# Comprehensive 16S rRNA gene sequencing and meta-transcriptomic analyses of the female reproductive tract microbiota: two molecular profiles with different messages

## TL;DR

This study compares DNA and RNA sequencing methods to analyze microbes in the female reproductive tract, finding that RNA-based methods reveal more accurate and detailed microbial activity.

## Contribution

The study demonstrates that RNA-based meta-transcriptomic analysis provides higher resolution and functional insights into the endometrial microbiota compared to DNA-based 16S rRNA sequencing.

## Key findings

- RNA-based analysis detects higher microbial resolution and active pathogen presence in low-biomass environments like the endometrium.
- Lactobacillus DNA in the endometrium may originate from the lower reproductive tract, not active colonization.
- DNA-based methods may underestimate microbial diversity and functionality in low-microbial-biomass tissues.

## Abstract

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

DNA vs RNA-based microbial analysis techniques demonstrated significant microbial compositional differences and lack of transcriptionally active lactobacilli in the endometrium.

Our understanding of the endometrial microbiome is primarily based on DNA-based 16S rRNA gene profiling, but DNA detection does not imply the presence of living microbes. While this method is cost-effective and widely used, it has notable limitations, including the underestimation of microbial diversity, abundance, and functionality, as well as limited species-level resolution. While the microbiome reflects DNA-based characterization, the microbiota more precisely captures metabolically active communities. In this context, meta-transcriptomic analysis, an RNA-based approach, addresses these shortcomings by capturing functional transcripts that are actively expressed in living microbes.

This cross-sectional study consisted of 49 reproductive-aged women (27–42 years old) who were receiving ART. By simultaneously analysing the microbial composition and gene expression within female reproductive tract samples, we sought to provide a more comprehensive understanding of the microbiota and functional potential of these samples.

Vaginal swabs, endometrial brushing, and endometrial biopsy samples were collected from 49 participants during the mid-secretory phase of their menstrual cycle, 6–9 days after the luteinizing hormone surge for parallel 16S rRNA gene sequencing and meta-transcriptome analyses. For DNA-based analysis, the 16S rRNA gene V4 region was sequenced. For RNA-based analysis, total RNA was extracted followed by ribosomal RNA depletion. Strand-specific total RNA sequencing libraries were prepared and sequenced. Taxonomy was assigned by using Kraken2 (v2.2.1), and Bracken (v2.7).

Our findings suggest that in low-microbial-biomass environments such as the endometrium, the correlation between 16S rRNA gene sequencing and meta-transcriptomics is relatively weak. This highlights the limitations of microbial analysis of low-microbial-biomass samples. Alternatively, microbial functions and genome activity may be tissue-specific and dependent on the host tissue environment. Moreover, RNA-based analysis provides higher resolution in detecting certain pathogens, even within the endometrium.

The data presented in the study are deposited in the NCBI SRA Database, accession number PRJNA1247240.

High levels of host RNA and the low abundance of microbial reads in the endometrium complicate microbial identification. Our findings indicate that RNA-seq enables precise profiling of the vaginal microbiome and, in cases of dysbiosis, reveals higher pathogen activity than DNA-based approaches. However, the limited sample size restricts the generalization of these conclusions.

Contrary to the general belief of the dominance of Lactobacillus 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 indicate a need to re-consider/re-analyse the endometrial microbiome in health and disease.

This work was supported by the projects Endo-Map PID2021-127280OB-I00, ROSY CNS2022-135999, and ENDORE SAF2017-87526-R funded by MICIU/AEI/10.13039/501100011033 and by FEDER, EU. This work was also supported by the Estonian Research Council grants (PSG1082 and PRG1076), Swedish Research Council grant no. 2024-02530 and Novo Nordisk Foundation grant no. NNF24OC0092384. Additionally, A.S.L. and I.P.P. acknowledge Becas Fundación Ramón Areces para Estudios Postdoctorales—Convocatorias XXXV and XXXVI, para Ampliación de Estudios en el Extranjero en Ciencias de la Vida y de la Materia. A.S. is supported by Horizon Europe (NESTOR, grant no. 101120075) and the Ministry of Education and Research Centres of Excellence grant TK214 name of CoE. All the authors declare no conflict of interest.