Effects of vitrification solutons and equilibration times on the morphology of cynomolgous ovarina tissues vitrified ultra-rapidly by direct plungging into liquid nitrogen

Abstract

OBJECTIVE: Cryopreservation of preantral follicles in ovarian tissues has been expected to be an effective measure for preserving young women who need to undergo cytotoxic therapy. However, cryopreservation protocol has not yet been established. In this study, we assessed effects of vitrification solutions and equilibration times on morphologies of cynomolgus ovarian tissues vitrified ultra-rapidly using newly developed vitrification system which is consisted of 4 fine needles (Cryosupport). DESIGN: Experimental animal study. MATERIALS AND METHODS: Ovarian cortical sections (1 cm x 1cm x 0.1-0.2 cm) of 7 cynomolgus monkeys (85-137 months old) were randomly allocated to 6 groups. Ovarian sections were vitrified using 2 different vitrification solutions (35% (v/v) ethyleneglycol (EG) + 5% (w/v) PVP + 0.5 M sucrose (EG35) vs. 20% (v/v) EG + 20% (v/v) DMSO + 0.5 M sucrose (VSED)) after 3 different exposure times (5, 10 and 20 min). Ovarian sections put on the Cryosupport were vitrified ultra-rapidly by a direct application into liquid nitrogen. After warming, follicles were analyzed using light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: Five-minute exposure to EG35 brought in better follicle morphology (93%) compared with other conditions (77-89%) by LM and better mitochondrial morphology (88%) compared with other conditions by TEM (40-81%). Especially, the proportions of morphologically-normal follicles (77%) and of normal structure of mitochondria in oocytes (40%) of preantral follicles vitrified using VSED after 5 min were lower (P<0.01) than those achieved from follicles vitrified by other conditions (follicles: 87-94% and mitochondria: 78-88%, respectively). CONCLUSIONS: Results of the present study indicate that a combination of 35% EG with 5% PVP can support the morphology of vitrified preantral follicles and mitochondria in oocytes vitrified after the exposure for 5 min. Shorter exposure time to cryoprotectant might be expected to decrease the cytotoxic effect on the ovarian tissues.