In Vitro Maturation, In Vitro Oogenesis, and Ovarian Longevity.

This paper will review a remarkable new approach to in vitro maturation "IVM" of oocytes from ovarian tissue, based on our results with in vitro oogenesis from somatic cells. As an aside benefit we also have derived a better understanding of ovarian longevity from ovary transplant. We have found that primordial follicle recruitment is triggered by tissue pressure gradients. Increased pressure holds the follicle in meiotic arrest and prevents recruitment. Therefore recruitment occurs first in the least dense inner tissue of the cortico-medullary junction. Many oocytes can be obtained from human ovarian tissue and mature to metaphase 2 in vitro with no need for ovarian stimulation. Ovarian stimulation may only be necessary for removing the oocyte from the ovary, but this can also be accomplished by simple dissection at the time of ovary tissue cryopreservation. By using surgical dissection of the removed ovary, rather than a needle stick, we can obtain many oocytes from very small follicles not visible with ultrasound. A clearer understanding of ovarian function has come from in vitro oogenesis experiments, and that explains why IVM has now become so simple and robust. Tissue pressure (and just a few "core genes" in the mouse) direct primordial follicle recruitment and development to mature oocyte, and therefore also control ovarian longevity. There are three distinct phases to oocyte development both in vitro and in vivo: in vitro differentiation "IVD" which is not gonadotropin sensitive (the longest phase), in vitro gonadotropin sensitivity "IVG" which is the phase of gonadotropin stimulation to prepare for meiotic competence, and IVM to metaphase II. On any given day 35% of GVs in ovarian tissue have already undergone "IVD" and "IVG" in vivo, and therefore are ready for IVM.

In vitro germ cell induction from fertile and infertile monozygotic twin research participants.

Human induced pluripotent stem cells (hiPSCs) enable reproductive diseases to be studied when the reproductive health of the participant is known. In this study, monozygotic (MZ) monoamniotic (MA) twins discordant for primary ovarian insufficiency (POI) consent to research to address the hypothesis that discordant POI is due to a shared primordial germ cell (PGC) progenitor pool. If this is the case, reprogramming the twin's skin cells to hiPSCs is expected to restore equivalent germ cell competency to the twins hiPSCs. Following reprogramming, the infertile MA twin's cells are capable of generating human PGC-like cells (hPGCLCs) and amniotic sac-like structures equivalent to her fertile twin sister. Using these hiPSCs together with genome sequencing, our data suggest that POI in the infertile twin is not due to a genetic barrier to amnion or germ cell formation and support the hypothesis that during gestation, amniotic PGCs are likely disproportionately allocated to the fertile twin with embryo splitting.

In-vitro maturation and transplantation of cryopreserved ovary tissue: understanding ovarian longevity.

RESEARCH QUESTION: Is it possible to use experience gained from 24 years of frozen ovarian transplantation, and from recent experience with in-vitro gametogenesis to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? DESIGN: A total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24-year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back. RESULTS: Every woman had a return of ovarian function 5 months after transplant, similar to previous observations. As observed before, anti-Müllerian hormone (AMH) concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had had leukaemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes. CONCLUSIONS: The authors concluded the following. First, ovary tissue cryopreservation is a robust method for preserving fertility even for women with leukaemia, without a need to delay cancer treatment. Second, many mature oocytes can often be obtained from ovary tissue with simple media and no need for ovarian stimulation. Third, ovarian stimulation only be necessary for removing the oocyte from the ovary, which can also be accomplished by simple dissection at the time of ovary freezing. Finally, pressure and just eight 'core genes' control primordial follicle recruitment and development.

Generation of three human induced pluripotent stem cell sublines (UCLAi004-A, UCLAi004-B, and UCLAi004-C) for reproductive science research.

Three induced pluripotent stem cell sublines (hiPSCs) were generated from human dermal human dermal fibroblasts (HDFs) derived from a human skin punch biopsy. The biopsy was donated from a woman with known infertility due to ovarian failure. The hiPSC sublines were created using Sendai virus vectors and were positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was verified using PluriTest analysis and in vitro differentiation using Taqman Real-Time PCR assays for somatic lineage markers. This participant's monozygotic twin sister also donated a skin-punch biopsy, whose resulting hiPSC lines were published previously as a resource.

Generation of three human induced pluripotent stem cell sublines (UCLAi005-A, UCLAi005-B and UCLAi005-C) for reproductive science research.

We generated three human induced pluripotent stem cell (hiPSC) sublines from human dermal fibroblasts (HDF) (MZT05) generated from a skin biopsy donated from a previously fertile woman. The skin biopsy was broadly consented for generating hiPSC lines for biomedical research, including unique consent specifically for studying human fertility, infertility and germ cell differentiation. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal. Pluripotency was further verified using PluriTest analysis and in vitro differentiation was tested using Taqman Real-Time PCR assays. These sublines serve as controls for hiPSC research projects aimed at understanding the cell and molecular regulation of female fertility.

Generation of six human induced pluripotent stem cell sublines (MZT01E, MZT01F, MZT01N and MZT02D, MZT02G and MZT02H) for reproductive science research.

Six human induced pluripotent stem cell sublines (hiPSCs) were generated from human dermal fibroblasts (HDFs) derived from skin biopsies donated from monozygotic twin women wherein one woman had proven fertility and her sister was infertile due to ovarian failure. Three hiPSC sublines were created from each twin's HDFs. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was further verified using PluriTest. We show here that the hiPSC lines created from the twins are equivalent in measures of pluripotency and self-renewal, despite their differential diagnosis.

Fresh and cryopreserved ovarian tissue from deceased young donors yields viable follicles.

OBJECTIVE: Ovarian tissue cryopreservation is one of the crucial options for fertility preservation. Transplantation of cryopreserved ovarian tissue was proven to restore ovarian endocrine function in patients with premature ovarian insufficiency. Ovaries from deceased donors potentially serve as an excellent and readily available tissue for the translational and basic research. In this study, we used ovaries obtained from 5 deceased donors aged 18-26 years, to evaluate the number and quality of ovarian follicles isolated before and after cryopreservation. DESIGN: Preclinical. SETTING: Academic biomedical research laboratory. PATIENTS: De-identified deceased human donors. INTERVENTIONS: Slow-freeze cryopreservation and thawing. MAIN OUTCOME MEASURES: Follicle count, follicle density, follicle viability using immunohistochemical staining (TUNEL). RESULTS: The follicle density negatively correlated with age in both cryopreserved/thawed and fresh group. A total of 2803 follicles from fresh and 1608 follicles from cryopreserved tissues were classified and analyzed using Hematoxylin and eosin staining. There was no significant difference in the percent of morphologically normal follicles between two groups. TUNEL assay indicated no higher DNA damage in the follicles and the stroma cells after cryopreservation. Morphologically normal preantral follicles were enzymatically isolated from both fresh and cryopreserved tissue with 88.51 ± 5.93% (mean ± SD) of the isolated follicles confirmed viable using LIVE/DEAD evaluation. CONCLUSIONS: Our results indicate the ovarian tissue from deceased donors maintain high quality after long time extracorporeal circulation and transportation from the hospital to the laboratory. High survival rate of follicles at different developmental stages suggested tolerance to the cryopreservation process. Human ovarian tissues obtained from deceased donors is an ample source tissue and can be applied to promoting research and future clinical applications.

When "facts" are not facts: what does p value really mean, and how does it deceive us?

The recent paper in JAMA alleging that frozen embryo transfer causes twice the risk of childhood cancer in the offspring is an excellent example of the erroneous use of statistical tests (and the misinterpretation of p value) that is common in much of the medical literature, even in very high impact journals. These myths backed by misleading statements of "statistical significance" can cause far-reaching harm to patients and doctors who might not understand the pitfalls of specious statistical testing.

Generation of three human induced pluripotent stem cell sublines (MZT04D, MZT04J, MZT04C) for reproductive science research.

We generated three human induced pluripotent stem cell (hiPSC) sublines from human dermal fibroblasts (HDFs) (MZT04) generated from a skin biopsy donated from a previously fertile woman. The skin biopsy was broadly consented for generating hiPSC lines for biomedical research, including unique consent specifically for studying human fertility, infertility and germ cells. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was further verified using teratomas and PluriTest. These sublines serve as controls for hiPSC research projects aimed at understanding the cell and molecular regulation of female fertility and infertility.

Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.

PURPOSE: To report the results of cryopreserved ovary tissue transplantation for leukemia and other cancers, in a single US center. METHODS: One hundred eight females between age 6 and (median age 24) 35 were referred for possible ovary tissue cryopreservation over a 20-year period, with either slow freeze or vitrification. Thus far 13 patients returned up to 18 years later to have their tissue transplanted back. RESULTS: All 13 patients had return of ovarian function 5 months post transplant with regular menstrual cycling. AMH rose to very high levels as the FSH declined to normal. Four months later, the AMH again declined to very low levels. Nonetheless, the grafts remained functional for up to 5 years or longer. Ten of the 13 (77%) became spontaneously pregnant at least once, resulting in 13 healthy babies. A total of 24 healthy babies have been born 11 from fresh transplanted ovarian tissue and 13 from cryopreserved transplanted ovarian tissue. CONCLUSIONS: (1) Ovary tissue cryopreservation is a robust method for preserving a woman's fertility. (2) Cortical tissue pressure may be a key regulator of primordial follicle arrest, recruitment, and ovarian longevity. (3) This is the only such series yet reported in the USA.

Unraveling transcriptome dynamics in human spermatogenesis.

Spermatogenesis is a dynamic developmental process that includes stem cell proliferation and differentiation, meiotic cell divisions and extreme chromatin condensation. Although studied in mice, the molecular control of human spermatogenesis is largely unknown. Here, we developed a protocol that enables next-generation sequencing of RNA obtained from pools of 500 individually laser-capture microdissected cells of specific germ cell subtypes from fixed human testis samples. Transcriptomic analyses of these successive germ cell subtypes reveals dynamic transcription of over 4000 genes during human spermatogenesis. At the same time, many of the genes encoding for well-established meiotic and post-meiotic proteins are already present in the pre-meiotic phase. Furthermore, we found significant cell type-specific expression of post-transcriptional regulators, including expression of 110 RNA-binding proteins and 137 long non-coding RNAs, most of them previously not linked to spermatogenesis. Together, these data suggest that the transcriptome of precursor cells already contains the genes necessary for cellular differentiation and that timely translation controlled by post-transcriptional regulators is crucial for normal development. These established transcriptomes provide a reference catalog for further detailed studies on human spermatogenesis and spermatogenic failure.

Intrinsic fertility of human oocytes.

OBJECTIVE: To study the intrinsic fertility of the human oocyte. DESIGN: A large retrospective study of natural cycle single embryo transfer (ET) IVF cycles. SETTING: Private IVF clinic, university, and private hospital. PATIENT(S): Patients were enrolled consecutively over an 8-year period in a single ET natural cycle protocol. INTERVENTION(S): A total of 13,949 oocyte retrievals with natural IVF single ET. Software package R (version 3.2.5) was used for statistical calculations. MAIN OUTCOME MEASURE(S): Live baby rate per oocyte according to age. RESULT(S): A total of 14,185 natural cycle oocytes resulted in 1,913 live babies from single ET. The number of oocytes required to make one live baby in this large series varied with the age of the female partner. For those under 35, the live baby born per oocyte was 26%. For over age 42 it decreased to 1%. These results fit very robustly with a logistic function curve, which is at first steady (horizontal), followed by a linear decline after age 35 with a 10% loss every year until age 43, and then a flattening out (horizontal) by age 44. CONCLUSION(S): The intrinsic fertility per oocyte in natural cycle is far greater than reported in hyperstimulated cycles, varying robustly from 26% to 4% with age from <35 to 42 years. The curve is relatively flat until age 34, and then declines rapidly 10% per year thereafter.

Live birth derived from oocyte spindle transfer to prevent mitochondrial disease.

Mutations in mitochondrial DNA (mtDNA) are maternally inherited and can cause fatal or debilitating mitochondrial disorders. The severity of clinical symptoms is often associated with the level of mtDNA mutation load or degree of heteroplasmy. Current clinical options to prevent transmission of mtDNA mutations to offspring are limited. Experimental spindle transfer in metaphase II oocytes, also called mitochondrial replacement therapy, is a novel technology for preventing mtDNA transmission from oocytes to pre-implantation embryos. Here, we report a female carrier of Leigh syndrome (mtDNA mutation 8993T > G), with a long history of multiple undiagnosed pregnancy losses and deaths of offspring as a result of this disease, who underwent IVF after reconstitution of her oocytes by spindle transfer into the cytoplasm of enucleated donor oocytes. A male euploid blastocyst wasobtained from the reconstituted oocytes, which had only a 5.7% mtDNA mutation load. Transfer of the embryo resulted in a pregnancy with delivery of a boy with neonatal mtDNA mutation load of 2.36-9.23% in his tested tissues. The boy is currently healthy at 7 months of age, although long-term follow-up of the child's longitudinal development remains crucial.

Parallel evolution of male germline epigenetic poising and somatic development in animals.

Changes in gene regulation frequently underlie changes in morphology during evolution, and differences in chromatin state have been linked with changes in anatomical structure and gene expression across evolutionary time. Here we assess the relationship between evolution of chromatin state in germ cells and evolution of gene regulatory programs governing somatic development. We examined the poised (H3K4me3/H3K27me3 bivalent) epigenetic state in male germ cells from five mammalian and one avian species. We find that core genes poised in germ cells from multiple amniote species are ancient regulators of morphogenesis that sit at the top of transcriptional hierarchies controlling somatic tissue development, whereas genes that gain poising in germ cells from individual species act downstream of core poised genes during development in a species-specific fashion. We propose that critical regulators of animal development gained an epigenetically privileged state in germ cells, manifested in amniotes by H3K4me3/H3K27me3 poising, early in metazoan evolution.

Live birth rates after MESA or TESE in men with obstructive azoospermia: is there a difference?

STUDY QUESTION: How do live birth rates compare after intracytoplasmic sperm injection (ICSI) for men with obstructive azoospermia when using sperm derived from testicular sperm extraction (TESE) versus microsurgical epididymal sperm aspiration (MESA)? SUMMARY ANSWER: Our study suggests that proximal epididymal sperm (from MESA) result in higher live birth rates as compared with testicular sperm (from TESE) in couples where the man has obstructive azoospermia due to congenital bilateral absence of the vas deferens (CBAVD) or vasectomy. WHAT IS KNOWN ALREADY: For couples with obstructive azoospermia, MESA (epididymal sperm) and TESE (testicular sperm) have generally been assumed to be equivalent for use in ICSI. But this assumption has never been confirmed, and this view has important clinical and basic scientific consequences. STUDY DESIGN, SIZE, DURATION: This was a retrospective study of a consecutive cohort of 374 men with obstructive azoospermia and normal spermatogenesis, who underwent IVF and ICSI using either epididymal sperm or testicular sperm in the period 2000-2009. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included men undergoing MESA or TESE at St. Luke's Hospital for obstructive azoospermia due to CBAVD or vasectomy. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 280 couples underwent MESA and 94 underwent TESE with ICSI. The live birth rate was 39% after MESA-ICSI and 24% after TESE-ICSI. The MESA-ICSI cycles also resulted in a significantly higher implantation rate and significantly higher clinical and ongoing pregnancy rates than the TESE-ICSI cycles. There was no significant difference in results between fresh or frozen sperm for both MESA and TESE. When adjusted for the available confounders, the odds ratio for live birth was significantly in favour of MESA-ICSI versus TESE-ICSI (OR 1.82; 95% CI 1.05-3.67). The only significant confounders were female age and ovarian reserve. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective cohort study and not a randomized clinical trial. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that some aspect of sperm maturation after the sperm leaves the testicle to enter the epididymis is required for the most optimal results, even when ICSI is used for fertilization. STUDY FUNDING/COMPETING INTERESTS: No funding was used and there are no competing interests.

Applying clinically proven human techniques for contraception and fertility to endangered species and zoo animals: a review.

Reversible contraception that does not alter natural behavior is a critical need for managing zoo populations. In addition to reversible contraception, other fertility techniques perfected in humans may be useful, such as in vitro fertilization (IVF) or oocyte and embryo banking for endangered species like amphibians and Mexican wolves (Canis lupus baileyi). Furthermore, the genetics of human fertility can give a better understanding of fertility in more exotic species. Collaborations were established to apply human fertility techniques to the captive population. Reversible vasectomy might be one solution for reversible contraception that does not alter behavior. Reversible approaches to vasectomy, avoiding secondary epididymal disruption, were attempted in South American bush dogs (Speothos venaticus), chimpanzees (Pan troglodytes), gorillas (Gorilla gorilla), Przewalski's horse (Equus przewalski poliakov), and Sika deer (Cervus nippon) in a variety of zoos around the world. These techniques were first perfected in > 4,000 humans before attempting them in zoo animals. In vitro fertilization with gestational surrogacy was used to attempt to break the vicious cycle of hand rearing of purebred orangutans, and egg and ovary vitrification in humans have led to successful gamete banking for Mexican wolves and disappearing amphibians. The study of the human Y chromosome has even explained a mechanism of extinction related to global climate change. The best results with vasectomy reversal (normal sperm counts, pregnancy, and live offspring) were obtained when the original vasectomy was performed "open-ended," so as to avoid pressure-induced epididymal disruption. The attempt at gestational surrogacy for orangutans failed because of severe male infertility and the lack of success with human ovarian hyperstimulation protocols. Vitrification of oocytes is already being employed for the Amphibian Ark Project and for Mexican wolves. Vasectomy can be a reversible contraception option in zoo animals, even in endangered species. Ongoing use of gamete and embryo freezing may salvage vanishing species.