Survival of primordial follicles in vitrified-thawed whole ovary of New Zealand white rabbit

Abstract

One of the greatest challenges in cryobiology is the cryopreservation of entire organs. In spite of its difficulty, whole ovary freezing will have been one of developed tools of reproductive medicine for the preservation of the reproductive function in young women who are faced with premature ovarian failure through cancer therapy. The purpose of present study was to establish the cryopreservation method for whole ovary by using vitrification following perfusion with cryoprotective.agent (CPA). Effect of the CPA concentration and its penetration time during whole ovary perfusion on the survival of ovarian tissue after vitrification-thawing. For whole ovary vitrification, New Zealand white rabbits (2month, 2.5kg) were anaesthetized using sodium pentobarbital. Ovaries and the related blood vessels were removed by cut of aorta and vena cava. We perfused for each 10 or 20 min with CPA (VS40 (40%); PBS containing 3.1 M of diemethyl sulfoxide and 2.2 M 1.2-propanediol) increased concentrations gradient (10♢40%; 10♢ 20♢40%). And whole ovaries and reproductive organs in container after perfusion were cooled ultra-rapidly by placing on liquid nitrogen. After storing for several days at -80°C, the container was quickly thawed in 37°C water bath and the CPA was removed from whole ovaries and reproductive organs by re-perfusion with a reversed concentration gradient. Vitrified-thawed ovaries were sliced and cultured in vitro for 0 or 6 hours. In order to check their viability, cultured ovarian tissues were fixed and examined by H/E staining and immunohistochemical analysis using PCNA (cellular proliferation marker). Even though vitrified-thawed ovary showed poor morphology compared to fresh one, most of stromal tissues were survived by H/E staining. At thawing and rehydration, survival rate of primordial follicles was not different in all groups. However, in 6hours after in vitro culture, survival of follicles in perfusion group for 20 min interval of 3step (10–20-40%) was higher than others group. Also, the localization of PCNA was observed in a same pattern. These results demonstrated that rabbit's whole ovary can be, in part, successfully cryopreserved by vitrification conjunction with perfusion. Therefore, this method of whole ovary freezing could be a fertility option for women and children who will have been undergoing sterilizing chemotherapy in the future. However, further research for improving the survival rate of follicles will be needed in order to apply for clinical trial.