N-乙酰半胱氨酸对冻融人卵巢组织的保护作用

doi:10.3969/j.issn.1006-7795.2024.04.003

首都医科大学学报 ›› 2024, Vol. 45 ›› Issue (4): 573-582.doi: 10.3969/j.issn.1006-7795.2024.04.003

• 更年期生殖内分泌与生育力保护 • 上一篇下一篇

N-乙酰半胱氨酸对冻融人卵巢组织的保护作用

李扬璐¹，阮祥燕^1，2*，李妍秋¹，谷牧青¹，杜娟¹，王泽铖¹，程姣姣¹，金凤羽¹，蒋玲玲¹，杨瑜¹， Alfred O. Mueck^1,2

1.首都医科大学附属北京妇产医院/北京妇幼保健院内分泌科，北京 100026； 2.德国图宾根大学妇产医院妇女健康部与妇女健康研究中心，图宾根 D-72076

收稿日期:2024-05-04 出版日期:2024-08-21 发布日期:2024-07-08
通讯作者: 阮祥燕 E-mail:ruanxiangyan@ccmu.edu.cn
基金资助:
国家自然科学基金项目（82101822），“北京市属医院科研培育计划”项目（PX2021053），首都医科大学附属北京妇产医院中青年学科骨干培养专项（FCYY202015），首都医科大学校培育基金（PYZ20058），北京市自然科学基金项目（7202047）。

Protective effect of N-Acetyl-L-Cysteine on cryopreserved-thawed human ovarian tissue

Li Yanglu¹, Ruan Xiangyan^1，2*, Li Yanqiu¹, Gu Muqing¹, Du Juan¹, Wang Zecheng¹, Cheng Jiaojiao¹, Jin Fengyu¹, Jiang Lingling¹, Yang Yu¹，Alfred O. Mueck^1,2

1.Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University/Beijing Maternal and Child Health Care Hospital, Beijing 100026, China; 2.Department of Women's Health, Research Centre for Women's Health and University Women's Hospital of Tuebingen, University Hospitals of Tuebingen, Tuebingen D-72076, Germany

Received:2024-05-04 Online:2024-08-21 Published:2024-07-08
Supported by:
This study was supported by National Natural Science Foundation of China(82101822), Beijing Municipal Administration of Hospitals Incubating Program (PX2021053), Beijing Obstetrics and GynecologyHospital, Capital Medical University (No.FCYY202015), Capital Medical University (PYZ20058), Natural Science Foundation of Beijing (7202047).

摘要/Abstract

摘要： 目的以首都医科大学附属北京妇产医院生育力保护中心现有冻存复苏方案为基础，以雌性去势裸鼠为模型，通过人冻融卵巢组织异种移植探讨抗氧化剂N-乙酰半胱氨酸（N-Acetyl-L-Cysteine，NAC）对人卵巢组织的保护作用。方法将4例患者的卵巢组织按照原有冻融方案和改良方案（加入NAC）进行冻融，所有患者取材后留取新鲜卵巢组织进行钙黄绿素AM（Calcein acetoxymethyl ester，Calcein-AM）和苏木精-伊红 (hematoxylin-eosin staining，HE)染色评估卵泡活性并计数。将36只雌性去势裸鼠随机分为4组，将复苏后的卵巢组织移植于双侧裸鼠肾被膜下，按照移植卵巢组织的冻融方案分为原有冻融方案组（control group），改良方案组（NAC group），卵巢去势组（ovariectomy group）和正常对照组（normal group，不进行手术）。分别在移植第3天、第7天、第21天处死裸鼠，取血清及卵巢组织移植物。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）法检测血清雌二醇（estradiol, E2）、卵泡刺激素（follicle stimulating hormone, FSH）和抗苗勒管激素（anti-mullerian hormone，AMH）水平，取卵巢组织移植物1片用于HE染色观察卵泡发育情况，1片用于总抗氧化能力（total antioxidant capability, TAC）的检测。结果移植后各时间点两移植组卵泡发育分级差异无统计学意义（P>0.05）。NAC组总抗氧化能力显著高于control组（P<0.05），与新鲜卵巢组织相比差异无统计学意义（P>0.05）。移植第3天 control 组裸鼠血清中E2水平显著高于卵巢去势组（P<0.05），余各时间点两移植组与卵巢去势组差异无统计学意义（P>0.05）。移植后第3天和第21天两移植组裸鼠血清中FSH水平低于卵巢去势组（P<0.05），与正常对照组差异无统计学意义（P>0.05）。移植后第3天 control 组AMH显著高于卵巢去势组（P<0.05），与其余各组差异无统计学意义（P>0.05），移植第7天两移植组AMH水平下降，显著低于正常对照组（P<0.05），移植第21天两移植组AMH水平均显著高于卵巢去势组（P<0.05），与正常对照组差异无统计学意义（P>0.05）。结论原有冻存方案及改良冻存方案均能有效恢复卵巢组织的内分泌功能。与原有冻存方案相比，NAC改良方案能够提高移植卵巢组织的抗氧化能力，并减少移植初期原始卵泡的激活，保存更多的原始卵泡数量。

关键词: N-乙酰半胱氨酸, 原始卵泡, 卵泡激活, 卵泡丢失, 卵巢储备

Abstract: Objective To investigate the protective effect of antioxidant N-Acetyl-L-Cysteine (NAC) on human ovarian tissue by human ovarian tissue xenotransplantation based on the current human ovarian tissue cryopreservation and thawing protocol in Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Methods The ovarian tissues from 4 patients were cryopreserved and thawed according to the currently-used protocol or the modified protocol (adding with 5 mm NAC). the fresh ovarian tissues underwent Calcein acetoxymethyl ester （Calcein-AM ）staining and hematoxylin-eosin (HE) staining to evaluate follicle activity and follicle counting. Thirty-six female nude mice who underwent ovariectomy were randomly divided into 4 groups. The thawing ovarian tissues were transplanted under the bilateral renal capsules of the nude mice. According to the different freezing-thawing protocol, the four group were divided as current freezing-thawing protocol group (control group), the modified protocol group (NAC group), ovariectomy group and normal group (no surgery). The nude mice were killed on day 3, day 7 and day 21 respectively, and serum and ovarian tissue grafts were taken. Serum samples were detected by enzyme-linked immunosorbent assay (ELISA) for estradiol (E2), follicle stimulating hormone (FSH) and anti-mullerian hormone (AMH). One ovarian graft was taken for HE staining to observe follicle development and one graft for total antioxidant capability (TAC). Results There was no significant difference in follicle development stages between the two transplantation groups at each time point after transplantation (P>0.05). The TAC level of NAC group was significantly higher than that of control group (P<0.05), but no difference was found between NAC group and fresh ovarian tissue (P>0.05). The level of serum E2 in control group was significantly higher than that in ovariectomy group on the 3rd day of transplantation (P<0.05), and there was no significant difference between the two transplantation groups and ovariectomy group at other time points (P>0.05). The level of FSH at day 3 and 21 after transplantation was lower than that in ovariectomy group (P<0.05), and had no significant difference from that in normal group (P>0.05). On the 3rd day after transplantation, AMH in the control group was significantly higher than that in the ovarian ovariectomy group (P<0.05), but no significant difference was found between the control group and the other groups (P>0.05). On 7th day after transplantation, AMH level in the two transplantation groups decreased, and was significantly lower than that in the normal group (P<0.05). On the 21st day after transplantation, AMH levels in both transplantation groups were significantly higher than those in ovariectomy group (P<0.05), but had no significant difference with normal group (P>0.05). Conclusions Both the currently-use protocol and the modified protocol can effectively restore the endocrine function of ovarian tissue. Compared with the currently-use cryopreservation regimen, the modified regimen can improve the antioxidant capacity of transplanted ovarian tissue, reduce the primordial follicle activation at the initial stage of transplantation, and preserve more primordial follicles.

Key words: N-Acetyl-L-Cysteine, primordial follicle, follicle activation, follicle loss, ovarian reserve

中图分类号:

R711

李扬璐, 阮祥燕, 李妍秋, 谷牧青, 杜娟, 王泽铖, 程姣姣, 金凤羽, 蒋玲玲, 杨瑜, Alfred O. Mueck. N-乙酰半胱氨酸对冻融人卵巢组织的保护作用[J]. 首都医科大学学报, 2024, 45(4): 573-582.

Li Yanglu, Ruan Xiangyan, Li Yanqiu, Gu Muqing, Du Juan, Wang Zecheng, Cheng Jiaojiao, Jin Fengyu, Jiang Lingling, Yang Yu, Alfred O. Mueck. Protective effect of N-Acetyl-L-Cysteine on cryopreserved-thawed human ovarian tissue[J]. Journal of Capital Medical University, 2024, 45(4): 573-582.

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N-乙酰半胱氨酸对冻融人卵巢组织的保护作用

Protective effect of N-Acetyl-L-Cysteine on cryopreserved-thawed human ovarian tissue

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