ENDOPAIN4D assesment and platelet mediators in adenomyosis insights from thromboxane A2 and platelet activating factor

Adenomyosis is a common benign uterine disorder in women of reproductive age, often associated with pelvic pain, abnormal uterine bleeding, and infertility. Platelet-derived mediators such as thromboxane A2 (TXA 2) and platelet-activating factor (PAF) have been implicated in disease pathophysiology. However, their relation - ship with pain perception in adenomyosis remains incompletely understood. The cross-sectional nature of the available evidence limits conclusions regarding temporal or causal relationships between platelet-derived mediators and pain. This study aims to compare TXA 2 and PAF levels in adenomyosis and non adenomyosis uterine tissue. This cross-sectional study included 64 women aged 20–49 years, divided into an adenomyosis (n = 32) and non-adenomyosis uterine tissue (n = 32) groups. The comparison group consisted of women under - going gynecological surgery whose uterine tissue showed no histopathological evidence of adenomyosis. Pain perception was assessed using the ENDOPAIN-4D questionnaire. Tissue levels of TXA 2 and PAF were mea - sured using ELISA. Statistical analyses included t-tests or Mann–Whitney tests for group comparisons. TXA2 levels were significantly higher in adenomyosis tissue (287.26 ± 29.57 ng/L) compared to non-adenomyosis uterine tissue (177.33 ± 15.75 ng/L; p = 0.0001). PAF levels were also significantly higher in adenomyosis tissue (200.97 ± 22.83 ng/mL) compared to non-adenomyosis uterine tissue (128.52 ± 24.68 ng/mL; p = 0.0001). The observed differences indicate an association between adenomyosis and higher tissue levels of TXA2 and PAF but do not establish causality. TXA2 and PAF levels are elevated in adenomyosis tissue compared with non- adenomyosis uterine tissue. These findings suggest that TXA2 and PAF may be involved in the pathophysiol - ogy of adenomyosis; however, further studies are needed to clarify their mechanistic roles.

Adenomyosis · TXA2 · PAF · Pelvic pain · ENDOPAIN-4D · Platelet mediators Abbreviations BMI Body mass index COX Cyclooxygenase Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 E + P Estrogen + progesterone EMID Endometrial-myometrial interface disruption ELISA Enzyme-linked immunosorbent assay ENDOPAIN-4D Endometriosis pain 4-dimensional questionnaire GnRH Gonadotropin-releasing hormone NSAID Non-steroidal anti-inflammatory drug PAF Platelet-activating factor PBS Phosphate-buffered saline ROS Reactive oxygen species TXA2 Thromboxane A2 TXS Thromboxane synthase V AS Visual analog scale NRS Numerical rating scale VRS Verbal rating scale PAR-1 Protease-activated receptor

Adenomyosis is a frequently encountered pathological condition of the uterus, affecting approximately 20% of women of reproductive age and commonly associated with pelvic pain, abnormal uterine bleeding and infertil - ity. Adenomyosis is histopathologically characterized by the presence of ectopic endometrial tissue (endometrial glands and/or stroma) within the myometrium surrounded by hyperplastic and hypertrophic smooth muscle1,2 . The pathogenesis of adenomyosis is considered multifactorial and remains incompletely understood. One cur- rent theory mechanism is the Endometrial-Myometrial Interface Disruption (EMID) theory, in which disrup - tion or injury at the endometrial-myometrial interface may initiates platelet activation, inflammation, and local hypoxia. These processes may promote the release of inflammatory mediators, alterations in local estrogen sig - naling, tissue remodeling, and neuroangiogenesis, which have been proposed to contribute to adenomyotic lesion development and pain1,3. Although these mechanism have been proposed In previous studies, individual compo- nents of this pathway should be distinguished from findings directly measured in the present study. Thromboxane A2 (TXA2) and platelet-activating factor (PAF) are platelet associated mediators that participate in vascular, inflammatory, and cellular signaling pathway. TXA2 is generated during platelet activation and has been implicated in vasoconstriction, platelet aggregation, and inflammatory signaling. Previous studies have been proposed that platelet-related pathways may contribute to tissue remodeling and neuroangiogenesis in adeno - myosis, potentially influencing nociceptive signaling 1,4–7. PAF is bioactive phospholipid mediator involved in platelet activation and inflammatory responses and may influence vascular permeability, cellular recruitment, and uterine activity1. These biological pathways provide a plausible rationale for investigating TXA 2 and PAF in adenomyosis. However the present study does not directly measure neuroangiogenesis, fibrosis, or other downstream mecha - nisms. Therefore, any proposed relationship between TXA 2/PAF and these processes should be regarded as a mechanistic hypothesis supported by previous literature rather than as a mechanism demonstated by present study. Platelet-derived mediators such as TXA 2 and PAF are therefore hypothesized to play a role in pain percep - tion in adenomyosis. However, relatively in relation to patient reported pain 1,4–7. In particular, the relationship between tissue and TXA2 levels and multidimensional pelvic remains insufficiently characterized. An additional consideration is the frequent coexistence of adenomyosis and endometriosis. Endometriosis itself may cause pel- vic pain and may involve inflammatory and platelet-related pathways. Consequently, concomitant endometriosis Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 represents a potential confounder when evaluating the relationship between TXA2 and pain in women with ade- nomyosis. Accordingly, associations between TXA 2 and pain should be interpreted with consideration of the possible independent contribution of endometriosis. Various pain assessment tools have been used in endometriosis and adenomyosis, including the visual ana - log scale (V AS), numerical rating scale (NRS), and verbal rating scale (VRS). While these tools quantify pain intensity, they do not adequately capture the multidimensional and heterogeneous nature of pain. Comprehensive assessment of pain perception requires evaluation across multiple domains. The ENDOPAIN-4D questionnaire was selected for the present study because it provides a multidimensional assessment of pain across several clinically relevant domains, including spontaneous pelvic pain and dysmen - orrhea, dyspareunia, bowel-related pain, and other pain-related symptoms. This multidomain structure allows characterization of different pain manifestations rather than relying solely on a single global pain-intensity score8. Therefore, ENDOPAIN-4D was used in the present study as a patient-reported measure of pain burden and symp- tom distribution, rather than as a disease-specific diagnostic instrument for adenomyosis. The ENDOPAIN-4D has been previously validated as a reliable multidimensional instrument for assessing pain in women with endometriosis, demonstrating good internal consistency, construct validity, and responsive- ness to clinical change. However, its validation in the Indonesian population remains limited4. Therefore, this study aimed to compare TXA 2 and PAF levels in adenomyosis and non-adenomyosis uterine tissue and to characterize patient-reported pain using the ENDOPAIN-4D questionnaire. The findings may pro - vide a basis for future prospective and mechanistic studies evaluating the biological and potential clinical rel - evance of platelet-derived mediators in adenomyosis.

Study design This analytic observational study employed a cross-sectional design. The study population consisted of patients diagnosed with adenomyosis who presented to Dr. Hasan Sadikin General Hospital Bandung and Bandung Kiwari Hospital between January and March 2025. The study was designed to compare tissue TXA2 and PAF levels between patients with adenomyosis and a non-adenomyosis surgical comparison group. Pain character - istics were additionally assessed using the ENDOPAIN-4D questionnaire to describe the clinical pain profile of the study population. Given the cross-sectional design, the study was not intended to establish temporal or causal relationships between TXA2, PAF, and adenomyosis-related clinical manifestations. All procedures were conducted in accordance with relevant guidelines and regulations and were approved by the Ethics Committee of Hasan Sadikin General Hospital, Faculty of Medicine Universitas Padjadjaran (reference number: DP.04.03/D.XIV .6.5/34/2025). Ethical approval was obtained before initiation of participant recruitment in January 2025. The recruitment period was January–March 2025. All participants provided written informed consent before enrollment. Data collection The inclusion criteria for adenomyosis group were women aged 20–49 years with a normal body mass index (BMI) ranging from 18.5 to 24.9 kg/m2, based on the WHO Asia–Pacific classification, with a diagnosis of adeno- myosis confirmed by transvaginal ultrasonography and histopathological examination. Patients presented with pain symptoms with or without coexisting endometriosis, and underwent surgical interventions by laparoscopic or laparotomy approaches. The presence of concomitant endometriosis was recorded at baseline because endometriosis may indepen - dently contribute to pelvic pain and inflammatory or platelet-mediated pathways. Therefore, pain-related findings Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 in the adenomyosis group were not assumed to be attributable exclusively to adenomyosis. Endometriosis status was determined based on the clinical, imaging, intraoperative, and/or histopathological findings available in the medical records. Restriction to normal BMI was applied to reduce potential confounding effects of adiposity on inflammatory and platelet-related mediators, including TXA2 and PAF. The non-adenomyosis group consisted of women aged 20–49 years with normal BMI who underwent hyster - ectomy or other gynecological surgery. The non-adenomyosis group was not intended to represent a completely healthy population. Rather, it comprised women undergoing gynecological surgery for benign conditions in whom the uterine tissue analyzed showed no histopathological evidence of adenomyosis. Some non-adenomyo- sis participants had benign gynecological conditions, including uterine fibroids, non-endometriotic ovarian cysts, endometrial polyps, or uterine prolapse. This approach was selected because obtaining uterine tissue from healthy women without an indication for surgery would not be ethically or clinically feasible. Only uterine tissue without histopathological evidence of adenomyosis was included in the non-adenomyosis -tissue analysis. Exclusion criteria included the use of hormonal therapy within the previous 3 months, including progestins, combined estrogen–progestin preparations, and gonadotropin-releasing hormone agonists; use of anti-inflamma- tory medications, including non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids; active infection; coagulation disorders; and a history of immunological, endocrine, or metabolic diseases. Medication history, including NSAID used, was obtained through patient self-report and medical-record review where available. Because medication exposure was not assessed prospectively, the possibility of recall or exposure misclassification bias cannot be completely excluded. Sampling was performed using a consecutive non-probability sampling method. Participants meeting the eligibility criteria were enrolled sequentially during the study period until the required sample size was achieved. The sample size was calculated based on a two-group comparison of the primary outcome between the adeno- myosis and non-adenomyosis groups with a minimum of 26 participants per group was required, corresponding to a minimum total sample of 52 participants. The relatively small sample size was considered in the interpreta - tion of the findings and is acknowledged as a limitation of the studys. Therefore, no multivariable analysis of the relationship between TXA2 or PAF levels and pain perception was performed. Pain-related findings should consequently be interpreted as descriptive rather than as evidence of an independent biomarker–pain association. Pain assessment Pain perception was assessed using the ENDOPAIN-4D questionnaire, which consists of 21 items across four domains: spontaneous pelvic pain and dysmenorrhea (questions 1–10), dyspareunia (questions 11–13), bowel- related pain (questions 14–16), and other pain-related symptoms (questions 17–21). Each item is initially assessed dichotomously (yes/no), followed by a numerical rating scale (0–10) if the response is positive. ENDOPAIN-4D was selected because it provides a multidimensional assessment of pelvic pain, including spontaneous pelvic pain and dysmenorrhea, dyspareunia, bowel-related pain, and other pain-related symptoms, rather than assessing pain intensity alone. This multidimensional structure was considered relevant to the pres - ent study because pelvic pain in adenomyosis may present with multiple overlapping symptom patterns. The ENDOPAIN-4D questionnaire is copyrighted by Université de Versailles Saint-Quentin-en-Yvelines and is used under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0). Permission/documentation regarding the use of the questionnaire has been obtained from the relevant rights holder/authors and is provided as a supplementary file. Tissue sampling and processing Tissue samples were obtained intraoperatively following completion of the clinically indicated gynecological surgical procedure. In participants with adenomyosis, uterine tissue representing the adenomyotic lesion was Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 obtained during laparoscopic or laparotomic surgery. In non-adenomyosis participants, uterine tissue without histopathological evidence of adenomyosis was obtained from the corresponding surgical specimen. Tissue sampling was performed using sterile surgical instruments, with care taken to minimize contamination and avoid excessive blood contamination. Approximately 1 cm 3 of tissue was collected from each participant. The specimens were immediately placed in sterile containers and transported to the Laboratory of Molecular Genetics, Faculty of Medicine, Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital, for processing. Adenomyotic and non-adenomyosis tissue samples obtained after laparotomy or laparoscopy were rinsed in ice-cold phosphate-buffered saline (PBS; pH 7.4) to remove excess blood and were weighed before homogeniza- tion. The tissues were then minced and homogenized in PBS at a ratio of 1 g tissue to 9 mL PBS using a glass homogenizer on ice. To further disrupt the cells, the suspension was subjected to sonication using an ultrasonic cell disruptor. The homogenate was subsequently centrifuged at 5000 × g for 5 min, and the supernatant was collected for subsequent analysis. Samples intended for use within five days were stored at 2–8 °C. For longer-term storage, samples were stored at − 20 °C for up to one month or at − 80 °C for up to six months. Repeated freeze–thaw cycles were avoided to preserve sample integrity. The same tissue-processing protocol was applied to adenomyo- sis and non-adenomyosis specimens to minimize pre-analytical variation between groups. Laboratory examination TXA2 levels was performed using the enzyme-linked immunosorbent assay (ELISA) method with the Human Thromboxane A2 reagent, TXA2ELISA KIT 96 wells BZ-08126611-EB and PAF levels using the ELISA method with the Human Platelet Activating Factor reagent, PAF ELISA KIT 96 wells BZ-08126511-EB. The examination was conducted at the Laboratory of Molecular Genetics, Faculty of Medicine, Universitas Padjadjaran /RSUP Dr. Hasan Sadikin Bandung. The processed tissue supernatants were used for quantitative analysis of TXA2 and PAF according to the manufacturer's instructions. Protein concentrations were determined by comparison with the corresponding standard curves. Statistical analysis Data were analyzed using SPSS version 26.0. Group comparisons were performed using independent t-tests or Mann–Whitney tests as appropriate. Statistical significance was set at p < 0.05. Ethical considerations The study was conducted in accordance with the Declaration of Helsinki and relevant institutional regulations. Ethical approval was obtained from the Ethics Committee of Hasan Sadikin General Hospital, Faculty of Medi - cine, Universitas Padjadjaran (reference number: DP.04.03/D.XIV .6.5/34/2025). Written informed consent was obtained from all participants before enrollment. The ethics approval date and recruitment period have been reviewed to ensure chronological consistency.

A total of 64 patients were included in this study, consisting of 32 patients with adenomyosis and 32 patients in the non-adenomyosis surgical comparison group. The participant selection process is presented in Fig. 1. The comparison group included patients with benign gynecological conditions such as uterine fibroids (n = 21), non- endometriotic ovarian cysts (n = 6), uterine prolapse (n = 4), and endometrial polyps (n = 1). Tissue samples were obtained from patients undergoing laparotomy or laparoscopy. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 Baseline demographic and clinical characteristics are summarized in Table 1. The mean age in the adenomyo- sis group was 37.47 ± 7.397 years, while in the non-adenomyosis group it was 41.44 ± 8.33 years. The mean BMI was comparable between groups. Infertility was more frequent in the adenomyosis group (59.4%) compared to non-adenomyosis (21.9%). These baseline differences may act as potential confounders and should be considered when interpreting the results. As shown in Table 2, the mean TXA2 level in the adenomyosis group was significantly higher in the adenomyosis group than in the non-adenomyosis comparison group (287.26 ± 29.57 ng/L vs 177.33 ± 15.75 ng/L; p = 0.0001). Similarly, the mean PAF level was significantly higher in the adenomyosis group than in the comparison group Fig. 1 Participant Flow Chart Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 (200.97 ± 22.83 ng/mL vs. 128.52 ± 24.68 ng/mL; p = 0.0001). These findings demonstrate higher tissue levels of TXA2 and PAF in adenomyosis compared with the non-adenomyosis surgical comparison group. The present study was not designed to determine the diagnostic accuracy of TXA2 or PAF. No receiver operat- ing characteristic analysis, sensitivity, specificity, positive predictive value, or negative predictive value was per- formed. Accordingly, the observed elevation of TXA2 and PAF should not be interpreted as evidence that either mediator is an established diagnostic biomarker. Variables Group P-value Adenomyosis Non-adenomyosis n = 32 n = 32 TXA2Level (ng/L) 0.0001** Mean ± Std 287.26 ± 29.570 177.33 ± 15.747 Median 285.70 171.22 Range (min–max) 243.83–357.55 148.88–201.22 PAF levels (ng/mL) 0.0001** Mean ± Std 200.97 ± 22.826 128.52 ± 24.682 Median 193.75 134.32 Range (min–max) 172.85–262.31 52.03–155.22 Table 2 Comparison of Mean TXA2 and PAF Levels in the Adenomyosis and Non-adenomyosis Groups. Variables Group Adenomyosis Non-adenomyosis n = 32 n = 32 Age (years) Mean ± Std 37.47 ± 7.397 41.44 ± 8.328 Median 37.00 44.00 Range (min–max) 26.00–49.00 23.00–49.00 Height (cm) Mean ± Std 154.63 ± 5.015 152.47 ± 4.127 Median 155.50 154.00 Range (min–max) 141.00–164.00 145.00–161.00 Body Weight (kg) Mean ± Std 55.00 ± 4.635 53.81 ± 4.154 Median 54.50 53.00 Range (min–max) 48.00–64.00 48.00–63.00 BMI (kg/m2) Mean ± Std 22.97 ± 1,254 23.11 ± 1.011 Median 23.15 23.50 Range (min–max) 18.70–24.50 20.80–24.50 Pelvic Pain Score Mean ± Std 107.06 ± 3.741 6.09 ± 1.594 Median 107.00 6.00 Range (min–max) 101.00–118.00 2.00–9.00 Infertility Yes 19(59.4%) 7(21.9%) No 13(40.6%) 25(78.1%) Parity Nullipara 19(59.4%) 7(21.9%) Primiparous 3(9.4%) 3(9.4%) Multiparous 10(31.3%) 22(68.8%) Curettage History Yes 12(37.5%) 5(15.6%) No 20(62.5%) 27(84.4%) Table 1 Comparison of Characteristics of Study Patients in Adenomyosis and non-adenomyosis Groups. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 Within the adenomyosis group, the mean pain perception score in the adenomyosis group was 107.06 ± 3.74. Focal adenomyosis was present in 53.1% of patients, while 46.9% had diffuse adenomyosis. Pure adenomyosis accounted for 34.4% of cases, whereas 65.6% had concomitant endometriosis, which may contribute to pain perception as a confounding factor (Table 3). In the subgroup analysis, mean TXA2 and PAF levels were higher in the pure adenomyosis group compared with the group with concomitant endometriosis; however, these differences were not statistically significant (p > 0.05). Given the relatively small sample sizes in each subgroup (n = 11 and n = 21), this finding may reflect limited statistical power rather than a true absence of difference. Overall, there were no statistically significant differences in mean TXA2 and PAF levels or pelvic pain perception between patients with pure adenomyosis and those with adenomyosis with concomitant endometriosis (Table 4).

Adenomyosis is a common benign disorder in women during the reproductive period with pelvic pain is one of the frequent clinical manifestations as the main reason for seeking medical care 2. The mechanisms underly - ing pelvic pain in adenomyosis are complex, involving nerve-derived factors, inflammatory mediators, PGF2α, and other contributors 2. Pelvic pain may also be associated with pathologic changes surrounding pelvic struc - tures, such as inflammatory adhesions to accessory organs or the peritoneum, reflecting the intricate nociceptive Variables Adenomyosis P-value Pure With Concomitant n = 11 n = 21 TXA2Level (ng/L) 0.132 Mean ± Std 298.21 ± 32.508 281.52 ± 26.951 Median 295.83 272.10 Range (min–max) 249.44–357.55 243.83–331.01 PAF levels (ng/mL) 0.525 Mean ± Std 205.20 ± 29.955 198.75 ± 18,544 Median 191.79 195.27 Range (min–max) 176.98–262.31 172.85–239.42 Degree of Pelvic Pain 0.794 Mean ± Std 106.82 ± 5.036 107.19 ± 2.994 Median 106.00 107.00 Range (min–max) 101.00–118.00 103.00–112.00 Table 4 Comparison of TXA2 levels, PAF levels, and pain perception between patients with pure adenomy- osis and those with adeno- myosis with concomitant endometriosis. Variables n = 32 Degree of pain perception Mean ± Std 107.06 ± 3.741 Median 107.00 Range (min–max) 101.00–118.00 Types of adenomyosis Focal 14(43.8%) Diffuse 18(56.3%) Pure concomitant Pure 11(34.4%) Concomitant 21(65.6%) Severity Level Weight 20(62.5%) Medium 12(37.5%) Table 3 Overview of the Degree of Pain Perception, Type of Adenomyosis, Pure / Comorbidities and Severity in Adenomyosis Patients. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 pathways of the pelvic organs, often experienced as radiating pain 1,2. Additionally, pelvic pain can result from nerve-derived factors that directly induce spasmodic uterine contractions or from inflammatory cytokines and chemical neurotransmitters that indirectly activate nerve endings9–11. In the present study, TXA 2 levels in adenomyosis tissue were significantly higher than in non-adenomyosis uterine tissue. This finding demonstrates an association between adenomyosis and increased tissue TXA 2 lev- els but does not establish that TXA 2 causes adenomyosis-related symptoms or pain 1. The TXA2 is an unstable arachidonic acid metabolite that mediates a various of physiological and pathophysiological processes, includ - ing platelet aggregation and smooth muscle contraction 1. It is generated through the metabolism of arachidonic acid by cyclooxygenase (COX) followed by thromboxane synthase (TXS)1. Platelets are nucleated cells derived from cytoplasmic fragmentation of megakaryocytes mainly in the bone marrow. During activation, platelet sur - face receptors change conformation to bind exposed sub-endothelial matrix proteins and von Willebrand Fac - tor (vWF), leading to morphological changes such as pseudopod formation and granule release, which initiate aggregation1. Platelets also bind fibrinogen to form plugs that stop bleeding from small injuries. Furthermore, they release ADP from dense granules and produce TXA2 from membrane phospholipids, both of which amplify platelet activation. Beyond hemostasis, platelets contribute to tissue repair and the inflammatory response. How- ever, excessive or uncontrolled platelet activation can promote chronic inflammation and pathological conditions, including fibrosis12,13. In adenomyosis, stromal cells secrete thrombin and TXA2, which may induce platelet activation in a density- dependent manner. Activated platelets, in turn have been suggested to contribute to the progression of adeno - myosis through fibrogenic processes12. Therefore, the increased TXA2 levels observed in this study may reflect enhanced platelet activation and inflammatory activity within adenomyotic tissue. However, fibrosis was not directly measured or quantified in the present study. Consequently, the present findings cannot be interpreted as direct evidence that increased TXA 2 promotes fibrogenesis. The proposed relationship between TXA 2 and fibrogenesis should be regarded as a hypothesis supported by previous literature rather than a mechanism dem - onstrated by this study1,14. Platelet-Activating Factor (PAF) is a bioactive molecule released by activated basophils that induces plate - let aggregation15. Elevated PAF levels are observed in various pathological conditions involving inflammation and cellular damage, and evidence suggests its role in processes affecting the nervous system 15. One proposed mechanism involves pro-oxidative stressors generating oxidized glycerophosphocholine with PAF-like activity, which stimulates further PAF synthesis via PAF receptors. Chronic oxidative stress and related inflammation are recognized contributors to fibrosis in multiple organs. Similar to endometriosis, adenomyosis tissues experience recurrent bleeding during menstruation, where extracellular hemoglobin, heme, and free iron act as oxidants, inducing reactive oxygen species (ROS) formation, DNA damage, and increased cellular susceptibility to oxida- tive stress. Sublethal oxidative stress can also promote abnormal endometrial cell growth, facilitating adenomyo- sis development15,16. In the present study, PAF levels were significantly higher in adenomyosis tissue than in non-adenomyosis uter- ine tissue. These findings suggest an association between adenomyosis and increased local PAF levels. However, because of the cross-sectional design, the present study cannot determine whether elevated PAF contributes to disease development, represents a consequence of the disease process, or reflects another underlying inflamma - tory process. Recent studies suggest that activated platelets and their aggregation may contribute to adenomyosis develop - ment, consistent with the endometrial-myometrial interface disruption (EMID) theory. Tissue injury disrupts local blood vessels, causing blood extravasation, platelet aggregation, and clot formation 1. Wound healing then proceeds through four overlapping phases: hemostasis, inflammation, proliferation, and resolution or remodeling. Platelets, acting as first responders, aggregate at the injury site to form a fibrin clot composed of insoluble fibrin fibers. They release ADP from dense granules and generate TXA2 from membrane phospholipids, which further amplify platelet activation1. Activated platelets also release platelet-activating factor (PAF), a lipid mediator that participates in pro-inflammatory processes15,16. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 Both PAF and TXA2 are potent lipid mediators released by various cell types—including platelets, neutrophils, macrophages, monocytes, lymphocytes, endothelial cells, and smooth muscle cells—in response to diverse stim - uli. Experimental studies indicate that PAF receptor activation can trigger TXA2 release, and modulation of TXA2 synthesis or receptor activity can influence PAF responses, although some studies report opposite effects10,17. The elevated levels of both mediators observed in adenomyosis tissue in this study may support their involve- ment in the inflammatory processes associated with adenomyosis. However, the present study did not directly evaluate the molecular interaction between TXA 2 and PAF and cannot establish a causal relationship between these mediators or determine whether one mediator regulates the other. Pain is a primary reason patients with adenomyosis seek medical care. Common manifestations include dys - menorrhea, chronic pelvic pain, dyspareunia, and anorectal pain, although none are sufficiently specific to serve as definitive predictors. Multiple mechanisms contribute to pelvic pain in adenomyosis, including increased uterine contractility due to heightened contraction amplitude and oxytocin receptor expression, elevated pros - taglandin synthesis from inflammatory mediators released during tissue injury, and neurogenic factors such as TXA2 9,14 Previous studies have reported that adenomyotic lesions exhibit high levels of inflammatory and neuro- genic mediators, and the degree of lesion fibrosis correlates with pain severity. However, fibrosis was not directly assessed in the present study, and these observations are based on prior literature. TXA2 has also been suggested to function as a neurotrophic factor that may enhance innervation and potentially contribute to pain1,17. Neverthe- less, the present study did not directly assess the association between tissue TXA2 levels and pain perception. Several pain assessment scales have limitations, as they typically measure only symptom intensity without considering symptom heterogeneity. Pain perception can be more accurately evaluated using validated question- naires that capture this heterogeneity, such as the ENDOPAIN-4D, which assesses overall pain in endometriosis and adenomyosis4. Although TXA2 and PAF levels were elevated in adenomyosis tissue in the present study, these findings should not be interpreted as evidence of a direct relationship between either mediator and pain perception. Pain in adeno- myosis is multifactorial and may be influenced by coexisting endometriosis, inflammatory processes, neuroan - giogenesis, uterine contractility, and other factors. Further studies are needed to clarify the specific contribution of TXA2 and PAF to adenomyosis-related pain. Although the molecular mechanisms underlying adenomyosis-related pelvic pain remain incompletely under- stood, they are thought to involve increased uterine contractility, hyperinnervation, elevated lesion-derived pain mediators, and central sensitization. Platelet activation and Tissue Factor induction in adenomyosis lesions may lead to thrombin release, which may enhance uterine contractions via PAR-1 and contribute to dysmenorrhea. Activated platelets also release PAF, which has been implicated in dysmenorrhea and pelvic pain in endometrio- sis. Experimental studies show that PAF receptor agonists may induce visceral pain and pelvic hyperalgesia by reducing uterine perfusion and increasing contractility. However, these mechanisms are based primarily on previ- ous experimental and clinical studies and cannot be directly inferred from the present findings1,3. Strengths and limitation Research limitations This study has several limitations. First, its cross-sectional design precludes causal inference. Second, the com - parison group consisted of non-adenomyosis uterine tissue obtained from patients with benign gynecological conditions rather than completely healthy uterine tissue. These underlying conditions may have influenced inflammatory or platelet-related pathways and may have introduced residual confounding. Third, concomitant endometriosis was present in a substantial proportion of patients with adenomyosis. Because endometriosis is an independent cause of pelvic pain and may also influence inflammatory pathways, it may confound interpretation of the clinical pain characteristics. Although exploratory subgroup analysis was performed, the small subgroup Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 sizes limit definitive conclusions. Fourth, fibrosis within adenomyotic lesions was not directly measured, limiting interpretation of its relationship with pain. Finally, potential confounding factors such as differences in baseline characteristics and medication use may have influenced the results. Finally, the sample size was calculated based on the planned comparison of biomarker levels between the adenomyosis and non-adenomyosis groups. There - fore, the study was not specifically powered to evaluate the association between TXA2 or PAF levels and pain perception. The pain-related findings should consequently be interpreted as descriptive, and larger studies specifi- cally designed to assess biomarker–pain associations are warranted. Future research Future studies should include larger and prospectively recruited populations to improve statistical power and gen- eralizability. Studies should also incorporate healthy or more homogeneous comparison groups where ethically feasible and should systematically characterize concomitant endometriosis. Future research should specifically evaluate the independent association between TXA2, PAF, and pain using adequately powered multivariable and sensitivity analyses. Mechanistic studies incorporating histological fibrosis assessment, markers of neuroangio - genesis, inflammatory mediators, and platelet activation pathways may further clarify the biological mechanisms underlying adenomyosis. Diagnostic studies using ROC analysis and prospective validation cohorts are required before TXA2 or PAF can be considered candidate biomarkers. Similarly, interventional studies are necessary to determine whether modulation of platelet-derived pathways has therapeutic potential.

TXA2 and PAF levels were significantly higher in adenomyosis tissue than in non-adenomyosis uterine tissue. These findings demonstrate an association between adenomyosis and increased tissue levels of these platelet- derived mediators but do not establish a causal relationship between TXA2 or PAF and pain or disease progres - sion. TXA2 and PAF may represent potential pathways for further investigation, but the present study does not establish either mediator as a validated biomarker or therapeutic target. Further large-scale prospective, mecha - nistic, diagnostic, and interventional studies are required to determine their clinical relevance and potential appli- cations in adenomyosis. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1038/ s41598-026-76964-1.

The authors acknowledge Université de Versailles Saint-Quentin-en-Yvelines (UVSQ) and Professor Arnaud Fauconnier for permission to use the ENDOPAIN-4D questionnaire. The ENDOPAIN-4D questionnaire is copy - righted by Université de Versailles Saint-Quentin-en-Yvelines and is used under the Creative Commons Attribution-Non - Commercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0). Author contributions RA as Ruswana Anwar DA as Doni Aprialdi AR as Anita Rachmawati WP as Wiryawan Permadi HHS as Hanom Husni Syam MNAR as Mulyanusa Amarullah Ritonga JYA as Jimmy Yanuar Annas NT as Nurvita Triana- sari ASN as Aisyah Shofiatun Nisa Conceptualization RA, DA, AR Data curation RA, DA, AR Formal analysis RA, DA, NT Funding acquisition RA, DA Investigation DA Methodology RA, DA, AR, SP, HHS, MNAR Resources RA Supervision RA, AR, SP, HHS, MNAR, JYA Validation RA, AR Writing an original draft DA, ASN All authors reviewed the manuscript. Funding We declare that we do not receive external funding for this study. Data availability The dataset used and /or analysed during the current study is available from the corresponding author on reasonable request Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 Declarations Competing interests The author(s) declare that they have no conflict of interest in this study. Ethics approval The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Ethics Committee Review Board of Hasan Sadikin General Hospital – Faculty of Medicine, Universitas Padjadjaran (protocol date DP.04.03/D.XIV .6.5/34/2025 and date of approval 12 February 2025). All study participants provided written informed consent prior to engaging in any study-related procedures. All authors hereby declare that all patients were examined in accordance with the ethical standards specified in the 1964 Declaration of Helsinki. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

1. Guo, S.-W. The role of platelets in the pathogenesis and pathophysiology of adenomyosis. J. Clin. Med. 12(3), 842 (2023). 2. d’Otreppe, J. et al. Exploring the endocrine mechanisms in adenomyosis: From pathogenesis to therapies. Endocrines 5(1), 46–71 (2024). 3. Guo, S.-W. Cracking the enigma of adenomyosis: An update on its pathogenesis and pathophysiology. Reproduction 164(5), R101–R121 (2022). 4. Puchar, A. et al. The ENDOPAIN 4D questionnaire: A new validated tool for assessing pain in endometriosis. J. Clin. Med. 10(15), 3216 (2021). 5. García-Solares, J. et al. Pathogenesis of uterine adenomyosis: Invagination or metaplasia?. Fertil. Steril. 109(3), 371–379 (2018). 6. Antero, M.F., et al. Pathology and pathogenesis of adenomyosis. in Seminars in Reproductive Medicine. Thieme Medical Publishers, Inc (2020) 7. Zhu, B. et al. Anti-platelet therapy holds promises in treating adenomyosis: Experimental evidence. Reprod. Biol. Endocrinol. 14(1), 66 (2016). 8. Rosenberger, D. C. et al. A systematic literature review on patient-reported outcome domains and measures in nonsurgical efficacy trials related to chronic pain associated with endometriosis: An urgent call to action. Pain 165(11), 2419–2444 (2024). 9. Chen, Q. et al. Clinical manifestations of adenomyosis patients with or without pain symptoms. J. Pain Res. h t t p s : / / d o i . o r g / 1 0 . 2 1 4 7 / J P R . S 2 1 2 1 1 7 (2019). 10. Orazov, M. et al. Proangiogenic features in chronic pelvic pain caused by adenomyosis. Gynecol. Endocrinol. 32(sup2), 7–10 (2016). 11. Jiang, C. & Cheng, Z. Update of recent studies of adenomyosis-associated dysmenorrhea. Gynecol. Minim. Invasive Ther. 5(4), 137–140 (2016). 12. Mosele, S. et al. Investigation of the role of platelets in the aetiopathogenesis of adenomyosis. Reprod. Biomed. Online 42(4), 826–834 (2021). 13. Yan, D., Liu, X. & Guo, S.-W. Endometriosis-derived thromboxane A2 induces neurite outgrowth. Reprod. Sci. 24(6), 829–835 (2017). 14. Vannuccini, S. et al. Pathogenesis of adenomyosis: An update on molecular mechanisms. Reprod. Biomed. Online 35(5), 592–601 (2017). 15. Travers, J. B., Rohan, J. G. & Sahu, R. P. New insights into the pathologic roles of the platelet-activating factor system. Front. Endocrinol. 12, 624132 (2021). 16. Kobayashi, H., Kishi, Y . & Matsubara, S. Mechanisms underlying adenomyosis-related fibrogenesis. Gynecol. Obstet. Invest. 85(1), 1–12 (2020). 17. Carrarelli, P. et al. Expression of inflammatory and neurogenic mediators in adenomyosis: A pathogenetic role. Reprod. Sci. 24(3), 369–375 (2017). Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76964-1 Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors and Affiliations Ruswana Anwar1 · Doni Aprialdi1 · Anita Rachmawati1 · Wiryawan Permadi1 · Hanom Husni Syam1 · Mulyanusa Amarullah Ritonga1 · Jimmy Yanuar Annas2 · Nurvita Trianasari3 · Aisyah Shofiatun Nisa1 Ruswana Anwar [email protected] 1 Department of Obstetrics and Gynaecology, Faculty of Medicine, Dr. Hasan Sadikin General Hospital, Universitas Padjadjaran, Pasteur No. 38, Bandung 40161, West Java, Indonesia 2 Department of Obstetrics and Gynaecology, Faculty of Medicine, Dr. Soetomo Hospital Surabaya, Universitas Airlangga, Surabaya, Indonesia 3 School of Economics and Business, Telkom University, Bandung, Indonesia