FISH assessment of aneuploidy frequencies in mature and immature human spermatozoa classified by the absence or presence of cytoplasmic retention*

Ertug Kovanci1, Tamas Kovacs1, Elena Moretti1,3, Lynne Vigue1, Patricia Bray-Ward2, David C. Ward2 and Gabor Huszar1,4

1 The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, and 2 Department of Human Genetics, Yale University School of Medicine, New Haven, CT, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Previously, a relationship has been found between diminished cellular maturity of human spermatozoa and low-level expression of the testis-specific chaperone protein, HspA2. Because HspA2 is a component of the synaptonemal complex in rodents, and assuming that this is also the case in men, it was postulated that the frequency of chromosomal aneuploidies would be higher in immature versus mature spermatozoa. This question was examined in spermatozoa from semen and from 80% Percoll pellets (enriched for mature spermatozoa) of the same ejaculate in 10 oligozoospermic men. Immature spermatozoa with retained cytoplasm, which signifies spermiogenetic arrest, were identified by immunocytochemistry. Using fluorescence in-situ hybridization (FISH), ~7000 sperm nuclei were evaluated in each of the 20 fractions (142 086 spermatozoa in all) using centromeric probes for the X, Y and 17 chromosomes. The proportions of immature spermatozoa were 45.4 ± 3.4 versus 26.6 ± 2.2% in the two semen versus the Percoll groups (medians: 48.2 versus 25%, P < 0.001, n = 300 spermatozoa per fraction, total 6000 spermatozoa). There was also a concomitant decline in total disomy, total diploidy and total aneuploidy frequencies in the 80% Percoll versus semen fractions (0.17 versus 0.54%, 0.14 versus 0.26% and 0.31 versus 0.81% respectively, P < 0.001 in all comparisons). The mean decline of aneuploidies was 2.7-fold. With regard to the hypothesis that aneuploidies are related to sperm immaturity, there was a close correlation between the incidence of immature spermatozoa and disomies (r = 0.7, P < 0.001) but no correlation with diploidies (r = 0.03), indicating that disomies originate primarily in immature spermatozoa. It is suggested that the common factor underlying sperm immaturity and aneuploidies is the diminished expression of HspA2. In addition, the lack of this chaperone may also cause diminished cellular transport of proteins, such as DNA-repair enzymes or of the retention of cytoplasm that is extruded from normally maturing spermatozoa during spermiogenesis.

Key words: cellular maturity/chromosomal aneuploidy/diploidy frequency variations/HspA2 chaperone protein


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In an attempt to develop an objective assessment of male fertility, for the past 15 years we have pursued biochemical markers of sperm maturity and function. We have established that semen specimens with high sperm creatine phosphokinase B isoform (CK-B) concentrations have diminished fertility. Further studies showed that this relationship stems from the fact that the high content of CK-B, which is a cytoplasmic enzyme, is a reflection of cytoplasmic retention in spermatozoa, which in turn is caused by incomplete extrusion of the cytoplasm during the last phase of spermiogenesis (Clermont, 1963Go; Huszar et al., 1988Go; Huszar and Vigue, 1993Go). Thus, spermatozoa with high CK-B and high cytoplasmic content are of diminished maturity and, consequently, of diminished function. We have shown further that a protein with unique properties, which is thought to be a germ-cell specific CK-M isoform but recently identified as HspA2, a member of the 70 kDa testis-specific chaperone protein family (Huszar et al., 2000Go), is developmentally regulated and synthesized simultaneously with cytoplasmic extrusion. The presence of HspA2 is a characteristic of mature spermatozoa (Huszar and Vigue, 1990Go). Consequently, in semen samples, the relative concentrations of the chaperone protein and the CK-B isoform, or chaperone ratio (formerly CK-M ratio), expressed as [% HspA2/(HspA2+CK-B)], reflect the proportion of mature and immature spermatozoa. The negative predictive value of high CK activity and low chaperone ratio for the occurrence of pregnancies in couples treated with intrauterine insemination and IVF (Huszar et al., 1990Go, 1992Go) was demonstrated. This predictive value is related to the finding that immature spermatozoa with cytoplasmic retention show diminished binding to the zona pellucida (Huszar et al., 1994Go). Further, the proportion of mature and immature spermatozoa shows inter-individual variations even in normal men, and this proportion is largely independent from the sperm concentrations in the ejaculates.

The potential correlation between increased rates of sperm chromosomal abnormalities and male infertility has been explored by various investigators. The advent of fluorescence in-situ hybridization (FISH) with chromosome-specific DNA probes has facilitated the detection of aneuploidies of the X and Y chromosomes and of several autosomes in sperm samples. Most of the publications have focused upon three questions: (i) the rate of aneuploidies in fertile men; (ii) the rate of aneuploidies in infertile men; and (iii) variations in aneuploidy rates of autosomal versus sex chromosomes. An overview of these publications indicates that evidence is inconclusive, and the results show discrepancies (Robbins et al., 1993Go; Williams et al., 1993Go; Meschede et al., 1995Go; Moosani et al., 1995Go; Griffin et al., 1996Go; Downie et al., 1997Go; Bernardini et al., 1998Go; Rubes et al., 1998Go). In normozoospermic human populations the frequency of disomy reported for 10 autosomal chromosomes was a combined 0.13%, while the disomy rate for the sex chromosomes was higher, at 0.43% (0.07% for XX, 0.21% for YY and 0.15% for XY disomies; Spriggs et al., 1995; Martin et al., 1996). Other studies, however, have found no differences in disomy rates among autosomes and sex chromosomes in normal males (Guttenbach et al., 1994Go; Lu et al., 1994Go; Downie et al., 1997Go). Discrepancies in X and Y disomy rates have also been reported in spermatozoa from oligozoospermic males. Some studies showed no differences between infertile and fertile men: disomy rates for X and Y chromosomes in infertile men were 0.16% and 0.11%, and for the fertile group 0.13% and 0.08% respectively (Miharu et al., 1994Go). Others, however, found increases in selected autosomal and sex chromosome disomies in spermatozoa from infertile versus fertile males, or in men with low and high sperm concentrations (Moosani et al., 1995Go; Finkelstein et al., 1998Go). Based on data using biochemical markers of sperm maturity and function, it is suggested that the casual relationship between aneuploidy rates and infertility is the consequence of an inadequate definition of male infertility. Indeed, this classification is either based on the seminal sperm concentration and motility, or on the fertility history of the couple; whereas in a substantial percentage of oligozoospermic or even normozoospermic men, the proportion of mature spermatozoa will provide a truer indication of sperm fertilizing potential and infertility.

In previous studies, information regarding the relationship between sperm cellular maturity and chromosomal aneuploidies can be obtained from the following. First, the sperm enzyme lactate dehydrogenase C isoform (LDHC4), which is expressed in the developing male germ cell at the time of the commencement of the meiotic process (Wheat and Goldberg, 1977Go; Salehi-Ashtiani and Goldberg, 1993Go). It was found that ~40% of men who have an increased proportion of immature spermatozoa with cytoplasmic retention and diminished HspA2 chaperone ratios, also show low sperm LDHC4 concentrations, indicating defects in spermatogenesis that are initiated in the meiotic division stage (Lalwani et al., 1996Go). Second, the fact that the former CK-M was identified as HspA2, the human homologue of the HSP70-2 mouse chaperone protein, is important because HspA2 in human male germ cells is expressed at two critical points: in spermatocytes (although the presence of HspA2 in human synaptonemal complexes is not yet verified), and in terminal spermiogenesis (Huszar et al., 1990Go, 2000Go). Accordingly, the lack of this protein may be connected to both the defects of the meiotic process and to failure of cytoplasmic extrusion which is, according to our hypothesis, likely to be chaperoned by HspA2. Third, in the rodent model the 70 kDa chaperone forms part of the synaptonemal complex, and in HSP70-2 knock-out mice the meiotic process was disturbed in males (Allen et al., 1996Go; Dix et al., 1996Go). There is good evidence that synaptic anomalies during meiosis, chromosomal abnormalities and male infertility are related (Egozcue et al., 1983Go; MacDonald et al., 1994Go; Martin et al., 1996Go; Vendrell et al., 1999Go).

These observations led us to investigate the incidences of chromosomal aneuploidies in spermatozoa that originate in semen (lower proportion of mature spermatozoa) and in the 80% Percoll density gradient fractions (higher proportion of mature spermatozoa) of the same semen specimens. The enhancement of mature spermatozoa in 80% Percoll fractions is based on the lower specific gravity of immature spermatozoa with cytoplasmic retention compared with mature spermatozoa, which contain only the nucleus, mitochondrion and the sperm membrane (Huszar and Vigue, 1993Go). The experimental design relied on the observation that both mature and immature spermatozoa are present in virtually all semen samples. Thus, instead of characterizing men as `oligozoospermic' or `normozoospermic', or adhering to undefined clinical paradigms of `fertility' and `infertility', which may include couples with various origins of infertility, sperm fractions were prepared from the same semen samples containing higher and lower proportions of mature spermatozoa. The differences in constituent mature and immature spermatozoa were assessed by using CK-immunocytochemistry, a marker of cytoplasmic retention. The immature spermatozoa were identified by the presence of retained cytoplasm. Centromeric chromosome probes and FISH were used to detect aneuploidies in the two sperm populations. In line with our hypothesis, in mature and immature spermatozoa there were differences in aneuploidy frequencies, and the proportions of immature spermatozoa with cytoplasmic retention and frequency of aneuploidies were related.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Preparation of sperm fractions
Semen samples from 10 individuals were utilized. For the study of the semen fractions, 7–10 µl of neat semen were used to prepare each sperm smear on a laboratory glass slide. In order to prepare the corresponding 80% Percoll sperm fraction, an aliquot of the same semen sample was centrifuged through 2.0 ml of an 80% single-phase Percoll gradient at 500 g for 20 min at room temperature. The sperm pellet was resuspended in 2 ml human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) and re-centrifuged at 600 g for 10 min in order to eliminate the residual Percoll. The pellet was resuspended in HTF to a concentration of ~30–40x106 spermatozoa/ml, and smears were prepared on glass slides. The smears were fixed with methanol:acetic acid (3:1 ratio) for 10 min, air-dried, dehydrated in a series of 70, 80 and 100% ethanol, and stored at –70° C for the FISH experiments. Other sperm aliquots were subjected to CK-immunocytochemistry in order to determine the proportion of sperm with cytoplasmic retention. For the assessment of aneuploidy frequencies, ~7000 spermatozoa were evaluated in each sample (142 068 sperm nuclei in the 20 fractions of the 10 subjects). For the determination of the proportion of immature spermatozoa, 3x100 spermatozoa were assayed in each fraction (total 6000 spermatozoa). These studies were approved by the Human Investigation Committee of Yale School of Medicine.

CK-immunocytochemistry of individual spermatozoa
The procedures used have been described previously (Huszar and Vigue, 1993Go; Huszar et al., 1994Go). The washed spermatozoa were allowed to settle onto polylysine-treated microscope slides overnight in a humidity box at 5°C. The overlying solution was carefully pipetted off and replaced by 1% formalin in phosphate buffer/sucrose (PB-suc) for 20 min at 37°C. After removal of the formalin, the slide was allowed to air-dry. The spermatozoa were then blocked with 3% bovine serum albumin in PB-suc at 37°C, and treated with a 1:1000 dilution of polyclonal anti-CK-B antiserum overnight at 4°C (Chemicon Co., Temecula, CA, USA). After further PB-suc washes, the slide was processed with a biotinylated second antibody conjugated with horseradish peroxidase. The brown colour representing the CK-content of spermatozoa was developed by the ABC method (Vector, Burlingame, CA, USA and Sigma, St Louis, MO, USA). On each slide 300 spermatozoa were evaluated by two investigators and characterized as either mature (no cytoplasmic retention) or immature (CK-staining in spermatozoa, indicating cytoplasmic retention) (Figure 1a and bGo).



View larger version (95K):
[in this window]
[in a new window]
 
Figure 1. Visualization of the residual cytoplasm in mature and immature spermatozoa by CK-immunochemistry (Huszar and Vigue, 1993Go; Huszar et al., 1994Go). (A) Semen sperm fraction. (B) 80% Percoll fraction. Mature spermatozoa have clear heads; immature spermatozoa show various patterns of cytoplasmic retention (arrows). Spermatozoa with cytoplasmic retention are immature, regardless of the extent of the residual cytoplasm (original magnification, x700).

 
Preparation of sperm nuclei
Sperm slides were warmed to room temperature, and in order to render the sperm chromatin accessible to DNA probes, were first treated with 10 mmol/l dithiothreitol (DTT; Sigma) in 0.1 mol/l Tris–HCl, pH 8.0 for 30 min and then with 10 mmol/l lithium diiodosalicylate (LIS; Sigma) in Tris–HCl for 1–3 h.

DNA probes
The FISH studies were carried out using three probes: (i) a 20 kb assigned to the Xp11-Xp21 region of chromosome X (pXBR-1; Yang et al., 1982Go); (ii) Vysis (Downers Grove, IL, USA) alpha satellite rhodamine-labelled probe for the Y chromosome; and (iii) alpha-satellite sequence-specific probe for chromosome 17 (p17H8; Waye and Willard, 1986). The DNA probes for chromosome X and 17 were labelled indirectly with a hapten-conjugated nucleotide (biotin-16-dUTP or digoxigenin-11-dUTP) by nick translation (Rigby et al., 1977Go), and hybridized to metaphase chromosome spreads to develop optimal conditions for probe binding.

In-situ hybridization
Since multicolour FISH was necessary for the study of the frequencies of disomy and diploidy in the sex chromosomes, the probes for chromosomes X, Y and 17 were hybridized simultaneously. In these experiments, the chromosome 17 probe was combinatorially labelled with both biotin and digoxigenin nucleotides so that its fluorescence profile would be a combination of red and green (yellow/orange). A 12 µl sample of hybridization mixture (50% formamide, 10% dextran sulphate, 2xSSC) containing the probes was denatured at 75–80°C for 8 min and applied to the slide specimens previously denatured in 70% formamide, 2xSSC for 8 min at 70°C. The hybridization was carried out at 37°C in a moist chamber for 12–24 h.

Post-hybridization washes were performed with 50% formamide-2xSSC three times at 42°C and another three times with 0.1xSSC at 60°C in order to remove excess probe reagents. After a blocking step in 4xSSC/3% bovine serum albumin/0.1% Tween-20 for 30 min at 37°C, the sperm nuclei were incubated for 30 min at 37° C with avidin-FITC (fluorescence green; Roche Biochemicals, Indianapolis, IN, USA) for biotin labelled-probes, and anti-digoxigenin-rhodamine (fluorescence red; Roche Biochemicals) for digoxigenin-labelled probes. The slides were then washed with 4xSSC/0.1%Tween-20 at 42°C three times, and after staining with 4'-6 diamidino-2-phenylindole (DAPI; Sigma), they were mounted with an antifade solution Vectashield (Vector).

Scoring criteria and data collection
For each patient, two slides of both the initial and the mature sperm fractions were scored by two independent investigators, totalling >14 000 spermatozoa on the four slides. The overall hybridization efficiency in these experiments was >98%. Sperm nuclei were scored according to published criteria (Martin and Rademaker, 1995Go). Nuclei were eliminated from the scoring if they overlapped, or if they displayed no signal due to hybridization failure. In the case of aneuploidy, the presence of the sperm tail was confirmed. A spermatozoon was considered disomic when it showed two fluorescent domains of the same colour, comparable in size and brightness in the approximately same focal plane, and clearly positioned inside the edge of the sperm head and at least one domain apart. Diploidy was recognized by the presence of two double fluorescence domains with the above criteria. Scoring was performed on an Olympus AX70 epifluorescence microscope primarily with a triple bandpass filter for DAPI, FITC and rhodamine (Chroma Technologies Co., Brattleboro, VT, USA), with monochrome filters for DAPI, FITC and rhodamine for improved signal resolution. Aneuploid spermatozoa were always examined with all of the above filters, and also with a phase-contrast objective in order to verify the presence of the tail and to exclude apparent diploidy in two spermatozoa in close proximity.

Statistical analysis
Statistical analyses were performed using SigmaStat 2.0 (Jandel Corporation, San Rafael, CA, USA). Differences in disomy and diploidy frequencies were analysed using {chi}2 analysis of contingency tables. Because of the multiple comparisons in the determination of each nucleus for the various disomies and diploidies, P <= 0.02 was considered as the level of significance. Correlation between the proportion of immature spermatozoa and aneuploidy frequencies was examined with Spearman rank correlation.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Semen characteristics, CK-immunocytochemistry and proportion of immature spermatozoa in semen and 80% Percoll sperm fractions
In order to test the hypothesis that aneuploidies are found primarily in immature spermatozoa, 10 moderately oligozoospermic men were studied [mean (± SEM) sperm concentration: 13.3 ± 1.4x106 spermatozoa/ml, motility: 50.3 ± 3.4%]. The selection of this patient population was based on studies in which a relationship among cytoplasmic retention, lack of HspA2 expression and sperm immaturity was established. The sperm population from semen contains a higher proportion of immature spermatozoa with brown CK-immunostaining (Figure 1aGo) than the sperm fraction prepared from the 80% Percoll pellet (Figure 1bGo), which clearly shows a higher number of spermatozoa with clear heads without cytoplasmic retention. Accordingly, the proportion of immature spermatozoa in the initial semen and 80% Percoll fractions was 45.4 ± 3.4% versus 26.6 ± 2.2% (medians: 48.2 versus 25%, P < 0.001, n = 10).

XY ratios
Using the probes for the X, Y and 17 chromosomes allowed study of the parameters of X/Y ratio, disomies, diploidies, total disomies, total diploidies and total aneuploidies in the 20 samples (total of 142 068 spermatozoa evaluated). The X/Y ratios were somewhat higher in the 80% Percoll fractions compared with semen, but the differences did not reach significance (1.08 versus 1.05, ranges: 1.0–1.19 versus 0.98–1.08, medians: 0.97 versus 1.12 respectively).

Aneuploidy and diploidy frequencies within and between the semen and 80% Percoll groups
There were substantial differences in aneuploidy and diploidy frequencies between the sperm nuclei arising from semen and from 80% Percoll fractions. Each individual had significant differences (using the stricter level of P <= 0.02) between one or more of the aneuploidy and diploidy categories (Table IGo). Subject #8 had one difference (total disomy), while subjects #5 and #9 had two differences (total disomy and aneuploidy), subjects #3, #4 and #10 had three differences, subjects #1, #2 and #7 had four differences, and subject #6 had five differences in aneuploidy frequencies between their spermatozoa originating in semen and in 80% Percoll. Among the 31 significantly different comparisons within the 10 individuals, 13 were at the level of P < 0.001. Twelve of these differences occurred among the disomy comparisons; diploidy XX in subject #2 was the only diploidy difference. The most frequent significantly different comparison between the semen and 80% Percoll sperm fractions in individuals was the total disomy (in all subjects) and total aneuploidy and diploidy (nine of the 10 subjects). Total diploidy frequencies were different only in subjects #6 and #7. With respect to wide variations in disomy and diploidy frequencies within the semen or Percoll fractions among the subjects, there were only two of note: XY disomies (0.03–0.68%, subjects #8 and #1) and XY diploidies (0.01–0.52%, subjects #10 and #5) in the semen fractions. Another aspect to note is the frequency differences in X, Y and XY disomies in spermatozoa of the initial semen (X versus Y: P = 0.023; Y versus XY: P < 0.001). The cumulative data of the 10 subjects (Table IGo) indicate that the aneuploidy and diploidy frequencies are significantly lower in the 80% Percoll fractions compared with the semen spermatozoa fractions (P < 0.001, n = 70 683 and 71 385 spermatozoa). The exception is YY diploidy.


View this table:
[in this window]
[in a new window]
 
Table I. Frequency of the various X, Y and 17 chromosome disomies and diploidies in the semen and 80% Percoll sperm fractions
 
Analysis of the total disomy, total aneuploidy (disomy + diploidy) categories illustrates well the differences in spermatozoa from semen compared with the 80% Percoll (Table IGo). There are two major findings: First, there is a significant decline in total disomy (0.54 versus 0.17%), total diploidy (0.26 versus 0.14%) and total disomy and diploidy (0.81 versus 0.31%, P < 0.001 in all comparisons). Second, the inter-individual variations in aneuploidy frequencies are also diminished in the 80% Percoll fractions. The distribution of the values is closer, as detected by the ranges of the point plot (Figure 2Go). These findings are in line with the enhancement of mature spermatozoa in the 80% Percoll fraction. Also, the 80% Percoll fraction is more homogeneous from the point of view of sperm maturity, and the aneuploidy and diploidy frequencies are also similar to those in normal men.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 2. Distribution of aneuploidy frequencies and the proportion of immature spermatozoa in the semen and 80% Percoll fractions in the 10 individuals.

 
The total sperm aneuploidy (disomy+diploidy) frequencies in the initial semen compared with 80% Percoll fractions were reduced 2.7-fold (range: 1.0–6.3). These data do not include the nine instances in which frequencies in the Percoll fractions were reduced to 0% (four disomies and five diploidies). The mean decline in the 10 sample pairs was more distinct in the comparison of disomies (3.2-fold, range: 2.4–5.1) than of diploidies (2.0-fold, range: 0.7 –3.0). Thus, disomies are more related to the elimination of immature spermatozoa from the semen than are diploidies.

Proportions of immature spermatozoa and sperm concentrations in the samples
It has been shown previously that the biochemical parameters of sperm maturity (CK activity, chaperone ratio and proportion of mature/immature spermatozoa) are independent of the sperm concentrations in the samples (Huszar et al., 1988Go, 1990Go; Huszar and Vigue, 1993Go). The data of the current study, although the group is small, support this observation well (Table IIGo). If the 10 semen samples are divided according to the five lower and five higher sperm concentrations, the group of five men with lower sperm density (subjects 1, 2, 3, 7 and 10) have an average sperm concentration of 10.0 ± 0.6x106 spermatozoa/ml, whereas in the other men (subjects 4, 5, 6, 8 and 9) the average is 16.5 ± 1.2x106 spermatozoa/ml, which is closer to the normozoospermic range (20x106 spermatozoa/ml) range. However, the proportion of immature spermatozoa is higher in the group with sperm concentrations of 16.5x106 versus 10x106/ml (49.8 ± 4.5% versus 41.4 ± 5.2%). This inverse relationship is also evident in the men with the lowest and highest sperm concentrations (subjects 2 versus 5 with 8x106 and 19x106 spermatozoa/ml), in whom the proportions of immature spermatozoa are 37% and 59% respectively. Finally, the lack of a consistent relationship between sperm maturity and concentrations is best demonstrated by the three men with sperm concentrations of 10x106 spermatozoa/ml (subjects 1, 7 and 10). The proportions of immature sperm in these three semen samples are 42, 24 and 55% respectively, bridging the entire range of the 10 men.


View this table:
[in this window]
[in a new window]
 
Table II. Sperm concentrations and proportions of immature spermatozoa in the 10 semen samples
 
Relationship between the proportions of immature spermatozoa and chromosomal aneuploidies
In order to substantiate further a potential relationship between the incidence of immature spermatozoa and of aneuploidies/diploidies, correlation analyses were performed between the proportion of immature spermatozoa with cytoplasmic retention and the frequencies of total disomes, total diploidies and total disomies+diploidies, respectively. In line with our hypothesis, the data indicated that there was a close correlation between the incidence of cytoplasmic retention and disomies in the 20 samples (r = 0.7, P < 0.001). Among the various disomies, the Y disomy correlated best with the incidence of immature spermatozoa in the samples (r = 0.78). However, there was no correlation at all between the immature spermatozoa and the incidence of diploidies (r = 0.03). Finally, due to the lack of contribution by the diploidies, there was a less consistent—but still significant—correlation between the incidences of immature spermatozoa and total disomies+diploidies (r = 0.48, P < 0.01). These data suggest that our hypothesis was correct, and that most of the disomies are found in immature spermatozoa with cytoplasmic retention.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chromosomal aneuploidy, disomy or diploidy occurs when a sperm cell possesses more or less than a single copy of each autosomal or sex chromosome, or more than one copy of the entire genome. As reviewed in the Introduction, the previously reported sperm aneuploidy frequencies between `fertile' and `infertile' men are inconsistent, because in some studies too few spermatozoa were evaluated and because normally occurring variations in aneuploidy frequencies among certain chromosomes are not fully recognized (Williams et al., 1993Go; Hassold, 1998Go; Egozcue et al., 2000Go). Our concepts and data on sperm maturation provides a third line of evidence. We suggest that the primary cause of the variability is the fact that the `fertile' and `infertile' designations are based on non-objective criteria, i.e. lack of pregnancies in the couples and/or sperm concentration parameters. In couples with oligozoospermic or asthenozoospermic husbands who otherwise have adequate concentrations of mature and fertile spermatozoa, the wives' ovulatory patterns, tubal patency, antisperm antibody status and similar conditions contributing to infertility are frequently overlooked because of the poor semen parameters and presumed male factor infertility.

The present study was designed to establish the incidence of chromosomal aneuploidies in mature and immature spermatozoa originating in the same semen specimens. Semen and 80% Percoll sperm fractions were studied in which the incidence of immature spermatozoa with cytoplasmic retention, as assessed by CK-immunocytochemistry, was lower. Percoll centrifugation takes advantage of the lower specific gravity of spermatozoa with cytoplasmic retention compared to that of mature spermatozoa (Huszar and Vigue, 1993Go; Aitken et al., 1994Go). In the 10 subjects we reconfirmed the inter-individual variations in the proportion of mature and immature spermatozoa, independently of their sperm concentrations (Table IIGo). It is of note that in addition to cytoplasmic retention and lack of plasma membrane remodelling, there are also nuclear features of sperm immaturity, such as delay in the histone-protamine transition, or retention of high levels of lysine-rich histones. Indeed, a relationship was reported between aniline blue staining, and the frequency of some chromosomal aneuploidies (Morel et al., 1998Go).

The goals of this study were as follows: (i) to assess the frequencies of chromosomal aneuploidies in sperm fractions with different proportions of mature and immature spermatozoa; (ii) to examine the relationships between frequencies of either disomy or diploidy and the incidence of immature spermatozoa with cytoplasmic retention; and (iii) to test our hypothesis regarding the potential relationship between sperm immaturity and the incidence of chromosomal aneuploidies, as they may be related to adverse upstream meiotic events of spermatogenesis. We are assuming here that in man (similar to rodents) the 70 kDa HspA2 chaperone is part of the synaptonemal complex. Thus, we anticipated that the common factor leading to increased rates of aneuploidy and arrested spermiogenetic maturation is the diminished expression of the HspA2.

In the mature versus immature sperm fractions there was a somewhat (but not significantly) higher X/Y ratio, very close to the 50–50% range. In the 80% Percoll sperm fractions compared with the semen fractions of individual men (Table IGo), there was a significant decline in disomy and diploidy frequencies. Among all categories there were 31 such differences, 13 of these were significant at the P < 0.001 level, and the others at P < 0.1 and P <= 0.02. In addition, the man-to-man variation in aneuploidies and in the proportion of the immature spermatozoa also attenuated in the 80% Percoll compared with semen sperm fractions (Figure 2Go).

With respect to aneuploidy frequencies, it was found that the immature versus mature sperm fractions had substantially higher rates of aberrations, whether considering the X, Y or autosomal chromosome disomies or diploidies. All comparisons were different at the level of P < 0.001. The rates for the sex and autosomal chromosome abnormalities were similar, both within the immature (mean of X, Y, XY disomies and diploidies: 0.10%, 17 disomy: 0.10%) and mature spermatozoa (mean of X,Y,XY disomies and diploidies: 0.04%, 17 disomy: 0.04%; Table IGo), although the XY disomy and XY diploidy rates showed high variability among men. The overall frequencies of disomy plus diploidy provide the aggregate picture. With the exception of the YY diploidies, in which the difference did not reach significance, the other frequencies showed a decline by 1.5- to 4-fold (mean 2.7-fold) in the 80% Percoll fractions. The highest mean aneuploidy rates occurred in the XY disomies. In the 80% Percoll versus the semen fractions, total disomy and total diploidy declined >3-fold and >2-fold respectively (0.54 versus 0.17% and 0.81 versus 0.31%).

In line with our hypothesis, the frequency of aneuploidies and the proportion of immature spermatozoa indicated a significant correlation (r = 0.48, P < 0.001, n = 20 fractions). These data further strengthen the hypothesis that the majority of aneuploidies are found in immature spermatozoa. Further analysis of this correlation yielded a very interesting new finding. The correlation between all disomies and the proportion of immature spermatozoa was r = 0.70 (with Y disomies alone, r = 0.78), whereas the relationship with diploidies was random (r < 0.1). This is in spite of the fact that the diploidy frequencies were lower (P < 0.001) in the 80% Percoll compared with the semen sperm fractions. These differences in relationship between immature spermatozoa versus disomies or diploidies indicate there is a higher frequency of impaired meiotic division among immature spermatozoa. However, diploidies—which do not correlate with the incidence of immature spermatozoa in the samples—are likely to arise by diverse cellular mechanisms (Egozcue et al., 2000Go).

Data from other laboratories in men with presumptive mature and diminished maturity sperm populations, in which at least 10 000 nuclei per normozoospermic man were evaluated, showed disomy and diploidy rates which were similar to our data for spermatozoa in the 80% Percoll pellets (Martin et al., 1996Go; Downie et al., 1997Go). For instance, in the former of these studies the mean disomy rates for chromosomes 1, 12, X, Y and XY were 0.07–0.16, while in the latter study the respective values for chromosomes 3, 7, 16, X, Y and XY were between 0.05% and 0.20%, similar to the 0.03% to 0.14% for chromosomes X, Y, XY and 17 in the current study.

Several recent reports have dealt with semen samples of severe oligoozospermia and/or high incidence of abnormal sperm morphology, both of which are indicators of sperm immaturity. In one such study (Storeng et al., 1998Go), aneuploidy rates were studied in 19 men who were triaged to IVF or intracytoplasmic sperm injection (ICSI), based on their higher and lower sperm concentrations. The overall disomy rates, although different in absolute values, were about 20-fold higher in the ICSI group compared with the IVF group. The extremely high rate of aneuploidies in men who have few spermatozoa, and thus are likely to have a high proportion of immature spermatozoa, was also documented (In't Veld et al., 1997Go). According to others (Bernardini et al., 1998Go), in oligoozospermic samples with a high incidence of abnormal sperm morphology, there were significantly higher incidences of sperm aneuploidies. These authors suggest that a direct relationship may exist between the impairment of the spermatogenetic process and increased rates of aneuploidy. This idea accords well with our combined evidence of diminished spermiogenetic maturation and infertility that we developed, based on the high sperm CK content and low levels of HspA2 expression (CK-M at the time) which predicted the lack of pregnancies (Huszar et al., 1990Go, 1992Go). Further evidence was developed (Aran et al., 1999Go; Pang et al., 1999Go) following investigations of aneuploidy frequencies in male infertility patients. In subjects who had higher sperm density, and presumably higher proportions of mature spermatozoa, there were lower rates of disomies and diploidies compared with ICSI patients. Thus, sperm immaturity is related to meiotic defects indicated by the diminished expression of the HspA2 and LDHC4 (Lalwani et al., 1996Go; Huszar et al., 2000Go), and there was an increased frequency of aneuploidies in men treated with ICSI compared with normal men. There is also an association between sperm immaturity and the increased rates of lipid peroxidation along with the aneuploidies. Heavy smokers, who exhibit antioxidant depletion and increased sperm lipid peroxidation, showed elevated frequencies of disomies as well as an increased proportion of `round-headed' sperm which, as discussed, is the hallmark of cytoplasmic retention and diminished sperm maturity (Huszar and Vigue, 1993Go, 1994Go; Aitken et al., 1994Go; Gomez et al., 1996Go; Rubes et al., 1998Go; Twigg et al., 1998Go).

Considering the clinical aspects of male infertility, an association between oligoozospermia and synaptic anomalies during meiosis has already been suggested in 46,XY males, resulting in aneuploid and diploid spermatozoa (Egozcue et al., 1983Go; Martin et al., 1996Go; Aran et al., 1999Go; Vendrell et al., 1999Go). It is also known that trisomies, originating in non-disjunction during meiosis, are found in ~5% of clinically evidenced pregnancies, although trisomies of paternal origin are rare in newborns and in abortion materials (Hassold et al., 1992Go; MacDonald et al., 1994Go). The variability of non-disjunction rates among men is well demonstrated in the present study by the wide fluctuations in XY disomy frequencies (0.03–0.68%; Table IGo) among the 10 subjects. Another interesting aspect of the association between poor sperm parameters, reflecting sperm immaturity, and increased frequency of aneuploidies, is the further relationship with abnormal karyotypes (Vegetti et al., 2000Go). Thus, diminished HspA2 concentrations may also cause structural chromosomal aberrations as well as aneuploidies.

The relationship between increased aneuploidy frequencies and sperm immaturity, based on the diminished expression of HspA2, are also supported by a rodent model. Targeted disruption of the HSP70-2 gene (the homologue of HspA2 in the mouse) resulted in failed meiosis because HSP70-2 is a component of the mouse synaptonemal complex during the meiotic prophase, and HSP70-2 disruption results in synaptic abnormalities (Allen et al., 1966Go; Dix et al., 1996Go). The potential connections between aneuploidies and synaptic (meiotic pairing) defects have been suspected earlier (Hultén and Pearson, 1971Go; Skakkebaek et al., 1973; Egozcue et al., 1983Go; Hassold, 1998Go; Ashley, 2000Go). Our hypothesis, and the close correlation between disomy and the proportion of immature spermatozoa in the samples (r = 0.70–0.78), are consistent with a model in which the diminished presence of HspA2 chaperone in the male germ cell leads to various defects during the meiotic phase and during spermiogenetic maturation (delivery of DNA repair enzymes, cytoplasmic extrusion, plasma membrane remodelling), all of which may be interrupted in the absence of the HspA2. The common origin of defects in both synaptic/meiotic events and sperm maturation is one testable idea, though the alternative possibility that the lack of HspA2 expression and arrested cytoplasmic extrusion and plasma membrane remodelling are corollary consequences of an impaired spermatogenetic programme could also be valid.

In addition to providing a hypothesis for further investigations of the cell biology and the genetic aspects of spermatogenesis, the current study is of particular interest for clinicians who practice ICSI (Palermo et al., 1993Go). Immature spermatozoa, which have a 2- to 4-fold higher rate of chromosome abnormalities than mature spermatozoa (based only on the X, Y and 17 chromosomes), are not likely to be part of the fertilizing pool, because immature spermatozoa that have not completed the spermiogenetic plasma membrane remodelling are deficient in zona-binding site(s) (Huszar et al., 1994Go, 1997Go). However, in ICSI, immature spermatozoa may be used for fertilization. One of the consequences is the approximately 4- to 5-fold higher rate of sex chromosome aberrations in offspring of ICSI pregnancies (Bonduelle et al., 1998Go).

In summary, in line with the diminished expression of LDHC4 and HspA2 in immature human spermatozoa, indicating upstream defects of the meiotic process during spermatogenesis (Lalwani et al., 1996Go; Huszar et al., 2000Go), we have now shown that the occurrence of X, Y, XY and 17 chromosome aneuploidies is 1.5- to 4-fold higher in immature compared with mature spermatozoa. Further, a close correlation between the proportion of immature spermatozoa and the frequencies of total aneuploidies and disomies (but not of diploidies) was demonstrated, indicating that disomies primarily occur in immature spermatozoa. Because the proportion of mature spermatozoa show substantial inter-individual variation, independent of sperm concentrations (Huszar et al., 1988Go, 1990Go, 1992Go), we suggest that the uncertainty among reports regarding the chromosomal aneuploidy frequencies is due to the fact that infertility is poorly defined by the sperm concentrations, or by the fertility history of couples.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors gratefully acknowledge the helpful comments and discussions with Professors Mary Ann Handle, Baccio Bacceti and Terry Ashley. They are also grateful to Professor Andy Wyrobek for welcoming Dr Ertug Kovanci for a training visit in his laboratory. This research was supported by the NIH (HD-19505 and HD-32902 to G.H.), by the Italian Research Council to E.M., and by POI-GM057672–01 to P.B.W. and D.W.


    Notes
 
3 Present address: Institute for Germ Cell Biology, University of Sienna, Sienna, Italy Back

4 To whom correspondence should be addressed at: The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8063, USA. E-mail: gabor.huszar{at}yale.edu Back

* Presented in part at the 1999 Meeting of the American Society of Reproductive Medicine, Toronto, Canada, September 25–30, 1999. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Aitken, R.J., Krausz, C. and Buckingham, D. (1994) Relationship between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Mol. Reprod. Dev., 39, 268–279.[ISI][Medline]

Allen, R.L., Dix, D.J., Collins, B.W. et al. (1996) HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes. Chromosoma, 104, 414–421.[ISI][Medline]

Aran, B., Blanco, J., Vidal, F. et al. (1999) Screening for abnormalities of chromosomes X, Y and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization-intracytoplasmic sperm injection program. Fertil. Steril., 72, 699–701.

Ashley, T. (2000) An integration of old and new perspectives of mammalian meiotic sterility. Results Probl. Cell Differ., 28, 131–173.[Medline]

Bernardini, L., Borini, A., Preti, S. et al. (1998) Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men. Hum. Reprod., 13, 3406–3413.[Abstract]

Bonduelle, M., Aytoz, A. and Van Assche, E. (1998) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum. Reprod., 13, 781–782.[Free Full Text]

Clermont, Y. (1963) The cycle of the seminiferous epithelium in man. Am. J. Anat., 112, 35–51.[ISI]

Dix, D.J., Allen, J.W., Collins, B.W. et al. (1996) Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc. Natl Acad. Sci. USA, 93, 3264–3268.[Abstract/Free Full Text]

Downie, S.E., Flaherty, S.P., Swann, N.J. et al. (1997) Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in spermatozoa from 10 normozoospermic men using fluorescence in-situ hybridization. Mol. Hum. Reprod., 3, 815–819.[Abstract]

Egozcue, J., Templado, C., Vidal, F. et al. (1983) Meiotic studies in a series of 1100 infertile and sterile males. Hum. Genet., 65, 185–188.[ISI][Medline]

Egozcue, S., Blanco, J., Vendrell, J.M. et al. (2000) Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Hum. Reprod. Update, 6, 93–105.[Abstract/Free Full Text]

Finkelstein, S., Mukamel, E., Yavetz, H. et al. (1998) Increased rate of nondisjunction in sex cells derived from low-quality semen. Hum. Genet., 102, 129–137.[ISI][Medline]

Gomez, E., Buckingham, D.W., Brindle, J. et al. (1996) Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J. Androl., 17, 276–287.[Abstract/Free Full Text]

Griffin, D.K., Abruzzo, M.A., Millie, E.A. et al. (1996) Sex ratio in normal and disomic sperm: evidence that the extra chromosome 21 preferentially segregates with the Y chromosome. Am. J. Hum. Genet., 59, 1108–1113.[ISI][Medline]

Guttenbach, M., Schakowski, R. and Schmid, M. (1994) Incidence of chromosome 3, 7, 10, 11, 17 and X disomy in mature human sperm nuclei as determined by nonradioactive in situ hybridization. Hum. Genet., 93, 7–12.[ISI][Medline]

Hassold, T. (1998) Non-disjunction in the human male. In Handel, M.A. (ed.), Meiosis and Gametogenesis. Academic Press, San Diego, pp. 383–406.

Hassold, T.J., Pettay, D., Robinson, A. and Uchida, I. (1992) Molecular studies of paternal origin and mosaicism in 45,X conceptuses. Hum. Genet., 89, 647–652.[ISI][Medline]

Hultén, M. and Pearson, P.L. (1971) Fluorescent evidence for spermatocytes with two Y chromosomes in an XXY male. Ann. Hum. Genet., 34, 273–276.[ISI][Medline]

Huszar, G. and Vigue, L. (1990) Spermatogenesis related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa. Mol. Reprod. Dev., 25, 258–262.[ISI][Medline]

Huszar, G. and Vigue, L. (1993) Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentrations and abnormal head morphology. Mol. Reprod. Dev., 34, 292–298.[ISI][Medline]

Huszar, G. and Vigue, L. (1994) Correlation between the rate of lipid peroxidation and cellular maturity as measured by creatine kinase activity in human spermatozoa. J. Androl., 15, 71–77.[Abstract/Free Full Text]

Huszar, G., Corrales, M. and Vigue, L. (1988) Correlation between sperm creatine phosphokinase activity and sperm concentrations in normozoospermic and oligozoospermic men. Gamete Res., 19, 67–75.[ISI][Medline]

Huszar, G., Vigue, L. and Corrales, M. (1990) Sperm creatine kinase activity in fertile and infertile oligozoospermic men. J. Androl., 11, 40–46.[Abstract/Free Full Text]

Huszar, G., Vigue, L. and Morshedi, M. (1992) Sperm creatine phosphokinase M-isoform ratios and fertilizing potential of men: a blinded study of 84 couples treated with in vitro fertilization. Fertil. Steril., 57, 882–888.[ISI][Medline]

Huszar, G., Vigue, L. and Oehninger, S. (1994) Creatine kinase immunocytochemistry of human hemizona-sperm complexes: selective binding of sperm with mature creatine kinase-staining pattern. Fertil. Steril., 61, 136–142.[ISI][Medline]

Huszar, G., Sbracia, M., Vigue, L. et al. (1997) Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane ß-1,4,-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios. Biol. Reprod., 56, 1020–1024.[Abstract]

Huszar, G., Stone, K., Dix, D. and Vigue, L. (2000) The putative sperm creatine kinase M-isoform is identified as the 70 kDa testis expressed chaperone protein HspA2. Biol. Reprod., 63, 925–932.[Abstract/Free Full Text]

In't Veld, P., Brandenburg, H., Verhoeff, A. et al. (1997) ICSI and chromosomally abnormal spermatozoa. Hum. Reprod., 12, 752–754.[Abstract]

Lalwani, S., Sayme, N., Vigue, L. and Huszar, G. (1996) Biochemical markers of early and late spermatogenesis: relationship between LDHC4 and CK-M isoform concentrations in human sperm. Mol. Reprod. Dev., 43, 495–502.[ISI][Medline]

Lu, P.Y., Hammitt, D.G., Zinsmeister, A.R. and DeWald, G.W. (1994) Dual color fluorescence in situ hybridization to investigate aneuploidy in sperm from 33 normal males and a man with a t(2;4;8)(q23;q27;p21). Fertil. Steril., 62, 394–399.[ISI][Medline]

MacDonald, M., Hassold, T., Harvey, J. et al. (1994) The origin of 47,XXY and 47,XXX aneuploidy: heterogeneous mechanisms and role of aberrant recombination. Hum. Mol. Genet., 3, 1365–1371.[Abstract]

Martin, R.H. and Rademaker, A. (1995) Reliability of aneuploidy estimates in human sperm: results of fluorescence in situ hybridization studies using two different scoring criteria. Mol. Reprod. Dev., 42, 89–93.[ISI][Medline]

Martin, R.H., Spriggs, E. and Rademaker, A.W. (1996) Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225 846 sperm from 10 normal men. Biol. Reprod., 54, 394–398.[Abstract]

Meschede, D., Nieschlag, E. and Horst, J. (1995) The importance of clinical documentation in genetic studies of male infertility. Hum. Genet., 96, 500–501.[ISI][Medline]

Miharu, N., Best, R.G. and Young, R.S. (1994) Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization. Hum. Genet., 93, 502–506.[ISI][Medline]

Moosani, N., Cox, D.M., Pattinson, H.A. et al. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil. Steril., 64, 811–817.[ISI][Medline]

Morel, F., Mercier, S., Roux, C. et al. (1998) Interindividual variations of the disomy frequencies of human spermatozoa and their correlation with nuclear maturity as evaluated by aniline blue staining. Fertil. Steril., 69, 1122–1127.[ISI][Medline]

Palermo, G.D., Cohen, J., Alikani, M. et al. (1993) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil. Steril., 63, 1231–1240.

Pang, M.G., Hoegerman, S.F., Cuticcha, A.J. et al. (1999) Detection of aneuploidy for chromosomes 4,6,7,8,9,10,11,12,13,17,18,21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 1266–1273.[Abstract/Free Full Text]

Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol., 113, 237–251.[ISI][Medline]

Robbins, W.A., Segraves, R., Pinkel, D. and Wyrobek, A.J. (1993) Detection of aneuploid human sperm by fluorescence in situ hybridization: evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y. Am. J. Hum. Genet., 52, 799–807.[ISI][Medline]

Rubes, J., Lowe, X., Moore, D. et al. (1998) Smoking cigarettes is associated with increased sperm disomy in teenage men. Fertil. Steril., 70, 715–723.[ISI][Medline]

Salehi-Ashtiani, K. and Goldberg, E. (1993) Differences in regulation of testis specific lactate dehydrogenase in rat and mouse occur at multiple levels. Mol. Reprod. Dev., 35, 1–7.[ISI][Medline]

Skakkabaek, N.E., Bryant, J.I. and Philip, J. (1973) Studies on meiotic chromosomes in infertile men and controls with normal karyotypes. J. Reprod. Fertil., 35, 23–36.[Medline]

Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1995) Aneuploidy in human sperm: results of two- and three-color fluorescence in situ hybridization using centromeric probes for chromosomes 1, 12, 15, 18, X, and Y. Cytogenet. Cell Genet., 71, 47–53.[ISI][Medline]

Storeng, R.T., Plachot, M., Theopile, D. et al. (1998) Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol. Acta Obstet. Gynecol. Scand., 77, 191–197.[ISI][Medline]

Twigg, J.P., Irvine, D.S. and Aitken, R.J. (1998) Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum. Reprod., 13, 1864–1871.[Abstract]

Vegetti, W., Van Assche, E., Frias, A. et al. (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum. Reprod., 15, 351–365.[Abstract/Free Full Text]

Vendrell, J.M., Garcia, F. and Veiga, A. (1999) Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Hum. Reprod., 14, 375–378.[Abstract/Free Full Text]

Waye, J.S. and Willard, H.F. (1986) Structure, organization and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome. Mol. Cell. Biol., 6, 3156–3165.[ISI][Medline]

Wheat, T.E. and Goldberg, E. (1977) An allelic variant of the sperm-specific lactate dehydrogenase C (LDH-X) isozyme in humans. J. Exp. Zool., 202, 425–430.[ISI][Medline]

Williams, B.J., Ballenger, C.A., Malter, H.E. et al. (1993) Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes. Hum. Mol. Genet., 2, 1929–1936.[Abstract]

Yang, P.T., Hansen, S.K., Oishi, K.K. et al. (1982) Characterization of a cloned repetitive DNA sequence concentrated of the human X chromosome. Proc. Natl Acad. Sci. USA, 79, 6593–6597.[Abstract]

Submitted on December 13, 2000; accepted on March 12, 2001.