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

doi:10.3969/j.issn.1006-7795.2013.04.006

首都医科大学学报 ›› 2013, Vol. 34 ›› Issue (4): 506-511.doi: 10.3969/j.issn.1006-7795.2013.04.006

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

田玄玄¹, 阮祥燕¹, Montag Markus², Liebenthron Jana³, Alfred O. Mueck⁴

1. 首都医科大学附属北京妇产医院内分泌科, 北京 100026;
2. 海德堡大学妇产医院生殖医学研究中心, 海德堡 69115;
3. 波恩大学妇产医院卵巢组织冻存中心, 波恩 53012;
4. 德国图宾根大学妇产医院内分泌与绝经研究中心, 图宾根 D-72076

收稿日期:2013-06-10 出版日期:2013-08-21 发布日期:2013-07-20
通讯作者: 阮祥燕 E-mail:ruanxiangyan@163.com
基金资助:
北京市卫生系统高层次卫生技术人才培养计划项目(2009-3-52)和国家外专局引智项目(20121100017,20131100006)。

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

TIAN Xuanxuan¹, RUAN Xiangyan¹, Montag Markus², Liebenthron Jana³, Alfred O. Mueck⁴

1. Department of Gynecological Endocrinology, Beijing Obsterics and Gynecology Hospital, Capital Medical University, Beijing 100026, China;
2. Reproductive Biology Laboratory, Women's Hospital, University of Heidelberg, Heidelberg 69115, Germany;
3. Ovarian Tissue Cryobank, Women's Hospital, University of Bonn, Bonn 53012, Germany;
4. Department of Endocrinology, University Women's Hospital of Tuebingen, Tuebingen D-72076, Germany

Received:2013-06-10 Online:2013-08-21 Published:2013-07-20
Supported by:
This study was supported by Beijing Municipality Health Technology High-level Talent(2009-3-52) and China Administration of Foreign Experts Affairs "YinZhi" Project(20121100017, 20131100006).

摘要/Abstract

摘要：

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。

关键词: 卵巢组织, 程序冷冻, 体外培养

Abstract:

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 mm×1 mm×1 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. Secondary follicles have greater potential of development than that of primordial and primary follicles of ovarian tissues cultured in vitro.

Key words: ovarian tissue, programmable cryopreservation, in vitro culture

中图分类号:

R339.2

田玄玄, 阮祥燕, Montag Markus, Liebenthron Jana, Alfred O. Mueck. 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[J]. 首都医科大学学报, 2013, 34(4): 506-511.

TIAN Xuanxuan, RUAN Xiangyan, Montag Markus, Liebenthron Jana, Alfred O. Mueck. Influence of programmable cryopreservation and in vitro culture on follicular viability and estradiol secretion of human ovarian tissue[J]. Journal of Capital Medical University, 2013, 34(4): 506-511.

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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

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