Cross Sectional and Longitudinal Imaging Reveals Spatiotemporal Divergence in Morphogenesis and Cell Lineage Specification between in-vivo and in-vitro Mouse Embryo during Pre- and Peri-implantation

In-vitro culture is an essential step in assisted reproductive technology. Previous studies using sectional approaches have reported the impacts of in-vitro technologies on mammalian embryogenesis and offspring development. However, systematic and longitudinal comparisons between in-vivo and in-vitro spatiotemporal morphogenesis and cell lineage specification remain incomplete. Moreover, the phototoxicity of laser confocal microscopes on embryos is not fully evaluated. This study aims to bridge this gap using 4D live imaging via a laser scanning confocal microscopy to capture dynamic embryo development from a CAG::H2B-GFP mouse line. The results showed that in-vitro culture under a confocal microscopy cumulatively slowed the increase in embryonic cell number and embryos size over time. Furthermore, in-vitro embryo compaction, cavitation, primitive cell migration and hatching deferred in both spatial pattern and temporal progression. Additionally, in-vitro and in-vivo cell lineages differed in spatial distribution and expansion rate. These findings demonstrate that in-vitro culture delays embryo development, alters morphological events and disrupts cell lineage specification at both spatial and temporal scales during pre- and early peri-implantation. This work provides insights on the detailed extent of similarities and differences between in-vivo and in-vitro embryogenesis, beneficial when translating in-vitro findings into clinical, industrial and ecological applications.