Figure 4. Establishment of a co-culture model recapitulates the molecular signature of the human menstrual cycle in vitro.

(A) Schematic and representative images of the co-cultures. EEOs (10 intact organoids/µL) and ESC (10k cells/µL) are expanded separately and embedded in the synthetic ECM, cultured as 3 µl droplets and maintained for 15 days of culture. A dual-adhesion peptide synthetic ECM (3wt% 20kDa-PEG) functionalized with GFOGER (1.5 mM) and PHSRN-K-RGD (1.5mM) supports the establishment of the EEOs and ESC co-culture model. (B) Representative 3D images of day-15 endometrial EEOs (EpCAM = green) and ESC (Vimentin = red) in the co-cultures (scale = 100 µm). Images acquired from Video S3 in Data File 3. (C) Schematic and timeframe of the idealized 28-day menstrual cycle in vivo. (D) Experimental design for the validation of the co-culture endometrial model. Treatment groups are designed to mimic the hormonal changes in the phases of the menstrual cycle, including a pharmacologic induction of the menstruation using a PGR antagonist (RU-486, 10 µM). ‘Inflamed’ groups were co-treated with IL1B (1ng/mL) throughout the length of the experiment. (E-G) Transcriptomic (bulk RNAseq) analysis of the co-culture models (n= 7). (E) Gene ontology (GO) analysis of the top significantly enriched GO terms that are downregulated or upregulated in response to progestin treatment. Cocultures treated with E2 + MPA (“PE”) have gene ontology (GO) expression profiles that align with the secretory phase of the menstrual cycle. GSEA analysis of hallmark pathway gene sets show pathway changes consistent with corresponding phases of the menstrual cycle. All images and transcriptomic analysis were performed on day-15 co-cultures in the synthetic 3wt% 20kDa-PEG-Mix hydrogels (N=8). Data is shown with the −log of their P values.