Background: Successful placentation requires delicate communication between endometrium and trophoblast. The invasion and integration of trophoblasts into the endometrium during early pregnancy is crucial to placentation. Dysregulation of these functions is associated with various pregnancy complications, e.g. miscarriage and preeclampsia. The endometrial microenvironment exerts important influence on trophoblast cell functions. The precise effect and mechanism of decidual glandular epithelial cells on trophoblast functions remain uncertain. Hypothesis: We hypothesized that the secretome of human endometrial gland is involved in regulating trophoblast migration and invasion. Methods:Human endometrial tissues were obtained from endometrial biopsies with written consent. The endometrial organoids were established in matrix gel with a defined culture condition. They were treated with hormones mimicking the environment of the proliferative phase (Estrogen, E2), the secretory phase (E2+ Progesterone, P4) and early pregnancy (E2+P4+Human Chorionic Gonadotropin, hCG). RNA-seq and miRNA-seq were performed on the treated organoid to compare their transcriptomes. Organoid secretions were also collected for mass spectrometric analysis. The viability and invasion/migration of the trophoblasts were determined by XTT assay and transwell assay respectively. Results: Endometrial organoid model with high expressions of endometrial gland markers were successfully established. Spent medium from E2+P4+hCG-treated endometrial organoids, which mimics the pregnant decidual microenvironment, significantly enhanced the invasion and migration of the trophoblast when compared to those obtained from proliferative/secretary phases. All the treatments have no effect on trophoblast viability. Our results further demonstrated that hormone treatment up-regulated the S100A9 protein production by the endometrial gland via mir-3194 suppression, and thereby promoted trophoblast invasion and migration. Conclusion: By using a human endometrial organoid model, we demonstrated that hormonal stimulation of endometrial gland secretome during pregnancy may be crucial to regulate the functions of human trophoblasts.