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

Immunostaining of the tested proteins was performed on samples of eutopic endometrium from control and endometriosis groups, both in the proliferative and secretory phases of the menstrual cycle. The results of immunolocalization of investigated proteins were similar regardless of the investigated group and phase of the cycle. Therefore, we decided to show representative images from control eutopic endometrium from the secretory phase. As seen in Fig.  1 , 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. Fig. 1 Immunohistochemical localization of ZEB-1 and ZEB-2 expression in eutopic endometrium at secretory phase from a representative control woman. Original magnification x200 Immunohistochemical localization of ZEB-1 and ZEB-2 expression in eutopic endometrium at secretory phase from a representative control woman. Original magnification x200 On the contrary, a relatively strong reaction was found in the nuclei of epithelial and glandular cells in the case of SNAI1 and SNAI2 (Fig.  2 ). SNAI1 and SNAI2 immunoreactivity was also noted in the nuclei of endometrial stromal cells. Fig. 2 Immunohistochemical localization of SNAI1 and SNAI2 expression in eutopic endometrium at secretory phase from a representative control woman. Original magnification x200 Immunohistochemical localization of SNAI1 and SNAI2 expression in eutopic endometrium at secretory phase from a representative control woman. Original magnification x200 Expression of CDH1 was seen only in epithelial cells (Fig.  3 ). 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 a sparse positive reaction was also seen in some endometrial stromal cells (Fig.  3 ). N-cadherin reactivity pattern in epithelial cells was like that of E-cadherin. Fig. 3 Immunohistochemical localization of CDH1 and CDH2 expression in eutopic endometrium at the secretory phase from a representative control woman. Original magnification x200 Immunohistochemical localization of CDH1 and CDH2 expression in eutopic endometrium at the secretory phase from a representative control woman. Original magnification x200 Expression of mRNA and miRNA specific for all studied EMT-related genes was detected in all endometrial tissue samples from both control women and women with endometriosis. The results of semiquantitative mRNA evaluations of all studied EMT-related genes in the control group and endometriosis patients are summarized in Tables  2 and 3 , respectively. As seen in Table  2 , endometrium from control women expressed ca. ten times more TGFB1 than TGFB2 mRNA during the proliferatory phase of the cycle. Expression of TGFB1 mRNA was also few times higher in the secretory phase; however, this difference did not reach statistical significance due to a high result variability. Expression of SNAI2 mRNA was 10–20 times higher than the SNAI1 gene, and this difference was highly statistically significant, irrespective of the phase of the menstrual cycle. There were no significant differences in expression between the ZEB1 and ZEB2 genes. Expression of the CDH1 gene was about 3–6 times higher than CDH2 . An increased miRNA MIR200C to MIR200B gene expression ratio was observed only during the secretory phase of the endometrial cycle (Table  2 ). Table 2 Comparisons of expression levels of related EMT-associated gene mRNAs and MiRNAs in endometrium of healthy women Gene Proliferatory phase Secretory phase N Median (range) P N Median (range) P TGFB1 13 0.0474 (0.029–0.1768) 0.0005 16 0.0359 (0.0096–0.0718) ns TGFB2 13 0.0042 (0.0003–0.0544) 16 0.0053 (0.0002–0.1340) ZEB1 14 0.0292 (0.0156–0.1250) ns 16 0.0372 (0.0168–0.0825) ns ZEB2 14 0.0388 (0.0127–0.1340) 16 0.0222 (0.0118–0.0884) SNAI1 11 0.0025 (0.0015–0.0136) 0.0010 16 0.0024 (0.0002–0.0118) < 0.0001 SNAI2 11 0.0254 (0.0111–0.0670) 16 0.0583 (0.0180–0.4061) CDH1 11 0.1806 (0.0625–1.2510) 0.0049 16 0.0947 (0.0385–0.9659) 0.0002 CDH2 11 0.0583 (0.0059–0.2679) 16 0.0151 (0.0026–0.0625) MIR200B 12 19.31 (0.06–2048.00) ns 11 24.25 (1.41–724.10) 0.0020 MIR200C 12 58.55 (0.57–9410.00) 11 34.30 (3.03–5943.00) All results are shown as medians (range) of relative mRNA expression (2 -ΔCt ). N, number of cases. P -values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant. Comparisons of expression levels of related EMT-associated gene mRNAs and MiRNAs in endometrium of healthy women All results are shown as medians (range) of relative mRNA expression (2 -ΔCt ). N, number of cases. P -values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant. Relative mRNA expression pattern in samples from endometriosis patients (Table  3 ) followed that observed in the control group. The ratio of the expression levels of TGFB1 and TGFB2 genes was elevated in both phases of the menstrual cycle, and a similar pattern was observed for SNAI2 relative to the SNAI1 gene (Table  3 ). No evident differences in ZEB1 to ZEB2 expression levels were detected in samples from the endometriosis group. Predominant CDH1 gene expression relative to CDH2 was also observed in this group. The gene expression ratio for MIR200B and MIR200C in the endometriosis group was similar to that observed in control subjects (Table  3 ). Table 3 Comparisons of expression levels of related EMT-associated gene mRNAs and MiRNAs in the endometrium of women with endometriosis Gene Proliferatory phase Secretory phase N Median (range) P N Median (range) P TGFB1 16 0.0372 (0.0206–0.0625) 0.0005 27 0.0412 (0.0156–0.1649) 0.0032 TGFB2 16 0.0040 (0.0002–0.0146) 27 0.0063 (0.0007–0.1649) ZEB1 16 0.0263 (0.0059–0.0412) ns 30 0.0323 (0.0073–0.1539) ns ZEB2 16 0.0282 (0.0136–0.0583) 30 0.0313 (0.0090–0.1340) SNAI1 14 0.0024 (0.0011–0.0136) 0.0010 26 0.0046 (0.0005–0.0313) < 0.0001 SNAI2 14 0.0313 (0.0032–0.1166) 26 0.0544 (0.0180–0.1649) CDH1 14 0.1091 (0.0313–0.4665) 0.0049 25 0.1253 (0.0335–0.8123) 0.0006 CDH2 14 0.0545 (0.0048–0.1895) 25 0.0474 (0.0042–0.3078) MIR200B 12 17.19 (0.08–1552) ns 26 1.71 (0.12–1261) 0.0163 MIR200C 12 27.57 (0.44–8192.00) 26 8.29 (0.00–9410.00) All results are shown as medians (range) of relative mRNA expression (2 -ΔCt ). N, number of cases. P -values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant. Comparisons of expression levels of related EMT-associated gene mRNAs and MiRNAs in the endometrium of women with endometriosis All results are shown as medians (range) of relative mRNA expression (2 -ΔCt ). N, number of cases. P -values were computed by Wilcoxon matched-pairs signed rank test. ns, not significant. Changes in specific expression of endometrial mRNA and miRNA for the investigated EMT-related genes between the proliferative and secretory phases of the menstrual cycle in control women are shown in Fig.  4 . As seen, the 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. Fig. 4 Expression 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 U -test. ns, not significant Expression 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 U -test. ns, not significant Fig. 5 Expression 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 U -test. ns, not significant Expression 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 U -test. ns, not significant A significant 2-fold upregulation of SNAI2 mRNA expression in the secretory phase was also observed in the endometriosis group (Fig.  5 ). Furthermore, a decreased expression was also noted in the case of both MIR200B and MIR200C genes. Expression of the MIR200B gene was reduced tenfold, and expression of MIR200C was over 3 times lower compared to the proliferatory phase; however, due to a great variability of the results, this difference did not reach statistical significance. There were no differences in the expression of other studied genes. As Fig.  6 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. Fig. 6 Comparison of expression of EMT-related gene mRNAs and miRNAs between eutopic endometrium from the healthy control group and eutopic endometrium from the 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 the Mann-Whitney U -test. ns, not significant Comparison of expression of EMT-related gene mRNAs and miRNAs between eutopic endometrium from the healthy control group and eutopic endometrium from the 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 the Mann-Whitney U -test. ns, not significant In the secretory phase (Fig.  7 ), the level of CDH2 mRNA was 3-fold higher in the endometrium of women with endometriosis as compared to the control group, and this difference was statistically significant. A statistically significantly higher expression of the SNAI1 gene was also observed in the endometriosis group; however, this difference was at the borderline significance. There were no differences in the level of expression of all other investigated EMT-related genes. Fig. 7 Comparison 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 U -test. ns, not significant Comparison 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 U -test. ns, not significant

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 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 [ 40 ]. 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. Control subjects did not present any symptoms typical of endometriosis, such as pelvic pain, and their routine transvaginal ultrasound examination yielded negative results. 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 before 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 the 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 Demographic and clinical characteristics of endometriosis patients and the control group 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 Pipelle ® 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™ 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. 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 were cut into 5 µm 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 the respective primary antibody for 60 min at room temperature. All primary antibodies, their specification, origins, and working dilutions are listed in Supplementary Table 1. Primary antibodies were detected by incubation with Novolink™ Polymer Detection Reagent (Leica Biosystems, RE7140-CE) for 30 min at room temperature according to the manufacturer’s instructions. The peroxidase reaction was visualized using the DAB chromogen provided in the kit. 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 s. 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 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min. cDNA samples were stored at −20 °C until qRT-PCR assays were performed. 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 mRNA probes used in this study is shown in Supplementary Table 2. 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 s and 60 °C for 1 min. Each sample was run in triplicate. GAPDH and RNU43 served as endogenous controls 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 ΔCt method, and the results are presented as relative units calculated from the 2 − ΔCt formula. 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.

To our best knowledge, this is the first study aimed at evaluating EMT-related gene expression in the 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 [ 24 , 32 , 41 ]. 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 . It should be stressed, however, that the difference between TGFB1 and TGFB2 in the secretory phase in the control group did not reach statistical significance, probably due to the very high range of expression of the latter one. 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 [ 35 , 42 ]. 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 [ 24 , 41 ]. 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 [ 43 – 45 ]. However, putative factors that may be responsible for the regulation of EMT/MET endometrial balance remain obscure. To address this point, we evaluated changes of mRNA levels of EMT-related genes in the endometrium during proliferatory and secretory phases 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 the SNAI2 gene. Similar upregulation of SNAI2 mRNA was also found in eutopic endometrium from women with endometriosis which strongly implies that SNAI2 may indeed play some role in physiological endometrial changes in the 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 [ 46 – 49 ]. Both SNAI1 and SNAI2 can stimulate EMT by repression of E-cadherin transcription in epithelial cells [ 48 – 50 ]; however, unlike the members of the 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 [ 48 , 51 ]. Accordingly, it is tempting to speculate that upregulated SNAI2 expression may contribute to extensive growth and maturation of endometrial glands and stroma during the secretory phase. This, however, needs further elucidation. It is also not clear what the mechanism is of increased expression of SNAI2 expression during the 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 [ 50 ]. Accordingly, expression of TWIST has also been reported in normal endometrium and endometrium from women with endometriosis [ 32 , 52 ] thus, it is plausible that it may play a part in the regulation of endometrial SNAIs expression. Changes in SNAI2 expression and EMT/MET might also be dependent on menstrual cycle-associated hormonal changes. Many reports indicate a role of 17-β-oestradiol (E2) in the induction of EMT and EMT-related factors, especially in hormone-dependent tumours [ 53 – 55 ]. It has also been claimed that E2 may play a part in the induction of EMT in the course of endometriosis via upregulation of the β-catenin/SNAIL pathway [ 41 ]. Therefore, some role of oestrogens in the 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 [ 56 ]. WNT/β‐catenin signalling is known to downregulate E-cadherin expression and stimulate cell migration via activation of SNAIs [ 50 ], which 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 [ 57 ], and a similar phenomenon was also observed in in vitro model of decidualization following stromal cell treatment with progesterone and cAMP [ 58 ]. 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 , 59 ]. 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 a 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 the 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 phases. This strongly suggests that the menstrual cycle has no important effect on the phenomena that may be mediated by these EMT-related factors. TGF-β is considered one of the most important factors participating in the pathogenesis of endometriosis [ 60 , 61 ]. In particular, TGF-β is considered a principal inducer of different EMT mechanisms, including TGF-β/ZEB/miR200 loop [ 37 , 39 , 62 , 63 ] or TGF-β/TWIST/SNAIL pathway [ 28 , 31 ]. Lack of significant changes in TGFB1/TGFB2 expression profile may suggest that these pathways are not involved in physiological changes of the 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 a 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 the already discussed difference in CDH2 gene expression, there were also some differences in TGFB1 and SNAI1 expression, respectively, in proliferatory and secretory phases. 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 a few other studies based on immunohistochemical evaluations [ 26 ] and microarray analyses performed on the secretory endometrium [ 64 , 65 ]. Moreover, no differences were observed in the level of CDH1 mRNA expression in the endometrium from infertile endometriosis patients compared to healthy controls [ 66 ]. 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 [ 67 ]. 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 [ 68 ]. Present data argue for a limited role of EMT in eutopic endometrium in the pathogenesis of endometriosis and suggest that hypothetical endometrial epithelial cells that underwent a transition into mesenchymal-like cells are unlikely to be responsible for the 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 the secretory phase of the menstrual cycle, thus suggesting its role in the physiology of the cyclic endometrial changes. This role remains unknown and requires further investigation. Furthermore, we were unable to reveal any significant and biologically relevant differences in the 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 the view that EMT is not actively involved in the 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 [ 24 , 25 , 32 – 35 , 52 , 69 ] seems to be rather a secondary phenomenon, depending e.g., on the effects of a local peritoneal milieu.

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 an autoimmune disorder [ 1 – 4 ]. It may manifest as dysmenorrhea and pelvic pain and is considered one of the major causes of female infertility [ 5 , 6 ]. Endometriosis affects ca. 10% of women of 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 the retrograde flow of endometrial cells shed in the course of menstruation [ 9 ]. Appearance of distant lesions, such as in the lung, heart, liver, or brain may be due to dissemination by the 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 abrogated elimination by the cells of the local immune surveillance system [ 15 ]. Lesion formation may be also facilitated by a decreased susceptibility to apoptotic cell death [ 16 – 18 ], an increased adhesiveness and invasiveness [ 16 , 19 , 20 ]. The latter one may involve collective cell migration that has been described both in endometriosis [ 21 ] and adenomyosis [ 22 , 23 ]. Pathogenesis of endometriosis may also involve a phenomenon of epithelial-to-mesenchymal transition (EMT) [ 24 – 26 ]. EMT is defined as a process characterized by morphological and functional changes from epithelial to mesenchymal cell phenotype [ 27 , 28 ]. Typically, epithelial cells lose their polarized phenotype and intercellular connections and acquire a fibroblastic shape with enhanced migratory and invasive potential. These changes are related to downregulation of E-cadherin and increased N-cadherin expression [ 27 , 28 ]. EMT appears to be one of the fundamental mechanisms involved in the development of multicellular organisms, playing a role in embryo- and morphogenesis [ 27 , 28 ]. On the other hand, EMT appears to be crucial for a variety of pathological phenomena, including fibrosis and tumour invasion and metastasis [ 29 – 31 ]. 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 [ 24 , 32 – 35 ]. It has also been suggested that EMT in endometriotic cells may also depend on abrogated expression of miRNAs of the miR200 family, which are major regulators of ZEB expression [ 36 – 39 ]. However, the exact role of EMT in the development of endometriotic lesions remains poorly understood. There is also little known about the expression and a putative role of EMT-related genes in eutopic endometrium in the course of the menstrual cycle. Therefore, the present study was aimed at investigating the expression of major EMT-related genes coding for the members of TGF-β, ZEB, SNAIL, CDH, and miR200 family in eutopic endometrium of women with and without endometriosis in the proliferative and secretory phase of the menstrual cycle.

Supplementary Material 1 Supplementary Material 1