人卵巢组织程序化冷冻及体外培养对卵泡活性和雌二醇分泌的影响

Influence of programmable cryopreservation and in vitro culture on follicular viability and estradiol secretion of human ovarian tissue

目的探讨程序化冷冻保存及组织体外培养对人卵巢皮质内卵泡活性和雌二醇分泌的影响。方法取手术切除的人卵巢皮质组织,修剪成1 cm3大小组织块,采用抽签法随机分为新鲜组、程序冷冻组、新鲜培养组、冻融后培养组。应用程序化冷冻法冻存卵巢组织,冷冻前后分别对卵巢组织进行体外培养,电化学发光免疫分析法检测上清液中雌二醇水平。冷冻前后及培养前后分别用Clacein AM进行卵泡荧光染色,各级卵泡计数。结果①冷冻前后各级卵泡数量及构成比差异无统计学意义(P>0.05);②新鲜组织和冻融组织体外培养都具有持续分泌E2的功能,开始培养阶段冻融组织分泌的雌二醇水平偏低(P<0.05),之后组间上清液雌二醇水平差异无统计学意义(P>0.05);③体外培养前后始基卵泡和初级卵泡数量及其构成比差异无统计学意义(P>0.05);体外培养后次级卵泡数量及构成比增加,差异有统计学意义(P<0.05)。结论程序冷冻法可以较好地保存人卵巢皮质内的窦前卵泡,不影响体外培养的卵巢组织的雌二醇分泌。卵巢组织体外培养情况下,次级卵泡的生长优于始基卵泡和初级卵泡的生长。

Objective To investigate the influence of programmable cryopreservation and in vitro culture on follicular viability and estradiol secretion of human ovarian tissue. Methods Pieces of human ovarian cortex were prepared, the specimens were round in shape and were about 1 mmxl mmxl mm, and were divided into fresh group, frozen group, fresh culturing group and freezing-thawing culturing group. Pieces of human ovarian cortex were frozen with the method of programmable cryopreservation and cultured in vitro before freezing and after thawing. The level of estradiol in medium was measured with electro-chemiluminescence immunoassay. Healthy follicles stained with Clacein AM were conducted under fluorescence microscopy after digestion for all ovarian tissues. Results The number of follicles and their proportions in freezing group had no significant difference from that in fresh group（P〉0.05 ）. Estradiol was secreted continuously throughout in-vitro culture in both of fresh tissues and freezing-thawing tissues. The level of estradiol in freezing-thawing culturing group was lower than that in fresh culturing group in the first 2 d（P〈0.05）. The difference of the estradiol level was not significant（P〉0.05） after 2 d. The number of primordial and primary follicles and their proportions had no significant difference after in vitro culture （ P〉0.05 ） ; the number of secondary follicles and its proportion were higher in tissues cultured in vitro for 14 days （ P 〈 0.05 ）. Conclusion The method of programmable cryopreservation can maintain the viability of preantral follicles and E2 secretion of ovarian tissues cultured in vitro. Secondaryfollicles have greater potential of development than that of primordial and primary follicles of ovarian tissues cultured in vitro.