BEZ235-Mediated PI3K/mTOR dual inhibition improves ovarian follicle survival in a preclinical model.

BACKGROUND: Follicular loss after ovarian tissue cryopreservation and autotransplantation (OTCTP) remains a major challenge due to follicle activation and ischemia. We evaluated BEZ235, a dual PI3K/mTOR inhibitor, as a strategy to improve follicle survival in a preclinical model. Its effects were evaluated during ovarian culture, cryopreservation, and transplantation, including the potential benefit of post-grafting VEGF/G-CSF injections. METHODS: Murine ovaries, organotipically cultured with chemotherapeutic treatment (4-HC, 2 µM), with or without supplementation with BEZ235 (1 µM), rapamycin (1 µM), LY294002 (25 µM), or AMH (200 ng/ml) were used to evaluate follicle activation. For cryopreservation studies, those inhibitors were added to the freezing medium, and pathways activation were assessed via Western blot. In vivo, ovaries cryopreserved with or without BEZ235 or rapamycin were autotransplanted under the kidney capsule of mice. A subset of mice received intraperitoneal VEGF/G-CSF injections for five days post-transplantation. Follicle quantification, proliferation and activation marker assessment, and fibrosis evaluation were performed three weeks post-grafting. RESULTS: In vitro, BEZ235 significantly counteracted chemotherapy-induced activation of both Akt and mTOR pathways, whereas rapamycin and LY29400 inhibited only mTOR or Akt, respectively. Similarly, during cryopreservation, only BEZ235 significantly reduced activation of both pathways. AMH did not enhance BEZ235's protective effects. In vivo, ovaries slow-frozen with BEZ235 retained a higher percentage of primordial follicles and showed reduced follicle proliferation and activation compared to both control and rapamycin three weeks after transplantation. Additionally, post-grafting injection of VEGF/G-CSF did not further enhance follicle preservation or reduce fibrosis. CONCLUSION: Dual inhibition of PI3K/mTOR with BEZ235 provides superior protection of the primordial follicle pool by maintaining follicle dormancy, in both in vitro and in vivo models. These findings highlight BEZ235's potential to enhance OTCTP outcomes, extend graft longevity and improve fertility preservation strategies in women.