Intra-individual and inter-individual variations in sperm aneuploidy frequencies in normal men

Intra-individual and inter-individual variations in sperm aneuploidy frequencies in normal men Helen G. Tempest, Ph.D.,a Evelyn Ko, B.Sc.,a Alfred Rademaker, Ph.D.,b Peter Chan, M.D.,c Bernard Robaire, Ph.D.,d and Ren ee H. Martin, Ph.D.a a Department of Medical Genetics, University of Calgary, Calgary, Alberta, Canada; b Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois; c Department of Urology, Obstetrics and Gynaecology, McGill University Health Centre, Montreal, Quebec, Canada; and d Department of Pharmacology and Therapeutics, Obstetrics and Gynaecology, McGill University, Montreal, Quebec, Canada

Objective: To investigate whether there are intra-individual and/or inter-individual variations in sperm aneuploidy frequencies within the normal male population, and, if this is the case, whether they are sporadic or time-stable variants. Design: Prospective study. Setting: University research laboratory. Patient(s): Ten men aged 18–32 years. Intervention(s): None. Main Outcome Measure(s): Fluorescence in situ hybridization was used to investigate sperm aneuploidy frequencies for chromosomes X, Y, 13, and 21 in serial semen samples collected over a period of 12–18 months. Result(s): Intra-individual and inter-individual variations were investigated by comparing serial samples from the same donor and by comparing the donors with each other, respectively. Intra-individual variations were found in all 10 donors for at least one investigated chromosome; variations tended to be sporadic events affecting only one time point. Inter-individual variations were found for all chromosomes (except XX and YY disomy and disomy 21), with three men identified as stable variants, consistently producing higher levels of aneuploidy for at least one of the following aneuploidies: sex chromosome nullisomy; disomy 13, or diploidy. Conclusion(s): These results suggest that there are a number of factors and mechanisms that have the potential to sporadically or consistently affect sperm aneuploidy. (Fertil Steril 2009;91:185–92. 2009 by American Society for Reproductive Medicine.) Key Words: Sperm aneuploidy, nondisjunction, FISH, intra-individual variation, inter-individual variation

Chromosome abnormalities, in particular chromosome aneuploidy (trisomy or monosomy), is the leading cause of pregnancy loss, developmental disabilities, mental retardation, and infertility in humans (1). Chromosome aneuploidy can arise as a de novo event either during meiosis (maternal or paternal) or during mitosis shortly after fertilization. Trisomies involving all chromosomes have been observed in spontaneous abortions (2, 3). It has been reported that approximately 1 in 300 live births are aneuploid (4). The vast majority of aneuploidies, however, are lethal and are lost during early embryonic development, with a few exceptions: trisomy 13, 18, 21, and aneuploidy for the sex chromosomes can survive to full term. As a result of the lethality of aneuploidy, the estimated frequency of chromosomal abnormalities at conception is estimated to be significantly higher Received September 11, 2007; revised November 5, 2007; accepted November 6, 2007. Funding for this work was provided by the Institute for Human Development, Child and Youth Health of the Canadian Institutes of Health Research (CIHR). R.H.M. holds a Canada Research Chair in Genetics. H.G.T. is a recipient of a CIHR Strategic Training Fellowship in Genetics, Child Development and Health, the Petro Canada Young Innovators Award, and the Champion Technologies Award. e H. Martin, Ph.D., Department of Medical GenetReprint requests: Rene ics, University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada (FAX: 403-210-7931; E-mail: [email protected]).

0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2007.11.002

than that seen in newborns, with estimates varying enormously, from 5% to 50% (1, 5). The advent of fluorescence in situ hybridization (FISH) analysis using DNA probes for specific chromosomes in the early 1990s revolutionized the study of aneuploidy in male gametes. Fluorescence in situ hybridization is relatively simple, cheap, and rapid to perform, enabling large numbers of cells to be analyzed at one time. It should be noted that FISH does have several disadvantages, because it is only able to generate data on the chromosomes investigated, and the detection of structural aberrations is limited at best (6). The advantages, however, outweigh these disadvantages, and as a result this technique has been rapidly adopted in laboratories worldwide to investigate aneuploidy. More than 35 studies have used FISH in human sperm to address a number of questions. These studies have been reviewed in detail by Shi and Martin (7) and Tempest and Griffin (8). In brief, aneuploidy frequencies in study subjects have been shown to exhibit low baseline frequencies for all chromosomes. Evidence has also been provided to suggest that chromosome nondisjunction does not equally affect all chromosomes, with the sex chromosomes and chromosome 21 being more prone to nondisjunction than other autosomes (7). A number of factors have also been implicated in increasing chromosome aneuploidy in sperm, including male factor infertility (in particular,

Fertility and Sterility Vol. 91, No. 1, January 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

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oligoasthenoteratozoospermia), smoking, alcohol, caffeine, drugs, pesticides, and chemotherapy (7–15). It is also clear from these studies that there is a great deal of variation in aneuploidy frequencies between studies. This may be due, in part, to technical differences such as sperm decondensation, scoring criteria, FISH method, storage of samples, and the criteria for inclusion of patients and controls in studies (8). What has been hitherto poorly investigated is whether there are variations in aneuploidy frequencies between repeat semen samples from the same individual over time (intraindividual variations) and/or whether differences occur between individuals (inter-individual variations). Should this be the case, the question remains as to whether intraindividual and/or inter-individual variations are sporadic or time-stable events. To date, there have been a number of studies that report on inter-individual differences (16–29), with only two studies reporting on intra-individual differences (16, 26). However, it should be noted that the vast majority of these studies are based on a single or a few samples per individual and thus do not examine the stability and persistence of any changes over time. However, individuals can be examined with confidence only in studies that analyze repeated samples from the same individual (26). As a result, only studies with repeated samples from the same individual will be considered further. Because the majority of studies have focused on interindividual differences (in most cases in only one sample), the focus of this study was to investigate both inter-individual and intra-individual variations in sperm aneuploidy frequencies. Chromosomes 13, 21, X, and Y were investigated in repeated (three to four) sperm samples from 10 healthy men over a period of 12–18 months. Intra-individual and inter-individual variations for chromosome 13 have not previously been reported. MATERIAL AND METHODS Ten healthy male donors of unproven fertility were recruited from the McGill University Health Centre, Montreal (age range, 18–32 years; average age, 24.9 years at the commencement of the project). All recruited donors gave informed consent, provided detailed lifestyle histories, and this study was approved by institutional ethics committees. Aneuploidy frequencies were analyzed in sperm from donors A–J at 0 months, 6 months, and 12 months and also from donors A–D at 18 months (34 samples total). To ensure blind scoring of samples, sperm specimens were coded before shipping on ice to Calgary for FISH analysis. Aneuploidy for chromosomes 13, 21, X, and Y was analyzed by two-color FISH for chromosomes 13 and 21 and threecolor FISH for the sex chromosomes (chromosome 1 was used as an internal autosomal control to distinguish diploidy from sex chromosome disomy). Approximately 5,000 sperm/ patient/chromosome probe sets were scored by one trained researcher, adhering to strict scoring criteria. These methods have been previously described in detail elsewhere (30). 186

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The proportion of aneuploid sperm (disomic, nullisomic, and diploid) was analyzed for the investigated chromosomes, to identify differences in the frequency of aneuploidy within the same individuals across time (intra-individual differences) and across donors (inter-individual differences). Statistical analysis for intra-individual variation was performed with a generalized linear model procedure (SAS; SAS Institute, Cary, NC) for dichotomous outcomes that analyzes the mean value for abnormalities at each time point. Because testing was carried out for multiple significance, the Bonferroni correction was applied to control overall false positives; therefore, results with a P value of %.0167 were considered significant. To determine whether there was donor heterogeneity (inter-individual variations), a two-way analysis of variance using time and donor as the two factors was performed (SAS); donor heterogeneity was confirmed at P%.05. Pairwise comparisons between the donors was also performed to identify any time-stable variants. As with the intra-individual variation analysis, multiple significance was tested; therefore, the Bonferroni correction was applied to control against false positives. Accordingly, results with a P value of %.00091 were considered significant. RESULTS A total of 340,534 sperm was scored: 170,417 for chromosomes 13 and 21 (with an average of 5,012 per donor) and 170,117 for the sex chromosomes (with an average of 5,003 per donor). The aneuploidy frequencies and total number of sperm scored per donor at each time point are presented in Table 1 for the sex chromosomes and Table 2 for chromosomes 13 and 21. Intra-individual Variations in Sperm Aneuploidy The intra-individual aneuploidy results for each donor examined across the different time points are presented in Table 3 for the sex chromosomes and Table 4 for chromosomes 13 and 21. When comparing the different time points within each donor, all 10 donors were found to have a significantly increased aneuploidy frequency for at least one chromosome at one time point. Aneuploidy variation within donors was found for all chromosomes, with the exception of XX and YY disomy. These intra-individual aneuploidy results exemplify that in the majority of cases, most differences within the donors are restricted to a single time point. The results can be split into several categories. There are examples of individuals with a significantly increased aneuploidy frequency at a given time point compared with other time points (e.g., Table 3, donor C, 12 months and Table 4; Fig. 1, donor A, 6 months). There are also examples of individuals with significant increases at different time points, compared, for the most part, with a single time point (e.g., Table 3; Fig. 2, donor A, compared with 0 months and Table 4, donor C compared with 18 months). Donors A and C had a slight variation on this second example, with significantly increased frequencies of sex chromosome nullisomy and 13/21 diploidy, respectively, compared with two time points. Only donor J

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TABLE 1 Sex chromosome aneuploidy results for the 10 control donors across the different time points. Disomy

Donor A A A A Average B B B B Average C C C C Average D D D D Average E E E Average F F F Average G G G Average H H H Average I I I Average J J J Average

Time point 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo

X

Y

XX

XY

YY

Nullisomy

Diploid

Total aneuploidy

49.39 48.93 49.37 49.63 49.33 50.81 50.35 50.76 49.35 50.16 47.69 48.89 47.79 49.49 48.47 48.09 49.41 47.29 48.99 48.45 48.55 49.89 49.01 49.15 48.99 46.81 47.81 47.87 49.89 50.02 49.60 49.84 50.35 50.11 50.21 50.22 47.93 48.45 50.49 48.96 49.07 49.55 50.53 49.72

50.17 49.81 49.51 49.71 49.80 48.89 49.19 49.14 49.87 49.27 51.73 50.55 50.91 49.83 50.76 50.51 48.95 51.69 50.23 50.35 50.49 49.29 50.03 49.94 49.95 52.13 51.13 51.07 49.79 49.80 49.70 49.76 48.77 49.37 48.93 49.02 50.81 50.81 48.61 50.08 50.19 50.05 49.19 49.81

0.00 0.02 0.14 0.02 0.05 0.02 0.04 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.02 0.04 0.02 0.00 0.02 0.04 0.00 0.00 0.01 0.00 0.02 0.00 0.01 0.00 0.00 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.08 0.02 0.03

0.08 0.38 0.26 0.18 0.23 0.04 0.14 0.08 0.26 0.13 0.02 0.12 0.18 0.16 0.12 0.24 0.14 0.36 0.14 0.22 0.16 0.14 0.12 0.14 0.06 0.06 0.06 0.06 0.16 0.02 0.14 0.11 0.44 0.04 0.26 0.25 0.18 0.10 0.14 0.14 0.42 0.18 0.14 0.25

0.00 0.04 0.00 0.02 0.02 0.04 0.00 0.00 0.02 0.02 0.00 0.04 0.02 0.02 0.02 0.02 0.00 0.02 0.00 0.01 0.00 0.02 0.02 0.01 0.00 0.02 0.00 0.01 0.00 0.00 0.02 0.01 0.04 0.00 0.02 0.02 0.04 0.02 0.00 0.02 0.02 0.00 0.00 0.01

0.36 0.82 0.72 0.44 0.59 0.20 0.28 0.00 0.48 0.24 0.54 0.38 1.08 0.48 0.62 1.14a 1.48a 0.62a 0.64a 0.97a 0.76 0.64 0.82 0.74 0.96a 0.98a 1.00a 0.98a 0.16 0.14 0.54 0.28 0.40 0.48 0.56 0.48 1.02 0.60 0.74 0.79 0.30 0.14 0.12 0.19

0.06 0.36 0.44 0.24 0.28 0.00 0.06 0.04 0.10 0.05 0.06 0.10 0.32 0.20 0.17 0.28 0.26 0.36 0.08 0.25 0.06 0.16 0.22 0.15 0.24 0.14 0.26 0.21 0.12 0.08 0.08 0.09 0.24 0.02 0.14 0.13 0.17 0.20 0.22 0.20 0.22 0.12 0.18 0.17

0.50 1.62 1.56 0.90 1.17 0.30 0.52 0.14 0.88 0.47 0.64 0.66 1.62 0.88 0.95 1.70 1.90 1.38 0.86 1.47 1.02 0.98 1.18 1.06 1.30 1.20 1.32 1.27 0.44 0.26 0.78 0.50 1.12 0.54 1.00 0.89 1.43 0.94 1.12 1.17 0.96 0.52 0.46 0.65

No. of sperm scored 4,998 5,013 5,017 5,007 5,009 4,995 4,998 4,996 5,000 4,997 4,998 5,000 5,011 5,005 5,004 5,009 5,008 5,013 4,999 5,007 4,998 5,003 5,006 5,002 5,007 5,002 5,008 5,006 5,001 4,998 5,000 5,000 5,007 4,996 5,002 5,002 5,002 5,005 5,006 5,004 5,006 5,001 5,002 5,003

Note: Values are percentages unless otherwise noted. a Time-stable variant. Tempest. Sperm aneuploidy variation in normal men. Fertil Steril 2009.

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TABLE 2 Aneuploidy results for chromosomes 13 and 21 in each of the ten control donors across the different time points. Chromosome 13

Donor A A A A Average B B B B Average C C C C Average D D D D Average E E E Average F F F Average G G G Average H H H Average I I I Average J J J Average

Chromosome 21

No. of Time Total sperm point Unisomy Disomy Nullisomy Unisomy Disomy Nullisomy Diploid aneuploidy scored 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 18 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo 0 mo 6 mo 12 mo

99.44 99.06 99.28 99.32 99.23 99.84 99.82 99.84 99.92 99.86 99.54 99.66 99.64 99.36 99.56 99.58 99.46 99.44 99.78 99.57 99.36 99.62 99.30 99.43 99.62 99.78 99.56 99.46 99.62 99.72 99.86 99.73 99.70 99.70 99.72 99.71 99.52 99.68 99.72 99.64 99.90 99.70 99.78 99.79

0.46a 0.58a 0.48a 0.58a 0.52a 0.08 0.10 0.12 0.04 0.09 0.14 0.06 0.10 0.20 0.13 0.14 0.16 0.10 0.14 0.14 0.26 0.28 0.42 0.32 0.08 0.10 0.10 0.09 0.02 0.12 0.02 0.05 0.04 0.00 0.08 0.04 0.36 0.00 0.08 0.15 0.02 0.06 0.18 0.09

0.10 0.36 0.24 0.10 0.20 0.08 0.08 0.04 0.04 0.06 0.32 0.28 0.26 0.44 0.33 0.28 0.38 0.46 0.08 0.30 0.38 0.10 0.28 0.25 0.30 0.12 0.34 0.25 0.06 0.16 0.12 0.11 0.26 0.30 0.20 0.25 0.12 0.32 0.20 0.21 0.08 0.24 0.04 0.12

99.74 99.26 99.74 99.76 99.63 99.84 99.66 99.72 99.78 99.76 99.82 99.48 99.64 99.96 99.73 99.58 99.50 99.68 99.84 99.65 99.88 99.84 99.72 99.81 99.90 99.40 99.76 99.69 99.84 99.90 99.90 99.88 99.44 99.60 99.56 99.53 99.86 99.32 99.38 99.52 99.80 99.62 99.80 99.74

0.20 0.44 0.10 0.20 0.24 0.14 0.22 0.16 0.12 0.16 0.08 0.26 0.20 0.02 0.14 0.28 0.30 0.12 0.12 0.21 0.04 0.00 0.04 0.03 0.08 0.52 0.16 0.25 0.04 0.06 0.04 0.05 0.42 0.22 0.26 0.30 0.10 0.44 0.42 0.32 0.14 0.22 0.10 0.15

0.06 0.36 0.16 0.04 0.16 0.02 0.12 0.12 0.10 0.09 0.10 0.26 0.16 0.02 0.14 0.14 0.20 0.20 0.04 0.15 0.08 0.16 0.24 0.16 0.02 0.08 0.08 0.06 0.12 0.04 0.06 0.07 0.14 0.18 0.18 0.17 0.04 0.24 0.20 0.16 0.06 0.16 0.10 0.11

0.18a 0.64a 0.32a 0.40a 0.39a 0.08 0.12 0.06 0.08 0.09 0.06 0.16 0.38 0.12 0.18 0.34a 0.81a 0.46a 0.32a 0.48a 0.40 0.26 0.32 0.33 0.20 0.16 0.18 0.18 0.08 0.02 0.12 0.07 0.28 0.28 0.18 0.25 0.26 0.20 0.48 0.31 0.18 0.20 0.21 0.20

1.00 2.38 1.30 1.32 1.51 0.40 0.64 0.50 0.38 0.49 0.70 1.02 1.10 0.80 0.92 1.18 1.85 1.34 0.70 1.28 1.16 0.80 1.30 1.09 0.68 0.98 0.86 0.83 0.32 0.40 0.36 0.35 1.14 0.98 0.90 1.01 0.88 1.20 1.38 1.15 0.48 0.88 0.63 0.67

5,004 5,027 5,011 5,015 5,014 4,999 5,046 4,998 4,999 5,011 4,998 5,003 5,014 5,001 5,004 5,012 5,086 5,018 5,011 5,032 5,015 5,008 5,011 5,011 5,005 5,003 5,004 5,004 4,999 4,997 5,001 4,999 5,010 5,009 5,004 5,008 5,008 5,005 5,019 5,010 5,004 5,005 5,118 5,042

Note: Values are percentages unless otherwise noted. a Time-stable variant. Tempest. Sperm aneuploidy variation in normal men. Fertil Steril 2009.

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TABLE 3 Significant intra-individual increases over time in sex chromosome aneuploidy. Donor

Time point

A A B C D D D G H H J

XX disomy

XY disomy

6 mo 12 mo 18 mo 12 mo 0 mo 6 mo 12 mo 12 mo 0 mo 12 mo 0 mo

YY disomy

0 mo 0 mo 0 mo

Nullisomy

Diploid

0, 18 mo 0 mo

0 mo 0 mo

0, 6, 18 mo 12, 18 mo 12, 18 mo

0, 6 mo

18 mo 0, 6 mo 6 mo 6 mo 12 mo

6 mo

Note: Only time points with a significant increase in aneuploidy (a P value of < .0167) are indicated in the different aneuploidy categories. For example, the frequency of aneuploidy in donor A at 6 mo was significantly higher than that for XY disomy and diploidy at 0 mo and for nullisomy at 0 and 18 mo. Tempest. Sperm aneuploidy variation in normal men. Fertil Steril 2009.

for chromosomes 13 and 21 and donor D for the sex chromosomes do not fit into these two categories. Inter-individual Variations in Sperm Aneuploidy The statistical analyses for inter-individual variation revealed donor heterogeneity for the following aneuploidies: XY disomy, sex chromosome nullisomy, disomy and nullisomy for chromosome 13, nullisomy for chromosome 21, and diploidy. In the majority of cases the overall donor heterogeneity test was significant; however, for many aneuploidies (XY disomy, nullisomy for chromosomes 13 and 21, and diploidy for the sex chromosomes) differences between donors were not

found using the Bonferroni correction. However, three donors with consistently elevated aneuploidy frequencies compared with the other donors in this study have been identified: donors D and F for sex chromosome nullisomy (Table 1), donor A for disomy 13, and donors A and D for diploidy frequencies scored for chromosomes 13 and 21 (Table 2).

DISCUSSION In theory, intra-individual and inter-individual differences could be due to technical artefact. In this study, however, all samples were stored in the same manner, coded before

TABLE 4 Significant intra-individual increases over time in chromosome 13 and 21 aneuploidy. Donor A C C D D E F I I J J

Time point 6 mo 6 mo 12 mo 6 mo 12 mo 0 mo 6 mo 6 mo 12 mo 6 mo 12 mo

Disomy 13

Disomy 21

Nullisomy 13

12 mo 18 mo 18 mo

0, 18 mo

Nullisomy 21 0, 18 mo 18 mo

12 mo 0, 18 mo 0, 18 mo

18 mo 18 mo 6 mo 0, 12 mo 0 mo 0 mo

Diploid

0 mo 6 mo 12 mo

0 mo

Note: Only time points with a significant increase in aneuploidy (a P value of < .0167) are indicated in the different aneuploidy categories. For example, the frequency of aneuploidy in donor A at 6 mo was significantly higher than that for disomy 21 and diploidy at 12 mo and for nullisomy 13 and 21 at 0 and 18 mo. Tempest. Sperm aneuploidy variation in normal men. Fertil Steril 2009.

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FIGURE 1

FIGURE 2

Demonstration of significantly higher intra-individual chromosome 13 and 21 aneuploidy frequencies at a single time point (6 months) compared with all other time points for donor A. Indicated (6 month) time point is significantly higher (P%.0167) than at 12 months (*) and 0 and 18 months (y).

Demonstration of significantly higher intra-individual sex chromosome aneuploidy frequencies at several time points compared, for the most part, with a single time point (0 months) for donor A. Time point indicated is significantly higher (P%.0167) than at 0 months (*) and 0 and 18 months (*y).

Donor A aneuploidy frequencies for chromosomes 13 & 21

Donor A aneuploidy freqeuncies for the sex chromosomes

Disomy 21 Nullisomy 13 Nullisomy 21 Diploidy (13, 21)

0.9

0.7

*

0.6 0.5

* † †

0.4 0.3 0.2 0.1

*

0.8

Percentage anueploidy

Percentage aneuploidy

0.8

XY disomy 0.9

†

Sex chr nullisomy Diploidy (sex chr)

*

0.7 0.6 0.5 0.4

*

0.3

*

*

0.2 0.1

0 0

6

12

18

0

Sampling time (months)

0

Indicated (6 month) time point is significantly higher (p =