Hum. Reprod. Advance Access originally published online on October 5, 2006
Human Reproduction 2007 22(2):477-484; doi:10.1093/humrep/del383
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Semen quality in a residential, geographic and age representative sample of healthy Chinese men
1 Shanghai Institute of Planned Parenthood Research, Shanghai, P.R. China 2 OMNI Research Group, Department of Obstetrics & Gynecology 3 Clinical Epidemiology Program, Ottawa Health Research Institute 4 McLaughlin Centre for Population Health Risk Assessment, Institute of Population Health and 5 Department of Epidemiology and Community Medicine, University of Ottawa, Ottawa, Ontario, Canada
6 To whom correspondence should be addressed at: OMNI Research Group, Department of Obstetrics & Gynecology, University of Ottawa, 501 Smyth Road, Box 241, Ottawa, Ontario, Canada K1H 8L6. E-mail: swwen{at}ohri.ca
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Abstract |
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BACKGROUND: Population-based study of semen quality is rare in literature. METHODS: Healthy men aged 20–60 years from six Chinese provinces were invited to participate in the study between December 2000 and November 2002. Posters were distributed in the participating counties to enroll 200 subjects from each province. Medians, percentiles, and proportions below lower threshold of the WHO criteria for semen parameters were calculated. Generalized linear models were used to examine the determinants of semen quality. RESULTS: Semen samples from 1191 healthy Chinese men were collected and analysed. The medians (5th and 95th percentiles) were 2.3 ml (1.0–4.5) for semen volume, 65 x 106/ml (20–150) for semen concentration, 154 x 106/ejection (29–421) for sperm count, 19% (5–32) for rapid progressive motility, 46% (29–66) for progressive motility, 67% (47–81) for total motile spermatozoa, 70% (48–88) for sperm viability and 39% (23–76) for normal morphology. Many healthy Chinese men had semen parameter values below the lower threshold of the WHO criteria. Region, age, abstinence duration and season were important determinants of semen quality. CONCLUSIONS: Chinese men have lower values of semen parameters according to WHO standard, and a lower threshold for normal semen parameters for Chinese men should be considered.
Key words: semen parameters/semen quality/healthy men/risk factor/Chinese
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Routine and basic semen measurements remain the most powerful parameters to assess the male fertility in reproductive medicine (Andrade-Rocha, 2003
). For the convenience of recruitment, studies on semen quality have often relied on samples taken from infertility clinics and sperm donor banks (Auger et al., 1995; Bujian et al., 1996). There are major differences in demographic characteristics between infertile patients, semen donors and the general population (Lalos et al., 2003; Muller et al., 2004).
Semen quality has been considered as one of the most important indicators of environmental pollution (Moline et al., 2000). Previous retrospective studies have indicated that semen quality has been declining in the past several decades (Carlsen et al., 1992; Auger et al., 1995; Fisch et al., 1996; Auger and Jouannet, 1997; Becker and Berhane, 1997; Swan et al., 1997; Andrade-Rocha, 2003), suggesting a deterioration in the quality of environments. However, semen quality is affected by various factors other than physical environments, such as age (Kidd et al., 2001), occupation (Thonneau et al., 1996, 1997; Bigelow et al., 1998), cigarette smoking (Vine, 1996; Vine et al., 1994) and other lifestyle factors (Tiemessen et al., 1996). Most previous studies have used semen samples taken from fertility laboratories, although several recent studies (Jorgensen et al., 2002; Punab et al., 2002) from Northern European countries have investigated well-defined groups representing the general population of young men. Because there have been dramatic changes in population profiles and other non-physical environmental factors during the last several decades, it is difficult to attribute the decline in semen quality to the deterioration of the environment based on studies from fertility laboratories. Other limitations of previous studies, mainly those published before 1995, include the lack of comparability of laboratory techniques and inappropriate statistical analysis. The objective of this study is to make a comprehensive assessment of semen quality in a residential, geographic and age representative sample of healthy Chinese men.
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Materials and methods |
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Study design and study population
To make a comprehensive assessment of semen quality in a residential, geographic and age representative sample of healthy Chinese men, the Chinese Population and Family Planning Commission (CPFPC) made a contract with the Shanghai Institute for Planned Parenthood Research (SIPPR) in 2000 to conduct a nationwide study. To achieve the goal of geographic representativeness, SIPPR negotiated and reached an agreement with six provincial family planning institutes which are geographically and demographically representative of the country: Hebei (northern China), Henen and Shanxi (central China), Zhejiang and Qindao (east coast) and Guizhou (south-west China).
The field work of the study was carried out between December 2000 and November 2002.
A two-stage sampling strategy was used. At the first stage of sampling, 15 counties with mixed urban and rural locations were selected from the six participating provinces. At the second stage of sampling, volunteers from the 15 participating counties were recruited to participate in the study. According to the protocol, posters were distributed to each family by the local office of China Family Planning Network in the 15 participating counties to enroll 200 subjects in each province. The subjects who came to the Family Planning clinics were recruited if they met the eligibility criteria of the study. All men of 20
60 years of age with no major chronic disease or known reproductive disorders or identifiable history of infertility and who were permanent residents of the participating counties were considered eligible for the study. The known reproductive disorders which were considered as exclusion criteria included any one of the following reproductive or urological diseases diagnosed by physicians: hydrocele of tunica vaginalis, hematocele, hernia, torsion of spermatic cord, torsion of testicular appendage, varicocele II or more severe seminal vesiculitis, sexually transmitted disease, gangrene on skin of scrotum, cryptorchidism, small testis (<12 ml), congenital absence of vas deferens and tuberculosis of epididymis. To ensure the representativeness of Chinese men with different age and rural/urban residence, equal sample sizes were allocated to the four age groups: 20–29, 30–39, 40–49 and 50–60 with 50% from rural areas and 50% from urban areas. The recruitment was on first come first service basis. When the number of participants in each specific age/geographic group was reached in the participating province, the recruitment was stopped for that particular target population. The provincial family planning institutes were in charge of the recruitment, interview, physical examination and semen analysis. The staff at the family planning institutes explained to the eligible study subjects of the details of the purpose of the study and possible benefits and risks of participating in the study, and the study subjects were asked to sign a consent form if they agreed to participate. Each study subject was asked to provide detailed information on demography, life style, living conditions, sexual behavior and reproductive history using a structured questionnaire by a qualified interviewer. A general and reproductive health examination including scrotal palpation and Prader orchidometer, which was used to assess the testicular volume, was conducted by a physician. The study participants were also asked to provide two semen samples within 1530 days, after 27 days abstinence between the two donations. The semen specimens were collected within the local family planning institution using masturbation into a wide-mouth plastic container and delivered to laboratory in the same building immediately. The room temperature at masturbation was kept between 18 and 25°C.
Semen analysis and quality assurance procedures
The semen analyses were carried out following the recommendations of WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction (third version) (World Health Organization, 1992). Semen quality parameters that were assessed included appearance, semen volume, viscosity, agglutination, liquefaction time, pH value, semen density, sperm motility, percentage motile sperm, sperm morphology, sperm velocity and non-sperm cells. Semen volume was evaluated by semen weight, assuming a density of 1.0 g per ml. The container was weighed before and after sample collection; the difference between the weights was recorded as volume. The pH value was measured by pH paper and compared with the calibration strip to read. Graded sperm motility percentage was measured by known volume of specimen which was placed on one clean glass slide covered by a coverslip using positive phase-contrast microscopy at a magnification of x400. With the help of ocular grid, the proportion in the four motility categories was assessed: fast progressive sperm (rapid progressive), slow progressive sperm, non-progressive sperm and immotile sperm. A 1-ml sample was diluted (1:20) with formaldehyde in each laboratory for examining the sperm concentration by improved Neubauer haemocytometer. Four to six fields were randomly chosen and then counted. For morphology classification of the spermatozoa, 100 spermatozoa were counted by high-quality phase-contrast microscope (magnification of x600). The thin and well-spread smear was air-dried, fixed and stained according to Papanicolaou method (Papanicolaou, 1942). The criteria of the classifications including ‘head shape/size defects’, ‘neck and midpiece defects’, ‘tail defects’ and ‘cytoplasmic droplets ‘was based on the manual published by WHO (World Health Organization, 1992). The above procedure was manipulated twice, and the disparity should have been less than 10%, if the difference greater than 10%, the third measurement from a new aliquot was made and the mean of the closer two was used.
Two technical training workshops were organized by SIPPR before and after the pilot study according to the third version of the WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction (World Health Organization, 1992). One technician in each province performed all semen analyses to avoid intervariation between laboratories. At the end of the second workshop, the record of six semen parameters (including sperm concentration, percentage of rapid progressive motility, slow or sluggish progressive motility, non-progressive motility, immotility and normal morphology) from the six technicians was documented and used as the reference value to amend final data. Detailed procedures were as follows: first, the mean value (two tests were performed for each parameter) was computed as the result of every technician. Second, the mean of all technicians on each parameter was quoted as ‘Golden Standard’. Third, the value of each center was adjusted by coefficient, which was calculated by ‘Golden Standard’ divided by mean of the values each of the six technicians reported. However, the results obtained from data with or without the above quality control measures were the same; so, we used original data for the final analysis. Laboratory equipments including inspective machines and reagents were all supplied uniformly by same certificated company to minimize the equipment-related variation.
Statistical methods
Only about one-third of the study participants provided two semen samples after 2–7 days of abstinence. To evaluate the consistency of our duplicated tests, we performed the mixed random effect model to calculate intraclass coefficients (ICC) on seven semen parameters. The procedures were as follows. First, we generated four models which allow different covariance structure according to different situations; the first model had center x sequence interaction term additional to main effect (center and sequence of two tests) and allowed heteroscedasticity between two tests for any participant; the second model only left main effect and removed the interaction; the third model reclaimed the interaction term but imposed a constraint of homoscedasticity and the fourth model removed the interaction on the base of model three. Second, the appropriate model was identified from above models to draw the covariance of different terms through the likelihood ratio tests on different hypothesis. Third, the intraclass correlation was derived in terms of the above-mentioned four models (Kao et al., 2003). The threshold point of ICC > 0.75 was chosen a priori as an indication of acceptable reliability (Lee et al., 1989). Because of the high agreement between the two donations (Table I), we used the first donation samples from all 1191 participants in our analysis.
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Although the sample size (1191) was quite large, percentiles were calculated on seven semen parameters because the distributions for these parameters were not normal. Percentages coincident with the criteria of WHO (World Health Organization, 1992
) were also performed. The data were summarized using median (5th and 95th percentiles), stratified by fertility status, duration of sexual abstinence before ejection and age. We used Kruskal–Wallis analysis of variance to compare medians between groups.
A generalized linear model was used to examine the independent effect of risk factors on semen parameters. A full model that included all risk factors to be examined in the final regression was used. Independent variables were entered into the regression model as dummy variables as follows: (i) age: 20, 25, 30, 35, 40, 45, 50, with 20 as reference; (ii) education: primary school and below, junior school, high school, college and higher, with primary school and below as reference; (iii) geographic variation: Henan province, Shanxi province, Guizhou province, Zhejiang province, Qingdao province and Hebei province, as reference; (iv) duration of abstinence: 
3 days, 47 days, 
8 days, as reference; (v) seasonal variation: spring (March to May), summer (June to August), autumn (September to November) and winter (December to February), as reference; (vi) time lag between semen sample taken and laboratory investigation: 60 min and >60 min, as reference; (vii) tobacco and alcohol consumption: No and Yes, as reference; (viii) cola and coffee intake in three months before the study: No and Yes, as reference and (ix) body mass index: 18.5 and <25, <18.5 or 25, as reference. The level of significance was established at 0.05. All semen parameters were log-transformed to improve the normality as dependent variables in the generalized linear models.
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Results |
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A total of 1191 healthy men who were permanent residents of the participating counties/provinces were recruited into the study. The demographic characteristics of the study population are summarized in Table II. Most of the study subjects had high school education; about half had a household income of >5000 RMB per year; nearly half of the subjects were smokers and 83.3% of the study subjects had caused a pregnancy (Table II).
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The semen parameters of the study subjects are described in Table III. A large proportion of the study subjects had semen parameter values below the lower threshold of the WHO criteria, especially for rapid progressive motility, sperm progressive motility and sperm viability (Table III).
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The difference in semen parameters between the proven and unproven fertility subjects are summarized in Table IV. Sperm concentration and sperm count were higher in the ‘proven fertility’ group than in the ‘unproven fertility’ group.
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Variations in semen quality after different lengths of abstinence are summarized in Table V. Sperm viability decreased with increasing days of abstinence. On the contrary, total sperm counts and rapid progressive motility increased with duration of abstinence. Semen volume and sperm concentration were increased in the group of 4
7 days abstinence. Percentages of progressive motility and normal morphology were markedly increased in the group of 8 days abstinence.
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The semen parameters according to age groups are tabulated in Table VI. In the 20
25 and 4559 years of age groups, sperm concentration, sperm count, sperm progressive motility, total motile spermatozoa, sperm viability and normal morphology were lower than in the other age groups (Table VI).
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The results of multiple regression analyses on semen parameters after log-transformation are summarized in Table VII. The coefficients listed were converted back to the original measure to reflect the relative differences compared with the reference group. There were significant differences in semen quality among different age groups, geographic regions and seasons. Percentages of rapid progressive motility, progressive motility, total motile spermatozoa, sperm viability and normal morphology were inversely related to advancing age. Sperm concentration, sperm count and percentage of normal morphology were sensitive to the period of abstinence. The semen quality in the winter appeared better than in the other three seasons. Sperm concentration and sperm count decreased obviously if the time from ejection to analysis was longer than 60 min.
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Discussion |
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In this study, we analysed the semen parameters in 1191 healthy Chinese men. To recruit subjects as representative as possible, we restricted the participants by residence and age in six geographically representative provinces. To our knowledge, this is the first research on semen quality with residential, geographic and age representative Chinese population.
Only a few previous studies have examined semen quality in oriental populations. Wang et al. (1985) examined the semen quality of 1239 men attending fertility clinics for semen donation, premarital check-up or prevasectomy screening. The values of semen volume, sperm concentration, total sperm count, motility and normal morphology in that study were higher than those in our study. The discrepancies between the study by Wang et al. and ours may be due to the younger population and longer abstinence in Wang’s study. Junqing et al. (2002) examined semen quality in 562 Chinese young men who attended for premarital check-up. Our results were in agreement with Junqing et al. except for the percentage of normal morphology. The percentage of normal morphology in Junqing’s study was 78.23%, double the value observed in our study (39%). The reason for this difference might be the difference of age distribution between the two studies. More recently, Liu et al. (2004) evaluated the semen quality in sperm donors and found that 48% donors reached all the WHO reference values of semen parameters, which was higher than that found by Junqing et al. (42.3%) and by ourselves (29.2%). The gaps between the semen quality of Chinese men and the WHO standard could be caused by ethnic variations, regional differences, lifestyle factors and environmental differences. Compared with the studies of young European men aged from 18 to 20 from the compulsory military medical examination (Andersen et al., 2000; Jorgensen et al., 2002; Richthoff et al., 2002; Tsarev et al., 2005; Jorgensen et al., 2006), semen volume in Chinese men was also lower. However, the 20–30% lower sperm concentration among the young men from these Nordic-Baltic countries compared with Chinese men was remarkable. The sperm count of Chinese men was similar to Finnish and Estonian men but was higher than Norwegian and Danish men. Whether the low concentration and low counts in these young European populations were caused by immaturity remains speculative (Carlsen et al., 2005). Iwamoto et al. (2006) compared the semen quality in Japanese fertile men with European populations and found that total sperm count, percentage of motile sperm and percentage of normal sperm were significantly lower in Japanese men than those of European population (except only for motile sperm in men from Paris). The finding by Chia et al. (2001) showed that Chinese, Malays and Indians had low sperm counts. Studies on semen quality showed apparent geographic variations, but it is unclear whether these variations should be attributable to true population differences or variations in study subjects sampling or semen analysis methods. Auger and Jouannet (1997) found that there were significant differences among study centers in seminal volume, sperm concentration, total number of spermatozoa and percentage of motile spermatozoa, although the same procedures and methods for semen analysis have been used in these centers. In our study, we found that there were regional differences in all semen parameters after adjustment for potential confounders, which were consistent with the findings by Junqing et al. (2002). We speculate that these regional variations may be attributable, at least in part, to lifestyle and environmental differences. Many studies (Jorgensen et al., 2001; Eskenazi et al., 2003; Carlsen et al., 2005) adjusted ejection-to-analysis time as a confounder when the semen parameters were analysed by multiple regression models. However, the only one study we found which specifically investigated the effect of delay time on semen parameters (Mortimer et al., 1982) indicated that there is no association between ejection-to-analysis time and semen quality. The association between ejection-to-analysis time and semen parameters observed in our study may be caused by the small number of subjects with time longer than 60 min [24 in 1191 samples (2%)].
Our study is a cross-sectional one with nearly 20% (219 subjects) older than 50. The proportion who had caused a pregnancy in these 219 subjects was 99.1% which means that almost all had formerly made their partner pregnant. However, this did not mean they could make their partner pregnant when they donated the semen samples. On the contrary, the proportion who had caused a pregnancy in younger group was relatively lower, which may not have been caused by poor semen quality but the result of unstable sexual relationships indicated by the lower proportion of married men. The main studies on fertile subjects have generally analysed the semen sample from men whose wives were in the process of giving birth (Iwamoto et al., 2006) or from the couples who were planning to have a baby and their wives were proven to be pregnant at the end of the prospective study (Bonde et al., 1998). Differences in the definition of ‘fertile’ between our study and other studies make it difficult to reconcile these results.
Our study found that age and season had significant effects on semen quality. A recent review suggests that increased age was associated with a decrease in semen volume, percentage normal sperm and percentage sperm motility (Kidd et al., 2001). There were several reasons to explain seasonal variation on semen parameters. Some investigators consider that semen quality is subjected to circannual changes because of temperature changes (Politoff et al., 1989; Chia et al., 2001). Others suggest that the length of daylight might be a factor (Snyder, 1990). The most recent report suggested that ejaculatory frequency may be the key reason for seasonal variation on sperm concentration (Carlsen et al., 2004).
Our study has several strengths. The most important strength of this study is that it is a residential, geographic and age representative sample of healthy men in China, the most populous country in the world. In our study, the participating provinces are located in different regions, representing central, south-west, east coast and northern China. The study subjects were recruited from selected counties from the participating provinces, with no payment. On the contrary, in most previous studies, subjects have been selected from fertility clinics and sperm donor banks or volunteers recruited by posters or bulletins locally who provide their semen samples for various reasons such as profit or willingness to receive an examination on fertility. Scientists (Larsen et al., 1998; Lalos et al., 2003; Muller et al., 2004) have found that there are major differences in demographic characteristics between the semen donors and the general population. Second, the sample size of 1191 healthy men is larger than that of most previous studies. Third, the age of subjects in our study ranged from 20 to 60 years, with a large number (219) of men 50 years, which is rare in previous studies.
The limitations of our study should not be overlooked. We made comparison of demography between our study sample and the census data and found that our study subjects had higher education and income levels and a higher drinking rate but lower smoking rate than the general male population in China (Ma et al., 2005; National Bureau of Statistical of China, 2005; Yang et al., 2005). There are two reasons for the discrepancies between our sample and the national census. First, although we have tried to make our study residential, geographic and age representative, we cannot randomly choose our samples even at cluster level (e.g. province/county). Some factors, such as collaboration of local governments and family planning units and so on, have played roles in deciding the study sites. As a result, some discrepancies with the national census are expected. Second, to ensure even rural versus urban distribution and equal number of subjects of the four age groups, we have determined a priori of the number of subjects in each stratum. The age and residence distribution in our sample was quite different from the census and therefore the demography. We have not been able to collect data on non-participants. Most subjects who came to the recruitment office but did not participate were those who did not meet the requirement on residence and age of our study. Another potential selective bias was the possible under-representativeness of infertile men in our sample. There is no definitive figure in Chinese literature regarding male infertility rate. Two studies based on small samples in 1985–1990 indicated that the male infertility rate varied between 2.01 and 3.80% (Yang and Zhang, 2005; Zhang, 2005). In our 1191 subjects, 64 couples had a history of infertility, and only 11 of these incidences of infertility were caused by the male, which means the male infertility rate here was 0.92%. The lower male infertility rate in our sample may be caused by the population chosen for study. The data on rapid progressive motility of 206 men from Zhejiang province were much lower (only about 10%) when compared with the other centers. The reason for such a large deviation is unknown. We have therefore elected to exclude the 206 subjects in the analyses of rapid progressive motility, progressive motility, and total motile spermatozoa. In our study, a certain degree of intercenter variations on semen analysis may remain, although the same procedures and methods were used, and training was provided. Semen analysis is a rather subjective technique and associated with interlaboratory variation (Jorgensen et al., 1997), which makes it difficult to compare assessment performed by different laboratories. Keel reported an instance of sperm concentration on one sample ranging from 3 to 492 x 106/ml (Keel et al., 2000). Such remarkable interlaboratory variation is likely due to a combination of differences in measurements and technicians. To improve the validity of semen analysis, internal quality control (IQC) and external quality assessment (EQA) for andrology laboratory procedures are considered necessary (Burdock et al., 1963). Although good consistency could be obtained if ICC was larger than 0.75 (Burdock et al., 1963; Lee et al., 1989), some investigators have suggested that an ICC > 0.5 is acceptable. ICCs of half of the eight semen parameters in our study were larger than 0.75, and all of them were larger than 0.5, suggesting high quality of our data.
In summary, our study examined the current status of semen quality among healthy Chinese men and identified several potential factors that may affect semen quality in them. The main findings that Chinese men had lower values of semen parameters according to WHO standard in our large residential, geographic and age representative sample of healthy Chinese men could be useful in the practice of reproductive medicine and policy development regarding male reproductive health. The information could also serve for comparison purposes in future studies of semen quality in Chinese or other ethnic populations.
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Acknowledgements |
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This study was supported by a grant from Shanghai Science and Technical Committee (02DJ14053) and a grant from Ministry of Science and Technology, China (9902). Dr Jun Gao is a recipient of International Fellowship of University of Ottawa. Dr Yang is a recipient of CIHR/STIRRHS Post-PhD fellowship. Drs Wen and Walker are recipients of New Investigator’s Award from CIHR. The authors thank the staff at the six provincial Planned Parenthood Research Institutes (Guizhou, Henan, Hebei, Qingdao, Shanxi and Zhejiang) and study participants for their support to the study.
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References |
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Submitted on March 6, 2006; resubmitted on May 31, 2006; resubmitted on July 28, 2006; accepted on August 21, 2006.
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