Aberrant progesterone receptor (PGR) expression is associated with estrogen receptor 1 (ESR1) expression in the endometrium from infertile women with endometriosis

To examine the levels of ESR1 and PGR, we performed immunohistochemical analysis in endometrium samples of 9 fertile women without endometriosis (controls) and 11 infertile women with endometriosis. Our immunohistochemistry results showed lower ESR1 protein levels in the endometrial stromal cells from infertile women with endometriosis compared to controls ( Figure 1A ). Semi-quantification analysis confirmed that ESR1 levels were significantly decreased in endometrial stroma from infertile women with endometriosis (mean ± SD; 54.10 ± 29.98) compared to controls (127.56 ± 12.60, p < 0.001) ( Figure 1B ). In contrast, the expression of ESR1 in endometrial epithelium was not different between controls (125.72 ± 22.02) and infertile women with endometriosis (129.7 ± 66.75) ( Figure 1B ). To further evaluate the relationship between ESR1 and PGR expression, we performed immunohistochemical analysis of PGR on serial sections from the same samples. As expected, epithelial PGR was not detected in endometrium of controls, whereas marked epithelial PGR expression was observed in the endometrium of infertile women with endometriosis ( Figure 1C ). Semi-quantitative analysis revealed a trend toward reduced stromal PGR expression in infertile women with endometriosis (105.40 ± 61.20) compared with controls (144.72 ± 27.29), although this difference did not reach statistical significance ( p = 0.092) ( Figure 1D ). In contrast, epithelial PGR levels were significantly increased in the infertile group (141.55 ± 88.45) relative to controls (31.61 ± 26.03, p = 0.002) ( Figure 1D ). Notably, two abnormal patterns of PGR expression were observed among infertile women with endometriosis. Four of eleven samples (36.4%) exhibited weak stromal PGR expression, while eight of eleven samples (72.7%) displayed strong epithelial PGR expression ( Figure 1D ). Next, we performed a correlation analysis of ESR1 and PGR expression in the endometrial stroma and epithelium from control women and infertile women with endometriosis ( Figure 1E ). Our analysis revealed a significant positive correlation (correlation coefficient = 0.4509, p < 0.01) between ESR1 and PGR in endometrial stroma. Although ESR1 levels in the endometrial epithelium were not significantly different between controls and infertile women with endometriosis, a positive correlation was observed between ESR1 and PGR expression in the endometrial epithelium (correlation coefficient = 0.2227, p < 0.05). These findings suggest that aberrant overexpression of PGR may be associated with ESR1 in epithelial cells from infertile women with endometriosis. Multiplexed immunofluorescence is an imaging platform that enables the simultaneous detection of multiple proteins, allowing for visualization of signal activity within tissues ( Gerdes et al. , 2013 ). To investigate the relationship between aberrant PGR overexpression and ESR1 expression in epithelial cells from infertile women with endometriosis, we analyzed the endometrial cell states of ESR1 and PGR in the human endometrium. Specifically, three endometrial samples from infertile women with endometriosis were selected for multiplex immunofluorescence analysis based on the presence of strong epithelial PGR expression, as determined by prior immunohistochemical evaluation. ESR1 expression was not used as a selection criterion, as ESR1 upregulation was analyzed as an outcome variable rather than a predefined stratification parameter, and because of limited availability of additional endometriosis samples suitable for multiplex analysis. We then performed multiplex immunofluorescence assay on endometrial tissue from control (n = 3) and infertile women with endometriosis (n = 3) at mid-secretory phase using antibodies for Pan-Keratin (epithelial marker), CD10 (stromal marker), PGR and ESR1. First, endometrial epithelial cells and stromal cells were defined with Pan-Keratin (yellow) and CD10 (cyan), respectively. Our results of multiplex immunofluorescence analysis revealed distinct patterns of PGR and ESR1 expression between control and infertile women with endometriosis. In the control group ( Figure 2A ), PGR (green) was strongly detected in the stromal cells and ESR1 (red) was detected in both stroma and epithelium. While there was remarkable colocalization of PGR and ESR1 in endometrial stromal cells, there was no endometrial epithelial cell with colocalization of PGR and ESR1 ( Figure 2A ). In infertile women with endometriosis group ( Figure 2B ), aberrant overexpression of PGR and ESR1 was observed in endometrial epithelial cells. Interestingly, aberrantly strong PGR expression was associated with strong ESR1 expression in epithelium ( Figure 2B , displayed as a yellow merged signal) of infertile women with endometriosis. In addition, there were only a few endometrial stromal cells with colocalization of PGR and ESR1. For enhanced precision, we performed quantitative and correlation analysis of ESR1 and PGR in our multiplex immunofluorescent assay using digital image analysis software powered by artificial intelligence (AI) - based deep learning. Initially, ROIs were manually defined to segment epithelial and stromal cells. The investigated specimen size was 0.160 ± 0.025 mm 2 per sample for the control group and 0.150 ± 0.023 mm 2 per sample for the endometriosis group. Subsequently, an AI deep learning model was applied to accurately detect and analyze nuclei within these ROIs. The algorithm accurately identified individual nuclei and quantified the absolute fluorescence intensity (marked as ESR1 and PGR) for each nucleus. We examined a quantitative analysis of 4,951 stromal cells (mean ± SD: 1,475 ± 274 per sample) and 2,378 epithelial cells (654 ± 147 per sample) from the control group and 3,593 stromal cells (1,051 ± 185 per sample) and 2,877 epithelial cells (827 ± 218 per sample) from the infertile women with endometriosis at mid-secretory phase. The data were analyzed at the single-cell level using advanced quantification methods. Stromal and epithelial cell populations were visualized, showing distinct clusters with clear positive trends for ESR1 and PGR co-expression. We found positive correlations between ESR1 and PGR in stromal and epithelial cells. In stromal cells, the correlation coefficient was r = 0.4546 for controls and r = 0.6291 for infertile women with endometriosis group ( Figure 2C ). In epithelial cells, the correlation coefficient was r = 0.05631 for control and r = 0.6676 for infertile women with endometriosis group ( Figure 2D ). Notably, the correlation strength was higher in infertile women with endometriosis group compared to controls, particularly in epithelial cells, suggesting enhanced co-regulation of ESR1 and PGR in endometriosis pathological conditions. These findings suggest a potential mechanism by which dysregulation of the hormone receptor pathways involving ESR1 and PGR may contribute to the development of endometriosis and its associated infertility.

The study was approved by the Institutional Review Board of Atrium Health Wake Forest Baptist (Human Protocol: IRB00057549). Informed written consent was obtained from all participants. The study design and conduct complied with all relevant regulations regarding the use of human participants and were conducted in accordance with the criteria set by the Declaration of Helsinki. For experiments examining expression patterns of ESR1 and PGR in the endometrium, we used samples of human eutopic endometrium from infertile women with endometriosis (n = 11) and fertile disease-free control women (n = 9) during the mid-secretory phase. Endometrial biopsies were obtained at the time of surgery from women between the ages of 18 and 45 years with regular menstrual cycles. The presence or absence of endometriosis was confirmed during surgery. All women in the endometriosis-positive group carried a diagnosis of infertility, and none of the control women had infertility despite undergoing surgery for non-endometriosis indications. Control endometrial tissues were laparoscopically confirmed to be negative for endometriosis and had not been exposed to any hormonal therapies for at least three months prior to surgery. All patients with endometriosis-associated infertility underwent endometrial sampling prior to the surgical removal of endometriosis. Use of an intrauterine device or hormonal therapies in the 3 months preceding surgery was exclusionary for this study. Histological dating of endometrial samples was done based on the criteria of Noyes (50) and confirmed by subsequent histopathological examination by an experienced fertility specialist (B.A.L.). Endometrial biopsies were transferred into 10% neutral buffered formalin immediately after surgical collection. Tissues were fixed for approximately 18–24 hours at room temperature, then processed through graded ethanol and xylene, paraffin-embedded and sectioned at 5 μm. Paraffin sections were mounted on positively charged glass slides and dried before immunostaining. Sections were deparaffinized, and rehydrated in a series of graded alcohols [Xylene 3 × 5min (Fisher, Pittsburgh, PA, USA), 100% ethanol 3 × 3min (Fisher), 95% ethanol 2 × 2min, 70% ethanol 2 × 2min]. Antigen retrieval was performed using an antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) diluted 1:100 in distilled water. Slides were microwaved for 5 min at 70% power and 10 min at 30% power, followed by cooling at room temperature for 30 min. Alternatively, antigen retrieval was performed using an electric steamer (preheated for 3 min) for 4 min at high pressure. They were then incubated with 10% normal goat serum (NGS) (Vector Laboratories, Burlingame, CA, USA; S1000–20) in phosphate-buffered saline (PBS) (pH 7.5) for 2 h at room temperature in a humid chamber. Sections of endometrial tissues from n=11 infertile women with endometriosis and n=9 controls were then exposed to primary antibodies, ESR1 (Cell Signaling Technology Inc., Danvers, MA, USA; #8644) and PGR (Cell Signaling Technology Inc.; #8757S), which were diluted 1:500 in 10% NGS in PBS (pH 7.5) and incubated overnight at 4 °C. On the second day, sections were washed using PBS and incubated with secondary antibody (Vector Laboratories; Biotinylated anti-rabbit IgG BA-9200) for 1 h at room temperature. Next, horseradish peroxidase (HRP) (1:1000 dilution in sterile-filtered 1× PBS) streptavidin conjugate (Invitrogen, Carlsbad, CA, USA; 43–4323) was applied for 45 min at room temperature, followed by using the Vectastain Elite DAB kit (Vector Laboratories, Cat. #SK-4100) for signal development. Slides were then counterstained with hematoxylin (Fisher Scientific Inc., Hampton, NH, USA; Volu-sol; #VMH-032) and 1% lithium carbonate (Poly Scientific; #S127–1GL), followed by dehydration through an ethanol series [2 × 80% ethanol, 1 min each, 2 × 90% ethanol, 1 min each, 3 × 100% ethanol, 1 min each, 3× xylene, 1 min each]. After dehydration, images were acquired using the Akoya Biosciences PhenoImager HT (Akoya Biosciences, Marlborough, MA, USA), an automated imaging system for performing whole slide scans. The tissue sections were scanned in brightfield mode using a 20× objective at a resolution of 0.5 μm/pixel, generating high-resolution image files for subsequent analysis. For the multiplex immunofluorescence analysis, three control samples were randomly selected, whereas three endometriosis samples were selected based on the presence of distinct epithelial PGR expression profiles identified in prior immunohistochemical analyses. Multiplex immunofluorescence was conducted to examine the expression of Pan-Keratin, CD10, ESR1, and PGR using the Opal ™ 7-Color IHC Kit (Akoya Biosciences; NEL811001KT) according to the manufacturer’s instructions. Uterine tissue sections (5 μm) were deparaffinized in xylene and hydrated with graded alcohols, followed by fixation in 10% neutral buffered formalin (Epredia, Kalamazoo, MI, USA; #9400–1) for 20 minutes. After rinsing with distilled water, the sections were treated in an appropriate concentration of 10X AR6 buffer (Akoya Biosciences; #AR600250ML) at 95°C for 15 minutes using a microwave, then rinsed in Tris buffered saline with Tween 20 (TBST). The sections were cooled down at room temperature for 30 minutes. Subsequently, the sections were incubated with 1X Antibody Diluent/Block solution (Akoya Biosciences; #ARD1001EA) at room temperature for 10 minutes. The sections were then exposed to primary antibodies diluted in 1X Antibody Diluent/Block solution (Akoya Biosciences; #ARD1001EA), including CD10 (1:100, Cell Signaling Technology Inc.; #65534), Pan-Keratin (1:100, Cell Signaling Technology Inc.; #4545), ESR1 (1:100, Cell Signaling Technology Inc.; #13258), and PGR (1:200, Cell Signaling Technology Inc.; #8757) and incubated for 1 hour at room temperature. After washing the section with TBST, the sections were treated with 1X Opal Anti-MS + Rb HRP (Akoya Biosciences; #ARH1001EA) for 10 minutes at room temperature and then rinsed in TBST. Following this, the sections were incubated for 10 minutes at room temperature with Opal 480 (Akoya Biosciences; #FP1500001KT), 520 (Akoya Biosciences; #FP1487001KT), 570 (Akoya Biosciences; #FP1488001KT), and 690 (Akoya Biosciences; #FP1497001KT) fluorophores diluted 1:200 in 1X Plus Manual Amplification Diluent (Akoya Biosciences; #FP1498) and then rinsed in TBST. The sections were again treated in an appropriate concentration of 10X AR6 buffer (Akoya Biosciences; #AR600250ML) at 95°C for 15 minutes using a microwave, rinsed in TBST, and exposed to 10X Spectral DAPI (Akoya Biosciences; #FP1490) diluted in TBST for 5 minutes and rinsed with TBST and distilled water at room temperature. Finally, the sections were mounted using ProLong ™ Diamond Antifade Mountant (Invitrogen; # P36970 ) and scanned with the Phenolmager ® HT 2.0 (Akoya Biosciences). For semiquantitative analysis of IHC, DAB-positive (positive) cells were classified into DAB staining intensity (+1(weak), +2(moderate), or +3(strong)). H-score was calculated using the following formula: H-score value = (1* percentage of weak staining cells (+1)) + (2 * percentage of moderate staining cells (+2)) + (3 * percentage of strong staining cells (+3)), varying from 0 to 100% of each grade cell population. The overall score of data ranged from 0 to 300 ( Budwit-Novotny et al. , 1986 ). For quantitative analysis of multiplex immunofluorescence, Visiopharm (Visiopharm Integrator System, Hoersholm, Denmark), which is a digital image analysis software using an artificial intelligence algorithm, was used for the semiquantitative grading system (H-score) for IHC results. The deep learning application in Visiopharm ver. 2025.08.1 (Visiopharm Integrator System, Hoersholm, Denmark) was used to recognize nuclei stained for ESR1 and PGR and to generate cell population data in both the epithelium and stroma. During the post-processing step, IHC-stained cells were classified as DAB-positive (positive) or hematoxylin-positive (negative) and further assigned to epithelial or stromal compartments. Endometrial epithelial and stromal cells were manually segmented with the region of interest (ROI) drawing tool ( Supplementary Figure S1 ). To distinguish epithelial and stromal compartments, regions of interest (ROIs) were manually annotated using the built-in drawing tools in Visiopharm. Epithelial areas were marked with blue solid lines, and stromal regions with red solid lines ( Fig. S1A ). Nuclear segmentation was performed using the “Nuclear Detection, AI (Fluorescence)” App (#10169, Visiopharm, Hoersholm, Denmark), guided by DAPI counterstaining ( Fig. S1B – C ). Each segmented nucleus was defined as an individual object, enabling per-cell analysis of marker expression. To quantify fluorescence intensity for ESR1 and PGR, we used the object-level output variable “MP: Multiplexing of All Labels in Classified image” to extract absolute fluorescence values for each nucleus. Specific fluorophores used were Opal 520 for PGR and Opal 690 for ESR1. For each nucleus, we calculated the mean fluorescence intensity of ESR1 and PGR signals within the nuclear region. These values were normalized within each image to minimize variability caused by regional staining differences or section thickness. Only cell objects that met predefined size and shape criteria were included, ensuring that partial or artifact-prone nuclei were excluded. The extracted single-cell data were used to generate co-expression plots of ESR1 and PGR (Fig. 4), allowing detailed assessment of hormone receptor dynamics in a cell type-specific context. An overview of tiled whole-slide DAPI image was used to visualize the global segmentation coverage ( Fig. S1D ). Epithelial areas were marked with blue solid lines, and stromal regions with red solid lines. Nuclear segmentation was performed using the “Nuclear Detection, AI (Fluorescence)” App (Visiopharm Integrator System; #10169), guided by DAPI counterstaining. Each segmented nucleus was defined as an individual object, enabling per-cell analysis of marker expression. To quantify fluorescence intensity for ESR1 and PGR, we used the object-level output variable “MP: Multiplexing of All Labels in Classified image” to extract absolute fluorescence values for each nucleus. Specific fluorophores used were Opal 520 for PGR and Opal 690 for ESR1. For each nucleus, we calculated the mean fluorescence intensity of ESR1 and PGR signals within the nuclear region. These values were normalized within each image to minimize variability caused by regional staining differences or section thickness. Only cell objects that met predefined size and shape criteria were included, ensuring that partial or artifact-prone nuclei were excluded. The extracted single-cell data were used to generate co-expression plots of ESR1 and PGR, allowing detailed assessment of hormone receptor dynamics in a cell type-specific context. An overview of tiled whole-slide DAPI image was used to visualize the global segmentation coverage and then analyzed over 2,000 single cells using multiplex immunofluorescence imaging to investigate the correlation between ESR1 and PGR. Cells were selected based on predefined criteria, including minimal gland size, presence of lumen, and absence of artifacts, which were discarded prior to analysis. To assess the correlation coefficient between fluorescence signals, we calculated the Pearson correlation coefficient (PCC). PCC was calculated using GraphPad Prism (version 10, San Diego, CA, USA). To assess statistical significance of parametric data, we used unpaired, two-tailed Student’s t -tests for two groups. Statistical analyses were performed by GraphPad Prism. A p-value < 0.05 was considered statistically significant.

Our study systematically examined expression profiles of ESR1 in infertile women with endometriosis and fertile women without disease during the mid-secretory phase. By integrating immunohistochemistry with multiplex immunofluorescence and AI-assisted single-cell quantification, we identified a significant positive correlation between ESR1 and PGR in both epithelial and stromal compartments. Although alterations in ESR1 or PGR have been individually described in endometriosis, the coordinated expression of these receptors within the same cellular environments, particularly in infertile women, has not been well characterized. Our findings provide new evidence linking receptor co-regulation with endometriosis-associated infertility and highlight the importance of disrupted steroid hormone signaling in the nonreceptive endometrium. Despite considerable progress in understanding endometriosis, the mechanisms underlying endometriosis-associated infertility remain incompletely understood and are likely multifactorial ( Bonavina and Taylor, 2022 ). Diagnostic delays of 4 to 11 years ( Koninckx et al. , 1991 ) further complicate timely management. Among the proposed mechanisms, P4 resistance in the eutopic endometrium is one of the most widely recognized contributors to impaired receptivity. Reduced responsiveness to P4, together with dysregulated PGR expression, has been consistently documented in women with endometriosis ( Marquardt et al., 2019 , Moberg et al. , 2015 ). In our cohort, infertile women with endometriosis exhibited decreased stromal ESR1 expression, aberrant epithelial ESR1 expression, with a notable bimodal distribution in approximately four samples, and marked epithelial PGR overexpression, consistent with previous reports. The strong positive correlation between ESR1 and PGR in epithelial cells of infertile women with endometriosis suggests that aberrant ESR1 activity may contribute to inappropriate induction of PGR in this compartment, a feature associated with impaired receptivity. Normally, ESR1 is highly expressed during the proliferative phase and downregulated during the window of implantation, while PGR becomes enriched in the stroma and downregulated in the epithelium to support receptivity ( Lessey and Kim, 2017 , Lessey et al., 2006 ). Disruption of this cyclical regulation compromises the cellular transitions necessary for implantation. Our findings align with prior studies demonstrating reduced stromal ESR1 and increased epithelial ESR1 in infertile women with endometriosis ( Lessey et al., 2006 ). Overexpression of ESR1 is associated with reduced integrin beta 3 expression, that is a well-established marker of uterine receptivity ( Lessey et al., 2006 ). Therefore, dysregulated ESR1 expression may represent a key step in the establishment of P4 resistance and implantation failure. ( Lessey et al., 2006 ; Lessey and Kim, 2017 ). Given that ESR1 transcriptionally induces PGR during the proliferative phase, and considering our observed correlation patterns, ESR1 may play a contributory role in driving aberrant PGR expression in epithelial cells. However, we acknowledge that receptor regulation may be reciprocal and that our data do not establish causality. Future mechanistic studies are needed to determine whether ESR1 directly drives epithelial PGR overexpression in endometriosis or whether additional upstream factors modulate both receptors. We also acknowledge that our study did not distinguish between PGR isoforms (PR-A and PR-B), which have distinct and sometimes opposing functions. The PR-B/PR-A ratio represents the relative expression of PGR isoforms, with PR-A typically having inhibitory roles and PR-B activating gene transcription (Conneely et al., 2001; Lydon et al., 1995). The PR-A/PR-B ratio may therefore influence receptor correlations and downstream signaling. Although beyond the scope of the present study, investigating how ESR1 relates to PR-A/PR-B balance in endometriosis-associated infertility is an important next step. Our data were derived exclusively from mid-secretory phase samples, ensuring appropriate cycle matching among participants. However, subtle molecular heterogeneity within the mid-secretory interval may still exist, and this should be considered a limitation. The study did not include fertile women with endometriosis, which would help determine whether the receptor changes observed are driven by endometriosis itself or are specific to endometriosis-associated infertility. However, obtaining eutopic endometrial biopsies from fertile women with surgically confirmed endometriosis is extremely challenging, because these women rarely undergo surgery or endometrial sampling in the absence of infertility or significant pelvic pain. Future studies incorporating such a cohort would allow clearer distinction between disease-related and infertility-specific alterations. Additionally, multiplex immunofluorescence was performed on a selected subset of samples enriched for epithelial PGR overexpression to enable detailed assessment of receptor co-localization. Expanding multiplex analyses to include samples with a broader range of PGR and ESR1 expression patterns will be an important direction for future work. In summary, our study demonstrates a positive correlation between ESR1 and PGR expression in the eutopic endometrium of both women with and without endometriosis. Notably, aberrant PGR overexpression is strongly associated with elevated ESR1 levels in endometrial epithelial cells from infertile women with endometriosis. These findings highlight the importance of understanding the regulatory mechanisms of steroid hormone signaling in endometriosis-associated infertility. A clearer definition of these molecular pathways is essential for the development of targeted diagnostic tools and therapeutic interventions, as progress in these areas has been limited by the lack of well-characterized mechanisms underlying the disease.

Endometriosis is a chronic, estrogen-dependent disorder characterized by the presence of endometrial-like tissue outside the uterine cavity, affecting approximately 10% of reproductive-age women ( Wang et al. , 2020 ). Up to 30–50% of infertile women are diagnosed with endometriosis, making it one of the most common causes of infertility ( Macer and Taylor, 2012 ). Beyond pelvic adhesions and anatomical distortion, endometriosis disrupts the peritoneal and endometrial environments through persistent inflammation, altered immune responses, and aberrant steroid hormone signaling. The eutopic endometrium of women with endometriosis frequently exhibits progesterone (P4) resistance, characterized by reduced expression of progesterone receptor (PGR) and impaired decidualization, and diminished endometrial receptivity ( Lessey and Kim, 2017 , Marquardt et al. , 2019 , Marquardt et al. , 2023 ). These molecular and cellular alterations contribute to implantation failure and infertility, even in women without overt anatomical distortion. Therefore, defining how hormonal signaling pathways are disrupted in the eutopic endometrium in endometriosis is critical for understanding endometriosis-associated infertility. Endometrial receptivity is essential for embryo implantation and successful pregnancy ( Cha et al. , 2012 ). Endometrial receptivity is orchestrated by the cyclic actions of P4 and estrogen (E2), which act primarily through their nuclear receptors, estrogen receptor 1 (ESR1) and PGR ( Marquardt et al., 2019 , Vasquez and DeMayo, 2013 ). In the normal menstrual cycle, ESR1 expression is high during the proliferative phase, driving epithelial growth and inducing PGR expression. During the mid-secretory phase, the window of implantation, PGR becomes enriched particularly in the stromal compartment and is downregulated in the epithelial compartment, a coordinated pattern that supports differentiation and receptivity ( Lessey et al. , 1988 ). This tightly regulated, cell-type–specific interplay between ESR1 and PGR is essential for the transition from a proliferative to a receptive endometrium ( Moustafa and Young, 2020 ). In endometriosis, these spatiotemporal patterns of steroid receptor signaling are disrupted. Several studies describe reduced stromal PGR, attenuated P4-responsive gene expression, and blunted P4-induced decidualization that is collectively referred to as P4 resistance ( Burney et al. , 2007 , Fox et al. , 2016 , Kao et al. , 2003 , Lessey and Kim, 2017 , Lessey and Young, 2014 ). The aberrant overexpression or persistent presence of epithelial PGR during this critical period is associated with a non-receptive endometrium and infertility ( Lessey et al. , 1996 , Marquardt et al., 2019 ). In parallel, endometriotic stromal cells exhibit reduced ESR1 expression ( Yilmaz and Bulun, 2019 ), which can amplify E2-driven inflammation and prostaglandin production ( Chantalat et al. , 2020 ). These receptor alterations contribute to an endocrine environment dominated by E2 and insufficiently responsive to P4, ultimately compromising implantation ( Dorostghoal et al. , 2018 , Lessey and Kim, 2017 , Lessey and Young, 2014 , Marquardt et al., 2019 , Patel et al. , 2017 ). However, despite extensive studies of ESR1 or PGR individually, the relationship between these two receptors, particularly their coordinated expression within the same cellular compartments of the eutopic endometrium in infertile women with endometriosis, remains poorly defined. A deeper understanding of how ESR1 and PGR are co-regulated in mid-secretory phase endometrium may offer critical insight into the molecular basis of P4 resistance. Although prior studies have described receptor expression changes separately, no study has directly quantified ESR1 and PGR simultaneously within the same tissue sections using multiplex immunofluorescence and artificial intelligence–based single-cell analysis. Such an approach enables precise evaluation of receptor co-localization and correlation at the cellular level, which may reveal new aspects of hormone-receptor dysregulation in endometriosis. In this study, we examined expression profiles of ESR1 and PGR in mid-secretory phase endometrial tissue from infertile women with endometriosis compared to fertile women without endometriosis using immunohistochemistry (IHC) and multiplex immunofluorescence assays. Quantitative analyses revealed a strong correlation between ESR1 and PGR proteins in both epithelial and stromal compartments, with distinct expression patterns in the infertile endometriosis group. These findings highlight a potential molecular link between ESR1 and PGR dysregulation in endometriosis-associated infertility and provide new insights into the disrupted hormonal environment of the eutopic endometrium.