Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection.

Recent evidence suggests that infertile males donating semen for intracytoplasmic sperm injection (ICSI) may be at an increased risk of transmitting numerical (predominantly sex chromosome) abnormalities to their offspring. The present study was designed to determine aneuploidy in spermatozoa from oligoasthenoteratozoospermic (OAT) patients undergoing ICSI. Aneuploidy frequencies of 12 autosomes and the sex chromosomes were determined by fluorescence in-situ hybridization (FISH) on spermatozoa from fresh ejaculate of nine severe OAT patients and four proven fertile donors. FISH, using directly labelled (fluorochrome-dUTP) satellite or contig DNA probes specific for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X, and Y, was performed on decondensed spermatozoa. Per chromosome disomy frequencies for autosomes and sex chomosomes in OAT males were 0-5. 4%. In contrast, the disomy frequencies in controls were 0.05-0.2%. The frequency of diploid spermatozoa in OAT patients was 0.4-9.6%; controls showed a mean of 0.04%. Using recently developed formulae, the total aneuploidy in our OAT patient population was estimated to be 33-74%. In contrast, estimates of mean total aneuploidy in the spermatozoa of controls ranged from 4.1 to 7.7%, depending upon method of calculation. Six series of ICSI were performed on five of the OAT patients. Four resulted in no establishment of pregnancy; the others failed to establish ongoing pregnancies. Our cytogenetic data show significantly elevated frequencies of diploidy, autosomal disomy and nullisomy, sex chromosome aneuploidy, and total aneuploidy in OAT patients, which may contribute to the patients' infertility.     

Reliability of estimates of diploid human spermatozoa using multicolour fluorescence in-situ hybridization [published erratum appears in Hum Reprod 1997 May;12(5):1118]

Multicolour fluorescence in-situ hybridization (FISH) analysis permits distinction between disomic and diploid spermatozoa. Thus estimates of the frequency of diploid spermatozoa can be obtained for human semen samples. The issue of the accuracy and reliability of these diploidy estimates has been addressed by analysing diploidy frequencies in 10 men using the same sperm sample to estimate diploidy twice-once during two-colour FISH analysis of disomy for chromosomes 1 and 12 and a second independent analysis of three-colour FISH for disomy estimates for chromosomes X and Y (with chromosome 1 used as the autosomal control). A minimum of 10,000 spermatozoa per hybridization per male was counted for a total of over 200,000 spermatozoa analysed. The mean frequency of diploid spermatozoa was 0.13% for the autosomal study and 0.14% for the sex chromosomal study, which were not significantly different. One donor had extremely divergent values of diploidy in the two studies. Analysis of values in the other nine donors demonstrated no significant difference in the two diploidy estimates. These results indicate that the FISH technique is an accurate and reliable method for determining diploid frequencies in human spermatozoa.      

Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in-situ hybridization

There is concern that intracytoplasmic sperm injection (ICSI) may lead to offspring with a high frequency of chromosomal abnormalities. Accordingly, we studied spermatozoa sampled from eight infertile men with oligoasthenoteratozoospermia (OAT) by multi-colour fluorescence in-situ hybridization (FISH), using DNA probes for chromosomes 13, 18, 21, X and Y. Results were compared with those of spermatozoa sampled from 10 healthy men with normal semen profiles. Analysis of the diploidy values was repeated twice in each of the 18 men. There was no significant difference in the two diploidy estimates; thus the FISH technique appeared to be accurate and reliable for determining aneuploidy in human spermatozoa. We found the average frequencies of disomy for chromosomes 13, 18, 21 and X or Y to be 0.13, 0.12, 0.24 and 0.59% respectively for the OAT group and 0.09, 0.13, 0.19 and 0.38% respectively for the control group. The diploidy rate was 0.29% in the OAT group, and 0.16% in the control group. Thus, the OAT group showed a significantly higher frequency of disomy for chromosomes 13 (P < 0.001), 21 (P < 0.05), sex (P < 0.001), and diploidy (P < 0.005) than the control group. This finding suggests there may be some risk of aneuploidy in the offspring conceived by the ICSI technique.     

Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound

Spermatozoa-zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0. 14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0. 32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.     

Oligonucleotide probes for the analysis of specific repetitive DNA sequences by fluorescence in situ hybridization.

Five non-isotopically labeled oligonucleotides have been designed and synthesized to facilitate the analysis of specific human and murine repetitive DNA sequences by fluorescence in situ hybridization (FISH). Three of the oligonucleotides contain alphoid DNA sequences; one hybridizes to the centromeres of all human and mouse chromosomes except the Y, while the other two are specific for human chromosomes 2 and 12, respectively. The fourth oligomer, containing sequences from the spacer region of a human 5S rDNA repeat, was used to confirm the map location of a approximately 100 copy 5S rDNA tandem repeat locus. The fifth oligomer, specific to the Alu family of repeats, generates a sharp R-banding pattern on human metaphase chromosomes, suitable for FISH karyotyping. These probes permit highly specific chromosome enumeration and aneuploidy detection with hybridization times as short as 30 minutes.     

Acrocentric cryptic translocation associated with nondisjunction of chromosome 21

Down syndrome is the most frequent autosome aneuploidy in live newborns. It was recently proposed that pericentromeric cryptic translocations might be a cause of chromosome nondisjunction. We describe here a phenotypically normal subject with a cryptic translocation involving the short arms of chromosomes 13 or 21 and 22, who had a son with Down syndrome. Fluorescent in situ hybridization (FISH) on paternal metaphase chromosomes showed a chromosome 22 centromere positive for both 13/21 and 14/22 centromeric probes. The same probes hybridized on different and contiguous sites of chromatin fibers, eliminating cross-hybridization artifacts. This confirmed the presence of a cryptic translocation generating a dicentric chromosome 22: fib ish dic(21;22)(21 pter --> 21q10::22q10 --> 22 qter)(D13/21Z1+;D14/22Z1+). Microsatellite STR segregation analysis confirmed the paternal origin of the additional chromosome 21 in the Down syndrome patient. To determine whether the father showed a higher-than-normal frequency of chromosome 21 nondisjunction, FISH analysis of spermatozoa was performed using a sequence specific probe (21q22.13-q22.2). The frequency of disomy 21 spermatozoa was twofold higher in the cryptic translocation carrier as compared to normal subjects (P < 0.014), suggesting that the rearrangement favored the nondisjunction of chromosome 21. This is the first report associating a pericentromeric cryptic translocation of acrocentric chromosomes with the generation of aneuploidy, supporting the hypothesis that this type of rearrangement may contribute to abnormal chromosomal segregation.     

Disomy rates for chromosomes 14 and 21 studied by fluorescent in-situ hybridization in spermatozoa from three men over 60 years of age

In order to further investigate the paternal-age effect on meiotic non- disjunction rates for the chromosomes 14 and 21, we examined spermatozoa from three men aged > 60, using multicolour fluorescent in- situ hybridization (FISH). More than 10,000 sperm cells were analysed for each of the three subjects (A, B and C), by simultaneously hybridizing two YAC probes specific for chromosomes 14 and 21 respectively using two-colour FISH. The results show that the disomy 21 rates observed in the spermatozoa of two out of the three men aged > 60 years were higher (1.02 and 1.17% respectively) than the rates observed in eight control adults aged < 30 years (mean frequency 0.48%) analysed under similar conditions. These results suggest that there may be a small effect of age on male non-disjunction rates for chromosome 21. However, before any firm conclusions could be drawn, a much bigger sample of older men would have to be compared with a paired control population using the same FISH experimental approach.      

Chromosome aneuploidy in the spermatozoa of two men with globozoospermia

The objective of this study was to determine the aneuploidy level in spermatozoa in two men with globozoospermia. Sperm nuclei were analysed by fluorescence in-situ hybridization (FISH) in two infertile males with globozoospermia. Dual FISH for chromosomes 7 and 9, 13 and 21, and triple FISH for chromosomes X, Y, and 18 was performed. The main outcome measured was meiotic segregation differences between both globozoospermic men and controls. A statistically significant difference in disomies 13 and 21 was found between patients 1 and 2. The diploidy rate of spermatozoa of patient 1 (0.876%) was significantly increased compared with that of patient 2 (0.304%) and control men (0.293%). In conclusion there seems to be a slightly increased frequency of aneuploidy in round-headed spermatozoa. However, it is unlikely that these aneuploid spermatozoa would be used in assisted reproduction techniques.     

Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in spermatozoa from 10 normospermic men using fluorescence in-situ hybridization

Fluorescence in-situ hybridization (FISH) is a fast and efficient method of estimating aneuploidy in human spermatozoa. In this study, we have estimated baseline disomy frequencies in spermatozoa from a group of 10 normospermic men, using stringent scoring criteria. A triple- probe FISH procedure was used for chromosomes 3, X and Y, while a double-probe FISH method was used for chromosomes 7 and 16. A total of 101273 spermatozoa were scored for chromosomes 3, X and Y, resulting in 97.83% haploidy (3X or 3Y), 0.39% disomy (33X, 33Y, 3XX, 3YY or 3XY) and 0.35% diploidy (33XX, 33YY or 33XY). A total of 100760 spermatozoa were scored for chromosomes 7 and 16, giving 98.9% haploidy (716), 0.11% disomy (7716 or 71616) and 0.27% diploidy (771616). Disomy frequencies for individual chromosomes differed (chromosome 3, 0.20%; chromosome 7, 0.05%, chromosome 16, 0.06%; X + Y, 0.19%). The frequency of disomy 3 was significantly higher than disomy 7 (P = 0.019) and disomy 16 (P = 0.022), while the frequency of sex chromosome disomy was significantly higher than disomy 7 (P = 0.0058) and disomy 16 (P = 0.0067), but not disomy 3 (P = 0.73). The disomy and diploidy (0.27- 0.35%) estimates obtained for this normospermic population were generally low and were similar to other recent reports.      

Sensitive detection of chromosome copy number aberrations in prostate cancer by fluorescence in situ hybridization.

The pattern of chromosomal aberrations and their significance in prostate cancer are poorly understood. We studied 23 prostate cancer and 10 benign prostatic hyperplasia (BPH) specimens by fluorescence in situ hybridization (FISH) using pericentromeric repeat-specific probes for 10 different chromosomes. The aims of the study were: 1) to compare the sensitivity of FISH and DNA flow cytometry in aneuploidy detection, 2) to determine which chromosome copy number changes are most common, and 3) which probe combinations would be most effective in aneuploidy diagnosis. Disaggregated tumor cells from formalin-fixed, paraffin-embedded tissues were pretreated with our newly developed method based on hot glycerol solution to improve probe penetration. All BPH specimens were diploid by DNA flow cytometry and showed no numerical chromosome aberrations by FISH. In prostate cancer, flow cytometry showed abnormal DNA content in 35% of cases, whereas 74% were abnormal by FISH. Aberrant copy number of chromosomes 8 (48% of cases), X (43% of cases), and 7 (39% of cases) were most common. Ninety-four percent of all aneuploid cases would have been detected with these three probes alone. Simple chromosome losses were uncommon but in DNA tetraploid tumors relative losses (trisomy or disomy) of several chromosomes were often found, suggesting progression of prostate cancer through tetraploidization followed by losses of selected chromosomes. In conclusion, our results indicate that FISH using three selected chromosome-specific probes is two to three times more sensitive than flow cytometric DNA content analysis in aneuploidy detection.     

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

The use of chromosome specific DNA probes labelled with fluorochromesand especially the combination of several probes has been usedto indirectly study the chromosome constitution in condensedsperm nuclei by fluorescence in-situ hybridization (FISH), andhas allowed to include this test in the protocol of study ofinfertile males. Still, if the test is to be valid, severalstrict conditions must be met, and some specific characteristicshave to be taken into account. This becomes evident when comparingearlier results with more recent ones. The basic technical factorsto be taken into account are the methods of chromatin decondensation,the number of spermatozoa and of individuals to study, the useof internal controls, the scoring criteria, the specificityof the probes and the possible existence of polymorphisms thatmay interfere with the detection of flourescent signals. Inthe last 7 or 8 years, a large number of papers has been published,describing the incidence of aneuploidies in controls, in individualsin whom a tendency to non-disjunction was suspected and in infertilemales. 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-satellite polymorphisms when using centromere probes. In thecontrol population, the frequency of aneuploidy per haploidset has been estimated at 6%. The incidence of aneuploidiesin sperm nuclei for some of the chromosomes more frequentlyinvolved in trisomies is considerably higher than the incidenceof these trisomies established through epidemiological datausing the global incidence of chromosome abnormalities duringthe peri-implantation stage. In infertile males and in maleswith sex-chromosome abnormalities (usually with very low numbersof spermatozoa) the results show an increased incidence of sexchromosome aneuploidies and diploid (multi-aneuploid?) spermnuclei. The results could be related to the higher incidenceof chromosome abnormalities (especially sex-chromosome aneuploidies)observed in children conceived by intracytoplasmic sperm injection(ICSI).     

Isolation of DNA probes specific for rat chromosomal regions 19p, 19q and 4q and their application for the analysis of diethylstilbestrol-induced aneuploidy in binucleated rat fibroblasts

DNA probes specific for rat chromosomes 19p, 19q and 4q were isolated, characterized and used for the detection and analysis of diethylstilbestrol(DES)-induced aneuploidy. By denaturing and partially reassociating total genomic DNA a new rat repetitive DNA family was isolated, which was located on chromosome 19p21. Sequencing of a number of subclones from cos76-1 and other clones of this so-called 76-family revealed that the repeat units are interrupted with large areas of other (unique) DNA. Consequently, after fluorescence in situ hybridization (FISH) the signals in interphase nuclei are large and spread out. The other two probes, cos25 (chromosome 4q) and cos42-47 (chromosome 19q), were isolated by screening cosmid libraries with probes isolated previously in our laboratory. The repeat unit of cos25 is a 2174 bp long EcoRI unit that contains three Sau3A sites and is tandemly organized. Sequencing of subclones of cos42-47 revealed that this probe was in fact the 5S RNA gene, located on 19q12. In order to determine if these probes were suitable probes for aneuploidy detection, two series of dual colour FISH with the combinations cos25/cos76-1 (4q/19p) and cos42-47/cos76-1 (19q/19p) were carried out on slides from an in vitro micronucleus assay with DES. With all three probes used, an increase in binucleated cells with non-disjunction or chromosome loss was observed in the DES-treated cultures. Scoring of additional micronucleated cells on slides hybridized with the cos25/cos76-1 (4q/19p) probes revealed that the hybridization signal of probe cos25 (4q) was over-represented in the micronuclei of the control cultures. The simultaneous use of the 19q and 19p probes is a particularly valuable tool for the detection of aneuploidy, since it allows distinction between aneugenic and clastogenic events in binucleated cells. Results of this analysis showed that apart from aneuploidy, DES also induced structural chromosome aberrations, although to a lesser extent.     

精子大头多尾畸形与性染色体异常

目的 探讨精子畸形与精子染色体异常的关系,了解畸形精子的病理学改变,方法 应用光学和电子显微镜,性染色体特异性探针荧光原位杂交（FISH）技术研究罕见的大头多尾畸形精子。结果 巴氏染色后观察的精子畸形率达98．75％（油镜下测量头部畸形达100％）,精子多尾率达60．25％（最多达8尾）,电镜观察证实,精子头部表面凹凸不平,核型极不规则,有大量细胞质结构,尾部除数量异常外,尚有中心粒,线粒体和鞭毛结构的异常,FISH结果证实,性染色体多体率为61．4％,与精子多尾的组成比有大致的平行关系。结论 尽管体细胞染色体正常,畸形精子人可伴有严重染色体异常。     

Maternal cell contamination of buccal smear samples in nursing neonates

Buccal smear analysis is a non-invasive method which is being popularized by new fluorescence in situ hybridization (FISH) techniques. It is frequently used for gender identification and detection of sex chromosome aneuploidy in neonates. We attempted to determine whether or not buccal smears of nursing infants can be contaminated by maternal cells from breast feeding. FISH involving centromere specific directly labeled, multicolor probes for chromosomes X, Y and 18 were used for analysis of buccal smear samples. Buccal smear samples from 22 breast fed and 20 formula fed male neonates were analyzed in a blinded fashion. Twenty-seven percent of samples from breast fed infants had some (0.5–2.5%) XX signal pattern while the samples from formula fed infants had no XX signal pattern (difference statistically significant, p < 0.02, at 95% confidence interval). Our results indicate that breast feeding can cause maternal cell contamination of buccal smear samples that can lead to misinterpretation of results involving FISH analysis or other DNA based diagnostic studies. We have also modified the FISH technique to suit the neonates.     

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (chi2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.     

Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization

Analysis of small numbers of chromosomes using interphase fluorescent in-situ hybridization (FISH) probes has revealed that 50% of human preimplantation embryos contain abnormal cells. Detection of high levels of mosaicism with so few probes has led some researchers to extrapolate that a full analysis of all 23 pairs of chromosomes would reveal that all human embryos contain a proportion of abnormal cells. However, existing cytogenetic protocols cannot achieve such an analysis due to technical limitations. We have developed a novel technique based on whole genome amplification and comparative genomic hybridization (CGH), which for the first time allows the copy number of every chromosome to be assessed in almost every cell of a cleavage-stage embryo. We have successfully analysed 64 cells (blastomeres) derived from 12 embryos and have detected unusual forms of aneuploidy, high levels of chromosomal mosaicism, non-mosaic aneuploidy and chromosome breakage. This is the first report of a comprehensive assessment of chromosome copy number in human embryos and indicates that, despite high levels of mosaicism, some embryos do have normal chromosome numbers in every cell. Such embryos may have a superior developmental potential, and their low frequency may explain correspondingly low success rates of natural and assisted conception in humans.     

Mitotic non-disjunction as a mechanism for in vitro aneuploidy induction by X-rays in primary human cells

A collaborative study of three laboratories compared the inductionof aneuploidy by X-rays in human lymphocytes and fibroblasts.The induction of non-disjunction versus chromosome loss by X-rayswas investigated using a variety of aneuploidy detection methods.Chromosome loss was determined by fluorescence in situ hybridization(FISH) with pan-centromeric probes in cytochalasin-B-blockedbinucleated cells. Chromosome non-disjunction was estimatedby FISH with chromosome-specific centromeric probes in binucleatedinterphase cells. Chromosomes were counted in parallel in lymphocytemetaphase cells; chromosome counts of the whole karyotype andcounts of chromosomes 2 and 8 using chromosome paints. A majorobservation in spontaneous non-disjunction frequencies concernedthe clear difference in frequencies observed between the twopainted chromosomes in the same primary cells. When cells wereirradiated elevated frequencies were observed for all the differentcytogenetic endpoints. Although only a small number of the micronucleiwere positive for the centromeric signal and presumably containedwhole chromosomes, the absolute number     

Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia

Infertile men with azoospermia now have the possibility of fathering children by testicular sperm extraction combined with intracytoplasmic sperm injection. However, there are concerns about the risk of chromosomal abnormalities in their spermatozoa. We have studied aneuploidy frequencies for chromosomes 13, 21, X and Y by multicolour fluorescence in-situ hybridization (FISH) in testicular spermatozoa extracted from three men with non-obstructive azoospermia. The men were 34-37 years of age and had normal follicle-stimulating hormone (FSH) concentrations and normal 46,XY somatic karyotypes. A total of 3324 spermatozoa was analysed. The infertile patients had an elevated frequency of disomy for chromosomes 13, 21, XY disomy compared to controls but none of these reached statistical significance. Also there was no significant difference in the sex ratio or the frequency of diploidy in azoospermic patients compared to normal control donors. This first report on chromosomal aneuploidy in spermatozoa extracted from testes of patients with non-obstructive azoospermia suggests that some azoospermic men do not have a substantially increased risk of chromosomally abnormal spermatozoa.     

Role of multicolor fluorescence in situ hybridization (FISH) in simultaneous detection of probe sets for chromosome 18, X and Y in uncultured amniotic fluid cells.

Major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. The purpose of this study was to evaluate the role of multicolor fluorescence in situ hybridization in simultaneous detection of probe sets for chromosome 18, X, and Y in uncultured amniotic fluid cells as a safer alternative method for aneuploidy detection prenatally. Fifty amniotic fluid samples were analyzed by FISH and standard cytogenetics. Mean time to obtain results was three days for fluorescence in situ hybridization and 20 days for karyotype. Fluorescence in situ hybridization was informative in 43 samples (86%), and within this group, two aneuploidies were correctly identified. This evaluation demonstrates that FISH with X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory.     

Development of a non-human primate sperm aneuploidy assay tested in the rhesus macaque (Macaca mulatta)

Numerical chromosome aberrations in germ cells are important factors contributing to abnormal reproductive outcomes. Fluorescence in situ hybridization onto spermatozoa (sperm-FISH) has allowed the study of the influence of a wide range of biological factors and chemical exposure on aneuploidy incidences in human sperm as well as in mouse and rat animal models. The assay presented here extends the applicability of the sperm-FISH method to non-human primates and was tested in the prevalent model species, the rhesus macaque. The assay provides probes for macaque chromosomes 17, 18, 19, 20, X and Y, the homologues of human chromosomes 13, 18, 19, 16, X and Y, respectively. The analysis of 11 000 spermatozoa each from five individuals revealed spontaneous sex chromosomal disomy frequencies (X: 0.08%; Y: 0.09%) and an average autosomal disomy frequency (0.03%) coinciding with some of the lowest incidences scored in human studies. The non-human primate sperm-FISH assay provides a fast and efficient tool complementing the available analysis methods in non-human primate exposure studies. Since the assay employs large locus-specific FISH probes representing evolutionary conserved DNA sequences, it can be expected that the assay is also applicable to other cercopithecoid and hominoid non-human primate species.