Institute of Reproductive Medicine, Westphalian Wilhelms-University, Domagkstr. 11, D-48149 Muenster, Germany
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Abstract |
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Key words: age/aneuploidy/FISH/hormone levels/male infertility
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Introduction |
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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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Materials and methods |
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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 MannWhitney 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. KruskallWallis 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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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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In the younger subject group, a median of 50.4% (range 42.957.8) of sperm bore an X chromosome, whereas in the older group a median of 50.2% (range: 48.755.9%) 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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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 1829 and 3039 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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Notes |
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References |
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Baumgartner, A., Van Hummelen, P., Lowe, X.R., Adler, I.D. and Wyrobek, A. (1999) Numerical and structural chromosomal abnormalities detected in human sperm with a combination of multicolor FISH assays. Environ. Mol. Mutagen., 33, 4958.[ISI][Medline]
Bernardini, L., Borini, A., Preti, S., Conte, N., Flamigni, C., Capitanio, G.L. and Venturini, P.L. (1998) Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men. Hum. Reprod., 13, 34063413.[Abstract]
Bonduelle, M., Wilikens, A., Buysse, A., Van Assche, E., Wisanto A., Devroey P., Van Steirteghem, A.C. and Liebaers, I. (1996) Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum. Reprod., 11 (Suppl. 4), 131155; discussion 156159.[ISI][Medline]
Bosch, M., Rajmil, O., Martinez-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, 533538.[ISI][Medline]
Brinkworth, M.H. (2000) Paternal transmission of genetic damage: findings in animals and humans. Int. J. Androl., 23, 123135.[ISI][Medline]
Calogero, A.E., De Palma, A., Grazioso, C., Barone, N., Romeo, R., Rappazzo G. and D'Agata, R. (2001) Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters. Hum. Reprod., 16, 11721179.
Colombero, L.T., Hariprashad, J.J., Tsai, M.C., Rosenwaks, Z. and Palermo, G.D. (1999) Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil. Steril., 72, 9096.[ISI][Medline]
Cooper, T.G., Neuwinger, J., Bahrs, S. and Nieschlag, E. (1992) Internal quality control of semen analysis. Fertil. Steril., 58, 172178.[ISI][Medline]
Cooper, T.G., Atkinson, A.D. and Nieschlag, E. (1999) Experience with external quality control in spermatology. Hum. Reprod., 14, 765769.
Downie, S.E., Flaherty, S.P. and Matthews, C.D. (1997) Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization. Mol. Hum. Reprod., 3, 585598.[Abstract]
Estop, A.M., Cieply, K.M. and Aston, C.E. (1997) The meiotic segregation pattern of a reciprocal translocation t(10;12)(q26.1;p13.3) by fluorescence in situ hybridization sperm analysis. Eur. J. Hum. Genet., 5, 7882.[ISI][Medline]
Griffin, D.K., Abruzzo, M.A., Millie, E.A. Sheean, L.A., Feingold, E., Sherman, S.L. and Hassold, T. (1995) Non-disjunction in human sperm: evidence for an effect of increasing paternal age. Hum. Mol. Genet., 4, 22272232.[Abstract]
Guttenbach, M., Engel, W. and Schmid, W. (1997) Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review. Hum. Genet., 100, 121.[ISI][Medline]
Härkönen, K., Viitanen, T., Larsen, S.B. Bonde, J.P. and Lahdetie, J. (1999) Aneuploidy in sperm and exposure to fungicides and lifestyle factors. Environ. Mol. Mutagen., 34, 3946.[ISI][Medline]
Härkönen, K., Suominen, J. and Lähdetie, J. (2001) Aneuploidy in spermatozoa of infertile men with teratozoospermia. Int. J. Androl., 24, 197205.[ISI][Medline]
Hassold, T., Hunt, P. and Sherman, S. (1993) Trisomy in humans: incidence, origin, and etiology. Curr. Opin. Genet. Dev., 3, 398403.[Medline]
In't Veld, P.A., Broekmans, F.J., de France, H.F., Pearson, P.L., Pieters, M.H.E.C. and Van Kooij, R.J. (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum. Reprod., 12, 752754.[Abstract]
Kinakin, B., Rademaker, A. and Martin, R. (1997) Paternal age effect of YY aneuploidy in human sperm, as assessed by fluorescence in situ hybridization. Cytogenet. Cell Genet., 78, 116119[ISI][Medline]
Liebaers, I., Bonduelle, M., Van Assche, E., Devroey, P. and Van Steirteghem, A. (1995) Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet, 346, 1095.
Martin, R.H., Spriggs, E., Ko, E. and Rademaker, A.W. (1995) The relationship between paternal age, sex ratios, and aneuploidy frequencies in human sperm, as assessed by multicolor FISH. Am. J. Hum. Genet., 57, 13951399.[Medline]
McInnes, B., Rademaker, A., Greene, C.A., Ko, E., Barclay, L. and Martin, R. (1998a) Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men. Hum. Reprod., 13, 27872790.
McInnes, B., Rademaker, A. and Martin, R. (1998b) Donor age and the frequency of disomy for chromosomes 1, 13, 21 and structural abnormalities in human spermatozoa using multicolour fluorescence in-situ hybridization. Hum. Reprod., 13, 24892494.[Abstract]
Nieschlag, E. (2000) Scope and goals of andrology. In Nieschlag, E. and Behre, H.M. (eds), Andrology: Male Reproductive Health and Dysfunction, 2nd edn. Springer-Verlag, Heidelberg, pp. 18.
Pang, M.G., Hoegerman, S.F., Cuticchia, A.J., Moon, S.Y., Doncel, G.F., Acosta, A.A. and Kearns, W. (1999) 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 oligoathenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 12661273.
Pfeffer, J., Pang, M.-G., Hoegerman, S.F. Osgood, C.J., Stacey, M.W., Mayer, J., Oehninger, S. and Kearns, W.G. (1999) Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil. Steril., 72, 472478.[ISI][Medline]
Plas, E., Berger, P., Hermann, M. and Pfluger, H. (2000) Effects of aging on male fertility? Exp. Gerontol., 35, 543551.[ISI][Medline]
Rives, N., Mazurier, S., Bellet, D., Joly, G. and Mace, B. (1998) Assessment of autosome and gonosome disomy in human sperm nuclei by chromosome painting. Hum. Genet., 102, 616623.[ISI][Medline]
Robbins, W.A., Baulch, J.E., Moore, D., Weier, H.U., Blakey, D. and Wyrobek, A.J. (1995) Three-probe fluorescence in situ hybridization 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, 799809.[ISI][Medline]
Robbins, W.A., Vine, M.F., Truong, K.Y. and Everson, R.B. (1997) Use of fluorescence in situ hybridization (FISH) to assess effects of smoking, caffeine, and alcohol on aneuploidy load in sperm of healthy men. Environ. Mol. Mutagen., 30, 175183.[ISI][Medline]
Rolf, C. and Nieschlag, E. (2001) Reproductive functions, fertility and genetic risks of ageing men. Exp. Clin. Endocrinol. Diabetes, 109, 6874.[ISI][Medline]
Rousseaux, S., Hazzouri, M., Pelletier, R., Monteil, M., Usson, Y. and Sele, B. (1998) Disomy rates for chromosomes 14 and 21 studied by fluorescent in-situ hybridization in spermatozoa from three men over 60 years of age. Mol. Hum. Reprod., 4, 695699.[Abstract]
Storeng, R.T., Plachot, M., Theophile, D., Mandelbaum, J., Belaisch-Allart, J. and Vekemans, M. (1998) Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol. Acta Obstet. Gynecol. Scand., 77, 191197.[ISI][Medline]
Tarlatzis, B.C. and Bili, H. (2000) Intracytoplasmic sperm injection. Survey of world results. Ann. NY Acad. Sci., 900, 336344.
Vegetti, W., Van Assche, E., Frias, A., Verheyen, A., Bianchi, G., Bonduelle, M.M., Liebaers, M. and Van Steirteghem, A. (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum. Reprod., 15, 351365.
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge, 128 pp.
Williams, B.J., Ballenger, C.A., Malter, H.E., Bishop, F., Tucker, M., Zwingman, T.A. and Hassold, T.J. (1993) Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes. Hum. Mol. Genet., 2, 19291936.[Abstract]
Yurov, Y.B., Saias, M.J., Vorsanova, S.G., Erny, R., Soloviev, I.V., Sharonin, V.O., Guichaoua, M.R. and Luciani, J.M. (1996) Rapid chromosomal analysis of germ-line cells by FISH: an investigation of an infertile male with large-headed spermatozoa. Mol. Hum. Reprod., 2, 665668.[Abstract]
Submitted on November 1, 2001; resubmitted on January 15, 2002; accepted on March 4, 2002.