Chromosomes of human sperm: Variability among normal individuals

Summary The chromosomal constitution of 2468 human sperm cells been investigated by fusion of human sperm with hamster eggs. The overall frequency of cells with structural aberrations was 7.7%, ranging from 1.9% to 15.8%, and varying significantly among individuals. The highest frequency occurred in sperm from the oldest donor (49 years), who also had had a vasectomy reversal three years prior to sampling. The overall aneuploidy frequency was 1.7%, ranging from 0.6% to 3.1%. In nine out of ten donors from whom blood samples were available the frequency of sperm cells with structural aberrations was higher than that for lymphocytes. Two previously reported donors (Brandriff et al. 1984) were resampled after an interval of 14 and 16 months respectively, and were each found to have similar frequencies of sperm chromosome abnormalities at both sampling times. A father-son pair included in the study had several chromosome breakpoints in common, although no more frequently than unrelated individuals.     

An analysis of human sperm chromosome breakpoints.

Sperm chromosome analysis of 19 sperm donors with either normal or balanced karyotypes was carried out in order to explore the nature of sperm chromosome structural aberrations. A total of 2,389 cells (range 36-298/donor) were karyotyped after in vitro penetration of hamster eggs. The median percentage of sperm structural aberrations was 9.3% (SD +/- 4.7; range 0%-17.8%), with a total of 247 breakpoints, of which 220 could be characterized fully. Two sets of donors were studied in two different centers: center 1 (United States) and center 2 (Spain). The frequencies of nonrejoined and rejoined chromosome-type aberrations were very similar between center 1 and center 2: 83.6% and 10.0%, and 75.0% and 10.3%, respectively. Chromatid-type aberrations were more frequent in center 2 (14.7%) than in center 1 (6.4%) (P = .037). Chromosome 4 had less than the expected number of breakpoints (P < .001). A positive significant correlation was found between sperm breakpoints reported in this study and sites of balanced chromosome de novo rearrangements detected at prenatal diagnosis and reported in the literature (P = .0001).     

Simultaneous detection of structural and numerical chromosome abnormalities in sperm of healthy men by multicolor fluorescence in situ hybridization

Both structural and numerical chromosome aberrations in sperm represent important categories of paternally transmitted genetic damage. Therefore, a new multiprobe fluorescence in situ hybridization (FISH) method, using DNA probes for three targets (centromere and telomere of chromosome 1, centromere of chromosome 8), was developed to detect human sperm carrying three types of chromosomal defects: (1) terminal duplications or deletions in chromosome 1p, (2) aneuploidy involving chromosomes 1 or 8, and (3) diploidy. Baseline frequencies were determined for three healthy donors who had been previously evaluated for sperm cytogenetics by the human-sperm/hamster-oocyte cytogenetic technique (hamster technique). Among ∼120 000 sperm analyzed by the new FISH method, the average baseline frequencies of sperm carrying telomeric duplications and deletions of 1p were 3.2 ± 1.9 and 2.9 ± 3.6 per 10⁴, respectively. Diploid sperm was found in an average frequency of 6.6 ± 4.0 per 10⁴. Average frequencies of disomic sperm for chromosomes 1 or 8 were 1.7 ± 2.2 and 1.9 ± 2.3 per 10⁴, respectively. Inter-individual differences were observed for deletions of 1p but not for the other sperm phenotypes. A good correlation was obtained between the frequencies of sperm with structural chromosome aberrations detected with the new assay and the frequency of sperm carrying premeiotic or meiotic cytogenetic damage detected with the hamster technique. The observed levels of numerical aberrations with the new FISH assay were within range of the baseline frequencies reported by the hamster technique. The newly developed FISH assay has promising applications in genetic, clinical, physiological and toxicological studies. Received: 26 February 1996 / Revised: 6 May 1996     

Segregation analysis in a man heterozygous for a pericentric inversion of chromosome 7 (p13;q36) by sperm chromosome studies.

We have analyzed 140 sperm chromosome complements from a subfertile man heterozygous for an inv(7)(p13;q36). Seventy-five percent of the chromosome complements were not recombinant: 37.9% contained the normal chromosome 7, and 37.1% contained the inverted chromosome 7. Twenty-five percent of the 140 were recombinant: 7.1% carried a recombinant chromosome 7 with a duplication p and deletion q, 17.1% carried a recombinant chromosome 7 with a duplication q and deletion p, and 0.7% carried both recombinant chromosomes. The frequency of structural chromosomal aberrations unrelated to the inversion was 11.4%, and the frequency of aneuploidy was 2.9%. Both frequencies were not significantly different from those in control donors. Two sperm complements with a second independent, contiguous inversion involving one of the original breakpoints (q36) were observed (1.4%). The risk of producing chromosomally abnormal offspring or spontaneous abortions would be 34.3%. The proportion of X-bearing and Y-bearing sperm was 46.8% and 53.2%, respectively, not significantly different from the expected 1:1 ratio.     

Chromosome analysis of human sperm

Summary A modified technique has been developed for the visualization of the chromosomes in human sperm. The cytogenetic analysis of 129 G-banded human sperm metaphases of 6 normal donors showed an incidence of structural and numerical chromosome abnormalities of 7.8%. Two out of 129 spermatozoa were aneuploid (1.6%). The frequency of sperms with chromatid-type aberrations was 2.3% (3/129). Chromosome-type aberrations were found in 5 out of 129 (3.9%) spermatozoa. X to Y ratio did not differ significantly from the expected one-to-one ratio. Twenty-six sperm complements from a patient 18–20 months after testes exposure to 30 Gy were examined. A significant increase of numerical and structural chromosome abnormalities was not observed. Chromatidtype aberrations were found in two sperm complements (7.7%) and chromosome-type aberrations in one sperm complement (3.9%). The cytogenetic analysis of 15 human sperms from a cancer patient 26 months after chemotherapy showed an increased frequency of aberrant sperm complements (33.4%). One chromatid-type (6.7%), three chromosometype aberrations (20.0%) and one (6.7%) hyperploid sperm complement could be observed. The sample size is still too small to answer the question whether chemical mutagens may increase the frequency of chromosomal abnormalities in human sperm.     

An analysis of structural aberrations in human sperm chromosomes

We have analyzed structural aberrations in 5,000 sperm chromosome complements obtained from 20 men over a 5-yr period by fusion of human sperm with hamster eggs. Detailed data are presented on 366 abnormal cells with 379 analyzable breakpoints. The frequency of cells with structural aberrations ranged from 1.9% to 14.5% among donors; this interindividual variability was statistically significant (p less than 0.0001). In contrast, repeat samples from individual men showed no significant variation over time. The number of sperm chromosome sets processed per hamster egg had no effect on the frequency with which structural aberrations occurred, nor were sperm chromosome abnormalities altered by varying capacitation or culture conditions. The spectrum of structural aberrations observed in human sperm chromosomes and a chi-square analysis of breakpoints based on DNA content are presented. Although human sperm chromosome abnormalities were visualized with a cross-species system, we believe that they represent an inherent, biologically significant phenomenon.     

A new FISH assay to simultaneously detect structural and numerical chromosomal abnormalities in mouse sperm

De novo aberrations in chromosome structure represent important categories of paternally transmitted genetic damage. Unlike numerical abnormalities, the majority of de novo structural aberrations among human offspring are of paternal origin. We report the development of a three-color fluorescence in situ hybridization (FISH) assay (CT8) to detect mouse sperm carrying structural and numerical chromosomal abnormalities. The CT8 assay uses DNA probes for the centromeric and telomeric regions of chromosome 2, and a probe for the subcentromeric region of chromosome 8. The CT8 assay was used to measure the frequencies of sperm carrying certain structural aberrations involving chromosome 2 (del2ter, dup2ter, del2cen, dup2cen), disomy 2, disomy 8, and sperm diploidy. Analysis of approximately 80,000 sperm from eight B6C3F1 mice revealed an average baseline frequency of 2.5 per 10,000 sperm carrying partial duplications and deletions of chromosome 2. Extrapolated to the entire haploid genome, approximately 0.4% of mouse sperm are estimated to carry structural chromosomal aberrations, which is more than fivefold lower than the spontaneous frequencies of sperm with chromosome structural aberrations in man. We validated the CT8 assay by comparing the frequencies of abnormal segregants in sperm of T(2;14) translocation carriers detected by this assay against those detected by chromosome painting cytogenetic analysis of meiosis II spermatocytes. The CT8 sperm FISH assay is a promising method for detecting structural chromosome aberrations in mouse sperm with widespread applications in genetics, physiology, and genetic toxicology.     

Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.     

Cytogenetic studies in motile sperm from normal men

Summary Most studies on human sperm chromosomes from normal men involve the heterologous fertilization of zona free hamster eggs by unselected human sperm. In this work, we have performed cytogenetic studies of highly motile sperm, selected by a swim-up method. A total of 505 motile human sperm complements from three normal donors was analysed. The total frequency of sperm with chromosomal abnormalities (10.9%; 6.9% structural aberrations and 4.0% aneuploidy) and the sex ratio (50.4% X49.6% Y) were similar to those obtained from whole semen samples. Our results indicate that the selection of motile sperm does not imply chromosomal selection.     

Frequency and distribution of chromosome abnormalities in human spermatozoa

This study reviews the frequency and distribution of numerical and structural chromosomal abnormalities in spermatozoa from normal men obtained by the human-hamster system and by multicolor-FISH analysis on decondensed sperm nuclei. Results from large sperm karyotyping series analyzed by chromosome banding techniques and results from multicolor FISH in sperm nuclei (of at least 10(4) spermatozoa per donor and per probe) were reviewed in order to establish baseline values of the sperm chromosome abnormalities in normal men. In karyotyping studies, the mean disomy frequency in human sperm is 0.03% for each of the autosomes, and 0.11% for the sex chromosomes, lower than those reported in sperm nuclei by FISH studies using a similar methodology (0.09% and 0.26%, respectively). Both types of studies coincide in that chromosome 21 and sex chromosomes have a greater tendency to suffer segregation errors than the rest of the autosomes. The mean incidence of diploidy, only available from multicolor FISH in sperm nuclei, is 0.19%. Inter-donor differences observed for disomy and diploidy frequencies among FISH studies of decondensed sperm nuclei using a similar methodology could reflect real differences among normal men, but they could also reflect the subjective application of the scoring criteria among laboratories. The mean frequency of structural aberrations in sperm karyotypes is 6.6%, including all chromosome types of abnormalities. Chromosome 9 shows a high susceptibility to be broken and 50% of the breakpoints are located in 9q, between the centromere and the 9qh+ region. Structural chromosome aberrations for chromosomes 1 and 9 have also been analyzed in human sperm nuclei by multicolor FISH. Unfortunately, this assay does not allow to determine the specific type of structural aberrations observed in sperm nuclei. An association between advancing donor age and increased frequency of numerical and structural chromosome abnormalities has been reported in spermatozoa of normal men.     

Chromosomal damage in sperm of patients surviving Hodgkin's disease following MOPP (nitrogen mustard,vincristine, procarbazine,and prednisone) therapy with and without radiotherapy

Following fusion with hamster eggs, human sperm chromosomes from six Hodgkin's disease patients were analyzed to determine the genotoxic effects of therapy. Each patient had received two to six cycles of MOPP (nitrogen mustard, vincristine, procarbazine, and prednisone), with or without radiotherapy, from 3 to 20 years before the study. A total of 571 cells from the six patients were analyzed; 9.8% of the cells had structural aberrations, and 1.6% were hyperhaploid. Analysis of 5998 metaphases from a control group of 24 male donors revealed only 6.9% of cells with structural aberrations and 0.8% aneuploidy. The increase in hyperhaploidy in the patients was statistically significant. Thus, results of this study suggest that the MOPP regimen, with or without radiotherapy, is capable of causing chromosome abnormalities in the sperm of Hodgkin's disease patients.     

Chromosome aberrations in 450 sperm complements from eight controls and lack of increase after chemotherapy in two patients

Summary Four hundred fifty sperm complements from eight controls were analyzed. A conservative estimate of aneuploidy was 1.8% with a hyperhaploid rate of 0.9% (4/450). The overall frequency of structural aberrations was 8.9% (40/450). The proportion of X-bearing (47.5%) and Y-bearing (52.5%) sperm did not differ significantly. Sperm complements were analyzed from a cancer patient 9 months after poly chemotherapy (n = 63) and from a patient being treated with Imurek (azathioprine) (n = 30). There was no significant increase in the incidence of numerical and structural chromosome aberrations in the sperm of either patient. The percentages of X-bearing and Y-bearing sperm were not significantly different from the expected 50%.     

Analysis of sperm chromosome complements from a man heterozygous for a pericentric inversion of chromosome 1

Human sperm chromosomes were studied in a man heterozygous for a pericentric inversion of chromosome (1)(p31q12). Q-banded pronuclear chromosomes were analyzed after in vitro penetration of golden hamster oocytes. A total of 159 sperm were examined: 54% bearing the inverted chromosome 1 and 46% the normal chromosome 1. These frequencies are not significantly different from the theoretical 11 ratio. There were no recombinant sperm with duplications or deficiencies, suggesting that a pairing loop failed to form or that crossing-over was suppressed. The frequency of abnormalities unrelated to the inversion was 5% for numerical, 8.8% for structural, 2.5% for numerical and structural, values not significantly different from control donors studied in our lab. The frequencies of X- and Y-bearing sperm were 46% and 54%, respectively, not significantly different from the expected value of 50%. This is the fifth pericentric inversion studied by human sperm chromosome analysis; recombinant chromosomes have been observed in two of the five cases. Some of the factors associated with an increased risk of recombinant sperm appear to be inversion size greater than 30% of the chromosome and chromosome breakpoints in G-light bands.     

Chromosome aberrations in mouse embryos and fetuses produced by assisted reproductive technology

The occurrence of structural chromosome aberrations in mouse one-cell embryos produced by intracytoplasmic sperm injection (ICSI) with mature spermatozoa was dependent on the type of sperm incubation medium and sperm incubation time. When cauda epididymal spermatozoa were used following incubation in bicarbonate-buffered TYH medium for 0h (no incubation) and 0.5h, the chromosome aberration rates (6.9% and 7.4%, respectively) in the resultant embryos were significantly higher than that (2.3%) in the IVF embryos. However, when the spermatozoa were incubated for 2-2.5h and 6h in the same medium, the chromosome aberration rates were reduced to the IVF embryo level (3.8% and 4.3%, respectively). When spermatozoa incubated in Hepes-buffered H-mCZB and phosphate-buffered PB1 media were used for ICSI, chromosome aberration rates in embryos were significantly high (8.6-28.1%) and increased in a time-dependent manner. On the other hand, when immature testicular spermatozoa were incubated in those three media for 0.5h and 6h, the incidences of resultant embryos with structural chromosome aberrations ranged between 7.4% and 11.7%, and there was no medium- and time-dependent change in these aberration rates. To evaluate transmissible risk of chromosome aberrations to offspring, two- or four-cell embryos derived from cauda epididymal spermatozoa were transferred into the oviducts of pseudopregnant females and chromosomes of live fetuses were examined on gestational day 16. One (2.0%) mosaic fetus was found when spermatozoa were incubated in TYH for 2-2.5h, and there were four (6.7%) fetuses displaying a structurally abnormal karyotype when spermatozoa were incubated in H-mCZB for 2-2.5h, indicating that structural chromosome aberrations generated in ICSI one-cell embryos are transmissible to offspring. The causal mechanism of structural chromosome aberrations in ICSI one-cell embryos is discussed in relation to the acrosomal plasma membrane cholesterol and the acrosome.     

Spontaneous frequencies of aneuploid and diploid sperm in 10 normal Chinese men: assessed by multicolor fluorescence in situ hybridization

Many studies have been published establishing the background frequencies of disomic and diploid sperm in normal men by fluorescence in situ hybridization (FISH) analysis, with highly significant variance among the reports. Besides interdonor heterogeneity and differences in the experimental protocols used, the question of inherent differences in chromosome malsegregation and meiotic arrest among different geographic and ethnic groups of donors has been raised. In this study, multicolor FISH analysis was carried out on semen samples from 10 nonsmoking, nondrinking Chinese men from the People's Republic of China. The results were compared to FISH data on 10 nonsmoking, nondrinking Canadians under the same experimental conditions, in the same laboratory. A total of 200,497 sperm was scored in the Chinese donors and compared to 202,320 sperm from Canadian donors. Approximately 10,000 sperm per chromosome probe per donor were analyzed. The mean hybridization efficiency was 99.99%. The frequencies of X-bearing and Y-bearing sperm were not significantly different from the expected 50% for each individual and for the combined data from all donors (49.73% vs. 49.46%, P = 0.3946). The mean disomy frequencies (range) were 0.07% (0.02%-0.12%) for chromosome 13, 0.18% (0.09%-0.19%) for chromosome 21, 0.05% (0. 01%-0.09%) for 24,XX, 0.02% (0.01%-0.06%) for 24,YY, and 0.29% (0. 13%-0.49%) for 24,XY. The mean diploidy frequency (range) was 0.38% (0.22%-0.73%) for 13-21 hybridizations and 0.32% (0.07%-0.70%) for XY hybridizations. Highly significant interdonor heterogeneity was found for diploidy (P = 0.0000) and for XY disomy (P = 0.0011), but no age effect was observed in any category of disomic or diploid sperm. The data reported here show no marked differences in disomy and diploidy frequencies between the mainland Chinese and Canadian groups, if donor heterogeneity is taken into account.     

Sperm chromosome complements in a 47,XYY man

Summary Human sperm chromosomes from a 47,XYY male were examined using the direct method of sperm chromosome analysis with two modifications in the semen processing. A total of 75 sperm complements was karyotyped and all of these contained one sex chromosome. The percentages of X-and Y-bearing sperm were 53% and 47%, respectively. There were 10 sperm with autosomal chromosomal abnormalities. The frequencies of numerical (4.0%), structural (10.6%), and total (13.3%) abnormalities were not significantly different from the frequencies observed in normal donors in our laboratory. Our results do not support the suggestion that XYY males have an increased risk of aneuploid progeny as a result of secondary non-disjunction or interchromosomal effects. They do support the hypothesis that one Y chromosome is eliminated in the germ cells of XYY males. However since our study provides the first information on sperm chromosomes in an XYY male, further studies on other XYY men are required.     

Meiotic segregation of human sperm chromosomes in translocation heterozygotes: report of a t(9;10)(q34;q11) and a review of the literature

Meiotic segregation products were studied in sperm from a man who was heterozygous for a reciprocal translocation, t(9;10)(q34;q11). A total of 171 sperm chromosome complements were studied by in vitro fertilization of hamster eggs. All possible 2:2 and 3:1 meiotic segregations were observed with the following frequencies: alternate, 41%; adjacent-1, 48%; adjacent-2, 5%; 3:1, 6%. Within alternate segregations, the number of normal sperm (35) was not significantly different from the number of sperm carrying a balanced form of the translocation (33), as expected. The proportion of sperm with an unbalanced form of the translocation was 60%. There was no evidence for an interchromosomal effect, since the frequencies of numerical (8%) and structural (15%) chromosomal abnormalities (both unrelated to the translocation) were within the normal range of control donors. The literature on a total of 10 translocation heterozygotes studied by sperm chromosome analysis was reviewed.     

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

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.     

Biological monitoring of workers in the rubber industry. I. Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of vulcanizers

To evaluate the possible genetic consequences of the industrial exposure among the vulcanizers of a rubber plant we measured the in vivo levels of chromosomal aberrations and sister-chromatid exchanges in peripheral lymphocytes of 34 vulcanizers and in an adequate control population. The observed chromosomal aberration frequencies were 1.9 +/- 1.4 aberrations/100 cells in the exposed group and 2.1 +/- 1.5 aberrations/100 cells in the controls. No difference was found between the two groups for the mean value of sister-chromatid exchanges (5.2 +/- 1.3 in the exposed, 5.2 +/- 0.7 in the control group). Cigarette-smoking was clearly associated with increased sister-chromatid exchange frequencies both in the exposed and in the control groups, while chromosomal aberration frequencies were not correlated with smoking habits.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation

The induction of chromosome aberrations in eggs of mice fertilized with X-irradiated sperm was performed by using an in vitro fertilization technique. Capacitated mature sperm was irradiated with various doses of X-rays and cytological analysis of the first cleavage metaphase of in vitro fertilized eggs was made. The frequencies of chromosome aberrations increased exponentially with dose and the dose-response relationship for overall breaks fitted well to a quadratic equation. The chromosome aberrations were mainly chromosome-type (82.1%), and the majority of aberrations were fragments.