metalloids and semen quality, sperm apoptosis and DNA integrity

metalloids and semen quality, sperm apoptosis and DNA integrity

Environmental Pollution xxx (2017) 1e11 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/...

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Environmental Pollution xxx (2017) 1e11

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity* Yi-Xin Wang a, b, Peng Wang a, b, Wei Feng a, b, Chong Liu a, b, Pan Yang a, b, Ying-Jun Chen a, b, Li Sun a, b, Yang Sun a, b, Jing Yue c, Long-Jie Gu c, Qiang Zeng a, b, Wen-Qing Lu a, b, * a

Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental Protection, State Key Laboratory of Environmental Health (incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China c Reproductive Medicine Center, Tongji Hospital, Wuhan, Hubei, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 September 2016 Received in revised form 12 January 2017 Accepted 15 January 2017 Available online xxx

This study aimed to investigate the relationships between environmental exposure to metals/metalloids and semen quality, sperm apoptosis and DNA integrity using the metal/metalloids levels in seminal plasma as biomarkers. We determined 18 metals/metalloids in seminal plasma using an inductively coupled plasma-mass spectrometry among 746 men recruited from a reproductive medicine center. Associations of these metals/metalloids with semen quality (n ¼ 746), sperm apoptosis (n ¼ 331) and DNA integrity (n ¼ 404) were evaluated using multivariate linear and logistic regression models. After accounting for multiple comparisons and confounders, seminal plasma arsenic (As) quartiles were negatively associated with progressive and total sperm motility using multivariable linear regression analysis, which were in accordance with the trends for increased odds ratios (ORs) for below-reference semen quality parameters in the logistic models. We also found inverse correlations between cadmium (Cd) quartiles and progressive and total sperm motility, whereas positive correlations between zinc (Zn) quartiles and sperm concentration, between copper (Cu) and As quartiles and the percentage of tail DNA, between As and selenium (Se) quartiles and tail extent and tail distributed moment, and between tin (Sn) categories and the percentage of necrotic spermatozoa (all Ptrend<0.05). These relationships remained after the simultaneous consideration of various elements. Our results indicate that environmental exposure to As, Cd, Cu, Se and Sn may impair male reproductive health, whereas Zn may be beneficial to sperm concentration. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Apoptosis DNA integrity Epidemiology Semen quality Seminal plasma metals/metalloids

1. Introduction Studies have reported a decline in human semen quality over the past several decades (Almagor et al., 2003; Carlsen et al., 1992; Feki et al., 2009; Jorgensen et al., 2011; Rolland et al., 2013; Sk et al., 2008). This constitutes a severe public health concern, although the conclusions remain controversial (Bonde et al., 2011; Jorgensen

*

This paper has been recommended for acceptance by Dr. Chen Da. * Corresponding author. Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China. E-mail address: [email protected] (W.-Q. Lu).

et al., 2012). Exposure to environmental toxicants, including heavy metals/metalloids, is likely a contributing factor to this decline in semen quality. Heavy metals/metalloids are ubiquitously present in the environment due to natural process and anthropogenic activities, and high levels of metals/metalloids have been detected in the airborne particulate matter, soil, food and drinking water in China (Lu et al., 2015; Wu et al., 2016; Zhao et al., 2016). The principle sources of exposure to the general population are through the inhalation of polluted air and intake of contaminated food and water (Khan et al., 2008; Roychowdhury et al., 2003; Zheng et al., 2010). Low levels of toxic elements, including aluminum (Al), arsenic (As), cadmium (Cd), lead (Pb), antimony (Sb), thallium (Tl) and uranium (U), have been demonstrated to pose detrimental effects on reproductive

http://dx.doi.org/10.1016/j.envpol.2017.01.083 0269-7491/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wang, Y.-X., et al., Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.01.083

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Y.-X. Wang et al. / Environmental Pollution xxx (2017) 1e11

health of rats (Asadi et al., 2014; Babaei and Abshenas, 2013; Llobet et al., 1995; Oliveira et al., 2009; Pant et al., 2001). Essential elements, including chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), zinc (Zn), selenium (Se), molybdenum (Mo), tin (Sn) and tungsten (W), have also been associated with male reproductive health problems after exposure above specific levels in animals (Baltaci et al., 2014; Chandra et al., 2007; Lyubimov et al., 2004; Mitra et al., 2014; Pedigo et al., 1988; Wellejus et al., 2000). Some studies of non-occupationally exposed population have reported associations of As and Cd with lower sperm motility (Benoff et al., 2009; Meeker et al., 2008; Pant et al., 2003; Telisman et al., 2000; Wang et al., 2016b), Mo with declined sperm concentration and normal morphology (Meeker et al., 2008) and Pb with damaged DNA integrity (Hernandez-Ochoa et al., 2005), while others have not (Bonde et al., 2002; Mendiola et al., 2011; Zeng et al., 2015). Given that metals/metalloids are distributed differently in seminal plasma versus other bodily fluids in men (e.g., whole blood and urine) (Mendiola et al., 2011), using metal/metalloids measures in the blood or urine as biomarkers may not directly reflect the exposure to the male reproductive system. Epididymis is the place where sperm undergoes the process of maturation and acquires motility (Pant et al., 2004). The seminal vesicle also plays an important role in sperm fertility, and a removal of seminal vesicles from mice can result in declined fertility (Araki et al., 2016). Cd, Mo and Zn have been found to accumulate in the testes and epididymis of males (Oldereid et al., 1993; Pandey and Singh, 2002; Sorensen et al., 1999) indicating that metals/metalloids in the seminal plasma may be a more direct index for the exposure status of the human reproductive tract. As a result, the measurements of Al, As, Mn, Co, Ni, Se, Cu, Zn, Mo, Cd, Sn and Pb in seminal plasma have been tested for their exposure effects on human semen quality (Jeng et al., 2015; Kim et al., 2014; Zafar et al., 2015). However, the limited sample sizes (<200 males) in these prior investigations prevented precise conclusions. Additionally, no studies to date have evaluated the associations between seminal plasma metals/metalloids and human sperm apoptosis and DNA integrity. To fill the data gap, in this study we measured 18 metals/metalloids (i.e., Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Sn, Sb, W, Tl, Pb and U) in the seminal plasma of non-occupationally exposed Chinese men and comprehensively evaluated their associations with semen quality, sperm apoptosis and DNA integrity. 2. Materials and methods 2.1. Study subjects This study was approved by the committees on research ethics of Tongji Medical College. Eligible subjects were the males between 18 and 55 years of age in subfertile couples who came to Wuhan reproductive medicine center for examination and had no prior knowledge of male factor infertility (Wang et al., 2016b). A total of 1247 men were ultimately enrolled in this study in 2013 (participation rate: 84%). Each participant completed a questionnaire and provided a semen sample on their clinic visiting day. Questions included information on demographic features, lifestyle, history of medical treatment and occupational exposure. After excluding volunteers with azoospermia (n ¼ 58), an occupational exposure to metals (n ¼ 16) or self-reported disease that may adversely affect reproductive system or urinary excretion of chemicals (n ¼ 121), 1052 participants remained. We additionally excluded 306 men because of their inadequate semen volumes for metals/metalloids determinations. Thus, samples from a total of 746 were used in this study. The reported abstinence time, sperm volume, concentration

and total count differed between the men retrained in this study and those excluded (all P < 0.05; Table S1). 2.2. Semen collection and analysis After an abstinence period of 2e7 days, each man provided a semen sample into a trace element-free polypropylene specimen cup by masturbation in a semen collection room at the reproductive medicine center. A semen analysis was performed by two welltrained professional technicians following guidelines of World Health Organization (WHO, 2010) (Wang et al., 2016b). The semen volume was determined using a trace element-free polypropylene pipette. The progressive sperm motility, non-progressive motility and sperm concentration were analyzed using a computer-aided semen analyzer (WLJX 9000, Weili New Century Science & Tech Dev., Beijing, China). We calculated the total motility (progressive sperm motility þ non-progressive sperm motility) and total sperm count (sperm concentration  semen volume). Sperm morphology was determined by the high-power magnification (1000) on fixed and Papanicolaou stained smears based on the WHO (2010) guidelines. Each participant had two slides. The technicians evaluated no less than 200 sperm per replicate and compared the replicate values to see if they were acceptably close. External quality controls were established according to the WHO (2010) guidelines, and the differences in quality control results reported by the technicians were not significant. 2.3. Annexin V/PI and comet assay Cryopreservation was found to damage sperm DNA and induce spermatozoa apoptosis (Duru et al., 2001; Li et al., 2007). Therefore, we used semen samples collected within 1 hour (hr) to determine sperm apoptosis and DNA integrity. Because Annexin V/PI and neutral comet assays could not be simultaneously conducted for all the samples within a day, approximately 15 semen samples were randomly selected each day for the determinations. No significant differences were found between the subgroup and the entire study population regarding their demographic characteristics (Wang et al., 2016c). After assessing semen quality parameters, the remaining semen samples were promptly transported to our lab using a cooler filled with ice packs. The analytical procedure for Annexin V/PI and comet assays have been illustrated previously (You et al., 2015). We used flow cytometry to determine the percentage of necrotic spermatozoa (PI positive), apoptotic spermatozoa (Annexin V positive, PI negative) and viable spermatozoa (Annexin V negative, PI negative), and used the Comet Assay Software Project Lab image analysis system to calculate the percentage of tail DNA (tail %), tail extent and the tail distributed moment (TDM) (You et al., 2015). For quality control, we repeatedly determined 2 randomly selected semen sample each analysis run. The coefficients of variance for sperm DNA integrity and spermatozoa apoptotic measures were less than 10%. 2.4. Metals/metalloids measurements The remaining semen samples were centrifuged, and the resulting seminal plasma was stored at 80  C until metal measurements (within 5 months). Seminal plasma levels of 18 metals/ metalloids were analyzed using our modified method that was validated for the analysis of elements in urine (Feng et al., 2015; Wang et al., 2016a). Briefly, a 0.4-mL of seminal plasma was transferred to a trace element-free polyethylene tube, diluted to 4.0 mL with 1.2% HNO3 (v/v), and then stored in a freezer overnight at 4  C. Metal/metalloids levels in seminal plasma were detected using an inductively coupled plasma-mass spectrometry (Agilent

Please cite this article in press as: Wang, Y.-X., et al., Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.01.083

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7700, Agilent Technologies, USA). For quality control, we used certified reference materials ClinChek no. 8883 and 8884 (human plasma controls), the standard reference material 1640a and a spiked pooled sample. The measurements of Al, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Sn and Sb were found to be within the range of ClinChek certified values. To assess instrument performance, the standard reference material 1640a were always analyzed every 20 samples. Spiked seminal plasma samples were analyzed for their low and high concentrations of each elements to evaluate the method's accuracy. The spiked recoveries of the metals/metalloids ranged from 82% to 114%; the within-day and between-day variations were less than 10%. Each batch of samples also included one blank tube that contained deionized water; the analyte levels in all blank samples were lower than the limits of quantification (LOQ). The LOQs for Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Sn, Sb, W, Tl, Pb and U were 0.25, 0.032, 0.060, 0.41, 0.0040, 0.036, 0.13, 0.49, 0.014, 0.047, 0.033, 0.0037, 0.020, 0.0030, 0.055, 0.0026, 0.0063, and 0.0020 mg/L, respectively. Participants with measurements < LOQ had their values replaced with LOQ/√2, as has been commonly utilized in prior epidemiological studies (Hornung and Reed, 1990). 2.5. Statistics Statistical Analysis Software (SAS) (SAS Institute Inc., Cary, USA) was used for all Statistical analyses. We performed descriptive statistics for the population features, distributions of seminal plasma metals/metalloids and outcome measures. Spearman's Rank Correlation Test were applied to assess the correlations among the seminal plasma elements. Associations of the seminal plasma metals/metalloids with continuous outcome measures were assessed using multivariable linear regression. Data for progressive sperm motility, total motility, sperm morphology and the percentage of viable spermatozoa were modeled untransformed because they showed normal distributions. All other parameters were natural logarithm (ln) transformed to achieve a normal residual distribution; resulting estimates and 95% CIs on the log scale were then back-transformed to obtain the percent changes using the formula 100  (expestimate-1). The metals/metalloids in the seminal plasma were categorized into two, three or four groups based on the observed distributions to investigate dose-dependent relationships. Mn, Fe, Co, Cu, Zn, As, Se, Mo, Cd, Sb and Tl were divided into quartiles. Because Cr, Sn and Pb were not detected in a high proportion of the samples, an ordinal three-category variable was constructed: low-exposure group with levels < LOQ, and equally sized middle- and high-exposure groups among the samples with detectable levels. The detection rates of Al, Ni, W and U were 17%, 3.9%, 3.9% and 19%, respectively. To avoid biased estimates in data using imputed values for measurements < LOQ (Windham et al., 2015), data on Al, Ni, W and U were not analyzed further, although for exploratory purpose we also examined the associations of Al, Ni, W and U with semen quality parameters, apoptotic markers and DNA damage measures by dividing these elements into two groups by LOQ (see Sensitivity Analyses). Tests for trends in ordinal metal categories were conducted using regression models with integer values. In addition, we used multivariable logistic models to assess the correlations between metal/metalloids categories and dichotomous outcomes. Participants were dichotomized based on the WHO reference levels for progressive sperm motility (32% motile sperm), total motility (40% motile sperm), total count (39 million) and concentration (15 million/mL) (WHO, 2010). Participants had values for all four parameters that were equal to or greater than the reference levels were treated as the comparison. Covariates were considered to be retained in the multivariable

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regression models based on statistical and biological considerations. We used the method recommended by Greenland (1989) to decide which covariates should be included in the multivariable models. The confounders were retained if they caused a >10% change in the effect of metal levels on each outcome. Age, body mass index (BMI), time between semen ejaculation and analysis and cigarette consumption were examined as continuous variables. Having ever fathered a pregnancy (yes or no), education category (below high school or high school and above) and alcohol consumption (yes or no) were used as dichotomous variables. Income (<3000, 3000e6000, >6000 Yuan/month) and abstinence duration (3, 3e5, >5 days) were used as three-level ordinal variables. Smoking history (current and former vs. never) was used to create two dummy variables. Models for each set of reproductive outcome accounted for the same confounders to maintain consistency. The false-discovery rate (FDR) correction was implemented for adjustment of multiple tests. We calculated the FDR corrected Pvalues using the available spreadsheet file developed by Pike (2011), based on the classical one-stage method (Benjamini and Hochberg, 1995). Because various metals/metalloids may affect human reproductive health together (Feng et al., 2015; Meeker et al., 2008; Wang et al., 2016c), we further constructed multipleelement models by considering various metals/metalloids and covariates simultaneously for the single element models that showed significant associations after accounting for multiple comparisons. Backward elimination procedure with an alpha of 0.10 were used to confirm if the confounders should be reserved in the multiple-element models; we then individually added each covariate that was not retained in the multiple-element models to further determine evidence of confounding if they changed the effect estimates for metals/metalloids in the multiple-element model >10% (Meeker et al., 2008). We calculated the variance inflation factor to assess multicollinearity of the various metals/ metalloids in the multiple-element models. A variance inflation factor <10 indicates that multicollinearity may not affect the estimations (Neter et al., 1989). We finally explored the interaction between different metals/ metalloids in association with male reproductive function. We only included metals/metalloids that were statistically significant after controlling for multiple tests in the single-element models as well as in the multiple-element models. The interaction was tested by introducing a multiplicative interaction of quartiles/categories of toxic elements (e.g., As, Cd, Sn) and binary variable of a metal (e.g., Zn, Cu, Se) that may be beneficial to male reproductive health in the multivariable linear and logistic regression models. For all analyses, we regarded P-values <0.05 as statistically significant. 3. Results 3.1. Characteristics of participants According to the WHO reference values, 200 men (27%), 238 men (32%), 56 men (8.8%) and 66 men (9.0%) had progressive motilities, total motilities, total counts and concentrations less than the reference values, respectively (Table 1). Furthermore, 482 men (65%) had values for all four parameters that were equal to or greater than the reference values. The mean time between semen ejaculation and analysis significantly differed between the belowreference group and the comparison for progressive and total motility. The reported smoking status and the percentage of men having ever fathered a pregnancy significantly differed between the below-reference groups and the comparison for sperm concentration. No statistically significant differences in semen quality parameters were observed between the below-reference groups and the comparison for the other demographic characteristics.

Please cite this article in press as: Wang, Y.-X., et al., Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.01.083

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Table 1 Demographic categories [n (%) or mean ± SD] according to semen quality parameters. Category

All subjectsd, n ¼ 746

Age, years 32 ± 5.1 23 ± 3.2 BMI, kg/m2 Time between ejaculation and semen 18 ± 18 a analysis, minutes Daily cigarette consumption 7.2 ± 8.5 Ever fathered a pregnancyb Yes 300 (41) No 441 (59) Race Han 728 (98) Other 18 (2.0) Abstinence time, days <3 72 (10) 3-5 493 (66) >5 180 (24) Education level Less than high school 275 (37) High school and above 463 (63) c Smoking history Never-smoker 299 (40) Ever-smoker 447 (60) Former 90 (12) Current 357 (48) Alcohol consumption Yes 458 (39) No 288 (61) Income, RMB yuan/month 3000 323 (43) 3000-6000 293 (40) 6000 130 (17) a b c d

Comparison group, Progressive motility n ¼ 482 <32%, n ¼ 200

Total motility <40%, n ¼ 238

Total count <3.9  107, n ¼ 56

Concentration <1.5  107/mL, n ¼ 66

32 ± 5.1 23 ± 3.4 17 ± 18

32 ± 5.0 23 ± 3.1 21 ± 21

32 ± 5.2 23 ± 3.0 21 ± 21

32 ± 4.6 24 ± 2.9 16 ± 17

31 ± 4.8 23 ± 2.8 18 ± 18

7.3 ± 8.6

6.2 ± 7.4

6.8 ± 8.6

7.4 ± 11

6.4 ± 8.2

202 (42) 277 (58)

80 (40) 119 (60)

92 (39) 145 (61)

17 (31) 38 (69)

19 (29) 47 (71)

468 (97) 14 (3.0)

196 (98) 4 (2.0)

234 (98) 4 (2.0)

55 (98) 1 (2.0)

65 (98) 1 (2.0)

42 (9.0) 326 (68) 113 (23)

20 (10) 124 (62) 56 (28)

25 (11) 150 (63) 63 (26)

9 (16) 38 (68) 9 (16)

8 (12) 44 (67) 14 (21)

183 (38) 295 (62)

71 (36) 126 (64)

87 (37) 147 (63)

19 (35) 36 (65)

28 (44) 36 (56)

181 (38) 301 (62) 57 (12) 244 (50)

91 (46) 109 (54) 24 (12) 85 (42)

109 (46) 129 (54) 28 (12) 101 (42)

28 (50) 28 (50) 9 (16) 19 (34)

32 34 12 22

177 (37) 305 (63)

85 (43) 115 (57)

100 (42) 138 (58)

23 (41) 33 (59)

27 (41) 39 (59)

205 (43) 185 (38) 92 (19)

82 (41) 86 (43) 32 (16)

103 (43) 102 (43) 33 (14)

29 (52) 21 (38) 6 (10)

32 (49) 25 (38) 9 (13)

(49) (51) (18) (33)

Differed between the comparison group and the below-reference group for progressive sperm motility and total motility (p < 0.05). Differed between the comparison group and the below-reference group for sperm concentration (p < 0.05). Differed between the comparison group and below-reference group for sperm concentration (p < 0.05). A total of 2 patients had missing information for age, 5 for the history of a successful pregnancy, 8 for education level and 1 for abstinence time.

3.2. Seminal plasma metals/metalloids and reproductive outcome measures Table S2 presented the seminal plasma metal/metalloids concentrations of the participants according to semen quality parameters. We found that men with abnormal progressive motilities, total motilities, total counts or concentrations had higher levels of Al, As and Sb than men with normal semen quality parameters (See Table S2). Cr, Sn and Pb were detected in 57%, 43% and 72% of the subjects, respectively. Al, Ni, W and U were detected in <20% of the subjects. The other eleven metals were detected in >80% of the subjects. Spearman's rank correlation analyses revealed that most of the elements in the seminal plasma positively correlated with each other (p < 0.05) (Table S3). Semen quality parameters, apoptotic markers and DNA integrity measures for the study participants are shown in Table S4. 3.3. Seminal plasma metals/metalloids and semen quality In the single-element linear models that adjusted for multiple comparisons and confounders, we found significantly inverse dosedependent relationships between Mn, As, Cd and Sb levels and progressive motility and between As and Cd levels and total motility, whereas positive relationships were detected between the Fe, Co, Zn and Se levels and the sperm concentration (all FDRadjusted Ptrend <0.05; Fig. 1). In the multiple-element models, only the associations of As and Cd levels with progressive and total sperm motility, and Zn levels with sperm concentration remained significant (Table 2). When the semen quality parameters were

modeled as dichotomous variables in multivariate logistic models, As quantities were significantly associated with increased risk of below-reference progressive and total sperm motility, and Sb quantities were associated with increased risk of below-reference total motility after accounting for multiple tests (all FDR-adjusted Ptrend <0.05; Fig. S1). In accordance with the single-element models, As remained a predictor of progressive and total sperm motility in multiple-element models (both P < 0.05; Table S5). The variance inflation factors in the multiple-element linear and logistic models were less than 2 (Table 2 and Table S5). 3.4. Seminal plasma metals/metalloids and apoptotic markers The associations of seminal plasma metals/metalloids with apoptotic markers based on single-element linear regression models are shown in Fig. 2. After accounting for multiple comparisons, Cr quartiles showed a significant inverse association with percentage of necrotic spermatozoa, while Zn, Sn, Sb and Tl quartiles showed a positive association with percentage of necrotic spermatozoa (all FDR-adjusted Ptrend <0.05). After adjusting for multiple metals/metalloids and covariates, however, only the association of Sn with percentage of necrotic spermatozoa was confirmed (Table 3). The variance inflation factors in the multipleelement models were less than 1.5 (Table 3). 3.5. Seminal plasma metals/metalloids and sperm DNA integrity The associations of seminal plasma metals/metalloids with sperm DNA damage based on single-element linear models are

Please cite this article in press as: Wang, Y.-X., et al., Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.01.083

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Fig. 1. Regression coefficients or percentage changes (95% CIs) in semen quality parameters associated with seminal plasma metals/metalloids based on single-element linear regression models, adjusting for age, BMI, abstinence duration, liquefaction time, alcohol use, smoking status and daily cigarette consumption (n ¼ 746). aFDR-adjusted p-value for trends. bTransformed by the natural logarithm and back-transformed to obtain the percent change.

shown in Fig. 3. After adjustment for multiple comparisons, we observed positive dose-dependent relationships for Cu, As and Se levels with one or more sperm DNA integrity measures (all FDRadjusted Ptrend <0.05). In multiple-element models, the associations of Cu and As levels with tail%, and As and Se with tail extent and TDM remained significant (Table 3). The variance inflation factors in the multiple-element models were all less than 1.5 (Table 3). Evidence for interaction was explored by introducing a multiplicative interaction of quartiles/categories of As, Cd, Cu, Se and Sn and binary variable of a metal that may be beneficial to male reproductive health in the multivariable linear and logistic regression models. We did not find any evidence of interaction

between different metals/metalloids in relation to semen quality parameters, sperm apoptosis and DNA integrity measures (results not shown). 3.6. Sensitivity analyses We reanalyzed all of the multivariable models by excluding subjects with abnormal progressive motilities, total motilities, total counts, or concentrations. The results were largely unchanged (results not shown). We also examined the associations of seminal plasma Al, Ni, W and U with semen quality parameters, apoptotic markers and DNA damage measures by dividing these elements into two groups by LOQ. After controlling for multiple tests, we

Please cite this article in press as: Wang, Y.-X., et al., Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.01.083

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Table 2 Adjusted regression coefficients or percentage changes (95% CIs) in semen quality parameters associated with seminal plasma metals/metalloids based on multiple-element models (n ¼ 746). Semen quality parameters Progressive motilitya As <25th 25the50th 50the75th >75th Cd <25th 25the50th 50the75th >75th Total motilityb As <25th 25the50th 50the75th >75th Cd <25th 25the50th 50the75th >75th Concentrationc Zn <25th 25the50th 50the75th >75th a b c

Regression coefficients or percentage changes (95% CIs)

Ptrend

Variance inflation factor

0.00 0.27 (3.8, 3.2) 2.3 (5.8, 1.2) 5.3 (8.9, 1.8)

0.001

1.0

0.00 2.9 (6.4, 0.71) 3.6 (7.2, 0.010) 5.3 (9.6, 2.2)

0.002

1.1

0.00 0.23 (4.2, 3.8) 2.4 (6.4, 1.5) 5.7 (9.8, 1.7)

0.002

1.0

0.00 2.4 (6.4, 1.6) 3.0 (7.1, 1.1) 5.1 (9.3, 0.87)

0.02

1.1

0.00 13% (2.2, 28%) 23% (7.7, 42%) 25% (8.0, 43%)

0.001

e

Adjusted for age, smoking status and daily cigarette consumption. Adjusted for age, BMI, smoking status and daily cigarette consumption. Adjusted for abstinence time.

found a significantly positive association of seminal plasma U with percentage of necrotic spermatozoa in single-element linear models (Table S6). However, none of these four metals were retained in the multiple-element models. 4. Discussion Among our subject population of non-occupationally exposed adults, we observed inverse dose-dependent relationships for seminal plasma As levels with progressive and total sperm motility using multivariable linear models, which were in accordance with the trends for increased ORs for below-reference semen quality parameters in the logistic models. In addition, we found inverse relationships for Cd levels with progressive and total sperm motility, whereas positive relationships for Zn levels with sperm concentration, Cu and As levels with tail %, As and Se levels with tail extent and TDM, and Sn levels with percentage of necrotic spermatozoa. These relationships were maintained when we corrected for multiple comparisons, and they persisted after the simultaneous consideration of various elements. As and Cd are well-documented male reproductive toxicants (Oliveira et al., 2009; Pant et al., 2001). In the present study, seminal plasma As and Cd were associated with reduced sperm motility in a linear dose-dependent manner, which is consistent with our recently published data on urinary metals/metalloids as biomarkers in an overlapping population (Wang et al., 2016b). In support of our findings, Meeker et al. (2008) reported that elevated blood As levels (median: 8.10 mg/L) was associated with an increased risk for low sperm motility among 219 American adults. The inverse association of Cd levels with sperm motility was consistently revealed in several prior studies of occupationally exposed males (n ¼ 149), infertility clinic patients (n ¼ 140) and healthy adults (n ¼ 100) (Benoff et al., 2009; Pant et al., 2003;

Telisman et al., 2000). As and Cd may suppress sperm motility by disrupting the spermatozoa antioxidant system (Asadi et al., 2014; Samikkannu et al., 2003). In addition to oxidative damage, As may immobilize spermatozoa by binding to thiols in epididymis and inhibiting enzymes (sorbitol dehydrogenase, acid phosphatase and 17p-hydroxysteroid dehydrogenase) in testes that are vital for the development of spermatocytes and spermatids (Pant et al., 2004). Zhu et al. (1999) observed decreased cellular adenosine triphosphate levels in several lymphocyte cell lines after exposure to As compounds. We found a positive dose-dependent trend between seminal plasma Zn levels and sperm concentration. The concentration of Zn in the male testis and prostate is much higher than that in other tissues (Ebisch et al., 2006). Human studies showed that oral Zn supplementation improves sperm concentration in subfertile males (Ebisch et al., 2006). There are several mechanisms by which Zn may be beneficial to sperm function. Zn is a cofactor for several hundred metalloenzymes, particularly the enzymes that are responsible for protein synthesis and have the capacity to enhance the quality of seminal fluids and protect sperm against damage (Di Leo et al., 2001). Additionally, the Zn levels in seminal plasma were related to inhibited superoxide anion production and maintenance of sperm chromatin stability (Kjellberg et al., 1992). A decline in Zn levels was linked to hypogonadism, decreased testis volume, atrophy of seminiferous tubules and inadequate development of secondary sexual characteristics (Baltaci et al., 2014). Inconsistent with previous findings from non-occupationally exposed men (Kim et al., 2014; Meeker et al., 2008; Telisman et al., 2007; Wang et al., 2016b), we detected no association between seminal plasma Mo and Pb levels and semen quality parameters. Telisman et al. (2007) reported a positive correlation between percentage of abnormal sperm and blood Pb levels among 240 Croatian men. Meeker et al. (2008) revealed that blood Mo

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Y.-X. Wang et al. / Environmental Pollution xxx (2017) 1e11

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Fig. 2. Regression coefficients or percentage changes (95% CIs) in apoptotic markers associated with seminal plasma metals/metalloids based on single-element linear regression models, adjusting for age, BMI, abstinence duration, liquefaction time, alcohol use, smoking status and daily cigarette consumption (n ¼ 331). aFDR-adjusted p-value for trends. b Transformed by the natural logarithm and back-transformed to obtain the percent change.

levels were inversely associated with sperm concentration and normal morphology among 219 American adults. These previous studies used the concentration of metals in the blood or urine as markers to assess individual-level exposure but not the exposure

status of the male reproductive tract, potentially leading to biased risk estimations. Kim et al. (2014) found that seminal plasma Pb concentrations were associated with lower total motile sperm among 30 American men using in vitro fertilization. Differences in

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Y.-X. Wang et al. / Environmental Pollution xxx (2017) 1e11

Table 3 Adjusted percentage changes (95% CIs) in spermatozoa apoptosis markers (n ¼ 331) and DNA damage measures (n ¼ 401) associated with seminal plasma metals/metalloids based on multiple-element models. Apoptosis or DNA damage measures Necrotic spermatozoaa Zn <25th 25the50th 50the75th >75th Sn <60th 60the80th >80th Tail%b Cu <25th 25the50th 50the75th >75th As <25th 25the50th 50the75th >75th Tail extentc As <25th 25the50th 50the75th >75th Se <25th 25the50th 50the75th >75th TDMc As <25th 25the50th 50the75th >75th Se <25th 25the50th 50the75th >75th a b c

Percentage changes (95% CIs)

Ptrend

Variance inflation factor

0.00 14% (9.2, 41%) 20% (4.3, 48%) 21% (3.1, 51%)

0.09

1.1

0.00 17% (4.1, 42%) 35% (12, 63%)

0.002

1.0

0.00 0.10% (5.9, 6.0%) 2.5% (2.9, 8.2%) 5.8% (0.20, 12%)

0.02

1.0

0.00 3.4% (2.4, 9.3%) 3.8% (1.9, 9.7%) 10% (4.1, 17%)

0.001

1.0

0.01

1.1

0.00 4.0% (2.5, 11%) 2.8% (4.0, 10%) 10% (2.9, 18%)

0.01

1.1

0.00 0.30% (11, 12%) 6.5% (4.8, 19%) 16% (3.6, 31%)

0.004

1.1

0.00 6.0% (5.0, 19%) 3.6% (7.9, 16%) 17% (4.4, 31%)

0.02

1.1

0.00 1.4% (8.2, 5.3%) 6.1% (0.80, 13%) 6.7% (0.50, 15)

Adjusted for abstinence time, smoking status, daily cigarette consumption and education levels. Adjusted for age and abstinence time. Adjusted for abstinence time.

study sample size and exposure levels may also contribute to these discrepancies. The median seminal plasma levels of Pb (0.66 mg/L) in the study of Kim et al. (2014) were 2.5 times higher than that found in the present study (0.26 mg/L). The findings of this study are also inconsistent with our previous results from an overlapping population (Wang et al., 2016b), where urinary Mo and Pb were associated with a decreasing percentage of normal morphology. The reported abstinence time, sperm volume, concentration and total count differed between the men retrained in this study and those excluded from the entire study population of our prior study (Wang et al., 2016b), indicating different composition of study population (e.g., percentage of fertile and infertile men). Our data indicate that exposure to As, Cu and Se can damage sperm DNA integrity. In toxicological studies, As induced DNA damage in human melanocytes, keratinocytes and dendritic cells (Graham-Evans et al., 2004). The essential elements Cu and Se have also been found to induce DNA strand breakage both in vitro and in vivo at high levels (Guecheva et al., 2001; Zhou et al., 2003). As, Cu and Se may act as catalyzers in the initiation of free radical reactions (Guecheva et al., 2001; Pant et al., 2004), which generates

reactive oxygen species that attack DNA. Of interest, we found that seminal plasma Cu and Se levels were not associated with semen quality parameters, which supports previous evidence that DNA integrity measures may be objective and independent markers of spermatozoa function (Han et al., 2011). Few prior human studies investigated the association of metal/metalloids exposure with sperm DNA integrity. In our published data among an overlapping population, no consistent associations were revealed between urinary elements and DNA damage in spermatozoa (Wang et al., 2016c). This probably reflects the fact that As, Cu and Se in seminal plasma are more direct indices for the exposure status of the human reproductive tract than that in urine. In a cross-sectional study of 68 Mexican men, Pb in seminal fluid was related to increased sperm chromatin condensation (Hernandez-Ochoa et al., 2005). The geometric mean for the seminal plasma levels of Pb (2.02 mg/L) in the study of Hernandez-Ochoa et al. (2005) were 10 times higher than the concentrations observed in our current study (0.22 mg/L). However, Bonde et al. (2002) found that blood Pb levels were not related to abnormal sperm chromatin structures in 141 reference workers.

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Y.-X. Wang et al. / Environmental Pollution xxx (2017) 1e11

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Fig. 3. Percentage changes (95% CIs) in DNA damage associated with seminal plasma metals/metalloids based on single-element linear regression models, adjusting for age, BMI, abstinence duration, alcohol use, smoking status and daily cigarette consumption (n ¼ 404). aFDR-adjusted p-value for trends. bTransformed by the natural logarithm and backtransformed to obtain the percent change.

Apoptosis markers are useful indexes of male fertility status (Han et al., 2011). In this study, we found a significant dosedependent relationship between Sn levels and an increasing percentage of necrotic spermatozoa, again contrasting with our

previous study that observed a suggestively positive association between urinary Mn with percentage of apoptotic spermatozoa and a negative association between Fe with percentage of necrotic spermatozoa (Wang et al., 2016c). As discussed for the findings on

Please cite this article in press as: Wang, Y.-X., et al., Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.01.083

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Y.-X. Wang et al. / Environmental Pollution xxx (2017) 1e11

semen quality, the disagreements between studies may be attributable to the variations in sample size, composition of study population and the exposure biomarkers (i.e., metals in seminal plasma vs. urine). In support of our findings, organotin compound exposure induced cell death in various cell types, such as PC12 cells and rodent Leydig cells (Mitra et al., 2014; Nakatsu et al., 2006); furthermore, these effects may be mediated by altered Ca2þ homeostasis and redox imbalance (Mitra et al., 2014; Nakatsu et al., 2006). However, our findings should be cautiously interpreted because the detectable rate of Sn in the seminal plasma was low. The major strength of this study was that we measured various metals/metalloids, both essential and nonessential, in the seminal plasma to estimate the exposure status of the male reproductive tract; in addition, we comprehensively evaluated the associations of these elements with semen quality, sperm apoptosis and DNA integrity. Lastly, we used fresh semen samples to conduct assays of neutral comet and Annexin V/PI without cryopreservation. Nevertheless, several limitations of our study should also be mentioned. First, we recruited participants from a reproductive medicine center. Such a study design may limit the generalization of our findings to the overall population, since there is likely a higher percentage of affected men among couples seeking infertility treatment than the general adults. However, a restriction of our subjects to those with normal sperm motilities, total counts and concentrations has little impact on our findings. Second, we collected single semen sample form each participant. If the elements levels in seminal plasma vary greatly over time due to variable exposure and short elimination half-life, using a single measurement of metals/metalloids in the seminal plasma could probably result in exposure misclassification, potentially attenuating risk estimates. Third, the absence of speciated measurements of some elements in seminal plasma (e.g., As, Cr) can also result in exposure misclassification because inorganic and organic species may vary in their toxicity (Yokel et al., 2006). Finally, temporality of associations cannot be established between exposure and outcome. This study is limited by its cross-sectional design that restricts our ability to establish a causal relationship between metal/metalloids exposures and impaired outcomes. 5. Conclusion Environmental exposure to As, Cd, Cu, Se and Sn may exert detrimental effects on human reproductive health, including impaired semen quality, spermatozoa apoptosis and damaged sperm DNA, whereas Zn exposure may be beneficial to sperm concentration. Considering the widespread human exposure to metals/metalloids, future large-scale prospective epidemiological investigations are urgently required to verify our findings. Competing financial interests None declared. Acknowledgments This study was supported by the National Natural Science Foundation of China (grant number: 81472946); and the Fundamental Research Funds for the Central Universities (HUST: No. 2015QN107). We sincerely thank all the recruited subjects who donated semen samples. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2017.01.083.

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