Uterine microbiota plasticity during the menstrual cycle: differences between healthy controls and patients with recurrent miscarriage or implantation failure

Objective: In contrast to the former notion of a sterile womb, sequencing techniques have proven a bacterial colonization of the uterus and the placenta. The presence of a non-lactobacillus-dominated microbiota of the uterus, especially increased levels of Gardnerella and Streptococcus seem to be associated with lower live birth rates. However, timing of microbiota analysis with regard to possible intra-cycle variations as well as specific alterations in patients with recurrent miscarriage (RM) or recurrent implantation failure (RIF) remain unknown.Study Design: In total, n=20 RM patients, n=20 RIF patients and n=10 healthy controls (no former pregnancy) were included. All patients had a regular 28-day menstrual cycle. Uterine flushing was performed during follicular, ovulatory and luteal phase. Bacterial DNA was isolated and 16S amplicon sequencing analysis of the V3-V4 region was carried out. Diversity measures were compared between samples from the RIF and RM disease groups and the control group separately for each timepoint of the menstrual cycle and overtime. Abundance of ASVs was compared using a negative binomial model.Results: In the control group a significant decrease of species richness and evenness was shown at ovulation which remained at this lower level during the luteal phase (Shannon index), indicating a more uniform distribution of microbiota (p < 0.05). This loss of diversity during the menstrual cycle was not apparent in RIF and RM patients. A higher similarity was seen in taxonomic distribution between RM and RIF patients compared to the control group. Longitudinal dynamics included increases in Firmicutes (controls and RM only) and a concomitant loss of Proteobacteria. Notably, the highest amounts of Bacteroides were detected in RIF patients. Actinobacteria were more frequent in RM and RIF as compared to controls.Conclusions: To our knowledge, this is the first study to show longitudinal intra-cycle-dependent changes in the endometrial microbiota of healthy controls. While in controls, relative abundance of Lactobacillaceae increases throughout the menstrual cycle, this was not present in RIF and RM patients. This increase in diversity in both patients groups could lead to a micro-environment that is more prone to pregnancy failures. Keywords: recurrent miscarriage, recurrent implantation failure, microbiota, uterine flushing