MiR-29c-3p Impairs the Adhesion of Endometrial Epithelial Cells via COL4A1/β-catenin in Endometriosis

This study was conducted in accordance with the Declaration of Helsinki, and approved by the Research Ethics Committee of Peking University Shenzhen Hospital (No. 2024047). Before being recruited, all patients signed a written informed consent form.

The final version of the paper has been reviewed by all co‐authors, who have all agreed to submit it for publication.

This research was supported by the Guangdong Basic and Applied Basic Research Foundation (2022A1515220164, 2024A1515030140), Sanming Project of Medicine in Shenzhen (No. SZSM202211043), and the National Natural Science Foundation of China (82271684).

The baseline clinical characteristics of all participants are summarized in Table  2 . The endometriosis and control groups were comparable with respect to BMI, cycle day at biopsy, and all hormonal profiles measured (all p > 0.05). However, the control group was significantly younger than the endometriosis group (28.4 ± 3.9 vs. 33.6 ± 3.9 years, p = 0.037), as formal age matching was not performed in this exploratory study. To investigate the potential involvement of miR‐29c‐3p in endometriosis‐associated infertility, we first quantified its expression in clinical samples.When comparing secretory‐phase HEECs from endometriosis to controls, qRT‐PCR indicated that miR‐29c‐3p levels were highly upregulated (Figure  1a ). To delineate the functional impact of this upregulation on embryo implantation, we modulated miR‐29c‐3p levels in Ishikawa cells using specific mimics and inhibitors. Transfection efficiency was confirmed by qRT‐PCR, demonstrating robust overexpression and knockdown, respectively (Figure  1b and c ). In spheroid attachment assays, the adhesion of JAr spheroids to the Ishikawa cell monolayer was significantly decreased by overexpressing miR‐29c‐3p (Figure  1d and e ). Given the pivotal role of E‐cadherin in establishing a receptive endometrium, we subsequently assessed its protein expression levels. According to Western blot analysis, overexpression of miR‐29c‐3p markedly reduced the expression of E‐cadherin (Figure  1f ). Given that miR‐29c‐3p is not anticipated to directly target E‐cadherin, these results imply that miR‐29c‐3p modulates endometrial receptivity by regulating upstream signaling networks that control E‐cadherin protein stability or expression. Baseline clinical characteristics of study participants. Abbreviation: AMH, anti‐Müllerian hormone, BMI, body mass index, E2, estradiol, FSH, follicle‐stimulating hormone, IQR, interquartile range, LH, luteinizing hormone, PRL, prolactin, SD, standard deviation. Cycle day was calculated as the interval between the last menstrual period and biopsy date. P values were calculated using Student's unpaired t‐test for normally distributed data or Mann‐Whitney U test for non‐normally distributed data. Normality was assessed by Shapiro‐Wilk test. ASRM, American Society for Reproductive Medicine. miR‐29c‐3p impairs the receptivity of endometrial epithelial cells. (a) Relative expression of miR‐29c‐3p in secretory‐phase endometrial tissues from patients with endometriosis and normal controls. (b, c) Validation of miR‐29c‐3p overexpression and knockdown in Ishikawa cells following transfection with mimics or inhibitors. (d, e) Representative images and quantitative analysis of JAr spheroid attachment to Ishikawa cells. (f) Western blot analysis of E‐cadherin protein expression levels in Ishikawa cells following modulated expression of miR‐29c‐3p. Data are presented as mean ± SEM ( n = 3). Abbreviations: EM, endometriosis, Con, control, mimic, miR‐29c‐3p mimic, inhibitor, miR‐29c‐3p inhibitor, mNC, mimic negative control, iNC, inhibitor negative control. ** p < 0.01, *** p < 0.001, ns, no significant. To systematically delineate the downstream regulatory network of miR‐29c‐3p, we employed a multi‐database computational approach combining TargetScan and miRDB. The intersection of these predictive algorithms yielded a pool of 765 putative target genes (Figure  2a ). Subsequent functional annotation via KEGG pathway enrichment analysis showed a strong clustering of these candidates within signaling cascades governing cell adhesion and extracellular matrix interactions (Figure  2b ). Notably, gene family analysis highlighted the collagen superfamily as a significant target hub. Among these, type IV collagen (COL4A1) emerged as a prominent candidate (Figure  2c ) due to its consistent prediction across algorithms, high confidence scores, functional relevance to cell adhesion, and inverse expression correlation with miR‐29c‐3p in clinical samples. These bioinformatic insights strongly suggested that miR‐29c‐3p may modulate endometrial function by targeting key adhesion‐related molecular components. Bioinformatic identification of miR‐29c‐3p targets. (a) Venn diagram illustrating the intersection of potential miR‐29c‐3p targets predicted by TargetScan and miRDB databases. (b) KEGG pathway enrichment analysis of the 765 predicted target genes showing significant enrichment in adhesion‐related pathways. (c) Heatmap visualization showing the functional association between candidate genes and key adhesion‐related pathways identified through bioinformatic analysis. Based on the bioinformatic prioritization, COL4A1 was selected for further empirical validation. Initially, we used a dual‐luciferase reporter assay to confirm the directly interaction between miR‐29c‐3p and the COL4A1 transcript. Co‐transfection results demonstrated that miR‐29c‐3p significantly suppressed luciferase activity when binding to the specific target site within the COL4A1 3'‐UTR (Figure  3a ). To establish the clinical relevance of this interaction, we subsequently examined patient tissues. Consistent with a negative regulatory mechanism, the endometrial epithelium of patients with endometriosis was discovered to have significantly lower levels of COL4A1 expression (both at the mRNA and protein levels) than controls, exhibiting a robust inverse correlation with the elevated miR‐29c‐3p levels observed previously (Figure  3b and c ). For Western blot and qPCR analyses of COL4A1 expression in clinical HEECs, each biological replicate ( n = 3) represents primary HEECs isolated from an individual patient, rather than pooled samples. Furthermore, gain‐ and loss‐of‐function assays in Ishikawa cells corroborated this post‐transcriptional silencing mechanism: transfection with miR‐29c‐3p mimics markedly attenuated COL4A1 expression levels, whereas inhibitor treatment restored its expression (Figure  3d–f ). Collectively, these findings provide definitive evidence that miR‐29c‐3p directly targets the function of COL4A1 in endometrial epithelial cells. miR‐29c‐3p negatively regulates COL4A1 expression. (a) Predicted miR‐29c‐3p binding sites within the COL4A1 3'‐UTR. (b, c) COL4A1 protein and mRNA expression levels in secretory‐phase HEECs isolated from patients with endometriosis and controls. (d, e) Relative COL4A1 mRNA expression in Ishikawa cells following transfection with miR‐29c‐3p mimics or inhibitors compared to the controls. (f) Western blot analysis demonstrating COL4A1 protein expression changes following miR‐29c‐3p modulation. Data are presented as mean ± SEM from three independent biological replicates ( n = 3), with each replicate representing primary HEECs isolated from a distinct patient (3 patients per group). * p < 0.05, **** p < 0.0001, ns, no significant. To determine whether COL4A1 downregulation recapitulates the phenotype observed with miR‐29c‐3p overexpression, we utilized siRNAs targeting COL4A1. Since siCOL4A1‐2 had the greatest knockdown efficiency out of the three siRNA candidates, it was chosen for further testing (Figure  4a and  b ). Silencing of COL4A1 in Ishikawa cells significantly impaired JAr spheroid adhesion (Figure  4c and  d ), effectively mirroring the effects previously observed with miR‐29c‐3p mimics. This functional defect was accompanied by a concomitant reduction in E‐cadherin protein expression (Figure  4e ). Consistent with our in vitro results, clinical endometrial samples from patients with endometriosis exhibited significantly reduced E‐cadherin protein levels compared to controls (Figure  4f ). These findings suggest that COL4A1 is essential for maintaining endometrial adhesiveness, most likely by sustaining E‐cadherin expression at the protein level. COL4A1 regulates JAr spheroid adhesion and E‐cadherin expression. (a, b) Validation of COL4A1 knockdown efficiency at both the mRNA and protein levels. (c, d) Representative images and quantification of JAr spheroid attachment to COL4A1‐silenced Ishikawa cells compared to control cells. (e) Western blot analysis of E‐cadherin protein expression following COL4A1 knockdown. (f) E‐cadherin protein expression levels in clinical endometrial samples from patients with endometriosis and controls. Data are presented as mean ± SEM ( n = 3). *** p < 0.001, **** p < 0.0001, ns, no significant. Beyond its role in adhesion, we further evaluated the impact of COL4A1 silencing on cellular state. Wound healing and CCK‐8 tests showed that COL4A1 knockdown markedly increased both the migration (Figure  5a and  b ) and proliferation capacity (Figure  5c ) of Ishikawa cells. Taken together with the reduced adhesion rates previously observed (Figure  4c and  d ), these results indicate that loss of COL4A1 shifts endometrial epithelial cells toward a dynamic proliferative and migratory phenotype, thereby compromising the stationary, adhesive state that is required for successful embryo implantation. COL4A1 knockdown promotes migration and proliferation of Ishikawa cells. (a) Representative images of wound healing assays in COL4A1‐silenced versus control cells at 0 and 24 h. (b) Quantitative analysis and statistical comparison of wound closure rates. (c) Cell proliferation kinetics assessed by CCK‐8 assay over a 72 h period. Data are presented as mean ± SEM ( n = 3). *** p < 0.001, **** p < 0.0001, ns, no significant. To decipher the molecular pathway linking COL4A1 loss to impaired endometrial receptivity, we comprehensively profiled the expression of key receptivity and EMT markers. While other receptivity genes remained largely unchanged (Figure  6a and  b ), COL4A1 knockdown specifically induced the mRNA expression of SNAI1, a master transcription factor that orchestrates epithelial‐mesenchymal transition (EMT) (Figure  6b ). This finding was further confirmed at the protein level: COL4A1 silencing significantly upregulated the mesenchymal markers SNAI1 and N‐cadherin, while concurrently downregulating the epithelial marker E‐cadherin (Figure  6c ), strongly indicative of an active EMT process. Mechanistically, we investigated the Wnt/ β ‐catenin pathway, a well‐established regulator of EMT. Western blot analysis revealed that COL4A1 knockdown significantly increased the expression of β ‐catenin and its downstream transcriptional targets, c‐Myc and Cyclin D1 (Figure  6d ). These findings suggest that the miR‐29c‐3p/COL4A1 axis may regulate endometrial receptivity at least in part through modulation of Wnt/ β ‐catenin signaling and EMT, thereby potentially contributing to the maintenance of epithelial integrity that is requisite for successful embryo implantation. However, we acknowledge that c‐Myc and Cyclin D1 are not uniquely specific to Wnt/ β ‐catenin signaling, and definitive confirmation of pathway activation would require evaluation of β ‐catenin and GSK3 β phosphorylation status. COL4A1 silencing induces EMT via the Wnt/ β ‐catenin pathway. (a, b) Relative mRNA expression of various adhesion‐related genes following COL4A1 knockdown, specifically note the dramatic and specific upregulation of SNAI1. (c) Western blot analysis of protein expression levels of canonical EMT markers (E‐cadherin, N‐cadherin, SNAI1) following COL4A1 silencing. (d) Western blot analysis of protein expression levels of Wnt/ β ‐catenin pathway components ( β ‐catenin, c‐Myc, Cyclin D1) in control and COL4A1‐silenced cells. Data are presented as mean ± SEM from three independent experiments ( n = 3). **** p < 0.0001, ns, no significant.

Women between the ages of 20 and 40 who had normal menstrual periods and a normal ovarian reserve were recruited for this study. The study group ( n = 7) was composed of women with pathologically confirmed ovarian endometriomas who had not received any hormonal treatment within three months prior to surgery. The control group ( n = 7) comprised women undergoing evaluation for male‐factor infertility, with no detectable gynecological abnormalities as confirmed by comprehensive clinical assessment, including transvaginal ultrasonography and hysteroscopy. Exclusion criteria encompassed perimenopause, uterine structural anomalies, hydrosalpinx, infectious or inflammatory conditions, autoimmune disorders, malignancies, and other endometrial pathologies. Endometrial tissues were obtained during the secretory phase, confirmed by ultrasound‐documented ovulation followed by serum progesterone measurement, under the supervision of experienced reproductive endocrinologists at the Reproductive Medicine Center of Peking University Shenzhen Hospital. A formal a priori power calculation was not performed for this exploratory mechanistic study, as the sample size was determined by the availability of fresh biopsies meeting the inclusion criteria. Before enrollment, all attendees signed a form requesting their informed consent, and the study procedures were approved by Peking University Shenzhen Hospital's Research Ethics Committee (Approval Number: 2024047). Fresh endometrial tissues were immediately immersed in phosphate‐buffered saline (PBS; Gibco, China) and washed thoroughly to remove blood clots and debris. Tissues were finely minced and subjected to enzymatic digestion with type I collagenase and deoxyribonuclease I (Biosharp, China) in PBS for 60 min at 37°C. DMEM/F12 (Gibco, China) supplemented with 10% fetal bovine serum (FBS; Gibco, Australia) and 1% penicillin‐streptomycin (Gibco, USA) was added to stop the digestion process. The resulting suspension was passed through a 40‐µm cell strainer to separate and remove stromal components. Epithelial glands retained on the filter were collected, resuspended in complete medium, and cultivated overnight at 37°C in a humidified atmosphere with 5% CO 2 . Non‐adherent cells were removed the next day, and the adherent HEECs were maintained in fresh medium [ 5 , 13 ]. The purity of primary HEECs was assessed by morphological examination under an inverted phase‐contrast microscope. HEECs were identified by their characteristic polygonal or cobblestone‐like appearance and typical growth in epithelial cell clusters, whereas contaminating stromal cells exhibited a spindle‐shaped, fibroblast‐like morphology.Primary HEECs were used at passage 0 (P0) without further subculture. Cells were cultured for 48–72 h after isolation prior to RNA or protein extraction. Cells from different culture dishes were not pooled; instead, each dish was processed independently as a separate biological replicate. Ishikawa cells (Pricella, China) were cultivated at 37°C in a humidified environment with 5% CO 2 in DMEM (Gibco, China) accompanied with 10% FBS and 1% penicillin‐streptomycin. According to the package recommendations, cells were transfected utilizing Lipofectamine 3000 (Invitrogen, USA) with miR‐29c‐3p mimics, miR‐29c‐3p inhibitors, negative controls (NC), or COL4A1 small interfering RNAs (siRNAs). Subsequent assays were performed 48 h post‐transfection. Protease inhibitor‐containing RIPA lysate (Beyotime, China) was used to retrieve the total protein from Ishikawa cells and primary HEECs. Equal amounts of protein were then separated by SDS‐PAGE (Biotides, China) and moved onto PVDF membranes (Millipore, Ireland). The membranes were blocked with 5% non‐fat milk in TBST (0.1% Tween‐20; Solarbio, China) for 1 h at room temperature, primary antibodies against COL4A1, E‐cadherin, N‐cadherin (Abcam, UK), β ‐catenin, SNAI1, c‐Myc, GAPDH (Proteintech, China), and Cyclin D1 (Selleck, USA) were then incubated overnight at 4°C. Following the wash, membranes were incubated with HRP‐conjugated secondary antibodies for 1 h at ambient temperature prior to visualization of protein bands using enhanced chemiluminescence (ECL) reagents (Invitrogen, USA). Total RNA, including miRNA, was isolated from primary HEECs and transfected Ishikawa cells using the miRNeasy Micro Kit (QIAGEN, Germany). cDNA synthesis was performed using the Evo M‐MLV Reverse Transcription Kit for mRNA and a specific miRNA First‐Strand Synthesis Kit (AG, China). qRT‐PCR was performed on a Roche 480 real‐time PCR system using the SYBR Green Pro Taq HS qPCR Kit II (AG, China). Table  1 lists the particular primers that were used. The 2^ (‐ΔΔCt) method was used to calculate relative expression levels, and internal reference controls for mRNA and miRNA were GAPDH and U6, respectively. Primer Sequences for qPCR. The targets of miR‐29c–3p were predicted from TargetScan and miRDB databases with the species set to Homo sapiens. Gene symbols of the common targets were converted to Entrez IDs using the bitrfunction from the clusterProfiler package in R software (version 4.5.0), with the org.Hs.eg.db as the annotation database. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted using the enrichKEGG functions in clusterProfiler, with the whole human genome as the background. The intersection of these databases was obtained via Venn diagram. Subsequent functional annotation via KEGG pathway enrichment analysis. Potential miR‐29c‐3p binding sites in the COL4A1 3'‐untranslated region (3'‐UTR) were predicted using TargetScan. Wild‐type and mutant COL4A1 3'‐UTR sequences were cloned into pmirGLO dual‐luciferase vectors (GenePharma, China). High‐glucose DMEM was used to support Ishikawa cells, and Lipofectamine 3000 was used to co‐transfect them with reporter plasmids (1 µg) and miR‐29c‐3p mimics or NC. After 48 h, the cells were lysed, and the Dual‐Glo Luciferase Assay System (Servicebio, China) was used to measure the Firefly and Renilla luciferase activities one after the other. Firefly luciferase activity was normalized to Renilla luciferase signals to control for transfection efficiency. Transfected Ishikawa cells were seeded into 96‐well plates (2,000 cells/well) and cultivated for 0, 24, 48, or 72h. Cell viability was assessed using the Cell Counting Kit‐8 (CCK‐8; Proteintech, China). Each well received 10µl of CCK‐8 reagent at each time point, and the cells were cultivated for 2h at 37°C. The absorbance was measured and analyzed at 450nm using a microplate reader. In 6‐well plates, cells were cultivated to 90–100% confluence. A sterile pipette tip was used to make a linear scratch wound. The remaining cells were cultivated in serum‐free DMEM for 24 h after detached cells were gently washed away. Using an optical microscope, the same fields were imaged at 0 and 24 h to track wound closure. The migration rate was computed as follows: (initial width ‐ 24 h width)/initial width × 100. Data represent the average of three randomly selected fields. The adhesion test was conducted in accordance with earlier directions [ 6 , 14 , 15 ]. In short, blastocyst surrogates were made from JAr choriocarcinoma cells. To generate spheroids, JAr cells were resuspended in RPMI‐1640 medium containing 10% FBS and plated into ultra‐low attachment 96‐well plates for 24 h. The generated spheroids were then transferred onto an adherent monolayer of transfected Ishikawa cells. The overall number of spheroids was counted (time = 0 h). After 6 h of co‐culture, non‐adherent spheroids were removed by washing three times with PBS. Next, the amount of spheroids that were attached was tallied. The attachment rate was defined as the percentage of attached spheroids relative to the total number initially seeded. At least three separate biological replicates were used in each test. Continuous variables are presented as mean±standard deviation (SD) or median (interquartile range, IQR) as appropriate. Normality of distribution was assessed using the Shapiro‐Wilk test. Comparisons between two groups were performed using Student's unpaired t‐test for normally distributed data or the Mann‐Whitney U test for non‐normally distributed data. Categorical variables were compared using Fisher's exact test. A two‐sided p ‐value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25.0 or GraphPad Prism 8.0. The prism data were presented as mean ± standard error of the mean (SEM). Significance levels were defined as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns denotes no significant differences.

In summary, this study elucidates a potential regulatory mechanism underlying defective endometrial receptivity in endometriosis (Figure 7 ). Our findings suggest that COL4A1 is suppressed by aberrant enhanced expression of miR‐29c‐3p, which may stimulates Wnt/ β ‐catenin signaling and triggers pathological EMT. This molecular cascade ultimately results in the loss of E‐cadherin expression and compromised embryo adhesion. The discovery of the miR‐29c‐3p/COL4A1/ β ‐catenin axis not only advances our molecular understanding of endometriosis‐related infertility but also highlights this pathway as a promising possible treatment target for restoring endometrial receptivity and may represent a potential experimental approach for future investigation of implantation regulation. However, this would require further evaluation of β ‐catenin and GSK3 β phosphorylation status. Schematic illustration of the proposed miR‐29c‐3p/COL4A1/Wnt/ β ‐catenin/EMT axis in endometriosis‐associated infertility.

Endometriosis is a debilitating chronic illness that impairs fertility and pelvic health due to aberrant molecular signaling. It is becoming more widely acknowledged that ectopic and eutopic endometrial tissues are characterized by altered gene expression and miRNA‐mediated post‐transcriptional regulatory, especially involving miRNAs. For instance, miR‐29c‐3p and miR‐143‐3p are markedly upregulated [ 16 , 17 ], while miR‐449b‐3p and miR‐518c‐3p are downregulated in ectopic lesions [ 16 , 18 ]. Clinically, infertility in endometriosis is frequently underpinned by defective endometrial receptivity—which represents the limiting factor for successful implantation. Recent evidence underscores the pivotal role of miRNAs in governing this receptive state. For example, diminished miR‐543 expression [ 19 ] and aberrant upregulation of miR‐182‐5p [ 20 ] have been linked to impaired receptivity and implantation failure. Conversely, miR‐23b‐3p promotes receptivity by modulating epithelial adhesion [ 15 ]. Collectively, these findings suggest that a delicate miRNA regulatory network is essential for the development of a receptive endometrium. In the current investigation, we discover that miR‐29c‐3p is a critical negative regulator of endometrial receptivity. We have realized that miR‐29c‐3p is significantly upregulated in the secretory‐phase endometrial epithelium of patients with endometriosis (Figure  1a ), which inversely correlates with E‐cadherin expression (Figure  4f ). Functionally, miR‐29c‐3p overexpression attenuated JAr spheroid adhesion and suppressed E‐cadherin protein levels in vitro (Figure  1e and f ). E‐cadherin is a cornerstone of epithelial integrity and a key regulator of EMT, a process intimately linked to endometrial receptivity. Notably, it seems that miR‐29c‐3p's function in EMT is very context‐dependent. In certain tumors and biliary atresia, it acts as a metastasis promoter by upregulating EMT markers [ 21 , 22 , 23 ]. In contrast, it functions as a tumor suppressor in other malignancies [ 24 , 25 , 26 , 27 , 28 ] by inhibiting EMT. Our findings suggest that in the context of the endometrium, miR‐29c‐3p promotes an EMT‐like phenotype (loss of E‐cadherin) that is detrimental to embryo attachment. By demonstrating that miR‐29c‐3p disrupts receptivity via the COL4A1/Wnt/ β ‐catenin axis, our study resolves previous ambiguities regarding its function in the reproductive tract and offers a novel mechanistic explanation for endometriosis‐associated infertility. The secretory phase represents a critical window for endometrial remodeling. We discovered that COL4A1 expression is markedly suppressed in the endometrium of patients with endometriosis during this period (Figure  3b and c ). Mechanistically, our data indicate that COL4A1 serves as a guardian of the epithelial phenotype. Its knockdown recapitulated the defects caused by miR‐29c‐3p overexpression, leading to reduced spheroid adhesion (Figure  4d ) and activation of EMT. This aligns with previous reports suggesting that COL4A1 downregulation impairs epithelial adhesion in infertility [ 12 ]. However, the function of COL4A1 is also pleiotropic; in gastric and hepatocellular carcinomas, COL4A1 overexpression drives invasion and metastasis via PI3K‐Akt signaling [ 29 , 31 ], and its inhibition prevents abdominal aortic aneurysm progression [ 30 ]. These disparities highlight that the biological outcome of COL4A1 signaling is strictly dictated by the tissue microenvironment. In the endometrium, COL4A1 appears to be crucial for maintaining the stability of the basement membrane and preventing premature or pathological EMT via the Wnt/ β ‐catenin pathway. Loss of COL4A1 triggers an unwanted EMT, shifting the cells toward a proliferative and migratory state that is hostile to embryo implantation. Several limitations of this study should be acknowledged. First, the sample size is modest ( n = 7 per group), and no formal power calculation was performed, as this was an exploratory mechanistic study. Formal ASRM staging was not available for the endometriosis cohort, and infertility duration was not systematically recorded, although all cases were pathologically confirmed as ovarian endometriomas. Additionally, the control group was not age‐matched and was evaluated by transvaginal ultrasonography and hysteroscopy rather than by diagnostic laparoscopy, the gold standard for endometriosis diagnosis. Second, primary HEECs were assessed for purity by morphological examination rather than by immunocytochemical staining with epithelial and mesenchymal markers, and mycoplasma testing was not routinely performed. The evidence for Wnt/ β ‐catenin pathway activation is suggestive rather than definitive, as phosphorylation of β ‐catenin and GSK3 β was not evaluated. Third, isolation of primary secretory‐phase HEECs is constrained by clinical sample availability and the technical challenges of long‐term culture, which limited our ability to perform rescue experiments and extensive genetic manipulations. Fourth, while the JAr spheroid assay is a widely accepted model for embryo attachment, it lacks the complex stromal‐epithelial and immune interactions present in the human uterus. Future studies utilizing endometrial organoid models, phosphorylation‐specific antibody validation, and appropriate in vivo models will be essential to confirm and extend our findings.

Endometriosis is a debilitating chronic gynecological condition that affects about 190 million women of reproductive age globally [ 1 , 2 ]. Characterized by infertility, pelvic pain, and dysmenorrhea, it is a major cause of reproductive dysfunction and is found in as many as 50% of infertile women [ 3 ]. Central to successful pregnancy establishment is endometrial receptivity, a transient physiological state that permits embryo attachment and invasion. This window of implantation (WOI) typically encompasses the mid‐secretory phase (days 6–10 post‐ovulation, or cycle days 20–24) [ 4 , 5 ]. During this critical period, the endometrium undergoes extensive structural and functional remodeling—including decidualization—to support embryo implantation [ 6 , 7 ]. Accumulating evidence indicates that patients with endometriosis, exhibit significant functional abnormalities in the endometrium during the WOI, resulting in impaired receptivity and implantation failure. However, it is still unclear exactly what molecular processes underlie these deficiencies. Small non‐coding RNAs with a magnitude of about 22 nucleotides, known as microRNAs (miRNAs), are crucial post‐transcriptional modulators in a number of physiological processes, including endometrial remodeling [ 7 , 8 ]. The dysregulation of miRNAs is increasingly recognized as a characteristic feature of endometriosis pathophysiology [ 9 ]. In particular, miR‐29c has attracted considerable attention for its role in endometrial function, although findings remain somewhat contradictory. While certain studies implicate miR‐29c in the broader pathogenesis of endometriosis [ 10 ], others demonstrate its aberrant upregulation contributes to progesterone resistance [ 11 ] and is significantly increased in the endometrial epithelium of infertile women during the secretory phase [ 12 ]. Although Griffiths et al. previously established a correlation between miR‐29c overexpression, COL4A1 downregulation, and reduced endometrial epithelial adhesion in infertile endometrium [ 12 ], the downstream molecular mechanisms linking this axis to impaired adhesion remained entirely unexplored. Their study did not investigate any signaling pathways downstream of COL4A1, nor did it address the cellular processes through which reduced COL4A1 leads to diminished adhesive capacity. Furthermore, previous studies have not definitively characterized the expression profile of miR‐29c in secretory human endometrial epithelial cells (HEECs) from endometriosis patients. Additionally, the mechanisms by which miR‐29c compromises receptivity remain unclear. To address this knowledge gap, the current study assessed the expression of miR‐29c‐3p in HEECs from patients with and without endometriosis during the WOI. Furthermore, our goal was to analyze the molecular processes that underlie the control of embryo adhesion by miR‐29c‐3p, thereby providing a theoretical foundation for potential targeted interventions to restore endometrial receptivity and enhance the chances of conception in women who are infertile because of endometriosis.

The authors declare no conflicts of interest.