Hum. Reprod. Advance Access originally published online on September 30, 2005
Human Reproduction 2006 21(2):524-528; doi:10.1093/humrep/dei321
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Sperm aneuploidy in fathers of Klinefelter’s syndrome offspring assessed by multicolour fluorescent in situ hybridization using probes for chromosomes 6, 13, 18, 21, 22, X and Y
1 Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Bellaterra and 2 Servei d’Andrologia, Fundació Puigvert, 08025 Barcelona, Spain
3 To whom correspondence should be addressed at: Unitat de Biologia Cel·lular, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. E-mail: carme.nogues{at}uab.es
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Abstract |
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BACKGROUND: It is still unclear if a recurrence risk would exist in fathers of an aneuploid offspring of paternal origin. We have studied disomy frequencies in spermatozoa from fathers having Klinefelter syndrome (KS) offspring or miscarriages. The effect of paternal age on sperm disomy percentages is also analysed. METHODS: Parental origin of 17 KS patients was carried out by amplification of X chromosome polymorphisms. Spermatozoa from their fathers were studied by multicolour fluorescent in situ hybridisation (FISH) using probes for chromosomes 6, 13, 18, 21, 22, X and Y. RESULTS: In 53% of KS cases studied the additional X chromosome was of paternal origin. The paternally transmitted KS group of fathers showed significantly higher frequencies for XY disomy sperm as compared to fathers of the maternal-origin group. A correlation between paternal age and XY disomy frequencies was only found in the paternally derived cases. In contrast, similar disomy frequencies for all autosomes analysed were found in both groups of fathers. CONCLUSIONS: XY disomy frequencies increase with advancing paternal age only in fathers with paternally inherited KS offspring.
Key words: FISH analysis/Klinefelter syndrome/parental origin/paternal age/sperm aneuploidy
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Introduction |
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Trisomies are the most frequent chromosomal abnormalities in human newborns (0.3%) (Hassold et al., 1996
), and Klinefelter’s syndrome (KS) is the most common sex-chromosome trisomy in humans (0.05% in newborns) (Thomas and Hassold, 2003). Most KS cases (98%) appear as a result of non-disjunction during meiosis and 
50% of them are paternally derived by an error during meiosis I (reviewed by Hassold and Hunt, 2001; Thomas and Hassold, 2003).
Fluorescent in situ hybridisation (FISH) studies in sperm have shown an increase in the production of disomic sperm in fathers of aneuploid children of paternal origin. Increased frequencies of XY disomy and 21 disomy have been reported in Turner’s syndrome fathers (Martínez-Pasarell et al., 1999; Tang et al., 2004) and in Down’s syndrome fathers (Blanco et al., 1998) respectively. However, in similar studies on KS fathers, no differences in XY disomy frequencies between paternally and maternally inherited KS children have been found (Eskenazi et al., 2002).
Recently, Soares et al. (2001a,b) described a generalized tendency to meiotic non-disjunction in fathers with Down’s or Turner’s syndrome children of paternal origin. These authors have enlarged their FISH studies to other chromosomes (4, 13, 18, 21, 22, X and Y) in addition to the chromosomes related to the aneuploid offspring and they have found increased disomy frequencies for chromosomes 13, 21, 22 and XY in both Down’s and Turner’s syndrome fathers.
The association between paternal age and fathering aneuploid offspring is still unclear. Lorda-Sanchez et al. (1992), using southern blots of restriction fragment-length polymorphisms (RFLP), reported that fathering a KS offspring might be associated with advanced paternal age. However, these results have not been confirmed by other authors (Thomas et al., 2000; Thomas and Hassold, 2003). In fathers with a KS son, an association has also been found between higher XY disomy frequencies in sperm and advanced paternal age, but for both paternally and maternally KS-transmitted cases (Eskenazi et al., 2002).
The objective of the present research is to determine whether: (i) fathers with KS offspring of paternal origin present an increase in disomy for chromosomes 6, 13, 18, 21, 22, X and Y; and (ii) an association between paternal age and increased disomy frequencies in sperm can be established.
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Materials and methods |
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Subjects
The study was carried out in 17 families with a 47,XXY fetus or offspring (aged 0–33 years). Chromosome analysis was performed in amniocentesis cells (seven cases) when an altered triple screen or advanced maternal age was identified, and in blood samples (10 cases) when an abnormal male secondary sexual development or male infertility was detected. All 17 fathers analysed were healthy, normozoospermic and aged 33–59 years, and none of them had been exposed to known mutagens or radiation. FISH analysis results in sperm from individuals C01, C02, C03, C04, C05, C06, C07 and C08 have been previously published (Arnedo et al., 2005
). All samples were collected after obtaining informed consent from each subject or their parents, in the case of a child. The study was approved by our institutional Ethics Committee.
PCR analysis
DNA from Klinefelter patients and their parents was extracted from peripheral blood samples using a standard salt procedure (Miller et al., 1988). When a 47,XXY karyotype was detected during pregnancy, DNA was extracted using the same protocol from fetal tissue or amniotic cell cultures. Ten polymorphic microsatellite markers along the X chromosome were amplified by PCR to determine the parental origin of the extra X chromosome present in the KS patients: DXS1283E, DYSII, DMD49, MAO, DXS991, AR, DXS1196, DXS101, DXS1192 and DXS8377 (details about these markers can be downloaded at http://www.gdb.org).
For each PCR reaction, 0.4–1 µg of DNA were amplified in a final reaction volume of 50 µl containing 1% of standard PCR buffer (Ecogen), 250 µmol/l of each dNTP (Amersham Pharmacia Biotech Inc.), 1.5 mmol/l MgCl2 (Ecogen), 0.8 µmol/l of each primer (Research Genetics) and 0.5 IU Taq polymerase (Ecogen). DNA was amplified in a Perkin Elmer thermal cycle. Specific PCR conditions for each primer are summarized in Table I. Samples were loaded onto a 6% acrylamide:bisacrylamide (19:1) gel. Electrophoresis was performed at 8–14 mA for 7–12 h. The gel was then stained with ethidium bromide.
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Sperm preparation
Semen samples were washed in KCl 0.075 mol/l, fixed in methanol:acetic acid (3:1) and smeared onto clean slides.
Probes
DNA centromeric probes for chromosomes 6 (1:1 mix of CEP6-Spectrum green and CEP6-Spectrum orange; Vysis Inc.), 18 (CEP18-Spectrum aqua; Vysis Inc.) and X (CEPX-Spectrum green; Vysis Inc.); locus-specific probes for chromosomes 13 (LSI13-Spectrum orange, locus RB-1; Vysis Inc.), 21 (LSI21-Spectrum orange, loci q22.14-q22.3; Vysis Inc.) and 22 (LSI22-Spectrum green, locus bcr; Vysis Inc.) and a DNA satellite probe for the Y chromosome (Y satIII-Spectrum aqua; Vysis Inc.) were used for the FISH analysis.
Hybridization procedure
Details of sperm preparation and the hybridization procedure have been described elsewhere (Arnedo et al., 2005). Briefly, sperm nuclei were decondensed by slide incubation in a solution of 5 mmol/l dithiothreitol and 1% Triton X-100 for 8–15 min, and denatured in 70% formamide at 73°C. Probes were denatured at 73°C for 5 min. Slides were hybridized in a dark chamber for 12–72 h at 37°C. Post-hybridization washes were performed according to the manufacturer’s instructions (Vysis Inc.). DAPI-III counterstain (Vysis Inc.) and antifade were applied to slides prior to observation. Probes for chromosomes 6, 21, X and Y were hybridized simultaneously on one slide and probes for chromosomes 13, 18 and 22 were hybridized on another slide. Autosomes 13, 18 and 21 were chosen because they are involved in the most common autosomal trisomies among newborns (Hassold et al., 1996) and chromosome 22 because only few studies have been published with a wide range of disomy frequencies (from 0.06 to 1.21%) (Martin and Rademaker, 1999; Soares et al., 2001a,b). Chromosome 6 provided an internal control.
Data collection and scoring criteria
A minimum of 10 000 sperm for each hybridization and donor was scored. Only slides with a hybridization efficiency of 
99% were accepted for analysis. Parental origin of the syndrome was performed by PCR only when sperm FISH analysis was finished; thus, sperm nuclei scoring was blind. The scoring criteria applied have been described previously (Arnedo et al., 2005). Briefly, chromosome disomy was considered when two signals of the same colour, comparable in brightness and size and separated from each other by a distance longer than the diameter of each one, were present. Slides were analysed under an Olympus AX70 epifluorescence microscope (Olympus Optical Co; Hamburg, Germany) equipped with a fluorescein isothiocyanate (FITC)/Texas Red/4,6-diamino-2-phenylindole (DAPI) triple-band pass filter, and a single-band pass filter for FITC, Texas Red and DAPI. Images were analysed with a Cytovision system (Applied Imaging; Sunderland, UK).
Statistical analysis
To determine if there were any significant differences in disomy and diploidy between individuals and between paternal and maternal groups, a two-tailed Fisher’s exact test was used. The Bonferroni correction was applied to post hoc multiple comparisons. Linear regression analysis was performed between parternal age and XY disomy percentage for each parental-origin group. Covariance analysis with interaction was used to compare the ages’ slopes between the paternal origin (PO) and maternal origin (MO) father groups.
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Results |
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The parental origin of the additional X chromosome in KS offspring or miscarriages was paternal in nine cases (53%) and maternal in eight cases (47%). All families were informative for at least one polymorphism in Xp and one in Xq. The most informative polymorphism was DYS-II (13 of 17 patients) and the least informative was DXS991 (two of 17 patients).
A total of 356 535 decondensed sperm nuclei was analysed by three- and four-colour FISH: 189 837 from the PO group of fathers and 166 698 from the MO group of fathers (Table II). Disomy frequencies for chromosomes 6, 13, 18, 21, 22, X and Y from fathers of the PO group (mean age 47.7 years) were compared with those from fathers of the MO group (mean age 43.4 years). Diploidy frequencies obtained from both FISH analyses were not statistically different. The ratio of X-bearing sperm versus Y-bearing sperm was 1:1 in both groups, as expected. Disomy frequencies for any chromosome studied and for diploidy were similar among individuals in fathers of the MO group. In contrast, the PO group presented inter-individual differences only for XY disomy. The PO group of fathers showed significantly increased (P < 0.05) mean frequencies for XY disomic sperm (0.51%), as compared to MO fathers’ mean frequencies (0.27%). No significant differences were found between PO and MO groups, neither for autosomal disomy frequencies (6, 13, 18, 21 and 22) nor for diploidy (see Table II), except for individual C15, who presented increased frequencies for disomy XY, disomy 18 and diploidy.
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In order to determine whether there was a correlation between paternal age and XY disomy frequencies, in both groups, a covariance analysis with interaction was applied. In maternally derived cases the slope of the linear regression analysis was 0.003, close to 0 (P = 0.489), whereas in paternally derived cases the slope was 0.031, significantly different from 0 (P = 0.044) (Figure 1). The differences between the two slopes approach significance (P = 0.087).
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Discussion |
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FISH studies in sperm from fathers of 47,XXY offspring were carried out to analyse disomy frequencies for sex chromosomes and for autosomes 6, 13, 18, 21 and 22 in paternally and maternally transmitted KS cases.
Our results on parental-origin analysis of the syndrome indicated that 53% of the cases studied were of paternal origin. According to the literature, 50% of 47,XXY males are derived from paternal non-disjunction (Hassold et al., 1991; Thomas and Hassold, 2003).
In the present study, an age effect in XY disomy frequencies was found in fathers with KS offspring of paternal origin, but not in fathers with KS offspring of maternal origin. Other authors (Eskenazi et al., 2002) studying sperm aneuploidy in fathers of boys with Klinefelter’s syndrome did not find significantly higher frequencies of disomy for chromosomes X, Y or 21 in the paternal group. Even if the frequency of XY disomy in PO fathers (0.51%) reported in the present work is clinically low, it is, on average, almost twice the percentage of the MO donors (0.28%). It is well known that the sex bivalent is the most prone to non-disjunction during male meiosis because of the existence of a single chiasma located in the pseudo-autosomic region (PAR1) (reviewed by Thomas and Hassold, 2003). Several studies have related the lack of recombination in the PAR1 region to an increase in the susceptibility of the XY bivalent to non-disjunction and aneuploid sperm (Shi et al., 2001; Sun et al., 2004). A significant reduction in recombination for the XY chromosome pair has been detected in paternally derived KS cases (Hassold et al., 1991; Lorda-Sanchez, 1992; Thomas et al., 2000), and also in maternally transmitted 47, XXY cases (reviewed by Thomas and Hassold, 2003).
Increased disomy frequencies for the chromosome pair implicated in the aneuploid offspring have also been detected in fathers of Down’s and Turner’s syndromes offspring (Blanco et al., 1998; Martinez-Pasarell et al., 1999; Tang et al., 2004). Moreover, in these fathers, a generalized tendency to meiotic non-disjunction for chromosomes 13, 21, 22 and XY has been described, but not for chromosome 18 (Soares et al., 2001a,b). Our results did not show an increase in disomy for any of the autosomes analysed, except for individual C15, indicating that the susceptibility to meiotic non-disjunction, in fathers with a 47,XXY offspring, is limited to the XY chromosome pair. There are several reasons that could explain this discrepancy: the difference in the sample size used in both studies and, as mentioned by the same authors (Soares et al., 2001b), the inter-individual variations in the degree of susceptibility to meioitic errors.
When a linear regression analysis between paternal age and XY disomy frequencies was considered, only the PO fathers group presented an age-effect increase in the rate of XY disomy. Lowe et al. (2001) found an association between paternal age and increased frequencies of 24,XY sperm in fathers with KS sons, but this increase was observed in both paternally and maternally derived cases. One year later, Eskenazi et al. (2002), apparently using the same data and dividing fathers according to parental origin, attributed the XY disomy increase to paternal age but not to paternal origin. The difference between their results and ours could be attributed to the paternal-age mean (42.4 versus 47.7, respectively) and to the inter-individual differences described in the general population for sperm disomy frequencies (reviewed by Templado et al., 2005). Some studies carried out in sperm samples from healthy men, using FISH, have demonstrated a donor age-effect specifically for the sex chromosomes (Robbins et al., 1995; Griffin et al., 1995; Martin et al., 1995; Kinakin et al., 1997; reviewed by Bosch et al., 2001 and Templado et al., 2005). A correlation between increased paternal age and reduced recombination in the XY bivalent has been reported by Lorda-Sanchez et al. (1992) but not in other studies (Thomas and Hassold, 2003). In women, an association has also been described between increased age and reduced recombination but only for some specific chromosomes (reviewed by Thomas and Hassold, 2003). Furthermore, some authors have described, in idiopathic infertile patients, the possibility that an age-related dysfunction in the X-chromosome centromere may explain the increase of XY disomy in sperm as age increases (Asada et al., 2000). Even though very little is known about the influence of genetic factors in baseline levels of sperm aneuploidy and how ageing may result in stable increases over time, it is possible that meiotic dysfunctions could increase over time and could be only statistically perceptible some years later.
In conclusion, XY disomy frequencies increase with advancing paternal age in fathers with paternally inherited KS offspring, whereas this age-related increase is not observed in fathers with maternally inherited KS offspring. However, in order to further reinforce this conclusion, the number of cases studied should be increased. This susceptibility to non-disjunction has not been detected in the autosomes analysed (6, 13, 18, 21 and 22). Nevertheless, more studies would be necessary to demonstrate the association between paternal age and increase in disomy and to establish the risk of fathering an aneuploid child in fathers of KS offspring.
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Acknowledgements |
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Financial support was given by the Ministerio de Ciencia y Tecnología (Project BFI2002-01193) and the Generalitat de Catalunya (2001SGR-00201 and 2001SGR-00202), Spain. Núria Arnedo was the recipient of a grant from the Universitat Autònoma de Barcelona during 2001. The English of this manuscript was corrected by Chuck Simons.
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References |
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Arnedo N, Nogués C, Bosch M and Templado C (2005) Mitotic and meiotic behaviour of a naturally transmitted ring Y chromosome: reproductive risk evaluation. Hum Reprod 20,462–468.
Asada H, Sueoka K, Hashiba T, Kuroshima M, Kobayashi N and Yoshimura Y (2000) The effects of age and abnormal sperm count on the nondisjunction of spermatozoa. J Assist Reprod Genet 17,51–59[CrossRef][Web of Science][Medline]
Blanco J, Gabau E, Gómez D, Baena N, Guitart M, Egozcue J and Vidal F (1998) Chromosome 21 disomy in the spermatozoa of the fathers of children with trisomy 21 in a population with a high prevalence of Down syndrome. Increased incidence in cases of paternal origin. Am J Hum Genet 63,1067–1072.[CrossRef][Web of Science][Medline]
Bosch M, Rajmil O, Martínez-Pasarell O, Egozcue J and Templado C (2001) Linear increase of diploidy in human sperm with age: a four-colour FISH study. Eur J Hum Genet 9,533–538.[CrossRef][Web of Science][Medline]
Eskenazi B, Wyrobek AJ, Kidd SA, Lowe X, Moore II D, Weisiger K and Aylstock M (2002) Sperm aneuploidy in fathers of children with paternally and maternally inherited Klinefelter syndrome. Hum Reprod 17,576–583.
Griffin DK, Abruzzo MA, Millie EA, Sheean LA, Feingold E, Sherman SL and Hassold TJ (1995) Non-disjunction in human sperm: evidence for an effect of increasing paternal age. Hum Mol Genet 4,2227–2232.
Hassold TJ and Hunt PA (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nature Rev Genet 2,280–291.[CrossRef][Web of Science][Medline]
Hassold TJ, Sherman SL, Pettay D, Page DC and Jacobs PA (1991) XY-chromosome nondisjunction in man is associated with diminished recombination in the pseudoautosomal region. Am J Hum Genet 49,253–260.[Web of Science][Medline]
Hassold T, Abruzzo M, Adkins K, Griffin D, Merrill M, Millie E, Saker D, Shen J and Zaragoza M (1996) Human aneuploidy: incidence, origin and etiology. Environ Mol Mut 28,167–175.[CrossRef][Web of Science][Medline]
Kinakin B, Rademaker A and Martin RH (1997) Paternal age effect of YY aneuploidy in human sperm, as assessed by fluorescence in situ hybridisation. Cytogenet Cell Genet 78,116–119.[Web of Science][Medline]
Lorda-Sanchez I, Binkert F, Maechler M, Robinson WP and Schinzel AA (1992) Reduced recombination and paternal age effect in Klinefelter syndrome. Hum Genet 89,524–530.
Lowe X, Eskenazi B, Nelson DO, Kidd S, Alme A and Wyrobek AJ (2001) Frequency of XY sperm increases with age in fathers of boys with Klinefelter syndrome. Am J Hum Genet 69,1046–1054.[CrossRef][Web of Science][Medline]
Martin RH and Rademaker A (1999) Nondisjunction in human sperm: comparison of frequencies in acrocentric chromosomes. Cytogenet Cell Genet 86,43–45.[CrossRef][Web of Science][Medline]
Martin RH, Spriggs E, Ko E and Rademaker AW (1995) The relationship between paternal age, sex ratios, and aneuploidy frequencies in human sperm, as assessed by multicolour FISH. Am J Hum Genet 57,1395–1399.[Web of Science][Medline]
Martínez-Pasarell O, Nogués C, Bosch M, Egozcue J and Templado C (1999) Analysis of sex chromosome aneuploidy in sperm from fathers of Turner syndrome patient. Hum Genet 104,345–349.[CrossRef][Web of Science][Medline]
Miller SA, Dykes DD and Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucl Acid Res 16,1215.
Robbins WA, Baulch JE, Moore D 2nd, Weier HU, Blakey D and Wyrobek AJ (1995) Three-probe fluorescence in situ hybridisation to assess chromosome X, Y and 8 aneuploidy in sperm of 14 men from two healthy groups: evidence for a paternal age effect on sperm aneuploidy. Reprod Fertil Dev 7,799–809.[CrossRef][Medline]
Shi Q, Spriggs E, Field LL, Ko E, Barclay L and Martin RH (2001) Single sperm typing demonstrates that reduced recombination is associated with the production of aneuploid 24, XY human sperm. Am J Med Genet 99,34–38.[CrossRef][Web of Science][Medline]
Soares SR, Templado C, Blanco J, Egozcue J and Vidal F (2001a) Numerical chromosome abnormalities in the spermatozoa of the fathers of children with trisomy 21 of paternal origin: generalised tendency to meiotic non-disjunction. Hum Genet 108,134–139.[CrossRef][Web of Science][Medline]
Soares SR, Vidal F, Bosch M, Martínez-Pasarell O, Nogués C, Egozcue J and Templado C (2001b) Acrocentric chromosome disomy is increased in spermatozoa from fathers of Turner syndrome patients. Hum Genet 108,499–503.[CrossRef][Web of Science][Medline]
Sun F, Oliver-Bonet M, Liehr T, Starke H, Ko E, Rademaker A, Navarro J, Benet J and Martin RH (2004) Human male recombination maps for individual chromosomes. Am J Hum Genet 74,521–531.[CrossRef][Web of Science][Medline]
Tang SS, Gao H, Robinson WP, Ho Yuen B and Ma S (2004) An association between sex chromosomal aneuploidy in sperm and an abortus with 45, X of paternal origin: possible transmission of chromosomal abnormalities through ICSI. Hum Reprod 19,147–151.
Templado C, Bosch M and Benet J (2005) Frequency and distribution of chromosome abnormalities in human spermatozoa. Cytogenet Genoma Res, 111: (DOI:10.1159/000086890).
Thomas NS and Hassold TJ (2003) Aberrant recombination and the origin of Klinefelter syndrome. Hum Reprod Update 9,309–317.
Thomas NS, Collins AR, Hassold TJ and Jacobs PA (2000) A reinvestigation of non-disjunction resulting in 47, XXY males of paternal origin. Eur J Hum Genet 8,805–808.[CrossRef][Web of Science][Medline]
Submitted on June 10, 2005; resubmitted on August 31, 2005; accepted on September 1, 2005.
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