人类卵巢组织玻璃化冷冻及复苏后形态学及组织增殖活性观察

Morphology and Celluar Proliferation of Follicles from Cryopreserved Human Ovarian Tissues

目的探讨玻璃化冷冻对成人卵巢组织中原始卵泡与初级卵泡的形态学及细胞增殖状态的影响。方法采用液氮直接覆盖玻璃化冷冻(direct cover vitrification,DCV)方法冻存成人卵巢组织块,液氮保存2周后复苏组织。观察和比较冻融前后人卵巢组织中原始卵泡与初级卵泡组织形态学改变;免疫组化染色测定经体外培养48 h后新鲜和冻融卵巢组织中增殖细胞核抗原(PCNA)的表达情况。结果冻融前后人卵巢组织内原始卵泡和初级卵泡分布差异无统计学意义(P>0.05);新鲜卵巢组织中,形态异常的原始卵泡比例与初级卵泡比例差异无统计学意义(P>0.05),冻融卵巢组织中,形态异常的原始卵泡比例低于形态异常的初级卵泡比例(P<0.05);新鲜组、新鲜及冻融后培养组均有PCNA的阳性表达,PCNA阳性表达可见于中间型卵泡和初级卵泡的卵母细胞和颗粒细胞、卵巢组织间质细胞。结论利用DCV方法对人卵巢组织进行冻存能够保存原始卵泡和初级卵泡,冻融过程对原始卵泡和初级卵泡形态结构都可能造成一定程度的损伤,冻融后组织内卵泡有体外生长发育的潜力。

Objective To compare the morphology and proliferation of primordial and primary follicles in fresh and vitrificated human ovarian tissues.Methods Human ovarian tissues were cryopreserved by direct cover vitrification（DCV） for 2 weeks.The morphology of the primordial and primary follicles from the frozen-thawed tissues was compared with those from the fresh tissues.Both fresh and cryopreservation tissues were cultured for 48 hours before the tissues were embedded in paraffin block for immunohistochemical staining for PCNA.Results The distribution of primordial and primary follicles in the fresh ovarian tissues was not different from that in the frozen tissues.The cryopreserved tissues had less abnormal morphology in primordial follicles than in primary follicles,but no difference was found between the cryopreserved tissues and fresh tissues.Positive staining on PCNA expression in granulsa cells and oocyte of transitional follicles and primary follicles as well as stromal cells were found in fresh,fresh cultured and cryopreserved cultured ovarian tissues.The fresh tissues had less positive staining on PCNA in the follicle than in the fresh cultured and cryopresered cultured tissues. Conclusion Cryopreserved human ovarian tissues by DCV can maintain partial primordial and primary follicles.Follicles in cryopreserved ovarian tissues can initiate development in vitro culture.