The human lower reproductive tract harbors a diverse microbiota, which can ascend to the upper reproductive tract through direct migration and potentially cause infections (Cheng et al., 2021). Khan et al. proposed that some Gram-negative bacteria, such as E. coli, infect the uterine wall after migrating up from the vagina to contaminate menstrual blood. Their study found that women with endometriosis had higher levels of E. coli in their menstrual blood than the control group (Khan et al., 2010). Combined with the theory of menstrual reflux, menstrual blood may flow from the fallopian tubes into the pelvic cavity (Halme et al., 1984). Bacterial lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria such as E. coli. LPS is constantly shed by living bacteria and is released when the bacteria die (Raetz and Whitfield, 2002). Khan et al. considered that E. coli contamination in her menstrual blood was a continuous source of LPS in the peritoneal cavity. Elevated LPS levels in peritoneal fluid may promote Toll-like receptor 4 (TLR4)-mediated progression of endometriosis (Khan et al., 2010). Another possible explanation for the residual accumulation of bacterial endotoxins in the pelvic environment is the transfer of E. coli or endotoxins from the intestine through intestinal cells and into the pelvic cavity (Deitch et al., 1989; Alexander et al., 1990). Dysbiosis of the intestinal microbiota can lead to an increase in Gram-negative bacteria, causing a large amount of LPS to enter the circulatory system inducing chronic inflammation throughout the body (He et al., 2019). Studies have shown that patients with endometriosis have elevated intestinal and serum LPS levels (Ni et al., 2020). Primary inflammation caused by LPS promotes the secretion of the NF-κB mediator in the peritoneal cavity through TLR4 present on macrophages, neutrophils, and other immune cells, followed by a cascade of reactions such as the production of significant levels of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8), thus promoting the development of endometriosis (Khan et al., 2018; Jeljeli et al., 2020). Although this theory provides a new strategy for treating EMS by targeting bacterial endotoxin or TLR4 to suppress inflammation, it is currently not feasible for clinical application.

Another possible mechanism by which the gut microbiota is involved in the development of endometriosis is through the regulation of estrogen (Baker et al., 2017). Estrogen plays an important role in maintaining the development of the female reproductive system and is closely associated with the development of endometriosis (Jeon et al., 2016; Galvankar et al., 2017). Studies have found that estrogen can induce proliferative diseases such as endometriosis, endometrial cancer, and hysteromyoma by stimulating the proliferation of female genital epithelial cells (Xu et al., 2019; Somasundaram et al., 2020). Endometriosis has been associated with alterations in estrogen signaling (Laux-Biehlmann et al., 2015). Endometriotic women have a heightened proinflammatory and anti-apoptotic response to estradiol (Reis et al., 2013). This attributed to changes in nuclear estrogen receptor expression. Endometriotic lesions express higher levels of estrogen receptor-β (ER-β), whose signaling promotes lesion growth by inhibiting TNF-α-induced apoptosis, activating an inflammasome which increases IL-1β, and enhancing cellular adhesion and proliferation (Han et al., 2015). The gut microbiota plays an important role in estrogen metabolism, and the gene pool in the gut microbiota capable of metabolizing estrogen is called the “estrogen group” (Salliss et al., 2021). It has been observed that the use of antibiotics leads to a decrease in estrogen levels (Chadchan et al., 2019). Estrogen metabolism takes place mainly in the liver. Estrogens are inactivated in the liver into less active estrone and estriol and bind to glucuronic acid to promote their excretion in bile, urine, and feces (Adlercreutz, 1974). However, beta-glucuronidase secreted by gut microbes can metabolize estrogen from the bound form to the unbound form (Ervin et al., 2019; Sui et al., 2021). Free estrogens can be absorbed into the circulatory system, resulting in increased levels of free estrogens, and can be transported through the blood to distal mucosal sites, such as the endometrium. Estrogen binds to estrogen receptors, which are subsequently internalized and function as transcription factors promoting the transcription of genes involved in cellular growth (Dabek et al., 2008; Baker et al., 2017; Salliss et al., 2021). Studies have shown that multiple genera in the gut microbiota, including Bifidobacteria, Bacteroidetes, Escherichia coli and Lactobacillus, can encode β-glucuronidase (Leonardi et al., 2020). When the intestinal flora is dysregulated, the flora producing β-glucuronidase in the gut increases, leading to elevated circulating estrogen levels and promoting the proliferation of ectopic endometrial lesions (Baker et al., 2017). Some studies have found that the levels of Bifidobacteria and Escherichia in the endometriosis group are higher than those in the control group (Yuan et al., 2018). In addition, elevated levels of β-glucuronidase expression were observed in endometriosis lesions compared to normal endometrium (Wei et al., 2023). Metabolites produced by the gut microbiota also affect estrogen metabolism and affect endometriosis. For example, short-chain fatty acids produced by some bacteria can affect estrogen metabolism by regulating the expression of estrogen-metabolizing enzymes in the liver and other tissues, affecting estrogen levels and hormone balance in endometriosis (Ervin et al., 2019). In summary, the gut microbiota can lead to increased circulating estrogen levels, which can promote the development of endometriosis (Qi et al., 2021). However, whether specific gut microbiota constituents stimulate β-glucuronidase production and whether the gut microbiota has synergistic effects on the pathogenesis of EMS remain to be studied further.

Immunity and inflammation play an integral role in the pathogenesis of endometriosis. The results of the sustained growth of endometrial lesions in ovariectomized animals suggest that the innate immune system in the pelvic environment can also influence the growth of endometriosis lesions in addition to the influence of sex hormones (Novella-Maestre et al., 2012). One study demonstrated that the number of macrophages, monocytes, and cytokines in the peritoneal fluid of women with endometriosis was increased, and the levels of the pro-inflammatory factors IL-1β, IL-18 and TGF-β are increased (Zhou et al., 2019; Agostinis et al., 2020). Increased numbers of macrophages help to secrete proinflammatory cytokines, such as transforming growth factor-β1 (TGF-β1), IL-8, IL-6, TNF-α, and IL-1β (Jeljeli et al., 2020). These cytokines are expressed mainly by macrophages in the peritoneal cavity through increasing the activation of the NF-κB pathway. A local cytokine surge can further promote the recruitment of immune cells and the secretion of additional cytokines, and stimulate the production of vascular endothelial growth factor (VEGF), which is conducive to angiogenesis (Becker and D'Amato, 2007; Symons et al., 2018). VEGF is a major regulator of vascularization and can induce endometrial cell proliferation (Young et al., 2015). In addition, when the inflammatory activity in the peritoneal fluid decreases, the level of the anti-inflammatory factor IL-37 increases. When inflammatory substances cannot be removed, an inflammatory response may occur, which spreads throughout the body, with long-term effects on the immune system (Symons et al., 2018; Zhou et al., 2019).

At present, many studies have confirmed the close relationship between gut microbiota and inflammation (Buford, 2017; Shi et al., 2017; Clemente et al., 2018; Brandsma et al., 2019). Jiang et al. hypothesized that changes in the gut and genital tract microbiota would lead to changes in the immune response. Gut microbiota dysbiosis destroys normal immune function and leads to the elevation of proinflammatory cytokines, which impairs immune surveillance in the pelvic region and changes the immune cell spectrum, creating an inflammatory microenvironment to promote the persistence of endometriosis (Jiang et al., 2021). Yuan et al. showed that the gut microbiota may interact with cytokines abnormally expressed in endometriotic lesions, peritoneal fluid and peripheral blood (Yuan et al., 2018). The effect of gut microbiota on host inflammatory response is mediated by a variety of mechanisms. Dysregulation of the gut microbiome can digest the protective mucus layer of the gut and interact directly with intestinal cells, leading to increased local and systemic inflammation (Blander et al., 2017). For example, segmented filamentous bacteria can directly stimulate T lymphocytes to differentiate into helper T lymphocytes (Th17) to drive autoimmune arthritis (Wu et al., 2010). Many studies have shown that Th17 cells and their cytokines are significantly increased in the peritoneal fluid of women with endometriosis (Gogacz et al., 2016; Tarokh et al., 2019). As previously mentioned, intestinal microbiota imbalance can also lead to LPS leakage by increasing intestinal permeability, which activates the innate immune system through TLR4 and increases the expression of pro-inflammatory cytokines, thus promoting the occurrence and development of endometriosis (Khan et al., 2010; Bruner-Tran et al., 2013). In addition, short-chain fatty acids act as a medium of communication between the gut microbiome and the immune system, serving as an energy source for intestinal cells or being transported into the blood, not only acting in the gut, but also regulating immune responses in distant tissues (Parada Venegas et al., 2019; Chadchan et al., 2021). These are described in detail later in Chapter 6. However, the molecular mechanisms that explain the microbiome’s interaction with immune inflammation are not fully understood, and more research is needed to explore the complex role of the gut microbiome in the multi-factorial etiology of EMS, including impaired cytokine and immune function.

The literature clearly highlights that gut microbes play a key role in the metabolism of short-chain fatty acids (Mirzaei et al., 2022). In the gut, SCFAs are the main beneficial metabolites produced by gut microbes through the metabolism of indigestible dietary fiber. The most abundant SCFA is acetate, which is produced mainly by Bacteroides, one of the largest flora in the gut. Acetate can be produced from pyruvate via the acetyl-CoA or Wood-Ljungdahl pathways (Ragsdale and Pierce, 2008). Another major SCFA, propionate, is produced by a few dominant bacterial genera, such as Ruminococcus bromii, Veillonella parvula and Bacteroides (van der Hee and Wells, 2021). Propionate is mainly produced from succinate via the succinate pathway or from lactate via the acrylate pathway (Koh et al., 2016). The third major SCFA, butyrate, is mostly produced by Firmicutes, such as Faecalibacterium, Eubacterium rectale and Eubacterium hallii (Vital et al., 2014). Butyrate is formed from acetyl-CoA and butyryl-CoA (Koh et al., 2016). There are metabolic linkages between different types of bacteria. For example, acetate produced by Bacteroides can be utilized by Firmicutes to produce butyrate. The diversity and quantity of host gut microbes are the main factors affecting the production of SCFAs. One randomized trial revealed that resistant starch was able to regulate the gut microbiome, such as increased Bifidobacteria, altered Firmicutes to bacteria ratio, and observed an increase in the relative abundance of butyrate (Alfa et al., 2018). In addition, a new study examined whether Bifidobacteria can synthesize SCFAs in vivo by LC−MS/MS and revealed that Bifidobacteria mice and mice without a specific pathogen exhibit high concentrations of acetic acid and propionate. Bifidobacteria colonization and changes in the gut microbiota can affect the abundance of intestinal short-chain fatty acids (Horvath et al., 2022). The above studies showed that diet and the gut microbiota are the influencing factors of SCFAs metabolism.

While gut microbes regulate the metabolism of short-chain fatty acids, SCFAs produced in the gut can inhibit the growth of harmful intestinal bacteria, which play an important role in maintaining the stability of the gut microbiota. Studies have shown that SCFAs can remodel antibiotic-induced disordered gut microbiota by increasing the number of beneficial bacteria and reducing the number of harmful bacteria. After the administration of SCFAs, the relative abundance of Firmicutes and Bacteroides in septic mice increased significantly, and the number of Enterobacteriaceae, such as Proteobacteria and Escherichia Shigella, was significantly reduced and returned to levels comparable to those in healthy mice (Lou et al., 2022). Oral administration of butyrate to mice can significantly enhance the antibacterial activity of colonic macrophages, for example, by conferring better killing effects against Salmonella enterica and Citrobacterrodentium (Flemming, 2019). SCFAs inhibit the growth of harmful bacteria mainly by releasing H⁺, reducing the intestinal pH, competing for energy, blocking the biosynthesis of harmful bacteria, and forming antibacterial peptides to achieve intestinal microecological balance ( Table 2 ). In the intestine, some SCFAs are protonated by the carboxyl and phosphate groups of lipopolysaccharides on the bacterial outer membrane, destroying the integrity of the bacterial outer membrane; in addition, another part of SCFAs dissociates in bacterial cells to produce large amounts of RCOO^- and H⁺. H⁺ disrupts intracellular pH homeostasis; RCOO^- increases intracellular osmotic pressure, forcing these cells to excrete precursors or accessory substances necessary for their growth to balance osmotic pressure, thus inhibiting the growth of harmful bacteria (Ma et al., 2022). Moreover, the continuous accumulation of RCOO^- changes the homeostatic environment in bacterial cells, thereby interfering with a series of physiological and biochemical characteristics inside bacteria and thereby blocking the biosynthesis of harmful bacteria. In addition, SCFAs trigger host cells to produce certain antibacterial peptides by activating and regulating the host immune system, which in turn achieves bacterial inhibition by lysing the bacterial cell membrane, resulting in leakage of the contents and death of the bacteria (Raqib et al., 2006; Jakubowski, 2019).

SCFAs can exert anti-inflammatory effects on many cell types through the activation of G protein-coupled receptors, thereby inhibiting the development of EMS. Among them, GPR43 (also known as FFAR2), GPR41 (also known as FFAR3) and GPR109A are the most important receptors for SCFAs in the GPCR family. FFAR2 is widely expressed in neutrophils, monocytes, intestinal Treg cells, eosinophils and other immune cells (Smith et al., 2013). The FFAR2 receptor reduces NF-κB levels through the β-arrestin-2 signaling pathway and reduces the number of NF-κB subunits, p65 and p50, in the nucleus, thereby inhibiting the transcription of proinflammatory cytokines (IL-1β and IL-6) (Lee et al., 2013). SCFAs can inhibit the expression of IL-6, IL-1β and TNF-α through FFAR2, thereby exerting their anti-inflammatory effects (Nakajima et al., 2017; Pirozzi et al., 2018; Mizuta et al., 2019). Consistent with its function in FFAR2, FFAR3 also plays important roles in inflammation. C3 can inhibit the expression of IL-4, IL-5 and IL-17A through FFAR3, and C4 can inhibit the expression of nitric oxide synthase (iNOS), TNF-α, IL-6 and monocyte chemoattractant protein-1 (MCP-1) through FFAR3 (Li et al., 2018). In contrast to FFAR2 and FFAR3, GPR109A is activated by longer SCFAs, mainly C4 (Offermanns, 2017). Li et al. reported that GPR109A exerts an anti-inflammatory effect by inhibiting the Akt/mTOR signaling pathway in MIN6 pancreatic β cells (Priyadarshini et al., 2018).

As a protease, histone deacetylases (HDACs) play important roles in the regulation of gene expression and the modification of chromosome structure. SCFAs are considered to be natural inhibitors of HDACs. Among all short-chain fatty acids, butyrate is the most effective inhibitor of HDAC activity. SCFAs may directly act on HDACs to enter cells through transporters or indirectly act on HDACs through GPCR activation. For example, C4 inhibits LPS-induced production of nitric oxide and inflammatory cytokines (such as IL-6 and IL-12) but directly inhibits HDACs and is unrelated to GPCRs (Chang et al., 2014). The authors of this study believe that C4 acts directly on HDACs. This is because SCFAs can be coupled to monocarboxylate transporter 1 (SMCT-1) through sodium transporter proteins rather than through membrane receptors, thereby entering cells, occupying the active sites of HDACs and inhibiting HDACs (Sun et al., 2017). In another report, the authors found that activation of FFAR3 could repress histone acetylation in a Chinese hamster ovary cell line through the inhibition of HDACs (Wu et al., 2012). In addition, FFAR3, FFAR2 and GPR109 may all be involved in the inhibition of HDACs by SCFAs. However, how SCFAs inhibit HDAC activity through GPCRs or directly has not been determined, and additional studies are needed to determine the underlying mechanisms involved.

SCFAs produced by the gut microbiota are transported throughout the body and can maintain systemic homeostasis by inhibiting inflammatory immune cells and promoting the differentiation of immune cells. We describe in detail how SCFAs regulate various immune cells, including macrophages (Macs), neutrophils, Tregs, and dendritic cells (DCs), thereby suppressing EMS-related inflammation.

Probiotics and prebiotics are two components of new therapies based on the gut microbiota. Probiotic intervention is an effective method for treating EMS. The potential effects of probiotics on immune regulation, inflammation and intestinal flora imbalance are all associated with the development of EMS (Bodke and Jogdand, 2022; Hashem and Gonzalez-Bulnes, 2022). Oral lactic acid bacteria have been shown to reduce pain associated with endometriosis (Khodaverdi et al., 2019). It also reduces endometriotic lesions in mice by enhancing NK cell activity and increasing the IL-12 concentration (Itoh et al., 2011). Dysbiota and endometriosis-related inflammation lead to impaired NK cell activity, and probiotic treatment reversed this immune dysregulation. In a rat model, probiotic treatment was also found to inhibit the growth of endometriosis (Uchida and Kobayashi, 2013). In addition, as mentioned previously, SCFAs can inhibit endometriosis. In vitro and in vivo studies have confirmed the positive effect of probiotics on increasing short-chain fatty acids (Markowiak-Kopeć and Śliżewska, 2020). The use of different types of probiotics has been found to increase SCFAs levels in patients with different diseases (such as colorectal cancer and respiratory diseases) (Hou et al., 2022; Williams et al., 2022). It was speculated that probiotics may also inhibit EMS by increasing the production of SCFAs.

To increase the effectiveness of probiotics, they can be combined with prebiotics. Although studies specifically focusing on the effects of prebiotics on endometriosis are limited, prebiotics can promote the growth of beneficial bacteria, reduce the translocation of harmful bacteria and their products into the blood, and help maintain the ecological balance of the intestinal microbiota. Subsequently, it may affect systemic inflammation and the immune response, thereby affecting EMS-associated symptoms (You et al., 2022). In addition, prebiotics are widely used to increase SCFAs levels (Gibson et al., 2017). One study showed that the use of high doses of arabinoxylan oligosaccharides could increase butyrate, acetate, propionate, and total SCFAs (François et al., 2012). Therefore, prebiotics may have potential benefits for treating endometriosis. Although probiotics and prebiotics have shown good potential in treating EMS, there is still a lack of guidelines outlining or supporting the standard use of probiotics and prebiotics in EMS management, and additional studies are needed to evaluate the effects of probiotics and prebiotics on EMS in the future.

FMT involves the transfer of feces from healthy people to the gastrointestinal tract of patients with imbalanced flora to reconstruct the gut microbiota and treat related diseases (Nigam et al., 2022). Currently, FMT has been applied to treat many diseases, such as gastrointestinal diseases, neuropsychiatric diseases, and blood diseases (Kim and Gluck, 2019). In addition, FMT has become a mature therapy for recurrent Clostridium difficile infection (Cammarota et al., 2015). Recently, studies have shown that FMT can be used to treat female reproductive tract diseases (Quaranta et al., 2019; Huang et al., 2021a). A study by Chadchan et al. revealed that after endometriosis mice were transplanted with feces from healthy mice, the number of uterine lesions decreased (Chadchan et al., 2021, 2023). Ni et al. recently reported similar findings that ZO-2 expression was reduced after the administration of feces from endometriotic mice to healthy mice (Ni et al., 2021). These preliminary studies highlighted the potential of FMT to relieve EMS through the reinforcement of barrier integrity. In addition, several pieces of evidence suggest that an increase in SCFAs may be a key determinant in the treatment of different diseases by FMT (Chen et al., 2019; El-Salhy et al., 2021; Xu et al., 2023). Taken together, these findings indicate that FMT might be a potential treatment for EMS. However, this strategy is limited by the presence of unknown microorganisms and pathogenic bacteria in the transplanted feces.

One study revealed that SCFAs play an important role in maintaining the intestinal barrier and suppressing immune function (Rekha et al., 2022). Among them, butyric acid played a particularly significant role. Butyrate can also suppress the immune response caused by intestinal microbial imbalance (Ma et al., 2020). A recent study showed that gut microbiota-derived butyrate could inhibit endometriosis in mice through the regulation of G protein-coupled receptors (Chadchan et al., 2021). In addition, another type of microbiota metabolite that has therapeutic effects on EMS is secondary bile acids. A study on the correlation between fecal metabolomics and the gut microbiota in endometriosis mice revealed that bile acid and chenodeoxycholic acid (CDCA) levels were increased in the intestines of endometriosis mice (Ni et al., 2020). The above studies showed that changes in the metabolite levels of the gut microbiota may play an important role in the development of EMS. Although several metabolites of the gut microbiota are maypotentially useful for treating EMS, the functional diversity of these metabolites needs to be overcome to design effective treatment strategies.

Dietary intervention may lead to different changes in the gut microbiota and SCFAs levels. Diet provides substrates that enable the manipulation of microbial composition. For example, the Mediterranean diet can increase the availability of substrates available for specific bacteria, thereby increasing the formation of SCFAs (Wagenaar et al., 2021; Yalçın Bahat et al., 2022). A fiber-rich diet can increase the levels of SCFAs, especially acetate and butyrate (So et al., 2018). Rinninella et al. evaluated the use of a ketogenic diet and reported that the total number and abundance of several beneficial bacteria and bacteria, were reduced. A ketogenic diet can significantly affect the characteristics of the gut microbiota and may lead to a reduction in short-chain fatty acids (Rew et al., 2022; Rinninella et al., 2023). A plant-based diet has been shown to lead to the dominance of SCFAs-producing Firmicutes, while an animal-based diet increases the number of Bacteroides (Tomova et al., 2019). Recently, a large number of studies have shown that diet is a potential risk factor for endometriosis (Jurkiewicz-Przondziono et al., 2017). Women who consume large amounts of fruits, vegetables, dairy products, or omega-3 fatty acids (PUFAs) have a reduced risk of developing endometriosis (Samaneh et al., 2019). A high intake of omega-3 polyunsaturated fatty acids can reduce the risk of endometriosis in women, which may be attributed to potential changes in the gut microbiota (Watson et al., 2018). A diet rich in ω-3 polyunsaturated fatty acids can reduce inflammation in a mouse model of endometriosis, as indicated by reduced TNF-α and IL-6 inflammatory marker levels (Pereira et al., 2019). In addition, some studies have shown that linolenic acid (ALA) can reduce inflammation by inhibiting the accumulation of prostaglandin E2 (PGE2) and nitrite. ALA can also reduce LPS levels and improve the abdominal inflammatory environment in endometriosis mice (Ni et al., 2021). Moreover, it can inhibit the inflammatory response of M1 macrophages (Pauls et al., 2018). However, the consumption of products and red meat rich in trans-unsaturated fatty acids can increase the risk of endometriosis (Marcinkowska and Górnicka, 2023). In conclusion, reasonable diet adjustment may help prevent EMS.

In summary, the gut microbiota and SCFAs can be modulated by probiotics and prebiotics, fecal microbiota transplantation, gut microbiota metabolites, or dietary regimens. These methods can greatly increase the SCFAs produced in the colon, inhibiting EMS. However, the clinical efficacy of these methods needs further study.