Marilen Benner

CHAPTER 6 152 While we believe that it is unlikely that the distribution profiles from endometrial swabs are merely a representation of vaginal colonization, the possibility of carrying over traces from vaginal microbiota onto the sample cannot be neglected. Likewise, insertion of a sampling device or speculum could carry over bacteria from the lower reproductive tract into the uterus. It is also unclear how surgical access of the uterus coincided with disruption of the cervical barrier allowing passage of bacteria that would normally not reside in the uterus as a result of the pre- operative preparation. Live bacteria? It cannot be ignored that, without further measures, sequencing of 16S rRNA does not differentiate between living bacteria or (dead) bacterial fragments. Chen et al. studied cultures of freshly collected peritoneal fluid samples from the Pouch of Douglas (the lowest part of the abdominal cavity, between uterus and rectum), a site shown to be similar in microbial diversity, and even lower in terms of microbial abundance, as compared to the endometrium (23). Eight different isolates were cultured belonging to seven genera, such as Lactobacillus, Staphylococcus, and Actinomyces, in five out of 15 samples, whereas various negative controls did not lead to bacterial growth when cultured. These results indicate that the detected 16S reads do not solely reflect dead bacterial fragments. Conclusions on the validity of the current evidence Based on the current study designs, no information on bacterial detection and distributions of a “core uterine microbiome” can be extracted. Contamination during sampling by surrounding microbiota and reagent kits have not been addressed systematically on a large cohort of healthy, fertile women yet, in order to allow any conclusions on the continuous baseline colonization of the uterus independent of pathologies. Future studies also need to address the question of whether detected 16S results from bacterial fragments rather than live organisms by using a more robust approach than culture in vitro since (even though proving that culture is not impossible) often no growth can be established in cases of these ultra-low biomass samples. The variability in culturing success probably also results from variation between women in species composition and biomass of live bacteria. Aside from this, even 16S rRNA data, reflecting a proportion of the overall community that is dead or fragmented, represent ligands for the host cells to recognize and act upon. Thereby, these inactive bacterial fragments can still contribute to a physiologic interaction with host cells. We conclude that well-setup large cohort studies are needed to define a healthy uterine microbiome. We want to stress that even though no uterus-typical bacterial profile can be established yet, existing data are not to be underrated as evidence towards a natural microbial presence within the uterus. A number of the presented studies do employ negative controls and/or additional vaginal swabs. Since species abundance of these controls was not identical