Characterization of menstrual fluid from healthy donors. (A) Sample preparation for the FACS assays. (B) Total sample volumes in mL. (C) Frequency of the number of donations per donor. (D) Volume in mL of MF across 2-4 donations from the same donor. (E) Million of live cells per mL of flow-through. (F) Percentage of CD45+ cells in flow-through.

Characterisation of menstrual fluid from healthy donors using single-cell RNA-seq (scRNA-seq). (A) Sample preparation for the scRNA-seq assays. (B) Single-cell UMAP of ciliated, glandular and exhausted epithelial cells, mesenchymal and decidualized stroma, smooth muscle/endothelial and immune cells. (C) Marker genes used for the cell-type annotation. (D) Cell-type abundances in each MF sample (c - clumps, f - flow-through). (E) UMAP of the MF samples integrated with endometrial biopsies from Wang et al. (20). (F) Cell-type abundances in MF and biopsies. (G) Gene expression correlation between the MF and biopsies. (H) Differential gene expression between stromal cells in MF and biopsies. Gene names are shown for the top 15 genes with largest absolute log2 fold changes and for genes with -log10 adjusted p-values greater than 20. Colors indicate if genes are members of the pathways indicated. The pathways chosen were significantly over-represented (FDR q-value <= 0,00027) in gene set enrichment analysis.

Comparison of menstrual fluid volume, cell number and cell-type proportions between endometriosis patients and healthy controls. (A) Sample preparation procedure. (B) MF volume in controls - C, and endometriosis stages 2 to 4 (E2-E4). Points are coloured by donor. (C) Cell number, viability, fraction of CD45+ cells and fraction of CD45+CD105+ cells in endometriosis - E, and controls - C.

Demographic and disease characteristics of the endometriosis and healthy controls.

Cell-type composition of MF samples by cell-type enrichment method. (A) Sample preparation procedure. (B) Principal component analysis of all samples using the full transcriptome. Shown in parentheses on the axes is the percentage of variance explained by each of the principal components. (C) Over-representation analysis of the top 30 genes positively associated with PC1 in the cell-type marker gene-sets from PanglaoDB(54). P-values were calculated using Fisher’s exact test and corrected for multiple testing using the Benjamini-Hochberg procedure. (D) Gene expression heatmap of the 10 most significantly differentially expressed genes between CD45-ex vivo and all in vitro samples. (E) Principal component analysis of ex vivo samples. (F) In silico cell-type deconvolution of the bulk RNA-seq samples using MuSiC. pDC - plasmacytoid dendritic cells, uNK – uterine NK cells. (G) Spearman correlation of the whole CD45+ ex vivo transcriptomes for donors that donated more than once. The row-side dendrogram (identical to the column-side one) was omitted.

Comparison of the transcriptomes between endometriosis patients and healthy controls. (A) Gene expression heatmap of 150 genes differentially expressed genes in CD45-in vitro samples (columns). The genes shown are either 1) members of pathways significantly enriched among differentially expressed genes, or 2) top most differentially expressed genes according to p-value and absolute log2 fold-change (“Other”). (B) Gene expression heatmap of 150 genes differentially expressed genes in CD45+ ex vivo samples (columns). The genes shown are either 1) members of pathways significantly enriched among differentially expressed genes, or 2) top most differentially expressed genes according to p-value and absolute log2 fold change (“Other”). (C) Log2 fold-change of the inflammatory signaling pathway activity between endometriosis patients – E, and controls – C in the different cell-types defined in Shih. et al. Horizontal bars show standard errors and asterisks indicate significance level of a t-test performed for each cell-type independently. P-values were adjusted for multiple testing with the method from Benjamini-Hochberg. (D) Log2 fold-change of the expression of selected ligand-receptors involved in inflammatory pathways between endometriosis – E and controls – C. (E) Volcano plot of the differential expression of endometriosis - E - versus healthy controls – C using all available samples. Points above the horizontal blue line indicate genes with multiple-testing adjusted p-values below 0.05. (F) Normalised expression levels of MTRNR2L1 in endometriosis - E and control - C samples. (G) Normalised expression levels of HBG2 in endometriosis - E and control - C samples.