Assessment of different isolation procedures for blastomeres from two-cell mouse embryos

As an extension of in-vitro fertilization and embryo transfer,detection of genetic and metabolic defects prior to implantationmight be possible in the future. The objective of pre-implaiitationdiagnosis would be to sample a minimal amount of cellular materialof the conceptus for diagnosis prior to transfer. Differentprotocols for isolating individual blastomeres from two-cellmouse embryos were evaluated. Two-cell mouse embryos were collectedand the zona pdlucida was removed by enzyme treatment (pronase),by exposure to acid Tyrode (pH = 2.5) or by mechanical force(suction into a small pipette, removal with a microblade). Individualblastomeres were obtained by exposure to a chelating agent (EDTA-glycmemixture), to Ca²⁺-Mg²⁺-free PBS or after isolation by mechanicalforce (bisection with a microblade or suction in a small pipette).The isolated blastomeres were then cultured in vitro withoutzonae pellucidae. All isolation procedures had a negative impacton the growth patterns of the isolated blastomeres. Differentabnormalities could be observed at the blastocyst stage includingembryos lacking visible compaction features, embryos with doubleblastocoeJk cavities and embryos with no inner cell mass (trophoblasticvesicles).