{"paper_id":"3a6c502a-c9b1-4062-899c-dd4872c2bb56","body_text":"Microbial dysbiosis and disease pathogenesis of endometriosis, could there\nbe a link?\nJessica Puca1 and Gerard F. Hoyne2,3,4,5*\n1School of Arts and Sciences, University of Notre Dame Australia, Fremantle, Western Australia, Australia\n2School of Health Sciences, University of Notre Dame Australia, Fremantle, Western Australia, Australia\n3Institute of Health Research, University of Notre Dame Australia, Western Australia, Australia\n4Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology and Harry Perkins\nInstitute of Medical Research, University of Western Australia, Nedlands, Australia\n5Institute for Respiratory Health, Centre for Respiratory Health, School of Medicine and Pharmacology, University of\nWestern Australia, Nedlands, Australia\nAbstract\nEndometriosis is an estrogen-dependent inflammatory condition in women that is characterised by the\nectopic growth of endometrial glands and stroma outside of the uterine cavity. Although there exists\nmany theories for the pathogenesis of endometriosis, none has been successively confirmed as a direct\ncause for disease development. The human body comprises a diverse microflora across all tissues that\ncan have fundamental roles in health and disease. The microbial flora in a healthy individual can vary\nremarkably between anatomical sites due to the physical and chemical properties of specific tissues.\nThis includes the female reproductive tract, notably the vagina, which harbors a microbiota\ndominated by Lactobacilli species. In addition, a core unique microbiome has been defined for the\nendometrium that also includes Lactobacilli spp. In this review we examine the possibility that\nendometriosis could result from microbial dysbiosis, whereby significant changes to the natural\nmicroflora within the endometrium could reduce mucosal immune regulation in this tissue with\nconcomitant expansion of pathogenic bacteria that trigger local tissue inflammation that could\nperpetuate the development of endometrial disease.\nKeywords: Endometriosis, Retrograde menstruation, Lactobacilli spp., Microbial dysbiosis,Gynaecological diseases.\nAccepted on 28 December, 2016\nIntroduction\nEndometriosis is a disorder characterised by benign, ectopic\ngrowth of estrogen-dependent endometrial tissue outside of the\nuterine cavity, commonly in the pelvic region. The prevalence\nof endometriosis is more frequent in women of reproductive\nage and those who exhibit pelvic pain and infertility.\nEndometriosis seems to be most common in women aged\naround 25 to 45 years [1-4]. Moreover, 45 to 49% of women\nwho presented with pelvic pain, 33% with dysmenorrhea and\n42% of women aged between 25 to 34 years with infertility had\nendometriosis [2,5]. The disorder is generally associated with\ndysmenorrhoea (painful menstruation), dyspareunia (painful\nsexual intercourse) dysuria (painful urination), pelvic pain and\ninfertility [6-9]. In addition, risk factors for endometriosis\ninclude early menarche and late menopause, short menstrual\ncycle and heavy menstrual bleeding, along with prolonged\nexposure to endogenous estrogen and exposure to chemicals\nthat disrupt normal endocrine homeostasis within the\nreproductive tract [7,9,10].\nEndometriosis can be classified based on the anatomical\nlocation of lesions and severity and this can assist clinicians\nwith sequential treatment and management of the condition.\nDiagnostic methods for the early identification of\nendometriosis are still lacking which means that clinicians must\nrely heavily on invasive surgical procedures for confirmation of\nthe disease [11,12]. In addition, there a few treatment and\nmanagement options for patients with endometriosis, and\nsurgical intervention remains the main option for most patients.\nCurative treatments for the disease are absent and this is due\nmainly to a poor understanding of the cellular and molecular\nbasis of disease pathogenesis.\nSeveral studies have attempted to measure the impact of\nendometriosis on the quality of life of affected women and the\nsubsequent cost across countries and ethnicities [13-20]. These\nstudies reveal that the disease bears a significant social,\nphysical, psychological and economic burden on those affected,\ngiven that it negatively impacts an affected woman’s health\nrelated quality of life, reproductive capacity and work\nproductivity. Severe pelvic pain is the predominant contributor\nto a loss of work productivity among affected women and this\ncan greatly impede other daily activities [19]. Endometriosis\nthus can have a significant negative impact on a woman’s life\nin a multi-factorial manner which urges a closer examination of\nthe pathogenesis of the disease.\nReview http://www.alliedacademies.org/allied-journal-of-medical-research/\nAllied J Med Res 2017 Volume 1 Issue 11\n\nSeveral theories have been proposed for the pathogenesis of\nendometriosis, however the contribution of microbial dysbiosis\nto the development of the disease has been poorly examined. It\nis now understood that the human body has an extensive\nmicrobial flora which is established early in life [21] and each\ntissue displays a unique microbial flora which is determined by\nboth the physical and chemical properties of the individual\ntissue [22-24]. Scientists have discovered that the normal\nmicroflora can have direct health benefits to the host and if the\nbalance between healthy bacteria and pathogens ensues (i.e.\nmicrobial dysbiosis), this can have a direct impact on disease\npathogenesis [21]. In addition, the role of the microbiota has\nextended from the gut and the skin which are the two major\nmucosal sites of microbial inhabitation. It is now apparent that\nthe microbiota plays an important role in both health and\ndisease in humans and can impact on various body tissues, to\nthe extent that is has been implicated in diseases such as type 2\ndiabetes [28], autoimmune diseases like rheumatoid arthritis\nand multiple sclerosis [29,30] and for metabolic diseases such\nas kwashiorkor [21]. This review will provide an overview of\nsome recent studies which have examined the makeup of the\nmicrobial flora of the female reproductive tract but also\nexplore how microbial dysbiosis could provide a link to the\ndevelopment of endometriosis.\nTable 1. Current theories on the pathogensis of endometrosis.\nTheory Proposed mechanism of action References\nRetrograde\nmenstruation\nReflux of endometrial tissue and cells\nthrough the fallopian tubes to the ovaries. [31]\nStem cell\nimplantation\nSomatic stem cells: epithelial progenitor cells\n(eEPC) and endometrial mesenchymal stem\ncell (eMSP) populations undergo retrograde\nmigration into the peritoneum, because of\ncervical obstruction by mucus plug. After\nmigration, they lie dormant until menarche,\nwhen estrogen levels rise and stimulate the\ngrowth of endometriosis. [94,95]\nCoelomic\nmetaplasia\nStrongly accepted for the pathogenesis of\novarian endometriosis. Here metaplastic\nchange occurs to the coelomic epithelium,\ncovering the ovary and the serosa of the\nperitoneum, such that peritoneal tissue\ntransforms into endometrial-like tissue. [96]\nMüllerian\nremnant\nabnormalities\nAbnormal differentiation or migration of the\nembryonic Müllerian ducts (which develop\ninto the uterus, fallopian tubes and upper\nvagina) cause cells to spread to atypical\npelvic locations, particularly the uterosacral\nligaments and pouch of Douglas. [97-99]\nDisease Pathogenesis\nEndometriosis is identified as a complex disease given that it\nlacks a clear process of disease pathogenesis, which\nsubsequently impedes on diagnosis and treatment. The most-\nwidely supported theory for disease pathogenesis of\nendometriosis is that of retrograde menstruation (Table 1) [31].\nThis theory supports the notion that during the normal process\nof menstruation, there is a reflux of endometrial tissue and\ncells through the fallopian tubes to the ovaries, where it\nsubsequently enters into the peritoneal cavity and grafts\nectopically to genitourinary tissue in the peritoneal cavity.\nBurney and Giudice [32] explained that menstrual blood is\nquite commonly found in the peritoneal fluid of healthy\nwomen and this can be a common occurrence in adolescent\ngirls with congenital outflow obstruction. Moreover, retrograde\nmenstruation has been induced in non-human primates, the\nPapio anubis baboon, through supracervical ligation, which\nresulted in histologically-confirmed endometriosis [33,34].\nHowever, in challenge to the retrograde flow theory, it is\nobserved that approximately 90% of women are known to\nexhibit retrograde menstruation, whilst only 15% of women\nhave endometriosis [9]. This implies that there are other factors\nthat contribute to the pathogenesis of the disease.\nThus, given that the retrograde menstruation theory is not\nconclusive, many other theories have been hypothesised. These\ninclude the stem cell implantation theory, the coelomic\nmetaplasia theory and the Müllerian remnant abnormalities\ntheory summarised in Table 1. Although these theories are\nsupported to a degree by scientific evidence, they lack an\nabsolute association to the development of endometriosis.\nDespite these proposed theories, a clear definite pathogenesis\nof endometriosis has yet to be established.\nEndometriosis is characterised as an inflammatory condition,\ngiven that the peritoneal fluid of women with the disease has a\nheightened number of activated macrophages, as established\nthrough immune-histochemical analysis of endometrial tissue,\nplasma and peritoneal fluid among women with and without\nendometriosis [35]. Further associated to the inflammatory-\nstate of endometriosis is an increase in a range of soluble\nmediators including:\nChemokines: Macrophage inhibitory factor (MIF), MCP-1,\nRANTES [32,36]\nProinflammatory cytokines: TNF- α, IL-6, IL-1 β, INF- γ IL-8,\nIL-9, IL-17 [37,38].\nGrowth factors: Platelet-derived growth factor (PDGF), nerve\ngrowth factor (NGF) and fibroblast growth factor (FGF), also\nangiogenic and neurogenic factors, G-CSF [38].\nIncreased nuclear factor kappa beta (NF-kB) activation has\nbeen observed in peritoneal macrophages and peritoneal\nendometriotic lesions of patients with endometriosis resulting\nin up-regulation of inflammation and cell proliferation and\ndown-regulation of endometrial cell apoptosis [39-41].\nGenome-wide association studies have established certain\nsingle nucleotide polymorphisms (SNPs) associated with the\ndisease, namely those found on chromosomes near Wnt4,\nGreb1, Vezt and Kdr genes as summarised in Table 2 [42-50].\nHow these putative susceptibility genes impact on the\nestablishment of ectopic tissue growth or on the immune\ninflammatory responses within the affected individual is\ncurrently not understood. With significant correlations found\nbetween SNPs and endometriosis, genetic factors are regarded\nas important contributors to the development of the disease.\nAnimal Models of Endometriosis\nOne of the major barriers to understanding the cellular,\nmolecular and genetic basis of disease pathogenesis for\nCitation: Puca J, Hoyne GF . Microbial dysbiosis and disease pathogenesis of endometriosis, could there be a link?. Allied J Med Res\n2017;1(1):1-9.\n2Allied J Med Res 2017 Volume 1 Issue 1\n\nendometriosis is the lack of a suitable animal model. Non-\nhuman primates have been used extensively as a model of the\ndisease and as preclinical models, due to their spontaneous\ndevelopment of endometriosis. Moreover, endometriosis can\nalso be established in non-human primates through the\ninduction of retrograde menstruation [51]. Non-human\nprimates are considered the most suitable model for the study\nof endometriosis, yet there are ethical and high-cost limitations\nthat limit their use. Murine models offer an alternative for the\nstudy of endometriosis as they are more cost-effective and\neasily maintained. However, mice are unable to develop\nendometriosis spontaneously as they lack the ability to\nmenstruate.\nTable 2. Single nucleotide polymorphisms (SNPS) in genes associated\nwith endometrosis.\nGene Role\nSNPs in\nendometrio\nsis\nRole in\nendometriosis\nWNT4\nA ligand of the Wnt signalling\npathway. Associated with the\nnormal development of the\nfemale reproductive tract,\nfollicular development,\nsteroidogenesis and with\nendometrium proliferation,\ndecidualisation and\nimplantation.\nrs16826658\n(noncoding)\nrs3820282\n(noncoding)\nWNT4 protein\nexpression\ndownregulated\nGREB1-\nGrowth\nRegulatio\nn by\nEstrogen\nin Breast\nCancer 1\nEncodes a protein that is a co-\nactivator of estrogen receptor- α\n(ER-α) transcription factor.\nPromotes estrogen induced\ngrowth.\nrs13394619\n(noncoding)\nrs1898003\n(noncoding)\nrs11674184\n(noncoding)\nrs1865574\n(noncoding)\nrs2884374\n(noncoding)\nIncreased gene\nexpression in\nectopic\nendometrial tissue\nVEZT\nEncodes the adherens junction\ntransmembrane protein vezatin\nthat plays a role in cell-cell\nadhesion during\nembryogenesis. The protein can\nalso enter the nucleus and\nregulate the expression of\ntarget genes for cell adhesion\nand invasion. It has also been\nsuggested as a tumour\nsuppressive gene.\nrs10859871\n(noncoding)\nIncreased VEZT\nprotein expression\nin blood and\nendometrium\nKDR-\nKinase\nInsert\nDomain\nReceptor\nEncodes vascular endothelial\ngrowth factor (VEGF) receptor\n2. This protein is the main\nsignal transducer in the VEGF/\nVEGF receptor signaling\npathway, responsible for\ninducing angiogenesis.\nrs17773813\n(noncoding)\nIncreased\nexpression of\nVEGF receptor 2\nin blood vessels in\nendometrium\nIn order to replicate the disease in mice, recipient endometrial\ntissue must be introduced into mice, either from syngeneic\nanimals or through xenogeneic donor tissue [51,52]. However,\nrecent discoveries have identified the spiny mouse ( Acomys\ncahirinus) as the first rodent species known to menstruate\nspontaneously, with subsequent cyclic endometrial shedding\nand repair [53]. This provides a more suitable, yet still\naccessible and cost-effective murine model for the future study\nof endometriosis. A significant limitation with the spiny mouse\nstrain is that it is an outbred strain which would limit the\ncapacity to transfer cells or tissues between the spiny mouse\nand other inbred strains. The development of gene editing\ntechnology through the CRISPR-Cas9 system would offer one\npotential method by which specific gene mutations could be\nintroduced into the spiny mouse strain and the mutations could\nbe evaluated for their ability to induce endometriosis and its\nimpact on innate and adaptive immune responses in vivo.\nHost Microbiota\nThe healthy human body comprises of a unique, diverse and\nrelatively stable habitation of microorganisms (bacteria,\neukaryotes, archaea and viruses), whose symbiotic relationship\nwith the host contributes to general health and wellbeing.\nThese microorganisms that reside in and on the human body\nare collectively termed ‘microbiota,’ with their assembled\ngenomic sequences termed ‘microbiome’. The importance of\nmicrobiota to human health and physiology is essential, so\nmuch so that an individual’s collective microbial community\nhas previously been regarded as a ‘neglected’ and ‘forgotten’\norgan [22-24]. The significance of an individual’s microbiota\nstems from its physiological, immunological and metabolic\nfunctional capacity [21].\nTo further define the importance of an individual’s microbiota,\nan imbalance or disruption to an otherwise commensal or\nmutualistic relationship with the human host, results in a state\nof microbial dysbiosis which can affect host biology and\ncontribute to disease. Following the direction of Robert Koch\nin 1890, with his postulations that microorganisms were\ncausative agents for disease, many contemporary diseases have\nbeen associated with microbial level-changes [25]. For\ninstance, microbial dysbiosis has been associated with\ninflammatory bowel disease including Crohn’s disease and\nulcerative colitis [26], metabolic disease such as obesity [27]\nand type 2 diabetes [28], asthma [54], breast cancer [55],\nautoimmune disease [29,30], allergies [56] and autism\nspectrum disorder[57]. These growing correlations between\nmicrobial dysbiosis and disease are a product of an early 21st\ncentury scientific momentum to re-evaluate the role of\nmicrobiota in human health [58]. This topical approach to\nunderstanding disease has been made possible through\nadvancements in high-throughput metagenomic sequencing\ntechnology alongside global efforts to characterise the healthy\nhuman microbiome [58].\nGenitourinary Microbiota and Dysbiosis of the\nMicroflora\nThe colonisation of microorganisms in and on body surfaces\noccurs at birth, with the in-utero environment considered\naxenic (germ free) [59-61]. It has been reported that infants are\ninitially exposed to microorganisms upon birth, with maternal\ncervical mucus and immunoglobulins, as well as the placenta,\nproviding a degree of barrier defence and antimicrobial activity\nfrom ascending vaginal infective agents [62].\nPuca/Hoyne\nAllied J Med Res 2017 Volume 1 Issue 13\n\nRecent scientific efforts to characterise the human microbiome\nhave determined key microorganisms that constitute the\nvaginal flora. Lactobacilli species were the most dominant\nspecies and their ability to produce lactic acid subsequently\ncontributes to the low pH of the vagina (Figure 1) [63].\nFurthermore, hormonal changes associated with the menstrual\ncycle have been shown to alter the composition of the vaginal\nmicrobiome. Varying levels of estrogen and progesterone have\nbeen reported to impede on microbial flora stability,\nparticularly during menses when the flora presents with a lack\nof stability. However, hormonally fluctuated microbial\ncommunity changes occur without effecting the functional and\nmetabolic capacity of the vaginal flora [64].\nFigure 1. Summary of the microbial dysbiosis that occurs in response\nto endometriosis and bacterial vaginosis. The pie charts show\nrepresentations of various microbial species within the endometrium\nand vagina of females in health disease – For simplicity we have\nfocused on 4 main species Lactobacilli (purple), Gardnerella\nvaginalis (green) Atopobium vaginae (brown) and E. coli/ Shigella\nwhich are members of the Enterobactericiae family (red) that are\ndiscussed in the text. The microbiota of women with endometriosis\nshow a large expansion of E. coli/Shigella which are pathogenic and a\nreduction in the proportion of Lactobacilli spp. compared to healthy\nendometrium. In women with bacterial vaginosis there is an\nexpansion of Gardnerella vaginalis and Atopobium vaginae species at\nthe expense of Lactobacilli which could facilitate local tissue\ninflammation. The charts highlight that both the endometrium and\nvagina have a distinct microflora during normal tissue homeostasis,\nbut this can change during the onset of disease such as endometriosis\nor bacterial vaginosis respectively.\nMicrobial dysbiosis is not an alien term when discussing\ndiseases of the female reproductive tract. In fact, particular\ngynaecological diseases have been shown to develop in\nresponse to bacterial imbalances that can lead to bacterial\nvaginosis. Bacterial vaginosis is a bacterial infection of the\nvagina that commonly presents as abnormal grey vaginal\ndischarge with a strong unusual ‘fishy’ odour. This disease can\ncause discomfort and unease in affected women and can reduce\nthe frequency of successful pregnancy in patients undergoing\nin-vitro fertilisation (IVF) [65] and is also associated with\npremature births [66,67]. Lactobacilli spp. are the most\nabundant commensal bacteria in the vagina and the clinical\nonset of bacterial vaginosis is characterised by a decrease in\nthe population of Lactobacilli species [65]. In contrast there is\na substantial increase in growth of residing anaerobic or\nfacultative anaerobic bacteria, such as Gardnerella vaginalis\nand Atopobium vaginae (Figure 1). This microbial population\nshift introduces heterogeneity into the resident microbial\ncommunity, resulting in microbial imbalance and subsequent\ndisease [65,67].\nTable 3. Bacterial species identified in the endometrium.\nBacterial Phylotypes\nAcidovorax*\nAerococcus\nAtopobium vaginae\nBacteroides fragilis*\nBacteroides thetaiotaomicron*\nBacteroides ovatus*\nBacteroides vulgatus*\nBacteroides xylanisolvens*\nBetaproteobacteria*\nBifidobacterium\nCaulobacter*\nChitinophagaceae*\nClostridium\nEscherichia/Shigella*\nFlavobacterium ᶲ\nGardnerella vaginalis ᶲ\nLactobacillus crispatus ᶲ\nLactobacillus iners ᶲ\nLactobacillus jensenii\nPelomonas*\nPrevotella ᶲ\nPseudomonas*\nSphingomonas\nStenotrophomonas\nStreptococcus\nVeillonella\n*Suggested as part of the uterine core microbiome [68].\nᶲ=Abundant in at least one of the studies [68-71].\nRecently Verstraelen et al. [68] examined the resident\nmicrobiota of the endometrium through 16S ribosomal RNA\n(16S rRNA) metagenomic sequencing of endometrial samples.\nThey identified 183 different bacterial phylotypes, of which 15\nhad an abundance greater than 1% among the test subjects. The\nauthors identified a ‘uterine core microbiome’ that not only\nenforces the presence of microorganisms in the endometrium,\nbut further suggests a consistency of bacterial phylotypes\namong individuals. Belonging to this uterine core microbiome\nwere bacteria from the Proteobacteria, Firmicutes and\npredominantly Bacteroidetes phyla (Figure 1 and Table 3).\nThese findings have been supported by three independent\nCitation: Puca J, Hoyne GF . Microbial dysbiosis and disease pathogenesis of endometriosis, could there be a link?. Allied J Med Res\n2017;1(1):1-9.\n4Allied J Med Res 2017 Volume 1 Issue 1\n\nanalyses that examined the composition of the uterine\nmicrobiota [69-71]. Brewster, et al. [72] examined the bacterial\nmicrobiome of fallopian tubes, fimbriae and ovaries and\nshowed these various tissues harboured a significantly unique\nbacterial microbiome. Additionally, Pelzer et al. [73] revealed\nthe characteristics of the microbiome of follicular fluid which\nprovided insight into the microbial colonisation of the ovaries.\nAlthough contamination is common among the\nmicroorganisms of the vagina and follicular fluid, Pelzer et al.\n[73] identified that the microbiota of follicular fluid was\ndistinct and unique to that of the vagina in some patients.\nPredominant species identified were Lactobacillus iners,\nActinomyces spp., Corynebacterium auromucosum,\nFusobacterium spp., Peptinophilus asaccharolyticus,\nPeptostreptococcus spp., Propionibacterium spp. Prevotella\nspp., Staphylococcus spp., and the yeast Candida parapsilosi.\nThe Role of Lactobacillus species in the Vaginal\nMicroflora\nResident vaginal Lactobacilli spp. have been shown to reduce\ninfection of pathogens and protect against microbial dysbiosis\n[74]. This protection is achieved through the general ability of\nLactobacilli spp. to biosynthesise products of lactic acid,\nhydrogen peroxide (H 2O2) and antimicrobial compounds,\nwhich act to inhibit the growth of pathogens (Figure 2) [75].\nLactic acid is the primary microbicide agent produced by\nLactobacilli spp. and is responsible for maintaining a low pH\nenvironment, creating an unfavourable milieu for many\nbacterial species [76]. The acidification driven by Lactobacilli\nspp. has been shown to directly inhibit the growth of pathogens\n[74,75,77]. Accompanying lactic acid, is the production H 2O2,\nwhich further acts to inhibit the growth of pathogens. H 2O2\ngenerates reactive oxygen species and induces oxidative stress\nwhich results in DNA damage and bacterial lethality [78]. It\nseems that Lactobacilli spp. are themselves protected from this\nH2O2 through the expression of anti-oxidative enzymes\nincluding catalase, superoxide dismutase 2 and glutathione\nperoxidase-1 [79]. Furthermore, H 2O2 enhances host\nproduction of antimicrobial peptides secreted by epithelial\ncells, namely muramidase and lactoferrin (Figure 2).\nMuramidase is an enzyme that hydrolyses and thus cleaves the\nbacterial cell wall component peptidoglycan, inhibiting\nbacterial growth and survival, particularly that of gram positive\nbacteria [80]. Many, but not all, Lactobacilli spp. have a\nconstitutively expressed proteinaceous surface layer (S-layer),\nthat non-covalently binds to peptidoglycan and envelopes the\nentire cell wall [81,82]. This S-layer is often associated with a\nprotective function and could thus infer protection from\nmuramidase activity and other bacteriolytic enzymes [82]. On\nthe other hand, lactoferrin, a host-derived glycoprotein, acts by\nbinding to lipopolysaccharide (LPS), a component of the cell\nwall of gram negative that is absent in gram positive\nLactobacilli spp., destabilising it and increasing the\npermeability of the bacterial outer membrane to surrounding\nintrinsic bactericidal agents [83]. Interestingly, decreased\nlactoferrin levels have been detected in the peritoneal fluid of\nwomen with endometriosis [84]. Not only does this support\nreduced Lactobacilli spp. abundance in women with\nendometriosis, it further reveals that women with\nendometriosis have a reduced anti-bactericidal capacity, which\ncould further promote microbial dysbiosis. However, it remains\nunclear whether decreased lactoferrin levels indicate reduced\nLactobacilli spp. abundance in the endometrium or the\nperitoneal cavity, making it difficult to ascertain where the\nmicrobial dysbiosis takes place.\nFigure 2. Mucosal immune regulation induced by the microbial flora\nwithin the female reproductive system. Lactobacilli spp. dominate the\nnormal microflora in the vagina and endometrium of healthy women.\nThese organisms can secrete lactic acid to reduce the local pH as well\nas H 2O2 and bacteriocins that reduce the growth of microbial\nspecies.In addition, the presence of Lactobacilli can induce the\nsecretion of antimicrobial peptides (AMPs) that include human beta\ndefensins from epithelial cells that can directly impact on the\ncomposition and diversity of the local microflora. Emergence of\npathogenic E. coli/Shigella spp. can lead to the release of\nlipopolysaccharide (LPS) which can bind to pattern recognition\nreceptors (e.g. Toll-like receptor 4) on the surface of epithelial cellsto\ninduce signalling and the release of pro-inflammatory cytokines\n(TNF-a, Type 1 IFN and IL-6) and chemokines that promote\nrecruitment of inflammatory cells to the site of infection. If the\nmicrobial dysbiosis is not corrected then this could lead to chronic\ninflammation in the reproductive tract.\nIn addition to lactic acid and H2O2, Lactobacilli spp. have been\nfound to produce a range of bacteriocins (bacteria-derived\nantimicrobial agents) that further inhibit the growth of\nsurrounding bacterial and fungal pathogens [85,86]. For\ninstance, L. acidophilus, produces the bacteriocin Acidophillin\n801 which has a narrow inhibitory spectrum of activity against\ngram negative bacteria as well as some other Lactobacilli spp.\n[81] (Figure 2). L. acidophilus  contains a S-layer that is\nbelieved to infer protection from biosynthesised bacteriocins\n[81]. Whilst different species vary in their capacity to produce\nthese antimicrobial agents, the core microbiota Lactobacilli\nspp. population provides a collective effort to protect against\npathogens and prevent subsequent microbial dysbiosis. These\nfindings assert the importance of Lactobacilli spp. in the\nendometrium and their corresponding biosynthesised products\nin protecting against pathogens.\nBacterial Vaginosis and Microbial dysbiosis\nGiven that endometrial ectopic tissue has been found in the\nsurrounding genitourinary region, as a result of retrograde\nmenstruation, it raises a hypothesis that maybe the\ndevelopment of endometriosis could arise from a microbial\ndysbiosis. This concept has been identified more frequently\nand characterised in more detail with gastrointestinal diseases\nsuch as Crohn’s Disease and Ulcerative Colitis [26]. Given the\nPuca/Hoyne\nAllied J Med Res 2017 Volume 1 Issue 15\n\nproximity of the vagina to the uterus, and the recent\ncharacterisation of the microbiota along with different areas of\nthe reproductive tract, it warrants consideration to the influence\nthat a microbial dysbiosis might have on disease pathogenesis\nof endometriosis. Gynaecological diseases such as bacterial\nvaginosis have been shown to develop in response to an\nimbalance of bacterial species. As seen in Figure 1, bacterial\nvaginosis specifically involves a decrease in Lactobacilli spp.\nand an increase in growth of residing anaerobic or facultative\nanaerobic bacteria, including Gardnerella vaginalis and\nAtopobium vaginae. The ascent, introduction and domination\nof certain sexually transmitted microorganisms including\nChlamydia trachomatis and Neisseria gonorrhoeae, pathogenic\nEscherichia coli , or bacterial vaginosis-related species act to\nalter the reproductive tract microbiota, enforcing microbial\ndysbiosis and subsequent disease [87-89].\nAlthough microbial dysbiosis has been associated with other\ngynaecological diseases, it has been poorly investigated in\nrelation to endometriosis. Nonetheless, several studies have\nattempted to characterise the endometrial microbiome of\nwomen with endometriosis and investigate the role of bacteria\nin relation to the disease. Khan et al. [90], utilising 16s rRNA\ngenomic sequencing, identified a significant increase in\nStreptococcaceae in samples from women with endometriosis,\ncompared to control samples. The same study also recognized\na slight increase in Moraxellaceae and a modest decrease in\nLactobacillaceae in women with endometriosis, though these\ndifferences were statistically insignificant.\nKhan et al. [91] found that women with endometriosis had a\nsignificant increase in E. coli in menstrual blood and endotoxin\nlevels in menstrual and peritoneal fluid. Following this, Khan\net al. [92] further revealed that endometriosis was accompanied\nby an increase in Gardnerella, Group A-Streptococcus,\nEnterococci and E. coli  upon the culturing of endometrial\nsamples on culture medium. Although quantity of bacterial\ngrowth was measured, rather than specificity of bacterial\nspecies, culture-dependent techniques still warrant caution\nwhen accepting these results. Nonetheless, excess growth of\ncertain bacterial species or an imbalance of commensal\nbacteria could contribute to the development of endometriosis.\nMoreover, Khan et al. [92] also found that women with\nendometriosis were more predisposed to a higher vaginal pH\n(≥ 4.5) than in control subjects, inferring that this greater\ndiversity of bacterial species in women with endometriosis was\npermitted due to an altered vaginal environment. This suggests\nthat women with endometriosis could have a reduced lactic-\nacid producing bacterial population, namely Lactobacilli spp.,\nincreasing the susceptibility to microbial imbalance and\ndysbiosis. As aforementioned, although the results were not\nstatistically significant, Khan et al. [90] did note a decrease in\nLactobacillaceae in women with endometriosis. This decrease\nwas also commonly seen in women with bacterial vaginosis,\nsupporting the notion that perhaps altered Lactobacilli spp.\nlevels contribute to the development of endometriosis.\nStudies investigating endometriosis in non-human primates\nhave shed similar insights into a dysbiosis of endometrium.\nBailey and Coe [93] examined the concentration of popular\nbacterial species in the shed endometrium of female rhesus\nmonkeys with endometriosis by means of culture-dependent\ntechniques. It was found that rhesus monkeys with\nendometriosis had reduced Lactobacilli spp. concentrations,\nwhilst presenting an increased concentration of gram negative\nanaerobic and facultative anaerobic bacteria. As samples were\nnot taken directly from the endometrium, but instead from shed\nendometrium, this might not be an accurate characterisation of\nendometriosis-associated endometrium microbiota. However,\ndespite the method of sample obtainment and the results being\nobtained from non-human primates, they resemble the findings\npresented in human studies [92,90].\nConclusion\nWhilst microbial dysbiosis in relation to the development of\nendometriosis is quite a novel concept, the collective and\ncomprehensive analysis of several direct and multiple indirect\nstudies have allowed for an evaluation of the topic. It has been\ndemonstrated that the endometrium harbours a microbiota and\nis thus susceptible to dysbiosis, although further microbiome\ncharacterisation of the peritoneal cavity and deeper\ngenitourinary regions is poor. Additionally, microbial dysbiosis\nhas been shown to be responsible for other gynaecological\ndiseases, making it reasonable to deduce that microbial\ndysbiosis could contribute to the disease in question.\nMoreover, key microbes were identified in women with\nendometriosis, implying that endometriosis or at least the\ninflammatory property of endometriosis is a result of microbial\nimbalance. Of particular interest was reduced Lactobacilli spp.\nabundance accompanied by a high pH environment as well as\nan increase in gram negative bacteria abundance, commonly E.\ncoli. This shift in the microbial population was shown to have\nthe capacity to reduce host immunological capability and\ninduce host susceptibility to pathogens. This inference supports\nthe retrograde menstruation theory crediting the probability of\nretrograde migration of pathogenic bacteria into the\nendometrium and ectopic endometrial tissue to atypical sites\nand further appreciates that genetic susceptibility may also play\na role in the development of the disease, in conjunction with\nmicrobial dysbiosis. Ultimately, it seems plausible that the\npathogenesis of endometriosis could be related to microbial\ndysbiosis, predominately centred around a reduced\ncommensal-associated immune capability as well as an\nascending microbial infection. This could have important\nimplications for the treatment of endometriosis in the future.\nAcknowledgements\nWe would like to acknowledge grant support of the Juvenile\nDiabetes Research Foundation (4-2006-1025) and Diabetes\nResearch Foundation of Western Australia awarded to GFH.\nReferences\n1. Gylfason  JT, Kristjansson KA, Sverrisdottir G, et al. 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Gynecol Obstet\nInves. 1995;40:261-4.\n*Correspondance to\nGerard Hoyne,\nSchool of Health Sciences,\nUniversity of Notre Dame Australia,\nWestern Australia.\nPuca/Hoyne\nAllied J Med Res 2017 Volume 1 Issue 19","source_license":"CC0","license_restricted":false}