Appropriate Osmotic Balance Duration for Different Volumes of Ovarian Tissue in Vitrification Solution: A Study of Ovary Tissue Vitrification and Transplantation in Sheep

BACKGROUND: Auto-transplantation of cryopreserved ovarian tissue has become a promising method for fertility preservation and standardization of the process is crucial for practical applications. OBJECTIVE: Here we used different size of large sheep ovarian cortex to study the most suitable osmotic balance durations in the vitrification solution for large piece ovary cortex. MATERIALS AND METHODS: The ovarian cortices from six-month old female sheep were divided into 40 mm³, 80 mm³ and 160 mm³ volume, A two-step osmotic balance method was used based on the results from morphological and histological study, we detected the expression of VEGF after thawing, the percentage of follicles that survived and serum E2 levels, together with apoptosis test by TUNEL. RESULTS: the morphology of follicles and stromal cells were the best when the osmotic balance duration was 7 min, 11 min and 19 min, respectively. Osmotic balance time(T) is proportional to the area(S), we deduced that areas(S) of large pieces of ovarian cortex in vitrification fluid conformed to the standardized osmotic balance time(T) formula; i.e., T= (S+15)/5 ( 22∼24°C, sample thickness 1-2 mm), In vitro culture of vitrification-thawed large cortex ovaries and xenogenic heterotopic transplantation by using the standardized osmotic balance duration formula were both successful. CONCLUSION: Formula T= (S+15)/5 can be used to calculate optimal osmotic balance duration for different size of ovarian cortexes during vitrification.