卵巢组织玻璃化冷冻移植效果及体外受精结局的动物实验研究

Experimental animal studies on the effect of ovarian tissue vitrification-thawed-transplantation and in vitro fertilization outcome

目的探讨玻璃化冷冻和组织移植对小鼠卵巢组织的影响。方法将雌性ICR小鼠分为3组:正常对照组(卵母细胞体外培养及受精,n=9)、新鲜移植组(卵巢组织移植后卵母细胞体外培养及受精,n=9)、冷冻移植组(卵巢组织玻璃化冷冻复苏-移植、卵母细胞体外培养及受精,n=9)。同时,为了更为直接地说明玻璃化冷冻对于卵泡数量的影响和卵巢移植后内分泌功能的改变情况,本研究还设置了冷冻复苏组(卵巢组织玻璃化冷冻复苏,n=6)和卵巢去势组(卵巢组织切除,n=6)。卵巢组织移植3周后,通过苏木精-伊红(hematoxylin-eosin,HE)染色计算各组小鼠的卵泡数量、CD31免疫组织化学染色观察卵巢组织新生血管形成、Masson染色观察卵巢组织纤维化、酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清性激素水平,并统计获卵数、体外受精数量和形成囊胚数量。结果新鲜移植组和冷冻移植组小鼠的卵巢组织中总卵泡数量均较正常对照组明显减少(均P<0.001),且都明显少于冷冻复苏组(均P<0.001);新鲜移植组小鼠卵巢组织CD31阳性率明显高于正常对照组(P=0.044),冷冻移植组小鼠高于正常对照组,但差异无统计学意义(P=0.162);新鲜移植组和冷冻移植组小鼠卵巢组织纤维化面积百分率均明显高于正常对照组(P=0.004;P=0.005);新鲜移植组和冷冻移植组小鼠血清雌二醇水平均明显低于正常对照组(P=0.005;P=0.001),但明显高于卵巢去势组(P=0.011;P=0.035),血清卵泡刺激素(follicle-stimulating hormone,FSH)水平均明显高于正常对照组(P=0.040;P=0.012),但明显低于卵巢去势组(P=0.001;P=0.004);新鲜移植组和冷冻移植组小鼠的获卵数均较正常对照组明显减少(均P<0.001),新鲜移植组小鼠的获卵数高于冷冻移植组,但差异无统计学意义(P=0.272);新鲜移植组和冷冻移植组卵母细胞的体外受精数量和囊胚数均明显低于正常对照组(均P<0.001)。新鲜移植组和冷冻移植组小鼠在总卵泡数量、CD31阳性率、纤维化面积百分率、血清雌二醇和FSH水平、受精数和形成囊胚数上差异均无统计学意义(均P>0.05)。结论小鼠卵巢组织在接受玻璃化冷冻复苏-移植后能够恢复卵泡生长、内分泌功能以及生育功能,证实利用玻璃化冷冻技术进行卵巢组织冻存可以作为女性生育力保存的一个有效手段。玻璃化冷冻和组织移植均会造成卵巢组织中的卵泡损失并影响生育能力,移植后阶段是卵巢组织冷冻复苏-移植过程中卵泡损失的主要阶段,组织移植是影响卵巢组织冷冻复苏-移植效果的主要因素。

Objective To investigate the effects of vitrification and transplantation on mouse ovarian tissues.Methods Female ICR mice were divided into three groups:fresh-control group(in vitro oocyte maturation and fertilization,n=9),fresh-transplanted group(ovarian tissue transplantation,in vitro oocyte maturation and fertilization,n=9),frozen-transplanted group(ovarian tissue vitrification-thawed-transplantation,in vitro oocyte maturation and fertilization,n=9).Additionally,frozen-thawed group(ovarian tissue vitrification-thawed,n=6)and ovariectomy group(ovariectomy,n=6)were also set up,to more directly explain the effects of vitrification and freezing on the number of follicles and the changes of endocrine function after ovarian transplantation.We determined the number of follicles by hematoxylin-eosin(HE)staining,neovascularization by CD31 immunohistochemical staining,tissue fibrosis by Masson staining,and serum sex hormone levels by enzyme-linked immunosorbent assay(ELISA)three weeks following ovarian tissue transplantation.In addition,we counted the number of oocytes obtained,oocytes in vitro fertilized,as well as blastocysts.Results The number of total follicles in both the fresh-transplanted group and the frozen-transplanted group significantly decreased compared with the fresh-control group(all P<0.001)and the frozen-thawed group(all P<0.001).The CD31-positive rate of ovarian tissues in the fresh-transplanted group was significantly higher than that in the fresh-control group(P=0.044).Although the CD31-positive rate was higher in the frozen-transplanted group than in the fresh-control group,there was no statistical distinction(P=0.162).The fibrosis area percentage of ovarian tissues in both the fresh-transplanted group and the frozen-transplanted group increased significantly compared with the fresh-control group(P=0.004;P=0.005).Serum estradiol level in the fresh-transplanted group and the frozen-transplanted group was significantly lower than that in fresh-control group(P=0.005;P=0.001),significantly higher than that in the ovariectomy group(P=0.011;P=0.035).Serum follicle-stimulating hormone(FSH)level in the fresh-transplanted group and the frozen-transplanted group was significantly higher than that in the fresh-control group(P=0.040;P=0.012),significantly lower than that in the ovariectomy group(P=0.001;P=0.004).In comparison to the fresh-control group,the number of oocytes retrieved in the fresh-transplanted group and the frozen-transplanted group decreased significantly(all P<0.001).Furthermore,the number of oocytes retrieved in the fresh-transplanted group was higher than that in the frozen-transplanted group,and yet there was no statistical difference(P=0.272).And the number of oocytes in vitro fertilized and blastocysts in the fresh-transplanted group and the frozen-transplanted group were significantly lower than those in the fresh-control group(all P<0.001).The number of total follicles,CD31-positive rate,fibrosis area rate,serum estradiol,and FSH levels,the number of oocytes in vitro fertilized and blastocysts were no substantially distinction between the fresh-transplanted group and the frozen-transplanted group(all P>0.05).Conclusion​After ovarian tissue vitrification-thawed and transplantation,follicular growth,endocrine function,and fertility were restored in the mouse model,confirming that ovarian tissue vitrification is an effective method for female fertility preservation.Both vitrification and transplantation could cause follicles to be lost and fertility to decrease.And post-transplantation stage is the primary stage of follicle loss during ovarian tissue frozen-thawed-transplantation,and transplantation is the predominant factor affecting the effectiveness of ovarian tissue frozen-thawed-transplantation.