Endometriosis, defined as the presence of endometrial tissue outside the uterine cavity, typically lines various locations such as the ovaries, fallopian tubes, vagina, or other parts of the uterus (Vercellini et al., 2014), manifests with ectopic endometrial tissue that develops and detaches during the menstrual cycle, leading irregular bleeding. While the prevalence of endometriosis in fertile women ranges from 10% to 15%, the incidence rate significantly rises to 20%–50% in cases of infertility (Tanbo and Fedorcsak, 2017; Zondervan et al., 2020). The widely accepted theory of menstrual retrograde posits, proposed by Sampson (Sampson, 2024), that endometrial fragments retrograde through the fallopian tube, contributing significantly to the etiology of endometriosis. This process can induce inflammation and scar tissue formation, resulting in pelvic pain, sexual discomfort, menstrual irregularities, and even infertility (Tanbo and Fedorcsak, 2017; Peiris et al., 2018). Research has revealed differences in both the quantity and activation status of immune cells within the endometrium between patients with endometriosis and normal females (Agic et al., 2006; Saraswat et al., 2017; Patel et al., 2018; Vallvé-Juanico et al., 2019; Bunis et al., 2022). The aberrant expression of these immune cells may contribute to the development and progression of endometriosis, exerting adverse effects on embryo implantation and reproductive outcomes (Saraswat et al., 2017). Additionally, the ectopic endometrium of endometriosis patients harbors a spectrum of immune cells associated with both innate and adaptive immune systems, collectively creating a conducive environment for the ectopic endometrial growth (Vallvé-Juanico et al., 2019; Bunis et al., 2022).

The symbiotic association between the host and microbiota is characterized by mutual benefits (Shi et al., 2017). The host serves as a vital habitat, supplying essential nutrients to sustain the microbiome, while the gut microbiota, in turn, aid in the development of the metabolic system and facilitate the maturation of the immune system through the provision of advantageous nutrients (Kau et al., 2011). Nevertheless, alterations in the equilibrium of gut microbiota communities can lead to dysbiosis, triggering diseases (Rooks and Garrett, 2016; Shi et al., 2017). Numerous studies have underscored the correlation between alterations in gut microbiota and diverse disorders, emphasizing their impact on systemic inflammation and immune cell function (Karmarkar and Rock, 2013; Costello et al., 2015). Certain studies have specifically explored the correlation between the intestinal flora and the etiology of endometriosis (Chadchan et al., 2021; Salliss et al., 2021). Particularly noteworthy is the study by Ata et al., which identified elevated levels of Gardnerella, Streptococcus, Escherichia, Shigella, and Ureoplasma in women with endometriosis (Ata et al., 2019). Additionally, Acidovorax, Devosia, Methylobacterium, Phascolarctobacterium, and Streptococcus abundance in the peritoneal fluid of endometriosis patients surpassed that in controls (Yuan et al., 2022). The imbalance of gut microbiota can lead to an imbalance of the immune system, resulting in a dysregulation of immune cells and their corresponding pathways. While the microbiota composition in the vaginal, cervical, and intestinal regions exhibits similarities between ASRM (American Society for Reproductive Medicine) stage 3/4 endometriosis patients and controls, variations at the genus level are evident (Author Anonymous, 1997; Ata et al., 2019). They substantiated an association between gut microbiota, serum hormones, and inflammatory factors in endometriosis (Shan et al., 2021). Current primary treatment methods for endometriosis include medication and surgery. Hormonal therapies, such as GnRH agonists and Dienogest, are commonly employed to manage symptoms but come with side effects like mood instability, perimenopausal symptoms, and decreased bone density (Vercellini et al., 2014). Surgery, often utilized to remove endometriosis lesions and scar tissue, is associated with a high recurrence rate (Shakiba et al., 2008; Guo, 2009; Horne and Missmer, 2022). Frequent surgeries not only pose a risk to surrounding organs but also impose substantial economic and psychological burdens on patients. In addition, Zizolfi et al. have provided a comprehensive review of the current research on the interplay between microbiota and endometriosis, and suggested potential therapeutic interventions, including antibiotics, probiotics, and prebiotics, as well as novel approaches such as fecal, vaginal, or uterine microbial transplantation, to restore a dysbiotic state to a more favorable genital microenvironment (Zizolfi et al., 2023).

In this study, we explored the potential relationship between the microbiota and endometriosis and emphasizing this perspective as a focal point in future research to uncover underlying mechanisms, holds promise for paving the way towards the development of new approaches in preventing and treating endometriosis. This would bring hope to patients who bear the burden of endometriosis.

Mendelian randomization (MR), a novel statistical approach utilizing genetic variants as instrumental variables, provides a robust method to explore the causal link between exposure and outcome. This study employed Mendelian randomization analysis to explore the connection between endometriosis and gut microbiota. The analysis considered nine phyla, 16 classes, 20 orders, 32 families, and 119 genera (excluding 3 unknown families and 12 unknown genera). Endometriosis GWAS summary statistics from the FinnGen consortium R8 release data of European ancestry and gut microbiota GWAS summary statistics from the MiBioGen collaboration, predominantly of European ancestry, were collected for this purpose.

Studies indicated that changes in the composition of gut microbiota may affect the development and progression of endometriosis. Comprehending these links could assist in identifying potential biomarkers for swift diagnosis and in developing personalized treatment approaches for patients with endometriosis. Furthermore, understanding the associations between gut microbiota and endometriosis might lead to the development of innovative treatment methods and improve clinical outcomes for individuals affected by this condition.

Recently, Muraoka et al. found that Fusobacterium infection activates the transforming growth factor-β (TNF-β) signal, promoting the progression of endometriosis. Antibiotic treatment effectively prevented the disease’s progression in a mouse model (Muraoka et al., 2023). Lipopolysaccharide (LPS) derived from gut microbiota can activate Toll-like receptor 4 (TLR4) and induce an inflammatory response, promoting the growth of endometriotic lesions. The production of TNF-α and IL-8, triggered by LPS activating TLR4, is crucial for endometrial tissue adhesion and angiogenesis (Nothnick, 2001). Meanwhile, Iba et al. found that treating endometrial stromal cells (ESCs) obtained from ovarian endometriosis with an NF-kB inhibitor resulted in reduced production of TNF-α and IL-8 and decreased proliferation (Iba et al., 2004). Khan et al. compared peritoneal fluid and menstrual fluid samples in endometriosis patients and controls, showing higher levels of LPS in the menstrual fluid of individuals with endometriosis (Khan et al., 2010). The presence of LPS may contribute to TLR4-mediated growth of endometriosis.

The gut microbiota can impact the structure and function of the intestinal epithelium and has been linked to various diseases, including hypertension, Parkinson’s disease, and autoimmune diseases (Sampson et al., 2016; Li et al., 2017; Janney et al., 2020; Mu et al., 2020; Christovich and Luo, 2022). The precise mechanism by which gut microbiota affects endometriosis remains uncertain. In this study, gut microbiota had causal effects on endometriosis, specifically Holdemania and Ruminococcaceae UCG002, which are associated with different stages of endometriosis.

Certain bacterial families, including Burkholderiales, Oxalobacteraceae, Porphyromonadaceae, and Desulfovibrio, may play a role in the development of endometriosis through the LPS-TLR4 pathway. They can produce LPS which is an essential component of the outer membrane. LPS activates immune cells or immune cell receptors, causing systemic inflammation responses and contributing to the development of endometriosis (Iba et al., 2004; Clemente et al., 2018; Gao et al., 2023).

LPS can activate the PD-L1 pathway, promote overexpression of PD-1 and PD-L1, and suppress T cell activation and proliferation. On the other hand, the Clostridialesvadin BB60 group lead to decreased PD-L1 levels, which can, in turn, suppress the growth of ectopic endometrial tissue in the pelvic cavity (Huang et al., 2023). As a result, the Clostridialesvadin BB60 group may reduce the incidence of endometriosis or alleviate its symptoms. The ectopic endometrial tissue can lead to inflammation and immune dysregulation, which can in turn affect the microbiome composition.

Xue Q et al. found that the abundance of Haemophilus increased significantly in patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) (Xue et al., 2023). AECOPD is characterized by Haemophilus enrichment and a high level of TNF-α. Haemophilus is a respiratory pathogen specific to neutrophils (Sethi and Murphy, 2001). Neutrophils both promote tumor growth and inhibit tumor progression (Gungabeesoon et al., 2023). It exhibits anti-tumorigenic characteristics in the state of acute inflammation (Hirschhorn et al., 2023). Surprisingly, in this study, Haemophilus showed a protective effect in endometriosis. We hypothesize that endometriosis leads to inflammation in the body, and Haemophilus, through neutrophils, restricts the growth of ectopic endometrial tissue.

Holdemania and Ruminococcaceae UCG002 are involved in gut butyrate production (Sampson et al., 2016). Butyrate plays a vital role in maintaining intestinal homeostasis and anti-inflammation. It can regulate the interaction between dendritic cells and DC-T cells and promote Treg T cell differentiation, thereby maintaining immune balance. This is achieved by HDACi suppressing the expression of NF-κB and inducing anti-inflammatory gene transcription to activate dendritic cells (Park et al., 2015).

We further investigated the causal link between the genus of gut microbiota and the different stages of endometriosis, revealing that Holdemania acted as a protective factor against stage 3–4 endometriosis. Conversely, higher levels of Ruminococcaceae UCG002 increased the risk of stage 1–2 endometriosis. These findings were consistent with previous analyses and suggested that Holdemania and Ruminococcaceae UCG002 may play essential roles in developing endometriosis. As mentioned, Holdemania and Ruminococcaceae UCG002 participated in gut butyrate production (Gomez-Arango et al., 2018). These findings further indicated that gut microbiota worked in the occurrence and development of endometriosis. Notably, Ruminococcaceae UCG002 is positively correlated with Treg cells (Zhi et al., 2022), and its abundance is closely related to the levels of pro-inflammatory cytokines. Exploring the detailed mechanisms by which Holdemania and Ruminococcaceae UCG002 influence the progression of endometriosis should be focus on future research. The results are different between endometriosis states 1 and 2 and stages 3 and 4. We thought the lifestyle and treatment methods of patients with different stages of endometriosis may lead to changes in the composition of intestinal flora. Recent research has shown that lifestyle, diet, and other factors can affect gut microbiota composition. A comparison of the Hadza hunter and population in Nepal and California has revealed the impact of these factors (Carter et al., 2023).

In conclusion, these risk factors in gut microbiota mainly affect endometriosis through the LPS-TRL4 pathway. They induce local or systemic inflammation via releasing LPS, the concentrations of inflammatory mediators, cytokines, and chemokines are increased, and TRL4 is activated. This process promotes ectopic endometrial tissue angiogenesis and, thus, colonization. As for protective factors, they can reduce PD-1L levels, prevent angiogenesis, and inhibit the growth of ectopic endometrial tissue. Our study offers a new approach to treating endometriosis, reducing financial burden and relieving pain compared to conventional treatments. This benefits both patients and advances understanding of the condition.

There are several strengths in this study. Firstly, our study examines the relationship between gut microbiota and endometriosis at the genetic level, which reduces confounding bias and reverse causality. This study provides a new perspective for exploring the pathogenesis of endometriosis. Secondly, we have ensured two-sample Mendelian randomization (MR) analyses by selecting separate samples for exposure and outcome data. Thirdly, we have conducted multiple supplementary analyses, including heterogeneity, pleiotropy, leave-one-out sensitivity analyses, and MR-PRESSO, to ensure the robustness of our results.

Of course, several limitations also need to be acknowledged. Firstly, there are no data available at the species level. We cannot explicitly identify which bacteria affect endometriosis. Secondly, the summary statistic of gut microbiota includes multiple ancestries, which may introduce interference from population stratification in our results. Thirdly, the GWAS summary statistic for gut microbiota was not restricted to female participants. Although the exposure data have excluded the sex chromosomes, potential bias due to sex cannot be avoided entirely. In this study, some results were significant for only the IVW analysis. According to the supplementary analysis, there is horizontal pleiotropy or (and) heterogeneity. However, further MR-PRESSO analysis did not find any significant outliers. Therefore, it is necessary to verify our results through further clinical and basic research.

This two-sample MR analysis revealed a causal association between certain gut microbiotas and endometriosis. These microbiotas primarily influence endometriosis through the LPS-TRL4 pathway and inflammatory factors. Moving forward, it is essential to consider the design of randomized controlled trials (RCTs) and fundamental studies to elucidate further the risk and protective factors associated with endometriosis and its underlying mechanisms.