P–444 Presence of pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR signalling pathways during cryopreservation and organotypic cultures of murine ovaries limits early primordial follicle depletion

Study question Which signalling pathways are implicated in primordial follicle activation induced by cryopreservation and/or organotypic culture? Is it possible to limit this activation through pharmacological inhibitors? Summary answer Our findings provide support for the hypothesis that mTOR and PI3K inhibitors might represent an attractive tool to delay cryopreservation- and culture-induced primordial follicle activation. What is known already Cryopreservation of ovarian tissue containing immature primordial follicles followed by auto-transplantation (OTCTP) is the only option available to preserve the fertility of prepubertal patients or patients requiring urgent therapy for aggressive malignancies. However, a major obstacle in this process is follicular loss immediately after grafting, possibly due to slow neovascularization, apoptosis and/or massive follicular recruitment. In vitro and in vivo studies indicate that the PI3K/PTEN/Akt and mTOR signalling pathways are involved in follicle activation. The transplantation process seems to be the major cause of primordial follicle activation after OTCTP but information about how cryopreservation itself impacts follicle activation is sparse. Study design, size, duration Whole murine ovaries (4–8-weeks old) were cryopreserved by slow freezing and exposed to LY294002 (a powerful PI3K inhibitor) or rapamycin (a specific mTOR inhibitor) during cryopreservation and/or organotypic in vitro culture for a 24 h or 2 days. Participants/materials, setting, methods Western Blot and immunofluorescence analyses were used to determine the activation of PI3K/PTEN/Akt and mTOR signalling pathways in murine ovaries cryopreserved and/or organotypically cultured with/without inhibitors.Follicles were quantified according to their maturation degree on H&E stained histological sections. Main results and the role of chance Ratio of phosphorylated Akt or rps6 to total proteins (p-Akt/Akt and p-rps6/rps6) was increased in slow-frozen murine ovaries compared to control fresh ovaries, indicating an activation of the PI3K/PTEN/Akt and mTOR signalling pathways. The use of pharmacological inhibitors of follicle signalling pathways (LY294002 (25µM) and rapamycin (1µM)) during the cryopreservation process decreased p-Akt/Akt and p-rps6/rps6 ratios. In vitro organotypic culture for 24 h increased only the activation of the PI3K/PTEN/Akt pathway, as shown by increased p-Akt/Akt ratio in fresh ovaries cultured for 24 h compared to fresh non-cultured ovaries. This activation can be counteracted by cryopreservation of murine ovaries with rapamycin followed by in vitro culture for 24 h in the presence of LY294002. Follicle density quantifications indicated that when cryopreserved ovaries were maintained in culture for 2 days, a decrease of primordial follicle density concomitant with an increase of secondary and more mature follicles were found in comparison to slow-frozen/thawed ovaries without culture. Supplementation of the culture medium with LY294002 and rapamycin for 24 h or 2 days preserved primordial follicle densities compared to ovaries cultured without inhibitors. Limitations, reasons for caution This study is an in vitro study using murine ovaries. To analyze the efficiency of LY294002 and rapamycin to limit cryopreservation and transplantation induced follicle recruitment, these inhibitors should be tested in an in vivo model. Furthermore, these findings will need to be confirmed with human samples. Wider implications of the findings: We showed for the first-time that the sequential use of pharmacological inhibitors, rapamycin during the slow freezing process followed by organotypic culture supplemented with LY294002, is effective to limit early primordial follicle depletion. Trial registration number /