Marilen Benner

CHAPTER 6 150 MICROBIOTA IN THE UTERUS - REAL OR ARTIFACT? Until now, available 16S data on uterine microbiota do not allow conclusions on a “core uterine microbiome” due to a number of limitations. When investigating the questioned low-biomass microbiome, studies need to be designed with a special focus on possible contamination. In the following paragraph, we outline the pitfalls of the presented studies. Contamination acquired during sample acquisition and processing It is currently highly debated whether microbiota found to be involved in reproduction, such as in the placenta, are in fact merely the result of contamination and an artifact of the study design (60). Contamination always has to be a concern when studying 16S data. Especially when investigating a suspected low-biomass microbiome, as found within the uterus, the impact of various (contaminating) handling steps makes it hard to detect low-abundance microbes originating only from the sampling site (61-63). To overcome the hurdles associated with the need for highly sensitive detection, the 16S studies on endometrial colonization employed different controls (Table I). To examine the possible contribution of contamination when studying the placenta, Lauder and colleagues examined the results from “air swabs” waved in the laboratory space, as well as unused sterile swabs, in comparison to placental tissue samples (64). They could not distinguish between contamination controls and placental samples. Compositions of microbiota in negative controls are known to be associated with the DNA extraction kits (65). Misrepresentation of data needs to be minimized by using special DNA isolation kits for low microbial biomass samples and committing to one kit type for all samples (65). Low-biomass microbial communities are sensitive to contamination and misinterpretation Lauder et al. suggest the presence of a placental microbiome below their level of detection as a reason for not being able to distinguish between air swabs and sampled material (64). Detection of a low-biomass microbiome (where for 1g of placental tissue only 20 – 2000 ng of bacterial DNA is estimated to be extracted) is a major issue when assessing endometrial microbiota (66). Chen et al. included an assessment of the bacterial biomass in their approach. Copy numbers, as calculated from qPCR on vaginal typical Lactobacillus species, were related to the relative abundance of the species as found in 16S rRNA sequencing, showing that microbial numbers decrease from vagina (10 10 – 10 20 copies/sample) through the cervix (10 8 – 10 10 copies/sample) to the uterus (10 2 – 10 3 copies/sample) (23). As the authors point out, this still represents higher numbers than those that can potentially be detected in background noise (62) or in their controls of sterile PBS, sterile saline, and ultrapure water. Accordingly, even though the detected biomass is small, the detected species are not an artifact or contamination.