Efficiency Of Three Methods Applied In The Human Ovarian Tissue Vitrification

Objective:Cryopreservation of ovarian tissue is now a promising strategy for female fertility preservation. Up to date ovarian tissue cryopreservation is considered to be an experimental technique for fertility preservation, there is no standard preservation program.This study by observing the effect of different frozen carriers on vitrification outcome, focusing on established a feasible human ovarian tissue vitrification program. Materials and methods:From March to September 2014, forty patients who underwent operation for benign ovarian cysts in Tianjin central hospital of gynecology obstetrics were enrolled in this study. The ovarian tissues were transferred to PBS and send to the laboratory on ice in half an hour. Then the ovarian cortices were cut into pieces as 5×1×1mm3, then each patients’ ovarian tissues were randomly distributed into four groups: one as fresh group and vitrificated the remaining three groups. depending on the different carriers used in the program, the remaining three groups are also called micro-drop method group, cryovials method group and nylon strainer method group. In this study, we chosen low concentration ethylene glycol and low concentration diemethylsulphoxide as cryoprotectants, Observing the number of all levels of follicles, normal morphology ratio of primordial follicles by morphological, detection of cell apoptosis by TUNEL assay, evaluate the cell proliferation by immunohistochemical stainging, then collected and compared the four groups data. Results:1. There was no difference for the number of all levels of follicles and primordial follicle’s portion between micro-drop method group and fresh group, nylon strainer method group and fresh group(P> 0.05). While, the number in cryovials method group was lower than fresh group(P﹤0.05). Normal morphology ratio of primordial follicles in fresh group was significantly higher than micro-drop2. method group, cryovials method group and nylon strainer method group(c2=6.956, P=0.008; c2=27.038, P=0.000; c2=10.963, P=0.001), while the ratio in micro-drop method group and nylon strainer method group was significantly higher than cryovials method group(c2=8.444, P=0.004; c2=5.309, P=0.021), there is no significant difference between micro-drop method group and nylon strainer method group(c2=0.507, P=0.476).2. TUNEL assay indicates that no TUNEL staining was found in all levels of follicles of fresh and frozen/thawed ovarian tissue, but there was an indication of apoptosis of some stromal cells.3. There was no difference for the number of ki67 positive cells between micro-drop method group and fresh group, nylon strainer method group and fresh group(P> 0.05). While this outcome in cryovials method group was significantly lower than the fresh group(P﹤0.05). There was no difference for the number of ki67 positive cells between micro-drop method group and nylon strainer method group(P> 0.05). Conclusions:1.The research project is the first that focus on different cryoprotectants and different carriers system. My research compared the effect of three different carriers system on human ovarian tissue cryopreservation.2.Micro-drop method group and nylon strainer method has a better effect on ovarian tissue vitrification process than cryovials method group judging from the number of all levels of follicles, ovarian tissue cell apoptosis and cell prolification.3.Nylon strainer may be a feasible carrier for human ovarian tissue vitrification.4.Preparing for the future human ovarian tissue vitrification used in clinical on technical aspect.