Study On Effect Of Difference Freezing Methods Of The Human Ovarian Tissues

Part I Cryopreservation of the Human Ovarian Tissues with Two MethodsObject We preserved human ovarian tissues with the methods of programmed cryopreservation and vitrification, and compared the morphology of the primordial follicles after thawing between the two methodsMethods Collecting the ovarian tissues and do some pretreatment. We Divided the ovarian tissues into three groups which were the fresh group, the vitrification group and the slow programmed cryopreservation group,respectively. The fresh group was soaked in the liquor of 10% formalin and examined through HE staining and tissue section. The second group is frozen with vitrification and the last one is treated with slow cryopreservation. The last two groups were then preserved in the liquid nitrogen for two months. We thawed the tissues of the two groups by rapid rewarming and examined through HE staining and tissue section. We compared the influences on the original follicles from the two methods by the histological analyses.Results Morphological analysis was done with 572 primodial follicles from the fresh group(C), the Vitrification Freezing group (A) and the programmed cryopreservation group (B). The percent of normal follicles in group C was 96.9% while group A was 85.6% and group B was 83.9%. These three numbers have statistical differences(χ~2 = 19.19 and P< 0.01). The percentage of normal follicles in group A and C have the statistical differences (χ~2 = 15.36 and P<0.01) while group B and C have the statistical differences(χ~2 = 18.54 and P<0.01). The normal follicle percentages in Group A and B are less than C and Group A and B, which have no statistical differences (χ~2 = 0.21(p>0.05).Conclusions Although the normal follicle percentage in the frozen groups are lower than the fresh group, the programmed cryopreservation and Vitrification Freezing can both preserve the human ovarian tissues well, so both of them can be adopted.Part II Research on the Co-culture of thawed human ovarian tissues and somatic cellsObject To set up a co-culture system for human ovary tissues and decidual monolayer cells in the human early pregnancy. By measuring the level of estrogen and progesterone, the influences on the secretion function from different cryopreservation methods and the effect on the development of the follicles by the system were evaluated.Methods We set up a co-culture system for human ovarian tissues with decidual monolayer cells of the human early pregnancy. There were four groups in the experiment: group A, the thawed ovarian tissues after vitrification were co-cultured with the decidua monolayer cells; group B: Co-culturing the thawed ovarian tissues after slow-freezing with the decidua monolayer cells; group C: Culturing the thawed ovarian tissues after vitrification alone; group D: Culturing the thawed ovarian tissues after slow-freezing alone. The culture medium was collected every other day during in–vitro culture for 14 days , and the level of estrogen and progesterone were measured and compared among the groups. After 14 days' culturing, we analyzed the tissues stained by hematoxylin and eosin ,and compared the total follicle survival rate(TFSR) and the grow follicle rate (GFR)among the groups. Results The total follicle survival rate(TFSR) and the grow follicle rate (GFR)among the groups has statictical differences (P<0.01). There are no differences between before and after the freezing in group A (P > 0.05)on grow follicle rate (GFR%), but the grow follicle rate (GFR%) has statistical differences(P<0.01). The same results exists in group B. There are no statistical differences in TFSR and GFR between group A and B after culturing. In group C and D, the two indexes both have statistical differences before and after culturing, but no differences exists between group C and D after culturing. Comparing the two indexes between A and C, A and D, B and C,B and D, the differences were significant.The estrogen and progesterone level of the culture medium are significant different among the four groups after analysis of variance(P =0.000).The differences are not significant both between group A and B, and group C and D(P > 0.05). The differences are not significant both between thawed ovarian tissues after slow-freezing and vitrification by t-test. The estrogen and progesterone level of the culture medium are significant different among the four groups after analysis repeat measurement.Conclusions1. The endocrine function of the ovarian tissues is not influenced after treating with slow-freezing or vitrification.2. Decidual monolayer cells of the human early pregnancy can be co-cultured with the thawed ovarian tissues.3. The original follicles have different development in culture system. If the thawed ovarian tissues are co-cultured with the decidual monolayer cells of the human early pregnancy, the result is better than culturing alone.