Research Of Application And Mechanism Of Hyperbaric Oxygen Therapy On Ovarian Tissue Transplantation

Declines in reproductive gland function and fertility have been discovered in female malignant tumor patients who receive chemotherapy or radiotherapy frequently.With the advancement of assisted reproductive technology and cryobiology,clinicians and researchers can help women preserving fertility through surgery,medication or laboratory techniques.For prepubertal women,women who require treatment for malignant tumors immediately,and women with hormone-sensitive tumors,ovarian tissue cryopreservation is the only effective fertility preservation option currently.The operator puts ovarian tissue which has been froze into liquid nitrogen or mechanical freezer for cryopreservation.When the woman recovers,operator thaws cryopreserved ovarian tissue and auto-transplants it into the female body to restore female reproductive function and ovarian endocrine function.About 50-90%of total follicles in ovarian tissue have been lost after transplantation,and the pregnancy rates and live birth rates of women who receive ovarian tissue transplantation are low.How to improve the effectiveness has been a problem needed to be resolved immediately.The treatment which makes patients inhale pure oxygen in a hyperbaric oxygen chamber environment above 1 ATA（atmosphere absolute）is referred to as HBOT（hyperbaric oxygen therapy）.HBOT has been used in clinical practice widely.HBOT can increase oxygen content and dispersion distance in the blood to boost tissue oxygen supply.HBOT can alleviate tissue ischemia-reperfusion injury,and promote wound healing by improving fibroblast activity and collagen synthesis.HBOT has been studied in organ and tissue transplantation by animal experiments or clinic trial,such as liver transplantation,islet transplantation,flap transplantation.The beneficial results of HBOT have been reported.The procedure of ovarian tissue froze-thawed-transplantation consist of froze-thawed phase and post-transplanted phase.In the first part of this study,we managed mouse ovarian tissue by freeze-thawing,fresh transplantation,freeze-thaw-transplantation,and ovariectomy respectively to assess follicle loss.We found that the total number of follicles and the total normal morphological follicles percentage in the ovarian tissues which were frozen-thawed,fresh-transplanted,and freeze-thaw-transplanted reduced significantly compared with normal ovarian tissue.The total number of follicles and the total normal morphological follicles percentage were comparable between fresh-transplanted ovarian tissue and freeze-thaw-transplanted tissue,but both of them reduced significantly compared with frozen-thawed ovarian tissue.We found that there were no significant differences in the serum E₂（estradiol）concentration and FSH（follicle stimulating hormone）concentration between ovarian tissue fresh-transplanted mice and ovarian tissue freeze-thaw-transplanted mice.But the serum E₂ concentration of ovarian tissue fresh-transplanted mice and ovarian tissue freeze-thaw-transplanted mice increased significantly than ovariectomy mice,and the serum FSH concentration reduced significantly.These results indicate that follicle loss occurs in both the froze-thawed phase and post-transplanted phase,and follicle loss is more pronounced in the post-transplanted phase.In the second part of this study,we modeled ovarian tissue fresh-auto-transplanted mouse to observe the effect of HBOT on ovarian tissue transplantation.We found that on the10th day after transplantation,the total number of follicles and the total normal morphological follicles percentage in mice treated with HBOT for 5 days and mice treated with HBOT for 10 days increased significantly compared with the mice that were not treated with HBOT.There was no significant difference in the total number of follicles between HBOT-treated mice and mice without HBOT treatment,the total normal morphological follicles percentage was still improved significantly on the 21st day after transplantation.The recovery of estrous cycles in HBOT-treated mice was earlier than mice without HBOT treatment.The serum E₂ concentration of HBOT-treated mice increased significantly compared with mice without HBOT treatment on the 21st day after transplantation,and the serum FSH concentration reduced significantly.The results indicated that HBOT can improve ovarian endocrine function after transplantation.We found the content of HIF-1α（hypoxia inducible factor-1α）,lipid peroxide product MDA（malondialdehyde）,pro-inflammatory cytokine IL-1β（interleukin-1β）,pro-apoptotic factor Bax and cleaved caspase-3,and the CD31 positive area reduced significantly in HBOT-treated mouse ovarian tissues compared with the ovarian tissue of mice without HBOT treatment.The antioxidant SOD（superoxide dismutase）activity,and the content of anti-inflammatory cytokine IL-10and anti-apoptosis factor Bcl-2 increased significantly.There was no significant difference in the ovarian tissue fibrosis area among them.HBOT can improve the oxygen supply of ovarian tissue significantly after transplantation,and reduce oxidative stress damage,inflammatory response and apoptosis of ovarian tissue significantly.The process of tissue neovascularization delayed significantly by HBOT.We also found that the expression of Nrf2 and HO-1 in the ovarian tissues of HBOT-treated mice increased significantly,suggesting that HBOT may improve the effectiveness of ovarian tissue transplantation through the Nrf2/HO-1 signaling pathway.The expression of HIF-1αin ovarian tissue of mice with HBOT treated for 10 days reduced significantly compared with mice treated for 5days on 21 days after transplantation,indicating that HBOT improved tissue hypoxia status treatment time dependently.There were no significant differences in other tested items between them,indicating that prolonging HBOT treatment time could not improve the effectiveness further.In summary,the post-transplanted stage is the main stage of follicle loss during the procedure of ovarian tissue froze-thawed-transplantation.HBOT can improve the total normal morphology follicles percentage and ovarian endocrine function after transplantation significantly.HBOT can improve the effectiveness of ovarian transplantation significantly.HBOT can reduce hypoxia,apoptosis,oxidative stress damage and inflammatory response,angiogenesis after transplantation significantly,but has no significant impact on fibrosis.HBOT may improve effectiveness of ovarian transplantation by upregulating the Nrf2/HO-1 signaling pathway.Prolonging HBOT treatment time does not improve the effectiveness further.