{"paper_id":"7f31a7a3-e926-4ffb-9fa7-4f3534daa931","body_text":"Endometriosis is a chronic inflammatory pain condition believed to impact the lives of one in ten women and those assigned female at birth ( Horne & Missmer 2022 ). Whilst endometriosis is defined by the presence of endometrial-like tissue growing as ‘lesions’ outside the uterus, patients can present with a range of seemingly unrelated symptoms, leading to new, sometimes controversial, reframing of endometriosis as a body-wide disorder ( Hickey  et al.  2020 ). For example, whilst pain (cyclical or constant) and infertility are common symptoms, patients with endometriosis often present at clinics reporting a range of other problems. These include mood disorders (anxiety, depression) and symptoms affecting their digestive system, such as abdominal bloating and those mirroring irritable bowel syndrome (IBS) ( Saunders & Horne 2021 ,  Saunders & Horne 2023 ).\nThe gut microbiota is the collection of bacteria, viruses, and archaea within the gastrointestinal (GI) tract which produce essential metabolites, hormones, and neurotransmitters. Evidence for the impact of diet on the gut microbiota and the importance of the microbiome to general health is rapidly expanding ( Cryan  et al.  2019 ). Notably, metabolic products of the microbiota can affect the immune system and influence inflammation, leading to increased interest in how changes in the microbiota could impact on the severity of symptoms in disorders associated with aberrant immune responses, such as endometriosis ( Saunders & Horne 2021 ). A breakthrough in our understanding of the importance of the bidirectionality in signalling between the gut and brain has been informed by results of studies on symptoms including stress, pain, and mood disorders ( Rea  et al.  2019 ,  Wilmes  et al.  2021 ). Studies such as these have linked gut dysbiosis (an ‘imbalance’ in the gut microbial community) to the severity of symptoms and vice versa.\nResearch into the relationship between the gut microbiota and endometriosis is still limited in scope, with a focus on endometriotic lesion development and disease progression, rather than its potential influence on symptomology ( Chadchan  et al.  2023 ,  Wei  et al.  2023 ). In this narrative review, we will provide a brief overview of the symptoms of endometriosis that may be altered by signalling within the microbiota–gut–brain (MGB) axis, briefly consider the existing primary research exploring the function of the gut microbiota in endometriosis lesion development, which has been explored in depth elsewhere ( Talwar  et al.  2022 ,  Chadchan  et al.  2023 ), before focussing on the potential role of dialogue between the gut microbiota, inflammatory response, and pain pathways in promoting/mitigating the body-wide symptoms associated with the disorder. To increase the range of evidence we will summarise findings from other chronic inflammatory pain conditions, to demonstrate the potential mechanisms of interaction between the gut microbiota and key symptoms of endometriosis: pain and inflammation; GI symptoms; and mood disorders. Finally, we will discuss promising therapeutic opportunities, such as dietary intervention, supplements, probiotics, and antibiotics, to alleviate symptoms via manipulation of the gut microbiota, providing exciting opportunities for future research with the priority of improving symptomology and patients’ quality of life (QoL), some of which have been conducted in cohorts of endometriosis patients.\nNotably, as our understanding of the role(s) of other microbiomes has increased, researchers have also begun to explore whether the vaginal, endometrial, oral, and peritoneal microbiomes are altered in endometriosis patients, but results to date are variable. For the purposes of the current narrative review, we have focussed on the evidence that the gut microbiome, acting as part of a gut–brain bidirectional signalling system, can impact on symptoms of endometriosis, as well as evidence from studies on endometriosis patients and other disorders often co-morbid with endometriosis, that regulation of the microbiome could be a target for symptom relief.\nA comprehensive literature review identified articles and reviews through PubMed by searching for specific keywords including endometriosis, (chronic) pain, gut microbiome/metabolites, diet, supplements, IBS, mood, and other relevant related terms.\n\nThe exact cause of endometriosis is currently undetermined, although evidence shows genetic changes may increase the risk of developing the disorder ( Zondervan  et al.  2018 ,  Saunders 2022 ). A defining hallmark of endometriosis is considered the presence of ‘lesions’ resembling endometrial tissue, most commonly detected in the peritoneal cavity ( Saunders & Horne 2021 ). Our understanding of the mechanisms resulting in the establishment and survival of lesions has evolved from the theory of retrograde menstruation – the concept of menstrual debris entering the pelvic cavity via the Fallopian tubes during menstruation and implanting into the peritoneum, complemented by other routes including transfer via the vasculature ( Yovich  et al.  2020 ). In the past 20 years, evaluation of clinical samples and preclinical models have provided evidence to support a role for steroid hormone regulation of cell proliferation, inflammation, and neuroangiogenesis, with nerve projections connecting the lesions to the central nervous system (CNS), promoting the survival of lesion tissue and the development of pain symptoms (extensively reviewed elsewhere) ( Zondervan  et al.  2018 ,  Zondervan  et al.  2020 ,  Saunders & Horne 2021 ).\nCurrent therapeutic options for people with endometriosis are limited ( Saunders & Horne 2021 ). Surgical removal of lesions, hormonal therapies, and analgesics are the most common strategies, all of which can be associated with detrimental side effects, with over 50% of patients having repeat surgeries within 5 years ( Saraswat  et al.  2018 ). Many endometriosis patients have reported trialling a selection of self-management strategies, including dietary interventions and dietary supplements, likely in response to the combination of diagnostic delays and the narrow range of therapeutic options currently available.\nAn altered immune response and changes in immune-cell phenotype are reported in patients with endometriosis.  Table 1  summarises endometriosis-associated changes in key immune cells. For example, several studies have shown increased infiltration of neutrophils and macrophages in the peritoneal fluid and lesions, with altered M1–M2 macrophage polarisation, alongside suppressed natural killer (NK) cell activity and increased numbers of T helper 17 cells (Th17) ( Symons  et al.  2018 ). Importantly, these changes are associated with increased levels of proinflammatory cytokines in the lesion microenvironment, whose downstream effects include increased inflammation, angiogenesis, and cell proliferation, all of which contribute to survival/growth of lesions ( Herington  et al.  2011 ,  Symons  et al.  2018 ). Studies such as these have supported the argument that endometriosis should be considered as an inflammatory disorder, and therapies sought to blunt/normalise these responses (see  Saunders & Horne 2021 ).\n Table 1 Summary of immune-cell changes in peritoneal fluid of endometriosis patients. Immune cell type Cellular changes Cytokine and chemokine production Downstream effects Reference Neutrophils ↑ PF infiltration; ↓ Apoptosis ↑ TNF-α; ↑ VEGF ↑ Inflammation; ↑ Angiogenesis Symons  et al.  (2018) Macrophages ↑ Infiltration in PF and lesion microenvironment; ↓ Phagocytosis; ↑ Activation of transcription factor NF- κ B; Co-localisation with nerve fibres; altered M1–M2 polarisation ↑ IL-1β; ↑ IL-6; ↑ IL-10; ↑ TNF-α; ↑ TGF-β; ↑VEGF ↑ Inflammation; ↑ Angiogenesis; ↑ Stromal cell proliferation and invasiveness Herington  et al.  (2011) ,  Symons  et al.  (2018) NK cells ↓ Activity ↑ TNF-α ↓ Cytotoxicity; ↑ Inflammation Herington  et al.  (2011) ,  Symons  et al.  (2018) T cells ↑ Th17:Treg ratio ↑ IL-17 leads to: ↑ IL-8 and ↑ COX-2 ↑ Inflammation; ↑ Angiogenesis; ↑ Stromal cell proliferation; attracts and activates neutrophils Symons  et al.  (2018) NK, natural killer.\nSummary of immune-cell changes in peritoneal fluid of endometriosis patients.\nNK, natural killer.\nIndividuals with endometriosis report a variety of different types of pain ( Fig. 1A ). Mechanisms that initiate or promote endometriosis-associated pain symptoms remain the subject of intense research activity, with some evidence suggesting the role of nerve growth within lesions, which may occur in parallel with angiogenesis ( Asante & Taylor 2011 ). Many studies have reported a lack of correlation between pain intensity and the number, location, or type of lesions ( Vercellini  et al.  2007 ), indicating other mechanisms also contribute to pain experience in addition to the extent/presence of lesion neurogenesis ( Fig. 1B ).\n Figure 1 Pain mechanisms in endometriosis . A global assessment of pain mechanisms that may differ in endometriosis patients has identified alternations in chronic pain pathways associated with hypersensitivity to stimuli. (A) Commonly reported pain symptoms in endometriosis include dysmenorrhea (pain during menstruation), dyspareunia (pain during sex), and pain on defecation and urination ( Saunders and Horne, 2021 ). (B) Inflammation and innervation of endometrial lesions contribute to pain experience via a connection with the CNS and recruitment of immune cells ( Tokushige  et al.  2010 ,  Liu  et al.  2012 ). (C) Differences have also been identified in the brains of endometriosis patients, with reduced grey matter volume in the left thalamus (pale yellow), right putamen (orange), left cingulate gyrus (dark yellow), and right insula of those with chronic pelvic pain (CPP) ( As-Sanie  et al.  2012 ), as well as changes identified in the extent and location of activation in response to painful stimuli in those with dysmenorrhea ( Tu  et al.  2009 ). (D) Central sensitisation occurs when pain pathways are persistently amplified, enabling them to be ‘learnt’ by the CNS, thus reducing the level of stimuli required to trigger pain, eventually leading to sensitivity to non-noxious stimuli and at sites distal to the inflammation ( Berkley  et al.  2005 ,  Neziri  et al.  2010 ). In CPP, this manifests as visceral hypersensitivity ( Vincent  et al.  2011 ,  Kaya  et al.  2013 ) and has been demonstrated in endometriosis ( Weiwei  et al.  2010 ,  Aredo  et al.  2017 ,  Zheng  et al.  2019 ). The red arrow represents the bidirectional relationship between pain pathways triggered by local inflammation and immune responses in the peritoneum (or alternative lesion locations) and the structural and learnt changes in the brain which result in exacerbated pain experiences. Created with BioRender.com.\nPain mechanisms in endometriosis . A global assessment of pain mechanisms that may differ in endometriosis patients has identified alternations in chronic pain pathways associated with hypersensitivity to stimuli. (A) Commonly reported pain symptoms in endometriosis include dysmenorrhea (pain during menstruation), dyspareunia (pain during sex), and pain on defecation and urination ( Saunders and Horne, 2021 ). (B) Inflammation and innervation of endometrial lesions contribute to pain experience via a connection with the CNS and recruitment of immune cells ( Tokushige  et al.  2010 ,  Liu  et al.  2012 ). (C) Differences have also been identified in the brains of endometriosis patients, with reduced grey matter volume in the left thalamus (pale yellow), right putamen (orange), left cingulate gyrus (dark yellow), and right insula of those with chronic pelvic pain (CPP) ( As-Sanie  et al.  2012 ), as well as changes identified in the extent and location of activation in response to painful stimuli in those with dysmenorrhea ( Tu  et al.  2009 ). (D) Central sensitisation occurs when pain pathways are persistently amplified, enabling them to be ‘learnt’ by the CNS, thus reducing the level of stimuli required to trigger pain, eventually leading to sensitivity to non-noxious stimuli and at sites distal to the inflammation ( Berkley  et al.  2005 ,  Neziri  et al.  2010 ). In CPP, this manifests as visceral hypersensitivity ( Vincent  et al.  2011 ,  Kaya  et al.  2013 ) and has been demonstrated in endometriosis ( Weiwei  et al.  2010 ,  Aredo  et al.  2017 ,  Zheng  et al.  2019 ). The red arrow represents the bidirectional relationship between pain pathways triggered by local inflammation and immune responses in the peritoneum (or alternative lesion locations) and the structural and learnt changes in the brain which result in exacerbated pain experiences. Created with BioRender.com.\nReduced pain thresholds, along with increased activity in several brain regions associated with pain perception, have been identified in both cynomolgus monkeys with naturally occurring endometriosis ( Yano  et al.  2019 ) and a rat model of the disease ( Zheng  et al.  2020 ). Coupled with the fact that the diagnostic delay experienced by individuals with endometriosis may increase the likelihood of developing chronic/persistent pain that is resistant to standard therapies, it is clear that new approaches to pain management are required. In women with chronic pelvic pain (CPP), including those with endometriosis, changes in brain structures have been detected that appear consistent with an amplified/abnormal response to stimuli ( Fig. 1C ) ( Brawn  et al.  2014 ). Additional data have detected altered brain chemistry associated with these physical changes, consistent with amplification of pain signals and so-called central sensitisation augmenting signals from peripheral enteric nerves ( Fig. 1D ) ( As-Sanie  et al.  2016 ).\nThere is increased awareness of the impact of GI symptoms on the wellbeing of endometriosis patients ( Maroun  et al.  2009 ). A large study found women with endometriosis had increased risk of inflammatory bowel disease, Crohn’s disease, and ulcerative colitis ( Jess  et al.  2012 ). A two-fold higher incidence of IBS in individuals with endometriosis, compared to the general population ( Chiaffarino  et al.  2021 ,  Aupetit  et al.  2022 ), suggests overlapping mechanisms between the conditions, recently corroborated by evidence of shared genetic risk factors ( Yang  et al.  2023 ). IBS is characterised by chronic gut inflammation, bloating, and visceral pain – symptoms common in patients with a diagnosis of endometriosis ( Saunders & Horne 2021 ,  Deepak Kumar  et al.  2023 ). Crucially, patients highlight abdominal bloating as an important topic of unmet need ( Horne  et al.  2017 ).\nWomen with endometriosis have a higher incidence of psychiatric comorbidities and mood disorders, including depression and anxiety ( Gete  et al.  2023 ). Mechanisms to explain these comorbidities are likely to involve the hypothalamic–pituitary–adrenal (HPA) axis, a neuroendocrine signalling pathway with a critical role in hormone regulation ( Oyola & Handa 2017 ), whose dysregulation is involved in mood disorders ( Bao & Swaab 2019 ). When activated, the HPA axis causes the adrenal cortex to release the glucocorticoid, cortisol, making it the primary coordinator of the stress response. Chronic pain can lead to dysregulation of the HPA axis, and this has been demonstrated in a range of inflammatory conditions ( Kuehl  et al.  2010 ). A small study recently found an association between a dysregulated HPA axis and menstrual pain severity in endometriosis patients ( Ortiz  et al.  2020 ). In a study of 26 women with endometriosis and CPP, physical and psychological therapy normalised cortisol levels, reduced perceived stress, and improved physical functioning ( Friggi Sebe Petrelluzzi  et al.  2012 ). There is an increasing appreciation of the role of the stress response and cortisol in modulating the MGB axis at multiple levels, including gut function and composition of the gut microbiota. A recent comprehensive review on the connections between the HPA and MGB axis was published by  Rusch  et al.  (2023) .\n\nIn the following section, to provide a framework for considering the role of the gut microbiome and its metabolites in endometriosis, we provide a brief overview and references to some recent relevant papers.\nThe gut microbiota is the community of microorganisms, including bacteria, viruses, and archaea, residing in the GI tract. The bacterial community is dominated by the phyla Firmicutes and Bacteroidetes, which comprise approximately 90% of the total gut bacteria ( Sommer & Bäckhed 2013 ), with Fusobacteriota and Verrucomicrobiota present in low abundance ( Cryan  et al.  2019 ). Human gut microbiota have been broadly grouped into separate enterotypes, depending on levels of three specific genera: Bacteroides, Prevotella, and Ruminococcus. Differing enterotypes are associated with the consumption of certain diets: the Bacteroides and Prevotella enterotypes are associated with high-fat/high-protein diets and high-carbohydrate diets, respectively ( Cryan  et al.  2019 ).\nBacterial diversity can be differentiated by α- and β-diversity indices, with the former focussed on diversity within a single sample and the latter comparing population diversity between different samples ( Wagner  et al.  2018 ). Increased diversity is generally considered to be associated with improved health outcomes ( Valdes  et al.  2018 ). The gut microbiota plays essential roles in promoting gut health, including maintaining intestinal barrier function, and priming and maintenance of the immune system. The bacterial community regulates and activates both peripheral and resident immune cells, either via direct contact or compounds secreted through the mucus layer and gut epithelium, as well as signalling to the brain via nerve stimulation.\nThe MGB axis is a two-way communication pathway linking the CNS and gut bacteria. Gut bacteria produce an assortment of vital metabolites including: bile acids, short-chain fatty acids (SCFAs), hormones, and neurotransmitters; which can signal to the brain and regulate a range of functions throughout the body ( Liu  et al.  2022 ).\nSignalling between cells in the gut and brain is mediated via neural pathways involving the vagus nerve and the enteric/parasympathetic nervous system, as well as immunological and hormonal factors, including those contributing to the HPA axis (see the comprehensive review by ( Cryan  et al.  2019 )). Briefly, associations have been found between altered parasympathetic nerve activity, pain, and bacterial composition, including evidence from CNS disorders ( Wang & Kasper 2014 ). The production and utilisation of metabolic products, such as tryptophan and serotonin, by certain gut bacteria provide a secondary mechanism for their role in mood disorders via activation of the HPA axis ( O’Mahony  et al.  2015 ).\nGut microbiota also play a role in the maturation and maintenance of microglia, CNS-resident immune cells, which function in neuroinflammation and pain processing ( Erny  et al.  2015 ). Activation of these neuroimmune cells is considered one of the key mechanisms in central sensitisation due to the production of proinflammatory mediators, including IL-1β, interferon-γ, and TNF-α ( Guo  et al.  2019 ). These, amongst other cytokines and chemokines, disrupt the ratio of glutamate versus γ-aminobutyric acid (GABA) in synaptic transmission, leading to decreased pain thresholds ( Ustianowska  et al.  2022 ).\nSCFAs are an important product of bacterial metabolism, produced by certain bacterial species as a by-product of dietary fibre fermentation. They modulate the inflammatory status of the gut by regulating the immune response ( Liu  et al.  2022 ) and maintain the mucosal barrier by promoting the proliferation of intestinal epithelial cells ( Vinolo  et al.  2011 ). SCFAs act via two primary mechanisms: activation of G-protein-coupled receptors (GPCRs), GPR41 and GPR43, expressed on neutrophils and monocytes, and throughout the GI tract; and inhibition of histone deacetylases ( Tan  et al.  2014 ). SCFAs can promote peripheral Treg generation ( Arpaia  et al.  2013 ) and have been found to regulate neuroinflammation via the GPCR HCAR2 ( Boccella  et al.  2019 ), expressed during pain in the hypothalamus ( Li  et al.  2020 ).\nThe estrobolome is the collection of gut bacteria capable of altering the concentrations of bioactive steroids, including oestrogens, by enzymatic activities that cleave side chains from conjugated steroids ( Fig. 2A ). Examples include metabolism of oestrone-3-glucuronide and oestradiol-17-glucuronide, to oestrone (E1) and oestradiol (E2), respectively ( Ervin  et al.  2019 ). This has been further evidenced by a correlation between microbial diversity and higher E2 levels ( Shin  et al.  2019 ), with the bidirectionality of this relationship shown by the microbiota changes caused by ovariectomy ( O'Mahony  et al.  2017 ).\n Figure 2 (A) The estrobolome.Certain gut bacteria, including Bacteroides, Bifidobacterium, Escherichia, and Lactobacillus, are capable of impacting circulating oestrogen concentrations. These bacterial genera have β-glucuronidase and β-glucosidase activity, enzymes that deconjugate endogenous oestrogen and exogenous phytoestrogens in the gut ( Kwa  et al. , 2016 ). A higher prevalence of these bacteria, or increased activity of the enzymes, leads to increased concentration of biologically active oestrogen metabolites in circulation, which may influence cell proliferation or immune responses ( Symons  et al. , 2018 ). On the other hand, decreased activity will diminish circulating free oestrogen leading to increased excretion of conjugated parent oestrogens, oestrone (E1) and oestradiol (E2) ( Spichak  et al. , 2018 ). Created with BioRender.com. (B) Summary of potential symptom-management strategies via manipulation of the gut microbiome.A broad range of interventions have been trialled in endometriosis patients for their potential beneficial impact on symptoms. Different dietary modifications have been the most extensively researched strategies, with antibiotic and probiotic treatments currently in their infancy. *Trials conducted only in patients with IBS not endometriosis. Created with BioRender.com.\n(A) The estrobolome.Certain gut bacteria, including Bacteroides, Bifidobacterium, Escherichia, and Lactobacillus, are capable of impacting circulating oestrogen concentrations. These bacterial genera have β-glucuronidase and β-glucosidase activity, enzymes that deconjugate endogenous oestrogen and exogenous phytoestrogens in the gut ( Kwa  et al. , 2016 ). A higher prevalence of these bacteria, or increased activity of the enzymes, leads to increased concentration of biologically active oestrogen metabolites in circulation, which may influence cell proliferation or immune responses ( Symons  et al. , 2018 ). On the other hand, decreased activity will diminish circulating free oestrogen leading to increased excretion of conjugated parent oestrogens, oestrone (E1) and oestradiol (E2) ( Spichak  et al. , 2018 ). Created with BioRender.com. (B) Summary of potential symptom-management strategies via manipulation of the gut microbiome.A broad range of interventions have been trialled in endometriosis patients for their potential beneficial impact on symptoms. Different dietary modifications have been the most extensively researched strategies, with antibiotic and probiotic treatments currently in their infancy. *Trials conducted only in patients with IBS not endometriosis. Created with BioRender.com.\nGut dysbiosis, resulting from disturbances in normal microbiota communities, can be caused by many factors, such as stress, physical illness, antibiotics, and dietary changes ( Valdes  et al.  2018 ). Dysbiosis and inflammation can increase permeability of the intestinal barrier via weakening of the tight junctions, allowing movement of bacteria and pathogenic-associated molecular patterns (PAMPs) into circulation ( Gieryńska  et al.  2022 ). Subsequent recognition of PAMPs by TLRs triggers proinflammatory cytokine production, inducing activation of transcription factors, such as NF-κB ( Kawai & Akira 2010 ). The downstream effects of these pathways result in both local and systemic low-grade inflammation.\n\nAnimal models have been developed to simulate aspects of the aetiology and symptomology of endometriosis, including some using behavioural endpoints as a surrogate for pain ( Tejada  et al.  2023 ). The impact of the gut microbiota has been investigated using rodent models, complemented by studies in primates with naturally occurring endometriosis.\nTo date, the majority of studies have focussed on characterisation of gut microbiota following artificial induction of endometriosis ( Table 2A ). Unfortunately, the results reported in the different studies were not consistent, potentially due to the lack of standardised methods and outcome measures. Some studies reported positive effects of antibiotics ( Chadchan  et al.  2019 ), n-butyrate ( Chadchan  et al.  2021 ), or alpha-linolenic acid (ALA) ( Ni  et al.  2021 ), but no behavioural measurements were included, meaning it is impossible to assess whether there was any impact on pain.\n Table 2 Studies in animal models (A) and human studies (B) investigating the role of gut microbiota and metabolites on endometriosis disease progression. Findings Reference A) Animal models  Rhesus monkeys with naturally occurring endometriosis - Higher concentrations of gram-negative bacteria Bailey & Coe (2002) - Elevated levels of intestinal inflammation  Endometriotic mouse model – i.p. injection Findings 42 days after induction: Yuan  et al.  (2018)  - No significant differences in gut microbiota α-diversity  - Higher gut microbiota β-diversity  - Dysbiosis led to enriched Firmicutes  Endometriotic mouse model – surgical Findings 21 days after induction: Chadchan  et al.  (2019)  - Significantly lower gut microbiota α-diversity  - Higher abundance of Bacteroidetes and lower abundance of Firmicutes  - Microbiota depletion (MD) using broad-spectrum antibiotics significantly decreased the size of endometriotic lesions and the number of proliferative cells associated with a decrease in inflammatory markers  - MD followed by oral gavage with faeces from endometriotic, but not vehicle-treated, mice re-established lesion growth and inflammation  Endometriotic mouse model – surgical - No significant differences in gut microbiota α- or β-diversities after 7 and 21 days Hantschel  et al.  (2019)  Endometriotic rat model – surgical Findings 28 days after induction: Cao  et al.  (2020)  - Significantly lower gut microbiota α-diversity  - Higher abundance of Firmicutes and lower abundance of Bacteroidetes and Proteobacteria  - Gut microbiota β-diversity showed significant differences in species composition  Endometriotic mouse model – i.p. injection Findings 21 days after induction: Ni  et al.  (2020)  - Significantly lower gut microbiota α-diversity  - Increased abundance of Proteobacteria and decreased abundance of Firmicutes and Bacteroidetes  - Significantly increased abundance of  Akkermansia muciniphila  - Four differentially abundant metabolites identified: chenodeoxycholic acid, ursodeoxycholic acid, alpha-linolenic acid (ALA), and 12,13s-epoxy-9z,11,15z-octadecatrienoic acid (12,13-EOTrE)  Endometriotic mouse model – i.p. injection - Significant reduction in butyrate concentration in faeces Chadchan  et al.  (2021) - n-butyrate supplementation significantly decreased the size of endometriotic lesions and the number of proliferative cells and macrophages  Endometriotic mouse model – i.p. injection - Higher abundance of Firmicutes and lower abundance of Bacteroidetes Ni  et al.  (2021) - Supplementation with ALA restored the abundance of Firmicutes and Bacteroidetes, enhanced the intestinal barrier, and reduced levels of LPS and macrophages  Endometriotic olive baboon model – surgical - Significant differences in gut microbiota α- and β-diversities after 3 months, with α-diversity recovering by 15 months Le  et al.  (2022) - Changes in α-diversity positively correlated with circulating Treg populations  Endometriotic mouse model – surgical - Six differentially abundant metabolites identified: quinic acid; cytosine; 1-methyl-histidine;  N G , N G -dimethyl  l -arginine; 2-aminoheptanoic acid; and n-acetyl aspartic acid Chadchan  et al.  (2023) - Supplementation with quinic acid resulted in significantly larger endometriotic lesions  Endometriotic mouse model – i.p. injection - Significantly lower gut microbiota α-diversity at 28, but not 14, days after induction Wei  et al.  (2023) - Differences in β-diversity at 14 and 28 days - Significantly higher levels of LPS in PF - Injection of β-glucuronidase led to significant increases in: concentration of LPS in PF; number of macrophages; and number and size of endometrial lesions B) Clinical studies  Stage 3/4 endometriosis patients ( n  = 14) - No difference in gut microbiota α- and β-diversities Ata  et al.  (2019)  Endometriosis patients ( n  = 35) - No difference in gut microbiota α- and β-diversities Perrotta  et al.  (2020)  Endometriosis patients ( n  = 21) - Significantly reduced gut microbiota α- and β-diversities Huang  et al.  (2021)  Stage 3/4 endometriosis patients ( n  = 12) - Non-significantly reduced gut microbiota α-diversity compared to controls Shan  et al.  (2021) - Increased Firmicutes/Bacteroidetes ratio - Higher abundance of  Prevotella - Higher circulating levels of PGE2 and IL-8  Endometriosis patients ( n  = 66) - Significantly reduced gut microbiota α- and β-diversities Svensson  et al.  (2021) - Correlation between  Prevotella  abundance and GI-associated symptoms  Endometriosis patients ( n  = 35) - No difference in gut microbiota α- and β-diversities Wei  et al.  (2023) - Significantly higher serum levels of β-glucuronidase - Significantly increased β-glucuronidase expression in endometrial lesions compared to normal endometrium\nStudies in animal models (A) and human studies (B) investigating the role of gut microbiota and metabolites on endometriosis disease progression.\nHuman studies exploring the gut microbiota in endometriosis patients are also limited, with notable inconsistency in findings. For example, whilst some studies found no differences in bacterial diversity in endometriosis patients compared to controls, others identified significant changes ( Table 2B ). Interestingly, there was a correlation between the abundance of  Prevotella  and GI symptoms, but variations in genetic, dietary, and environmental factors limit interpretation/detection of disease-specific differences, making it difficult to draw robust conclusions.\n\nThe influence of the MGB axis on the immune system has been investigated in several chronic inflammatory pain conditions. In this section we consider data from studies on conditions and symptoms relevant to endometriosis.\nStudies in animal models have provided evidence to support the two-way connection between pain pathways and the gut microbiota ( Table 3 ). For example, germ-free mice lacking gut bacteria had increased visceral hypersensitivity, which was normalised following microbial re-colonisation ( Luczynski  et al.  2017 ). The phenotype was found to be transferable by faecal microbiota transfer (FMT) ( Yang  et al.  2019 ,  Lucarini  et al.  2022 ), and attenuated by treatment with antibiotics ( Aguilera  et al.  2021 ,  Ding  et al.  2021 ), potentially in an inflammasome-dependent manner ( Scuderi  et al.  2020 ,  Aguilera  et al.  2021 ). Taken together, these studies support a complex role involving the gut microbiota and immune interactions in pain responses.\n Table 3 Research studies in animal models investigating the relationship between the gut microbiota, pain, and inflammation. Model Findings Reference Mouse model of PI-IBS and PI-IBS treated with  Bifidobacterium longum - VH was significantly lower in  B. longum -treated mice compared to PI-IBS. Gu  et al.  (2016) - IL-18 and IL-1β expression were significantly lower in  B. longum -treated mice compared to PI-IBS. -  B. longum  may inhibit NLRP3 inflammasome. MD rat model (male) - Significantly increased VH but no significant difference in total pain scores. Hoban  et al.  (2016) - More depressive-like behaviours. - Decreased gut microbiota β-diversity. Germ-free mice - Significantly increased VH, which normalised following microbial colonisation. Luczynski  et al.  (2017) - Altered volume of pain-processing brain structures: smaller anterior cingulate cortex and larger periaqueductal grey. MS mouse model vs TLR4 KO-MS mouse model - Increased VH in MS but not in TLR4 KO-MS mice. Tang  et al.  (2017) - LPS-treatment induced VH in MS via TLR4. - VH blocked by inhibition of TLR4 signalling. Mouse model of alcohol-induced neuroinflammation (female) - Neuroinflammation and increased intestinal proinflammatory cytokines attenuated with Ab treatment. Lowe  et al.  (2018) - Ab-treatment increased mRNA expression of some inflammasome components. Rat model of spared nerve injury (male) - Depression-susceptible rats had significantly decreased gut microbiota α-diversity. Yang  et al.  (2019) - FMT to MD-mice transferred a painful phenotype and depression-like behaviours. Rat model of IBS - Inflammasome inhibition reduced NF-ΚB expression and inflammation, and restored IBS-associated tight junction alterations. Scuderi  et al.  (2020) Mouse model of inflammasome inhibition* - Increased abundance of Bifidobacterium in  Casp1  KO compared to WT. Aguilera  et al.  (2021) - Antibiotics reduced immune and inflammatory marker expression in  Casp1  KO but not WT. - Antibiotics reduced pain in WT but not  Casp1  KO. Mouse model of neuropathic pain † - Gut microbiota induced pain by influencing pro- and anti-inflammatory T cells. Ding  et al.  (2021) - Pain was attenuated by antibiotics. Rat model of colitis (male) - Unique bacterial profile and increased F/B ratio compared to controls. Lucarini  et al.  (2022) - FMT to healthy rats transferred VH. - FMT recipient rats had increased acetate but decreased butyrate. - FMT recipients had increased plasma IL-6 and TGF-β. * Casp1  knock-out;  † Chronic-constriction injury of sciatic nerve. Ab, antibiotic; F/B, firmicutes/bacteroidetes ratio; FMT, faecal microbiota transfer; IBS, irritable bowel syndrome; MD, microbiota-depleted; MS, maternal separation; PI, postinfectious; VF, visceral hypersensitivity.\nResearch studies in animal models investigating the relationship between the gut microbiota, pain, and inflammation.\n* Casp1  knock-out;  † Chronic-constriction injury of sciatic nerve.\nAb, antibiotic; F/B, firmicutes/bacteroidetes ratio; FMT, faecal microbiota transfer; IBS, irritable bowel syndrome; MD, microbiota-depleted; MS, maternal separation; PI, postinfectious; VF, visceral hypersensitivity.\nThe potential impact of estrobolome-contributing microbial populations on pain sensitivity has also been demonstrated in a recent study: visceral sensitivity fluctuated throughout the estrous cycle in wild-type mice and increased following a reduction in ovarian steroids as a consequence of ovariectomy. Notably, neither of these effects were seen in germ-free mice, suggesting one mechanism of microbial influence on pain occurs in an oestrogen-dependent manner ( Tramullas  et al.  2021 ).\nThe potential role of the gut microbiota in the symptomology of IBS has been researched in some depth in both patients and animal models ( Table 4 ). IBS patients are reported to have significant differences in microbial diversity compared to healthy controls. The inverse association between  Akkermansia muciniphila  and pain intensity ( Cruz-Aguliar  et al.  2019 ) is of interest due to its association with improved intestinal barrier function ( Cani & de Vos 2017 ). FMT has provided further supporting evidence, with transfer of the phenotype from patients to mice, and transfer from healthy donors to patients reducing symptoms and re-diversifying the gut microbiota. To date there are no reports of trials using FMT to treat endometriosis patients.\n Table 4 Research studies investigating the relationship between the gut microbiota and gastrointestinal symptoms. Model/cohort Findings Reference IBS patients - Microbial signatures clustered into two groups: normal microbiota vs. increased F/B ratio. Jeffery  et al.  (2012) - IBS with normal microbiota were more likely to have depression. - Suggests potential differing triggers for IBS-like symptoms. IBS patients and MD mouse model (male) - FMT from IBS patients to MD-mice transferred phenotype. Ge  et al.  (2017) - SCFAs and secondary bile acids were subsequently decreased in recipient mice. IBS patients and GF mouse model - FMT from IBS patients to GF-mice transferred phenotype including intestinal barrier dysfunction, innate immune activation, and anxiety-like behaviour. De Palma  et al.  (2017) IBS patients and healthy donor - FMT from healthy donor to IBS patients reduced abdominal pain symptoms and increased α- and β-diversities. Cruz-Aguliar  et al.  (2019) - Patients with microbiota mostly like the donor had the greatest reduction in pain. - Abundance of  Akkermansia muciniphila  inversely correlated with pain intensity. Review – multiple cohorts - Potential influence of  Lactobacillaceae  and  Bacteroides  metabolites on inflammation and bloating. Pittayanon  et al.  (2019) IBS patients - Significant differences in microbiome and metabolome profiles compared to controls. Jeffery  et al.  (2020) - Faecal metabolomes could differentiate IBS patients with and without bile acid malabsorption. - Decreased bacterial diversity. Meta-analysis of IBS patients - FMT from healthy donors significantly decreased IBS symptoms and improved QoL. Wang  et al.  (2023) Meta-analysis of IBS patients - FMT from healthy donors significantly decreased IBS symptoms but decreased QoL. Halkjær  et al.  (2023) Review of IBS patients - Increased F/B ratio in IBS. Shaikh  et al.  (2023) - No specific microbial signature. FMT, faecal microbiota transfer; IBS, irritable bowel syndrome; SCFA, short-chain fatty acid; QoL, quality of life.\nResearch studies investigating the relationship between the gut microbiota and gastrointestinal symptoms.\nFMT, faecal microbiota transfer; IBS, irritable bowel syndrome; SCFA, short-chain fatty acid; QoL, quality of life.\nComorbidity of GI issues and mood disorders is common. However, clinical evidence to support the role of the MGB axis remains limited. Similar to other areas of gut–brain research, differences in methodology and outcome measures have created challenges when comparing data. Although several studies have identified differences in both α- and β-diversities in the gut microbiota of people with depression and anxiety compared to controls, these findings are not consistent ( Simpson  et al.  2021 ). Additionally, there were no uniform findings in the differing abundance of specific bacterial species associated with neither depression nor anxiety ( Simpson  et al.  2021 ).\n\nClinical trials of dietary intervention for disease management are challenging to implement and standardise, with a plethora of variables likely to affect the outcomes ( Nap & De Roos 2022 ). Currently, most research into associations between diet and endometriosis is focussed on risk of disease development, rather than adapting diet for symptom-management ( Nap & De Roos 2022 ). However, there is anecdotal evidence within the endometriosis community for the benefit of dietary modifications as a self-management strategy and some preliminary clinical evidence to support these ideas. Diets (or specific foods) believed to increase bacterial diversity and growth of bacterial species associated with good health are often referred to as ‘prebiotics’, examples include diets rich in fibre and fermented foods ( Valdes  et al.  2018 ). Health benefits of these diets have been described for various conditions, including IBS ( Salmeri  et al.  2023 ); however, to date there has been no comprehensive randomised control trial (RCT) in endometriosis patients.\nMore generally, other research into the impact of Western diet, including the increased consumption of ultra-processed foods, is gaining momentum, with reports of associations with increased low-grade inflammation ( Tristan Asensi  et al.  2023 ). There is concern that diets high in ultra-processed foods may exacerbate symptoms in those with existing chronic inflammatory conditions, which may also include endometriosis.\nRecent surveys investigating the popularity of different diets used by people with endometriosis, and perceived effects on symptoms and QoL, have reported that, although no single dietary intervention appeared to be uniquely effective, many respondents found their chosen modification to be beneficial ( Krabbenborg  et al.  2021 ,  Armour  et al.  2021 ). In an Australian survey, 163 respondents had used dietary intervention, 69.0% of whom reported a reduction in the use of pharmaceutical medication ( Armour  et al.  2021 ). In a Dutch study, 55.5% of the 157 respondents reported nutrition affecting their symptoms and 46.5% were currently following a diet ( Krabbenborg  et al.  2021 ). These surveys were consistent in finding gluten -free, dairy -free/low lactose, and low fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs) as the most popular diets, although both studies are caveated by their relatively small participant numbers and limited geographical reach. The largest survey to date received 1385 responses, predominantly from the UK, of which 52.2% had tried adapting their diet to manage their endometriosis-associated gut symptoms ( Deepak Kumar  et al.  2023 ). Again, gluten-free was one of the most popular diets; however, only 0.6% were following a low-FODMAP diet, highlighting the strength of a larger dataset.\nFODMAPs are fermentable oligosaccharides, disaccharides, monosaccharides and polyols, which in high doses can cause inflammation and visceral pain ( Zhou  et al.  2017 ). A low-FODMAP diet is popular for management of IBS, with symptoms shown to improve after three weeks, alongside a reduction in serum levels of proinflammatory cytokines ( Hustoft  et al.  2017 ). However, long-term use of the low-FODMAP diet may have a negative impact on the gut microbiome ( Staudacher 2017 ). One culprit for inflammatory responses to certain foods is histamine, released by mast cells present in the gut mucosa and further stimulated in a positive feedback loop with oestrogen ( Theoharides 2017 ). Three weeks on a low-FODMAP diet ( n  = 19) was shown to reduce histamine levels eight-fold, in comparison to a high-FODMAP diet ( n  = 18) ( McIntosh  et al.  2017 ). Histamine mast cells express oestrogen receptors ( De Leo  et al.  2017 ) and have been implicated in both pathogenesis and pain mechanisms of endometriosis ( Kirchhoff  et al.  2012 ,  Mariuzzi  et al.  2016 ), providing a mechanistic link between mast cell activation and intestinal inflammation.\nIn a study of 160 women, those with both endometriosis and IBS were three-fold more likely to find a low-FODMAP diet effective for improving symptoms, compared to those with IBS alone ( Moore  et al.  2017 ). This could suggest the cause of IBS-type symptoms frequently reported by endometriosis patients may differ from those with IBS alone and be more receptive to dietary intervention.\nA gluten-free diet is frequently adopted by people with endometriosis; however, there is currently no clinical evidence to support this practice. There has been one retrospective observational study of women with endometriosis who followed a gluten-free diet for 12 months, 75% of whom reported significant pain improvement ( Marziali  et al.  2012 ). However, 88 of the original 295 participants withdrew within two weeks due to associated abdominal side effects.\nAnti-inflammatory diets, such as the typical Mediterranean diet, consisting of fruit, vegetables, whole grains, and oily fish, with low quantities of dairy and red meat, have been proven to decrease inflammatory markers including IL-6 and C-reactive protein ( Tristan Asensi  et al.  2023 ). Five months on a Mediterranean diet was found to significantly improve pain in 68 women with endometriosis; however, the study had no control group ( Ott  et al.  2012 ). A diet high in fermented foods has been shown to increase microbial diversity and decrease inflammatory markers in 18 healthy adults ( Wastyk  et al.  2021 ).\nSignorile  et al.  compared 3 months of an anti-inflammatory dietary supplement, a linseed oil/calcium salt combination, or a placebo, with 30 endometriosis patients in each group ( Signorile  et al.  2018 ). However, all participants also increased their fibre and omega-3 consumption and cut out soy, aloe, and oats. There was a significant decrease in reported pain symptoms associated with the anti-inflammatory supplement and a significant reduction in serum inflammatory markers (PGE2, CA-125). However, the potential impact of the dietary regime is unclear. A small double-blind RCT compared endometriosis patients taking an eight-week supplement of omega-3 ( n  = 17) versus olive oil ( n  = 16) ( Abokhrais  et al.  2020 ). Improvements in pelvic pain and QoL scores were seen in both arms; however, there were no significant differences. The use of olive oil in the control arm may explain the results as it has endogenous anti-inflammatory properties ( Cicerale  et al.  2012 ), ( Cicerale  et al.  2012 ); therefore, a larger trial is now required with an alternative placebo. Another study also found no benefits of omega-3 over 6 months when comparing fish oil ( n  = 20) to a placebo ( n  = 22) ( Nodler  et al.  2020 ). Taken together, these data make it difficult to say whether omega-3 supplementation is beneficial.\nAn earlier study looked at postoperative pain in endometriosis comparing 6 months of hormonal therapy ( n  = 77), dietary therapy ( n  = 35), or placebo ( n  = 110) ( Sesti  et al.  2007 ). At the 12-month follow-up, all groups reported lower scores for menstrual pain compared to baseline, though these were significantly lower with hormonal, but not dietary, therapy when compared to placebo. On the other hand, following both therapies, non-menstrual pelvic pain was significantly lower than placebo.\nA broad range of vitamins, minerals, and nutritional supplements have been associated with inflammation and immunity, a few of which have been investigated for their potential benefits in endometriosis. Vitamins are important for the normal functioning of the immune system, as well as having antioxidant and anti-inflammatory properties ( Carr & Maggini 2017 ,  Lewis  et al.  2019 ). The vitamin D receptor is expressed in reproductive tissues, leading to suggestions it may be involved in the aetiology of endometriosis ( Barnard  et al.  2023 ). A recent online survey with 1385 respondents found 381 (27.5%) took a vitamin D supplement ( Deepak Kumar  et al.  2023 ).\nOne study compared 12 weeks of vitamin D ( n  = 19) to a placebo ( n  = 19) and found no difference in reported pelvic pain or dysmenorrhea ( Almassinokiani  et al.  2016 ). Conversely, another paper reported 12 weeks of vitamin D treatment resulted in significantly decreased pelvic pain, compared to a placebo ( n  = 25 each group) ( Mehdizadehkashi  et al.  2021 ). A trial by Nodler  et al. , comparing vitamin D ( n  = 27), fish oil ( n  = 20), and a placebo ( n  = 22), found a significant reduction in ‘worst pain’ associated with vitamin D compared to the other groups ( Nodler  et al.  2020 ). This study recruited adolescent girls with a mean age of 19.7 – lower than the mean ages of 29.9 and 35.2 in the other two studies. These methodological differences, alongside the small participant numbers, provide a potential explanation for their inconsistency. Other vitamins have also shown promise: women receiving a combination of vitamins C and E ( n  = 30) had significantly lower pain scores for dysmenorrhea, dyspareunia, and CPP after 8 weeks, compared to a placebo ( n  = 30) ( Amini  et al.  2021 ).\nCurcumin is the active ingredient of turmeric with recognised anti-inflammatory properties ( Tabrizi  et al.  2019 ). Studies into its use in several health conditions have found reductions in oestrogen concentrations and proinflammatory mediators, as well as inhibition of angiogenesis ( Piecuch  et al.  2022 ). Following 2 months of daily curcumin supplementation, 33 women with endometriosis experienced significant improvements in pelvic pain, dysmenorrhea, and dyspareunia, with a 48% reduction in the number of participants using nonsteroidal anti-inflammatory drugs ( Fadin  et al.  2020 ).\nProbiotic treatment, based on ingestion of specific strain(s) of ‘beneficial’ bacteria, is still a relatively new field, with only a few strains available due to culturing and shelf-life constraints. Furthermore, the complex and diverse nature of the gut microbiome means there is still doubt as to whether the added presence of select strains in the form of supplement probiotics can have a significant impact on dysbiosis, considering their unique and sometimes temporary effects on the gut microbiome ( Leeming  et al.  2019 ).\nTwo RCTs have been conducted using probiotics to treat endometriosis. For 8 weeks, 16 women with endometriosis were given a combination of four different Lactobacillus strains:  Lactobacillus acidophilus; Lactobacillus plantarum; Lactobacillus fermentum;  and  Lactobacillus gasseri ; compared to a placebo ( n  = 16) ( Khodaverdi  et al.  2019 ). Both groups saw decreases in pain scores for CPP and dyspareunia, and for dysmenorrhea the change was significantly greater in the treatment arm. However, all pain scores had increased by the four-week follow-up which, though they had not reverted to baseline, suggests a potential lack of longevity for the probiotic combination.\nIn the second study, 29 women were treated with  L. gasseri  for 12 weeks and experienced a significant reduction in pain scores compared to placebo ( n  = 33) ( Itoh  et al.  2011 b \n ). However, there were no follow-up data and therefore no indication of the long-term impact of the probiotic. The influence of  L. gasseri  on endometriosis has been investigated in rodent models, with an apparent reduction in lesion growth and activation of NK cells ( Itoh  et al.  2011 a \n ,  Uchida and Kobayashi 2013 ). These data suggest the necessity for additional larger RCTs to investigate the use of  L. gasseri  as a treatment, with a focus on duration of response and the impact of repeated courses of supplementation.\nThe use of probiotics as a treatment strategy for IBS has been well documented, with a variety of different strains improving symptom severity, including pain and bloating ( Cryan  et al.  2019 ,  Francavilla  et al.  2019 ,  Wilmes  et al.  2021 ). Studies in animal models have demonstrated the alleviation of visceral pain following probiotic treatment ( Zhao  et al.  2018 ,  Zhang  et al.  2019 ,  Li  et al.  2019 ), though this was not always replicated ( Huang  et al.  2019 ). One study of 118 IBS patients showed treatment with  L. gasseri  reduced the mean abdominal pain score by 54.2% and attenuated symptoms in 85.0% of participants ( Ait Abdellah  et al.  2022 ). However, this was not placebo controlled.\nResearch into the use of probiotics to treat mood disorders provides inconsistent findings ( Taylor and Holscher 2020 ), with varying strains and methodologies used, meaning accurate comparisons are difficult.  Lactobacillus rhamnosus  reduced depression- and anxiety-like behaviour in mice ( Bravo  et al.  2011 ). Interestingly, in a double-blind RCT, pregnant women treated with  L. rhamnosus  ( n  = 212) reported significantly lower post-partum depression and anxiety scores compared to controls ( n  = 211) ( Slykerman  et al.  2017 ).\nAntibiotics have a strong influence on the gut, and their use in early life has been shown to have detrimental effects on the gut microbiota in adulthood ( O’Mahony  et al.  2014 ). Nonetheless, the preliminary research discussed below raises the potential that symptoms of endometriosis might be treated with antibiotics.\nThe theoretical benefits for treating endometriosis with antibiotics are multifaceted: perturbation of the gut microbiota could improve pain perception and mood disorders by altering signalling within the MGB axis and even influence disease progression if it blunted the immune response and/or reduced deconjugation of steroids by the estrobolome. Additionally, if bacterial infection is proven to have a causal role in lesion development ( Khan  et al.  2018 ), then specific antibiotics could provide a defence mechanism against further lesion growth. Some of the studies reviewed below provide support for both these lines of enquiry.\nThe effect of antibiotics on endometriosis was investigated using an endometriotic mouse model treated with a combination of vancomycin, neomycin, metronidazole, and ampicillin for 3 weeks. Antibiotic-treated mice had smaller lesions with fewer proliferative cells and lower concentrations of proinflammatory cytokines, compared to vehicle-treated controls. However, the gut microbiota in the antibiotic-treated mice had decreased α- and β-diversity, dominated by the phylum Proteobacteria, with negligible abundance of Bacteroidetes and Firmicutes ( Chadchan  et al.  2019 ). Further analysis of individual treatments with metronidazole or neomycin identified only the former as able to reduce lesion growth ( Chadchan  et al.  2019 ). The authors suggested this was due to susceptibility of the Bacteroides genus to metronidazole but not neomycin. Importantly, neomycin is a nonabsorbable antibiotic, meaning its influence is restricted to the gut, whereas metronidazole can move into circulation and even interact with the CNS. Therefore, the explanation for these differences could be a result of metronidazole having activities outside the gut.\nIn a recent study of ovarian endometriosis patients, Muraoka  et al.  reported 64% had  Fusobacterium nucleatum  in their endometrium compared to 7% of controls ( n  = 42 each group) ( Muraoka  et al.  2023 ). They also used a mouse model of endometriosis combined with injection of  F. nucleatum  and tested the impact of both metronidazole and chloramphenicol. Presence of the bacteria increased lesion size, whereas treatment with antibiotics largely prevented lesion formation and reduced the size of established lesions ( Muraoka  et al.  2023 ). This appeared to be due to activation of TGF-β1 signalling by the bacteria. Whilst these are new data that must be replicated by others, they do provide strong evidence that antibiotic treatment might be beneficial in some patients.\nTo date, there has only been one clinical trial investigating the impact of antibiotics on endometriosis patients. In a double-blind RCT, women with stage III/IV endometriosis found 6 months of broad-spectrum antibiotic, clarithromycin ( n  = 129), was no more effective than a placebo ( n  = 160) for reducing pain after surgical removal of lesions ( Alborzi  et al.  2019 ). Additionally, there was no difference in serum levels of inflammatory biomarkers, including TNF-α, between the two groups.\nAntibiotics, such as rifaximin, improve symptom severity in patients with IBS ( Vicari  et al.  2017 ) and decrease visceral pain in animal models ( Aguilera  et al.  2015 ,  Hoban  et al.  2016 ). Research into the use of antibiotics for chronic pain with an unknown aetiology is limited, although a recent study into chronic lower back pain found no clinical effect following treatment with amoxicillin ( Bråten  et al.  2019 ).\n\nThere is increasing, and robust, evidence that the gut microbiota and its metabolites play a key role in the bidirectional signalling pathway between the gut and brain, that can regulate pain, GI symptoms and mood disorders. As these symptoms are common in patients with endometriosis, there is increasing interest in exploring the contribution of the gut microbiota to the manifestation and exacerbation of symptoms, and subsequently whether the use of diet, supplements, probiotics, or antibiotics, all of which may alter the bacterial species in an individual’s microbiome, could be used to improve symptoms and QoL.\nWhilst data from endometriosis patients are limited, a large body of work on other chronic conditions has highlighted the impact of the microbiome on mechanisms known to be involved in aetiology, pathogenesis, and symptoms associated with endometriosis. These include immune education and regulation; biosynthesis of bacterial metabolites that interact with immune cells and nerves (enteric and CNS); and steroid metabolism/activation ( Cryan  et al.  2019 ,  Shin  et al.  2019 ,  Liu  et al.  2022 ,  Gieryńska  et al.  2022 ).\nIf we are to realise the full potential of the MGB axis as a therapeutic target in endometriosis, it will be essential to develop standardised experimental methodology and to undertake large, well-controlled clinical trials, including careful phenotyping of patients regarding diet, symptoms, and disease stage, complemented by an in-depth analysis of microbial diversity, plus inflammatory and metabolic profiling, allowing comparisons to be made between international cohorts – a technique that has led to breakthroughs in the genetics of endometriosis ( Saunders and Horne 2023 ).\nThe benefits of large-scale studies on the MGB would be two-fold. They could offer an opportunity to develop a microbial/biomarker profile that could be used to advise patients on personalised self-management strategies, such as the use of diet and probiotics, alongside pharmaceutical and surgical approaches ( Fig. 2B ). Secondly, if studies on the putative role of bacterial infection in disease progression can be replicated in diverse populations, this could provide a rationale for testing antibiotic treatments for endometriosis. However, these must be approached with caution as antibiotics may also upset the balance of beneficial versus dysbiotic resident gut microbiota.\nTo summarise, the impact of the gut microbiota on both the aetiology and symptomology of endometriosis is a rapidly expanding field, with some promising avenues for future research focussed on its manipulation to improve patients’ QoL.\n\nFHY, PTKS, and SO have no conflicting interests. AWH is a Co-Editor-in-Chief of  Reproduction and Fertility . AWH was not involved in the review or editorial process for this paper, on which he is listed as an author. AWH’s institution (The University of Edinburgh) has received payment for consultancy and grant funding from Roche Diagnostics to assist in the early development of a possible blood diagnostic biomarker for endometriosis. AWH’s institution has received payment for consultancy fees from Gesynta and Joii. AWH has received payment for a presentation from Theramex. AWH’s institution has received grant funding from the MRC, NIHR, CSO, and Wellbeing of Women for endometriosis research. AWH is listed as a co-inventor on a UK Patent Application (No. 2217921.2).\n\nArticle conception, FHY, AWH, and PTKS; literature survey and writing, FHY; editing and reviewing, FHY, AWH, PTKS, and SO; supervision, AWH, PTKS, and SO.","source_license":"CC-BY-4.0","license_restricted":false}