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

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.

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.

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 slow-freezing 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).

The 10-min exposure group for vitrification showed better results compared with other conditions and the slow-freezing group.