The Relationship Between 1,25(OH)2D3 Levels and Interleukin-32 (IL-32) and Vascular Endothelial Growth Factor (VEGF) Levels in Endometriosis Cyst Tissue

The Relationship Between 1,25(OH)2D3 Levels and Interleukin-32 (IL-32) and Vascular Endothelial Growth Factor (VEGF) Levels in Endometriosis Cyst Tissue | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Relationship Between 1,25(OH)2D3 Levels and Interleukin-32 (IL-32) and Vascular Endothelial Growth Factor (VEGF) Levels in Endometriosis Cyst Tissue Mirza Ismail, Hanom Husni Syam, Mulyanusa Amarullah Ritonga, Anita Rachmawati, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4759160/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Endometriosis involves the presence of endometrial-like tissue outside the uterus, driven by inflammatory pathways including elevated Interleukin-32 (IL-32) and Vascular Endothelial Growth Factor (VEGF). One potential anti-inflammatory agent is active vitamin D (1,25(OH)2D3). This research investigates the correlation among 1,25(OH)2D3 levels, IL-32, and VEGF in 46 confirmed endometriosis cases. Tissue samples were collected from patients at several hospitals, and assays were conducted at the Clinical Pathology Laboratory of Dr. Hasan Sadikin General Hospital using enzyme-linked immunosorbent assay (ELISA). The study revealed significant negative correlations between 1,25(OH)2D3 and both IL-32 (r= -0.346; p = 0.019) and VEGF levels (r= -0.316; p = 0.032). Conversely, there was a significant positive correlation between VEGF and IL-32 (r = 0.490; p = 0.001). These findings underscore the role of active vitamin D in mitigating inflammation associated with endometriosis by reducing IL-32 and VEGF, critical factors in inflammatory responses and blood vessel formation. Moreover, the positive association between VEGF and IL-32 highlights their collaborative role in the inflammatory processes underlying endometriosis. This study contributes to understanding how vitamin D may modulate key inflammatory pathways implicated in the pathogenesis of this condition. Health sciences/Diseases Health sciences/Diseases/Reproductive disorders Health sciences/Diseases/Reproductive disorders/Infertility calcitriol endometriosis interleukin-32 vascular endothelial growth factor Figures Figure 1 Figure 2 Figure 3 Introduction Endometriosis is a chronic inflammatory disease characterized by the presence of tissue similar to the endometrium outside the uterus 1 . The age range of patients diagnosed with endometriosis is between 20–42 years. The prevalence of endometriosis is estimated to be between 2–10% in the female population worldwide, and up to 50% of infertility cases are associated with endometriosis 2 . Potential origins of endometriosis lesions include the transplantation of endometrial tissue through retrograde menstruation and in situ metaplasia of coelomic epithelium. Lesions outside the pelvis may be caused by vascular or lymphatic metastasis 2 . Superficial and deep infiltrating endometriosis lesions form through molecular mechanisms involving interactions between adhesion, cell proliferation, systemic and local steroidogenesis, chronic inflammation, immune dysregulation, vascularization, and innervation 3 . The chronic inflammatory response in endometriosis involves both innate and adaptive immune responses influenced by estrogen hormones 4 . The initial innate immune response involves macrophage phagocytosis mediated by scavenger receptors and regulated by cytokines. Patients with endometriosis exhibit abnormalities in their innate immune response, where macrophages fail to perform phagocytic functions on endometriosis lesions, particularly during the mediation process by scavenger receptors 5 . The adaptive immune system becomes involved when the innate immune response is insufficient to handle the pathological conditions. The adaptive immune system consists primarily of T and B lymphocytes. In patients with endometriosis, there is an increase in cytokine modulation by T cells and the formation of autoantibodies by B cells 6 . Cytokines are proteins produced by various cells including stromal cells, fibroblasts, and endothelial cells. Interleukin-32 (IL-32) is one among proinflammatory cytokines thought to be involved in the pathogenesis of endometriosis. IL-32 has diverse functions such as regulating the body's immune response during infection, contributing to autoimmune diseases, cancer, and inflammatory reactions 7 . IL-32 is recognized as a proinflammatory cytokines and a potent inducer similar to other proinflammatory cytokines such as TNF-α and IL-8. IL-32 activates specific cytokine signaling pathways including NF-ĸB and p38 mitogen-activated protein kinases. Activation of the NF-ĸB pathway leads to inflammation, invasion of endometriosis cells, angiogenesis, proliferation, and inhibition of apoptosis processes within endometriosis cells 8 . Another cytokine pathway activated by IL-32 is the p38 mitogen-activated protein kinases pathway, which results in increased cytokine production, proliferation of endometriosis cells, and reduced apoptosis processes 9 . VEGF is a vasoactive substance involved in various physiological processes in the body, including wound healing and endometrial revascularization. Increased VEGF levels trigger excessive angiogenesis, allowing endometriosis lesions to persist and progress. Activation of VEGF receptors in endothelial cells initiates processes such as endothelial cell proliferation, migration, increased blood vessel permeability, and induction of blood vessel remodeling, ultimately leading to the formation of new blood vessels 10 . Another factor involved in endometriosis pathogenesis is vitamin D deficiency 6 . Vitamin D has anti-inflammatory benefits by reducing cytokine production in the body. The most active metabolite of vitamin D is 1,25(OH)2D3, which has been shown to have significant anti-proliferative effects in preclinical models, inhibiting cell differentiation processes, angiogenesis, and inducing apoptosis 11,12 . The role of vitamin D in relation to IL-32 is indirect, involving inhibition of NF-ĸB by direct binding to IKKβ, thereby reducing IL-32 levels 11 . Additionally, evidence suggests that vitamin D can affect the normal function of endothelial cells in angiogenesis by suppressing VEGF production through autocrine Wnt/β-catenin pathway inhibition 13 . Methods Study design This observational analytic correlational study measures independent and dependent variables simultaneously. The included subjects were patients aged 20–42 years diagnosed with endometriosis confirmed by histopathological examination. On the other hand, patients were excluded if they used hormone therapy within the last 3 months (progestin only, combination, leuprolide acetate), patients with ovarian infections, and ovarian malignancies. Sample selection was done using consecutive sampling, where endometriosis patients who visited Dr. Hasan Sadikin General Hospital Bandung, Limijati Mother and Child Hospital (RSIA), and Cibabat Regional General Hospital from January to May 2024 who met the criteria were included until the minimum sample size was met. Data collection Patients who met the criteria were informed about the study and given informed consent. Endometriosis diagnosis was established based on medical history, physical examination, transvaginal ultrasound, operative findings by Obstetrics and Gynecology specialists, and histopathological diagnostic examination by Pathology specialists. Endometriosis tissue diagnosed based on histopathological findings were sent to the Clinical Pathology Laboratory of Dr. Hasan Sadikin General Hospital Bandung. Tissue specimens underwent homogenization, protein extraction, and total protein concentration measurement, with focus on target protein concentrations. The concentrations of target proteins (1,25(OH)2D3, VEGF, and IL-32) extracted were quantified using ELISA method and spectrophotometric techniques, and results were compared with standard curves for normalization to obtain quantitative data. Statistical methods Data collected will be analyzed using SPSS version 25. A p-value less than 0.05 is considered statistically significant. Normality of numeric variables was assessed using the Shapiro-Wilk test. If the data were normally distributed, the correlation between the levels of 1,25(OH)2D3, IL-32, and VEGF levels would be analyzed using Pearson correlation. Conversely, if the data were not normally distributed, Spearman rank correlation will be used. Ethical issues This study was reviewed by the Health Research Ethics Committee of Hasan Sadikin General Hospital, number DP.04.03/D.XIV.6.5/13/2024. Permission was obtained from all participating respondents prior to initiating the study. Subject confidentiality was maintained during and after the study. All methods were performed in accordance with the relevant guidelines and regulations. Financial support This research is funded through an internal study grant from Universitas Padjadjaran ( Hibah Internal Riset Universitas Padjadjaran tahun 2024 ), with grant number 1670/UN6.3.1/PT.00/2024. Results A total of 46 patients diagnosed with endometriosis cysts, who underwent surgery either through laparoscopy or laparotomy at Dr. Hasan Sadikin General Hospital and Cibabat Regional Hospital from January to May 2024, were included in the study analysis. Table 1. Baseline Characteristic Characteristic n (%) 1. Age (years) n (%) 20–25 26–30 31–36 37–42 Mean (SD) 2. Body mass index (kg/m 2 ) Mean (SD) 3. Smoking n (%) Yes (Number/days) No 4. Infertility Yes (>12 months) No 9 (19,6) 13 (28,3) 16 (34,8) 8 (17,4) 30,8 (5,7) 24,12 (4,31) - 46 (100,0) 41 (89,1) 5 (10,9) Table 1 shows most endometrial cyst patients were found in the age group of 31—36 years (34.8%). The average age was 30.8 years (SD ± 5.7 years), indicating a wide variation in ages around the average. The average body mass index (BMI) of the patients reached 24.12 kg/m² ± 4.31 kg/m², showing that most of the BMI levels are classified as normoweight. All study subjects were recorded as non-smokers (100%). Out of all the patients in this study, it was found that only about 10.9%, or five patients, did not have infertility issues. The rest of the subjects were diagnosed with infertility. Subsequently, further bivariate analyses were conducted to uncover potential correlations among the various variables encompassed in the study, as delineated below. Table 2 Correlation between 1,25(OH) 2 D 3 levels with IL-32 in endometriosis cysts Correlation of 1,25(OH) 2 D 3 Levels with Correlation Coefficient (r) p-value IL-32 levels -0.346 0.019 *r = Spearman rank correlation Table 2 and Fig. 1 show that the levels of 1,25(OH)2D3 and IL-32 in endometriosis cysts were found to be significantly and negatively correlated in all measurements (P-value < 0.005). The correlation coefficient (r) was − 0.346 meaning that an increase in the levels of 1,25(OH)2D3 will be followed by a decrease in the levels of IL-32, and vice versa. Table 3 Correlation between 1,25(OH) 2 D 3 levels with VEGF level in endometriosis cysts Correlation of 1,25(OH) 2 D 3 Levels with Correlation Coefficient (r) p-value VEGF levels -0.316 0.032 *r = Spearman rank correlation Meanwhile, the correlation between 1,25(OH) 2 D 3 levels and VEGF in endometriosis cysts is shown in Table 3 . The analysis revealed a correlation coefficient (r) of -0.316, indicating a negative correlation between 1,25(OH) 2 D 3 and VEGF levels. This suggests that an increase in 1,25(OH) 2 D 3 levels tends to be followed by a decrease in VEGF levels, and vice versa. The correlation is weak to moderate represented by Fig. 2. However, the correlation is found to be statistically significant (p < 0.05). Table 4 Correlation between IL-32 levels with VEGF levels in endometriosis cysts Correlation of IL-32 Levels with Correlation Coefficient (r) p-value VEGF levels 0.490 0.001 *r = Spearman rank correlation Further, the correlation between IL-32 levels and VEGF in endometriosis cysts is presented in Table 4 . The analysis found a correlation coefficient (r) of 0.490 and a p-value of 0.001. This indicates a positive correlation between IL-32 and VEGF levels. Figure 3 shows that as IL-32 levels increase, VEGF levels also tend to increase, and vice versa. The p-value of 0.001 suggests that this correlation is highly statistically significant. Table 5 Correlation between IL-32 levels with BMI and duration of infertility in endometriosis cysts Correlation of IL-32 Levels with Correlation Coefficient (r) p-value BMI 0.065 0.666 Duration of infertility -0.005 0.978 *r = Spearman rank correlation Table 5 shows the correlation between IL-32 levels and BMI has a coefficient (r) of 0.065, indicating a weak positive correlation. The p-value of 0.666 shows that this result is not statistically significant. The correlation between IL-32 levels and the duration of infertility has a coefficient (r) of -0.005, indicating a very weak negative correlation, with a p-value of 0.978, showing that this result is also not statistically significant. Table 6 Correlation between IL-32 levels with VEGF levels in endometriosis cysts Correlation of VEGF Levels with Correlation Coefficient (r) p-value BMI 0.234 0.117 Duration of infertility -0.005 0.975 *r = Spearman rank correlation Table 6 shows the correlation between VEGF levels and BMI has a coefficient (r) of 0.234, indicating a weak positive correlation. The p-value of 0.117 suggests that this result is not statistically significant. The correlation between VEGF levels and the duration of infertility has a coefficient (r) of -0.005, indicating a very weak negative correlation, with a p-value of 0.975, showing that this result is also not statistically significant. Discussion Endometriosis is a chronic inflammatory disease characterized by the presence of tissue resembling endometrium outside the uterus 1 . The pathogenesis of endometriosis is complex. The growth of endometriosis lesions involves interactions among endocrine, immunological, pro-inflammatory, and pro-angiogenic processes 14 . Endometriosis can cause symptoms such as dysmenorrhea, dyspareunia, dysuria, dyschezia, rectal bleeding with pain, hematuria, catamenial pneumothorax, and infertility 1 . These issues arise due to inflammatory processes being activated, involving cytokine pathways such as increased IL-32 and the angiogenesis factor VEGF. One factor that can inhibit inflammation is vitamin D in its active form, 1,25(OH) 2 D 3 . Vitamin D plays a molecular role as an anti-proliferative, anti-inflammatory, and immunomodulatory agent 15 . In this study, subjects selected for research sample material were patients with histopathologically confirmed endometriosis cysts, ranging in age from 20 to 42 years. The majority of subjects were in the age group of 31–36 years, comprising 34.8%, followed by the age group of 26–30 years at 28.3%, and the age group of 20–25 years at 19.6%. The smallest age group range was 37–42 years, accounting for 17.4%. This is consistent with the theory that endometriosis has the highest incidence between the ages of 30 and 34 years 2 . Another characteristic examined in this study was BMI. The average BMI in the study sample was 24.12 kg/m² with a standard deviation of 4.31 kg/m², indicating that on average, subjects fell within the normal BMI category (18.5–24.9 kg/m²), with some variations towards the overweight category (25–29.9 kg/m²). These study findings align with those reported by Carson et al. 16 , indicating an average body mass index of 25 kg/m² among endometriosis patients, and all subjects in the sample were non-smokers, thereby concluding that smoking was not a confounding variable in this study. The study also investigated the duration of infertility (months) and found that 89.1% of the research subjects experienced infertility for more than 12 months, while only 10.9% did not experience infertility. These research findings are consistent with theories proposed by Taylor et al. 17 , suggesting that more than 50% of endometriosis patients experience infertility issues. This study explains that there is a negative correlation between the levels of 1,25(OH) 2 D 3 and IL-32 in endometriosis cyst tissue (r = -0.346; p = 0.019). This indicates that an increase in 1,25(OH) 2 D 3 levels is followed by a decrease in IL-32 levels, and vice versa, with a significance value of p < 0.05, showing that the results of this study are statistically significant. The findings of this study are consistent with those proposed by Bharali et al. 11 , suggesting that vitamin D can reduce IL-32 levels by suppressing the IκB kinase β (IKKβ) pathway. Another theory proposed by Setiabudiawan et al. 12 states that one of the benefits of vitamin D in reducing inflammation is by lowering cytokine levels produced by the body. This study also found a negative correlation between the levels of 1,25(OH) 2 D 3 and VEGF in endometriosis cyst tissue (r= -0.316; p = 0.032). This indicates that an increase in 1,25(OH) 2 D 3 levels tends to be followed by a decrease in VEGF levels, with a significance value of p < 0.05, demonstrating that these results are statistically significant. Vitamin D is capable of suppressing the expression of VEGF, a crucial angiogenic factor that plays a significant role in the formation of new blood vessels 18 . Similar findings have been reported in studies on patients with colorectal cancer, indicating that 1,25(OH) 2 D 3 suppresses VEGF through the Wnt/b-catenin pathway in cell nuclei indirectly by inhibiting the Notch pathway in angiogenesis processes 19 . Another study by Irani et al. 20 also showed that vitamin D3 supplementation in patients with polycystic ovary syndrome (PCOS) can reduce VEGF levels. In this study, the correlation between VEGF and IL-32 levels in endometriosis cyst tissue was also analyzed, revealing a positive correlation (r = 0.490; p = 0.001). This indicates a direct relationship between IL-32 and VEGF levels, meaning that an increase in IL-32 levels is associated with an increase in VEGF levels, and vice versa. The significance value of p < 0.05 shows that this correlation result is highly statistically significant. These findings align with the theory proposed by Shim et al. 21 , stating that IL-32 plays a crucial role in both innate and adaptive immune responses and can induce various cytokines such as TNF-α, IL-1β, IL-6, IL-8, and VEGF. Other studies by Nishida et al. 22 and Jafrin et al. 23 have explained that IL-32 can enhance angiogenesis by stimulating the production of VEGF and MMP, which affect cell migration and invasion. The confounding variables examined in this study include the correlation between IL-32 levels and BMI, which showed a positive correlation (r = 0.065; p = 0.666). This indicates a positive relationship between IL-32 levels and BMI, but because the p-value is > 0.05, these results are not statistically significant. Research by Catalan et al. 24 states that IL-32 increases in obesity and can induce inflammation processes in adipose tissue and the extracellular matrix. IL-32 is regulated in obesity, promoting inflammation and remodeling of the extracellular matrix in adipose tissue, inducing the expression of pro-inflammatory cytokines and genes related to extracellular matrix in human adipocytes 25 . This study also analyzed another confounding variable regarding the correlation between IL-32 levels and infertility duration (r= -0.005; p = 0.978). The correlation coefficient (r) being very close to zero indicates that there is almost no linear relationship between IL-32 levels and infertility duration. In this context, changes in IL-32 levels are not related to changes in infertility duration, and with a p-value > 0.05, these results are not statistically significant. This differs from the findings presented by Ehsani et al. 26 , who suggest that IL-32 can indirectly affect cytokine levels, disrupting the balance between pro-inflammatory and anti-inflammatory responses that may lead to infertility. An increase in the ratio of pro-inflammatory to anti-inflammatory cytokines occurs in women with unexplained infertility. Conclusions This study concluded that there is a statistically significant negative correlation between the levels of 1,25(OH) 2 D 3 and IL-32, as well as between the levels of 1,25(OH) 2 D 3 and VEGF. Additionally, there is a statistically significant positive correlation between VEGF and IL-32 levels. These findings are consistent with the anti-inflammatory properties of vitamin D, which can also suppress VEGF expression, thereby potentially reducing angiogenesis processes in endometriosis. The data from this study also indicates an interaction between inflammatory pathways and angiogenesis in endometriosis tissue, where increased VEGF levels are associated with elevated IL-32 levels. This study is the first to investigate the relationship between 1,25(OH) 2 D 3 levels and IL-32 and VEGF levels in endometriosis cyst tissue in Indonesia. Limitation of studies Limitations of this study include a limited sample size involving only 46 subjects, high variability in data for several variables such as infertility duration ranging from 12 to 240 months, and VEGF levels ranging from 0.01 to 2.93 ng/mL. Additionally, the observational analytical correlational design is another factor contributing to the limitations of this study. Declarations Conflict of Interest None. Author Contribution MI, HHS, MAR, and AR conceived the study. MI, SW, and MAR collected the data. WP and RA analyzed the data. MI and SW drafted the manuscript. All authors approved of this version for publication. Acknowledgements Not applicable. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Becker CM, Bokor A, Heikinheimo O, Horne A, Jansen F, Kiesel L et al. ESHRE guideline: endometriosis. 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Jafrin S, Abdul Aziz M, Islam MS. Impact of interleukin-32 germ-line rs28372698 and intronic rs12934561 polymorphisms on cancer development: A systematic review and meta-analysis. Int Immunopharmacol 2021; 99: 107964. Catalán V, Gómez-Ambrosi J, Rodríguez A, Ramírez B, Ortega VA, Hernández-Lizoain JL et al. IL-32α-induced inflammation constitutes a link between obesity and colon cancer. Oncoimmunology 2017;: e1328338. Catalán V, Gómez-Ambrosi J, Rodríguez A, Ramírez B, Valentí V, Moncada R et al. Increased Interleukin-32 Levels in Obesity Promote Adipose Tissue Inflammation and Extracellular Matrix Remodeling: Effect of Weight Loss. Diabetes 2016; 65: 3636–3648. Ehsani M, Mohammadnia-Afrouzi M, Mirzakhani M, Esmaeilzadeh S, Shahbazi M. Female Unexplained Infertility: A Disease with Imbalanced Adaptive Immunity. J Hum Reprod Sci 2019; 12: 274–282. Additional Declarations No competing interests reported. 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01:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4759160/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4759160/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62661303,"identity":"1b75d71e-18ce-48f2-9608-0e3c27cb6fc0","added_by":"auto","created_at":"2024-08-17 02:39:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20962,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels with IL-32 levels in endometriosis cysts\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4759160/v1/af34d42e3c9b6ceb46121dca.jpg"},{"id":62661751,"identity":"3912845a-8fe8-4321-a449-1096f90973b5","added_by":"auto","created_at":"2024-08-17 02:47:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22005,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels with VEGF level in endometriosis cysts\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4759160/v1/e9a46d9fdfd386a9c9e6cc8c.jpg"},{"id":62661301,"identity":"e3364ad3-4f58-4c32-a44f-5610032b8a14","added_by":"auto","created_at":"2024-08-17 02:39:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18256,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between IL-32 levels with VEGF level in endometriosis cysts\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4759160/v1/3aa85f2cb59ec986345aa125.jpg"},{"id":63099985,"identity":"f33a033a-b24d-4d7f-9d34-f0b2f0fafcd4","added_by":"auto","created_at":"2024-08-23 06:47:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":522932,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4759160/v1/d26dcbf4-a7f7-44a7-a08a-758a79b0cb20.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Relationship Between 1,25(OH)2D3 Levels and Interleukin-32 (IL-32) and Vascular Endothelial Growth Factor (VEGF) Levels in Endometriosis Cyst Tissue","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is a chronic inflammatory disease characterized by the presence of tissue similar to the endometrium outside the uterus \u003csup\u003e1\u003c/sup\u003e. The age range of patients diagnosed with endometriosis is between 20\u0026ndash;42 years. The prevalence of endometriosis is estimated to be between 2\u0026ndash;10% in the female population worldwide, and up to 50% of infertility cases are associated with endometriosis \u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePotential origins of endometriosis lesions include the transplantation of endometrial tissue through retrograde menstruation and in situ metaplasia of coelomic epithelium. Lesions outside the pelvis may be caused by vascular or lymphatic metastasis \u003csup\u003e2\u003c/sup\u003e. Superficial and deep infiltrating endometriosis lesions form through molecular mechanisms involving interactions between adhesion, cell proliferation, systemic and local steroidogenesis, chronic inflammation, immune dysregulation, vascularization, and innervation \u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe chronic inflammatory response in endometriosis involves both innate and adaptive immune responses influenced by estrogen hormones \u003csup\u003e4\u003c/sup\u003e. The initial innate immune response involves macrophage phagocytosis mediated by scavenger receptors and regulated by cytokines. Patients with endometriosis exhibit abnormalities in their innate immune response, where macrophages fail to perform phagocytic functions on endometriosis lesions, particularly during the mediation process by scavenger receptors \u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe adaptive immune system becomes involved when the innate immune response is insufficient to handle the pathological conditions. The adaptive immune system consists primarily of T and B lymphocytes. In patients with endometriosis, there is an increase in cytokine modulation by T cells and the formation of autoantibodies by B cells \u003csup\u003e6\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCytokines are proteins produced by various cells including stromal cells, fibroblasts, and endothelial cells. Interleukin-32 (IL-32) is one among proinflammatory cytokines thought to be involved in the pathogenesis of endometriosis. IL-32 has diverse functions such as regulating the body's immune response during infection, contributing to autoimmune diseases, cancer, and inflammatory reactions \u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIL-32 is recognized as a proinflammatory cytokines and a potent inducer similar to other proinflammatory cytokines such as TNF-α and IL-8. IL-32 activates specific cytokine signaling pathways including NF-ĸB and p38 mitogen-activated protein kinases. Activation of the NF-ĸB pathway leads to inflammation, invasion of endometriosis cells, angiogenesis, proliferation, and inhibition of apoptosis processes within endometriosis cells \u003csup\u003e8\u003c/sup\u003e. Another cytokine pathway activated by IL-32 is the p38 mitogen-activated protein kinases pathway, which results in increased cytokine production, proliferation of endometriosis cells, and reduced apoptosis processes \u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVEGF is a vasoactive substance involved in various physiological processes in the body, including wound healing and endometrial revascularization. Increased VEGF levels trigger excessive angiogenesis, allowing endometriosis lesions to persist and progress. Activation of VEGF receptors in endothelial cells initiates processes such as endothelial cell proliferation, migration, increased blood vessel permeability, and induction of blood vessel remodeling, ultimately leading to the formation of new blood vessels \u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother factor involved in endometriosis pathogenesis is vitamin D deficiency \u003csup\u003e6\u003c/sup\u003e. Vitamin D has anti-inflammatory benefits by reducing cytokine production in the body. The most active metabolite of vitamin D is 1,25(OH)2D3, which has been shown to have significant anti-proliferative effects in preclinical models, inhibiting cell differentiation processes, angiogenesis, and inducing apoptosis \u003csup\u003e11,12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe role of vitamin D in relation to IL-32 is indirect, involving inhibition of NF-ĸB by direct binding to IKKβ, thereby reducing IL-32 levels \u003csup\u003e11\u003c/sup\u003e. Additionally, evidence suggests that vitamin D can affect the normal function of endothelial cells in angiogenesis by suppressing VEGF production through autocrine Wnt/β-catenin pathway inhibition \u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis observational analytic correlational study measures independent and dependent variables simultaneously. The included subjects were patients aged 20\u0026ndash;42 years diagnosed with endometriosis confirmed by histopathological examination. On the other hand, patients were excluded if they used hormone therapy within the last 3 months (progestin only, combination, leuprolide acetate), patients with ovarian infections, and ovarian malignancies. Sample selection was done using consecutive sampling, where endometriosis patients who visited Dr. Hasan Sadikin General Hospital Bandung, Limijati Mother and Child Hospital (RSIA), and Cibabat Regional General Hospital from January to May 2024 who met the criteria were included until the minimum sample size was met.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData collection\u003c/h2\u003e \u003cp\u003ePatients who met the criteria were informed about the study and given informed consent. Endometriosis diagnosis was established based on medical history, physical examination, transvaginal ultrasound, operative findings by Obstetrics and Gynecology specialists, and histopathological diagnostic examination by Pathology specialists. Endometriosis tissue diagnosed based on histopathological findings were sent to the Clinical Pathology Laboratory of Dr. Hasan Sadikin General Hospital Bandung.\u003c/p\u003e \u003cp\u003eTissue specimens underwent homogenization, protein extraction, and total protein concentration measurement, with focus on target protein concentrations. The concentrations of target proteins (1,25(OH)2D3, VEGF, and IL-32) extracted were quantified using ELISA method and spectrophotometric techniques, and results were compared with standard curves for normalization to obtain quantitative data.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical methods\u003c/h3\u003e\n\u003cp\u003eData collected will be analyzed using SPSS version 25. A p-value less than 0.05 is considered statistically significant. Normality of numeric variables was assessed using the Shapiro-Wilk test. If the data were normally distributed, the correlation between the levels of 1,25(OH)2D3, IL-32, and VEGF levels would be analyzed using Pearson correlation. Conversely, if the data were not normally distributed, Spearman rank correlation will be used.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEthical issues\u003c/h2\u003e \u003cp\u003e This study was reviewed by the Health Research Ethics Committee of Hasan Sadikin General Hospital, number DP.04.03/D.XIV.6.5/13/2024. Permission was obtained from all participating respondents prior to initiating the study. Subject confidentiality was maintained during and after the study. All methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFinancial support\u003c/h2\u003e \u003cp\u003eThis research is funded through an internal study grant from Universitas Padjadjaran (\u003cem\u003eHibah Internal Riset Universitas Padjadjaran tahun 2024\u003c/em\u003e), with grant number 1670/UN6.3.1/PT.00/2024.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 46 patients diagnosed with endometriosis cysts, who underwent surgery either through laparoscopy or laparotomy at Dr. Hasan Sadikin General Hospital and Cibabat Regional Hospital from January to May 2024, were included in the study analysis.\u003c/p\u003e\u003cp\u003eTable 1. Baseline Characteristic\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"602\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"58.23627287853577%\"\u003e\u003cstrong\u003eCharacteristic\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"41.76372712146423%\" valign=\"top\"\u003e\u003cstrong\u003en (%)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"58.23627287853577%\" valign=\"top\"\u003e1. Age (years) n (%)\u003cbr\u003e20\u0026ndash;25\u003cbr\u003e26\u0026ndash;30\u003cbr\u003e31\u0026ndash;36\u003cbr\u003e37\u0026ndash;42\u003cbr\u003eMean (SD)\u003cbr\u003e2. Body mass index (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003cbr\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Mean (SD)\u003cbr\u003e3. Smoking n (%)\u003cbr\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Yes (Number/days)\u003cbr\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;No\u003cbr\u003e4. Infertility\u0026nbsp;\u003cbr\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Yes (\u0026gt;12 months)\u003cbr\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;No\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"41.76372712146423%\" valign=\"top\"\u003e\u0026nbsp;\u003cbr\u003e9 (19,6)\u003cbr\u003e13 (28,3)\u003cbr\u003e16 (34,8)\u003cbr\u003e8 (17,4)\u003cbr\u003e30,8 (5,7)\u003cbr\u003e\u0026nbsp;\u003cbr\u003e24,12 (4,31)\u003cbr\u003e\u0026nbsp;\u003cbr\u003e-\u003cbr\u003e46 (100,0)\u003cbr\u003e\u0026nbsp;\u003cbr\u003e41 (89,1)\u003cbr\u003e5 (10,9)\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cp\u003e Table 1 shows most endometrial cyst patients were found in the age group of 31\u0026mdash;36 years (34.8%). The average age was 30.8 years (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7 years), indicating a wide variation in ages around the average. The average body mass index (BMI) of the patients reached 24.12 kg/m\u0026sup2; \u0026plusmn; 4.31 kg/m\u0026sup2;, showing that most of the BMI levels are classified as normoweight. All study subjects were recorded as non-smokers (100%). Out of all the patients in this study, it was found that only about 10.9%, or five patients, did not have infertility issues. The rest of the subjects were diagnosed with infertility. Subsequently, further bivariate analyses were conducted to uncover potential correlations among the various variables encompassed in the study, as delineated below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels with IL-32 in endometriosis cysts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorrelation of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e Levels with\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation Coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-32 levels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*r\u0026thinsp;=\u0026thinsp;Spearman rank correlation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;1 show that the levels of 1,25(OH)2D3 and IL-32 in endometriosis cysts were found to be significantly and negatively correlated in all measurements (P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.005). The correlation coefficient (r) was \u0026minus;\u0026thinsp;0.346 meaning that an increase in the levels of 1,25(OH)2D3 will be followed by a decrease in the levels of IL-32, and vice versa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels with VEGF level in endometriosis cysts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorrelation of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e Levels with\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation Coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF levels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*r\u0026thinsp;=\u0026thinsp;Spearman rank correlation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMeanwhile, the correlation between 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels and VEGF in endometriosis cysts is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The analysis revealed a correlation coefficient (r) of -0.316, indicating a negative correlation between 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and VEGF levels. This suggests that an increase in 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels tends to be followed by a decrease in VEGF levels, and vice versa. The correlation is weak to moderate represented by Fig.\u0026nbsp;2. However, the correlation is found to be statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between IL-32 levels with VEGF levels in endometriosis cysts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorrelation of IL-32 Levels with\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation Coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF levels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*r\u0026thinsp;=\u0026thinsp;Spearman rank correlation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFurther, the correlation between IL-32 levels and VEGF in endometriosis cysts is presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The analysis found a correlation coefficient (r) of 0.490 and a p-value of 0.001. This indicates a positive correlation between IL-32 and VEGF levels. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that as IL-32 levels increase, VEGF levels also tend to increase, and vice versa. The p-value of 0.001 suggests that this correlation is highly statistically significant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between IL-32 levels with BMI and duration of infertility in endometriosis cysts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorrelation of IL-32 Levels with\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation Coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.666\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of infertility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.978\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*r\u0026thinsp;=\u0026thinsp;Spearman rank correlation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the correlation between IL-32 levels and BMI has a coefficient (r) of 0.065, indicating a weak positive correlation. The p-value of 0.666 shows that this result is not statistically significant. The correlation between IL-32 levels and the duration of infertility has a coefficient (r) of -0.005, indicating a very weak negative correlation, with a p-value of 0.978, showing that this result is also not statistically significant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between IL-32 levels with VEGF levels in endometriosis cysts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorrelation of VEGF Levels with\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation Coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of infertility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.975\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*r\u0026thinsp;=\u0026thinsp;Spearman rank correlation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the correlation between VEGF levels and BMI has a coefficient (r) of 0.234, indicating a weak positive correlation. The p-value of 0.117 suggests that this result is not statistically significant. The correlation between VEGF levels and the duration of infertility has a coefficient (r) of -0.005, indicating a very weak negative correlation, with a p-value of 0.975, showing that this result is also not statistically significant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis is a chronic inflammatory disease characterized by the presence of tissue resembling endometrium outside the uterus \u003csup\u003e1\u003c/sup\u003e. The pathogenesis of endometriosis is complex. The growth of endometriosis lesions involves interactions among endocrine, immunological, pro-inflammatory, and pro-angiogenic processes \u003csup\u003e14\u003c/sup\u003e. Endometriosis can cause symptoms such as dysmenorrhea, dyspareunia, dysuria, dyschezia, rectal bleeding with pain, hematuria, catamenial pneumothorax, and infertility \u003csup\u003e1\u003c/sup\u003e. These issues arise due to inflammatory processes being activated, involving cytokine pathways such as increased IL-32 and the angiogenesis factor VEGF. One factor that can inhibit inflammation is vitamin D in its active form, 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e. Vitamin D plays a molecular role as an anti-proliferative, anti-inflammatory, and immunomodulatory agent \u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, subjects selected for research sample material were patients with histopathologically confirmed endometriosis cysts, ranging in age from 20 to 42 years. The majority of subjects were in the age group of 31\u0026ndash;36 years, comprising 34.8%, followed by the age group of 26\u0026ndash;30 years at 28.3%, and the age group of 20\u0026ndash;25 years at 19.6%. The smallest age group range was 37\u0026ndash;42 years, accounting for 17.4%. This is consistent with the theory that endometriosis has the highest incidence between the ages of 30 and 34 years \u003csup\u003e2\u003c/sup\u003e. Another characteristic examined in this study was BMI. The average BMI in the study sample was 24.12 kg/m\u0026sup2; with a standard deviation of 4.31 kg/m\u0026sup2;, indicating that on average, subjects fell within the normal BMI category (18.5\u0026ndash;24.9 kg/m\u0026sup2;), with some variations towards the overweight category (25\u0026ndash;29.9 kg/m\u0026sup2;). These study findings align with those reported by Carson et al. \u003csup\u003e16\u003c/sup\u003e, indicating an average body mass index of 25 kg/m\u0026sup2; among endometriosis patients, and all subjects in the sample were non-smokers, thereby concluding that smoking was not a confounding variable in this study. The study also investigated the duration of infertility (months) and found that 89.1% of the research subjects experienced infertility for more than 12 months, while only 10.9% did not experience infertility. These research findings are consistent with theories proposed by Taylor et al. \u003csup\u003e17\u003c/sup\u003e, suggesting that more than 50% of endometriosis patients experience infertility issues.\u003c/p\u003e \u003cp\u003eThis study explains that there is a negative correlation between the levels of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and IL-32 in endometriosis cyst tissue (r = -0.346; p\u0026thinsp;=\u0026thinsp;0.019). This indicates that an increase in 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels is followed by a decrease in IL-32 levels, and vice versa, with a significance value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, showing that the results of this study are statistically significant. The findings of this study are consistent with those proposed by Bharali et al. \u003csup\u003e11\u003c/sup\u003e, suggesting that vitamin D can reduce IL-32 levels by suppressing the IκB kinase β (IKKβ) pathway. Another theory proposed by Setiabudiawan et al. \u003csup\u003e12\u003c/sup\u003e states that one of the benefits of vitamin D in reducing inflammation is by lowering cytokine levels produced by the body. This study also found a negative correlation between the levels of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and VEGF in endometriosis cyst tissue (r= -0.316; p\u0026thinsp;=\u0026thinsp;0.032). This indicates that an increase in 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels tends to be followed by a decrease in VEGF levels, with a significance value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, demonstrating that these results are statistically significant. Vitamin D is capable of suppressing the expression of VEGF, a crucial angiogenic factor that plays a significant role in the formation of new blood vessels \u003csup\u003e18\u003c/sup\u003e. Similar findings have been reported in studies on patients with colorectal cancer, indicating that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e suppresses VEGF through the Wnt/b-catenin pathway in cell nuclei indirectly by inhibiting the Notch pathway in angiogenesis processes \u003csup\u003e19\u003c/sup\u003e. Another study by Irani et al. \u003csup\u003e20\u003c/sup\u003e also showed that vitamin D3 supplementation in patients with polycystic ovary syndrome (PCOS) can reduce VEGF levels.\u003c/p\u003e \u003cp\u003eIn this study, the correlation between VEGF and IL-32 levels in endometriosis cyst tissue was also analyzed, revealing a positive correlation (r\u0026thinsp;=\u0026thinsp;0.490; p\u0026thinsp;=\u0026thinsp;0.001). This indicates a direct relationship between IL-32 and VEGF levels, meaning that an increase in IL-32 levels is associated with an increase in VEGF levels, and vice versa. The significance value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 shows that this correlation result is highly statistically significant. These findings align with the theory proposed by Shim et al. \u003csup\u003e21\u003c/sup\u003e, stating that IL-32 plays a crucial role in both innate and adaptive immune responses and can induce various cytokines such as TNF-α, IL-1β, IL-6, IL-8, and VEGF. Other studies by Nishida et al. \u003csup\u003e22\u003c/sup\u003e and Jafrin et al. \u003csup\u003e23\u003c/sup\u003e have explained that IL-32 can enhance angiogenesis by stimulating the production of VEGF and MMP, which affect cell migration and invasion. The confounding variables examined in this study include the correlation between IL-32 levels and BMI, which showed a positive correlation (r\u0026thinsp;=\u0026thinsp;0.065; p\u0026thinsp;=\u0026thinsp;0.666). This indicates a positive relationship between IL-32 levels and BMI, but because the p-value is \u0026gt;\u0026thinsp;0.05, these results are not statistically significant. Research by Catalan et al. \u003csup\u003e24\u003c/sup\u003e states that IL-32 increases in obesity and can induce inflammation processes in adipose tissue and the extracellular matrix. IL-32 is regulated in obesity, promoting inflammation and remodeling of the extracellular matrix in adipose tissue, inducing the expression of pro-inflammatory cytokines and genes related to extracellular matrix in human adipocytes \u003csup\u003e25\u003c/sup\u003e. This study also analyzed another confounding variable regarding the correlation between IL-32 levels and infertility duration (r= -0.005; p\u0026thinsp;=\u0026thinsp;0.978). The correlation coefficient (r) being very close to zero indicates that there is almost no linear relationship between IL-32 levels and infertility duration. In this context, changes in IL-32 levels are not related to changes in infertility duration, and with a p-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05, these results are not statistically significant. This differs from the findings presented by Ehsani et al. \u003csup\u003e26\u003c/sup\u003e, who suggest that IL-32 can indirectly affect cytokine levels, disrupting the balance between pro-inflammatory and anti-inflammatory responses that may lead to infertility. An increase in the ratio of pro-inflammatory to anti-inflammatory cytokines occurs in women with unexplained infertility.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study concluded that there is a statistically significant negative correlation between the levels of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and IL-32, as well as between the levels of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and VEGF. Additionally, there is a statistically significant positive correlation between VEGF and IL-32 levels. These findings are consistent with the anti-inflammatory properties of vitamin D, which can also suppress VEGF expression, thereby potentially reducing angiogenesis processes in endometriosis. The data from this study also indicates an interaction between inflammatory pathways and angiogenesis in endometriosis tissue, where increased VEGF levels are associated with elevated IL-32 levels. This study is the first to investigate the relationship between 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e levels and IL-32 and VEGF levels in endometriosis cyst tissue in Indonesia.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLimitation of studies\u003c/h2\u003e \u003cp\u003eLimitations of this study include a limited sample size involving only 46 subjects, high variability in data for several variables such as infertility duration ranging from 12 to 240 months, and VEGF levels ranging from 0.01 to 2.93 ng/mL. Additionally, the observational analytical correlational design is another factor contributing to the limitations of this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eNone.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMI, HHS, MAR, and AR conceived the study. MI, SW, and MAR collected the data. WP and RA analyzed the data. MI and SW drafted the manuscript. All authors approved of this version for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during 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\u003eBecker CM, Bokor A, Heikinheimo O, Horne A, Jansen F, Kiesel L \u003cem\u003eet al.\u003c/em\u003e ESHRE guideline: endometriosis. \u003cem\u003eHum Reprod Open\u003c/em\u003e 2022; 2022. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/hropen/hoac009\u003c/span\u003e\u003cspan address=\"10.1093/hropen/hoac009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOral E (ed.). \u003cem\u003eEndometriosis and Adenomyosis\u003c/em\u003e. 1st ed. 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Int Immunopharmacol 2021; 99: 107964.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCatal\u0026aacute;n V, G\u0026oacute;mez-Ambrosi J, Rodr\u0026iacute;guez A, Ram\u0026iacute;rez B, Ortega VA, Hern\u0026aacute;ndez-Lizoain JL \u003cem\u003eet al.\u003c/em\u003e IL-32α-induced inflammation constitutes a link between obesity and colon cancer. Oncoimmunology 2017;: e1328338.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCatal\u0026aacute;n V, G\u0026oacute;mez-Ambrosi J, Rodr\u0026iacute;guez A, Ram\u0026iacute;rez B, Valent\u0026iacute; V, Moncada R \u003cem\u003eet al.\u003c/em\u003e Increased Interleukin-32 Levels in Obesity Promote Adipose Tissue Inflammation and Extracellular Matrix Remodeling: Effect of Weight Loss. Diabetes 2016; 65: 3636\u0026ndash;3648.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEhsani M, Mohammadnia-Afrouzi M, Mirzakhani M, Esmaeilzadeh S, Shahbazi M. 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J Hum Reprod Sci 2019; 12: 274\u0026ndash;282.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"calcitriol, endometriosis, interleukin-32, vascular endothelial growth factor","lastPublishedDoi":"10.21203/rs.3.rs-4759160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4759160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEndometriosis involves the presence of endometrial-like tissue outside the uterus, driven by inflammatory pathways including elevated Interleukin-32 (IL-32) and Vascular Endothelial Growth Factor (VEGF). One potential anti-inflammatory agent is active vitamin D (1,25(OH)2D3). This research investigates the correlation among 1,25(OH)2D3 levels, IL-32, and VEGF in 46 confirmed endometriosis cases. Tissue samples were collected from patients at several hospitals, and assays were conducted at the Clinical Pathology Laboratory of Dr. Hasan Sadikin General Hospital using enzyme-linked immunosorbent assay (ELISA). The study revealed significant negative correlations between 1,25(OH)2D3 and both IL-32 (r= -0.346; p\u0026thinsp;=\u0026thinsp;0.019) and VEGF levels (r= -0.316; p\u0026thinsp;=\u0026thinsp;0.032). Conversely, there was a significant positive correlation between VEGF and IL-32 (r\u0026thinsp;=\u0026thinsp;0.490; p\u0026thinsp;=\u0026thinsp;0.001). These findings underscore the role of active vitamin D in mitigating inflammation associated with endometriosis by reducing IL-32 and VEGF, critical factors in inflammatory responses and blood vessel formation. Moreover, the positive association between VEGF and IL-32 highlights their collaborative role in the inflammatory processes underlying endometriosis. This study contributes to understanding how vitamin D may modulate key inflammatory pathways implicated in the pathogenesis of this condition.\u003c/p\u003e","manuscriptTitle":"The Relationship Between 1,25(OH)2D3 Levels and Interleukin-32 (IL-32) and Vascular Endothelial Growth Factor (VEGF) Levels in Endometriosis Cyst Tissue","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-17 02:39:47","doi":"10.21203/rs.3.rs-4759160/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b6328c0-f131-4f89-88f4-ec8a60ca9563","owner":[],"postedDate":"August 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35127465,"name":"Health sciences/Diseases"},{"id":35127466,"name":"Health sciences/Diseases/Reproductive disorders"},{"id":35127467,"name":"Health sciences/Diseases/Reproductive disorders/Infertility"}],"tags":[],"updatedAt":"2024-08-23T06:47:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-17 02:39:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4759160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4759160","identity":"rs-4759160","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}