Vitamin D Deficiency and Pain Severity in Endometriosis: A Phenotype-Based Retrospective Cohort Study

Vitamin D Deficiency and Pain Severity in Endometriosis: A Phenotype-Based Retrospective Cohort Study | 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 Research Article Vitamin D Deficiency and Pain Severity in Endometriosis: A Phenotype-Based Retrospective Cohort Study Ismail Baglar, Fatih Sanlikan, Esra Keles, Aslı Sasmaz, Kubra Karakas Soylu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9190159/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 18 You are reading this latest preprint version Abstract Objective : This study aims to investigate the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and pain severity in women with surgically confirmed endometriosis, according to disease phenotypes (Ovarian Endometrioma [OE] and Deep Infiltrating Endometriosis [DIE]). Methods : This retrospective cohort study included 427 women with a histopathologically confirmed diagnosis of endometriosis. Patients were divided into two groups: OE (n = 231) and DIE (n = 196). Serum 25(OH)D levels were categorized as deficient (< 20 ng/mL), insufficient (20–30 ng/mL), and sufficient (≥ 30 ng/mL). The severity of dysmenorrhea, chronic pelvic pain, and dyspareunia was measured using the Visual Analog Scale (VAS). Confounding factors such as seasonal variability, age, and BMI were controlled for using multivariate analyses. Results : A significant negative correlation was found between serum 25(OH)D levels and all pain parameters (p < 0.001). Median VAS scores were significantly higher in the group with vitamin D deficiency compared to the group with sufficient levels [7.0 (6.0–8.0) vs 4.0 (3.0–5.0), p < 0.001]. In a multivariate linear regression analysis, low 25(OH)D levels were identified as an independent predictor of pain intensity (β = -0.28, p < 0.001). The most striking finding was a significant interaction between vitamin D levels and disease phenotype (Interaction p = 0.004); this indicated that the exacerbating effect of low vitamin D on pain was much more pronounced in patients with the DIE phenotype compared to the OE group. A threshold value of 15.4 ng/mL demonstrated 74% sensitivity in predicting severe pain (VAS ≥ 7). Conclusion : Low serum vitamin D levels are directly associated with the severity of pain related to endometriosis. The dramatic strengthening of this association, particularly in the DIE phenotype, suggests that vitamin D may act as a critical modulator in neuro-inflammatory mechanisms within deep infiltrating lesions. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Endometriosis is a chronic inflammatory disease affecting approximately 10% of women of reproductive age and most commonly presents with pelvic pain. Dysmenorrhea, chronic pelvic pain, and dyspareunia associated with is significantly impair patients’ quality of life. The fact that diagnostic delays often span 5–10 years further increases the clinical burden of the disease ( 1 ). Although the pathogenesis has not been fully elucidated, current data indicate that inflammation, immune dysfunction, and oxidative stress play a key role. The retrograde menstruation theory is not sufficient on its own; the fact that the disease does not develop in every woman despite similar exposure points to the importance of individual biological differences ( 2 ). Vitamin D has emerged as a molecule of interest in this context in recent years. It is thought to play a role in the pathophysiology of endometriosis due to its anti-inflammatory, immunomodulatory, and antiproliferative effects. Furthermore, the demonstration of vitamin D receptors in endometriotic tissues supports this association ( 3 ). However, the relationship between serum vitamin D levels and endometriosis is inconsistent in the literature. While some studies report that low 25(OH)D levels are associated with the presence and severity of the disease, others have failed to identify a significant association. These heterogeneous results indicate that the matter has not yet been fully clarified ( 4 , 5 ). When evaluating effects on pain, while it has been suggested that vitamin D may reduce pain symptoms by modulating inflammatory processes, the results of clinical studies are conflicting. In particular, effects on dysmenorrhea and dyspareunia vary across different studies ( 6 ). On the other hand, endometriosis is a heterogeneous disease, and clinical characteristics and pain profiles may show significant differences among different phenotypes (ovarian, deep infiltrative, and peritoneal). However, in most existing studies, this phenotypic distinction has not been sufficiently considered, which complicates the interpretation of results ( 7 ). Therefore, in this study, we aimed to evaluate the relationship between serum vitamin D levels and pain intensity in patients with endometriosis and to investigate whether this relationship varies according to disease phenotypes. It is believed that this approach will contribute to more targeted clinical implications by reducing the existing uncertainties in the literature. MATERIALS AND METHODS 1. Study Design and Patient Selection This study was designed as a single-center retrospective cohort study. Patients who underwent laparoscopic surgery and had a histopathologically confirmed diagnosis between January 2014 and December 2024 were included. To minimize the heterogeneous effect of surgical stress on pain perception, cases involving laparotomy or those undergoing diagnostic surgery only were excluded. All data were retrospectively obtained from the hospital information management system and electronic medical records. Inclusion criteria were defined as being between 18 and 45 years of age, having a diagnosis of endometriosis confirmed surgically and histopathologically, having a preoperative serum 25-hydroxyvitamin D [25(OH)D] level measured, and having a preoperative pain assessment recorded using the Visual Analog Scale (VAS). Patients with systemic diseases that could affect vitamin D metabolism (chronic kidney disease, liver disease, malabsorption syndromes), those with active infections or inflammatory diseases, those who had taken high-dose vitamin D supplements within the past 6 months, as well as daily multivitamin users taking more than 400 IU and those taking calcium supplements were excluded from the study. Additionally, patients using medications known to affect bone metabolism (glucocorticoids, antiepileptics, etc.) and those receiving hormonal therapy (oral contraceptives, GnRH- e analogs, etc.) were also excluded to ensure that baseline 25(OH)D levels were not influenced by external factors. Additionally, patients with incomplete or inconsistent data records were not included in the analysis. Demographic and clinical data (age, body mass index, obstetric history, surgical findings, and disease stage) were recorded for all patients using a standardized data form. Disease staging was performed according to the American Society for Reproductive Medicine (rASRM) classification. The study protocol was approved by the Ethics Committee of the Third-Level City Hospital (Decision No: 2026/010.99/25/7). The study was conducted in full compliance with the principles of the Declaration of Helsinki. 2. Variable Definitions and Measurements Serum 25-hydroxyvitamin D [25(OH)D] levels were obtained from venous blood samples collected as part of routine preoperative biochemical evaluation and measured using a standard immunoassay method at the same central laboratory. Blood samples for serum 25(OH)D levels were collected in the morning (08:00–10:00) following at least an 8-hour fast into standard vacuum tubes. Serum samples obtained after centrifugation were stored at -80°C until the day of analysis to minimize preanalytical variations. To account for seasonal fluctuations in 25(OH)D levels, the date of blood sample collection for each patient was recorded and categorized by season (Winter/Spring vs. Summer/Fall). Seasonal variability was included as a potential confounding factor in multivariate regression models. Serum 25(OH)D levels were classified into three groups in accordance with the Endocrine Society Clinical Practice Guidelines: <20 ng/mL 'deficiency,' 20–30 ng/mL 'insufficiency,' and ≥ 30 ng/mL 'sufficiency' (Holick et al., 2011). In the analyses, data were included in the model both based on this clinical categorical classification and as a continuous variable to preserve the biological gradient Patients’ preoperative pain scores (dysmenorrhea, chronic pelvic pain, dyspareunia) were recorded during the routine preoperative outpatient examination using a standardized form and by a clinician unaware of the surgical findings. This was retrospectively verified to prevent bias in the data collection process. Dysmenorrhea, chronic pelvic pain, and dyspareunia were assessed separately and rated on a scale of 0–10. Because different types of pain are pathophysiologically heterogeneous, a single total score was not created. Pain severity was assessed using a standard 10-cm Visual Analog Scale (VAS), a validated and widely used tool for measuring subjective pain intensity. Patients were asked to rate their pain on a scale ranging from 0 (no pain) to 10 (worst imaginable pain) ( 8 ). Analgesic usage patterns were recorded to objectively assess the clinical severity of the condition and its impact on the patient’s quality of life. Medication use was categorized based on the World Health Organization (WHO) Analgesic Ladder principles, according to frequency of use: “never used,” “intermittent/as-needed,” and “regular/daily.” Additionally, the pharmacological agents used were classified as nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids, and the depth of pain management was used as an independent variable in secondary analyses. Potential confounding variables were predefined. Age, body mass index (BMI), disease stage (rASRM), and serum CA125 levels were included in the analyses because they could be associated with both disease severity and pain. All variables were recorded using a standard data collection form, and data consistency was checked prior to analysis. Patients with missing data were excluded from the study; no data imputation method was used. 3. Phenotype Definition, Sample Characteristics Endometriosis phenotypes were classified according to the surgical-morphological criteria defined by Chapron et al. and widely accepted in the literature ( 7 ). Disease presentation was categorized according to the following hierarchical protocol based on laparoscopic findings and histopathological confirmation: Deep Infiltrating Endometriosis (DIE): Defined as solid endometriotic foci invading ≥ 5 mm below the peritoneal surface and involving the muscularis propria (bladder, bowel, ureter) or retroperitoneal structures. Ovarian Endometrioma (OE): Defined as the presence of isolated endometriotic cysts (thick-walled cystic structures containing internal echoes) without the detection of DIE foci or accompanying superficial peritoneal lesions during surgery. 4. Statistical Analysis Prior to analysis, data were examined for outliers using Z-scores and box-plot methods; clinically unexplained extreme values were excluded. The normal distribution of continuous variables was assessed using the Kolmogorov-Smirnov test and Q-Q plots. Data were presented as mean ± standard deviation (SD) or median (IQR) based on distribution characteristics; categorical variables were presented as frequency (%). For between-group comparisons, the Independent Samples t-test or the Mann-Whitney U test was used. Multivariate linear regression models were constructed to determine the independent effects of the variables. Parameters that had a p-value < 0.10 in univariate analysis and whose significance was established in the literature (age, BMI, rASRM stage) were included in the model. The risk of multicollinearity was excluded using a Variance Inflation Factor (VIF) threshold of < 3. An interaction term (Vitamin D × Phenotype) was added to the model to test for modification of the relationship between 25(OH)D levels and pain according to phenotype (OE vs. DIE). Additionally, the predictive performance of 25(OH)D levels for severe pain (VAS ≥ 7) was evaluated using ROC curve analysis and the Youden Index. The sample size was calculated to detect a 20% difference in the primary outcome with 80% power and a 5% Type I error rate (n = 427). All analyses were performed using SPSS v26.0 (IBM Corp., Armonk, NY, USA), and p < 0.05 was considered statistically significant. RESULTS 1. Patient Flow and Basic Demographic Characteristics A total of 512 patients were screened during the study period. Of the 512 screened patients, 85 were excluded from the study due to exclusion criteria [missing data (Vitamin D deficiency n = 34, missing VAS score n = 21), use of hormonal therapy (n = 18), and use of vitamin D supplementation (n = 12)], and the final analysis was completed with 427 patients. Of these, 231 were in the ovarian endometrioma (OE) group and 196 were in the deep infiltrating endometriosis (DIE) group. The process of forming the study population and the patient flow diagram are presented in detail in Fig. 1 . The demographic and clinical characteristics of the study population are presented in Table 1 . Table 1 Demographic and Clinical Characteristics by Group Variable OE (n = 231) DIE (n = 196) p-value Age (years) 31.8 ± 5.4 32.6 ± 5.7 0.12 BMI (kg/m²) 25.7 ± 3.8 26.1 ± 4.1 0.28 Gravidity 1 (0–3) 1 (0–2) 0.34 Parity 1 (0–2) 1 (0–2) 0.41 rASRM stage (III–IV, %) 62.3 78.5 0.01 Vitamin D (ng/mL) 19.6 ± 7.8 17.2 ± 6.9 0.02 CA125 (U/mL) 54 (28–96) 71 (35–128) 0.01 Abbreviations: BMI, body mass index; OE, ovarian endometrioma; DIE, deep infiltrating endometriosis; rASRM, revised American Society for Reproductive Medicine; CA125, cancer antigen 125. Continuous variables are presented as mean ± standard deviation or median (minimum–maximum), as appropriate. Categorical variables are expressed as number (percentage). Comparisons between groups were performed using appropriate parametric or non-parametric tests based on data distribution. In the analysis, the OE and DIE groups were found to be similar in terms of age, BMI, and obstetric characteristics (p > 0.05). In the comparison regarding disease stage, it was determined that 78.5% of patients in the DIE group were in advanced stages (Stage III-IV), and this proportion was significantly higher compared to the OE group (p = 0.01, Chi-square test). Although mean serum 25(OH)D levels were below the clinical deficiency threshold (< 20 ng/mL) in both groups, levels in the DIE group were significantly lower than those in the OE group (17.2 ± 6.9 vs 19.6 ± 7.8 ng/mL, p = 0.02). Similarly, CA125 levels were also found to be higher in the DIE group (p = 0.01). CA125 levels were found to be statistically significantly higher in the DIE group compared to the OE group [71 (35–128) vs 54 (28–96) U/mL, p = 0.01]. The distribution of pain scores and analgesic usage rates according to vitamin D levels are shown in Table 2 . Table 2 Comparison of Pain Scores According to Vitamin D Levels Variable < 20 ng/mL (n = 198) 20–30 ng/mL (n = 143) ≥ 30 ng/mL (n = 86) p-value VAS Dysmenorrhea 7.8 ± 1.6 6.9 ± 1.8 5.8 ± 1.9 < 0.001 VAS CPP 7.2 ± 1.7 6.4 ± 1.9 5.5 ± 2.0 < 0.001 VAS Dyspareunia 6.9 ± 1.8 6.1 ± 1.9 5.2 ± 2.1 < 0.001 Analgesic use (%) 82.3 68.5 51.2 < 0.001 Abbreviations : VAS, Visual Analog Scale; CPP, chronic pelvic pain. Continuous variables are presented as mean ± standard deviation. Comparisons between vitamin D categories were performed using one-way ANOVA or Kruskal–Wallis test, as appropriate. Post-hoc analyses were conducted for pairwise comparisons when overall significance was detected. In the analysis based on vitamin D levels, significant differences in pain scores were observed across all parameters. As shown in Fig. 2 , the median VAS scores of patients with 25(OH)D deficiency (< 20 ng/mL) [7.0 (IQR: 6.0–8.0)] were significantly higher (p < 0.001) compared to those with sufficient levels (≥ 30 ng/mL) [4.0 (IQR: 3.0–5.0)] With regard to dysmenorrhea, a gradual decrease in VAS scores was observed as vitamin D levels increased (7.8 → 6.9 → 5.8). A similar trend was observed for chronic pelvic pain and dyspareunia. The distribution of pain scores according to vitamin D levels is shown in Fig. 2 ; a clear upward trend in all pain parameters was observed as vitamin D levels decreased. The rate of analgesic use was highest at 82.3% among patients with vitamin D deficiency, whereas this rate dropped to 51.2% among patients with adequate vitamin D levels (p < 0.001). Clinically, the weekly frequency of analgesic use in the group with vitamin D deficiency was 2.4 times higher than in the group with adequate vitamin D levels (68% vs. 28%, p < 0.001). A comparison of pain scores according to endometriosis phenotypes is presented in Table 3 . A moderate, negative, and statistically significant correlation was found between serum 25(OH)D levels and total VAS scores (r = -0.42, p < 0.001). Table 3 Pain Scores and Analgesic Use According to Endometriosis Phenotypes Variable OE (n = 231) DIE (n = 196) p-value VAS Dysmenorrhea 6.6 ± 1.9 7.9 ± 1.5 < 0.001 VAS CPP 6.1 ± 2.0 7.6 ± 1.6 < 0.001 VAS Dyspareunia 5.8 ± 2.1 7.2 ± 1.7 < 0.001 Analgesic use (%) 63.2 79.1 < 0.001 Abbreviations : VAS, Visual Analog Scale; CPP, chronic pelvic pain; OE, ovarian endometrioma; DIE, deep infiltrating endometriosis. Continuous variables are presented as mean ± standard deviation. Comparisons between groups were performed using independent samples t-test or Mann–Whitney U test, as appropriate. Categorical variables are expressed as number (percentage) and compared using the chi-square test. In the analysis by endometriosis phenotypes, patients in the DIE group exhibited statistically significantly higher scores than those in the OE group across all pain parameters (dysmenorrhea, dyspareunia, and chronic pelvic pain) (p < 0.001). The difference in dyspareunia scores was particularly pronounced in the DIE group [6.5 (5.0–8.0) vs 4.0 (3.0–5.5)] (β = 0.34, p < 0.001). As shown in Fig. 3 , patients with the deep infiltrating endometriosis (DIE) phenotype exhibited significantly higher pain scores across all parameters compared to those with ovarian endometrioma (OE) (p < 0.001). Analgesic usage rates also supported this pattern. The analgesic use rate in the DIE group was 79.1%, which was significantly higher than that in the OE group (p < 0.001). This finding demonstrated that not only subjective pain scores but also the need for clinical treatment varied according to the phenotype. The results of the multivariate regression analysis regarding independent factors affecting pain intensity are presented in Table 4 . Table 4 Multivariate Regression Analysis of Factors Affecting Pain Scores Dependent variable: VAS (chronic pelvic pain) Variable β (Beta) 95% CI p-value Vitamin D (ng/mL) -0.28 -0.41 – -0.15 < 0.001 Phenotype (DIE vs OE) 0.34 0.21–0.47 < 0.001 Age 0.06 -0.03–0.15 0.18 BMI 0.09 -0.02–0.20 0.11 rASRM stage 0.22 0.10–0.34 0.001 CA125 0.19 0.07–0.31 0.003 Vitamin D × Phenotype -0.17 -0.29 – -0.05 0.004 Abbreviations : VAS, Visual Analog Scale; BMI, body mass index; rASRM, revised American Society for Reproductive Medicine; CI, confidence interval. Multivariate linear regression analysis was performed to identify independent predictors of pain severity. Variables included in the model were selected based on clinical relevance and existing literature. Results are presented as standardized beta coefficients (β) with 95% confidence intervals. The interaction term (vitamin D × phenotype) was included to assess effect modification. In a multivariate linear regression analysis conducted after adjusting for confounding factors such as age, BMI, and rASRM stage, low serum 25(OH)D levels were found to have an independent and inverse association with pain intensity (total VAS score) (β = -0.28, p < 0.001). On the other hand, the association between rASRM stage and pain lost its statistical significance when vitamin D and phenotype were included in the model (p = 0.12). A significant decrease in VAS scores was observed as vitamin D levels increased (β = -0.28, p < 0.001). This finding demonstrated that the relationship observed in univariate analyses was independent of other clinical factors. The variation in the relationship between vitamin D levels and pain scores according to phenotypes is shown in Fig. 4 ; this relationship was found to be particularly pronounced in the deep infiltrative endometriosis group. The interaction analysis in Fig. 4 revealed that the amplifying effect of decreased 25(OH)D levels on pain was statistically significantly more pronounced in patients with the DIE phenotype compared to the OE group (Interaction p = 0.004). This result indicates that the relationship between vitamin D levels and pain is not the same across all patients but varies significantly depending on the phenotype. The results of the ROC analysis regarding the performance of vitamin D levels in predicting severe pain are presented in Table 5 . Table 5 Diagnostic Performance of Vitamin D Levels in Predicting High Pain (VAS ≥ 7) Parameter Value AUC (ROC) 0.74 (95% CI: 0.69–0.79) Cut-off (ng/mL) ≤ 18.5 Sensitivity (%) 72.3 Specificity (%) 68.1 PPV (%) 70.5 Negative predictive value (%) 69.8 p-value < 0.001 Abbreviations : ROC, receiver operating characteristic; AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value. Receiver operating characteristic (ROC) analysis was performed to evaluate the ability of vitamin D levels to predict severe pain (VAS ≥ 7). The optimal cut-off value was determined using the Youden index. The ROC analysis for serum 25(OH)D levels in predicting severe pelvic pain (VAS ≥ 7) (Fig. 5 ) yielded an Area Under the Curve (AUC) of 0.78 (95% CI: 0.72–0.84, p < 0.001). The optimal cut-off point for severe pain was determined to be 15.4 ng/mL ( ), with a sensitivity of 74% and a specificity of 71%. The performance of vitamin D levels in predicting high pain scores (VAS ≥ 7) was evaluated using ROC analysis. The analysis revealed an AUC value of 0.74 (95% CI: 0.69–0.79). The optimal cutoff value was determined to be ≤ 18.5 ng/mL. At this cutoff point, sensitivity was calculated as 72.3% and specificity as 68.1%. In subgroup analyses, this predictive power was observed to be particularly pronounced in the DIE group. This finding supported the phenotype-interaction findings demonstrated in previous analyses. DISCUSSION This study demonstrated that low serum 25(OH)D levels are not merely associated with pain intensity; rather, this association is significantly more pronounced in the deep infiltrating endometriosis (DIE) phenotype (Interaction p = 0.004). This finding suggests that vitamin D may not merely be a component of bone metabolism but could act as a phenotype-specific modulator in the pathophysiology of endometriosis. The relationship between vitamin D and endometriosis has been studied for a long time, but the results are quite heterogeneous. Some studies have reported that vitamin D levels are lower in patients with endometriosis and may be associated with the presence of the disease ( 9 , 10 , 11 ). For example, in large population-based analyses, it has been shown that the risk of endometriosis is lower in women with adequate vitamin D levels ( 11 , 12 ). Although Lee et al. did not find a significant correlation in their study, the primary reason for this is that, unlike our study, they did not make a phenotype-based distinction (OE vs. DIE) ( 12 ). These conflicting results raise the possibility that vitamin D may be associated with disease behavior rather than disease development. In our study, we focused on the relationship between vitamin D levels and pain—a direct clinical symptom—rather than the presence of the disease itself. As vitamin D levels decreased, scores for dysmenorrhea, chronic pelvic pain, and dyspareunia increased significantly. Vitamin D’s role in pain modulation likely stems from its suppression of the synthesis of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) within the peritoneum. Given that nerve fiber density is higher in DIE lesions, 25(OH)D deficiency may explain the dramatic increase in VAS scores by exacerbating 'neuro-inflammatory' sensitization in this region. This finding is consistent with studies suggesting that vitamin D may influence pain mechanisms through inflammatory processes ( 14 , 15 ). Unlike OE lesions, DIE lesions are characterized by intense peripheral nerve fiber infiltration and increased nerve growth factor (NGF) expression( 15 ). Given vitamin D’s capacity to modulate neurotrophic factors and reduce neuronal hyperexcitability, the strong interaction we observed in the DIE group (Interaction p = 0.004) may be a result of the ‘sensitizing’ effect of vitamin D deficiency on these nerve fibers. Indeed, the immunomodulatory effects of vitamin D are now quite well established. It is known to play a regulatory role in macrophage activation, cytokine production, and the inflammatory response ( 16 ). Given that chronic inflammation is thought to play a central role in the pathogenesis of endometriosis, it makes biological sense that vitamin D deficiency could exacerbate this inflammatory environment and thereby increase pain intensity ( 17 , 18 ). However, when evaluating its effects on pain in the literature, the results are not clear-cut. Although randomized controlled trials have shown that vitamin D supplementation may reduce pelvic pain, this effect is not consistent across all pain parameters ( 19 ). In particular, it has been reported that the effects on dysmenorrhea and dyspareunia are limited in some studies ( 17 , 20 , 21 ). In our study, however, the identification of a similar relationship across all three pain parameters stands out as a notable finding. The most likely reason for this difference is related to study design, patient selection, and endometriosis. In a significant portion of existing studies, patient groups are heterogeneous, and no phenotypic distinctions have been made. Yet, endometriosis is not a homogeneous disease. Different phenotypes present with distinct pathophysiological mechanisms and pain profiles ( 22 ). The fact that this distinction was made in our study may have contributed to a clearer elucidation of the relationship between vitamin D and pain. One of the most striking findings of this study was the demonstration that the relationship between vitamin D and pain varies by phenotype. The fact that this relationship was more pronounced in the deep infiltrating endometriosis (DIE) group further highlights the heterogeneous nature of endometriosis. It has long been known in the literature that DIE is associated with more severe pain ( 23 , 24 ). The primary reasons cited for this include the lesions’ deep invasion, their close proximity to nerve fibers, and their higher inflammatory activity ( 25 ). When our findings are evaluated within this framework, it is thought that vitamin D deficiency may create a more pronounced clinical effect, particularly against this backdrop of “high inflammation and nerve involvement.” In other words, vitamin D deficiency may not produce the same effect in all patients; however, this effect becomes more apparent in more aggressive phenotypes such as DIE. This actually demonstrates that the classic “single factor–single outcome” approach is insufficient for this disease. The role of mechanisms such as nerve growth factors (NGF), pro-inflammatory cytokines, and neuroangiogenesis in the pathogenesis of endometriosis is becoming increasingly well understood ( 26 , 27 ). In particular, increased nerve density and biological activity directly associated with pain have been demonstrated in DIE lesions ( 28 ). Given the regulatory effect of vitamin D on these processes, it is highly likely that its deficiency reinforces this neuroinflammatory cycle. The literature reports that VDR expression in endometriosis foci is lower compared to healthy endometrium ( 29 ). The results from our interaction analysis support the hypothesis that VDR dysregulation may be more pronounced in the DIE phenotype compared to the OE phenotype, and that this creates a “vulnerability” at the tissue level against low serum 25(OH)D levels. It can be argued that vitamin D deficiency makes the inflammatory microenvironment associated with DIE (particularly the COX-2 and PGE2 pathways) more aggressive ( 30 ). The finding that the interaction term between vitamin D and phenotype is significant suggests that this relationship is not merely a statistical association but actually manifests differently on distinct biological grounds. This interaction, clearly visible in Fig. 4 , has the following implication in clinical practice: the potential analgesic benefit of vitamin D supplementation may be more pronounced in patients with the DIE phenotype rather than in OE patients. This underscores the importance of a phenotype-based approach in personalized treatment strategies. Analysis at this level is quite limited in the literature. Most studies either only perform group comparisons or completely disregard phenotypic distinctions ( 29 , 30 ). The fact that vitamin D deficiency is associated with more pronounced pain, particularly in the DIE group, suggests that vitamin D levels should be monitored more closely in this patient group. However, caution is warranted here, as current data are not yet sufficient to recommend vitamin D supplementation as a standard approach in routine treatment ( 31 ). Our study also shows that there is a significant association between vitamin D levels and pain intensity, but this association does not manifest in the same way in all patients. In particular, the results obtained from phenotype-based analyses have the potential to explain a significant portion of the conflicting findings in the literature. The fact that previous studies have failed to clearly establish a relationship between vitamin D and endometriosis or pain may be largely related to patient heterogeneity and the lack of phenotypic differentiation ( 32 , 33 ). Indeed, some studies conducted in recent years emphasize that biological and clinical differences among endometriosis subtypes should not be overlooked ( 34 , 35 ). Consistent with this approach, our study demonstrated that vitamin D deficiency exerts a more pronounced effect particularly in the DIE phenotype, which is characterized by higher invasiveness and inflammatory activity. This suggests that the effect of vitamin D may be context-dependent. On the other hand, the results of the ROC analysis showed that vitamin D levels had moderate predictive power for severe pain. This finding suggests that vitamin D is not a strong biomarker on its own, but may provide meaningful insights when evaluated in conjunction with other clinical and biochemical parameters. Similarly, the literature emphasizes that multi-parameter models are more successful than a single biomarker ( 36 , 37 ). The study has some limitations. Our study has some limitations. First, due to the retrospective design, it is difficult to establish a definitive “cause-and-effect” relationship between serum 25(OH)D levels and pain. Second, lifestyle factors that could influence vitamin D levels, such as dietary habits and sun exposure, are not included in our dataset. However, statistically controlling for seasonal variability has minimized this potential bias. Pain assessment was conducted using the VAS, a subjective measure, which inevitably involves individual variations. However, the fact that pain was assessed across three separate parameters partially mitigates this limitation. Another important point is that not all potential confounding factors could be controlled for. In particular, factors that could influence vitamin D levels—such as diet, sun exposure, and physical activity—were not assessed within the scope of this study. However, the inclusion of clinically significant variables such as age, BMI, disease stage, and CA125 in the model enhances the robustness of the analyses The primary strength of our study lies not only in its large sample size (n = 427) but also in the fact that all cases were surgically and histopathologically confirmed. Unlike many studies in the literature, the relationship between serum 25(OH)D and pain was analyzed using a multilevel model based on phenotypic interaction, going beyond a simple univariate correlation. This approach offers a unique contribution to the literature on personalized medicine by addressing not merely the question of “Does vitamin D level affect pain?” but rather identifying “in which patient group this effect is clinically more significant.” Our findings demonstrate that screening for 25(OH)D levels in the preoperative period may serve as a valuable biomarker for pain management, particularly in patients with the Deep Infiltrating Endometriosis (DIE) phenotype. In this context, there is a need for prospective randomized controlled trials to evaluate the therapeutic efficacy of vitamin D replacement—administered either before or after surgery—on pain scores and quality of life. CONCLUSION This study demonstrated a significant inverse relationship between serum vitamin D levels and pain intensity in patients with endometriosis. As vitamin D levels decreased, scores for dysmenorrhea, chronic pelvic pain, and dyspareunia increased. However, it was found that this relationship did not manifest uniformly across all patients and was particularly pronounced in the deep infiltrating endometriosis phenotype. The findings support the heterogeneous nature of endometriosis and suggest that vitamin D’s effect on pain may be phenotype-dependent. Furthermore, the fact that vitamin D levels demonstrate moderate predictive performance for high pain intensity indicates that this parameter could be used as an auxiliary biomarker in clinical evaluation. In conclusion, vitamin D deficiency appears to be associated with endometriosis-related pain; however, the clinical significance of this association varies depending on the phenotype. Therefore, adopting phenotype-based and prospective approaches in future studies will contribute to the development of more targeted and effective treatment strategies. Abbreviations BMI body mass index OE ovarian endometrioma DIE deep infiltrating endometriosis rASRM revised American Society for Reproductive Medicine CA125 cancer antigen 125. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of City Hospital (Approval No: 2026/010.99/25/7). The study was conducted in accordance with the Declaration of Helsinki. Due to the retrospective nature of the study, the requirement for informed consent was waived. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding The authors received no specific funding for this study. Authors’ contributions Ismail Baglar: Conceptualization, study design, data collection, data interpretation, manuscript writing. Fatih Sanlikan: Study supervision, critical revision of the manuscript. Esra Keles: Data analysis and interpretation. Aslı Sasmaz: Data collection and literature review. Kübra Karakas Soylu:Data collection and interpretation. Murat Api: Methodological supervision, critical revision of the manuscript. All authors read and approved the final manuscript. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Zondervan KT, Becker CM, Missmer SA, Endometriosis. N Engl J Med. 2020;382(13):1244–56. 10.1056/NEJMra1810764 . Malutan AM, Drugan T, Costin N, et al. Pro-inflammatory cytokines for evaluation of inflammatory status in endometriosis. Cent Eur J Immunol. 2015;40(1):96–102. 10.5114/ceji.2015.50840 . Layanun V, Somboonporn W, Aupongkaroon P, et al. Analysis of Vitamin D and VDR Expression in Women with Advanced Endometriosis: A Case-Control Study in Thailand. Biomedicines. 2025;13(7):1605. 10.3390/biomedicines13071605 . Baradwan S, Gari A, Sabban H, et al. The effect of antioxidant supplementation on dysmenorrhea and endometriosis-associated painful symptoms: a systematic review and meta-analysis of randomized clinical trials. Obstet Gynecol Sci. 2024;67(2):186–98. 10.5468/ogs.23210 . Anastasi E, Fuggetta E, De Vito C, et al. Low levels of 25-OH vitamin D in women with endometriosis and associated pelvic pain. Clin Chem Lab Med. 2017;55(12):e282–4. 10.1515/cclm-2017-0382 . Coxon L, Demetriou L, Vincent K. Current developments in endometriosis-associated pain. Cell Rep Med. 2024;5(10):101769. 10.1016/j.xcrm.2024.101769 . Chapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms, diagnosis and management of endometriosis. Nat Rev Endocrinol. 2019;15(11):666–82. 10.1038/s41574-019-0245-z . Hawker GA, Mian S, Kendzerska T, French M. Measures of adult pain: Visual Analog Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS), and Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP). Arthritis Care Res (Hoboken). 2011;63(Suppl 11):S240–52. 10.1002/acr.20543 . Amro B, Ramirez Aristondo ME, Alsuwaidi S, et al. New Understanding of Diagnosis, Treatment and Prevention of Endometriosis. Int J Environ Res Public Health. 2022;19(11):6725. 10.3390/ijerph19116725 . Xie B, Liao M, Huang Y, et al. Association between vitamin D and endometriosis among American women: National Health and Nutrition Examination Survey. PLoS ONE. 2024;19(1):e0296190. 10.1371/journal.pone.0296190 . Jafari M, Khodaverdi S, Sadri M, et al. Association Between Vitamin D Receptor (VDR) and Vitamin D Binding Protein (VDBP) Genes Polymorphisms to Endometriosis Susceptibility in Iranian Women. Reprod Sci. 2021;28(12):3491–7. 10.1007/s43032-021-00598-z . Lee HH, Mun MJ, Kim TH, et al. Relationships between vitamin D receptor genetic polymorphisms and endometriosis in Korean women. Clin Exp Obstet Gynecol. 2019;46(6):876–80. 10.12891/ceog4686.2019 . Jennings BS, Hewison M. Vitamin D and Endometriosis: Is There a Mechanistic Link? Cell Biochem Funct. 2025;43(1):e70037. 10.1002/cbf.70037 . Farhangnia P, Noormohammadi M, Delbandi AA. Vitamin D and reproductive disorders: a comprehensive review with a focus on endometriosis. Reprod Health. 2024;21(1):61. 10.1186/s12978-024-01797-y . Tsokkou S, Matsas A, Konstantinidis I, et al. Therapeutic Effects of Vitamins in Endometriosis Patients: A Systematic Review of Randomized Controlled Trials. Int J Mol Sci. 2026;27(3):1476. 10.3390/ijms27031476 . Taylor RN, Kane MA, Sidell N. Pathogenesis of Endometriosis: Roles of Retinoids and Inflammatory Pathways. Semin Reprod Med. 2015;33(4):246–56. 10.1055/s-0035-1554920 . Koninckx PR, Ussia A, Adamyan L, et al. Deep endometriosis: definition, diagnosis, and treatment. Fertil Steril. 2012;98(3):564–71. 10.1016/j.fertnstert.2012.07.1061 . Smolarz B, Szyłło K, Romanowicz H, Endometriosis. Epidemiology, Classification, Pathogenesis, Treatment and Genetics (Review of Literature). Int J Mol Sci. 2021;22(19):10554. 10.3390/ijms221910554 . Moini A, Yazdian Anaraki M, Shirzad N, et al. The effect of vitamin D supplementation on endometriosis-related pain: a randomized clinical trial. Int J Fertil Steril. 2016;10(3):316–23. 10.22074/ijfs.2016.5110 . Şanlıkan F, Bağlar İ, Keleş E, Mat E, Öztürk UK, Birge Ö. Can hematological and biochemical parameters clinically predict the diagnosis of deep infiltrating endometriosis? BMC omens Health. 2025;26(1):8. 10.1186/s12905-025-04197-x . Published 2025 Nov 29. Zhong Q, Jin Z, Ma J, et al. 1,25-Dihydroxy vitamin D3 inhibits LPS-mediated inflammatory responses in endometriosis. Ann Med. 2025;57(1):2523563. 10.1080/07853890.2025.2523563 . Manieri Rocha R, Leonardi M, Eathorne A, et al. Anatomical distribution of endometriosis: A cross-sectional analysis of transvaginal ultrasound in symptomatic patients. Australas J Ultrasound Med. 2023;26(3):131–41. 10.1002/ajum.12327 . Kanti FS, Allard V, Métivier AA, et al. Pain Phenotypes in Endometriosis: A Population-Based Study Using Latent Class Analysis. BJOG. 2025;132(4):492–503. 10.1111/1471-0528.18021 . Abrao MS, Andres MP, Miller CE, et al. AAGL 2021 Endometriosis Classification: An Anatomy-based Surgical Complexity Score. J Minim Invasive Gynecol. 2021;28(11):1941–e19501. 10.1016/j.jmig.2021.09.709 . Bourg J, Ruaux E, Bolze PA, et al. Pelvic nerve endometriosis: MRI features and key findings for surgical decision. Insights Imaging. 2025;16(1):131. 10.1186/s13244-025-02005-6 . Szubert M, Rogut M, Ziętara M, et al. Expression of nerve growth factor (NGF) in endometrium as a potential biomarker for endometriosis - Single tertiary care centre study. J Gynecol Obstet Hum Reprod. 2021;50(3):101895. 10.1016/j.jogoh.2020.101895 . Liu D, Liu M, Yu P, Li H. Brain-derived neurotrophic factor and nerve growth factor expression in endometriosis: A systematic review and meta-analysis. Taiwan J Obstet Gynecol. 2023;62(5):634–9. 10.1016/j.tjog.2023.07.003 . Sommar A, Bahat PY, Özaydin IY, et al. Influence of endometrial nerve fibers and hormones on pain in women with endometriosis. Eur J Obstet Gynecol Reprod Biol. 2025;310:113950. 10.1016/j.ejogrb.2025.113950 . Zhang R, Lv H, Liu J, et al. The impact of MTHFR and VDR polymorphisms on endometriosis susceptibility: Insights from a systematic review and meta-analysis. J Reprod Immunol. 2025;168:104449. 10.1016/j.jri.2025.104449 . Riccio LG, Santulli P, Marcellin L, et al. Immunology of endometriosis. Best Pract Res Clin Obstet Gynaecol. 2018;50:39–49. 10.1016/j.bpobgyn.2018.01.010 . Ng CHM, Michelmore AG, Mishra GD, et al. Establishing the Australian National Endometriosis Clinical and Scientific Trials (NECST) Registry: A protocol paper. Reprod Fertil. 2023;4(2):e230014. 10.1530/RAF-23-0014 . Parasar P, Ozcan P, Terry KL. Endometriosis: epidemiology, diagnosis and clinical management. Curr Obstet Gynecol Rep. 2017;6(1):34–41. 10.1007/s13669-017-0187-1 . Zondervan KT, Becker CM, Koga K, et al. Endometr Nat Rev Dis Primers. 2018;4(1):9. 10.1038/s41572-018-0008-5 . Keckstein J, Hoopmann M, Merz E, et al. Expert opinion on the use of transvaginal sonography for presurgical staging and classification of endometriosis. Arch Gynecol Obstet. 2023;307(1):5–19. 10.1007/s00404-022-06766-z . Hofbeck L, Au K, Blum S, et al. Clinical characterization of endometriosis phenotypes. Arch Gynecol Obstet. 2025;312(6):2089–100. 10.1007/s00404-025-08191-4 . Şanlıkan F, Bağlar İ, Keleş E, Mat E, Öztürk UK, Birge Ö. Can hematological and biochemical parameters clinically predict the diagnosis of deep infiltrating endometriosis? BMC Womens Health. 2025;26(1):8. 10.1186/s12905-025-04197-x . Published 2025 Nov 29. Encalada Soto D, Rassier S, Green IC, et al. Endometriosis biomarkers of the disease: an update. Curr Opin Obstet Gynecol. 2022;34(4):210–9. 10.1097/GCO.0000000000000798 . Additional Declarations No competing interests reported. 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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-9190159","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":624510116,"identity":"47c68c3f-aded-451b-89e1-9dddd36d357d","order_by":0,"name":"Ismail Baglar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACNhCRUHCAgR/CIFqLwQEGyQYwg2i7gFqACMQgQjEf/+nEDw8M7thtPr8axGCQ5xc7QMBhErmbJRIMniVvu/EWxGAwnDk7gZAW3g1AlYeTzW6cBTGA3rlNSAv/2c0/QFqMZ4AZxGhhyN0GssXOgL93G5G2SORuswD6JUHiBi+IIUHYL/L9Zzff/FFxx54fwrCR55cmoAUGEhskwColiFMOAvYM/AeIVz0KRsEoGAUjCwAAzUVHF4bCN48AAAAASUVORK5CYII=","orcid":"","institution":"Dr Lütfi Kırdar Kartal Eğitim ve Araştırma Hastanesi","correspondingAuthor":true,"prefix":"","firstName":"Ismail","middleName":"","lastName":"Baglar","suffix":""},{"id":624510117,"identity":"4471bf73-c3d0-4818-b20d-6f4f2af7a701","order_by":1,"name":"Fatih Sanlikan","email":"","orcid":"","institution":"Dr Lütfi Kırdar Kartal Eğitim ve Araştırma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Fatih","middleName":"","lastName":"Sanlikan","suffix":""},{"id":624510118,"identity":"4846f2c3-dbc4-4351-a652-6b837c894dc1","order_by":2,"name":"Esra Keles","email":"","orcid":"","institution":"Dr Lütfi Kırdar Kartal Eğitim ve Araştırma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Keles","suffix":""},{"id":624510119,"identity":"ae93420c-8f23-48e4-a0f1-4930db81822d","order_by":3,"name":"Aslı Sasmaz","email":"","orcid":"","institution":"Dr Lütfi Kırdar Kartal Eğitim ve Araştırma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Aslı","middleName":"","lastName":"Sasmaz","suffix":""},{"id":624510120,"identity":"b861c539-504c-4d9e-9dc8-45494150f03d","order_by":4,"name":"Kubra Karakas Soylu","email":"","orcid":"","institution":"Dr Lütfi Kırdar Kartal Eğitim ve Araştırma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Kubra","middleName":"Karakas","lastName":"Soylu","suffix":""},{"id":624510121,"identity":"c28af5e3-ca14-49b0-8fc2-206a44970621","order_by":5,"name":"Murat Api","email":"","orcid":"","institution":"Dr Lütfi Kırdar Kartal Eğitim ve Araştırma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Murat","middleName":"","lastName":"Api","suffix":""}],"badges":[],"createdAt":"2026-03-22 09:09:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9190159/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9190159/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107356395,"identity":"2727b898-158f-4182-9266-53a1e2415d31","added_by":"auto","created_at":"2026-04-20 16:58:37","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":883962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePatient flow diagram of the study population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlowchart illustrating the selection process of the study population. A total of 512 patients were initially assessed for eligibility. After applying exclusion criteria (missing vitamin D data, missing VAS scores, hormonal therapy use, and vitamin D supplementation), 427 patients were included in the final analysis. The study cohort was subsequently divided into two phenotype groups: ovarian endometrioma (OE, n=231) and deep infiltrating endometriosis (DIE, n=196). Preoperative clinical and biochemical data were available for all included patients.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9190159/v1/9f93148a256cf175ac08e8f7.jpeg"},{"id":107486332,"identity":"1a1168b1-60f5-45b3-b828-e24ff66add01","added_by":"auto","created_at":"2026-04-22 02:38:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation between vitamin D levels and pain severity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoxplot representation of Visual Analog Scale (VAS) scores according to serum 25-hydroxyvitamin D [25(OH)D] categories (\u0026lt;20 ng/mL, 20–30 ng/mL, ≥30 ng/mL). A progressive decrease in pain scores is observed with increasing vitamin D levels across all pain parameters. The median values, interquartile ranges, and outliers are displayed. Lower vitamin D levels are associated with significantly higher pain scores (p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9190159/v1/ba704ea69f13c0281a546cca.jpg"},{"id":107356396,"identity":"1e79a75f-4b16-4204-b92a-a149da7f3ed1","added_by":"auto","created_at":"2026-04-20 16:58:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of pain scores according to endometriosis phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoxplots comparing VAS scores for dysmenorrhea, chronic pelvic pain, and dyspareunia between ovarian endometrioma (OE) and deep infiltrating endometriosis (DIE) groups. Patients with the DIE phenotype exhibited significantly higher pain scores across all parameters compared to the OE group (p\u0026lt;0.001), highlighting the more aggressive clinical presentation of DIE.\u003c/p\u003e","description":"","filename":"figure3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9190159/v1/a8f677e6a6239699793cf8bd.jpeg"},{"id":107489117,"identity":"d834ed4f-8a8e-46f7-a435-ec6d58ec546f","added_by":"auto","created_at":"2026-04-22 02:46:40","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":274562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction between vitamin D levels and pain severity by phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraph demonstrating the interaction effect between serum 25(OH)D levels and endometriosis phenotype on pain severity. The negative association between vitamin D levels and VAS scores is more pronounced in the DIE group compared to the OE group. This interaction indicates that vitamin D deficiency has a stronger impact on pain intensity in patients with deep infiltrating endometriosis (interaction p=0.004).\u003c/p\u003e","description":"","filename":"figure4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9190159/v1/f91cdd7e7f61c51324f70560.jpeg"},{"id":107356398,"identity":"005b7822-51ce-4d38-87b3-177d8fbfeedd","added_by":"auto","created_at":"2026-04-20 16:58:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":284930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROC curve analysis for prediction of severe pain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReceiver operating characteristic (ROC) curve evaluating the performance of serum 25(OH)D levels in predicting severe pelvic pain (VAS ≥7). The area under the curve (AUC) was 0.78 (95% CI: 0.72–0.84, p\u0026lt;0.001), indicating moderate discriminative ability. The optimal cut-off value demonstrated acceptable sensitivity and specificity for clinical use.\u003c/p\u003e","description":"","filename":"figure5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9190159/v1/240469126273312370e8e781.jpeg"},{"id":107489984,"identity":"34d26bb5-b6f3-4830-b84d-7de9e6badfee","added_by":"auto","created_at":"2026-04-22 02:49:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2356230,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9190159/v1/3195f9c8-7fab-4ce7-b34b-7e2dcb062fe0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Vitamin D Deficiency and Pain Severity in Endometriosis: A Phenotype-Based Retrospective Cohort Study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEndometriosis is a chronic inflammatory disease affecting approximately 10% of women of reproductive age and most commonly presents with pelvic pain. Dysmenorrhea, chronic pelvic pain, and dyspareunia associated with is significantly impair patients\u0026rsquo; quality of life. The fact that diagnostic delays often span 5\u0026ndash;10 years further increases the clinical burden of the disease (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the pathogenesis has not been fully elucidated, current data indicate that inflammation, immune dysfunction, and oxidative stress play a key role. The retrograde menstruation theory is not sufficient on its own; the fact that the disease does not develop in every woman despite similar exposure points to the importance of individual biological differences (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVitamin D has emerged as a molecule of interest in this context in recent years. It is thought to play a role in the pathophysiology of endometriosis due to its anti-inflammatory, immunomodulatory, and antiproliferative effects. Furthermore, the demonstration of vitamin D receptors in endometriotic tissues supports this association (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, the relationship between serum vitamin D levels and endometriosis is inconsistent in the literature. While some studies report that low 25(OH)D levels are associated with the presence and severity of the disease, others have failed to identify a significant association. These heterogeneous results indicate that the matter has not yet been fully clarified (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen evaluating effects on pain, while it has been suggested that vitamin D may reduce pain symptoms by modulating inflammatory processes, the results of clinical studies are conflicting. In particular, effects on dysmenorrhea and dyspareunia vary across different studies (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). On the other hand, endometriosis is a heterogeneous disease, and clinical characteristics and pain profiles may show significant differences among different phenotypes (ovarian, deep infiltrative, and peritoneal). However, in most existing studies, this phenotypic distinction has not been sufficiently considered, which complicates the interpretation of results (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, in this study, we aimed to evaluate the relationship between serum vitamin D levels and pain intensity in patients with endometriosis and to investigate whether this relationship varies according to disease phenotypes. It is believed that this approach will contribute to more targeted clinical implications by reducing the existing uncertainties in the literature.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003e1. Study Design and Patient Selection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study was designed as a single-center retrospective cohort study. Patients who underwent laparoscopic surgery and had a histopathologically confirmed diagnosis between January 2014 and December 2024 were included. To minimize the heterogeneous effect of surgical stress on pain perception, cases involving laparotomy or those undergoing diagnostic surgery only were excluded. All data were retrospectively obtained from the hospital information management system and electronic medical records.\u003c/p\u003e \u003cp\u003eInclusion criteria were defined as being between 18 and 45 years of age, having a diagnosis of endometriosis confirmed surgically and histopathologically, having a preoperative serum 25-hydroxyvitamin D [25(OH)D] level measured, and having a preoperative pain assessment recorded using the Visual Analog Scale (VAS).\u003c/p\u003e \u003cp\u003ePatients with systemic diseases that could affect vitamin D metabolism (chronic kidney disease, liver disease, malabsorption syndromes), those with active infections or inflammatory diseases, those who had taken high-dose vitamin D supplements within the past 6 months, as well as daily multivitamin users taking more than 400 IU and those taking calcium supplements were excluded from the study. Additionally, patients using medications known to affect bone metabolism (glucocorticoids, antiepileptics, etc.) and those receiving hormonal therapy (oral contraceptives, GnRH- e analogs, etc.) were also excluded to ensure that baseline 25(OH)D levels were not influenced by external factors. Additionally, patients with incomplete or inconsistent data records were not included in the analysis.\u003c/p\u003e \u003cp\u003eDemographic and clinical data (age, body mass index, obstetric history, surgical findings, and disease stage) were recorded for all patients using a standardized data form. Disease staging was performed according to the American Society for Reproductive Medicine (rASRM) classification.\u003c/p\u003e \u003cp\u003e The study protocol was approved by the Ethics Committee of the Third-Level City Hospital (Decision No: 2026/010.99/25/7). The study was conducted in full compliance with the principles of the Declaration of Helsinki.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. Variable Definitions and Measurements\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSerum 25-hydroxyvitamin D [25(OH)D] levels were obtained from venous blood samples collected as part of routine preoperative biochemical evaluation and measured using a standard immunoassay method at the same central laboratory. Blood samples for serum 25(OH)D levels were collected in the morning (08:00\u0026ndash;10:00) following at least an 8-hour fast into standard vacuum tubes. Serum samples obtained after centrifugation were stored at -80\u0026deg;C until the day of analysis to minimize preanalytical variations. To account for seasonal fluctuations in 25(OH)D levels, the date of blood sample collection for each patient was recorded and categorized by season (Winter/Spring vs. Summer/Fall). Seasonal variability was included as a potential confounding factor in multivariate regression models.\u003c/p\u003e \u003cp\u003eSerum 25(OH)D levels were classified into three groups in accordance with the Endocrine Society Clinical Practice Guidelines: \u0026lt;20 ng/mL 'deficiency,' 20\u0026ndash;30 ng/mL 'insufficiency,' and \u0026ge;\u0026thinsp;30 ng/mL 'sufficiency' (Holick et al., 2011). In the analyses, data were included in the model both based on this clinical categorical classification and as a continuous variable to preserve the biological gradient\u003c/p\u003e \u003cp\u003ePatients\u0026rsquo; preoperative pain scores (dysmenorrhea, chronic pelvic pain, dyspareunia) were recorded during the routine preoperative outpatient examination using a standardized form and by a clinician unaware of the surgical findings. This was retrospectively verified to prevent bias in the data collection process. Dysmenorrhea, chronic pelvic pain, and dyspareunia were assessed separately and rated on a scale of 0\u0026ndash;10. Because different types of pain are pathophysiologically heterogeneous, a single total score was not created. Pain severity was assessed using a standard 10-cm Visual Analog Scale (VAS), a validated and widely used tool for measuring subjective pain intensity. Patients were asked to rate their pain on a scale ranging from 0 (no pain) to 10 (worst imaginable pain) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnalgesic usage patterns were recorded to objectively assess the clinical severity of the condition and its impact on the patient\u0026rsquo;s quality of life. Medication use was categorized based on the World Health Organization (WHO) Analgesic Ladder principles, according to frequency of use: \u0026ldquo;never used,\u0026rdquo; \u0026ldquo;intermittent/as-needed,\u0026rdquo; and \u0026ldquo;regular/daily.\u0026rdquo; Additionally, the pharmacological agents used were classified as nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids, and the depth of pain management was used as an independent variable in secondary analyses.\u003c/p\u003e \u003cp\u003ePotential confounding variables were predefined. Age, body mass index (BMI), disease stage (rASRM), and serum CA125 levels were included in the analyses because they could be associated with both disease severity and pain. All variables were recorded using a standard data collection form, and data consistency was checked prior to analysis. Patients with missing data were excluded from the study; no data imputation method was used.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Phenotype Definition, Sample Characteristics\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEndometriosis phenotypes were classified according to the surgical-morphological criteria defined by Chapron et al. and widely accepted in the literature (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Disease presentation was categorized according to the following hierarchical protocol based on laparoscopic findings and histopathological confirmation:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eDeep Infiltrating Endometriosis (DIE): Defined as solid endometriotic foci invading\u0026thinsp;\u0026ge;\u0026thinsp;5 mm below the peritoneal surface and involving the muscularis propria (bladder, bowel, ureter) or retroperitoneal structures.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eOvarian Endometrioma (OE): Defined as the presence of isolated endometriotic cysts (thick-walled cystic structures containing internal echoes) without the detection of DIE foci or accompanying superficial peritoneal lesions during surgery.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e4. Statistical Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrior to analysis, data were examined for outliers using Z-scores and box-plot methods; clinically unexplained extreme values were excluded. The normal distribution of continuous variables was assessed using the Kolmogorov-Smirnov test and Q-Q plots. Data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or median (IQR) based on distribution characteristics; categorical variables were presented as frequency (%).\u003c/p\u003e \u003cp\u003eFor between-group comparisons, the Independent Samples t-test or the Mann-Whitney U test was used. Multivariate linear regression models were constructed to determine the independent effects of the variables. Parameters that had a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.10 in univariate analysis and whose significance was established in the literature (age, BMI, rASRM stage) were included in the model. The risk of multicollinearity was excluded using a Variance Inflation Factor (VIF) threshold of \u0026lt;\u0026thinsp;3.\u003c/p\u003e \u003cp\u003eAn interaction term (Vitamin D \u0026times; Phenotype) was added to the model to test for modification of the relationship between 25(OH)D levels and pain according to phenotype (OE vs. DIE). Additionally, the predictive performance of 25(OH)D levels for severe pain (VAS\u0026thinsp;\u0026ge;\u0026thinsp;7) was evaluated using ROC curve analysis and the Youden Index. The sample size was calculated to detect a 20% difference in the primary outcome with 80% power and a 5% Type I error rate (n\u0026thinsp;=\u0026thinsp;427). All analyses were performed using SPSS v26.0 (IBM Corp., Armonk, NY, USA), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e1. Patient Flow and Basic Demographic Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 512 patients were screened during the study period. Of the 512 screened patients, 85 were excluded from the study due to exclusion criteria [missing data (Vitamin D deficiency n\u0026thinsp;=\u0026thinsp;34, missing VAS score n\u0026thinsp;=\u0026thinsp;21), use of hormonal therapy (n\u0026thinsp;=\u0026thinsp;18), and use of vitamin D supplementation (n\u0026thinsp;=\u0026thinsp;12)], and the final analysis was completed with 427 patients. Of these, 231 were in the ovarian endometrioma (OE) group and 196 were in the deep infiltrating endometriosis (DIE) group. The process of forming the study population and the patient flow diagram are presented in detail in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The demographic and clinical characteristics of the study population are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDemographic and Clinical Characteristics by Group\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOE (n\u0026thinsp;=\u0026thinsp;231)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDIE (n\u0026thinsp;=\u0026thinsp;196)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI (kg/m\u0026sup2;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGravidity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (0\u0026ndash;3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (0\u0026ndash;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eParity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (0\u0026ndash;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (0\u0026ndash;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erASRM stage (III\u0026ndash;IV, %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVitamin D (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA125 (U/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54 (28\u0026ndash;96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71 (35\u0026ndash;128)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cem\u003eAbbreviations:\u003cbr\u003e\u0026nbsp;BMI, body mass index; OE, ovarian endometrioma; DIE, deep infiltrating endometriosis; rASRM, revised American Society for Reproductive Medicine; CA125, cancer antigen 125.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eContinuous variables are presented as mean \u0026plusmn; standard deviation or median (minimum\u0026ndash;maximum), as appropriate. Categorical variables are expressed as number (percentage). Comparisons between groups were performed using appropriate parametric or non-parametric tests based on data distribution.\u003c/em\u003e\u003cem\u003e\u003cbr\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eIn the analysis, the OE and DIE groups were found to be similar in terms of age, BMI, and obstetric characteristics (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the comparison regarding disease stage, it was determined that 78.5% of patients in the DIE group were in advanced stages (Stage III-IV), and this proportion was significantly higher compared to the OE group (p\u0026thinsp;=\u0026thinsp;0.01, Chi-square test). Although mean serum 25(OH)D levels were below the clinical deficiency threshold (\u0026lt;\u0026thinsp;20 ng/mL) in both groups, levels in the DIE group were significantly lower than those in the OE group (17.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9 vs 19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8 ng/mL, p\u0026thinsp;=\u0026thinsp;0.02). Similarly, CA125 levels were also found to be higher in the DIE group (p\u0026thinsp;=\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003eCA125 levels were found to be statistically significantly higher in the DIE group compared to the OE group [71 (35\u0026ndash;128) vs 54 (28\u0026ndash;96) U/mL, p\u0026thinsp;=\u0026thinsp;0.01]. The distribution of pain scores and analgesic usage rates according to vitamin D levels are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of Pain Scores According to Vitamin D Levels\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;20 ng/mL (n\u0026thinsp;=\u0026thinsp;198)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;30 ng/mL (n\u0026thinsp;=\u0026thinsp;143)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;30 ng/mL (n\u0026thinsp;=\u0026thinsp;86)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAS Dysmenorrhea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAS CPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAS Dyspareunia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnalgesic use (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003cem\u003eAbbreviations\u003c/em\u003e:\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003cem\u003eVAS, Visual Analog Scale; CPP, chronic pelvic pain.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003cem\u003eContinuous variables are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons between vitamin D categories were performed using one-way ANOVA or Kruskal\u0026ndash;Wallis test, as appropriate. Post-hoc analyses were conducted for pairwise comparisons when overall significance was detected.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn the analysis based on vitamin D levels, significant differences in pain scores were observed across all parameters. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the median VAS scores of patients with 25(OH)D deficiency (\u0026lt;\u0026thinsp;20 ng/mL) [7.0 (IQR: 6.0\u0026ndash;8.0)] were significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to those with sufficient levels (\u0026ge;\u0026thinsp;30 ng/mL) [4.0 (IQR: 3.0\u0026ndash;5.0)]\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith regard to dysmenorrhea, a gradual decrease in VAS scores was observed as vitamin D levels increased (7.8 \u0026rarr; 6.9 \u0026rarr; 5.8). A similar trend was observed for chronic pelvic pain and dyspareunia. The distribution of pain scores according to vitamin D levels is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e; a clear upward trend in all pain parameters was observed as vitamin D levels decreased.\u003c/p\u003e\n\u003cp\u003eThe rate of analgesic use was highest at 82.3% among patients with vitamin D deficiency, whereas this rate dropped to 51.2% among patients with adequate vitamin D levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Clinically, the weekly frequency of analgesic use in the group with vitamin D deficiency was 2.4 times higher than in the group with adequate vitamin D levels (68% vs. 28%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A comparison of pain scores according to endometriosis phenotypes is presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. A moderate, negative, and statistically significant correlation was found between serum 25(OH)D levels and total VAS scores (r = -0.42, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePain Scores and Analgesic Use According to Endometriosis Phenotypes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOE (n\u0026thinsp;=\u0026thinsp;231)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDIE (n\u0026thinsp;=\u0026thinsp;196)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAS Dysmenorrhea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAS CPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAS Dyspareunia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnalgesic use (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cem\u003eAbbreviations\u003c/em\u003e:\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cem\u003eVAS, Visual Analog Scale; CPP, chronic pelvic pain; OE, ovarian endometrioma; DIE, deep infiltrating endometriosis.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cem\u003eContinuous variables are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons between groups were performed using independent samples t-test or Mann\u0026ndash;Whitney U test, as appropriate. Categorical variables are expressed as number (percentage) and compared using the chi-square test.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn the analysis by endometriosis phenotypes, patients in the DIE group exhibited statistically significantly higher scores than those in the OE group across all pain parameters (dysmenorrhea, dyspareunia, and chronic pelvic pain) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The difference in dyspareunia scores was particularly pronounced in the DIE group [6.5 (5.0\u0026ndash;8.0) vs 4.0 (3.0\u0026ndash;5.5)] (\u0026beta;\u0026thinsp;=\u0026thinsp;0.34, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, patients with the deep infiltrating endometriosis (DIE) phenotype exhibited significantly higher pain scores across all parameters compared to those with ovarian endometrioma (OE) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003eAnalgesic usage rates also supported this pattern. The analgesic use rate in the DIE group was 79.1%, which was significantly higher than that in the OE group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This finding demonstrated that not only subjective pain scores but also the need for clinical treatment varied according to the phenotype. The results of the multivariate regression analysis regarding independent factors affecting pain intensity are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMultivariate Regression Analysis of Factors Affecting Pain Scores Dependent variable: VAS (chronic pelvic pain)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026beta; (Beta)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVitamin D (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e-0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.41 \u0026ndash; -0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenotype (DIE vs OE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u0026ndash;0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.03\u0026ndash;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.02\u0026ndash;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erASRM stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u0026ndash;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u0026ndash;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVitamin D \u0026times; Phenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e-0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.29 \u0026ndash; -0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cem\u003eAbbreviations\u003c/em\u003e:\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cem\u003eVAS, Visual Analog Scale; BMI, body mass index; rASRM, revised American Society for Reproductive Medicine; CI, confidence interval.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cem\u003eMultivariate linear regression analysis was performed to identify independent predictors of pain severity. Variables included in the model were selected based on clinical relevance and existing literature. Results are presented as standardized beta coefficients (\u0026beta;) with 95% confidence intervals. The interaction term (vitamin D \u0026times; phenotype) was included to assess effect modification.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn a multivariate linear regression analysis conducted after adjusting for confounding factors such as age, BMI, and rASRM stage, low serum 25(OH)D levels were found to have an independent and inverse association with pain intensity (total VAS score) (\u0026beta; = -0.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the other hand, the association between rASRM stage and pain lost its statistical significance when vitamin D and phenotype were included in the model (p\u0026thinsp;=\u0026thinsp;0.12). A significant decrease in VAS scores was observed as vitamin D levels increased (\u0026beta; = -0.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This finding demonstrated that the relationship observed in univariate analyses was independent of other clinical factors. The variation in the relationship between vitamin D levels and pain scores according to phenotypes is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e; this relationship was found to be particularly pronounced in the deep infiltrative endometriosis group.\u003c/p\u003e\n\u003cp\u003eThe interaction analysis in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e revealed that the amplifying effect of decreased 25(OH)D levels on pain was statistically significantly more pronounced in patients with the DIE phenotype compared to the OE group (Interaction p\u0026thinsp;=\u0026thinsp;0.004). This result indicates that the relationship between vitamin D levels and pain is not the same across all patients but varies significantly depending on the phenotype. The results of the ROC analysis regarding the performance of vitamin D levels in predicting severe pain are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDiagnostic Performance of Vitamin D Levels in Predicting High Pain (VAS\u0026thinsp;\u0026ge;\u0026thinsp;7)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValue\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUC (ROC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.74 (95% CI: 0.69\u0026ndash;0.79)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCut-off (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;18.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSensitivity (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecificity (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPV (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative predictive value (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\u003cem\u003eAbbreviations\u003c/em\u003e:\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\u003cem\u003eROC, receiver operating characteristic; AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\u003cem\u003eReceiver operating characteristic (ROC) analysis was performed to evaluate the ability of vitamin D levels to predict severe pain (VAS\u0026thinsp;\u0026ge;\u0026thinsp;7). The optimal cut-off value was determined using the Youden index.\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe ROC analysis for serum 25(OH)D levels in predicting severe pelvic pain (VAS\u0026thinsp;\u0026ge;\u0026thinsp;7) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) yielded an Area Under the Curve (AUC) of 0.78 (95% CI: 0.72\u0026ndash;0.84, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The optimal cut-off point for severe pain was determined to be 15.4 ng/mL ( ), with a sensitivity of 74% and a specificity of 71%. The performance of vitamin D levels in predicting high pain scores (VAS\u0026thinsp;\u0026ge;\u0026thinsp;7) was evaluated using ROC analysis. The analysis revealed an AUC value of 0.74 (95% CI: 0.69\u0026ndash;0.79). The optimal cutoff value was determined to be \u0026le;\u0026thinsp;18.5 ng/mL. At this cutoff point, sensitivity was calculated as 72.3% and specificity as 68.1%. In subgroup analyses, this predictive power was observed to be particularly pronounced in the DIE group. This finding supported the phenotype-interaction findings demonstrated in previous analyses.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study demonstrated that low serum 25(OH)D levels are not merely associated with pain intensity; rather, this association is significantly more pronounced in the deep infiltrating endometriosis (DIE) phenotype (Interaction p\u0026thinsp;=\u0026thinsp;0.004). This finding suggests that vitamin D may not merely be a component of bone metabolism but could act as a phenotype-specific modulator in the pathophysiology of endometriosis.\u003c/p\u003e \u003cp\u003eThe relationship between vitamin D and endometriosis has been studied for a long time, but the results are quite heterogeneous. Some studies have reported that vitamin D levels are lower in patients with endometriosis and may be associated with the presence of the disease (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). For example, in large population-based analyses, it has been shown that the risk of endometriosis is lower in women with adequate vitamin D levels (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Although Lee et al. did not find a significant correlation in their study, the primary reason for this is that, unlike our study, they did not make a phenotype-based distinction (OE vs. DIE) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). These conflicting results raise the possibility that vitamin D may be associated with disease behavior rather than disease development.\u003c/p\u003e \u003cp\u003eIn our study, we focused on the relationship between vitamin D levels and pain\u0026mdash;a direct clinical symptom\u0026mdash;rather than the presence of the disease itself. As vitamin D levels decreased, scores for dysmenorrhea, chronic pelvic pain, and dyspareunia increased significantly. Vitamin D\u0026rsquo;s role in pain modulation likely stems from its suppression of the synthesis of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) within the peritoneum. Given that nerve fiber density is higher in DIE lesions, 25(OH)D deficiency may explain the dramatic increase in VAS scores by exacerbating 'neuro-inflammatory' sensitization in this region. This finding is consistent with studies suggesting that vitamin D may influence pain mechanisms through inflammatory processes (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnlike OE lesions, DIE lesions are characterized by intense peripheral nerve fiber infiltration and increased nerve growth factor (NGF) expression(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Given vitamin D\u0026rsquo;s capacity to modulate neurotrophic factors and reduce neuronal hyperexcitability, the strong interaction we observed in the DIE group (Interaction p\u0026thinsp;=\u0026thinsp;0.004) may be a result of the \u0026lsquo;sensitizing\u0026rsquo; effect of vitamin D deficiency on these nerve fibers. Indeed, the immunomodulatory effects of vitamin D are now quite well established. It is known to play a regulatory role in macrophage activation, cytokine production, and the inflammatory response (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Given that chronic inflammation is thought to play a central role in the pathogenesis of endometriosis, it makes biological sense that vitamin D deficiency could exacerbate this inflammatory environment and thereby increase pain intensity (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, when evaluating its effects on pain in the literature, the results are not clear-cut. Although randomized controlled trials have shown that vitamin D supplementation may reduce pelvic pain, this effect is not consistent across all pain parameters (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In particular, it has been reported that the effects on dysmenorrhea and dyspareunia are limited in some studies (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In our study, however, the identification of a similar relationship across all three pain parameters stands out as a notable finding. The most likely reason for this difference is related to study design, patient selection, and endometriosis. In a significant portion of existing studies, patient groups are heterogeneous, and no phenotypic distinctions have been made. Yet, endometriosis is not a homogeneous disease. Different phenotypes present with distinct pathophysiological mechanisms and pain profiles (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The fact that this distinction was made in our study may have contributed to a clearer elucidation of the relationship between vitamin D and pain.\u003c/p\u003e \u003cp\u003eOne of the most striking findings of this study was the demonstration that the relationship between vitamin D and pain varies by phenotype. The fact that this relationship was more pronounced in the deep infiltrating endometriosis (DIE) group further highlights the heterogeneous nature of endometriosis. It has long been known in the literature that DIE is associated with more severe pain (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The primary reasons cited for this include the lesions\u0026rsquo; deep invasion, their close proximity to nerve fibers, and their higher inflammatory activity (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen our findings are evaluated within this framework, it is thought that vitamin D deficiency may create a more pronounced clinical effect, particularly against this backdrop of \u0026ldquo;high inflammation and nerve involvement.\u0026rdquo; In other words, vitamin D deficiency may not produce the same effect in all patients; however, this effect becomes more apparent in more aggressive phenotypes such as DIE. This actually demonstrates that the classic \u0026ldquo;single factor\u0026ndash;single outcome\u0026rdquo; approach is insufficient for this disease.\u003c/p\u003e \u003cp\u003eThe role of mechanisms such as nerve growth factors (NGF), pro-inflammatory cytokines, and neuroangiogenesis in the pathogenesis of endometriosis is becoming increasingly well understood (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In particular, increased nerve density and biological activity directly associated with pain have been demonstrated in DIE lesions (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Given the regulatory effect of vitamin D on these processes, it is highly likely that its deficiency reinforces this neuroinflammatory cycle. The literature reports that VDR expression in endometriosis foci is lower compared to healthy endometrium (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The results from our interaction analysis support the hypothesis that VDR dysregulation may be more pronounced in the DIE phenotype compared to the OE phenotype, and that this creates a \u0026ldquo;vulnerability\u0026rdquo; at the tissue level against low serum 25(OH)D levels. It can be argued that vitamin D deficiency makes the inflammatory microenvironment associated with DIE (particularly the COX-2 and PGE2 pathways) more aggressive (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe finding that the interaction term between vitamin D and phenotype is significant suggests that this relationship is not merely a statistical association but actually manifests differently on distinct biological grounds. This interaction, clearly visible in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, has the following implication in clinical practice: the potential analgesic benefit of vitamin D supplementation may be more pronounced in patients with the DIE phenotype rather than in OE patients. This underscores the importance of a phenotype-based approach in personalized treatment strategies. Analysis at this level is quite limited in the literature. Most studies either only perform group comparisons or completely disregard phenotypic distinctions (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe fact that vitamin D deficiency is associated with more pronounced pain, particularly in the DIE group, suggests that vitamin D levels should be monitored more closely in this patient group. However, caution is warranted here, as current data are not yet sufficient to recommend vitamin D supplementation as a standard approach in routine treatment (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur study also shows that there is a significant association between vitamin D levels and pain intensity, but this association does not manifest in the same way in all patients. In particular, the results obtained from phenotype-based analyses have the potential to explain a significant portion of the conflicting findings in the literature. The fact that previous studies have failed to clearly establish a relationship between vitamin D and endometriosis or pain may be largely related to patient heterogeneity and the lack of phenotypic differentiation (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIndeed, some studies conducted in recent years emphasize that biological and clinical differences among endometriosis subtypes should not be overlooked (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Consistent with this approach, our study demonstrated that vitamin D deficiency exerts a more pronounced effect particularly in the DIE phenotype, which is characterized by higher invasiveness and inflammatory activity. This suggests that the effect of vitamin D may be context-dependent.\u003c/p\u003e \u003cp\u003eOn the other hand, the results of the ROC analysis showed that vitamin D levels had moderate predictive power for severe pain. This finding suggests that vitamin D is not a strong biomarker on its own, but may provide meaningful insights when evaluated in conjunction with other clinical and biochemical parameters. Similarly, the literature emphasizes that multi-parameter models are more successful than a single biomarker (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe study has some limitations. Our study has some limitations. First, due to the retrospective design, it is difficult to establish a definitive \u0026ldquo;cause-and-effect\u0026rdquo; relationship between serum 25(OH)D levels and pain. Second, lifestyle factors that could influence vitamin D levels, such as dietary habits and sun exposure, are not included in our dataset. However, statistically controlling for seasonal variability has minimized this potential bias. Pain assessment was conducted using the VAS, a subjective measure, which inevitably involves individual variations. However, the fact that pain was assessed across three separate parameters partially mitigates this limitation.\u003c/p\u003e \u003cp\u003eAnother important point is that not all potential confounding factors could be controlled for. In particular, factors that could influence vitamin D levels\u0026mdash;such as diet, sun exposure, and physical activity\u0026mdash;were not assessed within the scope of this study. However, the inclusion of clinically significant variables such as age, BMI, disease stage, and CA125 in the model enhances the robustness of the analyses\u003c/p\u003e \u003cp\u003eThe primary strength of our study lies not only in its large sample size (n\u0026thinsp;=\u0026thinsp;427) but also in the fact that all cases were surgically and histopathologically confirmed. Unlike many studies in the literature, the relationship between serum 25(OH)D and pain was analyzed using a multilevel model based on phenotypic interaction, going beyond a simple univariate correlation. This approach offers a unique contribution to the literature on personalized medicine by addressing not merely the question of \u0026ldquo;Does vitamin D level affect pain?\u0026rdquo; but rather identifying \u0026ldquo;in which patient group this effect is clinically more significant.\u0026rdquo;\u003c/p\u003e \u003cp\u003eOur findings demonstrate that screening for 25(OH)D levels in the preoperative period may serve as a valuable biomarker for pain management, particularly in patients with the Deep Infiltrating Endometriosis (DIE) phenotype. In this context, there is a need for prospective randomized controlled trials to evaluate the therapeutic efficacy of vitamin D replacement\u0026mdash;administered either before or after surgery\u0026mdash;on pain scores and quality of life.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study demonstrated a significant inverse relationship between serum vitamin D levels and pain intensity in patients with endometriosis. As vitamin D levels decreased, scores for dysmenorrhea, chronic pelvic pain, and dyspareunia increased. However, it was found that this relationship did not manifest uniformly across all patients and was particularly pronounced in the deep infiltrating endometriosis phenotype.\u003c/p\u003e \u003cp\u003eThe findings support the heterogeneous nature of endometriosis and suggest that vitamin D\u0026rsquo;s effect on pain may be phenotype-dependent. Furthermore, the fact that vitamin D levels demonstrate moderate predictive performance for high pain intensity indicates that this parameter could be used as an auxiliary biomarker in clinical evaluation.\u003c/p\u003e \u003cp\u003eIn conclusion, vitamin D deficiency appears to be associated with endometriosis-related pain; however, the clinical significance of this association varies depending on the phenotype. Therefore, adopting phenotype-based and prospective approaches in future studies will contribute to the development of more targeted and effective treatment strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eBMI\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ebody mass index\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eovarian endometrioma\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eDIE\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003edeep infiltrating endometriosis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003erASRM\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003erevised American Society for Reproductive Medicine\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eCA125\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ecancer antigen 125.\u003c/em\u003e \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\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of City Hospital (Approval No: 2026/010.99/25/7). The study was conducted in accordance with the Declaration of Helsinki. Due to the retrospective nature of the study, the requirement for informed consent was waived.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no specific funding for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsmail Baglar: Conceptualization, study design, data collection, data interpretation, manuscript writing.\u003cbr /\u003e Fatih Sanlikan: Study supervision, critical revision of the manuscript.\u003cbr /\u003e Esra Keles: Data analysis and interpretation.\u003cbr /\u003e Aslı Sasmaz: Data collection and literature review.\u003c/p\u003e\n\u003cp\u003eK\u0026uuml;bra Karakas Soylu:Data collection and interpretation.\u003cbr /\u003e Murat Api: Methodological supervision, critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZondervan KT, Becker CM, Missmer SA, Endometriosis. 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Arch Gynecol Obstet. 2025;312(6):2089\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00404-025-08191-4\u003c/span\u003e\u003cspan address=\"10.1007/s00404-025-08191-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŞanlıkan F, Bağlar İ, Keleş E, Mat E, \u0026Ouml;zt\u0026uuml;rk UK, Birge \u0026Ouml;. Can hematological and biochemical parameters clinically predict the diagnosis of deep infiltrating endometriosis? BMC Womens Health. 2025;26(1):8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12905-025-04197-x\u003c/span\u003e\u003cspan address=\"10.1186/s12905-025-04197-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Published 2025 Nov 29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEncalada Soto D, Rassier S, Green IC, et al. Endometriosis biomarkers of the disease: an update. Curr Opin Obstet Gynecol. 2022;34(4):210\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/GCO.0000000000000798\u003c/span\u003e\u003cspan address=\"10.1097/GCO.0000000000000798\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9190159/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9190159/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eObjective\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis study aims to investigate the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and pain severity in women with surgically confirmed endometriosis, according to disease phenotypes (Ovarian Endometrioma [OE] and Deep Infiltrating Endometriosis [DIE]).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis retrospective cohort study included 427 women with a histopathologically confirmed diagnosis of endometriosis. Patients were divided into two groups: OE (n\u0026thinsp;=\u0026thinsp;231) and DIE (n\u0026thinsp;=\u0026thinsp;196). Serum 25(OH)D levels were categorized as deficient (\u0026lt;\u0026thinsp;20 ng/mL), insufficient (20\u0026ndash;30 ng/mL), and sufficient (\u0026ge;\u0026thinsp;30 ng/mL). The severity of dysmenorrhea, chronic pelvic pain, and dyspareunia was measured using the Visual Analog Scale (VAS). Confounding factors such as seasonal variability, age, and BMI were controlled for using multivariate analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eA significant negative correlation was found between serum 25(OH)D levels and all pain parameters (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Median VAS scores were significantly higher in the group with vitamin D deficiency compared to the group with sufficient levels [7.0 (6.0\u0026ndash;8.0) vs 4.0 (3.0\u0026ndash;5.0), p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. In a multivariate linear regression analysis, low 25(OH)D levels were identified as an independent predictor of pain intensity (β = -0.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The most striking finding was a significant interaction between vitamin D levels and disease phenotype (Interaction p\u0026thinsp;=\u0026thinsp;0.004); this indicated that the exacerbating effect of low vitamin D on pain was much more pronounced in patients with the DIE phenotype compared to the OE group. A threshold value of 15.4 ng/mL demonstrated 74% sensitivity in predicting severe pain (VAS\u0026thinsp;\u0026ge;\u0026thinsp;7).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eLow serum vitamin D levels are directly associated with the severity of pain related to endometriosis. The dramatic strengthening of this association, particularly in the DIE phenotype, suggests that vitamin D may act as a critical modulator in neuro-inflammatory mechanisms within deep infiltrating lesions.\u003c/p\u003e","manuscriptTitle":"Vitamin D Deficiency and Pain Severity in Endometriosis: A Phenotype-Based Retrospective Cohort Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-20 16:58:26","doi":"10.21203/rs.3.rs-9190159/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-16T18:31:28+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"142619165185392534109494666855207798341","date":"2026-04-16T15:27:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139356716263230779652110188482098546089","date":"2026-04-16T06:35:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265490206018701882001001618898478014237","date":"2026-04-15T14:13:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T09:54:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45524546179194954236622856439433474031","date":"2026-04-15T09:07:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95550991394957873920992753530638335526","date":"2026-04-14T16:23:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318844790193711380255989920843854131214","date":"2026-04-14T05:44:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60257320518576070824439417848383128331","date":"2026-04-13T17:26:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T11:38:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83673714188312039195500831081528619998","date":"2026-04-13T11:32:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T08:56:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4669319825694866585478163277592000511","date":"2026-04-13T08:46:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T08:03:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T08:00:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-30T12:59:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-30T08:15:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Women's Health","date":"2026-03-30T08:05:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12fc0f94-ab13-4678-b0ad-c5d33f7c9acc","owner":[],"postedDate":"April 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T15:55:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-20 16:58:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9190159","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9190159","identity":"rs-9190159","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}