{"paper_id":"8f3992dd-beb6-4a39-8255-68721179714b","body_text":"9\nORIGINAL RESEARCH    MICROBIOLOGY\nBULLETIN OF RSMU   3, 2023   VESTNIKRGMU.RU| |\nGumenyuk LN    , Zemlyanaya IA, Rami Almasoud, Badula ES, Ismailov AR, Seroshtanov NA, Kokareva SS, Cheremisova AA, Kupreichyuk YuR\nGUT MICROBIOTA ALTERATIONS AND THEIR ASSOCIATION WITH IL6, IL8 AND TNFα LEVELS IN \nPATIENTS WITH EXTERNAL GENITAL ENDOMETRIOSIS \nToday, the association of gut microbiota with external genital endometriosis (EGE) is of special scientific interest. The study was aimed to assess alterations of the \ngut microbiota taxonomic composition and explore their correlations with plasma levels of IL6, IL8 and TNFα at the species level in patients with EGE. The cross-\nsectional comparative study involved 50 patients with EGE (index group) and 50 healthy women (control group). The changes in the gut microbiota taxonomic \ncomposition and plasma levels of IL6, IL8 and TNFα were assessed. A significant decrease in the abundance of such species, as Coprococcus catu (р = 0.009), \nTuricibacter sanguinis (р = 0.008) and Ruminococcus gnavus (р < 0.001), along with the increase in the abundance of Eubacterium ramulus (р = 0.040), Bacterioides \ndorei (р = 0.001), Prevotella divia (р = 0.008) and  Shigella flexneri (р < 0.001) were found in the gut microbiota taxonomic composition in patients with EGE. \nSignificant correlations between the IL6 levels and the abundance of Turicibacter sanguinis (r = –0.92; р = 0.001), IL8 levels and the abundance of Shigella flexneri \n(r = 0.72; р < 0.001), TNFα levels and the abundance of Prevotella divia (r = 0.77; р = 0.001) were revealed. The findings add to the available literature data on the \nfeatures of gut microbiota alterations and their association with some inflammation biomarkers in individuals with EGE, which can justify further research in this area \nand probably open up new approaches to treatment of the disease. \nKeywords: external genital endometriosis, gut microbiota, IL6,IL8, TNFα.\nCorrespondence should be addressed: Lesya N. Gumenyuk\nBulvar Lenina, 5/7295006, Simferopol, Republic of Crimea, Russia; leya.sorokina@mail.ru\nSI Georgievsky Medical Academy, VI Vernadsky Crimean Federal University, Simferopol, Russia \nReceived: 16.05.2023 Accepted: 01.06.2023 Published online: 15.06.2023\nDOI: 10.24075/brsmu.2023.018\nAuthor contribution: Gumenyuk LN — study concept and design; Zemlyanaya IA, Rami A, Seroshtanov NA — data acquisition, analysis, and interpretation; \nBadula ES, Ismailov AR — statistical data processing; Kokareva SS, Cheremisova AA, Kupreichyuk YuR — manuscript writing.\nCompliance with ethical standards: the study was approved by the Ethics Committee of the SI Georgievsky Medical Academy, VI Vernadsky Crimean Federal \nUniversity (protocol № 10 of 14 November 2021), planned and conducted in accordance with the Declaration of Helsinki. The informed consent was obtained \nfrom all study participants. \nЛ. Н. Гуменюк    , И. А. Земляная, Алмасуд Рами, Е. С. Бадула, А. Р . Исмаилов, Н. А. Сероштанов, С. С. Кокарева, А. А. Черемисова, \nЮ. Р . Купрейчук\nИЗМЕНЕНИЯ МИКРОБИОТЫ КИШЕЧНИКА И ИХ СВЯЗЬ С ПОКАЗАТЕЛЯМИ IL6, IL8 И TNF α \nУ ПАЦИЕНТОК С НАРУЖНЫМ ГЕНИТАЛЬНЫМ ЭНДОМЕТРИОЗОМ \nАссоциация микробиоты кишечника и наружного генитального эндометриоза (НГЭ) на сегодняшний день представляет собой особый научный интерес. \nЦелью исследования было оценить изменения таксономического состава микробиоты кишечника и изучить на уровне видов их взаимосвязь с \nпоказателями IL6, IL8 и TNFα в плазме крови у пациенток с НГЭ. В одномоментное сравнительное исследование было включено 50 пациенток с НГЭ \n(основная группа) и 50 здоровых женщин (контрольная группа). Оценивали изменения таксономического состава микробиоты кишечника и уровни IL6, \nIL8 и TNF α в плазме крови. У пациенток с НГЭ в таксономическом составе микробиоты кишечника обнаружены статистически значимое cнижение \nпредставленности видов Coprococcus catu (р = 0,009), Turicibacter sanguinis (р = 0,008) и Ruminococcus gnavus (p < 0,001), повышение представленности \nвидов Eubacterium ramulus (р = 0,040), Bacterioides dorei (р = 0,001), Prevotella divia (р = 0,008) и Shigella flexneri (р < 0,001). Выявлены статистически \nзначимые корреляции показателя IL6 с представленностью Turicibacter sanguinis (r = –0,92; р = 0,001), IL8 и Shigella flexneri (r = 0,72; р < 0,001), TNFα с \nпредставленностью Prevotella divia (r = 0,77; р = 0,001). Полученные результаты дополняют имеющиеся литературные сведения о специфике изменений \nмикробиоты кишечника и их сопряженности с некоторыми биомаркерами воспаления при НГЭ, что может стать обоснованием для продолжения \nисследований в этом направлении и, возможно, открывает новые подходы к лечению этого заболевания.\nКлючевые слова:  наружный генитальный эндометриоз, микробиота кишечника, IL6, IL8, TNFα\nДля корреспонденции: Леся Николаевна Гуменюк \nбульвар Ленина, 5/7295006, г . Симферополь, Республика Крым, Россия; leya.sorokina@mail.ru\nМедицинская академия имени С. И. Г еоргиевского (структурное подразделение ФГАОУ ВО «КФУ имени В. И. Вернадского»), Симферополь, Россия\nСтатья получена: 16.05.2023 Статья принята к печати: 01.06.2023 О публикована онлайн: 15.06.2023\nDOI: 10.24075/vrgmu.2023.018\nВклад авторов:  Л. Н. Гуменюк — замысел и дизайн исследования; И. А. Земляная, А. Рами, Н. А. Сероштанов — сбор, анализ и интерпретация данных; \nЕ. С. Бадула, А. Р . Исмаилов — статистическая обработка данных; С. С. Кокарева, А. А. Черемисова, Ю. Р . Купрейчук — подготовка статьи.\nСоблюдение этических стандартов:  исследование одобрено этическим комитетом Крымской медицинской академии имени С. И. Г еоргиевского \nФГАОУ ВО «Крымский федеральный университет им. В.И. Вернадского» (протокол № 10 от 14 ноября 2021 г.), спланировано и проведено в соответствии \nс Хельсинской декларацией. Все лица, включенные в исследование, подписали добровольное информированное согласие. \nEndometriosis is a significant issue of modern gynecology, \nit remains under active consideration over the decades. \nAccording to the aggregate data, more than 176 million women \nall over the world have endometriosis [1], and its prevalence \nrate grows steadily in recent years. It is important to note that \nendometriosis is associated with infertility in 50–80% of cases \nand chronic pelvic pain in 50% of cases [1, 2]. These conditions \nworsen the patients’ mental and physical health, as well as \ntheir quality of life [3]. The difficulties in differential diagnosis of \nendometriosis often result in the diagnosis delay of 4–11 years, \nand 65% of women are misdiagnosed [3, 4], which results \nin the disease progression and grave consequences [5]. The \ntoday’s pharmacological and surgical approaches to treatment \nof endometriosis are associated with the risk of severe side \n\n10\nОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ    МИКРОБИОЛОГИЯ\nВЕСТНИК РГМУ   3, 2023   VESTNIKRGMU.RU| |\nTable 1. Characteristics of patients with external genital endometriosis and healthy women\nParameter EGE patients (n = 50) Control group (n = 50)\nAverage age, years, median [25%; 75%] 37,0 [32,0; 44,0] 37,7 [32,7; 43,2]\nBody mass index, kg/m2, median [25%; 75%] 23,0 [21,0; 24,3] 22,06 [20,8; 24,1]\nStage I–II EGE, n (%) 14 (28,0%) –\nStage III–IV EGE, n (%) 36 (70,0%) –\neffects and show insufficient efficiency. The relapse rate is still \nhigh: it reaches 15–21% [3]. That is why the search for new \npathophysiological mechanisms underlying external genital \nendometriosis (EGE), as well as for safe and efficient methods \nfor prevention and treatment of the disease, is still relevant.\nEGE, characterized by proliferation of endometrial tissue \noutside of the uterine cavity, is conventionally considered as \na chronic estrogen-dependent immune inflammatory disease \nthat is limited to the pelvis [6]. However, today EGE is more and \nmore often considered as a systemic inflammatory disorder \noften associated with heterogeneous multiple organ dysfunction \n[3, 7]. It is believed that aberrant cytokine production \naccompanied by the immune response dysregulation plays \na vital part in pathophysiology of systemic inflammation \nassociated with EGE. In this regard, pro-inflammatory \ninterleukins (IL6, IL8) and tumor necrosis factor alpha (TNF α) \nare considered to be among the most important. Assessment \nof the cytokine profile in blood of patients with EGE made it \npossible to detect the elevated levels of IL6, IL8 and TNF α \n[8–10]. Furthermore, elevated plasma IL6 levels were associated \nwith the pain severity [11], disease severity [12], and relapse \nrate [11] in patients with EGE. While plasma levels of IL8 were \nassociated with the size of active lesions [13] and infertility [14], \nthe levels of TNF α were associated with the severity of EGE \nclinical manifestations, disease activity and depth [15]. \nCurrent research suggests that gut microbiota is involved in \nEGE pathophysiology, which can be explained by its fundamental \nrole in maintaining the immune homeostasis and direct association \nwith the development of numerous inflammatory diseases [16]. \nThe experiments involving the heterologous surgical injection \nmurine model of endometriosis showed that gut microbiota \naffected the EGE course and progression [17, 18] via modulation \nof various immune system components [18]. Particularly, \nadministration of normal murine fecal microbiota to mice with \nexperimentally induced endometriosis and gut microbiota \ndepletion was associated with the decline in the endometriotic \nlesion growth, while administration of fecal microbiota \nobtained from mice with endometriosis resulted in the disease \nprogression. Furthermore, depletion of intestinal microbiota \nreduces the severity of inflammatory response associated with \nendometriosis [17] and modulates the abundance of immune \ncells in the peritoneum [18]. Finally, the papers provide strong \nevidence of changes in the intestinal microbiota profile in mice \n[18–20] and humans [18, 19]. At the same time, clinical data \non the gut microbiota species composition in patients with EGE \nare fragmentary, contradictory and insufficient for unambiguous \nconclusions. Thus, among 16 studies, focused on assessing the \nrelationship between EGE and microbiome, only six involved the \nanalysis of gut microbiota, and only four involved assessment \nof human intestinal microbiome [23]. It is also important to note \nthat, among the reviewed papers there are no studies focused \non assessing gut microbiota alterations in patients with EGE of \nSlavic ethnic background. In particular, there is little information \non the association between gut microbiota and inflammatory \nbiomarkers in patients with EGE.\nThe study was aimed to assess alterations of the gut \nmicrobiota taxonomic composition and explore their correlations \nwith plasma levels of IL6, IL8 and TNF α at the species level in \npatients with EGE.\nMETHODS\nThe cross-sectional comparative study was performed in \nthe Saint Luke Multidisciplinary Clinic (Simferopol, Republic \nof Crimea). The study involved 50 patients aged 18–45 with \nthe confirmed diagnosis of stage I–IV EGE admitted to the \nGynecology Department (index group) and 50 age-matched \nhealthy women who underwent preventive medical examination \n(control group). All EGE patients and healthy women submitted \nthe informed consent to study participation. \n Inclusion criteria for the index group: age 18–45 years; \nthe diagnosis of EGE verified by laparoscopy and histological \nassessment. \nNon-inclusion criteria for the index group: age < 18 of > 45 \nyears; body mass index >24.9 kg/m2; pregnancy and lactation; \ntype I or II diabetes mellitus, concomitant chronic systemic \nand somatic disorders; history of mental and behavioral \ndisorders; verified functional and inflammatory disorders \nof the gastrointestinal tract, hepatobiliary system; history \nof inflammatory disorders within a month before the study; \nhistory of stool problems (constipation/diarrhea) within a month \nbefore the study; taking hormonal oral birth control or anti-\ninflammatory drugs, antibiotics, probiotics, prebiotics, antiviral \ndrugs, symbiotics or acid–suppression medications within \nthree months before inclusion in the study;  taking medications \naffecting the stool passage within eight weeks before inclusion \nin the study; refusal to participate in research. \nInclusion criteria for the control group: age 18–45 years;  \nbody mass index < 24.9 kg/m2; no somatic disorders or allergy; \nno infectious or acute disorders within two months before \ninclusion in the study; no history of mental and behavioral \ndisorders; no stool problems (constipation/diarrhea) within a \nmonth before inclusion in the study; taking no hormonal oral \nbirth control or anti-inflammatory drugs, antibiotics, probiotics, \nprebiotics, antiviral drugs, symbiotics or acid–suppression \nmedications within three months before inclusion in the study; \ntaking no medications affecting the stool passage within eight \nweeks before inclusion in the study. \nNon-inclusion criteria for the control group: body \ntemperature above 36.9 °С.  \nThe characteristics of patients with EGE and controls \nare provided in Table 1. The groups were matched for age \n(р = 0.94; χ2) and body mass index ( р = 0.052; χ2). A total of \n36 patients (70.0%) had stage III–IV EGE. \nThe diagnosis of endometriosis was verified during surgery \nin accordance with the criteria of the American Society for \nReproductive Medicine (ASRM) classification. \nTo analyze the taxonomic composition of the gut microbiota \nof patients with EGE and healthy women, fecal samples were \ncollected in the morning (8 a.m. to 10 a.m.), and in EGE patients \nsampling was performed on the day of hospital admission. \nThe samples were frozen and stored in disposable plastic \ncontainers at a temperature of –80 °C prior to metagenomic \nanalysis. Isolation of total DNA was performed by phenol-based \n\n11\nORIGINAL RESEARCH    MICROBIOLOGY\nBULLETIN OF RSMU   3, 2023   VESTNIKRGMU.RU| |\nFig. 1. Phylogenetic composition of gut microbiota in patients with external genital endometriosis (EGE) and healthy women. CG — control group\nEGE \n500 500\n300\n700 700\n400\n900 900\n500 550 600\n600 600\n350\n800 800\n450\n1000 1000\n650\nEGE \nEGE \nCG CG\np = 0.014 p = 0.053\np = 0.051\nCG\nChao1 ACE\nSobs\nFig. 2. Species composition of gut microbiota in patients with external genital endometriosis (EGE) and healthy women. CG — control group\nEGE CG\n0 6 10 14 182 4 8 12 16 20\nextraction; the DNA nucleotide sequence was determined \nby shotgun sequencing using the SOLiD5500 Wildfire high-\nthroughput sequencing system (AppliedBiosystems; USA) [24]. \nThe reads were filtered based on their quality, and the \ntaxonomic classification was performed using the QIIME ver. \n1.9.1 software [25]. Taxonomic assignment of the reads was \nbased on the data taken from two taxonomic databases: \nduring the first phase the reference set of bacterial operational \ntaxonomic units (OTUs) was selected based on matching \nthe acquired reads of 16S rRNA genes with the GreenGenes \ndatabase, ver. 13.5 [26]. During the second phase taxonomic \nassignment of these OTUs was performed using the RDP \nalgorithm based on the specialized HITdb human intestinal \nmicrobiota database [27].\nThe qualitative and quantitative assessment of gut \nmicrobiota composition was performed by identification of \nmicrobial species, genera, and phyla; the microbial community \nα-diversity was assessed by calculating the Chao1 index, the \nnumber of taxa observed (Sobs), and the indicator of species \nrichness (ACE) using the Mothur v.1.22.0 software (http:// \nwww.mothur.org).\nBlood samples of EGE patients and healthy volunteers to be \nused for immunosorbent assay were collected by venipuncture \nin the morning in a fasting state at rest (for at least 15 min). \nPlasma levels of IL6, IL8 and TNFα were assessed by enzyme-\nlinked immunosorbent assay (ELISA) using the test system \n(Vector-Best; Novosibirsk, Russia). The tubes containing blood \nserum were frozen and stored at a temperature of –20 °С. \nStatistical data processing was performed using the \nSTATISTICA 8.0 software package (StatSoft.Inc.; USA). As for \nquantitative indicators, the distribution type was determined \nusing the Kolmogorov–Smirnov test. Given that the majority of \nShigella dysenteriae\nCoprococcus catu\nRuminococcus gnavus\nBacteroides dorei\nPrevotella bivia\nTuricibacter sanguinis\nEubacterium ramulus\n\n12\nОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ    МИКРОБИОЛОГИЯ\nВЕСТНИК РГМУ   3, 2023   VESTNIKRGMU.RU| |\nTable 2. Comparative analysis of plasma IL6, IL8 and TNF α levels in patients with external genital endometriosis (EGE) and healthy women. CG — control group; \nр — significance of differences between the values of patients with EGE and the CG\nParameter EGE patients (n = 50) Control group (n = 50) Р\nIL6, pg/mL, median [25%; 75%] 14,7 [8,1; 18,3] 3,8 [2,0; 6,6] < 0,001\nIL8, pg/mL, median [25%; 75%] 14,6 [9,6; 28,8] 2,2 [1,4; 6,8] < 0,001\nTNFα, pg/mL, median [25%; 75%] 17,9 [9,3; 26,5] 5,2 [2,8; 7,6] < 0,001\nquantitative indicators were not normally distributed, the median \n(Ме) and interquartile range (25 th percentile; 75 th percentile) \nwere calculated. As for qualitative traits, the percentage and \nabsolute values were determined. The chi-squared test ( χ2) \nwas used to compare qualitative traits, and quantitative traits \nwere compared using the Mann–Whitney U test. Spearman's \nrank correlation was applied to assess correlations between \nthe factors. The significance level for comparison of qualitative \nand quantitative traits, as well as for correlation analysis was \nset as р < 0.05. \nRESULTS\nAssessment of the gut microbiota taxonomic composition \nrevealed a significant decrease in the bacterial community \nα-diversity (Chao1 index р = 0.014) in patients with EGE \ncompared to healthy women. Furthermore, patients with \nEGE had lower ACE and Sobs indices than healthy women, \nhowever there were no significant differences between groups \n(р = 0.053; р = 0.051, respectively) (Fig. 1). \nComparative analysis of the gut microbiota species \ncomposition in patients with EGE relative to healthy \nwomen revealed a significant decrease in the abundance \nof Coprococcus catu  ( р = 0.009), Ruminococcus gnavus \n(р < 0.001) and Turicibacter sanguinis (р = 0.008) along with the \nincreased abundance of such bacterial species, as Eubacterium \nramulus (р = 0.040), Bacterioides dorei (р = 0.001), Prevotella \ndivia (р = 0.008), and Shigella flexneri (р < 0.001) (Fig. 2).\nThe IL6, IL8 and TNF α plasma levels of patients with EGE \nwere significantly higher than that of healthy women (Table 2).\nAt the same time we revealed a strong negative correlation \nbetween the abundance of Turicibacter sanguinis and the IL6 \nlevels ( r = –0.92; р = 0.001); there was a strong significant \npositive correlation between the increase in abundance \nof Shigella flexneri bacteria and the levels of IL8 ( r = 0.72; \np < 0.001). Furthermore, a strong positive correlation between \nthe TNFαlevels and the abundance of Prevotella divia (r = 0.77; \nр = 0.001) was reported.  \nDISCUSSION\nGut microbiota is associated with many inflammatory \ndisorders, including EGE [16–19]. However, today there are \njust a few human studies on the issue, the results of which \ndo not allow any consensus-based conclusions. Given the \nlack of knowledge of the issue, the primary objective of our \nstudy was to refine the gut microbiota taxonomic composition \nalterations in the group of patients with EGE. Our study has \nconfirmed that gut microbiota composition of EGE patients is \nquite different from that of healthy women. The findings show \nthat the lower bacterial α-diversity relative to healthy women \nis typical for patients with EGE, which is a common distinctive \nfeature of chronic inflammatory disorders [28]. Our findings \nare consistent with the data of the earlier reported study \n[22], but do not confirm other data [21], according to which \npatients with EGE are characterized by the decrease in both \nα- and β-diversity. The results of our study have also shown \nthat dysbiotic intestinal alterations in patients with EGE are \ncharacterized by the decrease in the abundance of bacteria \nhaving the potential for immunomodulation: Coprococcus \ncatu and Turicibacter sanguinis species representatives that \nare known to produce short-chain fatty acids (SCFAs), i.e. \nendogenous signaling molecules essential for maintaining \nthe host’s immune homeostasis, and Ruminococcus gnavus. \nMoreover, the decrease in the levels of SCFAs results in the \nincreased abundance of Gram-negative bacteria, and therefore \nlipopolysaccharide (LPS) levels [29]. There is evidence that \nfeces of mice with endometriosis have low levels of SCFAs, \nspecifically butyrate, while butyrate administration inhibits \nendometriotic cell growth  in vitro  and  in vivo  via inhibition of \nhistone deacetylase activity and activation of expression of the \nRap1GAP protein that inactivates the Rap1 intracellular signaling \nprotein [19]. In addition, we have detected the increased \nabundance of Eubacterium ramulus , Bacterioides dorei , \nPrevotella divia and Shigella flexneri. Among these the presence \nof Shigella flexneri should be noted, since these bacteria have \nbeen earlier detected in the fecal samples of patients with stage \nIII–IV EGE in the study [30]. It is suggested that this species \nplays a role of the trigger that initiates the immune alterations \nresulting in the development and progression of endometriosis \n[31]. Our findings are partially in line with the data of the number \nof other studies. For example, one of the studies has shown \nthat the decrease in the abundance of Coprococcus along \nwith the increase in the abundance of Bacterioides is typical for \npatients with EGE [21]. The other study has shown that patients \nwith EGE are characterized by the increase in abundance of \nEubacterium and  Bacterioides  [22]. The data obtained may \nbe inconsistent due to the fact that, firstly, the studies involved \npatients of different ethnic groups, and secondly, in contrast \nto the listed above researchers, we did not enroll overweight \npatients with EGE (since the effects of this factor on gut \nmicrobiota alterations was proven) and the patients taking \nhormonal, birth control and anti-inflammatory drugs in order to \navoid their effects on the study results. \nAs stated earlier, patients with EGE demonstrate a significant \nincrease in plasma levels of IL6, IL8 and TNF α, the role of \nwhich in the disease development and progression to severe \nforms has been proven [8–10]. Our study has also revealed \nsignificantly higher levels of IL6, IL8 and TNF α compared to \nhealthy women in patients with EGE. Meanwhile, intestinal \ndysbiosis, that is more and more often considered to be a \nfactor of inflammation, autoimmune and immune-mediated \ndisorders, can trigger the inflammatory immune response \nassociated with elevation of pro-inflammatory cytokine levels \nat the whole-body level [32]. That is why the second objective \nof the study was to assess the association of gut microbiota \ncomposition at the species level with plasma levels of IL6, IL8 \nand TNFα in the group of patients with EGE. We have found \nthat some intestinal microbial species of patients with EGE are \nassociated with plasma levels of the studied cytokines, which \ncan indicate the association of gut microbiota composition with \nEGE. In particular, a negative correlation between the elevated \nIL6 levels and the abundance of Turicibacter sanguinis bacteria \nhas been revealed. We have found a probable explanation for \n\n13\nORIGINAL RESEARCH    MICROBIOLOGY\nBULLETIN OF RSMU   3, 2023   VESTNIKRGMU.RU| |\nthis correlation in the literature. As is well known, the Turicibacter \nbacteria are involved in production of metabolites having a \nprotective effect on the intestinal epithelium and reproductive \nsystem, specifically such SCFAs, as acetic, valeric and \nbutyric acids. The decrease in the levels of the latter leads to \nactivation of histone deacetylase and the related NF-kB nuclear \ntranscription factor, as well as to inhibition of the GPR41, GPR43 \nand GPR109А G protein-coupled receptors, thereby inducing \nexpression of the genes responsible for synthesis of pro-\ninflammatory cytokines, including IL6 [33], and promoting the \ndevelopment of chronic inflammation [16]. The earlier reported \n[34] association of the IL8 levels with the abundance of bacteria \nof genus Subdoligranulum in patients with EGE has not been \nconfirmed in our study. According to our findings, a positive \ncorrelation of the IL8 levels with the abundance of  Shigella \nflexneri bacteria is typical for patients with EGE, which can be \nmediated by the ability of the latter to induce persistent NF-kB \ninhibitory kinase complex (IKK) activation and subsequent I-kB \ndegradation via initiation of the pattern recognition receptors \nTLR4. This, in turn, promotes the release of NF- κB with \nsubsequent translocation into the nucleus and triggering the \nIL8 transcription [35]. The literature reports such associations \nin patients with confirmed Shigella infection ( shigellosis) that \nhave been confirmed by strong positive correlations between \nthe abundance of Shigella flexneri and the levels of IL8 in blood \nplasma [36]. As we have already stated, the contrast between \nour findings and the results of the study these are compared \nwith may be due to the differences in design, specifically to \nthe fact of selective enrollment of normal-weight EGE patients \nhaving no extragenital comorbidities in our study, while in the \nother study [34] these characteristics were not considered as \nexclusion criteria. Furthermore, the differences may result from \nthe fact that we enrolled patients with stage I–IV EGE, while \nthe study [34] involved patients with stage III–IV EGE. This fact \ncould also affect the differences between the associations of \nIL8 with gut microbiota representatives in EGE patients and the \nassociations reported in the literature. The small sample size \n(12 patients) used in the earlier reported study should be also \nnoted [34]. Moreover, our study revealed a strong positive \ncorrelation between the TNFα blood levels and the abundance of \nPrevotella divia. We have found no reports of the research focused \non studying this subject in patients with EGE. However, it has been \npreviously shown that treatment of monocytic cell line with LPS \nfrom Prevotella results in simultaneous activation of three basic \nsignaling pathways of mitogen-activated protein kinase (MAPK) \n(extracellular signaling kinase 1/2 (ERK1/2), c-Jun N-terminal \nkinase 1/2 (JNK1/2), and p38) with subsequent induction of the \nTNFα mRNA expression and TNFα secretion stimulation [37]. \nOur findings suggest that gut microbiota plays a vital part \nin EGE immunogenesis. Apparently, the causal relationships \nbetween gut microbiota and blood levels of pro-inflammatory \ncytokines in individuals with EGE require a more detailed study \nand further research in this area. \nCONCLUSIONS\nSignificant alterations in the gut microbiota abundance and \ntaxonomic composition have been found in patients with EGE. \nFurthermore, the significant correlations of some bacterial \nspecies with plasma levels of IL6, IL8 and TNFα we have revealed \nsuggest the association of the gut mictobiota abundance and \ncomposition with the EGE immunopathogenesis. Further \nresearch is required to confirm the role of gut microbiota in the \nEGE pathophysiology. The targeted effects on gut microbiota \nmay contribute to the efficiency of approaches to treatment \nof EGE.\nReferences\n1. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, \nViganò P . Endometriosis. Nat Rev Dis Primers. 2018; 4: 9. \n2. Saunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, \nand therapeutic prospects. Cell. 2021; 184 (11): 2807–24. \n3. Bao C, Wang H, Fang H. Genomic Evidence Supports the \nRecognition of Endometriosis as an Inflammatory Systemic Disease \nand Reveals Disease-Specific Therapeutic Potentials of Targeting \nNeutrophil Degranulation. Front Immunol. 2022; 23 (13): 758440. \n4. Greene R, Stratton P , Cleary SD, Ballweg ML, Sinaii N. Diagnostic \nexperience among 4,334 women reporting surgically diagnosed \nendometriosis. Fertility and sterility. 2009; 91 (1): 32–39. \n5. Greenbaum H, Bat-El L, Galper BEL, Decter DH, Eisenberg VH. \nEndometriosis and autoimmunity: Can autoantibodies be used as \na non-invasive early diagnostic tool? Autoimmun Rev. 2021; 20 \n(5): 102795. \n6. Clement Philip B. The Pathology of Endometriosis: A Survey of \nthe Many Faces of a Common Disease Emphasizing Diagnostic \nPitfalls and Unusual and Newly Appreciated Aspects. Advances \nin Anatomic Pathology. 2007; 14 (4): 241–60. \n7. Taylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic \nsystemic disease: clinical challenges and novel innovations. \nLancet. 2021; 27: 839–52.  \n8. Yarmolinskaya MI. Citokinovyi ̆ profil' peritoneal'noi ̆ zhidkosti \ni perifericheskoi ̆ krovi bol'nyh s naruzhnym genital'nym \nehndometriozom. Zhurnal akusherstva i zhenskih bolezneĭ. 2008; \n57 (3): 30–34. Russian.\n9. Sikora J, Smycz-Kuban ́ska M, Mielczarek-Palacz A, Kondera-\nAnasz Z. Abnormal peritoneal regulation of chemokine activation — the \nrole of IL8 in pathogenesis of endometriosis. American Journal of \nReproductive Immunology. 2017; 77 (4).  \n10. Cameron MJ, Kelvin DJ. Cytokines and chemokines — their \nreceptors and their genes: an overview. Advances in Experimental \nMedicine and Biology. 2003; 520: 8–32. \n11. Somigliana E, Vigan ò P , Tirelli AS, Felicetta I, Torresani E, Vignali \nM, et al. Use of the concomitant serum dosage of CA 125, CA \n19-9 and interleukin-6 to detect the presence of endometriosis. \nResults from a series of reproductive age women undergoing \nlaparoscopic surgery for benign gynaecological conditions. \nHuman Reproduction. 2004; 19 (8): 1871–6. \n12. Dong Hao Lu, Song H, Shi G. Anti-TNFα treatment for pelvic pain \nassociated with endometriosis. Cochrane database of systematic \nreviews. 2010; 3 (3): CD008088. \n13. Li A, Dubey S, Varney ML, Dave BJ, Singh RK. IL8 directly \nenhanced endothelial cell survival, proliferation, and matrix \nmetalloproteinases production and regulated angiogenesis. \nJournal of Immunology. 2003; 170 (6): 3369–76. \n14. Malvezzi H, Hernandes C, Piccinato CA, Podgaec S. Interleukin in \nendometriosis-associated infertility-pelvic pain: systematic review \nand meta-analysis. Reproduction. 2019; 158 (1): 1–12.  \n15. Scholl B, Bersinger NA, Kuhn A. Correlation between symptoms \nof pain and peritoneal fluid inflammatory cytokine concentrations \nin endometriosis. Gynecol Endocrinol. 2009; 25 (11): 701–6.  \n16. Wu HJ, Wu E. The role of gut microbiota in immune homeostasis \nand autoimmunity. Gut Microbes. 2012; 3 (1): 4–14. \n17. Chadchan SB, Cheng M, Parnell LA, Yin Y, Schriefer A, \nMysorekar IU, et al. Antibiotic therapy with metronidazole reduces \nendometriosis disease progression in mice: a potential role for gut \nmicrobiota. Hum Reprod. 2019; 34: 1106–16.\n18. Chadchan SB, Naik SK, Popli P , et al. Gut microbiota and \nmicrobiota-derived metabolites promotes endometriosis. Cell \n\n14\nОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ    МИКРОБИОЛОГИЯ\nВЕСТНИК РГМУ   3, 2023   VESTNIKRGMU.RU| |\nЛитература\n1. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, \nViganò P . Endometriosis. Nat Rev Dis Primers. 2018; 4: 9. \n2. Saunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, \nand therapeutic prospects. Cell. 2021; 184 (11): 2807–24. \n3. Bao C, Wang H, Fang H. Genomic Evidence Supports the \nRecognition of Endometriosis as an Inflammatory Systemic \nDisease and Reveals Disease-Specific Therapeutic Potentials of \nTargeting Neutrophil Degranulation. Front Immunol. 2022; 23 (13): \n758440. \n4. Greene R, Stratton P , Cleary SD, Ballweg ML, Sinaii N. Diagnostic \nexperience among 4,334 women reporting surgically diagnosed \nendometriosis. Fertility and sterility. 2009; 91 (1): 32–39. \n5. Greenbaum H, Bat-El L, Galper BEL, Decter DH, Eisenberg VH. \nEndometriosis and autoimmunity: Can autoantibodies be used as \na non-invasive early diagnostic tool? Autoimmun Rev. 2021; 20 \n(5): 102795. \n6. Clement Philip B. The Pathology of Endometriosis: A Survey of \nthe Many Faces of a Common Disease Emphasizing Diagnostic \nPitfalls and Unusual and Newly Appreciated Aspects. Advances \nin Anatomic Pathology. 2007; 14 (4): 241–60. \n7. Taylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic \nsystemic disease: clinical challenges and novel innovations. \nLancet. 2021; 27: 839–52.  \n8. Ярмолинская М. И. Цитокиновый профиль перитонеальной \nжидкости и периферической крови больных с наружным \nгенитальным эндометриозом. Журнал акушерства и женских \nболезней. 2008; 57 (3): 30–34.\n9. Sikora J, Smycz-Kuban ́ska M, Mielczarek-Palacz A, Kondera-\nAnasz Z. Abnormal peritoneal regulation of chemokine activation — the \nrole of IL8 in pathogenesis of endometriosis. American Journal of \nReproductive Immunology. 2017; 77 (4).  \n10. Cameron MJ, Kelvin DJ. Cytokines and chemokines — their \nreceptors and their genes: an overview. Advances in Experimental \nMedicine and Biology. 2003; 520: 8–32. \n11. Somigliana E, Vigan ò P , Tirelli AS, Felicetta I, Torresani E, Vignali \nM, et al. Use of the concomitant serum dosage of CA 125, CA \n19-9 and interleukin-6 to detect the presence of endometriosis. \nResults from a series of reproductive age women undergoing \nlaparoscopic surgery for benign gynaecological conditions. \nHuman Reproduction. 2004; 19 (8): 1871–6. \n12. Dong Hao Lu, Song H, Shi G. Anti-TNFα treatment for pelvic pain \nassociated with endometriosis. Cochrane database of systematic \nreviews. 2010; 3 (3): CD008088. \n13. Li A, Dubey S, Varney ML, Dave BJ, Singh RK. IL8 directly \nenhanced endothelial cell survival, proliferation, and matrix \nmetalloproteinases production and regulated angiogenesis. \nJournal of Immunology. 2003; 170 (6): 3369–76. \n14. Malvezzi H, Hernandes C, Piccinato CA, Podgaec S. Interleukin in \nendometriosis-associated infertility-pelvic pain: systematic review \nand meta-analysis. Reproduction. 2019; 158 (1): 1–12.  \n15. Scholl B, Bersinger NA, Kuhn A. Correlation between symptoms \nof pain and peritoneal fluid inflammatory cytokine concentrations \nin endometriosis. Gynecol Endocrinol. 2009; 25 (11): 701–6.  \n16. Wu HJ, Wu E. The role of gut microbiota in immune homeostasis \nand autoimmunity. Gut Microbes. 2012; 3 (1): 4–14. \n17. Chadchan SB, Cheng M, Parnell LA, Yin Y, Schriefer A, \nMysorekar IU, et al. Antibiotic therapy with metronidazole reduces \nendometriosis disease progression in mice: a potential role for gut \nmicrobiota. Hum Reprod. 2019; 34: 1106–16.\n18. Chadchan SB, Naik SK, Popli P , et al. Gut microbiota and \nmicrobiota-derived metabolites promotes endometriosis. Cell \nDeath Discov. 2023; 9: 28. \n19. Chadchan SB, Popli P , Ambati CR, Tycksen E, Han SJ, Bulun SE, et \nal. Gut microbiota-derived short-chain fatty acids protect against \nthe progression of endometriosis. Life Sci Alliance. 2021; 30; 4 \n(12): e202101224. \n20. Ni Z, Sun S, Bi Y, Ding J, Cheng W, Yu J, et al. Correlation of fecal \nmetabolomics and gut microbiota in mice with endometriosis. Am \nDeath Discov. 2023; 9: 28. \n19. Chadchan SB, Popli P , Ambati CR, Tycksen E, Han SJ, Bulun SE, et \nal. Gut microbiota-derived short-chain fatty acids protect against \nthe progression of endometriosis. Life Sci Alliance. 2021; 30; 4 \n(12): e202101224. \n20. Ni Z, Sun S, Bi Y, Ding J, Cheng W, Yu J, et al. Correlation of fecal \nmetabolomics and gut microbiota in mice with endometriosis. Am \nJ Reprod Immunol. 2020; 84: e13307. \n21. Svensson A, Brunkwall L, Roth B, Orho-Melander M, Ohlsson B. \nAssociations Between Endometriosis and Gut Microbiota. Reprod \nSci. 2021; 28 (8): 2367–77. \n22. Chen S, Gu Z, Zhang W, Jia S, Wu Y, Zheng P , et al. The study \nof endometriosis and adenomyosis related microbiota in female \nlower genital tract in Northern Chinese population. Gynecology \nand Obstetrics Clinical Medicine. 2021; 1 (3): 119–29.  \n23. Ser H-L, Au Yong S-J, Shafiee MN, Mokhtar NM, Ali RAR. Current \nupdates on the role of microbiome in endometriosis: a narrative \nreview. Microorganisms. 2023; 11 (2): 360. \n24. Mitra S, Förster-Fromme K, Damms-Machado A, Scheurenbrand T, \nBiskup S, Huson, DH, et al. Analysis of the intestinal microbiota \nusing SOLiD16S rRNA gene sequencing and SOLiD shotgun \nsequencing. BMC Genomics. 2013; 14 (5): 16.\n25. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, \nCostello EK, et al. QIIME allows analysis of high-throughput \ncommunity sequencing data. Nat Methods. 2010; 7 (5): 335–6.\n26. DeSantis TZ, Hugenholtz P , Larsen N. Greengenes, a \nchimerachecked 16S rRNA gene database and workbench \ncompatible with ARB. Appl Environ Microbiol. 2006; 72: 5069–72.\n27. Ritari J, Saloj ärvi J, Lahti L, de Vos WM. Improved taxonomic \nassignment of human intestinal 16S rRNA sequences by a dedicated \nreference database. BMC Genomics. 2015; 16 (1): 1056.\n28. Vallejo V, Ilagan JG. A Postpartum Death Due to Coronavirus \nDisease 2019 (COVID-19) in the United States. ObstetGynecol. \n2020; 136 (1): 52–55.\n29. Kumari R, Ahuja V, Jaishree P . Fluctuations in butyr-ate-producing \nbacteria in ulcerative colitis patients of North India. World J \nGastroenterol. 2013; 19: 3404–14. \n30. Ata B, Yildiz S, Turkgeldi E, Brocal VP , Dinleyici EC, Moya A, et al. \nThe Endobiota Study: Comparison of Vaginal, Cervical and Gut \nMicrobiota Between Women with Stage 3/4 Endometriosis and \nHealthy Controls. Sci Rep. 2019; 9 (1): 2204. \n31. Kodati VL, Govindan S, Movva S, Ponnala S, Hasan Q. Role \nof Shigella infection in endometriosis: a novel hypothesis. Med \nHypotheses. 2008; 70 (2): 239–43. \n32. Gumenyuk LN, Golod MV, Silaeva NV, Sorokina LE, Ilyasov SS, \nAndroschyuk NA, et al. Gut microbiota alterations and their relationship \nto the disease severity and some cytokine profile indicators in patients \nwith COVID-19. Bulletin of RSMU. 2022; 1: 22–9. \n33. Liu P , Gao M, Liu Z, Zhang Y, Tu H, Lei L, et al. Gut microbiome \ncomposition linked to inflammatory factors and cognitive functions \nin first-episode, drug-naive major depressive disorder patients. \nFront Neurosci. 2022; 28 (15): 800764. \n34. Shan J, Ni Z, Cheng W, Zhou L, Zhai D, Sun S, et al. Gut microbiota \nimbalance and its correlations with hormone and inflammatory \nfactors in patients with stage 3/4 endometriosis. Arch Gynecol \nObstet. 2021; 304: 1363–73. \n35. Philpott DJ, Yamaoka S, Israël A, Sansonetti PJ. Invasive Shigella \nflexneri activates NF-kappa B through a lipopolysaccharide-\ndependent innate intracellular response and leads to IL8 \nexpression in epithelial cells. J Immunol. 2000; 165 (2): 903–14.\n36. Raqib R, Wretlind B, Andersson J, Lindberg AA. Cytokine secretion \nin acute shigellosis is correlated to disease activity and directed \nmore to stool than to plasma. J Infect Dis. 1995; 171: 376–384. \n37. Kim SJ, Choi EY, Kim EG, Shin SH, Lee JY, Choi JI, et al. \nPrevotella intermedia lipopolysaccharide stimulates release of \ntumor necrosis factor-alpha through mitogen-activated protein \nkinase signaling pathways in monocyte-derived macrophages. \nFEMS Immunol Med Microbiol. 2007; 51 (2): 407–13. \n\n15\nORIGINAL RESEARCH    MICROBIOLOGY\nBULLETIN OF RSMU   3, 2023   VESTNIKRGMU.RU| |\nJ Reprod Immunol. 2020; 84: e13307. \n21. Svensson A, Brunkwall L, Roth B, Orho-Melander M, Ohlsson B. \nAssociations Between Endometriosis and Gut Microbiota. Reprod \nSci. 2021; 28 (8): 2367–77. \n22. Chen S, Gu Z, Zhang W, Jia S, Wu Y, Zheng P , et al. The study \nof endometriosis and adenomyosis related microbiota in female \nlower genital tract in Northern Chinese population. Gynecology \nand Obstetrics Clinical Medicine. 2021; 1 (3): 119–29.  \n23. Ser H-L, Au Yong S-J, Shafiee MN, Mokhtar NM, Ali RAR. Current \nupdates on the role of microbiome in endometriosis: a narrative \nreview. Microorganisms. 2023; 11 (2): 360. \n24. Mitra S, Förster-Fromme K, Damms-Machado A, Scheurenbrand T, \nBiskup S, Huson, DH, et al. Analysis of the intestinal microbiota \nusing SOLiD16S rRNA gene sequencing and SOLiD shotgun \nsequencing. BMC Genomics. 2013; 14 (5): 16.\n25. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, \nCostello EK, et al. QIIME allows analysis of high-throughput \ncommunity sequencing data. Nat Methods. 2010; 7 (5): 335–6.\n26. DeSantis TZ, Hugenholtz P , Larsen N. Greengenes, a \nchimerachecked 16S rRNA gene database and workbench \ncompatible with ARB. Appl Environ Microbiol. 2006; 72: 5069–72.\n27. Ritari J, Saloj ärvi J, Lahti L, de Vos WM. Improved taxonomic \nassignment of human intestinal 16S rRNA sequences by a \ndedicated reference database. BMC Genomics. 2015; 16 (1): 1056.\n28. Vallejo V, Ilagan JG. A Postpartum Death Due to Coronavirus \nDisease 2019 (COVID-19) in the United States. ObstetGynecol. \n2020; 136 (1): 52–55.\n29. Kumari R, Ahuja V, Jaishree P . Fluctuations in butyr-ate-producing \nbacteria in ulcerative colitis patients of North India. World J \nGastroenterol. 2013; 19: 3404–14. \n30. Ata B, Yildiz S, Turkgeldi E, Brocal VP , Dinleyici EC, Moya A, et al. \nThe Endobiota Study: Comparison of Vaginal, Cervical and Gut \nMicrobiota Between Women with Stage 3/4 Endometriosis and \nHealthy Controls. Sci Rep. 2019; 9 (1): 2204. \n31. Kodati VL, Govindan S, Movva S, Ponnala S, Hasan Q. Role \nof Shigella infection in endometriosis: a novel hypothesis. Med \nHypotheses. 2008; 70 (2): 239–43. \n32. Гуменюк Л. Н., Г олод М. В., Силаева Н. В., Сорокина Л. Е., \nИльясов С. С., Андрощук Н. А. и др. Изменения микробиоты \nкишечника и их связь с тяжестью заболевания и некоторыми \nпоказателями цитокинового профиля у пациентов с \nCOVID-19. Вестник РГМУ. 2022; 1: 23–30. \n33. Liu P , Gao M, Liu Z, Zhang Y, Tu H, Lei L, et al. Gut microbiome \ncomposition linked to inflammatory factors and cognitive functions \nin first-episode, drug-naive major depressive disorder patients. \nFront Neurosci. 2022; 28 (15): 800764. \n34. Shan J, Ni Z, Cheng W, Zhou L, Zhai D, Sun S, et al. Gut microbiota \nimbalance and its correlations with hormone and inflammatory \nfactors in patients with stage 3/4 endometriosis. Arch Gynecol \nObstet. 2021; 304: 1363–73. \n35. Philpott DJ, Yamaoka S, Israël A, Sansonetti PJ. Invasive Shigella \nflexneri activates NF-kappa B through a lipopolysaccharide-\ndependent innate intracellular response and leads to IL8 \nexpression in epithelial cells. J Immunol. 2000; 165 (2): 903–14.\n36. Raqib R, Wretlind B, Andersson J, Lindberg AA. Cytokine secretion \nin acute shigellosis is correlated to disease activity and directed \nmore to stool than to plasma. J Infect Dis. 1995; 171: 376–384. \n37. Kim SJ, Choi EY, Kim EG, Shin SH, Lee JY, Choi JI, et al. \nPrevotella intermedia lipopolysaccharide stimulates release of \ntumor necrosis factor-alpha through mitogen-activated protein \nkinase signaling pathways in monocyte-derived macrophages. \nFEMS Immunol Med Microbiol. 2007; 51 (2): 407–13.","source_license":"CC0","license_restricted":false}