Aluminium, lead and cadmium concentrations in seminal plasma and ...

Human Reproduction vol.13 no.1 pp. 115–119, 1998

Aluminium, lead and cadmium concentrations in seminal plasma and spermatozoa, and semen quality in Finnish men

Outi Hovatta1,4, Eija-Riitta Vena¨la¨inen2, Leea Kuusima¨ki2, Jorma Heikkila¨3, Timo Hirvi2 and Ilkka Reima1 1Infertility

Clinic, The Family Federation of Finland, Kalevankatu 16, FIN-00100 Helsinki, 2National Veterinary Institute, PO Box 386, FIN-00101 Helsinki and 3Neste Oy, Neste Works, Porvoo Local Services, PO Box 310, FIN-06101 Porvoo, Finland 4To

whom correspondence should be addressed

Aluminium, cadmium and lead concentrations in the spermatozoa and seminal plasma of 27 employees of two industrial companies, a refinery and a polyolefin factory, and 45 consecutive sperm donor candidates at a sperm bank were studied using atomic absorption measurements. The relationship between metal concentration and parameters of semen analysis was studied. A high concentration of aluminium in spermatozoa was correlated with decreased sperm motility. The concentrations of cadmium and lead were low and did not show any correlation with parameters of semen analysis. Aluminium may be one of the environmental pollutants causing impaired semen quality. The mean sperm concentrations were similar in the factory employees (96H106/ml), in the sperm donor candidates of the comparison group (104H106/ml) and in 352 donor candidates at the sperm bank of the Family Federation of Finland (107H106/ml) between May 1993 and May 1995. Key words: aluminium/cadmium/lead/semen analysis/semen quality

Introduction A decline in sperm quality in several European countries during the past 50 years has been suggested (Carlsen et al., 1992; Irvine, 1994, 1996; Auger et al., 1995; Adamopoulos et al., 1996; Bujan et al., 1996; Irvine et al., 1996; Van Waeleghem et al., 1996; Pajarinen et al., 1997), although it has also been disputed (Bromwich et al., 1994; Farrow, 1994; Olsen et al., 1995). In Finland, high and unchanged sperm counts have been found (Suominen and Vierula, 1993; Vierula et al., 1996). On the other hand, in two necropsy series of middle-aged Finnish men, an increase in the incidence of disorders of spermatogenesis between the years 1981 and 1991 has been found (Pajarinen et al., 1997). Decreased fertility in Britain compared with that in Finland, based on differences in the time to conception, has been reported (Joffe, 1996). Local differences are also evident in the USA, with the highest mean sperm concentration being found in New York (131.03106/ml) and © European Society for Human Reproduction and Embryology

the lowest in California (72.73106/ml) (Fisch et al., 1996). A small increase in sperm concentration between the years 1972 and 1993 was seen in Seattle, Washington (Paulsen et al., 1996). Environmental factors have been suggested to have caused the decline observed in Europe, especially environmental oestrogens (Sharpe and Skakkebaek, 1993). Oestrogens have also been suggested to have caused the increase seen in the incidence of cryptorchidism and testicular cancer (Sharpe and Skakkebaek, 1993; Skakkebaek and Keiding, 1994). Heavy metals are other possible pollutants that may be harmful to sperm production. Occupational exposure to several metals is known to impair sperm quality (Schrag and Dixon, 1985). Lead and cadmium concentrations have been measured in human semen, seminal plasma, spermatozoa, blood and urine, and high concentrations have been found to be related to impaired sperm quality (Lancranjan et al., 1975; Plechaty et al., 1977; Braunstein et al., 1978; Pleban and Mei, 1983; Stanwell-Smith et al., 1983; Thomas and Borgan, 1983; Saaranen et al., 1987, 1989; Chia et al., 1992; Hu et al., 1992; Xu et al., 1993). In experimental studies, many metals have also been shown to be harmful with regard to testicular function and sperm production (Alabi et al., 1985). Aluminium may be one of the potential pollutants, because it reduces the weight of the testes and causes decreased epididymal sperm counts in the mouse (Llobet et al., 1979). Because there are apparent geographical variations in sperm quality, and because factors causing the decline are not clear, we studied semen quality and the concentrations of aluminium, cadmium and lead in spermatozoa and seminal plasma in a group of employees of a refinery and a polyolefin factory and compared the results with data obtained from samples from sperm bank donor candidates over the same time period. Materials and methods Study populations The concentrations of aluminium, cadmium and lead were measured in the spermatozoa and seminal plasma of two groups of men. Group I consisted of 27 volunteers who were employees of a refinery and a polyolefin factory (Neste/Borealis) located 40 km east of Helsinki, Finland. They had been recruited through a newsletter delivered to the personnel of the companies. Group II consisted of 45 consecutive sperm donor candidates presenting at the sperm bank of the Family Federation of Finland during the same period of time in 1994. All these men completed a questionnaire concerning their history of disorders that may affect semen quality, possible infertility, consumption of alcohol and smoking habits. They signed an informed consent document for this study, which was approved by the ethical committee of the Family Federation of Finland. Semen samples collected for the measurement of metal concentrations were also used for analyses

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Table I. Concentrations (mean 6 SD) of aluminium, cadmium and lead, and results of semen analyses (mean 6 SD) among the refinery and polyolefin factory employees (group I) and sperm bank donor candidates (group II) Parameter Cadmium (mg/kg) Spermatozoa Seminal plasma Lead (mg/kg) Spermatozoa Seminal plasma Aluminium (mg/kg) Spermatozoa Seminal plasma Concentration (3106/ml) Percentage motility (WHO categories a 1 b) Normal morphology (% normal spermatozoa)

Groups I 1 II

Group I

Group II

t-test value

df

P value

0.04 6 0.005 (71) 0.002 6 0.003 (71)

0.003 6 0.002 (27) 0.001 6 0.001 (27)

0.005 6 0.006 (44) 0.003 6 0.004 (44)

2.10 3.70

50 45

, 0.05 , 0.001

0.07 6 0.10 (72) 0.03 6 0.02 (72)

0.03 6 0.01 (27) 0.02 6 0.01 (27)

0.10 6 0.13 (45) 0.03 6 0.03 (45)

3.32 3.58

50 60

, 0.01 , 0.001

1.93 6 3.37 (72) 0.74 6 1.00 (72)

0.93 6 0.69 (27) 0.54 6 0.61 (27)

2.52 6 4.14 (45) 0.87 6 1.17 (45)

2.52 1.55

48 69

, 0.05 NS

109 6 56 (72) 62 6 14 (72)

96 6 54 (27) 61 6 18 (27)

117 6 56 (45) 63 6 12 (45)

1.51 0.58

57 39

NS NS

38 6 16 (71)

34 6 17 (26)

40 6 15 (45)

1.42

46

NS

Values in parentheses are numbers of participants. NS 5 not significant; WHO 5 World Health Organization.

Table II. Pearson’s correlation coefficients between the different parameters in the whole study population (groups I and II)

1. 2. 3. 4. 5. 6. 7. 8. 9.

Cd, spermatozoa Cd, seminal plasma Pb, spermatozoa Pb, seminal plasma Al, spermatozoa Al, seminal plasma Concentrations Motility Normal morphology

2

3

4

5

6

–0.04 (68)

0.26 (69)a –0.03 (69)

–0.11 (69) 0.60 (69)c 0.02 (70)

–0.05 0.30 0.03 0.41

(69) (69)a (70) (70)c

–0.08 0.03 –0.02 0.24 0.47

(69) (69) (70) (70)a (70)c

7

8

0.10 (69) –0.002 (69) 0.09 (70) –0.10 (70) 0.01 (70) 0.01 (70)

0.07 –0.04 0.07 –0.07 –0.28 –0.14 0.21

9 (69) (69) (70) (70) (70)a (70) (70)

0.00 (68) 0.11 (68) 0.05 (69) 0.11 (69) –0.18 (69) –0.04 (65) 0.36 (69)a 0.44 (69)b

Values in parentheses are df. Al 5 aluminium; Cd 5 cadmium; Pb 5 lead. a0.01 , P , 0.05; b0.001 , P , 0.01; cP , 0.0001.

of semen parameters. The mean age of the factory employees was 34 years (range 27–46) and that of the sperm donor candidates was 28 years (range 20–45). Three of the factory employees and two of the sperm donor candidates had experienced infertility (a minimum of 12 months of unprotected intercourse without achieving pregnancy). In three couples the cause of infertility was endometriosis of the female partners, and in two couples it had not been examined. Smoking habits and alcohol consumption were similar in both groups. The results of the semen analyses of 352 donor candidates (group III) at the sperm bank from May 1993 to May 1995 were also studied to check that the smaller population of donor candidates was representative with regard to the semen analyses. The mean age of these men was 27 years (range 18–44). Semen analyses Semen analyses were carried out according to World Health Organization (WHO, 1992) guidelines. The criteria for normozoospermia was a concentration of ù203106/ml, with grade A motility in 25% or grade A 1 B motility in 50% of spermatozoa, and normal morphology (in stained preparations) in at least 30% of the spermatozoa. Analyses of the concentrations and motility were carried out using a Makler chamber at room temperature. The samples were obtained by masturbation after 2–7 days of sexual abstinence.

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Metal analyses For metal analyses the spermatozoa were separated from the seminal plasma by centrifuging the semen (600 g, 10 min). The samples were frozen and stored at –20°C until analysed using atomic absorption spectrometry. The samples of spermatozoa and seminal plasma were dry-ashed and acid-leached as described by Niemi et al. (1991). Determinations of the concentrations of aluminium, cadmium and lead were performed by graphite furnace atomic absorption spectrometry (Perkin-Elmer 5100 PC AAS, Zeeman furnace module, Furnace cooling System and Autosampler AS-70, Norwalk, USA) using pyrolytic transverseheated graphite atomizer tubes. Aluminium was measured at 309.3 nm, cadmium at 228.8 nm and lead at 283.3 nm with hollow cathode lamps. For the determination of lead, a matrix modifier was used [0.2% Mg(NO3)2 1 4% NH4H2PO4/0.1 M HNO3]. The determinations were performed using direct comparison with standard solutions in 0.1 M nitric acid. The methods used (except that for aluminium) have been accredited by the Centre of Metrology and Accreditation of Finland. The laboratory fulfils the requirements of standards SFS-EN 45001 and ISO/IEC Guide 25. Standard reference materials (Community Bureau of Reference: bovine muscle) were analysed in duplicate, together with each sample

Semen quality in Finnish men

Table III. Linear analysis of regression between aluminium (Al), cadmium (Cd) and lead (Pb) concentrations in the seminal plasma, and semen analysis parameters among the sample quartile of men having the highest metal concentrations (minimum for Al 0.88 mg/kg, Cd 0.02 mg/kg, Pb 0.03 mg/kg) Dependent parameters

Independent parameters

b

r2 (%)

F

df

P

Concentration of spermatozoa

Cd, seminal plasma Pb, seminal plasma Al, seminal plasma Cd, Pb, Al, seminal plasmaa Cd, seminal plasma Pb, seminal plasma Al, seminal plasma Cd, Pb, Al, seminal plasmaa Cd, seminal plasma Pb, seminal plasma Al, seminal plasma Cd, Pb, Al, seminal plasmaa

–0.01 –0.08 0.10

0.0 0.6 1.0 2.1

0.003 0.178 0.318 0.203

1.31 1.31 1.31 3.29

0.958 0.676 0.577 0.894

0.02 0.04 –0.15

0.1 0.2 2.4 2.5

0.017 0.051 0.756 0.249

1.31 1.31 1.31 3.29

0.897 0.823 0.391 0.861

–0.04 0.07 –0.19

0.2 0.5 3.8 6.8

0.046 0.160 1.218 0.704

1.31 1.31 1.31 3.29

0.831 0.692 0.278 0.557

Motility of spermatozoa

Normal morphology of spermatozoa

aAll

samples with higher heavy metal concentrations (Cd, Pb, Al) pooled together.

Table IV. Linear analysis of regression between aluminium (Al), cadmium (Cd) and lead (Pb) concentrations in the spermatozoa, and semen analysis parameters among the sample quartile of men having the highest metal concentrations (minimum for Al 1.89 ng/kg, Cd 0.05 mg/kg, Pb 0.07 mg/kg) Dependent parameters

Independent parameters

b

r2 (%)

F

df

P

Concentration of spermatozoa

Cd, spermatozoa Pb, spermatozoa Al, spermatozoa Cd, Pb, Al, spermatozoaa

0.06 0.03 –0.08

0.4 0.1 0.7 0.9

0.108 0.036 0.210 0.086

1.31 1.31 1.31 3.29

0.744 0.851 0.650 0.967

Motility of spermatozoa

Cd, spermatozoa Pb, spermatozoa Al, spermatozoa Cd, Pb, Al, spermatozoaa

0.15 0.19 –0.51

2.4 3.4 26.5 27.5

0.752 1.107 11.175 3.669

1.31 1.31 1.31 3.29

0.392 0.301 0.002c 0.024b

Normal morphology of spermatozoa

Cd, spermatozoa Pb, spermatozoa Al, spermatozoa Cd, Pb, Al, spermatozoaa

0.01 0.11 –0.36

0.0 1.2 12.8 13.6

0.004 0.383 4.568 1.518

1.31 1.31 1.31 3.29

0.952 0.540 0.044b 0.231

aAll samples with higher heavy metal concentrations (Cd, Pb and Al) pooled together. Significance of regression b0.01 , P , 0.05 and c0.001 , P , 0.01.

series. Blank samples were also analysed with each sample to check for contamination. Calibrations of all the metal analyses were checked continuously using standard samples at different concentrations. Statistical analyses were carried out using a linear regression analysis, Pearson’s correlation coefficient with log transformation, analysis of variance and Student’s t-test, as appropriate.

Results All the metal concentrations were significantly lower in the 27 refinery and polyolefin factory employees (group I) than in the 45 sperm donor candidates (group II) in both sperm and seminal plasma samples (Table I), except for aluminium in seminal plasma. The concentrations, motilities and morphologies of the spermatozoa were not different between these two groups. Correlations between the parameters of semen analysis and the metal concentrations were studied after combining the data from groups I and II because there were no differences between the semen analysis results of these

groups. Pearson’s correlation coefficients for these data are given in Table II. There was no correlation between the concentrations of lead in the spermatozoa and that in seminal plasma; a similar situation held for cadmium. The concentrations of cadmium and lead in seminal plasma were correlated with each other. The aluminium concentration in the spermatozoa was significantly correlated with that in the seminal plasma. The concentrations of cadmium and lead were not correlated with any parameter of the semen analysis. There was a significant correlation between the aluminium concentration in the spermatozoa and sperm motility (P , 0.01). The variation between samples of aluminium concentrations in the spermatozoa and seminal plasma was high; the highest concentration was 215 times the lowest concentration in the spermatozoa, while in the seminal plasma the relative difference was 93 times. There was less variation in the concentrations of cadmium and lead. A linear analysis of regression (Tables III and IV) showed that relatively high concentrations of 117

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aluminium in the spermatozoa were correlated with the motility (0.001 , P , 0.01) and morphology (0.01 , P , 0.05) of the spermatozoa, but there was no correlation between the concentration of aluminium in the seminal plasma and semen analysis parameters. The semen analyses of the three men with clearly the highest aluminium concentrations in their spermatozoa (from 8.7 to 21.5 mg/kg) showed asthenozoospermia in all three cases (A1B motilities from 16 to 46%). Semen analysis parameters and metal concentrations were no different between smokers and non-smokers (Student’s t-test). The mean sperm concentration among the 352 sperm donor candidates from May 1993 to May 1995 (1073106/ml) did not differ from those of the other study groups. Discussion The concentrations of aluminium, cadmium and lead in spermatozoa and seminal plasma differed significantly between the refinery and polyolefin factory employees and the sperm bank donor candidates, being higher among the donor candidates. This reflects the good quality of occupational protection in these factories, but it also reflects other environmental factors. The factories lie in a rural area, and most of the employees live in the countryside. The sperm bank donor candidates came from the urban Helsinki area in Finland. It appears to be important to measure metal concentrations in the spermatozoa, not just those in seminal plasma, serum or other tissues. It has been reported that there is no correlation between semen quality and cadmium and lead concentrations measured in sources of tissue and fluid other than semen (Chia et al., 1992; Hu et al., 1992; Xu et al., 1993). LewisJones et al. (1996) studied the effects of zinc and fructose concentrations in seminal plasma on sperm activity. They found that seminal plasma components are poor indicators of andrological parameters. The cadmium and lead concentrations in seminal plasma measured in our study were a little higher or similar to those measured previously (Pleban and Mei, 1983; Saaranen et al., 1987). The concentrations of cadmium and lead measured in spermatozoa in our study were clearly lower than those measured previously in a healthy non-exposed population (Plechaty et al., 1977). The low concentrations of these metals in the spermatozoa, reflecting low exposure, may be one explanation for the relatively good sperm quality of Finnish men, as also observed in our study. The high concentrations of aluminium in the spermatozoa and seminal plasma of some men, and their correlation with decreased sperm motility, was a new finding. Aluminium may therefore be one of the environmental factors affecting sperm quality. It is important to determine the concentrations of aluminium in spermatozoa in areas with poorer sperm quality. The concentration of xeno-oestrogens in Finland is also a subject that remains to be studied. Acknowledgements We thank Helena Korpelainen for statistical assistance and Nicholas Bolton for revising the English. This study was supported by a grant from the Yrjo¨ Jahnsson Foundation, Helsinki, Finland.

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