The Research Of Ovarian Tissue Culture In Vitro

Objective:The aim of this research was to explore the survival of oocytes and growth of follicles, as well as the new angiogenesis after the thawed human ovarian tissue and fresh mouse ovarian were cultured in vitro used different culture solution. This study searched the optimized conditions of the freezing-threwed human ovarian tissue cultured in vitro and of the oocytes in vitro maturation, addition to the situation of the new angiogenesis was observed, so expect to decrease the ischemia injure after the transplantation of ovarian tissue.Methods:the human ovarian cortical tissue was acquired from two patients undergoing gynecological operation and was abnormal in pathological examination. The ovarian samples were performed cryopreservation with programmable freezing and rapidly thawed protocols. Meanwhile, mouse’s ovarian were obtained from twenty female KunMing mouse aged5to6weeks. The human ovarian cortex tissue and mouse’s ovarian were randomly divided into four groups and cultured in vitro in different culture medium respectively. The human follicular fluid (HFF) was acquired from the patients who undergoing controlled ovarian superstimulation and ultrasound-guided oocyte absorb by vagina puncturing, and was centrifuged, inactivated by high temperature and filtered for prepare. The group A was cultured by basic medium, The group B was cultured in50%HFF, the group C was cultured in basic medium additive recombinant human vascular endotheliing growth factor165(VEGF). the group D was cultured in50%HFF additive VEGF. Both the human ovarian and the mouse ovarian samples were cultured in vitro for21days, and a pieces of cultured ovarian sample from each group were randomly chosen for histological analysis every7days. And the levels of estrogen(E) in the replaced culture medium was detected. The survival of the oocytes,the growth of follicles and the situation of new angiogensis were observed.Results:1),No statisfically significant differences of the survival of oocytes and growth of the follicles were observed between the four groups of cultured human ovarian tissue samples.2), Significant increased new angiogenesis was found when compared the group C, group D with group A,group B of cultured human ovarian tissue samples.3), Apoptosis and necrosis was observed in the group B and group D of mouse ovarian samples which were cultured using50%HFF from day7. In the day14and day21survival cell were found only in the cortex, the fibrous tissue like necrotic and the iragmenntation or cell nucleus was tound in rest of the ovary including ovarian medulla.4), After mouse’ovaries were cultured21days,the proportion of follicles in developmental phase was significant more group A and group C than in fresh mouse ovaries(group A p=0.000, group C p=0.002). but not significant difference was observed between group A and group C about the proportion of follicles in developmental phase (p=0.659in day21).The levels of E2in the replaced cultured medium in each ovarian sample in the group A and group C were gradually increased along with the cultured longer, but not significant difference was observed between the two groups(p=0.241).5), The new angiogenesis was not found in group C in mouse’ovarian samples which were cultured in VEGF.Conclusion:1),50%HFF may be used in culture the human ovarian tissue in vitro, not growth follicles were found after cultured in mediu including50%HFF mabey because that the patients of the ovarian sample acquired were too old in this research, and there need futher studies to demonstrate it.2), VEGF can promote the new angiogenesis in vitro culture the human thawed ovarian tissue but not obvious in the mouse’ ovarian.3),50%HFF could not be used in culture mouse ovarian in vitro, maybe because chemical substances in HFF have toxic effect in the mouse ovarian.4),The mouse’ ovariy were cultured in vitro can acquired follicles in development phrase, but used VEGF could not accelerating the development of the follicles.