P-36: Addition of Zinc in The Vitrification Medium Improves In Vitro Maturation of Oocytes Derived from Vitrified-Warmed Follicles
Follicle cryopreservation has been proposed as an alternative fertility preservation option. Follicles can be cryopreserved in intact ovarian tissue pieces or after isolation of individual follicles from the fresh ovarian tissue using enzymatic or mechanical techniques. Researchers have used different cryoprotectants and various techniques to improve cryopreservation. It is plausible that inclusion of an antioxidant in the cryopreservation solutions may help in maintaining embryo viability after cryopreservation by reducing the harmful effects of oxygen radicals during the cryopreservation procedure. Zinc is an essential trace element. It has been known as a non adequate level of zinc can alter not only gene expression but also a variety of cellular functions.The present study was designed to determine the effects of different zinc concentrations in the follicle vitrification solutions on incidence of follicles viability and in vitro maturation of oocytes derived from vitrified-warmed follicles.
Materials and methods
Ovaries of 2-4 week-old NMRI mice were removed from the animals after being killed and placed in alpha-minimum essential medium (α-MEM; supplemented with 10% (FBS) and 100 IU/ml penicillin +100 μg/ml streptomycin). The ovaries were mechanically dissected using fine hypodermic needles and follicles randomly assigned to following groups :V0,V1,V2 ,V3 (vitrified-warmed follicles with 0, 100,150 and 200 μg/dl zinc concentration in vitrification solution). Follicles were vitrified sequentially by immersion into two vitrification solutions VS1: 7.5% ethylene glycol (EG) and 7.5% DMSO in α-MEM supplemented with 20% FBS for 3 minutes and VS2: 15% EG and 15% DMSO in α-MEM supplemented with 20% FBS and 0.5 M sucrose for 30 seconds. They were placed in the pulled straw with a minimum volume of vitrification medium and then plunged into liquid nitrogen for 1 week. Warming was performed in α-MEM including 20% FBS that supplemented with descending concentrations of sucrose (1, 0.5, 0.25 M) at room temperature for 3 minutes. After 2 hours of culture, in each experiment 20 preantral follicles per group were stained with 0.4% trypan blue for 1 min at room temperature. The follicular viability was assessed under an inverted microscope. 1.5 IU/ ml recombinant human chorionic gonadotrophin (rhCG) and 5 ng/ml recombinant epidermal growth factor (rEGF) were added to the cultures as in vitro ovulatory stimulus. Optimal nuclear maturation rate was achieved after 18-20 hours of induction.
Our results showed that the rate of viability and metaphase II increased in presence of zinc in vitrification medium. The viability rate of follicles after vitrification-warming was 70.3 , 81.2 , 88.5 and 98.0 % also percentage rates of oocytes reaching to MII stage were 35.5, 40.12, 47 and 58.3 % respectively in groups V0, 1, 2, 3 (ANOVA, P value<0.05).
The high percentage of survival and maturation rates proves the success of the vitrification procedure. The results also demonstrated zinc supplementation of vitrification medium has a positive effects on the the viability of follicles and maturation rate.