A fluorescent in situ hybridization analysis of X chromosome pairing in early human female meiosis

Fluorescent in situ hybridization (FISH) utilizing an X chromosome whole library probe was used directly to assess the rate of aneuploidy and pairing behavior of the X chromosome in human female meiosis. Over 3000 meiotic cells obtained from fetal ovaries (gestational age 13–22 weeks) were scored for meiotic stage and evaluated for pairing abnormalities. No pairing anomalies were observed in 832 pachytenes. Twenty-two percent (88/398) of cells in zygotene were partially paired, but nonhomologous pairings could not be identified. One aneuploid preleptotene oocyte, presumably from mitotic nondisjunction was detected. To our knowledge, this is the first report of the use of FISH utilizing whole chromosome probes to evaluate the pairing behavior of chromosomes in human female meiosis. The application of this technique to study the relationship between nondisjunction and chromosome pairing behavior in maternal-age-related aneuploidy is discussed.     

Increased sensitivity to bleomycin in upper aerodigestive tract mucosa of head and neck squamous cell carcinoma patients

Previous studies on lymphocytes have suggested that patients with head and neck squamous cell carcinoma (HNSCC) have an increased susceptibility for chromosomal damage induced by bleomycin, a known radiomimetic mutagen. However, it has so far not been possible to study whether this genetic instability is present also in the epithelial component of the upper aerodigestive tract mucosa, the tissue from which HNSCC originates. In the present study, we have successfully cultured epithelial cells and fibroblasts isolated from non-neoplastic mucosa samples of 30 HNSCC patients and 56 controls. All cell cultures were exposed to bleomycin and chromosome instability was assessed by analysis of chromosome breakage in cells harvested after 2h of exposure and subsequent removal of bleomycin. Furthermore, the status of the fragile histidine triad gene (FHIT) in chromosome band 3p14.2 was studied by fluorescence in situ hybridization (FISH) in epithelial cells that had been cultured after removal of bleomycin. Chromosomal damage, in the form of chromosomal breaks and gaps, was seen in all cell cultures harvested 2h after exposure to bleomycin. In epithelial cells, the frequency of chromosome breakage was significantly higher among HNSCC patients than among controls [mean breaks per cell (b/c) 1.02 vs. 0.77, p=0.02]. When subdivided according to smoking status, age, and sex, a significantly higher frequency of chromosome breakage was still found in HNSCC patients (smokers, p=0.01, age相似文献   

Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization

Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization.     

Aneuploidy detection in porcine embryos using fluorescence in situ hybridization

In contrast to human embryos, there are very few studies published on the frequency of chromosomal aneuploidy in farm animals. The objectives of this study were to apply a three-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in porcine embryos using chromosome-specific DNA probes, establish baseline frequencies of aneuploidy in embryos and compare the results with our previous findings of aneuploidy in spermatozoa and oocytes. The embryos were collected from superovulated gilts, which were slaughtered 48 h after insemination. FISH was performed using probes specific for the centromeric regions of porcine chromosomes 1, 10 and Y. Altogether 403 blastomeres from 114 porcine embryos were successfully investigated. Diploidy was observed in 101 (88.6%) embryos, triploidy in 2 (1.8%) embryos, mosaicism/mixoploidy in 9 (7.9%) embryos, and trisomy for chromosomes 1 or 10 in 2 (1.8%) embryos. No blastomere showed aneuploidy for chromosome Y. These findings correspond with the frequencies of aneuploidy we have found previously in porcine germ cells.     

Is there a relationship between sperm chromosome abnormalities and sperm morphology?

This review explores the relationship between sperm chromosomal constitution and morphology. With the advent of techniques for obtaining information on the chromosome complements of spermatozoa, this relationship has been studied in fertile men and in men with a high frequency of chromosomal abnormalities. Using human sperm karyotype analysis, no relationship between sperm chromosome abnormalities and morphology was found in fertile men, translocation carriers or post-radiotherapy cancer patients. Fluorescence in situ hybridization (FISH) analysis has not generally revealed a specific association between morphologically abnormal sperm and sperm chromosome abnormalities, but has indicated that teratozoospermia, like other forms of abnormal semen profiles (aesthenozoospermia, oligozoospermia) is associated with a modest increase in the frequency of sperm chromosome abnormalities. However, FISH studies on some infertile men and mouse strains have suggested that certain types of morphologically abnormal spermatozoa, such as macrocephalic multitailed spermatozoa, are associated with a very significantly increased frequency of aneuploidy. Thus, there may be an association between sperm morphology and aneuploidy in infertile men with specific abnormalities.     

Individual variation in the frequency of sperm aneuploidy in humans

To examine interindividual differences in sperm chromosome aneuploidy, repeated semen specimens were obtained from a group of ten healthy men, aged 20-21 at the start of the study, and analyzed by multi-color fluorescence in situ hybridization (FISH) analysis to determine the frequencies of sperm aneuploidy for chromosomes X, Y, 8, 18 and 21 and of diploidy. Semen samples were obtained three times over a five-year period. Statistical analysis examining the stability of sperm aneuploidy over time by type and chromosome identified two men who consistently exhibited elevated frequencies of sperm aneuploidy (stable variants): one with elevated disomy 18 and one with elevated MII diploidy. Differences among frequencies of aneuploidy by chromosome were also seen. Overall, disomy frequencies were lower for chromosome X, 8 and 18 than for chromosomes 21 or Y and for XY aneuploidy. The frequency of chromosome Y disomy did not differ from XY sperm frequency. Also, the frequency of meiosis I (XY) and II (YY + XX) sex chromosome errors did not differ in haploid sperm, but the frequency of MII errors was lower than MI errors in diploid sperm. Frequencies of sperm aneuploidy were similar between the first sampling period and the second, two years later. However, the frequency of some types of aneuploidy (XY, disomy Y, disomy 8, total autosomal disomies, total diploidy, and subcategories of diploidy) increased significantly between the first sampling period and the last, five years later, while others remained unchanged (disomy X, 21 and 18). These findings confirm inter-chromosome differences in the frequencies of disomy and suggest that some apparently healthy men exhibit consistently elevated frequencies of specific sperm aneuplodies. Furthermore, time/age-related changes in sperm aneuploidy may be detected over as short a period as five years in a repeated-measures study.     

Simultaneous detection of structural and numerical chromosome abnormalities in sperm of healthy men by multicolor fluorescence in situ hybridization

Both structural and numerical chromosome aberrations in sperm represent important categories of paternally transmitted genetic damage. Therefore, a new multiprobe fluorescence in situ hybridization (FISH) method, using DNA probes for three targets (centromere and telomere of chromosome 1, centromere of chromosome 8), was developed to detect human sperm carrying three types of chromosomal defects: (1) terminal duplications or deletions in chromosome 1p, (2) aneuploidy involving chromosomes 1 or 8, and (3) diploidy. Baseline frequencies were determined for three healthy donors who had been previously evaluated for sperm cytogenetics by the human-sperm/hamster-oocyte cytogenetic technique (hamster technique). Among ∼120 000 sperm analyzed by the new FISH method, the average baseline frequencies of sperm carrying telomeric duplications and deletions of 1p were 3.2 ± 1.9 and 2.9 ± 3.6 per 10⁴, respectively. Diploid sperm was found in an average frequency of 6.6 ± 4.0 per 10⁴. Average frequencies of disomic sperm for chromosomes 1 or 8 were 1.7 ± 2.2 and 1.9 ± 2.3 per 10⁴, respectively. Inter-individual differences were observed for deletions of 1p but not for the other sperm phenotypes. A good correlation was obtained between the frequencies of sperm with structural chromosome aberrations detected with the new assay and the frequency of sperm carrying premeiotic or meiotic cytogenetic damage detected with the hamster technique. The observed levels of numerical aberrations with the new FISH assay were within range of the baseline frequencies reported by the hamster technique. The newly developed FISH assay has promising applications in genetic, clinical, physiological and toxicological studies. Received: 26 February 1996 / Revised: 6 May 1996     

Aneuploidy in human spermatozoa

We reviewed the frequency and distribution of disomy in spermatozoa obtained by multicolor-FISH analysis on decondensed sperm nuclei in (a) healthy men, (b) fathers of aneuploid offspring of paternal origin and (c) individuals with Klinefelter syndrome and XYY males. In series of healthy men, disomy per autosome is approximately 0.1% but may range from 0.03 (chromosome 8) to 0.47 (chromosome 22). The great majority of authors find that chromosome 21 (0.18%) and the sex chromosomes (0.27%) have significantly elevated frequencies of disomy although these findings are not universal. The total disomy in FISH studies is 2.26% and the estimated aneuploidy (2× disomy) is 4.5%, more than double that seen in sperm karyotypes (1.8%). Increased disomy levels of low orders of magnitude have been reported in spermatozoa of some normal men (stable variants) and in men who have fathered children with Down, Turner and Klinefelter syndromes. These findings suggest that men with a moderately elevated aneuploidy rate may be at a higher risk of fathering paternally derived aneuploid pregnancies. Among lifestyle factors, smoking, alcohol and caffeine have been studied extensively but the compounding effects of the 3 are difficult to separate because they are common lifestyle behaviors. Increases in sex chromosome abnormalities, some autosomal disomies, and in the number of diploid spermatozoa are general features in 47,XXY and 47,XYY males. Aneuploidy of the sex chromosomes is more frequent than aneuploidy of any of the autosomes not only in normal control individuals, but also in patients with sex chromosome abnormalities and fathers of paternally derived Klinefelter, Turner and Down syndromes.     

Exploring bacterial flora in oral squamous cell carcinoma: a microbiological study

The oral cavity contains a unique and diverse micro?ora. While most of these organisms exhibit commensalism, shifts in bacterial community dynamics cause pathological changes within the oral cavity and at distant sites. We assessed the microbial flora using cultured saliva and oral swabs from subjects with oral squamous cell carcinoma (OSCC) and healthy controls. Microbial samples were collected from the carcinoma site, contralateral healthy mucosa, and saliva of the study group and samples were collected from healthy mucosa and saliva of controls. Samples were stored on ice and transported to the laboratory for culture. The median number of colony forming units (CFU)/ml at carcinoma sites was significantly greater than at the contralateral healthy mucosa. Similarly,?in saliva of carcinoma subjects, the median number of CFU/ml was significantly greater than in saliva of control subjects.     

Chromosomal mosaicism in spontaneous abortions: Analysis of 650 cases

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase MFISH and DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy. Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (∼ 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.     

A combination of the micronucleus assay and a FISH technique for evaluation of the genotoxicity of 1,2,4-benzenetriol

The cytokinesis-block micronucleus (CBMN) assay has emerged as one of the preferred methods for assessing chromosome damage. Micronuclei (MN) are small, extranuclear bodies that are formed in mitosis from acentric chromosomal fragments or chromosomes that are not included in each daughter nucleus. Thus, MN contain either chromosomal fragments or whole chromosomes. The CBMN assay, together with a fluorescence in situ hybridization (FISH) technique using specific centromeric probes for chromosomes 7 and 8, were employed in mitogen-stimulated human lymphocytes pretreated with the benzene metabolite, 1,2,4-benzenetriol (BT). Treatment of human lymphocytes resulted in the induction of MN in a dose-dependent manner. The frequency of MN in control lymphocytes was 4.5 per 1000 binucleated (BN) cells and this increased to 9.5, 14, 28 and 40 per 1000 BN cells at 10, 25, 50 and 100 microM BT, respectively. The frequency of aneuploidy 7 and 8 in BN cells also increased at each concentration. Aneuploidy 8 was more frequent than aneuploidy 7, suggesting that chromosome 8 is more sensitive to aneuploidy induction by BT. The frequency of MN containing centromere positive signals for chromosomes 7 and 8 increased with the concentration of BT. The frequency of MN with centromere positive signals was higher for chromosome 8 than for chromosome 7, also suggesting a greater sensitivity of chromosome 8 to this agent. These results suggest that combined application of the CBMN assay with a FISH technique, using chromosome-specific centromeric probes, would allow the detection of aneuploidy in human lymphocytes and identify the mechanistic origin of MN induced by a clastogen or aneugen.     

Meiotic segregation, recombination, and gamete aneuploidy assessed in a t(1;10)(p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm.

Meiotic segregation, recombination, and aneuploidy was assessed for sperm from a t(1;10)(p22.1;q22.3) reciprocal translocation carrier, by use of two multicolor FISH methods. The first method utilized three DNA probes (a telomeric and a centromeric probe on chromosome 1 plus a centromeric probe on chromosome 10) to analyze segregation patterns, in sperm, of the chromosomes involved in the translocation. The aggregate frequency of sperm products from alternate and adjacent I segregation was 90.5%, and the total frequency of normal and chromosomally balanced sperm was 48.1%. The frequencies of sperm products from adjacent II segregation and from 3:1 segregation were 4.9% and 3.9%, respectively. Reciprocal sperm products from adjacent I segregation deviated significantly from the expected 1:1 ratio (P < .0001). Our assay allowed us to evaluate recombination events in the interstitial segments at adjacent II segregation. The frequencies of sperm products resulting from interstitial recombination in chromosome 10 were significantly higher than those resulting from interstitial recombination in chromosome 1 (P < .006). No evidence of an interchromosomal effect on aneuploidy was found by use of a second FISH method that simultaneously utilized four chromosome-specific DNA probes to quantify the frequencies of aneuploid sperm for chromosomes X, Y, 18, and 21. However, a significant higher frequency of diploid sperm was detected in the translocation carrier than was detected in chromosomally normal and healthy controls. This study illustrates the advantages of multicolor FISH for assessment of the reproductive risk associated with translocation carriers and for investigation of the mechanisms of meiotic segregation of chromosomes.     

Aneuploidy frequency in sperm of fertile men

Analysis of sperm aneuploidy in 11 healthy men using two-or three-color FISH permitted to determine the average frequency of disomy for chromosomes 13 and 21 (0.11% and 0.2%, respectively), disomy for chromosome 18 (0.05%) and to reveal gonosomal aneuploidy variants and their frequency. The frequency of XX disomy was 0.04%; XY, 0.17%; YY, 0.06%; and gonosomal nullisomy, 0.29%. We assessed the frequency of meiotic nondisjunction of 13, 21, 18, X, and Y chromosomes and the frequency of XX, XY, and YY diploid spermatozoa. The XY variant prevailed in gonosomal aneuploidy and diploidy and was associated with abnormal chromosomal segregation in meiotic anaphase I. The contribution of human sperm chromosomal imbalance to early embryonic lethality and to some forms of chromosomal abnormalities in the off-spring is discussed.     

Collecting human spermatozoa onto filters for FISH. Application to the study of extreme oligozoospermia

OBJECTIVE: To describe a simple method of fluorescence in situ hybridization (FISH) performed on germ cells collected on filters that allows the study of aneuploid sperm frequency in cases of extreme oligozoospermia. STUDY DESIGN: The aneuploidy rates for chromosomes X, Y and 18 were analyzed in three groups to test the reliability of the filtration-FISH procedure when compared to controls and to evaluate the frequency of aneuploid spermatozoa in five oligospermic men. RESULTS: Filtration of semen samples before FISH does not modify the basal rate of aneuploidy even after various sperm dilutions. In oligospermic men, the frequency of gonosomal aneuploidies is significantly increased. CONCLUSION: The filtration-FISH technique is a powerful method of analyzing chromosomal abnormalities in sperm nuclei in men with extreme oligozoospermia.     

Human sperm chromosome complements in chemotherapy patients and infertile men

Our studies of human sperm karyotypes and interphase sperm analyzed by fluorescence in situ hybridization (FISH) have both yielded estimates of disomy frequencies of approximately 0.1% per chromosome with an overall aneuploidy frequency in human sperm of approximately 5%–6%. However, the distribution of aneuploidy in sperm is not even, as our data from sperm karyotypes and multicolour FISH analyses both demonstrate a significant increase in the frequency of aneuploidy for chromosome 21 and the sex chromosomes. We have studied men at increased risk of sperm chromosomal abnormalities including cancer patients and infertility patients. Testicular cancer patients were studied before and 2–13 years after chemotherapy (CT) with BEP (bleomycin, etoposide, cisplatin). Sperm karyotype analysis on 788 sperm demonstrated no significant difference in the frequency of numerical or structural chromosomal abnormalities post-CT vs pre-CT. Similarly, multicolour FISH analysis for chromosomes 1, 12, XX, YY and XY in 161,097 sperm did not detect any significant differences in the frequencies of disomy before and after treatment. However, recent evidence has suggested a significant increase in the frequency of disomy and diploidy during CT. We have found that infertile men, who would be candidates for intracytoplasmic sperm injection, have an increased frequency of chromosomally abnormal sperm karyotypes. Also, FISH analysis for chromosomes 1, 12, 13, 21, XX, YY and XY in 255,613 sperm demonstrated a significant increase in chromosomes 1, 13, 21, and XY disomy in infertile men compared with control donors. Received: 4 July 1998; in revised form: 7 September 1998 / Accepted: 8 September 1998     

Molecular Cytogenetic Characteristics of Chromosome Imbalance in Spontaneous Human Abortion Cells with Low Proliferative Activity in Vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ovum and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ovum, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously aborted embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1–2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Stable variants of sperm aneuploidy among healthy men show associations between germinal and somatic aneuploidy

Repeated semen specimens from healthy men were analyzed by sperm fluorescence in situ hybridization (FISH), to identify men who consistently produced elevated frequencies of aneuploid sperm and to determine whether men who were identified as stable variants of sperm aneuploidy also exhibited higher frequencies of aneuploidy in their peripheral blood lymphocytes. Seven semen specimens were provided by each of 15 men over a 2-year period and were evaluated by the X-Y-8 multicolor sperm FISH method (i.e., approximately 1,050,000 sperm were analyzed from 105 specimens). Three men were identified as stable aneuploidy variants producing significantly higher frequencies of XY, disomy X, disomy Y, disomy 8, and/or diploid sperm over time. In addition, one man and three men were identified as sperm-morphology and sperm-motility variants, respectively. Strong correlations were found between the frequencies of sperm with autosomal and sex-chromosome aneuploidies and between the two types of meiosis II diploidy; but not between sperm aneuploidy and semen quality. A significant association was found between the frequencies of sex-chromosome aneuploidies in sperm and lymphocytes in a subset of 10 men (r2=0.67, P=.004), especially between XY sperm and sex-chromosome aneuploidy in lymphocytes (r2=0.70, P=.003). These findings suggest that certain apparently healthy men can produce significantly higher frequencies of both aneuploid sperm and lymphocytes. Serious long-term somatic and reproductive health consequences may include increased risks of aneuploidy-related somatic diseases and of having children with paternally transmitted aneuploidies, such as Klinefelter, Turner, triple-X, and XYY syndromes.     

Molecular cytogenetic characteristics of chromosome imbalance in cells of spontaneous human abortion fetuses with low proliferative activity in vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ova and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ova, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously perished embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1-2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Analysis of the frequency of spontaneous aneuploidy in human somatic cells using interphase cytogenetic technology

The frequency of spontaneous aneuploidy of the four autosomes and sex chromosomes in the interphase nuclei of cultivated and uncultured lymphocytes from clinically healthy men was examined by use of two-color fluorescent in situ hybridization (FISH). It was shown that in uncultured cells from the individuals examined autosomal nullosomies were practically not detected (the frequency 0 to 0.01%). At the same time, the frequency of such cells with either Y, or X nullosomy was at least an order of magnitude higher (about 0.15%). This frequency was comparable with the level of Y- or X-disomic cells, and also with autosomal monosomies, precluding from considering the X-nullosomic cells as hybridization artifacts. During lymphocyte cultivation, a statistically significant increase in the total frequency of Y- or X-nullosomic cells was observed already after the first cell division cycle. Thus, interphase FISH analysis is a sufficiently sensitive method enabling detection of higher, compared to the autosomes, loss of sex chromosomes in the process of cell division, a phenomenon observed during replicative cell aging, as well as during natural aging of the organism. Male cells with the de novo lost singular X chromosome, probably, switch to apoptosis and do not survive during further life of a cell population. The frequency of total aneuploidy in human somatic cells with the correction for the resolution capacity of the interphase FISH analysis constituted 5.62 and 6.90% for uncultured and cultivated cells, respectively. This aneuploidy level is close to that in spermatozoa. The data obtained can serve as the basis for the examination of the aneugenic (aneuploidy-inducing) genotoxic effects and for the analysis of interindividual genetic instability.     

An Evaluation of the Frequency of Spontaneous Aneuploidy in Human Somatic Cells Using the Technology of Interphase Cytogenetics

The frequency of spontaneous aneuploidy of the four autosomes and both sex chromosomes in the interphase nuclei of cultured and noncultured lymphocytes from clinically healthy men was examined by use of two-color fluorescent in situ hybridization (FISH). It was shown that in noncultured cells from the individuals examined autosomal nullisomies were practically not detected (the frequency 0 to 0.01%). At the same time, the frequency of such cells with either Y, or X nullisomy was at least an order of magnitude higher (about 0.15%). This frequency was comparable with the level of Y- or X-disomic cells, and also with autosomal monosomies, precluding from consideration of X-nullisomic cells as hybridization artifact. During lymphocyte cultivation, a statistically significant increase in the total frequency of Y- or X-nullisomic cells was observed already after the first cell division cycle. Thus, interphase FISH analysis is a sufficiently sensitive method enabling detection of higher, compared to the autosomes, loss of sex chromosomes in the process of cell division, a phenomenon observed during replicative cell aging, as well as during natural aging of the organism. Male cells with the de novo lost single X chromosome, probably, switch to apoptosis and do not survive during further life of a cell population. The frequency of total aneuploidy in human somatic cells with the correction for the resolution capacity of the interphase FISH analysis was estimated to be 5.62 and 6.90% for noncultured and cultured cells, respectively. This aneuploidy level is close to that in spermatozoa. The data obtained can serve as the basis for the examination of the aneugenic (aneuploidy-inducing) genotoxic effects and for the analysis of interindividual genetic instability.