Volume 18, Issue 2 – Oct 2016 by The Journal of Clinical Embryology™

The Journal of

Clinical Embryology

The Journal of Clinical Embryologyâ&#x201E;˘

Volume 18, Issue 2 â&#x20AC;˘ August 2016

Volume 18, Issue 2

ISSN 1941-1901

A Journal for Clinical and Research Embryologists and Other Human Assisted Reproductive Technology and Medical Professionals w w w.embr yolog i st s .com #

The Journal of Clinical Embryology™

Volume 18, Issue 2

The Journal Of Clinical Embryology™ Editorial Board Editor-in-Chief Jason E. Swain, PhD, HCLD Corporate Laboratory Director CCRM IVF Laboratory Network Lone Tree, CO, USA

Associate Editor Charles L. Bormann, PhD, HCLD IVF Laboratory Director Massachusetts General Hospital Boston, MA, USA Editorial Board

Gloria Calderón, PhD Director & Co-founder Embryotools Barcelona, Spain H. Nadir Ciray, PhD Lecturer, Division of Reproduction and Early Development Leeds Institute of Cardiovascular and Metabolic Medicine Leeds, United Kingdom Dr. Basak Balaban Head of IVF Laboratory VKF American Hospital Istanbul, Turkey Alexander Lagunov, MS Director, IVF Laboratory CCRM Toronto Toronto, ON, Canada T. Arthur Chang, PhD, HCLD Director, IVF Laboratory University of Texas Health Science Center San Antonio, TX, US

Kersi M Avari, PhD Program Director, Embryology Academy for Research & Training Dahisar, Mumbai, India

Matthew VerMilyea, PhD, HCLD Director, Ovation Fertility Austin Embryology and Andrology Laboratories Scientific Director Texas Fertility Centers Austin, TX, USA Dr. Ulrich Schneider, DVM IVF International GmbH Hannoverschestr, Germany Markus Montag, PhD, Dipl Biol Founder, CEO Ilabcomm GmbH Augustin, Germany Daniel Franken, PhD Research Director Affiliated Lecturer Obstetrics and Gynaecology Faculty Health Sciences University of the Free State Republic of South Africa Dr. Alpesh Doshi, M.Sc, Dip RC Path Head of Embryology Centre for Reproductive and Genetic Health (CRGH) London, United Kingdom Professor Joyce Harper Embryology, IVF and Reproductive Genetics Group Institute for Women’s Health University College London 86-96 Chenies Mews London, WC1E 6HX

The Journal of Clinical Embryology™

Volume 18, Issue 2

Table of Contents Changes in the Field of IVF ��1 Jason E. Swain, PhD, HCLD, Editor-in-Chief

The Changing Landscape of ART Clinic Platforms: Where Are We Headed with Private Equity and Consolidations? ��2 Matthew (Tex) VerMilyea, PhD, HCLD/CC; Nate Snyder

Induced Pluripotent Stem Cell (iPSC)-Based Therapies – A New Hope in Fighting Infertility ��5 Yana A. Kisarova, MD, PhD; T. Arthur Chang, PhD, HCLD; Jason E. Swain, PhD, HCLD

Technique to Isolate Individual Cells of the Human Blastocyst and Reconstruct a Virtual Image of their Location �� 10 Tyl H. Taylor, MS; Darren K. Griffin, PhD; Seth L. Katz, MD; Jack L. Crain, MD; Lauren Johnson, MD, MSCE; Susan A. Gitlin, PhD

Supplementation of Embryo Transfer Medium with GM-CSF: A Prospective Randomized Controlled Trial �� 18 Mohamed Fawzy

Inside the IVF Lab (Getting to know your colleagues) �� 23 Why I Work in the IVF Field �� 24 Dara S. Berger, MPH, PhD, HCLD

Upcoming Meetings for the Embryologist �� 26

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THE JOURNAL OF CLINICAL EMBRYOLOGY™ MISSION STATEMENT: The Journal of Clinical Embryology™ is committed to reporting significant, accurate and up-to-date scientific articles and information concerning issues of importance to clinical laboratory embryologists, andrologists and those professionals engaged in the science of human assisted reproductive technology (ART) and infertility medicine.

The Journal of Clinical Embryology™

Volume 18, Issue 2

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Addresses Editor-in-Chief: Jason E. Swain, PhD, HCLD Corporate Laboratory Director CCRM IVF Laboratory Network Lone Tree, CO, USA Published by: Mrs. Akshara Nitinchandra Patel 1401 Eaton Court Danbury, CT 06811, USA akshara85@gmail.com

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The Journal of Clinical Embryology™

Volume 18, Issue 2

Changes in the Field of IVF Jason E. Swain, PhD, HCLD

Editor-in-Chief

his issue of the JC E highlight s several areas that could represent potential paradigm shifts for the field of ART. Research and innovation is a cornerstone to our profession and this is highlighted by the manuscripts within. Drs. Kisarova and Chang succinctly review the current status of iPSC research and the potential to create gametes within the laboratory. With research in both mouse and human creating sperm and eggs, this offers potential for a dramatic change in how some infertile couples may receive treatment to obtain a genetically related child. We also see an enlightening commentary from Dr. Matthew “Tex” Vermilyea about the growing trend of consolidation of IVF centers and formations of “IVF Networks”. While this approach has been present in the field of IVF in other countries, such as Australia, for several years; this is a more recent concept adopted in the United States. This creates an interesting landscape, not only for patients, but also for clinic and lab staff. With companies such as RMA, CCRM, Ovation, Vivere Health, Aspire, IntegraMed and others, this appears to be rapidly progressing shift in the field of assisted reproduction. As discussed in the commentary, different approaches

are utilized by these networks; with some striving for consistency between labs, some looking to employ a uniform practice management/business model, some drawing upon brand/name recognition, while others look to utilize unique physician recruitment paradigms and draw upon experience from other medical fields. One assumes the ultimate goal of these various approaches is to increase access to high quality fertility treatment at a reasonable price to the growing patient population. We are also introduced to two interesting research studies. The first examines a unique method of isolating cells of the blastocyst to create a “map”. This carries implications for detecting mosaicism within the trophectoderm, which could have a significant impact on CCS or PGS 3.0, as highlighted in the last issue of the JCE. What does this mean for accuracy of CCS results? Can laboratory biopsy technique be modified to address this potential issue? Additionally, an interesting study examining the impact of GM-CSF supplementation of embryo transfer medium is presented. Various studies have examined the potential benefit of adding of key compounds in embryo transfer medium (hyaluronan, hCG, etc). Prior studies have demonstrated a potential benefit of GM-CSF when supplemented in embryo culture medium in select patient populations. However, this is the first report of a potential benefit of GM-CSF when included in embryo transfer medium. Finally, we are introduced to Dr. Dara Berger and gain insight into her journey into the profession of IVF. n

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Volume 18, Issue 2

The Changing Landscape of ART Clinic Platforms: Where Are We Headed with Private Equity and Consolidations? Matthew (Tex) Vermilyea, PhD, HCLD/CC1, Nate Snyder2 Laboratory Director, Ovation Feritility 2 CEO, Ovation Fertility Email: tex@ovationfertility.com

segment, it is no wonder that we are experiencing this recent consolidation boom. IVF was established in the late 1970s with the birth of Louise Joy Brown but did not become an established practice until a decade or so later, when pregnancy success rates began to take hold. From there, demand for ART services started to grow, and to meet demand, reproductive endocrinology practices sprouted up across the country. Today approximately 90 percent of practices report to SART, with some metropolitan markets hosting dozens of clinics competing for the patient population. As competition heats up, ART clinics are constantly searching for new ways to gain market share. Some groups differentiate themselves by offering special financing or risk-share programs, whereas others offer low-cost solutions through “IVF lite” services or advanced fertility-preservation techniques. Recently, groups have begun seeking a competitive edge through consolidation, believing they can outpace their stand-alone peers with the backing of a well-heeled national entity.

new epoch of ART service is upon both patients and providers. Although laboratory and/or clinical consolidation is not entirely new to the ART industry, the speed at which it appears to be gaining momentum, both nationally and globally, is certainly unprecedented. Private-equity investors seeking to create centers of excellence by consolidating ART laboratories, reference-testing facilities, egg and sperm-donor services, surgery centers and clinical practices have transformed the field of reproductive medicine and brought about new opportunities and challenges. Partnerships between investors and clinicians can take many forms, but at the core of most is a desire to improve patient outcomes and create a competitive edge over independent, stand-alone practices and labs. Consolidation in the healthcare space is not a novel concept. Companies like DaVita, Surgical Care Affiliates, North American Partners in Anesthesia (NAPA) and US Oncology are just a few of the billion-dollar companies that have revolutionized their respective markets through consolidation. The co-existence of certain market factors tends to kick off a wave of consolidation within a given healthcare segment. For starters, consolidation generally takes place in healthcare segments that have become established, with well-documented, successful outcomes and widespread acceptance within patient communities. Second, healthcare segments ripe for consolidation generally have an overabundance of providers, creating fierce competition for a fixed and limited patient population. Third, it is helpful if capital markets are flush, a condition often associated with favorable interest rates and a strong national economy. If you consider the reproductive-medicine healthcare

Why Consolidators Can Be Successful There are several reasons why consolidation strategies have worked so well in other healthcare specialties. For one, they provide an opportunity to provide more-efficient care than stand-alone providers. Due to their size, consolidators benefit from scale, which enables them to negotiate lower pricing from suppliers and equipment manufacturers. Additionally, consolidated entities can more effectively manage administrative functions—such as billing, accounting, call centers and human resources—across multiple sites of service. These efficiencies give consolidators a competitive edge in the form of lower costs, which they

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Not All Consolidators are Created Equal

can pass on to their patients and/or retain as profits for their shareholders. Consolidators have also been successful in other markets because they have an opportunity to provide more-effective care than stand-alone entities can. Anyone who has spent time in a successful ART lab knows it is neither the facilities nor the equipment that creates successful outcomes, but rather the team of scientists within those facilities—using state-of-the-art equipment—that produces results. A typical stand-alone ART lab employs anywhere from one to six full-time scientists, depending on the productivity of its affiliated practice(s). Therefore the clinical ability of most stand-alone labs depends on the collective knowledge of one to six people working in operational silos. “Two heads are better than one” holds true in ART as it does elsewhere. It stands to reason that the collective knowledge of 60 experienced scientists working through a consolidator stands a better chance of creating more insightful best practices than one to six scientists working in a stand-alone lab. The trick, of course, is to coordinate the efforts of a larger group, as this model poses a risk of becoming disorganized if too many opinions are being considered. To be successful, consolidators need a corporatemanagement team that can effectively harness the collective knowledge of many independent hard-working scientists. A third benefit that consolidators enjoy over stand-alone practices and labs is the ability to control a larger portion of the patient experience. Consolidators are typically supported by professional management teams that, along with their private-equity partners, seek to grow successful companies. One way to enhance a consolidator’s value is to develop in-house service offerings such as egg banking, procedure financing, genetic testing, long-term storage and international marketing. This approach is called vertical integration, and it enables consolidators to streamline treatment processes by eliminating duplicate patient intake and billing forms, which are often required when stand-alone clinics rely on third parties to deliver fertility care. Vertically integrated consolidators also tend to have better control over more of the patient experience. When third-party vendors interact with patients, they may then influence the infertility-care experience. By consolidating and controlling more of these ancillary services, physicians and scientists can help ensure a consistent experience throughout more of their patients’ fertility journeys.

A defining characteristic that differentiates fertility consolidators is focus. Consolidator profiles in the market today include those who seek to acquire the practice and the ART lab, and those who focus on the ART lab, thereby excluding the practice from the equation. In other healthcare segments, consolidators who acquire a physician practice tend to turn the physicians into employees. This approach can be attractive to physicians who wish to offload practice-management responsibility and enjoy a steady paycheck. A potential downside may be the loss of identity and the ability to make decisions about one’s own practice. Consolidators who focus on ART labs tend to leave the practice alone, enabling physicians to retain a sense of independence at the local practice level while enjoying the benefits of consolidation at the lab level. Another differentiator pertains to a consolidator’s clinical management style. Some consolidators take a top-down universal approach to defining best practices. Typically this approach occurs when a single physician or practice within the consolidated entity is significantly more powerful or serves as the face of the company. In these cases, best practices can be established by a subset of the company’s brain trust, thereby failing to capture the collective knowledge of the entire community. A potential benefit of the single-voice strategy is that it creates consistency in the patient experience, enabling every practice and lab to function similarly, regardless of geographic location. Alternatively, consolidators comprised of multiple, balanced practices and labs may be more open to embracing input from all of their constituents. As a result, this meritocratic approach tends to produce multiple ways of achieving similar outcomes, enabling researchers to compare and contrast methodologies and identify the drivers of clinical effectiveness. A third type of consolidator has little to no unified management style, limiting its influence over its network practices and labs to a shared name— for marketing purposes. Additional questions will continue to surface as we ride the wave of consolidation that has taken hold in the industry. Questions you should ask yourself as you consider your group’s position in the market one, five or ten years down the road include the following: • What happens to profit margins when consolidators enter the market with a lower supply cost?

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• What is your group’s approach to developing best practices, and how does it compare to that of a consolidator entering your market?

and laboratories remain unanswered, and many more will arise as the field continues to rapidly change. From a laboratory-personnel perspective, it is necessary to ask in which consolidated structure do laboratory directors, embryologists, andrologists and technicians sense better job security and have more opportunities for professional development. Some larger centers of excellence may become more restrictive about whom they hire, funneling fresh graduates into internships to prove their interest in and reliability in regard to the tasks at hand. Others may invest in highly skilled senior staff who share their knowledge by rotating through network laboratories to confirm the consistency of protocols and techniques. Some personnel may thrive on the open exchange of concepts and viewpoints, whereas others may struggle with such freedom, wanting only to follow official consolidated rules and procedures. Whether employees feel safer under the umbrella of a consolidator or a stand-alone entity, what appears undeniable is that consolidation is underway and that there is an emerging new frontier for fertility-care providers. n

• What is the future of third-party reimbursement in infertility? How will smaller stand-alone clinics effectively negotiate with large third-party payers rather than a national consolidator? • What are the defining characteristics of the major consolidators in the field today? How are they similar? What sets them apart? • Are any consolidators known for a single founding physician or practice? If so, how might that distinction affect your group’s ability to make an impact? • What happens if your group does not team with a consolidator and then one enters your local market?

What Does Consolidation Mean for Laboratories and Personnel? Many questions about consolidations of IVF clinics

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Induced Pluripotent Stem Cell (iPSC)-Based Therapies â&#x20AC;&#x201C; A New Hope in Fighting Infertility Yana A. Kisarova, MD, PhD1, T. Arthur Chang, PhD, HCLD2, Jason E. Swain, PhD, HCLD3,4 Houston IVF, Houston, TX, 77024 University of Texas Health Science Center, San Antonio, TX 78229 3 Colorado Center for Reproductive Medicine, Lone Tree, CO, 80124 4 CCRM IVF Network, Lone Tree, CO 80124 Correspondence to: Yana Kisarova, 929 Gessner Rd., Suite 2300, Houston, TX, 77024 Email: ykisarova@houstonivf.net 1

lthough there are a great variety of cells in the human body, they all can be divided into three major cell types: somatic cells, stem cells and germ cells. Somatic cells are the predominant group that make up organs, transport oxygen and carbon dioxide in the blood and ensure immune defense. In general, somatic cells are highly specialized in their function and have a set of unique structural features making them distinct from one another (for instance, liver cells are different from heart cells). All somatic cells, except some in the immune system, are considered to be terminally differentiated, i.e., have obtained certain structural and functional characteristics to become specialized. Stem cells, unlike somatic cells, do not have distinctive functional specialization and serve as cells-precursors that can differentiate into somatic cells to repair the damaged tissues. Stem cells are thought to have unlimited replicative capacity and the capability of differentiating into various downstream cell lineages (pluripotency), whereas somatic cells become senescent and stop reproducing themselves after a certain number of divisions. The long-standing paradigm that terminally differentiated somatic cells cannot change their specialization has been recently shifted. Current approaches on deriving artificial gametes from somatic cells include three major types of technologies. First, differentiation from embryonic

stem cell (ESC) in combination with somatic cell nuclear transfer (SCNT) [1]. Second, direct transdifferentiation where somatic cells of one type can be directly converted to another type bypassing the intermediate pluripotent stage [2]. Third, differentiation from induced pluripotent cells (iPSC). Among these three technologies, iPSC has been considered a simpler approach, more easily performed in many laboratories without the need for complex and expensive equipment, and with least ethical concerns as no human embryonic cell required. Since 2007, scientists have successfully applied various transcriptional factors (factors that bind to DNA and activate and/or suppress expression of sets of genes) to convert multiple types of somatic cells into iPSC lines [3, 4]. The current iPSC protocols, if given appropriate stimuli, such as growth factors and signaling molecules, are able to differentiate to virtually any somatic cell. Various approaches, including viral vector-based transduction of transcription factors, non-integrating vectors, direct input of small molecules, mRNA, proteins, and the latest report of using defined chemicals, have been shown to be effective for iPSC production [5, 6]. These discoveries have opened new venues for stem cell-based therapies, although designing the signaling cocktails and optimizing the culturing protocols to achieve full identity of transdifferentiated cells to their somatic matches remains an area of extensive investigation.

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Interestingly, many efforts are currently directed at using induced pluripotency and transdifferentiation approaches to derive not only somatic cells, but also germ cells. Germ cells are the cells that give rise to gametes (sperms and oocytes). Their precursors are primordial germ cells; seen as a distinct cell line in early embryos that migrate out and back into the embryo to finally colonize the area where gonads are developing. Notably, both sperm and oocytes have a common precursor cell line. For patients with infertility caused by defective development or absence of gametes, iPSC-derived or somatic cell-derived germ cells could give hope of having genetically-related children. Successful attempts of deriving germ cell-like cells from human iPSCs have already been reported [7]. However, these cells must undergo further differentiation and maturation in order to become fertile gametes, and determining favorable culture and stimulation conditions to achieve their maturity remains a current challenge, especially in oocyte derivation [8]. Thus far, differentiation protocols for male germ cells are more developed than for female cells, likely due to gender differences in gametogenesis. Spermatozoa precursors (spermatogonial stem cells) are arrested in mitosis until puberty and then resume proliferation before producing mature spermatozoa through meiotic division, whereas oocyte precursors (oogonia) proliferate, enter and become arrested in meiotic division in utero as primary oocytes. Meiosis is the process of final division of germ cells which results in haploid number of chromosomes (23) possessed by functional gametes. This type of division is unique for germ cells and its recapitulation in vitro has remained the major obstacle toward generation of mature gametes. The studies aimed to generate functional artificial gametes have been reviewed recently [9, 10] and are summarized in Table 1. They emphasized the importance of somatic cells present in gonads and their embryonic precursors (gonadal ridges) for proper gamete development and survival. Hence, the current approaches to maintain and differentiate iPSC-derived germ cell-like cells include co-culture with components of gonadal tissue or/and implantation into gonads. The latter may also represent a feasible approach to restore gametogenesis in infertile individuals. The differentiation of male germ cells derived from autologous iPSCs and injected into seminiferous

tubules has already been demonstrated in mice and resulted in spermatozoa formation [11]. Furthermore, the cells transplanted into murine testis to mature were later collected from the testicular tissue and produced fertile offspring following use in intracytoplasmic sperm injection (ICSI) [12]. Similarly, fully grown oocytes that produced healthy offspring were generated in mice using iPSCs derived from embryonic fibroblasts [13]. Those iPSCs were first differentiated into primordial cell-like cells, then aggregated with reconstituted ovaries and transplanted into ovarian bursa to mature. Although results of the aforementioned and other studies are promising, there are a number of obstacles which need to be overcome before iPSC-derived gametes can safely be used for clinical applications. Among them are an incomplete understanding of gametogenesis in vivo that limits development of efficient differentiation protocols, low yield of iPSCs-derived germ cells, and genetic and epigenetic instability of iPSCs. While generated and cultured, iPSCs acquire numerical and structural chromosome abnormalities that compromise integrity of both nuclear DNA and mitochondrial DNA (trisomies and small copy number variations are most common to occur) [14]. In addition, mutations were reported to occur in some protein-coding genes associated with cancer. Furthermore, iPSCs have DNA methylation patterns and other epigenetic expressions distinct from those of embryonic stem cells, another type of pluripotent cell which derived from the inner cell mass (ICM) [15]. A presence of differentially methylated DNA regions as well as hypermethylated regions in iPSCs suggests that these cells do not completely mimic embryonic stem cells and may also possess somatic memory, i.e. they do not totally loose epigenetic signature of somatic cells from which they there originated [16]. Considering rapid advances in technology and stem cell research, there is a very good likelihood that all these obstacles mentioned above will be overcome in the future. This will permit gametes to be produced from human autologous somatic cells in vitro, eventually offering the potential for these gametes to be used in fertility clinics to help couples seeking fertility treatment to have geneticallyrelated children. n

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Table 1. Overview of studies demonstrating differentiation of iPSCs into germ cells

Description

Species

References

human

Park et al., 2009 [17]

human

Panula et al., 2011 [18]

human

Medrano et al., 2011 [19]

human

Eguizabal et al., 2011 [20]

human

Easley et al., 2012 [21]

Male germline Skin fibroblasts -> primordial germ cells. Human fetal gonadal stromal cells significantly improved differentiation efficiency. iPSC lines derived from adult and fetal somatic cells -> primordial germ cells and formed post-meiotic haploid cells (spermatids). iPSC lines derived from adult and fetal somatic cells -> primordial germ cells with maturation and progression through meiosis. iPSCs derived from keratinocytes and cord blood cells -> haploid cells. iPSCs derived from foreskin fibroblasts -> haploid spermatids with gene imprints similar to fertile sperm.

Human undifferentiated iPSCs were transplanted into murine seminiferous tubules and differentiated extensively to germ cell- human / mice like cells.

Ramanthal et al., 2014 [22]

iPSCs -> epiblast-like cells -> primordial germ cell-like cells. After transplantation into murine testes, maturation, cell extraction and ICSI, produced fertile offspring.

mice

Hayashi et al., 2011 [12]

iPSC line -> spermatogonial stem cells and then transplanted into testes. They were able to differentiate into male germ cells ranging from spermatogonia to round spermatids.

mice

Zhu et al., 2012 [11]

iPSCs -> male germ cells in vitro, mixed with testicular cells and transplanted into dorsal skin. Dissected xenografts grew into a testis-like tissue and iPSC-derived germ cells settled in at basement membranes of reconstituted seminiferous tubules.

mice

Yang et al., 2012 [23]

iPSCs -> primordial germ cells that were able reconstitute seminiferous tubules ectopically when transplanted together with neonatal testicular cells.

mice

Cai et al., 2013 [24]

mice

Li et al., 2013 [25]

mice

Li et at., 2014 [26]

iPSCs -> male germ cells in vitro. iPSCs derived from adult tail-tip fibroblasts -> primordial germ cell-like cells and then spermatogonial stem cells.

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Table 1. Overview of studies demonstrating differentiation of iPSCs into germ cells (continued) Female germline

iPSC lines derived from adult and fetal somatic cells -> primordial germ cells and formed post-meiotic haploid cells (spermatids). iPSC lines derived from adult and fetal somatic cells -> primordial germ cells with maturation and progression through meiosis. iPSCs derived from keratinocytes and cord blood cells -> haploid cells. Skin-derived stem cells -> oocyte-like cells. They were aggregated with ovarian cells, transplanted under the kidney capsule and produce early oocytes. Embryonic fibroblasts -> primordial cell-like cells, then aggregated with reconstituted ovaries (exhibit meiotic potential). Upon transplantation into ovarian bursa, cells developed into fully grown oocytes that contributed to healthy offspring.

References

human

Panula et al., 2011 [18]

human

Medrano et al., 2011 [19]

human

Eguizabal et al., 2011 [20]

mice

Dyce et al., 2011 [27]

mice

Hayashi et al., 2012,2013 [13, 28]

2015;6:e1906. 11. Zhu Y, Hu HL, Li P, Yang S, Zhang W, Ding H et al. Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study. Asian J Androl 2012;14:574-9. 12. Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011;146:519-32. 13. Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 2012;338:971-5. 14. Martins-Taylor K, Xu RH. Concise review: Genomic stability of human induced pluripotent stem cells. Stem Cells 2012;30:22-7. 15. Doi A, Park IH, Wen B, Murakami P, Aryee MJ, Irizarry R et al. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat Genet 2009;41:1350-3. 16. Ohi Y, Qin H, Hong C, Blouin L, Polo JM, Guo T et al. Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells. Nat Cell Biol 2011;13:541-9. 17. Park TS, Galic Z, Conway AE, Lindgren A, van Handel BJ, Magnusson M et al. Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal cells. Stem Cells 2009;27:783-95. 18. Panula S, Medrano JV, Kee K, Bergstrom R, Nguyen HN, Byers B et al. Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells. Hum Mol Genet 2011;20:752-62.

1. Tachibana M, Amato P, Sparman M, Gutierrez NM, TippnerHedges R, Ma H et al. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 2013;153:1228-38. 2. Cohen DE, Melton D. Turning straw into gold: directing cell fate for regenerative medicine. Nat Rev Genet 2011;12:243-52. 3. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861-72. 4. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917-20. 5. Gonzalez F, Boue S, Izpisua Belmonte JC. Methods for making induced pluripotent stem cells: reprogramming a la carte. Nat Rev Genet 2011;12:231-42. 6. Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A et al. A comparison of non-integrating reprogramming methods. Nat Biotechnol 2015;33:58-63. 7. Chen D, Clark AT. Human germline differentiation charts a new course. EMBO J 2015;34:975-7. 8. Handel MA, Eppig JJ, Schimenti JC. Applying â&#x20AC;&#x153;gold standardsâ&#x20AC;? to in-vitro-derived germ cells. Cell 2014;157:1257-61. 9. Ishii T. Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential. J Clin Med 2014;3:1064-83. 10. Ge W, Chen C, De Felici M, Shen W. In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells. Cell Death Dis

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Volume 18, Issue 2 24. Cai H, Xia X, Wang L, Liu Y, He Z, Guo Q et al. In vitro and in vivo differentiation of induced pluripotent stem cells into male germ cells. Biochem Biophys Res Commun 2013;433:286-91. 25. Li P, Hu H, Yang S, Tian R, Zhang Z, Zhang W et al. Differentiation of induced pluripotent stem cells into male germ cells in vitro through embryoid body formation and retinoic acid or testosterone induction. Biomed Res Int 2013;2013:608728. 26. Li Y, Wang X, Feng X, Liao S, Zhang D, Cui X et al. Generation of male germ cells from mouse induced pluripotent stem cells in vitro. Stem Cell Res 2014;12:517-30. 27. Dyce PW, Shen W, Huynh E, Shao H, Villagomez DA, Kidder GM et al. Analysis of oocyte-like cells differentiated from porcine fetal skin-derived stem cells. Stem Cells Dev 2011;20:809-19. 28. Hayashi K, Saitou M. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nature protocols 2013;8:1513-24.

19. Medrano JV, Ramathal C, Nguyen HN, Simon C, Reijo Pera RA. Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro. Stem Cells 2012;30:44151. 20. Eguizabal C, Montserrat N, Vassena R, Barragan M, Garreta E, Garcia-Quevedo L et al. Complete meiosis from human induced pluripotent stem cells. Stem Cells 2011;29:1186-95. 21. Easley CAt, Phillips BT, McGuire MM, Barringer JM, Valli H, Hermann BP et al. Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells. Cell reports 2012;2:440-6. 22. Ramathal C, Durruthy-Durruthy J, Sukhwani M, Arakaki JE, Turek PJ, Orwig KE et al. Fate of iPSCs derived from azoospermic and fertile men following xenotransplantation to murine seminiferous tubules. Cell reports 2014;7:1284-97. 23. Yang S, Bo J, Hu H, Guo X, Tian R, Sun C et al. Derivation of male germ cells from induced pluripotent stem cells in vitro and in reconstituted seminiferous tubules. Cell Prolif 2012;45:91-100.

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Technique to Isolate Individual Cells of the Human Blastocyst and Reconstruct a Virtual Image of their Location Tyl H. Taylor, MS1,2*,^, Darren K. Griffin, PhD2, Seth L. Katz, MD1,

Jack L. Crain, MD1, Lauren Johnson, MD, MSCE1, Susan A. Gitlin, PhD3 1

Reproductive Endocrinology Associates of Charlotte Charlotte, North Carolina, USA, 28277

University of Kent School of Biosciences Centre for Interdisciplinary Studies of Reproduction Canterbury, CT2 7NJ, UK 2

Eastern Virginia Medical School Jones Institute for Reproductive Medicine Norfolk, Virginia, USA, 23501 3

Email: *tyltaylor@gmail.com ^Author’s current address: Illumina Inc. • 5200 Illumina Way • San Diego, CA 92122

Abstract

were isolated from the first and second blastocyst, respectively. From the first blastocyst, 20 cells were isolated from the trophectoderm, 18 (90.0%) contained DNA while two (10.0%) did not. A total of 6 cells were isolated from the ICM, all six (100%) contained DNA. From the second blastocyst, a total of 20 cells were analyzed from the trophectoderm, nine (45.0%) contained DNA and 11 (55.0%) did not. Of the three cells analyzed from the ICM, two (66.7%) contained and one (33.3%) did not. This novel technique, which disassociates biopsied sections of the blastocysts into individual cells was shown to be feasible. Since the blastocyst was biopsied into quadrants, it was possible to estimate the location of individual cells in regards to the ICM. This provides proof of concept that will allow for a more complete examination of chromosomal mosaicism in the human blastocyst.

method that would allow individual cells from the blastocyst to be isolated and reconstituted in a virtual image of the blastocyst would allow “mapping” of the location of individual cells with respect to the inner cell mass (ICM) or what will become the fetus. Moreover, the chromosomal status of each cell could be determined, rendering the first chromosomal “map” of the human blastocyst and determine the true incidence and mechanisms of chromosomal mosaicism in the human blastocyst. To this end, two poor quality blastocysts had their ICM removed and were further biopsied into quadrants. The biopsied quadrants were placed in Calcium/Magnesium free medium with serum for 20 minutes. A holding pipette was used to aspirate the quadrants thereby separating them into individual cells. The isolated cells were identified and placed into tubes and the presence or absence of DNA was confirmed. A total of 26 and 23 individual cells

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Keywords

into a microcentrifuge tube for molecular analysis (1). The cells within the tube undergo a test to analyze all the chromosomes referred to as comprehensive chromosome screening (CCS). One of the pitfalls of CCS is the phenomenon of mosaicism or the presence of two or more distinct cell lines. Regardless of the number of cells in the tube, the most common method for CCS (array CGH) will only provide one diagnosis. For example, if ≥50% of the cells are abnormal, the CCS result is called abnormal or aneuploid. If ≥50% of the cells are normal, the call will be a normal or euploid result (2).

IVF, mosaicism, PGS, blastocyst, aneuploidy

Introduction Preimplantation genetic screening (PGS) refers to an in-vitro fertilization procedure in which cells from the developing embryo are removed and analyzed for chromosome copy number. During blastocyst biopsy (day 5-6 of human embryological development), approximately 5-10 cells are removed from the outer portion of the embryo referred to as the trophectoderm and placed

1:circles White represent circles represent the trophectoderm and dark circles represent thecell inner cell(ICM). mass (ICM). Figure 1:Figure White the trophectoderm and dark circles represent the inner mass (A) The whole (A)prior The whole blastocyst to biopsy. The created blastocyst a hole created by removed. the laser and the ICM without blastocyst to biopsy. (B) Theprior blastocyst with(B) a hole by with the laser and the ICM (C) Blastocyst the ICMremoved. showing(C) theBlastocyst lines at which further biopsies were (D) further The separated, individual quadrants of the without the ICM showing theperformed. lines at which biopsies were performed. trophectoderm. (D) The separated, individual quadrants of the trophectoderm. A.

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Mosaicism can lead to both false positive and false negative outcomes, discarding potentially viable embryos in the former instance, or transferring embryos that were not correctly diagnosed as euploid in the latter (3,4). The ability to detect mosaicism accurately is determined by the technology used and number of cells analyzed. Even if mosaicism is present, the impact on subsequent development varies depending upon which chromosome is involved, the mechanism of chromosomal separation, and at what stage the chromosomal abnormality presents itself (5). Utilizing the blastocyst biopsy procedure as previously described, the mosaicism rate has been documented to range from 16% to 69% (6, 7). In order to determine the true rate of chromosomal mosaicism, one individual cell (not multiple cells) from the trophectoderm must be placed into each microcentrifuge tube and each tube must then undergo the CCS procedure. Thus, the true rate of chromosomal mosaicism within the human blastocyst is currently unknown because individual cells from the blastocyst have yet to be isolated in a manner that can produce a CCS diagnosis. Next generation sequencing (NGS) for CCS is more sensitive than previous CCS tests and able to detect mosaicism within a biopsied sample. As a result, when 25-49% of the cells are abnormal the NGS would not be diagnosed as “abnormal” but rather would be diagnosed as mosaic, indicating that some normal cells are present with the abnormal cells. The number of cells analyzed can also influence mosaicism rates. For example, if one examines eight cells and one is normal and seven are abnormal, the result would be abnormal. If only three cells are examined and two are normal and one is abnormal, the result would be normal. Therefore, the detection of mosaicism is dependent not only on the technology used but also the number of cells analyzed. The examination of individual cells of the blastocyst will yield the true rate of mosaicism, provide the approximate location of the mosaic cell lines, and offer insights into possible origins and mechanisms of mosaicism, such as non-disjunction, endoreduplication, anaphase lagging, uniparental disomy, and their prevalence during preimplantation development (1, 8, 9). Here we report a novel technique to isolate individual cells of the human blastocyst with the ability to potentially “map” their position. Using this technique, the blastocyst is biopsied into five distinct sections: four sections of the

trophectoderm and the inner cell mass (ICM) which will become the eventual fetus. The trophectoderm sections were recorded with reference to their proximity to the ICM so that a precise reconstruction of the cells within the trophectoderm could be “mapped” (Figure 1).

Methods This study was approved by an institutional review board (WIRB #1138244) as well as The University of Kent Research Ethics Advisory Group. Two blastocysts from a 37 year old patient were donated to research and underwent the following procedure. To ease separation of individual cells, the blastocysts were placed into Calcium/Magnesium free (Ca2+/Mg2+ free) medium (Origio, Trumbull, Connecticut, United States) with 10% serum substitute supplement (SSS; Irvine Scientific, Santa Ana, California, United States) and overlayed by oil (Irvine Scientific, Santa Ana, California, United States) to prevent evaporation. Using an inverted microscope and a micromanipulation setup, the blastocysts were held in place with gentle suction via a holding pipette (G32806, Cook Medical, Limerick, Ireland), positioning the ICM at the 9 o’clock position (Figure 2A). A small diode, non-contact laser (Zilos-TK, Origio, Trumbull, Connecticut, United States) was used to create a hole in the trophectoderm at the 3 o’clock position (Figure 2B). A biopsy pipette (G32799, Cook Medical, Limerick, Ireland) with an inner diameter of 35 µm and an outer diameter of 49 µm, was inserted into the cavity of the blastocyst (the blastocoel) and Ca2+ /Mg2+ free was gently expelled. Using the same pipette, gentle section was applied to the ICM thereby removing it from the surrounding trophectoderm (Figure 2B). Using this technique, Capalbo and colleagues (10) demonstrated minimal contamination of trophectoderm cells being removed along with the ICM cells. While still holding the blastocyst, the ICM was removed from the Ca2+/Mg2+ free drop and placed into another drop of Ca2+/ Mg2+ free with 10% SSS, overlayed with oil. The remaining trophectoderm was biopsied into four individual quadrants. The first biopsy removed a section of the trophectoderm from the upper right position. The second biopsy removed a section of the trophectoderm from the lower right quadrant. The third biopsy removed a section of the upper left quadrant. The fourth and final biopsy isolated the trophectoderm from the lower left quadrant (Figure 1C). After each biopsy, the biopsy needle

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was changed and the trophectoderm piece biopsied was pipetted out of the media drop and into an individual drop of Ca2+/Mg2+ free media + 10% SSS for 20 minutes. This process was repeated after each section so there was no cross contamination or mislabeling during the procedure. After 20 minutes, a holding pipette was used to gently aspirate the sections of the blastocysts (Figure 2F). Doing so allowed the sections of the blastocyst to break apart into smaller pieces. Therefore, multiple, individual cells were obtained from each quadrant (Figure 2G). The cells of the blastocyst were identified under a dissecting scope. Individual cells were rinsed in a wash solution provided by the reference lab to remove any Ca2+/

Mg2+ free that may have remained. The cells were visualized under an inverted microscope, loaded into a microcentrifuge tube and sent to a reference lab (Foundation of Embryonic Competence, Morristown, New Jersey) to detect the presence or absence of DNA.

Results A total of 26 and 23 cells were sent for analysis from the first and second blastocyst, respectively. In the first blastocyst, 22 of 26 (84.6%) tubes contained DNA. Of the 20 cells isolated from the trophectoderm, 16 (80.0%) contained DNA while four (20.0%) did not. A total of 6 cells were isolated from the ICM, all six (100%) contained

Figure 2: (A) The whole blastocyst with the quadrants and inner cell mass (ICM) marked prior to biopsy. (B) Blastocyst undergoing ICM removal, the dotted lines mark the quadrants. (C) The blastocyst during the biopsy of the lower right quadrant. The upper right quadrant has already been biopsied. (D) The blastocyst after the biopsy of ICM and upper and lower right quadrants. (E) A quadrant of the blastocyst prior to separation into single cells. (F) A quadrant being pipetted through the holding pipette. (G) Individual cells of the quadrant.

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Figure 3: Reconstructed trophectoderm and inner cell mass (ICM) of blastocyst number one. Figure 3: Reconstructed trophectoderm and inner cell mass (ICM) of blastocyst number one.

DNA. Figure 3 represents the virtual reconstruction of the first blastocyst. In the second blastocyst, 15 of 23 (65.2%) tubes contained DNA. A total of 20 cells were analyzed from the trophectoderm, 13 (65.0%) contained DNA and 7 (35.0%) did not. Of the three cells analyzed from the ICM, two (66.7%) contained DNA and one (33.3%) did not. Figure 4 represents the virtual reconstruction of the second blastocyst.

arose. For example, a non-disjunction event would lead to a trisomy in one cell with the corresponding reciprocal monosomy in an adjacent cell. An anaphase lagging would lead to the presence of a monosomy chromosome. Finally, an endoreplication event would lead to the presence of a trisomy chromosome. Although multiple studies have examined mosaicism at the blastocyst stage, these studies are confounded by utilizing pieces of the trophectoderm that contain multiple cells (11-13). Examining these large of sections would not allow the chromosome constitution of individual cells within the blastocyst to be determined and therefore might under-report the true incidence of mosaicism. Ozawa and Hansen (14) were able to desegregate bovine blastocysts by exposure to trypsin and pipetting the blastocysts through a small glass pulled pipette. Similarly, we utilized a holding pipette designed for holding the oocyte or

Discussion We herein describe a novel approach that we believe to be the first to isolate individual human blastocyst cells which could be utilized for CCS. Although not performed in this study, this powerful approach can be used to determine the extent of chromosomal mosaicism in the human blastocyst and infer the mechanism through which mosaicism

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Figure 4: Reconstructed trophectoderm and inner cell mass (ICM) of blastocyst number two. Figure 4: Reconstructed trophectoderm and inner cell mass (ICM) of blastocyst number two.

embryo during micromanipulation procedures. This pipette had a very small bore size and assisted in the separation of cells from the trophectoderm. Our technique could also prove valuable for human embryonic stem cells (hESC). Often times these cells are in clumps and clusters and the isolation of single hESC may be desired for hESC culture. Prowse et al. (15) performed a similar process by which clumps of hESC were washed with Ca2+/Mg2+ free phosphate buffer solution. After the wash, Prowse and colleagues (15) added trypsin to help in the dissociation of cells. Similarly, Hasegawa and colleagues (16) also disassociated clumps of hESC into individual cells utilizing trypsin. We did not add trypsin to our cells and it is unknown if this would have aided in our separation. In these studies, trypsin was used on hESC whereas our study dealt with trophectoderm cells and trypsin may not separate trophectoderm cells as easily as hESC cells. We utilized Ca2+/Mg2+ free media because it was readily available and has been used in conjunction with CCS tests and embryo biopsy for years and its influence

on CCS results would be minimal (17). Our study utilized only equipment and supplies found in the typical in-vitro fertilization lab. Finally, this technique does not require any special training or any additional supplies, making our approach more feasible with human blastocysts. The average embryo biopsy contains five cells and from our biopsies (quadrants) an average 3.8 cells were isolated (18). The difference in cell numbers could be attributed to blastocyst quality, as the ones used in our study were poor quality and had fewer cells and those used in the later study were of optimal quality and had more cells. Another problem is the difficulty in the visualization of the cells after isolation. One suggestion could be the addition of a hypotonic solution to the isolated cells, thereby allowing them to swell and become more easily distinguishable under a microscope (19). Another technique referred to as optical tweezing allows for the control of small particles and possibly could be used to isolate individual cells (20, 21). Finally, staining the cells to distinguish trophectoderm

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from inner cell mass cells could aid in visualization by making the cells more distinguishable (14). This technique would require an ultra violet light which was not available in our lab. Moreover, it is possible the UV light would cause damage to the DNA. All of these techniques would require an expensive piece of equipment and/or training, neither of which our technique requires. Previous studies have been able to identify the chromosome status of individual cells within the trophectoderm by spreading the entire trophectoderm on a glass slide (22). This method utilized fluorescence in-situ hybridization (FISH) and did not allow for the virtual reconstruction of the blastocyst. Moreover, FISH is usually limited to 9 or 12 chromosomes and does not allow for the analysis of all 24 chromosomes. Due to cost, only the presence of DNA and not the chromosomal status of the cell(s) were performed in this study. The reference lab merely indicated if DNA was present or not in the PCR tube. It is possible that even though DNA is present a CCS result could not be determined due to possible damage from the procedure (23). Further studies are needed to determine if CCS is possible with this technique. Some tubes did not contain DNA and this is likely due to the fact that individual cells are incredibly small and visualization of cells into the tubes is extremely difficult. The failure of DNA detection may be due to the lack of genetic material and not the detection procedure as the sensitivity of this procedure has been documented to be >97% (24). By isolating individual cells within the blastocyst we could have the capability to accurately determine the true rate of mosaicism and the mechanism by which it arose, but that was not performed here. Moreover, by recording the position of the trophectoderm in relationship to the ICM, it is possible to virtually reconstruct the blastocysts and to properly determine the mechanisms of mosaicism at the blastocyst stage. n

LJ, provided guidance, proofread manuscript, and provided source material for experiments SAG, provided guidance and proofread manuscript

Acknowledgments The authors would like to thank Dr. Nathan Treff of the Foundation for Embryonic Competence for the confirmation of DNA in the tubes. Conflict of Interest The authors declare no competing interests.

References

1. Taylor TH, Patrick JL, Gitlin SA, Crain JL, Wilson JM, Griffin DK. 2014. Blastocyst euploidy and implantation rates in a young (<35 years) and old (≥35 years) presumed fertile and infertile patient population. Fertil Steril 102:1318-23. 2. Harton GL, Munne S, Surrey M, Grifo J, Kaplan B, McCulloh DH, et al. 2013. Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization. Fertil Steril 100:1695-703. 3. Haddad G, He W, Gill J, Witz C, Wang C, Kaskar K, Wang W. 2013. Mosaic pregnancy after transfer of a “euploid” blastocyst screened by DNA microarray. J Ovarian Res. 6:70. 4. Wener MD, Leondires MP, Schoolcraft WB, Miller BT, Copperman AB, Robins ED, Arrendondo F, Hickman TN, Gutmann J, Schillings WJ, Levy B, Taylor D, Treff NR, Scott RT Jr. 2014. Clinically recognizable error rate after the transfer of comprehensive chromosomal screened euploid embryos is low. Fertil Steril. 102:1613-8. 5. Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK. 2014. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Hum Reprod Update. 20;571-81. 6. Northrop LE, Treff NR, Levy B, Scott RT Jr. 2010. SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts. Mol Hum Reprod. 16:590-600. 7. Liu J, Wang W, Sun X, Liu L, Jin H, Li M, Witz C, Williams D, Griffith J, Skorupski J, Haddad G, Gill J. 2012. DNA microarray reveals that high proportions of human blastocyst from women of advanced maternal age are aneuploid and mosaic. Biol Reprod. 87:148. 8. Florentino F, Bono S, Biricik A, Nuccitelli A, Cotroneo E, Cottone G, et al. 2014. Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles. Hum Reprod 29:280213. 9. Greco E, Minasi MG, Fiorentino F. 2015. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. NEJM. 373:2089-90. 10. Capalbo A, Wright G, Elliott T, Ubaldi FM, Rienzi L, Nagy ZP. 2013. Fish reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocyst shows high accuracy of diagnosis and no major impact of mosaicism at the blastocyst stage. Hum Reprod. 28:2298-307. 11. Liu J, Wang W, Sun X, Liu L, Jin H, Li M, Witz C, Williams D, Griffith J, Skorupski J, Haddad G, Gill J. 2012. DNA microarray reveals

Author Contributions THT, performed the experiments and wrote the manuscript DKG, provided guidance and proofread manuscript SLK, provided guidance, proofread manuscript, and provided source material for experiments JLC, provided guidance, proofread manuscript, and provided source material for experiments

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Volume 18, Issue 2 MG, Wells D. 2010. Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril 94:1700-1706. 19. Drey LL, Graber MC, Bieschke J. 2013. Counting unstained, confluent cells by modified bright-field microscopy. BioTechniques 55:28-33. 20. Grier DG. 2003. A revolution in optical manipulation. Nature 424:810-6. 21. Prada I, Amin L, Furlan R, Legname G, Verderio C, Cojoc D. 2016. A new approach to follow a single extracellular vesicle-cell interaction using optical tweezers. BioTechniques 60:35-41. 22. Magli MC, Jones GM, Gras L, Gianaroli, Korman I, Trounson AO. 2000. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocyst in vitro. Hum Reprod 15:1781-86. 23. Bevilacqua C, Makhzami S, Helbling JC, Defrenaix P, Martin P. 2010. Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection. BMC Cell Biol 11:95. 24. Treff NR, Tao X, Ferry KM, Su J, Taylor D, Scott RT. 2012. Development and validation of an accurate quantitative real-time polymerase chain reaction-based assay for human blastocyst comprehensive chromosomal aneuploidy screening. Fertil Steril 97:819824.

that high proportions of human blastocyst from women of advanced maternal age are aneuploid and mosaic. Biol Reprod. 87:148. 12. Fragouli E and Wells D. Aneuploidy in the human blastocyst. 2011. Cytogenetic Genome Res. 133:149-59. 13. Northrop LE, Treff NR, Levy B, Scott RT Jr. 2010. SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts. Mol Hum Reprod. 16:590-600. 14. Ozawa M and Hansen PJ. 2011. A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting. Fert Stert. 95:799-802. 15. Prowse A, Wolvetang E, Gray P. 2009. A rapid, cost-effective method for counting human embryonic stem cells numbers as clumps. BioTechniques 47:599-606. 16. Haseqawa K, Fujika T, Nakamura Y, Nakatsuji N, Suemori H. 2006. A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells 24:2649-60. 17. Orris JJ, Taylor TH, Gilchrist JW, Hallowell SV, Glassner MJ, Wininger JD. 2010. The utility of embryo banking in order to increase the number of embryos available for preimplantation genetic screening in advanced maternal age patients. J Assist Reprod Genet 27:729-33. 18. Schoolcraft WB, Fragouli E, Stevens J, Munne S, Katz-Jaffe

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Supplementation of Embryo Transfer Medium with GM-CSF: A Prospective Randomized Controlled Trial Mohamed Fawzy, PhD

IVF Laboratory Director

IbnSina IVF center, Banon IVF Center and Ajyal IVF Center, Egypt Email: drfawzy001@me.com

Abstract

the human blastocyst-uterine interaction, and a handful of failed pregnancies are due to implantation failure (1). Recently, there has been renewed interest in understanding implantation by using mice models before human. The issue of cytokine role in regulating implantation has received considerable critical attention and painstakingly proved. Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is one of the most frequently stated cytokines that may have a role in the mysterious embryo-Endometrial Cross-Talk (2, 3). Human GM-CSF synthesis is at its highest level during the embryo implantation period (4, 5). The GM-CSF specific receptor GM-Ra is readily identifiable in the pre-implantation embryo and placental trophoblastic cells in both mice and humans, suggesting a throughout critical role during implantation process (6, 7). However, No previous translational study has provided conclusive information on GM-CSF role in implantation. In Assisted reproduction, previous studies have failed to demonstrate a link between embryo transfer medium and clinical outcomes. To date, there has been some evidence that favors hyaluronic acid enriched-transfer-medium (EmbryoGlue®, Vitrolife). What remains unclear is the role of GM-CSF enriched medium in regulating implantation process. The aim of this paper is to highlight the importance of enriching transfer medium with GM-CSF and monitoring the outcomes.

upplementation of embryo transfer medium with various compounds has been explored as a potential means of improving implantation and outcomes following IVF. One of the most studied compounds used for supplementation of transfer medium is hyaluronan. Supplementation of culture medium is also a widely explored area of research when attempting to improve upon success rates. Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a compound that has been shown to help improve outcomes in certain patient populations when included during embryo culture. Interestingly, GM-CSF plays a role in endometrial signalling and could have a potential impact if supplied during embryo transfer. However, no data exists examining the impact of GM-CSF supplementation in conjunction with hyaluronan when utilized during embryo transfer. Our study demonstrates that inclusion of GM-CSF during embryo transfer in a hyaluronan enriched medium improves pregnancy and implantation rate compared to controls. Future research is required to elucidate the mechanism of action and to confirm these findings.

Introduction Implantation process in humans involves close interaction between the embryo and the endometrium. Many restrictions have delayed and even repressed studying

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Material and Method

previous successful attempt). However, participants have not met the inclusion were excluded. 21 patients were not undergone fresh embryo transfer due to hyperstimulation syndrome or poor endometrium, and they were excluded from the results. The group I participants (experimental group) were transferred in the GM-CSF-enriched EG at a concentration of “500 ng/ml GM-CSF”. Conversely, the second group (control group) was transferred in traditional EmbryoGlue (EG).

A prospective randomized controlled trial was adopted to evaluate the effectiveness of GM-CSF enriched transfer medium. The study was conducted at two private centres (IbnSina IVF Centre, Sohag, and Banon IVF Centre, Asyut, Egypt), between June 2014 and June 2015. Prior to commencing the study, ethical clearance was sought from the centres ethical committee. On obtaining written informed consent from the patients, they received a comprehensive explanation of the study purpose. The study was registered at ClinicalTrials.gov (Identifier: NCT02422134). A cohort of 400 women was divided into two groups (200 each) according to a computer randomization method. The subjects were selected on the basis of a degree of homogeneity of their age (18-37 years), response (normal [> seven oocytes], hyper responder), causes of infertility (unexplained, tubal factor, male factor [mild to moderate]). Likewise, normal endometrium (tri-laminar of 8 mm thickness at HCG trigger day), and cycle attempt (first or

Stimulation Protocol A standard mid-luteal pituitary down-regulation protocol was used for all subjects. On day 21 of the preceding cycle, GnRH-a (Decapeptyl® 0.1mg, Ferring, Switzerland) was administrated to all women. On cycle day two, pituitary down-regulation was confirmed, and gonadotropin injection (Gn: rFSH, Puregon, MSD, USA and HMG, Menogon, Ferring, Switzerland) was initiated. When three follicles reached 18 mm (mean diameter), final

Figure 1. Study Design

Eligible (N = 400)

Randomized (N = 400)

Allocated in Group 1 (N = 200)

Allocated in Group 11 (N = 200)

-191 were transferred in EG+GM-CSF

-188 were transferred in EG

OHSS no transfer (N = 5) Poor Endometrium no transfer (N = 4)

OHSS no transfer (N = 7) Poor Endometrium no transfer (N = 5)

191 fresh transfer cycle in EG+GM-CSF

188 fresh transfer cycle in EG

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EG+GM-CSF under oil) were overnight incubated in a triple gas incubator (pH ~ 7.25). GM-CSF supplemented-EG was in-house fabricated to a concentration of 500 ng/ml, using a Yeast-Derived GM-CSF (Sargramostim, Leukine, Sanofi, Aventis, USA). The selected blastocysts were incubated in the transfer medium for 20 min before transfer. Two blastocysts (3AA or better - Gardner classification) were transferred in 30 microliters using Sidney-Transfer-Set (Cook, USA) using no-air-interrupt-technique. The endometrial thickness and pattern were assessed at HCG triggering and revealed similarity in both groups (>8 mm and tri-laminar). ResultsSPSS “Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA, version 16 for Microsoft Windows” was used to analyse all data. Student t-test for independent samples was used of mean ± standard deviation (± SD), or frequencies (number of cases) to analyse patient’s demographic and related features. Chi-square (χ2) test was performed to compare categorical data such as oocyte maturation, fertilization, top quality embryos “day 3 and day 5”, blastocyst formation, transferred cycles (fresh), pregnancy and implantation rates. Figure 1 presents an overview of the study design. A total of four hundred women were recruited and randomly allocated into two groups (200 each). As regard group I, nine patients was undergone no transfer due to ovarian hyperstimulation syndrome (OHSS) and poor Endometrium. Likewise, Twelve underwent no transfer for group II due to the same reasons as group I. This resulted in 379 women underwent fresh embryo transfer. The results of analysis are set out in Table 1. It shows a

maturation was induced by 10,000 IU of hCG (Choriomon, IBSA, Switzerland) 36 hours before oocyte retrieval. Intracytoplasmic sperm injection (ICSI) was adopted for all patients.

Ovum Pickup and Culture Protocol Ultrasound-guided-oocytes-collection was done on “one ml HEPES-Buffered-Medium (Lifeglobal)” using Wallace aspiration needle (Wallace, UK). Follicular fluid (FF) was conducted at 37 degrees Celsius (Cook test tube heater, Cook, USA) all the time. The aspirate was handled with a battery-charged test-tube-warmer (Thermocell, Labotect, Germany).The oocytes were gently removed from the follicular fluid using (SMZ800, Nikon, Japan), and washed twice with Global HEPES, then in bicarbonate-buffered medium (Global Total®, Lifeglobal). Concurrently, they were incubated in a C-Top incubator (Labotect, Germany)(pH = 7.25±0.02). Subsequently, one hour later, the oocytes were denuded using hyaluronidase (Lifeglobal) and a stripper (Vitrolife). Mature oocytes were injected using Nikon TIU (Nikon) and Integra3 micromanipulator (Research Instrument, UK). Eventually, Injected oocytes were cultured in 20 microliters (Global Total) overlaid by 5ml oil (Ovoil, Vitrolife) in a micro-droplet-culture-dish (Vitrolife) throughout the 5-days of culture in the C-Top incubator.

Embryo Transfer 379 subjects were undergone Day-5 fresh-embryotransfer under ultrasound-guidance (group I [191] and group II [188]). The transfer-dishes (0.5 ml EG or Table 1: Demographic Features of The Study Population Group I (GM-CSF+EG)

Group II (EG)

P Value

Age

28.4 ± 4.08

27.02 ± 4.06

BMI

26.5 ± 4.82

26.6 ± 4.71

FSH/HMG Dosage

2996 ± 1343

3008 ± 1352

Stimulation Days/ Cycle

12.4 ± 2.44

12.2 ± 2.63

Oocytes Collected/ Cycle

12.6 ± 5.9

12.4 ± 6.2

1.456 ± 0.534

1.457 ± 0.533

# Blastocysts Transferred /cycle

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similarity concerning patient’s demographic features and parameters of controlled ovarian stimulation as regard ages, BMI, amount of FSH/HMG used in both groups. Likewise, days of stimulation, oocyte collected and the blastocysts transferred average showed the same similarity. Table 2 illustrate no statistically significant difference in regard to fertilization, top quality embryos (day 3 and 5), blastocyst formation, and transferred cycle rates. There was a significant positive correlation between GM-CSF-Supplemented-EG (group I) with pregnancy and implantation rates (P value = 0.0321 and 0.0214 respectively). The pregnancy rate represents the number of cycles with positive HCG divided by the number of cycles transferred. The implantation rate was calculated by dividing the number of gestational sacs on ultrasonography by the number of embryos transferred. DiscussionIn Vitro Fertilization (IVF) gains a broad background for treating many cases of infertile couples and despite its dynamic advances, the pregnancy rate remains not optimal. Prior studies have noted that these suboptimal results may be due to nonphysiological in vitro culture. Implantation is considered one of the most crucial points in the overall cycle outcome. There is a limited time

frame for the implantation window in which a competent blastocyst is superimposed on a receptive endometrium. Merely, incoordination leads to failure or defective implantation. In humans, 75% of non-pregnancies are due to implantation failure (2, 3). Likewise, the receptivity window is also transient in humans. In reviewing the literature, respectable trials of understanding the signalling network for receptive and implantation-ready endometrium have been found (8). Of note, a close relationship between molecular expression, their potential functions and uterine receptivity and implantation has been reported in the literature. Additionally, One interesting finding is GM-CSF expression during blastocyst and luminal epithelium interaction (9). Very little information is found in the literature on the clinical solutions of this puzzling implantation circumstances. This study set out with the aim of assessing the importance of embryo transfer medium supplementation with GM-CSF determining its potential effect on pregnancy and implantation basing on Prior studies that have noted the importance of GM-CSF variety regarding pregnancy physiology, including implantation and fetal-placental

Table 2: Embryological and Clinical Outcomes of The Study Population Group I

(GM-CSF+EG

Group II (EG)

P Value

Metaphase II Rate

85.3%

86.4%

Fertilization Rate

72.4%

71.8%

Top Quality Day 3 Rate

78.4%

76.6%

Blastocyst Rate at Day 5

58.6%

59.2%

Top Quality Blastocyst Rate (3BB or better)

42.4%

43.7%

Transferred Cycles

96% (191/200)

94% (188/200)

Pregnancy Rate

69% (131/191)

58% (109/188)

0.0321

Implantation Rate

56% (152/273)

46% (128/279)

0.0213

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Acknowledgments

growth and differentiation (8). It is recognized that knockoutÂ mice GM-CSF genes have precise effects on early embryonic development and maintenance of gestation. Besides, GM-CSF expression occurs primarily by uterine LE and GE in humans during implantation (9, 10). Moreover, in humans and mice, GM-Ra (receptor) expression is readily detectable at the time of fertilization and expressed in blastocyst trophectoderm and inner cell mass (7, 11-13). In the present study, we show that supplementing the EG with 500 ng/ml GM-CSF may be considered as a promising new modality that may improve IVF outcomes. Likewise, one interesting finding is the significant improvement in pregnancy rate that has been shown in the experimental arm of the study. Moreover, perhaps the most clinically relevant finding is the marked improvement in the implantation rate. These results agree with the findings of other studies, in which GM-CSF is a cytokine that playing a crucial role in regulating implantation in human. However, this result has not previously been described. These results are likely to be related to a relatively high concentration of GM-CSF (500 ng/ml) compared to the dose that has been used for embryo culture (2 ng/ml). Hence, it could conceivably be hypothesized that cytokines may play a pivotal role in improving assisted reproduction techniques (ART). Despite these promising results, further studies are required to explore the mechanisms behind GM-CSF role in implantation. There are still many unanswered questions about the role of cytokines in improving ART outcome. However, more research on this topic needs to be undertaken before giving a solid conclusion. In conclusion, supplementing the embryo transfer medium with GM-CSF has correlated with a significant improvement in pregnancy and implantation rates. Taken together, these findings suggest a role for GM-CSF in improving ART outcomes. A further study could assess the long-term effects of GM-CSF on the resulting offspring. More broadly, research is also needed to determine the other cytokines effect on ART outcomes.

The author thanks his team that work in the IVF laboratories; A. Alaboudy, M. Gad, H. Morsy, M. Alaa, M. Emad, A. Ahmed and A. Khalaf. Their dedication and meticulous work are always making the deference.

References

1. Cha J, Sun X, Dey SK. Mechanisms of implantation: strategies for successful pregnancy. Nat Med 2012;18:1754-67. 2. Norwitz ER, Schust DJ, Fisher SJ. Implantation and the survival of early pregnancy. N Engl J Med 2001;345:1400-8. 3. Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 1999;340:1796-9. 4. Giacomini G, Tabibzadeh SS, Satyaswaroop PG, Bonsi L, Vitale L, Bagnara GP et al. Epithelial cells are the major source of biologically active granulocyte macrophage colony-stimulating factor in human endometrium. Hum Reprod 1995;10:3259-63. 5. Zhao Y, Chegini N. The expression of granulocyte macrophagecolony stimulating factor (GM-CSF) and receptors in human endometrium. Am J Reprod Immunol 1999;42:303-11. 6. Robertson SA, Sjoblom C, Jasper MJ, Norman RJ, Seamark RF. Granulocyte-macrophage colony-stimulating factor promotes glucose transport and blastomere viability in murine preimplantation embryos. Biol Reprod 2001;64:1206-15. 7. Sjoblom C, Wikland M, Robertson SA. Granulocyte-macrophage colony-stimulating factor (GM-CSF) acts independently of the beta common subunit of the GM-CSF receptor to prevent inner cell mass apoptosis in human embryos. Biol Reprod 2002;67:1817-23. 8. Robertson SA, Seamark RF, Guilbert LJ, Wegmann TG. The role of cytokines in gestation. Crit Rev Immunol 1994;14:239-92. 9. Robertson SA. GM-CSF regulation of embryo development and pregnancy. Cytokine Growth Factor Rev 2007;18:287-98. 10. Rapoport AP, Abboud CN, DiPersio JF. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF): receptor biology, signal transduction, and neutrophil activation. Blood Rev 1992;6:43-57. 11. Baldwin GC, Golde DW, Widhopf GF, Economou J, Gasson JC. Identification and characterization of a low-affinity granulocytemacrophage colony-stimulating factor receptor on primary and cultured human melanoma cells. Blood 1991;78:609-15. 12. Hayashida K, Kitamura T, Gorman DM, Arai K, Yokota T, Miyajima A. Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad Sci U S A 1990;87:9655-9. 13. Martinez-Moczygemba M, Huston DP. Biology of common beta receptor-signaling cytokines: IL-3, IL-5, and GM-CSF. J Allergy Clin Immunol 2003;112:653-65; quiz 66.

The Journal of Clinical Embryology™

Volume 18, Issue 2

Inside the IVF Lab

(Getting to know your colleagues)

n this issue of the JCE, we are introduced to Dara Berger, MPH, PhD, HCLD. Dr. Berger was introduced to the field of ART in a “less traditional” method than the usual Animal Science major or other similar background training programs common amongst embryologists. This unique career path has served Dara well, as she now directs two academic IVF programs and has the opportunity to be

involved in cutting edge research programs and involvement with training REI fellows and others; educating them in the nuances of clinical embryology. If you would like to be featured in a future issue of the JCE with fellow embryologists, please submit a professional photo and brief paragraph describing yourself to jceembryology@gmail.com. n

The Journal of Clinical Embryology™

Volume 18, Issue 2

Why I Work in the IVF Field Dara S. Berger, MPH, PhD, HCLD Penn Fertility Care Philadelphia, PA, USA

Email: Dara.Berger@uphs.upenn.edu

am originally from northern NJ, however, since high school I have moved around for my education and career. Currently, I live in Philadelphia, PA and work as the onsite laboratory director at Penn Fertility Care at the University of Pennsylvania. I also regularly travel to Raleigh, NC to work as the offsite laboratory director at UNC Fertility at the University of North Carolina. Especially for those who may be interested in a career in assisted reproduction, this may lead one may ask “How did you get here?”. I would like to say it was all strategically planned, but it wasn’t. That is surely a surprise to those who know me, because I am a planner. My first introduction to laboratory science and biomedical research was as an undergraduate student performing research at Case Western Reserve University in Cleveland, OH, where I investigated interleukin-6 production in a mouse model of ischemia/reperfusion injury. For me, the best part of this experience was the laboratory. While I wasn’t the biggest fan of the mice, I did appreciate the science of learning new procedures and conducting experiments. After 4 years of a well-rounded education, I found myself wanting more technical training in order to find better career opportunities. At the time of graduation I was not yet prepared for a first job, so I enrolled at the University of Illinois in the master’s of public health (MPH) program. As an MPH graduate student, I worked in a genetics laboratory studying genetic markers associated with gambling and addiction. I processed samples and ran gels, and similar to my undergraduate research, found I liked the bench work. As part of the MPH core curriculum, I was pushed into a class called “Law and Public Health.” This is when I first saw a picture of a human embryo. In an

invited lecture on stem cells, the visiting professor showed slides of blastocysts and liquid nitrogen dewars filled with embryos. While I was less interested in the legal focus of the talk, I was drawn to the laboratory science aspect. We discussed the laws prohibiting stem cell research and the numerous clinically-derived embryos that were donated, but legally unable to be used for research. This was in 2001, around the time President George W. Bush blocked NIH funding for research on embryonic stem cell lines derived after August 9, 2001. The MPH curriculum required a practicum, which I fulfilled through an internship at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. While at the CDC, my project focused on Cystic Fibrosis testing. I was at the intersection of the fields of public health and human genetics, and I appreciated the direct link between DNA and disease. As a result, I enrolled at the University of Pittsburgh to pursue a PhD in human genetics. My research focused on skewed X-inactivation and the sequencing of two gene fragments associated with recurrent miscarriage. I also investigated the immunological etiology of recurrent spontaneous abortion. After my PhD I began a postdoctoral fellowship at the Cleveland Clinic. My principal investigator’s focus was the role of RNA splicing in muscle development. Given my previous research experience with DNA mutations and disease I was eager to apply my background in genetics to RNA transcript modification and function research. My project used a mouse model to relate changes in RNA splicing with muscle development abnormalities. Specifically, Myotonic dystrophy (DM) is a multi-system disease, including not only myotonia but also infertility.

The Journal of Clinical Embryology™

Volume 18, Issue 2

While some symptoms were already known to result from alternative splicing, I used DM as a model to investigate the role of RNA splicing in infertility. While I still didn’t like working with mice, I did learn that I could bribe my coworkers with Starbucks coffee to help with the animal care aspect, and the project allowed me to weave together my interests in genetics and infertility. As my postdoctoral research progressed, I found myself unable to shake the last residue of those core curriculum classes. I realized that instead of bench science and pure research responsibilities, I was interested in a direct clinical perspective of science. And I desired a career that would combine the fields of public health, genetics and reproduction. Being a clinical embryologist seemed to offer all of those elements. And as an added bonus, I didn’t have to work with mice. So, while conducting my postdoctoral research, I enrolled in the Eastern Virginia Medical School (EVMS) master’s of science program in embryology and andrology. My EVMS graduation coincided with the end of my postdoctoral fellowship. With my commitment to research, I focused my job search on academic programs where I could both learn embryology as well as continue to participate in research projects. In 2009 I started in an embryology position at Montefiore Medical Center’s Institute for Reproductive Medicine and Health (IRMH), an affiliate of Albert Einstein College of Medicine. My first embryology job proved to be the perfect interface between genetics, reproductive health and clinical laboratory science that I wanted. I had found the field I was looking for. I was fully trained as an embryologist and supervisor at IRMH. With exposure to their academic atmosphere, skilled staff, and supportive

mentors, I received a fantastic embryology education. In addition, IRMH takes two fellows a year and all fellows have research projects. I joined a granulosa cell project that relied on academic embryology and faculty support. I enjoyed being a part of the research and also a part of fellowship education. After five years at IRMH, I obtained my high complexity laboratory director (HCLD) certification. At that juncture, I felt prepared to take the next step to become an independent IVF laboratory director. Because my experience with the fellowship program was so positive, I continued to focus my search on academic IVF programs. I was fortunate enough to find a position in an affiliate of the University of North Carolina at UNC Fertility, a unique program that is also an Integramed practice. UNC takes one fellow a year and is also very focused on education and research. It was a perfect fit for my career goals and I enjoyed collaborating with the fellows as much as I had hoped. In 2015 I was able to join another academic IVF program at Penn Fertility Care, with the ability to help train additional fellows and gain a stronger connection to scientific research. From my journey into the field of IVF, I have concluded that to become an IVF laboratory director, a PhD with a focus in genetics is ideal groundwork and postdoctoral experience adds further value. I have also discovered many remarkable people in this field. When dealing with sex and gametes, I guess that an accumulation of smart and witty people isn’t too surprising. I have been fortunate to share good times and interesting stories with my colleagues over the past several years…and look forward to many more. I am still impressed everyday by the process of what we do in the IVF laboratory. n

The Journal of Clinical Embryology™

Volume 18, Issue 2

Upcoming Meetings for the Embryologist Name of Event

Dates

Location

FSA 2016 Fertility Society of Australia

4th - 7th September, 2016

Crown Burswood, Perth, Western Australia

22nd IFFS World Congress on Fertility

21st - 25th September, 2016

Delhi, India

CFAS Canadian Fertility and Andrology Society

22nd - 24th September, 2016

Toronto, ON, Canada

New York Metropolitan Embryology Society

27th September, 2016

New York, NY, USA

ASRM American Society for Reproductive Medicine

15th - 19th October, 2016

Salt Lake City, UT, USA

The 61st Annual Meeting of the Japan Society for Reproductive Medicine

3rd - 4th November, 2016

Yokohama-shi, Kanagawa Prefecture, Japan

Translational Reproductive Biology and Clinical Reproductive Endocrinology

17th - 20th November, 2016

New York, NY, USA

Fertility 2017 (Joint Conference of the UK Fertility Societies: the Association of Clinical Embryologists, British Fertility Society and the Society for Reproduction & Fertility)

5th - 7th January, 2017

Edinburgh, UK

Best Practices of ASRM and ESHRE

23rd - 25th February, 2017

Paris, France

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