Ovarian Tissue Preservation after Various Cryopreservation Modes with the Help of DMSO

The purpose of the study was to investigate the follicular integrity and dynamics of thiobarbituric acid reactive substances (TBARS) accumulation in ovarian tissue fragments after cryopreservation depending on the final concentration of DMSO as well as the freezing mode. Material and methods. The research objects were the fragments of ovarian tissue. As cryoprotective agent (CPA) there was used dimethylsulfoxide (DMSO) in the concentrations of 1.5 M and 3,0 M in DMEM supplemented with 200 mM sucrose. The saturation was carried out step by step: 1.5 M (0 M → 1.5 M); 3.0 M in two stages (0 M → 1.5 M → 3.0 M). The tissue was incubated in CPA solution at 22°C for 30 min in each stage. Freezing was carried out in two modes. Mode 1: cooling at a rate of 1 deg / min to the start temperature of the initiation of crystal formation (IC), IC, further cooling at a rate of 1 deg / min to -40 °C and immersion into liquid nitrogen. Mode 2: cooling at a rate of 1 deg / min to -40 °C and subsequent immersion into liquid nitrogen. Samples were thawed in a water bath at 37 °C until the liquid phase appeared. The CPA was removed stepwise replacing the cryopreservation medium to DMEM solution containing mannitol. The number of normal and degenerative follicles was expressed as a percentage of the total number of follicles in the sample. The intensity of lipid peroxidation in the homogenate of cryopreserved tissue was assessed immediately after washing and after 3 and 24 hours of incubation in a DMEM medium in a CO2 incubator at 37 °C. Results and discussion. We noticed a decrease in the number of normal follicles by 20-30% at the stage of incubation after freezing under protection of 3.0 M DMSO (mode 2). This happened due to the presence of latent damage in the structure of ovarian tissue because of the toxic effect of this CPA. When 1.5 M DMSO was used, regardless of the freezing mode, such damage was absent, both after removal of the CPA and subsequent incubation of the tissue. It can be explained by uniform penetration of DMSO at 22°C throughout the tissue volume, which significantly reduces the effect of the mechanical factor of cell damage during crystallization. Conclusions. The obtained results indicate that the final concentration of DMSO determines the degree of damage to the ovarian tissue structure, depending on the freezing mode. It is necessary to select the cooling mode depending on the type and the final concentration of CPA while conducting cryopreservation of ovarian tissue.