Evaluation of chorionic girdle organoids for ultrastructure and equine Chorionic Gonadotropin production

The equine chorionic girdle (CG) is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (Day 0). The CG cells differentiate into binucleate endometrial cup cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG), which has LH-like activity in the horse but variable LH- and FSH-like activity in other species. eCG has been utilized for these properties both in vivo for estrous cycle manipulation and in vitro. To collect this hormone, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare. Attempts to produce eCG in vitro using long-term culture of CG explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and remain genetically and phenotypically stable throughout long-term (i.e. months) culture. Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin long-term. The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. CG was collected from Day 33 equine conceptuses (n=3). CG was isolated manually using a dissection microscope and then enzymaticallydigested and cultured as organoids using human trophoblast organoid medium. A monolayer culture was performed as a control 2D culture model for comparison. CG organoids were assessed morphologically using brightfield microscopy, immunohistochemistry (IHC) with an anti-horse trophoblast antibody, and transmission electron microscopy (TEM), and all cultures were assessed physiologically by quantifying equine LH (as a proxy for eCG) via radioimmunoassay of conditioned media. The dense, trophoblast organoid phenotype, as described for human trophoblast organoids, was maintained for 5 passages, a period of 6 weeks. TEM of organoids revealed characteristic binucleate cells in both passages 1 and 4 and exhibited the presence of girdle cell processes, which have been described previously in endometrial cups in vivo. Furthermore, eCG was secreted at high concentrations (up to 67,000 ng/mL) up to passage 4 in organoids but only for passage 0 in monolayer cultures (up to 1,100 ng/mL). Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for early equine pregnancy. Funding was provided by Foundation for the Horse.