Advertisement

Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics

  • Kevin R. Theis
    Correspondence
    Corresponding author: Kevin R Theis, PhD.
    Affiliations
    Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI

    Perinatal Research Initiative in Maternal, Perinatal and Child Health, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI
    Search for articles by this author
  • Roberto Romero
    Correspondence
    Corresponding author: Roberto Romero, MD, DMedSci.
    Affiliations
    Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI

    Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, MI
    Search for articles by this author
  • Andrew D. Winters
    Affiliations
    Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI

    Perinatal Research Initiative in Maternal, Perinatal and Child Health, Wayne State University School of Medicine, Detroit, MI
    Search for articles by this author
  • Jonathan M. Greenberg
    Affiliations
    Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI

    Perinatal Research Initiative in Maternal, Perinatal and Child Health, Wayne State University School of Medicine, Detroit, MI
    Search for articles by this author
  • Nardhy Gomez-Lopez
    Affiliations
    Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI

    Perinatal Research Initiative in Maternal, Perinatal and Child Health, Wayne State University School of Medicine, Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI
    Search for articles by this author
  • Ali Alhousseini
    Affiliations
    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Department of Physiology, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI
    Search for articles by this author
  • Janine Bieda
    Affiliations
    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI
    Search for articles by this author
  • Eli Maymon
    Affiliations
    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
    Search for articles by this author
  • Percy Pacora
    Affiliations
    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI
    Search for articles by this author
  • Jennifer M. Fettweis
    Affiliations
    Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA

    Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA
    Search for articles by this author
  • Gregory A. Buck
    Affiliations
    Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA

    Center for Microbiome Engineering and Data Analysis, Virginia Commonwealth University, Richmond, VA
    Search for articles by this author
  • Kimberly K. Jefferson
    Affiliations
    Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA
    Search for articles by this author
  • Jerome F. Strauss III
    Affiliations
    Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA
    Search for articles by this author
  • Offer Erez
    Affiliations
    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Maternity Department “D” and Obstetrical Day Care Center, Division of Obstetrics and Gynecology, Soroka University Medical Center, Ben Gurion University of the Negev, Beer-Sheva, Israel
    Search for articles by this author
  • Sonia S. Hassan
    Affiliations
    Perinatal Research Initiative in Maternal, Perinatal and Child Health, Wayne State University School of Medicine, Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Department of Physiology, Wayne State University School of Medicine, Detroit, MI

    Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, and Detroit, MI
    Search for articles by this author

      Background

      The human placenta has been traditionally viewed as sterile, and microbial invasion of this organ has been associated with adverse pregnancy outcomes. Yet, recent studies that utilized sequencing techniques reported that the human placenta at term contains a unique microbiota. These conclusions are largely based on the results derived from the sequencing of placental samples. However, such an approach carries the risk of capturing background-contaminating DNA (from DNA extraction kits, polymerase chain reaction reagents, and laboratory environments) when low microbial biomass samples are studied.

      Objective

      To determine whether the human placenta delivered at term in patients without labor who undergo cesarean delivery harbors a resident microbiota (“the assemblage of microorganisms present in a defined niche or environment”).

      Study Design

      This cross-sectional study included placentas from 29 women who had a cesarean delivery without labor at term. The study also included technical controls to account for potential background-contaminating DNA, inclusive in DNA extraction kits, polymerase chain reaction reagents, and laboratory environments. Bacterial profiles of placental tissues and background technical controls were characterized and compared with the use of bacterial culture, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and metagenomic surveys.

      Results

      (1) Twenty-eight of 29 placental tissues had a negative culture for microorganisms. The microorganisms retrieved by culture from the remaining sample were likely contaminants because corresponding 16S ribosomal RNA genes were not detected in the same sample. (2) Quantitative real-time polymerase chain reaction did not indicate greater abundances of bacterial 16S ribosomal RNA genes in placental tissues than in technical controls. Therefore, there was no evidence of the presence of microorganisms above background contamination from reagents in the placentas. (3) 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and background technical controls. (4) Most of the bacterial sequences obtained from metagenomic surveys of placental tissues were from cyanobacteria, aquatic bacteria, or plant pathogens, which are microbes unlikely to populate the human placenta. Coprobacillus, which constituted 30.5% of the bacterial sequences obtained through metagenomic sequencing of placental samples, was not identified in any of the 16S ribosomal RNA gene surveys of these samples. These observations cast doubt as to whether this organism is really present in the placenta of patients at term not in labor.

      Conclusion

      With the use of multiple modes of microbiologic inquiry, a resident microbiota could not be identified in human placentas delivered at term from women without labor. A consistently significant difference in the abundance and/or presence of a microbiota between placental tissue and background technical controls could not be found. All cultures of placental tissue, except 1, did not yield bacteria. Incorporating technical controls for potential sources of background-contaminating DNA for studies of low microbial biomass samples, such as the placenta, is necessary to derive reliable conclusions.

      Key words

      Related editorial, page 213.
      Culture-independent sequencing technologies provide insight into the diversity of microbial communities that inhabit the human body
      • Costello E.K.
      • Lauber C.L.
      • Hamady M.
      • Fierer N.
      • Gordon J.I.
      • Knight R.
      Bacterial community variation in human body habitats across space and time.
      Human Microbiome Project Consortium
      Structure, function and diversity of the healthy human microbiome.
      • Knight R.
      • Callewaert C.
      • Marotz C.
      • et al.
      The microbiome and human biology.
      as well as other ecosystems such as soil
      • Fierer N.
      Embracing the unknown: disentangling the complexities of the soil microbiome.
      • Delgado-Baquerizo M.
      • Oliverio A.M.
      • Brewer T.E.
      • et al.
      A global atlas of the dominant bacteria found in soil.
      and oceans.
      • Venter J.C.
      • Remington K.
      • Heidelberg J.F.
      • et al.
      Environmental genome shotgun sequencing of the Sargasso Sea.
      • Sunagawa S.
      • Coelho L.P.
      • Chaffron S.
      • et al.
      Ocean plankton: structure and function of the global ocean microbiome.
      • Mende D.R.
      • Bryant J.A.
      • Aylward F.O.
      • et al.
      Environmental drivers of a microbial genomic transition zone in the ocean’s interior.
      Studies derived from the Human Microbiome Project indicate that different human body sites are populated by site-specific microbiota (“the assemblage of microorganisms present in a defined niche or environment”
      • Marchesi J.R.
      • Ravel J.
      The vocabulary of microbiome research: a proposal.
      ).
      • Costello E.K.
      • Lauber C.L.
      • Hamady M.
      • Fierer N.
      • Gordon J.I.
      • Knight R.
      Bacterial community variation in human body habitats across space and time.
      Human Microbiome Project Consortium
      Structure, function and diversity of the healthy human microbiome.
      For example, the microbiota of the vagina
      • Lamont R.F.
      • Sobel J.D.
      • Akins R.A.
      • et al.
      The vaginal microbiome: new information about genital tract flora using molecular based techniques.
      • Ravel J.
      • Gajer P.
      • Abdo Z.
      • et al.
      Vaginal microbiome of reproductive-age women.
      • Romero R.
      • Hassan S.S.
      • Gajer P.
      • et al.
      The vaginal microbiota of pregnant women who subsequently have spontaneous preterm labor and delivery and those with a normal delivery at term.
      • Romero R.
      • Hassan S.S.
      • Gajer P.
      • et al.
      The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women.
      • Stout M.J.
      • Zhou Y.
      • Wylie K.M.
      • Tarr P.I.
      • Macones G.A.
      • Tuuli M.G.
      Early pregnancy vaginal microbiome trends and preterm birth.
      is different from that of the gut
      • Turnbaugh P.J.
      • Hamady M.
      • Yatsunenko T.
      • et al.
      A core gut microbiome in obese and lean twins.
      • Falony G.
      • Joossens M.
      • Vieira-Silva S.
      • et al.
      Population-level analysis of gut microbiome variation.
      and oral cavity.
      • Dewhirst F.E.
      • Chen T.
      • Izard J.
      • et al.
      The human oral microbiome.
      • Xu X.
      • He J.
      • Xue J.
      • et al.
      Oral cavity contains distinct niches with dynamic microbial communities.
      The microbial burden of each of these body sites is large,
      • Evaldson G.
      • Heimdahl A.
      • Kager L.
      • Nord C.E.
      The normal human anaerobic microflora.
      • Jespers V.
      • Menten J.
      • Smet H.
      • et al.
      Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests.
      • Sender R.
      • Fuchs S.
      • Milo R.
      Revised estimates for the number of human and bacteria cells in the body.
      and samples derived from these niches are considered to have a high microbial biomass.
      • Sender R.
      • Fuchs S.
      • Milo R.
      Revised estimates for the number of human and bacteria cells in the body.
      • de Goffau M.C.
      • Lager S.
      • Salter S.J.
      • et al.
      Recognizing the reagent microbiome.
      Results obtained with sequencing technologies of these samples are largely consistent, qualitatively, with those derived from cultivation techniques (ie, although molecular surveys of these sites typically capture far more microbial diversity than culture-based surveys, many of the prominent microbes in the molecular surveys have also been recovered through culture from these same sites).
      • Srinivasan S.
      • Munch M.M.
      • Sizova M.V.
      • et al.
      More easily cultivated than identified: classical isolation with molecular identification of vaginal bacteria.
      • Browne H.P.
      • Forster S.C.
      • Anonye B.O.
      • et al.
      Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation.
      • Lau J.T.
      • Whelan F.J.
      • Herath I.
      • et al.
      Capturing the diversity of the human gut microbiota through culture-enriched molecular profiling.
      • Sizova M.V.
      • Hohmann T.
      • Hazen A.
      • et al.
      New approaches for isolation of previously uncultivated oral bacteria.
      • Thompson H.
      • Rybalka A.
      • Moazzez R.
      • Dewhirst F.E.
      • Wade W.G.
      In vitro culture of previously uncultured oral bacterial phylotypes.
      In contrast, samples derived from sites with a low microbial biomass can give results that are difficult to distinguish from DNA present in reagents used for extraction, amplification, and sequence library preparation for molecular microbiology studies.
      • de Goffau M.C.
      • Lager S.
      • Salter S.J.
      • et al.
      Recognizing the reagent microbiome.
      • Salter S.J.
      • Cox M.J.
      • Turek E.M.
      • et al.
      Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.
      • Glassing 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.
      • Kim D.
      • Hofstaedter C.E.
      • Zhao C.
      • et al.
      Optimizing methods and dodging pitfalls in microbiome research.
      • Marsh R.L.
      • Nelson M.T.
      • Pope C.E.
      • et al.
      How low can we go? The implications of low bacterial load in respiratory microbiota studies.

      Why was this study conducted?

      • To examine whether there was evidence to support the existence of a microbiota in placentas delivered at term without labor via cesarean section.

      Key Findings

      • Placentas did not have a microbial DNA abundance exceeding that of background technical controls.
      • 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between samples of the placenta and technical controls.
      • Cultures were negative in 28 of 29 placentas.
      • Metagenomic analysis of placental tissues largely yielded bacterial sequences from cyanobacteria, aquatic bacteria, and plant pathogens, which are microbes ecologically unlikely to populate the human placenta.

      What does this add to what is known?

      • The findings of this study do not support the existence of a placental microbiota in patients who delivered at term without labor.
      Several reports have demonstrated that commercially available kits used to characterize the microbiota contain microbial DNA similar to that found in soil or water samples
      • Salter S.J.
      • Cox M.J.
      • Turek E.M.
      • et al.
      Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.
      • Glassing 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.
      and that this can affect the results of studies of low microbial biomass samples based on 16S ribosomal RNA (rRNA) gene amplicon or metagenomic sequencing.
      • de Goffau M.C.
      • Lager S.
      • Salter S.J.
      • et al.
      Recognizing the reagent microbiome.
      • Salter S.J.
      • Cox M.J.
      • Turek E.M.
      • et al.
      Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.
      • Glassing 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.
      • Marsh R.L.
      • Nelson M.T.
      • Pope C.E.
      • et al.
      How low can we go? The implications of low bacterial load in respiratory microbiota studies.
      • Lauder A.P.
      • Roche A.M.
      • Sherrill-Mix S.
      • et al.
      Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.
      • Bender J.M.
      • Li F.
      • Adisetiyo H.
      • et al.
      Quantification of variation and the impact of biomass in targeted 16S rRNA gene sequencing studies.
      DNA contamination of reagents is unavoidable, given the ubiquity of microorganisms and the fact that many reagents are products of microbial processes and engineering.
      • Kim D.
      • Hofstaedter C.E.
      • Zhao C.
      • et al.
      Optimizing methods and dodging pitfalls in microbiome research.
      Therefore, the claim that body sites with a low microbial biomass have bacteria, based on the analysis of 16S rRNA gene surveys and metagenomic studies, requires rigorous exclusion of reagent contamination to avoid experimental artifacts and incorrect conclusions.
      • de Goffau M.C.
      • Lager S.
      • Salter S.J.
      • et al.
      Recognizing the reagent microbiome.
      • Kim D.
      • Hofstaedter C.E.
      • Zhao C.
      • et al.
      Optimizing methods and dodging pitfalls in microbiome research.
      • Marsh R.L.
      • Nelson M.T.
      • Pope C.E.
      • et al.
      How low can we go? The implications of low bacterial load in respiratory microbiota studies.
      The challenge of studying low microbial biomass samples is important, particularly in the female reproductive tract, because several investigators have viewed the endometrial cavity,
      • Butler B.
      Value of endometrial cultures in sterility investigation.
      • Teisala K.
      Endometrial microbial flora of hysterectomy specimens.
      amniotic cavity,
      • Stroup P.E.
      Amniotic fluid infection and the intact fetal membrane.
      • Harwick H.J.
      • Iuppa J.B.
      • Fekety Jr., F.R.
      Microorganisms and amniotic fluid.
      • Prevedourakis C.
      • Papadimitriou G.
      • Ioannidou A.
      Isolation of pathogenic bacteria in the amniotic fluid during pregnancy and labor.
      • Prevedourakis C.N.
      • Strigou-Charalabis E.
      • Kaskarelis D.B.
      Bacterial invasion of amniotic cavity during pregnancy and labor.
      • Lewis J.F.
      • Johnson P.
      • Miller P.
      Evaluation of amniotic fluid for aerobic and anaerobic bacteria.
      • DiGiulio D.B.
      • Romero R.
      • Amogan H.P.
      • et al.
      Microbial prevalence, diversity and abundance in amniotic fluid during preterm labor: a molecular and culture-based investigation.
      • DiGiulio D.B.
      • Gervasi M.
      • Romero R.
      • et al.
      Microbial invasion of the amniotic cavity in preeclampsia as assessed by cultivation and sequence-based methods.
      • DiGiulio D.B.
      • Gervasi M.T.
      • Romero R.
      • et al.
      Microbial invasion of the amniotic cavity in pregnancies with small-for-gestational-age fetuses.
      • DiGiulio D.B.
      • Romero R.
      • Kusanovic J.P.
      • et al.
      Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes.
      • Gervasi M.T.
      • Romero R.
      • Bracalente G.
      • et al.
      Viral invasion of the amniotic cavity (VIAC) in the midtrimester of pregnancy.
      • Gervasi M.T.
      • Romero R.
      • Bracalente G.
      • et al.
      Midtrimester amniotic fluid concentrations of interleukin-6 and interferon-gamma-inducible protein-10: evidence for heterogeneity of intra-amniotic inflammation and associations with spontaneous early (<32 weeks) and late (>32 weeks) preterm delivery.
      • Romero R.
      • Miranda J.
      • Chaiworapongsa T.
      • et al.
      A novel molecular microbiologic technique for the rapid diagnosis of microbial invasion of the amniotic cavity and intra-amniotic infection in preterm labor with intact membranes.
      • Romero R.
      • Miranda J.
      • Chaiworapongsa T.
      • et al.
      Sterile intra-amniotic inflammation in asymptomatic patients with a sonographic short cervix: prevalence and clinical significance.
      • Combs C.A.
      • Gravett M.
      • Garite T.J.
      • et al.
      Amniotic fluid infection, inflammation, and colonization in preterm labor with intact membranes.
      • Romero R.
      • Miranda J.
      • Chaiworapongsa T.
      • et al.
      Prevalence and clinical significance of sterile intra-amniotic inflammation in patients with preterm labor and intact membranes.
      • Romero R.
      • Miranda J.
      • Chaemsaithong P.
      • et al.
      Sterile and microbial-associated intra-amniotic inflammation in preterm prelabor rupture of membranes.
      • Oh K.J.
      • Kim S.M.
      • Hong J.S.
      • et al.
      Twenty-four percent of patients with clinical chorioamnionitis in preterm gestations have no evidence of either culture-proven intraamniotic infection or intraamniotic inflammation.
      • Pacora P.
      • Romero R.
      • Erez O.
      • et al.
      The diagnostic performance of the beta-glucan assay in the detection of intra-amniotic infection with Candida species.
      • Rowlands S.
      • Danielewski J.A.
      • Tabrizi S.N.
      • Walker S.P.
      • Garland S.M.
      Microbial invasion of the amniotic cavity in midtrimester pregnancies using molecular microbiology.
      • Gomez-Lopez N.
      • Romero R.
      • Panaitescu B.
      • et al.
      Inflammasome activation during spontaneous preterm labor with intra-amniotic infection or sterile intra-amniotic inflammation.
      • Lim E.S.
      • Rodriguez C.
      • Holtz L.R.
      Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community.
      • Rehbinder E.M.
      • Lødrup Carlsen K.C.
      • Staff A.C.
      • et al.
      Is amniotic fluid of women with uncomplicated term pregnancies free of bacteria?.
      and placenta
      • Lauder A.P.
      • Roche A.M.
      • Sherrill-Mix S.
      • et al.
      Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.
      • Aquino T.I.
      • Zhang J.
      • Kraus F.T.
      • Knefel R.
      • Taff T.
      Subchorionic fibrin cultures for bacteriologic study of the placenta.
      • Romero R.
      • Kim Y.M.
      • Pacora P.
      • et al.
      The frequency and type of placental histologic lesions in term pregnancies with normal outcome.
      as being typically sterile.
      • Dominguez-Bello M.G.
      • Costello E.K.
      • Contreras M.
      • et al.
      Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns.
      • Shin H.
      • Pei Z.
      • Martinez 2nd, K.A.
      • et al.
      The first microbial environment of infants born by C-section: the operating room microbes.
      • Dominguez-Bello M.G.
      • De Jesus-Laboy K.M.
      • Shen N.
      • et al.
      Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer.
      • Perez-Munoz M.E.
      • Arrieta M.C.
      • Ramer-Tait A.E.
      • Walter J.
      A critical assessment of the “sterile womb” and “in utero colonization” hypotheses: implications for research on the pioneer infant microbiome.
      With the application of molecular microbiologic techniques, the sterility of these sites, apart from cases of infection, has been questioned,
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      • Doyle R.M.
      • Alber D.G.
      • Jones H.E.
      • et al.
      Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery.
      • Antony K.M.
      • Ma J.
      • Mitchell K.B.
      • Racusin D.A.
      • Versalovic J.
      • Aagaard K.
      The preterm placental microbiome varies in association with excess maternal gestational weight gain.
      • Zheng J.
      • Xiao X.
      • Zhang Q.
      • Mao L.
      • Yu M.
      • Xu J.
      The placental microbiome varies in association with low birth weight in full-term neonates.
      • Bassols J.
      • Serino M.
      • Carreras-Badosa G.
      • et al.
      Gestational diabetes is associated with changes in placental microbiota and microbiome.
      • Collado M.C.
      • Rautava S.
      • Aakko J.
      • Isolauri E.
      • Salminen S.
      Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid.
      • Prince A.L.
      • Ma J.
      • Kannan P.S.
      • et al.
      The placental membrane microbiome is altered among subjects with spontaneous preterm birth with and without chorioamnionitis.
      • Doyle R.M.
      • Harris K.
      • Kamiza S.
      • et al.
      Bacterial communities found in placental tissues are associated with severe chorioamnionitis and adverse birth outcomes.
      • Gomez-Arango L.F.
      • Barrett H.L.
      • McIntyre H.D.
      • Callaway L.K.
      • Morrison M.
      • Nitert M.D.
      Contributions of the maternal oral and gut microbiome to placental microbial colonization in overweight and obese pregnant women.
      • Parnell L.A.
      • Briggs C.M.
      • Cao B.
      • Delannoy-Bruno O.
      • Schrieffer A.E.
      • Mysorekar I.U.
      Microbial communities in placentas from term normal pregnancy exhibit spatially variable profiles.
      • Zheng J.
      • Xiao X.H.
      • Zhang Q.
      • et al.
      Correlation of placental microbiota with fetal macrosomia and clinical characteristics in mothers and newborns.
      • Leon L.J.
      • Doyle R.
      • Diez-Benavente E.
      • et al.
      Enrichment of clinically relevant organisms in spontaneous preterm delivered placenta and reagent contamination across all clinical groups in a large UK pregnancy cohort.
      • Mitchell C.M.
      • Haick A.
      • Nkwopara E.
      • et al.
      Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.
      • Franasiak J.M.
      • Werner M.D.
      • Juneau C.R.
      • et al.
      Endometrial microbiome at the time of embryo transfer: next-generation sequencing of the 16S ribosomal subunit.
      • Moreno I.
      • Codoñer F.M.
      • Vilella F.
      • et al.
      Evidence that the endometrial microbiota has an effect on implantation success or failure.
      • Verstraelen H.
      • Vilchez-Vargas R.
      • Desimpel F.
      • et al.
      Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene.
      • Chen C.
      • Song X.
      • Wei W.
      • et al.
      The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.
      • Tao X.
      • Franasiak J.
      • Zhan Y.
      • et al.
      Characterizing the endometrial microbiome by analyzing the ultra-low bacteria from embryo transfer catheter tips in IVF cycles: Next generation sequencing (NGS) analysis of the 16S ribosomal gene.
      • Kyono K.
      • Hashimoto T.
      • Nagai Y.
      • Sakuraba Y.
      Analysis of endometrial microbiota by 16S ribosomal RNA gene sequencing among infertile patients: a single-center pilot study.
      and functional hypotheses for potential mutualistic relationships between a microbiota and its human host are being considered.
      • Moreno I.
      • Codoñer F.M.
      • Vilella F.
      • et al.
      Evidence that the endometrial microbiota has an effect on implantation success or failure.
      • Prince A.L.
      • Chu D.M.
      • Seferovic M.D.
      • Antony K.M.
      • Ma J.
      • Aagaard K.M.
      The perinatal microbiome and pregnancy: moving beyond the vaginal microbiome.
      • Giudice L.C.
      Challenging dogma: the endometrium has a microbiome with functional consequences!.
      • Moreno I.
      • Franasiak J.M.
      Endometrial microbiota-new player in town.
      • Pelzer E.
      • Gomez-Arango L.F.
      • Barrett H.L.
      • Nitert M.D.
      Review: maternal health and the placental microbiome.
      • Benner M.
      • Ferwerda G.
      • Joosten I.
      • van der Molen R.G.
      How uterine microbiota might be responsible for a receptive, fertile endometrium.
      With respect to the placenta, microorganisms can invade the amnion and chorion
      • Pankuch G.A.
      • Appelbaum P.C.
      • Lorenz R.P.
      • Botti J.J.
      • Schachter J.
      • Naeye R.L.
      Placental microbiology and histology and the pathogenesis of chorioamnionitis.
      • Quinn P.A.
      • Butany J.
      • Taylor J.
      • Hannah W.
      Chorioamnionitis: its association with pregnancy outcome and microbial infection.
      • Hillier S.L.
      • Krohn M.A.
      • Kiviat N.B.
      • Watts D.H.
      • Eschenbach D.A.
      Microbiologic causes and neonatal outcomes associated with chorioamnion infection.
      • Hecht J.L.
      • Onderdonk A.
      • Delaney M.
      • et al.
      Characterization of chorioamnionitis in 2nd-trimester C-section placentas and correlation with microorganism recovery from subamniotic tissues.
      • Kim M.J.
      • Romero R.
      • Gervasi M.T.
      • et al.
      Widespread microbial invasion of the chorioamniotic membranes is a consequence and not a cause of intra-amniotic infection.
      • Kim C.J.
      • Romero R.
      • Chaemsaithong P.
      • Chaiyasit N.
      • Yoon B.H.
      • Kim Y.M.
      Acute chorioamnionitis and funisitis: definition, pathologic features, and clinical significance.
      • Garmi G.
      • Okopnik M.
      • Keness Y.
      • Zafran N.
      • Berkowitz E.
      • Salim R.
      Correlation between clinical, placental histology and microbiological findings in spontaneous preterm births.
      • Sweeney E.L.
      • Kallapur S.G.
      • Gisslen T.
      • et al.
      Placental infection with ureaplasma species is associated with histologic chorioamnionitis and adverse outcomes in moderately preterm and late-preterm infants.
      • Lannon S.M.R.
      • Adams Waldorf K.M.
      • Fiedler T.
      • et al.
      Parallel detection of lactobacillus and bacterial vaginosis-associated bacterial DNA in the chorioamnion and vagina of pregnant women at term.
      and the villous tree.
      • Garcia A.G.
      Fetal Infection in chickenpox and alastrim, with histopathologic study of the placenta.
      • Purtilo D.T.
      • Bhawan J.
      • Liao S.
      • Brutus A.
      • Yang J.P.
      • Balogh K.
      Fatal varicella in a pregnant woman and a baby.
      • Garcia A.G.
      • Marques R.L.
      • Lobato Y.Y.
      • Fonseca M.E.
      • Wigg M.D.
      Placental pathology in congenital rubella.
      • Garcia A.G.
      • Basso N.G.
      • Fonseca M.E.
      • Outani H.N.
      Congenital echo virus infection--morphological and virological study of fetal and placental tissue.
      • Qureshi F.
      • Jacques S.M.
      Maternal varicella during pregnancy: correlation of maternal history and fetal outcome with placental histopathology.
      • Benirschke K.
      • Coen R.
      • Patterson B.
      • Key T.
      Villitis of known origin: varicella and toxoplasma.
      • Satosar A.
      • Ramirez N.C.
      • Bartholomew D.
      • Davis J.
      • Nuovo G.J.
      Histologic correlates of viral and bacterial infection of the placenta associated with severe morbidity and mortality in the newborn.
      • Onderdonk A.B.
      • Delaney M.L.
      • DuBois A.M.
      • Allred E.N.
      • Leviton A.
      Detection of bacteria in placental tissues obtained from extremely low gestational age neonates.
      • Onderdonk A.B.
      • Hecht J.L.
      • McElrath T.F.
      • et al.
      Colonization of second-trimester placenta parenchyma.
      • Cardenas I.
      • Mor G.
      • Aldo P.
      • et al.
      Placental viral infection sensitizes to endotoxin-induced pre-term labor: a double hit hypothesis.
      • Stout M.J.
      • Conlon B.
      • Landeau M.
      • et al.
      Identification of intracellular bacteria in the basal plate of the human placenta in term and preterm gestations.
      • Whidbey C.
      • Harrell M.I.
      • Burnside K.
      • et al.
      A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta.
      • Mouillet J.F.
      • Ouyang Y.
      • Bayer A.
      • Coyne C.B.
      • Sadovsky Y.
      The role of trophoblastic microRNAs in placental viral infection.
      • Bayer A.
      • Delorme-Axford E.
      • Sleigher C.
      • et al.
      Human trophoblasts confer resistance to viruses implicated in perinatal infection.
      • Jurado K.A.
      • Simoni M.K.
      • Tang Z.
      • et al.
      Zika virus productively infects primary human placenta-specific macrophages.
      • Quicke K.M.
      • Bowen J.R.
      • Johnson E.L.
      • et al.
      Zika virus infects human placental macrophages.
      • Rosenberg A.Z.
      • Yu W.
      • Hill D.A.
      • Reyes C.A.
      • Schwartz D.A.
      Placental pathology of zika virus: viral infection of the placenta induces villous stromal macrophage (Hofbauer cell) proliferation and hyperplasia.
      • Sheridan M.A.
      • Yunusov D.
      • Balaraman V.
      • et al.
      Vulnerability of primitive human placental trophoblast to Zika virus.
      • Stanek J.
      Placental infectious villitis versus villitis of unknown etiology.
      • Arora N.
      • Sadovsky Y.
      • Dermody T.S.
      • Coyne C.B.
      Microbial vertical transmission during human pregnancy.
      This is often associated with complications of pregnancy, such as preterm labor,
      • Romero R.
      • Mazor M.
      • Wu Y.K.
      • et al.
      Infection in the pathogenesis of preterm labor.
      • Romero R.
      • Emamian M.
      • Quintero R.
      • et al.
      The value and limitations of the Gram stain examination in the diagnosis of intraamniotic infection.
      • Romero R.
      • Roslansky P.
      • Oyarzun E.
      • et al.
      Labor and infection: II, bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor.
      • Romero R.
      • Avila C.
      • Santhanam U.
      • Sehgal P.B.
      Amniotic fluid interleukin 6 in preterm labor: association with infection.
      • Romero R.
      • Jimenez C.
      • Lohda A.K.
      • et al.
      Amniotic fluid glucose concentration: a rapid and simple method for the detection of intraamniotic infection in preterm labor.
      • Romero R.
      • Shamma F.
      • Avila C.
      • et al.
      Infection and labor: VI, prevalence, microbiology, and clinical significance of intraamniotic infection in twin gestations with preterm labor.
      • Romero R.
      • Yoon B.H.
      • Mazor M.
      • et al.
      The diagnostic and prognostic value of amniotic fluid white blood cell count, glucose, interleukin-6, and gram stain in patients with preterm labor and intact membranes.
      • Gravett M.G.
      • Witkin S.S.
      • Haluska G.J.
      • Edwards J.L.
      • Cook M.J.
      • Novy M.J.
      An experimental model for intraamniotic infection and preterm labor in rhesus monkeys.
      • Kundsin R.B.
      • Leviton A.
      • Allred E.N.
      • Poulin S.A.
      Ureaplasma urealyticum infection of the placenta in pregnancies that ended prematurely.
      • Espinoza J.
      • Goncalves L.F.
      • Romero R.
      • et al.
      The prevalence and clinical significance of amniotic fluid ‘sludge’ in patients with preterm labor and intact membranes.
      • Kusanovic J.P.
      • Espinoza J.
      • Romero R.
      • et al.
      Clinical significance of the presence of amniotic fluid ‘sludge’ in asymptomatic patients at high risk for spontaneous preterm delivery.
      • Romero R.
      • Schaudinn C.
      • Kusanovic J.P.
      • et al.
      Detection of a microbial biofilm in intraamniotic infection.
      • Cardenas I.
      • Means R.E.
      • Aldo P.
      • et al.
      Viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor.
      • Cao B.
      • Stout M.J.
      • Lee I.
      • Mysorekar I.U.
      Placental microbiome and its role in preterm birth.
      • Allen-Daniels M.J.
      • Serrano M.G.
      • Pflugner L.P.
      • et al.
      Identification of a gene in Mycoplasma hominis associated with preterm birth and microbial burden in intraamniotic infection.
      • Chaemsaithong P.
      • Romero R.
      • Korzeniewski S.J.
      • et al.
      A point of care test for the determination of amniotic fluid interleukin-6 and the chemokine CXCL-10/IP-10.
      • Chaemsaithong P.
      • Romero R.
      • Korzeniewski S.J.
      • et al.
      A rapid interleukin-6 bedside test for the identification of intra-amniotic inflammation in preterm labor with intact membranes.
      • Park J.Y.
      • Romero R.
      • Lee J.
      • Chaemsaithong P.
      • Chaiyasit N.
      • Yoon B.H.
      An elevated amniotic fluid prostaglandin F2alpha concentration is associated with intra-amniotic inflammation/infection, and clinical and histologic chorioamnionitis, as well as impending preterm delivery in patients with preterm labor and intact membranes.
      • Boldenow E.
      • Gendrin C.
      • Ngo L.
      • et al.
      Group B streptococcus circumvents neutrophils and neutrophil extracellular traps during amniotic cavity invasion and preterm labor.
      • Racicot K.
      • Kwon J.Y.
      • Aldo P.
      • et al.
      Type I interferon regulates the placental inflammatory response to bacteria and is targeted by virus: mechanism of polymicrobial infection-induced preterm birth.
      • Gomez-Lopez N.
      • Romero R.
      • Garcia-Flores V.
      • et al.
      Amniotic fluid neutrophils can phagocytize bacteria: a mechanism for microbial killing in the amniotic cavity.
      • Gomez-Lopez N.
      • Romero R.
      • Xu Y.
      • et al.
      Are amniotic fluid neutrophils in women with intraamniotic infection and/or inflammation of fetal or maternal origin?.
      • Oh K.J.
      • Hong J.S.
      • Romero R.
      • Yoon B.H.
      The frequency and clinical significance of intra-amniotic inflammation in twin pregnancies with preterm labor and intact membranes.
      • Chaemsaithong P.
      • Romero R.
      • Docheva N.
      • et al.
      Comparison of rapid MMP-8 and interleukin-6 point-of-care tests to identify intra-amniotic inflammation/infection and impending preterm delivery in patients with preterm labor and intact membranes.
      • Kusanovic J.P.
      • Romero R.
      • Martinovic C.
      • et al.
      Transabdominal collection of amniotic fluid “sludge” and identification of Candida albicans intra-amniotic infection.
      • Varrey A.
      • Romero R.
      • Panaitescu B.
      • et al.
      Human beta-defensin-1: a natural antimicrobial peptide present in amniotic fluid that is increased in spontaneous preterm labor with intra-amniotic infection.
      preterm premature rupture of membranes,
      • Romero R.
      • Quintero R.
      • Oyarzun E.
      • et al.
      Intraamniotic infection and the onset of labor in preterm premature rupture of the membranes.
      • Romero R.
      • Ghidini A.
      • Mazor M.
      • Behnke E.
      Microbial invasion of the amniotic cavity in premature rupture of membranes.
      • Gomez R.
      • Romero R.
      • Edwin S.S.
      • David C.
      Pathogenesis of preterm labor and preterm premature rupture of membranes associated with intraamniotic infection.
      • Chaemsaithong P.
      • Romero R.
      • Korzeniewski S.J.
      • et al.
      A point of care test for interleukin-6 in amniotic fluid in preterm prelabor rupture of membranes: a step toward the early treatment of acute intra-amniotic inflammation/infection.
      cervical insufficiency,
      • Romero R.
      • Gonzalez R.
      • Sepulveda W.
      • et al.
      Infection and labor: VIII, microbial invasion of the amniotic cavity in patients with suspected cervical incompetence: prevalence and clinical significance.
      • Hassan S.
      • Romero R.
      • Hendler I.
      • et al.
      A sonographic short cervix as the only clinical manifestation of intra-amniotic infection.
      • Romero R.
      • Kusanovic J.P.
      • Espinoza J.
      • et al.
      What is amniotic fluid ‘sludge’?.
      • Lee S.E.
      • Romero R.
      • Park C.W.
      • Jun J.K.
      • Yoon B.H.
      The frequency and significance of intraamniotic inflammation in patients with cervical insufficiency.
      • Chaiworapongsa T.
      • Hong J.S.
      • Hull W.M.
      • Romero R.
      • Whitsett J.A.
      Amniotic fluid concentration of surfactant proteins in intra-amniotic infection.
      • Oh K.J.
      • Lee S.E.
      • Jung H.
      • Kim G.
      • Romero R.
      • Yoon B.H.
      Detection of ureaplasmas by the polymerase chain reaction in the amniotic fluid of patients with cervical insufficiency.
      • Paules C.
      • Moreno E.
      • Gonzales A.
      • Fabre E.
      • González de Agüero R.
      • Oros D.
      Amniotic fluid sludge as a marker of intra-amniotic infection and histological chorioamnionitis in cervical insufficiency: a report of four cases and literature review.
      • Kim Y.M.
      • Park K.H.
      • Park H.
      • Yoo H.N.
      • Kook S.Y.
      • Jeon S.J.
      Complement C3a, but not C5a, levels in amniotic fluid are associated with intra-amniotic infection and/or inflammation and preterm delivery in women with cervical insufficiency or an asymptomatic short cervix (≤25 mm).
      clinical chorioamnionitis,
      • Gibbs R.S.
      • Blanco J.D.
      • St. Clair P.J.
      • Castaneda Y.S.
      Quantitative bacteriology of amniotic fluid from women with clinical intraamniotic infection at term.
      • Hauth J.C.
      • Gilstrap 3rd, L.C.
      • Hankins G.D.
      • Connor K.D.
      Term maternal and neonatal complications of acute chorioamnionitis.
      • Romero R.
      • Nores J.
      • Mazor M.
      • et al.
      Microbial invasion of the amniotic cavity during term labor: prevalence and clinical significance.
      • Newton E.R.
      Chorioamnionitis and intraamniotic infection.
      • Gibbs R.S.
      Management of clinical chorioamnionitis at term.
      • Tita A.T.
      • Andrews W.W.
      Diagnosis and management of clinical chorioamnionitis.
      • Romero R.
      • Chaiworapongsa T.
      • Savasan Z.A.
      • et al.
      Clinical chorioamnionitis is characterized by changes in the expression of the alarmin HMGB1 and one of its receptors.
      • Romero R.
      • Miranda J.
      • Kusanovic J.P.
      • et al.
      Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques.
      • Romero R.
      • Chaemsaithong P.
      • Korzeniewski S.J.
      • et al.
      Clinical chorioamnionitis at term II: the intra-amniotic inflammatory response.
      • Romero R.
      • Chaemsaithong P.
      • Korzeniewski S.J.
      • et al.
      Clinical chorioamnionitis at term III: how well do clinical criteria perform in the identification of proven intra-amniotic infection?.
      • Mazaki-Tovi S.
      • Vaisbuch E.
      Clinical chorioamnionitis: an ongoing obstetrical conundrum.
      • Romero R.
      • Chaemsaithong P.
      • Docheva N.
      • et al.
      Clinical chorioamnionitis at term IV: the maternal plasma cytokine profile.
      • Romero R.
      • Chaemsaithong P.
      • Docheva N.
      • et al.
      Clinical chorioamnionitis at term V: umbilical cord plasma cytokine profile in the context of a systemic maternal inflammatory response.
      • Romero R.
      • Chaemsaithong P.
      • Docheva N.
      • et al.
      Clinical chorioamnionitis at term VI: acute chorioamnionitis and funisitis according to the presence or absence of microorganisms and inflammation in the amniotic cavity.
      • Maddipati K.R.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome.
      • Martinez-Varea A.
      • Romero R.
      • Xu Y.
      • et al.
      Clinical chorioamnionitis at term VII: the amniotic fluid cellular immune response.
      • Fouks Y.
      • Many A.
      • Orbach R.
      • et al.
      Is there a role for placental cultures in cases of clinical chorioamnionitis complicating preterm premature rupture of membranes?.
      • Chaiyasit N.
      • Romero R.
      • Chaemsaithong P.
      • et al.
      Clinical chorioamnionitis at term VIII: a rapid MMP-8 test for the identification of intra-amniotic inflammation.
      and congenital infections.
      • Garcia A.G.
      Fetal Infection in chickenpox and alastrim, with histopathologic study of the placenta.
      • Purtilo D.T.
      • Bhawan J.
      • Liao S.
      • Brutus A.
      • Yang J.P.
      • Balogh K.
      Fatal varicella in a pregnant woman and a baby.
      • Garcia A.G.
      • Marques R.L.
      • Lobato Y.Y.
      • Fonseca M.E.
      • Wigg M.D.
      Placental pathology in congenital rubella.
      • Garcia A.G.
      • Basso N.G.
      • Fonseca M.E.
      • Outani H.N.
      Congenital echo virus infection--morphological and virological study of fetal and placental tissue.
      • Qureshi F.
      • Jacques S.M.
      Maternal varicella during pregnancy: correlation of maternal history and fetal outcome with placental histopathology.
      • Mostoufi-Zadeh M.
      • Driscoll S.G.
      • Biano S.A.
      • Kundsin R.B.
      Placental evidence of cytomegalovirus infection of the fetus and neonate.
      • Yoon B.H.
      • Jun J.K.
      • Romero R.
      • et al.
      Amniotic fluid inflammatory cytokines (interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha), neonatal brain white matter lesions, and cerebral palsy.
      • Yoon B.H.
      • Kim C.J.
      • Romero R.
      • et al.
      Experimentally induced intrauterine infection causes fetal brain white matter lesions in rabbits.
      • Yoon B.H.
      • Romero R.
      • Kim C.J.
      • et al.
      High expression of tumor necrosis factor-alpha and interleukin-6 in periventricular leukomalacia.
      • Gomez R.
      • Romero R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Berry S.M.
      The fetal inflammatory response syndrome.
      • Yoon B.H.
      • Romero R.
      • Park J.S.
      • et al.
      The relationship among inflammatory lesions of the umbilical cord (funisitis), umbilical cord plasma interleukin 6 concentration, amniotic fluid infection, and neonatal sepsis.
      • Euscher E.
      • Davis J.
      • Holzman I.
      • Nuovo G.J.
      Coxsackie virus infection of the placenta associated with neurodevelopmental delays in the newborn.
      • Kim C.J.
      • Yoon B.H.
      • Romero R.
      • et al.
      Umbilical arteritis and phlebitis mark different stages of the fetal inflammatory response.
      • Yoon B.H.
      • Romero R.
      • Shim J.Y.
      • Shim S.S.
      • Kim C.J.
      • Jun J.K.
      C-reactive protein in umbilical cord blood: a simple and widely available clinical method to assess the risk of amniotic fluid infection and funisitis.
      • Gotsch F.
      • Romero R.
      • Kusanovic J.P.
      • et al.
      The fetal inflammatory response syndrome.
      • Korzeniewski S.J.
      • Romero R.
      • Cortez J.
      • et al.
      A “multi-hit” model of neonatal white matter injury: cumulative contributions of chronic placental inflammation, acute fetal inflammation and postnatal inflammatory events.
      • Kim S.M.
      • Romero R.
      • Lee J.
      • Chaemsaithong P.
      • Docheva N.
      • Yoon B.H.
      Gastric fluid versus amniotic fluid analysis for the identification of intra-amniotic infection due to Ureaplasma species.
      • Lee J.
      • Romero R.
      • Lee K.A.
      • et al.
      Meconium aspiration syndrome: a role for fetal systemic inflammation.
      • Mor G.
      Placental inflammatory response to zika virus may affect fetal brain development.
      • Kapisi J.
      • Kakuru A.
      • Jagannathan P.
      • et al.
      Relationships between infection with Plasmodium falciparum during pregnancy, measures of placental malaria, and adverse birth outcomes.
      The concept that most placentas have a microbial community emerged after a pioneering study that utilized sequencing techniques to analyze a large number of placentas.
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      Shortly after this report, questions were raised about this claim,
      • Kliman H.J.
      Comment on “the placenta harbors a unique microbiome.”.
      yet other investigators who used high-throughput sequencing strategies also reported the presence of a microbiota in the placenta.
      • Doyle R.M.
      • Alber D.G.
      • Jones H.E.
      • et al.
      Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery.
      • Antony K.M.
      • Ma J.
      • Mitchell K.B.
      • Racusin D.A.
      • Versalovic J.
      • Aagaard K.
      The preterm placental microbiome varies in association with excess maternal gestational weight gain.
      • Zheng J.
      • Xiao X.
      • Zhang Q.
      • Mao L.
      • Yu M.
      • Xu J.
      The placental microbiome varies in association with low birth weight in full-term neonates.
      • Bassols J.
      • Serino M.
      • Carreras-Badosa G.
      • et al.
      Gestational diabetes is associated with changes in placental microbiota and microbiome.
      • Collado M.C.
      • Rautava S.
      • Aakko J.
      • Isolauri E.
      • Salminen S.
      Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid.
      • Prince A.L.
      • Ma J.
      • Kannan P.S.
      • et al.
      The placental membrane microbiome is altered among subjects with spontaneous preterm birth with and without chorioamnionitis.
      • Doyle R.M.
      • Harris K.
      • Kamiza S.
      • et al.
      Bacterial communities found in placental tissues are associated with severe chorioamnionitis and adverse birth outcomes.
      • Gomez-Arango L.F.
      • Barrett H.L.
      • McIntyre H.D.
      • Callaway L.K.
      • Morrison M.
      • Nitert M.D.
      Contributions of the maternal oral and gut microbiome to placental microbial colonization in overweight and obese pregnant women.
      • Parnell L.A.
      • Briggs C.M.
      • Cao B.
      • Delannoy-Bruno O.
      • Schrieffer A.E.
      • Mysorekar I.U.
      Microbial communities in placentas from term normal pregnancy exhibit spatially variable profiles.
      • Zheng J.
      • Xiao X.H.
      • Zhang Q.
      • et al.
      Correlation of placental microbiota with fetal macrosomia and clinical characteristics in mothers and newborns.
      • Leon L.J.
      • Doyle R.
      • Diez-Benavente E.
      • et al.
      Enrichment of clinically relevant organisms in spontaneous preterm delivered placenta and reagent contamination across all clinical groups in a large UK pregnancy cohort.
      The interpretation of these data has become a subject of controversy,
      • de Goffau M.C.
      • Lager S.
      • Salter S.J.
      • et al.
      Recognizing the reagent microbiome.
      • Lauder A.P.
      • Roche A.M.
      • Sherrill-Mix S.
      • et al.
      Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.
      • Perez-Munoz M.E.
      • Arrieta M.C.
      • Ramer-Tait A.E.
      • Walter J.
      A critical assessment of the “sterile womb” and “in utero colonization” hypotheses: implications for research on the pioneer infant microbiome.
      • Hornef M.
      • Penders J.
      Does a prenatal bacterial microbiota exist?.
      • Willyard C.
      Baby’s first bacteria: the womb was thought to be sterile; some scientists argue it’s where the microbiome begins.
      given the recognition that reagents used in molecular microbiologic techniques have their own microbiome (termed the kitome).
      • de Goffau M.C.
      • Lager S.
      • Salter S.J.
      • et al.
      Recognizing the reagent microbiome.
      • Salter S.J.
      • Cox M.J.
      • Turek E.M.
      • et al.
      Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.
      • Glassing 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.
      • Kim D.
      • Hofstaedter C.E.
      • Zhao C.
      • et al.
      Optimizing methods and dodging pitfalls in microbiome research.
      • Lauder A.P.
      • Roche A.M.
      • Sherrill-Mix S.
      • et al.
      Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.
      • Weiss S.
      • Amir A.
      • Hyde E.R.
      • Metcalf J.L.
      • Song S.J.
      • Knight R.
      Tracking down the sources of experimental contamination in microbiome studies.
      Recently, investigators have called for the application of rigorous and systematic methods to address DNA contamination in low microbial biomass samples.
      • de Goffau M.C.
      • Lager S.
      • Salter S.J.
      • et al.
      Recognizing the reagent microbiome.
      • Kim D.
      • Hofstaedter C.E.
      • Zhao C.
      • et al.
      Optimizing methods and dodging pitfalls in microbiome research.
      • Marsh R.L.
      • Nelson M.T.
      • Pope C.E.
      • et al.
      How low can we go? The implications of low bacterial load in respiratory microbiota studies.
      The objective of this study was to determine whether a microbiota exists in term placentas, delivered by cesarean section without labor, using multiple complementary modes of microbiologic inquiry: cultivation, quantitative real-time polymerase chain reaction (qPCR), 16S rRNA gene sequencing, and metagenomics.

      Materials and Methods

      Study design

      This was a cross-sectional study in which the placenta was sampled from women not in labor at term (February–June 2016). The inclusion criteria were (1) cesarean delivery without labor at term (≥38 weeks), (2) singleton gestation, and (3) no antibiotic administration in the month before delivery, as determined by history and review of medical records. Each subject, however, did receive intraoperative prophylaxis before cesarean delivery (cefazolin or, if allergic, gentamicin and clindamycin), given the evidence that antimicrobial administration reduces perioperative complications.
      • Elkomy M.H.
      • Sultan P.
      • Drover D.R.
      • Epshtein E.
      • Galinkin J.L.
      • Carvalho B.
      Pharmacokinetics of prophylactic cefazolin in parturients undergoing cesarean delivery.
      • Smaill F.M.
      • Grivell R.M.
      Antibiotic prophylaxis versus no prophylaxis for preventing infection after cesarean section.
      • Maggio L.
      • Nicolau D.P.
      • DaCosta M.
      • Rouse D.J.
      • Hughes B.L.
      Cefazolin prophylaxis in obese women undergoing cesarean delivery: a randomized controlled trial.
      Exclusion criteria consisted of multiple gestation, preterm delivery, fetal anomalies, and evidence of clinical infection.
      The presence of bacteria in the placenta was determined using (1) cultivation, (2) 16S rRNA gene qPCR, (3) 16S rRNA gene sequencing, and (4) metagenomic sequencing. Placental histopathologic examinations were conducted according to protocols established by the Perinatology Research Branch.
      • Romero R.
      • Kim Y.M.
      • Pacora P.
      • et al.
      The frequency and type of placental histologic lesions in term pregnancies with normal outcome.
      • Kim C.J.
      • Romero R.
      • Chaemsaithong P.
      • Chaiyasit N.
      • Yoon B.H.
      • Kim Y.M.
      Acute chorioamnionitis and funisitis: definition, pathologic features, and clinical significance.
      The collection of samples and their use for research was approved by the Human Investigation Committee of Wayne State University and the Institutional Review Board of the Eunice Kennedy Shriver National Institute of Child Health and Human Development; all subjects provided written informed consent for participation.

      Sample collection

      After cesarean delivery, the placenta was placed in a sterile collection container with a sealed cover (Medline Standard C-Section Pack-LF, Mundelein, IL) within the sterile operating field. The placenta was taken directly to a biologic safety cabinet located within 1 of 2 nearby rooms in Hutzel Women’s Hospital, wherein study personnel (A.D.W., K.R.T.), wearing sterile surgical gowns, full hoods, and powder-free examination gloves (Kimberly-Clark, Roswell, GA) and using individually packaged, sterile, disposable scalpels (Surgical Design, Lorton, VA), forceps (TWD Scientific, Pleasant Prairie, WI), and surgical scissors (Sklar Instruments, West Chester, PA), collected a 1.5-cm2 core sample from the placenta (ie, amnion and chorionic plate through to basal plate). The tissue sample was taken halfway between the umbilical cord insertion point and the edge of the placental disk, along the line that represented the longest distance from the cord insertion point to the edge of the disk. The tissue sample was transferred to a sterile polystyrene Petri dish (FB0875712; Fisher Scientific, Waltham, MA) and divided into 3 approximately equal aliquots, with each aliquot traversing the amnion, chorionic plate, villous tree, and basal plate. One aliquot was placed in a sterile 5.0-mL conical tube (Denville Scientific, Holliston, MA) on ice and stored at −80°C within 1 hour of initial placental collection. The 2 remaining aliquots were placed into Anaerobic Transport Medium Surgery Packs (Anaerobe Systems, Morgan Hill, CA) and 0.85% sterile saline solution tubes (Thermo Scientific, Waltham, MA) for anaerobic and aerobic cultures, respectively.

      Bacterial culture of placental tissues

      Placental tissue aliquots within anaerobic and aerobic transport containers were delivered to the Detroit Medical Center University Laboratories Microbiology Core, wherein they were processed the same day. To assess viability of a placental microbiota, placental tissues were homogenized and inoculated on growth media (trypticase soy agar with 5% sheep blood, chocolate agar, MacConkey’s agar) under aerobic and anaerobic conditions and used in an assay for genital mycoplasmas. Detailed information on the cultivation protocols and taxonomic characterization of resultant bacterial cultivars is available in Supplemental Methods (Section 1).

      DNA extraction from placental tissues

      DNA extraction was performed to identify bacteria with molecular microbiologic techniques. During the process, study personnel wore sterile surgical gowns, gloves, and surgical masks (Soft Touch II; Kimberly-Clark, Roswell, GA) and used individually packaged, sterile, and disposable scalpels and forceps (DF 8988P-SPT; TWD Scientific, Pleasant Prairie, WI). For each placental tissue specimen, the amnion and chorionic plate (including a minimal amount of villous tissue) were separated from the placental villous tree, which remained attached to the basal plate. Genomic DNA was extracted from blocks of tissue that contained the amnion and chorionic plate and the villous tree and basal plate. The extraplacental chorioamniotic membranes were not sampled. DNA was extracted from the placental tissues (0.1–0.2 g) and background technical controls with the use of the MoBio PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA), according to the manufacturer’s protocol. The DNA extraction kit and the mass of placental tissue from which DNA was extracted were similar to those used in previous studies that addressed the issue of a placental microbiota.
      • Lauder A.P.
      • Roche A.M.
      • Sherrill-Mix S.
      • et al.
      Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      Background technical controls included extractions performed on (1) DNA extraction kits without placental tissue, processed exactly as the placental samples (n=6), (2) extraction kits with bead tubes that had been exposed to a biologic safety cabinet for 20 minutes during placental biopsy collection or processing (n=16 samples from 3 biosafety cabinets), and (3) extraction kits with bead tubes that had been exposed for 20 minutes to an operating room or microbiology laboratory used in this study (n=21 samples from 3 operating rooms and 3 laboratories). These control samples therefore represented either 5 or 6 technical controls that reflected each potential source of background DNA contamination (ie, extraction kits, 3 biosafety cabinets, 3 laboratories, and operating rooms), with the 3 contiguous operating room environments being treated as a single potential contamination source. DNA concentrations of placental tissue and background technical control samples were 42.0±18.5 (standard deviation) ng/μL and ≤0.03 ng/μL, respectively. Purified DNA was stored at −20°C.

      16S rRNA gene sequencing of DNA extracted from placental tissue and background technical control samples

      The 16S rRNA gene is used widely as a phylogenetic marker to identify bacterial types present in clinical samples. A table of PCR 16S rRNA gene primers used in this study is available in Supplemental Methods (Section 2). We initially used the standard PCR and Illumina MiSeq (San Diego, CA) protocols described later; however, this approach did not produce sufficient quantities of amplified DNA to generate sequence libraries from placental tissue or technical controls and thus for 16S rRNA gene profile comparisons (Supplemental Methods Section 3; Supplemental Figure 1). Therefore, because of the very low microbial biomass in these human tissue samples, purified bacterial DNA was amplified with the use of a nested PCR approach.
      • Fan Z.-Y.
      • Li X.-R.
      • Mao D.-P.
      • Zhu G.-F.
      • Wang S.-Y.
      • Quan Z.-X.
      Could nested PCR be applicable for the study of microbial diversity?.
      • Yu G.
      • Fadrosh D.
      • Goedert J.J.
      • Ravel J.
      • Goldstein A.M.
      Nested PCR biases in interpreting microbial community structure in 16S rRNA gene sequence datasets.
      Nested PCR has been used recently to characterize low biomass microbiota in the lungs of mice,
      • Kostric M.
      • Milger K.
      • Krauss-Etschmann S.
      • et al.
      Development of a stable lung microbiome in healthy neonatal mice.
      sheep,
      • Glendinning L.
      • Wright S.
      • Pollock J.
      • Tennant P.
      • Collie D.
      • McLachlan G.
      Variability of the sheep lung microbiota.
      and chickens,
      • Glendinning L.
      • McLachlan G.
      • Vervelde L.
      Age-related differences in the respiratory microbiota of chickens.
      and in the middle ear fluid of children.
      • Neeff M.
      • Biswas K.
      • Hoggard M.
      • Taylor M.W.
      • Douglas R.
      Molecular microbiological profile of chronic suppurative otitis media.
      • Sillanpaa S.
      • Kramna L.
      • Oikarinen S.
      • et al.
      Next-generation sequencing combined with specific PCR assays to determine the bacterial 16S rRNA gene profiles of middle ear fluid collected from children with acute otitis media.
      The first round in the nested PCR process included 20 cycles. Each reaction contained 0.4 μM (micromolar) each of the 16S rRNA gene broad-range primers 27f-CM (5ʹ-AGA GTT TGA TCM TGG CTC AG-3ʹ) and 1492R (5ʹ-ACG GCT ACC TTG TTA CGA CTT -3ʹ),
      • Weisburg W.G.
      • Barns S.M.
      • Pelletier D.A.
      • Lane D.J.
      16S Ribosomal DNA amplification for phylogenetic study.
      • Frank J.A.
      • Reich C.I.
      • Sharma S.
      • Weisbaum J.S.
      • Wilson B.A.
      • Olsen G.J.
      Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes.
      12.5 μL of 2X GoTaq Green Master Mix (Promega, Madison, WI), and 3.0 μL purified DNA. Thermocycling was initiated by a 5-minute incubation at 95°C. Cycling parameters were 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 120 seconds. Products were then diluted 1:15 in nuclease-free water (Promega).
      Amplification and sequencing of the V4 region of the 16S rRNA gene was performed at the University of Michigan’s Center for Microbial Systems (Ann Arbor, MI) with the use of the dual indexing sequencing strategy developed by Kozich et al.
      • Kozich J.J.
      • Westcott S.L.
      • Baxter N.T.
      • Highlander S.K.
      • Schloss P.D.
      Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.
      Sequencing was performed on the Illumina MiSeq platform, with a MiSeq Reagent Kit V2 (500-cycle format; MS102-2003; Illumina), according to the manufacturer’s instructions with modifications found in Kozich et al
      • Kozich J.J.
      • Westcott S.L.
      • Baxter N.T.
      • Highlander S.K.
      • Schloss P.D.
      Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.
      and Caporaso et al.
      • Caporaso J.G.
      • Lauber C.L.
      • Walters W.A.
      • et al.
      Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms.
      AccuPrime High Fidelity Taq (12346094; Life Technologies) was used instead of AccuPrime Pfx SuperMix. Each PCR reaction (20 μL) contained 1.0 μM of each primer, 2.5 μL template DNA, 0.15 μL AccuPrime HiFi Polymerase, and DNase-free water to produce a final volume of 20 μL. PCR was performed under the following conditions: 95°C for 2 minutes, followed by 30 cycles at 95°C for 20 seconds, 55°C for 30 seconds, and 72°C for 5 minutes, with an additional elongation at 72°C for 10 minutes. Sequencing libraries were prepared according to Illumina’s protocol for Preparing Libraries for Sequencing on the MiSeq (15039740 Rev. D) for 2 nM or 4 nM libraries. FASTQ files were generated for paired end reads. Sample-specific MiSeq run files have been deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (BioProject ID PRJNA397876).

      Processing of 16S rRNA gene sequence data

      Mothur software (version 1.39.5) was used to assemble paired-read contiguous sequences, to trim, filter, and align sequences, to identify and remove chimeras, to assign sequences to bacterial taxonomies, and to cluster sequences into operational taxonomic units (OTUs) based on the percentage of nucleotide similarity (97% and 99%).
      • Schloss P.D.
      • Westcott S.L.
      • Ryabin T.
      • et al.
      Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.
      Detailed information on sequence processing is available in Supplemental Methods (Section 4).
      Sequencing of DNA extracts of all samples and controls yielded 5,316,687 sequences. They clustered into 480 (209 singletons) and 35,503 (23,892 singletons) OTUs with the use of 97% and 99% sequence similarity cutoffs, respectively. The mean number of sequences for the placental tissue and technical control samples was 50,783 (range, 509–92,052) and 55,145 (2572–111,361), respectively. All raw count data for this study are available as supplemental material (Supplemental Data 1).
      With the use of a 97% OTU nucleotide similarity cutoff, the Good’s coverage values of all but 1 placental sample exceeded 99.7%; the exception was 98.8% (sample 25AC). Good’s coverage values of all technical control samples exceeded 99.8%. For analyses of alpha diversity (microbial diversity within a sample), individual sample libraries were subsampled to the depth of the second least-represented sample (1997 sequences), and the least-represented sample (509 sequences for 25AC) was excluded. After subsampling for alpha diversity analyses, Good’s coverage values of placental and technical control samples exceeded 99.4%.

      qPCR of the 16S rRNA genes in DNA extracts of placental tissues and background technical controls

      Bacterial DNA abundance within the samples was determined via qPCR amplification of the V1–V2 region of the 16S rRNA gene as described by Dickson et al,
      • Dickson R.P.
      • Erb-Downward J.R.
      • Freeman C.M.
      • et al.
      Changes in the lung microbiome following lung transplantation include the emergence of two distinct Pseudomonas species with distinct clinical associations.
      with minor modifications. These included the use of a degenerative forward primer (27f-CM: 5ʹ-AGA GTT TGA TCM TGG CTC AG-3ʹ) and a degenerate probe containing locked nucleic acids (+) (BSR65/17:5ʹ-56FAM-TAA +YA+C ATG +CA+A GT+C GA-BHQ1-3ʹ). Amplifications were performed with an annealing temperature of 50°C to minimize amplification bias and to allow for a greater number of potential bacterial types, such as Lactobacillus and Gardnerella species.
      • Frank J.A.
      • Reich C.I.
      • Sharma S.
      • Weisbaum J.S.
      • Wilson B.A.
      • Olsen G.J.
      Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes.
      Detailed information on the qPCR protocols are provided in the Supplemental Methods (Section 5).

      Metagenomic sequencing of extracted DNA from placental samples and background technical controls

      By contrast with sequencing surveys that target a specific bacterial gene (eg, 16S rRNA gene), a metagenomic survey entails sequencing all of the genes in a clinical sample and assigning the protein-coding genes of bacterial origin to particular bacterial taxa. Nine placental and 11 technical control samples underwent metagenomic sequencing with the use of the Illumina HiSeq 4000, 150-base paired-end read protocol at the University of Michigan’s DNA Sequencing Core (Ann Arbor, MI). The placental samples included amnion and chorionic plate as well as villous tree and basal plate samples from each of 4 subjects (subjects 14, 15, 22, and 30), and a villous tree and basal plate sample from 1 subject (subject 19). The technical control samples included 8 biologic safety cabinet and 3 blank extraction kit samples. Metagenomic sequence data were processed with MG-RAST.
      • Meyer F.
      • Paarmann D.
      • D’Souza M.
      • et al.
      The metagenomics RAST server: a public resource for the automatic phylogenetic and functional analysis of metagenomes.
      Bacterial taxonomic assignments were made with the use of the GenBank database and the default MG-RAST parameters. Detailed information on metagenomic sequencing and sequence data processing protocols are available in Supplemental Methods (Sections 6 and 7). All raw genus-level count data are available as supplemental material (Supplemental Data 2).

      Secondary DNA extractions and molecular analyses of placental tissues

      After the primary 16S rRNA gene sequencing analyses did not yield evidence of a placental microbiota (see Results), secondary analyses were conducted to ensure that the primary sequencing results were not due to cross-contamination between DNA extracted from placental tissues and background technical controls during processing, or exclusively because of the use of a nested PCR approach for bacterial DNA amplification.
      Secondary DNA extractions were performed on the collective villous tree and basal plate portion of each of the 29 placental samples. The extraction protocol was the same as that described earlier, except that at least 4 blank extraction kit controls were included in each of 4 rounds of extractions of the placental samples. Specifically, in the first 3 rounds of extractions, we processed 8 placental and 4 technical control samples. In the fourth round, we processed 5 placental and 5 technical control samples. Additionally, we completed a fifth round of extractions composed entirely of 12 blank extraction kit controls, which were not exposed to the atmospheres of the biologic safety cabinets or the laboratories; they were processed exactly as the placental samples. DNA concentrations of placental tissue and blank extraction control samples were 56.0±24.3 ng/μL and ≤0.03 ng/μL, respectively. Purified DNA was stored at −20°C.
      The secondary DNA extractions were used for 16S rRNA gene sequencing with the use of 3 amplification approaches: standard PCR, nested PCR, and touchdown PCR. For standard PCR, we aimed to generate the 16S rRNA gene profiles of DNA extracted from placental samples and background technical controls using 30, 35, and 40 amplification cycles. For nested PCR, we used a different primer pair for the first round of amplifications from that used in the primary analysis in this study and aimed to generate 16S profiles for these samples using 5, 10, and 20 cycles in the first round of amplification. The different primer set, 341F/1061R (Supplemental Methods, Section 2), was used for the first round of nested PCR in an attempt to eliminate potential underrepresentation
      • Klindworth A.
      • Pruesse E.
      • Schweer T.
      • et al.
      Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.
      or selection against single bacterial species or groups of species
      • Wang Y.
      • Qian P.Y.
      Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.
      in placental samples. Specifically, in silico studies querying these selected primers against taxonomically diverse sequences in 3 popular 16S rRNA gene databases (ie, Greengenes,
      • DeSantis T.Z.
      • Hugenholtz P.
      • Larsen N.
      • et al.
      Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.
      Ribosomal Database Project,
      • Cole J.R.
      • Wang Q.
      • Cardenas E.
      • et al.
      The Ribosomal Database Project: improved alignments and new tools for rRNA analysis.
      and SILVA
      • Pruesse E.
      • Quast C.
      • Knittel K.
      • et al.
      SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB.
      ) have shown these selected primers to be highly conserved.
      • Klindworth A.
      • Pruesse E.
      • Schweer T.
      • et al.
      Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.
      • Ong S.H.
      • Kukkillaya V.U.
      • Wilm A.
      • et al.
      Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.
      Last, we aimed to generate 16S rRNA gene profiles for these samples using touchdown PCR.
      • Don R.H.
      • Cox P.T.
      • Wainwright B.J.
      • Baker K.
      • Mattick J.S.
      ‘Touchdown’ PCR to circumvent spurious priming during gene amplification.
      • Bassis C.M.
      • Erb-Downward J.R.
      • Dickson R.P.
      • et al.
      Analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals.
      • Dickson R.P.
      • Erb-Downward J.R.
      • Freeman C.M.
      • et al.
      Spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography.
      Touchdown PCR can increase the sensitivity of PCR reactions in cases of very low microbial biomass and high background concentrations of host DNA.
      • Don R.H.
      • Cox P.T.
      • Wainwright B.J.
      • Baker K.
      • Mattick J.S.
      ‘Touchdown’ PCR to circumvent spurious priming during gene amplification.
      • Bassis C.M.
      • Erb-Downward J.R.
      • Dickson R.P.
      • et al.
      Analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals.
      • Dickson R.P.
      • Erb-Downward J.R.
      • Freeman C.M.
      • et al.
      Spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography.
      Touchdown PCR was used recently to characterize the microbiota of the lung,
      • Bassis C.M.
      • Erb-Downward J.R.
      • Dickson R.P.
      • et al.
      Analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals.
      • Dickson R.P.
      • Erb-Downward J.R.
      • Freeman C.M.
      • et al.
      Spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography.
      • Venkataraman A.
      • Bassis C.M.
      • Beck J.M.
      • et al.
      Application of a neutral community model to assess structuring of the human lung microbiome.
      • Dickson R.P.
      • Singer B.H.
      • Newstead M.W.
      • et al.
      Enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome.
      • Dickson R.P.
      • Erb-Downward J.R.
      • Freeman C.M.
      • et al.
      Bacterial topography of the healthy human lower respiratory tract.
      brain,
      • Singer B.H.
      • Dickson R.P.
      • Denstaedt S.J.
      • et al.
      Bacterial dissemination to the brain in sepsis.
      and blood
      • Dickson R.P.
      • Singer B.H.
      • Newstead M.W.
      • et al.
      Enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome.
      of mice and/or humans. The PCR cycle started with 2 minutes at 95°C, followed by (1) a touchdown PCR for 20 seconds at 95°C, 15 seconds at the annealing temperature (60°C in the first cycle and decreased by 0.3°C with each additional cycle), and 5 minutes at 72°C, and then (2) 20 cycles of a standard PCR with 20 seconds at 95°C, 15 seconds at 55°C, and 5 minutes at 72°C, with a final elongation step at 72°C for 10 minutes.
      All template DNA was diluted 3-fold and transferred to the University of Michigan’s Center for Microbial Systems for sequence library processing. Sequence library construction was done with the use of the dual indexing sequencing strategy developed by Kozich et al.
      • Kozich J.J.
      • Westcott S.L.
      • Baxter N.T.
      • Highlander S.K.
      • Schloss P.D.
      Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.
      All reactions included 4 μL of template DNA. Based on visual inspections of amplified products that use gel electrophoresis, sequence library generation was unsuccessful with 30 and 35 cycles of standard PCR. Sequence library generation was also unsuccessful with 5 and 10 cycles in the initial amplification round for nested PCR. Therefore, for the secondary 16S rRNA gene analyses, we generated sequence libraries for placental samples and background technical controls using 40 rounds of standard PCR, nested PCR with 20 initial rounds of amplification, and touchdown PCR. Sample-specific MiSeq run files have been deposited into the NCBI Sequence Read Archive (BioProject ID PRJNA397876), and all raw count data for the secondary analyses are provided as supplemental material (Supplemental Data 3). Sequence data processing for the secondary analyses proceeded as described earlier; see Supplemental Methods (Section 4). The analyses presented herein are of sequence data clustered into OTUs based on a nucleotide similarity percentage of 97%. Results did not substantively differ with a 97% or 99% nucleotide similarity; therefore, only the results that used 97% similarity are presented for the secondary analyses. Raw data from sequence clustering based on a nucleotide similarity percentage of 99% are provided in Supplemental Data 3.
      The abundances of 16S rRNA gene copies in each placental sample and blank extraction control in this secondary analysis were determined with qPCR, as described earlier, with minor alterations. Specifically, all samples were diluted 3-fold before analysis; each sample reaction was performed in triplicate, and, if a sample did not pass the threshold of quantification by 40 cycles, its cycle of quantification (Cq) value was assigned as 40.

      Statistical analysis

      16S rRNA gene profile alpha and beta diversity

      Alpha diversity (ie, diversity within a single sample) was assessed with the use of Chao1 richness and Simpson heterogeneity indices.

      Magurran AE. Measuring biological diversity. Malden (MA): Blackwell Publishing; 2004.

      Magurran AE. Biological diversity: frontiers in measurement and assessment. New York: Oxford University Press; 2011.

      Alpha diversity indices were calculated with Mothur software (version 1.39.5)
      • Schloss P.D.
      • Westcott S.L.
      • Ryabin T.
      • et al.
      Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.
      and statistically evaluated with Kruskal-Wallis tests and Mann-Whitney pairwise comparisons, if applicable, using Paleontological Statistics software (version 2.17c).
      • Hammer O.
      • Harper DAT
      • Ryan P.D.
      PAST: PAleontological STatistics software package for education and data analysis.

      Quinn GP, Keough MJ. Experimental design and data analysis for biologists. Cambridge: Cambridge University Press; 2002.

      Gotelli NJ, Ellison AM. A Primer of Ecological Statistics. Sunderland (MA): Sinauer Associates, Inc; 2004.

      Beta diversity (ie, diversity between 2 samples) was assessed with Jaccard and Bray-Curtis similarity indices to reflect 16S rRNA gene profile composition and structure, respectively. Bray-Curtis values were calculated with the use of percent relative abundance data for OTUs within samples. Beta diversity was visualized through principal coordinates analyses and heat maps and statistically evaluated with nonparametric multivariate analysis of variance (NPMANOVA),

      Quinn GP, Keough MJ. Experimental design and data analysis for biologists. Cambridge: Cambridge University Press; 2002.

      Gotelli NJ, Ellison AM. A Primer of Ecological Statistics. Sunderland (MA): Sinauer Associates, Inc; 2004.

      • Anderson M.J.
      A new method for non-parametric multivariate analysis of variance.
      with 9999 permutations. Principal coordinates analyses plots and NPMANOVA tests were conducted with Paleontological Statistics software (versions 2.17c and 3.14),
      • Hammer O.
      • Harper DAT
      • Ryan P.D.
      PAST: PAleontological STatistics software package for education and data analysis.
      and heat maps were generated via Matrix2png.
      • Pavlidis P.
      • Noble W.S.
      Matrix2png: a utility for visualizing matrix data.
      Linear discriminant analysis effect size (LEfSe)
      • Segata N.
      • Izard J.
      • Waldron L.
      • et al.
      Metagenomic biomarker discovery and explanation.
      was used to identify any OTUs that differed in relative abundance between the placental tissue and background technical control samples. Sourcetracker (version 1.0)
      • Knights D.
      • Kuczynski J.
      • Charlson E.S.
      • et al.
      Bayesian community-wide culture-independent microbial source tracking.
      was used to estimate the percentage of OTUs in placental samples whose origin could be explained by their distribution in the background technical controls. For this analysis, we removed doubleton and singleton OTUs from the dataset.

      16S rRNA gene qPCR

      To assess differences in 16S rDNA abundance between the amnion and chorionic plate and the villous tree and basal plate samples among the 29 subjects, differences in the Cq were evaluated with paired t-tests. To assess variation in bacterial burden among individual sample types (ie, amnion and chorionic plate, villous tree and basal plate, operating rooms and laboratories, biosafety cabinets, and blank DNA extraction kits), analysis of variance tests, or Welch F tests in the case of unequal variances, were used for global assessment of variation in Cq, followed by Tukey’s pairwise comparisons.

      Quinn GP, Keough MJ. Experimental design and data analysis for biologists. Cambridge: Cambridge University Press; 2002.

      Gotelli NJ, Ellison AM. A Primer of Ecological Statistics. Sunderland (MA): Sinauer Associates, Inc; 2004.

      When data were not distributed normally, we used Kruskal-Wallis tests and Mann-Whitney pairwise comparisons. Statistical analyses were performed using Paleontological Statistics software (version 2.17c).
      • Hammer O.
      • Harper DAT
      • Ryan P.D.
      PAST: PAleontological STatistics software package for education and data analysis.

      Results

      Patient characteristics

      Table 1 describes the demographic and clinical characteristics of the patients in this study. None of the placentas included in this study presented fetal or maternal inflammatory lesions, defined as stage 3 and/or grade 2 maternal and/or fetal inflammatory responses.
      • Romero R.
      • Kim Y.M.
      • Pacora P.
      • et al.
      The frequency and type of placental histologic lesions in term pregnancies with normal outcome.
      • Khong T.Y.
      • Mooney E.E.
      • Ariel I.
      • et al.
      Sampling and definitions of placental lesions: Amsterdam Placental Workshop group consensus statement.
      Table 1Descriptive and clinical characteristics of the 29 study participants
      VariableMedianInterquartile range
      Age, y29.025.5–33.0
      Body mass index, kg/m2
      Unreported for 7 subjects
      32.824.7–36.1
      Parity21–2
      Gestational age at delivery, wk39.139.0–39.3
      Birthweight, g34503063–3905
      Race, n (%)
      Race was self-reported by subjects; 3 subjects chose not to report.
       African American21 (80.8)
       Caucasian5 (19.2)
      Clinical indications, n (%)
       Repeat elective cesarean delivery23 (79.3)
       Large for gestational age fetus3 (10.3)
       Breech presentation2 (6.9)
       Myoclonus dystonia1 (3.4)
      Theis et al. Lack of evidence for a microbiota in the human placenta at term. Am J Obstet Gynecol 2019.
      a Unreported for 7 subjects
      b Race was self-reported by subjects; 3 subjects chose not to report.

      Bacterial culture of placental tissues

      Twenty-eight of the 29 placental tissue samples did not yield any bacterial cultivars. One tissue sample (subject 25) yielded 3 colonies in the primary zone of the 5% sheep blood agar plate incubated aerobically: Bacillus circulans, B pumilus, and Brevibacterium casei. It did not yield colonies on other media under aerobic or anaerobic conditions or yield growth of genital mycoplasmas. Exact matches (ie, 100% nucleotide similarity) to the V4 region of the 16S rRNA genes of the 3 isolates recovered on the sheep blood agar plate were not found among any of the sequences from the primary (13,766 sequences; Good’s coverage >99.9%) or the secondary (98,392 sequences; Good’s coverage >99.9%) MiSeq 16S rRNA gene surveys of subject 25’s placental tissues.

      16S rRNA gene surveys of placental tissue and background technical control samples

      Alpha diversity

      There was no variation in OTU richness among the amnion and chorionic plate samples and the room, hood, and blank extraction kit controls (Chao1 index; Kruskal-Wallis test; H=4.114; P=.248), nor was there variation among the villous tree and basal plate samples and the various controls (H=3.871; P=.274). There was also no variation in OTU heterogeneity between the placental and technical control samples (Simpson index; amnion and chorionic plate: H=3.384; P=.336; villous tree and basal plate: H=2.531; P=.470).

      Beta diversity

      There was no variation in the composition or structure of 16S rRNA gene profiles among the 3 biologic safety cabinets (NPMANOVA; Jaccard: F=0.846; P=.781; Bray-Curtis: F=0.880; P=.572), or among the different rooms used for sample processing (Jaccard: F=0.882, P=.833; Bray-Curtis: F=0.916; P=.602). Profile similarities among the amnion and chorionic plate samples, the villous tree and basal plate samples, and the 3 different types of technical controls (ie, blank extraction kits, biosafety cabinets, rooms) are illustrated in Figure 1. 16S rRNA gene profiles did not consistently vary among the amnion and chorionic plate samples, blank extraction kits, biologic safety cabinets, and processing rooms (Figure 1; Table 2). Similarly, 16S rRNA gene profiles did not vary among the villous tree and basal plate samples, blank extraction kits, biologic safety cabinets, and room controls (Figure 1; Table 2). Neither the 16S rRNA gene profiles of the amnion and chorionic plate samples nor those of the villous tree and basal plate samples differed specifically from those of the blank extraction kits (Table 2). These same patterns were found when an OTU nucleotide similarity cutoff of 99% was used (Supplemental Figure 2; Supplemental Table 1).
      Figure thumbnail gr1
      Figure 1Principal coordinates analyses illustrating similarity in 16S ribosomal RNA gene profiles among the amnion and chorionic plate, villous tree and basal plate, and technical control samples (i.e., blank DNA extraction kits (“Kit”), biological safety cabinets (“Hood”), and the operating rooms and laboratories (“Room”) used in the study)
      A, Plot of similarity in profile composition among placental and control samples based on the Jaccard index. B, Plot of similarity in profile structure among placental and control samples based on the Bray-Curtis index. Operational taxonomic units were generated with a 97% sequence similarity cutoff and the primary 16S ribosomal RNA gene nested polymerase chain reaction data set.
      Theis et al. Lack of evidence for a microbiota in the human placenta at term. Am J Obstet Gynecol 2019.
      Table 2Nonparametric multivariate analysis of variance shows lack of variation in 16S ribosomal RNA gene profiles among the amnion and chorionic plate, villous tree and basal plate, and room, hood, and blank extraction kit technical control samples
      VariableCompositionStructure
      FP valueFP value
      Placenta: Amnion and chorionic plate
       Global1.080.2611.128.270
       Rooms1.367.0602.211.028 (.077)
       Hoods1.310.1081.190.275
       Kits1.018.4120.545.873
      Placenta: Villous tree and basal plate
       Global1.051.3351.222.189
       Rooms1.450.037 (.223)2.513.007 (.043)
       Hoods1.149.2311.072.351
       Kits0.944.5520.875.529
      Operational taxonomic units were generated with a 97% sequence similarity cutoff. 16S profile composition and structure were characterized with the use of Jaccard and Bray-Curtis indices, respectively. Results of overall global effect analyses are presented along with the results of pairwise comparisons that involve placental samples. Probability values for these permutation tests were not adjusted for multiple pairwise comparisons, because this can be overly conservative. However, for pairwise tests that were statistically significant, we present the Bonferroni corrected probability value in parentheses.
      Theis et al. Lack of evidence for a microbiota in the human placenta at term. Am J Obstet Gynecol 2019.
      Sixteen of the 18 prominent OTUs (ie, those having an average relative abundance ≥1%) among the placental samples were classified confidently at the genus level (Figure 2). These OTUs were Achromobacter, Delftia, Phyllobacterium, Clostridium, Propionibacterium, Stenotrophomonas, Acinetobacter, Blastomonas, Methylobacterium, Sphingomonas, Paracoccus, Ralstonia, Staphylococcus, Leucobacter, and Ureaplasma. These 18 prominent OTUs accounted for 90.0% and 86.4% of total sequences obtained from the placental tissue samples and background technical controls, respectively. Fourteen of these 18 prominent placental OTUs were also prominent among the control samples (Figure 2). The 4 exceptions (OTUs classified as Acinetobacter, Paracoccus, Propionibacterium, and Ureaplasma) were OTUs that were either widely present among the technical control samples but at low relative abundances or abundant in only 1 to a few placental tissue samples. A full description of the distribution and relative abundances of these OTUs among placental samples and technical controls is provided in the Supplemental Results (Section 1).
      Figure thumbnail gr2
      Figure 2Heat map illustrating similarity in percent relative abundances of prominent operational taxonomic units among placental samples and technical controls
      Prominent operational taxonomic units were defined as those having an average relative abundance ≥1% among the placental samples. Operational taxonomic units were generated with a 97% sequence similarity cutoff and the primary 16S ribosomal RNA gene nested polymerase chain reaction data set. Asterisks indicate operational taxonomic units prominent in placental samples but not in controls.
      Theis et al. Lack of evidence for a microbiota in the human placenta at term. Am J Obstet Gynecol 2019.
      LEfSe indicated that 4 OTUs (classified as Achromobacter, Blastococcus, Methylobacterium, and Caldalkalibacillus) were more relatively abundant among the amnion and chorionic plate samples than the technical controls and that 3 OTUs (classified as Achromobacter, Burkholderiales, and Herbaspirillum) were more relatively abundant among the villous tree and basal plate samples than the controls (Supplemental Figure 3). The distribution and relative abundances of these OTUs among placental samples and technical controls is discussed in detail in Supplemental Results (Section 2).
      SourceTracker analyses indicated that a median of 99.7% (interquartile range [IQR], 50.7%) and 99.9% (IQR, 8.2%) of OTUs in the amnion and chorionic plate and the villous tree and basal plate samples, respectively, could be attributed confidently to contaminating DNA in blank extraction kits, PCR reagents, and/or the rooms used for sample processing. Furthermore, when defining the core microbiota as those OTUs present in at least one-half of the samples of a particular sampling group,
      • Collado M.C.
      • Rautava S.
      • Aakko J.
      • Isolauri E.
      • Salminen S.
      Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid.
      • Gomez-Arango L.F.
      • Barrett H.L.
      • McIntyre H.D.
      • Callaway L.K.
      • Morrison M.
      • Nitert M.D.
      Contributions of the maternal oral and gut microbiome to placental microbial colonization in overweight and obese pregnant women.
      every core OTU in the amnion and chorionic plate and the villous tree and basal plate samples was also a core OTU in the hood and blank extraction kit control samples (Supplemental Results, Section 3).

      Real-time qPCR assays of 16S rRNA gene copy abundances in the placental tissues and background technical controls

      Analysis of Cq values generated for broad-range standard curves and included across all qPCR runs indicated that the average amplification efficiency of the assay was 85.44%±1.91%. The regression curves were linear over a range of 101 to 106 gene copies, with slopes ranging from −3.88 to −3.62 and R2 values ≥0.980 (Figure 3, B). Analysis of Cq values generated for the narrow-range standard curve that ranged from 2.01×104 to 1.57×102 revealed that standard deviation values reached 0.506 cycles for the most dilute replicate reactions (Figure 3, C), indicating that the limits of detection and quantification for the assay were between 1.57×102 and 3.14×102 copies (Figure 3, C).
      Figure thumbnail gr3
      Figure 3Quantitative polymerase chain reaction analyses illustrating similarity in 16S ribosomal RNA gene abundance among the amnion and chorionic plate, villous tree and basal plate, and technical control samples
      A, Comparison of quantification cycle values (mean±standard deviation) of serially diluted placental genomic DNA samples spiked with equal concentrations (5.7×103 copies per reaction) of genomic DNA from Escherichia coli ATCC 25922 illustrate that amplification inhibition is eliminated by diluting samples with nuclease-free water by a factor of ≥1:3. B, Standard curves for 3 10-fold dilution series (2.82×106 to 2.82×101 copies, 2.12×106 to 2.12×101 copies, and 2.97×106 to 2.97×101 copies) of E coli ATCC 25922 16S ribosomal DNA (mean quantification cycle values across all quantitative polymerase chain reaction runs). C, Standard curve for a 2-fold dilution series (mean quantification cycle values) of E coli ATCC 25922 DNA illustrate a limit of detection for the quantitative polymerase chain reaction assay between 1.57×102 and 3.14×102 16S ribosomal DNA copies per reaction (20 μL), as indicated by a standard deviation of replicate dilution samples >0.5 cycles. D, Comparison of mean 16S ribosomal DNA quantitative polymerase chain reaction quantification cycle values for placental and control samples. E, Amplification curves from placental samples, technical controls, and the serial dilution series of E coli DNA described in B.
      Cq, quantification cycle; PCR, polymerase chain reaction; Rn, normalized fluorescence.
      Theis et al. Lack of evidence for a microbiota in the human placenta at term. Am J Obstet Gynecol 2019.
      qPCR revealed that 16S rDNA abundances within the majority of the placental and background technical control samples were beyond the detection and quantification limits of the qPCR assay (Figure 3, D and E). There were no differences in Cq between the amnion and chorionic plate and the villous tree and basal plate samples (paired t-test: n=29; t=–0.485; P=.631). For the background technical control samples, there was no variation in Cq values among the location-specific control samples from the rooms (analysis of variance: n=21; F=0.008; P=.999) or from the individual biological safety cabinets (n=16; F=0.063; P=.939). Therefore, these samples were combined within their respective groups for comparison to the amnion and chorionic plate and the villous tree and basal plate samples. Variation in Cq values was observed among the amnion and chorionic plate samples and the room, hood, kit, and water samples (Welch F test: n=81; F=7.683; P=.0005), and among the villous tree and basal plate samples and controls (F=9.572; P=.0001). In both cases, the variation was due to the room control samples having lower Cq values (ie, higher rDNA abundances) than the placental and water samples (Tukey’s pairwise comparisons; amnion and chorionic plate vs rooms: Q=4.544; P=.016; villous tree and basal plate vs rooms: Q=5.108, P=.005; water vs rooms: Q=5.773; P=.001). Cq values did not differ between amnion and chorionic plate samples and blank extraction kits (t-test; t=–1.093; P=.282). They also did not differ between villous tree and basal plate samples and blank extraction kits (t=–1.535; P=.134). When a subset (n=32/43) of total control samples was diluted 1:9, there were no differences between the amnion and chorionic plate samples, villous tree and basal plate samples, and technical controls (t-tests: amnion and chorionic plate: t=–0.296; P=.768; villous tree and basal plate: t=0.048; P=.962). Differences were then also absent between the placental tissue samples and a subset (n=13/21) of room control samples (t-tests; amnion and chorionic plate: t=0.018; P=.985; villous tree and basal plate: t=0.354; p=.725).

      Metagenomic surveys of placental tissues

      At least 43,000,000 sequence reads were obtained from each of 9 placental tissue samples (61,027,678±9,572,214). On average, 0.05% of these sequences were classified as bacterial in origin. Good’s coverage values (99.6%±0.004%) indicated that the bacterial profiles of these samples were characterized thoroughly from a taxonomic standpoint. The survey identified 267 bacterial genera, with 19 having an average relative abundance of ≥0.1% (Figure 4). Only 5 genera had an average relative abundance ≥1.0%: Cyanothece, Coprobacillus, “Candidatus phytoplasma,” Chlorobium, and Streptomyces. Escherichia was present in each placental sample, with an average relative abundance of 0.05%. The functions of bacterial genes were characterized broadly as metabolism (amino acid, carbohydrate, vitamin, and energy metabolism), genetic (DNA translation, replication, repair, and degradation), and environmental (membrane transport and signal transduction) processing.
      Figure thumbnail gr4
      Figure 4Heat map illustrating relative abundances of prominent bacterial genera among placental sample profiles as determined by metagenomic sequencing
      Prominent genera are defined as those having an average relative abundance ≥0.1% among the placental sample profiles. AC indicates amnion and chorionic plate samples, and V indicates villous tree and basal plate samples, respectively.
      Theis et al. Lack of evidence for a microbiota in the human placenta at term. Am J Obstet Gynecol 2019.
      Given the necessary differences in metagenomic library preparation for the placental tissue and technical control samples, their broad bacterial profiles cannot be compared in a quantitative manner. However, it is reasonable to inquire if there are genera consistently identified in placental tissue samples that were not also widely present in the sequenced background technical controls. There were 36 genera present in all 9 sequenced placental tissue samples, and 89 genera present in at least one-half. Each of these genera was present in all 11 sequenced background technical controls. Of the 267 total genera, or approximate genus-level taxa, that were identified in placental tissue samples, only 1 was not found in every control sample: an unclassified Myxococcales, present in 1 placental sample with an abundance <0.01%.
      Of the prominent genera identified in the primary 16S rRNA gene sequencing analysis (Figure 2), only Clostridium was present in placental metagenomic bacterial profiles at an average relative abundance ≥0.1% (Figure 4). Achromobacter, Clostridium, Propionibacterium, Staphylococcus, and Stenotrophomonas were present in the metagenomic profiles of at least one-half of the placental samples. However, each of these genera was also present in the metagenomic profiles of all 11 sequenced background technical controls.

      Secondary 16S rRNA gene sequencing and qPCR analyses

      16S rRNA gene surveys with standard PCR

      The median number of sequences obtained from the 29 placental samples was 89 (IQR, 15–3210), and no blank extraction kit controls yielded >100 quality sequences. Of the 29 placental samples, only 31% (9/29) yielded at least 1000 quality sequences and had Good’s coverage values exceeding 99%. Their microbial profiles included 8 prominent OTUs (ie, average relative abundance ≥1%; Supplemental Figure 4). Pelomonas and Sphingomonas were most consistently abundant. These genera represented 2 of the 3 OTUs present in at least one-half of the 9 placental samples. The remaining core OTU (OTU001) was Escherichia, present in each of the 9 placental samples with a median relative abundance of 0.07% (IQR, 0.02–0.13%). Although the blank extraction kits had poor sequence yield, their bacterial profiles were dominated by Escherichia (median, 67%; IQR, 41–100%). Indeed, OTU001 was detected in 27 of 28 blank extraction kit controls that yielded sequence data.
      Neither Pelomonas nor Sphingomonas was detected in the bacterial profiles of the 9 placental tissues characterized through metagenomic sequencing in the primary analyses described earlier.

      16S rRNA gene surveys with the use of nested PCR

      Fifty-seven of 58 placental and blank extraction kit control samples yielded ≥1000 sequences with a Good’s coverage value that exceeded 99%. One blank extraction kit sample yielded 423 sequences and was excluded from analyses. The remaining placental samples and technical controls yielded 80,492±27,721 and 77,670±79,160 quality sequences, respectively. These sequences clustered into 207 OTUs. For alpha diversity analyses, each sample was subsampled to a depth of 4020 sequences. Alpha diversity did not differ between blank extraction controls processed alongside (n=16) or independent of (n=12) placental samples (Mann-Whitney; Chao1: U=67.5; P=.192; Simpson: U=90.0; P=.799). The richness (U=10.5; P<.0001) and heterogeneity (U=67.0; P=.0001) of blank extraction kit control samples, although very low, were greater than those of placental tissue samples (Supplemental Figure 5).
      Extraction controls processed alongside placental samples did not have a different bacterial profile than those processed alone (NPMANOVA; Jaccard: F=0.863; P=.810; Bray Curtis: F=0.577; P=.940), which indicates that bacterial signals obtained from blank extraction kit samples were not simply due to DNA cross-contamination from placental tissue samples during processing. The bacterial profiles of placental tissue samples and blank extraction kit controls differed in both composition and structure (Supplemental Table 2; Supplemental Figure 6). However, OTU001, classified as Escherichia, accounted for 99.0% and 97.6% of the sequences obtained from placental samples and extraction controls, respectively. OTU009, an Enterococcus, was also found in all samples, with an average relative abundance of 0.11% and 0.33% among placental samples and blank extraction kit controls, respectively. OTU102, a Clostridium, was the only other OTU with an average relative abundance ≥0.1% among the placental tissue samples, and it was detected in only 3 of 29 of these samples. In addition to the 2 Escherichia and Enterococcus OTUs, OTU185, a Shewanella, was a third core OTU (ie, present in at least one-half of the samples) among placental tissue samples. LEfSe analyses indicated that OTU001, Escherichia, was the only OTU that was more relatively abundant among placental samples than technical controls. SourceTracker analysis indicated that a median of 100% (IQR, 99–100%] of the OTUs present in the 16S rRNA gene profiles of placental samples could be explained by their distribution among the profiles of technical controls.

      16S rRNA gene surveys with the use of touchdown PCR

      Twenty-four of 29 placental tissue samples and 28 of 29 blank extraction kit controls yielded ≥1000 sequences with Good’s coverage values exceeding 99%. The other samples were excluded from analyses. The remaining placental and extraction control samples yielded 14,602±12,641 and 38,817±35,710 quality sequences, respectively. These sequences clustered into 350 OTUs. For alpha diversity analyses, each sample was subsampled to a depth of 1060 sequences. Alpha diversity did not differ between controls processed alongside (n=17) or independent of (n=11) placental samples (Mann-Whitney; Chao1: U=81.5; P=.587; Simpson: U=78.0; P=.480). Alpha diversity also did not differ between placental samples and extraction controls (Chao1: U=168.5; P=.354; Simpson: U=190.0; P=.728).
      The bacterial profiles of background extraction controls did not differ between controls processed alongside placental samples or alone (NPMANOVA; Jaccard: F=1.216; p=0.083; Bray Curtis: F=0.867, p=0.672). There was variation in the composition of bacterial profiles based on sample type and round of extraction (Supplemental Table 3; Supplemental Figure 7). Specifically, there was a modest observed difference in bacterial profile composition between placental sample and blank extraction controls in the first round of extractions (F=1.506; P=.040), but not in the second (F=1.032; P=.394), third (F=1.211; P=.122), or fourth (F=0.900; P=.734) round of extractions. In the first round, 5 of 6 and 4 of 6 of the placental samples contained OTU015 (Ralstonia) and OTU034 (an unclassified Enterobacteriaceae), respectively. These OTUs were not present in any of the 4 blank extraction controls processed in round 1. There was no difference in the structure of bacterial profiles between placental tissue samples and blank extraction controls (Supplemental Table 3; Supplemental Figure 7).
      There were 21 prominent OTUs (ie, average relative abundance ≥1%) among placental samples (Supplemental Figure 8). Eight of these OTUs were also prominent among blank extraction control samples. None of the 13 OTUs prominent among placental samples, but not prominent among technical control samples, was present in >21% (5/24) of the placental samples. LEfSe indicated that 3 OTUs were more relatively abundant among placental samples than blank extraction controls (Supplemental Figure 9). These OTUs were 15 (Ralstonia), 17 (Chthoniobacter), and 41 (Anaerococcus). OTUs 15 and 17 were among the prominent OTUs for blank extraction control samples. OTU041 was not prominent among either placental or technical control samples. It was present in 5 of 24 placental samples, with an average relative abundance of 1.79%. OTU041 was not present in any of the 17 blank extraction control samples processed alongside placental samples. However, it did account for 6.4% of the sequences from 1 blank extraction control processed independently of placental samples.
      There were 5 core OTUs (ie, present in at least one-half of samples) among placental samples. Three of the 5 were also core OTUs among blank extraction controls (OTUs 1, 2, and 3). The exceptions were OTU015 (Ralstonia) and OTU017 (Chthoniobacter), which were nonetheless prominent among technical controls. Neither Ralstonia nor Chthoniobacter was detected in the bacterial profiles of the 9 placental tissues characterized through metagenomic sequencing in the primary analyses described earlier.
      SourceTracker analyses indicated that a median of 24% (IQR, 0–76%) of OTUs in the placental samples could be attributed to background DNA contamination in the extraction kits and/or PCR reagents. The large degree of observed variation was due to whether the bacterial profiles of placental samples were dominated by 1 of the 4 most prominent OTUs among the placental samples (OTUs 3, 8, 15, and 2; Supplemental Figure 8). Among the 12 of 24 placental samples that derived at least 25% of their sequences from 1 of these 4 OTUs, 75% (IQR, 55–95%) of their OTUs could be attributed to background DNA contamination. The profiles of the 12 remaining placental samples were each dominated by a different OTU (Supplemental Figure 8). These OTUs were only sporadically present among the technical controls, so their distribution among the placental samples could not be attributed to background DNA contamination based on SourceTracker analyses (median, 0; IQR, 0).

      qPCR

      The secondary qPCR analysis did not indicate the presence of bacteria in placental samples. Although an increase in overall reaction efficiency was observed (96.7%) for the secondary qPCR analysis compared to the primary analysis, the sensitivity of the assay remained approximately 150 copies. As in the primary qPCR analysis, the vast majority of the placental and background technical control samples were beyond the detection limits of the assay. Mean Cq values for both placental sample and background technical controls were >37 cycles (Supplemental Figure 10). There was no difference in Cq values between blank extraction kit controls processed alongside (n=17) or independent of (n=12) placental samples (t-test: t=1.579; P=.126). Therefore, bacterial signals in blank extraction kit samples were not simply due to DNA cross-contamination from placental tissue samples during processing.

      Comment

      Principal findings of the study

      Our principal findings were that (1) cultivation of the placental tissues did not yield viable bacteria in 28 of 29 cases; in the case in which it did, the microorganisms were not detected by 16S rRNA gene sequencing; (2) qPCR did not indicate a greater abundance of bacterial 16S rRNA genes in placental tissues than in technical controls (laboratory environments and reagents); (3) 16S rRNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and technical controls, and (4) metagenomic surveys of placental tissues largely yielded bacterial sequences from cyanobacteria, aquatic bacteria, and plant pathogens, which are microbes ecologically unlikely to populate the human placenta. The identification of Coprobacillus, Streptomyces, and other potentially clinically relevant genera in the metagenomic data, although intriguing, was not consistent with their absence or extreme rarity in the multiple 16S rRNA gene surveys of these samples. Overall, we did not find consistent evidence that the human placenta harbors a unique microbiota because microbial signals derived from placental tissues were similar to those observed in technical controls.

      The claim that “the placenta harbors a unique microbiome”

      In 2014, a key publication reported the results of a study of 320 placentas that used 16S rRNA gene sequencing and of a subset of these (n=48) that also underwent metagenomic sequencing.
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      The authors characterized “a unique placental microbiome niche composed of nonpathogenic commensal microbiota from the Firmicutes, Tenericutes, Proteobacteria, Bacterioidetes, and Fusobacteria phyla.”
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      Placental microbiota profiles were more similar to those of the human oral cavity than those of the vagina, gut, and skin (Figure 1 in Aagaard et al
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      ). Escherichia coli was most abundant in the placenta, followed by Bacteroides spp, P acnes, Neisseria lactamica, and S epidermidis (Figure 2 in Aagaard et al
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      ). However, cultures were not used in this study; therefore, there is no information about the viability of the microbes from which sequences were detected. qPCR was also not part of the study; nonetheless, the authors emphasized that the placenta was a low microbial biomass site.
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      This publication stimulated research into the existence of a placental microbiota. Twelve additional studies (Table 3) have interrogated placental samples at term with the use of sequence-based techniques to determine, at least in part, whether there is a placental microbiota.
      • Lauder A.P.
      • Roche A.M.
      • Sherrill-Mix S.
      • et al.
      Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.
      • Doyle R.M.
      • Alber D.G.
      • Jones H.E.
      • et al.
      Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery.
      • Antony K.M.
      • Ma J.
      • Mitchell K.B.
      • Racusin D.A.
      • Versalovic J.
      • Aagaard K.
      The preterm placental microbiome varies in association with excess maternal gestational weight gain.
      • Zheng J.
      • Xiao X.
      • Zhang Q.
      • Mao L.
      • Yu M.
      • Xu J.
      The placental microbiome varies in association with low birth weight in full-term neonates.
      • Bassols J.
      • Serino M.
      • Carreras-Badosa G.
      • et al.
      Gestational diabetes is associated with changes in placental microbiota and microbiome.
      • Collado M.C.
      • Rautava S.
      • Aakko J.
      • Isolauri E.
      • Salminen S.
      Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid.
      • Prince A.L.
      • Ma J.
      • Kannan P.S.
      • et al.
      The placental membrane microbiome is altered among subjects with spontaneous preterm birth with and without chorioamnionitis.
      • Doyle R.M.
      • Harris K.
      • Kamiza S.
      • et al.
      Bacterial communities found in placental tissues are associated with severe chorioamnionitis and adverse birth outcomes.
      • Gomez-Arango L.F.
      • Barrett H.L.
      • McIntyre H.D.
      • Callaway L.K.
      • Morrison M.
      • Nitert M.D.
      Contributions of the maternal oral and gut microbiome to placental microbial colonization in overweight and obese pregnant women.
      • Parnell L.A.
      • Briggs C.M.
      • Cao B.
      • Delannoy-Bruno O.
      • Schrieffer A.E.
      • Mysorekar I.U.
      Microbial communities in placentas from term normal pregnancy exhibit spatially variable profiles.
      • Zheng J.
      • Xiao X.H.
      • Zhang Q.
      • et al.
      Correlation of placental microbiota with fetal macrosomia and clinical characteristics in mothers and newborns.
      • Leon L.J.
      • Doyle R.
      • Diez-Benavente E.
      • et al.
      Enrichment of clinically relevant organisms in spontaneous preterm delivered placenta and reagent contamination across all clinical groups in a large UK pregnancy cohort.
      Eleven of these studies have concluded that there is evidence of a placental microbiota at term based on 16S rRNA gene sequencing and/or metagenomics.
      • Doyle R.M.
      • Alber D.G.
      • Jones H.E.
      • et al.
      Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery.
      • Antony K.M.
      • Ma J.
      • Mitchell K.B.
      • Racusin D.A.
      • Versalovic J.
      • Aagaard K.
      The preterm placental microbiome varies in association with excess maternal gestational weight gain.
      • Zheng J.
      • Xiao X.
      • Zhang Q.
      • Mao L.
      • Yu M.
      • Xu J.
      The placental microbiome varies in association with low birth weight in full-term neonates.
      • Bassols J.
      • Serino M.
      • Carreras-Badosa G.
      • et al.
      Gestational diabetes is associated with changes in placental microbiota and microbiome.
      • Collado M.C.
      • Rautava S.
      • Aakko J.
      • Isolauri E.
      • Salminen S.
      Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid.
      • Prince A.L.
      • Ma J.
      • Kannan P.S.
      • et al.
      The placental membrane microbiome is altered among subjects with spontaneous preterm birth with and without chorioamnionitis.
      • Doyle R.M.
      • Harris K.
      • Kamiza S.
      • et al.
      Bacterial communities found in placental tissues are associated with severe chorioamnionitis and adverse birth outcomes.
      • Gomez-Arango L.F.
      • Barrett H.L.
      • McIntyre H.D.
      • Callaway L.K.
      • Morrison M.
      • Nitert M.D.
      Contributions of the maternal oral and gut microbiome to placental microbial colonization in overweight and obese pregnant women.
      • Parnell L.A.
      • Briggs C.M.
      • Cao B.
      • Delannoy-Bruno O.
      • Schrieffer A.E.
      • Mysorekar I.U.
      Microbial communities in placentas from term normal pregnancy exhibit spatially variable profiles.
      • Zheng J.
      • Xiao X.H.
      • Zhang Q.
      • et al.
      Correlation of placental microbiota with fetal macrosomia and clinical characteristics in mothers and newborns.
      • Leon L.J.
      • Doyle R.
      • Diez-Benavente E.
      • et al.
      Enrichment of clinically relevant organisms in spontaneous preterm delivered placenta and reagent contamination across all clinical groups in a large UK pregnancy cohort.
      Thus, the existence of a placental microbiota has become a majority view in perinatal microbiology at this time.
      Table 3Description of previous 16S ribosomal RNA gene or metagenomic studies of the human placental microbiota at term
      StudyAuthorYearCentral research questionsMode of delivery (sample size)Type of sampleMolecular microbiology methodsWas culture used?Were DNA contamination controls included?
      1Aagaard et al
      • Aagaard K.
      • Ma J.
      • Antony K.M.
      • Ganu R.
      • Petrosino J.
      • Versalovic J.
      The placenta harbors a unique microbiome.
      2014Is there a placental microbiota? Does it vary with antenatal infection and preterm birth?Term cesarean (n=53); term vaginal (n=178); preterm cesarean (n=20); preterm vaginal (n=69)Villous tree; collected <1 hour after delivery16S ribosomal RNA gene sequencing; metagenomic sequencing (subset of 48 subjects)NoOne blank extraction kit processed per 11 placental samples; these blanks did not generate noticeable bands of amplified DNA and thus were not sequenced routinely; reagents from a limited number of blanks were sequenced, and their bacterial profiles reflected airway or nonhuman sources (data not provided)
      Conclusions: There is a placental microbiota at term and preterm, regardless of mode of delivery; the placental microbiota shares similarity with the microbiota of the oral cavity; the placental microbiota differs between women who deliver preterm and at term; the placental microbiota differs between women with and without a remote history of antenatal infection.
      2Doyle et al
      • Doyle R.M.
      • Alber D.G.
      • Jones H.E.
      • et al.
      Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery.
      2014Does the placental microbiota differ between preterm and term deliveries?Term cesarean without labor (n=4); term vaginal (n=6); preterm vaginal (n=14)Amnion and chorion; time between delivery and processing not provided16S ribosomal RNA gene sequencingNoNo
      Conclusions: There is a placental microbiota at term and preterm, regardless of mode of delivery; nevertheless, the microbial profiles of placental tissues differ between cesarean and vaginal deliveries; the placental microbiota differs between term and preterm deliveries.
      3Antony et al
      • Antony K.M.
      • Ma J.
      • Mitchell K.B.
      • Racusin D.A.
      • Versalovic J.
      • Aagaard K.
      The preterm placental microbiome varies in association with excess maternal gestational weight gain.