Marilen Benner

MICROBIOTA AND ENDOMETRIAL HEALTH 143 6 vagina. However, the need of these studies to select specific probes targeting a certain species is intrinsically biased. Rather than evaluating the presence and/or abundance of a certain species, amplicon sequencing of the hypervariable 16S region of the ribosomal RNA makes it possible to identify species present in a sample (50). The metagenomic molecular approach of high-throughput 16S rRNA sequencing allows for a more complete view, reflecting the diversity and relative abundance of microbiota. However, also this technique comes with its limitations as outlined below. While shotgun whole genome sequencing (WGS) offers advantages to amplicon 16S sequencing (51, 52), as yet this approach has not been employed to study endometrial microbiota. Evidence of endometrial microbiota by 16S sequencing In the Human Microbiome Project (HMP), efforts are made to understand more about the natural colonization of various body sites, its physiological importance and implications for disease (53). In the context of the HMP, data on gut and vaginal colonization increase, but the upper reproductive tract has not been characterized extensively yet (54). However, the importance of microbiota for human reproduction is increasingly acknowledged (55). Recently, several studies targeted a putative endometrial microbiome through 16S rRNA sequencing, and each of the studies documented the presence of uterine microbiota (15-23). An overview of these recent studies assessing human uterine microbial composition based on 16S rRNA sequencing is given in Table I. Tips used for embryo transfer, swabs, biopsies or aspirates were employed to obtain endometrial samples. The studies used next-generation sequencing and assigned the sequence reads to different operational taxonomic units (OTUs). Phylotypes or, depending on the fit against the database used for taxonomic annotation, genotype or species could be assigned to the 16S rRNA gene amplicon sequence. Depending on the study, varying 16S rRNA regions were targeted simultaneously, since depending on which hypervariable V1 to V9 region is targeted, more differentiation at genus or species level is possible (56). Alpha (within a subject) and beta (between different subjects) diversity were assessed using different diversity indices (Hugerth and Andersson,2017). One of the classifications often used to express alpha diversity and species richness (i.e. how different species are sequenced in total) is the Shannon Diversity Index. Another often-used index is Chao1, using rare classes of OTUs (e.g. Franasiak et al., 2016; Moreno et al. , 2016). Beta diversity is often expressed through the Bray-Curtis dissimilarity (e.g. Walther-Antonio, et al .,2016). Taxonomic distribution profiles of identified genera or species are markedly different between the studies.