Damage to fetal bovine ovarian tissue caused by cryoprotectant exposure and vitrification is mitigated during tissue culture

Abstract

Purpose: The objective of this study is to characterize the impact of exposure to cryoprotectants followed by vitrification on primordial follicle survival and activation using a fetal bovine model. Methods: In the first study, fetal bovine cortical pieces were exposed to cryoprotectants with or without sucrose and cultured up to 7 days in the presence or absence of insulin. In the second study, cortical pieces were exposed to cryoprotectants with or without sucrose, vitrified, and cultured up to 7 days after warming in the presence or absence of insulin. Viability and morphology of follicles, as well as proliferation and/or DNA repair in ovarian tissue were analyzed. Results: When compared to non-exposed controls, normal follicular morphology was affected in groups exposed to cryoprotectants only immediately post-exposure and after 1 day of culture, but improved by day 3 and did not significantly differ by day 7. Similarly, normal follicular morphology was compromised in vitrified groups after warming and on day 1 compared to controls, but improved by days 3 and 7. Proliferation and/or DNA repair in ovarian tissue was not affected by vitrification in this model. Cryoprotectant exposure and vitrification of ovarian tissue did not impair the activation of primordial follicles in response to insulin, although activation was delayed relative to non-exposed controls. Interestingly, sucrose had no noticeable protective effect. Conclusion: Vitrified fetal bovine ovarian tissue has the intrinsic capacity to mitigate the immediate damage to primordial follicles’ morphology and retains the capacity to activate. These findings provide a basis for a successful cryopreservation protocol for ovarian cortical tissue in other species including humans.