Optimal condition of vitrification method for cryopreservation of human ovarian cortical tissues

Aim: In order to find the optimal exposure time of cryoprotectant, we performed a comparison of vitrification versus slow freezing according to the degree of normal morphology and apoptosis of human ovarian follicles. Materials and Methods: Eleven patients aged 20–41 years who underwent operative laparoscopy for benign ovarian cysts or cesarean section were enrolled in this study. We carried out a prospective parallel comparison of survival and morphology of follicles after freezing (slow freezing and vitrification) and thawing. The ovarian strips were vitrified with two-step exposure to equilibration and vitrification solutions at room temperature. After various exposure times of cryoprotectant solution (5 min, 10 min, and 20 min, respectively), cryoprotectant-filled cryovials with pretreated cortical tissues were immediately plunged into liquid nitrogen. Results: In total, 336 follicles were analyzed by light microscopy to assess the morphology. The distribution of follicles was as follows: primordial, primary, and secondary follicles were 55.7% (187/336), 36.9% (124/336), and 7.4% (25/336), respectively. Vitrification in the 10-min exposure group preserved the follicles most effectively (ratio of grade 1 follicle: 3.6%, 34.7%, 13.8%, and 20.0% in the 5-min, 10-min, 20-min, and slowfreezing groups, respectively). Fewer terminal-deoxynucleotidyl-transferase-dUTP-nick-end-labeling-positive cells were found in vitrification in the 10-min equilibrium group compared with the other cryopreserved– thawed groups (52.1%, 31.5%, 53.1%, and 46.7% in the 5-min, 10-min, 20-min, and slow-freezing groups, respectively). The stromal cells were also better preserved in the 10-min group than the others (P < 0.05). Conclusions: The 10-min exposure group for vitrification showed better results compared with other conditions and the slow-freezing group.