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

doi:10.1016/j.ajog.2018.10.018

American Journal of Obstetrics and Gynecology

Volume 220, Issue 3, March 2019, Pages 267.e1-267.e39

https://doi.org/10.1016/j.ajog.2018.10.018 Get rights and content

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.

Section snippets

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),

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.59, 231

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

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

Acknowledgments

We acknowledge the contributions of Dr Paul Lephart, Mr Timothy Burger, and Ms Maureen Taylor of the Detroit Medical Center’s Microbiology Laboratory, who made possible the conduct of cultivation assays; Ms Judy Opp of the University of Michigan was helpful with the sequencing of 16S rRNA gene amplicons and metagenomic surveys.

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: Supported, in part, by the 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 (NICHD/NIH/DHHS), and, in part, with federal funds from the NICHD/NIH/DHHS under Contract No. HHSN275201300006C; this research was also supported by the Wayne State University Perinatal Research Initiative in Maternal, Perinatal and Child Health.

: Dr. Roberto Romero has contributed to this work as part of his official duties as an employee of the U.S. Federal Government.

: The authors report no conflict of interest.

: Cite this article as: Theis KR, Romero R, Winters AD, et al. Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics. Am J Obstet Gynecol 2019;220:267.e1-39.

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

Background

Objective

Study Design

Results

Conclusion

Section snippets

Study design

Patient characteristics

Bacterial culture of placental tissues

Principal findings of the study

Acknowledgments

Am J Obstet Gynecol

Fertil Steril

Eur J Obstet Gynecol Reprod Biol

Am J Obstet Gynecol

Am J Obstet Gynecol

Am J Obstet Gynecol

Am J Obstet Gynecol

Am J Obstet Gynecol

Placenta

Am J Obstet Gynecol

Am J Obstet Gynecol

Am J Obstet Gynecol

Am J Obstet Gynecol

Human Microbiome Journal

Am J Obstet Gynecol

Fertil Steril

Placenta

Am J Obstet Gynecol

Am J Obstet Gynecol

Lab Invest

Am J Obstet Gynecol

Am J Obstet Gynecol

Placenta

Bacterial community variation in human body habitats across space and time

Science

Structure, function and diversity of the healthy human microbiome

Nature

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Annu Rev Genomics Hum Genet

Embracing the unknown: disentangling the complexities of the soil microbiome

Nat Rev Microbiol

A global atlas of the dominant bacteria found in soil

Science

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Microbiome

The vaginal microbiome: new information about genital tract flora using molecular based techniques

BJOG

Vaginal microbiome of reproductive-age women

Proc Natl Acad Sci U S A

The vaginal microbiota of pregnant women who subsequently have spontaneous preterm labor and delivery and those with a normal delivery at term

Microbiome

The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women

Microbiome

A core gut microbiome in obese and lean twins

Nature

Population-level analysis of gut microbiome variation

Science

The human oral microbiome

J Bacteriol

Oral cavity contains distinct niches with dynamic microbial communities

Environ Microbiol

The normal human anaerobic microflora

Scand J Infect Dis Suppl

Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests

BMC Microbiol

Revised estimates for the number of human and bacteria cells in the body

PLoS Biol

Recognizing the reagent microbiome

Nat Microbiol

More easily cultivated than identified: classical isolation with molecular identification of vaginal bacteria

J Infect Dis

Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation

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