O-268 Re-considering the endometrial microbiome: a comparison of DNA- and RNA-based sequencing techniques within the same cohort
Authors: Sola-Leyva A , Canha-Gouveia A , Pérez-Prieto I , Salas-Espejo E , Molina N  M , Vargas E , Terrón-Camero L , Andrés-León E , Fontes-Jiménez J , Castilla J  A , Vela-Moreno S , Modhukur V , Saare M , Salumets A , Altmäe S 
Published: July 2024

Abstract

Study question
Does the analysis of the endometrial microbiome provide the same information when using DNA or RNA sequencing-based techniques?

Summary answer
DNA vs. RNA-based microbiome analysis techniques demonstrate significant microbial compositional differences, meaning that the previous endometrial 16S rRNA gene-based microbiome analysis results might be overestimated.

What is known already
In recent years, high-throughput sequencing technologies have revolutionised the field of reproductive microbiome research. Our understanding of the composition of endometrial microbiome predominantly relies on DNA-based 16S rRNA gene profiling method. Regardless of its effectiveness and low cost, its underestimation of microbial diversity, abundance, or functionality and lack of detection precision on species level have been highlighted. The meta-transcriptome analysis, RNA-based method, overcomes these limitations and identifies functional genes that are actively expressed by the alive microbes. This study aims to elucidate for the first time the endometrial microbiome using a dual approach of 16S rRNA gene-method and meta-transcriptomic analyses.

Study design, size, duration
In total forty-seven women with infertility at ages 27-42 years old were enrolled in the present study. Paired endometrial samples, endometrial brushing and Pipelle biopsy were collected from each participant at the mid-secretory menstrual phase (LH + 7/9).

Participants/materials, setting, methods
Endometrial samples were collected using Tao Brush for 16S rRNA analysis (DNA analysis) and Pipelle endometrial curette for meta-transcriptomic analysis (total RNA analysis). QIAamp UCP Pathogen Mini Kit was used for DNA extraction and 16S rRNA gene V3-V4 regions were sequenced on MiSeq. RNA was extracted using miRNeasy Micro kit, with rRNA removal by RiboZero kit and Libraries were generated using Stranded Total RNA Prep. Taxonomy was assigned by using Kraken2 (v2.2.1), Bracken (v2.7).

Main results and the role of chance
To the best of our knowledge, this is the first study to compare endometrial microbial identification using both metagenomics (16S rRNA sequencing) and meta-transcriptomics (meta-RNA sequencing) within the same cohort. Analysis of the composition of the microorganisms by bacterial 16S rRNA gene sequencing revealed that the most abundant bacterial genus in the endometrium was Lactobacillus. In the meta-transcriptome analysis, no microorganisms belonging to the Lactobacillus genus were detected in high abundance. This indicates that the relative abundance of this genus at the DNA level does not necessarily imply microbial activity or the presence of viable Lactobacillus in the endometrium. The meta-transcriptomics analysis detected microbial taxa such as Staphylococcus, Bacillus, Streptococcus, and Burkholderia as the most dominant. The results suggest that DNA-based detection of microorganisms in the endometrium may be overestimated due to the presence of naked DNA sequences or microorganisms from the vagina and cervix. Furthermore, RNA-based analysis demonstrates a population of active microorganisms different from the image shown by DNA analysis. Altogether, our study results indicate that the uterine microenvironment, as previously identified through DNA sequencing, may partly reflect the vaginocervical microenvironment, with the uterine tissue being a low biomass site not dominated by lactobacilli.

Limitations, reasons for caution
The endometrial samples were paired; however, DNA was analysed in the endometrial brushing sample while RNA in the tissue biopsy, which might result in some differences in microbial composition.

Wider implications of the findings
Contrary to the general belief of the Lactobacillus dominance in the human endometrium, our study suggests that the endometrial microenvironment may be harbouring DNA fragments and/or cells of lactobacilli originating from the lower reproductive tract. Our study results call out to re-consider/re-analyse the endometrial microbiome in health and disease.

Trial registration number
not applicable