In ovo versus ex ovo incubation differentially shapes chorioallantoic membrane maturation, angiogenesis, and tumor growth.

The chorioallantoic membrane (CAM) is a highly vascularized avian extraembryonic membrane, widely used as an in vivo model for angiogenesis, with predominant application in tumor research. CAM assays are performed under in ovo or ex ovo conditions; however, a clear rationale for model selection is lacking, and comparative morphological data remain limited. In this study, we compared chicken embryo CAM development under both incubation conditions. Morphological analyses revealed pronounced incubation-dependent differences in CAM maturation, with the mesoderm showing the most dynamic alterations. Ex ovo CAMs exhibited early mesodermal thickening, and peaks in angiogenesis; indicative of accelerated but less coordinated tissue and vascular development. In ovo CAMs showed a gradual and well-coordinated increase in mesodermal thickness and vascularization, peaking at later developmental stages. Differences were also observed in epithelial organization: ex ovo CAMs exhibited disrupted ectodermal architecture, whereas in ovo CAMs maintained stable epithelial stratification. Functional assessment using GFP-labelled human fibrosarcoma cells (HT1080) demonstrated significantly higher embryo survival and larger tumor growth in the in ovo model, while ex ovo conditions were associated with increased mortality and reduced tumor size. These findings demonstrate that incubation conditions significantly influence CAM morphology and functional performance, highlighting the importance of careful model selection in angiogenesis research.