Transcriptomic insights into ovine ovarian response to extended in vitro culture and cryopreservation

PURPOSE: Sheep ovarian tissue is a valuable model for studying folliculogenesis, cellular interactions, and fertility preservation strategies. The impact of culture duration, culture conditions, and cryopreservation techniques on the transcriptome of ovarian tissue remains incompletely understood. METHODS: Ovarian fragments were subjected to conventional or bioreactor culture for up to 12 weeks, with or without prior cryopreservation by slow freezing or vitrification. RNA was extracted at multiple time points and analyzed using RNA sequencing to identify differentially expressed genes and enriched pathways. RESULTS: Prolonged conventional culture induced metabolic reprogramming, increased glycolytic and biosynthetic activity, and triggered stress, inflammatory, and even apoptosis pathways, whereas bioreactor culture limited stress responses and better-preserved cell proliferation and development. Ovarian tissue culture in bioreactor allowed for extended in vitro culture periods of up to 12 weeks, whereas conventional culture did not allow survival of the samples. Cryopreservation had minor effects on gene expression, with slow freezing and vitrification yielding largely similar transcriptomic profiles. Folliculogenesis-related genes showed a trend towards downregulation. CONCLUSIONS: Bioreactor culture seems to provide a more physiologically relevant environment for maintaining ovarian tissue viability and function, and cryopreservation seems to have limited impact on the transcriptome compared to culture conditions. These findings offer valuable insights for optimizing fertility preservation and translational approaches in reproductive medicine.