Genetics and human conception: DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease

We report the world's first clinical pregnancy resulting fromDNA-based enrichment for X-bearing human spermatozoa, for preventionof X-linked hydrocephalus. Sperm separation was followed byembryo biopsy and nested multiplex polymerase chain reaction(PCR) for gender determination. Enriched populations of X-bearingspermatozoa ranging from 80 to 89% pure as determined by fluorescencein-situ hybridization (FISH) resulted in in-vitro fertilization(IVF) rates indistinguishable from normal IVF procedures (65%).In two separate biopsy procedures, 7/9 and 15/16 of the resultingembryos were determined to be female by multiplex PCR. Embryotransfer resulted in a karyotypically normal female fetus. Thistechnique should be widely applicable to gender selection forthe prevention of genetic disorders.     

Acrosomal integrity assessed by flow cytometry in men with variable sperm quality.

The binding of a specific, fluorescent acrosomal marker (FITC-labelled peanut lectin) to spermatozoa from men exhibiting differences in sperm quality by conventional criteria was quantitated by flow cytometry. When a standard number of cells was analysed, binding of the lectin diminished and became more variable as the degree of sperm pathology increased. In general, ejaculates with total sperm counts exceeding 120 x 10(6) cells exhibited a stable level of binding within relatively narrow limits. In two normozoospermic men, low levels of acrosomal fluorescence were demonstrated. The significance of these observations with regard to prognosis in assisted fertilization programmes is discussed.     

Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays

BACKGROUND: Sperm DNA integrity (SDI) is an important factor in the prognosis of male fertility. Here we compare the toluidine blue (TB) image cytometry test, recently proposed by us for SDI assessment, with two other tests-the sperm chromatin structure assay (SCSA) and the terminal nick-end labelling (TUNEL) assay. METHODS: Sperm samples from 35 men were evaluated for standard sperm parameters and subjected to the TB test and SCSA. Eighteen of the 35 samples were also subjected to the TUNEL assay. RESULTS: The proportion of sperm cells with abnormal DNA integrity assayed by the TB test correlated strongly with the proportion of abnormal cells detected by the SCSA and TUNEL assay (rho=-0.84 and rho=0.80, P<0.001, respectively). Furthermore, the fractions of abnormal cells by the TB test corresponded closely to the sum of two SCSA parameters, the DNA fragmentation index (DFI) and the fraction of highly DNA-stainable cells (HDS) (medians 33.0 versus 32.0%, P=0.6). CONCLUSIONS: Abnormal cells in a TB test correspond to the sum of DFI and HDS fractions in the SCSA. TB-positive cells may represent sperm with fragmented DNA and/or abnormal chromatin structure. Because the TB test is an easy and inexpensive method, its potential use as a routine test for sperm DNA integrity, complementary to standard semen parameters, should be investigated further.     

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).     

Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI

BACKGROUND: Although ICSI provides a way of treating azoospermic men, concern has been raised about the potential risk for transmission of genetic abnormalities to the offspring. We quantified the incidence of chromosomal abnormalities in epididymal and testicular sperm retrieved from azoospermic patients undergoing ICSI. METHODS: Individual testicular sperm were collected from testicular biopsies with an ICSI pipette, and epididymal sperm were retrieved by microsurgical epididymal sperm aspiration. Samples were processed by fluorescent in-situ hybridization (FISH) for chromosomes 18, 21, X and Y and the results compared with those from normal ejaculated samples. RESULTS: The overall aneuploidy rate of 11.4% in men with non-obstructive azoospermia was significantly higher (P = 0.0001) than the 1.8% detected in epididymal sperm from men with obstructive azoospermia and also the 1.5% found in ejaculated sperm. No significant difference was found between the epididymal and ejaculated samples. When the chromosomal abnormalities were analysed, gonosomal disomy was the most recurrent abnormality in both obstructive and non-obstructive azoospermic patients, while autosomal disomy was the most frequent in ejaculated sperm. CONCLUSIONS: Sperm of non-obstructive azoospermic men had a higher incidence of chromosomal abnormalities, of which sex chromosome aneuploidy was the most predominant. Genetic counselling should be offered to all couples considering infertility treatment by ICSI with testicular sperm.     

Genetics: Discontinuous Percoll gradients enrich X-bearing human spermatozoa: a study using double-label fluorescence int-situ hybridization

The aim of this study was to evaluate the efficacy of enrichingX-bearing human spermatozoa using 12-step (25–80%) discontinuousPercoll gradients. X- and Y-bearing spermatozoa were simultaneouslyidentified in neat semen (controls) and in 80% Percoll fractionsfrom the same samples using double-label fluorescence in-situhybridization and chromosome-specific DNA probes. Hybridizationand labelling efficiencies of 95–99% were obtained inall samples. The mean ratio of X- to Y-bearing spermatozoa inthe controls was 49.0: 48.2, whereas there was a significantenrichment (P < 0.0001) of X-bearing spermatozoa in the Percollfractions (mean X:Y ratio was 55.1:41.1 or 1.35). The ratiovaried from 1.1–1.5 in individual Percoll samples. Therewere no significant differences in the proportions of aneuploidspermatozoa (XX, YY, XY) between the control and Percoll fractions.We conclude that 12-step discontinuous Percoll gradients enrichX-bearing spermatozoa, but the degree of enrichment is insufficientfor use in preconceptional sex selection.     

Smoking habits of parents and male: female ratio in spermatozoa and preimplantation embryos

BACKGROUND: Previous observations have addressed a decreased male:female ratio associated with smoking. Our aim was to assess whether this effect is observed at the spermatozoa or at the early embryo development. METHODS: We retrospectively assessed smoking intake habits of 56 couples included in our preimplantation genetic diagnosis (PGD) program. Three groups were established according to male or female cigarette consumption per day: non-smokers, smokers (1-19 cigarettes per day) and heavy smokers (> or =20 cigarettes per day). Fluorescence in-situ hybridization (FISH) was performed on ejaculated sperm samples to analyse chromosomes X and Y. On day 3, embryos were also analysed. Additionally, sperm samples from four heavy smoking and four non-smoking donors were prospectively analysed before and after capacitation. RESULTS: FISH on spermatozoa revealed no statistical differences in the Y:X ratio between the three groups. However, in the PGD study, in male heavy smokers, the XY:XX embryo ratio was decreased compared with non-smokers (22:47 versus 80:71; P = 0.0057). The smoking condition of the female partner had no significant effect on embryo XY:XX ratio, but for non-smoking females with a heavy smoking partner, the ratio was decreased (P = 0.0018) compared with non-smoking males. In heavy smoking donors a decreased of Y:X ratio was observed after swim-up with a statistically significant difference of ratios (P = 0.021). CONCLUSIONS: Smoking habits of males do not have an effect on the percentage of X- and Y-bearing spermatozoa on ejaculated samples. However, male heavy smokers produce an increased incidence of female embryos that could be related to an enrichment of X spermatozoa after swim-up in patients with high tobacco consumption.     

Fluorescence in-situ hybridization of sex chromosomes in spermatozoa and spare preimplantation embryos of a Klinefelter 46,XY/47,XXY male

It has been suggested recently that 47,XXY germ cells are able to progress through meiosis to produce hyperhaploid spermatozoa. We report on a 46,XY/47,XXY Klinefelter patient whose spermatozoa were recovered from the ejaculate and used for intracytoplasmic sperm injection (ICSI). Fluorescence in-situ hybridization (FISH) analysis of the patient's spermatozoa and of spare preimplantation embryos with DNA probes specific for chromosomes X, Y and 18 revealed sex chromosome hyperploidy in 3.9% of the sperm nuclei analysed (2.23% XY18, 1.12% XX18, 0.56% YY18), while only three out of 10 spare embryos analysed were normal for chromosomes tested. The abnormalities included two diploid mosaic embryos with the majority of the blastomeres normal for the chromosomes tested, and five embryos with mostly abnormal blastomeres and chaotic chromosome X, Y and 18 patterns. None of the embryos analysed showed a XXY1818 or XXX1818 chromosome complement. The frequency of sex chromosome hyperploidy in the spermatozoa of the mosaic Klinefelter patient was higher than the mean reported for karyotypically normal males, supporting the hypothesis that 47,XXY germ cells are able to complete meiosis and produce aneuploid spermatozoa. However, most of the spermatozoa analysed were normal for sex chromosomes, and ICSI of the patient's spermatozoa did not result in a spare embryo with a uniform 47,XXY or 47,XXX chromosome complement. Instead, fertilization produced a high percentage of mosaic embryos with chaotic chromosome arrangements.     

Seminal haploid cell detection by flow cytometry in non-obstructive azoospermia: a good predictive parameter for testicular sperm extraction

BACKGROUND: Testicular sperm extraction (TESE) associated with ICSI gives patients suffering from non-obstructive azoospermia (NOA) the possibility of becoming a father. The success rate of TESE based on sperm recovery is approximately 50%, and the commonly used non-invasive parameters are not predictive enough. Only the invasive testis biopsy has a good prognostic value. The aim of this study was to evaluate the prognostic value of the detection of seminal haploid cells by flow cytometry (FCM) in order to avoid unnecessary testicular biopsy. METHODS: For 37 NOA patients undergoing testicular biopsy, we measured testis size, serum FSH and inhibin B levels and carried out seminal cytology, seminal FCM analysis and histological examination. RESULTS: Sperm were found in 18 biopsies. These results were correlated with cytology, FCM analysis and the histological examination. FCM was more sensitive than cytology (100 versus 59%) but less specific (67 versus 83.5%) whereas the histological observation of complete spermatogenesis appeared to be less sensitive (50%) but more specific (100%). CONCLUSION: Detection of seminal haploid cells by FCM appears to be an interesting non-invasive technique which can predict TESE results and improve the management of NOA patients.     

Ultrastructural nuclear defects and increased chromosome aneuploidies in spermatozoa with elongated heads

BACKGROUND: Cellular and molecular mechanisms leading to elongated sperm heads are not known. We have analysed the nuclear status of spermatozoa with elongated heads. METHODS: Fourteen men with at least 30% of spermatozoa with an elongated nucleus were studied and compared with five fertile men as controls. Sperm morphology was analysed by a quantitative ultrastructural analysis. Sperm chromosomal content was assessed by three-colour fluorescence in-situ hybridization (chromosomes X, Y, 18). Y chromosome microdeletion and karyotype were analysed. RESULTS: Elongated sperm head rates of the patients were 46.9% (30-75 versus 0-2% in the control group) by light microscopy and 34.4% by electron microscopy. In all patients, the chromatin was poorly condensed in elongated sperm heads (50% of elongated nuclei). No anomalies of sperm biochemical markers were found. All the men showed normal karyotype (46,XY) and absence of Y chromosome microdeletion. Aneuploidy rates of gonosomes and chromosome 18 were significantly increased in patients (1.64- and 3.6-fold, P = 0.006 and 0.026, respectively). CONCLUSIONS: This study demonstrates that impaired chromatin compaction and slightly increased chromosome aneuploidies are found in spermatozoa with an elongated head, suggesting possible mechanisms such as meiotic non-disjunctions or spermiogenesis anomalies.     

Sperm aneuploidy in fathers of children with paternally and maternally inherited Klinefelter syndrome

BACKGROUND: It is unclear whether frequency of sperm aneuploidy is associated with risk of fathering children with trisomy. METHODS: We recruited 36 families with a boy with Klinefelter syndrome (KS), interviewed the fathers about their exposures and medical history, received a semen sample from each father, and collected blood samples from the mother, father and child. We applied a multicolour fluorescent in-situ hybridization assay to compare the frequencies of sperm carrying XY aneuploidy and disomies X, Y and 21 in fathers of maternally and paternally inherited KS cases. RESULTS: Inheritance of the extra X chromosome was paternal in 10 and maternal in 26 families. Fathers of paternal KS cases produced higher frequencies of XY sperm (P = 0.02) than fathers of maternal KS cases. After controlling for age, the major confounding variable, the difference between the two groups was no longer significant (P less-than-or-equal 0.2). Also, there were no significant differences between the parental origin groups for disomy X, Y or 21. CONCLUSIONS: Men who fathered a child with a Klinefelter syndrome produced higher frequencies of XY sperm aneuploidy, which is explained, in part, by both paternal age and parent of origin.     

Influence of global sperm DNA methylation on IVF results

BACKGROUND: In cases of male infertility, routine analysis for sperm characteristics is a poor predictive factor for the segmentation rate and embryo development in assisted reproductive technologies. It is assumed that epigenetic factors could have an influence on the embryo's quality. The aim of this work was to determine the relationship between sperm DNA methylation level and fertilization and pregnancy rates according to the assisted reproduction technique performed. METHODS: A prospective study was undertaken. Ejaculates were obtained from men (n = 63) undergoing an assisted reproduction procedure. 5-Methylcytosine was immunostained with a polyclonal antibody and revealed by fluorescein isothiocyanate. The DNA methylation level was then quantified by flow cytometry. RESULTS: Sixty-three conventional IVF cycles were performed, 760 oocytes were retrieved, an average of 8.1 +/- 4.8 embryos was obtained, and 2.4 embryos were transferred. Neither the fertilization rate nor the rate of good quality embryos was correlated with the DNA methylation level (r = -0.1 and r = -0.08 respectively; not significant). When sperm DNA methylation was >555 arbitrary units, the pregnancy rate was 33.3% compared with 8.3% in the lower (<555) group (P<0.05). CONCLUSION: DNA methylation level in human sperm could represent a new approach to study the ability of sperm to lead to pregnancy in an assisted reproduction procedure, especially when sperm samples with normal characteristics are used.     

Female sex selection using clomiphene citrate and albumin separation of human sperm

BACKGROUND: This study was undertaken to assess whether the use of clomiphene citrate in conjunction with albumin-separated sperm would alter the sex ratio (expressed as the proportion of males) towards females and, if so, whether this skewing was due solely to the induction of ovulation. METHODS: The sex ratios of 184 single and 42 twin births at five assisted reproduction biology clinics were determined. The normal approximation to the binomial distribution was used to determine significant differences between these sex ratios and the established sex ratios for single, twin and combined (single and twin) non- and ovulation-induced births. RESULTS: The non-ovulation-induced sex ratios for singletons (51.4%) and twins (50.2%) were greater than the treatment singleton (27.7%; P < 0.001) and twin (33.3%; P < 0.01) sex ratios respectively. Correspondingly, the non-induced sex ratio for combined births (51.4%) was greater than the treatment sex ratio (28.8%; P < 0.001). The previously established induced singleton and twin sex ratios (48.1%) were lower than the non-induced sex ratio (51.4%), but higher than the treatment singleton (27.7%; P < 0.001) or twin (33.3%; P < 0.03) sex ratios. The ovulation-induced combined ratio (48.1%) was less than the non-induced combined (51.4%) sex ratio, although greater than the treatment combined sex ratio (28.8%; P < 0.001). CONCLUSION: Clomiphene citrate in conjunction with albumin-separated sperm decreased the sex ratio; a reduction that was not exclusively due to induction of ovulation.     

Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications

BACKGROUND: Sperm DNA integrity is an important factor in the prognosis of male fertility. In this study, we investigated intra-individual variation of sperm chromatin structure assay (SCSA) parameters in infertility patients undergoing assisted reproductive techniques (ARTs). METHODS: Retrospective study of 282 consecutive patients referred for ART [intrauterine insemination (IUI), IVF or ICSI] with repeated (between 2 and 5) SCSA measurements. RESULTS: Mean coefficient of variation (CV) of DNA Fragmentation Index (DFI) for repeated SCSA measurements was 29%. A high proportion [37%; 95% confidence interval (CI): 27%, 49%] of patients with DFI >30% in the first test had DFI <30% in the second test. Also, a considerable proportion (27%; 95% CI : 16%, 40%) of patients with 21-30% DFI values in the first test had DFI >30% in the second test. CONCLUSIONS: Intra-individual variability in DFI is significant, therefore repeated SCSA measurements are recommended. The biological mechanisms behind these variations remain to be elucidated.     

Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters

A large proportion of patients with oligoasthenoteratozoospermia (OAT) have an abnormal karyotype and hence they produce aneuploid gametes. However, a normal karyotype does not exclude the chance of having germ cell aneuploidy, since an altered intra-testicular environment not only damages spermatogenesis, but may also disrupt the mechanisms controlling chromosomal segregation during meiosis. Therefore, this study was undertaken to evaluate the rate of aneuploidy in the spermatozoa of selected patients with abnormal sperm parameters. For this purpose, sperm aneuploidy rate for chromosomes 8, 12, 18, X and Y was evaluated by multicolour fluorescence in-situ hybridization (FISH) in nine patients with teratozoospermia alone and 19 OAT patients of presumably testicular origin. Thirteen normozoospermic healthy men served as controls. Patients with teratozoospermia or OAT had significantly greater disomy and diploidy rates compared with controls, whereas the rate of nullisomy was similar. XY disomy was very low in all groups, suggesting that chromosomal non-disjunction occurs mainly during the second meiotic division. Autosome 12 disomy rate was low in both patients and controls. There was a marked variability of total sperm aneuploidy rate in both groups of patients. Sperm aneuploidy rate was negatively correlated with sperm concentration and particularly with the percentage of normal forms. In conclusion, patients with teratozoospermia or OAT have an increased rate of sperm aneuploidy. This increase is similar in both groups, suggesting that teratozoospermia may be the critical sperm parameter associated with aneuploidy.     

Chromosomal status of uni-pronuclear human zygotes following in-vitro fertilization and intracytoplasmic sperm injection

Uni-pronuclear embryos (n = 42) were analysed by fluorescencein-situ hybridization (FISH) with two to four chromosome pair-specificprobes. Half of these embryos resulted from conventional inseminationand half from intracytoplasmic sperm injection (ICSI). The majorityof uni-pronuclear embryos from conventional insemination werenormally diploid (61.9%) whereas only 9.5% of uni-pronuclearICSI embryos (P < 0.001) were diploid. In addition, a significantlyhigher number of uni-pronuclear embryos from conventional inseminationhad a Y chromosome (10/21, 47.6%) when compared with ICSI embryos(2/21, 9.5%) (P = 0.015). It is concluded that the majorityof uni-pronuclear embryos following regular in-vitro fertilizationare fertilized, whereas those from ICSI are parthenogeneticallyactivated. The latter embryos should not be considered for embryoreplacement.     

双色FISH检测人精子染色体非整倍体方法的建立

目的　建立用双色FISH检测人精子染色体非整倍体的方法。方法　采用双色荧光原位杂交 (FISH)方法取适量精子标本用EDTA/PBS处理 ,然后用二硫苏糖醇 (DTT) ,使精子去凝集。固定后滴片 ,然后与双色荧光直接标记探针杂交。结果　在OLYMPUS荧光显微镜下可以清楚看到精子头部的蓝色杂交信号 ,头部有 1个绿色荧光杂交信号的精子为X染色体精子 (X精子 ) ,有 1个红色荧光杂交信号的精子为Y染色体精子 (Y精子 )。精子头部有 2个荧光杂交信号的精子为染色体数目异常精子。结论　双色荧光原位杂交 (FISH)方法可以用于测定人精子染色体非整倍体率的变化。     

10, 15 reciprocal translocation in an infertile man: ultrastructural and fluorescence in-situ hybridization sperm study: case report

BACKGROUND: Peculiar sperm defects are described in a sterile man heterozygous for a balanced translocation t(10;15) (q26;q12). As this structural reorganization was absent in the parents, the translocation must have appeared de novo in the present patient. METHODS: Spermatozoa were analysed under light and transmission electron microscopy (TEM). Fluorescence in-situ hybridization (FISH) was performed on the lymphocyte karyotype. Aneuploidy frequencies of chromosomes 18, X and Y in sperm nuclei, not involved in the translocation, were investigated using three-colour FISH. Dual- colour FISH was used to evaluate segregation of chromosomes 10, 15 in decondensed sperm nuclei. Moreover, three-colour FISH, using telomeric probes for chromosomes 10, 15 was performed in order to distinguish balanced and unbalanced gametes. RESULTS AND CONCLUSIONS: Overall, structural characteristics indicate general immaturity of the germinal cells. FISH sperm analysis detected an increase in chromosome 18 disomy (0.81%) suggesting an interchromosomal effect. A high frequency of diploidies, particularly 18,18,X,X and 18,18,X,Y, was also found. FISH segregation analysis for chromosomes 10, 15 indicated that 32.8% were balanced gametes, whereas 68.2% were unbalanced. Taken together, these data demonstrate in a male carrier of a reciprocal translocation t(10;15) the presence of diffuse ultrastructural sperm alterations and a high frequency of sperm aneuploidies. The existence of a correlation among these factors is proposed.     

Chromosome Painting in Marsupials: Genome Conservation in the Kangaroo Family

In order to deduce the ancestral genome arrangement in the karyotypically diverse marsupial family Macropodidae, and to assess chromosome change in this family, chromosome-specific paints from the tammar wallaby (2n = 16) were hybridized to metaphase spreads from the two species proposed to represent the 2n = 22 ancestral karyotype, as well as species with derived 2n = 20 and 2n = 14 karyotypes. Identical patterns were observed in the two 2n = 22 species, from which the rearrangements to form the three derived karyotypes may be easily deduced to be 1, 3 and 4 different fusions, respectively. The identical Thylogale and Dorcopsis genomes may both be used to represent the pleisiomorphic macropodid chromosome complement. Variation in the X chromosome was also investigated by hybridizing an X-Y shared tammar wallaby 12-kb repeat element to chromosomes from the other four macropodid species, finding that it hybridized only to the most closely related species, and therefore is of recent origin. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.