Vitrification preserves oocyte transcriptome in a 3D in vitro follicle development and oocyte maturation system

Vitrification is increasingly used to cryopreserve gametes and embryos in assisted reproductive technology (ART). Our prior research demonstrates that vitrification preserves the viability and functionality of ovarian follicles. However, its impact on follicle-enclosed oocyte remains unknown. The current study investigates whether vitrification, combined with a 3D encapsulated in vitro follicle growth (eIVFG) system, maintains oocyte transcriptome during in vitro follicle development and oocyte maturation. Immature mouse preantral follicles were vitrified and cultured in eIVFG for 8 days to grow to the preovulatory stage, followed with the induction of ovulation and oocyte maturation on day 9, with fresh follicles as the control. Oocytes at germinal vesicle (GV) stage from grown preovulatory follicles on day 8 and oocytes at metaphase II (MII) upon ovulation on day 9 were collected for single-oocyte Smart-Seq2 RNA sequencing analysis. The principal component analysis (PCA) separated GV and MII oocytes into two distinct clusters, but oocytes from fresh and vitrified follicles were largely overlapped. Differential gene expression (DEG) analysis revealed that GV or MII oocytes from fresh and vitrified follicles had comparable expression of maternal effect genes and other genes related to oocyte meiotic and developmental competence. There was a significant transcriptomic change during the GV-to-MII transition. Gene ontology (GO) and KEGG analysis identified DEGs between GV and MII oocytes related to cell cycle, RNA processing, mitochondria, and ribosome. In summary, our study demonstrates that vitrification preserves oocyte transcriptome during in vitro follicle development and oocyte maturation, supporting its potential in fertility preservation. Moreover, our single-oocyte RNA sequencing analysis identifies key DEGs upon GV-to-MII transition, indicating their potential functions in underpinning oocyte meiotic and developmental competence. Immature mouse preantral follicles were vitrified and cultured in eIVFG for 8 days to grow to the preovulatory stage, followed with the induction of ovulation and oocyte maturation on day 9, with fresh follicles as the control. Oocytes at germinal vesicle (GV) stage from grown preovulatory follicles on day 8 and oocytes at metaphase II (MII) upon ovulation on day 9 were collected for single-oocyte Smart-Seq2 RNA sequencing analysis.