Rescue in vitro maturation using ovarian support cells of human oocytes from conventional stimulation cycles yields oocytes with improved nuclear maturation and transcriptomic resemblance to in vivo matured oocytes.

PURPOSE: Determine if the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes are bidirectionally influenced by co-culture during in vitro maturation (IVM). METHODS: Fertility patients aged 25 to 45 years old undergoing conventional ovarian stimulation donated denuded immature oocytes for research. Oocytes were randomly allocated to either OSC-IVM culture (intervention) or Media-IVM culture (control) for 24-28 h. The OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione, and doxycycline supplementation. The Media-IVM control lacked OSCs and contained the same supplementation. A limited set of in vivo matured MII oocytes were donated for comparative evaluation. Endpoints consisted of MII formation rate, morphological and spindle quality assessment, and gene expression analysis compared to in vitro and in vivo controls. RESULTS: OSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the Media-IVM control, with no apparent effect on morphology or spindle assembly. OSC-IVM MII oocytes displayed a closer transcriptomic maturity signature to IVF-MII controls than Media-IVM control MII oocytes. The gene expression profile of OSCs was modulated in the presence of oocytes, displaying culture- and time-dependent differential gene expression during IVM. CONCLUSION: The OSC-IVM platform is a novel tool for rescue maturation of human oocytes, yielding oocytes with improved nuclear maturation and a closer transcriptomic resemblance to in vivo matured oocytes, indicating a potential enhancement in oocyte cytoplasmic maturation. These improvements on oocyte quality after OSC-IVM are possibly occurring through bidirectional crosstalk of cumulus-free oocytes and ovarian support cells.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

STUDY QUESTION: Can in vitro maturation (IVM) and developmental competence of human oocytes be improved by co-culture with ovarian support cells (OSCs) derived from human-induced pluripotent stem cells (hiPSCs)? SUMMARY ANSWER: OSC-IVM significantly improves the rates of metaphase II (MII) formation and euploid Day 5 or 6 blastocyst formation, when compared to a commercially available IVM system. WHAT IS KNOWN ALREADY: IVM has historically shown highly variable performance in maturing oocytes and generating oocytes with strong developmental capacity, while limited studies have shown a positive benefit of primary granulosa cell co-culture for IVM. We recently reported the development of OSCs generated from hiPSCs that recapitulate dynamic ovarian function in vitro. STUDY DESIGN, SIZE, DURATION: The study was designed as a basic science study, using randomized sibling oocyte specimen allocation. Using pilot study data, a prospective sample size of 20 donors or at least 65 oocytes per condition were used for subsequent experiments. A total of 67 oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without hCG triggers and retrieved cumulus-oocyte complexes (COCs) were allocated between the OSC-IVM or control conditions (fetal-like OSC (FOSC)-IVM or media-only IVM) in three independent experimental design formats. The total study duration was 1 April 2022 to 1 July 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte donors between the ages of 19 and 37 years were recruited for retrieval after informed consent, with assessment of anti-Mullerian hormone, antral follicle count, age, BMI and ovarian pathology used for inclusion and exclusion criteria. In experiment 1, 27 oocyte donors were recruited, in experiment 2, 23 oocyte donors were recruited, and in experiment 3, 17 oocyte donors and 3 sperm donors were recruited. The OSC-IVM culture condition was composed of 100 000 OSCs in suspension culture with hCG, recombinant FSH, androstenedione, and doxycycline supplementation. IVM controls lacked OSCs and contained either the same supplementation, FSH and hCG only (a commercial IVM control), or FOSCs with the same supplementation (Media control). Experiment 1 compared OSC-IVM, FOSC-IVM, and a Media control, while experiments 2 and 3 compared OSC-IVM and a commercial IVM control. Primary endpoints in the first two experiments were the MII formation (i.e. maturation) rate and morphological quality assessment. In the third experiment, the fertilization and embryo formation rates were assessed with genetic testing for aneuploidy and epigenetic quality in blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE: We observed a statistically significant improvement (∼1.5×) in maturation outcomes for oocytes that underwent IVM with OSCs compared to control Media-IVM and FOSC-IVM in experiment 1. More specifically, the OSC-IVM group yielded a MII formation rate of 68% ± 6.83% SEM versus 46% ± 8.51% SEM in the Media control (P = 0.02592, unpaired t-test). FOSC-IVM yielded a 51% ± 9.23% SEM MII formation rate which did not significantly differ from the media control (P = 0.77 unpaired t-test). Additionally, OSC-IVM yielded a statistically significant ∼1.6× higher average MII formation rate at 68% ± 6.74% when compared to 43% ± 7.90% in the commercially available IVM control condition (P = 0.0349, paired t-test) in experiment 2. Oocyte morphological quality between OSC-IVM and the controls did not significantly differ. In experiment 3, OSC-IVM oocytes demonstrated a statistically significant improvement in Day 5 or 6 euploid blastocyst formation per COC compared to the commercial IVM control (25% ± 7.47% vs 11% ± 3.82%, P = 0.0349 logistic regression). Also in experiment 3, the OSC-treated oocytes generated blastocysts with similar global and germline differentially methylated region epigenetic profiles compared commercial IVM controls or blastocysts after either conventional ovarian stimulation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: While the findings of this study are compelling, the cohort size remains limited and was powered on preliminary pilot studies, and the basic research nature of the study limits generalizability compared to randomized control trials. Additionally, use of hCG-triggered cycles results in a heterogenous oocyte cohort, and potential differences in the underlying maturation state of oocytes pre-IVM may limit or bias findings. Further research is needed to clarify and characterize the precise mechanism of action of the OSC-IVM system. Further research is also needed to establish whether these embryos are capable of implantation and further development, a key indication of their clinical utility. WIDER IMPLICATIONS OF THE FINDINGS: Together, these findings demonstrate a novel approach to IVM with broad applicability to modern ART practice. The controls used in this study are in line with and have produced similar to findings to those in the literature, and the outcome of this study supports findings from previous co-culture studies that found benefits of primary granulosa cells on IVM outcomes. The OSC-IVM system shows promise as a highly flexible IVM approach that can complement a broad range of stimulation styles and patient populations. Particularly for patients who cannot or prefer not to undergo conventional gonadotropin stimulation, OSC-IVM may present a viable path for obtaining developmentally competent, mature oocytes. STUDY FUNDING/COMPETING INTEREST(S): A.D.N., A.B.F., A.G., B.P., C.A., C.C.K., F.B., G.R., K.S.P., K.W., M.M., P.C., S.P., and M.-J.F.-G. are shareholders in the for-profit biotechnology company Gameto Inc. P.R.J.F. declares paid consultancy for Gameto Inc. P.C. also declares paid consultancy for the Scientific Advisory Board for Gameto Inc. D.H.M. has received consulting services from Granata Bio, Sanford Fertility and Reproductive Medicine, Gameto, and Buffalo IVF, and travel support from the Upper Egypt Assisted Reproduction Society. C.C.K., S.P., M.M., A.G., B.P., K.S.P., G.R., and A.D.N. are listed on a patent covering the use of OSCs for IVM: U.S. Provisional Patent Application No. 63/492,210. Additionally, C.C.K. and K.W. are listed on three patents covering the use of OSCs for IVM: U.S. Patent Application No. 17/846,725, U.S Patent Application No. 17/846,845, and International Patent Application No.: PCT/US2023/026012. C.C.K., M.P.S., and P.C. additionally are listed on three patents for the transcription factor-directed production of granulosa-like cells from stem cells: International Patent Application No.: PCT/US2023/065140, U.S. Provisional Application No. 63/326,640, and U.S. Provisional Application No. 63/444,108. The remaining authors have no conflicts of interest to declare.

The use of GnRH-agonist trigger for the final maturation of oocytes in normal and low responders undergoing planned oocyte cryopreservation.

STUDY QUESTION: Does GnRH-agonist trigger offer similar maturity rate (MR) in low and normal responders compared to high responders in women undergoing planned oocyte cryopreservation, for whom even a small risk of ovarian hyperstimulation syndrome (OHSS) may not be acceptable? SUMMARY ANSWER: GnRH-agonist is an appropriate choice for final maturation of oocytes in planned oocyte cryopreservation, regardless of response to stimulation or risk of ovarian hyperstimulation syndrome. WHAT IS KNOWN ALREADY: Numerous studies have demonstrated the utility of GnRH-agonist trigger for the prevention of ovarian hyperstimulation in high-responder in vitro fertilization cycles. Limited data exist supporting its use in normal or low responders, or in non-infertile women undergoing planned oocyte cryopreservation. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study of 1189 subjects including all planned oocyte cryopreservation cycles performed at a large, single center, oocyte cryopreservation program from April 2016 to December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1680 cycles were included in the study. A total of 57.1% (959/1680) utilized GnRH-agonist for trigger. Demographic and clinical data were collected from the medical record. Maturation rate was calculated for the entire cohort, and by trigger type, using the quotient of Metaphase II (MII) oocytes and retrieved oocytes. A sub-cohort of GnRH-agonist trigger cycles were categorized by peak estradiol (E2) levels and maturation rates compared between groups. Associations were made using Student's t test, ANOVA, Mann-Whitney U and Kruskal-Wallis, where appropriate. A sample size calculation for 90% power with a significance of 5% to detect non-inferiority of <0.05 from a 0.75 maturity rate between subjects with E2 > 3000 pg/mL and E2 < 3000 pg/mL demonstrated the need for at least 116 cycles per group. MAIN RESULTS AND THE ROLE OF CHANCE: Mean MR was 0.71 ± 0.19 overall, and 0.73 ± 0.18 in the sub-cohort of GnRH-agonist trigger cycles. A total of 611 cycles (63.7%) had peak E2 < 3000, and 331 (34.5%) had E2 > 3000. No significant difference in maturity rate was noted between cycles with E2 levels >3000 pg/mL and <3000 pg/mL (0.72 ± 0.19 vs. 0.74 ± 0.14, P = 0.18), confirming the non-inferiority of maturity rates with GnRH-agonist triggers in cycles with peak E2 < 3000 pg/mL. While lower mean oocytes retrieved and mean MII oocytes were associated with lower peak E2 levels, maturity rate did not significantly differ amongst E2 level groups. Cycles with E2 < 1000 pg/mL had lower MR irrespective of trigger type. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature cannot entirely exclude selection biases, confounding factors or additional variables that could not be accounted for or were not collected by the electronic medical record. Given the nature of planned oocyte cryopreservation, studies of ongoing pregnancy rates and birth outcomes will naturally be delayed. Lastly, the study population was limited to women undergoing planned oocyte cryopreservation; therefore, the results may not be generalizable to women undergoing in vitro fertilization. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study specifically comparing the efficacy of GnRH-agonist in patients at lower risk for OHSS to those at high risk, as well the first study evaluating GnRH-agonist's efficacy specifically in planned oocyte cryopreservation cycles. STUDY FUNDING/COMPETING INTEREST(S): Study support provided by departmental funds from the Center for Fertility Research and Education-Extend Fertility Medical Practice. BLM discloses personal fees from Ferring Pharmaceuticals and Merck KgAA, unrelated to the submitted work. C.S., M.G., L.R. and J.K. have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

Likelihood of achieving a 50%, 60%, or 70% estimated live birth rate threshold with 1 or 2 cycles of planned oocyte cryopreservation.

PURPOSE: To characterize the likelihood of cryopreserving enough oocytes for 50%, 60%, or 70% estimated live birth rate (eLBR) with 1-2 planned oocyte cryopreservation (Pl-OC) cycles. METHODS: We performed a retrospective cohort study utilizing all patients completing ≥ 1 Pl-OC cycle from 2016 to 2018 at a large single-center OC program. Subjects were categorized by age at retrieval and number of cycles. We extrapolated age-based oocyte thresholds for 50%, 60%, or 70% eLBR from previously published data. We calculated the proportion of subjects overall, and for each age group, whose number of frozen oocytes was greater than or equal to their age-based threshold for a 50%, 60%, or 70% eLBR after 1 and 2 cycles. OR for 60% eLBR with one cycle was calculated for age and AMH cutoff values and corroborated with logistic regression. RESULTS: A total of 1241 subjects, completing 1799 Pl-OC cycles, were included. With one cycle, 66% (819/1241) achieved ≥ 50% eLBR and 51% (634/1241) achieved 70% eLBR. With two cycles, 79.6% (988/1241) attained ≥ 50% eLBR and 65.5% (813/1241) achieved 70% eLBR. Achieving 50%, 60%, or 70% eLBR with 1-2 cycles was significantly associated with both age (p < 0.001) and AMH (p < 0.001). Age < 37.5 and AMH > 1.995 were independently associated with attaining 60% eLBR with one cycle (age: OR 13.73; 95%CI 9.16-20.57, p < 0.001; AMH: OR 7.32; 95% CI 5.50-9.76, p < 0.001). CONCLUSIONS: Younger age and higher AMH were associated with achieving 50%, 60%, or 70% eLBR thresholds with Pl-OC. Nevertheless, almost all subjects were successfully able to preserve enough oocytes for ≥ 50% eLBR in 1-2 cycles.

Ethics in egg donation: past, present, and future.

Since the advent of clinical human egg donation just over 25 years ago, ethical considerations have been central to its successful application and popular acceptance. Early in its history, "essentialist" arguments questioning the moral validity of the practice altogether were commonplace. More recently, most academic discussion has been focused on "consequentialist" issues relating to practical approaches to egg donation that minimize ethically troublesome consequences. Three issues that have attracted a bulk of the attention in this regard are compensation, postmenopausal pregnancy, and egg sharing. Although much consensus has been reached on some very controversial issues, the enormous potential of increasingly successful oocyte cryopreservation, as well as emerging stem cell technologies, is very likely to provide abundant fuel for the continued debate of provocative and contentious ethical issues in human egg donation.

In vitro fertilization outcomes after transfer of embryos contaminated with yeast.

In this retrospective, matched-paired study, yeast in the embryo culture medium was associated with a trend toward decreased developmental competency that was more pronounced when observed early in culture. Because live births occurred after transfer of embryos in the yeast-contaminated group, we concluded that yeast contamination is not a reason to cancel embryo transfer (ET).