Figure 5. Functional validation of the co-culture model recapitulates morphologic and biochemical cycle-dependent reproductive processes.

(A) Representative histological (IF) characterization of the proliferative and secretory phases of the menstrual cycle in vivo reveals morphologic and biochemical changes. Vimentin (red), F-Actin (Green), DAPI (blue). (B) Characterization of the epithelial morphology in co-cultures at day 15 of culture by IF reveals glandular maturation and invagination of EEOs in response to progestin treatment groups. (C) IF staining of PAEP (glycodelin) in co-cultures recapitulates the epithelial secretory phenotype. (D) Stromal decidualization was assessed by measuring prolactin secretion in the medium across 15 days of culture. PRL secretion is induced by PE treatment but suppressed in menses treatment group. (E) PGR staining in the co-cultures at day 15 of treatment. (F) Temporal profiles of decidualization normalized to fold change relative to day 0 were performed by measuring spent media (N=12) and can be suppressed by IL1B treatment (1 ng/mL, E+, PE+). (G-H) Cleaved caspase-3 (Casp-3) IF in the co-cultures as a marker of apoptosis (G) and quantified in as positive staining per organoid area (H). All images are of day-15 co-cultures in the synthetic ECM 3wt% 20kDa-PEG-Mix hydrogels. Significance is indicated as *p<0.05, **p<0.01, ***p<0.001.