No microorganism was detected in amniotic fluid of healthy pregnancies from the second trimester to the delivery

Abstract Background The early colonization and establishment of the microbiome in newborn is a crucial step in the development of immune system and host metabolism. However, the exact timing of initial microbial colonization remains a subject of ongoing debate. While numerous studies have attempted to determine the presence or absence of intrauterine bacteria, the majority of them have drawn conclusions based on sequencing data from maternal or infant samples taken at a single time point. In this study, we aimed to investigate the microbial signals in amniotic fluid (AF) from the second trimester until the time of delivery using multiple microbiological methods. Methods AF samples were collected during the second trimester (19-21 gestational weeks) and at the time of delivery. Cohort 1 included 51 women who underwent the term and elective cesarean section, with both their second trimester and delivery AF samples (n=55, respectively) analyzed.Cohort 2 contained of 22 women who experienced preterm birth and/or infection, with only their second trimester AF samples (n=24) examined. Additionally, multiple procedural negative controls and technical positive controls were applied into this study to remove potential contamination. Microbial profiles were assessed through cultivation, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and cytokine analysis. Results In cohort 1, the bacterial load and community structure in the second trimester AF samples were indistinguishable from negative controls. Although marginally higher bacterial loads and different bacterial communities were observed in the delivery AF samples compared to negative controls, these bacterial DNA signals were not considered biologically functional due to the absence of maternal inflammatory responses. In cohort 2, the bacterial load and community structure of the second trimester AF samples differed significantly from those of negative controls, with Ureaplasma and Lactobacillus identified as the most prevalent genera against negative controls. Conclusions Our study demonstrates that no microorganisms were detected in the AF of healthy pregnancies from the second trimester to the delivery. The presence of Ureaplasma and Lactobacillus in the second trimester AF may be associated with preterm birth and intra-amniotic infection.