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 newborns is a crucial step in the development of the 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 population 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 22 women who experienced infection-related adverse pregnancy outcomes (including preterm birth, histological chorioamnionitis, and stillbirth), with only their second trimester AF samples (n = 24) examined. Additionally, multiple procedural negative controls and technical positive controls were applied to 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 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 infection-related adverse pregnancy outcomes. Video Abstract.

Keywords: Amniotic fluid; Bacteria; Infection; Pregnancy; Preterm birth.

Fig. 1

The comparison of 16S rRNA gene copy number among various sample types. a Standard curves for tenfold diluting series (10⁷ copies to 10 copies) of Escherichia coli 16S ribosomal DNA. b Comparison of 16S rRNA gene copy numbers among various sample types. Statistical significance was assessed by the Mann–Whitney U test.  Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Del AF, delivery amniotic fluid in cohort 1; Sec AF in cohort 2, second trimester amniotic fluid in cohort 2; SC, sampling controls; EC, extraction controls; AC, amplification controls; VS, vaginal swab

Fig. 2

The analysis of 16S rRNA gene sequencing. a The comparison of 16S rRNA read counts between sample types. b The comparison of 16S rRNA OTU numbers between sample types. c The comparison of alpha diversity (Chao1, Shannon, and Simpson indices) between sample types. d Principal coordinates analysis based on unweighted UniFrac distance is shown along the first two principal coordinate (PC) axes, and percentages are the percent variation explained by each PC axis. e The comparison of between-group UniFrac distance of second trimester AF and delivery AF in cohort 1. Statistical significance was assessed by the Mann–Whitney U test. Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Del AF, delivery amniotic fluid in cohort 1; Sec AF in cohort 2, second trimester amniotic fluid in cohort 2; SC, sampling controls; EC, extraction controls; AC, amplification controls; VS, vaginal swab

Fig. 3

The distribution of specific bacterial genera in second trimester AF and delivery AF in cohort 1. a Bacterial genera predominant and high in second trimester AF samples are depicted as “second trimester AF-specific genera”. b Bacterial genera predominant and high in delivery AF samples are depicted as “delivery AF-specific genera”. Dot-plot showing the distribution and abundance of each specific genera in both second trimester and delivery AF samples. Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Del AF in cohort 1, delivery amniotic fluid in cohort 1

Fig. 4

The analysis of 16S rRNA gene sequencing. a Principal coordinates analysis based on unweighted UniFrac distance is shown along the first two principal coordinates (PC) axes, and percentages are the percent variation explained by each PC axis. b the comparison of between-group UniFrac distance of second trimester AF in cohort 1 and second trimester AF in cohort 2. Statistical significance was assessed by the Mann–Whitney U test. Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Sec AF in cohort 2, second trimester amniotic fluid in cohort 2; SC, sampling controls; EC, extraction controls; AC, amplification controls; VS, vaginal swab