OBJECTIVE: Sperm aneuploidy is associated with ICSI failure, pregnancy loss and aneuploid conceptions. However, routine screening has not been readily adopted, in part hindered by the large number of sperm recommended to be scored, (minimum 5000). We have investigated the feasibility of scoring a reduced number whilst maintaining accuracy.DESIGN: Prospective study.MATERIALS AND METHODS: Fluorescence in-situ hybridisation in 11 sperm samples for chromosomes 13, 18, 21 and 22 and 7 sperm samples for chromosomes X, Y and 15. To determine the minimum number of scored sperm needed for reliable estimates of sperm aneuploidy, 5000 sperm were scored in increments of 100. Using Fishers exact test incremental levels of sperm aneuploidy were compared to levels obtained at 5000 to determine at what point differences, if any, could be observed.Table 1# sperm scored% autosome disomy (range)% gonosome disomy (range)% diploidy (range)10000.55 (0.2-1.2)0.33 (0.1-0.6)0.31 (0-1.5)50000.49 (0.18-1.02)0.34 (0.23-0.58)0.33 (0.01-1.6) Open table in a new tab CONCLUSIONS: Our data suggest reliable sperm aneuploidy estimates are obtained after screening 1000 sperm. Additional counts provide little additional information. Assuming a current gold standard of 5000 sperm counted per sample these findings suggest that: (i) reduced test turn around time, less technical time and reduced costs may lead to increased test volumes (ii) the opportunity to analyse all chromosomes for each sample within a total of 5000 sperm using commercially available probes for all 24 chromosomes. OBJECTIVE: Sperm aneuploidy is associated with ICSI failure, pregnancy loss and aneuploid conceptions. However, routine screening has not been readily adopted, in part hindered by the large number of sperm recommended to be scored, (minimum 5000). We have investigated the feasibility of scoring a reduced number whilst maintaining accuracy. DESIGN: Prospective study. MATERIALS AND METHODS: Fluorescence in-situ hybridisation in 11 sperm samples for chromosomes 13, 18, 21 and 22 and 7 sperm samples for chromosomes X, Y and 15. To determine the minimum number of scored sperm needed for reliable estimates of sperm aneuploidy, 5000 sperm were scored in increments of 100. Using Fishers exact test incremental levels of sperm aneuploidy were compared to levels obtained at 5000 to determine at what point differences, if any, could be observed. CONCLUSIONS: Our data suggest reliable sperm aneuploidy estimates are obtained after screening 1000 sperm. Additional counts provide little additional information. Assuming a current gold standard of 5000 sperm counted per sample these findings suggest that: (i) reduced test turn around time, less technical time and reduced costs may lead to increased test volumes (ii) the opportunity to analyse all chromosomes for each sample within a total of 5000 sperm using commercially available probes for all 24 chromosomes.

OBJECTIVE: Fluorescence in situ hybridisation (FISH) for up to 12 chromosomes is the gold standard for Preimplantation Genetic Screening but some women may benefit from comprehensive 24-chromosome screening. We demonstrate that reanalysis of embryos identified as aneuploid by conventional FISH analysis of 8 chromosomes at cleavage stages is comparable using either SNP genotyping or comparative genomic hybridisation (aCGH) microarrays. DESIGN: Prospective and blinded. MATERIALS AND METHODS: Aneuploidy screening by cleavage stage embryo biopsy and FISH analysis (X, Y, 22, 21, 18, 16, 15, 13) was performed for advanced maternal age (41yr). Ten embryos identified as aneuploid were analysed with informed consent. After zona removal, whole embryos were placed in PBS and frozen at -20°C. Lysis and whole genome amplification (WGA) was carried out according to manufacturers instructions (Genomplex, Sigma) and parental genomic DNA and WGA products processed for either (i) genome-wide genotyping of ∼ 350K SNPs (Human CNV370 Quad v3; Illumina) analysed using dedicated Karyomapping software or (ii) array CGH using optimised BAC arrays (Cytochip v2) and BlueFuse software (BlueGnome, Cambridge, UK). RESULTS: Two embryos (20%) diagnosed as aneuploid by single blastomere FISH analysis were normal for all chromosomes by Karyomapping and aCGH. One embryo had 11 separate maternal monosomies and a maternal trisomy (identified by both array methods). Overall, 19 separate aneuploidies were detected by karyomapping and 20 by aCGH with 17 concordant results. CONCLUSIONS: Both SNP genotyping and array CGH approaches gave reliable aneuploidy results, identifying two FISH false-positive results, and were highly concordant when identifying multiple aneusomies in specific embryos. Array CGH is rapid (∼24 hr), reliable and commercially available while SNP genotyping (combined with karyomapping) identifies parental origin of aneusomies (providing clinically significant information and improving accuracy) and contamination.

The karyotypes of 10 lung cancer cell lines of the NCI-H series were analyzed with spectral karyotyping (SKY): 7 non-small lung cancer (NSCLC) lines and 3 small cell lung cancer (SCLC) lines. Modal chromosome number ranged from 42 (NCI-H2171) to 72 (NCI-H2126). All lines showed at least six structural abnormalities, and most had amplifications visible as double minutes or homogeneously staining regions (HSRs). Four reciprocal translocations were found: t(1;17)(p10;p10) in NCI-H82, t(3;6)(q24;q21) and t(12;17)(p10;p10) in NCI-H2009, and a complex t(2;6) in NCI-H1437. NCI-H1770 had a striking HSR containing many copies of the NMYC region. Karyotypes showed a wide range of relationship between numerical and structural change. Two of the lines showed little numerical change but many structural rearrangements (NCI-H209 with mode 46, but 12 rearrangements, and NCI-H2009 with mode 48 but 27 rearrangements). A second group had karyotypes that appeared to have evolved by unbalanced translocation leading to proportionate loss of chromosomes, with or without endoreduplication. In other lines, notably NCI-H2122, the structurally abnormal chromosomes appeared to have been added to a near-diploid karyotype. The karyotypes contribute to a full genomic characterization of these lines, almost all of which have matching normal lymphoblastoid cell lines.

A large proportion of epithelial cancers show the chromosome-instability phenotype, in which they have many chromosome abnormalities. This is thought to be the result of mutations that disrupt chromosome maintenance, but the causative mutations are not known. We identified cell lines known to have mutations that might cause chromosome instability, and examined their karyotypes. Two cell lines, the breast cancer line HCC1937 and the pancreatic cancer line CAPAN-1, that have mutations respectively in BRCA1 and BRCA2, had very abnormal karyotypes, with many structural and numerical chromosome changes and substantial variation between metaphases. However, two colorectal cancer lines with mutations in BUB1, a spindle checkpoint protein involved in chromosome segregation, had rather simple near-tetraploid karyotypes, with minimal loss or gain of chromosomes other than the endoreduplication event, and minimal structural change. Apart from tetraploidy, these karyotypes were typical of colorectal lines considered to be chromosomally stable. Two lines derived from the same tumour, DLD-1 and HCT-15, with bi-allelic mutation of CHK2, had karyotypes that were typical of near-diploid colorectal lines considered chromosomally stable. The karyotypes observed supported the proposed role for BRCA1 and BRCA2 mutations in chromosomal instability, but showed that the tested mutations in BUB1 and CHK2 did not result in karyotypes that would have been predicted if they were sufficient for chromosomal instability.

The aim of the present study was to evaluate the genotoxic effects of hexavalent chromium (Cr(VI)) in vivo in exposed Bulgarian chromium platers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes and exfoliated buccal cells. No significant difference was observed between the exposed workers and the controls with regard to the frequency of cells with chromosome aberrations (CAs) using conventional Giemsa staining and in the frequency of sister chromatid exchanges (SCEs). However, there was a significant increase in the number of cells with micronuclei (MN) in peripheral lymphocytes from chromium exposed workers as compared to the controls. In the buccal cells from these workers, this increase was even more pronounced. Cytosine arabinoside (AraC), an inhibitor of DNA synthesis and repair, was found to significantly increase the levels of MN in vitro in the lymphocytes of both groups. The increase was more expressed in the lymphocytes of chromium exposed workers. Both centromere positive (C+) as well as centromere negative (C−) MN were observed by the fluorescence in situ hybridization (FISH) technique in both of the cell types studied. No difference between C+ and C− MN frequencies was found in the lymphocytes as well as in the buccal cells. Thus, Cr(VI) appears to have both clastogenic as well as aneugenic effects in humans.

Werner syndrome (WS) is a rare autosomal disorder characterized by premature aging exhibiting chromosome instability and predisposition to cancer. Cells derived from WS patients show a variety of constitutionally stable chromosomal aberrations as detected by conventional chromosome banding techniques. We have employed the fluorescence in situ hybridization (FISH) technique using painting probes for 12 different chromosomes to detect stable chromosome exchanges in three WS cell lines and three control cell lines. WS cell lines showed increased frequencies of both stable and unstable chromosome aberrations detected by FISH and Giemsa staining, respectively. One WS lymphoblastoid cell line (KO375) had a 5/12 translocation in all the cells and approximately 60% of the cells had an additional translocated chromosome 12. A high frequency of aneuploid cells was found in all the WS cell lines studied. Though WS cells are known to be chromosomally unstable, unlike other chromosome instability syndromes they are not sensitive to mutagenic agents. We studied the frequencies of X-ray-induced chromosomal aberrations in two WS cell lines and found an approximately 60% increase in the frequencies of fragments and no consistent increase in the frequencies of exchanges.

For the initial estimate of absorbed radiation dose, following the Goiania accident, the frequencies of unstable chromosome exchanges (dicentrics and rings) in blood lymphocytes stimulated in vitro were determined. Using a dose response curve for induction of unstable aberrations following in vitro irradiation with Cs{sup 137} gamma-rays, dose estimates were made. Several of the victims have been followed up till 1993 and the frequencies of dicentrics have been determined (Ramalho et al., 1994). Using the chromosome 'painting' technique, the frequencies of stable chromosomal aberrations (translations) have been determined. The validity of using the frequencies of translocations for retrospective biological dosimetry of past radiation exposures has been examined and the results are presented. (author)

Thermomagnetic properties of fine CoFeO4 particles with average sizes of 43, 51, 66, 195 and 465 Å were investigated between 5 and 800 K in fields up to 140 kOe. The particles with sizes of 43 and 51 Å behave superparamagnetically at room temperature, but the others exhibit mixed superparamagnetic and ferrimagnetic behavior. The concentration of the ferrimagnetic phases and their magnetic properties were evaluated.

Purpose: To investigate the induction and persistence of chromosome aberrations in mouse bone marrow cells after X-ray exposure and to detect differential involvement of individual chromosomes in translocations.Materials and methods: Male and female Swiss mice were exposed to 1 and 3 Gy of X-rays. Chromosome aberrations in bone marrow cells were analysed at 1, 7, 21 and 100 days following irradiation by means of fluorescence in situ hybridization (FISH) with mouse chromosome-specific DNA libraries (#1,13; #2,8; #6,15 and X,Y). In total, 38% of mouse genome was painted and examined.Results: Pooled data indicate that the frequencies of dicentrics and fragments decreased with time and reached to the control level at day 21 after exposure. Following exposure to 1 Gy of X-rays, the frequencies of translocations were not significantly lower between days 7 and 100 than observed at day 1. However, the frequencies of translocations for the 3 Gy group were significantly (about 40%) lower at day 7, then remained constant up to day 100. After exposure to 3 Gy of X-rays, the frequencies of nonreciprocal translocations decreased with time, whereas reciprocal translocations between days 7 and 100 were not significantly less frequent than at day 1. A comparison of observed and expected numbers of translocations involving individual chromosomes showed that at day 1 after irradiation, distribution of X-ray-induced translocations among the painted chromosomes was proportional to their DNA content. However, at day 100 after exposure, the observed translocations involving chromosome 2 were more frequent than expected, those involving chromosomes 8 and 15 were less frequent than expected, while chromosomes 1, 6, X and Y were involved as frequently as expected.Conclusion: Among induced translocations, non-reciprocal translocations are relatively unstable, especially after exposure to high-dose X-rays. While the initial distribution of X-ray-induced translocations is proportional among the painted chromosomes, the persistence of these translocations is heterogeneous.

The magnetization processes of small (350 Å) and nearly spherical particles of -Fe2O3 were studied at room temperature using the initial, hysteresis and remanence magnetization curves. The effective interparticle interactions were estimated for a number of virgin and alternating current demagnetized samples of -Fe2O3-SiO2 mixtures with different magnetic phase concentrations. A non-monotonous dependence of the interactions on the magnetic phase concentration was observed. It was related to the changes in the domain state of some of the particles from the single-domain to the multi-domain.

It has been shown repeatedly that following irradiation of human lymphocytes in the G0 stage, more translocations are induced than dicentrics. To check the role of DNA double-strand breaks (DSB) alone for the induction of symmetrical and asymmetrical chromosome aberrations, the frequencies of induced exchange aberrations by the restriction enzyme AluI were analyzed. The enzyme was introduced into cells using the pellet pipetting technique. Frequencies of induced translocations and dicentrics were determined using a chromosome painting assay with chromosome-specific DNA libraries for chromosomes 1, 4 and X (representing 16.8% of the human genome). The number of translocations detected was approximately 3-fold higher than the number of dicentrics, indicating that the increased frequency of translocations compared with dicentrics found in irradiated human lymphocytes does not result from DNA lesions other than DSB but from differential processing of DSB.

This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The authors present results of broadband femtosecond transient absorption and broadband nanosecond optical limiting studies of C{sub 60} and derivatized C{sub 60}. They have investigated both solutions and solid-state mixed materials (sol-gel glass hosts doped with fullerene guests). They show that derivatized fullerenes provide enhanced solubility and processability, with a ground-state absorption extended into the infrared compared with C{sub 60}. They have extensively studied both the dynamic optical response and the excited-state absorption cross sections of solutions and solids for multiple wavelengths in the visible to near infrared. Wavelength-dependent studies show that the optical limiting response improves monotonically at longer wavelengths, demonstrating broadband limiting in all 6,6 mono-adducts and neat C{sub 60}. The authors report new approaches to processing sol-gel glass/fullerene composites to improve the optical limiting performance of solid-state materials to approach the response of solution limiters.

CoFe2O4 fine powders with particle sizes between 43Å (4.3nm) and 465Å (46.5nm) were produced by a method of `gentle' chemistry. It was found that a surface layer with thickness of about 10Å does not contribute to the magnetization in the fields used. Maximum coercivity of 14kOe and reduced remanence of 0.64 were measured at 5K after magnetization in 50kOe indicating a cubic-type magnetocrystalline anisotropy.

PURPOSE:To estimate the frequencies of radiation- (low and high LET) induced chromosome aberrations in Chinese hamster splenocytes by two-colour fluorescence in situ hybridization using DNA painting probes specific for chromosomes 2, 3, 8, X and Y and to determine (1) the ratio of radiation-induced translocations and dicentrics; (2) the spectrum of exchange aberrations induced by X-rays and neutrons; and (3) the relative involvement of the different chromosomes in the formation of aberrations.MATERIALS AND METHODS:Isolated splenocytes from the Chinese hamster were irradiated in vitro with different doses of 200 kV X-rays (0.75, 1.5, 3.0 Gy) and 1 MeV fast neutrons (0.25, 0.5, 1.0 Gy). Conventional analysis of chromosome aberrations was carried out in Giemsa-stained preparations. Chromosome aberrations involving chromosomes 2, 3, 8, X and Y were analysed in first division metaphases using two-colour FISH.RESULTS:The results indicate that when all types of translocations are taken into account both X-rays and neutrons induce more translocations than dicentrics, the ratio between the two types of exchanges being 1.4 and 1.8 respectively. The ratio of 'apparently simple' reciprocal translocations and reciprocal complete dicentrics was close to 1 for both types of radiation. The RBE of neutrons for induction of exchanges was found to be between 5 and 8. Neutron irradiation was more efficient at inducing insertions. Among the chromosomes studied, an increased involvement was observed for chromosome 8 in dicentrics and translocations than that expected on the basis of its chromosome length. The high content of interstitial telomeric sequences in chromosome 8 may be responsible for the observed sensitivity of this chromosome.CONCLUSIONS:The results obtained in this study indicate that: (1) more translocations are found than dicentrics; (2) heterogeneity exists among Chinese hamster chromosomes for involvement in radiation-induced exchanges; (3) the spectrum and distribution of exchange aberrations are different between X-rays and neutrons; and (4) the relative frequencies of insertions could be used as a 'fingerprint' for exposure to high LET radiation.

It is not yet resolved as to what type of DNA double strand break repair operates in G0 lymphocytes. We have employed Down syndrome (DS) lymphocytes with three copies of chromosome #21 to answer the question whether the presence of Three copies reduces the frequency of exchange aberrations involving this chromosome in comparison to normal cells with two copies of #21. Peripheral blood lymphocytes from three DS patients and two normal individuals were X-irradiated with 1 and 3 Gy. The frequencies of unstable aberrations were found to be higher in DS lymphocytes than normal lymphocytes after 3 Gy of X-rays. FISH studies employing chromosome specific DNA libraries for chromosomes #21 and #22 indicated that the frequencies of exchange aberrations per chromosome are similar in both disomic and trisomic condition. This indicates that the presence of an extra copy of chromosome #21 does not alter the yield, suggesting that homologous recombination does not play a major role in the repair of DNA strand breaks in human G0 lymphocytes.

The radiation accident in focus here occurred in a section of Goiânia (Brazil) where more than a hundred individuals were contaminated with 137Cesium on September 1987. In order to estimate the absorbed radiation doses, initial frequencies of dicentrics and rings were determined in 129 victims [A.T. Ramalho, PhD Thesis, Subsidios a tecnica de dosimetria citogenetica gerados a partir da analise de resultados obtidos com o acidente radiologico de Goiânia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 1992]. We have followed some of these victims cytogenetically over the years seeking for parameters that could be used as basis for retrospective radiation dosimetry. Our data on translocation frequencies obtained by fluorescence in situ hybridization (FISH) could be directly compared to the baseline frequencies of dicentrics available for those same victims. Our results provided valuable information on how precise these estimates are. The frequencies of translocations observed years after the radiation exposure were two to three times lower than the initial dicentrics frequencies, the differences being larger at higher doses (>1 Gy). The accuracy of such dose estimates might be increased by scoring sufficient amount of cells. However, factors such as the persistence of translocation carrying lymphocytes, translocation levels not proportional to chromosome size, and inter-individual variation reduce the precision of these estimates.