OVARIAN TISSUE CRYOPRESERVATION Effect of freezing and environment on follicle activation

Details

Supervisors

Dolmans, Marie-Madeleine

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

While cancer incidence remains stable, increasing numbers of both adults and children are now surviving their disease. However, several proposed treatments are gonadotoxic and pose a real threat to female fertility. For adult women who cannot postpone anti-cancer therapy and young girls of pre-pubertal age, cryopreservation of ovarian tissue (OT) is the only way to preserve their fertility. Cryopreserved OT reimplantation makes it possible to restore fertility for 5-7 years following the procedure. Unfortunately, after reimplantation, there is a massive follicle burnout through an abnormal follicle activation and death process which is probably related to the hypoxic conditions in the early post-grafting period. This study aims to (i) investigate the impact of cryopreservation on follicle activation in frozen-thawed (FT) OT by comparing it with fresh OT using the in vitro culture (IVC) technique, and (ii) explore the impact of the immediate environment on follicle activation in cryopreserved OT by comparing the IVC and the chick chorioallantoic membrane (CAM) grafting methods. To this end, we studied follicle activation in fresh IVC, in FT IVC, and in FT CAM-grafted groups during 0, 1 and 6 days. Histological analyses were performed to assess follicle density and follicle classification. Immunohistochemistry (IHC) was used to assess follicle proliferation, follicle apoptosis, and follicle autophagy by labelling Ki67, cCasp3 and Lc3b, respectively. Follicle activation was evaluated via the PI3K/AKT signaling pathway by targeting pAKT and p-rpS6 (by IHC) and cytoplasmic Foxo-1 (by immunofluorescence) which are the active protein forms stimulating follicle growth. Gene expression of oocyte-specific genes involved in oocyte growth, such as Lhx8 and GDF9, and inhibitory factors of the PI3K/AKT pathway, such as p27 and Tsc1, were studied by ddPCR to conclude on follicle activation. The ultrastructure of the follicles was scrutinized by transmission electron microscopy to study morphological changes occurring in follicles subjected to 6 days of IVC and CAM. Results obtained so far showed a significant decrease in the proportion of primordial follicles associated with a significant increase in the proportion of growing follicles after 6 days of both IVC and CAM. The analysis of follicle proliferation and apoptosis confirmed the primordial follicle pool burn-out occurring in both fresh and FT OT after 6 days of IVC. This suggests that cryopreservation does not affect the behavior of follicles within the tissue. However, follicle growth was significatively slowed down in FT OT grafted on CAM compared to FT OT cultured in vitro, with a better preservation of the primordial follicle pool in the CAM group. This suggests that the immediate environment probably plays a role in regulating follicle behavior. Furthermore, follicle vacuolization increased significantly in all groups across IVC and CAM-grafting. Nevertheless, further investigations showed that vacuolated follicles were not more predisposed to undergo apoptosis nor autophagy than normally shaped follicles. Understanding the mechanisms underlying follicle activation in cryopreserved OT is the first step in developing future strategies capable of sustaining the primordial follicle pool in order to extend the fertile lifespan of women requiring OT cryopreservation and transplantation.