Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation

Abstract

To investigate whether needle immersed vitrification (NIV) can further lower the concentration of cryoprotective agent (CPA). Experimental cross-sectional controlled in vitro study. University teaching hospital. Human ovarian biopsy tissues were obtained from ten women undergoing gynecology operations. Ovarian cortical tissues were cryopreserved using slow freezing or vitrification. The vitrification solutions used were as follows: group A: 2.69 mol/L ethylene glycol (EG)+2.11 mol/L dimethylsulfoxide (DMSO)+0.5 mol/L sucrose; group B: 2.42 mol/L EG+1.90 mol/L DMSO+0.5 mol/L sucrose; group C: 2.15 mol/L EG+1.69 mol/L DMSO+0.5 mol/L sucrose; and group D: 1.88 mol/L EG+1.48 mol/L DMSO+0.5 mol/L sucrose. Histologic evaluations were performed using light and electron microscopy. Apoptosis was assessed by TUNEL staining. Tissue damage after cryopreservation was measured by the levels of lactate dehydrogenase (LDH) in culture. The proportion of normal ultrastructure of granulosa cells and stromal cells in groups B and C was higher than that in group A. The proportion of TUNEL-positive primordial follicles and stromal cells in the NIV groups decreased with reduction of concentration. Additionally, LDH levels in groups B and C were lower than in group A. The NIV method could further lower the concentration of CPA. Therefore, we can use the CPA of group C as an optimal concentration for NIV.