Molecular Human Reproduction, Vol. 5, No. 4, 331-337,
April 1999
© 1999 European Society of Human Reproduction and Embryology
Zinc, magnesium and calcium in human seminal fluid: relations to other semen parameters and fertility
1 Laboratory of Reproductive Toxicology, Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark, 2 Department of Occupational Environmental Medicine, Institute of Laboratory Medicine, University of Lund, SE-221 85, Lund, Sweden and 3 Department of Occupational Medicine, Aarhus University Hospital, DK-8000 Aarhus C, Denmark
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Abstract |
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The effects of zinc, magnesium and calcium in seminal plasma on time-to-pregnancy (TTP) in healthy couples, on conventional semen parameters and computer-assisted semen analysis (CASA) parameters were evaluated. The localization of chelatable zinc ions in seminal plasma and spermatozoa were assessed by autometallography (AMG). Differences in chelatable zinc localization in samples with high and low total zinc were evaluated. Semen samples from 25 couples with short TTP and 25 couples with long TTP were subjected to conventional semen analysis, CASA, zinc and magnesium measurements by inductively coupled plasma mass spectrometry, and calcium by flame atomic absorption spectrometry. The cations were strongly inter-correlated, but no correlation with TTP or conventional semen parameters was found. Semen samples with high zinc concentrations exhibited statistically significant poorer motility assessed by the CASA parameters straight line velocity and linearity than samples with low zinc content. Calcium concentration also showed statistically significant differences for the same parameters, but the effect was removed by entering zinc concentration into a multiple regression model. Semen samples with high total zinc exhibited stronger staining of the seminal plasma at AMG. It is suggested that high seminal zinc concentrations have a suppressing effect on progressive motility of the spermatozoa (`quality of movement'), but not on percentage of motile spermatozoa (`quantity of movement').
autometallography/calcium/CASA/semen/spermatozoa/zinc
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Introduction |
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The total zinc content in semen from mammals is high, and zinc has been found to be critical to spermatogenesis. Deficiency of zinc is associated with hypogonadism and insufficient development of secondary sex characteristics in humans (Prasad, 1991
), and can cause atrophy of the seminiferous tubules in the rat and hence failure in spermatogenesis (Millar et al., 1958; Underwood, 1977; Endre et al., 1990). High zinc concentrations have been reported to depress oxygen uptake in the sperm cell (Huacuja et al., 1973; Foresta et al., 1990), and albumin-induced acrosome reaction (Foresta et al., 1990). Head–tail attachment/detachment and nuclear chromatin condensation/decondensation is also influenced by seminal zinc (Kvist, 1980; Bjorndahl and Kvist, 1982). There have been conflicting reports on the effect of seminal zinc on sperm motility (Stankovic and Mikac-Devic, 1976; Danscher et al., 1978; Caldamone et al., 1979; Lewis-Jones et al., 1996). It has been demonstrated that chelation of zinc ions affects sperm motility (Saito et al., 1967; Danscher and Rebbe, 1974), and it has been suggested that bioavailable zinc bound to vesicular high molecular weight proteins rather than total seminal zinc should be a measure of the effect of zinc on sperm function (Bjorndahl et al., 1991; Carpino et al., 1998). This study focuses primarily on zinc. The association of seminal zinc, and to a certain degree magnesium and calcium, with time-to-pregnancy (TTP) in healthy couples was evaluated. Furthermore, the correlation between these divalent cations to conventional semen parameters and computer-assisted semen analysis (CASA) parameters was assessed.
Autometallography (AMGZn) is a histochemical method for the detection of zinc ions and loosely bound zinc ions (chelatable zinc) in tissue. Differences in zinc ion localization at light microscopic and electron microscopic levels in the spermatozoa and seminal plasma from men with high total zinc and low total zinc were investigated.
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Study population
A total of 430 couples were recruited among 52 255 members of four trade unions. Semen samples were obtained from the 430 couples, the motility was assessed manually and by CASA (see later), and the samples were stored frozen (–20°C) until further analysis. Couples without earlier reproductive experience were enrolled when they discontinued contraception and were followed for a minimum of six complete menstrual cycles or until pregnancy was recognized [for further details please see Bonde et al. (1998)]. Of the 430 couples, 50 couples who became pregnant were selected for the present study: the 25 couples with the shortest TTP (S-TTP, median 1 month, range 1–2 months), and the 25 couples with the longest TTP (L-TTP, median 10 months, range 7–28 months). Semen samples from these 50 couples were subjected to the below-mentioned analyses.
Conventional semen characteristics
Semen samples were obtained by masturbation into 50 ml sterile polystyrene jars after recommended 3 days of abstinence. After liquefaction the samples were analysed at room temperature according to the World Health Organization criteria (1992).
Volume was measured in a 10 ml graduated Falcon glass tube (0.1 ml accuracy). Manual sperm motility assessments were performed in a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). Each spermatozoon encountered was graded as follows: a: rapid progressive motility, b: slow or sluggish progressive motility, c: non-progressive motility, d: immotility. At least 2x100 scores were performed. If the difference between two consecutive counts exceeded 10% two new counts were performed. The percentage of motile sperm cells was defined as groups `a + b + c'. Concentration was measured in a Makler counting chamber, and morphology was classified according to the Tygerberg `strict' criteria described by Kruger et al. (1986) on air-dried smears fixed in ethanol and ether, stained using Papanicolaou's technique (World Health Organization, 1992). Scoring of morphology was undertaken by one single trained laboratory technician. Sperm viability was determined on eosin-negrosin stained smears.
Computer-assisted semen analysis
Material for CASA was obtained as follows: 4.5 µl of fresh, well-mixed spermatozoa [one undiluted sample, and one sample diluted to ~40x106/ml with a non-capacitating medium, MAR (Bie & Berntsen, Højbjerg, Denmark)] was transferred with a pipette to a Makler counting chamber with a depth of 10 µm. The sample was placed in an Olympus BH-2 phase-contrast microscope (Olympus Denmark A/S, Glostrup, Denmark) with a heating plate (37°C) at x200 magnification. A Sony video camera DXC-107 (Sony Corp., Tokyo, Japan) transferred the images to a Sony PVM-1440QM colour video monitor (Sony Corp., Tokyo, Japan). Recordings of the images were made on a JVC HR-D560EG/E video cassette recorder (JVC Victor Company of Japan, Tokyo, Japan). Recordings were later analysed on a Hobson Sperm Tracker (Hobson Tracking Systems Ltd, Sheffield, UK) at an acquisition frequency of 25 Hz, tracking time 2 s (total of 50 frames), field of view 300x300 µm (allowing all straight line velocity values of up to 150 µm/s to be detected). 100 spermatozoa were analysed per sample.
The following parameters were derived from the analyses: curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), average path velocity (VAP), and amplitude of lateral head displacement (ALH).
Determination of zinc, magnesium, and calcium in semen
Seminal zinc, magnesium, and calcium were measured in all 50 samples. Zinc and magnesium concentrations in semen were determined by inductively coupled plasma mass spectrometry (ICP-MS). The instrument was a PQ2+ from Fisons Elemental (Winsford, Cheshire, UK). An aliquot of 20 µl was diluted 100-fold with a solution containing 5 g/l of 25% ammonia (ARISTAR; Merck, Poole, UK), 0.5 g/l EDTA (Janssen Chimica, Geel, Belgium), and 0.5 g/l Triton X-100 (Sigma, St Louis, MO, USA) in 18 MW Millipore water. As an internal standard, scandium (AccuStandard, New Haven, CT, USA) was added to a final concentration of 100 µg/l. All samples were prepared in duplicate. Calibration was carried out using blank samples, to which zinc and magnesium (AccuStandard) had been added to final concentrations of 1, 2 and 3 mg/l, corresponding to concentrations of 100, 200, and 300 mg/l in the original semen samples. This analysis was carried out in peak jumping mode with measurements at 24Mg, 66Zn and 45Sc.
Determination of calcium was made in the same preparations using flame atomic absorption spectrometry (FAAS). The instrument was a Perkin-Elmer 306 (Norwalk, CT, USA). Calibration was carried out for concentrations corresponding to 100, 200 and 400 mg/l in the original semen samples. Samples with high calcium concentrations were diluted three times further to fall within the calibration curve.
The detection limits, calculated as three times the standard deviation for 10 blank samples prepared on the same occasion as the samples, were 1 mg/l for zinc, 3 mg/l for magnesium, and 2 mg/l for calcium (expressed as concentrations in the undiluted semen samples). The overall coefficients of variation in the determinations, as calculated from the results of the duplicate analyses, were 18% for zinc, 32% for magnesium, and 17% for calcium.
Preparations were also made for determination of zinc by flame atomic absorption spectrometry (FAAS) in ten of the samples. Linear regression analysis of ICP-MS versus FAAS results gave a slope of 0.79 (95% confidence interval: 0.58–1.00), an intercept of 13 µg/l and r2 of 0.90. The somewhat higher results for the ICP-MS analyses were thus not statistically significant.
Autometallographical (AMG) development of semen smears for light microscopic analysis
Five samples with high (242–308 mg/l) and 5 with low (38–57 mg/l) zinc content as determined by ICP-MS were incubated in 0.5% sodium sulphide (Bie & Berntsen) for 30 min to create zinc sulphide lattices. Smears of the ejaculate/sulphide solution were made on Farmer rinsed glass slides [nine parts 10% sodium thiosulphate (Superfoss, Vedbæk, Denmark) and one part 10% potassium ferrocyanide (Merck, Darmstadt, Germany)]. The smears were air dried, fixed in 3% glutaraldehyde (Bie & Berntsen) for 30 min, and placed in 0.1 M phosphate buffer for 3x2 min and once in distilled water for 2 min.
The smears were then AMG developed to silver amplificate the zinc sulphide lattices. The developer consisted of 60 ml filtered gum arabic solution (1 kg dissolved in 2 l distilled water; Bidinger A/S, Aarhus, Denmark), 10 ml sodium citrate buffer [25.5 g citric acid monohydrate (Merck, Darmstadt, Germany) + 23.5 g sodium citrate dihydrate (Merck, Darmstadt, Germany) and 100 ml distilled water] and 0.85 g hydroquinone (Merck, Darmstadt, Germany) dissolved in 15 ml warm, distilled water. Immediately before use, 0.11 g silver lactate (Fluka, Buchs, Switzerland) in 15 ml distilled water was added and the solution was mixed thoroughly.
Farmer rinsed jars containing the semen smears were placed in a 26°C water bath and transferred to a light-tight box. The newly prepared AMG developer was poured into the jars, and the smears were developed for 30 min in the dark box.
After development smears were rinsed in running de-ionized water for 10 min, placed in 5% sodium thiosulphate for 5 min, washed with running de-ionized water for 2 min, post-fixed with 70% ethanol for 30 min, and finally washed with running de-ionized water for 2 min. Counterstaining was performed with a 0.1% aqueous toluidine blue solution, pH 4.0 (1 g of toluidine blue in 1000 ml buffer: 614.5 ml 0.1 M citric acid monohydrate and 385.5 ml 0.2 M di-sodium hydrogen phosphate dihydrate; Merck, Darmstadt, Germany). The smears were placed for 2 x 2 min in distilled water, 3 x 3 min in 99% ethanol, 3x3 min in xylol, and finally mounted with DEPEX (Merck, Darmstadt, Germany).
Preparations of semen smears for electron microscopic analysis
Smears of semen samples were prepared as described above, except that they were not mounted with DEPEX. After the procedure 0.5% osmium tetroxide [1 ml 0.2 M buffer + 0.5 ml 2% osmium tetroxide (Johnson Matthey S.A., Paris, France) + 0.5 ml distilled water] was added for 30 min, and the smears were placed for 3x2 min in buffer and 1x2 min in distilled water. The osmium-contrasted smears were studied in the light microscope, and areas of interest for further electron microscopical analyses were marked with a diamond knife.
The selected smears were dehydrated in graded ethanol solutions and embedded in Epon (Bie & Berntsen). Epon blocks placed on top of the previously marked areas of interest were kept at 60°C for 24 h and then broken off the glass smears. Semi-thin (3 µm) sections were cut and placed on glass slides. After light microscopic analyses, selected sections were re-embedded in Epon, and ultra-thin sections were made and counterstained with lead citrate [26.6 g lead nitrate (Merck, Darmstadt, Germany), 3.52 g tri-sodium citrate dihydrate (Merck, Darmstadt, Germany), 16 ml. 1 N NaOH (Bie & Berntsen) in 100 ml distilled water), and saturated uranyl acetate [15 g of uranyl acatate dihydrate (Merck, Darmstadt, Germany) in 200 ml distilled water, stored overnight in ultrasound bath] before examination in a JEOL 100S electron microscope.
Statistical methods
Multiple regression analyses were applied to the data to detect the impact of the individual parameters on the outcome. Spearman's rank correlation coefficients were computed for several correlations. For comparison of groups the Wilcoxon rank-sum test for difference in medians was performed.
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Results |
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In Table I
zinc, magnesium and calcium contents are presented together with the relative proportion of the cations in seminal plasma. Comparison of medians for these parameters in the two groups S-TTP versus L-TTP by two-sample t-tests did not reveal any statistically significant differences for any of these parameters. A strong positive inter-correlation was found between zinc, magnesium and calcium (Spearman's rank correlation coefficients were 0.79–0.86, P < 0.01) (Figure 1).
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) and total calcium (
) in seminal fluid from 50 healthy men. Mg: r = 0.86, P < 0.001. Ca: r = 0.79, P < 0.001.Neither cation was closely correlated to any of the conventional semen parameters. When performing multiple regression analysis on all the data only zinc content came out with a P value below 0.05 for the following CASA parameters: VSL 0.04, and LIN 0.008. Entering either magnesium or calcium into the model did not improve values.
The semen samples were divided into two groups of equal size according to cation status of each cation. The following dividing points (medians for all 50 samples) were identified: zinc 112 mg/l, magnesium 98 mg/l and calcium 476 mg/l. A number of statistically significant differences between the two groups were detected (Table II). The P-values for the zinc groups indicated the biggest differences, the calcium groups having intermediate differences, and magnesium groups exhibiting only poor difference (except for LIN).
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For the relative proportion between the cations (Table III
) the dividing points were: zinc:magnesium 1.26, and zinc: calcium 0.22. The samples with a zinc:calcium ratio above 0.22 showed statistically significant lower values for the CASA parameters VSL, LIN and STR compared to those samples with a ratio below 0.22. The correlation zinc:magnesium is 0.86 (Spearman's rank correlation coefficient), and for zinc:calcium is 0.79. Zinc content seems to be more closely associated with magnesium than calcium contents, which could explain the difference between the groups.
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When evaluating zinc ion content in AMG preparations, a difference was detected at light microscopic level in samples with low total zinc compared to samples with high total zinc. Figure 2
shows differences in AMG staining in a sample with 299 mg/l and a sample with 53 mg/l seminal zinc. Staining around the acrosome, the tail and in the seminal plasma was found to be sparse in the samples with low total zinc. It was not possible to confirm these findings at electron microscopic level (Figure 3A), and in particular no difference in intracellular staining of the spermatozoa was seen. Figures 3B and 4 show the zinc ion localization in the tail of the spermatozoa. It is found throughout the tail concentrated in the flagellar accessory fibres and the membrane. In the seminal plasma micrometer-sized bodies were found to be rich in zinc ion content (Figure 5).
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Discussion |
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This is to the best of our knowledge the first study to evaluate the impact of seminal zinc, magnesium and calcium on TTP and CASA parameters in healthy human beings. An association was found between high zinc concentrations and low linearity of sperm movements as expressed by a decrease in VAP, VSL, STR and LIN. Magnesium and calcium concentrations were closely correlated to zinc concentration, but were not as strongly associated with the CASA parameters. The motile concentration was found to be unaffected by the total concentration of zinc, magnesium and calcium. However, differences were found between high versus low zinc content and some CASA parameters (Table II
). When comparing the 25 semen samples with the lowest total zinc (median 72 mg/l) to the 25 samples with the highest total seminal zinc (median 187 mg/l) the Wilcoxon rank-sum test revealed a statistically significant difference in medians for VSL (P = 0.004) as well as LIN (P = 0.001). For VAP the difference was also significant (P = 0.02). No difference was found for VCL. As there were no differences in percentage motile or motile concentration in the low and high zinc samples, it therefore seems that a zinc-rich environment makes the sperm cells move in a more random and less forward fashion. It does not seem to reduce the number of motile sperm cells. VSL expresses how far straight ahead the sperm cell moves in a certain amount of time, and it is most likely, from a clinical point of view, to be the most important CASA parameter. A study by Moore and Akhondi (1996) on fertilizing capacity of epididymal rat spermatozoa showed a decline in VSL to be highly negatively correlated to the outcome of fertilization in vitro.
Over the years there has been much debate on the role of seminal plasma zinc on sperm functions. The sperm head accumulates a manyifold higher concentration than seminal plasma, and zinc is essential to chromatin stability and the ability for the chromatin to decondense at the appropiate time (Kvist, 1982; Kvist and Bjorndahl, 1985; Kvist et al., 1987, 1988), and a physiological role for zinc as a preserver for an inherent mechanism for head–tail detachment has been suggested (Bjorndahl and Kvist, 1982). There have been conflicting reports on the effect of seminal zinc on sperm motility, though, and most studies have dealt with quantitative assessments. In a study by Lewis-Jones et al. (1996) 1178 patients referred for fertility treatment had zinc and fructose concentrations in the seminal plasma measured, but for zinc no statistically significant correlation to motility was found. Abou-Shakra et al. (1989) have measured several trace elements in seminal plasma using ICP-MS, but did not detect any correlation of seminal zinc concentration to either sperm density or motility in normospermic, oligospermic, or azoospermic males. The concentrations of zinc in their study were similar to the levels presented in this study. Danscher et al. (1978) have reported high zinc concentrations to be associated with depressed sperm motility, while others have reported high zinc content in seminal plasma to be associated with a high degree of sperm cell motility (Stankovic and Mikac-Devic, 1976; Caldamone et al., 1979).
In this study we have dealt with both quantitative assessments of total zinc and qualitative determination of zinc ion localization in the sperm cell and seminal fluid. Although no differences in zinc ion content of the spermatozoa were detected at ultrastructural level (Figure 2A) among those samples with high or low total zinc, the features seen at light microscopic level (Figure 1) might reflect a relation between total amount of zinc and free zinc ions in seminal plasma. In recent studies Lewis-Jones et al. (1996) and Carpino et al. (1998) have concluded that total seminal zinc is an unreliable marker of spermatogenic activity, and suggest bioavailable zinc ions bound to vesicular high molecular weight proteins to be a better index. The AMG method here presented demonstrates chelatable zinc, i.e. zinc ions in the plasma or zinc loosely bound to macromolecules. Zinc bound firmly to proteins will not be detected by AMG and cannot be chelated. We agree with the mentioned studies that it is the bioavailable (hence chelatable) zinc that exerts functions on sperm cells, including motility. This study indicates that total zinc and chelatable zinc concentrations are related. Further studies are necessary, and computerized image analysis e.g. of AMG preparations could be an important tool.
In contrast to the effects of zinc, high magnesium concentration in itself did not appear to have any inhibitory effect on sperm motility. A negative effect on LIN was found, but it was not large enough to have any impact on the other CASA parameters. It has been reported that human seminal plasma contains secretory granules and vesicles of prostatic origin which might have a regulatory effect on sperm motility by modulating the concentration of essential cations in their environment (Stegmayr et al., 1982). Membranes of these organelles contain Mg2+- and Ca2+-dependent ATPase competitively inhibited by Zn2+ (Ronquist et al., 1978a,b). High positive correlation between zinc and magnesium in seminal plasma has previously been reported (Papadimas et al., 1983; Umeyama et al., 1986), and in this study similar strong intercorrelations between all three cations were found (r = 0.79–0.86, P < 0.01).
Calcium is important for sperm physiology including motility (Morton et al., 1974; Lindemann et al., 1987), metabolism (Peterson and Freund, 1976), acrosome reaction, and fertilization (Yanagimachi and Usui, 1974; Yanagimachi, 1981). The role of seminal calcium in sperm motility is, however, not fully understood. Thomas and Meizel (1988) found chelation of extracellular calcium ions with EGTA to inhibit acrosome reaction, but at the same time to have no effect on motility. Addition of the acrosome reaction inducing divalent cation ionophore ionomycin had no effect on motility either, but increased the number acrosome reacting sperm cells significantly. Magnus et al. (1990) found no association between ionized calcium concentrations and the proportion of spermatozoa displaying progressive movement. Arver and Sjöberg (1982) have reported low ionized calcium to be associated with more and better progressive motile spermatozoa. Prien et al. (1990) compared sperm motility, velocity, and progressive movement with total and ionized calcium in patients with normal (>60%) and decreased (<60%) sperm motility. No difference in total calcium was found, but a statistically significant decrease in seminal ionized calcium was found in those men with decreased motility. In this study total calcium was measured but the calcium-binding capacity of the seminal plasma is not known. As for calcium, no impact on motile concentration was detected, and the correlations to CASA parameters were weaker than those for zinc (Table II). Both VSL and LIN showed inverse significant correlations to total calcium concentration (P = 0.02 for both), while VCL was unaffected. But adding zinc concentration into a multiple regression analysis removed the effects of total calcium concentration on motility. This is in accordance with the previously mentioned study by Prien et al. (1990). In this study samples with a low zinc:calcium ratio exhibited statistically significant better CASA values (VSL, LIN, and STR) compared to those with a high ratio. This is mainly due to differences in zinc concentration.
The precision of zinc, magnesium and calcium determinations in this study were relatively poor, with coefficients of variation of 18, 32 and 17%, respectively. The reason is probably inhomogeneity of the samples, which in combination with the small sample volumes (20 µl) may have impaired the precision. Despite the large imprecision, the variation generated in zinc and magnesium determinations was minor compared to the large concentration range in the samples.
It has previously been demonstrated that chelation of zinc ions affects sperm motility in man (Danscher and Rebbe, 1974), rat and dog (Saito et al., 1967; Stoltenberg et al., 1997). Studies with intra- and extracellular chelation of zinc ions are currently being carried out, and this might reveal the importance of zinc ion location in seminal fluid and the sperm cell.
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Acknowledgments |
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The authors wish to thank Ms H.Brandstrup, Ms D.Jensen, Ms K.Lunding, Ms K.Wiedemann, Ms Anna Akantis and Mr A.Meier for skilful technical assistance. The Danish Fecundity Study Group supported this study which is part of a collaborative follow-up study on the environmental and biological determinants of fertility. The project is coordinated by the Steno Institute of Public Health, University of Aarhus, and is undertaken in collaboration with the Department of Growth and Reproduction, National University Hospital in Copenhagen. The study was mainly supported by a grant from the Aarhus University Research Foundation (J 1994-7430-1). Additional support was also given by the Danish Medical Research Council (J 12-2042-1), the Danish Health Insurance Foundation (J 11/243-91, J 11/236-93), Fonden til Lægevidenskabens Fremme (A.P.Møller) and The Ciconia Foundation.
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Notes |
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4 To whom correspondence should be addressed
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References |
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Submitted on July 28, 1998; accepted on December 16, 1998.
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