Ovarian survival 6 years after whole organ cryopreservation and transplantation

Abstract

It was previously reported (Arav et. al.2005) that ovarian function continued for up to 3 years after whole organ cryopreservation. Now we report the longest functional term survival (6 years) of autotransplanted frozen/thawed whole sheep ovaries. Descriptive research to perfect whole ovary cryopreservation/thawing procedures. Nine month old Assaf sheep were used for in vivo experiments (n = 3). Briefly, under general anesthesia one of the ovaries was excised leaving the contralateral in place. The ovarian artery was perfused with 10 ml of cold (4°C) UW supplemented with 10% DMSO for 3 minutes and then inserted into a glass freezing tube for cryopreservation. Unidirectional solidification freezing methodology reduced the temperature to –30°C at the very slow speed of 0.01 mm/sec, resulting in a cooling rate of −0.3°C/min after which the tubes were plunged into liquid nitrogen. Thawing was performed by placing the tubes into a 37°C water bath. Cryoprotectants were rinsed out from the thawed ovary via the ovarian artery using 10 ml Hepes-Talp medium supplemented with 0.5 M sucrose and 10 iu/ml heparin. Ovarian auto transplantation was performed by microscopic end-to-end vascular (artery and vein) reanastomoses to the contralateral ovarian vascular pedicle using 10–0 sutures at the same time that the intact ovary was removed. 6 years following transplantation sheep were sacrificed and the ovaries were excised, visually inspected, fixed in Bouin's liquid and embedded in paraffin for histology. Progesterone levels were measured in transplanted sheep up to 6 years post transplantation, twice per week indicating normal ovulation. Transplanted ovaries were of normal size and shape showing no necrotic lesions and one ovary clearly showed a recent corpus luteum. A total of 36 antral follicles were observed by transillumination and after sectioning, there were 4 GV's oocytes that matured in vitro to MII stage. Histological evaluation using Eosin & Hematoxylene revealed normal tissue architecture including follicles at various developmental stages and intact blood vessels. This report documents the longest term survival of whole ovary cryopreserved and then re-transplanted. Cryopreservation of whole ovaries using directional freezing and microvascular anastomoses is a promising method for preserving long-term reproductive capacity and endocrine function.