CSIRO PUBLISHING
Reproduction, Fertility and Development, 2016, 28, 1376–1381 http://dx.doi.org/10.1071/RD14063
Human sperm aneuploidy after exposure to polycyclic aromatic hydrocarbons Michał Radwan A,E, Joanna Jurewicz B,E, Wojciech Sobala B, Sławomir Brzez´nicki C, Paweł Radwan A, Lucjusz Jakubowski D, Wanda Hawuła D, Anna Ulan´ska D and Wojciech Hanke B A
Department of Gynaecology and Reproduction, Gameta Hospital 34/36 Rudzka Street, 95-030, Rzgo´w, Poland. B Department of Environmental Epidemiology, Nofer Institute of Occupational Medicine, 8 Teresy Street, 91-348 Lodz, Poland. C Department of Chemical Safety, Nofer Institute of Occupational Medicine, 8 Teresy Street, 91-348 Lodz, Poland. D Department of Medical Genetics, Polish Mother’s Memorial Hospital – Research Institute, 281/289 Rzgowska Street, 93-338 Lodz, Poland. E Corresponding authors. Emails: [email protected]; [email protected]
Abstract. The purpose of this cross-sectional study was to investigate whether environmental exposure to polycyclic aromatic hydrocarbons (PAHs) was associated with sperm aneuploidy. A sample of 181 men who attended an infertility clinic for diagnostic purposes and who had a normal semen concentration of 20–300 106 spermatozoa mL1 or slight oligozoospermia (semen concentration of 15–20 106 spermatozoa mL1; WHO 1999) provided urine and semen samples. Analysis of the level of PAH biomarker 1-hydroxypyrene (1-OHP) in urine was performed using highperformance liquid chromatography. Sperm aneuploidy was assessed using multicolour florescence in situ hybridisation (FISH) using DNA probes specific for chromosomes X, Y, 18, 13 and 21. Positive associations were observed between the level of 1-OHP in urine and total sex-chromosome disomy (P ¼ 0.03) and chromosome-18 disomy (P ¼ 0.03). These results suggest that environmental exposure to PAHs may be associated with sperm aneuploidy. This is the first epidemiological study to investigate the relationship between environmental exposure to PAHs and sperm aneuploidy. Therefore, these findings require further replication in other populations using different biomarkers of PAH exposure. Additional keywords: environmental exposure to PAH, FISH, level of 1-OHP, male fertility, PAH exposure, sperm disomy. Received 17 February 2014, accepted 29 January 2015, published online 10 March 2015 Introduction Studies in infertile men have demonstrated that 2–14% have constitutional chromosomal abnormalities (Meschede et al. 1997). The incidence of chromosomal aberrations is dependent on the definition of ‘infertility’ and is observed in ,2% in males with combined factors of infertility (Meschede et al. 1997) and 14% in azoospermic men (Johnson 1998). These frequencies are all considerably higher than the 0.7% population incidence seen in newborns (Thompson et al. 1986). For humans, aneuploidy is identified in at least 5% of all clinically recognised pregnancies (Lamb and Hassold 2004) and is associated with approximately one-third of all pregnancy losses. The majority of aneuploid liveborns are attributed to parental gametes carrying abnormal numbers of chromosomes (Hassold and Hunt 2001). It has been estimated that 2% or more of all spermatozoa have missing or additional chromosomes (Hassold and Hunt 2001). Journal compilation Ó CSIRO 2016
It should be noted that in animal species (pigs and cattle) sperm aneuploidy rates are similar to those in humans (Rubesˇ et al. 1999). It has been suggested that one reason might be human or animal exposure to various environmental agents. Several factors have been implicated in increasing chromosome aneuploidy in spermatozoa, including smoking, alcohol, caffeine, drugs, pesticides, air pollution and chemotherapy (Robbins et al. 1997, 1999, 2005; Rubes et al. 1998; Shi et al. 2001; Tempest et al. 2004; Ha¨rko¨nen 2005; Wyrobek et al. 2005; Martin 2006). Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals that are formed during the incomplete burning of organic substances. There are more than 100 different kinds of PAHs (Agency for Toxic Substances and Disease Registry (ATSDR) et al. 1995). Because PAHs are ubiquitous in daily life, the potential consequences of human exposure to PAHs www.publish.csiro.au/journals/rfd
PAH exposure and sperm aneuploidy
have raised concerns in the general population (Santodonato 1997). In particular, PAHs may have endocrine-disrupting chemical properties that may affect the reproductive and developmental processes in humans (Piskorska-Pliszczynska et al. 1986; Tran et al. 1996; Clemons et al. 1998); however, there are very few epidemiological studies on the effect of exposure to PAHs on male reproduction. Most of the studies are focussed on semen quality (Selevan et al. 2000; Hsu et al. 2006; Xia et al. 2009; Jeng et al. 2013) and sperm DNA damage (Selevan et al. 2000; Hsu et al. 2006; Han et al. 2011; Jeng et al. 2013). Those studies suggest a harmful effect of PAH exposure on the male reproductive system. According to our knowledge, no previous studies have assessed the relationship between environmental exposure to PAHs and sperm aneuploidy. The present study was designed to investigate whether environmental exposure to PAHs, assessed by 1-hydroxypyrene (1-OHP) level in urine, is associated with an altered frequency of sperm aneuploidy in adult men. Materials and methods Study population Between January 2008 and April 2011 men aged under 45 years of age with normal semen concentration of 20–300 106 spermatozoa mL1 or with slight oligozoospermia (semen concentration of 15– 20 106 spermatozoa mL1; WHO 1999) were recruited from an infertility clinic in Lodz, Poland. The protocol and informed consent form were approved by the Nofer Institute of Occupational Medicine Bioethical Committee Board. After obtaining a written informed consent from each participant, information was collected during an in-person interview on their occupational history, socio-demographic characteristics, reproductive, medical history and lifestyle factors. Additionally, smoking status was verified by measuring cotinine levels in saliva using high-performance liquid chromatography coupled with tandem mass spectrometry–positive electrospray ionisation (LC-ESIþMS/MS) and the isotope dilution method. Full details of the study have been described elsewhere (Jurewicz et al. 2014). Among 334 men who agreed to participate, a subset of 181 men (52.6%) were eligible for this sub-study and had available semen samples for florescence in situ hybridisation (FISH) analysis and enough urine sample for the measure of 1-hydroxypyrene in urine. Sperm aneuploidy analysis Sperm aneuploidy was measured by multicolour FISH analysis using DNA probes specific for chromosomes 13, 18, 21, X and Y (AneuVysion DNA Probe Kit; VYSIS, Abbott Molecular Inc., IL, USA) and the slides were viewed by fluorescence microscopy on a Nikon Eclypse 80i (Nikon Instruments Europe, Amsterdam, The Netherlands) equipped with LUCIA Cytogenetics-Karyo/ FISH software (Ushijima et al. 2000; Laboratory Imaging, Praha, Czech Republic) as previously described (Jurewicz et al. 2013a). Briefly, frequency of aneuploidy was analysed in every sample in two hybridisation areas. In the first area the number of fluorescent signals from chromosomes X, Y and 18 were counted and in the second, signals from chromosome 13 and 21 were counted. One thousand cells were examined in
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each area. Six types of sperm disomy were examined: XY spermatozoa (sperm FISH genotype: X-Y-18), disomy X (XX-18), disomy Y (Y-Y-18), disomy 18 (X-18-18 or Y-18-18), disomy 21 (X-21-21 or Y-21-21) and disomy 13 (X-13-13 or Y-13-13). The overall hybridisation efficiency in our study was from 98.91 to 99.36%, which is higher than the hybridisation efficiency (97%) reported in other studies (Johannisson et al. 2002; Tiido et al. 2005; Martin 2006; McAuliffe et al. 2012). Measurement of 1-hydroxypyrene in urine The 1-OHP was analysed using high-performance liquid chromatography (HPLC). The analytical procedure used in this study was based on the method described by Jongeneelen et al. (1987). The sensitivity of the method is estimated to be 0.2 mg L1. The full details of the method used for measure of 1-hydroxypyrene in urine are presented elsewhere (Jurewicz et al. 2013). Statistical analysis Descriptive statistics for demographic characteristics, sperm aneuploidy and level of 1-OHP in urine were calculated. The relationship between the level of 1-OHP and each type of sperm aneuploidy was evaluated using negative binomial regression modelling for disomy X and disomy 13 and generalised linear mixed model with a Poisson distribution for disomy Y, XY, 18, 21, total sex-chromosome disomy and total chromosome disomy. Models were selected based on Akaike Information Criteria (AIC). We compared the distributions of variables from the questionnaire among men with 1-OHP levels by Chi-square test in order to identify potential confounders. Data were analysed using multivariate analysis. Covariates in the final models were: sexual abstinence (days), age (years), smoking (yes/no), season of the year during semen collection (May–September, October–April), past diseases (yes/no). 1-OHP was included as a continuous variable in the models. Sperm aneuploidies were log-transformed to obtain a normal distribution. We used R 2.15.1 statistical program for all data analysis (R Core Team 2013; http://www.R-project.org/). Results Demographic and other characteristics of the 181 study participants are presented in Table 1. Overall, participants had mean age 32.1 4.6 years. The mean duration of abstinence was 5 2.3 days. Most of the study participants had secondary (39.8%) and higher education (37.0%) and were non-smokers (72%). As shown in Table 2 the sperm concentration was 50.71 49.33 106 mL1, the percentage of motile sperm cells and the percentage of spermatozoa with normal morphology were 56.00 51.30 and 46.00 78.60 respectively. The unadjusted mean frequency and s.e.m. of sperm disomy among men in our study was 1.30 0.99 for XY spermatozoa, 0.28 0.26 for disomy X, 0.61 0.43 for disomy Y, 1.13 0.23 for total sexchromosome disomy, 0.54 0.38 for disomy 13, 0.85 0.67 for disomy 18, 0.98 0.95 for disomy 21 and 1.72 0.92 for aggregate aneuploidy.
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Environment is 0.5 mg L1 (Schulz et al. 2012), which is a little lower than the 95th percentile of the urinary concentration of 1-OHP in our study 0.68 mg L1. The unadjusted geometric mean of 1-OHP in urine was 0.35 0.35 mg L1 (range 0.04–1.95 mg L1) and adjusted for creatinine 0.29 0.27 mg g1 creatinine (range 0.04–2.03 mg g1 creatinine; Table 2). Table 3 summarises the relationships between the level of 1-OHP in urine and sperm aneuploidy, crude and adjusted for potential confounders. Positive associations were observed between the level of 1-OHP in urine and total sex-chromosome disomy (P ¼ 0.04; Table 3). When the model was adjusted for sexual abstinence, age, smoking, season of the year during semen collection and past diseases the total sex-chromosome disomy (P ¼ 0.03) remained significant. Additionally, an increase in the frequency of disomy 18 (P ¼ 0.03) was related to the level of 1-OHP in urine.
The reference values (95th percentile of the values measured for the substance concentration) of 1-OHP levels in urine for non-smoking general population aged between 3 and 69 years established by the Commission of the German Federal Table 1. Characteristics of the study population s.e.m., standard error of the mean; n, number of participants Characteristics
n (%)
Education Vocational 42 (23.2) Secondary 72 (39.8) Higher 67 (37.0) Smoking determined by cotinine level No 131 (72.4) Yes 47 (26.0) Missing data 3 (1.6) Past diseases, which may have impact on semen quality No 161 (89.0) Yes 20 (11.0) Duration of couple’s infertility (years) 1–2 70 (38.7) 2–3 57 (31.5) 3–5 28 (15.4) .5 26 (14.4) Abstinence (days) ,3 19 (10.5) 3–7 134 (74.0) .7 28 (15.5) mean s.e.m. 5.0 2.3 median (min–max) 5.0 (0.0–20.0) Age (years) mean s.e.m. 32.1 4.6 median (min–max) 31.8 (22.7–44.8) Season (3 months before semen collection) May–September 68 (37.6) October–April 113 (62.4)
Discussion This is the first epidemiological study to investigate the relationship between environmental exposure to PAHs and sperm aneuploidy. Our results suggest a significant increase in the frequency of total sex-chromosome disomy and chromosome18 disomy after adjusting for potential confounders. Because no prior studies have investigated 1-hydroxypyrene in urine and sperm aneuploidy, comparisons of these findings with other studies are limited. Six epidemiological studies have evaluated the harmful effects of air pollution and PAH exposure on semen quality: sperm concentration, sperm number per ejaculate (Xia et al. 2009), abnormal morphology (Selevan et al. 2000; Hsu et al. 2006), motility (Jeng et al. 2013), sperm DNA damage (Selevan et al. 2000; Hsu et al. 2006; Han et al. 2011; Jeng et al. 2013) and sperm aneuploidy (Robbins et al. 1999). The latter found an association between exposure to seasonal air pollution and increased YY disomy (Robbins et al. 1999).
Table 2. Percentage of sperm aneuploidy and level of 1-OHP in urine s.e.m., standard error of the mean; n ¼ 181
Sperm aneuploidy (%) X-Y-18 X-X-18 Y-Y-18 Sex-chromosome disomy 13-13 18-18 21-21 Total chromosome disomy Sperm parameters Sperm concentration (106 mL1) Total motility (%) Normal sperm morphology (%) Level of 1-OHP in urine 1-OHP mg L1 1-OHP mg g1 creatinine
Minimum
25%
50%
75%
95%
0 0 0 0.60 0 0 0 0
0.50 0.10 0.30 0.97 0.30 0.71 0.60 0.70
0.76 0.20 0.50 1.11 0.49 0.82 0.98 0.82
1.50 0.40 0.80 1.27 0.70 1.52 1.44 0.91
1.99 0.83 1.23 1.74 1.50 2.03 5.12 1.89
15.00 6.00 89.00
21.20 46.30 67.50
45.70 54.20 52.80
81.70 65.30 31.50
0.04 0.04
0.15 0.12
0.24 0.22
0.31 0.44
Maximum
Mean
s.e.m.
2.34 1.16 2.12 2.00 2.48 2.61 8.21 1.20
1.30 0.28 0.61 1.13 0.54 0.85 0.98 1.72
0.99 0.26 0.43 0.23 0.38 0.67 0.95 0.92
115 81.20 25.88
360 97.00 22.00
50.71 56.00 46.00
49.33 51.30 78.60
0.68 0.72
1.95 2.03
0.35 0.29
0.35 0.27
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Gaspari et al. (2003) observed that occupational exposure to PAH, but not smoking, was significantly associated with higher levels of PAH–DNA adducts, especially among infertile men (P ¼ 0.04). In a study performed in China, increased urinary 2-hydroxynaphthalene (2-OHNa) levels were associated with increased comet parameters, including the percentage of DNA in the tail length and tail distribution (Han et al. 2011). Higher rates of DNA denaturation in spermatozoa were observed among workers occupationally exposed to PAHs (Hsu et al. 2006). Selevan et al. (2000) reported an association between air pollution episodes of elevated PAH and abnormal chromatin in human spermatozoa, whereas Jeng et al. (2013) did not find an association between DNA fragmentation and 1-OHP exposure. The results of those studies are in line with our findings regarding the impact of air pollutants and PAH exposure on sperm sex-chromosome disomy. Additionally we found an increase risk of chromosome-18 disomy in relation to the level of 1-hydroxypyrene in urine. These results suggest that chromosome 18 and sex chromosomes are more susceptible than other chromosomes to nondisjunction during male meiosis induced by environmental exposure to PAHs (Robbins et al. 1997). The mean frequencies of sperm aneuploidy for sex chromosomes among men in our study were higher than reported in other studies ((XX, 0.03–0.37; YY, 0.04–0.21; XY, 0.06–0.42) for men from the general population (Egozcue et al. 1997; Templado et al. 2005) and similar to studies performed among andrology clinic patients (Tempest et al. 2010; McAuliffe et al. 2012). In the case of chromosomes 21, 13 and 18, the frequencies of disomy were similar to previously reported values (0.095–2.3%, 0.55–2.2% and 0.04–1.1%, respectively; Smith et al. 2004; Gianaroli et al. 2005; Young et al. 2008). The variation in aneuploidy frequencies between studies may be due, in part, to technical differences such as sperm decondensation, scoring criteria, FISH method, storage of samples and their criteria for inclusion of men in the studies (Tempest et al. 2004). Male susceptibility is a function of competing, interacting or additive effects related to age, nutrition, stress, work place and recreational exposures as well as a myriad of
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other health factors that can affect normal meiosis and increase the risk for aneuploid spermatozoa (Robbins et al. 2005). The design of our study does not, however, allow us to clarify the mechanisms behind the observed associations. One hypothesis could be that reactive metabolites of PAHs might reach the testes and epididymis and then react with sperm DNA to form adducts, causing DNA damage (Gaspari et al. 2003). Additionally, compounds resulting from the oxidation of PAHs have the ability to enter redox cycles, which increased the formation of reactive oxygen species (ROS) (Farmer et al. 2003) and thus caused sperm DNA damage (Barroso et al. 2000). There are limitations to the use of urine levels of 1-OHP as biomarkers of PAH exposure in humans and rodents. Some studies suggest that urinary 1-OHP level is an appropriate surrogate biomarker for total PAH exposure of human populations (Jongeneelen et al. 1987; Siwin´ska et al. 1998; Jacob and Seidel 2002). On the other hand, some authors claim that 1-OHP may not represent the numerous PAH metabolites (Al-Saleh et al. 2013). The men in this study were attending an infertility clinic for diagnostic purposes. They were different from the general population, but all men in our study had normal semen parameters or slight oligozoospermia based on WHO classification (WHO 1999). Currently, there is no evidence showing that they would differ in ways that would alter their response to PAHs (McAuliffe et al. 2012). Our study only used a single semen sample; this could be a limitation of this study. However, there are no data in the literature on possible temporal variation in chromosomal frequencies (Tiido et al. 2005). Any such variability, if it exists, would most likely dilute and not magnify the associations found in the present study (Tiido et al. 2005). This study has several strengths. Multicolour FISH was carried out using DNA probes specific for different chromosomes (13, 18, 21, X, Y) among a large number of subjects (n ¼ 181). Smoking status was assessed using the level of cotinine in saliva. In addition, detailed questionnaire information on demographics, medical and lifestyle risk factors allowed for control of confounding factors in the statistical models. The results of the present study suggest that environmental exposure
Table 3. Association between sperm aneuploidy and level of 1-OHP in urine Multivariate model adjusted for sexual abstinence, age, smoking, season of the year during semen collection, past diseases. Statistically significant at the level 0.05. CI, confidence interval Disomy
X-Y-18 X-X-18 Y-Y-18 Sex-chromosome disomy 13-13 18-18 21-21 Total chromosome disomy
Crude (1-OHP–creatinine)
Adjusted (1-OHP–creatinine)
b coefficient
95% CI
P
b coefficient
95% CI
P
1.08 1.31 1.25 1.43 1.02 1.30 1.17 1.06
0.80–1.46 0.86–1.98 0.88–1.77 1.02–2.00 0.79–1.32 0.96–1.76 0.85–1.60 0.78–1.27
0.62 0.21 0.21 0.04 0.89 0.09 0.33 0.98
1.11 1.26 1.36 1.46 0.97 1.22 1.06 1.02
0.81–1.51 0.83–1.91 0.96–1.92 1.05–2.03 0.83–1.14 1.02–1.63 0.77–1.46 0.80–1.30
0.52 0.27 0.09 0.03 0.74 0.03 0.72 0.85
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to PAHs may be associated with sperm disomy. As the men were from an infertility clinic, further studies are needed to confirm the findings in other populations and using different biomarkers of PAH exposure. Acknowledgements This study was performed under the project ‘Epidemiology of reproductive hazards – multicentre study in Poland’ supported by National Centre for Research and Development in Poland from grant no. PBZ-MEiN-/8/2//2006; contract no. K140/P01/2007/1.2.1.2. This study was performed under the project ‘Epidemiology of reproductive hazards – multicentre study in Poland’ supported by the National Centre for Research and Development in Poland from grant no. PBZ-MEiN-/8/2//2006; contract no. K140/P01/2007/ 1.2.1.2 and the project financed with a grant for statutory activity IMP 10.23.
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R Core Team (2013). ‘R: a language and environment for statistical computing’. (R Foundation for Statistical Computing: Vienna.) Available at http://www.R-project.org/ [Verified 15 September 2013]. Robbins, W. A., Vine, M. F., Truong, K. Y., and Everson, R. B. (1997). Use of fluorescence in situ hybridisation (FISH) to assess effects of smoking, caffeine and alcohol on aneuploidy load in spermatozoa of healthy men. Environ. Mol. Mutagen. 30, 175–183. doi:10.1002/(SICI)1098-2280 (1997)30:2,175::AID-EM10.3.0.CO;2-A Robbins, W. A., Rubes, J., Selevan, S. G., and Perreault, S. D. (1999). Air pollution and sperm aneuploidy in healthy young men. Int. J. Hyg. Environ. Health 1, 125–131. Robbins, W. A., Elsshoff, D. A., Xun, L., Jia, J., Li, N., Wu, G., and Wei, F. (2005). Effect of lifestyle exposures on sperm aneuploidy. Cytogenet. Genome Res. 111, 371–377. doi:10.1159/000086914 Rubes, J., Lowe, X., Moore, D., 2nd, Perreault, S., Slott, V., Evenson, D., Selevan, S. G., and Wyrobek, A. J. (1998). Smoking cigarettes is associated with increased sperm disomy in teenage men. Fertil. Steril. 70(4), 715–723. doi:10.1016/S0015-0282(98)00261-1 Rubesˇ, J., Vozdova´, M., and Kubı´cˇkova´, S. (1999). Aneuploidy in pig spermatozoa: multicolour fluorescence in situ hybridisation using probes for chromosome 1, 10 and Y. Cytogenet. Cell Genet. 85, 200– 204. doi:10.1159/000015293 Santodonato, J. (1997). Review of the oestrogenic and antioestrogenic activity of polycyclic aromatic hydrocarbons – relationship to carcinogenicity. Chemosphere 34, 835–848. doi:10.1016/S0045-6535(97)00012-X Schulz, C., Wilhelm, M., Heudorf, U., and Kolossa-Gehring, M. (2012). Reprint of ‘Update of the reference and HBM values derived by the German Human Biomonitoring Commission’. Int. J. Hyg. Environ. Health 215(2), 150–158. doi:10.1016/J.IJHEH.2012.01.003 Selevan, S. G., Borkovec, L., Slott, V. L., Zudova, Z., Rubes, J., Evenson, D. P., and Perreault, S. D. (2000). Semen quality and reproductive health of young Czech men exposed to seasonal air pollution. Environ. Health Perspect. 108, 887–894. doi:10.1289/EHP.00108887 Shi, Q., Ko, E., Barclay, L., Hoang, T., Rademaker, A., and Martin, R. (2001). Cigarette smoking and aneuploidy in human spermatozoa. Mol. Reprod. Dev. 59(4), 417–421. doi:10.1002/MRD.1048 Siwin´ska, E., Mielzyn´ska, D., Smolik, E., Bubak, A., and Kwapulin´ski, J. (1998). Evaluation of intra- and inter-individual variation of urinary 1-hydroxypyrene, a biomarker to polycyclic aromatic hydrocarbons. Sci. Total Environ. 217, 175–183. doi:10.1016/S0048-9697(98)00186-7 Smith, J. L., Garry, V. F., Rademaker, A. W., and Martin, R. H. (2004). Human sperm aneuploidy after exposure to pesticides. Mol. Reprod. Dev. 67, 353–359. doi:10.1002/MRD.20022 Tempest, H. G., Homa, S. T., Dalakiouridou, M., Christopikou, D., Wright, D., Zhai, X. P., and Griffin, D. K. (2004). The association
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between male infertility and sperm disomy: evidence for variation in disomy levels among individuals and a correlation between particular semen parameters and disomy of specific chromosome pairs. Reprod. Biol. Endocrinol. 2, 82. doi:10.1186/1477-7827-2-82 Tempest, H. G., Cheng, S. Y., Gillott, D. J., Handyside, A. H., Thornhill, A. R., and Griffin, D. K. (2010). Scoring of sperm chromosomal abnormalities by manual and automated approaches: qualitative and quantitative comparisons. Asian J. Androl. 12, 257–262. doi:10.1038/ AJA.2009.85 Templado, C., Bosch, M., and Benet, J. (2005). Frequency and distribution of chromosome abnormalities in human spermatozoa. Cytogenet. Genome Res. 111, 199–205. doi:10.1159/000086890 Thompson, M. W., Mclnnes, R. R., and Willad, H. F. (1986). Clinical cytogenetics: general principles and autosomal abnormalities. In ‘Genetics in Medicine’. 5th edn. (Ed. M. W. Thompson.) pp. 201–229. (WB Saunders: Toronto.) Tiido, T., Rignell-Hydbom, A., Jo¨nsson, B., Giwercman, Y. L., Rylander, L., Hagmar, L., and Giwercman, A. (2005). Exposure to persistent organochlorine pollutants associates with human sperm Y:X chromosome ratio. Hum. Reprod. 20, 1903–1909. doi:10.1093/HUMREP/DEH855 Tran, D. Q., Ide, C. F., McLachlan, J. A., and Arnold, S. F. (1996). The antioestrogenic activity of selected polynuclear aromatic hydrocarbons in yeast expressing human oestrogen receptor. Biochem. Biophys. Res. Commun. 229(1), 102–108. doi:10.1006/BBRC.1996.1764 Ushijima, C., Kumasako, Y., Kihaile, P. E., Hirotsuru, K., and Utsunomiya, T. (2000). Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in situ hybridisation. Hum. Reprod. 15(5), 1107–1111. doi:10.1093/HUMREP/15.5.1107 World Health Organization (1999) ‘WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction’. 4th edn. (Cambridge University Press: Cambridge.) Wyrobek, A. J., Schmid, T. E., and Marchetti, F. (2005). Relative susceptibilities of male germ cells to genetic defects induced by cancer chemotherapies. J. Natl. Cancer Inst. Monogr. 2005, 31–35. doi:10.1093/ JNCIMONOGRAPHS/LGI001 Xia, Y., Han, Y., Zhu, P., Wang, S., Gu, A., Wang, L., Lu, C., Fu, G., Song, L., and Wang, X. (2009). Relation between urinary metabolites of polycyclic aromatic hydrocarbons and human semen quality. Environ. Sci. Technol. 43, 4567–4573. doi:10.1021/ES9000642 Young, S. S., Eskenazi, B., Marchetti, F. M., Block, G., and Wyrobek, A. J. (2008). The association of foliate, zinc and antioxidant intake with sperm aneuploidy in health non-smoking men. Hum. Reprod. 23, 1014–1022. doi:10.1093/HUMREP/DEN036
www.publish.csiro.au/journals/rfd
Reproduction, Fertility and Development, 2016, 28, 1376–1381 http://dx.doi.org/10.1071/RD14063
Human sperm aneuploidy after exposure to polycyclic aromatic hydrocarbons Michał Radwan A,E, Joanna Jurewicz B,E, Wojciech Sobala B, Sławomir Brzez´nicki C, Paweł Radwan A, Lucjusz Jakubowski D, Wanda Hawuła D, Anna Ulan´ska D and Wojciech Hanke B A
Department of Gynaecology and Reproduction, Gameta Hospital 34/36 Rudzka Street, 95-030, Rzgo´w, Poland. B Department of Environmental Epidemiology, Nofer Institute of Occupational Medicine, 8 Teresy Street, 91-348 Lodz, Poland. C Department of Chemical Safety, Nofer Institute of Occupational Medicine, 8 Teresy Street, 91-348 Lodz, Poland. D Department of Medical Genetics, Polish Mother’s Memorial Hospital – Research Institute, 281/289 Rzgowska Street, 93-338 Lodz, Poland. E Corresponding authors. Emails: [email protected]; [email protected]
Abstract. The purpose of this cross-sectional study was to investigate whether environmental exposure to polycyclic aromatic hydrocarbons (PAHs) was associated with sperm aneuploidy. A sample of 181 men who attended an infertility clinic for diagnostic purposes and who had a normal semen concentration of 20–300 106 spermatozoa mL1 or slight oligozoospermia (semen concentration of 15–20 106 spermatozoa mL1; WHO 1999) provided urine and semen samples. Analysis of the level of PAH biomarker 1-hydroxypyrene (1-OHP) in urine was performed using highperformance liquid chromatography. Sperm aneuploidy was assessed using multicolour florescence in situ hybridisation (FISH) using DNA probes specific for chromosomes X, Y, 18, 13 and 21. Positive associations were observed between the level of 1-OHP in urine and total sex-chromosome disomy (P ¼ 0.03) and chromosome-18 disomy (P ¼ 0.03). These results suggest that environmental exposure to PAHs may be associated with sperm aneuploidy. This is the first epidemiological study to investigate the relationship between environmental exposure to PAHs and sperm aneuploidy. Therefore, these findings require further replication in other populations using different biomarkers of PAH exposure. Additional keywords: environmental exposure to PAH, FISH, level of 1-OHP, male fertility, PAH exposure, sperm disomy. Received 17 February 2014, accepted 29 January 2015, published online 10 March 2015 Introduction Studies in infertile men have demonstrated that 2–14% have constitutional chromosomal abnormalities (Meschede et al. 1997). The incidence of chromosomal aberrations is dependent on the definition of ‘infertility’ and is observed in ,2% in males with combined factors of infertility (Meschede et al. 1997) and 14% in azoospermic men (Johnson 1998). These frequencies are all considerably higher than the 0.7% population incidence seen in newborns (Thompson et al. 1986). For humans, aneuploidy is identified in at least 5% of all clinically recognised pregnancies (Lamb and Hassold 2004) and is associated with approximately one-third of all pregnancy losses. The majority of aneuploid liveborns are attributed to parental gametes carrying abnormal numbers of chromosomes (Hassold and Hunt 2001). It has been estimated that 2% or more of all spermatozoa have missing or additional chromosomes (Hassold and Hunt 2001). Journal compilation Ó CSIRO 2016
It should be noted that in animal species (pigs and cattle) sperm aneuploidy rates are similar to those in humans (Rubesˇ et al. 1999). It has been suggested that one reason might be human or animal exposure to various environmental agents. Several factors have been implicated in increasing chromosome aneuploidy in spermatozoa, including smoking, alcohol, caffeine, drugs, pesticides, air pollution and chemotherapy (Robbins et al. 1997, 1999, 2005; Rubes et al. 1998; Shi et al. 2001; Tempest et al. 2004; Ha¨rko¨nen 2005; Wyrobek et al. 2005; Martin 2006). Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals that are formed during the incomplete burning of organic substances. There are more than 100 different kinds of PAHs (Agency for Toxic Substances and Disease Registry (ATSDR) et al. 1995). Because PAHs are ubiquitous in daily life, the potential consequences of human exposure to PAHs www.publish.csiro.au/journals/rfd
PAH exposure and sperm aneuploidy
have raised concerns in the general population (Santodonato 1997). In particular, PAHs may have endocrine-disrupting chemical properties that may affect the reproductive and developmental processes in humans (Piskorska-Pliszczynska et al. 1986; Tran et al. 1996; Clemons et al. 1998); however, there are very few epidemiological studies on the effect of exposure to PAHs on male reproduction. Most of the studies are focussed on semen quality (Selevan et al. 2000; Hsu et al. 2006; Xia et al. 2009; Jeng et al. 2013) and sperm DNA damage (Selevan et al. 2000; Hsu et al. 2006; Han et al. 2011; Jeng et al. 2013). Those studies suggest a harmful effect of PAH exposure on the male reproductive system. According to our knowledge, no previous studies have assessed the relationship between environmental exposure to PAHs and sperm aneuploidy. The present study was designed to investigate whether environmental exposure to PAHs, assessed by 1-hydroxypyrene (1-OHP) level in urine, is associated with an altered frequency of sperm aneuploidy in adult men. Materials and methods Study population Between January 2008 and April 2011 men aged under 45 years of age with normal semen concentration of 20–300 106 spermatozoa mL1 or with slight oligozoospermia (semen concentration of 15– 20 106 spermatozoa mL1; WHO 1999) were recruited from an infertility clinic in Lodz, Poland. The protocol and informed consent form were approved by the Nofer Institute of Occupational Medicine Bioethical Committee Board. After obtaining a written informed consent from each participant, information was collected during an in-person interview on their occupational history, socio-demographic characteristics, reproductive, medical history and lifestyle factors. Additionally, smoking status was verified by measuring cotinine levels in saliva using high-performance liquid chromatography coupled with tandem mass spectrometry–positive electrospray ionisation (LC-ESIþMS/MS) and the isotope dilution method. Full details of the study have been described elsewhere (Jurewicz et al. 2014). Among 334 men who agreed to participate, a subset of 181 men (52.6%) were eligible for this sub-study and had available semen samples for florescence in situ hybridisation (FISH) analysis and enough urine sample for the measure of 1-hydroxypyrene in urine. Sperm aneuploidy analysis Sperm aneuploidy was measured by multicolour FISH analysis using DNA probes specific for chromosomes 13, 18, 21, X and Y (AneuVysion DNA Probe Kit; VYSIS, Abbott Molecular Inc., IL, USA) and the slides were viewed by fluorescence microscopy on a Nikon Eclypse 80i (Nikon Instruments Europe, Amsterdam, The Netherlands) equipped with LUCIA Cytogenetics-Karyo/ FISH software (Ushijima et al. 2000; Laboratory Imaging, Praha, Czech Republic) as previously described (Jurewicz et al. 2013a). Briefly, frequency of aneuploidy was analysed in every sample in two hybridisation areas. In the first area the number of fluorescent signals from chromosomes X, Y and 18 were counted and in the second, signals from chromosome 13 and 21 were counted. One thousand cells were examined in
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each area. Six types of sperm disomy were examined: XY spermatozoa (sperm FISH genotype: X-Y-18), disomy X (XX-18), disomy Y (Y-Y-18), disomy 18 (X-18-18 or Y-18-18), disomy 21 (X-21-21 or Y-21-21) and disomy 13 (X-13-13 or Y-13-13). The overall hybridisation efficiency in our study was from 98.91 to 99.36%, which is higher than the hybridisation efficiency (97%) reported in other studies (Johannisson et al. 2002; Tiido et al. 2005; Martin 2006; McAuliffe et al. 2012). Measurement of 1-hydroxypyrene in urine The 1-OHP was analysed using high-performance liquid chromatography (HPLC). The analytical procedure used in this study was based on the method described by Jongeneelen et al. (1987). The sensitivity of the method is estimated to be 0.2 mg L1. The full details of the method used for measure of 1-hydroxypyrene in urine are presented elsewhere (Jurewicz et al. 2013). Statistical analysis Descriptive statistics for demographic characteristics, sperm aneuploidy and level of 1-OHP in urine were calculated. The relationship between the level of 1-OHP and each type of sperm aneuploidy was evaluated using negative binomial regression modelling for disomy X and disomy 13 and generalised linear mixed model with a Poisson distribution for disomy Y, XY, 18, 21, total sex-chromosome disomy and total chromosome disomy. Models were selected based on Akaike Information Criteria (AIC). We compared the distributions of variables from the questionnaire among men with 1-OHP levels by Chi-square test in order to identify potential confounders. Data were analysed using multivariate analysis. Covariates in the final models were: sexual abstinence (days), age (years), smoking (yes/no), season of the year during semen collection (May–September, October–April), past diseases (yes/no). 1-OHP was included as a continuous variable in the models. Sperm aneuploidies were log-transformed to obtain a normal distribution. We used R 2.15.1 statistical program for all data analysis (R Core Team 2013; http://www.R-project.org/). Results Demographic and other characteristics of the 181 study participants are presented in Table 1. Overall, participants had mean age 32.1 4.6 years. The mean duration of abstinence was 5 2.3 days. Most of the study participants had secondary (39.8%) and higher education (37.0%) and were non-smokers (72%). As shown in Table 2 the sperm concentration was 50.71 49.33 106 mL1, the percentage of motile sperm cells and the percentage of spermatozoa with normal morphology were 56.00 51.30 and 46.00 78.60 respectively. The unadjusted mean frequency and s.e.m. of sperm disomy among men in our study was 1.30 0.99 for XY spermatozoa, 0.28 0.26 for disomy X, 0.61 0.43 for disomy Y, 1.13 0.23 for total sexchromosome disomy, 0.54 0.38 for disomy 13, 0.85 0.67 for disomy 18, 0.98 0.95 for disomy 21 and 1.72 0.92 for aggregate aneuploidy.
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Environment is 0.5 mg L1 (Schulz et al. 2012), which is a little lower than the 95th percentile of the urinary concentration of 1-OHP in our study 0.68 mg L1. The unadjusted geometric mean of 1-OHP in urine was 0.35 0.35 mg L1 (range 0.04–1.95 mg L1) and adjusted for creatinine 0.29 0.27 mg g1 creatinine (range 0.04–2.03 mg g1 creatinine; Table 2). Table 3 summarises the relationships between the level of 1-OHP in urine and sperm aneuploidy, crude and adjusted for potential confounders. Positive associations were observed between the level of 1-OHP in urine and total sex-chromosome disomy (P ¼ 0.04; Table 3). When the model was adjusted for sexual abstinence, age, smoking, season of the year during semen collection and past diseases the total sex-chromosome disomy (P ¼ 0.03) remained significant. Additionally, an increase in the frequency of disomy 18 (P ¼ 0.03) was related to the level of 1-OHP in urine.
The reference values (95th percentile of the values measured for the substance concentration) of 1-OHP levels in urine for non-smoking general population aged between 3 and 69 years established by the Commission of the German Federal Table 1. Characteristics of the study population s.e.m., standard error of the mean; n, number of participants Characteristics
n (%)
Education Vocational 42 (23.2) Secondary 72 (39.8) Higher 67 (37.0) Smoking determined by cotinine level No 131 (72.4) Yes 47 (26.0) Missing data 3 (1.6) Past diseases, which may have impact on semen quality No 161 (89.0) Yes 20 (11.0) Duration of couple’s infertility (years) 1–2 70 (38.7) 2–3 57 (31.5) 3–5 28 (15.4) .5 26 (14.4) Abstinence (days) ,3 19 (10.5) 3–7 134 (74.0) .7 28 (15.5) mean s.e.m. 5.0 2.3 median (min–max) 5.0 (0.0–20.0) Age (years) mean s.e.m. 32.1 4.6 median (min–max) 31.8 (22.7–44.8) Season (3 months before semen collection) May–September 68 (37.6) October–April 113 (62.4)
Discussion This is the first epidemiological study to investigate the relationship between environmental exposure to PAHs and sperm aneuploidy. Our results suggest a significant increase in the frequency of total sex-chromosome disomy and chromosome18 disomy after adjusting for potential confounders. Because no prior studies have investigated 1-hydroxypyrene in urine and sperm aneuploidy, comparisons of these findings with other studies are limited. Six epidemiological studies have evaluated the harmful effects of air pollution and PAH exposure on semen quality: sperm concentration, sperm number per ejaculate (Xia et al. 2009), abnormal morphology (Selevan et al. 2000; Hsu et al. 2006), motility (Jeng et al. 2013), sperm DNA damage (Selevan et al. 2000; Hsu et al. 2006; Han et al. 2011; Jeng et al. 2013) and sperm aneuploidy (Robbins et al. 1999). The latter found an association between exposure to seasonal air pollution and increased YY disomy (Robbins et al. 1999).
Table 2. Percentage of sperm aneuploidy and level of 1-OHP in urine s.e.m., standard error of the mean; n ¼ 181
Sperm aneuploidy (%) X-Y-18 X-X-18 Y-Y-18 Sex-chromosome disomy 13-13 18-18 21-21 Total chromosome disomy Sperm parameters Sperm concentration (106 mL1) Total motility (%) Normal sperm morphology (%) Level of 1-OHP in urine 1-OHP mg L1 1-OHP mg g1 creatinine
Minimum
25%
50%
75%
95%
0 0 0 0.60 0 0 0 0
0.50 0.10 0.30 0.97 0.30 0.71 0.60 0.70
0.76 0.20 0.50 1.11 0.49 0.82 0.98 0.82
1.50 0.40 0.80 1.27 0.70 1.52 1.44 0.91
1.99 0.83 1.23 1.74 1.50 2.03 5.12 1.89
15.00 6.00 89.00
21.20 46.30 67.50
45.70 54.20 52.80
81.70 65.30 31.50
0.04 0.04
0.15 0.12
0.24 0.22
0.31 0.44
Maximum
Mean
s.e.m.
2.34 1.16 2.12 2.00 2.48 2.61 8.21 1.20
1.30 0.28 0.61 1.13 0.54 0.85 0.98 1.72
0.99 0.26 0.43 0.23 0.38 0.67 0.95 0.92
115 81.20 25.88
360 97.00 22.00
50.71 56.00 46.00
49.33 51.30 78.60
0.68 0.72
1.95 2.03
0.35 0.29
0.35 0.27
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Gaspari et al. (2003) observed that occupational exposure to PAH, but not smoking, was significantly associated with higher levels of PAH–DNA adducts, especially among infertile men (P ¼ 0.04). In a study performed in China, increased urinary 2-hydroxynaphthalene (2-OHNa) levels were associated with increased comet parameters, including the percentage of DNA in the tail length and tail distribution (Han et al. 2011). Higher rates of DNA denaturation in spermatozoa were observed among workers occupationally exposed to PAHs (Hsu et al. 2006). Selevan et al. (2000) reported an association between air pollution episodes of elevated PAH and abnormal chromatin in human spermatozoa, whereas Jeng et al. (2013) did not find an association between DNA fragmentation and 1-OHP exposure. The results of those studies are in line with our findings regarding the impact of air pollutants and PAH exposure on sperm sex-chromosome disomy. Additionally we found an increase risk of chromosome-18 disomy in relation to the level of 1-hydroxypyrene in urine. These results suggest that chromosome 18 and sex chromosomes are more susceptible than other chromosomes to nondisjunction during male meiosis induced by environmental exposure to PAHs (Robbins et al. 1997). The mean frequencies of sperm aneuploidy for sex chromosomes among men in our study were higher than reported in other studies ((XX, 0.03–0.37; YY, 0.04–0.21; XY, 0.06–0.42) for men from the general population (Egozcue et al. 1997; Templado et al. 2005) and similar to studies performed among andrology clinic patients (Tempest et al. 2010; McAuliffe et al. 2012). In the case of chromosomes 21, 13 and 18, the frequencies of disomy were similar to previously reported values (0.095–2.3%, 0.55–2.2% and 0.04–1.1%, respectively; Smith et al. 2004; Gianaroli et al. 2005; Young et al. 2008). The variation in aneuploidy frequencies between studies may be due, in part, to technical differences such as sperm decondensation, scoring criteria, FISH method, storage of samples and their criteria for inclusion of men in the studies (Tempest et al. 2004). Male susceptibility is a function of competing, interacting or additive effects related to age, nutrition, stress, work place and recreational exposures as well as a myriad of
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other health factors that can affect normal meiosis and increase the risk for aneuploid spermatozoa (Robbins et al. 2005). The design of our study does not, however, allow us to clarify the mechanisms behind the observed associations. One hypothesis could be that reactive metabolites of PAHs might reach the testes and epididymis and then react with sperm DNA to form adducts, causing DNA damage (Gaspari et al. 2003). Additionally, compounds resulting from the oxidation of PAHs have the ability to enter redox cycles, which increased the formation of reactive oxygen species (ROS) (Farmer et al. 2003) and thus caused sperm DNA damage (Barroso et al. 2000). There are limitations to the use of urine levels of 1-OHP as biomarkers of PAH exposure in humans and rodents. Some studies suggest that urinary 1-OHP level is an appropriate surrogate biomarker for total PAH exposure of human populations (Jongeneelen et al. 1987; Siwin´ska et al. 1998; Jacob and Seidel 2002). On the other hand, some authors claim that 1-OHP may not represent the numerous PAH metabolites (Al-Saleh et al. 2013). The men in this study were attending an infertility clinic for diagnostic purposes. They were different from the general population, but all men in our study had normal semen parameters or slight oligozoospermia based on WHO classification (WHO 1999). Currently, there is no evidence showing that they would differ in ways that would alter their response to PAHs (McAuliffe et al. 2012). Our study only used a single semen sample; this could be a limitation of this study. However, there are no data in the literature on possible temporal variation in chromosomal frequencies (Tiido et al. 2005). Any such variability, if it exists, would most likely dilute and not magnify the associations found in the present study (Tiido et al. 2005). This study has several strengths. Multicolour FISH was carried out using DNA probes specific for different chromosomes (13, 18, 21, X, Y) among a large number of subjects (n ¼ 181). Smoking status was assessed using the level of cotinine in saliva. In addition, detailed questionnaire information on demographics, medical and lifestyle risk factors allowed for control of confounding factors in the statistical models. The results of the present study suggest that environmental exposure
Table 3. Association between sperm aneuploidy and level of 1-OHP in urine Multivariate model adjusted for sexual abstinence, age, smoking, season of the year during semen collection, past diseases. Statistically significant at the level 0.05. CI, confidence interval Disomy
X-Y-18 X-X-18 Y-Y-18 Sex-chromosome disomy 13-13 18-18 21-21 Total chromosome disomy
Crude (1-OHP–creatinine)
Adjusted (1-OHP–creatinine)
b coefficient
95% CI
P
b coefficient
95% CI
P
1.08 1.31 1.25 1.43 1.02 1.30 1.17 1.06
0.80–1.46 0.86–1.98 0.88–1.77 1.02–2.00 0.79–1.32 0.96–1.76 0.85–1.60 0.78–1.27
0.62 0.21 0.21 0.04 0.89 0.09 0.33 0.98
1.11 1.26 1.36 1.46 0.97 1.22 1.06 1.02
0.81–1.51 0.83–1.91 0.96–1.92 1.05–2.03 0.83–1.14 1.02–1.63 0.77–1.46 0.80–1.30
0.52 0.27 0.09 0.03 0.74 0.03 0.72 0.85
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to PAHs may be associated with sperm disomy. As the men were from an infertility clinic, further studies are needed to confirm the findings in other populations and using different biomarkers of PAH exposure. Acknowledgements This study was performed under the project ‘Epidemiology of reproductive hazards – multicentre study in Poland’ supported by National Centre for Research and Development in Poland from grant no. PBZ-MEiN-/8/2//2006; contract no. K140/P01/2007/1.2.1.2. This study was performed under the project ‘Epidemiology of reproductive hazards – multicentre study in Poland’ supported by the National Centre for Research and Development in Poland from grant no. PBZ-MEiN-/8/2//2006; contract no. K140/P01/2007/ 1.2.1.2 and the project financed with a grant for statutory activity IMP 10.23.
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