The influence of menstrual cycle and endometriosis on endometrial expression of epithelial-to-mesenchymal transition (EMT)-related genes

The influence of menstrual cycle and endometriosis on endometrial expression of epithelial-to-mesenchymal transition (EMT)-related genes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The influence of menstrual cycle and endometriosis on endometrial expression of epithelial-to-mesenchymal transition (EMT)-related genes Agata Góźdź, Marta Żeberkiewicz, Izabela Janiuk, Anna Hyc, Anna Iwan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7227864/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Endometriosis is a common chronic gynaecological disorder related to the presence of ectopic foci of endometrial-like tissue mostly in the pelvic cavity. Pathogenesis of this disease may be associated with epithelial-to-mesenchymal transition (EMT), a phenomenon defined by morphological and functional changes from epithelial to mesenchymal cell phenotype. The role of EMT in development of endometriotic lesions remains poorly understood. There is also little known about a role of EMT in eutopic endometrium in course of the menstrual cycle. Therefore, the present study was aimed at investigating expression of major EMT-related genes of TGF-b, ZEB, SNAIL, CDH and miR200 families in eutopic endometrium of women with and without endometriosis in proliferative and secretory phase of the menstrual cycle. Methods: The study included 46 women with endometriosis and 30 control women without symptoms of the disease. Eutopic endometrial tissue samples were collected during mid-proliferative and mid-secretory phase. Tissue localization of the tested factors was detected by immunohistochemical staining. Expression of specific RNAs was evaluated by quantitative RT-PCR. Differences between groups were determined using the Student’s t -test, Wilcoxon matched-pairs signed rank test or Mann–Whitney U -test. Results: All investigated factors were expressed in eutopic endometrium both at the protein and mRNA level. Comparison of mRNA expression during different cycle phases has revealed a significant upregulation of SNAI2 mRNA in the secretory phase in both endometriosis and control group. Secretory phase was also associated with a decreased expression of CDH2 mRNA in the control group. However, similar difference was not revealed in the endometriosis patients. There were no differences in mRNA levels of the other tested EMT-related factors between endometriosis and control group regardless the cycle phase. Conclusions: The present study shows that SNAI2 expression is upregulated during secretory phase of the menstrual cycle thus suggesting its role in physiology of the cyclic endometrial changes. However, our data argue for a limited role of EMT in eutopic endometrium in the pathogenesis of endometriosis. These findings put a new light on the physiology of endometrium and the role of EMT in the pathogenesis of endometriosis. Endometrium Endometriosis Menstrual cycle Epithelial-to-mesenchymal transition (EMT) TGF-β ZEB SNAIL E-cadherin N-cadherin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Endometriosis is a common chronic gynaecological disorder related to the presence of ectopic foci of endometrial-like tissue (glandular and stromal cells) mostly in the pelvic cavity. The disease is associated with chronic pelvic inflammatory reactions and may also display some features of autoimmune disorder [1–4]. It may manifest by dysmenorrhea and pelvic pain and is considered as one of the major causes of the female infertility [5,6]. Endometriosis affects ca. 10% of women in reproductive age and has a significant impact on patients’ life quality and constitutes an important clinical and social problem. Etiopathology of endometriosis appears to be very complex and remains obscure [7,8]. According to the most accepted Sampson’s theory, endometriosis develops because of retrograde flow of endometrial cells shed in course of menstruation [9] . Appearance of distant lesions such as in lung, heart, liver or brain may be due to dissemination by lymphatic or blood system [10,11]. Ectopic endometriotic-like tissue may also develop because of coelomic metaplasia of Müllerian system remnants [12]. The evidence accumulates that endometriosis may originate in part from disseminating stem-like epithelial precursor cells [13,14] . However, the mechanisms responsible for survival, implantation and progression of endometriotic cells in the peritoneal cavity remain poorly recognized. It may be partially due to their decreased susceptibility to apoptotic cell death [15–17] , an increased adhesiveness and invasiveness [15,18,19] , as well as their abrogated elimination by the cells of the local immune surveillance system [20]. Pathogenesis of endometriosis may also involve a phenomenon of epithelial-to-mesenchymal transition (EMT) [21–23]. EMT is defined as a process characterized by morphological and functional changes from epithelial to mesenchymal cell phenotype [24,25]. Typically, epithelial cells lose their polarized phenotype and intercellular connections and acquire fibroblastic shape with enhanced migratory and invasive potential. These changes are related to downregulation of E-cadherin and an increased N-cadherin expression [24,25]. EMT appears to be one of the fundamental mechanisms involved in development of multicellular organisms playing a role in embryo- and morphogenesis [24,25]. On the other hand, EMT appears to be crucial for a variety of pathological phenomena including fibrosis and tumour invasion and metastasis [26–28]. It has been reported that endometriotic epithelial cells display a decreased expression of E-cadherin and endometriosis is associated with an increased expression of EMT inducing factors such as members of ZEB and SNAIL families of transcription factors [21,29–32]. It has also been suggested that EMT in endometriotic cells may also depend on abrogated expression of miRNAs of miR200 family, that are major regulators of ZEB expression [33–36]. However, the exact role of EMT in development of endometriotic lesions remains poorly understood. There is also little known about expression and a putative role of EMT-related genes in eutopic endometrium in course of the menstrual cycle. Therefore, the present study was aimed at investigating expression of major EMT-related genes coding for the members of TGF-b, ZEB, SNAIL, CDH and miR200 family in eutopic endometrium of women with and without endometriosis in proliferative and secretory phase of the menstrual cycle. Material and methods Patients and controls All patients enrolled in the present study were diagnosed at the Departments of Obstetrics and Gynaecology, Medical University of Warsaw and the Department of Gynaecology, Military Institute of Medicine, Warsaw, Poland between January 2010 and December 2015. All participants gave an informed consent to the study and the investigations were approved by the Institutional Bioethical Review Board of the Medical University of Warsaw and Military Institute of Medicine, Poland (permissions no. WUM/KB/223/2009, 49/WIM/2011, 37/WIM/2013) and conducted according to the Helsinki Declaration guidelines. The study included 46 women with laparoscopically and histologically confirmed endometriosis. The severity of the disease has been classified according to the revised American Society of Reproductive Medicine (rASRM) criteria [37]. The control group consisted of 30 women without any clinical symptoms of endometriosis who underwent removal of a cervical polyp or were subjected to a cervical biopsy due to a positive result of the Pap smear. Women with histopathologically confirmed cervical dysplasia were not included. None of the patients with endometriosis or the control subjects had any other chronic diseases, and none had received hormonal treatment for at least three months prior to the study. Detailed demographic and clinical characteristics of the patients with endometriosis and the control subjects are presented in Table 1. Table 1. Demographic and clinical characteristics of endometriosis patients and healthy control group. Characteristics Control Endometriosis Total Proliferative phase Secretory phase Total Proliferative phase Secretory phase Number of cases (N) 30 14 (46.7%) 16 (53.3%) 46 16 (34.8%) 30 (65.2%) Age, years (mean ± SD) 34.7 ± 6.6 34.8 ± 6.2 34.6 ± 7.0 32.3 ± 5.6 29.9 ± 3.7* 33.5 ± 6.1 BMI, kg/m 2 (mean ± SD) 22.4 ± 4.4 22.5 ± 5.0 22.3 ± 3.7 21.6 ± 3.2 21.4 ± 2.6 21.7 ± 3.5 rASRM I (minimal) na na na 7 (15.2%) 2 (12.5%) 5 (16.7%) II (mild) na na na 5 (10.9%) 0 5 (16.7%) III (moderate) na na na 22 (47.8%) 9 (56.3%) 13 (43.3%) IV (severe) na na na 12 (26.1%) 5 (31.2%) 7 (23.3%) Lesion localization Ovarian na na na 37 (80.4%) 14 (85.5%) 22 (73.3%) Peritoneal na na na 35 (76.1%) 13 (81.3%) 22 (73.3%) Both na na na 25 (54.3%) 11 (68.8%) 14 (46.7%) na, not applicable; *Different from Endometriosis secretory phase group at P =0.0176 and from Control proliferative phase group at P =0.0133 as judged by Student- t test. Tissue samples were collected from the participants during mid-proliferative and mid-secretory phase of the menstrual cycle. A phase of the menstrual cycle was established basing on the date of the last menstrual bleeding and was confirmed by pelvic ultrasound examination. Endometrial tissue samples were obtained by an aspiration biopsy with a PipelleÒ catheter (Pipelle de Cornier, Laboratoire C.C.D., Paris, France). For immunohistochemical examinations the samples were routinely fixed in 10% buffered formalin whereas for evaluation of mRNA expression, the specimens were immediately placed in 5 volumes of RNAlaterÔ solution (TermoFisher Scientific, Waltham, Massachusetts, USA), kept at 4°C for one day and then stored frozen at -70°C until RNA isolation was performed. Immunohistochemical staining Expression of ZEB1, ZEB2, SNAI1, SNAI2, E-cadherin (CDH1) and N-cadherin (CDH2) was detected by routine immunohistochemical staining. In brief, formalin-fixed paraffin-embedded tissue samples of eutopic endometrium and endometriotic cysts were cut into 5 mm sections, deparaffinized, rehydrated in graded alcohol series and permeabilized in 10 mM citrate buffer (pH 6.0) at 95°C. Endogenous peroxidase was blocked by Novolink Peroxide Block (Leica Biosystems, RE7140-CE). Then the sections were incubated with respective primary antibody for 60 minutes at room temperature. All primary antibodies, their specification, origin and working dilution are listed in Table 2. Table 2. A list of primary antibodies used for immunohistochemical staining of endometrial samples. Antibody Origin/clone Working dilution Source Anti-ZEB1 Mouse monoclonal IgG2a/clone OTI3G6 1:150 Abcam, Cambridge, UK Anti-ZEB2 Mouse monoclonal IgG2a/ clone 6E5 1:250 Sigma-Aldrich, USA Anti-SNAI1 Rabbit polyclonal 1:180 Affinity Biosciences, USA Anti-SNAI2 Rabbit polyclonal 1:200 Affinity Biosciences, USA Anti-CDH1 Mouse monoclonal IgG1/clone 4A2 1:150 Abcam, Cambridge, UK Anti-CDH2 Mouse monoclonal IgG1/clone 8C11 1:250 Abcam, Cambridge, UK Primary antibodies were detected by incubation with Novolink™ Polymer Detection Reagent (Leica Biosystems, RE7140-CE) for 30 minutes at room temperature according to the manufacturer’s instructions. The peroxidase reaction was visualized using the DAB chromogen provided in the kit. RNA isolation and quantitative reversed transcription PCR (qRT-PCR) Large and small RNAs from tissue samples (5 mm 3 ) were isolated with the NucleoSpin® miRNA Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. The quantity and quality of the isolated RNA was evaluated spectrophotometrically using NanoDrop2000 spectrophotometer with software for analysis of nucleic acids (ThermoFisher Scientific). Reverse transcription of 2 μg of large RNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cheshire, UK), according to the manufacturer’s protocol in an Eppendorf Mastercycler gradient at 25°C for 10 min, 37°C for 120 min and 85°C for 5 sec. Reverse transcription of 10 ng of small RNA was performed using the TaqMan MicroRNA Transcription Kit and specific hsa-miR-200b-3p (Assay ID 00225), hsa-miR-200c-3p (Assay ID 002300) and RNU43 (Assay ID 001095) TaqMan MicroRNA assays (Applied Biosystems), respectively for miRNA200b, miRNA200c and RNU43. Reaction was performed according to the manufacturer’s protocol in an Eppendorf Mastercycler gradient at 16ºC for 30 min, 42ºC for 30 min and 85ºC for 5 min. cDNA samples were stored at -20°C until qRT-PCR assays were performed. Table 3. A list of TaqMan primers for used for qRT-PCR. Factor Gene TaqMan probe TGF-b1 TGFB1 Hs00998133_m1 TGF-b2 TGFB2 Hs 00234244_m1 ZEB-1 ZEB1 Hs00232783_m1 ZEB-2 ZEB2 Hs00207691_m1 SNAIL SNAI1 Hs00195591_m1 SLUG SNAI2 Hs00161904_m1 E-cadherin CDH1 Hs01023894_m1 N-cadherin CDH2 Hs00983056_m1 GAPDH GAPDH Hs99999905_m1 Hsa-miR200b-3p MIR200B Hs04231483_s1 Hsa-miR200c-3p MIR200C Hs04231534_s1 RNU43 RNU43 Hs00504439_CE Real time qRT-PCR was performed in the ABI PRISM 7500 thermocycler (Applied Biosystems) using specific TaqMan expression assays (Applied Biosystems) with FAM-labelled probes and 96-well optical plates. A list of specific mRNAs probes used in this study is shown in Table 3. The reactions were run in a total volume of 20 μL including TaqMan Universal Master Mix, the appropriate primer set, MGB probe and 50 ng of cDNA template with an initial denaturation at 95°C for 10 min, amplification for 50 cycles at 95°C for 15 sec and 60°C for 1 min. Each sample was run in triplicate. GAPDH and RNU43 served as endogenous control for large and small RNAs, respectively. Relative RNA expression was computed by the Sequence Detection System (SDS) v 1.2 software (Applied Biosystems) using the DCt method and the results are presented as relative units calculated from the 2 - D Ct formula. Statistical evaluation All statistical analyses and graphical presentations were generated using GraphPad Prism 8.2.0 (GraphPad Software, San Diego, CA, USA). The study groups were characterized using descriptive statistics consistent with the distribution of variables. Differences between groups were determined using the Student’s t -test, Wilcoxon matched-pairs signed rank test or Mann–Whitney U -test when applicable. Probability ( P ) values of < 0.05 were considered statistically significant. The results are presented as mean ± SD or medians with range or interquartile range. Results Endometrial immunolocalization of ZEB1, ZEB2, SNAI1, SNAI2, CDH1 and CDH2 The results of immunolocalization of investigated proteins are shown on Fig. 1. As seen, expression of ZEB1 and ZEB2 was primarily localized in the nuclei of endometrial stromal cells. No significant immunoreactivity was seen in the epithelium and glands. On the contrary, a relatively strong reaction was found in the nuclei of epithelial and glandular cells in case of SNAI1 and SNAI2. SNAI1 and SNAI2 immunoreactivity was also noted in nuclei of endometrial stromal cells. Expression of CDH1 was seen only in epithelial cells. It was localized mostly in cell membranes and only mild diffuse staining was seen in the cytoplasm. N-cadherin was found mostly in epithelial cells, but sparse positive reaction was also seen in some endometrial stromal cells. N-cadherin reactivity pattern in epithelial cells was like that of E-cadherin. Evaluation of endometrial mRNA and miRNA expression of investigated EMT-related genes Expression of mRNA and miRNA specific for all studied EMT-related genes was detected in all endometrial tissue samples from both control healthy women and women with endometriosis. The results of semiquantitative mRNA and miRNA evaluations showed that, irrespectively of the phase of the menstrual cycle, the endometrium from control healthy women expressed significantly much more TGFB1 and SNAI2 mRNA as compared to their related TGFB2 and SNAI1 genes , respectively (Table 4). Accordingly, expression of TGFB1 was ca. 6-10 times higher than expression of TGFB2 , and expression of SNAI2 was ca. 10-20 times higher than expression of SNAI1 . Expression of CDH1 and MIR200C was slightly higher (about 3 times) than CDH2 and MIR200B , respectively, whereas there were no significant differences in expression between ZEB1 and ZEB2 genes. Exactly similar results were seen in case of endometrium samples from women with endometriosis (Table 5). Table 4. Comparisons of expression levels of related EMT-associated gene mRNAs and miRNAs in endometrium of healthy women. Gene Proliferatory phase P Secretory phase P TGFB1 0.0474 (0.029-0.1768) 0.0005 0.0359 (0.0096-0.0718) 0.0507 TGFB2 0.0042 (0.0003-0.0544) 0.0053 (0.0002-0.1340) ZEB1 0.0292 (0.0156-0.1250) ns 0.0372 (0.0168-0.0825) ns ZEB2 0.0388 (0.0127-0.1340) 0.0222 (0.0118-0.0884) SNAI1 0.0025 (0.0015-0.0136) 0.0010 0.0024 (0.0002-0.0118) <0.0001 SNAI2 0.0254 (0.0111-0.0670) 0.0583 (0.0180-0.4061) CDH1 0.1806 (0.0625-1.2510) 0.0049 0.0947 (0.0385-0.9659) 0.0002 CDH2 0.0583 (0.0059-0.2679) 0.0151 (0.0026-0.0625) MIR200B 19.31 (0.06-2048.00) ns 24.25 (1.41-724.10) 0.0020 MIR200C 58.55 (0.57-9410.00) 34.30 (3.03-5943.00) All results are shown as medians (range) of relative mRNA expression (2 - D Ct ). P -values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant. Table 5. Comparisons of expression levels of related EMT-associated gene mRNAs and miRNAs in endometrium of women with endometriosis. Gene Proliferatory phase P Secretory phase P TGFB1 0.0372 (0.0206-0.0625) <0.0001 0.0412 (0.0156-0.1649) 0.0032 TGFB2 0.0040 (0.0002-0.0146) 0.0063 (0.0007-0.1649) ZEB1 0.0263 (0.0059-0.0412) 0.0136 0.0323 (0.0073-0.1539) ns ZEB2 0.0282 (0.0136-0.0583) 0.0313 (0.0090-0.1340) SNAI1 0.0024 (0.0011-0.0136) 0.0001 0.0046 (0.0005-0.0313) <0.0001 SNAI2 0.0313 (0.0032-0.1166) 0.0544 (0.0180-0.1649) CDH1 0.1091 (0.0313-0.4665) 0.0580 0.1253 (0.0335-0.8123) 0.0006 CDH2 0.0545 (0.0048-0.1895) 0.0474 (0.0042-0.3078) MIR200B 17.19 (0.08-1552) ns 1.71 (0.12-1261) 0.0163 MIR200C 27.57 (0.44-8192.00) 8.29 (0.00-9410) All results are shown as medians (range) of relative mRNA expression (2 - D Ct ). P -values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant. Menstrual cycle-dependent changes in expression of endometrial mRNA and miRNA for EMT-related genes Changes in specific expression of endometrial mRNA and miRNA for the investigated EMT-related genes between proliferative and secretory phase of the menstrual cycle in control women are shown in Fig. 2. As seen, secretory phase was associated with a significant 2-fold upregulation in SNAI2 mRNA expression and a significant 5-fold decrease of CDH2 mRNA level. No differences were observed in mRNA or miRNA expression of other EMT-related genes. A significant 2-fold upregulation of SNAI2 mRNA expression in the secretory phase was also observed in the endometriosis group (Fig. 3). Furthermore, a decreased expression was also noted in case of both MIR200B and MIR200C genes. Expression of MIR200B gene was reduced ten-fold, and expression MIR200C was over 3 times lower compared to proliferatory phase; however, due to a great variability of the results this difference did not reach a statistical significance. There were no differences in expression of other studied genes. Differences in endometrial mRNA expression of EMT-related genes between healthy control women and women with endometriosis As Fig. 4 shows, comparison of endometrial mRNA and miRNA expression of EMT-related genes during proliferatory phase of the menstrual cycle between healthy women and endometriosis patients did not reveal any important differences except a slight but significantly lower level of TGFB1 gene expression in the endometriosis group. In the secretory phase (Fig. 5) the level of CDH2 mRNA was 3-fold higher in the endometrium of women with endometriosis as compared to control group and this difference was statistically significant. A statistically significantly higher expression of SNAI1 gene was also observed in the endometriosis group; however, this difference was at the significance borderline. There were no differences in the level of expression of all other investigated EMT-related genes. Discussion To our best knowledge, this is the first study aimed at evaluation of EMT-related gene expression in endometrium from healthy women and women with endometriosis in relation to the phase of the menstrual cycle. Our present results show that normal endometrium as well as endometrium from women with endometriosis constitutively express mRNA of all investigated EMT-related genes and this is consistent with previously published observations [21,29,38]. Interestingly, we report for the first time that expression of TGFB1 and SNAI2 mRNA was many times higher than expression of their respective family members, TGFB2 and SNAI1 . On the contrary, the differences in expression of CDH1 and CDH2, as well as MIR200C and MIR200B were not so much pronounced. Expression of the investigated EMT-related factors was confirmed by immunohistochemical staining of endometrial tissue samples. Both ZEB1and ZEB2 proteins were localized in the nuclei, and their expression was limited to stromal cells. This localization of ZEBs in eutopic endometrium is consistent with previous observations [32,39]. Expression of SNAI1 and SNAI2 was also localized in the nuclei; however, unlike ZEBs, it was present in epithelial and glandular cells as well as in some stromal cells. Presence of SNAIs both in epithelial and stromal cells has been reported previously [21,38] . Expression of CDH1 and CDH2 in epithelial and glandular cells was found in the intercellular junctions. Some CDH2 expression was also seen in the stromal cells. EMT as well as a reverse process, mesenchymal to epithelial transition (MET) are believed to play an important role in endometrium regeneration, receptivity and in vitro decidualization [40–42]. However, putative factors that may be responsible for regulation of EMT/MET endometrial balance remain obscure. To address this point, we evaluated changes of mRNA levels of EMT-related genes in endometrium during proliferatory and secretory phase of the menstrual cycle. We found for the first time that in healthy control endometrium the secretory phase was related to a significant several-fold upregulation of SNAI2 gene. Similar upregulation of SNAI2 mRNA was also found in eutopic endometrium from women with endometriosis that strongly implies that SNAI2 may indeed play some role in physiological endometrial changes in course of the menstrual cycle. This assumption may be additionally supported by observation that SNAI1 and SNAI2 expression localizes in the nuclei thus suggesting their transcriptional activity. SNAI2, a member of the SNAIL family of transcriptional regulators is C2H2-type zinc finger transcription factor [43–46]. Both, SNAI1 and SNAI2 can stimulate EMT by repression of E-cadherin transcription in epithelial cells [45,47,46]; however, unlike the members of ZEB family they are not considered as up-regulators of mesenchymal markers. Nevertheless, they may increase matrix metalloproteinase expression and activity and mediate cell motility and invasion, proliferation as well as cellular senescence and apoptosis [45,48]. Accordingly, it is tempting to speculate that upregulated SNAI2 expression may contribute to extensive growth and maturation of endometrial glands and stroma during secretory phase. This, however, needs further elucidation. It is also not clear what is the mechanism of increased expression of SNAI2 expression during secretory phase. Upregulation of SNAIs is mediated by TWIST, the basic helix-loop-helix (bHLH) transcription factor that is considered as an indirect EMT inducer [47]. Accordingly, expression of TWIST has also been reported in normal endometrium and endometrium from women with endometriosis [29,49] thus it is plausible that it may play a part in regulation of endometrial SNAIs expression. Changes in SNAI2 expression and EMT/MET might be also dependent on menstrual cycle-associated hormonal changes. There are many reports indicating a role of 17b-oestradiol (E2) in induction of EMT and EMT-related factors, especially in hormone-dependent tumours [50–52]. It has also been claimed that E2 may play a part in induction of EMT in course of endometriosis via upregulation of β‐catenin/SNAIL pathway [38]. Therefore, some role of oestrogens in regulation of endometrial EMT/MET cannot be excluded. Interestingly, it has been demonstrated that decidualization of human endometrial stromal cells was associated with the WNT/β‐catenin pathway [53]. WNT/β‐catenin signalling is known to downregulate E-cadherin expression and stimulate cell migration via activation of SNAIs [47] that may suggest a role of SNAIs and EMT in decidualization. On the other hand, however, overexpression of WNT promoted MET in human endometrial stromal cells [54], and similar phenomenon was also observed in in vitro model of decidualization following stromal cell treatment with progesterone and cAMP [55] . Thus, the hypothetical role of EMT/MET and SNAI2 in decidualization awaits further elucidation. Interestingly, we also found a significant downregulation of CDH2 gene expression in the secretory phase of the menstrual cycle in healthy control endometrium. Similar change was not seen in endometriosis patients. CDH2 expression is localized mainly in epithelial cells in basalis endometrial layer and is considered as a marker of endometrial progenitor/stem cells participating in tissue regeneration following menstruation [14,56]. Differentiating glandular cells lose their progenitor phenotype, it is therefore possible that a decreased level of CDH2 expression reflects a lower proportion of CDH2 + progenitor cells in the whole population of endometrial cells in the secretory phase of the cycle. Lack of similar decrease in endometrium from patients with endometriosis may be explained by an increased proportion and increased expression of markers of CDH2 + endometrial epithelial progenitor cells. A decreased level of CDH2 mRNA in the secretory phase in healthy control endometrium may also account for a relative increase in level of CDH2 mRNA observed in secretory phase in endometrium from endometriosis patients when compared to secretory phase control endometrial tissue. Analysis of expression of the remaining TGFB1 , TGFB2 , ZEB1 , ZEB2 , SNAI1 , CDH1 , MIR200B and MIR200C genes in both healthy control endometrium and endometrium from women with endometriosis did not reveal any significant differences between proliferatory and secretory phase. This strongly suggest that the menstrual cycle has no important effect on the phenomena that may be mediated by these EMT-related factors. TGF-b is considered as one of the most important factors participating in the pathogenesis of endometriosis [57,58]. In particular, TGF-b is considered as a principal inducer of different EMT mechanisms including TGF-b/ZEB/miR200 loop [34,36,59,60] or TGF-b/TWIST/SNAIL pathway [25,28]. Lack of significant changes in TGFB1/TGFB2 expression profile may suggest that these pathways are not involved in physiological changes of endometrium during the menstrual cycle. Therefore, upregulation of SNAI2 expression during the secretory phase seems to be related to a different yet unrevealed mechanism, e.g. WNT/β‐catenin pathway, as suggested before. As discussed above both normal healthy endometrium and endometrium from endometriosis patients display the very similar pattern of expression of EMT-related genes. Similarly, direct comparison of the levels of investigated mRNAs or miRNAs from normal healthy endometrium and endometrium from endometriosis patients stratified according to the phase of the menstrual cycle did not reveal any significant differences. Although, in addition to already discussed difference in CDH2 gene expression there were also some differences in TGFB1 and SNAI1 expression respectively in proliferatory and secretory phase. However, despite statistical significance, considering that expression of SNAI1 was ca. 10-20 times lower than SNAI2 and that these differences were low, it may be concluded that the biological meaning of these disparities may be negligible. Our observation that there are no significant differences in expression of EMT-related factors between normal endometrium and endometrium from patients with endometriosis is consistent with the results of few other studies based on immunohistochemical evaluations [23] and microarray analyses performed on the secretory endometrium [61,62]. Moreover, no differences were observed in the level of CDH1 mRNA expression in the endometrium from infertile endometriosis patients compared to healthy controls [63]. On the other hand, in women without a diagnosis of endometriosis, endometrial CDH1 mRNA expression in the secretory phase was lower than during the proliferative phase [64]. Additionally, Yun et al. reported decreased CDH2 mRNA levels in the mid-secretory endometrium of women with endometriosis, accompanied by an increase in CDH1 expression [65]. Present data argue for a limited role of EMT in eutopic endometrium in the pathogenesis of endometriosis and suggests that hypothetical endometrial epithelial cells that underwent a transition into mesenchymal-like cells are unlikely to be responsible for formation of distant endometrioid lesions. In conclusion, the results of the present study show for the first time that SNAI2 expression is significantly upregulated during secretory phase of the menstrual cycle thus suggesting its role in physiology of the cyclic endometrial changes. This role remains unknown and requires further investigations. Furthermore, we were unable to reveal any significant and biologically relevant differences in expression of EMT-related genes between normal healthy endometrium and endometrium from endometriosis patients. This observation implies that EMT is not differentially regulated in eutopic endometrium in endometriosis as compared to control women and strongly supports a view that EMT is not actively involved in development of endometriosis at the early stages of the disease. Absence of immunoreactive ZEBs in epithelial and glandular cells may also argue against active EMT. Thus, a significant upregulation of EMT-related genes in ectopic endometrioid lesions that was repeatedly reported by many studies [21,22,22,29–32,49,66] seems to be rather a secondary phenomenon depending e.g. on the effects of a local peritoneal milieu. References Matarese G, De Placido G, Nikas Y, Alviggi C. Pathogenesis of endometriosis: natural immunity dysfunction or autoimmune disease? Trends in Molecular Medicine. 2003;9:223–8. Tomassetti C, Meuleman C, Pexsters A, Mihalyi A, Kyama C, Simsa P, et al. Endometriosis, recurrent miscarriage and implantation failure: is there an immunological link? 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JCM. 2019;8:1468. Bartley J, Jülicher A, Hotz B, Mechsner S, Hotz H. Epithelial to mesenchymal transition (EMT) seems to be regulated differently in endometriosis and the endometrium. Arch Gynecol Obstet. 2014;289:871–81. Yang Y-M, Yang W-X. Epithelial-to-mesenchymal transition in the development of endometriosis. Oncotarget. 2017;8:41679–89. Konrad L, Dietze R, Riaz MA, Scheiner-Bobis G, Behnke J, Horné F, et al. Epithelial–Mesenchymal Transition in Endometriosis—When Does It Happen? JCM. 2020;9:1915. Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Invest. 2009;119:1438–49. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420–8. Thompson EW, Newgreen DF. Carcinoma Invasion and Metastasis: A Role for Epithelial-Mesenchymal Transition? Cancer Research. 2005;65:5991–5. Thiery JP, Acloque H, Huang RYJ, Nieto MA. Epithelial-Mesenchymal Transitions in Development and Disease. Cell. 2009;139:871–90. Nieszporek A, Skrzypek K, Adamek G, Majka M. Molecular mechanisms of epithelial to mesenchymal transition in tumor metastasis. Acta Biochim Pol [Internet]. 2019 [cited 2025 Feb 11]; Available from: https://www.frontierspartnerships.org/articles/10.18388/abp.2019_2899/pdf Proestling K, Birner P, Gamperl S, Nirtl N, Marton E, Yerlikaya G, et al. Enhanced epithelial to mesenchymal transition (EMT) and upregulated MYC in ectopic lesions contribute independently to endometriosis. Reprod Biol Endocrinol. 2015;13:75. Furuya M, Masuda H, Hara K, Uchida H, Sato K, Sato S, et al. ZEB1 expression is a potential indicator of invasive endometriosis. Acta Obstet Gynecol Scand. 2017;96:1128–35. Ntzeros K, Mavrogianni D, Blontzos N, Soyhan N, Kathopoulis N, Papamentzelopoulou M-S, et al. Expression of ZEB1 in different forms of endometriosis: A pilot study. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2023;286:121–5. Wu R-F, Chen Z-X, Zhou W-D, Li Y-Z, Huang Z-X, Lin D-C, et al. High expression of ZEB1 in endometriosis and its role in 17β-estradiol-induced epithelial-mesenchymal transition. Int J Clin Exp Pathol. 2018;11:4744–58. Eggers JC, Martino V, Reinbold R, Schäfer SD, Kiesel L, Starzinski-Powitz A, et al. microRNA miR-200b affects proliferation, invasiveness and stemness of endometriotic cells by targeting ZEB1, ZEB2 and KLF4. Reproductive BioMedicine Online. 2016;32:434–45. Brabletz S, Brabletz T. The ZEB/miR‐200 feedback loop—a motor of cellular plasticity in development and cancer? EMBO Reports. 2010;11:670–7. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10:593–601. Hill L, Browne G, Tulchinsky E. ZEB/miR‐200 feedback loop: At the crossroads of signal transduction in cancer. Intl Journal of Cancer. 2013;132:745–54. American Society For Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertility and Sterility. 1997;67:817–21. Xiong W, Zhang L, Liu H, Li N, Du Y, He H, et al. E 2 ‐mediated EMT by activation of β‐catenin/Snail signalling during the development of ovarian endometriosis. J Cellular Molecular Medi. 2019;23:8035–45. Konrad L, Gronbach J, Horné F, Mecha EO, Berkes E, Frank M, et al. Similar Characteristics of Endometrial and Endometriotic Epithelial Cells. Reprod Sci. 2019;26:49–59. Zhang X-H, Liang X, Liang X-H, Wang T-S, Qi Q-R, Deng W-B, et al. The mesenchymal-epithelial transition during in vitro decidualization. Reprod Sci. 2013;20:354–60. Ran J, Yang H-H, Huang H-P, Huang H-L, Xu Z, Zhang W, et al. ZEB1 modulates endometrial receptivity through epithelial-mesenchymal transition in endometrial epithelial cells in vitro. Biochemical and Biophysical Research Communications. 2020;525:699–705. Patterson AL, Zhang L, Arango NA, Teixeira J, Pru JK. Mesenchymal-to-Epithelial Transition Contributes to Endometrial Regeneration Following Natural and Artificial Decidualization. Stem Cells and Development. 2013;22:964–74. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer. 2007;7:415–28. Chiang C, Ayyanathan K. Snail/Gfi-1 (SNAG) family zinc finger proteins in transcription regulation, chromatin dynamics, cell signaling, development, and disease. Cytokine & Growth Factor Reviews. 2013;24:123–31. Cano A, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, Del Barrio MG, et al. The transcription factor Snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000;2:76–83. Kielbik M, Szulc-Kielbik I, Klink M. Snail transcription factors – Characteristics, regulation and molecular targets relevant in vital cellular activities of ovarian cancer cells. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2024;1871:119705. Tang H, Massi D, Hemmings BA, Mandalà M, Hu Z, Wicki A, et al. AKT-ions with a TWIST between EMT and MET. Oncotarget. 2016;7:62767–77. Hemavathy K, Ashraf SI, Ip YT. Snail/slug family of repressors: slowly going into the fast lane of development and cancer. Gene. 2000;257:1–12. Proestling K, Birner P, Balendran S, Nirtl N, Marton E, Yerlikaya G, et al. Enhanced expression of the stemness-related factors OCT4, SOX15 and TWIST1 in ectopic endometrium of endometriosis patients. Reprod Biol Endocrinol. 2016;14:81. Bouris P, Skandalis SS, Piperigkou Z, Afratis N, Karamanou K, Aletras AJ, et al. Estrogen receptor alpha mediates epithelial to mesenchymal transition, expression of specific matrix effectors and functional properties of breast cancer cells. Matrix Biology. 2015;43:42–60. Qureshi R, Picon-Ruiz M, Sho M, Van Booven D, Nunes De Paiva V, Diaz-Ruano AB, et al. Estrone, the major postmenopausal estrogen, binds ERa to induce SNAI2, epithelial-to-mesenchymal transition, and ER+ breast cancer metastasis. Cell Reports. 2022;41:111672. Park S-H, Cheung LWT, Wong AST, Leung PCK. Estrogen Regulates Snail and Slug in the Down-Regulation of E-Cadherin and Induces Metastatic Potential of Ovarian Cancer Cells through Estrogen Receptor α. Molecular Endocrinology. 2008;22:2085–98. Duncan WC, Shaw JLV, Burgess S, McDonald SE, Critchley HOD, Horne AW. Ectopic Pregnancy as a Model to Identify Endometrial Genes and Signaling Pathways Important in Decidualization and Regulated by Local Trophoblast. Schönbach C, editor. PLoS ONE. 2011;6:e23595. Liang Y-X, Hu W, Jin Z-Y, Diao H-L, Liu L, Yang Y, et al. Nucleolar stress regulates stromal–epithelial transition via NPM1 during decidualization. Reproduction. 2020;160:491–500. Huang Z, Mao X, Lin D, Hong Y, Liang G, Chen Q, et al. Establishment and characterization of immortalized human eutopic endometrial stromal cells. American J Rep Immunol [Internet]. 2020 [cited 2025 Jul 22];83. Available from: https://onlinelibrary.wiley.com/doi/10.1111/aji.13213 Cousins FL, O DF, Gargett CE. Endometrial stem/progenitor cells and their role in the pathogenesis of endometriosis. Best Practice & Research Clinical Obstetrics & Gynaecology. 2018;50:27–38. Omwandho COA, Konrad L, Halis G, Oehmke F, Tinneberg H-R. Role of TGF- s in normal human endometrium and endometriosis. Human Reproduction. 2010;25:101–9. Soni UK, Chadchan SB, Kumar V, Ubba V, Khan MTA, Vinod BSV, et al. A high level of TGF-B1 promotes endometriosis development via cell migration, adhesiveness, colonization, and invasiveness†. Biology of Reproduction. 2019;100:917–38. Gregory PA, Bracken CP, Smith E, Bert AG, Wright JA, Roslan S, et al. An autocrine TGF-β/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition. Bronner-Fraser M, editor. MBoC. 2011;22:1686–98. Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S, et al. A reciprocal repression between ZEB1 and members of the miR‐200 family promotes EMT and invasion in cancer cells. EMBO Reports. 2008;9:582–9. Fassbender A, Verbeeck N, Börnigen D, Kyama CM, Bokor A, Vodolazkaia A, et al. Combined mRNA microarray and proteomic analysis of eutopic endometrium of women with and without endometriosis. Human Reproduction. 2012;27:2020–9. Sherwin JRA, Sharkey AM, Mihalyi A, Simsa P, Catalano RD, D’Hooghe TM. Global gene analysis of late secretory phase, eutopic endometrium does not provide the basis for a minimally invasive test of endometriosis. Human Reproduction. 2008;23:1063–8. Matsuzaki S, Darcha C, Maleysson E, Canis M, Mage G. Impaired Down-Regulation of E-Cadherin and β-Catenin Protein Expression in Endometrial Epithelial Cells in the Mid-Secretory Endometrium of Infertile Patients with Endometriosis. The Journal of Clinical Endocrinology & Metabolism. 2010;95:3437–45. Fujimoto J, Ichigo S, Hori M, Tamaya T. Alteration of E-cadherin, α-and β-catenin mRNA expression in human uterine endometrium during the menstrual cycle. Gynecological Endocrinology. 1996;10:187–91. Yun BS, Yun NY, Lee JE, Go M, Jang HY, Park JE, et al. Endometrial E-cadherin and N-cadherin Expression during the Mid-Secretory Phase of Women with Ovarian Endometrioma or Uterine Fibroids. JPM. 2024;14:920. Zeitvogel A, Baumann R, Starzinski-Powitz A. Identification of an Invasive, N-Cadherin-Expressing Epithelial Cell Type in Endometriosis Using a New Cell Culture Model. The American Journal of Pathology. 2001;159:1839–52. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7227864","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491642499,"identity":"3da2aa92-d9a5-4db4-913a-d36b217ba732","order_by":0,"name":"Agata Góźdź","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Agata","middleName":"","lastName":"Góźdź","suffix":""},{"id":491642500,"identity":"5113c904-7959-4773-90b6-0244f4d7674b","order_by":1,"name":"Marta Żeberkiewicz","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Żeberkiewicz","suffix":""},{"id":491642501,"identity":"e78703fe-5ce4-44e3-bf49-78ecedd9bdf2","order_by":2,"name":"Izabela Janiuk","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Izabela","middleName":"","lastName":"Janiuk","suffix":""},{"id":491642502,"identity":"30fcea36-d2e0-4794-9d90-a27bca976bcf","order_by":3,"name":"Anna Hyc","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Hyc","suffix":""},{"id":491642503,"identity":"6ccae980-fa43-4ed2-8657-003343adfe66","order_by":4,"name":"Anna Iwan","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Iwan","suffix":""},{"id":491642504,"identity":"417f46ab-7e80-4eda-89ea-1bf95f616de3","order_by":5,"name":"Aneta Zwierzchowska","email":"","orcid":"","institution":"Cardinal Stefan Wyszyński University","correspondingAuthor":false,"prefix":"","firstName":"Aneta","middleName":"","lastName":"Zwierzchowska","suffix":""},{"id":491642505,"identity":"c5d5617d-2369-4714-9efd-ab996db48d62","order_by":6,"name":"Radosław Maksym","email":"","orcid":"","institution":"Centrum Medyczne Kształcenia Podyplomowego","correspondingAuthor":false,"prefix":"","firstName":"Radosław","middleName":"","lastName":"Maksym","suffix":""},{"id":491642506,"identity":"6560332e-73d5-4430-935e-a7a70f80a5c0","order_by":7,"name":"Kateryna Shevchenko","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Kateryna","middleName":"","lastName":"Shevchenko","suffix":""},{"id":491642507,"identity":"8ff798ee-0f4a-459c-9f6c-ccff2382287a","order_by":8,"name":"Paweł Włodarski","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Paweł","middleName":"","lastName":"Włodarski","suffix":""},{"id":491642509,"identity":"ce324bbc-4cf7-46b5-9055-cc503c0f0e50","order_by":9,"name":"Ewa Barcz","email":"","orcid":"","institution":"Cardinal Stefan Wyszyński University","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Barcz","suffix":""},{"id":491642511,"identity":"6ef31ecb-03b0-446b-bc40-9d5fcd9536ce","order_by":10,"name":"Jacek Malejczyk","email":"data:image/png;base64,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","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":true,"prefix":"","firstName":"Jacek","middleName":"","lastName":"Malejczyk","suffix":""}],"badges":[],"createdAt":"2025-07-27 18:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7227864/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7227864/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12958-025-01486-w","type":"published","date":"2025-11-18T15:58:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87900899,"identity":"2959326d-d59a-47d1-9f1e-7a335a751e2c","added_by":"auto","created_at":"2025-07-30 08:13:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":318278,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical localization of ZEB-1, ZEB-2, SNAIL, SLUG, E-Cadherin and N-Cadherin expression in eutopic endometrium from healthy women. Original magnification x200.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7227864/v1/21c75bba5b5e27ebbcf5a976.png"},{"id":87901789,"identity":"3b33ae73-9221-4a85-8771-3b88512137a4","added_by":"auto","created_at":"2025-07-30 08:21:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83673,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of EMT-related gene mRNAs and miRNAs in eutopic endometrium from healthy control group in proliferative and secretory phase of the menstrual cycle. The results are shown as medians with interquartile range. Differences between groups were computed by Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test. ns, not significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7227864/v1/c363a8999dd9f059440742d6.png"},{"id":87900896,"identity":"ea3260e7-010c-44c3-b819-c8dd42890135","added_by":"auto","created_at":"2025-07-30 08:13:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96157,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of EMT-related gene mRNAs and miRNAs in eutopic endometrium from endometriosis group in proliferative and secretory phase of the menstrual cycle. The results are shown as medians with interquartile range. Differences between groups were computed by Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test. ns, not significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7227864/v1/020872cb9d96ab41be81ad9e.png"},{"id":87901790,"identity":"b7dfae28-30d2-4575-b5b7-b658e6247b36","added_by":"auto","created_at":"2025-07-30 08:21:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91023,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of expression of EMT-related gene mRNAs and miRNAs between eutopic endometrium from healthy control group and eutopic endometrium from endometriosis group in the proliferative phase of the menstrual cycle. The results are shown as medians with interquartile range. Differences between groups were computed by Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test. ns, not significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7227864/v1/eab5e22b8f7ddb8127f3f9c0.png"},{"id":87900898,"identity":"b7ca462e-eef3-455e-ae9d-3eed16e5e616","added_by":"auto","created_at":"2025-07-30 08:13:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98914,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of expression of EMT-related gene mRNAs and miRNAs between eutopic endometrium from control group and eutopic endometrium from endometriosis group in the secretory phase of the menstrual cycle. The results are shown as medians with interquartile range. Differences between groups were computed by Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test. ns, not significant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7227864/v1/24b6ddab746e3fcb44016843.png"},{"id":96650335,"identity":"ac149df9-05f9-4afd-b05f-58c303a8ca7b","added_by":"auto","created_at":"2025-11-24 16:11:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1532236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7227864/v1/3f4a328d-9452-4632-a183-b1bbbf05744f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The influence of menstrual cycle and endometriosis on endometrial expression of epithelial-to-mesenchymal transition (EMT)-related genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is a common chronic gynaecological disorder related to the presence of ectopic foci of endometrial-like tissue (glandular and stromal cells) mostly in the pelvic cavity. The disease is associated with chronic pelvic inflammatory reactions and may also display some features of autoimmune disorder\u0026nbsp;[1–4]. It may manifest by dysmenorrhea and pelvic pain and is considered as one of the major causes of the female infertility [5,6].\u0026nbsp;Endometriosis affects ca. 10% of women in reproductive age and has a significant impact on patients’ life quality and constitutes an important clinical and social problem.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEtiopathology of endometriosis appears to be very complex and remains obscure [7,8].\u0026nbsp;According to the most accepted Sampson’s theory, endometriosis develops because of retrograde flow of endometrial cells shed in course of menstruation\u0026nbsp;[9]\u0026nbsp;.\u0026nbsp;Appearance of distant lesions such as in lung, heart, liver or brain may be due to dissemination by lymphatic or blood system\u0026nbsp;[10,11]. Ectopic endometriotic-like tissue may also develop because of coelomic metaplasia of Müllerian system remnants [12].\u003c/p\u003e\n\u003cp\u003eThe evidence accumulates that endometriosis may originate in part from disseminating stem-like epithelial precursor cells\u0026nbsp;[13,14]\u0026nbsp;. However, the mechanisms responsible for survival, implantation and progression of endometriotic cells in the peritoneal cavity\u0026nbsp;remain\u0026nbsp;poorly recognized. It may be partially due to\u0026nbsp;their decreased susceptibility to apoptotic cell death\u0026nbsp;[15–17]\u0026nbsp;,\u0026nbsp;an increased adhesiveness and invasiveness\u0026nbsp;[15,18,19] , as well as their abrogated elimination by the cells of the local immune surveillance system [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePathogenesis of endometriosis may also involve a phenomenon of epithelial-to-mesenchymal transition (EMT) [21–23]. EMT is defined as a process characterized by morphological and functional changes from epithelial to mesenchymal cell phenotype [24,25]. Typically, epithelial cells lose their polarized phenotype and intercellular connections and acquire fibroblastic shape with enhanced migratory and invasive potential. These changes are related to downregulation of E-cadherin and an increased N-cadherin expression\u0026nbsp;[24,25]. EMT appears to be one of the fundamental mechanisms involved in development of multicellular organisms playing a role in embryo- and morphogenesis\u0026nbsp;[24,25]. On the other hand, EMT appears to be crucial for a variety of pathological phenomena including fibrosis and tumour invasion and metastasis\u0026nbsp;[26–28].\u003c/p\u003e\n\u003cp\u003eIt has been reported that endometriotic epithelial cells display a decreased expression of E-cadherin and endometriosis is associated with an increased expression of EMT inducing factors such as members of ZEB and SNAIL families of transcription factors\u0026nbsp;[21,29–32]. It has also been suggested that EMT in endometriotic cells may also depend on abrogated expression of miRNAs of miR200 family, that are major regulators of ZEB expression\u0026nbsp;[33–36].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, the exact role of EMT in development of endometriotic lesions remains poorly understood. There is also little known about expression and a putative role of EMT-related genes in eutopic endometrium in course of the menstrual cycle. Therefore, the present study was aimed at investigating expression of major\u0026nbsp;EMT-related\u0026nbsp;genes coding for the members of TGF-b, ZEB, SNAIL, CDH and miR200 family in eutopic endometrium of women with and without endometriosis in proliferative and secretory phase of the menstrual cycle.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients enrolled in the present study were diagnosed at the Departments of Obstetrics and Gynaecology, Medical University of Warsaw and the Department of Gynaecology, Military Institute of Medicine, Warsaw, Poland between January 2010 and December 2015.\u0026nbsp;All participants gave an informed consent to the study and the investigations were approved\u0026nbsp;by the Institutional Bioethical Review Board of the Medical University of Warsaw and Military Institute of Medicine, Poland (permissions no. WUM/KB/223/2009, 49/WIM/2011, 37/WIM/2013) and conducted according to the Helsinki Declaration guidelines.\u003c/p\u003e\n\u003cp\u003eThe study included 46 women with laparoscopically and histologically confirmed endometriosis. The severity of the disease has been classified according to the revised American Society of Reproductive Medicine (rASRM) criteria [37]. The control group consisted of 30 women without any clinical symptoms of endometriosis who underwent removal of a cervical polyp or were subjected to a cervical biopsy due to a positive result of the Pap smear. Women with histopathologically confirmed cervical dysplasia were not included. None of the patients with endometriosis or the control subjects had any other chronic diseases, and none had received hormonal treatment for at least three months prior to the study. Detailed demographic and clinical characteristics of the patients with endometriosis and the control subjects are presented in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eDemographic and clinical characteristics of endometriosis patients and healthy control group.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eEndometriosis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProliferative phase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSecretory phase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProliferative phase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSecretory phase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eNumber of cases (N)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14 (46.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16 (53.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16 (34.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30 (65.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAge, years (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.7 \u0026plusmn; 6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.8 \u0026plusmn; 6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.6 \u0026plusmn; 7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e32.3 \u0026plusmn; 5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e29.9 \u0026plusmn; 3.7*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e33.5 \u0026plusmn; 6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eBMI, kg/m\u003csup\u003e2\u003c/sup\u003e (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22.4 \u0026plusmn; 4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22.5 \u0026plusmn; 5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22.3 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e21.6 \u0026plusmn; 3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e21.4 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e21.7 \u0026plusmn; 3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003erASRM\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eI (minimal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7 (15.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2 (12.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5 (16.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eII (mild)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5 (10.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5 (16.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIII (moderate)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22 (47.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9\u0026nbsp;(56.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e13 (43.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIV (severe)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12 (26.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5 (31.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7 (23.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eLesion localization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOvarian\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e37 (80.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14 (85.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22 (73.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePeritoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35 (76.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e13 (81.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22 (73.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBoth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25 (54.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e11 (68.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14 (46.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ena, not applicable; *Different from Endometriosis secretory phase group at \u003cem\u003eP\u003c/em\u003e=0.0176 and from Control proliferative phase group at \u003cem\u003eP\u003c/em\u003e=0.0133\u0026nbsp;as judged by Student-\u003cem\u003et\u003c/em\u003e test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTissue samples were collected from the participants during mid-proliferative and mid-secretory phase of the menstrual cycle. A phase of the menstrual cycle was established basing on the date of the last menstrual bleeding and was confirmed by pelvic ultrasound examination. Endometrial tissue samples were obtained by an aspiration biopsy with a Pipelle\u0026Ograve; catheter (Pipelle de Cornier, Laboratoire C.C.D., Paris, France). For immunohistochemical examinations the samples were routinely fixed in 10% buffered formalin whereas for evaluation of mRNA expression, the specimens were immediately placed in 5 volumes of RNAlater\u0026Ocirc; solution (TermoFisher Scientific, Waltham, Massachusetts, USA), kept at 4\u0026deg;C for one day and then stored frozen at -70\u0026deg;C until RNA isolation was performed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression of ZEB1, ZEB2, SNAI1, SNAI2, E-cadherin (CDH1) and N-cadherin (CDH2) was detected by routine immunohistochemical staining. In brief, formalin-fixed paraffin-embedded tissue samples of eutopic endometrium and endometriotic cysts were cut into \u003cs\u003e5\u0026nbsp;\u003c/s\u003emm sections, deparaffinized, rehydrated in graded alcohol series and permeabilized in 10 mM citrate buffer (pH 6.0) at 95\u0026deg;C. Endogenous peroxidase was blocked by Novolink Peroxide Block (Leica Biosystems, RE7140-CE). Then the sections were incubated with respective primary antibody for 60 minutes at room temperature. All primary antibodies, their specification, origin and working dilution are listed in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eA list of primary antibodies used for immunohistochemical staining of endometrial samples.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrigin/clone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWorking dilution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eAnti-ZEB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMouse monoclonal IgG2a/clone OTI3G6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1:150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eAnti-ZEB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMouse monoclonal IgG2a/\u0026nbsp;clone 6E5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1:250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eAnti-SNAI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003ch4\u003eRabbit polyclonal\u003c/h4\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1:180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eAffinity Biosciences, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eAnti-SNAI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eRabbit polyclonal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eAffinity Biosciences, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eAnti-CDH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMouse monoclonal IgG1/clone 4A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1:150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eAnti-CDH2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eMouse monoclonal IgG1/clone 8C11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1:250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ePrimary antibodies were detected by incubation with Novolink\u0026trade; Polymer Detection Reagent (Leica Biosystems, RE7140-CE) for 30 minutes at room temperature according to the manufacturer\u0026rsquo;s instructions. The peroxidase reaction was visualized using the DAB chromogen provided in the kit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and quantitative reversed transcription PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLarge and small RNAs from tissue samples (5 mm\u003csup\u003e3\u003c/sup\u003e) were isolated with the NucleoSpin\u0026reg; miRNA Kit (Macherey-Nagel, D\u0026uuml;ren, Germany) according to the manufacturer\u0026rsquo;s protocol. The quantity and quality of the isolated RNA was evaluated spectrophotometrically using NanoDrop2000 spectrophotometer with software for analysis of nucleic acids (ThermoFisher Scientific).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReverse transcription of 2 \u0026mu;g of large RNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cheshire, UK), according to the manufacturer\u0026rsquo;s protocol in an Eppendorf Mastercycler gradient at 25\u0026deg;C for 10 min, 37\u0026deg;C for 120 min and 85\u0026deg;C for 5 sec. Reverse transcription of 10 ng of small RNA was performed using the TaqMan MicroRNA Transcription Kit and specific hsa-miR-200b-3p (Assay ID 00225), hsa-miR-200c-3p (Assay ID 002300) and RNU43 (Assay ID 001095) TaqMan MicroRNA assays (Applied Biosystems), respectively for miRNA200b, miRNA200c and RNU43. Reaction was performed according to the manufacturer\u0026rsquo;s protocol in an Eppendorf Mastercycler gradient at 16\u0026ordm;C for 30 min, 42\u0026ordm;C for 30 min and 85\u0026ordm;C for 5 min. cDNA samples were stored at -20\u0026deg;C until qRT-PCR assays were performed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eA list of TaqMan primers for used for qRT-PCR.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFactor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTaqMan probe\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eTGF-b1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eTGFB1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs00998133_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eTGF-b2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eTGFB2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs 00234244_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eZEB-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eZEB1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs00232783_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eZEB-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eZEB2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs00207691_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eSNAIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eSNAI1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs00195591_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eSLUG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eSNAI2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs00161904_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eE-cadherin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eCDH1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs01023894_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eN-cadherin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eCDH2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs00983056_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs99999905_m1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHsa-miR200b-3p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eMIR200B\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs04231483_s1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHsa-miR200c-3p\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eMIR200C\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs04231534_s1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eRNU43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e\u003cem\u003eRNU43\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eHs00504439_CE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eReal time qRT-PCR was performed in the ABI PRISM 7500 thermocycler (Applied Biosystems) using specific TaqMan expression assays (Applied Biosystems) with FAM-labelled probes and 96-well optical plates. A list of specific mRNAs probes used in this study is shown in Table 3. The reactions were run in a total volume of 20 \u0026mu;L including TaqMan Universal Master Mix, the appropriate primer set, MGB probe and 50 ng of cDNA template with an initial denaturation at 95\u0026deg;C for 10 min, amplification for 50 cycles at 95\u0026deg;C for 15 sec and 60\u0026deg;C for 1 min. Each sample was run in triplicate. GAPDH and RNU43 served as endogenous control for large and small RNAs, respectively. Relative RNA expression was computed by the Sequence Detection System (SDS) v 1.2 software (Applied Biosystems) using the DCt method and the results are presented as relative units calculated from the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003eD\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e formula.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses and graphical presentations were generated using GraphPad Prism 8.2.0 (GraphPad Software, San Diego, CA, USA). The study groups were characterized using descriptive statistics consistent with the distribution of variables. Differences between groups were determined using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, Wilcoxon matched-pairs signed rank test or Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e-test when applicable. Probability (\u003cem\u003eP\u003c/em\u003e) values of \u0026lt; 0.05 were considered statistically significant. The results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or medians with range or interquartile range.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEndometrial immunolocalization of ZEB1, ZEB2, SNAI1, SNAI2, CDH1 and CDH2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of immunolocalization of investigated proteins are shown on Fig. 1. As seen, expression of ZEB1 and ZEB2 was primarily localized in the nuclei of endometrial stromal cells. No significant immunoreactivity was seen in the epithelium and glands. On the contrary, a relatively strong reaction was found in the nuclei of epithelial and glandular cells in case of SNAI1 and SNAI2. SNAI1 and SNAI2 immunoreactivity was also noted in nuclei of endometrial stromal cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExpression of CDH1 was seen only in epithelial cells. It was localized mostly in cell membranes and only mild diffuse staining was seen in the cytoplasm. N-cadherin was found mostly in epithelial cells, but sparse positive reaction was also seen in some endometrial stromal cells. N-cadherin reactivity pattern in epithelial cells was like that of E-cadherin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of endometrial mRNA and miRNA expression of investigated EMT-related\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression of mRNA and miRNA\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003especific for all studied EMT-related genes was detected in all endometrial tissue samples from both control healthy women and women with endometriosis. The results of semiquantitative mRNA and miRNA evaluations showed that, irrespectively of the phase of the menstrual cycle, the endometrium from control healthy women expressed significantly much more \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eSNAI2\u003c/em\u003e mRNA as compared to their related \u003cem\u003eTGFB2\u003c/em\u003e and \u003cem\u003eSNAI1\u003c/em\u003e genes\u003cem\u003e,\u0026nbsp;\u003c/em\u003erespectively (Table 4). Accordingly, expression of \u003cem\u003eTGFB1\u003c/em\u003e was ca. 6-10 times higher than expression of \u003cem\u003eTGFB2\u003c/em\u003e, and expression of \u003cem\u003eSNAI2\u003c/em\u003e was ca. 10-20 times higher than expression of \u003cem\u003eSNAI1\u003c/em\u003e. Expression of \u003cem\u003eCDH1\u003c/em\u003e and \u003cem\u003eMIR200C\u003c/em\u003e was slightly higher (about 3 times) than\u003cem\u003e\u0026nbsp;CDH2\u003c/em\u003e and \u003cem\u003eMIR200B\u003c/em\u003e, respectively, whereas there were no significant differences in expression between \u003cem\u003eZEB1\u003c/em\u003e and \u003cem\u003eZEB2\u003c/em\u003e genes. Exactly similar results were seen in case of endometrium samples from women with endometriosis (Table 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Comparisons of expression levels of related EMT-associated gene mRNAs and miRNAs in endometrium of healthy women.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProliferatory phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecretory \u0026nbsp;phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eTGFB1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0474 (0.029-0.1768)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0359 (0.0096-0.0718)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.0507\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eTGFB2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0042 (0.0003-0.0544)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0053 (0.0002-0.1340)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eZEB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0292 (0.0156-0.1250)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0372 (0.0168-0.0825)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eZEB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0388 (0.0127-0.1340)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0222 (0.0118-0.0884)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eSNAI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0025 (0.0015-0.0136)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0024 (0.0002-0.0118)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eSNAI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0254 (0.0111-0.0670)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0583 (0.0180-0.4061)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eCDH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.1806 (0.0625-1.2510)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0947 (0.0385-0.9659)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eCDH2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.0583 (0.0059-0.2679)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e0.0151 (0.0026-0.0625)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eMIR200B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e19.31 (0.06-2048.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 76px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e24.25 (1.41-724.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.0020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eMIR200C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e58.55 (0.57-9410.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e34.30 (3.03-5943.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll results are shown as medians (range) of relative mRNA expression (2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003eD\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e). \u003cem\u003eP\u003c/em\u003e-values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u003c/strong\u003e Comparisons of expression levels of related EMT-associated gene mRNAs and miRNAs in endometrium of women with endometriosis.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 188px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProliferatory phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 187px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecretory \u0026nbsp;phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cem\u003eTGFB1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.0372 (0.0206-0.0625)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.0412\u0026nbsp;(0.0156-0.1649)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.0032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cem\u003eTGFB2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.0040 (0.0002-0.0146)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.0063 (0.0007-0.1649)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eZEB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.0263 (0.0059-0.0412)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.0136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.0323 (0.0073-0.1539)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eZEB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.0282 (0.0136-0.0583)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.0313 (0.0090-0.1340)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eSNAI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.0024 (0.0011-0.0136)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.0046 (0.0005-0.0313)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eSNAI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.0313 (0.0032-0.1166)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.0544 (0.0180-0.1649)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eCDH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.1091 (0.0313-0.4665)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0.0580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.1253 (0.0335-0.8123)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.0006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eCDH2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e0.0545 (0.0048-0.1895)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e0.0474 (0.0042-0.3078)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eMIR200B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e17.19 (0.08-1552)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 70px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e1.71 (0.12-1261)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.0163\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eMIR200C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e27.57 (0.44-8192.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e8.29 (0.00-9410)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll results are shown as medians (range) of relative mRNA expression (2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003eD\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e). \u003cem\u003eP\u003c/em\u003e-values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMenstrual cycle-dependent changes in expression of endometrial mRNA and miRNA for EMT-related\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egenes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in specific expression of endometrial mRNA and miRNA\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor the investigated EMT-related\u0026nbsp;genes\u0026nbsp;between proliferative and secretory phase of the menstrual cycle in control women are shown in Fig. 2. As seen, secretory phase was associated with a significant 2-fold upregulation in\u0026nbsp;\u003cem\u003eSNAI2\u0026nbsp;\u003c/em\u003emRNA expression and a significant 5-fold decrease of \u003cem\u003eCDH2\u003c/em\u003e mRNA level. No differences were observed in mRNA or\u0026nbsp;miRNA\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eexpression of other EMT-related genes.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A significant 2-fold upregulation of \u003cem\u003eSNAI2\u0026nbsp;\u003c/em\u003emRNA expression in the secretory phase was also observed in the endometriosis group (Fig. 3). Furthermore, a decreased expression was also noted in case of both \u003cem\u003eMIR200B\u003c/em\u003e and \u003cem\u003eMIR200C\u003c/em\u003e genes. Expression of \u003cem\u003eMIR200B\u003c/em\u003e gene was reduced ten-fold, and expression \u003cem\u003eMIR200C\u003c/em\u003e was over 3 times lower compared to proliferatory phase; however, due to a great variability of the results this difference did not reach a statistical significance. There were no differences in expression of other studied genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferences in endometrial mRNA expression of EMT-related\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egenes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;between healthy control women and women with endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs\u0026nbsp;Fig. 4 shows,\u0026nbsp;comparison of endometrial mRNA and miRNA\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eexpression of EMT-related genes during proliferatory phase of the menstrual cycle between healthy women and endometriosis patients did not reveal any important differences except a slight but significantly lower level of \u003cem\u003eTGFB1\u003c/em\u003e gene expression in the endometriosis group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the secretory phase (Fig. 5) the level of \u003cem\u003eCDH2\u003c/em\u003e mRNA was 3-fold higher in the endometrium of women with endometriosis as compared to control group and this difference was statistically significant. A statistically significantly higher expression of \u003cem\u003eSNAI1\u0026nbsp;\u003c/em\u003egene\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas also observed in the endometriosis group; however, this difference was at the significance borderline. There were no differences in the level of expression of all other investigated EMT-related genes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo our best knowledge, this is the first study aimed at evaluation of EMT-related gene expression in endometrium from healthy women and women with endometriosis in relation to the phase of the menstrual cycle. Our present results show that normal endometrium as well as endometrium from women with endometriosis constitutively express mRNA of all investigated EMT-related genes and\u0026nbsp;this is consistent with previously published observations\u0026nbsp;[21,29,38]. Interestingly, we report for the first time that expression of\u0026nbsp;\u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eSNAI2\u003c/em\u003e mRNA was many times higher than expression of their respective family members, \u003cem\u003eTGFB2\u003c/em\u003e and \u003cem\u003eSNAI1\u003c/em\u003e. On the contrary, the differences in expression of \u003cem\u003eCDH1\u003c/em\u003e and \u003cem\u003eCDH2,\u003c/em\u003e as well as\u0026nbsp;\u003cem\u003eMIR200C\u003c/em\u003e and \u003cem\u003eMIR200B\u003c/em\u003e were not so much pronounced.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Expression of the investigated EMT-related factors was confirmed by immunohistochemical staining of endometrial tissue samples. Both ZEB1and ZEB2 proteins were localized in the nuclei, and their expression was limited to stromal cells. This localization of ZEBs in eutopic endometrium is consistent with previous observations [32,39]. Expression of SNAI1 and SNAI2 was also localized in the nuclei; however, unlike ZEBs, it was present in epithelial and glandular cells as well as in some stromal cells. Presence of SNAIs both in epithelial and stromal cells has been reported previously [21,38] .\u0026nbsp;Expression of CDH1 and CDH2 in epithelial and glandular cells was found in the intercellular junctions. Some CDH2 expression was also seen in the stromal cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEMT as well as a reverse process, mesenchymal to epithelial transition (MET) are believed to play an important role in endometrium regeneration, receptivity and \u003cem\u003ein vitro\u003c/em\u003e decidualization\u0026nbsp;[40\u0026ndash;42]. However, putative factors that may be responsible for regulation of EMT/MET endometrial balance remain obscure. To address this point, we evaluated changes of mRNA levels of\u0026nbsp;EMT-related genes\u0026nbsp;in endometrium during proliferatory and secretory phase of the menstrual cycle. We found for the first time that in healthy control endometrium the secretory phase was related to a significant several-fold upregulation of \u003cem\u003eSNAI2\u003c/em\u003e gene. Similar upregulation of\u003cem\u003e\u0026nbsp;SNAI2\u003c/em\u003e mRNA was also found in eutopic endometrium from women with endometriosis that strongly implies that SNAI2 may indeed play some role in physiological endometrial changes in course of the menstrual cycle. This assumption may be additionally supported by observation that SNAI1 and SNAI2 expression localizes in the nuclei thus suggesting their transcriptional activity.\u003c/p\u003e\n\u003cp\u003eSNAI2, a member of the SNAIL family of transcriptional regulators is C2H2-type zinc finger transcription factor\u0026nbsp;[43\u0026ndash;46]. Both, SNAI1\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eSNAI2\u003cem\u003e\u0026nbsp;\u003c/em\u003ecan stimulate EMT by repression of E-cadherin transcription in epithelial cells\u0026nbsp;[45,47,46]; however, unlike the members of ZEB family they are not considered as up-regulators of mesenchymal markers. Nevertheless, they may increase matrix metalloproteinase expression and activity and mediate cell motility and invasion, proliferation as well as cellular senescence and apoptosis\u0026nbsp;[45,48]. Accordingly, it is tempting to speculate that upregulated \u003cem\u003eSNAI2\u003c/em\u003e expression may contribute to extensive growth and maturation of endometrial glands and stroma during secretory phase. This, however, needs further elucidation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is also not clear what is the mechanism of increased expression of \u003cem\u003eSNAI2\u003c/em\u003e expression during secretory phase. Upregulation of SNAIs is mediated by TWIST, the basic helix-loop-helix (bHLH) transcription factor that is considered as an indirect EMT inducer [47]. Accordingly, expression of TWIST has also been reported in normal endometrium and endometrium from women with endometriosis [29,49] thus it is plausible that it may play a part in regulation of endometrial SNAIs expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChanges in \u003cem\u003eSNAI2\u003c/em\u003e expression and EMT/MET might be also dependent on menstrual cycle-associated hormonal changes. There are many reports indicating a role of 17b-oestradiol (E2) in induction of EMT and EMT-related factors, especially in hormone-dependent tumours [50\u0026ndash;52]. It has also been claimed that E2 may play a part in induction of EMT in course of endometriosis via upregulation of \u0026beta;‐catenin/SNAIL pathway\u0026nbsp;[38]. Therefore, some role of oestrogens in regulation of endometrial EMT/MET cannot be excluded. Interestingly, it has been demonstrated that decidualization of human endometrial stromal cells was associated with the WNT/\u0026beta;‐catenin pathway\u0026nbsp;[53]. WNT/\u0026beta;‐catenin signalling is known to downregulate E-cadherin expression and stimulate cell migration via activation of SNAIs\u0026nbsp;[47]\u0026nbsp;that may suggest a role of SNAIs and EMT in decidualization. On the other hand, however, overexpression of WNT promoted MET in human endometrial stromal cells\u0026nbsp;[54], and similar phenomenon was also observed in \u003cem\u003ein vitro\u003c/em\u003e model of decidualization following stromal cell treatment with progesterone and cAMP\u0026nbsp;[55]\u0026nbsp;. Thus, the hypothetical role of EMT/MET and SNAI2 in decidualization awaits further elucidation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, we also found a significant downregulation of \u003cem\u003eCDH2\u003c/em\u003e gene expression in the secretory phase of the menstrual cycle in healthy control endometrium. Similar change was not seen in endometriosis patients. CDH2 expression is localized mainly in epithelial cells in basalis endometrial layer and is considered as a marker of endometrial progenitor/stem cells participating in tissue regeneration following menstruation\u0026nbsp;[14,56]. Differentiating glandular cells lose their progenitor phenotype, it is therefore possible that a decreased level of \u003cem\u003eCDH2\u003c/em\u003e expression reflects a lower proportion of CDH2\u003csup\u003e+\u003c/sup\u003e progenitor cells in the whole population of endometrial cells in the secretory phase of the cycle. Lack of similar decrease in endometrium from patients with endometriosis may be explained by an increased proportion and increased expression of markers of CDH2\u003csup\u003e+\u003c/sup\u003e endometrial epithelial progenitor cells. A decreased level of \u003cem\u003eCDH2\u003c/em\u003e mRNA in the secretory phase in healthy control endometrium may also account for a relative increase in level of \u003cem\u003eCDH2\u003c/em\u003e mRNA observed in secretory phase in endometrium from endometriosis patients when compared to secretory phase control endometrial tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Analysis of expression of the remaining \u003cem\u003eTGFB1\u003c/em\u003e, \u003cem\u003eTGFB2\u003c/em\u003e, \u003cem\u003eZEB1\u003c/em\u003e, \u003cem\u003eZEB2\u003c/em\u003e, \u003cem\u003eSNAI1\u003c/em\u003e, \u003cem\u003eCDH1\u003c/em\u003e, \u003cem\u003eMIR200B\u003c/em\u003e and \u003cem\u003eMIR200C\u003c/em\u003e genes in both healthy control endometrium and endometrium from women with endometriosis did not reveal any significant differences between proliferatory and secretory phase. This strongly suggest that the menstrual cycle has no important effect on the phenomena that may be mediated by these EMT-related factors. \u0026nbsp;TGF-b\u0026nbsp;is considered as one of the most important factors participating in the pathogenesis of endometriosis\u0026nbsp;[57,58]. In particular, TGF-b\u0026nbsp;is considered as a principal inducer of different EMT mechanisms including TGF-b/ZEB/miR200 loop\u0026nbsp;[34,36,59,60]\u0026nbsp;or TGF-b/TWIST/SNAIL pathway\u0026nbsp;[25,28]. Lack of significant changes in \u003cem\u003eTGFB1/TGFB2\u003c/em\u003e expression profile may suggest that these pathways are not involved in physiological changes of endometrium during the menstrual cycle. Therefore, upregulation of \u003cem\u003eSNAI2\u003c/em\u003e expression during the secretory phase seems to be related to a different yet unrevealed mechanism, e.g. WNT/\u0026beta;‐catenin pathway, as suggested before.\u003c/p\u003e\n\u003cp\u003eAs discussed above both normal healthy endometrium and endometrium from endometriosis patients display the very similar pattern of expression of EMT-related genes. Similarly, direct comparison of the levels of investigated mRNAs or miRNAs from normal healthy endometrium and endometrium from endometriosis patients stratified according to the phase of the menstrual cycle did not reveal any significant differences. Although, in addition to already discussed difference in \u003cem\u003eCDH2\u003c/em\u003e gene expression there were also some differences in \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eSNAI1\u003c/em\u003e expression respectively in proliferatory and secretory phase. However, despite statistical significance, considering that expression of \u003cem\u003eSNAI1\u003c/em\u003e was ca. 10-20 times lower than \u003cem\u003eSNAI2\u003c/em\u003e and that these differences were low, it may be concluded that the biological meaning of these disparities may be negligible. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur observation that there are no significant differences in expression of EMT-related factors between normal endometrium and endometrium from patients with endometriosis is consistent with the results of few other studies based on immunohistochemical evaluations\u0026nbsp;[23]\u0026nbsp;and microarray analyses performed on the secretory endometrium\u0026nbsp;[61,62]. Moreover, no differences were observed in the level of \u003cem\u003eCDH1\u003c/em\u003e mRNA expression in the endometrium from infertile endometriosis patients compared to healthy controls [63]. On the other hand, in women without a diagnosis of endometriosis, endometrial \u003cem\u003eCDH1\u003c/em\u003e mRNA expression in the secretory phase was lower than during the proliferative phase [64].\u0026nbsp;Additionally, Yun et al. reported decreased \u003cem\u003eCDH2\u003c/em\u003e mRNA levels in the mid-secretory endometrium of women with endometriosis, accompanied by an increase in \u003cem\u003eCDH1\u003c/em\u003e expression\u0026nbsp;[65]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePresent data argue for a limited role of EMT in eutopic endometrium in the pathogenesis of endometriosis and suggests that hypothetical endometrial epithelial cells that underwent a transition into mesenchymal-like cells are unlikely to be responsible for formation of distant endometrioid lesions.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the results of the present study show for the first time that \u003cem\u003eSNAI2\u003c/em\u003e expression is significantly upregulated during secretory phase of the menstrual cycle thus suggesting its role in physiology of the cyclic endometrial changes. This role remains unknown and requires further investigations. Furthermore, we were unable to reveal any significant and biologically relevant differences in expression of EMT-related genes between normal healthy endometrium and endometrium from endometriosis patients. This observation implies that EMT is not differentially regulated in eutopic endometrium in endometriosis as compared to control women and strongly supports a view that EMT is not actively involved in development of endometriosis at the early stages of the disease. Absence of immunoreactive ZEBs in epithelial and glandular cells may also argue against active EMT. Thus, a significant upregulation of EMT-related genes in ectopic endometrioid lesions that was repeatedly reported by many studies [21,22,22,29\u0026ndash;32,49,66] seems to be rather a secondary phenomenon depending e.g. on the effects of a local peritoneal milieu.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMatarese G, De Placido G, Nikas Y, Alviggi C. Pathogenesis of endometriosis: natural immunity dysfunction or autoimmune disease? Trends in Molecular Medicine. 2003;9:223\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eTomassetti C, Meuleman C, Pexsters A, Mihalyi A, Kyama C, Simsa P, et al. Endometriosis, recurrent miscarriage and implantation failure: is there an immunological link? Reproductive BioMedicine Online. 2006;13:58\u0026ndash;64.\u003c/li\u003e\n \u003cli\u003eRiccio LDGC, Santulli P, Marcellin L, Abr\u0026atilde;o MS, Batteux F, Chapron C. Immunology of endometriosis. 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The transcription factor Snail controls epithelial\u0026ndash;mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000;2:76\u0026ndash;83.\u003c/li\u003e\n \u003cli\u003eKielbik M, Szulc-Kielbik I, Klink M. Snail transcription factors \u0026ndash; Characteristics, regulation and molecular targets relevant in vital cellular activities of ovarian cancer cells. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2024;1871:119705.\u003c/li\u003e\n \u003cli\u003eTang H, Massi D, Hemmings BA, Mandal\u0026agrave; M, Hu Z, Wicki A, et al. AKT-ions with a TWIST between EMT and MET. Oncotarget. 2016;7:62767\u0026ndash;77.\u003c/li\u003e\n \u003cli\u003eHemavathy K, Ashraf SI, Ip YT. Snail/slug family of repressors: slowly going into the fast lane of development and cancer. Gene. 2000;257:1\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eProestling K, Birner P, Balendran S, Nirtl N, Marton E, Yerlikaya G, et al. Enhanced expression of the stemness-related factors OCT4, SOX15 and TWIST1 in ectopic endometrium of endometriosis patients. Reprod Biol Endocrinol. 2016;14:81.\u003c/li\u003e\n \u003cli\u003eBouris P, Skandalis SS, Piperigkou Z, Afratis N, Karamanou K, Aletras AJ, et al. Estrogen receptor alpha mediates epithelial to mesenchymal transition, expression of specific matrix effectors and functional properties of breast cancer cells. Matrix Biology. 2015;43:42\u0026ndash;60.\u003c/li\u003e\n \u003cli\u003eQureshi R, Picon-Ruiz M, Sho M, Van Booven D, Nunes De Paiva V, Diaz-Ruano AB, et al. Estrone, the major postmenopausal estrogen, binds ERa to induce SNAI2, epithelial-to-mesenchymal transition, and ER+ breast cancer metastasis. Cell Reports. 2022;41:111672.\u003c/li\u003e\n \u003cli\u003ePark S-H, Cheung LWT, Wong AST, Leung PCK. Estrogen Regulates Snail and Slug in the Down-Regulation of E-Cadherin and Induces Metastatic Potential of Ovarian Cancer Cells through Estrogen Receptor \u0026alpha;. Molecular Endocrinology. 2008;22:2085\u0026ndash;98.\u003c/li\u003e\n \u003cli\u003eDuncan WC, Shaw JLV, Burgess S, McDonald SE, Critchley HOD, Horne AW. Ectopic Pregnancy as a Model to Identify Endometrial Genes and Signaling Pathways Important in Decidualization and Regulated by Local Trophoblast. Sch\u0026ouml;nbach C, editor. PLoS ONE. 2011;6:e23595.\u003c/li\u003e\n \u003cli\u003eLiang Y-X, Hu W, Jin Z-Y, Diao H-L, Liu L, Yang Y, et al. Nucleolar stress regulates stromal\u0026ndash;epithelial transition via NPM1 during decidualization. Reproduction. 2020;160:491\u0026ndash;500.\u003c/li\u003e\n \u003cli\u003eHuang Z, Mao X, Lin D, Hong Y, Liang G, Chen Q, et al. Establishment and characterization of immortalized human eutopic endometrial stromal cells. American J Rep Immunol [Internet]. 2020 [cited 2025 Jul 22];83. Available from: https://onlinelibrary.wiley.com/doi/10.1111/aji.13213\u003c/li\u003e\n \u003cli\u003eCousins FL, O DF, Gargett CE. Endometrial stem/progenitor cells and their role in the pathogenesis of endometriosis. Best Practice \u0026amp; Research Clinical Obstetrics \u0026amp; Gynaecology. 2018;50:27\u0026ndash;38.\u003c/li\u003e\n \u003cli\u003eOmwandho COA, Konrad L, Halis G, Oehmke F, Tinneberg H-R. Role of TGF- s in normal human endometrium and endometriosis. Human Reproduction. 2010;25:101\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eSoni UK, Chadchan SB, Kumar V, Ubba V, Khan MTA, Vinod BSV, et al. A high level of TGF-B1 promotes endometriosis development via cell migration, adhesiveness, colonization, and invasiveness\u0026dagger;. Biology of Reproduction. 2019;100:917\u0026ndash;38.\u003c/li\u003e\n \u003cli\u003eGregory PA, Bracken CP, Smith E, Bert AG, Wright JA, Roslan S, et al. An autocrine TGF-\u0026beta;/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition. Bronner-Fraser M, editor. MBoC. 2011;22:1686\u0026ndash;98.\u003c/li\u003e\n \u003cli\u003eBurk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S, et al. A reciprocal repression between ZEB1 and members of the miR‐200 family promotes EMT and invasion in cancer cells. EMBO Reports. 2008;9:582\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eFassbender A, Verbeeck N, B\u0026ouml;rnigen D, Kyama CM, Bokor A, Vodolazkaia A, et al. Combined mRNA microarray and proteomic analysis of eutopic endometrium of women with and without endometriosis. Human Reproduction. 2012;27:2020\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eSherwin JRA, Sharkey AM, Mihalyi A, Simsa P, Catalano RD, D\u0026rsquo;Hooghe TM. Global gene analysis of late secretory phase, eutopic endometrium does not provide the basis for a minimally invasive test of endometriosis. Human Reproduction. 2008;23:1063\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eMatsuzaki S, Darcha C, Maleysson E, Canis M, Mage G. Impaired Down-Regulation of E-Cadherin and \u0026beta;-Catenin Protein Expression in Endometrial Epithelial Cells in the Mid-Secretory Endometrium of Infertile Patients with Endometriosis. The Journal of Clinical Endocrinology \u0026amp; Metabolism. 2010;95:3437\u0026ndash;45.\u003c/li\u003e\n \u003cli\u003eFujimoto J, Ichigo S, Hori M, Tamaya T. Alteration of E-cadherin, \u0026alpha;-and \u0026beta;-catenin mRNA expression in human uterine endometrium during the menstrual cycle. Gynecological Endocrinology. 1996;10:187\u0026ndash;91.\u003c/li\u003e\n \u003cli\u003eYun BS, Yun NY, Lee JE, Go M, Jang HY, Park JE, et al. Endometrial E-cadherin and N-cadherin Expression during the Mid-Secretory Phase of Women with Ovarian Endometrioma or Uterine Fibroids. JPM. 2024;14:920.\u003c/li\u003e\n \u003cli\u003eZeitvogel A, Baumann R, Starzinski-Powitz A. Identification of an Invasive, N-Cadherin-Expressing Epithelial Cell Type in Endometriosis Using a New Cell Culture Model. The American Journal of Pathology. 2001;159:1839\u0026ndash;52.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Endometrium, Endometriosis, Menstrual cycle, Epithelial-to-mesenchymal transition (EMT), TGF-β, ZEB, SNAIL, E-cadherin, N-cadherin","lastPublishedDoi":"10.21203/rs.3.rs-7227864/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7227864/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Endometriosis is a common chronic gynaecological disorder related to the presence of ectopic foci of endometrial-like tissue mostly in the pelvic cavity. Pathogenesis of this disease may be associated with epithelial-to-mesenchymal transition (EMT), a phenomenon defined by morphological and functional changes from epithelial to mesenchymal cell phenotype. The role of EMT in development of endometriotic lesions remains poorly understood. There is also little known about a role of EMT in eutopic endometrium in course of the menstrual cycle. Therefore, the present study was aimed at investigating expression of major EMT-related genes of TGF-b, ZEB, SNAIL, CDH and miR200 families in eutopic endometrium of women with and without endometriosis in proliferative and secretory phase of the menstrual cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The study included 46 women with endometriosis and 30 control women without symptoms of the disease. Eutopic endometrial tissue samples were collected during mid-proliferative and mid-secretory phase. Tissue localization of the tested factors was detected by immunohistochemical staining. Expression of specific RNAs was evaluated by quantitative RT-PCR. Differences between groups were determined using the Student’s \u003cem\u003et\u003c/em\u003e-test, Wilcoxon matched-pairs signed rank test or Mann–Whitney \u003cem\u003eU\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e All investigated factors were expressed in eutopic endometrium both at the protein and mRNA level. Comparison of mRNA expression during different cycle phases has revealed a significant upregulation of \u003cem\u003eSNAI2\u003c/em\u003emRNA in the secretory phase in both endometriosis and control group. Secretory phase was also associated with a decreased expression of CDH2 mRNA in the control group. However, similar difference was not revealed in the endometriosis patients. There were no differences in mRNA levels of the other tested EMT-related factors between endometriosis and control group regardless the cycle phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e The present study shows that \u003cem\u003eSNAI2\u003c/em\u003e expression is upregulated during secretory phase of the menstrual cycle thus suggesting its role in physiology of the cyclic endometrial changes. However, our data argue for a limited role of EMT in eutopic endometrium in the pathogenesis of endometriosis. These findings put a new light on the physiology of endometrium and the role of EMT in the pathogenesis of endometriosis.\u003c/p\u003e","manuscriptTitle":"The influence of menstrual cycle and endometriosis on endometrial expression of epithelial-to-mesenchymal transition (EMT)-related genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 08:13:48","doi":"10.21203/rs.3.rs-7227864/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-21T08:48:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T17:53:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T15:20:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296183837351879641104761817482708376296","date":"2025-08-14T22:46:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207360053542255939450701484139559065533","date":"2025-08-04T15:53:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-01T07:30:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T12:50:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-30T05:35:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2025-07-27T18:20:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ad66eb4-2c61-436a-a861-c48d9a0c6232","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:06:23+00:00","versionOfRecord":{"articleIdentity":"rs-7227864","link":"https://doi.org/10.1186/s12958-025-01486-w","journal":{"identity":"reproductive-biology-and-endocrinology","isVorOnly":false,"title":"Reproductive Biology and Endocrinology"},"publishedOn":"2025-11-18 15:58:06","publishedOnDateReadable":"November 18th, 2025"},"versionCreatedAt":"2025-07-30 08:13:48","video":"","vorDoi":"10.1186/s12958-025-01486-w","vorDoiUrl":"https://doi.org/10.1186/s12958-025-01486-w","workflowStages":[]},"version":"v1","identity":"rs-7227864","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7227864","identity":"rs-7227864","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}