Experimental Study On Cryopreservation And Autotransplantation Of Rabbit Ovarian Tissue

Ovary is the important endocrine organ,and closely linked to endocrine and physiological functions of women.Chemotherapy and radiotherapy,as well as genetic, infectious and autoimmune factors may cause premature ovarian failure and infertility. Ovarian tissue cryopreservation and transplantation may be a promising clinical technique because it avoids ovarian stimulation and provides the opportunity for preserving gonadal function in prepubertal,as well as adult patients.A certain number of ovarian follicles is the foundation of maintaining ovarian function.The aim of cryopreservation of ovarian transplantation is to get more survival follicles,which will conduce to the recovery and longevity of ovarian grafts,by constantly improving the technology of cryopreservation and transplantation.Researches focus on:how to optimize the protocol of freezing and thawing,which transplanted site is most favorable,how about the expression of gonadotropin receptors and how to produce fully grown and developmentally competent oocytes from cryopreserved tissues after autotransplantation.Primordial follicles are the most abundant stage of ovarian follicles present in the ovary.Primordial follicles are relatively quiescent,have a low metabolic rate,and lack zona pellucida and cortical granules.Due to these characteristics,they appear to be more tolerant to freezing and thawing.However,intracellular ice crystal formation and salt deposits will cause freezing injury.Vitrification is a freezing procedure that traps all of the aqueous solutions in a solid phase,described as amorphous or vitreous, preventing any ice crystal formation.The phenomenon can be regarded as an extreme increase of viscosity and requires either rapid cooling rates or the use of cryoprotectant solutions,which depress ice crystal formation and increase viscosity at low temperature.Vitrification freezing becomes the hot spot in the field because of cost efficiency,simplicity and speed of the procedure.In the human field,vitrification freezing has been applied in the cryopreservation of oocytes and embryos.But there are only a few reports about vitrification freezing of ovarian tissue.This study was to explore the efficiency of vitrification freezing for rabbit ovarian tissue.The purpose of ovarian tissue cryopreservation is to restore the ovarian endocrine function and fertility after transplanted to the recipient.Ovarian transplantation can be divided into autograft,allograft and xenograft according to the relationship between the donor and the recipient.The main problem about allograft and xenograft is immune rejection reactions,which limit their clinical application.Autologous ovarian transplantation can be applied to patients with serious gynecological diseases or cancer but ovary has not been involved.Ovarian tissue can be cryopreserved prior to radiotherapy and chemotherapy,and then autotranplantated the frozen-thawed ovarian tissue after treatment to restore the endocrine as well as gametogenic functions. Transplantation site is important to the survival of ovarian tissue and the restoration of ovarian function.The optimal site for transplantation should provide favourable conditions for tissue survival,follicle monitoring,oocyte harvesting or spontaneous ovulation.It remains unknown which site is most effective and practical.Ovarian autotransplantation can be divided into orthotopic transplantation and heterotopic transplantation in accordance with the transplanted sites of recipient.Orthotopic transplantation refers to transplant the ovarian tissue in the cortex of ovary or the peritoneum beneath the ovarian hilum.However,the possibility of ovarian tissue transplantation in the remaining native ovary was limited because of the small size of the atrophic organ.For heterotopic transplantation,the ovarian tissues were often introduced to the renal capsule,the subcutaneous tissue of arm and abdomen, peritoneum and rectus muscle.Renal capsule is considered to be one of the optimal sites of transplant because of its rich blood supply.But the pressure from the capsule may limit the growth of follicles.Ovarian tissues transplanted under the skin of forearm or abdomen will be at a risk of exposure to suboptimal temperatures or to mechanical stress.The mesometrium is the mesentery of the uterus and,in humans, constitutes the majority of the broad ligament of the uterus.The clinical application of heterotopic ovarian autotransplantation in the mesometrium in women might be feasible because of the spongier texture,abundant vessels,and large surface area at this site.In this study,small pieces of cryopreserved ovarian tissues were transplanted into different sites(mesometrium,renal capsule and abdomen subcutaneous)in rabbits,and the histology,ultrastructure and physiological function of the grafts were assessed to choose the optimal site.After ovarian transplantation,whether follicles can accept the stimulation of FSH (Follicle stimulating hormone)and LH(Luetinizing hormone)to develop depending on whether there are FSH receptor(Follicle stimulating hormone receptor,FSHR)and LH receptor(Luetinizing hormone receptor,LHR).To research the expression of gonadotropin receptor after cryopreservation and transplantation will help understanding the initiation and development adjustment mechanism of primordial follicles.This study was to compare the expression of LHR and FSHR before and after the ovarian cryopreservation and autotransplantation.The results of this study may provide theoretical basis for the mechanism of action of gonadotropin in grafts. In recent years,autotransplantation of frozen-thawed ovarian tissue has proven to be a feasible method to restore endocrine function and fertility in various animals and humans.However,it is still unkown whether cryopreservation and transplantation will affect the developmental potency of oocytes retrieved from grafts.In this study, frozen-thawed ovarian tissues were autologously transplanted into mesometrium to investigate the effects of transplantation of frozen-thawed ovarian tissues on the maturation,fertilization and further developmental potency of oocytes retrieved from grafts.PartⅠThe comparison between vitrification freezing and slow freezing of rabbit ovarian tissueObjective:To explore the efficiency of vitrification freezing for rabbit ovarian tissue. Methods:30 New Zealand female rabbits were bilateral ovariectomized and each ovary were randomly divided into three parts,Group l(n=10),control group;Group 2(n=10),conventional slow freezing group;Group 3(n=10),vitrification freezing group.Morphology,ultrastructure and DNA fragmentation were compared between fresh and the frozen-thawed tissue.Results:The morphological integral follicles rates were 87.4%,82.0%and 82.7%for control group,slow freezing group and vitrification freezing group,respectively;The morphological integral follicles rates in slow freezing and vitrification freezing groups were lower than those of the control group(P＜0.05);But no significant differences were observed between the two types of freezing protocols(P＞0.05);The percentages of TUNEL-positive oocytes were 21.4%,13.5%and 17.1%for control group,slow freezing group and vitrification freezing group,respectively,no significant differences were observed among them(P＞0.05).Conclusion:The vitrification freezing may be an efficient,simple and feasible cryopreservation method of ovarian tissue.PartⅡThe effect of different sites for rabbit ovarian autotransplantation and the expression of gonadotrophin receptors after freeze thawing and transplantationObjective To compare the effect of different transplantation sites for ovarian tissues, and to study the expression of gonadotrophin receptors after ovarian freeze thawing and transplantation.Methods 30 bilateral ovariectomized New Zealand rabbits were randomly divided into three groups.Ovarian tissues thawed from vitrification freezing were transplanted into mesometrium(GroupⅠ),renal capsule(GroupⅡ)and abdomen subcutaneous (GroupⅢ).30 days later,grafts were removed aseptically and ovarian histological analysis was used to confirm the effect of cryopreservation.Vaginal cytology was used to assess the secretary function of transplanted ovarian tissues.At the same time, the expressions of FSHR and LHR in grafts were determined by immunohistochemical ABC techniques,analyzing the positive sections with an image analysis system.Results All rabbits in different groups started to reveal comified epithelial cells in Vaginal smears from day 14 to 20 after transplantation.The recovery rate of grafts in abdomen subcutaneous group was lower than that in mesometrium and renal capsule groups.No significant differences were observed in the proportions of normal and morphologically changed follicles among different tranplantation groups(P＞0.05). The percentages of normal follicle in 3 transplantation groups were lower than that of the fresh ovarian tissues(P＜0.05).No significant differences were observed in the proportions of follicles at different growth stages among different transplantation groups and fresh ovarian tissues(P＞0.05).The ultrastructure of ovarian tissue and follicles did not show any obviously changes after cryopreservation and transplantation.There were no significant differences in the mean optical density of FSHR positive follicles and LHR positive follicles after cryopreservation and transplantation(P＞0.05).Conclusions No significant morphological and ultrastructural changes were found and the follicles survived and developed well after grafting.Mesometrium and renal capsule are all favourable transplantation sites.Cryopreservation and transplantation do not affect the expression of FSHR and LHR of grafts.PartⅢStudy on the capability of maturation,fertilization and further development of ooeytes retrieved from rabbit ovarian tissue treated with cryopreservation and autotransplantationObjective To investigate the effects of transplantation of frozen-thawed ovarian tissues on the maturation,fertilization and further developmental potency of oocytes retrieved from grafts.Methods Twenty five New Zealand White female rabbits were divided into three groups randomly,Group 1(n=5),control group;Group 2(n=10),fresh ovarian tissues were autologously transplanted into mesometrium;Group 3(n=10),frozen-thawed ovarian tissues were autologously transplanted into mesometrium.Three months after the transplantation,rabbits were stimulated with FSH and oocytes were retrieved from antral follicles 13 hours after HCG injection.Oocytes matured in vivo or in vitro were then fertilized by conventional in vitro fertilization(IVF)or intracytoplasmic sperm injection(ICSI),followed by observation and evaluation of fertilization rate,cleavage rate and blastocyst formation rateResults The number of follicles per high power field of ovarian tissue was markedly reduced in two transplanted groups than that of the control group(P＜0.05);There were no significant differences both in the percentage of immature oocytes and the maturation rate after IVM among 3 groups(P＞0.05);Among 3 groups or in each group,the fertilization rate,cleavage rate and blastocyst formation rate had no difference no matter oocytes matured in vivo or in vitro(P＞0.05);The blastocyst formation rate derived from oocytes matured in vitro was significantly lower than oocytes matured in vivo(P＜0.05).Conclusions Rabbit ovarian follicles can survival and reinitiating development after cryopreservation and autotransplantation,and oocytes recovered from ovarian grafts can develop to blastocyst stage by IVM and IVF/ICSI.Thus,cryopreservation and transplantation of ovarian tissue and subsequent Assisted Reproductive Technologies(ART)may provide possible means of fertility preservation.SummaryThe vitrification freezing may be an efficient,simple and feasible cryopreservation method for rabbit ovarian tissue.Mesometrium and renal capsule are favourable transplantation sites.Cryopreservation and transplantation do not affect the expression of FSHR and LHR of grafts.Rabbit ovarian follicles can survival and initiating development after cryopreservation and autologous transplantation,and oocytes recovered from ovarian grafts can develop to blastocyst stage by IVM and IVF/ICSI. Thus,cryopreservation and transplantation of ovarian tissue and subsequent Assisted Reproductive Technologies(ART)may provide possible means of fertility preservation.