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.