{"paper_id":"b4e1c03f-ac75-4513-893b-1f3d411e7eea","body_text":"Abstract\nEndometriosis remains a challenge to understand and to diagnose. This is an observational cross-sectional pilot study to characterize the gut and vaginal microbiome profiles among endometriosis patients and control subjects without the disease and to explore their potential use as a less-invasive diagnostic tool for endometriosis. Overall, 59 women were included, n = 35 with endometriosis and n = 24 controls. Rectal and vaginal samples were collected in two different periods of the menstrual cycle from all subjects. Gut and vaginal microbiomes from patients with different rASRM (revised American Society for Reproductive Medicine) endometriosis stages and controls were analyzed. Illumina sequencing libraries were constructed using a two-step 16S rRNA gene PCR amplicon approach. Correlations of 16S rRNA gene amplicon data with clinical metadata were conducted using a random forest-based machine-learning classification analysis. Distribution of vaginal CSTs (community state types) significantly differed between follicular and menstrual phases of the menstrual cycle (p = 0.021, Fisher’s exact test). Vaginal and rectal microbiome profiles and their association to severity of endometriosis (according to rASRM stages) were evaluated. Classification models built with machine-learning methods on the microbiota composition during follicular and menstrual phases of the cycle were built, and it was possible to accurately predict rASRM stages 1–2 verses rASRM stages 3–4 endometriosis. The feature contributing the most to this prediction was an OTU (operational taxonomic unit) from the genus Anaerococcus. Gut and vaginal microbiomes of women with endometriosis have been investigated. Our findings suggest for the first time that vaginal microbiome may predict stage of disease when endometriosis is present.\nSimilar content being viewed by others\nData Availability\nThe raw sequencing data of the vaginal and rectal samples used in this study as well as the associated metadata have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject PRJNA424567.\nReferences\nBurney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril. 2012;98(3):511–9.\nLaganà AS, Vitale SG, Salmeri FM, Triolo O, Ban Frangež H, Vrtačnik-Bokal E, et al. Unus pro omnibus, omnes pro uno: a novel, evidence-based, unifying theory for the pathogenesis of endometriosis. Med Hypotheses. 2017 Jun;103:10–20.\nRiccio LDGC, Santulli P, Marcellin L, Abrão MS, Batteux F, Chapron C. Immunology of endometriosis. Best Pract Res Clin Obstet Gynaecol. 2018 Feb 8. pii: S1521–6934(18)30028–2.\nSenturk LM, Arici A. Immunology of endometriosis. J Reprod Immunol. 1999;43(1):67e83.\nChristodoulakos G, Augoulea A, Lambrinoudaki I, Sioulas V, Creatsas G. Pathogenesis of endometriosis: the role of defective 'immunosurveillance'. Eur J Contracept Reprod Health Care. 2007;12(3):194e202.\nAbrao MS, Gonçalves MO, Dias JA Jr, Podgaec S, Chamie LP, Blasbalg R. Comparison between clinical examination, transvaginal sonography and magnetic resonance imaging for the diagnosis of deep endometriosis. Hum Reprod. 2007 Dec;22(12):3092–7.\nDunselman GA, Vermeulen N, Becker C, Calhaz-Jorge C, D'Hooghe T, De Bie B, Heikinheimo O, Horne AW, Kiesel L, Nap A, Prentice A, Saridogan E, Soriano D, Nelen W; European society of human reproduction and embryology. ESHRE guideline: management of women with endometriosis. Hum Reprod 2014 Mar;29(3):400–412.\nO DF, Flores I, Waelkens E, D'Hooghe T. Noninvasive diagnosis of endometriosis: review of current peripheral blood and endometrial biomarkers. Best Pract Res Clin Obstet Gynaecol 2018 Jul;50:72–83. doi: https://doi.org/10.1016/j.bpobgyn.2018.04.001. Epub 2018 Apr 13.\nNisenblat V, Prentice L, Bossuyt PM, Farquhar C, Hull ML, Johnson N. Combination of the non-invasive tests for the diagnosis of endometriosis. Cochrane Database Syst Rev. 2016 Jul 13;7:CD012281. https://doi.org/10.1002/14651858.CD012281 Review.\nBorrelli GM, Abrão MS, Mechsner S. Can chemokines be used as biomarkers for endometriosis? A systematic review. Hum Rep. 2014;29(2):253–66.\nPeterson CT, Sharma V, Elmen L, Peterson SN. Immune homeostasis, dysbiosis and therapeutic modulation of the gut microbiota. Clin Exp Immunol. 2015;179:363–77.\nRogers GB, Bruce KD. Next-generation sequencing in the analysis of human microbiota: essential considerations for clinical application. Mol Diagn Ther. 2010 Dec 1;14(6):343-.\nClemente JC, Manasson J, Scher JU. The role of the gut microbiome in systemic inflammatory disease. BMJ. 2018 Jan 8;360:j5145.\nKiely CJ, Pavli P, O'Brien CL. The role of inflammation in temporal shifts in the inflammatory bowel disease mucosal microbiome. Gut Microbes. 2018 Mar;15:1–25.\nOpazo MC, Ortega-Rocha EM, Coronado-Arrázola I, Bonifaz LC, Boudin H, Neunlist M, et al. Intestinal microbiota influences non-intestinal related autoimmune diseases. Front Microbiol. 2018 Mar 12;9:432.\nNarayanan V, Peppelenbosch MP, Konstantinov SR. Human fecal microbiome-based biomarkers for colorectal cancer. Cancer Prev Res (Phila). 2014 Nov;7(11):1108–11.\nQuigley EMM. Gut microbiome as a clinical tool in gastrointestinal disease management: are we there yet? Nat Rev Gastroenterol Hepatol. 2017 May;14(5):315–20.\nChen C, Song X, Wei W, Zhong H, Dai J, Lan Z, et al. The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases. Nat Commun. 2017 Oct 17;8(1):875.\nCampos GB, Marques LM, Rezende IS, Barbosa MS, Abrão MS, Timenetsky J. Mycoplasma genitalium can modulate the local immune response in patients with endometriosis. Fertil Steril. 2018 Mar;109(3):549–60.\nPodgaec S, Abrao MS, Dias JA, Rizzo LV, De Oliveira RM, Baracat EC. Endometriosis: an inflammatory disease with a Th2 immune response component. Hum Reprod. 2007;22(5):1373–9.\nKvaskoff M, Mu F, Terry KL, Harris HR, Poole EM, Farland L, et al. Endometriosis: a high-risk population for major chronic diseases? Hum Reprod Update. 2015 Jul-Aug;21(4):500–16.\nMaybin JA, Critchley HOD. Menstrual physiology: implications for endometrial pathology and beyond. Hum Reprod Update. 2015;21(6):748–61.\nPreheim SP, Perrotta AR, Martin-Platero AM, Gupta A, Alm EJ. Distribution-based clustering: using ecology to refine the operational taxonomic unit. Appl Environ Microbiol. 2013 Nov;79(21):6593–603.\nWang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73(16):5261–7.\nBrooks JP, Buck GA, Chen G, Diao L, Edwards DJ, Fettweis JM, et al. Changes in vaginal community state types reflect major shifts in the microbiome. Microb Ecol Health Dis. 2017 Apr 10;28(1):1303265.\nRavel J, Gajer P, Abdo Z, Schneider GM, Koenig SSK, Mcculle SL, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci. 2010;108 (Supplement 1):4680–7.\nPapa E, Docktor M, Smillie C, Weber S, Preheim SP, Gevers D, et al. Non-invasive mapping of the gastrointestinal microbiota identifies children with inflammatory bowel disease. PLoS One. 2012 Jan 29;7(6):e39242.\nBeste MT, Pfäffle-Doyle N, Prentice EA, Morris SN, Lauffenburger DA, Isaacson KB, et al. Molecular network analysis of endometriosis reveals a role for c-Jun—regulated macrophage activation. Sci Transl Med. 2014;6(222):222ra16--222ra16.\nQuast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2012;gks1219.\nLaschke MW, Menger MD. The gut microbiota: a puppet master in the pathogenesis of endometriosis? Am J Obstet Gynecol. 2016 Jul;215(1):68.e1–4.\nYuan M, Li D, Zhang Z, Sun H, An M, Wang G. Endometriosis induces gut microbiota alterations in mice. Hum Reprod. 2018 Apr 1;33(4):607–16.\nZhou X, Hansmann MA, Davis CC, Suzuki H, Brown CJ, Pierson JD, et al. The vaginal bacterial communities of Japanese women resemble those of women in other racial groups. FEMS Immunol Med Microbiol. 2010;58(2):169–81.\nAnahtar MN, Byrne EH, Fichorova RN, Kwon DS, Anahtar MN, Byrne EH, et al. Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital article cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract. Immunity. 2015;42(5):965–76.\nRomero R, Hassan SS, Gajer P, Tarca AL, Fadrosh DW, Nikita L, et al. The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women. Microbiome. 2014;2(1):4.\nGajer P, Brotman RM, Bai G, Sakamoto J, Schütte UME, Zhong X, et al. Temporal dynamics of the human vaginal microbiota. Sci Transl Med. 2012;4(132):132ra52-132ra52.\nFarage M, Maibach H. Lifetime changes in the vulva and vagina. Arch Gynecol Obstet. 2006;273(4):195–202.\nNunn KL, Forney LJ. Unraveling the dynamics of the human vaginal microbiome. Yale J Biol Med. 2016;89:331–7.\nEslami S, Hadjati J, Motevaseli E, Mirzaei R, Farashi Bonab S, Ansaripour B, et al. Lactobacillus crispatus strain SJ-3C-US induces human dendritic cells (DCs) maturation and confers an anti-inflammatory phenotype to DCs. APMIS. 2016 Aug;124(8):697–710.\nPodgaec S, Rizzo LV, Fernandes LF, Baracat EC, Abrao MS. CD4(+) CD25(high) Foxp3(+) cells increased in the peritoneal fluid of patients with endometriosis. Am J Reprod Immunol. 2012 Oct;68(4):301–8.\nDavid LA, Materna AC, Friedman J, Campos-Baptista MI, Blackburn MC, Perrotta A, et al. Host lifestyle affects human microbiota on daily timescales. Genome Biol. 2015;15(7).\nAcknowledgments\nThe authors would like to acknowledge the support of Marta Privato for providing invaluable organizational and communication support over the course of this research.\nThe authors would like to acknowledge the support of Pawel Gajer and Jacque Ravel for providing access to speciateIT and McClassifier, the trained support vector machine learning model utilized for CST assignment in this study.\nFunding\nLibrary preparation and sequencing efforts through the MIT BioMicro Center were funded by the National Institute of Environmental Health Sciences of the NIH under award P30-ES002109.\nThis study was funded by grants from the Brazilian Council for Scientific and Technological Development (CNPq). Specifically, the Massachusetts Institute of Technology (MIT)/Brazil Seed Fund (Process number: CNPq-MIT 457125/2012-8).\nAuthor information\nAuthors and Affiliations\nEthics declarations\nThe Internal Review Boards of the University of Sao Paulo (CAPPesq #814.743) and the Massachusetts Institute of Technology (COUHES protocol # 1107004572) approved this protocol. Written informed consent was obtained from all participants.\nAdditional information\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nElectronic Supplementary Material\nESM 1 (download DOCX )\n(DOCX 19 kb)\nRights and permissions\nAbout this article\nCite this article\nPerrotta, A.R., Borrelli, G.M., Martins, C.O. et al. The Vaginal Microbiome as a Tool to Predict rASRM Stage of Disease in Endometriosis: a Pilot Study. Reprod. Sci. 27, 1064–1073 (2020). https://doi.org/10.1007/s43032-019-00113-5\nReceived:\nAccepted:\nPublished:\nVersion of record:\nIssue date:\nDOI: https://doi.org/10.1007/s43032-019-00113-5","source_license":"public-domain-us","license_restricted":false}