1 The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 Department of Genetics Yale University School of Medicine, New Haven, CT, USA and 3 Department of Obstetrics and Gynecology, University of Debrecen, Hungary (Attilasufrevacpt1otthon)
4 To whom correspondence should be addressed. e-mail: gabor.huszar{at}yale.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: aneuploidy/chromosomal aberrations/diminished maturity/FISH/swim-up
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The introduction of fluorescence in-situ hybridization (FISH) with chromosome-specific DNA probes has facilitated the detection of sperm with chromosomal aberrations. Publications dealing with FISH and swim-up have primarily focused upon two questions, without reaching a consensus: (i) what are the X and Y sex ratios in initial semen and swim-up fractions; and (ii) what is the elimination rate of sperm with aneuploidies and diploidies in swim-up fractions. There is substantial diversity in study findings, which may, in part, result from inconsistent patient selection and experimental design. However, the most significant confounding factor is the variation in number of sperm nuclei evaluated. For a reliable assessment of aneuploidy and diploidy rates, considering the mean frequency of 0.1 to 0.5% per chromosome, or one to five aberrant sperm nuclei per 1000, one should evaluate 500010 000 sperm in each sample. In the papers that dealt with sperm swim-up as discussed below, the range of sperm per sample studied was 1706000. In five of the papers cited, the number of sperm evaluated equalled 1000 or less.
Regarding the X and Y ratios between the initial semen and swim-up sperm fractions, some reports indicated no differences (Benet et al., 1992; Han et al., 1993
; Samura et al., 1997
; Pfeffer et al., 1999
; Calogero et al., 2001
). Other authors found an increase in the proportion of Y-bearing sperm after swim-up (Martinez-Pasarell et al., 1997
; Li and Hoshai, 1998
). With respect to elimination of aneuploid sperm, in two studies there were no differences between the initial and swim-up fractions (Samura et al., 1997
; Van Dyk et al., 2000
), while others reported a reduction of diploid sperm (Han et al., 1993
; Martinez-Pasarell et al., 1997
; Li and Hoshiai, 1998
). In comparing sperm selection by three methods, swim-up, glass wool filtration and two-phase discontinuous Percoll gradient centrifugation, Samura et al. (1997)
found no difference between initial semen and treated sperm fractions with respect to frequencies of diploidy and disomy. The sex chromosome aneuploidy and diploidy rates after swim-up were found to be unchanged by Martinez-Pasarell et al. (1997)
, whereas two other studies indicated a reduction of diploidy frequencies in swim-up fractions (Han et al., 1993
; Li and Hoshiai, 1998
).
There is general agreement that severely oligospermic men, who are candidates for ICSI, have higher rates of aneuploidies than normospermic men (Colombero et al., 1999; Pang et al., 1999
; Zeyneloglu et al., 2000
; Calogero et al., 2001
). In two swim-up studies of ICSI patients, the overall aneuploidy frequencies were higher than in normospermic men, but the X/Y ratios and rates of numerical chromosomal abnormalities were similar to those of the initial semen (Benet et al., 1992
; Calogero et al., 2001
).
Our interest in the efficacy of the swim-up method in elimination of sperm with aneuploidies and diploidies stems from our previous study, in which we addressed the relationship between the proportions of sperm with diminished cellular maturity and the frequencies of sperm with numerical chromosomal aberrations (Kovanci et al., 2001). In that study, as in the present work, immature sperm were monitored by the presence of cytoplasmic retention, which signifies arrest in terminal spermiogenesis, when the surplus cytoplasm of the elongated spermatids is extruded (Clermont, 1963
; Huszar et al., 1988a
; 1990
; Huszar and Vigue, 1993
). After 80% Percoll gradient centrifugation, the incidences of immature sperm and sperm with aneuploidy and diploidy were reduced in the Percoll pellet compared with the initial semen. The rate of reduction, or clearance, was 3.2x for disomies (range 2.45.1x in case of the autosomal- and sex-chromosomes) and 2.0x (range 0.73.0x) for diploidy frequencies (Kovanci et al., 2001
).
There was also a close correlation between the proportion of immature sperm and chromosomal disomes (with an r value of 0.7 for all disomies, and 0.78 for Y disomies, both P < 0.001; Kovanci et al., 2001). This relationship between the frequencies of chromosomal aneuploidies and diminished sperm maturity is probably based on the finding that in sperm with cytoplasmic retention and diminished maturity, there is a low expression of the 70 kDa testis-specific chaperone protein, HspA2 (Huszar et al., 2000
). As was shown in the mouse, the HSP70-2, a homologue of the HspA2 chaperone protein, is a component of the synaptonemal complex and also facilitates the intracellular movement of proteins (Dix et al., 1996
; Eddy, 1999
). This association may explain the relationship between meiotic errors, thus aneuploidies, and perhaps the presence of surplus cytoplasm in sperm of diminished maturity. Low sperm HspA2 [previously sperm creatine kinase M subunit (CK-M)] levels were also predictive for failure of pregnancies in two blinded studies of IVF couples (Huszar et al., 1992
; Ergur et al., 2002
).
In the present study we examined the efficiency of the swim-up technique in eliminating aneuploid or diploid sperm. In addition to the insight based upon the role of HspA2, the experience with the gradient centrifugation study (Kovanci et al., 2001) helped us to an improved experimental design. (i) In general, in samples that are oligospermic or are in the 2030 x 106/ml range there are higher proportions of immature sperm (Huszar et al., 1988a
; b
; 1990
; Huszar and Vigue, 1993
). For this reason, we have utilized samples primarily with <20 x 106sperm/ml, but also have included two normospermic men. (ii) We monitored by CK immunocytochemisty the proportions of sperm with diminished maturity in semen and in the swim-up fractions. (iii) We studied motile sperm yield, or the recovery of motile sperm, in the swim-up fractions. (iv) In order to validate further our study methods and results, we utilized five chromosome probes, and independently employed two- and three-colour FISH in evaluating at least 20 000 sperm in each patient.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of sperm for the FISH studies
Smears of the initial and swim-up fractions 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 20°C until FISH was performed.
CK immunochemistry of individual sperm
The procedures used were as described previously (Huszar and Vigue, 1993; Huszar et al., 1994
; Kovanci et al., 2001
). The washed sperm (whether from the initial semen or swim-up fractions) were allowed to settle onto polylysine-treated microscope slides overnight in a humidity chamber at 5°C. After a 20 min fixation with 1% formalin at 37°C, the overlying solution was carefully replaced with phosphate buffer/sucrose (PB-suc). The sperm 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). Furthermore, the slide was processed with a biotinylated second antibody conjugated with horseradish peroxidase. The brown colour representing the CK content of sperm was developed by the avidinbiotin complex (ABC) method (Vector, Burlingame, CA, USA and Sigma, St Louis, MO, USA). On each slide 300 sperm were evaluated by two investigators and characterized as either mature (no cytoplasmic retention) or immature (CK staining in sperm, indicating cytoplasmic retention).
Preparation of sperm nuclei for FISH
For decondensation, the sperm slides were warmed to room temperature, and in order to render the sperm chromatin accessible to DNA probes they were first treated with 10 mmol/l dithiothreitol (DTT; Sigma) in 0.1 mol/l TrisHCl, pH 8.0, for 30 min, and then with 10 mmol/l lithium diiodosalicylate (LIS; Sigma) in TrisHCl for 13 h.
DNA probes
The FISH studies were carried out using five probes: (i) a 20 kb repeated family probe assigned to Xp11Xp21 region of chromosome X (pXBR-1; Yang et al., 1982); (ii) microdissected probes for the Y chromosome (Guan et al., 1996
); alpha-satellite sequence specific centromeric probes for (iii) chromosome 17 (p17H8; Waye and Willard, 1986
), (iv) chromosome 10 (p
10RP8; Devilee et al., 1988
) and (v) chromosome 11 (pLC11A; Waye et al., 1987
). The DNA probes were labelled indirectly with a hapten-conjugated nucleotide (biotin-11-dUTP for chromosome 10, 17 and X probes, or digoxigenin-11-dUTP for chromosome 11, 17 and Y probes) by nick translation (Rigby et al., 1977
), and added to metaphase chromosome spreads to develop optimal conditions for hybridization.
FISH
In each individual, the initial and migrated fractions were examined using both two-colour and multicolour FISH. In order to detect the frequency of autosomal disomy and diploidy using chromosome 10 and 11 probes, two-colour FISH was utilized (1011 assay). Since three probes are necessary to study the frequencies of disomy and diploidy in the sex chromosomes, multicolour FISH was performed when chromosome X, Y and 17 were hybridized together (X-Y-17 assay). In the triple-probe FISH experiments, chromosome 17 was combinatorially detected with both biotin-labelled and digoxigenin-labelled probes, so that its fluorescence profile would be the combination of two colours (in our case red and green resulted in orange/yellow). A 12 µl sample of hybridization mixture (50% formamide, 10% dextran sulphate, 2x SSC) containing the probes was denatured at 7580°C for 8 min and applied to the slide specimens previously denatured in 70% formamide, 2x SSC for 8 min at 70°C. The hybridization was carried out at 37°C in a moist chamber for 1214 h. Post-hybridization washes were performed with 50% formamide/2x SSC three times at 42°C and another three times with 0.1x SSC at 60°C in order to remove the excess probe reagents. After a blocking step in 4x SSC/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 avidinFITC (fluorescence green; Roche Biochemicals, Indianapolis, IN, USA) for biotin-labelled probes, and anti-digoxigeninrhodamine (fluorescence red) for digoxigenin-labelled probes. The slides were then washed with 4x SSC/0.1%Tween-20 at 42°C three times, and after staining with 4'-6' diamino-2-phenylindole (DAPI; Sigma), they were mounted with an antifade solution (Vectashield; Vector Laboratories).
Scoring criteria and data collection
For each patient, two slides (double-probe FISH and triple-probe FISH) of both the initial and the swim-up sperm fractions were scored by two independent investigators, totalling >20 000 sperm on the four slides. The overall hybridization efficiency in these experiments was >98%. Sperm nuclei were scored according to published criteria (Martin and Rademaker, 1995). Since it is hard to interpret whether an absence of a signal indicates nullisomy or failure of hybridization, nullisomies were disregarded, as per generally accepted methods (Egozcue et al., 1997
). 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 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 approximately the same focal plane, 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 the triple pass filter for DAPI, FITC and rhodamine (Chroma Technologies Co., Brattleboro, VT, USA), with monochrome filters for DAPI, FITC and rhodamine for improved signal resolution and distinction. If extra intracellular chromosome signal was observed with the triple bandpass filter, it was always examined with the monochrome filters for DAPI (blue only), FITC (green only) and rhodamine (red only) to confirm the existence of an extra chromosome. Aneuploid or diploid sperm were always examined also with a phase-contrast objective in order to verify the presence of the tail and to exclude apparent diploidy in two sperm in close proximity.
For the assessment of aneuploidy frequencies, 10 000 sperm were evaluated in each sample (207 987 sperm nuclei in the 20 fractions from 10 subjects). For the determination of the proportion of immature sperm, 3x 100 sperm were assayed in each of the 20 samples (a total of 6000 sperm).
Statistical analysis
Statistical analyses were performed using SigmaStat 2.0 (Jandel Corporation, San Rafael, CA, USA). Differences in disomy and diploidy frequencies, as well as immature sperm rates were analysed using the 2 analysis of contingency tables. MannWhitney rank sum test were used to analyse the motility differences between the fractions. Correlations between the motility, the proportion of immature sperm and aneuploidy frequencies were examined with the Pearson correlation test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
There was no relationship between sperm concentrations and frequencies of aneuploidies. For instance, the five men in the upper sperm concentration range (patients 2, 3, 5, 6 and 10) and the five men in the lower sperm concentration range (patients 1, 4, 7, 8 and 9) had disomy frequencies of 0.33% (range 0.120.64) and 0.31% (range 0.250.39). This finding is in agreement with our previous studies, which demonstrated that both the proportions of immature sperm and the frequencies of aneuploidies were independent of sperm concentrations in semen samples (Huszar et al., 1988a; 1990
; 1992
; Kovanci et al., 2001
). However, in line with the association between diminished sperm maturity and the frequencies of aneuploidies, there was a moderate correlation between the incidences of sperm with cytoplasmic retention and disomies in the initial semen and in the swim-up fractions (r = 0.46, P < 0.05, n = 20).
Although disomy frequencies showed a declining trend in the swim-up fractions, these changes did not reach statistical significance for any of the five chromosomes investigated, except in one patient (no. 6). In this patient, the total frequency of 17, X and Y chromosome disomies was significantly higher in semen and lower in swim-up fraction (0.64 versus 0.34%; P < 0.05). Considering the data from all five probes following the swim-up procedure, the overall disomy frequencies decreased from 0.61% (range 0.341.13%) to 0.42% (range 0.210.59), with a reduction rate of 1.5x. In spite of variations in aneuploidy frequencies among the chromosomes studied, the reductions of the various disomies were proportional, as indicated by the correlations between aneuploidy frequencies in the swim-up versus initial semen fractions for Y disomy (r = 0.75, P < 0.01), 11 disomy (r = 0.76, P = 0.01), 17 disomy (r = 0.9, P < 0.001) and all five disomies (r = 0.75, P = 0.01). Conversely, the motile sperm yield was related to the clearance factor of eliminating disomies from the swim-up fractions (r = 0.65, P < 0.05, n = 10).
Diploidy frequencies in sperm of semen and swim-up fractions
The diploidy frequencies and reductions in the swim-up fractions showed outcomes quite different from those of the disomies (Table III). With respect to the three-colour FISH approach, there was a significant reduction in six of the 10 patients, with a 3-fold decline in diploidy rates for the group of 10 patients. Similarly, with probes 10 and 11, there was a significant decline in six of the 10 patients. Thus, the decline in diploidy frequencies was statistically significant in eight of the 10 samples at the level of P < 0.01. Five of these men showed declines with both the two- and three-colour FISH probes, whereas diploidy frequencies with the X, Y and 17 probes were reduced in patient 2, and with the two-colour FISH in patient 1. There was also considerable inter-individual variations, with a mean 0.65% (range 0.131.76%) in the initial semen, which decreased significantly to 0.24% (0.030.79%) in the swim-up fractions (P < 0.001). This decline represents a 2.7-fold reduction rate for diploidies.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We optimized the experimental design of the swim-up studies based on our experience with gradient centrifugation experiments. (i) In general, men having oligospermia or low normal sperm concentrations have a higher proportion of sperm with diminished maturity. Thus, we primarily used semen samples with sperm concentrations around the 20 x 106 sperm/ml range. However, since swim-up separation required a sufficient number of motile sperm, this precluded the use of severely oligospermic and/or asthenospermic samples. (ii) We monitored motile sperm yield, a parameter that reflects recovery of motile sperm in the swim-up fraction. The motile sperm yield is related to the clearance rate of disomic sperm (r = 0.65, P < 0.001, n = 10). (iii) We used five different chromosomal probes on two independent slides: X, Y and 17 as three-colour FISH, and 10 and 11 as two-colour FISH (10 000 sperm in each fraction and 20 000 sperm in each man evaluated). Our results were further validated by the correlation of r > 0.9 between the comparable data with three-colour and two-colour FISH. (iv) Finally, in addition to monitoring chromosomal aberrations, we also assessed the proportions of immature sperm both in initial semen and in the swim-up fractions. Sperm with diminished maturity were identified by the presence of retained cytoplasm, as highlighted by CK immunocytochemistry. The proportions of sperm with diminished maturity and with chromosomal aberrations showed a correlation (r = 0.46, P < 0.05, n = 20). This correlation was not as close as that for the gradient centrifugation study, in which the proportion of immature sperm and the frequencies of sperm with disomies were correlated at r = 0.7 for all disomies, and r = 0.78 for the Y disomies (Kovanci et al., 2001). This difference indicates that sperm motility is less discriminatory than sperm density and buoyancy, which are the bases of the gradient fractionation.
In comparing the efficiency of the swim-up and gradient methods in eliminating aneuploid and diploid sperm, one has to consider whether, despite moderate differences in actual sperm concentrations and motilities (20.3 ± 3.8 versus 13.3 ± 1.4 x 106 sperm/ml, 45.2 ± 2.4 versus 50.3 ± 3.4%, respectively), the two study populations are similar. We suggest that this is the case, and this notion is supported by three factors. (i) The motile sperm concentrations, which are the important determinants regarding the efficiency of sperm separation by either swim-up or gradient centrifugation, are similar in the two groups (8.8 ± 1.6 versus 6.7 ± 0.8 x 106 sperm/ml; P = not significant; medians: 6.0 and 6.6, respectively). (ii) The proportions of diminished maturity sperm, which are related to the frequencies of aneuploidies, are virtually identical in the initial semen samples (45.5 ± 3.6 versus 44.4 ± 4.3%). (iii) The third argument, that sperm concentrations are not a reliable measure of sperm quality and maturity, is based on the various biochemical marker experiments, and is evident in the present study (see inconsistencies between sperm concentrations and percentage diminished maturity sperm in patients 2 and 10, or patients 7 and 8; Table I).
The relationships between diminished sperm maturity and chromosomal disomies and diploidies may be explained by the roles of the HspA2 chaperone in both supporting meiosis as a component of the synaptonemal complex, and in facilitating cellular movement of proteins, a function that we believe may involve cytoplasmic extrusion (Eddy, 1999; Huszar et al., 2000
). Thus, sperm with diminished maturity and low HspA2 expression level may show both increased frequencies of meiotic errors, causing numerical chromosomal aberrations and cytoplasmic retention, which in turn affect shape and density of sperm (due to the fact that the excess cytoplasm is lighter than DNA and the nuclear components). Relationships between synaptic anomalies during meiosis, chromosomal abnormalities and male infertility were recognized earlier (Egozcue et al., 1983
; Vendrell et al., 1999
).
Our data indicate that the swim-up step eliminates sperm with disomies and diploidies, and sperm with diminished maturity with an overall significant reduction at the level of P < 0.001. However, the results were not consistent. Of the 10 patients there were seven that reached significant declines in proportions of immature sperm, and only one in the proportion of disomic sperm (Tables I and II). Regarding diploidies, six of the 10 men reached a significant reduction in the swim up fraction, and this pattern was similar whether we considered data from three- or two-colour FISH (Table III). This outcome is different from that reported in the previous gradient centrifugation study, in which all 10 patients showed a decline in disomy frequencies, and only two in diploidy frequencies in the Percoll pellet versus semen sperm fractions (Kovanci et al., 2001). In addition, the difference between the present swim-up study and the gradient centrifugation approach was evident considering the clearance rates for disomic sperm of 1.51.4-fold (the three- and two-colour FISH) and 3.2-fold, respectively. It is of further interest that diploid sperm showed much higher rates of clearance with swim-up (2.7-fold) compared with those for disomic sperm (1.51.4-fold). In line with our swim-up results in a recent study, utilizing swim-up fractionation of sperm, a reduction in frequencies of disomies and diploidies with a clearance rate of
1.5-fold was reported (Ong et al., 2002
).
The higher efficiency of gradient centrifugation versus swim-up is due to the fact that, in the Percoll gradient, sperm with cytoplasmic retention do not reach the pellet, whereas, in swim-up fractionation, the differing swimming efficiency of the mature versus diminished maturity sperm (particularly diploid sperm), by virtue of sperm head shape and swimming pattern, is a likely contributory factor. Differences in sperm velocity between sperm with normal and abnormal morphology have been recognized previously (Katz et al., 1985). We have found previously that cytoplasmic retention, as evidenced by CK immunocytochemistry, is related to abaxial insertion of the tail, a larger and rounder sperm head size and to an increased proportion of amorphous sperm heads (Huszar and Vigue, 1993
). Furthermore, in a study of objective sperm morphometry, sperm midpiece shape, tail length and the ratio of tail length/large head axis were directly related to CK activity and HspA2 ratios within sperm fractions (Gergely et al., 1999
). Further supporting evidence for the relationship between sperm immaturity, sperm shape and chromosomal aneuploidies was provided by the demonstration of increased frequencies of sperm disomy and diploidy in teratozoospermic men (Harkonen et al., 2001
).
In addition to cytoplasmic retention as indicative of the relationship between HspA2 expression and sperm maturity, nuclear attributes such as aniline blue staining, which is a marker of persisting histones in immature sperm, have been explored previously. Selection of mature sperm, by binding to immobilized hyaluronan, eliminated sperm cells with aniline blue staining or with cytoplasmic retention (Huszar et al., 2003). This result is in agreement with an earlier study, in which a relationship was found between frequencies of sperm with disomies and aniline blue staining in semen samples (Morel et al., 1998
).
Regarding assisted reproduction, the presence of aneuploid sperm with diminished levels of plasma membrane remodelling and zona-binding sites in sperm preparations is not an important problem with conventional fertilization via IUI or IVF, since these sperm also have diminished fertilizing capacity (Huszar et al., 1997). However, the issue has became prominent with the introduction of ICSI, in which the zona pellucida selection barrier is overridden upon fertilization. The power of zona sperm selection is well demonstrated by a small scale study (500 sperm per slide scored), in which disomy rates in sperm from men treated with ICSI were determined both in the swim-up and hemizona-bound sperm fractions. As one might expect, based on the relationship among HspA2 expression, sperm membrane remodelling and formation of the zona-binding site(s), the combined aneuploidy frequency for 18, X, Y and XY disomies was
1.1% in semen and in the swim-up fractions, whereas in hemizona-bound sperm, the rates were <0.4% (Huszar et al., 1997
; 2000
; Van Dyk et al., 2000
).
In summary, we have found that swim-up fractionation results in a reduction in sperm having disomies and diploidies and in sperm with diminished maturity. Our work using gradient centrifugation (Kovanci et al., 2001) showed an even more efficient elimination of disomic sperm. Gradient centrifugation, in which density of the sperm cell is the major factor, is very efficient in reducing sperm with disomies, but is not as efficient for eliminating diploidies. On the other hand, the swim-up method is very efficient in reducing the proportion of diploid sperm: the large-headed diploid sperm remain preferentially in the lower phase, due to their swimming inefficiency. In addition to providing additional experimental support for the efficacy of swim-up, the present results, based on the assessment of 200 000 sperm, have resolved the inconsistencies reported in the earlier publications that are reviewed in the Introduction. Our data suggest that the discrepancies were primarily related to inadequate numbers of sperm nuclei examined.
![]() |
Acknowledgements |
---|
![]() |
FOOTNOTES |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calogero, A.E., De Palma, A., Grazioso, C., Barone, N., Burrello, N., Palermo, I., Gulisano, A., Pafumi, C. and DAgata, R. (2001) High sperm aneuploidy rate in unselected infertile patients and its relationship with intracytoplasmic sperm injection outcome. Hum. Reprod., 16, 14331439.
Clermont, Y. (1963) The cycle of the seminiferous epithelium in man. Am. J. Anat., 112, 3551.[ISI]
Colombero, L.T., Hariprashad, J.J., Tsai, M.C., Rosenwaks, Z. and Palermo, G.D. (1999) Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil. Steril., 72, 9096.[CrossRef][ISI][Medline]
Devilee, P., Kievits, T., Waye, J.S., Pearson, P.L. and Willard, H.F. (1988) Chromosome-specific alpha satellite DNA: isolation and mapping of a polymorphic alphoid repeat from human chromosome 10. Genomics, 3, 17.[Medline]
Dix, D.J., Allen, J.W., Collins, B.W., Mori, C., Nakamura, N., Poorman-Allen, P., Goulding, E.H. and Eddy, E.M. (1996) Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis and male infertility. Proc. Natl Acad. Sci. USA, 93, 32643268.
Eddy, E.M. (1999) Role of heat shock protein HSP70-2 in spermatogenesis. Rev. Reprod., 4, 2330.
Egozcue, J., Templado, C., Vidal, F., Navarro, J., Morer-Fargas, F. and Marina, S. (1983) Meiotic studies in a series of 1100 infertile and sterile males. Hum. Genet., 65, 185188.[ISI][Medline]
Egozcue, J., Blanco, J. and Vidal, F. (1997) Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH). Hum. Reprod. Update, 3, 441452.
Ergur, A.R., Dokras, A., Giraldo, J.L., Habana, A., Kovanci, E. and Huszar, G. (2002) Sperm maturity and treatment choice of in vitro fertilization (IVF) or intracytoplasmic sperm injection: diminished sperm HspA2 chaperone levels predict IVF failure. Fertil. Steril., 77, 910918.[CrossRef][ISI][Medline]
Gergely, A., Kovanci, E., Senturk, L., Cosmi, E., Vigue, L. and Huszar, G. (1999) Morphometric assessment of mature and diminished-maturity human sperm: sperm regions that reflect differences in maturity. Hum. Reprod., 14, 20072014.
Guan, X.Y., Zhang, H., Bittner, M., Jiang, Y., Meltzer, P. and Trent, J. (1996) Chromosome arm painting probes. Nature Genet., 12, 1011.[ISI][Medline]
Han, T.L., Flaherty, S.P., Ford, J.H. and Matthews, C.D. (1993) Detection of X- and Y-bearing human sperm after motile sperm isolation by swim-up. Fertil. Steril., 60, 10461051.[ISI][Medline]
Harkonen, K., Suominen, J. and Lahdetie, J. (2001) Aneuploidy in sperm of infertile men with teratozoospermia. Int. J. Androl., 24, 197205.[CrossRef][ISI][Medline]
Huszar, G. and Vigue, L. (1993) Incomplete development of human sperm is associated with increased creatine phosphokinase concentration and abnormal head morphology. Mol. Reprod. Dev., 34, 292298.[ISI][Medline]
Huszar, G., Corrales, M. and Vigue, L. (1988a) Correlation between sperm creatine phosphokinase activity and sperm concentrations in normospermic and oligospermic men. Gamete Res., 19, 6775.[ISI][Medline]
Huszar, G., Vigue, L. and Corrales, M. (1988b) Sperm creatine phosphokinase activity as a measure of sperm quality in normospermic, variablespermic and oligospermic men. Biol. Reprod., 38, 10611066.[Abstract]
Huszar, G., Vigue, L. and Corrales, M. (1990) Sperm creatine kinase activity in fertile and infertile oligospermic men. J. Androl., 11, 4046.
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, 882888.[ISI][Medline]
Huszar, G., Vigue, L. and Oehninger, S. (1994) Creatine kinase immunocytochemistry of human spermhemizona complexes: selective binding of sperm with mature creatine kinase-staining pattern. Fertil. Steril., 61, 136142.[ISI][Medline]
Huszar, G., Sbracia, M., Vigue, L., Miller, D.J. and Shur, B.D. (1997) Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase and creatine phosphokinase isoform ratios. Biol. Reprod., 56, 10201024.[Abstract]
Huszar, G., Stone, K., Dix, D. and Vigue, L. (2000) Putative creatine kinase M-isoform in human sperm is identified as the 70-kilodalton heat shock protein HspA2. Biol. Reprod., 63, 925932.
Huszar, G., Celik-Ozenci, C., Cayli, S., Zavaczki, Z., Hansch, E. and Vigue, L. (2003) Hyaluronic acid binding by human sperm indicates cellular maturity, viability and unreacted acrosomal status. Fertil. Steril., 79 (5), 16161624.[CrossRef][ISI][Medline]
Katz, D.F., Davis, R.O., Delandmeter, B.A. and Overstreet, J.W. (1985) Real-time analysis of sperm motion using automatic video image digitization. Comput. Methods Programs Biomed., 21, 173182.[CrossRef][ISI][Medline]
Kovanci, E., Kovacs, T., Moretti, E., Vigue, L., Bray-Ward, P., Ward, D.C. and Huszar, G. (2001) FISH assessment of aneuploidy frequencies in mature and immature human sperm classified by the absence or presence of cytoplasmic retention. Hum. Reprod., 16, 12091217.
Li, P. and Hoshiai, H. (1998) Detection of numerical chromosome abnormalities in human sperm by three-color fluorescence in situ hybridization. J. Obstet. Gynecol. Res., 24, 385392.
Makler, A., Murillo, O., Huszar, G., Tarlatzis, B., DeCherney, A. and Naftolin, F. (1984) Improved techniques for separating motile sperm from human semen. II. An atraumatic centrifugation method. Int. J. Androl., 7, 7178.[ISI][Medline]
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, 8993.[ISI][Medline]
Martinez-Pasarell, O., Marquez, C., Coll, M.D., Egozcue, J. and Templado, C. (1997) Analysis of human sperm-derived pronuclei by three-colour fluorescent in-situ hybridization. Hum. Reprod., 12, 641645.[Abstract]
Morel, F., Mercier, S., Roux, C., Elmrini, T., Clavequin, M.C. and Bresson, J.L. (1998) Interindividual variations in the disomy frequencies of human sperm and their correlation with nuclear maturity as evaluated by aniline blue staining. Fertil. Steril., 69, 11221127.[CrossRef][ISI][Medline]
Ong, T.D., Xun, L., Perreault, S.D. and Robbins, W.A. (2002) Aneuploidy and chromosome breakage in swim-up versus unprocessed semen from twenty healthy men. J. Androl., 23, 270277.
Pang, M.G., Hoegerman, S.F., Cuticchia, A.J., Moon, S.Y., Doncel, G.F., Acosta, A.A. and Kearns, W.G. (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 sperm from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 12661273.
Pfeffer, J., Pang, M.G., Hoegerman, S.F., Osgood, C.J., Stacey, M.W., Mayer, J., Oehninger, S. and Kearns, W.G. (1999) Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil. Steril., 72, 472478.[CrossRef][ISI][Medline]
Rigby, P.W., 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, 237251.[ISI][Medline]
Samura, O., Miharu, N., He, H., Okamoto, E. and Ohama, K. (1997) Assessment of sex chromosome ratio and aneuploidy rate in motile sperm selected by three different methods. Hum. Reprod., 12, 24372442.[Abstract]
Van Dyk, Q., Lanzendorf, S., Kolm, P., Hodgen, G.D. and Mahony, M.C. (2000) Incidence of aneuploid sperm from subfertile men: selected with motility versus hemizona-bound. Hum. Reprod., 15, 15291536.
Vendrell, J.M., Garcia, F., Veiga, A., Calderon, G., Egozcue, S., Egozcue, J. and Barri, P.N. (1999) Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Hum. Reprod., 14, 375378.
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, 31563165.[ISI][Medline]
Waye, J.S., Greig, G.M. and Willard, H.F. (1987) Detection of novel centromeric polymorphisms associated with alpha satellite DNA from human chromosome 11. Hum. Genet., 77, 151156.[ISI][Medline]
Yang, T.P., Hansen, S.K., Oishi, K.K., Ryder, O.A. and Hamkalo, B.A. (1982) Characterization of a cloned repetitive DNA sequence concentrated on the human X chromosome. Proc. Natl Acad. Sci. USA, 79, 65936597.[Abstract]
Zeyneloglu, H.B., Baltaci, V., Ege, S., Haberal, A. and Batioglu, S. (2000) Detection of chromosomal abnormalities by fluorescent in-situ hybridization in immotile viable sperm determined by hypo-osmotic sperm swelling test. Hum. Reprod., 15, 853856.
Submitted on November 1, 2002; resubmitted on March 10, 2003; accepted on March 26, 2003.