Association and potential mechanisms between maternal amniotic fluid microbiota heterogeneity and fetal complex congenital heart disease

Numerous studies have found that dysbiosis of the microbiota is associated with cardiovascular diseases in humans and shows certain links to congenital heart disease (CHD) in children. However, direct evidence regarding whether maternal gut microbiota is related to fetal heart development remains lacking. This study aims to collect amniotic fluid samples during cesarean sections from pregnant women, compare the differences in microbiota between fetuses with complex CHD and normal fetuses, and explore the role of maternal gut microbiota as an environmental pathogenic factor in complex CHD. Amniotic fluid samples were collected from patients undergoing cesarean sections at a single center between November 2024 and October 2025. The study included 6 cases in the fetal complex congenital heart disease (CCHD) group and 9 cases in the fetal normal (NOR) group. Microbial 16 S rRNA sequencing was performed on these samples. A total of 1,908,431 paired-end reads were obtained, which were assembled into 1,897,612 tags, corresponding to 4,057 ASVs/OTUs. No significant differences in microbial diversity were observed between the two groups. However, in the complex congenital heart disease (CCHD) group, the abundance of certain microbial taxa associated with diabetes, inflammatory bowel disease, and periodontal disease was significantly elevated. ( p < 0.01). In this exploratory cohort, no statistically significant differences in alpha or beta diversity were detected between the CCHD and NOR groups, whereas several taxa showed higher abundance in the CCHD group. Although this study had a small sample size and low-biomass amniotic fluid samples are susceptible to contamination, these findings provide hypotheses for further large-sample studies with rigorous contamination control.