{"paper_id":"28d83dbb-67a7-4306-8afc-9fc3dce14fd8","body_text":"Vol.:(0123456789)1 3\nArchives of Gynecology and Obstetrics (2020) 301:1003–1011 \nhttps://doi.org/10.1007/s00404-020-05472-y\nGENERAL GYNECOLOGY\nLocalization of claudin‑2 and claudin‑3 in eutopic and ectopic \nendometrium is highly similar\nAlena Hoerscher1 · Fabian Horné1 · Raimund Dietze1 · Eniko Berkes2 · Frank Oehmke1 · Hans‑Rudolf Tinneberg3 · \nIvo Meinhold‑Heerlein1 · Lutz Konrad1 \nReceived: 18 November 2019 / Accepted: 22 February 2020 / Published online: 5 March 2020 \n© The Author(s) 2020, corrected publication 2021\nAbstract\nPurpose Claudins as the major components of tight junctions are important in maintaining cell–cell integrity and thus \nfunction as a barrier. Dysregulation of the claudins is often associated with loss of the epithelial phenotype, a process called \nepithelial–mesenchymal transition (EMT), which most often results in gain of migrative and invasive properties. However, \nthe role of claudins in the endometrium or endometriosis has only rarely been examined.\nMethods In this study, we investigated localization of claudin-2 and claudin-3 in the eutopic and ectopic endometrium with \nimmunohistochemistry. A detailed quantification with HSCORE was performed for claudin-2 and claudin-3 in endometrium \nwithout endometriosis and in cases with endometriosis compared to the three endometriotic entities: peritoneal, ovarian, \nand deep-infiltrating endometriosis.\nResults We found a preferential localization of both claudins in the glandular and the luminal epithelial cells in the endo-\nmetrium with and without endometriosis. Quantification of localization of both claudins showed no differences in eutopic \nendometrium of control cases compared to cases with endometriosis. Furthermore, both claudins are localized highly similar \nin the ectopic compared to the eutopic endometrium, which is in clear contrast to previously published data for claudin-3.\nConclusion From our results, we conclude that localization of claudin-2 and claudin-3 is highly stable in eutopic and ectopic \nendometrium without any loss of the epithelial phenotype and thus do not contribute to the pathogenesis of endometriosis.\nKeywords Endometrium · Endometriosis · Claudin-2 · Claudin-3\nIntroduction\nEndometriosis is characterized by the presence of endome-\ntrial glands and stroma outside the normal localization, how-\never, irrespective of location, the histological appearance of \nendometriotic glands always resembles uterine endometrial \nglands [1]. Despite the histological similarities, it has been \nsuggested that peritoneal endometriosis, endometriomas \nand deep-infiltrating endometriosis (DIE) are three dis -\ntinct entities, which do not share a common pathogenesis \n[2]. Retrograde menstruation followed by implantation of \nthe endometrial tissue on distinct surfaces most often in \nthe pelvic or abdominal cavity is generally accepted as the \nmain cause of endometriosis [3 ]. Despite the high rate of \nretrograde menstruation ranging from 76 to 90% [4, 5], only \napproximately 0.8–2.0% of women in their reproductive age \nacquire endometriosis as shown in large population-based \nstudies of low-risk patients [6 –10]. This discrepancy sug-\ngests secondary factors like immune dysfunction for the \nestablishment of endometriotic lesions [11].\nClaudins are the major components of tight junctions \n(TJ), and as transmembrane proteins are mostly located in \nthe apicolateral membranes of epithelial and endothelial \ncells [12]. The critical contributions of claudins to TJs are to \nstrand formation and the fence and barrier function, although \nmany other proteins are also involved in the structure of \nTJ complexes. The composition of claudins determines the \nproperties of epithelial barriers such as sealing claudins \n * Lutz Konrad \n lutz.konrad@gyn.med.uni-giessen.de\n1 Department of Gynecology and Obstetrics, Justus Liebig \nUniversity, University of Giessen, Feulgenstr. 10-12, \n35392 Giessen, Germany\n2 Department of Gynecology, UKE Hamburg, Hamburg, \nGermany\n3 Gynecological Hospital, Frankfurt, Germany\n\n Archives of Gynecology and Obstetrics (2020) 301:1003–1011\n1 3\n1004\n(claudin-1, -3, -5, -11, -14, and -18) predominating in tight \nepithelia [13].\nIn the human endometrium expression of claudin-1, -2, \n-3, -4, -5, -7, and 10 was found in contrast to claudin-6, -8, \n-9, -11, -14, and 16–18 [14– 18]. In endometriotic lesions, \nclaudin-3 seems to be downregulated [15, 16] similar to \nclaudin-7 [15], whereas claudin-5 mRNA was decreased \nbut the protein levels increased compared to eutopic endo-\nmetrium [15]. However, as recently published by us, clau-\ndin-7 is not downregulated in endometriosis in contrast to \nsubtle changes in localization of claudin-11 [18]. Of note, \noverexpression of claudin-3 markedly inhibited migration, \ninvasion and epithelial–mesenchymal transition (EMT) of \nlung squamous cell carcinoma [19].\nSimilarly, mRNA expression and localization of clau-\ndin-4 in eutopic and ectopic endometrium were described \ncontroversially. Whereas some researchers found downregu-\nlated mRNA and protein expression of claudin-4 in ectopic \nendometrium [16], others demonstrated no differences [15] \nor even an increased mRNA expression in ectopic lesions \n[20]. In endometrial cancer, downregulated claudin-7 has \nbeen described to be associated with increased proliferation \nand metastasis [21].\nDuring the menstrual cycle, an increased abundance of \nclaudin-1 and -5 protein was described for the secretory \nphase [14]. In contrast, claudin-2, -3 and -4 were similarly \nabundant throughout the menstrual cycle [14, 16, 17, 22] \nwith the exception that claudin-4 mRNA was significantly \nhigher expressed in the secretory phase compared to the \nproliferative phase [14]. Intriguingly, claudin-3 protein was \nexpressed in the endothelial cells of the decidual vessels \nwith a possible role in decidual angiogenesis [17]. Progester-\none but not estradiol induced claudin-1, -3, -4, and -7 protein \nexpression in human endometrial epithelial cells resulted in \ndownregulation of the barrier, but not fence function [23].\nThe loss of epithelial cell-to-cell contacts is considered \nto be one of the hallmarks of EMT, which was suggested to \nbe involved also in the pathogenesis of endometriosis [24]. \nRecently, we showed no decrease in claudin-7 and only \nsubtle changes in the localization of claudin-11 in ectopic \nendometrium and suggested that only a partial EMT might \nbe involved in the pathogenesis of endometriosis [18]. Thus, \nin this study, we examined claudin-2 and claudin-3 expres-\nsion in eutopic and ectopic endometrium to further clarify \nthe role of cell-to-cell contacts in endometriosis.\nMaterials and methods\nPatients\nThis study has been approved by the Ethics Committee \nof the Medical Faculty of the Justus-Liebig-University, \nGiessen, Germany (registry number 95/09). The participants \ngave written informed consent. All specimens (Table 1) were \nobtained by hysterectomy (uteri, n = 51 patients) or laparos-\ncopy (endometriotic tissues, n = 55 with n = 59 lesions) from \npatients mainly suffering from pain. The intraoperative find-\nings were classified according to the revised American Soci-\nety for Reproductive Medicine score (rASRM) and ENZIAN \nscore [25]. Dating of the endometrial tissue was based on the \ndates of the last menstrual period and histological evaluation \nby the pathologist. Although the pathogenesis and definition \nof DIE is still highly unclear [26], we rely for classification \non MRI images and the ENZIAN score [25] which classifies \nDIE during operation.\nSpecimens were fixed in Bouin’s solution (and partly in \nformaldehyde for the histological evaluation by the patholo-\ngist) and embedded in paraffin. After staining 5 µm sections \nwith hematoxylin and eosin, the histological evaluation was \nperformed.\nImmunohistochemical analysis and quantification\nSerial sections of 5 µm were cut to ensure that in most cases, \nthe same lesions could be examined. Immunohistochemis-\ntry (IHC) of bouin-fixed or formalin-fixed specimens was \nperformed as published previously [27]. The EnVision Plus \nSystem (cat-no K4002, DAKO, Hamburg, Germany) was \nused according to the manufacturer’s instructions. Briefly, \nantigen retrieval was performed with a citrate buffer (pH \n6, DAKO) and then the jars containing the slides were put \ninto a steamer (Braun, Multi Gourmet) at 100 °C for 20 min \nand remained in the steamer for cooling for 20 min. Pri-\nmary antibodies against claudin-2 (diluted 1:200, cat-no \n32-5600, Thermo Fisher, Waltham, MA, USA) or claudin-3 \n(diluted 1:100, cat-no 34-1700, Invitrogen, Waltham, MA, \nUSA) were added and incubation was done in a humidi-\nfied chamber overnight at 4 °C. After washing with PBS, \nincubation with the appropriate secondary antibody (cat-no \nK4002, DAKO) was done for 30 min at room temperature. \nThe staining was visualized with diaminobenzidine (Liq -\nuid DAB K3467, DAKO). Counterstaining was performed \nwith Mayer’s hematoxylin (Waldeck, Germany) and after \ndehydration in ethanol, slides were mounted with Eukitt. \nNegative controls for IHC were prepared by omission of \nthe primary antibody. Digital images were obtained with \nLeica DM 2000/Leica MC170/Leica application suite LAS \n4.9.0 and then processed with Adobe Photoshop CS6. IHC \nquantification was done by use of the HSCORE (0, no stain-\ning; 1 + , weak, but detectable; 2 + , moderate or distinct; \n3 + , intense) which was calculated for each tissue by sum-\nming the percentages of cells grouped in one intensity cat-\negory and multiplying this number with the intensity of the \nstaining. All glands or cysts were used for evaluation of the \nHSCORE.\n\nArchives of Gynecology and Obstetrics (2020) 301:1003–1011 \n1 3\n1005\nStatistics\nAll values are presented as mean ± standard error of the \nmean (SEM) or median. HSCORE values between the differ-\nent groups were analysed using one-way analysis of variance \n(ANOVA). Then, comparison between more than two groups \nwas done with the test of Kruskal–Wallis. P values ≤  0.05 \nwere considered to be significant. GraphPad Prism 6.01 \n(www. graph pad. com) was used for the statistics.\nResults\nAnalysis of claudin-2 in patients with and without endome-\ntriosis showed a preferential apical localization for glandu-\nlar and luminal eutopic epithelial cells in the majority of \nepithelial cells and glands in both the proliferative and the \nsecretory phase (Fig.  1a−d). We found a high similarity of \nthe HSCORE between patients with and without endome-\ntriosis as well as proliferative and secretory phases (Table 2).\nPositivity for claudin-2 was further identified in the \nmajority of endometriotic epithelial cells and lesions irre-\nspective of the three endometriotic entities: ovarian (Fig. 2a), \nperitoneal (Fig.  2b) or deep-infiltrating endometriosis \n(Fig.  2c). As the HSCORE showed no significant differences \nbetween eutopic endometrium with and without endometrio-\nsis (Table  2), we merged both datasets and found no signifi-\ncant differences between the eutopic and ectopic endome-\ntrium (Table  3).\nLocalization of claudin-3 in patients with and without \nendometriosis was strong in the membranes of glandular \neutopic epithelial cells, nearly approaching 100% of all epi-\nthelial cells in both the proliferative and the secretory phase \n(Fig.  3a–d). A strong membrane localization was also found \nin the luminal cells (Fig. 3e), whereas the negative control of \nthe same patient showed no staining (Fig. 3f). Quantification \nof the staining showed a high similarity of the HSCORE \nbetween patients with and without endometriosis as well as \nproliferative and secretory phases (Table  2).\nPositivity for claudin-3 was further identified in almost all \nectopic endometriotic epithelial cells and lesions irrespective \nof the three endometriotic entities: ovarian (Fig.  4a), peri-\ntoneal (Fig.  4b) or deep-infiltrating endometriosis (Fig.  4c). \nAs the HSCORE showed no significant differences between \neutopic endometrium with and without endometriosis \n(Table  2), we merged both datasets and found no significant \ndifferences between the eutopic and ectopic endometrium \n(Table  3).\nTable 1  Overview of the tissue \nsamples used for claudin-3\ne.g. n = 19 (20) means 20 lesions from 19 patients\nlig, ligament; DIE, deep-infiltrating endometriosis\nTissues Endometrium Ovarian\nendometriosis\nPeritoneal endome-\ntriosis\nDIE\nAll samples n = 51 n = 19 (20) n = 17 (18) n = 19 (21)\nMedian age ± SD 43 ± 7.1 33 ± 4.0 33 ± 4.2 32 ± 5.2\nProliferative (median age) n = 23 (44 ± 8.5)\nSecretory (median age) n = 28 (42 ± 5.9)\nLeiomyoma n = 28\nAdenomyosis n = 10\nBladder 2 1\nUterosacral lig 1 3\nOvarian fossa 3\nPouch of Douglas 3\nRound lig of uterus 1\nPeritoneum 1\nPelvic wall 2\nRectum 7\nRectosigmoid 2\nRectovaginal septum 1 4\nParaurethral 1 1\nSigmoid colon 1 1\nVagina 1\nIntestine 1\nMesovarium 1\nLig latum uteri 1\n\n Archives of Gynecology and Obstetrics (2020) 301:1003–1011\n1 3\n1006\nDiscussion\nIn this study, we analyzed epithelial cells of eutopic endo-\nmetrial glands and endometriotic lesions in the ovary, \nperitoneum, and DIE with claudin-2 and claudin-3. Our \nresults demonstrate convincingly that both claudins are \nubiquitously expressed in both the eutopic as well as the \nendometriotic lesions with a highly similar pattern and \nabundance.\nClaudin-2 is expressed in epithelial layers with high \nparacellular permeability such as proximal tubules and the \nintestine [28]. Mostly, an overexpression of claudin-2 in \ngynecological tumors has been observed [29]. In the human \nendometrium, claudin-2 is expressed similarly in the prolif-\nerative and secretory phase [14] as described in our study. \nFurthermore, we identified a stable expression of claudin-2 \nalso in the ectopic endometrium compared to the eutopic \nendometrium which is according to our knowledge, a new \nfinding. The observed preferential localization in the apical \nregion is similar to the apical localization in intestinal cell \norganoids [30].\nFig. 1  Representative microphotographs of claudin-2 in prolifera-\ntive (a) and secretory (b) endometrium without endometriosis and in \nproliferative (c) and secretory (d) endometrium with endometriosis. \nSimilarly, the luminal cells are also stained (a). Most samples showed \na preferential apical staining. An example of a negative control can be \nfound in Fig. 3f. Scale bars 100 µm, magnification × 200\nTable 2  Comparison of localization of claudin-2 and claudin-3 in \nendometrium with and without endometriosis by the HSCORE\nns, not significant\nEndometrium without \nendometriosis\nEndometrium with \nendometriosis\nProliferative Secretory Proliferative Secretory\nClaudin-2\n N (median age) 6 (45.5) 6 (46.5) 6 (32.5) 8 (42)\n Mean 165 187 193 185\n SEM 22.2 24.2 9.5 6.0\n P ns ns ns\nClaudin-3\n N (median age) 9 (46) 10 (40.5) 14 (41.5) 18 (42.5)\n Mean 268 253 275 265\n SEM 11.2 12.0 8.5 9.4\n P ns ns ns\n\nArchives of Gynecology and Obstetrics (2020) 301:1003–1011 \n1 3\n1007\nIn the human endometrium with endometriosis, an \nimpaired expression of claudin-3, -5 and -7 has previ-\nously been identified [15]. In another study, claudin-3 \nand claudin-4 have been described to be downregulated in \nectopic endometrium [16]. Both suggested that the downreg-\nulation of various members of the claudin family might con-\ntribute to endometrial cell detachment and increase the num-\nber of cells invading pelvic organs [15, 16]. These data are \nin clear contrast to our results which did not show any dys-\nregulation of claudin-3 in endometriotic lesions. However, \nin our study, we demonstrated localization of claudin-3 in \nthe endometrial luminal epithelial cells which is in accord-\nance to observations by Schumann et al. [17], whereas in the \nother studies, it was not mentioned [14– 16]. Furthermore, \nwe recently could not confirm downregulation of claudin-7 \nin endometriotic lesions as published by Gaetje et al. [15], \nbut found a moderate non-significant increased presence in \nperitoneal and DIE lesions instead [ 18]. Although we sup -\nposed that this might be due to different detection systems \nand antibodies used, we found that tissue fixation with Bouin \nwas superior to formalin in some cases of claudin detection \n(unpublished observation).\nIn numerous types of human cancer, the number of \ncell–cell junctions decreases, permitting the escape of can-\ncer cells from their primary sites, along with the acquisition \nFig. 2  Representative microphotographs of claudin-2 in ovarian \nendometriosis (a), peritoneal endometriosis (b, bladder), and DIE \n(c, rectovaginal septum). In Fig.  2b, the preferential apical staining \nis best visible. PE, peritoneal endometriosis; DIE, deep-infiltrating \nendometriosis. Scale bars 100 µm, magnification × 200\nTable 3  Comparison of localization of claudin-2 and claudin-3 in \neutopic and ectopic endometrium by the HSCORE\nN = number of lesions; EM, endometriosis; ns, not significant\nEndome-\ntrium\nOvarian EM DIE Peritoneal EM\nClaudin-2\n N (median \nage)\n26 (42) 6 (33) 6 (35.5) 7 (33)\n Mean 183 179 209 220\n SEM 7.8 25.1 25.2 31.2\n P ns ns ns\nClaudin-3\n N (median \nage)\n51 (43) 20 (33) 25 (32) 17 (32)\n Mean 266 266 286 263\n SEM 5.0 8.6 4.6 9.5\n P ns ns ns\n\n Archives of Gynecology and Obstetrics (2020) 301:1003–1011\n1 3\n1008\nof invasive and metastatic properties [31, 32]. However, in \nendometrioid endometrial cancer, an upregulation of clau -\ndin-2 [29] as well as of claudin-3 [29, 33–35] was described. \nThus, it remains unclear why in endometriosis, a downregu-\nlation of claudin-3 [15, 16] should contribute to the dissemi-\nnation of endometrial cells.\nEMT is a biological process characterized by two hall-\nmarks, loss of the epithelial phenotype and gain of properties \nof mesenchymal cells [ 31, 32, 36]. This process requires a \nseries of complex changes in cell architecture and behavior \nwhich is driven by various cellular signals. The molecu-\nlar changes of this transition include the loss of epithelial \nmarkers such as E-cadherin, keratins, desmoplakin, mucin-\n1, occludin and claudins and gain of mesenchymal mark -\ners like N-cadherin, α-smooth muscle actin, vimentin and \nfibronectin. Alterations of these pathways are associated \nwith enhanced migration, invasiveness and resistance to \napoptosis [31, 32, 36].\nFig. 3  Representative microphotographs of claudin-3 in proliferative \n(a) and secretory (b) endometrium without endometriosis and in pro-\nliferative (c) and secretory (d) endometrium with endometriosis. In \nall samples, a strong membranous staining is visible. Similarly, the \nluminal cells are also stained (e); the negative control showed no \nstaining (f). Scale bars 100 µm, magnification × 200\n\nArchives of Gynecology and Obstetrics (2020) 301:1003–1011 \n1 3\n1009\nRecently, EMT was described to be involved in endo-\nmetriosis [24, 37]. Although endometriotic cells share \nsome similarities with metastatic tumor cells in terms of \ndissemination and invasion [24], endometrial cells do not \nnecessarily need to undergo EMT, as menstruation allows \na physiological detachment of cells. Additionally, irrespec-\ntive of the location of endometriotic lesions such as in the \novary, peritoneum, or DIE, ectopic endometriotic glands \nnearly always have an overtly endometrioid appearance and \nhistologically resemble uterine endometrial glands, clearly \nindicating no loss of the epithelial phenotype [1 ]. Recently, \nwe observed no loss of the endometrial epithelial phenotype \nas defined by expression of keratins and claudins in endome-\ntriosis and suggested that if at all only a partial EMT might \nbe involved in the pathogenesis of endometriosis [ 18, 27]. \nThe current study further corroborates this assumption by \nshowing a stable expression of claudin-2 and claudin-3 in \neutopic and ectopic endometrium.\nAlthough many authors stress the differences of eutopic \nand ectopic endometrial cells, however, we and others [38] \nbelieve that these differences may be explained as a direct \nconsequence of the different environments, such as the \nperitoneal fluid and the intraovarian microenvironment of \nthe lesions, in relation to the intrauterine environment. \nWe suggest that the changes in the eutopic endometrium \nat the beginning of the disease are quite subtle and that the \nmajority of differences can be observed after the implan-\ntation. Based on our observations about the similarities \nbetween eutopic and ectopic endometrial epithelial and \nstromal cells [27, 39], we propose to focus on the interplay \nof endometrial cells with, for example, peritoneal cells at \nthe site of implantation for future research on the patho-\ngenesis of endometriosis [40].\nIn summary, in our study, we could clearly show no loss \nof cell-to-cell contacts characterized by a stable localiza-\ntion of claudin-2 and claudin-3 in epithelial cells of both \neutopic and ectopic endometrium. Thus, the epithelial phe-\nnotype is definitely not lost and only a partial EMT might \ncontribute to the pathogenesis of endometriosis.\nAcknowledgements We thank Cornelia Hof and Ursula Schneider for \ntechnical assistance and the medical staff of the Dept. of Gynecology \nand Obstetrics for data and sample collection.\nFig. 4  Representative microphotographs of claudin-3 in ovarian \nendometriosis (a), peritoneal endometriosis (b, Douglas), and DIE (c, \nrectum). In all samples, a strong membranous staining is visible. PE, \nperitoneal endometriosis; DIE, deep-infiltrating endometriosis. Scale \nbars 100 µm, magnification × 200\n\n Archives of Gynecology and Obstetrics (2020) 301:1003–1011\n1 3\n1010\nAuthor contributions AH, FH: performed all experiments, RD: pro-\ntocol/project development/Manuscript writing, EB, FO, HRT, IMH: \noperations and collection of patient samples, LK: project/protocol \ndevelopment, data analysis, statistics, manuscript writing.\nFunding Open Access funding enabled and organized by Projekt \nDEAL. Funder name: Justus-Liebig-Universität Gießen (3114).\nCompliance with ethical standards \nConflict of interest The author(s) declared no potential conflicts of in-\nterest with respect to the research, authorship, and/or publication of \nthis article.\nEthical approval This study has been approved by the Ethics Com-\nmittee of the Medical Faculty of the Justus-Liebig-University, Gies-\nsen, Germany (registry number 95/09). The participants gave written \ninformed consent.\nOpen Access This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article’s Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a \ncopy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\nReferences\n 1. 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Hum Reprod \nUpdate 19:558–569\nPublisher’s Note Springer Nature remains neutral with regard to \njurisdictional claims in published maps and institutional affiliations.","source_license":"CC0","license_restricted":false}