Preimplantation genetic diagnosis (PGD) is a technology that allows for the selection and transfer of embryos unaffected by genetic disease. The limited number of cells available for genetic testing is a weakness of PGD and has been solved by means of the development of various strategies such as polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and cell recycling. A confounding factor in PGD is the existence of preimplantation embryos with severe chromosomal abnormalities. Therefore, genetic analysis should be performed with the assumption that embryos have severe chromosomal abnormalities. The visualization of metaphase plates allows screening for numerical chromosomal abnormality and several kinds of structural chromosomal abnormality. In addition, in vitro culture of single isolated blastomeres makes it possible to reexamine samples to ensure accuracy of the results and to obtain additional genetic information.

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Duchenne muscular dystrophy (DMD), caused by mutations of the dystrophin gene, is a severe X-linked recessive neuromuscular disorder. Preimplantation diagnosis of DMD includes three approaches. The first approach is gender determination of embryos by either polymerase chain reaction (PCR) or the fluorescence in situ hybridization (FISH)-based method. While each method is well established, the FISH method has some advantages over PCR in gender determination. The second approach is diagnosis of specific gene mutation. The partial deletions are diagnosed by the PCR with primers constructed to amplify the deletion exons. The partial duplication cannot be detected by now available strategies. The small mutations can be diagnosed by the specific PCR based assay. The third approach is linkage analysis by means of linked markers. CA repeats have been shown to be highly polymorphic and to be of great diagnostic utility because they can be easily assayed by PCR.

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Chorionic villus sampling (CVS) is a technique for prenatal diagnosis of the fetal karyotype through cytogenetic analysis, and of Mendelian inherited diseases through molecular or biochemical analysis. Because the sampling technique can be performed in the first-trimester of pregnancy and diagnostic results can be obtained earlier than with amniocentesis, it has been utilized by clinicians in Europe and the US since 1982. CVS includes two methods: a transcervical approach and a transabdominal approach. Both are performed in the 10^th week of pregnancy under careful ultrasound guidance to prevent adverse effects, but in comparison with amniocentesis, a slightly higher risk of pregnancy loss has been reported. Moreover, diagnostic accuracy is often disturbed by maternal cell contamination and chromosomal mosaicism of the placenta. Therefore, clinicians must give patients sufficient information on such technical and diagnostic trouble through counseling in order to obtain fully informed consent.

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The incidence of major chromosome abnormalities in newborns is about 0.7 percent and increases with maternal age. Amniocentesis is the most common invasive prenatal procedure for the detection of fetal chromosomal abnormalities. Amniocentesis is a relatively safe procedure and fetal loss related to amniocentesis is about 0.5%. An advanced maternal age is the most common reason for using amniocentesis. The use of amniocentesis because of abnormal fetal ultrasound findings has increased recently. Fluorescence in situ hybridization (FISH) is currently a powerful tool in the area of prenatal cytogenetics. The number of amniocentesis procedures in Japan is about ten thousand per year and it is generally recognized to be a great benefit for pregnant women who have a risk of fetal chromosomal abnormalities.

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The effect of polyvinyl alcohol (PVA) on the development of mouse preimplantation embryos was examined. In Hoppe and Pitts medium supplemented with 0.1 or 1.0 mg/ml PVA, a high percentage (comparable to BSA-supplemented medium) of mouse 1-cell embryos developed to the expanded blastocyst stage. However, in medium supplemented with 10.0 mg/ml PVA, no embryos developed to the blastocyst stage. These results indicate that, at optimum concentration, PVA supports embryo development from the 1-cell to the expanded blastocyst stage, and that the PVA-supplemented medium is a valuable chemically defined medium for mouse embryonic culture. Also, in this study, in the PVA-supplemented medium, percentages and speeds of embryo development from each stage (1-cell, 2-cell, 8-cell and early blastocyst) to the expanded blastocyst stage were almost the same as those in the BSA-supplemented medium. These results suggest that these embryos developed to expanded blastocysts via normal processes, and that PVA supports embryo development in each stage up to the expanded blastocyst stage. Incidence of partial hatching and complete hatching of blastocysts was clearly decreased in cultures of each embryo from 1-cell to the early blastocyst stage in the PVA-supplemented medium. It has been considered that protease may participate in the hatching process of blastocysts in vitro, thus, it is probable that the low hatching rate of blastocysts in the PVA-supplemented medium is due to a decline in protease synthesis and/or secretion.

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Changes in the number of lipid droplets during meiotic maturation, fertilization and early development were histochemically examined in cultured porcine oocytes and embryos. The oocytes and embryos possessed Sudanophilic lipids composed of small (<2.5 μm), medium (2.5-4.9 μm) and large (≥5.0 μm) droplets. In oocytes soon after collection, the numbers of Sudanophilic lipid droplets with small and medium sizes were few and the number of those with large size was 148 ± 11.36. After being cultured for 22 and 44 hrs, the number of lipid droplets with large size remarkably decreased, while the number of those with small and medium sizes increased. The numbers of lipid droplets of each size in the oocytes 4 and 8 hrs after insemination were similar to those in oocytes 44 hrs after maturation culture. On the other hand, the number of lipid droplets in embryos did not vary greatly between the pronuclear and the 16-cell stages, but gradually decreased after the morula stage. Expanded blastocysts had few small and medium lipid droplets and 11 ± 1.68 large ones. The present findings confirmed that lipid droplets contained in oocytes become smaller in size and larger in number. Since the smaller lipid droplets appear not to be used in the process of fertilization, we presume that they are mainly used as an energy source for the formation and expansion of blastocysts.

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