Table 1 Historical evidence of the female intrauterine microbiome in pregnancy

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]