Vitrification preserves oocyte transcriptome in a 3D <i>in vitro</i> follicle development and oocyte maturation system

Vitrification is increasingly used to cryopreserve gametes and embryos in assisted reproductive technology. Our prior research demonstrates that vitrification preserves the viability and functionality of follicles. However, its impact on oocyte remains unknown. The current study investigates whether vitrification maintains the oocyte transcriptome during in vitro follicle development and oocyte maturation. Immature mouse preantral follicles were vitrified, then warmed and cultured in vitro for 8 days to grow to the preovulatory stage, followed with 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) post-ovulation on day 9 were collected for single-oocyte Smart-Seq2 RNA sequencing. Principal component analysis separated GV and MII oocytes into two distinct clusters, but oocytes from fresh and vitrified follicles largely overlapped. Differentially expressed genes (DEG) analysis revealed that oocytes from fresh and vitrified follicles, at either GV or MII stage, had comparable expression of maternal effect genes and other genes related to oocyte meiotic and developmental competence. There was a significant transcriptomic change in oocytes during GV-to-MII transition. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified DEGs between GV and MII oocytes related to cell cycle, RNA processing, mitochondrion, and ribosome. In summary, our study demonstrates that vitrification preserves oocyte transcriptome during in vitro follicle development and oocyte maturation, supporting its potential for fertility preservation. Moreover, key DEGs identified during GV-to-MII transition indicate their potential functions in oocyte meiotic and developmental competence.