Upregulation of SOX9 in the eutopic endometrium of adenomyosis and its association with epithelial-mesenchymal transition

Upregulation of SOX9 in the eutopic endometrium of adenomyosis and its association with epithelial-mesenchymal transition | 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 Upregulation of SOX9 in the eutopic endometrium of adenomyosis and its association with epithelial-mesenchymal transition Jiajia Wei, Ping He, Li Fan, Jingjing Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9059335/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Adenomyosis is an estrogen-dependent benign gynecological disorder, although its exact pathophysiology is still mostly unclear. SRY-box transcription factor 9 (SOX9) is an essential transcription factor regulating epithelial-mesenchymal transition (EMT) in various diseases. This study investigated SOX9 expression in the eutopic endometrium of adenomyosis and its association with the EMT process, providing new insights into the underlying pathological mechanisms. Methods: A retrospective case-control study was conducted, including 60 patients with adenomyosis and 60 control patients with uterine leiomyomas. Eutopic endometrial tissue samples were collected from all participants. The expression levels of SOX9 and key EMT-related markers ((including E-cadherin, N-cadherin, Vimentin, Pan-keratin, and Cytokeratin 7) were evaluated using immunohistochemistry (IHC) and quantitative real-time PCR (RT-qPCR). Furthermore, multivariate logistic regression analysis was performed to assess the association between SOX9 expression and the risk of adenomyosis. Results: Both SOX9 protein levels and mRNA expression were significantly upregulated in the eutopic endometrium of the adenomyosis group compared to the control group (both with P < 0.001). Elevated SOX9 levels positively correlated with the mesenchymal marker Vimentin, and negatively correlated with the epithelial marker E-cadherin. Multivariate analysis indicated that a higher SOX9 score was independently associated with adenomyosis (OR = 1.144, 95% CI 1.008-1.298, P < 0.05). Conclusions: SOX9 is significantly overexpressed in the eutopic endometrium of adenomyosis patients and is closely associated with the EMT process. These findings suggest that SOX9-associated EMT may play a critical role in the pathogenesis of adenomyosis, highlighting SOX9 as a potential biomarker or therapeutic target for this disease. Adenomyosis SOX9 Epithelial-Mesenchymal Transition Eutopic endometrium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Summary Elevated SOX9 expression in the eutopic endometrium is independently associated with adenomyosis and specific EMT profiles, identifying it as a potential novel clinical biomarker. Background Adenomyosis is a prevalent estrogen-dependent gynecological condition marked by the infiltration of endometrial glands and stroma into the myometrium, along with adjacent myometrial hypertrophy and fibrosis (1). It mainly affects women of childbearing age, with an estimated prevalence of 5% to 70%. The highest prevalence is observed in women aged over 40 years (2). The main clinical manifestations include dysmenorrhea, heavy menstrual bleeding, chronic pelvic pain, and infertility, which substantially impair patients’ quality of life; approximately one-third of patients may be asymptomatic (3). Histopathological examination remains the gold standard for definitive diagnosis, whereas imaging modalities such as transvaginal ultrasound and magnetic resonance imaging (MRI) lack standardized diagnostic criteria and show considerable variability in accuracy (4). The current treatment primarily depends on progestin-based medications, such as the levonorgestrel-releasing intrauterine system (LNG-IUS) and dienogest. However, many individuals develop resistance to progesterone, rendering it less effective and increasing the likelihood of recurrence (5, 6). Recent research indicates that KRAS mutations may diminish progesterone receptor expression by facilitating the methylation of its promoter, thereby attenuating the anti-proliferative effects of dienogest (7). In women desiring fertility preservation, both medical treatments and conservative surgeries frequently fail to achieve long-term symptom relief, while hysterectomy, although curative, is unsuitable for those who wish to retain the uterus (8). These therapeutic challenges underscore the urgent need to elucidate the pathogenesis of adenomyosis, identify novel biomarkers, and discover new therapeutic targets. The pathophysiology of adenomyosis is not yet fully elucidated. The two primary concepts are the hypothesis of basal endometrium invagination and the metaplasia theory. The invagination theory is the most widely accepted perspective. This concept asserts that repeated tissue injury and repair (TIAR) is crucial, shaped collectively by uterine hyperperistalsis, a localized hyperestrogenic milieu, inflammation, and the disruption of the endometrial-myometrial interface (EMI) (9, 10) . EMT is regarded as a core mechanism facilitating the migration and myometrial invasion of endometrial cells: epithelial cells lose their polarity and adhesion molecules (downregulation of E-cadherin, Pan-keratin, and Cytokeratin 7) and acquire mesenchymal characteristics (upregulation of N-cadherin and Vimentin), thereby enhancing their migratory and invasive abilities (11) . Estrogen-induced EMT has been confirmed in both tissue samples from patients with adenomyosis and in animal models (11) . SOX9 (SRY-related HMG-box gene 9) is a key transcription factor of the SOX family that plays important roles in organogenesis, stem cell maintenance, and cell fate determination (12). In various malignant tumors, SOX9 promotes EMT, invasion, and metastasis by activating multiple signaling pathways, including Wnt/β-catenin, TGF-β/Smad, and Notch (13, 14). Previous studies have demonstrated that SOX9 is involved in the development and progression of endometrial hyperplasia, endometriosis, and endometrial cancer (15-17). Recent single-cell RNA sequencing analyses have shown that Wnt-related genes, including SOX9, are significantly upregulated in ectopic endometrium of adenomyosis (18). Our previous studies indicated that downregulation of SOX9 predicts the response to progestin therapy in endometrial hyperplasia (19). Nonetheless, the expression profile of SOX9 in eutopic endometrium associated with adenomyosis and its relationships with EMT markers are not well-defined. This study seeks to compare SOX9 expression in eutopic endometrium between adenomyosis patients and control subjects, while also examining its associations with significant EMT markers (E-cadherin, Cytokeratin 7, Pan-keratin, N-cadherin, and Vimentin), thereby offering novel insights into the pathogenesis of adenomyosis and identifying potential therapeutic targets. Methods Study design and patient recruitment All specimens collected received thorough histological validation. Two senior pathologists independently examined the postoperative slides to verify the accuracy of the diagnosis and to ensure that all endometrial samples were in the proliferative phase of the menstrual cycle. Based on the definitive pathological results, patients were categorized into two groups: the adenomyosis group (n = 60) and the control group (n = 60). To identify the intrinsic molecular alterations that may drive myometrial invasion, this study focused on the eutopic endometrium of AM patients. We utilized patients with uterine leiomyoma as a clinically relevant control to account for the shared estrogen-dependent proliferative environment. This design allows for the identification of specific molecular drivers (e.g., SOX9) and the assessment of the EMT-associated 'priming' state of the endometrium, distinct from general benign muscular hyperplasia. The study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Guangxi Medical University (approval number: KY2024001) and the Medical Ethics Committee of Liuzhou Hospital of Guangzhou Women and Children's Medical Center (approval number: Quick Review-Research-2024-118). All patients provided written informed consent prior to surgery. Inclusion and exclusion criteria The inclusion criteria included: (i) Patients aged 18–50 years; (ii) Histologically verified proliferative-phase endometrium; and (iii) Histopathologically confirmed adenomyosis in the adenomyosis group and uterine leiomyomas (FIGO types IV–VI) in the control group. The exclusion criteria included: (i) Incomplete clinical data; (ii) Concurrent endometrial hyperplasia, endometriosis, or reproductive system malignancies; (iii) Use of hormonal therapy, antiplatelet drugs, or oral contraceptives within 3 months prior to surgery; (iv) Presence of pelvic inflammatory disease, genital tract malformations, endocrine disorders, or autoimmune conditions; and (v) Pregnancy, lactation, or history of long-term exposure to toxins or carcinogens. Clinical data collection Peripheral blood samples were collected on days 2–4 of the menstrual cycle prior to surgery to measure serum levels of estradiol (E₂), prolactin (PRL), carbohydrate antigen (CA125), activated partial thromboplastin time (APTT), hemoglobin, and platelet count. Patient characteristics, including age, body mass index (BMI), disease duration, gravidity and parity, history of uterine surgery, severity of dysmenorrhea (assessed using the visual analog scale [VAS], 0–10 points), and menstrual blood loss (quantified by the pictorial blood assessment chart [PBAC] score), were recorded. Preoperative uterine dimensions were measured in a blinded manner by two senior ultrasound physicians using the same ultrasound equipment. Uterine volume was calculated using the formula 0.52 × anteroposterior diameter × longitudinal diameter × transverse diameter. Tissue specimen collection and processing During surgery, the uterus was incised along the anterior wall with a “Y”-shaped incision, and eutopic endometrial tissue from the uterine cavity was collected. Fresh specimens were divided into two parts: one part was immediately fixed in 10% neutral buffered formalin (volume ratio ≥10:1, fixation time ≤1 week) for immunohistochemistry (IHC); the other part was cut into 1–2 mm³ pieces, rapidly frozen in liquid nitrogen, and then transferred to a −80°C freezer for storage until used for reverse transcription quantitative real-time PCR (RT-qPCR). IHC Paraffin-embedded sections were prepared by experienced pathologists (thickness 5 μm). The primary antibodies and their dilutions were as follows: SOX9 monoclonal antibody (1:200), E-cadherin polyclonal antibody (1:6000), N-cadherin polyclonal antibody (1:200), Cytokeratin 7 (CK7) specific polyclonal antibody (1:3000), pan-keratin polyclonal antibody (1:3000), and Vimentin polyclonal antibody (1:3000) (all purchased from Servicebio Biotechnology, Wuhan, China). The staining was performed using the streptavidin-peroxidase two-step method, followed by the development of the 3,3'-diaminobenzidine chromogen and counterstaining with hematoxylin. The H-score semi-quantitative method was employed to analyze the results. The staining intensity was classified as 0 (negative), 1 (weakly positive), 2 (moderately positive), or 3 (highly positive). The H-score was calculated by summing the staining intensity and the percentage of positive cells at that intensity. The score can range from 0 to 300. Four random fields at ×200 magnification were selected per slide, and the average score was used as the final value. Two independent observers performed the scoring in a blinded manner. RT-qPCR. We extracted total RNA from the eutopic endometrium of the control group and the adenomyosis group using the TRIzol method. RNA was reversely transcribed into cDNA (42°C for 15 min and 95°C for 3 min) by the FastKing gDNA Dispelling RT SuperMix kit (TIANGEN, Catalog No. KR118-02, China). The expression levels of SOX9 in the eutopic endometrium of the control group and the adenomyosis group were assessed utilizing SuperReal PreMix (SYBR Green) (TIANGEN, China) according to the manufacturer’s instructions. And then, qRT-PCR analysis was done on the 7500 Real-Time PCR System (Applied Biosystems, USA) by the following two-step PCR amplification procedure: 1 cycle of pre-denaturation at 95°C for 15 min and 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 60°C for 30 s. GAPDH was utilized as an internal reference to normalize gene expression. Primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. Relative expression levels of mRNA were calculated using the 2⁻ΔΔCt method. Primers were as follows: (i) SOX9 forward: 5’-AGCGAACGCACATCAAGAC-3’ Reverse: 5’-CTGTAGGCGATCTGTTGGGG-3’ (ii) GAPDH forward: 5’-ATCTTCCAGGAGCGAGATCCC-3’ Reverse: 5’-TGAGTCTTCCACGATACCAA-3’ Statistical analysis Statistical analyses were performed using SPSS version 26.0 and GraphPad Prism version 10.0 software. Normally distributed continuous data are presented as mean ± standard deviation (SD) and compared between groups using the independent samples t-test. Non-normally distributed data are expressed as medians (25th, 75th percentiles) and compared using the Mann-Whitney U test. Categorical variables were analyzed using the chi-square test or Fisher’s exact test as appropriate. Correlations were assessed using Spearman’s rank correlation coefficient. Multivariate logistic regression analysis was conducted to identify independent risk factors associated with AM. A P value < 0.05 was considered statistically significant. Results A total of 120 patients were included in this study, with 60 patients in the AM group and 60 in the control group. There were no statistically significant differences between the two groups in age, BMI, PBAC score, APTT, uterine volume, E₂ level, disease duration, number of previous uterine surgeries, or parity (P > 0.05). Compared with the control group, the AM group showed significantly higher dysmenorrhea VAS scores [4 (3, 6) vs. 1 (0, 2)], CA125 levels [110.4 (81.95, 173.00) U/mL vs. 17.5 (13.30, 24.60) U/mL], and PRL levels (22.89 ± 8.40 ng/mL vs. 19.68 ± 7.88 ng/mL) (P < 0.05). The H-scores of epithelial markers (E-cadherin, Cytokeratin 7, and Pan-keratin) were significantly lower in the AM group than in the control group, while the H-scores of mesenchymal markers (Vimentin and N-cadherin) were significantly higher (P < 0.001) (Table 1). Immunohistochemical staining revealed that SOX9 was mostly localized in the nuclei of glandular epithelial and stromal cells, exhibiting yellowish to brownish granular expression. In the eutopic endometrium of the AM group, the intensity of SOX9 staining and the proportion of positive cells were significantly elevated compared to the control group, exhibiting H-scores of 160.60 ± 18.55 versus 136.00 ± 14.20 (P < 0.001). RT-qPCR further confirmed that SOX9 mRNA relative expression levels were significantly upregulated in the AM group (P < 0.001), consistent with the immunohistochemical results (Figures 1-3). EMT-related markers were expressed in the eutopic endometrium of both groups. In the AM group, SOX9 H-score was negatively correlated with E-cadherin (r = -0.2610, P = 0.0440) and positively correlated with Vimentin (r = 0.3676, P = 0.0039), while no significant correlations were found with Pan-keratin, Cytokeratin 7, or N-cadherin (all P > 0.05) (Figure 4, Supplementary Figure 1). Multivariate logistic regression analysis included variables that were statistically significant in univariate analysis (SOX9 H-score, CA125 level, dysmenorrhea VAS score, PRL level, and gravidity). The findings indicated that SOX9 H-score (OR = 1.144, 95% CI: 1.008–1.298), CA125 level (OR = 1.075, 95% CI: 1.035–1.117), and dysmenorrhea VAS score (OR = 2.812, 95% CI: 1.136–6.961) were autonomous risk factors for adenomyosis (all P 0.05) (Table 2). To evaluate the diagnostic efficacy of the dysmenorrhea VAS score, serum CA125, SOX9 expression score, and the combined DCS model (dysmenorrhea VAS score + CA125 + SOX9) for adenomyosis, we performed a receiver operating characteristic (ROC) curve analysis. All four indicators demonstrated significant diagnostic value. Among them, the DCS model exhibited the highest Area Under the Curve (AUC) of 0.993 (95% CI: 0.983–1.000), followed by CA125 with an AUC of 0.984 (95% CI: 0.968–1.000), both showing exceptionally high diagnostic accuracy. The AUC values for the dysmenorrhea VAS score and SOX9 were 0.889 and 0.871, respectively. Subsequent pairwise comparisons demonstrated that the diagnostic efficiency of the DCS model was markedly superior to both the dysmenorrhea VAS score (AUC difference = -0.103, P < 0.001) and SOX9 (AUC difference = -0.122, P < 0.001). Similarly, the diagnostic effectiveness of CA125 significantly surpassed that of the dysmenorrhea VAS score (AUC difference = -0.095, P = 0.002) and SOX9 (AUC difference = 0.113, P = 0.001). However, there was no statistically significant difference between the AUC values of the DCS model and CA125 alone (P = 0.174), suggesting similar overall diagnostic effectiveness. (Figure 5). Discussion The research indicated that individuals with adenomyosis exhibited markedly elevated dysmenorrhea VAS scores, serum CA125 concentrations, and PRL levels compared to the control group (P < 0.05). Additionally, the AM group's eutopic endometrium exhibited a significant elevation in SOX9 protein and mRNA expression. Changes in EMT markers indicated a significant EMT process in the eutopic endometrium of AM patients. SOX9 expression showed a negative association with E-cadherin and a positive correlation with Vimentin, indicating that SOX9 plays a crucial role in the pathogenesis of AM via regulating EMT. A multivariate logistic regression analysis showed the SOX9 H-score, CA125 level, and dysmenorrhea VAS score as independent risk factors for adenomyosis. The results demonstrate that the eutopic endometrium in AM patients undergoes "molecular priming" or transcriptional reprogramming (20), with SOX9 acting as a crucial coordinator, imparting migratory properties to endometrial cells necessary for invasion. The investigation revealed that the AM group had significantly higher dysmenorrhea VAS scores, CA125 levels, and PRL levels than the control group (P < 0.05). Findings from further studies support this result (3, 21). In particular, elevated dysmenorrhea and CA125 levels have long been recognized as typical clinical features of adenomyosis (3, 21). Furthermore, the increase in PRL levels in the AM group aligns with findings from animal models in which hyperprolactinemia induced adenomyosis (22). This observation strongly supports recent clinical data and single-cell RNA sequencing studies, which emphasize that locally produced PRL and its receptor (PRLR) act as core drivers and promising therapeutic targets in the pathogenesis of AM (23, 24). Given that PRL signaling is recognized to enhance endometrial cell proliferation, invasion, and EMT, we hypothesize that the increased PRL levels observed in our study may function upstream of SOX9. This suggested PRL/PRLR-SOX9 regulatory axis might collaborate to create a pro-invasive milieu, hence facilitating endometrial cell infiltration into the myometrium. The two groups did not show any significant differences in their hemoglobin levels, PBAC scores, E₂ levels, previous uterine surgeries, or number of children. These results are in opposition to recent research that found previous uterine surgery, multiparity, and heavy monthly bleeding to be independent risk factors for adenomyosis (25, 26). The primary reason may be because the control group in this study comprised patients with uterine leiomyoma, an estrogen-dependent condition associated with a heightened risk of previous surgery, menorrhagia, and uterine volume. This investigation led to comparably balanced baseline clinical characteristics among the groups; however, the markedly increased SOX9 expression in the adenomyosis group indicates that SOX9-mediated EMT serves as a unique pathological driver for myometrial invasion, offering a potential molecular basis for the differential diagnosis of these two prevalent uterine disorders (27). Immunohistochemical and RT-qPCR results showed that SOX9 protein and mRNA expression were both significantly higher in the eutopic endometrium of the AM group compared with the control group (P < 0.001), with predominant nuclear localization in glandular epithelial cells and stromal cells. The AM group exhibited markedly reduced levels of epithelial markers (E-cadherin, Cytokeratin 7, and Pan-keratin) and elevated levels of mesenchymal markers (N-cadherin and Vimentin) (P < 0.001), signifying a robust association with EMT (28).A correlation analysis indicated that in the AM group, the SOX9 H-score exhibited a negative association with E-cadherin (r = -0.2610, P = 0.0440) and a positive correlation with Vimentin (r = 0.3676, P = 0.0039), ), but showed no significant correlation with Pan-keratin, Cytokeratin 7, and N-cadherin. This suggests that in the specific microenvironment of the adenomyosis eutopic endometrium, SOX9 might primarily drive EMT through the E-cadherin and Vimentin pathways, rather than the N-cadherin pathway. The specific molecular mechanisms regulating this selective regulation require more investigation. Our comprehensive analysis of these markers provides an in-depth representation of the mesenchymal transition in the eutopic endometrium, reinforcing the hypothesis that the endometrium in AM patients is 'pre-conditioned' for invasion before the onset of morphological alterations (29, 30). SOX9 is instrumental in modulating EMT in many malignancies by activating signaling pathways, including Wnt/β-catenin and HGF/c-Met-FRA1 (31, 32). While direct evidence for adenomyosis is still emerging, recent studies suggest that upstream regulators such as lncRNA TUG1 and TBX3 may promote the persistent elevation of SOX9 levels in the endometrium. Elevated SOX9 appears to facilitate the conversion of endometrial epithelial cells into mesenchymal cells via coordinating these regulatory networks (33, 34). Elevated SOX9 appears to facilitate the conversion of endometrial epithelial cells into mesenchymal cells via coordinating these regulatory networks. This modification enhances cellular infiltration into the myometrium, aggravating adenomyosis, which can lead to increased pain and menstrual irregularities in affected individuals (11, 12). Beyond its biological role in driving EMT, we evaluated the clinical relevance and diagnostic potential of SOX9 in adenomyosis. Multivariate logistic regression analysis demonstrated that the SOX9 H-score (OR = 1.144, 95% CI: 1.008–1.298), CA125 level (OR = 1.075, 95% CI: 1.035–1.117), and dysmenorrhea VAS score (OR = 2.812, 95% CI: 1.136–6.961) were independent risk factors for adenomyosis. According to these findings, ROC curve analysis indicated that the SOX9 score possesses significant diagnostic value (AUC = 0.871), comparable to the dysmenorrhea VAS score. The integration of these three distinct risk factors into a singular DCS model yielded almost ideal diagnostic accuracy (AUC = 0.993). Serum CA125 exhibited substantial diagnostic efficacy (AUC = 0.984) without a statistically significant difference relative to the DCS model; yet, their clinical implications are divergent. Serum CA125 alone exhibited substantial diagnostic efficacy (AUC = 0.984), revealing no statistically significant difference when compared to the DCS model; nonetheless, their clinical implications are divergent. While CA125 is a readily obtainable systemic measure, its elevation can result from various factors unrelated to the uterus. Conversely, SOX9 immediately indicates the inherent pathogenic changes occurring in the eutopic endometrium. Therefore, the integration of local molecular signals (SOX9), systemic markers (CA125), and clinical symptoms (dysmenorrhea) provides a comprehensive multidimensional perspective. This illustrates the importance of SOX9 not merely as an auxiliary diagnostic tool but as a crucial biological marker intricately linked to the disease mechanism. Despite the significant findings, our study has several limitations that must be acknowledged. First, the present research is primarily observational. Although we found a strong statistical association between SOX9 upregulation and EMT markers in the eutopic endometrium, we did not perform in vitro functional assays (such as SOX9 knockdown or overexpression in primary endometrial cells) or in vivo animal experiments. Therefore, the direct causal relationship and the exact molecular mechanisms by which SOX9 drives EMT in adenomyosis remain to be fully elucidated in future studies. Second, our study's control group comprised patients with uterine leiomyomas. This selection aimed to regulate a common estrogen-dependent proliferative microenvironment; nevertheless, leiomyomas may intrinsically modify the local uterine environment and endometrial condition. The lack of a strictly healthy control group, comprising women without any hormone-dependent gynecological issues, may result in potential selection bias. Finally, this is a single-center retrospective study with a relatively small sample size. Due to this, we did not subclassify adenomyosis into focal or diffuse types, making it impossible to assess the heterogeneity of SOX9 expression across different subtypes of AM. Future prospective, multicenter studies employing strictly normal endometrial samples, precise disease subtyping, and comprehensive in vitro mechanistic analyses are important to validate and augment our current findings. Conclusion In summary, this study provides robust clinical evidence that SOX9 is significantly upregulated in the eutopic endometrium of patients with adenomyosis and is closely associated with the EMT process. In addition to confirming its increased expression, we identified the SOX9 H-score, together with the CA125 and dysmenorrhea VAS scores, as independent risk factors for the condition. These findings identify SOX9 as a promising biomarker for early risk assessment and a potential therapeutic target for intercepting the progression of adenomyosis. Future multicenter studies and functional analyses are essential to comprehensively evaluate its therapeutic relevance. Abbreviations AM Adenomyosis APTT Activated Partial Thromboplastin Time BMI body mass index CA125 Carbohydrate Antigen 125 E2 Estradiol EMI Endometrial-Myometrial Interface EMT Epithelial-Mesenchymal Transition IHC Immunohistochemistry LNG-IUS Levonorgestrel-releasing Intrauterine System PBAC Pictorial Blood Loss Assessment Chart PBS Phosphate Buffered Saline PRL Prolactin RT-qPCR Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction SOX9 SRY-related high mobility group-box gene 9 TIAR Tissue Injury and Repair VAS Visual Analogue Scale Declarations Acknowledgements Not applicable. Author contributions Ping He and Jiajia Wei contributed equally to this work and share first authorship. They were responsible for the methodology, investigation (including sample collection and processing), data curation, formal analysis, and writing of the original draft. Li Fan and Jingjing Li contributed equally to this work and share corresponding authorship. They were responsible for the conceptualization and design of the study, supervision of the research, project administration, and critical review and revision of the manuscript. They also verified the underlying data. All authors reviewed and approved the final version of the manuscript for publication. Funding This study was funded by the Guangxi Health Commission Self-funded Project (Western Medicine) (Grant No. Z-B20241323), the Guangxi Science and Technology Plan Project under a Grant from the Guangxi Clinical Research Center for Obstetrics and Gynecology (Grant No. GuiKe AD22035223), and the Key Research and Development Program of Guangxi (Grant No. Guike AB18126056). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Ethics approval and consent to participate The study was approved by the Ethics Committee of The Fourth Affiliated Hospital of Guangxi Medical University (approval number: KY2024001) and the Medical Ethics Committee of Liuzhou Hospital of Guangzhou Women and Children's Medical Center (approval number: Quick Review-Research-2024-118). The research was conducted in strict compliance with the Declaration of Helsinki. All patients provided written informed consent prior to surgery. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Ferenczy A. Pathophysiology of adenomyosis. Human reproduction update. 1998;4(4):312-22. Chao X, Chen X, Su H, Shang X, Wu H, You Y, et al. Whole genome doubling in adenomyosis. Clinical and translational medicine. 2024;14(8):e1809. Wu HM, Tsai TC, Liu SM, Pai AH, Chen LH. 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Perturbations of the endometrial immune microenvironment in endometriosis and adenomyosis: their impact on reproduction and pregnancy. Seminars in immunopathology. 2025;47(1):16. Wang X, Cai W, Liang T, Li H, Gu Y, Wei X, et al. The matrix stiffness is increased in the eutopic endometrium of adenomyosis patients: a study based on atomic force microscopy and histochemistry. European journal of histochemistry : EJH. 2024;68(4). Liu X, Shen M, Qi Q, Zhang H, Guo SW. Corroborating evidence for platelet-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis. Human reproduction (Oxford, England). 2016;31(4):734-49. Guo C, Qi Y, Qu J, Gai L, Shi Y, Yuan C. Pathophysiological Functions of the lncRNA TUG1. Current pharmaceutical design. 2020;26(6):688-700. Yuan B, Wang W, Zhao H, Wang L. Role of lncRNA TUG1 in Adenomyosis and its Regulatory Mechanism in Endometrial Epithelial Cell Functions. Endocrinology. 2022;163(5). Mengqi L, Ting L, Tingting J, Yi C, Cheng L, Qiheng L, et al. Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of TBX3 and the EMT pathway to mediate the occurrence of adenomyosis. Research Square (Research Square). 2023. Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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06:05:24","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20199,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9059335/v1/c80b70ac76b2a71c9a4b6cd6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Upregulation of SOX9 in the eutopic endometrium of adenomyosis and its association with epithelial-mesenchymal transition","fulltext":[{"header":"Summary","content":"\u003cp\u003eElevated SOX9 expression in the eutopic endometrium is independently associated with adenomyosis and specific EMT profiles, identifying it as a potential novel clinical biomarker.\u003c/p\u003e\n"},{"header":"Background","content":"\u003cp\u003eAdenomyosis is a prevalent estrogen-dependent gynecological condition marked by the infiltration of endometrial glands and stroma into the myometrium, along with adjacent myometrial hypertrophy and fibrosis (1). It mainly affects women of childbearing age, with an estimated prevalence of 5% to 70%. The highest prevalence is observed in women aged over 40 years (2). The main clinical manifestations include dysmenorrhea, heavy menstrual bleeding, chronic pelvic pain, and infertility, which substantially impair patients\u0026rsquo; quality of life; approximately one-third of patients may be asymptomatic (3). Histopathological examination remains the gold standard for definitive diagnosis, whereas imaging modalities such as transvaginal ultrasound and magnetic resonance imaging (MRI) lack standardized diagnostic criteria and show considerable variability in accuracy (4). The current treatment primarily depends on progestin-based medications, such as the levonorgestrel-releasing intrauterine system (LNG-IUS) and dienogest. However, many individuals develop resistance to progesterone, rendering it less effective and increasing the likelihood of recurrence (5, 6). Recent research indicates that KRAS mutations may diminish progesterone receptor expression by facilitating the methylation of its promoter, thereby attenuating the anti-proliferative effects of dienogest (7). In women desiring fertility preservation, both medical treatments and conservative surgeries frequently fail to achieve long-term symptom relief, while hysterectomy, although curative, is unsuitable for those who wish to retain the uterus (8). These therapeutic challenges underscore the urgent need to elucidate the pathogenesis of adenomyosis, identify novel biomarkers, and discover new therapeutic targets.\u003c/p\u003e\n\u003cp\u003eThe pathophysiology of adenomyosis is not yet fully elucidated. The two primary concepts are the hypothesis of basal endometrium invagination and the metaplasia theory. The invagination theory is the most widely accepted perspective. This concept asserts that repeated tissue injury and repair (TIAR) is crucial, shaped collectively by uterine hyperperistalsis, a localized hyperestrogenic milieu, inflammation, and the disruption of the endometrial-myometrial interface (EMI) (9, 10) . EMT is regarded as a core mechanism facilitating the migration and myometrial invasion of endometrial cells: epithelial cells lose their polarity and adhesion molecules (downregulation of E-cadherin, Pan-keratin, and Cytokeratin 7) and acquire mesenchymal characteristics (upregulation of N-cadherin and Vimentin), thereby enhancing their migratory and invasive abilities (11) . Estrogen-induced EMT has been confirmed in both tissue samples from patients with adenomyosis and in animal models (11) .\u003c/p\u003e\n\u003cp\u003eSOX9 (SRY-related HMG-box gene 9) is a key transcription factor of the SOX family that plays important roles in organogenesis, stem cell maintenance, and cell fate determination (12). In various malignant tumors, SOX9 promotes EMT, invasion, and metastasis by activating multiple signaling pathways, including Wnt/\u0026beta;-catenin, TGF-\u0026beta;/Smad, and Notch (13, 14). Previous studies have demonstrated that SOX9 is involved in the development and progression of endometrial hyperplasia, endometriosis, and endometrial cancer (15-17). Recent single-cell RNA sequencing analyses have shown that Wnt-related genes, including SOX9, are significantly upregulated in ectopic endometrium of adenomyosis (18). Our previous studies indicated that downregulation of SOX9 predicts the response to progestin therapy in endometrial hyperplasia (19). Nonetheless, the expression profile of SOX9 in eutopic endometrium associated with adenomyosis and its relationships with EMT markers are not well-defined. This study seeks to compare SOX9 expression in eutopic endometrium between adenomyosis patients and control subjects, while also examining its associations with significant EMT markers (E-cadherin, Cytokeratin 7, Pan-keratin, N-cadherin, and Vimentin), thereby offering novel insights into the pathogenesis of adenomyosis and identifying potential therapeutic targets.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy design and patient recruitment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll specimens collected received thorough histological validation. Two senior pathologists independently examined the postoperative slides to verify the accuracy of the diagnosis and to ensure that all endometrial samples were in the proliferative phase of the menstrual cycle. Based on the definitive pathological results, patients were categorized into two groups: the adenomyosis group (n = 60) and the control group (n = 60). To identify the intrinsic molecular alterations that may drive myometrial invasion, this study focused on the eutopic endometrium of AM patients. We utilized patients with uterine leiomyoma as a clinically relevant control to account for the shared estrogen-dependent proliferative environment. This design allows for the identification of specific molecular drivers (e.g., SOX9) and the assessment of the EMT-associated \u0026apos;priming\u0026apos; state of the endometrium, distinct from general benign muscular hyperplasia. The study was approved by the Ethics Committee of\u0026nbsp;the Fourth Affiliated Hospital of Guangxi Medical University (approval number: KY2024001) and the Medical Ethics Committee of Liuzhou Hospital of Guangzhou Women and Children\u0026apos;s Medical Center (approval number: Quick Review-Research-2024-118). All patients provided written informed consent prior to surgery.\u003c/p\u003e\n\u003cp\u003eInclusion and exclusion criteria\u003c/p\u003e\n\u003cp\u003eThe inclusion criteria included: (i) Patients aged 18\u0026ndash;50 years; (ii) Histologically verified proliferative-phase endometrium; and (iii) Histopathologically confirmed adenomyosis in the adenomyosis group and uterine leiomyomas (FIGO types IV\u0026ndash;VI) in the control group.\u003c/p\u003e\n\u003cp\u003eThe exclusion criteria included: (i) Incomplete clinical data; (ii) Concurrent endometrial hyperplasia, endometriosis, or reproductive system malignancies; (iii) Use of hormonal therapy, antiplatelet drugs, or oral contraceptives within 3 months prior to surgery; (iv) Presence of pelvic inflammatory disease, genital tract malformations, endocrine disorders, or autoimmune conditions; and (v) Pregnancy, lactation, or history of long-term exposure to toxins or carcinogens.\u003c/p\u003e\n\u003cp\u003eClinical data collection\u003c/p\u003e\n\u003cp\u003ePeripheral blood samples were collected on days 2\u0026ndash;4 of the menstrual cycle prior to surgery to measure serum levels of estradiol (E₂), prolactin (PRL), carbohydrate antigen (CA125), activated partial thromboplastin time (APTT), hemoglobin, and platelet count. Patient characteristics, including age, body mass index (BMI), disease duration, gravidity and parity, history of uterine surgery, severity of dysmenorrhea (assessed using the visual analog scale [VAS], 0\u0026ndash;10 points), and menstrual blood loss (quantified by the pictorial blood assessment chart [PBAC] score), were recorded. Preoperative uterine dimensions were measured in a blinded manner by two senior ultrasound physicians using the same ultrasound equipment. Uterine volume was calculated using the formula 0.52 \u0026times; anteroposterior diameter \u0026times; longitudinal diameter \u0026times; transverse diameter.\u003c/p\u003e\n\u003cp\u003eTissue specimen collection and processing\u003c/p\u003e\n\u003cp\u003eDuring surgery, the uterus was incised along the anterior wall with a \u0026ldquo;Y\u0026rdquo;-shaped incision, and eutopic endometrial tissue from the uterine cavity was collected. Fresh specimens were divided into two parts: one part was immediately fixed in 10% neutral buffered formalin (volume ratio \u0026ge;10:1, fixation time \u0026le;1 week) for immunohistochemistry (IHC); the other part was cut into 1\u0026ndash;2 mm\u0026sup3; pieces, rapidly frozen in liquid nitrogen, and then transferred to a \u0026minus;80\u0026deg;C freezer for storage until used for reverse transcription quantitative real-time PCR (RT-qPCR).\u003c/p\u003e\n\u003cp\u003eIHC\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eParaffin-embedded sections were prepared by experienced pathologists (thickness 5 \u0026mu;m). The primary antibodies and their dilutions were as follows: SOX9 monoclonal antibody (1:200), E-cadherin polyclonal antibody (1:6000), N-cadherin polyclonal antibody (1:200), Cytokeratin 7 (CK7) specific polyclonal antibody (1:3000), pan-keratin polyclonal antibody (1:3000), and Vimentin polyclonal antibody (1:3000) (all purchased from Servicebio Biotechnology, Wuhan, China). The staining was performed using the streptavidin-peroxidase two-step method, followed by the development of the 3,3\u0026apos;-diaminobenzidine chromogen and counterstaining with hematoxylin. The H-score semi-quantitative method was employed to analyze the results. The staining intensity was classified as 0 (negative), 1 (weakly positive), 2 (moderately positive), or 3 (highly positive). The H-score was calculated by summing the staining intensity and the percentage of positive cells at that intensity. The score can range from 0 to 300. Four random fields at \u0026times;200 magnification were selected per slide, and the average score was used as the final value. Two independent observers performed the scoring in a blinded manner.\u003c/p\u003e\n\u003cp\u003eRT-qPCR.\u003c/p\u003e\n\u003cp\u003eWe extracted total RNA from the eutopic endometrium of the control group and the adenomyosis group using the TRIzol method. RNA was reversely transcribed into cDNA (42\u0026deg;C for 15 min and 95\u0026deg;C for 3 min) by the FastKing gDNA Dispelling RT SuperMix kit (TIANGEN, Catalog No. KR118-02, China). The expression levels of SOX9 in the eutopic endometrium of the control group and the adenomyosis group were assessed utilizing SuperReal PreMix (SYBR Green) (TIANGEN, China) according to the manufacturer\u0026rsquo;s instructions. And then, qRT-PCR analysis was done on the 7500 Real-Time PCR System (Applied Biosystems, USA) by the following two-step PCR amplification procedure: 1 cycle of pre-denaturation at 95\u0026deg;C for 15 min and 40 cycles of denaturation at 95\u0026deg;C for 5 s and annealing and extension at 60\u0026deg;C for 30 s. GAPDH was utilized as an internal reference to normalize gene expression. Primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. Relative expression levels of mRNA were calculated using the 2⁻\u0026Delta;\u0026Delta;Ct method.\u0026nbsp;Primers were as follows:\u003c/p\u003e\n\u003cp\u003e(i) SOX9 forward: 5\u0026rsquo;-AGCGAACGCACATCAAGAC-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eReverse: 5\u0026rsquo;-CTGTAGGCGATCTGTTGGGG-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003e(ii) GAPDH forward: 5\u0026rsquo;-ATCTTCCAGGAGCGAGATCCC-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eReverse: 5\u0026rsquo;-TGAGTCTTCCACGATACCAA-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using SPSS version 26.0 and GraphPad Prism version 10.0 software. Normally distributed continuous data are presented as mean \u0026plusmn; standard deviation (SD) and compared between groups using the independent samples t-test. Non-normally distributed data are expressed as medians (25th, 75th percentiles) and compared using the Mann-Whitney U test. Categorical variables were analyzed using the chi-square test or Fisher\u0026rsquo;s exact test as appropriate. Correlations were assessed using Spearman\u0026rsquo;s rank correlation coefficient. Multivariate logistic regression analysis was conducted to identify independent risk factors associated with AM. A P value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 120 patients were included in this study, with 60 patients in the AM group and 60 in the control group. There were no statistically significant differences between the two groups in age, BMI, PBAC score, APTT, uterine volume, E₂\u0026nbsp;level, disease duration, number of previous uterine surgeries, or parity (P \u0026gt; 0.05). Compared with the control group, the AM group showed significantly higher dysmenorrhea VAS scores [4 (3, 6) vs. 1 (0, 2)], CA125 levels [110.4 (81.95, 173.00) U/mL vs. 17.5 (13.30, 24.60) U/mL], and PRL levels (22.89 \u0026plusmn; 8.40 ng/mL vs. 19.68 \u0026plusmn; 7.88 ng/mL) (P \u0026lt; 0.05). The H-scores of epithelial markers (E-cadherin, Cytokeratin 7, and Pan-keratin) were significantly lower in the AM group than in the control group, while the H-scores of mesenchymal markers (Vimentin and N-cadherin) were significantly higher (P \u0026lt; 0.001) (Table 1).\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining revealed that SOX9 was mostly localized in the nuclei of glandular epithelial and stromal cells, exhibiting yellowish to brownish granular\u0026nbsp;expression. In the eutopic endometrium of the AM group, the intensity of SOX9 staining and the proportion of positive cells were significantly elevated compared to the control group, exhibiting H-scores of 160.60 \u0026plusmn; 18.55 versus 136.00 \u0026plusmn; 14.20 (P \u0026lt; 0.001). RT-qPCR further confirmed that SOX9 mRNA relative expression levels were significantly upregulated in the AM group (P \u0026lt; 0.001), consistent with the immunohistochemical results (Figures\u0026nbsp;1-3).\u003c/p\u003e\n\u003cp\u003eEMT-related markers were expressed in the eutopic endometrium of both groups. In the AM group, SOX9 H-score was negatively correlated with E-cadherin (r = -0.2610, P = 0.0440) and positively correlated with Vimentin (r = 0.3676, P = 0.0039), while no significant correlations were found with Pan-keratin, Cytokeratin 7, or N-cadherin (all P \u0026gt; 0.05) (Figure 4, Supplementary Figure 1).\u003c/p\u003e\n\u003cp\u003eMultivariate logistic regression analysis included variables that were statistically significant in univariate analysis (SOX9 H-score, CA125 level, dysmenorrhea VAS score, PRL level, and gravidity). The findings indicated that SOX9 H-score (OR = 1.144, 95% CI: 1.008\u0026ndash;1.298), CA125 level (OR = 1.075, 95% CI: 1.035\u0026ndash;1.117), and dysmenorrhea VAS score (OR = 2.812, 95% CI: 1.136\u0026ndash;6.961) were autonomous risk factors for adenomyosis (all P \u0026lt; 0.05). Gravidity and PRL level were not independently correlated with adenomyosis following the correction for additional variables (P \u0026gt; 0.05) (Table 2).\u003c/p\u003e\n\u003cp\u003eTo evaluate the diagnostic efficacy of the dysmenorrhea VAS score, serum CA125, SOX9 expression score, and the combined DCS model (dysmenorrhea VAS score + CA125 + SOX9) for adenomyosis, we performed a receiver operating characteristic (ROC) curve analysis. All four indicators demonstrated significant diagnostic value. Among them, the DCS model exhibited the highest Area Under the Curve (AUC) of 0.993 (95% CI: 0.983\u0026ndash;1.000), followed by CA125 with an AUC of 0.984 (95% CI: 0.968\u0026ndash;1.000), both showing exceptionally high diagnostic accuracy. The AUC values for the dysmenorrhea VAS score and SOX9 were 0.889 and 0.871, respectively. Subsequent pairwise comparisons demonstrated that the diagnostic efficiency of the DCS model was markedly superior to both the dysmenorrhea VAS score (AUC difference = -0.103, P \u0026lt; 0.001) and SOX9 (AUC difference = -0.122, P \u0026lt; 0.001). Similarly, the diagnostic effectiveness of CA125 significantly surpassed that of the dysmenorrhea VAS score (AUC difference = -0.095, P = 0.002) and SOX9 (AUC difference = 0.113, P = 0.001). However, there was no statistically significant difference between the AUC values of the DCS model and CA125 alone (P = 0.174), suggesting similar overall diagnostic effectiveness. (Figure 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe research indicated that individuals with adenomyosis exhibited markedly elevated dysmenorrhea VAS scores, serum CA125 concentrations, and PRL levels compared to the control group (P \u0026lt; 0.05). Additionally, the AM group\u0026apos;s eutopic endometrium exhibited a significant elevation in SOX9 protein and mRNA expression. Changes in EMT markers indicated a significant EMT process in the eutopic endometrium of AM patients. SOX9 expression showed a negative association with E-cadherin and a positive correlation with Vimentin, indicating that SOX9 plays a crucial role in the pathogenesis of AM via regulating EMT. A multivariate logistic regression analysis showed the SOX9 H-score, CA125 level, and dysmenorrhea VAS score as independent risk factors for adenomyosis. The results demonstrate that the eutopic endometrium in AM patients undergoes \u0026quot;molecular priming\u0026quot; or transcriptional reprogramming (20), with SOX9 acting as a crucial coordinator, imparting migratory properties to endometrial cells necessary for invasion.\u003c/p\u003e\n\u003cp\u003eThe investigation revealed that the AM group had significantly higher dysmenorrhea VAS scores, CA125 levels, and PRL levels than the control group (P \u0026lt; 0.05). Findings from further studies support this result\u0026nbsp;(3, 21). In particular, elevated dysmenorrhea and CA125 levels have long been recognized as typical clinical features of adenomyosis (3, 21). Furthermore, the increase in PRL levels in the AM group aligns with findings from animal models in which hyperprolactinemia induced adenomyosis (22). This observation strongly supports recent clinical data and single-cell RNA sequencing studies, which emphasize that locally produced PRL and its receptor (PRLR) act as core drivers and promising therapeutic targets in the pathogenesis of AM (23, 24). Given that PRL signaling is recognized to enhance endometrial cell proliferation, invasion, and EMT, we hypothesize that the increased PRL levels observed in our study may function upstream of SOX9. This suggested PRL/PRLR-SOX9 regulatory axis might collaborate to create a pro-invasive milieu, hence facilitating endometrial cell infiltration into the myometrium. The two groups did not show any significant differences in their hemoglobin levels, PBAC scores, E₂\u0026nbsp;levels, previous uterine surgeries, or number of children. These results are in opposition to recent research that found previous uterine surgery, multiparity, and heavy monthly bleeding to be independent risk factors for adenomyosis\u0026nbsp;(25, 26). The primary reason may be because the control group in this study comprised patients with uterine leiomyoma, an estrogen-dependent condition associated with a heightened risk of previous surgery, menorrhagia, and uterine volume. This investigation\u0026nbsp;led to comparably balanced baseline clinical characteristics among the groups; however, the markedly increased SOX9 expression in the adenomyosis group indicates that SOX9-mediated EMT serves as a unique pathological driver for myometrial invasion, offering a potential molecular basis for the differential diagnosis of these two prevalent uterine disorders\u0026nbsp;(27).\u003c/p\u003e\n\u003cp\u003eImmunohistochemical and RT-qPCR results showed that SOX9 protein and mRNA expression were both significantly higher in the eutopic endometrium of the AM group compared with the control group (P \u0026lt; 0.001), with predominant nuclear localization in glandular epithelial cells and stromal cells. The AM group exhibited markedly reduced levels of epithelial markers (E-cadherin, Cytokeratin 7, and Pan-keratin) and elevated levels of mesenchymal markers (N-cadherin and Vimentin) (P \u0026lt; 0.001), signifying a robust association with EMT (28).A correlation analysis indicated that in the AM group, the SOX9 H-score exhibited a negative association with E-cadherin (r = -0.2610, P = 0.0440) and a positive correlation with Vimentin (r = 0.3676, P = 0.0039), ), but showed no significant correlation with Pan-keratin, Cytokeratin 7, and N-cadherin. This suggests that in the specific microenvironment of the adenomyosis eutopic endometrium, SOX9 might primarily drive EMT through the E-cadherin and Vimentin pathways, rather than the N-cadherin pathway. The specific molecular mechanisms regulating this selective regulation require more investigation.\u0026nbsp;Our comprehensive analysis of these markers provides an in-depth representation of the mesenchymal transition in the eutopic endometrium, reinforcing the hypothesis that the endometrium in AM patients is \u0026apos;pre-conditioned\u0026apos; for invasion before the onset of morphological alterations\u0026nbsp;(29, 30). SOX9 is instrumental in modulating EMT in many malignancies by activating signaling pathways, including Wnt/\u0026beta;-catenin and HGF/c-Met-FRA1\u0026nbsp;(31, 32). While direct evidence for adenomyosis is still emerging, recent studies suggest that upstream regulators such as lncRNA TUG1 and TBX3 may promote the persistent elevation of SOX9 levels in the endometrium. Elevated SOX9 appears to facilitate the conversion of endometrial epithelial cells into mesenchymal cells via coordinating these regulatory networks\u0026nbsp;(33, 34). Elevated SOX9 appears to facilitate the conversion of endometrial epithelial cells into mesenchymal cells via coordinating these regulatory networks. This modification enhances cellular infiltration into the myometrium, aggravating adenomyosis, which can lead to increased pain and menstrual irregularities in affected individuals\u0026nbsp;(11, 12).\u003c/p\u003e\n\u003cp\u003eBeyond its biological role in driving EMT, we evaluated the clinical relevance and diagnostic potential of SOX9 in adenomyosis. Multivariate logistic regression analysis demonstrated that the SOX9 H-score (OR = 1.144, 95% CI: 1.008\u0026ndash;1.298), CA125 level (OR = 1.075, 95% CI: 1.035\u0026ndash;1.117), and dysmenorrhea VAS score (OR = 2.812, 95% CI: 1.136\u0026ndash;6.961) were independent risk factors for adenomyosis. According to these findings, ROC curve analysis indicated that the SOX9 score possesses significant diagnostic value (AUC = 0.871), comparable to the dysmenorrhea VAS score. The integration of these three distinct risk factors into a singular DCS model yielded almost ideal diagnostic accuracy (AUC = 0.993). Serum CA125 exhibited substantial diagnostic efficacy (AUC = 0.984) without a statistically significant difference relative to the DCS model; yet, their clinical implications are divergent. Serum CA125 alone exhibited substantial diagnostic efficacy (AUC = 0.984), revealing no statistically significant difference when compared to the DCS model; nonetheless, their clinical implications are divergent. While CA125 is a readily obtainable systemic measure, its elevation can result from various factors unrelated to the uterus. Conversely, SOX9 immediately indicates the inherent pathogenic changes occurring in the eutopic endometrium. Therefore, the integration of local molecular signals (SOX9), systemic markers (CA125), and clinical symptoms (dysmenorrhea) provides a comprehensive multidimensional perspective. This illustrates the importance of SOX9 not merely as an auxiliary diagnostic tool but as a crucial biological marker intricately linked to the disease mechanism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite the significant findings, our study has several limitations that must be acknowledged. First, the present research is primarily observational. Although we found a strong statistical association between SOX9 upregulation and EMT markers in the eutopic endometrium, we did not perform in vitro functional assays (such as SOX9 knockdown or overexpression in primary endometrial cells) or in vivo animal experiments. Therefore, the direct causal relationship and the exact molecular mechanisms by which SOX9 drives EMT in adenomyosis remain to be fully elucidated in future studies. Second, our study\u0026apos;s control group comprised patients with uterine leiomyomas. This selection aimed to regulate a common estrogen-dependent proliferative microenvironment; nevertheless, leiomyomas may intrinsically modify the local uterine environment and endometrial condition. The lack of a strictly healthy control group, comprising women without any hormone-dependent gynecological issues, may result in potential selection bias. Finally, this is a single-center retrospective study with a relatively small sample size. Due to this, we did not subclassify adenomyosis into focal or diffuse types, making it impossible to assess the heterogeneity of SOX9 expression across different subtypes of AM. Future prospective, multicenter studies employing strictly normal endometrial samples, precise disease subtyping, and comprehensive in vitro mechanistic analyses are important to validate and augment our current findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study provides robust clinical evidence that SOX9 is significantly upregulated in the eutopic endometrium of patients with adenomyosis and is closely associated with the EMT process. In addition to confirming its increased expression, we identified the SOX9 H-score, together with the CA125 and dysmenorrhea VAS scores, as independent risk factors for the condition. These findings identify SOX9 as a promising biomarker for early risk assessment and a potential therapeutic target for intercepting the progression of adenomyosis. Future multicenter studies and functional analyses are essential to comprehensively evaluate its therapeutic relevance.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eAM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eAdenomyosis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eAPTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eActivated Partial Thromboplastin Time\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eBMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003ebody mass index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eCA125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eCarbohydrate Antigen 125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eEstradiol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eEMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eEndometrial-Myometrial Interface\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eEMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eEpithelial-Mesenchymal Transition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eIHC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eImmunohistochemistry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eLNG-IUS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eLevonorgestrel-releasing Intrauterine System\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003ePBAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003ePictorial Blood Loss Assessment Chart\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003ePhosphate Buffered Saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003ePRL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eProlactin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eRT-qPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eReal-Time Quantitative Reverse Transcription Polymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eSOX9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eSRY-related high mobility group-box gene 9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eTIAR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eTissue Injury and Repair\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eVAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eVisual Analogue Scale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePing He and Jiajia Wei contributed equally to this work and share first authorship. They were responsible for the methodology, investigation (including sample collection and processing), data curation, formal analysis, and writing of the original draft. Li Fan and Jingjing Li contributed equally to this work and share corresponding authorship. They were responsible for the conceptualization and design of the study, supervision of the research, project administration, and critical review and revision of the manuscript. They also verified the underlying data. All authors reviewed and approved the final version of the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Guangxi Health Commission Self-funded Project (Western Medicine) (Grant No. Z-B20241323), the Guangxi Science and Technology Plan Project under a Grant from the Guangxi Clinical Research Center for Obstetrics and Gynecology (Grant No. GuiKe AD22035223), and the Key Research and Development Program of Guangxi (Grant No. Guike AB18126056). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of The Fourth Affiliated Hospital of Guangxi Medical University (approval number: KY2024001) and the Medical Ethics Committee of Liuzhou Hospital of Guangzhou Women and Children\u0026apos;s Medical Center (approval number: Quick Review-Research-2024-118). The research was conducted in strict compliance with the Declaration of Helsinki. All patients provided written informed consent prior to surgery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFerenczy A. Pathophysiology of adenomyosis. Human reproduction update. 1998;4(4):312-22.\u003c/li\u003e\n\u003cli\u003eChao X, Chen X, Su H, Shang X, Wu H, You Y, et al. Whole genome doubling in adenomyosis. Clinical and translational medicine. 2024;14(8):e1809.\u003c/li\u003e\n\u003cli\u003eWu HM, Tsai TC, Liu SM, Pai AH, Chen LH. 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Perturbations of the endometrial immune microenvironment in endometriosis and adenomyosis: their impact on reproduction and pregnancy. Seminars in immunopathology. 2025;47(1):16.\u003c/li\u003e\n\u003cli\u003eWang X, Cai W, Liang T, Li H, Gu Y, Wei X, et al. The matrix stiffness is increased in the eutopic endometrium of adenomyosis patients: a study based on atomic force microscopy and histochemistry. European journal of histochemistry : EJH. 2024;68(4).\u003c/li\u003e\n\u003cli\u003eLiu X, Shen M, Qi Q, Zhang H, Guo SW. Corroborating evidence for platelet-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis. Human reproduction (Oxford, England). 2016;31(4):734-49.\u003c/li\u003e\n\u003cli\u003eGuo C, Qi Y, Qu J, Gai L, Shi Y, Yuan C. Pathophysiological Functions of the lncRNA TUG1. Current pharmaceutical design. 2020;26(6):688-700.\u003c/li\u003e\n\u003cli\u003eYuan B, Wang W, Zhao H, Wang L. Role of lncRNA TUG1 in Adenomyosis and its Regulatory Mechanism in Endometrial Epithelial Cell Functions. Endocrinology. 2022;163(5).\u003c/li\u003e\n\u003cli\u003eMengqi L, Ting L, Tingting J, Yi C, Cheng L, Qiheng L, et al. Abnormal activation of the Wnt3a/\u0026beta;-catenin signaling pathway promotes the expression of TBX3 and the EMT pathway to mediate the occurrence of adenomyosis. Research Square (Research Square). 2023.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"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":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adenomyosis, SOX9, Epithelial-Mesenchymal Transition, Eutopic endometrium","lastPublishedDoi":"10.21203/rs.3.rs-9059335/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9059335/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAdenomyosis is an estrogen-dependent benign gynecological disorder, although its exact pathophysiology is still mostly unclear. SRY-box transcription factor 9 (SOX9) is an essential transcription factor regulating epithelial-mesenchymal transition (EMT) in various diseases. This study investigated SOX9 expression in the eutopic endometrium of adenomyosis and its association with the EMT process, providing new insights into the underlying pathological mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA retrospective case-control study was conducted, including 60 patients with adenomyosis and 60 control patients with uterine leiomyomas. Eutopic endometrial tissue samples were collected from all participants. The expression levels of SOX9 and key EMT-related markers ((including E-cadherin, N-cadherin, Vimentin, Pan-keratin, and Cytokeratin 7) were evaluated using immunohistochemistry (IHC) and quantitative real-time PCR (RT-qPCR). Furthermore, multivariate logistic regression analysis was performed to assess the association between SOX9 expression and the risk of adenomyosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBoth SOX9 protein levels and mRNA expression were significantly upregulated in the eutopic endometrium of the adenomyosis group compared to the control group (both with P \u0026lt; 0.001). Elevated SOX9 levels positively correlated with the mesenchymal marker Vimentin, and negatively correlated with the epithelial marker E-cadherin. Multivariate analysis indicated that a higher SOX9 score was independently associated with adenomyosis (OR = 1.144, 95% CI 1.008-1.298, P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eSOX9 is significantly overexpressed in the eutopic endometrium of adenomyosis patients and is closely associated with the EMT process. These findings suggest that SOX9-associated EMT may play a critical role in the pathogenesis of adenomyosis, highlighting SOX9 as a potential biomarker or therapeutic target for this disease.\u003c/p\u003e","manuscriptTitle":"Upregulation of SOX9 in the eutopic endometrium of adenomyosis and its association with epithelial-mesenchymal transition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 06:05:14","doi":"10.21203/rs.3.rs-9059335/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-16T09:37:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T19:46:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T17:19:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44419748323416827491718919475866718409","date":"2026-04-08T15:04:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101007531922183444034266204542070269737","date":"2026-03-30T15:37:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313763538473079497485964126375967462482","date":"2026-03-29T21:09:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-20T09:25:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-20T09:21:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-11T08:22:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T20:36:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Women's Health","date":"2026-03-10T14:29:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d33e6bd8-918d-4c42-9097-a6e9c0c2fbc0","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-06-13T06:24:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 06:05:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9059335","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9059335","identity":"rs-9059335","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}