The role of calcium homeostasis in endometriosis: a comprehensive study of multiple types of Mendelian randomization

The role of calcium homeostasis in endometriosis: a comprehensive study of multiple types of Mendelian randomization | 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 The role of calcium homeostasis in endometriosis: a comprehensive study of multiple types of Mendelian randomization Zhi-Min Deng, Fang-Fang Dai, Rui-Qi Wang, Xiao Yang, Yan-Xiang Cheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3273922/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 Background: Endometriosis is considered to be a complex gynecological disorder that affects the health and quality of life of affected women. The etiology and pathogenesis of endometriosis remain unclear, and few modifiable risk factors have been identified. It is generally believed that endometriosis is a chronic inflammatory disease, which can cause local immune disorders. And calcium homeostasis of the body is closely related to inflammation and immunity. Although observational studies have assessed the association between calcium homeostasis regulatory factor levels and endometriosis risk, the conclusions have been inconsistent. Therefore, the aim of this study was to explore the causal relationship between calcium homeostasis regulators and endometriosis risk using publicly available genome-wide association studies (GWAS) aggregated statistics. Methods: The Mendelian randomization (MR) analysis was performed using GWAS data, including calcium (N = 315,153), serum 25-Hydroxyvitamin D (25(OH)D) (N = 496,946), parathyroid hormone (PTH) (N = 3,301), endometriosis (N = 77,257), endometriosis of ovary (N = 72,200), endometriosis of rectovaginal septum and vagina (N = 70,329), endometriosis of intestine (N = 69,146), endometriosis of fallopian tube (N = 69,085), endometriosis of pelvic peritoneum (N = 71,922), endometriosis of uterus (N = 71,341), and Unspecified/other endometriosis (N = 70,404). Four levels of MR analysis were undertaken, starting with single univariate MR and multivariate MR to test the correlation between calcium homeostasis regulatory factors and endometriosis, followed by inverse MR to explore the effect of endometriosis on body calcium homeostasis. And further two-sample MR to probe the relationship between calcium levels and endometriosis subtypes. Cochran's Q test, MR-Egger intercept test, leave-one-out analysis and funnel plot were utilized for sensitivity analysis. Results: The two-sample MR analysis revealed a strong positive causal relationship between genetically predicted calcium levels and endometriosis risk (IVW: OR = 1.15, 95% CI: 1.02-1.29, p = 0.018). Notably, the results of MVMR analysis demonstrated that the positive correlation of calcium levels on endometriosis still held even after correction for 25(OH)D and PTH (OR = 1.14, 95% CI: 1.02-1.28, p = 0.026). After removing outliers using MR-PRESSO to ensure that horizontal pleiotropy was eliminated, MVMR analysis was performed again, and the causal association between calcium levels and endometriosis remained significant (OR = 1.13, 95% CI: 1.01-1.27, p = 0.033). The inverse MR analysis discovered a causal association between endometriosis and 25(OH)D (β = 0.01, 95% CI: 0.00-0.02, p = 0.007) and calcium (β = 0.02, 95% CI: 0.00-0.04, p = 0.035). The two-sample MR analysis we employed to further investigate that calcium levels were positively and causally associated only with endometriosis of uterus (IVW: OR = 1.23, 95% CI: 1.01-1.49, p = 0.038), with no suggestion of a causal relationship with the risk of other types of endometriosis subtypes. Conclusion：The comprehensive study of multiple types of MR provides genetic evidence for a causal relationship between calcium homeostasis and endometriosis risk, demonstrating that calcium levels are a risk factor for endometriosis. It also emphasizes the importance of monitoring calcium levels in patients suffering from endometriosis, which may provide dietary guidance for patients with endometriosis. Mendelian randomization calcium parathyroid hormone 25-Hydroxyvitamin D dietary guidance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Endometriosis is a common women's disease, causing dysmenorrhea and chronic pelvic pain, and even making many women of childbearing age infertile, seriously affecting their health and quality of life[ 1 ]. As the name implies, endometriosis is a condition in which endometrial tissue, which originally grows in the uterine cavity, grows outside the uterine cavity, such as ovaries, fallopian tubes, uterine surface, cavum douglasi, vagina, pelvic peritoneum, intestine, etc., with the characteristics of proliferation, diffusion, and metastasis (Fig. 1 A). While many factors, including hormones, inflammation, genetic factors, and environmental factors, are thought to fuel the development of endometriosis[ 2 ], its etiology and pathogenesis have not been fully elucidated, and few modifiable risk factors have been identified. There remains an unmet need to improve understanding of the pathophysiological mechanisms of endometriosis to develop more effective treatments. Calcium is an important ion in cell signaling, hormone regulation, and bone health, and performs important physiological functions within cells, such as excitatory regulation of neurons, muscle contraction, and apoptosis. Under normal circumstances, the intestinal tract, especially the small intestine, is the main part of calcium absorption in the body, and vitamin D can promote the absorption of calcium and phosphorus from the intestinal mucosal cells[ 3 ]. Parathyroid hormone (PTH) regulates the metabolism of calcium and phosphorus through small intestine, bone and kidney[ 4 ], in which bone relies on the balance of osteoblasts and osteoclasts to regulate the level of calcium, while kidney regulates the excretion of blood calcium through reabsorption and filtration mechanisms[ 5 ]. 25-Hydroxyvitamin D (25(OH)D) is the form of vitamin D stored in the liver and transported in the blood, and is the basis for clinical determine of vitamin D levels[ 6 ]. Overall, vitamin D (primarily 25 (OH) D), PTH, and serum calcium act synergistically to regulate extracellular calcium homeostasis in humans (Fig. 1 B). Imbalances of calcium homeostasis can lead to a variety of diseases, such as cardiovascular diseases[ 7 ], neurological diseases[ 8 ] and abnormal bone metabolism[ 9 ]. Studies on calcium homeostasis and endometriosis are relatively scarce. In a large prospective cohort study of 1,385 patients with laparoscopically confirmed endometriosis, investigators observed a significantly lower incidence of endometriosis in women with higher levels of predicted plasma 25(OH)D as well as the amount of calcium and vitamin D ingested from food[ 10 ]. At the molecular mechanisms level, researches have attempted to alter calcium homeostasis to achieve inhibition of endometriosis at the cellular level[ 11 , 12 ]. Although observational and basic laboratory studies have suggested that endometriosis risk may be influenced by organismal calcium homeostasis, the causal relationship between the two remains unclear. The existence of residual confounding and potential reverse causality in traditional observational studies poses a major challenge to exploring the causal effect of the two. Thus, elucidating the causal relationship between body calcium homeostasis and endometriosis risk may have important implications for the prevention and treatment of endometriosis. Mendelian randomization (MR) is an emerging statistical method for assessing the causal relationship between an exposure of interest and an outcome using genetic variants (i.e., single nucleotide polymorphisms (SNPs)) as instrumental variables (IVs). It takes advantage of genetic variation that exists in nature to classify individuals into different genotypes, thus simulating the effects of randomized trials to assess whether the effect of a factor on a disease is causal[ 13 ]. This approach avoids the effects of confounding factors and reverse causality in observational studies, thereby increasing the confidence of causal inference[ 14 ]. Utilizing the existing genome-wide association studies (GWASs) databases, our study determined to answer the following three key questions by applying MR analysis: (1) what is the correlation between calcium homeostasis regulatory factors (calcium, 25 (OH) D, and PTH) and endometriosis; (2) presumptive causal relationship between calcium homeostasis regulatory factors (calcium, 25 (OH) D, and PTH) and endometriosis; (3) what is the effect of calcium levels on different types of endometrioses? 2. Materials and methods 2.1 Study design We conducted two-sample MR analysis using the publicly available GWAS datasets on calcium homeostasis regulators (calcium, 25 (OH) D, and PTH) and endometriosis. To improve the confidence of the results, in addition to single univariate MR (SVMR) analysis of SNPs for each calcium homeostasis regulators separately, we also performed multivariate MR (MVMR). Meanwhile, reverse MR analysis was also applied to explore the effects of endometriosis on calcium homeostasis in patients' organisms. This reverse causality analysis will help to further determine the deep relationship between the two. In addition, the level of calcium, a key to calcium homeostasis, and different types of endometrioses was further explored, including endometriosis of ovary, abbreviated as EO, endometriosis of rectovaginal septum and vagina (EV), endometriosis of intestine (EI), endometriosis of fallopian tube (ET), endometriosis of pelvic peritoneum (EP), unspecified/other endometriosis (EUO), endometriosis of uterus (EU). An overview and ideas of the research design is shown in Fig. 2 . 2.2 Data source We obtained the GWAS of PTH from the previously published GWAS dataset for European pedigrees[ 15 ]. And GWAS data for serum 25(OH)D levels was available from the European Bioinformatics Institute (EBI)[ 16 ], while calcium GWAS data were gain from the UK Biobank (UKBB). Data on endometriosis and its sub phenotypes were derived from the FinnGen Research Program's Finnish Biobank, including endometriosis (8,288 cases, 68,969 controls), EO (3,231 cases, 68,969 controls), ER (1,360 cases, 68,969 controls), EI (177 cases, 68,969 controls), ET (116 cases, 68,969 controls), EP (2,953 cases, 68,969 controls), EUO (1,435 cases, 68,969 controls), and EU (2,372 cases, 68,969 controls). There was no sample overlap between the exposure and outcome datasets, and all data samples were of European ethnicity. Ethical approval and informed consent records were granted for all original studies. Details of the data sources used are shown in Table 1 . Table 1 Basic characteristics for selected summary-level GWASs applied in MR study. GWAS ID Traits Year Ethnicity Sample size Cases SNP size Author/consortium Link ebi-a-GCST90000618 Serum 25-Hydroxyvitamin D levels 2020 European 496,946 NA 6,896,093 Revez JA https://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90000618/ ukb-d-30680_irnt Calcium 2018 European 315,153 NA 13,585,309 Neale lab https://gwas.mrcieu.ac.uk/datasets/ukb-d-30680_irnt/ prot-a-2431 Parathyroid hormone 2018 European 3,301 NA 10,534,735 Sun BB https://gwas.mrcieu.ac.uk/datasets/prot-a-2431/ finn-b-N14_ENDOMETRIOSIS Endometriosis 2021 European 77,257 8,288 16,377,306 FinnGen team Trait: Endometriosis - IEU OpenGWAS project (mrcieu.ac.uk) finn-b-N14_ENDOMETRIOSIS_OVARY Endometriosis of ovary 2021 European 72,200 3,231 16,376,686 FinnGen team Trait: Endometriosis of ovary - IEU OpenGWAS project (mrcieu.ac.uk) finn-b-N14_ENDOMETRIOSIS_RECTPVAGSEPT_VAGINA Endometriosis of rectovaginal septum and vagina 2021 European 70,329 1,360 16,376,472 FinnGen team https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_RECTPVAGSEPT_VAGINA/ finn-b-N14_ENDOMETRIOSIS_INTESTINE Endometriosis of intestine 2021 European 69,146 177 16,376,157 FinnGen team https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_INTESTINE/ finn-b-N14_ENDOMETRIOSIS_FALLOPIAN_TUBE Endometriosis of fallopian tube 2021 European 69,085 116 16,376,156 FinnGen team https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_FALLOPIAN_TUBE/ finn-b-N14_ENDOMETRIOSIS_PELVICPERITONEUM Endometriosis of pelvic peritoneum 2021 European 71,922 2,953 16,376,599 FinnGen team Trait: Endometriosis of pelvic peritoneum - IEU OpenGWAS project (mrcieu.ac.uk) finn-b-N14_ENDOMETRIOSIS_NOS Unspecified/other endometriosis 2021 European 70,404 1,435 16,376,331 FinnGen team https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_NOS/ finn-b-N14_ENDOMETRIOSIS_UTERUS Endometriosis of uterus 2021 European 71,341 2,372 16,376,529 FinnGen team Trait: Endometriosis of uterus - IEU OpenGWAS project (mrcieu.ac.uk) 2.3 IVs selection IVs were obtained from the exposure dataset for MR analysis through the following quality control steps to ensure the accuracy of the causal conclusions of the exposure factors against the outcome factors. 1) Select genome-wide significant SNPs for exposure traits (Two-sample MR and inverse MR: p < 5×10 − 8 for calcium/25(OH)D/endometriosis, p < 5×10 − 6 for PTH; MVMR: p < 5×10 − 6 for calcium/25(OH)D/PTH/endometriosis); 2) The selected SNPs were tested for linkage disequilibrium (LD) to ensure the validity of the data. We employed a clumping procedure to filter independent SNPs (clumping distance = 10,000 kb and r 2 < 0.001 threshold; 3) Excluded SNPs with F-statistics < 10. The formula for F- statistic was shown as following: $$F=\frac{{R}^{2}\times \left(\text{N}-\text{K}-1\right)}{\text{K}\times \left(1-{R}^{2}\right)}$$ where N denotes the sample size of the included GWAS data, K indicates the number of SNPs subjected to MR analysis, and R 2 represents the variance corresponding to each exposed SNP; 4) Excluded SNPs associated with confounders/outcomes at the genome-wide significance level ( p < 1×10 − 5 ) using the Phenoscanner tool[ 17 ] ( http://www.Phenoscanner.cam.ac.uk/ ). After obtaining the IVs, we then extracted essential information of SNP instruments from the GWAS outcome data. 5) Finally, we proceeded data harmonization, those palindromic and ambiguous SNPs with intermediate allele frequencies (that is allele frequencies lying between 0.01 and 0.30) and were excluded[ 18 ]. By rigorously screening for IV, we reduce the weak associations between potential confounders and genetic variants. 2.4 Mendelian randomization analysis The validity of the MR analysis relies on three key assumptions[ 19 ]: 1) Correlation hypothesis: the SNPs selected as IVs should be strongly correlated with the exposure, thus avoiding the problem of weak instrumental bias in the model. The criteria for determining correlation are the p and LD values of the SNPs, while determining strong correlation is to satisfy the F-statistic > 10 on the basis of correlation[ 20 ]; 2) Exclusivity hypothesis: each IVs must influence the outcome only through exposure factors and no through any other means; 3) Independence assumption: the selected IVs need to be independent of any potential confounding factors affecting exposure and outcomes. The assumptions of the bidirectional Mendelian randomization study in this paper are shown in Fig. 3 . Herein, we adopted Inverse variance weighted (IVW) as the primary method to explore the potential causal relationship between calcium homeostasis regulators and endometriosis. Four validated methods, namely MR-Egger, Weighted median, Weighted mode and Simple mode, are also applied to estimate the robust effects. Zhang et al. provided an explanatory note on these 5 approaches[ 21 ]. We further applied MVMR to dissect the effect of the levels of potential calcium homeostasis regulators on the causal estimation of endometriosis. SNPs associated with specific calcium homeostasis regulators were selected as candidate IVs for further MVMR analysis, with p < 5×10 − 6 as the cutoff value. The remaining screening criteria were as described previously. For the horizontal pleiotropy test under MVMR, we utilized the MR-PRESSO method, and if the p value of global test in MR- PRESSO results was greater than 0.05, indicating that there is no horizontal pleiotropy. If it exists, the outliers should be removed and performed MR-PRESSO analysis again until the p value is less than 0.05, which is followed by MVMR analysis. 2.5 Sensitivity analyses Next, sensitivity analyses were carried to check whether the results of MR analysis were reliable. Firstly, Cochran's Q test was applied to quantify the heterogeneity among SNPS, where a p -value greater than 0.05 indicates the absence of heterogeneity. Secondly, MR-Egger regression test was employed to confirm the existence of horizontal pleiotropy through the intercept index, and if horizontal pleiotropy was detected in selected SNPs, the analysis was repeated after the removal of those pleiotropy SNPs. Finally, leave-one-out analysis was done to assess the impact of individual SNPs on the population, verifying whether there are outliers that strongly influence the results. All MR analyses were undertaken in R (version 4.2.2) software using the R packages "TwoSampleMR" and "MRPRESSO". Scatter plots, forest plots, and funnel plots were used to visualize MR analysis results. Estimates were expressed as an odds ratio (OR) if the outcome indicator was a binary variable, whereas if it was a continuous variable, the estimate was expressed as a beta value (the effect size of the SNP on the phenotype). 3. Results 3.1 Selection of instrumental variables Based on our aforementioned rigorous screening criteria, a total of 189 significant IVs were used for two-sample Mendelian randomization between calcium levels and endometriosis and its various sub phenotypes. Meanwhile, 115 and 14 independent and genome-wide significant SNPs were applied to construct IVs for two-sample Mendelian randomization of 25(OH)D and PTH on endometriosis, separately. 11 SNPs of endometriosis were found in the analysis of inverse MR. See Supplementary Data Sheet 1 for detail. 3.2 Calcium homeostasis regulator levels and endometriosis (including SVMR and MVMR MR) We first performed a two-sample MR analysis to estimate the causal effects of three calcium homeostasis regulators on endometriosis risk. The MR estimates for the different methods are shown in the orange module of Supplementary Data Sheet 2. The two-sample MR analysis revealed a strong causal relationship between genetically predicted calcium levels and endometriosis risk (IVW: OR = 1.15, 95% CI: 1.02–1.29, p = 0.018) (Fig. 4 ). However, no significant association was observed in the analysis of the remaining four models, and there was no significant causal effect of the other calcium regulators (25(OH)D and PTH) on endometriosis ( p > 0.05). The findings suggest a positive causal relationship between calcium levels and endometriosis. We did not detect significant evidence of horizontal pleiotropy in the MR Egger intercept test (P-intercept = 0.553) (Fig. 8 A, upper left panel). Heterogeneity was assessed by Cochrane's Q test, which demonstrated that there was heterogeneity in endometriosis for calcium (MR Egger: Cochran's Q = 220.64, P-heterogeneity = 0.047; IVW: Cochran's Q = 221.06, P-heterogeneity = 0.050), although the macroscopic funnel diagram shows a relatively symmetrical left and right side (Fig. 8 A, lower left panel). Further leave-one-out analyses were performed to identify potential outliers in the instrumental variables, and the leave-one-out chart suggested that the positive causality between calcium levels and endometriosis was highly stable and unlikely to be influenced by some individual SNPs (Fig. 8 A, right panel). Detailed information on each of these sensitivity analyses can be found in the orange module of Supplementary Data Sheet 2. Notably, the results of MVMR analysis demonstrated that the positive correlation of calcium levels on endometriosis still held even after correction for 25(OH)D and PTH (Fig. 5 A and 5 B, OR = 1.14, 95% CI: 1.02–1.28, p = 0.026). To ensure the reliability of MVMR, we conducted MR-PRESSO for further analysis, setting the value of NbDistribution to 10,000 times, and found that horizontal pleiotropy existed (Fig. 5 C, RSSobs of Global Test in MR-PRESSO results = 381.591, P value of Global Test in MR-PRESSO results = 0.042), and an outlier was identified (rs28520334). We therefore performed MR-PRESSO again after removing the outlier, at which point horizontal pleiotropy was eliminated (Fig. 5 D, RSSobs of Global Test in MR-PRESSO results = 370.957, P value of Global Test in MR-PRESSO results = 0.078), on the basis of which we reran the secondary MVMR analysis, and the causal association between calcium levels and endometriosis remained significant after correcting for other calcium homeostatic modifiers (Fig. 5 A and 5 B, OR = 1.13, 95% CI: 1.01–1.27, p = 0.033) (see blue module of Supplementary Data Sheet 2). Combining SVMR and MVMR analyses results, we were able to draw a strong conclusion that genetically predicted calcium levels are causally associated with an elevated risk of endometriosis, and that calcium levels are a risk factor for the development of endometriosis. 3.3 Endometriosis and calcium homeostasis regulatory levels (Inverse MR) We carried inverse MR analysis to explore the effects of endometriosis on body calcium homeostasis. Endometriosis was considered as exposure, and calcium levels, 25 (OH) D, and PTH as outcomes. A total of 11 SNPs ( p < 5× 10 − 8 ) that were closely associated with endometriosis risk were identified. And we discovered a causal association between endometriosis and 25(OH)D (β = 0.01, 95% CI: 0.00-0.02, p = 0.007) and calcium (β = 0.02, 95% CI: 0.00-0.04, p = 0.035). The inverse causality analysis of endometriosis and PTH did not reach significance (Fig. 6 ) (details in yellow module of Supplementary Data Sheet 2). A sensitivity analysis was similarly performed and no significant evidence of horizontal pleiotropy in calcium were detected in either calcium (P-intercept = 0.621) (scatter plot in Fig. 8 B) or 25(OH)D (P-intercept = 0.244) (scatter plot in Fig. 8 C). Heterogeneity in endometriosis for calcium (MR Egger: Cochran's Q = 27.27, P-heterogeneity = 0.001; IVW: Cochran's Q = 28.07, P-heterogeneity = 0.002) were revealed, whereas no heterogeneity was present in 25(OH)D (MR Egger: Cochran's Q = 4.84, P-heterogeneity = 0.775; IVW: Cochran's Q = 6.42, P-heterogeneity = 0.698). Due to the small number of IVs included in the analysis during the inverse MR process, the funnel plot results are not intuitive (funnel plots in Fig. 8 B and 8 C). The leave-one-out analysis showed a high overall stability (the red line representing the overall effect did not cross the null line) (leave-one plots in Fig. 8 B and 8 C). Details of the above results can be viewed in the yellow module of Supplementary Data Sheet 2.) 3.4 Calcium levels and endometriosis sub phenotypes Since endometriotic lesions can occur in different locations in the body, we collected GWAS aggregated statistics from the FinnGen database for six sub phenotypes of endometriosis, covering EO, EV, EI, ET, EP, EU, and EUO. Building on previous research, we employed two-sample MR analysis to further investigate the causal effects of genetically predicted calcium levels on different subtypes of endometriosis. As can be seen from the results (Fig. 7 ), calcium levels were positively and causally associated only with EU, i.e., endometriosis of uterus (IVW: OR = 1.23, 95% CI: 1.01–1.49, p = 0.038), with no suggestion of a causal relationship with the risk of other types of endometriosis subtypes (green module of Supplementary Data Sheet 2). 4. Discussion Currently, it is widely recognized that endometriosis is a chronic inflammatory disease in which the glands and stroma of the ectopic endometrium are monitored by the body's immune system leading to chronic inflammation[ 22 ]. There is a complex relationship between the body's calcium homeostasis and inflammation, for example, hypocalcemia resulting from an imbalance of calcium homeostasis is commonly appears in systemic inflammatory diseases such as burns and sepsis[ 23 ]. It has been demonstrated that receptors of calcium homeostasis, calcium sensing receptors (CaSR), are both promoters and responders of inflammation[ 24 ]. Not only that, the high incidence of infertility in women with pelvic endometriosis has also brought immunological factors into the field of endometriosis research. In endometriosis, the disturbance of local immunity has been recognized by most researchers, and the oxidative stress accompanied by inflammation further contributes to the disorder of immune response[ 25 ]. Improvement of the pelvic immune microenvironment after endometriosis lesion resection remains an effective therapeutic measure for chronic pelvic pain and infertility[ 26 ]. Vitamin D, as a nutrient that maintains the body's immune balance, regulates immune sensitivity and prevents the production of autoantibodies, prevents allergic reactions of the immune system, and may play a role in the prevention of autoimmune diseases and cancer[ 6 ]. In addition, evidence for the emerging role of CaSR in controlling intestinal fluid homeostasis and immune homeostasis has been provided[ 27 ]. All these studies emphasize the possible close relationship between calcium homeostasis and endometriosis. Our study is the first to investigate the causal relationship between calcium homeostasis regulatory factors and endometriosis risk based on large-scale abstract level GWAS genetic data. Although several observational studies have been conducted previously, those conclusions have been inconsistent. For example, Delbandi et al., by including 56 healthy women and 54 patients with endometriosis, found that subjects with low serum 25 (OH) D levels were at higher risk of endometriosis[ 28 ], and others have found low levels of 25(OH)D in women with endometriosis[ 29 ]. However, a 2012 systematic review found that 25(OH)D levels were positively associated with endometriosis[ 30 ]. Delbandi et al. also noted significantly lower calcium levels and significantly higher PTH in patients with endometriosis[ 28 ], but in another prospective cohort study that included 53 women with endometriosis and 25 controls, a trend toward elevated calcium levels was observed in the patients[ 31 ]. At the same time, a retrospective analysis of studies found no significant difference in calcium levels between case and control groups of women with endometriosis[ 32 ]. Recent studies on endometriosis and extracellular calcium homeostasis have focused on the effects of therapeutic measures (e.g., gonadotropin-releasing hormone agonists) on bone mineral density and calcium levels in endometriosis[ 33 , 34 ], and observational studies on the causal relationship between calcium levels and endometriosis have not yielded uniform results, leaving much room for exploration and research in this field. We obtained pooled statistics on serum 25(OH)D, calcium, endometriosis and its sub phenotypes, and PTH from EBI, UKBB, FinnGen databases, and published GWAS literature. Using these data, we performed four levels of MR analysis, including SVMR, MVMR, inverse MR and two-sample MR (exploring subtypes of endometriosis). Our results showed that genetically predicted calcium levels were positively associated with endometriosis, and inverse MR analysis indicated that endometriosis was associated with levels of 25(OH)D and calcium. In addition, MR analysis categorized by ectopic location identified calcium levels as a risk factor for endometriosis of uterus. These findings highlight the importance of monitoring calcium levels in patients with endometriosis. Since a large source of calcium and vitamin D in the body is obtained from food, the results of this study may provide dietary guidance for endometriosis patients and have important implications for the development of endometriosis prevention strategies and treatments. While fewer previous studies have examined associations between diet and endometriosis, Harris et al conducted a 14-year prospective cohort study and found that higher predicted serum 25(OH)D levels and higher dairy intake were associated with a reduced risk of endometriosis[ 10 ], which results were also demonstrated by Joanna et al.[ 35 ]. However, our study does not suggest a causal relationship between serum 25(OH)D levels and endometriosis risk, and additional experimental validation is needed in the future to more fully understand the role of these calcium homeostasis regulators in endometriosis risk. The strengths herein are as follows. The first is the advantage of MR, a statistical modality, which is not susceptible to confounding bias and reverse causation [ 36 ], which was further amplified by our comprehensive study of multiple types of MR. Next, rather than examining causality between calcium levels and endometriosis alone, we comprehensively included two other common regulators of calcium homeostasis (25 (OH) D and PTH). Overall, the analysis of multiple data and multiple MR methods improved the confidence of our results. Finally, we employed multiple rigorous methods for screening of IVs and used multiple complementary sensitivity analyses, including heterogeneity, pleiotropy and leave-one-out analyses, to verify the reliability of the MR Findings. Although numerous strengths exist, there are also some shortcomings in the study. First of all, based on the genome-wide statistical significance threshold (5×10 − 8 ), IVs that meet the criteria for PTH cannot be obtained for further studies. Therefore, we expanded the cutoff value to 5×10 − 6 when screening the IVs of PTH. Second, endometriosis is a female-specific disease, but the GWAS datasets for calcium homeostasis regulators included in the study were gender-biased, which may bias our findings. In addition, all the data in this study were limited to European ancestry, and it remains to be verified whether the findings can be applied to other populations. 5. Conclusion This is the first comprehensive multi-type MR study to examine the causal relationship between calcium homeostasis and endometriosis risk. Our findings support a causal relationship between calcium levels and endometriosis risk, demonstrating that calcium levels are a risk factor for endometriosis. It also emphasizes the importance of monitoring calcium levels in patients suffering from endometriosis, which may provide dietary guidance for patients with endometriosis and may have important implications for endometriosis prevention and treatment. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The data used in this study can be accessed through the links provided in Table 1. Additional data are included in the article/supplementary material. Further queries can be directed to the corresponding author. Competing interests The authors declare that they have no competing interests. Funding This work was supported by cross-innovation talent project in Renmin Hospital of Wuhan University (grant number JCRCZN-2022-016); Undergraduate education quality construction comprehensive reform project (grant number 2022ZG282) and the National Natural Science Foundation of China (grant number 82071655). Authors' contributions FFD and YXC contributed to the conceptualization and design of the study, RQW and XY collected and initially screened the data. ZMD performed a visual analysis of the data and was the main contributor to the manuscript. All authors read and approved the final manuscript. 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Davey Smith G, Hemani G: Mendelian randomization: genetic anchors for causal inference in epidemiological studies. Hum Mol Genet 2014, 23(R1):R89-98. Richmond RC, Davey Smith G: Mendelian Randomization: Concepts and Scope. Cold Spring Harb Perspect Med 2022, 12(1). Sun BB, Maranville JC, Peters JE, Stacey D, Staley JR, Blackshaw J, Burgess S, Jiang T, Paige E, Surendran P et al : Genomic atlas of the human plasma proteome. Nature 2018, 558(7708):73–79. Revez JA, Lin T, Qiao Z, Xue A, Holtz Y, Zhu Z, Zeng J, Wang H, Sidorenko J, Kemper KE et al : Genome-wide association study identifies 143 loci associated with 25 hydroxyvitamin D concentration. Nat Commun 2020, 11(1):1647. Kamat MA, Blackshaw JA, Young R, Surendran P, Burgess S, Danesh J, Butterworth AS, Staley JR: PhenoScanner V2: an expanded tool for searching human genotype-phenotype associations. Bioinformatics 2019, 35(22):4851–4853. Chen X, Liu Z, Cui J, Chen X, Xiong J, Zhou W: Circulating adipokine levels and preeclampsia: A bidirectional Mendelian randomization study. Front Genet 2022, 13:935757. Davies NM, Holmes MV, Davey Smith G: Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians. BMJ 2018, 362:k601. Bowden J, Del Greco MF, Minelli C, Davey Smith G, Sheehan NA, Thompson JR: Assessing the suitability of summary data for two-sample Mendelian randomization analyses using MR-Egger regression: the role of the I2 statistic. Int J Epidemiol 2016, 45(6):1961–1974. Zhang D, Hu Y, Guo W, Song Y, Yang L, Yang S, Ou T, Liu Y, Zhang Y: Mendelian randomization study reveals a causal relationship between rheumatoid arthritis and risk for pre-eclampsia. Front Immunol 2022, 13:1080980. Wang Y, Nicholes K, Shih IM: The Origin and Pathogenesis of Endometriosis. Annu Rev Pathol 2020, 15:71–95. Iamartino L, Brandi ML: The calcium-sensing receptor in inflammation: Recent updates. Front Physiol 2022, 13:1059369. Hendy GN, Canaff L: Calcium-sensing receptor, proinflammatory cytokines and calcium homeostasis. Semin Cell Dev Biol 2016, 49:37–43. Fukui A, Mai C, Saeki S, Yamamoto M, Takeyama R, Kato T, Ukita Y, Wakimoto Y, Yamaya A, Shibahara H: Pelvic endometriosis and natural killer cell immunity. Am J Reprod Immunol 2021, 85(4):e13342. Maksym RB, Hoffmann-Mlodzianowska M, Skibinska M, Rabijewski M, Mackiewicz A, Kieda C: Immunology and Immunotherapy of Endometriosis. J Clin Med 2021, 10(24). Tang L, Cheng CY, Sun X, Pedicone AJ, Mohamadzadeh M, Cheng SX: The Extracellular Calcium-Sensing Receptor in the Intestine: Evidence for Regulation of Colonic Absorption, Secretion, Motility, and Immunity. Front Physiol 2016, 7:245. Delbandi AA, Torab M, Abdollahi E, Khodaverdi S, Rokhgireh S, Moradi Z, Heidari S, Mohammadi T: Vitamin D deficiency as a risk factor for endometriosis in Iranian women. J Reprod Immunol 2021, 143:103266. Anastasi E, Fuggetta E, De Vito C, Migliara G, Viggiani V, Manganaro L, Granato T, Benedetti Panici P, Angeloni A, Porpora MG: Low levels of 25-OH vitamin D in women with endometriosis and associated pelvic pain. Clin Chem Lab Med 2017, 55(12):e282-e284. Lerchbaum E, Obermayer-Pietsch B: Vitamin D and fertility: a systematic review. Eur J Endocrinol 2012, 166(5):765–778. Somigliana E, Panina-Bordignon P, Murone S, Di Lucia P, Vercellini P, Vigano P: Vitamin D reserve is higher in women with endometriosis. Hum Reprod 2007, 22(8):2273–2278. Lu BC, Zhang XM: [Associations of metabolism of lipid, calcium and phosphate in endometriosis]. Zhonghua Fu Chan Ke Za Zhi 2008, 43(3):185–188. Stodtmann S, Nader A, Polepally AR, Suleiman AA, Winzenborg I, Noertersheuser P, Ng J, Mostafa NM, Shebley M: Validation of a quantitative systems pharmacology model of calcium homeostasis using elagolix Phase 3 clinical trial data in women with endometriosis. Clin Transl Sci 2021, 14(4):1611–1619. Veth VB, van de Kar MM, Duffy JM, van Wely M, Mijatovic V, Maas JW: Gonadotropin-releasing hormone analogues for endometriosis. Cochrane Database Syst Rev 2023, 6(6):CD014788. Jurkiewicz-Przondziono J, Lemm M, Kwiatkowska-Pamula A, Ziolko E, Wojtowicz MK: Influence of diet on the risk of developing endometriosis. Ginekol Pol 2017, 88(2):96–102. Verduijn M, Siegerink B, Jager KJ, Zoccali C, Dekker FW: Mendelian randomization: use of genetics to enable causal inference in observational studies. Nephrol Dial Transplant 2010, 25(5):1394–1398. Additional Declarations No competing interests reported. Supplementary Files SupplementaryDataSheet1.xlsx Supplementary Table 1. Independent and genome-wide significant SNPs for constructing instrumental variables for two-sample bidirectional Mendelian randomization of calcium homeostasis regulators and endometriosis. SupplementaryDataSheet2.xlsx Supplementary Table 2. Results of different types and different MR analysis methods and corresponding sensitivity analysis results. SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3273922","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":228160704,"identity":"0077d8d0-1ef7-43d3-bc1f-4cbccb0e13b3","order_by":0,"name":"Zhi-Min Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACNvb245//VNjU88s/PkCcFj6eM2kMQJwg2ZCWQJwWOYkEMwbelsMJBgdyDIh0GM+BtAeSDYfzDA6c+XjjDYOdnG4DIS3sjccNDHekF0se7N1sOYch2djsAGFbEiQSz1gz9h3m3SbNw3AgcRtBLRIJBhIH25gZG47xPCNai5lkY5tz4oQzPGxEauE5k2zMcCbNWHIGm7HlHAMi/CLf3n7wMUOFjRy/BPPDG28q7OQIakEBEjxERg2yFlJ1jIJRMApGwYgAAOsMQ/tRjZOAAAAAAElFTkSuQmCC","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Zhi-Min","middleName":"","lastName":"Deng","suffix":""},{"id":228160705,"identity":"2cab18d0-2d0b-4252-91b9-239a90ffd2df","order_by":1,"name":"Fang-Fang Dai","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Fang-Fang","middleName":"","lastName":"Dai","suffix":""},{"id":228160706,"identity":"69f35986-bf38-4826-9604-ec0be85bcaed","order_by":2,"name":"Rui-Qi Wang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Rui-Qi","middleName":"","lastName":"Wang","suffix":""},{"id":228160707,"identity":"232ab490-75d0-448a-b455-7b156d4c3943","order_by":3,"name":"Xiao Yang","email":"","orcid":"","institution":"Peking University People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Yang","suffix":""},{"id":228160708,"identity":"ceb5dfd8-bc86-4a26-89c4-32d4c479c1ef","order_by":4,"name":"Yan-Xiang Cheng","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Yan-Xiang","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2023-08-18 04:15:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3273922/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3273922/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42165473,"identity":"b81ddc44-452c-4b68-8775-6e76c1ab3ba3","added_by":"auto","created_at":"2023-08-25 21:13:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":449971,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Classification of endometriosis sub phenotypes included in this article;\u003cstrong\u003e \u003c/strong\u003e(B) Schematic diagram of\u003cstrong\u003e \u003c/strong\u003ecalcium, 25 hydroxyvitamin D (25(OH)D), and parathyroid hormone (PTH) exert calcium homeostatic regulation through the blood, small intestine, parathyroid glands, liver, bone, and kidney.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/650e8633bc39f63b3d4418b8.jpg"},{"id":42165827,"identity":"f603c35c-4ffb-4bc5-aa44-8447381d4c0a","added_by":"auto","created_at":"2023-08-25 21:21:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1178132,"visible":true,"origin":"","legend":"\u003cp\u003eAn overview and ideas of the research design.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/03d5132f300c2df4fcaef914.jpg"},{"id":42165830,"identity":"ccb31e29-0e43-4792-bbed-3950f0fb94d5","added_by":"auto","created_at":"2023-08-25 21:21:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":664773,"visible":true,"origin":"","legend":"\u003cp\u003eThe assumptions of the bidirectional Mendelian randomization study in this paper. Red lines represent forward Mendelian randomization and blue lines represent reverse Mendelian randomization.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/c1557007f7fccff1eeec35ce.jpg"},{"id":42165480,"identity":"0101ac9b-e717-40fb-9b74-a0482dd5d6d1","added_by":"auto","created_at":"2023-08-25 21:13:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":460722,"visible":true,"origin":"","legend":"\u003cp\u003eThe table and forest plot of causal estimates between the levels of three calcium homeostasis regulators (25(OH)D, PTH, and calcium) and endometriosis. The charts mainly present the results obtained by IVW and MR Egger. The horizontal bars present 95% confidence interval (CI). Significant P value was highlighted in orange.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/ad6f5ee56288d8d6c5070387.jpg"},{"id":42165481,"identity":"dfb42d66-6206-484c-8e55-959da4d0b2c9","added_by":"auto","created_at":"2023-08-25 21:13:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1030855,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B) Results of multivariate Mendelian randomization analysis before and after performing MR-PRESSO and corresponding forest plot; (C) Results of the first MR-PRESSO analysis, showing the presence of horizontal pleiotropy; (D) Results of the second MR-PRESSO analysis after removing the outliers IVs, showing that horizontal pleiotropy has been removed.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/12d83449639a1db580135f17.jpg"},{"id":42165475,"identity":"212b72fa-89de-461e-bf71-6676b9f23af2","added_by":"auto","created_at":"2023-08-25 21:13:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":460805,"visible":true,"origin":"","legend":"\u003cp\u003eInverse Mendelian randomization analysis. The table and forest plot of causal estimates between endometriosis and levels of three calcium homeostasis regulators (25(OH)D, PTH, and calcium). The charts mainly present the results obtained by IVW and MR Egger. The horizontal bars present 95% confidence interval (CI). Significant P value was highlighted in yellow.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/0149a164c9790e30f94face6.jpg"},{"id":42165483,"identity":"90069720-610d-4bd4-bd0f-1a58b90758e0","added_by":"auto","created_at":"2023-08-25 21:13:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":841381,"visible":true,"origin":"","legend":"\u003cp\u003eSub phenotypic Mendelian randomization analysis. The table and forest plot depict the causal estimates between calcium level and different sub phenotypes of endometriosis, including endometriosis of ovary (EO), endometriosis of rectovaginal septum and vagina (EV), endometriosis of intestine (EI), endometriosis of fallopian tube (ET), endometriosis of pelvic peritoneum (EP), endometriosis of uterus (EU) and Unspecified/other endometriosis (EUO). The charts mainly present the results obtained by IVW and MR Egger. The horizontal bars present 95% confidence interval (CI). Significant P value was highlighted in green.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/09aa4ddd8ae833ceb56d0776.jpg"},{"id":42165482,"identity":"97d42ab6-bd6d-4d1f-be8a-d9d0b18c4843","added_by":"auto","created_at":"2023-08-25 21:13:21","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2959513,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of sensitivity analyses in significant MR analyses, including scatter plots (horizontal pleiotropy), funnel plots (heterogeneity), and leave-one-out analyses (stability). (A) Results of sensitivity analysis of two-sample Mendelian randomization between calcium levels and endometriosis; (B) Results of sensitivity analysis of inverse Mendelian randomization between endometriosis and calcium levels; (C) Results of sensitivity analysis of inverse Mendelian randomization between endometriosis and 25(OH)D levels; (D) Results of sensitivity analysis of two-sample Mendelian randomization between calcium levels and endometriosis of uterus.\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/d7667176bc6bb1e98011e578.jpg"},{"id":42176728,"identity":"3252325a-f5c2-4aa1-b5be-cb85beb6918e","added_by":"auto","created_at":"2023-08-26 08:37:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1144953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/cc717a25-2bbf-42f0-974e-72869354037c.pdf"},{"id":42166641,"identity":"f693d88a-0ad8-4145-81b1-86ffe31ee3ce","added_by":"auto","created_at":"2023-08-25 21:29:21","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1.\u003c/strong\u003e Independent and genome-wide significant SNPs for constructing instrumental variables for two-sample bidirectional Mendelian randomization of calcium homeostasis regulators and endometriosis.\u003c/p\u003e","description":"","filename":"SupplementaryDataSheet1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/33817f3b23f9a7caa5ec9541.xlsx"},{"id":42165828,"identity":"472fa825-cfb4-4faa-bfa8-e698a9b327a3","added_by":"auto","created_at":"2023-08-25 21:21:21","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2.\u003c/strong\u003e Results of different types and different MR analysis methods and corresponding sensitivity analysis results.\u003c/p\u003e","description":"","filename":"SupplementaryDataSheet2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/fc9fbd08ad89fee4c688367b.xlsx"},{"id":42165478,"identity":"70b59d1d-9d9d-420c-b8a7-f8bbd7439c65","added_by":"auto","created_at":"2023-08-25 21:13:21","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14172,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3273922/v1/33f8d5b9dce47f14448277ba.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The role of calcium homeostasis in endometriosis: a comprehensive study of multiple types of Mendelian randomization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEndometriosis is a common women's disease, causing dysmenorrhea and chronic pelvic pain, and even making many women of childbearing age infertile, seriously affecting their health and quality of life[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As the name implies, endometriosis is a condition in which endometrial tissue, which originally grows in the uterine cavity, grows outside the uterine cavity, such as ovaries, fallopian tubes, uterine surface, cavum douglasi, vagina, pelvic peritoneum, intestine, etc., with the characteristics of proliferation, diffusion, and metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). While many factors, including hormones, inflammation, genetic factors, and environmental factors, are thought to fuel the development of endometriosis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], its etiology and pathogenesis have not been fully elucidated, and few modifiable risk factors have been identified. There remains an unmet need to improve understanding of the pathophysiological mechanisms of endometriosis to develop more effective treatments.\u003c/p\u003e \u003cp\u003eCalcium is an important ion in cell signaling, hormone regulation, and bone health, and performs important physiological functions within cells, such as excitatory regulation of neurons, muscle contraction, and apoptosis. Under normal circumstances, the intestinal tract, especially the small intestine, is the main part of calcium absorption in the body, and vitamin D can promote the absorption of calcium and phosphorus from the intestinal mucosal cells[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Parathyroid hormone (PTH) regulates the metabolism of calcium and phosphorus through small intestine, bone and kidney[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], in which bone relies on the balance of osteoblasts and osteoclasts to regulate the level of calcium, while kidney regulates the excretion of blood calcium through reabsorption and filtration mechanisms[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 25-Hydroxyvitamin D (25(OH)D) is the form of vitamin D stored in the liver and transported in the blood, and is the basis for clinical determine of vitamin D levels[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Overall, vitamin D (primarily 25 (OH) D), PTH, and serum calcium act synergistically to regulate extracellular calcium homeostasis in humans (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eImbalances of calcium homeostasis can lead to a variety of diseases, such as cardiovascular diseases[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], neurological diseases[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and abnormal bone metabolism[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Studies on calcium homeostasis and endometriosis are relatively scarce. In a large prospective cohort study of 1,385 patients with laparoscopically confirmed endometriosis, investigators observed a significantly lower incidence of endometriosis in women with higher levels of predicted plasma 25(OH)D as well as the amount of calcium and vitamin D ingested from food[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. At the molecular mechanisms level, researches have attempted to alter calcium homeostasis to achieve inhibition of endometriosis at the cellular level[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although observational and basic laboratory studies have suggested that endometriosis risk may be influenced by organismal calcium homeostasis, the causal relationship between the two remains unclear. The existence of residual confounding and potential reverse causality in traditional observational studies poses a major challenge to exploring the causal effect of the two. Thus, elucidating the causal relationship between body calcium homeostasis and endometriosis risk may have important implications for the prevention and treatment of endometriosis.\u003c/p\u003e \u003cp\u003eMendelian randomization (MR) is an emerging statistical method for assessing the causal relationship between an exposure of interest and an outcome using genetic variants (i.e., single nucleotide polymorphisms (SNPs)) as instrumental variables (IVs). It takes advantage of genetic variation that exists in nature to classify individuals into different genotypes, thus simulating the effects of randomized trials to assess whether the effect of a factor on a disease is causal[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This approach avoids the effects of confounding factors and reverse causality in observational studies, thereby increasing the confidence of causal inference[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUtilizing the existing genome-wide association studies (GWASs) databases, our study determined to answer the following three key questions by applying MR analysis: (1) what is the correlation between calcium homeostasis regulatory factors (calcium, 25 (OH) D, and PTH) and endometriosis; (2) presumptive causal relationship between calcium homeostasis regulatory factors (calcium, 25 (OH) D, and PTH) and endometriosis; (3) what is the effect of calcium levels on different types of endometrioses?\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.1 Study design\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWe conducted two-sample MR analysis using the publicly available GWAS datasets on calcium homeostasis regulators (calcium, 25 (OH) D, and PTH) and endometriosis. To improve the confidence of the results, in addition to single univariate MR (SVMR) analysis of SNPs for each calcium homeostasis regulators separately, we also performed multivariate MR (MVMR). Meanwhile, reverse MR analysis was also applied to explore the effects of endometriosis on calcium homeostasis in patients' organisms. This reverse causality analysis will help to further determine the deep relationship between the two. In addition, the level of calcium, a key to calcium homeostasis, and different types of endometrioses was further explored, including endometriosis of ovary, abbreviated as EO, endometriosis of rectovaginal septum and vagina (EV), endometriosis of intestine (EI), endometriosis of fallopian tube (ET), endometriosis of pelvic peritoneum (EP), unspecified/other endometriosis (EUO), endometriosis of uterus (EU). An overview and ideas of the research design is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.2 Data source\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWe obtained the GWAS of PTH from the previously published GWAS dataset for European pedigrees[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. And GWAS data for serum 25(OH)D levels was available from the European Bioinformatics Institute (EBI)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], while calcium GWAS data were gain from the UK Biobank (UKBB). Data on endometriosis and its sub phenotypes were derived from the FinnGen Research Program's Finnish Biobank, including endometriosis (8,288 cases, 68,969 controls), EO (3,231 cases, 68,969 controls), ER (1,360 cases, 68,969 controls), EI (177 cases, 68,969 controls), ET (116 cases, 68,969 controls), EP (2,953 cases, 68,969 controls), EUO (1,435 cases, 68,969 controls), and EU (2,372 cases, 68,969 controls).\u003c/p\u003e \u003cp\u003eThere was no sample overlap between the exposure and outcome datasets, and all data samples were of European ethnicity. Ethical approval and informed consent records were granted for all original studies. Details of the data sources used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic characteristics for selected summary-level GWASs applied in MR study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGWAS ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTraits\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthnicity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSample size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSNP size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAuthor/consortium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLink\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eebi-a-GCST90000618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum 25-Hydroxyvitamin D levels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e496,946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6,896,093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRevez JA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90000618/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90000618/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eukb-d-30680_irnt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e315,153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13,585,309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNeale lab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/ukb-d-30680_irnt/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/ukb-d-30680_irnt/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eprot-a-2431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParathyroid hormone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3,301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10,534,735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSun BB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/prot-a-2431/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/prot-a-2431/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e77,257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8,288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,377,306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTrait: Endometriosis - IEU OpenGWAS project (mrcieu.ac.uk)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS_OVARY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis of ovary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72,200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3,231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,376,686\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTrait: Endometriosis of ovary - IEU OpenGWAS project (mrcieu.ac.uk)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS_RECTPVAGSEPT_VAGINA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis of rectovaginal septum and vagina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70,329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1,360\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,376,472\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_RECTPVAGSEPT_VAGINA/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_RECTPVAGSEPT_VAGINA/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS_INTESTINE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis of intestine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e69,146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,376,157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_INTESTINE/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_INTESTINE/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS_FALLOPIAN_TUBE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis of fallopian tube\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e69,085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,376,156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_FALLOPIAN_TUBE/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_FALLOPIAN_TUBE/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS_PELVICPERITONEUM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis of pelvic peritoneum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71,922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2,953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,376,599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTrait: Endometriosis of pelvic peritoneum - IEU OpenGWAS project (mrcieu.ac.uk)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS_NOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnspecified/other endometriosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70,404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1,435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,376,331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_NOS/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/finn-b-N14_ENDOMETRIOSIS_NOS/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efinn-b-N14_ENDOMETRIOSIS_UTERUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometriosis of uterus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71,341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2,372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16,376,529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFinnGen team\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTrait: Endometriosis of uterus - IEU OpenGWAS project (mrcieu.ac.uk)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 IVs selection\u003c/h2\u003e \u003cp\u003eIVs were obtained from the exposure dataset for MR analysis through the following quality control steps to ensure the accuracy of the causal conclusions of the exposure factors against the outcome factors.\u003c/p\u003e \u003cp\u003e1) Select genome-wide significant SNPs for exposure traits (Two-sample MR and inverse MR: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e for calcium/25(OH)D/endometriosis, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e for PTH; MVMR: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e for calcium/25(OH)D/PTH/endometriosis);\u003c/p\u003e \u003cp\u003e2) The selected SNPs were tested for linkage disequilibrium (LD) to ensure the validity of the data. We employed a clumping procedure to filter independent SNPs (clumping distance\u0026thinsp;=\u0026thinsp;10,000 kb and r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 threshold;\u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e3) Excluded SNPs with F-statistics \u003c 10. The formula for F- statistic was shown as following:\u003c/p\u003e\n\u003cp\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$F=\\frac{{R}^{2}\\times \\left(\\text{N}-\\text{K}-1\\right)}{\\text{K}\\times \\left(1-{R}^{2}\\right)}$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere N denotes the sample size of the included GWAS data, K indicates the number of SNPs subjected to MR analysis, and R\u003csup\u003e2\u003c/sup\u003e represents the variance corresponding to each exposed SNP;\u003c/p\u003e \u003cp\u003e4) Excluded SNPs associated with confounders/outcomes at the genome-wide significance level (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e) using the Phenoscanner tool[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.Phenoscanner.cam.ac.uk/\u003c/span\u003e\u003cspan address=\"http://www.Phenoscanner.cam.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). After obtaining the IVs, we then extracted essential information of SNP instruments from the GWAS outcome data.\u003c/p\u003e \u003cp\u003e5) Finally, we proceeded data harmonization, those palindromic and ambiguous SNPs with intermediate allele frequencies (that is allele frequencies lying between 0.01 and 0.30) and were excluded[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. By rigorously screening for IV, we reduce the weak associations between potential confounders and genetic variants.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Mendelian randomization analysis\u003c/h2\u003e \u003cp\u003eThe validity of the MR analysis relies on three key assumptions[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]: 1) Correlation hypothesis: the SNPs selected as IVs should be strongly correlated with the exposure, thus avoiding the problem of weak instrumental bias in the model. The criteria for determining correlation are the \u003cem\u003ep\u003c/em\u003e and LD values of the SNPs, while determining strong correlation is to satisfy the F-statistic\u0026thinsp;\u0026gt;\u0026thinsp;10 on the basis of correlation[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]; 2) Exclusivity hypothesis: each IVs must influence the outcome only through exposure factors and no through any other means; 3) Independence assumption: the selected IVs need to be independent of any potential confounding factors affecting exposure and outcomes. The assumptions of the bidirectional Mendelian randomization study in this paper are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eHerein, we adopted Inverse variance weighted (IVW) as the primary method to explore the potential causal relationship between calcium homeostasis regulators and endometriosis. Four validated methods, namely MR-Egger, Weighted median, Weighted mode and Simple mode, are also applied to estimate the robust effects. \u003cem\u003eZhang\u003c/em\u003e et al. provided an explanatory note on these 5 approaches[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We further applied MVMR to dissect the effect of the levels of potential calcium homeostasis regulators on the causal estimation of endometriosis. SNPs associated with specific calcium homeostasis regulators were selected as candidate IVs for further MVMR analysis, with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e as the cutoff value. The remaining screening criteria were as described previously. For the horizontal pleiotropy test under MVMR, we utilized the MR-PRESSO method, and if the \u003cem\u003ep\u003c/em\u003e value of global test in MR- PRESSO results was greater than 0.05, indicating that there is no horizontal pleiotropy. If it exists, the outliers should be removed and performed MR-PRESSO analysis again until the \u003cem\u003ep\u003c/em\u003e value is less than 0.05, which is followed by MVMR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sensitivity analyses\u003c/h2\u003e \u003cp\u003eNext, sensitivity analyses were carried to check whether the results of MR analysis were reliable. Firstly, Cochran's Q test was applied to quantify the heterogeneity among SNPS, where a \u003cem\u003ep\u003c/em\u003e-value greater than 0.05 indicates the absence of heterogeneity. Secondly, MR-Egger regression test was employed to confirm the existence of horizontal pleiotropy through the intercept index, and if horizontal pleiotropy was detected in selected SNPs, the analysis was repeated after the removal of those pleiotropy SNPs. Finally, leave-one-out analysis was done to assess the impact of individual SNPs on the population, verifying whether there are outliers that strongly influence the results.\u003c/p\u003e \u003cp\u003eAll MR analyses were undertaken in R (version 4.2.2) software using the R packages \"TwoSampleMR\" and \"MRPRESSO\". Scatter plots, forest plots, and funnel plots were used to visualize MR analysis results. Estimates were expressed as an odds ratio (OR) if the outcome indicator was a binary variable, whereas if it was a continuous variable, the estimate was expressed as a beta value (the effect size of the SNP on the phenotype).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Selection of instrumental variables\u003c/h2\u003e \u003cp\u003eBased on our aforementioned rigorous screening criteria, a total of 189 significant IVs were used for two-sample Mendelian randomization between calcium levels and endometriosis and its various sub phenotypes. Meanwhile, 115 and 14 independent and genome-wide significant SNPs were applied to construct IVs for two-sample Mendelian randomization of 25(OH)D and PTH on endometriosis, separately. 11 SNPs of endometriosis were found in the analysis of inverse MR. See Supplementary Data Sheet 1 for detail.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Calcium homeostasis regulator levels and endometriosis (including SVMR and MVMR MR)\u003c/h2\u003e \u003cp\u003eWe first performed a two-sample MR analysis to estimate the causal effects of three calcium homeostasis regulators on endometriosis risk. The MR estimates for the different methods are shown in the orange module of Supplementary Data Sheet 2. The two-sample MR analysis revealed a strong causal relationship between genetically predicted calcium levels and endometriosis risk (IVW: OR = 1.15, 95% CI: 1.02–1.29, \u003cem\u003ep\u003c/em\u003e = 0.018) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, no significant association was observed in the analysis of the remaining four models, and there was no significant causal effect of the other calcium regulators (25(OH)D and PTH) on endometriosis (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). The findings suggest a positive causal relationship between calcium levels and endometriosis.\u003c/p\u003e\u003cp\u003eWe did not detect significant evidence of horizontal pleiotropy in the MR Egger intercept test (P-intercept = 0.553) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, upper left panel). Heterogeneity was assessed by Cochrane's Q test, which demonstrated that there was heterogeneity in endometriosis for calcium (MR Egger: Cochran's Q = 220.64, P-heterogeneity = 0.047; IVW: Cochran's Q = 221.06, P-heterogeneity = 0.050), although the macroscopic funnel diagram shows a relatively symmetrical left and right side (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lower left panel). Further leave-one-out analyses were performed to identify potential outliers in the instrumental variables, and the leave-one-out chart suggested that the positive causality between calcium levels and endometriosis was highly stable and unlikely to be influenced by some individual SNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, right panel). Detailed information on each of these sensitivity analyses can be found in the orange module of Supplementary Data Sheet 2.\u003c/p\u003e\u003cp\u003eNotably, the results of MVMR analysis demonstrated that the positive correlation of calcium levels on endometriosis still held even after correction for 25(OH)D and PTH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, OR = 1.14, 95% CI: 1.02–1.28, \u003cem\u003ep\u003c/em\u003e = 0.026). To ensure the reliability of MVMR, we conducted MR-PRESSO for further analysis, setting the value of NbDistribution to 10,000 times, and found that horizontal pleiotropy existed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, RSSobs of Global Test in MR-PRESSO results = 381.591, P value of Global Test in MR-PRESSO results = 0.042), and an outlier was identified (rs28520334). We therefore performed MR-PRESSO again after removing the outlier, at which point horizontal pleiotropy was eliminated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, RSSobs of Global Test in MR-PRESSO results = 370.957, P value of Global Test in MR-PRESSO results = 0.078), on the basis of which we reran the secondary MVMR analysis, and the causal association between calcium levels and endometriosis remained significant after correcting for other calcium homeostatic modifiers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, OR = 1.13, 95% CI: 1.01–1.27, \u003cem\u003ep\u003c/em\u003e = 0.033) (see blue module of Supplementary Data Sheet 2).\u003c/p\u003e\u003cp\u003eCombining SVMR and MVMR analyses results, we were able to draw a strong conclusion that genetically predicted calcium levels are causally associated with an elevated risk of endometriosis, and that calcium levels are a risk factor for the development of endometriosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Endometriosis and calcium homeostasis regulatory levels (Inverse MR)\u003c/h2\u003e \u003cp\u003eWe carried inverse MR analysis to explore the effects of endometriosis on body calcium homeostasis. Endometriosis was considered as exposure, and calcium levels, 25 (OH) D, and PTH as outcomes. A total of 11 SNPs (\u003cem\u003ep\u003c/em\u003e \u0026lt; 5× 10\u003csup\u003e− 8\u003c/sup\u003e) that were closely associated with endometriosis risk were identified. And we discovered a causal association between endometriosis and 25(OH)D (β = 0.01, 95% CI: 0.00-0.02, \u003cem\u003ep\u003c/em\u003e = 0.007) and calcium (β = 0.02, 95% CI: 0.00-0.04, \u003cem\u003ep\u003c/em\u003e = 0.035). The inverse causality analysis of endometriosis and PTH did not reach significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e) (details in yellow module of Supplementary Data Sheet 2).\u003c/p\u003e \u003cp\u003eA sensitivity analysis was similarly performed and no significant evidence of horizontal pleiotropy in calcium were detected in either calcium (P-intercept = 0.621) (scatter plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) or 25(OH)D (P-intercept = 0.244) (scatter plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Heterogeneity in endometriosis for calcium (MR Egger: Cochran's Q = 27.27, P-heterogeneity = 0.001; IVW: Cochran's Q = 28.07, P-heterogeneity = 0.002) were revealed, whereas no heterogeneity was present in 25(OH)D (MR Egger: Cochran's Q = 4.84, P-heterogeneity = 0.775; IVW: Cochran's Q = 6.42, P-heterogeneity = 0.698). Due to the small number of IVs included in the analysis during the inverse MR process, the funnel plot results are not intuitive (funnel plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The leave-one-out analysis showed a high overall stability (the red line representing the overall effect did not cross the null line) (leave-one plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Details of the above results can be viewed in the yellow module of Supplementary Data Sheet 2.)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Calcium levels and endometriosis sub phenotypes\u003c/h2\u003e \u003cp\u003eSince endometriotic lesions can occur in different locations in the body, we collected GWAS aggregated statistics from the FinnGen database for six sub phenotypes of endometriosis, covering EO, EV, EI, ET, EP, EU, and EUO. Building on previous research, we employed two-sample MR analysis to further investigate the causal effects of genetically predicted calcium levels on different subtypes of endometriosis. As can be seen from the results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e), calcium levels were positively and causally associated only with EU, i.e., endometriosis of uterus (IVW: OR = 1.23, 95% CI: 1.01–1.49, \u003cem\u003ep\u003c/em\u003e = 0.038), with no suggestion of a causal relationship with the risk of other types of endometriosis subtypes (green module of Supplementary Data Sheet 2).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCurrently, it is widely recognized that endometriosis is a chronic inflammatory disease in which the glands and stroma of the ectopic endometrium are monitored by the body's immune system leading to chronic inflammation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. There is a complex relationship between the body's calcium homeostasis and inflammation, for example, hypocalcemia resulting from an imbalance of calcium homeostasis is commonly appears in systemic inflammatory diseases such as burns and sepsis[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been demonstrated that receptors of calcium homeostasis, calcium sensing receptors (CaSR), are both promoters and responders of inflammation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Not only that, the high incidence of infertility in women with pelvic endometriosis has also brought immunological factors into the field of endometriosis research. In endometriosis, the disturbance of local immunity has been recognized by most researchers, and the oxidative stress accompanied by inflammation further contributes to the disorder of immune response[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Improvement of the pelvic immune microenvironment after endometriosis lesion resection remains an effective therapeutic measure for chronic pelvic pain and infertility[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Vitamin D, as a nutrient that maintains the body's immune balance, regulates immune sensitivity and prevents the production of autoantibodies, prevents allergic reactions of the immune system, and may play a role in the prevention of autoimmune diseases and cancer[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, evidence for the emerging role of CaSR in controlling intestinal fluid homeostasis and immune homeostasis has been provided[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. All these studies emphasize the possible close relationship between calcium homeostasis and endometriosis.\u003c/p\u003e\u003cp\u003eOur study is the first to investigate the causal relationship between calcium homeostasis regulatory factors and endometriosis risk based on large-scale abstract level GWAS genetic data. Although several observational studies have been conducted previously, those conclusions have been inconsistent. For example, \u003cem\u003eDelbandi\u003c/em\u003e et al., by including 56 healthy women and 54 patients with endometriosis, found that subjects with low serum 25 (OH) D levels were at higher risk of endometriosis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and others have found low levels of 25(OH)D in women with endometriosis[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, a 2012 systematic review found that 25(OH)D levels were positively associated with endometriosis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. \u003cem\u003eDelbandi\u003c/em\u003e et al. also noted significantly lower calcium levels and significantly higher PTH in patients with endometriosis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but in another prospective cohort study that included 53 women with endometriosis and 25 controls, a trend toward elevated calcium levels was observed in the patients[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. At the same time, a retrospective analysis of studies found no significant difference in calcium levels between case and control groups of women with endometriosis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Recent studies on endometriosis and extracellular calcium homeostasis have focused on the effects of therapeutic measures (e.g., gonadotropin-releasing hormone agonists) on bone mineral density and calcium levels in endometriosis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and observational studies on the causal relationship between calcium levels and endometriosis have not yielded uniform results, leaving much room for exploration and research in this field.\u003c/p\u003e\u003cp\u003eWe obtained pooled statistics on serum 25(OH)D, calcium, endometriosis and its sub phenotypes, and PTH from EBI, UKBB, FinnGen databases, and published GWAS literature. Using these data, we performed four levels of MR analysis, including SVMR, MVMR, inverse MR and two-sample MR (exploring subtypes of endometriosis). Our results showed that genetically predicted calcium levels were positively associated with endometriosis, and inverse MR analysis indicated that endometriosis was associated with levels of 25(OH)D and calcium. In addition, MR analysis categorized by ectopic location identified calcium levels as a risk factor for endometriosis of uterus. These findings highlight the importance of monitoring calcium levels in patients with endometriosis. Since a large source of calcium and vitamin D in the body is obtained from food, the results of this study may provide dietary guidance for endometriosis patients and have important implications for the development of endometriosis prevention strategies and treatments. While fewer previous studies have examined associations between diet and endometriosis, \u003cem\u003eHarris\u003c/em\u003e et al conducted a 14-year prospective cohort study and found that higher predicted serum 25(OH)D levels and higher dairy intake were associated with a reduced risk of endometriosis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which results were also demonstrated by \u003cem\u003eJoanna\u003c/em\u003e et al.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, our study does not suggest a causal relationship between serum 25(OH)D levels and endometriosis risk, and additional experimental validation is needed in the future to more fully understand the role of these calcium homeostasis regulators in endometriosis risk.\u003c/p\u003e\u003cp\u003eThe strengths herein are as follows. The first is the advantage of MR, a statistical modality, which is not susceptible to confounding bias and reverse causation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which was further amplified by our comprehensive study of multiple types of MR. Next, rather than examining causality between calcium levels and endometriosis alone, we comprehensively included two other common regulators of calcium homeostasis (25 (OH) D and PTH). Overall, the analysis of multiple data and multiple MR methods improved the confidence of our results. Finally, we employed multiple rigorous methods for screening of IVs and used multiple complementary sensitivity analyses, including heterogeneity, pleiotropy and leave-one-out analyses, to verify the reliability of the MR Findings.\u003c/p\u003e\u003cp\u003eAlthough numerous strengths exist, there are also some shortcomings in the study. First of all, based on the genome-wide statistical significance threshold (5×10\u003csup\u003e− 8\u003c/sup\u003e), IVs that meet the criteria for PTH cannot be obtained for further studies. Therefore, we expanded the cutoff value to 5×10\u003csup\u003e− 6\u003c/sup\u003e when screening the IVs of PTH. Second, endometriosis is a female-specific disease, but the GWAS datasets for calcium homeostasis regulators included in the study were gender-biased, which may bias our findings. In addition, all the data in this study were limited to European ancestry, and it remains to be verified whether the findings can be applied to other populations.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis is the first comprehensive multi-type MR study to examine the causal relationship between calcium homeostasis and endometriosis risk. Our findings support a causal relationship between calcium levels and endometriosis risk, demonstrating that calcium levels are a risk factor for endometriosis. It also emphasizes the importance of monitoring calcium levels in patients suffering from endometriosis, which may provide dietary guidance for patients with endometriosis and may have important implications for endometriosis prevention and treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study can be accessed through the links provided in Table 1. Additional data are included in the article/supplementary material. Further queries can be directed to the corresponding author.\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\u003eThis work was supported by cross-innovation talent project in Renmin Hospital of Wuhan University (grant number JCRCZN-2022-016); Undergraduate education quality construction comprehensive reform project (grant number 2022ZG282) and the National Natural Science Foundation of China (grant number 82071655).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFFD and YXC contributed to the conceptualization and design of the study, RQW and XY collected and initially screened the data. ZMD performed a visual analysis of the data and was the main contributor to the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTaylor HS, Kotlyar AM, Flores VA: Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet 2021, 397(10276):839\u0026ndash;852.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChapron C, Marcellin L, Borghese B, Santulli P: Rethinking mechanisms, diagnosis and management of endometriosis. Nat Rev Endocrinol 2019, 15(11):666\u0026ndash;682.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTebben PJ, Singh RJ, Kumar R: Vitamin D-Mediated Hypercalcemia: Mechanisms, Diagnosis, and Treatment. 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Nature 2018, 558(7708):73\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRevez JA, Lin T, Qiao Z, Xue A, Holtz Y, Zhu Z, Zeng J, Wang H, Sidorenko J, Kemper KE \u003cem\u003eet al\u003c/em\u003e: Genome-wide association study identifies 143 loci associated with 25 hydroxyvitamin D concentration. Nat Commun 2020, 11(1):1647.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamat MA, Blackshaw JA, Young R, Surendran P, Burgess S, Danesh J, Butterworth AS, Staley JR: PhenoScanner V2: an expanded tool for searching human genotype-phenotype associations. Bioinformatics 2019, 35(22):4851\u0026ndash;4853.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Liu Z, Cui J, Chen X, Xiong J, Zhou W: Circulating adipokine levels and preeclampsia: A bidirectional Mendelian randomization study. 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J Clin Med 2021, 10(24).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang L, Cheng CY, Sun X, Pedicone AJ, Mohamadzadeh M, Cheng SX: The Extracellular Calcium-Sensing Receptor in the Intestine: Evidence for Regulation of Colonic Absorption, Secretion, Motility, and Immunity. Front Physiol 2016, 7:245.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelbandi AA, Torab M, Abdollahi E, Khodaverdi S, Rokhgireh S, Moradi Z, Heidari S, Mohammadi T: Vitamin D deficiency as a risk factor for endometriosis in Iranian women. J Reprod Immunol 2021, 143:103266.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnastasi E, Fuggetta E, De Vito C, Migliara G, Viggiani V, Manganaro L, Granato T, Benedetti Panici P, Angeloni A, Porpora MG: Low levels of 25-OH vitamin D in women with endometriosis and associated pelvic pain. Clin Chem Lab Med 2017, 55(12):e282-e284.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLerchbaum E, Obermayer-Pietsch B: Vitamin D and fertility: a systematic review. Eur J Endocrinol 2012, 166(5):765\u0026ndash;778.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSomigliana E, Panina-Bordignon P, Murone S, Di Lucia P, Vercellini P, Vigano P: Vitamin D reserve is higher in women with endometriosis. Hum Reprod 2007, 22(8):2273\u0026ndash;2278.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu BC, Zhang XM: [Associations of metabolism of lipid, calcium and phosphate in endometriosis]. Zhonghua Fu Chan Ke Za Zhi 2008, 43(3):185\u0026ndash;188.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStodtmann S, Nader A, Polepally AR, Suleiman AA, Winzenborg I, Noertersheuser P, Ng J, Mostafa NM, Shebley M: Validation of a quantitative systems pharmacology model of calcium homeostasis using elagolix Phase 3 clinical trial data in women with endometriosis. Clin Transl Sci 2021, 14(4):1611\u0026ndash;1619.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeth VB, van de Kar MM, Duffy JM, van Wely M, Mijatovic V, Maas JW: Gonadotropin-releasing hormone analogues for endometriosis. Cochrane Database Syst Rev 2023, 6(6):CD014788.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJurkiewicz-Przondziono J, Lemm M, Kwiatkowska-Pamula A, Ziolko E, Wojtowicz MK: Influence of diet on the risk of developing endometriosis. Ginekol Pol 2017, 88(2):96\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerduijn M, Siegerink B, Jager KJ, Zoccali C, Dekker FW: Mendelian randomization: use of genetics to enable causal inference in observational studies. Nephrol Dial Transplant 2010, 25(5):1394\u0026ndash;1398.\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":"Mendelian randomization, calcium, parathyroid hormone, 25-Hydroxyvitamin D, dietary guidance","lastPublishedDoi":"10.21203/rs.3.rs-3273922/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3273922/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Endometriosis is considered to be a complex gynecological disorder that affects the health and quality of life of affected women. The etiology and pathogenesis of endometriosis remain unclear, and few modifiable risk factors have been identified. It is generally believed that endometriosis is a chronic inflammatory disease, which can cause local immune disorders. And calcium homeostasis of the body is closely related to inflammation and immunity. Although observational studies have assessed the association between calcium homeostasis regulatory factor levels and endometriosis risk, the conclusions have been inconsistent. Therefore, the aim of this study was to explore the causal relationship between calcium homeostasis regulators and endometriosis risk using publicly available genome-wide association studies (GWAS) aggregated statistics.\u003c/p\u003e\n\u003cp\u003eMethods: The Mendelian randomization (MR) analysis was performed using GWAS data, including calcium (N = 315,153), serum 25-Hydroxyvitamin D (25(OH)D) (N = 496,946), parathyroid hormone (PTH) (N = 3,301), endometriosis (N = 77,257), endometriosis of ovary (N = 72,200), endometriosis of rectovaginal septum and vagina (N = 70,329), endometriosis of intestine (N = 69,146), endometriosis of fallopian tube (N = 69,085), endometriosis of pelvic peritoneum (N = 71,922), endometriosis of uterus (N = 71,341), and Unspecified/other endometriosis (N = 70,404). Four levels of MR analysis were undertaken, starting with single univariate MR and multivariate MR to test the correlation between calcium homeostasis regulatory factors and endometriosis, followed by inverse MR to explore the effect of endometriosis on body calcium homeostasis. And further two-sample MR to probe the relationship between calcium levels and endometriosis subtypes. Cochran's Q test, MR-Egger intercept test, leave-one-out analysis and funnel plot were utilized for sensitivity analysis.\u003c/p\u003e\n\u003cp\u003eResults: The two-sample MR analysis revealed a strong positive causal relationship between genetically predicted calcium levels and endometriosis risk (IVW: OR = 1.15, 95% CI: 1.02-1.29, \u003cem\u003ep\u003c/em\u003e = 0.018). Notably, the results of MVMR analysis demonstrated that the positive correlation of calcium levels on endometriosis still held even after correction for 25(OH)D and PTH (OR = 1.14, 95% CI: 1.02-1.28, \u003cem\u003ep\u003c/em\u003e = 0.026). After removing outliers using MR-PRESSO to ensure that horizontal pleiotropy was eliminated, MVMR analysis was performed again, and the causal association between calcium levels and endometriosis remained significant (OR = 1.13, 95% CI: 1.01-1.27, \u003cem\u003ep \u003c/em\u003e= 0.033). The inverse MR analysis discovered a causal association between endometriosis and 25(OH)D (β = 0.01, 95% CI: 0.00-0.02, \u003cem\u003ep\u003c/em\u003e = 0.007) and calcium (β = 0.02, 95% CI: 0.00-0.04, \u003cem\u003ep\u003c/em\u003e = 0.035). The two-sample MR analysis we employed to further investigate that calcium levels were positively and causally associated only with endometriosis of uterus (IVW: OR = 1.23, 95% CI: 1.01-1.49, \u003cem\u003ep\u003c/em\u003e= 0.038), with no suggestion of a causal relationship with the risk of other types of endometriosis subtypes.\u003c/p\u003e\n\u003cp\u003eConclusion：The comprehensive study of multiple types of MR provides genetic evidence for a causal relationship between calcium homeostasis and endometriosis risk, demonstrating that calcium levels are a risk factor for endometriosis. It also emphasizes the importance of monitoring calcium levels in patients suffering from endometriosis, which may provide dietary guidance for patients with endometriosis.\u003c/p\u003e","manuscriptTitle":"The role of calcium homeostasis in endometriosis: a comprehensive study of multiple types of Mendelian randomization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-25 21:13:16","doi":"10.21203/rs.3.rs-3273922/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":"52ea6b5e-f2ac-4853-af1a-03f95713060c","owner":[],"postedDate":"August 25th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-08-26T08:29:12+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-25 21:13:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3273922","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3273922","identity":"rs-3273922","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}