Fetal environment and fetal intestine are sterile during the third trimester of pregnancy

Recent next generation sequencing studies on host-associated microbiomes generated debatable conclusions regarding the central dogma of fetal gut sterility. These observations challenge the concepts that microbial colonization of the gut begins during and after birth as well as the concept of antigen-independent prenatal maturation of mucosal-associated lymphoid tissue in ruminants and humans. The placental barrier varies markedly among mammalian species with mice and humans having haemochorial placentas (fetal tissue in direct contact with maternal blood) versus epitheliochorial placentation (maternal and fetal blood separated by six tissue layers) in ruminants. Therefore, this study re-examined the question of fetal gut sterility using the fetal lamb as a model ruminant species with the most complete placental barrier. Use of PCR and quantitative real-time PCR with three different pairs of universal bacterial primers (27 F and 1492R, HDA1 and HDA2, U2F and U2R) to amplify 16S rRNA gene did not generate detectable PCR products from samples collected from the fetal environment (placenta, amniotic fluid) and fetal intestine during the third trimester of pregnancy. Procedures to further enrich microbial DNA from total extracted DNA also resulted in no detectable genomic DNA. Moreover, use of 16S amplicon sequencing confirmed the absence of bacteria in the fetal environment during the third trimester of pregnancy. A 'No Template' control containing only PCR reagents generated sequences that could be clustered into OTUs at 97% similarity and assigned to bacterial genera, including Staphylococcus, Lactobacillus and Escherichia-Shigella. Use of multiple molecular-based approaches to profile fetal environment-associated microbiota supports the conclusion that the fetal environment and fetal intestine remain sterile during the third trimester of pregnancy. The use of appropriate controls, both positive and no template, revealed inherent contamination in reagents and that variations in the data analysis pipeline can produce artificial microbial profiles from host tissues containing low microbial biomass. Finally, these findings confirm that extensive development of gut-associated lymphoid tissue in the ruminant fetal intestine, characterized by active B cell proliferation and immunoglobulin V gene somatic mutation, is not associated with exposure to bacterial DNA and antigens.