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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE292062 | GEO | 2025/04/02

REPOSITORIES: GEO

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Project description:Vitrification is a method for long-term biological sample cryopreservation without causing intra- and extra-cellular ice formation. We recently established a novel closed vitrification system to cryopreserve mouse ovarian follicles. Using the 3D alginate hydrogel encapsulated in vitro follicle growth (eIVFG) method, we have demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes. Our recent study further demonstrated the faithful preservation of molecular signatures of follicle-stimulating hormone (FSH)-stimulated follicle maturation in vitrified follicles. However, it is unknown whether ovulation, another crucial gonadotropin-dependent follicular event, and involved ovulatory gene regulatory pathways are well conserved in vitrified follicles. Fresh and vitrified follicles grown for 8 days by eIVFG were collected at 0, 1, 4 and 8-hour post human chorionic gonadotropin (hCG) treatment for the single-follicle RNA sequencing. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Furthermore, the expression of several genes essential for ovulation were comparable between vitrified and fresh follicles. In summary, these results demonstrate that our closed vitrification system preserves follicular transcriptomic dynamics during ex vivo ovulation. Together with our previous findings that vitrification preserves FSH-stimulated follicle maturation, the integration of vitrification of immature follicles and eIVFG has a great potential to serve as an additional fertilization preservation approach for young cancer patients and endangerers species. Moreover, follicle vitrification enables a high-content ovarian follicle biobank, which can greatly improve the throughput of eIVFG for studying ovulation biology, anovulatory disease, toxicology, and novel contraceptive drug development targeting ovulation.

Project description:Vitrification is a method for long-term biological sample cryopreservation that transforms cells into a glass-like state by cooling without causing intra- and extra-cellular ice formation, which is a major driver of cell cryoinjury. Using an alginate hydrogel encapsulated in vitro follicle growth (eIVFG) system, we have recently demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes as well as comparable expression levels of several genes that are essential for gonadotropin-dependent folliculogenesis and oogenesis. However, it is unknown whether vitrification preserves the molecular signatures of folliculogenesis at the whole transcriptomic level, which is the primary research focus in this study. Multilayered secondary follicles with a diameter of 130 -160 µm were mechanically isolated from 16-day-old CD-1 female mice and vitrified using the closed vitrification method. After a 2-week storage in liquid nitrogen, vitrified follicles were warmed, and together with freshly-harvested follicles, cultured for 8 days using the eIVFG method. Next, mature antral follicles from day 8 were collected for single-follicle RNA sequencing using the SMART-seq RNA-seq method. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Using the criterion of fold change >2 or <0.5 and a false discovery rate (FDR) adjusted p-value < 0.05, there were 35 differentially expressed genes out of a total of 11,806 detected genes between vitrified and fresh follicles, among which 17 were up-regulated and 18 were down-regulated in vitrified follicles. To the best of our knowledge, none of those differentially expressed genes identified between vitrified and fresh follicles have been shown to be critical to folliculogenesis and oogenesis. Meanwhile, gene ontology (GO) and KEGG pathway analysis over these 35 genes using the functional annotation tool DAVID revealed that no GO terms or signaling pathways were significantly enriched. Furthermore, the expression of several genes essential for the gonadotropin-dependent folliculogenesis and oogenesis were comparable between vitrified and fresh follicles, including granulosa cell-specific genes (Cyp19a1, Amh, Inha, Inhba, Inhbb, and Fshr), theca cell-specific genes (Cyp17a1, Col1a2, and Col3a1), and oocyte-specific genes (Gdf9, Bmp15, Zp1, Zp2, and Zp3). Equally important, Lhcgr, the gene that is primarily expressed in theca cells of preantral follicles and then greatly induced in mural granulosa cells in antral follicles to prepare for the luteinizing hormone (LH) surge and ovulation, also had comparable mRNA expression levels between vitrified and fresh follicles. Taken together, these results demonstrate that vitrification preserves the follicular cell transcriptome and molecular signatures of gonadotropin-dependent folliculogenesis in the eIVFG system. In conclusion, our study re-evaluates individual follicle cryopreservation by using the closed vitrification method in a more in-depth perspective and proves that vitrification preserves the follicular cell transcriptome in the eIVFG system, thus providing a robust model for fertility preservation, conservation of endangered species, and also establishing a high-content ovarian follicle biobank for studying ovarian biology and female reproductive toxicology.

Project description:Oocyte competence for early embryo development relies on intercellular communication between the maturing oocyte and preovulatory follicle. Preovulatory follicle maturity, as indicated by serum estradiol concentration or follicle diameter, has previously been linked to pregnancy, follicular fluid metabolites, cumulus-oocyte metabolism, and oocyte competency for embryo development. Such relationships indicate metabolic and developmental programming of the oocyte based on the preovulatory follicle’s physiological status, but downstream impacts on the molecular signature of blastocysts have not been examined. We hypothesized that supplementing maturing oocytes with follicular fluid originating from preovulatory follicles of greater or lesser maturity would impact the transcriptome of resulting blastocysts and indicate metabolic programming of the embryo that originated from the oocyte’s maturation environment. The objective was to investigate the effect of follicle maturity on metabolic preparation of the oocyte by examining the functional genome of blastocysts originating from oocytes matured in the presence of follicular fluid from preovulatory follicles of greater or lesser maturity. In vitro maturing oocytes were supplemented with follicular fluid collected from preovulatory follicles of greater or lesser maturity. Following identical embryo culture procedures, RNA-sequencing was performed on pools of 2 blastocysts (Greater, n = 12; Lesser, n = 15; all with stage code = 7 and quality code = 1). A total of 12,310 gene transcripts were identified in blastocysts after filtering to remove lowly abundant transcripts. There were 113 transcripts that differed in abundance between blastocysts originating from oocytes matured in greater versus lesser maturity follicular fluid (eFDR < 0.01). Although no pathways were significantly enriched with differentially abundant transcripts, transcriptome profiles suggested improved Wnt/β-catenin signaling, metabolism, and protection from oxidative stress in blastocysts derived from oocytes matured in greater maturity follicular fluid, while unregulated cell growth presented in blastocysts resulting from the lesser follicle maturity treatment.

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

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