Adjunctive vitamin D supplementation in women receiving dienogest therapy for endometriosis: a randomized controlled trial

Background: Endometriosis is a chronic, estrogen-dependent inflammatory disease that causes debilitating pain and significantly impairs quality of life. Due to its anti-inflammatory and immunomodulatory properties, vitamin D has been proposed as a potential therapeutic agent, but clinical evidence remains inconsistent. This study aimed to evaluate the effect of vitamin D supplementation on pain symptoms and quality of life in women with endometriosis.

This randomized, double-blind, placebo-controlled clinical trial was conducted on 66 women with symptomatic endometriosis and vitamin D levels below 30 ng/mL. Participants were randomly assigned to either the intervention group (n=33), receiving 4000 IU of vitamin D every other day, or the control group (n=33), receiving a matching placebo for eight weeks. Crucially, all participants in both groups continued their standard dienogest (Verogest) therapy. The primary outcomes were pain severity, assessed using the Visual Analogue Scale (VAS) and the ENDOPAIN-4D questionnaire, and quality of life as secondary outcome, assessed with the Endometriosis Health Profile-30 (EHP-30). Outcomes were measured at baseline and after the eight-week intervention.

All 66 participants completed the trial. Despite baseline imbalances in socio-demographic characteristics (quantified via standardized mean differences), adjusting for these variables as covariates did not yield significant differences between groups. Regarding the ENDOPAIN-4D scores, for the "usual level of pain," the post-intervention mean score was 38.8 (SD = 6.5) in the vitamin D group compared to 50.1 (SD = 7.4) in the placebo group (adjusted mean difference [AMD]: -11.3; 95% Confidence Interval [CI]: -26.2 to 3.5; P = 0.136). For the "worst level of pain," the post-intervention mean score was 23.6 (SD = 3.1) in the vitamin D group and 25.6 (SD = 3.5) in the placebo group (AMD: -2.0; 95% CI: -9.0 to 4.9; P = 0.492). The adjusted mean post- ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS intervention VAS score was 5.8 (SD = 0.3) in the vitamin D group versus 6.2 (SD = 0.4) in the placebo group (AMD: -0.3; 95% CI: -1.3 to 0.5; P = 0.113). No significant differences were observed in the ENDOPAIN-4D scores or in any of the five domains of the EHP-30 questionnaire (all P > 0.05).

In women under hormonal suppression with dienogest, eight weeks of adjunctive vitamin D did not yield statistically or clinically significant improvements in pain or quality of life. These null findings likely reflect a "floor effect" from the baseline hormonal therapy and are limited by the lack of biochemical confirmation of repletion. These findings are specific to adjunctive use and may not reflect the potential of vitamin D as a monotherapy.

Vitamin D, Endometriosis, Pain, Quality of Life Trial registration: Iranian Registry of Clinical Trials (IRCT): IRCT20120718010324N78. Date of first registration: 2023-05-25. URL: https://irct.behdasht.gov.ir/trial/68131

Endometriosis is a chronic, estrogen-dependent inflammatory condition characterized by the presence of endometrial-like glands and stroma outside the uterine cavity [1]. While most commonly found in the pelvic peritoneum and ovaries, lesions can occur at extra-pelvic sites, including the bowel and diaphragm [2]. The disease manifests through a spectrum of symptoms, most notably dysmenorrhea, dyspareunia, and chronic pelvic pain, but it is also a primary driver of female infertility and adhesion-related complications [3]. It affects approximately 10% (190 million) of women and girls of reproductive age [4]. Clinical management traditionally prioritizes medical therapy as first-line treatment, with surgical intervention reserved for cases of medical failure, deep-seated endometriomas, or compressive symptoms [5, 6]. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS In contemporary clinical practice, the diagnostic paradigm has shifted. While laparoscopy with histological confirmation remains the gold standard [7], current international guidelines from ESHRE (2022) and NICE now advocate for a clinical diagnosis based on history, physical examination, and high-quality imaging (ultrasound or MRI) in the majority of cases; laparoscopy is no longer strictly required for the initiation of treatment [8, 9]. The exact etiopathogenesis of endometriosis remains complex and multifactorial. Beyond the classical theory of retrograde menstruation [10], research highlights the roles of inflammation, immune dysregulation, and oxidative stress in lesion survival and proliferation [11, 12]. Mechanistically, oxidative stress triggers pathways such as the Mitogen-Activated Protein (MAP) kinase and ERK pathways, which enhance the survival of ectopic tissue [13]. Furthermore, emerging evidence suggests that symptom severity is modulated not only by peripheral inflammation but also by central neurobiological and psychosocial factors, including shared neurobiology with other comorbid conditions [14, 15]. Vitamin D, a fat-soluble secosteroid, has emerged as a potential modulator of these pathways [16]. Endogenous synthesis occurs primarily in the lower layers of the epidermis, where UVB radiation facilitates the conversion of 7-dehydrocholesterol into pre-vitamin D3, which is subsequently hydroxylated in the liver and kidneys to its active form, calcitriol [17]. Calcitriol binds to vitamin D receptors (VDR) expressed throughout the female reproductive system, influencing gene transcription related to immune function and inflammation [18, 19]. The biological plausibility for vitamin D in endometriosis is supported by preclinical data. In murine models, vitamin D3 administration has demonstrated a 48.8% reduction in the area of endometriotic lesions and a significant decrease in fibrosis [20]. In vitro, vitamin D3 has been shown to suppress interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) responses in human ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS endometrial stromal cells [21]. Despite these promising findings, clinical evidence remains inconsistent. Given the high prevalence of vitamin D deficiency in this population and the need for non-hormonal adjuncts, this study aimed to evaluate whether vitamin D supplementation provides additional benefit for pain (primary outcome) and quality of life (secondary outcome) in women already receiving standard dienogest therapy.

Type of study and participants This study was a double-blind, randomized clinical trial conducted at the Al-Zahra educational and therapeutic center between 2023 and 2024. Crucially, as the primary aim was to assess the effect of Vitamin D correction, the study population was composed exclusively of women with confirmed vitamin D insufficiency (25-OH vitamin D < 30 ng/mL). Inclusion and Exclusion Criteria The inclusion criteria for participation were as follows: 1. A confirmed diagnosis of endometriosis by a specialist gynecologist based on a combination of clinical history, physical examination, and imaging (Transvaginal Ultrasound or MRI) according to ESHRE 2022 guidelines, or prior laparoscopic confirmation. 2. Presence of symptomatic endometriosis (VAS ≥ 4). 3. Maintenance of a stable regimen of 2 mg daily dienogest (Verogest) for at least three months prior to enrollment. This duration was selected to ensure that any initial therapeutic response to hormonal therapy had plateaued, thereby allowing for the assessment of Vitamin D as a true adjunctive (add-on) therapy. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS 4. Marital status (married and sexually active), required for the accurate assessment of the dyspareunia domain within the ENDOPAIN-4D instrument. 5. Serum vitamin D level < 30 ng/mL. Exclusion criteria included a history of regular vitamin D supplementation within the past three months. Furthermore, patients with comorbid conditions such as diabetes, thyroid disorders, hypertension, hyperprolactinemia, or Cushing’s syndrome were excluded. This was necessary to minimize confounding variables, as these metabolic and endocrine disorders can independently alter systemic inflammatory markers, calcium metabolism, and the subjective perception of chronic pain. Study Groups and Intervention Eligible participants were randomly assigned to one of two groups: the intervention group, which received 4000 IU of vitamin D every other day, and the control group, which received a placebo following the same schedule. It is a key component of this study that all patients in both groups continued to receive their standard routine treatment, which consisted of a daily 2 mg dose of dienogest (Verogest). Sample size The sample size for this study was calculated using G-Power software, based on the variable of painful symptoms of endometriosis. According to the findings of a study by Alizadeh et al. [22] concerning this variable, and considering M1= 6.60 (mean score of painful menstrual symptoms), M2= 5.28 (assuming a 20% reduction in the score following the intervention), SD1 = SD2 = 1.77, a two-sided α of 0.05, and a power of 80%, the required sample size was calculated to be 30 ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS individuals. Factoring in a potential 10% attrition rate, the final sample size was considered 33 participants per group. Sampling The trial was conducted on women referred to the Al-Zahra educational and therapeutic hospital in Tabriz. Sampling commenced after obtaining approval from the Ethics Committee and the Vice- Chancellor for Research and Technology of Tabriz University of Medical Sciences, and after the trial was registered with the Iranian Registry of Clinical Trials (IRCT). All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the Helsinki declaration and its amendments. The study was performed on a total of 66 women with endometriosis attending the gynecology clinic at Al-Zahra hospital. An initial convenience sampling method was employed. The researcher approached potential participants at the Al-Zahra therapeutic centers whose endometriosis diagnosis was confirmed by a collaborating gynecologist. The objectives and procedures of the study were explained, and individuals were assessed against the eligibility criteria. For those who were eligible and willing to participate, written informed consent was obtained. Subsequently, participants completed a socio-demographic questionnaire, the Painful Endometriosis Symptoms questionnaire (ENDOPAIN 4D), the Endometriosis Health Profile-30 (EHP-30) questionnaire, and the Visual Analogue Scale (VAS) before being allocated to a group. A blood sample was also collected to measure serum vitamin D levels. Only individuals whose serum vitamin D level was reported to be less than 30 ng/mL were enrolled in the study. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Participants were provided with a Visual Analog Scale (VAS) and instructed to mark their pain severity during the first three days of their next menstrual cycle and to begin their assigned medication on the fourth day of that cycle. They were also asked to continue recording their pain severity on the VAS scale during the first three days of the following two menstrual cycles. Eight weeks after the start of the intervention, the ENDOPAIN 4D and EHP-30 questionnaires were completed again by the participants. This follow-up assessment was conducted between days 7 and 12 of the menstrual cycle. Randomization and Allocation Concealment Participants who met the inclusion criteria were randomly assigned to either the intervention group or the control group using a block randomization method. The randomization sequence was generated by an independent person using random block sizes of four and six to ensure a balanced distribution of participants between the two groups throughout the recruitment period. A 1:1 allocation ratio was used. To ensure allocation concealment, the pharmacy prepared the study medication in sequentially numbered, identical bottles. The bottles containing either the vitamin D or placebo tablets were the same in appearance, size, and labeling. Each bottle was labelled with a unique participant number corresponding to the randomization list. Intervention The intervention group received 4000 IU vitamin D tablets, and the control group received a placebo; both were administered every other day for eight weeks. The vitamin D and placebo tablets, manufactured by Dana Pharmaceutical Company, were identical in appearance. Each participant received a sealed envelope containing their assigned medication at the beginning of the intervention and was informed that the next follow-up would be eight weeks later. Throughout the ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS eight-week intervention period, participants received a weekly phone call to remind them to take their medication and to complete a medication adherence checklist. Participants were also instructed to report any side effects, which were assessed via a checklist. In the event of a serious adverse event, the intervention was discontinued for that individual, though they were included in the final analysis. It is important to note that all patients continued to receive their routine treatment, which consisted of 2 mg of Verogest daily. Data Collection Tools Data for this study were collected by the researcher through structured interviews and self- administered questionnaires. The following instruments were used: Eligibility Criteria Checklist: This checklist was used to screen potential participants based on the predefined inclusion and exclusion criteria to ensure they were eligible for allocation into the study groups. Socio-Demographic Questionnaire: This researcher-developed questionnaire gathered data on participants' background characteristics, including age, level of education, obstetric history, occupation, and household income. Endometriosis Health Profile-30 (EHP-30): This disease-specific questionnaire was used to measure the quality of life of patients with endometriosis. The EHP-30 consists of 30 items across five core domains: pain (11 items), control and powerlessness (6 items), emotional well-being (6 items), social support (4 items), and self-image (3 items). All items were rated on a 5-point Likert scale, where a score of 1 represented the best health status and 5 represented the worst. The raw scores for each domain were transformed to a scale of 0 to 100, where 0 indicates the best possible quality of life and 100 indicates the worst. The EHP-30 does not provide a single total score; ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS instead, a separate score is calculated for each domain. This instrument was originally designed and validated by Jones et al. at the University of Oxford in 2001 [23, 24] and has been validated in multiple countries. The Persian version was validated by Nojomi et al. in 2011, demonstrating high internal consistency with Cronbach's alpha coefficients ranging from 0.80 to 0.93 [25]. ENDOPAIN-4D Questionnaire: This instrument was used to assess pelvic and gynecological pain symptoms. It contains 21 items divided into four domains: spontaneous pelvic pain (10 items), pain during sexual intercourse (dyspareunia) (3 items), bowel pain and related symptoms during menstruation (3 items), and other symptoms (5 items), including pain during urination, right shoulder pain during menstruation, and infertility. The psychometric properties of this instrument were previously assessed and confirmed by the principal investigator in a separate study [26]. Visual Analogue Scale (VAS): The VAS was used to measure pain intensity. It consists of a 10 cm horizontal line anchored by "no pain" at the 0 cm mark and "unbearable pain" at the 10 cm mark. Pain scores were interpreted as follows: 0 indicated no pain, 1–3 as mild pain, 4–6 as moderate pain, 7–9 as severe pain, and a score above 9 as very severe pain. The VAS is a standard and widely used tool for pain assessment in research [27]. Medication Adherence Checklist: Participants used this checklist to self-report their intake of the assigned medication (vitamin D or placebo) on the scheduled days. Adverse Events Checklist: This checklist was used to systematically document any adverse events experienced by participants during the intervention period, including the nature and severity of the event. Data Analysis ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Data were analyzed using SPSS software (version 25.0) through both descriptive and inferential statistics. The normality of the distribution for quantitative variables was assessed using the Kolmogorov-Smirnov test. To evaluate the success of randomization and quantify the extent of baseline imbalances between groups, Standardized Mean Differences (SMDs) were calculated for all demographic and clinical variables. An SMD > 0.1 was considered a meaningful imbalance, necessitating a covariate-adjusted approach for the final analysis. An independent t-test compared baseline scores of outcomes between groups. After intervention, to account for the identified imbalances in socio-demographic characteristics, VAS scores (normally distributed), ENDOPAIN-4D, and EHP-30 scores (non-normally distributed) were analyzed using Generalized Linear Model. Results are presented as adjusted means and standard deviations, with adjusted mean differences (AMD) and 95% confidence intervals (CI) as effect sizes. A Bonferroni correction addressed multiplicity across EHP-30 and ENDOPAIN-4D domains. Intention-to-treat (ITT) analysis was performed, and a corrected p-value < 0.05 was considered statistically significant.

Participant Characteristics Initially, 87 women were assessed for eligibility. Of these, 21 were excluded for not meeting the inclusion criteria (n=17) or declining to participate (n=4). The remaining 66 participants were randomly allocated to either the vitamin D group (n=33) or the placebo group (n=33). Regarding adverse effects, one participant reported nausea, four reported dizziness, four experienced fatigue, nine had constipation, and one had diarrhea. All reported conditions were mild, and no serious ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS adverse events were noted. All participants completed the eight-week follow-up, and their data were included in the final analysis (Figure 1). The baseline socio-demographic and clinical characteristics are presented in Table 1. The mean age of participants was 30.7 (SD=7.0) years in the vitamin D group and 35.3 (SD=5.8) years in the placebo group. Mean BMI was 24.4 (SD=3.5) in the vitamin D group and 24.7 (SD=2.6) in the placebo group. Evaluation of baseline comparability using Standardized Mean Differences (SMD) identified several imbalances, most notably in age (SMD=0.7). Additionally, various socio- demographic characteristics with SMDs ranging from 0.2 to 0.5 exceeded the pre-specified threshold (>0.1) and were therefore incorporated as covariates in the adjusted analysis. Pain Symptoms (ENDOPAIN-4D and VAS) For the ENDOPAIN-4D scores (Table 2), after adjusting for baseline values and socio- demographic characteristics with SMD > 0.1, no statistically significant difference was found between groups. For the "usual level of pain," the post-intervention mean score was 38.8 (SD=6.5) in the vitamin D group compared to 50.1 (SD=7.4) in the placebo group (AMD: -11.3, 95% CI: - 26.2 to 3.5; P=0.136). Similarly, for the "worst level of pain," the post-intervention mean score was 23.6 (SD=3.1) in the vitamin D group and 25.6 (SD=3.5) in the placebo group (AMD: -2.0, 95% CI: -9.0 to 4.9; P=0.492). Regarding the VAS score for pain intensity (Table 3), the mean score at baseline was 8.12 (SD=1.5) in the vitamin D group and 8.3 (SD=1.6) in the placebo group. After the eight-week intervention and adjusting for the pre-specified covariates, the mean VAS score was 5.8 (SD=0.3) in the vitamin D group and 6.2 (SD=0.4) in the placebo group. This difference was not statistically significant (AMD: -0.3, 95% CI: -1.3 to 0.5; P=0.113). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Quality of Life (EHP-30) The quality of life scores across five domains are detailed in Table 4. After adjusting for baseline scores and prognostic variables, no statistically significant differences were observed between the vitamin D and placebo groups in any domain (all P > 0.05): •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; P=0.577). •Control and powerlessness: Vit D 21.4 (SD=4.3) vs. Placebo 30.0 (SD=4.8); (AMD: -8.6, 95% CI: -18.4 to 1.2; P=0.143). •Social support: Vit D 10.4 (SD=3.9) vs. Placebo 13.8 (SD=4.4); (AMD: -3.4, 95% CI: -12.4 to 5.6; P=0.625). •Emotional well-being: Vit D 19.1 (SD=2.9) vs. Placebo 19.9 (SD=3.3); (AMD: -0.8, 95% CI: -7.6 to 5.9; P=0.814). •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; P=0.242). To evaluate the impact of baseline imbalances, a sensitivity analysis compared the crude and adjusted models. The lack of statistical significance persisted across all primary and secondary outcomes after adjustment, confirming that the initial demographic differences did not substantively bias the study findings.

This randomized, double-blind, placebo-controlled trial evaluated the adjunctive potential of vitamin D3 for managing endometriosis-associated pain and quality of life. Our findings indicate that 4000 IU of vitamin D every other day does not provide a statistically or clinically significant ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS benefit when added to a stable regimen of dienogest. By utilizing adjusted ANCOVA models, we were able to account for the notable baseline imbalances in age and education. This rigorous approach confirmed that the improvements observed across both study arms were primarily a function of the underlying hormonal therapy rather than the nutraceutical intervention. The divergence between our results and studies reporting positive outcomes [28, 29] likely stems from our specific focus on vitamin D as an add-on therapy. In this context, a "floor effect" appears to be the most plausible explanation for the null findings. When patients are already stable on a potent progestin like dienogest, the inflammatory milieu may be sufficiently suppressed to a degree that an additional anti-inflammatory agent yields no detectable change. This is further substantiated by our analysis of the Minimal Clinically Important Difference (MCID). The adjusted mean differences for the VAS and EHP-30 domains remained well below the thresholds of 1.2 point and 11.1 points, respectively [30, 31], suggesting that even the minor fluctuations observed were clinically negligible. While the anti-proliferative and anti-fibrotic potential of vitamin D is well-documented in laboratory settings [20, 21], our trial highlights the challenges of clinical translation. One significant barrier may be the lack of phenotypic stratification. Endometriosis is a heterogeneous disease, and the sensitivities of the Vitamin D Receptor (VDR) may vary significantly between peritoneal, ovarian, and deep infiltrating (DIE) lesions [32, 33]. Without a stratified analysis, any benefit specific to a particular disease subtype may have been obscured. Furthermore, the selection of a fixed dosing regimen without titration based on individual baseline levels may have contributed to a heterogeneous therapeutic response across the trial. Finally, the management of endometriosis-related pain requires a broader conceptual framework that moves beyond peripheral inflammation. Our findings likely reflect a mechanistic mismatch in ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS this regard. As highlighted in contemporary literature, symptom severity is deeply intertwined with central neurobiological sensitization and shared neuropsychological comorbidities [34, 35]. While Vitamin D acts primarily as a peripheral anti-inflammatory and immunomodulator [36], chronic pain in endometriosis is increasingly recognized as a state of central sensitization [37], where the central nervous system maintains a state of high reactivity despite peripheral changes. Consequently, a peripheral intervention like Vitamin D may be insufficient to reverse the complex neurobiological remodeling and "pain memory" established in long-term endometriosis patients. When pain is shaped by these complex central determinants, a nutraceutical approach targeting only the inflammatory component may be insufficient to achieve a meaningful clinical shift. This emphasizes the need for a more personalized, multidisciplinary approach in future research, where interventions are tailored not only to the physical stage of the disease but also to the broader neurobiological and biochemical profile of the patient. Strengths and Limitations The strengths of this trial include its randomized, double-blind design, a 100% participant retention rate, and the use of validated, disease-specific instruments (EHP-30 and ENDOPAIN-4D). However, several limitations warrant caution. First, the concurrent use of dienogest likely created a "floor effect," where the high efficacy of hormonal therapy masked potential incremental benefits from Vitamin D. Second, the eight-week duration may be insufficient to elicit significant immunomodulatory changes. Crucially, the absence of post-intervention serum 25(OH)D measurements prevents confirmation of biochemical repletion, which is a major caveat of this study. We cannot definitively confirm if the 4000 IU dose administered every other day was sufficient to reach optimal therapeutic ranges in all participants, particularly given the potential for malabsorption. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Generalizability is limited by the lack of stratification by endometriosis subtype (e.g., DIE vs. ovarian) and the inclusion of only married, sexually active women. Extrinsic variables like diet and UV exposure were not controlled. Finally, our null results must be viewed through a biopsychosocial lens; endometriosis pain is heavily modulated by central neurobiological and psychosocial factors. A single nutraceutical intervention may have limited detectable impact when symptoms are shaped by such complex comorbidities.

In conclusion, this trial found that the administration of adjunctive Vitamin D3 (4000 IU every other day) to standard dienogest therapy for eight weeks did not result in a statistically or clinically significant improvement in pain symptoms or quality of life compared to dienogest alone. These findings suggest a notable "floor effect," where the high efficacy of baseline hormonal therapy likely masked any incremental benefits of the nutraceutical intervention. Importantly, as our conclusions are based on the administration of the supplement rather than the achievement of a confirmed serum 25(OH)D threshold, the absence of biochemical confirmation of repletion means these null results cannot definitively rule out the biological potential of Vitamin D. While our study does not support the routine use of short-term Vitamin D as an adjunctive therapy for women already stable on dienogest, its role as a standalone treatment or over longer durations in specific endometriosis phenotypes warrants further investigation in more robustly powered and stratified clinical trials. Abbreviations DIE Deep Infiltrating Endometriosis EHP-30 Endometriosis Health Profile-30 ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS ENDOPAIN-4D Endometriosis Pain 4D Questionnaire ERK Extracellular Signal-related Kinase MAP Mitogen-Activated Protein VAS Visual Analogue Scale Authors’ contributions All authors contributed to the conception and design of the study and participated in the revision of the manuscript. FS authored the manuscript under the direct supervision of MM, the Corresponding Author. MM conducted the statistical analysis. All authors reviewed and approved the final version of the manuscript. Funding Tabriz University of Medical Sciences funded this research but did not participate in its design, execution, or manuscript submission decisions. Data availability The data used to support the findings of this study can be obtained from the corresponding author upon request. Declarations Ethics approval and consent to participate This study received approval from the Ethics Committee of Tabriz University of Medical Sciences under the code IR.TBZMED.REC.1402.109. Before participating, the researcher provided a detailed explanation of the study's objectives and methodologies to all participants. Informed ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS written consent was obtained from each participant, confirming their voluntary involvement and understanding of the study's purpose and procedures. Competing interests N/A Acknowledgments The authors appreciate the assistance and cooperation of the participants of this study.

1. Arafah M, Rashid S, Akhtar M: Endometriosis: a comprehensive review. Advances in anatomic pathology 2021, 28(1):30-43. 2. Charatsi D, Koukoura O, Ntavela IG, Chintziou F, Gkorila G, Tsagkoulis M, Mikos T, Pistofidis G, Hajiioannou J, Daponte A: Gastrointestinal and urinary tract endometriosis: a review on the commonest locations of extrapelvic endometriosis. Advances in medicine 2018, 2018(1):3461209. 3. Gruber TM, Mechsner S: Pathogenesis of endometriosis: the origin of pain and subfertility. Cells 2021, 10(6):1381. 4. Xu S, Zhang Y, Ye P, Huang Q, Wang Y, Zhang Y, Yang C, Ding J: Global, regional, and national burden of endometriosis among women of childbearing age from 1990 to 2021: a cross- sectional analysis from the 2021 global burden of disease study. Int. J. Surg. 2025, 111(9):5927- 5940. 5. Mick I, Freger SM, van Keizerswaard J, Gholiof M, Leonardi M: Comprehensive endometriosis care: a modern multimodal approach for the treatment of pelvic pain and endometriosis. Therapeutic advances in reproductive health 2024, 18:26334941241277759. 6. Ortega-Gutiérrez M, Muñoz-Gamez A, Girón-Prieto MdlS: Primary care approach to endometriosis: diagnostic challenges and management Strategies—A narrative review. J. Clin. Med. 2025, 14(13):4757. 7. Pascoal E, Wessels J, Aas‐Eng M, Abrao MS, Condous G, Jurkovic D, Espada M, Exacoustos C, Ferrero S, Guerriero S: Strengths and limitations of diagnostic tools for endometriosis and relevance in diagnostic test accuracy research. Ultrasound in obstetrics & gynecology 2022, 60(3):309- 327. 8. Becker CM, Bokor A, Heikinheimo O, Horne A, Jansen F, Kiesel L, King K, Kvaskoff M, Nap A, Petersen K: ESHRE guideline: endometriosis. Human reproduction open 2022, 2022(2):hoac009. 9. Norton W, Holloway D: Understanding the NICE guidance on endometriosis. Practice nursing 2020, 31(1):8-16. 10. Hegazy AA: A new look at the theoretical causes of endometriosis: narrative review. Int. J. Reprod. BioMed. 2024, 22(5):343. 11. Ansariniya H, Yavari A, Javaheri A, Zare F: Oxidative stress‐related effects on various aspects of endometriosis. Am. J. Reprod. Immunol. 2022, 88(3):e13593. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS 12. Chen S, Liu Y, Zhong Z, Wei C, Liu Y, Zhu X: Peritoneal immune microenvironment of endometriosis: Role and therapeutic perspectives. Frontiers in immunology 2023, 14:1134663. 13. Liu Y, Wang J, Zhang X: An update on the multifaceted role of NF-kappaB in endometriosis. Int. J. Biol. Sci. 2022, 18(11):4400. 14. Alexander KG, Nordahl EJ, Newton TI, Georgiou C: Neuropsychiatric and cognitive manifestations of endometriosis: insights into the ‘endometriosis brain’. Academia Mental Health and Well- Being 2025, 2(4). 15. Pszczołowska M, Walczak K, Kołodziejczyk W, Kozłowska M, Kozłowski G, Gachowska M, Leszek J: Understanding deep endometriosis: from molecular to neuropsychiatry dimension. Int. J. Mol. Sci. 2025, 26(2):839. 16. Voiculescu VM, Nelson Twakor A, Jerpelea N, Pantea Stoian A: Vitamin D: beyond traditional roles—insights into its biochemical pathways and physiological impacts. Nutrients 2025, 17(5):803. 17. Raczyk M, Carlberg C: From Sunlight to Signaling: Evolutionary Integration of Vitamin D and Sterol Metabolism. Metabolites 2026, 16(1):74. 18. Grzesiak M, Tchurzyk M, Socha M, Sechman A, Hrabia A: An overview of the current known and unknown roles of vitamin D3 in the female reproductive system: lessons from farm animals, birds, and fish. Int. J. Mol. Sci. 2022, 23(22):14137. 19. Oliynyk S: The regulatory and pleiotropic role of vitamin D. Regulatory Mechanisms in Biosystems 2026, 17(2):1-8. 20. Abbas MA, Taha MO, Disi AM, Shomaf M: Regression of endometrial implants treated with vitamin D3 in a rat model of endometriosis. Eur. J. Pharmacol. 2013, 715(1-3):72-75. 21. Almassinokiani F, Khodaverdi S, Solaymani-Dodaran M, Akbari P, Pazouki A: Effects of Vitamin D on Endometriosis-Related Pain: A Double-Blind Clinical Trial. Med Sci Monit 2016, 22:4960- 4966. 22. Alizad S, Mirghafourvand M, Oskouei BS, Bani S: Endometriosis painful symptoms and its relationship with quality of life in women referring to educational centers of Tabriz University of Medical Sciences. Current Women's Health Reviews 2024, 20(1):3-12. 23. Jones G, Kennedy S, Barnard A, Wong J, Jenkinson C: Development of an endometriosis quality-of- life instrument: The Endometriosis Health Profile-30. Obstetrics & Gynecology 2001, 98(2):258-264. 24. Khong S-Y, Lam A, Luscombe G: Is the 30-item Endometriosis Health Profile (EHP-30) suitable as a self-report health status instrument for clinical trials? Fertility and sterility 2010, 94(5):1928-1932. 25. Nojomi M, Bijari B, Akhbari R, Kashanian M: The assessment of reliability and validity of Persian version of the endometriosis health profile (EHP-30). Iran. J. Med. Sci. 2011, 36(2):84. 26. Ahmadpour P, Jahangiry L, Bani S, Iravani M, Mirghafourvand M: Validation of the Iranian version of the ENDOPAIN-4D questionnaire for measurement of painful symptoms of endometriosis. J. Obstet. Gynaecol. 2022, 42(6):2341-2348. 27. Phumdoung S, Rattanaparikonn A, Maneechot K: Pain during the first stage of labor. 2010. 28. Mehdizadehkashi A, Rokhgireh S, Tahermanesh K, Eslahi N, Minaeian S, Samimi M: The effect of vitamin D supplementation on clinical symptoms and metabolic profiles in patients with endometriosis. Gynecological Endocrinology 2021, 37(7):640-645. 29. Nodler JL, DiVasta AD, Vitonis AF, Karevicius S, Malsch M, Sarda V, Fadayomi A, Harris HR, Missmer SA: Supplementation with vitamin D or ω-3 fatty acids in adolescent girls and young women with endometriosis (SAGE): a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr. 2020, 112(1):229-236. 30. Almendra R, Egas Araujo E, Sousa DN, Nogueira-Silva C: Translation, cultural adaptation, and validation of the Portuguese version of ENDOPAIN-4D questionnaire. J Psychosom Obstet Gynaecol. 2026, 47(1):2643521. 31. Koo M, Yang S-W: Visual Analogue Scale. Encyclopedia 2025, 5(4):190. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS 32. Imperiale L, Nisolle M, Noël J-C, Fastrez M: Three types of endometriosis: pathogenesis, diagnosis and treatment. State of the art. J. Clin. Med. 2023, 12(3):994. 33. Saunders PT, Horne AW: Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell 2021, 184(11):2807-2824. 34. Delanerolle G, Pathiraja V, Mudalige T, Wijamuni N, Rathnayake N, Haddadi M, Phiri P, Eleje GU, Elneil S: Reproductive Health and Brain Function: An Integrative Review of the Neurological Burden of Common Gynaecological Conditions. 2025. 10.20944/ preprints 202509.0728.v1. 35. Di Michele S, Camoglio C, Chieppa P, Incognito GG, Caiazzo A, Cabras A, Picci F, Angioni S: Endometriosis and eating disorders: epidemiology, shared neurobiology, and clinical implications. Archives of Gynecology and Obstetrics 2026, 313(1):58. 36. Liu Q, Li Z, Li S, Li Y, Pan H, Tao Y: Vitamin D3 as an immunomodulatory agent: molecular mechanisms, clinical translation, and precision therapeutic strategies. Frontiers in Immunology 2026, 17:1770141. 37. Wang J, Mao X, Zhu L, Zhang X: Unravelling the Intricate Link: Mast Cells and Estrogen-Induced Pain Sensitization in Endometriosis. Int. J. Biol. Sci. 2025, 21(13):5891. Figure 1: Flow chart of the study Enrollment Assessed for eligibility (n= 87) Excluded (n= 21) •Unwillingness to participate (n= 4) •Comorbid conditions (n=10) •Daily intake of supplements containing vitamin D (n=7) Randomization (n=66) Allocated to Placebo Group (n=33) Lost to follow-up (n= 0) Lost to follow-up (n= 0) Analyzed (n= 33) Analyzed (n= 33)Analysis Allocated to Vitamin D Group (n=33) Allocation ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Table 1: Socio-demographic characteristics of the participants (n=66) a Standard deviation Characteristic Vitamin D (n= 33) Placebo (n= 33) Mean (SDa) Mean (SD) Age (Year) 30.7 (7.0) 35.3 (5.8) Spouse age (Year) 35.4 (6.2) 39.0 (6.0) Marriage age (Year) 22.3 (4.2) 24.4 (3.7) First gravida age (Year) 25.0 (4.1) 26.4 (4.2) First menstruation age (Year) 12.7 (1.0) 13.0 (1.0) Vitamin D level 27.8 (7.2) 29.7 (8.0) BMI 24.4 (3.5) 24.7 (2.6) Number (Percent) Number (Percent) Education Illiterate 1 (3.0) 0 (0.0) Primary 1 (3.0) 7 (21.2) Secondary 2 (6.1) 2 (6.1) High school 8 (24.2) 5 (15.2) Diploma 11 (33.3) 6 (18.2) University 10 (30.3) 28 (75.7) Spouse Education Primary 2 (6.1) 0 (0.0) Secondary 1 (3.0) 4 (12.1) High school 0 (0.0) 3 (9.1) Diploma 16 (48.5) 13 (39.4) University 14 (42.4) 13 (39.4) Spouse job Unemployed 0 (0.0) 1 (3.0) Worker 4 (12.1) 8 (24.2) Employee 14 (42.4) 11 (33.3) Freelance 15 (45.5) 13 (39.4) Income sufficiency Insufficient 0 (0.0) 7 (21.9) Somewhat sufficient 24 (80.0) 16 (50.0) Completely sufficient 6 (20.0) 9 (28.1) Infertility history Yes 4 (12.1) 4 (12.1) No 29 (87.9) 29 (87.9) Medication type No additional analgesics 14 (42.4) 11 (33.3) Hormonal 15 (45.5) 22 (66.7) Pain reliever 4 (12.1) 0 (0.0) Medication effect Low 6 (31.6) 5 (22.7) Intermediate 5 (26.3) 9 (40.9) High 8 (42.1) 8 (36.4) Gravida 1 15 (48.4) 17 (51.5) 2 15 (48.4) 8 (24.2) 3+ 1 (3.2) 8 (24.2) Para 1 19 (61.3) 21 (63.6) 2+ 12 (38.7) 12 (36.4) ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Table 2: Comparison of the mean score of the ENDOPAIN-4D among study groups Variable N Vitamin D Mean (SD†) N Placebo Mean (SD†) Mean Difference (95% Confidence Interval) P-valueb ENDOPAIN-4D (Score range: 0 to 210) Usual level of pain Before intervention 33 81.5 (32.9) 33 76.1 (35.2) 5.4 (-11.3 to 22.2) 0.521 After intervention 33 38.8 (6.5) 33 50.1 (7.4) -11.3 (-26.2 to 3.5) 0.136 Worst level of pain Before intervention 33 42.6 (19.7) 33 40.7 (20.1) 1.9 (-7.8 to 11.7) 0.698 After intervention 33 23.6 (3.1) 33 25.6 (3.5) -2.0 (-9.0 to 4.9) 0.492 † Standard Deviation; b Generalized Linear Model The independent t-test was used before the intervention and Generalized Linear Model after the intervention by adjusting baseline values and socio-demographic characteristics with SMD>0.1. Table 3: Comparison of the mean score of the VAS score among study groups Variable N Vitamin D Mean (SD†) N Placebo Mean (SD†) Mean Difference (95% Confidence Interval) P-valueb VAS (Score range: 0 to 10) Before intervention 33 8.12 (1.5) 33 8.3 (1.6) -0.2 (-1.0 to 0.5) 0.496 After intervention 33 5.8 (0.3) 33 6.2 (0.4) -0.3 (-1.3 to 0.5) 0.113 † Standard Deviation; b General Linear Model The independent t-test was used before the intervention and Generalized Linear Model after the intervention by adjusting baseline values and socio-demographic characteristics with SMD>0.1. Table 4: Comparison of the mean score of the EHP-30 among study groups Variable N Vitamin D Mean (SD†) N Placebo Mean (SD†) Mean Difference (95% Confidence Interval) P-valueb EHP-30 (Score range: 0 to 100) Pain Before intervention 33 52.6 (17.7) 33 52.9 (20.5) -0.2 (-9.7 to 9.1) 0.954 After intervention 33 33.8 (3.6) 33 35.2 (4.1) -1.3 (-9.6 to 6.9) 0.577 Control and powerlessness Before intervention 33 43.8 (23.3) 33 44.6 (25.5) -0.8 (-12.9 to 11.1) 0.884 After intervention 33 21.4 (4.3) 33 30.0 (4.8) -8.6 (-18.4 to 1.2) 0.143 Social support Before intervention 33 31.6 (23.4) 33 24.8 (21.1) 6.8 (-4.1 to 17.7) 0.219 After intervention 33 10.4 (3.9) 33 13.8 (4.4) -3.4 (-12.4 to 5.6) 0.625 Emotional well-being ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Before intervention 33 38.0 (23.1) 33 33.0 (19.5) 4.9 (-5.6 to 15.4) 0.354 After intervention 33 19.1 (2.9) 33 19.9 (3.3) -0.8 (-7.6 to 5.9) 0.814 Self-image Before intervention 33 20.4 (23.8) 33 20.4 (22.1) 0.0 (-11.3 to 11.3) 1.000 After intervention 33 7.3 (3.5) 33 1.6 (3.9) -3.3 (-11.3 to 4.7) 0.242 † Standard Deviation; b Generalized Linear Model The independent t-test was used before the intervention and Generalized Linear Model after the intervention by adjusting baseline values and socio-demographic characteristics with SMD>0.1. 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