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Table 1 Historical evidence of the female intrauterine microbiome in pregnancy

From: Maternal and early life exposures and their potential to influence development of the microbiome

Year Endometrium or decidua Placenta parenchyma Chorion Amnion Amniotic fluid Meconium or fetal intestine Culture-independent Detection Culture-dependent Detection a Ref.
Method Conclusion Method Conclusion
1926       n/a n/a Aerobic 38% of meconium samples are culture-positive [158]
1934       Histology (Löffler’s methylene blue and Gram stains) 6% of meconium samples contained recognized bacteria Aerobic: EMB lactose, Blood, Dextrose broth, Deep iron brain 38% of meconium samples are culture-positive [159]
1936       Histology (Cover slip and Gram stain) No bacteria detected Aerobic: EMB, Blood, Brain or glucose broth. Anaerobic: Blood slants 10% of meconium samples collected within 30 min after delivery are culture-positive [160]
1982        n/a n/a Aerobic: Broth and blood agar Aerobic bacteria have been cultured from the chorion in the absence of histologic chorioamnionitis [161]
1997        16S rRNA polymerase chain reaction (PCR) of the V6-8 HVRs 16S rRNA is detected in 94% of culture-positive and 36% of culture-negative amniotic fluid from cases of preterm labor with intact membranes Traditional clinical culture 20% of amniotic fluid samples are culture-positive and 80% are culture-negative [162]
2004        16S rRNA sequencing of the V6-8 HVRs using Sequenase or Cycle Sequencing 16S rRNA identified in culture-negative amniotic fluid is mapped to Leptotrichia sanguinegens, Fusobacterium nucleatum, Ureaplasma urealyticum Traditional clinical culture 20% of amniotic fluid samples are culture-positive and 80% are culture-negative [163]
2007        Porphyromonas gingivalis -specific PCR and 16S rRNA PCR of V5-8 HVRs P. gingivalis DNA is detected in 30% of amniotic fluid from cases of threatened preterm labor Anaerobic: non-selective Columbia blood All amniotic fluid samples are culture-negative for P. gingivalis [164]
2007     Gram stain of amniotic fluid samples 10% of culture-negative amniotic fluid samples yield a positive Gram stain Traditional clinical culture Diverse microbes are recovered from term placentas without histological or clinical evidence of inflammation [165]
2008       16S rRNA PCR of the V3-5 HVRs, histology (Gram stain), and gas-liquid chromatography of glucose fermentation products There is a PCR inhibitor in the placenta. At higher concentrations of placental tissue, 16S rRNA PCR products are no longer detected from known culture-positive placental samples Aerobic: Chocolate, TSA blood. Anaerobic: A-7, Brucella blood 51% of placental samples from Cesarean-delivered and 75% from vaginally delivered preterm births are culture-positive [166]
2008        16S rRNA end-point PCR (V1-V4 and V3-5 HVRs) and 16S rRNA real-time PCR (V1-2 and V3-5 HVRs). Plus 18S fungal targets Microbial rRNA is detected from culture-negative amniotic fluid of preterm labor with intact membranes Aerobic and anaerobic 9.6% of amniotic fluid samples are culture-positive [167]
2008       PCR of genetically labeled Enterococcus fecium HA1 (that was orally inoculated into pregnant mice) Meconium from term murine pups contains the genetic label of the E. fecium fed to their dams during pregnancy Aerobic: BHI, VRB, CNA. Anaerobic: Wilkins-Chalgren, MRS Enterococcus and Staphylococcus are cultured from meconium [168]
2009        Bifidobacterium and Lactobacillus -specific PCR Bifidobacterium and Lactobacillus spp. DNA is detected in 91–97% of placental tissue Anaerobic: Blood liver, LB All samples are culture-negative for Bifidobacterium spp. and Lactobacillus spp. [169]
2010        16S rRNA PCR (primers A17F and 1512R) After human oral bacteria are injected into murine tail vein, the 16S rRNA is detected in the murine placenta n/a n/a [170]
2010        16S rRNA end-point PCR (V1-V4 and V3-5 HVRs) and 16S rRNA real-time PCR (V1-2 and V3-5 HVRs). Plus 18S fungal targets 16S rRNA is detected from culture-negative amniotic fluid in preterm pre-labor rupture of membranes Aerobic and anaerobic 34% of amniotic fluid samples were culture-positive [171]
2013       Histology (H&E, Gram, Hema 3 modified Geimsa, Brown-Hopps modified Gram stains) Intracellular bacteria are detected by histology in the non-inflamed maternal basal plate of the placenta n/a n/a [172]
2013       16S rRNA PCR of the V6-8 HVRs analyzed by Denaturing Gradient Gel Electrophoresis and 16S rRNA Human Intestinal Tract Chip analysis of the V1-6 HVRs Meconium microbiome is characterized by abundant Firmicutes and Proteobacteria, and is lower diversity than 3-week old infant stool Aerobic: MRS, MacConkey, BP, SDC, BHI, CNA. Anaerobic: WC, MRS with L-cysteine 78% of meconium samples are culture-positive [173]
2014        16S rRNA pyrosequencing of the V1-3 HVRs with comparative WGS metagenomic sequencing Distinct placental microbiome composed of nonpathogenic commensals, most similar to the human oral microbiome n/a n/a [56]
2014       16S rRNA pyrosequencing of the V1-2 and V5-6 HVRs Common placental genera regardless of delivery mode n/a n/a [174]
2014       Histology (Brown-Hopps modified Gram stain) Bacteria are detected within extravillous trophoblasts of the placental basal plate Inoculation of the placental basal plate ex vivo Inoculated bacteria are detected within extravillous trophoblasts of the placental basal plate [175]
2015        16S rRNA sequencing of the V3-4 HVRs using Illumina MiSeq Lower diversity and lower Lactobacillus percentage in placenta associated with low birthweight versus full term n/a n/a [176]
2015        16S rRNA pyrosequencing of the V1-3 HVRs with comparative WGS metagenomic sequencing Distinct placental microbiome between women with and without excess gestational weight gain n/a n/a [61]
2016      16S rRNA pyrosequencing of the V1-3 HVRs with comparative quantitative PCR and denaturing gradient gel electrophoresis Placental and amniotic fluid microbiomes both characterized by low richness, low diversity, and high Proteobacteria, which suggests microbial transfer at the fetal-maternal interface Anaerobic: Gifu, LB 50% of taxa identified by culture-independent methods were recovered through culture [177]
2016        WGS metagenomic sequencing Distinct placental microbiome in spontaneous preterm birth that further differentiates by severity of chorioamnionitis Traditional clinical culture for Ureaplasma and Mycoplasma Some chorioamnionitis-positive patients were negative for Ureaplasma. Some chorioamnionitis-negative patients were positive for Ureaplasma [178]
2016        16S rRNA sequencing of the V3-4 HVRs using Illumina MiSeq and TaqMan gene expression using RT-PCR Distinct placental microbiome associated with gestational diabetes n/a n/a [179]
2017       16S rRNA sequencing of the V6-8 HVRs using Illumina MiSeq Fetal side of placenta harbors microbiome resembling the gravidae’s oral (not fecal) microbiome n/a n/a [180]
2017      16S rRNA sequencing of the V5-7 HVRs using Illumina MiSeq with comparative qPCR Only specific bacteria found in placental tissue is associated with chorioamnionitis and low-birthweight neonates n/a n/a [181]
2017        16S rRNA sequencing of the V3-4 HVRs using Illumina MiSeq Distinct placental microbiome associated with fetal macrosomia n/a n/a [182]
2017    16S rRNA sequencing of all V1-9 HVRs using Illumina MiSeq with comparative qPCR (V4 region and Ralstonia insidiosa) Composition of the placental microbiome differs between maternal, fetal-maternal, and fetal spaces, independent of delivery mode n/a n/a [183]
2018        16S rRNA pyrosequencing of the V4 HVR Amniotic fluid and placenta contain low-diversity microbiomes dominated by Enterobacteriaceae phylotype Anaerobic: BHI, Columbia blood 0% of amniotic fluid samples and 20% of placenta samples are culture-positive [184]
2018      16S rRNA sequencing of the V3-4 HVR using Illumina MiSeq with comparative qPCR 16S rRNA is detected in 49% of placental samples, with equal richness and diversity between HPV-positive and negative groups n/a n/a [185]
2019    16S rRNA ISH with comparative 16S rRNA sequencing of the V4 HVR using Illumina MiSeq and histology (H&E, Warthin-Starry, and Gram stains) Detection of low-abundance bacterial RNA in the placental villi and chorion Aerobic: Blood, MacConkey, Chocolate, A-7. Anaerobic: Brucella, PEA, K-V No detectable growth [186]
2019      16S rRNA sequencing using PacBio SMRT cell 100% of meconium and 84% of amniotic fluid samples contained bacterial 16S rRNA n/a n/a [187]
2019        GFP plasmid DNA PCR and immunohistochemical detection of fluorescent-tagged Staphylococcus aureus Fluorescent-tagged S. aureus injected into the maternal bloodstream is recovered in the placenta BHI, TSA No detectable growth [188]
2019       16S rRNA sequencing of the V5-6 HVRs using Illumina MiSeq Bacterial DNA detected in endometrial biopsies following elective Cesarean delivery n/a n/a [189]
2019   16S rRNA sequencing of the V4-5 HVRs using Illumina MiSeq Bacterial DNA is detected in meconium, placenta, and fetal membranes independent of delivery method n/a n/a [190]
2019     16S rRNA ISH with comparative 16S rRNA sequencing of the V4 HVR using Illumina MiSeq Bacterial DNA present in meconium partially aligns to DNA found in the in utero environment Aerobic and Anaerobic: BHI, MRS, Chocolate, MacConkey Early gestational samples are more likely to be culture-positive in murine fetus [191]
2019        16S rRNA sequencing of the V3-4 HVR using Illumina MiSeq with comparative qPCR Neonatal oral microbiota most resembles the 16S rRNA profile of the placenta n/a n/a [192]
2020       n/a n/a Aerobic and Anerobic: Blood 86.5% of meconium and 57% of placental samples are culture-positive [193]
2020       16S rRNA sequencing of the V4 HVR using Illumina MiSeq with comparative qPCR Bacterial DNA profiles of placental samples without histological evidence of chorioamnionitis are distinctly different than negative controls n/a n/a [194]
2020     ISH and qPCR of the 16S rRNA inter-HVR segment between the V2-3 HVRs and Fusobacterium-specific 16S segment Preeclampsia-exposed placenta has increased total bacterial and Fusobacterium DNA load n/a n/a [195]
2020       16S rRNA sequencing of the V4 HVR using Illumina MiSeq with comparative V6 HVR qPCR, eubacterial fluorescence ISH, and scanning electron microscopy Sparse but viable bacteria are visualized in human meconium and detected by 16S rRNA signal Aerobic: Micrococcus-specific isolation in BHI Guided by molecular identification, viable Micrococcus spp. isolates were cultured from meconium [196]
2021        16S rRNA sequencing of the V4 HVR using Illumina MiSeq following 16S Specific Enrichment Enrichment preceding DNA sequencing allows for resolution between placental microbes and air swab controls n/a n/a [197]
2021       WGS metagenomic and metatranscriptomic sequencing using Illumina HiSeq A low-diversity, low-biomass microbiome was identified in the prenatal fetal gut of lambs n/a n/a [198]
2021   16S rRNA sequencing of the V4-5 HVRs from nested PCR using Illumina HiSeq, 16S RNA-ISH, and scanning electron microscopy Few but consistently present microbes are detected in healthy human fetuses in the 2nd trimester. Bacterial cocci-like shapes are localized along mucin-like structures of the fetal ileum. Aerobic and Anaerobic: pre-reduced anaerobic blood- supplemented basal media Tissue from 75% of fetuses produced reproducible colonies with heterogenous distribution suggesting true signal from specific organs. [199]
  1. a Culture Media Abbreviations: A-7 Shepard’s Differential Agar Base, BHI Brain Heart Infusion, BP Baird Parker, CNA Columbia Colistin-Nalidixic Acid, EMB Eosin Methylene Blue, K-V Kanamycin-Vancomycin, LB Luria-Bertani, MRS De Man, Rogosa, and Sharpe, PEA Phenylethyl Alcohol Agar, PPLO PleuroPneumonia-Like Organism, PRAS Pre-Reduced Anaerobically Sterilized, PYG Peptone Yeast Extract Glucose, SDC Sabouraud Dextrose Agar with Chloramphenicol, TSA Tryptic Soy Agar, VRB Violet Red Bile, WC Wilkins-Chalgren