Angiogenesis stimulation of cryopreserved ovarian tissue

Introduction: Ovarian tissue (OT) cryopreservation is one of the most promising methods to address female fertility preservation. Maintenance of follicular viability during the freezing/warming process and after transplantation is a major challenge. Therefore, graft survival and lifespan are limited by the initial induced ischemia, causing the loss of 50% of primordial follicles. Aims of the study: Improve early angiogenesis in cryopreserved-thawed OT. Methods: In vitro and in vivo studies were performed. Initially, frozen/thawed OT was divided into 5 groups: OT immediately after thawing, cryopreserved OT cultured for 4-hours in the absence and presence of hMG, of VEGF+bFGF and of the association of the three products. Considering in vitro results, we performed auto-transplantation for 7 and 21-days of frozen/thawed OT that was previously cultured within an alginate matrix for 4-hours. Rat OT was cryopreserved and thawed with validated protocols. Follicular analysis was conducted after H&E staining. Apoptosis, proliferating and microvessel density were assessed by immunohistochemistry. Angiogenesis stimuli was analysed also by gene and protein expression in the OT and supernatant. Results: A decrease in primordial follicles density was observed in the in vitro study. However, the groups with hMG supplementation (hMG and hMG+VEGF+bFGF) show high degree of follicular proliferation. No differences were observed in follicular atresia and apoptosis. The 4-hour culture with angiogenic factors had similar levels of microvessel density, but there were differences in the expression of pro-angiogenic genes and proteins. In the in vivo study, the tissue submitted to the triple treatment (hMG+VEGF+bFGF) showed greater levels of microvessel density, 21-days after transplantation. The primordial follicular count remained stable, as well as the percentage of follicles and stromal cells with Ki67 and caspase-3 staining. Conclusion: Culture medium enriched with angiogenic factors (VEGF, bFGF and hMG) improved vessel area in the graft, with no impact on follicular density, proliferation and apoptosis.