Microbial profiling of endometrioma and eutopic endometrium using 16S rRNA sequencing

The involvement of microbiota in the pathogenesis of endometriosis has been increasingly suggested; however, existing evidence remains inconsistent, largely owing to the challenges of analyzing extremely low-biomass samples. To clarify whether distinct microbial signatures exist among the non-endometriosis endometrium (nEM), eutopic endometrium with endometriosis (eEM), and endometrioma, we performed a comparative microbiome analysis using 16 S ribosomal RNA (rRNA) gene sequencing of the V4–V5 region on an Illumina NextSeq 1000 platform. Of 48 samples collected, 37 (10 nEM, 18 eEM, and 9 endometrioma) yielded data suitable for analysis. No significant differences were observed in alpha or beta diversity. Lactobacillus was the predominant genus in both nEM and eEM, although no genus differed significantly between groups after correction for multiple comparisons. Sequence-level examination of all 146 amplicon sequence variants (ASVs) showed that background-associated sequences accounted for 41% of the dataset. Because no extraction blank was processed, contamination among the remaining sequences could not be excluded. Inter-individual variation exceeded any difference between groups, and no disease-specific microbial signature was identified. These findings indicate that 16S rRNA gene sequencing of low-biomass endometrial tissue is unlikely to yield a usable biomarker for endometriosis on its own.