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

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 et al. Am J Obstet Gynecol. 2019 Mar.

Abstract

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.

Keywords: bacteria; bacterial culture; contamination; low microbial biomass sample; microbiome; microorganism; pregnancy; tissue.

Published by Elsevier Inc.

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Conflict of interest statement

Disclosure statement: The authors report no conflicts of interest.

Figures

Figure 1.
Figure 1.. Principal Coordinates Analyses (PCoA) illustrating similarity in 16S rRNA gene profiles among amnion & chorionic plate, villous tree & basal plate, and technical control samples:

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 (OTUs) were generated using a 97% sequence similarity cutoff and the primary 16S rRNA gene nested PCR data set.

Figure 2.
Figure 2.. Heat map illustrating similarity in percent relative abundances of prominent operational taxonomic units (OTUs) among placental samples and technical controls.

Prominent OTUs were defined as those having an average relative abundance ≥ 1% among the placental samples. OTUs were generated using a 97% sequence similarity cutoff and the primary 16S rRNA gene nested PCR data set. Asterisks indicate OTUs that were prominent in placental samples but not in controls.

Figure 3.
Figure 3.. Quantitative PCR (qPCR) analyses illustrating similarity in 16S rRNA gene abundance among amnion & chorionic plate, villous tree & basal plate, and technical control samples:

a. Comparison of quantification cycle (Cq) values (mean ± SD) of serially diluted placental genomic DNA samples spiked with equal concentrations (5.7 × 103 copies per reaction) of genomic DNA from Echerichia coli ATCC 25922, illustrating that amplification inhibition is eliminated by diluting samples with nuclease-free water by a factor of 1:3 or more; b. Standard curves for three 10-fold dilution series (2.82 × 106 – 2.82 × 101 copies, 2.12 × 106 – 2.12 × 101 copies, and 2.97 × 106 – 2.97 × 101 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 102 and 3.14 × 102 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq 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 Figure panel b.

Figure 4.
Figure 4.. Heat map illustrating relative abundances of prominent bacterial genera among placental samples as determined by metagenomic sequencing.

Prominent genera were here defined as those having an average relative abundance ≥ 0.1% among the placental samples. AC and V indicate amnion & chorionic plate and villous tree & basal plate samples, respectively.

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References

    1. Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R. Bacterial community variation in human body habitats across space and time. Science 2009;326:1694–7. - PMC - PubMed
    1. Human Microbiome Project C. Structure, function and diversity of the healthy human microbiome. Nature 2012;486:207–14. - PMC - PubMed
    1. Knight R, Callewaert C, Marotz C, et al. The Microbiome and Human Biology. Annu Rev Genomics Hum Genet 2017;18:65–86. - PubMed
    1. Fierer N Embracing the unknown: disentangling the complexities of the soil microbiome. Nat Rev Microbiol 2017;15:579–90. - PubMed
    1. Delgado-Baquerizo M, Oliverio AM, Brewer TE, et al. A global atlas of the dominant bacteria found in soil. Science 2018;359:320–25. - PubMed

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