Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.     

Pathologic characterization of hypotensive C57BL/6J-agt: angiotensinogen-deficient C57BL/6J mice

Most studies on preconception diagnosis published so far have used polymerase chain reaction (PCR) analysis to identify single gene defects. Although fluorescent DNA probes have been used to obtain a partial cytogenetic diagnosis of aneuploidies in first polar bodies without defined chromosome structures, the analysis of structural chromosome anomalies in the interphase nucleus is not adequate. We describe a procedure to obtain first polar body chromosome complements from hamster and human oocytes. In 63.6% (105 of 165) of hamster first polar bodies the chromosome complement showed a defined chromosome morphology and in 94.1% (16 of 17) of human oocytes fixed after follicular puncture it was possible to obtain high quality, well spread chromosome complements. First polar body chromosomes are fuzzy and shorter than oocyte chromosomes, but fluorescent in-situ hybridization results obtained in human first polar bodies clearly show that it is possible to detect whole chromosomes, centromeres and unique sequences, including the terminal regions of small chromosomes. This suggests that in fresh oocytes, DNA loss resulting from apoptotic chromosome fragmentation has not yet occurred. Using the procedure described, first polar bodies could be used to analyse the meiotic segregation of maternal structural abnormalities and to detect numerical chromosome anomalies in humans.     

Cytogenetic analysis of spermatozoa in the father of a child with a de-novo reciprocal translocation t(7;9) (q22;p23)

Analysis of sperm chromosomes was carried out in the father of a child with a de-novo reciprocal translocation t(7;9) (q22;p23) by G-banding and chromosome painting. Sperm metaphases were obtained using the zona-free hamster oocyte-human sperm fusion technique. A total of 138 complements were sequentially analysed by G-banding and fluorescence in-situ hybridization (FISH). The frequency of spermatozoa with structural chromosome abnormalities (5.1%) and the estimated conservative aneuploidy (1.4%) were within the range obtained in our control donors (6.9 and 4%). The sex ratio (45.3% X versus 54.7% Y) was not significantly different from the theoretical 1:1. A total of 309 sperm complements was analysed by FISH, 138 sequentially analysed by G-banding-FISH and another 171 analysed by FISH only. The frequencies of structural chromosome abnormalities for chromosomes 7 and 9 (0.6 and 0% respectively) were not significantly different from those obtained in our control donors (0.6 and 0.8%). No spermatozoa with the t(7;9) (q22;p23) were observed, showing no evidence for a germ-cell mosaicism. A statistically significant, positive association between sperm breakpoints and fragile sites (P = 0.0225) was observed. However, the coincidence between fragile sites and sperm breaks (80%) was not significantly different from that obtained in our control donors (79.2%). These results suggest that in this case the risk of structural chromosome abnormalities in further offspring is not increased, although an association between fragile sites and sperm chromosome breaks in the father does exist.     

Evaluation and long-term treatment of aberrant behavior displayed by young children with disabilities

We have studied the distribution and methylation of CpG islands on human chromosomes, using the novel technique of self-primed in situ labeling (SPRINS). The SPRINS technique is a hybrid of the two techniques primed in situ labeling (PRINS) and nick translation in situ. SPRINS detects chromosomal DNA breaks, as in nick translation in situ, and not annealed primers, as is the case in PRINS. We analyzed in situ-generated DNA breaks induced by the restriction enzymes HpaII and MspI. These restriction enzymes enable the detection of chromosomal CpG islands. Both HpaII- and MspI-SPRINS produce a banding pattern resembling R-banding, indicating a higher level of CpG islands in R-positive bands than in R-negative bands. Our SPRINS banding observations also indicate differences in sequence copy number in the satellites of homologous acrocentric chromosomes. Furthermore, a comparison of homologous HpaII-SPRINS-banded X chromosomes of females from lymphocyte cultures grown without methotrexate or bromodeoxyuridine revealed methylation difference between them. The same comparison of homologous X chromosomes from the cell line GM01202D, which has four X chromosomes, one active and three inactive, revealed the active X chromosome to be hypermethylated.     

Prognosis of operable squamous cell carcinoma of the esophagus. Relationship with clinicopathologic features and DNA ploidy

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines

To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.     

Assessment of sex chromosome ratio and aneuploidy rate in motile spermatozoa selected by three different methods

Using three-colour fluorescence in-situ hybridization, sex chromosome ratios and frequencies of diploidy and disomy for chromosomes X, Y and 18 were compared in spermatozoa of good and poor motility after separation by swim-up, glass-wool and two-layer discontinuous Percoll methods. Semen samples were collected from seven normal males aged 26-31 years. A minimum of 6000 sperm nuclei per sample were evaluated for each chromosome for a total of 308,432 sperm nuclei. Hybridization efficiency was 99.8%. A slight change in the ratio of X- to Y-bearing spermatozoa was noted after Percoll separation (from 49.3:49.5 to 50.0:48.9; P = 0.036 and P = 0.046), but not after separation by the other two methods. We did not observe significant differences in the disomy rates for sex chromosomes or chromosome 18 or in the diploidy rate between spermatozoa with good and poor motility after separation by any of the three methods. Our data indicate that separation of motile spermatozoa does not alter the ratio of X- to Y-bearing spermatozoa to a degree that represents sex chromosome selection.     

Anti-seminal plasma antibodies associated with infertility: II. Comparative investigation of human antibody binding to normo-, astheno-, and azoospermic seminal plasma antigens

We present a new method to detect epididymal sperm aneuploidy (ESA) in mice using simultaneous fluorescence in situ hybridization (FISH) with DNA probes specific for mouse chromosomes X, Y and 8. The method was applied to Robertsonian (Rb) translocation (8.14) heterozygotes and homozygotes as well as the chromosomally normal B6C3F1. The sex ratios of sperm did not differ from the expected 1:1 and the hybridization efficiencies were approximately 99.7% for over 60 000 sperm analyzed. Mice heterozygous for Rb (8.14) produced about tenfold higher rates of sperm with chromosome 8 hyperhaploidy than did Rb (8.14) homozygotes or chromosomally normal mice, while frequencies of sperm with hyperhaploidies for chromosomes X and Y were unaffected in all three lines of mice. Hyperhaploid frequencies obtained with the ESA method were consistent with those of the previous testicular FISH method and were validated by published data obtained by conventional cytogenetic analyses (meiotic metaphase II and first cleavage). Thus, the mouse three-chromosome ESA assay together with the previously developed aneuploidy assay for human sperm constitute a promising pair of interspecific biomarkers for comparative studies of the genetic and physiologic mechanisms of the induction and persistence of aneuploidy in male germ cells.     

Aneuploidy detection in human sperm nuclei using PRINS technique

Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a < 2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28-0.34%. There were no significant interindividual or interchromosomal differences in disomy rates.     

Segregational fidelity of chromosomes in human thyroid tumour cells

Using fluorescence in situ hybridisation (FISH) we have analysed the segregational fidelity of all the human chromosomes during mitotic cell division. The losses and gains of chromosomes were analysed in human polyploid cell lines derived from a well-differentiated papillary thyroid cancer. These thyroid cells can be cultured for more than 300 population doublings. For the purpose of our study the polyploid nature of the cells may act as a protective buffer against the cell-lethal effects of the loss of individual chromosomes. To evaluate the role of the p53 gene product in maintaining the fidelity of chromosome segregation we compared the frequencies of chromosome loss and gain in cultures with wild-type p53 activity (K1E7neo3) and cultures transfected with plasmids expressing a mutant p53 product (K1E7scx6). Cultures were analysed for the presence of both structurally normal and rearranged chromosomes at both early and late passages. Cell cultures with defective p53 activity showed progressive chromosome loss from a median chromosome number of 87-97 to 75-86. Cell growth in cultures with wild-type p53 activity showed the loss of chromosomes 6, 7, and 8 and the gain of 17 and 20. Cultures expressing mutant p53 activity showed the loss of chromosomes 2, 5, 14 and 17 and the gain of 4 and 22. The combination of defective p53 and growth resulted in further destabilisation with the additional losses of chromosomes 3, 11, 15, 16 and 21. Chromosomes 1, 9, 10, 12, 13, 18, 19, X and Y segregated stably under all the culture conditions as did the structurally rearranged marker chromosomes. The study has demonstrated variation in the fidelity of mitotic chromosome segregation and the influence of p53 gene activity upon the segregation of individual human chromosomes.     

Hamster origin of metaphases with multiple chromosome rearrangements in first cleavage human-hamster embryos

Laboratories using the human sperm-hamster egg fertilization system to analyse sperm chromosomes obtain, sporadically, metaphases with multiple aberrations. Due to the high number of aberrations, these metaphases cannot be fully karyotyped. In some of them, one or several human chromosomes can be identified, guaranteeing the human origin of the whole metaphase. However, in others, none of the chromosomes can be recognized as human. This latter type of grossly rearranged metaphases is characterized by complex chromatid exchanges, multifragmented chromosomes and pulverized chromosome material. Using fluorescent in-situ hybridization techniques (FISH) with either human or hamster genomic DNA probes, we examined the origin of this second type of metaphase with multiple chromatid exchanges and fragmented chromosomes. Our study demonstrates that all of them hybridize with hamster genomic DNA probes and not with human DNA, proving their hamster origin. Since some of these metaphases seem to be diploid, we suggest that they may arise from hamster eggs that have failed to complete meiosis and have not extruded the second polar body.     

Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double-blind study

In the present study we have assessed the cytogenetic abnormalities of unfertilized oocytes from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programmes during a one year period (July 1995 to July 1996) with the cytogenetic analysis being carried out in a double-blind manner. A total of 88 unfertilized ICSI and 85 unfertilized IVF oocytes were used for the study and of these 51 and 62 oocytes, in each respective group, were suitable for analysis. The haploidy, diploidy and aneuploidy rates between ICSI (62.7, 7.8 and 5.9%) and IVF (61.3, 9.7 and 14.5%) groups were similar. A significant inter-patient variation in the incidence of hypohaploidy was observed within the IVF group. Chromosomal fragmentation or breakage was observed at a similar rate in both groups of unfertilized oocytes (23.5 and 14.5% for ICSI and IVF respectively). A significantly higher proportion of ICSI oocytes contained sperm nuclei (27/51, 52.9%) than did IVF oocytes (20/62, 32.3%, P < 0.01). The distribution and state of sperm head chromatin in relation to oocyte chromosomal complement was studied in both groups. ICSI oocytes contained decondensed or swollen sperm nuclei in association with haploid oocyte chromosomes (12/27, 44.4%) or condensed sperm heads in oocytes showing no chromosomal complements (7/27, 25.9%). In IVF oocytes sperm heads were either arrested in the condensed state (5/20, 25%), metaphase stage (3/20, 15%) or had undergone premature chromosome condensation (PCC; 6/20, 30%) in association with haploid oocyte chromosomes. The incidence of PCC was similar in the two groups. A marked variation in the incidence of total chromosomal abnormality was observed between patients within both ICSI (0-75%) and IVF (0-71%) groups indicating a possible similarity in oocyte quality between the majority of male factor and tubal infertility patients. The type of sperm used in the two fertilization procedures showed an increased incidence of chromosomal breakage with ICSI-MESA (microepididymal sperm aspiration) spermatozoa (4/6, 67%) compared to the ICSI-ejaculated (6/35, 17.1%; P < 0.05), ICSI-testicular biopsy (2/10, 20%) and IVF-normospermic (9/62, 14.5%; P < 0.01) spermatozoa. Chromosomal fragmentation may be associated with the degree of difficulty experienced at sperm injection, especially with sperm retrieved from the reproductive tract. Thus chromosomal fragmentation in ICSI may need further investigation using a larger sample size in order to assess the possible causative factors.     

Interspecies comparative genome hybridization and interspecies representational difference analysis reveal gross DNA differences between humans and great apes

Comparative chromosome G-/R-banding, comparative gene mapping and chromosome painting techniques have demonstrated that only few chromosomal rearrangements occurred during great ape and human evolution. Interspecies comparative genome hybridization (CGH), used here in this study, between human, gorilla and pygmy chimpanzee revealed species-specific regions in all three species. In contrast to the human, a far more complex distribution of species-specific blocks was detected with CGH in gorilla and pygmy chimpanzee. Most of these blocks coincide with already described heterochromatic regions on gorilla and chimpanzee chromosomes. Representational difference analysis (RDA) was used to subtract the complex genome of gorilla against human in order to enrich gorilla-specific DNA sequences. Gorilla-specific clones isolated with this technique revealed a 32-bp repeat unit. These clones were mapped by fluorescence in situ hybridization (FISH) to the telomeric regions of gorilla chromosomes that had been shown by interspecies CGH to contain species-specific sequences.     

Chromosome spatial order in human cells: evidence for early origin and faithful propagation

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked "haploid sets" containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes.     

Cytogenetic effects of cryopreservation on human sperm: assessment using an improved method for analyzing human sperm chromosomes

OBJECTIVE: To evaluate any cytogenetic effects of cryopreservation on human sperm by comparing the frequencies of sperm chromosome anomalies and sex ratios before and after freezing. METHODS: Using in vitro fertilization of zona-free hamster oocytes, analysis of sperm chromosomes was first performed on portions of fresh human semen samples. The residual semen was then analyzed for sperm chromosomes after cryopreservation for several weeks. Sperm donors were 5 healthy men aged 26-38 years. RESULTS: A total of 166 sperm karyotypes were analyzed, 94 before freezing and 72 after freezing. The results indicated no significant differences between fresh and frozen sperm in either frequencies of aneuploidy (fresh: 0%, frozen: 2.8%) or structural anomalies (fresh: 7.5%, frozen: 9.7%). The sex ratios did not differ from the expected 1:1 ratio under either condition. CONCLUSIONS: The results of these studies indicate that cryopreservation does not exert any cytogenetic mutagenicity on human spermatozoa or alter X/Y ratio of human sperm.     

Chromosomal mapping of two novel human FGF genes, FGF11 and FGF12

The fibroblast growth factor (FGF) family comprises to date 12 members, which are involved in various physiological processes throughout embryogenesis and adult life. Two novel members of the family have been identified recently (FGF11 and FGF12). Using in situ hybridization on metaphasic chromosomes, we have been able to assign FGF11 to band p12-p13 of human chromosome 17 and FGF12 to band q28 of human chromosome 3.     

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.     

Functional heterogeneity of pituitary gonadotropes in response to a variety of neuromodulators

We have investigated the yields of radiation-induced translocations in several human tumor cell lines and in normal diploid human fibroblasts by fluorescence in situ hybridization. The translocation yields were determined with respect to chromosome no. 1 in all cell lines investigated, and moreover in chromosomes nos. 2, 4 and 9 in fibroblasts and one tumor cell line. The chromosomes were "painted' with fluorescent whole chromosome-hybridization probes. The clonogenic survival of the studied cell lines was determined by standard colony-formation assay. We observed a higher frequency of reciprocal translocations in the radiosensitive cells MCF-7 and MDA-MB-436 as compared with the radioresistant cells CaSki and normal skin fibroblasts. Thus, the results suggest a possibility to predict the intrinsic tumor radiosensitivity on the basis of reciprocal translocation yield determined in cells irradiated in vitro. The correlation was observed in spite of the trisomy no. 1 which occurred in all three investigated tumor cell lines. On the other hand, the results obtained with different chromosomes in MCF-7 cells suggest that only chromosomes with relatively low "spontaneous' translocation yields are suitable for this kind of analysis.     

Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH)

The use of chromosome specific DNA probes labelled with fluorochromes and especially the combination of several probes has been used to indirectly study the chromosome constitution of decondensed sperm nuclei by fluorescence in-situ hybridization (FISH), and has allowed to include this test in the protocol of study of infertile males. Still, if the test is to be valid, several strict conditions must be met, and some specific characteristics have to be taken into account. This becomes evident when comparing earlier results with more recent ones. The basic technical factors to be taken into account are the methods of chromatin decondensation, the number of spermatozoa and of individuals to study, the use of internal controls, the scoring criteria, the specificity of the probes and the possible existence of polymorphisms that may interfere with the detection of fluorescent signals. In the last 7 or 8 years, a large number of papers has been published, describing the incidence of aneuploidies in controls, in individuals in whom a tendency to non-disjunction was suspected and in infertile males. Studies in controls have shown a considerable intra- and inter-individual variability in the frequency of aneuploidies, the tendency of some chromosomes to undergo non-disjunction (chromosome 21 and the sex chromosomes) and the importance of alpha-satellite polymorphisms when using centromere probes. In the control population, the frequency of aneuploidy per haploid set has been estimated at approximately 6%. The incidence of aneuploidies in sperm nuclei for some of the chromosomes more frequently involved in trisomies is considerably higher than the incidence of these trisomies established through epidemiological data using the global incidence of chromosome abnormalities during the peri-implantation stage. In infertile males and in males with sex-chromosome abnormalities (usually with very low numbers of spermatozoa) the results show an increased incidence of sex chromosome aneuploidies and diploid (multi-aneuploid?) sperm nuclei. The results could be related to the higher incidence of chromosome abnormalities (especially sex-chromosome aneuploidies) observed in children conceived by intracytoplasmic sperm injection (ICSI).