Rapamycin preserves primordial follicles during closed‑system vitrification of mouse ovarian tissue

Ovarian tissue cryopreservation is an essential fertility preservation technique. Two primary methods are used for ovarian tissue cryopreservation: slow freezing and vitrification. Recently, vitrification has been favored over slow freezing, and a closed system is recommended to prevent cross-contamination in liquid nitrogen. Follicular loss during freezing and thawing remains a major challenge. We investigated whether rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR) pathway, could mitigate primordial follicle loss during closed-system vitrification and thawing of mouse ovarian tissue.

Mouse ovaries were vitrified and thawed with or without 750 nanomolar rapamycin, then immediately analyzed or cultured for 5 days. Activation of the mTOR pathway was assessed using phosphorylated S6 kinase immunostaining, and follicle survival and development were evaluated by histological analysis.

Closed-system vitrification did not induce apoptosis in primordial follicles. The median phosphorylated S6K-positive rate in primordial follicles was 7.1% in fresh controls, 87.9% in the rapamycin-free group, and 19.0% in the rapamycin-treated group (fresh-control vs. rapamycin-free and rapamycin-free vs. rapamycin-treated, both p<0.001). Rapamycin treatment suppressed this activation, resulting in significantly higher primordial follicle counts after culture (605 vs. 289 follicles per ovary, p<0.05) and a lower ratio of primary to primordial follicles, indicating reduced follicle activation.

These findings demonstrate that rapamycin preserves the primordial follicle pool by preventing follicle activation during cryopreservation and thawing. Incorporating rapamycin into closed-system vitrification protocols may improve ovarian tissue cryopreservation outcomes and enhance fertility preservation for patients with cancer.