P-97: A Novel Staining Method of MouseOvarian Tissue for Comparison of Two Different Methods of Vitrification

Background: Different cryoprotectants are used for Cryopreservation of ovarian tissue in patients who are at risk of infertility. Ethylene glycol (EG), dimethyl sulfoxide (DMSO) and propanediol (PROH) were chosen as the basic permeable cryoprotectants due to their less glass-formation characteristic compared to the others. In the present study, the effects of two different vitrification methods on whole mouse ovarian tissue by using a novel staining method (Trypan Blue) was evaluated. Materials and Methods: Ovaries of 8 days-old NMRI mice were isolated and distributed into control, Vitrification 1 (Vit1) and Vitrification 2 (Vit2). Vit1 and Vit2 solutions were composed of (α-MEM+ 20% FBS + 15% EG + 15% DMSO) and (α-MEM+ 15% FBS +20% EG + 20% PROH) respectively.Vit1 and Vit2 procedures were done in 4°C and room temperature respectively. Warming was performed in α-MEM+ 20% FBS that supplemented with 1M sucrose in Vit 1 group and α-MEM+ 15% FBS and descending concentration of sucrose (1,0.5,0.25 M) in Vit2 group. Control and vitrified warmed Ovaries were put in α-MEM that supplemented by 4% trypan blue for 15 minutes, then stained ovaries were fixed in bouin’s fixative, serially sectioned in paraffin wax and finally quantitatively evaluated under light microscope. Results: The highest percentage of primordial follicles was observed in control group (59.73 ± 5.87) and a significant difference was seen between control and Vit1, also between Vit1 group (26.27 ± 3.04) and Vit2 group (45.98 ± 6.38, p<0.05). No significant difference was observed in primary and preantral follicles between control and vitrification groups. Conclusion: Trypan blue staining is a faster and easier method for evaluation of the ovarian tissue and also vitrification 2 protocol is better for preservation of follicles reserve in ovary.