Endometrial microbiome: new hope, or hype?

Abstract

Technological advances in genomics and bioinformatics have resulted in an explosion of studies defining microbial communities in different body sites by sequencing the unique bacterial 16S ribosomal RNA gene, providing an analysis of the genomes of the microbes present, termed the microbiome. Increasingly, organs and tissues thought once to be sterile are shown to host commensal bacteria in the absence of infection, including the upper female reproductive tract and the placenta (Baker et al., 2018Baker J.M. Chase D.M. Herbst-Kralovetz M.M. Uterine Microbiota: Residents, Tourists, or Invaders?.Front. Immunol. 2018; 9: 208Crossref PubMed Scopus (87) Google Scholar; Benner et al., 2018Benner M. Ferwerda G. Joosten I. van der Molen R.G. How uterine microbiota might be responsible for a receptive, fertile endometrium.Hum. Reprod. Update. 2018; 24: 393-415Crossref PubMed Scopus (64) Google Scholar; Evans et al., 2016Evans J. Salamonsen L.A. Winship A. Menkhorst E. Nie G. Gargett C.E. Dimitriadis E. Fertile ground: human endometrial programming and lessons in health and disease.Nat. Rev. Endocrinol. 2016; 12: 654-667Crossref PubMed Scopus (108) Google Scholar; Koedooder et al., 2019Koedooder R. Mackens S. Budding A. Fares D. Blockeel C. Laven J. Schoenmakers S. Identification and evaluation of the microbiome in the female and male reproductive tracts.Hum. Reprod. Update. 2019; 25: 298-325Crossref PubMed Scopus (37) Google Scholar). The revolutionary studies by Mitchell et al. (Mitchell et al., 2015Mitchell C.M. Haick A. Nkwopara E. Garcia R. Rendi M. Agnew K. Fredricks D.N. Eschenbach D. Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.Am. J. Obstet. Gynecol. 2015; 212 (e1-611.e9): 611Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), Franasiak et al. (Franasiak et al., 2016Franasiak J.M. Werner M.D. Juneau C.R. Tao X. Landis J. Zhan Y. Treff N.R. Scott R.T. Endometrial microbiome at the time of embryo transfer: next-generation sequencing of the 16S ribosomal subunit.J. Assist. Reprod. Genet. 2016; 33: 129-136Crossref PubMed Scopus (102) Google Scholar), Moreno et al. (Moreno et al., 2016Moreno I. Codoñer F.M. Vilella F. Valbuena D. Martinez-Blanch J.F. Jimenez-Almazán J. Alonso R. Alamá P. Remohí J. Pellicer A. Ramon D. Simon C. Evidence that the endometrial microbiota has an effect on implantation success or failure.Am. J. Obstet. Gynecol. 2016; 215: 684-703Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), Verstraelen et al. (Verstraelen et al., 2016Verstraelen H. Vilchez-Vargas R. Desimpel F. Jauregui R. Vankeirsbilck N. Weyers S. Verhelst R. De Sutter P. Pieper D.H. Van De Wiele T. Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene.PeerJ. 2016; 4: e1602Crossref PubMed Scopus (133) Google Scholar) and Chen et al. (Chen et al., 2017Chen C. Song X. Wei W. Zhong H. Dai J. Lan Z. Li F. Yu X. Feng Q. Wang Z. Xie H. Chen X. Zeng C. Wen B. Zeng L. Du H. Tang H. Xu C. Xia Y. Xia H. Yang H. Wang Jian Wang Jun Madsen L. Brix S. Kristiansen K. Xu X. Li J. Wu R. Jia H. The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.Nat. Commun. 2017; 8: 875Crossref PubMed Scopus (224) Google Scholar) among others, have paved the way of microbiome research in the reproductive medicine, highlighting the possible influence of the microbiome in endometrial function. Special focus has been on detecting sequences from Lactobacillus species, as Lactobacillus dominance in the sample (defined as ≥90% or ≥80% Lactobacillus species) has been associated with increased implantation, pregnancy, ongoing pregnancy and live birth rates, while Lactobacillus species depletion and increased abundance of Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus, Streptococcus, Aptopobium, Bifidobacterium, and Chryseobacterium species has been associated with clinical miscarriage or no pregnancy (Kyono et al., 2019Kyono K. Hashimoto T. Kikuchi S. Nagai Y. Sakuraba Y. A pilot study and case reports on endometrial microbiota and pregnancy outcome: An analysis using 16S rRNA gene sequencing among IVF patients, and trial therapeutic intervention for dysbiotic endometrium.Reprod. Med. Biol. 2019; 18: 72-82Crossref PubMed Scopus (36) Google Scholar; Moreno et al., 2021Moreno, I., Garcia-Grau, I., Perez-Villaroya, D., Gonzalez-Monfort, M., Bahçeci, M., Barrionuevo, M.J., Taguchi, S., Puente, E., Dimattina, M., Wei Lim, M., Meneghini, G., Aubuchon, M., Leondires, M., Izquierdo, A., Perez-Olgiati, M., Chavez, A., Seethram, K., Bau, D., Gomez, C., Valbuena, D., Vilella, F., Simon, C., 2021. Endometrial microbiota composition is associated with reproductive outcome in infertile patients. medRxiv.Google Scholar, Moreno et al., 2016Moreno I. Codoñer F.M. Vilella F. Valbuena D. Martinez-Blanch J.F. Jimenez-Almazán J. Alonso R. Alamá P. Remohí J. Pellicer A. Ramon D. Simon C. Evidence that the endometrial microbiota has an effect on implantation success or failure.Am. J. Obstet. Gynecol. 2016; 215: 684-703Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Nevertheless, several studies have not detected any association between the microbiome and gynaecological/reproductive outcomes (reviewed in Molina et al., 2021Molina N.M. Sola-Leyva A. Haahr T. Aghajanova L. Laudanski P. Castilla J.A. Altmäe S. Analysing endometrial microbiome: methodological considerations and recommendations for good practice.Hum. Reprod. 2021; 36: 859-879Crossref PubMed Scopus (7) Google Scholar), and have questioned Lactobacillus dominance within the endometrium (Sola-Leyva et al., 2021Sola-Leyva A. Andrés-León E. Molina N.M. Terron-Camero L.C. Plaza-Díaz J. Sáez-Lara M.J. Gonzalvo M.C. Sánchez R. Ruíz S. Martínez L. Altmäe S. Mapping the entire functionally active endometrial microbiota.Hum. Reprod. 2021; 36Crossref Scopus (2) Google Scholar; Verstraelen et al., 2016Verstraelen H. Vilchez-Vargas R. Desimpel F. Jauregui R. Vankeirsbilck N. Weyers S. Verhelst R. De Sutter P. Pieper D.H. Van De Wiele T. Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene.PeerJ. 2016; 4: e1602Crossref PubMed Scopus (133) Google Scholar; Winters et al., 2019Winters A.D. Romero R. Gervasi M.T. Gomez-Lopez N. Tran M.R. Garcia-Flores V. Pacora P. Jung E. Hassan S.S. Hsu C.-D. Theis K.R. Does the endometrial cavity have a molecular microbial signature?.Sci. Rep. 2019; 9: 9905Crossref PubMed Scopus (40) Google Scholar), and whether microbes are detectable in all endometrial samples (Oberle et al., 2021Oberle A. Urban L. Falch-Leis S. Ennemoser C. Nagai Y. Ashikawa K. Ulm P.A. Hengstschläger M. Feichtinger M. 16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis.Reprod. Biomed. Online. 2021; 42 (in this issue): 1097-1107Abstract Full Text Full Text PDF Scopus (1) Google Scholar; Winters et al., 2019Winters A.D. Romero R. Gervasi M.T. Gomez-Lopez N. Tran M.R. Garcia-Flores V. Pacora P. Jung E. Hassan S.S. Hsu C.-D. Theis K.R. Does the endometrial cavity have a molecular microbial signature?.Sci. Rep. 2019; 9: 9905Crossref PubMed Scopus (40) Google Scholar). In all previous endometrial microbiome studies, other than two metagenome shotgun sequencing studies (Garcia-Grau et al., 2019Garcia-Grau I. Perez-Villaroya D. Bau D. Gonzalez-Monfort M. Vilella F. Moreno I. Simon C. Taxonomical and Functional Assessment of the Endometrial Microbiota in A Context of Recurrent Reproductive Failure: A Case Report.Pathogens. 2019; 8: 4-6Crossref Scopus (13) Google Scholar; Li et al., 2018Li F. Chen C. Wei W. Wang Z. Dai J. Hao L. Song L. Zhang X. Zeng L. Du H. Tang H. Liu N. Yang H. Wang J. Madsen L. Brix S. Kristiansen K. Xu X. Li J. Wu R. Jia H. The metagenome of the female upper reproductive tract.Gigascience. 2018; 7: giy107Crossref Scopus (27) Google Scholar), short-read sequencing of one or multiple hypervariable regions (V1–V9) of the bacterial 16S rRNA gene has been analysed. However, full-length sequencing of the whole 16S rRNA gene (∼1500 bp) has been shown to facilitate microbiome identification by yielding a deeper level of taxonomic resolution (Johnson et al., 2019Johnson J.S. Spakowicz D.J. Hong B.-Y. Petersen L.M. Demkowicz P. Chen L. Leopold S.R. Hanson B.M. Agresta H.O. Gerstein M. Sodergren E. Weinstock G.M. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis.Nat. Commun. 2019; 10: 5029Crossref PubMed Scopus (212) Google Scholar). The current issue of RBMO presents a novel study by Oberle and colleagues where a new platform for detecting endometrial microbiome is proposed: in-situ 16S rRNA gene long-read sequencing using portable nanopore sequencing technology MinION (Oxford Nanopore Technologies, Oxford, UK) (Oberle et al., 2021Oberle A. Urban L. Falch-Leis S. Ennemoser C. Nagai Y. Ashikawa K. Ulm P.A. Hengstschläger M. Feichtinger M. 16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis.Reprod. Biomed. Online. 2021; 42 (in this issue): 1097-1107Abstract Full Text Full Text PDF Scopus (1) Google Scholar). The study protocol demonstrates accurate assessment of the endometrial microbiome which could serve as a template for a fast, inexpensive and portable diagnostic workflow. While being an initial pilot study, it is nevertheless carefully designed with appropriate positive and negative controls for bias and contamination evaluation, using validation approaches with different platforms and centres. The results are very promising and could lead to feasible and accessible solutions for any clinic as MinION does not require high initial investment outlay and could be used in remote places to obtain data quickly and on site (Oberle et al., 2021Oberle A. Urban L. Falch-Leis S. Ennemoser C. Nagai Y. Ashikawa K. Ulm P.A. Hengstschläger M. Feichtinger M. 16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis.Reprod. Biomed. Online. 2021; 42 (in this issue): 1097-1107Abstract Full Text Full Text PDF Scopus (1) Google Scholar). However, despite the potential of this new technology, there is still a gap in knowledge between the molecular analyses and clinical applicability. We need to establish what we are looking for – a molecular marker for predicting infertility treatment outcomes or to identify dysregulated microbial composition and tailor treatment accordingly? Lactobacillus species dominance seems to serve as potential biomarker predicting IVF success (Kyono et al., 2019Kyono K. Hashimoto T. Kikuchi S. Nagai Y. Sakuraba Y. A pilot study and case reports on endometrial microbiota and pregnancy outcome: An analysis using 16S rRNA gene sequencing among IVF patients, and trial therapeutic intervention for dysbiotic endometrium.Reprod. Med. Biol. 2019; 18: 72-82Crossref PubMed Scopus (36) Google Scholar; Moreno et al., 2016Moreno I. Codoñer F.M. Vilella F. Valbuena D. Martinez-Blanch J.F. Jimenez-Almazán J. Alonso R. Alamá P. Remohí J. Pellicer A. Ramon D. Simon C. Evidence that the endometrial microbiota has an effect on implantation success or failure.Am. J. Obstet. Gynecol. 2016; 215: 684-703Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), and specific bacteria have been associated with gynaecological disorders (reviewed in Molina et al., 2020Molina N.M. Sola-Leyva A. Saez-Lara M.J. Plaza-Diaz J. Tubić-Pavlović A. Romero B. Clavero A. Mozas-Moreno J. Fontes J. Altmäe S. New Opportunities for Endometrial Health by Modifying Uterine Microbial Composition: Present or Future?.Biomolecules. 2020; 10: 593Crossref Scopus (19) Google Scholar). In addition, a recent multi-centre study analysing 342 infertile patients, the biggest cohort studied to date, concluded that endometrial microbial composition before embryo transfer is a useful biomarker to predict reproductive outcome, offering an opportunity to further improve diagnosis and treatment strategies (Moreno et al., 2021Moreno, I., Garcia-Grau, I., Perez-Villaroya, D., Gonzalez-Monfort, M., Bahçeci, M., Barrionuevo, M.J., Taguchi, S., Puente, E., Dimattina, M., Wei Lim, M., Meneghini, G., Aubuchon, M., Leondires, M., Izquierdo, A., Perez-Olgiati, M., Chavez, A., Seethram, K., Bau, D., Gomez, C., Valbuena, D., Vilella, F., Simon, C., 2021. Endometrial microbiota composition is associated with reproductive outcome in infertile patients. medRxiv.Google Scholar). Nevertheless, further studies with large patient cohorts are required to establish the sensitivity and specificity of microbial biomarkers for clinical application, as the findings between different studies conducted so far are barely comparable and occasionally contradictory, mainly due to the varying and limited study designs (Molina et al., 2021Molina N.M. Sola-Leyva A. Haahr T. Aghajanova L. Laudanski P. Castilla J.A. Altmäe S. Analysing endometrial microbiome: methodological considerations and recommendations for good practice.Hum. Reprod. 2021; 36: 859-879Crossref PubMed Scopus (7) Google Scholar). In the case of treating microbial dysbiosis, no consensus has been reached on the microbial profile of endometrial bacterial pathogens and the mechanisms by which they could interfere with endometrial functions. Furthermore, we are still lacking information on what is normal, what is the core uterine microbial composition in healthy fertile women. It is not clear whether dysbiosis within the uterus is a cause or a consequence of a pathology. Further, microbiome analysis using 16S rRNA gene sequencing is based on analysis of DNA, which does not necessarily equate with the presence of live bacteria. DNA molecules can persist for decades (Glassing et al., 2016Glassing A. Dowd S.E. Galandiuk S. Davis B. Chiodini R.J. Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples.Gut. Pathog. 2016; 8: 24Crossref PubMed Scopus (195) Google Scholar), thus DNA sequences may be present in microbial breakdown products (e.g. DNA from dead bacteria) or be present as background DNA contamination (de Goffau et al., 2018de Goffau M.C. Lager S. Salter S.J. Wagner J. Kronbichler A. Charnock-Jones D.S. Peacock S.J. Smith G.C.S. Parkhill J. Recognizing the reagent microbiome.Nat. Microbiol. 2018; 3: 851-853Crossref PubMed Scopus (111) Google Scholar). Thereby detection of bacterial DNA is not the same as the detection of a living microbe; DNA can be used to characterise a microbiome but not to establish its existence (Willyard, 2018Willyard C. Could baby's first bacteria take root before birth?.Nature. 2018; 553: 264-266Crossref PubMed Scopus (52) Google Scholar). In short, in order to treat ‘dysbiosis’ with antibiotics and/or pro-/pre-/synbiotics (treatment strategies summarised in Molina et al., 2020Molina N.M. Sola-Leyva A. Saez-Lara M.J. Plaza-Diaz J. Tubić-Pavlović A. Romero B. Clavero A. Mozas-Moreno J. Fontes J. Altmäe S. New Opportunities for Endometrial Health by Modifying Uterine Microbial Composition: Present or Future?.Biomolecules. 2020; 10: 593Crossref Scopus (19) Google Scholar), we have to first identify living microbes within the uterus before offering any treatment strategies for patients. As less than 1% of microbes can grow and form colonies on agar plates (Wade, 2002Wade W. Unculturable bacteria–the uncharacterized organisms that cause oral infections.J. R. Soc. Med. 2002; 95: 81-83Crossref PubMed Scopus (104) Google Scholar), one alternative would be to analyse the sequences of mRNA molecules that reflect microbial functional activity (meta-transcriptomics), bacterial proteins (meta-proteomics) or bacterial metabolites (meta-metabolomics) within the sample (see Sola-Leyva et al., 2021Sola-Leyva A. Andrés-León E. Molina N.M. Terron-Camero L.C. Plaza-Díaz J. Sáez-Lara M.J. Gonzalvo M.C. Sánchez R. Ruíz S. Martínez L. Altmäe S. Mapping the entire functionally active endometrial microbiota.Hum. Reprod. 2021; 36Crossref Scopus (2) Google Scholar; Molina et al., 2021Molina N.M. Sola-Leyva A. Haahr T. Aghajanova L. Laudanski P. Castilla J.A. Altmäe S. Analysing endometrial microbiome: methodological considerations and recommendations for good practice.Hum. Reprod. 2021; 36: 859-879Crossref PubMed Scopus (7) Google Scholar for techniques). As it stands, we are facing more questions than answers, and the question of whether the endometrial microbiome is a new hope in reproductive medicine needs to be clarified with future studies. The applicability of microbial composition and molecular biomarkers needs to be confirmed with large cohort studies, and the identification of living bacteria within the uterus, including the core profile of microbial composition in health and disease, need to be addressed before any treatment strategies for patients can be offered. Future studies will clarify how much endometrial microbes contribute to the cogwheels of hormones and physiological adaptations that are required for successful embryo implantation and pregnancy establishment, and whether, to what extent, and how, the microenvironment could be modified in endometrial microbial dysbiosis.