A primary study: high-throughput sequencing analysis of amniotic fluid microbiota in 50 high-risk pregnant women during the second trimester

Objective Whether bacteria exist in the pregnancy uterus without pathological infection has long been a controversial topic. Through this study, we aim to determine whether characteristic amniotic fluid microbiota exists in the uterus of high-risk pregnant women during the second trimester. Methods This study recruited high-risk pregnant women in the second trimester from September 1, 2024, to August 30, 2025, and recorded their age and gestational age. Amniotic fluid samples were obtained through amniocentesis under strictly sterile conditions, and 16S rRNA high-throughput sequencing was performed on the samples. The inclusion criteria mainly consisted of: advanced maternal age, non-invasive prenatal test results indicating chromosomal abnormalities, abnormal fetal ultrasound findings, history of adverse pregnancy outcomes, and high-risk Down syndrome screening results. Results This study included a total of 50 high-risk pregnant women who underwent amniocentesis. The results showed that bacteria were present in all amniotic fluid samples, primarily composed of Actinobacteriota and Proteobacteria . There was no difference in amniotic fluid microbiota diversity between pregnant women under 35 years old and those 35 years or older; however, the abundances of Cutibacterium and Leifsonia differed between the two groups. A slight difference in microbiota diversity was observed between women with gestational ages below 20 weeks and those at 20 weeks or above, but no significant difference was found in microbial composition between the two groups. Conclusion There was bacterial DNA in amniotic fluid of high-risk pregnant women in the second trimester, with Actinobacteriota and Proteobacteria being the predominant phyla, showing limited correlation with maternal age or gestational age. This study provided evidence for the presence of microorganisms in amniotic fluid during pregnancy and might offer some preliminary data for future research related to eugenics and reproductive health.