Clinical and experimental reproductive medicine · 2025
Rapamycin preserves primordial follicles during closed‑system vitrification of mouse ovarian tissue.
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Tanaka Yuji, Takebayashi Akie, Deguchi Mari, Amano Tsukuru, Nakamura Akiko, Inatomi Ayako, Tsuji Shunichiro, Murakami Takashi
Аннотация
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.