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

Abstract

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.

Keywords: BEZ235; Fertility preservation; Follicle activation; Organotypic ovarian culture; Ovarian tissue cryopreservation; Ovarian transplantation; PI3K/PTEN/Akt signaling; Preclinical mouse model; mTOR inhibition.

Fig. 1

Effects of In Vitro Organotypic Ovarian Culture under Chemotherapeutic Conditions on Follicle Pathway Activation. Fresh ovaries from 4-weeks-old C57BL/6 mice were cultured for 24 h in the presence of rapamycin (1 µM), LY294002 (25 µM), or BEZ235 (1 µM) under chemotherapeutic conditions with 4-HC (4-hydroperoxycyclophosphamide). A Fold change of phosphorylated to total protein form ratio of Akt and B Rps6, including representative blots. CT = control, LY = LY294002, RAPA = rapamycin, BEZ = BEZ235. n = 6 samples/group. * p ≤ 0.05

Fig. 2

Effects of Adding Rapamycin, LY294002, or BEZ235 During Cryopreservation on Follicle Activation in Murine Ovaries. Ovaries from 4–7-days-old C57BL/6 mice were cryopreserved with or without rapamycin (1 µM), LY294002 (25 µM), or BEZ235 (1 µM). A Fold change of phosphorylated to total protein form ratio of Akt and B Rps6, normalized for Actin, including representative blots. CT = control, LY = LY294002, RAPA = rapamycin, BEZ = BEZ235. n = 6 samples/group, with each sample comprising of 4 pooled ovaries. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Fig. 3

Effects of BEZ235 and AMH on Follicular Preservation Under Chemotherapeutic Conditions in an Organotypic Ovarian Culture. Frozen/thawed ovaries from 4-weeks-old C57BL/6 mice were cultured for 24 h under chemotherapeutic conditions (4-HC) with or without AMH (200 ng/ml) or BEZ235 (1 µM). A Fold change of phosphorylated to total protein form ratio of Akt and B Rps6, including representative blots. C Quantifications of primordial follicles and D health assessment. E Representative image with unhealthy primordial follicles represented by asterisks and healthy primordial follicles by arrowheads. 4-HC = 4 hydroxy cyclophosphamide, AMH = Anti-Müllerian hormone, BEZ = BEZ235. n = 4–6 ovaries/group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Fig. 4

In Vivo Effects of BEZ235 on Follicle Preservation After Cryopreservation and Transplantation. Experimental scheme (A). IHC assisted follicle quantification (B, DDX4), and analysis of follicle proliferation (C, Ki67) and activation (D, pAkt and E, pRps6), in ovaries slow-frozen in control medium or medium supplemented with rapamycin (1 µM) or BEZ235 (1 µM), autotransplanted under the kidney capsule of C57BL/6 mice, including representative images. SFct = slow-frozen/thawed ovaries in control medium, SFrapa = slow-frozen/thawed ovaries in medium supplemented with rapamycin, SFbez = slow-frozen/thawed ovaries in medium supplemented with BEZ235. n = 7–10 ovaries/group. Arrows: black = primordial follicle, red = primary follicle, purple = secondary or more growing follicle, white = negative staining, orange = positive staining. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Fig. 5

Effects of VEGF and G-CSF on Follicle Preservation in BEZ235-Treated Cryopreserved Ovaries Post-Transplantation. A Experimental scheme. B, C IHC-assisted primordial follicle quantification in ovaries slow-frozen in control medium (SFct) or medium supplemented with BEZ235 (SFbez, 1 µM), autotransplanted under the kidney capsule of C57BL/6 mice, followed by intraperitoneal (IP) injection of VEGF (8 µg/kg/day), G-CSF (50 µg/kg/day), or their combination. D Analysis of primordial and primary follicle proliferation via Ki67 immunostaining. n = 9–10 ovaries/group. * p ≤ 0.05, ** p ≤ 0.01