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

Islam M. Saadeldin^1,2*, Budur Alshehri^1,2,3, Maha AlThubyani¹, Ayman A. Swelum⁴, Serdar Coskun^1,2, Abdullah M. Assiri^1,2

¹Comparative Medicine Department, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia

²College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia

³King Fahd Medical City, Riyadh, Saudi Arabia

⁴Department of Animal Production, College of Agriculture and Life Science, King Saud University, Riyadh, Saudi Arabia

 

Abstract Text: This study represents the first successful generation and detailed characterization of trophoblastic organoids (TOs) and lacunoids/cystoids (TCs) derived from sheep intracytoplasmic sperm injection (ICSI) embryos. The novelty of this work lies in utilizing embryo-derived trophectoderm to generate naïve forms of trophoblastic organoids.
Trophoblast cells derived from ICSI embryos exhibited robust proliferation in monolayer culture, forming compact spheroid structures upon transfer to Matrigel and culture for 6–9 days. These TOs demonstrated plasticity, reverting to monolayer cultures when re-cultured on tissue culture dishes. Confocal microscopy confirmed the presence of trophoblast markers β-catenin, SOX2, and cytokeratin-18 in both monolayer and 3D TO structures.
In extended culture ( > day 12), cystic structures, termed trophoblastic lacunoids/cystoids (TCs), emerged from TOs, maintaining their morphology through multiple passages and exhibiting polarized cellular organization. These structures were also observed when individual blastocysts (n = 14) were trypsinized and cultured in Matrigel domes. Co-culture experiments with embryos revealed that TOs supported extended embryo development up to day 14, whereas control embryos cultured without TOs showed signs of degeneration at an earlier stage.
Additionally, endometrial cells formed spherical multicellular organoids (EOs) that grew in size and retained their morphology across multiple passages. Immunofluorescence analysis of EOs demonstrated that Ki-67, a proliferation marker, and Lamin, a nuclear envelope structural protein, were localized in the nucleus, while β-catenin, a cell adhesion protein, was localized in the cytoplasm.
Co-culture of TOs and EOs resulted in the formation of voids and cavities within both organoid types, suggesting reciprocal interactions. Immunofluorescence analysis revealed VEGFA expression in both TOs and EOs, VEGFR1 expression in EOs, and VEGFR2 expression in TOs. Furthermore, embryos (day 8) co-cultured with EOs aligned to attach to Matrigel, exhibiting lateral attachment and the formation of cysts or lacunae by the 4th day, which became more distinct by the 10th day. Subsequently, the embryonic structures attached to Matrigel propagated, forming trophoblastic cell roots with elongated cytoplasmic protrusions.
Overall, this study elucidates the morphological characteristics and functional implications of TOs and EOs, highlighting their potential in modeling early pregnancy interactions and supporting embryo development in vitro.