Investigation On Vitrification Of Mammalian Ovarian Tissues

With the development of malignant disease detection and treatment methods,chances of survival are increasing considerably,especially for those patients diagnosed with cancer in childhood or adolescent,and some patients can be completely cured.Unfortunately,the applied high-dose chemotherapy and radiotherapy treatment may create irreversible damage in reproductive cells and tissues resulting in menstrual disorders,amenorrhea,premature ovarian failure and infertility.Accordingly,preservation of the reproductive potential of female patients at or before the reproductive age should be part of the therapy of malignancies.As one of the important methods of fertility preservation,most western countries have established ovarian tissue banking or cryopreservation procedure.To date,more than 60 babies have been born after autotransplantation of cryopreserved ovarian tissue.However,restrained by various conditions,research on ovarian tissue cryopreservation in China is still regarded as experimental procedure requiring optimization,standardization and clinical trials to prove its efficiency,and more deep researches and discussions are required for clinical services.Objective: To explore the cryopreservation effect of three vitrification methods and a modified slow-rate freezing method(mSFM)on mice and human ovarian tissue,and to develop a superior vitrification system and evaluation system for human ovarian tissues,by testing the functional recovery of the xeno-transplanted human ovarian tissue after warming.Method: Twenty-four 6-8 weeks old female C57BL/6J mice,obtained their ovarian cortexes,then divided the ovarian tissues randomly into fresh,vitrification groups(VP1,VP2 and VP3)and modified slow-rate freezing group(mSFM),followed by cryopreservation of each group.After one week,thawing the ovarian tissues from each group,checked and recorded their morphology,follicular survival and the in-vitro development of the recovered ovarian tissues from each group,to provide references for recovering the human samples in the following studies.Then,we recruited 30 cases of human ovarian samples after gynaecological operations,which were tested normal in pathological examinations,to investigate the vitrification effect of human ovarian tissues and the transplantation recovery of the three vitrification methods.Results: 1)The VP1 vitrification technology optimized in our laboratory showed a good preservation of mice and human ovarian tissues.2)VP1 protocol showed a good preservation of human ovarian tissue and a good functional recovery of xeno-grafts in vivo.3)We established a series of standards for the in vitro and in vivo functional tests,which can provide comprehensive and systematic evaluations for the cryopreservation of human ovarian tissues.Conclusion: To date,there is no recognized standard for the clinical application of the ovarian tissues after freezed-thawing or vitrified-warmed.It is also not clear whether the ovarian tissues of female cancer patients are suitable for vitrification,or the cryopreserved ovarian tissue slices are suitable for transplantation.In this study,we developed a superior vitrification system for human ovarian tissues,and the vitrification effect was systematically evaluated in mice and human ovarian tissues by using various in vitro and in vivo indicators.Thus,an efficient vitrification system for preserving human ovarian tissue was established in this study.This systematic approach may help to select a better technique for future clinical application.