Emerging signal transduction pathways and the role of bacterial extracellular vesicles linked to the pathogenesis of endometriosis

Approximately 10% of reproductive-aged women are affected by endometriosis, which is defined by the presence of endometrial glands and the growth of stromal cells outside the endometrial cavity, most commonly in the peritoneum or ovaries 1 – 3 . Retrograde menstruation is generally accepted to cause endometriosis 4 . The latter involves the flow of menstrual blood back into the abdominal cavity, which may result in endometrial cells exiting the uterus through the fallopian tubes. This theory is supported by an association between short menstrual cycles, which induce retrograde menstruation more frequently, and the risk of developing endometriosis. However, retrograde menstruation is observed in almost all reproductive-aged women, whereas the prevalence of endometriosis reaches only 10%. This suggests that the establishment of endometriosis lesions requires additional factors, such as endometriosis-specific signaling in combination with retrograde menstruation. Normal signaling in eutopic endometrial cells differs significantly from disease-related signaling in ectopic endometriosis lesions. Although endometriosis is a benign disease, it is accompanied by abnormal cell proliferation and chronic inflammatory signals, as occurs in malignant diseases 5 . Endometriotic lesions comprise both epithelial and stromal cells, each of which contributes to disease pathogenesis through distinct signaling pathways. Signals related to the epithelial-mesenchymal transition (EMT) are present in epithelial cells, and signals related to cell fibrosis are present in stromal cells 6 , 7 . Cells of the immune system are also associated with the development of endometriosis 8 . In particular, macrophages have been linked to sustained peritoneal inflammation, which is a key pathological manifestation of endometriosis 9 . Recently, the association between microbiota and endometriosis has received considerable attention 10 – 12 . We recently reported that Fusobacterium is involved in the pathogenesis of endometriosis 12 , and future work aims to clarify the communication between bacteria and host cells. Endometriosis is often treated with surgery or hormones. However, each of these methods has limitations. Ovarian cystectomy, the surgical method in the treatment of ovarian endometriosis, is a surgical excision approach to remove the lesion. However, the surgical technique may impair ovarian function, and the high postoperative recurrence rate is problematic 13 – 15 . Because hormonal treatment aims to eliminate menstruation and reflux of menstrual blood, it cannot be used in patients who wish to become pregnant, and the side effects of hormonal treatment are often serious 4 . There is therefore interest in the development of new strategies to treat endometriosis that take into consideration mechanisms of disease pathophysiology. Here, we review the cell-to-cell signaling pathways that have been implicated in endometriosis pathophysiology, as well as the novel concept of the role of endometriosis-associated bacterial signaling focused on BEVs. Rethinking endometriosis signaling is expected to contribute significantly to the development of novel concepts of treatment and a more precise understanding of this disease. Here, we review the relationship between endometriosis and signal transduction, including classical hormonal, inflammatory, and angiogenic signaling pathways, in addition to emerging microbial and epigenetic pathways. We further discuss previous studies and future challenges from the perspective of molecular signal transduction tools, such as extracellular vesicles. Improved understanding of signal transduction in endometriosis will be valuable to develop novel therapies for endometriosis. Estrogen is the main hormonal signaling pathway in the endometrium and in endometriotic lesions. Estrogen affects the proliferation of endometrial cells by binding estrogen receptors (ERs) and G protein-coupled receptor 30 (GRP30) via the activation of phosphoinositide 3-kinases (PI3K) and mitogen-activated protein kinase (MAPK) through transactivation of the epidermal growth factor receptor (EGFR) 16 . Abnormal estrogen synthase expression has been reported in endometriosis 3 . Locally, the expression of aromatase (a key enzyme in the biosynthesis of estrogens) and steroidogenic acute regulatory proteins are increased to produce estrogen, which causes localized elevations of estrogen in endometriotic lesions 3 . In addition to increasing local estrogen, increased ER expression plays a role by enhancing estrogen signaling. There are two subtypes of ER, ERα and Erβ. Increased expression of ERβ promotes the growth of endometriosis lesions by enhancing the interleukin-1β (IL-1β) signal, which leads to both greater cellular adhesion to the peritoneum and enhanced endometrial cell proliferation 17 . Estrogen/ERβ signaling induces the Nod-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome, which is involved in the maturation of IL-1β formation from pro-IL-1β 18 . We revealed the therapeutic potential of an NLRP3 inhibitor as a non-hormonal therapy in a mouse model of endometriosis 19 , and this signaling cascade is important in mast cells from patients with endometriosis 20 . The IL-1β signaling pathway, with estrogen/ERβ as its main axis, is thought to play a key role in endometriosis formation 21 . Immune cell responsiveness to estradiol (E2) is another aspect of the pathophysiology of endometriosis. In a mouse model of endometriosis, E2 induced the infiltration of macrophages into endometriotic lesions and promoted an inflammatory response, which is the key inducer of endometriosis pathophysiology 22 . The synergistic effects of estrogen and inflammatory signaling are thought to influence endometriosis pathogenesis. Endometriosis is an estrogen-dependent disorder, and estrogen-suppressive medicine is a reasonable treatment option for women of reproductive age. However, the long-term use of ovarian function suppressors, such as combined oral contraceptives and gonadotropin-releasing hormone agonizts (GnRHa) or antagonists (GnRHant), may cause side effects. Therefore, the benefit of non-hormonal treatments that are more focused downstream in the signaling pathway should be further explored. Progesterone signaling inhibits the action of estrogen by repressing cell proliferation and initiating decidualization, which is a process that results in significant changes to cells of the endometrium before and during pregnancy. In the endometrium, progesterone binds to progesterone receptors (PRA and PRB), and Indian hedgehog (IHH)/chicken ovalbumen upstream promoter-transcription factor II (COUPTF II)/bone morphogenetic protein 2 (BMP2) signaling is activated to induce decidualization 23 . Another PR target is homeobox protein-A10 (HOXA10), which regulates the responsiveness to progesterone 24 . In endometriotic lesions, the expression of PRs is suppressed by the hypermethylation of their promoter regions 25 , inducing a progesterone-resistant state 26 . Moreover, ERβ, which is overexpressed in endometriosis, suppressed the expression of PR 27 . Endometriotic lesions are therefore thought to progress due to the inability of progesterone growth-inhibiting signaling to antagonize estrogen growth signaling. Dienogest has been widely used to target this hormonal signaling abnormality and functions via endometrial regression and ovarian suppression to inhibit the growth of endometriosis 4 , 26 . However, this treatment is also a hormonal therapy and cannot be used for women who wish to conceive; therefore, other signal-targeted treatment strategies are needed. Endometriosis is also considered an inflammatory disorder in which immune dysregulation induces endometriotic lesion development and proliferation. IL-6 is one of the main signaling molecules involved in endometriosis 28 . Macrophages are a source of IL-6 production, and peripheral fluid from endometriosis patients contains increased numbers of macrophages and high concentrations of inflammatory cytokines, such as IL-6 and IL-1β 29 . Inflammatory signals induce chronic inflammation and tissue fibrosis, similar to a “non-healing wound process”. Anti-IL-6 receptor monoclonal antibody (tocilizumab) was tested in a rat endometriosis model and significantly suppressed the volume of endometriotic lesions compared to non-treated rats 30 . This signal has also been identified in several autoimmune diseases, and therapies that block this signal may provide novel strategies for the treatment of endometriosis. Our group also focused on IL-6, which can be repressed by dienogest treatment before and after surgery for endometriosis 31 . Patients with endometriosis are constantly exposed to inflammation, and a postsurgical inflammatory response could damage preserved ovarian function. Blocking inflammatory cytokines, such as IL-6, may have a positive effect on the ovarian reserve as well as on the endometriotic lesions themselves. Prostaglandin E2 (PGE2) is another key inflammatory signaling mediator linked to angiogenesis, estrogen biosynthesis, and pain symptoms. Increased cyclooxygenase-2 (COX-2) has been widely reported in endometriotic lesions, inducing pain 32 . COX-2/PGE2 signaling is closely associated with endometriosis and induces various inflammatory processes 33 . Furthermore, COX-2/PGE2 signaling is associated with the direct proliferation of endometrial cells and indirect effects on endometriosis, such as angiogenesis and immunosuppression 34 . COX-2/PGE2 signaling is also upregulated by estrogen, which forms a positive feedback loop that affects COX-2/PGE2 production 35 . COX-2 inhibitors are nonsteroidal anti-inflammatory drugs (NSAIDs) and are effective in managing endometriosis-related pain. Endometriotic lesions are complex and are affected by several types of surrounding cells. Angiogenesis is essential to establish endometriotic lesions and to supply blood to the lesion microenvironment to allow cells to survive and grow 36 . Vascular endothelial cell growth factor (VEGF) promotes angiogenesis associated with hypoxia. Its expression is induced by the hypoxia inducible factor α (HIF1α). VEGF binds to the VEGF receptors, promotes vascular permeability, increases the migration and proliferation of endometrial cells, and contributes to the formation of new blood vessels 37 . Anti-VEGF medicines are approved as cancer therapies but are not indicated for endometriosis because it is a benign disease. Although it is difficult to inhibit angiogenesis locally because it occurs throughout the body, leuprolide acetate 38 , sorafenib 39 , 40 , pentoxifylline 41 , melatonin 42 , 43 , and metformin 44 , 45 reduce the expression of VEGF, leading to the suppression of endometriosis lesion growth. miRNAs are small non-coding RNAs that regulate post-transcriptional gene expression and participate in complex regulatory pathways that control development and maintain homeostasis 46 . Several miRNAs function as modulators of endometriosis-associated signaling pathways. In hormonal signaling, miR-34 regulates progesterone resistance and enhances the proliferation of endometrial cancer cells 47 . miR-135a and miR-135b target HOXA10 and modulate responsiveness to progesterone in endometriosis 48 . miR-199a-5p increases expression of COX-2, resulting in inflammation and cell proliferation in endometriosis lesions 49 . In angiogenesis signaling, miR-20a expression is increased in endometriosis lesions, and prolonged HIF1α activation leads to the induction of the expression of both VEGF and fibroblast growth factor 9 (FGF-9) 50 . These factors influence angiogenesis and endometrial stromal cell proliferation in endometriotic lesions. The following chapters summarize these endometriosis-related signals with a focus on the factors that transmit them. Previously, the only way to identify bacteria was by culturing them. This process can be complicated, and depending on the number of bacteria that can be collected from lesions, it is often impossible to culture disease-related bacteria in vitro 51 , 52 . The signaling association between disease cells and microbiota has recently received much attention owing to advances in next-generation sequencing (NGS). Such studies have revealed detailed bacterial distribution, even with only a small number of samples collected 53 . Dysbiosis is defined as a disruption to the composition of the normal microbiota and has been implicated in various human diseases, including intestinal diseases and cancer 54 – 56 . Several studies have reported that both direct infection by pathogens and dysbiosis can affect the surrounding microenvironment and contribute to disease development 57 , 58 . Several signaling-associated dysbiotic effects influence the surrounding microenvironment and the bacterial niche and lead us to focus on the endometriosis-related microbiota and the related signaling that influences disease pathogenesis. Khan et al. revealed that the endotoxin concentration in the menstrual blood and peritoneal fluid of women with endometriosis is higher than that in women without endometriosis, and the main contaminating bacteria were Escherichia Coli ( E. Coli ) 59 . This bacterial contamination in menstrual blood could be a constant source of bacterial endotoxins in the peritoneal fluid via retrograde menstruation. Bacterial lipopolysaccharide (LPS) signaling activates macrophages in the peritoneal fluid, triggering toll-like receptor (TLR) 4-mediated inflammation and promoting endometriosis. LPS/TLR4 signaling stimulates a cascade of intracellular adopter molecules that trigger expression of target molecules, such as cytokines IL-6, IL-8, and tumor necrosis factor alpha (TNF-α); chemokines, monocyte chemotactic protein 1 (MCP1), also known as C-C motif chemokine ligand 2 (CCL2); and growth factors, VEGF and hepatocyte growth factor (HGF), via activation of nuclear factor kappa-light chain-enhancer of activated B-cells (NF-kB) 60 . As mentioned, the NLRP3 inflammasome is activated by damage-associated molecular patterns and pathogen-associated molecular patterns (PAMPs) signaling. One of the PAMP signals is LPS, which drives macrophages to produce inflammation-related molecules 61 , 62 . Endometriosis pathogenesis may be mediated at least in part by such bacterial signals acting on macrophages. In endometriosis, cells signaling from macrophages, NF-kB also promotes cell proliferation and inhibits apoptosis 63 , 64 . Moreover, NF-kB signaling in endometriotic stromal cells induces macrophage infiltration via estrogen and MCP-1, which further contributes to the pathogenesis of endometriosis 65 . This microbiota-related inflammatory signaling induces a microenvironment promoting the growth of endometriotic lesions 66 . Bacterial signaling through macrophages has a strong influence on the progression of endometriotic lesions, and the interaction between macrophages and endometriotic cells through signaling further contributes to the exacerbation of endometriosis (Fig.  1 ). Fig. 1 Emerging microbial signaling and the relationship with the surrounding cells. Schematic showing an overview of the relationship between bacteria, macrophages, and endometriotic cells. Lipopolysaccharide-induced bacterial signaling activates macrophages to produce inflammatory cytokines. Several inflammatory cytokines influence endometriotic stromal cell signaling. In addition, signaling factors secreted by endometriotic stromal cells cause infiltration by macrophages. The interactions between these signals create the endometrial microenvironment. The abbreviations used in the figure are listed in Supplementary Table  1 . Schematic showing an overview of the relationship between bacteria, macrophages, and endometriotic cells. Lipopolysaccharide-induced bacterial signaling activates macrophages to produce inflammatory cytokines. Several inflammatory cytokines influence endometriotic stromal cell signaling. In addition, signaling factors secreted by endometriotic stromal cells cause infiltration by macrophages. The interactions between these signals create the endometrial microenvironment. The abbreviations used in the figure are listed in Supplementary Table  1 . The transforming growth factor (TGF) superfamily is one of the major signaling pathways contributing to endometriosis pathophysiology 67 , 68 . TGF-β is secreted from macrophages, especially M2 macrophages, which are known to be anti-inflammatory and involved in tissue repair. We revealed significant infiltration by M2 macrophages in the endometrium of patients with endometriosis and further found that signals induced by secreted TGF-β promoted the phenotypic transition of endometrial fibroblasts 69 . We monitored TGF-β/Smad2/3 signaling using the chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) method and observed that such signals promoted expression of myofibroblast-associated genes that induced quiescent fibroblasts towards a myofibroblastic phenotype. We detected Fusobacterium in the endometrium of patients with endometriosis and further demonstrated in an endometriosis mouse model that the presence of these bacteria causes downstream endometriosis-associated signaling by TGF-β/Smad2/3. TGF-β signaling also mediates fibrosis in endometriosis lesions via the wingless-type mouse mammary tumor virus integration site family (Wnt)/β-catenin cascade 70 – 72 . Endometriotic lesions are dominated by fibrotic tissue, which is the final pathological outcome of chronic inflammation. Therapies targeting fibrosis have been tested in other diseases; however, their application in endometriosis remains a challenge 73 – 75 . Emerging evidence suggests that crosstalk between genital microbiota is involved in the pathogenesis of endometriosis, and clinical studies have identified microbiota associated with endometriosis 76 – 80 . Advances in next-generation sequencing (NGS) have facilitated analysis of the female genital microbiota since 2016. This microbiota, particularly in the endometrium, was previously thought to be sterile but is increasingly being studied for its contribution to the development of endometriosis. Moreover, further analysis of the microbiota in the vagina and cervix, and the intestinal tract, is contributing to the identification of new biomarkers of endometriosis. However, confirmation that these bacteria contribute to the pathogenesis of endometriosis, and details regarding the signaling that drives this process, remain unknown. Differences between the microbiome of healthy and diseased states have been reported, particularly in cancer 55 , 56 . Several bacteria have been linked to the development of cancer via reprogramming of the tumor microenvironment. A particularly well-studied cancer-related microbe is Fusobacterium , which induces intestinal cancers. Fusobacterium is a Gram-negative bacterium found in the human oral cavity and gastrointestinal tract 81 . It is a strictly anaerobic bacterium that cannot tolerate oxygen and metabolizes sugars and amino acids to produce butyric acid as its primary end product 82 . In the oral cavity, it is known as a pathogen associated with periodontitis, and in the gastrointestinal tract, it has been reported to be involved in the development of colorectal cancer. This bacterium expresses on its surface the adhesion molecule FadA, which binds to E-cadherin on host cells to activate β-catenin signaling 83 . Furthermore, Fusobacterium can bind to pattern recognition receptors and activate TLR4/myeloid differentiation primary response gene 88 (MYD88)/NF-kB signaling pathway in colorectal cancer cells 84 . Bacteroides , which produce the B. fragilis toxin, can also stimulate E-cadherin cleavage and activate β-catenin signaling 85 . E-cadherin/β-catenin signaling and Wnt/β-catenin signaling induce EMT, which drives the development of endometriosis in epithelial cells 86 . Epithelial cells are important early targets of bacterial signals that induce cell proliferation, EMT, and chronic inflammation. We have previously shown that Fusobacterium infiltrates stromal lesions in the endometrium and endometriotic lesions, indicating that after breaking through the epithelial cell barrier, the bacteria remained in the stromal region and continue to affect the surrounding cells. Fusobacterium binds to endometrial epithelial cells before infiltrating the stroma. Because endometrial epithelial cells express specific glycan chains (Gal-GalNAc) 87 , Fusobacterium, which can bind to those chains, is able to selectively adhere to the endometrial epithelium, thereby enabling its infiltration into the stroma. Since Gal-GalNAc is expressed in secretory-phase endometrial epithelial cells 88 , it is possible that the adhesion and invasion of Fusobacterium also occur more frequently during the secretory phase. Although other bacteria have been implicated in the development of endometriosis, few have been studied in detail. Further research is urgently needed to clarify the relationships between bacteria and endometriotic cells and/or the surrounding immune cells in greater detail. The clarification of bacterial-specific signaling factors should lead to the development of bacteria-specific therapeutic targets. A therapeutic approach specifically targeting endometriosis-related bacteria without affecting other microbial flora could represent a more precise treatment option for endometriosis. According to Nezhat C et al., the female reproductive tract microbiome and its metabolic products, which influence the pathophysiology of endometriosis by modulating immune function, represent a key aspect of our understanding of the endometriosis condition 89 . EVs have a vesicular structure with a diameter of 30–160 nm lined by a lipid bilayer and carry nucleic acids, proteins, and lipids 90 . EVs transmit information from cells to the extracellular matrix mediated by the vesicle contents 91 . They can reach relevant target cells by direct fusion, endocytosis, receptor-ligand interactions, and other mechanisms 92 , 93 . The uptake of EVs can be local to the site of release or distant as they circulate in biological fluids. Once EVs reach the recipient cells, they can either trigger signaling by directly interacting with surface receptors, fusing with the plasma membrane, or by internalization 94 . The International Society for Extracellular Vesicles (ISEV) 2018 recommendations seek to standardize terminology and collection methods for EVs 95 . The roles of EVs have been proposed in the context of several key signaling pathways involved in endometriosis pathophysiology, such as immunomodulation in macrophages, proliferation and migration of ectopic endometriosis lesions, and angiogenesis 96 – 98 . Zhang et al. reported that peritoneal macrophages secrete miR-22-3p via EVs, which regulates NF-kB signaling, thereby enhancing ectopic stromal cell proliferation, migration, and invasion 96 . There is marked polarization towards the M2 phenotype in macrophages of patients with endometriosis, and there are a few reports of the involvement of EVs in this polarization. miR-301a-3p in EVs from endometriosis patients drives macrophage polarization via regulating PTEN-PI3K signaling 97 . In addition, miR-146a-5p within EVs derived from ectopic stromal cells promotes M2 macrophage polarization 99 . Thus, EV-loaded miRNAs convey information between macrophages and endometriosis stromal cells. Retrogradely transplanted endometriotic cells and tissues require a blood supply for proliferation at the ectopic site. Angiogenesis is affected by many pathological processes, and both miRNAs and long non-coding RNAs (lncRNAs) have been shown to play important roles in cell-to-cell communication via EVs during angiogenesis. miR-21 is expressed in EVs released from primary endometrial stromal cells and is associated with VEGF expression 98 . Moreover, exosomal miR-21-5p promotes angiogenesis by targeting TIMP3 signaling in endometriosis lesions 100 . EVs loaded with the lncRNA HOTAIR promote angiogenesis via the miR-761/HDAC1 signaling axis and lead to activation of STAT3-mediated inflammatory signals 101 . lncRNA aHIF has also been shown to promote angiogenesis in endometriosis patients, and its expression is increased in the serum of endometriosis patients 102 . BEVs containing bioactive components are derived from the parent bacteria 103 – 105 . Both Gram-positive and Gram-negative bacteria release BEVs containing various components, such as nucleic acids, proteins, lipids, and lipoproteins 106 . BEVs produced by Gram-positive bacteria are termed cytoplasmic membrane vesicles (CMVs), whereas BEVs secreted by Gram-negative bacteria are called outer-membrane vesicles (OMVs) due to the presence of an outer membrane 107 . BEVs also interact with host cells and induce downstream immune responses indirectly via surface ligand and receptor interactions, or directly via cargo delivery into the host cells 108 . Recently, BEVs have been identified as key mediators of bacteria-host interactions in disease pathogenesis, and investigators are exploring therapeutic opportunities that target BEV-associated signaling. Emerging evidence supports the role of microbiota in the development and progression of endometriosis, although studies have yet to focus on the role and importance of BEVs specifically. Endometriosis mouse models have shown that certain gut microbiota-derived metabolites promote the growth of endometriosis lesions 10 . Moreover, short-chain fatty acids (n-butyrate) from gut microbiota inhibit endometriosis lesion growth in a mouse model 109 . Lee et al. reported that gut microbiota-derived BEVs could traverse the intestinal barrier and directly affect the peritoneal environment, owing to their small size 110 . They identified significant alterations in the composition of microbiota BEVs in the peripheral fluid of women with endometriosis compared to that observed in the controls. However, their study lacks functional analyses revealing how BEVs affect the development of endometriosis. Further studies characterizing BEVs linked to endometriosis are needed to identify potential novel therapeutic approaches that target the direct bacterial effect of exosomes. In a previous study, we reported that Fusobacterium is an endometriosis-associated bacterium and validated the pathogenicity of Fusobacterium , focusing particularly on BEVs. We showed that BEVs from Fusobacterium nucleatum ( F. nuc .) significantly stimulated the migration ability of endometrial stromal cells 111 . Furthermore, exposure of macrophages to BEVs from F. nuc . enhanced cytokine secretion and appeared to induce the polarization of macrophages towards the M2 phenotype over the M1 phenotype. Our findings provide valuable insights into the direct influence of BEVs on endometriosis pathogenesis. We previously considered the possibility of an ascending route of infection by Fusobacterium in the endometrium; however, since Fusobacterium is also endemic in the oral cavity and BEVs can affect remote organs, we must also consider the possibility of a hematogenous route of infection from the oral cavity. Fusobacterium is an anaerobic and Gram-negative opportunistic bacterium that can cause colorectal cancer and periodontal diseases 112 . In colorectal cancer, F. nuc . promotes an NF-kB signaling to secrete the inflammatory cytokine, such as IL-6 113 . Several studies have shown that BEVs from F. nuc . contribute to the pathogenesis of colon cancers. Engevik et al. demonstrated that BEVs from F. nuc . subsp. polymorphum could activate TLR4/NF-kB signaling to promote proinflammatory cytokine production 114 . Another study revealed that BEVs from F. nuc . could promote NF-kB activation via TLR2 in a manner dependent upon FomA, the major outer membrane protein of F. nuc ., thereby inducing effects similar to those observed with whole F. nuc . bacteria 115 . Because of their surface structure and adhesion proteins, BEVs from F. nuc . could act remotely on distal cells. Furthermore, Zheng et al. demonstrated that BEVs from F. nuc . facilitated the adherence of F. nuc . to colorectal cancer via FomA 116 . This suggests that BEVs from F. nuc . establish a causal relationship and form a positive feedback loop promoting the colonization of F. nuc . in colorectal cancer tissue. BEVs from F. nuc . may facilitate the translocation of bacteria to distant tissues, thereby providing targets for bacterial adherence. Zhang et al. demonstrated the BEVs from F. nuc . could trigger a series of intracellular reactions brought about by the activation of NLRP3 inflammasome via NF-kB signaling in periodontitis 117 . Their study revealed that BEVs from F. nuc . contained multiple types of virulence factors that are correlated with the pathogenicity of periodontitis and showed integrated activation of cytoplasmic signaling via BEVs. According to a cohort study conducted in Europe, a correlation was observed between the prevalence of endometriosis and periodontitis; the study showed that women with periodontitis had a 1.07-fold higher odds ratio (OR) of being diagnosed with endometriosis 118 . Previous studies have also reported an association between the prevalence of periodontal disease and endometriosis 119 , 120 ; given the possibility that the causative bacteria for both conditions are the same, these findings are particularly intriguing. F. nuc . contributes to other systemic diseases, such as adverse pregnancy outcomes, respiratory tract infections, Alzheimer’s disease, cardiovascular disease, and rheumatoid arthritis 121 – 125 . Studies of these related diseases could lead to a better understanding of the contribution of Fusobacterium and its BEVs to the pathogenesis of various diseases. Endometriosis is primarily classified into three types based on the site of occurrence and depth of invasion: superficial peritoneal lesions, ovarian endometriosis, and deep endometriosis. Each type exhibits distinct signal transduction and characteristics 126 . First, regarding estrogen signaling, there is a tendency towards reduced ER expression and estrogen signaling as the lesion progresses deeper and fibrosis advances. This reduction is particularly pronounced in deep endometriosis 127 . In addition, the expression levels of transcription factor (TCF) 21, a key regulator of fibrosis, vary depending on the stromal cells that make up the lesion formation. Lesions located at greater depths are thought to be exposed to stronger pro-fibrotic signaling 128 . Taken together, because fibrotic signaling is stronger and estrogen signaling is weaker in deep endometriosis, this may lead to differences in treatment strategies. The mechanism underlying the development of these three morphologically distinct types of endometriosis is thought to involve the retrograde flow of endometrial tissue; however, it is believed that specific signal amplification occurs during the subsequent progression of the lesions, resulting in each type acquiring its own unique phenotype. The extent to which bacteria and BEVs are involved in these signaling changes remains unclear, making this is an important area for future research. We examined the association between signal transduction in endometriosis and its pathophysiology, especially focused on BEVs (Fig.  2 ). The pathogenesis of endometriosis is complex and involves several factors and signals that interact to contribute to disease pathogenesis. Recently, novel concepts related to microbial signaling, including BEVs, have been proposed to contribute to pathogenesis, and further research on new therapeutic targets for endometriosis signaling is required. First, our previous study investigated whether BEVs function as biomarkers. We confirmed the presence of BEV-derived nucleic acid sequences within blood samples and demonstrated that using six of these sequences enabled the efficient diagnosis of endometriosis [area under the curve (AUC) = 0.91] 111 . Based on the clinical application of these results, BEVs are considered diagnostic markers for endometriosis. In addition, regarding therapeutic applications of BEVs, we are focusing on their ability to function as a tool for intercellular communication. Consequently, BEVs are expected to find future applications in nucleic acid therapeutics, in which nucleic acids are encapsulated in a stabilized state within a lipid bilayer. Our previous research has demonstrated that BEVs derived from Fusobacterium significantly enhance the migratory ability of endometrial stromal cells 111 . However, the specific components within the BEVs responsible for this effect remain to be elucidated. Furthermore, it may be possible to develop anti-migratory agents targeting genes that inhibit migration, encapsulate them within lipid bilayer vesicles, and deliver them to endometrial stromal cells in the future. It is therefore urgent to apply biomarkers and treatment methods associated with BEVs to the clinical field. There is also considerable mechanistic overlap between endometriosis and fibrosis, the inflammatory status, and the microbial microenvironment. A better understanding of signaling in other diseases would be helpful in expanding the therapeutic armamentarium targeting endometriosis. Bacterial involvement and systemic signaling via BEVs is a novel concept involved in the pathogenesis of endometriosis, and this BEV-mediated signaling to distant organs mandates that endometriosis should be considered a systemic disease. Fig. 2 Possible systemic interactions between endometriosis-related signals via BEVs. Schematic showing that endometriosis is a systemic disease. Fusobacterium is present in the oral cavity and intestinal tract and produces BEVs. The BEVs produced can reach the endometrium in the bloodstream and exert effects in remote organs. Schematic showing that endometriosis is a systemic disease. Fusobacterium is present in the oral cavity and intestinal tract and produces BEVs. The BEVs produced can reach the endometrium in the bloodstream and exert effects in remote organs.

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