Apostolov, A. | Search bioRxiv

bioRxiv2025

Aberrant epithelialization: A plausible factor for the development of endometrial polyps

Endometrial polyps (EPs) are localized overgrowths of endometrial glands and stroma, common in reproductive-age and postmenopausal women, and can cause abnormal uterine bleeding and infertility. Here, we investigated the cellular heterogeneity and molecular mechanisms of EPs by integrating bulk and single-cell RNA sequencing (scRNA-seq) of EPs and adjacent endometrial tissues (adENs) from 12 women. Bulk RNA-seq revealed high transcriptional similarity, with few differentially expressed genes including upregulated KMT2B and DLEC1 and downregulated COL9A1 and RAB3C. ScRNA-seq identified eight major cell clusters, such as stromal, epithelial, endothelial, immune, perivascular, macrophage, B, and ciliated cells. Pseudotime analysis showed aberrant stromal-to-epithelial transitions in EPs, marked by MECOM and EYA2 intermediate clusters, indicating incomplete epithelial maturation. These altered differentiation trajectories may disrupt perivascular and endothelial cell development, contributing to abnormal vascular remodeling in EPs, despite minimal overall transcriptomic changes compared with adENs.

molecular biology↗

bioRxiv2025

Comprehensive 16S rRNA gene sequencing and meta-transcriptomic analyses in female reproductive tract microbiota: Two molecular profiles with different messages

In recent years, high-throughput sequencing technologies have revolutionised reproductive microbiome research. Our understanding of the endometrial microbiome is primarily based on DNA-based 16S rRNA gene profiling, but DNA detection does not imply alive microbe presence. 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. Meta-transcriptomic analysis, an RNA-based approach, addresses these shortcomings by capturing functional transcripts that are actively expressed in living microbes. This study aims to characterize the endometrial microbiota through a dual approach, by integrating 16S rRNA gene sequencing and meta-transcriptomic analyses. By simultaneously analysing microbial composition and gene expression within the female reproductive tract samples, we seek to provide a more comprehensive understanding of its microbiota and their functional potential. A total of 49 women, aged 27-42 years, were enrolled in the study. Vaginal swabs, endometrial brushings, and endometrial biopsy samples were collected from each participant during the mid-secretory menstrual cycle phase, 6-9 days post luteinizing hormone surge. Our findings suggest that in low-microbial-biomass environments like 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, it suggests that microbial functions and genome activity are tissue-specific and dependent on the host tissue environment. Moreover, RNA-based analysis provides higher resolution in detecting certain pathogens, even within the endometrium. In conclusion, our study underscores the need to integrate genomic and transcriptomic data in microbiota studies to better elucidate critical and health-related microbiota-host interactions.

genomics↗