Purpose To utilize a novel mitochondrial function assay with pooled granulosa cells to determine whether mitochondrial function would differ by patient demographics and embryo development. Methods This was a prospective pilot study in a hospital-based assisted reproductive program and public university. Mitochondrial metabolic substrate utilization was assessed in pooled granulosa cells from 40 women undergoing in vitro fertilization during 2018 and 2019. Results Assessment of mitochondrial substrate metabolism in pooled granulosa cells revealed higher citric acid, L-malic acid, and octanoyl-L-carnitine utilization with higher body mass index (BMI). Utilization of citric acid, cis-aconitic acid, D-alpha-keto-glutaric acid, L-glutamine, and alanine plus glycine was significantly lower as total dosage of FSH administered increased. Utilization of glycogen was significantly higher in patients with a higher percentage of fertilized oocytes. D-alpha-keto-glutaric acid utilization was significantly lower in patients with a higher percentage of good 8-cell embryos. L-glutamine utilization was significantly lower, with a higher percentage of blastocyst formation. Mitochondrial metabolic scores (MMS), which reflect overall mitochondrial activity of the granulosa pool, were significantly higher in patients with higher BMI and with greater numbers of mature oocytes retrieved. MMS in granulosa decreased as total FSH dose administered increased. Conclusions Granulosa cell utilization of substrates feeding into the citric acid cycle changed with total FSH dosage and BMI. Fertilization rate, 8-cell embryo quality, and blastocyst formation also associated with different energy substrate usage. Mitochondrial substrate utilization by granulosa cells from individual follicles could be further developed into a useful diagnostic tool.

Cryopreservation and associated cryostorage has become a well-established technique in both basic and clinical science. When the potentially lethal consequences of freezing itself are ameliorated, existence at cryogenic temperatures seems to be a form of true viable stasis that can persist for long periods of time. Natural cryopreservation and revival after long-term periods in cryostasis is reality in many species. While some evidence exists for imperfections in artificial cryopreservation protocols and storage, these protocols are for the most part successful and compatible with efficient restoration of vitality in a variety of biomaterial after freezing. Clinical protocols in use for cryopreserving and storing gametes and embryos in human-assisted reproduction are similarly well proven and supported by a large body of basic science and clinical outcome data.

Objective To evaluate if embryo mosaicism is linked to poorer clinic pregnancy rates.Design Retrospective case-control study between January 2013 and January 2017.Materials and Methods Complete Chromosomal Screening (CCS) was performed on embryos of couples attending our fertility clinic between January 2013 and January 2017.In the first phase, array-CGH (aCGH) analyses were performed using the Agilent SurePrint platform G3 8 × 60 K CGH.Cells from the trophectoderm of the embryos were biopsied at the blastocyst stage (D + 5 or D + 6).We analyzed 1923 blastocysts from 704 cycles of IVF.An embryo was considered mosaic when the percentage of mosaicism was >25% and <80%.Embryos >80% were classified as aneuploid.Subsequently, embryos, in single embryo transfers previously diagnosed by array-CGH as euploid, were reanalyzed by NGS (n = 102).The chromosomal analysis was performed using the Veri-Seq Illumina kit and bioinformatic analysis was performed with the BlueFuse Multi software program (Illumina).Here an

Micromanipulation describes a set of tools and techniques for cellular microsurgery and manipulation. Micromanipulation techniques have played an important role in basic research and the development of clinical techniques in assisted reproductive technology. This work provides a review of the development and current practices involving micromanipulation in the human clinical assisted reproduction laboratory.

Before there was public debate on mitochondrial replacement therapy (MRT) to treat mothers at risk of transmitting mitochondrial disease (Hyslop et al, 2016Hyslop L.A. Blakeley P. Craven L. Richardson J. Fogarty N.M. Fragouli E. Lamb M. Wamaitha S.E. Prathalingam N. Zhang Q. O'Keefe H. Takeda Y. Arizzi L. Alfarawati S. Tuppen H.A. Irving L. Kalleas D. Choudhary M. Wells D. Murdoch A.P. Turnbull D.M. Niakan K.K. Herbert M. Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.Nature. 2016; 534: 383-386https://doi.org/10.1038/nature18303Crossref PubMed Scopus (203) Google Scholar), and before there was a publicly traded company called OvaScience aiming to use nuclear transplantation and stem-cell technologies to treat infertility (Woods, Tilly, 2015Woods D.C. Tilly J.L. Autologous Germline Mitochondrial Energy Transfer (AUGMENT) in human assisted reproduction.Semin. Reprod. Med. 2015; 33: 410-421Crossref PubMed Scopus (74) Google Scholar), there was a solitary case report from China about the use of nuclear transplantation with donor oocyte-derived cytoplasm at the zygote stage to overcome cleavage arrest in a patient's embryos (Zhang et al, 2003Zhang J. Zhuang G. Zeng Y. Acosta C. Shu Y. Grifo J. Pregnancy derived from human pronuclear transfer.Fertil. Steril. 2003; 80: O-148Google Scholar). The landmark abstract, presented to the 2003 annual meeting of the American Society for Reproductive Medicine (ASRM), was received with excitement and concern, sentiments that promptly seeped into the lay press. This was not surprising considering the topic and the risk-sensitive audience of reproductive scientists listening to the presentation given by the lead author, Dr John Zhang. The case report described an IVF patient with normal fertilization whose zygotes divided only once. Early development arrest affected all the embryos of this patient in two consecutive cycles. Approximately 10% of cleaving embryos arrest in culture, but the repeated arrest of an entire cohort is indeed rare, and, based on experimental evidence, could be attributable to ooplasmic deficits. Ooplasmic developmental determinants are far from understood, but one possibility is the deregulation of proteins like the stress sensor p66Shc, which may affect signaling pathways for mitochondrial function (Betts, Madan, 2008Betts D.H. Madan P. Permanent embryo arrest: molecular and cellular concepts.Mol. Hum. Reprod. 2008; 14: 445-453https://doi.org/10.1093/molehr/gan035Crossref PubMed Scopus (108) Google Scholar). Complete developmental arrest represents an extreme manifestation of ooplasm-associated deficits that can occur during human assisted reproduction treatment cycles. The idea that ooplasmic deficits could be overcome by cytoplasmic replacement or augmentation using donor oocytes or embryos is based on work performed nearly three decades ago by Pratt, Muggleton-Harris, 1988Pratt H.P.M. Muggleton-Harris A.L. Cycling cytoplasmic factors that promote mitosis in the cultured 2-cell mouse embryo.Development. 1988; 104: 115-120PubMed Google Scholar in the mouse. These authors injected 8 pl of cytoplasm extracted from normally dividing 2-cell mouse embryos into single blastomeres of arrested 2-cell recipient embryos. Using various combinations of blocking and non-blocking mouse strains, 2-cell embryos were injected with the aid of a membrane relaxant in order to avoid lysis. The authors' aim was to study cell-cycle dependent blockage. They found that only 4% of the total donor blastomere cytoplasmic volume was sufficient to overcome the block. The ability of a small volume of presumably normal cytoplasm to change cell cycle events in early embryos became the basis for cytoplasmic transfer to treat oocytes of patients with multiple cycles of abnormal embryo development, using ooplasm from donor eggs during ICSI (Cohen et al, 1998Cohen J. Scott R. Alikani M. Schimmel T. Munné S. Levron J. Wu L. Brenner C. Warner C. Willadsen S. Ooplasmic transfer in mature human oocytes.Mol. Hum. Reprod. 1998; 4: 269-280Crossref PubMed Scopus (280) Google Scholar). A similar approach has been recently advocated using autologous transfer of mitochondria derived from egg precursor cells (AUGMENT™, OvaScience), which morphologically appear to resemble ooplasmic mitochondria (Woods, Tilly, 2015Woods D.C. Tilly J.L. Autologous Germline Mitochondrial Energy Transfer (AUGMENT) in human assisted reproduction.Semin. Reprod. Med. 2015; 33: 410-421Crossref PubMed Scopus (74) Google Scholar). In a follow-up paper by Zhang and coworkers in this issue of RBMOnline (Zhang et al, 2016Zhang J. Zhuang G. Zeng Y. Grifo J. Acosta C. Shu Y. Liu H. Pregnancy derived from human zygote pronuclear transfer in a patient who had arrested embryos after IVF.Reprod. Biomed. Online. 2016; 33: 529-533Google Scholar) the case performed in China in 2003 is presented in more detail. Why the 13-year hiatus between presentation of the abstract at a meeting and publication of a full paper? Perhaps the authors were discouraged by the tragic loss of the resulting triplet pregnancy and the criticism from colleagues and ethicists. Whatever the circumstances, the current paper describes the reconstitution protocol and the DNA analyses which showed that the fetuses had mitochondrial DNA from the oocyte donor and nuclear DNA from the mother. Unfortunately, other details are missing: no micrographs are provided of the nuclear transplantation procedure and there is essentially no data or micrographs from the two unsuccessful treatment attempts that led the authors to apply nuclear transplantation in the first place. None of the patient's zygotes were kept in culture as a control in parallel with the reconstituted zygotes in order to document repeated early arrest. Other questions remain unanswered about this case. What was the review and ethics approval process at Sun-Yat Sen University where this experiment was conducted? Was the demise of one fetus following premature rupture of membranes at 24 weeks of gestation, and cord prolapse and demise of the last fetus at 29 weeks, related to the manipulations or to the clinical management of this high-risk pregnancy? Could the procedure be considered successful from a technical standpoint since the majority of the reconstructed zygotes developed into apparently normal diploid embryos? This was indeed an important, if limited finding, considering the developmental history. Are there details that may shed more light on the technique itself and on whether this protocol differs significantly from those used by teams advocating MRT in more recent studies (Hyslop et al, 2016Hyslop L.A. Blakeley P. Craven L. Richardson J. Fogarty N.M. Fragouli E. Lamb M. Wamaitha S.E. Prathalingam N. Zhang Q. O'Keefe H. Takeda Y. Arizzi L. Alfarawati S. Tuppen H.A. Irving L. Kalleas D. Choudhary M. Wells D. Murdoch A.P. Turnbull D.M. Niakan K.K. Herbert M. Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.Nature. 2016; 534: 383-386https://doi.org/10.1038/nature18303Crossref PubMed Scopus (203) Google Scholar, Tachibana et al, 2013Tachibana M. Amato P. Sparman M. Woodward J. Sanchis D.M. Ma H. Gutierrez N.M. Tippner-Hedges R. Kang E. Lee H.S. Ramsey C. Masterson K. Battaglia D. Lee D. Wu D. Jensen J. Patton P. Gokhale S. Stouffer R. Mitalipov S. Towards germline gene therapy of inherited mitochondrial diseases.Nature. 2013; 493: 627-631Crossref PubMed Scopus (294) Google Scholar)? These and other questions should add to the ongoing debate and discussions in the literature and among legislative and regulatory bodies such as the UK parliament, UK Human Fertilisation and Embryology Authority (HFEA) and US Food and Drug Administration (FDA) on the application of nuclear transplantation technology for the purpose of preventing the transfer of mitochondrial disease (Cohen et al, 2015Cohen I.G. Savulescu J. Adashi E.Y. Transatlantic lessons in regulation of mitochondrial replacement therapy.Science. 2015; 348: 178-180Crossref PubMed Scopus (30) Google Scholar, Schandera, Mackey, 2016Schandera J. Mackey T.K. Mitochondrial replacement techniques: divergence in global policy.Trends Genet. 2016; 32: 385Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The 2003 nuclear transfer procedure seems to be very similar to those proposed for MRT. Is this experimental approach suitable for non-mitochondrial disease applications? The recent Institute of Medicine (IOM) report distinguishes between mitochondrial replacement therapy and cytoplasmic replacement for overcoming infertility (National Academies of Sciences, Engineering, and Medicine, 2016National Academies of Sciences, Engineering, and Medicine Mitochondrial Replacement Techniques: Ethical, Social, and Policy Considerations. The National Academies Press, Washington, DC2016Google Scholar). The HFEA seems to take a similar position, inferring that cytoplasmic replacement should not be used for treatment of infertility (Human Fertilisation and Embryology Authority, 2015Human Fertilisation and Embryology Authority World first as mitochondrial donation regulations come into force.http://www.hfea.gov.uk/9946.htmlDate: 2015Google Scholar). It seems that the ethical debate has focused on the risk factors. These were not discussed in depth in the current paper. Is it morally acceptable to carry out experimental procedures such as nuclear transplantation or spindle transfer to treat infertility patients? Medical ethics involve the presentation of a moral standard to the practice of medicine. This obviously implies the responsibility to ‘do no harm’, but also to avoid increased risk, in both routine and experimental treatments. This serious responsibility must always be balanced with the valid search for treatment options providing significant potential benefit even in the case of non-life threatening human disease like infertility. However, this could also be argued in relation to MRT for mitochondrial disease, as the egg, embryo and fetus do not exist prior to therapy and in some cases there are alternatives like preimplantation genetic diagnosis (PGD), egg donation or adoption, which could be considered. The suggestion that reducing the risk of transmitting mitochondrial disease is not the moral equivalent of overcoming infertility and that the latter requires a different decision-making process or legal framework would benefit from further scrutiny. A parallel can be drawn with PGD where testing for genetic mutations in embryos has been considered by many reproductive specialists as different to, and somehow more justified than, testing for numeric chromosome anomalies. With reduced error and diagnosis-failure rates, both applications seem to be more acceptable now and the delineation between the two testing types is becoming blurred. The same may be on the horizon for nuclear or cytoplasmic transfer. Biotechnology, pharmacology and genetic diagnosis are rapidly growing areas in China, but the presentation of guidelines regarding ethics and policy debates may be perceived as not exhaustive by Western standards. This is not always justified (Sipp, Pei, 2016Sipp D. Pei D. No wild east.Nature. 2016; 534: 465-467Crossref PubMed Scopus (6) Google Scholar). Modification of the embryonic genome may be allowed, but Chinese national guidelines have prohibited the transfer of such embryos since 2003. There is no national forum to which clinical experiments may be submitted for consideration, so examination of such proposals remains the purview of local hospital ethics boards. Although this is not unlike the familiar internal review board (IRB) process, in the USA, for instance, other governmental (e.g., the FDA) and non-governmental (e.g., NAS) forums representing the public interest and including professionals in diverse fields are also involved, debating the ethics of procedures such as cytoplasmic transfer, gene therapy, xenotransplantation and MRT. Observers in the West may wonder whether the clinical research climate in China encourages experimental approaches such as stem-cell therapies, nuclear transplantation, and other developing technologies without thorough discussion, but it is likely that such deliberations do take place but are screened from public view. In that context, it is not surprising that the first clinical applications of CRISPR-based technology for genome editing are reported by Chinese teams employing the process in abnormal dispermic human zygotes (Kang et al, 2016Kang X. He W. Huang Y. Yu Q. Chen Y. Gao X. Sun X. Fan Y. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.J. Assist. Reprod. Genet. 2016; 33: 581-588https://doi.org/10.1007/s10815-016-0710-8Crossref PubMed Scopus (197) Google Scholar, Liang et al, 2015Liang P. Xu Y. Zhang X. Ding C. Huang R. Zhang Z. Lv J. Xie X. Chen Y. Li Y. Sun Y. Bai Y. Songyang Z. Ma W. Zhou C. Huang J. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.Protein Cell. 2015; 6: 363-372https://doi.org/10.1007/s13238-015-0153-5Crossref PubMed Scopus (760) Google Scholar). The review process in 2003 of the case presented in this issue of RBMO may have been comparable with the ethics approval process in the West in the 1970s and 1980s. Views regarding fertilization and the status of the human embryo seem less complicated in China than they are in the West. In China, life is seen as a continuum, whereas in the West, many argue that conception represents a sacred moment (life and its sanctity begin at conception). Perhaps the zygote nuclear transplantation described by Zhang et al. should be viewed from this perspective, although renewed criticism is likely to follow given the experimental nature of this case, missing data, and the prolonged period between initial announcement of the work and publication of this paper. Pregnancy derived from human zygote pronuclear transfer in a patient who had arrested embryos after IVFReproductive BioMedicine OnlineVol. 33Issue 4PreviewNuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patient's pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Full-Text PDF

In this issue of RBMOnline, Zhang and Liu present their latest work involving mammalian germinal vesicle (GV) transfer (Zhang, Liu, 2015Zhang J. Liu H. Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically-arrested oocytes.Reprod. Biomed. Online. 2015; 31: 71-78Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). This study extends the prior work of these authors and others in examining this technique (Liu et al, 2003Liu H. Chang H.C. Zhang J. Grifo J. Krey L.C. Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term.Hum. Reprod. 2003; 18: 1903-1907Crossref PubMed Scopus (38) Google Scholar, Takeuchi et al, 2001Takeuchi T. Gong J. Veeck L.L. Rosenwaks Z. Palermo G.D. Preliminary findings in germinal vesicle transplantation of immature human oocytes.Hum. Reprod. 2001; 16: 730-736Crossref PubMed Scopus (51) Google Scholar, Zhang et al, 1999Zhang J. Wang C.W. Krey L. Liu H. Meng L. Blaszczyk A. Adler A. Grifo J. In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer.Fertil. Steril. 1999; 71: 726-731Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). One finding of developmental interest is the observation that when reciprocal GV transfers between human and mouse oocytes were performed, the maturation process and spindle formation followed the expected form of the species donating the ooplast. This phenomenon has been observed in prior cross-species GV transfer between rabbit and mouse (Li et al, 2001Li G.P. Chen D.Y. Lian L. Sun Q.Y. Wang M.K. Song X.F. Meng L. Schatten H. Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals.J. Exp. Zool. 2001; 289: 322-329Crossref PubMed Scopus (13) Google Scholar) but, as we know, early development often exhibits species-specific aspects so the finding that there is human GV breakdown in mouse oocytes has significance. The greater question however concerns the ultimate clinical utility of the GV transfer scenario. In theory, such manipulations could be used to somehow provide a “rejuvenated” ooplasmic environment for the maternal germline of advanced-age patients by creating hybrid oocytes in which their chromosomal complement is transferred to enucleated donor ooplasts at the GV stage (Zhang, 2015Zhang J. Revisiting germinal vesicle transfer as a treatment for aneuploidy in infertile women with diminished ovarian reserve.J. Assist. Reprod. Genet. 2015; 32: 313-317Crossref PubMed Scopus (17) Google Scholar). Such a process would potentially circumvent early developmental deficits in the aged maternal oocyte, leading to “healthier” mature oocytes for the patient. This concept has been pursued via basic research studies, as reported in the current paper, in a limited fashion over the past 15 years without significant advancement. The physical “success” of the basic transfer scenario seems established – and this is certainly a credit to the experience and skill of the authors and coworkers. However, the ultimate extension of such an experiment into the human clinical treatment arena must be considered. For one thing, the ‘real world’ requirements of a potential clinical scenario seem quite daunting and several open questions about clinical outcome remain. Synchronous cohorts of healthy, maturation-competent GV-stage oocytes would have to be obtained from both patient and donor. Then, the complex handling and manipulation involved with the GV transfer itself would have to be accomplished with an acceptable level of clinical efficiency. Based on the research protocols used to date, this would probably require exposing human patient material to potentially toxic cytoskeletal/membrane relaxants such as cytochalasin B and then to electro-fusion medium and a membrane-disruptive electric field pulse or the use of Sendai virus. While there is some evidence of electro-fusion related disturbance to oocyte activation, these manipulations seem compatible with overtly normal development in animal models (Liu et al, 2003Liu H. Chang H.C. Zhang J. Grifo J. Krey L.C. Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term.Hum. Reprod. 2003; 18: 1903-1907Crossref PubMed Scopus (38) Google Scholar, Mitalipov et al, 2007Mitalipov S.M. Zhou Q. Byrne J.A. Ji W.Z. Norgren R.B. Wolf D.P. Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling.Hum. Reprod. 2007; 22: 2232-2242Crossref PubMed Scopus (97) Google Scholar). However, their safety and downstream effects in the human remain an open question. Speculation has been raised that the necessary “mismatch” between donor-derived ooplasm and patient-derived genome entailed in the procedure could potentially result in downstream developmental consequences (Cheng et al, 2009Cheng Y. Wang K. Kellam L.D. Lee Y.S. Liang C.-G. Han Z. Mtango N.R. Latham K.E. Effects of ooplasm manipulation on DNA methylation and growth of progeny in mice.Biol. Reprod. 2009; 80: 464-472Crossref PubMed Scopus (34) Google Scholar). This seems unlikely, but admittedly it does remain an unknown and could be raised in any possible regulatory review of proposed clinical GV transfer treatment scenarios. Another open question is the actual implantation potential of embryos resulting from such reconstituted oocytes, since a reduction in implantation from the manipulations themselves (as might be expected) could negate any potential clinical “improvement” from the proposed oocyte rejuvenation. A final open question concerns the real ability of GV transfer to address the high rate of aneuploidy in the oocytes of advanced maternal age (AMA) candidate patients. While very limited evidence of normal chromosome complements following GV transfer from AMA oocytes has been observed in research to date, this by no means provides proof of principle that allowing meiosis to proceed in “young” ooplasm will truly and consistently alleviate age-associated oocyte aneuploidy. Considering the high rate of aneuploidy in potential GV transfer patients, this could be another issue reducing the efficiency of the overall scenario. A second consideration is the ongoing development of other, perhaps more practical, clinical protocols that could theoretically be used to “rejuvenate” human oocytes (or simply create healthy oocytes) and address AMA issues. During the decade in which GV transfer research has been pursued, considerable advancement has been made in our understanding of the developmental biology of human oocytes. It is now recognized that populations of quiescent immature oocytes and oocyte stem cells exist even in the ovaries of women of very advanced reproductive age (and post-menopause) (Johnson et al, 2004Johnson J. Canning J. Kaneko T. Pru J.K. Tilly J.L. Germline stem cells and follicular renewal in the postnatal mammalian ovary.Nature. 2004; 428: 145-150Crossref PubMed Scopus (985) Google Scholar, Tilly, Telfer, 2009Tilly J.L. Telfer E.E. Purification of germline stem cells from adult mammalian ovaries: a step closer towards control of the female biological clock?.Mol. Hum. Reprod. 2009; 15: 393-398Crossref PubMed Scopus (64) Google Scholar). Manipulation of these germ cell populations has allowed for the creation of apparently viable human eggs (White et al, 2012White Y.A.R. Woods D.C. Takai Y. Ishihara O. Seki H. Tilly J.L. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive age women.Nat. Med. 2012; 18: 413-421Crossref PubMed Scopus (527) Google Scholar). A “rejuvenation” strategy using such AMA patient stem cell-derived eggs as a source of ooplasmic augmentation is already in the clinical trial stage (Tilly, Sinclair, 2013Tilly J.L. Sinclair D.A. Germline energetics, aging, and female infertility.Cell Metab. 2013; 17: 838-850Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Another obvious, but currently speculative, treatment scenario would involve simply replacing the patient's existing, age-compromised oocytes with those derived from stem-cell manipulation. The true clinical utility of both scenarios remains to be seen but the existence of such oocyte stem cells and the demonstrated ability to manipulate these in AMA patients suggests that maternal age treatment models that avoid complex egg donor-based strategies such as GV transfer may be inevitable. We congratulate the authors on their current work and recognize that GV transfer may continue to play an important role in pursuing basic knowledge about oocyte development, maturation and meiotic aspects – and increased access to human oocytes from stem-cell manipulation can hopefully facilitate such pursuit.

Background: Polycystic ovarian syndrome (PCOS) is characterized by increased ovarian angiogenesis and vascularity. Accumulating evidence indicates that vascular endothelial growth factor (VEGF) is increased in PCOS and may play an important role in these vascular changes and the pathogenesis of this disease. Placental growth factor (PlGF), a VEGF family member, has not been previously characterized in PCOS women. We investigated levels and temporal expression patterns of PlGF and its soluble receptor sFlt-1 (soluble Fms-like tyrosine kinase) in serum and follicular fluid (FF) of women with PCOS during controlled ovarian stimulation.Methods: This was a prospective cohort study of 14 PCOS women (Rotterdam criteria) and 14 matched controls undergoing controlled ovarian stimulation. Serum was collected on day 3, day of hCG and day of oocyte retrieval. FF was collected on retrieval day. PlGF, sFlt-1 and anti-mullerian hormone (AMH) protein concentrations were measured using ELISA. Since sFlt-1 binds free PlGF, preventing its signal transduction, we calculated PlGF bioavailability as PlGF/sFlt-1 ratio.Results: Serum PlGF and sFlt-1 levels were constant throughout controlled ovarian stimulation, and no significant differences were observed in either factor in PCOS women compared with non-PCOS controls at all three measured time points. However, FF PlGF levels were increased 1.5-fold in PCOS women compared with controls (p < 0.01). Moreover, FF PlGF correlated positively with number of oocytes retrieved and the ovarian reserve marker anti-mullerian hormone (AMH) and negatively with age. In addition, FF sFlt-1 levels were decreased 1.4-fold in PCOS women compared to controls (p = 0.04). PlGF bioavailability in FF was significantly greater (2-fold) in PCOS women compared with non-PCOS controls (p < 0.01).Conclusions: These data provide evidence that FF PlGF correlates with ovarian stimulation and that its bioavailability is increased in women with PCOS undergoing controlled ovarian stimulation. This suggests that PlGF may play a role in PCOS pathogenesis and its angiogenic dysregulation.

OBJECTIVE: Serum Antimullerian hormone (AMH) levels are elevated in polycystic ovarian syndrome and have been shown to be useful in its diagnosis. However, the clinical significance of extremely high AMH levels is understudied. We aimed to characterize a population of women with elevated AMH (>5 ng/ mL).STUDY DESIGN: This was a retrospective cohort study of 134 women presenting to our fertility clinic for infertility evaluation and treatment who were found to have random serum AMH over 5 ng/mL. Women were divided into 3 groups according to AMH: 5-10 ng/mL, >10-14 ng/mL, and >14 ng/mL. Endocrine characteristics, polycystic ovarian syndrome (PCOS) phenotypes, fertilization rate, implantation rate, clinical pregnancy, and multiple pregnancy rates were compared between groups.RESULTS: AMH ranged between 5 to 48 ng/mL. Greater than 97% of women with ultrahigh AMH (>10 ng/mL) had PCOS. In addition, women with AMH >10 ng/mL had greater prevalence of polycystic ovarian morphology and oligoamenorrhea than women with AMH 5-10 ng/mL. Moreover, serum AMH correlated positively with luteinizing hormone, total testosterone, and dehydroepiandrosterone sulfate. Furthermore, AMH showed strong predictive ability for the presence of amenorrhea (area under the curve, 0.87; 95% confidence interval, 0.80-0.92; P < .0001). Despite similar age and mean number of transferred embryos, women with AMH >10 ng/mL showed higher rates of ovarian hyperstimulation syndrome and clinical pregnancy rates compared with women with AMH 5-10 ng/mL.CONCLUSION: These data characterize a population of women with elevated AMH levels, demonstrating that the vast majority of women with AMH >10 ng/mL have PCOS. Increased AMH levels correlated with PCOS severity and are associated with greater ovarian stimulation and higher clinical pregnancy rates following assisted reproductive technology.

OBJECTIVE:To evaluate the relationship between transforming growth factor (TGF)-β1 and its receptor, soluble endoglin (sENG), in the serum and follicular fluid of women with polycystic ovarian syndrome (PCOS) compared with that of non-PCOS normal ovulating women during controlled ovarian stimulation (COS).DESIGN:Prospective case-control study.SETTING:Academic-affiliated assisted reproductive technology unit.PATIENT(S):Fourteen PCOS and 14 matched non-PCOS control women undergoing COS.INTERVENTION(S):Serum was collected on day 3 (baseline), day of hCG, and day of retrieval. Follicular fluid (FF) was collected on day of oocyte retrieval. ELISA was performed to determine TGF-β1 and sENG protein levels.MAIN OUTCOME MEASURE(S):Serum and FF levels of TGF-β1 and sENG.RESULT(S):Serum TGF-β1 did not change significantly during COS but was increased in PCOS compared with non-PCOS women on day 3 and days of hCG administration and oocyte retrieval. Serum sENG increased after hCG administration only in the non-PCOS control group. In addition, serum sENG was decreased in PCOS compared with non-PCOS control women on the days of hCG and retrieval. Accordingly, the bioavailability of TGF-β1 (TGF-β1/sENG ratio) was increased in women with PCOS compared with non-PCOS controls at all three time points. No differences in either factor were noted in FF between groups.CONCLUSION(S):The increased TGF-β1 bioavailability in PCOS is not only due to increased TGF-β1 levels but also to decreased levels of its receptor, sENG. These data suggest that increased TGF-β1 bioavailability may contribute to the pathogenesis of PCOS and its increased risk for ovarian hyperstimulation.

Polycystic ovarian syndrome (PCOS) ovaries are characterized by increased angiogenesis and hypervascularity. While angiopoietin-1 (Ang-1) and its antagonist, angiopoietin-2 (Ang-2), are essential for ovarian function and angiogenesis, the levels of Ang-1 and Ang-2 in PCOS are unknown. This was a prospective cohort study of 14 PCOS women and 14 matched controls undergoing controlled ovarian stimulation (COS). Serum was collected on day 3, hCG and retrieval days. Follicular fluid (FF) was collected on retrieval day. Serum Ang-1 and Ang-2 levels were constant throughout COS, but serum Ang-1 levels were increased at all time points in PCOS women compared with controls (p < 0.05). No differences between groups were found in serum Ang-2 levels or FF Ang-1 levels. However, FF Ang-2 levels were increased almost 2-fold in PCOS women compared with controls (p < 0.01), and correlated positively with number of oocytes retrieved (r = 0.65, p < 0.0001). This study is the first to provide evidence of an alteration in the Ang-1/Ang-2 system in PCOS women. The biological role of Ang-2 in promoting capillary leakage, the increased Ang-2 FF level in PCOS, and its correlation with number of oocytes suggest that Ang-2 may play an important role in the increased risk of ovarian hyperstimulation in PCOS.

ObjectiveVascular endothelial growth factor (VEGF) is increased in PCOS and its levels correlate with ovarian hyperstimulation syndrome (OHSS). Besides VEGF, the role of other angiogenic factors in PCOS is unknown. The angiopoietin family consists of angiopoietin-1 (Ang1) which promotes angiogenesis, and its antagonist, angiopoietin-2 (Ang2), which destabilizes blood vessels causing capillary leakage. Our specific aim was to characterize levels of Ang1 and Ang2 in follicular fluid (FF) of PCOS women compared to non-PCOS control women during controlled ovarian stimulation (COS), and assess for correlation between their levels and stimulation cycle parameters.DesignProspective case-control.Materials and MethodsFF was collected on day of oocyte retrieval from 14 PCOS and 14 matched non-PCOS control women undergoing COS. ELISA was performed to determine Ang1 and Ang2 protein levels. Correlations were assessed between Ang protein levels and various cycle parameters.ResultsNo differences were found in FF Ang1 levels between PCOS and non-PCOS women (0.43 ± 0.06 vs. 0.40 ± 0.07 ng/ml). In contrast, FF Ang2 levels were 1.9-fold greater in PCOS women compared with non-PCOS controls (19.6 ± 2.6 vs. 10.5 ± 1.2 ng/ml, p < 0.01). Furthermore, FF Ang2 levels positively correlated with the number of oocytes retrieved and number of embryos obtained in both PCOS and non-PCOS groups (r =0.65, and r =0.50, p<0.0001 and p<0.05, respectively). No significant correlation was found between Ang2 levels and FSH, estrogen on day of hCG or total gonadotropin dose. No significant correlations were found between Ang1 levels and either cycle parameter.ConclusionAng2 destabilizes blood vessels, inducing capillary permeability and vessel leakage. We report for the first time that Ang2 is increased in FF of PCOS women and correlates with ovarian stimulation, suggesting that Ang2 may play a role in ovarian hyperstimulation and may contribute to the increased risk of OHSS in PCOS women. ObjectiveVascular endothelial growth factor (VEGF) is increased in PCOS and its levels correlate with ovarian hyperstimulation syndrome (OHSS). Besides VEGF, the role of other angiogenic factors in PCOS is unknown. The angiopoietin family consists of angiopoietin-1 (Ang1) which promotes angiogenesis, and its antagonist, angiopoietin-2 (Ang2), which destabilizes blood vessels causing capillary leakage. Our specific aim was to characterize levels of Ang1 and Ang2 in follicular fluid (FF) of PCOS women compared to non-PCOS control women during controlled ovarian stimulation (COS), and assess for correlation between their levels and stimulation cycle parameters. Vascular endothelial growth factor (VEGF) is increased in PCOS and its levels correlate with ovarian hyperstimulation syndrome (OHSS). Besides VEGF, the role of other angiogenic factors in PCOS is unknown. The angiopoietin family consists of angiopoietin-1 (Ang1) which promotes angiogenesis, and its antagonist, angiopoietin-2 (Ang2), which destabilizes blood vessels causing capillary leakage. Our specific aim was to characterize levels of Ang1 and Ang2 in follicular fluid (FF) of PCOS women compared to non-PCOS control women during controlled ovarian stimulation (COS), and assess for correlation between their levels and stimulation cycle parameters. DesignProspective case-control. Prospective case-control. Materials and MethodsFF was collected on day of oocyte retrieval from 14 PCOS and 14 matched non-PCOS control women undergoing COS. ELISA was performed to determine Ang1 and Ang2 protein levels. Correlations were assessed between Ang protein levels and various cycle parameters. FF was collected on day of oocyte retrieval from 14 PCOS and 14 matched non-PCOS control women undergoing COS. ELISA was performed to determine Ang1 and Ang2 protein levels. Correlations were assessed between Ang protein levels and various cycle parameters. ResultsNo differences were found in FF Ang1 levels between PCOS and non-PCOS women (0.43 ± 0.06 vs. 0.40 ± 0.07 ng/ml). In contrast, FF Ang2 levels were 1.9-fold greater in PCOS women compared with non-PCOS controls (19.6 ± 2.6 vs. 10.5 ± 1.2 ng/ml, p < 0.01). Furthermore, FF Ang2 levels positively correlated with the number of oocytes retrieved and number of embryos obtained in both PCOS and non-PCOS groups (r =0.65, and r =0.50, p<0.0001 and p<0.05, respectively). No significant correlation was found between Ang2 levels and FSH, estrogen on day of hCG or total gonadotropin dose. No significant correlations were found between Ang1 levels and either cycle parameter. No differences were found in FF Ang1 levels between PCOS and non-PCOS women (0.43 ± 0.06 vs. 0.40 ± 0.07 ng/ml). In contrast, FF Ang2 levels were 1.9-fold greater in PCOS women compared with non-PCOS controls (19.6 ± 2.6 vs. 10.5 ± 1.2 ng/ml, p < 0.01). Furthermore, FF Ang2 levels positively correlated with the number of oocytes retrieved and number of embryos obtained in both PCOS and non-PCOS groups (r =0.65, and r =0.50, p<0.0001 and p<0.05, respectively). No significant correlation was found between Ang2 levels and FSH, estrogen on day of hCG or total gonadotropin dose. No significant correlations were found between Ang1 levels and either cycle parameter. ConclusionAng2 destabilizes blood vessels, inducing capillary permeability and vessel leakage. We report for the first time that Ang2 is increased in FF of PCOS women and correlates with ovarian stimulation, suggesting that Ang2 may play a role in ovarian hyperstimulation and may contribute to the increased risk of OHSS in PCOS women. Ang2 destabilizes blood vessels, inducing capillary permeability and vessel leakage. We report for the first time that Ang2 is increased in FF of PCOS women and correlates with ovarian stimulation, suggesting that Ang2 may play a role in ovarian hyperstimulation and may contribute to the increased risk of OHSS in PCOS women.

Vascular endothelial growth factor (VEGF) is increased in PCOS, and is a key mediator of ovarian hyperstimulation syndrome (OHSS). Besides VEGF, the role of other angiogenic factors in PCOS is unknown. Our specific aim was to characterize the angiogenic factors platelet-derived growth factor-B (PDGF-B), angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) in PCOS during controlled ovarian stimulation (COS). Prospective case-control. Serum was collected from PCOS (Rotterdam criteria) (n=9) or control (n=10) patients undergoing COS on day 3, hCG day, and retrieval day. Follicular fluid (FF) was collected on retrieval day. ELISA was performed to measure the concentration of each protein. Statistical analysis was performed using Student's t-test or Mann-Whitney test. Serum PDGF-B was increased in PCOS women vs. controls on day 3 and on day of retrieval (1.7 and 1.9-fold, P=0.01 and P=0.02, respectively) and was undetectable in FF. Ang1 and Ang2 serum levels did not change throughout COS. Ang1 and Ang2 were elevated in FF of PCOS vs. control women (1.5 and 1.6-fold, P=0.04 and P=0.03, respectively). In addition, Ang1 was increased in serum of PCOS women at all time points (1.4, 1.3 and 1.4-fold, P=0.01, P=0.05 and P=0.04, respectively), while Ang2 was not. Furthermore, Ang2/Ang1 ratio in FF was 32 and 30 in PCOS and control groups, respectively, in contrast to the Ang2/Ang1 serum ratio of 0.03 for both groups (P<0.0001). Serum PDGF-B and Ang1 as well as FF Ang1 and Ang2 are increased in PCOS women, consistent with a proangiogenic state. These data suggest Ang2 is locally produced within the stimulated follicle in much greater concentrations than Ang1. Ang2 is a known antagonist of Ang1 and its dominance results in destabilizing blood vessel endothelium leading to capillary permeability. The role of Ang2 in the ovary and its upregulation in PCOS suggests Ang2 may contribute to the increased risk of OHSS in PCOS during COS.

Transforming growth factor-β1 (TGF-β1) is involved in cell proliferation and angiogenesis and was recently shown to be increased in PCOS women. Soluble endoglin (sENG) is its circulating receptor. Their binding leads to decreased TGF-β1 bioavailability. Decreased TGF-β1/sENG ratio is implicated in preeclampsia pathogenesis. sENG expression has not been characterized in PCOS, neither has its relationship to TGF-β1. We examined the relationship of TGF-β1 and sENG in PCOS compared to control patients during controlled ovarian stimulation (COS). Prospective case-control. Serum was collected from PCOS (Rotterdam Criteria) (n=9) or control (n=10) patients undergoing COS on day 3, day of hCG, and day of retrieval. Follicular fluid (FF) was collected on day of retrieval. ELISA was performed to measure the levels of each factor. Statistical analysis was performed using Student's t-test or Mann-Whitney test. Serum TGF-β1 was increased in PCOS vs. control women on days 3, hCG and retrieval (1.3, 1.5 and 1.5-fold, respectively). Serum sENG was decreased in PCOS vs. control patients on days of hCG and retrieval (0.8-fold). The bioavailability of TGF-β1 (TGF-β1/sENG ratio) was increased in PCOS patients vs. controls on days 3, hCG and retrieval (1.3, 1.9, 1.9-fold, P=0.02, P<0.01, P<0.01, respectively). No differences in either factor were noted in FF between groups.Tabled 1Angiogenic FactorGroupDay 3hCG DayRetrieval DayFFTGF-b1 (ng/ml)PCOS29.333.035.40.74Control22.821.725.80.8Fold change1.31.51.50.93P-value0.040.00010.04NSsENG (ng/ml)PCOS3.12.63.21.4Control3.23.14.11.4Fold change0.970.80.81.0P-valueNS0.040.02NS Open table in a new tab In contrast to preeclampsia, TGF-β1 is increased while sENG is decreased in serum of stimulated PCOS patients. The increased TGF-β1/sENG ratio results in increased bioavailability of TGF-β1. This dysregulation may contribute to the pathogenesis of PCOS and its increased risk for ovarian hyperstimulation.

This study evaluated anti-Müllerian hormone (AMH) as a possible predictor of twin pregnancy in women undergoing fresh cycles who had more than one embryo transferred. A retrospective study was performed of 139 patients undergoing fresh non-donor cycles which resulted in either singleton or twin pregnancy between 2009 and 2010 in this fertility clinic. Random serum AMH and other clinically relevant variables were compared. For further analysis, the population was stratified by age (<34 and ⩾34 years). Random serum AMH concentrations were 1.4-times greater in women conceiving twins compared with singletons (P=0.03). In women aged ⩾34, the AMH concentration in twins was 1.8-fold greater than singletons (P=0.001). Multivariate analysis demonstrated that AMH was an independent predictor of twins. ROC curve analysis showed that AMH had a significant predictive ability for twin pregnancy in women aged ⩾34 (AUC 0.67, P=0.01). In contrast, in women aged <34, AMH was not different between twin and singleton pregnancies. In summary, random serum AMH is an independent predictor of twin gestation when more than one embryo is transferred in women aged ⩾34. Considering a woman's AMH before transferring more than one embryo may assist in reducing the incidence of twins. Transferring multiple embryos to increase the chance of pregnancy and live birth rate is a common practice among assisted reproduction providers but often results in a high proportion of multiple pregnancies. Identifying factors predictive of multiple pregnancies is of paramount importance for the successful development of strategies to minimize multiple gestations. Anti-Müllerian hormone (AMH) has been shown to be closely correlated with a woman's egg reserve. Aside from the strong association of serum AMH concentration with quantitative ovarian response, serum AMH concentration has been associated with qualitative aspects of assisted reproduction such as embryo quality and pregnancy rates. Our objective was to evaluate AMH as a possible predictor of twin pregnancy in women undergoing fresh cycles who had more than one embryo transferred. Our study included 139 patients undergoing fresh non-donor cycles which resulted in either a singleton or twin pregnancy between 2009 and 2010 in our fertility clinic. Random serum AMH concentrations were compared. For further analysis, the population was divided into two groups (age <34 and ⩾34). AMH concentrations were 1.4-times greater in women conceiving twins compared with singletons. In women aged ⩾34, the AMH concentration in twins was 1.8-fold greater than singleton pregnancies. AMH was an independent predictor of twins with a significant predictive ability for twin pregnancy in women aged ⩾34. In contrast, in women aged <34, AMH was not different between twin and singleton pregnancies. In conclusion, considering a woman's AMH before transferring more than one embryo may assist in reducing the incidence of twins.

In a previous issue of Reproductive BioMedicine Online, Chiaratti and co-workers presented a bovine model for ooplasmic transfer, which demonstrated a positive effect on early development. Developmental deficits resulting from artificial treatment of recipient eggs with a toxic compound were ameliorated by the addition of small volumes of healthy donor cytoplasm. This model provides an important advance in the understanding of ooplasmic effects in early development and addresses issues about the prior human trials in this area.

The cytoplasm of oocytes and early embryos is literally the "stuff of life." The mature mammalian oocyte is an enormous cell invested with all the components required for the initiation of development. Through embryonic cloning scenarios, it is now clear that such cytoplasm is a primary requirement—necessary and sufficient (with some limitations) for supporting term development even through the control of a previously quiescent somatic genome (1,2). However, the components and mechanisms at work in the cytoplasm of eggs and early embryos satisfying this requirement remain mysterious and are the subject of considerable research. Ooplasm harbors the factors responsible for the events of fertilization/activation, maternal and paternal genome remodeling, epigenetic programming, embryonic genome activation and early development. Research into these areas is not only of great importance to our understanding of basic developmental biology, but also has critical implications in animal husbandry and biotechnology (3), as well as in assisted reproductive medicine (4). Experimental manipulation of the cytoplasm of oocytes and early embryos has been a significant component of this research. Also, such manipulation has recently been used to directly address ooplasm-specific defects in human reproduction (5). This chapter presents concepts and techniques for the manipulation of cytoplasm in mammalian eggs and early embryos and provides a brief review of knowledge gained from such research.