A 3D endometrium-on-a-chip reveals the role of conceptus-derived factors CAPG and PDI in conceptus-endometrial communication

Early embryo loss affects all mammalian species, including humans and agriculturally important food-producing mammals such as cattle. The developing conceptus (embryo and extra-embryonic membranes) secretes factors which modify the endometrium and can be critical for early pregnancy processes such maternal recognition of pregnancy (MRP) and enhancing uterine receptivity to implantation. For example, a competent bovine conceptus secretes IFNT to initiate MRP. The bovine conceptus also secretes other proteins at the time of MRP, including CAPG and PDI, which are highly conserved among placental mammals. We have previously shown that these proteins act upon the endometrium to modulate receptivity, embryo development, and implantation in species with different implantation strategies (humans and cattle). We hypothesise that developing a novel 3D bovine endometrium on a chip system will enhance our understanding of the role of conceptus-derived factors in altering the endometrium and/or ULF secretion. Here we have developed a 3D bovine endometrium on a chip system, comprising both stromal and epithelial cell culture combined with culture medium flow better mimics the in vivo endometrium and exposure to conceptus-derived factors than conventional 2D endometrial cell culture. We have demonstrated that the conceptus-derived proteins CAPG and PDI modulate the endometrial transcriptome and secretory response to promote pathways associated with early pregnancy and alter ULF composition. This work highlights the critical need for more robust and in vivo -like culture systems to study endometrial-conceptus interactions in vitro to further investigate the role of conceptus derived factors for pregnancy success. SIGNIFICANCE STATEMENTWe have developed an in vitro 3D bovine endometrium-on-a-chip system comprising both primary stromal cells under static conditions and epithelial cells under flow conditions to mimic the in vivo endometrial environment from the conceptuses perspective. The secretome of the 3D endometrium-on-a-chip was characterised, was found to contain proteins associated with cell adhesion and tissue development, and contained proteins previously identified in in vivo uterine luminal fluid. PDI and CAPG (previously identified conceptus-derived factors) altered the transcriptome and secretome of cells within the system. Exposure to CAPG or PDI altered the secretome of proteins previously identified in pregnant uterine luminal fluid or associated with early pregnancy, and exposure to CAPG or PDI also altered the transcriptome to support processes such as immune response, secretion, proliferation, and adhesion related pathways. This data supports previously published works and highlights the need for the use of more in vivo-like in vitro models to study conceptus-endometrial interactions.