Successful fertility following optimized perfusion and cryopreservation of whole ovary and allotransplantation in a premature ovarian insufficiency rat model

Background: Fertility preservation by whole ovary cryopreservation and transplantation (WOCP&TP) with vascular anastomosis requires successful cryopreservation. In this study, we investigated the possibility of restoring ovarian function and natural fertility after WOCP&TP in a premature ovarian insufficiency (POI) rat model. The influence of cryopreservation on the offspring of rats following WOCP&TP was also explored.

Method: Rats aged 8-10 weeks were used as donors and recipients for allotransplantation. Fifteen rat whole ovaries were divided into three groups: the optimized group, the conventional group, and the fresh group. Different perfusion modes were used before cryopreservation and after thawing. Whole ovaries were observed by morphologic analysis, immunohistochemical staining, and transferase-mediated deoxyuridine triphosphate nick end-labeling assay. Ovarian function and fertility after WOCP&TP were then observed in 25 cyclophosphamide-induced POI rats for 8 months. Ovarian function was assessed by vaginal smears and blood hormone levels. Fertility restoration was quantified as the live birth rate after mating. The filial generation of rats was mated at 8-10 weeks of age. Offspring were observed for birth defect.

Results: Histological evaluation demonstrated intact morphology of follicles in all groups, with 77.6% of the total number of follicles identified as intact in the optimized group. The apoptotic rates of ovarian cells in the optimized group were significantly lower than that in the conventional group. Of the 20 live POI rats, 14 (70%) began to recover ovarian function after 2 weeks of transplantation, with normal hormone levels achieved 4 weeks after transplantation. Four of 14 rats were pregnant and delivered live offspring. One rat had a second pregnancy and delivered a second litter of live offspring. When the offspring matured, they were mated, and second and third generations of rats were born. All offspring had no abnormalities in appearance.

Conclusions: High rates of restoration of ovarian function and natural fertility with multiple generations of offspring were obtained following WOCP&TP in a cyclophosphamide-induced POI rat model by utilizing optimized perfusion. Cryopreservation did not affect the viability of successive generations.