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.