Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies

Abstract

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid, placenta) are stably colonized by microbes in a healthy pregnancy remains the subject of debate. Here, we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbiology, bioinformatics, immunology, clinical microbiology, and gnotobiology, and assess possible mechanisms by which the fetus might interact with microbes. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples, DNA extraction, and DNA sequencing. Further, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology, and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and also illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a "fetal microbiome" serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls, also incorporating biological, ecological, and mechanistic concepts.

The Blood Microbiome and Health: Current Evidence, Controversies, and Challenges

“…Several studies have concluded that circulating bacteria, especially those found in healthy volunteers, are either traces of microbial DNA from external sources or organic remnants from non-living bacteria [ 79 , 80 ]. For similar reasons, the findings of the microbiome in other low-biomass environments such as the womb during pregnancy, brain and tumors have also been brought into question [ 81 , 82 ]. Indeed, NGS sequencing is very sensitive to capturing minute amounts of microbial DNA contaminants from extraction kits, storage containers, sequencing reagents and even the sequencer itself.…”

“…Thirdly, there is an urgent need to establish the best practices and a standardized workflow for sample collection and processing, negative controls, and post-sequencing decontamination pipeline to enable data-sharing and inter-study comparison [ 81 ]. The gut microbiome is highly dynamic and labile to host and environmental factors such as host genetics, diet, medication, lifestyle and geographic regions [ 24 , 96 ].…”

Comparing Vaginal and Endometrial Microbiota Using Culturomics: Proof of Concept

“…It has to be acknowledged that analysis of the endometrial microbiome is hampered by multiple technical limitations and the low accessibility to healthy controls. Sequencing bias, originating from DNA/RNA contamination in laboratory reagents and extraction kits, is a major drawback in sequence-based studies, especially when studying low-biomass microbiota like that of the endometrium [ 16 , 17 , 18 ]. Sequencing of microbiota often gives only genus-level identifications with no information on which species are present.…”

Human microbiome research: Growing pains and future promises

“…Additionally, contaminations and annotation errors occurring at each step along the microbiome processing and analysis cascade may introduce artifacts and biases that are challenging to tackle and complicate the distinction between true and spurious biological signals. Understanding the scope, limitations, and confounders associated with each of these steps is essential for separating true signal from confounding factors, particularly in tissues with low or no microbial abundance [ 6 ]. Better harmonization of these methodologies, coupled with inclusion of multiple technical and biological controls, could enable more accurate interpretation of studies that can be generalized and reproduced across populations, geographies, genders, and ethnicities.…”

“…Avoiding some of the field’s early technical missteps [ 6 ] while allowing innovative research to progress necessitates an appreciation of the extraordinary complexity and modularity of host–microbiome interactions, coupled with a much-needed modification of expectations. It is increasingly realized that diagnostic and therapeutic microbiome utilization in human disease will require a continued and, at times, painstaking and exhaustive exploration of molecular mechanisms and regulation, from overarching descriptive community associations to the microscale function of discrete bioactive therapeutic targets.…”