1 Department of Molecular and Clinical Endocrinology and Oncology, 2 Department of Preventive Medical Sciences and 3 Department of Internal Medicine, University of Naples Federico II, Italy
4 To whom correspondence should be addressed at: Department of Molecular and Clinical Endocrinology and Oncology, University of Naples Federico II, via S. Pansini 5, 80131 Naples, Italy. e-mail: miderosa{at}unina.it
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Abstract |
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Key words: environmental pollution/lead/methaemoglobin/semen analysis/spermatogenesis
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Introduction |
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We conducted this observational, analytical, prospective casecontrol study to evaluate whether continuous exposure to traffic-derived environmental pollutants adversely affects male fertility.
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Subjects and methods |
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Study protocol
The study was performed from January 2000 to January 2002. All 170 subjects were interviewed about the family lifestyle. They were asked the number of months of unprotected intercourse before the couples first pregnancy (time to pregnancy; TTP). Serum FSH, LH and total testosterone levels and semen analysis were measured to evaluate gonadal function. The concentrations of carbon monoxide (CO), nitrogen oxide (NO), sulphur oxide (SO) and Pb were measured at eight tollgates and at eight sites in the urban area where the 170 subjects live. Blood levels of methaemoglobin (MHb), sulphaemoglobin (SHb), carboxyhaemoglobin (COHb), Pb, zinc protoporphyrin (ZnPP), which are biological markers of environmental pollution, were assayed in all subjects.
Hormonal assay
Serum levels of FSH (reference range = 0.711.1 IU/l), LH (reference range = 0.87.6 IU/l) and testosterone (reference range = 2.815.1 µg/l) were determined with a chemiluminescent enzyme immunometric assay using commercially available kits and the Immulite® automated analyser (Diagnostic Products Corporation, USA; Medical System, Italy). Intra- and inter-assay coefficients of variation were respectively: FSH: 5.4 and 6.5%; LH: 4.8 and 8.1%; testosterone: 4.3 and 6.5%.
Semen analysis
Semen was analysed according to the World Health Organization (World Health Organization, 1999) guidelines. Seminal fluid samples were obtained by masturbation after 35 days of sexual abstinence. After collection, the ejaculates were left to liquefy for 30 min at room temperature. Sperm count and motility were evaluated with the Makler Counting Chamber; sperm morphology was evaluated at the optical microscope (x40 magnification) after dilution of sperm (1:1) in phosphate-buffered saline and Giemsa staining.
The live sperm count (normal range 75%) was evaluated with the eosin test, sperm membrane function (normal range
60%) with the hypo-osmotic swelling test (HOS), the sperm kinetic index (normal range
30 mm) within bovine cervical mucus penetration test (CMPT), and sperm nuclear DNA integrity (normal range
70%) with the acridine orange test.
Sperm kinetics were evaluated by computer-assisted sperm analysis with the Cell Track/S® apparatus (Motion Analysis Corporation, USA) to determine sperm curvilinear velocity (VCL) (normal range 46 µm/s), sperm linear velocity (VSL) (normal range
25 µm/s), the linearity of sperm motion (LIN = VSL/VCL) (normal range
58%) and the amplitude of lateral movement of the sperm head (ALH) (normal range
1.5 µm).
Environmental pollutants assay
The gaseous pollutants NO and SO were measured with specific analysers (Babuc, L.S.I., Italy) for 24 h in summer (June and July) and in winter (December and January) at eight tollgates and at eight sites in the area were the 170 subjects lived. Atmospheric Pb was concentrated on porous filters by a suction pump (Bravo; TCR Tecpora, Italy) and the filters were analysed in atomic absorption (Branderberger, 1967). The maximum occupational exposure to these pollutants according to Italian legislation, is <80 <200 µg/m3 for 1 h/24 h for NO; <120 µg/m3/24 h for CO; <40 mg/m3/24 h for Pb; <2 µg/m3/24 h for SO.
Toxicological evaluation
Absorption of environmental pollutants was evaluated by specific dose-and-effect gauges: MHb for nitrogen dioxide, SHb for sulphur dioxide, COHb for carbonium oxide, circulating blood Pb and ZnPP for environmental Pb (Schifman and Finley, 1981). The percentage concentrations of MHb (normal range <1.5% Hb), SHb (normal range <1.2% Hb) and COHb (normal range: for smokers <8% Hb; for non smokers <2.5% Hb) were measured by the oxymetric method (OSM3; Radiometer, Denmark) (Kelner and Bailey, 1985
). Environmental Pb levels (normal range <20 µg/dl) were measured by spectral analysis in atomic absorption, and ZnPP levels (normal range <40 µg/dl) with the fluorimetric method.
Statistical analysis
Continuous data were expressed as mean ± SEM and data obtained in the study and control groups were compared by the two-tailed t-test for unpaired data. Categorical data (married subjects in the control and study groups) were expressed as percentages and compared by the Yates corrected 2-test. Based on values established by the World Health Organization (WHO) (see above), the study group was divided into two subsets: 30 subjects with normal total sperm motility (subset 1) and 52 with deranged sperm total motility (subset 2). We used linear regression analysis to correlate seminal variables and environmental pollutants and toxicological markers in the study group as a whole and in the two subsets. P < 0.05 was considered significant.
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Results |
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Semen analysis
Sperm parameters (excluding sperm volume and count) were below the WHO reference range in the study group (Table II), and sperm total motility, forward progression, functional tests and sperm kinetics were significantly lower than in controls (P < 0.0001). The mean sperm count was similar in the two groups (Table II).
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Discussion |
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The peculiarity of this study is that it was conducted with a homogeneous population of young to middle-aged subjects equally and constantly exposed to occupation-related environmental pollutants. In fact, they were exposed to vehicle-discharged gases at their workplace, i.e. a small motorway tollbooth. The occupational exposure lasts 6 h a day in various work shifts. Consequently, all tollgate workers were exposed to the same environmental pollutants for the same length of time.
As expected, on-site measurements (data not shown) showed that they were exposed to higher amounts of environmental pollutants (NO, SO, CO, Pb) than age-matched controls. Higher circulating levels of MHb, SHb, Pb and ZnPP support this finding. The environmental concentrations of Pb, which were significantly higher in the study group versus control subjects, were consistent with blood Pb concentrations almost three times higher in the former. This finding is probably due to the tollgate workers continuous exposure to pollution. Prolonged sitting has also been implicated in poor semen quality. Figà-Talamanca et al. (1996) found that prolonged sitting could increase temperature in the pelvic region, which in turn could determine semen deterioration. More recently, Bujan et al. (2000
) reported elevated scrotal temperatures in car drivers. However, both our study group and control group had sedentary jobs.
There is a body of evidence linking blood Pb concentrations with sperm alterations. Lead exposure resulted in a decreased intertubular tissue volume in the testes of adult rats and a significant reduction of plasma testosterone (Thoreux-Manlay et al., 1995). In cynomolgus monkey testes, chronic Pb treatment caused impairment of sperm chromatin (Foster et al., 1996
) and ultrastructural alterations of Sertoli cells and of spermatogenetic cells (Foster et al., 1998
). In mice, low doses of Pb reduced the number of sperm within the epididymis, and high doses reduced the sperm count percentage of motile sperm and increased the percentage of abnormal sperm within the epididymis (Wadi and Ahmad, 1999
). The negative relationship between Pb and Zn was confirmed by the finding that Zn supplementation ameliorates Pb-induced testicular damage at cellular and sub-cellular levels in rats (Batra et al., 1998
). More recently, it has been demonstrated that concomitant administration of Zn reduces Pb levels in rat testes, and improves the spermatogenesis arrested by Pb alone (Batra et al., 2001
). Earlier findings showed asthenozoospermia and teratozoospermia in Pb-exposed workers (Lerda, 1992
), and decreased sperm count, suppressed FSH levels and decreased ventral prostate weight in experimental animals exposed to Pb (Sokol et al., 1998
). Blood Pb levels >40 µg/dl increased the risk of impaired sperm concentration in men employed at a Pb smelter compared with individuals whose blood values were <15 µg/dl (Alexander et al., 1996
). Increased circulating Pb levels are also associated with adverse changes in sperm count, ejaculate volume, percentage of motile sperm, swimming velocity and morphology (Moorman et al., 1998
). Total sperm count and concentration were shown to decrease with increasing concentrations of blood Pb (Alexander et al., 1998
). Blood Pb levels are also inversely correlated with the percentage of live sperm (Dawson et al., 1998
). Telisman et al. (2000
) found that even moderate exposure to Pb significantly reduced human semen quality but they did not find any conclusive evidence for Pb-related derangement of male reproductive endocrine function.
Interestingly, Pb accumulates in male reproductive organs; human testes and sperm contain numerous potassium channels through which metallic toxicants can enter into mature sperm (Benoff et al., 2000). Pb can compete with, or even replace the Zn in human protamine at two different sites, so causing a conformational change in the protein. This interaction adversely affects sperm chromatin condensation (Quintanilla-Vega et al., 2000
). Bonde et al. (2002
) recently observed that sperm concentration was reduced by
49% in men with a blood Pb concentration >50 µg/dl; however, there was no indication of a linear trend of lower sperm concentration with increasing blood Pb values, and sperm chromatin deterioration was not correlated with blood Pb concentration. Circulating Pb concentrations were higher in our tollgate workers (20.1 ± 0.6 µg/dl) than in controls but lower than those reached in workers at a Pb smelter (Alexander et al., 1996
). Sperm count did not differ significantly between our study group and controls, whereas functional parameters, motility, functional test and sperm kinetics were significantly lower in the study group. This finding is in line with the report that spermatogenesis is impaired in men with high circulating Pb levels (Alexander et al., 1996
). In fact, in our subjects who have intermediate blood Pb levels, sperm functional activity was diminished. The difference between tollgate and Pb smelter workers is probably due to the fact that, although exposure to Pb was less intense in the former, it was continuous.
In agreement with previous findings (Ng et al., 1991), serum FSH levels were significantly higher in our study group than in controls; the mean sperm count was normal (World Health Organization, 1999) and similar to controls. However, blood Pb levels in the study group were inversely correlated with sperm count, suggesting that the absorption of environmental Pb at lower doses with respect to occupationally Pb-exposed workers induced impairment of the ejaculates because of damage to the germinal epithelium and a subsequent increased pituitary FSH response. This is confirmed by the finding that most qualitative parameters (motility, viability, membrane function, DNA integrity, and kinetics) were impaired in the tollgate workers and were inversely related to MHb levels in the subset with abnormal parameters. Methaemoglobin level, which is a marker of NOx exposure, was increased in the study group. Our tollgate workers were undoubtedly exposed to high NOx levels and their increased MHb levels match this observation. The impaired patterns of sperm function correlated to MHb levels suggest that NO damages sperm (see Table III). In addition, the TTP was significantly longer in tollgate workers than in controls.
The usefulness of TTP data has been questioned. Baird et al. (1986) used TTP in epidemiological studies to evaluate the effects of pollutants on fertility. On the other hand it has been suggested that TTP is influenced by factors that could change over time or may differ among populations (Basso et al., 2000
). Moreover, Slama et al. (2002
) noted that morphologically normal sperm influences TTP regardless of sperm concentration. Similarly, Andersen et al. (2002
) did not find an association between TTP and sperm count or sperm motility. In contrast, Sallmen (2001
) demonstrated that male exposure to Pb is associated with delayed conception. Although, Apostoli et al. (2000
) found no difference in fertility between Pb-exposed and non-exposed subjects, TTP was significantly longer at Pb exposure levels >40 µg/dl. Time to first pregnancy was higher in our tollgate workers than in controls even though blood Pb levels did not reach those reported by Apostoli et al. (2000
). Our finding reinforces the concept that blood Pb levels do not necessarily reflect the effects induced by long-term Pb exposure.
In conclusion, our study demonstrates that continuous exposure to traffic pollutants impairs sperm quality in young/middle-aged men. The comparative evaluation of sperm parameters, absorption markers and environmental concentrations indicates that Pb is probably causing the impaired spermatogenesis. Alteration of sperm function could be considered a precocious marker of detrimental toxicological effects. The analysis of potential fertility of these workers after they have been removed from tollgate duty will add other important information to this intriguing issue. Although longitudinal data are necessary to identify the specific modifications induced by each pollutant on the male endocrine and reproductive system, our results will hopefully prompt clinical and epidemiological studies of male infertility in other work categories exposed to similar levels of environmental pollutants. Lastly, given the effect of pollution on our tollgate workers, health authorities should be alert to the insidious health effects of environmental pollution.
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Acknowledgements |
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References |
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Submitted on July 27, 2001; resubmitted on December 23, 2002; accepted on February 5, 2003.