Multicolor FISH analysis of chromosomal breaks, duplications, deletions, and numerical abnormalities in the sperm of healthy men

Abstract

Transmitted de novo structural chromosomal abnormalities, the majority of which are paternally derived, can lead to abnormal reproductive outcomes as well as genetic diseases in offspring. We developed and validated a new multicolor FISH procedure (sperm ACM, which utilizes DNA probes specific for the alpha [1cen], classical, [1q12], and midi [1p36.3] satellites of chromosome 1) which utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical abnormalities plus two categories of structural aberrations: (1) duplications and deletions of 1pter and 1cen, and (2) chromosomal breaks within the 1cen-1q12 region. In healthy men, the average frequencies of sperm with duplications and deletions were (a) 4.5 +/- 0.5 and 4.1 +/- 1.3 per 10(4) involving 1pter and (b) 0.9 +/- 0.4 and 0.8 +/- 0.3 per 10(4) involving 1cen, respectively. The frequency of sperm exhibiting breaks within the 1cen-1q12 region was 14.1 +/- 1.2 per 10(4). Structural aberrations accounted for 71% of the abnormalities detected by sperm ACM, which was significantly higher than numerical abnormalities (P=2x10-8). Our findings also suggest that, for healthy men, (a) sperm carrying postmeiotic chromosomal breaks appear to be more prevalent than those carrying products of premeiotic or meiotic breakage or rearrangements, (b) the high frequency of chromosome breaks measured after "fertilization" by the hamster-egg cytogenetic method already appear to be present and detectable within human sperm by FISH, and (c) there are nonrandom and donor-specific distributions of breakpoint locations within 1q12 in sperm. FISH facilitates the analysis of much larger numbers of sperm than was possible when the hamster-egg method was used. Therefore, FISH-based procedures for simultaneously detecting chromosomal breaks, rearrangements, and numerical abnormalities in sperm may have widespread applications in human genetics, genetic toxicology, and reproductive medicine.

Figure 1

ACM FISH methodology for human sperm. DNA probes were hybridized to three target regions on chromosome 1 (1cen, 1q12, and 1p36.3) to detect three major types of abnormalities: 1, breakage or rearrangements involving 1p in stem cells, spermatogonia, or spermatocytes can produce multiple sperm carrying partial chromosomal duplications or deletions of 1pter (fig. 2B) or 1cen; 2, chromosome breaks at various locations within 1cen-1q12 (fig. 2C–E); and 3, meiotic nondisjunction of chromosome 1 (fig. 2A).

Figure 2

Photomicrographs and classification scheme for ACM FISH methodology. A, Sperm containing two copies of chromosome 1 (bottom) can represent sperm diploidy or chromosome 1 disomy. A normal sperm is shown on top. B, Sperm carrying a terminal duplication of 1p. C, Fluorescence phenotypes and scoring criteria used to classify sperm according to the relative location of chromosomal breaks within 1q12 (see Material and Methods section and fig. 3). D, Example of a sperm carrying a chromosomal break directly between 1cen and 1q12 (i.e., group 1). E, Example of a sperm carrying a chromosomal break within 1q12 (i.e., group 4). Sperm were imaged at 1,000× under fluorescence and phase-contrast microscopy.

Figure 3

Distribution of breakpoint locations within 1cen-1q12 of sperm (see fig. 2C). A, Average profile of the frequencies of sperm (per 10⁴) in each breakpoint group across donors. B, Profiles of the raw number of sperm in each breakpoint group per donor (D1–D4). Stacked bars indicate the number of breaks observed during the first (gray), second (white), and third (hatched) coded scoring analyses for each donor. Short bars that cross the X-axis represent zero values.

Figure 4

Genomewide comparison of the sperm ACM and hamster-egg cytogenetic methods. Bars A–E show the frequency of structural aberrations per 100 sperm metaphases evaluated in the following hamster-egg studies: A, Brandriff et al. (1988); B, Estop et al. (1995); C, Jenderny et al. (1992); D, Martin et al. (1987); E, Benet et al. (1992). The contribution made by three types of damage are shown, top to bottom: hatched, fragments of unknown origin (200/814 aberrations); unblackened, chromosome breaks (428/814 aberrations); and black, rearrangements (186/814 aberrations). The weighted mean (number of aberrations/total sperm ± SE) is also shown (sixth bar). Bars F (Van Hummelen et al. 1996) and G (Baumgartner et al. 1999) show the estimated frequencies of sperm carrying rearrangements in two previous studies that used a more limited FISH method. The ACM bar represents the overall frequency of sperm carrying structural abnormalities estimated from the data of the current study. The arrow reflects the estimate that 1q12 to 1pter represents 4%–5% of the haploid genome (see Discussion section). The portion contributed by partial chromosomal duplications and deletions is represented in black.

Figure 5

Comparison of the frequencies of sperm with breaks in 1cen-1q12, as determined by FISH and hamster-egg methods. The first bar represents the mean frequency (±SE) of sperm with breaks in 1cen-1q12 in a hamster-egg study (11 total donors, 2,468 sperm [Brandriff et al. 1984, 1985]) that included the same men evaluated in the current study. The second bar shows the average of three other labs: those of Martin et al. (1987) (30 donors, 1,582 sperm), Estop et al. (1991) (7 donors, 555 sperm), and Jenderny et al. (1992) (8 donors, 450 sperm). The mean represents the weighted average of all four hamster-egg studies.