Effects of different biopsy methods on the development of preimplantation mouse embryos, in vitro and in vivo: implication for preimplantation genetic diagnosis.

The aim of this study was to investigate the efficacy of two different embryo biopsy techniques, direct aspiration and partial zona dissection (PZD)-push, on subsequent in vitro and in vivo development of 8-cell stage mouse embryos. It was found that the rates of normal blastocyst formation and hatching blastocysts of direct aspiration, PZD-push, solution control and control embryos were not significantly different (80.8%, 81.6%, 84.5%, 86.7% and 71.9%, 72.3% and 74.6%) respectively. There was, however, a significant reduction in rate of complete hatching blastocysts (P < 0.1) (72.9% aspiration versus 85.2 per cent solution control and 86.4% control) and rate of live-born fetuses (24.2% aspiration versus 43.3% solution control and 41.2% control) (P < 0.05) in the direct aspiration group but no significant difference in the PZD-push group (80.3% of complete hatched blastocysts and 33.8% of live-born fetuses). These findings indicated that embryo biopsy with PZD-push was superior to the direct aspiration method. This mouse embryo biopsy model was useful in advancing development of biopsy technique for human preimplantation genetic diagnosis.

Reliable sex determination of mouse preimplantation embryos by PCR amplification of male-specific genes in single blastomeres.

OBJECTIVE: To assess the reliability of sex determination in mouse preimplantation embryos using the two-step polymerase chain reaction method. SETTING: Division of Immunology, Department of Microbiology and Division of Reproductive Medicine, Department of OB/GYN. METHODS: The Sry and Zfy genes, known to be present in the sex-determining region of mouse Y chromosome, were selected for Y-specific target sequences and DXNds 3 locus located on mouse X chromosome was served as the internal control sequence. DNAs extracted from heart blood of male and female mice were used to test the correctness and specificity of the selected primers using the two-step PCR method. The same experimental conditions were then used to amplify the single copy genes in single mouse blastomeres with two pairs of primers for each of the target sequences. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. RESULTS: All male and female blood DNA sample results confirmed the correct sex identification of the origin (100%). Nineteen of 20 single blastomeres showed the accurate diagnosis when compared with theirs 7/8 embryos. The sex of 36 of 37 mouse pups born from biopsied male and female embryos agreed with the predicted sex. CONCLUSION: The reliable genetic analysis of sex chromosome- specific sequences in single cell is possible by the two-step PCR method and could be applied for diagnosis of defective genes of human preimplantation embryos derived from the in vitro fertilization program.