Gut microbiota, inflammatory cytokines and endometriosis: a mediation Mendelian randomization study

Gut microbiota, inflammatory cytokines and endometriosis: a mediation Mendelian randomization study | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 24 February 2025 V1 Latest version Share on Gut microbiota, inflammatory cytokines and endometriosis: a mediation Mendelian randomization study Authors : Lele Pan , Yuying Chen , Yuzhen Cao , Biqi Huang , Xiaohui Huang , and Ying Ma [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174037893.33966505/v1 352 views 151 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Endometriosis (EM) is a prevalent and complex disease affecting women of reproductive age, with significant individual and societal burdens. Despite extensive research, the underlying etiology of EM remains poorly understood. Previous studies suggest a potential link between gut microbiota and EM, but the causal relationship between the two, as well as the role of inflammatory cytokines as a potential mediator, remains uncertain. Methods: Using publicly available genetic data, we employed Mendelian randomization (MR) to examine the causal relationships between gut microbiota, cytokines, and EM. A sensitivity analysis was performed to evaluate the robustness, heterogeneity, and potential horizontal pleiotropy of the results. Furthermore, we investigated whether inflammatory cytokines function as mediators in the pathway linking gut microbiota to EM. Results: Our results indicated that 20 gut microbiota and 7 inflammatory cytokines were causally linked to EM. In contrast, EM was found to affect the abundance of 14 gut microbiota and the levels of 2 inflammatory cytokines. Additionally, our data suggest that the impact of 4 gut microbiota on EM may be mediated through four distinct inflammatory factors. Acidaminococcus fermentans promoted the occurrence of EM through the mediating factor Cystatin D（CST5）, accounting for 3.8% of the total effect.Actinobacteria exerted protective effects against EM through the mediating factor Macrophage colony-stimulating factor 1 （CSF-1）, accounting for 7.6% of the total effect.Gut microbiota GCA-900066495 promoted the occurrence of EM through the mediating factor C-C motif chemokine 4（CCL4）, accounting for 8.3% of the total effect. Gut microbiota V9D3004 promoted the occurrence of EM through the mediating factor Interleukin-6（IL-6）, accounting for 17.6% of the total effect. Sensitivity analyses detected no significant heterogeneity or pleiotropy. Conclusion: Our Mendelian randomization study provides evidence of causal relationships between specific gut microbiota taxa and inflammatory cytokines in the context of EM. We identify potential inflammatory mediators that may facilitate the linkage between gut microbiota and EM. These insights underline the importance of targeting gut microbiota and cytokine pathways in future therapeutic strategies for EM management. not-yet-known not-yet-known not-yet-known unknown 1. Introduction Endometriosis is a prevalent gynecological condition, affecting an estimated 10% of women, which corresponds to around 190 million individuals worldwide.1 It is characterized by the presence of tissue resembling endometrial lining in locations outside the uterus.1 This condition is associated with symptoms that significantly deteriorate the quality of life for patients, including intense pelvic pain, dysfunction of pelvic organs, infertility, and secondary psychological issues.2 Despite the substantial effects of endometriosis on both the physical and mental health of women, the fundamental mechanisms that contribute to its pathogenesis are not well elucidated. The etiology of endometriosis is intricate and involves multiple factors, such as retrograde menstruation, dysregulation of the immune system, benign metastasis, hormonal fluctuations, coelomic metaplasia, contributions from stem cells, alterations in epigenetic regulation, and environmental factors.1-3 Presently, a plethora of theories and supporting evidence exists for each of these factors. However, no singular pathophysiological or molecular mechanism has been identified that comprehensively accounts for all cases of the disease. A thorough understanding of the mechanisms underlying this condition is essential for the development of more effective treatment options, which remain limited at present. The gut microbiota is considered the largest microbial ecosystem in the human body, containing around 10^14 microorganisms.4 These microbes are vital for maintaining physiological balance and regulating metabolism, offering a wide range of health benefits to the host. These benefits include modulating the immune system, reinforcing epithelial barriers, synthesizing vitamins, and enhancing nutrient absorption, which is why they are often referred to as the ”forgotten organ”.4, 5 In recent years, numerous studies have demonstrated variations in the diversity of gut microbiomes between women with endometriosis and those without the condition. Svensson et al. demonstrated a significant difference in the abundance of 12 bacterial species from the classes Bacilli, Bacteroidia, Clostridia, Coriobacteriia, and Gammaproteobacteria between patients with endometriosis and the control group.6 Yu et al. also found that, compared to the control group, the EM group exhibited lower α diversity of the gut microbiota and a higher Firmicutes/Bacteroidetes ratio.7 They observed significant differences in the abundances of various taxa, including Actinobacteria, Tenericutes, Blautia, Bifidobacterium, Dorea, and Streptococcus, between the two groups. Ata et al. observed that the composition of the gut microbiota was altered in patients with endometriosis compared to the control group.8 Specifically, they found that a higher proportion of women in the stage 3/4 endometriosis group exhibited a stool microbiome dominated by Shigella/Escherichia. Our research group also reported an increase in the relative abundance of the harmful microorganisms Eggerthella lenta and Eubacterium dolichum in the gut of women with endometriosis, alongside a significant decrease in the relative abundance of other protective microorganisms, particularly Clostridia, Ruminococcus, and Lachnospiraceae.9 Although numerous studies have been conducted, evidence for the causal relationship between the gut microbiome and endometriosis remains insufficient. Furthermore, due to the critical role of inflammatory immune responses in the development and progression of endometriosis, this condition is also classified as a chronic inflammatory disease. Evidence indicates that aberrant immunologic and inflammatory responses, particularly elevated levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-17A, are present in the serum, peritoneal fluid, and ectopic lesions of patients with endometriosis.10-12 Furthermore, an increasing body of research suggests that altered expression of anti-inflammatory cytokines, including IL-4, IL-10, and transforming growth factor-β1, occurs in women with endometriosis, including in serum, peritoneal fluid, and ectopic lesions.13 In addition to these inflammatory mediators, other cytokines, such as macrophage migration inhibitory factor (MIF), IL-3, IL-5, IL-8, IL-13, IL-25, IL-33, IL-36, and IL-37, have also been implicated in the pathogenesis and progression of the disease.14 These inflammatory cytokines play crucial roles by promoting the survival, growth, invasion, differentiation, angiogenesis, and immune evasion of endometriotic lesions. In summary, these cytokines provide valuable insights for the future exploration of potential serum biomarkers for the diagnosis of endometriosis and are emerging as promising therapeutic targets for its treatment. Previous research has also highlighted the critical role of gut microbiota in regulating various inflammatory and proliferative conditions.15 Disruption of immune homeostasis, driven by microbial imbalances, can result in chronic inflammation, thereby fostering an environment that promotes enhanced adhesion and angiogenesis—key processes involved in the development of endometriosis.16 Evidence suggests that dysbiosis can impair normal immune function, leading to elevated levels of pro-inflammatory cytokines, weakened immune surveillance, and altered immune cell profiles, all of which may contribute to the pathogenesis of endometriosis.17 Yu et al. also observed a positive correlation between serum IL-17A levels and the abundance of Bacteroides, while a negative correlation was found between IL-17A levels and the abundances of Streptococcus and Bifidobacterium.7 Additionally, serum IL-8 levels were significantly negatively correlated with the abundance of Subdoligranulum. A recent study suggests that intestinal dysbiosis contributes to elevated estrogen levels, which subsequently strongly activate IL-17 and IL-33.18 This supports the concept of a gut–endometrial axis as a key factor in infertility associated with endometriosis. Therefore, it is plausible to hypothesize that causal relationships may exist between the gut microbiota, inflammatory cytokines, and endometriosis. Our study seeks to explore these potential connections and identify specific inflammatory cytokines that could serve as important biomarkers for early diagnosis and possible clinical intervention. Although traditional observational studies may contribute to understanding the causal relationships between gut microbiota or inflammatory cytokines and endometriosis, they are often limited by challenges such as confounding factors and reverse causation. Mendelian randomization (MR) is a genetic epidemiological approach that uses genetic variants as instrumental variables (IVs) to assess causal relationships between exposure factors and outcomes. Compared to traditional observational methods, MR is less prone to residual confounding and reverse causation. In this study, we performed a comprehensive MR analysis to investigate the causal relationships between the gut microbiome, inflammatory cytokines, and endometriosis. We also explored the role of inflammatory cytokines as mediators in the pathway linking the gut microbiota to EM. Additionally, through reverse causality analysis, we assessed whether genetic predisposition to endometriosis influences the gut microbiota and inflammatory cytokines. This approach will contribute to a deeper understanding of the roles of the gut microbiome and inflammatory cytokines in the pathogenesis of endometriosis, offering new perspectives for clinical diagnosis and treatment. 2. Methods 2.1 Study design Our study investigated the genetic associations between gut microbiota, inflammatory cytokines, and endometriosis using publicly available genome-wide association studies (GWAS) data. The study design consisted of three main steps: First, we applied bi-directional two-sample univariable MR to examine the causal relationships between 473 gut microbiota species and endometriosis. Second, we utilized a similar approach to assess the causal links between 91 inflammatory cytokines and endometriosis. Third, we conducted a mediation analysis to explore the role of inflammatory cytokines in the pathway from gut microbiota to endometriosis. Following the framework proposed by Bowden et al., 19 our MR analysis was based on three key assumptions: (1) the instrumental variables (IVs) were associated with the exposure variable, (2) the IVs were not correlated with unmeasured confounders affecting the exposure, and (3) the IVs impacted the outcome exclusively through the exposure variable without any alternative causal pathways. A schematic of the study design is shown in Figure 1. 2.2 Data sources The data on 473 gut microbiota samples (GCST90032172–GCST90032644) were extracted from GWAS examining 2801 microbial taxa and 7,967,866 human genetic variants in a cohort of 5959 European individuals from the FR02 study, which is based on the Finnish population. 20 The genome-wide association analysis of gut microbial taxa identified 567 independent SNP-taxon associations. The inflammatory cytokine dataset used in this study was obtained from the GWAS Catalog database (ID: GCST90274758-GCST90274848) . 21 Additionally, the EM data were sourced from the FinnGen consortium’s R11 release dataset (https://r11.finngen.fi/), which includes 18,260 case samples and 119,468 control samples, all of European ancestry. 22 Our study involved human participants and was based on a secondary analysis of previously published data, which had already obtained ethical approval and informed consent. Therefore, no additional ethical clearance or informed consent was required. 2.3 Instrumental variables selection Consistent with the methodologies employed in previous studies, multiple IVs were carefully selected for gut microbiota, each inflammatory cytokine, and EM within the framework of our MR analysis. 23, 24 Single nucleotide polymorphisms (SNPs) with statistically significant associations to gut microbiota and inflammatory cytokines were identified at the genome-wide significance threshold ( p < 5 × 10⁻⁶, r² = 0.001, genetic distance = 10,000 kb). A more lenient threshold was applied to the gut microbiota and inflammatory cytokines to ensure a sufficient number of SNPs for subsequent sensitivity analyses. In the reverse MR analysis assessing the impact of endometriosis on gut microbiota or inflammatory cytokines, EM was treated as the ”exposure,” and the significance threshold was adjusted to p < 5 × 10⁻⁸, r² = 0.001, genetic distance = 10,000 kb. The clumping procedure utilized the European reference panel from the 1000 Genomes Project to estimate linkage disequilibrium (LD) among SNPs. A critical step in the MR analysis involved verifying that the effects of the SNPs on the exposure aligned with the same allele as their effects on the outcome. After aligning the outcome data, palindromic SNPs (those with A/T or G/C alleles, where the forward and reverse strands are identical) were excluded to ensure accuracy. The R² value for each SNP was calculated to quantify the proportion of exposure variance explained by the IVs. To minimize potential bias from weak instruments, we assessed the strength of IV correlations using the F-statistic and included only SNPs with an F -statistic greater than 10 in the MR analysis. 2.4 MR analysis and mediation analysis To estimate the causal effects of gut microbiota and inflammatory cytokines on EM, we conducted a two-sample bidirectional MR analysis. The primary method used was Inverse Variance Weighted (IVW), with four additional methods (Weighted Median, MR-Egger regression, Weighted Mode, and Sample Mode) employed as supplementary analyses. Through the two-sample analysis, we identified gut microbiota and inflammatory cytokines that exert significant causal effects on EM, which were subsequently included in the mediation analysis to examine whether inflammatory cytokines mediate the causal pathway from gut microbiota to the EM outcome. The overall effect was decomposed into an indirect effect (via mediators) and a direct effect (without mediators). 25 Specifically, the total effect of gut microbiota on EM was partitioned into: 1) the direct effects of gut microbiota on EM (β0 in Figure 1, Step C); 2) the indirect effects mediated by gut microbiota through the mediator (β1 × β2 in Figure 1, Step C). The proportion of the effect mediated by the mediator was calculated by dividing the indirect effect by the total effect ([β1 × β2] / β0). Additionally, 95% confidence intervals were calculated using the method. 2.5 Sensitivity analysis We performed sensitivity analyses using MR-Egger regression, the leave-one-out method, and the MR-PRESSO approach. Heterogeneity among individual SNPs was assessed using Cochran’s Q statistic. A fixed-effects model was employed when heterogeneity was minimal (P < 0.05), while a random-effects model was applied in the presence of substantial heterogeneity. To assess potential horizontal pleiotropy, we utilized both MR-Egger regression and the MR-PRESSO method. MR-PRESSO was used to identify significant outliers and adjust for horizontal pleiotropy by removing these outliers. The P-value from the intercept test in MR-Egger regression was used to evaluate the presence of horizontal pleiotropy. Additionally, a ”leave-one-out” sensitivity analysis was performed to examine whether any individual SNP could have biased the overall causal inference. Scatter plots were used to demonstrate the absence of outlier influence on the results, and funnel plots provided evidence for the robustness of the association, free from heterogeneity. For this study, MR analysis results for binary outcomes are presented as odds ratios (ORs), while results for continuous outcomes are presented as Beta coefficients, with the sign of the Beta value indicating the direction of the association. No corrections for multiple testing were made in this exploratory analysis. All statistical analyses were two-sided and conducted at a significance level of 0.05. Analyses were performed using R (v4.3.2) statistical software, and MR analysis was conducted with the R package ”TwoSampleMR.” 3. Results 3.1 Casual relationship between gut microbiota and EM To examine the causal relationship between gut microbiota and EM, a bidirectional two-sample MR analysis was performed. The comprehensive results from the MR analysis, shown in Figure 2, indicate that 20 gut microbiota have a significant causal effect on EM. Among these, 10 species (Acetobacterales, Acidaminococcus fermentans, CAG-180 sp000432435, CAG-495, CAG-510 sp002432425, Faecalibacterium prausnitzii E, GCA-900066495, Pauljensenia sp000411415, Terrisporobacter, V9D3004) increased the risk of EM (OR values ranging from 1.060 to 2.353) (Figure 2), while 10 species (Actinobacteria, Actinomycetales, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium ruminantium, Clostridium M sp001304855, Enterococcus B, Enteroscipio, Paceibacteria, UBA6398 sp002451695) had protective effects against EM (OR values ranging from 0.484 to 0.935) (Figure 2). The reverse MR analysis conducted in our study revealed that EM, as an exposure factor, exerts an influence on 14 gut microbiota, as shown in Figure 2. Employing the IVW analysis, we determined that the incidence of EM correlates with heightened abundance of 5 gut microbiota species (Bifidobacteriaceae, Bifidobacterium adolescentis, Bifidobacterium ruminantium, Bifidobacterium, UBA6398) (OR values ranging from 1.065 to 1.117) (Figure 2), while decreased abundance of 9 gut microbiota species (Bacteroides stercoris, Enterococcus B, Firmicutes E, K10, Sporomusales, Succinivibrionaceae, Succinivibrio, Syntrophorhabdaceae, Syntrophorhabdia) (OR values ranging from 0.906 to 0.986) (Figure 2). Other Mendelian randomization approaches also corroborated our findings, revealing a similar trend of change (Supplementary Figure). The robustness of the identified causal relationships was further confirmed by the results obtained from alternative methods and sensitivity analyses, as shown in Supplementary Table S1. Additionally, scatter plots and funnel plots offered visual confirmation of the consistency and reliability of the results, as illustrated in Supplementary Figure. 3.2 Casual relationship between inflammatory cytokines and EM Based on the Mendelian randomization analysis, we obtained the following results (Figure 3). The IVW analysis indicated that genetically predicted higher levels of Macrophage colony-stimulating factor 1 （CSF-1）, Cystatin D（CST5）, and IL-6 (OR = 1.107, 95% CI = 1.021-1.199, p = 0.013; OR = 1.05, 95% CI = 1.006-1.084, p = 0.020; OR = 1.163, 95% CI = 1.016-1.332, p = 0.027), along with lower levels of C-C motif chemokine 4（CCL4）, C-X-C motif chemokine 11（CXCL11）, Leukemia inhibitory factor receptor（LIF-R）, and Programmed cell death 1 ligand 1 （PD-L1）( OR = 0.923, 95% CI = 0.887-0.960, p = 8.058e-05; OR = 0.897, 95% CI = 0.826-0.975, p = 0.010;OR = 0.866, 95% CI = 0.802-0.936, p = 0.0002;OR = 0.873, 95% CI = 0.801-0.952, p = 0.002), are associated with an increased risk of endometriosis. Additionally, we investigated the impact of endometriosis as an exposure factor on two inflammatory cytokines. The incidence of endometriosis was found to be positively correlated with elevated levels of CCL19 and IL-8 (OR = 1.053, 95% CI = 1.000-1.109, p = 0.047; OR = 1.060, 95% CI = 1.000-1.123, p = 0.048). To validate the robustness of these associations, we employed four additional methods and conducted a sensitivity analysis (Supplementary Table). Cochran’s Q test and MR-Egger regression intercept analysis showed no significant heterogeneity or horizontal pleiotropy among the IVs. Further confirmation of our findings was provided by scatter plots, funnel plots, and leave-one-out analyses. Additionally, MR-PRESSO analysis revealed no detectable outliers. 3.3 Mediating role of inflammatory cytokines between gut microbiota and EM Through a two-step Mendelian randomization analysis, our mediation MR results revealed the following findings (Figure 4 and Table 1). Specifically, we identified that four cytokines act as mediators in the pathway linking gut microbiota to endometriosis, playing a promotive role in the effect of specific gut microbiota on endometriosis. Sensitivity analyses showed no significant heterogeneity or pleiotropy (Supplementary Figures and Table). Acidaminococcus fermentans promoted the occurrence of EM through the mediating factor CST5. The mediating effect of CST5 was 0.0071, with a mediating ratio of 3.8%. Actinobacteria exerted protective effects against EM through the mediating factor CSF-1. The mediating effect of CSF-1 was -0.0224, with a mediating ratio of 7.6%. Gut microbiota GCA-900066495 promoted the occurrence of EM through the mediating factor CCL4. The mediating effect of CCL4 was 0.0164, with a mediating ratio of 8.3%. Gut microbiota V9D3004 promoted the occurrence of EM through the mediating factor IL-6. The mediating effect of IL-6 was 0.0236, with a mediating ratio of 17.6%. 4. Discussion In this study, we conducted bidirectional two-sample Mendelian randomization and mediation analyses, providing evidence for a causal relationship between gut microbiota and endometriosis, with inflammatory cytokines acting as mediators. We identified four specific gut microbial taxa that exert their effects on endometriosis through mediation by corresponding inflammatory cytokines. This research underscores the importance of gut microbiota and inflammatory cytokines in the pathogenesis of EM and highlights potential therapeutic targets for intervention. CST5 is a member of the cystatin superfamily, which consists of inhibitors of cathepsins—cysteine proteases that degrade various substrates, including adhesion proteins, matrix components, and other proteases. 26 Human cystatin D, encoded by the CST5 gene, inhibits cathepsins H, S, and L, but not cathepsin B, and was initially purified from saliva 27 . Furthermore, cystatins have been implicated in a range of biological processes, such as cell proliferation, differentiation, survival, migration, as well as the production of interleukins and nitric oxide. 28 Prior to the present study, the relationship between CST5, gut microbiota, and endometriosis had not been elucidated. Acidaminococcus fermentans is a strictly anaerobic, gram-negative coccus that is capable of fermenting glutamate into ammonia, CO2, acetate, butyrate, and hydrogen (H2) . 29, 30 Previous studies have indicated that Acidaminococcus fermentans is positively associated with left ventricular diastolic dysfunction (LVDD). 31 Zhou et al. also identified Bacteroides coprophilus, Parabacteroides distasonis, Eubacterium siraeum, Acidaminococcus fermentans, and Prevotella copri as specific microbiota markers enriched in the gut of patients with ankylosing spondylitis, along with enhanced pathways related to oxidative phosphorylation, LPS biosynthesis, and glycosaminoglycan degradation. 32 However, research on the relationship between Acidaminococcus fermentans and endometriosis is currently very limited. Our findings indicate that Acidaminococcus fermentans promotes the development of endometriosis through the mediating factor CST5. These results offer new insights for the design of future experimental studies, although further investigation into the underlying mechanisms is still required. CSF-1, or macrophage colony-stimulating factor（M-CSF）, initiates a signaling cascade upon binding to its receptor, CSF-1R (also known as CD115 or c-fms), thereby enhancing the differentiation, proliferation, survival, recruitment, and functional activity of macrophages. 33, 34 Previous studies have suggested that CSF-1 plays a role in sustaining the chronic inflammatory response associated with endometriosis. Elevated levels of CSF-1 have been detected in the peritoneal fluid of individuals with endometriosis, indicating its involvement in the disease’s pathophysiology. 35 Acting in an autocrine manner, CSF-1 directly influences the proliferation of endometrial epithelial cells and their attachment to peritoneal mesothelial cells, which are key early steps in the formation of endometriotic lesions on the peritoneum. 36 Budrys et al. further investigated and confirmed that elevated CSF-1 levels may play a role in the formation and progression of endometriotic lesions. 33 The study indicated that the increase in CSF-1 following the coculture of endometrial cells with PMCs suggests that the endometrial tissue could serve as a source of peritoneal CSF-1. Additionally, the study revealed that the upregulation of C-FMS expression in endometrial cells from women with endometriosis, when cocultured with PMCs, implies that the endometrial tissue involved in lesion formation is highly responsive to CSF-1 signaling. This study reveals the relationship between CSF-1 and Actinobacteria in endometriosis. Actinobacteria are a large and diverse group of bacteria known to produce a wide range of secondary metabolites, many of which exhibit significant biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. 37 Research indicates that, in contrast to healthy endometrium, the endometrial microbiota in patients with endometriosis shows an increased abundance of Actinobacteria. 38 Another study suggests that the cervical microbiome can be altered during the development and progression of endometriosis, with a tendency toward decreased levels of Actinobacteria. 39 A recent study compared the gut microbiota between the EM and control groups, and the results demonstrated a significant increase in the abundance of Actinobacteria in the EM group. 7 Additionally, the study confirmed the association between the gut microbiota and serum hormones as well as inflammatory factors in EM. Our findings indicate that the bidirectional Mendelian randomization analysis between CSF-1 and endometriosis reveals that CSF-1 is a risk factor for the development of endometriosis, while CSF-1 also acts as a mediating factor in the protective effects of Actinobacteria on endometriosis. Previous studies, along with our own, have elucidated the complex causal relationship between the gut microbiota, inflammatory cytokines, and the pathogenesis of endometriosis. Further in-depth research is still required in the future. CCL4, formerly known as macrophage inflammatory protein-1β (MIP-1β), is an inflammatory chemokine within the CC chemokine family. 40 CCL4 exerts diverse effects on both immune and non-immune cells through its interaction with the specific receptor CCR5, in concert with related CC chemokines such as CCL3 and CCL5, which also bind to CCR5. 41 Research has shown that CCL4 can promote tumor development and progression by recruiting regulatory T cells and pro-tumor macrophages, as well as influencing other resident cells within the tumor microenvironment, such as fibroblasts and endothelial cells, to enhance their pro-tumorigenic functions. 41 Conversely, in certain contexts, CCL4 may enhance tumor immunity by recruiting cytolytic lymphocytes and macrophages with phagocytic activity. 41, 42 The dual role of CCL4 in both immune activation and suppression highlights its complex involvement in immune dynamics. Recent studies have increasingly focused on the role of CCL4 (MIP-1β) in the pathogenesis of endometriosis. In the study conducted by Geethadevi et al., CCL4 levels were assessed in the conditioned medium of isolated endometrial stromal cells derived from primary infertile patients without endometriosis, as well as from those with stage IV ovarian endometriosis, utilizing a multiplex assay. 43 The researchers discovered that CCL4 was detectable exclusively in the conditioned media of stromal cells obtained from the ovarian endometriosis group. Monsanto et al. investigated the impact of endometriotic lesion removal on local and systemic inflammation. 44 They found that, compared to pre-surgery tissue, macrophage inflammatory protein 1β was expressed at higher levels in eutopic tissue than in ectopic tissue. A previous case-control study, including 126 women with endometriosis and 45 control women, found that during the menstrual phase, peritoneal fluid from endometriosis patients showed upregulation of MIF-1β compared to other phases. 45 However, no such upregulation was observed in the peritoneal fluid of control women. Our study also indicates that the gut microbiota GCA-900066495 promotes the development of endometriosis through the mediator CCL4. However, research on the relationship between gut microbiota GCA-900066495, CCL4, and endometriosis remains limited. Our findings provide new insights and perspectives for further investigating the role of GCA-900066495 and CCL4 in the pathogenesis of endometriosis. IL-6 is a multifunctional cytokine that exerts a range of biological activities through its receptor (IL-6R) and plays a pivotal role in various biological processes, including immune response, inflammation, and hematopoiesis 46 . IL-6 also plays a critical role in reproductive processes, such as steroid hormone production by the ovaries, folliculogenesis, and oocyte maturation, facilitated by ovarian angiogenesis and the enhancement of vascular permeability. Increasing evidence suggests that IL-6 is a key mediator in the inflammatory processes associated with endometriosis. 47 Studies have demonstrated that IL-6 levels are significantly elevated in the serum, follicular fluid, and peritoneal fluid (PF) of women with endometriosis compared to healthy controls. 48, 49 Macrophages are the predominant source of IL-6 secretion in the PF. 50 Further studies have shown that mature activated T cells, ectopic endometrial stromal cells, and epithelial cells can also produce IL-6, influencing its concentration in the PF of endometriosis patients. 48 IL-6 acts as an activator of macrophages, amplifying angiogenesis and modulating the immune microenvironment of the endometrium. 47 Consequently, IL-6 is believed to contribute to the infertility often observed in women with endometriosis. Additionally, research indicates that IL-6 can influence the composition and function of the gut microbiota, while the gut microbiota can also modulate IL-6 production, establishing a bidirectional relationship that is crucial for maintaining gut health and overall immune function. 51-53 Currently, research on the relationship between IL-6 and gut microbiota dysbiosis in endometriosis remains limited. Our study suggests that IL-6 plays a crucial role in promoting the development of endometriosis via the gut microbiota strain V9D3004. This strain, classified as Lachnospiraceae bacterium, has not been extensively studied in relation to endometriosis and IL-6. Further investigation is needed to deepen our understanding of this connection, which could pave the way for novel therapeutic approaches in the treatment of endometriosis. Our study provides genetic evidence linking gut microbiota to inflammatory cytokines in the context of endometriosis. However, several limitations in our analyses should be acknowledged. First, subgroup analyses were constrained due to the lack of comprehensive demographic information, such as age. Future studies should aim to incorporate a more diverse and detailed demographic dataset to facilitate more robust subgroup analyses. Second, as the GWAS cohort predominantly consisted of individuals of European descent, the generalizability of our findings to other populations may be limited. Third, our study primarily focused on genetic associations and did not account for potential confounding factors. Future research should integrate comprehensive clinical and lifestyle data—such as BMI, age, treatment history, lifestyle factors, family history, reproductive history, menopausal status, and comorbidities—to address this limitation. Furthermore, the absence of clinical validation, coupled with the complex interplay between gut microbiota and inflammatory cytokines, warrants caution in interpreting these results, as they require further confirmation through clinical studies. Additionally, the relatively small sample size in our study may limit the generalizability of our conclusions. Replication of our findings in larger, more ethnically diverse cohorts will be essential to validate our results and ensure the consistency of the observed associations across various populations and settings. 5. Conclusion To our knowledge, this is the first comprehensive study evaluating the causal relationships between gut microbiota, inflammatory cytokines, and EM. Our research demonstrates a causal relationship between specific gut microbiota taxa and inflammatory cytokines in EM, revealing critical inflammatory mediators that link these factors. The robust findings underscore the significance of exploring gut microbiota and cytokine pathways for future therapeutic interventions in EM management. However, further investigations are necessary to validate these relationships clinically and to elucidate the underlying biological mechanisms at play. Supplementary Materials: The following supporting information can be downloaded at: Supplementary FigureS1: Results of sensitivity analysis and visualization of the MR results. Supplementary Table S1: Summary of 473 gut microbiota and 91 inflammatory cytokine data, along with the results of the MR analysis. Author Contributions: The authors’ contributions are as follows: P.L., C.Y.Y., C.Y.Z., H.X., and M.Y. developed the concept and design; P.L. and C.Y.Y. collected the data; P.L., C.Y.Y., and C.Y.Z.,H.B. performed the statistical analysis and data interpretation; P.L. drafted the manuscript; P.L., C.Y.Y., C.Y.Z.,H.B.,H.X., and M.Y. conducted critical revisions of the manuscript; and all authors were involved in data interpretation, document revision, and final approval of the manuscript. Funding: This work was supported by Guangdong Basic and Applied Basic Research Foundation (Grant Nos. 2022A1515011880, 2023A1515011688), and the President Foundation of Zhujiang Hospital, Southern Medical University (Grant No. yzjj2022ms18) to Ying Ma. This work was also supported by the Clinical Study on the Treatment of Dysmenorrhea with Fire Dragon Cupping Combined with Feng’s Endometriosis Formula (Grant No. 202300067) to Xiaohui Huang. Data Availability Statement: All data presented in this manuscript were previously deposited in publicly accessible repositories and are freely available for download. The GWAS summary statistics for gut microbiota and each inflammatory cytokine are publicly available from the GWAS Catalog (gut microbiota IDs: GCST90301941-GCST90302173, and inflammatory cytokine IDs: GCST90274758-GCST90274848) (https://www.ebi.ac.uk/gwas/).The GWAS summary statistics for EM are available on the FinnGen consortium’s R11release dataset(https://r11.finngen.fi/). Acknowledgments: We extend our sincere gratitude to the participants and researchers of the FinnGen study for their efforts in making the GWAS data publicly available. We also appreciate the GWAS Catalog for providing the summary statistics of plasma circulating metabolites. Declarations Ethics approval and consent to participate： As the GWAS data is publicly accessible, ethical approval was not required. 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Figure 1 Hypothesis and design of bidirectional and mediated Mendelian randomization analyses. β 0 represents the total effect of gut microbiota on endometriosis; β 2 denotes the causal effect of inflammatory cytokines on endometriosis; β 1 reflects the causal effect of gut microbiota on inflammatory cytokines. Figure 2. Mendelian randomization analyses of the causal effects between gut microbiota and endometriosis Figure 3. Mendelian randomization analyses of the causal effects between inflammatory cytokines and endometriosis Figure 4. Mendelian randomization analyses of the causal effects between gut microbiota and inflammatory cytokines. Exposure Mediator Total effect Mediating effect direct effect Proportion mediated Acidaminococcus fermentans CST5 0.1845 0.0071 0.1774 0.038 Actinobacteria CSF-1 -0.2923 -0.0224 -0.2699 0.076 GCA-900066495 CCL4 0.1966 0.0164 0.1802 0.083 V9D3004 IL-6 0.134 0.0236 0.1104 0.176 Table 1. Mediation analysis of the causal effects between gut microbiota, inflammatory cytokines, and endometriosis. Information & Authors Information Version history V1 Version 1 24 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cytology: endometrial endocrinology endometriosis: basic science general gynaecology Authors Affiliations Lele Pan Zhujiang Hospital of Southern Medical University Department of Obstetrics and Gynecology View all articles by this author Yuying Chen Zhujiang Hospital of Southern Medical University Department of Obstetrics and Gynecology View all articles by this author Yuzhen Cao Zhujiang Hospital of Southern Medical University Department of Obstetrics and Gynecology View all articles by this author Biqi Huang Zhujiang Hospital of Southern Medical University Department of Obstetrics and Gynecology View all articles by this author Xiaohui Huang Guangdong Women and Children Hospital View all articles by this author Ying Ma [email protected] Zhujiang Hospital of Southern Medical University Department of Obstetrics and Gynecology View all articles by this author Metrics & Citations Metrics Article Usage 352 views 151 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Lele Pan, Yuying Chen, Yuzhen Cao, et al. 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