Ovarian cryopreservation with rapamycin improves fertility restoration in a murine orthotopic transplantation model

Currently, the only fertility preservation option of prepubertal patients is ovarian tissue cryopreservation followed by autotransplantation (OTCTP). Once in remission and patients desire to conceive, autotransplantation of frozen/thawed tissue is performed. A major issue of this technique is follicular loss directly after transplantation, mainly due to follicle activation. Our previous research showed that adding rapamycin to the freezing medium counteracted follicle proliferation and activation induced by OTCTP in heterotopic autotransplantation of ovaries in mice. Our current study aimed to test the potential of this approach to improve fertility restoration in mice. Forty 4-week-old female C57BL/6 mice underwent unilateral oophorectomy followed by slow-freezing of ovaries with or without rapamycin. After chemically disabling the remaining ovary, orthotopic autotransplantation was performed. After recovery, estrous cycle analysis was conducted using daily vaginal smears. The mice were mated with males for 4 months, and pregnancy outcomes were recorded. After mating, half the females were super-ovulated for oocyte quantification and ovarian analysis, while the others had their ovaries collected for analysis of remaining primordial follicles using immunohistochemistry. Female mice whose ovaries were cryopreserved with rapamycin prior to chemically disabling the remaining ovary and orthotopic autotransplantation, gave birth to more pups (102 rapamycin, 48 control). The live birth rate was also higher (P = 0.0025) when ovaries were cryopreserved in rapamycin compared to control medium. Additionally, more mice in the rapamycin group gave birth (13 rapamycin, 8 control) with a higher average litter size (P = 0.0837). More mice had primordial follicles left at the end of the experiment in the rapamycin group (P = 0.0397). Superovulation showed a similar number of oocytes collected (P = 0.4462). While rapamycin did not influence cyst formation after autotransplantation, mice that developed ovarian cysts gave birth to fewer pups per dam (P = 0.0119) with a lower live birth rate compared to mice without ovarian cysts (P = 0.0032). The use of rapamycin improved fertility restoration in mice. Using rapamycin during OTCTP in humans could potentially resolve the massive follicular loss directly after grafting, and thus eventually lead to better opportunities for women to become pregnant.