Summary: Microbes found in the placenta may play a key role in shaping the developing fetal immune system.
Source: Baylor College of Medicine
Researchers at Baylor College of Medicine previously found evidence that the placenta harbors a sparse but still present community of microorganisms, which they and other researchers speculate may contribute to key functions in pregnancy, including immunity.
“There has been some debate about our and others’ findings in the placenta. Because it is a sparse, or low biomass, community, it is a fair question to ask how much of what we identify as the microbiome is actually bacteria and how much is potentially environmental contamination, or maternal blood in the placenta,” said senior author Dr. Kjersti Aagaard, professor and the Henry and Emma Meyer Chair of obstetrics and gynecology at Baylor.
“Previously, bacteria were found using metagenomics or microbiome sequencing, and now we have confirmed that signal based on our ability to label the bacterial RNA with a florescent ‘tag’ and actually see them,” said Dr. Maxim Seferovic, instructor in obstetrics and gynecology at Baylor and lead author in the study. “We leveraged a powerful new imaging technology to add greater specificity in the signal of bacterial RNA, which helped us to see bacteria within the microarchitecture of the placental tissue.”
Researchers examined microbes in term and preterm gestations using a signal amplified 16S universal in situ hybridization probe designed for bacterial rRNA, along with several other histologic methods. Seferovic said the study was carefully designed to control for contamination as best as possible, so that these sparse bacteria could be accurately attributed to their location in the placenta.
“We did not see quantitative or numerical differences between preterm or full-term births, nor did we see them localizing to different substrata. But we do see differences in what genera of bacteria are there in preterm or full term, and this supported our and other’s past findings as well,” said Aagaard.
A sparse community
Seferovic said the study was designed to determine if past studies were, in fact, accurate and truly did look at a low biomass community of microbes that could be reliably distinguished from environmental contamination. This work, when combined with that of several other labs, should give researchers confidence that not only can they sequence these microbes but also that they can see the bacteria in very predictable locations in different placentas.
Seferovic and Aagaard suggest that this boosts their team’s and others’ confidence that they can begin to look more toward the role of microbes in the intrauterine environment in shaping the developing immune system in the fetus, and what role things like the mom’s diet or preterm birth may play in that development.
“At some point, we all acquire trillions of bacteria in our bodies that we do not reject with an immune inflammatory response. We are speculating that these low biomass communities may play a key role in shaping the developing fetal immune system to help educate it on which microbes may be beneficial and which might not,” Aagaard said.
Both Aagaard and Seferovic agree that there is still a lot of work ahead to be done in this exciting area of the developing microbiome and microbiome science. It is their hope that the techniques and tools developed for this study will lend a hand to other researchers similarly working in challenging low biomass communities.
Others who took part in the study include Dr. Ryan M. Pace, Dr. Matthew Carroll, Benjamin Belfort, Angela M. Major, Dr. Derrick M. Chu, Dr. Diana A. Racusin, Dr. Eumenia C. C. Castro, Dr. Kenneth L. Muldrew and Dr. James Versalovic, all with Baylor College of Medicine. Aagaard also is professor in the Departments of Molecular and Human Genetics, Molecular and Cellular Biology, and Molecular Physiology and Biophysics.
Funding: Funding is from the March of Dimes Preterm Birth Research Initiative, the Burroughs Welcome Fund Preterm Birth Initiative and the NIH (1R01NR014792, 6R01DK089201, R01HD091731, NICHD N01-HD-80020 NCS Formative Research.
Baylor College of Medicine
Graciela Gutierrez – Baylor College of Medicine
The image is in the public domain.
Original Research: Closed access
“Visualization of microbes by 16S in situ hybridization in term and preterm placentae without intraamniotic infection”. Kjersti Aagaard et al.
American Journal of Obstetrics & Gynecology. doi:10.1016/j.ajog.2019.04.036
Visualization of microbes by 16S in situ hybridization in term and preterm placentae without intraamniotic infection
Numerous reports have documented bacteria in the placental membranes and basal plate decidua in the absence of immunopathology using histologic techniques. Similarly, independent metagenomic characterizations have identified an altered taxonomic makeup in association with spontaneous preterm birth. Here we sought to corroborate these findings by localizing presumptive intact bacteria using molecular histology within the placental microanatomy.
Here we examined for microbes in term and preterm gestations using a signal-amplified 16S universal in situ hybridization probe set for bacterial rRNA, alongside traditional histologic methods of Warthin–Starry and Gram stains, as well as clinical culture methodologies. We further sought to differentiate accompanying 16S gene sequencing taxonomic profiles from germ-free (gnotobiotic) mouse and extraction and amplicon contamination controls.
Placentae were collected from a total of 53 subjects, composed of term labored (n = 4) and unlabored cesarean deliveries (n = 22) and preterm vaginal (n = 18) and cesarean deliveries (n = 8); a placenta from a single subject with clinical and histologic evident choriomanionitis was employed as a positive control (n = 1). The preterm cohort included spontaneous preterm birth with (n = 6) and without (n = 10) preterm premature rupture of membranes, as well as medically indicated preterm births (n = 10). Placental microbes were visualized using an in situ hybridization probe set designed against highly conserved regions of the bacterial 16S ribosome, which produces an amplified stable signal using branched DNA probes. Extracted bacterial nucleic acids from these same samples were subjected to 16S rRNA metagenomic sequencing (Illumina, V4) for course taxonomic analysis, alongside environmental and kit contaminant controls. A subset of unlabored, cesarean-delivered term pregnancies were also assessed with clinical culture for readily cultivatable pathogenic microbes.
Molecular in situ hybridization of bacterial rRNA enabled visualization and localization of low-abundance microbes after systematic high-power scanning. Despite the absence of clinical or histologic chorioamnionitis in 52 of 53 subjects, instances of 16S rRNA signal were confidently observed in 13 of 16 spontaneous preterm birth placentas, which was not significantly different from term unlabored cesarean specimens (18 of 22; P > .05). 16S rRNA signal was largely localized to the villous parenchyma and/or syncytiotrophoblast, and less commonly the chorion and the maternal intervillous space. In all term and unlabored cesarean deliveries, visualization of evident placental microbes by in situ hybridization occurred in the absence of clinical or histologic detection using conventional clinical cultivation, hematoxylin–eosin, and Gram staining. In 1 subject, appreciable villous bacteria localized to an infarction, where 16S microbial detection was confirmed by Warthin–Starry stain. In all instances, parallel sample principle coordinate analysis using Bray-Cutis distances of 16S rRNA gene sequencing data demonstrated consistent taxonomic distinction from all negative or potential contamination controls (P = .024, PERMANOVA). Classification from contaminant filtered data identified a distinct taxonomic makeup among term and preterm cohorts when compared with contaminant controls (false discovery rate <0.05).
Presumptively intact placental microbes are visualized as low-abundance, low-biomass and sparse populations within the placenta regardless of gestational age and mode of delivery. Their taxonomic makeup is distinct from contamination controls. These findings further support several previously published findings, including our own, which have used metagenomics to characterize low-abundance and low-biomass microbial communities in the placenta.