{"paper_id":"091c73fe-1118-4f8a-8d39-b0a121e764a0","body_text":"ARTICLE | Article in Press\nScientific Reports https://doi.org/10.1038/s41598-026-76964-1\nScientific Reports\nThis Article in Press is shared early to give you faster access to new research. It is citable and carries a permanent DOI. \nThe final edited version will replace it automatically.\nENDOPAIN4D assesment and platelet mediators in \nadenomyosis insights from thromboxane A2 and \nplatelet activating factor\nRuswana Anwar · Doni Aprialdi · Anita Rachmawati · Wiryawan Permadi ·  \nHanom Husni Syam · Mulyanusa Amarullah Ritonga · Jimmy Yanuar Annas · \nNurvita Trianasari · Aisyah Shofiatun Nisa\nReceived: 31 August 2025 / Accepted: 16 October 2026\n© The Author(s) 2026\nAbstract\nAdenomyosis is a common benign uterine disorder in women of reproductive age, often associated with pelvic \npain, abnormal uterine bleeding, and infertility. Platelet-derived mediators such as thromboxane A2 (TXA 2) \nand platelet-activating factor (PAF) have been implicated in disease pathophysiology. However, their relation -\nship with pain perception in adenomyosis remains incompletely understood. The cross-sectional nature of \nthe available evidence limits conclusions regarding temporal or causal relationships between platelet-derived \nmediators and pain. This study aims to compare TXA 2 and PAF levels in adenomyosis and non adenomyosis \nuterine tissue. This cross-sectional study included 64 women aged 20–49 years, divided into an adenomyosis \n(n = 32) and non-adenomyosis uterine tissue (n = 32) groups. The comparison group consisted of women under -\ngoing gynecological surgery whose uterine tissue showed no histopathological evidence of adenomyosis. Pain \nperception was assessed using the ENDOPAIN-4D questionnaire. Tissue levels of TXA 2 and PAF were mea -\nsured using ELISA. Statistical analyses included t-tests or Mann–Whitney tests for group comparisons. TXA2 \nlevels were significantly higher in adenomyosis tissue (287.26 ± 29.57 ng/L) compared to non-adenomyosis \nuterine tissue (177.33 ± 15.75 ng/L; p = 0.0001). PAF levels were also significantly higher in adenomyosis tissue \n(200.97 ± 22.83 ng/mL) compared to non-adenomyosis uterine tissue (128.52 ± 24.68 ng/mL; p = 0.0001). The \nobserved differences indicate an association between adenomyosis and higher tissue levels of TXA2 and PAF \nbut do not establish causality. TXA2 and PAF levels are elevated in adenomyosis tissue compared with non-\nadenomyosis uterine tissue. These findings suggest that TXA2 and PAF may be involved in the pathophysiol -\nogy of adenomyosis; however, further studies are needed to clarify their mechanistic roles.\nKeywords Adenomyosis · TXA2 · PAF · Pelvic pain · ENDOPAIN-4D · Platelet mediators\nAbbreviations\nBMI  Body mass index\nCOX  Cyclooxygenase\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nE + P  Estrogen + progesterone\nEMID  Endometrial-myometrial interface disruption\nELISA  Enzyme-linked immunosorbent assay\nENDOPAIN-4D  Endometriosis pain 4-dimensional questionnaire\nGnRH  Gonadotropin-releasing hormone\nNSAID  Non-steroidal anti-inflammatory drug\nPAF  Platelet-activating factor\nPBS  Phosphate-buffered saline\nROS  Reactive oxygen species\nTXA2  Thromboxane A2\nTXS  Thromboxane synthase\nV AS  Visual analog scale\nNRS  Numerical rating scale\nVRS  Verbal rating scale\nPAR-1  Protease-activated receptor\nIntroduction\nAdenomyosis is a frequently encountered pathological condition of the uterus, affecting approximately 20% of \nwomen of reproductive age and commonly associated with pelvic pain, abnormal uterine bleeding and infertil -\nity. Adenomyosis is histopathologically characterized by the presence of ectopic endometrial tissue (endometrial \nglands and/or stroma) within the myometrium surrounded by hyperplastic and hypertrophic smooth muscle1,2 .\nThe pathogenesis of adenomyosis is considered multifactorial and remains incompletely understood. One cur-\nrent theory mechanism is the Endometrial-Myometrial Interface Disruption (EMID) theory, in which disrup -\ntion or injury at the endometrial-myometrial interface may initiates platelet activation, inflammation, and local \nhypoxia. These processes may promote the release of inflammatory mediators, alterations in local estrogen sig -\nnaling, tissue remodeling, and neuroangiogenesis, which have been proposed to contribute to adenomyotic lesion \ndevelopment and pain1,3. Although these mechanism have been proposed In previous studies, individual compo-\nnents of this pathway should be distinguished from findings directly measured in the present study.\nThromboxane A2 (TXA2) and platelet-activating factor (PAF) are platelet associated mediators that participate \nin vascular, inflammatory, and cellular signaling pathway. TXA2 is generated during platelet activation and has \nbeen implicated in vasoconstriction, platelet aggregation, and inflammatory signaling. Previous studies have been \nproposed that platelet-related pathways may contribute to tissue remodeling and neuroangiogenesis in adeno -\nmyosis, potentially influencing nociceptive signaling 1,4–7. PAF is bioactive phospholipid mediator involved in \nplatelet activation and inflammatory responses and may influence vascular permeability, cellular recruitment, and \nuterine activity1.\nThese biological pathways provide a plausible rationale for investigating TXA 2 and PAF in adenomyosis. \nHowever the present study does not directly measure neuroangiogenesis, fibrosis, or other downstream mecha -\nnisms. Therefore, any proposed relationship between TXA 2/PAF and these processes should be regarded as a \nmechanistic hypothesis supported by previous literature rather than as a mechanism demonstated by present \nstudy.\nPlatelet-derived mediators such as TXA 2 and PAF are therefore hypothesized to play a role in pain percep -\ntion in adenomyosis. However, relatively in relation to patient reported pain 1,4–7. In particular, the relationship \nbetween tissue and TXA2 levels and multidimensional pelvic remains insufficiently characterized. An additional \nconsideration is the frequent coexistence of adenomyosis and endometriosis. Endometriosis itself may cause pel-\nvic pain and may involve inflammatory and platelet-related pathways. Consequently, concomitant endometriosis \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nrepresents a potential confounder when evaluating the relationship between TXA2 and pain in women with ade-\nnomyosis. Accordingly, associations between TXA 2 and pain should be interpreted with consideration of the \npossible independent contribution of endometriosis.\nVarious pain assessment tools have been used in endometriosis and adenomyosis, including the visual ana -\nlog scale (V AS), numerical rating scale (NRS), and verbal rating scale (VRS). While these tools quantify pain \nintensity, they do not adequately capture the multidimensional and heterogeneous nature of pain. Comprehensive \nassessment of pain perception requires evaluation across multiple domains.\nThe ENDOPAIN-4D questionnaire was selected for the present study because it provides a multidimensional \nassessment of pain across several clinically relevant domains, including spontaneous pelvic pain and dysmen -\norrhea, dyspareunia, bowel-related pain, and other pain-related symptoms. This multidomain structure allows \ncharacterization of different pain manifestations rather than relying solely on a single global pain-intensity score8. \nTherefore, ENDOPAIN-4D was used in the present study as a patient-reported measure of pain burden and symp-\ntom distribution, rather than as a disease-specific diagnostic instrument for adenomyosis.\nThe ENDOPAIN-4D has been previously validated as a reliable multidimensional instrument for assessing \npain in women with endometriosis, demonstrating good internal consistency, construct validity, and responsive-\nness to clinical change. However, its validation in the Indonesian population remains limited4.\nTherefore, this study aimed to compare TXA 2 and PAF levels in adenomyosis and non-adenomyosis uterine \ntissue and to characterize patient-reported pain using the ENDOPAIN-4D questionnaire. The findings may pro -\nvide a basis for future prospective and mechanistic studies evaluating the biological and potential clinical rel -\nevance of platelet-derived mediators in adenomyosis.\nMaterial and methods\nStudy design\nThis analytic observational study employed a cross-sectional design. The study population consisted of patients \ndiagnosed with adenomyosis who presented to Dr. Hasan Sadikin General Hospital Bandung and Bandung \nKiwari Hospital between January and March 2025. The study was designed to compare tissue TXA2 and PAF \nlevels between patients with adenomyosis and a non-adenomyosis surgical comparison group. Pain character -\nistics were additionally assessed using the ENDOPAIN-4D questionnaire to describe the clinical pain profile of \nthe study population. Given the cross-sectional design, the study was not intended to establish temporal or causal \nrelationships between TXA2, PAF, and adenomyosis-related clinical manifestations.\nAll procedures were conducted in accordance with relevant guidelines and regulations and were approved by \nthe Ethics Committee of Hasan Sadikin General Hospital, Faculty of Medicine Universitas Padjadjaran (reference \nnumber: DP.04.03/D.XIV .6.5/34/2025). Ethical approval was obtained before initiation of participant recruitment \nin January 2025. The recruitment period was January–March 2025. All participants provided written informed \nconsent before enrollment.\nData collection\nThe inclusion criteria for adenomyosis group were women aged 20–49 years with a normal body mass index \n(BMI) ranging from 18.5 to 24.9 kg/m2, based on the WHO Asia–Pacific classification, with a diagnosis of adeno-\nmyosis confirmed by transvaginal ultrasonography and histopathological examination. Patients presented with \npain symptoms with or without coexisting endometriosis, and underwent surgical interventions by laparoscopic \nor laparotomy approaches.\nThe presence of concomitant endometriosis was recorded at baseline because endometriosis may indepen -\ndently contribute to pelvic pain and inflammatory or platelet-mediated pathways. Therefore, pain-related findings \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nin the adenomyosis group were not assumed to be attributable exclusively to adenomyosis. Endometriosis status \nwas determined based on the clinical, imaging, intraoperative, and/or histopathological findings available in the \nmedical records. Restriction to normal BMI was applied to reduce potential confounding effects of adiposity on \ninflammatory and platelet-related mediators, including TXA2 and PAF.\nThe non-adenomyosis group consisted of women aged 20–49 years with normal BMI who underwent hyster -\nectomy or other gynecological surgery. The non-adenomyosis group was not intended to represent a completely \nhealthy population. Rather, it comprised women undergoing gynecological surgery for benign conditions in \nwhom the uterine tissue analyzed showed no histopathological evidence of adenomyosis. Some non-adenomyo-\nsis participants had benign gynecological conditions, including uterine fibroids, non-endometriotic ovarian cysts, \nendometrial polyps, or uterine prolapse. This approach was selected because obtaining uterine tissue from healthy \nwomen without an indication for surgery would not be ethically or clinically feasible. Only uterine tissue without \nhistopathological evidence of adenomyosis was included in the non-adenomyosis -tissue analysis.\nExclusion criteria included the use of hormonal therapy within the previous 3 months, including progestins, \ncombined estrogen–progestin preparations, and gonadotropin-releasing hormone agonists; use of anti-inflamma-\ntory medications, including non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids; active infection; \ncoagulation disorders; and a history of immunological, endocrine, or metabolic diseases.\nMedication history, including NSAID used, was obtained through patient self-report and medical-record \nreview where available. Because medication exposure was not assessed prospectively, the possibility of recall \nor exposure misclassification bias cannot be completely excluded. Sampling was performed using a consecutive \nnon-probability sampling method. Participants meeting the eligibility criteria were enrolled sequentially during \nthe study period until the required sample size was achieved.\nThe sample size was calculated based on a two-group comparison of the primary outcome between the adeno-\nmyosis and non-adenomyosis groups with a minimum of 26 participants per group was required, corresponding \nto a minimum total sample of 52 participants. The relatively small sample size was considered in the interpreta -\ntion of the findings and is acknowledged as a limitation of the studys. Therefore, no multivariable analysis of \nthe relationship between TXA2 or PAF levels and pain perception was performed. Pain-related findings should \nconsequently be interpreted as descriptive rather than as evidence of an independent biomarker–pain association.\nPain assessment\nPain perception was assessed using the ENDOPAIN-4D questionnaire, which consists of 21 items across four \ndomains: spontaneous pelvic pain and dysmenorrhea (questions 1–10), dyspareunia (questions 11–13), bowel-\nrelated pain (questions 14–16), and other pain-related symptoms (questions 17–21). Each item is initially assessed \ndichotomously (yes/no), followed by a numerical rating scale (0–10) if the response is positive.\nENDOPAIN-4D was selected because it provides a multidimensional assessment of pelvic pain, including \nspontaneous pelvic pain and dysmenorrhea, dyspareunia, bowel-related pain, and other pain-related symptoms, \nrather than assessing pain intensity alone. This multidimensional structure was considered relevant to the pres -\nent study because pelvic pain in adenomyosis may present with multiple overlapping symptom patterns. The \nENDOPAIN-4D questionnaire is copyrighted by Université de Versailles Saint-Quentin-en-Yvelines and is used \nunder the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License \n(CC BY-NC-ND 4.0). Permission/documentation regarding the use of the questionnaire has been obtained from \nthe relevant rights holder/authors and is provided as a supplementary file.\nTissue sampling and processing\nTissue samples were obtained intraoperatively following completion of the clinically indicated gynecological \nsurgical procedure. In participants with adenomyosis, uterine tissue representing the adenomyotic lesion was \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nobtained during laparoscopic or laparotomic surgery. In non-adenomyosis participants, uterine tissue without \nhistopathological evidence of adenomyosis was obtained from the corresponding surgical specimen.\nTissue sampling was performed using sterile surgical instruments, with care taken to minimize contamination \nand avoid excessive blood contamination. Approximately 1 cm 3 of tissue was collected from each participant. \nThe specimens were immediately placed in sterile containers and transported to the Laboratory of Molecular \nGenetics, Faculty of Medicine, Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital, for processing.\nAdenomyotic and non-adenomyosis tissue samples obtained after laparotomy or laparoscopy were rinsed in \nice-cold phosphate-buffered saline (PBS; pH 7.4) to remove excess blood and were weighed before homogeniza-\ntion. The tissues were then minced and homogenized in PBS at a ratio of 1 g tissue to 9 mL PBS using a glass \nhomogenizer on ice. To further disrupt the cells, the suspension was subjected to sonication using an ultrasonic \ncell disruptor.\nThe homogenate was subsequently centrifuged at 5000 × g for 5 min, and the supernatant was collected for \nsubsequent analysis. Samples intended for use within five days were stored at 2–8 °C. For longer-term storage, \nsamples were stored at − 20 °C for up to one month or at − 80 °C for up to six months. Repeated freeze–thaw \ncycles were avoided to preserve sample integrity. The same tissue-processing protocol was applied to adenomyo-\nsis and non-adenomyosis specimens to minimize pre-analytical variation between groups.\nLaboratory examination\nTXA2 levels was performed using the enzyme-linked immunosorbent assay (ELISA) method with the Human \nThromboxane A2 reagent, TXA2ELISA KIT 96 wells BZ-08126611-EB and PAF levels using the ELISA method \nwith the Human Platelet Activating Factor reagent, PAF ELISA KIT 96 wells BZ-08126511-EB. The examination \nwas conducted at the Laboratory of Molecular Genetics, Faculty of Medicine, Universitas Padjadjaran /RSUP \nDr. Hasan Sadikin Bandung. The processed tissue supernatants were used for quantitative analysis of TXA2 and \nPAF according to the manufacturer's instructions. Protein concentrations were determined by comparison with \nthe corresponding standard curves.\nStatistical analysis\nData were analyzed using SPSS version 26.0. Group comparisons were performed using independent t-tests or \nMann–Whitney tests as appropriate. Statistical significance was set at p < 0.05.\nEthical considerations\nThe study was conducted in accordance with the Declaration of Helsinki and relevant institutional regulations. \nEthical approval was obtained from the Ethics Committee of Hasan Sadikin General Hospital, Faculty of Medi -\ncine, Universitas Padjadjaran (reference number: DP.04.03/D.XIV .6.5/34/2025). Written informed consent was \nobtained from all participants before enrollment. The ethics approval date and recruitment period have been \nreviewed to ensure chronological consistency.\nResults\nA total of 64 patients were included in this study, consisting of 32 patients with adenomyosis and 32 patients in \nthe non-adenomyosis surgical comparison group. The participant selection process is presented in Fig. 1. The \ncomparison group included patients with benign gynecological conditions such as uterine fibroids (n = 21), non-\nendometriotic ovarian cysts (n = 6), uterine prolapse (n = 4), and endometrial polyps (n = 1). Tissue samples were \nobtained from patients undergoing laparotomy or laparoscopy.\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nBaseline demographic and clinical characteristics are summarized in Table 1. The mean age in the adenomyo-\nsis group was 37.47 ± 7.397 years, while in the non-adenomyosis group it was 41.44 ± 8.33 years. The mean BMI \nwas comparable between groups. Infertility was more frequent in the adenomyosis group (59.4%) compared to \nnon-adenomyosis (21.9%). These baseline differences may act as potential confounders and should be considered \nwhen interpreting the results.\nAs shown in Table 2, the mean TXA2 level in the adenomyosis group was significantly higher in the adenomyosis \ngroup than in the non-adenomyosis comparison group (287.26 ± 29.57 ng/L vs 177.33 ± 15.75 ng/L; p = 0.0001). \nSimilarly, the mean PAF level was significantly higher in the adenomyosis group than in the comparison group \nFig. 1 Participant Flow Chart\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\n(200.97 ± 22.83 ng/mL vs. 128.52 ± 24.68 ng/mL; p = 0.0001). These findings demonstrate higher tissue levels of \nTXA2 and PAF in adenomyosis compared with the non-adenomyosis surgical comparison group.\nThe present study was not designed to determine the diagnostic accuracy of TXA2 or PAF. No receiver operat-\ning characteristic analysis, sensitivity, specificity, positive predictive value, or negative predictive value was per-\nformed. Accordingly, the observed elevation of TXA2 and PAF should not be interpreted as evidence that either \nmediator is an established diagnostic biomarker.\nVariables Group P-value\nAdenomyosis Non-adenomyosis\nn = 32 n = 32\nTXA2Level (ng/L) 0.0001**\nMean ± Std 287.26 ± 29.570 177.33 ± 15.747\nMedian 285.70 171.22\nRange (min–max) 243.83–357.55 148.88–201.22\nPAF levels (ng/mL) 0.0001**\nMean ± Std 200.97 ± 22.826 128.52 ± 24.682\nMedian 193.75 134.32\nRange (min–max) 172.85–262.31 52.03–155.22\nTable 2 Comparison of \nMean TXA2 and PAF Levels \nin the Adenomyosis and \nNon-adenomyosis Groups.\n \nVariables Group\nAdenomyosis Non-adenomyosis\nn = 32 n = 32\nAge (years)\nMean ± Std 37.47 ± 7.397 41.44 ± 8.328\nMedian 37.00 44.00\nRange (min–max) 26.00–49.00 23.00–49.00\nHeight (cm)\nMean ± Std 154.63 ± 5.015 152.47 ± 4.127\nMedian 155.50 154.00\nRange (min–max) 141.00–164.00 145.00–161.00\nBody Weight (kg)\nMean ± Std 55.00 ± 4.635 53.81 ± 4.154\nMedian 54.50 53.00\nRange (min–max) 48.00–64.00 48.00–63.00\nBMI (kg/m2)\nMean ± Std 22.97 ± 1,254 23.11 ± 1.011\nMedian 23.15 23.50\nRange (min–max) 18.70–24.50 20.80–24.50\nPelvic Pain Score\nMean ± Std 107.06 ± 3.741 6.09 ± 1.594\nMedian 107.00 6.00\nRange (min–max) 101.00–118.00 2.00–9.00\nInfertility\nYes 19(59.4%) 7(21.9%)\nNo 13(40.6%) 25(78.1%)\nParity\nNullipara 19(59.4%) 7(21.9%)\nPrimiparous 3(9.4%) 3(9.4%)\nMultiparous 10(31.3%) 22(68.8%)\nCurettage History\nYes 12(37.5%) 5(15.6%)\nNo 20(62.5%) 27(84.4%)\nTable 1 Comparison of \nCharacteristics of Study \nPatients in Adenomyosis and \nnon-adenomyosis Groups.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nWithin the adenomyosis group, the mean pain perception score in the adenomyosis group was 107.06 ± 3.74. \nFocal adenomyosis was present in 53.1% of patients, while 46.9% had diffuse adenomyosis. Pure adenomyosis \naccounted for 34.4% of cases, whereas 65.6% had concomitant endometriosis, which may contribute to pain \nperception as a confounding factor (Table 3).\nIn the subgroup analysis, mean TXA2 and PAF levels were higher in the pure adenomyosis group compared \nwith the group with concomitant endometriosis; however, these differences were not statistically significant \n(p > 0.05). Given the relatively small sample sizes in each subgroup (n = 11 and n = 21), this finding may reflect \nlimited statistical power rather than a true absence of difference. Overall, there were no statistically significant \ndifferences in mean TXA2 and PAF levels or pelvic pain perception between patients with pure adenomyosis and \nthose with adenomyosis with concomitant endometriosis (Table 4).\nDiscussion\nAdenomyosis is a common benign disorder in women during the reproductive period with pelvic pain is one \nof the frequent clinical manifestations as the main reason for seeking medical care 2. The mechanisms underly -\ning pelvic pain in adenomyosis are complex, involving nerve-derived factors, inflammatory mediators, PGF2α, \nand other contributors 2. Pelvic pain may also be associated with pathologic changes surrounding pelvic struc -\ntures, such as inflammatory adhesions to accessory organs or the peritoneum, reflecting the intricate nociceptive \nVariables Adenomyosis P-value\nPure With Concomitant\nn = 11 n = 21\nTXA2Level (ng/L) 0.132\nMean ± Std 298.21 ± 32.508 281.52 ± 26.951\nMedian 295.83 272.10\nRange (min–max) 249.44–357.55 243.83–331.01\nPAF levels (ng/mL) 0.525\nMean ± Std 205.20 ± 29.955 198.75 ± 18,544\nMedian 191.79 195.27\nRange (min–max) 176.98–262.31 172.85–239.42\nDegree of Pelvic Pain 0.794\nMean ± Std 106.82 ± 5.036 107.19 ± 2.994\nMedian 106.00 107.00\nRange (min–max) 101.00–118.00 103.00–112.00\nTable 4 Comparison of \nTXA2 levels, PAF levels, \nand pain perception between \npatients with pure adenomy-\nosis and those with adeno-\nmyosis with concomitant \nendometriosis.\n \nVariables n = 32\nDegree of pain perception\nMean ± Std 107.06 ± 3.741\nMedian 107.00\nRange (min–max) 101.00–118.00\nTypes of adenomyosis\nFocal 14(43.8%)\nDiffuse 18(56.3%)\nPure concomitant\nPure 11(34.4%)\nConcomitant 21(65.6%)\nSeverity Level\nWeight 20(62.5%)\nMedium 12(37.5%)\nTable 3 Overview of the \nDegree of Pain Perception, \nType of Adenomyosis, Pure \n/ Comorbidities and Severity \nin Adenomyosis Patients.\n \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\npathways of the pelvic organs, often experienced as radiating pain 1,2. Additionally, pelvic pain can result from \nnerve-derived factors that directly induce spasmodic uterine contractions or from inflammatory cytokines and \nchemical neurotransmitters that indirectly activate nerve endings9–11.\nIn the present study, TXA 2 levels in adenomyosis tissue were significantly higher than in non-adenomyosis \nuterine tissue. This finding demonstrates an association between adenomyosis and increased tissue TXA 2 lev-\nels but does not establish that TXA 2 causes adenomyosis-related symptoms or pain 1. The TXA2 is an unstable \narachidonic acid metabolite that mediates a various of physiological and pathophysiological processes, includ -\ning platelet aggregation and smooth muscle contraction 1. It is generated through the metabolism of arachidonic \nacid by cyclooxygenase (COX) followed by thromboxane synthase (TXS)1. Platelets are nucleated cells derived \nfrom cytoplasmic fragmentation of megakaryocytes mainly in the bone marrow. During activation, platelet sur -\nface receptors change conformation to bind exposed sub-endothelial matrix proteins and von Willebrand Fac -\ntor (vWF), leading to morphological changes such as pseudopod formation and granule release, which initiate \naggregation1. Platelets also bind fibrinogen to form plugs that stop bleeding from small injuries. Furthermore, \nthey release ADP from dense granules and produce TXA2 from membrane phospholipids, both of which amplify \nplatelet activation. Beyond hemostasis, platelets contribute to tissue repair and the inflammatory response. How-\never, excessive or uncontrolled platelet activation can promote chronic inflammation and pathological conditions, \nincluding fibrosis12,13.\nIn adenomyosis, stromal cells secrete thrombin and TXA2, which may induce platelet activation in a density-\ndependent manner. Activated platelets, in turn have been suggested to contribute to the progression of adeno -\nmyosis through fibrogenic processes12. Therefore, the increased TXA2 levels observed in this study may reflect \nenhanced platelet activation and inflammatory activity within adenomyotic tissue. However, fibrosis was not \ndirectly measured or quantified in the present study. Consequently, the present findings cannot be interpreted \nas direct evidence that increased TXA 2 promotes fibrogenesis. The proposed relationship between TXA 2 and \nfibrogenesis should be regarded as a hypothesis supported by previous literature rather than a mechanism dem -\nonstrated by this study1,14.\nPlatelet-Activating Factor (PAF) is a bioactive molecule released by activated basophils that induces plate -\nlet aggregation15. Elevated PAF levels are observed in various pathological conditions involving inflammation \nand cellular damage, and evidence suggests its role in processes affecting the nervous system 15. One proposed \nmechanism involves pro-oxidative stressors generating oxidized glycerophosphocholine with PAF-like activity, \nwhich stimulates further PAF synthesis via PAF receptors. Chronic oxidative stress and related inflammation are \nrecognized contributors to fibrosis in multiple organs. Similar to endometriosis, adenomyosis tissues experience \nrecurrent bleeding during menstruation, where extracellular hemoglobin, heme, and free iron act as oxidants, \ninducing reactive oxygen species (ROS) formation, DNA damage, and increased cellular susceptibility to oxida-\ntive stress. Sublethal oxidative stress can also promote abnormal endometrial cell growth, facilitating adenomyo-\nsis development15,16.\nIn the present study, PAF levels were significantly higher in adenomyosis tissue than in non-adenomyosis uter-\nine tissue. These findings suggest an association between adenomyosis and increased local PAF levels. However, \nbecause of the cross-sectional design, the present study cannot determine whether elevated PAF contributes to \ndisease development, represents a consequence of the disease process, or reflects another underlying inflamma -\ntory process.\nRecent studies suggest that activated platelets and their aggregation may contribute to adenomyosis develop -\nment, consistent with the endometrial-myometrial interface disruption (EMID) theory. Tissue injury disrupts \nlocal blood vessels, causing blood extravasation, platelet aggregation, and clot formation 1. Wound healing then \nproceeds through four overlapping phases: hemostasis, inflammation, proliferation, and resolution or remodeling. \nPlatelets, acting as first responders, aggregate at the injury site to form a fibrin clot composed of insoluble fibrin \nfibers. They release ADP from dense granules and generate TXA2 from membrane phospholipids, which further \namplify platelet activation1. Activated platelets also release platelet-activating factor (PAF), a lipid mediator that \nparticipates in pro-inflammatory processes15,16.\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nBoth PAF and TXA2 are potent lipid mediators released by various cell types—including platelets, neutrophils, \nmacrophages, monocytes, lymphocytes, endothelial cells, and smooth muscle cells—in response to diverse stim -\nuli. Experimental studies indicate that PAF receptor activation can trigger TXA2 release, and modulation of TXA2 \nsynthesis or receptor activity can influence PAF responses, although some studies report opposite effects10,17.\nThe elevated levels of both mediators observed in adenomyosis tissue in this study may support their involve-\nment in the inflammatory processes associated with adenomyosis. However, the present study did not directly \nevaluate the molecular interaction between TXA 2 and PAF and cannot establish a causal relationship between \nthese mediators or determine whether one mediator regulates the other.\nPain is a primary reason patients with adenomyosis seek medical care. Common manifestations include dys -\nmenorrhea, chronic pelvic pain, dyspareunia, and anorectal pain, although none are sufficiently specific to serve \nas definitive predictors. Multiple mechanisms contribute to pelvic pain in adenomyosis, including increased \nuterine contractility due to heightened contraction amplitude and oxytocin receptor expression, elevated pros -\ntaglandin synthesis from inflammatory mediators released during tissue injury, and neurogenic factors such as \nTXA2\n9,14 Previous studies have reported that adenomyotic lesions exhibit high levels of inflammatory and neuro-\ngenic mediators, and the degree of lesion fibrosis correlates with pain severity. However, fibrosis was not directly \nassessed in the present study, and these observations are based on prior literature. TXA2 has also been suggested \nto function as a neurotrophic factor that may enhance innervation and potentially contribute to pain1,17. Neverthe-\nless, the present study did not directly assess the association between tissue TXA2 levels and pain perception.\nSeveral pain assessment scales have limitations, as they typically measure only symptom intensity without \nconsidering symptom heterogeneity. Pain perception can be more accurately evaluated using validated question-\nnaires that capture this heterogeneity, such as the ENDOPAIN-4D, which assesses overall pain in endometriosis \nand adenomyosis4.\nAlthough TXA2 and PAF levels were elevated in adenomyosis tissue in the present study, these findings should \nnot be interpreted as evidence of a direct relationship between either mediator and pain perception. Pain in adeno-\nmyosis is multifactorial and may be influenced by coexisting endometriosis, inflammatory processes, neuroan -\ngiogenesis, uterine contractility, and other factors. Further studies are needed to clarify the specific contribution \nof TXA2 and PAF to adenomyosis-related pain.\nAlthough the molecular mechanisms underlying adenomyosis-related pelvic pain remain incompletely under-\nstood, they are thought to involve increased uterine contractility, hyperinnervation, elevated lesion-derived pain \nmediators, and central sensitization. Platelet activation and Tissue Factor induction in adenomyosis lesions may \nlead to thrombin release, which may enhance uterine contractions via PAR-1 and contribute to dysmenorrhea. \nActivated platelets also release PAF, which has been implicated in dysmenorrhea and pelvic pain in endometrio-\nsis. Experimental studies show that PAF receptor agonists may induce visceral pain and pelvic hyperalgesia by \nreducing uterine perfusion and increasing contractility. However, these mechanisms are based primarily on previ-\nous experimental and clinical studies and cannot be directly inferred from the present findings1,3.\nStrengths and limitation\nResearch limitations\nThis study has several limitations. First, its cross-sectional design precludes causal inference. Second, the com -\nparison group consisted of non-adenomyosis uterine tissue obtained from patients with benign gynecological \nconditions rather than completely healthy uterine tissue. These underlying conditions may have influenced \ninflammatory or platelet-related pathways and may have introduced residual confounding. Third, concomitant \nendometriosis was present in a substantial proportion of patients with adenomyosis. Because endometriosis is an \nindependent cause of pelvic pain and may also influence inflammatory pathways, it may confound interpretation \nof the clinical pain characteristics. Although exploratory subgroup analysis was performed, the small subgroup \n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nsizes limit definitive conclusions. Fourth, fibrosis within adenomyotic lesions was not directly measured, limiting \ninterpretation of its relationship with pain. Finally, potential confounding factors such as differences in baseline \ncharacteristics and medication use may have influenced the results. Finally, the sample size was calculated based \non the planned comparison of biomarker levels between the adenomyosis and non-adenomyosis groups. There -\nfore, the study was not specifically powered to evaluate the association between TXA2 or PAF levels and pain \nperception. The pain-related findings should consequently be interpreted as descriptive, and larger studies specifi-\ncally designed to assess biomarker–pain associations are warranted.\nFuture research\nFuture studies should include larger and prospectively recruited populations to improve statistical power and gen-\neralizability. Studies should also incorporate healthy or more homogeneous comparison groups where ethically \nfeasible and should systematically characterize concomitant endometriosis. Future research should specifically \nevaluate the independent association between TXA2, PAF, and pain using adequately powered multivariable and \nsensitivity analyses. Mechanistic studies incorporating histological fibrosis assessment, markers of neuroangio -\ngenesis, inflammatory mediators, and platelet activation pathways may further clarify the biological mechanisms \nunderlying adenomyosis. Diagnostic studies using ROC analysis and prospective validation cohorts are required \nbefore TXA2 or PAF can be considered candidate biomarkers. Similarly, interventional studies are necessary to \ndetermine whether modulation of platelet-derived pathways has therapeutic potential.\nConclusion\nTXA2 and PAF levels were significantly higher in adenomyosis tissue than in non-adenomyosis uterine tissue. \nThese findings demonstrate an association between adenomyosis and increased tissue levels of these platelet-\nderived mediators but do not establish a causal relationship between TXA2 or PAF and pain or disease progres -\nsion. TXA2 and PAF may represent potential pathways for further investigation, but the present study does not \nestablish either mediator as a validated biomarker or therapeutic target. Further large-scale prospective, mecha -\nnistic, diagnostic, and interventional studies are required to determine their clinical relevance and potential appli-\ncations in adenomyosis.\nSupplementary Information  The online version contains supplementary material available at https://doi.org/10.1038/\ns41598-026-76964-1.\nAcknowledgements The authors acknowledge Université de Versailles Saint-Quentin-en-Yvelines (UVSQ) and Professor \nArnaud Fauconnier for permission to use the ENDOPAIN-4D questionnaire. The ENDOPAIN-4D questionnaire is copy -\nrighted by Université de Versailles Saint-Quentin-en-Yvelines and is used under the Creative Commons Attribution-Non -\nCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0).\nAuthor contributions RA as Ruswana Anwar DA as Doni Aprialdi AR as Anita Rachmawati WP as Wiryawan Permadi \nHHS as Hanom Husni Syam MNAR as Mulyanusa Amarullah Ritonga JYA as Jimmy Yanuar Annas NT as Nurvita Triana-\nsari ASN as Aisyah Shofiatun Nisa Conceptualization RA, DA, AR Data curation RA, DA, AR Formal analysis RA, DA, \nNT Funding acquisition RA, DA Investigation DA Methodology RA, DA, AR, SP, HHS, MNAR Resources RA Supervision \nRA, AR, SP, HHS, MNAR, JYA Validation RA, AR Writing an original draft DA, ASN All authors reviewed the manuscript.\nFunding We declare that we do not receive external funding for this study.\nData availability The dataset used and /or analysed during the current study is available from the corresponding author on \nreasonable request\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nDeclarations\nCompeting interests The author(s) declare that they have no conflict of interest in this study.\nEthics approval The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional \nReview Board (or Ethics Committee) of Ethics Committee Review Board of Hasan Sadikin General Hospital – Faculty of \nMedicine, Universitas Padjadjaran (protocol date DP.04.03/D.XIV .6.5/34/2025 and date of approval 12 February 2025). All \nstudy participants provided written informed consent prior to engaging in any study-related procedures. All authors hereby \ndeclare that all patients were examined in accordance with the ethical standards specified in the 1964 Declaration of Helsinki.\nOpen Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, \nsharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The \nimages or other third party material in this article are included in the article’s Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your \nintended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly \nfrom the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\nReferences\n 1. Guo, S.-W. The role of platelets in the pathogenesis and pathophysiology of adenomyosis. J. Clin. Med.  12(3), 842 \n(2023).\n 2. d’Otreppe, J. et al. Exploring the endocrine mechanisms in adenomyosis: From pathogenesis to therapies. Endocrines \n5(1), 46–71 (2024).\n 3. Guo, S.-W. Cracking the enigma of adenomyosis: An update on its pathogenesis and pathophysiology. Reproduction \n164(5), R101–R121 (2022).\n 4. Puchar, A. et al. The ENDOPAIN 4D questionnaire: A new validated tool for assessing pain in endometriosis. J. Clin. \nMed. 10(15), 3216 (2021).\n 5. García-Solares, J. et al. Pathogenesis of uterine adenomyosis: Invagination or metaplasia?. 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Reprod. \nSci. 24(3), 369–375 (2017).\n\n\nArticle in Press\nScientific Reports\nhttps://doi.org/10.1038/s41598-026-76964-1\nPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional \naffiliations.\nAuthors and Affiliations\nRuswana Anwar1 · Doni Aprialdi1 · Anita Rachmawati1 · Wiryawan Permadi1 ·  \nHanom Husni Syam1 · Mulyanusa Amarullah Ritonga1 · Jimmy Yanuar Annas2 · \nNurvita Trianasari3 · Aisyah Shofiatun Nisa1\n Ruswana Anwar\nruswana.anwar@unpad.ac.id\n1 Department of Obstetrics and Gynaecology, Faculty of Medicine, Dr. Hasan Sadikin General Hospital, Universitas \nPadjadjaran, Pasteur No. 38, Bandung 40161, West Java, Indonesia\n2 Department of Obstetrics and Gynaecology, Faculty of Medicine, Dr. Soetomo Hospital Surabaya, Universitas \nAirlangga, Surabaya, Indonesia\n3 School of Economics and Business, Telkom University, Bandung, Indonesia","source_license":"CC0","license_restricted":false}