A multicenter randomized placebo-controlled trial evaluating daidzein-rich isoflavone aglycones in endometriosis-associated dysmenorrhea

A multicenter randomized placebo-controlled trial evaluating daidzein-rich isoflavone aglycones in endometriosis-associated dysmenorrhea | 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 Article A multicenter randomized placebo-controlled trial evaluating daidzein-rich isoflavone aglycones in endometriosis-associated dysmenorrhea Osamu Takaoka, Hiroyuki Okimura, Yosuke Tarumi, Mari Kawamata, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9799296/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Endometriosis-associated dysmenorrhea is commonly managed with hormonal therapies that suppress ovulation, limiting their use in women desiring fertility. Daidzein-rich isoflavone aglycones (DRIAs), a food-derived compound with estrogen receptor-mediated anti-inflammatory and proliferative effects, may represent a provide a novel therapeutic option. Methods This multicenter, randomized, double-blind, placebo-controlled trial enrolled Japanese women aged 20–50 years with endometriosis-associated dysmenorrhea (visual analog scale (VAS) ≥ 20). Participants were randomized to receive DRIAs (30 mg/day) or placebo for 4 months. The primary endpoint was change in dysmenorrhea VAS score, analyzed using a baseline-adjusted linear mixed model. Secondary outcomes included changes in endometriotic cyst diameter, other pain symptoms, and safety parameters. Results: A total of 81 participants were randomized, and 71 were included in the modified intention-to-treat analysis. Dysmenorrhea VAS scores improved significantly over time in both groups (p < 0.001). although the group-by-time interaction was not statistically significant, a significant between-group difference favoring DRIAs was observed at Month 3 (− 13.53, 95% CI − 23.8 to − 3.22; adjusted p = 0.041). The direction of effect consistently favored DRIAs across time points. The responder rate (≥ 30% reduction at Month 4) was higher in the DRIAs group (73.5% vs 56.8%), although not statistically significant. No serious adverse events or clinically relevant laboratory abnormalities were observed. Conclusions: DRIAs may provide a safe, non-hormonal therapeutic option for endometriosis-associated dysmenorrhea without suppression of ovarian function. The observed magnitude and consistency of pain reduction suggest a clinically meaningful effect, supporting further evaluation in adequately powered studies. Health sciences/Diseases Health sciences/Endocrinology Health sciences/Health care Health sciences/Medical research endometriosis phytoestrogen fertility preservation chronic pelvic pain Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Endometriosis is a chronic inflammatory disease characterized by the growth of endometrial-like tissue outside the uterus. It affects approximately 10% of women of reproductive age and significantly impairs quality of life due to chronic pelvic pain and infertility. Although the precise etiology remains unclear, immunological abnormalities ( 1 , 2 ), inflammation ( 3 ), and endocrine factors are thought to contribute to its onset and progression ( 4 ). Current treatments for endometriosis include surgical interventions and a range of hormonal therapies. Gonadotropin-releasing hormone (GnRH) agonists/antagonists, the synthetic progestin dienogest, and oral contraceptives/low-dose estrogen–progestin (OC/LEP) combinations are widely used and recommended by several international guidelines, including those from European Society of Human Reproduction and Embryology (ESHRE) and Japan Society of Obstetrics and Gynecology (JSOG) ( 5 , 6 ). These therapies are based on the estrogen-dependent nature of endometriosis and exert their effects mainly through the suppression of estrogen signaling pathways. However, all of these agents could suppress ovulation, presenting a significant limitation for patients with infertility, as pregnancy is not possible during treatment, and these therapies do not improve pregnancy rates ( 7 ). Therefore, the development of new treatment options that do not suppress ovulation is urgently needed. Isoflavones are a class of naturally occurring polyphenols predominantly found in legumes. Because their chemical backbone resembles that of endogenous estrogens, they are capable of interacting with estrogen receptors (ERs), albeit with substantially lower potency than 17β-estradiol ( 8 ). Compounds such as daidzein therefore belong to the group of phytoestrogens. Despite their comparatively weak intrinsic estrogenic activity, estimated to be several orders of magnitude lower than that of estradiol, isoflavones may function as selective estrogen receptor modulators, exerting either estrogenic or anti-estrogenic effects depending on the hormonal milieu ( 9 , 10 ). In premenopausal women, where circulating estrogen levels are relatively high, competitive receptor interactions have been proposed as a potential mechanism underlying reported anti-estrogenic actions ( 11 ). Epidemiological and experimental studies have suggested that habitual intake of isoflavone-rich foods may contribute to a reduced incidence of hormone-dependent malignancies, including breast and prostate cancers, as well as colorectal cancer ( 11 , 12 ). In addition, isoflavones have been investigated for their ability to mitigate menopausal symptoms, particularly vasomotor disturbances and bone loss ( 13 , 14 ). Emerging clinical evidence further indicates a possible role in reproductive medicine, with some reports describing improved luteal function, implantation rates, and pregnancy outcomes among women undergoing assisted reproductive technologies ( 15 , 16 ). However, the biological effects of isoflavones are not uniformly beneficial. Concerns have been raised regarding potential stimulation of endometrial tissue under certain conditions, with some studies linking high intake to an elevated risk of endometrial hyperplasia or cancer ( 17 ). Isoflavones occur in both glycosylated and aglycone forms, the latter generally demonstrating superior bioavailability following oral ingestion ( 18 ). Daidzein-rich aglycone isoflavones (DRIAs) commercially available as AglyMax®, have been developed as dietary supplements. Clinical investigations have reported improvements in menopausal vasomotor symptoms and implantation outcomes without serious adverse events observed to date, although continued safety evaluation remains warranted ( 19 ). In our previous experimental studies, we demonstrated that DRIAs exert anti-proliferative and anti-inflammatory effects in endometriotic stromal cells derived from ovarian endometriomas (OESCs). Specifically, DRIAs suppressed cell proliferation and reduced the expression of inflammatory mediators, including IL-6, IL-8, COX-2, and aromatase, without affecting normal endometrial stromal cells ( 20 ). Importantly, these inhibitory effects were mediated through estrogen receptor β (ERβ), as pharmacologic blockade or siRNA knockdown of ERβ reversed the anti-proliferative effects of DRIAs ( 20 ). In contrast, the precursor compounds daidzein and equol did not reproduce these effects, suggesting that the biological activity is specific to the aglycone-rich formulation. Furthermore, we recently clarified that DRIAs modulate local estrogen metabolism in endometriosis. DRIAs significantly suppressed 17β-hydroxysteroid dehydrogenase type 1 (HSD17β1) expression and enzymatic activity, an enzyme that is highly expressed in endometriotic tissues, while increasing estrogen sulfotransferase (EST) levels in OESCs ( 21 , 22 ). Immunohistochemical analysis of ovarian endometrioma specimens from patients treated with DRIAs demonstrated reduced HSD17β1 expression compared with untreated controls. These findings indicate that DRIAs may attenuate local estradiol biosynthesis not only via aromatase inhibition but also through coordinated regulation of estrogen-metabolizing enzymes ( 20 , 21 ). Based on these preclinical findings, we conducted a pilot open-label clinical study (ERB-1202-1) involving 17 patients with endometriosis-associated pain at our institution. Participants received DRIAs for four months according to the same protocol as the present study. No treatment-related adverse events were reported. After four months, the mean reduction in dysmenorrhea VAS was 24.4 ± 25.8 points (p < 0.01), with scores decreasing from 49.0 ± 9.4 at baseline to 24.6 ± 8.2. In addition, the mean diameter of endometriotic ovarian cysts decreased from 37.1 ± 2.5 mm to 32.7 ± 3.2 mm (p < 0.05). No significant changes were observed in VAS scores for dyschezia, dysuria or dyspareunia. Although limited by the small size and open-label design, these preliminary findings suggested potential clinical benefits of DRIAs in reducing menstrual pain and cyst size without evident safety concerns. Therefore, building upon our preclinical data and pilot observations, the present study was designed as a randomized, placebo-controlled, double-blind clinical trial to rigorously evaluate the efficacy and safety of DRIAs in patients with endometriosis-associated pain. Material and Method Study design The study was a multicenter, randomized, double-blind, placebo-controlled clinical trial conducted in Japanese women with clinically diagnosed endometriosis. The trial was performed in accordance with the ethical principles of the Declaration of Helsinki and complied with the Clinical Trials Act in Japan. The protocol was approved by the institutional review board at each participating center. Written informed consent was obtained from all participants prior to enrollment. The trial was registered with the Japan Registry of Clinical Trials (jRCT; registration number: jRCTs051200074, https://jrct.mhlw.go.jp/en-latest-detail/jRCTs051200074 , Registration Date: 2020/10/27). This study is reported in accordance with the CONSORT guidelines. This study was also approved by the Clinical Research Review Board of Kyoto Prefectural University of Medicine (Protocol No. 2019031-8). Participants were recruited from Kyoto Prefectural University of Medicine Hospital, Japanese Red Cross Kyoto Daiichi Hospital, North Medical Center Kyoto Prefectural University of Medicine. Eligible patients were centrally registered using anonymized case registration forms. Participants were randomly assigned to receive either aglycone-type isoflavone (DRIAs) or placebo. Randomization was performed using a minimization method with dynamic allocation to ensure balance between groups. Allocation adjustment factors were: ( 1 ) baseline dysmenorrhea visual analog scale (VAS) score (< 50 vs. ≧ 50), ( 2 ) ovarian endometriotic cyst diameter ( 5 cm), and ( 3 ) serum CA125 level (< 35 U/mL vs. ≧ 35 U/mL). The study was conducted under double-blind conditions. The allocation manager, who was not involved in patient care or outcome assessment, generated the randomization list and ensured identical appearance, packaging, and labeling of study medications. The allocation list was securely stored until completion of data collection and unblinding. Sample size calculation In a prior open-label pilot study (ERB-1202-1) involving 17 patients with endometriosis-associated pain, the mean reduction in dysmenorrhea visual analogue scale (VAS) score after four months of DRIAs treatment was 24.4 points, with a standard deviation (SD) of 25.8 (change from baseline). Given the substantial placebo response reported in previous placebo-controlled trials in endometriosis, the placebo effect was conservatively estimated at 40% of the observed treatment response. Accordingly, the expected mean improvement in the placebo group was set at 9.8 points (24.4 × 0.4), resulting in an anticipated between-group difference of 14.6 points. Assuming a common SD of 25.8, a two-sided α level of 0.05, and 80% power, 50 participants per group were required using a two-sample t-test. Allowing for an estimated 10% dropout rate, the planned total sample size was 110 participants. Recruitment was conducted between April 2020 and March 2024. However, enrollment was substantially affected by the COVID-19 pandemic, which led to reduced outpatient visits and slower-than-anticipated recruitment. Enrollment was therefore closed at the prespecified end of the recruitment period before reaching the planned sample size. Analyses were performed using all enrolled participants. As the target sample size was not achieved, the study may have been underpowered, particularly for secondary endpoints and later time-point comparisons. Participants Women aged 20–50 years with clinically diagnosed endometriosis were eligible. Diagnosis was established based on laparotomy or laparoscopy, transvaginal ultrasonography, magnetic resonance imaging (MRI), and/or gynecological examination according to the Japan Society of Obstetrics and Gynecology (JSOG) Guidelines for the Management of Endometriosis ( 6 ). Participants were required to have a regular menstrual cycle (25–38 days) and Chronic pelvic pain with a dysmenorrhea VAS score ≧ 20. Participants were excluded if they ( 1 ) had taken isoflavone-containing dietary supplements within 3 months prior to screening, ( 2 ) had received GnRH agonists/antagonists, progestins, estrogen-containing preparations, low-dose oral contraceptives, testosterone derivatives, or Kampo, Chinese traditional, medicines for endometriosis within 3 months, ( 3 ) had undergone surgical treatment for endometriosis within 3 months, ( 4 ) were pregnant or suspected to be pregnant, ( 5 ) became pregnant during the study, ( 6 ) were breastfeeding, ( 7 ) were deemed inappropriate for participation by investigators for safety or other reasons. Intervention Participants in the intervention group received DRIAs at a daily dose equivalent to 30 mg/day of aglycone, consistent with supplemental upper intake level recommended by the Food Safety Commission of Japan. The control group received a matching placebo. Study medication was administered orally twice daily. Treatment began one month after registration and continued for four months. Outcomes The primary endpoint was change in dysmenorrhea severity VAS score from baseline, assessed by the visual analog scale (VAS). Secondary endpoints included ( 1 ) change in endometriotic cyst diameter measured by transvaginal ultrasonography. ( 2 ) change in dyspareunia, ( 3 ) change in dysuria/dyschezia, ( 4 ) changes in hematological parameters, liver function tests, ( 5 ) change in serum CA125 levels, and incidence of adverse events. Adverse events were assessed at each outpatient visit through structured interviews. Statistical analysis The primary endpoint, longitudinal changes in dysmenorrhea VAS scores, and the secondary endpoint of ovarian endometriotic cyst diameter were evaluated using a linear mixed model (LMM) to account for repeated measures and handle missing data. The model included treatment group, time (categorical: Months 1, 2, 3, and 4), and the group-by-time interaction as fixed effects, with the baseline value included as a covariate. Patient ID was included as a random effect. Between-group comparisons at each post-baseline time point were performed using estimated marginal means derived from the LMM, and p-values were adjusted using the Holm method to control for multiple comparisons. For serum CA125 levels, due to the right-skewed distribution, the values were natural log-transformed prior to analysis. An analysis of covariance (ANCOVA) was performed on the log-transformed Month 4 values, adjusting for the log-transformed baseline values, to evaluate the between-group difference. Additionally, an overall paired t-test was conducted on the log-transformed data to assess the main effect of time across both groups. Categorical variables were compared using the chi-square test or Fisher’s exact test, as appropriate. All statistical tests were two-sided with a significance level of 0.05. Secondary endpoints were considered exploratory. Analyses were conducted according to a modified intention-to-treat principle, including all randomized participants who received at least one dose of study medication and had at least one post-baseline assessment. In addition, an exploratory analysis was performed. A responder was defined as a patient who achieved a ≥ 30% reduction in dysmenorrhea VAS score from baseline to Month 4 ( 23 ). The proportion of responders was compared between groups using the chi-square test. Results Participants Between April 2020 and March 2024, 81 women were randomized to receive DRIAs (n = 40) or placebo (n = 41). In the DRIAs group, six participants discontinued the study (four withdrew consent and two discontinued due to a desire for pregnancy). In the placebo group, four participants discontinued (two withdrew consent and two discontinued due to a desire for pregnancy). A total of 71 patients (34 in the DRIAs group and 37 in the placebo group) were included in the modified intention-to-treat analysis (Fig. 1 ). Baseline characteristics were comparable between groups (Table 1). No statistically significant differences were observed in demographic or clinical variables, including age, BMI, baseline dysmenorrhea severity, cyst diameter, or serum CA125 levels. Of the planned 110 participants, 81 were enrolled before recruitment was closed at the prespecified end of the study period due to COVID-19-related constraints. Outcome Mean baseline dysmenorrhea VAS scores were 59.4 in the DRIAs group and 60.7 in the placebo group, with no significant difference between groups (Fig. 2 ). A baseline-adjusted linear mixed model was used to evaluate dysmenorrhea VAS. A highly significant main effect of time was observed (p = 0.00006), indicating an overall improvement in both groups over the four-month period. In the between-group comparison (DRIAs-control) at each time point, the DRIAs group showed a significantly lower value at Month 3 (p = 0.0104). However, the group-by-time interaction was not significant (p = 0.344). Post hoc pairwise comparisons, adjusted using the Holm method, revealed that the VAS score was significantly lower in the DRIAs group than in the placebo group at Month 3 only (estimated difference: -13.53, 95% CI: -23.8 to -3.22, Holm-adjusted p = 0.041) (Fig. 2 ). Changes in the maximum diameter of ovarian endometriomas over the 4-month period were evaluated using a baseline-adjusted linear mixed model. There was no significant main effect of time (p = 0.539), nor was there a significant group-by-time interaction (p = 0.965) (Fig. 3 ). Furthermore, post-hoc comparisons between the DRIAs and placebo groups at each time point revealed no significant differences (Holm-adjusted p = 1.000 for all time points). Exploratory analyses of dysuria VAS, dyschezia VAS, and dyspareunia VAS were also performed using baseline-adjusted linear mixed models. No significant group-by-time interactions were observed for any of these endpoints. No significant between-group differences were observed after Holm adjustment. As an exploratory analysis, responder rates were assessed at Month 4. The responder rate at Month 4 was 73.5% (25/34) in the DRIAs group and 56.8% (21/37) in the placebo group. Although the between-group difference was not statistically significant (p = 0.2134), the DRIAs group showed a numerically higher proportion of patients with clinically meaningful improvement. Log-transformed serum CA125 levels decreased significantly from baseline to Month 4 across both groups (p = 0.035). However, baseline-adjusted ANCOVA revealed no significant between-group difference at Month 4 (p = 0.574) (Fig. 4 ). Safety One patient in the DRIAs group developed a skin rash during the fourth month of treatment and discontinued the study medication. No other adverse events were reported. No clinically significant changes were observed in hematological parameters or liver function tests during the study period. Discussion In this multicenter, randomized, double-blind, placebo-controlled trial, administration of DRIAs resulted in a significant reduction in endometriosis-associated dysmenorrhea compared with placebo. Although pain scores decreased over time in both groups, statistical superiority was observed at Month 3. The observed between-group difference exceeded commonly reported minimal clinically important differences for chronic pain outcomes, supporting the clinical relevance of the treatment effect. These findings extend our previous pilot observations and provide controlled clinical evidence supporting the analgesic effect of aglycone-rich isoflavones in reproductive-aged women with endometriosis. A placebo response is frequently observed in randomized trials evaluating endometriosis-associated pain, and clinically meaningful reductions have been reported in placebo arms of large hormonal therapy trials ( 24 – 26 ). Consistent with these observations, VAS scores in the present study showed modest improvement in the placebo group. Nevertheless, the greater magnitude of improvement in the DRIAs group suggests a genuine treatment effect beyond placebo response. Although statistical significance was not maintained at Month 4, the magnitude and direction of the treatment effect remained consistent, with between-group differences of similar magnitude at Months 3 and 4. In addition, an exploratory responder analysis showed a numerically higher proportion of patients achieving clinically meaningful pain reduction (≥ 30% from baseline) in the DRIAs group compared with the placebo group at Month 4, although the difference was not statistically significant. This finding supports the possibility that DRIAs may provide clinically relevant benefit at the individual patient level. The absence of statistical significance at the later time point may reflect limited statistical power rather than attenuation of therapeutic efficacy, particularly as the study did not reach the originally planned sample size due to COVID-19–related recruitment constraints. Despite improvement in dysmenorrhea, no significant reduction in ovarian endometriotic cyst diameter was observed. Pain severity in endometriosis does not necessarily correlate with lesion size, and inflammatory signaling is considered a key contributor to symptom generation. In our previous experimental studies, DRIAs suppressed IL-6, IL-8, COX-2, aromatase, and HSD17β1 expression in endometriotic stromal cells via ERβ-dependent mechanisms, thereby attenuating local estradiol production and inflammatory activity ( 27 – 31 ). These molecular effects may contribute to symptomatic relief without inducing rapid macroscopic regression of cysts within a four-month treatment period. Serum CA125 levels showed a decreasing trend in the DRIAs group; however, the change did not reach statistical significance. Phytoestrogens have attracted attention as potential modulators of endometriosis because of their structural similarity to estradiol and preferential binding to ERβ, which is highly expressed in endometriotic lesions ( 9 , 32 ). Experimental studies have reported anti-inflammatory and anti-proliferative effects of several compounds, including resveratrol and isoflavones ( 33 , 34 ). However, clinical evidence remains limited, and many previous human studies evaluated these agents as adjuncts to hormonal therapies rather than as independent treatments. To our knowledge, no randomized trials have assessed phytoestrogens as monotherapy in women with endometriosis. In this context, the present randomized, double-blind, placebo-controlled trial provides evidence supporting the clinical utility of an aglycone-rich isoflavone formulation administered without concomitant ovulation suppression. Current standard medical treatments for endometriosis primarily rely on suppression of ovulation and reduction of estrogen levels ( 5 , 6 ). While effective for pain control, these approaches prevent conception during treatment and may be associated with hypoestrogenic or progestin-related adverse effects ( 35 ). In contrast, DRIAs were administered at a dose corresponding to the supplemental upper intake level considered acceptable by the Food Safety Commission of Japan and did not induce ovulatory suppression ( 36 ). No serious adverse events were observed, and no clinically relevant abnormalities in laboratory parameters were detected. One participant developed a skin rash that resolved after discontinuation. These findings indicate favorable short-term tolerability and suggest potential utility in women wishing to preserve reproductive function. The possibility of symptom relief without suppression of ovulation is clinically meaningful. Many patients seek alternatives to surgery or long-term hormonal therapy because of concerns about fertility preservation or adverse effects ( 5 , 37 ). Although pregnancy outcomes were not evaluated in this study, the absence of endocrine suppression suggests that DRIAs may be compatible with attempts at conception. Further investigation is required to determine their impact on fertility outcomes. Several limitations should be considered. The study did not reach the planned sample size, which may have limited statistical power, particularly for secondary endpoints and later time points. The treatment duration was limited to four months, and long-term outcomes after discontinuation were not evaluated. Quality-of-life measures were not comprehensively assessed, and symptoms such as dyspareunia, dyschezia, and dysuria were not sufficiently prevalent to allow robust analysis. Larger trials with adequate statistical power and extended follow-up are required to confirm durability of effect and to evaluate potential impacts on fertility outcomes. Conclusions DRIAs demonstrated a consistent trend toward reduction in endometriosis-associated dysmenorrhea and were well tolerated without suppression of ovarian function. As a food-derived therapy with a favorable safety profile, DRIAs may represent a clinically relevant treatment option, particularly for women seeking to preserve fertility for whom conventional hormone-based therapies are not suitable. Although further adequately powered studies are required, this mechanism-informed approach offers a promising alternative strategy in the management of endometriosis-associated pain. Abbreviations DRIAs Daidzein-rich isoflavone aglycones VAS visual analog scale Declarations Ethics approval and consent to participate The protocol was approved by the institutional review board at each participating center. Written informed consent was obtained from all participants prior to enrollment. The trial was registered with the Japan Registry of Clinical Trials (jRCT; registration number: jRCTs051200074, https://jrct.mhlw.go.jp/en-latest-detail/jRCTs051200074 ). This study is reported in accordance with the CONSORT guidelines. Competing interests All authors have no conflict of interest. Funding This study was supported in part by Grants-in Aid for Scientific Research (22K16839) from the Ministry of Education, Culture, Science, and Technology (Japan). Author Contribution OT developed the manuscript design, collected data from participants, performed statistical analysis and drafted the manuscripts. HO and YT, FI, AK, IK collected data from participants. MK performed statistical analysis. JK and TM developed study design and revised the manuscript. All authors read and approved the final manuscript. Acknowledgement We thank Mr. Akihiro Kitatani, Mrs. Ayaka Miura, and Mrs. Mutsumi Kazui for data management. This study was supported in part by Grants-in Aid for Scientific Research (22K16839) from the Ministry of Education, Culture, Science, and Technology (Japan). Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Hooper, L. et al. Effects of soy protein and isoflavones on circulating hormone concentrations in pre- and post-menopausal women: a systematic review and meta-analysis. Hum. Reprod. Update . 15 (4), 423–440 (2009). Vercellini, P., Vigano, P., Somigliana, E. & Fedele, L. Endometriosis: pathogenesis and treatment. Nat. reviews Endocrinol. 10 (5), 261–275 (2014). Additional Declarations No competing interests reported. Supplementary Files DRIAsRCTTable1BMCWH.pdf ClinicalResearchProtocolEnglishversion.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 04 Jul, 2026 Reviewers invited by journal 03 Jul, 2026 Editor assigned by journal 08 Jun, 2026 Editor invited by journal 28 May, 2026 Submission checks completed at journal 27 May, 2026 First submitted to journal 27 May, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9799296","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":673143704,"identity":"d17ad658-796b-4db8-8d21-4cd7dfc3b22f","order_by":0,"name":"Osamu Takaoka","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Osamu","middleName":"","lastName":"Takaoka","suffix":""},{"id":673143705,"identity":"7d055d90-92a9-4041-ba86-8b069d4c77b2","order_by":1,"name":"Hiroyuki Okimura","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Okimura","suffix":""},{"id":673143706,"identity":"2c2a06a5-2f12-414b-8de6-5473c11fe296","order_by":2,"name":"Yosuke Tarumi","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yosuke","middleName":"","lastName":"Tarumi","suffix":""},{"id":673143707,"identity":"ea45ba9e-769d-467f-865f-09a56baac6fd","order_by":3,"name":"Mari Kawamata","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mari","middleName":"","lastName":"Kawamata","suffix":""},{"id":673143709,"identity":"a0ed298e-1695-4819-a28a-219676a958c9","order_by":4,"name":"Fumitake Ito","email":"","orcid":"","institution":"Kyoto Prefectural University of 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12:23:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9799296/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9799296/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":114077852,"identity":"b7ccd530-8266-495f-a886-a429e3d57f49","added_by":"auto","created_at":"2026-07-07 12:20:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":397830,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"DRIAsRCTFig.1BMCWH.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9799296/v1/f68c76c7947473ff88cf14b5.jpg"},{"id":114076236,"identity":"18bb7d4f-49a8-474c-b9d5-5661b50bf364","added_by":"auto","created_at":"2026-07-07 12:14:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":685646,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"DRIAsRCTFig.2BMCWH.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9799296/v1/83d3263c674bb67c0d685cdd.jpg"},{"id":114076223,"identity":"5c6f52d0-c267-4264-bdf3-b44c2bc86db3","added_by":"auto","created_at":"2026-07-07 12:14:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":578747,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"DRIAsRCTFig.3BMCWH.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9799296/v1/0616971dec9ca16c7b24ea37.jpg"},{"id":114076999,"identity":"13827521-3951-41d3-8654-37a791873bef","added_by":"auto","created_at":"2026-07-07 12:17:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":944925,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"DRIAsRCTFig.4BMCWH.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9799296/v1/32d3b90151a5a0a505bde882.jpg"},{"id":114103578,"identity":"913de61a-0fd1-4c1e-8bd3-28e651189b21","added_by":"auto","created_at":"2026-07-07 15:58:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2811719,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9799296/v1/e4606581-f13e-427b-8744-4aa1868f1960.pdf"},{"id":114077069,"identity":"2e7c97e7-110c-475e-b0ca-efe53537f5e7","added_by":"auto","created_at":"2026-07-07 12:17:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":332778,"visible":true,"origin":"","legend":"","description":"","filename":"DRIAsRCTTable1BMCWH.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9799296/v1/952e4c05e538ac6d77b8a20e.pdf"},{"id":114077885,"identity":"c1a1f9c5-0668-48f8-bada-25368aeb290a","added_by":"auto","created_at":"2026-07-07 12:20:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":72976,"visible":true,"origin":"","legend":"","description":"","filename":"ClinicalResearchProtocolEnglishversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-9799296/v1/ceec4ae433be09f75671340d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A multicenter randomized placebo-controlled trial evaluating daidzein-rich isoflavone aglycones in endometriosis-associated dysmenorrhea","fulltext":[{"header":"Background","content":"\u003cp\u003eEndometriosis is a chronic inflammatory disease characterized by the growth of endometrial-like tissue outside the uterus. It affects approximately 10% of women of reproductive age and significantly impairs quality of life due to chronic pelvic pain and infertility. Although the precise etiology remains unclear, immunological abnormalities (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), inflammation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and endocrine factors are thought to contribute to its onset and progression (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrent treatments for endometriosis include surgical interventions and a range of hormonal therapies. Gonadotropin-releasing hormone (GnRH) agonists/antagonists, the synthetic progestin dienogest, and oral contraceptives/low-dose estrogen\u0026ndash;progestin (OC/LEP) combinations are widely used and recommended by several international guidelines, including those from European Society of Human Reproduction and Embryology (ESHRE) and Japan Society of Obstetrics and Gynecology (JSOG) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These therapies are based on the estrogen-dependent nature of endometriosis and exert their effects mainly through the suppression of estrogen signaling pathways. However, all of these agents could suppress ovulation, presenting a significant limitation for patients with infertility, as pregnancy is not possible during treatment, and these therapies do not improve pregnancy rates (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, the development of new treatment options that do not suppress ovulation is urgently needed.\u003c/p\u003e \u003cp\u003eIsoflavones are a class of naturally occurring polyphenols predominantly found in legumes. Because their chemical backbone resembles that of endogenous estrogens, they are capable of interacting with estrogen receptors (ERs), albeit with substantially lower potency than 17β-estradiol (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Compounds such as daidzein therefore belong to the group of phytoestrogens. Despite their comparatively weak intrinsic estrogenic activity, estimated to be several orders of magnitude lower than that of estradiol, isoflavones may function as selective estrogen receptor modulators, exerting either estrogenic or anti-estrogenic effects depending on the hormonal milieu (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In premenopausal women, where circulating estrogen levels are relatively high, competitive receptor interactions have been proposed as a potential mechanism underlying reported anti-estrogenic actions (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Epidemiological and experimental studies have suggested that habitual intake of isoflavone-rich foods may contribute to a reduced incidence of hormone-dependent malignancies, including breast and prostate cancers, as well as colorectal cancer (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In addition, isoflavones have been investigated for their ability to mitigate menopausal symptoms, particularly vasomotor disturbances and bone loss (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Emerging clinical evidence further indicates a possible role in reproductive medicine, with some reports describing improved luteal function, implantation rates, and pregnancy outcomes among women undergoing assisted reproductive technologies (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, the biological effects of isoflavones are not uniformly beneficial. Concerns have been raised regarding potential stimulation of endometrial tissue under certain conditions, with some studies linking high intake to an elevated risk of endometrial hyperplasia or cancer (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Isoflavones occur in both glycosylated and aglycone forms, the latter generally demonstrating superior bioavailability following oral ingestion (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Daidzein-rich aglycone isoflavones (DRIAs) commercially available as AglyMax\u0026reg;, have been developed as dietary supplements. Clinical investigations have reported improvements in menopausal vasomotor symptoms and implantation outcomes without serious adverse events observed to date, although continued safety evaluation remains warranted (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our previous experimental studies, we demonstrated that DRIAs exert anti-proliferative and anti-inflammatory effects in endometriotic stromal cells derived from ovarian endometriomas (OESCs). Specifically, DRIAs suppressed cell proliferation and reduced the expression of inflammatory mediators, including IL-6, IL-8, COX-2, and aromatase, without affecting normal endometrial stromal cells (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Importantly, these inhibitory effects were mediated through estrogen receptor β (ERβ), as pharmacologic blockade or siRNA knockdown of ERβ reversed the anti-proliferative effects of DRIAs (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In contrast, the precursor compounds daidzein and equol did not reproduce these effects, suggesting that the biological activity is specific to the aglycone-rich formulation. Furthermore, we recently clarified that DRIAs modulate local estrogen metabolism in endometriosis. DRIAs significantly suppressed 17β-hydroxysteroid dehydrogenase type 1 (HSD17β1) expression and enzymatic activity, an enzyme that is highly expressed in endometriotic tissues, while increasing estrogen sulfotransferase (EST) levels in OESCs (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Immunohistochemical analysis of ovarian endometrioma specimens from patients treated with DRIAs demonstrated reduced HSD17β1 expression compared with untreated controls. These findings indicate that DRIAs may attenuate local estradiol biosynthesis not only via aromatase inhibition but also through coordinated regulation of estrogen-metabolizing enzymes (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on these preclinical findings, we conducted a pilot open-label clinical study (ERB-1202-1) involving 17 patients with endometriosis-associated pain at our institution. Participants received DRIAs for four months according to the same protocol as the present study. No treatment-related adverse events were reported. After four months, the mean reduction in dysmenorrhea VAS was 24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;25.8 points (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with scores decreasing from 49.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 at baseline to 24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2. In addition, the mean diameter of endometriotic ovarian cysts decreased from 37.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 mm to 32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 mm (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant changes were observed in VAS scores for dyschezia, dysuria or dyspareunia. Although limited by the small size and open-label design, these preliminary findings suggested potential clinical benefits of DRIAs in reducing menstrual pain and cyst size without evident safety concerns. Therefore, building upon our preclinical data and pilot observations, the present study was designed as a randomized, placebo-controlled, double-blind clinical trial to rigorously evaluate the efficacy and safety of DRIAs in patients with endometriosis-associated pain.\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThe study was a multicenter, randomized, double-blind, placebo-controlled clinical trial conducted in Japanese women with clinically diagnosed endometriosis. The trial was performed in accordance with the ethical principles of the Declaration of Helsinki and complied with the Clinical Trials Act in Japan. The protocol was approved by the institutional review board at each participating center. Written informed consent was obtained from all participants prior to enrollment. The trial was registered with the Japan Registry of Clinical Trials (jRCT; registration number: jRCTs051200074, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jrct.mhlw.go.jp/en-latest-detail/jRCTs051200074\u003c/span\u003e\u003cspan address=\"https://jrct.mhlw.go.jp/en-latest-detail/jRCTs051200074\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Registration Date: 2020/10/27). This study is reported in accordance with the CONSORT guidelines. This study was also approved by the Clinical Research Review Board of Kyoto Prefectural University of Medicine (Protocol No. 2019031-8).\u003c/p\u003e \u003cp\u003e Participants were recruited from Kyoto Prefectural University of Medicine Hospital, Japanese Red Cross Kyoto Daiichi Hospital, North Medical Center Kyoto Prefectural University of Medicine. Eligible patients were centrally registered using anonymized case registration forms. Participants were randomly assigned to receive either aglycone-type isoflavone (DRIAs) or placebo. Randomization was performed using a minimization method with dynamic allocation to ensure balance between groups. Allocation adjustment factors were: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) baseline dysmenorrhea visual analog scale (VAS) score (\u0026lt;\u0026thinsp;50 vs. ≧ 50), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) ovarian endometriotic cyst diameter (\u0026lt;\u0026thinsp;5 cm vs. \u0026gt; 5 cm), and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) serum CA125 level (\u0026lt;\u0026thinsp;35 U/mL vs. ≧ 35 U/mL).\u003c/p\u003e \u003cp\u003eThe study was conducted under double-blind conditions. The allocation manager, who was not involved in patient care or outcome assessment, generated the randomization list and ensured identical appearance, packaging, and labeling of study medications. The allocation list was securely stored until completion of data collection and unblinding.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample size calculation\u003c/h3\u003e\n\u003cp\u003eIn a prior open-label pilot study (ERB-1202-1) involving 17 patients with endometriosis-associated pain, the mean reduction in dysmenorrhea visual analogue scale (VAS) score after four months of DRIAs treatment was 24.4 points, with a standard deviation (SD) of 25.8 (change from baseline). Given the substantial placebo response reported in previous placebo-controlled trials in endometriosis, the placebo effect was conservatively estimated at 40% of the observed treatment response. Accordingly, the expected mean improvement in the placebo group was set at 9.8 points (24.4 \u0026times; 0.4), resulting in an anticipated between-group difference of 14.6 points. Assuming a common SD of 25.8, a two-sided α level of 0.05, and 80% power, 50 participants per group were required using a two-sample t-test. Allowing for an estimated 10% dropout rate, the planned total sample size was 110 participants.\u003c/p\u003e \u003cp\u003eRecruitment was conducted between April 2020 and March 2024. However, enrollment was substantially affected by the COVID-19 pandemic, which led to reduced outpatient visits and slower-than-anticipated recruitment. Enrollment was therefore closed at the prespecified end of the recruitment period before reaching the planned sample size. Analyses were performed using all enrolled participants. As the target sample size was not achieved, the study may have been underpowered, particularly for secondary endpoints and later time-point comparisons.\u003c/p\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eWomen aged 20\u0026ndash;50 years with clinically diagnosed endometriosis were eligible. Diagnosis was established based on laparotomy or laparoscopy, transvaginal ultrasonography, magnetic resonance imaging (MRI), and/or gynecological examination according to the Japan Society of Obstetrics and Gynecology (JSOG) Guidelines for the Management of Endometriosis (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Participants were required to have a regular menstrual cycle (25\u0026ndash;38 days) and Chronic pelvic pain with a dysmenorrhea VAS score\u0026thinsp;≧\u0026thinsp;20.\u003c/p\u003e \u003cp\u003eParticipants were excluded if they (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) had taken isoflavone-containing dietary supplements within 3 months prior to screening, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) had received GnRH agonists/antagonists, progestins, estrogen-containing preparations, low-dose oral contraceptives, testosterone derivatives, or Kampo, Chinese traditional, medicines for endometriosis within 3 months, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) had undergone surgical treatment for endometriosis within 3 months, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) were pregnant or suspected to be pregnant, (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) became pregnant during the study, (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) were breastfeeding, (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) were deemed inappropriate for participation by investigators for safety or other reasons.\u003c/p\u003e\n\u003ch3\u003eIntervention\u003c/h3\u003e\n\u003cp\u003eParticipants in the intervention group received DRIAs at a daily dose equivalent to 30 mg/day of aglycone, consistent with supplemental upper intake level recommended by the Food Safety Commission of Japan. The control group received a matching placebo. Study medication was administered orally twice daily. Treatment began one month after registration and continued for four months.\u003c/p\u003e\n\u003ch3\u003eOutcomes\u003c/h3\u003e\n\u003cp\u003eThe primary endpoint was change in dysmenorrhea severity VAS score from baseline, assessed by the visual analog scale (VAS). Secondary endpoints included (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) change in endometriotic cyst diameter measured by transvaginal ultrasonography. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) change in dyspareunia, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) change in dysuria/dyschezia, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) changes in hematological parameters, liver function tests, (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) change in serum CA125 levels, and incidence of adverse events. Adverse events were assessed at each outpatient visit through structured interviews.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe primary endpoint, longitudinal changes in dysmenorrhea VAS scores, and the secondary endpoint of ovarian endometriotic cyst diameter were evaluated using a linear mixed model (LMM) to account for repeated measures and handle missing data. The model included treatment group, time (categorical: Months 1, 2, 3, and 4), and the group-by-time interaction as fixed effects, with the baseline value included as a covariate. Patient ID was included as a random effect. Between-group comparisons at each post-baseline time point were performed using estimated marginal means derived from the LMM, and p-values were adjusted using the Holm method to control for multiple comparisons. For serum CA125 levels, due to the right-skewed distribution, the values were natural log-transformed prior to analysis. An analysis of covariance (ANCOVA) was performed on the log-transformed Month 4 values, adjusting for the log-transformed baseline values, to evaluate the between-group difference. Additionally, an overall paired t-test was conducted on the log-transformed data to assess the main effect of time across both groups. Categorical variables were compared using the chi-square test or Fisher\u0026rsquo;s exact test, as appropriate. All statistical tests were two-sided with a significance level of 0.05. Secondary endpoints were considered exploratory. Analyses were conducted according to a modified intention-to-treat principle, including all randomized participants who received at least one dose of study medication and had at least one post-baseline assessment. In addition, an exploratory analysis was performed. A responder was defined as a patient who achieved a\u0026thinsp;\u0026ge;\u0026thinsp;30% reduction in dysmenorrhea VAS score from baseline to Month 4 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The proportion of responders was compared between groups using the chi-square test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eBetween April 2020 and March 2024, 81 women were randomized to receive DRIAs (n\u0026thinsp;=\u0026thinsp;40) or placebo (n\u0026thinsp;=\u0026thinsp;41). In the DRIAs group, six participants discontinued the study (four withdrew consent and two discontinued due to a desire for pregnancy). In the placebo group, four participants discontinued (two withdrew consent and two discontinued due to a desire for pregnancy). A total of 71 patients (34 in the DRIAs group and 37 in the placebo group) were included in the modified intention-to-treat analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Baseline characteristics were comparable between groups (Table\u0026nbsp;1). No statistically significant differences were observed in demographic or clinical variables, including age, BMI, baseline dysmenorrhea severity, cyst diameter, or serum CA125 levels. Of the planned 110 participants, 81 were enrolled before recruitment was closed at the prespecified end of the study period due to COVID-19-related constraints.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOutcome\u003c/h2\u003e \u003cp\u003eMean baseline dysmenorrhea VAS scores were 59.4 in the DRIAs group and 60.7 in the placebo group, with no significant difference between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A baseline-adjusted linear mixed model was used to evaluate dysmenorrhea VAS. A highly significant main effect of time was observed (p\u0026thinsp;=\u0026thinsp;0.00006), indicating an overall improvement in both groups over the four-month period. In the between-group comparison (DRIAs-control) at each time point, the DRIAs group showed a significantly lower value at Month 3 (p\u0026thinsp;=\u0026thinsp;0.0104). However, the group-by-time interaction was not significant (p\u0026thinsp;=\u0026thinsp;0.344). Post hoc pairwise comparisons, adjusted using the Holm method, revealed that the VAS score was significantly lower in the DRIAs group than in the placebo group at Month 3 only (estimated difference: -13.53, 95% CI: -23.8 to -3.22, Holm-adjusted p\u0026thinsp;=\u0026thinsp;0.041) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Changes in the maximum diameter of ovarian endometriomas over the 4-month period were evaluated using a baseline-adjusted linear mixed model. There was no significant main effect of time (p\u0026thinsp;=\u0026thinsp;0.539), nor was there a significant group-by-time interaction (p\u0026thinsp;=\u0026thinsp;0.965) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, post-hoc comparisons between the DRIAs and placebo groups at each time point revealed no significant differences (Holm-adjusted p\u0026thinsp;=\u0026thinsp;1.000 for all time points). Exploratory analyses of dysuria VAS, dyschezia VAS, and dyspareunia VAS were also performed using baseline-adjusted linear mixed models. No significant group-by-time interactions were observed for any of these endpoints. No significant between-group differences were observed after Holm adjustment. As an exploratory analysis, responder rates were assessed at Month 4. The responder rate at Month 4 was 73.5% (25/34) in the DRIAs group and 56.8% (21/37) in the placebo group. Although the between-group difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.2134), the DRIAs group showed a numerically higher proportion of patients with clinically meaningful improvement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLog-transformed serum CA125 levels decreased significantly from baseline to Month 4 across both groups (p\u0026thinsp;=\u0026thinsp;0.035). However, baseline-adjusted ANCOVA revealed no significant between-group difference at Month 4 (p\u0026thinsp;=\u0026thinsp;0.574) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSafety\u003c/h2\u003e \u003cp\u003eOne patient in the DRIAs group developed a skin rash during the fourth month of treatment and discontinued the study medication. No other adverse events were reported. No clinically significant changes were observed in hematological parameters or liver function tests during the study period.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this multicenter, randomized, double-blind, placebo-controlled trial, administration of DRIAs resulted in a significant reduction in endometriosis-associated dysmenorrhea compared with placebo. Although pain scores decreased over time in both groups, statistical superiority was observed at Month 3. The observed between-group difference exceeded commonly reported minimal clinically important differences for chronic pain outcomes, supporting the clinical relevance of the treatment effect. These findings extend our previous pilot observations and provide controlled clinical evidence supporting the analgesic effect of aglycone-rich isoflavones in reproductive-aged women with endometriosis.\u003c/p\u003e \u003cp\u003eA placebo response is frequently observed in randomized trials evaluating endometriosis-associated pain, and clinically meaningful reductions have been reported in placebo arms of large hormonal therapy trials (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Consistent with these observations, VAS scores in the present study showed modest improvement in the placebo group. Nevertheless, the greater magnitude of improvement in the DRIAs group suggests a genuine treatment effect beyond placebo response. Although statistical significance was not maintained at Month 4, the magnitude and direction of the treatment effect remained consistent, with between-group differences of similar magnitude at Months 3 and 4. In addition, an exploratory responder analysis showed a numerically higher proportion of patients achieving clinically meaningful pain reduction (\u0026ge;\u0026thinsp;30% from baseline) in the DRIAs group compared with the placebo group at Month 4, although the difference was not statistically significant. This finding supports the possibility that DRIAs may provide clinically relevant benefit at the individual patient level. The absence of statistical significance at the later time point may reflect limited statistical power rather than attenuation of therapeutic efficacy, particularly as the study did not reach the originally planned sample size due to COVID-19\u0026ndash;related recruitment constraints.\u003c/p\u003e \u003cp\u003eDespite improvement in dysmenorrhea, no significant reduction in ovarian endometriotic cyst diameter was observed. Pain severity in endometriosis does not necessarily correlate with lesion size, and inflammatory signaling is considered a key contributor to symptom generation. In our previous experimental studies, DRIAs suppressed IL-6, IL-8, COX-2, aromatase, and HSD17β1 expression in endometriotic stromal cells via ERβ-dependent mechanisms, thereby attenuating local estradiol production and inflammatory activity (\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). These molecular effects may contribute to symptomatic relief without inducing rapid macroscopic regression of cysts within a four-month treatment period. Serum CA125 levels showed a decreasing trend in the DRIAs group; however, the change did not reach statistical significance.\u003c/p\u003e \u003cp\u003ePhytoestrogens have attracted attention as potential modulators of endometriosis because of their structural similarity to estradiol and preferential binding to ERβ, which is highly expressed in endometriotic lesions (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Experimental studies have reported anti-inflammatory and anti-proliferative effects of several compounds, including resveratrol and isoflavones (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). However, clinical evidence remains limited, and many previous human studies evaluated these agents as adjuncts to hormonal therapies rather than as independent treatments. To our knowledge, no randomized trials have assessed phytoestrogens as monotherapy in women with endometriosis. In this context, the present randomized, double-blind, placebo-controlled trial provides evidence supporting the clinical utility of an aglycone-rich isoflavone formulation administered without concomitant ovulation suppression.\u003c/p\u003e \u003cp\u003eCurrent standard medical treatments for endometriosis primarily rely on suppression of ovulation and reduction of estrogen levels (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). While effective for pain control, these approaches prevent conception during treatment and may be associated with hypoestrogenic or progestin-related adverse effects (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In contrast, DRIAs were administered at a dose corresponding to the supplemental upper intake level considered acceptable by the Food Safety Commission of Japan and did not induce ovulatory suppression (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). No serious adverse events were observed, and no clinically relevant abnormalities in laboratory parameters were detected. One participant developed a skin rash that resolved after discontinuation. These findings indicate favorable short-term tolerability and suggest potential utility in women wishing to preserve reproductive function. The possibility of symptom relief without suppression of ovulation is clinically meaningful. Many patients seek alternatives to surgery or long-term hormonal therapy because of concerns about fertility preservation or adverse effects (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Although pregnancy outcomes were not evaluated in this study, the absence of endocrine suppression suggests that DRIAs may be compatible with attempts at conception. Further investigation is required to determine their impact on fertility outcomes.\u003c/p\u003e \u003cp\u003eSeveral limitations should be considered. The study did not reach the planned sample size, which may have limited statistical power, particularly for secondary endpoints and later time points. The treatment duration was limited to four months, and long-term outcomes after discontinuation were not evaluated. Quality-of-life measures were not comprehensively assessed, and symptoms such as dyspareunia, dyschezia, and dysuria were not sufficiently prevalent to allow robust analysis. Larger trials with adequate statistical power and extended follow-up are required to confirm durability of effect and to evaluate potential impacts on fertility outcomes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDRIAs demonstrated a consistent trend toward reduction in endometriosis-associated dysmenorrhea and were well tolerated without suppression of ovarian function. As a food-derived therapy with a favorable safety profile, DRIAs may represent a clinically relevant treatment option, particularly for women seeking to preserve fertility for whom conventional hormone-based therapies are not suitable. Although further adequately powered studies are required, this mechanism-informed approach offers a promising alternative strategy in the management of endometriosis-associated pain.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDRIAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDaidzein-rich isoflavone aglycones\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evisual analog scale\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThe protocol was approved by the institutional review board at each participating center. Written informed consent was obtained from all participants prior to enrollment. The trial was registered with the Japan Registry of Clinical Trials (jRCT; registration number: jRCTs051200074, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jrct.mhlw.go.jp/en-latest-detail/jRCTs051200074\u003c/span\u003e\u003cspan address=\"https://jrct.mhlw.go.jp/en-latest-detail/jRCTs051200074\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This study is reported in accordance with the CONSORT guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eAll authors have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported in part by Grants-in Aid for Scientific Research (22K16839) from the Ministry of Education, Culture, Science, and Technology (Japan).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eOT developed the manuscript design, collected data from participants, performed statistical analysis and drafted the manuscripts. HO and YT, FI, AK, IK collected data from participants. MK performed statistical analysis. JK and TM developed study design and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Mr. Akihiro Kitatani, Mrs. Ayaka Miura, and Mrs. Mutsumi Kazui for data management. This study was supported in part by Grants-in Aid for Scientific Research (22K16839) from the Ministry of Education, Culture, Science, and Technology (Japan).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKnez, J., Kovačič, B. \u0026amp; Goropevšek, A. The role of regulatory T-cells in the development of endometriosis. \u003cem\u003eHum. Reprod.\u003c/em\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, Y. et al. Exacerbation of Endometriosis Due To Regulatory T-Cell Dysfunction. \u003cem\u003eJ. Clin. 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Effects of soy protein and isoflavones on circulating hormone concentrations in pre- and post-menopausal women: a systematic review and meta-analysis. \u003cem\u003eHum. Reprod. Update\u003c/em\u003e. \u003cb\u003e15\u003c/b\u003e (4), 423\u0026ndash;440 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVercellini, P., Vigano, P., Somigliana, E. \u0026amp; Fedele, L. Endometriosis: pathogenesis and treatment. \u003cem\u003eNat. reviews Endocrinol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (5), 261\u0026ndash;275 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"endometriosis, phytoestrogen, fertility preservation, chronic pelvic pain","lastPublishedDoi":"10.21203/rs.3.rs-9799296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9799296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEndometriosis-associated dysmenorrhea is commonly managed with hormonal therapies that suppress ovulation, limiting their use in women desiring fertility. Daidzein-rich isoflavone aglycones (DRIAs), a food-derived compound with estrogen receptor-mediated anti-inflammatory and proliferative effects, may represent a provide a novel therapeutic option.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis multicenter, randomized, double-blind, placebo-controlled trial enrolled Japanese women aged 20\u0026ndash;50 years with endometriosis-associated dysmenorrhea (visual analog scale (VAS)\u0026thinsp;\u0026ge;\u0026thinsp;20). Participants were randomized to receive DRIAs (30 mg/day) or placebo for 4 months. The primary endpoint was change in dysmenorrhea VAS score, analyzed using a baseline-adjusted linear mixed model. Secondary outcomes included changes in endometriotic cyst diameter, other pain symptoms, and safety parameters.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eA total of 81 participants were randomized, and 71 were included in the modified intention-to-treat analysis. Dysmenorrhea VAS scores improved significantly over time in both groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). although the group-by-time interaction was not statistically significant, a significant between-group difference favoring DRIAs was observed at Month 3 (\u0026minus;\u0026thinsp;13.53, 95% CI\u0026thinsp;\u0026minus;\u0026thinsp;23.8 to \u0026minus;\u0026thinsp;3.22; adjusted p\u0026thinsp;=\u0026thinsp;0.041). The direction of effect consistently favored DRIAs across time points. The responder rate (\u0026ge;\u0026thinsp;30% reduction at Month 4) was higher in the DRIAs group (73.5% vs 56.8%), although not statistically significant. No serious adverse events or clinically relevant laboratory abnormalities were observed.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eDRIAs may provide a safe, non-hormonal therapeutic option for endometriosis-associated dysmenorrhea without suppression of ovarian function. The observed magnitude and consistency of pain reduction suggest a clinically meaningful effect, supporting further evaluation in adequately powered studies.\u003c/p\u003e","manuscriptTitle":"A multicenter randomized placebo-controlled trial evaluating daidzein-rich isoflavone aglycones in endometriosis-associated dysmenorrhea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-07-07 11:58:52","doi":"10.21203/rs.3.rs-9799296/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"26409281563029948996516883595237471037","date":"2026-07-04T04:49:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-07-03T09:20:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-06-08T12:41:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-05-28T18:27:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-27T13:56:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-05-27T10:44:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f2959a0-2de3-4630-aa6c-8010e183a495","owner":[],"postedDate":"July 7th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"26409281563029948996516883595237471037","date":"2026-07-04T04:49:21+00:00","index":66,"fulltext":""},{"type":"reviewersInvited","content":"11","date":"2026-07-03T09:20:05+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":71584447,"name":"Health sciences/Diseases"},{"id":71584448,"name":"Health sciences/Endocrinology"},{"id":71584449,"name":"Health sciences/Health care"},{"id":71584450,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-07-07T11:58:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-07-07 11:58:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9799296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9799296","identity":"rs-9799296","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}