Marilen Benner

MICROBIOTA AND ENDOMETRIAL HEALTH 151 6 Another source of misinterpretation that is gaining attention is the approach in which species distribution is related to the obtained sequencing library. Disease-associated changes in Prevotella and Bacteroides abundances found in Crohn’s disease were found to be rooted in proportional profiling and had to be ascribed to a general decrease in microbiota richness in Crohn’s disease (67). If abundance is interpreted as a relative proportion, a change in the presence of a certain species presents as increase or decrease of a possibly unrelated species. Low-biomass microbiota, as detected in the uterus, are highly sensitive to this misrepresentation of data. A quantitative assessment of genus abundance proportional to cell counts and microbial load of the sample would allow a more precise representation of data (67). Vaginal microbiota as a source of contamination Due to its high biomass relative to the uterinemicrobiome and the high abundance of Lactobacillus in the vagina , misrepresentation of species distribution cannot be excluded. Especially when using a transcervical approach, contamination by vaginal bacteria needs to be controlled for. Moreno and colleagues compared endometrial and vaginal samples, and only two of the 26 sample pairs (two pairs per woman) of EF and vaginal aspirate showed the same microbial profile (19). While in some samples only minor differences were observed, six paired samples exhibited totally different profiles when women belonged to the group of non- Lactobacilli dominated subjects. In nine of the 13 women, Lactobacillus consistently reflected the dominant genus with more than 90% colonization in the pre-receptive and receptive phase of endometrium and vagina. Notably, some common species, such as A. vaginae, G. vaginalis, Prevotella, and Sneathia, of both endometrium and vagina were identified. Also, the study that circumvented this risk, by sampling from uteri obtained during hysterectomy, showed a correlation between the vaginal and uterine microbiomes (20). However, it is unclear how the uterine microenvironment is contaminated by transfer of cervicovaginal bacteria into the excised uterus. As manipulation of the uterus during surgery might enable passage of bacteria from the lower reproductive tract, it cannot be excluded that the correlation of vaginal and uterine microbiota is just an artifact. This correlation was also seen by Verstraelen et al. (2016), who detected endometrial phylotypes known to be associated with the vagina. L. crispatus was present in 12 of the 19 women, and most abundant in five of 19 individuals (range from 17.1% to 79.1% of the total number of sequence reads). Also, species associated with vaginal microbiota imbalance, such as Prevotella ( P. amnii, Prevotella sp ., P. timonensis, P. disiens ), were abundantly expressed in two subjects (18.4% and 55.4% of total sequence reads) (47, 68, 69). Likewise, G. vaginalis was present in six women, but at an abundance of less than 1%. While trying to avoid vaginal contamination through the use of a sheath-protected sampling surface and trying to prevent cervicovaginal contact of the sampling tool, it cannot be excluded that insertion or extraction of the device facilitates bacterial carry-over. Verstraelen et al. pointed out that, even though a certain degree of species overlap exists, it is not to be assumed that endometrial samples reflect vaginal colonization, since the diversity found in endometrial samples is higher than in the vagina (16).