Sperm chromosome analysis of two males heterozygous for a t(2;17)(q35;p13) and t(3;8)(p13;p21) reciprocal translocation

Summary Sperm chromosome complements from two males, one heterozygous for the reciprocal translocation t(2;17)(q35;p13) (n = 18) and one for t(3;8) (p13;p21) (n = 73), were analyzed. Only 2:2 segregations were observed with t(2;17): alternate, 56%; adjacent-I, 33%; adjacent-II, 11%. Both 2:2 and 3:1 meiotic segregations occurred in t(3;8): alternate, 34.2%; adjacent-I, 43.8%; adjacent-II, 20.5% and 3:1, 1.4%. A significant excess of chromosomally normal versus balanced sperm complements was observed with both translocation heterozygotes. The frequencies of other chromosome aberrations unrelated to the translocations were 16.7% for t(2;17) and 8.2% for t(3;8). The ratio of X-bearing to Y-bearing sperm was not different from the theoretically expected ratio of 1:1.     

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 segregation in a man heterozygous for a pericentric inversion, inv(9)(p11q13), analyzed by using sperm karyotyping and two-color fluorescence in situ hybridization on sperm nuclei

Analysis of sperm karyotypes and two-color fluorescent in situ hybridization (FISH) on sperm nuclei were carried out in a man heterozygous for the pericentric inversion inv(9)(p11q13). Sperm chromosome complements were obtained after in vitro fusion of zona-free hamster oocytes and donor sperm. A total of 314 sperm complements was analyzed: 153 (48.7%) carried the inverted chromosome 9 and 161 (51.3%) carried the normal one. None of the sperm complements contained a recombinant chromosome 9, suggesting that no chiasmata were formed in the heterochromatic region. The frequency of structural chromosome aberrations unrelated to the inversion (8.3%) and the frequency of conservative aneuploidy (3.2%) were within the limits observed in our control donors. The proportions of X-bearing (47.3%) and Y-bearing sperm (52.7%) were not significantly different from the expected 1:1 ratio. The percentage of disomy for chromosome 21 was analyzed by two-color FISH in 10 336 sperm nuclei. The disomy rate for chromosome 21 (0.30%) was not significantly different from that found in our controls. These results suggest that the risk for this man of producing chromosomally abnormal offspring or spontaneous abortions was not increased, and do not support the existence of an interchromosomal effect for chromosome 21. Received: 28 October 1996     

Sperm chromosome analysis of a man heterozygous for a pericentric inversion of chromosome 20

Summary The sperm chromosomes of a man heterozygous for inv(20)(p13q11.2) were analyzed. Twenty-six sperm chromosome complements were examined, of which fourteen contained the normal chromosome, and twelve the inverted chromosome. None of the sperm complements contained a recombinant chromosome 20. The frequency of structural chromosomal aberrations unrelated to the inversion was 11.5% (3/26). Numerical aberrations were not observed. The percentages of X- and Y-bearing sperm were 56% and 44%, respectively, which was similar to the expected 11 ratio.     

Sex ratio distortion in bovine sperm correlates to recombination in the pseudoautosomal region

A total of 2122 single sperm from 35 bulls belonging to six different paternal half-sib groups were analysed with respect to two markers in the bovine pseudoautosomal region (PAR) and sex-specific loci on the X and Y chromosomes, respectively. A segregation ratio significantly different from 1:1 was observed in a test over all families, with a higher proportion of X-bearing gametes (53.5%). The analysis of recombination conducted separately for X- and Y-bearing sperm showed that X-bearing sperm cells possess highly significant individual and between-family variability in recombination rate, whereas Y-bearing sperm show linkage homogeneity. To test whether the two phenomena are related, different logistic regression models were fitted to the data. The results show that sex ratio significantly correlates with changes in recombination rate among X-bearing but not among Y-bearing sperm. Different hypotheses to explain these observations are discussed.     

Variation in the frequency and type of sperm chromosomal abnormalities among normal men

Summary The chromosomal constitution of 1582 human sperm from 30 normal men of proven fertility was investigated after sperm penetration of hamster eggs. A minimum of 30 sperm chromosome complements were analysed per donor so that the distribution and variation in the frequency and type of sperm chromosomal abnormalities could be assessed. The mean frequency of sperm chromosomal abnormalities in individual men was 10.4% (±6.0%) with a range of 0–24.7%. For numerical abnormalities the mean was 4.7% (±2.9%) with a range of 0–10% and for structural abnormalities the mean was 6.2% (±6.0%) with a range of 0–23.1%. The 95% confidence intervals for the mean of an individual male were 0–10.5% for numerical abnormalities, 0–18.2% for structural abnormalities, and 0–22.4% for total abnormalities. There was a significant excess of hypohaploid complements compared with hyperhaploid complements. Since hypohaploid complements could be caused by technical artefact, a conservative estimate of aneuploidy was obtained by doubling the frequency of hyperhaploid sperm, yielding an estimate of 2.4% aneuploidy. The proportion of X-bearing (53%) and Y-bearing (47%) sperm did not differ significantly. These results were compared to the other two large studies of sperm chromosome complements from normal men.     

The murine Rb(6.16) translocation: evidence for sperm selection and a modulating effect of aging

Summary The segregation products of the Rb(6.16) translocation were studied at first cleavage metaphase. Male mice heterozygous for the translocation mated at 3- and 14-day intervals to superovulated random-bred ICR females. Chromosome preparations of the recovered one-cell embryos were sequentially G- and C-banded and male and female complements analyzed cytogenetically. Of the 309 zygotes analyzed from both mating groups, no unbalanced segregants of the translocation were observed. In the 3-day group there was a highly significant difference (P<0.001) from the expected 1:1 ratio between sperm with normal (70.5%) and balanced segregants (26.2%) of alternate segregation. In the 14-day group the level of significance for the difference was ten times lower (P<0.01) as normal segregants were observed in 56.4% of the sperm and balanced ones in 36.5%. A comparison of the two groups using a 2×2 contingency table showed that segregant type was related to mating frequency (P<0.05). There was a highly significant distortion (P<0.01) of the sex ratio, with 178 Y-bearing and 131 X-bearing sperm in the combined populations. When sex ratio was analyzed according to mating intervals, the distortion was significant (P<0.01) only for the 3-day group. An analysis of the sex ratio according to the segregant type showed no significant deviation from 1:1 for type 1 segregants, which had normal chromosomes, while type 2 segregants, with the translocation, had a deficiency of X-bearing sperm. This deficiency was significant (P<0.05) only for the 3-day population. Analysis of meiotic preparations showed no association between the translocation trivalent and the X-Y bivalent. Our results, obtained under physiological conditions, provide definitive evidence for sperm selection and support previous findings that aging of sperm can modify the effect of selection.     

Direct chromosomal analysis of human spermatozoa: preliminary results from 18 normal men.

Chromosomal analysis of 240 spermatozoa from 18 normal men was performed using in vitro fertilization of zona-free golden hamster eggs. The frequency of chromosome abnormalities in this population was 9.2% (22/240). Of the abnormal complements, 18 were aneuploid (13 hyperploid and five hypoploid) and four had a chromosome break. The sex ratio of Y-bearing to X-bearing sperm was .68. The frequency and type of sperm chromosome abnormalities is compared with those seen in spontaneous abortions.     

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.     

Cytogenetic analysis of sperm from a man heterozygous for a pericentric inversion, inv (3) (p25q21).

Human sperm chromosomes were studied in a man heterozygous for a pericentric inversion of chromosome 3(p25q21). The pronuclear chromosomes were analyzed after in vitro penetration of golden hamster eggs. A total of 144 sperm were examined: 69.2% were chromosomally balanced and 30.8% were recombinant. Of the balanced complements, the proportion with a normal chromosome 3 (37.6%) was approximately equal to the proportion with an inverted 3 (31.6%). Of the recombinant complements, the proportion of sperm with a duplication q/deletion p (17.3%) was approximately equal to the reciprocal event of duplication p/deletion q (13.5%). The recombinant chromosome 3 with a duplication q and deletion p has been observed in several abnormal children, but the duplication p/deletion q has never been reported. My results demonstrate that both recombinant chromosomes are produced as expected from an unequal number of crossovers within an inversion loop. In all likelihood the duplication p/deletion q chromosome is an early embryonic lethal because of the amount of genetic material deleted. The proportions of X-bearing (48.9%) and Y-bearing sperm (51.1%) were not significantly different from the expected 1:1 ratio. There was no evidence for an interchromosomal effect. Of the three inversions studied by human sperm chromosome analysis, recombinant chromosomes have been observed only in this case.     

Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting

Intact, viable X and Y chromosome-bearing sperm populations of the rabbit were separated according to DNA content with a flow cytometer/cell sorter. Reanalysis for DNA of an aliquot from each sorted population showed purities of 86% for X-bearing sperm and 81% for Y-bearing sperm populations. Sorted sperm were surgically inseminated into the uterus of rabbits. From does inseminated with sorted X-bearing sperm, 94% of the offspring born were females. From does inseminated with sorted Y-bearing sperm from the same ejaculates, 81% of the offspring were males. The probability of the phenotypic sex ratios differing from 50:50 were p less than 0.0003 for X-sorted sperm and p less than 0.004 for Y-sorted sperm. Thus, the phenotypic sex ratio at birth was accurately predicted from the flow-cytometrically measured proportion of X- and Y-bearing sperm used for insemination.     

The chromosome constitution of 1000 human spermatozoa

Summary Chromosomal analysis of 1000 spermatozoa from 33 normal men was performed using in vitro fertilization of zonafree golden hamster eggs. The frequency of abnormal sperm complements was 8.5%: 5.2% were aneuploid and 3.3% had a structural chromosome abnormality. The frequencies of hyperhaploid (2.4%) and hypohaploid (2.7%) sperm complements were not significantly different and all chromosome groups were represented among the aneuploid complements. The majority (22/33) of structurally abnormal complements had a chromosome break. The percentages of X and Y-bearing sperm were 53.9% and 46.1%, which is significantly different from the expected one to one ratio.     

Estimates of sperm sex chromosome disomy and diploidy rates in a 47,XXY/46,XY mosaic Klinefelter patient

A 47,XXY/46,XY male was investigated for the incidence of aneuploidy in sperm sex chromosomes using a three-colour X/Y/18 fluorescence in situ hybridisation (FISH) protocol. A total of 1701 sperm nuclei were analysed. The ratio of X-bearing to Y-bearing sperm did not differ from the expected 1 : 1 ratio although there were more 23,Y sperm than 23,X sperm (844 vs 795). There was a significantly increased proportion of disomy XY and XX sperm compared with normal controls (0.41% vs 0.10%, P < 0.001 and 0.29% vs 0.04%, P < 0.01). However, the incidence of YY sperm was similar to the controls (0.06% vs 0.02%). The diploidy rate was also significantly increased (1.7% vs 0.13%, P < 0.0001), as was disomy 18 (0.71% vs 0.01%) and 25,XXY (0.47% vs 0%). The results support the hypothesis that some 47,XXY cells are able to undergo meiosis and produce mature spermatozoa. Patients with mosaic Klinefelter syndrome with severe oligozoospermia have significantly elevated incidences of disomy XY and XX sperm and may be at a slightly increased risk of producing 47,XXX and 47,XXY offspring. Additionally, they may be at risk of producing offspring with autosomal trisomies. Hence, patients with Klinefelter mosaicism scheduled for intracytoplasmic sperm injection intervention should first undergo FISH analysis of their sperm to determine their risk. Received: 16 November 1998 / Accepted: 16 February 1999     

Increased frequency of X-bearing sperm in males from an infertility clinic: analysis by two-color fluorescence in situ hybridization

Semen samples from 34 men visiting the Lübeck infertility clinic were investigated using a two-color FISH method to determine the ratio of X- and Y-bearing sperm. The overall ratio was significantly shifted to a preponderance of X-containing sperm. A statistical comparison with seven reports from the literature which included 53 normal probands demonstrated in our patients a significant tendency of a preponderance of X-bearing sperm and significantly less Y-bearing sperm. Furthermore, the Lübeck sperm samples are remarkably more heterogeneous in respect to their variability of X- and Y-bearing spermatozoa than in the other mentioned studies with normal probands. These phenomena have to be evaluated in further studies on groups of infertile males showing similar infertility histories.     

Sex ratio in normal and disomic sperm: evidence that the extra chromosome 21 preferentially segregates with the Y chromosome.

In humans, deviations from a 1:1 male:female ratio have been identified in both chromosomally normal and trisomic live births: among normal newborns there is a slight excess of males, among trisomy 18 live borns a large excess of females, and among trisomy 21 live borns an excess of males. These differences could arise from differential production of or fertilization by Y- or X-bearing sperm or from selection against male or female conceptions. To examine the proportion of Y- and X-bearing sperm in normal sperm and in sperm disomic for chromosomes 18 or 21, we used three-color FISH (to the X and Y and either chromosome 18 or chromosome 21) to analyze >300,000 sperm from 24 men. In apparently normal sperm, the sex ratio was nearly 1:1 (148,074 Y-bearing to 148,657 X-bearing sperm), and the value was not affected by the age of the donor. Certain of the donors, however, had significant excesses of Y- or X-bearing sperm. In disomy 18 sperm, there were virtually identical numbers of Y- and X-bearing sperm; thus, the excess of females in trisomy 18 presumably is due to selection against male trisomic conceptions. In contrast, we observed 69 Y-bearing and 44 X-bearing sperm disomic for chromosome 21. This is consistent with previous molecular studies, which have identified an excess of males among paternally derived cases of trisomy 21, and suggests that some of the excess of males among Down syndrome individuals is attributable to a nondisjunctional mechanism in which the extra chromosome 21 preferentially segregates with the Y chromosome.     

The murine Rb(6.16) translocation: alterations in the proportion of alternate sperm segregants effecting fertilization in vitro and in vivo

Summary The segregation products of the mouse Rb(6.16)24 Lub male translocation carrier were analyzed at first cleavage metaphase to determine whether the proportion of normal, balanced, and unbalanced sperm segregants differ in fertilizations occurring in vivo and in vitro. From 34 males, the sperm genomes in 268 firstcleavage mouse embryos were analyzed cytogenetically: 137 and 131 following in vivo and in vitro fertilization, respectively. Both systems demonstrated a preponderance of alternate (67.2% and 54.2%) as compared to adjacent segregation (10.2% and 13.7% as estimated). A contingency table showed that the distribution of reciprocal alternate segregants differed significantly between the two fertilization environments (x ²=20.64, P<0.0005). Whereas chromosomally normal sperm were 3.6 times more likely than the balanced reciprocals to fertilize in vivo (78.3% normal:21.7% balanced), 11 ratios were recovered following in vitro fertilization (43.7% normal: 56.3% balanced). The data also showed an excess of Y-bearing sperm with the translocation in both in vivo and in vitro fertilization groups. In the latter these segregants were 3 times more likely than X-bearing ones to effect fertilization. These data suggest a phenotypic disadvantage of translocation-X-bearing sperm, possibly mediated through altered haploid gene expression on chromosome 6 and gene expression on the Y. The results show clear evidence for prezygotic selection in vivo and indicate that the environment in which fertilization occurs significantly affects the transmission frequency of this specific translocation.     

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.     

On the selective elimination of Y-bearing sperm

The potential use of antibodies that selectively recognize either X-bearing or Y-bearing sperm is self-evident. Thus our attention was directed to the fact that under optimal conditions, H-Y antibody lyses 50% of mouse spermatozoa. Accordingly, we asked whether expression of H-Y antigen is haploid in spermatozoa from XY male mice heterozygous for the autosomal dominantSxr gene, for if H-Y expression were haploid, H-Y antibody would be expected to kill 75% of spermatozoa derived from these XY,Sxr/- males. However, maximal lysis remained at the 50% level, which indicates that haploid expression of H-Y antigen and the potential immunoselection of Y-(or X-) bearing spermatozoa are unlikely.     

Analysis of aneuploidy for chromosomes 13, 21, X and Y by multicolour fluorescence in situ hybridisation (FISH) in a 47,XYY male.

The frequency of aneuploid sperm was assessed by fluorescence in situ hybridisation (FISH) in a 47,XYY male previously studied by sperm karyotyping. A total of 20,021 sperm were studied: 10,017 by two-colour FISH for chromosomes 13 and 21 and 10,002 by three-colour FISH for the sex chromosomes using chromosome 1 as an autosomal control for diploidy and lack of hybridisation. Results were compared with more than 500,000 sperm from 18 normal men. The frequencies of X-bearing (49.4%) and Y-bearing sperm (49.8%) were not significantly different from 50% as shown in our sperm karyotyping study. There was no significant increase in the frequency of diploid sperm compared with control donors. There was a significant increase in the frequency of disomy for chromosome 13 (p < 0.0001) and XY disomy (p = 0.0008) compared with control donors. However, since the frequency of disomy was 0.40% for chromosome 13 and 0.55% for XY disomy, it is not surprising that these increases were not discovered previously in our analysis of 75 sperm karyotypes. Our results suggest that the extra Y chromosome is eliminated during spermatogenesis in the majority of cells but that there may be a small but significant increase in the frequency of aneuploid sperm in these men.     

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.