Leon, LJ; Doyle, R; Diez-Benavente, E; Clark, TG; Klein, N; Stanier, P; Moore, GE; (2018) Enrichment of clinically relevant organisms in spontaneous preterm delivered placenta and reagent contamination across all clinical groups in a large UK pregnancy cohort. Applied and Environmental Microbiology , 84 (14) 10.1128/AEM.00483-18.

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Abstract

In this study differences in the placental microbiota of term and preterm deliveries from a large UK pregnancy cohort were studied using 16S targeted amplicon sequencing. The impact of contamination from DNA extraction, PCR reagents, as well as those from delivery itself were also examined. A total of 400 placental samples from 256 singleton pregnancies were analysed and differences investigated between spontaneous preterm, non-spontaneous preterm, and term delivered placenta. DNA from recently delivered placenta was extracted, and screening for bacterial DNA was carried out using targeted sequencing of the 16S rRNA gene on the Illumina MiSeq platform. Sequenced reads were analysed for presence of contaminating operational taxonomic units (OTUs) identified via sequencing of negative extraction and PCR blank samples. Differential abundance and between sample (beta) diversity metrics were then compared. A large proportion of the reads sequenced from the extracted placental samples mapped to OTUs that were also found in negative extractions. Striking differences in the composition of samples were also observed, according to whether the placenta was delivered abdominally or vaginally, providing strong circumstantial evidence for delivery contamination as an important contributor to observed microbial profiles. When OTU and genus level abundances were compared between the groups of interest, a number of organisms were enriched in the spontaneous preterm cohort, including organisms that have been previously associated with adverse pregnancy outcomes, specifically Mycoplasma spp., and Ureaplasma spp.. However, analyses of overall community structure did not reveal convincing evidence for the existence of a reproducible 'preterm placental microbiome'. IMPORTANCE: Preterm birth is associated with both psychological and physical disabilities and is the leading cause of infant morbidity and mortality worldwide. Infection is known to be an important cause of spontaneous preterm birth, and recent research has implicated variation in the 'placental microbiome' with preterm birth risk. Consistent with previous studies, the abundance of certain clinically relevant species differed between spontaneous preterm and non-spontaneous preterm or term delivered placenta. These results support the view that a proportion of spontaneous preterm births have an intra-uterine infection component. However, an additional observation from this study was that a substantial proportion of reads sequenced were contaminating reads, rather than DNA from endogenous, clinically relevant species. This observation warrants caution in the interpretation of sequencing output from such low biomass samples as the placenta.

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