Human Reproduction, Vol. 17, No. 7, 1826-1832,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Sperm aneuploidy rates in younger and older men
Institute of Reproductive Medicine, Westphalian Wilhelms-University, Domagkstr. 11, D-48149 Muenster, Germany
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Abstract |
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BACKGROUND: In order to assess the possible risk of chromosomal abnormalities in offspring from older fathers, we investigated the effects of age on the frequency of chromosomal aneuploidy rates of human sperm. METHODS AND RESULTS: Semen samples were collected from 15 men aged <30 years (24.8 ± 2.4 years) and from eight men aged >60 years (65.3 ± 3.9 years) from the general population. No significant differences in ejaculate volume, sperm concentration and sperm morphology were found, whereas sperm motility was significantly lower in older men (P = 0.002). For the hormone values, only FSH was significantly elevated in the older men (P = 0.004). Multicolour fluorescence in-situ hybridization was used to determine the aneuploidy frequencies of two autosomes (9 and 18); and of both sex chromosomes using directly labelled satellite DNA probes on decondensed sperm nuclei. A minimum of 8000 sperm per donor and >330 000 sperm in total were evaluated. The disomy rates per analysed chromosomes were 0.1–2.3% in younger men and 0.1–1.8% in older men. The aneuploidy rate determined for both sex chromosomes and for the autosomes 9 and 18 were not significantly different between the age groups. CONCLUSIONS: The results suggest that men of advanced age still wanting to become fathers do not have a significantly higher risk of procreating offspring with chromosomal abnormalities compared with younger men.
Key words: age/aneuploidy/FISH/hormone levels/male infertility
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Inherited numerical chromosomal aberrations can be deleterious to human reproduction and to the viability and health of the offspring. Chromosomal aneuploidy is detected in ~26% of spontaneous abortions or intrauterine fetal deaths and in 0.3% of newborns (Hassold et al., 1993
). In germ cells, aneuploidy most often arises from non-disjunction of the chromosomes during meiosis. In trisomies where the fetus can survive to term, paternal contributions have been shown to vary between 10% (trisomy 21, trisomy 18, trisomy 13; triple-X) and 100% (XYY) (Baumgartner et al., 1999). In Western societies, parental age has significantly increased (Nieschlag, 2000) and the possibilities of conceiving offspring by assisted reproduction have been greatly extended. It has been shown that the incidence of sex chromosome aberrations is significantly higher in fetuses conceived by ICSI compared with IVF (Liebaers et al., 1995; Bonduelle et al., 1996; Tarlatzis and Bili, 2000). Moreover, genetic diseases have been shown to be positively associated with increasing paternal age (Brinkworth, 2000). The increased use of assisted reproduction together with advanced parental age (not only maternal age but also paternal age) could lead to a higher incidence of defects due to an increase of sperm abnormality (Plas et al., 2000; Rolf and Nieschlag, 2001).
The possibility of paternal inheritance of aneuploidies has triggered various investigations on numerical chromosome aberrations in human sperm (Guttenbach et al., 1997; Downie et al., 1997). Most of these were performed using fluorescence in-situ hybridization (FISH) which allows counting of chromosomes in interphase cells via the numbers of signals emitted by fluorescently labelled, chromosome-specific DNA probes. Recent publications reported an increase of aneuploid sperm in oligoasthenoteratozoospermia (OAT) by a factor of 1.5 to 3 compared with fertile controls (McInnes et al., 1998a; Colombero et al., 1999; Pang et al., 1999; Pfeffer et al., 1999; Vegetti et al., 2000). To date, several investigators have tried to correlate male age with the numbers of aneuploid sperm (Table I) (Griffin et al., 1995; Martin et al., 1995; Robbins et al., 1995; Kinakin et al., 1997; McInnes et al., 1998b; Rousseaux et al., 1998; Asada et al., 2000; Bosch et al., 2001). According to the diverging results, in five of six studies the frequencies of sexual aneuploidies were partly positively correlated with increasing male age, whereas the analysis of autosomal aneuploidy gave varying results for chromosomes 1 and 21, and no correlation for the other chromosomes analysed (6, 8, 12, 13, 14 and 18). As a consequence, and because of the low number of subjects tested so far, more studies on aneuploidy in sperm of aged healthy males are needed.
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In this study, using multicolour FISH techniques, we tried to elucidate the effect of male age on chromosome numbers in sperm. We investigated the rates of sperm aneuploidy for chromosomes 9, 18, X and Y in 15 men aged <30 years and in eight men aged >60 years. The autosomes 9 and 18 were chosen because it has been shown (Guttenbach et al., 1997
) that these two chromosomes are most likely to have the highest hyperploidy rate in their chromosome size group. |
Materials and methods |
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Subjects
Twenty-six men were recruited from the general population for the study. All men filled in a questionnaire about their health and employment situation. The study was approved by the Ethics Committee of the Medical Faculty and the State Medical Board and informed consent was obtained from all volunteers.
Semen analysis and serum hormones
Semen samples were provided by 16 men aged <30 years (group 1), and 11 men aged >60 years (group 2). The semen samples were first analysed to evaluate concentration, motility and morphology according to published parameters (World Health Organization, 1999) under internal and external quality control (Cooper et al., 1992, 1999). We also obtained peripheral blood for analysis of hormone values. FSH, LH, estradiol, prostate-specific antigen (PSA), and sex hormone-binding globulin (SHBG) were analysed by immunofluorometric assays (Autodelfia; Wallac, Inc., Freiburg, Germany). Serum testosterone was measured by radioimmunoassay (Diagnostic Systems Laboratories, Inc., Sinsheim, Germany).
Sperm preparation and fixation
Semen samples were frozen at –20°C and then thawed and hybridized by the following procedure. Sperm were washed three times in phosphate-buffered saline (PBS), pH 7.2, centrifuged at 280 g for 10 min and the sediment was then fixed in methanol:acetic acid (3:1). The fixed specimens were stored at –20°C until further processing. The fixed sperm were spread on SuperFrost Plus slides (Langenbrink, Emmendingen, Germany) and maintained at –20°C. At least four slides were prepared for each volunteer.
Nuclei decondensation procedure
Slides were washed in 2x standard saline citrate solution (SSC) and incubated for 30 min in 0.1 mol/l Tris buffer, pH 7.6, containing 10 mmol/l dithiothreitol (DTT) at 0°C and for another 90 min in 0.1 mol/l Tris buffer, pH 7.6, containing 4 mmol/l 3,5-diiodo salycylic acid (LIS) at room temperature. After decondensation, the slides were washed once in 2xSSC and air-dried.
The commercially available kits CEP (chromosome enumeration probe) 18 SpectrumAqua, CEP 9 SpectrumGreen, CEP X SpectrumAqua, and CEP Y SpectrumOrange (Vysis, Downers Grove, IL, USA) were used.
The first probe mixture consisted of probes for chromosome 9 (Green), chromosome X (Aqua) and chromosome Y (Orange), and the second probe mixture consisted of probes for chromosome 18 (Aqua), chromosome 9 (Green), and chromosome Y (Orange) as an internal control. This control was used to determine the hybridization efficiency by checking for all fluorescence signals of the autosomes. Counting of the aberrant chromosome numbers of the double-hybridized chromosomes (9 and Y) was performed with one of the two probe mixtures.
Multicolour FISH
The FISH procedure was performed according to the protocol recommended by Vysis: slides were denatured for 5 min in a 70% formamide 20xSSC solution pre-warmed to 73 ± 1°C in a waterbath. Slides were then dehydrated through an ethanol series and air-dried. Ten µl of each probe mixture was added to a slide and covered with the other slide. The slides were sealed with rubber cement and hybridization took place overnight in a humidified chamber at 37°C. Post-hybridization washes were carried out as follows: the slides were immersed immediately in a 2xSSC/50% formamide solution at 45 ± 1°C for 10 min in a waterbath and then in a 2xSSC solution for 10 min at 37°C and finally in 2xSSC solution for 10 min at room temperature. The slides were counterstained with 15 ng/ml 4',6-diamidino-2-phenyl-indole (DAPI) and mounted in Vectashield antifade medium (Vector Laboratories Inc., Burlingame, CA, USA).
Scoring of sperm nuclei
Slides were observed using a fluorescence microscope (Axioskop, Zeiss, Oberkochen, Germany) with the appropriate filter sets: single band pass filter (Aqua, FITC, TRITC) and a triple band pass filter (DAPI/FITC/TRITC). Only slides with a hybridization rate of 99% were analysed and at least 8000 nuclei per patient were scored by two investigators in a blinded analysis design. Hybridization efficiency was checked by controlling all scored nuclei with the triple band pass filter after the scoring process and the previous scoring was assessed only if every nucleus contained at least one of the three different fluorescence signals. The quality of decondensation and the rate of the FISH failure were comparable among all subjects and only one semen specimen (subject no. 14) did not allow scoring of the signals due to sperm fixation problems.
For slide scoring, we applied the stringent scoring criteria (Williams et al., 1993). Only intact sperm bearing a similar degree of decondensation and clear hybridization signals were scored; disrupted or overlapping sperm were excluded from analysis. Sperm were regarded as abnormal if they presented two (or more) distinct hybridization signals for the same chromosome, each equal in intensity and size to the single signal found in normal monosomic nuclei. We considered only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head.
Statistical analysis
Student's t-test was used to test the homogeneity of mean ages between groups. The non-parametric Mann–Whitney rank sum test was performed to analyse statistical differences in sperm parameters, serum levels, and aneuploidy rates. Spearman correlation was used to correlate total chromosomal abnormalities and sperm parameters. Kruskall–Wallis analysis of variance (ANOVA) on ranks was used to analyse interchromosomal variations. The test was applied to the deviation of X- and Y-bearing sperm from the expected 1:1 ratio and to test the homogeneity among groups of disomy rates for all the different chromosomes analysed (SPSS 10.0 for Windows; SPSS Inc., Carey, NC, USA).
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Results |
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Of the 16 younger men, 14 were normozoospermic, but among the group of elderly men only three had normal sperm forms, three showed asthenozoospermia, and two oligozoospermia. The lifestyle of the younger men was very different compared with the older men. Most of the older men smoked or consumed alcohol, whereas only two of the younger men consumed cigarettes or alcohol. The profiles of the sperm and hormone parameters are shown in Table II
. Although sperm morphology was similar between the two groups, the younger men had a significantly higher percentage of progressive motile sperm (P = 0.003) and of total sperm count (P = 0.02). The elderly men had significantly higher FSH (P = 0.002) and PSA (P = 0.002) values and a significantly lower free testosterone level (P = 0.002). None of the other values tested differed significantly between age groups.
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A minimum of 8000 sperm nuclei per subject were scored. A total of 234 103 sperm nuclei were analysed in the younger group (n = 15) and 101 562 sperm nuclei in the older group (n = 8). The missing subjects were excluded from chromosome analysis because among the younger group one subject (no. 2) showed azoospermia and among the older group one subject (no. 27) was azoospermic, one (no. 9) had too few sperm for analysis and the sperm of one subject (no. 14) did not attach to glass slides for hybridization.
The aneuploidy rates of each subject for chromosomes 9, 18, X and Y tested are presented in Table III. The frequency of aberrant sperm nuclei for the four chromosomes is summarized in Figure 1. To evaluate any correlation between age and aneuploidy frequencies, the two age groups were compared and no significant difference was found (Table IVb).
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Interchromosomal deviations
The aberration rates were evaluated to check for meiotic non-disjunctions occurring differently among the four chromosomes analysed. The disomy rates for autosomes 9 and 18 were not statistically different (not significant; Mann–Whitney test). The sex chromosomes, however, were significantly less affected by chromosomal aberrations than the autosomes (P < 0.05, Kruskall–Wallis ANOVA on ranks). The disomy rates of the X chromosome were significantly higher than the Y chromosome in both groups (P < 0.05, t-test).
In the younger subject group, a median of 50.4% (range 42.9–57.8) of sperm bore an X chromosome, whereas in the older group a median of 50.2% (range: 48.7–55.9%) of sperm carried an X chromosome (Table III
). A comparison of sperm bearing Y and X chromosomes showed no statistically significant difference from the expected 1:1 ratio.
Aneuploidy correlated with sperm and serum parameters
To check for any correlation between aneuploidy rates and sperm parameters or serum values, we correlated all values with each other (Table IV). Only progressive motility correlated with the FSH levels as well as PSA levels, whereby the latter two were significantly different between the two age groups (Table I). Although none of the tested serum levels and semen parameters correlated significantly with the aneuploidy rates, age was negatively correlated with progressive motility (r = –0.591; P = 0.0031) and positively correlated with hormone values of SHBG (r = 0.497; P = 0.0259) and FSH (r = 0.525; P = 0.0123), as well as with the PSA value (r = 0.615; P = 0.0024) (Table IVa).
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Discussion |
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Previous studies have shown a slight effect of age on numerical aberrations of the Y chromosome (Griffin et al., 1995
; Martin et al., 1995; Robbins et al., 1995; Kinakin et al., 1997; Asada et al., 2000). With one exception (Martin et al., 1995), these studies could not detect age-related effects on tested autosomes in sperm. Although these studies correlated age with aneuploidy rates, they all performed the analysis on sperm from men aged <60 years. The only study with men aged <30 (n = 8) or >60 (n = 3) years showed an age-related increase for chromosome 21 aneuploidy (Rousseaux et al., 1998). This contradicts a study (McInnes et al., 1998b) which found no such age-related aneuploidy increase involving chromosome 21. In a recent study of sperm from men between 24 and 74 years (Bosch et al., 2001) male age was also correlated with the aneuploidy rate of the sex chromosomes and two autosomes. Despite a slight tendency of sex chromosome disomy augmentation with increasing age, no significant differences were observed.
The present study is the first to investigate a possible correlation of aneuploidy and age in men aged <30 and >60 years on a wider scale with different chromosomes. Because of increased numbers of volunteers, the present study might be more sensitive in detecting age-related aneuploidy shifts in sperm, thus helping to solve questions which remained open in preceding investigations. This study verifies most previous results for the X chromosome and confirms the latest study (Bosch et al., 2001) for the Y chromosome, demonstrating no significant difference between groups of different age. Nor did our results for the autosomal chromosomes 9 and 18 show any significant differences between age groups. Surprisingly, a non-significant decrease of aneuploidy rates for chromosome 18 was detected. This was also found by comparing two groups of men aged 18–29 and 30–39 years (Griffin et al., 1995). In our study, we also detected a non-significant higher aneuploidy rate of the tested autosomes compared with the sex chromosomes (chromosome 9, P = 0.560; chromosome 18, P = 0.847), but this has already been shown for some autosomes (9, 16 and 21) (Guttenbach et al., 1997) and two recent studies (Baumgartner et al., 1999; Pang et al., 1999) for other autosomes.
Our proportions for sperm aneuploidy are relatively high compared with other studies, but, taking into account that a minor correlation between aberrations and age was found in only a few studies, our determination of aneuploidy frequencies indicate a non-existent or minor risk for an increase of chromosomal aneuploidies in sperm of elderly men. The previous studies merely stress slight differences for only very few chromosomes (Y and 21). The varying results for different aneuploidy values might be due to different criteria for subject enlistment. Yet other reasons include different FISH probes, which rely on varying protocols with different sperm decondensation techniques, and scoring criteria (Vegetti et al., 2000).
It has been shown that the sperm aneuploidy rate correlates with cigarette and alcohol consumption (Robbins et al., 1997; Härkönen et al., 1999). To verify that the measured aneuploidy rates were due to age, the men were asked for their smoking habits and alcohol consumption. In the group of younger men, one smoked moderately and had elevated aneuploidy rates in his sperm. Two of three younger men with elevated aneuploidy rates had contact with organic solvents or mineral oil products on a regular basis, but neither had any other health-related problems. One elderly man admitted to smoking up to 20 cigarettes daily and the aneuploidy rate for chromosome 9 of his sperm was elevated compared with the other men of the same age (subject no. 4; 1.75%). Five of the older men were using medication such as drugs to normalize blood circulation, cholesterol levels, and to support their immune system. One subject took Madopar and Selegelin to suppress his Parkinson's disease and a second subject used an 
1-adrenoreceptor antagonist to suppress symptoms caused by prostate hyperplasia. None of the medications are known to be correlated with aneuploidies. Only one who also drank alcohol regularly had elevated aneuploidy rates for the four tested chromosomes (subject no. 1).
One study showed that patients undergoing ICSI have a significantly higher incidence of sex chromosomal aneuploidy compared with IVF patients (Storeng et al., 1998). Sperm morphology seems especially to be significantly correlated with aneuploidy frequencies (Yurov et al., 1996; Estop et al., 1997; In't Veld et al., 1997; Bernardini et al., 1998; Calogero et al., 2001). Two studies (Rives et al., 1998; Pang et al., 1999) found an elevated aneuploidy rate for the sex chromosomes; the latter group also for autosomal chromosomes in oligoasthenozoospermia patients. The range of aneuploidy is between 0 and 5.4% versus <0.2% in controls. Recently it was demonstrated that sperm morphology is directly associated with the number of chromosomes in sperm by studying sperm divided into four distinct morphology groups (Härkönen et al., 2001). Our results do not allow a correlation between normal sperm morphology, progressive motility and total aneuploidy rate (Table IV). Although the authors state that patients with higher aneuploidy rates for chromosomes 13, 18, 21, X and Y have low sperm motility and low sperm concentrations, our data do not support such correlations, possibly due to the small subject numbers in both subgroups.
In conclusion, no severe risk for aneuploidies appears to be borne by elderly fathers. To find a clear correlation between male age and sperm aneuploidy frequencies, a much larger group of men would have to be evaluated and each of the 46 chromosomes would have to be tested separately.
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Acknowledgements |
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We thank I.Upmann for technical assistance, and Dr Trevor Cooper, PhD and Susan Nieschlag, MA for language editing of the manuscript.
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Notes |
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1 To whom correspondence should be addressed. E-mail: Nieschl{at}uni-muenster.de
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References |
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Submitted on November 1, 2001; resubmitted on January 15, 2002; accepted on March 4, 2002.
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 Y. Xia, S. Cheng, Q. Bian, L. Xu, M. D. Collins, H. C. Chang, L. Song, J. Liu, S. Wang, and X. Wang Genotoxic Effects on Spermatozoa of Carbaryl-Exposed Workers Toxicol. Sci., May 1, 2005; 85(1): 615 - 623. [Abstract] [Full Text] [PDF]
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ESHRE Capri Workshop Group Fertility and ageing Hum. Reprod. Update, May 1, 2005; 11(3): 261 - 276. [Abstract] [Full Text] [PDF]
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P. Lenz, C.M. Luetjens, A. Kamischke, B. Kuhnert, I. Kennerknecht, and E. Nieschlag Mosaic status in lymphocytes of infertile men with or without Klinefelter syndrome Hum. Reprod., May 1, 2005; 20(5): 1248 - 1255. [Abstract] [Full Text] [PDF]
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B. Kuhnert and E. Nieschlag Reproductive functions of the ageing male Hum. Reprod. Update, July 1, 2004; 10(4): 327 - 339. [Abstract] [Full Text] [PDF]
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N. Nikolettos, B. Asimakopoulos, and I. Hatzissabas Intrafamilial sperm donation: ethical questions and concerns Hum. Reprod., May 1, 2003; 18(5): 933 - 936. [Abstract] [Full Text] [PDF]
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 A. Kamischke, A. Baumgardt, J. Horst, and E. Nieschlag Clinical and Diagnostic Features of Patients With Suspected Klinefelter Syndrome J Androl, January 1, 2003; 24(1): 41 - 48. [Abstract] [Full Text] [PDF]
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