The Experimental Studies On Cryopreservation And Heterologous Ectopic Transplantation Of Ovarian Tissue Of Rabbits And Human

With the development of society, the improvement of people’s living standards and increasing concerning of their own health, much more critical demands on the preservation of women’s reproductive capacity are proposed recently. This has greatly promotes the development and deeper of the study on the cryopreserved technology of human ovary tissue and clinical application. A study of World Health Organization (WHO) found that700thousand people of1.4million newly increased cancer patients were women in2008. And it will lead to the loss of young women’s reproductive by large dose of radiotherapy, chemotherapy and ovarian surgery. However, cryopreserved ovarian tissue can provide a kind of compensation method for those patients, because of keeping large number of follicles. In early1950s, Parkes and other scholars using the method of slow freezing to make the rats ovarian tissue slice stored in-79℃glycerol mixture, and carried on the autologous transplantation after rapid thaw recovery. Then the growth and development of follicle in the transplantation ovarian tissue was observed. In1990s, along with the development and improvement of science and technology as well as a variety of antifreeze, the freezing technology of ovarian tissue has been developing rapidly, Many experimental study about ovarian tissue frozen of mice, sheep and non-human mammals was reported Constantly, as well as successful reports about freezing and thawing ovary tissue transplant. Sheep ovaries is similar with mankind no matter in the ovarian tissue structure or the ovulation cycle. Therefore the successful pregnancy after freezing thawing and transplantation of ovary tissue in sheep provide a meaningful model for technology development and application of human ovarian tissue freezing and thawing, and laid the solid experimental basis. Donnez J carried on autologous transplantation by laparoscopic surgery for a Hodgkin’s lymphoma patient whose ovarian tissue has been frozen for six years,after six months, the patient restored the menstrual cycle, cyclical ovulation and corpus luteum generation.11months after surgery, HCG determination and ultrasonic confirmed that patients with successful pregnancy and a smooth delivery. Ovarian tissue freezing technology in our country is still in the exploratory stage, Zijiang Chen has gained some experience in frozening human ovarian cortex, studing at follicle morphology and ovarian cortex tissue in vitro culture.The advantage of ovarian cryopreservation technology is freezing the immature cortical primordium follicles, which have the character of relatively quiet, small in size and lack oolemma and cortical granule, also tolerate the damage of freeze and recovery. Cryopreserved ovarian tissue has abundant primate follicles, complete structure, energy and viability. Successful cryopreservation and reviving will open up a new treatment way for the patiens with premature ovarian failure and malignancy. In order to survive threatened and endangered animals and to maintain gene diversity, gene group stock centers around the world are now collecting and freezing the ovarian tissue from these animals.About human ovarian cryopreservation and recovery, there are still many problems to be resolved, although ovarian tissue cryopreservation technologies in rodents and large primates have already born offspring and achieved stage research. The application of this technology in clinical is difficult. The main difficulty is to establish the best frozen measure. Many factors will influence the effect of ovarian cryopreservation technology, including cryoprotectant choose, seeding temperature, thickness of tissue slice and process of freeze and thaw, the skill level of the operator, hardware conditions, etc. There are no best appropriate frozen measure for less injury now.The key of recovery reproductive capacity is survival situation of ovarian tissue after transplantation. Firstly, partial reperfusion injury after transplantation. In the autologous transplantation model experiments of sheep ovarian tissue, Aubard and some scholars found there was only5%primordial follicle alive after transplantation in their experiment. It is likely because ovary lack vascular anastomosis and has no its own blood supply after transplantation. It needs at least48h to revascularization. During this time it has caused irreversible damage to ovarian, so how to protect the transplanted tissue out of hypoxic-ischemic damage after freezing and thawing is a problem which should be solved quickly. Secondly, to find the best transplantation site. So far, it is unknown where is the best site, however what should be clear is that the site should has fine live condition for tissue and be easy for follicle check and puncture oocyte retrieval. At last, solution of the problem of security after the transplantation of ovary tissue with autologous. With the development of frozen and transplant of ovary tissue, it brings new hope to the cancer patients who want to restore its own ovarian function and preservation of fertility, but it still has a certain risk about the transplantation of ovary tissue. Freezing and thawing ovarian tissue allograft mainly in order to save the fertility of cancer patients, some scholars have questioned that transplant the frozen ovarian tissue containing the tiny cancer lesions will lead the patiens to the occurrence of disseminated tumor cells after transplantation or recurrence. The experimental study by Mueller A,etc. found that the rat after heterotopic transplantation, the occurrence of a few of the sex cord-stromal tumors rate of100%. Dror and other studies have shown that using RT-PCR can detection the ovarian tissue remnants of small lesions of cancer patients sensitively, thereby enhancing the safety of the transplant. Before transplantation, using advanced detection methods, such as polymerase chain reaction (PCR), FISH technology, gene chip technology, detect the ovarian tissue remnants of small lesions to reduce tumor cell metastasis risk of ovarian transplant, benefit to improve the effectiveness and safety of ovarian tissue transplant. The samples of ovarian tissue we used to study cry op reservation, recovery and transplantation are mostly obtained from the patients who need to carry on gynecological operation because of ovary disease. Because of their age are generally large, whole flock ovarian follicle in some region of ovary was exhausted and combined with ovary pathological changes such as endometrial implantation cyst, sufficient samples are hardly to obtain. Moreover ovarian cryopreservation, revive and transplantation all related to ethic problems, so the number of the samples were limited. It is proved that there are no obvious difference between species about primordial follicles. This article chose the ovary tissue of rabbit to study in the early stage, because of their popular prices, broad sources, short breeding cycle and the similar size and the minuteness structure of the meiosis ovary. Basic to the early study, this article did a research on the human ovarian cryopreservation, revive and transplantation which come off the ovary operation.Our research study includes four stages:The experimental studies of the frozen and thawed of rabbit ovarian tissue; The experimental study of heterologous ectopic transplantation of rabbit ovarian tissue; The experimental study of frozen and thawed of human ovarian; The experimental study of heterologous ectopic transplantation of human ovarian tissue. We used different frozen procedure (including rapid vitrification, slow-freezing protocols), different frozen carrier (vasculature, microsphere and hard surface method), different frozen reagent and compatibility (DMSO, EG, PROH) to study ovary tissue of human and rabbit in frozen-thawed, in vitro culture, allotransplantation etc. In order to find appropriate ovary tissue frozen-thawed technology and transplantation site, to provide experimental data for developing human ovarian tissue frozen and transplantation, to make basement for founding ovarian tissue frozen bank. Part I:The experimental studies of the frozen and thawed of rabbit ovarian tissuePurposeApplication PROH, DMSO and EG alone or compatibility in slow programmed freezing or vitrification to frozen and recovery the rabbit ovarian tissue, then study the follicle shape of ovarian tissue, c-kit, ki67and MHC-Ⅱ expression, in order to determine a variety of frozen rabbit ovarian tissue morphology, cell proliferation activity, ovarian tissue antigenicity and immunogenicity to explore relatively suitable for freezing ovarian tissue freezing recovery program.Methods and results1The effect of4different frozen methods on rabbit ovarian morphology1.1Objects and groups:Choose12healthy female Japanese white rabbits with big ear. The rabbits are randomly divided into4groups, laparotomy after anesthesia to take ovarian tissue and diced, according to group to take rabbits ovarian tissue blocks with slowly programmed freezing recovery (two subgroups, respectively using PROH and DMSO as frozen liquid, labeled A2and B2)), and rapid vitrification frozen recovery (two subgroups, respectively DMSO+PROH, labeled C2and DMSO+EG, labeled D2), frozen and thawed ovarian tissue; Prior to freezing, take a small amount blocks of fresh ovarian tissue from each group, as controls group.1.2Contents:By histological examination of the ovarian tissue, observing ovarian tissue follicles proportion morphological structure and normal rate in the fresh control and recovery. 1.3Results1.3.1The characteristics of fresh ovarian tissue of rabbits morphological:Slices of fresh ovarian tissue blocks from rabbits, the total number of follicles are1236under the microscope in the slices of fresh ovarian tissue blocks from rabbits, which accounted for88.2%of primordial follicles and the normal form rate of primordial follicles are all the highest91.4%,, the primary follicles8.4%, the secondary follicles accounted for only33.4%. Follicles are954which accounting from the three set of frozen-thawed ovarian tissue slice,857of them are primordial follicles account for89.8%, the primary follicles7.3%, the secondary follicles accounted for only2.8%.1.3.2Follicles morphological changes in rabbit ovarian tissue after different freezing program and refrigerant:In frozen group, slowly programmed freezing PROH group of primordial follicles has the highest normal rate of80.1%, significantly higher than the programmed DMSO group (70.5%), rapid vitrification DMSO+PROH group (67.6%) and rapid vitrification DMSO+EG group (69.7%), the difference has statistically significant; make a calculation about all frozen groups at all levels of follicle, primordial follicles with normal morphology (72.7%) was significantly higher than the primary follicles morphologically normal rate (55.7%), due to the total number of secondary follicles is so exile, statistical analysis isn’t be implemented.1.3.3The effect of frozen to rabbit ovarian tissue structure:Observing the rabbit fresh ovarian tissue under the microscope to observe the rabbit fresh ovarian tissue,we can see the follicles packed by row close rules ovarian stromal cells tightly (Fig1-1). All the frozen resuscitation group ovarian tissue can visible the damage which mainly are primary follicles and secondary follicles. These follicles are isolated with surrounding stromal cells,but primordial follicles are less affected; interstitial cells in various frozen ovaries are disorders and the connection is loose and formation of cracks (Fig.1-2～1-6)2Effects of slow-frozen method and vitrification of value-added activity of rabbit ovarian follicle cells 2.1Objects and groups:9healthy female Japanese white rabbits aged4-5month were chosed. The rabbits are randomly divided into3groups (fresh control group, PROH slow-freezing-thawing, DMSO+EG Rapid vitrification group). Get ovarian tissue as previously described, diced standby.2.2Contents2.2.1The expression of rabbit ovarian tissue MHC-2antigen:the variation of the two sets of frozen rabbit ovarian tissue MHC-2antigen expression was detected by SP immunohistochemical and judge the impact of frozen to the ovarian tissue antigen through observe the average absorbance value of the MHC-2antigen.2.2.2Effect of freezing and thawing to Proliferation activity of all follicle cells: Separation the follicles in the fresh control and freezing/thawing ovarian tissue, cultured in vitro, then transferred to a liquid scintillation vial machine to measure cpm to observe the effect of freezing to all levels of follicle cells proliferative activity.2.3Results2.3.1The effect of procedures and vitrification to MHC-2expression of rabbit ovarian tissue:Immunohistochemistry results show that MHC-2mainly expressed in ovarian interstitial cells, secondary follicles oocytes, oocytes and granulosa cells which in primordial follicles and primary follicles had no expression; Vitrification DMSO+EG group compared with program PROH group and the control group-of ovarian tissue MHC-antigen,average absorbance values significantly lower; PROH group has no statistically significant in absorbance value compared to the control group.2.3.1The effect to proliferative activity of rabbits follicle cells:After culture of follicle cells in vitro with program PROH and vitrification DMSO+EG frozen program, use thymidine marked by3H incorporation test to study the proliferative activity and found that DMSO+EG vitrification frozen program and program PROH has no statistically significant in cpm value differences with fresh group of small OGC, and then the cpm value of the big OGC significantly lower; there are no statistically significant between them.3The effect of different vitrification carriers to c-kit and ki67expression of rabbit ovarian tissue3.1Objects and Group:12healthy female Japanese white rabbits were chose,,according to different vitrification frozen carriers randomly divided into4groups:Control group(A), straw method group(B), microsphere group(C), SSV group(D). Taking ovary tissue as what mentioned before, make the ovarian tissue diced stand.3.2Contents:According to observe the frozen-thawed ovarian tissue histology and test the expression of frozen-thawed ovary tissue c-kit and ki67which adopt immunohistochemical method, to judge the impact of frozen to ovarian tissue proliferation activity and development potential.3.3Results:The effect of the expression of c-kit and Ki67in rabbit ovary tissue with different vitrification frozen carrier, the immunohistochemistry results demonstrated that the ki67protein in all levels of follicular oocytes membrane and/or cytoplasm ki67protein in rabbit ovarian tissue both showed positive expression in brown or tan,. Be frozen by straw, microsphere, SSV method,the expression rate of Ki67and c-kit has no statistically significant compared with the fresh group.Conclusion:1. The method of PROH slow programmed freezing of rabbit ovarian tissue achieved the higher primordial follicles normal morphology and cell proliferative activity, may be a better freezing methods2. Vitrification method can reduce the primordial follicles morphologically normal rate,make primordial follicles good proliferative activity, and rabbit ovarian tissue immunogenicity is decreased significantly. And the vitrification method is simple, economical and practical, possible be a future research directions3. There were no significant influence in the form of the structural integrity of small follicles cell viability about the freezing of PROH programmed and DMSO+EG vitrification. Part Ⅱ:The experimental study of heterologous ectopic transplantation of rabbit ovarian tissuePurposeAfter heterotopic transplantation of the fresh and vitrification ovarian tissue into the neck of the castrated nude mice subcutaneously, to observe the nude mice estrous cycle hormone secretion, changes of mice uterine morphology and transplanted tissues, in order to evaluate the effect of freezing.Methods1Material and groupsWe choose4cleanly female Japanese big ear white rabbits,and cut their ovarian tissue into block, then divided into3groups to observe fresh tissue,fresh transplants, frozen and thawed transplant; Transplant host using8to10weeks of age, of SPF grade mature female Nu/Nu nude mice36.The mouse were divided to4groups which have9mouse. Except the control,3other groups were used for fresh tissue transplantation, freeze-thaw transplantation and castration control after castrated.2Methods2.1The freezing recovery method of ovarian tissue:DG+DMSO in lower concentration as cryoprotectant solution was used in the rapid recovery after ovarian tissue vitrification, SSV method be used as a carrier.2.2Ovarian tissue transplant:After anesthetizing nude mice, we disinfected and cut the skin of nude mice on the left side of the neck, and forceps gripping transplanted rabbit ovarian tissue which was fresh multi-point or freezing and thawing subcutaneously, then suture and mark transplantation part. 2.3The evaluation of transplant effect of freezing recovery rabbit ovarian tissue2.3.1The observation of vaginal exfoliative cytology about Nude mice:observing the changes about the vaginal smear epithelial cells of nude mice to determine estrous cycle and its duration.2.3.2The detection of estrogens in nude mice’s blood:Both normal control group and the two transplant groups of nude mice have estrous cycle, the castration that not transplant group don’t have estrous cycle. Testing serum estrogen (E2) level of each group respectively to determine the function of ovary.2.3.3Uterine morphological observation and ovarian graft observation of Nude mouse:observing morphological changes of uterus removed from each group by the naked eye and microscope respectively; Cut the neck skin of the fresh and frozen ovary transplantation group to remove graft, then have histological observation.Results1Estrous cycle conditions of Nude mice:Normal control group have regular estrous cycle;7/9of fresh transplant group have estrous cycle, while the frozen transplant group is6/9. There are no difference between the two transplanted group in recovering estrous cycle days, as well estrous duration between three groups.2The detection of estrogens in nude mice blood:Determining the E2levels in blood of all nude mice and ovariectomized which occur estrous cycle, there is no significant difference about the E2levels in blood between the two transplant group and the normal control group, but they were significantly higher than castrated group.3The observation of uterus morphology about nude miceOvariectomized group’uterus displayed some special characters, such as atrophy, pale, thin endometrium, less glands, surface covered with cubic or simple squamous epithelial cells, which were significantly different with the other groups; then control group and transplanted group had a ruddy uterus with full shap thickness of coarse glands, the amount is more abundant cytoplasm of the glandular cells were high columnar or pseudostratified layer of the surface coated with a single layer of high columnar, endometrial epithelial cells.4Morphological observation of the ovarian tissue graftRemoving subcutaneous graft from the nude mouse to be observed, it can be seen that most grafts grew into a pale yellow round or oval body, and its volume increased significantly. the surface of part transplanted tissue blocks were covered uneven beaded protrusions. Surviving transplants have rich capillaries by objective lens and follicles within the cortexConclusionsThe low concentration of DMSO+EG vitrification method can save the structure and function of the ovarian tissue, fresh and freeze-thaw nude heterotopic ovarian tissue can live and maintain endocrine function after transplantation. Part Ⅲ:The experimental study of frozen and thawed of human ovarianPurposeWith different concentration of compatibility DMSO+EG as vitrification frozen protective agent, vitrified freeze ovarian tissue separately using straw method, microsphere method, SSV method, then observeing follicles’morphological change of human ovary tissue before and after frozen, apoptosis rate detection and E2determination in supernate of ovary tissue blocks cultivate in vitro after recovery, which help to evaluate the influence of different frozen-thawed methods.Methods1ObjectsWe collected20residual organization from patients’ ovarian cyst wall, all of them accepted the laparoscopic surgery because of ovarian diseases, during2008.7～2008.12. The study used discarded ovarian organization came from of20patients, Signed consent form was obtained from each participant. The study protocols were approved by the Ethics Committee on Human Research. Cut the collected ovarian tissue into2x1x1mm3blocks.2Contents2.1Freezing and thawing of ovary tissue:using different concentration DMSO and EG to prepare two sets of vitrification agent(low concentration I group, high concentration II group). Steeping Ovarian tissue blocks separately into equilibrium liquid and two kinds of vitrification frozen agent for a period of time, then by means of straw method, microsphere method, SSV method to do vitrified frozen, rapid thawed method to recover human ovary tissue fragment.2.2Human ovary tissue cultivation in vitro after recovery:regularly collecting tissue culture supernate and detecting E2level,14d later fished out ovarian tissue blocks and settled, coloured them then histology detection.2.3Frozen recovery effect judgment of human ovary tissue:2.3.1The morphological analysis of follicles of ovary tissue before and after frozen:before and after frozen and cultivation14d later in vitro, observing follicle density and distribution of tissue cortex and follicle morphological changes, count the ratio of Normal form and anomaly change, detect follicle density and distribution of tissue cortex of every group and proportion changes of level of follicles. 2.3.2Apoptosis situation of all kinds of cells in thawing ovarian tissue:using DNA in situ end labeling method, to detect apoptosis situation of all kinds cells in thawing ovarian tissue at DNA level.2.3.3Estradiol detection of supernate culture in vitro:collect supernate of ovary tissue culture in vitro every48hours, keep it at-80℃after centrifugation. Detecting the level of E2in supernate by RIA method.Results1Follicles morphological change and proportion changes of each levels of follicle of human ovary tissue before and after frozenThe normal form rate (43.7%) of DMSO+EG high compatibility groups II group primordial follicles has a dramatic decline than fresh control group (75.5%) and low concentration group I group (69.4%). Among these three groups, the proportion of follicle of each levels has no significance difference, and the highest proportion is primordial follicles.2Morphological changes of human ovary tissue structure in post-thawInterstitial cells of fresh ovary tissue arranged regularly and wrapped, follicles of each level. Two kinds of vitrification refrigerating fluid and use partly three carriers to cryopreserved human ovary tissue, there is no obvious influence on primordial follicles, but it is clear that part of the interstitial cells of ovary tissue arrange disorder, loose and even separate.3Apoptosis of various cell of human ovary tissue after freezing and thawingThere are no brown or sepia apoptosis cells which are positive expressed in nucleus of interstitial cells. With one-way analysis of variance, there is no statistical significance on apoptotic rate of interstitial cells between vitrification and fresh control group. 4The changes of E2secretion of ovary tissue culture in vitro after frozenIn carriers groups of low concentration compatibility I group E2of ovary tissue secret higher than carriers groups of high concentration compatibility II groups, there is significant difference on SSV method between two carriers, while no significance among carriers of frozen groups.Conclusions1. The tolerance of primordial follicles to frozen damage is higher than primary follicles.2. It is better for saving ovary tissue when use using low concentration frozen protective agent combined SSV vitrification frozen method. Part IV:The experimental study of heterologous ectopic transplantation of human ovarian tissuePurposeOn the basis of preliminary study, we used DMSO+EG with lower concentration as frozen protective agent, SSV method as vitrification freezing and thawing, heterotopia allograft to the Cervical subcutaneous of ovariectomized nude mice. To observe exfoliocytology of vagina, the secretion of E2in serum and morphology change of uterus to charge frozen effect.Methods1. Objects and groups 2009.11～2010.3,20cases of ovarian disease and line laparoscopic surgery stripping of ovarian cyst wall residual organization were collected to study. Through Ethics committee review and make patients informed consent. Cut the collected ovarian tissue into2×1×1mm3blocks. Transplantation host is healthy sexual maturity Nu/Nu nude mice, group them in accordance with whether to transplant into four: fresh transplantation group, cryopreservation group, ovariectomized group and common control group. The former three group cut Bilateral uterine.2. Contents2.1Freezing and thawing:use lower concentration DMSO+EG, vitrification frozen ovarian tissue by SSV method and reviving it by rapid thawing method for reviving.2.2Transplantation method of ovarian tissue:fresh and frozen-thawed ovary tissue separately multipoint transplanted into the Cervical subcutaneous of ovariectomized nude mice in relevant group.2.3Indicator for further observation:2.3.1viginal exfoliocytology observation:observe estrous cycle from the fifth day after transplantation through viginal exfoliocytology.2.3.2Estrogen level measurement:detecting blood serum E2level of transplanted group in estrus to charge the ovarian function; ovariectomized nude mice draw blood detection before death.2.3.3Morphology observation and transplant collection of nude mice:settle removed nude ovary by methanal, stain sections and observe morphology of endometrial glands and mesenchyme in light microscope; cutting the transplanted site in neck of transplanted mice to find graft.Results1Recovery condition of oestrous cycle of transplanted nude miceControl group keep regular estrogen periodic change,4/9of fresh transplanted group recovered oestrous cycle,3/9of frozen transplanted group recovered; in ovariectomized group epithelial cells with nuclear and keratinocyte fade away, white blood cells largely increased, cast-off cells did not seen cyclic variation. Frozen transplanted group Restore estrus cycle time for the first time [(22.33±1.20)d] later than fresh transplanted group [(19.00±1.58) d], but compare Estrus days of two groups [(5.67±1.15)d,(5.0±0.82) d] to common control group [(4.83±0.75) d], there was no significant difference(P>0.05).2Uterine morphological changes of transplanted groupTransplanted group mice’s uterine thickened and became ruddy, permanently resiliency, which was same to the common control group. There are no difference on uterine wet weight with transplanted group and control group, overweight than ovariectomized group obviously. By light microscopy transplanted group endometrial interstitial loose, endometrial glands bulky and abundance, gland cell is tall columnar, cytoplasm rich, false stratified epithelium cells, no significant difference with normal endometrial morphology.Nude mice in ovariectomized control group uterine atrophy, become thin, colour etiolation, endometrial interstitial density. Endometrial atrophy thin, the quantity of glands is less, gland cavity smaller, gland cell is resting stage performance.Though in transplanted group mice had similar E2secretion level with normal control group, no obvious grafts were found in neck graft sites.3The average level of serum E2of nude miceFresh and frozen transplanted group have no significance difference in average level of serum E2, but both higher than ovariectomized control group.Conclusions1. Using low concentration ratio DMSO+EG cryogen, SSV vitrification freeze human ovarian tissue can got the same effect with fresh ovarian tissue transplantation.2. It is proved that the transplantation of human ovarian tissue ectopic nude mouse cervical subcutaneous without vascular anastomosis can survive and recover nude mice oestrous cycle and endocrine function.