offered genetic counseling before their spermatozoa are used for assisted reproductive techniques. This article provides a contemporary review of the evaluation of the azoospermic male. KEYWORDS: Azoospermia; Male Infertility; Testis Biopsy; Testicular Biopsy. Gudeloglu A, Parekattil SJ. Update in the evaluation of the azoospermic male. Clinics. 2013;68(S1):27-34. Received for publication on June 25, 2012; Accepted for publication on July 10, 2012 E-mail: sijo.parekattil@winterhavenhospital.org Tel.: 1-863-292-4652
and the complete absence of spermatozoa is defined as azoospermia (1). The etiologic classification of azoospermia is divided into three primary categories: pretesticular, testicular, and posttesticular. Although the pretesticular and post-testicular causes of azoospermia are generally curable, the testicular causes of azoospermia are generally not. Pretesticular azoospermia can be caused by endocrine abnormalities that are characterized by low levels of sex steroids and abnormal gonadotropin levels. These abnormalities can be congenital (e.g., Kallmann syndrome), acquired (e.g., hypothalamic or pituitary disorders) or secondary (e.g., an adverse effect from a medication). Testicular causes include congenital, acquired or idiopathic disorders that lead to spermatogenic failure. Congenital testicular causes consist of anorchia, testicular dysgenesis (cryptorchidism), genetic abnormalities (Y chromosome deletions), germ cell aplasia (Sertoli cell-only syndrome) and spermatogenic arrest (maturation arrest). Acquired testicular causes include trauma, torsion, infection (mumps orchitis), testicular tumors, medications, irradiation, surgery (compromising vascularization of testis), systemic diseases (cirrhosis, renal failure) and varicocele (5). Post-testicular causes include ejaculatory disorders or obstructions, which impair the transport of spermatozoa from the testis. These obstructions can also be congenital, caused by a bilateral absence of the vas deferens (CBAVD), or acquired because of infection or surgery (vasectomy or an iatrogenic injury). Obstructive azoospermia (OA) is also classified according to the localization of the obstruction: epididymal (postinfection), vasal (vasectomy, CBAVD) or ductal (Mu¨llerian cysts) (5). Azoospermia may also be clinically classified as obstructive (post-testicular) and nonobstructive (pretesticular or testicular). Obstructive azoospermia (OA) is less common than nonobstructive azoospermia (NOA) and occurs in 15 to 20% of men with azoospermia (5). Although NOA indicates
& INTRODUCTION Azoospermia is defined as the absence of sperm in at least two different ejaculate samples (including the centrifuged sediment) (1,2). In the general population, 10 to 15% of couples suffer from infertility issues (3,4). Approximately 50% of these cases can be attributable to a male issue. Of these infertile men, 10 to 20% (or 1% of all men in the general population) suffer from azoospermia (3). A detailed history, a physical examination, a hormone profile, imaging and genetic counseling are important to determine the specific clinical classification of the azoospermia. This distinction is important given that, for example, obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) require different treatment approaches. In this chapter, we will provide a contemporary review of the evaluation process for males with azoospermia.
& EVALUATION OF THE AZOOSPERMIC MALE Definition and classification of azoospermia Azoospermia is defined as the absence of spermatozoa in the semen. If no spermatozoa are observed in the wet preparation, the World Health Organization (WHO) recommends an examination of the centrifuged sample (3000 X g or greater for 15 minutes). If no sperm are observed in the centrifuged sample, the semen analysis should be repeated. The presence of a small number of spermatozoa in either of the centrifuged samples is defined as cryptozoospermia,
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)04
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low ejaculate volume. A postejaculation urine evaluation can identify patients with retrograde ejaculation.
impaired sperm production of the entire testis by definition, it has been observed that focal normal spermatogenesis can be observed in 50 to 60% of men with NOA (6,7).
Initial evaluation of the azoospermic male A complete medical history, physical examination and hormonal investigation are the principal components of the initial evaluation of males with azoospermia (8).
Semen analysis of the azoospermic male In 2010, the WHO updated the lower reference limits for semen characteristics, which are provided in Table 1 (1). In the absence of vasal agenesis or testicular atrophy, semen volume and serum FSH are key factors in determining the cause of azoospermia. Azoospermic men with a normal ejaculate volume may have either epididymal/vasal obstruction or an abnormality of spermatogenesis. In such cases, a hormonal evaluation would aid in the differential diagnosis. Azoospermia in men with a low semen volume and normal-sized testes can be caused by ejaculatory dysfunction; however, the most likely cause is ejaculatory duct obstruction (EDO) (8). Semen volume, pH and fructose levels are generally within normal ranges in NOA; however, these parameters can be lower in OA, depending on the location of the obstruction. Although there are no pathognomonic findings for the ejaculate evaluation of patients with EDO, the absence of spermatozoa in a centrifuged semen sample accompanied by a low ejaculate volume (,2.0 ml), a pH below 7.2, and the absence of fructose in the seminal fluid suggest EDO (9). The classic clinical presentation of EDO includes postejaculatory pain, hematospermia and infertility. This triad of clinical presentation warrants a transrectal ultrasound examination. The presence of dilated seminal vesicles and/or dilated ejaculatory ducts upon transrectal ultrasound examination, combined with a normal hormonal profile, support a diagnosis of EDO. A congenital bilateral absence of the vas deferens (CBAVD) is responsible for 2 to 6% of cases of OA. CBAVD classically presents as a nonpalpable vas segment, low semen volume and low pH and fructose levels caused by an obstructed epididymis and the atrophia or congenital absence of the seminal vesicles (10,11). Nevertheless, ejaculatory disorders and retrograde ejaculation should be eliminated as a diagnosis for azoospermic patients with a
Medical history It is important to obtain a complete infertility medical history for both partners. The azoospermic male’s history should include such conditions as trauma, torsion, cryptorchidism; history of pelvic, inguinal or scrotal surgeries; any potentially compromising testicular vascularization; vasal patency; and ejaculatory function. Additionally, a late onset of puberty can indicate hypogonadotropic hypogonadism. Any prior fertility history can be beneficial in distinguishing between OA and NOA. If there is a history of vasectomy, the duration since the procedure and any reversal attempts may be helpful in developing a treatment plan. Genitourinary infections, such as urethritis and epididymitis can cause OA, while late pubertal mumps orchitis can cause NOA. It is important to inquire about systemic diseases, given that diabetes mellitus, cirrhosis or chronic renal insufficiency can affect sperm production or transport. Previous malignancies, especially if treated with cytotoxic chemotherapy or radiotherapy, should be identified. Medications that include gonadotoxic agents, such as cimetidine, nitrofurantoin and calcium channel blockers, should be documented.
Physical examination The physical examination should begin with the inspection of the body habitus, given that hair distribution, gynecomastia and a eunuchoid stature can indicate a testosterone deficiency or hormonal imbalance. Possible disorders include low serum testosterone level, hyperprolactinemia, abnormalities in the estrogen-to-testosterone ratio, adrenal dysfunction, and genetic syndromes that are associated with subvirilization, such as Klinefelter’s syndrome. Additionally, disproportionately long extremities (caused by testosterone deficiency at the time of puberty, leading to delayed closure of the epiphyseal plates) should be noted. The genital examination should also include an inspection of the external genitalia. Penile curvature, hypospadias and surgical scars should be noted. Penile curvature and hypospadias may cause improper sperm placement into the vagina. Surgical scars may indicate possible injuries to the testicular blood supply and/or vas deferens. The scrotal contents should be assessed via manual palpation to determine the testis size and consistency and the presence of testicular mass or asymmetry. An orchidometer, calipers or sonographic measurement techniques may be utilized to measure testicle size. The normal adult testicle should be 463 cm or approximately 20 ml (12). Although smaller and softer testes can signal inadequate sperm production, men with Klinefelter’s syndrome have small (approximately 5 ml), firm testes that are devoid of germ cells (13). Epididymal enlargement, induration and cysts should be assessed and can be considered a cause of OA. The presence of the vas deferens and varicose veins (Figure 1) should be ascertained while palpating the
Table 1 - The 2010 WHO lower reference limits (5th percentiles and 95% confidence intervals) for semen analysis (PR = progressive; NP = nonprogressive; MAR = mixed antiglobulin reaction). Parameter Semen volume (ml) Total sperm number (106 per ejaculate) Sperm concentration (106 per ml) Total motility (PR+NP) Progressive motility (PR, %) Vitality (live spermatozoa, %) Sperm morphology (normal forms, %) Other consensus threshold values: pH Peroxidase-positive leukocytes (106 per ml) MAR test (motile spermatozoa with bound particles, %) Immunobead test (motile spermatozoa with bound beads, %) Seminal zinc (mmol/ejaculate) Seminal fructose (mmol/ejaculate) Seminal neutral glucosidase (mU/ejaculate)
Lower reference limit (range) 1.5 (1.4-1.7) 39 (33-46) 15 (12-16) 40 (38-42) 32 (31-34) 58 (55-63) 4 (3.0-4.0) .7.2 ,1.0 ,50 ,50 $2.4 $13 $20
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spermatic cord. The Valsalva maneuver may aid in the identification of low-grade varicocele. Examiners should bear in mind that vasal agenesis may be accompanied by genetic or renal abnormalities. The physical examination findings for patients with CBAVD should be normal-sized and consistent testis; full and firm caput epididymis caused by obstructed efferent ducts (with the presence or absence of the distal two thirds of the epididymis); nonpalpable vas deferens; atrophic, dysfunctional or congenitally absent seminal vesicle; and normal male external genitalia (14). A complete physical examination of males with azoospermia must include a rectal examination to exclude ejaculatory duct obstruction. Midline prostatic cysts, such as Mu¨llerian duct cysts and dilated seminal vesicles, can be easily palpated, and prostatic induration or tenderness supports a diagnosis of prostatitis.
Hormonal analysis and hypogonadism Sperm production is controlled by the hypothalamopituitary-gonadal axis (Figures 2 and 3). Gonadotropinreleasing hormone is secreted by the hypothalamus and stimulates the anterior pituitary gland to release luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH and FSH stimulate Leydig cells and the germinal epithelium to produce testosterone and sperm, respectively. Testosterone is required for the completion of the meiotic division and spermatid development and thus plays an important role in the initiation and maintenance of spermatogenesis. FSH also stimulates Sertoli cells, which produce inhibin B. LH and FSH are under negative feedback control by testosterone and inhibin B, respectively (Figure 3) (15). Normal levels of LH and FSH are expected in OA; however, LH and FSH can be low or elevated in NOA. Hypogonadism is defined by impaired testicular function, which potentially affects spermatogenesis and/or testosterone synthesis. Sussman et al. reported that the respective incidences of hypogonadism in males who visited an infertility clinic were 35.3%, 45% and 16.7% for those with
Figure 2 - Illustration of the hypothalamo-pituitary-gonadal axis, Part 1: Regulatory pathways.
Figure 1 - Illustration of left-sided varicocele on physical examination.
Figure 3 - Illustration of the hypothalamo-pituitary-gonadal axis, Part 2: Feedback pathways.
normal sperm analysis, NOA and OA (15). Hypogonadism can be caused by primary testicular failure (hypergonadotropic hypogonadism) or secondary testicular failure resulting from a hypothalmo-pituitary deficiency (hypogonadotropic hypogonadism). Rarely, hypogonadism can occur in complete (testicular feminization) or partial (Reifenstein’s syndrome) androgen insensitivity syndrome. The end organs that are affected by hypogonadism are the external genitalia, the muscles, the bone, the skin and the brain. The symptoms include infertility, muscular hypotrophy, low bone mineral density, anemia, decreased acne, alterations in body hair distributions and decreased libido (5,15). In primary hypogonadism, normal or elevated LH and FSH levels accompany low serum testosterone levels. An FSH level greater than twice the normal upper limit is accepted as a clear elevation of the serum FSH level, and it is a reliable indicator of abnormal spermatogenesis (8). However, elevated FSH in men with azoospermia does not eliminate the possibility of obstruction and the capacity for fertility (16). Furthermore, a stronger correlation has been demonstrated between inhibin B and spermatogenesis
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compared with FSH and spermatogenesis (17). Primary hypogonadism can be observed in patients who exhibit testicular failure caused by congenital (anorchia, undescended testes and genetic abnormalities, such as Klinefelter’s syndrome or Y chromosome defects), acquired (trauma, tumor, torsion, orchitis or varicocele) or idiopathic causes. While evaluating testosterone levels, sex hormonebinding globulin should be considered, and an accurate calculation of free and bioavailable testosterone should be performed. Because of diurnal variation, blood samples used to measure testosterone should be taken prior to 10 o’clock in the morning. The Food and Drug Administration (FDA) defines the normal range of total testosterone levels as between 300 and 1000 ng/dL (5,15). Hypogonadotropic hypogonadism is characterized by low serum FSH levels in association with low serum testosterone levels and, generally, low LH levels. According to a University of Illinois study, nearly half of all men who have NOA suffer from hypogonadotropic hypogonadism. This finding suggests that hypogonadotropic hypogonadism may be considerably more prevalent in the infertile male population than was previously believed. Genetic hypothalamic disorders, such as Kallmann syndrome, and congenital or acquired pituitary deficiencies, such as empty sella syndrome or pituitary tumors (functional or nonfunctional), can cause hypogonadotropic hypogonadism. In men who have azoospermia with anosmia, decreased libido, gynecomastia, headaches or visual field deficits, hypogonadotropic hypogonadism should be suspected, and a complete endocrine work-up, including cranial imaging, should be performed.
patients with clinical varicocele. Doppler US is a promising method for assessing patients who are affected by azoospermia. This technique allows the differentiation of obstructive azoospermia (normal vessel distribution) from nonobstructive azoospermia (reduced or absent testicular vessels) (18). The benefit of evaluating the intratesticular blood vessel distribution prior to the performance of any method to retrieve intra-testicular spermatozoa for intracytoplasmic sperm injection has also been demonstrated (19). When evaluating the azoospermic male to diagnose EDO, TRUS is used for men with low ejaculate volume, but only rarely for those with normal ejaculates (8). A dilated seminal vesicle (anterior-posterior diameter $15 mm, Figure 4) and anechoic areas in the seminal vesicle are the TRUS abnormalities most frequently associated with EDO (20). TRUS is also able to identify other known and potentially correctable OA causes, such as Mu¨llerian (utricular) or Wolffian (ejaculatory duct) cysts, ejaculatory duct calcifications, congenital unilateral or bilateral absence of the vas deferens, and obstructing seminal vesicle cysts (21). TRUS-guided seminal vesiculography and seminal vesicle washout are other invasive imaging techniques used to investigate OA (22). In a prospective, comparative study, Purohit et al. observed that TRUS has a poor specificity for EDO evaluation when compared with vesiculography, seminal vesicle aspiration and duct chromotubation (23). Newly developed 3D-guided transrectal imaging devices enable easier visualization and needle guidance for vesiculography techniques (Figure 5). The combination of azoospermia, a testis biopsy revealing complete spermatogenesis and at least one palpable vas deferens strongly warrant a vasography. If necessary, the biopsy should be performed at the same time as the scrotal exploration and the definitive repair of obstruction. Stricture or obstruction at the vasography site, vasal blood supply injury, hematoma and sperm granuloma are potential complications of vasography (24). Endorectal coil magnetic resonance imaging assesses the relationships between the proximal prostatic urethra and the posterior wall of the ejaculatory ducts, which must be precisely known when endoscopic resection of the ejaculatory ducts is planned (25). An azoospermic male with hypogonadotropic hypogonadism may merit cranial imaging to identify hypothalamopituitary disorders, especially if prolactin levels are elevated.
& IMAGING TECHNIQUES Scrotal ultrasonography (US), transrectal US (TRUS), TRUS-guided seminal vesiculography, seminal tract washout, vasography, endorectal magnetic resonance imaging, abdominal US and cranial imaging are all imaging studies that can be used to evaluate males with azoospermia. Scrotal US is a first-line, basic imaging tool for all scrotal abnormalities, and it has also been demonstrated that testicular volume as measured by scrotal US is significantly correlated with testicular function. An increased resistive index and pulsatility index of the capsular branches of the testicular arteries on unenhanced color Doppler US examination may indicate impaired testicular microcirculation in
Figure 4 - Transrectal imaging of the seminal vesicles.
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Figure 5 - New 3D-guided transrectal imaging device (TargetScanH, Envisioneering Inc., St. Louis, MO).
provides couples with information about the nature, inheritance pattern, and implications of genetic disorders to help them make informed medical and personal decisions. The common genetic disorders that are associated with azoospermia are reviewed below.
Abdominal US imaging should be considered if there is a unilateral or bilateral congenital absence of the vas deferens. Because of the embryologic origins of the vas deferens and the kidney, anomalies in these organs tend to coexist. One study has demonstrated that 26% and 11% of men with unilateral or bilateral congenital absence of vas deferens, respectively also exhibit renal agenesis (26).
Kallmann syndrome Kallmann syndrome is an X chromosome-linked disorder characterized by isolated gonadotropin releasing hormone (GnRH) deficiency accompanied by complete or partial anosmia. Six X chromosome-linked autosomal dominant and recessive genes have been identified; of these, KAL1 is the gene that is most commonly associated with Kallmann syndrome. This syndrome is essentially a hormonal disorder in which the lack of GnRH secretion leads to testicular insufficiency (i.e., hypogonadotropic hypogonadism) (31). Kallmann syndrome is diagnosed clinically in the presence of anosmia, micropenis, cryptorchidism, diminished libido, erectile dysfunction and the absence of secondary sex characters. While serum testosterone level is low (,100 ng/ml) in patients with Kallmann syndrome, pituitary and hypothalamus imaging studies are normal. Adult males with Kallmann syndrome tend to exhibit prepubertal testicular volume (,4 ml) and have eunuchoid body habitus caused by delayed skeletal maturation (31). One study recently demonstrated that testicular morphology in patients with Kallmann syndrome can vary (32). However, spermatogenesis can be easily induced by hormonal stimulation (5). Depending on the type of gene mutation, nonreproductive phenotypes in men with Kallmann syndrome can include unilateral renal agenesis, dyskinesia and/or skeletal abnormalities, cleft lip/palate, ear/hearing defects, coloboma (eye defect) and hyperlaxity of the joints.
Testicular biopsy In the evaluation of azoospermic males who have normalsized testis and a normal hormone profile, a testicular biopsy is critical for distinguishing between OA and NOA. If feasible, it is best to plan for the cryopreservation of the sperm at the same time as this procedure. In NOA, the prognostic value of the testis biopsy is controversial. Although many groups have not demonstrated a relationship between testicular histology and sperm retrieval rate, certain groups argue that a prior testicular biopsy may help to determine the sperm retrieval success rate during a follow-up procedure, such as a microTESE (testicular sperm extraction) (27,28). It has also been demonstrated that sperm retrieval success rates are generally high in men with hypospermatogenesis, moderate in men with maturation arrest and limited in men with Sertoli cell-only syndrome. However, examiners should bear in mind that a single testicular biopsy is not representative of the entire testicle (29). A normal testicular biopsy implies obstruction at some level in the sperm transport system. Diagnostic testicular biopsies are of limited value in men with small testis size and elevated FSH levels (greater than twice the upper limit), which supports a diagnosis of NOA. Research recommends that the patient undergo a microTESE (combined with cryopreservation) in these cases and subsequent in vitro fertilization with intracytoplasmic sperm injection (ICSI); this procedure would enable several samples to be collected (5,8). A diagnostic testicular biopsy may be considered for select azoospermic individuals who have risk factor(s) for a testicular germ cell tumor, such as cryptorchidism, atrophic testis and a testicular germ cell history accompanied by testicular microlithiasis on ultrasound imaging (30). Again, if feasible, any sperm that are detected should be cryopreserved at the time of this biopsy.
Klinefelter’s syndrome Klinefelter’s syndrome has a wide spectrum of clinical presentations. It is a chromosomal disorder in which at least one additional X chromosome is observed in the male karyotype. Although there are several mosaic forms of this syndrome, most cases are of the nonmosaic form, 47, XXY. Klinefelter’s syndrome is the most common chromosome aneuploidy in human beings and the most common form of male hypogonadism, with a prevalence of 0.1 to 0.2% in the general population and up to 3.1% in the infertile population. The presence of the extra X chromosome sets in motion several undefined events that lead to spermatogenic and androgenic failure, gynecomastia, and learning difficulties (13,14,33). The extra X chromosome may originate from either the maternal or paternal side.
Genetic investigation Genetic factors occupy an important place in the evaluation and management of the azoospermic male. Such factors can be pretesticular (e.g., Kallmann syndrome), testicular (e.g., Klinefelter’s syndrome or Y chromosome microdeletions) or post-testicular (CBAVD). Genetic counseling
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sperm, a present or absent distal two-thirds of the epididymis and a bilateral absence of the vas deferens. Semen analysis reveals a low-volume (0.5 ml), acidic ejaculate that is devoid of fructose and seminal vesicle fluid because of atrophic, dysfunctional or absent seminal vesicles. The seminal vesicle anomalies can be confirmed with TRUS imaging (14). Most men with CBAVD exhibit normal spermatogenesis, but it has been observed that a large proportion exhibit impaired spermatogenesis. Prior to sperm harvesting, other potential coexisting causes of impaired spermatogenesis should be investigated (38). A careful abdominal US should be performed because as many as 10% of patients with CBAVD may also exhibit renal agenesis (26).
The clinical presentation of Klinefelter’s syndrome varies according to the age at diagnosis and the severity of the mosaicism. It is difficult to differentiate prepubertal boys with Klinefelter’s syndrome from normal boys based solely on their phenotype. Small, firm testes and varying symptoms of androgen deficiency characterize Klinefelter’s syndrome in adolescence and after puberty (13). On one end of the spectrum are boys who are identified as having Klinefelter’s syndrome because they have failed to undergo puberty and virilization as a result of nearly complete androgenic malfunction. These boys exhibit a eunuchoid appearance. On the opposite end of the spectrum are phenotypically normal boys who are diagnosed with Klinefelter’s syndrome during an evaluation for azoospermia (14). Although the exact mechanism of androgen deficiency is unknown, most patients with Klinefelter’s syndrome exhibit low serum testosterone concentrations and elevated FSH levels. This reflects spermatogenic compromise and the compensatory elevation of LH levels that results because the Leydig cells are being maximally stimulated and have a small reserve capacity (14). The majority of these patients also suffer from decreased libido and erectile dysfunction. Generally, the patients’ ejaculate presents with azoospermia. A testis biopsy reveals extensive fibrosis, hyalinization of seminiferous tubules and hyperplasia of the interstitium. However, the tubules may exhibit residual foci of spermatogenesis (34).
Y chromosome microdeletions The relationship between deletions on the Y chromosome and azoospermia was first recognized in 1976. With the elucidation of the molecular anatomy of the Y chromosome, specific microdeletions that are associated with azoospermia or severe oligospermia were discovered in the 1990s (39). Since this time, several case series have been published. A study with a large number of participants demonstrated that the prevalence of microdeletions was approximately 3% in unselected infertile men, 8% in men with NOA and 5.5% in men with severe OA (40). Although rare, these microdeletions have also been reported to occur in fertile men (41). Three microdeletions have been mapped to three different regions on the long arm of the Y chromosome. These regions are referred to as azoospermia factors (AZF)a, AZFb and AZFc and are observed proximally, centrally and distally on Yq11, respectively (42). Multiple genes are distributed throughout these regions; most are involved in spermatogenesis but are still poorly characterized. For example, the deleted-in-azoospermia (DAZ) gene is located in the AZFc region. This gene encodes a transcription factor that is generally present in men with normal fertility (43). The most frequently deleted region is AZFc (65-70%), and the least frequently deleted region is AZFa (5%). AZFb, AZFb+c and AZFa+b+c deletions are responsible for approximately 25 to 30% of Y microdeletions. It has been reported that Y microdeletions are observed nearly exclusively in patients with severe oligospermia (,1 million spermatozoa/ml) and are extremely rare in patients with sperm concentrations .5 million spermatozoa/ml (44). Genetic testing for Y microdeletions may predict the outcome of sperm retrieval techniques. One study has reported that sperm retrieval is possible in approximately 50% of patients with AZFc and partial AZFb deletions. The same study reported that the possibility of detecting mature spermatozoa in patients with complete AZFb deletions is virtually zero (45). In another study, Kamp et al. demonstrated a strong association between AZFa deletions and Sertoli cell-only syndrome (46). It is also important to know whether AZFc microdeletions are present in patients with oligospermia given the evidence of a progressive decrease in sperm count over time in such men. The cryopreservation of spermatozoa in these cases may avoid invasive sperm retrieval procedures in the future (45). Hormonal analysis (FSH and inhibin B levels) studies have not been reliable in discriminating between patients
Congenital bilateral absence of vas deference Congenital bilateral absence of vas deference (CBAVD) is observed in 2 to 6% of men with OA and is responsible for infertility in approximately 1% of infertile men (10,35). A strong association between CBAVD and the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been demonstrated (36). This gene is located on the short arm of chromosome 7 and encodes the CFTR protein, which is crucial for maintaining proper sodium/chloride balance in epithelial secretions. This balance is necessary to optimize the viscosity and fluidity of these secretions. Approximately 1,500 different mutations of the CFTR gene have been described. Nearly all male patients with clinically diagnosed cystic fibrosis have CBAVD, and approximately 80% of patients with CBAVD have mutations in at least one CFTR allele. The inability to identify a second mutation is presumed to result from the fact that these mutations are located elsewhere in the noncoding regions of the CFTR gene (5,8,37). Cystic fibrosis (CF) is characterized by elevated concentrations of electrolytes in the sweat, chronic pulmonary disease resulting from thickened respiratory epithelial secretions and pancreatic exocrine insufficiency secondary to thickened and occlusive ductal secretions. Both maternal and paternal mutant alleles must be present to cause clinical CF. However, the clinical presentation of CF depends on the severity of the mutations in each CFTR allele and/or in the noncoding regions of the genes (e.g., the 5T alleles). Thus, whereas a subset of patients with CFTR mutations suffer from severe pulmonary disease and pancreatic dysfunctions, the bilateral absence of the vas deferens may be the only observable effect in other patients (14,37). The physical examination of the patients with CBAVD reveals normal-sized and full testes, a full and firm caput epididymis caused by efferent ducts that are distended with
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with idiopathic and microdeletion-associated oligospermia and azoospermia (44). The male offspring of men with Y chromosome microdeletions are likely to inherit the same abnormality and may also be infertile. It is unclear whether Y chromosome microdeletions can cause additional health risks or affect the results of assisted reproductive techniques. Genetic counseling may be offered whenever a genetic abnormality is suspected in either the male or the female partner. Men with NOA should receive genetic counseling and should be offered karyotyping and Y chromosome microdeletion analysis before their sperm is used in assisted reproductive techniques (8).
with information about the nature, inheritance patterns, and implications of genetic disorders to help them make informed medical and personal decisions.
& ACKNOWLEDGMENTS We would like to thank Dr. Ashok Agarwal, Dr. Johannes Vieweg and Tom Crawford for their continued support of our program.
& AUTHOR CONTRIBUTIONS Gudeloglu A performed the research review, collected the data and wrote the preliminary draft of the manuscript. Parekattil S performed an additional research review and edited and revised the manuscript.
& EXPERT COMMENTARY
& REFERENCES
Azoospermic men constitute a significant portion of the infertile male population. A detailed and complete medical history, physical examination and hormonal profile are essential in the evaluation of the azoospermic male. Imaging studies, testicular biopsies (with cryopreservation) and genetic testing are also important. Men with NOA should be offered genetic counseling before their sperm is used in assisted reproductive techniques. The accurate classification and evaluation of the azoospermic male is important when determining the therapeutic approach that will be chosen for such patients.
1. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: World Health Organization; 2010. 2. Corea M, Campagnone J, Sigman M. The diagnosis of azoospermia depends on the force of centrifugation. Fertil Steril. 2005;83(4):920-2, http://dx.doi.org/10.1016/j.fertnstert.2004.09.028. 3. Jarow JP, Espeland MA, Lipshultz LI. Evaluation of the azoospermic patient. J Urol. 1989;142(1):62-5. 4. Stephen EH, Chandra A. Declining estimates of infertility in the United States: 1982-2002. Fertil Steril. 2006;86(3):516-23, http://dx.doi.org/10. 1016/j.fertnstert.2006.02.129. 5. Jungwirth A, Diemer T, Dohle GR, Giwercman A, Kopa Z, Krausz C, et al. European Association of Urology Guidelines on Male Infertility. 2012. 6. Colpi GM, Piediferro G, Nerva F, Giacchetta D, Colpi EM, Piatti E. Sperm retrieval for intra-cytoplasmic sperm injection in non-obstructive azoospermia. Minerva Urol Nefrol. 2005;57(2):99-107. 7. Esteves SC, Miyaoka R, Agarwal A. Sperm retrieval techniques for assisted reproduction. Int Braz J Urol. 2011;37(5):570-83. 8. Evaluation of the azoospermic male. Fertil Steril. 2008;90(5 Suppl):S74-7. 9. Smith JF, Walsh TJ, Turek PJ. Ejaculatory duct obstruction. Urol Clin North Am. 2000;35(2):221, viii-7. 10. Donat R, McNeill AS, Fitzpatrick DR, Hargreave TB. The incidence of cystic fibrosis gene mutations in patients with congenital bilateral absence of the vas deferens in Scotland. Br J Urol. 1997;79(1):74-7. 11. Grangeia A, Niel F, Carvalho F, Fernandes S, Ardalan A, Girodon E, et al. Characterization of cystic fibrosis conductance transmembrane regulator gene mutations and IVS8 poly(T) variants in Portuguese patients with congenital absence of the vas deferens. Hum Reprod. 2004;19(11):2502-8, http://dx.doi.org/10.1093/humrep/deh462. 12. Sabanegh E, Agarwal A. Male Infertility. In: Kavoussi LR, Novick AC, Partin AW, Peters CA, editors. Campbell-Walsh Urology. Tenth ed. United States of America: ELSEVIER SAUNDERS; 2012. p. 616-47. 13. Lanfranco F, Kamischke A, Zitzmann M, Nieschlag E. Klinefelter’s syndrome. Lancet. 2004;364(9430):273-83, http://dx.doi.org/10.1016/ S0140-6736(04)16678-6. 14. Oates RD. The genetic basis of male reproductive failure. Urol Clin North Am. 2008;35(2):257, ix-70. 15. Sussman EM, Chudnovsky A, Niederberger CS. Hormonal evaluation of the infertile male: has it evolved? Urol Clin North Am. 2008;35(2):147, vii-55. 16. Hauser R, Temple-Smith PD, Southwick GJ, de Kretser D. Fertility in cases of hypergonadotropic azoospermia. Fertil Steril. 1995;63(3):631-6. 17. Pierik FH, Vreeburg JT, Stijnen T, De Jong FH, Weber RF. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab. 1998;83(9):3110-4, http://dx.doi.org/10.1210/jc.83.9.3110. 18. Schurich M, Aigner F, Frauscher F, Pallwein L. The role of ultrasound in assessment of male fertility. Eur J Obstet Gynecol Reprod Biol. 2009;144 Suppl 1:S192-8, http://dx.doi.org/10.1016/j.ejogrb.2009.02.034. 19. Foresta C, Garolla A, Bettella A, Ferlin A, Rossato M, Candiani F. Doppler ultrasound of the testis in azoospermic subjects as a parameter of testicular function. Hum Reprod. 1998;13(11):3090-3, http://dx.doi. org/10.1093/humrep/13.11.3090. 20. Colpi GM, Negri L, Nappi RE, Chinea B. Is transrectal ultrasonography a reliable diagnostic approach in ejaculatory duct sub-obstruction? Hum Reprod. 1997;12(10):2186-91, http://dx.doi.org/10.1093/humrep/12.10. 2186. 21. Kuligowska E, Fenlon HM. Transrectal US in male infertility: spectrum of findings and role in patient care. Radiology. 1998;207(1):173-81. 22. Donkol RH. Imaging in male-factor obstructive infertility. World J Radiol. 2010;2(5):172-9. 23. Purohit RS, Wu DS, Shinohara K, Turek PJ. A prospective comparison of 3 diagnostic methods to evaluate ejaculatory duct obstruction. J Urol. 2004;171(1):232-5; discussion 5-6.
& KEY ISSUES a) Azoospermia is defined as the absence of sperm in at least two different ejaculate samples (including the centrifuged sediment) (1,2). Ten to fifteen percent of couples in the general population suffer from infertility issues (3,4). Approximately 50% of these cases can be attributable to a male issue. Ten to twenty percent of these men (or 1% of men in the general population) suffer from azoospermiainduced infertility (3). b) A complete medical history, physical examination and hormonal investigation are the principal components of the initial evaluation of the azoospermic male (8). c) Sperm production is controlled by the hypothalamopituitary-gonadal axis (Figures 2 and 3). d) Scrotal ultrasonography (US), transrectal US (TRUS), TRUS-guided seminal vesiculography, seminal tract washout, vasography, endorectal magnetic resonance imaging, abdominal US and cranial imaging studies can be performed when evaluating the azoospermic male. e) In the evaluation of azoospermic patients who have normal-sized testes and a normal hormone profile, testicular biopsy has a critical role in distinguishing between OA and NOA. It is best to plan for the cryopreservation of sperm at the time of the biopsy, if feasible. f) Diagnostic testicular biopsies are of limited value in men with small testes and elevated FSH levels (greater than twice the upper limit), which support a diagnosis of NOA. It is recommended that such patients undergo a microTESE (combined with cryopreservation) and subsequent in vitro fertilization via intracytoplasmic sperm injection (ICSI); this procedure would enable several samples to be taken (5,8). g) Genetic factors are important in the evaluation and management of azoospermic males. These factors can be pretesticular (Kallmann syndrome), testicular (Klinefelter’s syndrome or Y chromosome microdeletions) or posttesticular (CBAVD). Genetic counseling provides couples
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24. Goldstein M. Vasography. In: Goldstein M, editor. Surgery of Male Infertility. 1st ed., United States of America: SAUNDERS; 1995. p. 26-31. 25. Cornud F, Belin X, Delafontaine D, Amar T, Helenon O, Moreau JF. Imaging of obstructive azoospermia. Eur Radiol. 1997;7(7):1079-85, http://dx.doi.org/10.1007/s003300050258. 26. Schlegel PN, Shin D, Goldstein M. Urogenital anomalies in men with congenital absence of the vas deferens. J Urol. 1996;155(5):1644-8. 27. Hauser R, Yogev L, Paz G, Yavetz H, Azem F, Lessing JB, et al. Comparison of efficacy of two techniques for testicular sperm retrieval in nonobstructive azoospermia: multifocal testicular sperm extraction versus multifocal testicular sperm aspiration. J Androl. 2006;27(1):28-33. 28. Ramasamy R, Schlegel PN. Microdissection testicular sperm extraction: effect of prior biopsy on success of sperm retrieval. J Urol. 2007;177(4):1447-9. 29. Dohle GR, Elzanaty S, van Casteren NJ. Testicular biopsy: clinical practice and interpretation. Asian J Androl. 2012;14(1):88-93. 30. van Casteren NJ, Looijenga LH, Dohle GR. Testicular microlithiasis and carcinoma in situ overview and proposed clinical guideline. Int J Androl. 2009;32(4):279-87. 31. Pallais JC, Au M, Pitteloud N, Seminara S, Crowley WF. Kallmann Syndrome. In: Pagon RA, Bird TD, Dolan CR, Stephens K, Adam MP, editors. GeneReviews. Seattle (WA). 1993. 32. Nishio H, Mizuno K, Moritoki Y, Kamisawa H, Kojima Y, Mizuno H, et al. Clinical features and testicular morphology in patients with Kallmann syndrome. Urology. 2012;79(3):684-6, http://dx.doi.org/10. 1016/j.urology.2011.10.032. 33. Visootsak J, Graham JM, Jr. Klinefelter syndrome and other sex chromosomal aneuploidies. Orphanet J Rare Dis. 2006;1:42. 34. Wikstrom AM, Dunkel L. Klinefelter syndrome. Best Pract Res Clin Endocrinol Metab. 2011;25(2):239-50, http://dx.doi.org/10.1016/j.beem. 2010.09.006. 35. Jequier AM, Ansell ID, Bullimore NJ. Congenital absence of the vasa deferentia presenting with infertility. J Androl. 1985;6(1):15-9. 36. Anguiano A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, et al. Congenital bilateral absence of the vas deferens.A primarily genital form of cystic fibrosis. JAMA. 1992;267(13):1794-7, http://dx.doi.org/10.1001/ jama.1992.03480130110034.
37. Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med. 1995;332(22):1475-80. 38. Meng MV, Black LD, Cha I, Ljung BM, Pera RA, Turek PJ. Impaired spermatogenesis in men with congenital absence of the vas deferens. Hum Reprod. 2001;16(3):529-33, http://dx.doi.org/10.1093/humrep/16. 3.529. 39. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet. 1976;34(2):119-24, http://dx.doi.org/10.1007/BF0027 8879. 40. Ferlin A, Arredi B, Speltra E, Cazzadore C, Selice R, Garolla A, et al. Molecular and clinical characterization of Y chromosome microdeletions in infertile men: a 10-year experience in Italy. J Clin Endocrinol Metab. 2007;92(3):762-70. 41. Pryor JL, Kent-First M, Muallem A, Van Bergen AH, Nolten WE, Meisner L, et al. Microdeletions in the Y chromosome of infertile men. N Engl J Med. 1997;336(8):534-9. 42. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet. 1996;5(7):93343, http://dx.doi.org/10.1093/hmg/5.7.933. 43. Jobling MA, Tyler-Smith C. The human Y chromosome: an evolutionary marker comes of age. Nat Rev Genet. 2003;4(8):598-612, http://dx.doi. org/10.1038/nrg1124. 44. Krausz C, Forti G, McElreavey K. The Y chromosome and male fertility and infertility. Int J Androl. 2003;26(2):70-5. 45. Krausz C, Quintana-Murci L, McElreavey K. Prognostic value of Y deletion analysis: what is the clinical prognostic value of Y chromosome microdeletion analysis? Hum Reprod. 2000;15(7):1431-4, http://dx.doi. org/10.1093/humrep/15.7.1431. 46. Kamp C, Huellen K, Fernandes S, Sousa M, Schlegel PN, Mielnik A, et al. High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome. Molecular Hum Reprod. 2001;7(10):987-94, http://dx.doi.org/10.1093/molehr/7.10.987.
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REVIEW
The importance of semen analysis in the context of azoospermia Nabil Aziz Liverpool Women’s Hospital & The University of Liverpool, Liverpool, United Kingdom
Azoospermia is a descriptive term referring to ejaculates that lack spermatozoa without implying a specific underlying cause. The traditional definition of azoospermia is ambiguous, which has ramifications on the diagnostic criteria. This issue is further compounded by the apparent overlap between the definitions of oligospermia and azoospermia. The reliable diagnosis of the absence of spermatozoa in a semen sample is an important criterion not only for diagnosing male infertility but also for ascertaining the success of a vasectomy and for determining the efficacy of hormonal contraception. There appears to be different levels of rigor in diagnosing azoospermia in different clinical situations, which highlights the conflict between scientific research and clinical practice in defining azoospermia. KEYWORDS: Azoospermia; Male; Infertility; Semen Analysis; Oligozoospermia. Aziz N. The importance of semen analysis in the context of azoospermia. Clinics. 2013;68(S1):35-38. Received for publication on August 1, 2012; Accepted for publication on August 5, 2012 E-mail: n.f.aziz@liverpool.ac.uk Tel.: 44 151 7089988
of a centrifuged sample’’ (3). The American Urological Association offers the following, more detailed definition: ‘‘no sperm after centrifugation at 3000 x g for 15 minutes and examination of the pellet’’ (4). The aim of the examination of the pelleted semen is to exclude cryptospermia, which is the presence of a very small number of live sperm in a centrifuged pellet but not in a standard semen analysis. Thus, the accurate assessment of very low sperm counts is particularly important to avoid labeling severely oligospermic men as azoospermic. In one study, centrifuging semen at the low speed of 200 x g for 10 minutes revealed that 18.6% of men diagnosed with ‘obstructive azoospermia’ and 22.8% of men diagnosed with ‘nonobstructive azoospermia’ had motile and non-motile spermatozoa in the semen pellet (5). In addition to these laboratory considerations, the need for a change in the clinical definition of azoospermia to include its etiology, treatment, and prognosis has been repeatedly expressed (6,7).
Azoospermia is a descriptive term referring to ejaculates that lack spermatozoa without implying a specific underlying cause. The condition is almost always an unforeseen finding when semen analysis is performed for any indication. Only in a few cases is azoospermia expected prior to semen analysis, such as in cystic fibrosis, Klinefelter’s syndrome and previous vasectomy cases. Such azoospermic semen samples are found in up to 2% of the adult male population and 5-59% of infertile men (1). It is important for azoospermia to be distinguished from aspermia; specifically, the latter indicates the lack of semen formation or the lack of ejaculation, such as in the case of total retrograde ejaculation.
& THE DEFINITION AND DIAGNOSIS OF AZOOSPERMIA The appropriateness of the term azoospermia and the reliability of diagnosing the absence of spermatozoa have been the focus of debate over the past decade. The traditional definition of azoospermia is ambiguous, which has ramifications on the diagnostic criteria. The 5th edition of the World Health Organization (WHO) manual (2010) (2) adopted the following definition that was first proposed by Eliason in 1981: ‘‘no spermatozoa are found in the sediment
& THE ROUTINE ASSESSMENT OF SPERM COUNT The total number of sperm in an ejaculate is influenced by testicular sperm production, the integrity of the conducting system, the presence of retrograde ejaculation (partial or total), and the duration of abstinence before the analysis. The WHO laboratory manual for the examination and processing of human semen includes standards to enhance the accuracy and precision of the sperm number estimates to make them reproducible. Special attention is required to control patient-related factors, such as the optimal abstinence duration of 2-7 days and the complete collection of ejaculate. Frequent ejaculation within a short period of time may deplete the epididymal stores, resulting in hardly any
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)05
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detectable sperm in the semen sample. Similarly, losing the first portion of the ejaculate, which is the sperm-rich portion, will significantly affect the accuracy of the assessment of sperm number. The last portion of the ejaculate is comprised mainly of seminal vesicular fluid (8). Thus, patients need to receive clear instructions, both verbal and written, on how to collect the entire ejaculate and to report the loss of any fraction of the sample. Sound laboratory techniques are needed to reduce the amount of analytical error and enhance the precision of the sperm count. These techniques include the adequate mixing of the ejaculate, appropriate semen dilution if needed, and comparison of the replicate counts to determine if they are acceptably close (2). In addition to these technical details, there is a high biological variation in semen quality, including transient azoospermia, that may influence the clinical interpretation of seminal parameters (9). Transient azoospermia is also encountered secondary to toxic, environmental, infectious or iatrogenic conditions. Given this observation, repeating the examination of ejaculates on two to three occasions is helpful to obtain baseline data (9,10,11).
the pH of liquefied semen is 7.2 (2). In CBAVD, the semen pH is characteristically low (,6.8) as a consequence of dysplasia or the absence of the seminal vesicles. When the fructose-rich alkaline secretion of the seminal vesicles is lost, the seminal plasma is formed mainly from the relative scanty and acidic prostatic secretion.
& THE DIAGNOSTIC VALUE OF MACROSCOPIC FEATURES OF SEMEN IN AZOOSPERMIA Semen volume and pH are important for determining the differential diagnosis of the cause of azoospermia. In patients with low-volume, acidic, azoospermic samples, the differential diagnosis is CBAVD or bilateral complete ejaculatory duct obstruction (EDO). A fructose assay is not needed because the volume coupled with the pH indicates no contribution from the seminal vesicles. Azoospermic ejaculates with a normal volume and alkaline pH indicate functional seminal vesicles and patent ejaculatory ducts. The differential diagnosis includes spermatogenic failure or an obstruction at the level of the more proximal vas deferens or epididymis but does not include CBAVD or bilateral EDO. In azoospermic cases with an alkaline, low-volume ejaculate, the seminal vesicles are present and functional, and at least one ejaculatory duct is open. Therefore, in cases of azoospermia, attention to the details of semen volume and pH may be quite helpful in establishing the diagnosis.
& THE MACROSCOPIC FEATURES OF AZOOSPERMIC SEMEN SAMPLES Opacity of the ejaculate A normal liquefied semen sample with a normal cellular content will have a homogenous grey-opalescent appearance. Azoospermic samples and those with very low sperm counts appear less opaque.
& MICROSCOPIC EXAMINATION OF CENTRIFUGED SAMPLES TO DETECT SPERMATOZOA When no spermatozoa are observed in replicate wet preparations, the semen sample can be centrifuged, and the pelleted semen can then be examined to determine if any spermatozoa are present. Whether spermatozoa are found in the pellet depends on the centrifugation time and speed and on how much of the pellet is examined (5,19).
Semen volume The seminal vesicles contribute up to 70% of the normal ejaculate volume. The lower reference limit for semen volume is 1.5 ml (5th percentile, 95% confidence interval 1.4–1.7) (2). Although a low sperm volume is more likely to be due to the incomplete collection of the ejaculate, it may also be due to obstruction of the ejaculatory duct or congenital bilateral absence of the vas deferens (CBAVD) (12,13). In a study of 105 males diagnosed with CBAVD, the mean ejaculate volume was 0.7 ml (14). In CBAVD, there is dysplasia or absence of the seminal vesicles with the loss of its contribution to the semen volume. Retrograde ejaculation and frequent orgasms may lead to what is described in lay terms as ‘dry ejaculation’. In these situations, there will be hardly any seminal fluid containing sperm. Retrograde ejaculation should be suspected in any case of azoospermia and when the seminal fluid volume is ,1 ml. The diagnosis is confirmed by finding spermatozoa in the post-ejaculatory urine. Spermatozoa found in the pellet after urinary centrifugation will mostly be dead due to the combined effects of osmotic stress, low pH and urea toxicity (15). The recovery of high-quality sperm after the induced modification of the urine composition and pH to facilitate its use in the intracytoplasmic sperm injection technique (ICSI) has been described (16,17).
Centrifugation time and speed In the literature, there are different recommendations for the speed and time of centrifugation (Table 1). These recommendations appear not only inconsistent but also indecisive when terms such as ‘at least’ and ‘less than’ are used. In one study that attempted to resolve this confusion, 25 ejaculates from ‘azoospermic men’ were centrifuged at 600 x g for 10 minutes, and no sperm were found in the pellets (20). However, when supernatants resulting from the 600 x g centrifugation of the samples were centrifuged at 1000 x g for 15 minutes, spermatozoa were detected. Because no more sperm-containing pellets were detected by centrifuging the 1000 x g supernatant at 3000 x g for 15 minutes, the authors concluded that a minimum of 1000 x g for 15 minutes was adequate for the detection of azoospermia. The same study (20) demonstrated that centrifugation at 3000 x g for 15 minutes did not remove spermatozoa from the supernatant of 23 of 25 normozoospermic samples. Another study examined the interplay between the centrifugation speed and duration and demonstrated a dramatic increase in the appearance of spermatozoa in the pellet with both increasing time (10-15 minutes) and speed (600-3600 x g) of centrifugation (21). Thus, the accuracy of any centrifugation protocol of less than 3000 x g in pelleting all the spermatozoa in an ejaculate is uncertain. However, after high-speed centrifugation (3000 x g), motility may be lost
Semen pH The balance between the alkaline secretion of the seminal vesicles and the acidic prostatic secretion determines the semen pH. The importance of assessing the semen pH and its physiological reference range has been a matter of intense debate (18). The consensus lower reference value of
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Semen analysis and azoospermia Aziz N
inherently larger volume, such as the improved Neubauer chamber (2,22). Because this technique utilizes a fixative to immobilize the sperm cells, it is not suitable when the semen sample is examined for the potential harvesting of sperm, if found, for ICSI treatment. The absence of spermatozoa from the examined aliquot does not necessarily indicate their absence from the remainder of the sample. If no spermatozoa are found in replicate assessments, the WHO manual (2010) recommends that the sample be reported as ‘‘No spermatozoa were seen in the replicates, too few for accurate determination of concentration’’ (2). Another alternative is the use of fluorescence microscopy using an aliquot of semen diluted 1:2 with a fixative containing Hoechst 33342 bisbenzimide fluorochrome (1 mg/l) to label the sperm nuclei (2,22). Again, this method is not suitable when the identified sperm are used for ICSI treatment.
Table 1 - Different centrifugation speeds have been recommended to examine azoospermic ejaculates. Some of these recommendations appeared indecisive when terms such as ‘at least’ and ‘less than’ are used (italics are used to highlight such terms). Reference Mortimer (1994) (23) the Nordic Association for Andrology (24)
Recommended centrifugation 1000 x g for 15 minutes At least 1000 x g for 15 minutes
WHO manual (1999) (25)
600 x g for 15 minutes to concentrate samples with low sperm counts (less than 2 sperm per 400x field) Less than 3000 x g for 15 minutes for all samples in which spermatozoa are not detected
Corea et al. (2005) (20)
A minimum of 1000 x g for 15 minutes was adequate for the detection of azoospermia
WHO manual (2010) (2)
& FUTURE DEVELOPMENTS
3000 x g for 15 minutes for all samples in which no spermatozoa are detected
The standard method of semen analysis is to assess the macroscopic appearance and cellular content of the ejaculate. Investigation of the molecular composition of seminal plasma to explain the cause of sperm and seminal plasma abnormalities has thus far been limited to the evaluation of the fructose content and, in later years, the level of reactive oxygen species in the plasma (26,27,28)). More recently, the study of the seminal plasma proteome appears to offer the potential to identify biomarkers that may aid in the diagnosis of the causes of azoospermia. Many of the proteins in the seminal plasma are expressed in the testis and epididymis and are linked to fertility. Some of these proteins may be useful as noninvasive biomarkers to discriminate non-obstructive azoospermia from obstructive azoospermia (29). The reliable diagnosis of the absence of spermatozoa in a semen sample is important for diagnosing male infertility, ascertaining the success of vasectomy, and determining the efficacy of hormonal contraception. From the laboratory point of view, inconsistent approaches to studying semen and imprecision can handicap both research and clinical practice (30). These technical issues are further compounded by the apparent overlap between the definitions of oligospermia and azoospermia. This overlap is present in the WHO manual (2010), and although appearing unjustified, it echoes the different levels of rigor in diagnosing azoospermia in different clinical situations and highlights the conflict between scientific research and clinical practice in defining azoospermia.
(23), and the concentration will therefore be underestimated (22). This literature survey indicates that the replication of results among laboratories using different centrifugal forces is unlikely to be consistent (22). The most recent WHO manual (2010) suggested that when assessing an apparently azoospermic sample, consideration must be given to whether subjective data on the presence and motility of spermatozoa are sufficient or whether accurate spermatozoa counts are required. For example, when motile spermatozoa are sought in a postvasectomy semen sample, the high-speed centrifugation of spermatozoa must be avoided, and only an aliquot of the undiluted sample can be assessed. The microscopic examination in this procedure can take longer (up to 10 minutes) than low-speed centrifugation because the sample will have a high cellular background. When no spermatozoa are observed in replicate assessments of pelleted semen, the WHO manual (2010) recommends reporting the sample as ‘‘No spermatozoa were seen in the replicates, too few for accurate determination of concentration’’. This guarded reporting takes into account the errors of counting (2,22) and the possibility that the absence of spermatozoa from the examined aliquot does not necessarily indicate their absence from the remainder of the sample. This approach is adopted when a case of apparent azoospermia is examined further to determine if there are enough spermatozoa to fertilize a limited number of eggs using the ICSI technique. However, when the aim of pelleting semen samples is to obtain an accurate assessment of the sperm number, highspeed centrifugation should be used (3000 x g). This approach is necessary for male hormone contraception research and for diagnostic purposes, such as in CBAVD and the confirmation of sperm clearance after a vasectomy. In these situations, rendering spermatozoa immotile and promoting reactive oxygen species-induced sperm damage as a result of the high-speed centrifugation are irrelevant.
& REFERENCES 1. Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, Lansac J, et al. Incidence and Main Causes of Infertility in a Resident Population (1850000) of 3 French Regions (1988-1989). Hum Reprod. 1991;6(6):811-6. 2. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th ed. Geneva: World Health Organization; 2010. 3. Eliasson R. Analysis of semen. In: Burger H, de Kretser D, eds. The testis. New York: Raven Press; 1981. p 381-99. 4. Male Infertility Best Practice Policy Committee of the American Urological Association; Practice Committee of the American Society for Reproductive Medicine. Report on optimal evaluation of the infertile male. Fertil Steril. 2006;86(5 Suppl 1):S202-9. 5. Jaffe TM, Kim ED, Hoekstra TH, Lipshultz LI. Sperm pellet analysis: A technique to detect the presence of sperm in men considered to have azoospermia by routine semen analysis. J Urology. 1998;159(5):1548-50. 6. Sharif K. Reclassification of azoospermia: the time has come? Hum Reprod. 2000;15(2):237-8, doi: 10.1093/humrep/15.2.237.
Alternatives to centrifugation One alternative to centrifugation is the use of a low semen dilution (1+1 [1:2]) to evaluate larger volumes by either preparing more chambers or using chambers with an
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7. Ezeh UIO, Moore HMD. Redefining azoospermia and its implications. Fertil Steril. 2001;75(1):213-4, doi: 10.1016/S0015-0282(00)01657-5. 8. Bjorndahl L, Kvist U. Sequence of ejaculation affects the spermatozoon as a carrier and its message. Reprod Biomed Online. 2003;7(4):440-8, doi: 10. 1016/S1472-6483(10)61888-3. 9. Castilla JA, Alvarez C, Aguilar J, Gonzalez-Varea C, Gonzalvo MC, Martinez L. Influence of analytical and biological variation on the clinical interpretation of seminal parameters. Hum Reprod. 2006;21(4):847-51. 10. Carlsen E, Petersen JH, Andersson AM, Skakkebaek NE. Effects of ejaculatory frequency and season on variations in semen quality. Fertil Steril. 2004;82(2):358-66, doi: 10.1016/j.fertnstert.2004.01.039. 11. Keel BA. Within- and between-subject variation in semen parameters in infertile men and normal semen donors. Fertil Steril. 2006;85(1):128-34, doi: 10.1016/j.fertnstert.2005.06.048. 12. de la Taille A, Rigot JM, Mahe P, Gervais R, Dumur V, Lemaitre L, et al. [Correlation of genitourinary abnormalities, spermiogram and CFTR genotype in patients with bilateral agenesis of the vas deferens]. Prog Urol. 1998;8(3):370-6. 13. Daudin M, Bieth E, Bujan L, Massat G, Pontonnier F, Mieusset R. Congenital bilateral absence of the vas deferens: clinical characteristics, biological parameters, cystic fibrosis transmembrane conductance regulator gene mutations, and implications for genetic counseling. Fertil Steril. 2000;74(6):1164-74, doi: 10.1016/S0015-0282(00)01625-3. 14. Weiske WH, Salzler N, Schroeder-Printzen I, Weidner W. Clinical findings in congenital absence of the vasa deferentia. Andrologia. 2000;32(1):13-8. 15. Mortimer D. Semen analysis and other standard laboratory tests. In: Hargreave T, ed. Male Infertility. 2nd ed. London: Springer; 1994. p. 55-6. 16. Aust TR, Brookes S, Troup SA, Fraser WD, Lewis-Jones DI. Development and in vitro testing of a new method of urine preparation for retrograde ejaculation; the Liverpool solution. Fertil Steril. 2008;89(4):885-91, doi: 10. 1016/j.fertnstert.2007.04.042. 17. Jefferys A, Siassakos D, Wardle P. The management of retrograde ejaculation: a systematic review and update. Fertil Steril. 2012;97(2):306U551, doi: 10.1016/j.fertnstert.2011.11.019. 18. Meacham R. From Androlog. J Androl. 2002;23(3):330-1. 19. Lindsay KS, Floyd I, Swan R. Classification of Azoospermic Samples. Lancet. 1995;345(8965):1642, doi: 10.1016/S0140-6736(95)90150-7.
20. Corea M, Campagnone J, Sigman M. The diagnosis of azoospermia depends on the force of centrifugation. Fertil Steril. 2005;83(4):920-2, doi: 10.1016/j.fertnstert.2004.09.028. 21. Lindsay KS, Floyd I, Swan R. Classification of azoospermic patients. Lancet. 1995; 345(8965):1642, doi: 10.1016/S0140-6736(95)90150-7. 22. Cooper TG, Hellenkemper B, Jonckheere J, Callewaert N, Grootenhuis AJ, Kersemaekers WM, et al. Azoospermia: virtual reality or possible to quantify? J Androl. 2006;27(4):483-90. 23. Mortimer D. Practical Laboratory Andrology. New York: Oxford University Press; 1994. 24. NAFA and ESHRE-SIGA. Manual on Basic Semen Analysis (2002). Accessed 2012. Available from: http://www.ki.se/org/nafa/manual/ Manual2002.pdf. 25. World Health Organisation. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed. Cambridge: Cambridge University Press; 1999. 26. Aziz N, Saleh RA, Sharma RK, Lewis-Jones I, Esfandiari N, Thomas AJ, et al. Novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index. Fertil Steril. 2004;81(2):349-54, doi: 10.1016/j.fertnstert.2003.06 .026. 27. Aziz N, Agarwal A. Evaluation of sperm damage: beyond the World Health Organization criteria. Fertil Steril. 2008;90(3):484-5, doi: 10.1016/j. fertnstert.2007.07.1287. 28. Aziz N, Novotny J, Oborna I, Fingerova H, Brezinova J, Svobodova M. Comparison of chemiluminescence and flow cytometry in the estimation of reactive oxygen and nitrogen species in human semen. Fertil Steril. 2010;94(7):2604-8, doi: 10.1016/j.fertnstert.2010.03.022. 29. Batruch I, Smith CR, Mullen BJ, Grober E, Lo KC, Diamandis EP, et al. Analysis of Seminal Plasma from Patients with Non-obstructive Azoospermia and Identification of Candidate Biomarkers of Male Infertility. J Proteome Res. 2012;11(3):1503-11, doi: 10.1021/pr200812p. 30. Riddell D, Pacey A, Whittington K. Lack of compliance by UK andrology laboratories with World Health Organization recommendations for sperm morphology assessment. Hum Reprod. 2005;20(12):3441-5, doi: 10.1093/humrep/dei230.
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REVIEW
A comprehensive review of genetics and genetic testing in azoospermia Alaa J. Hamada,I Sandro C. Esteves,II Ashok AgarwalI I
Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio, USA. II ANDROFERT – Andrology & Human Reproduction Clinic, Campinas, Sa˜o Paulo, Brazil.
Azoospermia due to obstructive and non-obstructive mechanisms is a common manifestation of male infertility accounting for 10-15% of such cases. Known genetic factors are responsible for approximately 1/3 of cases of azoospermia. Nonetheless, at least 40% of cases are currently categorized as idiopathic and may be linked to unknown genetic abnormalities. It is recommended that various genetic screening tests are performed in azoospermic men, given that their results may play vital role in not only identifying the etiology but also in preventing the iatrogenic transmission of genetic defects to offspring via advanced assisted conception techniques. In the present review, we examine the current genetic information associated with azoospermia based on results from search engines, such as PUBMED, OVID, SCIENCE DIRECT and SCOPUS. We also present a critical appraisal of use of genetic testing in this subset of infertile patients. KEYWORDS: Azoospermia; Male Infertility; Genetic Testing; Genetic Diseases; Klinefelter Syndrome; Y Chromosome. Hamada AJ, Esteves SC, Agarwal A. A comprehensive review of genetics and genetic testing in azoospermia. Clinics. 2013;68(S1):39-60. Received for publication on September 5, 2012; Accepted for publication on September 6, 2012 E-mail: agarwaa@ccf.org Tel.: 1 216 444-9485
assisted reproduction therapeutic tools, clinical pregnancy and live birth rates were reported to range from 26-57% and 18-55% for NOA and OA, respectively (10-16). Azoospermia of a genetic origin is primarily caused by a wide array of genetic disorders, such as chromosomal abnormalities, monogenic disorders, multifactorial genetic diseases, and epigenetic disorders. These conditions constitute the genetic basis of reproductive failure. Table 1 summarizes the genetic basis of azoospermia at the posttesticular (obstructive azoospermia), pre-testicular and testicular (non-obstructive azoospermia) levels. The aim of this article is to review the genetic causes of azoospermia and to critically appraise the available types of genetic testing and the utility of such tests for the diagnosis and management of azoospermic males.
& INTRODUCTION Infertility refers to failure of a couple to conceive following 12 months of unprotected regular intercourse, and this problem affects 10-15% of couples in the United States (1). Male factor infertility is partially or fully responsible for approximately 30-55% of cases of infertility (2,3). It was recently reported that 1 in 13 men of reproductive age requests medical assistance to have a child (4). Azoospermia, which is the complete absence of sperm in the ejaculate, accounts for 10-15% of male infertility cases and generally affects 1% of the male population (3,5,6). Azoospermia is divided into two major categories: obstructive azoospermia (OA), in which there is genital tract outflow obstruction, blocking passage of the sperm, and non-obstructive azoospermia (NOA), in which the testicle fails to produce mature sperm in the ejaculate. Although some reports indicate a higher incidence of NOA than OA (60 vs. 40%) (6) and (85.2 vs. 12.9%) (7), others have reported the opposite. (8). Genetic factors explain 21-29% of azoospermia (9), whereas 12-41% of azoospermic cases are idiopathic and most likely related to unknown genetic factors (7). Using a series of advanced diagnostic and
& OBSTRUCTIVE AZOOSPERMIA OF GENETIC ORIGIN Obstructive azoospermia comprises the genetic and acquired diseases that cause the obstruction of the reproductive tract pathway. The genetic causes of OA account for approximately 30% of cases (17). In a study that evaluated 179 men with OA, congenital bilateral absence of the vas deferens (CBAVD) was the most frequent condition linked to genetic abnormalities (17). Four genetic posttesticular diseases have been characterized and are described below. Young syndrome is another condition that is associated with OA and that may have a genetic background. Nevertheless, no definitive causative mutations have been discovered.
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)06
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the vas deferens. This protein is composed of 1,480 amino acids and has a molecular weight of 168,173 Da. Several reports noted the role of CFTR in sperm maturation in the epididymis, as this protein is necessary for fluid absorption and the facilitation of sperm capacitation and fertilization ability (21-23). The level of functionally active CFTR protein determines the clinical phenotypes of CFTR mutations. Normal individuals should have an active CFTR protein level of 50-100%, whereas individuals with CF exhibit CFTR activity that is lower than 10% of normal levels (24). Only 2-3% of men with classic CF are fertile, and the majority are infertile (98%) due to congenital bilateral absence or atresia of the vas deferens, which results in azoospermia (25). Epididymal malformations are also common manifestation of CF and are characterized by the absence of the body and tail and blindended efferent ductules. Seminal vesicles in these men exhibit a spectrum of anomalies, ranging from atresia, hypoplasia and cystic dilatation. Obstructed ejaculatory ducts are also common. Patients with CF may exhibit varying combinations of these abnormalities. In most patients with CF, testicular histology indicates normal spermatogenesis. A subset of patients, however, exhibit impaired spermatogenesis, and such impairment may be attributed to the role of the CFTR protein in gametogenesis (26,27). Abnormal sperm morphology has also been described. It is attributed to defective spermiogenesis due to a lack of CFTR protein, which is also expressed in spermatozoa (28). The vast majority of patients with CF carry two major mutations in chromosome 7 (88%), whereas 11% exhibit compound heterozygous mutations, consisting of a severe mutation in one chromosome and a mild mutation in the other (18,29). The types of severe mutations that are frequently encountered in patients with CF encompass F508del (30%), G542X (3.4%) and G551D (2.4%) (30). The prevalence of these specific mutations vary with the ethnic background of the patient. For example, nF508 is the most common mutation in 70-90% of men with CF in North America and Northern Europe, compared with 50% in Southern Europe and less than 30% in Asians and Indians (31). CFTR mutations are also implicated in isolated seminal duct abnormalities. These abnormalities are referred to as primary genital or atypical forms of CF.
Table 1 - Genetic diseases and abnormalities that result in azoospermia at the post-testicular (obstructive azoospermia), pre-testicular and testicular (nonobstructive azoospermia) levels. Obstructive Azoospermia of Genetic Origin Cystic Fibrosis Congenital Bilateral Absence of the Vas Deferens Congenital Unilateral Absence of the Vas Deferens (CUAVD) Congenital Bilateral Epididymal Obstruction and Normal Vasa Young Syndrome Nonobstructive Azoospermia of Genetic Origin Genetic Pre-testicular Causes of NOA Hypothalamic HH Congenital HH Adult-onset genetic hypothalamic HH Pituitary Disorders Associated with Hypogonadism Generalized anterior pituitary hormone deficiency Selective gonadotropin deficiency Genetic Testicular Disorders Affecting Spermatogenesis and Androgen Production Klinefelter syndrome XX male syndrome Mutation in X-linked USP 26 X-linked SOX3 mutation Bilateral anorchia Noonan syndrome 45 X/46XY mosaicism (mixed gonadal dysgenesis) Affecting Spermatogenesis Y chromosome microdeletion Autosome translocations Monogenic disorders Multifactorial disorders (e.g., cryptorchidism) Affecting Androgen Production or Action Androgen receptor mutation Steroidogenic acute regulatory protein StAR mutation 3BHSD type 2 deficiency SRD5A2 mutation Dysfunctional Cell Regulatory Pathways Epigenetic Defects Genetic Abnormities at the Primordial Germ Cell Level
Cystic fibrosis Cystic fibrosis (CF) is a life-threatening autosomal recessive disease that affects 1/2,500 Caucasian newborns and has a carrier frequency of 4% (18). CF is characterized by the presence of thick and viscid secretions in the lungs, pancreas, intestines, and liver. Such abnormal secretions result in the formation of plugs that obstruct the ductal lumens in these organs, causing dilatations, frequent infections, and fibrosis. The cause of the viscid secretions is a failure of epithelial cells to transport chloride, sodium ions and water to the lumen of the epithelial tubes. This failure is due to a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel protein. The CFTR gene is located on chromosome 7 q31.2 (19) and has been implicated in the formation of excurrent seminal ducts (20). The CFTR gene is 190 kb in length and consists of 27 exons. Over 1,800 mutations in this gene have been reported (20). The CFTR protein is a glycosylated transmembrane ATPase protein that functions as a plasma membrane channel for sugars, peptides, inorganic phosphate, chloride, and metal cations. CFTR is expressed in the epithelial cells of exocrine tissues, such as the lungs, the pancreas, sweat glands, the head of the epididymis and
Congenital bilateral absence of the vas deferens The congenital bilateral absence of the vas deferens (CBAVD) invariably results in azoospermia. CFTR is mutated in 60-90% of patients with CBAVD (32). CBAVD accounts for at least 6-25% of cases of obstructive azoospermia and approximately 2% of infertility cases (33,34). Men with CBAVD generally exhibit either a single or two mild mutations in the CFTR gene (12%) or a combination of a severe and a mild mutation (88%) (18,35,36). Five patterns of CFTR mutations have been described: i) class I, which is characterized by a defect in protein synthesis, with a premature termination that results in a nonsense or truncated protein; ii) class II, which is caused by a defect in protein processing and localization of CFTR protein to the apical plasma membrane, e.g., DF508; iii) class III, which is characterized by a defect in cAMP regulation of the channel opening, e.g., G551D (replacement of glycine with aspartic acid at position 551); iv) class IV, which is caused by a partial decline in chloride conductance; and v) class V, which is characterized by reduced levels of functional CFTR
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patent contralateral vas exhibited mutations in the CFTR gene, although three of these patients had ipsilateral renal agenesis (41). Other authors have reported that CFTR mutations may be observed even in men with CUAVD who have normal patent contralateral vas. Radpour et al. demonstrated that in the absence of renal agenesis, five of seven (70%) Iranian men with CUAVD and a patent contralateral vas had either single or compound heterozygous CFTR mutations (42). Studies from Canada (43), Spain (44), Germany (45), France (37), and Portugal (43) also reported variable rates of CFTR mutations among men with CUAVD (20%, 27%, 60-100%, and 75%, respectively). However, a critical point with respect to these data sets, besides the small sample sizes, is the lack of information regarding the patency of the contralateral vas deferens.
protein. Classes I, II and III have been associated with a complete lack of functional CFTR and severe CF manifestations, including pancreatic insufficiency. Class IV and V mutations cause a mild CF phenotype due to residual CFTR protein activity. Men with compound heterozygosity (i.e., a mild and a severe mutation, such as DF508) may have either CBAVD or a mild form of CF. The common CFTR mutations found in men with CBAVD are the following: i) DF508, in which the three nucleotides that encode the phenylalanine at position 508 are missing in the protein’s amino acid sequence. DF508 is a class II mutation observed in 21-40% of men with CBAVD; ii) polymorphisms within intron 8 (5T, 7T). Such polymorphisms reduce the production of the CFTR protein, resulting in a reduction in the splicing efficiency of the CFTR gene (accounts for 19-37% of CBAVD cases); iii) a missense R117H mutation in exon 4 (14% of CBAVD cases); and iv) a combination of DF508/R117H, which represents the most common mutation in patients with CBAVD (40%) (36,37). A recent meta-analysis by Yu et al. revealed that 78% of men with CBAVD carry a minimum of one CFTR mutation. In the aforementioned study, 46% and 28% of men carried two mutations and a single mutation, respectively (38). Moreover, compound heterozygous DF508/5T and DF508/ R117H mutations were present in 17% and 4% of CBAVD cases, respectively. The three most common mutations are 5T (25%), DF508 (17%), and R117H (3%) (38). NonCaucasian men exhibit a higher incidence of single rather than double mutations (68 vs. 50%, p = 0.012), a higher frequency of 5T mutations (31 versus 20%, p = 0.009) and a ¨ F508 mutations (8 versus 22%, lower frequency of A p = 0.001) compared with Caucasian men (38). Intracytoplasmic sperm injection (ICSI) is a useful method for the treatment of azoospermic men with the CFTR mutation. Partners who both carry the mutation should be advised to have a preimplantation genetic diagnosis (PGD) performed to avoid passing the abnormality to their offspring (39).
Congenital bilateral epididymal obstruction and normal vasa CFTR mutations have been implicated in bilateral epididymal obstruction in azoospermic men in the presence of normal, bilaterally palpable vasa. Mak et al. identified CFTR mutations in 14/56 (25%) men with idiopathic epididymis obstruction (46). The most common identified mutations were IVS8-5T, DF508, R117H and L206W. Similarly, Jarvi et al. reported that as many as 47% of patients with bilateral epididymal obstruction carried CFTR mutations, such as IVS8-5T, DF508, and R117H (47).
Young syndrome Young syndrome is a rare disease primarily characterized by a constellation of three components, that is, bilateral epididymal obstruction with azoospermia, bronchiectasis, and chronic sinusitis. The estimated prevalence is unknown, with newly discovered cases being described as case reports. Unfortunately, the origin of this disease is also unknown, although childhood exposure to mercury and genetic etiologies have been suggested (48,49). Its familial incidence in one case and its association with medullary sponge kidney in another suggest its inheritability (50,51); however, mutations have not been identified. Male infertility is attributed to bilateral epididymal head dilatation and blockage by an expressible amorphous mass that is attributed to poor epididymal mucociliary clearance (49). The diagnosis of Young syndrome is made by the exclusion of the two other similar syndromes, namely, CF (screened for by testing for CFTR mutations) and immotile cilia syndrome, which is confirmed by prolonged nasal mucociliary clearance of the tested material (saccharine) (49,52). Functional rather than subtle ultrastructural epididymal and nasal ciliary defects are considered to be the basic mechanism of the disease, and epididymal aspirations revealed motile spermatozoa (52). Interestingly, epididymal obstruction often occurs in middle-aged men; therefore, previously successful parenthood may be anticipated in such syndromes (52,53).
Congenital unilateral absence of the vas deferens (CUAVD) This condition affects 0.5-1.0% of the male population and represents a heterogeneous disorder with respect to its etiology and clinical presentations. Whereas most men with CUAVD are fertile, a subgroup exhibits azoospermia or oligozoospermia. In the embryonic period, the vas deferens arises from the mesonephric duct. At the 7th week of development, this duct gives rise to the ureteric bud, which in turn induces the development of the kidney from the metanephros. An embryological insult to a single mesonephric duct at or prior to the seventh week of development can result in unilateral vasal aplasia and ipsilateral renal agenesis. It has been estimated that 79% of individuals with CUAVD have an absent ipsilateral kidney (40). CFTR mutations have been observed in men with CUAVD who exhibit no renal agenesis. Kolettis et al. reported either single CFTR mutations, such as Negative/ 621_G-T, or compound heterozygous mutations, such as DF508/5T and DF508/7T/9T, in three out of four men with CUAVD and a distally obstructed contralateral vas deferens. The fourth patient, who was negative for a CFTR mutation, exhibited contralateral renal agenesis (41). In the aforementioned series, none of six men with CUAVD and
& NON-OBSTRUCTIVE AZOOSPERMIA OF GENETIC ORIGIN Non-obstructive azoospermia (NOA) is a heterogeneous disorder that is characterized by various testicular tissue alterations. Such changes result in poor and/or absent spermatogenesis within the testes and the absence of sperm
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this hormone. Moreover, there is a brief postnatal surge of GnRH during the infantile period, which lasts for a few months and allows for the proper diagnosis of a suspected deficiency at an early age (57). Genetic HH is primarily divided into two general categories based on the age of onset: congenital and adultonset HH. Congenital HH is subdivided by the presence of an intact olfactory sense: anosmic HH (Kallmann syndrome) and congenital normosmic isolated HH (IHH). Prader-Willi syndrome is also a congenital disorder that presents with HH.
in the ejaculate. NOA accounts for approximately 60% of men with azoospermia and represents the most severe form of male factor infertility. Although NOA is caused by a multitude of factors, such as heat, radiation, drugs, varicocele, infections and cancer, genetic etiologies contribute significantly to the development of this disorder in 21-28% of cases (7,9,40,57-59). NOA is didactically divided into two major categories, i.e., pre-testicular and testicular. The genetic pre-testicular etiology encompasses hereditary hypothalamic-pituitary abnormalities, which lead to insufficient gonadotropin action on testicular cells. This defect results in small testes that exhibit an immature histological pattern. In these cases, immature Sertoli cells or spermatogonia type A (which primarily reside in the center of seminiferous tubules) and the absence of Leydig cells are often observed. Genetic testicular causes of NOA include the following: i) chromosomal abnormalities, ii) Y chromosome microdeletions, iii) failure of the primordial germ cells to reach the developing gonads, iv) lack of differentiation of the primordial germ cells to spermatogonia, and v) male germ line mutations that affect spermatogenesis. The lattermost cause is further divided into mutations that control transcription, signal transduction, apoptosis, cell response to stress factors, cytokines (cross-talk), immune sensitization of germ cells, meiotic divisions, and epigenetic factors. Genetic mutations of androgen receptors are also included in this category.
Congenital HH This disease is primarily characterized by early-onset hypogonadism due to the dysfunctional release or action of GnRH. This defect results in delayed or absent pubertal development, with low sex steroid levels in the setting of low or normal gonadotropin levels. Normal hypothalamic pituitary gland anatomy on magnetic resonance imaging and the absence of other causes of HH, such as hemochromatosis, are prerequisites for diagnosis (58). The congenital incidence of HH is 1-10/100,000 live births, with approximately 2/3 being due to Kallmann syndrome and 1/3 to normosmic HH (59). Kallmann syndrome (KS) is characterized by the presence of complete or partial anosmia in association with congenital HH. The failure of the migration of GnRH neurons from the olfactory placode to their destination in the hypothalamus and the olfactory lobe is the basic embryological defect that characterizes this syndrome (59). However, the genetic bases of this condition have not been fully elucidated. Sporadic (2/3) and familial (1/3) varieties of this condition have been described (60). Hereditary studies reveal that familial KS is heterogenetic, with variable modes of inheritance (autosomal dominant, autosomal recessive, and X-linked) being observed. X-linked inheritance is the most common mode. It is not only the genotypic characteristics but also the phenotypic features of this syndrome that are variable. A diverse spectrum of physical manifestations is observed. Males are affected five times more frequently than females, and its incidence in males is approximately 1/8,000 (61). Unfortunately, the genetic origin of only 50% of familial cases and 10% of sporadic cases has been clarified (62). Six known genes account for only 25-35% of all cases of KS (60). These genes are as follows: KAL-1, FGFR-1, PROK-2, PROKR-2, CHD-7, and FGF-8. However, other genetic abnormalities have been described, such as chromosomal translocation 46 XY, t(10,12) (63) and copy number variations (CNVs) (64). Regions in which CNVs are observed account for less than 12% of the human genome and are defined as large segments of DNA on a particular chromosome that have been deleted or duplicated (65,66). Five distinctive chromosomal regions have been implicated in Kallmann syndrome, and the majority of these CNVs involve the intronic regions of a particular gene, reflecting a possible disturbance in splicing mechanisms. These regions include the following: 1p21.1, 2q32.2, 8q21.13, 14q21.2, and Xp22.31 (64). KAL-1 was the first gene that was discovered in Kallmann syndrome patients. This gene maps to the X chromosome (Xp22.32) and consists of 14 exons (59,67). It encodes an 840-amino acid protein that is referred to as anosmin 1. This is an extracellular adhesion protein that has a potential role in orchestrating GnRH neuron adhesion and axonal
& GENETIC PRE-TESTICULAR CAUSES OF NOA Pre-testicular causes of NOA result from either hypothalamic or pituitary disorders.
Hypothalamic hypogonadotropic hypogonadism Genetic hypothalamic disorders essentially fall under the classification of hypothalamic hypogonadotropic hypogonadism (HH), which encompasses a broad spectrum of diseases characterized by various genotypes. A deficiency of gonadotropin-releasing hormone (GnRH) or its receptor is the fundamental endocrine abnormality that is detected in this disease. GnRH is a decapeptide that is synthesized by a loose network of neurons located in the medial basal hypothalamus (MBH) and in the arcuate nucleus of the hypothalamus. A subset of GnRH neurons is observed outside of the hypothalamus in the olfactory organ, reflecting the common embryological origin of these neurons (54). Developmentally, GnRH neurons originate from the olfactory placode/vomeronasal organ of the olfactory system and migrate along the vomeronasal nerves to the hypothalamus. Here, these cells extend processes to the median eminence and the pituitary gland (55). GnRH is synthesized as a precursor, 92-amino acid hormone and is then cleaved into a 69-amino acid prohormone. This prohormone is further cleaved at the nerve terminals to form the active decapeptide (55). GnRH receptors are plasma membrane-associated receptors that promote increases in intracellular calcium concentrations, which acts as a second messenger, upon binding to GnRH (55). The essential function of GnRH is to stimulate the secretion of LH and FSH from the anterior pituitary gland at the time of puberty (56). The hypothalamic pulse generator triggers the pulsatile release of GnRH and is considered to be a regulatory mechanism of the action of
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migration (59,67). The majority of the observed mutations in the KAL-1 gene are either nucleotide insertions or deletions that result in a frame shift mutation or a premature stop codon (59). However, in fewer than 20% of cases, these nucleotide insertions or deletions may cause amino acid substitution, disrupting the tertiary structure of anosmin 1 and attenuating its function (68). Rarely, contiguous gene syndrome, which includes the deletion of terminal regions of the short arm of the X chromosome (Xp22), may contribute to KS (69,70). Such deletions may cause, in addition to KS, short stature, chondrodysplasia punctata, mental retardation, and steroid sulfatase deficiency (69,70). The KAL-1 gene is responsible for the X-linked recessive mode of inheritance in familial KS and accounts for 10-20% of all cases of KS (71-73). Specifically, KAL-1 mutations account for 30-60% of familial cases and 10-15% of sporadic cases of KS (73-75). Nevertheless, this gene has not been observed in female KS patients or in isolated normosmic HH cases (76). Interestingly, several phenotypic characteristics have been associated with KAL-1 mutations, such as mid-facial clefting, unilateral renal agenesis in 30% of cases, specific neurological abnormalities such as synkinesis (mirror movement), cerebellar dysfunction, deafness, eye abnormalities and mental retardation (60). The fibroblast growth factor receptor 1 (FGFR-1 or KAL-2, 8p11.2-p11.1) gene is the second most common genetic mutation that is associated with KS (77,78). This gene encodes a tyrosine kinase-linked membrane glycoprotein receptor that binds to extracellular acidic and basic fibroblast growth factors (79). A potential function of fibroblast growth factor is to facilitate GnRH neuron migration, differentiation and survival (80,81). The dysfunction of the FGF receptor results in improper migration and localization of GnRH neurons, potentially explaining the contribution of this mutation to normosmic HH. FGFR-1 mutations are observed in 10% of patients with KS (78,82). Mutations in this gene are observed in 11% of sporadic cases and 8% of familial cases (83). More importantly, the observed mode of inheritance in familial KS is autosomal dominant, with variable expressivity, incomplete penetrance and an even male-to-female ratio (84). Variable expression of the FGFR-1 gene is reflected by the occurrence of anosmia alone, hypogonadism alone, or both in family members of the proband (i.e., the affected individual). Moreover, mutations in the FGFR-1 gene do not always result in KS (incomplete penetrance); this phenomenon may highlight the requirement of a loss of function mutation in the development of KS. More than 70% of mutations in FGFR-1 are missense point mutations that result in amino acid substitutions in the immunoglobulin-like or tyrosine kinase domains. Other mutations are either nonsense, frame shift or splice mutations (61). FGFR-1 mutation-induced KS is characterized by variably severe hypogonadism (from mild to complete) and certain morphogenic abnormalities, such as mid-facial clefting, synkinesis (20% of patients) and missing teeth (61). Fibroblast growth factor 8 (FGF-8) is considered to be one of the ligands for FGFR-1 and is hypothesized to facilitate the migration and differentiation of GnRH neurons to the hypothalamus, as was described above. Mutations in the gene that encodes this protein can cause KS and normosmic HH (85,86). This gene has been mapped to chromosome 10q24 and is responsible for fewer than 2% of all cases of KS (60). An autosomal dominant mode of inheritance is also
demonstrated in familial cases with variable penetrance. Other genes that may be affected in KS include PROK2, PROKR2, and CHD7. Prokineticin 2 is an 81-amino acid protein that is encoded by the PROK2 gene, which has been mapped to chromosome 3p13. This protein has a putative role in the chemoattraction of GnRH neurons in their migration to and differentiation in the hypothalamus (59). This protein acts by binding to a specific G-protein-linked receptor that is encoded by PROKR2 and is located at 20p12.3 (59,61,62). Frame-shift mutations in PROK2 and missense mutations in PROKR2 account for 5-10% of cases of KS (59,61). Mutations in either of these genes exhibit homozygous (autosomal recessive), heterozygous (autosomal dominant) and compound heterozygous modes of inheritance (87). Furthermore, these mutations are associated with variable phenotypic manifestations, such as fibrous dysplasia, severe obesity, sleep problems and synkinesis. Similarly, mutations in these genes have been described in normosmic HH (88). Chromodomain helicase DNA binding protein 7 (CHD7) is a member of a family of proteins whose function is to organize chromatin remodeling (packaging), a process that regulates gene expression (89,90). Tightly arranged chromatin is characterized by lower gene expression compared with loosely arranged chromatin. CHD7 is ubiquitously expressed in fetal tissues, the brain, the eyes, the inner ear, olfactory neural tissue and GnRH neurons. The gene that encodes this protein is located on chromosome 8q12.2 (62). Mutations in this gene have been linked to several diseases, such as KS, normosmic HH and CHARGE syndrome. CHARGE syndrome is associated with eye coloboma, heart defects, atresia of the nasal choana, retarded growth, genitourinary abnormalities, anosmia, and hypogonadism (KS) (89-91). The CHD7 protein is postulated to be an essential factor in the migration and differentiation of GnRH neurons. Seven mutations have been described for this gene in sporadic and familial KS and normosmic HH (59,92). CHD7 accounts for 6% of all cases of KS and 6% of sporadic KS cases. Moreover, familial KS due to CHD7 mutations exhibits an autosomal dominant mode of inheritance (92). Normosmic HH, which is also referred to as isolated or idiopathic HH, is defined as a lack of GnRH secretion or function in the setting of i) normal or low pituitary gonadotropins and ii) the absence of anatomical or functional hypothalamic abnormalities. Patients with normosmic HH present with low levels of sex steroids, normal MRIs and a normal olfactory sense. This disease contributes to 40% of cases of hypothalamic HH. Two-thirds of cases of normosmic HH are considered to be sporadic, whereas 1/3 of cases are familial (60). This disease frequently overlaps with KS in terms of both clinical presentation and the involved genes. Familial cases exhibit X-linked, autosomal dominant and recessive modes of inheritance. The pathogenetic mechanism is attributed to a failure of differentiation or development of normally migrating GnRH neurons into the hypothalamus, resulting in a lack of GnRH secretion or apulsatile secretion (59). A wide array of causative mutations have been identified by nucleotide sequence studies. Nevertheless, the genetic etiology of this condition is unknown for more than 50% of patients. Genes that have been previously reported to be mutated in KS, including FGFR-1, FGF8, PROK2, PROKR2, and CHD7, have also been implicated in the pathogenesis of normosmic HH. The other implicated mutations include those in GnRH,
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GnRHR, KISS1R, TAC3, TACR3, and DAX1 (59,60,76). However, chromosomal abnormalities (generally sporadic) have been observed in 3% of patients with normosmic HH. Such abnormalities include 46,XY/46,X,inv(Y)(p11.2q11.2) and mos46,XY,t(3;12)(p13;p13)/46,XY (76,93). Mutations in the GnRH gene were recently determined to be a rare cause of normosmic HH in two human studies of familial HH (94,95). The gene maps to chromosome 8q21p11.2 and encodes a large 92-amino acid precursor protein. An autosomal recessive mode of inheritance for this trait has been described, and homozygous frame-shift mutations have been observed in probands (94,95). However, mutations in the gene that encodes the G-protein-coupled receptor for GnRH (GnRHR1) are the most common genetic abnormality that is detected in patients with this disorder, accounting for 5-40% of normosmic HH cases (3.5-16% of sporadic cases and as many as 40% of familial cases) (93). This gene maps to chromosome 4q13.2-3, spans over 18.9 kb and encodes a 328-amino acid protein (96). Autosomal recessive homozygous or compound heterozygous mutations have been reported in familial cases (97-103). The majority of GnRHR1 mutations are missense mutations that cause single amino acid substitutions. Such mutations result in variable functional effects, ranging from mild impairment to complete inactivation of the receptor (104,105). Recent studies have identified other ligand proteins and their receptors, such as Kisspeptin and neurokinin B, the functions of which are to regulate the differentiation of GnRH neurons and to initiate their function during puberty. The Kisspeptin (KSS1) gene has been mapped to chromosome 1q32, and a missense mutation in this gene causes autosomal recessive inherited normosmic HH (62,106). Moreover, Kisspeptin receptor (KSSR1, chromosome 19p13.3) mutations also exhibit autosomal recessive patterns of inheritance, and both KISS1 and KISSR1 mutations contribute to fewer than 5% of normosmic HH cases (107). Mutations in neurokinin B (TAC3, chromosome 12q13-q21) and its receptor (TACR3, 4q25) have also been implicated in the pathogenesis of normosmic HH and exhibit autosomal recessive inheritance patterns (108-110). Convertase 1 is an endopeptidase that is encoded by the PCKS1 gene and is involved in i) the post-translational modification of precursor GnRH and ii) the release of mature and active GnRH (59). Mutations in the gene that encodes this protein have been linked to anosmic HH, diabetes and obesity (111). Finally, an X-linked mode of inheritance of normosmic HH has been linked to DAX1 mutations, which cause congenital X-linked adrenal hypoplasia (112). Prader-Willi syndrome (PWS) is a complex genetic disorder that is associated with various degrees of systemic involvement. This condition is caused by the lack of expression of paternally derived imprinted genes on chromosome 15q11-q13. This lack of expression is due either to the deletion of these genes, maternal uniparental disomy of chromosome 15 or the disruption of the paternally inherited chromosome 15 (9,90). Genomic imprinting refers to a phenomenon in which certain genes are expressed in a parent in an origin-specific manner. In this situation, an allele from a given parent is silenced to allow for the expression of non-imprinted genes from the other parent. Genomic imprinting is observed in fewer than 1% of genes. Obesity, hyperphagia, growth retardation, mild to moderate mental retardation, dysmorphic facial features, and sleep abnormalities are the characteristic
features of Prader-Willi syndrome. Hypothalamic HH is a consistent feature of all men with this syndrome (9). During infancy, 80-90% of affected children exhibit cryptorchidism, with a poorly developed scrotum and micropenis. Most adolescents will exhibit delayed or incomplete puberty; however, precocious puberty has been described in 4% of patients (9,24,90). Maternally derived supernumerary marker chromosome (SMC) 15 is the most frequently observed supernumerary chromosome marker in humans (50%). This dicentric chromosome fragment arises from the two homologous chromosomes 15 and is now referred to as dic(15). This genetic defect was formerly referred to as inverted duplication of chromosome 15 or inv dup(15). The size of the fragment is variable; long fragments may contain the PWS critical region and may lead to the development of PWS, whereas short fragments do not contain this critical region, in which case PWS is not observed. However, azoospermia and mild facial dysmorphism, such as mandibular anomalies, have been reported in men that carry the short fragment. Further genetic analysis of the genes on chromosome 15 is lacking but may aid in our understanding of the role of these genes in male infertility.
Adult-onset genetic hypothalamic HH This disease category has recently been described and is restricted to men who successfully completed pubertal development (and who may already have children) and who subsequently exhibited disruption of the HPG axis. Testicular size in such patients is normal, but serum testosterone and gonadotropins levels are low. Moreover, an apulsatile pattern of LH secretion is observed in these patients. A single study of 10 men with adult-onset HH revealed a heterozygous PROKR2 mutation in one patient. However, a good prognosis is expected in these men following treatment with respect to their future fertility potential and androgenization status (9).
Pituitary disorders that are associated with hypogonadism Male hypogonadism that is attributed to genetic pituitary diseases is rare, and such conditions are divided into two major categories: a) generalized or combined anterior pituitary hormone deficiency, and b) selective gonadotropin deficiency.
Generalized anterior pituitary hormone deficiency Several mutations have been observed in men with combined anterior pituitary hormone deficiency (CPHD). Most of these mutations involve genes that code for signaling molecules and transcription factors. Transcription factors are DNA-binding proteins that facilitate the transcription of mRNA from DNA. The affected hormones in CPHD include growth hormone, prolactin, thyroid-stimulating hormone, and gonadotropins (LH and FSH). ACTH may or may not be involved. Such mutations may interfere with the early or late embryonic development of the pituitary gland from Rathke’s pouch. Certain mutations may give rise to various syndromes, such as septo-optic hypoplasia and craniofacial abnormalities (113). In addition to phenotypic variability, the appearance of the pituitary gland on MRI is also variable in CPHD and ranges from enlarged in cases with PROP1 mutations (113,114) to normal or hypoplastic in patients with SOX2 mutations
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of testosterone. Although reported in only five men, recessively inherited missense mutations in the beta subunit (121 amino acids) of the LH gene (19q13.3) result in delayed or absent pubertal development and oligozoospermia or azoospermia (129-131). The hormonal profile of such patients indicates normal FSH levels, high LH immunoreactivity and low testosterone (129,130). The detected LH does not exhibit biological activity. A variety of autosomal recessive mutations (missense, insertion, deletion and nonsense) have been observed in the gene that encodes the G-protein-linked LH receptor. Such mutations result in variable phenotypic traits and infertility. Interestingly, LH glycoprotein receptors (674 amino acids) respond not only to LH but also to hCG and are thus occasionally referred to as LhCGRs. These receptors are present not only in Leydig cells but also in sperm, seminal vesicles, the skin, the thyroid and other organs, where they have an unidentified physiological significance. The gene for the LH receptor has been mapped to chromosome 2p21 and consists of 11 exons and 10 introns (132). This genomic location is close to that of the FSHR gene, and mutations in the LH receptor gene result in Leydig cell hypoplasia (LCH) or agenesis (115,133). LCH exhibits a wide spectrum of manifestations, ranging from male pseudohermophroditism in cases of 46,XY (female external genitalia, undescended abdominal testes, absent breast development) to selective, milder undervirilization defects, such as micropenis, hypospadias, and cryptorchidism (115).
(114). Specifically, the implicated mutated transcription factor genes that cause hypogonadism are the following: PROP1 (most common), LHX and SOX2. Familial and sporadic cases that exhibit autosomal recessive patterns of inheritance are the most common forms of CPHD (114). To accurately differentiate between hypothalamic and pituitary HH, a GnRH stimulation test is performed. In hypothalamic disorders, significant increases in gonadotropin levels are observed, whereas no response is observed in patients with pituitary hypogonadism.
Selective gonadotropin deficiency FSH and LH are glycoproteins that are secreted by anterior pituitary gonadotropes to stimulate testicular spermatogenesis and testosterone production, respectively. Each glycoprotein molecule is composed of a and b chains. The a chain represents a common chain for LH, FSH, human chorionic gonadotropin (hCG), and TSH, whereas the different b chains of these hormones confer immunological and biological hormone specificity. Selective gonadotropin deficiency includes mutations in the genes that code for the synthesis of FSH and LH and their receptors. Specifically, mutations that affect hormone synthesis generally involve the b chain genes, given that mutations in the a chain gene (CGA, 6q12-21) are generally embryonically lethal due to the lack of placental HCG synthesis (115). FSH exerts its action through Sertoli cell receptors, stimulating their proliferation in the immature testis. FSH also stimulates and maintains spermatogenesis. FSH is composed of an a chain, which is composed of 92 amino acids and is non-covalently bound to the b chain (111 amino acids). Rare b subunit (chromosome 11p13) mutations result in isolated FSH deficiency, delayed or normal puberty and small or normal-sized testes in association with severe oligozoospermia or non-obstructive azoospermia (116-118). The b subunit gene is composed of three exons and two introns. To date, two missense and three stop codon mutations with autosomal recessive modes of inheritance have been detected (117,119-121). In contrast, FSH b knockout mice are not infertile, indicating differential regulatory mechanisms in humans and mice (122,123). Furthermore, rare mutations in the gene that encodes the FSH receptor (FSHR), which is expressed in Sertoli cells, have variable effects on spermatogenesis. In a study of five men who were homozygous for FSHR mutations, none exhibited normal semen parameters. Specifically, three patients exhibited severe oligozoospermia and one exhibited moderate oligozoospermia; the fifth patient exhibited a low semen volume and teratozoospermia despite having a normal sperm count (124). The FSHR gene has been mapped to chromosome 2p21-16 and consists of 10 exons and 9 introns (125,126). This gene encodes the mature form of a G-protein-linked glycoprotein receptor, which is composed of 678 amino acids and is exclusively expressed in Sertoli cells (125). Single missense inactivating mutations that result in a valine to alanine substitution at position 189 (A189V) have been reported; these mutations exhibit an autosomal recessive mode of inheritance. Preliminary studies by Simoni et al. (127) and Ahda et al. (128) have revealed differences in the FSHR polymorphisms between fertile and infertile men. Further research is required to clarify the genetic background of FSHR mutations. LH initiates male pubertal development through its effect on LH receptors on Leydig cells, which stimulate the release
Genetic testicular disorders (GTDs) Genetic testicular disorders that cause male infertility can be divided into three categories according to the specific altered function: 1) genetic testicular disorders that primarily affect spermatogenesis and androgen production; 2) genetic testicular disorders that primarily affect spermatogenesis; and 3) genetic testicular disorders linked to androgen synthesis or action.
GTDs that affect spermatogenesis and androgen production Klinefelter syndrome. KLFS is the most common cause of hypogonadism and infertility in males (1 in 500). Klinefelter syndrome is also the most common chromosomal aneuploidy that is observed in azoospermic men (10%) (134); specifically, azoospermia is detected in 74% of men with KLFS (134). The mechanisms of infertility associated with KLFS include a lack of the potential for testicular growth, premature degeneration of the primordial germ cells before puberty and the early or late maturation arrest of spermatogenesis at the primary spermatocyte stage. Later stages of sperm development can also be affected (39). Generally, two forms of KLFS are observed: non-mosaic, (47,XXY, 85% of cases) and mosaic (47,XXY/ 46,XY, 15% of cases). Twenty-five percent of patients with non-mosaic KLFS have sperm in their ejaculate (32). Rare cases of KLFS are caused by isochromosome Xq i(Xq) or X-Y translocations in 0.3-0.9% of males with X chromosome polysomies (135,136). Residual spermatogenesis is observed in men with both mosaic and non-mosaic forms of KLFS. In addition to very small (1-3 ml) and firm testes, gynecomastia (40%) and features of male hypogonadism are also present, such as sparse facial and pubic hair growth, loss of libido and erectile dysfunction. Low testosterone is observed in as many as 80% of men, an effect that is
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acid DNA binding domain that is characteristic of a large protein superfamily that is referred to as the high mobility group (HMG) due to the high migration rate of these proteins in polyacrylamide gels (150). SOX proteins specifically bind to the DNA minor groove and regulate gene expression by acting either as transcriptional activators or repressors (150). The name SOX is derived from the firstdiscovered Y-linked SOX gene, i.e., sex-determining region Y (SRY). The other genes in this family are therefore referred to as SRY-box (SOX) genes. The SOX genes are divided into several groups, from A to H, with group A consisting only of the Y-linked SRY (150). SOX3 is X-linked (Xq26.3) and belongs to the SOX B1 group. The SOX3 gene is specifically expressed in developing testicular and neural tissues and encodes the SOX3 transcriptional activator (151,152). Solmon et al. and Woods et al. correlated genetic mutations in the SOX3 gene with hypopituitarism and mental retardation (153,154). Recessively inherited polymorphic mutations in SOX3 have been observed in men with idiopathic oligozoospermia and in mice with severely impaired sperm production and hypogonadism (155,156). Bilateral anorchia. Bilateral anorchia is a rare congenital disease with an estimated prevalence of 1 in 20,000 males. This condition is characterized by the absence of testicular tissue in 46,XY individuals (157). Because male infants who are born with this disorder exhibit normal genital differentiation, the absence of testicular tissue is most likely attributed to testicular regression that occurs in the second half of gestation. A micropenis is observed in half of these cases (157). The exact etiology of this disease is unknown; however, a subset of cases exhibit familial clustering, suggesting a genetic etiology. Philibert et al. recently noted the role of mutated steroidogenic factor-1 (NR5A1), which is a member of the nuclear receptor family, in the disease etiology. These genes regulate the transcription of other genes that control the development of adrenal and gonadal tissues (158). NR5A1 has been mapped to chromosome 9q33 and has been correlated with other human diseases, such as male infertility, hypospadias, ovarian insufficiency, and others (158). Noonan syndrome. Noonan syndrome is a relatively common heterogeneous genetic disorder that results in a wide array of clinical manifestations and genotypic abnormalities. Its incidence ranges from 1:1,000 to 1:2,500 live births, and it is inherited in an autosomal dominant manner (150). To date, nine genes have been implicated in Noonan or Noonan-associated syndromes (PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, MEK1, and CBL) (159). The basic cellular abnormalities that are caused by mutations in these genes are defective signal transduction pathways, particularly the RAS-GTPase and mitogenactivated protein kinase (MAPK) signaling cascades (159). The typical phenotypic features of Noonan syndrome include a short stature, a webbed neck, facial dysmorphism, congenital pulmonic stenosis and other manifestations. Unilateral and bilateral cryptorchidism are frequent in this syndrome and are observed in as many as 77% of patients (160). Moreover, delayed or absent pubertal development in males with this syndrome is attributed to testicular failure (161). Therefore, altered spermatogenesis with oligozoospermia or azoospermia is multifactorial due to the basic genetic defect itself and its association with cryptorchidism.
attributed to small testicular growth despite the presence of Leydig cell hyperplasia. Several studies have demonstrated that KLFS patients exhibit a high incidence of aneuploid gametes, rendering them at risk for producing offspring with chromosomal abnormalities (137). Interestingly, the successful fathering of 60 children has been achieved by testicular sperm extraction (TESE) and ICSI in men with KLFS. Karyotype studies that were performed in approximately 50 children revealed no chromosomal abnormalities (138,139). These genetic findings have been further clarified in a study by Sciurano et al., who detected spermatogenesis foci in testicular biopsies of 6/11 men with nonmosaic KLFS (140). Whereas the majority of seminiferous tubules are devoid of germ cells, 8-24% do contain germ cells. Sciurano et al. examined the chromosomal complements of 92 meiotic spermatocytes using fluorescent in situ hybridization (FISH). These spermatocytes exhibited euploidy (normal chromosomal constituents) and the ability to form haploid gametes (140). These novel findings may explain the high rate of normal children who are born following testicular sperm extraction and ICSI in men with KLFS. Nonetheless, even with this high rate of normally born children, there is still a risk of genetic defects in the offspring; therefore, it is advised that a preimplantation genetic diagnosis (PGD) be offered prior to ART to ensure that the offspring are not aneuploid (39). XX males. This genetic disorder is very rare, with an estimated prevalence of 1:10,000-1: 20,000 (141). The genetic event that causes this disorder involves the translocation of genetic material of the testis-determining region of SRY (or SOX A, a gene that lies on the Y chromosome) to the X chromosome during paternal meiosis. SRY encodes a transcription factor of approximately 204 amino acids (142). This translocation results in the successful differentiation of indeterminate gonads into testes; however, the lack of other genes involved in the initiation of spermatogenesis renders these males azoospermic. Furthermore, SRY-negative variants have also been described, which are characterized by severe undervirilization defects, such as undescended testes, hypospadias and bifid scrota (143). Phenotypically, these men are very similar (but with smaller statures) to patients with KLFS (144). Mutations in X-linked USP26. The USP26 gene is a single-exon gene that maps to chromosome Xq26.2. This gene encodes the USP26 protease (913 amino acids), which is a deubiquitinating enzyme (145,146). The ubiquitination and deubiquitination of macromolecules are essential for the regulation of the cell cycle, maintenance of chromosomal structure and gene silencing (145). The removal of histones and the regulation of protein turnover during meiosis are important functions of this protein. More than twenty mutations in this gene have been reported. These mutations result in the severe impairment of spermatogenesis, and several result in hypogonadism (147). Studies have demonstrated a relationship between this gene and certain cases of non-obstructive azoospermia (148,149). X-linked SOX3 mutations. The SOX genes are essential for development and control of embryonic ontogenesis in the human testes, neural tissues, cartilage, and neural crest cells. These genes are present only in vertebrates and give rise to SOX proteins that have a role in both the developing and adult gonads. These proteins share a common 79-amino
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(mixed
gonadal
dysgenesis).
deletions of the DBY gene can result in SCOS and azoospermia. The USP9Y gene is a single-copy gene that encodes a ubiquitin-specific protease (a deubiquitinating enzyme). This enzyme binds to ubiquitin-protein conjugates and hydrolyzes the ubiquitin peptide chain. Such action confers stability to cellular proteins and protects them from cellular degradation by the proteasome complex (173). The expression of USP9Y is restricted to spermatids in humans and mice (174,175). USP9Y expression in male germ cell lines may lead to the stability of certain cellular proteins that are synthesized in primordial germ cells (PGCs) and that are important for germ cell survival. Point mutations in the USP9Y gene result in maturation arrest at the spermatid stage (176), oligozoospermia (177), oligoasthenozoospermia, and asthenozoospermia (178). The AZFb subregion spans approximately 6 Mb and is located in the distal portion of interval 5 and the proximal portion of interval 6 (subinterval 5O-6B) (179). The meiotic arrest of spermatogenesis at the primary spermatocyte stage is usually observed when AZFb is deleted. AZFb contains 32 genes and overlaps with AZFc (179). As such, AZFb deletions often remove certain genes from the AZFc region (e.g., DAZ1 and DAZ2), as well as one copy each of BPY2, CDY1, and PRY. The primary protein-encoding genes in AZFb are RBMY and PRY. Six copies of RBMY are located in the distal portion of AZFb and are only expressed in germ cells (180). RBMY encodes four types of testis-specific RNAbinding proteins that are involved in mRNA processing, transport, and splicing (181). Due to its proximity to AZFc, certain deletions within AZFc can remove several copies of RBMY genes. Two copies of PRY are located in the AZFb region and presumably regulate apoptosis (167). Hypospermatogenesis is observed when all of the AZFb genes are deleted except for RBMY and PRY. Conversely, spermatogenesis is completely arrested when both the RBMY and PRY genes are deleted (182,183). AZFc spans over 3.5 Mb and contains a large number of amplicons that are arranged as direct repeats, inverted repeats, or palindromes. Examples of these repeats include b, g, r, and P repeats. Seven distinct gene families, encompassing 23 genes, are observed in the AZFc region. These families include PRY (two copies), TTY (eight copies), BPY (three copies), DAZ (four copies), GOLGA2LY (two copies), CSPYG4LY (two copies), and CDY (two copies). Deletions in the AZFc region alone or deletions in this region that are combined with deletions in other AZF regions are the most common types and account for as many as 87% of Yq microdeletions. The incidence of these deletions is 1/4,000 males. Although AZFa and AZFb deletions result in azoospermia, deletions in the AZFc region can result in either azoospermia or oligozoospermia (184). AZFc deletions can explain approximately 12% of non-obstructive azoospermia and 6% of severe oligozoospermia cases (185). Two scenarios have been described regarding the cause of complete AZFc deletions, namely, a new deletion in addition to a preexisting partial deletion or a complete deletion of a preexisting normal gene. AZFc deletions may jeopardize Y chromosome integrity, resulting in its loss and sex reversal. As such, AZFc deletions predispose one’s offspring to the 45,X0 karyotype (186) and to the mosaic phenotype 45,X/46,XY (187). Two types of AZFc deletions have been described. The classic AZFc deletion is denoted by b2/b4. This deletion encompasses four DAZ genes, resulting in azoospermia.
Normal male external genitalia are observed in 90% of males with 45,X/46,XY mosaicism; abnormal, ambiguous and female genitalia are observed in the other 10%. Mixed gonadal dysgenesis (a streak gonad on one side and a testis on the other) is observed in 10-30% of patients with this type of mosaicism (162). Abnormal gonadal development results in azoospermia and low testosterone levels (163).
GTDs that affect spermatogenesis Y chromosome microdeletion. The long and short arms of the Y chromosome contain many genes that regulate spermatogenesis and testes development, respectively. Microdeletions on the long arm of the Y chromosome (Yq) are well correlated with male infertility. Yq microdeletions are detected in approximately 13% of men with nonobstructive azoospermia and in 5% of men with severe oligozoospermia (sperm counts lower than 5 million/mL) (164,165). A microdeletion is defined as a chromosomal deletion that spans several genes but that is small in size and cannot be detected using conventional cytogenetic methods (e.g., karyotyping). Y chromosome microdeletions are clustered in intervals 5 and 6 of the long arm of the Y chromosome. This region at Yq11 is referred to as the ‘‘Azoospermia Factor’’ (AZF) region. The AZF region is further subdivided into three subregions that are termed AZFa, AZFb, and AZFc (Figure 1). The most common aberrations in the AZF region are multiple gene deletions in the AZFb and AZFc sub regions (166), which can produce a wide range of infertility phenotypes. The genes on the Y chromosome are generally divided into three types: ‘‘X transposed’’, X degenerated, and amplicons. Eight palindromes are recognized in the AZF region and six of these are related to male fertility. Six of the genes that are located in the AZF regions are expressed exclusively in the testes and are therefore referred to as ‘‘AZF candidate genes’’. The AZFa region is the smallest portion of the AZF and spans approximately 400-600 kb of DNA. It is located in the proximal portion of interval 5. The AZFa region is characterized by a non-repetitive structure and a low deletion frequency. This subregion contains three genes: USP9Y, DBY (DDX3Y) and UTY. Two protein-coding genes are directly related to male infertility: USP9Y and DBY (recently termed DDX3Y). Complete and partial deletions of AZFa have been described. Complete deletions that remove both genes cause Sertoli cell-only syndrome (SCOS) and bilateral small-sized testes (167,168). It is estimated that 955% of SCOS cases are caused by deletions in the AZFa region (169-171). Partial deletions have also been reported, with particular involvement of USP9Y. The DBY gene is the major single-copy gene in the AZFa region and belongs to the DEAD BOX RNA helicase family. This family consists of a group of genes that encode proteins that specifically regulate RNA transcription, translation, and splicing in the G1 phase of the cell cycle. DBY expression is observed in the male germ line, whereas its expression in other tissues is uncertain (172). DBX, a DBY homologue, belongs to the same family as DBY and is expressed in male germ cells. Its expression pattern has been elucidated by immunohistochemistry techniques. The DBY gene and protein are specifically expressed in premeiotic germ cells, whereas the DBX gene and protein are expressed in postmeiotic germ cells (172). This differential distribution explains why
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Figure 1 - Schematic representation of the Y chromosome that depicts the AZF region, the AZF subregions and the primary genes within each subregion. Adapted from: O’Flynn, O’Brien KL, Varghese AC, Agarwal A. The genetic causes of male factor infertility: a review, pages 1-12, copyright 2010, Fertil Steril 93, with permission from Elsevier via the Copyright Clearance Center (Order Detail ID: 62879217).
Partial or sub-deletions are denoted by gr/gr, b1/b3, and b2/b3. The gr/gr subdeletion removes nearly half of the AZFc region and results in different phenotypes in various populations. Such variability suggests that other confounding factors may be involved, such as ethnicity and environmental effects on gene expression. Although certain studies have recognized gr/gr deletions as a risk factor for impaired spermatogenesis (168,188-190), others failed to conclusively demonstrate this relationship (191-193). The deletion of b1/b3 removes the proximal portion of AZFc. Two of the six copies of RBMY1, the two functional copies of PRY (194), one of the three copies of BPY2 and two of the four DAZ genes are deleted. Nevertheless, large 1.8-Mb deletions have been observed in normozoospermic fertile individuals (195), whereas other researchers have identified this deletion in azoospermic men (196,197). The deletion of b2/b3 removes nearly half of the AZFc region (1.8-Mb DNA segment) and 12 genes, including two copies of DAZ and two copies of BPY. With respect to b1/b2, no conclusive impact on spermatogenesis has been determined (198,199). The coding genes in AZFc include DAZ and CDY. DAZ plays important roles throughout germ cell development from embryonic life to adulthood; there are four copies of this gene on the Y chromosome (210,211). Postnatally, DAZ encodes proteins that have RNA recognition motifs (RRMs),
which are involved in the regulation of RNA translation (200) and control of meiosis. DAZ is essential for the maintenance of the PGC during embryogenesis (201). DAZ2 and DAZ3 each have a single RRM, whereas DAZ1 has two and DAZ4 has three RRMs. There are two autosomal homologues of DAZ: DAZL on chromosome 3q24.3 and BOULE on chromosome 2q33. DAZ gene expression was observed to be reduced in azoospermic patients (202), and partial deletions of DAZ genes appear to be related to oligozoospermia. Two copies of the chromodomain protein Y-linked (CDY) gene are located in the AZFc region. CDY is expressed exclusively in germ cells, where it encodes a protein that contains a chromodomain and a histone acetyltransferase catalytic domain, which is primarily observed in the nucleus of late spermatids. (167). This protein regulates histone hyperacetylation, which is essential to proceed from a histone- to a protamine-based chromatin structure in spermatid nuclei. Other Y chromosome genes, excluding those that are clustered in intervals 5 and 6 of the long arm, may participate in spermatogenesis. Copies of the TSPY gene have been detected on both Yp and Yq. The protein product of this gene is expressed in spermatogonia and is believed to play a role in the timing of spermatogenesis by signaling to spermatogonia to enter meiosis (168,203). A study of copy
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genetic factors. Mutations in the INSL3 gene (insulin-like 3 on chromosome 19) and its receptor LGR8 (relaxin/insulinlike family peptide receptor 2 on chromosome 13) occur in approximately 5% of men with cryptorchidism and have been linked to the disease (215). Additionally, the first phase of normal testicular descent is controlled by INSL3 (216). The INSL3 gene may also participate in testicular dysgenesis syndrome (TDS) (217), which comprises a variety of disorders that may present alone or in combination. Such disorders include cryptorchidism, hypospadias, an elevated risk of testicular cancer, and infertility. It has been suggested that TDS results from a combination of genetic, environmental, and lifestyle factors (218).
number variations of the TSPY gene revealed that more copies were present in infertile patients (204). This finding warrants further investigation of TSPY to characterize its role in azoospermia. Autosome translocations. Translocations can cause loss of genetic material at gene breakpoints, thereby corrupting the genetic message (205). Autosomal translocations were determined to be 4-10 times more likely in infertile males than in normal males (206,207). Robertsonian translocations, which occur when two acrocentric chromosomes fuse, are the most frequent structural chromosomal abnormalities in humans (32). Although the prevalence of Robertsonian translocations is only 0.8% in infertile males, this prevalence is 9 times higher than that in the general population (39,208). Such translocations can result in a variety of sperm production phenotypes, from normal spermatogenesis to azoospermia (39). Robertsonian translocations are more common in oligozoospermic and azoospermic men, with rates of 1.6 and 0.09%, respectively (209,210). Given the risk of passing on the translocations to offspring, preimplantational genetic screening may be advisable for couples who undergo ART (41). Monogenic disorders. DAZL is a single-copy gene that is located on chromosome 3. DAZL belongs to the DAZ gene family and is the autosomal homologue of the DAZ gene that is located on the Y chromosome. DAZL is expressed in fetal primordial germ cells, fetal gonocytes and adult male and female germ cells in both the nucleus and cytoplasm. The DAZL protein may regulate protein synthesis and meiosis (211). Although mice with DAZL null mutations are sterile in both sexes and the male germ cells of these animals are arrested at the leptotene stage, no conspicuous mutations in human DAZL that result in sterility have been recognized. Nevertheless, Teng et al., when examining a group of azoospermic and oligozoospermic men in Taiwan, reported that a subset of these men were heterozygous for the single nucleotide polymorphism 386A_G (212). However, these results have not been confirmed in Caucasian men (228), and more studies are required in different populations to examine the role of DAZL mutations and polymorphisms in azoospermia. The methylenetetrahydrofolate reductase (MTHFR) gene, which is located on the short arm of chromosome 1, encodes an enzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. This reaction is important for methionine and S-adenosylmethionine (SAM) synthesis from homocysteine, which is a toxic product, as well as for the synthesis of thymidine. SAM serves as a methyl donor for DNA methyltransferase, which controls DNA methylation, an important process in germ cell development. A recent meta-analytic study revealed that individuals who were homozygous for the single nucleotide polymorphism (SNP) 1298CC (i.e., the CC vs. AA genotype) or who carried a recessive allele (CC vs. AA/ AC) were at an increased risk of azoospermia (OR = 1.66 for CC vs. AA; OR = 1.67 for CC vs. AA/AC genotype) (213). This SNP inserts an alanine at position 429. In another metaanalysis, the presence of a MTHFR 677T mutation, in which a valine substitution occurs at amino acid 222 and thus encodes a thermolabile enzyme with reduced activity, is associated with a significantly increased risk of azoospermia (214). Multifactorial disorders. Cryptorchidism causes an infertile phenotype and appears to be influenced by
GTDs that affect androgen action or production Androgen receptor mutations. The androgen receptor (AR) gene is a single-copy gene that is located on Xq11-q12. This gene encodes a cytoplasmic protein that binds specifically to testosterone, and the resultant complex can activate the expression of certain DNA segments. AR is essential for meiosis, in which spermatocytes are converted into round spermatids, and for the appearance of secondary sex characteristics. The AR gene consists of eight exons. Exon 1 encodes the transactivation domain, which activates transcription; exons 2-3 encode the DNA binding domain (DBD); exons 5-8 encode the ligand-binding domain (LBD); and exon 4 encodes the hinge region that connects the DBD and the LBD. From 2004 to the present, the number of reported mutations in the AR gene increased from 605 to 1,029; these mutations have been linked to prostate cancer, male infertility and breast cancer (219). Point mutations, insertions or deletions, and altered CAG repeats can severely impair the amount, structure and function of the AR gene, causing androgen insensitivity syndrome (AIS). The phenotypic manifestation of AIS includes ambiguous genitalia, partial labialscrotal fusions, hypospadias, bifid scrota and gynaecomastia (220). Testosterone and LH levels are consistently elevated in patients with AIS. Although no mutations have been identified in more than 40% of patients, others reported point mutations and polymorphisms in the AR gene in azoospermic men who exhibit normal external genitalia. Alterations in two polymorphic trinucleotide repeats (CAG and GGC) in the 5’ region of exon 1 have been implicated in male infertility and azoospermia. Mirfakhraie et al. identified the transversion of 1510CRA in exon 1 of the AR gene in a single patient with SCOS (221). Hose et al. discovered the novel mutation 212ARG in the CAG repeat that resulted in a glutamine-arginine substitution in men with SCOS (222). Although GGC polymorphisms or repeat length alterations are inversely correlated with the transactivation ability of the receptor (223), no marked effects on male fertility potential have been observed. Steroidogenic acute regulatory protein (StAR) mutations. Testosterone is the most important male
androgen, and the major site of its production is the Leydig cells, which account for 75% of testosterone synthesis. Steroidogenic acute regulatory protein (StAR) facilitates the transfer of cholesterol from the outer to the inner mitochondrial membranes; this is the rate-limiting step of testosterone synthesis (224). Congenital lipoid hyperplasia is a rare disease that is caused by frame-shift, missense and nonsense mutations in the StAR gene. This
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Epigenetic defects. Disturbed germ cell nuclear histone acetylation and single-stranded DNA break repair have been implicated in SCOS and maturation arrest (258).
gene spans 8 kb, consists of seven exons and six introns and maps to 8p11.2 (225). Because StAR is the first essential step in the synthesis of all of the steroid hormones in all steroidsynthesizing cells, the classic deficiency of StAR results in a lack of corticosteroids, mineralocorticoids and testosterone. Males with the classic form are born with feminized external genitalia. Given that the condition is life-threatening, the delayed administration of proper hormonal replacement may lead to fatal outcomes shortly after birth. However, a non-classic form has also been described and is characterized by partial protein activity. In such a form, males may be born with external genitalia; nonetheless, they may exhibit compromised fertility potential and azoospermia (226). 3BHSD type 2 deficiency. 3BHSD type 2 is one of the intracellular adrenal and gonadal enzymes that is necessary for the synthesis of all steroids. Mutations in the 3BHSD type 2 gene (located at 1p11-13) result in the salt-losing or non-salt-losing forms, which are characterized by femalelike genitalia at birth in 46,XY males. These males may develop secondary sexual characteristics at puberty (227). SRD5A2 mutation. SRD5A2 is a gene that maps to 2p23 and encodes 5-alpha reductase type 2. This enzyme is present in the external genitalia and prostate. Autosomal recessive homozygous mutations in this gene result in feminized external genitalia in 46,XY males. At the time of puberty, signs of masculinization develop due to the activity of 5-alpha reductase type 1 (SRD5A1) in the skin and liver. However, other features, such as prostatic hypoplasia, less body hair and a female frontal hair line, remain. These men are often infertile due to prostatic underdevelopment. Nevertheless, fertility has been reported in certain men due to partial enzymatic activity, which can be conferred by different types of mutations. Epidemiologically, this disease has been reported more frequently in an isolated area in the Dominican Republic (228).
Genetic abnormalities at the primordial germ cell level The specification, formation, and migration of primordial germ cells toward the developing gonads are all under complex genetic control (287-290). Moreover, several genes regulate the settlement process of primordial germ cells within the testes, the protection of these cells from apoptosis, the formation of gonocytes and the differentiation of germ cells into spermatogonia (291). Spermatogonia undergo proliferative phases that are regulated by many external and internal factors that are encoded by specific genes. Certain genes are activated in each germ cell stage, whereas others are repressed. Selective mutations in the genes that regulate human primordial cell lines are likely to affect male fertility status and may also explain certain cases of non-obstructive azoospermia.
& GENETIC TESTING IN AZOOSPERMIA There are three groups of genetic tests used to detect genetic diseases in azoospermic men: a) cytogenetic tests that detect chromosomal aneuploidy and structural alterations, such as conventional karyotyping; b) polymerase chain reaction to detect Y chromosome microdeletions; and c) specific gene sequencing for mutational analysis of a specific gene. Conventional karyotyping involves the collection of heparinized peripheral blood samples (approximately 5 ml) from the patient and the isolation of a plasma lymphocyte suspension. Lymphocytes are then transferred to culture media (RPMI) containing a mitotic stimulator (PHA) and incubated for 72 hours. After 70 hours, cell division is arrested at the metaphase stage using colchicine. The cells are then subjected to hypotonic treatment (KCl) and fixed with Karnovsky fixative overnight at 4 ËšC. Finally, the cells are spread on a clean, grease-free wet slide and subjected to GTG banding for karyotyping (292). The standard protocols are available in various practical guidelines and should be optimized for different laboratory conditions (293). Cytogenetic analysis is the most frequently used diagnostic test in the evaluation of patients with azoospermia (294,295). The Y chromosome microdeletion (YCMD) assay is a PCR-based blood test that detects the presence or absence of defined sequence-tagged sites (STSs). This technique therefore enables the detection of the presence or absence of any clinically relevant microdeletion. Yq microdeletion analysis is generally performed using multiplex polymerase chain reaction (PCR) to amplify the AZFa, AZFb, and AZFc loci in the long arm of the Y chromosome. The set of PCR primers that are used to amplify the AZF regions is important in the identification of deletions (296). The PCR should be performed at least twice in the presence of an internal control (SRY), as well as positive and negative controls, to confirm the presence of deletions. PCR is a rapid method for the detection of submicroscopic Y chromosome deletions, which conventional cytogenetic analysis is unable to resolve. To obtain uniform results, it is necessary to follow the same guidelines for the entire examined population. The European Association of Andrology (EAA) recommends the
Dysfunctional cell regulatory pathways Genetic defects at the levels of germ or Sertoli cells in azoospermic men may result from impaired cellular regulatory pathways, culminating in the loss of the germ cells or the arrest of spermatogenesis (229-286). First, exaggerated apoptotic signals due to mutations in the genes that encode for enzymes with crucial roles in apoptosis, such as inducible nitric oxide synthase (iNOS), Fas, FasL, and active caspase 3, affect germ cell proliferation and may result in azoospermia with the SCOS phenotype (245-250). Second, impaired cross-talk or increased immune injury due to mutations in the genes that encode interleukins may lead to maturation arrest or SCOS (229-232). Third, the disruption of Sertoli cell cytoskeletal integrity can result in an inefficient supportive role for spermatogenesis. An intact germ cell cytoskeleton is essential for the regulation of germ cell development; maturation arrest and SCOS are observed when the cell cytoskeleton is disrupted (233-243). Fourth, the cell cycle in mitosis or meiosis should pass through four important phases: G1, S, G2 and M. A defect in any of these phases can result in cell cycle arrest or apoptosis (244-249). Fifth, impaired signal transduction may undermine the response of testicular cells to activating factors, such as hormones and growth factors (248-257). Finally, selective defects in the genes encoding transcription factors in germ and/or supporting cells, such as DAX-1 and nuclear export factor (NF2), have been implicated in NOA (283,284).
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use of a set of seven markers in men with idiopathic infertility who present with severe oligozoospermia or azoospermia: six STSs in the AZF locus, and one STS (sY14) in the SRY locus, the latter of which is the internal control. Y chromosome infertility is inherited in a Y-linked manner. For specific gene sequencing and mutational analyses, the ‘‘dye terminator sequence’’ method is performed. This method is a high-throughput, automated, more efficient and more rapid method than the original Sanger method of sequencing. The principle, which is similar to that of Sanger’s method, depends on the premature termination of four separate sequencing reactions that contain all four of the standard deoxynucleotides (dATP, dGTP, dCTP, and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), which are chain-terminating nucleotides that lack the 3’-OH group required for the formation of a phosphodiester bond between two nucleotides. Thus, DNA strand extension is terminated, resulting in DNA fragments of varying lengths. Next, these labeled DNA fragments are separated by gel electrophoresis on a denaturing polyacrylamide-urea gel and read in a specific manner from the shortest to the longest (297). Table 2 summarizes the currently available genetic tests for azoospermic men who seek fertility counseling.
& EXPERT COMMENTARY The aim of this article was to review the current knowledge of the genetic basis of azoospermia and to highlight the requirement for genetic testing in such conditions. Thousands of single or multiple genes are involved in establishing the male fertility potential, and many others are yet to be revealed. Currently, genetic testing ranges from the chromosomal level to specific gene mutations. Single gene disorders are important for understanding the etiology of male infertility. Moreover, testing can aid in the improved management of couples who seek genetic counseling prior to conception. The initial evaluation of infertile men generally commences with history taking and a thorough physical examination. These steps are followed by an initial seminal fluid analysis and, if required, endocrine profile testing and imaging analysis. Until approximately two decades ago, the understanding of the genetic basis of infertility was of limited value for it provided only a diagnosis. In the current era of ART, however, genetic testing have emerged as tools of paramount importance in helping clinicians not only to explore the specific genetic background of a disease but also to take the necessary precautions to prevent the transmission of the disease to the offspring via assisted conception. Certain genetic defects are associated with increased morbidity, childhood cancer and genital ambiguity. As such,
Table 2 - Genetic testing that is currently available for the investigation of azoospermia Phenotype Post-testicular azoospermia (obstructive azoospermia)
Genetic point of interest
Type of testing
CFTR gene
Mutational analysis of the CFTR gene in cases of CAVD, congenital epididymal obstruction and normal vasa, and Young syndrome
Long arm of the Y chromosome Testicular azoospermia (non-obstructive azoospermia)
Autosome and sexual chromosomes Androgen receptor gene
Pre-testicular azoospermia (HH)
X-linked USP 26, X-linked SOX3, LH and FSH receptors, X-linked TAF7L, DAZL, MTHFR, ER1, ER2, and FSH
Mutational analysis of the specific gene
KAL1, FGFR, PROK2, PROKR2, FGF8, CDH7, KISS1, GPR54, TAC3, TACR3, GnRH, and GnRHR
Mutational analysis of genes related to Kallmann syndrome and normosmic HH
PROP1, LHX, and SOX2 FSH and LH genes, and FSH and LH receptor genes
Complex/multifactorial genetic disorders
Polymerase chain reaction to detect Y chromosome microdeletion Cytogenetic analysis to detect chromosomal aneuploidy and structural alterations CAG repeat/AR mutation analysis for androgen insensitivity syndrome (AIS)
NR5A1 gene INSL3 gene and its receptor PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, MEK1, and CBL genes Chromosome 15
Steroid 5-alpha-reductase 2 (SRD5A2) gene
Mutational analysis of genes related to generalized pituitary insufficiency Mutational analysis of the genes that code for the secretion of FSH and LH, and their receptors, in cases of selective gonadotropin deficiency Mutational analysis of the specific gene in cases of bilateral anorchia Mutational analysis of the specific genes in cryptorchidism Mutational analysis of the specific genes in Noonan syndrome Cytogenetic/FISH analysis to detect deletions, maternal uniparental disomy of chromosome 15q11-q13 or the disruption of paternally inherited chromosome 15 in PraderWilli syndrome Mutation analysis of the specific gene in cases of SRD5A2 deficiency
CAVD: congenital agenesis of the vas deferens; CFTR: cystic fibrosis transmembrane regulator protein, FISH: fluorescence in situ hybridization; FSH: follicle-stimulating hormone; LH: luteinizing hormone; GnRH: gonadotropin-releasing hormone; HH: hypogonadotropic hypogonadism.
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genetic testing in azoospermic males allows couples to make educated decisions regarding their choice to use a sperm donor or to opt for advanced assisted conception techniques. Such techniques can be coupled with preimplantation genetic diagnosis (PGD) if an abnormal result is obtained. Compiling the data from the initial evaluation that include history and physical examination may segregate azoospermic men into four major groups based on their probability of harboring male fertilityrelated genetic diseases. For the first group, who present with a history and a physical examination that is consistent with cryptorchidism, Noonan syndrome, bilateral anorchia or Prader-Willi syndrome, certain genetic tests are available; however, these tests are not routinely used. In the second group, azoospermic men with clear evidence of seminal duct obstruction based on physical examination (CBAVD, Young syndrome) require testing for CFTR mutations. The third group encompasses men with non-obstructive azoospermia. For such individuals, after the exclusion of acquired testicular pathologies and a history of exposure to gonadotoxins, such as radiation and chemotherapy, genetic testing, including karyotype and Yq microdeletion analyses, should be set as minimum standards (Table 2). Lastly, for men with feminized or ambiguous external genitalia, the genetic workup should also include androgen receptor genetic analysis. Chromosomal defects are the most common genetic abnormalities in infertile men with azoospermia and may account for as many as 15% of cases (298-302). The most common chromosomal aberrations that are associated with severe spermatogenic defects are sex chromosome aneuploidies and chromosomal translocations. Among the various cytogenetic abnormalities, Klinefelter syndrome is the major cytogenetic/sex chromosome/numerical anomaly that is detected in infertile men, followed by translocations, deletions and inversions. Cytogenetic abnormalities may predispose to malsegregation and/or abnormal embryonic development (303). Moreover, the occurrence of aneuploid embryos after IVF and ICSI will not only decrease the success rate of the treatment (304) but also increase the risk of both an unbalanced translocation and altered amount of genetic material in the offspring. Thus, PGD may be useful for certain couples undergoing IVF treatment. Y microdeletion screening is mandatory in infertile men with azoospermia of unknown origin who opt for ART. Y chromosome microdeletions are observed in as many as 15% of men with NOA (59,296,297). Yq microdeletion screening may not only identify the cause of azoospermia but also predict the probability of sperm retrieval in ART candidates. In cases that involve AZFa and/or AZFb microdeletions, sperm retrieval is currently not recommended because there is no evidence that testicular sperm can be found, irrespective of the retrieval method (56-59). Conversely, sperm can be retrieved from the testes in approximately 70% of cases that involve AZFc Yq microdeletions (56-59,305). In such cases, ICSI can be performed. The probability of fatherhood by ICSI is unaltered by the presence of AZFc microdeletions (305). However, ARTderived male offspring will inherit the Yq microdeletion, potentially resulting in subsequent infertility. The probability of whole Y chromosome deletion also increases in such cases, which may lead to genital ambiguity in the offspring (306). Female fetuses from a father with a Y chromosome deletion have no increased risk of congenital
abnormalities or infertility. Given that males with deletion of the AZF regions of the long arm of the Y chromosome are infertile, the deletions are generally de novo and are therefore absent from the father of the proband. Rarely, within a family, the same deletion of the Y chromosome can cause infertility in certain males but not in others; thus, certain fertile males with a deletion in the AZF regions have fathered sons who are infertile (57-59). In pregnancies that are conceived via assisted reproduction and that are known to include the risk of producing a male with a Y chromosome deletion, specific prenatal testing or preimplantation testing may be performed to determine the sex of the fetus and/or the presence of the Y chromosome deletion (292). Mutations in the cystic fibrosis gene are the most common genetic mutations that result in azoospermia. Congenital bilateral absence of the vas deferens (CBAVD) is a syndromic disorder that is characterized by the absence of the vas deferens and accounts for at least 6% of cases of obstructive azoospermia and approximately 2% of infertility cases. CFTR mutations are responsible for CBAVD in at least 95% of men, and CFTR analysis is one of the most important genetic tests in infertility cases (obstructive azoospermia) (307). Patients with CAVD due to CFTR mutations are at risk of having both male and female offspring with cystic fibrosis and male offspring with CAVD, given the relatively high carrier rate of CFTR mutations (e.g., 4% in the Caucasian population) (259). Screening for CFTR mutations may also be recommended for men with Young syndrome and for those with unilateral vasal agenesis before attempting to conceive (40). Ideally, both partners should be screened prior to assisted reproductive techniques (ART) to determine the risk of transmitting CFTR mutations to the offspring (41). Up-to-date information on CFTR mutations can be found at http:// www. genet.sickkids.on.ca/cftr/app, which is a CFTR mutation database. Different techniques, such as Westernblotting, in situ hybridization, fluorescence in situ hybridization, single-strand conformation polymorphism analysis, heteroduplex analysis, PCR, and real time PCR followed by direct sequencing, have been developed to screen for CFTR mutations. PGD has been regarded as a useful tool to identify the presence of CFTR mutations in in vitro-derived embryos, assuming that both the male and female partners have been screened for genetic mutations. In azoospermia, genetic testing using karyotyping, Yq chromosome microdeletion analysis and CFTR mutation screening reveals a genetic etiology in approximately 30% of cases. A summary of the current recommendations for genetic testing in azoospermia is presented in Table 3. Incorporating novel techniques, such as genomics, proteomics, and metabolomics, into infertility research may assist in the creation of a complete portrait of the genes that are involved in infertility and would allow for improvements in ART and the development of more targeted solutions (297). Microarrays are emerging as valuable tools for the determination of the gene expression profiles of infertile phenotypes (308). Microarray technology is also useful in the examination of spermatogenesis. An analysis of gene expression over time may be performed to identify the genes that are involved in each spermatogenic stage. Genomic analyses can also be used to determine differentially transcribed genes (309). An enhanced understanding of transcriptional regulation could aid geneticists in the discovery of the mechanisms
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Table 3 - The current recommendations for genetic testing in azoospermia based on clinical phenotypes Genetic test Cytogenetic analysis Yq microdeletion analysis Cystic fibrosis transmembrane conductance regulator (CFTR) mutation analysis KAL1 mutation analysis CAG repeat/AR mutation analysis Steroid 5-alpha-reductase 2 (SRD5A2) mutation analysis Lutenizing hormone (LH ) receptor mutation analysis Gonadotropin-releasing hormone (GnRH) mutation analysis DAZL/MTHFR mutation analysis
Phenotype
Recommendation
Non-obstructive azoospermia Non-obstructive azoospermia Obstructive azoospermia and congenital absence of the vas deferens (CAVD) or congenital bilateral epididymal obstruction and normal vasa Kallmann syndrome (KS) Androgen insensitivity syndrome (AIS) SRD5A2 deficiency
Mandatory Mandatory
Recommended Recommended Recommended
Pseudohermaphroditism , azoospermia, micropenis, delayed puberty and arrest of spermatogenesis
Suggested
Low serum LH and FSH levels Non-obstructive azoospermia
Suggested Suggested
by which different expression patterns impact a patient’s fertility status. However, because the results of gene expression microarray studies are variable, it would be necessary to determine global gene expression patterns of RNA samples from the testis before this type of analysis become clinically relevant. Grouping the expressed genes into functional categories may allow the characterization of a gene expression signature for normal human spermatogenesis, which could be used as a diagnostic marker. Proteins are identified using two-dimensional electrophoresis and mass spectrometry techniques, and the results are used to create proteome maps in relation to sperm and seminal plasma (310,311). The identification of protein biomarkers for male factor infertility will allow for unbiased comparisons of fertile and infertile males and will clarify the pathophysiology of the disease. An advantageous characteristic of genomic and proteomic technology is that the results provide a definitive characterization of infertile phenotypes. The use of technologies such as genomics and proteomics is a step toward creating personalized medical diagnoses by determining the individual causes of infertility (309). Finally, metabolomics involves the measurement of metabolite expression. Metabolites are small biomarkers that indicate the functionality of a cell and characterize certain diseases or physiological states. The determination of the human metabolome will reveal the functional phenotype of the system being examined, whether it is a single cell or an entire organism. Mass spectroscopy, nuclear magnetic resonance spectroscopy, and other chromatography methods can be used to create metabolite profiles. Pathway or cluster analyses are used to identify subsets of metabolites that can facilitate more accurate diagnosis (309-311). By identifying differences in the metabolome of infertile phenotypes, novel noninvasive methods for the diagnosis and treatment of male factor infertility can be developed.
N N
N
N
N N N
N
& KEY POINTS
N
Highly Recommended
Approximately 2,000 genes have been implicated in male fertility. There is a genetic basis for both the hypothalamic pituitary gonadal axis control of sperm production and the molecular events that characterize this process. Moreover, genes control the formation of
N
53
the ductal system and orchestrate sperm function during fertilization. Currently, genetic abnormalities explain approximately 1/3 of azoospermia cases. Although 12-41% of azoospermic cases are idiopathic, it is likely that these cases have unknown genetic causes. Azoospermia of genetic origin primarily encompasses men with any of a wide variety of genetic disorders, including chromosomal abnormalities, monogenic disorders, multifactorial genetic diseases, and epigenetic disorders. Sex and numerical chromosomal abnormalities (Klinefelter syndrome and Robertsonian translocations), gene deletions (Yq chromosome microdeletions) and mutations (CF mutations) are the most common abnormalities in the context of azoospermia. Three groups of genetic tests are used to detect genetic diseases in azoospermic men: cytogenetic tests (karyotyping) to detect chromosomal aneuploidy and structural alterations, PCR to detect Y chromosome microdeletions, and specific gene sequencing analyses (mutational analysis of a specific gene). A high frequency of CFTR mutations is detected in men with obstructive azoospermia who present with congenital bilateral or unilateral absence of the vas deferens and bilateral epididymal obstruction. Non-obstructive azoospermia is primarily due to pretesticular and testicular defects; each of these conditions has a multitude of genetic causes. Depending on whether the azoospermia is obstructive (OA) or non-obstructive (NOA), one can determine which genes require analysis. CFTR gene analysis is recommended in OA, whereas karyotyping and Yq microdeletion analyses are recommended in NOA. Yq microdeletion screening may not only identify the cause of azoospermia but also predict the probability of sperm retrieval in ART candidates. ART-derived male offspring will inherit the Yq microdeletion, potentially resulting in subsequent infertility. Genetic testing in infertile males allows couples to make educated decisions regarding whether to use a sperm donor or to opt for advanced ART and a preimplantation genetic diagnosis if an abnormal result is revealed.
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18. Radpour R, Gourabi H, Dizaj AV, Holzgreve W, Zhong XY. Genetic investigations of CFTR mutations in congenital absence of vas deferens, uterus, and vagina as a cause of infertility. J Androl. 2008;29(5):506-13. 19. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. 1989;245(4922):1059-65, http://dx.doi.org/10.1126/ science.2772657. 20. Radpour R, Gourabi H, Gilani MA, Dizaj AV. Molecular study of (TG)m(T)n polymorphisms in Iranian males with congenital bilateral absence of the vas deferens. J Androl. 2007;28(4):541-7. 21. Wong PY. CFTR gene and male fertility. Mol Hum Reprod. 1998;4(2):107-10, http://dx.doi.org/10.1093/molehr/4.2.107. 22. Patrizio P, Salameh WA. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in normal and pathological adult human epididymis. J Reprod Fertil Suppl. 1998;53:261-70. 23. 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Morral N, Llevadot R, Casals T, Gasparini P, Macek M, Jr., Dork T, et al. Independent origins of cystic fibrosis mutations R334W, R347P, R1162X, and 3849 + 10kbC–.T provide evidence of mutation recurrence in the CFTR gene. Am J Hum Genet. 1994;55(5):890-8. 32. Ferlin A, Raicu F, Gatta V, Zuccarello D, Palka G, Foresta C. Male infertility: role of genetic background. Reprod Biomed Online. 2007;14(6):734-45, http://dx.doi.org/10.1016/S1472-6483(10)60677-3. 33. Oates RD, Amos JA. The genetic basis of congenital bilateral absence of the vas deferens and cystic fibrosis. J Androl. 1994;15(1):1-8. 34. Patrizio P, Leonard DG. Mutations of the cystic fibrosis gene and congenital absence of the vas deferens. Results Probl Cell Differ. 2000;28:175-86, http://dx.doi.org/10.1007/978-3-540-48461-5_7. 35. Gervais R, Dumur V, Rigot JM, Lafitte JJ, Roussel P, Claustres M, et al. High frequency of the R117H cystic fibrosis mutation in patients with congenital absence of the vas deferens. 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The inability to identify a specific genetic etiology in idiopathic azoospermia carries an increased risk of the transmission of the same trait to the offspring. Incorporating novel techniques, such as genomics, proteomics, and metabolomics, into infertility research could assist in the creation of a complete portrait of the genes that are involved in infertility and would allow for improvements in ART and the development of more targeted solutions.
& AUTHOR CONTRIBUTIONS All of the authors were involved in the drafting and revision of the manuscript. All of the authors have read and approved the final version.
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REVIEW
Obstructive azoospermia: reconstructive techniques and results Karen Baker, Edmund Sabanegh Jr. Center for Male Fertility, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio, United States.
Obstructive azoospermia is a common cause of male infertility and can result from infection, congenital anomalies, or iatrogenic injury. Microsurgical vasal reconstruction is a suitable treatment for many cases of obstructive azoospermia, although some couples will require sperm retrieval paired with in-vitro fertilization. The various causes of obstructive azoospermia and recommended treatments will be examined. Microsurgical vasovasostomy and vasoepididymostomy will be discussed in detail. The postoperative patency and pregnancy rates for surgical reconstruction of obstructive azoospermia and the impact of etiology, obstructive interval, sperm granuloma, age, and previous reconstruction on patency and pregnancy will be reviewed. KEYWORDS: Obstructive Azoospermia; Wolffian Duct Abnormalities; Iatrogenic Injury; Microsurgery; Vasovasostomy. Baker K, Sabanegh E. Obstructive azoospermia: reconstructive techniques and results. Clinics. 2013;68(S1):61-73. Received for publication on February 24, 2012; Accepted for publication on March 3, 2012 E-mail: bakerk8@ccf.org Tel.: 1 216 445 1103
with spontaneous conception. The rate of birth defects is higher, albeit slightly, with IVF/ICSI. Multiple gestations occur in over 30% of all IVF/ICSI pregnancies (9) and carry risks to the mother and unborn children, such as prematurity and low birth weight. In the following chapter, we will describe the various etiologies for obstructive azoospermia and discuss the treatment options. The factors that influence the success of vasal reconstruction will be reviewed, and the microsurgical techniques for vasovasostomy (VV) and vasoepididymostomy (VE) will be described in detail.
& INTRODUCTION Obstructive azoospermia (OA) is defined as the absence of spermatozoa in the ejaculate despite normal spermatogenesis. OA is a common urologic condition and accounts for 6.1% (1) to 13.6% (2) of patients presenting for fertility evaluation. Vasectomy is a frequent cause of OA; however, alternate etiologies represent 19% (3) to 69% (4) of patients undergoing surgical exploration for OA. Infection, iatrogenic injury, and genetic and congenital conditions are all possible causes of OA. While some of these conditions are amenable to curative surgery, others will require sperm retrieval and in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). Is correction of OA necessary when couples could elect to proceed directly to sperm retrieval and IVF/ICSI? While treatment should be tailored to the individual couple, there is an excellent rationale for recommending surgical reconstruction for OA. First, treatment may obviate the need for IVF/ ICSI and thereby eliminate the risks and costs associated with advanced assisted reproductive techniques. Cost analyses reveal that vasectomy reversal is less expensive than IVF/ ICSI (5-8). IVF/ICSI also subjects the spouse to risks, such as ovarian hyperstimulation syndrome, that are not present
Etiologies Ejaculatory duct obstruction. Ejaculatory duct obstruction (EDO) is a rare cause of OA and accounts for approximately 1% of patients presenting with male infertility. EDO is an evolving topic, and a discussion regarding partial vs. complete EDO and functional vs. anatomic obstruction is beyond the scope of this chapter. The diagnosis of complete EDO should be suspected when the patient has low-volume, acidic semen that contains no sperm. An absence of fructose in the semen supports the diagnosis, as fructose is present in the secretions from the seminal vesicles. Occasionally, pain at the time of ejaculation is reported. Physical examination may reveal enlarged seminal vesicles or a midline nodule in the prostate, but frequently, the rectal exam is unremarkable. Testicular volume is usually normal, and the vasa deferentia are present. Laboratory studies will confirm normal gonadotropin and testosterone levels. Retrograde ejaculation should be rule out by examining post-ejaculatory urine for sperm. Transrectal ultrasound is a useful tool for confirming the diagnosis and further defining the causative factor. A 7 to 10 mHz endocavitary probe provides excellent visualization
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)07
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decreasing incidence of Young’s syndrome in men born after mercury-containing teething powder and worm medications were banned in the United Kingdom (18). There is building evidence that modern day Young’s syndrome may be genetic variations of Kartagener’s syndrome (19,20) or CFTR gene mutations (21,22). Genetic testing should be performed in patients presenting with signs and symptoms of Young’s syndrome. Surgical reconstruction, while technically feasible, has resulted in poor outcomes in traditional cases of Young’s syndrome (18). Sperm retrieval paired with IVF/ICSI is the best option for these couples. Infection. Epididymitis is a common genitourinary condition, and an infectious etiology should always be considered in men with this diagnosis. Gonorrhea, chlamydia, trichomonas, brucellosis, BCG, ureaplasma, mycoplasma, coliforms bacteria, adenovirus, and enterovirus have all been reported as causes of epididymitis. Regardless of the etiology, epididymitis can cause an intense inflammatory reaction, leading to secondary scarring and obstruction of the epididymis. Physical examination may reveal enlarged or indurated epididymides and a transition point suggesting the site of obstruction. Semen volumes are typically normal, and white cells are not necessarily present in the ejaculate or urine outside of the period of acute infection. In cases of tuberculosis, the vas deferens may be nodular and enlarged, and a low-volume ejaculate may be present when the disease involves the prostate and seminal vesicles. Infection was the proposed etiology for obstructive azoospermia in 8-46% of patients undergoing vasal reconstruction in several large series (2,4,23,24). The incidence of post-infectious epididymal obstruction is thought to be low in developed countries due to prompt treatment, but it may account for a disproportionately large percentage of OA in the developing world (25). Twenty-three percent of infertile men presenting to a Nigerian teaching hospital had findings consistent with chronic infectious epididymitis, and 14% of infertile men had azoospermia attributed to post-infectious obstruction of the vas or epididymis (26). In a retrospective review of couples with male factor fertility evaluated at a tertiary hospital in Nigeria, Eke et al. reported sexually transmitted disease as the cause of infertility in 29.4% of men, with the majority of these men presenting with signs and/or symptoms consistent with active infection (27). OA due to infection was diagnosed in 8.6% of men with male factor infertility, and an additional 4.3% of men were diagnosed with an infection of the male accessory sex glands in a study of infertility in Western Siberia (28). A study of infertile couples in Mongolia found post-infectious obstructive azoospermia and male accessory gland infection in 8.4% and 6.7% of men, respectively (29). A high percentage of these men (44.2%) reported previous treatment for sexually transmitted infections (STI), with 59%, 9.1%, and 1.2% reporting treatment for gonorrhea, trichomonas, and chlamydia, respectively. Vigil et al. found chlamydia in 38.6% of males in couples presenting for fertility evaluation in Chile but detected no statistically significant differences in semen parameters between men with and without active chlamydia infections (30). Scrotal exploration and microsurgical reconstruction is a viable option for post-infectious epididymal obstruction, and the outcomes and surgical techniques are described later in this chapter. A notable exception is genitourinary tuberculosis. Outcomes for surgical reconstruction for
of the prostate and other accessory sex organs. Dilation of the seminal vesicles to greater than 1.5 cm in the anteroposterior axis is consistent with EDO, and the presence of 10 or more sperm per high-powered field in the seminal vesicle aspirate confirms the diagnosis. To aid diagnosis, the patient should be instructed to ejaculate within the 24 hours prior to the ultrasound. Sonography can also demonstrate dilation of the ejaculatory ducts, calcifications within the ejaculatory ducts, or prostate, utricle, or Mu¨llerian duct cysts that can occlude the ejaculatory ducts. Traditional treatment consists of transurethral resection of the ejaculatory ducts (TURED). Yurdakul et al. retrospectively reviewed the outcomes of 12 azoospermic men with EDO who underwent TURED. Sperm appeared in the ejaculate of 11 of 12 patients. Of these 11 patients, 42% (n = 5) had a postoperative sperm concentration of.20 mil/ml. The authors reported spontaneous pregnancy in three couples, pregnancy by IUI in one couple, and one couple with sperm concentration less than five million per ml who obtained a pregnancy through ICSI using fresh ejaculated sperm (10). Emerging data suggests that vesiculoscopy paired with ejaculatory duct dilation and/or calculi extraction may also be a viable treatment option for patients with EDO (11,12). Wolffian duct abnormalities. Congenital bilateral absence of the vas deferens (CBAVD) is often caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This condition is suspected based on the absence of palpable vas deferens at the time of physical exam. The caput of the epididymis is present, and the testicles should be a normal size and consistency; however, the seminal vesicles are absent or hypoplastic in the majority of patients (13). CBAVD can also be associated with unilateral renal agenesis in a minority of patients (14). Unilateral absence or hypoplasia of the vas deferens is derived from failure of organogenesis of the Wolffian ducts system, and its association with renal agenesis has been well described. Unilateral or bilateral vasal hypoplasia or unilateral absence of the vas may be an indicator of obstructive azoospermia, as a high percentage of these patients will have anomalies of the contralateral seminal vesicle. Raviv et al. published their experience with TRUS in the evaluation of low-volume azoospermic men and noted that 10 of 12 patients (83%) with unilateral absence of the vas demonstrated contralateral abnormalities of the seminal vesicles or ejaculatory ducts (15). Partial vasal agenesis has also been described. Anger and Goldstein published a series of three men found to have segmental dysplasia of the vas deferens during scrotal exploration for possible microsurgical reconstruction (16). Surgical reconstruction may be a viable treatment for some patients with unilateral vasal agenesis or hypoplasia. CBAVD is not amenable to surgical reconstruction, but sperm is readily retrievable from these patients via percutaneous (PESA) or microsurgical (MESA) epididymal aspiration, testicular sperm aspiration (TESA), or simple open biopsy (TESE). Young’s syndrome. Young’s syndrome is obstructive azoospermia associated with chronic sinopulmonary infections. Initially described in 1950 by Dr. David Young, the relative rarity of this condition in the modern era has caused some to call the existence of this syndrome into question (17). Mercury exposure is one proposed etiology for Young’s syndrome, and in an elegant argument supporting this theory, Hendry et al. demonstrated the
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vasectomy and frequency of divorce, the demand for this procedure will continue to grow. Reconstruction is less expensive than sperm retrieval paired with IVF/ICSI, avoids the risks associated with IVF/ICSI, and offers the possibility of natural conception and multiple pregnancies over time without additional expense (38). Sperm retrieval may be a reasonable option in couples likely to require IVF for concomitant female factor due to advanced age or tubal disease. In these cases, sperm can readily be obtained through PESA performed with local anesthetic or sedation. Men with previous paternity and normal genitourinary exams do not require additional fertility evaluation before proceeding to surgery. Patients should be queried about previous inguinal, scrotal, or pelvic surgeries, as these procedures may complicate vasal reconstruction. The physical exam should confirm normal testicles bilaterally. Enlarged epididymides are common, and the location and size of the vasectomy defects should be noted. Couples should be counseled about the option of intraoperative sperm retrieval for cryopreservation, and it is our practice to recommend this step to couples with a higher likelihood of requiring VE.
tuberculosis are particularly poor due to scarring at multiple levels of the male reproductive tract (31). Once identified, prompt treatment for genitourinary tuberculosis should be initiated, as early treatment may resolve the inflammation and return sperm to the ejaculate (32). Sperm retrieval paired with IVF/ICSI should be considered for patients who remain azoospermic despite adequate treatment for tuberculosis (31). Iatrogenic injury. Injury to the vas during surgical procedures has been well described and presents a unique challenge to fertility specialists. Vasal injury has been attributed to a variety of inguinal, scrotal, and pelvic surgeries, including herniorrhaphy, hydrocelectomy, appendectomy, and renal transplant. Trauma is a rare cause of vasal obstruction (4). The author has experience with one patient with proven paternity who was unreconstructable at the time of vasectomy reversal due to obstruction of the pelvic vas, which was presumably due to a history of multiple pelvic fractures suffered during a blast injury. Transection, compression, fibrosis, and ischemic injury are all possible mechanisms for vasal injury. There are no large series establishing the most common mechanisms of injury; however, transection of the vas is thought to account for no more than 25% of the cases of postsurgical obstruction based on surgical series and examination of pathological specimens (33,34). Obstruction was attributed to iatrogenic injury in 8-19% of patients in several series examining the outcomes of VE (4,23,24). Sheynkin et al. reviewed 472 men who underwent scrotal exploration for obstructive azoospermia and found that 7.2% of men had findings consistent with iatrogenic injury to the vas deferens. Pediatric inguinal hernia repair was the most common cause (59%), followed by adult inguinal hernia repair (29%), renal transplant (6%), appendectomy (3%), and spermatocelectomy (3%) (35). Fifty-six percent of the cases had a history of bilateral procedures that were exclusively herniorrhaphy. A history of a unilateral surgery was noted in 44% of the cohort; however, all of these patients had a contralateral abnormalities that included vasal and/or epididymal obstruction, testicular atrophy, absent testis, congenital epididymal aplasia, and congenital absence of the vas deferens. The likelihood of vasal obstruction after inguinal hernia repair may be influenced by the surgical approach (laparoscopic vs. open), pediatric vs. adult hernia repair, the surgical method, and the material used to bolster the repair (36). Of particular concern to fertility specialists is the use of polypropylene mesh. Shin et al. described 14 patients from eight infertility centers diagnosed with vasal obstruction secondary to polypropylene mesh herniorrhaphy. Obstruction at the hernia repair was confirmed with vasography, and exploration revealed dense fibrosis encasing and in some cases obliterating the vas deferens (37). In theory, newer, lightweight mesh material causes less of an inflammatory reaction and may decrease the chances of vasal obstruction secondary to hernia repair (36). Surgical reconstruction is possible in many cases of iatrogenic injury to the vas in the scrotum or inguinal canal; however, the surgeon should be prepared to perform VE and other complex repairs. Elective sterilization. Prevalence of vasectomy varies by country and is influenced by affluence, religion, and culture. Vasal reconstruction is the preferred method for restoration of fertility after a vasectomy, and due to the popularity of
& INFLUENTIAL FACTORS The majority of the literature exploring the factors that influence the success of vasal reconstruction is derived from the outcomes after vasectomy reversal. Some factors, such as age and obstructive interval, are likely to impact postoperative outcomes after vasal reconstruction for other etiologies of OA. Obstructive interval. An obstructive interval of 10 years or longer was thought to portend a poor outcome after vasal reconstruction. Recent publications have reported minimal decline in patency among couples with obstructive intervals of 10 years or longer, though pregnancy rates are lower in couples with longer obstructive intervals. The Vasovasostomy Study Group reported that patency and pregnancy rates decreased with increasing time since the vasectomy; however, pregnancy rates declined more rapidly. When grouped by obstructive interval, patency and pregnancy rates were 97% and 76%, respectively, for less than three years, 88% and 53%, respectively, for 3 to 8 years, 79% and 44%, respectively, for 9 to 14 years, and 71% and 30%, respectively, for intervals of 15 years or more (39). In 2004, Boorjian reviewed 213 patients randomly selected from a single surgeon’s 17-year operative experience of bilateral vasal reconstructions. Patency (defined as the presence of any intact sperm in the ejaculate) was achieved in 90% of patients, and there was no statistically significant difference in patency rates based on obstructive interval. Pregnancy rates, however, decreased dramatically from 85% for obstructive intervals of 15 years or less to 44% for intervals of more than 15 years (p,0.05) (40). Magheli reviewed a single surgeon’s series of 334 vasectomy reversals and reported that patency and pregnancy rates were not influenced by the length of obstructive interval, although multivariate analysis revealed that longer obstructive intervals were associated with an increased tendency to perform VE (41). Kolettis found favorable patency and pregnancy rates in 74 patients with obstructive intervals of 10 years or longer drawn from the experiences of three surgeons. Patency and pregnancy rates were 74% and 40%, 87% and 36%, and 75% and 27% for obstructive intervals of 10 to 15 years, 16 to 19 years, and 20 or more years, respectively
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postoperative patency. The Vasovasostomy Study Group found no difference in postoperative patency or pregnancy when comparing patients with and without histologically confirmed sperm granulomas (39). Magheli found that the presence of sperm granuloma did not improve postoperative patency or pregnancy rates (41). Boorjian examined the outcomes of 213 vasectomy reversals and determined that the presence of sperm granuloma was associated with a lower incidence of VE but that it was not associated with an increase in patency or pregnancy rates (40). Conversely, Bolduc found the presence of a sperm granuloma correlated with patency in a series of 747 vasectomy reversals (51). Hinz reported the presence of a sperm granuloma improved patency but not pregnancy rates in a review of 351 vasectomy reversals (52). Previous reconstruction. Repeat vasal reconstruction yields encouraging patency and pregnancy rates. Piack reported patency and natural birth rates of 92% and 52%, respectively, in 62 patients who underwent repeat vasal reconstruction after failed VV. Of note, unless technically unfeasible, the authors performed VV regardless of the quality of the intravasal fluid or presence of sperm (43). Hollingsworth reported patency and pregnancy rates of 85% and 44%, respectively, in 49 patients undergoing repeat vasectomy reversal. Unilateral or bilateral VE was performed in 34% of the patients. An obstructive interval of 10 years or more did not predict an increased need to perform VE or negatively impact the rates of patency or pregnancy after repeat reconstruction in this series (53). Hernandez and Sabanegh reported overall patency and pregnancy rates of 79% and 31%, respectively, in 41 couples who underwent repeat vasectomy reversal after one or more attempts at vasal reconstruction. Unilateral or bilateral VE was performed in 73% of these couples (49). Pasqualotto reported patency and spontaneous pregnancy in 66.7% and 25%, respectively, in 18 couples who underwent bilateral (n = 8) or isolated unilateral (n = 10) VE after failure of previous VE (54). The literature supports repeat vasal reconstruction, though the surgeon should be comfortable performing VE, as the need to perform VE is frequently reported.
(42). The authors concluded that pregnancy rates after obstructive intervals of up to 20 years were on par with IVF/ICSI. Age. Age of the female partner significantly impacts postoperative pregnancy rates, as female fertility potential drops profoundly in women over 40 years of age (41,43). Gerrard demonstrated a precipitous decline in postoperative pregnancy rates after age 40 in a series of 249 vasal reconstructions. Postoperative patency and pregnancy rates were 90% and 67%, respectively, for females age 20-24, 89% and 52%, respectively, for those age 25-29, 90% and 57%, respectively, for those age 30-34, 86% and 54%, respectively, for those age 35-39, and 83% and 14%, respectively, for those age 40 and older (44). Kolettis examined the outcomes of 46 vasectomy reversals in men with female partners aged 35 or older and found a marked decline in pregnancy rates when the female partner was 40 years of age or older. Overall patency and pregnancy rates were 81% and 35%, respectively, though the pregnancy rate was 46% for women 35 to 39 and only 14% for women 40 or older (45). Hinz examined 212 vasectomy reversals performed by a single surgeon and found female age to be an independent predictor of postoperative pregnancy on multivariate analysis, with age 40 and older associated with significantly poor pregnancy rates compared with the younger age groups (42% vs. 74%, respectively) (46). In contrast, the age of the male does not appear to independently influence patency or pregnancy after vasectomy reversal. In some series, older male age is associated with longer obstructive intervals, which in turn may be associated with increased need to performed VE (41,47). Couples with previous conceptions together. Several studies have demonstrated that a history of previous conception together increases a couple’s chance of pregnancy after vasectomy reversal. The Vasovasostomy Study Group reported 86% patency and 75% pregnancy rates in couples citing death of a child as the reason for vasectomy reversal (39). Chan compared the outcomes of 27 couples with a history of previous children together to 100 historical controls. Postoperative patency and pregnancy rates for couple with previous children together were 100% and 86%, respectively. Patency was not significantly different between the study group and historical cohort, but the pregnancy rate in the study group far exceeded the 54% pregnancy rate in the historical controls. Notably, the mean age of the study group was statistically older than the historical control (37.2 vs. 29.9, p,0.01) (48). Hernandez and Sabanegh examined the outcomes of 41 couples undergoing repeat vasal reconstruction and found couples with a history of children together had a pregnancy rate of 80% compared with couples married to new partners, in whom the pregnancy rate was only 17% (49). In contrast, Kim et al. examined the outcomes of 44 couples undergoing repeat vasectomy reversal and found no significant difference in pregnancy rates between couples with a history of children together and couples married to new partners. The authors determined that female age of less than 35 years was the only significant predictor of pregnancy after repeat VV (50). Granuloma. Much attention has been paid to the impact of the presence of sperm granuloma on the success of vasectomy reversal. The published literature provides conflicting results, but the balance of evidence suggests that sperm granuloma does not impact postoperative pregnancy rates and does not exert a strong influence on
& SURGICAL TECHNIQUE In 1977, Silber and Owen independently described the microsurgical approach to vasectomy reversal and thereby ushered in the modern era of vasal reconstruction. The microsurgical approach is the gold standard for vasal reconstruction, as the precise mucosal-to-mucosal anastomosis is believed to result in superior outcomes. Series comparing microsurgical, Loupe magnification, and unmagnified VV have revealed that patency and pregnancy rates are better when higher magnification is used (55-57). To date, there have only been two published reports to detail the outcomes of robot-assisted vasovasostomies in humans. Neither report included pregnancy rates, and patency after robot-assisted reconstruction was either inferior or on par with traditional microsurgery (58,59). Given these results and the expense of robot-assisted surgery, we advocate a traditional microsurgical approach to vasal reconstruction. General or spinal anesthesia is typically favored for vasal reconstruction due to the possible length of these cases. Patients should be positioned in a supine position, the scrotal hair clipped, and the clippings thoroughly removed
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distal to the vasectomy defect or sperm granuloma are identified. Careful dissection with limited electrocautery is used to mobilize the vas deferens. Battery-powered thermal cautery units are a useful adjunct, as these devices have a very limited area of collateral tissue damage. To aid with retraction and identification, the vas may be encircled with vessel loops, or fine-stay sutures may be placed in the adventitia. The vascular pedicles of the vas are ligated at the expected level of the anastomosis using a fine suture, such as a 6-0 prolene (Figure 2). In the instances in which a longer distance must be bridged to affect a tension-free anastomosis, the distal vas can be mobilized on its vascular pedicle all the way into the inguinal canal, affording several additional centimeters of length (Figure 3). Once the vasa are mobilized and vascular pedicles controlled, the operative microscope is brought over the operative field and focused. Patency and pregnancy rates are equivalent between anastomosis to the straight and convoluted portions of the proximal vas; therefore, the surgeon should concentrate on the identifying healthyappearing vas (60,61). The proximal vas is incised with a small knife, such as a Beaver or supersharp eye blade. The division may be performed free-hand against a surface, such as a tongue blade, or with the aid of a cutting guide. The cut should be clean and perpendicular to the long axis of the vas. Gentle milking of the vas and/or epididymis may yield effluent that is examined for the presence and motility of sperm with a bench-top microscope. A touch prep of the effluent can be prepared by touching a sterile slide to the proximal vas. Alternatively, the effluent can be aspirated through a small angiocatheter attached with a sterile tuberculin syringe preloaded with a small amount of saline.
Figure 1 - Initial dissection of the vas. The incision can be minimized by fixing the vasectomy defect below the scrotal skin with a towel clamp or vas deferens clamp.
to prevent interference with visualization of the suture. The patient should be positioned and padded appropriately to prevent iatrogenic injuries, such as occipital alopecia, brachial plexus injuries, and myolysis. Sequential compression devices and prophylactic perioperative antibiotics are recommended. Vasovasostomy. The vasectomy defect is identified and swept to the anterior scrotal wall (Figure 1). A small vertically oriented incision, typically no longer than 2 cm, is made through the anterior scrotal skin, and the dartos is divided until the vas deferens is identified. Healthy appearing portions of the vas immediately proximal and
Figure 2 - Vascular control. Once the vas is isolated and mobilized, the vascular pedicle is ligated with 6-0 prolene just below the expected level of the anastomosis (inset). Monopolar electrocautery should be avoided in close proximity to the vas deferens.
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the literature, and patency rates between the two techniques are comparable (39,66). The formal two-layer approach allows for direct mucosal-to-mucosal anastomosis. This technique may prove advantageous when there is a marked difference in the diameter of the lumens. The anastomosis begins by opposing the adventitia and muscularis of the vasal ends on the posterior side of the anastomosis with one to two interrupted 9-0 nylon stitches. Once the ends are approximated, interrupted 10-0 sutures are placed in the mucosal layer of the vas beginning on the far side of the anastomosis (Figure 4A). The stitches should be placed so that the knots reside outside the lumen. Shorter doublearmed stitches may prove useful. Care is taken to gently approximate the delicate mucosal layers, and the first few stitches can be tied as they are placed. Six to eight luminal stitches are typically required to create a well-spaced anastomosis (Figure 4B). The second layer is completed by placing an additional 4 to 5 interrupted 9-0 nylon stitches through the serosal and muscularis layers of the anterior vas (Figure 4C). If desired, additional stitches can be used to oppose the perivasal tissues (Figure 4D). The modified two-layer anastomosis diverges from the formal two-layer technique in the size of the suture and layers incorporated in the luminal stitches. Full-thickness 90 interrupted nylon stitches incorporating the serosa, muscularis and mucosa are used to approximate the two vasal ends (Figure 5A). The surgeon may begin the anastomosis on the deep side of the field and move circumferentially. Alternatively, one may begin on the near side of the anastomosis and rotate or ‘‘flip’’ the vas after the first two to three stitches are placed. A microsurgical vas clamp can assist with positioning and rotating the vasal ends. A total of four to six full-thickness stitches are typically required to create a well-spaced anastomosis. The stitches should be placed such that the knots will reside on the serosa. The first few stitches are tied immediately after placement, and the remaining two to three stitches are tied once all remaining stitches have been placed to allow visualization of the lumen and confirmation of good suture placement (Figure 5B). Once the lumens are approximated, a second layer of 9-0 stitches incorporating the adventitia and small amount of muscularis is then placed in between the full-thickness stitches to affect a water-tight anastomosis (Figure 5C). Vasoepididymostomy. The absence of effluent or poor quality effluent containing no recognizable sperm parts is consistent with obstruction of the proximal vas, and a VE should then be considered. Modern techniques for VE center upon identifying and then directly anastomosing a single patent epididymal tubule to the much larger and sturdier lumen of the vas deferens. The patient preparation and initial dissection for the VE are as described for the VV. Careful attention should be paid to patient positioning during VE, as iatrogenic injuries can be avoided through simple measures, such as ensuring correct positioning and padding. The vertically oriented incision described in the VV technique is extended, and the testis is delivered. The tunica vaginalis is then opened, and the epididymis is inspected. On occasion, a transition point marking the level of epididymal obstruction can be readily identified proximal to where the epididymal tubules will be engorged. The presence of lipofuchsin, a blue-brown discoloration caused by the breakdown of extravasated sperm, may also indicate
Figure 3 - Mobilization of the distal vas. A tension-free anastomosis is essential. Gentle blunt dissection paired with judicious use of thermal cautery can be used to mobilize the distal vas to the external ring. When necessary, the scrotal incision can be extended cranially to aid in the dissection. In extreme cases, the inguinal canal can be opened, and the vas can be mobilized through the level of the internal ring. These maneuvers provide several centimeters of additional length.
The presence of numerous sperm, in whole or in part, or the presence of copious clear fluid, even in the absence of intravasal sperm, is associated with excellent postoperative patency (39,62-64). When these findings are present, the surgeon should proceed with VV. A healthy portion of the distal vas is then sharply divided perpendicular to its long axis, and the lumen is assessed. Some favor dilating the lumen with lacrimal duct probes or the tips of microforceps. In instances in which the surgeon is suspicious of obstruction distal to the vasostomy, either a saline vasogram or traditional radiographic vasogram can be performed. Patency can also be directly assessed by flushing the vas with methylene blue-tainted saline while performing simultaneous cystoscopy to visualize the ejaculatory ducts. The level of obstruction can be assessed by cannulating the distal vas with a 0-nylon. The ends of the vas can be held in proximity with gentle traction on stay stitches or by use of a vasovasostomy clamp. When necessary, tissue adjacent to the vas can be approximated with a stitch to allow for a tension-free vasal anastomosis. A fine-point operative marker can be used to mark the sites of the anastomosis sutures, which is a step that can help ensure even spacing between the sutures during the anastomosis (65). The vasal anastomosis can be performed with a one- or two-layer anastomosis. The formal two-layer and modified two-layer are the techniques most commonly described in
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Figure 4 - Two-layer vasovasostomy.
the level of obstruction, though in some instances, the level of obstruction will not be apparent by inspection alone. The distal vas may require additional mobilization to ensure a tension-free anastomosis. Additional length can be obtained by mobilizing the vas into the inguinal canal (Figure 3). Frequently, this maneuver can be performed through the scrotal incision, but when necessary, the vertically oriented incision can be extended to the level of the external inguinal ring. Care should be taken to ensure the vascular pedicle remains intact and that hemostasis is ensured. The caudal portion of the epididymis can also be mobilized by dividing the relatively avascular plane of the lateral sulcus at the junction of the epididymis and testis (Figure 6A). This maneuver will allow the tail of the
epididymis to swing posteriorly. Additional distance can be bridged by rotating the entire testis and epididymis on its horizontal axis, in essence, inverting the testis and epididymis (Figure 6B). The testis should be inspected to assure adequate perfusion, but when carefully applied, these maneuvers should not jeopardize the blood supply. A tension-free anastomosis is critical to the success of a VE. The adventitia of the distal vas should be secured to the tunica albuginea in a location and orientation that allows for minimal angulation of the completed anastomosis. Bringing the distal vas through a separate, small aperture at the posterior-lateral reflection of the tunica vaginalis may provide the best lie for the vas deferens. Outcome studies for VE suggest better patency and pregnancy rates for more
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Figure 5 - Modified two-layer vasovasostomy.
distal anastomoses. As such, exploration for a candidate tubule should begin as distally as is feasible. All modern VE techniques are modifications of the end-to-side anastomosis wherein the end of the vas deferens is anastomosed to the side of the epididymal tubule. A 5-mm incision is made through the tunica albuginea overlying the target tubules (Figure 7A). Careful sharp dissection under high magnification (25X) is used to mobilize a candidate tubule. Visualization is aided by gentle irrigation with saline through an angiocatheter. Judicious use of bipolar cautery or handheld low-temperature cautery units can aid with
hemostasis. The adventitia of the vas is secured to the tunica albuginea of the epididymis with a 9-0 nylon stitch. The number of sutures and timing for opening the epididymis vary with the different anastomotic techniques. For conventional end-to-side anastomosis, a microknife or microscissors are used to make a K- to 1-mm aperture in the side of a candidate tubule (Figure 7A). A 10-0 nylon suture is placed through the lip of the tubule to aid in identification of the mucosal edge. Alternately, dilute methylene blue can be painted on the tubule to highlight the mucosal edge. The effluent is then assessed and whole, preferably motile,
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Figure 6 - Mobilization of the epididymis. A) Division of the tunica albuginea through the avascular potions of the lateral epididymal sulcus allows posterior rotation of the epididymal body. B) Additional length can be gained by pivoting the testis and epididymis superiorly and thereby swinging the epididymis into a cranial position. In extreme cases, the testis may be inverted.
Figure 7 - Conventional end-to-side vasoepididymostomy.
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wall of the tubule, but the throw is not completed, thus anchoring the body of the needle in the epididymal tubule (Figure 8A). An additional one to two sutures are similarly passed depending on whether the 2-suture or triangulation technique is used (Figures 8A and B). The epididymal tubule is then sharply incised, and the fluid is assessed. The needle throws are then completed by drawing the needle completely through the epididymal wall. Each double-armed needle is then passed inside to out through the mucosa of the vas in its corresponding sector (Figure 8C). The 10-0 sutures are gently pulled to invaginate and then are tied to secure the epididymal aperture inside the lumen of the vas (Figure 8D). Additional 9-0 nylon sutures are placed to secure the vas to the tunica albuginea. The tunica vaginalis, dartos, and skin are closed in accordance with standard practice with careful attention to proper hemostasis.
sperm should be found. An additional two to three 10-0 sutures are evenly spaced around the tubotomy to triangulate or quadrangulate the epididymal opening (Figure 7B). Single-arm or double-armed sutures may be used in accordance with the surgeon’s preference, and all knots should lie outside the lumen. The 10-0 sutures are passed through their corresponding sectors in the vasal mucosa, and the sutures are tied while supporting the vas in close proximity to the epididymal tubule. Additional supporting 9-0 nylon sutures are placed circumferentially between the adventitia and tunica albuginea (Figure 7C). The epididymal tubule can also be drawn into the vasal lumen using an intussusception technique. Precise needle placement may be easier with this technique, as the needles are all placed before the tubule is opened and has decompressed. As with conventional end-to-side anastomosis, a candidate tubule is selected and then widely mobilized so that it can be drawn into the vasal lumen. The adventitia of the vas is secured to the tunica albuginea of the epididymis with an interrupted 9-0 nylon suture. The first needle of a double-armed 10-0 nylon stitch is passed in and out of the
& OUTCOMES Microsurgical vasectomy reversal delivers excellent patency and pregnancy outcomes. The Vasovasostomy
Figure 8 - Intussusception end-to-side vasoepididymostomy.
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Study Group reported overall patency of 86% and pregnancy in 52% of patients in their series of 1,469 microsurgical vasectomy reversals (39). Bolduc et al. reviewed 747 microsurgical vasovasostomies and reported overall patency of 86% and a pregnancy rate of 53% (51). Hinz et al. reviewed 212 vasectomy reversals performed by a single surgeon and reported overall patency of 93% and a pregnancy rate of 72% (46). Adherence to good microsurgical techniques with special attention to selecting healthy vas segments, ensuring a tension-free anastomosis, and performing precise mucosalto-mucosal anastomosis will result in excellent outcomes for couples electing vasectomy reversal. The results of vasal reconstruction for etiologies other than vasectomy are limited, though the majority of the literature suggests that while surgery may be challenging, postoperative patency is acceptable in the instances wherein reconstruction is technically feasible. Tuberculosis is a notable exception, as the extensive inflammatory reaction is not amenable to surgical reconstruction (see above). VE and/or complex repairs requiring transseptal, inguinal, or pelvic approaches are frequently required for success. Simultaneous sperm retrieval and cryopreservation should be discussed with couples. Berardinucci and Jarvi reviewed the etiologies, intraoperative findings, and outcomes of 80 men without a history of vasectomy who underwent surgical exploration for obstructive azoospermia. All patients had normal ejaculatory volume or transrectal ultrasound not diagnostic for EDO, at least one palpable vas deferens, and active spermatogenesis, as documented by testicular biopsy. The etiologies for obstruction were idiopathic in 73% (n = 59), infectious in 18% (n = 14), and surgical in 8% (n = 7) of subjects. Twenty-eight patients (35%) had findings that precluded surgical repair, which included 24 patients with either bilateral or unilateral intra-testicular obstruction defined as the absence of sperm in the epididymal fluid. The remaining 52 patients underwent reconstruction consisting of unilateral VE in 22 patients, bilateral VE in 27 patients, and cross VVs in three patients. Overall patency was 62%. The likelihood of reconstruction and subsequent patency were higher for patients with obstruction attributed to an infectious or surgical etiology compared with those with an idiopathic cause (93% and 77%, respectively, for those with infectious causes, 79% and 60%, respectively, for those with surgical causes, and 58% and 55%, respectively, for those with idiopathic causes) (23). Kim et al. reported 49 patients with no history of vasectomy who underwent bilateral or isolated unilateral VE. Etiologies of obstruction included idiopathic 43% (n = 21), inflammatory 39% (n = 19), unreported 12% (n = 2), congenital 4% (n = 2), and traumatic 2% (n = 1) causes. Overall patency was 81%, and the pregnancy rate was 37%; however, the outcomes were not stratified by etiology. The authors noted that a more proximal anastomosis (caput) favored patency, while a more distal anastomosis favored pregnancy (3). Similarly, Ho et al. reviewed the outcomes of 22 patients with OA not attributable to surgery or vasectomy who underwent VE. Overall patency was 57%, while patency was 60% for post-infectious obstruction and 50% for idiopathic obstruction (25). Shiff et al. reviewed the outcomes of four surgical techniques in 153 men undergoing VE. Of the men with postoperative semen analysis, the etiology for obstruction was vasectomy in 45, infection in 47, iatrogenic injury in eight, and unknown etiology in
two of the subjects. Patency was higher in men with iatrogenic etiology for obstruction (75% iatrogenic vs. 44% for vasectomy, 45% for infection, and 0% for unknown), though the difference did not reach statistical significance (24). Hopps and Goldstein reported eight patients with a history of hydrocelectomy who underwent reconstruction for OA due to injury to the epididymis (n = 6) or scrotal vas (n = 2). The injury was bilateral in four patients, while the remaining four had a unilateral injury that was associated with a contralateral abnormality, such as atrophy, absence of the testis, or obstruction from hernia repair. Sperm was detected in the ejaculate in five of six patients who submitted a semen sample, though only one spontaneous pregnancy was reported, and three of the remaining couples elected to proceed to IVF. (67) These outcomes prompted the authors to recommend couples be counseled about the likelihood of VE and option of sperm retrieval paired with IVF/ICSI in lieu of reconstruction when hydrocelectomy or spermatocelectomy is suspected as the cause for obstructive azoospermia. OA after herniorrhaphy can represent a difficult challenge. Shyenkin reported overall patency and natural pregnancy rates of 65% and 39%, respectively, in a review of 34 patients who underwent vasal reconstruction after iatrogenic injury to the vas deferens, the majority of which were attributed to prior herniorrhaphy (35). Pasqualotto reported overall patency and pregnancy rates of 65% and 40%, respectively, in 13 men who underwent open microsurgical vasal reconstruction for azoospermia or severe oligospermia attributed to herniorrhaphy (68). Of note, six patients required a second procedure, and half of these men remained azoospermic despite repeat reconstruction, which is an outcome that speaks to the technical difficulty of the anastomosis in this situation. Shaeer and Shaeer reviewed their experience with laparoscopic mobilization of the pelvic vas after unilateral or bilateral post-herniorrhaphy vasal obstruction confirmed by vasography. Once mobilized, the freed end of the vas is tunneled through the conjoint tendon and/or abdominus rectus, and an open microsurgical vasal reconstruction is then performed. The authors reported an overall patency of 68%, with bilateral repairs faring better than unilateral repairs (80% patency vs. 60% patency, respectively) (69). Sabanegh and Thomas reported equivalent patency but lower mean sperm concentrations in cross transseptal vasoepididymostomies performed in four patients with unilateral vasal agenesis, as opposed to six patients with other etiologies (70). The mean postoperative sperm concentration was 37.8 mil/ml in agenesis patients vs. 136 mil/ml in the remaining patients (p,0.05) (70). In a series of 18 patients requiring repeat vasoepididymostomies; however, patients with congenital etiologies fared slightly better, with postoperative patency of 85.7% compared with patients with inflammatory or post-vasectomy obstruction (patency 43% and 75%, respectively) (54). Microsurgery reconstruction is a viable option in many causes of obstructive azoospermia and remains the gold standard for vasal reconstruction. Postoperative patency and pregnancy rates are excellent after VV. Vasoepididymostomy or other complex repairs may be required, especially if vasectomy is not the cause of the obstruction or if the surgery is a repeat reconstruction. Reasonable patency and pregnancy rates are reported after complex repairs and repeat
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22. Goeminne PC, Dupont LJ. The sinusitis-infertility syndrome: Young’s saint, old devil. Eur Respir J. 2010;35(3):698, http://dx.doi.org/10.1183/ 09031936.00163809. 23. Berardinucci D, Zini A, Jarvi K. Outcome of microsurgical reconstruction in men with suspected epididymal obstruction. J Urol. 1998;159(3):831-4. 24. Schiff J, Chan P, Li PS, Finkelberg S, Goldstein M. Outcome and late failures compared in 4 techniques of microsurgical vasoepididymostomy in 153 consecutive men. J Urol. 2005;174(2):651-5. 25. Ho KL, Wong MH, Tam PC. Microsurgical vasoepididymostomy for obstructive azoospermia. Hong Kong Med J. 2009;15(6):452-7. 26. Ahmed A, Bello A, Mbibu NH, Maitama HY, Kalayi GD. Epidemiological and aetiological factors of male infertility in northern Nigeria. Niger J Clin Pract. 2010;13(2):205-9. 27. Eke AC, Okafor CI, Ezebialu IU. Male infertility management in a Nigerian tertiary hospital. Int J Gynaecol Obstet. 2011;114(1):85-6. 28. Philippov OS, Radionchenko AA, Bolotova VP, Voronovskaya NI, Potemkina TV. Estimation of the prevalence and causes of infertility in western Siberia. Bull. World Health Organ. 1998;76(2):183-7. 29. Bayasgalan G, Naranbat D, Tsedmaa B, Tsogmaa B, Sukhee D, Amarjargal O, et al. Clinical patterns and major causes of infertility in Mongolia. J Obstet Gynaecol Res. 2004;30(5):386-93, http://dx.doi.org/ 10.1111/j.1447-0756.2004.00217.x. 30. Vigil P, Morales P, Tapia A, Riquelme R, Salgado AM. Chlamydia trachomatis infection in male partners of infertile couples: incidence and sperm function. Andrologia. 2002;34(3):155-61, http://dx.doi.org/10. 1046/j.1439-0272.2002.00472.x. 31. Paick J, Kim SH, Kim SW. Ejaculatory duct obstruction in infertile men. BJU Int. 2000;85(6):720-4. 32. Shah RS. Obstructive azoospermia following genital tuberculosis may be reversible with medical therapy. AUA 2004 Abstract 1600. Available from: http://www.abstracts2view.com/aua_archive/view.php?nu=200 4001503. 33. Ridgway PF, Shah J, Darzi AW. Male genital tract injuries after contemporary inguinal hernia repair. BJU Int. 2002;90(3):272-6, http:// dx.doi.org/10.1046/j.1464-410X.2002.02844.x. 34. Steigman CK, Sotelo-Avila C, Weber TR. The incidence of spermatic cord structures in inguinal hernia sacs from male children. Am J Surg Pathol. 1999;23(8):880-5. 35. Sheynkin YR, Hendin BN, Schlegel PN, Goldstein M. Microsurgical repair of iatrogenic injury to the vas deferens. J Urol. 1998;159(1):139-41. 36. Junge K, Binnebo¨sel M, Rosch R, Ottinger A, Stumpf M, Mu¨hlenbruch G, et al. Influence of mesh materials on the integrity of the vas deferens following Lichtenstein hernioplasty: an experimental model. Hernia. 2008;12(6):621-6, http://dx.doi.org/10.1007/s10029-008-0400-2. 37. Shin D, Lipshultz LI, Goldstein M, Barme´ GA, Fuchs EF, Nagler HM, et al. Herniorrhaphy with polypropylene mesh causing inguinal vasal obstruction: a preventable cause of obstructive azoospermia. Ann Surg. 2005;241(4):553-8, http://dx.doi.org/10.1097/01.sla.0000157318.13975.2a. 38. Baker K, Sabanegh ES Jr. The Role of Microsurgical Reconstruction in the Era of ICSI. In: Sandlow JI (ed). Microsurgery for Fertility Specialists: A Practical Text. New York: Springer Science+Business Media; 2013.p.15366. 39. Belker AM, Thomas AJ, Fuchs EF, Konnak JW, Sharlip ID. Results of 1,469 microsurgical vasectomy reversals by the Vasovasostomy Study Group. J Urol. 1991;145(3):505-11. 40. Boorjian S, Lipkin M, Goldstein M. The impact of obstructive interval and sperm granuloma on outcome of vasectomy reversal. J Urol. 2004;171(1):304-6. 41. Magheli A, Rais-Bahrami S, Kempkensteffen C, Weiske WH, Miller K, Hinz S. Impact of obstructive interval and sperm granuloma on patency and pregnancy after vasectomy reversal. Int J Androl. 2010;33(5):730-5. 42. Kolettis PN, Sabanegh ES, D’amico AM, Box L, Sebesta M, Burns JR. Outcomes for vasectomy reversal performed after obstructive intervals of at least 10 years. Urology. 2002;60(5):885-8, http://dx.doi.org/10. 1016/S0090-4295(02)01888-5. 43. Paick J-S, Park JY, Park DW, Park K, Son H, Kim SW. Microsurgical vasovasostomy after failed vasovasostomy. J Urol. 2003;169(3):1052-5. 44. Gerrard ER, Sandlow JI, Oster RA, Burns JR, Box LC, Kolettis PN. Effect of female partner age on pregnancy rates after vasectomy reversal. Fertil Steril. 2007;87(6):1340-4, http://dx.doi.org/10.1016/j.fertnstert.2006.11. 038. 45. Kolettis PN, Sabanegh ES, Nalesnik JG, D’Amico AM, Box LC, Burns JR. Pregnancy outcomes after vasectomy reversal for female partners 35 years old or older. J Urol. 2003;169(6):2250-2. 46. Hinz S, Rais-Bahrami S, Kempkensteffen C, Weiske WH, Schrader M, Magheli A. Fertility rates following vasectomy reversal: importance of age of the female partner. Urol Int. 2008;81(4):416-20, http://dx.doi.org/ 10.1159/000167839. 47. Parekattil SJ, Kuang W, Agarwal A, Thomas AJ. Model to predict if a vasoepididymostomy will be required for vasectomy reversal. J Urol. 2005;173(5):1681-4. 48. Chan PTK, Goldstein M. Superior outcomes of microsurgical vasectomy reversal in men with the same female partners. Fertil Steril. 2004;81(5):1371-4, http://dx.doi.org/10.1016/j.fertnstert.2003.09.066.
reconstructions. CBAVD and obstruction due to tuberculosis should be treated with sperm retrieval.
& AUTHOR CONTRIBUTIONS Baker K authored the outline, researched relevant articles and co-authored the manuscript. Sabanegh E conceptualized the manuscript, researched relevant articles, and co-authored and revised the manuscript. Both authors attest that they participated sufficiently in the work to take public responsibility for authorship.
& REFERENCES 1. Aziz N, Agarwal A, Nallella KP, Thomas AJ Jr. Relationship between epidemiological features and aetiology of male infertility as diagnosed by a comprehensive infertility service provider. Reprod Biomed. 2006;12(2):209-14, http://dx.doi.org/10.1016/S1472-6483(10)60863-2. 2. Jequier AM. Obstructive azoospermia: a study of 102 patients. Clin Reprod Fertil. 1985;3(1):21-36. 3. Kim ED, Winkel E, Orejuela F, Lipshultz LI. Pathological epididymal obstruction unrelated to vasectomy: results with microsurgical reconstruction. J Urol. 1998;160(6 Pt 1):2078-80. 4. Chan PTK, Brandell RA, Goldstein M. Prospective analysis of outcomes after microsurgical intussusception vasoepididymostomy. BJU Int. 2005;96(4):598-601, http://dx.doi.org/10.1111/j.1464-410X.2005.05691.x. 5. Pavlovich CP, Schlegel PN. Fertility options after vasectomy: a costeffectiveness analysis. Fertil Steril. 1997;67(1):133-41, http://dx.doi.org/ 10.1016/S0015-0282(97)81870-5. 6. Donovan JF Jr, DiBaise M, Sparks AE, Kessler J, Sandlow JI. Comparison of microscopic epididymal sperm aspiration and intracytoplasmic sperm injection/in-vitro fertilization with repeat microscopic reconstruction following vasectomy: is second attempt vas reversal worth the effort? Hum Reprod. 1998;13(2):387-93, http://dx.doi.org/10.1093/humrep/13. 2.387. 7. Kolettis PN, Thomas AJ Jr. Vasoepididymostomy for vasectomy reversal: a critical assessment in the era of intracytoplasmic sperm injection. J Urol. 1997;158(2):467-70. 8. Lee R, Li PS, Goldstein M, Tanrikut C, Schattman G, Schlegel PN. A decision analysis of treatments for obstructive azoospermia. Hum Reprod. 2008;23(9):2043-9, http://dx.doi.org/10.1093/humrep/den200. 9. Clinic Summary Report [Internet]. [cited 2011 Sep 15]; Available from: https:// www.sartcorsonline.com/rptCSR_PublicMultYear.aspx?ClinicPKID = 0. 10. Yurdakul T, Gokce G, Kilic O, Piskin MM. Transurethral resection of ejaculatory ducts in the treatment of complete ejaculatory duct obstruction. Int Urol Nephrol. 2008;40(2):369-72, http://dx.doi.org/10. 1007/s11255-007-9273-z. 11. Xu B, Niu X, Wang Z, Li P, Qin C, Li J, et al. Novel methods for the diagnosis and treatment of ejaculatory duct obstruction. BJU Int. 2011;108(2):263-6, http://dx.doi.org/10.1111/j.1464-410X.2010.09775.x. 12. Wang H, Ye H, Xu C, Liu Z, Gao X, Hou J, et al. Transurethral Seminal Vesiculoscopy Using a 6F Vesiculoscope for Ejaculatory Duct Obstruction. J Androl. 2012;33(4):637-43. 13. Kuligowska E, Fenlon HM. Transrectal US in male infertility: spectrum of findings and role in patient care. Radiology. 1998;207(1):173-81. 14. Daudin M, Bieth E, Bujan L, Massat G, Pontonnier F, Mieusset R. Congenital bilateral absence of the vas deferens: clinical characteristics, biological parameters, cystic fibrosis transmembrane conductance regulator gene mutations, and implications for genetic counseling. Fertil Steril. 2000;74(6):1164-74, http://dx.doi.org/10.1016/S0015-0282 (00)01625-3. 15. Raviv G, Mor Y, Levron J, Shefi S, Zilberman D, Ramon J, et al. Role of transrectal ultrasonography in the evaluation of azoospermic men with low-volume ejaculate. J Ultrasound Med. 2006;25(7):825-9. 16. Anger JT, Goldstein M. Intravasal ‘‘toothpaste’’ in men with obstructive azoospermia is derived from vasal epithelium, not sperm. J Urol. 2004;172(2):634-6. 17. Arya AK, Beer HL, Benton J, Lewis-Jones I, Swift AC. Does Young’s syndrome exist? J Laryngol Otol. 2009;123(5):477-81, http://dx.doi.org/ 10.1017/S0022215109004307. 18. Hendry WF, A’Hern RP, Cole PJ. Was Young’s syndrome caused by exposure to mercury in childhood? BMJ. 1993;307(6919):1579-82, http:// dx.doi.org/10.1136/bmj.307.6919.1579. 19. Ichioka K, Kohei N, Okubo K, Nishiyama H, Terai A. Obstructive azoospermia associated with chronic sinopulmonary infection and situs inversus totalis. Urology. 2006;68(1):204.e5-7, http://dx.doi.org/10. 1016/j.urology.2006.01.072. 20. Shiraishi K, Ono N, Eguchi S, Mohri J, Kamiryo Y, Takihara H. Young’s syndrome associated with situs inversus totalis. Arch Androl. 2004;50(3):169-72, http://dx.doi.org/10.1080/01485010490425511. 21. Hirsh A, Williams C, Williamson B. Young’s syndrome and cystic fibrosis mutation delta F508. Lancet. 1990;342(8863):118.
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49. Hernandez J, Sabanegh ES. Repeat vasectomy reversal after initial failure: overall results and predictors for success. J Urol. 1999;161(4):1153-6. 50. Kim SW, Ku JH, Park K, Son H, Paick J-S. A different female partner does not affect the success of second vasectomy reversal. J Androl. 2005;26(1):48-52. 51. Bolduc S, Fischer MA, Deceuninck G, Thabet M. Factors predicting overall success: a review of 747 microsurgical vasovasostomies. Can Urol Assoc J. 2007;1(4):388-94. 52. Hinz S, Rais-Bahrami S, Weiske WH, Kempkensteffen C, Schrader M, Miller K, et al. Prognostic value of intraoperative parameters observed during vasectomy reversal for predicting postoperative vas patency and fertility. World J Urol. 2009;27(6):781-85. 53. Hollingsworth MR, Sandlow JI, Schrepferman CG, Brannigan RE, Kolettis PN. Repeat vasectomy reversal yields high success rates. Fertil Steril. 2007;88(1):217-9, http://dx.doi.org/10.1016/j.fertnstert.2006.11.077. 54. Pasqualotto FF, Agarwal A, Srivastava M, Nelson DR, Thomas AJ. Fertility outcome after repeat vasoepididymostomy. J Urol. 1999; 162(5):1626-8. 55. Jee SH, Hong YK. One-layer vasovasostomy: microsurgical versus loupeassisted. Fertil Steril. 2010;94(6):2308-11, http://dx.doi.org/10.1016/j. fertnstert.2009.12.013. 56. Dewire DM, Lawson RK. Experience with macroscopic vasectomy reversal at the Medical College of Wisconsin. Wis Med J. 1994; 93(3):107-9. 57. Lee L, McLoughlin MG. Vasovasostomy: a comparison of macroscopic and microscopic techniques at one institution. Fertil Steril. 1980;33(1):54-5. 58. Parekattil SJ, Atalah HN, Cohen MS. Video technique for human robotassisted microsurgical vasovasostomy. J Endourol. 2010;24(4):511-4, http://dx.doi.org/10.1089/end.2009.0235. 59. Parekattil SJ, Cohen MS. Robotic microsurgery 2011: male infertility, chronic testicular pain, postvasectomy pain, sports hernia pain and
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REVIEW
Medical management of non-obstructive azoospermia Rajeev Kumar All India Institute of Medical Sciences, Department of Urology, New Delhi, India.
Non-obstructive azoospermia is diagnosed in approximately 10% of infertile men. It represents a failure of spermatogenesis within the testis and, from a management standpoint, is due to either a lack of appropriate stimulation by gonadotropins or an intrinsic testicular impairment. The former category of patients has hypogonadotropic hypogonadism and benefits from specific hormonal therapy. These men show a remarkable recovery of spermatogenic function with exogenously administered gonadotropins or gonadotropin-releasing hormone. This category of patients also includes some individuals whose spermatogenic potential has been suppressed by excess androgens or steroids, and they also benefit from medical management. The other, larger category of non-obstructive azoospermia consists of men with an intrinsic testicular impairment where empirical medical therapy yields little benefit. The primary role of medical management in these men is to improve the quantity and quality of sperm retrieved from their testis for in vitro fertilization. Gonadotropins and aromatase inhibitors show promise in achieving this end point. KEYWORDS: Infertility; Drug Therapy; Gonadotropins; Hypogonadism; Aromatase Inhibitors. Kumar R. Medical management of non-obstructive azoospermia. Clinics. 2013;68(S1):75-79. Received for publication on February 29, 2012; Accepted for publication on March 2, 2012 E-mail: rajeev02@gmail.com Tel.: +91-11-26594884
hormone administration, severe febrile illnesses, chemotherapy/radiation or prolonged antibiotic use. Hormone analysis forms the cornerstone of the further evaluation and management of NOA and serves two important functions. The first function is to identify a distinct subset of men who have hypogonadotropism (low FSH), in which azoospermia results from an inadequate stimulation of the testis by gonadotropins. The inherent spermatogenic potential of the testis may be partially recoverable, and the management and prognosis of infertility in these men differ from all other subsets. The second function is to predict the success of medical therapy and of surgical sperm retrieval. Based on these initial hormone studies, the two broad categories are hypogonadotropic hypogonadism and hypergonadotropic hypogonadism or eugonadism (Table 1). There is considerable overlap in the hormone statuses of men who do not have hypogonadotropism, with similar etiologies producing a spectrum of hormonal changes. The American Urological Association recommends an estimation of serum FSH and testosterone as the initial hormonal assessment (1). However, endocrine abnormalities are a rare cause of male infertility and account for less than 3% of all cases. Additional hormone analysis, including luteinizing hormone (LH), estradiol and prolactin evaluations, is performed based on the likelihood of their abnormality and potential impact on management.
& INTRODUCTION Non-obstructive azoospermia (NOA) is generally considered a non-medically manageable cause of male infertility. These patients, who constitute up to 10% of all infertile men, have abnormal spermatogenesis as the cause of their azoospermia. The establishment of in vitro fertilization using intracytoplasmic sperm injection (ICSI) as a standard treatment modality has resulted in a number of these men successfully fathering a child through surgically retrieved sperm from the testis. The challenge, however, is to improve their spermatogenic function to enable the appearance of sperm in their ejaculate or to improve the chances of a successful retrieval from the testis for ICSI. The initial evaluation aims at resolving the following issues: (1) confirming azoospermia, (2) differentiating obstructive from non-obstructive etiology, (3) assessing for the presence of reversible factors and (4) evaluating for the presence of genetic abnormalities. An elevated folliclestimulating hormone (FSH) level or an absence of normal spermatogenesis by testicular histology in the presence of azoospermia is generally considered sufficient evidence of a non-obstructive etiology. The most common reversible factors that need to be ruled out include recent exogenous
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
& HYPOGONADOTROPIC HYPOGONADISM Hypogonadotropic hypogonadism (HH) is a condition of low serum testosterone due to a decrease in the secretion of FSH and LH from the pituitary gland. HH may be congenital, acquired or idiopathic. The congenital forms
No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)08
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patients with intact pituitary glands. GnRH can be administered subcutaneously, intravenously or intranasally, but the 2-hour dosing regimen makes the intravenous and intranasal routes impractical. The standard method is 5-20 micrograms administered every 2 hours subcutaneously. In a large cohort of men with idiopathic hypogonadotropic hypogonadism (IHH), a bihourly pulsatile administration of GnRH subcutaneously through an infusion pump for 12-24 months resulted in the induction of spermatogenesis in 77% of initially azoospermic men (9). The authors reported a more rapid return of spermatogenesis in men who had achieved at least partial puberty at the time of initiating GnRH therapy, with all such subjects responding within six months. GnRH therapy reliably corrected the hypogonadism, with a reversal of azoospermia in a majority of the men, but a subset of men may exhibit pituitary resistance or atypical response (10). Other studies have also reported the successful initiation of pregnancy using GnRH therapy (11,12). Pulsatile GnRH therapy is more cumbersome and expensive than gonadotropin therapy, and this precludes its routine use. However, in men with IHH who fail to respond to gonadotropin therapy, GnRH therapy may be an option. GnRH therapy is also not possible in men who do not have functional pituitary glands. Congenital abnormalities are likely to have been noticed early during adolescence, and a number of these men would have received testosterone therapy near the onset of puberty to induce the development of secondary sexual characteristics. This prior treatment with testosterone at the time of puberty does not interfere with the response to gonadotropins when attempting a pregnancy. A number of these men will produce sperm in their ejaculate but with total counts below the reference range. However, these men are often able to initiate a pregnancy with these low counts (13). Therapeutic interventions may be associated with a significant delay in the appearance of sperm or achieving a sufficient quantity or quality to imitate a spontaneous pregnancy, and some men may benefit from assisted reproduction techniques if an adequate response is not observed at the end of one year (14,15).
Table 1 - Non-obstructive azoospermia classification Hypogonadotropic hypogonadism
NN N
Low FSH, Low LH, Low testosterone Congenital: Kallmann syndrome (hypothalamic GnRH deficiency) Acquired: Pituitary tumors
Hypergonadotropic hypogonadism/eugonadism
NN NN NN NN
High/normal FSH, Normal/high LH, Normal/low testosterone Congenital: Genetic abnormalities (Chromosomal) Acquired: Varicocele Orchitis Gonadotoxins (chemotherapy/radiation) Trauma/torsion Idiopathic
are classically syndromic, such as Kallmann syndrome, Prader-Willi syndrome and Laurence-Moon syndrome. Acquired HH usually results from the destruction of normal pituitary function following radiotherapy, trauma or a pituitary tumor. Another form of acquired HH is due to excess exogenous steroids or androgens. Hyperprolactinemia may also cause infertility by inhibiting the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) and also through a direct inhibition of the binding of LH to the Leydig cells in the testis. Congenital hypogonadotropic hypogonadism typically occurs in association with anosmia and is known as Kallmann syndrome. It has varying genetic modes of transmission, including via both sex chromosomal (Xlinked) and autosomal abnormalities. Hypogonadism results in abnormal secondary sexual characteristics, including gynecomastia, cryptorchidism and micropenis. The primary abnormality is decreased GnRH secretion from the hypothalamus with consequent low levels of gonadotropins (FSH and LH) from the pituitary. Hypogonadotropic hypogonadism is one of the few causes of NOA that have shown a consistent response to medical management (2,3). Gonadotropin therapy is begun at the time the patient wishes to father a child, and three to six months of treatment are usually sufficient to induce spermatogenesis (4,5). Therapy is initiated with human chorionic gonadotropin (hCG) at 2,000 IU subcutaneously three times per week or 2,500 IU twice a week and supplemented with FSH (menopausal, purified or recombinant) at 37.5-150 IU three times a week after three to six months. hCG is sufficient to initiate spermatogenesis, but FSH is required to complete the spermiogenesis, particularly in patients with congenital abnormalities. In one of the largest reported series, Burgues et al. (6) evaluated self-administered highly purified follicle-stimulating hormone in 60 men with hypogonadotropic hypogonadism who showed an adequate response to an initial hCG therapy. Subjects self-administered 150 IU of FSH three times per week and 2,500 IU of hCG twice a week for at least six months, with non-responders continuing treatment for a further period. By the end of the treatment, 80% of these azoospermic/aspermic men had sperm in their ejaculate, with a median time to sperm presence of five months. Similar data reported from other studies support the specific role of hormone replacement therapy in these men (7,8). An alternative method for treating hypogonadotropic hypogonadism is with a pulsatile injection of GnRH in
Androgen and steroid excess Androgen excess through exogenous administration or pituitary/adrenal or testicular tumors can also lead to the suppression of spermatogenesis and NOA that is amenable to medical management. This is a form of hypogonadotropic hypogonadism, where the excess androgens produce feedback inhibition on normal pulsatile gonadotropin secretion, and suppression of excess steroids results in the recovery of spermatogenesis. Congenital adrenal hyperplasia is a diagnosis of childhood. However, a number of these men will present as adults with infertility. Infertility is particularly common in the presence of associated testicular adrenal rest tumors (TARTs). Exogenous steroid administration results in suppression of adrenocorticotropic hormone (ACTH) with a consequent decrease in the size of the TARTs and the possible return of fertility (16,17). Devoto et al. (18) reported a 29-yearold male with azoospermia and congenital adrenal hyperplasia with a 21-hydroxylase deficiency who had normal serum hormones but a testicular biopsy suggesting hypogonadism. The patient was treated with exogenous corticosteroids that resulted in normal testicular development, improvement in sperm counts and spontaneous paternity
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significant improvements in T/E ratios. However, while the oligospermic men demonstrated significant improvements in total sperm counts and motility, none of the 12 azoospermic men who completed three months of treatment showed any return of sperm in the ejaculate. In a direct comparison between testolactone and anastrozole, Raman and Schlegel treated 140 men with low T/E ratios with testolactone and/or anastrozole (23). Some men received both therapies sequentially; they overlapped in the study population. Ultimately, 74 men received 50 to 100 mg of testolactone twice daily for a mean of six months, and 104 men received 1 mg of anastrozole daily for a mean of 4.7 months. The 12 azoospermic men did not exhibit any improvement. Similar to their experience with testolactone, while the T/E ratios improved in all men, none of the 14 azoospermic men experienced a return of sperm to the ejaculate. These studies argue against the use of aromatase inhibitors in NOA men. However, there may be a potential role in improving the quality or quantity of testicular sperm, thus improving the outcomes of sperm retrieval using testicular sperm extraction (TESE) and ICSI. Schiff et al. reported their experience with pretreatment with testolactone, hCG and anastrozole in men with non-mosaic Klinefelter’s syndrome prior to TESE/ICSI (24). Among their cohort of 42 men, 36 had low pre-treatment levels of testosterone. These men received either an aromatase inhibitor alone or in combination with hCG until the testosterone or T/E ratio normalized. TESE was successful in all men who received either anastrozole alone or in combination with hCG, while it succeeded in 74% of those receiving only testolactone and 54% of those receiving testolactone and hCG. The authors did not have a control group, and it is difficult to assess the contribution of the pretreatment on the successful outcomes, particularly because all six men who did not require pretreatment had successful outcomes. However, the success rates in these men were higher than historical controls. In a more recent update of their data, this group identified no significant impact of the pretreatment on a successful outcome (25). However, they did note an improved outcome if the pretreatment resulted in an improved serum testosterone level prior to performing the TESE.
on two occasions. The authors cautioned that hormone analysis in these men might not suggest hypogonadism because the excess testosterone production from the adrenals masks the intra-testicular hypogonadism. A reversal of azoospermia may depend on the type of steroid used and its dose. Claahsen-van der Grinten (19) noted inadequate suppression with 30 mg of hydrocortisone but a significant improvement after changing to an equivalent dose of dexamethasone in a patient who had azoospermia but went on to father two children through spontaneous conception after this change. These outcomes have been supported by other reports on the reversal of azoospermia with exogenous dexamethasone treatment (20,21). Excess androgens present in anabolic steroids or testosterone itself are potent causes for the suppression of endogenous hormone production. The initial step in the management of these men is the cessation of the use of all such hormones. It may take a year or longer for endogenous production to return to normal after the cessation of exogenous therapy. Spermatogenesis may then be spontaneously restored, or there may then be evidence of additional endocrine abnormalities that will need to be addressed.
& HYPERGONADOTROPIC HYPOGONADISM/ EUGONADISM Aromatase inhibitors High intra-testicular levels of testosterone are necessary for spermatogenesis. An imbalance between the circulating testosterone and estrogen levels has been investigated as a potential therapeutic target in men with NOA. The enzyme aromatase, which is present in the adipose tissue, liver, testis and skin, is responsible for converting testosterone and other androgens to estradiol in men. Estradiol suppresses pituitary LH and FSH secretion and also directly inhibits testosterone biosynthesis. This results in an imbalance in the testosterone and estradiol (T/E) ratio, which may be reversible. Aromatase inhibitors have the potential to block the conversion of androgens to estradiol. The two types of aromatase inhibitors are steroidal (testolactone) and non-steroidal (anastrozole, letrozole). Both of these groups of agents have been studied for potential therapeutic roles in NOA. In 2001, Pavlovich et al. reported one of the first studies on the potential role of low-dose oral testolactone in men with NOA or severe oligospermia (22), in which 63 men (NOA or sperm density below 10 million/mL) were evaluated. This study included 43 men with NOA that was documented by a testis biopsy. Data from these men were compared with those from 40 age-matched fertile controls. All men underwent a baseline hormone profile, and 45 out of 63 men had a testosterone-to-estradiol ratio below the 20th percentile of normal, as determined from the healthy controls. These 45 men included patients with Klinefelter’s syndrome, chromosomal anomalies, varicoceles, etc. as the cause for NOA. Subjects received 50 to 100 mg of oral testolactone twice a day for a mean of five months. Hormone profiles were obtained at one month of therapy, and the dose of testolactone was increased from 50 to 100 mg if the ratios remained low. A semen analysis was performed after at least three months of stable drug therapy. The authors reported a decline in estradiol or an increase in testosterone with drug therapy in all patients, with
Antiestrogens Clomiphene citrate and tamoxifen are non-steroidal antiestrogens that have long been used as empirical options in the management of idiopathic oligospermia. Clomiphene blocks the feedback inhibition of estrogen on the pituitary, resulting in increased FSH and LH secretion and a consequent rise in testosterone. There is little scientific evidence in favor of using clomiphene empirically. However, it continues to be a popular choice and has been used in men with NOA. Hussein et al. treated 42 men with NOA and either hypospermatogenesis or maturation arrest based on a biopsy with clomiphene citrate, which was initially 50 mg every alternate day for two weeks and then increased serially until the testosterone levels reached between 600-800 ng/dL (26). Semen analysis was performed at regular intervals, and if azoospermia persisted beyond six months of treatment, a testis biopsy was performed. The results revealed that 64% of the men experienced a return of sperm in their ejaculate, with sperm density ranging from 1-16 million/mL. Among those who remained azoospermic, sperm retrieval for ICSI was successful
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in all cases. Of note, the authors excluded all men with Sertoli cell-only syndrome or a low testicular volume and a mean FSH at baseline of 7.21 mIU/mL. Further, this remains the only series on the successful use of clomiphene in azoospermic men, and its results have not been replicated by other investigators.
a remarkable improvement in all subjects but was hampered by a poorly documented methodology (33). Among men with NOA, gonadotropin therapy for hypogonadotropic hypogonadism is the only specific indication that has universally shown an improvement in semen analysis and pregnancy rates. Gonadotropins (hCG and rFSH) in combination constitute a standard therapy, with GnRH therapy reserved for non-responders. The medical management of other forms of NOA remains empirical. Drug therapy with aromatase inhibitors and gonadotropins shows potential promise in improving outcomes in men requiring surgical sperm retrieval, but there is lack of level I clinical evidence for this indication.
Gonadotropins The use of gonadotropins to induce the appearance of sperm in the ejaculate or to increase the success rates of TESE in men with NOA has been based on the rationale that high levels of exogenous gonadotropins decrease the endogenous gonadotropin secretion. This in turn allows a ‘‘resetting’’ of the FSH and LH receptors in the Leydig and Sertoli cells, whose functions then improve (27). Selman et al. reported a 32-year-old man with Y chromosome microdeletions who received recombinant FSH and hCG before a successful ICSI procedure with ejaculated sperm that numbered in the few thousands (28). Similarly, Efesoy et al. reported sperm in the ejaculate of 2 of 11 azoospermic men with maturation arrest who were treated with recombinant FSH (29). Shiraishi et al. reviewed their data on repeat TESE in men with NOA who had no sperm retrieved during the first TESE (30). Of 48 such men with no chromosomal anomalies and normal serum testosterone levels, 28 received 5,000 IU of hCG three times a week for three months. The men whose FSH declined significantly received additional recombinant FSH of 150 IU three times a week for two months, while the other men continued to receive hCG until the repeat TESE. Another 20 men in this cohort refused hormonal therapy and underwent a second TESE at a mean of 17 months after the first surgery. None of the 48 men experienced a return of sperm in their ejaculate. However, the second TESE was successful in 21% of men who received hormonal therapy, while it failed in all those who did not. By evaluating a more select group of men with normal baseline hormone profiles, testicular volumes and genetic analyses, Selman et al. treated 49 men with rFSH, which was initially at 75 IU on alternate days for two months, followed by 150 IU for two months and then an additional 2,000 IU of hCG for two months (31). All men remained azoospermic at the end of treatment; however, while none had mature sperm in the pre-treatment testis biopsy, sperm were found in the biopsies of 22% men after treatment. Similarly, Ramasamy et al. reported improved outcomes of primary TESE following gonadotropin therapy in men with NOA and Klinefelter’s syndrome (25).
& REFERENCES 1. American Urological Association. The optimal evaluation of the infertile male. Accessed February 22, 2012. Available from: http://www.auanet. org/content/media/optimalevaluation2010.pdf. 2. Hoffman AR, Crowley WF. Induction of Puberty in Men by Long-Term Pulsatile Administration of Low-Dose Gonadotropin-Releasing Hormone. New Engl J Med. 1982;307(20):1237-41, http://dx.doi.org/10.1056/ NEJM198211113072003. 3. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E. Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone. Science. 1978;202(4368):631-3, http:// dx.doi.org/10.1126/science.100883. 4. Vicari E, Mongioi A, Calogero AE, Moncada ML, Sidoti G, Polosa P, et al. Therapy with human chorionic gonadotrophin alone induces spermatogenesis in men with isolated hypogonadotrophic hypogonadism—longterm follow-up. International journal of andrology. 1992;15(4):320-9, http://dx.doi.org/10.1111/j.1365-2605.1992.tb01131.x. 5. Finkel DM, Phillips JL, Snyder PJ. Stimulation of spermatogenesis by gonadotropins in men with hypogonadotropic hypogonadism. N Engl J Med. 1985;313(11):651-5. 6. Burgues S, Calderon MD. Subcutaneous self-administration of highly purified follicle stimulating hormone and human chorionic gonadotrophin for the treatment of male hypogonadotrophic hypogonadism. Spanish Collaborative Group on Male Hypogonadotropic Hypogonadism. Hum Reprod. 1997;12(5):980-6, http://dx.doi.org/10.1093/humrep/12.5.980. 7. Liu PY, Gebski VJ, Turner L, Conway AJ, Wishart SM, Handelsman DJ. Predicting pregnancy and spermatogenesis by survival analysis during gonadotrophin treatment of gonadotrophin-deficient infertile men. Hum Reprod. 2002;17(3):625-33, http://dx.doi.org/10.1093/humrep/17.3.625. 8. Efficacy and safety of highly purified urinary follicle-stimulating hormone with human chorionic gonadotropin for treating men with isolated hypogonadotropic hypogonadism. European Metrodin HP Study Group. Fertil Steril. 1998;70(2):256-62. 9. Pitteloud N, Hayes FJ, Dwyer A, Boepple PA, Lee H, Crowley WF, Jr. Predictors of outcome of long-term GnRH therapy in men with idiopathic hypogonadotropic hypogonadism. The Journal of clinical endocrinology and metabolism. 2002;87(9):4128-36, http://dx.doi.org/ 10.1210/jc.2002-020518. 10. Sykiotis GP, Hoang XH, Avbelj M, Hayes FJ, Thambundit A, Dwyer A, et al. Congenital idiopathic hypogonadotropic hypogonadism: evidence of defects in the hypothalamus, pituitary, and testes. J Clin Endocrinol Metab. 2010;95(6):3019-27, http://dx.doi.org/10.1210/jc.2009-2582. 11. Buchter D, Behre HM, Kliesch S, Nieschlag E. Pulsatile GnRH or human chorionic gonadotropin human menopausal gonadotropin as effective treatment for men with hypogonadotropic hypogonadism: a review of 42 cases. Eur J Endocrinol. 1998;139(3):298-303. 12. Delemarre-Van de Waal HA. Induction of testicular growth and spermatogenesis by pulsatile, intravenous administration of gonadotrophin-releasing hormone in patients with hypogonadotrophic hypogonadism. Clin Endocrinol (Oxf). 1993;38(5):473-80. 13. Burris AS, Clark RV, Vantman DJ, Sherins RJ. A Low Sperm Concentration Does Not Preclude Fertility in Men with Isolated Hypogonadotropic Hypogonadism after Gonadotropin Therapy. Fertility and sterility. 1988;50(2):343-7. 14. Tachiki H, Ito N, Maruta H, Kumamoto Y, Tsukamoto T. Testicular findings, endocrine features and therapeutic responses of men with acquired hypogonadotropic hypogonadism. International journal of urology : official journal of the Japanese Urological Association. 1998;5(1):80-5, http://dx.doi.org/10.1111/j.1442-2042.1998.tb00244.x. 15. Liu PY, Handelsman DJ. The present and future state of hormonal treatment for male infertility. Human reproduction update. 2003;9(1):923, http://dx.doi.org/10.1093/humupd/dmg002.
Antioxidants The role of antioxidant therapy in male subfertility continues to be debated. While there is a significant amount of data documenting raised oxidative stress and low antioxidant capacity in the seminal plasma of infertile men with abnormal semen parameters, including azoospermia, there is little high-quality evidence in support of using antioxidants in the treatment of these men (32). This is primarily because such therapy has not resulted in significant improvements in pregnancy rates, although statistically significant changes in anti-oxidant capacity and improvements in measured seminal parameters have occurred. Furthermore, most of the existing literature involves men with oligo/asthenospermia and not azoospermia. One of the few studies using antioxidants on azoospermic men reported
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Drug therapy for NOA Kumar R 25. Ramasamy R, Ricci JA, Palermo GD, Gosden LV, Rosenwaks Z, Schlegel PN. Successful fertility treatment for Klinefelter’s syndrome. J Urol. 2009;182(3):1108-13. 26. Hussein A, Ozgok Y, Ross L, Niederberger C. Clomiphene administration for cases of nonobstructive azoospermia: a multicenter study. J Androl. 2005;26(6):787-91; discussion 92-3. 27. Foresta C, Bettella A, Spolaore D, Merico M, Rossato M, Ferlin A. Suppression of the high endogenous levels of plasma FSH in infertile men are associated with improved Sertoli cell function as reflected by elevated levels of plasma inhibin B. Hum Reprod. 2004;19(6):1431-7, http://dx.doi.org/10.1093/humrep/deh255. 28. Selman HA, Cipollone G, Stuppia L, De Santo M, Sterzik K, El-Danasouri I. Gonadotropin treatment of an azoospermic patient with a Ychromosome microdeletion. Fertility and sterility. 2004;82(1):218-9, http://dx.doi.org/10.1016/j.fertnstert.2003.11.055. 29. Efesoy O, Cayan S, Akbay E. The efficacy of recombinant human folliclestimulating hormone in the treatment of various types of male-factor infertility at a single university hospital. J Androl. 2009;30(6):679-84. 30. Shiraishi K, Ohmi C, Shimabukuro T, Matsuyama H. Human chorionic gonadotrophin treatment prior to microdissection testicular sperm extraction in non-obstructive azoospermia. Hum Reprod. 2012;27(2):3319, http://dx.doi.org/10.1093/humrep/der404. 31. Selman H, De Santo M, Sterzik K, Cipollone G, Aragona C, El-Danasouri I. Rescue of spermatogenesis arrest in azoospermic men after long-term gonadotropin treatment. Fertil Steril. 2006;86(2):466-8, http://dx.doi. org/10.1016/j.fertnstert.2005.12.055. 32. Esteves SC, Agarwal A. Novel concepts in male infertility. Int Braz J Urol. 2011;37(1):5-15. 33. Singh AK, Tiwari AK, Singh PB, Dwivedi US, Trivedi S, Singh SK, et al. Multivitamin and micronutrient treatment improves semen parameters of azoospermic patients with maturation arrest. Indian J Physiol Pharmacol. 2010;54(2):157-63.
16. Augarten A, Weissenberg R, Pariente C, Sack J. Reversible male infertility in late onset congenital adrenal hyperplasia. J Endocrinol Invest. 1991;14(3):237-40. 17. Tiitinen A, Valimaki M. Primary infertility in 45-year-old man with untreated 21-hydroxylase deficiency: successful outcome with glucocorticoid therapy. The Journal of clinical endocrinology and metabolism. 2002;87(6):2442-5, http://dx.doi.org/10.1210/jc.87.6.2442. 18. Devoto CE, Madariaga AM, Fernandez W. [Congenital adrenal hyperplasia causing male infertility. Report of one case]. Revista medica de Chile. 2011;139(8):1060-5, http://dx.doi.org/10.4067/S0034-98872011000800012. 19. Claahsen-van der Grinten HL, Otten BJ, Sweep FC, Hermus AR. Repeated successful induction of fertility after replacing hydrocortisone with dexamethasone in a patient with congenital adrenal hyperplasia and testicular adrenal rest tumors. Fertility and sterility. 2007;88(3):705 e5-8. 20. Nicopoullos JD, Ramsay JW, Cassar J. From zero to one hundred million in six months: the treatment of azoospermia in congenital adrenal hyperplasia. Archives of andrology. 2003;49(4):257-63, http://dx.doi. org/10.1080/713828165. 21. Iwamoto T, Yajima M, Tanaka H, Minagawa N, Osada T. [A case report: reversible male infertility due to congenital adrenal hyperplasia]. Nihon Hinyokika Gakkai zasshi The japanese journal of urology. 1993;84(11): 2031-4. 22. Pavlovich CP, King P, Goldstein M, Schlegel PN. Evidence of a treatable endocrinopathy in infertile men. J Urol. 2001;165(3):837-41. 23. Raman JD, Schlegel PN. Aromatase inhibitors for male infertility. J Urol. 2002;167(2 Pt 1):624-9. 24. Schiff JD, Palermo GD, Veeck LL, Goldstein M, Rosenwaks Z, Schlegel PN. Success of testicular sperm extraction [corrected] and intracytoplasmic sperm injection in men with Klinefelter syndrome. J Clin Endocrinol Metab. 2005;90(11):6263-7, http://dx.doi.org/10.1210/jc.2004-2322.
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REVIEW
Hypogonadotropic Hypogonadism Revisited Renato Fraietta,I Daniel Suslik Zylberstejn,I Sandro C. EstevesII I Division of Urology, Department of Surgery, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo/SP, Brazil. Reproduction Clinic, Campinas, Sa˜o Paulo/SP, Brazil.
II
ANDROFERT – Andrology & Human
Impaired testicular function, i.e., hypogonadism, can result from a primary testicular disorder (hypergonadotropic) or occur secondary to hypothalamic-pituitary dysfunction (hypogonadotropic). Hypogonadotropic hypogonadism can be congenital or acquired. Congenital hypogonadotropic hypogonadism is divided into anosmic hypogonadotropic hypogonadism (Kallmann syndrome) and congenital normosmic isolated hypogonadotropic hypogonadism (idiopathic hypogonadotropic hypogonadism). The incidence of congenital hypogonadotropic hypogonadism is approximately 1-10:100,000 live births, and approximately 2/3 and 1/3 of cases are caused by Kallmann syndrome (KS) and idiopathic hypogonadotropic hypogonadism, respectively. Acquired hypogonadotropic hypogonadism can be caused by drugs, infiltrative or infectious pituitary lesions, hyperprolactinemia, encephalic trauma, pituitary/brain radiation, exhausting exercise, abusive alcohol or illicit drug intake, and systemic diseases such as hemochromatosis, sarcoidosis and histiocytosis X. The clinical characteristics of hypogonadotropic hypogonadism are androgen deficiency and a lack/delay/stop of pubertal sexual maturation. Low blood testosterone levels and low pituitary hormone levels confirm the hypogonadotropic hypogonadism diagnosis. A prolonged stimulated intravenous GnRH test can be useful. In Kallmann syndrome, cerebral MRI can show an anomalous morphology or even absence of the olfactory bulb. Therapy for hypogonadotropic hypogonadism depends on the patient’s desire for future fertility. Hormone replacement with testosterone is the classic treatment for hypogonadism. Androgen replacement is indicated for men who already have children or have no desire to induce pregnancy, and testosterone therapy is used to reverse the symptoms and signs of hypogonadism. Conversely, GnRH or gonadotropin therapies are the best options for men wishing to have children. Hypogonadotropic hypogonadism is one of the rare conditions in which specific medical treatment can reverse infertility. When an unassisted pregnancy is not achieved, assisted reproductive techniques ranging from intrauterine insemination to in vitro fertilization to the acquisition of viable sperm from the ejaculate or directly from the testes through testicular sperm extraction or testicular microdissection can also be used, depending on the woman’s potential for pregnancy and the quality and quantity of the sperm. KEYWORDS: Male Infertility; Hypogonadism; Endocrine System Abnormalities; Azoospermia; Review. Fraietta R, Zylberstejn DS, Esteves SC. Hypogonadotropic Hypogonadism Revisited. Clinics. 2013;68(S1):81-88. Received for publication on June 20, 2012; Accepted for publication on June 25, 2012 E-mail: fraietta@uol.com.br Tel.: 55 11 55764488
gonadotropin releasing hormone (GnRH). The effect of LH and FSH on germ cell development is mediated by the androgen and FSH receptors that are present on Leydig and Sertoli cells, respectively. Whereas FSH acts directly on the germinal epithelium, LH stimulates the secretion of testosterone by Leydig cells. Testosterone stimulates sperm production and virilization, in addition to providing feedback to the hypothalamus and pituitary to regulate GnRH secretion. FSH stimulates Sertoli cells to support spermatogenesis and secrete inhibin B, which negatively regulates FSH secretion. The GnRH pulse generator is the main regulator of puberty, and the production of GnRH starts early in fetal life. As a result, gonadotropin levels change drastically during fetal development, childhood, puberty and adulthood. Male infants exhibit what is called a ‘‘window period’’ during the first six months of life, during which gonadal function can be clinically detected in response to gonadotropin stimulation. After that period, serum gonadotropin levels drop and can only be detected again with the onset of puberty (1).
& INTRODUCTION The hypothalamus, the pituitary, and the testes form an integrated system that is responsible for the adequate secretion of male hormones and normal spermatogenesis. The endocrine components of the male reproductive system are integrated in a classic endocrine feedback loop. The testes require stimulation by the pituitary gonadotropins, i.e., luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are secreted in response to hypothalamic
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)09
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Figure 1 - A schematic representation of the components of the hypothalamic-pituitary-testicular axis and the endocrine regulation of spermatogenesis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are secreted by the pituitary in response to hypothalamic gonadotropin releasing hormone (GnRH). Whereas FSH acts directly on the germinal epithelium, LH stimulates the secretion of testosterone from Leydig cells. Testosterone stimulates sperm production and also feeds back to the hypothalamus and pituitary to regulate GnRH secretion. FSH stimulates Sertoli cells to support spermatogenesis and secrete inhibin B, which negatively regulates FSH secretion. 82
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Hypogonadotropic hypogonadism and fertility Fraietta R et al.
This paper aims to review the causes of hypogonadotropic hypogonadism, the implications of this condition depending on the period in which it occurs and the different types of treatment available.
that plays a possible role in orchestrating GnRH neuron adhesion and axonal migration. Most KAL-1 mutations are nucleotide insertions or deletions that result in frame shift mutations or a premature stop codon. Mutations in this gene lead to a GnRH migration and olfactory neuron disorder (8). The failure of GnRH neurons to migrate from the olfactory placode to their destination in the hypothalamus and olfactory lobe represents the basic embryological defect of this syndrome. The KAL-1 gene accounts for the Xlinked recessive mode of inheritance of familial KS and 1020% of all KS cases. The FGR1, GNRHR, NELF, GPRS54, PROK-2, PROKR-2, CHD-7 and FGF-8 genes have also been linked to this syndrome. These genes can act alone or in combination, and mutations in all of these genes lead to impaired GnRH production (9). Some genetic diseases, such as Prader-Willi syndrome, Laurence-Moon-Biedl syndrome, and Moebius syndrome, are also related to HH (6). IHH is characterized by low levels of gonadotropins and sex steroids in the absence of anatomical or functional abnormalities of the hypothalamic–pituitary–gonadal axis. The pathogenetic mechanism of IHH involves the failure of GnRH neurons in the hypothalamus to differentiate or develop, which results in a lack of GnRH secretion or apulsatile GnRH secretion. Acquired hypogonadotropic hypogonadism can be caused by drugs (e.g., sex steroids and gonadotropinreleasing hormone analogues), infiltrative or infectious pituitary lesions, hyperprolactinemia, encephalic trauma, pituitary/brain radiation, exhausting exercise, and abusive alcohol or illicit drug intake (3). Systemic diseases such as hemochromatosis, sarcoidosis and histiocytosis X are also associated with HH (6). The major causes of HH are listed in Table 1 (3).
& DEFINITIONS AND PREVALENCE Male hypogonadism is characterized by impaired testicular function, which can affect spermatogenesis and/or testosterone synthesis. Although it is a common endocrine disorder, the exact prevalence of this disease is unknown. Male hypogonadism can result from a primary testicular disorder or occur secondary to hypothalamic-pituitary dysfunction. Hypergonadotropic hypogonadism is also known as primary hypogonadism and is the most frequent form of hypogonadism found in adult men. The Massachusetts Male Aging Study (MMAS) reported a crude incidence rate of 12.3 cases per 1,000 individuals per year, leading to an estimated prevalence of 481,000 new cases of late-onset hypogonadism (LOH) per year in American men 40 to 69 years of age (2). The symptoms of this disorder can include decreased libido, impaired erectile function, muscle weakness, increased adiposity, depressed mood, and decreased vitality. Primary hypogonadism is characterized by low testosterone production and elevated levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (3). Klinefelter’s syndrome is the most common congenital manifestation of primary hypogonadism and affects approximately one in every 500 men. The acquired forms of hypergonadotropic hypogonadism include male aging, which affects approximately 4.1% of men between the ages of 40-49 years and 9.3% of men between the ages of 60-70 years, and exposure to gonadotoxic agents such as those used in chemotherapy and radiotherapy treatments. The latter agents cause gonadal failure by adversely impacting Leydig and Sertoli cell function (4). In contrast to primary hypogonadism, male hypogonadotropic hypogonadism (HH) is a consequence of congenital or acquired diseases that affect the hypothalamus and/or the pituitary gland (3). In HH, secretion of gonadotropin releasing hormone (GnRH) is absent or inadequate. Isolated lack of production or inadequate biosynthesis of pituitary gonadotropins may also result in HH (5). The prevalence of this form of hypogonadism has been estimated to range from 1:10,000 to 1:86,000 individuals (6).
& THE ROLE OF GONADOTROPIN RELEASING HORMONE (GNRH) IN THE PHYSIOLOGICAL REGULATION OF THE HYPOTHALAMICPITUITARY-GONADAL AXIS GnRH is a decapeptide that is synthesized by a loose network of neurons located in the medial basal hypothalamus (MBH) and the arcuate nucleus of the hypothalamus. Some GnRH neurons are found outside the hypothalamus in the olfactory lobe, reflecting their common embryological origin. Developmentally, GnRH neurons originate from the olfactory placode/vomeronasal organ of the olfactory system and migrate along the vomeronasal nerves to the
& ETIOLOGY Table 1 - Etiologies of Hypogonadotropic Hypogonadism (3).
HH can be congenital or acquired. Congenital HH is divided in two main subdivisions depending on the presence of an intact sense of smell: anosmic HH (Kallmann syndrome) and congenital normosmic isolated hypogonadotropic hypogonadism (idiopathic HH [IHH]). The incidence of congenital HH is approximately 1-10:100,000 live births, with approximately 2/3 and 1/3 of cases arising from Kallmann syndrome (KS) and idiopathic HH, respectively (7). Congenital HH can have a genetic origin. The KAL1 gene has been linked to KS and is the best-characterized gene related to GnRH deficiency (6). This gene has been mapped to X-chromosome region Xp22.32 and consists of 14 exons. KAL1 encodes the protein anosmin-1, which has a length of 840 amino acids and is an extracellular adhesion protein
Hyperprolactinemia Pituitary lesions (tumor, granuloma, abscess) Cushing syndrome Drug use (opiates, alcohol abuse) Anabolic steroids use Severe or chronic illness Pituitary irradiation, trauma or surgery Iron overload Kallmann syndrome Idiopathic hypogonadotropic hypogonadism Other genetic mutations Prader Willi syndrome Modified from Darby E, Anawalt BD. Male hypogonadism: an update on diagnosis and treatment. Treat Endocrinol. 2005;4(5):293-309.
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hypothalamus, where they extend processes to the median eminence and pituitary gland. GnRH is synthesized as a precursor hormone that contains 92 amino acids and is then cleaved to a prohormone with a length of 69 amino acids. The prohormone is further cleaved in the nerve terminals to form the active decapeptide. GnRH activation is achieved when specific receptors (i.e., the KiSS1-derived peptide receptor, also known as GPR54 or the kisspeptin receptor) are occupied by kisspeptin protein, which is also produced in the hypothalamus. Androgens (testosterone and dihydrotestosterone) and estrogens exert negative feedback by activating specific receptors that are located on the kisspeptin-secreting neurons of the arcuate nucleus. Other substances also influence GnRH secretion. Noradrenaline and leptin have stimulatory effects, whereas prolactin, dopamine, serotonin, gamma-aminobutyric acid (GABA) and interleukin-1 are inhibitory (10). GnRH has a pulsatile secretion and a half-life of approximately 10 minutes, and it is secreted into the hypothalamic-hypophyseal portal blood system, which carries it to the pituitary gland (11). Once secreted, GnRH binds to specific pituitary cell membrane receptors, which results in the production of diacylglycerol and inositol triphosphate, intracellular calcium increase (by mobilization from intracellular stores and extracellular influx) and the activation of protein kinase C. As a consequence, gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]) are released by exocytosis. The GnRHreceptor complex undergoes intracellular degradation; thus, the cell requires some time to replace the receptors, which is reflected by the 60-90 minute interval between GnRH pulses (12). The essential function of GnRH is to stimulate the secretion of LH and FSH from the anterior pituitary gland (Figure 1). LH and FSH are glycoproteins consisting of alpha and beta polypeptide chains (a and b subunits). They have identical alpha subunits but differ in their beta subunit which determines receptor-binding specificity. Once synthesized, LH and FSH are stored in granules in the pituitary gland. GnRH induces exocytosis of the granules and the release of these hormones into the circulation. A low GnRH pulse frequency tends to preferentially release FSH, whereas higher frequencies are associated with preferential secretion of LH (13). Sialization allows FSH to have a longer (2 hours) half-life than LH (20 minutes). FSH and LH target specific membrane receptors whose internalization produces cAMP and protein kinase A. LH initiates male pubertal development by binding to LH receptors on Leydig cells, thereby stimulating the release of testosterone. Sertoli cells have receptors for FSH and testosterone. It is therefore believed that both FSH and testosterone support the initiation of spermatogenesis and that both are necessary for the maintenance of quantitatively normal spermatogenesis. Testosterone or its metabolite dihydrotestosterone binds to androgen receptors on Sertoli cells and then modulates gene transcription. Functional Sertoli cell androgen receptors are required for normal spermatogenesis. Intratesticular testosterone levels are ,50 times higher than serum testosterone levels; therefore, it has been suggested that the androgen receptors in the normal testis are fully saturated (14). FSH, in contrast, binds to FSH receptors on Sertoli cells and initiates signal transduction events that ultimately lead to the production of inhibin B, which is a
marker of Sertoli cell activity. Inhibin B and testosterone in turn regulate pituitary FSH secretion (Figure 1). FSH receptors are expressed in the regions of the seminiferous tubules that are involved in the proliferation of spermatogonia. The dual hormonal dependence of normal spermatogenesis can be appreciated in males with hypogonadotropic hypogonadism. Sperm production is restored to approximately 50% of the normal level with either FSH or hCG (as a surrogate for LH) alone; only the combination of hCG plus FSH leads to full quantitative restoration (15). It has been suggested that testicular function is also regulated by other factors. For instance, Sertoli cells are influenced by factors secreted by the germ cells. Estrogen receptors are found in the efferent ducts, Sertoli cells and most germ cell types. The testes are a major site of estrogen production; however, direct evidence for a role of estrogen in spermatogenesis has not yet been identified. The thyroid hormone receptor is important for Sertoli cell development (16).
& DIAGNOSIS Clinical The clinical characteristics of HH are androgen deficiency and a lack/delay/stop in pubertal sexual maturation. Moreover, hypogonadotropic hypogonadism is considered idiopathic (IHH) when there is an isolated GnRH secretion deficiency in individuals over 18 years of age. Below that age, HH is more properly defined as pubertal delay (1,5). It is important to consider that the first endocrinological change to occur during puberty is an increase in LH, which initially occurs more in amplitude than in frequency and only at night; subsequently, both LH and FSH levels increase at night and during the day until adult levels are reached. It is difficult to differentiate between HH and delayed puberty, as low gonadotropin and testosterone levels are found in both conditions. Therefore, a definitive HH diagnosis must be confirmed only after the patient is 18 years of age (1,17). When the symptoms are associated with anosmia or hyposmia without findings such as harelip and cleft palate, neuro-sensorial deafness, cerebellar ataxia and renal agenesia, a diagnosis of Kallmann syndrome should be confirmed. The appearance of clinical characteristics depends on when HH begins. When GnRH deficiency occurs in the late fetal or early neonatal periods and is caused by a lack of the first intrinsic GnRH peak, which lasts until six months of age, the incidence of cryptorchidism and/or micro penis is high (5). Men presenting with HH that started in the prepubertal phase and was triggered by the intrinsic second GnRH peak exhibit eunuchoid body proportions, a delay in the development of secondary sexual characteristics, a highpitched voice, pre-pubertal testicles, and delayed bone maturation. Men with an initially delayed HH condition present with diminished libido, considerable weight gain, sexual impotence, hot flashes, and infertility. Infertility is one of the most frequent complaints among these patients and has a negative effect on their quality of life (3,6). Table 2 shows the most prevalent symptoms of pre- and postpubertal hypogonadism (3).
Laboratory and Imaging Every hypogonadism diagnosis must start with a confirmation of low blood testosterone levels, preferably the free testosterone level, which is based on the total
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Table 2 - Signs and symptoms of pre- and pos-pubertal hypogonadism (3). Pre-pubertal hypogonadism
Post-pubertal hypogonadism
Eunuchoidal stature Small testes (usually ,6cm3) Small penis (,5cm) Lack of normal scrotal rugae and pigmentation Small prostate Scant facial, axillary and pubic hair High pitched voice Gynecomastia Infertility Lack of libido Low bone mineral density Low muscle mass, high percentage of body fat Mild anemia
Normal stature Testes volume normal to slightly low (.10cm3); soft Penis normal size Normal scrotal rugae and pigmentation Normal prostate Thinning of facial, axillary and pubic hair Normal voice Gynecomastia Infertility Loss of libido Low bone mineral density Low muscle mass, high percentage of body fat Mild anemia Hot flashes Lack of male pattern baldness Decrease sense of well-being Erectile disfunction
Modified from Darby E, Anawalt BD. Male hypogonadism: an update on diagnosis and treatment. Treat Endocrinol. 2005;4(5):293-309.
replacement is indicated for men who already have children or have no desire for children, and testosterone therapy is used to reverse the symptoms and signs of hypogonadism. The presence of androgens has been linked to a good sexual life, with preserved libido and erections. The preservation of muscular strength and lean body mass are related to androgens, as is bone hemostasis, which prevents osteoporosis. Improvements in humor and well-being are generally the first clinical signs mentioned by patients who begin hormone replacement. Conversely, GnRH or gonadotropin therapies are the best options for men who wish to have children. In such cases, the stimulation of sperm production requires treatment with human chorionic gonadotropin (hCG) alone or combined with recombinant FSH, urinary FSH or human menopausal gonadotropins (hMG). In the rare cases of ‘fertile eunuchs’, who produce sufficient FSH but not LH, treatment with hCG alone may be sufficient to stimulate sperm production and achieve normal testosterone levels (18).
testosterone level, albumin level and SHBG level (sexual hormone-binding globulin). The free and bioavailable testosterone level can be calculated from the total testosterone and SHBG levels (http://www.issam.ch/freetesto. htm). These laboratory tests should preferably be performed before 10 am to account for the circadian rhythm of male hormones. If testosterone levels are low, a new test must be performed because of high physiological variations. When low testosterone levels are found, the gonadotropin (FSH and LH) levels must be analyzed. A low pituitary hormone level confirms the HH diagnosis (3,5). HH is characterized as an isolated secretion disorder. However, some of these releasing gonadotropin hormone characteristics make direct laboratory tests impossible, as GnRH is confined within the pituitary portal system and exhibits a short half-life of 2 to 4 minutes. Therefore, GnRH levels are better checked by indirect measures such as the total or partial absence of LH pulses, the regularization of pituitary and gonadal function, a response to exogenous GnRH replacement and hormonal reserve tests, which check the integrity of the hypothalamic-pituitary system’s control mechanism. A prolonged stimulated intravenous GnRH test (100 mcg followed by 500 mcg) can be useful: in hypothalamic GnRH deficiency, LH and FSH gradually appear, whereas hyporesponsiveness occurs in the pituitary cases (5). It is important to note that the diagnosis can only be concluded after magnetic resonance imaging (MRI) is used for pituitary, prolactinoma, and craniopharyngioma tumor exclusion tests (1,6). In Kallmann syndrome, cerebral MRI can show anomalous morphology or the absence of the olfactory bulb, and it therefore plays a pivotal role in presumptive diagnoses.
Treatment for Infertility in Hypogonadotropic Hypogonadism Most hypogonadal young men want to be fertile. Thus, it is important to remember that the fertility of men with hypogonadotropic hypogonadism is only reduced and that fertility may be restored through hormone therapy. Men who present with hypergonadotropic hypogonadism do not respond to hormone medication because the disorder is caused by primary testicular failure (3,4). In contrast, in secondary hypogonadism, the Sertoli cells are deprived of the stimulus necessary for spermatogenesis. Nevertheless, it is important to remember that high intra-testicular testosterone levels are necessary for spermatogenesis (4). The fertility of patients with HH can be restored through the use of GnRH when cases have a hypothalamic origin or, more commonly, with the use of gonadotropins. Various gonadotropins, either urinary or recombinant, are presently available. The urinary gonadotropin forms are produced from the urine of menopausal women and include human menopause gonadotropin (hMG), which contains urinary FSH and LH. Another commonly used urinary gonadotropin is highly purified urinary FSH (3,4,6).
& THERAPEUTIC MANAGEMENT The therapy for HH depends on the patient’s desire for future fertility. Normal androgen levels and the subsequent development of secondary sex characteristics (in cases where the onset of hypogonadism occurred before puberty) and a eugonadal state can be achieved by androgen replacement alone. Hormone replacement with testosterone is the classic treatment for hypogonadism. Androgen
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(21). Therefore, if a spontaneous pregnancy does not occur after 20 months (or eight months after achieving a sperm concentration of 56106/mL) assisted reproductive technologies may be considered to achieve pregnancy (22). Intrauterine insemination (IUI) is a good option for men who have achieved good spermatogenesis (a sperm concentration higher than 56106/mL) with hormone therapy but failed to impregnate their partner. In these cases, hysterosalpingography should be performed on the partner to confirm tubal patency. IUI is a less expensive and more natural way to conceive. Intracytoplasmic sperm injection (ICSI) is the treatment of choice for patients who have completed at least one year of therapy and exhibit sperm concentrations of ,16106/mL or patients who have sperm concentrations .56106/mL but have failed to achieve impregnation after 20 months. In addition to male infertility, the reproductive potential of the female partner should also be investigated. It is common to find concomitant female infertility in these cases (22). Bakircioglu et al. evaluated 22 ICSI attempts with a pregnancy rate of 54.5% (22). Zorn et al. reported four men with HH who underwent 10 ICSI cycles after hormone treatment. This group achieved a 67% fertilization rate and a 30% pregnancy rate per cycle (23). Until recently, remaining childless, adoption or sperm donation were the only options for HH patients with persistent azoospermia despite long periods of hormone therapy. For these patients, testicular sperm extraction (TESE) could be an excellent option to achieve a pregnancy (19,22). Some authors have published results with TESE in azoospermic men with HH; for example, Fahmy et al. used TESE to successfully recover spermatozoa in 11 out of 15 patients (73%) and, more recently, Akarsu et al. found sufficient spermatozoa for ICSI and cryopreservation for future cycles in all cases (19,24). Assisted reproductive techniques (ART) are an important tool for achieving pregnancy in couples where the male partner has HH. Even couples who achieve spermatogenesis with hormonal therapy may not achieve pregnancy or may require extended periods to achieve pregnancy. This situation can cause low compliance, anxiety, and discomfort in the patients and can increase the financial burden (19). Evaluation of the female partner’s fertility can save time for the couple, as can using ART to achieve a pregnancy as soon as possible.
Regardless of the hormone used for treatment, the total number of sperm usually remains below the normal threshold. This finding does not eliminate the potential for these patients to become fathers, and impregnation rates can reach 50 to 80% with a sperm concentration of 5 million per mL (4,5). The predictors of treatment success are described as an increased baseline testicular volume, no history of cryptorchidism, a history of sexual maturation, and no previous testosterone replacement therapy (5). Even individuals with a testicular volume of 3 mL can benefit from treatment, although these patients may need two years of hormonal therapy before spermatogenesis is triggered (4,5). Because a long period is necessary to restore spermatogenesis, it is advised that every man who aims to become a father start treatment 6 to 12 months before attempting to conceive (4,5).
GnRH pump treatment: A portable infusion pump administers pulses of GnRH into the subcutaneous tissue of the abdominal wall every 2 hours, with doses ranging from 100 to 400 ng/kg. This treatment lasts approximately four months and is usually shorter than gonadotropin therapy. However, GnRH usage is restricted to specialized tertiary hospitals and has a high cost, in addition to interfering with the patient’s everyday life (4,5).
Gonadotropin treatment: This treatment can be administered in all cases of secondary hypogonadism and is compulsory in cases with pituitary lesions or a defective GnRH receptor. The b-chains of LH and hCG are very similar and exhibit similar properties, such as affinity for receptors on Leydig cells. Currently, urinary gonadotropins are highly purified and can be injected subcutaneously, which avoids the induction of antibodies against the medication (4,5). Gonadotropic treatment starts with the administration of 1,000 to 2,500 international units (IU) of isolated hCG twice a week for 8 to 12 weeks. This initial phase is the induction phase, which is crucial for allowing testosterone levels to increase. In certain cases, hCG alone can induce spermatogenesis. In individuals who do not have sufficient endogenous FSH, treatment can continue with the coadministration of 75 to 150 IU hMG three times per week for up to 18 months, as the presence of FSH is crucial for stimulating spermatogenesis. Recombinant FSH can be used in place of hMG, with patients receiving 150 IU three times per week for the same period of time. This combined treatment provides considerable testicular growth in most patients, in addition to spermatogenesis in up to 90% of patients (4,5). These treatments demand rigid discipline and perseverance because of their duration. Financial support is also necessary because the medication utilized is expensive.
& EXPERT COMMENTARY Conventionally, gonadotropin therapy in the form of human chorionic gonadotropin (hCG) alone or combined with human menopausal gonadotropin (hMG) or folliclestimulating hormone (FSH) is indicated to restore spermatogenesis in HH men wishing to father a child. The glycoprotein hormones FSH, LH, hCG are composed of two non–covalently linked protein subunits: the alpha and beta subunits. The alpha subunits of the hormones are identical, whereas the beta subunits are distinct and confer the unique biological and immunological properties and receptor specificity of each of these glycoproteins (25). The beta subunit of LH contains the same amino acid sequence as the beta subunit of hCG, but the hCG beta subunit contains an additional 23 amino acids. The two hormones differ in the composition of their carbohydrate moieties,
Reproductive outcomes after treatment for infertility in HH Treatment with gonadotropins has been shown to be effective in males affected by hypogonadotropic hypogonadism, but the final response to hormonal treatment varies widely from patient to patient (19,20). Spontaneous conception can be achieved within 6-9 months after beginning gonadotropin treatment but can require up to two years
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considered an alternative to intramuscularly injected, urinary-derived hCG for HH men seeking fertility and normal androgenic status. Because of the cost, gonadotropin treatment for fertility restoration should be used until pregnancy is achieved. Ideally, the medication should be continued until the beginning of the second trimester, after which the risk of miscarriage is low (5). It is also advisable to offer sperm cryopreservation to such patients as an option for preserving future fertility. Classically, the duration of gonadotropin treatment for restoring spermatogenesis is greater than three months. Older studies estimated the duration of spermatogenesis (from the differentiation of pale spermatogonia to the ejaculation of mature spermatozoa) to be approximately 74 days (29). This concept was recently challenged by Misell et al. (2006), who showed that the appearance of new sperm in the semen occurred after a mean duration of 64 days. In their study, men with normal sperm concentrations ingested deuterated (heavy) water (2H2O) daily and provided semen samples every two weeks for up to 90 days. The incorporation of the 2H2O label into sperm DNA was quantified by gas chromatography/mass spectrometry, which allowed the percentage of new cells to be calculated. The mean overall time to the detection of labeled sperm in the ejaculate was 64¡8 days (range 4276). Biological variability was also observed, which contradicts the current belief that the duration of spermatogenesis is fixed. All of the subjects exhibited more than 70% new sperm in the ejaculate by day 90, but plateau labeling was not attained in the majority of the subjects, which suggests the rapid washout of old sperm in the epididymal reservoir (30). These data also suggest that in normal men, the sperm released from the seminiferous epithelium enter the epididymis in a coordinated manner, with little mixing of old and new sperm before subsequent ejaculation. This information is useful as a counseling tool for doctors who rely on gonadotropin treatment for HH males, in the sense that monitoring using semen analysis can be tailored accordingly. Assisted reproductive techniques can also be used for couples who are unable to attain an unassisted pregnancy. Intrauterine insemination (IUI) and in vitro fertilization (IVF) techniques are available, depending on the woman’s potential for pregnancy and the quality and quantity of sperm. In cases where viable spermatozoa are not obtained by clinical treatment, they are likely to be obtained directly from the testes through testicular sperm extraction (TESE) or testicular microdissection as part of an in-vitro fertilization program with intracytoplasmic sperm injection (ICSI). These techniques should be applied before considering the use of donor sperm. Contraception is advisable for cases where pregnancy is achieved, as spermatogenesis may continue after therapy stops (5). In up to 10% of cases, the patient exhibits a sustained sperm response and adequate serum testosterone levels even after the complete withdrawal of medication (9). This situation is called hypogonadotropic hypogonadism reversal. The mechanism underlying these cases has not been completely explained, but there appears to be neuronal plasticity in GnRH-producing cells. Sex steroid production is thought to be responsible for the net neuronal stimulus, which has been linked to the secretion of GnRH
which affect bioactivity and half-life. The half-life of LH is 20 minutes, whereas the half-life of hCG is 24 hours (26). The history underpinning the development of gonadotropin therapy spans close to 100 years and provides an example of how basic research and technological advances have progressed to clinical application. Originally, gonadotropins were derived from animal (pregnant mare serum) or human (post-mortem pituitary gland) sources, but these preparations were abandoned because of safety concerns. The modern gonadotropin era started in the 1940s with the extraction of hCG and hMG from urine. Improvements in purification methods led to the production of urinary gonadotropins containing FSH only in the 1980s and 1990s. Advances in DNA technology in the end of the last century enabled the development of recombinant gonadotropins. Recombinant human chorionic gonadotropin (rec-hCG), FSH (rec-hFSH) and LH (rec-hLH), which have the advantage of being devoid of other gonadotropin hormones and contaminants of human origin, have become available, and the use of recombinant FSH combined with recombinant LH preparations in anovulatory women suffering from hypogonadotropic hypogonadotropism has shown to be an effective way to promote follicular development (27). However, data are limited on the effectiveness of these preparations to treat HH males. In a recent study, the clinical efficacy, safety, and tolerability of recombinant human chorionic gonadotropin (rec-hCG) in restoring spermatogenesis and androgen status were assessed in a group of men with hypogonadotropic hypogonadism seeking fertility (28). Eleven men with adultonset HH were treated with a single weekly subcutaneous (SC) injection of 250 mcg of recombinant hCG for a minimum of 12 weeks. The patients self-administered the rec-hCG with a ready-to-inject, prefilled syringe. In this study, the causes of secondary HH were pituitary tumor, long-term exogenous steroid use that did not respond to discontinuation, and cranioencephalic trauma. All of the patients presented with clinical signs of hypoandrogenism and were azoospermic. The testis histopathology results (available for six patients) revealed peritubular fibrosis and maturation arrest. The mean¡SD baseline (pretreatment) hormone levels were as follows: FSH = 0.46¡ 0.28 mUI/mL, LH = 0.39¡0.32 mUI/mL, and total testosterone = 41.3¡26.9 ng/dL. All but one of the patients (with a history of cryptorchidism) exhibited restored spermatogenesis after a mean treatment duration of 12 weeks. The average total motile sperm count was 396106 (range 0.0156.96106) at the 12th treatment week. Two unassisted pregnancies and one assisted (via in vitro fertilization-ICSI) pregnancy were obtained during the follow-up period of five months. The mean¡SD testosterone levels were 647.5¡219.0 ng/dL at the completion of treatment. Marked improvements in virilization, libido and erectile function were also observed after treatment, and the mean combined testis volume increased from 24 mL before treatment to 33 mL after treatment. Headache, gynecomastia, and increased estradiol levels were observed in one man who did not recover spermatogenesis. All of the patients reported subcutaneous hCG self-administration with minimal to no local side effects and/or discomfort. In this study, a single weekly injection of rec-hCG effectively restored spermatogenesis and androgen production in most adultonset HH males. In light of the favorable efficacy, safety, and tolerability profile of rec-hCG, this treatment may be
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11. Finkelstein JS, Whitcomb RW, O’Dea LS, Longcope C, Schoenfeld DA, Crowley WF, Jr. Sex steroid control of gonadotropin secretion in the human male. I. Effects of testosterone administration in normal and gonadotropin-releasing hormone-deficient men. J Clin Endocrinol Metab. 1991;73(3):609-20, http://dx.doi.org/10.1210/jcem-73-3-609. 12. Schwarting GA, Wierman ME, Tobet SA. Gonadotropin-releasing hormone neuronal migration. Semin Reprod Med. 2007;25(5):305-12, http://dx.doi.org/10.1055/s-2007-984736. 13. Ferris HA, Shupnik MA. Mechanisms for pulsatile regulation of the gonadotropin subunit genes by GNRH1. Biol Reprod. 2006;74(6):993-8, http://dx.doi.org/10.1095/biolreprod.105.049049. 14. Raivio T, Wikstrom AM, Dunkel L. Treatment of gonadotropin-deficient boys with recombinant human FSH: long-term observation and outcome. Eur J Endocrinol. 2007;156(1):105-11. 15. Matsumoto AM, Karpas AE, Bremner WJ. Chronic human chorionic gonadotropin administration in normal men: evidence that folliclestimulating hormone is necessary for the maintenance of quantitatively normal spermatogenesis in man. J Clin Endocrinol Metab. 1986; 62(6):1184-92, http://dx.doi.org/10.1210/jcem-62-6-1184. 16. Maffei L, Murata Y, Rochira V, Tubert G, Aranda C, Vazquez M, et al. Dysmetabolic syndrome in a man with a novel mutation of the aromatase gene: effects of testosterone, alendronate, and estradiol treatment. J Clin Endocrinol Metab. 2004;89(1):61-70, http://dx.doi. org/10.1210/jc.2003-030313. 17. Bouligand J, Ghervan C, Tello JA, Brailly-Tabard S, Salenave S, Chanson P, et al. Isolated familial hypogonadotropic hypogonadism and a GNRH1 mutation. N Engl J Med. 2009;360(26):2742-8. 18. Burris AS, Rodbard HW, Winters SJ, Sherins RJ. Gonadotropin therapy in men with isolated hypogonadotropic hypogonadism: the response to human chorionic gonadotropin is predicted by initial testicular size. J Clin Endocrinol Metab. 1988;66(6):1144-51, http://dx.doi.org/10.1210/ jcem-66-6-1144. 19. Akarsu C, Caglar G, Vicdan K, Isik AZ, Tuncay G. Pregnancies achieved by testicular sperm recovery in male hypogonadotrophic hypogonadism with persistent azoospermia. Reprod Biomed Online. 2009;18(4):455-9, http://dx.doi.org/10.1016/S1472-6483(10)60119-8. 20. De Leo V, Musacchio MC, Di Sabatino A, Tosti C, Morgante G, Petraglia F. Present and future of recombinant gonadotropins in reproductive medicine. Curr Pharm Biotechnol. 2012;13(3):379-91, http://dx.doi.org/ 10.2174/138920112799361918. 21. Resorlu B, Abdulmajed MI, Kara C, Unsal A, Aydos K. Is intracytoplasmic sperm injection essential for the treatment of hypogonadotrophic hypogonadism? A comparison between idiopathic and secondary hypogonadotrophic hypogonadism. Hum Fertil (Camb). 2009;12(4):204-8, http://dx.doi.org/10.3109/14647270903331139. 22. Bakircioglu ME, Erden HF, Ciray HN, Bayazit N, Bahceci M. Gonadotrophin therapy in combination with ICSI in men with hypogonadotrophic hypogonadism. Reprod Biomed Online. 2007; 15(2):156-60, http://dx.doi.org/10.1016/S1472-6483(10)60703-1. 23. Zorn B, Pfeifer M, Virant-Klun I, Meden-Vrtovec H. Intracytoplasmic sperm injection as a complement to gonadotrophin treatment in infertile men with hypogonadotrophic hypogonadism. Int J Androl. 2005; 28(4):202-7. 24. Fahmy I, Kamal A, Shamloul R, Mansour R, Serour G, Aboulghar M. ICSI using testicular sperm in male hypogonadotrophic hypogonadism unresponsive to gonadotrophin therapy. Hum Reprod. 2004;19(7):155861, http://dx.doi.org/10.1093/humrep/deh243. 25. Vaitukaitis JL, Ross GT, Braunstein GD, Rayford PL. Gonadotropins and their subunits: basic and clinical studies. Recent Prog Horm Res. 1976;32:289-331. 26. Gonadotropin preparations: past, present, and future perspectives. Fertil Steril. 2008;90(5 Suppl):S13-20. 27. Bosch E. Recombinant human follicular stimulating hormone and recombinant human luteinizing hormone in a 2:1 ratio combination. Pharmacological characteristics and clinical applications. Expert Opin Biol Ther. 2010;10(6):1001-9, http://dx.doi.org/10.1517/14712598.2010.485607. 28. Esteves S, Papanikolaou V. Clinical Efficacy, Safety and Tolerability of Recombinant hCG to Restore Spermatogenesis and Androgenic Status of Hypogonadotropic Hypogonadism Males. Fertil Steril. 2011;96(3):S230, http://dx.doi.org/10.1016/j.fertnstert.2011.07.884. 29. Amann RP. The cycle of the seminiferous epithelium in humans: a need to revisit? J Androl. 2008;29(5):469-87. 30. Misell LM, Holochwost D, Boban D, Santi N, Shefi S, Hellerstein MK, et al. A stable isotope-mass spectrometric method for measuring human spermatogenesis kinetics in vivo. J Urol. 2006;175(1):242-6; discussion 6.
to generate sustained reversal of hypogonadotropic hypogonadism (9).
& KEY ISSUES
N
N N N N N
Male hypogonadotropic hypogonadism (HH) is defined as the failure of the testes to produce androgens and sperm and is a consequence of congenital or acquired diseases that affect the hypothalamus and/or the pituitary gland. The signs and symptoms of HH vary according to age. Diagnosis requires the determination of serum folliclestimulating hormone levels, luteinizing hormone levels and testosterone levels. MRI scans of the brain and sella should be considered. Androgen replacement therapy should not be used for the treatment of hypogonadotropic hypogonadal males desiring fertility. HH represents one of the rare conditions in which specific medical treatment can reverse infertility. The induction and maintenance of both spermatogenesis and androgen production are achieved by the exogenous administration of gonadotropins.
& AUTHOR CONTRIBUTIONS All the authors were involved in the drafting and revision of the manuscript.
& REFERENCES 1. Layman LC. Hypogonadotropic hypogonadism. Endocrinol Metab Clin North Am. 2007;36(2):283-96, http://dx.doi.org/10.1016/j.ecl.2007.03. 010. 2. Araujo AB, O’Donnell AB, Brambilla DJ, Simpson WB, Longcope C, Matsumoto AM, et al. Prevalence and incidence of androgen deficiency in middle-aged and older men: estimates from the Massachusetts Male Aging Study. J Clin Endocrinol Metab. 2004;89(12):5920-6, http://dx.doi. org/10.1210/jc.2003-031719. 3. Darby E, Anawalt BD. Male hypogonadism : an update on diagnosis and treatment. Treat Endocrinol. 2005;4(5):293-309, http://dx.doi.org/ 10.2165/00024677-200504050-00003. 4. Zitzmann M, Nieschlag E. Hormone substitution in male hypogonadism. Mol Cell Endocrinol. 2000;161(1-2):73-88, http://dx.doi.org/10.1016/ S0303-7207(99)00227-0. 5. Han TS, Bouloux PM. What is the optimal therapy for young males with hypogonadotropic hypogonadism? Clin Endocrinol (Oxf). 2010;72(6): 731-7. 6. Hayes FJ, Seminara SB, Crowley WF, Jr. Hypogonadotropic hypogonadism. Endocrinol Metab Clin North Am. 1998;27(4):739-63, vii, http://dx. doi.org/10.1016/S0889-8529(05)70039-6. 7. Bianco SD, Kaiser UB. The genetic and molecular basis of idiopathic hypogonadotropic hypogonadism. Nat Rev Endocrinol. 2009;5(10):56976, http://dx.doi.org/10.1038/nrendo.2009.177. 8. Viswanathan V, Eugster EA. Etiology and treatment of hypogonadism in adolescents. Pediatr Clin North Am. 2011;58(5):1181-200, x, http://dx. doi.org/10.1016/j.pcl.2011.07.009. 9. Raivio T, Falardeau J, Dwyer A, Quinton R, Hayes FJ, Hughes VA, et al. Reversal of idiopathic hypogonadotropic hypogonadism. N Engl J Med. 2007;357(9):863-73. 10. Popa SM, Clifton DK, Steiner RA. The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction. Annu Rev Physiol. 2008;70:213-38, http://dx.doi.org/10.1146/annurev.physiol.70.113006. 100540.
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REVIEW
The role of varicocele treatment in the management of non-obstructive azoospermia Kubilay Inci,I Levent Mert GunayII I
Hacettepe University, Faculty of Medicine, Department of Urology, Ankara, Turkey. II Sistem Surgical Medical Center, Mersin, Turkey.
The literature on male reproductive medicine is continually expanding, especially regarding the diagnosis and treatment of infertility due to non-obstructive azoospermia. The advent of in vitro fertilization with intracytoplasmic sperm injection has dramatically improved the treatment of male infertility due to nonobstructive azoospermia. Assisted reproduction using testicular spermatozoa has become a treatment of hope for men previously thought to be incapable of fathering a child due to testicular failure. In addition, numerous studies on non-obstructive azoospermia have reported that varicocelectomy not only can induce spermatogenesis but can also increase the sperm retrieval rate; however, the value of varicocelectomy in patients with non-obstructive azoospermia still remains controversial. The purpose of this review is to present an overview of the current status of varicocele repair in men with non-obstructive azoospermia. KEYWORDS: Varicocele; Varicocele Repair; Male Infertility; Non-Obstructive Azoospermia; Sperm Retrieval. Inci K, Gunay LM. The role of varicocele treatment in the management of non-obstructive azoospermia. Clinics. 2013;68(S1):89-98. Received for publication on February 20, 2012; Accepted for publication on March 19, 2012 E-mail: kuinci@hacettepe.edu.tr Tel.: +90 312 3051970
article provides an overview (from varying perspectives) of the role of varicocelectomy in patients with NOA-related infertility, based on the most current data.
& INTRODUCTION A varicocele is an abnormally dilated pampiniform plexus, which is the venous network that drains blood from the testicles. The varicocele prevalence in the general population is estimated to be 15%; however, the prevalence is 35% among men with primary infertility and 81% among men with secondary infertility (1,2). The detrimental effects of varicoceles on fertility and the benefit gained by their repair have been debated among andrologists for almost 60 years. Since Tulloch reported the first unassisted pregnancy following varicocele repair in an azoospermic man in 1952, the effect of varicocelectomy on male infertility has become a hotly debated topic (3). Azoospermia renders spontaneous pregnancy nearly impossible. The only treatment option for men with nonobstructive azoospermia (NOA) who desire to be biological parents is testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI). One of the primary benefits of varicocelectomy in NOA patients is that it has the potential to produce motile sperm; however, the value of varicocelectomy in patients with NOA remains unclear. Nonetheless, cumulative data reveal that varicocelectomy can improve spermogram results (4-18). The present review
& THE RELATIONSHIP BETWEEN VARICOCELES AND INFERTILITY Varicoceles are diagnosed primarily during physical examinations and are graded based on the Dubin system: grade 1, varicose veins in the scrotum are palpable with the Valsalva maneuver; grade 2, veins are palpable without the Valsalva maneuver; and grade 3, varicose veins are observed in the scrotum without any maneuver or manipulation. Varicoceles that are detected via physical examination are referred to as clinical varicoceles, whereas those that are .3 mm in diameter and observed only via Doppler ultrasound with the Valsalva maneuver are considered subclinical varicoceles. Most studies on varicoceles are based on the Dubin system classification; thus, interobserver variation in the diagnosis of grade poses an obvious problem.
The pathophysiology of varicocele-related infertility Rather than address the classical theories of varicocele formation, the present review focuses on theories concerning the mechanisms by which dilated scrotal veins impair spermatogenesis and cause infertility. The literature primarily includes studies on the progressive toxic effects of varicoceles, namely elevated temperature, adrenal hormone reflux, gonadotoxic metabolite reflux, altered testicular blood flow, antisperm antibody formation, alterations in the hypothalamic-pituitary-gonadal axis, and oxidative stress. Because the detrimental effects of varicoceles on
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)10
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another important theory for explaining the negative effects of varicoceles on testicular function; this explanation is gaining more support over time (31,32). In addition to other gonadotoxic factors associated with varicoceles, ROS also oxidize fatty acids in spermatozoa membranes and cause DNA damage and fragmentation of the sperm (33). There are several theories regarding the pathophysiological mechanism of the accumulation of ROS in tissues, the most widely accepted of which is the elevated venous pressure theory. Increases in venous pressure decrease testicular blood flow and cause hypoxia, which in turn leads to the accumulation of ROS (28). Several studies have revealed that infertile men with varicoceles have higher levels of seminal oxidative stress markers (e.g., ROS, lipid peroxidation, oxidative DNA damage) than fertile men and infertile men without varicoceles (27,34-36). Total antioxidant capacity (TAC) measurements are reportedly low in men with varicoceles compared with fertile controls (37). Agarwal et al. identified 23 human studies on the role of oxidative stress in varicocele-associated infertility and selected four that measured similar types of reactive oxygen species (ROS) using similar methods (37). This meta-analysis confirmed that ROS-induced oxidative stress, lipid peroxidation, and a low TAC may play a role in the etiology of varicocele-related infertility. Sperm DNA fragmentation is greater in infertile men with varicoceles than in fertile men without varicoceles, which was similarly observed in adolescents with bilateral varicoceles (38-40). Blummer et al. reported impaired mitochondrial activity and DNA fragmentation in the spermatozoa of men with varicoceles (41). The levels of oxidative stress biomarkers decrease following varicocelectomy, suggesting that, in men with a varicocele, oxidative stress in the seminal fluid is primarily caused by the varicocele itself (27). Additionally, after varicocele repair, an increase in the semen antioxidant capacity is observed (42). Several prospective and retrospective studies on the effect of varicocelectomy on sperm DNA damage have demonstrated that varicocele repair is associated with reduced sperm DNA damage (43-46). However, no randomized controlled trial has been conducted regarding the role of varicocelectomy in sperm DNA integrity.
spermatogenesis are apparently related to several factors that may act synergistically, it is difficult to explain the mechanism of action using only one theory. In healthy males, the scrotal temperature is 2 ËšC lower than the core body temperature. A testicular temperature that is identical to the core body temperature is associated with a decrease in the sperm count and sperm quality. Although the exact mechanism by which the temperature influences spermatogenesis is not clearly known, the most commonly accepted theory is thermal damage to the DNA and proteins in the nucleus of spermatic tubule cells and/or Leydig cells (19,20). It has been reported that men with varicoceles and impaired sperm quality have elevated scrotal temperatures and that varicocelectomy leads to a normal scrotal temperature (19,21); however, these results are limited by the fact that the studies were not designed to address factors other than varicoceles that can affect scrotal temperature, such as external exposure to heat and daily postural changes. The reflux of catecholamines and their metabolites from the adrenal gland into left-sided varicoceles is reported to cause vasoconstriction and reduced testicular function; however, these results have not been consistently observed (19). Venous hypertension, caused by the exertion of pressure on the gonadal venous valves by a hydrostatic column can cause chronic vasoconstriction of testicular arterioles, thereby reducing testicular function (22). This phenomenon leads to persistent hypoperfusion, stasis, hypoxia, and subsequent dysfunction of the spermatic epithelium (23). Additional research is necessary to determine if the reflux of renal or adrenal metabolites contributes to the mechanism of injury observed with varicoceles. Antisperm antibody formation is another theory for explaining varicocele-related male infertility. Infertile men have higher levels of testicular autoantibodies in their serum than fertile men. Currently, based on animal experiments, artificial varicocele induction does not cause rupture of the blood-testis barrier and is not correlated with an increase in antibody levels (24). Moreover, based on direct immunobead assays, varicoceles in infertile men do not alter the autoantibody level (25). This theory has yet to accumulate sufficient evidence-based support. Another debatable pathophysiological theory of varicocele-related infertility is that varicoceles negatively affect the hypothalamic-pituitary-gonadal axis. Some patients with varicoceles were reported to have low testosterone levels and sperm quality, which were reversed via varicocelectomy (26,27). The mechanism of effect of varicoceles on the hypothalamic-pituitary-gonadal axis is related to Leydig cell injury and an increase in the heat-associated malfunction of intratesticular enzymes acting on spermatogenesis (28). Additionally, it has been suggested that a low testosterone level negatively affects sperm maturation and increases apoptosis. In contrast, some studies report that there is no association between varicocelectomy and an increase in testosterone levels or sperm quality (29,30). The major criticism of these studies that examined the varicocele/ hypothalamic-pituitary-gonadal axis theory is that blood samples used for testosterone measurement were obtained peripherally, which is not the most reliable sampling method (28). The contemporary andrology literature lacks research on intratesticular testosterone levels. Oxidative stress secondary to elevated scrotal temperatures and the formation of reactive oxygen species (ROS) is
The effects of varicocele repair on infertility The current data in the medical literature concerning the effect of varicocelectomy on infertility are so inconsistent that a definitive interpretation of the findings is difficult, primarily due to differences in the treatment and outcome parameters. Whereas interventional radiologists percutaneously treat varicoceles using sclerotherapy or angioembolization, andrologists use open or endoscopic surgery. Open surgery can be performed via inguinal, subinguinal, or retroperitoneal approaches, with or without microscopic assistance. Retroperitoneal laparoscopic surgery is being replaced by inguinal robot-assisted procedures at some centers. A recent comparative review that included over 5,000 patients concluded that open, microsurgical, inguinal, or sub-inguinal varicocelectomy techniques resulted in higher spontaneous pregnancy rates (44.75%; range 33.851.5%) with lower recurrence and hydrocele-formation rates (2.07 and 0.72%, respectively) compared with laparoscopic, radiological embolization, and macroscopic inguinal or retroperitoneal varicocelectomy techniques (47).
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group underwent high ligation one year later, and the oneand two-year post-surgery pregnancy rates were 44 and 22%, respectively. In a recent RCT comparing subinguinal microsurgical varicocelectomy (n = 73) with observation (n = 72), Abdel-Meguid et al. found that the pregnancy rate was significantly higher in the treatment group (32.9% vs. 13.9%, OR 3.04, 95% CI = 1.33-6.95) (18). All of the semen parameters significantly improved in the treatment arm (p,0.0001), while none of these parameters changed significantly in the control arm (sperm concentration [p = 0.18], progressive motility [p = 0.29], and normal morphology [p = 0.05]). The Cochrane Review (2004) analyzed randomized clinical trials investigating the effects of surgery and embolization for varicoceles in subfertile men (53). The combined Peto OR favoring treatment over no-treatment was 1.10 (95% CI = 0.73-1.68), indicating no benefit of varicocele treatment over observational management in subfertile couples in which a varicocele in the male partner is the only abnormal finding. This meta-analysis concluded that surgical and radiological treatment of varicoceles in men with otherwise unexplained infertility could not be recommended; however, the meta-analysis was criticized because it included studies of men with normal semen parameters and subclinical varicoceles. Ficarra et al. published a meta-analysis including only three randomized clinical trials that did not include patients with normal spermogram findings or those with subclinical varicoceles (50,52,54). The researchers reported that these studies were methodologically deficient and heterogeneous and that pooling them does not result in a high-quality meta-analysis. Marmar et al. conducted a metaanalysis that included only randomized controlled studies of men with palpable varicoceles and abnormal semen parameters and found significantly increased odds of pregnancy after varicocele treatment (OR: 2.87; 95% CI = 1.33-6.20) (55). The Cochrane Review was subsequently updated in 2009, reporting that the treatment of varicoceles in men with no other cause of infertility does not increase the possibility of conception (56). Eight studies were included in the metaanalysis, which examined a total of 607 men. However, trials that included men with subclinical varicoceles and men with normal semen analyses were included in the treatment arms. When the analysis was limited to include only the studies of men who had abnormal semen parameters with clinical varicoceles, the OR suggested a possible benefit of varicocele treatment, although the statistical power was reduced (OR: 2.08; 95% CI = 0.60-4.25) (57). A recent meta-analysis, which was published in 2011, concluded that varicocelectomy improves seminal parameters and reduces sperm DNA damage and seminal oxidative stress in subfertile men with clinical varicoceles; however, there is insufficient evidence to demonstrate a beneficial effect of varicocele repair on spontaneous pregnancy rates (58). Nevertheless, the American Urological Association (AUA) and the American Society of Reproductive Medicine (ASRM) still recommend varicocele repair for patients with palpable varicoceles and at least one abnormal semen parameter (59,60).
The infertility-related treatment endpoints that are commonly analyzed following varicocele repair are semen parameters (i.e., concentration, motility, and/or morphology), sperm DNA integrity, and pregnancy rate (PR). Most published studies consider semen parameters (specifically the sperm density, motility, and morphology) to be the primary outcome parameters of varicocele repair.
Repair of clinical varicoceles Clinical varicoceles are among the most extensively studied urological issues; however, the findings have been inconsistent, perhaps due to differences in the study designs. The first randomized controlled trial (RCT) to report that varicocele repair did not improve fertility was conducted by Nilsson et al. (48). The study randomized 96 infertile patients with visible left-sided varicoceles to receive high ligation (51 patients) or no treatment (46 patients), followed by 36-74 months of follow-up. The patients underwent semen analysis and a pregnancy inquiry every six months. The semen analysis findings and the reported pregnancy rates did not indicate that the varicocele repairs were effective. The report was criticized because some of the patients had normal sperm parameters preoperatively, and the sperm analysis findings were not stratified by the postoperative month. Another prospective RCT, which included 96 patients who were followed-up for four years, also reported that varicocele repairs did not positively affect the semen parameters or pregnancy rates (49). The pregnancy rate in the untreated group (22/41, or 53.7%) was higher than that in the group receiving treatment (13/ 38, or 34.2%). However, 26 of the 38 men who were randomized to treatment underwent open surgery, and 12 were treated with sclerotization or embolization. The use of multiple treatment methods associated with different success rates is a major limitation of the study. A multicenter, prospective RCT investigating varicocele treatment that was performed in Germany examined the effect of sclerotization of dilated veins on pregnancy rates (50). The researchers reported that there was no increase in pregnancy rates in the treatment group. The conception rate was 30% within 12 months after sclerotization among the treated patients and 16.2% among the untreated patients (p = 0.189). Although the number of cases necessary to achieve the study’s goal was calculated to be 460, only 67 patients were randomized at the end of the three-year follow-up period. Therefore, poor recruitment and follow-up were the main limitations of the investigation. Studies reporting a positive effect of varicocelectomy on fertility have similar drawbacks, which are primarily related to study design and execution. Performing meta-analyses of RCTs is challenging because of the wide variation in study designs, methodologies, and populations. The following are some examples of larger-scale RCTs: A Japanese study compared 141 patients who underwent high ligation and 83 patients who did not receive treatment (51). The researchers reported a significant improvement in the concentration and motility of the sperm and higher pregnancy rates in the surgically treated group. Madgar et al. studied 45 couples in which the male partner was infertile (with oligozoospermia) and in which clinical varicoceles were the only observable factor causing infertility (52). In total, 25 of the men underwent high ligation, and their partners had a 60% pregnancy rate one year post surgery, whereas the non-treatment group had a pregnancy rate of 10%. The men in the non-treatment
The effect of varicocele repair in azoospermic patients NOA significantly reduces a man’s potential for fertility. Historically, spermatogenesis has been induced and stimulated in some men following varicocele repair. Tulloch was the first to report a spontaneous post-varicocelectomy pregnancy in a couple with an azoospermic male (3). Since
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sperm in their ejaculate after undergoing varicocele repair to avoid TESE (16). Schlegel and Kaufmann reported that seven of 31 patients (22%) produce sperm, as measured by a postvaricocelectomy semen analysis at an average follow-up of 14.7 months, and only 9.6% have sufficient motile sperm in the ejaculate to enable the use of ICSI and avoid TESE. However, positive changes in the semen parameters following varicocele repair do not last forever. Some researchers recommend the cryopreservation of semen samples that contain motile sperm because azoospermia may recur (12,14). Pasqualotto et al. treated 27 azoospermic men with microsurgical repair (14). Semen samples obtained six months post-surgery revealed that nine patients had sperm in their semen; however, a 12-month post-surgery semen sample analysis revealed that five patients (55.6%) were again azoospermic, which the researchers posited may be a temporary effect due to the induction of spermatogenesis. They also stated the possibility that the men had intermittent sperm production and that the findings were thus unrelated to the surgery. Most of the above-mentioned studies were small, retrospective, uncontrolled case series that examined pre- and post-varicocele-repair sperm parameters and reported only short-term outcomes. As such, there is a need for evidence obtained from well-designed randomized or non-randomized controlled trials and from meta-analyses of primary studies to enhance the objectivity and validity of the findings. A recent meta-analysis examined the ability of varicocele repair to improve the semen parameters and pregnancy rates in patients with NOA, focusing on factors that may predict treatment success (63). The analysis included only English-language reports on surgical varicocelectomy or internal spermatic vein embolization in men with NOA. Studies that included patients with obstructive azoospermia, severe oligospermia, and cryptozospermia, as well as investigations including patients with subclinical varicoceles, individual case reports, and studies with a follow-up of ,4 months, were excluded from the analysis. There were no prospective or randomized trials investigating the treatment of men with NOA. Eleven studies on varicocele repair in men with NOA (a total of 233 patients) were included in the meta-analysis. Following varicocele repair, motile sperm were observed in the semen of 91 of the 233 (39.1%) men, and there were 14 (6%) spontaneous pregnancies and 10 pregnancies with the assistance of in vitro fertilization (IVF). The post-operative sperm density was 1.66106¡1.26106 per milliliter and the mean sperm motility was 20.1¡18.5%. In total, 11 (4.6%) post-surgical patients with motile sperm in their semen relapsed into azoospermia within two-six months after treatment. As reported by the researchers, the primary limitation of the meta-analysis was the lack of prospective studies and RCTs on varicocele repair in men with NOA; all of the included studies were retrospective and non-randomized. Additionally, only 20 small-scale reports were included, and the pregnancy rates in this meta-analysis could have been higher if studies with longer follow-up periods had been included.
then, varicocelectomy has become the most frequently performed surgery for the treatment of male infertility. Azoospermia or severe oligospermia occurs in 4-13% of men with clinical varicoceles (28). Matthews et al. studied a cohort of 78 infertile men; 22 were azoospermic and 56 were oligoasthenospermic (13). All of the patients underwent microvaricocelectomy. Post-operative semen analyses revealed that 55% of the azoospermic patients and 82% of the oligoasthenospermic patients had motile sperm. The pregnancy rate in the azoospermic group was 14% (versus 38% in the oligoasthenospermic group), and two spontaneous pregnancies occurred in the azoospermic group. The researchers also reported that the presence of testicular atrophy at the initial examination has no prognostic value for fertility; however, the study was limited by the lack of a control group. Beginning four months after varicocele-repair surgery, Kim et al. examined 28 men with azoospermia and bilateral or unilateral varicoceles (11). Of the 28 men, 12 (43%) had sperm in their ejaculates, with a mean sperm count of 1.2¡3.66106/ml at 24 months of follow-up. They reported two pregnancies following assisted reproductive technology (ART) treatment; however, there were no spontaneous pregnancies. In another case-control study that evaluated the treatment outcomes and benefits of varicocelectomy in men with NOA and severe oligospermia, spermatogenesis was induced, and 0.2 million motile sperm were produced by two of the six men with NOA (9). It was also reported that 28.6% of virtual azoospermia patients had motile sperm counts above five million following microsurgical varicocele repair, and spontaneous pregnancy occurred in three (21.4%) patients (10). The researchers also reported that 28.6% of patients with virtual azoospermia were spared ICSI procedures as an initial therapeutic option and were given the opportunity to conceive children without bypassing the usual process of natural selection. Gat et al. observed a significant improvement in the concentration, motility, and morphology of sperm in 56.2% of azoospermic men following internal spermatic vein embolization (8). The mean sperm concentration increased to 3.81¡1.696106/ml after embolization. The authors concluded that if azoospermia is not too long-standing, the treatment of varicoceles may significantly improve spermatogenesis or renew sperm production. In addition, adequate treatment may spare 50% of azoospermic patients from TESE in preparation for ICSI. In a recently published prospective noncontrolled study, Taha A. Abdel-Meguid reported the recovery of motile sperm in the ejaculate of 10 of 31 (32.3%) men with NOA and clinically palpable varicoceles following subinguinal microsurgical varicocelectomy (61). Since 1952, numerous reports of changes in the sperm parameters and pregnancy rates in patients with NOA after varicocele repair have been published. Based on these reports, 21-56% of men have motile sperm and 0-15% of their partners have spontaneous pregnancies following varicocele repair; however, none of these studies included a control group (Table 1) (4-18). On one hand, control groups are not considered to be necessary because the control group is expected to remain azoospermic for the duration of any study; on the other hand, NOA patients can exhibit the spontaneous return of spermatozoa in their ejaculate, indicating the necessity for a control group (62). In contrast, some studies indicate that men with clinical varicoceles that are associated with NOA rarely have an adequate number of
The effect of varicocele repair on the sperm retrieval rate Although varicocele repair improves spermatogenesis in 39.1% of patients, TESE is inevitable due to inadequate numbers of sperm in some patients’ ejaculates and to
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19
6
10
Lee, 2007 (12)
Ishikawa, 2008 (9) Cocuzza, 2009 (5)
Abdel-Meguid, 2012 (61)
31
19.3
7.4
12
27
79 (51 complete, 28 virtual azoospermia)
24.8
14
Youssef, 2009 (17)
12
32
Gat, 2005 (8) Poulakis, 2006 (15) Pasqualotto, 2006 (14)
18.9
17
9
13
Esteves, 2005 (7)
14.7
31
Schlegel, 2004 (16) Cakan, 2004 (4)
13.4
15
28
24
10.3
22
35
32
29
N/A
32
30
33
34
32
29
N/A
30
35
35
Patient age Follow-up (months) (years)
Kadioglu, 2001 (10)
Matthews, 1998 (13) Kim, 1999 (11)
Reference
No. of patients
Microsurgery/ subinguinal
High ligation
Microsurgical/ inguinal Microsurgery/ subinguinal
Microsurgical/ inguinal
Percutaneous embolization Anterograde sclerotherapy Microsurgery/ subinguinal
Microsurgery/ inguinal
Varicocelectomy/ inguinal
N/A
Microsurgery/ inguinal
Varicocelectomy/ inguinal
Microsurgery
Procedure/ approach
61.3
73.4
100
33
36
56
87
88
65
15
94
71
71
77
18.1¡7.3
16.56¡8.32
21¡15.2
14.6¡10.5
20.8¡12.3
17.0¡12.4
17.8¡4.8
N/A
14.6
35¡2.8
N/A
12.3¡7.1
20.0¡16.0
19.6¡4.5
SCO: 10 MA: 8 HS: 13
SCO: 22 MA: 26 HS: 23 Normal: 8
SCO: 4 MA: 4 HS: 2
SCO: 10 MA: 6 HS: 3
SCO:3 MA: 5 HS:4 SCO:10 MA: 8 HS: 9
N/A
SCO: 6 MA: 5 HS: 6
SCO: 5 MA: 3 HS: 5
N/A
SCO: 7 MA: 14 HS: 3
SCO: 3 MA: 13 HS: 18
N/A
Pre-op Bilateral mean¡ SD repair FSH (mIU/ml) Histopathology (%)
Table 1 - Literature review of varicocele repair performed in men with NOA
SCO: 0 MA: 3 HS: 7 Overall: 10 (32.3%)
SCO: 1 MA: 0 HS: 2 Overall: 3/10 (30%) SCO: 2 MA: 6 HS: 13 Normal: 6 Complete: 14/51 (28%) Virtual: 13(46%)
2/6 (33%)
SCO: 1 MA: 4 HS: 2 Overall: 7/19 (36%)
SCO: 4 MA: 3 HS: 2 Overall: 9/27 (33%)
7/14 (50%)
SCO: 0 MA: 0 HS: 3 Overall: 3/13 (23%) SCO: 0 MA: 3 HS: 5 Overall: 8/17 (47%) 18/32 (56%)
12/22 (55%) SCO: MA: 5 HS: 9 Overall: 14/28 (50%) SCO: 3 MA: 1 HS: 1 Overall: 5/24 (21%) 7/31 (22%)
Return of motile sperm to the ejaculate
SCO :0 MA: 40 HS: 35 Overall: 37 SCO: 33 MA: 49 HS: 41 Normal: 41 Overall: 42
15
2.3¡1.7
N/A
47
19
2
1
N/A
11
N/A
14
19
55
Post-op sperm motility (%)
SCO: 0 MA: 12.2 HS: 2.25 Overall: 5.5 SCO: 0.65 MA: 4.6 HS: 2.77 Normal:5.21 Overall: 3.56
0.2
0.36
0.87
3.1
3.8
0.8
0.7
N/A
0.04
1.20
2.20
Post-op sperm density (mean x106/ml)
SCO: 0 MA: 2 HS: 2 Normal: 2 Complete: 2/ 51 (4%) Virtual: 4/ 28(14%) Overall: 6/79 (7.6%) N/A
N/A
SCO: 0 MA: 1 HS: 0 Overall: 1/9 (11%) SCO: 0 MA: 0 HS: 1 Overall: 1/19 (5%) 0/6 (0%)
2/14 (14%)
4/18 (12%)
1/17 (6%)
0/13 (0%)
0/31 (0%)
0/24 (0%)
2/28 (7%)
3/22 (15%)
N/A
N/A
N/A
N/A
N/A
N/A
N/A
5
N/A
N/A
N/A
N/A
2
3
SCO: 0 MA: 1 HS: 1 Overall: 2/10 (20%)
N/A
N/A
SCO: 4 MA: 1 HS: 2 Overall: 7/9 (78%) 2/7 (29%)
7/18 (39%) N/A
N/A
0/13 (0%)
4/7 (57%)
N/A
N/A
N/A
Spontaneous pregnancy Pregnancies Relapse rate with ART rate
CLINICS 2013;68(S1):89-98 Role of varicocelectomy in men with NOA Inci K and Gunay LM
Role of varicocelectomy in men with NOA Inci K and Gunay LM
CLINICS 2013;68(S1):89-98
couple used fresh ejaculate for ICSI, and another couple underwent TESE with ICSI. Kadioglu et al. detected motile sperm in the ejaculate of five of 24 patients who underwent microsurgical varicocele repair (10). Based on the histopathological findings, the motile sperm rates in the patients with the SCO pattern, maturation arrest at the spermatocyte stage, maturation arrest at the spermatid stage, the SCO pattern with focal spermatogenesis, and hypospermatogenesis were 0 (0/5), 0 (0/6), 37.5 (3/8), 50 (1/2), and 33.3% (1/3), respectively. Esteves and Glina reported that eight of 17 azoospermic patients had sperm in their ejaculate following microsurgical subinguinal repair. In total, five of six patients with hypospermatogenesis and three of five patients with maturation arrest had sperm in their ejaculate, but none of the six SCO syndrome patients had sperm in their ejaculate (7). Lee et al. analyzed their patients’ semen three months after microsurgical inguinal varicocelectomy, and the presence of sperm based on the histopathological pattern was as follows: hypospermatogenesis, two of three patients; maturation arrest, four of six patients; and SCO pattern, one of ten patients (12). One couple in the hypospermatogenesis group had a spontaneous pregnancy. In the study of AbdelMeguid, sperm were recovered in patients with HS (seven of 13, 53.8%) and late MA (three of six, 50%), whereas no sperm could be recovered from the ejaculate of patients with early MA or SCO. Research findings on the potential for inducing spermatogenesis following varicocele repair in azoospermic men with the SCO pattern are inconsistent. Pasqualotto et al. reviewed the medical records of 27 azoospermic men who underwent testis biopsy and microsurgical repair of clinical varicoceles (14). The microsurgical repair was bilateral in 15 patients and unilateral in 12 patients, and it was performed using a subinguinal approach. Each patient underwent an open, diagnostic testicular biopsy during the varicocele repair, which was performed under general anesthesia. Biopsies were performed on both testes. Germ cell aplasia was identified in 10 of the patients, hypospermatogenesis was identified in nine patients, and early maturation arrest was identified in eight patients. Induction of spermatogenesis was achieved in nine of the patients (33.3%), six of whom had bilateral varicoceles (40%, 6/15) and three of whom had unilateral varicoceles (25%, 3/12). Four (40%) of the nine patients exhibited the SCO pattern, three (33%) had maturation arrest, and two (22%) had hypospermatogenesis. The researchers concluded that, because a single testis biopsy showing germ cell aplasia may not indicate the overall testis histology, varicocele repair must be considered for all men with azoospermia and a palpable varicocele, regardless of their testicular histopathology. A meta-analysis that compared the outcomes of varicocele repair in men with NOA, based on histopathology, indicated that, compared with men with SCO, there is a higher probability for the successful induction of spermatogenesis in men with late maturation arrest or hypospermatogenesis (63). The rates of success in 156 patients, defined as sperm in the ejaculate or spontaneous pregnancy, were 42.1% in the patients with maturation arrest and 54.5% in those with hypospermatogenesis, which were significantly higher than that in subjects with the SCO pattern (11.3%) (patients with the SCO pattern vs. maturation arrest: p,0.001; patients with the SCO pattern vs. hypospermatogenesis: p,0.001). Patients with late maturation arrest
azoospermia relapse following the recovery of spermatogenesis in other patients (14,16,63). There are few studies on the effect of varicocele repair on the results of TESE. In a retrospective study, Schlegel and Kaufmann evaluated the sperm retrieval rates in varicocele repair and non-repair groups, dividing them into subgroups based on histopathological abnormalities (16). In patients with the Sertoli cellonly (SCO) pattern, the sperm retrieval rate by TESE was 26% with repair and 38% without repair; in subjects with the maturation-arrest pattern, the retrieval rate was 53% with repair and 47% without repair; and in patients with the hypospermatogenesis pattern, the rate was 96% with repair and 96% without repair. The total sperm retrieval rate was 60% in 68 patients who underwent varicocelectomy and same (60%) in 70 patients with untreated varicoceles. Patients with subclinical varicoceles were also included in the analysis, which may explain why the sperm retrieval rates were not affected by a history of varicocelectomy, as treatment for subclinical varicoceles is of questionable benefit (64,65). One of our earlier studies (conducted at Hacettepe University Hospital, Ankara, Turkey), which compared the sperm retrieval rate based on micro-TESE and ICSI outcomes in 96 patients with NOA (including 66 patients who previously underwent successful varicocelectomy for clinical varicoceles and 30 patients who had unrepaired varicoceles), demonstrated that varicocele repair significantly increases the sperm retrieval rate in patients with clinical varicoceles and NOA (53% versus 30%, OR = 2.63, 95% CI = 1.05-6.60, p = 0.036) (66). In that study, we also compared 2PN fertilization rate, the high-quality embryo rate, the mean number of transferred embryos, and the clinical pregnancy rate; however, the parameters were similar in the treated and untreated groups. Haydardedeoglu et al. also compared the sperm retrieval rates and ICSI outcomes in treated and untreated varicocele groups and found that the sperm retrieval rate was higher in the varicocele repair group (60.81 and 38.46%, respectively, p = 0.01) (67). They also reported that the clinical pregnancy rate and the live-birth rate were significantly higher in the varicocelectomy group (74.2% versus 52.3% and 64.5% versus 41.5%, respectively, p,0.05). Notably, patients with spermatozoa, as measured by post-operative semen analyses, were excluded from these two studies. Although this exclusion represents a bias in favor of the non-treatment groups, the sperm retrieval rate in the varicocele repair groups was still higher than that in the non-treatment groups.
The role of testicular histopathology The utility of testicular biopsy findings in predicting which patients are most likely to exhibit improved semen parameters after varicocele repair has been studied. Reportedly, patients with late-stage maturation arrest and hypospermatogenesis are more likely to exhibit improved semen parameters and pregnancy rates (7,10-12). Kim et al. observed sperm in 12 of 28 men with azoospermia following varicocele repair (11). When they divided the group based on the biopsy results, nine men with severe hypospermatogenesis and five with maturation arrest at the spermatid stage exhibited improved sperm densities. No improvement was noted in three men with the SCO pattern or in three others with maturation arrest at the spermatocyte stage; furthermore, no spontaneous pregnancies occurred. One
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Role of varicocelectomy in men with NOA Inci K and Gunay LM
more important predictive factor for sperm retrieval than is a coincident varicocele (16). Similarly, in men with Klinefelter syndrome, a history of varicocele repair does not appear to change the outcome of TESE (16). Therefore, Y chromosome mapping and karyotype analyses are essential in the work-up of men with varicoceles and azoospermia and may be predictive of the varicocele repair outcomes. Additionally, couples should be aware of the genetic anomalies associated with karyotype abnormalities an Y chromossome deletions and should undergo genetic counseling to ascertain the risk of transmitting these mutations to their offspring.
(n = 24) had a higher success rate (45.8%) than subjects with early maturation arrest (n = 11.0%) (p = 0.007). The validity of the results of this meta-analysis is limited by the small total study population (n = 156); however, the researchers concluded that testicular histopathology based on testicular biopsy could be used to determine whether patients with NOA may benefit from varicocele repair. Several factors should also be considered when interpreting the results. As the researchers stated, unilateral and bilateral testicular biopsies were performed in a heterogeneous manner, based on the preferences of each researcher. None of these eight studies reported a predominant or favorable histopathology or other types of histopathology, nor did the studies examine the number of seminiferous tubules per biopsy. Therefore, the results should be interpreted with caution, as a single testis biopsy is not representative of the entire organ. As such, azoospermic patients who exhibit the SCO pattern based on a single, large testis biopsy may exhibit improvements in semen quality following varicocelectomy. Another concern regarding the diagnostic testicular biopsy is its invasiveness and associated potential risks, such as inflammatory changes, hematoma, parenchymal fibrosis, and permanent devascularization of the testis, as well as the potential to remove foci of spermatogenesis in an already compromised testicle; therefore, there is no consensus regarding the indications for a diagnostic testicular biopsy before varicocele repair.
The cost effectiveness of varicocele repair in NOA patients Decision models have been constructed based on predefined assumptions and have been used to predict outcomes when multiple complex treatments are available. Several decision analysis models have been used to calculate the cost of pregnancy associated with initial surgical or initial ART treatment in men with infertility caused by varicoceles. In 1997, Schlegel published a cost-effectiveness analysis of the value of varicocele repair versus ART treatment in couples with varicocele-associated infertility (74). Only results from controlled trials of varicocelectomy were used. Varicocelectomy-associated costs were determined based on surgeon and anesthesiologist fees, as well as hospitalassociated charges. All of the costs were obtained from published sources, including the Medicare Resource-Based Relative Value Scale (MRBRVS). Time off from work and the costs of treating complications (hydrocelectomy and exploration for bleeding) were estimated based on published studies on men who underwent varicocelectomy. The cost of a basic evaluation for the presence of a varicocele, including a comprehensive office consultation, follicle stimulating hormone (FSH) and testosterone blood tests, two semen analyses, and a follow-up visit, was also included in the analysis. The treatment of varicocele-associated male infertility using varicocelectomy was reported to be a cost-effective alternative to treatment with ART. The mean cost of a live birth following varicocelectomy and the mean cost of a live delivery following ICSI were calculated to be $26,268 (95% CI = $19,138-$44,656) and $89,091 (95% CI = $78,720-$99,462), respectively. Another decision analysis study of the cost of treatment of male infertility compared the cost-effectiveness of varicocelectomy and ART treatment in patients with varicoceles (75). The cost per pregnancy and the pregnancy rate in each study arm were calculated and compared. Overall, the initial surgical repair of varicoceles was more cost-effective than ART was; however, intrauterine insemination (IUI) yielded a lower cost per pregnancy than varicocelectomy in men with a preoperative total motile sperm count (TMC) between 10 and 20 million sperm ($9,000 versus $11,333, respectively). In men with low sperm counts (TMC ,10 million) who qualified for sperm retrieval/ICSI but not IUI, a varicocelectomy was more cost-effective than sperm retrieval/ICSI when the post-operative pregnancy rate was greater than the 14% threshold. In men with high sperm counts (TMC.10 million) who qualified for IUI but not sperm retrieval/ICSI, a varicocelectomy was more costeffective than IUI only when the pregnancy rate was greater than the 45% threshold.
The effect of genetic anomalies on the outcome of varicocele repair The presence of Y microdeletions or karyotype abnormalities is clinically significant. Karyotype abnormalities or Y chromosome deletions are observed in 16.6% of azoospermic men (68). The data from studies on Y chromosome microdeletions in men with varicoceles clearly indicate that genetic defects and varicoceles can coexist (69,70). Rao et al. compared chromosomal abnormalities and Y chromosome microdeletions in infertile men with varicoceles and idiopathic infertility (71). The frequencies of chromosomal defects in the individuals with varicoceles and idiopathic infertility were 19.3 and 8.76%, respectively, whereas the frequencies of Y chromosome microdeletions were 5.26 and 3.60%, respectively. This close association has made it necessary to investigate the effect of coexisting genetic anomalies on varicocele repair; however, only a few studies have addressed the results of varicocelectomy in infertile men with coexisting genetic infertility. Cayan et al. reported the results of varicocelectomy in oligospermic infertile men who presented varicoceles either with or without genetic anomalies (72). Among the 19 patients who were included, five had a genetic anomaly (abnormal karyotype [n = 3] or Y chromosome microdeletions [n = 2]). In the genetic anomaly group, all five patients exhibited improvements in their semen quality following varicocele repair. A similar study evaluated the effect of Y chromosome microdeletions on the varicocele repair outcome in five patients who harbored a Yq microdeletion and in four who had no microdeletions (73). A post-operative semen analysis revealed that the five patients with Yq microdeletions exhibited no improvement in their semen parameters, whereas the semen parameters were significantly improved in the patients without microdeletions. The Y chromosome plays a crucial role in the control of spermatogenesis. The site of Y chromosome deletion is a
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Role of varicocelectomy in men with NOA Inci K and Gunay LM
CLINICS 2013;68(S1):89-98
Varicocelectomy not only results in the induction of spermatogenesis, rendering testicular sperm extraction/ retrieval unnecessary but also increases the micro-TESE sperm-retrieval rate in men who remain azoospermic following varicocele repair. However, because of the possibility of azoospermia relapse following an initial post-varicocelectomy improvement in the semen quality, patients should be informed of the option for sperm cryopreservation. The testicular histopathology may predict the success of varicocele repair. The value of varicocele repair in men with azoospermia and the SCO pattern is questionable. In patients with NOA, the testicular histology is often heterogeneous, so a single testis biopsy may not indicate the overall testis histology. Therefore, azoospermic patients with the SCO pattern based on a single large testis biopsy may exhibit improvements in their semen quality following varicocelectomy. There is a strong association between genetic defects and varicocele-related infertility in men. Because it is possible that these defects can be transmitted to offspring, Y chromosome mapping and karyotype analysis are crucial for the evaluation of men with varicocele-related infertility. Informing surgical candidates about underlying genetic abnormalities and the potential for a poor response to surgery would be extremely helpful in their decisionmaking process. Additionally, if a genetic abnormality is identified, the couple should undergo genetic counseling. In contrast to earlier reports, a recent cost-effectiveness analysis has revealed that the use of varicocelectomy for the specific treatment of varicocele-associated male infertility is not more cost-effective than assisted reproduction using ICSI; however, cost-effectiveness should not necessarily be the primary concern for clinicians who treat infertile couples. Additionally, an analysis of cost-effectiveness should be conducted in an institution-specific manner. Although several studies have evaluated the role of varicoceles in NOA, these investigations were poorly designed studies that lacked controls; therefore, properly designed and carefully randomized controlled trials are necessary to precisely assess the impact of varicocelectomy on fertility outcomes in NOA patients. Nonetheless, in light of the currently available data, varicocele repair should be considered before TESE/ICSI in all azoospermic men who have clinically palpable varicoceles.
Although these studies examined the cost-effectiveness of varicocele repair and ART, they were not specific to varicocele-associated NOA patients. A recent analysis specifically investigated the cost-effectiveness of ART for varicocele-associated NOA (76). In this study, the cost per live birth associated with varicocelectomy and microsurgical TESE was calculated and examined over time. The authors estimated the contribution of direct-cost elements and the impact of indirect costs, such as time off work while recovering from male-related and IVF-related interventions; maternal complications, including ovarian hyperstimulation syndrome (OHSS), pelvic hemorrhage, infection, stroke, myocardial infarction, and ovarian cancer; complications from male interventions, including interventions for bleeding, infection, and testicular atrophy, as well as those associated with anesthesia-related complications; and multiple-gestation pregnancies associated with IVF/ICSI. In contrast to previous studies, this decision analysis model was based on outcome data from the Society for Assisted Reproductive Technology (SART) database, the peerreviewed literature, the MRBRVS, and high-volume IVF centers in the US, and the model revealed that microsurgical TESE is more cost-effective than varicocelectomy for the treatment of varicocele-related NOA when indirect costs are considered. The costs of varicocelectomy and TESE were calculated to be $79,576 and $69,731, respectively. Varicocelectomy was more cost-effective than TESE when the rate of spontaneous pregnancy after varicocelectomy was .40% or when the live birth rate following IVF/ICSI was ,10%; however, several factors were not considered in the analysis. As the researchers reported, it is possible that some of the patients in the TESE group also had obstructive azoospermia or oligospermia, which may have caused an upward bias in the live delivery rate. Moreover, the live birth rate associated with the use of freshly ejaculated sperm following varicocele repair and the live birth rate associated with the use of sperm retrieved via TESE following unsuccessful varicocele repair should have been included in the analysis because varicocelectomy increases the sperm retrieval rate (66,67,76). The analysis included only four studies of varicocele repair in men with NOA, resulting in a lower rate of viable sperm in the post-operative ejaculates (10%) and a lower spontaneous pregnancy rate (2.8%) than those reported in a recent meta-analysis (63). The researchers also did not consider the reimbursements offered by most insurance companies for varicocele repair, the various costs based on geography, the limited availability of IVF/ ICSI, and the cost of complications related to IVF/ICSI (63). Nevertheless, consideration of the cost-effectiveness of treatment options should not necessarily be the primary concern for a clinician who is treating infertile couples. Costeffectiveness analyses should be considered in relation to institution-specific data, thus yielding institution-specific results.
& AUTHOR CONTRIBUTIONS Inci K and Gunay LM reviewed the current literature in detail and wrote this manuscript. Both authors approved the final version of the text.
& REFERENCES 1. Gorelick JI, Goldstein M. Loss of Fertility in Men with Varicocele. Fertil Steril. 1993;59(3):613-6. 2. Saypol DC. Varicocele. J Androl. 1981;2(2):61-71. 3. Tulloch WS. Consideration of sterility; subfertility in the male. Edinburg Med J. 1952(59):29-34. 4. Cakan M, Altug U. Induction of spermatogenesis by inguinal varicocele repair in azoospermic men. Arch Androl. 2004;50(3):145-50, http://dx. doi.org/10.1080/01485010490425250. 5. Cocuzza M, Pagani R, Lopes RI, Athayde KS, Lucon AM, Srougi M, et al. Use of subinguinal incision for microsurgical testicular biopsy during varicocelectomy in men with nonobstructive azoospermia. Fertil Steril. 2009;91(3):925-8, http://dx.doi.org/10.1016/j.fertnstert.2007.12.065. 6. Czaplicki M, Bablok L, Janczewski Z. Varicocelectomy in Patients with Azoospermia. Arch Androl. 1979;3(1):51-5, http://dx.doi.org/10.3109/ 01485017908985048.
& EXPERT COMMENTARY Varicoceles are very common in infertile males and exhibit a progressive pathology. Although the precise pathophysiology remains unknown, varicocele repair can successfully reverse the negative effects of varicoceles on testicular function. Although the role of varicocele repair in NOA patients has been evaluated in several studies, the value of varicocelectomy in these cases remains unclear.
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The effect of microsurgical varicocelectomy on serum follicle stimulating hormone, testosterone and free testosterone levels in infertile men with varicocele. BJU Int. 1999;84(9):1046-9. 27. de Catalfo GEH, Ranieri-Casilla A, Marra FA, de Alaniz MJT, Marra CA. Oxidative stress biomarkers and hormonal profile in human patients undergoing varicocelectomy. Int J Androl. 2007;30(6):519-30. 28. Will MA, Swain J, Fode M, Sonksen J, Christman GM, Ohl D. The great debate: varicocele treatment and impact on fertility. Fertil Steril. 2011;95(3):841-52, http://dx.doi.org/10.1016/j.fertnstert.2011.01.002. 29. Swerdloff RS, Walsh PC. Pituitary and Gonadal Hormones in Patients with Varicocele. 1975;26(10):1006-12. 30. Su LM, Goldstein M, Schlegel PN. The Effect of Varicocelectomy on Serum Testosterone Levels in Infertile Men with Varicoceles. J Urology. 1995;154(5):1752-5. 31. Shiraishi K, Takihara H, Matsuyama H. Elevated scrotal temperature, but not varicocele grade, reflects testicular oxidative stress-mediated apoptosis. World J Urol. 2010;28(3):359-64. 32. Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol. 2008;59(1):2-11. 33. Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ. Analysis of the impact of intracellular reactive oxygen species generation on the structural and
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REVIEW
An update on sperm retrieval techniques for azoospermic males Sandro C. Esteves,I Ricardo Miyaoka,I Jose´ Eduardo Orosz,I,II Ashok AgarwalIII I
ANDROFERT – Andrology & Human Reproduction Clinic, Campinas, Sa˜o Paulo, Brazil. II Comissa˜o de Ensino e Treinamento da Sociedade Brasileira de Anestesiologia, Pontifı´cia Universidade Cato´lica de Campinas, Campinas, Sa˜o Paulo, Brazil. III Center for Reproductive Medicine, Cleveland Clinic Foundation, Cleveland, Ohio, USA.
The use of non-ejaculated sperm coupled with intracytoplasmic sperm injection has become a globally established procedure for couples with azoospermic male partners who wish to have biological offspring. Surgical methods have been developed to retrieve spermatozoa from the epididymides and the testes of such patients. This article reviews the methods currently available for sperm acquisition in azoospermia, with a particular focus on the perioperative, anesthetic and technical aspects of these procedures. A critical analysis of the advantages and disadvantages of these sperm retrieval methods is provided, including the authors’ methods of choice and anesthesia preferences. KEYWORDS: Sperm Retrieval; Assisted Reproductive Techniques; Male Infertility; Azoospermia; Anesthesia; Review. Esteves SC, Miyaoka R, Orosz JE, Agarwal A. An update on sperm retrieval techniques for azoospermic males. Clinics. 2013;68(S1):99-110. Received for publication on June 25, 2012; Accepted for publication on June 28, 2012 E-mail: s.esteves@androfert.com.br Tel.: 55 19 3295-8877
to be a cost-effective treatment that allows for natural conception in selected cases of OA, such as post-vasectomy (10). Despite being highly successful, ductal recanalization may not be an option for some infertile couple or may be impossible in certain cases of congenital obstructions and post-infectious obstruction or failed vasectomy reversals. Spermatozoa can be retrieved from the epididymides or testicles in almost all cases of OA, irrespective of the technique used for sperm collection and the cause of obstruction. Nonobstructive azoospermia (NOA), on the other hand, is a consequence of spermatogenic failure and is the cause of most cases of azoospermia (8). NOA has congenital and acquired etiologies other than hypothalamic-pituitary disease and obstruction of the male genital tract. Unlike men with OA, men with NOA have no treatment options other than attempting testicular sperm retrieval. In such cases, spermatogenesis may be focal, which means that spermatozoa can be found and used for ICSI in approximately 30-60% of men with NOA (1). Testicular sperm extraction (TESE) is the technique of choice for NOA (1,11), and the use of microsurgery for TESE seems to increase retrieval rates (1,12). Three main goals should be accomplished during sperm retrieval: (i) the acquisition of an adequate number of sperm for both immediate use and cryopreservation, (ii) the retrieval of the highest quality of sperm, and (iii) minimizing the damage to the reproductive tract, thus preserving the option of future retrieval attempts and testicular function (13). A list of the candidates eligible for sperm retrieval is provided in Table 1. The aim of this review is to update readers on the methods currently available for sperm acquisition in azoospermia, focusing in particular on the operative and technical aspects
& INTRODUCTION Sperm retrieval techniques (SRTs) are surgical methods that have been developed to obtain spermatozoa from the epididymides and testicles of azoospermic men seeking fertility treatment (1). After sperm acquisition, intracytoplasmic sperm injection (ICSI) is used instead of standard in vitro fertilization (IVF) because ICSI has been shown to result in a significantly higher fertilization rate (2). Alternatively, the retrieved sperm can be cryopreserved for use in future sperm injection attempts (3,4). The use of non-ejaculated sperm and ICSI has become an established procedure for couples whose male partner has azoospermia to obtain biological offspring (5-7). The method of choice for sperm retrieval (SR) is based on the type of azoospermia, which can be obstructive or nonobstructive, and the attending surgeon’s preferences and experience. Obstructive azoospermia (OA) is associated with the inability to detect spermatozoa in the ejaculate and post-ejaculate urine after centrifugation due to the bilateral obstruction of the seminal ducts (8,9). Obstruction of the male reproductive system can be congenital or acquired. Microsurgical ductal reconstruction is generally considered
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)11
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Table 1 - Candidates for sperm retrieval, grouped according to the type and etiology of azoospermia. Obstructive Azoospermia
Non-obstructive Azoospermia (Testicular Failure)
Congenital Ductal Obstructions: Congenital bilateral absence of the vas deferens Young’s syndrome (clinical triad of chronic sinusitis, bronchiectasis, and obstructive azoospermia) Stenosis or atresia of the ejaculatory ducts Midline prostatic cysts (utricular and Mu¨llerian cysts) Ejaculatory duct cysts Seminal vesicle cysts
Congenital Testicular Failure: Testicular dysgenesis/cryptorchidism Genetic abnormalities (Klinefelter syndrome, Y chromosome microdeletions*) Germ cell aplasia (Sertoli cell-only syndrome) Spermatogenic (maturation) arrest
Acquired Ductal Obstructions: Post-infection (epididymitis, prostatitis, seminal vesiculitis) Post-vasectomy Post-surgical (epididymal cysts, hernia repair, scrotal surgery, bladder neck surgery, prostatectomy) Iatrogenic (urologic endoscopic instrumentation)
Acquired Testicular Failure: Testicular trauma Testicular torsion Post-inflammatory (e.g., mumps orchitis) Exogenous factors (steroid medications, cytotoxic drugs, irradiation, heat) Systemic diseases (liver cirrhosis, renal failure) Testicular tumor Varicocele Post-surgical (surgeries that may compromise testicular vascularization, resulting in testicular atrophy)
Idiopathic: Idiopathic epididymal obstruction *
Idiopathic (unknown etiology)
The likelihood of obtaining sperm at sperm retrieval is virtually zero when complete AZFa and/or AZFb Yq microdeletions are found.
of these procedures. Moreover, a critical expert analysis of the advantages and disadvantages of the sperm acquisition methods is provided, including the authors’ methods of choice and anesthesia preferences.
to retrieve spermatozoa from the epididymis or the testicle with or without microsurgery. Percutaneous retrievals, on the other hand, require a needle to be percutaneously inserted into the sperm source, i.e., the epididymis or the testicle. Irrespective of the method used, the goal of SR is to obtain the epididymal fluid or the seminiferous tubules and their contents. Table 2 lists the SR options available and their indications. Table 3 compares the advantages and disadvantages of the different SR methods.
& SPERM RETRIEVAL: AVAILABLE METHODS AND TECHNICAL ASPECTS The two general SR methods are open surgery and percutaneous acquisition. Open surgery can be performed
Table 2 - Sperm retrieval techniques, acronyms and indications. Technique
Acronym
Indications
Percutaneous epididymal sperm aspiration
PESA
Obstructive azoospermia
Microsurgical epididymal sperm aspiration
MESA
Obstructive azoospermia
Open epididymal fine-needle aspiration Percutaneous testicular sperm aspiration; percutaneous testicular fine-needle aspiration
Testicular sperm extraction (single or multiple biopsies)
Single seminiferous tubule biopsy
Microsurgical testicular sperm extraction
ND
Obstructive azoospermia
TESA; TEFNA
Obstructive azoospermia; Failed epididymal retrieval in OA cases; Epididymal agenesis in CAVD cases; Favorable testicular histopathology1 in NOA cases; Previous successful TESA/TEFNA attempt in NOA cases
TESE
Obstructive azoospermia; Failed epididymal retrieval in OA cases; Failed TESA/TEFNA in OA cases; Non-obstructive azoospermia
ND
Obstructive azoospermia; Failed epididymal retrieval in OA cases; Failed TESA/TEFNA in OA cases; Non-obstructive azoospermia
Micro-TESE
Non-obstructive azoospermia
OA: obstructive azoospermia; NOA: non-obstructive azoospermia. CAVD: congenital absence of the vas deferens. ND: not defined. Hypospermatogenesis.
1
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Table 3 - Advantages and disadvantages of sperm retrieval techniques. Advantages
Disadvantages
Fast and low cost; Minimal morbidity, repeatable; No microsurgical expertise required; Few instruments and materials; No open surgical exploration
Few sperm retrieved; Limited number of sperm for cryopreservation; Fibrosis and obstruction at the aspiration site; Risk of hematoma/spermatocele
Repeatable; No microsurgical expertise required; Relatively large number of sperm for cryopreservation; Few instruments and materials
Open surgical exploration required; Increased cost and time-demanding; Fibrosis and obstruction at the aspiration site; Postoperative discomfort; Not validated in a large series of patients
MESA
Large number of sperm retrieved; High number of sperm for cryopreservation; Reduced risk of hematoma; Reconstruction possible1
Open surgical exploration required; Increased cost and time-demanding; Operating microscope required; Microsurgical instruments and expertise required; Postoperative discomfort
TESA
Fast and low cost; Repeatable; No open surgical exploration; No microsurgical expertise required; Few instruments and materials; Minimal/mild postoperative discomfort
Relatively low success rate in NOA cases; Few sperm retrieved in NOA cases; Limited number of sperm for cryopreservation; Risk of hematoma/testicular atrophy
Fast and low cost; Repeatable; No open surgical exploration; No microsurgical expertise required; Few instruments and materials required; Minimal/mild postoperative discomfort
Few sperm retrieved in NOA cases; Limited number of sperm for cryopreservation; Risk of hematoma/testicular atrophy; Not validated in a large series of patients
No microsurgical expertise required; Repeatable
Increased cost and time-demanding; Open surgical exploration required; Relatively few sperm retrieved in NOA cases; Risk of testicular atrophy3; Risk of testicular androgen production impairment3; Postoperative discomfort
No microsurgical expertise required; Repeatable
Increased cost and time-demanding; Open surgical exploration required; Relatively few sperm retrieved in NOA; Postoperative discomfort; Not validated in a large series of patients
Higher success rates in NOA cases2; Larger number of sperm retrieved2; Relatively higher chance of sperm cryopreservation2; Low risk of complications
Surgical exploration required; Increased cost and time-demanding; Operating microscope required; Microsurgical instruments and expertise required; Postoperative discomfort
PESA
Open epididymal fine-needle aspiration
TEFNA
TESE
Single seminiferous tubule biopsy
Micro-TESE
PESA: percutaneous epididymal sperm aspiration; MESA: microsurgical epididymal sperm. aspiration; TESA: percutaneous testicular sperm aspiration; TESE: conventional testicular sperm extraction; micro-TESE: microsurgical testicular sperm extraction. 1in cases of post-vasectomy obstructions. 2compared with TESA and TESE in NOA cases. 3multiple biopsy-TESE.
Preoperative Considerations
Operating Room and Patient Preparation
The procedure, results, and potential complications should be reviewed and discussed with the patient and his spouse by experienced staff. The patient should sign an informed consent form prior to surgery and be instructed that someone should accompany him if SR is to be performed on an outpatient basis. In addition, aspirin and/or nonsteroidal anti-inflammatory drugs should be avoided for one week before surgery. Those patients taking anti-coagulating agents should discontinue the medication during the preoperative period. Scrotal hair shaving is required for open retrievals, and patients should be instructed to void the bladder prior to admission to the operating room.
All of the instruments and materials used during the sperm retrieval procedure should be assessed for availability and/ or operational conditions. For open procedures, a grounding pad should be available to allow the safe use of electrocautery. Ideally, an operating table with motorized control should be available for open procedures. The patient should be positioned on the operating table in a supine position. For microsurgical techniques, the operating microscope should be positioned and adjusted. The skin should be cleansed from mid-abdomen to mid-thigh using a povidone-iodine or similar solution. The surgical staff should scrub and gown properly. Sterile drapes should be positioned in a manner such that only the scrotum is exposed. A list of instruments
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Table 4 - Materials and instruments commonly used in sperm retrieval techniques. Sperm retrieval method All
PESA, TESA and TEFNA
Equipment and Supplies Basic instruments and materials:
NUnipolar coagulating generator (open retrievals) NBipolar coagulating generator (MESA and micro-TESE) NAntiseptic solution for skin cleaning N30-cc 1% xylocaine solution (spermatic cord anesthesia) N19- (40612) and 22- (2567) gauge hypodermic needles (spermatic cord anesthesia) NSterile towels NGauze sponges NSterile gowns NSurgical gloves NSurgical drapes NSurgery instrument table (optional) NMayo table NSterile drapes for tables N20-cc syringes (spermatic cord anesthesia) NSaline solution for irrigation (MESA and micro-TESE) NUnipolar cautery pen (MESA and micro-TESE; optional) NSharp-beveled fine needle (19-, 22-, 23- or 26-gauge, depending on the surgeon’s preference and technique) attached to a 1-mL tuberculin syringe (PESA) or to a 10- or 20-mL syringe coupled to a Cameco (or similar) syringe holder Tissue-cutting biopsy needle (e.g., Tru-cutTM needle or BioptyTM gun; optional)
N
TESE, micro-TESE, MESA, Open epididymal Non-microsurgical set: fine-needle aspiration, Single seminiferous Basic set of surgical instruments for delicate surgeries (including small needle holder, small smooth and tubule biopsy toothed forceps (Addison forceps), small suture scissors, small curved dissection scissors, a pair of small farabeuf retractors, scalpels, curved kelly clamps, straight mosquito clamps, backhaus clamps) Sutures (e.g., 4-0 vicryl with tapered needle, 4-0 catgut with tapered needle, 5-0 black monofilament nylon with tapered cut needle (micro-TESE), 9-0 black monofilament nylon with tapered needle (MESA))
N N
Micro-TESE and MESA
Microsurgical Set
NStraight non-toothed fine-tip forceps (13.5-cm long) NCurved non-toothed fine-tip forceps (13.5 cm long) NNon-locking needle holder with a rounded, finely curved tip NPair of straight or curved blunt dissecting scissors NBipolar cautery with fine-tipped forceps NSmall retractor NBlunt, long and rounded irrigating needle NMicrosurgical scalpel NAutoclavable case NSilicone tubing for protecting instrument tips
Micro-TESE and MESA
Operating Microscope: Operating microscope equipped with 200-, 300- and 350-mm objective lenses and motorized operated zoom system Note: The optical, mechanical and electrical microscope components should be checked before surgery to ensure that the operational conditions are adequate. A spare lamp should be readily available. A sterile microscope cover and/or handles should be available to allow for microscope adjustments during surgery.
All
Reagents and Laboratory Supplies: Sperm culture media (kept at 37 ˚C) 6-mL sterile centrifuge polystyrene tubes with caps 60615-mm center-well Petri dishes (micro-TESE)
N N
N N N
PESA: percutaneous epididymal sperm aspiration; TESA: testicular sperm aspiration; MESA: microsurgical epididymal sperm aspiration; TESE: testicular sperm extraction; micro-TESE: microdissection testicular sperm extraction.
and materials that are commonly used in sperm retrievals is provided in Table 4.
Conventional Open Sperm Retrieval Methods Open surgical SR can be used for both epididymal and testicular sperm collection. In both cases, a scrotal incision is made to approach the epididymis or the testis. Testicular delivery to facilitate the exposure of the epididymis or testis is optional, as the procedures can be carried out without testis delivery using the ‘‘window’’ technique (14). In the open epididymal sperm aspiration, the goal is to puncture an epididymal tubule and aspirate the epididymal fluid using a needle. In the open testicular sperm extraction (TESE) procedure, either a large single biopsy or multiple biopsies
Anesthesia Sperm retrievals are relatively simple surgeries that can be safely performed with general anesthesia or spinal blocks. However, because sperm retrievals are typically outpatient procedures, the latest trend is to employ local or locoregional anesthesia with or without intravenous sedation. A review of the anesthesia techniques used for SR is provided in a separate section below.
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Figure 2 - Percutaneous epididymal sperm retrieval. The epididymis is stabilized between the index finger, thumb and forefinger. A needle attached to a tuberculin syringe is inserted into the epididymis through the scrotal skin, and fluid is aspirated (see the text for a detailed description). Figure 1 - Conventional testicular sperm extraction (TESE). The illustration depicts TESE using a single open biopsy (see the text for a detailed description). Adapted from: Esteves SC, Agarwal A. Sperm retrieval techniques. In: Gardner DK, Rizk BRMB, Falcone T, Eds. Human assisted reproductive technology: future trends in laboratory and clinical practice. 1st. edition. Cambridge: Cambridge University Press 2011; pp. 41-53.
in the upper, middle and lower testicular poles in an organized manner for the sampling of different areas. The testicular specimens are sent to the laboratory for processing and immediate microscopic examination. The tunica albuginea is closed with a running, non-absorbable suture. Single Seminiferous Tubule Biopsy. This technique is a variation of TESE. The scrotum is opened, and the testis is exposed. An avascular area of the tunica is punctured with a 26-gauge needle. A microforceps tip is used to dilate the puncture site, thus allowing a loop of seminiferous tubule to emerge (15). The seminiferous tubule is pulled out using the microforceps and sent for microscopic examination. If sperm are seen, additional tubule is pulled out from the same site. If no sperm are found or the tubule appears fibrous, the procedure is repeated in a different area. Multiple sites can be sampled until sperm are found or the entire testicular surface has been explored. Albuginea openings are not sutured because these openings are very small. Like the open epididymal FNA, single seminiferous tubule biopsy does not require special equipment or training but has not been validated in a large patient series.
are performed to obtain seminiferous tubules and their contents. In both cases, the retrieved spermatozoa can be used for fresh sperm injection or cryopreserved for a single or multiple subsequent ICSI attempts. Open epididymal sperm aspirations are only indicated in OA cases, whereas open testicular extractions can be used in both OA and selected NOA cases (Table 2). Open Epididymal Fine-Needle Aspiration. The epididymis is exposed and a tubule is directly punctured through the tunica without any dissection (15). The epididymal fluid is aspirated using a 26-gauge needle; the epididymal fluid that continues to flow out of the punctured tubule upon needle withdrawal is also aspirated. The tubular opening is not closed. Epididymal fluid can be aspirated from different locations to maximize the number and quality of sperm retrieved. The procedure does not require special equipment or training, but it has not been validated in a large series of patients. Testicular Sperm Extraction (TESE). The extraction of the testicular parenchyma for sperm search and isolation was first described in 1995 (16). For conventional TESE, a standard open surgical biopsy technique is used to remove the testicular parenchyma without the aid of optical magnification. This procedure is usually carried out without delivering the testis (14). Briefly, a 2-cm transverse incision is made through the anterior scrotal skin, dartos and tunica vaginalis. A small selfretaining retractor can be used to ensure proper exposure of the tunica albuginea. A 1-cm incision is made in the albuginea, and gentle pressure is applied to the testis to aid the extrusion of the testicular parenchyma. A fragment of approximately 565 mm is excised with sharp scissors and placed in sperm culture media (Figure 1). Single or multiple specimens can be extracted from the same incision. Alternatively, individual albuginea incisions can be made
Percutaneous Sperm Retrieval Methods Since their description in 1994, the use of percutaneous approaches to retrieve sperm from the epididymis has gained popularity (1,5,17,18). Both the epididymal and testicular techniques share similar traits, as they require that a needle be percutaneously inserted into the sperm source (14). The goal of percutaneous epididymal sperm aspiration (PESA) is to obtain the epididymal fluid, which should contain sperm. In testicular sperm aspiration (TESA), the seminiferous tubules and their contents are removed. Percutaneous sperm retrieval may have either a diagnostic or a therapeutic role (19). Regarding the former role, the procedure confirms the presence of viable spermatozoa that can be cryopreserved for future use prior to ICSI. Regarding the latter role, the procedure is performed in conjunction with oocyte retrieval and permits the use of fresh sperm for sperm injections. In addition to offering a less invasive alternative for retrieving sperm, percutaneous techniques can generally be performed under local anesthesia on an outpatient basis. Percutaneous
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Figure 3 - Percutaneous testicular sperm aspiration. A 20-mL needle syringe connected to a Cameco holder is percutaneously inserted into the testis. Negative pressure is created, and the tip of the needle is moved within the testis to disrupt the seminiferous tubules and sample different areas. The testicular parenchyma is aspirated (see the text for a detailed description).
Figure 5 - Testicular fine-needle aspiration (TEFNA). A 23-gauge fine needle attached to a 10-mL syringe coupled to a Cameco syringe holder is percutaneously inserted into the testicle to map different areas. Negative pressure is applied, and the needle is moved in and out within the testis with no change in direction. A tissue fragment from each mapped area is expelled into a preidentified tube containing sperm culture medium.
testicular retrievals are indicated in OA cases, as well as selected cases of NOA. In contrast, percutaneous epididymal retrievals are only recommended in OA cases (Table 2). Percutaneous Epididymal Sperm Aspiration (PESA). The technical procedure for percutaneous epididymal sperm aspiration involves the insertion of a needle attached to a syringe through the scrotal skin into the epididymis (Figure 2). Originally, the use of a larger butterfly needle was described (17). Currently, most experts use a fine needle (26 gauge) attached to a tuberculin syringe containing sperm washing medium (1,5,14). After creating negative pressure by pulling the syringe plunger, the tip of the needle is gently and slowly moved in and out inside the epididymis until fluid is aspirated. If motile sperm are not obtained, PESA may be repeated at a different site (from the
cauda to caput epididymis) until an adequate number of motile sperm is retrieved. These aspirations are usually performed in the corpus epididymis and then in the caput epididymis if needed, as aspirates from the cauda are often rich in poor-quality senescent spermatozoa, debris and macrophages (13). Because PESA is a blind procedure, multiple attempts may be needed before high-quality sperm are found. If PESA fails to enable the retrieval of motile sperm, testicular sperm retrieval can be attempted during the same operation. Testicular Sperm Aspiration (TESA) and Testicular FineNeedle Aspiration (TEFNA). In TESA, a needle is inserted
through the scrotal skin into the testis (Figure 3). The needle is usually inserted into the anteromedial or anterolateral portion of the superior testicular pole at an oblique angle toward the medium and lower poles. These areas are the least likely to contain major branches of the testicular artery running superficially underneath the tunica albuginea. These aspirations are usually carried out using either fine (testicular fine-needle aspiration; TEFNA) or large-diameter needles attached to a syringe. The testicular parenchyma is aspirated by creating negative pressure, and the specimen is sent to the laboratory for microscopic examination (Figure 4). TESA can be carried out in the contralateral testis if an insufficient number of or no sperm are obtained during the first attempt. Alternatively, testicular parenchyma can be obtained percutaneously using a tissue-cutting biopsy needle (e.g., a Tru-cutTM needle or BioptyTM gun). For this procedure, the needle is placed against the testis and, upon release of the springer, the needle enters the parenchyma, cuts a piece of tissue and withdraws it into a sheath (21). Turek et al. proposed the use of systematic fine-needle aspiration of the testis (FNA mapping) as a diagnostic tool in cases of non-obstructive azoospermia (22). However, testicular fine-needle aspiration (TEFNA) can also be
Figure 4 - Photograph showing a tube containing one fragment of testicular tissue obtained by percutaneous testicular sperm aspiration (TESA). The fragment is immersed in sperm culture medium.
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Figure 6 - Microsurgical epididymal sperm aspiration (MESA). After exposure of the testis and epididymis, a dilated epididymal tubule is dissected and opened. The fluid is aspirated, diluted with sperm medium and sent to the laboratory for examination.
Figure 7 - Microdissection testicular sperm extraction (microTESE). Microsurgical techniques and instruments (A), including an operating microscope (B), are used throughout the procedure. After testis exteriorization, a single large incision is made in an avascular area of the albuginea (C), and the testicular parenchyma is widely exposed (D).
applied for therapeutic sperm retrieval in cases of obstructive and non-obstructive azoospermia (Table 2). The concept behind FNA is to map the testicle to direct biopsies to preidentified areas of sperm production, thus facilitating sperm retrieval in cases of non-obstructive azoospermia (NOA). Depending on the size of the testis, four to nine systematically placed aspiration sites are mapped (Figure 5). FNA mapping is performed with a sharp-beveled 23-gauge fine needle attached to a 10-mL syringe coupled with a Cameco syringe holder. Suction is applied, and the syringe holder is held steady as the needle is moved in and out within the testis with no change in direction. Twenty to 30 incursions are performed at a depth range of 8 to 12 mm. Suction is released before the needle is withdrawn from the testis. Tissue fragments are expelled from the needle onto a slide after air aspiration and fixed by immersion in 95% ethyl alcohol in the cases in which TEFNA is used for diagnostic purposes. In therapeutic SR, tissue fragments are expelled into pre-identified tubes containing sperm media.
incision. The epididymal tunica is incised, and an enlarged tubule is selected. Then, the epididymal tubule is dissected and opened with sharp microsurgical scissors. The fluid that flows out of the tubule is aspirated with the aid of a silicone tube or a needle attached to a tuberculin syringe (Figure 6). The aspirate is flushed into a tube containing warm sperm medium and is transferred to the laboratory for examination. MESA can be repeated at a different site on the same epididymis (from the cauda to caput regions) and/or the contralateral epididymis until an adequate number of motile sperm is retrieved (1,14). If MESA fails to retrieve motile sperm, TESA or TESE can be performed as part of the same procedure. However, MESA often provides enough sperm for cryopreservation. A single MESA procedure usually enables the retrieval of a large number of high-quality sperm that can be used for ICSI or intentionally cryopreserved for subsequent ICSI attempts (4,24). Microsurgical Testicular Sperm Extraction (micro-TESE). Microdissection testicular sperm extraction was originally described in 1999 in a successful combination of testicular sperm extraction with the assistance of an operating microscope (24). For micro-TESE, the scrotal skin is stretched over the anterior surface of the testis, after which a 2-3-cm transverse incision is made. Alternatively, a single midline scrotal incision can be used (25). The incision extends through the dartos muscle and the tunica vaginalis. The tunica is opened, and identifiable bleeders are cauterized. The testis is delivered extravaginally, and the tunica albuginea is examined. Then, a single, large, mid-portion incision is made in an avascular area of the tunica albuginea under 6-86 magnification, and the testicular parenchyma is widely exposed in its equatorial plane (Figure 7). The testicular parenchyma is dissected at 16-256 magnification to enable the search and isolation of seminiferous tubules that exhibit larger diameters (which are more likely to contain germ cells and eventually normal sperm production) in comparison to non-enlarged or collapsed counterparts (Figure 8). If needed, the superficial and deep testicular regions can be examined, and microsurgical-guided testicular biopsies are performed by carefully removing enlarged tubules
Microsurgical Sperm Retrieval Methods Microsurgical-guided sperm acquisition has been applied in both epididymal and testicular retrievals. The goal of microsurgical epididymal sperm aspiration (MESA) is to identify and open a single epididymal tubule to aspirate a sperm-rich, red blood cell-free fluid that can be used for fresh sperm injection or cryopreserved for a single or multiple later ICSI attempts. In microsurgical testicular sperm extraction (microdissection TESE; micro-TESE), the testicular parenchyma is dissected under magnification to search for enlarged seminiferous tubules, which are more likely to contain germ cells and foci of sperm production compared to non-enlarged or collapsed tubules. Such seminiferous tubules are removed rather than proceeding with the large single or multiple biopsies performed in conventional TESE. Microsurgical techniques and instruments, including an operating microscope, are used throughout both the MESA and micro-TESE procedures. MESA is indicated for cases of OA, whereas micro-TESE is recommended for the most severe cases of NOA (Table 2). Microsurgical Epididymal Sperm Aspiration (MESA). MESA was first described in 1985 (23). This surgical technique requires testis delivery through a 2-3-cm transverse scrotal
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Figure 8 - Photograph showing the micro-TESE intraoperative aspect (256 magnification). The seminiferous tubules with enlarged diameters (black arrow) are likely to contain active spermatogenesis, while the thin tubules usually contain Sertoli cells only (white arrow).
Figure 9 - Photograph showing a petri dish (left) containing seminiferous tubules obtained by microdissection testicular sperm extraction (micro-TESE) immersed in sperm culture medium. The specimen is mechanically minced under stereomicroscopy to release the content of the seminiferous tubules (right).
using microsurgical forceps. If enlarged tubules are not observed, any tubule that differs from the remaining tubules in size is excised. The excised testicular tissue specimens are placed into the inner well of a Petri dish containing sperm media, and are sent to the laboratory for processing and sperm search (1,13,14) (Figure 9). The tunicas albuginea and vaginalis are then closed in a running fashion using non-absorbable and absorbable sutures. The dartos muscle is closed with interrupted absorbable sutures, respectively. Immediately prior to complete closure, 3 cc of 1% xylocaine solution may be injected into the subcuticular layers. The skin is closed using a continuous subcuticular 4-0 vicryl suture. A fluffy-type scrotal dressing and scrotal supporter are placed.
In another study of 26 patients undergoing MESA, only 38% of the patients tolerated the procedure solely under spermatic cord block through the infiltration of 5-8 mL of 1% lidocaine; the remaining 62% required intravenous sedation (28). The percentage of patients who underwent a bilateral procedure and required intravenous sedation was as high as 75%. General anesthesia may offer comfort and the efficient management of anxiety. However, when performed with inhalational agents such as N2O and halogenated agents, this approach is associated with a high incidence of postoperative nausea and vomiting (29). These two complaints are among the most frequent causes of hospitalization and the inability to discharge patients scheduled for ambulatory procedures. Additionally, these symptoms are among the most feared by patients undergoing minor surgery, surpassing even postoperative pain (30). The opposite effect occurs when employing propofol (2,6diisopropylphenol), as this drug offers antiemetic effects (31). Propofol is a hypnotic intravenous drug that can be used both to induce and maintain general anesthesia and sedation. Moreover, propofol causes a gentle awakening compared with halogenated agents, as patients wake up with a feeling of well-being and a clear mental state. In addition, patients usually experience less postoperative confusion, recognizing the environment and the people around them more easily. Patients also tend to be more cooperative and show less agitation (32). Thus, patients undergoing general anesthesia combined with propofol have a lower incidence of postoperative complaints but still have a slower recovery compared with those receiving propofol alone for intravenous sedation. The combination of intravenous sedation and local anesthesia offers patients the analgesic effectiveness of local anesthetics combined with the comfort and effective control of anxiety provided by intravenous sedation. When this combination includes propofol, patients experience great satisfaction, recovering quickly and with minimal adverse effects. These patients may also benefit from the advantages of outpatient procedures.
& ANESTHESIA FOR SPERM RETRIEVAL PROCEDURES There are very few studies describing anesthesia techniques for SR. From the anatomy viewpoint, it is possible to provide efficient anesthesia simply by using local or locoregional anesthesia. However, most patients express great concern about the procedures, most likely because sperm retrievals are carried out in a very delicate part of the male body. For this and other reasons related to the high expectations associated with the procedure, patients undergoing SR have historically been very anxious on the day of surgery. In a study of 34 patients undergoing PESA and/or TESA, spermatic cord block was performed with 10 mL of 1% lidocaine without epinephrine (27). The authors reported block failure in 6% of the cases, which required the combination of intravenous sedation, and two cases of vasovagal reflex, which required the use of atropine for reversal. They also stated that 16% of the patients reported moderate but tolerable pain. The results of the aforementioned study highlight the fact that the chosen anesthesia technique enabled SR to be performed; however, this method cannot be considered a good-quality anesthesia technique because it did not offer enough comfort to a large proportion of the group studied. Furthermore, 35% of the patients complained about being very anxious preoperatively, which shows that over a third of the patients could have benefited from the coadministration of locoregional anesthesia and sedation.
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have diminished androgen production, such as Klinefelter syndrome patients (33). Nonetheless, testosterone levels return to pre-surgical values in most individuals within 12 months following surgery (39). In fact, Ramasamy et al. reported a return to 95% of the pre micro-TESE testosterone levels after 18 months (34). Given the potential serious postoperative complications of SR, it is recommended that these procedures be performed by surgeons who have specific training in the above-mentioned techniques (39).
& POSTOPERATIVE CARE AND COMPLICATIONS OF SPERM RETRIEVAL The vast majority of SR procedures are performed on an outpatient basis, with patients usually being discharged 23 hours after surgery. Patients should be examined for a scrotal hematoma prior to discharge. A companion should be available, and under no circumstances should the patient be allowed to leave the heath care facility alone or drive if general anesthesia or intravenous sedation has been used. After percutaneous retrievals, patients often resume their normal activities on the following day. Bed rest and the application of an ice pack to the scrotum is recommended for the first 48 hours, especially following open retrievals. For these procedures, patients are instructed to remove the scrotal dressing after 24 hours and are encouraged to take warm showers and wash the incision area with soap and water after 24 hours. Oral analgesics and non-steroidal antiinflammatory medications are routinely used for 3-5 days. Postoperative antibiotics are not routinely prescribed. Patients are instructed to resume a normal diet and increase their daily activities to a normal level over a 3- to 4-day period. The use of a scrotal supporter is strongly recommended for approximately one week after the procedure. The patient should abstain from sports activities, heavy lifting and sexual intercourse for approximately 10 days. Moreover, patients should be informed of the likelihood of scrotal swelling and ecchymosis at the wound site, as well as mild discomfort that should subside in approximately one week (14,19). After SR, patients are advised to report any adverse signs and symptoms, including fever, persistent pain or swelling, bleeding or excessive fluid leakage from the wound. A scrotal ultrasound may be indicated in cases with complications. The determination of hormone levels, including total and free testosterone, FSH, LH, and estradiol, is recommended six months after open testicular retrievals. The incidence of post-SR complications, including persistent pain, swelling, infection, hydrocele, and hematoma, ranges from 0-70% (33-41). The complication rates vary depending on the sperm retrieval technique. Percutaneous retrievals have an increased risk of hematoma compared with open techniques (5,37). Nevertheless, except for minor pain and local swelling, there have been no reports of clinically significant intra- or postoperative complications leading to medical treatment or hospital care when percutaneous techniques are used (1,38). Intratesticular hematomas have been observed on ultrasounds performed three months after surgery in most patients (up to 80%) who undergo TESE with single or multiple biopsies, but they often resolve spontaneously without compromising testicular function (35). Large-volume conventional TESE has been associated with a higher risk of a transient or even permanent decrease in serum testosterone levels due to testicular devascularization and excessive tissue removal (34,39). On the other hand, the incidence of complications is lower following micro-TESE compared to conventional TESE (11,25,34,36). In micro-TESE, the testicular vessels under the tunica albuginea are identified prior to the placement of an incision in the testis. In addition, the use of optical magnification and microsurgical techniques allows the preservation of the intratesticular blood supply (34). However, a significant decrease in serum testosterone has been documented following micro-TESE in men who already
& EXPERT COMMENTARY The literature is rich in studies focusing on different sperm retrieval methods. Both percutaneous and microsurgical methods have high success rates, in the range of 90100%, for OA (37,38,42-44). A series of studies on NOA has reported overall successful retrieval rates (SRRs) ranging from 30-60% (11-14,22,24,25,38,45-50), which means that 3060% of men with NOA have focal areas of sperm production within the testes. In a recent study, we reported a cumulative success rate of 97.3% for percutaneous retrievals in OA cases (38). Epididymal sperm retrievals were successful in 78.0% of the cases, and subsequent attempts at testicular retrieval were successful in the vast majority of failed epididymal retrievals. We concluded that percutaneous SR was a reliable method for obtaining sperm for ICSI in OA. Our overall complication rate following percutaneous retrievals was 5.5%, and we noted that complications, albeit of minimal morbidity, occurred more often in the patients undergoing TESA compared with those undergoing PESA. For this reason, we use percutaneous methods for sperm acquisition in OA and preferentially use PESA over TESA. In our recent study, sperm cryopreservation was possible in one-third of the cases. Although increased cryopreservation rates have been reported for open SR, the associated costs of this procedure are significantly higher (5). Percutaneous approaches, on the other hand, can be performed under local anesthesia on an outpatient basis, and, if needed, repeat percutaneous procedures may result in successful SR (37). It is still a matter of debate whether percutaneous retrievals are more cost-effective than MESA. No study has yet compared the cumulative pregnancy rates after repeated cycles of percutaneous retrievals and ICSI with a single MESA attempt for intentional sperm cryopreservation coupled with multiple subsequent ICSI cycles. Nonetheless, the ICSI outcomes using frozen-thawed or fresh sperm retrieved from men with OA are comparable (51). The testicular SRRs associated with the different etiological categories of non-obstructive azoospermia—namely, cryptorchidism, varicocele, orchitis, genetic, radio-/chemotherapy and idiopathic—are comparable (14,33,52-55). The efficiency of sperm retrieval in NOA males varies depending on the method of sperm collection. The TESA retrieval rates range from 10-30% (11,35,39,46,47,56,57) except in the favorable cases of a previous successful TESA or a testicular histopathology showing hypospermatogenesis. In such cases, the TESA SRRs are greater than 60% (14,53). A recent meta-analysis reported a mean TESE SRR of 49.5% (11). TESE with multiple biopsies has a higher SRR than fine-needle aspiration (TEFNA), especially in cases of Sertoli cell-only (SCO) syndrome and maturation arrest (11). The reported micro-TESE retrieval rates range
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Moreover, the testis is usually delivered, thus causing some degree of tension on the spermatic cord. As a result, a more intense nociceptive stimulus is expected, which requires an anesthesia technique capable of providing increased analgesia in addition to autonomic response blockade. Deep sedation with assisted ventilation or even general anesthesia with controlled ventilation using drugs with a short or ultrashort duration is the preferred technique. However, it is also possible to obtain sufficient anesthesia using spermatic cord block associated with mild to moderate sedation (32). For this purpose, we use a 4-mg.kg-1 induction dose of propofol followed by a 60- to 100-mg.kg-1.min-1 infusion under spontaneous or assisted ventilation using a face mask with 100% oxygen, according to the needs and characteristics of the patient. Small amounts of an opioid, such as fentanyl (1 to 2 mg.kg-1) or alfentanil (7 to 15 mg.kg-1), are added before the surgeon injects 1% lidocaine at the incision site. The cord block is achieved with 4 to 6 mL of the same local anesthetic, which is injected by the surgeon when the pampiniform plexus is exposed.
from 35-77% (5,12,14,25,33,36,41,47,49,51,54,55). Moreover, controlled studies demonstrated that micro-TESE performs better than conventional TESE or TESA (12,47-50). MicroTESE has been shown to minimize the damage to testicular tissue and maximize sperm recovery because the seminiferous tubules containing active foci of spermatogenesis can be better identified (34). Micro-TESE was shown to be particularly more effective than conventional TESE in recovering sperm from men with a testicular volume of less than 10 mL (42% vs. 27%) (58). It seems that the best chance of sperm recovery during micro-TESE is within the first 2 hours of the operation. However, more than four hours were required to achieve success in up to 37% of men (59). In a recent controlled study, we compared micro-TESE with conventional single-biopsy TESE in a group of 60 men with NOA (12). Overall, the SRRs were significantly higher when micro-TESE was used (45% vs. 25%). Furthermore, the results were in favor of micro-TESE after patient stratification by the histopathology categories of hypospermatogenesis (93% vs. 64%), maturation arrest (64% vs. 9%) and Sertoli cell-only syndrome (2% vs. 6%). In cases of NOA, our preference is to use micro-TESE over the other SRT. However, our patients exhibiting hypospermatogenesis on previous testicular histopathology or those with a history of a successful SR attempt are eligible for TESA as the firstchoice method if their testicular volume is larger than 10 cc. Our SRRs with TESA and micro-TESE have proven comparable (51%) with this treatment algorithm (14,53). Based on our ten-year experience in the management of azoospermic men seeking fertility treatment, the likelihood of a successful sperm retrieval is 43-fold higher (odds ratio [OR] = 43.0; 95% confidence interval [CI]: 10.3-179.5) in men with OA compared with men with NOA (60). Sperm retrieval procedures are always carried out in a restricted region. As such, it is often not difficult to implement local or locoregional anesthesia even for more extensive interventions, such as micro-TESE. Despite this fact, general anesthesia and spinal blocks are often used for open procedures because such modalities are safe and effective and, as a rule, most patients are too anxious on the day of the procedure to support the use of local anesthesia. However, simpler, less expensive, and less invasive techniques that offer higher patient satisfaction and quicker recovery are more suitable for outpatient procedures. Due to the brevity and small to moderate intensity of the pain stimulus in PESA, local anesthesia is not required when employing intravenous sedation. The use of propofol as a single agent under spontaneous and/or assisted ventilation with a face mask offers excellent results not only in terms of the surgeon’s working conditions during manual testis mobilization but also in terms of patient satisfaction by providing anxiolysis and comfort. The dosage should be tailored to each patient by the anesthetist to obtain the necessary sedation level and is usually in the range of 3 to 4 mg.kg-1. Small amounts of an opioid, such as fentanyl (1 to 3 mg.kg-1) or alfentanil (10 to 20 mg.kg-1), can be added if necessary. In TESA, it is appropriate to add local anesthetic infiltration to the intravenous propofol sedation, as TESA requires further manipulation of the testis. Our preference is to couple sedation with a percutaneous block of the spermatic cord by injecting 6 to 8 mL of 1% lidocaine without a vasoconstrictor at the external inguinal ring. In contrast, open procedures require an incision to be made.
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Sperm retrieval techniques (SRTs) are surgical methods that have been developed to retrieve spermatozoa from the epididymides and the testicles of azoospermic men seeking fertility treatment. After sperm acquisition, intracytoplasmic sperm injection (ICSI) is used in place of standard in vitro fertilization (IVF) because ICSI has been shown to result in a significantly higher fertilization rate. From the clinical standpoint, the goals of sperm retrieval are two-fold: i) to obtain an adequate number of the highest quality sperm possible, which can be immediately used for ICSI or alternatively cryopreserved for future ICSI attempts, and ii) to minimize damage to the reproductive tract, thus preserving the option of repeated retrieval attempts and testicular function. The two general SR methods are open surgery and percutaneous acquisition. Open surgery can be carried out to retrieve spermatozoa from the epididymis or the testicle with or without microsurgery. Percutaneous retrievals require a needle to be percutaneously inserted into the sperm source, i.e., the epididymis or the testicle. Irrespective of the method used, the goal is to obtain the epididymal fluid or the seminiferous tubules and their contents. Epididymal retrievals are only indicated in cases of obstructive azoospermia, whereas testicular extractions can be used in both obstructive and non-obstructive azoospermia cases. Sperm production is normal and gametes can be easily retrieved from the epididymis or testis in virtually all cases of obstructive azoospermia. In obstructive azoospermia, the choice of sperm retrieval technique should be based on the surgeon’s preferences and expertise, as there is no evidence that one particular method is superior to another. Although increased cryopreservation rates have been reported for open surgical retrieval methods, the costs of these methods are significantly higher. Percutaneous approaches, on the other hand, can be performed under local anesthesia on an outpatient basis and, if needed, be repeated to achieve a successful
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SR. In general, sperm retrieval in obstructive azoospermia is associated with low complication rates and minimal morbidity. Sperm production is markedly impaired or absent in men with non-obstructive azoospermia. In this clinical scenario, testicular sperm extraction is the method of choice for sperm retrieval. Overall, successful retrieval rates range from 30-60%, which means that 30-60% of the men with NOA have focal areas of sperm production within the testes. The efficiency of retrieval in non-obstructive azoospermia is related to the method of sperm acquisition. Percutaneous testicular aspiration retrieval rates range from 10-30% and are markedly lower than the 50% success rate reported for testicular sperm extractions. Microsurgical-guided sperm acquisition has been applied in both epididymal and testicular retrievals. The goal of microsurgical epididymal sperm aspiration (MESA) is to identify and open a single epididymal tubule to enable the aspiration of a sperm-rich, red blood cell-free fluid that can be used for fresh sperm injection or cryopreserved for a single or multiple later ICSI attempts. In microsurgical testicular sperm extraction (micro-TESE), the testicular parenchyma is dissected under magnification to search for enlarged seminiferous tubules, which are more likely to contain germ cells and foci of sperm production. MESA is indicated for OA cases, whereas micro-TESE is recommended for the most severe NOA cases. Microsurgical retrievals require microsurgical training, microsurgical instruments and an operating microscope. These techniques are associated with increased operative time and costs. Micro-TESE has superior sperm retrieval rates and requires the removal of much less tissue than conventional open testicular retrievals. Micro-TESE has been successfully used in different populations of men with testicular failure. Complications after testicular retrievals include intratesticular hematoma, pain, swelling, infection, and hydrocele. Most complications resolve spontaneously without compromising testicular function. The extraction of a large volume of testicular parenchyma may lead to a transient or permanent decrease in serum testosterone levels due to testicular devascularization and excessive tissue removal. The incidence of complications is lower following micro-TESE than conventional TESE because the former procedure enables the excision of a minimal amount of tissue and preserves the vasculature. Sperm retrieval techniques are relatively simple surgeries that can be safely completed with general anesthesia or spinal blocks. However, because these surgeries are typically outpatient procedures, the latest trend is to employ local or locoregional anesthesia with or without intravenous sedation. The combination of local anesthesia and intravenous sedation offers the patient the analgesic effectiveness of local anesthetics combined with the comfort and effective control of anxiety provided by intravenous sedation. When this combination includes propofol, patients experience greater satisfaction, recovering quickly and with minimal adverse effects. These patients may also benefit from the advantages of outpatient procedures.
& AUTHOR CONTRIBUTIONS All of the authors were involved in drafting and revising the manuscript, and all of the authors read and approved the final version.
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REVIEW
Predictive factors for sperm retrieval and sperm injection outcomes in obstructive azoospermia: Do etiology, retrieval techniques and gamete source play a role? Ricardo Miyaoka, Sandro C. Esteves ANDROFERT – Andrology & Human Reproduction Clinic, Referral Center for Male Reproduction, Campinas, Sa˜o Paulo, Brazil.
Obstructive azoospermia is a relatively common male infertility condition. The main etiologies of obstructive azoospermia include congenital, surgical-derived, traumatic and post-infectious cases. Although seminal tract reconstruction is a cost-effective treatment in most cases, this approach may not be feasible or desired in some cases. In such cases, assisted reproduction techniques offer a method for achieving pregnancy, notably via sperm retrieval and intracytoplasmic sperm injection. This process requires several considerations and decisions to be made, including the cause and duration of obstruction, which sperm retrieval technique to use, and whether to use fresh or frozen-thawed sperm. We present a review of obstructive azoospermia and assisted reproduction techniques, highlighting the most relevant aspects of the decision-making process for use in clinical practice. KEYWORDS: Male Infertility; Obstructive Azoospermia; Sperm Retrieval; Intracytoplasmic Sperm Injection; Outcomes; Review. Miyaoka R, Esteves SC. Predictive factors for sperm retrieval and sperm injection outcomes in obstructive azoospermia: Do etiology, retrieval techniques and gamete source play a role? Clinics. 2013;68(S1):111-119. Received for publication on April 8, 2012; Accepted for publication on April 11, 2012 E-mail: s.esteves@androfert.com.br Tel.: 55 19 3295-8877
or caused by trauma (surgical or non-surgical) or infection. Generally, spermatogenesis is fully preserved. The absence of sperm on routine examination must be confirmed by at least two semen samples collected 1-4 weeks apart and centrifuged at high speed (1500-1800 g for 15 min at room temperature) to distinguish azoospermia from cryptozoospermia; otherwise, an equivocal diagnosis may occur in up to 20% of cases (4,5). If retrograde ejaculation is suspected, a post-ejaculate urine specimen should be analyzed (6). Additional proposed criteria to diagnose obstructive azoospermia include a normal hormonal profile, normal-sized testis, and normal spermatogenesis, as evidenced by a testicular biopsy or an epididymal aspirate full of spermatozoa (7). Ninety-six percent of men with OA are found to have FSH levels #7.6 mIU/mL or a testicular long axis .4.6 cm, whereas 89% of men with non-obstructive azoospermia (NOA) have FSH levels .7.6 mIU/mL or a testicular long axis #4.6 cm (8). In patients with OA, although normal spermatogenesis is preserved, the quality of spermatozoa may be altered because the distal epididymis contains a high number of sperm fragments with macrophages (9). The number of macrophages progressively decreases toward the proximal epididymis and testis, while the quantity of motile sperm gradually increases. The concentration of motile spermatozoa in the epididymal fluid can be as high as 1 million sperm per mL (10).
& INTRODUCTION Azoospermia, defined as the complete absence of sperm from the ejaculate, affects approximately 1% of all men and 5 to 10% of all subfertile males seeking care (1,2). Although the majority of cases are secondary to an impairment of testicular function, a bilateral obstruction of the male genital tract causes azoospermia in up to 20 to 40% of cases (3). We review the currently available data regarding sperm retrieval in obstructive azoospermia (OA). Here, we present the current status of the literature concerning sperm retrieval and intracytoplasmic sperm injection (ICSI) outcomes in relation to the cause of obstruction, the method of sperm retrieval, and the gamete source.
& OBSTRUCTIVE AZOOSPERMIA: AN OVERVIEW Obstructive azoospermia is defined as the absence of spermatozoa in the ejaculate secondary to a physical disruption of the seminal tract, which may be congenital
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)12
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pregnancy rates with ART treatment might also decline (22). In a study by Sukcharoen et al. comprising 17 patients and 21 ICSI cycles within a period of two years, subjects were divided into three groups according to the time elapsed since vasectomy: 0-10 years, 11-20 years, or more than 20 years. No difference could be found regarding fertilization rates (FRs), implantation rates (IRs), and pregnancy rates (PRs) between the groups. The authors of the aforementioned study concluded that the interval between vasectomy and surgical sperm retrieval associated with ICSI treatment had no impact on pregnancy outcomes, although it should be noted that the analyzed sample was small and likely lacked statistical power to enable definitive conclusions (23). Congenital unilateral (CUAVD) and bilateral (CBAVD) absence of the vas deferens and congenital obstruction of the epididymides are all part of the spectrum of vasal aplasia. CBAVD affects 2% of infertile men and has been described as a primary genital form of cystic fibrosis (5). Approximately 80% of CBAVD patients exhibit definable mutations within at least one allele of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is located on the short arm of chromosome 7 (24). This gene encodes a protein that functions as an ion channel but also affects the formation of the distal 2/3 of the epididymis, vas deferens, seminal vesicle and ejaculatory duct. An additional 10 to 15% of men with vasal aplasia exhibit renal anomalies (e.g., agenesis, failure of assent, and ectopia). The diagnosis of vasal aplasia must be suspected in infertile men with a low semen volume and with a low pH as a consequence of the absence of the seminal vesicles. Spermatogenesis in CBAVD appears normal, but sperm derived from the caput epididymis are thought to have a low fertilizing capacity in conventional IVF cycles because of their short passage through the epididymis (25). All men with CBAVD should be assumed to harbor CFTR mutations, as a negative genetic test does not rule out the mutation with 100% accuracy due to the gene length and testing limitations. However, genetic screening should be offered to the female partner because a 25 to 50% risk of inheritance of cystic fibrosis exists if she also carries a mutation (26). Because spermatogenesis is not altered by this condition, sperm can be retrieved from both the testicle and the epididymis without difficulty. In an earlier study by Chen et al. (27), 31 patients with obstructive azoospermia underwent epididymal aspiration procedures associated with ART (including IVF, zygote intra-Fallopian transfer, subzonal insemination). The fertilization rate for caput spermatozoa was lower than that for spermatozoa derived from other areas of the epididymis (p,0.05). In the aforementioned study, the CBAVD group exhibited a higher probability to achieve pregnancy compared with the acquired obstruction group (20 versus 5.9%, p.0.05). Currently, ICSI is used rather than conventional IVF after sperm retrieval because sperm injection has been shown to result in a significantly higher fertilization rate (28). Ejaculatory duct abnormalities can also result in obstruction. Congenital ejaculatory duct obstruction (EDO) represents an anomaly caused by maldevelopment of the portion of the Wolffian ductal system that courses through the central zone of the prostate and enters the prostatic urethra. Mu¨llerian duct and prostatic utricle cysts can extrinsically compress the ejaculatory ducts and result in obstruction
Vasectomy comprises the main cause of OA, and 2 to 6% of vasectomized men will seek reversal to restore fertility. In these cases, microsurgical reconstruction by either vasovasostomy or vasoepididimostomy is the standard of care (1113). There is compelling evidence that vasectomy reversal is a more cost-effective approach than in vitro fertilization (IVF) for vasectomy-related OA (14,15). However, the final therapeutic decision should be individualized for each couple, as certain situations (e.g., significant female factor infertility or social factors) may favor sperm retrieval (SR) and IVF as more suitable treatment options. Both men with OA who are not candidates for reconstructive surgery and those who fail to achieve success with such procedures have been historically considered hopelessly infertile. However, with the first reports of successful pregnancies achieved by the use of IVF in the early 1990s, a new perspective emerged (16,17). Currently, IVF is an established assisted reproductive technique (ART) with consistent results. The Society of Assisted Reproductive Technology (SART) publishes yearly data on the effectiveness of IVF in different patient populations. A total of 17% of all IVF cycles from 2000 to 2008 were performed due to male factor infertility. The live birth rate associated with the use of IVF is increasing. In fact, the delivery rate across all age groups increased from 34.5% in 2003 to 37.3% in 2008. The use of ICSI for male factor infertility also increased from 84 to 87% during this time period. Data from SART, however, do not offer fine enough resolution to distinguish IVF treatments undertaken for obstructive versus non-obstructive azoospermia (15,18). In Latin America, the latest report from the Red Latinoamericana de Reproducio´n Asistida (RedLara) in 2009 noted ICSI and IVF procedure proportions of 85 and 15%, respectively; a total of 27,174 IVF/ICSI cycles were initiated, resulting in 7,141 live births (19). Again, it is not possible to determine the proportion of such treatments applied to men with OA. As the number of ART procedures performed is expected to increase with the anticipated increase in the current 15% incidence of infertility over the next 20 years (20), some questions have emerged regarding the state-of-the-art methods used to perform sperm retrieval for use in IVF/ ICSI.
& THE IMPACT OF ETIOLOGY ON RETRIEVAL SUCCESS AND REPRODUCTIVE OUTCOMES AFTER ICSI Common causes of OA include previous vasectomy, congenital bilateral absence of the vas deferens (CBAVD), infectious epididymitis/orchitis, Young’s Syndrome, and testicular trauma (21). Some authors include ejaculatory dysfunctions in the OA category, including retrograde ejaculation and anejaculation (7,21). The appropriateness of the inclusion of these conditions in the OA category is, however, debatable, as there is no anatomical barrier precluding sperm from being ejaculated in these conditions. The most common cause of iatrogenic ductal obstruction is vasectomy. Although vasectomy reversal is achievable in many cases, the scope of this review will be limited to vasectomized men who failed reconstruction or opted for sperm retrieval procedures associated with IVF treatment. After vasectomy, testicular histology changes, as noted by the presence of fibrosis and decreased spermatid number, which worsen with longer periods of obstruction; as such,
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(29). Acquired EDO causes include chronic prostatitis, prostatic calcification, prior prostatic biopsy, and iatrogenic damage to the ejaculatory ducts during urethral instrumentation (6,30). Transurethral resection of the ejaculatory ducts (TURED) has been the classic treatment for this disorder. Alternatively, sperm retrieval can be attempted with ICSI. Epididymal, testicular, and transperineal seminal vesical sperm retrievals have been applied in such cases with adequate yields of motile sperm for either immediate use with ICSI or cryopreservation (31). In cases of ejaculatory dysfunction, spermatogenesis is fully preserved, and gametes can be recovered either from the urine (in cases of retrograde ejaculation) or from the epididymides or testicles with any chosen surgical sperm retrieval technique (in cases of primary anorgasmia or failed urinary sperm recovery). In a recent study, Esteves et al. (32) reported a cumulative sperm retrieval success rate (SRR) of 97.9% using percutaneous epididymal sperm aspiration (PESA), in association or not with testicular sperm aspiration (TESA), in men with OA, regardless of the cause of obstruction. PESA alone was able to retrieve sperm in more than 80% of the cases. Reproductive outcomes after ICSI were not affected by the source (epididymis versus testicle) or etiology (congenital, failed vasectomy reversal, post-infectious disease). Successful epididymal sperm retrievals were achieved in all congenital obstructive cases, whereas testicular retrievals were needed in approximately 1/3 of the cases classified in the other etiology groups (vasectomy, post-infectious obstructions). In a meta-analysis involving 756 ICSI cycles using surgically retrieved sperm, Nicopoullos et al. compared the outcomes of ICSI between patients with congenital and acquired OA. The meta-analysis revealed no difference in either clinical pregnancy rate (relative risk [RR]: 1.03; 95% confidence interval [CI]: 0.75-1.31; p = 0.87) or live birth rate (RR: 1.03; 95% CI: 0.81-1.31; p = 0.80) between patients with congenital and acquired cases of OA. A significantly higher fertilization rate was noted in the acquired group (RR: 0.92; 95% CI: 0.84-1; p = 0.05), while a significantly higher miscarriage rate (MR) was noted in the congenital group (RR: 2.67). The authors concluded that in ICSI cycles for men with OA, the cause of OA appears to influence the outcome, with higher FRs and lower MRs observed in patients with acquired OA. However, tests of heterogeneity were significant, and it should be noted that the studies included had no power to detect clinically significant differences in the analyzed outcomes (21). A more recent and larger series conducted by Kamal et al. involved 1,661 ICSI cycles in 1,121 men with proven histological OA. The outcomes were compared according to the sperm retrieval source and cause of obstruction. The fertilization rates (68.0% versus 64.2%, p = 0.02), implantation rates (19.9% versus 20.8%, p = 0.41), and frequencies of clinical pregnancies (43.2% versus 42.3%, p = 0.84) and miscarriages (18.4% vs. 17.6%, p = 1.0) resulting from the use of testicular and epididymal spermatozoa, respectively, were comparable in ICSI. Similar rates were maintained after stratification according to the cause of obstruction (CBAVD versus acquired obstruction), suggesting that neither the origin of surgically retrieved spermatozoa nor the cause of obstruction have any significant effect on the success of IVF/ICSI (7).
& IMPACT OF THE METHOD OF COLLECTION ON RETRIEVAL SUCCESS AND REPRODUCTIVE OUTCOMES AFTER ICSI The method of choice for sperm retrieval is based on the attending surgeon’s preferences and the preferences of the embryologist involved in the patient’s care. In a metaanalysis by Van Peperstraten et al., the technique for sperm retrieval (microsurgical epididymal sperm aspiration [MESA] versus epididymal micropuncture with perivascular nerve stimulation and ultrasound-guided TESA versus conventional TESA) and the sperm source (testis, epididymis, vas deferens, or seminal vesicle) did not seem to play a role in the pregnancy rates achieved with IVF/ICSI (33). All methods have been shown to provide sufficient viable sperm for ICSI and often also for cryopreservation (34). The use of surgical techniques for sperm retrieval in OA men, as well as the use of retrieved spermatozoa in IVF treatments, dates back to the 1980s with the description of MESA by Temple-Smith et al. (35). Thereafter, many other techniques have been developed, including those meant to extract sperm from the epididymis and from the testis. Their use varies according to the azoospermia etiology and specific scenarios (36). Sperm retrieval should focus on three main goals: 1. To retrieve an adequate number of sperm for both immediate use and cryopreservation; 2. To obtain the highest quality sperm possible; 3. To minimize damage to the reproductive tract to not jeopardize future sperm retrieval attempts or testicular function (36,37). Several authors have examined the effectiveness of percutaneous procedures for sperm retrieval in OA. Sperm retrieval rates have been quoted at approximately 100% when percutaneous epididymal and testicular retrievals are combined (38-43) (Table 1). Glina et al. reported a series of 58 men with OA treated with ICSI who underwent percutaneous epididymal sperm retrievals (with rescue TESA whenever needed). The authors reported 100% recovery of motile sperm using these combined techniques. Successful repeated PESA was performed up to three times, with recovery of motile sperm in over 80% of the cases. Forty-three percent of PESA procedures yielded sufficient spermatozoa to allow cryopreservation (42). Esteves et al. reported a SRR of 97.9% among 142 men with OA. In these series, TESA as a rescue procedure after a failed PESA was performed in 17% of the cases. One-third of the retrievals yielded a sufficient number of spermatozoa for cryopreservation (32). Lin et al. analyzed 100 men with irreparable OA who underwent 109 ICSI cycles. The PESA SRR was 61%. MESA or testicular sperm extraction (TESE) were successfully performed if PESA failed. Fertilization and pregnancy rates were not significantly different for PESA-ICSI cycles (56 and 39%, respectively) and MESA-ICSI cycles (47 and 45%, respectively) (43). Despite the notably lower SRR for PESA the authors’ data corroborate the ability to perform rescue procedures in cases of initial failure. The reasons for epididymal retrieval failures include obstructions at the level of the rete testis, which, according to Pryor (44), can be found in up to 15% of OA men. Such individuals have normal spermatogenesis but no clinical findings suggestive of obstruction. Even grossly distended
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Table 1 - Studies reporting sperm retrieval efficacy and/or pregnancy outcomes in men with obstructive azoospermia. No. of Mean Paternal cycles Age (years)
Author
Country
Design
Rosenlund et al. 1998
Sweden
Retrospective
NR
37
Recovery of viable sperm on repeated PESA
Levine et al. 1998
USA
Retrospective
37
NR
Dohle et al. 1998
The Netherlands
Retrospective
39
38.1
Efficacy and safety of percutaneous sperm retrieval and ICSI outcomes (CPR) using retrieved sperm ICSI outcomes (ongoing pregnancy rates) using percutaneously or surgically-retrieved sperm
Janzen et al. 2000
USA
Retrospective
108
38.3
ICSI outcomes (CPR) using fresh or frozen epididymal sperm
Brazil
Retrospective
79
45
Success at obtaining sperm by PESA and CPR after ICSI
Westlander et al. 2001
Sweden
Retrospective
22
34.8
ICSI outcomes up to ongoing pregnancy/ delivery rates after TESA
Levine et al. 2003
USA
Retrospective
112
37.1
ICSI outcomes (CPR) after percutaneous sperm retrieval
Dozortsev et al. 2006
Brazil
Retrospective
185
NR
ICSI outcomes (ongoing pregnancy rates) with percutaneous sperm retrieval
Pasqualotto et al. 2006
Brazil
Retrospective
155
NR
ICSI outcomes according to etiology of OA
USA
Retrospective
38
39.1
ICSI outcomes (LBR) using TESA
1,661
39.2
ICSI outcomes (CPR) according to sperm source and etiology of obstruction
258
NR
ICSI outcomes (live birth rate) using fresh or frozen-thawed retrieved sperm
Glina et al. 2003
Garg et al. 2008
Kamal et al. Egypt, The Retrospective 2010 Netherlands, United Kingdom
Kalsi et al. 2010
United Kingdom
Retrospective
Outcome
Main findings
Conclusion
High sperm recovery PESA is simple, offers a high rates were found sperm recovery rate and can on repeated PESA be safely repeated in OA men, up to 4 times yielding similar SRR results. Efficacy and safety of PESA and TESA are more percutaneous sperm effective alternatives retrieval techniques (PESA, compared with the more TESA) were demonstrated invasive MESA approach. High fertilization rate Both percutaneously was obtained after ICSI and surgically retrieved regardless of the spermatozoa provide retrieval method adequate pregnancy outcomes. Both fresh and cryoComparable pregnancy thawed epididymal sperm outcomes for fresh and frozen epididymal sperm, harvested by MESA with logistic-related yielded similar CPR advantages for frozen sperm High SRR on repeated PESA is simple, offers a high PESA up to 4 times sperm retrieval rate and can be safely repeated in men with OA. TESA can be repeated Repeated TESA is safe and effective with no negative impact on the recovery of mature spermatozoa or pregnancy outcome PESA and TESA are Percutaneous sperm highly effective aspiration is effective, for sperm retrieval safe and reproducible and offer similar pregnancy outcomes with ICSI. Higher FR in the Embryo development was PESA group and higher significantly better when testicular sperm was implantation rate in the TESA group were reported; used for ICSI trends toward higher PR and lower miscarriage rate in the TESA group No impact of etiology Higher FR and of OA on CPR implantation rate in men with congenital OA; similar PR in all etiology categories TESA is highly effective TESA is an effective means of recovering mature in recovering motile spermatozoa and motile sperm which are offers adequate suitable for cryopreservation pregnancy outcomes. in most cases. The source of sperm and Similar pregnancy the etiology of obstruction outcomes for different do not seem to influence sperm sources (testicular pregnancy and miscarriage or epididymal) and rates. causes of obstruction (congenital or acquired) Higher pregnancy No negative impact of using and live birth rates frozen-thawed epididymal were reported for frozen- or testicular sperm for ICSI; a thawed compared with tendency for higher PR and live birth rate associated fresh sperm with frozen-thawed testicular sperm
SRR: sperm retrieval rate; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; PESA: percutaneous epididymal sperm aspiration; TESA: testicular sperm aspiration; MESA: microsurgical epididymal sperm aspiration; FR: fertilization rate; PR: pregnancy rate; CPR: clinical pregnancy rate; LBR: live birth rate; NR: not reported.
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regardless of the cause. In early ICSI series, the fertilization and pregnancy rates using surgically retrieved spermatozoa obtained from the epididymis or testis were comparable to the results of ejaculated sperm (51,52). As both sources were proven adequate for ICSI, the possible treatment outcome advantages of one over the other have been explored. Testicular sperm aspiration pregnancy rates with ICSI are reportedly as high as 31%, with a calculated delivery rate of 27% considering a miscarriage rate of 11.6% (38). Similarly, pregnancy rates with ICSI using epididymal sperm are reported to be as high as 43%, with a calculated delivery rate of 38% if a similar miscarriage rate is assumed (53-55). In a meta-analysis by Nicopoullos et al. (56), fertilization rates were reported to vary from 45 to 72% for epididymal and 34 to 81% for testicular sperm. No differences in cleavage, PRs or IRs were reported in any of the individual articles. Relative risk (RR) ratios of 1.08, 1.01, and 0.71 were described for the fertilization rate, clinical pregnancy rate and live birth rate, respectively, for epididymal compared with testicular sperm (p.0.05). The authors of the aforementioned studies therefore concluded that when a diagnosis of OA is made, epididymal aspiration should be the retrieval method of choice in view of the possible complications of testicular extraction, including inflammation, hematoma, devascularization (57) and decreased serum testosterone levels (58). Similar results were reported by Kamal et al. (7) in a large cohort involving 1,121 patients with OA undergoing ICSI. These authors argued that the preferential use of testicular sperm or epididymal sperm in cases of OA is unfounded, as neither the source nor the etiology seems to affect sperm injection outcomes. Conversely, Dozortsev et al. reported higher FRs using spermatozoa retrieved from the epididymis compared with the testis in OA cases (77.2 versus 67.5%, p = 0.0005). However, patients in the testicular sperm group exhibited significantly higher IR (20.8 versus 32.8%, p = 0.008), with a trend toward higher ongoing PR and lower miscarriage rate. These authors speculate that motile sperm randomly taken from the epididymis have lower reproductive potential than random sperm taken from the testicle, and argue that the prolonged presence of sperm within the epididymis may lead to structural chromosomal aberrations that can compromise the reproductive potential of the such cells (59). To corroborate these findings, recent studies have demonstrated that epididymal sperm exhibit more DNA damage than sperm retrieved directly from the testis. The frequency of terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL)-positive cells according to the sperm cell origin has been reported as follows: 9.3ยก2.3% in the testis, 17.4ยก4.0% in the epididymis, and 29.2ยก6.7% in the vas deferens (60,61). Despite these reports, sperm retrieved directly from the testis are generally limited in number and are often immotile or nonprogressively motile, although they are still viable and almost always functional for use in ART (48).
epididymides represent a poor clinical sign of obstruction and can be misleading. Successful epididymal sperm retrieval in these cases can be achieved in approximately 70% of cases (40). The PESA SRR is influenced by the anatomical conformation of the epididymis, and PESA may be more difficult in small and loosely attached epididymides, which can make isolation more difficult. Needle width can also affect the results and must be selected properly. Levine et al. reported their experience using a 23gauge butterfly needle to aspirate spermatozoa from the epididymis with satisfactory sperm quantity for ICSI use (45). In a study by Mallidis and Baker (46) comparing devices for fine needle tissue aspiration biopsy of the testis, the authors observed that the 20-gauge biopsy needles with stylet penetrated easier within the testis and caused the least amount of tissue distortion. Repeat percutaneous procedures have been associated with successful sperm retrieval. Repeated PESA up to three times on the same unilateral epididymis with equivalent fertilization rates was described as being successful by Rosenlund et al. in a retrospective series involving 27 men with OA or ejaculatory dysfunction (47). Sperm retrieval rates of 91, 89, and 86% were reported at the first, second and third PESA, respectively. Repeated TESA has also been reported to yield SRRs of 100 and 96% for first and second attempts, respectively. No significant impacts on fertilization rates were observed by the authors of the study. PESA and TESA have some disadvantages compared with open surgical sperm retrievals (36). Both methods have an increased risk of hematoma compared with open techniques (48). Nevertheless, except for minor pain and local swelling, there are no reports of clinically significant perioperative or postoperative complications leading to medical treatment or hospital care when percutaneous techniques are used. The time interval between the procedures (classified as less than three months, three to six months, and more than six months) did not influence the outcome (sperm recovery, fertilization, and pregnancy rates). It should be noted, however, that the TESA technique is not standardized in the published studies, and in most centers, TESA is considered a rescue procedure for OA cases (48,49). Other factors associated with the preference for one technique over the other include the quantity of retrieved spermatozoa and the ability to cryopreserve the excess retrieved sperm. PESA has been associated with better recovery of motile spermatozoa compared with testicular retrieval (TESA) (100% versus 39.3%, p,0.0001) in patients with azoospermia or severe oligozoospermia (33). However, the ability to cryopreserve excess retrieved sperm is lowest in percutaneous compared with open sperm retrievals (33). Interestingly, Garg et al. (50) reported a 97.5% success rate in motile sperm recovery that was adequate for intentional cryopreservation in 40 men with either OA or ejaculatory dysfunction using TESA. Cryopreservation is important because it prevents the need for future retrieval procedures in the event that ICSI fails.
& IMPACT OF GAMETE SOURCE (EPIDIDYMIS OR TESTICLE) ON REPRODUCTIVE OUTCOMES WITH ICSI
& IMPACT OF GAMETE STATUS (FRESH OR FROZEN-THAWED) ON REPRODUCTIVE OUTCOMES AFTER ICSI
As stated earlier, spermatozoa can be recovered from either the epididymis or the testicle with a high probability of success in men diagnosed with obstructive azoospermia,
The cryopreservation of epididymal and testicular sperm offers the advantage of a single retrieval procedure for
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sperm collection, thereby enabling the use of stored spermatozoa in multiple IVF cycles. Cryopreservation avoids the logistical difficulties associated with attempts to coordinate sperm retrieval and ART on the same day and permits infectious triage prior to insemination (48). However, cryopreservation has been shown to negatively affect motility, acrosome integrity, acrosin activity, and morphology of both testicular and ejaculated spermatozoa. For example, Prabakaran et al. noted a 25% decrease in postthaw testicular sperm motility (62). Decreased sperm morphology, mitochondrial function, and viability have also been described by O’Connell et al. after testicular sperm cryopreservation and thawing (63). The cryopreservation and thawing process has also been associated with the lipid peroxidation of sperm plasma membranes, which may facilitate free radical oxygen species access to sperm nuclei and ultimately disrupt DNA integrity (64). Despite these potential downsides, the clinical pregnancy rate of 141 ICSI cycles using frozen-thawed (n = 33) or fresh (n = 108) spermatozoa retrieved from men with OA were comparable (60.6% vs. 66.7%, respectively) in the series of Jansen et al. (65). Garg et al. (50) also reported similar fertilization rates and biochemical pregnancy rates resulting from the use of frozen-thawed or fresh testicular spermatozoa. Conversely, a significantly higher clinical pregnancy rate was observed for fresh epididymal sperm versus frozen-thawed counterparts in a meta-analysis study, despite the fact that the fertilization (RR: 1.02; 95% CI: 0.96-1.08; p = 0.61) and implantation (RR: 1.20; 95% CI: 1.01.42; p = 0.05) potential of both gametes were equivalent (33). Intriguingly, a recent retrospective study by Kalsi et al. compared 493 ICSI cycles with the use of fresh versus frozen sperm by stratifying results according to the testicular and epididymal sources. Unexpectedly higher PR (60.0% versus 32.1%, p,0.05) and live birth rate (60.0% versus 28.6%, p,0.05) were observed for frozen-thawed testicular sperm compared with fresh testicular sperm. The miscarriage rates were equivalent (25.0% versus 29.2% for fresh and frozenthawed sperm, respectively) (66).
& EXPERT COMMENTARY In a group of 2,383 infertile men attending our tertiary center for male reproduction, 835 (35%) were identified as having azoospermia; approximately 36% of those cases resulted from obstruction in the ductal system. The adoption of strict criteria to diagnose OA is crucial for obtaining a high retrieval success rate in the range of 90100% using percutaneous techniques. In our experience, percutaneous sperm retrieval is a highly effective method for collecting sperm in men with OA. Successful sperm retrieval was achieved in over 85% of the cases using PESA, but more than one aspiration was often required (32). In cases of failed PESA, TESA was adequate to obtain sperm in nearly all cases. Motile spermatozoa were obtained in approximately 73% of the cases after the first or second PESA aspiration, and TESA was performed as a rescue procedure after failed PESA in approximately 14% of the individuals (32). Our results show that ICSI outcomes using spermatozoa collected by PESA or TESA are similar, thus suggesting that the reproductive potential of those gametes is independent of their source in OA. However, epididymal spermatozoa are easier to handle in the IVF laboratory compared with testicular sperm, and it is more likely that there will be excess sperm for freezing if epididymal retrievals are performed. In our group of men with OA, fertilization and live birth rates did not differ between individuals who had vasectomy/failed reversal, CBAVD or infection as the cause of obstruction (32). Despite the fact that higher rates of sperm cryopreservation have been reported for open sperm retrieval compared to percutaneous retrieval, the costs of the former procedure are significantly higher. Cryopreservation is important because it prevents the need for future retrieval procedures in the case that ICSI fails. However, if needed, repeat percutaneous procedures result in successful sperm retrieval. This is encouraging because the need for open procedures is lessened. It is debatable whether percutaneous retrievals are more cost-efficient than MESA, and no study to date has compared the cumulative pregnancy rate per couple using repeat percutaneous retrievals and fresh sperm injections with a single MESA attempt and intentional sperm cryopreservation for use in multiple subsequent ICSI attempts. Percutaneous sperm retrieval techniques can be performed for both diagnostic and therapeutic purposes. For the latter purpose, sperm retrieval is often performed on the same day of oocyte retrieval or on the preceding day. Using PESA, our approach is to perform the first aspiration at the epididymis corpus and proceed to the caput if necessary because aspirates from the cauda are usually of poor quality and contain senescent spermatozoa, debris, and macrophages. Most cases of PESA failures are not necessarily technical failures because immotile spermatozoa is usually retrieved. As a diagnostic procedure, the presence of spermatozoa at the time of percutaneous epididymal retrieval has shown to have 93% sensitivity and 94% specificity to diagnose obstructive azoospermia (70). Usually, percutaneous sperm retrievals are performed on an outpatient basis. Patients are discharged one hour later and can return to normal activities the following day. Oral analgesics are prescribed, but pain complaints are minimal. The most common complication is fibrosis at the aspiration site. Other potential complications include hematoma,
& ASSESSMENT OF CHILDREN BORN WITH ICSI USING SPERM RETRIEVED FROM MEN WITH OA Concern has been raised regarding the risk of using nonejaculated spermatozoa in assisted reproduction. Epididymal sperm may be immature or senescent because of the long stay in the obstructed epididymis, which may lead to genetic risks if these sperm are used for fertilization (61). Testicular sperm, in turn, have the potential risk of incomplete genomic imprinting, incomplete chromatin condensation, and incomplete protamination (67,68). In a recent Dutch prospective, multicenter study by Woldringh et al. (69), 378 children born from ICSI cycles using retrieved epididymal sperm were evaluated. More than 1,000 children born as a result of ICSI or IVF using ejaculated sperm were available for comparison. Assessments were performed at birth and at 1 and 4 years of age using mailed questionnaires. Moreover, follow-up visits of 2-year-old children were carried out to evaluate motor performance and mental-language development. The epididymal sperm group did not exhibit a higher incidence of stillbirths, malformations or poor development compared with the reference group of children born after ICSI or IVF using ejaculated sperm.
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Due to the importance of obstructive azoospermia in the context of male infertility, there is a need for the execution of well-designed multi-center studies with adequate sample sizes and follow-up durations, the development of standard data sets to enable the differentiation of men into the various OA subgroups, the consideration of the cause of obstruction and the source of the male gamete, and the use of control groups of children conceived naturally or with ICSI using ejaculated sperm. For now, the continued monitoring of children born after ICSI using non-ejaculated sperm from men with OA should be set as the minimum standard.
bleeding and infection, but these complications are rare. On rare occasions, we perform MESA for sperm retrieval in OA men with coagulation disorders. While PESA is sufficient for the majority of patients, TESA is needed for more difficult cases. We noted that significantly fewer men with CBAVD required a TESA procedure compared with the post-infectious and vasectomy groups. Our results indicate that PESA is sufficient for sperm retrieval in CBAVD cases but that TESA will need to be performed in approximately 1/3 of cases in the other etiology groups (32). In our series, the higher percentage of vasectomy and post-infectious patients, compared to those with congenital obstruction, requiring a rescue TESA procedure after the initial failed epididymal attempt is likely due to the epididymis being obstructed after vasectomy or failed reversals or being severely damaged after infection, thus making it difficult to percutaneously retrieve motile sperm. The concept of cryopreservation may be used in association with sperm retrieval procedures. Epididymal and testicular spermatozoa can be cryopreserved using protocols routinely used for ejaculated sperm. Some centers prefer to retrieve and intentionally cryopreserve sperm for future use. If sperm are frozen, thawing can be performed at any time, thus obviating the need to organize two operations (oocyte and sperm retrieval) on the same day. Additionally, cryopreservation may be an interesting method for storing leftover specimens that would otherwise be discarded after ICSI, especially if the treatment cycle does not result in a pregnancy. Future ICSI attempts may be performed without repeated surgical retrievals. We routinely freeze excess motile epididymal spermatozoa that are not needed for the current ICSI cycle. Most often, motile sperm will be available after thawing in such cases, and ICSI outcomes using motile fresh or frozen epididymal sperm do not appear to differ. The best currently available evidence regarding the influence of the cause of obstruction on ICSI results is provided by a meta-analysis that was entirely based on a retrospective heterogenic series (21). Therefore, further studies are needed, particularly studies with a prospective design. Moreover, few studies have considered the impact of maternal age on fecundity. Vasectomy reversal may not be the first line therapy for couples in which the female partner is of advanced maternal age because sperm retrieval and ICSI expedites the time to pregnancy and delivery. Moreover, when analyzing studies that compare pregnancy outcomes, it must be noted that a significant proportion of them consider only the results achieved for the first ICSI cycle. These results can be misleading, as success can be achieved in subsequent cycles as a result of optimized ovarian stimulation of the female partner or the replacement of cryopreserved embryos. Finally, there is little high-quality evidence on short- and long-term pregnancy outcomes with ICSI using spermatozoa from men with OA. From the limited data available, no major differences have been reported in the short-term neonatal outcomes of children born from such fathers. ICSI with epididymal sperm does not lead to a higher incidence of stillbirths or congenital malformations compared with IVF and ICSI with ejaculated sperm and does not lead to poor childhood development (69). In this sense, the Dutch follow-up study is reassuring, but obtaining more data on fetal, neonatal and long-term outcomes should be identified as a major research priority.
& KEY ISSUES
N N N
N N N N N N
In men with OA, sperm production is normal, and gametes can be easily retrieved from the epididymis or testis in the vast majority of cases. The sperm retrieval technique and the cause of obstruction have little impact on sperm retrieval success rates. Percutaneous sperm retrievals are simple and effective methods for collecting epididymal or testicular spermatozoa in OA. TESA should be performed as a rescue procedure because TESA may impose a higher risk of complications. MESA may yield a higher number of motile sperm but is not demonstrably cost-effective and is more technically demanding compared with percutaneous retrieval methods. The time interval since vasectomy and the paternal age do not seem to affect the sperm retrieval success rate or pregnancy outcomes from sperm injections. The current evidence demonstrates equivalent pregnancy outcomes in OA men when comparing epididymal and testicular sperm, fresh and frozen-thawed sperm, and different causes of obstruction. Sperm chromatin integrity seems to be decreased toward the distal portion of an obstructed seminal tract. Motile sperm retrieved from men with OA should be cryopreserved whenever possible for future use, thereby sparing men from unnecessary procedures. No major difference has been reported in the short-term neonatal outcomes of children born from fathers with OA. ICSI performed with epididymal sperm does not lead to a higher rate of stillbirths or congenital malformations compared with IVF and ICSI with ejaculated sperm and does not lead to poor childhood development. However, the current data are limited, and the continued monitoring of children born to OA fathers is of utmost importance.
& AUTHOR CONTRIBUTIONS Miyaoka R and Esteves SC were involved in the acquisition and analysis of the data, as well as the drafting and revision of the manuscript.
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The role of testicular biopsy in the modern management of male infertility. J Urol. 2002;167(1):197-200. 9. Schlegel PN, Berkeley AS, Goldstein M, Cohen J, Alikani M, Adler A, et al. Epididymal Micropuncture with in-Vitro Fertilization and Oocyte Micromanipulation for the Treatment of Unreconstructable Obstructive Azoospermia. . Fertil Steril. 1994;61(5):895-901. 10. Schlegel PN. Causes of azoospermia and their management. Reprod Fert Develop. 2004;16(5):561-72, http://dx.doi.org/10.1071/RD03087. 11. Belker AM, Thomas AJ, Fuchs EF, Konnak JW, Sharlip ID. Results of 1,469 Microsurgical Vasectomy Reversals by the Vasovasostomy StudyGroup. J Urol. 1991;145(3):505-11. 12. Potts JM, Pasqualotto FF, Nelson D, Thomas AJ, Agarwal A. Patient characteristics associated with vasectomy reversal. J Urol. 1999;161(6):1835-9. 13. Sandlow JI, Westefeld JS, Maples MR, Scheel KR. Psychological correlates of vasectomy. Fertil Steril. 2001;75(3):544-8, http://dx.doi. org/10.1016/S0015-0282(00)01744-1. 14. Lee R, Li PS, Goldstein M, Tanrikut C, Schattman G, Schlegel PN. A decision analysis of treatments for obstructive azoospermia. Hum Reprod. 2008;23(9):2043-9, http://dx.doi.org/10.1093/humrep/den200. 15. Lee R, Li PS, Schlegel PN, Goldstein M. Reassessing reconstruction in the management of obstructive azoospermia: reconstruction or sperm acquisition? Urol Clin North Am. 2008;35(2):289-301, http://dx.doi. org/10.1016/j.ucl.2008.01.005. 16. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992;340(8810):17-8, http://dx.doi.org/10.1016/0140-6736(92)92425-F. 17. Vansteirteghem AC, Nagy Z, Joris H, Jiaen L, Staessen C, Smitz J, et al. High Fertilization and Implantation Rates after Intracytoplasmic Sperm Injection. Hum Reprod. 1993;8(7):1061-6. 18. Shridharani A, Sandlow JI. Vasectomy reversal versus IVF with sperm retrieval: which is better? Curr Opin Urol. 2010;20(6):503-9, http://dx. doi.org/10.1097/MOU.0b013e32833f1b35. 19. Red Latinoamericana de Reproducio´n Asistida (RedLara). Accessed in March 27th, 2012. Available from: http://www.redlara.com/aa_portugues/ registro_anual.asp?categoria=Registros%20Anuais&cadastroid=316#. 20. Stephen EH, Chandra A. Updated projections of infertility in the United States: 1995-2025. Fertil Steril. 1998;70(1):30-4, http://dx.doi.org/10. 1016/S0015-0282(98)00103-4. 21. Nicopoullos JDM, Gilling-Smith C, Ramsay JWA. Does the cause of obstructive azoospermia affect the outcome of intracytoplasmic sperm injection: a meta-analysis. BJU Int. 2004;93(9):1282-6, http://dx.doi.org/ 10.1111/j.1464-410X.2004.04817.x. 22. McVicar CM, O’Neill DA, McClure N, Clements B, McCullough S, Lewis SE. Effects of vasectomy on spermatogenesis and fertility outcome after testicular sperm extraction combined with ICSI. Hum Reprod. 2005;20(10):2795-800, http://dx.doi.org/10.1093/humrep/dei138. 23. Sukcharoen N, Sithipravej T, Promviengchai S, Chinpilas V, Boonkasemsanti W. No differences in outcome of surgical sperm retrieval with intracytoplasmic sperm injection at different intervals after vasectomy. Fertil Steril. 2000;74(1):174-5, http://dx.doi.org/10. 1016/S0015-0282(00)00579-3. 24. Oates RD, Amos JA. The Genetic-Basis of Congenital Bilateral Absence of the Vas-Deferens and Cystic-Fibrosis. J Androl. 1994;15(1):1-8. 25. Silber SJ, Balmaceda J, Borrero C, Ord T, Asch R. Pregnancy with Sperm Aspiration from the Proximal Head of the Epididymis - a New Treatment for Congenital Absence of the Vas-Deferens. Fertil Steril. 1988;50(3):525-8. 26. Anguiano A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, et al. Congenital bilateral absence of the vas deferens. A primarily genital form of cystic fibrosis. JAMA. 1992;267(13):1794-7. 27. Chen CS, Chu SH, Soong YK, Lai YM. Epididymal sperm aspiration with assisted reproductive techniques: difference between congenital and acquired obstructive azoospermia? Hum Reprod. 1995;10(5):1104-8. 28. Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey P, Van Steirteghem AC. Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum Reprod. 1994;9(9):1705-9.
29. Netto NR, Jr., Esteves SC, Neves PA. Transurethral resection of partially obstructed ejaculatory ducts: seminal parameters and pregnancy outcomes according to the etiology of obstruction. J Urol. 1998;159(6):204853. 30. Cornel EB, Dohle GR, Meuleman EJ. Transurethral deroofing of midline prostatic cyst for subfertile men. Hum Reprod. 1999;14(9):2297-300, http://dx.doi.org/10.1093/humrep/14.9.2297. 31. Cerruto MA, Novella G, Antoniolli SZ, Zattoni F. Use of transperineal fine needle aspiration of seminal vesicles to retrieve sperm in a man with obstructive azoospermia. Fertil Steril. 2006;86(6):1764-9. 32. Esteves SC, Lee W, Benjamin DJ, Seol B, Verza S Jr, Agarwal A. Reproductive potential of men with obstructive azoospermia undergoing percutaneous sperm retrieval and intracytoplasmic sperm injection according to the cause of obstruction. J Urol. 2013;189(1):232-7, http:// dx.doi.org/10.1016/j.juro.2012.08.084. 33. Van Peperstraten A, Proctor ML, Johnson NP, Philipson G. Techniques for surgical retrieval of sperm prior to ICSI for azoospermia. Cochrane Database Syst Rev. 2006(3):CD002807. 34. Practice Committee of the American Society for Reproductive Medicine in collaboration with the Society for Male Reproduction and Urology. The management of infertility due to obstructive azoospermia. Fertil Steril. 2008;90(Suppl.3):S121-4. 35. Temple-Smith PD, Southwick GJ, Devroey P,Van Steirteghem AC. Pregnancies after intracytoplasmic sperm injection of single spermatozoa into an oocyte. Lancet. 1992;2:17-8. 36. Esteves SC, Miyaoka R, Agarwal A. Sperm retrieval techniques for assisted reproduction.Int Braz J Urol. 2011;37:570-83. 37. Esteves SC, Miyaoka R, Agarwal A. Surgical treatment of male infertility in the era of intracytoplasmic sperm injection - new insights. Clinics. 2011;66(8):1463-78, http://dx.doi.org/10.1590/S1807-59322011000800026. 38. Belker AM, Sherins RJ, Dennison-Lagos L, Thorsell LP, Schulman JD. Percutaneous testicular sperm aspiration: A convenient and effective office procedure to retrieve sperm for in vitro fertilization with intracytoplasmic sperm injection. J Urol. 1998;160(6):2058-62. 39. Westlander G, Hamberger L, Hanson C, Lundin K, Nilsson L, Soderlund B, et al. Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men. Hum Reprod. 1999;14(1):118-22, http://dx. doi.org/10.1093/humrep/14.1.118. 40. Bromage SJ, Falconer DA, Lieberman BA, Sangar V, Payne SR. Sperm retrieval rates in subgroups of primary azoospermic males. Eur Urol. 2007;51(2):534-40, http://dx.doi.org/10.1016/j.eururo.2006.08.032. 41. Levine LA, Dimitriou RJ, Fakouri B. Testicular and epididymal percutaneous sperm aspiration in men with either obstructive or nonobstructive azoospermia. Urology. 2003;62(2):328-32, http://dx.doi. org/10.1016/S0090-4295(03)00374-1. 42. Glina S, Fragoso JB, Martins FG, Soares JB, Galuppo AG, Wonchockier R. Percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia. Int Braz J Urol. 2003;29(2):141-5, discussion 5-6. 43. Lin YM, Hsu CC, Kuo TC, Lin JS, Wang ST, Huang KE. Percutaneous epididymal sperm aspiration versus microsurgical epididymal sperm aspiration for irreparable obstructive azoospermia--experience with 100 cases. J Formos Med Assoc. 2000;99(6):459-65. 44. Pryor JP. Indications for vesiculography and testicular biopsy. An update. In: ColpiGM, PozzaD. Diagnosing Male Infertility: New Possibilities and Limits. Basel: Karger; 1992.p.130-5. 45. Levine LA, Lisek EW. Successful sperm retrieval by percutaneous epididymal and testicular sperm aspiration. J Urol. 1998;159(2):437-40. 46. Mallidis C, Baker HW. Fine needle tissue aspiration biopsy of the testis. Fertil Steril. 1994;61(2):367-75. 47. Rosenlund B, Westlander G, Wood M, Lundin K, Reismer E, Hillensjo T. Sperm retrieval and fertilization in repeated percutaneous epididymal sperm aspiration. Hum Reprod. 1998;13(10):2805-7, http://dx.doi.org/ 10.1093/humrep/13.10.2805. 48. The Practice Committee of the American Society for Reproductive Medicine: Sperm retrieval for obstructive azoospermia. Fertil Steril. 2008; 90(5 Suppl):S213-8. 49. Westlander G, Rosenlund B, Soderlund B, Wood M, Bergh C. Sperm retrieval, fertilization, and pregnancy outcome in repeated testicular sperm aspiration. J Assist Reprod Genet. 2001;18(3):171-7, http://dx.doi. org/10.1023/A:1009459920286. 50. Garg T, LaRosa C, Strawn E, Robb P, Sandlow JI. Outcomes after testicular aspiration and testicular tissue cryopreservation for obstructive azoospermia and ejaculatory dysfunction. J Urol. 2008;180(6):2577-80. 51. Silber SJ, Devroey P, Tournaye H, Vansteirteghem AC. FertilizingCapacity of Epididymal and Testicular Sperm Using Intracytoplasmic Sperm Injection (Icsi). Reprod Fert Develop. 1995;7(2):281-93, http://dx. doi.org/10.1071/RD9950281. 52. Mansour RT, Kamal A, Fahmy I, Tawab N, Serour GI, Aboulghar MA. Intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum Reprod. 1997;12(9):1974-9, http://dx.doi.org/10. 1093/humrep/12.9.1974. 53. Spandorfer SD, Davis OK, Barmat LI, Chung PH, Rosenwaks Z. Relationship between maternal age and aneuploidy in in vitro fertilization
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REVIEW
Prognostic factors for sperm retrieval in non-obstructive azoospermia Sidney Glina,I,II Marcelo VieiraI,III I
Projeto ALFA, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina do ABC, Santo Andre´/SP, Brazil.
III
Hospital Pe´rola Byngton, Sa˜o Paulo/SP, Brazil.
Testicular sperm retrieval techniques associated with intracytoplasmic sperm injection have changed the field of male infertility treatment and given many azoospermic men the chance to become biological fathers. Despite the current use of testicular sperm extraction, reliable clinical and laboratory prognostic factors of sperm recovery are still absent. The objective of this article was to review the prognostic factors and clinical use of sperm retrieval for men with non-obstructive azoospermia. The PubMed database was searched for the Medical Subject Headings (MeSH) terms azoospermia, sperm retrieval, and prognosis. Papers on obstructive azoospermia were excluded. The authors selected articles that reported successful sperm retrieval techniques involving clinical, laboratory, or parenchyma processing methods. The selected papers were reviewed, and the prognostic factors were discussed. No reliable positive prognostic factors guarantee sperm recovery for patients with non-obstructive azoospermia. The only negative prognostic factor is the presence of AZFa and AZFb microdeletions. KEYWORDS: Azoospermia; Testicular Sperm Retrieval; Male Infertility; Reproductive Techniques; Prognosis. Glina S, Vieira M. Prognostic factors for sperm retrieval in non-obstructive azoospermia. Clinics. 2013;68(S1):121-124. Received for publication on September 3, 2012; Accepted for publication on September 6, 2012 E-mail: glinas@terra.com.br Tel.: 55 11 3515 7999
etiology of infertility, and genetic alterations; however, the histological testicular pattern remains the best predictor of sperm retrieval, although with the inconvenience of a second invasive procedure (3). The objective of this article was to review the prognostic factors and clinical use of sperm retrieval for men with nonobstructive azoospermia.
& INTRODUCTION In the last 20 years, the most important development in male factor infertility has been the introduction of intracytoplasmic sperm injection (ICSI). ICSI brought hope for men with severe male factor infertility who wanted to become biological fathers. Since Devroye et al.’s (1) report on the use of testicular spermatozoa in an azoospermic male to fertilize human oocytes, physicians have sought a safe and simple method of testicular sperm extraction that is associated with a high recovery rate. Ideally, the best method would only cause minor testicular damage, present a high recovery rate, have a low cost and be reproducible. The first proposed technique was named testicular sperm extraction (TESE) and was simply a testicular biopsy. Further discussions about this technique involved the number of testicular fragments taken, the site of the extraction and in the last decade, the use of microsurgery to improve the results (2). Despite the current use of testicular sperm extraction, reliable clinical and laboratory prognostic factors of sperm recovery are missing. Prognostic factors have included testis size, follicle stimulating hormone (FSH), inhibin beta, the
& METHODS The PubMed database was searched for the Medical Subject Headings (MeSH) terms azoospermia, sperm retrieval, and prognosis. Papers on obstructive azoospermia were excluded. The authors selected articles that reported successful clinical, laboratory, or parenchyma processing methods. The selected papers were reviewed, and the prognostic factors were discussed. Spermatogenesis is a complex process that involves a mitotic and meiotic cellular division and depends on more than 40 enzymes. While the process occurs in the testicle, part of the process is completely isolated from the immunological system that is hidden behind the bloodtesticular barrier (4). Non-obstructive azoospermia is the most serious alteration of the spermatogenesis, and its evaluation and treatment remain a challenge. Although there are well-established treatment protocols, the chances of successful clinical or surgical treatment for non-obstructive azoospermia are small. For most patients, the remaining option is based on sperm retrieval and ICSI. Sperm retrieval is conducted with testicular aspiration or biopsy for testicular sperm extraction (TESE), and laboratory
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)13
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searches are conducted for viable sperm that can be used for ICSI. TESE is an invasive procedure and should be done with the intention of treatment. Sperm retrieval procedures are associated with uncertainties, high costs and the possibility of morbidity for women who suffer from ovarian hyper stimulation during the oocytes retrieval procedure. The prognostic factors of sperm retrieval are important to understand. Historical prognostic factors for sperm recovery in nonobstructive azoospermia are related to the clinical, laboratory, and surgical technique; the testicular tissue processing method in the gamete laboratory; and the histological pattern of the testis.
discussion about surgical treatment and in vitro fertilization (IVF) remains active in the literature. The benefits of treatment in terms of sperm quality are difficult to question, but the issue becomes complicated for patients with azoospermia associated with varicocele who want to naturally conceive after surgical correction (13,14). Lee et al. (14) analyzed the cost effectiveness of live birth after varicocele treatment for patients with azoospermia after testicular microdissection and ICSI. The authors concluded that ICSI was the most cost effective. The goal of the present article was to discuss whether varicocele treatment before TESE improves the chances of retrieving sperm and serves as a positive prognostic factor. Inci et al. retrospectively studied 96 patients with varicocele and non-obstructive azoospermia. Sixty-six patients had undergone a previous surgical varicocele treatment with TESE or ICSI at least five months previously, and 30 patients had not previously undergone treatment. The mean patient ages, testicular volumes, FSH levels, and partner ages were not significantly different between the two groups. The results showed a significantly better recovery rate for the treated group (53% versus 30%). There were no statistically significant differences in embryo quality, implantation rates, miscarriage rates, or live birth rates (15).
& CLINICAL Clinical aspects related to sperm recovery include the etiology of infertility and the age and testicular volume of the patient. For non-obstructive azoospermia, these three prognostic factors are typically considered alone or combined with endocrinologic data. For the etiology of azoospermia, there are data on Klinefelter syndrome, cryptorchidism, and varicocele. Klinefelter syndrome is a karyotype alteration that results from a supernumerary X chromosome that can be expressed in two forms: mosaic and non-mosaic. Klinefelter syndrome is usually associated with infertility. The sperm recovery rate in Klinefelter patients is approximately 50% (5-7). Previous literature suggests that age (5) and testicular volume (4) are prognostic factors, but a more recent study showed no correlation between clinical parameters and sperm retrieval (7). However, these studies used limited numbers of patients, ranging from 20 to 51. In 2010, a review of 13 papers involving 373 Klinefelter patients with azoospermia who underwent sperm retrieval showed that clinical parameters were not good prognostic factors (8). Cryptorchidism is one of the most common child malformations, and it affects 3% of full term male infants. Cryptorchidism is related to male infertility, testicular tumors, testicular torsion, and inguinal hernias (9). Studies on the clinical factors related to sperm retrieval in cryptorchid patients have small numbers of individuals, and the conclusions remain controversial. Negri et al. (10) compared 30 azoospermic cryptorchid men with 77 men with various causes of non-obstructive azoospermia and concluded that bilateral orchidopexy was a positive predictive factor for sperm finding after TESE. However, the authors did not compare unilateral and bilateral orchidopexy, which invalidates the final conclusion. In a study of 38 azoospermic men who had previously undergone surgical treatment for cryptochidism, Raman and Schlegel (11) found correlations between sperm recovery rate and age at orchidopexy and testicular volume. Men who had had surgery before they were ten years old had a better sperm recovery rate than men who had had orchidopexy at ten years of age or higher. More recently, Wiser et al. (12) studied 40 patients who had undergone orchidopexy and were presenting with azoospermia and found no statistically significant differences between men who had undergone surgery before or after ten years of age. The authors also found no significant relationship between sperm recovery rate and testicular volume. Varicocele is an important male infertility factor that has a high incidence. While surgical treatment is possible, the
& LABORATORY Laboratory investigations are based on tests that verify the hypothalamic-pituitary-gonadal axis by measuring follicle-stimulating hormone (FSH) levels and the feedback regulator inhibin B. Other tests include the genetic detection of chromosome alterations. An intact hypothalamic-pituitary-gonadal axis is necessary for correct sperm production. The FSH level was initially used as a predictor of sperm recovery, but its use remains controversial. Ramasamy et al. evaluated 792 men with non-obstructive azoospermia who underwent testicular microdissection. The men were divided into four groups according to FSH levels: less than 15 IU/mL, between 15 and 30 IU/mL, 31 to 45 IU/mL, and greater than 45 IU/mL. Compared with the group with less than 15 IU/mL FSH, the recovery rate for mature sperm was significantly higher in the groups with greater than 15 IU/mL FSH (16). Chen et al. conducted a prospective study with 208 patients who underwent an FSH test and TESA. To establish an FSH cutoff limit, the men were divided into two groups based on mature sperm recovery. The authors found a cut-off value of 19.4 mIU/mL. Sperm was not found in men with FSH levels at or above the cut-off limit (17). Both of these studies have a large number of patients and had the prime objective of analyzing the relationship between FSH levels and sperm finding; in the second study, the mean FSH levels for successful sperm recovery were in the normal range and did not characterize the histological findings in each group. A predominance of hypospermatogenesis in the successful group may explain the findings. Inhibins, anti-Mullerian hormone (AMH), and activins are glycoproteins that are transforming growth factors (TGF). These glycoproteins cause the pituitary gland to take part in the feedback mechanism at the hypothalamicpituitary-gonadal axis. For this reason, they can be used as spermatogenesis markers. Plasma levels of inhibin fraction B and seminal levels of AMH can be used as predictive parameters for sperm recovery in non-obstructive azoospermia
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In 2000, Amer et al. compared classic biopsy on one side and testicular microdissection on the other, in 116 nonobstructive azoospermic men. The study aimed to verify the sperm retrieval rates and parenchymal changes in the Doppler ultrasound during the first six months after the procedure. The sperm retrieval rate was significantly higher on the side that underwent testicular microdissection, and at the end of the follow-up, there were no permanent devascularization areas in the testes. Biopsies were performed on the same patients. Authors used 100 patients with similar histology for comparison, however, only one testicular fragment was retrieved in classic biopsy side and evaluated (22). In a retrospective comparative study, Okada et al. analyzed two groups of patients with obstructive and non-obstructive azoospermia who had undergone TESE and testicular microdissection. The authors reported a better sperm retrieval rate with testicular microdissection. This advantage was present for all patterns of testicular histology; however, TESE was performed using three 5-mm incisions (23). In a retrospective study with 46 patients who had undergone traditional TESE and whose sperm retrievals had failed, Tsujimura et al. found a 44% sperm retrieval rate with testicular microdissection and concluded that the technique was superior. When analyzing the results, the authors highlighted the difference between the sperm retrieval rates in patients with one (47%) or three (33%) testicular fragments; only nine patients had multiple bilateral fragments collected in the first TESE procedure, which suggests that the amount of tissue removed should be considered (24). In 2002, Tsujimura et al. compared several conventional TESE and testicular microdissection techniques in different patients and found no significant differences in the sperm retrieval rates between the groups (25).
(18). Inibine B and AHM can be measured in the plasma or seminal plasma, and their clinical use is debated in the literature (19). A prospective study of 139 men with nonobstructive azoospermia was conducted by Mitchell et al. The authors measured FSH, inhibin B in the plasma and AMH in the seminal plasma before TESE, and they compared the mean levels of successful sperm retrieval with the failed group. The recovery sperm rate was 43% (60/139), and there was a statistically significant correlation between serum and seminal plasma inhibin B levels and seminal plasma AMH and inhibin B levels. However, the mean inhibin B seminal plasma levels and mean AMH seminal plasma levels did not show statistically significant differences between the successful and failed sperm retrieval groups. On the contrary, the mean serum levels of FSH and serum inhibin B differed between the two groups; the statistical significance is difficult to explain and justify (19). Tiepolo and Zuffardi postulated the involvement of deletions in the long arm of the Y chromosome. Using a cytogenetic analysis, they identified a region known as azoospermia factor (AZF). That region was later subdivided into three regions (AZFa, AZFb, and AZFc), and the relationship with infertility was established. Y chromosome microdeletions occur in 6-8% of severely oligozoospermic men and 3-15% of azoospermic men. The importance of Y microdeletions as a prognostic factor for sperm recovery is based on the absence of mature sperm in azoospermic men with AZFa and AZFb microdeletions who underwent sperm retrieval techniques. Fortunately, AZFc is the Y microdeletion most often found in azoospermic men (60%), and sperm can be retrieved for these patients. For this reason, the presence of AZFa or AZFb is a negative predictive factor for sperm retrieval in azoospermic men (20).
& SURGICAL TECHNIQUES
& TESTICULAR TISSUE PROCESSING METHOD
To analyze the surgical technique as a prognostic factor, it is necessary to compare existing techniques. Sperm can be retrieved from testicles using percutaneous (testicular sperm aspiration, TESA; testicular fine needle aspiration, TFNA) or open (testicular sperm extraction, TESE; testicular microdissection, TM) methods (21). Comparisons between these techniques are difficult, as patients are not the same, and the efficacy of the method varies according to the testicular histology. Percutaneous methods are less invasive, but the laboratory has less tissue to search for sperm. Open techniques provide larger samples to work with but are more invasive. Finally, TFNA mapping attempts to combine the qualities of the anterior techniques by mapping the parenchyma and making a target opening, but an expert cytologist is still required to correctly identify mature sperm on the cytology sample. In general, percutaneous techniques are more successful in hypospermatogenesis than in other histological patterns (21). The sperm recovery rates range from 11-47% for TESA, 30-63% for TESE, and 43-63% for TM; the rate for TFNA is approximately 47% (21). Since Schlegel’s 1999 publication of the TM technique (2), studies have attempted to find its superiority over previous methods, but definitive conclusions are difficult to make. The comparative studies that found statistically significant differences between TESE and TM used different testicles (22), took one to three TESE samples per testicle (22,23) or did not explore two testicles in the previous TESE (24).
After tissue has been removed, two methods can be used to open the seminiferous tubules to separate the structural tissue from the viable sperm: enzymatic tissue digestion and mechanical tissue disruption. Mechanical preparation consists of using needles or scalps to mince and shred the tissue to open the tubules and separate the sperm from the tubular epithelium. The procedure is conducted in the operating room at the same time as the surgery and later in the manipulation laboratory, and the procedure is typically associated with a microdroplets search for viable spermatozoa (26). Enzymatic preparation uses a collagenase tissue exposure to liberate sperm from the tubules so that they can be manipulated in the laboratory (26). A comparison between the two techniques was conducted by a multicenter German study. The data were from eleven centers that used testicular sperm from obstructive and non-obstructive azoospermia, but the authors did not conduct separate analyses of the techniques; thus, the results are inconclusive (26). Enzymatic tissue digestion can be used in the laboratory after a mechanical preparation is conducted to rescue negative initial searches by digesting the testicular parenchyma and freeing sperm. A study of 501 negative testicular samples from testicular microdissection showed that an enzymatic preparation could rescue sperm in seven percent of men after a mechanical preparation (27).
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12. Wiser A, Raviv G, Weissenberg R, Elizur SE, Levron J, Machtinger R, et al. Does age at orchidopexy impact on the results of testicular sperm extraction? Reprod Biomed Online. 2009;19(6):778-83, http://dx.doi.org/ 10.1016/j.rbmo.2009.09.031. 13. Baazeem A, Belzile E, Ciampi A, Dohle G, Jarvi K, Salonia A, et al. Varicocele and Male Factor Infertility Treatment: A New Meta-analysis and Review of the Role of Varicocele Repair. Eur Urol. 2011;60(4):796808, http://dx.doi.org/10.1016/j.eururo.2011.06.018. 14. Lee R, Li PS, Goldstein M, Schattman G, Schlegel PN. A decision analysis of treatments for nonobstructive azoospermia associated with varicocele. Fertil Steril. 2009;92(1):188-96, http://dx.doi.org/10.1016/j.fertnstert. 2008.05.053. 15. Inci K, Hascicek M, Kara O, Dikmen AV, Gurgan T, Ergen A. Sperm Retrieval and Intracytoplasmic Sperm Injection in Men With Nonobstructive Azoospermia, and Treated and Untreated Varicocele. J Urology. 2009;182(4):1500-4. 16. Ramasamy R, Lin K, Gosden LV, Rosenwaks Z, Palermo GD, Schlegel PN. High serum FSH levels in men with nonobstructive azoospermia does not affect success of microdissection testicular sperm extraction. Fertil Steril. 2009;92(2):590-3, http://dx.doi.org/10.1016/j.fertnstert.2008. 07.1703. 17. Chen SC, Hsieh JT, Yu HJ, Chang HC. Appropriate cut-off value for follicle-stimulating hormone in azoospermia to predict spermatogenesis. Reprod Biol Endocrinol. 2010;8:108, http://dx.doi.org/10.1186/14777827-8-108. 18. Deffieux X, Antoine JM. [Inhibins, activins and anti-Mullerian hormone: structure, signalling pathways, roles and predictive value in reproductive medicine]. Gynecol Obstet Fertil. 2003;31(11):900-11, http://dx.doi. org/10.1016/j.gyobfe.2003.08.012. 19. Mitchell V, Boitrelle F, Pigny P, Robin G, Marchetti C, Marcelli F, et al. Seminal plasma levels of anti-Mullerian hormone and inhibin B are not predictive of testicular sperm retrieval in nonobstructive azoospermia: a study of 139 men. Fertil Steril. 2010;94(6):2147-50, http://dx.doi.org/10. 1016/j.fertnstert.2009.11.046. 20. Shefi S, Turek PJ. Sex chromosome abnormalities and male infertility: A clinical perspective. In: De Jonge C, Barrat C, editors. The Sperm Cell Production, Maturation, Fertilization, Regeneration. Cambridge: Cambridge University Press; 2006.p.261-78. 21. Harris SE, Sandlow JI. Sperm acquisition in nonobstructive azoospermia: what are the options? Urol Clin North Am. 2008;35(2):235-42, ix, http:// dx.doi.org/10.1016/j.ucl.2008.01.008. 22. Amer M, Ateyah A, Hany R, Zohdy W. Prospective comparative study between microsurgical and conventional testicular sperm extraction in nonobstructive azoospermia: follow-up by serial ultrasound examinations. Hum Reprod. 2000;15(3):653-6, http://dx.doi.org/10.1093/humrep/ 15.3.653. 23. Okada H, Dobashi M, Yamazaki T, Hara I, Fujisawa M, Arakawa S, et al. Conventional versus microdissection testicular sperm extraction for nonobstructive azoospermia. J Urology. 2002;168(3):1063-7. 24. Tsujimura A, Miyagawa Y, Takao T, Takada S, Koga M, Takeyama M, et al. Salvage microdissection testicular sperm extraction after failed conventional testicular sperm extraction in patients with nonobstructive azoospermia. J Urology. 2006;175(4):1446-9. 25. Tsujimura A, Matsumiya K, Miyagawa Y, Tohda A, Miura H, Nishimura K, et al. Conventional multiple or microdissection testicular sperm extraction: a comparative study. Hum Reprod. 2002;17(11):2924-9, http://dx.doi.org/10.1093/humrep/17.11.2924. 26. Baukloh V. Retrospective multicentre study on mechanical and enzymatic preparation of fresh and cryopreserved testicular biopsies. Hum Reprod. 2002;17(7):1788-94, http://dx.doi.org/10.1093/humrep/17.7. 1788. 27. Ramasamy R, Reifsnyder JE, Bryson C, Zaninovic N, Liotta D, Cook CA, et al. Role of tissue digestion and extensive sperm search after microdissection testicular sperm extraction. Fertil Steril. 2011;96(2):299302, http://dx.doi.org/10.1016/j.fertnstert.2011.05.033. 28. Meng MV, Cha IM, Ljung BM, Turek PJ. Relationship between classic histological pattern and sperm findings on fine needle aspiration map in infertile men. Hum Reprod. 2000;15(9):1973-7, http://dx.doi.org/10. 1093/humrep/15.9.1973. 29. Ramasamy R, Schlegel PN. Microdissection testicular sperm extraction: Effect of prior biopsy on success of sperm retrieval. J Urology. 2007;177(4):1447-9.
& HISTOLOGIC PATTERN The most reliable factor for predicting sperm retrieval in non-obstructive azoospermic patients is the testicular histological pattern. However, its use is limited, as the patient must undergo a prognostic biopsy, which adds a surgical procedure. The worst pattern for sperm recovery is Sertoly cell-only syndrome, which has a rate of sperm retrieval ranging from 4-51%; maturation arrest (8-80%) is affects sperm recovery adversely, while hypospermatogenesis produces high rates of sperm recovery (80-100%) (3,28,29). Non-obstructive azoospermia remains the most challenging diagnosis for andrologists, and there are no positive prognostic factors that guarantee sperm recovery for these patients. The only reliably negative prognostic factor is the presence of AZFa and AZFb microdeletions.
& AUTHOR CONTRIBUTIONS Glina S and Vieira M conducted the literature search and prepared the manuscript.
& REFERENCES 1. Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, et al. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod. 1995;10(6):145760, http://dx.doi.org/10.1093/HUMREP/10.6.1457. 2. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod. 1999;14(1):131-5, http://dx.doi.org/10.1093/humrep/14.1.131. 3. Glina S, Soares JB, Antunes N, Jr., Galuppo AG, Paz LB, Wonchockier R. Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients. Int Braz J Urol. 2005;31(4):338-41. 4. Sutovsky P, Manandhar G. Mammalian spermatogenesis and sperm structure: anatomical and compartmental analysis. In: De Jonge C, Barrat C, editors. The Sperm Cell Production, Maturation, Fertilization, Regeneration. Cambridge: Cambridge University Press. 2006.p.1-30. 5. Madgar I, Dor J, Weissenberg R, Raviv G, Menashe Y, Levron J. Prognostic value of the clinical and laboratory evaluation in patients with nonmosaic Klinefelter syndrome who are receiving assisted reproductive therapy. Fertil Steril. 2002;77(6):1167-9, http://dx.doi.org/ 10.1016/S0015-0282(02)03092-3. 6. Okada H, Goda K, Yamamoto Y, Sofikitis N, Miyagawa I, Mio Y, et al. Age as a limiting factor for successful sperm retrieval in patients with nonmosaic Klinefelter’s syndrome. Fertil Steril. 2005;84(6):1662-4, http:// dx.doi.org/10.1016/j.fertnstert.2005.05.053. 7. Koga M, Tsujimura A, Takeyama M, Kiuchi H, Takao T, Miyagawa Y, et al. Clinical comparison of successful and failed microdissection testicular sperm extraction in patients with nonmosaic Klinefelter syndrome. Urology. 2007;70(2):341-5, http://dx.doi.org/10.1016/j.urology.2007.03. 056. 8. Fullerton G, Hamilton M, Maheshwari A. Should non-mosaic Klinefelter syndrome men be labelled as infertile in 2009? Hum Reprod. 2010;25(3):588-97, http://dx.doi.org/10.1093/humrep/dep431. 9. Garrone G, Liguori R. Distopias Testiculares e Malformac¸o˜es Genitais. In: Nardozza Jr A, Zerati Filho M, Borges dos Reis R, editors. Urologia Fundamental. Sa˜o Paulo: Plamark; 2010.p.383-9. 10. Negri L, Albani E, DiRocco M, Morreale G, Novara P, Levi-Setti PE. Testicular sperm extraction in azoospermic men submitted to bilateral orchidopexy. Hum Reprod. 2003;18(12):2534-9, http://dx.doi.org/10. 1093/humrep/deg497. 11. Raman JD, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection is successful for the treatment of nonobstructive azoospermia associated with cryptorchidism. J Urology. 2003;170(4):1287-90.
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REVIEW
Laboratory processing and intracytoplasmic sperm injection using epididymal and testicular spermatozoa: what can be done to improve outcomes? Wana Popal, Zsolt P. Nagy Reproductive Biology Associates, Atlanta, GA, USA.
There are two main reasons why sperm may be absent from semen. Obstructive azoospermia is the result of a blockage in the male reproductive tract; in this case, sperm are produced in the testicle but are trapped in the epididymis. Non-obstructive azoospermia is the result of severely impaired or non-existent sperm production. There are three different sperm-harvesting procedures that obstructive azoospermic males can undergo, namely MESA (microsurgical epididymal sperm aspiration), PESA (percutaneous epididymal sperm aspiration), and TESA (testicular sperm aspiration). These three procedures are performed by fine-gauge needle aspiration of epididymal fluid that is examined by an embryologist. Additionally, one technique, called TESE (testicular sperm extraction), is offered for males with non-obstructive azoospermia. In this procedure, a urologist extracts a piece of tissue from the testis. Then, an embryologist minces the tissue and uses a microscope to locate sperm. Finding sperm in the testicular tissue can be a laborious 2- to 3-hour process depending on the degree of sperm production and the etiology of testicular failure. Sperm are freed from within the seminiferous tubules and then dissected from the surrounding testicular tissue. It is specifically these situations that require advanced reproductive techniques, such as ICSI, to establish a pregnancy. This review describes eight different lab processing techniques that an embryologist can use to harvest sperm. Additionally, sperm cryopreservation, which allows patients to undergo multiple ICSI cycles without the need for additional surgeries, will also be discussed. KEYWORDS: Intracytoplasmic Sperm Injection; Non-Obstructive Azoospermia; Obstructive Azoospermia; Testicular Biopsy; Human Spermatozoa. Popal W, Nagy ZP. Laboratory processing and intracytoplasmic sperm injection using epididymal and testicular spermatozoa: what can be done to improve outcomes? Clinics. 2013;68(S1):125-130. Received for publication on August 16, 2012; Accepted for publication on August 20, 2012 E-mail: wpopal15@hotmail.com Tel.: +1 404 257 1840
infertility has been attributed to complete azoospermia, which presents either in an obstructive or a non-obstructive form (2). There are four different harvesting techniques that can be used to obtain sperm for in vitro fertilization (IVF). Each procedure has advantages and disadvantages and is described below. Sperm-harvesting techniques used to obtain sperm from men with obstructive azoospermia are described in the following paragraphs. MESA (microsurgical epididymal sperm aspiration): Using an intraoperative stereomicroscope, a cut is made in a single tubule of the epididymis. The contents therein, which should contain sperm, are then aspirated with a finegauge needle. An embryologist examines the sample for the presence of motile sperm. If no motile spermatozoa are found at the first site, the maneuver is repeated. Typically, only a few microliters of epididymal fluid are retrieved because sperm are highly concentrated in the epididymal fluid (approximately 16106 sperm/ml). A MESA approach should provide more than adequate numbers of sperm for immediate use, as well as for cryopreservation. As reported by Dr. Shlegel and colleagues, who used MESA and ICSI in
& INTRODUCTION Azoospermia is defined as an absence of spermatozoa in the ejaculate (1), and it is classified as either obstructive or non-obstructive. Obstructive azoospermia is the result of a blockage in the male reproductive tract; therefore, sperm production in the testicle is normal, but the sperm are trapped inside the epididymis. The absence of sperm in the ejaculate can be due to an abnormality in the epididymis, vas deferens, or ejaculatory ducts. The obstruction can be caused by many factors, such as infection, surgery (vasectomies), or an absent vas deferens. Non-obstructive azoospermia is the result of severely impaired or non-existent sperm production. Approximately 10% of male factor
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)14
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performed even if no sperm are found in the TESE process and there is no donor sperm back-up. In this case, eggs can be cryopreserved to enable the couple to make future fertility decisions. Importantly, this new option is available because vitrification has significantly improved oocyte survival rates. For men who must undergo sperm-harvesting procedures to recover testicular or epididymal sperm, it is now possible to cryopreserve the excess sperm for future cycles if needed, thus eliminating the need for repeat surgery. However, some clinics still prefer to use freshly retrieved epididymal or testicular sperm instead of frozenthawed samples.
a group of men with obstructive azoospermia, clinical pregnancies were detected by a fetal heartbeat in 75% (57/ 76) of attempts, and healthy deliveries occurred in 64% (49/ 76) of attempts (3). PESA (percutaneous epididymal sperm aspiration): This simple technique can be performed under local anesthesia or mild sedation. Unlike MESA, where the surgeon is able to visualize the exposed epididymal tubules, PESA is a blind procedure. A fine needle is used to puncture the epididymis, and sperm are aspirated and given to an embryologist to examine. Again, if no motile spermatozoa are found at the first site, the surgeon will repeatedly draw samples for an embryologist to examine. TESA (testicular sperm aspiration): For obstructive azoospermia patients in whom sperm cannot be found in the epididymis, it is always possible to find sperm in the testis. TESA may be performed as a primary harvesting technique if there is an absent epididymis or severe epididymal fibrosis. This blind procedure is usually performed under local anesthesia or mild sedation. A wide-bore needle is pushed into the testis through the skin, suction is applied, and the contents of the needle are flushed into a petri dish containing culture media. Then, an embryologist examines the aspirate for motile or immotile spermatozoa with a stereomicroscope. Using fine needles, sperm are released from within the seminiferous tubules, where sperm are produced, and they are then dissected from the surrounding tissue. Next, the embryologist determines whether there is a sufficient number of sperm for treatment or cryopreservation for future use. If not, several more needle biopsies will be attempted by the surgeon in each testis to obtain a sufficient amount. TESE (testicular sperm extraction): This technique is reserved for men with non-obstructive azoospermia. This procedure is also routinely carried out under mild sedation or local anesthesia. The surgeon exposes a small area of the testis by making an incision in the scrotum. The seminiferous tubules are forced out through the incision by gently squeezing the testis, the tubules are excised and the biopsy sample is placed in a petri dish and given to an embryologist to examine. An approximately 500-g biopsy sample is rinsed in culture media to remove red blood cells and divided into small pieces, using fine needles, under a stereomicroscope to check for sperm (4). If no sperm are found, the surgeon will continue taking biopsies from different areas of the testis, and the embryologist will continue to examine the samples. Sperm can usually be easily obtained from infertile men with obstructive azoospermia for intracytoplasmic injection (ICSI); however, individuals who exhibit non-obstructive azoospermia have historically been difficult to treat (4). Examining biopsies for sperm under a microscope can be very time-consuming and difficult. Finding sperm in the testicular tissue can be a laborious 2- to 3-hour process depending on the degree of sperm production and the etiology of testicular failure, especially in men with partial testicular failure. For men with non-obstructive azoospermia, some IVF clinics ask that a TESE be performed a day before the egg retrieval process because they believe culturing the testicular tissue in an incubator overnight will help sperm to acquire motility. When sperm are not found after surgery, the couple must decide whether they want to cancel the egg retrieval, cancel the fertility cycle, or proceed with donor sperm. Alternatively, oocyte collection can be
& INTRACYTOPLASMIC SPERM INJECTION (ICSI) Two major breakthroughs have recently occurred in the area of male infertility (4). The first is the development of the intracytoplasmic sperm injection (ICSI) technique for the treatment of male factor infertility due to severely abnormal semen quality. The second is the extension of ICSI to azoospermic males and the demonstration that spermatozoa derived from either the epididymis or the testis are capable of producing normal fertilization and pregnancy (4). ICSI is now the primary technique used to treat male-factor infertility. ICSI has achieved consistent fertilization and high pregnancy rates in couples with suboptimal spermatozoa (5). Although the fertilization rates obtained with surgically retrieved spermatozoa were satisfactory, they were significantly lower than those achieved with fresh ejaculates (6). A concern associated with the use of suboptimal spermatozoa for ICSI is the potential for transmitting the genetic abnormalities responsible for male infertility to the offspring (4). Despite this concern, ICSI is the customary therapeutic approach, although genetic screening and patient counseling are still imperative for these patients. In IVF clinics, surgically retrieved sperm samples are most often prepared for use before a patient progresses through an ICSI cycle. This is a safe course of action in cases of obstructive azoospermia, but in cases of non-obstructive azoospermia, it is beneficial to undergo the surgery before the cycle because the female has already been stimulated with medication. At that time, a couple would have to decide whether to proceed with donor sperm or cryopreserve the oocytes for use at a later time. Epididymal aspirates are relatively easy to prepare in the lab, either for ICSI or for cryopreservation. Typically, an embryologist washes the sample with culture media to dilute any epididymal fluid or blood contamination. The most common issues are a high level of red blood cells, an absence of observed sperm, poor sperm motility, and slow progression (5).
& DIFFERENT METHODS FOR PROCESSING EPIDIDYMAL AND TESTICULAR SPERM SAMPLES Shredding method The preparation of testicular samples is more difficult if the sperm are contained within the seminiferous tubules. Many of the sperm found are immature, with a large cytoplasmic drop attached to the head, and some sperm are motile; however, most are still viable for ICSI. It is advantageous to perform the sperm retrieval procedure at least 24 hours in advance of the egg retrieval process, as
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cases of non-obstructive azoospermia. The main difficulty is due to the abundance of red blood cells in shredded testicular biopsy specimens. The presence of a very high concentration of erythrocytes and the very infrequent occurrence of sperm cells sometimes result in a much longer examination (10). Therefore, one option for testicular biopsy specimens is suspension in Hepes-buffered medium. This can then be centrifuged for 5 minutes at 300 g. Treatment of the testicular sperm pellet with erythrocyte lysing buffer is performed by resuspending the testicular sperm pellet in 2 to 4 ml of erythrocyte lysing buffer (155 mM NH4Cl, 10 mM KHCO3, and 2 mM ethylenediaminetetracetic acid; pH 7.2) for 10 minutes at room temperature. Then, 5 to 10 ml of Hepes-buffered medium supplemented with protein is added to the suspension in the same tube and then centrifuged for 10 minutes at 500 g. The pellet is then resuspended with 1.5 ml of culture media with protein. This resuspended pellet can be transferred to an Eppendorf tube and centrifuged again at 500 g for 5 minutes. After centrifugation, the pellet is resuspended in 50 ml of culture media supplemented with protein. This pellet is then placed in an injection dish that contains 20-30 droplets containing 5 ml of Hepes-buffered medium; the central droplet is composed of 10% PVP solution. Subsequently, the embryologist can search for spermatozoa in the sample that will be used for ICSI. This method is beneficial when there is a large amount of red blood cell contamination in the sample. This technique has been reported and described in more detail by Nagy and colleagues (10). One of the main difficulties in finding spermatozoa in a testicular sample is the abundance of erythrocytes (10).
motility improves with time in culture (7). Biopsies will always require incubation before motility is observed. In some cases, it was found that the sample needed to be cultured for at least 24 hours at 37 ËšC. A common procedure associated with the shredding method is the excision of testicular tissue by a surgeon and its subsequent examination by an embryologist. The tissue can be finely minced and teased apart with fine needles or glass slides in HEPES-buffered media. Then, the product is placed in a 5-ml Falcon tube and centrifuged for 5 minutes at 1,800 g. The supernatant is removed with a Pasteur pipette, and the pellet is resuspended in 0.2 ml of culture media. The number of spermatozoa in the droplet can be very low, as well as poor motility when visualized microscopically. An embryologist must retrieve sperm from the field of debris, red blood cells, and Sertoli cells for ICSI. Then, the sperm suspension can be incubated at 37 ËšC until the time of ICSI (8).
Squeezing method Seminiferous tubules are teased apart and rinsed to remove blood contamination, and they are then placed into a petri dish with fresh culture media. Tubules are then cut into short lengths (1-2 cm) with fine needles. A long, thin Pasteur pipette is pulled over a flame and then bent (ideally at an angle of 45Ëš). A second pipette (without a bend) should be heated and used to pick up the tubule contents. By holding one end of a cut tubule with the point of a needle, the bent pipette can be run along the length of the tubule while simultaneously pushing down against the base of the petri dish. This procedure squeezes the entire contents of the tubule into the medium. The contents can now be picked up with the second pulled pipette and placed in a test tube filled with clean sperm media or placed on a slide to look for sperm (9).
The hypoosmotic swelling test (HOS test) The hypoosmotic swelling test (HOS) was developed by Jeyandran et al. (1984) (11) to evaluate the functional integrity of the sperm membrane. Viable sperm with normal membrane function will exhibit tail swelling and curving due to the influx of water when exposed to hypoosmotic conditions. The use of this test for distinguishing viable from non-viable spermatozoa for ICSI was first proposed by Desmet et al. in 1994 (12). It has been shown that the fertilization rate after ICSI of randomly selected, immotile spermatozoa is usually very low, especially with ejaculated spermatozoa (13,14). There is a reduced success rate of fertilization for azoospermic males due to the injection of non-viable spermatozoa, which cannot be distinguished from viable spermatozoa with no motility. Application of the hypo-osmotic swelling test, however, seems to be a promising method of identifying live spermatozoa for ICSI (14). Casper et al. (1996) (15) conducted a study that showed a higher fertilization rate after injecting spermatozoa selected by the HOS test than after injecting randomly selected spermatozoa. Another study using the HOS test has also been reported. In that study, immotile spermatozoa were suspended in a microdroplet consisting of 50% culture medium and 50% Millipore-grade water. After a maximum of 10 seconds, the viable spermatozoa, whose tails were curved and swollen, were selected and transferred to another microdroplet of Hepes-buffered medium where they were washed three times to osmotically re-equilibrate them before transferring them to a PVP drop. The group found that the modified HOS test resulted in improved fertilization and pregnancy
Cell strainer A method for processing large biopsy samples that has the benefit of removing unwanted debris consists of the use of a cell strainer. First, the biopsy sample is teased apart and sliced with fine needles. These slices are rinsed in a series of petri dishes containing sperm preparation media to remove any blood contamination. The tubules are then placed in a cell strainer (Becton Dickinson & Company, Franklin Lakes, New Jersey, USA). Heat-treating the end of a clean, sterile Pasteur pipette produces a sphere-shaped tip approximately 5 mm in diameter. This pipette is used as a pestle to grind and break up the seminiferous tubules against a mesh strainer (9).
Tissue grinder method Another method for processing large tissue samples is to use a mini-tissue grinder. The tissue is teased apart, sliced with fine needles and placed in a test tube with fresh culture media and a glass pestle. With the glass pestle, the sample is ground at the bottom of a glass tube. Then, the sample is concentrated via centrifugation. After centrifugation, the pellet is resuspended with fresh culture media and placed on a Petri dish in 10-ml drops to look for sperm.
Erythrocyte lysing buffer method Sperm recovery from epididymal fluid is usually rapid and efficient, whereas the recovery of sperm cells from testicular biopsy specimens is more difficult, especially in
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rates compared with the regular HOS test and non-use of the HOS test (16).
the shredding method. This method can be considered successful in reducing sperm recovery failure and may increase the chance of selecting the highest quality spermatozoa in patients with non-obstructive azoospermia (19).
Pentoxyfylline method Pentoxyfylline is a phosphodiesterase inhibitor of the methylxantine group. It inhibits the breakdown of cyclic adenosine monophosphate (cAMP), a molecule known to play a role in sperm motility (17). It was found that adding pentoxyfylline to a testicular sperm sample caused sperm to be motile. This procedure is performed by adding pentoxyfylline to the sperm suspension at a 1:1 ratio so that the final concentration of pentoxyfylline in the sample is 0.5 mg/ml. The sample is incubated at 37 ˚C for 20 minutes. Next, the sample is observed for the identification and isolation of motile sperm. Some studies have shown that pentoxyfylline is toxic and should not be used (18). It has been suggested that a lower concentration of pentoxyfylline with shorter exposure times may be beneficial (17). One study found that this was a better and quicker identification and selection method for vital sperm for use with ICSI. This procedure also resulted in better fertilization rates and less time consuming for staff involved in TESA/TESE (17).
& CRYOPRESERVATION OF TESTICULAR AND EPIDIDYMAL SAMPLES Successful sperm cryopreservation allows patients to have multiple ICSI cycles without the need for additional sperm harvesting procedures. Because ICSI enables severely impaired viable sperm to fertilize an oocyte, cryopreserved sperm can achieve rates of fertilization and pregnancy similar to those achieved using fresh sperm (4). Studies have also found that the cryopreservation of testicular sperm is more problematic than the cryopreservation of epididymal sperm because testicular tissue yields a much lower number of spermatozoa with limited motility. Nevertheless, reports have indicated that it is possible to cryopreserve testicular biopsy tissue samples and subsequently extract spermatozoa after thawing, with at least isolated pregnancies being achieved (8). Although the use of cryopreserved testicular sperm for ICSI has several advantages, the data concerning the outcomes of IVF-ICSI procedures using frozen-thawed testicular sperm are still controversial. Some investigators claim that they have demonstrated, in obstructive and nonobstructive azoospermic men, that cryopreserved sperm can function as well as fresh sperm (20). Additionally, some labs have shown that the use of frozen-thawed testicular sperm for ICSI results in higher abortion rates and lower live birth rates compared with fresh testicular sperm (20). Without cryopreservation, testicular tissue and testicular sperm can only be used for one ICSI cycle. Lacking cryopreserved sperm, each cycle of ICSI in these couples requires repeated surgeries. Repeated testicular surgeries can cause permanent testicular damage, irreversible atrophy, deterioration of spermatogenic development, and even loss of endocrine function (2). The cryopreservation of samples allows for multiple ICSI cycles and minimizes the number of invasive testicular surgeries. Surgically recovered sperm from PESA can be cryopreserved in a manner similar to the cryopreservation of fresh ejaculates, although the post-thaw survival rates are usually very poor. Due to extreme variability in post-thaw survival rates, it is important to freeze a small aliquot for a post-thaw survival test. This test requires thawing of the sample the day before the egg retrieval. In cases of virtually zero postthaw survival, having results from the test sample allows time for the patient to consider what precautions to take if no viable sperm are identified on the day of their partner’s egg retrieval. Because there is never an absolute guarantee that cryopreserved sperm will thaw with appropriate viability, counseling patients regarding the use of donor sperm as a back-up should be considered, as well as options of repeat surgery or oocyte cryopreservation. Haberman described the following cryopreservation technique (20). The biopsy specimen is placed in a petri dish with HEPES buffer supplemented with protein at 37 ˚C. Under a dissecting microscope, the seminiferous tubules are teased apart using fine needles, and the contents are squeezed into the surrounding media. The tubules are transferred to a 15-ml conical tube containing 1 ml of fresh
Collagenase method In a study by Crabbe et al. in 1997 (19), the enzymatic treatment of testicular biopsies with collagenase type IV was applied in clinical ICSI cases where no spermatozoa had been found after the sample was minced using two fine needles. This procedure was performed to determine whether spermatozoa can be recovered from the residual tissue pieces. This method is performed by shredding the testicular biopsy sample with fine needles. Microscopic examination of the wet preparation is carried out at 400x magnification under an inverted microscope. Biopsy factions are further minced with two fine forceps in the Petri dish until tissue pieces of ,1 mm3 or free tubule pieces of a few millimeters in length are obtained (19). Spermatozoa are directly recovered from the pellet after centrifuging the supernatant of the shredded tissue at 300 g for 5 minutes. Then, erythrocyte-lysing buffer is used to increase the probability of visualizing any spermatozoa or elongated spermatids (19). The residual pieces are placed in 1 ml of pre-warmed HEPES medium supplemented with protein, 1.6 mM CACL2, 25 mL/ml DNase, and 1000 IU/ml collagenase Type IV. The tissue samples are then placed in an incubator at 37 ˚C for 1 hour to allow digestion to occur. To facilitate complete enzymatic digestion, the samples are shaken every 10-15 minutes during the incubation period. The digested tissue solution is gently centrifuged for 5 minutes at 50 g to remove any residual pieces or debris not dissolved by the enzymes. The remaining cell suspension (supernatant) containing loose cells is then washed twice with culture media and centrifuged for 5 minutes at 1000 g. The supernatant is removed, and the pellet is resuspended once more with 50-100 ml. A 5-ml drop of the suspension is examined on a glass slide with a coverslip and placed under an upright phase contrast microscope. When spermatozoa are found in one of the suspensions, multiple small droplets are added under oil in a Petri dish to further examine and retrieve spermatozoa for ICSI. This study concluded that the fertilization rate obtained using the samples treated with collagenase type IV was comparable to that obtained using the samples retrieved via
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media. The cell suspension is then transferred to a separate centrifuge tube. Both tubes are incubated at 37 ˚C for 15-30 minutes, and the supernatant (containing tubules) is combined within the cell suspension in the second tube. The suspension is centrifuged at 500 g for 5 minutes, and the pellet is resuspended in 1 ml of fresh media with protein. A cell count is performed, and the suspension is diluted or concentrated to 0.5-1.0 million sperm/ml. Before freezing, an aliquot is removed to assess sperm quality. At the time of cryopreservation, multiple aliquots of sperm are frozen whenever possible along with a test cryovial. The cell suspension is slowly mixed in a 1:1 ratio with test yolk buffer and 12% glycerol so that the final concentration of glycerol is 6%. The samples are slowly cooled at a rate of 0.5 ˚C/min to 4 ˚C and then packaged in 1-ml cryovials. The vials are frozen at a rate of -10 ˚C/min to -90 ˚C and are stored in liquid nitrogen at -196 ˚C (20). A study was conducted using this protocol for 29 patients with obstructive and non-obstructive azoospermia undergoing testicular sperm extraction for a total of 46 IVF-ICSI cycles (12 fresh, 34 frozen). No statistically significant differences were noted between the fresh and frozen-thawed testicular spermatozoa (20). Cohen et al. 1997 (2) described a method where cryopreservation and recovery of spermatozoa can be performed even in patients who have fewer than 100 spermatozoa present in the final testicular tissue homogenate. A porous capsule, such as an emptied zona pellucida, is used as a vessel to contain individual spermatozoa. Empty zona pellucida is prepared by microscopic dissection; specifically, small incisions are made in the zona using a microsuction device. The emptied zona pellucida is then maintained in Hepes-buffered media supplemented with protein. A sperm suspension in 10% PVP is then injected into the zona, placed in an 8% glycerol solution and cryopreserved using a standard freeze protocol with plastic straws. The use of an empty zona pellucida is very advantageous for samples with very low numbers of spermatozoa and also reduces the loss of motility associated with post-thaw dilution and sperm washing, which is observed when thawing frozen donor sperm (2). In cases where freezing intact testicular tissue is required, a method adapted from Allan and Cotman (1997) is used (21). Testicular tubules are rinsed in HEPES-buffered medium to eliminate or reduce red blood cell contamination. The tubules are sectioned with a scalpel blade into pieces of approximately 2-3 mm and placed into a 35-mm dish that contains HEPES-buffered medium and cryoprotectant medium at a 1:1 v/v ratio at room temperature. Then, after 30 minutes, the samples are loaded into as many cryostraws as required. The 30-minute room-temperature equilibration period is ongoing during the loading process. The straws are sealed and labeled with the patient’s name. At the end of the 30-minute room-temperature equilibration period, the straws are loaded into the goblets and cryocanes and placed horizontally in the freezer for 30 minutes. At the end of the 30-minute cooling period, the specimens are hung over liquid nitrogen, well below the frost line of the tank, for 30 minutes. Then, the specimens are plunged into liquid nitrogen for continued storage. It is recommended to cryopreserve a test straw and then thaw it to determine the success of the cryopreservation procedure. To thaw these samples, the straws must be taken out of the liquid nitrogen tank and placed in a room-temperature water bath for 10
minutes. The contents are then expelled into roomtemperature HEPES-buffered medium, rinsed with fresh HEPES-buffered medium and allowed to stand for 10 minutes before the sample is processed. The tissue is placed in a sterile Petri dish with a small volume of HEPESbuffered medium, sufficient to cover the tissue, and this sample is then homogenized with fine needles. Using a pulled pipette, a small volume of the homogenate is moved to the ICSI dish, and motile sperm are captured using an assisted hatching pipette. These sperm are then placed in a drop of PVP. Note that overnight incubation of the cryopreserved, thawed, and processed homogenate may increase the effective yield of the preparation (22).
& FUTURE DIRECTIONS Further studies and data will help to identify and define how to more effectively extract sperm from men with obstructive and non-obstructive azoospermia. A key issue identified by Silber et al. (2003) is that there is a high incidence of mosaicism in embryos derived from TESE. Sperm retrieved from men with non-obstructive azoospermia may have a higher rate of compromised or immature centrosome structures, which may lead to mosaicism of the embryo. Their results showed a significant difference in mosaicism rates between embryos from ICSI cycles using ejaculated sperm and embryos from ICSI cycles using TESE for non-obstructive azoospermia (23). Another key issue is the increased DNA fragmentation observed in testicular sperm samples from patients with azoospermia, due either to spermatogenic failure or duct obstruction. Several studies have shown that male fertility can be affected by DNA damage found in sperm (24,25). One study yielded two interesting conclusions resulting from the measurement of DNA fragmentation using the sperm chromatin dispersion (SCD) test (25). The first conclusion was that patients exhibiting low implantation rates and low embryo quality may benefit from this test, which determines the levels of DNA fragmentation. Second, the observation of nucleolus asynchrony in a zygote from a sperm sample with a high DNA fragmentation rate could indicate the possibility of an upcoming low-quality blastocyst cohort (25). In addition, sperm DNA fragmentation appears to be related to the ability of sperm to fertilize the oocyte (25,26). The use of fragmented sperm is still a matter of concern because of the long-term consequences on the development and behavior of the offspring. Still, studies have shown that oocytes may partially repair fragmented DNA, depending on the level of DNA fragmentation, producing blastocysts that are able to implant and produce live offspring (23). In addition, it has been shown that testicular cultures should not be cultured for more than 48 hours to increase motility. Aged spermatozoa may be more susceptible to the damaging effects of free oxygen radicals. Moreover, an increase in structural chromosomal abnormalities in in vitro-stored spermatozoa has been reported (27). The efficacy and safety associated with the retrieval of incompletely developed spermatozoa (i.e., spermatids) from men with non-obstructive azoospermia will also need to be clarified over time. Anecdotal pregnancies and deliveries from these clinical scenarios have been reported (28). However, spermatids usually cannot be extracted from the testis if TESE does not yield fully developed spermatozoa. However, other new therapies, including spermatogonial
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11. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJ. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil. 1984;70(1):219-28, http://dx.doi.org/10.1530/jrf.0.0700219. 12. Desmet B, Joris H, Nagy Z, Liu J, Bocken G, Vankelecom A, et al. Selection of vital immotile spermatozoa for intracytoplasmic injection by the hyposmotic swelling test. Hum Reprod. 1994;9(suppl 4):24-31. 13. Nagy ZP, Liu J, Verheyen G, Tournaye H, Camus M, Derde MP, et al. The Result of Intracytoplasmic Sperm Injection Is Not Related to Any of the 3 Basic Sperm Parameters. Hum Reprod. 1995;10(5):1123-9. 14. Verheyen G, Joris H, Crits K, Nagy Z, Tournaye H, VanSteirteghem A. Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection. Hum Reprod Update. 1997;3(3):195-203, http://dx.doi.org/ 10.1093/humupd/3.3.195. 15. Casper RF, Cowan L, Meriano JS, Lucato ML, Jarvi KA. The hypoosmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia. Fertil Steril. 1996;65(5):972-6. 16. Sallam HN, Farrag A, Agameya A, El-Garem Y, Ezzeldin F, Sallam A. Using the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in ICSI - A randomized controlled study. Fertil Steril. 2005;84:S373-S4, http://dx.doi.org/10.1016/j. fertnstert.2005.07.977. 17. Kovacic B, Vlaisavljevic V, Reljic M. Clinical use of pentoxifylline for activation of immotile testicular sperm before ICSI in patients with azoospermia. J Androl. 2006;27(1):45-52. 18. Tournaye H, Wieme P, Janssens R, Verheyen G, Devroey P, Vansteirteghem A. Incubation of Spermatozoa from Asthenozoospermic Semen Samples with Pentoxifylline and 2-Deoxyadenosine - Variability in Hyperactivation and Acrosome Reaction-Rates. Hum Reprod. 1994;9(11):2038-43. 19. Crabbe E, Verheyen G, Silber S, Tournaye H, Van de Velde H, Goossens A, et al. Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with nonobstructive azoospermia. Hum Reprod. 1998;13(10):2791-6, http://dx. doi.org/10.1093/humrep/13.10.2791. 20. Habermann H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L. In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa. Fertil Steril. 2000;73(5):955-60, http://dx.doi.org/10.1016/S0015-0282(00)00416-7. 21. Allan JA, Cotman AS. A new method for freezing testicular biopsy sperm: three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule. Fertil Steril. 1997;68(4):741-4, http://dx. doi.org/10.1016/S0015-0282(97)00272-0. 22. Schiewe M. Fertility Conundrums, Testicular Sperm and ICSI. 2011. Available from: http://ivfconundrums.com/node/137. 23. Silber S, Escudero T, Lenahan K, Abdelhadi I, Kilani Z, Munne S. Chromosomal abnormalities in embryos derived from testicular sperm extraction. Fertil Steril. 2003;79(1):30-8, http://dx.doi.org/10.1016/ S0015-0282(02)04407-2. 24. Tarozzi N, Bizzaro D, Flamigni C, Andrea B. Clinical relevance of sperm DNA damage in assisted reproduction. Reprod Biomed Online. 2007;14(6):746-57, http://dx.doi.org/10.1016/S1472-6483(10)60678-5. 25. Meseguer M, Santiso R, Garrido N, Gil-Salom M, Remohi J, Fernandez JL. Sperm DNA fragmentation levels in testicular sperm samples from azoospermic males as assessed by the sperm chromatin dispersion (SCD) test. Fertil Steril. 2009;92(5):1638-45, http://dx.doi.org/10.1016/j. fertnstert.2008.08.106. 26. Muriel L, Meseguer M, Fernandez JL, Alvarez J, Remohi J, Pellicer A, et al. Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study. Hum Reprod. 2006;21(3):738-44. 27. Angelopoulos T, Adler A, Krey L, Licciardi F, Noyes N, McCullough A. Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue. Fertil Steril. 1999;71(2):240-3, http://dx.doi.org/10. 1016/S0015-0282(98)00434-8. 28. Cornell. What’s New in Male Infertility Treatment at Cornell Microsurgical Retrieval of Epididymal Sperm. Cornell University; 1998. Available from: http://www.maleinfertility.org/new-retrieval2.html.
transplants, have been reported in animal models. Because many men with non-obstructive azoospermia may have significant genetic abnormalities, caution must be exercised before these fertility treatments can be accepted as standard therapies (28). In conclusion, the use of ICSI combined with spermharvesting techniques has transformed fertility treatment for azoospermic males. In both obstructive and nonobstructive azoospermia, the sperm retrieval technique itself seems to have no impact on the success rates of ICSI. Laboratory techniques that recover sperm from surgical specimens should be carried out with great care and caution. It is important to not jeopardize the potential of the sperm to fertilize the oocyte. The chances of retrieving and recovering spermatozoa and of achieving a live birth by ICSI are increased in couples in which the male partner has obstructive rather than non-obstructive azoospermia (5). Each laboratory technique described in this paper has advantages and disadvantages; the embryologist must determine which method yields the best chance of extracting viable sperm for ICSI and enabling cryopreservation of a patient’s sperm for future IVF cycles.
& AUTHOR CONTRIBUTIONS Popal W wrote the paper and Nagy ZP was responsible for proofreading the manuscript.
& REFERENCES 1. WHO. WHO Laboratory Manual for the Examination and processing of human semen: World Health Organization; 2010. 2. Gardner DK, Weissman A, Howles CM, Shoham Z. Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives, First Edition: Martin Dunitz; 2001. 3. Schlegel PN, Berkeley AS, Goldstein M, Cohen J, Alikani M, Adler A, et al. Epididymal micropuncture with in vitro fertilization and oocyte micromanipulation for the treatment of unreconstructable obstructive azoospermia. Fertil Steril. 1994;61(5):895-901. 4. Gardner DK, Lane M, Watson AJ. A laboratory guide to the mammalian embryo. New York: Oxford University Press; 2004.p.76-85. 5. David K. Gardner BRMBR, Tommaso Falcone, editor. Human Assisted Reproductive Technology: Future Trends in Laboratory and Clinical Practice. Cambridge: Cambridge University Press; 2011. 6. Nagy Z, Liu J, Cecile J, Silber S, Devroey P, Van Steirteghem A. Using ejaculated, fresh, and frozen-thawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil Steril. 1995;63(4):808-15. 7. Zhu J, Tsirigotis M, Pelekanos M, Craft I. In-vitro maturation of human testicular spermatozoa. Hum Reprod. 1996;11(1):231-2, http://dx.doi. org/10.1093/oxfordjournals.humrep.a019030. 8. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum Reprod. 1995;10(1):148-52, http://dx.doi.org/10.1093/humrep/10.1.148. 9. Fleming SCaS, Cooke S. Textbook of assisted reproduction for scientists in reproductive technology. Fremantle: Vivid Publishing, 2009. 10. Nagy ZP, Verheyen G, Tournaye H, Devroey P, Van Steirteghem AC. An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series. Fertil Steril. 1997;68(2):376-9, http://dx.doi.org/10.1016/S0015-0282(97)81534-8.
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REVIEW
Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception Bhushan K. Gangrade Center for Reproductive Medicine, Orlando, FL, USA.
The introduction of the technique of intracytoplasmic sperm injection to achieve fertilization, especially using surgically retrieved testicular or epididymal sperm from men with obstructive or non-obstructive azoospermia, has revolutionized the field of assisted reproduction. The techniques for the retrieval of spermatozoa vary from relatively simple percutaneous sperm aspiration to open excision (testicular biopsy) and the more invasive Micro-TESE. The probability of retrieving spermatozoa can be as high as 100% in men with obstructive azoospermia (congenital bilateral absence of the vas deferens, status post-vasectomy). However, in nonobstructive azoospermia, successful sperm retrieval has been reported in 10-100% of cases by various investigators. The surgical retrieval and cryopreservation of sperm, especially in men with non-obstructive azoospermia, to some extent ensures the availability of sperm at the time of intracytoplasmic sperm injection. In addition, this strategy can avoid unnecessary ovarian stimulation in those patients intending to undergo in vitro fertilization-intracytoplasmic sperm injection with freshly retrieved testicular sperm when an absolute absence of sperm in the testis is identified. Several different methods for the cryopreservation of testicular and epididymal sperm are available. The choice of the container or carrier may be an important consideration and should take into account the number or concentration of the sperm in the final preparation. When the number of sperm in a testicular biopsy sample is extremely low (e.g., 1-20 total sperm available), the use of an evacuated zona pellucida to store the cryopreserved sperm has been shown to be an effective approach. KEYWORDS: Azoospermia; Testicular Sperm Retrieval; Epididymal Sperm Aspiration; Sperm Cryopreservation; Intracytoplasmic Sperm Injection. Gangrade BK. Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception. Clinics. 2013;68(S1):131-140. Received for publication on July 24, 2012; Accepted for publication on July 28, 2012 E-mail: bkgangrade@hotmail.com Tel.: 407-756-1596
(3). The technique of testicular sperm extraction was first introduced in 1993 (2,4) to retrieve sperm from patients with obstructive azoospermia (OA). Pregnancies resulting from the testicular sperm of obstructive azoospermic patients were followed by reports of successful outcomes using the testicular sperm from patients with non-obstructive azoospermia (NOA) (5-11). Nowadays, ICSI using frozen testicular (12) and epididymal (13) sperm has become an effective and standard approach to treating infertility secondary to obstructive and non-obstructive azoospermia. The cryopreservation of mammalian sperm has been practiced for decades. In fact, ejaculated spermatozoa were the first successfully cryopreserved human cells (14). The use of cryopreserved ejaculated sperm in intrauterine insemination and IVF is a standard practice. The Food and Drug Administration enforces a quarantine period before sperm from anonymous donors can be used, thus requiring the cryopreservation and storage of donor sperm for at least six months. Generally, the large number of sperm (usually in the millions) in semen makes the cryopreservation of ejaculated sperm easy and feasible, as even after a loss in viability following the thawing of sperm, enough live and
& INTRODUCTION The introduction of intracytoplasmic sperm injection (ICSI), a technique for the fertilization of oocytes, has revolutionized the field of in vitro fertilization and assisted reproduction, especially in couples with male factor infertility. The report (1) of successful live births following the fertilization of metaphase II oocytes by the injection of a single sperm in couples for whom in vitro fertilization (IVF) and subzonal injection (SUZI) of sperm had previously failed represented a major milestone. Initially, these investigators used freshly ejaculated spermatozoa for ICSI, but soon thereafter, pregnancy and live births were reported using sperm retrieved from the testes (2) and epididymis
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)15
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With the advances in the field of cancer treatment, the survival rate for a variety of malignant diseases in recent years has significantly improved. A significant proportion of boys diagnosed with some form of childhood (pre-pubertal) cancer are cured and able to maintain a normal adult life. In male children (10-13 years of age) diagnosed with some form of malignancy, spermatogenesis may not be fully established, and ejaculated spermatozoa may not be available for banking. However, in such patients, isolated portions of the seminiferous tubules may contain enough sperm for cryopreservation and ICSI. Even in younger males in whom spermatogenesis has not yet been initiated, the cryopreservation of testicular tissue before starting chemo- or radiotherapy offers fertility potential in the future (17). In men with non-obstructive azoospermia (NOA), obtaining enough sperm for cryopreservation may pose a challenge. Depending on the etiology and severity of the underlying condition, none to a fair number of testicular sperm can be retrieved. In 10-15% of patients diagnosed with NOA, the condition is attributed to micro-deletions in the long arm of the Y chromosome (AZF region). The micro-deletion of two genes, USP9Y and DDX3Y, in the AZFa region invariably results in the appearance of Sertoli cell-only syndrome and the complete absence of spermatogenesis in the seminiferous tubules. Depending on the extent and size of the deletions in the AZFb region, complete absence of spermatogenesis or focal hypo-spermatogenesis in isolated seminiferous tubules can occur. Deletions in the distal AZFc region produce a wide array of aberrations in spermatogenesis, often resulting in mild to severe hypo-spermatogenesis and leading to severe oligospermia or even ejaculatory azoospermia. Other causes of NOA may be attributed to cryptorchidism, spermatic cord torsion, testicular or inguinal surgery/infection/trauma, history of radiation and chemical exposure, endocrine disruption, or, in some cases, the use of medications associated with the impairment of sperm production. The probability of recovering sperm obviously depends on the extent and severity of each case. In patients with NOA, it is difficult to predict a successful retrieval of testicular sperm, as no definitive markers of spermatogenesis have been described. Testicular measurements (e.g., size, volume, and plasma FSH concentrations) do not accurately predict the success of testicular biopsy in obtaining enough sperm for ICSI. The treatment of the female partner of NOA patients with gonadotropins in anticipation of oocyte retrieval and ICSI may be unnecessary in up to 50% of patients, as it is possible that no sperm may be available following the testicular biopsy. An exploratory testicular biopsy surgery with ‘‘possible testicular sperm freeze’’ is a valid option and should be offered to the couple. If there is evidence of rare sporadic hypo-spermatogenesis in the seminiferous tubules, the few spermatozoa that are retrieved may be frozen at this time. If it is doubtful whether enough spermatozoa will be available for ICSI post-thaw, the couple may be advised to undergo a fresh biopsy on the day before or on the morning of oocyte retrieval. Patients with NOA often have decreased testicular volumes, and multiple biopsies with the excision of excessive testicular parenchyma carry the risk of irreversible damage and atrophy. Couples hesitant to undergo a repeat biopsy may be offered donor sperm as a back-up in case the number of mature oocytes exceeds the number of available viable testicular sperm for ICSI. This strategy offers the patient and the healthcare provider the advantage of knowing if there
motile sperm are available for insemination or IVF. In comparison, the cryopreservation of surgically retrieved testicular sperm is more cumbersome and difficult because of the low number (total count) of sperm retrieved and their lack of motility. In addition, the testicular sperm preparations are almost always contaminated with a high proportion of cellular debris and blood cells. The basic principles and methods of sperm cryopreservation for testicular, epididymal, and ejaculated sperm are, however, similar and involve the use of glycerol as a cryoprotectant. The cryopreservation of testicular and epididymal sperm has now become a standard technique in the management of male factor infertility.
& REASONS FOR AND ADVANTAGES OF THE CRYOPRESERVATION OF TESTICULAR AND EPIDIDYMAL SPERM The complete absence of sperm in the ejaculate following two-three days of abstinence on at least two occasions is the standard used to confirm the diagnosis of azoospermia. This diagnosis is the primary reason for attempting sperm retrieval from the testis for use in IVF-ICSI. Azoospermia can be caused by epididymal pathology or an obstruction in the reproductive tract at the post-testicular/epididymal locus in men who have otherwise normal spermatogenesis. Obstructive azoospermia (OA) also occurs in men with vasectomy or pathological blockage of the vas deferens. Mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene are a relatively frequent cause of the congenital bilateral absence of the vas deferens (CBAVD) leading to azoospermia. In men with OA, the probability of acquiring sperm from testes is almost guaranteed, and the surgical retrieval of sperm can be scheduled at the convenience of the patient, urologist, and laboratory personnel. Thus, sperm can be cryopreserved in advance of the oocyte retrieval from the female partner. This flexibility not only avoids having to plan the testicular biopsy surgery to coincide with the egg retrieval but also avoids both partners having to undergo surgical procedures at approximately the same time. On rare occasions, men with an infertility diagnosis of ductal obstruction may present with a failure of spermatogenesis, and a fresh biopsy on the day of oocyte retrieval with no available sperm may cause undue psychological stress and a financial burden for the couple. The cryopreservation of spermatozoa in multiple vials/ aliquots confers the advantage of enabling multiple IVFICSI attempts without the male partner undergoing surgery for each attempt. Multiple testicular biopsies or the removal of an excessive amount of tissue can cause irreversible damage and in some cases may result in testicular atrophy (15). Patients who have had a vasectomy for personal reasons but wish to undergo vas reconstruction surgery to regain fertility are also candidates for the cryopreservation of testicular sperm. Testicular sperm can be retrieved and cryopreserved at the time of vas re-anastomosis. This strategy ensures the availability of sperm if the reconstruction fails (probability of 20-25%) (16) and avoids a repeat surgical sperm retrieval. Patients undergoing exploratory surgery to assess the cause of azoospermia should also be counseled to freeze testicular sperm at this time if possible, thus avoiding another surgical intervention.
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than that obtained by needle aspiration. A variation of TESA, testicular fine needle aspiration (TEFNA, FNA), involves the aspiration of tubules from multiple sites in the gonad. Multiple testicular fine needle aspirations have been attempted in men with NOA with varying success. In men with NOA, the success of sperm retrieval by FNA depends on the etiology and severity of the underlying cause of the azoospermia. In a prospective study comparing the efficacy of multiple needle biopsies with open testicular excision, spermatozoa were retrieved in only 14% of the patients with non-obstructive azoospermia by multiple needle aspirations and in 63% of patients following open excision biopsy (22). The probability of retrieving testicular sperm in NOA by various surgical techniques is presented schematically in Figure 1.
are sperm available and if alternate arrangements may be needed. Because the number of sperm retrieved for cryopreservation in NOA patients may be extremely low, the loss of a few sperm during the freeze/thaw cycle can be significant and is a very real disadvantage.
& SURGICAL TECHNIQUES FOR SPERM RETRIEVAL In patients with documented OA, sperm can be surgically retrieved from the testis or epididymis. In comparison, men with NOA are restricted to testicular surgery for the retrieval of male gametes. A variety of surgical approaches, ranging from percutaneous aspiration to open biopsy, have been successfully employed for sperm retrieval. Although enough sperm for IVF-ICSI in a fresh cycle may be retrieved by a particular surgical technique (such as needle aspiration), if the intent is to cryopreserve multiple aliquots for future use, techniques that yield higher numbers of sperm, such as open biopsy and micro-TESE, may be more suitable options.
Retrieval of epididymal sperm Percutaneous epididymal sperm aspiration (PESA). PESA involves the insertion of a 23-gauge butterfly needle through the scrotal skin into the epididymis. Negative suction pressure is applied using a 10-ml syringe containing a small volume of culture medium. The epididymal fluid mixed with culture medium is then examined for the presence of motile sperm. The procedure may involve multiple punctures at different locations in the epididymis until live sperm are found. PESA may be a good option for patients with ductal obstruction distal to the epididymis. Microsurgical epididymal sperm aspiration (MESA). The technique of MESA involves the surgical exposure of the epididymis and the aspiration of the effluent from the epididymal tubules under optical magnification. It is the technique of choice for some surgeons to surgically retrieve sperm because of the high concentration and quality of spermatozoa obtained compared with that of testicular sperm, especially in patients with irreparable epididymal obstruction. Epididymal spermatozoa are mature and progressively motile, and epididymal aspirates are much cleaner and devoid of the cellular debris that is seen in testicular sperm preparations. The motility of epididymal sperm makes sperm selection during ICSI easier without the introduction of additional steps (such as treatment with
Retrieval of testicular sperm Open testicular biopsy. This conventional method of surgical sperm extraction generally offers the best chance of retrieving spermatozoa, irrespective of the etiology of azoospermia. Open biopsy also allows the excision of a larger tissue mass, allowing access to a greater number of sperm available for freezing. The major drawback of open biopsy, from the point of view of the patient, is the size of the wound and the healing time compared with other forms of aspiration, namely needle aspiration. In men with NOA, open testicular biopsy is more effective than testicular sperm aspiration (TESA) or multiple fine needle aspirations (FNAs). Patients with focal spermatogenesis or hypospermatogenesis are also best served with open biopsy or, in some cases, with micro-TESE (or micro-dissection TESE). Micro-TESE is a more invasive surgical procedure involving a thorough examination under an operating microscope of the bifurcated gonad to locate and excise the seminiferous tubules exhibiting active spermatogenesis. The sperm retrieval rate by micro-TESE has been reported as superior to that of conventional TESE (18-20), especially in patients with focal and sporadic spermatogenesis. The excision of isolated seminiferous tubules with active spermatogenesis in micro-dissection TESE results in the removal of significantly less tissue (average of 9.4 mg) compared with a standard biopsy (720 mg) (18). The advantages of micro-TESE in addition to the superior sperm retrieval rate include the avoidance of complications such as hematoma, fibrosis, and impaired androgen production (18). Testicular sperm aspiration (TESA). Testicular sperm aspiration or percutaneous testicular sperm aspiration is a technique originally described as a diagnostic procedure used to evaluate spermatogenesis in azoospermic patients (21). In this technique, a 19- or 21-gauge needle attached to a 20-ml syringe is used to percutaneously puncture the testis, and negative pressure is applied to aspirate the tubules. Fragments of the seminiferous tubules are analyzed for the presence of sperm. Since the development of ICSI, in which a single viable sperm per mature oocyte is needed for fertilization, the availability of a few spermatozoa has been enough to offer a chance of pregnancy. In men with OA, testicular sperm aspiration invariably generates enough sperm for IVF-ICSI. In general, the number of sperm obtained by open biopsy (TESE) is significantly higher
Figure 1 - Probability of retrieving sperm in NOA by various surgical procedures (high to low).
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a conical tube (A) while leaving the larger tissue fragments in the dish. 3. Add 2.0 ml of the sperm-washing medium to the petri dish. Using a 3-cc syringe attached to a 21-gauge needle, gently aspirate the tissue suspension repeatedly. The repeated aspiration through the syringe needle dissociates the seminiferous tubules and releases the sperm from the tubular lumen. 4. Transfer the suspension to the same conical tube (A) used in step 2 and let it stand at room temperature for 5-10 minutes. The large tissue fragments tend to settle at the bottom. Transfer the supernatant to a new conical tube (B). 5. Add 1-2 ml of the sperm-washing medium to the tissue pellet and mix well with a Pasteur pipet. 6. Allow the tissue fragments to settle for 5 minutes and transfer the supernatant to the conical tube (B). 7. Centrifuge the conical tube (B) at 400 g for 10 minutes. 8. Gently aspirate and discard the supernatant, leaving the pellet at the bottom. 9. Add 3-4 ml of RBC Lysis Buffer to the pellet and mix with a Pasteur pipet. 10. Centrifuge at 400 g for 5 minutes. 11. Gently aspirate the supernatant and discard it without disturbing the pellet. 12. Re-suspend the pellet in 3.0 ml of the sperm-washing medium and centrifuge the conical tube at 400 g for 10 minutes to wash off the RBC Lysis Buffer. 13. Discard the supernatant and resuspend the pellet in 0.5-1.0 ml of the sperm-washing medium. 14. Add an equal volume of TEST-Yolk sperm cryopreservation medium to the sperm suspension and aliquot the mixture into 3-4 labeled cryovials. 15. Cool the cryovials to 4 ˚C (in the refrigerator) for 30-45 minutes. 16. Expose the vials to the liquid nitrogen vapor phase (810 inches above the liquid nitrogen level) for an hour. 17. Plunge the frozen vials into liquid nitrogen for storage.
motility-enhancing agents) that may be needed to identify viable gametes if testicular sperm are used. In a study comparing the two techniques of sperm retrieval from the epididymis (PESA and MESA), sperm were successfully retrieved by PESA in 61% of patients with obstructive azoospermia, compared with a 93% sperm retrieval rate by MESA (23).
& CRYOPRESERVATION OF TESTICULAR SPERM I. Materials and Equipment Sterile culture dishes Polystyrene conical tubes Sterile glass pipettes Syringes (50 ml) Syringes (3 ml) with 21-gauge needles Syringe filter (Nalgene, 0.22 mm) Microscope glass slides Coverslips Gloves Sterile pair of curved iris scissors Sterile pair of forceps Bench-top clinical centrifuge Microscope Refrigerator Weighing chemical balance Cryo vials Aluminum canes Plastic cryosleeves Liquid nitrogen dewar Liquid nitrogen Personal protective gear for handling liquid nitrogen
II. Reagents Quinn’s Sperm Washing Medium (modified human tubal fluid with human serum albumin, 5 mg/ml; SAGE IVF Inc., Trumbull, CT, USA). Sperm Freezing Medium (20% TEST Yolk buffer, 12% glycerol with gentamycin sulfate; Irvine Scientific, Santa Ana, CA, USA). Tissue culture-grade water (SAGE IVF Inc., Trumbull, CT, USA). RBC Lysis Buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 2 mM EDTA; pH 7.2).
& CRYOPRESERVATION OF EPIDIDYMAL SPERM 1. Once the presence of sperm is documented by microscopic examination of the epididymal aspirate, the fluid is transferred to a conical tube. 2. Add 1-2 ml of the sperm-washing medium to the conical tube containing the epididymal fluid. Mix gently. 3. Centrifuge the conical tube at 400 g for 10 minutes. 4. Discard the supernatant. If the pellet appears to be contaminated with erythrocytes, blood cells may be removed by washing the pellet with RBC Lysis Buffer as follows. 5. Re-suspend the pellet in 2 ml of RBC Lysis Buffer. Mix gently. 6. Centrifuge the conical tube at 400 g for 5 minutes. 7. Gently aspirate the supernatant and discard without disturbing the sperm pellet. 8. Wash the pellet with 1-2 ml of the sperm-washing medium by centrifuging the tube at 400 g for 10 minutes. 9. Re-suspend the pellet in 1.0 ml of the sperm-washing medium.
Preparation of RBC lysis buffer Ammonium chloride (NH4Cl) 0.829 g Potassium bicarbonate (NaHCO3) 0.100 g Ethylenediaminetetraacetic acid (EDTA) 0.074 g Tissue culture-grade water 100 ml Dissolve and adjust the pH to 7.2. Sterilize the buffer using a 0.2-mm syringe filter and store in the refrigerator at 4 ˚C for up to four weeks.
Procedure (The procedure can be performed at room temperature) 1. Rinse the tissue in sperm-washing medium to remove the blood and transfer it to a sterile petri dish. 2. Using a pair of sterile curved iris scissors, mince the tissue well. Keep the tissue moist with the sperm-washing medium during mincing. Add 1.0 ml of the spermwashing medium to the finely minced tissue. Tilt the dish, aspirate the medium, and transfer the suspension to
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10. Add an equal volume (1.0 ml) of TEST-Yolk sperm cryopreservation medium to the epididymal sperm suspension. 11. Aliquot the mixture into 3-4 labeled cryovials for cryostorage. 12. Place the cryovials in a 4 ËšC refrigerator for 30-45 minutes and then freeze the cryovials in liquid nitrogen vapor for 1 hour. 13. Plunge the vapor-frozen vials into liquid nitrogen for storage.
section is a standard technique when several hundred to thousands or a million sperm are available for preservation. For some patients with NOA, the retrieval of only a few (less than 100, and sometimes as few as 10 or less) sperm poses a technical challenge for cryopreservation and, later, at the time of warming for the successful retrieval of these few gametes for ICSI. Several investigators have attempted to cryopreserve extremely low number of sperm (and sometimes a single sperm) in small volumes using various carriers (Table 1). The two methods that have resulted in successful pregnancies and live births involve the use of empty zona (human, mouse, or hamster) and cryoloops. These methods are briefly summarized below.
& PROCEDURE FOR THAWING THE TESTICULAR AND EPIDIDYMAL SPERM
Cryopreservation of a single human sperm using a zona pellucida
Note: Use proper safety precautions and personal protective gear while handling liquid nitrogen.
Cohen and colleagues (24) were the first to report this unique method of the cryopreservation of a single spermatozoon inside empty zona obtained from mouse, hamster, or human oocytes. The oocytes were treated with hyaluronidase to remove the cumulus and corona cells. The denuded oocyte was held by a holding pipette, and two small holes were drilled into the zona (by mechanical breach, acid tyrodes solutions, or a laser). The ooplasm was aspirated by suction, leaving the zona empty of its contents. The testicular suspension was carefully examined under the microscope for the presence of spermatozoa, and once a twitching/moving sperm was found, it was transferred to a PVP (10%) droplet. This procedure was repeated until a few sperm had been retrieved. The next step involved the insertion of one or more (up to 15) sperm into empty zona using an ICSI needle. Injected zonae containing sperm were placed into an 8% glycerol solution in phosphate-buffered saline (PBS) containing human serum albumin (3%). The zonae were then frozen individually in sterile straws (0.25 ml). Each zona was placed in a column of cryopreservation medium sandwiched between two air bubbles. The straws were heat-sealed and exposed to liquid nitrogen vapor for two hours, followed by storage in liquid nitrogen. The spermatozoa were recovered by thawing the straw in a water bath (30 ËšC) for 30 seconds. One end of the straw was cut with a pair of sterile scissors, and the content of the
1. Before thawing, locate, identify, and confirm the patient information on the vial. 2. Take the vial out of the liquid nitrogen and thaw at room temperature for 10 minutes. 3. Transfer the contents of the vial into a conical tube. Add 1-2 ml of the sperm-washing medium slowly, drop by drop, to the thawed suspension. Mix the contents gently. 4. Centrifuge the tube at 400 g for 10 minutes. 5. Gently aspirate the supernatant and discard it without disturbing the sperm pellet. 6. Re-suspend the pellet in 1-2 ml of the sperm-washing medium and centrifuge the tube again at 400 g for 10 minutes. 7. Gently remove the supernatant. Re-suspend the sperm pellet in 50-100 ml of the sperm-washing medium. 8. The sperm suspension is ready for ICSI.
& METHODS FOR THE CRYOPRESERVATION OF SINGLE (OR FEW) SPERM The procedure for the cryopreservation of testicular and epididymal sperm as described in detail in the preceding
Table 1 - Carriers used for sperm cryopreservation in microquantities. Carrier Empty zona (mouse, hamster, or human)
Cryoloop Mini straws or openpulled straws ICSI pipette Microdroplets
Volvox globator algae Alginate beads or agarose microspheres Cryotop
Advantages
Disadvantages
Comments
References
Successful pregnancy 24,25,45,46 Human or non-human biological material; Laborintensive; Requires micromanipulation and inhouse evacuation and preparation of the zona envelope Commercially available Requires a micromanipulator to load sperm onto Successful pregnancy 27,28,47 the loop and is somewhat labor-intensive Easy and simple technique Not feasible for an extremely low volume/ Can be used to freeze severely 48 number of sperm oligospermic samples Commercially available; Fragile glass pipette; difficult to store and No reported pregnancies 49 found in every IVF lab handle in liquid nitrogen Easy and relatively simple Difficult to handle and store in liquid nitrogen; Has yielded successful 50-52 technique Culture dishes are fragile when stored in pregnancies; has not received liquid nitrogen; Variable recovery rates widespread acceptance Inexpensive; plentifully Non-human biological material; laborNot suitable for human clinical 53 available intensive advance preparation of algae spheres use Inert polymers used as Very labor-intensive technique No report of clinical 54,55 carriers pregnancies 56 Commercially available, Sperm recovery, survival easy to load and handle similar to that obtained using the empty zona method Easy to handle the zona envelope; good sperm recovery and survival
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Table 2 - Outcomes using testicular sperm in patients with obstructive azoospermia. Testicular Sperm Fresh Frozen-thawed Fresh Frozen-thawed Fresh Fresh Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed *
Fertilization (%)
Embryonic Cleavage (%)
Implantation (%)
Clinical Pregnancies (%)
Ongoing/Live births (%)
52 51 58 64 64 62 50 49 72 68 52 -
99 96 98 95 99 93 106* -
33 14 18 14 33 17 14 -
0 6 33 32 30 25 26 29 69 42 24 -
0 6 20 20 23 22 19 44 25 15 -
References 12 57 58 59 60 31 29
.100% because embryos that did not show normal signs of fertilization (2 pronuclei) but that did show cleavage are included.
for up to 30 minutes before being plunged into liquid nitrogen for storage. Sperm frozen on the cryoloop are thawed at room temperature using a dissecting binocular microscope. The cryoloop attached to the cap is unscrewed under liquid nitrogen and is swiftly shifted under the binoculars over a sterile culture dish. The loop is held so that it touches the surface of the dish, and it is cut with a scalpel from the metal shaft. Immediately, 2 ml of HEPES-buffered medium supplemented with albumin is laid on top of the loop, and the droplet is overlaid with mineral oil. Spermatozoa may be individually moved to a fresh drop of medium, washed, and then transferred to PVP before ICSI. The cryoloop method for the cryopreservation of a single spermatozoon is also labor-intensive and time-consuming and requires extensive training and experience. The cryopreservation of a single sperm, although it is feasible and has had documented success, is rarely practiced, and alternative options, such as fresh biopsy with donor sperm back-up, are more prevalent.
straw was expelled into a sterile dish. The zona was recovered and washed several times in HEPES-buffered medium. The zona containing the sperm was then transferred to a droplet of PVP (12%). Using a holding pipette, the zona envelope was positioned to permit the penetration of the ICSI needle through the slit (used earlier to evacuate the ooplasm). The ICSI needle was inserted into the zona, and the sperm was aspirated gently and released into the PVP droplet. The sperm was immobilized and injected into the metaphase II oocyte, held in a drop of HEPES-buffered medium. This technique of the cryopreservation of individual spermatozoa inside an empty zona, although quite labor-intensive and time-consuming, offers an opportunity to retrieve and store sperm in extreme cases of male factor infertility. Successful pregnancies and live births have been reported with this method (25).
Cryopreservation of sperm using the cryoloop method The use of a cryoloop as a carrier to contain the frozen embryos was initially proposed by Lane and colleagues (26). The premise of holding the embryo on a thin film of cryoprotectant solution on a small (0.5-0.7 mm) loop has the advantage of enabling the handling of a very small volume (2 ml) with excellent recovery rates. The use of cryoloops was thereafter extended to freeze a very small number of sperm (27). The method of cryopreservation on cryoloops was refined to enable the freezing of individual sperm by loading the sperm onto the cryoloop by a micromanipulation technique (28). The procedure of single sperm cryopreservation involves holding the cryoloop on a magnetic wand. The magnetic wand is attached to the micromanipulator on an inverted microscope and is lowered to visualize the loop. The spermatozoa are isolated using a micropipette (e.g., ICSI pipet) and concentrated onto a microdrop of HEPES-buffered medium, supplemented with 1% human serum albumin. Just before loading the sperm onto the cryoloop, the individual sperm is transferred to another microdrop of a 1:1 mixture of HEPES medium and sperm cryopreservation medium (TEST-Yolk buffer containing 12% glycerol; Irvine scientific, Santa Ana, CA). Using a micropipette, sperm are then loaded onto the film in the loop. The cryoloop attached to the cap is screwed onto the cryovial, and the vial is exposed to liquid nitrogen vapor
& OUTCOME Obstructive azoospermia The use of testicular and epididymal sperm in the treatment of male factor infertility secondary to OA has now become a standard approach. The probability of retrieving sperm in men with OA is close to 100%. In men with OA, the use of fresh testicular sperm for ICSI invariably offers fertilization, pregnancy, and live birth rates that are comparable to those derived from the use of ejaculated sperm in age-matched controls. The use of frozen testicular sperm from OA patients for ICSI offers fertilization, implantation, and clinical pregnancy rates that are equivalent to those derived from the use of freshly retrieved testicular sperm (Table 2). Fertilization and clinical pregnancy rates are also similar between spermatozoa of epididymal or testicular origin (29). MESA is the preferred method of sperm recovery if the obstruction is determined to be at a location distal to the epididymis. The use of either fresh or frozen epididymal sperm offers comparable fertilization, embryonic development, implantation, and clinical pregnancy rates (30). Fertilization and pregnancy rates across different sperm retrieval methods and obstruction etiologies are also comparable (23). The number of
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is more successful than needle aspiration. In approximately 13% of men diagnosed with NOA, the failure of spermatogenesis may be attributed to Y chromosome microdeletions. The probability of retrieving spermatozoa in men who have microdeletions in the AZFa and/or AZFb regions is close to zero, whereas men with AZFc microdeletions have an approximately 70% chance of having enough sperm available for ICSI (36). Although the reports detailing the rates of testicular sperm retrieval in men with NOA range from 1487% (Table 3), a conservative estimate of an overall 50-60% chance of successful sperm retrieval is appropriate. In general, the total number of sperm retrieved in NOA is significantly less than that obtained in OA. The cryopreservation of sperm in general results in a decrease in postthaw motility and vitality. This loss of viability can be of great concern in NOA because of the low number of sperm available; however, unique approaches to the freezing of single/individual sperm now offer excellent post-thaw recoveries. It is well documented that spermatozoa in men with oligoasthenoteratozoospermia exhibit an increased incidence of chromosomal abnormalities (37-40). Interestingly, a comparison of aneuploidy frequency between embryos derived from testicular sperm from men with OA and from
high-quality embryos obtained using frozen testicular sperm is similar to that obtained using fresh testicular sperm (31). Despite the high pregnancy rates achieved using fresh and frozen surgically retrieved sperm, there is some concern that the use of testicular sperm results in higher miscarriage rates compared with the use of ejaculated sperm (31-33). The rate of aneuploidy in testicular and epididymal spermatozoa in OA patients is similar to that observed in sperm from normal men (34), suggesting that the higher incidence of early miscarriages cannot be attributed to the paternal genome. Testicular sperm are generally immature but undergo maturation during their passage through the epididymis. Immature testicular sperm are easily recognizable by the presence of large cytoplasmic droplets attached to their middle pieces and necks. It has been suggested that reactive oxygen species in cytoplasmic droplets may cause irreversible DNA damage (35) in immature testicular sperm, resulting in a higher miscarriage rate (31).
Non-obstructive azoospermia The probability of sperm retrieval in NOA is dependent on two factors, namely the etiology of NOA and the surgical approach. In general, microdissection TESE or open biopsy
Table 3 - Rate of testicular sperm retrieval in men with non-obstructive azoospermia. Sperm Retrieval Technique
Rate of Sperm Retrieval
TESE Micro-TESE TESE TESE TESE Multiple FNAs TESE TESE TESE TESA TESE
13/15 (87%) 17/27 (63%) 15/25 (60%) 14/18 (78%) 22/35 (63%) 5/35 (14%) 33/55 (60%) 43/64 (67%) 15/42 (36%) 22/86 (26%) 5/48 (10%)
TESE TESE TESE Multiple TESE
10/22 10/17 18/31 13/37
Micro-TESE FNA
24/56 (43%) 35/51 (69%)
TESE TESE TESE If no sperm retrieved, patients had multiple repeat biopsies
Micro-TESE TESE TESE/Micro-TESE TESE Micro-TESE TESE+Micro-TESE Micro-TESE TESE Micro-TESE
(45%) (59%) (58%) (35%)
5/12 (42%) 23/30 (77%) 261/628 (42%) 77/103 (74%)
Comment
References 5 9
Compared TESE and Multiple FNAs
61 22 43 62 63 64
48 patients with failed TESA underwent TESE Compared TESE and Micro-TESE
Compared multiple TESE and Micro TESE 2 patients who had no sperm retrieved during the first FNA attempt had a positive sperm retrieval during the second FNA attempt Post-chemotherapy azoospermia First attempt Second attempt
28/34 (82%) 11/11 (100%) 5/6 (83%) 2/2 (100%) 384/784 (49%) 57% 32% 65/138 (47%) 87/258 (34%) 16/77 (21%) 131/258 (51%) 37/65 (57%) 26/68 (38%) 27/73 (37%)
67 68
69 31 70
Third attempt Fourth attempt Fifth Attempt Sixth attempt Overall Compared TESE and Micro-TESE
71
Compared TESE and Micro-TESE
72 73
Compared TESE and Micro-TESE Post-chemotherapy azoospermia
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18 65 66 67
20 20 74
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Table 4 - Outcome using testicular sperm in patients with non-obstructive azoospermia. Testicular Sperm
Fertilization (%)
Embryonic Cleavage (%)
Implantation (%)
48 39
78 68
19 25
60
20 -
47 44 66 58 54 51 52 56 58 55 67 68 58 52-60 64 -
93 89 97 97 75 94 93 93 93 85 93 -
9 11 13 18 13 9 13 24 7 11.3 -
26 27 30 50 27 22 33 67 32 29 29 13 25 40-52 8 -
22 9 20 13 18 13 22 31-39 7 -
Fresh Frozen-Thawed Fresh Frozen-Thawed Fresh Frozen-thawed Fresh Frozen-Thawed Fresh Frozen-thawed Fresh Frozen-Thawed Fresh Frozen-thawed Fresh Frozen-Thawed Fresh Frozen-Thawed Fresh Frozen-thawed Fresh Frozen-Thawed Fresh Frozen-thawed *
Clinical Pregnancies Ongoing pregnancies/ (%) Live births (%)
References 5 9 61 62 43 57 66* 58 70 44 74 75
Data represent a combination of OA (10 patients) and NOA (18 patients).
those with NOA revealed no difference between the two groups (41), suggesting the chromosomal normalcy of the testicular sperm. However, a study comparing surgically retrieved versus ejaculated sperm showed a significantly higher incidence of chromosomal abnormalities in surgically retrieved sperm from men with OA and NOA compared with normospermic ejaculated sperm (42). Despite differences in the study designs that make any comparison of outcome results between OA and NOA difficult, it appears that if sperm are retrieved and viable gametes are available for ICSI, the fertilization, implantation, and pregnancy rates resulting from the use of fresh sperm from men with NOA (Table 4) are in line with those resulting from the use of sperm from men with OA (Table 3). Therefore, the cryopreservation of sperm does not affect the fertilization and pregnancy outcomes.
epididymal) sperm are as effective as freshly retrieved sperm. In NOA patients with severe hypospermatogenesis or focal spermatogenesis, where very few sperm are retrieved and the cryopreservation of individual (few) sperm using specialized methods (such as the empty zona or cryoloop method) is not feasible, a fresh biopsy should be offered. The injection of sperm using non-motile spermatozoa results in a significantly lower fertilization and live birth rate, emphasizing the importance of the motility/viability of sperm (43). Currently, there are no definitive parameters, besides surgical testicular exploration, that are reliable in predicting the presence of sperm in men with NOA.
& REFERENCES 1. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992;340(8810):17-8, http://dx.doi.org/10.1016/0140-6736(92)92425-F. 2. Schoysman R, Vanderzwalmen P, Nijs M, Segal-Bertin G, van de Casseye M. Successful fertilization by testicular spermatozoa in an in-vitro fertilization programme. Hum Reprod. 1993;8(8):1339-40. 3. Tournaye H, Devroey P, Liu J, Nagy Z, Lissens W, Van Steirteghem A. Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil Steril. 1994;61(6):1045-51. 4. Craft I, Bennett V, Nicholson N. Fertilizing Ability of Testicular Spermatozoa. Lancet. 1993;342(8875):864, http://dx.doi.org/10.1016/ 0140-6736(93)92722-6. 5. Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, et al. Pregnancies after Testicular Sperm Extraction and Intracytoplasmic Sperm Injection in Nonobstructive Azoospermia. Hum Reprod. 1995;10(6):1457-60, http://dx.doi.org/10.1093/HUMREP/10.6.1457. 6. Silber SJ, Nagy Z, Liu J, Tournaye H, Lissens W, Ferec C, et al. The Use of Epididymal and Testicular Spermatozoa for Intracytoplasmic Sperm Injection - the Genetic-Implications for Male-Infertility. Hum Reprod. 1995;10(8):2031-43. 7. Silber SJ, Vansteirteghem AC, Devroey P. Sertoli-Cell Only Revisited. Human Reproduction. 1995;10(5):1031-2. 8. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum Reprod. 1995;10(1):148-52, http://dx.doi.org/10.1093/humrep/10.1.148.
& CONCLUSION Successful pregnancies resulting from the use of surgically retrieved spermatozoa for ICSI (2) were first reported in 1993. Various surgical techniques, ranging from less invasive percutaneous aspiration to highly invasive open testicular biopsy and micro-dissection TESE, are employed. In OA, the probability of obtaining sperm from the testis is almost guaranteed. In men with non-obstructive azoospermia, freshly retrieved sperm offers the best chance of pregnancy; however, in 30-50% of NOA patients, it is still possible that no sperm may be retrieved (43). In patients with frozen sperm, sometimes no viable sperm may be available at the time of ICSI. The incidence of complete nonviability or the inability to find any injectable sperm postthaw in NOA patients is reported to be approximately 20% (44). The availability of viable sperm, regardless of the source (testis or epididymis), at the time of ICSI largely dictates the outcome of fertilization. Frozen (testicular or
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Using Ejaculated, Fresh, and Frozen-Thawed Epididymal and Testicular Spermatozoa Gives Rise to Comparable Results after Intracytoplasmic Sperm Injection. Fertil Steril. 1995;63(4):808-15. 14. Smith AU, Polge C. Survival of Spermatozoa at Low Temperatures. Nature. 1950;166(4225):668-9, http://dx.doi.org/10.1038/166668a0. 15. Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod. 1997;12(8):1688-92, http://dx.doi.org/10.1093/ humrep/12.8.1688. 16. Kolettis PN, Sabanegh ES, D’Amico A M, Box L, Sebesta M, Burns JR. Outcomes for vasectomy reversal performed after obstructive intervals of at least 10 years. Urology. 2002;60(5):885-8, http://dx.doi.org/10. 1016/S0090-4295(02)01888-5. 17. Keros V, Hultenby K, Borgstrom B, Fridstrom M, Jahnukainen K, Hovatta O. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment. 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A prospective study of multiple needle biopsies versus a single open biopsy for testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod. 1998;13(11):307580, http://dx.doi.org/10.1093/humrep/13.11.3075. 23. Lin YM, Hsu CC, Kuo TC, Lin JS, Wang ST, Huang KE. Percutaneous epididymal sperm aspiration versus microsurgical epididymal sperm aspiration for irreparable obstructive azoospermia—experience with 100 cases. J Formos Med Assoc. 2000;99(6):459-65. 24. Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT. Cryopreservation of single human spermatozoa. Hum Reprod. 1997;12(5):994-1001, http://dx.doi.org/10.1093/humrep/12.5.994. 25. Walmsley R, Cohen J, Ferrara-Congedo T, Reing A, Garrisi J. The first births and ongoing pregnancies associated with sperm cryopreservation within evacuated egg zonae. Hum Reprod. 1998;13 Suppl 4:61-70, http://dx.doi.org/10.1093/humrep/13.suppl_4.61. 26. Lane M, Bavister BD, Lyons EA, Forest KT. 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Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod. 2002;17(11):2813-24, http://dx.doi.org/10.1093/humrep/17.11. 2813. Moosani N, Pattinson HA, Carter MD, Cox DM, Rademaker AW, Martin RH. Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil Steril. 1995;64(4):811-7. Bernardini L, Gianaroli L, Fortini D, Conte N, Magli C, Cavani S, et al. Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients. Hum Reprod. 2000;15(10):2165-72, http://dx.doi.org/10.1093/humrep/15.10.2165. Colombero LT, Hariprashad JJ, Tsai MC, Rosenwaks Z, Palermo GD. Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil Steril. 1999;72(1):90-6, http://dx. doi.org/10.1016/S0015-0282(99)00158-2. Pang MG, Hoegerman SF, Cuticchia AJ, Moon SY, Doncel GF, Acosta AA, et al. Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum Reprod. 1999;14(5):1266-73, http://dx.doi.org/10.1093/humrep/14.5.1266. Platteau P, Staessen C, Michiels A, Tournaye H, Van Steirteghem A, Liebaers I, et al. Comparison of the aneuploidy frequency in embryos derived from testicular sperm extraction in obstructive and nonobstructive azoospermic men. Hum Reprod. 2004;19(7):1570-4, http:// dx.doi.org/10.1093/humrep/deh306. Rodrigo L, Rubio C, Mateu E, Simon C, Remohi J, Pellicer A, et al. Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by fluorescence in-situ hybridization. Hum Reprod. 2004;19(1):118-23, http://dx.doi.org/10. 1093/humrep/deh012. Ben-Yosef D, Yogev L, Hauser R, Yavetz H, Azem F, Yovel I, et al. Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with nonobstructive azoospermia. Hum Reprod. 1999;14(7):1794-801, http://dx. doi.org/10.1093/humrep/14.7.1794. Verheyen G, Vernaeve V, Van Landuyt L, Tournaye H, Devroey P, Van Steirteghem A. Should diagnostic testicular sperm retrieval followed by cryopreservation for later ICSI be the procedure of choice for all patients with non-obstructive azoospermia? Hum Reprod. 2004;19(12):2822-30, http://dx.doi.org/10.1093/humrep/deh490. Fusi F, Calzi F, Rabellotti E, Papaleo E, Gonfiantini C, Bonzi V, et al. Fertilizing capability of frozen-thawed spermatozoa, recovered from microsurgical epididymal sperm aspiration and cryopreserved in oocytefree human zona pellucida. Hum Reprod. 2001;16:117-8. Hsieh YY, Tsai HD, Chang CC, Lo HY. Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae. Fertil Steril. 2000;73(4):694-8, http://dx.doi.org/10.1016/S0015-0282(99)006123. Desai N, Glavan D, Goldfarb J. A convenient technique for cryopreservation of micro quantities of sperm. Annual meeting program supplement. Fertil Steril. 1998;70:S197-8 Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F, et al. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10(3):350-4, http:// dx.doi.org/10.1016/S1472-6483(10)61795-6. Gvakharia M, Adamson GD. A method of successful cryopreservation of small numbers of human spermatozoa. Fertil Steril. 2001;76(3):S101, http://dx.doi.org/10.1016/S0015-0282(01)02297-X. Gil-Salom M, Romero J, Rubio C, Ruiz A, Remohi J, Pellicer A. Intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Mol Cell Endocrinol. 2000;169(1-2):15-9, http://dx.doi.org/10. 1016/S0303-7207(00)00345-2. Quintans CJ, Donaldson MJ, Asprea I, Geller M, Rocha MG, Pasqualini RS. Pregnancy after ICSI with Spermatozoa Cryopreserved with a Novel Technique Useful for the Cryostorage of Very Small Numbers of Sperm
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REVIEW
Reproductive outcomes, including neonatal data, following sperm injection in men with obstructive and nonobstructive azoospermia: case series and systematic review Sandro C. Esteves,I Ashok AgarwalII I
ANDROFERT – Andrology & Human Reproduction Clinic, Campinas, Sa˜o Paulo, Brazil. Foundation, Cleveland, Ohio, USA.
II
Center for Reproductive Medicine, The Cleveland Clinic
We compared pregnancy outcomes following intracytoplasmic sperm injection for the treatment of male infertility according to the type of azoospermia. First, we analyzed our data from 370 couples who underwent intracytoplasmic sperm injection using sperm from men with obstructive azoospermia and nonobstructive azoospermia, and the outcomes were compared to a group of 465 non-azoospermic infertile males. Then, we performed a systematic review of the published data on pregnancy and neonatal outcomes of children born after sperm injection using sperm from men with obstructive and nonobstructive azoospermia. Live birth rates were significantly lower in the nonobstructive azoospermia group (21.4%) compared with the obstructive azoospermia (37.5%) and ejaculated sperm (32.3%) groups. A total of 326 live births resulted in 427 babies born. Differences were not observed between the groups in gestational age, preterm birth, birth weight and low birth weight, although we noted a tendency towards poorer neonatal outcomes in the azoospermia categories. The overall perinatal death and malformation rates were 2.8% and 1.6%, respectively, and the results did not differ between the groups. We identified 20 published studies that directly compared pregnancy outcomes between obstructive azoospermia and nonobstructive azoospermia. Most of these studies were not designed to detect differences in live birth rates and had lower power to detect differences in less frequent outcomes, and the reporting of neonatal outcomes was unusual. The included studies reported either a decrease or no difference in pregnancy outcomes with intracytoplasmic sperm injection in cases of nonobstructive azoospermia and obstructive azoospermia. In general, no major differences were noted in short-term neonatal outcomes and congenital malformation rates between children from fathers with nonobstructive azoospermia and obstructive azoospermia. KEYWORDS: Male Infertility; Azoospermia; Intracytoplasmic Sperm Injection; Pregnancy Outcomes; Systematic Review. Esteves SC, Agarwal A. Reproductive outcomes, including neonatal data, following sperm injection in men with obstructive and nonobstructive azoospermia: case series and systematic review. Clinics. 2013;68(S1):141-149. Received for publication on April 9, 2012; Accepted for publication on April 11, 2012 E-mail: s.esteves@androfert.com.br Tel.: 55 19 3295-8877
male population, it has been estimated that 8% of men at reproductive ages seek medical assistance for infertilityrelated problems, and 1-10% of them have a condition that affects their reproductive potential (3). One of these conditions is azoospermia, defined as the complete absence of spermatozoa in the ejaculate after centrifugation, which occurs in 1-3% of the male population and approximately 10% of infertile males (4). Despite being associated with infertility, azoospermia does not necessarily imply sterility because many azoospermic men maintain sperm production at varying levels within the testes (5). In fact, two distinct clinical presentations are usually seen in men with azoospermia. In obstructive azoospermia (OA), spermatogenesis is normal, but either a mechanical blockage exists in the genital tract between the epididymis and the ejaculatory duct or the vasa deferentia are absent. Causes of
& INTRODUCTION Worldwide, an estimated 9% of couples meet the definition of infertility, with 50% to 60% of these couples seeking care (1). From a global perspective, these figures indicate that approximately 140 million individuals at reproductive age are unintentionally childless or have undergone treatment to reproduce (2). With regards to the
Copyright ß 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)16
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OA may be either acquired or congenital and include vasectomy, failure of vasectomy reversal, post-infectious diseases, surgical procedures in the scrotal, inguinal, pelvic or abdominal regions, cystic fibrosis, congenital absence of the vas deferens (CAVD), ejaculatory duct or prostatic cysts and Young’s syndrome. Unlike OA, men presenting with nonobstructive azoospermia (NOA) either lack or have severely impaired spermatogenesis. NOA comprises a spectrum of testicular histopathology resulting from various causes that include genetic and congenital abnormalities, infection, exposure to gonadotoxins, medications, varicocele, trauma, endocrine disorders, and idiopathic disorders (5). In a group of 2,383 infertile men attending the tertiary center for male reproduction of one of the authors (SE), 835 (35%) were identified as having azoospermia; approximately 36% of those cases resulted from obstruction in the ductal system, whereas over 60% were associated with testicular failure caused by different conditions (Table 1). Although selected cases of OA may be surgically correctable, treatment options for most couples with azoospermiarelated infertility will ultimately include assisted reproductive techniques (ART), which is a broad term used to define any procedure that involves handling of both sperm and oocytes outside the body, such as in vitro fertilization (IVF) and its variant, intracytoplasmic sperm injection (ICSI) (6). To this end, several sperm retrieval methods have been developed to collect epididymal and testicular sperm to be used in conjunction with ART for men with azoospermia. Briefly, either percutaneous (PESA) or microsurgical epididymal sperm aspiration (MESA) are used to retrieve sperm from the epididymis in men with obstructive azoospermia, and testicular sperm aspiration (TESA) or testicular sperm extraction (TESE) are used to retrieve sperm from the testes both in men with OA who fail PESA and those with NOA (7). The clinical application of ART has increased significantly over time. According to the International Committee for Monitoring Assisted Reproductive Technology (ICMART), an international non-profit organization that collects data on ART and monitors approximately 2/3 of ART treatments performed worldwide, the number of treatments has steadily increased since its first report in 1998 (8,9). Along the same trend, the number of babies born from such treatments rose from 84,594 in 1998 to approximately
180,000 in 2003 (an increase of 103%). Similarly, the proportion of total births resulting from ART increased from 0.37% in 1996 to slightly more than 1% in 2009 in industrialized countries such as the United States (10). Intracytoplasmic sperm injection, which is mainly intended to bypass severe male factor infertility, including azoospermia, has become the most used form of ART treatment (8). Although these treatments improve the chances that a couple become parents, they also carry risks, including multiple gestations and preterm delivery, which carries an increased risk of short- and long-term post-natal complications. Nevertheless, there has been a large number of babies born after ICSI in cases of severe male infertility, including azoospermia, and concerns still exist regarding whether the use of spermatozoa from such individuals might affect the health of offspring (11). The purpose of this study was to compare the pregnancy results due to intracytoplasmic sperm injection and neonatal outcomes of children born after ICSI using surgicallyretrieved sperm from men with obstructive and nonobstructive azoospermia with the results from non-azoospermic infertile males treated with sperm injection. In addition, we present a systematic review of published data comparing the pregnancy outcomes after ICSI for the treatment of male infertility due to obstructive and nonobstructive azoospermia and the short- and long-term safety of such interventions.
& METHODS Study Group Consecutive ICSI cycles involving fresh embryo transfers performed at Androfert from January 2004 to December 2010 were initially screened. A total of 471 ICSI cycles using fresh surgically-extracted sperm from men with azoospermia and 621 cycles using fresh ejaculated sperm from men with male factor infertility were reviewed in detail and included in the analysis. A complete male and female workup was conducted in all couples before enrollment in our ART program to both determine the cause of infertility and the treatment strategy as previously described (5). Semen analyses were performed on at least two different occasions according to the World Health Organization criteria (12). Azoospermic individuals were seen by the
Table 1 - Distribution of Diagnostic Categories and Frequency of Azoospermia in a Group of Infertile Men Attending a Tertiary Center for Male Reproduction. Category Varicocele Post-infectious1 Endocrine Ejaculatory dysfunction Systemic disease Idiopathic|| Immunologic Obstruction" Cancer Cryptorchidism Genetic{ TOTAL Obstructive Azoospermia; N (%) Nonobstructive Azoospermia; N (%)
Men Presenting with Azoospermia; Number and Relative Frequencies, N (%)
Number and Absolute Frequencies; N (%) 629 161 54 28 11 645 54 259 11 342 189 2,383
(26.4) (6.9) (2.3) (1.1) (0.4) (27.1) (2.2) (10.9) (0.4) (14.4) (7.9) (100.0)
1 include orchitis and sexually transmitted diseases; ||include testicular failure and idiopathic obstruction; obstruction; {include congenital absence of vas deferens, Yq microdeletion and Klinefelter syndrome.
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32 (5.1) 57 (35.4) 26 (48.1) NA 2 (18.1) 178 (27.6) None 244 (94.2) 4 (36.3) 174 (50.8) 118 (62.4) 835 (35.0) 302 (36.1) 507 (60.7) "
include vasectomy and ejaculatory duct
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Reproductive potential of azoospermic males in ART Esteves SC and Agarwal A
are annually reported to the Latin America ART Registry (REDLARA). Signed informed consent was obtained from patients to use both clinical and laboratory data for analysis with guarantees of confidentiality. For the statistical analysis, sperm injection cycles were grouped according to the source of sperm used for ICSI, i.e., ejaculated or surgically-retrieved. In ICSI cycles with nonejaculated sperm, a distinction was made between OA and NOA. The main pregnancy outcomes analyzed were clinical pregnancy, ectopic pregnancy, miscarriage, live birth, and the perinatal outcomes of babies born (parity, weight and gestational age at the time of delivery, mortality and birth defects). The qualitative variables were expressed as both absolute (n) and relative (%) frequencies. The relationship between the variables among groups was evaluated by the Chi-square test and Fisher’s exact test as appropriate. The quantitative variables were expressed as means and standard deviations. An analysis of variance for one factor (one-way ANOVA) was used to compare these variables when there was a normal distribution, and differences were analyzed by the Tukey multiple comparisons test. For the variables without normal distribution, comparisons were performed with the Kruskal–Wallis test, and the differences were compared using the Dunn multiple comparisons test. A pvalue below 0.05 was considered significant. All analyzes were processed using SPSSH, version 13.0 (SPSS Inc., Chicago, IL, USA). Multiple pregnancies were considered as a single event, and when one gestation produced one live birth and one abortion, the result was registered as a live birth.
urological team to diagnose whether the azoospermia was obstructive or nonobstructive. Of the 370 azoospermic men, 182 and 188 had OA and NOA, respectively. The distinction between obstructive and nonobstructive azoospermia took into account the history, physical examination, endocrine analysis, and genetic testing as appropriate (5). In addition, diagnostic sperm retrieval and testis biopsy for histopathology analysis were conducted in selected cases. The type of azoospermia was further confirmed by testicular histopathology in all testicular sperm retrievals performed at the time of sperm injections. Indications for ICSI were in accordance with the guidelines of the II Brazilian Consensus of Male Infertility (13). Laboratory and clinical protocols remained practically unchanged during these time periods. Ovarian stimulation, oocyte and sperm retrieval, sperm processing, and IVF were conducted as previously reported (14-17). Briefly, percutaneous epididymal (PESA) and/or testicular sperm aspiration (TESA) were performed in cases of obstructive azoospermia, and microsurgical testicular sperm extraction (micro-TESE) or testicular sperm aspiration (TESA) was used for sperm collection in nonobstructive azoospermia. Ejaculated spermatozoa were processed by discontinuous colloidal gradient. Oocytes were retrieved after ovarian stimulation with gonadotropins in association with either pituitary down-regulation with gonadotropin-releasing hormone (GnRH) agonists or luteinizing hormone (LH) surge suppression with GnRH antagonists. The cumuluscorona-oocytes complexes were stripped, classified according to nuclear maturity, and maintained in culture until sperm microinjection. Sperm injections were conducted under 4006 magnification using epididymal or testicular sperm in OA, testicular sperm in NOA and ejaculated sperm in non-azoospermic cases. The injected oocytes were transferred to a closed culture system and incubated for 1618 hours at 37 ˚C and 5.5% CO2 until fertilization was confirmed. Fertilized oocytes were maintained in culture, and embryo quality was scored daily according to the criteria described by Veeck for cleaving embryos (18) and by Gardner for blastocysts (19). The embryos were classified as top quality when they had 3-4 and 7-8 symmetrical blastomeres on the second and third days of culture, respectively, with no multinucleation, 0-20% of the perivitelline space occupied with fragments, and exhibited no abnormalities in the zona pellucida. Embryos kept in extended culture were considered top quality upon exhibiting full blastocyst formation and onwards. Selected embryos were transferred to the uterine cavity on the third or fifth day of embryo culture. Luteal support with a once-daily transvaginal application of progesterone gel was initiated in the day of oocyte retrieval and continued up to the 12th gestational week. Oocyte and sperm retrieval, micromanipulation of gametes, embryo culture, and the transfer of embryos to the uterine cavity were conducted in clean room environments (20). Pregnancy was first detected using quantitative serum beta-hCG testing and further confirmed clinically by observing the presence of gestational sac on the seventhweek ultrasound scan. In our program, clinical and laboratory ART data are systematically and continually entered into a database, and pregnancy follow-up is conducted by telephone interviews on a monthly basis. With confirmation of live birth, follow-ups continue with the registration of gestational ages, birth weights, neonatal disorders, and eventual malformations for a period of 30 days post-delivery. Pregnancy outcomes of all ART cycles
Systematic Review The review was structured around one key question involving short-term (including clinical pregnancy, spontaneous abortion, ectopic pregnancy, live birth and multiple pregnancy) and long-term (preterm delivery, low birth weight, neonatal and infant complications and longer-term physical and developmental problems) pregnancy outcomes for the fetus/child following sperm injections using fresh surgically-retrieved sperm from men with OA and NOA. Specifically, we intended to investigate if the pregnancy outcomes after ICSI differ according to the classification of azoospermia as obstructive or nonobstructive. We searched Pubmed, Scielo and Open J-gate for English-, Portuguese-, or Spanish-language studies published from January 1995 through March 2012. The search was supplemented by a hand search of reviews published by the Cochrane Library. The search strategy used the National Library of Medicine’s Medical Subject Headings (MeSH) keyword nomenclature. The keywords were ‘‘reproductive techniques, assisted;’’ ‘‘infertility, male;’’ ‘‘sperm injections, intracytoplasmic;’’ and ‘‘pregnancy’’ or ‘‘child.’’ Case reports and reviews were excluded. Articles were also excluded if the type of azoospermia (obstructive or nonobstructive) was not clearly stated or if only one azoospermia category was studied, regardless of whether or not a control group of sperm injections using ejaculated sperm had been included. If more than one paper presented data from the same group of patients, we selected the most recent paper.
Nomenclature description For the purpose of this study, pregnancy outcome nomenclature was defined as follows: i) The pregnancy was considered clinical if a gestational sac was visualized by ultrasonography on the seventh week of gestation. The
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clinical pregnancy rate was the ratio between the number of clinical pregnancies after embryo transfers and the number of initiated ICSI cycles (per cycle) or number of embryo transfers (per transfer). ii) Ectopic pregnancies were registered as clinical pregnancies occurring outside the uterine cavity. iii) Spontaneous abortion was defined as pregnancy loss anytime from the establishment of pregnancy to the completion of 20 gestational weeks. The miscarriage rate was the ratio of the number of spontaneous abortions and number of clinical pregnancies. iv) Live birth included deliveries that resulted in at least one live birth. The live birth rate was the ratio between the number of live birth deliveries and the number of initiated ICSI cycles (per cycle) or number of embryo transfers (per transfer). v) Multiple births were pregnancies resulting in more than one birth. vi) Preterm births were those taking place after 20 but before 37 gestational weeks. vii) Low birth weight and very low birth weight were defined as babies weighing less than 2,500 g and 1,500 g, respectively, at birth. viii) Perinatal mortality included stillbirths after 20 weeks of gestation and neonatal deaths (deaths within the first 28 days). ix) Birth defects were defined as structural, functional or developmental abnormalities presented at birth or later due to genetic or non-genetic factors acting before birth.
fertilization and high-quality embryos available for transfer were significantly lower in the group of men with NOA, but they did not differ if sperm injections were performed using non-ejaculated sperm from men with OA or ejaculated sperm from patients with male infertility. The rates of clinical pregnancy and live birth were lowest in the NOA group. Live birth rates differed between the NOA group (21.4%) compared with OA (37.5%) and ejaculated sperm (32.3%; p = 0.003). Miscarriage, ectopic pregnancy and multiple pregnancy rates did not differ among the groups. Three hundred and twenty-six live births resulted from 1,041 fresh embryo transfers (live birth rate of 31.3%). The distribution of live births in relation to parity, gestational age, and birth weight is shown in Table 3. A total of 427 babies was delivered and assessed. Overall, differences were not observed among groups for gestational age and birth weight in the three parity stratifications. Similarly, the rates of preterm birth, low birth weight and very low birth weight did not differ among groups in the studied parity stratifications. The preterm birth rates were greatest for singletons in OA (17.9%) and for twins in both OA (47.1%) and NOA (44.5%) compared with the ejaculated group (9.7% and 27%, respectively), but these differences were not significant (p = 0.15). We noted a tendency towards lower gestational age for twins in the OA group (35.6¡2.8) compared with the NOA (36.2¡2.4) and ejaculated groups (37.0¡2.3), but the numbers were relatively small to reach statistical significance (p = 0.06). The overall perinatal death and malformation rates were 2.8% and 1.6%, respectively, and the results did not differ among groups. The frequency of babies of the male gender was higher in the OA group (56.4%) compared with the NOA (41.4%) and ejaculated (39.7%) groups (p = 0.02).
& RESULTS Study Group A total of 1,092 ICSI cycles were performed in 835 patients. Patient characteristics and a comparison of laboratory and clinical ICSI outcomes are shown in Table 2. Groups were homogeneous in terms of the mean age of the female partners, female serum hormones, and the proportion of females with an associated fertility problem. There were no statistically significant differences in the numbers of retrieved oocytes and transferred embryos to the uterine cavity among the groups. The percentages of normal
Systematic Review We reviewed 373 abstracts relevant to ICSI, male infertility and pregnancy. To address the key question discussed in this
Table 2 - Patient Characteristics and Comparison of Laboratory and Clinical Outcomes after Sperm Injections in Azoospermic (Obstructive and Nonobstructive) and Non-azoospermic Infertile Males.
Patient Characteristics No. of patients Mean ¡ SD male age; years Mean ¡ SD female age; years Laboratory Outcomes No. of cycles Mean ¡ SD no. oocytes retrieved Mean ¡ SD% 2PN fertilization* Mean ¡ SD% high-quality embryo1 No. embryo transfers Mean ¡ SD no. embryos transferred Clinical Outcomes No. clinical pregnancies (%) No. ectopic pregnancies (%) No. miscarriages (%) No. live births (%) No. multiple births (%) *
Obstructive Azoospermia
Nonobstructive Azoospermia
Ejaculated Sperm
182 42.6¡9.0a 32.6¡5.8
188 37.0¡7.6b 32.4¡4.7
465 36.3¡8.9c 33.0¡6.8
243 11.8¡7.7 62.9¡22.3a 52.50¡30.2a 237 2.7¡1.3
228 12.7¡6.9 43.7¡27.9b 45.3¡33.6b 210 2.7¡1.6
621 11.7¡7.0 64.5¡35.8c 47.8¡32.4c 594 2.7¡1.4
116 (48.9)a 2 (1.7) 24 (21.0) 89 (37.5)a 22 (24.4)
60 (28.6)b 3 (5.0) 11 (19.2) 45 (21.4)b 13 (28.3)
248 (41.7)c 8 (3.2) 47 (19.6) 192 (32.3)c 46 (23.8)
p-value
,0.001(a vs. 0.26
b,c
)
0.07 ,0.001(b vs. a,c) = 0.01(b vs. a,c) 0.99 ,0.001(b vs. a,c) 0.22 0.75 = 0.003(b vs. a,c) 0.52
PN = pronuclei (normal fertilization after sperm injections was defined as the presence of two pronuclei and a second polar body); 2PN fertilization rate defined as the ratio of the number of normal 2PN fertilized oocytes and number of mature injected oocytes. 1Embryo quality was assessed according to the arrangement and number of blastomeres, presence or absence of multinucleation, and degree of cytoplasmic fragmentation. Embryos were considered high quality when 3-4 and 7-8 symmetrical blastomeres were seen on the second and third days of culture, respectively, with no multinucleation and no more than 20% of the perivitelline space occupied with cytoplasmic fragments. The high-quality embryo rate is the ratio of the number of high-quality embryos and number of embryos developed. Multiple births include live births resulting in more than one baby delivered. Each live birth is considered a single event.
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Table 3 - Neonatal Outcomes of Children Born Following Sperm Injection in Azoospermic and Non-azoospermic Infertile Males.
No. live birth singletons Mean ¡ SD gestational weeks at birth No. preterm births (%) Mean ¡ SD birth weight (grams) No. low birth weights (%) No. very low birth weights (%) No. live birth twins Mean ¡ SD gestational weeks at birth No. preterm births (%) Mean ¡ SD birth weight (grams) No. low birth weights (%) No. very low birth weights (%) No. live birth triplets Mean ¡ SD gestational weeks at birth No. preterm births (%) Mean ¡ SD birth weight (grams) No. low birth weights (%) No. very low birth weight (%) Total No. children born No. perinatal deaths* Gender No. boys (%) No. girls (%) No. unknown (%) No. birth defects (%) *
Obstructive Azoospermia
Nonobstructive Azoospermia
Ejaculated Sperm
67 37.5¡2.2 12 (17.9) 2,963¡480 7 (10.5) 2 (2.9) 17 35.6¡2.8 8 (47.1) 2,261¡594 11 (64.7) 2 (11.7) 5 32.6¡3.1 4 (80.0) 1,660¡624 4 (80.0) 2 (40.0) 117 3 (2.5)
32 37.8¡2.1 3 (9.4) 2,957¡667 3 (9.4) 2 (6.2) 9 36.2¡2.4 4 (44.5) 2,357¡403 6 (66.6) 1 (11.1) 4 32.3¡5.9 2 (50.0) 1,311¡471 3 (75.0) 3 (75.0) 63 4 (6.3)
145 38.0¡2.1 14 (9.7) 3,092¡579 10 (6.9) 4 (2.8) 37 37.0¡2.3 10 (27.0) 2,461¡672 18 (48.7) 4 (10.8) 9 32.6¡4.5 2 (22.2) 1,600¡642 8 (77.8) 3 (33.3) 247 5 (2.0)
66 (56.4)a 43 (36.8) 8 (6.8) 2 (1.7)
26 (41.4)b 35 (55.5) 2 (3.1) 2 (3.2)
98 (39.7)c 122 (49.4) 27 (10.9) 3 (1.2)
p-value
0.11 0.10 0.24 0.37 0.38 0.06 0.15 0.30 0.28 0.92 0.93 0.37 0.35 0.87 0.32 0.10 0.02 a vs. 0.02 a vs. 0.06 0.26
b,c b,c
Perinatal deaths included stillbirths (birth of fetuses with no sign of life that occur after 20 weeks of gestation) and neonatal deaths (deaths within the first 28 days). One stillbirth occurred in each group. Birth defects were defined as structural, functional or developmental abnormalities presented at birth or later due to genetic or non-genetic factors acting before birth.
men with OA and NOA (Table 5). In general, the data showed no major differences between children from fathers with NOA and OA. Of note, lower gestational age in singletons and increased frequency of premature twins were reported in one study in the NOA group. Similar congenital malformation rates, ranging from 1.3% to 5.2% and 0% to 4.2%, were observed in the OA and NOA groups, respectively. However, the data were based on a very limited population. We did not identify any study comparing long-term physical, neurological and developmental outcomes in these categories of male infertility.
study, 20 manuscripts that directly compared pregnancy outcomes between men with OA and NOA were included in our analysis. Most studies were retrospective in nature. Twelve studies provided data on pregnancy rates and/or live birth only (Table 4). Four studies reported on neonatal outcomes (Table 5). All studies were published in English.
Short-term pregnancy outcomes We identified 12 studies that compared pregnancy outcomes with ICSI using surgically-retrieved spermatozoa of men with OA and NOA (Table 4). Six of these reported decreased pregnancy rates (clinical or live birth) with sperm from men with NOA compared with OA, and the other half showed no difference in outcomes, regardless of the type of azoospermia. A control group of sperm injections using ejaculated sperm was available in six studies, and again, conflicting results were noted. Three studies found lower outcomes in the NOA group compared with either the OA or ejaculated sperm groups, whereas three reported similar pregnancy rates among the groups. One consistent finding was that pregnancy outcomes did not differ if ejaculated sperm or non-ejaculated sperm obtained from men with OA were used for ICSI. Only three studies compared miscarriage rates; two reported no difference in risk of miscarriage among the groups, whereas one showed a significant increase in loss rates with ICSI using sperm from NOA men. Ectopic pregnancy was occasionally reported but not compared. None reported multiple pregnancy rates.
Expert Commentary Currently, ICSI is widely used for patients with azoospermia, and several publications have reported the sperm injection outcomes with non-ejaculated sperm. It has been shown that neonatal outcomes, including karyotype results and malformation rates, of children conceived with nonejaculated sperm are comparable to those of counterparts conceived with the use of ejaculated sperm (35,36). Bonduelle et al. and Jozwiak et al. analyzed karyotype results of fetuses and newborns for which ejaculated and testicular sperm were used for ICSI. Abnormal karyotypes were found in 3.1% and 1.9% in the respective aforementioned studies when ICSI was performed with ejaculated sperm, and those results did not differ from those obtained by using testicular sperm (4.8% and 1.5%). However, Bonduelle et al. called attention to the fact that the frequency of de novo chromosomal anomalies was higher in the testicular sperm group, but the numbers were too small to allow definitive conclusions. Several investigators have also studied congenital anomalies in such groups (37-40). Ludwig et al. reported major malformation rates of 9.2%
Long-term pregnancy outcomes Four studies compared neonatal outcomes of children born after ICSI using surgically-retrieved spermatozoa in
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Table 4 - Studies Comparing Pregnancy Outcomes after Intracytoplasmic Sperm Injection using Surgically-retrieved Spermatozoa in Men with Obstructive and Nonobstructed Azoospermia.
Authors; Year (reference)
OA vs. NOA; Control group of number of ejaculated sperm; cycles yes/no; No. cycles
Most relevant pregnancy outcome assessed
Design
Region
Aboulghar et al.; 1997 (21)
Retrospective
Egypt
126 vs. 80
Yes; 102
Ghazzawi et al. 1998 (22)
Prospective
Jordan
19 vs. 30
Yes; 28
Ubaldi et al.; 1999 (23)
Prospective controlled
Italy
33 vs. 29
Yes; 62
Palermo et al.; 1999 (24)
Retrospective
USA
255 vs. 53
No
De Croo et al.; 2000 (25)
Retrospective
Belgium
139 vs. 54
No
Turkey
43 vs. 53
Yes; 780
Germany
300 vs. 414
No
LBR
Egypt
48 vs. 42
No
CPR
Italy
NA
NA
CPR
Brazil
39 vs. 54
Yes; 220
CPR
Bukulmez et Retrospective al.; 2001 (26) Schwarzer et Retrospective al. 2003 (27) Ghanem et al.; Case series and 2005 (28) meta-analysis of cohort studies La Sala et al.; Retrospective 2006 (29) Retrospective Verza Jr & Esteves; 2008 (15)
Main findings
Other findings
CPR
Lower CPR for NOA Miscarriage rate and compared with multiparity rates presented other groups but not compared Increased miscarriage rate LBR Lower LBR when testicular sperm from when testicular sperm from NOA men were used men with NOA was used (10%) compared with ejaculated (21%) or epididymis (22%) Ongoing Similar results Implantation rate lower in pregnancy and among groups NOA (13.4%) vs. ejaculated LBR sperm or OA (,26%) LBR Lower LBR with testicular Similar malformation sperm from NOA vs. rate between groups epididymal sperm from OA LBR Similar LBR between OA Miscarriage and (16.2%) and NOA (22.6%) multiparity described but not compared CPR No difference in outcome NR
SemiaËœoFrancisco et al.; 2010 (30)
Retrospective
Brazil
274 vs. 102
No
CPR
He et al., 2010 (31)
Retrospective
China
112 vs. 42
No
CPR
Lower LBR in NOA NR (19%) vs. OA (28%) Lower fertilization Similar CPR between OA (25%) and rate in NOA NOA (23.1%) Similar CPR in OA NR (12.9%) vs. NOA (15.4%) Lower pregnancy Miscarriage rates did not rates (25.9%) in NOA differ between groups compared with OA (51.3%) and ejaculated sperm (36.6%) No differences in Higher miscarriage rate CPR between groups in OA with the use of testicular sperm compared with epididymal sperm Lower CPR in NOA Similar miscarriage rates (21.4%) than OA (40.2%)
AO = obstructive azoospermia; NOA = nonobstructive azoospermia; LBR = live birth rate; CPR = clinical pregnancy rate; NR = not reported; NA = not available.
and 3.8% in children born after the use of testicular and epididymal spermatozoa, respectively; these values were not significantly different than the rate of 8.4% with ejaculated sperm (37). Other investigators reached similar conclusions and reported even lower rates of congenital anomalies in those groups (38-40). These publications, however, failed to discriminate between the subgroups of men with OA and NOA. Nonetheless, few studies to date have addressed outcomes by making a systematic distinction between OA and NOA. This prevents consideration regarding the severity of spermatic defects on ICSI results. Whereas men with OA have normal sperm production, those with NOA have severely defective spermatogenesis and a very limited amount of sperm within the testes, if any, which may result in an increased risk of genetic and epigenetic defects (41). In this study, we compared the reproductive potential of azoospermic men undergoing sperm injections according to the type of azoospermia. A subgroup of non-azoospermic infertile men treated with ICSI was included for comparison.
We noted that sperm injections with testicular sperm of men with NOA resulted in lower fertilization and embryo development compared with either the sperm of OA individuals or ejaculated sperm of non-azoospermic men. Moreover, clinical pregnancy and live birth rates were lowest in the NOA group, whereas no difference was observed between the groups of OA and ejaculated sperm. We also reported the neonatal profile of babies born with ICSI and showed similar outcomes from using NOA, OA or ejaculated sperm. Although we noted that the preterm birth rates were greatest for singletons from OA and twins from both OA and NOA and that the gestational age was lowest for twins from OA, our data involved a limited population. In our series, congenital malformation (1.6%) and perinatal death rates (2.8%) did not differ between groups and are in agreement with those reported in larger cohorts (32-34). Given the relative rarity of specific birth defects, identifying an association between a specific exposure and subsequent risk is difficult. Moreover, not all major malformations are found at birth, and a proportion of children were lost to follow-up
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Table 5 - Studies Comparing Neonatal and Developmental Outcomes of Children Born Following Intracytoplasmic Sperm Injection using Surgically-retrieved Spermatozoa from Men with Obstructive and Nonobstructed Azoospermia. Control group of children born with ejaculated sperm; yes/no
Outcomes assessed
Main findings Congenital malformation rate did not differ in OA (1.3%) compared with NOA (4.5%) Similar multiple birth, overall preterm delivery, low birth weight, perinatal death and malformation rates (3% OA; 4% NOA). Lower gestational age in singletons and increased frequency of premature twins in the NOA group Malformation rate in OA (4.0%); No malformations reported in NOA group Similar multiple birth, overall preterm delivery, low birth weight, perinatal death and malformation rates (5.2% OA; 4.2% NOA)
Design
Region
OA vs. NOA; Number of children
Palermo et al. 1999 (24)
Retrospective
USA
158 vs. 22
No
Malformation rate
Vernaeve et al. 2003 (32)
Retrospective
Belgium
196 vs. 61
No
Fedder et al 2007 (33)
Retrospective
Denmark
282 vs. 76
No
Belva et al.; 2011 (34)
Prospective
Belgium
474 vs. 193
No
Multiple pregnancy rates, gestational age, birth weight, preterm delivery, low birth weight, malformation rate Multiple pregnancy rates; congenital anomalies Multiple pregnancy rates, gestational age, birth weight, preterm delivery, low birth weight, malformation rate
Authors; Year (reference)
(Table 3). It is therefore possible that the number of malformations is underestimated. We also presented a systematic review of published data comparing the pregnancy outcomes following ICSI for the treatment of male infertility due to obstructive and nonobstructive azoospermia and the short- and long-term safety of such interventions. We noted that few studies have specifically compared sperm injection outcomes taking into account the type of azoospermia. Moreover, these studies had several shortcomings. The majority were retrospective case studies that only provided data on pregnancy rates (clinical or live birth). We were unable to identify follow-up studies on the physical, neurological, and developmental outcomes of children from fathers with these categories of male infertility. In general, the clinical pregnancy and live birth rates reported in the literature range from 26-57% and 18-55% in NOA and OA, respectively, and the results are similar to those reported with ICSI using ejaculated sperm (15,21-31). Although the assessment of fertilization and implantation rates was not the scope of this study, we noted that such parameters were lower with ICSI using testicular spermatozoa of men with NOA compared with ejaculated sperm or epididymal/testicular sperm of men with OA (15,23). Nonetheless, conflicting reports exist regarding whether clinical pregnancy and live birth rates are affected by the type of azoospermia. Studies have reported either a decrease (15,21,22,24,27,31) or no difference (23,25,26,28-30) in pregnancy outcomes with ICSI in cases of NOA and OA, respectively. Such decreased reproductive potential of men with NOA seen in some studies may be explained by the fact that testicular spermatozoa from men with severely impaired spermatogenesis have a higher tendency to carry deficiencies, such as the those related to the centrioles and genetic material, which ultimately affect the capability of the male gamete to activate the egg and trigger the formation and development of a normal zygote and viable embryo (32). Palermo et al. (1999), Vernaeve et al. (2003), and Belva et al. (2011) were among the few researchers that differentiated
between men with OA and NOA. In the first study, the frequency of congenital malformation did not differ in relation to the sperm source or type of azoospermia. In the study of Vernaeve et al., malformation rates of 4% and 3% were reported after the use of testicular sperm of NOA and OA patients, respectively. Recently, Belva et al. reported on the neonatal outcome of 724 children born after ICSI using non-ejaculated sperm and included a subgroup analysis of the type of azoospermia in which the frequencies of major malformation and karyotype anomalies were not different in OA and NOA. Despite the limited population analyzed, some differences observed in our study and that of Vernaeve et al. (32) regarding the gestational age and birth weight of babies born call for continuing monitoring. Because intrauterine growth is strongly dependent on placental function, these observations may suggest increased abnormalities of implantation/placentation in such pregnancies. The extent to which this is a function of treatment, maternal/ embryonic factors, or both is yet to be determined. This report has several limitations, including the restriction of studies to English, Portuguese and Spanish languages, the potential for missing relevant studies, and the lack of studies with large patient samples and metaanalysis. As noted, the relative lack of data on fetal, neonatal and long-term outcomes in the studied male infertility categories should be identified as a major research priority. As such, future research considerations should include the use of multi-center trials with adequate sample sizes, the development of standard data sets to differentiate between the groups of men with OA and NOA, and control groups of children conceived with ICSI using ejaculated sperm to facilitate meta-analyses and reach a consensus on significant clinical differences to aid sample size estimates, especially for less common outcomes.
Key issues
N 147
In our series of 1,092 ICSI cycles performed in 835 male infertility patients, live birth rates were lowest in the
Reproductive potential of azoospermic males in ART Esteves SC and Agarwal A
N
N
N
N
N N
N
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NOA group. Miscarriage, ectopic pregnancy and multiple pregnancy rates were not different between clinical pregnancies obtained using ejaculated or non-ejaculated sperm from men with OA or NOA. Of the 427 babies born following ICSI using sperm from non-azoospermic infertile fathers and azoospermic fathers with OA and NOA, the short-term neonatal outcomes were similar among groups, despite a tendency towards higher preterm births in both azoospermia categories and lower gestational age for twins from OA. The overall perinatal death and malformation rates were 2.8% and 1.6%, respectively, and our results did not differ regarding whether deliveries following ICSI used ejaculated or non-ejaculated sperm from men with OA or NOA. Most published studies that addressed pregnancy and neonatal outcome of children born after the use of nonejaculated sperm suffer from methodological shortcomings. The population included is small, and in general, no discrimination is made between OA and NOA. To date, few studies have directly compared pregnancy outcomes between OA and NOA, and the data are limited. Most of the studies were not designed to detect differences in pregnancy and live birth rates and had low power to detect differences in less-frequent outcomes, such as multiple births and complications. In general, clinical pregnancy and live birth rates reported in the literature range from 26-57% for NOA and 18-55% for OA, and the results are similar to those reported with ICSI using ejaculated sperm. Published studies have shown either a decrease or no difference in pregnancy outcomes with ICSI in cases of NOA and OA. No major difference was noted in short-term neonatal outcomes and congenital malformation rates between children from fathers with NOA and OA. However, these results are based on a very limited population, and tendencies towards lower gestational age and birth weight of babies born from azoospermic fathers call for continued monitoring. No follow-up study has yet compared the long-term physical, neurological and developmental outcomes of children born with ICSI using sperm from azoospermic men with OA and NOA. Due to the relative lack of data on fetal, neonatal and long-term outcomes of children born from azoospermic fathers, future studies should include the use of multicenter trials with adequate sample size and development of standard datasets to differentiate between the groups of men with OA and NOA. Efforts should also be made to reach a consensus on significant clinical differences regarding sample size estimates, especially for less common outcomes, thus facilitating meta-analyses. Currently, the limited evidence regarding pregnancy and postnatal outcomes of ICSI using surgically-derived sperm from azoospermic men is reassuring, but a call for continuous monitoring is of utmost importance to support the recommendation of sperm retrieval and ICSI in such male infertility categories.
& AUTHOR CONTRIBUTIONS Esteves SC was involved in the acquisition and analysis of the data; both authors were involved in the drafting and revision of the manuscript.
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& ACKNOWLEDGMENTS We thank Danielle T. Schneider for her contribution to the data collection.
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REVIEW
Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction? Veerle Vloeberghs, Greta Verheyen, Herman Tournaye Centre for Reproductive Medicine, University Hospital Brussels, Brussels, Belgium.
Intracytoplasmic injection with testicular spermatozoa has become a routine treatment in fertility clinics. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of immature germ cells for intracytoplasmic injection has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following intracytoplasmic injection using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells to more mature stages has been proposed as a means to improve this poor outcome. Several years after the introduction of intracytoplasmic injection with elongating and round spermatids, uncertainty remains as to whether this approach can be considered a safe treatment option. This review outlines the clinical and scientific data regarding intracytoplasmic injection using immature germ cells and in vitro matured germ cells. KEYWORDS: Round Spermatid; Elongating Spermatid; Elongated Spermatid; ICSI; In Vitro Maturation. Vloeberghs V, Verheyen G, Tournaye H. Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction? Clinics. 2013;68(S1):151156. Received for publication on August 10, 2012; Accepted for publication on August 14, 2012 E-mail: tournaye@uzbrussel.be Tel.: +32 2 477 66 99
patients with OA, and sufficient numbers of spermatozoa can be obtained for ICSI and/or cryopreservation. However, the recovery of fully elongated spermatids or spermatozoa fails in approximately 50% of NOA men (3). The only hope for these patients to father their own genetic children is the use of more immature germ cells for ICSI. Spermatids are the earliest cells in the male germ cell lineage with a haploid number of chromosomes. In various studies using mouse, hamster and rabbit models, two techniques, elongated spermatid injection (ELSI) and round spermatid injection (ROSI), have been successfully employed to fertilize a mature oocyte with such immature germ cells, resulting in the delivery of healthy offspring (4-7). Although the fertilization, pregnancy and live birth rates were low in these animal models, the results demonstrated the potential of spermatids to contribute to normal fertilization and embryonic development. Based on these observations, Edwards suggested the use of spermatids for ICSI in humans when sperm at more mature stages were not available (8). In humans, the injection of spermatids leading to fertilization and early cleavage was reported by Vanderzwalmen (9). The first reported successful births were by Tesarik using round spermatids from the ejaculate (10) and by Fishel using elongated spermatids extracted from the testis (11). The first reports of human pregnancies following round spermatid injection involved round spermatid nucleus injection (ROSNI), but all of these pregnancies ended in spontaneous abortions (12). In the midnineties, several IVF centers used testicular spermatids for ICSI, and most of the reported pregnancies were achieved using late spermatids. When round spermatids were used, the pregnancy rate was much lower. More than 15 years after
& INTRODUCTION Intracytoplasmic sperm injection (ICSI) has constituted a breakthrough in the treatment of severe male-factor infertility (1). This technique was initially introduced as a treatment for severe oligoasthenoteratozoospermia using ejaculated sperm. In 1993, the first successful pregnancy using spermatozoa that had been directly extracted from the testis of an azoospermic man was achieved (2). Azoospermia is present in 1% of the general male population and in 10 to 15% of infertile men. There are two major etiologic categories of azoospermia: obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). In OA, complete spermatogenesis is observed during histological analysis, whereas in NOA, either germ cell aplasia (a Sertoli cell-only pattern), maturation arrest or tubular sclerosis and atrophy is revealed by histological analysis. The histology of the latter three may or may not show focal spermatogenesis. The most mature stage of the male gamete at the end of spermiogenesis is the elongated spermatid. After spermiation, spermatozoa are released into the tubular lumen. These spermatozoa become functional during the passage through the epididymis. Testicular sperm retrieval is successful in virtually 100% of
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)17
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the first live birth achieved with ROSI, fewer than ten children have been born worldwide using this technique. The in vitro culture of immature germ cells to more mature stages of spermatogenesis has been suggested as an approach to improve the poor live birth rate associated with ROSI and as a tool to improve both the identification and the selection of spermatids used for ICSI. Despite a number of reports of the delivery of healthy offspring in humans following the use of immature germ cells for ICSI, several ethical concerns have been raised. In some countries, e.g., the United Kingdom, government regulations have banned the use of spermatids for ICSI.
treat men with NOA when no testicular spermatozoa could be retrieved. Either intact round spermatids (ROSI), round spermatid nuclei (ROSNI) or elongated spermatids (ELSI) are injected into the oocyte. Although ELSI, ROSI and ROSNI were introduced more than 15 years ago, the number of reported pregnancies has remained extremely limited.
ELSI A 2002 review summarized the literature on ICSI with elongated spermatids (ELSI) (14). Elongated spermatids isolated either from ejaculate or directly from testicular tissue have been successfully used. An analysis of the studies using late spermatids for injection indicated that this technique was associated with a low fertilization rate (48.4%) but an otherwise acceptable pregnancy rate (28.9%) (14). The overall results of ICSI using testicular elongated spermatids were comparable to those using testicular spermatozoa (14). The retrospective nature of most of the studies and the presence of patient selection bias and even publication bias may explain these results. Furthermore, some of the ELSI studies also included men who exhibited normal spermatogenesis.
& BASICS OF SPERMATOGENESIS AND SPERMIOGENESIS The term ‘‘spermatogenesis’’ refers to all of the processes and events involved in the production of mature gametes that occur within the seminiferous tubules of the testis. Spermatogenesis begins with the division of a testicular stem cell and ends with the formation of a mature spermatozoon, and it can be divided into three major stages (13): 1) The mitotic proliferation and differentiation of diploid germ cells (i.e., spermatogonia) into diploid (2n) primary spermatocytes. 2) The meiotic division of tetraploid germ cells (i.e., spermatocytes) into four haploid germ cells called spermatids; the first meiotic division produces two secondary spermatocytes, which are separated into haploid (1n) spermatids during the second meiotic division. The secondary spermatocytes contain a set of haploid chromosomes in duplicate form. The meiotic process is a critical event during gametogenesis because it involves the recombination of genetic material, a reduction in the chromosome number and the development of spermatids. 3) The transformation of the haploid germ cells (spermatids) into testicular sperm (i.e., spermiogenesis). Spermatids are mitotically inactive round cells that undergo a remarkable and complicated transformation leading to the ultimate production of differentiated elongated spermatids and spermatozoa. This transformation includes the condensation and structural shaping of the cell nucleus, the formation of the flagellum and the expulsion of a large portion of the cytoplasm. The overall process is termed ‘‘spermiogenesis.’’ When spermatogenesis is completed, the cytoplasmic connections between the sperm and the Sertoli cells are broken, followed by the release of the sperm from the germinal epithelium into the tubular lumen. This process is referred to as spermiation.
ROSI The first successful reports of ROSI in humans described seven azoospermic men who had round spermatids but no mature spermatozoa in their ejaculate (10). Round spermatids were used for ICSI and produced two viable pregnancies. Sousa et al. reviewed reports of ICSI with round spermatids isolated from either ejaculate or testicular tissue. Compared with ELSI, the success rates of ROSI are dramatically lower; the latter approach appears to be clinically inefficient, with a 21.8% fertilization rate and a 2.8% clinical pregnancy rate (14). Since 2002, no clinical pregnancies have been reported following ROSI (15-20). In summary, seven clinical pregnancies have been reported overall after the use of ROSI: three with spermatids from the ejaculate and four with spermatids extracted from the testis. Antinori is the only researcher to have reported three clinical pregnancies after ROSI in the absence of spermatozoa in the preliminary ejaculate or in biopsies (21,22). After the publication of Antinori’s work, no pregnancies have been reported following the use of round spermatids from patients with a complete absence of elongated spermatids or spermatozoa in preliminary ejaculate or diagnostic testicular biopsies. Based on the above results, it may be concluded that ROSI is an inefficient approach for treating infertility in azoospermic men with primary testicular failure who exhibit no spermatozoa in testicular biopsies.
The coordinating mechanism behind the processes of human spermatogenesis and spermiogenesis remains unclear. When maturation arrest occurs at the primary spermatocyte stage, the germ cells still contain a diploid number of chromosomes and are therefore not suitable for ICSI. When maturation arrest occurs at the round spermatid stage, the germ cells contain a haploid number of chromosomes and theoretically possess all of the genetic information needed to fertilize mature oocytes by ICSI.
ROSNI On the basis of animal experiments, the ROSNI technique has been suggested to overcome some of the disadvantages of ROSI: (1) the injecting micropipettes used for ROSNI have a smaller diameter, which reduces the risk of oocyte damage, and (2) the presence of a large amount of cytoplasm around the spermatid nucleus (in ROSI) may impede the transformation into the male pronucleus (23). Human experiments, however, have revealed that oocyte degeneration after ROSI is not adversely affected by the use of a larger microinjection pipette nor by the presence of a cytoplasmic layer surrounding the spermatid nucleus; the rupture of the cytoplasmic membrane and the nuclear
& ICSI WITH SPERMATIDS: CLINICAL EXPERIENCE Following the reports of results in animals, several groups have described the use of round or elongated spermatids, retrieved from either the ejaculate or testicular tissue, to
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round spermatids, this approach has diagnostic value only; it is not feasible in clinical practice (31,32). Yamanaka et al. have characterized round spermatids using confocal scanning laser microscopy (33); however, this equipment is expensive and therefore not accessible to all laboratories. The use of phase-contrast optics on an inverted microscope is a simple and reliable method of identifying round spermatids (29).
envelope is rapidly achieved after microinjection (14). Sousa reported a low oocyte degeneration rate (9%) in 33 ROSI cycles following the injection of 200 oocytes (14). Most human pregnancies reported after ROSNI ended in spontaneous abortion (12). To our knowledge, only one publication has reported live births (three) following ROSNI (23,24).
Secondary spermatocyte injection (SECSI) There have also been isolated reports of the successful use of human secondary spermatocytes for ICSI; including the delivery of a healthy boy (25). However, limited clinical benefits can be expected from this approach because most NOA men with secondary spermatocytes in their testicular biopsies also have more mature germ cells (23). Furthermore, no additional reports have confirmed the success of this approach in humans. As a result of the overall disappointing results of spermatid injection (ROSI, ROSNI and ELSI) following its initial hype, most IVF programs stopped using spermatid injection in the mid-nineties. Consequently, there have been no published reports of spermatid injection since that time period.
& ICSI WITH SPERMATIDS: IS THERE A TARGET GROUP? ROSI has been proposed as an ART technique for males with primary testicular failure resulting from complete maturation arrest. However, maturation arrest at the level of the round spermatid is rare; Shulze et al. (34) reviewed 1,418 stained biopsies from 766 azoospermic men and identified late maturation arrest (i.e., round spermatid arrest) in only seven men (0.9%). Silber and Johnson (35) reviewed 125 stained biopsies from men with unexplained NOA and found no evidence of arrest at the round spermatid stage. In biopsies where round spermatids were observed, elongated spermatids or even spermatozoa were invariably present as well. Furthermore, in our experience, an extensive search of wet preparations revealed identifiable round spermatids only in patients for whom the more mature stages of development were also present (29). Therefore, round spermatids may be observed in the testicular biopsies of only a limited number of patients from whom no spermatozoa or elongated spermatids can be recovered.
& ICSI WITH SPERMATIDS: IDENTIFICATION IS CRUCIAL In most of the reports of ICSI with spermatids, the spermatid stage that was used is unclear identified. The adoption of a clear terminology is needed to avoid confusion regarding success rates, so that reliable conclusions can be drawn and results can be compared between different centers (26). A critical problem in the use of spermatids for ICSI is the identification of spermatids within a heterogeneous population of round cells that have been obtained from either testicular tissue or the ejaculate. In an unstained wet preparation under an inverted microscope, haploid spermatids can be divided into four categories according to their shape, amount of cytoplasm and tail size: round (Sa, Sb1), elongating (Sb2), elongated (Sc, Sd1), and fully elongated (Sd2) spermatids (27). Elongating or elongated spermatids are easy to recognize, but the identification of round spermatids is more difficult. The presence of pathological conditions makes this identification even more difficult because the cells are retrieved from patients with abnormal spermiogenesis. Tesarik and Mendoza (28) have described a method to distinguish round spermatids from other round cells in the ejaculate (leucocytes, lymphocytes, monocytes, erythrocytes, Sertoli cells, spermatogonia and primary and secondary spermatocytes) according to the shape and size of the cell and its nucleus. A developing acrosomal granule can be recognized as a bright spot adjacent to the spermatid nucleus; however, this bright spot may be easily confused with a vacuole (29). Misidentification can lead to the injection of round cells that are not round spermatids, which might explain the low success rates of ROSI. Using the Hoffman modulation contrast microscopy, which is employed by most centers for the ICSI procedure, the identification of round spermatids is extremely difficult (29,30). Optimal identification techniques should be developed and applied in everyday clinical practice, allowing correctly selected round spermatids to be injected. Although staining procedures may clearly reveal the presence of
& IN VITRO MATURATION AND ART In vivo spermatogenesis is a long and complex process of germ cell development within the seminiferous tubules and is regulated not only by gonadotropins but also by interactions between spermatogenic cells and somatic Sertoli cells (36,37). In theory, in vivo arrest may be overcome by in vitro culture, either by co-culturing isolated germ cells on a somatic cell monolayer or by culturing isolated segments of seminiferous tubules. In vitro culture can target either the meiotic or postmeiotic maturation (spermiogenesis) of germ cells. Therefore, in vitro culture has been proposed to improve the selection against spermatids or spermatozoa carrying DNA damage and to overcome in vivo maturation arrest (38). Postmeiotic differentiation via the in vitro culture of round spermatids to the elongating stage may be a way to replace ROSI with ELSI, as the latter is technically much simpler and yields a superior outcome (28). Aslam and Fishel (39) observed flagellar growth in 22% of round spermatids during the first 4-8 h of culture in modified Eagle’s minimum essential medium with no hormonal supplementation. Follicle-stimulating hormone (FSH)-independent rapid flagellar growth in vitro by round spermatids has also been reported by others (40,41). In contrast to flagellar growth, the processes of nuclear condensation, the peripheral migration and protrusion of the spermatid nucleus, as well as the differentiation of the acrosome, are strictly dependent on the presence of FSH in the culture medium (41). Cremades et al. demonstrated that round spermatids could mature into elongating and elongated spermatids in vitro after seven days of culture at 32 ˚C in microdrops of Vero cell-conditioned
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injection of in vitro matured elongated spermatids from men with maturation arrest at the primary spermatocyte stage (53). These findings provide strong evidence that premeiotic arrest at the primary spermatocyte stage can be overcome in vitro under optimized culture conditions and can result in haploid cells with full reproductive capacity.
medium and that these cells could successfully fertilize oocytes to form blastocysts (42). The in vitro culture of germ cells may be useful for overcoming the inadvertent use of apoptotic spermatids in assisted reproduction and may therefore increase the fertilization rate (43). However, many authors have reported that both developmental capacity and clinical outcomes remain extremely poor after the use of in vitro-cultured round spermatids and have concluded that in vitro culture offers no clinical benefit apart from improving the fertilization rate (27,44,45). The births of two healthy babies have been reported following ICSI with elongated spermatids obtained after the in vitro culture of round spermatids from a patient with complete maturation arrest at the round spermatid stage (46,47). Later, the birth of two healthy babies (twins) was achieved by the same group using an in vitro culture system that supported the postmeiotic differentiation of round spermatids from patients with incomplete arrest of spermiogenesis and a high frequency of apoptosis among postmeiotic germ cells (48). Arrest at the primary spermatocyte stage is the most common type of maturation arrest in men with NOA (49). To overcome this type of arrest, the in vitro transmeiotic differentiation of primary spermatocytes has been proposed. Testicular biopsies of patients with obstructive azoospermia and normal spermatogenesis were cultured in vitro for 24 h by Tesarik et al. (41). Whole segments of the seminiferous tubules and Sertoli cells were cultured in the presence or absence of recombinant FSH and testosterone (T) at a temperature close to that of the human testis. The presence of recombinant FSH increased the proportions of primary and secondary spermatocytes undergoing meiotic progression and postmeiotic differentiation (41). The addition of testosterone enhanced the effects of FSH on meiosis and spermiogenesis by preventing the apoptosis of Sertoli cells in culture, whereas no effect was observed for the addition of T without FSH (50). Compared with the normal kinetics of the human germinal epithelium (51), a surprisingly rapid maturation (two days) was observed when culturing primary and secondary spermatocytes to elongated spermatids (50). Tanaka et al. were the first to report the in vitro development of four round spermatids derived from a single primary spermatocyte using co-culture with a Vero cell line (52). They observed in vitro meiotic division that was independent of the addition of FSH and T, in contrast to earlier reports by Tesarik et al. (50). The newly divided cells were confirmed to be round spermatids using chromosomal analysis (52). Although co-culture with Vero cells could replace the use of Sertoli cells as a support for in vitro meiosis, the Vero cells appeared unable to support postmeiotic differentiation to later stages, i.e., elongated spermatids or spermatozoa (52). The same in vitro culture conditions that had been previously recommended by Tesarik were used to culture testicular cells from five men with maturation arrest at the primary spermatocyte stage. Spermatids from two of the men were cultured after two days, and the injection of these in vitro cultured elongated spermatids successfully fertilized oocytes and resulted in normally developing embryos in both cases and healthy twin in one case (46). Later Tesarik et al. reported on the birth of a third healthy child after the
& ART WITH SPERMATIDS: A SAFE OPTION? Apart from its low overall success rate, the safety of ICSI with immature haploid germ cells has been questioned. One of the major concerns about any reproductive technology is the possibility of genetic and epigenetic risks to the offspring. There are concerns related to genomic imprinting following spermatid injection. Although genes are expressed equally from the two parental alleles, a small subgroup of genes are differentially expressed depending on whether they were inherited maternally or paternally. Genes that display inhibited expression when derived from the maternal germline are termed ‘‘maternally imprinted,’’ and genes with inhibited expression when transmitted by the father are termed ‘‘paternally imprinted.’’ The differential expression of the paternal and maternal alleles of imprinted genes is related to differential DNA methylation patterns in these genes. Genomic imprinting primarily occurs during gametogenesis and may be incomplete or defective in immature gametes or in gametes that have matured under abnormal conditions, e.g., in an in vitro culture (54). During spermatogenesis, the histone-to-protamine transition ensures protection from mutation of the spermatid DNA. However, in a knock-out mouse model with abnormal spermiogenesis, the round spermatids displayed increased levels of DNA damage caused by a deficiency in the histone-to-protamine transmission (55). In NOA men, a high frequency of DNA damage in round spermatids has been reported in patients with complete spermiogenesis failure (54), whereas abnormal chromatin packing in elongated spermatids has been reported (56). Spermatids differ from mature sperm in their chromatin structure, and this difference may affect the epigenetic behavior of the paternal genome (57). The strict control of DNA methylation in the preimplantation embryo is necessary for normal development. Preimplantation genetic diagnosis (PGD) has been proposed to increase both the implantation rate and safety of ROSI. Benkhalifa et al. reported the results of preimplantation genetic diagnosis (PGD) performed on embryos obtained by ROSI (20). Their data indicated that the failure of ROSI to produce pregnancy and live births occurs at both the prezygotic and postzygotic stages and is primarily caused by aneuploidy. Although Benkhalifa et al. did not report any increases in chromosomal or other genetic abnormalities in the rare pregnancies that occurred, they concluded that ROSI should not be used in ART programs. Additional concerns about ICSI with immature haploid germ cells are related to the immaturity of the cytoplasm. The cytoplasm of male gametes contains two factors that are important for normal embryonic development: the centrosomes and oocyte-activating factor (OAF). Abnormal or damaged centrosomes due to spermatid immaturity may cause abnormal spindle formation and may explain the arrest, mosaicism and anomalies observed in embryos that
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Intracytoplasmic spermatid injection and in vitro maturation Vloeberghs V et al. 10. Tesarik JN, Mendoza C, Testart J. Viable Embryos from Injection of Round Spermatids into Oocytes. New Engl J Med. 1995;333(8):525, http://dx.doi.org/10.1056/NEJM199508243330819. 11. Fishel S, Green S, Bishop M, Thornton S, Hunter A, Fleming S, et al. Pregnancy after Intracytoplasmic Injection of Spermatid. Lancet. 1995;345(8965):1641-2, http://dx.doi.org/10.1016/S0140-6736(95)901493. 12. Hannay T. New Japanese Ivf Method Finally Made Available in Japan. Nat Med. 1995;1(4):289-90, http://dx.doi.org/10.1038/nm0495-289. 13. Weinbauer G, Luetjens C, Simoni M, Nieschlag E. Physiology of testicular function. In: Nieschlag E, Behre HM, Nieschlag S. Andrology : male reproductive health and dysfunction. 3rd ed. Heidelberg; New York: Springer; 2010. 14. Sousa M, Cremades N, Silva J, Oliveira C, Ferraz L, Teixeira da Silva J, et al. Predictive value of testicular histology in secretory azoospermic subgroups and clinical outcome after microinjection of fresh and frozenthawed sperm and spermatids. Hum Reprod. 2002;17(7):1800-10, http:// dx.doi.org/10.1093/humrep/17.7.1800. 15. Balaban B, Urman B, Isiklar A, Alatas C, Aksoy S, Mercan R, et al. Progression to the blastocyst stage of embryos derived from testicular round spermatids. Hum Reprod. 2000;15(6):1377-82, http://dx.doi.org/ 10.1093/humrep/15.6.1377. 16. Levran D, Nahum H, Farhi J, Weissman A. Poor outcome with round spermatid injection in azoospermic patients with maturation arrest. Fertil Steril. 2000;74(3):443-9, http://dx.doi.org/10.1016/S0015-0282(00)006981. 17. Vicdan K, Isik AZ, Delilbasi L. Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure. J Assist Reprod Genet. 2001;18(2):78-86, http://dx.doi.org/ 10.1023/A:1026578507736. 18. Urman B, Alatas C, Aksoy S, Mercan R, Nuhoglu A, Mumcu A, et al. Transfer at the blastocyst stage of embryos derived from testicular round spermatid injection. Hum Reprod. 2002;17(3):741-3, http://dx.doi.org/ 10.1093/humrep/17.3.741. 19. Khalili MA, Aflatoonian A, Zavos PM. Intracytoplasmic injection using spermatids and subsequent pregnancies: Round versus elongated spermatids. J Assist Reprod Gen. 2002;19(2):84-6, http://dx.doi.org/10. 1023/A:1014447731630. 20. Benkhalifa M, Kahraman S, Biricik A, Serteyl S, Domez E, Kumtepe Y, et al. Cytogenetic abnormalities and the failure of development after round spermatid injections. Fertil Steril. 2004;81(5):1283-8, http://dx.doi. org/10.1016/j.fertnstert.2003.09.075. 21. Antinori S, Versaci C, Dani G, Antinori M, Pozza D, Selman HA. Fertilization with human testicular spermatids: Four successful pregnancies. Hum Reprod. 1997;12(2):286-91, http://dx.doi.org/10.1093/ humrep/12.2.286. 22. Antinori S, Versaci C, Dani G, Antinori M, Selman HA. Successful fertilization and pregnancy after injection of frozen-thawed round spermatids into human oocytes. Hum Reprod. 1997;12(3):554-6, http:// dx.doi.org/10.1093/humrep/12.3.554. 23. Sofikitis N, Miyagawa I, Yamamoto Y, Loutradis D, Mantzavinos T, Tarlatzis V. Micro- and macro-consequences of ooplasmic injections of early haploid male gametes. Human reproduction update. 1998;4(3):197212, http://dx.doi.org/10.1093/humupd/4.3.197. 24. Sofikitis N, Matzavinos T, Loutradis D, Antypas S, Miyagawa I, Tarlatzis V. Treatment of male infertility caused by spermatogenic arrest at the primary spermtocyte stage with ooplasmic injections of round spermatids or secondary spermatocystes isolated from foci of early haploid male gametes. Presented at the 13th Annual Meeting of The European Society of Human Reproduction and Embryology in Edinburgh, June 22-25, 1997. Hum Rep. 1997;12(S):81-82. 25. Sofikitis N, Mantzavinos T, Loutradis D, Yamamoto Y, Tarlatzis V, Miyagawa I. Ooplasmic injections of secondary spermatocytes for nonobstructive azoospermia. Lancet. 1998;351(9110):1177-8, http://dx.doi. org/10.1016/S0140-6736(05)79121-2. 26. Tesarik J. Sperm or spermatid conception? Fertility and sterility. 1997;68(2):214-6, http://dx.doi.org/10.1016/S0015-0282(97)81503-8. 27. Sousa M, Barros A, Takahashi K, Oliveira C, Silva J, Tesarik J. Clinical efficacy of spermatid conception: analysis using a new spermatid classification scheme. Hum Reprod. 1999;14(5):1279-86, http://dx.doi. org/10.1093/humrep/14.5.1279. 28. Tesarik J, Mendoza C. Spermatid injection into human oocytes .1. Laboratory techniques and special features of zygote development. Hum Reprod. 1996;11(4):772-9, http://dx.doi.org/10.1093/oxfordjournals.humrep. a019253. 29. Verheyen G, Crabbe E, Joris H, Van Steirteghem A. Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy. Hum Reprod. 1998;13(6):1570-7, http://dx.doi.org/10.1093/ humrep/13.6.1570. 30. Vanderzwalmen P, Zech H, Birkenfeld A, Yemini M, Bertin G, Lejeune B, et al. Intracytoplasmic injection of spermatids retrieved from testicular tissue: Influence of testicular pathology, type of selected spermatids and oocyte activation. Hum Reprod. 1997;12(6):1203-13, http://dx.doi.org/ 10.1093/humrep/12.6.1203.
develop after spermatid injection (58). The injection of human round spermatids into oocytes produces normal fertilization, which indicates that human spermatogenic cells have developed OAF- and calcium oscillation-inducing capabilities by the round spermatid stage (49), whereas the round spermatids of rodents require oocyte activation after injection (60). Aneuploidy may result from either immature centrosomes (which prevent the formation of normal microtubule-organizing centers) or abnormal oocyte activation. In 2000, Zech et al. reported two major congenital malformations in four pregnancies achieved using ELSI: one fetus exhibited the Arnold-Chiari malformation, and another fetus displayed hydrocephaly combined with trisomy 9 (47, XY,+9) in all of the amniotic cells (61). There have been no further published reports of abnormalities caused by imprinting disorders related to the use of immature germ cells. Epigenetic failure in germline cells is of great concern because it may only manifest later in life (such as through an increased predisposition to cancer) and/or be silently transmitted to the next generation (62). Finally, there are some concerns about the accelerated speed of the in vitro development of germ cells from men with in vivo maturation arrest. There are suspicions of a possible negative effect on DNA when some of the essential checkpoints of in vivo development are bypassed (47).
& AUTHOR CONTRIBUTIONS Vloeberghs V participated in the acquisition, analysis and interpretation of the data, as well as the drafting and final approval of the manuscript. Verheyen G participated in the analysis and interpretation of the data, as well as the revision and final approval of the manuscript. Tournaye H participated in the conception and design of the manuscript, as well as the analysis and interpretation of the data and the revision and final approval of the manuscript.
& REFERENCES 1. Palermo G, Joris H, Devroey P, Vansteirteghem AC. Pregnancies after Intracytoplasmic Injection of Single Spermatozoon into an Oocyte. Lancet. 1992;340(8810):17-8, http://dx.doi.org/10.1016/0140-6736(92) 92425-F. 2. Schoysman R, Vanderzwalmen P, Nijs M, Segalbertin G, Vandecasseye M. Successful Fertilization by Testicular Spermatozoa in an in-Vitro Fertilization Program. Hum Reprod. 1993;8(8):1339-40. 3. Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber S, et al. Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Human Reproduction. 1997;12(1):80-6, http:// dx.doi.org/10.1093/humrep/12.1.80. 4. Ogura A, Yanagimachi R, Usui N. Behaviour of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion. Zygote. 1993;1(1):1-8. 5. Ogura A, Matsuda J, Yanagimachi R. Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc Natl Acad Sci U S A. 1994;91(16):7460-2, http://dx.doi.org/10.1073/pnas.91.16. 7460. 6. Ogura A, Yanagimachi R. Round Spermatid Nuclei Injected into Hamster Oocytes Form Pronuclei and Participate in Syngamy. Biol Reprod. 1993;48(2):219-25, http://dx.doi.org/10.1095/biolreprod48.2. 219. 7. Sofikitis NV, Miyagawa I, Agapitos E, Pasyianos P, Toda T, Hellstrom WJG, et al. Reproductive Capacity of the Nucleus of the Male Gamete after Completion of Meiosis. J Assist Reprod Gen. 1994;11(7):335-41, http://dx.doi.org/10.1007/BF02214138. 8. Edwards RG, Tarin JJ, Dean N, Hirsch A, Tan SL. Are Spermatid Injections into Human Oocytes Now Mandatory. Hum Reprod. 1994;9(12):2217-9. 9. Vanderzwalmen P, Lejeune B, Nijs M, Segalbertin G, Vandamme B, Schoysman R. Fertilization of an Oocyte Microinseminated with a Spermatid in an in-Vitro Fertilization Program. Hum Reprod. 1995;10(3):502-3.
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48. Tesarik J, Cruz-Navarro N, Moreno E, Canete MT, Mendoza C. Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells. Fertil Steril. 2000;74(5):1044-6, http://dx.doi.org/10.1016/S0015-0282(00)015 69-7. 49. Martin-du Pan RC, Campana A. Physiopathology of spermatogenic arrest. Fertil Steril. 1993;60(6):937-46. 50. Tesarik J, Guido M, Mendoza C, Greco E. Human spermatogenesis in vitro: respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and Sertoli cell apoptosis. J Clin Endocrinol Metab. 1998;83(12):4467-73, http://dx.doi.org/10.1210/jc.83.12.4467. 51. Heller CH, Clermont Y. Kinetics of the Germinal Epithelium in Man. Recent progress in hormone research. 1964;20:545-75. 52. Tanaka A, Nagayoshi M, Awata S, Mawatari Y, Tanaka I, Kusunoki H. Completion of meiosis in human primary spermatocytes through in vitro coculture with Vero cells. Fertil Steril. 2003;79 Suppl 1:795-801, http:// dx.doi.org/10.1016/S0015-0282(02)04833-1. 53. Tesarik J. Overcoming maturation arrest by in vitro spermatogenesis: search for the optimal culture system. Fertil Steril. 2004;81(5):1417-9, http://dx.doi.org/10.1016/j.fertnstert.2003.12.018. 54. Tesarik J, Greco E, Cohen-Bacrie P, Mendoza C. Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol Hum Reprod. 1998;4(8):757-62, http://dx.doi.org/10.1093/molehr/4.8.757. 55. Jurisicova A, Lopes S, Meriano J, Oppedisano L, Casper RF, Varmuza S. DNA damage in round spermatids of mice with a targeted disruption of the Pp1cgamma gene and in testicular biopsies of patients with nonobstructive azoospermia. Mol Hum Rep. 1999;5(4):323-30, http://dx.doi. org/10.1093/molehr/5.4.323. 56. Francavilla S, Bianco MA, Cordeschi G, D’Abrizio P, De Stefano C, Properzi G, et al. Ultrastructural analysis of chromatin defects in testicular spermatids in azoospermic men submitted to TESE-ICSI. Hum Reprod. 2001;16(7):1440-8, http://dx.doi.org/10.1093/humrep/16. 7.1440. 57. Kimmins S, Sassone-Corsi P. Chromatin remodelling and epigenetic features of germ cells. Nature. 2005;434(7033):583-9, http://dx.doi.org/ 10.1038/nature03368. 58. Silber S, Escudero T, Lenahan K, Abdelhadi I, Kilani Z, Munne S. Chromosomal abnormalities in embryos derived from testicular sperm extraction. Fertil Steril. 2003;79(1):30-8, http://dx.doi.org/10.1016/ S0015-0282(02)04407-2. 59. Yazawa H, Yanagida K, Sato A. Human round spermatids from azoospermic men exhibit oocyte-activation and Ca2+ oscillation-inducing activities. Zygote. 2007;15(4):337-46. 60. Kimura Y, Yanagimachi R. Development of normal mice from oocytes injected with secondary spermatocyte nuclei. Biol Reprod. 1995;53(4):855-62, http://dx.doi.org/10.1095/biolreprod53.4.855. 61. Zech H, Vanderzwalmen P, Prapas Y, Lejeune B, Duba E, Schoysman R. Congenital malformations after intracytoplasmic injection of spermatids. Hum Reprod. 2000;15(4):969-71, http://dx.doi.org/10.1093/humrep/15. 4.969. 62. Nikolettos N, Asimakopoulos B, Papastefanou IS. Intracytoplasmic sperm injection--an assisted reproduction technique that should make us cautious about imprinting deregulation. J Soc Gynecol Investig. 2006;13(5):317-28, http://dx.doi.org/10.1016/j.jsgi.2006.04.002.
31. Mendoza C, Tesarik J. The occurrence and identification of round spermatids in the ejaculate of men with nonobstructive azoospermia. Fertil Steril. 1996;66(5):826-9. 32. Angelopoulos T, Krey L, McCullough A, Adler A, Grifo JA. A simple and objective approach to identifying human round spermatids. Hum Reprod. 1997;12(10):2208-16, http://dx.doi.org/10.1093/humrep/12.10. 2208. 33. Yamanaka K, Sofikitis NV, Miyagawa I, Yamamoto Y, Toda T, Antypas S, et al. Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia. J Assist Reprod Gen. 1997;14(1):55-62, http://dx.doi.org/10.1007/BF02765754. 34. Schulze W, Thoms F, Knuth UA. Testicular sperm extraction: comprehensive analysis with simultaneously performed histology in 1418 biopsies from 766 subfertile men. Hum Reprod. 1999;14:82-96, http:// dx.doi.org/10.1093/humrep/14.suppl_1.82. 35. Silber SJ, Johnson L. Are spermatid injections of any clinical value? ROSNI and ROSI revisited. Human Reproduction. 1998;13(3):509-15, http://dx.doi.org/10.1093/humrep/13.3.509. 36. Griswold MD. Interactions between Germ-Cells and Sertoli Cells in the Testis. Biol Reprod. 1995;52(2):211-6, http://dx.doi.org/10.1095/ biolreprod52.2.211. 37. Skinner MK. Cell-Cell Interactions in the Testis. Endocr Rev. 1991;12(1):45-77, http://dx.doi.org/10.1210/edrv-12-1-45. 38. Tesarik J, Mendoza C. Using the male gamete for assisted reproduction: Past, present, and future. J Androl. 2003;24(3):317-28. 39. Aslam I, Fishel S. Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception. Hum Reprod. 1998;13(3):634-8, http://dx.doi.org/10.1093/humrep/13.3.634. 40. Cremades N, Bernabeu R, Barros A, Sousa M. In-vitro maturation of round spermatids using co-culture on Vero cells. Hum Reprod. 1999;14(5):1287-93, http://dx.doi.org/10.1093/humrep/14.5.1287. 41. Tesarik J, Greco E, Rienzi L, Ubaldi F, Guido M, Cohen-Bacrie P, et al. Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum Reprod. 1998;13(10): 2772-81, http://dx.doi.org/10.1093/humrep/13.10.2772. 42. Cremades N, Sousa M, Bernabeu R, Barros A. Developmental potential of elongating and elongated spermatids obtained after in-vitro maturation of isolated round spermatids. Human Reproduction. 2001;16(9): 1938-44, http://dx.doi.org/10.1093/humrep/16.9.1938. 43. Tesarik J, Mendoza C, Greco E. In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil Steril. 1999;72(5):80913, http://dx.doi.org/10.1016/S0015-0282(99)00379-9. 44. Bernabeu R, Cremades N, Takahashi K, Sousa M. Successful pregnancy after spermatid injection. Hum Reprod. 1998;13(7):1898-900, http://dx. doi.org/10.1093/humrep/13.7.1898. 45. Barros A, Takahashi K, Bernabeu R. Spermatid injetracyotplasmic injection: report on 56 cycles. Fertil Steril. 1998;(Abstract Suppl.):S44. 46. Tesarik J, Bahceci M, Ozcan C, Greco E, Mendoza C. Restoration of fertility by in-vitro spermatogenesis. Lancet. 1999;353(9152):555-6, http://dx.doi.org/10.1016/S0140-6736(98)04784-9. 47. Tesarik J, Bahceci M, Ozcan C, Greco E, Mendoza C. In-vitro spermatogenesis - Reply. Lancet. 1999;353(9165):1708, http://dx.doi. org/10.1016/S0140-6736(05)77017-3.
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REVIEW
Biotechnological approaches to the treatment of aspermatogenic men Pedro Manuel Aponte,I,II Stefan Schlatt,III Luiz Renato de FrancaI I
Federal University of Minas Gerais, Department of Morphology, Minas Gerais, Brazil. II Central University of Venezuela, Department of Anatomy, Maracay, Venezuela. III Center for Reproductive Medicine and Andrology, Mu¨nster, Germany.
Aspermatogenesis is a severe impairment of spermatogenesis in which germ cells are completely lacking or present in an immature form, which results in sterility in approximately 25% of patients. Because assisted reproduction techniques require mature germ cells, biotechnology is a valuable tool for rescuing fertility while maintaining biological fatherhood. However, this process involves, for instance, the differentiation of preexisting immature germ cells or the production/derivation of sperm from somatic cells. This review critically addresses four potential techniques: sperm derivation in vitro, germ stem cell transplantation, xenologous systems, and haploidization. Sperm derivation in vitro is already feasible in fish and mammals through organ culture or 3D systems, and it is very useful in conditions of germ cell arrest or in type II Sertoli-cell-only syndrome. Patients afflicted by type I Sertoli-cell-only syndrome could also benefit from gamete derivation from induced pluripotent stem cells of somatic origin, and human haploid-like cells have already been obtained by using this novel methodology. In the absence of alternative strategies to generate sperm in vitro, in germ cells transplantation fertility is restored by placing donor cells in the recipient germ-cell-free seminiferous epithelium, which has proven effective in conditions of spermatogonial arrest. Grafting also provides an approach for ex-vivo generation of mature sperm, particularly using prepubertal testis tissue. Although less feasible, haploidization is an option for creating gametes based on biological cloning technology. In conclusion, the aforementioned promising techniques remain largely experimental and still require extensive research, which should address, among other concerns, ethical and biosafety issues, such as gamete epigenetic status, ploidy, and chromatin integrity. KEYWORDS: Spermatogenesis; Azoospermia; Assisted Reproductive Techniques; Transplantation; Spermatozoa; Biotechnology. Aponte PM, Schlatt S, Franca LR. Biotechnological approaches to the treatment of aspermatogenic men. Clinics. 2013;68(S1):157-167. Received for publication on August 21, 2012; Accepted for publication on August 30, 2012 E-mail: lrfranca@icb.ufmg.br Tel.: +55 31 3409 2816
outcome of absent or disturbed spermatogenesis, which leads to infertility. Human infertility is usually defined as the inability of couples to achieve pregnancy after 12 months of unprotected intercourse and is a problem that currently affects 10 to 15% of couples. An outstanding 50% of these cases are associated with male factors (1,2) Specific etiologies of male infertility include systemic diseases (e.g., endocrine, infectious, and cancer), varicoceles, obstructive syndromes, genetic/chromosomal factors, testicular failure/hypogonadism, and cryptorchidism. Of all of the causes reported, approximately 12% of the determinants of primary dysfunction in the male reproductive organs are of unknown origin and are usually confounded by the context of multicausal origins (Figure 1). Furthermore, it is clear that many cases of infertility are secondary to general systemic diseases or congenital defects, with reproductive consequences that may be treated when appropriate state-of-the-art procedures are implemented. Data in the literature show that 25 to 75% of cases (depending on the report/study) could theoretically be treated medically and/or surgically (Figure 1). In many cases, very few sperm are present in the testes, and they can
& INTRODUCTION Beyond the apparent anatomical simplicity of the male reproductive system, which has a basic design consisting of a pair of gonads with its corresponding excurrent ducts and associated accessory sexual glands, there lies an overwhelmingly complex system that is responsible for gamete production and transport into the female tract for ultimate sexual reproduction. Although many aspects of the endocrine regulation of testis function are well understood and therapeutic options for hypogonadal men are available, many aspects of the multiple physiological processes involved in gamete development inside the testis are often deregulated and out of homeostasis, with the subsequent
Copyright Ă&#x; 2013 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2013(Sup01)18
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Figure 1 - Frequency of male infertility etiological groups determined from reproductive clinic records. Percentages for each category are presented as ranges (minimum and maximum values in the reported literature) (3,4). Patients from Group A (27.8 to 73.8% of cases) can undergo medical or surgical treatments to address their infertility problems. An unknown percentage of cases within Group B (idiopathic azoospermia) and perhaps even some from Group C can benefit from ART, provided that their sperm can be retrieved. Men suffering from aspermatogenesis, with some type of premeiotic/meiotic spermatogenic disturbance, are included in Group C.
(MeSH database PubMed). However, in contrast to patients with complete SCO, these patients benefit from ART procedures, as their sperm can be retrieved by TESE (testicular sperm extraction). Type I SCO can have a genetically determined developmental origin that results in the disturbance of migration and/or colonization of primordial germ cells (PGCs) into the genital ridges. Because the diagnosis of type I or type II SCO depends on histopathologic analysis, which is of course prone to sampling error (because biopsies cover only a very small portion of the testis), the subtypes of SCO are often misdiagnosed. The situation would present less of a diagnostic challenge if a reliable, noninvasive method were available to distinguish cases of partial and complete absence of germ cells. The first steps in this direction involved the amplification of specific germ cells, seminal vesicles and prostate mRNA from cell-free seminal mRNA by using RT-PCR (7). Combinations of non-invasive diagnostic procedures with other procedures, such as hormonal profiles and histopathology, may unequivocally help to diagnose patients correctly. As type I SCO patients have no germ cells, the only possible way to generate offspring would be the use of their somatic cells to generate germline cells in vitro. Arrest represents a situation in which spermatogenesis stops at a specific stage of germ cell development. McLachlan et al. proposed a nomenclature in which the term arrest exclusively refers to histological samples in which no progression occurs beyond that specific stage in any seminiferous tubule (5). Arrest can represent an intermediate step on the way to more severe germ cell
be retrieved by biopsy and testicular sperm extraction from the tissue. Patients with access to assisted reproductive technologies (ART) can undergo ICSI (intracytoplasmatic sperm injection), which is routinely used in many fertility centers worldwide, to achieve pregnancy. However, the group of patients for whom ART procedures are not an option deserves special attention. These men present with azoospermia and varying degrees of germ cell pathology, from germinal aplasia to germ cell premeoitic/meiotic arrest. This group of testicular pathologies has a poor prognosis and holds the status of being untreatable and incurable from a medical point of view. We propose that men with incomplete spermatogenesis could be collectively classified as aspermatogenic, i.e., having a severe testicular pathology with a complete absence of spermatids. Such men comprise from 25 to 50% of andrological cases (3,4) (see Figure 1), and the only option currently available for these patients is adoption or artificial insemination using donor sperm. Histopathologic findings in biopsies from patients in the aspermatogenic group can be classified as: a) Sertoli cell only (SCO) syndrome, which strictly consists of testes presenting a complete absence of germ cells; b) arrest at the level of meiotic germ cells; or c) spermatogonia only (pre-meiotic arrest) (Figure 2). Sertoli cell only syndrome, i.e., type I SCO, refers to a situation in which testicular biopsy reveals an absence of germ cells in the seminiferous tubules (5). The presence of few seminiferous tubules showing active spermatogenesis has also been referred as to focal SCO (6) or type II SCO
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Figure 2 - Schematic summary of the types of seminiferous epithelium impairment that can lead to infertility in men. Group I summarizes pathologies in which germ cell arrest occurs at spermiogenesis. (a) Presents an apparently normal seminiferous tubule with apparently normal sperm and elongated spermatids as the most advanced cell types present. Several pathologies show a morphologically normal seminiferous epithelium, as depicted, but sperm can still present a pathological condition and therefore be unable to fertilize an egg. Many of these cases can be solved by using ART, i.e., testicular sperm extraction (TESE) combined with ICSI. (b) Arrest at the level of round spermatids, which can be used for ROSI (round spermatid injection). This type of arrest is rare. Group II represents arrest at the levels of (c) spermatocytes (12.5 % of cases) or (d) spermatogonial cells (1.7 % of cases) and (e) total absence of germ cells, as in SCO syndrome (estimated to occur in 16.5 % of cases, based on data from Tuttelmann (2010) (3), with detailed etiological entities including chromosomal aberrations, as in Klinefelter (11), XX males (12), translocations (13,14) and microdeletions at the level of the AZF regions of the Y chromosome (15,16). Prevalence data are from McLachlan et al. (2007) (5), except for the SCO data (3).
biology from several animal models are waiting to be translated into human reproductive medicine, provided that some technical issues and biosafety and ethical concerns are overcome. We propose four main groups of technologies that, alone or combined, could bring hope to men with severe cases of aspermatogenesis: i) sperm derivation in vitro; ii) germ stem cell transplantation; iii) xenologous systems; and iv) haploidization. These topics are addressed below.
losses. Arrest occurs often at the spermatocytes or spermatogonial level (12.5% and 1.7% of 534 patients, respectively) (5). Arrest during spermiogenesis is rare, and hypospermatogenesis (often misdiagnosed as arrest), in which a lower spermatid count is combined with the presence of all spermatogenic stages, which are reduced to some extent, accounts for up to 63% of cases (7). In any case, because hypospermatogenesis results in the presence of spermatids, ART procedures are applicable. Given that germ cells are present in cases of arrest, an interesting strategy would be to differentiate these cells in vitro to obtain sperm for ICSI or IVF (in vitro fertilization). Interestingly, in arrest at the level of meiotic cells, these cells can be injected into oocytes to produce viable embryos and even offspring (8-10). Nevertheless, intracytoplasmatic spermatocyte injection is only an experimental option with low efficiency. For instance, only 15% of mouse oocytes injected with secondary spermatocytes generated offspring, whereas those injected with primary spermatocytes generated none (8). Furthermore, in another set of experiments, from 0 to 9% of eggs ‘‘fertilized’’ by primary spermatocytes developed into adult fertile mice (9). In humans, one report showed an offspring-deriving efficiency of approximately 3% (10). For these reasons, the use of spermatocytes to compensate for a lack of more mature male germ cells will require further experimentation. Many patients believe that biological fatherhood has a very high value. In this respect, biotechnology offers, now more than ever, a basis on which to develop clinical applications for such cases, to restore fertility and consequently to obtain offspring. In this context, advances in cell
& SPERM DERIVATION IN VITRO The complex spatial and functional organization of the testis and its seminiferous epithelium make it difficult to achieve efficient spermatogenesis in artificial systems. The observation of at least a few aspects of spermatogenesis in culture dishes has been a scientific aim for decades (17,18). Spermatogenesis in vitro is not only a long-desired technique to gain deeper insight into the complex process of gametogenesis; it is also desired for clinical applications to broaden the therapeutic options for men with infertility problems. Given the current status of ART and the requirement of only one sperm to fertilize an oocyte through ICSI, the in vivo physiological situation, in which millions of sperm (many more than needed) are produced on a daily basis may be surpassed by in vitro strategies, which may be inefficient but which can generate a few fully mature sperm for ART procedures. Spermatogenesis in vitro was first successfully achieved in vertebrates by using a fish model that consisted of a tissue culture system that supported full spermatogenesis in Japanese eels (19,20), which opened the possibility for
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similar technological advances in higher vertebrates. Systems for in vitro spermatogenesis in fish seem to be very simple from a practical point of view. For example, one such system based on a spermatogonial cell line without Sertoli cells and involving medaka fish, allowed postmeiotic advances in the presence of a culture medium containing a crude embryo extract with no specific growth factors added (21), which introduced the idea that germ cells in lower vertebrates are more intrinsically programmed and less dependent on regulation by extrinsic signals. Currently, two main experimental approaches dominate the efforts to generate mammalian sperm in culture systems: organ culture of testes or testicular fragments that maintain the complex architecture of testicular tissue and its arrangement inside the epithelium. A recent article by Sato et al. convincingly demonstrated, in what has been described as a great scientific breakthrough, the feasibility of obtaining viable and apparently normal offspring from sperm generated through testicular tissue explants that initially contained immature germ cells (22). In this approach, the cytoarchitecture of the gonad remains widely preserved in the system, many endogenous factors are produced and released by the mostly intact seminiferous epithelium, and associated somatic cells regulate the germ cells in the system similarly to the in vivo condition. In dissected tissue, the supply of oxygen and nutrients is disturbed, and therefore the spermatogenic process is often disrupted and continues, at best, at low efficiency (22). Alternatively, enzymatically digested cell suspensions can be used for various culture systems. The supply of oxygen and nutrients to isolated cells is achieved by modern cell culture media under appropriate culture conditions. However, the seminiferous tubule microenvironment, with its different, functionally distinct compartments (e.g., interstitial, basal, intraepithelial, and adluminal), is destroyed. If these changes in the microenvironment during germ cell differentiation are significant, then spermatogenesis will be disrupted. Although many additional strategies of cell and tissue culture have been tested over the last 30 years, success has been limited (23-29). Recent studies have used monolayers other than those generated from Sertoli cells as sources of differentiating factors (30-33). Because the production of sperm in higher vertebrates involves an overwhelmingly intricate process of germ cell differentiation, further research efforts have been directed toward identifying signaling pathways associated with differentiation in the seminiferous epithelium during spermatogenesis. To incorporate this knowledge into the development of in vitro spermatogenesis systems, studies that have addressed the presence of substances such as retinol (34,35), kit ligand (stem cell factor) (36,37), insulin-like growth factor and transforming growth factor alpha (TGF-alpha) (38), as well as hormones such as testosterone (39) and FSH (40), among others, have been instrumental in recapitulating the entire differentiation process. These studies will become particularly valuable if the paradigm of spermatogenesis in vitro, as a model of the in vivo situation, is accepted. Therefore, other factors, such as scrotal temperature, the timing of cellular events and the presence of important culture media components, e.g., fetal calf serum, oxygen levels, pressure, substrates and the use of feeder layers, should be carefully evaluated and whenever possible translated from the in vivo situation to the artificial system. The use of Sertoli cells is controversial but, in principle, these cells are important
for the maintenance of germ cells in culture and to provide differentiating factors (41-44). Among the advantages of cell culture systems is that such systems (cell cultures started from dissociated germ cell suspensions) are much simpler than tissue cultures, in which cell function is tightly regulated and more complex, as in the in vivo situation. Cell cultures can be started with purified, selected, isolated cells, thereby allowing uncomplicated manipulation schemes because signal redundancy, which is observed in intact biological systems, is minimized. Furthermore, as the first step of the procedure, germ cells can be stimulated to proliferate in vitro, which creates the conditions suitable to obtain large numbers of spermatogonial stem cells before inducing them to differentiate during the process of gamete production. Recently, a novel three-dimensional testicular cell culture approach was described. The soft agar culture system (SACS), which was originally described for the culture of hematopoietic cells in vitro, has created the conditions necessary to enable the full development of murine male germ cells in vitro (45-47). This system provides options for manipulation, e.g., the testing of the effects of hormones, drugs, toxins or other compounds on germ cell development. We were able to demonstrate the progression of spermatogonia into meiosis and the cells’ further maturation into morphologically normal spermatozoa. However, the efficiency of sperm production with SACS is very low. The success of such strategies may depend on the reconstruction of small functional units of testicular cells inside the matrix, which occasionally fully resembles the microenvironment in the seminiferous tubules and thus results in small islands of complete spermatogenesis. To recapitulate the situation in the testis, researchers must sequentially and gradually provide the right substances/ conditions at the proper time, thereby emulating the in vivo processes that occur in an orderly fashion in different functional compartments (e.g., basal, meiotic, and adluminal) of the seminiferous epithelium during normal in vivo spermatogenesis. Apparently, the most challenging step of differentiation to achieve in an in vitro spermatogenic system is proceeding beyond meiosis (41). Hence, progress must be experimentally monitored by meiotic/postmeioticspecific markers, by immunohistochemistry and/or by PCR. Some examples are summarized in Table 1. The need to distinguish somatic cells renders the use of markers for this cell group useful (Table 2). These findings have greatly inspired further research involving humans in the hope of reversing cases of incomplete spermatogenesis. This type of approach would require biopsy procedures, which usually yield small testicular samples with limited numbers of germ cells. If the availability of testicular tissue is not an obstacle, then fertility patients with immature germ cell arrest could expect to overcome infertility by using this procedure. Moreover, cell culture systems for in vitro spermatogenesis would provide advantages not only to germ cell arrest patients but also to type II SCO syndrome cases or cases with other reproductive pathologies involving faulty spermatogonial stem cell niches. Current technology allows, at least on the theoretical level, SCO syndrome patients, who have no germ cells, to achieve the goal of producing offspring that retain their own genetic material. To this end, biotechnology must first provide gametes to help these men overcome their fertility
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Table 1 - Testicular germ cell markers. Cell type
Marker
Germ cell general Gonocytes Undifferentiated type A spermatogonia (single) Undifferentiated type A spermatogonia (single and paired) Undifferentiated type A spermatogonia (single, paired and aligned)
Spermatogonia (general) Pachytene spermatocytes Spermatocytes (general) Round spermatids Elongated spermatids Spermatids (general) Sperm
VASA (48) h ZBTB16 (49) m,h ID4 (50) m; CSF1R (51) m EFR3 (52) m THY1 (53) m; UTF1 (54) m; PLZF (55) m; LIN28 (TEX17) (56) m; NGN3 (57) m; NANOS3 (52) m; CD24 (53) m; GFRalpha1 (57) m; CDH1 (58) m; GPR125 (59) m; SOHLH2 (60) m; RET (61) m; BCL6B (62) m; SOX3 (63) m; SALL4 (64) p HSP60 (65) h; MAGEA-4 (66) h; UCHL1 (PGP9.5) (66) h; ITGA6 (alpha6 integrin) (66) h; ZBTB16 (66) h, (49) m,h; Kit (36) m testis specific histone (TH2B)(67) r; phosphoprotein P19 (68) r; SPTRX-3 (69) h SCP-3 (70) b, (71) h; GRP78 (65) h; Kit (66) h; MLH1 (71) h Transition proteins T1, T2 (72,73) h; protamine 2 (PRM-2) (74); CREM (75) h,m; GRP78 (65) h; Kit (66) h CRES (76) h SPANX (a subset) (77) h; KLF4 (78) h; SPTRX-3 (69) h; SP-10 (79) h; TGFbeta3 (80) h outer dense fibers (ODF-2) (70); acrosin (81) b; SPANX (77); HSP60 (65) h; GRP78 (65) h; CRES (76) h; DAZ2 (82) h
m, mouse; r, rat; d, dog; b, bovine; p, non-human primate; h, human.
The development of in vitro spermatogenesis has benefited from various animal models, including models that use farm animals. Some culture systems were developed in domestic animal models in which germ cells progressed to advanced steps of spermatogenesis. For instance, Izadyar et al. observed elongated spermatids in a long-term culture of bovine germ cells (70), whereas Dong et al. obtained elongated spermatids from fetal calf gonocyte cultures (100). In these species, the available procedures proved to be rather inefficient because not many terminally differentiated gametes could be derived. In parallel, murine models involving ES (embryonic stem) cells resulted in advances in the field. In principle, germ cell derivation from ES cells does not seem to be practical for infertility patients, as almost no man has access to his own embryonic cells. However, because iPS cells appear to be similar to ES cells (101), and in nature, technically derivable from somatic cells, the resulting scenario becomes very promising to drive them into the production of gametes in vitro. Efforts to generate germ cells and gametes from ES cells and iPS cells in murine models and humans are summarized on Figure 3. Briefly, three research groups have made advances in the derivation of germ cells from mouse ES cells. One of these groups produced gamete-like cells, and using ICSI as a functional assay, they managed to obtain embryos after fertilization had reached the blastocyst stage (102). Two laboratories were able to generate sperm in vitro (31,103), but only one of them obtained offspring by injecting the derived sperm into donor oocytes using ICSI (31). However, in this case, the offspring production had low efficiency (7 out of 65 embryos), and the mice that were born died
problems. Pluripotent stem cells could be a reliable and plentiful source of immature germ cells to provide a baseline for a differentiation process that eventually will yield spermatozoa in vitro. Pluripotency is the property of a cell to differentiate into somatic cells of ectodermal, endodermal and mesodermal lineages, which constitute the vast majority of the cells in the body (94). It has been demonstrated that some pluripotent stem cells can also generate germ cells. Pluripotent stem cells can be basically obtained from two sources: a) the epiblast region in blastodermic embryos, and b) adult somatic cells. The latter source provides an opportunity for success in the great challenge of obtaining pluripotent stem cells from adult patients, despite the conventional wisdom in cell biology that cells in tissues tend to progress into lineage restrictions during development, gradually reaching terminal differentiation. The idea of somatic pluripotentiation, or reprogramming, has been discussed for quite some time and found original success in mouse models (95). Efforts continued shortly thereafter using human cells (96,97). Only recently, independent research groups (98,99) went a step further to achieve the major breakthrough of defining four basic pluripotency-inducing factors, i.e., Oct4 (pouf1), Sox2, cMyc, and Klf4, which are required to switch somatic cells into a pluripotent state, thus opening a window for deeper understanding of pluripotency networks, as well as the possibility of generating germ cells, among other cell types, from somatic cells induced to be pluripotent (iPS cells). The availability of such personalized germ cells of somatic origin would be a prerequisite for an in vitro sperm-generating system for SCO patients. Table 2 - Testicular somatic cell markers. Cell type Sertoli immature Sertoli mature
Leydig cells Peritubular myoid cells
Marker AMH (anti-mullerian hormone) (83) p; WT1 (Wilms’ tumor gene)- transcription factor (83) r; aromatase (P450 enzymes) (83) r; NCAM (neural cell adhesion molecule) (83) r; cytokeratin 18 (83,84) h; M2A (83) h occludin (85) m,r,d; vimentin (86) r, (43,87) b; P27Kip 1 (cyclin-dependent kinase inhibitor) (83) m,r,h; WT1 (Wilms` tumor gene)- transcription factor (83) r; Dmrt1 (88) m; Gata 4 (88) m; Gata 1(83) m; AR (androgen receptor) (83) r,p,h; transferrin (88) r; ITGA6 (alpha6 integrin) (66) h ITGalpha6 (alpha6 integrin) (66) h; RLF (89) m; 3 beta-HSD (90) g, (91) r; TGF alpha (92) r alpha-smooth muscle actin (93) r, (43,87) b
m, mouse; r, rat; g, guinea pig; d, dog; b, bovine; p, non-human primate; h, human.
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Figure 3 - Recent advances in the derivation of germ cells / gametes from ES cells and iPS cells in mouse models (A) and in humans (B). ESC, embryonic stem cells; iPS, induced pluripotent stem cells; PGCs, primordial germ cells; SSC, spermatogonial stem cells; HL, haploidlike cells; ICSI, intracytoplasmatic sperm injection. In (A) arrows point into the cell types generated by each specific research group. Grey letter X and arrow in (B) indicate advances not yet accomplished.
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studies using human testes. Ultrasound-guided infusion of germ cell suspensions via the intratesticular rete testis appears to offer a simple and relatively non-invasive procedure for the autologous infusion of germ cells back into the patient (113). Our preclinical studies have demonstrated the feasibility of transplanting germ cell suspensions into the testes of non-human primates and men (113,114). A first clinical trial was initiated at the Christie Hospital in Manchester, UK, in 1999. The fertility outcomes of the patients in this study have not been yet published (115,116). In conclusion, important issues in achieving re-fertilization through germ cell transplantation include the retrieval of sufficient testicular tissue or in vitro expansion strategies for spermatogonia, prevention of ischemia in the dissected tissues and cells, cryopreservation and thawing of cell suspensions and sorting of tumor cells or enrichment of spermatogonial stem cells. The only issue solved is the ultrasound-guided non-invasive transfer of germ cell suspensions into the rete testis. These critical steps must be optimized by future research before this technique can offer a promising and safe option for the protection of male fertility in men with spermatogonial arrest. Testicular grafting is an extreme form of organ culture in which the tissue is transplanted into a host. The environment is well controlled, and also the blood supply to the grafted tissue is restored. Immature testicular tissue has a high regenerative capacity and can survive and develop as an auto- or xenograft. In the absence of alternative strategies to generate sperm in vitro, grafting provides an approach to ex-vivo generation of mature sperm. In mice, sperm generated in grafts were capable of generating healthy offspring. We and others have recently shown that xenografting of neonatal and prepubertal testicular tissue from a variety of species permitted the generation of sperm, whereas xenografting of more developed tissue resulted in the degeneration of testicular tissue. We have also shown that immature primate testes can be maintained on ice for 24 hours prior to xenografting and can also be cryopreserved. Grafting will create exciting, clinically applicable strategies for fertility preservation in immature patients. However, fertility problems are usually diagnosed in adulthood, when grafting appears to be a less useful tool for generating sperm. We therefore consider the application of grafting techniques to be more valuable for fertility preservation in children undergoing oncological treatment. An alternative to achieve germ cell development is the use of xenologous systems, which recapitulate the organogenesis of a functional testis [for review: Gassei and Schlatt, 2007, (117)]. The male pathway involves regulated cell differentiation of somatic cells within the gonadal primordium, including the migration of mesonephric cells and primordial germ cells. Finally, testis cords form and begin to elongate. Tissue engineering approaches that mimic male embryonic gonadogenesis could offer novel ways to study early testicular differentiation (29) and may, when eventually combined with xenografting approaches, also provide scenarios for the differentiation of male germ cells.
prematurely (five months after birth), which suggests developmental aberrations. Progress in germ cell derivation from mouse iPS cells was initially more limited because none of the groups involved could obtain offspring, although PGCs were obtained in one case (104) and gamete-like cells were also recently obtained (105). Recent advances have also been made in humans. PGCs were derived from human ES cells by several groups (30,106,107). Recently, Aflatoonian et al. not only generated PGCs but also derived human spermlike cells (haploid-like cells) for the first time (108). In another recent study, haploid-like cells were produced from human iPS cells (33). Nevertheless, up to now, no human embryos have been generated using these techniques, and the scientific community continues to work together with clinicians to develop rigorous, ethical and secure tests and protocols for these innovative techniques. The principles underlying these methods are simple enough to allow further progress from experimental to applied status. In summary, pluripotent ES cells are first given a differentiation environment in vitro to produce multiple cell lineages, and then, in a second phase, cells are selected for specific antigen expression or reporter germ cell genes. Also important is the addition of differentiating factors. The current stage of methods for the derivation of human haploid-like cells in vitro allows the generation of gametes with the correct epigenetic status (32,109,110). Taken together, all of the findings presented are genuine biotechnological advances with potential applications in cases of men who present with aspermatogenesis, but regardless of the tremendous potential of these techniques for the derivation of germ cells to produce gametes in vitro using culture techniques, much research remains to be conducted in this area.
& GERM STEM CELL TRANSPLANTATION AND XENOLOGOUS SYSTEMS In cases in which the testes of infertile men contain spermatogonia, the stem cell fraction among these premeiotic germ cells could be targeted for fertility preservation. The presence of male germline stem cells indicates high regenerative potential, as these cells can efficiently recolonize the seminiferous tubules, even when the starting population of spermatogonial stem cells is small. When the technique was first applied by Brinster and Zimmermann in 1994 (111), a crude, single-cell suspension containing spermatogonia was infused into the testis. The stem cells migrated into testicular stem cell niches spontaneously, whereas somatic and more differentiated germ cells were flushed out. Recolonization was initiated from individual spermatogonia, which slowly but steadily divided and migrated to repopulate all of the seminiferous tubules (111,112). The technique of germ cell transplantation could, in principle, be applied across species. Microinjection of germ cell suspensions from rats into the seminiferous tubules of mice led to the initiation of spermatogenesis from donor spermatogonial stem cells. Spermatogonia from less closely related species have the ability to repopulate the testis but will not differentiate, which currently renders the production of primate sperm in mouse testes impossible. Subsequent studies in rodents showed that spermatogonia can be cryopreserved and expanded by in vitro culture prior to germ cell transfer. The clinical potential of this technique was shown in non-human primates and in experimental
& HAPLOIDIZATION In previous sections, several prospective technologies were proposed to overcome the lack of gametes in men with aspermatogenesis. These technologies are based on differentiating preexisting or pluripotential stem cell-derived
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germ cells in vitro (spermatogenesis in vitro) or on in vivo systems (transplantation and xenografting) to produce sperm. Haploidization is an alternative approach to manufacture gametes based on biological cloning technology. In cloning, an entire genome is obtained from a single individual when he donates a somatic cell nucleus, which is transferred into an enucleated, developmentally young cell (MII oocyte, zygote, embryo) (118). In contrast, haploidization is a modified cloning technique in which there is a genetic haploid contribution from both parents, which is one reason why this technique has also been called semi-cloning (119,120). Although promising, this technology has not been successful for generating live normal offspring in murine experimental models, and therefore it remains largely experimental, with no immediate prospects for clinical application. For instance, in preliminary experiments, only 17 to 22% of mouse oocytes ‘‘fertilized’’ with somatic cells formed blastocysts, and no offspring were derived from them (121).
Azoospermic men with type II SCO would have the alternatives of germ cell derivation from a) somatic cells via induced pluripotency or b) haploidization. The most challenging differentiation step in an in vitro spermatogenic system is completion of meiosis which has been achieved in several animal models by ex vivo approaches or 3D culture systems. In the absence of alternative strategies, testicular grafting (auto- or xenograft) should be considered to exvivo generate mature sperm and would be more valuable for fertility preservation in children undergoing oncological treatment. Haploidization is a modified cloning technique, in which each parent genetically contributes with a haploid complement, but unfortunately this technique offers no immediate perspective for clinical application. Although extensive research has been and continues to be performed, biotechnologies described on this review article still remain largely experimental.
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& ACKNOWLEDGMENTS & EXPERT COMMENTS This study was supported by CNPq and FAPEMIG.
This review article provides an overview of several biotechnological approaches that have the potential to offer solutions for a range of reproductive pathologies that cause sterility in a significant number of azoospermic men, while at the same time providing new tools to contribute to our current knowledge of male reproductive physiology. Although they are thus far experimental, the proposed emerging technologies could work in combination with other already well-established reproductive technologies and take advantage of the requirement for just one healthy spermatozoon to ensure successful fertilization and, hopefully, normal offspring from these individuals. Thus far, only haploid-like cells, with an apparently correct epigenetic status, have been generated by several research groups. In the near future, we can expect the generation of sperm as the most natural, terminally differentiated cells to be used in assisted reproduction for men afflicted with aspermatogenesis. Regardless of the pervading optimism, remaining challenges include the verification of aspects such as normal cytogenetics, epigenetic stability, the tumorogenic potential of derived germ cells and even ethical aspects, which should guarantee the correct implementation of these new reproductive technologies. Additionally, the genetic basis for many conditions of aspermatogenesis will necessitate caution, as well as a consideration of the need to develop gene therapies that are specific for these reproductive problems to prevent their transmission to future generations.
& AUTHOR CONTRIBUTIONS All of the authors who are listed participated sufficiently in the present review article and therefore take public responsibility for its content.
& REFERENCES 1. Evers JL. Female subfertility. Lancet. 2002;360(9327):151-9, http://dx. doi.org/10.1016/S0140-6736(02)09417-5. 2. Tuttelmann F, Simoni M, Kliesch S, Ledig S, Dworniczak B, Wieacker P, et al. Copy number variants in patients with severe oligozoospermia and Sertoli-cell-only syndrome. PloS one. 2011;6(4):e19426, http://dx. doi.org/10.1371/journal.pone.0019426. 3. Tuttelmann F, Werny F, Cooper TG, Kliesch S, Simoni M, Nieschlag E. Clinical experience with azoospermia: aetiology and chances for spermatozoa detection upon biopsy. Int J Androl. 2011;34(4):291-8. 4. Hamada A, Esteves SC, Agarwal A. Unexplained male infertility: potential causes and management. Hum Androl. 2011;1:2-16, http://dx. doi.org/10.1097/01.XHA.0000397686.82729.09. 5. McLachlan RI, Rajpert-De Meyts E, Hoei-Hansen CE, de Kretser DM, Skakkebaek NE. Histological evaluation of the human testis approaches to optimizing the clinical value of the assessment: Mini Review. Hum Reprod. 2007;22(1):2-16. 6. Anniballo R, Brehm R, Steger K. Recognising the Sertoli-cell-only (SCO) syndrome: a case study. Andrologia. 2011;43(1):78-83, http://dx.doi. org/10.1111/j.1439-0272.2009.01030.x. 7. Li HG, Wu CL, Gu XL, Xiong CL. A novel application of cell-free seminal mRNA: non-invasive identification of the presence of germ cells or complete obstruction in men with azoospermia. Hum Reprod. 2012;27(4):991-7, http://dx.doi.org/10.1093/humrep/der481. 8. Kimura Y, Yanagimachi R. Development of Normal Mice from Oocytes Injected with Secondary Spermatocyte Nuclei. Biol Reprod. 1995; 53(4):855-62, http://dx.doi.org/10.1095/biolreprod53.4.855. 9. Ogura A, Suzuki O, Tanemura K, Mochida K, Kobayashi Y, Matsuda J. Development of normal mice from metaphase I oocytes fertilized with primary spermatocytes. P Natl Acad Sci USA. 1998;95(10):5611-5, http://dx.doi.org/10.1073/pnas.95.10.5611. 10. Sofikitis N, Mantzavinos T, Loutradis D, Yamamoto Y, Tarlatzis V, Miyagawa I. Ooplasmic injections of secondary spermatocytes for nonobstructive azoospermia. Lancet. 1998;351(9110):1177-8, http://dx.doi. org/10.1016/S0140-6736(05)79121-2. 11. Aksglaede L, Wikstrom AM, Rajpert-De Meyts E, Dunkel L, Skakkebaek NE, Juul A. Natural history of seminiferous tubule degeneration in Klinefelter syndrome. Hum Reprod Update. 2006;12(1):39-48. 12. Yamamoto M, Yokoi K, Katsuno S, Hibi H, Miyake K. A case of sex reversal syndrome with sex-determining region (XX male). Nagoya journal of medical science. 1995;58(3-4):111-5. 13. Shapiro CE. Unbalanced chromosomal translocation associated with Sertoli-cell-only histology. J Urol. 1991;145(3):563-4.
& KEY ISSUES
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At least 25% of azoospermic men are sterile because their testes either hold only immature or no germ cells at all, a condition that can be collectively termed as aspermatogenesis. Possible solutions to aspermatogenesis require biotechnological approaches to retain biological fatherhood. Azoospermic men with germ cell arrests at early stages of spermatogenesis would benefit from sperm derivation in vitro or ex vivo.
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