P-225: The Reactive Oxygen Spieces Production of Mice Vitrified Isolated Pre-Antral Follicles Using Cryotop Methods

s:2022:"Background: Cryopreservation is an important option for preserving the fertility. We examined the reactive oxygen species (ROS) production, total antioxidant capacity (TAC) and developmental competence, of vitrified isolated mice pre-antral follicles as an alternative method for restoring fertility. Materials and Methods: The 140-160 μm pre-antral follicles mechanically isolated from ovaries of 14-day-old mice were exposed to equilibration solution consisting of 7.5% ethylene glycol (EG) and 7.5%dimethyl sulfoxide (DMSO) and 20% FBS for 5min and then exposed to vitrification solution composed of 15%EG, 15%DMSO, 20%FBS and 0.5M sucrose for 30 seconds and then were vitrified by using cryotop methods. Cryoprotectants were removed by equilibrating vitrified follicles in "warming solutions" consisting of descending concentration of sucrose and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. In parallel, after 0, 24, 48, 72 and 96 hours of culture, ROS and TAC were analyzed by spectoflorometry and FRAP methods respectively. Results: The growth rates of follicles in the second and fourth days of cultivation were higher in control group than vitrified group (p<0.05). The rates of Survival and antrom formation of control groups were significantly higher (p<0.05) than those of vitrified isolated follicles. Significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than those of fresh controls (49.2 vs. 14.2%, p<0.05). ROS production, and TAC levels of culture of pre-antral follicles in vitrified and non-vitrified groups were decreased after 24 hours up to 96 hours, at iniate time of cultivation ROS production of vitrified follicle was significantly higher and TAC levels was significantly lower than non-vitrified samples (p<0.05) Conclusion: Vitrification and in vitro culture of pre-antral follicles inducing the reactive oxygen species production.";