Midtrimester amniotic fluid from healthy pregnancies has no microorganisms using multiple methods of microbiologic inquiry.

Abstract Background There is controversy about whether the amniotic fluid contains bacteria. Using sequencing-based methods, recent studies report that the amniotic fluid is colonized by microorganisms. However, background-contaminating DNA might lead to false-positive findings when such a low microbial biomass sample is examined. Objective To determine whether the midtrimester amniotic fluid of patients who subsequently had normal pregnancy outcomes contains a microbial signature. Study design In this prospective cohort study, 42 amniotic fluid samples were collected from 37 pregnancies (5 twin and 32 singletons) during genetic amniocentesis in the midtrimester. The subsequent pregnancy outcomes of all the participants were followed. Multiple methods were used to detect the presence of microorganisms in this study, including cultivation, quantitative real-time polymerase chain reaction (qPCR) and 16S ribosomal RNA (rRNA) gene sequencing. Multiple positive controls (n=16) served as quality controls, including 3 adult fecal samples, 4 vaginal swabs and 9 artificial bacterial communities, and run in parallel with negative controls (n=12), including 4 from the hospital operating room and 8 from the laboratory, to account for background-contaminating DNA during each step of the experiments. Results (1) No bacteria under anaerobic or aerobic conditions or genital mycoplasmas were cultured from any of the amniotic fluid samples. (2) Quantitative PCR did not reveal greater copy numbers of 16S rRNA gene in amniotic fluid samples than in negative controls. (3) 16S rRNA gene sequencing did not indicate significant difference in the microbial richness or community structures between amniotic fluid and negative controls. Conclusion With multiple methods of microbiologic inquiry, no microorganisms were identified in the midtrimester amniotic fluid of healthy pregnancies with a normal pregnancy outcome.