Effect of paternal age on human sperm chromosomes

Transcription

1 FERTILITY AND STERILITY VOL. 76, NO. 6, DECEMBER 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Effect of paternal age on human sperm chromosomes Elza Maria Prestes Sartorelli, Ph.D., a,b Luiz Fernando Mazzucatto, B.Sc., b and João Monteiro de Pina-Neto, M.D., Ph.D. b University of São Paulo, Ribeirão Preto, São Paulo, Brazil Received February 21, 2001; revised and accepted June 18, Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, São Paulo, Brazil, grant numbers 88/0751-2, 88/ , and 92/ Reprint requests: Elza Maria Prestes Sartorelli, Ph.D., Departamento BEG, Centro de Ciĕncias Biológicas, Universidade Federal de Santa Catarina, Campus Trindade, CEP Florianópolis, Santa Catarina, Brazil (FAX: ; elza@ccb.ufsc.br). a Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciĕncias Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil. b Departamento de Genética, Faculdade de Medicina de Ribeirão Preto /01/$20.00 PII S (01) Objective: To investigate whether increased age alters the frequency and type of chromosomal anomalies in human spermatozoa. Design: Semen specimens were collected from donors via masturbation; cytogenetic studies were performed on sperm chromosomes after heterologous (human-hamster) in vitro fertilization. Setting: Cytogenetics Laboratory, Genetics Department, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. Patients(s): Seven men ages (older group) and five men ages (control group). Main Outcome Measure(s): Frequency and types of chromosomal anomalies in older and control group donors. Result(s): The frequency of numerical and structural aberrations (acentric fragments and complex radial figures) was significantly greater in chromosomes of older donors when compared with those of the control group. Conclusion(s): The higher frequency of sperm chromosome aberrations in older men was mainly a result of increased nondisjunction, acentric fragments, and complex radial figures. (Fertil Steril 2001;76: by American Society for Reproductive Medicine.) Key Words: Cytogenetics, human spermatozoa, chromosomal aberrations Chromosomal anomalies passed on by germ line cells have a significant influence on gestational loss, infertility, congenital malformation, and mental deficiency. Several laboratories have determined the frequencies of abnormalities found in the sperm karyotypes of normal men (1 9) through the hamster oocyte/human spermatozoa in vitro fusion technique (10). In testing the hypothesis of the effect of paternal age on chromosomal abnormalities, some authors (11) found no evidence for an increased risk of numerical chromosomal aberrations with increased paternal age in the sperm chromosome complements from 30 men, grouped by age (22 55 years). However, they found a significant increase in the frequency of structural chromosomal aberrations: 2.8% in the youngest group (22 24 years) and 13.6% in the oldest (45 55 years). A small increase in the frequency of structural chromosomal aberrations was found in the spermatozoa of male partners (25 50 years) of couples with habitual abortion, when compared with the fertile control group (12). A trend toward a higher incidence of structural aberrations with increased paternal age was also suggested in another study (13), which compared men years old with men years old, although the differences were not significant. However, few normal donors older than 50 have been analyzed by the human-hamster technique. Since older couples may wish to have children, there is growing interest in age-related changes in male fertility (14). For this reason, we compared the frequency and types of chromosomal anomalies found in the haploid complements of sperm from donors over 59 years with those of a younger control group, years old. MATERIAL AND METHODS Donors The older group consisted of seven men years old (donors A G), and the control 1119

2 TABLE 1 Characteristics of semen specimens from older and control group donors (mean values). Donors Volume (ml) Count ( 10 6 ) a Motility (A% B%) Morphology (%) Vitality (%) Older Control a Count volume (ml) no. of spermatozoa/ml. group consisted of five men years old (donors C1 C5). Among the 12 donors, five (donors D, C1, C2, C4, and C5) had unknown fertility and three were smokers (donors B, F, and C1). All donors were karyotyped, with normal results (46, XY). All of them gave informed consent before the study and reported no unusual exposure to environmental hazards or to known mutagenic or clastogenic agents. This study was approved by the Ethics Committee of the Hospital das Clínicas de Ribeirão Preto. Semen Collection and Assessment All donors were instructed to produce a semen specimen via masturbation after 3 4 days of sexual abstinence. The semen samples were evaluated for volume (ml), total count ( 10 6 ), motility (%), vitality (%), and morphological characteristics (%) after complete liquefaction (30 minutes). A total of 100 spermatozoa per specimen were assessed according to World Health Organization guidelines (15), and the measurements were made under blinded conditions. Semen characteristics from older and control group donors are shown in Table 1 (mean values). Most sperm characteristics, including volume, total count, motility, and vitality, were significantly greater in control samples than in the older group. Human-Hamster Technique Human sperm metaphases were obtained after in vitro fusion of zona-free hamster oocytes with human sperm (16), with some modifications. Only liquefied, fresh human sperm samples were used. After the last wash of semen with BWW medium (17) with 0.3% of human serum albumin (HSA), 1 ml of BWW was added to the pellet and then placed for an hour in a CO 2 incubator (5%, 37 C) to recover the highly motile spermatozoa (swim-up method). The supernatant was recovered and centrifuged to permit the sperm count and adjustment to the ideal concentration, which was made with BWW medium containing 3.5% HSA (capacitation at this concentration was better for the older group). The capacitation time of the older group varied from 8 to 10.5 hours, with spermatozoa/ml of medium. Under the same experimental conditions (long period of capacitation time in 3.5% HSA), sperm concentration was decreased in semen samples in the control group (from 0.4 to spermatozoa/ml). We used Tarkowski s fixation technique (18) to obtain the sperm chromosome preparations. Sperm complements were analyzed after G banding (19). Cytogenetic Analysis Sperm metaphases containing less than 21 chromosomes were not considered. If a sperm complement had more than one numerical or structural anomaly, it was registered only once. The complement was registered twice only when it had one extra and one missing chromosome (hyper- and hypohaploidy) or when it had one numerical and one structural aberration. No case of diploidy was registered, since it was impossible to distinguish between diploidy and dispermy. As there were numerous hypohaploid complements, we used the conservative estimation of aneuploidy, doubling the hyperhaploid frequency to calculate the total frequency of aneuploidy. Analysis and classification of chromosome abnormalities were done according to ISCN nomenclature (20). Data Analysis A two-way 2 test was used to verify the expected oneto-one ratio of spermatozoa containing the X or Y chromosome. A Mann-Whitney U test was also used to compare the frequency of numerical and structural anomalies found in the older versus control groups. RESULTS A total of 1,270 sperm chromosome complements were studied, including 750 complements from the seven donors of the older group and 520 from the five control donors. The number of cells analyzed per donor was between 55 and 167. The results are shown in Table 2. The mean proportion of X- and Y-bearing sperm in the older group was not significantly different from the expected 1:1 ratio (47.4% and 52.6%, respectively). However, in the control group, this proportion was significantly different (44.5% and 55.5%, respectively; ; P.05). The frequencies of numerical and structural aberrations were significantly greater in the older than in the control group (P.0200 and P.0114, respectively). In the study of numerical abnormalities, the older donors (except donor A) had hyperhaploid complements, which involved chromosome 3 (two complements, 0.3%), group C 1120 Sartorelli et al. Paternal age and human sperm chromosomes Vol. 76, No. 6, December 2001

3 TABLE 2 No. and frequency of spermatozoa with chromosomal abnormalities in older and control group men. Donor Age (y) Total cells analyzed Sex ratio %X/%Y Hypo haploid n (%) Hyper haploid Aneuploidy (2 hype-haploidy) Structural aberrations Total a anomalies A / (16.7) 0 (0.0) 0 (0.0) 10 (8.3) 10 (8.3) B /50 13 (13.1) 2 (2.0) 4 (4.0) 8 (8.1) 12 (12.1) C /55 13 (11.9) 5 (4.6) 10 (9.2) 13 (11.9) 23 (21.1) D / (20.0) 2 (3.6) 4 (7.2) 5 (9.1) 9 (16.3) E /50 21 (12.6) 4 (2.4) 8 (4.8) 15 (9.0) 23 (13.8) F /54 16 (24.2) 3 (4.5) 6 (9.1) 6 (9.1) 12 (18.2) G / (11.9) 3 (2.2) 6 (4.5) 14 (10.4) 20 (14.0) Total / (14.7) 19 (2.5) 38 (5.0) b 71 (9.5) c 109 (14.5) C / (13.0) 1 (0.9) 2 (1.85) 2 (1.85) 4 (3.7) C / (9.6) 0 (0.0) 0 (0.0) 5 (4.8) 5 (4.8) C / (15.2) 1 (0.8) 2 (1.6) 4 (3.2) 6 (4.8) C /58 12 (11.9) 0 (0.0) 0 (0.0) 4 (4.0) 4 (4.0) C / (14.6) 0 (0.0) 0 (0.0) 6 (7.3) 6 (7.3) Total /55.5 d 67 (12.9) 2 (0.4) 4 (0.8) 21 (4.0) 25 (4.8) a Total anomalies 2 hyperhaploidy structural aberrations. b Significantly different (P.0200). c Significantly different (P.0114). d Significantly different ( , P.05). chromosomes (eight complements, 1.2%), group D and F chromosomes (one complement each, 0.13%), and group G chromosomes (five complements, 0.66%). Two extra-ring chromosomes (unknown origin) were also found. Only two young donors (control group) had hyperhaploid complements, involving group C and F chromosomes (one complement each). Among the structural chromosomal abnormalities, more than 50% of those found in the older group were chromosome-type aberrations, including acentric fragments (34 of 89) and chromosomal breaks (17 of 89). These types of structural anomalies were also frequent in the sperm of the control donors (7 of 25 and 10 of 25, respectively). The frequency of acentric fragments was significantly greater in the older group than in the control group (P.0457). Similar frequencies of chromatid breaks were found in the older and control groups (1.5% and 1.3%, respectively). However, significantly more complex radial figures (P.0221) were found in sperm metaphases of the older group. The specific types and frequencies of structural chromosomal aberrations in spermatozoa of both groups can be seen in Table 3. Figure 1 shows some of these structural anomalies. DISCUSSION There is little published data about sperm chromosome complements of men of advanced age. Our cytogenetic study was designed to compare the frequencies of sperm chromosome aberrations in younger and older men. Male fertility gradually declines with advancing age (14). Although it does not have a significant impact on fertility, the long spermatozoa capacitation time necessary to obtain TABLE 3 Type and frequency of structural chromosomal aberrations in spermatozoa of older and control group men. Structural aberrations Older donors Control group donors Chromosome type Acentric fragments 34 (4.5) a 07 (1.3) Chromosome breaks 17 (2.3) 10 (1.9) Deletion 01 (0.1) 0 (0.0) Rings 03 (0.4) 0 (0.0) Gaps 03 (0.4) 0 (0.0) Chromatid type Chromatid breaks 11 (1.5) 07 (1.3) Radial figures 07 (0.9) b 0 (0.0) Gaps 05 (0.7) 0 (0.0) Centric fissions 05 (0.7) 01 (0.2) Fragile site 03 (0.4) 0 (0.0) Total c 89 (11.9) 25 (4.8) a Significantly different (P.0457). b Significantly different (P.0221). c Some metaphases had more than one structural aberration and each event was considered here (older men n 750; control group men n 520). FERTILITY & STERILITY 1121

4 FIGURE 1 Some structural anomalies found in chromosomes of spermatozoa from the older group donors. The arrows show (A) a ring chromosome, (B) a chromosome break, (C) a complex figure involving chromosomes of groups B and C, and (D) a complex figure involving chromosomes of groups D and F. the penetration in the heterologous in vitro technique, in donors of the older group, could be explained by the low motility values and the early death rates of spermatozoa (short vitality), which were obtained by spermograms (Table 1). A decline in penetration rates with increasing age of donors has already been found by other authors (12). The frequency of aneuploidy was estimated by doubling the frequency of hyperhaploid complements (3). We found slightly more numerical anomalies in older men (5%) than in the upper border of the range proposed by other authors, who found 3% 4% (21) or 1% 4% (22) aneuploidy. In our study, they were significantly higher in older men when we compared both groups of data. This could be explained [1] by the greater incidence of disomy of chromosome 3, found only in the older group in our study; [2] by a higher incidence of extra group C chromosomes (including the X chromosome) and group G chromosomes; or [3] by extra group D and F chromosomes. A relatively high incidence of disomy of chromosome 3, compared with other autosomes, was also observed in some other studies, where authors (23) found a 0.41% and a 0.27% disomy frequency of this chromosome in young donors (32 and 31 years old, respectively) and a mean frequency of 0.2% chromosome 3 disomy in another study, which included 10 normospermic men, with a mean age of years (24). These studies determined the nondisjunction of this autosome through fluorescent in situ hybridation in a large number of sperm of younger donors. Although relatively few sperm metaphases were examined, we found a similar frequency of this disomy in the older group. Considering that [1] these authors (23, 24) did not study men of advanced age, [2] there is considerable variability in nondisjunction frequency among chromosomes, [3] some individuals may be predisposed to higher or lower frequencies of nondisjunction, and [4] older men, like older women, may have an increased likelihood of producing aneuploid offspring in comparison with their younger counterparts (25), we suggest that further studies in older men are necessary to determine if advancing paternal age affects segregation at meiosis and, in the case that it does, how it changes it. The reported incidences of structural aberrations in sperm complements (in healthy men) have a large range from 0% (10) to 13% (5), with a mean of 6% 7% (22). Two studies found frequent structural aberrations in men of advanced age, with 16.5% chromosomal abnormalities in 30 sperm complements of a 55-year-old man and 6.7% (2 of 30) cells with structural aberrations (3) and 14.5% (18 of 124) cells with structural abnormalities in a 65-year-old man (26). In our study, the significantly greater frequency of cells with structural aberrations found in older men was mainly due to an increase in [1] chromosome-type aberrations, such as acentric fragments and [2] chromatid-type aberrations, giving rise to complex radial figures. As discussed by other investigators (13, 26), chromosome-type anomalies must have originated sometime between the end of male meiosis and the beginning of the S-phase of the first cell cycle, otherwise fragments would have been lost. Preexistence of chromosomal breaks in human sperm before fertilization has also been recently suggested (27). The high frequency of acentric fragments observed in donors of advanced age in this study suggests that the spermatozoa chromatin of older men is more susceptible to chromosome breaks than that of younger men. There was also no selection at fertilization against sperm carrying chromosomal damage. In a study designed to determine the effect of age and lifestyle in 91 healthy people (from newborn to 79 years old), the authors also observed an age-related increase (2.9-fold) of acentric fragments in lymphocytes of adults aged 50 and over (28). Considering the chromatid-type events that induced a high frequency of complex radial figures in sperm metaphases of older men and knowing that these aberrations should have been formed during DNA synthesis, or later, in the hamster egg, we also suggest that the susceptibility of the chromatin of older men has an important role in inducing these aberrations, since these complex radial figures were only found in sperm metaphases of men of advanced age. More men over 60 should be studied to clarify if nondis Sartorelli et al. Paternal age and human sperm chromosomes Vol. 76, No. 6, December 2001

5 junction occurs differently in older men and to determine if the chromatin is more susceptible to damage with advancing paternal age. Although the medical literature does not yet provide a clear answer on these points, we suggest that paternal age should also be considered when older couples come for genetic counseling. Acknowledgments: The authors thank Dr. Cristina Templado, Departament de Biologia Celular y de Fisiologia, Universitat Autónoma de Barcelona, for her assistance and friendship during the development of this research and Marcelo Menezes Reis, Universidade Federal de Santa Catarina, for the statistical analysis. References 1. Martin RH, Lin CC, Balkan W, Burns K. Direct chromosomal analysis of human spermatozoa: preliminary results from 18 normal men. Am J Hum Genet 1982;34: Martin RH, Balkan W, Burns K, Rademaker AW, Lin CC, Rudd NL. The chromosome constitution of 1000 human spermatozoa. Hum Genet 1983;63: Martin RH, Rademaker AW, Hildebrand K, Long-Simpson L, Peterson D, Yamamoto J. Variation in the frequency and type of sperm chromosomal abnormalities among normal men. Hum Genet 1987;77: Brandriff BF, Gordon LA, Ashworth L, Watchmaker G, Carrano A, Wyrobek AJ. Chromosomal abnormalities in human sperm: comparisons among four healthy men. Hum Genet 1984;66: Kamiguchi Y, Mikamo K. An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova. Am J Hum Genet 1986;38: Jenderny J, Röhrborn G. Chromosome analysis of human sperm. I. First results with a modified method. Hum Genet 1987;76: Templado C, Benet J, Navarro J, Caballin MR, Miro R, Egozcue J. Human sperm chromosomes. Hum Reprod 1988;3: Estop AM, Cieply K, Van Kirk V, Munné S, Garver K. Cytogenetic studies in human sperm. Hum Genet 1991;87: Benet J, Genescà A, Navarro J, Egozcue J, Templado C. Cytogenetic studies in motile sperm from normal men. Hum Genet 1992;89: Rudak E, Jacobs PA, Yanagimachi R. Direct analysis of the chromosome constitution of human spermatozoa. Nature 1978;274: Martin RH, Rademaker AW. The effect of age on the frequency of sperm chromosomal abnormalities in normal men. Am J Hum Genet 1987;41: Rosenbusch B, Strehler E, Sterzik K. Cytogenetics of human spermatozoa: correlations with sperm morphology and age of fertile men. Fertil Steril 1992;58: Estop AM, Márquez C, Munné S, Navarro J, Cieply K, Van Kirk V, et al. An analysis of human sperm chromosome breakpoints. Am J Hum Genet 1995;56: Haidl G, Jung A, Schill WB. Ageing and sperm function. Hum Reprod 1996;11: World Health Organization. WHO Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge: Cambridge University Press, Martin RH. A detailed method for obtaining preparations of human sperm chromosomes. Cytogenet Cell Genet 1983;35: Biggers JD, Whitten WK, Wittingham DG. The culture of mouse embryos in vitro. In: Daniel JC, ed. Methods in mammalian embryology. San Francisco: Freeman, 1971: Tarkowski AK. An air drying method for chromosome preparation from mouse eggs. Cytogenetics 1966;5: Benet J, Genescà A, Navarro J, Egozcue J, Templado C. G-banding of human sperm chromosomes. Hum Genet 1986;73: ISCN. An international system for human cytogenetic nomenclature. Mittelman F, ed. Basel: S. Karger, Cytogenetics Cell Genetics, Martin RH, Ko E, Chan K. Detection of aneuploidy in human interphase spermatozoa by fluorescence in situ hybridization (FISH). Cytogenet Cell Genet 1993;64: Guttenbach M, Engel W, Schmid M. Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review. Hum Genet 1997; 100: Bischoff FZ, Nguyen DD, Burt JJ, Shaffer LG. Estimates of aneuploidy using multicolor fluorescence in situ hybridization on human sperm. Cytogenet Cell Genet 1994;66: Downie SE, Flaherty SP, Swann NJ, Matthews CD. Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in spermatozoa from 10 normospermic men using fluorescence in-situ hybridization. Mol Hum Reprod 1997;3: Griffin DK, Abruzzo MA, Millie EA, Sheean LA, Feingold E, Sherman SL, et al. Non-disjunction in human sperm: evidence for an effect of increasing paternal age. Hum Mol Genet 1995;4: Brandriff BF, Gordon LA, Moore II DH, Carrano A. An analysis of structural aberrations in human sperm chromosomes. Cytogenet Cell Genet 1988;47: Sloter ED, Lowe X, Moore II DH, Nath J, Wyrobek AJ. Multicolor FISH analysis of chromosomal breaks, duplications, deletions and numerical abnormalities in the sperm of healthy men. Am J Hum Genet 2000;67: Ramsey MJ, Moore II DH, Briner JF, Lee DA, Olsen LA, Senft JR, et al. The effects of age and lifestyle factors on the accumulation of cytogenetic damage as measured by chromosome painting. Mutat Res 1995;338: FERTILITY & STERILITY 1123