Single-cell characterization of menstrual fluid at homeostasis and in endometriosis

Progress in detecting and understanding endometrial conditions in women of fertile age, such as endometriosis, has been hampered by the invasiveness of the sample collection procedure. Menstrual fluid (MF) can be sampled non-invasively and could provide a unique opportunity to study the physiological state of tissues in the reproductive system. Despite this potential, the use of MF for diagnostics and research has been limited. Here we establish protocols and assess the feasibility of collecting and processing MF in an outpatient setting. We characterize the cellular contents of MF from 15 healthy women using flow cytometry and single-cell RNA-sequencing, and demonstrate the ability to recover millions of live cells from the different cellular fractions of interest (epithelial, stromal, endothelial, perivascular and blood). Through computational integration of MF with endometrial samples we show that MF sampling is a good surrogate for endometrial biopsy. In a proof-of-principle case-control study, we collect MF from a further 7 women with a diagnosis of endometriosis and 11 healthy controls. Through RNA sequencing of 93 MF samples from these women we highlight important differences between ex vivo and cultured cells, identify impaired decidualisation, low apoptosis, high proliferation, and both higher and lower inflammatory activity in different subsets of immune cells as distinguishing features of endometriosis patients. Finally, we identify potential novel pan-cell-type biomarkers for this neglected condition. ### Competing Interest Statement DA, PCS and MB conducted this work while employed at EPFL in Switzerland. DA is currently employed by and holds Alithea Genomics company stock. MB is currentl employed by Lonza AG. PCS is currently employed by and holds F. Hoffmann-La Roche Ltd. company stock. The cups (LadyCup) for the endometriosis cohort were donated by Ladyplanet GmbH. We do not hold any link with this provider. ### Funding Statement This study was funded by: - an "Early Detection Gynaecological Cancers Pump Priming award 2017" by CRUK to AG. - HFSP and Diversa Foundation Grant to PCS. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: South West - Frenchay Research Ethics Committee (IRAS project ID 221351) gave ethical approval for the healthy cohort work. Swiss Cantonal Authorities Vaud (CER-VD, project nr. 2016-00770) gave ethical approval for the endometriosis cohort work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript and available online at 10.5281/zenodo.11105267