This study is designed to explore the relationship between the ratio of differentiated cell types, inner cell mass (ICM) and trophectoderm, at the time of blastulation and the developmental potential of the embryo. Since cell position in the morula determines the direction of differentiation, a predictable relationship should exist between total cell number in the morula and ratio of ICM to trophectoderm in the blastocyst. A recently devised, vital method of cell counting, using the DNA dye bisbenzimide, which does not affect developmental potential of embryos should provide a simple, noninvasive means of determining embryonic potential once this relationship is established. Assessment of viability by embryo transfer and development to midgestation provides the most stringent test of normality which distinguishes between simple metabolic functioning and true developmental potential of the embryo as a whole. The relationship between cell number and ratio in the blastocyst and its relevance to developmental potential will first be examined in the mouse. Differential cell counts of ICM and trophectoderm will be made in embryos doubly dyed with bisbenzimide and propidium iodide (PI) following specific lysis of trophectoderm by immunosurgery. All cells are permeable to bisbenzimide whereas only nuclei of lysed cells will stain with PI. Microsurgery will be used to reduce cell number and effects of culture or development in vivo in an immature mouse oviduct as a surrogate environment will be examined to test cell ratio and viability relationships over a wide range of conditions. Ascertainment of the nature of developmental abnormalities encountered will contribute to the long term goal of relating specific fetal abnormalities to specific developmental parameters of the preimplantation embryo. The normal range of these parameters will then be examined in the pig to explore the possibility that inherent differences among early embryos could account for the high embryonic wastage characteristic of the pig and humans. Since ethical considerations limit experimental techniques applied to human embryos, it is important to test the general applicability of mechanisms in a range of species before making assumptions of similarity. A long term goal is that a greater understanding of factors that affect develpmental potential will aid in the interpretation of human embryonic development.

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Tufts University
Schools of Medicine
United States
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