Single-nucleus analysis of menstrual fluid highlights gene expression differences in epithelial cells of endometriosis donors

Background Endometriosis is a common complex gynecological condition that is difficult to efficiently diagnose and remains poorly understood. Menstrual fluid provides a non-invasive source of disease-relevant tissue and has been identified as promising for endometriosis diagnostics. Peripheral blood-based assays have yielded few clinically relevant targets, thus we hypothesize that cells specific to the uterus will exhibit more disease-related differences and may lead to insights into disease development and potential diagnostic markers.

We profiled 10 menstrual fluid samples, from 5 donors with endometriosis and 5 donors without, using both single-nucleus RNA sequencing and bulk RNA sequencing. We tested for differential abundance of cell types, differential gene expression within cell types, and differential cell communication between cell types by disease status. Finally, we compared the results of both single-nucleus and bulk RNA sequencing analyses to identify potential diagnostic targets.

We identified endometrial and immune cell types present in menstrual fluid, with large inter-individual heterogeneity in cell type representation. While most cells were immune, the cell types with the greatest number of differentially expressed genes were endometrial epithelial cells followed by stromal cells. Epithelial cells in particular recapitulated some gene expression differences previously described in the context of endometriosis, although most identified differences were novel. Cell-cell communication in endometriosis was characterized by a loss of interactions between epithelial and stromal cells related to developmental and growth genes WNT2B, IGF2 and TGFB2, but a gain of cell-cell communications between dendritic cells and other cell types, especially within the BMP signaling pathway. Bulk RNA sequencing revealed that some of the differentially expressed genes found in epithelial cells could be replicated in the whole tissue, highlighting the following genes as biomarker candidates: TIMP2, AKR1C2, DMBT1, FERMT1, and KCNK5.

We identified a set of promising genes that may contribute to endometriosis pathophysiology understanding and have potential as diagnosis biomarkers in whole menstrual blood. Further assessment of their stability over time within each patient and in a larger cohort will confirm whether this represents a viable non-invasive strategy to reduce diagnostic delays. Similar content being viewed by others Abbreviations - BMI: - Body Mass Index - DC: - Dendritic Cell - DEG: - Differentially Expressed Gene - DIE: - Deep Infiltrating Endometriosis - FDR: - False Discovery Rate - logFC: - log Fold Change - MRI: - Magnetic Resonance Imaging - OMA: - Ovarian endometriosis/endometrioMA - pDC: - plasmacytoid Dendritic Cell - RIN: - RNA Integrity Number - RNA-seq: - RNA sequencing - snRNA-seq: - single nucleus RNA sequencing - UMAP: - Uniform Manifold Approximation and Projection

We thank all the volunteers who participated in this research by donating menstrual fluid. We thank the scBiomarkers UTechS platform, C2RT, Institut Pasteur, France, for support in conducting this study. We thank M. Monot and the Biomics platform, C2RT, Institut Pasteur, France, supported by France Génomique (ANR-10-INBS-09) and IBISA. We thank the INVOLvE investigation center team from the Institut Pasteur, France (https://research.pasteur.fr/en/team/involve/), for their help with the sample collection. Funding This project was supported by Institut Pasteur (G5 package), Centre National de la Recherche Scientifique (CNRS UMR 3525), Institut National de la Santé et de la Recherche Médicale (INSERM UA12), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 851360 to C.B.), the Inception program (Investissement d’Avenir grant ANR-16-CONV-0005 to C.B.), the Fondation Schlumberger pour l’Education et la Recherche and the Fondation pour la Recherche Médicale (grant FSER202401018852), the Fondation pour la Recherche sur l’Endométriose (grant number FRE202112014887) and the EndoFrance patient organisation. E.L. is supported by a doctoral fellowship from Université Paris Cité (ED BioSPC 562). L.D. is supported by European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 101078556). Author information Authors and Affiliations Corresponding authors Ethics declarations Competing interests The authors declare no competing interests. Ethics approval and consent to participate The study is part of clinical trial C18-19 EVOMENS sponsored by Inserm. It was approved by the French National Institute for Health and Medical Research (INSERM, IDRCB 2019-A01089-48) and the Ethics Committee for the protection of individuals Est V (ID: 20/13 / SI 19.11.26.65936) on March 20, 2020. This study was also registered on the Clinical Trial website under the number NCT04085835. All study participants gave their informed, written consent to participation, in line with French ethical guidelines. Consent for publication Not applicable Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information 12916_2026_5020_MOESM1_ESM.xlsx (download XLSX ) Supplementary material 1: Additional File 1: Tables S1-S7. Table S1 – QC attributes of the snRNA-seq. Table S2 – QC attributes of the bulk RNA-seq. Table S3 – DEGs identified by NEBULA in bulk RNA-seq. Table S4 – KEGG pathways identified by pathfindR. Table S5 – Interactions identified by cellphoneDB. Table S6 – DEGs identified by DESeq2 in bulk RNA-seq. Table S7 - DEGs identified by both NEBULA and DESeq2 in bulk RNA-seq Rights and permissions Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. About this article Cite this article Leap, K., Lepelletier, A., Liorzou, E. et al. Single-nucleus analysis of menstrual fluid highlights gene expression differences in epithelial cells of endometriosis donors. BMC Med (2026). https://doi.org/10.1186/s12916-026-05020-6 Received: Accepted: Published: DOI: https://doi.org/10.1186/s12916-026-05020-6