Microbiota-metabolite axis in endometriosis: Pathogenic mechanisms and clinical implications

Growing  evidence  highlights  the  role  of  microbiota,  including  those  of  the  gut,  reproductive  tract,  and endometrial tissue, as critical functional drivers in the pathogenesis of endometriosis (EM). Studies have revealed a characteristic microbial imbalance in patients with EM, marked by a reduced abundance of beneficial bacteria and enrichment of opportunistic pathogens. These microbial communities exert their influence primarily through metabolic activity, as demonstrated by metabolomic studies showing their capacity to modulate host immune and endocrine responses. This imbalance leads to multiple key metabolic disturbances, including decreased levels of short-chain  fatty  acids,  particularly  butyrate;  a  shift  in  tryptophan  metabolism  toward  the  kynurenine  pathway; elevated  β-glucuronidase  activity;  increased  lipopolysaccharide  production;  and  altered  secondary  bile  acid profiles.  Functionally,  these  metabolic  alterations  converge  to  promote  EM  by  disrupting  immune  homeostasis, enhancing  estrogen  signaling,  and  driving  systemic  inflammation,  collectively  creating  a  permissive microenvironment  for  ectopic  lesion  growth  and  invasion.  Targeted  interventions,  such  as  probiotics,  high-fiber dietary  strategies,  fecal  microbiota  transplantation,  and  selective  modulation  of  microbial  or  host  metabolic enzymes,  are  emerging  as  promising  non-hormonal  therapeutic  approaches.  Nonetheless,  further  studies incorporating  longitudinal  cohort  designs  and  integrative  multi-omics  approaches  are  essential  to  establish causality and facilitate the development of precise diagnostic and personalized treatment strategies.

endometriosis, microbiota, metabolite

Endometriosis  (EM)  is  defined  as  the  presence  of endometrial-like glands and stroma outside the uterine cavity. Clinically, it presents as pelvic pain, infertility, or  pelvic  masses,  affecting  approximately  10%  of women  of  reproductive  age,  with  a  peak  incidence between  25  and  35  years[1].  Upon  estrogen stimulation,  ectopic  endometrial  glands  undergo hormone-dependent  cyclic  changes.  Depending  on ✉Corresponding  authors:  Zhen  Gong,  Email:  gongzhen@njmu. edu.cn,  ORCID,  0000-0003-1421-5830;  Dake  Li,  Email: [email protected];  ORCID,  0000-0001-6904-8529.;  Pengfei  Xu, Email: [email protected], ORCID, 0000-0002-0389-1182. Received:  15  December  2025;  Revised:  15  April  2026;  Accepted: 20 April 2026; CLC number: R711.71, Document code: A The authors reported no conflict of interests. This is an open access article under the Creative Commons Attribu- tion (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. Available online at www.jbr-pub.org.cn Open Access at PubMed Central Journal of Biomedical Research, 2026 40(0): 1–17 Review Article © 2026 by Journal of Biomedical Research. https://doi.org/10.7555/JBR.39.20250566 Unproofed their  location,  such  as  the  ovaries,  fallopian  tubes, pelvic  peritoneum,  or  ligaments,  they  can  cause  local bleeding,  pain,  fibrotic  nodule  formation,  or infertility[2,3].  EM  pathogenesis  is  complex  and multifactorial. The most widely accepted theory posits that  retrograde  menstruation  facilitates  the translocation  of  viable  endometrial  cells  to  the  pelvic cavity  and  other  sites[4].  Although  some  degree  of retrograde  menstruation  occurs  in  most  women,  only 1%–2%  develop  EM,  indicating  that  additional pathophysiological  mechanisms  are  involved[5]. Various  factors  have  been  implicated  in  the  initiation and  progression  of  EM,  including  inflammatory cytokines[6],  alterations  in  the  immunopathological microenvironment[7],  environmental  factors[8],  and microbiota[9]. In  recent  years,  several  studies  have  suggested  a significant  role  for  the  microbiota,  including  the  gut, reproductive  tract,  and  tissue-resident  microbiota,  in the  pathogenesis  and  progression  of  EM[9]. Furthermore, Mendelian randomization analyses have provided  novel  insights  into  the  potential  causal relationship  between  the  gut  microbiota  and  EM. Members  of  the  families  Christensenellaceae  and Ruminococcaceae,  as  well  as  Eubacterium ruminantium,  exhibit  protective  associations  against EM.  Conversely,  Anaerotruncus  and  Olsenella  have been  identified  as  risk-associated  taxa  that  increase susceptibility to EM, whereas taxa such as those in the order  Bacillales  and  the  family  Prevotellaceae  have been  implicated  in  elevated  disease  risk[10–12].  These microbial  communities  primarily  influence  EM progression  through  their  metabolites,  which  regulate key  processes  such  as  local  inflammation,  estrogen metabolism, and neurogenesis. These findings provide genetic  evidence  supporting  potential  causal  links between  the  gut  microbiota  and  EM.  Therefore,  this review  systematically  summatise  and  integrates current evidence on the roles of the gut, reproductive tract,  and  tissue-resident  microbiota  in  the development  and  progression  of  EM  from  the  dual perspectives of microbial composition and metabolite- mediated  mechanisms  and  explores  their  potential clinical implications.  Microbiome characteristics in EM Dysbiosis, particularly that of the gut microbiota, is associated  with  conditions  such  as  inflammatory bowel  disease[13],  arthritis[14],  and  certain  cancers[15], highlighting  its  role  in  regulating  systemic inflammation  via  immune-mediated  mechanisms. Given  the  importance  of  aberrant  inflammatory responses in EM pathogenesis, a potential microbiota- EM link has been suggested[16]. Dysbiosis may elevate systemic  estrogen  levels  by  influencing  hepatic metabolism  and  intestinal  β-glucuronidase  activity[17], thereby  promoting  the  growth  of  ectopic  lesions  and amplifying  inflammation[18].  In  addition  to  the  gut, alterations in the local microbiota of the reproductive tract and endometrial tissues have garnered increasing attention  in  EM  research.  Patients  with  EM  exhibit reduced  vaginal  microbiota  diversity  and  decreased relative  abundance  of  Lactobacillus[19],  accompanied by  an  increase  in  the  abundance  of  opportunistic pathogens  such  as  Gardnerella  and  Prevotella.  This dysbiotic state may promote the adhesion and survival of  ectopic  endometrial  tissues  by  altering  the  local immune  microenvironment  and  enhancing  the secretion of inflammatory factors[20]. The upper female reproductive  tract  is  now  recognized  as  non-sterile, and  the  microbiota  composition  of  the  endometrium and  peritoneal  fluid  exhibits  disease-specific alterations[21]. For instance, the enrichment of bacterial genera such as Fusobacterium in ectopic lesions may directly  contribute  to  lesion  establishment  and progression by activating growth factors and multiple inflammatory  pathways.  Furthermore,  bacterial translocation within the pelvic cavity, such as through retrograde  menstruation  or  intestinal  bacterial translocation,  may  further  interconnect  the  gut  and reproductive  tract  microenvironments,  forming  an integrated  microbial-immune  regulatory  network. Figure 1  illustrates  the  compositional  changes  of  the microbiome  and  the  potential  inter-compartmental microbial  translocation  across  the  gut,  reproductive tract,  and  ectopic  tissue  in  EM.  Based  on  these multifaceted  associations,  the  following  sections systematically  explore  the  characteristics  of  these microbial alterations.  Gut microbiota The  composition  of  the  gut  microbiota  in  patients with EM is significantly altered compared with that in healthy  individuals,  a  phenomenon  consistently observed  in  both  human  clinical  samples  and  animal models[22–26].  In  human  studies,  comparative  analyses of  fecal  microbiomes  from  patients  with  EM  and healthy  controls  have  revealed  significant  differences in  the  abundance  of  at  least  12  bacterial  genera[23]. Animal models provide robust and suportive evidence. For instance, in a rhesus macaque EM model, distinct alterations  were  observed  in  the  shed  fecal  microbial profiles of affected individuals[26]. In mouse models of EM,  although  some  studies  using  α-  and  β-  diversity analyses  did  not  show  significant  changes  in  overall community  diversity,  more  granular  taxonomic 2 et al. J Biomed Res, 2026, 40(0) Unproofed analyses  revealed  specific  compositional  and structural alterrations in the gut microbiota[22,24]. These findings support the conclusion that EM is associated with disease-specific gut microbial dysbiosis. The dysbiosis observed in EM does not manifest as a  global,  unidirectional  shift  across  bacterial  taxa  but presents  a  heterogeneous  and  context-dependent pattern.  At  the  phylum  level,  reported  trends  are highly  variable,  potentially  due  to  differences  in experimental  models,  host  species  or  strains,  dietary composition,  and  analytical  methodologies.  For example, one mouse study identified an enrichment of the  phyla  Firmicutes  and  Actinobacteria  in  the  EM group,  accompanied  by  a  significantly  elevated Firmicutes-to-Bacteroidetes  ratio,  a  metric  frequently regarded  as  an  indicator  of  gut  dysbiosis[22,27].  In contrast,  another  study  reported  increased Bacteroidetes  abundance  concomitant  with  reduced Firmicutes  in  EM  mice[28].  These  two  studies employed  different  modeling  approaches (intraperitoneal  injection  of  endometrial  fragments versus  surgical  autotransplantation  combined  with antibiotic  intervention),  which  may  substantially influence microbial outcomes. At  the  genus  level,  the  changes  were  more pronounced  and  taxonomically  specific.  The  relative abundance  of  several  taxa  with  putative  protective  or homeostatic  functions  was  reduced,  including depletion  of  Lactobacillus  observed  in  rhesus macaques  and  some  human  cohorts[23,26],  and  lower levels  of  Alloprevotella  and  Turicibacter  in  patients with  EM[23].  Conversely,  the  abundance  of  taxa associated  with  pro-inflammatory  signaling  and disease  progression  increased.  Representative examples  include  a  higher  prevalence  of Shigella/Escherichia-dominated  communities  in women  with  EM[25,29],  enrichment  of  Gram-negative bacteria  such  as  Escherichia coli  (E. coli)  and Enterobacter  in  a  rhesus  macaque  model[26],  and elevated  levels  of  Ruminococcaceae  UCG-014, Bifidobacterium,  and  Parasutterella  in  murine  EM models[22].  Taxon-specific  microbial  alterations  are summarized in Table 1. These  distinct  microbial  alterations  may  contribute to  the  pathogenesis  and  progression  of  EM  via multiple  interrelated  mechanisms.  Shifts  in  gut microbiota  composition  are  closely  associated  with perturbations in estrogen metabolism. Microbes, such   EM ↑ Tenericutes ↑ Firmicutes Gardnerella ↑ Atopobium ↓ Bacteroides ↑ Prevotellaceae ↓ Ruminococcus ↑ Lactobacillus ↓ Escherichia ↑ Shigella ↑ Others Reproductive Tract M icrobiota Gut M icrobiota Ectopic Tissue Fsetucimri Patodanomodues Batodioretca Aatotecymonitc ↑ Proteobacteria   Fig. 1     Microbiome characteristics in endometriosis (EM).  This  circular  schematic  illustrates  the  compositional  changes  and  potential inter-compartmental  microbial  translocation  across  three  anatomical  niches  in  EM.  The  central  gray  circle  denotes  the  disease  state  (EM), surrounded  by  three  anatomical  modules  representing  distinct  microbiota  habitats:  gut  microbiota  (purple),  reproductive  tract  microbiota (green),  and  ectopic  tissue  microbiota  (yellow).  Triangular  arrows  between  modules  indicate  putative  migration  routes  of  microorganisms among  these  compartments.  The  outermost  ring  comprises  four  phylum-level  modules  and  one  "Others"  module.  Each  phylum  module displays  representative  genera  with  directional  arrows  indicating  increased  (↑)  or  decreased  (↓)  abundance.  Created  in  BioRender.  Xu,  P. (2025) https://BioRender.com/a51kabp. Microbiota–metabolite axis in endometriosis 3 Unproofed as  those  belonging  to  the  phyla  Firmicutes  and Bacteroidetes and the genus Bifidobacterium, possess genes  encoding  β-glucuronidase,  an  enzyme  that catalyzes  the  deconjugation  of  estrogens  and facilitates  their  reabsorption  into  the  systemic circulation.  Consequently,  microbial  dysbiosis  may lead  to  elevated  in  circulating  bioactive  estrogen levels,  thereby  establishing  a  permissive  hormonal   Table 1 Characteristics of differentially abundant microbiota in endometriosis (EM) Phylum Bacterial name/Group Gram staining Abundance change (EM vs. Control) References Gut microbiota 　Actinomycetota Bifidobacterium G+ Up [22] 　Bacteroidota Bacteroidales S24-7 group G− Up [22] Bacteroides G− Up [23] Alloprevotella G− Down [23] Prevotellaceae UCG-001 group G− Up [22] Parabacteroides G− Up [23] 　Firmicutes Ruminobacter G+ Down [23] Coprococcus G+ Up [23] Lachnospiraceae NK4A136 group G+ Up [22] Oscillibacter G+ Up [23] Ruminococcaceae UGG-014 group G+ Up [22] Lactobacillus G+ Down [23,26] Turicibacter G+ Down [23] 　Pseudomonadota Parasutterella G- Up [22] Unclassified genus Typically G− Up [23] Shigella/Escherichia G− Up [25,29] Escherichia coli G− Up [26] Enterobacter G− Up [26] Reproductive tract microbiota 　Actinomycetota Gardnerella variable Up [19,25,34] Atopobium G+ Down [25] Corynebacterium G+ Up [25,32] 　Bacteroidota Prevotella G− Up [19] 　Firmicutes Lactobacillus spp. G+ Down [19] Streptococcus G+ Up [25,32,34] Enterococcus G+ Up [34] Veillonellaceae (family) G− Up [19,25] 　Pseudomonadota Escherichia coli/Shigella G− Up [25,29,34] Enterobacteriaceae (family) G− Up [25,32] Pseudomonas G− Up [25,32] 　Tenericutes Ureaplasma urealyticum G− Up [19,25] In the ectopic tissue 　Fusobacteria Fusobacterium nucleatum G− Up [38] Indifferent [42] 　Firmicutes Faecalibacterium prausnitzii G+ Up [39] 　Proteobacteria Pseudomonas G− Up (predominant in ascites) [41] 4 et al. J Biomed Res, 2026, 40(0) Unproofed milieu  that  supports  the  persistence,  growth,  and progression  of  endometriotic  lesions[17].  Furthermore, the enrichment of pro-inflammatory bacteria (Shigella and  Escherichia)  may  amplify  systemic  or  local inflammation,  whereas  the  depletion  of  protective commensal taxa may compromise immune regulatory capacity  and  inflammatory  resolution[25,26,29]. Anatomical  factors,  such  as  a  shorter  anogenital distance, may modulate these processes by increasing the  likelihood  of  bacterial  translocation  between anatomical  compartments[30].  Collectively,  the  gut microbiota  has  emerged  as  a  critical  determinant  of EM  pathophysiology  and  represents  a  promising target  for  dietary  modulation  and  microbiota-based therapeutic interventions[27].  Reproductive tract microbiota Studies on the lower reproductive tract microbiome in  patients  with  EM  have  revealed  a  heterogeneous and  nuanced  microbial  landscape.  Some  studies  have reported  no  significant  differences  in  the  cervix between  patients  with  EM  and  healthy  controls[31], whereas  others  have  identified  specific  microbial perturbations.  The  most  consistent  finding  observed was  a  marked  reduction  in  protective  Lactobacillus species,  which  are  essential  for  preserving  a  healthy, acidic  vaginal  environment  and  cervicovaginal microbial  homeostasis[19].  At  the  phylum  level,  this dysbiosis  often  corresponds  to  a  decreased  relative abundance of Firmicutes (to which Lactobacillus spp. belong)  and  an  increased  abundance  of  other  phyla such  as  Actinobacteria  and  Proteobacteria. Concurrently, there is a relative enrichment of a broad spectrum  of  genera,  many  of  which  are  associated with  bacterial  vaginosis  (BV)  or  pro-inflammatory states.  These  include  members  of  the  family Enterobacteriaceae,  genera  Streptococcus, Pseudomonas,  and  Corynebacterium[25,32],  family Veillonellaceae,  genus  Gardnerella,  and  species  E. coli/Shigella  and  Ureaplasma urealyticum[19,25]. Interestingly,  one  study  reported  absence  of Atopobium,  a  genus  associated  with  BV,  in  the cervical  and  vaginal  microbiota  of  patients  with EM[25].  In  contrast,  other  reports  indicate  that  non- Lactobacillus-dominant  microbial  communities, paticularly  those  enriched  in  Gardnerella  and Atopobium,  frequently  colonize  the  cervicovaginal epithelium  in  women  with  BV[33].  These  seemingly conflicting  findings  suggest  that  the  lower reproductive  tract  microbiota  in  EM  may  exhibit context-dependent  or  subtype-specific  dysbiosis patterns. Collectively, these observations indicate that the lower reproductive tract in patients with EM exists in  a  state  of  disease-associated  microbial  imbalance, which  may  contribute  to  disease  progression  through modulation  of  local  immune  responses  and inflammatory signaling. The  existence  of  distinct  endometrial  microbiota remains  controversial,  as  microbial  detection  may partially  reflect  contamination  by  cervicovaginal  or intestinal bacteria. Studies have reported an increased abundance  of  Gardnerella,  Streptococcus, Enterococcus,  and  E. coli  in  the  endometrium  of patients  with  EM[34],  which  have  previously  been associated  with  menopause  and  estrogen-depleted states[35]. As Enterococcus and E. coli are common gut colonizers,  these  findings  raise  the  possibility  of intestinal  bacterial  carryover  or  contamination  in certain  samples.  However,  other  investigations  have yielded  contrasting  results,  identifying  an  enrichment of  Prevotella,  Veillonellaceae,  and  Atopobium  in  the endometrium  of  women  with  EM;  these  genera  are frequently  detected  in  women  with  BV[19].  Such inconsistencies  likely  reflect  disease  heterogeneity, methodological  variability,  or  differences  in  disease stage or sampling strategies. In summary, the current evidence indicates a higher prevalence  of  BV-associated  bacteria  in  the cervicovaginal  and  endometrial  microbiota  of  women with EM. Functionally, this shift reflects the depletion of protective Lactobacillus alongside an enrichment of pro-inflammatory  taxa  (Gardnerella  and  E. coli), which  may  facilitate  disease  progression  by maintaining  a  chronic  inflammatory microenvironment  that  favors  ectopic  lesion establishment.  Despite  inter-study  variability  in specific bacterial compositions, certain BV-associated microbiota  have  consistently  been  associated  with EM.  Tissue-resident microbiota In  recent  years,  microbial  communities  in pathological  tissues  have  attracted  increasing attention. In human tumor tissues, bacteria have been identified as intratumoral residents within both cancer and  infiltrating  immune  cells[36].  Fusobacterium,  a common  commensal  bacterium  found  in  the  oral cavity  and  gastrointestinal  tract,  is  considered  an opportunistic  pathogen.  Species  within  the  genus Fusobacterium, such as Fusobacterium nucleatum (F. nucleatum),  participate  in  the  development  of periodontitis and may facilitate tumorigenic processes by eliciting the production of inflammatory cytokines, including  interleukin-6,  interleukin-8,  and  tumor necrosis  factor[37].  The  presence  of  tissue-resident microbiota  within  tumors  has  prompted  a  broader Microbiota–metabolite axis in endometriosis 5 Unproofed reconsideration  of  how  microbes  may  influence  non- malignant tumor-like pathological conditions. Despite its  histologically  benign  nature,  EM  shares  several hallmark  features  with  neoplastic  diseases,  including sustained  proliferation,  resistance  to  apoptosis,  local invasiveness,  angiogenesis,  and  immune  evasion. These  tumor-like  characteristics  suggest  that  ectopic endometrial  lesions  may  provide  a  permissive  niche for microbial colonization or persistence analogous to that observed in malignant tissues. Emerging  evidence  indicates  that  the  endometrium and  ectopic  lesions  of  patients  with  EM  show significant  enrichment  of  F. nucleatum[38]  and Faecalibacterium prausnitzii[39]  compared  with healthy controls. Among these, F. nucleatum activates transforming  growth  factor  beta  (TGF-β)  signaling, thereby  promoting  the  transformation  of  fibroblasts into  myofibroblasts  with  enhanced  proliferative, adhesive,  and  migratory  capabilities,  ultimately contributing  to  the  progression  of  endometrial lesions[38].  Under  certain  conditions,  this  enrichment may  be  associated  with  vaginal  dysbiosis[40]. Accordingly,  these  bacteria  may  gain  access  to  the upper  female  reproductive  tract  via  mechanisms  such as retrograde menstruation from the dysbiotic vaginal microenvironment,  leading  to  the  colonization  of ectopic  lesions.  Furthermore,  one  study  reported  that Pseudomonas  is  the  predominant  bacterial  genus  in the  peritoneal  fluid  of  patients  with  EM,  with approximately  95%  originating  from  the  intestine[41]. This  phenomenon  is  often  accompanied  by  elevated lipopolysaccharide  (LPS)  levels  and  disruption  of intestinal  barrier  integrity.  Through  fecal  microbiota transplantation  in  a  mouse  model,  researchers experimentally demonstrated the translocation process of  bacteria  along  the  "gut-peritoneal  cavity-lesion" axis, providing mechanistic evidence for the intestinal origin  of  the  microbiota  within  the  ectopic endometrium[41]. However,  a  recent  study  revealed  no  significant difference  in  the  presence  of  Fusobacterium  spp.  in the  normal  endometrium  between  patients  with  EM and  healthy  individuals[42].  This  contradiction  with previous findings is likely due to the heterogeneity of the  study  populations.  Moreover,  differences  in  host factors,  such  as  geographical  location,  dietary  habits, and ethnic background, may account for the divergent

regarding  F. nucleatum  enrichment, suggesting  that  its  potential  diagnostic  or  prognostic utility in EM warrants further investigation.  Microbiota-derived metabolites in EM Following  the  identification  of  gut  microbiota,  the reproductive tract, and ectopic tissue dysbiosis in EM, a  central  question  arises:  how  do  these  microbial perturbations influence host physiology? Women with EM exhibit gut dysbiosis and significant differences in stool  metabolites[43].  The  microbiota  functions  as  an active,  metabolically  integrated  biological  unit  that generates  metabolites  that  enter  the  systemic circulation  and  modulate  immune,  endocrine,  and metabolic  processes.  Therefore,  metabolomics,  which enables  the  comprehensive  profiling  of  small- molecule  metabolites,  provides  critical  insights  into upstream  microbial-host  interactions[44].  Accordingly, microbial  dysbiosis  in  EM  likely  drives  significant remodeling of the host metabolite landscape. In  the  context  of  EM,  gut  microbiota  imbalance leads  to  pronounced  changes  in  microbiota-derived metabolites, which primarily fall into three categories. First, dietary components are abnormally transformed by gut microbes. For example, short-chain fatty acids (SCFAs),  including  acetate,  propionate,  and  butyrate, are major fermentation products of undigested dietary fibers generated by colonic bacteria and play essential roles in maintaining intestinal homeostasis[45]. In EM, reduced  SCFA  levels  may  impair  anti-inflammatory and immunoregulatory functions, thereby promoting a pro-inflammatory  microenvironment  conducive  to lesion  establishment  and  growth.  Tryptophan  (Trp) metabolism  in  the  gastrointestinal  tract  is  highly dependent on the microbiota, which converts Trp into various  bioactive  compounds[46].  Altered  Trp metabolites  in  EM  can  modulate  immune  responses and  affect  pain  perception,  thereby  eliciting neurological  symptoms  associated  with  the  disease. Second,  host-synthesized  molecules  undergo microbial  modification  in  the  gut.  A  key  example  is secondary  bile  acids  (SBAs).  Primary  bile  acids produced in the liver are converted into SBAs, such as deoxycholic  acid  (DCA)  and  lithocholic  acid  (LCA), by  specific  gut  bacteria  (Clostridium  spp.)  through dehydroxylation  reactions,  a  process  tightly  regulated by  the  microbial  composition[47].  In  EM,  SBAs  may contribute  to  inflammation  and  cellular  proliferation, potentially  exacerbating  the  development  of  ectopic lesions.  Third,  certain  metabolites  originate  directly from  the  microorganisms  themselves.  LPS,  a  major component  of  the  outer  membrane  of  Gram-negative bacteria,  can  translocate  into  the  circulation  during dysbiosis  and  act  as  a  potent  inflammatory  activator, triggering  systemic  inflammation  via  Toll-like receptor 4 (TLR4) signaling[48]. Elevated LPS levels in EM are believed to drive chronic pelvic inflammation and  pain  while  promoting  the  adhesion  and  invasion of endometrial cells. Simultaneously, β-glucuronidase 6 et al. J Biomed Res, 2026, 40(0) Unproofed produced  by  certain  gut  bacteria  hydrolyzes conjugated  estrogen  into  its  free,  biologically  active form, enabling its reabsorption and disrupting normal enterohepatic  estrogen  cycling[49].  The  microbial reactivation  of  estrogen  in  EM  increases  local  and systemic  estrogen  levels,  which  are  key  factors  in stimulating  endometrial  tissue  growth  and  survival outside the uterus. These  three  dysbiosis-driven  metabolic  alterations constitute  the  critical  biochemical  basis  for  EM. Hence,  we  propose  the  microbiota–metabolite  axis  as a  pathological  framework  to  systematically  elucidate the  mechanisms  underlying  dysbiosis  in  the  gut  and vaginal microbiota through the modulation of specific metabolic  pathways,  thereby  reshaping  local  and systemic  immune  microenvironments,  thereby promoting the initiation and progression of EM. In the following  section,  drawing  on  the  five  metabolic reprogramming  pathways  (SCFAs,  Trp,  SBAs,  LPS, and  estrogen  metabolism)  outlined  in  Figure 2,  we explore  the  mechanisms  of  microbiota-derived metabolites  in  EM  from  the  perspective  of  altered metabolic profiles.  SCFA dysregulation The production of SCFAs is critically dependent on   Dietary fiber SCFA SCFAs Dysregulation Promote M2 Polarization, Suppress M1 and Limit Treg Expansion Trp Indole AhR KYNAhR IL-6 TNF-α IL-22 Treg Trp Metabolic Imbalance Inhibit Teff function and expand Tregs Pro-inflammatory Pro-inflammatory DCA,LCA Primary bile acids TGR5 cAMP PKA NF-κB TNF-α IL-17 IL-1β SBAs-Related Metabolism TLR4 NF-κB TNF-α IL-17 IL-1β LPS LPS-Related Metabolism Reprogramming of E2 Metabolism GnRH‑Mediated E2 Secretion and Promote lesion growth Pituitary gland IDO-1 SCFA Treg Me Ac Foxp3 IL-6 TNF-α GPR15 GPR43 CD4+T GPR109a DC mø Conjugated estrogen Unconjugated estrogen Unconjugated glucuronic acid Facilitation Inhibition Receptor G protein-coupled receptor Intestinal/Vaginal Epithelium Blood vessel Increase Reduce A B C D E   Fig. 2     Microbiota dysbiosis drives metabolic reprogramming in endometriosis (EM).  This  schematic  illustrates  five  interconnected pathways  through  which  microbial  imbalance  disrupts  host  metabolism,  immune  function,  and  estrogen  homeostasis  in  EM.  Panels  are arranged  horizontally.  (a)  SCFAs  dysregulation:  Reduced  SCFA-producing  bacteria  decrease  SCFAs,  impairing  Treg/M2  polarization  and enhancing  NF-κB-mediated  inflammation.  (b)  Tryptophan  metabolic  aberration:  Altered  microbiota  shifts  tryptophan  toward  kynurenine pathway, expanding Tregs while suppressing Teffs. Reduced AhR ligands (IPA, IAA) increase IL-6/TNF-α, fostering immune tolerance. (c) SBA-related  metabolic  alteration:  Reduced  SBA-producing  bacteria  lower  DCA/LCA,  diminishing  TGR5-cAMP/PKA  signaling,  which disinhibits  NF-κB  and  promotes  pro-inflammatory  cytokines.  (d)  LPS-related  metabolic  perturbation:  Gram-negative  bacterial  overgrowth elevates  LPS,  activating  NF-κB  via  TLR4  and  driving  TNF-α/IL-17/IL-1β  secretion,  contributing  to  chronic  pelvic  inflammation.  (e) Reprogramming  of  estrogen  metabolic  imbalance:  Microbial  β-glucuronidase  increases  free  E2  reabsorption,  elevating  local/systemic estrogen. Altered neuroactive metabolites disrupt the hypothalamic-pituitary-ovarian axis, creating a self-reinforcing cycle of estrogen excess and  inflammation.  Collectively,  these  dysbiosis-driven  metabolic  shifts  promote  ectopic  lesion  survival,  proliferation,  and  invasiveness  in EM. Abbreviations: AhR, aryl hydrocarbon receptor; DC, dendritic cell; DCA, deoxycholic acid; E2, Estradiol; IAA, indole-3-acetic acid; IDO-1,  indoleamine  2,3-dioxygenase  1;  IPA,  indole-3-propionic  acid;  KYN,  kynurenine;  LCA,  lithocholic  acid;  LPS,  lipopolysaccharide; SBAs, secondary bile acids; SCFA, Short-chain fatty acids; TGR5, Takeda G protein-coupled receptor 5; TLR4, Toll-like receptor 4; Treg, regulatory T cell; Trp, Tryptophan; mø, macrophage. Created in BioRender. Xu, P. (2025) https://BioRender.com/a51kabp. Microbiota–metabolite axis in endometriosis 7 Unproofed the  composition  of  the  gut  microbiota.  Key  SCFA- producing  genera  include  Bacteroides, Bifidobacterium,  Ruminococcus,  Lactobacillus, Faecalibacterium,  Prevotella,  and  Eubacterium[50].  In the context of EM, a dysbiotic state characterized by a reduced  abundance  of  specific  beneficial  taxa, particularly  Ruminococcus  and  Eubacterium  species, is  the  primary  cause  of  impaired  SCFA  generation. Beneficial bacteria, such as Eubacterium ruminantium (family  Ruminococcaceae),  are  positively  correlated with  SCFA  production  in  EM[51],  and  species  such  as Ruminococcus  faecis  play  a  pivotal  role  in butyrogenesis[52]. SCFAs,  principally  acetate  (60%),  propionate (20%),  and  butyrate  (20%),  are  produced  by  the  gut microbiota  through  anaerobic  fermentation  of  dietary fibers[45].  These  potent  SCFAs  exert  systemic immunomodulatory  effects  through  distinct mechanisms. Their primary immunoregulatory actions are mediated by activating specific G protein-coupled receptors (GPCRs) and inhibiting histone deacetylases (HDACs)[53].  Butyrate,  the  main  energy  source  for colonocytes[45]  and  recognized  for  its  anti- inflammatory  and  anticancer  properties[54,55],  acts  as  a key  regulator  of  colonic  regulatory  T  cell  (Treg) generation by directly promoting histone acetylation at the FOXP3 locus to drive naïve CD4+ T cells toward a Treg  phenotype[56]  or  by  indirectly  inducing  Treg differentiation  via  dendritic  cell  activation  through GPR109a[57].  Furthermore,  butyrate  enhances  Treg function  and  Foxp3  expression  by  inhibiting  HDACs (HDAC9)  [64],  thereby  promoting  FOXP3+  Treg expansion[58].  Propionate  and  acetate  recruit extraintestinal  Tregs  to  the  colon  via  GPR15  and GPR43,  respectively[59].  Beyond  Tregs,  SCFAs suppress  nuclear  factor  (NF)-κB-mediated inflammatory  responses  in  macrophages[60]  and promote  macrophage  polarization  toward  the  anti- inflammatory M2 type over the pro-inflammatory M1 phenotype. SCFA deficiency resulting from gut dysbiosis plays a  significant  role  in  the  pathogenesis  of  EM.  The consequent  reduction  in  systemic  anti-inflammatory capacity and compromised immunoregulation create a permissive environment for ectopic lesions. Decreased butyrate  levels  stemming  from  a  reduced  abundance of  key  producers  weaken  the  suppression  of  NF-κB- mediated  inflammation[60]  and  impair  Treg differentiation  and  function[56,61].  One  study  revealed that  experimental  depletion  of  Tregs  in  mice exacerbated  lesion  growth  and  inflammation[62]. Moreover, butyrate supplementation in mouse models significantly  reduced  the  endometriotic  lesion burden[63,64]. Butyrate exerts bidirectional regulation of steroidogenesis,  and  its  deficiency  may  impair  the correction  of  the  hyperestrogenic  state  in  EM[65]. Altogether,  gut  dysbiosis-induced  SCFA  deficiency impairs  systemic  anti-inflammatory  capacity  and intestinal barrier integrity, thereby facilitating immune imbalance,  the  diffusion  of  inflammatory  mediators, and  the  establishment  and  persistence  of  ectopic endometrial tissue[66].  Aberrations in tryptophan metabolism The dysbiotic gut microbiota profile is a key driver of the metabolic imbalance of Trp in EM. Dysbiosis is characterized  by  reduced  levels  of  Lactobacillus  and Bacillus species and an increase in that of E. coli. This reduction  affects  known  commensal  bacteria  capable of  producing  aryl  hydrocarbon  receptor  (AhR) ligands,  which  are  limited  to  Lactobacillus  species[67] and  Peptostreptococcus russellii[68].  Concurrently,  an increase in the abundance of E. coli, which, along with some  Lactobacillus  species,  increases  tryptophanase expression  to  convert  Trp  into  indole[69],  thereby redirecting  the  metabolic  flux.  The  decrease  in  the number  of  Bacillus  species  that  generate  tryptamine via  decarboxylation[70]  further  contributes  to  the  loss of  bioactive  molecules.  This  dysbiosis  is  reflected  by reduced  levels  of  the  bacterial  metabolite  4- hydroxyindole  (4HI)  in  feces,  which  correlates  with specific  microbial  shifts:  positively  with  beneficial genera  such  as  Faecalibacterium  and Lachnospiraceae  and  negatively  with  the  commensal bacterium  Dorea[43].  This  specific  microbial  shift fundamentally  disrupts  the  homeostasis  of  host  Trp metabolism. Trp  metabolism  in  the  gastrointestinal  tract  occurs mainly  through  three  pathways[46]:  (1)  direct conversion  of  Trp  by  the  gut  microbiota  into  AhR ligands and other bioactive molecules; (2) catabolism via  indoleamine  2,3-dioxygenase  1  (IDO-1)  in immune  and  epithelial  cells,  forming  the  kynurenine (KYN)  pathway;  and  (3)  synthesis  of  serotonin  by enterochromaffin  cells.  Microbial  metabolism  is  the primary source of AhR activation in the gut[71]. During microbial  tryptophan  metabolism,  the  oxidation  and reduction  pathways  yield  indole-3-acetic  acid  (IAA) and  indole-3-propionic  acid  (IPA),  both  of  which modulate  intestinal  permeability  and  immune function[72]. Indole, a key intermediate metabolite, acts as  an  interspecies  signaling  molecule  that  influences bacterial  physiology[73].  Thus,  by  shaping  Trp  flux across these pathways, gut microbes critically regulate host  barrier  integrity,  local  immunity,  and  systemic physiology. 8 et al. J Biomed Res, 2026, 40(0) Unproofed In  EM,  dysbiosis-driven  disruption  manifests  as  a pivotal  imbalance  between  the  KYN  and  indole pathways,  which  play  a  decisive  role  in  disease progression.  The  altered  microbiota  specifically enhances  the  KYN  pathway  while  suppressing  the indole  pathway.  The  KYN  pathway  is  markedly upregulated in patients with EM due to elevated IDO- 1  activity,  leading  to  increased  KYN  levels.  KYN promotes immune tolerance by suppressing effector T cells  and  expanding  Tregs,  thereby  facilitating  the immune evasion of ectopic lesions[74]. In contrast, the microbial  indole  pathway  was  suppressed.  Fecal metabolomic  analyses  revealed  significantly  reduced levels of beneficial metabolites, such as IPA. As a key AhR  ligand,  IPA  exerts  anti-inflammatory  and antioxidant  effects  by  activating  this  receptor  and inhibiting the production of pro-inflammatory factors, such  as  IL-6  and  TNF-α,  thereby  contributing  to  its anti-inflammatory  role[75].  The  decrease  in  the  levels of other immunomodulatory derivatives, such as IAA, indole-3-lactic  acid,  and  4HI,  further  weakens  host defense[43].  Quinic  acid  is  a  key  fecal  metabolite  that promotes lesion growth in EM[76]. This dual alteration, an enhanced immunosuppressive KYN pathway and a deficient  protective  AhR  pathway,  generates  an immunotolerant  microenvironment  conducive  to lesion survival and proliferation. The functional significance of AhR signaling in EM is  supported  by  direct  cellular  evidence  at  multiple levels.  Mechanistically,  AhR  is  expressed  in  mast cells  that  infiltrate  endometriotic  lesions;  upon  AhR activation, these mast cells produce IL-17 and reactive oxygen  species,  contributing  to  a  pro-inflammatory microenvironment that sustains lesion development[77]. Furthermore,  AhR  may  interact  with  other  receptors, including  classical  estrogen  receptors,  thereby modulating the course of estrogen-dependent diseases, such  as  EM[78].  Environmental  AhR  ligands,  such  as 2,3,7,8-tetrachlorodibenzo-p-dioxin,  exert  their  toxic effects  by  binding  to  AhR,  forming  an  activated heterodimer  with  transcriptional  activity  that  may promote an inflammatory state and facilitate menstrual processes  relevant  to  EM  pathogenesis[79].  These findings  provide  a  direct  mechanistic  link  between tryptophan metabolic disturbances and EM pathology. Consequently,  therapeutic  strategies  targeting  the Trp-AhR  axis,  such  as  probiotics,  prebiotics,  IDO-1 inhibitors,  and  AhR  modulators,  are  emerging  as promising  approaches  for  microbiota-based  immune modulation in EM[80].  Secondary bile acid-related metabolic alterations The  production  of  SBAs  in  the  intestine  is  directly governed by the gut microbiota. Primary bile acids are converted into SBAs, such as DCA and LCA, through microbial  transformations,  particularly dehydroxylation,  mediated  by  bacteria  such  as Clostridium difficile[47].  DCA  and  LCA  are  the  most prevalent SBAs. The abundance of specific beneficial bacteria  determines  SBA  levels.  A  previous  study indicated  a  significant  positive  correlation  between Ruminococcus abundance and SBA levels[66], whereas Eubacterium species have been shown to regulate bile acid  metabolism  and  mitigate  inflammation[51]. Therefore,  dysbiosis,  which  involves  a  decline  in  the abundance of these key genera, is the primary cause of reduced SBA production. As  important  signaling  molecules,  SBAs  can systemically  modulate  immune  balance  and inflammatory  responses  by  significantly  reducing  the production  of  multiple  inflammatory  chemokines  and cytokines, including TNF-α and IL-17[81]. Their potent anti-inflammatory effects are predominantly mediated by  the  activation  of  Takeda  G  protein-coupled receptor  5  (TGR5).  TGR5  inhibits  NF-κB  activation by modulating the cAMP/PKA signaling pathway. For instance, in a murine model of Staphylococcus aureus- induced  endometritis,  DCA  exerts  protective  effects by  significantly  upregulating  TGR5  and  PKA expression;  this  effect  is  abolished  by  TGR5  or  PKA inhibitors,  which  block  the  production  of  TNF-α  and IL-1β  and  suppress  NF-κB  activation[82].  LCA  and DCA  suppress  pro-inflammatory  cytokine  production by  human  peripheral  blood-derived  macrophages  via TGR5 activation[83]. In  the  context  of  EM,  gut  dysbiosis-induced  SBA deficiency  creates  a  permissive  pro-inflammatory milieu  that  fuels  disease  progression.  A  reduction  in the  abundance  of  beneficial  bacteria,  such  as Ruminococcus  and  Eubacterium,  leads  to  decreased SBA  levels.  This  deficiency  impairs  the  vital  TGR5- mediated  suppression  of  the  NF-κB  pathway  and  the production  of  pro-inflammatory  cytokines  (TNF-α, IL-1β,  and  IL-17)[81–83].  The  consequent  loss  of systemic anti-inflammatory signaling disrupts immune homeostasis,  allowing  unchecked  inflammation  that promotes  the  survival,  implantation,  and  growth  of ectopic  endometrial  tissue.  Direct  evidence  from human  endometriotic  stromal  cells  supports  the protective  role  of  TGR5  activation.  Activation  of TGR5  by  its  specific  agonist  INT-777  protects endometriotic  stromal  cells  from  TNF-α-induced inflammation and oxidative stress, reducing the levels of pro-inflammatory cytokines (IL-6, IL-8, and MCP- 1),  adhesion  molecules  (ICAM-1  and  VCAM-1),  and NADPH oxidase 4 (NOX4) expression. These effects Microbiota–metabolite axis in endometriosis 9 Unproofed are mediated through the inhibition of JNK/AP-1 and NF-κB  signaling[84].  Thus,  dysbiosis-driven suppression  of  SBA  production  represents  a  key mechanistic  link  between  gut  microbiota  alterations and the inflammatory pathogenesis of EM.  LPS and endotoxemia-related metabolic perturbations LPS, a major component of the outer membrane of Gram-negative bacteria, acts as a potent inflammatory activator  by  binding  to  TLR4  on  immune  cells, thereby  triggering  downstream  signaling  pathways, such  as  NF-κB.  This  cascade  activates  macrophages and  induces  the  release  of  pro-inflammatory cytokines,  including  IL-6  and  TNF-α[48].  In  EM,  an increased  abundance  of  Gram-negative  bacteria,  such as those from the Prevotellaceae family, in the gut or reproductive  tract,  leads  to  elevated  systemic  LPS levels.  Sustained  endotoxemia  drives  chronic inflammation  in  the  pelvic  and  systemic compartments, creating a favorable microenvironment for  the  adhesion,  proliferation,  and  progression  of ectopic  endometrial  cells[85].  In  the  context  of  EM, LPS enhances TLR4 expression and activates the NF- κB  pathway,  exacerbating  peritoneal  inflammation and promoting the development of endometriotic-like lesions  in  mouse  models[86].  Moreover,  menstrual blood  is  frequently  contaminated  with  high  levels  of E. coli,  and  microbial  colonization  within  the endometrium  has  been  documented,  both  of  which may serve as significant sources of systemic LPS and contribute to endotoxemia[87].  Reprogramming of estrogen metabolic imbalance Estrogen plays a pivotal role in the pathogenesis of EM,  and  its  homeostasis  is  regulated  by  the  gut microbiota.  First,  during  gut  dysbiosis,  which  is characterized by reduced microbial diversity, microbe- derived  β-glucuronidase  is  abnormally  upregulated. This  enzyme  deconjugates  estrogen,  which  is inactivated by hepatic glucuronidation, and converts it back  to  its  biologically  active  free  form[49].  These reactivated  estrogens  are  reabsorbed  via  the enterohepatic  circulation,  leading  to  abnormally elevated  circulating  estrogen  levels,  which  directly promote  the  growth,  shedding,  and  inflammatory activity of ectopic endometrial tissue[87]. Second, gut dysbiosis can influence the function of the  hypothalamic–pituitary–ovarian  axis  by  altering neuroactive  metabolites.  Dysbiosis-induced  changes in the metabolome can elevate the circulating levels of neurotransmitter-like  substances,  such  as  serotonin, glutamate,  SCFAs,  and  γ-aminobutyric  acid.  These metabolites  can  cross  the  blood–brain  barrier  and  act on  various  receptors,  including  those  expressed  on gonadotropin-releasing hormone neurons, triggering a cascade  of  hormonal  signals  that  lead  to  increased ovarian  estrogen  secretion[9].  Furthermore,  shifts  in microbial composition can modulate the production of SCFAs  and  LPS  in  response  to  estrogen  imbalance, whereas  fluctuations  in  estrogen  levels  can reciprocally influence microbial community structure. This bidirectional interaction contributes to the altered immune  microenvironment  observed  in  patients  with EM[49,88].  Other metabolic alterations In addition to the metabolites discussed previously, many other metabolites are associated with alterations in  the  gut  microbiota  in  EM.  Using  correlation analysis,  a  mouse  model  study  confirmed  that  L- methionine  and  L-cysteine  levels  were  increased  in the feces of mice with adenomyosis. Their abundance is  negatively  correlated  with  that  of  Bacteroides  but positively  correlated  with  Desulfovibrio,  suggesting  a statistical  association  between  specific  amino  acid metabolism  and  microbiota  composition[89]. Furthermore,  other  studies  using  the  same  disease model have depicted a broader landscape of metabolic dysregulation,  identifying  the  differential  expression of various metabolites, including quinic acid, cytosine, and L-methylhistidine, in the feces of affected mice[76]. Among  these,  quinic  acid  levels  increased significantly.  Functional  experiments  confirmed  the ability  of  quinic  acid  to  effectively  promote  the proliferation  of  immortalized  human  endometrial epithelial  cells,  providing  a  potential  molecular explanation  for  the  direct  involvement  of  microbial metabolites  in  the  growth  of  ectopic  lesions[76].  The effects of this metabolic reprogramming were evident in  lipid  and  primary  bile  acid  metabolism.  For instance,  in  the  feces  of  EM  model  mice,  the abundance  of  bile  acids,  such  as  chenodeoxycholic and  ursodeoxycholic  acids  is  increased,  whereas  the levels of alpha-linolenic acid and its metabolite 12,13- epoxyoctadecatrienoic  acid  are  decreased.  This  shift leads  to  significant  perturbations  in  enriched pathways,  including  secondary  bile  acid  biosynthesis and  alpha-linolenic  acid  metabolism[90].  Altered abundance  of  specific  metabolites  has  been  observed in  the  fecal  samples  of  women  with  EM.  These biomarkers  include  linoleic  acid,  adenine,  cytosine and  adenosine  (Table 2)[43].  Although  these  studies illustrate  extensive  alterations  in  the  gut  microbial metabolic  network  under  disease  conditions,  most analyses  remain  descriptive  and  have  yet  to  elucidate 10 et al. J Biomed Res, 2026, 40(0) Unproofed the causal relationship between microbial changes and specific alterations in metabolite levels.  Clinical applications of microbiota and their metabolites in EM Our  proposed  "microbiota–metabolite"  axis  has been substantiated, providing a systematic framework for understanding the underlying mechanisms of EM. This  model  highlights  how  microbial  dysbiosis  acts synergistically  across  multiple  levels,  including immune  regulation,  inflammatory  responses,  and hormonal  balance,  by  driving  disturbances  in  key metabolic  pathways  to  promote  disease  development and  persistence.  Given  the  central  role  of  the "microbiota–metabolite" axis in EM pathogenesis, the development  of  novel  diagnostic  and  therapeutic strategies  targeting  this  axis  has  emerged  as  a promising  research  direction.  These  translational approaches aim to address the critical unmet needs in current  clinical  practice,  namely  delayed  diagnosis and limited treatment options.  Diagnostic strategies Currently,  the  gold  standard  for  diagnosing  EM  is invasive  laparoscopic  surgery,  which  has  an  average diagnostic  delay  of  7–10  years[91].  Although  most biomarkers  are  still  in  the  exploratory  stage, metabolomics and microbiome profiling offer a robust foundation  for  developing  noninvasive  or  minimally invasive liquid biopsy approaches. Fecal microbiome- based diagnostic methods have been successfully used in  the  clinical  diagnosis  of  various  diseases[92], suggesting  that  fecal  microbial  markers  have  the potential for cross-disease diagnostic applications.   Table 2 Differentially abundant microbiota-derived metabolites in endometriosis (EM) Metabolite name Category Change in EM (abundance) References 12,13-Epoxyoctadecatrienoic acid Fatty acid derivative Down [87] 1-Methylhistidine Amino acid derivative Down [76] 20-Deoxyadenosine Nucleoside Down [43] 4-Hydroxyindole Tryptophan metabolite (indole pathway) Down [43] Acetate Short-chain fatty acid Down [43,45,66] Adenine Nucleobase Down [43] Adenosine Nucleoside Down [43] Alpha-linolenic acid Polyunsaturated fatty acid Down [87] Butyrate Short-chain fatty acid Down [43,45,51,52,66] Chenodeoxycholic acid Primary bile acid Up [87] Cytosine Nucleobase Down [43,76] Deoxycholic acid (DCA) Secondary bile acid Down [47,66,78-80] Free (deconjugated) estrogen Steroid hormone Up [49,83] Indole-3-acetic acid (IAA) Tryptophan metabolite (indole pathway) Down [43,72] Indole-3-lactic acid (ILA) Tryptophan metabolite (indole pathway) Down [43] Indole-3-propionic acid (IPA) Tryptophan metabolite (indole pathway) Down [43,75] Kynurenine Tryptophan metabolite (Kynurenine pathway) Up [74] L-cysteine Amino acid Up [86] Linoleic acid Polyunsaturated fatty acid Down [43] Lipopolysaccharide (LPS) Bacterial endotoxin Up [48,81–83] Lithocholic acid (LCA) Secondary bile acid Down [47,66,78,80] L-methionine Amino acid Up [86] N-formyl-L-methionine Amino acid derivative Up [43] Propionate Short-chain fatty acid Down [43,45,66] Quinic acid Tryptophan metabolite / Other Up [76] Ursodeoxycholic acid Primary bile acid Up [87] Microbiota–metabolite axis in endometriosis 11 Unproofed Multiple  studies  have  indicated  that  microbial alterations can serve as potential diagnostic targets. F. nucleatum  infiltration  is  detectable  in  the endometrium  of  64%  of  patients  with  EM[38], suggesting  its  potential  as  a  biomarker  for  the auxiliary  diagnosis  of  the  disease[93].  Importantly,  the diagnostic value of this bacterium has been confirmed in  colorectal  and  gastric  cancers,  with  area  under  the curve  (AUC)  values  of  0.80[94]  and  0.813[95], respectively,  providing  strong  evidence  for  its application in EM diagnosis. Fecal microbial markers also  demonstrate  diagnostic  potential.  The  ratio  of Prevotella  to  Bacteroides  in  the  gut  is  significantly higher in EM patients compared to controls[96]. Known as an important biomarker for obesity, its alteration in EM  further  supports  its  utility  as  a  non-specific indicator  reflecting  the  disease-associated microenvironment for auxiliary diagnosis[27]. Beyond  direct  microbial  markers,  microbe-derived metabolites  have  emerged  as  promising  non-invasive diagnostic  tools.  Diagnostic  approaches  based  on blood,  urine,  and  stool-derived  metabolites  for  EM have achieved AUC values exceeding 0.8[43]. Multiple untargeted  metabolomics  studies  have  consistently identified distinct metabolic signatures in the blood of EM patients[97], involving disturbances in amino acid, lipid,  and  energy  metabolism  pathways.  Notably, combined  metabolite  panels  have  shown  excellent performance. Studies have confirmed that a diagnostic model  incorporating  N-[1H-indol-3-ylacetyl]valine (odds ratio [OR]=1.601, a tryptophan metabolite) and hippuric  acid  (OR=0.368,  derived  from  gut  microbial metabolism  of  dietary  components)  along  with  other features  achieved  an  AUC  greater  than  0.9[98].  These metabolic  alterations  likely  reflect  the  functional output  of  microbial  dysbiosis.  Animal  studies  further suggest the diagnostic potential of related metabolites. Research  has  found  significant  differences  in  fecal metabolites  between  EM  model  mice  and  healthy controls,  with  quinic  acid,  lactic  acid,  and  N-acetyl aspartic  acid  showing  high  expression[76].  These differential  metabolites  hold  promise  as  candidate targets for non-invasive EM diagnosis. Although  microbe-derived  markers  show  great promise  for  non-invasive  EM  diagnosis,  cautious advancement  toward  clinical  application  is  needed. Current  findings  exhibit  some  heterogeneity,  and certain discoveries are based solely on animal models. Therefore,  large-scale,  multi-center  independent cohort  studies  are  still  required  to  systematically validate  the  diagnostic  performance  of  existing markers.  Therapeutic strategies Modulating the "microbiota–metabolite" axis opens up  a  new  frontier  for  potentially  safer  and  less  toxic adjunctive  therapies  for  EM.  The  following  sections provide  an  overview  of  these  therapies  and  their potential use in treating EM.  Probiotic interventions Future  therapeutic  strategies  for  EM  will  move beyond  generic  probiotic  supplementation  toward precision nutrition tailored to the individual microbial and  metabolic  profiles.  For  instance,  in  patients  with low  fecal  butyrate  levels,  the  targeted  administration of  butyrate-producing  strains  (Clostridium butyricum)[99]  or  prebiotics  rich  in fructooligosaccharides  and  inulin  may  be  more effective  than  conventional  Lactobacillus-based products.  Supplementation  with  Bifidobacterium bifidum  for  4  weeks  modulated  the  gut  microbiota  in healthy  adults,  reducing  the  abundance  of Prevotellaceae and Prevotella while increasing that of Ruminococcaceae  and  Lachnospiraceae,  along  with elevating  butyrate  concentrations[100].  Animal  models have provided proof-of-concept evidence that specific probiotics  can  reduce  lesion  size  and  alleviate  pain- related behaviors in EM mice[101].  Metabolite-based interventions Targeting  the  gut  microbiota–metabolite  axis represents  a  novel  therapeutic  approach  for  EM,  with the  direct  supplementation  of  key  metabolites demonstrating  significant  potential.  First,  butyrate (sodium  butyrate)  has  been  shown  to  effectively inhibit lesion growth in animal models by suppressing endometrial  cell  survival  through  multiple  pathways, including activation of GPCRs, inhibition of HDACs, and  upregulation  of  the  GTPase-activating  protein RAP1GAP[63].  Second,  bile  acid  metabolites  have regulatory  value;  chenodeoxycholic  acid  and  its derivatives  may  alleviate  EM  pathology  by modulating  the  activity  of  the  associated  metabolic enzymes[102].  Furthermore,  the  microbial  Trp metabolite 4-HI is found at reduced levels in patients with  EM,  and  its  supplementation  inhibits  disease progression, underscoring its potential as a therapeutic candidate[43].  In  summary,  interventions  targeting butyrate, bile acids, and Trp metabolites offer diverse and promising avenues for the metabolic management of EM.  Dietary modifications Encouraging  patients  with  EM  to  adopt  a  high- 12 et al. J Biomed Res, 2026, 40(0) Unproofed fiber, omega-3 fatty acid-rich Mediterranean diet[103] is a  safe,  cost-effective,  and  multi-beneficial  strategy. High-fiber  diets  are  fermented  by  the  gut  microbiota into  SCFAs,  which  exert  direct  anti-inflammatory effects, whereas omega-3 fatty acids are potent natural anti-inflammatory  agents[104].  Observational  studies have  identified  associations  between  dietary  patterns and  EM  risk[105],  although  large-scale  randomized controlled  trials  are  required  to  provide  high-level evidence.  Moreover,  cruciferous  vegetables  supply indole-3-carbinol,  which  is  converted  in  the  acidic gastric  environment  into  various  bioactive compounds —including  diindolylmethane[106]—that modulate  estrogen  metabolism  and  exhibit  anti- inflammatory  effects,  potentially  restricting  the progression of EM.  Fecal microbiota transplantation As the most radical method of microbial ecosystem reconstruction, fecal microbiota transplantation (FMT) has  strong  theoretical  potential  for  patients  with refractory  EM  who  fail  conventional  treatments  and exhibit  severe  dysbiosis[107].  For  example,  the transplantation  of  Lachnospiraceae,  which  produces SCFAs[50],  Lactobacillus  species  capable  of synthesizing AhR ligands[67], and Clostridium difficile, which  is  involved  in  the  conversion  of  SBAs[47]. However,  the  application  of  FMT  in  EM  remains entirely  theoretical,  and  no  clinical  studies  have evaluated its safety or efficacy. First, despite rigorous donor  screening,  potential  risks  include  the transmission  of  pathogens  (multidrug-resistant organisms) and adverse events, ranging from transient abdominal  discomfort  and  diarrhea  to  rare  severe infections[108,109].  Second,  ethical  considerations, including  informed  consent  procedures,  privacy protection,  and  sample  ownership,  raise  concerns, with  most  survey  respondents  expressing skepticism[110].  Third,  FMT  products  vary  widely  in their preparation methods, donor selection criteria, and administration  routes,  and  currently  lack  regulatory approval  for  non-Clostridioides difficile  infection indications[108]. Consequently, FMT for EM must first be  evaluated  in  well-designed  early-phase  clinical trials  (Phase  I/II)  to  thoroughly  assess  its  safety  and preliminary  efficacy  before  any  clinical  application and should not be attempted empirically.

EM  has  long  been  viewed  as  a  localized gynecological  condition;  however,  growing  evidence positions  it  within  a  broader  systemic  framework centered  on  the  "microbiota–metabolite"  axis.  This review  integrates  current  findings  to  reveal  a consistent  pathological  pattern  in  EM:  depletion  of beneficial  microbes  (butyrate  producers  such  as Ruminococcus  and  Eubacterium),  overgrowth  of  pro- inflammatory  taxa,  reduced  SCFA  levels,  a  shift  in Trp metabolism toward the immunosuppressive KYN pathway,  increased  β-glucuronidase  activity  driving estrogen  reactivation,  and  elevated  LPS  release promoting  inflammation.  These  alterations  disrupt immune  homeostasis,  enhance  estrogenic  stimulation, and foster a chronic inflammatory milieu that supports the persistence and progression of ectopic lesions. However,  despite  these  advances,  several  key challenges remain. Few findings from existing animal models have been translated into clinical applications in  humans.  The  limitations  of  the  animal  models discussed  in  this  study  include  exogenous  estrogen failing to mimic the natural human cycle, interspecies immune differences hindering mechanistic translation, surgical  transplantation  deviating  from  the  natural course  of  retrograde  menstruation,  and  disparities  in baseline  microbial  abundances  between  animals  and humans,  limiting  the  extrapolation  of  metabolic findings. Other key challenges remain, particularly in establishing  causality  and  overcoming  the heterogeneity  of  studies.  Therefore,  future  efforts should  focus  on  developing  standardized  protocols; integrating  multi-omics  data;  conducting  multi- regional,  multi-cohort  longitudinal  studies;  advancing lesion-based  stratification  studies  that  correlate microbial and metabolic profiles with EM phenotypes to  lay  the  groundwork  for  personalized  diagnostics and treatments; and designing metabolite intervention trials  to  validate  the  feasibility  of  using  microbiota- derived metabolites as therapeutic targets. Well-designed  clinical  trials  testing  microbiota- targeted interventions are essential for translating this paradigm  into  effective  diagnostic  and  therapeutic strategies. In redefining EM through the lens of host- microbe  metabolic  crosstalk,  we  gaindeeper mechanistic insight and transformative opportunity for early disease detection and the precision management of patients with EM.  Funding This  project  was  supported  by  Nanjing  Medical Science  and  Technique  Development  Foundation (Grant  No.  YKK24153  to  S.X.),  and  Research Innovation Program for Graduates of Jiangsu Province (SJCX24_0755 to N.Y.)  Microbiota–metabolite axis in endometriosis 13 Unproofed Author contributions Moyuan Li:  Conceptualization,  Writing–original draft,  Visualization.  Aiyuan Yue:  Formal  analysis. Lingfeng Gu:  Investigation.  Feiyang Li: Visualization.  Nuo Ye:  Investigation,  Funding acquisition.  Sujuan Xu:  Funding  acquisition.  Zhen Gong:  Supervision,  Project  administration.  Dake Li: Conceptualization.  Pengfei Xu:  Conceptualization, Project administration, Writing–review & editing.  Acknowledgments: The  figure  was  created  using  BioRender (https://www.biorender.com/)

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