We employed various approaches to assess the potential causal connections between gut microbiota and IBS. These methods included the fixed/random-effects inverse-variance weighted (IVW) method, weighted median method, MR-Egger regression, and MR pleiotropy residual sum and outlier (MR-PRESSO) test. The IVW method was our primary analysis due to its precise effect estimates and widespread use in MR-analysis [24–26]. Initially, the IVW method calculated ratio estimates for individual SNPs using the Wald estimator and Delta method. Then, these estimates were combined to derive the main causal estimate [27]. To evaluate heterogeneity among selected SNPs, we used Cochran’s Q test. If heterogeneity was present (p < 0.05), we applied the random-effects IVW method; otherwise, we used the fixed-effects IVW method [28].
To assess the robustness of associations considering valid instruments and potential pleiotropic effects, sensitivity analyses were conducted. Firstly, we utilized the weighted median method as it provides reliable estimates of causal effects when valid instruments are lacking [29]. This approach yields valid causal effect estimates even if less than 50% of information is derived from invalid instruments [29]. Secondly, MR-Egger regression was employed to examine potential horizontal pleiotropy. If the p-value of the intercept was below 0.05, it indicated possible horizontal pleiotropy of SNPs [30]. Finally, we performed the MR-PRESSO test to identify outliers among SNPs by conducting a global test of heterogeneity. After identifying and removing potential outliers, a corrected association result was obtained [31].
To further investigate potential directional pleiotropy, each SNP used as instrumental variables (IVs) was examined for their associations with secondary phenotypes using the GWAS Catalog (http://www.ebi.ac.uk/gwas, last accessed on November 22, 2022). MR analyses were then repeated after excluding SNPs associated with other phenotypes.
The associations between human gut microbiota and the risk of IBS were reported as odds ratios (ORs) with 95% confidence intervals (CIs). All MR analyses were conducted using R version 4.2.2 (https://www.r-project.org/) with the “Mendelian Randomization,” “TwoSampleMR,” and “MR-PRESSO” packages.
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