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

Abstract

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.

Ovarian support cell in vitro maturation (OSC-IVM) results in healthy murine live births with no evidence of reprotoxicology in a multigenerational study

“…This study was conducted as a pivotal toxicology assessment of the recently described OSC-IVM method to assess the impact of co-culturing human ovarian support cells (OSCs) with immature oocytes (Piechota et al 2023). To achieve this, fresh immature mouse oocytes from the B6/CBA hybrid strain were collected at the germinal vesicle (GV) stage from stimulated females.…”

“…Ovarian support cells (OSCs) were generated from a single line of human induced pluripotent stem cells (hiPSCs) using overexpression of the transcription factors GATA4 , NR5A1 and RUNX2 , as previously described (Pierson Smela et al, 2023, Piechota et al 2023). OSCs are defined as a pure population of CD82+, FOXL2+ mural granulosa-like cells, and are known to be steroid and growth factor producing in response to follicle stimulating hormone (FSH) and androstenedione (A4) stimulation.…”

“…Supplemented IVM media equilibrated overnight at 37.3°C, 6% CO 2 and 7% O 2 under mineral oil (Hypure oil Heavy, Kitazato) was used for final resuspension. OSCs were then plated in suspension in non-adherent culture dishes at a concentration of 100,000 OSCs per 100 µl, as in our previous preclinical human studies (Piechota et al, 2023). The IVM condition containing OSCs (test article) in supplemented IVM media is referred to as OSC-IVM.…”

“…However, ongoing research and advancements in IVM protocols are necessary to enhance success rates and broaden their clinical applicability. Current IVM protocols predominantly rely on supplementing cell culture media with growth factors, small molecules, and hormones known to influence oocyte meiotic progression (Akin et al, 2021; Braam et al, 2019; De Vos et al, 2011; Fadini et al, 2009; Guzman et al, 2012; Ma et al, 2020; Mochini et al, 2011; Mohsenzadeh et al, 2022; Piechota et al, 2023; Pongsuthirak et al, 2015; Sanchez et al, 2019; Sanchez et al, 2017; Shu-Chi et al, 2006; Vuong et al, 2020a, 2020b). However, these protocols have yielded various outcomes concerning oocyte maturation and embryo formation rates (Gilchrist et al, 2024), often attributed to challenges associated with the synchronization of nuclear-cytoplasmic maturation and the heterogeneity of oocyte disposition at retrieval (Ahmad et al, 2023).…”

“…Leveraging their functional resemblance to human granulosa cells, Fertilo OSCs enhance the nuclear and cytoplasmic maturation of human oocytes in vitro by supplementation into traditional IVM media. Consequently, OSC-IVM significantly improves human oocyte maturation rates and subsequent embryo development, yielding an improved blastocyst formation rate compared to traditional IVM options (Piechota et al, 2023; Giovannini et al 2023).…”