{"paper_id":"c0c8db36-eafd-404e-b6f7-e5e310c5bf31","body_text":"ARTICLE IN PRESS\nArticle in Press\nAdjunctive vitamin D supplementation in women \nreceiving dienogest therapy for endometriosis: a \nrandomized controlled trial\nScientific Reports\nReceived: 27 October 2025\nAccepted: 13 July 2026\nCite this article as: Shabani F., \nHajizadeh K., Hesami M. et al. Adjunctive \nvitamin D supplementation in women \nreceiving dienogest therapy for \nendometriosis: a randomized controlled \ntrial. Sci Rep (2026). https://doi.\norg/10.1038/s41598-026-62548-6\nFatemeh Shabani, Khadije Hajizadeh, Mahsa Hesami, Elnaz Shaseb, Vahideh \nRahmani, Marzieh Parizad Nasirkandy & Mojgan Mirghafourvand\nWe are providing an unedited version of this manuscript to give early access to its \nfindings. Before final publication, the manuscript will undergo further editing. Please \nnote there may be errors present which affect the content, and all legal disclaimers \napply.\nIf this paper is publishing under a Transparent Peer Review model then Peer \nReview reports will publish with the final article.\nhttps://doi.org/10.1038/s41598-026-62548-6\n© The Author(s) 2026. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International \nLicense, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit \nto the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do \nnot have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this \narticle 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 \narticle’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain \npermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.\n\nAdjunctive Vitamin D Supplementation in Women Receiving Dienogest Therapy for \nEndometriosis: A Randomized Controlled Trial\nFatemeh Shabani 1, Khadije Hajizadeh 2, Mahsa Hesami 3, Elnaz Shaseb 4, Vahideh Rahmani 5,  \nMarzieh Parizad Nasirkandy6, Mojgan Mirghafourvand7*\n1Midwifery Department, Faculty of Nursing and Midwifery, Tabriz University of medical \nSciences, Tabriz, Iran. Email: fatemehshabani7697@gmail.com\n2Midwifery Department, Faculty of Nursing and Midwifery, Tabriz University of Medical \nSciences, Tabriz, Iran. Email: hajizade_k@yahoo.com\n3Midwifery Department, Faculty of Nursing and Midwifery, Tabriz University of Medical \nSciences, Tabriz, Iran. Email: mahsahesami336@gmail.com\n4Department of Clinical Pharmacy, Faculty of Pharmacy, Tabriz University of Medical Sciences, \nTabriz, Iran. Email: shasebe@tbzmed.ac.ir\n5 Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. Email: \nVahideh_rahmani@yahoo.com\n6 Faculty of Medicine, Tabriz university of medical sciences, Tabriz, Iran. Email: \nparizad116@gmail.com\n7Social Determinants of Health Research Center, Faculty of Nursing and Midwifery, Tabriz \nUniversity of Medical Sciences, Tabriz, Iran. Email: mirghafourvand@gmail.com\n* Corresponding author: Mojgan Mirghafourvand, Social Determinants of Health Research \nCenter, Faculty of Nursing and Midwifery, Tabriz University of Medical sciences, Tabriz, Iran. E-\nmail: mirghafourvand@gmail.com, Phone: 00989143206121.\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nAbstract\nBackground: Endometriosis is a chronic, estrogen-dependent inflammatory disease that causes \ndebilitating pain and significantly impairs quality of life. Due to its anti-inflammatory and \nimmunomodulatory properties, vitamin D has been proposed as a potential therapeutic agent, but \nclinical evidence remains inconsistent. This study aimed to evaluate the effect of vitamin D \nsupplementation on pain symptoms and quality of life in women with endometriosis.\nMethods: This randomized, double-blind, placebo-controlled clinical trial was conducted on 66 \nwomen with symptomatic endometriosis and vitamin D levels below 30 ng/mL. Participants were \nrandomly assigned to either the intervention group (n=33), receiving 4000 IU of vitamin D every \nother day, or the control group (n=33), receiving a matching placebo for eight weeks. Crucially, \nall participants in both groups continued their standard dienogest (Verogest) therapy. The primary \noutcomes were pain severity, assessed using the Visual Analogue Scale (VAS) and the \nENDOPAIN-4D questionnaire, and quality of life as secondary outcome, assessed with the \nEndometriosis Health Profile-30 (EHP-30). Outcomes were measured at baseline and after the \neight-week intervention.\nResults: All 66 participants completed the trial. Despite baseline imbalances in socio-demographic \ncharacteristics (quantified via standardized mean differences), adjusting for these variables as \ncovariates did not yield significant differences between groups. Regarding the ENDOPAIN-4D \nscores, for the \"usual level of pain,\" the post-intervention mean score was 38.8 (SD = 6.5) in the \nvitamin D group compared to 50.1 (SD = 7.4) in the placebo group (adjusted mean difference \n[AMD]: -11.3; 95% Confidence Interval [CI]: -26.2 to 3.5; P = 0.136). For the \"worst level of \npain,\" the post-intervention mean score was 23.6 (SD = 3.1) in the vitamin D group and 25.6 (SD \n= 3.5) in the placebo group (AMD: -2.0; 95% CI: -9.0 to 4.9; P = 0.492). The adjusted mean post-\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nintervention VAS score was 5.8 (SD = 0.3) in the vitamin D group versus 6.2 (SD = 0.4) in the \nplacebo group (AMD: -0.3; 95% CI: -1.3 to 0.5; P = 0.113). No significant differences were \nobserved in the ENDOPAIN-4D scores or in any of the five domains of the EHP-30 questionnaire \n(all P > 0.05).\nConclusion: In women under hormonal suppression with dienogest, eight weeks of adjunctive \nvitamin D did not yield statistically or clinically significant improvements in pain or quality of \nlife. These null findings likely reflect a \"floor effect\" from the baseline hormonal therapy and are \nlimited by the lack of biochemical confirmation of repletion. These findings are specific to \nadjunctive use and may not reflect the potential of vitamin D as a monotherapy.\nKeywords: Vitamin D, Endometriosis, Pain, Quality of Life\nTrial registration: Iranian Registry of Clinical Trials (IRCT): IRCT20120718010324N78. Date \nof first registration: 2023-05-25. URL: https://irct.behdasht.gov.ir/trial/68131\nBackground\nEndometriosis is a chronic, estrogen-dependent inflammatory condition characterized by the \npresence of endometrial-like glands and stroma outside the uterine cavity [1]. While most \ncommonly found in the pelvic peritoneum and ovaries, lesions can occur at extra-pelvic sites, \nincluding the bowel and diaphragm [2]. The disease manifests through a spectrum of symptoms, \nmost notably dysmenorrhea, dyspareunia, and chronic pelvic pain, but it is also a primary driver \nof female infertility and adhesion-related complications [3]. It affects approximately 10% (190 \nmillion) of women and girls of reproductive age [4]. Clinical management traditionally prioritizes \nmedical therapy as first-line treatment, with surgical intervention reserved for cases of medical \nfailure, deep-seated endometriomas, or compressive symptoms [5, 6].\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nIn contemporary clinical practice, the diagnostic paradigm has shifted. While laparoscopy \nwith histological confirmation remains the gold standard [7], current international guidelines from \nESHRE (2022) and NICE now advocate for a clinical diagnosis based on history, physical \nexamination, and high-quality imaging (ultrasound or MRI) in the majority of cases; laparoscopy \nis no longer strictly required for the initiation of treatment [8, 9].\nThe exact etiopathogenesis of endometriosis remains complex and multifactorial. Beyond \nthe classical theory of retrograde menstruation [10], research highlights the roles of inflammation, \nimmune dysregulation, and oxidative stress in lesion survival and proliferation [11, 12]. \nMechanistically, oxidative stress triggers pathways such as the Mitogen-Activated Protein (MAP) \nkinase and ERK pathways, which enhance the survival of ectopic tissue [13]. Furthermore, \nemerging evidence suggests that symptom severity is modulated not only by peripheral \ninflammation but also by central neurobiological and psychosocial factors, including shared \nneurobiology with other comorbid conditions [14, 15].\nVitamin D, a fat-soluble secosteroid, has emerged as a potential modulator of these \npathways [16]. Endogenous synthesis occurs primarily in the lower layers of the epidermis, where \nUVB radiation facilitates the conversion of 7-dehydrocholesterol into pre-vitamin D3, which is \nsubsequently hydroxylated in the liver and kidneys to its active form, calcitriol [17]. Calcitriol \nbinds to vitamin D receptors (VDR) expressed throughout the female reproductive system, \ninfluencing gene transcription related to immune function and inflammation [18, 19].\nThe biological plausibility for vitamin D in endometriosis is supported by preclinical data. \nIn murine models, vitamin D3 administration has demonstrated a 48.8% reduction in the area of \nendometriotic lesions and a significant decrease in fibrosis [20]. In vitro, vitamin D3 has been \nshown to suppress interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) responses in human \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nendometrial stromal cells [21]. Despite these promising findings, clinical evidence remains \ninconsistent. Given the high prevalence of vitamin D deficiency in this population and the need \nfor non-hormonal adjuncts, this study aimed to evaluate whether vitamin D supplementation \nprovides additional benefit for pain (primary outcome) and quality of life (secondary outcome) in \nwomen already receiving standard dienogest therapy.\nMethods\nType of study and participants\nThis study was a double-blind, randomized clinical trial conducted at the Al-Zahra educational \nand therapeutic center between 2023 and 2024. Crucially, as the primary aim was to assess the \neffect of Vitamin D correction, the study population was composed exclusively of women with \nconfirmed vitamin D insufficiency (25-OH vitamin D < 30 ng/mL).\nInclusion and Exclusion Criteria\nThe inclusion criteria for participation were as follows:\n1. A confirmed diagnosis of endometriosis by a specialist gynecologist based on a \ncombination of clinical history, physical examination, and imaging (Transvaginal \nUltrasound or MRI) according to ESHRE 2022 guidelines, or prior laparoscopic \nconfirmation.\n2. Presence of symptomatic endometriosis (VAS ≥ 4).\n3. Maintenance of a stable regimen of 2 mg daily dienogest (Verogest) for at least three \nmonths prior to enrollment. This duration was selected to ensure that any initial therapeutic \nresponse to hormonal therapy had plateaued, thereby allowing for the assessment of \nVitamin D as a true adjunctive (add-on) therapy.\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n4. Marital status (married and sexually active), required for the accurate assessment of the \ndyspareunia domain within the ENDOPAIN-4D instrument.\n5. Serum vitamin D level < 30 ng/mL.\nExclusion criteria included a history of regular vitamin D supplementation within the past three \nmonths. Furthermore, patients with comorbid conditions such as diabetes, thyroid disorders, \nhypertension, hyperprolactinemia, or Cushing’s syndrome were excluded. This was necessary to \nminimize confounding variables, as these metabolic and endocrine disorders can independently \nalter systemic inflammatory markers, calcium metabolism, and the subjective perception of \nchronic pain.\nStudy Groups and Intervention\nEligible participants were randomly assigned to one of two groups: the intervention group, which \nreceived 4000 IU of vitamin D every other day, and the control group, which received a placebo \nfollowing the same schedule. It is a key component of this study that all patients in both groups \ncontinued to receive their standard routine treatment, which consisted of a daily 2 mg dose of \ndienogest (Verogest).\nSample size\nThe sample size for this study was calculated using G-Power software, based on the variable of \npainful symptoms of endometriosis. According to the findings of a study by Alizadeh et al. [22] \nconcerning this variable, and considering M1= 6.60 (mean score of painful menstrual symptoms), \nM2= 5.28 (assuming a 20% reduction in the score following the intervention), SD1 = SD2 = 1.77, \na two-sided α of 0.05, and a power of 80%, the required sample size was calculated to be 30 \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nindividuals. Factoring in a potential 10% attrition rate, the final sample size was considered 33 \nparticipants per group.\nSampling\nThe trial was conducted on women referred to the Al-Zahra educational and therapeutic hospital \nin Tabriz. Sampling commenced after obtaining approval from the Ethics Committee and the Vice-\nChancellor for Research and Technology of Tabriz University of Medical Sciences, and after the \ntrial was registered with the Iranian Registry of Clinical Trials (IRCT). All procedures performed \nin this study involving human participants were in accordance with the ethical standards of the \ninstitutional and national research committee and with the Helsinki declaration and its \namendments. The study was performed on a total of 66 women with endometriosis attending the \ngynecology clinic at Al-Zahra hospital.\nAn initial convenience sampling method was employed. The researcher approached \npotential participants at the Al-Zahra therapeutic centers whose endometriosis diagnosis was \nconfirmed by a collaborating gynecologist. The objectives and procedures of the study were \nexplained, and individuals were assessed against the eligibility criteria. For those who were eligible \nand willing to participate, written informed consent was obtained. Subsequently, participants \ncompleted a socio-demographic questionnaire, the Painful Endometriosis Symptoms questionnaire \n(ENDOPAIN 4D), the Endometriosis Health Profile-30 (EHP-30) questionnaire, and the Visual \nAnalogue Scale (VAS) before being allocated to a group. A blood sample was also collected to \nmeasure serum vitamin D levels. Only individuals whose serum vitamin D level was reported to \nbe less than 30 ng/mL were enrolled in the study.\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nParticipants were provided with a Visual Analog Scale (VAS) and instructed to mark their \npain severity during the first three days of their next menstrual cycle and to begin their assigned \nmedication on the fourth day of that cycle. They were also asked to continue recording their pain \nseverity on the VAS scale during the first three days of the following two menstrual cycles.\nEight weeks after the start of the intervention, the ENDOPAIN 4D and EHP-30 \nquestionnaires were completed again by the participants. This follow-up assessment was \nconducted between days 7 and 12 of the menstrual cycle.\nRandomization and Allocation Concealment\nParticipants who met the inclusion criteria were randomly assigned to either the intervention group \nor the control group using a block randomization method. The randomization sequence was \ngenerated by an independent person using random block sizes of four and six to ensure a balanced \ndistribution of participants between the two groups throughout the recruitment period. A 1:1 \nallocation ratio was used. To ensure allocation concealment, the pharmacy prepared the study \nmedication in sequentially numbered, identical bottles. The bottles containing either the vitamin \nD or placebo tablets were the same in appearance, size, and labeling. Each bottle was labelled with \na unique participant number corresponding to the randomization list.\nIntervention\nThe intervention group received 4000 IU vitamin D tablets, and the control group received a \nplacebo; both were administered every other day for eight weeks. The vitamin D and placebo \ntablets, manufactured by Dana Pharmaceutical Company, were identical in appearance. Each \nparticipant received a sealed envelope containing their assigned medication at the beginning of the \nintervention and was informed that the next follow-up would be eight weeks later. Throughout the \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\neight-week intervention period, participants received a weekly phone call to remind them to take \ntheir medication and to complete a medication adherence checklist. Participants were also \ninstructed to report any side effects, which were assessed via a checklist. In the event of a serious \nadverse event, the intervention was discontinued for that individual, though they were included in \nthe final analysis. It is important to note that all patients continued to receive their routine \ntreatment, which consisted of 2 mg of Verogest daily.\nData Collection Tools\nData for this study were collected by the researcher through structured interviews and self-\nadministered questionnaires. The following instruments were used:\nEligibility Criteria Checklist: This checklist was used to screen potential participants based on \nthe predefined inclusion and exclusion criteria to ensure they were eligible for allocation into the \nstudy groups.\nSocio-Demographic Questionnaire: This researcher-developed questionnaire gathered data on \nparticipants' background characteristics, including age, level of education, obstetric history, \noccupation, and household income.\nEndometriosis Health Profile-30 (EHP-30): This disease-specific questionnaire was used to \nmeasure the quality of life of patients with endometriosis. The EHP-30 consists of 30 items across \nfive core domains: pain (11 items), control and powerlessness (6 items), emotional well-being (6 \nitems), social support (4 items), and self-image (3 items). All items were rated on a 5-point Likert \nscale, where a score of 1 represented the best health status and 5 represented the worst. The raw \nscores for each domain were transformed to a scale of 0 to 100, where 0 indicates the best possible \nquality of life and 100 indicates the worst. The EHP-30 does not provide a single total score; \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\ninstead, a separate score is calculated for each domain. This instrument was originally designed \nand validated by Jones et al. at the University of Oxford in 2001 [23, 24] and has been validated \nin multiple countries. The Persian version was validated by Nojomi et al. in 2011, demonstrating \nhigh internal consistency with Cronbach's alpha coefficients ranging from 0.80 to 0.93 [25].\nENDOPAIN-4D Questionnaire: This instrument was used to assess pelvic and gynecological \npain symptoms. It contains 21 items divided into four domains: spontaneous pelvic pain (10 items), \npain during sexual intercourse (dyspareunia) (3 items), bowel pain and related symptoms during \nmenstruation (3 items), and other symptoms (5 items), including pain during urination, right \nshoulder pain during menstruation, and infertility. The psychometric properties of this instrument \nwere previously assessed and confirmed by the principal investigator in a separate study [26].\nVisual Analogue Scale (VAS): The VAS was used to measure pain intensity. It consists of a 10 \ncm horizontal line anchored by \"no pain\" at the 0 cm mark and \"unbearable pain\" at the 10 cm \nmark. Pain scores were interpreted as follows: 0 indicated no pain, 1–3 as mild pain, 4–6 as \nmoderate pain, 7–9 as severe pain, and a score above 9 as very severe pain. The VAS is a standard \nand widely used tool for pain assessment in research [27].\nMedication Adherence Checklist: Participants used this checklist to self-report their intake of \nthe assigned medication (vitamin D or placebo) on the scheduled days.\nAdverse Events Checklist: This checklist was used to systematically document any adverse \nevents experienced by participants during the intervention period, including the nature and severity \nof the event.\nData Analysis\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nData were analyzed using SPSS software (version 25.0) through both descriptive and inferential \nstatistics. The normality of the distribution for quantitative variables was assessed using the \nKolmogorov-Smirnov test. To evaluate the success of randomization and quantify the extent of \nbaseline imbalances between groups, Standardized Mean Differences (SMDs) were calculated for \nall demographic and clinical variables. An SMD > 0.1 was considered a meaningful imbalance, \nnecessitating a covariate-adjusted approach for the final analysis.\nAn independent t-test compared baseline scores of outcomes between groups. After intervention, \nto account for the identified imbalances in socio-demographic characteristics, VAS scores \n(normally distributed), ENDOPAIN-4D, and EHP-30 scores (non-normally distributed) were \nanalyzed using Generalized Linear Model. Results are presented as adjusted means and standard \ndeviations, with adjusted mean differences (AMD) and 95% confidence intervals (CI) as effect \nsizes. A Bonferroni correction addressed multiplicity across EHP-30 and ENDOPAIN-4D \ndomains. Intention-to-treat (ITT) analysis was performed, and a corrected p-value < 0.05 was \nconsidered statistically significant.\nResults\nParticipant Characteristics\nInitially, 87 women were assessed for eligibility. Of these, 21 were excluded for not meeting the \ninclusion criteria (n=17) or declining to participate (n=4). The remaining 66 participants were \nrandomly allocated to either the vitamin D group (n=33) or the placebo group (n=33). Regarding \nadverse effects, one participant reported nausea, four reported dizziness, four experienced fatigue, \nnine had constipation, and one had diarrhea. All reported conditions were mild, and no serious \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nadverse events were noted. All participants completed the eight-week follow-up, and their data \nwere included in the final analysis (Figure 1).\nThe baseline socio-demographic and clinical characteristics are presented in Table 1. The mean \nage of participants was 30.7 (SD=7.0) years in the vitamin D group and 35.3 (SD=5.8) years in the \nplacebo group. Mean BMI was 24.4 (SD=3.5) in the vitamin D group and 24.7 (SD=2.6) in the \nplacebo group. Evaluation of baseline comparability using Standardized Mean Differences (SMD) \nidentified several imbalances, most notably in age (SMD=0.7). Additionally, various socio-\ndemographic characteristics with SMDs ranging from 0.2 to 0.5 exceeded the pre-specified \nthreshold (>0.1) and were therefore incorporated as covariates in the adjusted analysis.\nPain Symptoms (ENDOPAIN-4D and VAS)\nFor the ENDOPAIN-4D scores (Table 2), after adjusting for baseline values and socio-\ndemographic characteristics with SMD > 0.1, no statistically significant difference was found \nbetween groups. For the \"usual level of pain,\" the post-intervention mean score was 38.8 (SD=6.5) \nin the vitamin D group compared to 50.1 (SD=7.4) in the placebo group (AMD: -11.3, 95% CI: -\n26.2 to 3.5; P=0.136). Similarly, for the \"worst level of pain,\" the post-intervention mean score \nwas 23.6 (SD=3.1) in the vitamin D group and 25.6 (SD=3.5) in the placebo group (AMD: -2.0, \n95% CI: -9.0 to 4.9; P=0.492).\nRegarding the VAS score for pain intensity (Table 3), the mean score at baseline was 8.12 \n(SD=1.5) in the vitamin D group and 8.3 (SD=1.6) in the placebo group. After the eight-week \nintervention and adjusting for the pre-specified covariates, the mean VAS score was 5.8 (SD=0.3) \nin the vitamin D group and 6.2 (SD=0.4) in the placebo group. This difference was not statistically \nsignificant (AMD: -0.3, 95% CI: -1.3 to 0.5; P=0.113).\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nQuality of Life (EHP-30)\nThe quality of life scores across five domains are detailed in Table 4. After adjusting for baseline \nscores and prognostic variables, no statistically significant differences were observed between the \nvitamin D and placebo groups in any domain (all P > 0.05):\n•Pain: Vit D 33.8 (SD=3.6) vs. Placebo 35.2 (SD=4.1); (AMD: -1.3, 95% CI: -9.6 to 6.9; \nP=0.577).\n•Control and powerlessness: Vit D 21.4 (SD=4.3) vs. Placebo 30.0 (SD=4.8); (AMD: -8.6, \n95% CI: -18.4 to 1.2; P=0.143).\n•Social support: Vit D 10.4 (SD=3.9) vs. Placebo 13.8 (SD=4.4); (AMD: -3.4, 95% CI: -12.4 \nto 5.6; P=0.625).\n•Emotional well-being: Vit D 19.1 (SD=2.9) vs. Placebo 19.9 (SD=3.3); (AMD: -0.8, 95% CI: \n-7.6 to 5.9; P=0.814).\n•Self-image: Vit D 7.3 (SD=3.5) vs. Placebo 1.6 (SD=3.9); (AMD: -3.3, 95% CI: -11.3 to 4.7; \nP=0.242).\nTo evaluate the impact of baseline imbalances, a sensitivity analysis compared the crude and \nadjusted models. The lack of statistical significance persisted across all primary and secondary \noutcomes after adjustment, confirming that the initial demographic differences did not \nsubstantively bias the study findings.\nDiscussion\nThis randomized, double-blind, placebo-controlled trial evaluated the adjunctive potential of \nvitamin D3 for managing endometriosis-associated pain and quality of life. Our findings indicate \nthat 4000 IU of vitamin D every other day does not provide a statistically or clinically significant \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nbenefit when added to a stable regimen of dienogest. By utilizing adjusted ANCOVA models, we \nwere able to account for the notable baseline imbalances in age and education. This rigorous \napproach confirmed that the improvements observed across both study arms were primarily a \nfunction of the underlying hormonal therapy rather than the nutraceutical intervention.\nThe divergence between our results and studies reporting positive outcomes [28, 29] likely stems \nfrom our specific focus on vitamin D as an add-on therapy. In this context, a \"floor effect\" appears \nto be the most plausible explanation for the null findings. When patients are already stable on a \npotent progestin like dienogest, the inflammatory milieu may be sufficiently suppressed to a degree \nthat an additional anti-inflammatory agent yields no detectable change. This is further \nsubstantiated by our analysis of the Minimal Clinically Important Difference (MCID). The \nadjusted mean differences for the VAS and EHP-30 domains remained well below the thresholds \nof 1.2 point and 11.1 points, respectively [30, 31], suggesting that even the minor fluctuations \nobserved were clinically negligible.\nWhile the anti-proliferative and anti-fibrotic potential of vitamin D is well-documented in \nlaboratory settings [20, 21], our trial highlights the challenges of clinical translation. One \nsignificant barrier may be the lack of phenotypic stratification. Endometriosis is a heterogeneous \ndisease, and the sensitivities of the Vitamin D Receptor (VDR) may vary significantly between \nperitoneal, ovarian, and deep infiltrating (DIE) lesions [32, 33]. Without a stratified analysis, any \nbenefit specific to a particular disease subtype may have been obscured. Furthermore, the selection \nof a fixed dosing regimen without titration based on individual baseline levels may have \ncontributed to a heterogeneous therapeutic response across the trial.\nFinally, the management of endometriosis-related pain requires a broader conceptual framework \nthat moves beyond peripheral inflammation. Our findings likely reflect a mechanistic mismatch in \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nthis regard. As highlighted in contemporary literature, symptom severity is deeply intertwined with \ncentral neurobiological sensitization and shared neuropsychological comorbidities [34, 35]. While \nVitamin D acts primarily as a peripheral anti-inflammatory and immunomodulator [36], chronic \npain in endometriosis is increasingly recognized as a state of central sensitization [37], where the \ncentral nervous system maintains a state of high reactivity despite peripheral changes. \nConsequently, a peripheral intervention like Vitamin D may be insufficient to reverse the complex \nneurobiological remodeling and \"pain memory\" established in long-term endometriosis patients. \nWhen pain is shaped by these complex central determinants, a nutraceutical approach targeting \nonly the inflammatory component may be insufficient to achieve a meaningful clinical shift. This \nemphasizes the need for a more personalized, multidisciplinary approach in future research, where \ninterventions are tailored not only to the physical stage of the disease but also to the broader \nneurobiological and biochemical profile of the patient.\nStrengths and Limitations\nThe strengths of this trial include its randomized, double-blind design, a 100% participant retention \nrate, and the use of validated, disease-specific instruments (EHP-30 and ENDOPAIN-4D). \nHowever, several limitations warrant caution. First, the concurrent use of dienogest likely created \na \"floor effect,\" where the high efficacy of hormonal therapy masked potential incremental benefits \nfrom Vitamin D. Second, the eight-week duration may be insufficient to elicit significant \nimmunomodulatory changes. Crucially, the absence of post-intervention serum 25(OH)D \nmeasurements prevents confirmation of biochemical repletion, which is a major caveat of this \nstudy. We cannot definitively confirm if the 4000 IU dose administered every other day was \nsufficient to reach optimal therapeutic ranges in all participants, particularly given the potential for \nmalabsorption.\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nGeneralizability is limited by the lack of stratification by endometriosis subtype (e.g., DIE vs. \novarian) and the inclusion of only married, sexually active women. Extrinsic variables like diet \nand UV exposure were not controlled. Finally, our null results must be viewed through a \nbiopsychosocial lens; endometriosis pain is heavily modulated by central neurobiological and \npsychosocial factors. A single nutraceutical intervention may have limited detectable impact when \nsymptoms are shaped by such complex comorbidities.\nConclusion\nIn conclusion, this trial found that the administration of adjunctive Vitamin D3 (4000 IU every \nother day) to standard dienogest therapy for eight weeks did not result in a statistically or clinically \nsignificant improvement in pain symptoms or quality of life compared to dienogest alone. These \nfindings suggest a notable \"floor effect,\" where the high efficacy of baseline hormonal therapy \nlikely masked any incremental benefits of the nutraceutical intervention.\nImportantly, as our conclusions are based on the administration of the supplement rather than the \nachievement of a confirmed serum 25(OH)D threshold, the absence of biochemical confirmation \nof repletion means these null results cannot definitively rule out the biological potential of Vitamin \nD. While our study does not support the routine use of short-term Vitamin D as an adjunctive \ntherapy for women already stable on dienogest, its role as a standalone treatment or over longer \ndurations in specific endometriosis phenotypes warrants further investigation in more robustly \npowered and stratified clinical trials.\nAbbreviations\nDIE Deep Infiltrating Endometriosis\nEHP-30 Endometriosis Health Profile-30\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nENDOPAIN-4D Endometriosis Pain 4D Questionnaire\nERK Extracellular Signal-related Kinase\nMAP Mitogen-Activated Protein\nVAS Visual Analogue Scale\nAuthors’ contributions\nAll authors contributed to the conception and design of the study and participated in the revision \nof the manuscript. FS authored the manuscript under the direct supervision of MM, the \nCorresponding Author. MM conducted the statistical analysis. All authors reviewed and approved \nthe final version of the manuscript.\nFunding\nTabriz University of Medical Sciences funded this research but did not participate in its design, \nexecution, or manuscript submission decisions.\nData availability\nThe data used to support the findings of this study can be obtained from the corresponding author \nupon request.\nDeclarations\nEthics approval and consent to participate\nThis study received approval from the Ethics Committee of Tabriz University of Medical Sciences \nunder the code IR.TBZMED.REC.1402.109. Before participating, the researcher provided a \ndetailed explanation of the study's objectives and methodologies to all participants. Informed \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nwritten consent was obtained from each participant, confirming their voluntary involvement and \nunderstanding of the study's purpose and procedures.\nCompeting interests\nN/A\nAcknowledgments\nThe authors appreciate the assistance and cooperation of the participants of this study.\nReferences\n1. Arafah M, Rashid S, Akhtar M: Endometriosis: a comprehensive review. Advances in anatomic \npathology 2021, 28(1):30-43.\n2. Charatsi D, Koukoura O, Ntavela IG, Chintziou F, Gkorila G, Tsagkoulis M, Mikos T, Pistofidis G, \nHajiioannou J, Daponte A: Gastrointestinal and urinary tract endometriosis: a review on the \ncommonest locations of extrapelvic endometriosis. Advances in medicine 2018, \n2018(1):3461209.\n3. Gruber TM, Mechsner S: Pathogenesis of endometriosis: the origin of pain and subfertility. Cells \n2021, 10(6):1381.\n4. Xu S, Zhang Y, Ye P, Huang Q, Wang Y, Zhang Y, Yang C, Ding J: Global, regional, and national \nburden of endometriosis among women of childbearing age from 1990 to 2021: a cross-\nsectional analysis from the 2021 global burden of disease study. Int. J. Surg. 2025, 111(9):5927-\n5940.\n5. Mick I, Freger SM, van Keizerswaard J, Gholiof M, Leonardi M: Comprehensive endometriosis care: \na modern multimodal approach for the treatment of pelvic pain and endometriosis. \nTherapeutic advances in reproductive health 2024, 18:26334941241277759.\n6. Ortega-Gutiérrez M, Muñoz-Gamez A, Girón-Prieto MdlS: Primary care approach to endometriosis: \ndiagnostic challenges and management Strategies—A narrative review. J. Clin. Med. 2025, \n14(13):4757.\n7. Pascoal E, Wessels J, Aas‐Eng M, Abrao MS, Condous G, Jurkovic D, Espada M, Exacoustos C, Ferrero \nS, Guerriero S: Strengths and limitations of diagnostic tools for endometriosis and relevance \nin diagnostic test accuracy research. Ultrasound in obstetrics & gynecology 2022, 60(3):309-\n327.\n8. Becker CM, Bokor A, Heikinheimo O, Horne A, Jansen F, Kiesel L, King K, Kvaskoff M, Nap A, \nPetersen K: ESHRE guideline: endometriosis. Human reproduction open 2022, \n2022(2):hoac009.\n9. Norton W, Holloway D: Understanding the NICE guidance on endometriosis. Practice nursing 2020, \n31(1):8-16.\n10. Hegazy AA: A new look at the theoretical causes of endometriosis: narrative review. Int. J. Reprod. \nBioMed. 2024, 22(5):343.\n11. Ansariniya H, Yavari A, Javaheri A, Zare F: Oxidative stress‐related effects on various aspects of \nendometriosis. Am. J. Reprod. Immunol. 2022, 88(3):e13593.\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n12. Chen S, Liu Y, Zhong Z, Wei C, Liu Y, Zhu X: Peritoneal immune microenvironment of \nendometriosis: Role and therapeutic perspectives. Frontiers in immunology 2023, 14:1134663.\n13. Liu Y, Wang J, Zhang X: An update on the multifaceted role of NF-kappaB in endometriosis. Int. \nJ. Biol. Sci. 2022, 18(11):4400.\n14. Alexander KG, Nordahl EJ, Newton TI, Georgiou C: Neuropsychiatric and cognitive manifestations \nof endometriosis: insights into the ‘endometriosis brain’. Academia Mental Health and Well-\nBeing 2025, 2(4).\n15. Pszczołowska M, Walczak K, Kołodziejczyk W, Kozłowska M, Kozłowski G, Gachowska M, Leszek \nJ: Understanding deep endometriosis: from molecular to neuropsychiatry dimension. Int. J. \nMol. Sci. 2025, 26(2):839.\n16. Voiculescu VM, Nelson Twakor A, Jerpelea N, Pantea Stoian A: Vitamin D: beyond traditional \nroles—insights into its biochemical pathways and physiological impacts. Nutrients 2025, \n17(5):803.\n17. Raczyk M, Carlberg C: From Sunlight to Signaling: Evolutionary Integration of Vitamin D and \nSterol Metabolism. Metabolites 2026, 16(1):74.\n18. Grzesiak M, Tchurzyk M, Socha M, Sechman A, Hrabia A: An overview of the current known and \nunknown roles of vitamin D3 in the female reproductive system: lessons from farm animals, \nbirds, and fish. Int. J. Mol. Sci. 2022, 23(22):14137.\n19. Oliynyk S: The regulatory and pleiotropic role of vitamin D. Regulatory Mechanisms in Biosystems \n2026, 17(2):1-8.\n20. Abbas MA, Taha MO, Disi AM, Shomaf M: Regression of endometrial implants treated with \nvitamin D3 in a rat model of endometriosis. Eur. J. Pharmacol. 2013, 715(1-3):72-75.\n21. Almassinokiani F, Khodaverdi S, Solaymani-Dodaran M, Akbari P, Pazouki A: Effects of Vitamin D \non Endometriosis-Related Pain: A Double-Blind Clinical Trial. Med Sci Monit 2016, 22:4960-\n4966.\n22. Alizad S, Mirghafourvand M, Oskouei BS, Bani S: Endometriosis painful symptoms and its \nrelationship with quality of life in women referring to educational centers of Tabriz \nUniversity of Medical Sciences. Current Women's Health Reviews 2024, 20(1):3-12.\n23. Jones G, Kennedy S, Barnard A, Wong J, Jenkinson C: Development of an endometriosis quality-of-\nlife instrument: The Endometriosis Health Profile-30. Obstetrics & Gynecology 2001, \n98(2):258-264.\n24. Khong S-Y, Lam A, Luscombe G: Is the 30-item Endometriosis Health Profile (EHP-30) suitable \nas a self-report health status instrument for clinical trials? Fertility and sterility 2010, \n94(5):1928-1932.\n25. Nojomi M, Bijari B, Akhbari R, Kashanian M: The assessment of reliability and validity of Persian \nversion of the endometriosis health profile (EHP-30). Iran. J. Med. Sci. 2011, 36(2):84.\n26. Ahmadpour P, Jahangiry L, Bani S, Iravani M, Mirghafourvand M: Validation of the Iranian version \nof the ENDOPAIN-4D questionnaire for measurement of painful symptoms of endometriosis. \nJ. Obstet. Gynaecol. 2022, 42(6):2341-2348.\n27. Phumdoung S, Rattanaparikonn A, Maneechot K: Pain during the first stage of labor. 2010.\n28. Mehdizadehkashi A, Rokhgireh S, Tahermanesh K, Eslahi N, Minaeian S, Samimi M: The effect of \nvitamin D supplementation on clinical symptoms and metabolic profiles in patients with \nendometriosis. Gynecological Endocrinology 2021, 37(7):640-645.\n29. Nodler JL, DiVasta AD, Vitonis AF, Karevicius S, Malsch M, Sarda V, Fadayomi A, Harris HR, \nMissmer SA: Supplementation with vitamin D or ω-3 fatty acids in adolescent girls and young \nwomen with endometriosis (SAGE): a double-blind, randomized, placebo-controlled trial. Am \nJ Clin Nutr. 2020, 112(1):229-236.\n30. Almendra R, Egas Araujo E, Sousa DN, Nogueira-Silva C: Translation, cultural adaptation, and \nvalidation of the Portuguese version of ENDOPAIN-4D questionnaire. J Psychosom Obstet \nGynaecol. 2026, 47(1):2643521.\n31. Koo M, Yang S-W: Visual Analogue Scale. Encyclopedia 2025, 5(4):190.\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n32. Imperiale L, Nisolle M, Noël J-C, Fastrez M: Three types of endometriosis: pathogenesis, diagnosis \nand treatment. State of the art. J. Clin. Med. 2023, 12(3):994.\n33. Saunders PT, Horne AW: Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell \n2021, 184(11):2807-2824.\n34. Delanerolle G, Pathiraja V, Mudalige T, Wijamuni N, Rathnayake N, Haddadi M, Phiri P, Eleje GU, \nElneil S: Reproductive Health and Brain Function: An Integrative Review of the Neurological \nBurden of Common Gynaecological Conditions. 2025. 10.20944/ preprints 202509.0728.v1.\n35. Di Michele S, Camoglio C, Chieppa P, Incognito GG, Caiazzo A, Cabras A, Picci F, Angioni S: \nEndometriosis and eating disorders: epidemiology, shared neurobiology, and clinical \nimplications. Archives of Gynecology and Obstetrics 2026, 313(1):58.\n36. Liu Q, Li Z, Li S, Li Y, Pan H, Tao Y: Vitamin D3 as an immunomodulatory agent: molecular \nmechanisms, clinical translation, and precision therapeutic strategies. Frontiers in \nImmunology 2026, 17:1770141.\n37. Wang J, Mao X, Zhu L, Zhang X: Unravelling the Intricate Link: Mast Cells and Estrogen-Induced \nPain Sensitization in Endometriosis. Int. J. Biol. Sci. 2025, 21(13):5891.\nFigure 1: Flow chart of the study\nEnrollment\nAssessed for eligibility (n= 87)\nExcluded (n= 21)\n•Unwillingness to participate (n= 4)\n•Comorbid conditions (n=10)\n•Daily intake of supplements \ncontaining vitamin D (n=7)\nRandomization (n=66)\nAllocated to Placebo \nGroup (n=33)\nLost to follow-up (n= 0) Lost to follow-up (n= 0)\nAnalyzed (n= 33) Analyzed (n= 33)Analysis\nAllocated to Vitamin D Group\n (n=33)\nAllocation\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nTable 1: Socio-demographic characteristics of the participants (n=66)\na Standard deviation\nCharacteristic Vitamin D\n(n= 33)\nPlacebo\n(n= 33)\nMean (SDa) Mean (SD)\nAge (Year) 30.7 (7.0) 35.3 (5.8)\nSpouse age (Year) 35.4 (6.2) 39.0 (6.0)\nMarriage age (Year) 22.3 (4.2) 24.4 (3.7)\nFirst gravida age (Year) 25.0 (4.1) 26.4 (4.2)\nFirst menstruation age (Year) 12.7 (1.0) 13.0 (1.0)\nVitamin D level 27.8 (7.2) 29.7 (8.0)\nBMI 24.4 (3.5) 24.7 (2.6)\nNumber (Percent) Number (Percent)\nEducation\nIlliterate 1 (3.0) 0 (0.0)\nPrimary 1 (3.0) 7 (21.2)\nSecondary 2 (6.1) 2 (6.1)\nHigh school 8 (24.2) 5 (15.2)\nDiploma 11 (33.3) 6 (18.2)\nUniversity 10 (30.3) 28 (75.7)\nSpouse Education\nPrimary 2 (6.1) 0 (0.0)\nSecondary 1 (3.0) 4 (12.1)\nHigh school 0 (0.0) 3 (9.1)\nDiploma 16 (48.5) 13 (39.4)\nUniversity 14 (42.4) 13 (39.4)\nSpouse job\nUnemployed 0 (0.0) 1 (3.0)\nWorker 4 (12.1) 8 (24.2)\nEmployee 14 (42.4) 11 (33.3)\nFreelance 15 (45.5) 13 (39.4)\nIncome sufficiency\nInsufficient 0 (0.0) 7 (21.9)\nSomewhat sufficient 24 (80.0) 16 (50.0)\nCompletely sufficient 6 (20.0) 9 (28.1)\nInfertility history\nYes 4 (12.1) 4 (12.1)\nNo 29 (87.9) 29 (87.9)\nMedication type\nNo additional analgesics 14 (42.4) 11 (33.3)\nHormonal 15 (45.5) 22 (66.7)\nPain reliever 4 (12.1) 0 (0.0)\nMedication effect\nLow 6 (31.6) 5 (22.7)\nIntermediate 5 (26.3) 9 (40.9)\nHigh 8 (42.1) 8 (36.4)\nGravida\n1 15 (48.4) 17 (51.5)\n2 15 (48.4) 8 (24.2)\n3+ 1 (3.2) 8 (24.2)\nPara\n1 19 (61.3) 21 (63.6)\n2+ 12 (38.7) 12 (36.4)\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nTable 2: Comparison of the mean score of the ENDOPAIN-4D among study groups\nVariable N Vitamin D\nMean (SD†)  \nN Placebo\nMean (SD†) \nMean Difference (95% \nConfidence Interval)\nP-valueb\nENDOPAIN-4D (Score range: 0 to 210)\nUsual level of pain\nBefore intervention 33 81.5 (32.9) 33 76.1 (35.2) 5.4 (-11.3 to 22.2) 0.521\nAfter intervention 33 38.8 (6.5) 33 50.1 (7.4) -11.3 (-26.2 to 3.5) 0.136\nWorst level of pain\nBefore intervention 33 42.6 (19.7) 33 40.7 (20.1) 1.9 (-7.8 to 11.7) 0.698\nAfter intervention 33 23.6 (3.1) 33 25.6 (3.5) -2.0 (-9.0 to 4.9) 0.492\n† Standard Deviation; b Generalized Linear Model\nThe independent t-test was used before the intervention and Generalized Linear Model after the intervention by adjusting baseline \nvalues and socio-demographic characteristics with SMD>0.1.\nTable 3: Comparison of the mean score of the VAS score among study groups\nVariable N Vitamin D\nMean (SD†)  \nN Placebo\nMean (SD†) \nMean Difference (95% \nConfidence Interval)\nP-valueb\nVAS (Score range: 0 to 10)\nBefore intervention 33 8.12 (1.5) 33 8.3 (1.6) -0.2 (-1.0 to 0.5) 0.496\nAfter intervention 33 5.8 (0.3) 33 6.2 (0.4) -0.3 (-1.3 to 0.5) 0.113\n† Standard Deviation; b General Linear Model\nThe independent t-test was used before the intervention and Generalized Linear Model after the intervention by adjusting baseline \nvalues and socio-demographic characteristics with SMD>0.1.\nTable 4: Comparison of the mean score of the EHP-30 among study groups\nVariable N Vitamin D\nMean (SD†)  \nN Placebo\nMean (SD†) \nMean Difference (95% \nConfidence Interval)\nP-valueb\nEHP-30 (Score range: 0 to 100)\nPain\nBefore intervention 33 52.6 (17.7) 33 52.9 (20.5) -0.2 (-9.7 to 9.1) 0.954\nAfter intervention 33 33.8 (3.6) 33 35.2 (4.1) -1.3 (-9.6 to 6.9) 0.577\nControl and powerlessness\nBefore intervention 33 43.8 (23.3) 33 44.6 (25.5) -0.8 (-12.9 to 11.1) 0.884\nAfter intervention 33 21.4 (4.3) 33 30.0 (4.8) -8.6 (-18.4 to 1.2) 0.143\nSocial support\nBefore intervention 33 31.6 (23.4) 33 24.8 (21.1) 6.8 (-4.1 to 17.7) 0.219\nAfter intervention 33 10.4 (3.9) 33 13.8 (4.4) -3.4 (-12.4 to 5.6) 0.625\nEmotional well-being\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\nBefore intervention 33 38.0 (23.1) 33 33.0 (19.5) 4.9 (-5.6 to 15.4) 0.354\nAfter intervention 33 19.1 (2.9) 33 19.9 (3.3) -0.8 (-7.6 to 5.9) 0.814\nSelf-image\nBefore intervention 33 20.4 (23.8) 33 20.4 (22.1) 0.0 (-11.3 to 11.3) 1.000\nAfter intervention 33 7.3 (3.5) 33 1.6 (3.9) -3.3 (-11.3 to 4.7) 0.242\n† Standard Deviation; b Generalized Linear Model\nThe independent t-test was used before the intervention and Generalized Linear Model after the intervention by adjusting baseline \nvalues and socio-demographic characteristics with SMD>0.1.\nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS","source_license":"public-domain-us","license_restricted":false}