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中华妇幼临床医学杂志(电子版)

Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition) ›› 2023, Vol. 19 ›› Issue (05): 530 -539. doi: 10.3877/cma.j.issn.1673-5250.2023.05.006

Original Article

Differences in epithelial mesenchymal transition-related biological markers between in-situ and ectopic endometrial epithelial cells in endometriosis patients

Dan Luo, Weimin Kong†(), Shuning Chen, Xiaoling Zhao, Yunkai Xie   

  1. Department of Gynecology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital, Beijing 100006, China
  • Received:2023-02-17 Revised:2023-09-02 Published:2023-10-01
  • Corresponding author: Weimin Kong
  • Supported by:
    National Natural Science Foundation of China(81871142)
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Objective

To investigate the expression characteristics of epithelial mesenchymal transition (EMT)-related biological markers in in-situ and ectopic endometrium of endometriosis (EMS) patients.

Methods

The EMS-related dataset GSE7305 was downloaded from the Gene Expression Omnibus (GEO) database via the GEOquery package in R language. The dataset was derived from ectopic endometrial tissues of 10 EMS patients and in-situ endometrial tissues of 10 subjects with normal endometrial tissues. EMT-related differential gene expression in this dataset was analyzed by bioinformatics method. A total of 27 EMS patients who received total hysterectomy (6 cases) or laparoscopic oophorocystectomy of affected side and hysteroscopy (8 cases) or laparoscopic oophorocystectomy of affected side (13 cases) and 14 patients with cervical intraepithelial neoplasia (CIN)3 and cervical carcinoma stage ⅠA1 who underwent total hysterectomy in Beijing Obstetrics and Gynecology Hospital, Capital Medical University from October 1, 2020 to March 31, 2022 were selected as study subjects. The patients were included in EMS ectopic endometrium group (n=27, ectopic endometrium in all EMS patients), EMS in-situ endometrium group (n=14, in-situ endometrium in EMS patients who received total hysterectomy or laparoscopic oophorocystectomy of affected side and hysteroscopy), and control group (n=14, normal endometrium in patients with CIN3 and cervical carcinoma stage ⅠA1), according to the diagnostic results of postoperative biopsy histopathology. The patients in EMS ectopic endometrium group were further divided into EMS ectopic endometrium proliferative subgroup (n=14) and EMS ectopic endometrium secretory subgroup (n=13) according to the menstrual cycle of patients in EMS ectopic endometrium group during which the endometrium was obtained. Immunohistochemistry (IHC) was used to detect the expression of EMT-related markers, such as E-cadherin, N-cadherin, vimentin, snail family (snail, slug) and twist transcription factors in endometrial tissues of patients in EMS ectopic endometrium group, EMS in-situ endometrium group, and control group. Their positive expression rates and histochemistry score (H-score) of these biological markers were compared by chi-square test, Fisher′s exact probability test and Kruskal-Wallis H test. Further pairwise comparisons were conducted by Bonferroni method to adjust the test level. The procedures followed in this study were in accordance with the regulations of the Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University and were approved by this Ethics Committee (Approval No. 2019-KY-002-01). All patients signed an informed consent form for the clinical study. There was no statistically significant difference in general clinical data among EMS ectopic endometrium group, EMS in-situ endometrium group, and control group, such as age, body mass index (BMI), and menstrual cycle (P>0.05).

Results

①In GSE7305 dataset, compared with normal endometrial tissues, ectopic endometrial tissues of EMS patients showed up-regulated expression of SNAI2 gene and down-regulated expression of SNAI1, TWIST2, and CDH1 genes. ②The positive expression rates of E-cadherin in EMS ectopic endometrium group, EMS in-situ endometrium group, and control group were 0, 35.7% (5/14), 42.9% (6/14), respectively, and the H-score values of E-cadherin of these three groups were 0.9 points (0.3 points, 2.5 points), 5.7 points (1.3 points, 11.6 points), 7.2 points (1.0 points, 24.5 points), respectively, the positive expression rates of vimentin of these three groups were 70.4% (19/27), 14.3% (2/14), and 21.4% (3/14), respectively, and the H-score values of vimentin of these three groups were 107.9 points (86.7 points, 122.8 points), 42.8 points (29.4 points, 75.4 points), and 51.8 points (37.8 points, 100.2 points), respectively. The overall comparison of E-cadherin, vimentin positive expression rate and H-score values among three groups of patients showed statistical significance (E-cadherin: P<0.001, H=20.14, P<0.001; vimentin: χ2=15.56, P<0.001, H=17.31, P=0.004). Further pairwise comparisons showed that the E-cadherin positive expression rate and H-score value in EMS ectopic endometrium group were significantly lower than those in the EMS in-situ endometrium group and control group, whereas the vimentin positive expression rate and H-score value were significantly higher than those in EMS in-situ endometrium group and control group, and all the differences were statistically significant (P<0.05). There was no statistically significant difference in the positive expression rates and H-score values of E-cadherin and vimentin between EMS in-situ endometrium group and control group (P>0.05). There was no statistically significant difference in the positive expression rates and H-score values of N-cadherin, slug, snail, and twist among three groups (P>0.05). ③The H-score value of twist in ectopic endothelial tissues of EMS patients in EMS ectopic endometrium proliferative subgroup was significantly higher than that of ectopic endothelial tissues of EMS patients in EMS ectopic endometrium secretory subgroup, and the difference was statistically significant (H=5.42, P=0.020).

Conclusions

Decreased expression of E-cadherin in EMS patients may promote the occurrence and development of EMS. The roles of vimentin, N-cadherin, slug, snail and twist in the pathogenesis of EMS have not been fully clarified so far, and still need to be further studied.

Key words: Endometriosis, Epithelial-mesenchymal transition, Biomarker, Differential gene, In-situ and ectopic endometrium in endometriosis, Women
图1 EMS患者中EMT相关差异基因(SNAI1、SNAI2、VIM、CDH1、CDH2、TWIST2基因)表达(图1A：GSE7305数据集差异基因与genecard数据库中EMT相关基因取交集的韦恩图；图1B：GSE7305数据集中6个EMT生物标志物相关基因表达热图；图1C：GSE7305数据集中6个EMT生物标志物相关基因表达箱型图)注：EMS为子宫内膜异位症，EMT为上皮间充质转化
表1 EMS异位内膜组、EMS在位内膜组与对照组受试者一般临床资料比较(±s)
组别 例数 年龄(岁) BMI(kg/m2) 月经周期(d)
EMS异位内膜组 27 37.8±6.4 22.2±2.5 29.4±3.3
EMS在位内膜组 14 41.5±6.3 23.9±3.5 28.7±3.6
对照组 14 35.8±6.1 22.9±3.4 29.2±5.3
F值   3.00 1.47 0.15
P值   0.059 0.240 0.857
图2 EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织EMT生物标志物IHC染色结果(图2A～2C：分别为EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织E-cadherin表达情况；图2D～2F：分别为EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织vimentin表达情况；图2G～2I：分别为EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织N-cadherin表达情况；图2J～2L：分别为EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织twist表达情况；图2M～2O：分别为EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织slug表达情况；图2P～2R：分别为EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织snail表达情况) (IHC-SP法染色，高倍)注：EMS为子宫内膜异位症，EMT为上皮间充质转化，IHC为免疫组织化学，SP为链霉菌抗生物素蛋白-过氧化物酶连结
表2 EMS异位内膜组、EMS在位内膜组与对照组患者子宫内膜组织EMT生物标志物阳性表达率与H-score值比较
组别 例数 阳性表达率[例数(%)]
E-cadherin vimentin N-cadherin twist slug snail
EMS异位内膜组 27 0(0) 19(70.4) 4(14.8) 14(51.9) 6(22.2) 4(14.8)
EMS在位内膜组 14 5(35.7) 2(14.3) 4(28.6) 4(28.6) 5(35.7) 1(7.1)
对照组 14 6(42.9) 3(21.4) 3(21.4) 9(64.3) 7(50.0) 6(42.9)
统计量   — χ2＝15.56 — — — —
P值   <0.001 <0.001 0.562 0.183 0.180 0.062
组别 例数 H-score值[分，M(Q1，Q3)]
E-cadherin vimentin N-cadherin twist slug snail
EMS异位内膜组 27 0.9(0.3，2.5) 107.9(86.7，122.8) 1.3(0.3，3.8) 1.9(0.8，47.5) 0.8(0.2，5.4) 0.6(0.3，1.7)
EMS在位内膜组 14 5.7(1.3，11.6) 42.8(29.4，75.4) 0.3(0.1，6.7) 1.0(0.3，17.4) 0.5(0.2，26.4) 0.2(0.1，0.7)
对照组 14 7.2(1.0，24.5) 51.8(37.8，100.2) 1.2(0.3，4.4) 19.8(0.8，54.3) 6.4(0.7，47.0) 1.4(0.2，35.4)
统计量   H＝20.14 H＝17.31 H＝1.37 H＝3.28 H＝3.48 H＝4.81
P值   <0.001 0.004 0.505 0.194 0.176 0.090
表3 EMS异位内膜增生期亚组与EMS异位内膜分泌期亚组EMS患者正常在位内膜组织中EMT相关生物标志物H-score值比较[分，M(Q1，Q3)]
组别 例数 E-cadherin N-cadherin vimentin twist slug snail
EMS异位内膜增生期亚组 14 6.3(1.0，22.2) 1.4(0.3，4.4) 46.8(25.8，56.1) 29.0(7.2，54.3) 3.0(0.5，37.2) 0.5(0.2，13.5)
EMS异位内膜分泌期亚组 13 5.9(1.3，10.3) 0.2(0.1，6.7) 44.5(28.8，75.5) 22.8(6.6，56.1) 0.8(0.2，34.8) 0.2(0.1，3.2)
H值   0.04 2.43 0.02 0.11 0.87 0.70
P值   0.848 0.119 0.886 0.737 0.350 0.401
表4 EMS异位内膜增生期亚组与EMS异位内膜分泌期亚组EMS患者异位内膜组织中EMT相关生物标志物H-score值比较[分，M(Q1，Q3)]
组别 例数 E-cadherin N-cadherin vimentin twist slug snail
EMS异位内膜增生期亚组 14 1.0(0.3，3.3) 1.4(0.6，5.0) 73.6(43.2，111.1) 6.8(1.2，77.1) 1.3(0.3，28.7) 0.9(0.3，1.8)
EMS异位内膜分泌期亚组 13 0.8(0.3，2.3) 0.9(0.2，2.9) 49.7(33.2，77.5) 0.8(0.4，7.5) 0.5(0.2，2.3) 0.5(0.2，3.1)
H值   0.08 1.85 1.73 5.42 1.85 0.53
P值   0.771 0.174 0.133 0.020 0.174 0.467
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