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Gynecologic health and disease in relation to the microbiome of the female reproductive tract

  • Katherine A. Green
    Affiliations
    Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland
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  • Shvetha M. Zarek
    Affiliations
    Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland
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  • William H. Catherino
    Correspondence
    Reprint requests: William H. Catherino, M.D., Ph.D., Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Building A, Room 3078, 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799.
    Affiliations
    Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

    Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, Maryland
    Search for articles by this author
      It is well established that the vagina is colonized by bacteria that serve important roles in homeostasis. Imbalances in the proportion of bacteria may lead to a predisposition to infection or reproductive complications. Molecular-based approaches demonstrated a greater degree of microbial diversity both within and between women than previously recognized. The vaginal microbiome may fluctuate during various states of health, such as during the menstrual cycle or after menopause, and there may be differences in the vaginal microbiome between women of different ethnicities. Furthermore, the specific composition of the vaginal microbiome may influence the predisposition to dysbiosis and the transmission of sexually transmitted infections. An understanding of the diversity of the vaginal microbial environment during states of health is essential for the identification of risk factors for disease and the development of appropriate treatment.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/greenk-gynecologic-health-microbiome/
      The maintenance of human health is dependent on a symbiotic relationship between humans and associated bacteria. There are approximately 10 times as many microbes associated with a human as there are human cells in the body (
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      A rendezvous with our microbes.
      ). Despite recognition of the importance of the interactions between the host human body and the bacteria it supports, there remain many unanswered questions regarding how the microbial environment varies within and among individuals in healthy and diseased states. Historically, bacteria have been identified using Gram stain or culture-based techniques. However, as few as 20% of bacteria closely associated with the human body can be cultivated via culture-based techniques (
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      ). Culture-based methods may therefore underestimate the diversity of the microbiome.
      Over the past decade there has been an explosion of interest in molecular-based, culture-independent techniques to study the microbiome. Molecular-based techniques primarily involve analysis of 16S ribosomal RNA (rRNA) with polymerase chain reaction (PCR), DNA hybridization or fingerprinting, and next-generation sequencing (
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      Comparison of the diversity of the vaginal microbiota in HIV-infected and HIV-uninfected women with or without bacterial vaginosis.
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      Pyrosequencing of the genital microbiotas of HIV-seropositive and -seronegative women reveals Lactobacillus iners as the predominant Lactobacillus Species.
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      ). Recognizing the potential of molecular techniques to further our understanding of human bacterial communities, the National Institutes of Health initiated the Human Microbiome Project (HMP) in 2007 (
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      Human Microbiome Project Consortium
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      ). In this consortium, samples from approximately 300 healthy human subjects from body sites including the skin, nose, mouth, lower gastrointestinal tract, and vagina were analyzed in an effort to more accurately characterize the normal human microbial environment (
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      The NIH Human Microbiome Project.
      ,
      Human Microbiome Project Consortium
      Structure, function and diversity of the healthy human microbiome.
      ,
      Human Microbiome Project Consortium
      A framework for human microbiome research.
      ,
      • Aagaard K.
      • Petrosino J.
      • Keitel W.
      • Watson M.
      • Katancik J.
      • Garcia N.
      • et al.
      The Human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters.
      ).
      The HMP targeted the genitourinary system because it has been established for more than a century that bacteria are present within the vagina and that an imbalance within this microbial environment may be associated with disease (
      • Doderlein A.
      Das scheidensekret und seine bedeutung fur puerperalfieber.
      ,
      • Gardner H.L.
      • Dukes C.D.
      Haemophilus vaginalis vaginitis: a newly defined specific infection previously classified non-specific vaginitis.
      ,
      • Spiegel C.A.
      • Amsel R.
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      • Eschenbach D.
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      Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations.
      ). Research has demonstrated that alterations in the vaginal microbiome affect susceptibility to gynecologic infections, including cervicovaginitis, postoperative infections, and human immunodeficiency virus (HIV) infection (
      • Schwebke J.R.
      Gynecologic consequences of bacterial vaginosis.
      ,
      • Soper D.E.
      • Bump R.C.
      • Hurt W.G.
      Bacterial vaginosis and trichomoniasis vaginitis are risk factors for cuff cellulitis after abdominal hysterectomy.
      ,
      • Plitt S.S.
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      • Gaydos C.A.
      • Strathdee S.A.
      • Sherman S.G.
      • Taha T.E.
      Prevalence and correlates of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis infections, and bacterial vaginosis among a cohort of young injection drug users in Baltimore, Maryland.
      ,
      • Cherpes T.L.
      • Meyn L.A.
      • Krohn M.A.
      • Lurie J.G.
      • Hillier S.L.
      Association between acquisition of herpes simplex virus type 2 in women and bacterial vaginosis.
      ,
      • Martin H.L.
      • Richardson B.A.
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      • Lavreys L.
      • Hillier S.L.
      • Chohan B.
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      Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition.
      ,
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      • Mayer K.H.
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      Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract.
      ,
      • Cohen C.R.
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      • Baeten J.M.
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      • Spiegel C.A.
      • Hong T.
      • et al.
      Bacterial vaginosis associated with increased risk of female-to-male HIV-1 transmission: a prospective cohort analysis among African couples.
      ). Nevertheless, data from molecular-based techniques suggest that many of the differences in the vaginal microbiome may represent normal variation and may not necessarily indicate disease.
      An important consideration when interpreting molecular-based microbiome studies is that molecular techniques primarily rely on the sequencing of bacterial RNA. Unless specific focus is placed on defining relative proportions of bacteria, the presence of a particular organism's genetic material does not imply that it is dominant or even present in significant concentration. Information obtained from both culture- and molecular-based methods are presented in this review, because a combination of the two methods may clarify both the role of bacteria in maintaining gynecologic health and how changes to the microbiome impact susceptibility to disease.

      Vaginal microbiome in the healthy state

      It is well established that the normal vaginal microbiome is dominated by Lactobacilli species (
      • Doderlein A.
      Das scheidensekret und seine bedeutung fur puerperalfieber.
      ,
      • Thomas S.
      Doderlein’s bacillus: Lactobacillus acidophilus.
      ,
      • Redondo-Lopez V.
      • Cook R.L.
      • Sobel J.D.
      Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora.
      ). Lactobacilli help prevent vaginal infection by producing lactic acid, hydrogen peroxide, bacteriocins, or through competitive exclusion of other bacteria (
      • O’Hanlon D.E.
      • Moench T.R.
      • Cone R.A.
      Vaginal pH and microbicidal lactic acid when lactobacilli dominate the microbiota.
      ,
      • Eschenbach D.A.
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      • Williams B.L.
      • Klebanoff S.J.
      • Young-Smith K.
      • Critchlow C.M.
      • et al.
      Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis.
      ,
      • McGroarty J.A.
      Probiotic use of lactobacilli in the human female urogenital tract.
      ,
      • Sobel J.D.
      • Schneider J.
      • Kaye D.
      • Levison M.E.
      Adherence of bacteria to vaginal epithelial cells at various times in the menstrual cycle.
      ). Studies utilizing 16S rRNA PCR have demonstrated that the relative proportions of specific vaginal bacteria may vary between healthy, asymptomatic women (
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • Schneider G.M.
      • Koenig S.S.
      • McCulle S.L.
      • et al.
      Vaginal microbiome of reproductive-age women.
      ,
      • Zhou X.
      • Bent S.J.
      • Schneider M.G.
      • Davis C.C.
      • Islam M.R.
      • Forney L.J.
      Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods.
      ). These genomic studies demonstrated that the vaginal microbial environment is usually dominated by one or two Lactobacilli species, most frequently Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, or Lactobacillus jensenii (
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • Schneider G.M.
      • Koenig S.S.
      • McCulle S.L.
      • et al.
      Vaginal microbiome of reproductive-age women.
      ,
      • Pavlova S.I.
      • Kilic A.O.
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      • So J.S.
      • Nader-Macias M.E.
      • Simoes J.A.
      • et al.
      Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences.
      ). However, a portion of asymptomatic, healthy women, particularly black and Hispanic women, host a polymicrobial vaginal environment dominated by bacteria other than Lactobacilli, including Prevotella, Gardnerella, Atopobium, and Megasphaera species (
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • Schneider G.M.
      • Koenig S.S.
      • McCulle S.L.
      • et al.
      Vaginal microbiome of reproductive-age women.
      ,
      • Zhou X.
      • Bent S.J.
      • Schneider M.G.
      • Davis C.C.
      • Islam M.R.
      • Forney L.J.
      Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods.
      ).
      Of the 73% of women with a Lactobacilli-dominant environment, the most frequently detected organism was L. iners, which was the predominant organism in 34% of women sampled. The second most common Lactobacilli-dominant environment was one in which L. crispatus was most prevalent (26.2% of women) (
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • Schneider G.M.
      • Koenig S.S.
      • McCulle S.L.
      • et al.
      Vaginal microbiome of reproductive-age women.
      ). The identification of an L. iners-dominant microbial environment in the majority of healthy women is in contrast to findings from culture-dependent and early molecular-based studies, which suggested other dominant Lactobacilli species, including Lactobacillus acidophilus, L. crispatus, and L. jensenii (
      • Antonio M.A.
      • Hawes S.E.
      • Hillier S.L.
      The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species.
      ,
      • Ocana V.S.
      • Bru E.
      • De Ruiz Holgado A.A.
      • Nader-Macias M.E.
      Surface characteristics of lactobacilli isolated from human vagina.
      ). The species of Lactobacilli that dominate the vaginal environment may have implications for gynecologic health: it seems that various species may differentially predispose to dysbiosis (
      • Santiago G.L.
      • Cools P.
      • Verstraelen H.
      • Trog M.
      • Missine G.
      • El Aila N.
      • et al.
      Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles.
      ,
      • Santiago G.L.
      • Tency I.
      • Verstraelen H.
      • Verhelst R.
      • Trog M.
      • Temmerman M.
      • et al.
      Longitudinal qPCR study of the dynamics of L. crispatus, L. iners, A. vaginae, (sialidase positive) G. vaginalis, and P. bivia in the vagina.
      ). For example, it has been suggested that an L. crispatus-dominant vaginal microbiome is more stable and less likely to transition to bacterial vaginosis (BV) than an L. iners or mixed-Lactobacilli environment (
      • Castro J.
      • Henriques A.
      • Machado A.
      • Henriques M.
      • Jefferson K.K.
      • Cerca N.
      Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells.
      ,
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ). However, recent studies have suggested that the increased proportion of L. iners in women with BV may be related to an inherent ability of L. iners to tolerate the conditions of BV (such as elevated pH) more than other species of Lactobacilli (
      • Lamont R.F.
      • Sobel J.D.
      • Akins R.A.
      • Hassan S.S.
      • Chaiworapongsa T.
      • Kusanovic J.P.
      • et al.
      The vaginal microbiome: new information about genital tract flora using molecular based techniques.
      ).
      Microscopy and culture-dependent methods demonstrated that the composition of normal vaginal flora may also fluctuate within an individual woman; for example, throughout the menstrual cycle or as a result of sexual activity (
      • Schwebke J.R.
      • Richey C.M.
      • Weiss H.L.
      Correlation of behaviors with microbiological changes in vaginal flora.
      ,
      • Eschenbach D.A.
      • Thwin S.S.
      • Patton D.L.
      • Hooton T.M.
      • Stapleton A.E.
      • Agnew K.
      • et al.
      Influence of the normal menstrual cycle on vaginal tissue, discharge, and microflora.
      ,
      • Brotman R.M.
      • Ravel J.
      • Cone R.A.
      • Zenilman J.M.
      Rapid fluctuation of the vaginal microbiota measured by Gram stain analysis.
      ). Molecular-based studies support these earlier findings that a woman's vaginal microbial environment is dynamic and may change in response to normal life stages or activities (
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ,
      • Srinivasan S.
      • Liu C.
      • Mitchell C.M.
      • Fiedler T.L.
      • Thomas K.K.
      • Agnew K.J.
      • et al.
      Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis.
      ). During menses there is a decrease in Lactobacilli and a relative increase in the proportion of bacteria associated with higher Nugent scores, though women may not report BV symptoms (
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ,
      • Srinivasan S.
      • Liu C.
      • Mitchell C.M.
      • Fiedler T.L.
      • Thomas K.K.
      • Agnew K.J.
      • et al.
      Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis.
      ). Recent sexual activity may also affect the microbial composition of the vagina by decreasing the proportion of Lactobacilli species present (
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ,
      • Jespers V.
      • van de Wijgert J.
      • Cools P.
      • Verhelst R.
      • Verstraelen H.
      • Delany-Moretlwe S.
      • et al.
      The significance of Lactobacillus crispatus and L. vaginalis for vaginal health and the negative effect of recent sex: a cross-sectional descriptive study across groups of African women.
      ), which may predispose to dysbiosis with the loss of the protective effects of Lactobacilli. Decreased Lactobacilli, particularly L. crispatus, as well as decreased bacterial diversity, have also been observed in postmenopausal women, specifically those with vaginal dryness or atrophy (
      • Petricevic L.
      • Domig K.J.
      • Nierscher F.J.
      • Sandhofer M.J.
      • Krondorfer I.
      • Kneifel W.
      • et al.
      Differences in the vaginal lactobacilli of postmenopausal women and influence of rectal lactobacilli.
      ,
      • Zhang R.
      • Daroczy K.
      • Xiao B.
      • Yu L.
      • Chen R.
      • Liao Q.
      Qualitative and semiquantitative analysis of Lactobacillus species in the vaginas of healthy fertile and postmenopausal Chinese women.
      ,
      • van de Wijgert J.H.
      • Borgdorff H.
      • Verhelst R.
      • Crucitti T.
      • Francis S.
      • Verstraelen H.
      • et al.
      The vaginal microbiota: what have we learned after a decade of molecular characterization?.
      ). It is important to note that studies may differ in the sampling and sequencing methods used, and specific molecular-based methods vary in the data produced. For example, whole-genome amplification yields species and strains, whereas 16S rRNA PCR provides information on genus and may yield species (
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • Schneider G.M.
      • Koenig S.S.
      • McCulle S.L.
      • et al.
      Vaginal microbiome of reproductive-age women.
      ,
      Human Microbiome Project Consortium
      Structure, function and diversity of the healthy human microbiome.
      ,
      Human Microbiome Project Consortium
      A framework for human microbiome research.
      ,
      • Aagaard K.
      • Petrosino J.
      • Keitel W.
      • Watson M.
      • Katancik J.
      • Garcia N.
      • et al.
      The Human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters.
      ). Therefore, variations in design and analysis should be considered before forming conclusions based on the direct comparison of different studies.
      The observed fluctuations throughout the menstrual cycle may be explained by evidence that high levels of E2 may favor a Lactobacilli-dominant environment, especially L. crispatus (
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ,
      • Jakobsson T.
      • Forsum U.
      Changes in the predominant human Lactobacillus flora during in vitro fertilisation.
      ,
      • Hickey R.J.
      • Abdo Z.
      • Zhou X.
      • Nemeth K.
      • Hansmann M.
      • Osborn T.W.
      • et al.
      Effects of tampons and menses on the composition and diversity of vaginal microbial communities over time.
      ). In states of relatively low estrogen, such as the beginning of a menstrual cycle or in postmenopausal women, L. crispatus levels may also be low (
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ,
      • Srinivasan S.
      • Liu C.
      • Mitchell C.M.
      • Fiedler T.L.
      • Thomas K.K.
      • Agnew K.J.
      • et al.
      Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis.
      ,
      • Petricevic L.
      • Domig K.J.
      • Nierscher F.J.
      • Sandhofer M.J.
      • Krondorfer I.
      • Kneifel W.
      • et al.
      Differences in the vaginal lactobacilli of postmenopausal women and influence of rectal lactobacilli.
      ,
      • Zhang R.
      • Daroczy K.
      • Xiao B.
      • Yu L.
      • Chen R.
      • Liao Q.
      Qualitative and semiquantitative analysis of Lactobacillus species in the vaginas of healthy fertile and postmenopausal Chinese women.
      ). However, despite evidence from both culture-dependent and -independent methods supporting the dynamic nature of the vaginal microbiome, some molecular-based studies suggest that the microbiome is relatively stable through periods of hormonal fluctuation, such as puberty or the menstrual cycle (
      • Hickey R.J.
      • Zhou X.
      • Settles M.L.
      • Erb J.
      • Malone K.
      • Hansmann M.A.
      • et al.
      Vaginal microbiota of adolescent girls prior to the onset of menarche resemble those of reproductive-age women.
      ,
      • Chaban B.
      • Links M.G.
      • Jayaprakash T.P.
      • Wagner E.C.
      • Bourque D.K.
      • Lohn Z.
      • et al.
      Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle.
      ). There have been a number of studies that have evaluated the vaginal microbiota in tandem by both culture-based and molecular techniques. These studies demonstrate that culture-dependent and -independent methods have a moderate level of concordance, thus providing similar but not identical vaginal microbiome profiles (
      • Balkus J.E.
      • Mitchell C.
      • Agnew K.
      • Liu C.
      • Fiedler T.
      • Cohn S.E.
      • et al.
      Detection of hydrogen peroxide-producing Lactobacillus species in the vagina: a comparison of culture and quantitative PCR among HIV-1 seropositive women.
      ,
      • Martínez-Peña M.D.
      • Castro-Escarpulli G.
      • Aguilera-Arreola M.G.
      Lactobacillus species isolated from vaginal secretions of healthy and bacterial vaginosis-intermediate Mexican women: a prospective study.
      ).
      The quantity and proportion of specific microorganisms in the vagina may vary between women of different ethnic origins. African American women may have increased L. iners and decreased L. crispatus levels compared with Caucasian or Asian women (
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ). This distinction is important because, as mentioned above, an L. iners dominated environment may predispose to BV (
      • Castro J.
      • Henriques A.
      • Machado A.
      • Henriques M.
      • Jefferson K.K.
      • Cerca N.
      Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells.
      ,
      • Gajer P.
      • Brotman R.M.
      • Bai G.
      • Sakamoto J.
      • Schütte U.M.
      • Zhong X.
      • et al.
      Temporal dynamics of the human vaginal microbiota.
      ). Molecular studies have also demonstrated that African American and Hispanic women are also more likely to harbor a vaginal microbiome dominated by bacteria other than Lactobacilli species compared with Caucasian women. These studies suggest that African American women may have higher levels of Gardnerella, Atopobium, Clostridiales, and BV-associated bacterial (BVAB) species or be more likely to harbor a polymicrobial environment compared with Caucasian women (
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • Schneider G.M.
      • Koenig S.S.
      • McCulle S.L.
      • et al.
      Vaginal microbiome of reproductive-age women.
      ,
      • Fettweis J.M.
      • Brooks J.P.
      • Serrano M.G.
      • Sheth N.U.
      • Girerd P.H.
      • Edwards D.J.
      • et al.
      Differences in vaginal microbiome in African American women versus women of European ancestry.
      ,
      • Zhou X.
      • Brown C.J.
      • Abdo Z.
      • Davis C.C.
      • Hansmann M.A.
      • Joyce P.
      • et al.
      Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women.
      ). Taken together, these data suggest that differences in the microbiome between women of various races may alter a woman's predisposition to infection and may at least in part explain the racial disparities in the incidence of BV and sexually transmitted infections (STIs) (
      • Koumans E.H.
      • Sternberg M.
      • Bruce C.
      • McQuillan G.
      • Kendrick J.
      • Sutton M.
      • et al.
      The prevalence of bacterial vaginosis in the United States, 2001–2004; associations with symptoms, sexual behaviors, and reproductive health.
      ,
      • Ness R.B.
      • Hillier S.
      • Richter H.E.
      • Soper D.E.
      • Stamm C.
      • Bass D.C.
      • et al.
      Can known risk factors explain racial differences in the occurrence of bacterial vaginosis?.
      ). In addition, there is increasing evidence that the differences in vaginal microbiota by ethnicities may be related to mitochondrial DNA (mtDNA) haplotypes, which are maternally determined genomic markers (
      • Ma J.
      • Coarfa C.
      • Qin X.
      • Bonnen P.E.
      • Milosavljevic A.
      • Versalovic J.
      • et al.
      mtDNA haplogroup and single nucleotide polymorphisms structure human microbiome communities.
      ,
      • Blekhman R.
      • Goodrich J.K.
      • Huang K.
      • Sun Q.
      • Bukowski R.
      • Bell J.T.
      • et al.
      Host genetic variation impacts microbiome composition across human body sites.
      ). These studies evaluated sequencing data from the HMP evaluating host mtDNA variants by haplotype and mtDNA nucleotide polymorphisms and demonstrated significant associations of specific vaginal microbiota profiles to several haplotypes, confirming previous findings on differences by race and ethnicity. These studies underscore the importance of host genetic variations in influencing the human microbiome and overall disease and health. These host genetic variations are theorized to vary the inflammatory response in an individual to dynamic levels of reactive oxygen species activity.

      Vaginal microbiome in pathologic states

       Bacterial Vaginosis

      For more than a century, the medical community accepted that a shift in the microbial environment of the vagina, specifically a decrease in Doderlein's rods (later identified as Lactobacilli), can lead to symptomatic vaginitis with vaginal discharge (
      • Doderlein A.
      Das scheidensekret und seine bedeutung fur puerperalfieber.
      ,
      • Thomas S.
      Doderlein’s bacillus: Lactobacillus acidophilus.
      ). Subsequent studies by Gardner and Dukes demonstrated that nonspecific vaginitis was associated with a relative increase in a rod-shaped bacteria on Gram stain, later identified as Gardnerella vaginalis (
      • Gardner H.L.
      • Dukes C.D.
      Haemophilus vaginalis vaginitis: a newly defined specific infection previously classified non-specific vaginitis.
      ,
      • Gardner H.L.
      • Dukes C.D.
      New etiologic agent in nonspecific bacterial vaginitis.
      ,
      • Piot P.
      • van Dyck E.
      • Goodfellow M.
      • Falkow S.
      A taxonomic study of Gardnerella vaginalis (Haemophilus vaginalis) Gardner and Dukes 1955.
      ). These studies also described the “clue-cells” characteristic of BV, resulting from vaginal epithelial cells with grainy cell borders due to adherent coccobacilli (
      • Gardner H.L.
      • Dukes C.D.
      Haemophilus vaginalis vaginitis: a newly defined specific infection previously classified non-specific vaginitis.
      ,
      • Gardner H.L.
      • Dukes C.D.
      New etiologic agent in nonspecific bacterial vaginitis.
      ).
      After these initial observations, researchers sought to implement standardized diagnostic criteria, including diagnosis based on clinical criteria (Amsel's criteria) and Gram stain (Nugent score) (
      • Amsel R.
      • Totten P.A.
      • Spiegel C.A.
      • Chen K.C.
      • Eschenbach D.
      • Holmes K.K.
      Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations.
      ,
      • Nugent R.P.
      • Krohn M.A.
      • Hillier S.L.
      Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation.
      ). Amsel's criteria requires three of four clinical conditions to be present: [1] thin, white, vaginal discharge; [2] “clue-cells” on microscopy; [3] vaginal pH >4.5; and [4] positive “whiff test” (10% KOH addition to sample produces fishy odor) (
      • Amsel R.
      • Totten P.A.
      • Spiegel C.A.
      • Chen K.C.
      • Eschenbach D.
      • Holmes K.K.
      Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations.
      ). The Nugent score is calculated by evaluating the proportion of large, Gram-positive rods (Lactobacilli), small, Gram-variable rods (Gardnerella), and curved, Gram-variable rods (Mobiluncus species) on Gram stain (
      • Nugent R.P.
      • Krohn M.A.
      • Hillier S.L.
      Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation.
      ). In one study the sensitivity and specificity of Amsel's criteria were estimated to be 70% and 94%, respectively, and of the Nugent score were 89% and 83%, respectively (
      • Schwebke J.R.
      • Hillier S.L.
      • Sobel J.D.
      • McGregor J.A.
      • Sweet R.L.
      Validity of the vaginal gram stain for the diagnosis of bacterial vaginosis.
      ). However, it is recognized that these diagnostic methods are partially subjective and may over-diagnose BV, and as many as 42% of women diagnosed with BV by Nugent score may be asymptomatic (
      • Klebanoff M.A.
      • Schwebke J.R.
      • Zhang J.
      • Nansel T.R.
      • Yu K.F.
      • Andrews W.W.
      Vulvovaginal symptoms in women with bacterial vaginosis.
      ).
      Culture-dependent studies of BV demonstrate increased diversity of vaginal bacteria (including an increase in facultative anaerobes such as Gardnerella, Mycoplasma, and Prevotella), with a simultaneous decrease of Lactobacilli (
      • Hillier S.
      • Holmes K.
      • Marrazzo J.
      Bacterial vaginosis.
      ). However, the diversity of bacteria both in the normal state and in BV may have been underestimated using culture techniques and Gram stain. It is apparent that the microbial environment of many women previously thought to have “asymptomatic BV” resembles what is now recognized as a normal vaginal microbiome according to molecular-based studies (
      • Ma B.
      • Forney L.J.
      • Ravel J.
      Vaginal microbiome: rethinking health and disease.
      ).
      With increasing use of molecular-based techniques to study the vaginal microbiome, bacteria that seemingly evaded detection using culture-based methods have now been associated with BV, including Atopobium vaginae, Clostridiales, and Megasphaera species (
      • Zhou X.
      • Bent S.J.
      • Schneider M.G.
      • Davis C.C.
      • Islam M.R.
      • Forney L.J.
      Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods.
      ,
      • Zhou X.
      • Brown C.J.
      • Abdo Z.
      • Davis C.C.
      • Hansmann M.A.
      • Joyce P.
      • et al.
      Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women.
      ,
      • Mendes-Soares H.
      • Krishnan V.
      • Settles M.L.
      • Ravel J.
      • Brown C.J.
      • Forney L.J.
      Fine-scale analysis of 16S rRNA sequences reveals a high level of taxonomic diversity among vaginal Atopobium spp.
      ,
      • Fredricks D.N.
      • Fiedler T.L.
      • Marrazzo J.M.
      Molecular identification of bacteria associated with bacterial vaginosis.
      ). The improvements in bacterial detection using cultivation-independent techniques may reveal why some women respond to standard treatment for BV and other women seem to be either resistant to treatment or prone to recurrent BV. Studies using PCR-based techniques have demonstrated that women with recurrent BV may harbor bacteria resistant to traditional metronidazole therapy, including Clostridiales species, Megasphaera, Peptoniphilus lacrimalis, various BV-associated bacterial species, Enterobacteriacae, and Staphylococci (
      • Devillard E.
      • Burton J.P.
      • Reid G.
      Complexity of vaginal microflora as analyzed by PCR denaturing gradient gel electrophoresis in a patient with recurrent bacterial vaginosis.
      ,
      • Bradshaw C.S.
      • Morton A.N.
      • Hocking J.
      • Garland S.M.
      • Morris M.B.
      • Moss L.M.
      • et al.
      High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence.
      ). Specifically, these studies demonstrate a minimum of six types of vaginal microbiota that are known as community state types (CSTs) (
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • Schneider G.M.
      • Koenig S.S.
      • McCulle S.L.
      • et al.
      Vaginal microbiome of reproductive-age women.
      ,
      • Zhou X.
      • Brown C.J.
      • Abdo Z.
      • Davis C.C.
      • Hansmann M.A.
      • Joyce P.
      • et al.
      Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women.
      ). Although the majority of the CST show a predominance of Lactobacillus species, two of the CST show a paucity of Lactobacillus. Not only is there a dearth of Lactobacillus in the vaginal microbiota of these CST, but instead they demonstrate a predominance of anaerobic bacteria seen with states of BV. Although these CST are found in healthy women, they produce higher Nugent scores suggestive of BV and may be a risk factor for gynecologic and obstetrics sequelae (
      • Fredricks D.N.
      • Fiedler T.L.
      • Marrazzo J.M.
      Molecular identification of bacteria associated with bacterial vaginosis.
      ).

       Sexually Transmitted Infection

      Increased bacterial diversity, as in BV, is associated with gynecologic infections, such as infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, human papillomavirus, and Herpes simplex virus-2 infection (
      • Cherpes T.L.
      • Meyn L.A.
      • Krohn M.A.
      • Lurie J.G.
      • Hillier S.L.
      Association between acquisition of herpes simplex virus type 2 in women and bacterial vaginosis.
      ,
      • Wiesenfeld H.C.
      • Hillier S.L.
      • Krohn M.A.
      • Landers D.V.
      • Sweet R.L.
      Bacterial vaginosis is a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection.
      ,
      • Dareng E.O.
      • Ma B.
      • Famooto A.O.
      • Akarolo-Anthony S.N.
      • Offiong R.A.
      • Olaniyan O.
      • et al.
      Prevalent high-risk HPV infection and vaginal microbiota in Nigerian women.
      ,
      • Brotman R.M.
      • Klebanoff M.A.
      • Nansel T.R.
      • Yu K.F.
      • Andrews W.W.
      • Zhang J.
      • et al.
      Bacterial vaginosis assessed by gram stain and diminished colonization resistance to incident gonococcal, chlamydial, and trichomonal genital infection.
      ). It is unclear whether it is the altered levels of bacteria themselves that predispose to infection, or whether the altered vaginal microbiome leads to BV, which predisposes to these states owing to altered pH (leading to less efficient neutralization of pathogens and decreased immune response). However, the assumption that BV, as diagnosed by traditional methods such as Gram stain or Nugent score, predisposes to STIs may be overly simplistic. Molecular-based methods have demonstrated that there is an increased risk of trichomoniasis in women with BV; however, this risk is actually greatest in women with intermediate Nugent scores (
      • Fettweis J.M.
      • Brooks J.P.
      • Serrano M.G.
      • Sheth N.U.
      • Girerd P.H.
      • Edwards D.J.
      • et al.
      Differences in vaginal microbiome in African American women versus women of European ancestry.
      ). The findings suggest that there may be a particular composition of vaginal microbes, such as Mycoplasma, that predispose to Trichomonas vaginalis infection and that were not previously identified using culture-based techniques.
      Numerous studies have demonstrated the association between BV and an increased risk of HIV acquisition (
      • Cu-Uvin S.
      • Hogan J.W.
      • Caliendo A.M.
      • Harwell J.
      • Mayer K.H.
      • Carpenter C.C.
      • et al.
      Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract.
      ,
      • Cohen C.R.
      • Lingappa J.R.
      • Baeten J.M.
      • Ngayo M.O.
      • Spiegel C.A.
      • Hong T.
      • et al.
      Bacterial vaginosis associated with increased risk of female-to-male HIV-1 transmission: a prospective cohort analysis among African couples.
      ,
      • Atashili J.
      • Poole C.
      • Ndumbe P.M.
      • Adimora A.A.
      • Smith J.S.
      Bacterial vaginosis and HIV acquisition: a meta-analysis of published studies.
      ,
      • Benning L.
      • Golub E.T.
      • Anastos K.
      • French A.L.
      • Cohen M.
      • Gilbert D.
      • et al.
      Comparison of lower genital tract microbiota in HIV-infected and uninfected women from Rwanda and the US.
      ). Hydrogen peroxide produced by Lactobacilli is known to have viricidal properties, and the relative decrease in Lactobacilli in cases of BV may increase susceptibility to HIV infection (
      • Klebanoff S.J.
      • Coombs R.W.
      Viricidal effect of Lactobacillus acidophilus on human immunodeficiency virus type 1: possible role in heterosexual transmission.
      ). In a prospective cohort study evaluating the relationship between the vaginal microbial environment and infection risk, the absence of Lactobacilli on culture and the presence of abnormal vaginal flora on Gram stain were associated with an increased risk of HIV acquisition, even after controlling for risk factors (hazard ratio 2.0 and 1.9, 95% confidence interval [CI] 1.2–3.5 and 1.1–3.1, respectively) (
      • Martin H.L.
      • Richardson B.A.
      • Nyange P.M.
      • Lavreys L.
      • Hillier S.L.
      • Chohan B.
      • et al.
      Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition.
      ). High-throughput pyrosequencing of bacterial 16S rRNA has facilitated the detection of specific micro-organisms that are associated with BV and HIV infection. An analysis of US and Rwandan women with and without HIV found that Mycoplasma was significantly more prevalent in HIV-positive Rwandan women (39%) compared with HIV-positive US women (6%), HIV-negative Rwandan women (9%), and HIV-negative US women (10%) (
      • Benning L.
      • Golub E.T.
      • Anastos K.
      • French A.L.
      • Cohen M.
      • Gilbert D.
      • et al.
      Comparison of lower genital tract microbiota in HIV-infected and uninfected women from Rwanda and the US.
      ). It is apparent that the specific composition of the vaginal microbiome affects predisposition to STIs, which is important not only at the individual level but has significant public health implications, as well. Newer, molecular-based techniques may facilitate the identification of bacteria implicated in these infections and appropriate treatment of dysbiosis.

       Upper Genital Tract Infection

      Just as alterations in the microbial environment affect vaginal health, these fluctuations may also predispose to upper genital tract infection, such as pelvic inflammatory disease (PID). Subclinical PID, defined as histologic evidence of endometritis, was detected in 27% and 26% of women with C. trachomatis and N. gonorrhoeae vaginal infections, respectively, and 15% of women with BV diagnosed by clinical criteria and Gram stain (
      • Wiesenfeld H.C.
      • Hillier S.L.
      • Krohn M.A.
      • Amortegui A.J.
      • Heine R.P.
      • Landers D.V.
      • et al.
      Lower genital tract infection and endometritis: insight into subclinical pelvic inflammatory disease.
      ). This association between lower genital tract bacterial composition and PID was also demonstrated in a multicenter study. Utilizing Gram stain and culture, women with vaginal samples that were consistent with BV by Gram stain and who were culture-positive for “BV-associated bacteria” (Gardnerella vaginalis, Mycoplasma hominis, anaerobic Gram-negative rods, and Ureaplasma urealyticum) were at increased risk for PID (adjusted rate ratio 2.03, 95% CI 1.16–3.53) (
      • Ness R.B.
      • Kip K.E.
      • Hillier S.L.
      • Soper D.E.
      • Stamm C.A.
      • Sweet R.L.
      • et al.
      A cluster analysis of bacterial vaginosis-associated microflora and pelvic inflammatory disease.
      ). These findings raise the possibility that specific vaginal microbes, including bacteria that are not directly responsible for cervicovaginitis, may increase the risk for upper genital tract infection. However, because the isolation of BV-associated bacteria in the vagina has been demonstrated to increase the risk of STI acquisition (
      • Wiesenfeld H.C.
      • Hillier S.L.
      • Krohn M.A.
      • Landers D.V.
      • Sweet R.L.
      Bacterial vaginosis is a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection.
      ,
      • Brotman R.M.
      • Klebanoff M.A.
      • Nansel T.R.
      • Yu K.F.
      • Andrews W.W.
      • Zhang J.
      • et al.
      Bacterial vaginosis assessed by gram stain and diminished colonization resistance to incident gonococcal, chlamydial, and trichomonal genital infection.
      ), the correlation of BV with PID may be related to altered vaginal flora that predisposes to STIs and subsequent ascending infection.
      Direct culture or sequencing of samples from upper genital tract structures has been performed less frequently than the evaluation of vaginal samples, but available data confirm that BV-associated bacteria can be isolated from the upper genital tract. In a study of 45 women with laparoscopically confirmed acute salpingitis (cases) and 44 women seeking bilateral tubal ligation (controls), 16S rDNA PCR detected bacteria in the fallopian tubes of 24% of cases and none of the controls (
      • Hebb J.K.
      • Cohen C.R.
      • Astete S.G.
      • Bukusi E.A.
      • Totten P.A.
      Detection of novel organisms associated with salpingitis, by use of 16S rDNA polymerase chain reaction.
      ). Several of the specimens contained bacteria associated with BV, such as Atopobium vaginae, as well as Leptotrichia species and N. gonorrhoeae. The identification of causal microorganisms in upper genital infection is important for understanding disease pathogenesis and provides insight into why some cases are resistant to conventional treatment.
      The presence of certain organisms in the vagina is physiologic, and bacteria may also exist in the upper cervix and uterus during states of health (
      • Mitchell C.M.
      • Haick A.
      • Nkwopara E.
      • Garcia R.
      • Rendi M.
      • Agnew K.
      • et al.
      Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.
      ). By performing quantitative PCR on endometrial and upper cervical swabs from 58 women undergoing hysterectomy for benign conditions, at least one bacterial species in the upper genital tract was found in 95% of women (
      • Mitchell C.M.
      • Haick A.
      • Nkwopara E.
      • Garcia R.
      • Rendi M.
      • Agnew K.
      • et al.
      Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.
      ). The most frequently detected species were L. iners (45%), Prevotella species (33%), and L. crispatus (33%) (
      • Mitchell C.M.
      • Haick A.
      • Nkwopara E.
      • Garcia R.
      • Rendi M.
      • Agnew K.
      • et al.
      Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.
      ). An important consideration is that the upper cervix and uterus were grouped together as the “upper genital tract; ” however, these sites may contain different bacterial species or proportions of bacteria. Furthermore, although the authors address the limitations of using selective PCR, some women with detectable bacterial DNA in the upper genital tract were positive for only one organism, and the inference of colonization based on these results may not be entirely accurate. Despite these limitations, the findings demonstrate that the specific composition and function of the uterine microbiome is incompletely understood. For instance, there was a statistically significant difference in the proportion of upper genital tract bacteria based on race. African American and Hispanic women were more likely to harbor an upper genital tract microbiome dominated by a non-Lactobacilli species (83% and 75%, respectively) compared with Caucasian women (54%). These results mirror those of vaginal microbiome in that non-Lactobacilli species were more common in African American and Hispanic women, but the clinical implications of these findings are unclear. Furthermore, there was no evidence of significant inflammation in the endometrial samples that contained bacteria typically found in the vaginal tract. Possible explanations for the lack of inflammation include vaginal contamination of the uterine samples or that molecular methods detected RNA of nonliving organisms that did not affect clinical status. Alternatively, it remains a possibility that certain bacteria in the upper cervix and uterus may serve important roles in maintaining homeostasis and may not necessarily represent pathology.

      Reproduction

      Similar to the vaginal microbiome, it seems plausible that an imbalance in the uterine microbiome may affect predisposition to disease, such as infertility and pregnancy complications. There are data to support that the composition and/or dysbiosis of the uterine microbiome may impact reproductive outcomes, including early and late miscarriage, preterm delivery, and postpartum endometritis (
      • Ralph S.G.
      • Rutherford A.J.
      • Wilson J.D.
      Influence of bacterial vaginosis on conception and miscarriage in the first trimester: cohort study.
      ,
      • Hay P.E.
      • Lamont R.F.
      • Taylor-Robinson D.
      • Morgan D.J.
      • Ison C.
      • Pearson J.
      Abnormal bacterial colonisation of the genital tract and subsequent preterm delivery and late miscarriage.
      ,
      • McDonald H.M.
      • O’Loughlin J.A.
      • Jolley P.
      • Vigneswaran R.
      • McDonald P.J.
      Prenatal microbiological risk factors associated with preterm birth.
      ,
      • Jacobsson B.
      • Pernevi P.
      • Chidekel L.
      • Jörgen Platz-Christensen J.
      Bacterial vaginosis in early pregnancy may predispose for preterm birth and postpartum endometritis.
      ). Studies focused on the microbiome and reproductive outcomes after IVF have also contributed to our knowledge of the bacterial environment of the upper genital tract. Pelzer et al (
      • Pelzer E.S.
      • Allan J.A.
      • Cunningham K.
      • Mengersen K.
      • Allan J.M.
      • Launchbury T.
      • et al.
      Microbial colonization of follicular fluid: alterations in cytokine expression and adverse assisted reproduction technology outcomes.
      ) cultured the follicular fluid from women undergoing transvaginal oocyte retrieval for IVF. All samples contained bacteria, and there was a difference in the specific bacterial composition according to diagnosis (polycystic ovarian syndrome and endometriosis) and between women with and without implantation. A comprehensive review of assisted reproductive and pregnancy outcomes related to the microbiome is not within the scope of this article; however, these findings suggest that the gynecologic microbial environment has widespread effects on reproductive health.
      In conclusion, although we have gained insight into diseases associated with imbalances in the vaginal microbiome, we still have limited information regarding the origins and mechanisms of dysbiosis. Furthermore, specific cause-and-effect relationships have yet to be determined; for example, does an L. iners-dominant environment predispose to BV, or is this particular species of Lactobacilli more resistant to the conditions of BV, such as elevated pH? Molecular-based methods have identified a substantial number of bacteria in the genital tract that previously eluded detection by Gram stain and culture; however, genetic material detected through sequencing may be derived from organisms that are nonviable or that do not exist in significant number to have clinical effect (
      • Romeika J.
      • Yan F.
      Recent advances in forensic DNA analysis.
      ). Determining what constitutes a normal vaginal microbiome and defining fluctuations through states of health and disease may best be accomplished using a combination of culture- and molecular-based techniques. Further identification of specific microbial imbalances that are associated with genital tract infections will allow for tailored therapy and potentially result in an individualized approach to treating gynecologic infections.

      Acknowledgments

      The authors thank Dr. Alan DeCherney and Dr. Richard Scott, Jr., for their advice during the preparation and completion of the manuscript.

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