Modeling Early Stages of Trophectoderm-Endometrium Interactions Using Trophoblastic and Endometrial Organoids and the Generation of Lacunoids/Cystoids.

This study presents the first successful generation and comprehensive characterization of trophoblastic organoids (TOs) and the derivation of three-dimensional cavity- or sac-like structures-termed lacunoids/cystoids-from sheep intracytoplasmic sperm injection (ICSI) embryos. TOs were generated from sheep ICSI embryos for the first time and were shown to express trophoblastic markers at levels comparable to those in embryonic tissue. Detailed morphological characterization was conducted for both the TOs and the derived lacunoids/cystoids. Additionally, the TOs' interactions with endometrial organoids (EOs), as well as those with preimplantation embryos, were investigated through co-culture experiments. The TOs expressed key trophoblastic markers, including CDX2, GATA3, syncytin-1, KRT18, KRT7, and Sox2, confirming their validity as a model for studying sheep trophoblast biology. The generation of lacunoids/cystoids from the TOs further revealed their structural and developmental characteristics, contributing valuable insights into early placental development and trophoblast-related pathologies. The TOs also supported extended embryonic development, and their co-culture with EOs induced dynamic changes in gene expression, particularly in angiogenesis-related genes, in both organoid types. This novel and reproducible in vitro model offers a reliable platform to study early placental development, effectively recapitulating the biological crosstalk between the trophectoderm and endometrium. The in-depth characterization of TOs and lacunoids/cystoids highlights their potential to advance our understanding of trophoblast differentiation and related developmental disorders.