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 implantation might be possible in the future. The objective of pre-implantation diagnosis would be to sample a minimal amount of cellular material of the conceptus for diagnosis prior to transfer. Different protocols for isolating individual blastomeres from two-cell mouse embryos were evaluated. Two-cell mouse embryos were collected and the zona pellucida 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). Individual blastomeres were obtained by exposure to a chelating agent (EDTA-glycine mixture), to Ca2+-Mg2+-free PBS or after isolation by mechanical force (bisection with a microblade or suction in a small pipette). The isolated blastomeres were then cultured in vitro without zonae pellucidae. All isolation procedures had a negative impact on the growth patterns of the isolated blastomeres. Different abnormalities could be observed at the blastocyst stage including embryos lacking visible compaction features, embryos with double blastocoelic cavities and embryos with no inner cell mass (trophoblastic vesicles).