Ovarian Support Cell in Vitro Maturation (OSC-IVM) Results in Healthy Murine Live Births with No Evidence of Reprotoxicology in a Multigenerational Study

Study question Does application of human stem cell-derived ovarian support cells (OSCs) for in vitro maturation (IVM) have a safe reproductive toxicity profile?Summary answer The use of OSC-IVM co-culture improves blastocyst formation in a mouse model and results in healthy live births with no evidence of reprotoxicity.What is known already Abbreviated stimulation to obtain immature oocytes combined with a successful IVM offers a promising alternative to traditional in vitro fertilization, reducing hormonal doses and making IVF shorter and safer. Recently, we developed an OSC platform derived from human induced pluripotent stem cells (hiPSCs) that replicate dynamic ovarian function in vitro , enhancing human oocyte maturation and yielding an improved blastocyst formation rate compared to commercial IVM options. However, the reproductive toxicity profile, commonly assessed via murine multigenerational models, for OSC-IVM remains unknown.Study design, size, duration A total of 70 B6/CBA 6–8-week-old stimulated female mice were used in this study to collect immature mouse oocytes (n=2,025) at the germinal vesicle (GV) stage. Half of these oocytes were retrieved denuded (denuded oocytes condition, n= 930), while the remaining oocytes were kept with the cumulus cells (COCs condition, n= 1,095) to simulate the two possible dispositions of oocytes during clinical practice. Oocytes from each condition, denuded oocytes and COCs, were randomly assigned to either commercially available traditional IVM media (MediCult-IVMTM, Origio) group (control group) or the same traditional IVM media supplemented with human OSCs (FertiloTM, Gameto Inc.) to form the OSC-IVM group (test group).Participants/materials, setting, methods Oocytes from each condition, denuded oocytes and COCs, were subjected to in vitro culture for 18-20 hours. After IVM, metaphase II (M2) oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and cultured to assess blastocyst formation in vitro . Embryos that reached the blastocyst stage on day five were vitrified using Kitazato’s protocol in preparation for embryo transfers. A group of M2 oocytes and blastocyst embryos were employed for quality analyses by immunofluorescence.Vitrified blastocysts were warmed and transferred to pseudopregnant females (4-5 embryos per uterine horn), evaluating the F1 offspring. Pup characteristics were tracked, including weight, length, sex ratio, and physiology. Weekly monitoring assessed mouse behavior and development. At reproductive age, select F1 mice were outbred to wildtype mice to produce the F2 generation, analyzing live births, sex ratio, morphology, and behavior across groups. Moreover, hormonal and organ histological analyses were performed in F1 mice to further explore the overall health of the progeny.Main results and the role of chance In contrast to findings in humans, in mice OSC-IVM generally led to a decreased maturation rate compared to Traditional-IVM (68.6% ± 14.1% versus 80.9% ± 5.9%, p=0.0101). Subsequent embryo culture yielded significantly different fertilization rates between the four groups (p=0.0055). Specifically, OSC-IVM with COCs significantly differed from Traditional-IVM with denuded oocytes (89.5 ± 10.5 versus 96.5 ± 4.8, p=0.0098). There were no differences in the cleavage rates (p=0.7547). However, there was a significant distinction in the blastocyst formation (p=0.0068), wherein OSC-IVM with COCs showed a greater formation rate compared to Traditional-IVM for both denuded oocytes and COCs (56.1% ± 19.2% versus 41.5% ± 15.9% and 38.0% ± 16.2%; p=0.0408, and p=0.0063). Spindle morphology analysis demonstrated normal spindle morphology in denuded oocytes and COCs under both Traditional-IVM and OSC-IVM. Moreover, embryo analysis showed no significant difference in inner cell mass count (p=0.1550).Following embryo transfers, analysis of live births showed no significant distinctions between groups regarding delivery, sex ratio, pup length, developmental and behavioral abnormalities, hormonal values or histopathological anomalies in the F1 generation. Evaluation of the F2 generation also showed no significant differences in live births, sex ratio, or developmental/behavioral abnormalities between groups, further validating the absence of long-term implications and transgenerational effects derived from OSC-IVM culture.Limitations, reasons for caution Although this study was conducted in compliance with European Medicines Agency (EMA) ICH E6 (R2) Good clinical practice scientific guidelines to demonstrate the OSC safety, human clinical studies evaluating in vivo and live birth outcomes are necessary to corroborate the findings of this study.Wider implications of the findings This study provides evidence of the safety of using the OSC-IVM system, as evidenced by the lack of adverse effects on in vitro embryo development post OSC-IVM and on the health and fertility of offspring across successive generations in vivo .Trial registration number N/A### Competing Interest StatementThis research was funded by the for-profit entity Gameto Inc., and did not receive any other specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Noblett, Paulsen, Kramme, Barrachina, Potts, Marchante, and Piechota are shareholders in the for-profit biotechnology company Gameto Inc. Kramme, Piechota, Marchante, Paulsen, Potts, and Noblett are listed on a patent covering the use of OSCs for in vitro maturation: U.S. Provisional Patent Application No. 63/492,210. Additionally, Kramme is listed on three patents covering the use of OSCs for in vitro maturation: U.S. Patent Application No. 17/846,725, U.S Patent Application No. 17/846,845, and International Patent Application No.: PCT/US2023/026012. Kramme is additionally listed on three patents for the TF-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.