[1]王婧,段世超.人绒毛膜促性腺激素对反复种植失败不孕患者子宫内膜容受性的影响[J].新乡医学院学报,2023,40(11):1024-1031.[doi:10.7683/xxyxyxb.2023.11.004]
　WANG Jing,DUAN Shichao.Effect of human chorionic gonadotropin on endometrial receptivity of infertile patients with repeated implantation failure[J].Journal of Xinxiang Medical University,2023,40(11):1024-1031.[doi:10.7683/xxyxyxb.2023.11.004]
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人绒毛膜促性腺激素对反复种植失败不孕患者子宫内膜容受性的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年11
页码:
1024-1031
栏目:
临床研究
出版日期:
2023-11-05

文章信息/Info

Title:
Effect of human chorionic gonadotropin on endometrial receptivity of infertile patients with repeated implantation failure
作者:
王婧1; 2; 段世超2
（1.河南省人民医院妇产科，河南　郑州　450003；2.河南省人民医院临床医学研究中心,河南　郑州　450003）
Author(s):
WANG Jing1; 2; DUAN Shichao2
（1.Department of Obstetrics and Gynecology，Henan Provincial People′s Hospital，Zhengzhou 450003,Henan Province,China;2.Center of Clinical Medical Research，Henan Provincial People′s Hospital，Zhengzhou 450003，Henan Province,China）
关键词:
人绒毛膜促性腺激素; 反复种植失败; 白血病抑制因子; 子宫内膜容受性
Keywords:
human chorionic gonadotropin; repeated implantation failure; leukemia inhibitory factor; endometrial receptivity
分类号:
R697.33
DOI:
10.7683/xxyxyxb.2023.11.004
文献标志码:
A
摘要:
目的　探讨人绒毛膜促性腺激素（HCG）对反复种植失败（RIF）不孕患者子宫内膜容受性的影响。
方法　收集2015年1月至2022年12月在河南省人民医院行辅助生殖助孕的20例RIF患者月经第14天的子宫内膜组织，分离培养获得40份子宫内膜细胞，同时复苏解冻40枚相同时间段行辅助生殖助孕并成功分娩、已无生育需求患者的实验室冻存废弃胚胎，构建40个子宫内膜和胚胎体外共培养模型，然后将体外共培养模型分为子宫内膜-胚胎-HCG共培养组（试验组）和子宫内膜-胚胎共培养组（对照组），置于培养箱中培养。培养第1天（D1），试验组加入1 mg·L-1的HCG 1 mL ，对照组加入1 mL含1 mg·L-1雌激素（E2） 和10 mg·L-1孕激素（P）的子宫内膜细胞培养液，连续培养7 d。收集培养D1、第3天（D3）、第5天（D5）、第7天（D7）试验组和对照组培养液各 1 mL，-20 ℃冻存。另于培养D7收集试验组培养液5 mL，分为阴性未干预组、1 mg·L-1 HCG组、2 mg·L-1 HCG组、4 mg·L-1 HCG组、5 mg·L-1 HCG组，分别加入0、1、2、4、5 mg·L-1 HCG各1 mL；同时，收集对照组培养液5 mL，分为阴性未干预组、1 mg·L-1 E2+10 mg·L-1 P组、2 mg·L-1 E2+20 mg·L-1 P组、4 mg·L-1 E2+40 mg·L-1 P组、5 mg·L-1 E2+50 mg·L-1 P组，加入分别含0、1、2、4、5 mg·L-1 E2和 0、10、20、40、50 mg·L-1 P的子宫内膜细胞培养液各1 mL；继续培养1 d后，收集试验组各组及对照组各组培养液各1 mL，-20 ℃冻存。取D1、D3、D5、D7的试验组和对照组冻存培养液，采用酶联免疫吸附试验(ELISA)法检测培养液中白血病抑制因子（LIF）水平，Western blot法检测培养液中LIF蛋白相对表达量。取不同浓度HCG或E2、P干预后试验组和对照组冻存培养液，采用ELISA法检测培养液中LIF水平，Western blot法检测培养液中LIF蛋白相对表达量。
结果　D1、D3、D5、D7时，试验组培养液中LIF水平均高于对照组（P<0.05）。试验组和对照组D3、D5、D7时培养液中LIF水平均高于D1时（P<0.05），D5、D7时培养液中LIF水平均高于D3时（P<0.05），D7时培养液中LIF水平均高于D5时（P<0.05）。D1、D3、D5、D7时，试验组培养液中LIF蛋白相对表达量均高于对照组（P<0.05）。试验组和对照组D3、D5、D7时培养液中LIF蛋白相对表达量均高于D1时（P<0.05），D5、D7时培养液中LIF蛋白相对表达量均高于D3时（P<0.05），D7时培养液中LIF蛋白相对表达量均高于D5时（P<0.05）。对照组阴性未干预组、1 mg·L-1 E2+10 mg·L-1 P组、2 mg·L-1 E2+20 mg·L-1 P组、4 mg·L-1 E2+40 mg·L-1 P组、5 mg·L-1 E2+50 mg·L-1 P组各组之间培养液中LIF水平比较差异无统计学意义（P>0.05）。试验组1 mg·L-1 HCG组、2 mg·L-1 HCG组、4 mg·L-1 HCG组、5 mg·L-1 HCG组培养液中LIF水平高于阴性未干预组（P<0.05），2 mg·L-1 HCG组、4 mg·L-1 HCG组、5 mg·L-1 HCG组培养液中LIF水平高于1 mg·L-1 HCG组（P<0.05），4 mg·L-1 HCG组、5 mg·L-1 HCG组培养液中LIF水平高于2 mg·L-1 HCG组（P<0.05），5 mg·L-1 HCG组培养液中LIF水平高于4 mg·L-1 HCG组（P<0.05）。试验组阴性未干预组培养液中LIF水平高于对照组阴性未干预组（P<0.05），1 mg·L-1 HCG组培养液中LIF水平高于1 mg·L-1 E2+10 mg·L-1 P组（P<0.05），2 mg·L-1 HCG组培养液中LIF水平高于2 mg·L-1 E2+20 mg·L-1 P组（P<0.05）， 4 mg·L-1 HCG组培养液中LIF水平高于4 mg·L-1 E2+40 mg·L-1 P组（P<0.05），5 mg·L-1 HCG组培养液中LIF水平高于5 mg·L-1 E2+50 mg·L-1 P组（P<0.05）。 对照组阴性未干预组、1 mg·L-1 E2+10 mg·L-1 P组、2 mg·L-1 E2+20 mg·L-1 P组、4 mg·L-1 E2+40 mg·L-1 P组、5 mg·L-1 E2+50 mg·L-1 P组各组之间培养液中LIF蛋白相对表达量比较差异均无统计学意义（P>0.05）。试验组1 mg·L-1 HCG组、2 mg·L-1 HCG组、4 mg·L-1 HCG组、5 mg·L-1 HCG组培养液中LIF蛋白相对表达量高于阴性未干预组（P<0.05），2 mg·L-1 HCG组、4 mg·L-1 HCG组、5 mg·L-1 HCG组培养液中LIF蛋白相对表达量高于1 mg·L-1 HCG组（P<0.05），4 mg·L-1 HCG组、5 mg·L-1 HCG组培养液中LIF蛋白相对表达量高于2 mg·L-1 HCG组（P<0.05），5 mg·L-1 HCG组培养液中LIF蛋白相对表达量高于4 mg·L-1 HCG组（P<0.05）。试验组阴性未干预组培养液中LIF蛋白相对表达量高于对照组阴性未干预组（P<0.05），1 mg·L-1 HCG组培养液中LIF蛋白相对表达量高于1 mg·L-1 E2+10 mg·L-1 P组（P<0.05），2 mg·L-1 HCG组培养液中LIF蛋白相对表达量高于2 mg·L-1 E2+20 mg·L-1 P组（P<0.05），4 mg·L-1 HCG组培养液中LIF蛋白相对表达量高于4 mg·L-1 E2+40 mg·L-1 P组（P<0.05）， 5 mg·L-1 HCG组培养液中LIF蛋白相对表达量显著高于5 mg·L-1 E2+50 mg·L-1 P组（P<0.05）。
结论　在体外共培养条件下，HCG能够促进RIF患者子宫内膜中LIF的分泌和表达，提高RIF患者的子宫内膜容受性。
Abstract:
Objective　To investigate the effect of human chorionic gonadotropin (HCG) on endometrial receptivity of infertile patients with repeated implantation failure (RIF).
Methods　Endometrial tissues of 20 patients with RIF on the 14th day of menstruation in Henan Provincial People′s Hospital from January 2015 to December 2022 were collected and 40 endometrial cells were isolated and cultured,and 40 laboratory frozen discarded embryos of patients who underwent assisted reproductive assistance during the same time period and successfully delivered with no fertility need were resuscitated.Forty endometrial and embryo co-culture models were constructed and divided into endometrium-embryo-HCG co-culture group(experimental group) and endometrium-embryo co-culture group(control group),and they were cultured in incubator.A total of 1 mL of 1 mg·L-1 HCG was added into the experimental group and 1 mL endometrial cell culture medium which containing 1 mg·L-1 estrogen（E2）and 10 mg·L-1 progesterone（P）was added into the control group on the first day (D1) and cultured for 7 days.A total of 1 mL culture medium of the experimental group and the control group on the D1,third day (D3),fifth day (D5) and seventh day (D7) of cultivation were collected and frozen storage at -20 ℃.On D7,5 mL culture medium of the experimental group were collected and divided into negative untreated group,1 mg·L-1 HCG group,2 mg·L-1 HCG group,4 mg·L-1 HCG group,5 mg·L-1 HCG group,and 1 mL of 0,1,2,4,5 mg·L-1 HCG were added,respectively;at the same time,5 mL culture medium of the control group were collected and divided into negative untreated group,1 mg·L-1 E2+10 mg·L-1 P group,2 mg·L-1 E2+20 mg·L-1 P group,4 mg·L-1 E2+40 mg·L-1 P group,5 mg·L-1 E2+50 mg·L-1 P group,and 1 mL endometrial cell culture medium which containing 0,1,2,4,5 mg·L-1 E2 and 0,10,20,40,50 mg·L-1 P were added,respectively;after continuous culture for 1 day,1 mL culture medium of the experimental group and the control group were collected and frozen storage at -20 ℃.On the D1,D3,D5,D7,the frozen culture medium in the experimental group and the control group were collected,and the level of leukemia inhibitory factor（LIF）was detected by enzyme linked immunosorbent assay(ELISA) and the relative expression of LIF protein was detected by Western blot.The frozen culture medium of the experimental group and the control group intervened with different concentration of HCG or E2 and P were collected,and the level of LIF in the culture medium was detected by ELISA,and the relative expression of LIF protein was detected by Western blot.
Results　On the D1,D3,D5,D7,the level of LIF in culture medium in the experimental group was significantly higher than that in the control group (P<0.05);the levels of LIF in culture medium in the experimental group and the control group on the D3,D5,D7 were significantly higher than those on the D1(P<0.05);the levels of LIF in culture medium in the experimental group and the control group on the D7 were significantly higher than those on the D5(P<0.05).On the D1,D3,D5,D7,the relative expression of LIF in culture medium in the experimental group was significantly higher than that in the control group (P<0.05）;the relative expression of LIF in culture medium in the experimental group and the control group on the D3,D5,D7 was significantly higher than that on the D1(P<0.05);the relative expression of LIF in culture medium in the experimental group and the control group on the D5,D7 was significantly higher than that on the D3(P<0.05);the relative expression of LIF in culture medium in the experimental group and the control group on the D7 was significantly higher than that on the D5 (P<0.05).The level of LIF in culture medium in 1 mg·L-1 HCG group,2 mg·L-1 HCG group,4 mg·L-1 HCG group,5 mg·L-1 HCG group was significantly higher than that in the negative untreated group in the experimental group (P<0.05);the level of LIF in culture medium in 2 mg·L-1 HCG group,4 mg·L-1 HCG group,5 mg·L-1 HCG group was significantly higher than that in 1 mg·L-1 HCG group(P<0.05);the level of LIF in culture medium in 4 mg·L-1 HCG group,5 mg·L-1 HCG group was significantly higher than that in 2 mg·L-1 HCG group(P<0.05);the level of LIF in culture medium in 5 mg·L-1 HCG group was significantly higher than that in 4 mg·L-1 HCG group(P<0.05).The level of LIF in culture medium in negative untreated group in the experimental group was significantly higher than that in negative untreated group in the control group (P<0.05);the level of LIF in culture medium in 1 mg·L-1 HCG group was significantly higher than that in 1 mg·L-1 E2+10 mg·L-1 P group(P<0.05).The level of LIF in culture medium in 2 mg·L-1 HCG group was significantly higher than that in 2 mg·L-1 E2+20 mg·L-1 P group (P<0.05);the level of LIF in culture medium in 4 mg·L-1 HCG group was significantly higher than that in 4 mg·L-1 E2+40 mg·L-1 P group (P<0.05);the level of LIF in culture medium in 5 mg·L-1 HCG group was significantly higher than that in 5 mg·L-1 E2+50 mg·L-1 P group (P<0.05).The relative expression of LIF protein in culture medium in 1 mg·L-1 HCG group,2 mg·L-1 HCG group,4 mg·L-1 HCG group,5 mg·L-1 HCG group was significantly higher than that in the the negative untreated group in experimental group (P<0.05);the relative expression of LIF protein in culture medium in 2 mg·L-1 HCG group,4 mg·L-1 HCG group,5 mg·L-1 HCG group was significantly higher than that in the 1 mg·L-1 HCG group(P<0.05);the relative expression of LIF protein in culture medium in 4 mg·L-1 HCG group,5 mg·L-1 HCG group was significantly higher than that in 2 mg·L-1 HCG group(P<0.05);the relative expression of LIF protein in culture medium in 5 mg·L-1 HCG group was significantly higher than that in 4 mg·L-1 HCG group(P<0.05).The relative expression of LIF protein in culture medium in negative untreated group in the experimental group was significantly higher than that in negative untreated group in the control group (P<0.05);the relative expression of LIF protein in culture medium in 1 mg·L-1 HCG group was significantly higher than that in 1 mg·L-1 E2+10 mg·L-1 P group (P<0.05);the relative expression of LIF protein in culture medium in 2 mg·L-1 HCG group was significantly higher than that in 2 mg·L-1 E2+20 mg·L-1 P group (P<0.05)；the relative expression of LIF protein in culture medium in 4 mg·L-1 HCG group was significantly higher than that in 4 mg·L-1 E2+40 mg·L-1 P group (P<0.05);the relative expression of LIF protein in culture medium in 5 mg·L-1 HCG group was significantly higher than that in 5 mg·L-1 E2+50 mg·L-1 P group (P<0.05).
Conclusion　HCG can promote the secretion and expression of LIF in the endometrium and improve the endometrial receptivity of RIF patients under the in vitro co-culture condition.

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更新日期/Last Update: 2023-11-05