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.

Keywords: in vitro maturation; abbreviated stimulation; blastocysts; embryos; euploidy; granulosa cells; oocyte quality; ovarian support cells; stem cells.

Figure 1.

Study design flow chart. Schematic representation of the three independent experiments performed in the study, depicting stimulation protocols and embryology workflows. (A) Experiment 1 refers to Fig. 2. (B) Experiment 2 refers to Fig. 3. (C) Experiment 3 refers to Figs 4 and 5. (D) Representative image of co-culture setup at time of plating containing human cumulus–oocyte complexes (COCs) (n = 5) and 100 000 ovarian support cells (OSCs). Scale bar: 100 µm. COCs with varying degrees of cumulus enclosure are seen with surrounding OSCs in suspension culture. COCs, cumulus–oocytes complexes; ICSI, intracytoplasmic sperm injection; IU, international units; rFSH, recombinant follicle stimulating hormone; Clomid, clomiphene citrate; hCG, human chorionic gonadotropin; IVM, in vitro maturation; LAG, pre-incubation medium (MediCult); PGT-A, pre-implantation genetic testing for aneuploidy; EM-Seq; enzymatic methylation sequencing.

Figure 2.

OSCs improve human oocyte maturation rates compared to supplemented IVM medium lacking OSCs or containing other cell types. (A) Maturation rate of oocytes after 24- to 28-h in vitro maturation (IVM) experiments in Experiment 1, including oocytes co-cultured with ovarian support cells (OSCs), fetal-like ovarian somatic cells (FOSCs) or in Media Control. n indicates the number of individual oocytes in each culture condition. Error bars indicate mean±SEM. P-value derived from unpaired t-test comparing OSC-IVM to control (Media Control) and FOSC-IVM to control (Media Control). (B) Total Oocyte Score (TOS) generated from imaging analysis of metaphase II (MII) oocytes after 24- to 28-h IVM experiments. n indicates the number of individual MII oocytes analyzed. Median (dashed line) and quartiles (dotted line) are indicated. A one way analysis of variance (ANOVA) indicated no significant difference between the means of the three groups. Due to low numbers of retrieved oocytes per donor, oocytes could not be consistently split between both conditions. Groups contain oocytes from predominantly non-overlapping donor cohorts thus pairwise comparisons are not utilized.

Figure 3.

OSC-IVM demonstrates improved oocyte maturation compared to a commercially available IVM system. (A) Maturation rate of oocytes after 28-h in vitro maturation (IVM) experiments in Experiment 2, including oocyte co-culture with ovarian support cells (OSCs) or in the commercial IVM Control. n indicates the number of individual oocytes in each culture condition. Error bars indicate mean±SEM. P-value derived from paired t-test comparing Experimental OSC-IVM to the commercial IVM Control. (B) Total Oocyte Score (TOS) derived from imaging analysis of metaphase II (MII) oocytes after 28-h IVM experiments. n indicates the number of individual MII oocytes analyzed. Median (dashed line) and quartiles (dotted line) are indicated. An unpaired t-test indicated no significant (P=0.9420) difference between the means. Cumulus–oocyte complexes (COCs) from each donor were randomly and equitably distributed between control and intervention to allow for pairwise statistical comparison.

Figure 4.

OSC-IVM assisted oocytes are developmentally competent for healthy embryo formation. (A) Euploid blastocyst (Day 5 or 6) formation rate per cumulus–oocyte complex (COC) in Experiment 3 after oocyte co-culture with ovarian support cells (OSCs) or in the commercial in vitro maturation (IVM) Control. n indicates the number of individual oocytes in each culture condition. Error bars indicate mean±SEM. P-value derived from logistic regression comparing Experimental OSC-IVM to the commercial IVM Control. (B) Representative images of embryo formation in OSC-IVM versus commercial IVM conditions at Days 5, 6, and 7 of blastocyst formation. Embryos that were of suitable vitrification quality are labeled as ‘biopsied’ and were utilized for trophectoderm biopsy and preimplantation genetic testing for aneuploidy (PGT-A).

Figure 5.

OSC-IVM blastocysts display similar global and germline methylation profiles to COS blastocysts. (A) 19 germline differentially methylated regions (gDMRs) were profiled for their methylation pattern in five ovarian support cell in vitro maturation (IVM) (OSC-IVM) Day 5 or 6 euploid blastocysts and eight donated Day 5 or 6 euploid blastocysts after conventional ovarian stimulation (COS). Additional control data were obtained from Saenz-de-Juano et al. (2019) for biphasic IVM (CAPA-IVM) and COS samples. Symbols represent gDMR percentages of individual embryos. Data are plotted as median with error bars representing 95% confidence interval (CI). Statistical significance testing was performed using one way analysis of variance (ANOVA) with multiple comparisons testing for post hoc analysis comparison to the external reference COS control. (B) Whole genome methylation analysis of 5-methylcytosine (5mC) levels for the five OSC-IVM blastocysts and eight donated COS blastocyst controls. Statistical significance testing was performed using two-way unpaired t-test comparison to the internal reference COS control.