{"paper_id":"49be5447-8f9a-47f9-b0a6-22670112f472","body_text":"ARTICLE IN PRESS\nhttps://doi.org/10.1038/s42003-026-10548-7\nReceived: 29 September 2025\nAccepted: 16 June 2026\nCite this article as: Gargaros, A.,\nRusidzé, M., Singla, P . et al.\nComprehensive transcript and\nprotein profiling reveals ERα rather\nthan ERβ as the predominant\nestrogen receptor in human\nendometriotic lesions. Commun Biol\n(2026). https://doi.org/10.1038/\ns42003-026-10548-7\nAdrien Gargaros, Mariam Rusidzé, Perrine Singla, Nathalie Van Acker, Ariane Weyl,\nAurélie Buffeteau , Claire Illac-Vauquelin, Krystyna Boriak, Philippe Lluel, Jean-François\nArnal, Coralie Fontaine, Lilian Basso, Élodie Chantalat & Françoise Lenfant\nWe are providing an unedited version of this manuscript to give early access to its\nfindings.  Before final  publication, the manuscript will undergo further editing. 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To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.\nCommunications Biology\nArticle in Press\nComprehensive transcript and protein profiling\nreveals ERα rather than ERβ as the predominant\nestrogen receptor in human endometriotic lesions\n\n\nARTICLE IN PRESS\nARTICLE IN PRESS\nComprehensive transcript and protein profiling reveals ERα rather than ERβ as the predominant \nestrogen receptor in human endometriotic lesions \nAdrien Gargaros1, Mariam Rusidze1*, Perrine Singla1*, Nathalie Van Acker2*,  Ariane Weyl1,3*, Aurélie \nBuffeteau1, Claire Illac-Vauquelin4, Krystyna Boriak1,5, Philippe Lluel6, Jean-François Arnal1, Coralie \nFontaine1, Lilian Basso7, Élodie Chantalat1,3, Françoise Lenfant1$\n1 INSERM U1297 - I2MC – Institut des maladies métaboliques et cardiovasculaires, Équipe EstER, Université de Toulouse , Toulouse, France. \n2Imag’IN Platform, Department of Pathology, CHU, IUCT-Oncopôle, Toulouse, France \n3 Département de chirurgie gynécologique, CHU Toulouse Rangueil, Toulouse, France \n4Department of Pathology, Claudius Regaud Institute, IUTC Oncopôle, Toulouse, France \n5 Poltava State Medical University, Poltava, Ukraine \n6Urosphere, Rue des Satellites, 31400 Toulouse, France \n7  INSERM U1291- INFINITY-Intitute for Infectious and Inflammatory Diseases -CNRS UMR5051, University of Toulouse, Toulouse, France. \n* Contributed equally\n$Corresponding Author \nFrançoise LENFANT, INSERM/UPS UMR 1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires \n1 avenue Jean Poulhes, BP 84225, 31432 Toulouse Cedex 4, France. Tel: +33 5 31 22 40 98 \nE-mail:  Francoise.lenfant@inserm.fr\nABSTRACT :  \nEndometriosis (EMS) is a chronic estrogen -dependent inflammatory disease. Although several studies \nhave suggested a key role for estrogen receptor ERβ in EMS lesion development, its detection has been \nchallenged by the lackof specificity of many ERβ antibo dies. To clarify the status of sex steroid receptors \nin the endometrium and matched EMS lesions , we perform RNAScope and immunohistochemistry  on a \ntissue microarray cohort, mapping the expression of estrogen receptors ERα and ERβ, as well as \nprogesterone and androgen receptors (PR and AR). \nWe find that ER α is the predominant estrogen receptor in epithelial and stromal compartments across \nlesion types, including ovarian endometriomas. By contrast, ER β expression remains low and is mainly \nrestricted to endoth elial cells. ER α expression is reduced in stromal cells across lesion types relative to \nmatched endometrium and in the epithelium of superficial peritoneal lesions. In deep endometriosis \nlesions, reduced stromal ER α expression is associated with a signific ant increase in epithelial PR and AR \nexpression, suggesting compartment-specific perturbations of ERα/PR signaling and broader remodeling \nof steroid hormone responses. Together, these findings identify ER α as the dominant but dysregulated \nestrogen receptor  in EMS lesions and support steroid receptor profiling as a framework for lesion \nstratification and improved endometriosis diagnosis.  \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nINTRODUCTION \nEndometriosis (EMS) is a chronic inflammatory gynecologic disease,  whose growth, progression and \npersistence are strongly influenced by estradiol -dependent. It affects  nearly 10% of reproductive -aged \nwomen, and globally impacting 190 million women 1-5,6. It is characterized by the presence of endometrial \ntissue (glands and stroma) outside the uterus, including the ovaries and other pelvic structures. This \ndisease can be classified into three subtypes depending on its localization: ovarian endometriomas (OMA), \nsuperficial peritoneal lesions (SPE), and deep endometriosis (DE ), also referred to as deep  infiltrating \nendometriosis (D IE), defined clinically as lesions that infiltrate >5 mm under the peritoneal surface). \nWomen with EMS experience severe pelvic pain leading to a major impairment in the quality of life, and \nEMS represents a major cause of infertility. The heterogeneity in the disease presentation makes it \ndifficult to diagnose, resulting in a diagnostic delay of  6 to 10 years1. \nThe human endometrium is a highly dynamic tissue with a remarkable regenerative capacity, \nundergoing cyclic phases of proliferation, differentiation, and shedding under the influence of ovarian \nsteroid hormones, particularly estrogen and progesterone7-10. These ovarian hormones exert their actions \nby binding to th eir respective estrogen receptors , namely estrogen receptor alpha (ERα), estrogen \nreceptor beta ( ERβ) and the progesterone receptor (PR). In addition to the classical nuclear estrogen \nreceptor, the G protein -coupled estrogen receptor (GPER, also known as GP R30) has been proposed to \nmediate rapid non -genomic estrogen signaling. However, accumulating  structural and functional \nevidence11 indicates that GPER does not function as a classical estrogen receptor, supporting the central \nrole of ERα and ERβ in mediating estrogen signaling in the endometrium. Current medical treatments for \nendometriosis such as oral contraceptive pills, gonadotropin -releasing hormone (GnRH) agonists, \nhormone-releasing intrauterine devices, and subdermal implants act by suppressing ovarian steroid \nproduction and/or modulating ho rmonal signaling, thereby reducing estrogenic stimulation of \nendometriotic lesions. However, these therapies do not eradicate the disease  and instead induce a \ntemporary, hormonally suppressed state, with symptoms frequently recurring after treatment \ndiscontinuation Given the hormo ne-dependent nature and marked  heterogeneity of endometriosis, \naccurately defining the steroid receptor  profile of endometrio sis lesions is essential for improving our \nunderstanding of disease pathophysiology and for the developmen t of  more effective therapeutic \nstrategies. \nIn the human endometrium of healthy women, ERα is strongly expressed in both epithelial and stromal \ncells during the proliferative phase and mediates the proliferative effects of estradiol⁶. Proteins encoded \nby human ESR2 splice variants (ER β2, ER β5) have also been detected in human endometrium 12,13 and \nendometrial cells14, although their functional significance remains incompletely understood. In contrast, \nthe expression patterns of ER α and ERβ receptors in endometriosis are variable and remain a matter of \ndebate 15. While increased expression of both ESR1  (encoding ER α protein) and ESR2  (encoding ER β \nprotein) mRNAs have been reported in eutopic and ectopic tissues from women with endometriosis  16, \nnumerous studies suggest a relative imbalance characterized by downregulation of ESR1 transcripts and \nupregulation of ESR2 transcripts in endometriotic lesions 17-20. Notably, Matsuzaki and colleagues 17 \ndemonstrated that cyclic ovarian hormone variations differentially regulate E SR1 and E SR2 mRNA \nexpression in endometriotic tissue and highlighted distinct expression patterns between ovarian and \nperitoneal lesions. These observations suggest that steroid receptor expression in endometriosis may be \ninfluenced not only by menstrual cycle -dependent hormonal f luctuations but also by lesion subtype \n(OMA, SPE, DE) and exposure to exogenous hormonal treatments21. A marked increase in ESR2 transcript \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nand ERβ protein levels have been particularly reported in ovarian lesions, especially within the stromal \ncompartment22-25, and elevated ER β levels have also been described in primary stromal cell cultur es \nderived from women with endometriosis24. Furthermore, higher ESR2 expression in OMA compared with \nSPE and DE lesions has been associated with differential responses to suppressive hormonal therapies²⁰.  \nNotably, a study published in Cell by Han et al. (2015)26 concluded that ERβ plays a central and driving role \nin the pathogenesis of endometriosis, promoting lesion survival and inflammatory signaling. This work, \nwhich has been extensively cited, has substantially shaped the prevailing view that ER β predominates \nover ERα in endometriotic tissue and represents a key therapeutic target. Given the broad impact of this \nconclusion on the field, a rigorous reassessment of ER β expression using validated and highly specific \nmethodological approaches appears part icularly warranted. While functional studies have suggested a \nrole for ERβ in lesion biology, accurate determination of its expression pattern in human tissues remains \nessential to correctly interpret these findings.  \nIn this context, rigorous validation of ERβ detection methods is essential. Accurate assessment of ERβ \nprotein expression has been  challenged by evidence showing that  many antibodies against ERβ lack \nspecificity, with the notable exception of the monoclonal antibody PPZ0506 27, which has demonstrated \nspecificity but has been rarely used in previous ly published studies.  Consequently, , many findings \nregarding ERβ protein levels may warrant reassessment using validated detection methods, and th e \nreported imbalance between ERα and ERβ remains uncertain. To unambiguously assess steroid receptor \nexpression in EMS, we performed a comprehensive analysis of ERα and ERβ expression at both RNA and \nprotein levels in the endometrium of 49 women with or without endometriosis and compared eutopic \nendometrium with endometriotic lesions. We also analyzed the expression of PR (progesterone receptor) \nand AR (androgen receptor), given their key roles in menstrual cycle regulation. Using tissue-microarrays \n(TMAs), composed of matched normal endometrium and EM S lesions, we performed multi-level testing \nof ERα, ERβ, PR, Ki-67 and AR expression in a cohort of patients operated for endometriosis in comparison \nto healthy women. Our analysis combines RNA fluorescent in situ hybridization (RNA -FISH) and \nimmunohistochemistry (IHC)on adjacent tissue sections, enabling parallel assess ment of  mRNA and \nprotein expression. This approach enabled the examination of matched patient samples, including eutopic \nendometrium and various types of endometriotic lesions, while accounting for the menstrual cycle phase \nand lesion heterogeneity (OMA, SPE and DE). \nIn contrast to previous stud ies, we detected a high prevalence of ERα and a strikingly low to \nundetectable expression of ERβ across all lesion types, including ovarian endometriomas with ER β \ndetectable only in endothelial cells of the endometrium. While ERα expression varies across the menstrual \ncycle, decreasing in late secretory phase, analysis of matched tissues demonstrated that both SPE and DE \nlesions exhibit reduced stromal ERα expression relative to the corresponding eutopic endo metrium and \nallowed direct comparison of ER transcript and protein expression within the same lesions . Strikingly, DE \nlesions exhibit the most pronounced differences, characterized by upregulated expression of \nprogesterone (PR) and androgen receptors (AR),  and were associated with decreased cellular \nproliferation. These findings highligh t the predominant expression of ERα in both epithelial and stromal \ncompartments of EM S lesions, while also suggesting a complex disruption of estrogenic homeostasis \nbetween these cell types, accompanied by significant alterations in hormonal paracrine signaling \npathways. \n \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nRESULTS: \nPredominant ESR1 expression with limited ESR2 transcript detection in endometrium and \nendometriotic lesions \nTo accurately characterize and compare ERα and ERβ expression in endometriotic lesions at different \nlocations (SPE, DE and OMA), biopsies were collected from women with (EM S) or without endometriosis \n(Healthy women, HW). These samples were used to generate TMAs, enabling the simultaneous analys is \nand comparison of expression across multiple tissue specimens on a single slide  (Fig. 1 A and \nSupplementary Fig. 1A-H).  \nESR1 and ESR2 mRNA expression w as first assessed using RNA in situ hybridization (RNA-FISH) with \nmultiplex fluorescent RNAscope probes, enabling single cell detection of both ESR1 (in green) and ESR2 \n(in purple) mRNAs simultaneously (Fig. 1B-D). Normal ovarian tissue containing an antral follicle was also \nincluded in two spots, as a positive control for ESR2 expression (Fig. 1B). In antral follicles, granulosa cells \nbecome involved in estradiol production, which is associated with strong ER α expression, while ER β \nremains detectable 28. This control further validated the reliable detection of both ESR1 and ESR2 \ntranscripts. H-score quantification of mRNAs signals was then performed across all tissue samples at single \ncell resolution, separating expression in epithelial and stromal c ompartments and taking the menstrual \nphase ( proliferative and secretory phases) into account , as determined by PAEP and CPM \nimmunofluorescence staining (Supplementary Fig. 1I). The maximum H-score of 300 corresponds to 100% \nof cells exhibiting more than 10 spots per cell or 20% of cells containing clusters whereas an H-score of 5 \nindicates that only 5% of cells exhibit a single spot (see Materials and Methods).  \nHigh levels of ESR1 mRNA expression (shown in green) were observed across all endometrial tissue \nsamples including both control (HW) and endometriotic (EM S) endometrium, as well as SPE and DE \nlesions. ESR1 mRNAs was also present in ovarian lesions, with a H-score of 132 for epithelium and 36 for \nstromal comp artment (Fig. 1 C). The cell distribution of mRNA confirms that ESR1 mRNA is the \npredominant transcript for ERs, expressed in both epithelial and stromal cells of the endometrium of \nhealthy (HW) or endometriotic (EM S) women with similar levels of expression. Specifically, ESR1 \nexpression had a very high H-score (close to the maximum possible score of 300) with values of 273.8±5.4 \nin epithelial cells of endometrium from healthy women (HW) versus 268.6±5.4 in those of endometriotic \nwomen (EMS); and 215.6±9 in stromal cells from HW versus 195.5±14 in EMS. Similarly, ESR1 mRNA was \nhighly expressed in endometriotic lesions, with H-scores of 236.4±20.5 in epithelial cells of SPE lesions and \n236.7±20.7 in those of DE lesion during the proliferative phase (Fig 1E-F).  Consistently, ESR1 expression \nwas significantly reduced throughout the menstrual cycle, with a 30-35% decrease in the secretory phase \ncompared with the  proliferative phase in the endometrium. No s ignificant differences were observed \nbetween among endometrium, HW or EMS tissues, nor between EMS endometrium and endometriotic \nlesions. However, significant differences between the proliferative and secretory p hases were observed \nfor all tissue types.   \nIn contrast, ESR2 mRNA expression (in purple) was barely detectable in both endometrial tissues and \nlesions (Fig. 1C-D), with only a few isolated spots observed in epithelial and stromal cells across all tissues \nexamined. The specificity of the ESR2 probe , however, was validated by s trong signal in the positive \ncontrol (ovarian tissues) where granulosa cells exhibited pronounced ESR2 expression (H -score = 111 in \novarian follicles, Fig. 1 B). As expected, ESR2 expr ession in the endometrium was low, with H -scores of \n7.6 ± 3 in epithelial cells of healthy women and 4.8 ± 0.8 in those of women with endometriosis. Similarly, \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nstromal cells showed low ESR2 expression, with H -scores of 11.1 ± 1 in HW and 5.9 ± 1.5 in EMS patients \nduring the proliferative phase. U nexpectedly, ESR2 was notably absent in the ovarian lesions  analyzed, \nshowing negligible ESR2 expression (H -score = 0 -3, Fig. 1 C). Notably, the rare ESR2-positive signals \ndetected in the endometrial stroma were freq uently distributed along aligned cells, particularly during  \nthe secretory phase, consistent with  a morphology suggestive of  endothelial cell s. This was further \nsupported by ERG immunostaining  (a nuclear endothelial marker ) on serial sections , which showed  \nspatially corresponding labeling (Fig. 2G).  \nTo independently confirm the predominant expression of ESR1 and not ESR2 transcripts in endometrial \ntissues, we took advantage of two publicly available single-cell RNA-sequencings atlases of endometriotic \ntissues, that regroup all the major cell types found in the endometrial tissues (from Fonseca aet al.29,  Fig. \n1H and Garcia-Alonso et al. 30, Fig. 1 I). These scRNAseq data confirmed the predominant expression of \nESR1 in both epithelial and stromal cells of all tissue types and almost no expression of ESR2. ESR1 was \nalso modulated along the menstrual cycle, with a decrease of expression during the secretory phase \ncompared to the proliferative phase (Fig. 1J). In agreement with our data, ESR2 transcript expression was \npresent at low levels in endothelial cells, and was nearby undetectable in e pithelial and stromal \ncompartments (Fig. 1K). \nOverall, these data demonstrate that ESR1 transcripts are highly expressed in endometriotic lesions \nand eutopic endometrium of EM S women whereas ESR2 transcripts remains lowly expressed, and \ndetectable in endometrial endothelial cells. \n \nERβ protein is expressed at low levels in endometriosis lesions, including ovarian lesions \nWe then performed immunohistochemistry, using different validated antibodies against ER α and ERβ \nto evaluate expression of the steroid hor mone receptors at the protein level. To ensure consistency and \nprotocol validation, the clinically approved ER α antibody (clone SP1, Roche Diagnostics) was used along \nwith the two validated monoclonal PPZ0506 27, and CWKF12 31 antibodies for ERβ immunolabeling. First, \nwe cross-validated the specificity of the anti-ERβ antibodies, using cell lines transfected or not with ERβ-\nexpressing plasmids (see Materials and Methods). ERβ expression was detected in both MDA-MB-231 and \nMCF7 transfected cells by RT -qPCR and Western blot (Supplementary Fig. 2A-B) as well as by \nimmunohistochemistry which showed strong immunolabeling, and thereby validated the accuracy of the \nantibodies used (Fig. 2A). In ovarian positive controls containing an antral follicle, in which ESR1 and ESR2 \ntranscripts were expressed at similar levels (Fig. 2B, left panel), strong ERα labeling was observed (middle \npanel), whereas both anti -ERβ antibodies produce only faint immunostaining, mainly in granulosa cells, \nwith comparable results for the two anti-ERβ antibodies (Fig. 2B, right panels).  \n Analysis of ER β expression in endometrial tissues  using the PPZ0506 antibody revealed only weak \nlabeling in rare cells from both control and EMS women, as well as in all lesions tested, with less than 2.5 \n% of cells being ERβ-positive, independently of their location ( SPE, DE or OMA , Fig. 2 C-D) and of the \nmenstrual phase. In the endometrium, both anti-ERβ antibodies showed similarly weak staining in a small \nsubset of cells with a morphology suggestive of endothelial cells, consistent with the relatively higher ESR2 \ntranscript expression detected in this cell population (Fig. 1 G). The presence of ERβ -positive cells \nremained very low, w ith H-scores of only 2.2 ± 1.4 in epithelial cells and 1.3 ± 0.9 in stromal cells of the \nendometrium from HW women during phase 2. No increased expression was observed in any \nendometriotic lesion, including OMA. These findings indicate that the percentage of ERβ-positive cells is \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nlow (between 2-3 %) and remains nearly undetectable in endometriotic tissues. Therefore, we focused on \nthe expression of ERα for the remainder of our study.  \n \nERα and Ki -67 expression  are unchanged  in control versus EM S endometrium, whereas PR is \ndecreased in the stroma of EMS women during the late secretory phase \nWe then assessed the variation of ERα expression in endometrium of both control and EM S women \ntogether with the progesterone receptor (PR) along the menstrual cycle, distinguishing between the \nproliferative and secretory phases (Fig. 3). Importantly, Ki -67 as a marker of cell proliferation, was highly \nexpressed in the epithelium of the endometrium in both control women and those with endometriosis, \nduring the proliferative phase, compared to the secretory phase, confirming the accurate identification of \nthe menstrual phase (Fig. 3A-B).  \n ERα protein was highly expressed in endometrium of control women, both in epithelial and stromal \ncells (Fig 3 A-B). Similar ERα expression levels and distribution were observed with two independent \nantibody clones (SP1, recognizing all ER α splice variants, and 6F11, specific for the full -length isoform32) \n(Supplementary Fig. 3). A marked and significant reduction in ERα protein expression was observed during \nthe late secretory phase in both epithelial and stromal cel ls of the endometrium, independently of the \ndisease status in HW and EMS women. In epithelial cells, ERα decreased from 98.1± 0.6 to 41.7±14 in HW \nwomen (P<0.0001) and from94.4±2 to 46.3±10.9 (P<0.01) in EMS women (Fig. 3B, right panel).  \nWe also quantified PR protein expression, a key hormonal regulator of the female reproductive system. \nIn epithelial cells, PR expression closely paralleled that of ERα expression, with a marked decrease during \nthe late secretory phase in both control and EM S endometrium. In contrast, stromal PR expression \nremained stable across the menstrual cycle in HW women (82.3% versus 80.2% in the proliferative and \nlate secretory phases, respectively) whereas it was significantly reduced in stromal cells from EMS women \nduring the late secretory phase  (49.1% versus 87.1 % in P and LS, respectively) , indicating a difference \nbetween healthy and EMS women.   \nOverall, ERα expression in the endometrium did not differ significantly between HW and women with \nendometriosis, whereas PR expression was reduced in the stromal compartment of EMS women \ncompared to HW controls.   \n \nERα and PR are broadly preserved but vary across endometriotic lesion types \nWe then compared ER α and PR expression in endometriotic lesions ( SPE, DE and OMA) with that of \neutopic endometrium within the same patients  (Fig. 4 A). No significant differences were observed \nbetween the proliferative and secretory phase, likely because early and late secretory phases were no \nlonger distinguished, except for the PR expressio n in eutopic endometrium and DE lesions. Overall, ERα \nand PR expression remained preserved across all tissue types in both epithelial and stromal cells (Fig. 4B). \nHowever, some heterogeneity was observed, particularly, a significant reduction in the percentage of ERα-\npositive cells in the stromal compartment, depending of lesion type. During the proliferative phase, 81.3% \nof stromal cells were ERα-positive in the endometrium , compared with  52% in SPE lesions, 74% in DE \nlesions and 43.8% in OMA lesions . PR expression was also significantly reduced during the proliferative \nphase in the epithelial cells of OMA lesions compared with endometrium, SPE and DE lesions. Moreover, \nthe percentage of PR-positive cells in the stromal compartments differ ed significantly, according to the \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nlesion types and menstrual phase. The proliferative marke r, Ki-67, also showed substantial variability \nacross samples.  \nWe also assessed AR expression on these TMAs, as androgen signaling play also critical roles in the \nfemale reproductive tract. We could confirm that AR expression is significantly the highest in the stromal \ncells of the upper functional layer of epithelium of control women during the beginning of the menstrual \ncycle (proliferative phase and early secretory phase), as already observed by Gibson and his collaborators \n(2020)8 (Supplementary  Fig. 4). AR expression remai ns high in the stromal cells of the lesions, and \nsurprisingly, is now also detected in the epithelial cells of the DE lesions, although at a level below 10%.  \n \nDE lesions show altered expression patterns of steroid receptors such as ERα, PR and AR, indicating \na potential disruption in paracrine signaling between stromal and epithelial cells \n To evaluate potential variations in steroid receptor expression patterns between lesions and matched \neutopic endometrium, we assessed the expression of ERα, PR, AR and Ki-67 markers in EM S lesions and \ntheir corresponding matched endometrium from the same patients (Supplementary Fig. 5). This paired \ncomparison was performed to minimize potential variability related to menstrual cycle phase. Overall, we \nobserved substantial inter-patient and intra -patient heterogeneity in the abundance of ER α- and PR-\npositive cells, particularly in the lesions. No consistent directional change (increase or decrease) was \nidentified between matched eutopic endometrium and lesion samples. \nTo a ddress the marked inter -patient variability in receptor expression, we performed paired \ncomparisons between lesions and their matched eutopic endometrium. The percentage of ER α-, PR-, Ki-\n67-, and AR-positive cells was therefore compared in matched lesional and eutopic tissues (left panels of \nFig. 5A, C, E, G for epithelial cells and Fig. 5B, D, F, H for stromal cells). To further quantify these paired \ndifferences, we also calculated the change in the percentage of positive cells between each lesion and its \nmatched eutopic endometrium (lesion – EUT). These values were then summarized by lesion subtype s \n(SPE, DE, OMA) to evaluate the average direction and magnitude of receptor expression changes relative \nto the matched endometrium (right panels  of Fig. 5A –H). Positive values indicate higher expression in \nlesions relative to the matched eutopic endometrium, whereas negative values indicate lower expression. \nERα expression was significantly decreased in both epithelial and stromal compartments of SPE lesions \n(P = 0.02 and P = 0.007, respectively)  whereas epithelial ERα expression remained unchanged  in DE \nlesions. In SPE lesions, ERα expression decreased by an average of 16.6% in epithelial cells  and 21% in  \nstromal compartments with reduction reaching up to 65% (Fig. 5A-B, left panels). Stromal ERα expression \nwas also significantly reduced across all lesion types, with the strongest decrease observed in OMA lesions \n(average 42%) . In DE lesions, the stromal decrease in ERα was associated with a small but significant \nincrease in epithelial PR expression compared to matched endometrium (P = 0.011, Fig. 5 C, left panel ) \nwith an average increase of 10% and values reaching up to with a +78%. Although no significant overall \nchange in stromal PR expression was observed (P=0.07), individual analysis showed that around 25% of \nDE lesions exhibited reductions of up to 40%. These changes were associated with a marked significant \ndecrease in Ki-67 expression in DE lesions (P<0.0001, Fig. 5E middle panel) with an average reduction of \n21% and a maximum decrease of up to 79%. In parallel, AR expression was significantly increased in \nepithelial cells of DE lesions (P=0.021, Fig. 5 G, left panel ) with an average increase of 3.9 % and values \nreaching up to 31%. Finally, OMA lesions showed a significant decrease in both ERα and PR expression in \nstromal cells.  \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nOverall, d espite the marked heterogeneity in steroid hormone receptor expression across lesions, \nthese findings highlight differences in ERα expression across the disease . In the SPE lesions, a small but \nsignificant decre ase in E Rα expression was observed in both epithelial and stromal cells  compared to \nmatched eutopic endometrium. In contrast , DE lesions exhibit an altered ERα/PR expression, \ncharacterized by decreased stromal ERα and small but significant increase in epithelial PR, indicating that \nthe hormonal signaling of these cells of endometrial origin is altered.  \nIn endometrium, ERα is highly expressed in epithelial and stromal cells thr oughout the estrogen -\ndominant phase and decline during the secretory phase, while PR is maintained in the endometrial stroma \nand decline in the epithelium 7. We then assessed the correlation between ERα expression and that of PR, \nKi-67 and AR across all tissue types, distinguishing expression in epithelial and stromal cells, respectively \n(Table 1 ). Spearman correlation analysis demonstrated a  positive correlation between ERα and PR \nexpression in both epithelial and stromal c ompartments of the endometrium and SPE lesions, whereas \nthis association was weaker in DE lesions. ERα and AR expression were also positively correlated, but only \nin stromal cells of the endometrium and SPE lesions, with no significant association in epithelial cells or \nDE lesions. In contrast, the correlation between ER α and Ki -67 was confined to epithelial cells of the \nendometrium and OMA lesions and was absent in all other cell types and tissues. These findings support \nthe conclusion that ER α remains the predominant estrogen receptor in endometriotic lesions, while its \ncoordinated expression with PR is preserved in endometrium and SPE lesions but diminished in DE lesions. \nTo further investigate  the altered ER α/PR relationship in endometriosis, we performed multiplex \nRNAscope for ESR1 and PR on the same tissue sections, to determine whether the differential correlations \nobserved at the protein level were also recapitulated at the transcript level ( Supplementary Fig. 6A). A \nsignificant positive correlation between ESR1 and PR transcripts was observed in both epithelial and \nstromal cells of the endometrium, regardless of whether the w omen had endometriosis (Supplementary \nFig. 6B). In contrast, this correlation was lost in all lesion types and in both cellular compartments.  \n Together, these results support the presence of altered ESR1 –PR transcriptional coordination in \nendometriotic lesions, consistent with changes in steroid receptor signaling, and suggest impaired \nERα/PR-mediated paracrine signaling between epithelial and stromal compartments in endometriosis.  \n \nDISCUSSION:  \n Millions of women are affected by endometriosis and receive hormonal treatments to suppress \nestrogen signaling, thereby reducing the stimulation of endometrial tissue. This tissue is dynamic and \nhighly sensitive to the effects of estrogen and progesterone because it expresses high -affinity hormone \nreceptors. However, resistance to the proposed hormonal treatments occur, and a comprehens ive \nanalysis of expression of these nuclear receptors ERα, ERβ, PR and A R is required to improve our \nunderstanding of the disease and develop more effective therapeutic strategies. The use of bulk tissue \nanalysis techniques, such as RT-PCR and Western blotting to unravel receptor expression in endometriotic \nlesions, has important limitations, as these methods provide only an average measure of expression \nacross the tissue and do not allow assessment of cell -specific localization or tissue distribution.   \nFurthermore, the use of non -specific antibodies for ERβ induced confusion with regards to the presence \nof ERβ receptor in endometriosis27. \nOur study, combin ing RNAscope multiplex technology and immunohistochemistry on TMAs, \ndemonstrates that estrogen receptor alpha (ERα) is the predominant estrogen receptor expressed at both \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nthe transcriptional and translational level in the endometrium as well as in all types of endometriotic \nlesions studied —SPE, DE and OMA. ERα expression was 10 - to 100 -fold higher than ER β, in line with \nprevious reports17,33. Consistently, ESR2 expression was minimal, whereas ESR1 levels remained markedly \nhigher in eutopic endometrium and endometriotic lesions. At the protein level, ERβ was detected only at \nvery low levels in epithelial and stromal cells from both endometrial and endometriotic tissues, with mean \nexpression below 2.5%. This near absence of ER β was reproducibly confirmed using two independently \nvalidated antibodies: PPZ0506, directed against amino acids 2 –8827 and CWK-F12, developed by B. \nKatzenellenbogen and independently validated by Nelson et al. (2017) 31, directed against amino-acids \n256–505 . The absence of ERβ expression was also observed in ovarian lesions, despite the possibility of \nresidual ERβ expression from  normal granulosa cells . Th ese results  contrast with previous studies \nreporting higher ESR2 mRNA expression in endometriomas than in superficial or deep infiltrating \nendometriosis lesions, or in normal uterine endometrium 18,23,24,34. Nevertheless, functional studies have \nshown that ERβ overexpression in a murine model of endometriosis was associated with enhanced \ndisease progression26. Overall, the discrepancies between our findings and previous reports are likely due \nto methodological differences, including the use of highly specific probes  for ESR2 transcripts and \nvalidated antibodies with confirmed ERβ specificity27,31.  \nMoreover, we detected low levels of ESR2 mRNA expression  in vascular cells of the endometrium \nfrom both control and EMS women, consistent with the presence of low ERβ protein levels.  Although ERα \nis well established as a key mediator of the vasculoprotective effects of estrogens , its role appears \nparticularly prominent in large arteries such as the aorta and carotid arteries , where it contributes to \ncardiovascular pro tection in females , by promoting vasodilation, angiogenesis, and anti -inflammatory \nresponses35,36. In contrast, ERβ has been detected in certain microvascular beds, and has been previously \nbeen identified  in primary human uterine microvascular endothelial cells 37,38. Andersson et al. 27 also \ndescribed ERβ expression in endothelial cells from endometrial cancer. Together, these independent \nobservations support the findings of our manuscript. Specifically, Tamura (2013)37 showed that estradiol \nincreases COX -2 protein expression and PGE2 production in these cells through estrogen receptor \nsignaling, most likely via ERβ, since ERα was not detected in these cells. Additionally, Greaves and her \ncollaborators38 reported that ERβ activation  differentially regulates  endothelial cell function in the \nendometrium and myometrium , with in some cases opposing effects. In endometrial endothelial cells, \nERβ activation was associated with reduced angiogenic activity. These findings suggest that ERβ may have \na specific role in endometrial endothelial cells, particularly at the end of the secretory phase, contributing \nto the complex regulation of uterine vasculature across the menstrual cycle. Future directed studies are \nnow warranted to determine how endothelial ERα and ERβ in these endothelial cells affect uterine \nphysiology and endometriosis.  \nBesides the predominant expression of ERα in all endometrial tissues, our findings also confirm the \nmarked heterogeneity of ERα expression in endometriotic lesions, a feature previously reported in the \nliterature21. Beyond the variability associated with lesion location and disease stage, a key contributing \nfactor is the dynamic fluctuation in receptor expression across the menstrual cycle , particularly the \npronounced decrease in ERα and PR levels in epithelium during the l ate secretory phase, which follows \nthe rise in progesterone. Although this temporal variation has long been recognized 7,8, it has not always \nbeen adequately considered in previous analyses, which may have contributed to inconsistencies in the \nliterature. \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nUsing tissue microarrays and matched samples, we direc tly compared endometriotic lesions and \neutopic endometrium under identical conditions, despite di sease heterogeneity. W e observed reduced \nERα expression in SPE lesions in both epithelial and stromal compartments, whereas in DE and OMA \nlesions, this decrease was predominantly stromal.  In DE lesions, this stromal loss of ERα may contribute \nto the reduced epithelial proliferation observed, consistent with evidence that estrogen-driven epithelial \nproliferation is mediated by ERα-positive stromal cells39. These alterations in ERα expression also suggest \nthat the estrogen-responsive stromal compartment is always affected across lesion types, which may in \nturn influence steroid receptor crosstalk and downstream hormonal responses. Accordingly, PR \nexpression displayed lesion - and compartment -specific variations, with a modest increase in the \nepithelium of DE lesions and a decrease in the stroma of OMA lesions. Together with previous reports of \nvariable PR expression 40,41, these findings highligh t the complex regulation of progesterone  signaling in \nendometriosis. Notably, we found a loss of coordinated ERα and PR expression in ectopic lesions at both \nthe transcriptional and translational levels, in contrast to the correlated expression pattern maintained in \nthe endometrium ( Table 1 and Supplementary Fig. 6B). Because ERα /PR regulatory feedback normally  \noperates across the menstrual cycle in endometrium 42, disruption of this relationship is consistent with  \naltered steroid receptor signaling and supports the concept of progesterone resistance in endometriosis. \nImportantly, our results indicate that PR expression is not uniformly reduced, but instead varies by lesion \ntype, menstrual phase and cellular compartment, highlighting the heterogeneity of progesterone \nsignaling acro ss endometriotic lesions. This heterogeneity may have clinical relevance, as altered \nprogesterone receptor expression has been associated with response to progestin therapy in \nendometriosis43. Together, these observations suggest that progesterone resistance in endometriosis may \nreflect not only changes in PR abundance  and signaling , but also altered estrogen receptor ER α \ncoordination and downstream signaling. \n Finally, while androgens play some role in epithelial and stromal cross talk 4, being normally \nexpressed in stromal cells during the early secretory phase  protecting these cells against stress or \napoptosis44, the present work also reported an elevated AR expression in epithelial cells of DE lesions (Fig. \n5G). Once more, this AR elevated expression in epithelial cells is the signs of strong dysregulated pathways \nindicating that these endometrial lesions, although endometrial -like in appearance, with epithelial and \nstromal cells, are not functionally the same.  Further studies on the role of these AR expression in the \nregulation of endometrial function are needed to better understand their actions in the pathogenesis of \nendometriosis.  \nOnly a small subset of lesions exhibited very low steroid receptor expression (having less than 20% \nERα and PR -positive cells ), confirming that ERα and PR remain key markers for both detection and \nstratification Notably, dysregulation of these receptors was more frequent in DE lesions compared with \nSPE lesions. The proportion of ERα- and PR-positive cells therefore represent a useful classification criteria. \nHowever, the marked heterogeneity of endometriotic lesions, as demonstrated by our findings and those \nof others, must be more fully acknowle dged, as it poses a challenge to establishing a straightforward \nclassification or prognostic model based on steroid receptor expression.  \nThese findings are of particular interest, since they are important to challenge response to \nhormonal treatments. Spec ific agonists and antagonists for ERα and ERβ have been respectively \ngenerated45 while ERα also acts via complex nuclear and membrane actions on a tissue -specific manner. \nSome ligands, such as estetrol, a natural estrogen have a specific pharmacological activation profile46, that \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nhave shown exciting differences in endometriosis that wi ll need further evaluation for endometriosis \ntreatment47. Notably, selective inhibition of ERβ activity was also found to significantly reduced ectopic \nlesion growth in a mouse model 26. However, while ERβ is not highly expressed in EMS lesions, this current \nwork completely rules out the possibility to use these selective antagonists to block the development of \nlesions. In contrast, ERα is the only form of ER receptor expressed in EMS lesions, highlighting that it is the \nprimary ER target to design therapeutic strategies.   \nOverall, our findings indicate that ERα is the predominant ER expressed in epithelial and stromal \ncells of the endometrium and lesions, with limited evidence for opposing ERβ activity. This expression \npattern may promote an environment that favors ERα -driven proliferative and inflammatory responses \nwhereas ERβ might have a more restricted role in endometrial vasculature. These results challenge the \nnotion of broad ER β expression in endometriosis and highlight the relevance of ERα and PR profiling for \nlesion stratification, diagnosis and development of hormone -based therapeutic strategies.  The \ndysregulated receptor landscape observ ed in stromal and epithelial compartments further emphasizes \nthe need for continued research to better understand estrogen signaling in endometriosis.  \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nMETHODS: \nPatient cohorts \nThe study involved biopsies from 15 healthy women and 34 women with endometriosis in different phases \nof the menstrual cycle: proliferative (n= 7 healthy and n= 17 endometriotic); early secretory (n= 3 healthy \nand n= 7 endometriotic; late secretory (n= 5 healthy and n= 10 endometriotic )). Cycle phase dating was \ndefined by surgeon, pathologists and confirmed by immunofluorescence staining using antibodies against \nCPM (proliferative phase) and PAEP (end of secretory phase) 48. The endometriotic biopsies from different \nlocations (classified as SPE, DE or OMA by the surgeon) were obtained from 34 endometriotic women who \nunderwent hysterectomy surgery or laparoscopic surgery in the Gynecological Surgery Department of the \nToulouse University Hospital. To compare with endometrium from healthy women, normal endometrium \nfrom 15 healthy women undergoing laparoscopic surgery for non-malignant gynecologic indications were \nused as controls. All samples were obtained from pre -menopausal women aged 22-49 years old (median \nage-35 years old), with regular menstrual cycles, and no hormonal contraceptives or no recent hormonal \nuterine device usage in the past 2 months (See Supplementary Table 1).  Endometrial biopsy was obtained \nusing the Pipelle de Cornier biopsy device. Written informed consent was received prior to participation. \nPatients’ samples were obtained after written informed consent prior to participation, in accordance with \nthe Declaration of Helsinki and stored at the “CRB Cancer des Hôpitaux de Toulouse (BB -0033-00014)” \ncollection. According to the French law, CRB Cancer collection has been declared to the Ministry of Higher \nEducation and Research (DC-2008-463 and DC-2020-4074) and obtained a transfer agreement (AC-2013-\n1955 and AC-2025-7615) after approval by the ethics committee (Comité de Protection des Personnes \nSudouest et outre mer II, CPP). Clinical and biological annotations of the samples have been declared to \nthe CNIL (Comité National Informatique et Libertés).  \n \nTissue Microarrays (TMAs) \n3 different TMAs were generated  from the endometrial biopsies or endometriotic lesions of women \nincluded in the cohort (described above),  using an automated tissue microarrayer (Ex cilone, Elancourt, \nFrance). Representative endometrial tissue -rich areas, previously annotated by a certifi ed pathologist, \nwere selected from the formalin -fixed paraffin -embedded (FFPE) patient tissue blocks. Each TMA \nconsisted of cores with a diameter of 2 mm randomly distributed and included duplicate cores per lesion \nper woman when possible. Tissue sections (thickness: 4μm) were prepared from the TMAs for RNAs-cope \nIn Situ Hybridization (TMA-1 and -2) or immunohistochemistry (all TMAs).  \n \nDetermination of menstrual phase by Immunofluorescence staining with CPM and PAEP antibodies \nThe TMA slides were deparaffinized using Toluene (386001 Carlo Erba), and an acid unmasking procedure \nwas performed (H -3300, Vector laboratories). Samples were permeabilized and blocked with blocking \nbuffer (1% BSA (Sigma), 4% normal goat serum (ab7481, Abcam), 0.1% TritonX -100 (Sigma) in PBS) 1h at \nRT. Slides were incubated with primary antibodies CPM (HPA002657 -100UL, Sigma , lot 27155, RRID: \nAB_1078398) and PAEP (ab17247, lot 1022890 -1, Abcam; RRID: AB_2159754) overnight at 4°C.  Then \nsections were incubated with the conjugated secondary antibodies Alexa Fluor 647 (Abcam) and the nuclei \nwere counterstained with DAPI (Invitrogen, Life Technologies). The samples were then mounted with \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nMounting Medium (S3023, DAKO) and acquired using a LSM900 Fluorescence Microscope (Zeis s). The \nimages were further processed using QuPath v0.5.0. \n \nCell culture and transfection, and preparation of cytoblocks \nMDA-MB-231 (ATCC-HTB-26) and MCF7 (ATCC-HTB-22) cells were transiently transfected with either the \npSG5puro-ERβ plasmid or the empty vector (gift of P. Balaguer, IRCM, Montpellier, France 49) as a control \nusing the Lipofectamine 3000 (L3000-015, Invitrogen, Villebon-Sur-Yvette, France). Cells were maintained \nin Dulbecco’s modified Eagle’s medium (DMEM/F12; P04 -41250, Dutscher , Ber nolsheim, France ) \nsupplemented with 5% fetal calf serum (S181W-500; Biowest , Nuaillé, France ) and antibiotics (P0781, \nSigma, Lezennes, France) at 37 °C under 5% CO2.  \nTransfected MDA -MB-231 cells were formalin -fixed and paraffin -embedded using the Shandon™ \nCytoblock™ Cell Block Preparation System, according to the manufacturer’s protocol.  \n \nGene expression analysis   \nCells were freeze -dried and then RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). 1000 ng of \nRNA was reverse transcribed (RT) at 25°C for 10 min and then at 37°C for 2 h using the High Capacity cDNA \nreverse transcriptase kit (Applied Biosystems). For gene expression, qPCR w as performed using SsoFast \nEvaGreen Supermix (Bio -Rad, Marnes -La coquette, France ) with primers efficiency validated using \nstandard curves method (95% < efficiency < 105%). Gene expression was quantified using the comparative \nCt (threshold cycle) method. HPRT gene was  used as housekeeping gene to normalize the mRNA. The \nprimer sequences were: ESR2: Forward: 5’ - GTC AGG CAT GCG AGT AAC AA - 3’; Reverse: 5’ - GGG AGC \nCCT CTT TGC TTT TA - 3’; HPRT: Forward: 5’ - TGC TTT CCT TGG TCA GGC AGT - 3’; Reverse: 5’ - CTT CGT \nGGG GTC CTT TTC ACC - 3’.  \n \nExploration of public scRNAseq datasets \nWe used publicly available datasets from Fonseca et al  29 (Accession number GSE213216)  and Garcia -\nAlonso et al. 30 (Accession numbers E-MTAB-10287). RDS files were download and processed using Seurat. \nFigures were generated using the FeaturePlot function in Seurat, or using ggplot2 package. \n \nWestern blot analysis \nThe cells were lysed using lysis buffer (150mM NaCl, 50mM Tris-HCl (pH 7,5), 1% NP40, 1mM EDTA, 5mM \nNaF, 1mM orthovanadate, 0,5mM DTT, proteinase inhibitors (Complete™ EDTA -free, Roche, Boulogne-\nBillancourt, France), 0,1% SDS)) Total proteins were separated on a 10% SDS/PAGE gel and transferred to \nnitrocellulose membranes using Trans Blot Turbo RTA Transfer Kit 0.2 µm Nitrocellulose.  The ladder was \nthe PageRuler Plus Prestained Protein ladder (10-180 kDa) from ThermoFisher Sientific , Illkirch, France . \nThe following primary antibodies  were used: anti-ERβ (clone PPZ0506, Cat#PP-PPZ0506-00, lot A -2, \nRRID:AB_2293861, R&D Systems , 1/1 000; anti-ERβ CWKF12 (DSHB depository, 1/200) and anti-GAPDH \n(clone 6C5,sc-32233, lot 71628,  RRID:AB_627679, Santa Cruz , dilution 1/2000 ). Then, revelation was \nperformed using HRP -conjugated secondary antibodies (Goat anti -mouse IgG (HRP), Cat# 91196, \nRRID:AB_2940774, Cell Signaling Technology ) and visualized by ECL detection according to the \nmanufacturer’s instructions (Amersham Biosciences/GE He althcare), using ChemiDoc  Imaging System \n(Bio-Rad). Bands were quantified using ImageLab. \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \n \nMultiplex Fluorescent RNAScope assay on BOND RX \nThe RNAScope LS Multiplex Fluorescent assay (ref. 322800, 32327, ACD, BioTechne, MN, USA) was used \naccording to manufacturer’s procedures (technical note: UM 322800/Rev B) and performed on the BOND \nRX research advanced automated slide stainer (Leica Biosystems, Nusslo ch, Germany). Formalin -fixed \nparaffin embedded (FFPE) TMA tissue sections of 4µm were prepared and air dried overnight. Quality of \nRNA of the tested samples was verified and validated using the RNAscope  2.5 LS 3-plex Positive Control \nProbe-Hs (ref. 320868, ACD, BioTechne, MN, USA). Non -specific staining was absent as confirmed by the \nuse of RNAscope 2.5 LS Multiplex Negative Control Probe dapB (Bacillus subtilis strain, ref. 320878, ACD, \nBioTechne, MN, USA). Probes targeting the widely expressed Peptidylprolyl Isomerase B (PPIB) and against \nDNA-directed RNA polymerase II subunit RPB1 (PolR2A) were used as positive controls, to ensure about \nthe RNA integrity. PPIB is a commonly used housekeeping gene for this purpose, while POLR2A serves as \nan alternative p ositive control, particularly in proliferating tissues. FFPE TMA tissue slides were heat -\npretreated using ER2 pretreatment solution (pH8, Leica Biosystems, Nussloch, Germany) for 15 minutes \nat 95°C and subsequently incubated using RNAscope  2.5 LS Protease  III (ref. 322800, ACD, BioTechne, \nMN, USA) during 15 minutes. The target probes used in this study were  were as follows: RNAscope™ 2.5 \nLS Probe - Hs-ESR1-C2 (ref. 310308 -C2 targeting region 1251 -2376, ACD, BioTechne, MN, USA) and \nRNAscope™ 2.5 LS Probe- Hs-ESR2 ( ref. 470158-C1 targeting region 456 - 1995, BioTechne SAS, MN, USA) \nor the combination of RNAscope™ 2.5 LS Probe - Hs-ESR1 (ref. 310308 targeting region 1251 -2376, Bio-\nTechne SAS, MN, USA)and RNAscope™ 2.5 LS Probe - Hs-PGR-C2 (ref. 589758-C2 targeting region 1609 - \n2579, BioTechne SAS, MN, USA)  These target probes recognized all splice variants and  were visualized \nusing OPAL TM dyes (AKOYA Biosciences, Marlborough, USA) with excitation and emission wavelengths \ncompatible with our whole slide im aging system were used: OPAL TM 570 and OPAL TM 650 for ESR2 and \nESR1, or OPAL TM 520 and OPAL TM 570 for ESR1 and PR (1/1500 in 1x Plus Automation Amplification \nDiluent, AKOYA Biosciences, Marlborough, USA). The tissue slides were counterstained using RNAscope \nLS Multiplex DAPI (AKOYA Biosciences, Marlborough, USA) and mounted with Invitrogen TM ProLongTM \nGold Antifade Mounting medium (Life Technologies, ThermoFisher Scientific, California, USA). \n \nMultispectral fluorescence imaging was performed using a n AxioScan Z1 (Carl Zeiss Microscopy, \nOberkochen, Germany) whole-slide scanner with appropriate narrow band -pass excitation and emission \nfilters and specific dichroic mirrors (Semrock Inc., Rochester, NY, USA), and with a multi -channel solid-\nstate light engine (Colibri 7, Carl Zeiss Microscopy, Oberkochen, Germany) equipped with 7 LEDs covering \nthe entire visible spectrum from UV to far -red (370 – 648 nm). Analog to digital image sampling was \nperformed at 16 -bit (65 536 grey levels, 37 000:1 dynamic range) with a high -resolution scientific \ncomplementary metal oxide semiconductor (sCMOS sensor with 2 048 x 2 048 cells of size 6.5 x 6.5 µm \neach) Peltier -cooled monochrome camera (Orca Flash 4.0 V3, Hamamatsu Photonics K.K., Japan), to \nachieve a final scan resolution of 0.32 µm/pixel.  \n \nRNAscope quantification \nQuantification of RNAscope  signals was performed at 20× magnification and the images were further \nprocessed and quantified using QuPath (v0.5.0) in a blinded manner. Quantification was conducted across \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nmultiple regions of each biopsy to ensure reliable assessment and to account for tissue heterogeneity. \nWhen duplicate biopsy spots were available, both spots were analyzed independently, and the mean of \nthe two measurements was used for subsequent analysis. Epithelium and stroma were differentiated in \nthe analysis. An H-score was used to determine the class of each cell and assign a value to each cell type \nfor each TMA spot. Different class of cells were distinguished, de pending of the RNA spot number per \ncells, i.e. Class 0 (0 spot/cell), Class 1 (1 -5 spots/cell); Class 2 (6-10 spots/cell) and Class 3 (>10 spots/cell \nor >20% cluster in cells). The following formula was then used to calculate the average number of \nRNA/cell: (0 x % cells class 0) + (1 x % cells class 1) + (2 x % cells class 2) + (3 x % cells class 3). So, the \nmaximum H-score is 300 if all cells are class 3. \n \nERβ Immunohistochemistry \nFormalin-fixed paraffin embedded (FFPE) tissue sections (TMA and cell block) of 4µm were prepared and \nair dried overnight. ERβ Immunohistochemistry was automated on the AS48 automated stainer (Agilent \nTechnologies, CA, USA). Dewaxing and antigen retrieval was performed using a PT Link pressure cooker \n(Agilent Technologies, CA, USA ) for 25 minutes at 97°C using En VisionTM FLEX target retrieval solution \n(pH6, ref. K800521, Agilent Technologies, CA, USA). Primary anti -ERβ antibodies ( clone PPZ0506, lots \nXF3616191 and 797560-25, RRID: AB_2717280, ThermoFisher Scientific, 1/200  and lot A-2, \nRRID:AB_2293861, R&D Systems , in Envision FLEX antibody diluent (Agilent technologies, CA, USA), and \nCWKF12 (DSHB depository, 1/200 in Envision  FLEX antibody diluent (ref. K800621, Agilent technologies, \nCA, USA)) were incubated for 30 minutes at room  temperature and visualized using the En VisionTM FLEX \nHRP detection system (ref. K800221 Agilent technologies, CA, USA). Slides were subsequently \ncounterstained using haematoxylin/eosin (ref. K800821, Agilent technologies, CA, USA), dehydrated and \nmounted using xylene-based mounting in a Tissue  Tek automated cover slipper (Sakura FineTek Europe, \nAV, The Netherlands).    \n \nStained slides were digitized with a Panoramic 250 Flash II digital microscope (3DHISTECH, Budapest, \nHungary) equipped with a Zeiss Plan -Apochromat 20X NA 0.8 objective and a CIS VCC -FC60FR19CL 4 -\nmegapixel CMOS sensor (unit cell size 5.5 x 5.5 µm) mounted on a 1.6X optical adaptor, to achieve a scan \nresolution of 0.24 μm/pixel in the final image (corresponds to 41.1X magnification at the hig hest optical \nresolution in traditional microscopy). \n \nERα, PR, AR and Ki67 Immunohistochemistry \nAutomated classical immunohistochemical (IHC) stain was performed using the Benchmark ULTRA (Roche, \nVentana Medical Systems, Innovation Park Drive Tucson, Arizona 85755 USA) on FFPE tissue sections \n(4µm). After dewaxing, tissue slides were heat pre-treated using a CC1 (pH8) buffer (05424569001, Roche \nDiagnostics, IN, USA) at 98°C. The slides were blocked for endogenous peroxidase activity and incubated \nwith primary anti-ERα (clone SP1, Cat# 06523838001, lot N11424, RRID: AB_2335977;  Roche Diagnostics, \nIN, USA or clone 6F11, Cat# MA1-80216, lot OL1796492, RRID: AB_930763, Thermo Fisher Scientific), anti-\nPR (clone 1E2, Cat# 05278392001, lot N09748, RRID: AB_2335976, Roche Diagnostics, IN, USA) ,  anti-AR \n(clone SP107, Cat# 06523838001, lot V0005472, RRID: AB_10903299    Roche Diagnostics, IN, USA) and \nanti-ERG (Cat# 790-4576, lot N16218, RRID:AB_2861321, Roche Diagnostics) antibodies. The targets were \nthen visualized sing the UltraView DAB Detection kit (05269806001, IN, USA). For Ki67 (clone 30 -9, Cat # \n05278384001, RRID:AB_2631262, lot N60087, Roche Diagnostics, IN, USA) targets were visualized using \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nthe OptiView DAB detection kit ( 06396500001, Roche Diagnostics, IN, USA). The tissue slides were \ncounterstained using hematoxylin II (05277965001, Roche Diagnostics, IN, USA) for 8 minutes followed by \npost-coloration using Bluing reagent for 4 minutes at room temperature (05266769001, Ro che \nDiagnostics, IN, USA). The slides were then dehydrated (ethanol and xylene) and mounted using xylene -\nbased mounting (Sakura Tissue -Tek®, Sakura Finetek Europe, AV, The Netherlands).  All antibodies used \nwere validated by the pathology lab, following ISO15189 recommendations.  \nStained slides were digitized with a Panoramic 250 Flash II digital microscope (3DHISTECH, Budapest, \nHungary) equipped with a Zeiss Plan -Apochromat 20X NA 0.8 objective and a CIS VCC -FC60FR19CL 4 -\nmegapixel CMOS sensor (unit cell size 5.5 x 5.5 µm) mounted on a 1.6X optical adaptor, to achieve a scan \nresolution of 0.24 μm/pixel in the final image (corresponds to 41.1X magnification at the highest optical \nresolution in traditional microscopy). \n \nStatistics and Reproducibility \nStatistical analysis was performed with the PRISM software v10.1.2 (GraphPad Software, San Diego, CA, \nUSA) (See Supplementary Data 1). Results are expressed as mean ± SEM. Statistical differences between \nendometrium and endometriotic lesions  were assessed using Wilcoxon matched-pairs signed-rank test, \ntwo-way ANOVA with Tukey’s multiple comparison tests, as appropriate.  Spearman’s correlation \ncoefficients and simple linear regression were used for correlation analyses (*: P < 0.05; **: P < 0.01; ***: \nP < 0.001). \n \nAuthor Contributions:  \nA.G., M.R., P.S performed the experiments, analyzed the data and contributed to the draft manuscript. \nAB and N.V.A performed immunostainings and RNAscope multiplex and helped with TMAs. K.B. helped \nwith immunofluorescence stainings. LB conducted the bioinformatic re -analysis of the scRNA -seq data. \nE.C., A.W provided material and helped to edit the manuscript. F.L., and E.C. analyzed and critically \nreviewed the data, obtained funding and wrote the manuscript. P.L. helps with funding. F .L and E.C. \ndesigned, conceived and supervised the study. F.L. wrote the manuscript with input of LB, CF and JFA, and \nediting was performed by all the authors. \n \nReporting summary \nFurther information on research design is available in the Nature  Portfolio Reporting Summary linked to \nthis article. \n \nData availability:  \nSupplementary Table 1 provides information on patient status, menstrual cycle phase, and the analyses \nin which each sample was included, namely RNAscope (Figure 1 and Supplementary Fig. 6 ) or \nimmunohistochemistry (IHC; Figures 2–5). The raw data underlying the charts and graphs are provided in \nthe corresponding Source Data file, as indicated in the relevant figure legends. Results of the statistical \nanalyses are presented in Supplementary Data 1. All results associated with the datasets used to generate \nthe figures are available in Supplementary Data2. \n \nConflict-of -interest: \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nThe authors have declared that no conflict of interest exists. \n \nFundings \nThe work at Inserm U1297 was supported by the National Institute of Health and Medical Research \n(INSERM), University Paul Sabatier - Université de Toulouse; University Hospital Center of Toulouse, \nRégion-Occitanie-Midi-Pyrénées-GRAINE-ENDOTREAT, Urosphere, the Endofrance Association  and ANR-\n23-CE17-0010-01 (EDISON) . A. Gargaros was funded by INSERM and Région -Occitanie Midi -Pyrénées \nCEBBOC. A. Buffeteau was funded by Société Française d'Endocrinologie (SFE). P. Singla was supported by \nANR-23-CE17-0010-01 (EDISON) and K. Boriak obtained funding from ANR-PAUSE (Ukraine). \n \nAcknowledgements  \nWe thank P. Balaguer for providing the plasmid pSG5puro-ERβ and its empty vector. We acknowledge A. \nLucas and C. Bernis from the We-Met Functional Biochemistry Facility (INSERM U1297, Toulouse, France), \nR. Florès-Florès from the imaging plateau of the TRI platform Genotoul (INSERM U1297, Toulouse, France), \nDr. P. Brousset and Dr. Anne Gomez-Mascard from the Department of Pathological Anatomy and Cytology \nof IUCT, Toulouse for their help on Tissue collection, immunostainings and RNAscope ® technology. We \ngratefully acknowledge François-Xavier Frenois from the Imag’IN Platform of the University institute of \nCancer (https://www.ibisa.net/plateformes/imag-in-368.html, Toulouse) for the slide scanning of TMAs, \nSophie Péries from the CRB (centre de ressources biologiques, CHU Toulouse) for their help in \nanatomopathological analysis and generating the TMAs, Estelle Michau d and Lou -Na Redoute, from \nInserm U1297, for their help with RNAscope quantification. We gratefully acknowledge all the surgeons \nin the Department of Gynecological Surgery at the Toulouse University Hospital Center for their \ncontribution to this work. We would like to thank all women who participated in this study.  \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nReferences: \n \n1 Horne, A. W. & Saunders, P. T. K. SnapShot: Endometriosis. Cell 179, 1677 -1677 e1671 (2019). \nhttps://doi.org/10.1016/j.cell.2019.11.033 \n2 Zondervan, K. T.  et al.  Endometriosis. Nature reviews. Disease primers  4, 9 (2018). \nhttps://doi.org/10.1038/s41572-018-0008-5 \n3 Zondervan, K. T., Becker, C. M. & Missmer, S. A. Endometriosis. N Engl J Med 382, 1244-1256 (2020). \nhttps://doi.org/10.1056/NEJMra1810764 \n4 Saunders, P. T. K. & Horne, A. W. Endometriosis: Etiology, pathobiology, and therapeutic prospects. \nCell 184, 2807-2824 (2021). https://doi.org/10.1016/j.cell.2021.04.041 \n5 Chapron, C., Marcellin, L., Borghese, B. & Santulli, P. 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J Clin Endoc rinol Metab  96, E1746 -1755 (2011). \nhttps://doi.org/10.1210/jc.2011-0272 \n45 Nilsson, S., Koehler, K. F. & Gustafsson, J. A. Development of subtype-selective oestrogen receptor-\nbased therapeutics. Nat Rev Drug Discov 10, 778-792 (2011). https://doi.org/10.1038/nrd3551 \n46 Davezac, M. et al. The different natural estrogens promote endothelial healing through distinct cell \ntargets. JCI insight 8 (2023). https://doi.org/10.1172/jci.insight.161284 \n47 Patino-Garcia, D.  et al.  Estetrol Increases Progesterone Genetic Response without Triggering \nCommon Estrogenic Effects in Endometriotic Cell Lines and Primary C ultures. Biomedicines 11 \n(2023). https://doi.org/10.3390/biomedicines11041169 \n48 Zieba, A.  et al.  The Human Endometrium -Specific Proteome Defined by Transcriptomics and \nAntibody-Based Profiling. OMICS 19, 659-668 (2015). https://doi.org/10.1089/omi.2015.0115 \n49 Escande, A. et al. Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen \nreceptor alpha or beta. Biochem Pharmacol  71, 1459 -1469 (2006). \nhttps://doi.org/10.1016/j.bcp.2006.02.002 \n \n \n \n \n \n \n \n \n \n \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \n \n \n \n \n \n \n \n \n \n \nTable 1: Spearman’s correlation coefficients between ERα/PR, ERα/Ki-67 and ERα/AR expressions across \ntissue type, distinguishing epithelial and stromal compartments – ND: Not determined \nColor scale indicates the strength and direction of the correlation: dark blue represents strong positive \ncorrelations, light blue moderate p ositive correlations, grey weak correlations, and red negative \ncorrelations. ND: not determined. \n \n \nTissu Epithelium / \nStroma \nCorrelation \nERα / PR \nCorrelation \nERα / Ki-67 \nCorrelation \nERα / AR \nEndometrium \n(EUT) \nEpithelium 0.85  \n(P<0.0001) \n0.8  \n(P<0.0001) \n0.56  \n(P<0.001) \nStroma 0.88 \n(P<0.0001) \n0.47   \n(P<0.05) \n0.85  \n(P<0.0001) \nSuperficial \nLesion (SUP) \nEpithelium 0.91  \n(P<0.0001) \n0.6                    \nns \n0.55              \nns \nStroma 0.72        \n(P<0.05) \n0.3                  \nns \n0.78  \n(p<0.05) \nDeep Lesion     \n(DIE) \nEpithelium 0.43       \n(P<0.05) -0.04 0.3               \nns \nStroma 0.54     \n(P<0.01) 0.07 0.67 \n(P<0.001) \nEndometrioma \n(OMA) \nEpithelium 0.8                    \nns \n0.8                 \nns ND \nStroma 0.7                    \nns -0.051 0.5                \nns \n \n \n \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \n \n \n \n \n \n \n \n \nFigure Legends:  \nFigure 1.  Predominant ESR1 expression with limited ESR2 transcript detection in endometrium and \nendometriotic lesions \n(A) Representative haematoxylin and eosin (H&E)-stained tissue microarray (TMA) spot prepared from a \nparaffin-embedded deep endometriotic lesion.  \n(B-D) Fluorescent RNAscope hybridization for ESR2 (pink dots) and ESR1 (green dots) transcripts with DAPI \n(grey) In (B) ovarian follicle, (C) ovarian lesion and (D) endometrium from healthy women (HW) and \nwomen with endometriosis (EMS), together with matched SPE and DE lesions in the proliferative (upper \npanels) and secretory (lower panels) phases. Right panels show higher magnification of the boxed regions \nin the left panels. In B, the theca (T) and granulosa (G) cells are delineated. Scale bar, 50 µm for full images \nand 25 µm for insets.  \n(E-F) Quantification of ESR1 and ESR2 mRNA transcripts using the histoscore (H-score) method in epithelial \n(upper panels) and stromal (lower panels) compartments. (E) Endometrium from HW (n = 6 in P and n = 5 \nin S) and EMS (n = 9 in P and n = 4 in S) women. (F) SPE (n = 3 in P and n = 4 in S) and DE (n = 6 in P and \nn = 5 in S) lesions. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 \nby two-way ANOVA. P, proliferative phase, S, secretory phase. \n(G) Representative fluorescent in situ hybridisation for ESR2 (yellow) with DAPI (white) in endometrium \n(upper panel), together with anti-ERG immunostaining on a serial section. Scale bar, 50 µm \n(H-I) UMAP visualisation of major cell types identified using single -cell RNA sequencing (left panels) by \nFonseca et al. (2023) (H) and Garcia-Alonso et al. (2023) (I), showing ESR1 (middle panels) and ESR2 (right \npanels) mRNA expression in endometrium and lesions of women with endometriosis. \n(J-K) Mean ESR1 and ESR2 transcript expression in epithelial cells across the menstrual cycle (J) and across \nthe major cell types analysed (K) using datasets from Fonseca et al. (2023). \n \nFigure 2. ERβ protein is expressed at low levels in endometriosis lesions, including ovarian lesions \n(A) Immunohistochemical staining of MDA -MB-231 cells transfected with ESR2 cDNA or control vector, \nused as a positive control, with two anti-ERβ antibodies (PPZ0506 and CWKF12). Scale bar, 50 µm.  \n(B) Left panel: fluorescent RNAscope hybridization of an ovarian follicle used as a positive control, showing \nESR2 (red dots) and ESR1 (green dots) transcripts with DAPI (blue). Middle and right panels: \nimmunohistochemical staining of an ovarian follicle using anti-ERα antibody (SP1) and anti-ERβ antibodies \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \n(CWKF12, PPZ0506) as indicated. The lower panels shows higher magnification of the boxed region. Brown \nindicates positive immunohistochemical staining a nd blue indicates haematoxylin counterstain. Arrows \nindicate examples of cells with positive nuclear staining. The theca (T) and granulosa (G) cells are \ndelineated. Scale bar, 50 µm for full images and 25 µm for insets.  \n(C) Representative images of immuno histochemical staining of endometrium from women with \nendometriosis (EMS) or without endometriosis (HW), and of SPE, DE and OMA lesions, using anti -ERβ \nantibody (PPZ0506). Insets show higher magnification of the boxed regions. Scale bar, 50 µm. Arrows \nindicate examples of cells with positive nuclear staining. Scale bar, 50 µm for full images and 25 µm for \ninsets.  \n(D) Quantification of ERβ-positive cells (%) in epithelial and stromal compartments of endometrium from \nwomen with endometriosis (EMS; n = 10 in P, n = 4 in ES and n = 4 in LS) or without endometriosis (HW; \nn = 6 in P, n = 3 in ES and n = 6 in LS) across the menstrual cycle (left panels), and in epithelial and stromal \ncompartments of SPE (n = 4 in P and n = 4 in S), DE (n = 13 in P and n = 9 in S) and OMA (n = 1 in P and n = 4 \nin S) lesions compared with endometrium from women with endometriosis (right panels).  \nP, proliferative phase; ES, early secretory phase; LS, late secretory phase and S, secretory phase (combined \nES + LS). Data are presented as mean ± SEM. Statistical differences between groups were assessed by two-\nway ANOVA. \n \nFigure 3. Hormone receptor expression and proliferation are largely unchanged in endometrium from \nwomen with or without endometriosis, except for stromal PR in the late secretory phase \n(A) Representative images of immunohistochemical staining of serial endometrial sections from women \nwithout endometriosis (left panels) or with endometriosis (right panels) using anti-ERα (SP1), anti-PR (1E2) \nand anti-Ki-67 (30-9) antibodies during the proliferative (P), early secretory (ES) and late secretory phase \n(LS). Scale bar, 50 µm.   \n(B) Quantification of ERα, PR and Ki -67-positive cells (%) in epithelial (l eft panels ) and stromal ( right \npanels) compartments of endometrium from women  without endometriosis (HW; n=15 including n=6 in \nP, n=3 in ES, n=6 in LS) or with endometriosis (EMS; n=21 or 22 including n=14 for ER α or Ki-67 or n=15 \nfor PR in P, n=7 in ES, and n=8 in LS).  \nData are depicted by mean ± SEM. Statistical differences between groups were assessed by 2-way ANOVA \nwith Tukey’s multiple comparison test. * P<0.05; ** P<0.01; ***P<0.001; **** P<0.0001 \n \nFigure 4. ERα remains the predominant estrogen receptor in endometriotic lesions \n(A) Representative images of immunohistochemical staining of matched eutopic endometrium (EUT) and \nlesions (SPE, DE, OMA) from the same patients with EMS during the proliferative (left panels) and \nsecretory (right panels) phases, using anti-ERα (SP1), anti-PR (1E2) and anti-Ki-67 (30-9) antibodies. Scale \nbar, 50 µm.   \n(B) Quantification of ERα, PR and Ki -67-positive cells (%) in epithelial (upper panels) and stromal (lower \npanels) compartments of eutopic endometrium (EUT), SPE, DE and OMA lesions from women with \nendometriosis across the proliferative  (P) and secretory (S) phases. Data are presented as mean ± SEM. \nStatistical differences between groups were assessed by two -way ANOVA. *P < 0.05; **P < 0.01; \n***P < 0.001; ****P < 0.0001. \nSample sizes were as follows: EUT, n = 14 for ERα and Ki-67 and n = 15 for PR in P, and n = 14 for all markers \nin S; SPE, n = 5 for ERα and PR and n = 14 for Ki-67 in P, and n = 5 for all markers in S; DE, n = 5 for ERα and \n\nARTICLE IN PRESS\nARTICLE IN PRESS\n \n \n \nPR and n = 14 for Ki-67 in P, and n = 16 for ERα and Ki-67 and n = 17 for PR in S; OMA, n = 3 for all markers \nin P and n = 4 for all markers in S. \n \nFigure 5. Paired comparison of ERα, PR, Ki -67 and PR expression between endometriotic lesions (SPE, \nDE and OMA) and matched eutopic endometrium from women with endometriosis  \nLesions were compared with matched eutopic endometrium obtained from the same patient. \n(A-H) Left panels: comparison of ERα, PR, Ki-67 and AR-positive cells (%) in epithelial (A,C,E,G) and stromal \n(B,D,F,H) compartments between eutopic endometrium and matched SPE lesions (n = 8 for ER α and PR; \nn = 6 for Ki -67; n = 7 for AR), DE lesions (n = 34 for ER α; n = 35 for PR; n = 32 for Ki -67; n = 33 for AR) or \nOMA lesions (n = 6 for ER α, PR and Ki -67; n = 7 for AR). Data are shown as individual paired values \npresented as mean ± SEM. Right panels:  difference in the percentage of ER α, PR, Ki -67 and AR -positive \ncells in epithelial and stromal compartments between lesions and matched eutopic endometrium from \nthe same patient (lesion − EUT). Number of paired samples: SPE, n = 8; DE, n = 22; OMA, n = 4.  Data are \nshown as individual paired values, with paired differences presented as mean ± SEM.  \nPositive values indicate higher expression in lesions relative to matched eutopic endometrium. Statistical \ndifferences were assessed by two -sided Wilcoxon signed -rank test. *P < 0.05; **P < 0.01; ***P < 0.001; \n****P < 0.0001. \n \n \n \n \n \n\nARTICLE IN PRESS\n0.2\n0.0\n0.1\n0.3\nP S P S P S P S\n0\n100\n200\n300\nHscore\nP S P S P S P S\n0\n100\n200\n300\nHscore\nEutopic endometrium and lesions (EMS)\nAProliferativeSecretory\nEndometrioma\nNucleus / ESR1 / ESR2\nNucleus / ESR1 / ESR2\nOvarian follicle\nHscore: Granulosa cells\nESR1: 139  &ESR2: 111\nHscore: Epithelium (ESR1: 132 & ESR2:0)\nStroma (ESR1: 36 and ESR2: 3)\nB\nD\nE\nI\nC\nF\nSuperficial lesion (SPE)\nNucleus / ESR1 / ESR2\nDeep lesion (DE)\nNucleus / ESR1 / ESR2\nEndometrium (EUT)\nNucleus / ESR1 / ESR2\nEndometrium\nNucleus / ESR1 / ESR2\nHeathly woman (HW)\nJ\nK\nESR1 ESR2\nOMAEUT\n0.000\n0.005\n0.010\n0.015\n0.0\n0.1\n0.2\n0.3\n0.4\n0.5\nmean_expression\nMenstrual.Cycle\nProliferative\nSecretory\nECT EUT ECT OMA\nH\nEndometriotic woman (EMS)\nEndometrium (HW vs EMS)\nSPEEUT DE\nG\nEpithelium\nESR2ESR1\nmean_expression\nESR2ESR1\nStromal \ncompartement\nESR2ESR1\nEMSHWn\nEpitheliumStromal \ncompartement\nPhase : **** ; Statut : ns; \nInteraction : ns\nPhase : ns ; Statut : ns; \nInteraction : ns\nPhase : *** ; Statut : ns; \nInteraction : ns\nPhase : ns ; Statut : *; \nInteraction : ns\nP S P S\n0\n5\n10\n15\n20\nHscore\nP S P S\n0\n5\n10\n15\n20\nHscore\nP S P S P S P S P S P S\n0\n100\n200\n300\nHscore\nP S P S P S P S P S P S\n0\n100\n200\n300\nHscore\nPhase : **** ; Tissue : ns; \nInteraction : ns\nPhase : ns ; Tissue : ns; \nInteraction : ns\nPhase : ** ; Tissue : ns; \nInteraction : ns\nPhase : ns ; Tissue : ns; \nInteraction : ns\nP S P S P S\n0\n5\n10\n15\nHscore\nP S P S P S\n0\n5\n10\n15\nHscore\nT\nG\nNucleus / ERG\nNucleus / ESR2\n50µm 50µm\n25µm25µm\n50µm\n50µm\n50µm\n50µm\n50µm\n50µm\n50µm\n50µm\n25µm\n25µm\n25µm\n25µm\n25µm\n25µm\n25µm\n25µm\nARTICLE IN PRESS\n\nARTICLE IN PRESS\nESR1 / ESR2\n ERα\n ERβ (PPZ0506)ERβ (CWK-F12)\nT\nG\nT\nG\nT\nG\nT\nG\nT\nG\nT\nG\nP ES LS P ES LS\n0\n2\n4\n6\n8\n10\n% ERβ+ cells\nP ES LS P ES LS\n0\n2\n4\n6\n8\n10\n% ERβ+ cells\nP S P S P S P S\n0\n2\n4\n6\n8\n10% ERβ+ cells\nP S P S P S P S\n0\n2\n4\n6\n8\n10% ERβ+ cells\nBA\nOvarian lesion (OMA)\nDeep lesion (DE)\nEndometrium\n Superficial lesion (SPE)\nEndometrium\nC Endometriotic woman (EMS)Heathly woman (HW)\nMDA-MB-231 ERβ+\n MDA-MB-231 ERβ-\nERβ (PPZ0506) ERβ (CWK-F12)\nD\nEpithelium Stromal \ncompartment Stromal compartmentEpithelium\nEndometrium\nEUT SPE DE OMA\nEutopic endometrium and endometriotic lesions\nHW EMS\nPhase : ns ; Statut : ns; \nInteraction : ns\nPhase : ns ; Statut : ns; \nInteraction : ns\nPhase : ns ; Statut : ns; \nInteraction : ns\nPhase : ns ; Statut : ns; \nInteraction : ns\nHW EMS EUT SPE DE OMA\n25µm 25µm 25µm 25µm\n25µm 25µm 25µm 25µm 25µm\nARTICLE IN PRESS\n\nARTICLE IN PRESS\nP ES LS P ES LS\n0\n25\n50\n75\n100\n% ERα+ cells\nP ES LS P ES LS\n0\n25\n50\n75\n100\n% PR+ cells\nP ES LS P ES LS\n0\n25\n50\n75\n100\n% Ki-67+ cells\nP ES LS P ES LS\n0\n25\n50\n75\n100\n% ERα+ cells\nP ES LS P ES LS\n0\n25\n50\n75\n100\n% PR+ cells\n✱✱\n✱✱✱\n✱✱✱\nP ES LS P ES LS\n0\n25\n50\n75\n100\n% Ki-67+ cells\nProliferative (P) Early secretory (ES) Late secretory (LS)\nHealthy woman (HW) Endometriotic woman (EMS)\nA\nB\nProliferative (P) Early secretory (ES) Late secretory (LS)\nERα\nPR\nKi-67\nStromal compartmentEpithelium\nERα PR Ki-67 ERα PR Ki-67\nHW \nEMS\nPhase : **** ; Statut : ns; \nInteraction : ns\nPhase : **** ; Statut : ns; \nInteraction : ns\nPhase : *** ; Statut : ns; \nInteraction : ns\nPhase : ** ; Statut : ns; \nInteraction : ns\nInteraction : * Phase : ns ; Statut : ns; \nInteraction : ns\nARTICLE IN PRESS\n\nARTICLE IN PRESS\nA Proliferative Secretory\nB\nERα PR Ki-67\nEUT\nOMA\nDE\nSPE\nERα PR Ki-67\nStromal \ncompartment Epithelium\nERα PR Ki-67\nEUT\nSPE\nDE\nOMA\nEUT\nSPE\nDE\nOMA\nP S P S P S P S\n0\n25\n50\n75\n100\n% ERα+ cells\nP S P S P S P S\n0\n25\n50\n75\n100\n% PR+ cells\n✱✱✱\n✱✱\n✱✱✱\n✱✱✱ ✱✱\nP S P S P S P S\n0\n25\n50\n75\n100\n% Ki-67+ cells\nP S P S P S P S\n0\n25\n50\n75\n100\n% ERα+ cells\nP S P S P S P S\n0\n25\n50\n75\n100\n% PR+ cells\nP S P S P S P S\n0\n25\n50\n100\n% Ki-67+ cells\nPhase : ns ; Tissue : ns; \nInteraction : ns\nInteraction : * Phase : p=0.065 ; Tissue : ns; \nInteraction : ns\nPhase : ns ; Tissue : *; \nInteraction : ns\nPhase : * ; Tissue : * ; \nInteraction : ns\nPhase : ns ; Tissue: ns; \nInteraction : ns\nARTICLE IN PRESS\n\nARTICLE IN PRESS\nARTICLE IN PRESS\n0\n20\n40\n60\n80\n100\n% ERα+ cells\n✱ p = 0.0625\n-100\n-50\n0\n50\n% ERα+ cells lesions - EUT\n0\n20\n40\n60\n80\n100\n% ERα+ cells\n✱✱✱✱✱\n-100\n-50\n0\n50\n100\n% ERα+ cells lesions - EUT\n0\n20\n40\n60\n80\n100\n% PR+ cells\n✱\n-50\n-25\n0\n25\n50\n100\n% PR+ cells lesions - EUT\n0\n20\n40\n60\n80\n100\n% PR+ cells\n✱\n-50\n-25\n0\n25\n50\n% PR+ cells lesions - EUT\n0\n20\n40\n60\n80\n100\n% Ki-67+ cells\n✱✱✱✱p=0.0625\n-100\n-50\n-50\n0\n50\n50\n100\n% Ki-67+ cells lesions - EUT\n0\n10\n20\n30\n40\n50\n50\n100\n% Ki-67+ cells\n✱✱\n-40\n-20\n0\n20\n40\n% Ki-67+ cells lesions - EUT\n0\n5\n10\n15\n20\n50100\n% AR+ cells\n✱\n0\n5\n10\n10\n30\n50\n% AR+ cells lesions - EUT\n0\n20\n40\n60\n80\n100\n% AR+ cells\np=0.0625\n-50\n-25\n0\n25\n50100\n% AR+ cells lesions - EUT\nEpithelium Stromal compartmentA\nC\nG\nE\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\nB\nF\nD\nH\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\nEUT\nSPE\nDE\nOMA\nSPE - EUT\nDE - EUT\nOMA - EUT\n\nARTICLE IN PRESS\nARTICLE IN PRESS","source_license":"public-domain-us","license_restricted":false}