1 Department of Occupational and Environmental Medicine, 3 Department of Clinical Chemistry, Institute of Laboratory Medicine, Lund University Hospital, SE-221 85 Lund and 2 Fertility Centre, Malmö University Hospital, Malmö, Sweden
4 To whom correspondence should be addressed. Email: anna.rignell-hydbom{at}ymed.lu.se
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Abstract |
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Key words: polychlorinated biphenyls/POP/p,p'-DDE/semen quality/sperm motility
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Introduction |
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It has been proposed that exposure to endocrine disrupters, such as certain POPs, may contribute to an impairment of sperm motility, concentration and volume, and affect reproductive function (Sharpe, 2001). Carlsen et al. (1992)
have published a meta-analysis that suggested a 40% decline in human sperm concentration between 1938 and 1990. This finding has been supported by some other studies (Auger et al., 1995
; Irvine et al., 1996
; Van Waeleghem et al., 1996
; Swan et al., 2000
), but in other studies no time-related changes in sperm concentration have been found (Olsen et al., 1995
; Bujan et al., 1996
; Fisch et al., 1996
). Sperm motility, together with concentration and morphology, is considered as one of the important predictors of male fertility in vivo. The percentage of motile sperm was found to be an independent predictor of the probability of achieving pregnancy in 358 normal Danish couples (Larsen et al., 2000
). In contrast to the findings for sperm concentration, no threshold value was found for the relationship between percentage motile sperm and fertility, indicating that any factors affecting sperm motility can also have an effect on the fertility potential of the male.
There is evidence from wildlife and laboratory animal studies that POPs can affect male reproductive function, mainly if the exposure takes place during fetal or early postnatal life (Sager, 1983; Sager et al., 1987
; Mably et al., 1992b
; Fry, 1995
; Gray et al., 1995
, 1997
; Kelce et al., 1995
; Cooke et al., 1996
; Sommer et al., 1996
; Faqi et al., 1998
; Huang et al., 1998
; Kim, 2001
; Hsu et al., 2003b
). There are only few reports on the effects of POPs on male reproduction in humans, mainly indicating weak negative effects on sperm motility (Bush et al., 1986
; Guo et al., 2000
; Dallinga et al., 2002
; Hauser et al., 2003
; Hsu et al., 2003a
; Richthoff et al., 2003
).
Fishermen from the Swedish east (by the Baltic Sea) and west coasts have been found to eat on average more than twice as much fatty fish than subjects from the general Swedish population (Svensson et al., 1995). Fatty fish species, mainly salmon and herring, from the Baltic Sea are much more contaminated with POPs than corresponding fish from the Swedish west coast (Bergqvist et al., 1989
). This was reflected in higher average plasma levels of dioxin-like POPs among east coast fishermen (290 pg/g lipid) than among west coast fishermen (139 pg/g lipid) and men from the general Swedish population (123 pg/g lipid) (Svensson et al., 1995
).
In the present study, we used 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) as a biomarker for POP exposure, because it correlates very well (r=0.9) with both total PCB concentration in plasma and serum (Grimvall et al., 1997; Glynn et al., 2000
) and the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalent (TEQ) in plasma from PCBs (all r
0.9) as well as the total POP-derived TEQ in plasma (r=0.74) (Gladen et al., 1999
). Another relevant biomarker is the anti-androgenic compound p,p'-DDE, which is still present in relatively high serum concentrations in men consuming fatty fish from the Baltic Sea (Sjödin et al., 2000
).
The aim of the present study was to assess whether exposure to CB-153 and.p,p'-DDE, through consumption of fatty fish from the Baltic Sea, affects human semen quantity, quality and reproductive hormones. Swedish fishermen constitute a socio-economically homogenous group with high fish consumption and are therefore a suitable study population. In order to ensure a sufficient variation in POP exposure, fishermen from both the Swedish east (more exposed) and west coasts (less exposed) formed the study base.
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Materials and methods |
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Non-participants
The non-participants from the original fishermen's cohort had a similar age distribution (median 51.8 years, range 2967) as the participants (median 50.6 years, range 2967). This was also the case with fishermen who had become members since 1988 (median 47 years, range 2067 versus median 46 years, range 2459). The participants had on average 2.0 children. We do not have any directly comparable fertility data for the non-participants, but a previous study showed that during 19731991, fishermen's wives, on average, gave birth to 2.0 infants (Rylander et al., 1995).
The men who wanted more information about the semen study had similar body mass index (BMI) values to those who did not want such information (median BMI 26, range 1741; and 26, range 1844, respectively). The percentage of men who smoked was 23% in both groups. The median age differed somewhat (51 years, range 2967; and 54 years, range 2967, respectively).
Questionnaire
Information on lifestyle, medical and reproductive history was collected through telephone interviews (Table II). The questionnaire was sent out to the participants a couple of weeks before telephone contact. In this way, the subjects had time to be acquainted with the questions. During the telephone contact, an agreement was reached on a time and date for collection of semen and blood samples at the subject's home. The participants received information on the procedures for collecting the semen samples in both verbal and written form. This study was approved by The Ethical Committee at Lund University.
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The blood and semen samples were collected at the subject's residence. The men were instructed to collect the semen sample within 30 min before the arrival of the mobile laboratory. The semen samples were analysed within 1 h (median 45 min) after collection. The ejaculate volume was estimated by weighing the collection tube with the semen sample and subsequently subtracting the pre-determined weight of the empty tube assuming that 1 ml of ejaculate weighs 1 g. The measurements were made using a Sartorius® scale and the results were expressed to one decimal place. For assessment of sperm motility, 6 µl of well mixed semen was placed on a clean glass slide, which had been kept at 37°C, and covered with an 18 x 18 mm coverslip. The preparation was placed on the heated stage (37°C) of the phase contrast microscope and immediately examined at a total magnification of x 40. Motility assessment was performed according to the guidelines of the World Health Organization (1999). Sperm were scored in categories A, B, C and D. A corresponded to rapid progressive motility, B to slow progressive motility, C to non-progressive motility and D were immotile sperm. Because there is considerable inter-observer variation in discriminating between A and B motile sperm, these two scores were pooled together and represent the percentage of progressively motile sperm. Previous studies have indicated that the percentage of A+B motile sperm is the most significant of the WHO categories in relation to fertility potential of a male (Jouannet et al., 1988
).
Sperm concentration was assessed with an improved Neubauer haemocytometer, and positive displacement pipettes were used for a proper dilution of the ejaculate. The appropriate dilution was determined after preliminary examination of the undiluted sample, and only spermatozoa with tails were counted. Furthermore, sperm concentration and the percentages of motile, locally motile and immotile sperm were assessed by use of a CRISMAS computer-aided sperm motility analyser (CASA), previously described by Larsen et al. (2000), with the only exception that a 20 am Microcell chamber (Leja, Oslo, Norway) was used, as recommended by the World Health Organization (1999)
. The percentage of motile sperm was based on the level of curvilinear velocity, with >25 µm/s at 37°C for motile spermatozoa, 525 µm/s for locally motile spermatozoa and <5 µm/s for immotile spermatozoa.
In order to reduce the variation in assessment of sperm characteristics, all analyses were performed by one investigator (A.R.H.) who was trained at the Fertility Center, Malmö University Hospital. The investigator had no knowledge about the exposure biomarker levels of the subjects when she analysed the semen samples, The Fertility Center is accredited by the European Academy of Andrology and participates in the Nordic Association of Andrology and the European Society of Human Reproduction and Embryology (ESHRE) quality control programme (Cooper et al., 2002). The intra-laboratory coefficient of variation for assessment of sperm concentration was found to be <8.5% The average discrepancies for sperm concentration and motility (sum of A+B grade) between the principal investigator (A.R.H.) and the laboratory staff at the Fertility Center in Malmö were 7 and 11%, respectively. Previous studies have demonstrated a coefficient of variation among trained technicians within one laboratory performing analysis of sperm concentration to be 11% (Cooper et al., 2002
).
Blood sampling
Venous blood samples were collected from 190 of the men. The samples were centrifuged and sera were frozen at 80°C for later analysis. The samples were collected between 7 a.m. and 9 p.m.
Hormone analyses
Serum concentrations of FSH, LH and estradiol were analysed with immunofluorometric techniques. The total assay coefficients of variation were 2.9, 2.6 and 8.1% respectively. Serum testosterone and sexual hormone-binding globulin (SHBG) were measured by commercially available immunoassays. The total assay variation coefficients were 5.5 and 4.6% respectively. Inhibin B levels were assessed using a specific immunometric method, as previously described (Groome et al., 1996), with a detection limit of 15 ng/l and intra-assay and total assay coefficients of variation below 7%
Determination of CB-153 and p,p'-DDE
The levels of CB-153 were determined as previously described by Richthoff et al. (2003). In addition, p,p'-DDE was analysed by the same method. Briefly, the CB-153 and p,p'-DDE were extracted from the serum by solid phase extraction (Isolute ENV+; IST, Hengoed, UK) using on-column degradation of the lipids and analysis by gas chromatographymass spectrometry. 13C12-labelled CB-153 and 13C12-labelled p,p'-DDE were used as internal standards. The selected ion monitoring of p,p'-DDE was performed at m/z 318, while m/z 330 was used for the internal standard. The relative standard deviations, calculated from samples analysed in duplicate on different days, was 7% at 0.6 ng/ml (n=76) and 5% at 1.5 ng/ml (n=37) for CB-153, and 12% at 0.6 ng/ml (n=56) and 7% at 2.4 ng/ml (n=50) for p,p'-DDE. The detection limits were 0.05 ng/ml for CB-153 and 0.1 ng/ml for p,p'-DDE. The analyses of CB-153 and p,p'-DDE are part of the Round Robin inter-comparison programme (Professor Dr Med Hans Drexler, Institute and Out-Patient Clinic for Occupational, Social and Environmental Medicine, University of Erlangen-Nuremberg), with analysis results within the reference limits.
Determination of lipids by enzymatic methods
Plasma concentrations of triglycerides, cholesterol and phospholipids were determined by enzymatic methods using reagents from Boehringer-Mannheim (triglycerides and cholesterol; Mannheim, Germany) and Waco Chemicals (phospholipids; Neuss, Germany). The total lipid concentration in plasma was calculated by summation of the amounts of triglycerides, cholesterol and phospholipids. In these calculations, the average molecular weights of triglycerides and phospholipids were assumed to be 807 and 714 Da. For cholesterol, we used an average molecular weight of 571 Da, assuming that the proportion of free and esterified cholesterol in plasma was 1:2.
Statistical analysis
Bivariate associations between lipid-adjusted serum concentrations of CB-153 and p,p'-DDE and semen and hormone characteristics were evaluated using Pearson's correlation coefficients. To ensure that linear associations were reasonable (and accordingly the use of Pearson's r), scatter plots were assessed for all bivariate comparisons. The semen characteristics included semen volume, sperm concentration, total sperm count and sperm motility, and the hormone parameters included FSH, LH, testosterone, SHBG, testosterone/SHBG, inhibin B and estradiol.
The effect of the exposure variables CB-153 and p,p'-DDE, respectively, on the sperm characteristics and hormone parameters was evaluated by linear regression models. The exposure variables were treated as continuous variables (untransformed and log transformed) as well as categorized into five equally sized groups (see categories in Tables III and IV). The categorized variables were entered as dummy variables in the models. Some of the outcome variables, e.g. sperm concentration, had somewhat skewed distributions. For these variables, we also evaluated whether log transformation better fulfilled the model assumptions, which were checked by means of residual analysis. Age (as a continuous variable), BMI (as a continuous variable), abstinence time (02, >24, >46 and >6 days), time from semen collection to analysis (as a continuous variable) and current smoking status (yes versus no) were considered as potential confounders for the semen characteristics. A confounder should, by definition, be associated with the outcome as well as with the exposure variables. In the present data, neither abstinence time, time from semen collection to analyses nor smoking habits showed any correlation with the POP variables (all P-values >0.5 when age was included in linear regression models). On the other hand, age (P<0.01) was clearly associated and BMI (P=0.11) tended to be associated with the POP variables. These two variables were therefore included, one at a time, together with the exposure variable in the models. If the adjusted effect estimate differed by at least 15%, the potential confounder was kept in the model. In addition, we evaluated whether these variables modified the effect of exposure by including an interaction term in the model. The procedure was the same when we evaluated the effect of the exposure variables on the hormone parameters.
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Results |
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Treating sperm motility and sperm concentration as dichotomized variables did not change the pattern. The subjects in the highest exposed quintile more often tended to have A+B motile sperm below 50% compared with the subjects in the lowest exposed quintile (OR 2.5, 95% CI 0.97.0, P=0.07). Including age in the model, the OR decreased to 1.7 (95% CI 0.55.4, P=0.40).
Associations between p,p'-DDE and sperm and hormone parameters
There was a significant negative correlation between serum concentrations of p,p'-DDE and the proportion of A+B motile sperm (Figure 4). An increase of p,p'-DDE levels by 100 ng/g lipid implied a mean decrease of A+B motile sperm of 1.0% (95% CI 0.12.0, P=0.04; Table III). However, when age was included in the model, the association was no longer significant (mean difference 0.6% 95% CI 0.4 to 1.7, P=0.22). No significant associations between p,p'-DDE levels and the other semen characteristics were found, irrespective of whether the p,p'-DDE values were treated as a continuous variable (data not shown) or categorized into quintiles. Moreover, no significant associations between p,p'-DDE levels and hormones were observed (data not shown).
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Discussion |
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The lack of associations between POP biomarkers and sex steroid hormone levels were in agreement with similar findings in another group of adult men with relatively high exposure levels (Hagmar et al., 2001). On the other hand, in adolescent males with lower exposure levels to CB-153 than the men in the present study, a weak negative association between CB-153 and free testosterone levels was observed (Richthoff et al., 2003
). The discrepancy in the results might be due to age-related susceptibility or chance findings.
As with all human semen studies, low participation rates and potential selection bias are of concern. Results from a study by Larsen et al. (1998) suggest that both age and demonstrated fertility have an impact on the participation rate in semen studies. In the present study, the age distributions as well as the average number of children were very similar among the participants and the non-participants, as were smoking habits and BMI. Therefore, we do not suspect that selection bias concerning these factors is of major concern for the interpretation of the results in the present study.
We used both the manual technique and CASA for sperm motility assessment. The CASA technique might, in a stable laboratory environment, be considered as a more reliable and accurate method than the manual one, and it could therefore be considered as an unexpected finding that the associations between POP exposure and motility were stronger with the manual technique than with the CASA. On the other hand, in the present field study, where both techniques were employed in a mobile laboratory unit in a rebuilt mini-van, the CASA technique was perhaps not obviously the best performing one.
The analytical accuracy for the determinations of CB-153 and p,p'-DDE was high, as determined from the results from the interlaboratory comparison programme, and so was the precision for semen analysis. Thus we do not consider analytical measurement error to be a matter of great concern in the present study. A more crucial issue is whether CB-153 and p,p'-DDE are good enough proxy biomarkers of all POPs that could potentially be hazardous for male fertility. Previous studies indicate that CB-153 is a good marker for total PCBs (r=0.9) (Grimvall et al., 1997; Glynn et al., 2000
), hydroxylated PCBs (OH-PCBs) (Åke Bergman, personal communication) and dioxin-like compounds (Gladen et al., 1999
). However, we cannot exclude that there might be associations between other POPs, for which CB-153 and p,p'-DDE are less good proxy markers, and semen characteristics.
Ideally, to minimize observer bias, the investigator should not be aware of the hypothesis of the study. Regarding the present study, it was not feasible for the investigator to not be aware of which coast the subjects were from, but the investigator had no knowledge of the subjects' CB-153 and p,p'-DDE serum levels. There was a considerable overlap in exposure between subjects from the two costal areas.
We regarded abstinence time and time from sample collection to sample processing as potential confounders, because they have been related to semen volume, sperm concentration and motility (Schwartz et al., 1979; Magnus et al., 1991
; Bonde and Storgaard, 2002
). However, in our data, we found no association at all between abstinence time, time from collection to analysis and our exposure variables, and therefore abstinence time and time to analysis were not included in the multivariate analysis. Age and smoking habits have shown an effect on sperm motility, sperm concentration and semen volume in some studies (Johnson et al., 1984
; Vine et al., 1994
, 1996
; Jung et al., 2002
; Chen et al., 2003
; Eskenazi et al., 2003
), but not in others (Haidl et al., 1996
; Wong et al., 2000
). In the present study, smoking habits were not associated with the exposure variables, and therefore were not included in the multivariate analyses. Age was clearly associated with serum levels of CB-153 and p,p'-DDE, and therefore exhibited a strong confounding potential. It was therefore not surprising that age adjustment weakened the observed associations between the POP biomarkers and the reproductive function characteristics.
A number of PCB congeners and DDT-derived compounds act as endocrine disrupters and are able to bind to estrogen or androgen receptors. Thus, it has been shown that CB-153 as well as some other non-ortho-PCBs exhibit receptor-mediated anti-estrogenic or anti-androgenic effects (Bonefeld-Jorgensen et al., 2001). Moreover, OH-PCBs have shown a higher oestrogen receptor binding capacity than the parent PCB compounds (Layton et al., 2002
). p,p'-DDT exhibit estrogenic effects (McLachlan, 2001
), whereas its major metabolite p,p'-DDE exhibits anti-androgenic effects (Kelce et al., 1995
). POP exposure may affect spermatogenesis during the post-meiotic phase by disturbances in hormone homeostasis, or by formation of reactive oxygen species (Iwasaki and Gagnon, 1992
; Sun et al., 1997
) derived from OH-PCBs (Schlezinger et al., 1999
; McLean et al., 2000
), found in human serum in relatively high concentrations (Sjodin et al., 2000
).
In wildlife, there are several examples of defects in the male reproductive system after exposure to POPs (Guillette et al., 1994, 1996
; Fry, 1995
). In experimental animals, decreased sperm production has been observed after exposure to low doses of TCDD (Mably et al., 1992a
; Gray et al., 1995
, 1997
; Sommer et al., 1996
; Faqi et al., 1998
; Huang et al., 1998
), and p,p'-DDE (Kelce et al., 1995
). Moreover, high doses of the commercial PCB mixture Aroclor 1254 resulted in impaired mating behaviour and fertility in male rats (Sager, 1983
), and in decreased capability of sperm to fertilize oocytes, but had no effect on sperm motility or morphology (Sager et al., 1987
).
There are a few previous reports on the effects of POPs on male reproductive function in humans. Accidental exposures to very high POP levels have been shown to be hazardous for sperm function. Boys who been exposed in utero to very high doses of PCBs and PCDFs showed abnormal sperm morphology, and reduced motility and capacity to penetrate hamster oocytes (Guo et al., 2000). Similar changes in semen quality were found in postnatally exposed men, except that the sperm motility was unaffected (Hsu et al., 2003a
). There are contradictory results regarding whether high exposure to DDT/DDE (two orders of magnitudes higher than observed among the Swedish fishermen in the present study) among malaria control workers has an effect on semen function (Ayotte et al., 2001
; Dalvie et al., 2003
). Earlier studies on background POP exposures have been performed on selected groups of males attending fertility clinics. PCB concentrations in semen samples were inversely correlated to sperm motility, but only in samples with a sperm concentration <20 x 106/ml (Bush et al., 1986
). In a small study from The Netherlands, sperm counts and motility were inversely correlated to the sum of PCB metabolites (probably hydroxylated) in serum, but not to the PCB or DDE levels (Dallinga et al., 2002
). Surprisingly, a positive correlation was seen between morphologically normal sperm and the PCB concentration. The median PCB concentration was only about half of that in the present study. In a study from the USA, there was evidence of an inverse doseresponse association between one of the non-ortho-PCBs and sperm concentration, motility and morphology, and limited evidence of an inverse relationship of sum-PCB with sperm motility and sperm morphology, as well as limited evidence of an inverse association between p,p'-DDE and sperm motility (Hauser et al., 2003
). When comparing the CB-153 concentration in serum, the median concentration for the US males was only a fifth of that observed in the present study of Swedish fishermen, while the p,p'-DDE concentrations were comparable. It should be borne in mind that men recruited through infertility clinics represent a selected population that might have a higher susceptibility to environmental toxicants than the general population.
Besides the present study, there is only one previous study of the association between background exposure to PCB and semen function in subjects not recruited from fertility clinics. In an examination of 305 Swedish adolescent military conscripts, a weak but significant negative correlation between CB-153 and sperm motility was observed (Richthoff et al., 2003). The median CB-153 level among the military conscripts was only a third of the median level in the present study. The estimated association between CB-153 and sperm motility was stronger in the present study but, due to fewer subjects, did not reach statistical significance.
It should be kept in mind that the serum level of CB-153 and p,p'-DDE reflects the current internal dose of these contaminants and also gives a good estimate of the exposure during recent years, but it does not give information about prenatal exposure. According to recent hypotheses and in agreement with experimental data, the most profound effect of endocrine disrupters can be expected following fetal exposure (Skakkebaek et al., 2001). However, a caveat with this conclusion is that there are very limited data concerning reproductive effects after dosage of endocrine disrupters to adult animals. Moreover, the hazardous effects of PCDFs and PCBs after accidental postnatal exposure (Hsu et al., 2003a) argue against the hypothesis that endocrine disruption is restricted to the fetal period only.
To summarize, very high exposures to PCBs and PCDFs seem undoubtedly able to affect various human semen functions in a negative way. It is not obvious, however, that there are adverse effects on semen quality and hormone levels among men with extraordinarily high serum levels of p,p'-DDE. The results from previous studies of POP levels occurring in the general population in the Western world give some indication of harmful effects on semen characteristics, especially on sperm motility (Dallinga et al., 2002; Hauser et al., 2003
; Richthoff et al., 2003
). The present study offers some support for the previous findings, although the study design does not allow any insight into the mechanisms behind the impairment of sperm motility due to POP exposure. The lack of any effect on levels of reproductive hormones and on the other sperm characteristics indicates that the limited impairment of sperm motility might not be mediated through endocrine regulation of spermatogenesis. Alternatively one can speculate whether impairment of epididymal/accessory sex gland functions or a negative effect on sperm DNA integrity, which correlates with sperm motility (Giwercman et al., 2003
), might be involved.
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Acknowledgements |
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Submitted on February 9, 2004; accepted on May 18, 2004.