Tsuyoshi Hashiba

The Effects of Age and Abnormal Sperm Count on the Nondisjunction of Spermatozoa

AbstractPurpose: The effect of paternal age on the nondisjunctionof sex chromosomes is controversial. Also, the prevalenceof chromosomal anomalies in infertile patients iscontroversial, it has been reported that the sex chromosomalaneuploidy rate following treatment with intracytoplasmic sperminjection (ICSI) is higher than in naturally conceivedpregnancies. We investigated the influence of paternal age andoligozoospermia on the nondisjunction of spermatozoa. Methods: We determined the rate of aneuploidy forgonosomes and autosomes, using two-color fluorescence in situhybridization (FISH) of the X and Y chromosomes andchromosomes 12 and 18 in 10 donors under 25 years of agewho had a normal sperm count (≤20 × 106/ml), 10 donorsover the age of 39 years with idiopathic infertility andnormozoospermia (≤20 × 106/ml), and 5 oligozoospermicdonors (<20 × 106/ml). Results: There was no obvious relationship betweenincreasing age and autosomal disomy (disomy 12 and disomy 18).Neither autosomal disomy nor diploidy was increased inany group. The frequency of X-, Y-, XX-, and YY-bearingsperm did not differ significantly among groups, but thefrequency of XY-bearing sperm was significantly higher inthe older infertile group than in the control donors. Conclusions: The incidence of nondisjunction of paternalsex chromosome in meiosis I was higher in older men withidiopathic infertility. The present results suggest that therisk of producing XXY fetuses is higher among men >39years of age with idiopathic infertility.

The development of novel quantification assay for mitochondrial DNA heteroplasmy aimed at preimplantation genetic diagnosis of Leigh encephalopathy

Purpose: To perform preimplantation genetic diagnosis (PGD) of Leigh encephalopathy, we developed a rapid and reliable quantification assay for the percentage of T8993G mtDNA mutation and analyzed various specimens. Methods: We prepared the standard curve by measuring serial proportion of 8993T/G cloned plasmid DNA using real-time PCR, and measured (1) mutant DNA (known proportions by PCR-RFLP), (2) single lymphocytes from 46% mutant carrier, (3) 123 blastomeres from 20 abnormal embryos. Results: (1) These were within −5∼+6% error range, (2) mean 44.3%(11–70%), (3) Five embryos harbored T8993G mutation (4–22%). Embryos from same person indicated different degrees of heteroplasmy, and blastomeres from same embryo demonstrated limited dispersion of heteroplasmy (2–11%). Conclusions: (1) This method provides rapid and reliable PGD for Leigh encephalopathy. (2) The variable heteroplasmy with somatic mitosis was suggested. (3) T8993G mutation was existed in undeveloped embryo, and the bottleneck theory was supported. The limited heteroplasmy dispersion of blastomeres from same embryo also supported reliability of PGD for T8993G mutation.

The "Spanning Protocol": A new DNA extraction method for efficient single-cell genetic diagnosis

Purpose: We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our “spanning protocol.”Methods: Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine.Results: With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males.Conclusion: The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

Accurate Multiplex Polymerase Chain Reaction Assay for Gender Determination from a Single Cell

For gender determination of preimplantation embryos or circulating fetal cells in maternal blood, we developed a multiplex polymerase chain reaction assay from a single cell. This assay which co-amplifies X (DXZ1)- and Y (DYZ1)-specific repeat sequences, yields a 308-bp band in females and two bands of 154 and 308 bp in males. In a randomized, blinded assay of 100 isolated single amniocytes, 99 (99%) were amplified successfully. All 50 of the XY cells were correctly diagnosed as male (100%), whereas 49 of the 50 XX cells were diagnosed as female (98%). This accurate and efficient assay may be applicable in these clinical settings.

An accurate and rapid gender determination assay in single cells by the capillary polymerase chain reaction method

Purpose:In preimplantation genetic diagnosis (PGD), a rapid and accurate assay has been required. We have therefore developed a capillary polymerase chain reaction (PCR) method using rapid thermal cycling programs to determine the gender of single amniocytes.Methods:Single amniocytes from each amniotic fluid sample were isolated by micromanipulation and their gender was determined by a multiplex PCR assay in a capillary tube, using primers that amplify a 308-bp DXZ1 and a 154-bp DYZ1 repeat sequence on the X and Y chromosomes, respectively.Results:All four thermal cycling programs, which took 180, 150, 120, and 90 min, were 100% accurate in diagnosing the gender of single amniocytes. No DNA contamination was observed in any samples.Conclusions:The multiplex PCR assay was rapid and accurate in diagnosing gender in single cells and may be clinically applicable in PGD.