Variation in crossover interference levels on individual chromosomes from human males.

Crossovers (COs) generated by homologous recombination ensure the proper segregation of chromosomes during meiosis. COs exhibit interference, which leads to widely spaced COs along chromosomes. Strong positive CO interference has been found in humans. However, little is known about the extent of human CO interference. In this study, variations in CO interference over the entire human genome and among individuals were analyzed by immunofluorescence combined with fluorescence in situ hybridization of testicular biopsies from 10 control men. These methods allow for direct identification of the frequency and location of COs in specific chromosomes of pachytene cells. The strength of CO interference was estimated by fitting the frequency distribution of inter-CO distances to the gamma model. Positive interference among CO on chromosomes was observed in these men, and the strength of inter-arm interference was significantly stronger than that for intra-arm CO. In addition, interference was observed to act across the centromere. Significant inter-individual and inter-chromosomal variations in the levels of interference were found, with smaller chromosomes exhibiting stronger interference. Discontinuous chromosome regions (gaps) and unsynapsed chromosome regions (splits) in chromosome 9 had both cis and trans effects on CO interference levels. This is the first report that the interference level varies significantly across the whole genome and that, at least in the human male, anomalies in chromosome synapsis play an important role in altering CO interference levels.

Immunofluorescence analysis of human spermatocytes.

New immunofluorescence techniques allow visual identification of human cells in various stages of meiotic prophase. Antibodies to the synaptonemal complex, the centromere and sites of recombination allow these stages of meiotic prophase to be identified. The progress of chromosome synapsis, recombination and associated phenomena such as interference can be studied in normal men, translocation heterozygotes and men with infertility problems. This has greatly stimulated research in human meiosis, leading to many exciting studies on the mechanisms underlying recombination and the generation of chromosome abnormalities.

Is there a relationship between sperm chromosome abnormalities and sperm morphology?

This review explores the relationship between sperm chromosomal constitution and morphology. With the advent of techniques for obtaining information on the chromosome complements of spermatozoa, this relationship has been studied in fertile men and in men with a high frequency of chromosomal abnormalities. Using human sperm karyotype analysis, no relationship between sperm chromosome abnormalities and morphology was found in fertile men, translocation carriers or post-radiotherapy cancer patients. Fluorescence in situ hybridization (FISH) analysis has not generally revealed a specific association between morphologically abnormal sperm and sperm chromosome abnormalities, but has indicated that teratozoospermia, like other forms of abnormal semen profiles (aesthenozoospermia, oligozoospermia) is associated with a modest increase in the frequency of sperm chromosome abnormalities. However, FISH studies on some infertile men and mouse strains have suggested that certain types of morphologically abnormal spermatozoa, such as macrocephalic multitailed spermatozoa, are associated with a very significantly increased frequency of aneuploidy. Thus, there may be an association between sperm morphology and aneuploidy in infertile men with specific abnormalities.

Analysis of aneuploidy in spermatozoa from testicular biopsies from men with nonobstructive azoospermia.

Testicular sperm biopsy combined with intracytoplasmic sperm injection (ICSI) allows men with azoospermia the possibility of fathering a child. However, little information exists on the risk of chromosome abnormalities in their sperm. Multicolor fluorescence in situ hybridization (FISH) analysis was used to determine the frequency of sperm diploidy and disomy for the sex chromosomes in six men with normal karyotypes and non-obstructive azoospermia. A new method using microwave decondensation and codenaturation of sperm nuclei yielded a much larger number of sperm nuclei for FISH analysis than our previous study of men with azoospermia. A total of 59916 sperm were analyzed; more than 9000 sperm were scored for each man. The men with nonobstructive azoospermia had an increased frequency of sperm chromosomal disomy for YY, XY, total sex chromosomal disomy, and diploidy compared with 18 normal controls, but only YY disomy reached statistical significance. One infertile man had a frequency of 3.8% XY disomy and 4.3% diploidy, which was 13-fold and 7-fold higher than control donors, respectively. Our results suggest that some men with nonobstructive azoospermia have a significantly increased frequency of sex chromosomal abnormalities than normal men, but that the overall frequency of abnormalities is similar to that found in infertile men with abnormal semen parameters.

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

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.

Analysis of sperm karyotypes in a patient treated with griseofulvin.

Griseofulvin is known to interfere with chromosome segregation by binding to microtubule-associated proteins. Studies in mouse germ cells have demonstrated that griseofulvin can induce aneuploidy (numerical chromosome abnormalities) at therapeutic concentrations. The aim of this study was to determine if chronic griseofulvin treatment led to an increased frequency of sperm chromosome abnormalities in one male subject. We analyzed 290 full sperm karyotypes using the human sperm-hamster oocyte fusion system. The frequency of X- and Y-bearing sperm was equal. There was no increase in the frequency of numerical (1.7%) or structural (9.3%) abnormalities in the subject compared to unexposed controls. Although reassuring, this is the first report on this subject and future studies are needed to assess the risk of griseofulvin.

Abnormal progression through meiosis in men with nonobstructive azoospermia.

OBJECTIVE: To study meiotic abnormalities in men with nonobstructive azoospermia. DESIGN: Analysis of synaptonemal complex and recombination in testicular tissue. SETTING: Research laboratory. PATIENT(S): Twenty-nine men with nonobstructive azoospermia and 12 men with normal spermatogenesis. INTERVENTION(S): Testicular tissues were processed with immunofluorescent staining using antibodies against proteins associated with synaptonemal complex and recombination events. MAIN OUTCOME MEASURE(S): Synaptonemal complex configuration and recombination in meiosis I. RESULT(S): In patients with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: nearly half of them had a complete absence of meiotic cells, one case had germ cells arrested at the zygotene stage of meiotic prophase, and, in general, the rest had impaired fidelity of chromosome synapsis and recombination in pachytene cells. Compared with controls, these patients had significantly more cells in leptotene/zygotene and higher frequencies of unpaired chromosome regions in pachytene. Significantly reduced recombination, an increased frequency of achiasmate autosome bivalents, and sex univalents in pachytene were also observed in these patients with nonobstructive azoospermia. CONCLUSION(S): Defects in chromosome synapsis and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to the unexplained infertility in these patients.

Variation in MLH1 distribution in recombination maps for individual chromosomes from human males.

Meiotic recombination is essential for the segregation of homologous chromosomes and the formation of normal haploid gametes. Little is known about patterns of meiotic recombination in human germ cells or the mechanisms that control these patterns. Documentation of the normal range of variability of recombination distribution over the genome among individuals is an essential prerequisite for understanding abnormal recombination patterns, which may be associated with non-disjunction and chromosome rearrangements. In this article, variation in recombination maps for individual chromosomes among 10 normal human males is examined for the first time. An immunocytogenetic approach allowed analysis of pachytene cells, using antibodies to detect the mature synaptonemal complex (SCP1/SCP3), the centromere (CREST) and sites of crossing over (MLH1). Individual bivalents were identified with centromere-specific multicolor fluorescence in situ hybridization. Significant heterogeneity in MLH1 focus frequency across donors was observed for larger chromosome arms (P<0.05, one-way ANOVA). Significant inter-donor variation in the overall crossover frequency per cell was also found (P<0.0001, one-way ANOVA). Furthermore, several chromosome arms showed significant differences in crossover distribution along the SCs among donors. Inter-individual variation in interference distances was observed for all chromosomes. The significance of altered recombination patterns among individuals and the role of interference are discussed.

Variation in meiotic recombination frequencies among human males.

Meiotic recombination is essential for the segregation of homologous chromosomes and the formation of normal haploid gametes. Little is known about patterns of meiotic recombination in human germ cells or the mechanisms that control these patterns. Here, newly developed immunofluorescence techniques, based on the detection of MLH1 (a DNA mismatch repair protein) foci on synaptonemal complexes (SCs) at prophase I of meiosis, were used to examine recombination in human spermatocytes. The mean number of MLH1 foci per cell in all donors was 48.0 with range from 21 to 65. Remarkable variation in the recombination frequency was noted among 11 normal individuals: the mean frequencies of chromosomal recombination foci ranged from a low of 42.5 to a high of 55.0 exchanges. Donor age did not contribute to this variation. There was no correlation between this variation and the frequency of gaps (discontinuities) or splits (unpaired chromosome regions) in the SCs. The mean percentage of cells with gaps was 35% (range: 20% to 58%) and with splits was 7% (range: 0% to 37%). Bivalents without a recombination focus were rare, with a frequency of only 0.3%. Thus, achiasmate chromosomes appear to be rare in human male meiosis.

Unusual segregation products in sperm from a pericentric inversion 17 heterozygote.

Chromosome segregation and interchromosomal effect were studied in spermatozoa from a carrier of a pericentric chromosome 17 inversion, 46,XY,inv(17)(p13.1q25.3). Sperm chromosome segregation, lymphocytes of the inversion carrier, and cells from his offspring were analysed by multicolour fluorescence in situ hybridization. The frequency of balanced sperm was 73%. An unusual segregation of recombinants was observed, viz. deletion of the p arm (14.6%) or duplication of the p arm with the presence of one q arm (8.4%), instead of the expected recombinants, viz. duplication of one arm with deletion of the other and vice versa. These unusual recombinants were explained by the position of the 17q breakpoint, which was between the q arm telomere-associated repeats and the unique q subtelomere region. The offspring of the donor were found to have a 17p deletion including the Miller-Dieker critical region, similar to the most frequent recombinant sperm class. The disomy frequency was significantly increased for chromosome 17 compared with other autosomes, suggesting that pairing and recombination of the inversion may predispose to non-disjunction. There was no significant difference between the frequencies of aneuploidy for chromosomes 13, 21, X and Y in the chromosome inversion heterozygote compared with controls. Thus, this unique pericentric inversion of chromosome 17 produces unusual recombinant products; no evidence was apparent of an interchromosomal effect in any of the tested chromosomes.

Discontinuities and unsynapsed regions in meiotic chromosomes have a cis effect on meiotic recombination patterns in normal human males.

During meiosis, homologous chromosome pairing is essential for subsequent meiotic recombination (crossover). Discontinuous chromosome regions (gaps) or unsynapsed chromosome regions (splits) in the synaptonemal complex (SC) indicate anomalies in chromosome synapsis. Recently developed immunofluorescence techniques (using antibodies against SC proteins and the crossover-associated MLH1 protein) were combined with fluorescence in situ hybridization (using centromere-specific DNA probes) to identify bivalents with gaps/splits and to examine the effect of gaps/splits on meiotic recombination patterns during the pachytene stage of meiotic prophase from three normal human males. Gaps were observed only in the heterochromatic regions of chromosomes 9 and 1, with 9q gaps accounting for 90% of these events. Most splits were also found in chromosomes 9 and 1, with 58% of splits occurring on 9q. Gaps and splits significantly altered the distribution of MLH1 foci on the SC. On gapped SC 9q, the frequency of MLH1 foci was decreased compared with controls, and single 9q crossovers tended toward a more distal distribution. Furthermore, the larger the gap the more distal the location of the MLH1 focus closest to the q arm's telomere. MLH1 foci on split SC 9 had distributions similar to those of gapped SC 9; however, splits did not change the frequencies of MLH1 foci on SC 9. This is the first demonstration that gaps and splits have an effect on meiotic recombination in humans.

Chromosome abnormalities in sperm from infertile men with asthenoteratozoospermia.

Research over the past few years has clearly demonstrated that infertile men have an increased frequency of chromosome abnormalities in their sperm. These studies have been further corroborated by an increased frequency of chromosome abnormalities in newborns and fetuses from pregnancies established by intracytoplasmic sperm injection. Most studies have considered men with any type of infertility. However, it is possible that some types of infertility have an increased risk of sperm chromosome abnormalities, whereas others do not. We studied 10 men with a specific type of infertility, asthenozoospermia (poor motility), by multicolor fluorescence in situ hybridization analysis to determine whether they had an increased frequency of disomy for chromosomes 13, 21, XX, YY, and XY, as well as diploidy. The patients ranged in age from 28 to 42 yr (mean 34.1 yr); they were compared with 18 normal control donors whose ages ranged from 23 to 58 yr (mean 35.6 yr). A total of 201 416 sperm were analyzed in the men with asthenozoospermia, with a minimum of 10 000 sperm analyzed per chromosome probe per donor. There was a significant increase in the frequency of disomy in men with asthenozoospermia compared with controls for chromosomes 13 and XX. Thus, this study indicates that infertile men with poorly motile sperm but normal concentration have a significantly increased frequency of sperm chromosome abnormalities.

Analysis of chromosome segregation in sperm from a chromosome 2 inversion heterozygote and assessment of an interchromosomal effect.

Using fluorescence in situ hybridization (FISH) analysis, the chromosome segregation of a pericentric inversion of chromosome 2 was studied in spermatozoa. An interchromosomal effect (ICE) was also determined for chromosomes 13, 21, X, and Y. This chromosome inversion included more than 2/3 of the total length of the chromosome and the breaks points were in G-light bands. The frequency of non-recombinant sperm was 55.9%, and that of recombinant sperm was 34.5% (with a 1:1 ratio of duplication of the p arm and deletion of the q arm and vice versa). There was a significantly increased frequency of disomy for chromosome 2 (0.6%) compared to the other autosomes, suggesting that pairing and recombination of the inversion may predispose to nondisjunction. There was no significant difference between the frequencies of aneuploidy for chromosomes 13, 21, X, and Y for the chromosome inversion heterozygote compared to control donors. Thus we did not find evidence for an ICE.

Absence of age effect on meiotic recombination between human X and Y chromosomes.

Recombination between the X and Y chromosomes is limited to the pseudoautosomal region and is necessary for proper segregation of the sex chromosomes during spermatogenesis. Failure of the sex chromosomes to disjoin properly during meiosis can result in individuals with a 47,XXY constitution, and approximately one-half of these result from paternal nondisjunction at meiosis I. Analysis of individuals with paternally derived 47,XXY has shown that the majority are the result of meiosis in which the X and Y chromosomes have failed to recombine. Our studies of sperm have demonstrated that aneuploid 24,XY sperm have a decreased recombination frequency, compared with that of normal sperm. Some studies have indicated a relationship of increased paternal age with 47,XXY offspring and with the production of XY disomic sperm, whereas others have failed to find such relationships. To determine whether there is a relationship between paternal age and recombination in the pseudoautosomal region, single-sperm genotyping was performed to measure the frequency of recombination between a sex-specific locus, STS/STS pseudogene, and a pseudoautosomal locus, DXYS15, in younger men (age < or =30 years) compared with older men (age > or =50 years). A total of 2,329 sperm cells were typed by single-sperm PCR in 20 men who were heterozygous for the DXYS15 locus (1,014 sperm from 10 younger men and 1,315 sperm from 10 older men). The mean recombination frequency was 39.2% in the younger men and 37.8% in the older men. There was no heterogeneity in the frequency of recombination rates. There was no significant difference between the recombination frequencies among the younger men and those among the older men, when analyzed by the clustered binomial Z test (Z=.69, P=.49). This result suggests that paternal age has no effect on the recombination frequency in the pseudoautosomal region.

A comparison of the frequency of sperm chromosome abnormalities in men with mild, moderate, and severe oligozoospermia.

Infertile men undergoing intracytoplasmic sperm injection have an increased frequency of chromosome abnormalities in their sperm. Men with low sperm concentration (oligozoospermia) have an increased risk of sperm chromosome abnormalities. This study was initiated to determine whether men with severe oligozoospermia (<10(6) sperm/ml) have a higher frequency of chromosome abnormalities in their sperm compared with men with moderate (1-9 x 10(6) sperm/ml) or mild (10-19 x 10(6) sperm/ml) oligozoospermia. Multicolor fluorescence in situ hybridization analysis was performed using DNA probes specific for chromosomes 13, 21, X, and Y (with chromosome 1 as an autosomal control for the sex chromosomes). Aneuploidy and disomy frequencies were assessed from a total of 603,011 sperm from 30 men: 10 in each of the categories. The mean frequencies of disomy for the patients with mild, moderate, and severe oligozoospermia were 0.17%, 0.24%, and 0.30%, respectively, for chromosome 13 and 0.22%, 0.44%, and 0.58%, respectively, for chromosome 21. For the sex chromosomes, the mean frequencies of disomy for mild, moderate, and severe oligozoospermia were 0.25%, 1.04%, and 0.68%, respectively, for XY, 0.047%, 0.08%, and 0.10%, respectively, for XX, and 0.04%, 0.06%, and 0.09%, respectively, for YY. The frequencies for diploidy also increased from 0.4% for mild to 1.20% for moderate to 1.24% for severe oligozoospermia. There was a significant inverse correlation between the frequency of sperm chromosome abnormalities and the sperm concentration for XY, XX, and YY disomy and diploidy. These results demonstrate that men with severe oligozoospermia have an elevated risk for chromosome abnormalities in their sperm, particularly sex chromosome abnormalities.

Human male recombination maps for individual chromosomes.

Meiotic recombination is essential for the segregation of chromosomes and the formation of normal haploid gametes, yet we know very little about the meiotic process in humans. We present the first (to our knowledge) recombination maps for every autosome in the human male obtained by new immunofluorescence techniques followed by centromere-specific multicolor fluorescence in situ hybridization in human spermatocytes. The mean frequency of autosomal recombination foci was 49.8+/-4.3, corresponding to a genetic length of 2,490 cM. All autosomal bivalents had at least one recombination focus. In contrast, the XY bivalent had a recombination focus in 73% of nuclei, suggesting that a relatively large proportion of spermatocytes may be at risk for nondisjunction of the XY bivalent or elimination by meiotic arrest. There was a very strong correlation between mean length of the synaptonemal complex (SC) and the number of recombination foci per SC. Each bivalent presented a distinct distribution of recombination foci, but in general, foci were near the distal parts of the chromosome, with repression of foci near the centromere. The position of recombination foci demonstrated positive interference, but, in rare instances, foci were very close to one another.