Assessing endometrial microbiota in endometriosis: culturomics and sequencing analysis of receptive-phase tissue

• First study to explore live microbes in endometrial samples in women with endometriosis • Combined culturomics and 16S sequencing to study low-biomass endometrial microbiota • Viable bacteria were recovered from receptive-phase endometrial biopsies • Direct plating enables quantitative analysis, while pre-incubation expands species recovery • Culturomics and sequencing provide complementary but method-dependent microbial profiles • Women present individual microbial profiles with no consistent endometriosis specific endometrial microbiota signature Endometriosis has been associated with alterations in the reproductive tract microbiota, yet studies focusing on the endometrial microbiome remain inconsistent, partly due to the low-biomass nature of this niche and methodological heterogeneity. In this prospective pilot study, we investigated whether the receptive-phase endometrial microbiota differs between women with and without endometriosis using a combined culturomics and 16S rRNA gene sequencing approach. Endometrial samples were collected from 20 women undergoing fertility assessment, including 10 women with a previous diagnosis of endometriosis and 10 controls with male factor infertility. Biopsies were used for culturomics and Tao brush samples for sequencing, both obtained during the mid-secretory phase. Viable bacterial growth was recovered from 19 of 20 biopsies, yielding approximately 2,500 colonies corresponding to 40 non-redundant species. Direct plating and aerobic pre-incubation shared around 45% of detected species, with pre-incubation enhancing species recovery but limiting quantitative comparisons. Bacterial load estimated from direct cultures showed wide inter-individual variability and no significant differences between groups, consistent with 16S rRNA gene sequencing results, which also revealed no clear differences in microbial diversity or overall community composition within this cohort. Only a limited overlap was observed between taxa detected by culture and sequencing, highlighting the complementarity and methodological biases of each approach. Overall, this study demonstrates the feasibility of recovering viable bacteria from receptive-phase endometrial samples and supports the combined use of culturomics and sequencing as complementary tools for studying microbiota in low-biomass uterine environments. These findings should, however, be interpreted considering the exploratory design and limited sample size.