1 Department of Health Risk Analysis and Toxicology, Faculty of Health Sciences, Maastricht University, P.O.Box 616, 6200 MD, Maastricht, 2 Department of Obstetrics and Gynecology and 3 Department of Clinical Genetics, University Hospital, Maastricht, The Netherlands
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Abstract |
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Key words: endocrine disruptor/hydroxy-PCB/organochlorine/semen quality/subfertility
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Introduction |
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The cause of involuntary childlessness in couples can be found in either the male or the female counterpart, or in both. From couples visiting the Maastricht University Hospital, males were selected to be included in either a group of subfertile men, based on extremely poor semen quality [male factor subfertility (MFS) group] or in a group of men with normal (good) semen quality [female factor subfertility (FFS) group]. The classification was based on semen analyses performed on three previous occasions, and progressively motile sperm concentration (PMSC) was used as the parameter to make the classification.
In order to determine whether presently occurring organochlorine levels play a role in the fertility defects of the MFS subgroup, various compounds, including several PCB and PCB metabolites, were analysed in blood samples of all volunteers and related to semen parameters. Moreover, the relationship between seminal plasma and blood levels of the unmetabolized organochlorine compounds was established by comparison of the samples of a small number of volunteers. Furthermore, to assess the potential role of genetic factors affecting PCB metabolism, polymorphic frequencies of the glutathione S-transferase (GST) GSTM1 and GSTT1 genes, encoding for these PCB-detoxifying enzymes, were determined in both the FFS and MFS groups.
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Materials and methods |
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Questionnaires were used to obtain additional information on smoking behaviour, possible occupational exposure, etc. and the ages of the volunteers at the time of sampling were recorded. The study was approved by the Medical Ethical Commission of the University and the University Hospital and informed consent was obtained from all volunteers.
Semen quality
The semen samples, produced on the same occasion that the blood samples were taken, were investigated with regard to volume, sperm concentration (sperm count), overall and progressive motility and morphology. Conventional methods were used to evaluate the volume, sperm concentration and motility. A Makler counting chamber was used for concentration and motility evaluation. Two groups of motile sperm were recognized (World Health Organization, 1999): sperm with rapid and linear progressive movements (group A, equal to WHO grade a), and sperm with all other types of movements: either slow, sluggish or hampered by a clearly visible morphological defect, non-progressive motility or immotility (group B, combining WHO grades b, c and d). The total number of sperm in group A was defined as the PMSC. The morphology was defined applying the morphology evaluation using strict criteria (Enginsu et al., 1992
) and expressed as a percentage of the total number of sperm counted.
Isolation of organochlorine compounds from blood
The blood samples of all volunteers were investigated with regard to the organochlorine compounds hexachlorobenzene (HCB), p,p-DDE:1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene p,p-DDT:1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethylene, 2,3',4,4',5-pentachlorobiphenyl (PCB-118), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153), 2,2',3,4,4',5-hexachlorobiphenyl (PCB-138) and 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB-180). These four PCB were selected, based on their relatively high abundance in humans (Safe, 1994) and the potential endocrine-disrupting activity of their metabolites, due to the ortho substitution (McKinney and Waller, 1994
). To this end, 3 ml samples were fortified with 50µl of an internal standard solution of PCB-143 (100 ng/ml) and hydrolysed for 2 h at 60°C with 3 ml ethanolic sodium hydroxide (1g NaOH in 25ml water and 25ml ethanol). The resulting solutions were extracted twice with 2 ml n-hexane, the combined hexane layers were dried over anhydrous sodium sulphate and further cleaned using deactivated silicon dioxide (SiO2) columns. The solvent was removed by evaporation and the residue redissolved in 50 µl iso-octane.
Metabolites were isolated separately, as follows. To 3 ml blood sample, 3 ml water and 3 ml methanol were added and the resulting solution was acidified with 0.5 mol/l sulphuric acid until pH <5. This solution was extracted with 3x3 ml n-hexane/methyl-t-butylether (1:1); the combined organic layers were evaporated and the residue was redissolved in 2 ml n-hexane. From this solution, the polar metabolites were extracted with 1 ml 1.0 mol/l KOH solution (H2O/CH3OH 1:1), and, after acidification with sulphuric acid until pH <2 and extraction with 2x1 ml n-hexane/methyl-t-butylether (1:1), the solvent was evaporated and the residue dissolved in 300 µl n-hexane. To this solution 200 µl of a diazomethane reagent was added and the resulting products were dissolved in n-hexane and cleaned over deactivated SiO2 columns. After removal of the solvent, the residue was redissolved in 50 µl iso-octane, containing PCB-143 as an internal standard (Bergman et al., 1994).
Isolation of organochlorine compounds from seminal plasma
Semen samples were centrifuged to remove the sperm and 1 ml of the resulting plasma was fortified with 50 µl of the internal standard and extracted as described above for the blood samples (unmetabolized organochlorine compounds). The quantities of the samples in combination with the low concentration of metabolites did not allow for metabolite isolation and determination in the seminal plasma.
Determination of organochlorine compounds
Aliquots (1 µl) of the extracts were introduced into a gas chromatograph (HRGC Mega 2 Series, 8560 gas chromatograph; Interscience, Breda, The Netherlands), equipped with a cold on-column injection port. The samples were analysed using a capillary, 25 m length, 0.25 mm inside diameter, 0.25 µm film thickness fused silica CP Sil-8 CB column, at a programmed temperature of 80270°C. Helium was used as the carrier gas and nitrogen as a make-up gas. An electron capture detector at 300°C was used to detect the organochlorine compounds in a sensitive and specific way Quantification was performed using the internal standard and based on peak area. Of the different metabolites, which were measured separately from the unmetabolized organochlorine compounds, one was identified by using the standard compound (metabolite c: 4-hydroxy-2,3,3',4',5-pentachloro biphenyl). The other seven metabolites were measured and compared using their relative retention times; these metabolites are bound to be hydroxy-PCB as well, because of the isolation and detection methods applied, analogous to other published methods (Bergman et al., 1994). The metabolites are labelled ah in the example chromatogram presented in Figure 1
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Statistical methods
Comparison of the groups defined as FFS and MFS was performed using the non-parametric MannWhitney U-test. This test was also used to examine potential effects of polymorphisms and smoking on sperm parameters and organochlorine contents. Quantitative relationships with regard to various organochlorine levels, sperm parameters and age were investigated using simple linear regression.
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Results |
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Correlation of age with other parameters
No relationship between the ages of the volunteers and any of the sperm parameters could be established, but the levels of organochlorines in blood appeared to be strongly positively related to the ages of the men. These relationships were found for most of the individual components, for all metabolites and for the combined PCB levels, combined metabolite levels and total organochlorine levels. As an example, the relationship between age and metabolite levels is shown in Figure 2.
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Organochlorine concentrations in blood and in seminal plasma
A significant relationship was found between the combined PCB levels in blood and the corresponding levels in seminal plasma (simple regression, n = 10, R2 = 0.38, P = 0.05), in which the blood levels appeared to be 20-fold higher than the seminal levels. When total organochlorine levels were compared, the relationship was similar, but not significant.
Comparison of the MFS and FFS groups
Comparison of the subgroups with regard to the levels of both metabolized and unmetabolized organochlorine compounds in blood using the MannWhitney U-test, revealed no significant differences in levels of individual components nor in combined PCB, metabolite or total organochlorine compounds. The PCB levels in seminal plasma were found to be higher in the samples of the FFS group than in the samples of the MFS-group, but these differences were not significant (MannWhitney U-test, n = 10, P = 0.06). The absence of significant differences in organochlorine levels for the two subgroups justifies the idea that the cause of the very poor sperm quality in the MFS subgroup has not resulted from exposure to organochlorine compounds, but from other, so far unknown, causes.
Sperm count and PCB metabolite concentrations in the FFS group
Within the FFS group, significant negative correlations between combined PCB metabolites concentration in blood and sperm count (n = 31, R2 = 0.14, P = 0.04) and between PCB metabolites and PMSC (n = 31, R2 = 0.17, P = 0.02) could be established (Figure 3). For the MFS group, corresponding (also negative) correlations were not significant (n = 34, R2 = 0.09, P = 0.08 and n = 33, R2 = 0.07, P = 0.13 respectively).
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Discussion |
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Perhaps not surprisingalthough previously unreportedis a positive correlation between organochlorine levels in blood and age. It appears that bioaccumulation in men has not reached an equilibrium at adulthood, but the levels of HCB, individual and combined PCB and metabolites continue to rise with age, whereas for p,p-DDE and p,p-DDT the relationships are less pronounced. Alternatively, the higher concentrations at an older age might be due to a greater exposure of the older persons at times that elevated levels of these compounds occurred. However, although the PCB concentrations in food are declining, this decline is not sharp; exposure to PCB from food is still the most important exposure route for man. From our data it is not possible to distinguish between the two explanations, but the effects would be the same for either of these two possibilities. The higher levels of organochlorine compounds in the FFS group, when compared with the MFS group, can be accounted for with this ageorganochlorine relationship: the mean age of the fertile men is ~2 years higher than that of the subfertile men. After correction of PCB levels for age, no difference between the two subgroups remains. The relationships shown in Figure 3 are not influenced by the agePCB concentration relationship; correction of the metabolite concentrations for age does not lead to different correlations.
The discussion concerning the potential relationship between environmental estrogens and decreasing semen quality is mainly focused on exposure in utero (Sharpe and Skakkebaek, 1993; Jensen et al., 1995
). In our hypothesis, an impact of environmental endocrine disruptors on spermatogenesis might also occur in adult males. However, in our study group, we found that semen quality parameters, such as sperm count, motility and morphology, appeared to be strongly positively correlated with the seminal plasma levels of organochlorine compounds. Enhanced organochlorine levels in semen without pathological findings when compared to sperm samples with various pathological findings were reported, but no relationship of these levels with sperm count, motility and morphology was found (Ensslen et al., 1990
). In another study, an inverse correlation between seminal PCB levels and motility was found (Bush et al., 1986
). Occupational exposure to 1,2-dibromo-3-chloropropane (a pesticide) was shown to be associated with a decreased sperm count (Whorton et al., 1977
; Glass et al., 1979
), chlordecone reversibly inhibited spermatogenesis (Guzelian, 1982
). Exposure to PCB was related to a non-significantly decreased sperm count (Emmett et al., 1988
). Recently, adverse effects of exposure to aromatic hydrocarbons on sperm count, motility and morphology were demonstrated (De Celis et al., 2000
).
In the relatively homogeneous FFS subgroup, we found a strong negative correlation between organochlorine metabolite levels in blood and sperm count. This is the first example of a demonstrable correlation between an environmentally widespread pollutant at a background level and parameters of human semen quality. The fact that, despite strong correlations between the blood levels of organochlorine compounds and metabolites of these compounds, such correlations were not found between the unmetabolized component concentrations and sperm parameters strengthens the idea that the PCB metabolites are the biologically active and thus interesting compounds. It seems that the combination of intake (predominantly from food) and individual metabolism governs the effect of organochlorine compounds on the reproductive performance of men. We could not demonstrate an influence of the GSTT1 and GSTM1 polymorphisms, but polymorphisms for other PCB metabolizing enzymes, such as CYP1A1 and CYP1A2, may be involved. To elucidate the role of their and other rather low frequency polymorphisms for PCB bioactivation and/or biodetoxification, much larger population studies are required. From the data presented, it appears that sperm count and progressive motility are the sperm parameters most affected by the organochlorine metabolite levels, since effects on overall motility and morphology could not be detected in the present dataset (FFS group). Whether such effects on sperm count and PMSC of organochlorine metabolites may affect the occurrence of pregnancies by natural conception remains to be elucidated.
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Acknowledgements |
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Notes |
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References |
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Submitted on April 17, 2001; resubmitted on December 14, 2001; accepted on April 19, 2002.