1 Department of Genetics and Reproduction, Veterinary Research Institute, Brno, Czech Republic, 2 National Center for Environmental Assessment, Office of Research and Development, US EPA, Washington, DC, 3 Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD, USA, 4 Institute of Hygiene, Brno, Czech Republic, 5 Center for Occupational and Environmental Health, University of California Los Angeles, Los Angeles, CA and 6 Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US EPA, Research Triangle Park, NC, USA
7 To whom correspondence should be addressed at: US EPA (MD-72), Research Triangle Park, NC 27711, USA. E-mail: Darney.sally{at}epa.gov
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Abstract |
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Key words: air pollution/computer-aided sperm analysis/human semen/sperm aneuploidy/sperm chromatin structure assay
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Introduction |
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Among the health effects studies initiated early in the Teplice Program was a male reproductive health study. Young men (aged 18 years) living in Teplice in 1993 and 1994 (Selevan et al., 2000) were sampled (one semen sample from each participant), either in the winter (when pollution was high) or the late summer (when pollution was low). This study found no significant associations between exposures to air pollution and measures of sperm production (sperm concentration or total sperm count). However, significant associations were found between the exposure to air pollution and some indicators of sperm quality, namely sperm morphology, sperm chromatin structure (Selevan et al., 2000
) and sperm aneuploidy (Robbins et al., 1999
) in men evaluated in the winter of 1993. Also, significant associations were found between exposure to air pollution and sperm morphology and motility in men evaluated in the winter of 1994. The authors pointed out that differences in results between years could be due to differences in levels of pollution, differences in the interval between the peaks of pollution and the specific day of semen sampling, and differences in the two groups of men examined.
This preliminary study (Selevan et al., 2000) had several limitations, including: data on only one semen sample per man, having only environmental exposure data (no internal markers of exposure), and having limited (infrequent) data on particulate material
10 µm in size (PM10) and its constituent polycyclic aromatic hydrocarbons (PAH). Nevertheless, the study suggested that exposure to ambient air pollution was associated with decreased semen quality, a finding that could negatively impact fertility, especially when considered on a population basis.
In light of these preliminary findings, the Czech government became interested in monitoring semen quality in young Teplice residents over longer periods of time during which actions were taken to reduce the air pollution. Therefore, a second study was initiated in 1995, designed to overcome the limitations of the first study. Accordingly, a cohort of men living in Teplice was sampled multiple times over 2 years, both during the winter when pollution was high, and during late summer after an extended period of much lower air pollution. This longitudinal design with repeated measures allows each man to serve as his own control. The study protocol also specified collection of serum samples to measure metals that may be associated with altered semen quality (reviewed by Robbins and Cousins, 1998), and urine to measure cotinine and thereby confirm self-reported smoking status. Air pollution monitoring, including measurement of PM10 and PAH, continued during the study period (Watts et al., 1994
; Lenicek et al., 2001
).
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Materials and methods |
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At entry, all participants provided written, informed consent, and passed a routine physical examination. For each of seven sampling sessions, the cohort of men was scheduled for appointments over 5 consecutive days; thus, the cohort had essentially the same exposure preceding each sampling. Sample 1, obtained in September 1995, served as the first reference or baseline sample. To capture exposures to episodes of high air pollution, men were sampled monthly during the winter of 1996, i.e. in January (sample 2), February (sample 3) and March (sample 4). This was followed by a second reference sample in September 1996 (sample 5), a fourth winter sample in February 1997 (sample 6) and a final reference sample in September 1997 (sample 7). Having this series of seven samples, four samples during periods of high air pollution and three after periods of relatively low air pollution, provided longitudinal data from which to examine potential associations between exposure to high versus low air pollution across two different winters, and to use each man as his own control. To remain in the final analysis, a participant had to have contributed at least one reference sample and two winter samples during the first year of the study.
Questionnaire
A structured questionnaire, similar to that used in the first study (Selevan et al., 2000), was administered at entry (September 1995). It collected information about each participants reproductive and general health, and about specific factors that might potentially impact semen quality, including: recent fever, use of medications and vitamins, smoking habits, alcohol and caffeine consumption, type of underwear worn (boxers or briefs), and exposures to solvents, pesticides or metals through work or hobby. The specific factors were asked with respect to the 90 days preceding semen sampling. This is considered a time span relevant to a given semen sample, since it encompasses the entire process of spermatogenesis, including epididymal maturation (Heller and Clermont, 1964
; Selevan et al., 2000
). A brief questionnaire was administered at each subsequent cycle to update information on lifestyle habits and medical/occupational exposures.
Semen collection and routine analyses
Semen samples were collected on-site by masturbation into clean glass containers (Kavalier, Sazava, CZ), and the abstinence interval recorded (an interval of 27 days having been requested). The semen was allowed to liquefy at room temperature for 30 min and processed within 1 h of collection. Sample volume was measured in a 15 ml graduated centrifuge tube and an aliquot removed to determine sperm concentration by haemocytometer (World Health Organization, 1992). For motility analysis, aliquots of semen were loaded into 20 µm deep chambers (Microcell; Fertility Technologies, Inc., Natick, MA, USA), mounted on a heated (37°C) microscope stage and videotaped (Selevan et al., 2000
). Samples with high sperm concentrations were diluted with homologous seminal fluid to achieve a concentration suitable for computer-aided sperm analysis (CASA). Aliquots of semen (7 µl) were smeared onto slides (four slides per sample), air-dried, fixed in 95% ethanol (15 min) and later stained with Papanicolaou stain for morphological evaluation (World Health Organization, 1992
). Finally, aliquots of semen were loaded into straws (0.5 ml) and frozen (80°C) without cryoprotectant for later analysis of genetic outcomes.
After all samples were obtained, videotapes were analysed for motility and motion characteristics by a single trained technician. The percentage of progressively motile cells, defined as World Health Organization (1992) grades a + b, was determined visually from the videotapes, based on
100 sperm in several fields. Videotaped sperm were analysed by CASA using Cell Motion Analyser (Version 2.0; Medical Technologies, Montreux SA, Switzerland) to evaluate the quality of sperm motion. Analyses were conducted at 60 Hz with maximum/minimum number of frames = 32/15. Sperm were considered motile when average path velocity was >10 µm/s. Typically, 100200 sperm tracks were analysed per sample, although a small number of samples (n = 11) had only 5198 sperm tracks on the videotape. For each sample, three CASA outcomes were reported: straightline velocity, an indication of sperm progression; curvilinear velocity, an indication of sperm vigour; and linearity, an indicator of the straightness of the sperm track. Of routinely obtained CASA outcomes, these measures are the least dependent upon instrument brand or software version (reviewed in Perreault, 2002
) and therefore most comparable with data in the preliminary study (Selevan et al., 2000
) and in the literature.
The percentage of morphologically normal sperm was determined by examining 300 sperm per sample at x1000 magnification under oil immersion and classifying them according to strict criteria as described by the World Health Organization (1992). The percentage of sperm with morphologically normal heads was also recorded. Two experienced technicians scored pre-coded slides so that they were read blind. Slides were pre-sorted so that all samples from a given participant were scored by one technician to preclude within-participant error due to inter-technician variability in scoring.
Sperm chromatin structure assay
Straws containing frozen semen were shipped to South Dakota State University for analysis of sperm chromatin structure using SCSA® (Evenson et al., 2002). Briefly, semen was thawed, diluted in acid buffer (pH 1.2) to potentially denature damaged nuclear DNA, stained with acridine orange and immediately evaluated by flow cytometry. Acridine orange is a metachromatic dye that fluoresces green when intercalated into double-stranded native DNA, but red when complexed with single-stranded DNA. Dual band flow cytometry was used to detect both green (515530 nm band pass filter) and red (630 nm long pass filter) fluorescence in
5000 sperm per sample. DNA denaturation was detected by a shift from green to red fluorescence and quantified by the expression DFI, defined as the ratio of red/(red + green) fluorescence. Cells with DNA fragmentation index (DFI) values above a threshold are considered abnormal and the percentage of such cells is termed %DFI.
Sperm aneuploidy
The sperm aneuploidy assay was conducted as previously described (Robbins et al., 1995; Rubes et al., 1998
, 2002
) for 15 of the participants selected because they contributed all seven samples and were either non-smokers or smoked <20 cigarettes/day. Briefly, air-dried smears of sperm were decondensed and hybridized immediately using fluorescent chromosome-specific
-satellite DNA probes for chromosomes X and 8, and satellite III DNA probe for chromosome Y (Vysis, Downers Grove, IL, USA). This method allows distinction between diploid and disomic sperm nuclei, and meiosis I and meiosis II errors in sex-chromosomal aneuploidy and diploidy. Slides were randomized and 10,000 sperm were scored per sample (~70000 sperm total per donor), using strict scoring criteria previously validated against human sperm/hamster oocyte data (Robbins et al., 1993
). The number of disomic sperm (exhibiting signals for XX8, YY8, XY8, X88 or Y88) per sample was recorded and the total number of disomic sperm per sample (per 10,000 cells) was calculated. The same was done for the number of diploid sperm (exhibiting signals for XX88, YY88 or XY88). Finally total disomies and total diploidies were summed.
Biomarkers of exposure to cigarette smoke and metals
Urine samples were obtained with samples 1 (September 1995), 2 (January 1996) and 4 (March 1996), and assayed for cotinine to confirm self-reported smoking status (Langone and Van Vunakis, 1982). Blood was collected by venipuncture with samples 1 (September 1995), 3 (February 1996), 5 (September 1996) and 6 (February 1997) and sent to the National Institute of Public Health (Prague, Czech Republic) for analysis of lead, mercury and cadmium, as an indication of possible exposure to metals from the air pollution or from occupational exposures or hobbies.
Ambient air pollution monitoring
Air pollution data, collected for 24 h periods as described (Pinto et al., 1998), included sulphur dioxide (SO2), nitrogen oxides (NOx), and particulate matter <10 µm in aerodynamic diameter (PM10). These data were collected daily except in summer months when PM10, and therefore PAH extracted from it, was measured only once or twice per week. Because air pollution is consistently low in the summer, as indicated by historic and ongoing monitoring, values obtained during the summer were considered representative of all days. PAH was extracted from the particulate matter, fractionated and analysed by gas chromatographymass spectroscopy to identify carcinogenic and non-carcinogenic species and to calculate the concentration of total PAH (Lenicek et al., 2001
). For each pollutant, the average value for the 90 days preceding each semen sampling was calculated in order to represent the average exposure to which those sperm had been exposed during their development from stem cells, through meiosis, spermiogenesis and epididymal transit (Clermont, 1963
; Heller and Clermont, 1964
).
Statistical analysis
Semen data were transformed when necessary to optimize distribution normalcy. Each semen outcome was then tested for association with exposure using Mixed models (PROC MIXED, SAS, 8.01, Cary, NC, USA) for repeated measures. This procedure permits inclusion of participants with incomplete data (fewer than seven semen samples). Exposure was dichotomized as low (relevant to the reference samples 1, 5 and 7) or high (relevant to the winter samples 2, 3, 4 and 6). Factors potentially associated with semen measures and categorized as previously described (Selevan et al., 2000) were considered in the regression model. These included: abstinence interval (<2 days versus longer); high fever (>38°C) within the last 3 months; wearing briefs versus loose-fitting underwear; alcohol consumption (025, 25199 or
200 ml ethanol/week); cigarette smoking (none, <1 or
1 pack/day); caffeine consumption (<
,
3 or
3 coffee cup equivalents/day); and working with solvents or metals (
10 h/week versus less). If a variable in the model was either of borderline statistical significance (P < 0.1) or a confounder (i.e. the associated
in the mixed model changed by >10%), it was retained in the final model. Because cigarette smoke contains chemicals also found in the air pollution, and has been associated with altered semen quality, including sperm chromatin structure (Potts et al., 1999
), all models were tested with smoking included. P < 0.05 was considered statistically significant.
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Results |
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Participants
Men from Teplice who had participated in the first study (n = 183) were mailed an invitation to participate in this study. Eighty-nine men were subsequently contacted by phone, while the remainder either could not be located (n = 47) or had moved out of the district (n = 45). Of those contacted, 60 agreed to participate in the study and 29 refused. Of the 60 who agreed, 12 did not show up for their initial appointment, leaving 48 men who were enrolled in the study. During the course of the study, 11 men were excluded because they moved or travelled out of the district (n = 6), or were lost to follow-up (n = 5). Also, one man was disqualified on medical grounds, because he had a varicocelectomy midway through the study. Of the 36 men who completed the study, 21 gave seven samples, 10 gave six, two gave five, two gave four and one gave three samples.
Characteristics of the study participants were similar to those in the preliminary study (Selevan et al. 2000), with only slight increases in cigarette, alcohol and caffeine consumption (Table I). About half of the participants were self-reported smokers, and all but one of these smoked less than a pack a day. Self-reported 24 h smoking agreed with urinary cotinine levels, e.g. Spearman correlation = 0.64, P < 0.001, for sample 1. No participants reported working with solvents and only three men reported working with metals.
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Blood levels of lead, cadmium and mercury did not differ comparing reference samples with those obtained mid-winter (reflecting exposure to higher air pollution) (P > 0.05, Wilcoxon test). Mean lead concentration (CI) in blood collected with sample 1 was 4.3 µg/dl [95% confidence intervals (CI): 3.2, 5.3] and only one participant had a blood lead >10 µg/dl. Mean cadmium concentration for these samples was 1.0 µg/l (95% CI: 0.7, 1.2) and individual values correlated with smoking status (r = 0.64, P < 0.001) as expected (reviewed by Robbins and Cousins, 1998). Mean mercury concentration for these samples was 0.7 µg/l (95% CI: 0.6, 0.9).
Semen outcomes and their association with air pollution
Descriptive statistics for semen data (means and 95% CI) are given in Table II, showing the time periods when air pollution levels were higher (winter: sampling periods 2, 3, 4 and 6) versus the reference sample periods (1, 5 and 7). Examination of values for the reference samples reveals that this cohort of young men consisted largely of normozoospermic individuals (reference values, World Health Organization, 1999) for semen volume, sperm concentration, and sperm motility. Mean values for percentage normal morphology, percentage normal head morphology and sperm motion characteristics (measures for which World Health Organization reference values are not provided) were comparable to those recorded in the first study (Selevan et al., 2000
). Mean values for SCSA%DFI were within the range associated with good fertility potential (Evenson et al., 1999
, 2002
) and values for total aneuploidy were within the range of those reported in the literature (reviewed by Shi and Martin, 2002
).
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Results of the repeated measures analysis categorized air pollution as low (all reference samples) versus high (all winter samples), and controlling for various factors other than exposure that might affect semen quality (Table III). is the slope of the regression line for the relationship between exposure and outcome, and is significant in the model when the 95% CI of the
does not include 0. Only mean SCSA%DFI was significantly associated with exposure (Table III). No significant associations were found between exposure to air pollution and any of the routine semen measures (volume, concentration, total count, percentage motile, or percentage normal morphology considering whole sperm or sperm head shape). Similarly, no significant associations were found between exposure and any of the three selected CASA measures, or total aneuploidy (Table III). Separating total aneuploidy into broad categories (total disomy or total diploidy) did not change the non-significant findings, nor did examination of frequencies of individual types of disomy or diploidy.
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Discussion |
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Consistent with the earlier study (Selevan et al., 2000), the present study found no significant associations between exposure and sperm concentration or total sperm counts, measures of sperm production. Although inter-individual variability in sperm concentration was high in this cohort, as is typical for humans, the repeated measures study design would be expected to detect consistent changes in this measure. It is likely therefore that intermittent exposure to air pollution, including the high levels encountered during thermal inversions in Teplice in the winter, is not a risk factor for decreased sperm concentrations or sperm counts. These results do not rule out an impact of continuously high air pollution on sperm production. Adamopoulos et al. (1996)
reported decreasing sperm concentrations in Athenian men during a period when air pollution was increasing and hypothesized that air pollution might contribute to secular trends in declining sperm counts.
Also consistent with the earlier study (Selevan et al., 2000), this study found a significant association between exposure to air pollution and the percentage of sperm with fragmented DNA (SCSA%DFI) thereby increasing the weight of evidence that exposure to high levels of air pollution may have damaging effects on sperm DNA. That this effect occurred in the absence of other changes in semen quality is not unexpected because SCSA is considered an independent measure of sperm function, namely sperm genetic integrity (Evenson et al., 1980
., 1991
, 1999
, 2002
). In clinical populations, routine semen measures of sperm production and physiology (sperm concentration, motility, morphology) are not strong predictors of SCSA. A biologically plausible hypothesis regarding the aetiology of the observed association between exposure to air pollution and increased SCSA%DFI is that reactive metabolites of PAH might reach the testes and react with sperm DNA to form adducts. Previous studies in the Teplice Program have shown that PAH found in the PM10 fraction can enter the body and form DNA adducts in at least two tissues, blood and placenta (Binkova et al., 1995
; Topinka et al., 1997
). Although DNA adducts in most germ cell stages should be repairable, DNA repair does not occur in condensed spermatids and epididymal sperm in which protamine has replaced somatic histones, rendering the DNA transcriptionally inert (reviewed by Baarends et al., 2001
). Thus, toxicant-induced DNA damage in this repair-deficient period of late spermiogenesis and epididymal sperm maturation, or about the last 10 days before ejaculation, would not be repaired and may be manifest as increased SCSA%DFI. A recent study documenting the presence of benzo(a)pyrene diol epoxideDNA adducts in sperm of men who smoke cigarettes (Zenzes et al., 1999
) lends further plausibility to this hypothesis. Furthermore, exposure to particulate matter containing PAH has been associated with increased rates of germ line mutation at expanded-simple-tandem-repeat DNA loci in mice (Somers et al., 2004
). The latter report suggests that PAH metabolites may reach the testes and induce mutations in pre-meiotic germ cells. The present results, with transient changes in SCSA%DFI associated with intermittent air pollution, are consistent with DNA damage to post-meiotic, late stage spermatids.
The change in SCSA%DFI associated with exposure in this study, although statistically significant, was relatively modest in magnitude. Mean baseline values for SCSA%DFI for this cohort were within a range considered normal (1215%) and increased to 1520% after exposure, which is still considered indicative of good fertility potential (Larson et al., 2000). Based on clinical studies, however, when SCSA%DFI approaches and exceeds 30% the risk for infertility and spontaneous miscarriage is considerable, even in men with otherwise good semen quality (Evenson et al., 1999
; Larson et al., 2000
; Spano et al., 2000
; Zini, 2002
; Larson-Cook et al., 2003
; Saleh et al., 2003
; Virro et al., 2004
). Although the change in average SCSA%DFI observed in this study may not have affected the fertility potential of these men, changes of this magnitude could impact fertility in men with higher baseline SCSA%DFI. Thus, when evaluating environmental risks to the general population, even modest increases in SCSA%DFI may impact fertility in those men at the higher end of the distribution of SCSA%DFI.
In contrast to the SCSA findings, associations between exposure to intermittent air pollution and sperm morphology or motility (percentage motile sperm) found in the earlier study were not replicated in the present study. Inconsistencies between studies, in which essentially the same laboratory methods were used, could be due to differences in the exposures. Indeed, remedial actions by the Czech government resulted in a decline in air pollution between 1993 and subsequent years (Pinto et al., 1998; Benes et al., 2001
). With specific reference to the two semen studies being discussed, mean SO2 levels for comparable 90 day intervals (late December to late March) were notably higher in 1993 (164.0 µg/m3, Selevan et al., 2000
) compared with 1996 (78.5µg/m3, from Figure 1, this study). The same was true for PM10 where the comparable 1993 mean was 184.7 µg/m3 (Selevan et al., 2000
) compared with 67.8 µg/m3for 1996 (from Figure 1, this study). Other differences that could affect the consistency of results include differences in the timing of peak exposures (during thermal inversions) with respect to semen sampling, and differences in the components of the particulate fraction which can change from year to year (Pinto et al., 1998
). Nevertheless, the lack of consistency between these two studies coupled with the lack of association with CASA outcomes (i.e. the quality of sperm motion) in both studies, weakens the weight of evidence for a convincing association between exposure to air pollution and these specific measures of sperm quality.
Exposure to high air pollution was not associated with increased sperm aneuploidy in this study, although an earlier study had found an association with increased incidence of YY disomy in a small group of non-smoking men sampled in Teplice in the winter of 1993 when exposures were higher (Robbins et al., 1999). This discrepancy suggests that intermittent air pollution may not be a significant risk factor for sperm aneuploidy. However, these results do not rule out possible interactions between exposures to mutagens in air pollution (intermittent or continuous) and in cigarette smoke that could contribute to genetic damage in sperm (such as increased aneuploidy or DNA damage). The present cohort included both non-smokers and smokers, although the latter were not heavy smokers, all but one smoking less than a pack a day. Smoking alone was not associated with SCSA%DFI (data not shown), but SCSA%DFI remained significantly associated with air pollution whether or not smoking was included in the model. Previous reports have suggested that smoking
1 pack of cigarettes a day may be associated with increased sperm aneuploidy (Rubes et al., 1998
), and smoking
10 cigarettes a day may be associated with DNA damage as measured by a variant of the SCSA (Potts et al., 1999
).
Despite the lack of association between air pollution and sperm aneuploidy in this study, these longitudinal data were useful for examining the stability of sperm aneuploidy (disomy and diploidy) in individuals over an extended period. Analysis demonstrated that three men in this cohort consistently exhibited unusually high levels of sperm aneuploidy over the 2 years (independent of exposure), suggesting that a common genetic defect may influence endogenous levels of sperm cell aneuploidy (Rubes et al., 2002). In the present study, the use of a repeated measures design allowed examination of the risks of exposure to air pollution in a small group of men despite this inter-individual variability for the aneuploidy measures.
The present study included measurement of blood lead, cadmium and mercury, as an indicator of possible occupational or environmental exposures, and because relatively high levels of lead (>40 µg/dl blood) have been associated with poor semen quality (reviewed by Apostoli et al., 1998; Robbins and Cousins, 1998
). Results indicated that the levels of these metals in the participants were within the range expected for the general US population (Centers for Disease Control, 2003
), and were well below those expected to be associated with poor semen quality (Apostoli et al., 1998
; Robbins and Cousins, 1998
; Bonde et al., 2002
). Furthermore, these blood metals were not associated with air pollution. Therefore, blood metal data do not suggest significant occupational or environmental metal exposures relevant to the study findings. To our knowledge, these studies in the Czech Republic (Selevan et al., 2000
and the present study) are the only environmental epidemiology studies to date reporting associations between exposure to ambient air pollution and altered semen quality in humans. However, a recent occupational health study in Italy found changes in semen quality in motorway tollgate workers exposed continuously (6 h per work day) to automobile exhaust (De Rosa et al., 2003
). Compared to an age-matched control group, these men had lower sperm viability, motility and velocity (measured by CASA), and fewer sperm with normal chromatin evaluated microscopically via acridine orange staining. Unlike our participants, however, these men had elevated blood lead levels (averaging 20.1 µg/dl) to which the authors attributed the seminal deficiencies. Although automobile exhaust contributed to the air pollution in Teplice, source signature models showed that combustion products of coal used for industry and home heating were the major components (Pinto et al., 1998
). Thus the present study differs from that of De Rosa et al. (2003)
with respect to both exposure composition and duration.
Taken together, this study and the previous study (Selevan et al., 2000) provide novel evidence that exposure to episodes of relatively high air pollution may have adverse effects on semen quality, specifically on sperm chromatin integrity. Because high SCSA%DFI (>30%) has been associated with clinical infertility and increased risk of spontaneous abortion (Evenson et al., 1999
; Larson et al., 2000
; Spano et al., 2000
; Zini et al., 2002
; Larson-Cook et al., 2003
; Saleh et al., 2003
; Virro et al., 2004
), the present findings may have implications for fertility on a population basis. Confirmation of similar changes in other study groups exposed to episodic or continuous air pollution, especially at levels that approach or exceed US air quality standards, would be of value for more detailed risk characterization.
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Acknowledgements |
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Submitted on February 16, 2005; resubmitted on May 4, 2005; accepted on May 6, 2005.
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