Semen phthalate metabolites, semen quality parameters and serum reproductive hormones: A cross-sectional study in China

Environmental Pollution 211 (2016) 173e182

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Semen phthalate metabolites, semen quality parameters and serum reproductive hormones: A cross-sectional study in China* Yi-Xin Wang a, b, Qiang Zeng a, b, Yang Sun a, b, Pan Yang a, b, Peng Wang c, Jin Li a, b, Zhen Huang a, b, Ling You a, b, Yue-Hui Huang a, b, Cheng Wang d, Yu-Feng Li d, 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, and 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 Department of Biostatistics, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong, PR China d Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 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 9 September 2015 Received in revised form 5 December 2015 Accepted 23 December 2015 Available online xxx

Exposure to phthalates has been found to have adverse effects on male reproductive function in animals. However, the findings from human studies are inconsistent. Here we examined the associations of phthalate exposure with semen quality and reproductive hormones in a Chinese population using phthalate metabolite concentrations measured in semen as biomarkers. Semen (n ¼ 687) and blood samples (n ¼ 342) were collected from the male partners of sub-fertile couples who presented to the Reproductive Center of Tongji Hospital in Wuhan, China. Semen quality parameters and serum reproductive hormone levels were determined. Semen concentrations of 8 phthalate metabolites were assessed using high-performance liquid chromatography and tandem mass spectrometry. Associations of the semen phthalate metabolites with semen quality parameters and serum reproductive hormones were assessed using confounder-adjusted linear and logistic regression models. Semen phthalate metabolites were significantly associated with decreases in semen volume [mono-n-butyl phthalate (MBP), mono-(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2ethyl-5-oxohexyl) phthalate (MEOHP)], sperm curvilinear velocity [monobenzyl phthalate (MBzP), MEHP, the percentage of di-(2-ethylhexyl)-phthalate metabolites excreted as MEHP (%MEHP)], and straight-line velocity (MBzP, MEHP, %MEHP), and also associated with an increased percentage of abnormal heads and tails (MBzP) (all p for trend <0.05). These associations remained suggestive or significant after adjustment for multiple testing. There were no significant associations between semen phthalate metabolites and serum reproductive hormones. Our findings suggest that environmental exposure to phthalates may impair human semen quality. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Biomarkers Epidemiology Semen phthalates Semen quality Hormones

Abbreviations: BBzP, butyl benzyl phthalate; DBP, di-n-butyl phthalate; DEHP, di-(2-ethylhexyl) phthalate; FAI, free androgen index; FDR, false-discovery rate; FSH, follicle-stimulating hormone; LH, luteinizing hormone; LIN, linearity; MBP, monobutyl phthalate; MBzP, monobenzyl phthalate; MEP, monoethyl phthalate; MEHP, mono(2-ethylhexyl) phthalate; MEHHP, mono(2-ethyl-5-hydroxyhexyl) phthalate; MEOHP, mono(2-ethyl-5-oxohexyl) phthalate; MMP, monomethyl phthalate; MOP, mono-n-octyl phthalate; SHBG, sex hormone-binding globulin; VCL, curvilinear velocity; VSL, straight-line velocity; %MEHP, the percentage of DEHP metabolites excreted as MEHP. * This paper has been recommended for acceptance by Eddy Y. Zeng * 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). http://dx.doi.org/10.1016/j.envpol.2015.12.052 0269-7491/© 2015 Elsevier Ltd. All rights reserved.

1. Introduction Phthalate esters are a family of chemicals whose structure only varies in the length of their ortho-positioned hydrocarbon chain 'arms'. Chain length varies from 1 to >10 carbons, in linear or branched format. Phthalates are used as plasticizers and solvents in a broad spectrum of industrial and commercial applications (ATSDR, 2001, 2002). High levels of phthalates have been detected in drinking water, as well as in the soils and vegetables in agricultural areas of China (Li et al., 2010; Ma et al., 2015; Sui et al., 2014). The general population are readily exposed to phthalates

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through ingestion, inhalation and dermal absorption, of which ingestion is the major source of exposure (Wittassek et al., 2011). Although phthalates are not bio-accumulative chemicals, constant contact results in daily exposures for the general population, and phthalate metabolites are consistently detected in human urine, serum and semen (Frederiksen et al., 2010; Joensen et al., 2012; You et al., 2015). Some authors have reported widespread declines in human semen quality and testosterone levels during the 20th century (Andersson et al., 2007; Carlsen et al., 1992; Jorgensen et al., 2011; Rolland et al., 2013), though the results remain inconclusive (Bonde et al., 2011; Jorgensen et al., 2012). Evidence of widespread exposure to phthalates, as well as toxicological studies demonstrating that certain phthalates [di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBzP) and di(2-ethylhexyl) phthalate (DEHP)] act as reproductive toxicants has led to an increasing concern that the chemicals may have caused a such decline. Animal studies have reported that adult rats exposed to BBzP and DBP cause testicular toxicity (Foster et al., 2001; Nagao et al., 2000). A recent study conducted by Desdoits-Lethimonier et al. (2012) revealed that DEHP and mono-(2-ethylhexyl) phthalate (MEHP) can inhibit testosterone synthesis in cultured adult human testicular tissue, with MEHP concentrations estimated to be of the same order of magnitude as those observed in the urine of men (Meeker et al., 2009; Pan et al., 2006). In humans, a number of studies have examined the associations between phthalate exposure at environmental levels and semen quality parameters and/or reproductive hormones (Axelsson et al., 2015; Bloom et al., 2015; Duty et al., 2005; Han et al., 2014; Hauser et al., 2006; Joensen et al., 2012; Jonsson et al., 2005; Liu et al., 2012; Meeker et al., 2009; Mendiola et al., 2011; Pant et al., 2014), but the results are inconsistent. Previous studies have primarily used measurements of phthalate metabolites in urine as biomarkers to estimate individual-level exposure. Although the urinary concentrations of phthalate metabolites represent an integrative measure of exposure to phthalates from multiple sources and routes, they may not effectively reflect the exposure status of male reproductive organs given that phthalate metabolites are distributed differently in the urine, serum and semen of men (Frederiksen et al., 2010). The concentrations of phthalates in human semen reflect the exposure status of target organ (Pant et al., 2008; You et al., 2015), because the male reproductive system is the main target organ of phthalates, as evidenced by plentiful animal data. For examples, phthalates such as DBP and DEHP have been found to adversely affect male fertility and reproduction in adult rats by altering the activities of testicular enzymes that were associated with the specific events of spermatogenesis (Parmar et al., 1986; Srivastava et al., 1990). A more recent study by Miura et al. (2007) revealed that DEHP-induced aspermatogenesis might be caused by the high sensitivity of the testicular tissues to MEHP rather than the specific accumulation or uptake of circulating MEHP into the testes. In the present study, we assessed 8 phthalate metabolites in semen [monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), MEHP, mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-n-octyl phthalate (MOP)] and examined the associations of semen phthalate metabolites with semen quality parameters and serum reproductive hormones among non-occupationally exposed Chinese men. To our knowledge, this study is the first investigation using semen phthalate metabolite concentrations as biomarkers to assess the associations between phthalate exposure and semen quality and reproductive hormones.

2. Materials and methods 2.1. Study design and participants The study was approved by the Ethics Committee of Tongji Medical College. Our study participants were male partners of subfertile couples who presented to the Reproductive Center of Tongji Hospital in Wuhan, China to seek semen examination without knowledge of their fertility status, as has previously been described (Wang et al., 2015; You et al., 2015). In brief, a total of 1490 men were invited to participate in the study between March and June 2013, of which 1247 (84%) ultimately enrolled. All participants provided written informed consent prior to participation. The participants were asked to provide a semen sample, a venous blood sample and two urine samples (at least 2 h apart), as well as to complete a questionnaire on the day of their clinic visit. The questionnaire assessed information regarding demographic characteristics, life habits, medical history and occupational exposure. Individuals who had consumed alcoholic beverages less than once per week over the past year were defined as non-drinkers and those who had smoked less than 100 cigarettes throughout their lifetime were defined as non-smokers (Wang et al., 2015). After excluding men with self-reported health conditions that might affect male reproductive health (e.g., epididymitis, vasectomy, varicocele, orchiditis, vesiculitis, testis injury and undescended testicle) and occupational exposure to synthetic materials that may have been a source of exposure to phthalates (e.g., polyvinyl chloride, lacquers, dyes, insecticides, synthetic leather and industrial solvents), 1040 participants remained. We also excluded 115 men due to inadequate semen volumes for phthalate metabolite measurements (<1.5 mL). Of the 925 eligible men, we randomly selected 687 for the current analysis. There were no significant differences in demographic characteristics between the subjects included in the present study and the remainder of the study population.

2.2. Semen sample collection and analysis All semen samples were collected from each participant by masturbation into a sterile polypropylene specimen container in a specialized semen collection room close to the semen laboratory. The samples were then kept at 37  C until liquefaction had occurred. After recording the time between ejaculation and semen analysis, semen quality parameters were assessed in accordance with World Health Organization (WHO, 2010) guidelines, as described previously in detail (Wang et al., 2015). In brief, semen volume was measured with a sterile polypropylene pipette. Sperm concentration, progressive motility, non-progressive motility and 3 sperm motion parameters [curvilinear velocity (VCL), straight-line velocity (VSL), and linearity (LIN)] were analyzed according to the WHO guidelines using a micro-cell slide and computer-aided semen analysis. We calculated total sperm count (semen volume  sperm concentration) and total motility (progressive motility þ non-progressive motility) (WHO, 2010). The percentage of sperm with normal morphology and the percentage of sperm with abnormal heads, midsections and tails were evaluated through manual analyses at a high-power magnification (1000) on fixed and stained smears using defined criteria with at least 200 sperm assessed per replicate. All semen analyses were performed by two professional technicians using the same instruments and methods without access to any information regarding the participants. The differences between the quality control results reported by the two technicians were not significant.

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2.3. Blood sample collection and hormone analyses Because diurnal variation of testosterone and other hormones in men has been well documented (Brambilla et al., 2009), blood samples were draw from the participants who presented to the Reproductive Center of Tongji Hospital for semen examination between 08:30 h and 11:30 h to minimize the diurnal variability in serum hormones. Of the 687 men, 342 had a blood sample drawn from their cubital vein. After centrifugation of the blood sample at 1000 rpm/min for 10 min, serum levels of estradiol, folliclestimulating hormone (FSH), luteinizing hormone (LH), sex hormone-binding globulin (SHBG) and total testosterone were measured using the direct chemiluminescence assay with commercial test kits on the day of collection in the Reproductive Center of Tongji Hospital (Zeng et al., 2013). Control samples adding the low and high levels of hormone standards to the serum sample were analyzed each day using the same analysis procedure (recovery range: 83%e108%). The coefficients of variance (inter-day variation) for estradiol, FSH, LH, SHBG and total testosterone were 2.9e9.3%, 2.4e4.9%, 3.2e8.4%, 3.7e4.3% and 5.4e8.2%, respectively. We calculated free androgen index (FAI) as the molar ratio of total testosterone to SHBG, and free testosterone was calculated from total testosterone and SHBG using a fixed albumin level of 44 g/L as suggested by Vermeulen et al. (1999). 2.4. Phthalate metabolite analysis After aliquots were taken for the assessment of semen quality parameters (mean time between ejaculation and semen analysis: 21 ± 21 min), the remaining sample was immediately placed in an ice cooler pack to inhibit the activity of hydrolysis enzyme. The samples were then shipped to the laboratory in an ice cooler and centrifuged at 1500 rpm/min for 5 min at 4  C. The supernatants were stored at 80  C within 1 h of collection until analysis of 8 phthalate metabolites (MMP, MEP, MBP, MBzP, MEHP, MEHHP, MEOHP and MOP) using a method described in detail in our previous study (You et al., 2015). Briefly, once it was thawed, 1-mL semen was vortexed with phosphoric acid to stop hydrolytic enzyme activity. Following a preceding enzymatic deconjugation of the samples, the target compounds were purified using solid-phase extraction cartridges, separated from other semen components by an Agilent 1290 high-performance liquid chromatograph and finally detected using an Agilent 6460 triple quad mass spectrometer. Each batch included one blank sampling tube containing 3-mL deionised water, which was analyzed using the same protocols as described for the semen samples. Analyte concentrations were below the limits of detection (LOD) in all blank samples. We also used a spiked pooled semen sample randomly selected from 100 men of present study as a quality control. The spike recoveries for the metabolites ranged from 82% to 117% and the coefficient of variance (intra-day and inter-day variation) was not higher than 10%. The LODs of the metabolites ranged from 8.0 to 43 ng/L. Values below the LOD were assigned a value of the LOD/√2. 2.5. Statistical analysis We calculated %MEHP as the percentage of total molar concentrations of three DEHP metabolites (MEHP, MEHHP and MEOHP) excreted as MEHP. Descriptive statistics were calculated for the population characteristics and for the distributions of semen quality parameters, serum reproductive hormones and semen phthalate metabolites. Correlations among the phthalate metabolites in semen were assessed using Spearman's Rank Correlation Test. We used multivariate linear regression models to assess the

175

associations between semen phthalate metabolites and continuous measurements of semen quality parameters and reproductive hormones. To achieve variance in homogeneity and normality in the distributions of the residuals, dependent variables were transformed using the natural logarithm for semen volume, sperm concentration, total count, FSH, LH, SHBG, total testosterone, free testosterone and FAI. Progressive and non-progressive sperm motility, sperm morphology parameters, sperm motion parameters and estradiol had close to normal distributions and were thus modeled as untransformed variables. Because MMP, MEP and MBzP were not detectable in a high proportion of semen samples, a threelevel ordinal variable was constructed as follows: the low-level group had concentrations < LOD, and the median- and high-level groups were divided equally among those with detectable concentrations. The levels of MBP, MEHP, MEHHP, MEOHP and %MEHP in semen were divided into tertiles. MOP was detectable in a limited numbers of semen samples (<15%), and thus data for this metabolite was not analyzed further. Tests for trend were conducted for ordinal phthalate metabolite categories in regression models using integer values (1, 2 and 3). Semen levels of phthalate metabolites and %MEHP were also entered into the linear regression models as continuous variables. We also constructed multivariable logistic regression models to assess the associations between semen metabolite categories and conventional sperm quality parameters. The study participants were classified as either being below or at/above the WHO reference levels for their progressive sperm motility (32% motile sperm), total motility (40% motile sperm), concentration (15 million/mL) and total count (39 million) (WHO, 2010). Participants with all four parameters at/above the reference levels were used as the reference group. Between-group differences in continuous or categorical variables (i.e., age, BMI, alcohol use, education level, having ever fathered a pregnancy, abstinence time, income and smoking status) were examined to explore potential confounding factors using parametric or non-parametric methods where appropriate. Covariates included in the multivariable regression models were based on biologic and statistical considerations. Age, body mass index (BMI) and time between ejaculation and semen analysis were considered for inclusion in the models as continuous variables. Education level (less than high school vs. high school and above) and having ever fathered a pregnancy (yes vs. no) were considered as dichotomous variables. Abstinence time (<3, 3, 4, 5 or >5 days), alcohol use (0, 1e3 or >3 times per week) and income (<3000, 3000e6000 or >6000 yuan per month) were examined as ordinal variables. Smoking status (current and former vs. never) was examined as a dummy variable. We used the “change-in-estimate method” to decide which potential confounders should be retained in the final models (Greenland, 1989). The confounders were retained if the effect estimates for semen phthalate metabolites and each outcome varied by more than 10% when they were included in the models. For consistency, models for each set of outcomes were adjusted for the same covariates. The problem with performing multiple simultaneous hypothesis tests is that, as the number of hypotheses increases, so too does the probability of wrongly rejecting a null hypothesis due to random chance. We therefore used the false-discovery rate (FDR) correction to account for multiple testing of the adjusted regression models using the available spreadsheet software developed by Pike (2011). To determine whether the findings are due to having a select clinically based sample, we conducted sensitivity analyses by simultaneously excluding men with progressive sperm motility, total motilities, concentrations, or total counts that were below the WHO reference values. Data analyses were performed using Statistical Analysis Software (SAS) version 9.2 (SAS Institute, Cary, NC). A p-value <0.05 was considered statistically significant, and p-

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values ranging from 0.05 to 0.10 were considered as being suggestive of statistical significance (You et al., 2015).

Table 2 Distribution of semen phthalate metabolites (mg/L) in the participants (n ¼ 687). Measures

% >LOD

Mean

Median

3. Results MMP MEP MBP MBzP MEHP MEHHP MEOHP MOP %MEHPa

3.1. Characteristics of the study population The participants were mostly Han (98%), and they had a mean age of 32 ± 5.5 years. According to the WHO reference values (WHO, 2010), 170 men (25%), 215 men (31%), 62 men (9.0%) and 48 men (6.9%) had progressive sperm motilities, total motilities, concentrations and total counts that were below the reference values, respectively; and 453 men (66%) had all the four semen parameters that were at or above the reference values (Table 1). The percentage of participants who had ever fathered a pregnancy was higher in the comparison than the below-reference group for sperm concentration (p < 0.05). The percentage of drinker (1e3 or >3 times per week) was higher in the comparison than the below-reference group for total sperm motility (p < 0.05). For the other demographic factors, no statistically significant differences were found among the semen quality groups. 3.2. Distribution of metabolite concentrations, semen quality and hormone levels

35 67 99 29 100 100 79 13 e

5.8 2.3 1.2 0.091 2.2 0.25 0.055 0.031 79


Selected percentiles 10%

25%

75%

90%



4.1 1.3 1.3
20.6 4.8 2.0 0.051 2.5 0.40 0.11 0.063 94

a %MEHP (%) ¼ [MEHP (nmol/L)/the sum of MEOHP (nmol/L), MEHHP (nmol/L) and MEHP (nmol/L)]  100.

the participants. MMP, MEP, MBzP and MEOHP were detected in 35%, 67%, 29% and 79% of the subjects, respectively, whereas MOP was detected in only 13% of the participants. The mean semen metabolite levels were the highest for MMP (5.8 ng/mL), followed by MEP (2.3 ng/mL), MEHP (2.2 ng/mL), MBP (1.2 ng/mL), MEHHP (0.25 ng/mL), MBzP (0.091 ng/mL) and MEOHP (0.055 ng/mL); they were lowest for MOP (0.031 ng/mL). Semen quality parameters and reproductive hormone levels of the participants are shown in Table 3.

The distributions of phthalate metabolites in semen are shown in Table 2. MBP, MEHP and MEHHP were detected in nearly 100% of

Table 1 Demographic categories [n (%) or mean ± SD] according to semen parameters (n ¼ 687)a. Category

Comparison subjectsd, n ¼ 455

Age, years 32 ± 5.4 BMI, kg/m2 23 ± 3.2 Ever fathered a pregnancyb Yes 190 (41.9) No 263 (58.1) Race Han 443 (97.4) Other 12 (2.6) Abstinence time, days <3 45 (9.9) 3-5 301 (66.3) >5 108 (23.8) Education level Less than high 177 (39.0) school High school and 277 (61.0) above Smoking status Never-smoker 170 (37.4) Ever-smoker 285 (62.6) Former 53 (11.6) Current 232 (51.0) Alcohol use, times/weekc 0 163 (35.2) 1-3 250 (54.9) >3 42 (9.9) Income, RMB yuan/month 3000 194 (42.7) 3000e6000 177 (39.0) 6000 83 (18.3)

Semen quality parametere Progressive motility <32%, n ¼ 170

Total motility <40%, n ¼ 215

Concentration <1.5  107/mL, n ¼ 62

Total count <3.9  107, n ¼ 48

32 ± 5.2 24 ± 3.1

33 ± 5.6 23 ± 3.0

31 ± 5.3 23 ± 2.8

33 ± 5.5 24 ± 2.8

67 (39.7) 102 (60.3)

83 (38.8) 131 (61.2)

17 (27.4) 45 (72.6)

14 (29.2) 34 (70.8)

166 (97.7) 4 (2.3)

211 (98.1) 4 (1.9)

61 (98.4) 1 (1.6)

47 (97.9) 1 (2.1)

15 (8.8) 103 (60.6) 52 (30.6)

17 (7.9) 137 (63.7) 61 (28.4)

8 (12.9) 40 (64.5) 14 (22.6)

8 (16.7) 30 (62.5) 10 (20.8)

62 (37.1)

78 (36.9)

26 (43.3)

18 (38.3)

105 (62.9)

133 (63.1)

34 (56.7)

29 (61.7)

75 95 18 77

(44.1) (55.9) (10.6) (45.3)

95 (44.2) 120 (55.8) 22 (10.2) 98 (45.6)

24 (38.7) 38 (61.3) 8 (12.9) 30 (48.4)

17 (35.4) 31 (64.6) 6 (12.5) 25 (52.1)

74 (43.5) 84 (49.4) 12 (7.1)

95 (44.2) 104 (48.4) 16 (7.4)

26 (41.9) 31 (50.0) 5 (8.1)

21 (43.7) 24 (50.0) 3 (6.3)

74 (43.5) 73 (42.9) 23 (13.6)

99 (46.1) 91 (42.3) 25 (11.6)

32 (51.6) 24 (38.7) 6 (9.7)

26 (54.2) 17 (35.4) 5 (10.4)

a Two patients were missing information on age, 3 were missing information on the history of a successful pregnancy, 5 patients were missing information on education level, 1 was missing information on abstinence time and 2 were missing information on income. b The percentage of participants who had ever fathered a pregnancy was higher in the comparison than the below-reference group for sperm concentration (p < 0.05). c The percentage of drinker (1e3 or >3 times per week) was higher in the comparison than the below-reference group for total sperm motility (p < 0.05). d The comparison group consists of men whose four semen parameters were all greater than the reference level. e A subject may have contributed to more than one category.

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177

Table 3 Semen quality parameters (n ¼ 687) and reproductive hormone levels (n ¼ 342) in the study population. Arithmetic mean ± SD

Measures Conventional semen quality Volume (mL) Progressive motility (%) Total motility (%) Concentration (106/mL) Total count (106/ejaculate) Semen morphology parameters Abnormal head (%) Abnormal midsection (%) Abnormal tail (%) Normal morphology (%) Sperm motion parameters VCL (mm/s) VSL (mm/s) LIN (%) Hormones Estradiol (pg/mL) FSH (mIU/mL) LH (mIU/mL) SHBG (nmol/L) Total testosterone (ng/dL) Free testosterone (ng/dL) FAI

Median (25th, 75th)

Range 1.5e12 0e84 0.51e94 3.4e218 9.3e828

3.3 43 50 50 154

± ± ± ± ±

1.5 17 20 33 108

3.0 (2.0, 4.0) 41 (32, 57) 49 (37, 66) 43 (26, 66) 129 (79, 198)

67 24 8.4 20

± ± ± ±

9.9 9.6 4.9 8.0

65 (59, 76) 22 (14, 32) 8.0 (4.0, 11) 21 (15, 24)

44 ± 8.2 28 ± 4.9 64 ± 6.9 36 5.1 4.2 34 411 8.3 46

± ± ± ± ± ± ±

11 2.9 1.8 15 147 2.6 18

0e98 0e56 0e36 0e43

45 (39, 50) 28 (25, 31) 63 (59, 68)

15e75 7.7e45 32e85

34 (29, 41) 4.3 (3.2, 6.3) 3.9 (3.0, 5.0) 32 (24, 42) 392 (304, 502) 7.9 (6.6, 9.8) 44 (34, 55)

12e83 0.84e24 0.81e16 6.3e97 78e1004 2.4e17 14e120

Abbreviations: VCL, curvilinear velocity; VSL, straight-line velocity; LIN, linearity; FSH, follicle-stimulating hormone; LH, luteinizing hormone; SHBG, sex hormone-binding globulin; FAI, free androgen index.

3.3. Correlations among the phthalate metabolites in semen The correlations among the phthalate metabolites in semen are shown in Table 4. We found that MBP, MBzP, MEHP, MEHHP and MEOHP were positively and significantly associated with each other (all p < 0.01). MMP was significantly positively associated with MBP and MBzP, but was negatively associated with MEHHP (all p < 0.05). 3.4. Semen phthalate metabolites and semen quality parameters Fig. 1 shows the associations between semen metabolite categories and conventional semen quality parameters, adjusted for age, abstinence duration, time between ejaculation and semen analysis, alcohol use, smoking status and ever having fathered a pregnancy. We found significant dose-response relationships of semen MBP, MEHP, MEHHP and MEOHP with decreasing semen volume (all p for trend <0.05). After adjusting for multiple testing, the associations remained significant for MBP, MEHP and MEOHP (FDR-adjusted p for trend <0.05), but became suggestive for MEHHP (FDR-adjusted p for trend ¼ 0.08). The men in the highest tertiles of MBP, MEHP, MEHHP and MEOHP showed significantly decreased percentages of semen volume of 17% (95% CI: 26, 9.2%), 10% (95% CI: 19, 2.7%), 8.1% (95% CI: 16, 0.70%) and 18% (95% CI: 26, 9.4%), respectively, Table 4 Spearman correlation coefficients among semen levels of phthalate metabolites (n ¼ 687). Measures

MMP

MEP

MBP

MBzP

MEHP

MMP MEP MBP MBzP MEHP MEHHP MEOHP

1.0 0.16** 0.032 0.081* 0.010 0.077* 0.044

1.0 0.060 0.029 0.041 0.033 0.062

1.0 0.12** 0.26** 0.34** 0.44**

1.0 0.16** 0.13** 0.23**

1.0 0.20** 0.31**

*Significant at the 0.05 level (2-tailed). **Significant at the 0.01 level (2-tailed).

MEHHP

MEOHP

compared with those of men in the lowest tertile. We did not find any significant associations of semen phthalate metabolites with progressive sperm motility, total motility, concentration or total count in either the multivariable linear (Fig. 1) or logistic regression models (see Supplemental Material, Fig. S1). The regression coefficients for sperm morphology parameters associated with semen phthalate metabolite categories adjusted for age, alcohol use, smoking status and ever having fathered a pregnancy are shown in Fig. 2. Semen MBzP levels were positively associated with percentages of abnormal heads and tails, but negatively associated with percentages of abnormal midsections (all p for trend <0.05). After adjustment for multiple testing, however, only suggestive associations of MBzP with percentages of abnormal heads and tails remained (FDR-adjusted p for trend ¼ 0.08 and 0.06, respectively). The men in the highest tertile of MBzP showed significant increases in the percentages of abnormal heads and tails of 2.8% (95% CI: 0.65, 5.0%) and 1.7% (95% CI: 0.62, 2.8%), respectively, compared with that of men in the lowest tertile. The regression coefficients for sperm-motion parameters associated with categories of semen phthalate metabolites adjusted for age, abstinence duration, time between ejaculation and semen analysis, alcohol use, smoking status and ever having fathered a pregnancy are shown in Fig. 3. We found significant dose-response relationships for MBzP, MEHP and %MEHP with VCL and VSL (all p for trend <0.05). After adjusting for multiple testing, the inverse associations of MBzP with VCL and VSL, and the associations of MEHP and %MEHP with VCL remained significant (FDR-adjusted p for trend <0.05); however, the association of MEHP and %MEHP with decreasing VSL became weaker and suggestive after FDRadjustment (both FDR-adjusted p for trend ¼ 0.08).

3.5. Semen phthalate metabolites and reproductive hormones 1.0 0.56**

1.0

Few significant dose-response relationships were detected in the regression models of associations between semen phthalate metabolite categories and reproductive hormones, adjusted for age, BMI, alcohol use and smoking status (Fig. 4). Estradiol was

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Fig. 1. Regression coefficients or percentage changes (95% CIs) in the conventional semen quality parameters associated with the metabolite concentrations (n ¼ 687) adjusted for age, abstinence duration, time between ejaculation and semen analysis, alcohol use, smoking status and ever having fathered a pregnancy. aTransformed by the natural logarithm and back-transformed {100[exp(beta)  1] to obtain the percent change. P: p-value for trend; Padj: FDR-adjusted p-value for trend.

positively associated with MMP (3.3 pg/mL higher estradiol in the highest MMP tertile compared with the lowest tertile, 95% CI: 0.24, 6.4 pg/mL; p for trend ¼ 0.03), LH was positively associated with MEP (12% higher LH in the highest MEP tertile compared with the lowest tertile, 95% CI: 0.90, 25%; p for trend ¼ 0.03), and total testosterone was negatively associated with MEOHP (10% lower total testosterone in the highest MEOHP tertile compared with the lowest tertile, 95% CI: 21, 0.60%; p for trend ¼ 0.04). However, these associations were not confirmed after adjusting for multiple testing (all FDR-adjusted p for trend >0.10). We repeated all of the above-mentioned analyses by simultaneously excluding men with abnormal progressive sperm motility, total motilities, concentrations, or total counts, and we found that the results did not change substantially (data not shown). The results of linear regression models were largely unchanged when semen levels of phthalate metabolites and %MEHP were modeled as continuous variables (see Supplemental Material, Tables S1, S2, S3 and S4). 4. Discussion As far as we are aware, this is the first human study to date to explore the associations between phthalate exposure and semen quality parameters and reproductive hormones using phthalate metabolites measured in semen as biomarkers to estimate individuals' exposure status of target reproductive organs. Taken together, we found significant dose-response relationships of

semen phthalate metabolites with decreased semen volume (MBP, MEHP, MEHHP, MEOHP), VCL (MBzP, MEHP, MEHP%) and VSL (MBzP, MEHP, MEHP%), as well as with increased percentages of abnormal heads and tails (MBzP). These associations remained suggestive or significant after adjustment for multiple testing, and they persisted when the analysis was restricted to men with normal conventional sperm quality parameters. It has been well established by animal studies that DBP, BBzP and DEHP act as endocrine disrupters that cause testicular toxicity (Foster et al., 2001; Gray et al., 2000). Toxicological studies of rodent species have reported that adolescence or adult exposure to DBP and DEHP cause reduced weight of accessory sex glands (Agarwal et al., 1986; Higuchi et al., 2003), which may be associated with a reduction in ejaculate volume (Sharpe and Skakkebaek, 2008). The mechanisms whereby sperm motion parameters may be impaired by phthalates, including BBzP and DEHP (Kasahara et al., 2002; Seo et al., 2004), could be related to oxidative stress and the production of reactive oxygen species and subsequent lipid peroxidation of the sperm plasma membrane (Aitken, 1997; Armstrong et al., 1999; Storey, 1997). However, we did not find any significant associations of semen phthalate metabolites with progressive sperm motility and total motility, supporting the hypothesis that sperm motion parameters may be more sensitive measures of reproductive toxicity than the conventional parameters (Perreault and Cancel, 2001). The data regarding a potential mechanism whereby BBzP acts on sperm malformation are scarce. In support of our findings of associations between semen

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Fig. 2. Regression coefficients (95% CIs) for changes in the sperm morphology parameters associated with the metabolite concentrations (n ¼ 687) adjusted for age, alcohol use, smoking status and ever having fathered a pregnancy. P: p-value for trend; Padj: FDR-adjusted p-value for trend.

phthalate metabolites and semen quality parameters, Specht et al. (2014) measured six metabolites of DEHP and diisononyl phthalate (DiNP) in the serum of 589 spouses of pregnant women and found significantly inverse associations between metabolites of DEHP with decreased semen volume; Duty et al. (2004) revealed suggestively dose-response relationships of urinary MEHP and MBzP with decreasing VCL and VSL among 220 men from an infertility clinic. The inverse association of MEHP with VSL was further confirmed in a recent meta-analysis (Cai et al., 2015). Sperm head abnormalities has been found to be related to fertilization failure and elevated degree of sperm DNA fragmentation (Daris et al., 2010). However, no previous studies in humans have examined phthalate exposure and percentages of abnormal heads. But a number of studies in rodent species have, in agreement with our findings, reported adverse effects of perinatal and adult exposure to BBzP on sperm morphology. For example, Ahmad et al. (2014) found significantly increased sperm head shape abnormalities in male offspring after pregnant rats were orally fed BBzP (100 mg/ kg). Tyrkiel et al. (2007) reported a dose-dependent increase in the frequency of abnormal sperm heads in male mice after exposure to 450e1800 mg/kg bw of BBzP. We did not find any significant associations between semen phthalate metabolites and progressive sperm motility, total motility, concentration or total count. Contrary to our findings, a number of previous human studies have reported negative associations between exposure to phthalates and these conventional parameters of semen quality (Axelsson et al., 2015; Hauser et al.,

2006; Jonsson et al., 2005; Pant et al., 2008, 2011, 2014; Specht et al., 2014), and exposure to DEHP, DBP and BBzP are the most consistently reported to be associated with a lower semen quality, according to a recent meta-analysis (Cai et al., 2015). The results of the present study were also inconsistent with our recently published data among an overlapping population of men (Wang et al., 2015), in which urinary MBP levels were associated with decreasing sperm concentration and total count, and MEHP and %MEHP were associated with increasing percentage of abnormal heads. Previous studies have used phthalate metabolites in urine or their prototypes in semen as biomarkers to estimate individual-level exposure. However, in the analysis of the correspondence between urine and semen samples, there were no significant correlations between the two sample types for most phthalate metabolites (Frederiksen et al., 2010). This implies that relying on measurements of phthalate metabolites in urine may not directly represent the real exposure status of target reproductive organs, which may have resulted in exposure measurement error for the target organs. The use of the parent compounds as markers of exposure has been questioned due to the possible contamination of the samples from phthalates in the surrounding environment (Calafat et al., 2013). The discrepancy between studies may also be related to study size, a different extent of exposure and composition of the study populations (e.g., fertile vs. infertile men). The semen volume of men in the present study was all above 1.5 mL, and thus this resulted in the inclusion of more fertile men compared with our previous study (Wang et al., 2015).

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Fig. 3. Regression coefficients (95% CIs) for changes in the sperm motion parameters associated with the metabolite concentrations (n ¼ 687) adjusted for age, abstinence duration, time between ejaculation and semen analysis, alcohol use, smoking status and ever having fathered a pregnancy. P: p-value for trend; Padj: FDR-adjusted p-value for trend. Abbreviations: VCL, curvilinear velocity; VSL, straight-line velocity; LIN, linearity.

We did not find any significant associations between semen levels of individual phthalate metabolites and serum reproductive hormones, which is consistent with a previous study of 881 healthy men (Joensen et al., 2012). However, Joensen et al. (2012) found inverse associations of urinary %MEHP with total testosterone and free testosterone, which were not confirmed in our study population. The results between studies may not be directly comparable because we did not include mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) in the calculation of standard %MEHP. Further, our findings differed from smaller studies by Pan et al. (2006), Meeker et al. (2009) and Mendiola et al. (2011) that found inverse associations of urinary DEHP metabolites with estradiol, total testosterone, free testosterone and FAI; additionally, Duty et al. (2005) reported an inverse association between urinary MBzP and FSH, and Pant et al. (2014) found inverse associations of semen DBP and DEHP with testosterone. As discussed for the observations on sperm quality parameters, the discrepancy between the present study and previous studies may be attributable to the differences in phthalate exposure levels, study size and, perhaps most importantly, the exposure biomarkers (i.e., phthalate metabolites in semen vs. urine samples). Measurements of phthalate metabolites in semen reflect the exposure status of male reproductive system (Pant et al., 2008). The mean semen levels of MEP (2.3 ng/mL), MBP (1.2 ng/mL), MBzP (0.091 ng/mL), MEHP (2.2 ng/mL), MEHHP (0.25 ng/mL) and MEOHP (0.063 ng/mL) in this study are similar to those reported

among Danish men for MEP (1.0 ng/mL), MBP (0.77 ng/mL), MBzP (0.35 ng/mL), MEHHP (0.12 ng/mL) and MEOHP (0.070 ng/mL), but they are slightly higher for MEHP (0.45 ng/mL) (Frederiksen et al., 2010), which may reflect regional variability in DEHP exposure levels. This comparison reinforces the importance of estimating the effects of phthalate exposure on human semen quality among different populations. Our study has several limitations. Most importantly, the men were selected from an infertility clinic. Such a study design allows us to increase the participation rate, but this may have resulted in the inclusion of more sub-fertile men and thereby limited the generalization of our results to the general population. However, restriction to participants with normal sperm quality parameters did not substantially change our findings. Secondly, we only collected a single semen sample from each man. Because phthalates are rapidly metabolized to their monoesters in the human body, a single measurement of phthalate metabolites in semen may provide only a brief snapshot in time of the exposure status of the testicle and thus may not reflect exposure during the etiologically relevant time window for effects on male reproductive function. Finally, as in many of the previous human studies, our study was limited by its cross-sectional design, and thus a causal relationship between phthalate exposure and semen quality cannot be established.

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181

Fig. 4. Regression coefficients or percentage changes (95% CIs) in the reproductive hormones associated with the metabolite concentrations (n ¼ 342) adjusted for age, BMI, alcohol use and smoking status. aTransformed by the natural logarithm and back-transformed {100  [exp(beta) - 1]} to obtain the percent change. P: p-value for trend; Padj: FDR-adjusted p-value for trend. Abbreviations: FSH, follicle-stimulating hormone; LH, luteinizing hormone; SHBG, sex hormone-binding globulin; T, testosterone.

5. Conclusion

Appendix A. Supplementary data

Despite these limitations, measurements of phthalate metabolites in semen reflect the exposure status of target organ. The associations of semen phthalate metabolites with a decrease in semen volume (MBP, MEHP, MEHHP, MEOHP), VCL (MBzP, MEHP, % MEHP) and VSL (MBzP, MEHP, %MEHP), as well as an increased percentage of abnormal heads and tails (MBzP), suggest that exposure to BBzP, DBP and DEHP at environmental levels may impair human semen quality. From the perspective of public health, more longitudinal epidemiological studies are needed to confirm our findings. Studies are also needed to elucidate the potential mechanisms underlying the adverse effects on semen quality of low-level phthalate exposure among humans.

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2015.12.052.

Competing financial interests None declared. Acknowledgments We sincerely thank all the participants of this study for providing the semen and urine samples. We also thank the technicians in the Reproductive Center of Tongji Hospital in Wuhan for analyzing the semen quality parameters. This study was supported by the National Natural Science Foundation of China (grants 81273020).

References Agarwal, D.K., Eustis, S., Lamb, JCt, Reel, J.R., Kluwe, W.M., 1986. Effects of di(2ethylhexyl) phthalate on the gonadal pathophysiology, sperm morphology, and reproductive performance of male rats. Environ. Health Perspect. 65, 343e350. Ahmad, R., Gautam, A.K., Verma, Y., Sedha, S., Kumar, S., 2014. Effects of in utero dibutyl phthalate and butyl benzyl phthalate exposure on offspring development and male reproduction of rat. Environ. Sci. Pollut. Res. 21, 3156e3165. Aitken, R.J., 1997. Molecular mechanisms regulating human sperm function. Mol. Hum. Reprod. 3, 169e173. Andersson, A.M., Jensen, T.K., Juul, A., Petersen, J.H., Jorgensen, T., Skakkebaek, N.E., 2007. Secular decline in male testosterone and sex hormone binding globulin serum levels in danish population surveys. J. Clin. Endocr. Metab. 92, 4696e4705. Armstrong, J.S., Rajasekaran, M., Chamulitrat, W., Gatti, P., Hellstrom, W.J., Sikka, S.C., 1999. Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radic. Biol. Med. 26, 869e880. ATSDR, 2001. Toxicological profile for di-n-butyl phthalate. Available. http:// wwwatsdrcdcgov/toxprofiles/tp135pdf (accessed 06.12.14.). ATSDR, 2002. Toxicological profile for di(2-ethylhexyl)phthalate (dehp). Available. http://wwwatsdrcdcgov/toxprofiles/tp9pdf (accessed 06.12.14.). Axelsson, J., Rylander, L., Rignell-Hydbom, A., Jonsson, B.A., Lindh, C.H., Giwercman, A., 2015. Phthalate exposure and reproductive parameters in young men from the general Swedish population. Environ. Int. 85, 54e60. Bloom, M.S., Whitcomb, B.W., Chen, Z., Ye, A., Kannan, K., Buck Louis, G.M., 2015. Associations between urinary phthalate concentrations and semen quality

182

Y.-X. Wang et al. / Environmental Pollution 211 (2016) 173e182

parameters in a general population. Hum. Reprod. 30, 2645e2657. Bonde, J.P., Ramlau-Hansen, C.H., Olsen, J., 2011. Trends in sperm counts: the saga continues. Epidemiology 22, 617e619. Brambilla, D.J., Matsumoto, A.M., Araujo, A.B., McKinlay, J.B., 2009. The effect of diurnal variation on clinical measurement of serum testosterone and other sex hormone levels in men. J. Clin. Endocr. Metab. 94, 907e913. Cai, H., Zheng, W., Zheng, P., Wang, S., Tan, H., He, G., et al., 2015. Human urinary/ seminal phthalates or their metabolite levels and semen quality: a metaanalysis. Environ. Res. 142, 486e494. Calafat, A.M., Koch, H.M., Swan, S.H., Hauser, R., Goldman, L.R., Lanphear, B.P., et al., 2013. Misuse of blood serum to assess exposure to bisphenol A and phthalates. Breast cancer Res. 15, 403. Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E., 1992. Evidence for decreasing quality of semen during past 50 years. BMJ 305, 609e613. Daris, B., Goropevsek, A., Hojnik, N., Vlaisavljevic, V., 2010. Sperm morphological abnormalities as indicators of DNA fragmentation and fertilization in icsi. Arch. Gynecol. Obstet. 281, 363e367. Desdoits-Lethimonier, C., Albert, O., Le Bizec, B., Perdu, E., Zalko, D., Courant, F., et al., 2012. Human testis steroidogenesis is inhibited by phthalates. Hum. Reprod. 27, 1451e1459. Duty, S.M., Calafat, A.M., Silva, M.J., Brock, J.W., Ryan, L., Chen, Z., et al., 2004. The relationship between environmental exposure to phthalates and computeraided sperm analysis motion parameters. J. Androl. 25, 293e302. Duty, S.M., Calafat, A.M., Silva, M.J., Ryan, L., Hauser, R., 2005. Phthalate exposure and reproductive hormones in adult men. Hum. Reprod. 20, 604e610. Foster, P.M., Mylchreest, E., Gaido, K.W., Sar, M., 2001. Effects of phthalate esters on the developing reproductive tract of male rats. Hum. Reprod. Update 7, 231e235. Frederiksen, H., Jorgensen, N., Andersson, A.M., 2010. Correlations between phthalate metabolites in urine, serum, and seminal plasma from young Danish men determined by isotope dilution liquid chromatography tandem mass spectrometry. J. Anal. Toxicol. 34, 400e410. Gray Jr., L.E., Ostby, J., Furr, J., Price, M., Veeramachaneni, D.N., Parks, L., 2000. Perinatal exposure to the phthalates DEHP, BBP, and DiBP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol. Sci. 58, 350e365. Greenland, S., 1989. Modeling and variable selection in epidemiologic analysis. Am. J. Public Health 79, 340e349. Han, X., Cui, Z., Zhou, N., Ma, M., Li, L., Li, Y., et al., 2014. Urinary phthalate metabolites and male reproductive function parameters in chongqing general population, china. Int. J. Hyg. Environ. Health 217, 271e278. Hauser, R., Meeker, J.D., Duty, S., Silva, M.J., Calafat, A.M., 2006. Altered semen quality in relation to urinary concentrations of phthalate monoester and oxidative metabolites. Epidemiology 17, 682e691. Higuchi, T.T., Palmer, J.S., Gray Jr., L.E., Veeramachaneni, D.N., 2003. Effects of dibutyl phthalate in male rabbits following in utero, adolescent, or postpubertal exposure. Toxicol. Sci. 72, 301e313. Joensen, U.N., Frederiksen, H., Jensen, M.B., Lauritsen, M.P., Olesen, I.A., Lassen, T.H., et al., 2012. Phthalate excretion pattern and testicular function: a study of 881 healthy Danish men. Environ. Health Perspect. 120, 1397e1403. Jonsson, B.A., Richthoff, J., Rylander, L., Giwercman, A., Hagmar, L., 2005. Urinary phthalate metabolites and biomarkers of reproductive function in young men. Epidemiology 16, 487e493. Jorgensen, N., Vierula, M., Jacobsen, R., Pukkala, E., Perheentupa, A., Virtanen, H.E., et al., 2011. Recent adverse trends in semen quality and testis cancer incidence among Finnish men. Int. J. Androl. 34, e37e48. Jorgensen, N., Joensen, U.N., Jensen, T.K., Jensen, M.B., Almstrup, K., Olesen, I.A., et al., 2012. Human semen quality in the new millennium: a prospective crosssectional population-based study of 4867 men. BMJ Open 2, e000990. Kasahara, E., Sato, E.F., Miyoshi, M., Konaka, R., Hiramoto, K., Sasaki, J., et al., 2002. Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl) phthalate. Biochem. J. 365, 849e856. Li, N., Wang, D., Zhou, Y., Ma, M., Li, J., Wang, Z., 2010. Dibutyl phthalate contributes to the thyroid receptor antagonistic activity in drinking water processes. Environ. Sci. Technol. 44, 6863e6868. Liu, L., Bao, H., Liu, F., Zhang, J., Shen, H., 2012. Phthalates exposure of Chinese reproductive age couples and its effect on male semen quality, a primary study. Environ. Int. 42, 78e83. Ma, T.T., Wu, L.H., Chen, L., Zhang, H.B., Teng, Y., Luo, Y.M., 2015. Phthalate esters contamination in soils and vegetables of plastic film greenhouses of suburb nanjing, china and the potential human health risk. Environ. Sci. Pollut. Res. Int. 22, 12018e12028. Meeker, J.D., Calafat, A.M., Hauser, R., 2009. Urinary metabolites of di(2-ethylhexyl)

phthalate are associated with decreased steroid hormone levels in adult men. J. Androl. 30, 287e297. Mendiola, J., Jorgensen, N., Andersson, A.M., Calafat, A.M., Silva, M.J., Redmon, J.B., et al., 2011. Associations between urinary metabolites of di(2-ethylhexyl) phthalate and reproductive hormones in fertile men. Int. J. Androl. 34, 369e378. Miura, Y., Naito, M., Ablake, M., Terayama, H., Yi, S.Q., Qu, N., et al., 2007. Short-term effects of di-(2-ethylhexyl) phthalate on testes, liver, kidneys and pancreas in mice. Asian J. Androl. 9, 199e205. Nagao, T., Ohta, R., Marumo, H., Shindo, T., Yoshimura, S., Ono, H., 2000. Effect of butyl benzyl phthalate in sprague-dawley rats after gavage administration: a two-generation reproductive study. Reprod. Toxicol. 14, 513e532. Pan, G., Hanaoka, T., Yoshimura, M., Zhang, S., Wang, P., Tsukino, H., et al., 2006. Decreased serum free testosterone in workers exposed to high levels of di-nbutyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP): a cross-sectional study in china. Environ. Health Perspect. 114, 1643e1648. Pant, N., Shukla, M., Kumar Patel, D., Shukla, Y., Mathur, N., Kumar Gupta, Y., et al., 2008. Correlation of phthalate exposures with semen quality. Toxicol. Appl. Pharm. 231, 112e116. Pant, N., Pant, A., Shukla, M., Mathur, N., Gupta, Y., Saxena, D., 2011. Environmental and experimental exposure of phthalate esters: the toxicological consequence on human sperm. Hum. Exp. Toxicol. 30, 507e514. Pant, N., Kumar, G., Upadhyay, A.D., Patel, D.K., Gupta, Y.K., Chaturvedi, P.K., 2014. Reproductive toxicity of lead, cadmium, and phthalate exposure in men. Environ. Sci. Pollut. Res. Int. 21, 11066e11074. Parmar, D., Srivastava, S.P., Seth, P.K., 1986. Effect of di(2-ethylhexyl)phthalate (dehp) on spermatogenesis in adult rats. Toxicology 42, 47e55. Perreault, S.D., Cancel, A.M., 2001. Significance of incorporating measures of sperm production and function into rat toxicology studies. Reproduction 121, 207e216. Pike, N., 2011. Using false discovery rates for multiple comparisons in ecology and evolution. Methods Ecol. Evol. 2, 278e282. Rolland, M., Le Moal, J., Wagner, V., Royere, D., De Mouzon, J., 2013. Decline in semen concentration and morphology in a sample of 26,609 men close to general population between 1989 and 2005 in France. Hum. Reprod. 28, 462e470. Seo, K.W., Kim, K.B., Kim, Y.J., Choi, J.Y., Lee, K.T., Choi, K.S., 2004. Comparison of oxidative stress and changes of xenobiotic metabolizing enzymes induced by phthalates in rats. Food Chem. Toxicol. 42, 107e114. Sharpe, R.M., Skakkebaek, N.E., 2008. Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. Fertil. Steril. 89, e33e38. Specht, I.O., Toft, G., Hougaard, K.S., Lindh, C.H., Lenters, V., Jonsson, B.A., et al., 2014. Associations between serum phthalates and biomarkers of reproductive function in 589 adult men. Environ. Int. 66, 146e156. Srivastava, S., Singh, G.B., Srivastava, S.P., Seth, P.K., 1990. Testicular toxicity of di-nbutyl phthalate in adult rats: effect on marker enzymes of spermatogenesis. Indian J. Exp. Biol. 28, 67e70. Storey, B.T., 1997. Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol. Hum. Reprod. 3, 203e213. Sui, H.X., Zhang, L., Wu, P.G., Song, Y., Yong, L., Yang, D.J., et al., 2014. Concentration of di(2-ethylhexyl) phthalate (dehp) in foods and its dietary exposure in china. Int. J. Hyg. Environ. Health 217, 695e701. Tyrkiel, E.J., Dobrzynska, M.M., Derezinska, E., Ludwicki, J.K., 2007. Effects of subchronic exposure of laboratory mice to benzylbutyl phthalate (bbp) on the quantity and quality of male germ cells. Rocz. Panstw. Zakl. Hig. 58, 677e686. Vermeulen, A., Verdonck, L., Kaufman, J.M., 1999. A critical evaluation of simple methods for the estimation of free testosterone in serum. J. Clin. Endocr. Metab. 84, 3666e3672. Wang, Y.X., You, L., Zeng, Q., Sun, Y., Huang, Y.H., Wang, C., et al., 2015. Phthalate exposure and human semen quality: results from an infertility clinic in china. Environ. Res. 142, 1e9. WHO, 2010. Who Laboratory Manual for the Examination and Processing of Human Semen, fifth ed. Available. http://whqlibdoc.Who.Int/publications/2010/ 9789241547789_eng.Pdf (accessed 06.12.14.). Wittassek, M., Koch, H.M., Angerer, J., Bruning, T., 2011. Assessing exposure to phthalates - the human biomonitoring approach. Mol. Nutr. Food Res. 55, 7e31. You, L., Wang, Y.X., Zeng, Q., Li, M., Huang, Y.H., Hu, Y., et al., 2015. Semen phthalate metabolites, spermatozoa apoptosis, and DNA damage: a cross-sectional study in china. Environ. Sci. Technol. 49, 3805e3812. Zeng, Q., Zhou, B., Feng, W., Wang, Y.X., Liu, A.L., Yue, J., et al., 2013. Associations of urinary metal concentrations and circulating testosterone in chinese men. Reprod. Toxicol. 41, 109e114.