Background:
During IVF, maximising oocyte yield through high dose ovarian stimulation produces more embryos for transfer but does not increase per oocyte conception rates. In fact, there is evidence excessive FSH stimulation impairs oocyte quality. Most aging studies use ovarian stimulation to obtain oocytes, so it is unclear whether reported changes with age reflect physiological aging or are confounded by stimulation. We hypothesised that ovarian stimulation produces lower-quality oocytes, and that with advanced age, stimulation masks the natural age-related changes in the oocyte transcriptome.
Methods:
Single-oocyte RNA sequencing libraries were generated from naturally ovulated young (3-month, n=40), aged (10-month, n=36), and ovarian-stimulated (n=9) mouse oocytes. Mitochondrial function was assessed using vital dyes (Mitotracker Green, TMRM) in young (n=46), ovarian stimulation (n=82) and aged (n=55) mouse oocytes. The transcriptomic data was analysed using DESeq2 to identify differentially expressed genes (DEGs; FDR-adjusted p<0.05) and gene ontology to compare treatment groups with young naturally ovulated oocytes.
Results:
Ovarian stimulation altered 2,771 genes relative to natural ovulation, enriched for processes involving chromosomal segregation, DNA-methylation, endocytosis, ATP-production and protein synthesis. Aging altered only 236 genes, enriched for ubiquitination, proteolysis and RNA metabolism. Fifty-five genes overlapped between the two DEG sets with a concordant change in expression. Ovarian stimulation resulted in altered mitochondrial dye uptake alongside changes in mitochondrial gene expression. However, aged oocytes showed altered mitochondrial function without corresponding transcriptomic changes, suggesting that previous reports of altered mitochondrial gene expression in aged oocytes may instead result from ovarian stimulation.
Conclusion:
This study identified shared changes in gene expression representing candidate regulators of the oocyte stress response, warranting further functional analysis. Although ovarian stimulation accurately reflects the context of assisted reproduction, this study suggests treatment introduces a confounding effect on oocyte biology. These findings support naturally ovulated oocytes as the more physiologically accurate model for studying oocyte aging.