Comparison of Two Different Immature Mouse Ovarian Vitrification Methods using Trypan Blue Staining

Different cryoprotectants are used for cryopreservation of ovarian tissue in patients at risk of infertility. Ethylene glycol (EG), dimethyl sulfoxide (DMSO) and propanediol (PROH) have been chosen as the basic permeable cryoprotectants due to their decreased glass-formation characteristics compared to other cryoprotectants. In the present study, the effects of two different vitrification methods on whole mouse ovarian tissue by the use of a novel staining method (trypan blue) has been evaluated. Ovaries of 8 day-old NMRI mice were isolated and divided among the control, vitrification 1 (Vit1) and vitrification 2 (Vit2) groups. The Vit1 solution was composed of α-MEM+ 20% FBS + 15% EG + 15% DMSO. The Vit 2 solution was composed of α-MEM+ 15% FBS +20% EG + 20% PROH. Vit1 and Vit2 procedures were performed at 4˚C and room temperature, respectively. Warming was performed in α-MEM+ 20% FBS supplemented with 1M sucrose in the Vit1 group and α-MEM+ 15% FBS with descending concentrations of sucrose (1, 0.5, 0.25 M) in the Vit2 group. Control and vitrified warmed ovaries were put in α-MEM supplemented by 0.4% trypan blue for 20 min, and then stained ovaries were fixed in Bouin’s fixative, serially sectioned in paraffin wax and finally quantitatively evaluated under a light microscope. The highest percentage of primordial follicles was observed in the control group. There was a significant difference between the control and Vit1 groups, and between the Vit1 and Vit2 groups (p<0.05). No significant difference was observed in primary and preantral follicles between the control and vitrification groups. Vitrification with EG and PROH are more suitable for preservation of follicle reserves in ovaries. Trypan blue staining is a faster and easier method for evaluation of ovarian tissue.