Culture model systems that can recapitulate the anatomy and physiology of reproductive organs, such as three-dimensional (3D) organoid culture systems, limit the cost and welfare concerns associated with a research animal colony and provide alternative approaches to study specific processes in humans and animals. These 3D models facilitate a greater understanding of the physiological role of individual cell types and their interactions than can be accomplished with traditional monolayer culture systems. Furthermore, 3D culture systems allow for the examination of specific cellular, molecular, or hormonal interactions, without confounding factors that occur with in vivo models, and provide a powerful approach to study physiological and pathological reproductive conditions. The goal of this paper is to review and compare organoid culture systems to other in vitro cell culture models, currently used to study female reproductive physiology, with an emphasis on the role of extracellular vesicle interactions. The critical role of extracellular vesicles for intercellular communication in physiological processes, including reproduction, has been well documented, and an overview of the roles of extracellular vesicles in organoid systems will be provided. Finally, we will propose future directions for understanding the role of extracellular vesicles in normal and pathological conditions of reproductive organs, utilizing 3D organoid culture systems.

Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze–thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of their normal physiology, including hormonal regulation. These organoids may provide an environment that mimics the in vivo equine oviduct and facilitate improved in vitro embryo production in equids.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle 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 can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.