大鼠卵巢组织直接覆盖式玻璃化冷冻保存和自体移植的研究
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摘要
随着恶性肿瘤诊治技术的不断进展,肿瘤患者预后明显改善,生存率显著提高。肿瘤治疗领域的进步很大程度上得益于大剂量联合化疗和放疗等治疗手段的不断改进。然而卵巢对具有细胞毒性的药物特别是烷化剂和电离辐射相当敏感,处于儿童期、青少年期和成年期的女性患者接受上述治疗后,卵泡大量丢失使卵巢储备功能受到极大的损伤。卵巢是女性重要的生殖内分泌器官,能够产生性激素、维持内分泌及生成卵子,它的损害会导致闭经、月经不调、性欲减退、第二性征不发育、卵巢早衰和不育等严重临床后果。卵巢组织的冷冻保存及移植正成为因大剂量放化疗等因素引起卵巢衰竭的患者保留卵巢内分泌及生殖功能最具前景的选择。目前,卵巢组织的保存主要有传统的慢速冻存法和玻璃化冷冻法。虽然前者己经取得了一系列的成果,但是这种方法需要程序冷冻仪精密控温,液氮消耗量大,所需时间长,费用较高,且卵巢组织结构较致密,含有多种不同类型的细胞,对冷冻平衡要求各不相同而且差异较大。玻璃化冷冻技术是近10多年来发展起来的一项冷冻保存新技术,它简单、经济,不会因为细胞内冰晶的形成而使细胞受到损伤,较传统的程序化冷冻有诸多优势。但玻璃化冷冻技术需要高浓度的冷冻保护剂(CPA),而高浓度的CPA对组织细胞有一定的毒性作用,CPA的细胞毒性作用是冷冻生物学的主要限制因素之一。如何既降低CPA的细胞毒性,减少作用的时间,又促进渗透性CPA充分进入组织和细胞内达到有效保护浓度,提高玻璃化冷冻效果,是当前的研究热点。Chen报道直接覆盖式玻璃化冷冻法—Direct cover vitrification(DCV),即用较低浓度冷冻保护液且组织与液氮直接接触,应用于保存小鼠卵巢组织并取得满意的效果,但在其他动物和人类的卵巢组织中的应用尚未见报道。
促性腺激素释放激素类似物(GnRHa)是人工合成的促性腺激素释放激素,具有较强的性腺抑制作用。近年来国内外有许多研究应用GnRHa旨在抑制卵巢排卵、卵泡发育,在未成熟卵泡占优势的情况,将抗癌药的影响减少到最低限度。目前认为化疗期间联合应用GnRHa有助于卵巢功能的保护并有利于今后生育功能的恢复。在卵巢组织冷冻保存的研究中发现,原始卵泡更能耐受冷冻复苏的过程,而初级卵泡等生长卵泡更易发生冷冻损伤。如果在获取卵巢组织前应用GnRHa,抑制卵泡的募集、发育,使卵泡尽可能地停留在原始卵泡阶段,是否有利于有利于更多的卵泡渡过低温冷冻期?从而减少卵巢组织的冷冻损伤?国内外的文献均无相关报道。
本研究以雌性性成熟SD大鼠为研究对象,采用直接覆盖式玻璃化冷冻方法,并设计了三种浓度的冷冻保护液,通过组织学、组织荧光活/死性染色(Live/DeadAssay)、TUNEL、透射电镜检测方法,比较与常规玻璃化冷冻方法对卵巢组织保存效果间的差别并探索直接覆盖式玻璃化冷冻最佳冷冻液浓度;初步探讨GnRHa预处理对大鼠卵巢组织的冷冻损伤的影响,以及自体移植后卵巢功能恢复、卵泡生长和血供建立可能产生的影响。
第一部分大鼠卵巢组织两种玻璃化冷冻保存方案的比较研究
目的:
比较大鼠卵巢组织直接覆盖式玻璃化冷冻和常规玻璃化冷冻法的冷冻效果,并探索直接覆盖式玻璃化冷冻的适宜浓度。
方法:
(1)将50只SD大鼠随机分为5组:新鲜对照组,常规玻璃化冷冻组(CV),直接覆盖式玻璃化冷冻组(DCV),采用3种冷冻保护液浓度分别为DCV1组,DCV2组,DCV3组。
(2)建立大鼠卵巢组织的直接覆盖式玻璃化冷冻(DCV)和常规玻璃化冷冻法(CV)保存方案。
(3)各组冷冻复苏后卵巢组织采用以下方法来评估冻存复苏效果:
①组织学观察各级卵泡形态的变化。
②荧光染色法观察卵泡的存活和坏死情况。
③TUNEL法检测卵巢组织中卵泡的凋亡情况。
④透射电镜观察卵泡超微结构的改变。
结果:
(1)组织学结果:新鲜组、CV、DCV1、DCV2、DCV3组形态正常的卵泡比例分别为95.04±2.87%,74.51±6.97%,69.24±5.71%,81.89±6.80%,75.11±5.33%。各冻存组形态正常的卵泡比例较新鲜组织组均有下降(P<0.05),DCV2组正常形态卵泡比例较其他各组高,差异有显著性(P<0.05),CV组与DCV3组间差异无显著性(P>0.05)。各组卵巢组织中形态正常的卵泡均为原始卵泡比例最高,初级卵泡其次,次级卵泡最最少。
(2)荧光染色法显示:新鲜组、CV、DCV1、DCV2、DCV3组存活卵泡比例为97.00±2.67%,75.09±5.49%,71.43±6.20%,83.60±5.33%,77.38±5.13%。各组卵巢组织经冷冻复苏后存活卵泡比例较新鲜组织组均有下降(P<0.05),各冷冻复苏组中,DCV2组存活卵泡比例较其他各组更高,差异有显著性(P<0.05),CV组与DCV1,DCV3组间差异无显著性(P>0.05)。
(3)TUNEL结果:在新鲜组和DCV1、DCV2、DCV3、CV组中,TUNEL阳性卵泡比例分别为7.72±0.65%,13.64±0.79%,14.24±1.55%,13.96±1.60%和13.14±1.67%。经冷冻保存后各组卵泡凋亡数有所增加(P<0.05),但在各冷冻组之间差异无显著性(P>0.05)。
(4)透射电镜结果:新鲜对照组、CV、DCV1、DCV2、DCV3组TEM总评分分别为90.25±5.31%,46.78±8.22%,50.92±6.52%,59.75±5.31%,55.57±5.55%,各冷冻组TEM总评分较新鲜对照组明显下降,DCV2组高于CV,DCV1组,(P<0.05)。卵母细胞的TEM评分DCV2组较其他冻存组增高(P<0.05),颗粒细胞及基质细胞TEM评分各冻存组间无显著差异。各组卵母细胞得分最高,颗粒细胞其次,基质细胞最低。
第二部分大鼠卵巢组织自体移植的实验研究
目的:
建立大鼠自体移植模型,观察新鲜卵巢组织片和各冻融组卵巢组织异位移植后的生长情况,分析不同冷冻方案对卵巢组织移植后生长的影响,从而进一步判断各冷冻方案的优劣。
方法:
(1)各组卵巢组织进行冷冻保存、复苏后,自体移植至两侧腹股沟区肌肉层内。A(CV移植组),B1(DCV1移植组),B2(DCV2移植组),B3(DCV3移植组),C(新鲜卵巢组织移植组),并设D(去势组),E(假手术组)作为对照。
(2)通过阴道上皮细胞观察动情周期恢复时间及恢复率。
(3)移植后4周左右测血清E2,FSH水平。
(4)移植后30天取出移植的卵巢,HE切片观察移植后卵巢卵泡形态和卵泡密度。
结果:
(1)A,B1,B2,B3组和C组动情周期的恢复率分别为77.8%(7/9),66.7%(6/9),80%(8/10),80%(8/10)和100%(10/10)。A、B1、B2、B3组动情周期开始的天数分别为:17.71±2.14,19.67±2.58,19.25±2.66,17.88±2.03天,各冻存移植组均长于C组(12.00±1.58),有显著性差异(P<0.05),各冻存移植组之间比较无显著差异(P>0.05)。
(2)移植前B1,B2,B3,A组卵泡密度(个/mm~2)为8.24±3.18,8.22±3.39,8.43±2.70,8.57±3.22,与C组之间无明显差异(P>0.05);但移植后B1,B2,B3,A组卵泡密度分别降为1.944±0.95,2.67±0.87,2.20±0.83,2.05±0.90,与F组的4.55±1.85比较均显著下降(P<0.05),其中DCV2组卵泡密度高于其他各冷冻组(P<0.05),CV、DCV1、DCV组之间无显著差异(P>0.05)。
(3)C组与E组比较,E2、FSH水平无显著差异(P<0.05);各冷冻保存组E2水平虽低于E组和C组(P<0.05),但较D组明显增高(P<0.01);各冷冻组FSH水平较C组和E组高(P<0.05),各冷冻保存组之间E2、FSH水平无显著差异。
第三部分GnRHa预处理对大鼠卵巢组织的冷冻保存及自体移植的影响
目的:
初步探讨GnRHa预处理对大鼠卵巢组织的冷冻损伤的影响,以及自体移植后卵巢功能恢复、卵泡生长和血供建立可能产生的影响。
方法:
(1)50只SD大鼠随机分为4组,A组(GnRHa组):术前5周每隔30天给予大鼠皮下注射GnRHa 0.25mg,共两次;B组(对照组):皮下注射注射生理盐水;C组:假手术组;D组:去势组。
(2)GnRHa用药后5周评估:血E2,FSH水平;HE切片观察卵泡数目和各级卵泡比例的变化。
(3)冷冻复苏后卵巢组织的评估:
①组织学观察卵泡形态和其中形态正常的各级卵泡比例。
②荧光染色法观察卵泡的存活和坏死情况。
③TUNEL法检测卵巢组织中卵泡的凋亡率。
(4)分别在移植后第3天,第7天,第14天,第30天,第60天回收移植物。
①HE切片观察卵巢组织切片中卵泡的形态,比较各时间点各组不同阶段卵泡所占的比例,动态观察移植物中卵泡启动生长的情况。
②免疫组化法检测移植物中各时间点VEGF蛋白的表达
结果:
(1)GnRHa用药二次后,A组大鼠血清E2:0.64±0.23 pg/ml,FSH:1.30±0.59mIu/ml,B组E2:38.05±4.41 pg/ml,FSH:6.64±1.51 mIu/ml,A、B组间有极显著差异(P<0.01)。
(2)GnRHa组原始卵泡比例(80.77±5.92%)较对照组(73.10±6.45%)显著增加(P<0.05),而初级卵泡(11.39±3.52a%)、次级卵泡比例(5.07±1.55%)下降,与对照组比较(16.24±3.75%,11.03±3.19%)有显著性差异(P<0.05)。
(3)冷冻复苏后,GnRHa组正常形态卵泡比例和形态改变卵泡比例为88.40±4.32%,10.21±3.71%,对照组分别为84.75±4.64%,13.09±3.18%,两组间有显著差异(P<0.05)。
(4)荧光染色法观察卵泡的存活率在GnRHa组与对照组中分别为82.10±5.15%,83.80±6.09%,差异无统计学意义(P>0.05)。
(5)GnRHa组卵泡的凋亡率为15.84±2.73%,对照组为13.63±2.56%,两组间有显著差异(P<0.05)。
(6)GnRHa组恢复动情周期为21.75±2.67天,对照组为17.90±2.67天,GnRHa组较对照组迟,两组有显著差异(P<0.05)。
(7)移植后两组移植物中均呈现早期原始卵泡为主,随着时间的推移,原始卵泡比例逐渐减少,初级、次级卵泡比例增高;GnRHa组与对照组比较,原始卵泡比例在各个时间点均无差异;至14天起,对照组次级卵泡的比例较GnRHa组增高(P<0.05);至60天时,两组差别更大。
(8)GnRHa组术后第3天,第7天,第14天,第30天,第60天VEGF表达水平分别为4.87±1.16,3.47±0.56,2.81±0.52,2.57±0.43,1.84±0.38;对照组分别为4.42±1.47,3.39±0.59,2.72±0.88,2.50±0.51,2.03±0.82。两组均呈现移植早期(1～3天)VEGF表达明显增强,第7天起表达水平平稳,第30天VEGF表达水平降低。GnRHa组各时间点VEGF表达水平与对照组比较,差异无统计学意义。
结论:
(1)以7.5%EG+7.5%DMSO+DPBS/15%EG+15%DMSO+DPBS为冷冻保护液结合直接覆盖式玻璃化技术(DCV)是适合大鼠卵巢组织冻存的理想方案,优于常规玻璃化冷冻法。
(2)各冻存组的卵巢组织自体移植后可以存活并具有与新鲜卵巢组织相接近的激素分泌能力。
(3)大腿根部肌肉层作为移植部位,简单易行,血供良好,便与监测。
(4)GnRHa预处理改变了卵泡池的各级卵泡的构成比,增加了原始卵泡的比例,减少易受冷冻损伤的生长卵泡的比例,有利于组织渡过冷冻复苏的过程。
(5)GnRHa预处理对移植物VEGF蛋白表达无影响。
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 autotransplantation
Objective: 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 autotransplantation
Objective:
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.
促性腺激素释放激素类似物(GnRHa)是人工合成的促性腺激素释放激素,具有较强的性腺抑制作用。近年来国内外有许多研究应用GnRHa旨在抑制卵巢排卵、卵泡发育,在未成熟卵泡占优势的情况,将抗癌药的影响减少到最低限度。目前认为化疗期间联合应用GnRHa有助于卵巢功能的保护并有利于今后生育功能的恢复。在卵巢组织冷冻保存的研究中发现,原始卵泡更能耐受冷冻复苏的过程,而初级卵泡等生长卵泡更易发生冷冻损伤。如果在获取卵巢组织前应用GnRHa,抑制卵泡的募集、发育,使卵泡尽可能地停留在原始卵泡阶段,是否有利于有利于更多的卵泡渡过低温冷冻期?从而减少卵巢组织的冷冻损伤?国内外的文献均无相关报道。
本研究以雌性性成熟SD大鼠为研究对象,采用直接覆盖式玻璃化冷冻方法,并设计了三种浓度的冷冻保护液,通过组织学、组织荧光活/死性染色(Live/DeadAssay)、TUNEL、透射电镜检测方法,比较与常规玻璃化冷冻方法对卵巢组织保存效果间的差别并探索直接覆盖式玻璃化冷冻最佳冷冻液浓度;初步探讨GnRHa预处理对大鼠卵巢组织的冷冻损伤的影响,以及自体移植后卵巢功能恢复、卵泡生长和血供建立可能产生的影响。
第一部分大鼠卵巢组织两种玻璃化冷冻保存方案的比较研究
目的:
比较大鼠卵巢组织直接覆盖式玻璃化冷冻和常规玻璃化冷冻法的冷冻效果,并探索直接覆盖式玻璃化冷冻的适宜浓度。
方法:
(1)将50只SD大鼠随机分为5组:新鲜对照组,常规玻璃化冷冻组(CV),直接覆盖式玻璃化冷冻组(DCV),采用3种冷冻保护液浓度分别为DCV1组,DCV2组,DCV3组。
(2)建立大鼠卵巢组织的直接覆盖式玻璃化冷冻(DCV)和常规玻璃化冷冻法(CV)保存方案。
(3)各组冷冻复苏后卵巢组织采用以下方法来评估冻存复苏效果:
①组织学观察各级卵泡形态的变化。
②荧光染色法观察卵泡的存活和坏死情况。
③TUNEL法检测卵巢组织中卵泡的凋亡情况。
④透射电镜观察卵泡超微结构的改变。
结果:
(1)组织学结果:新鲜组、CV、DCV1、DCV2、DCV3组形态正常的卵泡比例分别为95.04±2.87%,74.51±6.97%,69.24±5.71%,81.89±6.80%,75.11±5.33%。各冻存组形态正常的卵泡比例较新鲜组织组均有下降(P<0.05),DCV2组正常形态卵泡比例较其他各组高,差异有显著性(P<0.05),CV组与DCV3组间差异无显著性(P>0.05)。各组卵巢组织中形态正常的卵泡均为原始卵泡比例最高,初级卵泡其次,次级卵泡最最少。
(2)荧光染色法显示:新鲜组、CV、DCV1、DCV2、DCV3组存活卵泡比例为97.00±2.67%,75.09±5.49%,71.43±6.20%,83.60±5.33%,77.38±5.13%。各组卵巢组织经冷冻复苏后存活卵泡比例较新鲜组织组均有下降(P<0.05),各冷冻复苏组中,DCV2组存活卵泡比例较其他各组更高,差异有显著性(P<0.05),CV组与DCV1,DCV3组间差异无显著性(P>0.05)。
(3)TUNEL结果:在新鲜组和DCV1、DCV2、DCV3、CV组中,TUNEL阳性卵泡比例分别为7.72±0.65%,13.64±0.79%,14.24±1.55%,13.96±1.60%和13.14±1.67%。经冷冻保存后各组卵泡凋亡数有所增加(P<0.05),但在各冷冻组之间差异无显著性(P>0.05)。
(4)透射电镜结果:新鲜对照组、CV、DCV1、DCV2、DCV3组TEM总评分分别为90.25±5.31%,46.78±8.22%,50.92±6.52%,59.75±5.31%,55.57±5.55%,各冷冻组TEM总评分较新鲜对照组明显下降,DCV2组高于CV,DCV1组,(P<0.05)。卵母细胞的TEM评分DCV2组较其他冻存组增高(P<0.05),颗粒细胞及基质细胞TEM评分各冻存组间无显著差异。各组卵母细胞得分最高,颗粒细胞其次,基质细胞最低。
第二部分大鼠卵巢组织自体移植的实验研究
目的:
建立大鼠自体移植模型,观察新鲜卵巢组织片和各冻融组卵巢组织异位移植后的生长情况,分析不同冷冻方案对卵巢组织移植后生长的影响,从而进一步判断各冷冻方案的优劣。
方法:
(1)各组卵巢组织进行冷冻保存、复苏后,自体移植至两侧腹股沟区肌肉层内。A(CV移植组),B1(DCV1移植组),B2(DCV2移植组),B3(DCV3移植组),C(新鲜卵巢组织移植组),并设D(去势组),E(假手术组)作为对照。
(2)通过阴道上皮细胞观察动情周期恢复时间及恢复率。
(3)移植后4周左右测血清E2,FSH水平。
(4)移植后30天取出移植的卵巢,HE切片观察移植后卵巢卵泡形态和卵泡密度。
结果:
(1)A,B1,B2,B3组和C组动情周期的恢复率分别为77.8%(7/9),66.7%(6/9),80%(8/10),80%(8/10)和100%(10/10)。A、B1、B2、B3组动情周期开始的天数分别为:17.71±2.14,19.67±2.58,19.25±2.66,17.88±2.03天,各冻存移植组均长于C组(12.00±1.58),有显著性差异(P<0.05),各冻存移植组之间比较无显著差异(P>0.05)。
(2)移植前B1,B2,B3,A组卵泡密度(个/mm~2)为8.24±3.18,8.22±3.39,8.43±2.70,8.57±3.22,与C组之间无明显差异(P>0.05);但移植后B1,B2,B3,A组卵泡密度分别降为1.944±0.95,2.67±0.87,2.20±0.83,2.05±0.90,与F组的4.55±1.85比较均显著下降(P<0.05),其中DCV2组卵泡密度高于其他各冷冻组(P<0.05),CV、DCV1、DCV组之间无显著差异(P>0.05)。
(3)C组与E组比较,E2、FSH水平无显著差异(P<0.05);各冷冻保存组E2水平虽低于E组和C组(P<0.05),但较D组明显增高(P<0.01);各冷冻组FSH水平较C组和E组高(P<0.05),各冷冻保存组之间E2、FSH水平无显著差异。
第三部分GnRHa预处理对大鼠卵巢组织的冷冻保存及自体移植的影响
目的:
初步探讨GnRHa预处理对大鼠卵巢组织的冷冻损伤的影响,以及自体移植后卵巢功能恢复、卵泡生长和血供建立可能产生的影响。
方法:
(1)50只SD大鼠随机分为4组,A组(GnRHa组):术前5周每隔30天给予大鼠皮下注射GnRHa 0.25mg,共两次;B组(对照组):皮下注射注射生理盐水;C组:假手术组;D组:去势组。
(2)GnRHa用药后5周评估:血E2,FSH水平;HE切片观察卵泡数目和各级卵泡比例的变化。
(3)冷冻复苏后卵巢组织的评估:
①组织学观察卵泡形态和其中形态正常的各级卵泡比例。
②荧光染色法观察卵泡的存活和坏死情况。
③TUNEL法检测卵巢组织中卵泡的凋亡率。
(4)分别在移植后第3天,第7天,第14天,第30天,第60天回收移植物。
①HE切片观察卵巢组织切片中卵泡的形态,比较各时间点各组不同阶段卵泡所占的比例,动态观察移植物中卵泡启动生长的情况。
②免疫组化法检测移植物中各时间点VEGF蛋白的表达
结果:
(1)GnRHa用药二次后,A组大鼠血清E2:0.64±0.23 pg/ml,FSH:1.30±0.59mIu/ml,B组E2:38.05±4.41 pg/ml,FSH:6.64±1.51 mIu/ml,A、B组间有极显著差异(P<0.01)。
(2)GnRHa组原始卵泡比例(80.77±5.92%)较对照组(73.10±6.45%)显著增加(P<0.05),而初级卵泡(11.39±3.52a%)、次级卵泡比例(5.07±1.55%)下降,与对照组比较(16.24±3.75%,11.03±3.19%)有显著性差异(P<0.05)。
(3)冷冻复苏后,GnRHa组正常形态卵泡比例和形态改变卵泡比例为88.40±4.32%,10.21±3.71%,对照组分别为84.75±4.64%,13.09±3.18%,两组间有显著差异(P<0.05)。
(4)荧光染色法观察卵泡的存活率在GnRHa组与对照组中分别为82.10±5.15%,83.80±6.09%,差异无统计学意义(P>0.05)。
(5)GnRHa组卵泡的凋亡率为15.84±2.73%,对照组为13.63±2.56%,两组间有显著差异(P<0.05)。
(6)GnRHa组恢复动情周期为21.75±2.67天,对照组为17.90±2.67天,GnRHa组较对照组迟,两组有显著差异(P<0.05)。
(7)移植后两组移植物中均呈现早期原始卵泡为主,随着时间的推移,原始卵泡比例逐渐减少,初级、次级卵泡比例增高;GnRHa组与对照组比较,原始卵泡比例在各个时间点均无差异;至14天起,对照组次级卵泡的比例较GnRHa组增高(P<0.05);至60天时,两组差别更大。
(8)GnRHa组术后第3天,第7天,第14天,第30天,第60天VEGF表达水平分别为4.87±1.16,3.47±0.56,2.81±0.52,2.57±0.43,1.84±0.38;对照组分别为4.42±1.47,3.39±0.59,2.72±0.88,2.50±0.51,2.03±0.82。两组均呈现移植早期(1～3天)VEGF表达明显增强,第7天起表达水平平稳,第30天VEGF表达水平降低。GnRHa组各时间点VEGF表达水平与对照组比较,差异无统计学意义。
结论:
(1)以7.5%EG+7.5%DMSO+DPBS/15%EG+15%DMSO+DPBS为冷冻保护液结合直接覆盖式玻璃化技术(DCV)是适合大鼠卵巢组织冻存的理想方案,优于常规玻璃化冷冻法。
(2)各冻存组的卵巢组织自体移植后可以存活并具有与新鲜卵巢组织相接近的激素分泌能力。
(3)大腿根部肌肉层作为移植部位,简单易行,血供良好,便与监测。
(4)GnRHa预处理改变了卵泡池的各级卵泡的构成比,增加了原始卵泡的比例,减少易受冷冻损伤的生长卵泡的比例,有利于组织渡过冷冻复苏的过程。
(5)GnRHa预处理对移植物VEGF蛋白表达无影响。
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 autotransplantation
Objective: 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 autotransplantation
Objective:
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.
引文
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