The different methods for sperm preparation are used routinely to obtain a population of highly motile sperm for assisted reproductive technology. But, the study of chromosomal abnormalities in highly motile sperm obtained by sperm preparation is under debate. Using fluorescence in situ hybridization, we evaluated the fre quency of numerical sex chromosomal abnormalities in highly motile sperm obtained by swim-up method in the current study. Semen samples of 27 individuals, diagnosed as normal according to criterion of World Health Organization, were analyzed. These samples are classified as follows : swim-up group ; motile sperm obtained by swim-up method, pellet group ; sperm obtained by pellet, no treatment group ; no treatment sperm. At least 15000 sperm were analyzed for each sample. In swim-up group, the ratio of X-, Y-bearing, and sex chromoso mal numerical abnormality sperm were 47.6%, 52.2%, and 0.17%, respectively. In pellet group, the ratio of X-, Y bearing, and sex chromosomal numerical abnormality sperm were 52.2%, 47.3%, and 0.50%, respectively. In no treatment group, the proportion of X-, Y-bearing, and sex chromosomal numerical abnormality sperm were 50.0%, 49.7%, and 0.26%, respectively. There were not significant differences in the proportion of X- and Y bearing sperm among each group. The proportion of sex chromosomal numerical abnormality sperm in swim up group was significantly lower than that of pellet group and no treatment group. These results suggest that swim-up method can reduce the proportion of sperm with sex chromosomal numerical abnormality, but not absolutely. Therefore, it is necessary to study sperm selection more.
Preimplantation genetic diagnosis (PGD) is an established procedure of embryo genetic analysis, allows couples carrying monogenetic diseases to have an unaffected child, without invasive prenatal diagnosis and termination of pregnancy. PGD is performed with nested PCR, involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product. The scarcity of DNA is a limiting factor in many reseach fields. Multilocus simultaneous analyses from single cell is difficult. DNA microarrays manufactured to date are not able to analyze limited amount of DNA in a single cell. Development of a new gene amplification method which replaces PCR is desirable. Multiple displacement amplification method (MDA) is a non-PCR based whole genome amplification (WGA). We aimed to construct the PGD system using MDA especially for Duchenne muscular dystrophy (DMD) and to examine the presence of bias of amplification and the efficiency rate of amplification. The dilution samples were made with the extracted DNA from the lymphocytes of normal male, DMD patient and carrier, and used for the template of MDA. The amplification by MDA in all nine exons were confirmed from the result of following singleplex PCR and multiplex PCR using multiple primer of Chamberlain. As a result of the gene analyses by using realtime PCR with fluorescence image of TaqMan Probe, the agreement rate of the diagnosis using with 1ng and 100pg of template was 100%(14/14) and 86%(12/14), respectively. The agreement rate of the diagnosis with 10pg of template DNA was 71%(10/14). The MLPA techique was performed for the MDA products using exon-specific probes for the dystrophin gene and amplification of all 79 exons were examined. The amplification were confirmed in almost all exons when 100 pg of DNA was used as the template. Thirty out of 79 exons were not amplified when 10 pg of DNA was used. The bias of relative copy numbers calculated was examined. From the results, the combination of MDA and MLPA methods has not not been effective in analysis of quantification for the duplicated mutation, but effective for simultaneous qualitative analysis of the dystrophin gene. The strong potential of Genome-wide amplification by MDA has been demonstrated for PGD in case of more than 100 pg of template DNA.
Allogeneic immunization is thought to be concerned with recurrent pregnancy loss. A unique immune system make it possible for fetal tissues that have paternal major histocompatibility complex to implant into the maternal uterus. According to the immunotrophism hypothesis that maternal inmunne recognize paternal antigen and promote placental growth, a shift in the Th1/Th2 balance toward Th2 is important in the immune system around decidua and placenta. Indoleamine 2,3-dioxygenase (IDO) that inhbit an increase of the cytetoxic T cell to produce Th1 cytekines is received attention. Flowcytemetry is useful for measurement of these cytokines.
The introduction of in vitro fertilization (IVF) and the molecular analysis of single cells has made it possible to perform preimplantation screening of human embryos for a variety of genetic disorders. Preimplantation genetic diagnosis (PGD), a very early form of prenatal diagnosis, offers the advantage of allowing the selection and transfer of apparently normal embryos, so that couples at risk of transmitting a serious genetic disease or structural chromosomal abnormalities to their offsprings can start a pregnancy without the anxiety of a possible termination or miscarriage. Since its introduction in 1990, PGD has become a practical option world-wide, and by the end of 2007 about 22,000 cycles of PGD had been reported resulting in over 3,000 unaffected babies. In Japan, its clinical application for the couples with genetic diseases was allowed under the approval of Japan society of obstetrics and gynecology in 2004, and, in addition, the reciprocal translocation carriers with recurrent miscarriages were permitted as one of indications in 2006. Although PGD for translocation carriers, which is performed using FISH usually, has been reported to allow statistically significant reduction in spontaneous abortions, on the other hand, there are still some technical problems to be resolved, which are mainly based on single-cell genetic analysis and the high frequency of mosaicism observed at the cleavage stage embryo.
Aim. We are in charge of the central diagnosis and cell preservation as a part of childhood acute lymphoblastic leukemia treatment study in Tokyo children’s cancer study group. It is necessary to diagnose with a minimal quantity of specimen, to preserve leukemic cells effectively as possible. On the other hand, recent progress in multi-color flow cytometry enable to analyze cell marker of leukemia in more detail. We therefore we intended to perform a immuno-phenotypic diagnosis of childhood acute lymphoblastic leukemia by nine-color analysis with digital flow cytometer.
Methods. We examined cell markers of childhood acute lymphoblastic leukemia cells by nine-color analysis using digital flow cytometers. We decided the combination of the monoclonal antibodies for nine color analysis based on the recommendation of Japan Pediatric Leukemia/Lymphoma Study Group using commercially available fluorescence-laveled antibodies.
Results. Nine colors that we used in this study were fluorescein isothyocyanate, phycoerythrin (PE), phycoerythrin-Texas Red, Peridinin Chlorophyll Protein - cyanin (Cy) 5.5, PE-Cy7, allophycocyanin (APC), APC-Cy7, Pacific Blue, and Cascade Yellow. Using list mode compensation, nine color marker analysis of childhood leukemia was easy to perform.
Discussion. Although several problems need to be solved are present, nine-color analysis using digital flow cytometer is useful to obtain precise information of antigen expression as well as save precious specimen of childhood acute lymphoblastic leukemia.
The present study aimed to elucidate the roles of the mitogen-activated protein kinase kinase (MEK)/extra- cellular signal-regulated kinase (ERK) and phosphatidylinositol 3ʼ-kinase (PI3K)/Akt signaling pathways in reg- ulating cisplatin (CDDP)-induced cytotoxicity in ovarian carcinoma cells. We treated seven ovarian carcinoma cell lines (KF, KFr, KOC-2S, SK-OV-3, SHIN-3, TU-OS-3, and TU-OS-4) with CDDP combined with PI3K inhibitor [LY294002 (LY)], MEK inhibitor [PD98059 (PD)], or MEK/ERK activator [phorbol 12-myristate 13-acetate (PMA)], then assessed cell viability, expressions of Akt, phosphorylated (p)Akt, ERK, pERK, and cleaved caspase-3 protein by western blotting, cell cycle distribution, and apoptotic cells by flow cytometric analysis and annexin V staining. Proteins pMEK, pERK, and pAkt were expressed in all cell lines. The range of IC50 to CDDP was 2.4 to 26.9μM for those cell lines. CDDP combined with LY had an additive effect on inhibiting cell growth,whereas combined with PD had an antagonism for all cell lines. Interestingly, growth of cells was dramaticallysuppressed when CDDP was combined with PMA in the CDDP-resistant cells (KFr, SK-OV-3, SHIN-3, TU-OS- 3, TU-OS-4). Treatment with PMA up-regulated protein expression levels of pERK and cleaved caspase-3 only in the CDDP-resistant cells. CDDP combined with PMA increased the S-phase fraction and apoptotic cells inthe CDDP-resistant cells. These results indicate that MEK/ERK activated by PMA may overcome resistance to CDDP in ovarian carcinoma cells.