P-542 Bidirectional signaling between oocytes and ovarian support cells (OSCs) during in vitro maturation results in improved oocyte gene expression

Abstract

Abstract Study question Are the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes bidirectionally influenced by co-culture during in vitro maturation (IVM)? Summary answer OSC gene expression is modulated by oocyte presence and OSC-IVM matured oocytes display transcriptomic resemblance to in vivo matured oocytes potentially due to OSC-oocyte crosstalk. What is known already The application of IVM holds a significant interest to improve infertility treatment. We previously showed the development of human OSCs generated from human induced pluripotent stem cells (hiPSCs) with ability to improve the maturation rates of immature human cumulus oocyte complexes (COCs) and embryo formation rates from minimal ovarian stimulation cycles (Piechota et al. 2023). We recently demonstrated that OSC-IVM can also be used to improve the nuclear maturation rate of human denuded oocytes from conventional stimulation cycles. Study design, size, duration We aimed to determine how gene expression changed in oocytes and OSCs by OSC-IVM co-culture. Immature fresh denuded oocytes (n = 141) were collected and randomized for IVM without OSCs (Media-IVM) (MediCult; n = 59) or with OSCs (OSC-IVM) (OSCs+MediCult; n = 82) for 24-28h. Subject enrollment ranged from 12/01/2023-06/30/2023. OSCs were collected for analysis pre-IVM (time 0h; n = 3), and post-IVM with (n = 3) and without (n = 3) immature denuded oocyte co-culture. Participants/materials, setting, methods 47 fertility patients undergoing conventional ovarian stimulation donated immature denuded oocytes (GV or MI oocytes) for research, which were allocated between either the Media-IVM or OSC-IVM conditions. In vivo matured oocytes (IVF-MII) were used as controls. Single oocyte RNA sequencing (RNA-seq) was performed after IVM culture, including both mature oocytes or stalled immature oocytes, alongside IVF-MII oocytes. Bulk RNA-seq was performed on OSCs and primary cumulus cells were used as a reference control (n = 7). Main results and the role of chance RNA-seq analysis demonstrated in the absence of oocytes, OSCs retain their global transcriptomic signature during IVM, maintaining or upregulating expression of CYP19A1, MDK, NR2F2, FOXL2, and CD82 and downregulating FSHR. When co-cultured with oocytes, OSCs shifted their global transcriptomic profile to resemble primary cumulus cells. In OSCs, we identified upregulation of IGF2, EGF, and IGFBP4, and downregulation of AMHR2, EGFR and IGFBP2 in the presence of oocytes, indicating oocyte-directed remodeling of OSC gene expression. Differential gene expression analysis in oocytes comparing MIIs and GVs post-IVM identified 31 genes differentially enriched and 353 genes differentially depleted in MIIs after co-culture with OSCs compared to 37 and 230 genes respectively without OSCs. IVF MII gene expression did not differ from OSC-IVM MII oocytes but did significantly differ from Media-IVM MII gene expression (p = 0.0008). Pathway enrichment analysis demonstrated broad similarity between IVF and OSC-IVM MIIs for Unfolded Protein Response, Myc Targets, and Oxidative Phosphorylation, while Media-IVM uniquely showed enrichment in DNA Damage and Spindle Assembly pathways. Media-IVM produced MIIs enriched uniquely for genes involved in electron transport chain (ETC) and oxidative phosphorylation (OXPHOS), which were not found in OSC-IVM or IVF MIIs. Limitations, reasons for caution The number of oocytes utilized in this study was limited. Additionally, as this study used rescue IVM as a model, it is likely that approaches using cumulus enclosed oocytes and traditional IVM may differ. Wider implications of the findings Bidirectional signaling between OSCs and oocytes may contribute to an improved in vitro environment that supports oocyte nuclear and cytoplasmic maturation. Therefore, oocytes matured in the presence of OSCs are more similar to oocytes matured in vivo and display a pathway enrichment profile associated with a higher oocyte developmental competence. Trial registration number not applicable