Introduction/Background*Endometrial cancer has a dominant place among gynaecological cancers and is the fourth most common malignancy in women. Accumulating reports have associated gynaecological precancer and cancer with dysbiotic microenvironments. Our aim was to identify a microbial signature in endometrial cancer and explore its role in disease pathogenesis.

Methodology Eligibility criteria included patients undergoing total abdominal/laparoscopic hysterectomy for endometrial cancer or benign indications. Microbiome swabs were collected along the female genital tract (FGT) (vagina, external cervical os, endometrium, fallopian tubes and ovaries) and rectum. The V1-V2 hypervariable regions of 16S rRNA genes were sequenced (Illumina MiSeq platform), data were analysed with Mothur software package and OTU taxonomies were determined. Benign and malignant endometrial organoids were cultured and treated with increasing concentrations (10%, 20%, 30%) of L. crispatus- conditioned media. Proliferation was assessed by the CellTiter-GLo^® 3D cell viability assay and cytokine/chemokine secretion (IL-1β, IL-1ra, IL-2, IL-6, IL-8, IL-10, G- CSF, GM- CSF, IFN-γ, TNF-α, CCL4/MIP1beta, CCL5/RANTES) by the Magnetic Multiplex Cytokine Array (R&D systems).

Result(s)*Sixty-one women were recruited; 37 had endometrial cancer and 24 were benign controls. We confirmed the presence of a genuine, low– abundance microbiome above background contamination in the endometrium, fallopian tubes and ovaries in a subset of benign and endometrial cancer patients, which was one- four orders of magnitude lower than the heavily colonised vagina, cervix and rectum. In 75% (12/16) of benign patients, we found that the most abundant species of the lower genital tract could also be recovered from the whole length of the upper genital tract, while the microbial continuum was less cohesive in endometrial malignancy. We also demonstrated that Lactobacillus depletion and high microbial diversity along the genital tract are characteristic in endometrial cancer patients with concurrent enrichment of Porphyromonas, Prevotella, Peptoniphilus and Anaerococcus. Different histotypes and grades of endometrial cancer were not marked by microbial differences. L.crispatus, a FGT commensal that is depleted in endometrial cancer, was shown to reduce viability of endometrial cancer organoids at high concentrations and impact on cytokine secretion by benign and malignant endometrial organoids.

Conclusion*Endometrial cancer displays a distinct microbial signature. L.crispatus may exert an anti- proliferative effect in endometrial cancer and interfere with inflammatory pathways.