Evaluation of different biopsy methods of blastomeres from 2-cell mouse embryos.

Abstract

As an outgrowth of in-vitro fertilization and embryo transfer, detection of genetic and metabolic defects prior to implantation might be possible in the future. The objective for preimplantation diagnosis would be to sample a minimum of cell material of the conceptus for diagnosis prior to transfer. Different protocols for isolating individual blastomeres out of 2-cell mouse embryos were evaluated. 2-cell mouse embryos (from F1 hybrids C57B1 females x CBA males) were collected and the zona pellucida was removed by enzyme treatment (pronase), by exposure to Ca2+-Mg2+-free acid Tyrode (pH = 2.5) or by mechanical force. Individual blastomeres were obtained by exposure to an enzyme (pronase), to a chelating agent (EDTA-glycine mixture), to Ca2+-Mg2+-free PBS or after isolation by mechanical force. The biopsied blastomeres were then cultured in vitro as such or first replaced into a host zona pellucida. Evaluation was performed by culture in vitro up to the blastocyst stage and by transfer of embryos appearing morphologically normal into pseudopregnant foster mothers. A chromosomal study of the second mitotic division of the isolated blastomere was also performed. All isolation procedures had a negative impact on the in-vitro and in-vivo growth patterns of the isolated blastomeres. After culture in vitro to the blastocyst stage, different abnormalities could be observed: embryos lacking compaction, embryos with double blastocoelic cavities and embryos with no inner cell mass (trophoblastic vesicle). After replacement of the isolated blastomeres into a host zona pellucida, similar observations could be made. Chromosomal analysis did not reveal a clear influence of the different biopsy methods on the mitosis of the isolated blastomeres.