Study On Novel Direct Cover Vitrification For Rat Ovarian Tissue Cryopreservation And Autotransplantation

Recent advances in cancer therapy have resulted increased number of long-term cancer survivors.Unfortunately,aggressive chemotherapy and radiotherapy can destroy gonads and subsequently lead to a loss of fertility and premature menopause.The cryopreservation of ovarian tissue is a promising new method for preserving the fertility of women who are at risk of ovarian function failure because of pelvic diseases and chemotherapy or radiotherapy.Usually,there are two available techniques of ovarian cryopreservation:slow cooling/rapid thawing and vitrification.Though slow-freezing of human ovarian tissue pregnancies were successfully achieved,it is also problematic because the diversity of cell types demands different parameters to avoid ice crystallization damage,and this procedure is expensive and takes several hours. Vitrification has been concerned as the most promising approach to the cryopreservation of ovarian tissue.Vitrification is to replace the water in the cells by a cryoprotectant solution and requires high concentration of cryotectant to reduce the critical cooling rate. The critical cooling rate is the lowest rate that allows a given solution to vitrify,which decreases with increasing cryoprotectant concentration.Unfortunately,all the presently known cryoprotectants are toxic,and their toxicity increases with concentration. Toxicity of cryoprotective agents is a key limiting factor in cryobiology.How to reduce the toxicity of cryopretectants and promote the permeable cryopretecants get into tissues thus reach the effect pretecting concentration is the hot spot of study at present. Recently,Chen developed a new vitrification method-- novel direct cover vitrification (DCV),using less concentrated cryoprotectants and direct application of liquid nitrogen to ovarian tissue.He found the DCV method was highly efficient at increasing follicular survival and pregnancy of cryopreserved mouse ovarian tissues.However,to our knowledge,there is no report on using DCV methods for other animal and human ovarian tissue.Recently,many studies aimed at using Gonadotropin-releasing hormone(GnRHa) to inhibit the maturation of ovarian follicles from reaching the chemotharepy sensitive stage.Based on the available studies,GnRHa appear to improve ovarian function and the ability to achieve pregnancy following chemotherapy.In the investigation of ovarian tissue cryopreservation we found that the primordial follicles are more tolerant to freezing and thawing than growth follicles.So we speculated whether application of GnRHa before collected ovarian tissue is beneficial for more follicles sustain the cryopreservation period and diminish the frozen-thawed injury? No related report has been seen.Our study divided into three parts to investigate DCV protocol for cryopreservation of the rat ovarian tissues,comparing with conventional vitrification.We used three different concentration of CPA to explore the optimal one.To evaluate the efficiency of these two methods,we systematically examined their effects on rat ovarian tissue by histology,TUNEL assay,transmission electron microscopy(TEM) and heterotopic allograft.In addition,we preliminarily discuss the possible influence of GnRHa pretreatment on rat ovarian cryopreservation injury and autotranplantation.PartⅠThe comparision study of novel direct cover vitrification(DCV) and conventional vitrification(CV) on rat ovarian tissue. Objective:To compare the effect of novel direct cover vitrification(DCV) and conventional vitrification(CV) for rat ovarian tissue,and to explore the optimal concentration of cryoprotectants of DCV.Method:(1) 50 rats were randomly divided into 5 groups:Fresh tissue group,CV (conventional vitrification) group,DCV1(direct cover vitrification1) group,DCV2 group and DCV3 group.(2) Established novel direct cover vitrification(DCV) and conventional vitrification(CV) for rat ovarian tissue,and three different concentrations of CPA were used in DCV1,DCV2,DCV3 groups.(3) The methods to evaluate the efficiency of cropreservation:a.Histologically examined the morphological change of follicles.b.Observed the frozen-thawed ovarian tissue necorosis and survival condition by Live/dead Assay.c.Detectd the apoptosis positive follicles in ovarian tissue by TUNEL.d.Observed the ultrastructure change of follicles by Transmission electron microscopy.Results:(1) The percentages of morphologically normal follicles in Fresh,DCV1,DCV2, DCV3 and CV group were 95.04±2.87%,74.51±6.97%,69.24±5.71%,81.89±6.80% and 75.11±5.33%respectively.The proportion of morphologically normal follicles in frozen/thawed groups was lower than fresh tissue group(P<0.05),which in DCV2 group was significantly higher than DCV1,DCV3 and CV group(P<0.05),and no statistic difference was demonstrated between CV and DCV3 group(P>0.05).In all group,the highest proportion of morphologically normal follicle was the primordial follicle,second was the primary follicle,the lowest was the secondary follicle.(2) The percentages of viable follicles in fresh,DCV1,DCV2,DCV3 and CV group were 97.00±2.67%,75.09±5.49%,71.43±6.20%,83.60±5.33%and 77.38±5.13% respectively.The percentages of viable follicles in DCV2 group was significantly higher than other frozen/thawed groups(P<0.05),and there was no difference between CV, DCV1 and DCV3.(3) The percentages of TUNEL-positive follicles in fresh,DCV1,DCV2,DCV3 and CV group were 7.72±0.65%,13.64±0.79%,14.24±1.55%,13.96±1.60%and 13.14±1.67%respectively.The incidence of apoptotic follicles in frozen/thawed groups was significantly higher than fresh tissue(P<0.05).But there was no difference in various groups with cryopreservation.(4) The TEM total scores in fresh,CV,DCV1,DCV2,DCV3 group was 90.25±5.31 %,46.78±8.22%,50.92±6.52%,59.75±5.31%and 55.57±5.55%respectively,.The total scores of the frozen/thawed groups were obviously decreased compared with fresh group(P<0.05).The TEM total scores and the oocytes score of DCV2 group was higher than other frozen/thawed groups,(P<0.05) but the granulosa cells score and stromal cell score was no significant difference in all frozen/thawed groups.In each group,the oocyte score was the highest,the granulosa cell score was the second,and the stromal cell score was the lowest.PartⅡThe study on rat ovarian tissue autotransplantationObjective: To establish the rat autotransplantation model,and to investigate the growth of fresh or frozen/thawed ovarian tissue after transplantation with different cryopreservation method,then to judge the efficiency of cryopreservation further. Method:(1) Ovarian pieces after freezing and thawing were autografted at bilateral superficial muscles of groin area.The groups were as follows:A(CV),B1(DCV1),B2 (DCV2),B3(DCV3),C(fresh grafting group),D(Oophorectomized group) and E (Sham-operated group).(2) Observed the restoration rate and the interval of regular oestrous cyclic by vaginal smear.(3) Detected the serum level of E2 and FSH 4 weeks after transplantation.(4) Histologically investigated the follicular morphology and density in the grafts 30 days after transplantation.Results:(1) The restoration rate of regular oestrous cyclic in A,Bl,B2,B3 group was 66.7%(6/9),80%(8/10),80%(8/10) and 100%(10/10) respectively.The interval of regular oestrous cyclic in A,B1,B2,B3 group was 17.71±2.14,19.67±2.58,19.25±2.66 and 17.88±2.03 days,which was longer significantly than fresh control(P<0.05).There was no significant difference within all the frozen-thawed groups(P>0.05).(2) Before transplantation,the follicular density(n/mm~2) in A,B1,B2,B3 group was 8.24±3.18,8.22±3.39,8.43±2.70 and 8.57±3.22 respectively.A similar follicular density was observed in fresh and frozen-thawed ovarian tissue(P>0.05).After transplantation,the follicular density sharply reduced,which was 1.94±0.95,2.67±0.87, 2.20±0.83,2.05±0.90 in A,B1,B2,B3 group respectively.The follicle density of B2 was greater than B1,B3 and A groups(P<0.05),and no significant difference between other frozen-thawed groups(P>0.05).(3) The serum level of E2,FSH was no significant difference between group C and E(P>0.05).Though the E2 level of frozen-thawed grafting groups was lower than group C and E,but higher than group D(P<0.01);The FSH level of A,B1,B2,B3 was higher than group C and E(P<0.05).PartⅢThe effect of GnRHa pretreatment on rat ovarian tissue cryopreservation and autotransplantationObjective:To explore the possible effect of GnRHa pretreatment on rat ovarian tissue cryopreservation and autotransplantation.Method:(1) 50 rats were randomly divided into 4 groups:group A:injection of 2.5mg GnRHa was treated once four weeks twice before ovarectomy;group B:injection of saline was treated as control;group C:Sham-operated group;group D:Oophorectomized group.(2) Evaluation after GnRHa used 5 weeks later:serum E2,FSH level;the proportion and qauntity of various stage follicles by HE section.(3) Ealuation after DCV2 cropreservation:a.Histologically examined the morphological change of follicles and the proportion and qauntity of morphologically normal follicles.b.Observed the thawing ovarian tissue necorosis and survival condition by Live/dead Assay.c.Detected the apoptosis positive follicles in ovarian tissue by TUNEL.(4) Collect grafts 3 days,7 days,14days,30days and 60days after transplantation.a.Histologically examined the morphology of follicles and compare the the propotion of various stage follicles at different time,and investigate the follicular growth in grafts dynamically.b.Detected immunohistologially the expression of VEGF in grafts at different time.Results:(1) After treated with GnRHa twice,in group A,the serum E2 level was 0.64±0.23pg/ml,and the FSH level was 1.30±0.59mIu/ml,which were decreased very low compared with group B(P<0.01).(2) The proportion of primordial follicle in group A(80.77±5.92%)was significantly higher than group B(73.10±6.45%)(P<0.05),while the proportion of primary follicle and secondary follicle(16.24±3.75%,11.03±3.19%) was decreased compared to group B(P<0.05).(3) After freezing and thawing,the proportion of normal morphology follicle and morphologically changed follicle in group A were 88.40±4.32%and 10.21±3.71%,and those in group B were 84.75±4.64%and 13.09±3.18%.The difference between two groups was significant(P<0.05).(4) The percentages of viable follicles by live/dead assay of group A and group B were 82.10±5.15%and 83.80±6.09%.The difference between two groups was not significant(P<0.05).(5) The percentages of TUNEL-positive follicles of group A(15.84±2.73%) was higher than group B(13.63±2.56%)(P<0.05).(6) Interval of regular oestrous cyclic in group A(21.75±2.67 days) was significantly longer than group B(17.90±2.67 days).(P<0.05)(7) At the early stage after transplantation,the primordial follicle in grafts was dominant.With the passage of time,the percentage of primordial follicle decreased gradually,and the percentage of primary and secondary follicle increased.The proportion of primordial follicle did not differ between group A and B(P>0.05).Since 14 days after grafting,the percentage of secondary follicle in group B was higher than group A(P<0.05),till 60 days after grafting,the difference between two groups was more significant.(8) In group A,the expression of VEGF on d3,d7,d14,d30,d60 was 4.87±1.16, 3.47±0.56,2.81±0.52,2.57±0.43 and 1.84±0.38 respectively,which was in group B was 4.42±1.47,3.39±0.59,2.72±0.88,2.50±0.51 and 2.03±0.82 respectively.The expression of VEGF increased on day 3,stable on d 30,and decreased on day 30.No statistic difference was found between two groups at different time point(P>0.05).Conclusion:(1) The novel cover vitrification with optimal concentration of cryoprotectants (7.5%EG + 7.5%DMSO + DPBS / 15%EG + 15%DMSO + DPBS) is superior to conventional vitrification.It is suitable for rat ovarian tissue fragments with high efficiency and facility.(2) All frozen-thawed ovarian tissue after autotransplantation could survival and had the hormone secretion ability similar to fresh ovarian tissue.(3) Superficial muscle of groin area was a good implantation site for its convenience,plentiful blood supplying and easy monitoring.(4) The GnRHa pretreatment changed the proportion of the follicle pool with increasing percentage of primordial follicle and decreasing percentage of growth follicle. It is beneficial for more follicles sustain the cryopreservation period and diminish the frozen-thawed injury.(5) The GnRHa pretreatment did not influence the expression of VEGF protein.