Mouse ovarian follicle cryopreservation using vitrification or slow programmed cooling: Assessment of in vitro development, maturation, ultra-structure and meiotic spindle organization

Aim: To compare different outcomes of vitrification and slow freezing of isolated pre-antral follicles and to evaluate different cryo-devices for vitrification of isolated follicles. Methods: Pre-antral follicles were isolated from mouse ovaries and cryopreserved using vitrification and slow freezing.A preliminary experiment was carried out to select the optimal cryo-device for vitrification of isolated follicles. A total of 414 follicles were randomly distributed among four groups: control (CT) fresh (n = 100), nylon mesh (n = 96), electron microscopy grid (n = 102), and micro-capillary tips (n = 116). Subsequently, a total of 979 follicles were randomly assigned to three different groups: CT fresh (n = 256), vitrification (n = 399) and slow freezing (n = 324). CT and cryopreserved/thawed follicles were cultured in vitro and examined daily for development. Final maturation was triggered with human chorionic gonadotrophin and rates of oocyte maturation were calculated. The ultra-structure of cryopreserved/thawed follicles was studied using electron microscopy. Meiotic spindle presence and organization in mature oocytes were examined using the Oosight imaging system. Results: Micro-capillary tips resulted in poor immediate post-warming survival but no differences were observed in the subsequent in vitro development characteristics between different cryo-devices. Nylon mesh proved to be the easiest carrier, particularly when large numbers of follicles were to be vitrified. Compared to vitrification, slow freezing resulted in a significantly lower number of intact follicles at the end of the culture period (P < 0.0001). However all other outcome measures were comparable between both techniques. Conclusions: Isolated follicles were more vulnerable to cryodamage after slow freezing as compared to vitrification. © 2010 Japan Society of Obstetrics and Gynecology.