Dysregulation of Plasma Membrane Transition (PMT) in Endometrial Epithelium: Implications for Infertility in Endometriosis

Dysregulation of Plasma Membrane Transition (PMT) in Endometrial Epithelium: Implications for Infertility in Endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dysregulation of Plasma Membrane Transition (PMT) in Endometrial Epithelium: Implications for Infertility in Endometriosis Zihan Wang, Shuwei Li, Yanhong Mao, Shaoyuan Xu, Xinyu Liu, Yao Xiong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7614122/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background: Endometrial receptivity, a critical prerequisite for successful pregnancy, is impaired in women with endometriosis. Plasma membrane transformation (PMT), a key process involving dynamic remodeling of endometrial epithelial cells, is essential for establishing receptivity during the secretory phase. However, the status of PMT in the eutopic endometrium of women with endometriosis and its potential contribution to infertility are largely unknown. Methods: In this translational study, we investigated the PMT status in endometrial tissues from reproductive-age women with and without endometriosis. We further utilized human endometrial epithelial organoids derived from patient biopsies and a surgically induced mouse model of endometriosis to confirm our findings. PMT markers, including E-cadherin, p-ERM, F-actin, MUC2, and Occludin, and receptivity-associated molecules were analyzed using immunohistochemistry, immunofluorescence, RT-qPCR, Western blotting, and scanning electron microscopy. Results: We found that the dynamic changes in PMT markers observed in normal secretory endometrium, such as E-cadherin downregulation, p-ERM redistribution, and F-actin and MUC2 upregulation, were absent in the eutopic endometrium of women with endometriosis. Organoids derived from endometriosis patients also exhibited significantly reduced expression of receptivity-associated genes, diminished F-actin, persistent apical p-ERM, and aberrant mucin expression. Furthermore, the mouse model of endometriosis showed reduced implantation rates, atrophic pinopodes, and altered PMT markers, accompanied by a loss of induction of LIF and AREG. These findings were consistent across all three models. Conclusions: Our study demonstrates that plasma membrane transformation is aberrantly regulated in the eutopic endometrium of women with endometriosis, leading to impaired epithelial remodeling and compromised receptivity. This disruption represents a novel mechanism of infertility in endometriosis. The findings highlight PMT as a potential therapeutic target for improving endometrial receptivity and offer new insights into the pathological basis of endometriosis-associated infertility. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Endometriosis, prevalent in 10–15% of reproductive-age women, universally contributes to pelvic pain and infertility[ 1 ]. Current statistics indicate that over 50% of infertile women are affected by endometriosis[ 2 , 3 ]. Although impaired endometrial receptivity has long been proposed as a central mechanism linking endometriosis to infertility, the precise cellular and molecular basis remains incompletely understood. Successful embryo implantation depends on the coordinated dialogue between the embryo and endometrium[ 4 ]. Endometrial receptivity establishment is a critical process that allows the embryo to attach, invade, and develop[ 5 ]. Embryo implantation initiates with the uterine luminal epithelium, where an adhesive encounter between the trophoblast ectoderm's apical membrane and epithelial surface is essential[ 6 ]. Uterine epithelial cells typically do not adhere through apical membrane interactions, but a specific functional state is required for successful adhesion. During the establishment of endometrial receptivity, the coordinated events of epithelial cell proliferation cessation, apoptosis, and structural modifications are essential for preparing the uterine lining to accommodate embryo implantation[ 7 – 9 ]. Endometrial epithelial cells undergo a dynamic transition from their characteristic long columnar morphology to a cuboidal shape during embryo implantation, concurrently losing glandular polarity[ 10 ]. This process involves a reshaping of the epithelial cell cytoskeleton, which facilitates trophoblast cell adhesion and prepares the endometrium to receive the implanting embryo[ 11 ]. The transformation of endometrial epithelial cells encompasses a series of intricate changes, including a reduction in the thickness of the cell glycocalyx and alterations in cell surface charge which is accompanied by replacement of microvilli with bulbous protrusions known as pinopodes[ 12 ]. Notably, there is a loss of apicobasal polarity without compromising epithelial cell adhesion molecules, facilitating apical cell-cell interactions and promoting attachment between uterine epithelium and trophoblast[ 13 ]. This dynamic transformation involves significant changes in cell morphology, including loss of cell polarity and modifications in cell-cell adhesion, enabling the cells to become more mobile and responsive, as well as the acquisition of invasion and migratory capabilities, resembling of mesenchymal cells[ 14 , 15 ]. The remodeling of endometrial epithelial cells during the secretory phase, known as plasma membrane transformation (PMT), is hormonally regulated and shares similarities with epithelial-mesenchymal transition (EMT) in tumor cells[ 8 , 16 ]. Several cellular markers associated with epithelial-mesenchymal transition (EMT) in endometrial epithelial cells have been identified in previous studies[ 17 ]. The expression of these markers serves as indicators of the receptivity of the endometrium to support embryo implantation[ 18 ]. These markers include E-cadherin (CDH1), keratin, desmoplakin, mucin-1, and claudin, which are downregulated during EMT, while mesenchymal markers such as N-cadherin, vimentin, and fibronectin are upregulated[ 19 , 20 ]. In the meanwhile, the transition in cell differentiation and behavior during EMT is mediated by key transcription factors namely SNAIL, SLUG, TWIST, ZEB, and ZO-1[ 21 , 22 ]. These transcription factors play a crucial role in orchestrating the complex changes in gene expression profiles, including transcriptional, translational, and post-translational levels. PMT shares similarities with EMT in the loss of polarity, but differs in that epithelial structural integrity and adhesion are largely maintained. Numerous studies have indicated an association between endometriosis and impaired endometrial receptivity[ 3 , 23 , 24 ]. However, whether eutopic endometrium in women with endometriosis undergoes a normal PMT during the window of implantation remains unclear. To address this gap, we investigated the expression of PMT-related markers in mid-secretory eutopic endometrial tissues from endometriosis patients, as well as in a mouse model of endometriosis during the implantation window. Materials and methods Collection of human endometrial tissues This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (NO:2023039K), and informed consent was obtained from every patient according to the principles of the Declaration of Helsinki. From July 2020 to June 2022, eutopic endometrial biopsy samples used for IHC and IF were collected from EM patients with infertility and fertile women undergoing Hysteroscopy and Laparoscopy (for each group, n = 10, 5 in the proliferative phase, 5 in secretory phase) at the Gynecology and Obstetrics Department of Zhongnan Hospital of Wuhan University. From September 2022 to December 2024,eutopic endometrial biopsy samples used for endometrial epithelial organoids culture were collected from EM patients with infertility (n = 5) and fertile women without EM undergoing Hysteroscopy at Reproductive Center of Zhongnan Hospital. The women were all between the ages of 22 and 40. EM was confirmed by pathological results. Women who underwent tubal ligation and were laparoscopically free of EM were served as the control. All women had regular menstrual cycles and had no history of hormonal treatment within the previous 3 months. The menstrual cycle phase was confirmed by menstrual history combined with histological results. Patients who do not meet the conditions for specimen collection: 1. Hydrosalpinx, adenomyosis, Uterine submucosal fibroids, endometrial polyps, moderate to severe adhesions in the uterine cavity. 2. The thickness of the endometrium is less than 5.5mm. Immunohistochemistry Immunohistochemistry was performed on paraffin-embedded tissue sections. After being dewaxed, hydrated, repaired with EDTA buffer, and blocked with 5% BSA, tissues were incubated overnight at 4℃ with primary antibodies for the detection of the following: E-cadherin (ab40772, Abcam, USA) and p-ERM (3726S, Cell Signaling Technology, USA). After incubation with secondary antibody, slides were visualized using DAB-Substrate (Beyotime, China) and photographed using the Aperio ePathology Scanner (Leica, Germany). Image-pro Plus software was used to convert the image format and the grayscale units into optical density (IOD) units. Ten area, density and IOD were selected for measure according to the manufacturer’s protocol. RNA isolation, cDNA synthesis, and RT-qPCR Total RNA was isolated using Invitrogen™ TRIzol™ Reagent (Thermo Fisher Scientific, USA) and evaluated by the ratio of 260/280 and 260/230. cDNA was synthesized from total RNA using HiScript II Q RT SuperMix (R223-01, Vazyme, China) and DNA was removed by adding DNase. qPCR was performed on the Bio-Rad CFX96 (Bio-Rad Laboratories, USA) using SYBR Premix Ex Taq™ (Vazyme, China). The sequences of primers used for the RT-qPCR were as follows: for MAOA, 5’-CTGGGCAGAGTGAGATTT-3’(sense) and 5’-GCTTGTGGAGCTTTAGATG-3’(anti-sense); for LIF, 5’-CATGTGCCTTGCCGATGG-3’(sense) and 5’-GCGGGTGCAGCAGGTTCT-3’(anti-sense) and for PAEP, 5’-CATGTGCCTTGCCGATGG-3’(sense) and 5’-GCGGGTGCAGCAGGTTCT-3’(anti-sense). The quantity of each transcript was calculated based on the comparative threshold cycle (Ct), and the relative expression of the target gene was calculated using the 2-△△Ct method. Isolation of glands, derivation and culture of organoids from human uterine tissue samples Endometrial/decidual/carcinoma tissues were chopped using scalpels into approximately 0.5 mm3 cubes and enzymatically digested in 20–30 ml 1.25 U ml − 1 Dispase II (Sigma, D4693)/0.4 mg ml − 1 collagenase V (Sigma, C-9263) solution in RPMI 1640 medium (Thermo Fisher Scientific, 21875-034)/10% FCS (Biosera, FB-1001) with gentle shaking at 37°C for 30–60 min. The supernatant was passed through one or more 100 µm cell sieves (Corning, 431752) and the sieve washed several times with medium. The flow-through was collected for stromal cell culture in Advanced DMEM/F12 (Thermo Fisher Scientific, 12634010) + 10%FBS + pen/strep (Sigma, P0781) + L-glutamine (Sigma, 25030-024) for several days and subsequent analysis. The sieves were inverted over a Petri dish and retained glandular elements were backwashed from the sieve membranes, pelleted by centrifugation and resuspended in ice-cold Matrigel (Corning, 536231) at a ratio of 1:20 (vol:vol). Twenty-microlitre drops of Matrigel–cell suspension was plated into 48-well plates (Costar, 3548), allowed to set at 37°C and overlaid with 250 µl organoid Expansion Medium (ExM). See Supplementary Table 2 for ExM composition. The medium was changed every 2–3 d. Cultures were passaged by manual pipetting every 7–10 d. For freezing organoids, Matrigel was removed using Cell Recovery Solution (Corning, 354253) and organoids were resuspended in Recovery cell culture freezing medium (Thermo Fisher Scientific, 12648-010). A step-by-step protocol of the derivation and maintenance of human endometrial organoid cultures can be found at Nature Protocol Exchange. Differentiation of endometrial organoids For hormonal stimulation of organoids with β-oestradiol (E2, Sigma E4389), progesterone (P4, Sigma P7556) and 8-bromoadenosine 3′, 5′-cyclic monophosphate (cAMP, Sigma B7880), organoids were passaged routinely and after 4 d of growth in ExM, they were primed with 10 nM E2. After 48 h, medium was replaced with the following conditions: untreated (ExM); 10 nM E2; or 10 nM E2 + 1 µM P4 + 1 µM cAMP. After 96 h, the organoids were collected for downstream applications. Immunofluorescence For paraffin-embedded sections, after being dewaxed and hydrated, tissues were incubated overnight at 4℃ with primary antibodies for the detection of E-cadherin (ab40772, Abcam, USA), Phalloidin (CA1620, Solarbio, China), p-ERM (3726S, CellSignalingTechnology, USA), MUC2 (A14659, ABclonal Technology, China), MUC4 (35-4900, Thermo Fisher Scientific, USA), Cluadin3 (Santa Cruz Biotechnology, USA), MUC1 (), PAEP (), CK7 (GB12225-100, Servicebio Technology, China), Vimentin (10366-1-AP, Proteintech Group, USA), and Occludin (27260-1-AP, Proteintech Group, USA). Sections were then incubated with 488-conjugated Goat Anti-Rabbit IgG (H + L) (AS053, ABclonal Technology, China) or 594-conjugated Goat Anti-Rabbit IgG (H + L) (AS039, ABclonal Technology, China) for 30 min in a dark room. Subsequently, sections were then washed three times with PBS. Antifluorescence quenching Mounting medium which contains 4’,6-diamidino-2-phenylindole (DAPI) was added on the slides before covered with a coverslip. Images were acquired by Laser Scanning Confocal Microscope (Leica, Germany) and analyzed by Leica Application Suite X (Leica, Germany). Image-pro Plus software was used to convert the image format and the grayscale units into optical density (IOD) units. Ten area, density and IOD were selected for measure according to the manufactor’s protocol. Induction of EM in mice Five-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Beijing, China). Mice were housed in a facility with a 12 h light:12 h dark cycle and maintained at 25 ± 0.5℃ and 50%-60% humidity. All animal treatments were performed following the 3R principle of experimental animals and complied with the ethical regulations approved by the Center for Animal Experiments of Wuhan University. In this study, food was provided through a metal mesh and water was provided in glass bottles. All the apparatus used during the experiments were made of metal, glass or polypropylene. Before modeling, all mice were adaptively fed for 1 week. The donor mice were injected with estradiol (E2) (3µg/mouse, s. c.) for 1 week, then the donor mice were euthanized, the two horns were isolated and the endometrial tissue was scraped out and suspended in saline. Equal amounts of the endometrial fragment from one donor mouse were injected into the peritoneum of two recipient mice (0.5 mL/mouse). Mice injected with 0.5 mL saline without endometrial tissue were taken as the control. Mice were kept housing for 3 weeks. Hematoxylin and eosin (HE) staining was performed to confirm the success of modeling. After the successful establishment of EM in mice, estradiol (E2) (3µg/mouse, s. c.) was injected in mice of EM and control group to induce estrous phases consistently. Subsequently, female and male mice in a 3:1 ratio were mated in a cage, and the discovery of vaginal suppositories was recorded as the first day of pregnancy, denoted by GD0. In the next, the mice marked as GD0, GD3 and GD4 were executed to get the endometrium to conduct the next experiment and the uteruses of GD8 mice were taken to observe the changes in the number of embryo implants. Results were obtained from at least three independent experiments. Scanning electron microscopy On gestational day 3, the mice were euthanized, and the uteri were dissected and cut open longitudinally. Uteri were submerged in a fixative containing 2% paraformaldehyde/2.5% glutaraldehyde/0.15 M sodium phosphate, pH 7.4, and stored at 4℃ overnight to several days before processing. After several washes in buffer, the samples were dehydrated through an ethanol dilution series (30%, 50%, 75%, 90%, 100%, and 100%) and subsequently dried using a K850 critical point dryer (Quorum Technologies) with liquid carbon dioxide as the transition solvent. The tissue was then mounted onto aluminum scanning electron microscopy stubs with carbon adhesive tabs and was sputter coated with gold: palladium alloy (60:40) to a thickness of 20 nm using an Ion Sputter Coater (MC1000, Hitachi High-Tech Group). Specimens were visualized with an Energy Dispersive Spectrometer (ULTIMMAX 100, Oxford Instruments) and a Scanning Electron Microscope (SU8100, Hitachi High-Tech Group) using an accelerating voltage of 5 kV. Data Analysis Continuous data were shown as mean ± standard error of the mean (SEM). Student’s t-tests were used to analyze the difference between two groups, and one-way ANOVA was used for the comparisons among multiple groups. Categorical variables were analyzed using the Chi-square test. Data were analyzed by GraphPad Prism 10.0 (GraphPad Software, USA). P < 0.05 was considered as statistically significant. Results 1. Patient Enrollment and Baseline Characteristics Eutopic endometrial tissue samples were collected from cycling women aged 23–40 years (n = 57) undergoing laparoscopy for endometriosis, cervical lesions, or benign ovarian/fallopian tube lesions (Supplementary Table 1). The diagnosis of endometriosis was confirmed histopathologically and staged according to the revised American Society for Reproductive Medicine (ASRM) classification (1997). Participants completed a standardized questionnaire covering reproductive history and endometriosis-related symptoms, and written informed consent was obtained. Among women with endometriosis, 51.7% were classified as stage I–II, 6.8% as stage II–III, 37.9% as stage IV, and 6.8% were unstaged (Supplementary Table 1). No significant differences were observed between groups in age, average menstrual cycle length, dysmenorrhea severity, or reproductive history. However, women with endometriosis had significantly lower body mass index (BMI; P = 0.0073), prolonged menstrual cycles (P = 0.045), and a higher prevalence of infertility (P = 0.044) compared with control (Table 1 ). Table 1 Patient characteristics and reproductive history of study participants Control Endometriosis P-value Age at collection Mean ± SEM 32.43 ± 0.89 31.14 ± 1.07 0.357 BMI (kg/m2) Mean ± SEM 22.59 ± 0.46 20.95 ± 0.37 0.0073 Menstrual average cycle, days Mean ± SEM 28.71 ± 0.45 29.26 ± 0.78 0.548 Range 21–35 23–40 Period length, days Median; IQR, n 5.5; 5.0–7.0 (28) 6.5; 5.0–7.0 (29) 0.045 Range 3–10 5–7 Pain during period, n (%) 8 (28.57) 12 (41.38) 0.408 Pain during intercourse, n (%) 3 (10.71) 2 (6.9) 0.67 Pain outside period, n (%) 1 (3.57) 2(6.9) 1 Reproductive history Women with parity ≥ 1, n (%) 18 (94.7) 10 (83.3) 0.543 Women with parity 0, n (%) 1 (5.3) 2 (16.7) Infertility Women with infertility, n (%) 1 (1.8) 4(13.8) 0.044 Data are presented as mean ± SEM (number) for age, BMI, cycle length, median; interquartile range (IQR) for period length and number (proportion) for pain, reproductive history and infertility. Unpaired t-test (age, BMI, cycle length), Mann–Whitney (period length) or Fisher's exact test (pain, reproductive history and infertility) for differences. Data reported for patient samples where available; some women provided incomplete data. 2. Altered PMT Marker Profiles in Eutopic Endometrium of Endometriosis In the normal endometrial group, the expression of the pivotal membrane–cytoskeleton linker protein p-ERM was predominantly located to the apical surface of the glandular epithelium. Notably, its expression increased during the secretory phase, accompanied by a broadened localization pattern extending to the lateral membrane and the nucleus. In contrast, within the endometriosis group, p-ERM localization was restricted to the apical region of the glands, with no significant variation in its expression or distribution observed between the proliferative and secretory phases. (Fig. 1 A). Regarding E-cadherin, a marker of epithelial cell adhesion typically confined to epithelial cell membrane, showed a significant reduction during the secretory phase compared to the proliferative phase in the normal group. Conversely, in the endometriosis group, E-cadherin levels remained consistently throughout the secretory phase. (Fig. 1 B). Immunofluorescence staining analysis of E-cadherin and F-actin within the same tissue sections revealed distinct and noteworthy expression patterns. (Fig. 2 A). In normal endometrial epithelial cells during the secretory phase, E-cadherin expression was markedly decreased, whereas F-actin expression showed a corresponding increase. By contrast, in the eutopic endometrium of patients with endometriosis, E-cadherin levels during the secretory phase did not exhibit a significant decline, and F-actin expression remained relatively stable. (Fig. 2 B). Furthermore, immunofluorescence co-staining for Occludin and MUC2 demonstrated their presence in both glandular epithelium and stromal cells, with localization predominantly restricted to the cell membrane. (Fig. 2 C). In control endometrium, the integral membrane protein Occludin displayed a significant reduction in expression within glandular epithelial cells during the secretory phase compared to the proliferative phase. Conversely, endometriosis patients maintained high levels of Occludin expression in the glandular epithelium throughout both proliferative and secretory phases, with no significant interphase differences. Regarding the secretory mucin MUC2, its expression was significantly upregulated in the glandular epithelium of control endometrium during the secretory phase relative to the proliferative phase. In sharp contrast, MUC2 expression remained consistently low in the glandular epithelium of endometriosis patients during both phases, with secretory phase levels significantly reduced compared to controls. (Fig. 2 D). 3. PMT Protein Expression in 3D Endometrial Epithelial Organoids Endometrial epithelial organoids (EEOs) were successfully generated and maintained using a three-dimensional (3D) culture system derived from human endometrial biopsies. (Fig. 3 A). The identity and purity of these 3D cultures were confirmed by immunofluorescence co-staining for cytokeratin-7 (CK7) and vimentin. (Fig. 3 B). CK7, a well-established marker of glandular epithelium, was robustly expressed throughout the organoids, whereas vimentin, a marker characteristic of endometrial stromal cells, was completely absent. This clear differential staining demonstrated the high purity and epithelial specificity of the cultured organoids. Upon induction of secretory phase differentiation by simultaneous treatment with estradiol (E2), medroxyprogesterone acetate (MPA), and cAMP, as outlined in our experimental flowchart (Fig. 3 C), EEOs derived from reproductive-age healthy donors exhibited a marked upregulation of key endometrial receptivity genes including MAOA, LIF, and PAEP. (Fig. 3 D). Notably, expression of the transmembrane mucin MUC1 was significantly downregulated following hormonal stimulation, consistent with secretory phase progression, while the receptivity-associated glycoprotein PAEP displayed increased expression. (Fig. 3 E). Collectively, these findings indicate that the established organoid model faithfully recapitulates the functional changes characteristic of secretory phase transformation, underscoring its potential as a valuable in vitro platform for studying endometrial receptivity. Subsequently, we investigated the differential expression of endometrial receptivity markers, with a particular focus on PMT-associated proteins, following in vitro induction of the secretory phase transition in eutopic endometrium from endometriosis patients compared to normal controls. Immunofluorescence co-staining for phosphorylated ERM (p-ERM) and F-actin revealed distinct distribution patterns between groups. (Fig. 4 A). In secretory-phase EEOs derived from control endometrial biopsies, p-ERM displayed a relatively uniform distribution throughout the epithelial cells, accompanied by robust F-actin expression. In contrast, EEOs originating from the endometrium of endometriosis patients exhibited a pronounced polarization of p-ERM localization, predominantly concentrated at the apical tips of epithelial cells, coupled with noticeably reduced F-actin staining intensity compared to controls. Additionally, endometrial glandular organoids derived from control subjects and endometriosis patients exhibited divergent expression patterns of secretory mucin MUC2 versus membrane-tethered mucin MUC4. (Fig. 4 B). Following hormonally induced secretory-phase differentiation, endometriosis-derived organoids displayed significantly diminished MUC2 expression alongside elevated MUC4 levels compared to controls, revealing a transformation that appeared altered in the endometriosis-derived organoids. 4. Dysregulated PMT Markers During Peri-Implantation in Endometriosis Mice The mouse endometrium tissue transplantation model is a widely accepted experimental approach for studying endometriosis pathophysiology. To investigate whether plasma membrane transition (PMT) dysregulation occurs in this model, we established an endometriosis mouse model through ectopic implantation of endometrial tissue in the peritoneal cavity. Supplementary data confirm the successful establishment of ectopic lesions, with histological analysis via hematoxylin and eosin (HE) staining demonstrating well-preserved structural integrity of both glandular and stromal components within these lesions. This model thus provides a robust platform for evaluating eutopic endometrial receptivity during the peri-implantation period. Assessment of embryo implantation sites on gestational day 8 (GD8) revealed a significant reduction in the number of implantation sites in endometriosis-model mice compared to control animals. (Fig. 5 A). Ultrastructural examination by transmission electron microscopy at GD4 showed abundant and morphologically intact pinopodes on the luminal epithelium of the endometrial cavity in control mice, consistent with a receptive endometrium. (Fig. 5 B). In contrast, endometriosis-model mice exhibited pinopodes with a wrinkled morphology and an increased presence of microvilli, indicative of impaired epithelial receptivity. Quantitative real-time PCR (RT-qPCR) analysis of genes associated with endometrial receptivity further corroborated these morphological observations. (Fig. 5 C). Specifically, in control mice, expression of leukemia inhibitory factor (LIF) and amphiregulin (AREG) were significantly upregulated at GD4 relative to GD0, concomitant with a marked downregulation of mucin 1 (MUC1). Conversely, endometriosis-model mice displayed only modest increases in LIF and AREG expression at GD4, with levels significantly lower than those observed in controls. Additionally, MUC1 expression remained elevated at GD4 in endometriosis mice, contrasting with its suppression in control animals (Fig. 5 ). Collectively, these data demonstrate that eutopic endometrial receptivity is markedly compromised during the peri-implantation phase in the murine model of endometriosis. To further characterize the dynamics of plasma membrane transition (PMT) during embryo implantation, immunofluorescence co-staining of Occludin and MUC2 was performed, revealing distinct temporal expression patterns. (Fig. 5 D). In control mice, Occludin expression in the endometrial glandular epithelium was significantly downregulated at gestational day 4 (GD4) compared to GD0, whereas MUC2 expression exhibited a concomitant and significant upregulation. Conversely, in the endometriosis mouse model, Occludin expression remained persistently elevated at GD4 relative to GD0, with no significant decrease observed. Additionally, MUC2 expression in endometriosis mice increased only marginally at GD4 and remained significantly lower than that of controls at the same gestational stage. Parallel dual immunofluorescence analysis of Claudin-3, a key tight junction protein, and F-actin, a major cytoskeletal component, demonstrated that both proteins were markedly upregulated at GD4 compared to GD0 in control mice. (Fig. 5 E). In contrast, endometriosis-model mice exhibited consistently low expression levels of Claudin-3 and F-actin at both GD0 and GD4, with GD4 levels significantly reduced relative to controls. Collectively, these findings reveal a clear dysregulation of PMT-associated molecular markers in the eutopic endometrium of the endometriosis mouse model during the peri-implantation phase. Discussion Endometriosis-associated infertility has been linked to both molecular and structural alterations in the endometrium[ 25 ], yet the upstream regulatory pathways driving these changes remain insufficiently defined. Recent transcriptomic analyses indicate that the eutopic endometrium in endometriosis exhibits dysregulation of multiple receptivity-associated genes during the secretory phase, suggesting that impaired receptivity is not solely a downstream effect of inflammation or hormonal imbalance, but may also arise from intrinsic defects in epithelial remodeling programs[ 26 – 28 ]. By integrating human endometrial biopsies, hormonally differentiated endometrial organoids, and an in vivo implantation model, our study delineates a unified molecular signature linking epithelial remodeling defects to compromised embryo implantation. Our findings highlight disruptions in plasma membrane transformation (PMT)—a hormonally regulated remodeling process in glandular epithelium during the implantation window. Beyond the well-characterized morphological changes, we identify accompanying molecular reprogramming events, particularly the abnormal regulation of junctional and adhesion molecules (e.g., E-cadherin, occludin, ZO-1, claudins) and the dysregulation of mucins and cytoskeletal regulators. These abnormalities likely interfere with the precise epithelial depolarization required for optimal embryo attachment. Given that PMT partially overlaps but is not identical to epithelial–mesenchymal transition (EMT), the PMT defect in endometriosis may represent a hybrid state in which epithelial integrity is preserved while the receptive phenotype fails to develop. In healthy controls, secretory-phase remodeling involves coordinated E-cadherin downregulation to loosen adherens junctions and facilitate trophoblast invasion, along with dynamic phosphorylation of the ERM complex to reorganize the actin cytoskeleton. In endometriosis, these events are pathologically altered: E-cadherin levels remain abnormally high, reinforcing junctional integrity and potentially impeding embryo penetration; p-ERM shows aberrant apical accumulation in organoid models, indicating defective polarity remodeling despite preserved expression levels. Mechanistically, p-ERM participates in polarity remodeling by modulating Na⁺ channel proteins in endometrial cells. Its phosphorylation is regulated by signaling pathways such as Rac-1/PAK1(pPAK1)/ERM (pERM) and Sgk1, which facilitate cytoskeletal rearrangements during receptivity[ 29 ]. Moreover, loss of Rictor—a component of mTORC2—has been linked to altered p-ERM-mediated polarity remodeling, underscoring the intricate signaling networks involved in endometrial receptivity[ 30 ]. A distinctive molecular signature of mucin dysregulation was also observed. During PMT, the transmembrane mucin MUC4 at the apical membrane stabilizes architecture through cytoskeletal interactions; its downregulation promotes polarity remodeling. Secreted mucin MUC2, in turn, forms a dense glycocalyx between adjacent cells, enhancing adhesion. In controls, secretory differentiation induces reduced MUC4 and increased MUC2, optimizing surface architecture for embryo adhesion. In contrast, endometriosis tissues and organoids display the opposite pattern—elevated MUC4 with suppressed MUC2—disrupting the adhesive and structural microenvironment of the luminal surface. This “mucin switch,” together with mislocalized p-ERM, likely undermines coordinated epithelial responses during implantation. Tight junction remodeling further underscores PMT dysregulation. Occludin, a key transmembrane protein, interacts with claudins to form the structural backbone of the endometrial barrier and regulate adhesive properties; its deletion can lead to chronic inflammation in various epithelia[ 31 ]. The actin cytoskeleton, a dynamic F-actin filament network, coordinates with Occludin-based junctions to adapt epithelial architecture for implantation[ 32 ]. In controls, secretory-phase endometrium shows uniform Occludin downregulation with redistribution, facilitating barrier relaxation. In endometriosis, Occludin remains elevated with abnormal basolateral polarization, potentially maintaining an overly restrictive barrier. Similar defects in claudin expression and F-actin organization suggest a coordinated failure in junctional and cytoskeletal adaptation. The functional consequences of these molecular defects were validated in an endometriosis mouse model. Pinopodes—secretory-phase apical protrusions acting as receptivity sensors—were morphologically immature in diseased mice, with elongated microvilli, reduced expression of receptivity genes (LIF, AREG), and persistent MUC1 elevation, all indicative of a non-receptive state. Although implantation site counts trended lower, the morphological and transcriptional profiles strongly support a receptivity deficit. In summary, our study integrates clinical specimens, hormonally induced endometrial organoids, and an in vivo implantation model to define a reproducible PMT disruption signature in endometriosis, characterized by (i) pathological mucin reprogramming, (ii) aberrant junctional remodeling, and (iii) failed cytoskeletal adaptation. These findings provide mechanistic insight into how epithelial remodeling can be selectively impaired without loss of gross epithelial structure. While our data implicate signaling pathways such as mTORC2–Rictor and Rac1–PAK1, causal relationships remain to be validated by targeted perturbation. Future studies should assess whether pharmacologic or genetic modulation of mucin expression, junctional proteins, or ERM activation can restore receptivity in organoid and in vivo models, potentially informing new fertility-preserving therapies for women with endometriosis-associated infertility. Declarations Ethical conduct of research The study was approved by the Medical Ethics Committee, Zhongnan Hospital of Wuhan University (2023039K). Written informed consent was obtained from patients. All animal experiments were approved by Institutional Animal Care and Use Committee and following the 3R principle of experimental animals, and approved by Formal Review of Experimental Animal Welfare and Ethics of Zhongnan Hospital. (NO: ZN2022169) Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request. Consent for publication All authors consent for publication. Declaration of interest The authors declare that they have no competing interests. Funding This work was supported by National Nature Science Foundation of China (Grant NO.82201819, Grant NO.81771543) and Joint supported by Hubei Provincial Natural Science Foundation and Shiyan-of China (Grant No. 2025AFD199). Authors' contributions Zihan Wang performed experiments and wrote the paper. Shuwei Li performed data curation and provided software support. Yanhong Mao collected the endometrial samples. Yao Xiong designed the experiment and collected the tissue samples used in the study. Yuanzhen Zhang conceived the study and revised the draft. Xinyu Liu performed supervision and conduct investigation process. Shaoyuan Xu the validation and formal analysis. All authors discussed the results and contributed to the final manuscript. Acknowledgments The authors are grateful to the women who donated the endometrial tissue used in this study. References GL N, et al. Postoperative Imaging of Endometriosis. D – 8302501; 2024. - e230159. Guan Q, Velho RV, Sehouli J, Mechsner S. 2023 Endometriosis and Opioid Receptors: Are Opioids a Possible/Promising Treatment for Endometriosis? International journal of molecular sciences 24, http //10.3390/ijms24021633 P P, D, d. Z., JM A. - Endometrial receptivity in adenomyosis and/or endometriosis. D – 0372772; 2023. – 741–745. M K, et al. Human endometrial cell-type-specific RNA sequencing provides new insights into. D – 101722764; 2025. p. hoac043. BA L, SL Y. 2019 - What exactly is endometrial receptivity? - Fertil Steril. 2019;111(4):611–617. doi : 10.1016/j.fertnstert.2019.02. 009. , – 611–617. Ye X. Uterine Luminal Epithelium as the Transient Gateway for Embryo Implantation. Trends Endocrinol Metab. 2020;31:165–80. http//10.1016/j.tem.2019.11.008 . Wang HQ, et al. Maternal and embryonic signals cause functional differentiation of luminal epithelial cells and receptivity establishment. Dev Cell. 2023;58:2376–e23922376. http//10.1016/j.devcel.2023.08.004 . Whitby S, Zhou W, Dimitriadis E. 2020 Alterations in Epithelial Cell Polarity During Endometrial Receptivity: A Systematic Review. Frontiers in endocrinology 11, 596324, http //10.3389/fendo.2020.596324 W Z et al. 2024 - Dysregulated miR-124-3p in endometrial epithelial cells reduces endometrial. D – 7505876, - e2401071121. ER R et al. 2025 - Loss of PRICKLE1 leads to abnormal endometrial epithelial architecture, decreased. D – 9918367777906676 , - pgaf024. PT R et al. 2022 - Trophectoderm differentiation to invasive syncytiotrophoblast is promoted by. - Hum Reprod. 2022;37(4):777–792. doi : 10.1093/humrep/deac008. , – 777–792. Quinn KE, Matson BC, Wetendorf M, Caron KM. 2020 Pinopodes: Recent advancements, current perspectives, and future directions. Molecular and cellular endocrinology 501, 110644, http //10.1016/j.mce.2019.110644 X D, et al. ST6GALNAC1-mediated sialylation in uterine endometrial epithelium facilitates the. D – 101546952; 2025. – 197–212. S W, LA S, J E. - The Endometrial Polarity Paradox: Differential Regulation of Polarity Within. (2018). PT R, et al. Glucose influences endometrial receptivity to embryo implantation through. D – 100901225; 2024. - C634-C645. CR M. - Uterine receptivity and the plasma membrane transformation. D – 9425763; 2004. – 259–267. Y H et al. 2024 - Loss of KLF15 impairs endometrial receptivity by inhibiting EMT in endometriosis. D – 0375363 , T - epublish. M S, U PD, S., H T. 2024 - A Comprehensive Review of the Endometrial Receptivity Array in Euploid Embryo. D – 101596737 , - e63173. Tiwari A, Ashary N, Singh N, Sharma S, Modi D. 2021 Modulation of E-Cadherin and N-Cadherin by ovarian steroids and embryonic stimuli. Tissue & cell 73, 101670, http //10.1016/j.tice.2021.101670 Oghbaei F, et al. Epithelial-mesenchymal transition process during embryo implantation. Cell Tissue Res. 2022;388:1–17. http//10.1007/s00441-021-03574-w . Debnath P, Huirem RS, Dutta P, Palchaudhuri S. 2022 Epithelial-mesenchymal transition and its transcription factors. Bioscience reports 42, http //10.1042/bsr20211754 Bartley J, Jülicher A, Hotz B, Mechsner S, Hotz H. Epithelial to mesenchymal transition (EMT) seems to be regulated differently in endometriosis and the endometrium. Arch Gynecol Obstet. 2014;289:871–81. http//10.1007/s00404-013-3040-4 . Dahiphale SM et al. 2024 A Comprehensive Review of the Endometrial Receptivity Array in Embryo Transfer: Advancements, Applications, and Clinical Outcomes. Cureus 16, e67866, http //10.7759/cureus.67866 B A, E S. - Endometriosis, staging, infertility and assisted reproductive technology: time. – 103943: D – 101122473; 2024. G B, I B, & M H. 2014 - Structural and molecular features of the endomyometrium in endometriosis and. - Hum Reprod Update. 2014 May-Jun;20(3):386–402. doi : 10.1093/ humupd/dmt052. Epub , – 386–402. M S et al. 2022 - The expression pattern of endometrial receptivity genes is desynchronized between. - Reprod Biomed Online. 2022;45(4):713–720. doi : 10.1016/j.rbmo.2022.05. 028. , – 713–720. MAS F, et al. Single-cell transcriptomic analysis of endometriosis. D – 9216904; 2023. – 255–267. Q Q et al. 2025 - Update on the pathogenesis of endometriosis-related infertility based on. - Front Endocrinol (Lausanne). 2025;16:1558271. doi :, – 1558271. Tu Z, et al. Uterine RAC1 via Pak1-ERM signaling directs normal luminal epithelial integrity conducive to on-time embryo implantation in mice. Cell Death Differ. 2016;23:169–81. http//10.1038/cdd.2015.98 . Zhang Y et al. 2021 Rictor/mTORC2 is involved in endometrial receptivity by regulating epithelial remodeling. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 35, e21731, http //10.1096/fj.202100529RR G G, H HJHL, Y., K W. 2024 - Asiaticoside ameliorates uterine injury induced by zearalenone in mice by. D – 101153627 , – 118. H Z, et al. PAI-1 promotes human endometrial stromal decidualization via inhibiting. D – 8804484; 2024. p. e70233. Additional Declarations No competing interests reported. Supplementary Files SuppFig1.png Supplementary Figure Successful establishment and validation of the endometriosis mouse model A. Successful establishment of the endometriosis mouse model. Arrow 1 indicates the formation of ectopic lesions. B. Arrow 2 indicates the uterus of the endometriosis mouse. C. Hematoxylin–eosin staining of ectopic lesions. Left panels: 100× magnification, scale bar = 100 μm; right panels: 400× magnification, scale bar = 20 μm. SupplementaryTables.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 May, 2026 Reviews received at journal 23 Dec, 2025 Reviewers agreed at journal 01 Dec, 2025 Reviewers agreed at journal 28 Oct, 2025 Reviewers invited by journal 23 Sep, 2025 Editor assigned by journal 17 Sep, 2025 Submission checks completed at journal 16 Sep, 2025 First submitted to journal 14 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7614122","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":524067674,"identity":"7a2b12a9-f8b4-49bd-94e6-eb8722f06e1a","order_by":0,"name":"Zihan Wang","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Wang","suffix":""},{"id":524067675,"identity":"7714133a-dadd-499e-b016-beeeaedbef42","order_by":1,"name":"Shuwei Li","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan 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Immunohistochemical expression of E-cadherin in glandular epithelial cells and endometrial stromal cells of eutopic endometrium. B. Immunohistochemical expression of p-ERM in glandular epithelial cells and endometrial stromal cells of eutopic endometrium. Left panels：magnification 100 ×, scale bar=100 μm；right panels：magnification 400 ×, scale bar=20 μm.\u003cem\u003e *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"PMTFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/25151f56a56b79fef9c4018a.png"},{"id":92857217,"identity":"a7c3fa4c-3f7c-46f3-9265-ad51bfdeaf9b","added_by":"auto","created_at":"2025-10-06 11:42:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":817083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDysregulated expression of epithelial adhesion and cytoskeletal markers in endometriosis endometrium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Immunofluorescence co-staining of E-cadherin (red) and F-actin (green) in glandular epithelial cells, with nuclei counterstained using DAPI (blue). B. Quantification of E-cadherin and F-actin expression levels. C. Immunofluorescence co-staining of Occludin (green) and MUC2 (red) in glandular epithelial cells, with nuclei counterstained using DAPI (blue). D. Quantification of Occludin and MUC2 expression levels. Images were captured at 400× magnification; scale bar = 20 μm. \u003cem\u003e***p \u0026lt; 0.001; ***p \u0026lt; 0.0001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"PMTFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/5c9423ce759b15ab8c69d258.png"},{"id":92857214,"identity":"40c5e9ba-bb07-4ccc-8cd7-6b98301b71c4","added_by":"auto","created_at":"2025-10-06 11:42:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":596017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of a functional human endometrial epithelial organoid model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Bright-field image of three-dimensional (3D) endometrial epithelial organoids (EEOs). B. Immunofluorescence co-staining of CK7 (green) and Vimentin (red), with nuclei counterstained using DAPI (blue). C. Schematic illustration of the hormone-induced transformation model during the secretory phase. D. Quantitative RT-qPCR analysis of MAOA, LIF, and PAEP expression levels after transformation during the secretory phase. E. Immunofluorescence co-staining of MUC1 (red) and PAEP (green), with nuclei counterstained using DAPI (blue). Images were captured at 400× magnification; scale bar = 20 μm. \u003cem\u003e***p \u0026lt; 0.001; ***p \u0026lt; 0.0001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"PMTFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/ebc8694002140d96b91596ca.png"},{"id":92858880,"identity":"0d71e989-c705-4f13-97ed-ac4c87fd6e9b","added_by":"auto","created_at":"2025-10-06 11:58:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":319097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired plasma membrane transformation and mucin expression in endometriosis-derived organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Immunofluorescence co-staining of p-ERM (green) and F-actin (red), with nuclei counterstained using DAPI (blue). B. Immunofluorescence co-staining of MUC2 (green) and MUC4 (red), with nuclei counterstained using DAPI (blue). Images were captured at 400× magnification; scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"PMTFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/d51d7dc5efd5dbf4f59b3853.png"},{"id":92857222,"identity":"31d725ad-518f-4ad3-944d-b47a33948268","added_by":"auto","created_at":"2025-10-06 11:42:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1074564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired endometrial receptivity and altered PMT in a murine endometriosis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Statistical analysis of uterine morphology and the number of embryo implantation sites on gestational day 8 in control and endometriosis model mice. B. Scanning electron microscopy of the endometrium at day 3 of gestation. C. Quantitative RT-qPCR analysis of Lif, Muc1, and Areg expression levels. D. Immunofluorescence co-staining of Occludin (green) and MUC2 (red), with nuclei counterstained using DAPI (blue). E. Immunofluorescence co-staining of Claudin-3 (green) and F-actin (red), with nuclei counterstained using DAPI (blue). Images were captured at 1600× magnification; scale bar = 5 μm. \u003cem\u003e*p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ***p \u0026lt; 0.0001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"PMTFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/5df8c74575349adaf9665f17.png"},{"id":92860815,"identity":"03399e45-20bd-49a3-bec3-eadb2435126b","added_by":"auto","created_at":"2025-10-06 12:14:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4652696,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/4fd9b4d0-5aa9-480d-8c1f-dc0dd91e1200.pdf"},{"id":92857210,"identity":"78ee5255-82a0-496e-9711-da01ec4aff0d","added_by":"auto","created_at":"2025-10-06 11:42:09","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":684475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure Successful establishment and validation of the endometriosis mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Successful establishment of the endometriosis mouse model. Arrow 1 indicates the formation of ectopic lesions. B. Arrow 2 indicates the uterus of the endometriosis mouse. C. Hematoxylin–eosin staining of ectopic lesions. Left panels: 100× magnification, scale bar = 100 μm; right panels: 400× magnification, scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"SuppFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/a0401191b581f4afe7caf502.png"},{"id":92857211,"identity":"d4767e8c-5d73-446d-90ec-8fa5b11f103c","added_by":"auto","created_at":"2025-10-06 11:42:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27260,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7614122/v1/4d1da9b06ad214098fc7b864.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dysregulation of Plasma Membrane Transition (PMT) in Endometrial Epithelium: Implications for Infertility in Endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis, prevalent in 10\u0026ndash;15% of reproductive-age women, universally contributes to pelvic pain and infertility[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Current statistics indicate that over 50% of infertile women are affected by endometriosis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although impaired endometrial receptivity has long been proposed as a central mechanism linking endometriosis to infertility, the precise cellular and molecular basis remains incompletely understood.\u003c/p\u003e\u003cp\u003eSuccessful embryo implantation depends on the coordinated dialogue between the embryo and endometrium[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Endometrial receptivity establishment is a critical process that allows the embryo to attach, invade, and develop[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Embryo implantation initiates with the uterine luminal epithelium, where an adhesive encounter between the trophoblast ectoderm's apical membrane and epithelial surface is essential[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Uterine epithelial cells typically do not adhere through apical membrane interactions, but a specific functional state is required for successful adhesion. During the establishment of endometrial receptivity, the coordinated events of epithelial cell proliferation cessation, apoptosis, and structural modifications are essential for preparing the uterine lining to accommodate embryo implantation[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Endometrial epithelial cells undergo a dynamic transition from their characteristic long columnar morphology to a cuboidal shape during embryo implantation, concurrently losing glandular polarity[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This process involves a reshaping of the epithelial cell cytoskeleton, which facilitates trophoblast cell adhesion and prepares the endometrium to receive the implanting embryo[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The transformation of endometrial epithelial cells encompasses a series of intricate changes, including a reduction in the thickness of the cell glycocalyx and alterations in cell surface charge which is accompanied by replacement of microvilli with bulbous protrusions known as pinopodes[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, there is a loss of apicobasal polarity without compromising epithelial cell adhesion molecules, facilitating apical cell-cell interactions and promoting attachment between uterine epithelium and trophoblast[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This dynamic transformation involves significant changes in cell morphology, including loss of cell polarity and modifications in cell-cell adhesion, enabling the cells to become more mobile and responsive, as well as the acquisition of invasion and migratory capabilities, resembling of mesenchymal cells[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The remodeling of endometrial epithelial cells during the secretory phase, known as plasma membrane transformation (PMT), is hormonally regulated and shares similarities with epithelial-mesenchymal transition (EMT) in tumor cells[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Several cellular markers associated with epithelial-mesenchymal transition (EMT) in endometrial epithelial cells have been identified in previous studies[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The expression of these markers serves as indicators of the receptivity of the endometrium to support embryo implantation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These markers include E-cadherin (CDH1), keratin, desmoplakin, mucin-1, and claudin, which are downregulated during EMT, while mesenchymal markers such as N-cadherin, vimentin, and fibronectin are upregulated[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the meanwhile, the transition in cell differentiation and behavior during EMT is mediated by key transcription factors namely SNAIL, SLUG, TWIST, ZEB, and ZO-1[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These transcription factors play a crucial role in orchestrating the complex changes in gene expression profiles, including transcriptional, translational, and post-translational levels. PMT shares similarities with EMT in the loss of polarity, but differs in that epithelial structural integrity and adhesion are largely maintained.\u003c/p\u003e\u003cp\u003eNumerous studies have indicated an association between endometriosis and impaired endometrial receptivity[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, whether eutopic endometrium in women with endometriosis undergoes a normal PMT during the window of implantation remains unclear. To address this gap, we investigated the expression of PMT-related markers in mid-secretory eutopic endometrial tissues from endometriosis patients, as well as in a mouse model of endometriosis during the implantation window.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCollection of human endometrial tissues\u003c/h2\u003e\u003cp\u003e This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (NO:2023039K), and informed consent was obtained from every patient according to the principles of the Declaration of Helsinki.\u003c/p\u003e\u003cp\u003eFrom July 2020 to June 2022, eutopic endometrial biopsy samples used for IHC and IF\u003c/p\u003e\u003cp\u003ewere collected from EM patients with infertility and fertile women undergoing Hysteroscopy and Laparoscopy (for each group, n\u0026thinsp;=\u0026thinsp;10, 5 in the proliferative phase, 5 in secretory phase) at the Gynecology and Obstetrics Department of Zhongnan Hospital of Wuhan University. From September 2022 to December 2024,eutopic endometrial biopsy samples used for endometrial epithelial organoids culture were collected from EM patients with infertility (n\u0026thinsp;=\u0026thinsp;5) and fertile women without EM undergoing Hysteroscopy at Reproductive Center of Zhongnan Hospital. The women were all between the ages of 22 and 40. EM was confirmed by pathological results. Women who underwent tubal ligation and were laparoscopically free of EM were served as the control. All women had regular menstrual cycles and had no history of hormonal treatment within the previous 3 months. The menstrual cycle phase was confirmed by menstrual history combined with histological results. Patients who do not meet the conditions for specimen collection: 1. Hydrosalpinx, adenomyosis, Uterine submucosal fibroids, endometrial polyps, moderate to severe adhesions in the uterine cavity. 2. The thickness of the endometrium is less than 5.5mm.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry was performed on paraffin-embedded tissue sections. After being dewaxed, hydrated, repaired with EDTA buffer, and blocked with 5% BSA, tissues were incubated overnight at 4℃ with primary antibodies for the detection of the following: E-cadherin (ab40772, Abcam, USA) and p-ERM (3726S, Cell Signaling Technology, USA). After incubation with secondary antibody, slides were visualized using DAB-Substrate (Beyotime, China) and photographed using the Aperio ePathology Scanner (Leica, Germany). Image-pro Plus software was used to convert the image format and the grayscale units into optical density (IOD) units. Ten area, density and IOD were selected for measure according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eRNA isolation, cDNA synthesis, and RT-qPCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated using Invitrogen\u0026trade; TRIzol\u0026trade; Reagent (Thermo Fisher Scientific, USA) and evaluated by the ratio of 260/280 and 260/230. cDNA was synthesized from total RNA using HiScript II Q RT SuperMix (R223-01, Vazyme, China) and DNA was removed by adding DNase. qPCR was performed on the Bio-Rad CFX96 (Bio-Rad Laboratories, USA) using SYBR Premix Ex Taq\u0026trade; (Vazyme, China). The sequences of primers used for the RT-qPCR were as follows: for MAOA, 5\u0026rsquo;-CTGGGCAGAGTGAGATTT-3\u0026rsquo;(sense) and 5\u0026rsquo;-GCTTGTGGAGCTTTAGATG-3\u0026rsquo;(anti-sense); for LIF, 5\u0026rsquo;-CATGTGCCTTGCCGATGG-3\u0026rsquo;(sense) and 5\u0026rsquo;-GCGGGTGCAGCAGGTTCT-3\u0026rsquo;(anti-sense) and for PAEP, 5\u0026rsquo;-CATGTGCCTTGCCGATGG-3\u0026rsquo;(sense) and 5\u0026rsquo;-GCGGGTGCAGCAGGTTCT-3\u0026rsquo;(anti-sense). The quantity of each transcript was calculated based on the comparative threshold cycle (Ct), and the relative expression of the target gene was calculated using the 2-△△Ct method.\u003c/p\u003e\n\u003ch3\u003eIsolation of glands, derivation and culture of organoids from human uterine tissue samples\u003c/h3\u003e\n\u003cp\u003eEndometrial/decidual/carcinoma tissues were chopped using scalpels into approximately 0.5 mm3 cubes and enzymatically digested in 20\u0026ndash;30 ml 1.25 U ml\u0026thinsp;\u0026minus;\u0026thinsp;1 Dispase II (Sigma, D4693)/0.4 mg ml\u0026thinsp;\u0026minus;\u0026thinsp;1 collagenase V (Sigma, C-9263) solution in RPMI 1640 medium (Thermo Fisher Scientific, 21875-034)/10% FCS (Biosera, FB-1001) with gentle shaking at 37\u0026deg;C for 30\u0026ndash;60 min. The supernatant was passed through one or more 100 \u0026micro;m cell sieves (Corning, 431752) and the sieve washed several times with medium. The flow-through was collected for stromal cell culture in Advanced DMEM/F12 (Thermo Fisher Scientific, 12634010)\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;pen/strep (Sigma, P0781)\u0026thinsp;+\u0026thinsp;L-glutamine (Sigma, 25030-024) for several days and subsequent analysis. The sieves were inverted over a Petri dish and retained glandular elements were backwashed from the sieve membranes, pelleted by centrifugation and resuspended in ice-cold Matrigel (Corning, 536231) at a ratio of 1:20 (vol:vol). Twenty-microlitre drops of Matrigel\u0026ndash;cell suspension was plated into 48-well plates (Costar, 3548), allowed to set at 37\u0026deg;C and overlaid with 250 \u0026micro;l organoid Expansion Medium (ExM). See Supplementary Table\u0026nbsp;2 for ExM composition. The medium was changed every 2\u0026ndash;3 d. Cultures were passaged by manual pipetting every 7\u0026ndash;10 d. For freezing organoids, Matrigel was removed using Cell Recovery Solution (Corning, 354253) and organoids were resuspended in Recovery cell culture freezing medium (Thermo Fisher Scientific, 12648-010). A step-by-step protocol of the derivation and maintenance of human endometrial organoid cultures can be found at Nature Protocol Exchange.\u003c/p\u003e\n\u003ch3\u003eDifferentiation of endometrial organoids\u003c/h3\u003e\n\u003cp\u003eFor hormonal stimulation of organoids with β-oestradiol (E2, Sigma E4389), progesterone (P4, Sigma P7556) and 8-bromoadenosine 3\u0026prime;, 5\u0026prime;-cyclic monophosphate (cAMP, Sigma B7880), organoids were passaged routinely and after 4 d of growth in ExM, they were primed with 10 nM E2. After 48 h, medium was replaced with the following conditions: untreated (ExM); 10 nM E2; or 10 nM E2\u0026thinsp;+\u0026thinsp;1 \u0026micro;M P4\u0026thinsp;+\u0026thinsp;1 \u0026micro;M cAMP. After 96 h, the organoids were collected for downstream applications.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eFor paraffin-embedded sections, after being dewaxed and hydrated, tissues were incubated overnight at 4℃ with primary antibodies for the detection of E-cadherin (ab40772, Abcam, USA), Phalloidin (CA1620, Solarbio, China), p-ERM (3726S, CellSignalingTechnology, USA), MUC2 (A14659, ABclonal Technology, China), MUC4 (35-4900, Thermo Fisher Scientific, USA), Cluadin3 (Santa Cruz Biotechnology, USA), MUC1 (), PAEP (), CK7 (GB12225-100, Servicebio Technology, China), Vimentin (10366-1-AP, Proteintech Group, USA), and Occludin (27260-1-AP, Proteintech Group, USA). Sections were then incubated with 488-conjugated Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (AS053, ABclonal Technology, China) or 594-conjugated Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (AS039, ABclonal Technology, China) for 30 min in a dark room. Subsequently, sections were then washed three times with PBS. Antifluorescence quenching Mounting medium which contains 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) was added on the slides before covered with a coverslip. Images were acquired by Laser Scanning Confocal Microscope (Leica, Germany) and analyzed by Leica Application Suite X (Leica, Germany). Image-pro Plus software was used to convert the image format and the grayscale units into optical density (IOD) units. Ten area, density and IOD were selected for measure according to the manufactor\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInduction of EM in mice\u003c/h3\u003e\n\u003cp\u003eFive-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Beijing, China). Mice were housed in a facility with a 12 h light:12 h dark cycle and maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5℃ and 50%-60% humidity. All animal treatments were performed following the 3R principle of experimental animals and complied with the ethical regulations approved by the Center for Animal Experiments of Wuhan University. In this study, food was provided through a metal mesh and water was provided in glass bottles. All the apparatus used during the experiments were made of metal, glass or polypropylene. Before modeling, all mice were adaptively fed for 1 week.\u003c/p\u003e\u003cp\u003eThe donor mice were injected with estradiol (E2) (3\u0026micro;g/mouse, s. c.) for 1 week, then the donor mice were euthanized, the two horns were isolated and the endometrial tissue was scraped out and suspended in saline. Equal amounts of the endometrial fragment from one donor mouse were injected into the peritoneum of two recipient mice (0.5 mL/mouse). Mice injected with 0.5 mL saline without endometrial tissue were taken as the control. Mice were kept housing for 3 weeks. Hematoxylin and eosin (HE) staining was performed to confirm the success of modeling. After the successful establishment of EM in mice, estradiol (E2) (3\u0026micro;g/mouse, s. c.) was injected in mice of EM and control group to induce estrous phases consistently. Subsequently, female and male mice in a 3:1 ratio were mated in a cage, and the discovery of vaginal suppositories was recorded as the first day of pregnancy, denoted by GD0. In the next, the mice marked as GD0, GD3 and GD4 were executed to get the endometrium to conduct the next experiment and the uteruses of GD8 mice were taken to observe the changes in the number of embryo implants. Results were obtained from at least three independent experiments.\u003c/p\u003e\n\u003ch3\u003eScanning electron microscopy\u003c/h3\u003e\n\u003cp\u003eOn gestational day 3, the mice were euthanized, and the uteri were dissected and cut open longitudinally. Uteri were submerged in a fixative containing 2% paraformaldehyde/2.5% glutaraldehyde/0.15 M sodium phosphate, pH 7.4, and stored at 4℃ overnight to several days before processing. After several washes in buffer, the samples were dehydrated through an ethanol dilution series (30%, 50%, 75%, 90%, 100%, and 100%) and subsequently dried using a K850 critical point dryer (Quorum Technologies) with liquid carbon dioxide as the transition solvent. The tissue was then mounted onto aluminum scanning electron microscopy stubs with carbon adhesive tabs and was sputter coated with gold: palladium alloy (60:40) to a thickness of 20 nm using an Ion Sputter Coater (MC1000, Hitachi High-Tech Group). Specimens were visualized with an Energy Dispersive Spectrometer (ULTIMMAX 100, Oxford Instruments) and a Scanning Electron Microscope (SU8100, Hitachi High-Tech Group) using an accelerating voltage of 5 kV.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eContinuous data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Student\u0026rsquo;s t-tests were used to analyze the difference between two groups, and one-way ANOVA was used for the comparisons among multiple groups. Categorical variables were analyzed using the Chi-square test. Data were analyzed by GraphPad Prism 10.0 (GraphPad Software, USA). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1. Patient Enrollment and Baseline Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eEutopic endometrial tissue samples were collected from cycling women aged 23\u0026ndash;40 years (n\u0026thinsp;=\u0026thinsp;57) undergoing laparoscopy for endometriosis, cervical lesions, or benign ovarian/fallopian tube lesions (Supplementary Table\u0026nbsp;1). The diagnosis of endometriosis was confirmed histopathologically and staged according to the revised American Society for Reproductive Medicine (ASRM) classification (1997). Participants completed a standardized questionnaire covering reproductive history and endometriosis-related symptoms, and written informed consent was obtained.\u003c/p\u003e\n\u003cp\u003eAmong women with endometriosis, 51.7% were classified as stage I\u0026ndash;II, 6.8% as stage II\u0026ndash;III, 37.9% as stage IV, and 6.8% were unstaged (Supplementary Table\u0026nbsp;1). No significant differences were observed between groups in age, average menstrual cycle length, dysmenorrhea severity, or reproductive history. However, women with endometriosis had significantly lower body mass index (BMI; P\u0026thinsp;=\u0026thinsp;0.0073), prolonged menstrual cycles (P\u0026thinsp;=\u0026thinsp;0.045), and a higher prevalence of infertility (P\u0026thinsp;=\u0026thinsp;0.044) compared with control (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePatient characteristics and reproductive history of study participants\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEndometriosis\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge at collection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.357\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI (kg/m2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0073\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMenstrual average cycle, days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.548\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRange\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u0026ndash;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u0026ndash;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePeriod length, days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian; IQR, n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.5; 5.0\u0026ndash;7.0 (28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.5; 5.0\u0026ndash;7.0 (29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRange\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026ndash;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026ndash;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePain during period, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (28.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (41.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.408\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePain during intercourse, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (10.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (6.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePain outside period, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (3.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2(6.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReproductive history\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWomen with parity\u0026thinsp;\u0026ge;\u0026thinsp;1, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18 (94.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (83.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.543\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWomen with parity 0, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (5.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (16.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInfertility\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWomen with infertility, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (1.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4(13.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (number) for age, BMI, cycle length, median; interquartile range (IQR) for period length and number (proportion) for pain, reproductive history and infertility. Unpaired t-test (age, BMI, cycle length), Mann\u0026ndash;Whitney (period length) or Fisher\u0026apos;s exact test (pain, reproductive history and infertility) for differences. Data reported for patient samples where available; some women provided incomplete data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Altered PMT Marker Profiles in Eutopic Endometrium of Endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the normal endometrial group, the expression of the pivotal membrane\u0026ndash;cytoskeleton linker protein p-ERM was predominantly located to the apical surface of the glandular epithelium. Notably, its expression increased during the secretory phase, accompanied by a broadened localization pattern extending to the lateral membrane and the nucleus. In contrast, within the endometriosis group, p-ERM localization was restricted to the apical region of the glands, with no significant variation in its expression or distribution observed between the proliferative and secretory phases. (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Regarding E-cadherin, a marker of epithelial cell adhesion typically confined to epithelial cell membrane, showed a significant reduction during the secretory phase compared to the proliferative phase in the normal group. Conversely, in the endometriosis group, E-cadherin levels remained consistently throughout the secretory phase. (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining analysis of E-cadherin and F-actin within the same tissue sections revealed distinct and noteworthy expression patterns. (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). In normal endometrial epithelial cells during the secretory phase, E-cadherin expression was markedly decreased, whereas F-actin expression showed a corresponding increase. By contrast, in the eutopic endometrium of patients with endometriosis, E-cadherin levels during the secretory phase did not exhibit a significant decline, and F-actin expression remained relatively stable. (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, immunofluorescence co-staining for Occludin and MUC2 demonstrated their presence in both glandular epithelium and stromal cells, with localization predominantly restricted to the cell membrane. (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). In control endometrium, the integral membrane protein Occludin displayed a significant reduction in expression within glandular epithelial cells during the secretory phase compared to the proliferative phase. Conversely, endometriosis patients maintained high levels of Occludin expression in the glandular epithelium throughout both proliferative and secretory phases, with no significant interphase differences. Regarding the secretory mucin MUC2, its expression was significantly upregulated in the glandular epithelium of control endometrium during the secretory phase relative to the proliferative phase. In sharp contrast, MUC2 expression remained consistently low in the glandular epithelium of endometriosis patients during both phases, with secretory phase levels significantly reduced compared to controls. (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. PMT Protein Expression in 3D Endometrial Epithelial Organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eEndometrial epithelial organoids (EEOs) were successfully generated and maintained using a three-dimensional (3D) culture system derived from human endometrial biopsies. (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The identity and purity of these 3D cultures were confirmed by immunofluorescence co-staining for cytokeratin-7 (CK7) and vimentin. (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). CK7, a well-established marker of glandular epithelium, was robustly expressed throughout the organoids, whereas vimentin, a marker characteristic of endometrial stromal cells, was completely absent. This clear differential staining demonstrated the high purity and epithelial specificity of the cultured organoids. Upon induction of secretory phase differentiation by simultaneous treatment with estradiol (E2), medroxyprogesterone acetate (MPA), and cAMP, as outlined in our experimental flowchart (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC), EEOs derived from reproductive-age healthy donors exhibited a marked upregulation of key endometrial receptivity genes including MAOA, LIF, and PAEP. (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Notably, expression of the transmembrane mucin MUC1 was significantly downregulated following hormonal stimulation, consistent with secretory phase progression, while the receptivity-associated glycoprotein PAEP displayed increased expression. (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). Collectively, these findings indicate that the established organoid model faithfully recapitulates the functional changes characteristic of secretory phase transformation, underscoring its potential as a valuable in vitro platform for studying endometrial receptivity.\u003c/p\u003e\n\u003cp\u003eSubsequently, we investigated the differential expression of endometrial receptivity markers, with a particular focus on PMT-associated proteins, following in vitro induction of the secretory phase transition in eutopic endometrium from endometriosis patients compared to normal controls. Immunofluorescence co-staining for phosphorylated ERM (p-ERM) and F-actin revealed distinct distribution patterns between groups. (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). In secretory-phase EEOs derived from control endometrial biopsies, p-ERM displayed a relatively uniform distribution throughout the epithelial cells, accompanied by robust F-actin expression. In contrast, EEOs originating from the endometrium of endometriosis patients exhibited a pronounced polarization of p-ERM localization, predominantly concentrated at the apical tips of epithelial cells, coupled with noticeably reduced F-actin staining intensity compared to controls.\u003c/p\u003e\n\u003cp\u003eAdditionally, endometrial glandular organoids derived from control subjects and endometriosis patients exhibited divergent expression patterns of secretory mucin MUC2 versus membrane-tethered mucin MUC4. (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Following hormonally induced secretory-phase differentiation, endometriosis-derived organoids displayed significantly diminished MUC2 expression alongside elevated MUC4 levels compared to controls, revealing a transformation that appeared altered in the endometriosis-derived organoids.\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Dysregulated PMT Markers During Peri-Implantation in Endometriosis Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse endometrium tissue transplantation model is a widely accepted experimental approach for studying endometriosis pathophysiology. To investigate whether plasma membrane transition (PMT) dysregulation occurs in this model, we established an endometriosis mouse model through ectopic implantation of endometrial tissue in the peritoneal cavity. Supplementary data confirm the successful establishment of ectopic lesions, with histological analysis via hematoxylin and eosin (HE) staining demonstrating well-preserved structural integrity of both glandular and stromal components within these lesions. This model thus provides a robust platform for evaluating eutopic endometrial receptivity during the peri-implantation period.\u003c/p\u003e\n\u003cp\u003eAssessment of embryo implantation sites on gestational day 8 (GD8) revealed a significant reduction in the number of implantation sites in endometriosis-model mice compared to control animals. (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Ultrastructural examination by transmission electron microscopy at GD4 showed abundant and morphologically intact pinopodes on the luminal epithelium of the endometrial cavity in control mice, consistent with a receptive endometrium. (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). In contrast, endometriosis-model mice exhibited pinopodes with a wrinkled morphology and an increased presence of microvilli, indicative of impaired epithelial receptivity. Quantitative real-time PCR (RT-qPCR) analysis of genes associated with endometrial receptivity further corroborated these morphological observations. (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Specifically, in control mice, expression of leukemia inhibitory factor (LIF) and amphiregulin (AREG) were significantly upregulated at GD4 relative to GD0, concomitant with a marked downregulation of mucin 1 (MUC1). Conversely, endometriosis-model mice displayed only modest increases in LIF and AREG expression at GD4, with levels significantly lower than those observed in controls. Additionally, MUC1 expression remained elevated at GD4 in endometriosis mice, contrasting with its suppression in control animals (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Collectively, these data demonstrate that eutopic endometrial receptivity is markedly compromised during the peri-implantation phase in the murine model of endometriosis. To further characterize the dynamics of plasma membrane transition (PMT) during embryo implantation, immunofluorescence co-staining of Occludin and MUC2 was performed, revealing distinct temporal expression patterns. (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). In control mice, Occludin expression in the endometrial glandular epithelium was significantly downregulated at gestational day 4 (GD4) compared to GD0, whereas MUC2 expression exhibited a concomitant and significant upregulation. Conversely, in the endometriosis mouse model, Occludin expression remained persistently elevated at GD4 relative to GD0, with no significant decrease observed. Additionally, MUC2 expression in endometriosis mice increased only marginally at GD4 and remained significantly lower than that of controls at the same gestational stage. Parallel dual immunofluorescence analysis of Claudin-3, a key tight junction protein, and F-actin, a major cytoskeletal component, demonstrated that both proteins were markedly upregulated at GD4 compared to GD0 in control mice. (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). In contrast, endometriosis-model mice exhibited consistently low expression levels of Claudin-3 and F-actin at both GD0 and GD4, with GD4 levels significantly reduced relative to controls. Collectively, these findings reveal a clear dysregulation of PMT-associated molecular markers in the eutopic endometrium of the endometriosis mouse model during the peri-implantation phase.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis-associated infertility has been linked to both molecular and structural alterations in the endometrium[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], yet the upstream regulatory pathways driving these changes remain insufficiently defined. Recent transcriptomic analyses indicate that the eutopic endometrium in endometriosis exhibits dysregulation of multiple receptivity-associated genes during the secretory phase, suggesting that impaired receptivity is not solely a downstream effect of inflammation or hormonal imbalance, but may also arise from intrinsic defects in epithelial remodeling programs[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. By integrating human endometrial biopsies, hormonally differentiated endometrial organoids, and an in vivo implantation model, our study delineates a unified molecular signature linking epithelial remodeling defects to compromised embryo implantation.\u003c/p\u003e\u003cp\u003eOur findings highlight disruptions in plasma membrane transformation (PMT)\u0026mdash;a hormonally regulated remodeling process in glandular epithelium during the implantation window. Beyond the well-characterized morphological changes, we identify accompanying molecular reprogramming events, particularly the abnormal regulation of junctional and adhesion molecules (e.g., E-cadherin, occludin, ZO-1, claudins) and the dysregulation of mucins and cytoskeletal regulators. These abnormalities likely interfere with the precise epithelial depolarization required for optimal embryo attachment. Given that PMT partially overlaps but is not identical to epithelial\u0026ndash;mesenchymal transition (EMT), the PMT defect in endometriosis may represent a hybrid state in which epithelial integrity is preserved while the receptive phenotype fails to develop.\u003c/p\u003e\u003cp\u003eIn healthy controls, secretory-phase remodeling involves coordinated E-cadherin downregulation to loosen adherens junctions and facilitate trophoblast invasion, along with dynamic phosphorylation of the ERM complex to reorganize the actin cytoskeleton. In endometriosis, these events are pathologically altered: E-cadherin levels remain abnormally high, reinforcing junctional integrity and potentially impeding embryo penetration; p-ERM shows aberrant apical accumulation in organoid models, indicating defective polarity remodeling despite preserved expression levels. Mechanistically, p-ERM participates in polarity remodeling by modulating Na⁺ channel proteins in endometrial cells. Its phosphorylation is regulated by signaling pathways such as Rac-1/PAK1(pPAK1)/ERM (pERM) and Sgk1, which facilitate cytoskeletal rearrangements during receptivity[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Moreover, loss of Rictor\u0026mdash;a component of mTORC2\u0026mdash;has been linked to altered p-ERM-mediated polarity remodeling, underscoring the intricate signaling networks involved in endometrial receptivity[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA distinctive molecular signature of mucin dysregulation was also observed. During PMT, the transmembrane mucin MUC4 at the apical membrane stabilizes architecture through cytoskeletal interactions; its downregulation promotes polarity remodeling. Secreted mucin MUC2, in turn, forms a dense glycocalyx between adjacent cells, enhancing adhesion. In controls, secretory differentiation induces reduced MUC4 and increased MUC2, optimizing surface architecture for embryo adhesion. In contrast, endometriosis tissues and organoids display the opposite pattern\u0026mdash;elevated MUC4 with suppressed MUC2\u0026mdash;disrupting the adhesive and structural microenvironment of the luminal surface. This \u0026ldquo;mucin switch,\u0026rdquo; together with mislocalized p-ERM, likely undermines coordinated epithelial responses during implantation.\u003c/p\u003e\u003cp\u003eTight junction remodeling further underscores PMT dysregulation. Occludin, a key transmembrane protein, interacts with claudins to form the structural backbone of the endometrial barrier and regulate adhesive properties; its deletion can lead to chronic inflammation in various epithelia[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The actin cytoskeleton, a dynamic F-actin filament network, coordinates with Occludin-based junctions to adapt epithelial architecture for implantation[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In controls, secretory-phase endometrium shows uniform Occludin downregulation with redistribution, facilitating barrier relaxation. In endometriosis, Occludin remains elevated with abnormal basolateral polarization, potentially maintaining an overly restrictive barrier. Similar defects in claudin expression and F-actin organization suggest a coordinated failure in junctional and cytoskeletal adaptation.\u003c/p\u003e\u003cp\u003eThe functional consequences of these molecular defects were validated in an endometriosis mouse model. Pinopodes\u0026mdash;secretory-phase apical protrusions acting as receptivity sensors\u0026mdash;were morphologically immature in diseased mice, with elongated microvilli, reduced expression of receptivity genes (LIF, AREG), and persistent MUC1 elevation, all indicative of a non-receptive state. Although implantation site counts trended lower, the morphological and transcriptional profiles strongly support a receptivity deficit.\u003c/p\u003e\u003cp\u003eIn summary, our study integrates clinical specimens, hormonally induced endometrial organoids, and an in vivo implantation model to define a reproducible PMT disruption signature in endometriosis, characterized by (i) pathological mucin reprogramming, (ii) aberrant junctional remodeling, and (iii) failed cytoskeletal adaptation. These findings provide mechanistic insight into how epithelial remodeling can be selectively impaired without loss of gross epithelial structure. While our data implicate signaling pathways such as mTORC2\u0026ndash;Rictor and Rac1\u0026ndash;PAK1, causal relationships remain to be validated by targeted perturbation. Future studies should assess whether pharmacologic or genetic modulation of mucin expression, junctional proteins, or ERM activation can restore receptivity in organoid and in vivo models, potentially informing new fertility-preserving therapies for women with endometriosis-associated infertility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical conduct of research\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Medical Ethics Committee, Zhongnan\u0026nbsp;Hospital of Wuhan University (2023039K). Written informed consent was obtained from patients. All animal experiments were approved by Institutional Animal Care and Use Committee and following the 3R principle of experimental animals, and approved by Formal Review of Experimental Animal Welfare and Ethics of Zhongnan Hospital. (NO: ZN2022169)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors\u0026nbsp;consent\u0026nbsp;for\u0026nbsp;publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;National Nature Science Foundation of China (Grant NO.82201819, Grant NO.81771543) and Joint supported by Hubei Provincial Natural Science Foundation and Shiyan-of China (Grant No. 2025AFD199).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZihan Wang performed experiments and wrote the paper. Shuwei Li performed data curation and provided software support. Yanhong Mao collected the endometrial samples. Yao Xiong designed the experiment and collected the tissue samples used in the study. Yuanzhen Zhang conceived the study and revised the draft. Xinyu Liu performed supervision and conduct investigation process. Shaoyuan Xu the validation and formal analysis. All authors discussed the results and contributed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the women who donated the endometrial tissue used in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGL N, et al. Postoperative Imaging of Endometriosis. 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D \u0026ndash;\u0026thinsp;8804484; 2024. p. e70233.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7614122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7614122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Endometrial receptivity, a critical prerequisite for successful pregnancy, is impaired in women with endometriosis. Plasma membrane transformation (PMT), a key process involving dynamic remodeling of endometrial epithelial cells, is essential for establishing receptivity during the secretory phase. However, the status of PMT in the eutopic endometrium of women with endometriosis and its potential contribution to infertility are largely unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn this translational study, we investigated the PMT status in endometrial tissues from reproductive-age women with and without endometriosis. We further utilized human endometrial epithelial organoids derived from patient biopsies and a surgically induced mouse model of endometriosis to confirm our findings. PMT markers, including E-cadherin, p-ERM, F-actin, MUC2, and Occludin, and receptivity-associated molecules were analyzed using immunohistochemistry, immunofluorescence, RT-qPCR, Western blotting, and scanning electron microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe found that the dynamic changes in PMT markers observed in normal secretory endometrium, such as E-cadherin downregulation, p-ERM redistribution, and F-actin and MUC2 upregulation, were absent in the eutopic endometrium of women with endometriosis. Organoids derived from endometriosis patients also exhibited significantly reduced expression of receptivity-associated genes, diminished F-actin, persistent apical p-ERM, and aberrant mucin expression. Furthermore, the mouse model of endometriosis showed reduced implantation rates, atrophic pinopodes, and altered PMT markers, accompanied by a loss of induction of LIF and AREG. These findings were consistent across all three models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur study demonstrates that plasma membrane transformation is aberrantly regulated in the eutopic endometrium of women with endometriosis, leading to impaired epithelial remodeling and compromised receptivity. This disruption represents a novel mechanism of infertility in endometriosis. The findings highlight PMT as a potential therapeutic target for improving endometrial receptivity and offer new insights into the pathological basis of endometriosis-associated infertility.\u003c/p\u003e","manuscriptTitle":"Dysregulation of Plasma Membrane Transition (PMT) in Endometrial Epithelium: Implications for Infertility in Endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 11:42:04","doi":"10.21203/rs.3.rs-7614122/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-03T06:25:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-23T16:34:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218298777148645934294982655996984390677","date":"2025-12-01T15:36:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160448651676285992805958195791270010550","date":"2025-10-28T14:07:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-23T05:28:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-17T23:58:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-16T22:32:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2025-09-14T17:47:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"785aaf6b-3971-42ed-8233-f02127dfd871","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-31T16:24:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 11:42:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7614122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7614122","identity":"rs-7614122","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}