IVF NEWS.Direct! (International Edition) by iLogy iLogy

IVF NEWS.Direct! Volume 02, Issue 02

April - June 2010

Leading Peer-Reviewed Reproductive Medicine and ART Knowledge Resource

Embryo Selection: What is State of the ART?

EDITORIAL INTERNATIONAL EDITION

Pre-implantation Genetic Diagnosis The Way Forward Dr Pratap Kumar

MINI-REVIEWS and NEWS Vapor Phase Nitrogen Storage for Vitrified Human Oocytes Magnetic Resonance Spectroscopy for Non-obstructive Azoospermia

INTERVIEW

Cumulus-aided Transfer: A Novel Technique

Hydrotubation before IUI - New Evidence

Dr Firuza Parikh Non-cavity Distorting Myomas Impact on IVF Outcome

FEATURED MINI-REVIEW

Gonadotrophin Inhibitory Hormone Homologues - Can They Transform Fertility Endocrinology? ISSN 0975-6043

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CONTENTS IVF NEWS.Direct! www.ivfnewsdirect.com

Editorial

Pre-implantation Genetic Diagnosis - The Way Forward Dr Pratap Kumar

Review Article

Editorial Advisory Board Professor Alan Copperman Director, Division of Reproductive Endocrinology Vice-chairman, Department of ObGyn and Reproductive Science, Mount Sinai Medical Center Co-director, Reproductive Medicine Associates of New York Professor Zeev Blumenfeld Associate Professor, Reproductive Endocrinology, Department of ObGyn, Rambam Medical Center, Technion-Faculty of Medicine, Haifa, Israel Dr Peter Hollands Senior Lecturer in Biomedical Science, Department of Biosciences, University of Westminster, UK Professor Semra Kahraman Director, Istanbul Memorial Hospital ART and Genetics Center, Istanbul, Turkey Professor Pratap Kumar Professor and Head, Department of ObGyn, Director, Manipal Assisted Reproduction Centre, Manipal University, India Dr Luciano Nardo Consultant, St. Maryâ&#x20AC;&#x2122;s Hospital, CMFT University Hospitals, Manchester, UK Honorary Lecturer, University of Manchester, UK Director, North West Fertility, UK Professor Robert Norman Director, Robinson Institute, Adelaide Professor, Department of ObGyn, University of Adelaide, Australia Professor John C Petrozza Chief, Division of Reproductive Medicine & IVF Massachusetts General Hospital, Harvard Medical School, Boston, USA Professor Gamal Serour Director, International Islamic Center For Population Studies and Research, and Al Azhar ART Unit Department of ObGyn, Al Azhar University, Clinical Director, The Egyptian IVF & ET Center, Maadi, Egypt

Brief Review of State-of-the-Art Embryo Selection Techniques Dr Shylaja B Rajiv

Interview Cumulus-aided Transfer: A Novel Technique Dr Firuza R Parikh

Featured Mini Reviews

Discovery of Gonadotropin-Inhibitory Hormone Homologs to Transform Current Understanding of Human Reproductive Endocrinology

Vapor-phase Nitrogen System Superior to Traditional Liquid Nitrogen for Storing Vitrified Oocytes

ART

Study Reports Improved ICSI Outcome with Mid-follicular Recombinant LH Supplementation

Swedish Study Reiterates Benefits of Modified Natural and Mild IVF Cycles

Researchers Develop Model for Pre-empting Blastocyst Transfer Cycle Cancellation

Study Reports Higher Risk of Preterm Birth and Congenital Abnormalities after Blastocyst-stage Embryo Transfers

New Prospective Study Says Pre-IUI Hydrotubation May Not Benefit Unexplained Infertility Patients

INFERTILITY

Meta-analysis Highlights Negative Effect of Non-cavity-distorting Intramural Fibroids on IVF Outcome

Seminal Plasma Neopterin Levels May Correlate with Sperm Quality

Editorial Team

Managing Editor Dr B M John Assistant Content Editor Dr Shylaja B Rajiv

EMBRYOLOGY

Assistant Copy Editor Amoolya Moses Research Analysts Dhanya Mohan Shruthi V B Dr Raghavendra Rao Design Balamurugan M

SpermCheck速 May Serve as a Reliable Home Test to Predict Male Fertility Potential

Culture Systems Mimicking in vivo Biomechanical Environment may Enhance Embryonic Developmental Competence

CRYOPRESERVATION

Study Reiterates Efficacy of Long-term Liquid Nitrogen Storage for Preserving Sperm Motility

GENETICS

Lower CD4+CD25 Regulatory T Cells Linked to Unexplained Recurrent Spontaneous Abortions

TECHNOLOGY For contributions, author guidelines, and comments: editor@ivfnewsdirect.com For advertisements and reprints: sales@ivfnewsdirect.com Terms of use: http://www.ivfnewsdirect.com/?page_id=16 Editorial Process: http://www.ivfnewsdirect.com/?page_id=14

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Zona Pellucida Birefringence Useful to Predict Oocyte Developmental Competence

Case Series Findings Support PGD Safety in Singleton Pregnancies

NEWS

AMH Testing Effective for Individualizing Stimulation Protocols in Oocyte Donors

Recommendations for Lifestyle Modifications to Improve Fertility Outcomes

Retrospective Study Characterizes Factors Predictive of Pregnancy after IUI

Magnetic Resonance Spectroscopy as a Non-invasive Tool for Sperm Identification in Non-obstructive Azoospermia

Researchers Elucidate Role of Diverse Oocyte Proteins Influencing the Oocyte-Embryo Transition

Study Favors Transfer of Cryopreserved-thawed Embryos after Spontaneous Natural Cycles

In Vitro Culture Medium Shown to Influence Birthweight of Singleton Newborns

Magnetic-activated Cell Sorting (MACS) May Improve Sperm Fertilization Potential

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April - June 2010

EDITORIAL Pre-implantation Genetic Diagnosis - The Way Forward Introduction Pre-implantation Genetic Diagnosis (PGD) is a very controversial issue. Although the idea came up very strongly initially, it also made often conflicting social acceptability, in particular, due to its eugenic implications and also the doubtful place of the same. Indications for Pre-implantation Testing Indications for pre-implantation testing can be analysed in two broad areas. One, the couple is already known to be at risk of a genetic disease, usually a monogenic disease or chromosomal abnormality in the offspring due to a balanced chromosomal rearrangement in one of the partners. Conception can occur naturally in such individuals but they undergo in vitro fertilization so that embryos are selected for implantation only if they are found to be free of the genetic defect. Second, if there is an added infertility problem then in vitro fertilization may be the option, especially with advanced age.

Dr Pratap Kumar Head, Division of Reproductive Medicine, Kasturba Medical College and Kasturba Hospital Manipal, India

Preimplantation genetic testing is being reported for more and more single gene disorders. It has been successfully done for various conditions like beta thalassemia, cystic fibrosis, spinal muscular atrophy, myotonic dystrophy, Huntington disease, Marfan syndrome, etc. PGD aids in diagnosing many monogenic diseases. Aneuploidies are an important cause of abnormal embryos and reproductive failures. It can be thought that incorporation of aneuploidy screening in embryos can improve the success of in vitro fertilization. Review of Literature Two decades have elapsed since the first achievement of pregnancy after application of this technique in sex selection of human embryos at risk of an X-linked disease.1 Many studies, performed either at the cleavage or blastocyst stage, have addressed the impact of pre-implantation genetic screening in different groups of patients, with no benefit of performing.2 Since there are high levels of chromosomal mosaicism at cleavage stages, the cells which are checked may not be representing the status of the embryo.3,4 Moreover, lack of positive outcome from the randomized controlled trials (RCTs) may be due to the inability of PGD to test all the chromosomes. Present literature does not support the use of routine pre-implantation testing of the embryos in patients with advanced maternal age.5 Moreover, there is no conclusive data for problems like recurrent abortions, implantation failure, and severe male factor problem. A trial by European Society of Human Reproduction (ESHRE) done recently using polar body biopsy in patients with advanced maternal age may bring out some conclusive data in the coming years.6

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EDITORIAL

April - June 2010

Avoiding transgenerational risks of mitochondrial DNA disorders: a morally acceptable reason for sex selection? Pre-implantation testing for preventing mitochondrial DNA disorders may have a great future and this may happen through nuclear transfer called mitochondrial gene replacement. Sex selection though not permitted in certain countries may be looked into to check the prevention of the problem. However, this may raise several ethical questions in clinical practice. Until the time is set for permission, PGD for mitochondrial mutations should be reserved for scientific research.11

In a 2006 Cochrane study, PGD was not found to be effective in improving the live birth rates in IVF and intracytoplasmic sperm injection (ICSI) cycles. The researchers further suggested the need for more evidence before recommending the routine use of preimplantation diagnosis.7 Technical Limitations and Challenges • The short time available for analysis and less number of cells obtained by a biopsy of a blastomere seem to be a challenge. • False positive and false negatives may occur since one of the allele in a diploid cell (either normal or abnormal) fails to amplify and the results are interpreted on the basis of the other allele that is amplified. • It is estimated that the risk of transferring an affected embryo mistakenly identified as normal by PGD is approximately 2% for recessive disorders and 11% for dominant disorders.8

Conclusion Owing to the recent spectacular advancements in molecular genetics and assisted reproductive technology, the relatively new pre-implantation genetic diagnosis may give the sight for the future in certain indicated reasons. The direction to move would only be clearer in the years to come.

What next for Pre-implantation Genetic Testing? Fluorescence in situ hybridization (FISH), which is commonly adopted in PGD, involves the use of fluorescent labeled probes that bind to the target DNA in the biopsied cell. However, FISH cannot analyse all the loci of the chromosome and only a limited number of the loci may be analysed. This observation is thought to be the reason for the lack of increase in the take-home baby rates using FISH on cleavage stage embryos in randomized clinical trials. Recently, it has been suggested that comparative genome hybridization, a molecular-cytogenetic technique, identifies aneuploidy in all the chromosomes. The genome-wide array has some advantages, such as less invasiveness, ability to assess the whole complement of chromosomes, and more accurate, compared to conventional techniques (FISH). This progress may prove to be a welcome change.9

References 1.

Handyside A, Kontogianni E, Hardy K, Winston R. Pregnancies from biopsied human Preimplantation embryos sexed by Y-specific DNA amplification. Nature. 1990 Apr 19;344(6268):768-70.

Shahine LK, Cedars MI. Preimplantation genetic diagnosis does not increase pregnancy rates in patients at risk for aneuploidy. Fertil Steril. 2006 Jan;85(1):51-6.

Baart EB, Van Opstal D, Los FJ, Fauser BC, Martini E. Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day. Hum Reprod. 2004 Mar;19(3):685-93.

Coonen E, Derhaag JG, Dumoulin JC, et al. Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos. Hum Reprod. 2004 Feb;19(2):316-24.

Donoso P, Staessen C, Fauser BC, Devroey P. Current value of preimplantation genetic aneuploidy screening in IVF. Hum Reprod Update. 2007 Jan-Feb;13(1):15-25.

Human Leucocytic Antigen (HLA) Matching Among the future thoughts and experiments, HLA typing of the embryos could throw some light in the future. PGD of single gene disorders, combined with HLA matching, represents one of the most recent applications in reproductive medicine. This strategy has emerged as a tool for couples at risk of transmitting a genetic disease, to select unaffected embryos of a HLA tissue type compatible with that of an existing affected child. Stem cells from the newborn’s umbilical cord blood can be used to treat the affected sibling. Hence, HLA typing may be an important issue combined with PGD in those countries where the law permits it.10

Harper J, Coonen E, De Rycke M, et al. What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium steering committee. Hum Reprod. 2010 Apr;25(4):821-3.

Twisk M, Mastenbroek S, van Wely M, Heineman MJ, Van der Veen F, Repping S. Preimplantation genetic screening for abnormal number of chromosomes (aneuploidies) in in vitro fertilisation or intracytoplasmic sperm injection. Cochrane Database Syst Rev. 2006 Jan 25;(1):CD005291.

Lewis CM, Pinel T, Whittaker JC, Handyside AH. Controlling misdiagnosis errors in preimplantation genetic diagnosis: a comprehensive model encompassing extrinsic and intrinsic sources of error. Hum Reprod. 2001(Jan);16(1):43-50.

More references available online at www.ivfnewsdirect.com

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April - June 2010

REVIEW ARTICLE Brief Review of the State-of-the-Art Embryo Selection Techniques Dr Shylaja B Rajiv, Assistant Editor, IVF NEWS.Direct! Embryo selection is considered as the ‘holy grail’ of ART procedures. However, there exists difficulty in accurately predicting the embryo viability, which may lead to the use of defective or non-viable embryos, resulting in poor treatment outcomes and enhanced medical risk. In view of this, the practice of multiple embryo transfer was adopted, which increased the incidence of multiple births.1 The high multiple pregnancy rates in turn resulted in increased maternal, perinatal and neonatal adverse outcomes, leading to high health care expenditure.2

capacity • Decision making regarding the embryo transfer policy, after weighing the advantages and disadvantages of transferring embryos of moderate to-low quality Embryo Selection Methods Over the last two decades, there has been a dramatic change in the manner in which routine embryo examination is performed. The rapid advancements in the ART field have led to the development of different strategies for evaluating the competence of embryos; some of which have been briefed in the section given below.1

With the identification of single embryo transfer as an effective method of reducing such risk, the selection of the ‘right’ embryo with certain viability and high implantation potential became paramount.2 The main goals of the currently adopted ART methods include improvement in the rates of implantation and pregnancy, along with the reduction/prevention of the associated complications such as multiple gestations.3 The achievement of these primary objectives mandates the need for precise gamete/embryo testing and selection procedures.1

• Non-invasive microscopic embryo evaluation Since non-invasive techniques estimate embryos without causing any injury, ART specialists are required to be accustomed to these non-traumatic ways of evaluation.7 This precise evaluation method facilitates the choosing of the most potent embryo for transfer; thereby improving the success rates of the ART cycles.

Importance of Embryo Selection Owing to the invasiveness of the ART procedures, in addition to the increased human intervention and evasion of natural selection barriers, great care has to be taken during embryo selection to avert the probable complications in the potential offspring.4,5 Some of these concerns are enumerated below: • Chances of selecting defective gametes • Physical manipulation may potentially injure the gametes and subsequently the embryo, enhancing the chances of abnormally developed offspring • Candidates opting for ART may be at a high risk of transmitting genetic defects to offspring, particularly in advanced age patients4

Simple methodologies, such as the morphology and dynamics of embryo development, form an integral part of the non-invasive examination. Contrast-phase microscope with Hoffmann modulation contrast (HMC) or difference-interference contrast (DIC), is generally used to analyze the embryos without performing fixing and staining, thereby facilitating more accurate measurement. The assessment of various morphological features supplementing the routine examination further enables the prediction of the embryo’s developmental potential and chances of pregnancy in infertile couples. Literature provides evidence on diverse embryo qualityrelated factors, which discriminate the embryos based on their implantation rate. Studies also state that the markers of embryo quality should include morphological aspects of the embryo, underscoring the importance of morphological assessment in improving the outcome.1

Some studies have indicated that embryos should not be selected just based on their putative high competence; as the resulting cohort of stimulation cycles may not have any such embryos.6 It was observed that an evidence-based estimation of the potential viability of various embryos is necessary to present a qualitative and quantitative ranking, which will aid in the following: • Selecting the embryo with maximum implantation

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• Quality markers of gametes: The quality of the embryo depends on the quality of oocytes and sperms; hence it is vital to select the most

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competent gametes to ensure the availability of high quality embryos.6 Some of the parameters considered for assessing the quality of the gametes are listed in the table given below.

Oocyte Quality

Sperm Quality

Cumulus-coronal

Progressive sperm motility

Morphology

Sperm morphology

Oocyte morphology: perivitelline space, darkness or excessive cytoplasmic granularity, smooth endoplasmic reticulum clustering, presence of vacuoles or other cytoplasmic inclusions, refractile bodies

Concentration and count

First polar body morphology

Genetic Integrity

ATP content of oocytes

Dissolved oxygen content in follicular fluid

However, few other studies report contradictory findings with respect to the usefulness of the extended culture in improving the ART outcomes. A recent study further cautioned that day-5 blastocyst culture may be linked to a greater risk for increased monozygotic twinning rate.6 Embryos can be selected based on all the qualityrelated parameters noted at each stage of its development.6 Hence, the developmental stage forms the decisive factor in choosing the parameters to be considered. Although various parameters are available to select competence of embryos, further research is mandatory to clarify their significance at each developmental stage prior to them being accepted as useful tools. Initially, embryo quality-related parameters were the only factors considered, but large number of retrospective and prospective studies has identified other mophological features. In an earlier review, Bączkowski et al suggested some of the parameters, as indicated below, may impact the selection of good quality embryos.7 Parameters for Identifying Good Quality Embryos • Early cleavage • Pronuclear morphology • Appearance of zona pellucida and cytoplasm (vacuoles, pitting) • Blastomere count on particular days of culture • Compaction and expansion of blastomeres • Blastomere size, symmetry, and fragmentation • Presence of multinucleation in blastomere

• Quality markers of zygotes: Embryo quality could vary with changes in certain parameters such as oocyte polarity, nucleolar precursor bodies, presence of halo effect (cytoplasmic halo) in the zygote, and zygote score (based on size and relative positioning of pronucleoli, time of first cleavage, appearance of halo effect, etc.).6 • Quality markers of day-5 blastocysts / Prolonged embryo culture: Earlier, blastocyst quality was not considered for choosing the best embryo as it was assumed that the development to blastocyst stage is itself considered a sign of competence. However, recent evidence has demonstrated that blastocysts with characteristics, such as complete filling of the embryo with blastocoel and expanded volume greater than early cleaving embryo, have high implantation potential.6

• Electronic embryo evaluation Some of the drawbacks of the microscopic morphological embryo assessment are mentioned below. • Subjective in nature: it depends on the judgment of the operator; hence the results would vary from one laboratory to another. • Time acting as a limiting factor: Embryos cannot be kept outside the incubator for prolonged periods of time. Since light microscopic examination takes a long duration of time, it is difficult to assess the embryos in detail without compromising on the quality.1

Researchers suggest that the extension of the embryo culture till the blastocyst stage (from day3 to day-4 or -5) will help in selecting the most competent embryos, to segregate those that do not develop into blastocysts. Some opine that this extension of the embryo culture by an additional 1-2 days may serve as a natural process of selecting the best embryos.

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Previous studies have indicated that multinucleate embryos are linked to reduced implantation, pregnancy, birth rates, and raised chromosomal abnormality rates. It is hence important to ensure that embryo scoring systems determine nuclear status. However, analysis using the conventional light

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• Preimplantation genetic screening (PGS)/ Preimplantation genetic diagnosis (PGD) Since the morphology-based assessment is not accurate in detecting genetic defects, PGS/PGD was speculated to serve as an effective tool for embryo selection. Although the results of initial studies were promising, several randomized controlled trials do not support the use of the invasive technique and prefer the use of non-invasive methods over these techniques.10 Similar views have been echoed by the Practice Committee of American Society for Reproductive Medicine (ASRM).

microscopy is associated with one other disadvantage, i.e., difficulty in defining nuclear structures and differentiating them from other circular embryonic structures, like vacuoles, owing to the dynamic and diverse morphology of the nuclear composition.8 These limitations could be partly overcome by adopting computer-assisted embryo assessment and time lapse recordings that provide information pertaining to the early embryo’s kinetics. The development of computerized tools for capturing and analyzing high resolution images facilitates accurate, standardized, objective, and quick embryo selection.

• Metabolomic assessment Although cleavage rate and morphology form an important part of assessing embryos, numerous technologies that may be additional or alternative to this are being investigated.11 Recent evidence suggests the potential of metabolomic profiling of embryo culture media with optical and non-optical spectroscopies in predicting the reproductive potential of embryos.

Christina Hnida et al, in an earlier study, demonstrated that computer-controlled multilevel morphological analysis improved the nuclear status determination; and suggested it to be a powerful alternative tool to conventional microscopic evaluation.8 Imaging software, such as FertiMORPH®, help in assessing the quality of oocytes and embryos by providing a 3-dimensional morphological analysis. The system facilitates quicker and precise embryo selection through: • automated calculations of the morphologic data based on images • integration of the information of the whole embryonic space • provision for embryo scoring/grading along with the images9

Previous research has indicated the potential of metabolomics in indicating/detecting a metabolic problem or lesion with higher throughput at less cost compared to transcriptomics, proteomics, and genomics.1 In a prospective blinded study, Scott et al indicated that noninvasive metabolomic profiling of human embryo culture media using Raman spectroscopy has the potential to be developed as a quick and reliable tool for determining embryonic reproductive potential prior to transfer.12

A presentation by Hnida at the 2008 ESHRE Special Interest Group Embryology Workshop stated the following advantages of computer-based systems over conventional approach for embryo evaluation: • Attainment of critical and detailed morphologic information • Improved standardization and documentation • Quality control of scoring methods • Minimal storage time outside the incubator

A 2008 retrospective study was performed by Seli and coworkers to recognize the biomarkers affecting the reproductive outcome by metabolomic profiling of embryo culture media using proton nuclear magnetic resonance (1H NMR). The findings demonstrated substantially higher glutamate concentrations in the spent culture media of embryos, which resulted in pregnancy and delivery as opposed to those that failed to implant. Furthermore, it was noted that the viability of embryos was predicted by 1H NMR with a sensitivity and specificity of 88.2%.13

The researchers suggested the incorporation of computeraided assessment, along with traditional embryo evaluation to obtain accurate results. Also, standardized embryo scoring systems may aid in improving the embryo selection process.9

In a more recent study, Seli et al reported that metabolomic profiling using near infrared (NIR) spectroscopy is linked to the reproductive potential of embryos and is independent of embryo morphology.14 In an earlier study presented at the 23rd annual meeting of ESHRE, Vergouw et al noted

Apart from the morphological evaluation, the way forward would be to correlate embryonic development with quality culture media, stimulation criteria, and other parameters in order to increase the chances of selecting good prognostic embryos.

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and translation of proteins from a single genome. Currently, there exists little knowledge about the genome or proteome of gametes or preimplantation embryo. Deciphering this information is crucial in order to understand the complex reproductive physiology. Initially, proteomics was not introduced into ART owing to the lack of sensitivity; but now, with several improvements in the field, new protocols have been developed, with the ability to profile the following: • Proteome of embryos and individual gametes • Proteins in secretome

the effectiveness of metabolomic profiling with NIR spectroscopy of oxidative metabolism biomarkers in differentiating viable from non-viable embryos. They suggested that metabolomic profiles may serve as a useful, rapid, non-invasive tool for choosing embryos.2 Similar views were echoed in another review, which indicated that metabolomic changes in the culture medium of embryos or gametes correlate with embryo morphology and development. They further suggested that viability index on gametes/embryos as indicated by metabolomic tests could be a superior predictor of the potency of the embryo compared to morphological evaluation. Although the results were promising, the researchers reported the need to conduct further prospective studies to confirm on the findings. Furthermore, there is a need to specifically define the estimated advantages and the most appropriate applications of metabolomic profiling in the ART field.3

A recent review by Katz-Jaffe and Gardner noted that by identifying proteins involved in oocyte maturation, embryo development, and implantation, further advancements could be made to ART procedures in addition to the development of novel diagnostic tests for infertility. The researchers also suggested the probability of proteomics in creating a non-invasive viability assay, which would aid in choosing the best embryo prior to transfer.17

• Amino acid profiling Earlier trials noted a reduced or lower amino acid metabolism in embryos with higher viability compared to those that underwent growth arrest. A 2009 retrospective study speculated the increased usage of nutrients like amino acids by non-viable embryos for repair process (as they possess great cellular/molecular damage) to be the reason for this association. The scientists measured the amino acid profiles using high-performance liquid chromatography. Amino acid profiling of embryos were identified as non-invasive markers of DNA injury at the blastocyst stage; the findings consistent with the hypothesis. The results also suggest that the metabolic profiling (amino acids) is effective for the selection of a single competent embryo prior to transfer in IVF cycles.15

In a more recent study, scientists reported the potential of proteomics in estimating the embryos with highest implantation competence, thus indicating that, from a clinical standpoint, morphological assessment, along with non-invasive evaluation of secretome, may aid in the following: • Improvement in IVF success rates • Increased adoption of single embryo transfer (SET)18 Drawbacks of Embryo Selection Methods Although different grading methods based on morphology, are the mainstay of selecting the ‘most competent’ embryos, they have been unable to reduce the multiple birth rates. Now, various adjunctive technologies are being considered to overcome these drawbacks. Many non-invasive techniques have been proposed, including the assessment of embryos based on metabolic parameters, but most of these are cost-prohibitive as they need specialized equipments and trained laboratory staff and may not provide quick results as required for routine clinical practice. Also, there is a lack of sufficient blinded trials (using culture media) to validate the results of these techniques in terms of the correlation with embryo implantation potential after transfer.

An earlier review published in the Journal of Assisted Reproduction and Genetics highlights the importance of human leukocyte antigen G (HLA-G) for implantation, also indicating HLA-G as a marker for embryo selection in IVF cycles. The test is referred to as ‘Embryo Marker Expression Test (EMET)’. It was found that the selection of embryos with soluble HLA-G levels of ≥2 U/mL in the culture media, along with good morphologic grade, resulted in 65% pregnancy rate compared to 0% when the concentration was <2 U/mL.16 The lack of sufficient data to confirm these findings mandates the need for more research in order to validate the results.

The other main drawback of the non-invasive methods is the inability to detect chromosomal abnormalities or genetic disorders.7 Although the advent of genetic and micro-manipulation methods-enabled preimplantation diagnosis (PGD) aids in overcoming such limitations, they

• Proteomics Proteomics illustrates the variations in the expression

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are surrounded by several ethical, moral, and social issues. Also, PGD may help in identifying genetic characteristics of an embryo but not its intrinsic biological competence.1

April - June 2010

(CGH). The preliminary findings indicated that the PGS cycles using CGH had a substantial rise in the embryo implantation rates. The researchers projected that the techniques based on microarray platforms may provide similar results, but suggested the need for further studies to verify the accuracy and cost effectiveness.20

Embryo selection, unlike gene therapy, involves the testing of genetic characteristics in a blastomere, without altering the genetic makeup. However, the selection process may lead to the discarding of embryos, which do not possess ideal genetic features, thus raising ethical concerns.

Conclusion There is an ongoing debate whether to characterize only the top quality embryos or predict the implantation potential of all the embryos. It seems imperative to choose the best embryo to move towards the SET policy for averting complications; however, the ‘best’ embryo itself may be poorer than what was estimated.

Further, the process is effective mainly for single-gene defects, and becomes complicated if tested for multiple genes. Evidence through mathematical probability equations have shown the ineffectiveness of the selection methods in identifying any disease or trait that is regulated by >5 genes. Also, the process is a one-generation solution, i.e., it may aid in preventing the transmission of diseases to the immediate offspring, but does not confer protection to the subsequent generations.

Thus, it is desirable to determine the implantation potential of all embryos, which will help in treatment planning, taking into consideration the probability of achieving success with singleton pregnancy to the risks associated with multiple gestations.6

Ongoing Developments As choosing the best viable embryo is still challenging, alternative or additional techniques are needed to accomplish the optimal outcomes.3

A number of different parameters and tests are currently used, and new techniques being developed to determine the best embryo for transfer. Even with significant advancements in the field of assisted reproduction, there is a lack of welldefined analytical methods and biological measurements that can precisely identify the viability of the gametes and embryos.1 The absence of an effective single embryo evaluation leads to low implantation and unacceptably high multiple pregnancies; thereby limiting the treatment success. This calls for a consensus on the markers/parameters that are reliable, noninvasive, and objective, so as to optimize the probability of successful pregnancies in ART cycles, and progress towards a greater adoption of SET policy.

Many studies presented at the 64th Annual Meeting of ASRM (2008) in San Francisco demonstrated the effectiveness of several novel techniques in identifying healthy embryos without causing excessive stress. • Fragouli and coworkers observed that the analysis of chromosomes discarded in polar bodies may serve as a tool to detect approximately 2 times more maternallyderived abnormalities than routine PGS performed on blastomeres. The procedure has the potential to decrease embryo damage. • Dagan Wells and colleagues developed a technique, wherein trophectoderm cells analysis maximized the chromosomal information obtained from an embryo and minimized the risks on the embryo. Clinical pregnancy was noted in 70% of the embryo transfer cycles.19

References 1.

Kay Elder, Jacques Cohen, eds. Human Preimplantation Embryo Selection. Informa Healthcare;2007.

Vergouw, Lambalk, Lens, et al. Near Infrared Spectroscopy as a tool to predict embryo viability: a novel, non-invasive method for embryo selection. Paper presented at: Annual Meeting of European Society of Human Reproduction and Embryology;July 2,

Aneuploidy is one of the vital factors that impact embryo viability. However, standard morphological assessment cannot distinguish the embryos obtained from aneuploid gametes to their normal counterparts. The currently adopted PGS techniques that rely on biopsy followed by FISH, screen <50% of the chromosomes.

2007;France. 3.

Nagy ZP, Sakkas D, Behr B. Symposium: innovative techniques in human embryo viability assessment. Non-invasive assessment of embryo viability by metabolomic profiling of culture media (‘metabolomics’). Reprod Biomed Online. 2008 Oct;17(4):502-7.

Squires and Kaplan. Developmental Outcomes of Children Born After Assisted Reproductive Technologies. Infants & Young

In a 2008 review, investigators discussed the methods which could overcome these limitations and help in screening the entire chromosomal complement. One such method is the comparative genomic hybridization

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Children. 2007;20(1):2-10. 5.

Knoester M, Vandenbroucke JP, Helmerhorst FM, van der Westerlaken LA, Walther FJ, Veen S. Matched follow-up study of 5-8 year old ICSI-singletons: comparison of their neuromotor

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development to IVF and naturally conceived singletons. Hum

resonance correlates with reproductive potential of embryos

Reprod. 2007 Jun;22(6):1638-46.

in women undergoing in vitro fertilization. Fertil Steril. 2008

Jan Gerris, François Olivennes, Petra de Sutter, eds. Assisted

Dec;90(6):2183-9.

Reproductive Technologies: Quality and Safety. Parthenon

14. Seli E, Vergouw CG, Morita H, et al. Noninvasive metabolomic

Publishing;2003.

profiling as an adjunct to morphology for noninvasive embryo

Baczkowski T, Kurzawa R, Głabowski W. Methods of embryo

assessment in women undergoing single embryo transfer. Fertil

Hnida C, Agerholm I, Ziebe S. Traditional detection versus

15. Sturmey RG, Hawkhead JA, Barker EA, Leese HJ. DNA damage and

computer-controlled multilevel analysis of nuclear structures from

metabolic activity in the preimplantation embryo. Hum Reprod.

donated human embryos. Hum Reprod. 2005 Mar;20(3):665-71.

2009 Jan;24(1):81-91.

scoring in in vitro fertilization. Reprod Biol. 2004 Mar;4(1):5-22

Steril. 2009 Jul 7. [Epub ahead of print]

Hnida C. Multilevel morphometric imaging Using Computer

16. Roussev RG, Coulam CB. HLA-G and its role in implantation

assisted embryo analysis. Paper presented at: ESHRE Special

(review). J Assist Reprod Genet. 2007 Jul;24(7):288-95.

Interest Group Embryology Workshop;2008;France.

17. Katz-Jaffe MG, Gardner DK. Symposium: innovative techniques

10. Devroey P. IS PGS APPROPRIATE FOR EMBRYO SELECTION:

in human embryo viability assessment. Can proteomics help to

NO. Debate at: The 3rd International IVI Congress;May 14,

shape the future of human assisted conception? Reprod Biomed

2009;Madrid, Spain.

Online. 2008 Oct;17(4):497-501.

11. Botros L, Sakkas D, Seli E. Metabolomics and its application for

18. Katz-Jaffe MG, McReynolds S, Gardner DK, Schoolcraft WB. The

non-invasive embryo assessment in IVF. Mol Hum Reprod. 2008

role of proteomics in defining the human embryonic secretome.

Dec;14(12):679-90.

Mol Hum Reprod. 2009 May;15(5):271-7.

12. Scott R, Seli E, Miller K, Sakkas D, Scott K, Burns DH. Noninvasive

19. New Approaches to Screening Eggs and Embryos for Chromosomal

metabolomic profiling of human embryo culture media using

Defects are More Effective and Less Invasive. Press Release.

Raman spectroscopy predicts embryonic reproductive potential: a

ASRM. Last accessed February 3, 2010.

prospective blinded pilot study. Fertil Steril. 2008 Jul;90(1):77-83.

20. Wells D, Alfarawati S, Fragouli E. Use of comprehensive

13. Seli E, Botros L, Sakkas D, Burns DH. Noninvasive metabolomic

chromosomal screening for embryo assessment: microarrays and

profiling of embryo culture media using proton nuclear magnetic

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CGH. Mol Hum Reprod. 2008 Dec;14(12):703-10.

IVF NEWS.Direct!

April - June 2010

INTERVIEW Cumulus-aided Transfer: A Novel Technique Q: What is cumulus aided transfer and how does it improve IVF outcomes? A: Cumulus aided transfer (CAT) is an innovation in the field of infertility treatment developed by me and my team, to increase the success rates of assisted reproduction (IVF/ICSI).

Dr Firuza R Parikh, MD Director, Department of Assisted Reproduction and Genetics, Jaslok Hospital and Research Centre, Mumbai, India

Q: How did you conceptualize this procedure and when was it initiated/ developed? A: My husband Rajesh and I were taking a late night walk. We were talking about how wants can overpower ones needs. We were also talking about the bounty of nature and how nature can be used to harness energy and that one must not waste anything in life. At that point we started talking about the role people play in nurturing others. I mentioned to Rajesh that the life that we create in the IVF lab also has to be nurtured carefully for 3 to 4 days. It then struck us that if we could nurture the embryos in safer and a better way, perhaps that may reflect in better implantation. I spoke to him about the work one of my PhD students was doing on cumulus cells. Rajesh suggested that I use these cumulus cells to nurture the embryos in vitro and in vivo. Thus, the concept of CAT was born. Q: Who are the ideal candidates for this procedure? Is it suitable for all? A: This procedure can be safely used for all women undergoing IVF/ICSI. However, it should not be used in cases where the women have autoantibodies, such as antinuclear, antimicrosomial, antithyroid, and antiovarian antibodies. Women exhibiting lupus antibodies also should not have this procedure as the antibodies may be present in the cumulus cells and attack the embryo. Q: Can you explain the procedure in brief? A: In the technique of cumulus aided transfer, the embryos are first grown in a culture medium on top of a proliferating monolayer of the patient’s own cumulus cells, which are called “Feeder” or “Helper” cells. The feeder cells assist in the growth and development of the embryo. This technique is called cumulus co-culture. When a few of the feeder cells i.e., the cumulus cells on which the embryo is being cultured are transferred along with the embryo into the uterus, the technique is referred to as cumulus-aided transfer. The cumulus cells on which the embryos are co-cultured serve as a source of growth factors, which not only help to improve the embryo quality but also help in implantation when transferred along with the embryos. Q: How long will the CAT procedure take prior to the transfer? Does it require any special training? A: Cumulus-coculture is carried out simultaneously with the embryo culture and does not require any extra time. Perfecting the procedure of cumulusaided transfer would require a training of about 1-2 months.

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INTERVIEW

Q: How many success stories have been reported in India, and globally, using this technique? A: Our published data in the international scientific journal – Fertility and Sterility, October 2006, shows that the pregnancy rates were 47.6% and 34% in 267 and 250 women who underwent and who did not have the CAT procedure, respectively. We have seen an increase in the implantation and pregnancy rates at our centre using this technique. We have a collaboration with a French Centre and a centre in USA using this technique. Since they have just started using this technique we would have to wait for their data. I am aware that many IVF centres in India are using this technique and I would be happy if more people used it as this would translate into better pregnancy rates. We have trained about 4 centres in India in this technique. Q: What are the benefits and limitations of CAT? A: CAT produces better quality embryos, helps with implantation, and gives better pregnancy rates. It is particularly useful in couples who have previous failed IVF, in older women, and in those with less number of oocytes. The CAT procedure utilizes the mother’s own cells and hence does not pose any danger. Q: What is the difference between cumulus coculture and cumulus-aided embryo transfer? A: In IVF, the term “culture” means to grow or incubate embryos in a defined nutrient medium. In the technique of “cumulus co-culture”, embryos are grown in a culture medium on top of a proliferating monolayer of the patient’s own cumulus cells. These cumulus cells ensure the proper growth of the developing embryos. At the time of embryo transfer into the uterus, we add a few of the cumulus cells to the embryos so that the nutrients continued to be provided in vivo, thereby enhancing the pregnancy rates. This is the technique of cumulus aided transfer. Q: What is the role of cumulus cells in embryonic development? A: Use of cumulus cells in a co-culture has the following benefits, which in turn help in embryonic development • Cumulus cells synthesize cytokines, which play an important role in the in-vitro development of the embryo and aid its implantation into the endometrium. • Cumulus cells produce progesterone, an important steroid hormone of pregnancy, which helps prime the endometrium and also to create a microenvironment

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April - June 2010

Increased colonization with cumulus cells in close proximity of the embryo conducive for implantation. • Cumulus cells produce glutathione, a very important antioxidant, which neutralizes the reactive oxygen species (ROS); the culture media used in IVF is also a known source of ROS Q: Which are the cytokines and growth factors that contribute to the increased adhesiveness of the embryos? A: Cytokines involved are leukemia inhibitory factor (LIF), insulin-like growth factor (IGF-I) and interleukins like IL-4, IL-5, IL-6 and IL-10. The growth factors are vascular endothelial growth factor (VEGF) and transforming growth factor (TGF β, TGF α) Q: Will the overall cost of IVF treatment increase with CAT? A: The overall cost increases marginally as the amount of disposables and culture medium used increases. Q: What is the latest research on CAT? A: As the development of culture media for ART evolves, paralleling other advancements in the field, we find ourselves at a critical point at which increasing specialization and phase-specific design is the clear goal to help improve outcomes in IVF. This leads us to the question, is there an ideal culture medium? Clearly, the goal is to emulate the in vivo environment as closely as possible. Because this milieu is dynamic compared with the virtually static in vitro environment, incorporating critical molecules like LIF and others in the culture media is an ongoing endeavour.

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April - June 2010

FEATURED MINI REVIEW Discovery of Gonadotropin-Inhibitory Hormone Homologs to Transform Current Understanding of Human Reproductive Endocrinology In 2000, a study conducted by Japanese researchers on quail brains reported the existence of a gonadotropininhibitory hormone (GnIH), the first hypothalamic peptide noted in a vertebrate that directly blocks the synthesis and release of gonadotropins from the pituitary gland. Although several subsequent studies have identified the presence of the hormone in mice and sheep models, the existence of a similar hypothalamic gonadotropin-inhibiting system in humans was unclear. Now, in a groundbreaking study published in the recent issue of the journal PLoS One, researchers report the identification of human homologs of the GnIH hormone: RFamide-related peptide-1 (RFRP-1) and RFRP-3, in the human hypothalamus. The discovery of the new reproductive hormone mandates the revision of our current understanding of the central control mechanisms that regulate human reproductive functions.

in adrenal glucocorticoids contributes to increase in RFRP, which in turn causes a hypothlamic suppression of reproductive functions. The direct association noted by the researchers between stress and RFRP may aid in gaining better insight into stress-related reproductive dysfunction and infertility. Analogs of luteinizing-hormone-releasing hormone (LHRH), with the potential to modulate the hypothalamicpituitary-gonadal axis, have already been recognized as effective agents to treat diverse sex-steroid-dependent, benign and malignant disorders. Now, the novel GnIH hormones, having the ability to inhibit the action of gonadotropes, hold greater therapeutic implications as alternative or adjunct agents for treating: • Various hormone-dependent diseases, such as endometriosis, precocious puberty, uterine fibroids, and benign prostatic hyperplasia • Cancerous conditions, including prostatic and breast cancers

In addition to the existence of the GnIH hormone analogs, the study led by Takayoshi Ubuka from the Department of Integrative Biology, University of California, Berkeley, characterized the distribution and biological activity of these analogs in human hypothalamus. Using mass spectrometry, the researchers identified the peptide sequences of RFRP-1 and RFRP-3 as MPHSFANLPLRFNH2 and VPNLPQRF-NH2, respectively. Further, immunocytochemistry analysis demonstrated the presence of GnIH-immunoreactive neuronal cell bodies in the hypothalamic dorsomedial region with the axonal projections extending to the GnRH neurons present in the preoptic area and the median eminence.

The researchers also anticipate that the recent findings could pave the way for developing new contraceptive strategies. References 1.

New human reproductive hormone could lead to novel contraceptives. Press Release. University of California. Last accessed December 30, 2009.

Ubuka T, Morgan K, Pawson AJ, et al. Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis. PLoS One. 2009 Dec

Another recent study conducted by Clarke and colleagues (Endocrinology, 2008) showed the ability of the human RFRP3 in blocking the production of the gonadotropin hormone in sheep pituitary cell culture via the inhibition of Ca2+ mobilization. The study also demonstrated the expression of the GnIH receptor mRNA in the pituitary and hypothalamus; specifically in the luteinizing hormone producing cells (gonadotropes present in the anterior pituitory.

22;4(12):e8400. 3.

Clarke IJ, Sari IP, Qi Y, et al. Potent action of RFamiderelated peptide-3 on pituitary gonadotropes indicative of a hypophysiotropic role in the negative regulation of gonadotropin secretion. Endocrinology. 2008 Nov;149(11):5811-21.

Kirby ED, Geraghty AC, Ubuka T, Bentley GE, Kaufer D. Stress increases putative gonadotropin inhibitory hormone and decreases luteinizing hormone in male rats. Proc Natl Acad Sci U S A. 2009 Jul 7;106(27):11324-9.

Another research group from the University of Berkeley (PNAS, 2009) reported that the stress-induced increase

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FEATURED MINI REVIEW

April - June 2010

Vapor-phase Nitrogen System Superior to Traditional Liquid Nitrogen for Storing Vitrified Oocytes Any therapeutic intervention in reproductive biology must ensure complete protection of biological materials against a wide range of microorganisms. However, the liquid nitrogen (LN) used for cryostorage collects particulate contaminants from the atmosphere or from the container surface over a period of time, which could be a potential source of pathogenic microbes, such as herpes, papova, hepatitis, and vesicular stomatitis virus. Now, a recent study suggests that the storage of vitrified oocytes using vapor-phase nitrogen system serves as a safer alternative to the traditional LN storage tank as it avoids the risk of cross-contamination apart from retaining the developmental potential of these oocytes into healthy competent embryos. The findings of the prospective randomized trial have been published in the latest issue of Fertility and Sterility.

storage of vitrified oocytes in vapor-phase nitrogen system is capable of eliminating dangers of sample contamination that results from direct LN contact with blastocyst formation, implantation, and pregnancy rates similar to that noted in LN storage. In the recent years, several studies have provided information on various mechanisms of controlling crosscontamination. A study by Isachenko et al (Human Reproduction, 2005) suggested that vitrification of human pronuclear oocytes using open-pulled straws located inside hermetically closed containers ensured complete sealing in order to minimize the risk of crosscontamination. A more recent study by Parmegiani et al (Fertility and Sterility, 2009) reported that the rapid sterilization of LN was achievable with the use of ultraviolet irradiation, thereby making it possible for oocyte vitrification in open carriers.

Ana Cobo and colleagues, from the IVI-Universidad de Valencia, Spain, conducted the study on 44 oocyte donors and 46 recipients at a private infertility center to assess the efficacy of long duration storage of vitrified oocytes in vapor-phase nitrogen. Traditional LN freezer and vapor-phase nitrogen system were randomly used for the storage of oocytes vitrified using the Cryotop method. The surviving oocytes were later donated and evaluated for fertilization potential, embryo development, and clinical outcomes. The study results are listed in the table below:

Outcomes

A recent report by the Practice Committee of the American Society for Reproductive Medicine (ASRM) and the Society for Assisted Reproductive Technology (Fertility and Sterility, 2008) recommended the use of separate sample containers for specimens infected with microorganisms such as HIV in order to avoid significant cross contamination. In addition, the following measures were reported to reduce cross contamination of the cryopreserved samples stored in LN: • Storing specimens in the vapor phase of nitrogen in place of LN • Stocking samples in containers manufactured to resist freezing temperatures and thawing cycles • Using double bagging or sealing methods in order to eliminate direct contact of the storage tanks with LN • Employing techniques such as “sperm washing” before semen cryopreservation in order to reduce the viral load.

LN Storage Tank (%)

Vapor-phase Nitrogen System (%)

Survival rates

94.5

95.3

Fertilization rates

71.7

73.1

Cleavage rates on day 2

94.7

95.6

Cleavage rates on day 3

79.9

84.5

Blastocyst formation

53.9

54.7

Implantation rates

33.7

40.5

Clinical pregnancy rates

53.3

58.1

Remohí J. Storage of human oocytes in the vapor phase of

Ongoing pregnancy rates

46.6

48.8

nitrogen. Fertil Steril. 2010 Feb 4.

References 1. Cobo A, Romero JL, Pérez S, de Los Santos MJ, Meseguer M,

2. Isachenko V, Montag M, Isachenko E, et al. Aseptic technology

No statistically significant variations were found between the two storage systems with respect to the abovementioned outcomes. Based on the study findings,

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of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod. 2005 Feb;20(2):492-6.

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April - June 2010

ART Study Reports Improved ICSI Outcome with Mid-follicular Recombinant LH Supplementation A recent randomized controlled study highlights the increase in implantation and live birth rates with midfollicular recombinant human luteinizing hormone (r-HLH) supplementation in women aged 35 to 39 years, who were subjected to ovarian stimulation for intracytoplasmic sperm injection (ICSI) cycles. The findings have been published in the recent issue of the journal, Reproductive BioMedicine Online.

r-HFSH, and r-HLH. Observations demonstrated group II to have increased rates of the following parameters in comparison to group I: • Number of oocytes retrieved • Number of metaphase II oocytes • Fertilization rate • Average number of embryos produced and frozen for every ICSI cycle • Cumulative implantation rate

Roberto Matorras, Chairman of Obstetrics and Gynecology, Basque Country University, Spain, and coworkers, conducted the parallel group single-centre study in order to assess the effect of mid-follicular r-HLH as a supplement in women undergoing ICSI treatment cycles. The primary outcome measure was the number of oocytes retrieved at metaphase II stage. The study involved ovarian stimulation with 300–450 IU/day of recombinant human FSH (r-HFSH) following pituitary suppression with a gonadotropinreleasing hormone agonist. Of the total study population (n=131), 68 women were given r-HFSH alone, while the remaining 63 received r-HFSH and r-HLH from the 6th day of stimulation until its completion. Following final follicular maturation with 250 μg of recombinant human chorionic gonadotropin, oocytes were checked for nuclear maturity, and later fertilized by ICSI.

Studies have shown that r-HLH is equally efficacious, and significantly safer than human chorionic gonadotropin (hCG) in inducing final follicular maturation and triggering ovulation in assisted reproduction treatment. Also, the production of consistent, well-characterized r-HLH is tightly regulated, ensuring high-quality preparation. The Practice Committee of the American Society for Reproductive Medicine (Fertility and Sterility, 2008) has made rather conservative recommendations about the use of LH, suggesting that rHLH supplementation with FSH has the potential to increase follicular development in women with deficient LH levels. References 1.

The investigators observed no notable change in both groups in terms of oocyte or embryo quality and quantity. However, women receiving r-HLH supplementation demonstrated a notable improvement in embryo implantation and live birth rate when compared to the other group. Based on the findings, the researchers suggested that r-HLH supplementation may be beneficial in women aged 35 to 39 years undergoing ICSI procedure.

Matorras R, Prieto B, Exposito A, et al. Mid-follicular LH supplementation in women aged 35–39 years undergoing ICSI cycles: a randomized controlled study. Reprod Biomed Online. 2009 Dec;19(6):879-887.

Franco JG Jr, Baruffi RL, Oliveira JB, et al. Effects of recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-agonist protocol: a matched case-control study. Reprod Biol Endocrinol. 2009 Jun 4;7:58.

The Practice Committee of American Society for Reproductive Medicine, Birmingham, Alabama. Gonadotropin preparations:

Similar findings were reported in a study conducted by Franco et al (Reproductive Biology and Endocrinology, 2009). The researchers grouped 244 women without ovulatory dysfunction (age <40 years), undergoing first ICSI cycle, into two groups based on the ovulation stimulation protocol. Group I (n=122) underwent pituitary down-regulation with gonadotropin-releasing hormone agonist (GnRH-a) and r-HFSH stimulation alone, while group II (n=122) was administered with GnRH-a,

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past, present, and future perspectives. Fertil Steril. 2008 Nov;90(5 Suppl):S13-20. 4.

Gurgan T, Demirol A. Recombinant luteinizing hormone in assisted reproductive technology. Reviews in Gynaecological and Perinatal Practice. 2005 Mar;5(1):45-50.

Practice Committee of American Society for Reproductive Medicine. Use of exogenous gonadotropins in anovulatory women: a technical bulletin. Fertil Steril. 2008 Nov;90(5 Suppl):S7-12.

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ART

April - June 2010

Swedish Study Reiterates Benefits of Modified Natural and Mild IVF Cycles In order to reduce complications associated with the conventional IVF procedure, modified natural cycle IVF (mnc-IVF) and mild IVF (m-IVF) cycles have been adopted. However, there is speculation that the pregnancy and live birth rates may be lower with both these approaches compared to conventional cycles, probably due to the lower use of medicines and shorter treatment duration. Now, a 10-year Swedish study published in the journal, Reproductive BioMedicine Online highlights that mncIVF and m-IVF have comparable pregnancy rates to conventional cycles. Arthur Aanesen and coworkers from the IVF Unit, Queen Sophia Hospital, Stockholm, conducted the study on 43 patients (129 cycles) and 145 couples (250 cycles) undergoing mnc-IVF and m-IVF, respectively, between 1996 and 2007. The results were compared to patients who underwent conventional IVF cycles during the same period. The study findings are listed in the table given below. Parameters Tested

mnc-IVF

m-IVF

Conventional IVF

Ongoing pregnancy rate/embryo transfer (%)

26.7

27.2

34.3

Ongoing pregnancy rate/embryo transfer for patients ≥38 years (%)

17.5

27.0

Cancellation Rate (%)

53.5

39.6

13.7

It was also found that in comparison to the least expensive conventional IVF cycle, the medication costs were lower by 97.5% and 96.3% in the mnc-IVF and m-IVF groups, respectively. Based on the analysis, the researchers noted the following with respect to the two approaches when compared to conventional cycles. • Acceptable pregnancy rates per embryo transfer • Reduced medication cost • Substantially decreased risk of complications • Psychologically more acceptable to patients

open-label prospective trial, Heijnen et al (Nederlands Tijdschrift voor Geneeskunde, 2008) divided 404 subjects to receive either of the two: • m-IVF: gonadotrophin releasing hormone (GnRH) antagonist for ovarian stimulation in combination with single embryo transfer • standard treatment: prolonged stimulation using a GnRH agonist along with the transfer of two embryos It was observed that cumulative pregnancy rates, which resulted in full-term live births after 1 year, were 43.4% and 44.7% in the mild and standard groups, respectively. Also, the multiple pregnancy rates and the overall cost were substantially lowered with mild treatment. The researchers support the use of m-IVF and single embryo transfer in clinical practice. Similarly, Pelinck et al (Human Reproduction, 2007), in a single center study, indicated the mnc-IVF protocol as an effective treatment option for patients opting for IVF. An earlier report of the International Society for Mild Approaches in Assisted Reproduction (ISMAAR) noted growing concerns regarding the extensive use of conventional IVF treatment (high stimulation protocol) and its associated limitations such as safety, patient discomfort, and cost. In view of this, numerous research groups are developing new treatment protocols to ensure: • Lower risk of complications, such as ovarian hyperstimulation syndrome and multiple pregnancy • Improved endometrial and oocyte quality • Decreased financial and emotional burden to the patients With evidence indicating the advantages of mnc-IVF and m-IVF, there is a renewed interest and resurgence of these approaches in clinical practice. In an earlier review, Nargund and Frydman (Reproductive BioMedicine Online, 2007) indicated a trend towards a more patient-friendly approach in IVF, suggesting the likelihood of mnc-IVF or m-IVF replacing conventional treatment in downregulated cycles. References 1.

Nargund G, Frydman R. Towards a more physiological approach to IVF. Reprod Biomed Online. 2007 May;14(5):550-2.

Similar findings on the efficacy of mnc-IVF and m-IVF were reported in several other studies. In one such randomized

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April - June 2010

Researchers Develop Model for Pre-empting Blastocyst Transfer Cycle Cancellation Cycle cancellation has been identified as one of the main limitations of blastocyst transfer, as per a recent report of the Practice Committee of the American Society for Reproductive Medicine (ASRM) and Society for Assisted Reproductive Technology (Fertility and Sterility, 2008). In a new study, a group of French researchers have proposed a cycle-based model that has the potential to lower the incidence of day-5 blastocyst transfer cancellation in IVF cycles. The study findings have been published in the journal, Human Reproduction.

hoc conclusion that a decision to opt for blastocyst transfer on day 3, following oocyte retrieval, may lead to a significant reduction in the rate of cancellation. • A more recent study by Hayrinen and coworkers (Irish Journal of Medical Science, 2009) validated the efficacy of a coordinated two-stage transfer in a single IVF cycle to overcome the disadvantage. In this approach, embryos were transferred on day 3, with a subsequent second transfer (blastocyst) performed generally on day 5 or day 6.

Lionel Dessolle from the Service de biologie et médecine de la reproduction, Centre Hospitalier Universitaire de Nantes, France, and coworkers developed the model by analyzing 562 consecutive first IVF cycles and evaluated its predictive ability using an independent validation cohort. The patient and cycle characteristics were correlated with day-5 cultured blastocysts using multivariable logistic regression. The analysis was also used to construct a nomogram. Several thresholds were employed to measure the clinical utility of the model, whose performance was gauged by discrimination and calibration.

Substantial advancements in media culturing in the past few decades have contributed to a change in the practice of assisted conception from early cleavage embryo transfer to blastocyst stage transfer. The rationale for adopting the latter method is that it may result in higher implantation rates due to its ability to improve uterine and embryonic synchronicity, and also allow self-selection of viable embryos. However, based on the current evidence with respect to the blastocyst culture and transfer, the 2008 ASRM report summarized the lack of reliable criteria that could help in identifying those embryos that have the potential to develop into viable blastocysts. Hence, in addition to the use of predictive models for reducing the risk for cycle cancellation, extreme prudence is required in selecting appropriate candidates who will benefit from blastocyst culture during ART.

The study results demonstrated the independent association of blastocyst development with female age, fertilization technique, and number and quality ratio of day 3 embryos. The final prediction model was well calibrated, with the training set showing an area under the curve (AUC) of 0.75 (95% CI=0.73–0.77). The calibration of the model was acceptable in the validation data set, which had an AUC of 0.80 (95% CI=0.78–0.83). The negative predictive value and false positive rates were found to be 80.5% and 17%, respectively, at a decision threshold of 0.55. Based on the study findings, the researchers suggested the clinical relevance of the cycle-based model in decreasing the blastocyst transfer cancellation.

References 1.

Dessolle L, Fréour T, Barrière P, et al. A cycle-based model to predict blastocyst transfer cancellation. Hum Reprod. 2010 Mar;25(3):598-604.

Practice Committee of American Society for Reproductive Medicine; Practice Committee of Society for Assisted Reproductive Technology. Blastocyst culture and transfer in clinical-assisted reproduction. Fertil Steril. 2008 Nov;90(5 Suppl):S174-7.

Levitas E, Lunenfeld E, Har-Vardi I, et al. Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2-3 embryo transfer cycles: a prospective, randomized study.

Previous studies have suggested certain key strategies to avoid the risk of blastocyst transfer cancellation owing to culture failure. Some of them are enumerated below. • Levitas et al (Fertility and Sterility, 2004) made a post

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Fertil Steril. 2004 Mar;81(3):567-71. 4.

Hayrinen LH, Sills ES, Fogarty AO, Walsh DJ, Lutsyk AD, Walsh AP. First Irish delivery following sequential, two-stage embryo and blastocyst transfer. Ir J Med Sci. 2009 Oct 8. [Epub ahead of print]

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April - June 2010

Study Reports Higher Risk of Preterm Birth and Congenital Abnormalities after Blastocyst-stage Embryo Transfers A recent study by a team of Swedish researchers reports that extended embryo culturing to blastocyst stage raises the risk of preterm birth and congenital malformations among singletons. The findings of the study are published in the recent issue of Fertility and Sterility.

allow selection of embryos that have the capability for further development under embryonic genomic control. A report by the Practice Committee of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology (Fertility and Sterility, 2008), listed the following theoretical advantages of culturing embryos to blastocyst stage for assisted reproduction: • Increased implantation rate • Reduced the number of embryos needed to be transferred • Improved temporal synchronization between uterine endometrium and embryo at time of transfer • Allowed selection of viable embryos • Gave an opportunity to perform preimplantation genetic diagnosis (PGD) when necessitated

Bengt Källén, Professor, Tornblad Institute, University of Lund, Sweden, and co-workers, conducted the study to evaluate neonatal outcomes on transferring blastocyst and cleavage-stage embryos in IVF cycles. The study considered IVF births recorded in the Swedish Medical Birth Register between 2002 and 2006. Neonatal characteristics were compared in 1,311 and 12,562 infants born after blastocyst-stage and cleavage-stage embryo transfer, respectively. Furthermore, the comparison was done with all births (n=598,687) that took place between 2002 and 2007.

The report also documents the following disadvantages of routine blastocyst transfer in ART patients: • High incidence of monozygotic and dizygotic twinning • Risk of epigenetic mutations in the resulting offspring • Non-availability of suitable criteria for identifying embryos capable of developing into viable blastocysts in vitro • Improvement in birth rates only in patients with good prognosis, and the probable decrease in low prognosis patients (based on criteria such as age and quality, and number of embryos), unlike cleavage stage embryo transfers • Success with blastocyst cryopreservation differs with programs.

On adjustment of several parameters, such as birth year, maternal age, parity, body mass index, and smoking, it was noted that the risk of preterm birth and congenital malformations were higher among singletons born after blastocyst-stage embryo transfer compared to cleavagestage embryo transfer. Data limited to clinics doing blastocyst transfers estimated a higher risk for preterm birth, lower APGAR (appearance, pulse, grimace, activity, respiration) score, lower birth weight, and abnormal respiratory diagnoses, but the risk for congenital malformations remained the same. The researchers suggested that the increased risk associated with blastocyst transfer could be due to the longer duration of in vitro culturing, and may be partly dependant on the selection of women for blastocyst transfer.

References 1.

Källén B, Finnström O, Lindam A, Nilsson E, Nygren KG, Olausson PO. Blastocyst versus cleavage stage transfer in in vitro fertilization: differences in neonatal outcome? Fertil Steril. 2010 Feb 3. [Epub

An earlier retrospective study by Milki et al (Fertility and Sterility, 2003) showed that blastocyst-stage embryo transfer raises the incidence of monozygotic twinning (MZT), when compared to cleavage-stage embryo transfer. Transvaginal ultrasound showed 11 (5.6%) MZT incidences of 197 pregnancies, conceived after blastocyst transfer, compared to 7 (2%) MZT incidences out of 357 viable pregnancies achieved through cleavage-stage embryo transfer.

ahead of print] 2.

Milki AA, Jun SH, Hinckley MD, Behr B, Giudice LC, Westphal LM. Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer. Fertil Steril. 2003 Mar;79(3):503-6.

Mangalraj AM, Muthukumar K, Aleyamma T, Kamath MS, George K. Blastocyst stage transfer vs cleavage stage embryo transfer. J Hum Reprod Sci. 2009 Jan;2(1):23-6.

Practice Committee of American Society for Reproductive Medicine; Practice Committee of Society for Assisted Reproductive Technology.

However, several studies have shown that extending the duration of embryo culture to day 5 could aid in the development of chromosomally competent embryos, and

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Blastocyst culture and transfer in clinical-assisted reproduction. Fertil Steril. 2008 Nov;90(5 Suppl):S174-7.

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ART

April - June 2010

New Prospective Study Says Pre-IUI Hydrotubation May Not Benefit Unexplained Infertility Patients There are contradicting reports about the usefulness of hydrotubation prior to intrauterine insemination (IUI), a technique which is supposed to aid in removing luminal debris and decreasing reocclusion of the fallopian tube, and thereby improve chances of pregnancy in infertility patients. Now, a prospective randomized study highlights that hydrotubation done before intrauterine insemination (IUI), does not increase pregnancy rates in patients with unexplained infertility. The study findings have been published in the latest online issue of Reproductive BioMedicine Online.

(East African Medical Journal, 2009). The study analyzed the effect of hydrotubation in patients with tubal infertility and unexplained infertility. Some of the conditions, which indicated the need for tubal flushing were unexplained infertility, bilateral cornual blockage, bilateral perifimbrial adhesion (incomplete tubal occlusion), and bilateral fimbrial end occlusion. The researchers demonstrated the benefits of the tubal flushing procedure especially in infertile patients belonging to resource-poor countries, and suggested it to be an effective therapeutic strategy for unexplained infertility.

Mohamed Aboulghar, Professor, Faculty of Medicine, Cairo University, Egypt, and colleagues, conducted the study in 228 patients with unexplained infertility to determine the effectiveness of hydrotubation before IUI. The researchers stimulated the ovaries with 100 mg clomiphene citrate from the 3rd day of the menstrual cycle, and 1 ampoule human menopausal gonadotrophin from 6th cycle day, both for a period of 5 days. Subjects (n=15) who had poor response or with a stimulation of ≥3 follicles were cancelled from the study.

Earlier, Luttjeboer et al (Cochrane Database of Systemic Reviews, 2007) shortlisted 12 trials (subjects=2,079) from various databases, such as MEDLINE, EMBASE, and Cochrane, to investigate the impact of tubal flushing with oil- or water-soluble contrast media on treatment outcomes in patients with infertility. The review found that the odds of pregnancy and live birth were increased with hydrotubation using oil-soluble contrast media compared to no intervention. However, further robust randomized studies on the following approaches related to hydrotubation could be beneficial in developing guidelines for routine clinical practices: • Water-soluble media vs. no intervention • Oil-soluble vs. water-soluble media • Hydrotubation vs. other established infertility treatments.

Daily investigations of the urine levels of luteinizing hormone (LH), along with folliculometry were carried out till optimal LH concentrations were reported. Subsequently, the patients (n=213) were randomized to undergo or not receive hydrotubation (with 50 mL saline) followed by IUI. The ongoing pregnancy rates in the hydrotubation and non-hydrotubation groups were found to be 12.6% and 8.2%, respectively. The results did not show any statistically significant difference in the outcome (OR=1.66; 95% CI=0.62–4.63) between the two groups. Based on the findings, the scientists suggested that hydrotubation, before the insemination procedure, does not significantly enhance the treatment outcome.

References 1.

Aboulghar MA, Mourad LM, Al-Inany HG, Aboulghar MM, Mansour RT, Serour GA. Prospective randomized study for hydrotubation versus no hydrotubation before intrauterine insemination in unexplained infertility. Reprod Biomed Online. 2010 Jan 11.

Saini P, Kumar A. Pretreatment with hydrotubation in infertility management improves pregnancy rates. Int Congr Ser. 2004 Sep;1271:11-4.

On the contrary, Saini and Kumar (International Congress Series, 2004) demonstrated that hydrotubation is an effective, non-invasive, safe, and cost-effective method of improving pregnancy rates. Furthermore, the investigators recommended the routine use of the tubal flushing-out-patient procedure in all individuals opting for infertility treatment before undergoing IUI or IVF-ET.

Adesiyun AG, Cole B, Ogwuche P. Hydrotubation in the management of female infertility: outcome in low resource settings. East Afr Med J. 2009 Jan;86(1):31-6.

Luttjeboer F, Harada T, Hughes E, Johnson N, Lilford R, Mol BW. Tubal flushing for subfertility. Cochrane Database Syst Rev. 2007 Jul 18;(3):CD003718.

Practice Committee of the American Society for Reproductive Medicine. Effectiveness and treatment for unexplained infertility.

Similar views were echoed in a more recent prospective non-randomized observational study by Adesiyun et al

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Fertil Steril. 2006 Nov;86(5 Suppl 1):S111-4.

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April - June 2010

INFERTILITY Meta-analysis Highlights Negative Effect of Non-cavity-distorting Intramural Fibroids on IVF Outcome Uterine fibroids are the most common benign pelvic tumors in women of reproductive age. Although intramural fibroids distorting the uterine cavity are linked to reduced pregnancy rates, there exists controversy regarding this association when the uterine cavity is not altered by the fibroids. Now, a systematic review and meta-analysis published in the journal, Human Reproduction reports that non-cavity-distorting intramural fibroids may also result in adverse pregnancy outcomes in IVF cycles.

patients may not need pre-ART myomectomy. They also proposed that caution needs to be taken in patients with fibroids >4 cm, suggesting that they may probably need treatment before adopting assisted conception methods. However, the effectiveness of fibroid treatment in these subjects was not clear. Demonstrating similar results, in a more recent retrospective study, Vimercati et al (Reproductive BioMedicine Online, 2007) did not support the adoption on pre-IVF myomectomy in patients with small-tomoderate uterine fibroids, irrespective of their location.

Sesh Kamal Sunkara and colleagues from the Guy’s and St Thomas’ Hospitals NHS Foundation Trust, London, examined various databases such as MEDLINE, Cochrane, EMBASE, and Web of Science, to determine the relationship between non-cavity-distorting intramural fibroids and IVF outcome. Quality assessment was performed using the Newcastle-Ottawa Quality Assessment Scales.

According to a 2007 report prepared for the Agency for Healthcare Research and Quality (AHRQ), the cumulative incidence rates of uterine fibroids by the age of 50 years is around 70% and 80% among white American and African-American women, respectively. The underlying mechanism defining the association between intramural fibroids and adverse IVF outcomes is not clearly understood. A recent report by the Practice Committee of the American Society for Reproductive Medicine in collaboration with the Society of Reproductive Surgeons (Fertility and Sterility, 2008) suggested some of the probable ways through which the impact is exerted on the treatment outcome. • Uterine cavity distortion or enlargement • Cervix displacement, leading to decreased sperm exposure • Proximal fallopian tube obstruction • Altered uterine contractility • Endometrial inflammation, thinning, and atrophy, leading to altered implantation • Impaired endometrial blood flow • Changed tubo-ovarian anatomy

The scientists short-listed 19 observational studies that encompassed 6,087 IVF cycles. The results of the metaanalysis showed that patients with non-cavity-distorting intramural fibroids, as opposed to the group without fibroids, had a substantial reduction in the following outcome measures after IVF treatment: • Live birth rates: Relative risk (RR)=0.79; 95% CI=0.70– 0.88; P<0.0001 • Clinical pregnancy rates: RR=0.85; 95% CI=0.77–0.94; P=0.002 The authors concluded that there is a negative impact on treatment outcome due to intramural fibroids in assisted conception cycles, even when they do not alter the endometrial cavity. Similar findings, with respect to the association of small intramural fibroids (≤5cm) with reduced rates of cumulative pregnancy, ongoing pregnancy, and live birth following 3 IVF/ICSI cycles, were reported in a prospective study by Khalaf et al (Human Reproduction, 2006).

Although some studies have indicated no negative outcomes with intramural fibroids, most of them were retrospective in nature and evaluated the results in only one treatment cycle. The recent systemic review and meta-analysis, providing further credence to the probable correlation of non-cavity-distorting uterine fibroids with poor assisted reproductive outcome, serves as valuable data for counseling fibroid patients opting for IVF prior to treatment initiation.

On the contrary, Oliveira et al (Fertility and Sterility, 2004), in an earlier retrospective matched-control study, demonstrated that women with subserosal and intramural non-cavity distorting uterine fibroids (leiomyomas) of <4 cm in size, had similar IVF-ICSI outcome compared to women without the fibroids. The researchers suggested that such

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INFERTILITY

April - June 2010

References 1.

Sunkara SK, Khairy M, El-Toukhy T, Khalaf Y, Coomarasamy A. The effect of intramural fibroids without uterine cavity involvement

Steril. 2004 Mar;81(3):582-7. 4.

on the outcome of IVF treatment: a systematic review and metaanalysis. Hum Reprod. 2010 Feb;25(2):418-29. 2.

IVF outcomes? Reprod Biomed Online. 2007 Dec;15(6):686-91. 5.

Khalaf Y, Ross C, El-Toukhy T, Hart R, Seed P, Braude P. The effect

Viswanathan M, Hartmann K, McKoy N, et al. Management of uterine fibroids: an update of the evidence. Evid Rep Technol

of small intramural uterine fibroids on the cumulative outcome of assisted conception. Hum Reprod. 2006 Oct;21(10):2640-4.

Vimercati A, Scioscia M, Lorusso F, et al. Do uterine fibroids affect

Assess (Full Rep). 2007 Jul;(154):1-122. 6.

Practice Committee of American Society for Reproductive

Oliveira FG, Abdelmassih VG, Diamond MP, Dozortsev D, Melo

Medicine in collaboration with Society of Reproductive Surgeons.

NR, Abdelmassih R. Impact of subserosal and intramural uterine

Myomas and reproductive function. Fertil Steril. 2008 Nov;90(5

fibroids that do not distort the endometrial cavity on the outcome

Suppl):S125-30.

of in vitro fertilization-intracytoplasmic sperm injection. Fertil

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INFERTILITY

April - June 2010

Seminal Plasma Neopterin Levels May Correlate with Sperm Quality The oxidative stress exerted by seminal leukocytes via reactive oxygen species has been proposed as a significant factor in the etiology of defective sperm function. Previous studies have suggested the potential of activation markers and soluble factors generated by the leukocytes in enhancing the detection and treatment of male infertility caused by subclinical genital tract infections. However, the currently used cytological techniques, which measure only the number of leukocytes within the semen instead of their activity, are found to be less efficacious in assessing the sperm quality. Now, a new study published in the International Journal of Andrology reports seminal plasma neopterin concentration, a protein released by activated macrophages, as a potential marker for sperm quality.

The researchers also noted an increase in the number of macrophages upon analysis of semen samples with anti-sperm antibodies. One of the diverse mechanisms through which these antibodies interfere with the reproductive process includes the induction of leukocyte phagocytosis of antibody-opsonized spermatozoa. Standard procedures for semen analysis include the determination of the following sperm-linked factors: • Sperm count • Acrosomal status • Entire spermatozoal morphology and morphometry • Cell membrane integrity • Integrity of the nuclear chromatin • Motility and vitality • Sperm antibodies

Ozlem Tunc and Kelton Tremellen from the Research Centre for Reproductive Health, University of Adelaide, Australia, evaluated whether neopterin levels could be directly correlated with macrophage activity in the male genital tract. Analysis of diverse inflammatory markers was performed for semen samples obtained from 63 asymptomatic subjects suffering from male factor infertility and 11 fertile controls. The study showed a three-fold increase in the concentration of the protein in infertile men compared to the fertile group. Also, the findings showed a significant correlation between neopterin levels and sperm oxidative stress, DNA fragmentation, and apoptosis.

Determination of seminal factors, including round cells and leukocytes, is also done during standard seminal analysis. Although semen analysis is vital in evaluating the fertility status, studies contradict its efficacy as a sole predictor. Hence, it is mandatory to interpret the results of semen analysis in conjunction with more sophisticated molecular/immunological detection methods. The development of such techniques may also aid in clearly defining the underlying causes for reduced sperm quality and infertility in patients presenting with abnormalities that are not readily identifiable using current techniques. References

An earlier study by Solis et al (Actas Urológicas Españolas, 2003) ascertained the significance of macrophage immunosurveillance in assessing sperm quality. The study results showed a significant positive correlation between the log of phagocytic cells and increase in the number of sperms with DNA denaturation (P<0.05) in contrast to morphologically normal sperms (P=0.414).

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Tremellen K, Tunc O. Macrophage activity in semen is significantly correlated with sperm quality in infertile men. Int J Androl. 2010 Jan 28.

Solis EA, Bouvet BR, Brufman AS, Feldman R, Gatti VN. The possible macrophage role in seminal fluid. Actas Urol Esp. 2003 Mar;27(3):185-9.

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INFERTILITY

April - June 2010

SpermCheck® May Serve as a Reliable Home Test to Predict Male Fertility Potential Recently, scientists from the University of Twente’s MESA+ Institute for Nanotechnology have developed a novel ‘microfluidic fertility chip’ that has the potential to calculate sperm concentration by using electrical impedance measurements. The findings could serve as a vital step in developing an accurate pre-screening compact home test device for ascertaining male fertility status.

A new study reports that SpermCheck® Fertility (ContraVac), a simple, inexpensive, quick, and accurate immunochromatographic diagnostic test may enable men to assess their sperm count in the privacy of their homes. The device, touted to be the first home sperm test device, may aid couples planning for conception to check if the male partner requires subsequent comprehensive fertility status evaluation. The findings have been published in the online issue of the journal, Human Reproduction.

An earlier joint report by the American Urological Association and American Society for Reproductive Medicine (Fertility and Sterility, 2006) recommended that reproductive history and two accurately conducted semen analyses should form the basis for the initial assessment of male factor infertility. Although comprehensive semen analysis in the laboratory is the cornerstone for evaluating infertile men, and aids in detecting the severity of the male factor, certain disadvantages, such as time, privacy and cost, may serve as barriers for the method. This may in turn lead to a delay in the diagnosis and treatment initiation.

Michael A Coppola from the ContraVac, Inc., Charlottesville, Virginia, and coworkers, conducted the study in 225 semen samples to assess the ability of SpermCheck Fertility in determining sperm count. The researchers compared the results of the tests performed by untrained users with those from trained laboratory personnel. The ease-of-use of the device was gauged based on consumer feedback, which included the information obtained from 164 lay users. The findings demonstrated that the two-strip lateral flow immunodiagnostic device classified the samples as normozoospermic (2 x 107 sperm/mL), oligozoospermic (5 x 106 to 2 x 107 sperm/mL) or severely oligozoospermic (<5 x 106 sperm/mL) with an accuracy of >96%. The consumer studies reported that the device was simple and easy to use, with both the lay and laboratory groups independently concurring on the same result around 95% of the time. Furthermore, the correct response rate was found to be >97% on a 20-question survey.

Home-based tests, such as the SpermCheck Fertility, which have several key benefits, hold the potential to serve as effective and convenient initial screening methods of fertility status. Since self-diagnosis may raise ethical concerns, especially if people do not completely understand the implications of the results, home tests should only be considered as primary screening tools. Hence, it is important for patients to consult physicians for ascertaining the overall evaluation of their fertility health. References

SpermCheck Fertility, the product of the University of Virginia’s biotechnology start-up, ContraVac, uses a sperm-specific biomarker, SP-10 protein to categorize sperm count into fertile, subfertile, or infertile range, which is in compliance with World Health Organization standards. By adopting a hemacytometer procedure, it provides accurate results in less than 10 minutes of placing the semen sample in the device. All clinical trials with respect to the safety and efficacy of the product have concluded, and the device is currently pending for FDA approval.

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Coppola MA, Klotz KL, Kim KA, et al. SpermCheck® Fertility, an immunodiagnostic home test that detects normozoospermia and severe oligozoospermia. Hum Reprod. 2010 Feb 5. [Epub ahead of print]

RELIABLE HOME FERTILITY TEST IN SIGHT. Press Release. University of Twente. Last accessed February 17, 2010.

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 Nov;86(5 Suppl 1):S202-9.

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April - June 2010

EMBRYOLOGY Culture Systems Mimicking In Vivo Biomechanical Environment may Enhance Embryonic Developmental Competence oviductal contractions and ciliary currents.

Several factors related to embryo culturing, including the procedure and incubation time for both the gametes and embryos, are reported to significantly affect IVF treatment outcomes. Two independent studies have reported the development of two novel embryo culture systems, namely the tilting embryo culture system (TECS) and the microfluidic/microfunnel system, to mimic the in utero conditions in order to augment the development of the embryo and formation of blastocyst.

Heo et al from the Department of Biomedical Engineering, University of Michigan, Ann Arbor, evaluated mouse embryo models cultured under microdrop-static/ microfunnel-static controls, or microfunnel-dynamic conditions with microfluidics. The study results showed a substantial increase in the percentage of hatching or hatched blastocysts, indicating an augmentation of blastocyst developmental stage under dynamic microfunnel culture conditions. The blastocyst cell numbers obtained in the dynamic microfunnel cultured embryos were comparable to that in in vivo grown blastocysts. Also, the culture system contributed to considerable increase in the rates of implantation and ongoing pregnancy compared to static culture conditions.

TECS, developed by a group of Japanese researchers (Reproductive BioMedicine Online, 2010) applies mechanical stimuli to the developing embryos similar to that experienced in the fallopian tube prior to implantation. Koji Matsuura from the Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan, and colleagues, evaluated the images of embryos grown using the TECS, and reported that the velocities and shear stresses exerted by the system is comparable to that experienced in the oviduct. Additionally, the use of the culture system contributed to significant improvement in the development rates of embryos reaching blastocyst stage and an increase in the blastocyst cell number in mouse embryos (P<0.05).

One of the major challenges confronted by IVF practitioners is the increased incidence of higher order multiple pregnancies. This has compelled physicians to focus more on transferring less number of embryos with increased developmental competence. Recent advances in the knowledge of the physiology and development of early human embryos have resulted in the discovery of novel culture systems having the potential to generate highly competent and viable blastocysts. Further substantiation of the efficacy of the two novel culture systems in simulating the in vivo embryo developmental conditions hold the potential to revolutionize the currently adopted culturing strategies, and enhance the overall treatment outcomes of various ART techniques.

The study findings also demonstrated a statistically trivial increase in the development of thawed human embryos to blastocyst stage after culturing in TECS, when compared to static controls. Also, in low quality embryos and those grown under suboptimal conditions, the rates of blastocyst formation were reported to be substantially enhanced after culturing in TECS (P<0.05).

References The microfluidic/microfunnel system, developed by a US research team (Human Reproduction, 2010) holds the potential to improve the pregnancy outcomes, by minimizing inefficiencies in embryo generation across diverse fields, including ART and biomedical activity. The newly developed dynamic microfunnel embryo culture system simulates the in vivo fluid-mechanical and biochemical conditions that embryos experience from

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Matsuura K, Hayashi N, Kuroda Y, et al. Improved development of mouse and human embryos using a tilting embryo culture system. Reprod Biomed Online. 2010 Jan 19. [Epub ahead of print]

Heo YS, Cabrera LM, Bormann CL, Shah CT, Takayama S, Smith GD. Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates. Hum Reprod. 2010 Mar;25(3):613-22.

IVF NEWS.Direct!

April - June 2010

CRYOPRESERVATION Study Reiterates Efficacy of Long-term Liquid Nitrogen Storage for Preserving Sperm Motility Sperm banking has been recognized as an effective strategy to circumvent iatrogenic infertility and also retain the reproductive potential of adolescent and young adult males undergoing cancer therapy. However, the effect of long-term cryopreservation on the fertilization potential of sperm has been contentious. Now, a study performed by a group of Israeli researchers rules out any negative influence of prolonged storage in liquid nitrogen on the progressive motility concentration (PMC) of sperm. The results are published in the online issue of the journal Human Reproduction. Leah Yogev and colleagues from the Sackler Faculty of Medicine, Tel Aviv Sourasky Medical Center, Israel, investigated the effects of long-term liquid nitrogen cryostorage of sperm samples on PMC. The researchers evaluated 2,525 thawed semen samples obtained from 72 sperm donors for IUI, and noted the PMC prior to and after storing the specimens for a period of 0.5 to 14.4 years. The key findings were as follows: • The mean PMC (±SD) value observed after freezing/ thawing and prior to long-term storage was 10.8 ± 3.3 x 106/mL • The value noted after cryostorage and prior to using the sample for IUI was 12.3 ± 2.9 x 106/mL (P<0.0001) • Analysis of the specimens stored for different time periods showed that the duration of storage do not influence the PMC of the samples significantly (r=–0.03; P=0.08) • Period of storage did not cause substantial variation in the PMC of both raw and washed sperm samples The researchers also speculate that the high PMC values noted after the storage, when compared to the pre-storage values, could be an artifact due to the small amount of pre-storage specimens.

duration demonstrated good post-thaw motility and zona pellucida binding potential. Additionally, evaluation of four out of five specimens showed normal zona-induced acrosome reaction. Although sperm cryopreservation has been widely accepted as a simple and efficient method for preserving fertility of patients undergoing anticancer treatment, the number of patients being referred to a sperm bank by oncologists is comparatively low. A postal survey conducted by Schover et al (Journal of Clinical Oncology, 2002) investigated the oncologist’s attitudes, knowledge, and practice of referring patients for banking sperms prior to cancer treatment. The survey findings revealed that 91% of the physicians support sperm cryopreservation in cancer patients facing the risk of iatrogenic infertility prior to the initiation of the treatment. However, 48% of them agree that they either discuss this option to less than a quarter of eligible candidates ≥14 years of age, or not even mention the strategy to some patients. The recent study, suggesting the recovery of sperm fertilization capacity even after long-term storage, further underscore the importance of following recommendations for sperm banking put forth by the Royal College of Obstetricians and Gynaecologists. • All the male cancer patients confronting the enhanced risk for long-term gonadal toxicity should be referred to sperm banking • Facilities for long-term sperm banking should be fully funded and available universally to all candidates who are at an enhanced risk for future infertility References 1.

Yogev L, Kleiman SE, Shabtai E, et al. Long-term cryostorage of sperm in a human sperm bank does not damage progressive motility concentration. Hum Reprod. 2010 Feb 22. [Epub ahead of print].

Earlier, Clarke et al (Fertility and Sterility, 2006) concluded liquid nitrogen storage as an efficient cryopreservation protocol that preserves the sperms’ fertilization potential for a prolonged period of time. The researchers evaluated the motility and the ability of the sperm to bind with oocyte zona pellucida after cryopreserving the specimens in liquid nitrogen for 28 years. The samples stored for prolonged

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Clarke GN, Liu de Y, Baker HW. Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen. Fertil Steril. 2006 Sep;86(3):721-2. Epub 2006 Jun 30.

Schover LR, Brey K, Lichtin A, Lipshultz LI, Jeha S. Oncologists’ attitudes and practices regarding banking sperm before cancer treatment. J Clin Oncol. 2002 Apr 1;20(7):1890-7.

IVF NEWS.Direct!

April - June 2010

GENETICS Lower CD4+CD25 Regulatory T Cells Linked to Unexplained Recurrent Spontaneous Abortions A recent prospective clinical study reports that a reduction in the proportion of CD4+CD25 (high) regulatory T cells (Treg) and forkhead box P3 (FOXP3) expression in the peripheral blood and decidua could play a role in the pathogenesis of unexplained recurrent spontaneous abortions (URSA). The findings of the study are published in the recent issue of the journal Fertility and Sterility. FOXP3, belonging to the X chromosome-encoded forkhead transcription factor family, is essential for the development, differentiation, and function of Treg cells. Shanshan Mei at the Sun Yat-Sen University, People’s Republic of China, along with co-workers, evaluated the CD4+CD25 (high) T cell proportion and FOXP3 expression in peripheral blood and decidual samples of 125 URSA patients, 28 normal non-pregnant, and 35 normal early pregnant women. The following findings were noted: In the peripheral blood samples: • Considerably higher proportion of CD4+CD25 (high) T cells and expression of FOXP3 in normal early pregnant women, compared to the other two groups • Significantly lower CD4+CD25 (high) T cell proportion in URSA patients (non-pregnant) compared to normal non-pregnant women and URSA patients who experienced early miscarriages

to normal, early pregnant women. Jasper et al (Molecular Human Reproduction, 2006), in an earlier study, noted a two-fold reduction in the expression of FOXP3 mRNA in the endometrial biopsies of infertile women. In a more recent review, Guerin et al (Human Reproduction Update, 2009) investigated the role of Treg cells in testes, ovary, uterus and gestational tissues during pregnancy, and its association with pregnancy pathologies, miscarriage and infertility. The findings of the preclinical studies demonstrated the importance of Treg cells in controlling the maternal immune response towards the conceptus. Additionally, the increase in Treg cell pool, mediated via antigen-specific and non-specific pathways, contributes to the maternal tolerance during the vital peri-implantation phase. Results from clinical studies showed the accumulation of Treg cells in the decidua, and an increase in maternal blood numbers from the early stages of the first trimester. The researchers also observed that conditions such as pre-eclampsia, miscarriage, and infertility, were associated with reduced number of Treg cells.

In the decidual samples: Statistically substantial low proportions of CD4+CD25 (high) T cells and FOXP3 expression in URSA patients who experienced early miscarriages compared to normal early pregnant women

Several studies have shown that undefined immunological obstacles to normal placentation could be responsible for URSA. A 2003 report by the Royal College of Obstetricians and Gynaecologists (RCOG), suggests that abnormal functioning of immune effector cells; defects in cytokines, growth factors, and immunosuppressive factors at the feto-maternal interface could play a role in the etiopathology of implantation failure and early recurrent miscarriage.

Based on the study results, the researchers suggested that CD4+CD25 (high) T cells play a crucial role in sustaining normal pregnancy. The reduced count of these T cells is associated with reduced FOXP3 expression, and could be involved in URSA pathogenesis.

The current findings, signifying the role of CD4+CD25 Treg cells and FOXP3 gene in unexplained recurrent spontaneous abortion, could open up new avenues in the treatment of the URSA and other reproductive pathologies arising from disruption in immune tolerance.

Similar findings were reported in a prospective, casecontrolled study by Yang et al (Fertility and Sterility, 2008). The study found that the proportion of CD4+CD25 (bright) T cells were considerably lower in the decidua and peripheral blood samples of URSA patients compared

References

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The investigation and treatment of couples with recurrent miscarriage. Royal College of Obstetricians and Gynaecologists. Last accessed February 17, 2010.

More references available online at www.ivfnewsdirect.com

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April - June 2010

TECHNOLOGY Zona Pellucida Birefringence Useful to Predict Oocyte Developmental Competence The intrinsic optical property of zona pellucida (ZP), termed as ‘birefringence’, has been widely investigated to predict the developmental potential of a fertilized oocytes, as well as ART treatment outcomes. A recent prospective study published in the journal Fertility and Sterility further substantiates the efficacy of ZP birefringence (ZPB) in predicting the developmental competence of oocytes in the metaphase II stage.

pregnancy during ICSI cycles. The fertilization rate was found to be similar in both high and low ZPB groups when meiotic spindles were visualized. Additionally, the study results showed higher rates of implantation and pregnancy after the exclusive transfer of HB oocytes (P=0.041 and P=0.004, respectively). Also, the rates of miscarriage were significantly higher when embryos derived from low birefringence oocytes were used.

Daniela Paes de Almeida Ferreira Braga from the Assisted Fertilization Center Av. Brigadeiro Luis Antonio, Brazil, and coworkers, analyzed the ZPB of both in vitro and in vivo matured oocytes obtained from 30 ICSI patients after controlled ovarian stimulation. The researchers further correlated the ZPB with the oocyte developmental potential. Using a polarization imaging software module, the researchers categorized the mature and immature oocytes as high birefringence (HB) or low birefringence.

Birefringence imaging of subcellular structures, including ZP and meiotic spindles, using polarized light microscopy, is predicted to circumvent the limitations of conventional light microscopy in investigating the structure and morphology of oocytes. Some of the key benefits of zona imaging are as follows: • The use of polarization microscopy aids in the clear distinction of all the three layers of ZP • The automatic sampling of the obtained values assists in performing objective and user-independent scoring of the assessed oocytes in contrast to imaging of spindles • A high and consistent birefringence shown by the inner ZP layer corresponds to better developmental competence of oocytes

An increased percentage of HB was observed in immature oocytes compared to mature oocytes (40.1 vs. 23.6%). Also, in the immature oocyte group, the HB in prophase I stage oocytes was comparatively higher than those in metaphase I stage (50.7 vs. 25.0%). On comparison of oocytes before and after in vitro maturation, no significant difference was noted in the percentage of HB oocytes in the prophase I and metaphase I stages. Since no substantial correlation was noted between spontaneous in vitro maturation potential and ZPB, the researchers suggested that ZPB may not be indicative of molecular maturity. However, the study findings showed a positive association of ZPB with oocyte fertilization potential (OR=1.78) and embryo quality (OR=2.28), solely for metaphase II oocytes.

Further, substantiating the efficacy of this innovative imaging technique through research could enhance the currently adopted strategies for oocyte selection, as the technique is rapid, non-invasive, and not associated with any adverse effects on the oocytes. References 1.

Braga DP, Figueira RD, Queiroz P, Madaschi C, Iaconelli A Jr, Borges E Jr. Zona pellucida birefringence in in vivo and in vitro matured oocytes. Fertil Steril. 2010 Jan 14. [Epub ahead of print]

In another recent study, Madaschi et al (Reproductive BioMedicine Online, 2009) reported ZP and meiotic spindle imaging as effective strategies for embryo selection, which could substantially improve the rates of implantation and

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Madaschi C, Aoki T, de Almeida Ferreira Braga DP, et al. Zona pellucida birefringence score and meiotic spindle visualization in relation to embryo development and ICSI outcomes. Reprod Biomed Online. 2009 May;18(5):681-6.

IVF NEWS.Direct!

TECHNOLOGY

April - June 2010

Case Series Findings Support PGD Safety in Singleton Pregnancies In recent years, preimplantation genetic diagnosis (PGD) has emerged as a potential alternative to and compliments prenatal genetic diagnosis in preventing the birth of children with genetic/chromosomal abnormalities. Apart from congenital chromosomal and genetic abnormalities, PGD is currently used for the screening of late-onset diseases having a genetic predisposition, as well as preimplantation human leukocyte antigen (HLA) typing for the generation of savior siblings. However, certain researchers speculate that the technique is associated with adverse health effects including increased neurological defects in the offspring; probably due to the biopsy conducted in early stage embryos. Now, a recent prospective study published in the journal Human Reproduction rules out the risk for increased birth defects in singletons born after PGD.

an elevated risk for neurological disorders in offspring conceived following PGD. The study evaluated the preimplantation development efficiency, postnatal growth, and behavioral and physiological activity of embryos that had undergone blastomere biopsy at the four-cell stage. Mice grown from biopsied embryos demonstrated the occurrence of weight gain and memory decline when compared to the non-biopsied controls. Further analysis of the protein expression profiles of adult brains demonstrated altered expression pattern of proteins in the biopsied group, indicating the increased sensitivity of the nervous system to blastomere biopsy procedures. The study warrants further research to investigate the long-term effect of blastomere biopsy on the future development of offspring, and assess the overall safety of the PGD procedure.

Inge Liebaers, Head, Center for Medical Genetics, Brussels University Hospital, Belgium, and coworkers, carried out the prospective cohort study during 1992 to 2005 in order to analyze the effect of PGD on IVF/ICSIconceived children. Data for 581 post-PGD/PGS and 2,889 ICSI children were collected via questionnaires from physicians and parents during the time of conception and at delivery, and was based on the examination of infants at 2 months of age by clinical genetics experts.

In view of the contradictory findings regarding the benefits and the safety of PGD, extensive and comprehensive counseling is mandatory for the couples opting for the procedure. The recent recommendations put forth by the Genetics Committee of the Society of Obstetricians and Gynaecologists of Canada (SOGC) ascertains the need for conducting genetic counseling prior to the procedure; the guidelines directed towards conveying the following to the patients: • Enhanced risk of conceiving a child with genetic abnormality, and the impact of the condition on the future development of the offspring • Need for performing PGD to reduce the risk of having a child with genetic defects • Benefits and limitations of all the currently available options for genetic testing • Chances of false-negative results, and the lack of evidence to substantiate that PGD improves live birth rates.

The researchers did not find any statistically significant difference between post-PGD/PGS and ICSI children, with overall 2.13% and 3.38% (OR=0.62) rates for major malformation noted in the corresponding groups. However, they observed a significantly greater rate of perinatal death among post-PGD/PGS children when compared to the ICSI group (4.64% vs. 1.87%; OR=2.56). Additionally, further stratification based on multiple births showed that the perinatal death rate was more significant in post-PGD/PGS than in ICSI multiple pregnancies (11.73% vs. 2.54%; OR=0.83). The death rates were comparable in PGD/PGS and ICSI singletons (1.03% vs. 1.30%, respectively). Although the study concludes on the safety of blastomere biopsy for PGD, it cautions about an increased risk of perinatal mortality in multiple pregnancies. The researchers mandate long-term follow up to further substantiate the safety of the technique.

References 1.

Liebaers I, Desmyttere S, Verpoest W, et al. Report on a consecutive series of 581 children born after blastomere biopsy for preimplantation genetic diagnosis. Hum Reprod. 2010 Jan;25(1):275-82.

Yu Y, Wu J, Fan Y, et al. Evaluation of blastomere biopsy using a mouse model indicates the potential high risk of neurodegenerative disorders in the offspring. Mol Cell Proteomics. 2009 Jul;8(7):1490-500.

In a recent study conducted in mice models, Yu et al (Molecular & Cellular Proteomics, 2009) suggested

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Wilson RD, Allen V, Audibert F, et al. Preimplantation Genetic Testing. J Obstet Gynaecol Can. 2009;31(8):761–767.

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April - June 2010

NEWS AMH Testing Effective for Individualizing Stimulation Protocols in Oocyte Donors Although the efficacy of anti-Müllerian hormone (AMH) as a marker in IVF cycles has been established in various studies, there is insufficient evidence about its usefulness in oocyte donors. Now, a study has reported that AMH assessment in oocyte donors has the potential to ascertain the gonadotropin sensitivity and reduce/ prevent the occurrence of ovarian hyperstimulation syndrome (OHSS). The findings, indicating the benefits of testing AMH for individualizing stimulation protocols, have been published in the online issue of the journal, Reproductive BioMedicine Online.

the first evidence of AMH use in fertile population, and also its indication for screening oocyte donors. Earlier studies have investigated the role of AMH as a marker of ovarian reserve and dysfunction in ART cycles. In one such review, Visser et al (Reproduction, 2006) suggested various probable uses of AMH measurement: • Initial follicle recruitment and dominant follicle selection • Marker for ovarian aging, responsiveness, and pathophysiology • Diagnosis of polycystic ovary syndrome

Gary S Nakhuda and colleagues from the Columbia University College of Physicians and Surgeons, Center for Women’s Reproductive Care, New York, compared IVF outcomes and analyzed the levels of AMH in 104 anonymous oocyte donors, aged between 21 and 32 years. The scientists correlated AMH concentrations with the following parameters: • Number of retrieved oocytes (P=0.024) • Need for reducing gonadotropin dose to avert ovarian hyperstimulation syndrome (P=0.007) • Peak estradiol levels (P=0.024)

Many potential benefits of AMH assessment have been suggested in ART cycles including the increased understanding of the ovarian pathophysiology and the directing of clinical management of various conditions. However, in a recent review, La Marca et al, on behalf of ESHRE Special Interest Group for Reproductive Endocrinology – AMH Round Table, emphasized that vital questions on the basic AMH physiology and its clinical applications need to be addressed. References

Using receiver operating curve analysis, the researchers identified an AMH threshold that helped to predict the need for lowering gonadotropin dosing with a sensitivity and specificity of 70%. It was also noted that the AMH levels of the donors were not associated with clinical pregnancy rates of the recipients (77% per recipient). The researchers concluded that AMH testing helps to customize the stimulation protocol by assessing the gonadotropin sensitivity.

M. Anti-Müllerian hormone testing is useful for individualization of stimulation protocols in oocyte donors. RBM Online. January 2010;20(1) 42-47. 2.

Riggs R, Bocca S, Yin L, et al. Anti-mullerian hormone serum levels predict response to controlled ovarian hyperstimulation in oocyte donors. Paper presented at: 24th Annual Meeting of the European Society of Human Reproduction and Embryology;July 7-9, 2008;Barcelona, Spain.

Similarly, Riggs et al, in an earlier study, reported the association between AMH and the total number of mature oocytes retrieved and peak levels of estradiol, thereby suggesting the potential of the hormone as a predictor in fertile subjects. The findings that were presented at the 24th Annual Meeting of the ESHRE are touted to provide

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Nakhuda G, Douglas N, Thornton M, Guarnaccia M, Lobo R, Sauer

Visser JA, de Jong FH, Laven JS, Themmen AP. Anti-Müllerian hormone: a new marker for ovarian function. Reproduction. 2006 Jan;131(1):1-9.

La Marca A, Broekmans FJ, Volpe A, Fauser BC, Macklon NS; ESHRE Special Interest Group for Reproductive Endocrinology–AMH Round Table. Anti-Mullerian hormone (AMH): what do we still need to know? Hum Reprod. 2009 Sep;24(9):2264-75

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April - June 2010

Recommendations for Lifestyle Modifications to Improve Fertility Outcomes • Adverse impact on the seminal parameters of male offspring

Lifestyle modification to tackle obesity, tobacco smoking and alcohol consumption, have been proposed as crucial in influencing the outcome of both natural and assisted conceptions. A recent statement released by the European Society of Human Reproduction and Embryology (ESHRE) further substantiates this association.

Alcohol consumption: There is limited data substantiating an association between female alcohol use prior to the IVF cycle and adverse pregnancy outcomes, and male consumption and higher rate of miscarriages and reduced live births. Also, these associations were reported to be dose-dependent and considerably higher when the time of intake was close to the treatment cycles. The study linked prenatal alcohol consumption with enhanced risk for the following adverse effects: • Reduced chances of conception, low pregnancy rates, and increased miscarriages • Physical anomalies to behavioral and cognitive deficits in offspring, which can be included under the broad term, fetal alcohol spectrum disorders (FASD) • Fetal death, fetal growth restriction, and preterm birth

Some of the key statements published in the online issue of the journal Human Reproduction are as follows: Obesity: The reduced ovulation frequency and chances of conception observed in obese women could be attributed to the negative influence of obesity on hormonal and metabolic mechanisms. Such patients undergoing IVF possessed reduced chances of pregnancy (around 30%) as they required increased doses of gonadotropins for ovarian stimulation compared to women with recommended body mass index (BMI). The literature study reported enhanced risks for the following during both natural and assisted conceptions in obese patients:

Considering the negative impact of these lifestyle factors, the ESHRE task force has put forth five key recommendations for the physicians practicing in the ART field. • Fertility treatment should not be provided to women whose alcohol consumption is more than moderate levels, and to those unwilling or unable to reduce their consumption. • Special justification is needed to treat women who are severe or morbidly obese. Weight loss is reported to exert a positive effect on the reproductive potential. However, further data are mandatory to make the IVF treatment conditional to prior lifestyle modifications in obese and smoking women. • If the ART treatment is made conditional upon lifestyle changes, the physicians should assist the patients in achieving the required results. • Before initiating the treatment, physician counseling should be directed towards insisting patients to take serious efforts to reduce weight and stop smoking. However, this is not recommended in the case of women approaching the end of their reproductive years, due to implied time delay in attaining these results. • Further evidence and increased contribution from fertility specialists involved in scientific research are mandatory to substantiate the reproductive effects of alcohol consumption, obesity, smoking, and other lifestyle-related factors.

In pregnant women: • Increased miscarriage rate of around 30% • Pre-eclampsia • Gestational diabetes, with the risk ranging from 2-fold in overweight patients to 8-fold in those suffering from morbid obesity (BMI ≥40) In offspring: • Birth complications and perinatal death • Congenital abnormalities, including 80% increase in neural tube defects and 30% rise in cardiovascular anomalies Smoking: The study identified a strong association between the number of smoking years in a woman’s lifespan and the enhanced risk of conception failure after IVF. The adverse effect of smoking on IVF outcome was quite significant; and comparable to an increase in 10 years of female age. Additionally, the number of IVF cycles needed for successful conception was found to be two times more in women smokers than in nonsmokers. The study also reported an increased risk for the following in offspring born through spontaneous and assisted conception to smoker mothers: • Preterm birth, growth restriction, stillbirth, and early neonatal death • Low birth weight, oral facial clefts, and sudden infant death syndrome

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References available online at www.ivfnewsdirect.com

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April - June 2010

Retrospective Study Characterizes Factors Predictive of Pregnancy after IUI measuring >16 mm on the day of hCG administration, with estradiol level >500 pg/mL. The study findings also showed that the best treatment results can be achieved with the use of a soft catheter for performing IUI.

A considerable variation in pregnancy rates has been observed among patients undergoing intrauterine insemination (IUI) along with controlled ovarian stimulation. Also, several researchers have reported difficulties in evaluating IUI results due to the patient heterogeneity and the diverse stimulation protocols adopted. Now, a recent retrospective study published in the journal Fertility and Sterility defines the characteristics of couples that would determine the higher chances of pregnancy through IUI.

IUI along with controlled ovarian stimulation is the preferred first-line treatment for patients with patent fallopian tubes and diverse causes of infertility. However, the overall treatment success rate is still debated, and is reported to be influenced by several factors. Earlier, Rawal et al (Journal of Obstetrics and Gynecology, 2008) conducted a postal survey to investigate practitioners’ opinions regarding such factors. During the survey, the practitioners, including consultants, infertility nurse specialists, and other clinicians, were requested to fill up a questionnaire comprising of 22 parameters, and to rate each factor on a 1 to 10 scale. The factor that obtained the highest rating during the study was follicular number on the day of hCG administration. This was succeeded by follicular size, total sperm count, and the requirement of a standardized protocol for all the unit staff.

Philippe Merviel, from the Department of Gynecology, Obstetrics and Reproductive Medicine, Amiens University Medical Center, France, and colleagues, evaluated 1,038 IUI cycles conducted in 353 couples, to determine the factors leading to successful pregnancy. Ovarian stimulation was performed by administering daily subcutaneous injection of follicle stimulating hormone (FSH) or human menopausal gonadotropin (hMG). Following the stimulation of ovulation, IUI was performed after 36 hours after obtaining at least one follicle of >16 mm in size, with endometrial thickness of >7 mm. The clinical pregnancy rates were calculated based on the following factors: • Woman’s age • Type of infertility • Characteristics of spermogram • Total motile spermatozoa (TMS) count • Estradiol (E2) level before hCG injection • Number of mature follicles

The wide variations noticed in the IUI treatment approach have been attributed as the probable factor contributing to substantial inconsistencies in pregnancy rates. Hence, future prospective studies should be directed towards arriving at a consensus on the critical factors influencing the success of IUI and the standardization of treatment protocol. References

Based on the study results, the characteristics of the couples that would influence optimal treatment outcomes were described as follows: • Women below 30 years of age with cervical or anovulatory infertility • Men with TMS of ≥5 million

Merviel P, Heraud MH, Grenier N, Lourdel E, Sanguinet P, Copin H. Predictive factors for pregnancy after intrauterine insemination (IUI): An analysis of 1038 cycles and a review of the literature. Fertil Steril. 2010 Jan;93(1):79-88.

Rawal N, Drakeley A, Haddad N. Intrauterine insemination practice in the UK. J Obstet Gynaecol. 2008 Oct;28(7):738-41.

Additionally, the researchers reported that an ideal stimulation cycle may aid in the recruitment of two follicles

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April - June 2010

Magnetic Resonance Spectroscopy as a Non-invasive Tool for Sperm Identification in Non-obstructive Azoospermia Research is ongoing to develop new techniques for the assessment of quantitative and qualitative variations in the seminal plasma constituents in order to better understand the molecular profile changes that contribute to infertility. Several studies have suggested the ability of 1H magnetic resonance spectroscopy (1H-MRS) in detecting and localizing spermatogenesis in testis. A new study, published in the journal, Human Reproduction, has reported a novel application of the non-invasive metabolic scan in locating sperm in men with nonobstructive azoospermia (NOA), using a predictive model based on phosphocholine concentrations in the testes.

SCO and MA; and normal and MA groups. The researchers developed a predictive model for the presence of sperm, based on the PC levels, using 1H-MRS. Previously, many quantitative and qualitative studies have assessed various metabolites in seminal plasma, such as choline, citrate, lactate, and glycerophosphorylcholine, using 1H-MRS spectroscopy. The results have suggested that the analysis of metabolites may aid in differentiating various forms of azoospermia and also indicated that 1H-MRS may serve as a novel strategy for managing male infertility. The diagnosis of NOA is based on histopathological analysis, as clinical and endocrinal evaluation may not precisely differentiate between obstructive and nonobstructive azoospermia. Despite the advancements in the ART field, it is still a challenge to identify NOA men who may have some retrievable sperms. Multiple testicular sperm extraction is generally suggested in men with NOA as it may aid in accurately diagnosing absolute testicular failure and enhance the retrieval of sperm cells. Conventional methods to check for the presence of sperm in testicles, such as testicular biopsy, microdissection, etc., have some disadvantages like being invasive with a diagnostic capacity of around 60-65%. The new application of the non-invasive 1H-MRS in locating sperm in men with NOA, based on a unique metabolic signature has the potential to substitute the currently employed invasive techniques such as testicular biopsy.

The study, led by Paul J Turek, a male reproductive health expert at The Turek Clinic, San Francisco, and colleagues, was conducted on snap frozen testicular tissue samples obtained from 27 infertile men, to detect the metabolic signatures linked to different histological states of spermatogenesis. Based on quantitative high-resolution magic angle spinning spectroscopic analysis, the tissue specimens had three histological patterns: normal spermatogenesis (subjects undergoing vasectomy reversal), Sertoli-cell only (SCO), and early or late maturation arrest (MA). The scientists assessed the concentrations of 19 tissue metabolites from every biopsy specimen. Across the 3 histological groups, the changes in the metabolite levels were assessed using one-way analysis of variance (ANOVA).

References

The study results demonstrated a substantial variation in the concentrations of phosphocholine (PC) and taurine, between the SCO and normal groups. In comparison with SCO tissue, three times greater mean PC levels were found in those with normal testes (5.4Âą1.4 vs. 1.5Âą0.3 mmol/ kg). Also, changes in the PC concentrations were noted in the subsets of MA testes (with or without spermatids/ mature sperm) on subsequent histological stratification. However, the metabolite levels did not differ between

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Aaronson DS, Iman R, Walsh TJ, Kurhanewicz J, Turek PJ. A novel application of 1H magnetic resonance spectroscopy: noninvasive identification of spermatogenesis in men with nonobstructive azoospermia. Hum Reprod. 2010 Feb 2. [Epub ahead of print]

New Study Demonstrates Novel Use of Metabolic Imaging to Locate Sperm in Infertile Men. Press Release. The Turek Clinic. Last accessed February 9, 2010.

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April - June 2010

Researchers Elucidate Role of Diverse Oocyte Proteins Influencing the Oocyte-Embryo Transition Understanding the unique molecular mechanisms underlying preimplantation development, which includes a series of events such as final oocyte maturation, fertilization, and the oocyte to zygote transition, is crucial for improving ART success rates. Maternal transcripts and proteins stored in the oocytes during folliculogenesis have been proposed to play a crucial role in the regulation of embryogenesis. In a recent study published in the journal Reproduction, a group of US researchers have reported that diverse mammalian oocyte stored factors, termed as maternal effect structures (MESs), assist in the oocyte to embryo transition.

localized to oocyte specific subcortical maternal complex. Several studies have already suggested the association between the altered expression of proteins and transcripts coded by maternal genes, and the reduced developmental competence of oocytes. In recent years, a considerable amount of interest is being shown towards the identification of MEGs. Zygote arrest 1 (ZAR1), the first identified and well-characterized gene belonging to this group, is reported to be crucial for the completion of fertilization. In another study, Zuccotti et al (BMC Developmental Biology, 2008) identified octamer-4 (Oct-4) as a key maternal-effect factor that regulates the molecular events involved in the establishment of oocyte developmental competence. The study concluded that better understanding of the molecules and biochemical pathways involved in the early stage development of embryos may aid in preventing preimplantation developmental arrest, one of the major causes for the increased loss of embryos during IVF. Minami and Tsukamoto (Reproductive Medicine and Biology, 2006) also ascertained the need for evaluating oocyte-specific gene regulation to precisely identify the defective regulatory mechanisms associated with various reproductive disorders, including premature ovarian failure and infertility.

Piraye Yurttas, from the Cornell University, Weill Graduate School of Medical Sciences, New York, and colleagues, investigated the role of diverse maternal factors present in oocytes in the regulation of embryogenesis, using proteomics and transcriptional profiling. The study, summarizing the findings of previous studies, reported that the various proteins identified do not belong to the class of ubiquitous housekeeping proteins, but are factors uniquely located in oocytes. Further analysis in mouse models showed that many of these factors are transcription products of maternal effect genes (MEGs). The recent analysis of metaphase II-arrested mouse oocytes demonstrated the presence of a new panel of oocyte proteins. One such protein, the oocyte-restricted peptidylarginine deiminase (PADI6), is found to be significant for the formation of poorly characterized profuse filamentous structures called oocyte cytoplasmic lattices (CPL). CPL is attributed to function as a reserve for soluble tubulin, and in association with PAD16, is reported to play a crucial role in embryonic genome activation and the development of embryos beyond the two-cell stage. Proteomic screening also showed the presence of already identified MEG-derived abundant proteins that are

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References 1.

Yurttas P, Morency E, Coonrod S. Use of proteomics to identify highly abundant maternal factors that drive the egg to embryo transition. Reproduction. 2010 Jan 27. [Epub ahead of print]

Zuccotti M, Merico V, Sacchi L, et al. Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes. BMC Dev Biol. 2008 Oct 6;8:97.

Minami N, Tsukamoto S. Role of oocyte-specific genes in the development of mammalian embryos. Reprod Med Biol. 2006;5(3):175-182.

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April - June 2010

Study Favors Transfer of Cryopreserved-thawed Embryos after Spontaneous Natural Cycles Different regimens are used for priming the uterine environment before frozen-thawed embryo transfer (FET), including allowing spontaneous ovulatory cycles, ovulation induction using drugs, and the use of hormones for artificial preparation of endometrium, and there is no clear evidence regarding the superiority of one protocol over the other. A recent randomized controlled trial has reported that ongoing pregnancy rates are higher when frozen-thawed embryos are transferred after spontaneous, natural ovulatory cycles, compared to human chorionic gonadotropin (hCG)-induced natural cycles. The findings of the study have been published in the recent issue of Fertility and Sterility.

In order to compare the different cycle regimens for FET, Ghobara and Vandekerckhove (Cochrane Database of Systemic Reviews, 2008), in an earlier review, analyzed 7 randomized studies inclusive of 1,120 women. The scientists did not find any variation in the outcomes of natural cycle, artificial cycle, and ovulation induction cycle FET; and hence did not recommend the use of any particular intervention. The success rate of the FET procedure is associated with the precise synchronization of the endometrial maturation with embryo development. Although many trials have suggested that both, natural and artificial FET cycles can achieve the synchronization, besides having similar clinical outcomes, the latter is sometimes favored owing to the flexibility it offers with respect to the embryo transfer timing. In view of the contradictory findings with regard to the efficacy of these protocols, further studies appear to be of paramount importance to arrive at a consensus on the appropriate cycle regimen for optimizing the treatment outcomes of cryopreservedthawed ET cycles.

Human Mousavi Fatemi from the Center for Reproductive Medicine, Dutch-Speaking Free University Brussels, Belgium, and coworkers, enrolled 168 subjects undergoing FET to investigate the difference in the outcomes between spontaneous natural cycles and cycles in which ovulation was regulated by hCG administration. The researchers performed the analysis in 124 patients, of which 61 were in the natural cycle group, having a spontaneous LH-P rise, while the remaining belonged to the hCG group. The cryopreserved embryos were transferred 5 days after • LH surge in the spontaneous LH group • Administration of hCG (5000 IU) and also when the ultrasound assessment revealed an endometrial thickness of ≥7 mm and follicle size of ≥17 mm in the hCG group

References 1.

G, Devroey P. Cryopreserved-thawed human embryo transfer: spontaneous natural cycle is superior to human chorionic gonadotropin-induced natural cycle. Fertil Steril. 2010 Jan 22. [Epub ahead of print] 2.

The results demonstrated ongoing pregnancy rates of 31.1% and 14.3% (95% CI=4.4%-28.8%) in the spontaneous LH and the hCG groups, respectively; indicating a superiority of the former treatment protocol.

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Fatemi HM, Kyrou D, Bourgain C, Van den Abbeel E, Griesinger

Ghobara T, Vandekerckhove P. Cycle regimens for frozenthawed embryo transfer. Cochrane Database Syst Rev. 2008 Jan 23;(1):CD003414.

Clinic Summary Report. Society for Assisted Reproductive Technology. Last accessed February 1, 2010.

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April - June 2010

In Vitro Culture Medium Shown to Influence Birthweight of Singleton Newborns the in vitro culturing of preimplantation embryos to be a potential risk factor for subnormal fetal outcomes such as low or high birth weight. Earlier, a study by Khosla et al (Biology of Reproduction, 2001) demonstrated that the supplementation of fetal calf serum (FCS) into the M16 media for culturing mouse embryos could influence the regulation of imprinted gene expression and fetal development.

Numerous studies have documented that the birth weight of IVF-conceived newborns is lower than those conceived naturally, possibly attributable to factors such as multiple gestations and various causes of female infertility. A new study reports that the type of culture medium used for in vitro culturing of the preimplantation embryos could also influence the birthweight of babies born through IVF. The findings of the study are published in the latest issue of the journal, Human Reproduction.

Embryos require diverse culture conditions for their optimal development in utero as well as in vitro. Uterine and oviductal fluids contain complex undefined constituents, such as bioactive factors. Mimicking these exact conditions in the culture media is a great challenge. Further understanding of the growth and developmental requirements of embryos in vitro, and carrying out research to optimize the embryo culture media could help avoid developmental abnormalities attributed to embryo culture.

John C Dumoulin at the Maastricht University Medical Centre, Netherlands, along with co-workers, conducted the study, which considered 826 first IVF treatment cycles that utilized oocytes and embryos cultured in either of two commercially available sequential media brands (Vitrolife or Cook®). The team compared 110 live born singletons from the Vitrolife group and 78 from the Cook subset. Birthweight±SEM (3453±53 g vs. 3208±61 g; P=0.003) and birthweight after adjustment for gender and gestational age (mean z-score±SEM: 0.13±0.09 vs. -0.31±0.10; P=0.001) were considerably higher in the Vitrolife group. Analysis with multiple linear regression and numerous other variables that could influence birthweight, showed that the type of media used for culturing embryos and oocytes significantly affects birthweight (P=0.01).

References 1.

Dumoulin JC, Land JA, Van Montfoort AP, et al. Effect of in vitro culture of human embryos on birthweight of newborns. Hum Reprod. 2010 Mar;25(3):605-12.

Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of Preimplantation Mouse Embryos Affects Fetal Development and the Expression of Imprinted Genes. Biol Reprod. 2001

Several studies conducted in animal models have shown

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Mar;64(3):918-926.

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April - June 2010

Magnetic-activated Cell Sorting (MACS) May Improve Sperm Fertilization Potential the sperm motion characteristics. • Based on these results, the researchers suggested that apoptotic marker detection and application of MACS for spermatozoa preparation may help in planning the treatment for patients with UI and multiple IUI failure.

Numerous studies have documented apoptotic markers to be one of the major contributors to ART failure. Now, a study reports that the application of magnetic-activated cell sorting (MACS) for sperm preparation could reduce spermatozoa-associated apoptotic marker levels, and enhance acrosome reaction and fertilization potential in unexplained infertility (UI) and multiple intrauterine insemination (IUI) failure patients. The findings of the study are published in the recent issue of the journal, Human Reproduction.

Previously, a study by Said et al (Reproductive Biomedicine Online, 2006) reported that the application of MACS technology for sperm preparation demonstrated a sperm recovery rate of 73.8±12.1%. In another similar study by de Vantéry Arrighi C et al (Reproductive Biology and Endocrinology, 2009), MACS in combination with DGC brought about 70% and 60% mean reduction in EPS and MMP-disrupted spermatozoa, respectively. The researchers also observed a mean increase (50%) in sperm survival at 24 hours.

Tsung-Hsien Lee from Chung Shan Medical University, Taiwan, and co-workers, conducted the study to detect spermatozoa with apoptotic markers in patients with UI and IUI failure, and evaluate the efficacy of MACS in sperm preparation. The study recruited 60 UI couples with two IUI failures. Sperm samples collected from these patients were processed by density gradient centrifugation (DGC) and then separated into two aliquots. One of the aliquots was further subjected to MACS, while the other served as a control.

Conventional sperm preparation techniques such as DGC, select spermatozoa based on sperm DNA integrity and MMP, but do not eliminate apoptotic spermatozoa. MACS acts on the sperm at the molecular level to remove apoptotic spermatozoa, and compliments the routine sperm preparation protocols in enhancing the quality and function of sperms; in turn improving implantation and clinical pregnancy rates. MACS is reported to be a safe, fast, and reliable sperm preparation technique for IVF and IUI; however, additional prospective randomized studies are essential to validate the above findings, particularly in certain subgroups of male infertility.

Apoptotic markers, disrupted mitochondrial membrane potential (MMP), externalization of phosphatidylserine (EPS), and DNA fragmentation were identified by fluorescence-labeled dye and flow cytometry. Computeraided sperm analysis, induced acrosome reaction test (IART), and basic semen analysis were performed to assess the fertilization potential of the prepared spermatozoa samples. The following results were observed during the study: • On subjecting to DGC, the spermatozoa samples demonstrated 28.3% disrupted MMP, 18.6% EPS, and 13.5% DNA fragmentation. • The number of associated-spermatozoa of the apoptotic markers was considerably reduced in the aliquot subjected to DGC+MACS compared to DGC alone (P<0.001). • The IART notably improved after performing DGC+MACS (P<0.001), particularly in couples with normal hemizona assay, which is indicative of high fertilization potential of the prepared spermatozoa. • A slight decrease in sperm motility was noted after MACS; however, there was no impairment in most of

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References 1.

Lee TH, Liu CH, Shih YT, et al. Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility. Hum Reprod. 2010 Apr;25(4):839-46.

Said TM, Agarwal A, Grunewald S, Rasch M, Glander HJ, Paasch U. Evaluation of sperm recovery following annexin V magneticactivated cell sorting separation. Reprod Biomed Online. 2006 Sep;13(3):336-9.

de Vantéry Arrighi C, Lucas H, Chardonnens D, de Agostini A. Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential. Reprod Biol Endocrinol. 2009 Jan 8;7:1.