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Journal of Hazardous Materials 250– 251 (2013) 115– 121

Contents lists available at SciVerse ScienceDirect

Journal of Hazardous Materials

jou rn al h om epage: www.elsev ier .com/ loc ate / jhazmat

ssociation of exposure to phenols and idiopathic male infertility

injian Chena,b,1, Rong Tanga,b,1, Guangbo Fuc,1, Bin Xua,b, Pengfei Zhud, Shanlei Qiaoa,b,iaojiao Chena,b, Bo Xua,b, Yufeng Qina,b, Chuncheng Lua,b, Bo Hange, Yankai Xiaa,b,∗, Xinru Wanga,b,∗

State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029, ChinaKey Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029, ChinaHuai’an First Affiliated Hospital of Nanjing Medical University, Huai’an 223300, ChinaWuxi Centers for Disease Control and Prevention, Wuxi 214023, ChinaLife Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, United States

i g h l i g h t s

We examined relations between exposure to phenols and idiopathic male infertility.We determined urinary concentrations of various phenols.4-t-OP, 4-n-OP and 4-n-NP exposure increased risk of idiopathic male infertility.4-t-OP and 4-n-NP exposure increased risk of infertility with abnormal semen quality.We observed associations between sum APs exposure and idiopathic male infertility.

r t i c l e i n f o

rticle history:eceived 17 September 2012eceived in revised form 22 January 2013ccepted 25 January 2013vailable online 4 February 2013

eywords:lkylphenols

a b s t r a c t

Widespread human exposure to phenols has been documented recently, and some phenols which arepotential endocrine disruptors have demonstrated adverse effects on male reproduction in animal andin vitro studies. However, implications about exposure to phenols and male infertility are scarce inhumans. Case–control study of 877 idiopathic infertile men and 713 fertile controls was conducted. Uri-nary levels of bisphenol A, benzophenone-3, pentachlorophenol, triclosan, 4-tert-octylphenol (4-t-OP),4-n-octylphenol (4-n-OP) and 4-n-nonylphenol (4-n-NP) and semen parameters were measured. Aftermultivariate adjustment, we found 4-t-OP, 4-n-OP and 4-n-NP exposure was associated with idiopathic

ctylphenolonylphenolale infertility

emen parameter

male infertility (p-value for trend: <0.0001, 0.014 and 0.001, respectively). Aside from these associations,4-t-OP and 4-n-NP exposure was also associated with idiopathic male infertility with abnormal semenparameters. Moreover, we observed significant associations between sum alkylphenols (APs) exposureand idiopathic male infertility. There were no relationships between exposure to other phenols and idio-pathic male infertility in the present study. Our study provides the first evidence that exposure to APs(4-t-OP, 4-n-OP and 4-n-NP) is associated with idiopathic male infertility.

Abbreviations: BPA, bisphenol A; BP-3, benzophenone-3; APs, alkylphenols;-t-OP, 4-tert-octylphenol; 4-n-OP, 4-n-octylphenol; 4-n-NP, 4-n-nonylphenol;PEs, alkylphenol polyoxyethylene ethers; TCS, triclosan; PCP, pentachlorophe-ol; UPLC–MS/MS, ultra high performance liquid chromatography–tandem masspectrometry; LODs, limits of detection; CR, creatinine; WHO, World Health Orga-ization; BMI, body mass index; ORs, odds ratios; CI, confidence interval.∗ Corresponding authors at: State Key Laboratory of Reproductive Medicine,

nstitute of Toxicology, Nanjing Medical University, 140 Hanzhong Road, Nanjing10029, China. Tel.: +86 25 86862863; fax: +86 25 86662863.

E-mail addresses: yankaixia@njmu.edu.cn (Y. Xia), xrwang@njmu.edu.cnX. Wang).

1 These authors contributed equally to this work.

304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jhazmat.2013.01.061

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

The use of phenolic compounds is common worldwide. Bisphe-nol A (BPA) is widely used in the plastic industry and as adental sealant [1,2]. Benzophenone-3 (BP-3) is a common sun-screen agent, which is used in cosmetics and plastic surfacecoatings [3]. Alkylphenols (APs) such as 4-tert-octylphenol (4-t-OP), 4-n-octylphenol (4-n-OP) and 4-n-nonylphenol (4-n-NP) arethe major degradation products of alkylphenol polyoxyethyleneethers (APEs) which are used as non-ionic surfactants [1]. Triclosan(TCS) and pentachlorophenol (PCP) both possess antiseptic func-

tion. PCP has been used in wood preservations [4], and TCS hasbeen widely added to soaps, wound disinfections and toothpastes[5]. Phenols are mainly converted to glucuronide and sulfate conju-gate and excreted in urine in humans [6]. In the US population, these

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16 M. Chen et al. / Journal of Hazardous

henols are all detectable in human urine [7–11]. The determina-ion of urinary concentrations of phenols has been considered toe quite useful for the estimation of exposure to phenols [6], andas also been widely used in examining the relationships betweenhenol exposure and human diseases as well as laboratory abnor-alities [5,12,13].There is a heightened awareness of the potential role of environ-

ental factors in idiopathic male infertility [14]. Many compoundsntroduced into the environment by human activity are knowno impact the endocrine system of animals and humans. Someesearchers found that compounds with endocrine disruptingffects were associated with idiopathic male infertility and semenarameters [15–18]. Most chemicals with estrogenic activity char-cteristically possess a phenolic structure [19]. BPA, BP-3, APs, PCPnd TCS are all potential endocrine disruptors [1–3,5,20–22], andherefore their effect on male infertility needs particular attention.he results of animal and in vitro studies have suggested that expo-ure to phenols may impact male reproductive function [23–31].n recent years, epidemiological studies regarding this issue aremerging, but these studies have only examined the relationshipetween urinary BPA concentrations and male reproductive func-ion based on a small sample size [32,33]. As various phenols arexistent in day-to-day situation, there is a need to assess the rela-ionship between male reproductive function and exposure to aide range of phenolic compounds in a large sample of subjects.

o the best of our knowledge, although exposure to various phe-ols and its potential relation to male infertility is an important

ssue on the research agenda, so far, human data in this area aretill greatly limited.

Therefore, in this study, to investigate whether phenol exposures related to idiopathic male infertility in the general population, weetermined urinary phenol levels and conducted a case–controltudy including 877 idiopathic infertile men and 713 fertile con-rols. To gain broad insight into phenol exposure and idiopathic

ale infertility, we included BPA, BP-3, 4-t-OP, 4-n-OP, 4-n-NP,CP and TCS for analysis. The chemical structures are shown inig. S1 (Supplementary material).

. Materials and methods

.1. Study population

Study subjects were volunteers consecutively recruited fromffiliated hospitals of Nanjing Medical University between March005 and April 2010 (NJMU Infertility Study). The protocol andonsent form were approved by the Institutional Review Boardf Nanjing Medical University prior to the study. All activitiesnvolving human subjects were done under full compliance withovernment policies and the Helsinki Declaration. The participa-ion rates of cases and controls were 91.8% and 90.1%, respectively.ll participants provided informed consent. A questionnaire wassed to collect information including personal background, lifestyleactors, occupational and environmental exposures, genetic riskactors, sexual and reproduction status, medical history and physi-al activity. Cases included in this study were infertile patients withefinite idiopathic male infertility [34]. They were the male part-ers of couples who attended affiliated hospitals of NJMU becauseouples attempted conception for at least 12 months without suc-ess and no fertility problem were found in female partners, andhe men had undergone complete historical and physical exami-ations. 151 men with known causes of male infertility such as a

istory of orchitis, cryptorchidism, varicocele, obstruction of vaseferens and Y chromosome microdeletions were excluded fromhe study [16,17,34,35]. Controls in the present study were fertile

en recruited and sampled at the same period as the cases. They

rials 250– 251 (2013) 115– 121

were from the early pregnancy registry of the same hospitals whowere in the third month following a successful pregnancy. Theywere healthy men with normal reproductive function and con-firmed having healthy babies 6–8 months later [16]. All participantsfor final analysis, including 877 eligible cases and 713 eligible con-trols among whom urine sample was available, claimed that theirlife styles and environments had not changed for several monthsleading up to sample collection. All subjects were asked to firstclean their hands and then collected their urine in glass bottlesin the morning and the urine samples were frozen at −20 ◦C untilanalysis.

2.2. Measurement of urinary phenols

We measured total (free and conjugated) urinary concen-trations of phenols with a sensitive method as previouslydescribed [36]. Briefly, urine samples were incubated in 1 Mammonium acetate buffer solution (pH = 5.0) for hydrolyza-tion with �-glucuronidase/sulfatase (20,000 units/ml) overnight.�-glucuronidase/sulfatase type H-1 from Helix pomatia were pur-chased from Sigma–Aldrich (St. Louis, MO). After hydrolysis, thephenols were extracted and preconcentrated with solid phaseextraction (SPE) (500 mg/3 ml, Supelclean, USA), and determinedwith ultra high performance liquid chromatography (Waters,USA)–electrospray ionization tandem mass spectrometry (Waters,USA). The detection was conducted in the negative ion modeby multiple reaction monitoring. The limits of detection (LODs)were 0.36 ng ml−1 (BPA), 0.04 ng ml−1 (BP-3), 0.41 ng ml−1 (PCP),0.9 ng ml−1 (TCS), 0.34 ng ml−1 (4-t-OP), 0.02 ng ml−1 (4-n-OP) and0.02 ng ml−1 (4-n-NP). The sensitivity of this method was compara-ble to previous methods [9,11]. The intra- and inter-day precisionsfor these compounds were between 2% and 38% and the recov-eries were between 93% and 138% at spiked concentration of 2,20 ng ml−1. Blank controls (urine blank and reagent blank) andquality controls were analyzed in parallel with unknown samplesin each analytical batch. Urinary creatinine (CR) concentrationswere analyzed for correcting the variations of phenol concentra-tions caused by fluctuated urine concentration and dilution. CRdata were collected using an automated chemistry analyzer (7020Hitachi, Tokyo, Japan).

2.3. Semen analysis

Measurement of the semen parameters has been described pre-viously [16,17]. Briefly, the semen samples were collected in themorning on the same day of urine collection, which were obtainedin a private room by masturbation into a sterile wide-mouthand metal-free glass container after a recommended 2-day sexualabstinence. After liquefaction at 37 ◦C for 30 min, semen analysisincluding semen volume, sperm concentration and sperm numberper ejaculate was conducted by using the computer-aided semenanalysis (CASA, WLJY 9000, Weili New Century Science and TechDev.). Strict quality control measures were enforced throughoutthe whole study.

2.4. Statistical analysis

We performed data analysis using Stata statistical package (Ver-sion 9.2, Stata Corp, LP). For the comparisons of age and BMI bycase–control group, we used Welch’s t test. For the comparisonsof smoking and drinking status by case–control group, chi-squaretest was employed. Differences in semen volume, sperm number

per ejaculate and sperm concentration between cases and controlswere tested using Mann–Whitney test. According to the previ-ously reported method [37], semen parameters were dichotomizedbased on the WHO reference values (fifth edition) for semen

M. Chen et al. / Journal of Hazardous Materials 250– 251 (2013) 115– 121 117

Table 1Population characteristics among cases and controls (n = 1590).

Characteristic Control (n = 713) Case (n = 877)

Age (years, mean ± SD) 29.83 ± 3.86 28.50 ± 4.35*

BMI 23.87 ± 2.96 23.47 ± 3.34*

Smoking status [no. (%)]Never 380 (53.3) 461 (52.6)Ever 333 (46.7) 416 (47.4)

Drinking status [no. (%)]Never 381 (53.4) 458 (52.2)Ever 332 (46.6) 419 (47.8)

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Table 2Distribution of urinary concentrations of phenols (ng ml−1).a

Detectablerate (%)

Geometricmean

50th 75th 90th 95th

BPAControl 62.4 0.621 0.522 1.244 3.380 9.190Case 62.1 0.612 0.492 1.187 4.727 8.225

BP-3Control 56.0 0.070 0.056 0.184 0.443 1.028Case 53.2 0.070 0.052 0.188 0.471 0.898

PCPControl 27.1 b <LOD 0.464 1.423 2.011Case 27.9 b <LOD 0.464 1.133 2.120

TCSControl 47.8 1.593 <LOD 3.960 31.727 57.466Case 48.0 1.707 <LOD 5.014 32.219 65.916

4-t-OPControl 12.2 b <LOD <LOD 0.395 0.578Case 21.4 b <LOD <LOD 0.634 0.912

4-n-OPControl 28.2 b <LOD 0.023 0.047 0.066Case 34.2 b <LOD 0.029 0.057 0.079

4-n-NPControl 30.3 b <LOD 0.025 0.064 0.101Case 38.0 b <LOD 0.032 0.079 0.129

a No. (control) = 713; no. (case) = 877. The LODs are 0.36 ng ml−1 (BPA),0.04 ng ml−1 (BP-3), 0.41 ng ml−1 (PCP), 0.9 ng ml−1 (TCS), 0.34 ng ml−1 (4-t-OP),0.02 ng ml−1 (4-n-OP) and 0.02 ng ml−1 (4-n-NP). Samples with concentrations <LOD

* p < 0.05 for Welch’s t-test or two-sided chi-squared test for selected character-stics’ distributions between control and case groups.

olume (≥1.5 ml), total sperm number per ejaculate (≥39 × 106)nd sperm concentration (≥15 × 106 ml−1).

To analyze the associations between phenol exposure levelsnd idiopathic male infertility, multivariate logistic regressionas performed. Given the detectable rate and data characters,

ccording to former report [38], a three-level ordinal variable forhenol exposure was formed for statistical analysis: all samplesith concentrations <LOD were assigned to the lowest group,

nd two equally sized groups were formed among the samplesith detectable concentrations to generate the median- and high-

xposure groups. We included urinary CR as a continuous variableo adjust for urinary dilution [38]. In order to validate the robust-ess of the results, the sensitivity analyses were also conducted inhich the chemical values <LOD were imputed by a value equal to

ne half the LOD and subsequently chemical concentrations wereategorized into tertiles based on CR-adjusted values [16,37]. Inclu-ion of covariates in the multivariate logistic regression models wasased on statistical and biological considerations. We consideredge, body mass index (BMI), drinking status and smoking statuss potential covariates. Age and BMI were modeled as continuousndependent variables. Covariates were included if they were asso-iated with the outcomes at p ≤ 0.1 in separate bivariate models orf the coefficients changed by >10% upon removing the covariate.n order to simply the statistical analyses and the interpretation ofhe data, the set of covariates that met the above criteria for the

ajority of the outcomes was used in all models [39]. We includedhe set of covariates in all models as follows: age, age squared, BMI,MI squared. Tests for trend were performed for ordinal phenolsategories in the adjusted regression models with integer values0, 1, 2) [38]. We considered p < 0.05 statistically significant.

. Results

All of the 1590 participants were ethnic Han-Chinese, includ-ng 877 cases and 713 controls. As shown in Table 1, there wereignificant differences in age and BMI between cases and controls,hile no significant differences were identified between cases and

ontrols with smoking status and alcohol consumption.Distributions of urinary concentrations of BPA, BP-3, PCP, TCS

nd APs in the 1590 participants are presented in Table 2. Amonghese phenols, BPA, BP-3 and TCS exhibited relatively higheretectable rates. TCS showed highest urinary levels, followed byPA, PCP, BP-3 and APs. Among APs, 4-t-OP showed relativelyigher exposure levels than 4-n-OP and 4-n-NP.

Crude and adjusted odds ratios (ORs) for the relationshipetween phenol exposure levels and idiopathic male infertilityre presented in Fig. 1 and Table S1 (Supplementary material).he crude and adjusted results were consistent. Compared with

en in the lowest exposure group, men in both the median and

igh 4-t-OP and 4-n-NP exposure groups were more likely to suf-er from idiopathic male infertility {for 4-t-OP: adjusted ORs forncreasing exposure levels = 1.0, 1.47 [95% confidence interval (CI),

were given a value equal to LOD/2 for the statistical calculations.b Not calculated: proportion of results below LODs was too high to provide a valid

result.

1.01–2.13], 2.72 (95% CI, 1.81–4.09), p-value for trend <0.0001; for4-n-NP: adjusted ORs for increasing exposure levels = 1.0, 1.37 (95%CI, 1.03–1.82), 1.54 (95% CI, 1.17–2.03), p-value for trend = 0.001}.Similarly, men in the high 4-n-OP exposure group were more likelyto suffer from idiopathic male infertility [adjusted ORs for increas-ing exposure levels = 1.0, 1.30 (95% CI, 0.97–1.72), 1.37 (95% CI,1.02–1.82), p-value for trend = 0.014]. However, we did not find sig-nificant association between BPA, BP-3, PCP and TCS exposure andidiopathic male infertility.

The total sperm number per ejaculate and sperm concen-tration in the control group (median, 196.5 × 106; median,54.8 × 106 ml−1) were significantly higher than those in the casegroup (median, 102.0 × 106; median, 36.4 × 106 ml−1) (p < 0.05). Nosignificant difference was found in semen volume between cases(median, 3 ml) and controls (median, 3 ml) (p > 0.05). Moreover, wefound that semen parameters including semen volume, total spermnumber per ejaculate and sperm concentration obtained from thecontrol group were almost all (>95%) above or equal to WHO ref-erence values (fifth edition). In order to further identify possiblefactors contributing to the relationship between phenol exposureand idiopathic male infertility, the case group was stratified intothree subgroups in which the men possessed semen parametersbelow WHO reference values (Table 3). The crude and adjustedresults were nearly identical. 4-t-OP and 4-n-NP exposure was asso-ciated with idiopathic male infertility with below-reference spermconcentration and sperm number per ejaculate (p-value for trend<0.01). No relationships were observed between exposure to otherphenols and semen parameters based subgroups of idiopathic maleinfertility.

Based on the significant link between co-existing environmentalAPs (4-t-OP, 4-n-OP and 4-n-NP) and idiopathic male infertility, wecombined these APs and further explored the relationship betweenmixed exposure to these APs and idiopathic male infertility (Fig. 1and Tables S2 and S3, Supplementary material). Results obtained

from crude models were similar to those obtained from adjustedmodels. We found that, sum linear chain APs (the sum of 4-n-OP and 4-n-NP), sum OPs (the sum of 4-t-OP and 4-n-OP) andsum APs (the sum of 4-t-OP, 4-n-OP and 4-n-NP) exposure was

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aterials 250– 251 (2013) 115– 121

Table 3Crude and adjusted ORs (95% CI) for idiopathic male infertility with abnormal semen parameters by exposure levels of phenols.

Control(n = 713)

Idiopathic male infertility with abnormal semen parameters

Semen volume (<1.5 ml; n = 43) Sperm concentration (<15 × 106 ml−1; n = 170) Total sperm number per ejaculate (<39 × 106; n = 182)

No.a OR (95% CI) No.a OR (95% CI) No.a OR (95% CI)

Crude Adjustedb Crude Adjustedb Crude Adjustedb

BPA Low 268 17 1.0 (referent) 1.0 (referent) 58 1.0 (referent) 1.0 (referent) 63 1.0 (referent) 1.0 (referent)Median 216 11 0.80 (0.37–1.75) 0.85 (0.38–1.88) 45 0.96 (0.63–1.48) 0.94 (0.61–1.46) 49 0.97 (0.64–1.46) 0.95 (0.62–1.46)High 229 15 1.03 (0.50–2.11) 1.03 (0.49–2.15) 67 1.35 (0.91–2.00) 1.37 (0.91–2.07) 70 1.30 (0.89–1.91) 1.36 (0.91–2.03)p-Value for trend 0.950 0.957 0.133 0.136 0.181 0.144

BP-3 Low 314 24 1.0 (referent) 1.0 (referent) 78 1.0 (referent) 1.0 (referent) 86 1.0 (referent) 1.0 (referent)Median 205 9 0.57 (0.26–1.26) 0.60 (0.26–1.36) 43 0.84 (0.56–1.27) 0.82 (0.53–1.25) 45 0.80 (0.54–1.20) 0.78 (0.51–1.18)High 194 10 0.67 (0.32–1.44) 0.81 (0.37–1.79) 49 1.02 (0.68–1.52) 1.05 (0.69–1.59) 51 0.96 (0.65–1.42) 0.99 (0.66–1.48)p-Value for trend 0.229 0.472 0.974 0.917 0.728 0.825

PCP Low 520 27 1.0 (referent) 1.0 (referent) 126 1.0 (referent) 1.0 (referent) 136 1.0 (referent) 1.0 (referent)Median 86 12 2.69 (1.31–5.51)* 3.02 (1.43–6.41)* 26 1.25 (0.77–2.02) 1.36 (0.83–2.24) 27 1.20 (0.75–1.92) 1.33 (0.81–2.17)High 107 4 0.72 (0.25–2.10) 0.79 (0.26–2.36) 18 0.69 (0.41–1.19) 0.69 (0.40–1.20) 19 0.68 (0.40–1.15) 0.70 (0.41–1.19)p-Value for trend 0.700 0.555 0.365 0.424 0.290 0.406

TCS Low 372 25 1.0 (referent) 1.0 (referent) 90 1.0 (referent) 1.0 (referent) 103 1.0 (referent) 1.0 (referent)Median 183 7 0.57 (0.24–1.34) 0.59 (0.24–1.43) 37 0.84 (0.55–1.27) 0.84 (0.55–1.29) 36 0.71 (0.47–1.08) 0.71 (0.46–1.09)High 158 11 1.04 (0.50–2.16) 1.27 (0.59–2.70) 43 1.12 (0.75–1.69) 1.22 (0.80–1.86) 43 0.98 (0.66–1.47) 1.06 (0.70–1.60)p-Value for trend 0.842 0.776 0.734 0.500 0.662 0.904

4-t-OP Low 626 33 1.0 (referent) 1.0 (referent) 136 1.0 (referent) 1.0 (referent) 146 1.0 (referent) 1.0 (referent)Median 52 5 1.82 (0.68–4.87) 1.75 (0.62–4.91) 12 1.06 (0.55–2.04) 1.08 (0.55–2.12) 14 1.15 (0.62–2.14) 1.14 (0.60–2.16)High 35 5 2.71 (1.00–7.37) 2.52 (0.85–7.43) 22 2.89 (1.65–5.09)* 2.76 (1.52–5.02)* 22 2.70 (1.54–4.73)* 2.45 (1.35–4.45)*

p-Value for trend 0.029 0.061 0.001 0.002 0.001 0.006

4-n-OP Low 512 31 1.0 (referent) 1.0 (referent) 114 1.0 (referent) 1.0 (referent) 125 1.0 (referent) 1.0 (referent)Median 104 6 0.95 (0.39–2.34) 0.96 (0.38–2.42) 24 1.04 (0.64–1.69) 0.99 (0.60–1.64) 25 0.98 (0.61–1.59) 0.96 (0.59–1.58)High 97 6 1.02 (0.42–2.51) 1.17 (0.47–2.95) 32 1.48 (0.95–2.32) 1.49 (0.94–2.37) 32 1.35 (0.87–2.11) 1.35 (0.85–2.14)p-Value for trend 0.995 0.788 0.112 0.128 0.242 0.272

4-n-NP Low 497 30 1.0 (referent) 1.0 (referent) 96 1.0 (referent) 1.0 (referent) 109 1.0 (referent) 1.0 (referent)Median 106 4 0.63 (0.22–1.81) 0.69 (0.24–2.05) 29 1.42 (0.89–2.26) 1.51 (0.94–2.44) 28 1.20 (0.76–1.92) 1.28 (0.79–2.07)High 110 9 1.36 (0.63–2.94) 1.49 (0.67–3.29) 45 2.12 (1.41–3.19)* 2.23 (1.46–3.40)* 45 1.87 (1.25–2.79)* 1.97 (1.29–2.98)*

p-Value for trend 0.645 0.469 0.0003 0.0001 0.003 0.002

a The semen parameter categories were not mutually exclusive; a man could contribute data to more than one category.b ORs adjusted for age, BMI and CR.* p < 0.05.

M. Chen et al. / Journal of Hazardous Materials 250– 251 (2013) 115– 121 119

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ig. 1. Crude (hollow circles) and adjusted (solid circles) ORs (95% CIs) for idiopathnd sum APs (the sum of 4-t-OP, 4-n-OP and 4-n-NP) (adjusted for age, BMI and CR

ssociated with idiopathic male infertility and idiopathic malenfertility with abnormal total sperm number per ejaculate andbnormal sperm concentration (p-value for trend <0.05) (Fig. 1 andables S2 andS3, Supplementary material).

We conducted sensitivity analyses to test the robustness of theesults. Except for the relationships between idiopathic male infer-ility with abnormal semen parameters and 4-t-OP as well as sumPs which were attenuated and not statistically significant, otherssociations observed between APs exposure and idiopathic malenfertility as well as semen parameters based subgroups of idio-athic male infertility were unchanged and remained statisticallyignificant in the recalculated models (Tables S4–S7, Supplemen-ary material).

. Discussion

To the best of our knowledge, we present the first humantudy that explored the relationships between exposure to variousnvironmental phenols and idiopathic male infertility. By moni-oring urinary phenols with large samples, we found that urinary

le infertility by increasing exposure of BPA, BP-3, PCP, TCS, 4-t-OP, 4-n-OP, 4-n-NP

4-t-OP, 4-n-OP and 4-n-NP levels were associated with idiopathicmale infertility in the general population. Our study representsimportant advance in the knowledge of the potential effects ofexposure to phenols on male reproductive health in humans.

Our observation that exposure to APs (4-t-OP, 4-n-OP and 4-n-NP) showed stronger associations with male infertility than otherphenols examined in this study is consistent with the data that sug-gest these compounds possess higher male reproductive toxicitypotential [1,19,22,31]. It has been well known that xenoestrogenstend to be hydrophobic [19,22]. Moreover, lipophilic compoundsmay also slowly be released to provide a persistent exposure inblood; such continuous dosage may be most effective in stimulat-ing certain estrogenic responses [19]. It is reported that APs arelipophilic [40]. The large size of alkyl chain of APs would increasethe hydrophobicity of the molecule [22]. During the determina-tion, the three APs [alkyl chain carbons: 8 (OP); 9 (NP)] showed the

strongest retention than other phenols on reverse column, indicat-ing APs are more hydrophobic than other phenols. Besides, the paraposition of the alkyl group on the phenolic ring in the three APs withunhindered structure at the ortho position was also reported as the

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20 M. Chen et al. / Journal of Hazardous

ey feature for the estrogenic activity of AP compounds [19,22].mong BPA and APs, 4-n-NP showed higher relative potency thanPA and 4-n-OP in human estrogen receptor and aromatase assay,nd greater relative potency of 4-n-OP was observed than BPA inuman aromatase assay [1]. Additionally, for APs, the branchingn the �-carbon is also an important feature for their estrogenicctivity [22]. It has been reported that 4-t-OP possesses even higherstrogenic activity than 4-NP [22]. Aside from endocrine-disruptionotency, compound may perturb membrane structure and functionecause of its lipophilic property. Hydrophobicity is indicated as theey feature in relation to the Ca2+ pump inhibition potency of APs,hich induces cell death in Sertoli cells [31]. The lipophilic propertyay also result in accumulation of these compounds in the body,

eading to longer and higher levels of exposure in target organs. Aseported in previous work, 4-n-NP is likely to cause greater testesccumulation than BPA by oral administration in rats [41,42].

Animal studies have proved that 4-t-OP and 4-NP exposurempairs spermatogenesis in rats [23–25]. Additionally, it has beeneported that low dose OP is toxic to Sertoli cells (10−12 M) andpermatogenic cells (10−8 M) in in vitro studies, which will pro-uce a parallel dysfunction in sperm production [43]. Thus, it isiologically plausible that exposure to 4-t-OP and 4-n-NP mayompromise male fertility through changing semen quality, espe-ially sperm number. Among APs, the reason why 4-n-OP exposureas not associated with idiopathic male infertility with below-

eference semen parameters might be due to its lower exposureevel than 4-t-OP (Table 2) and lower endocrine disrupting potencyhan 4-n-NP [1]. Except for sperm number, animal studies havehowed that APs exposure may affect acrosome integrity and causexidative stress in epididymal sperm [25]. Previous studies alsondicated that sperm morphology appeared vulnerable to 4-t-OPxposure in rats and rams [24,26,27]. Moreover, the sperm mor-hology impairment occurred at low dose (10−9 M) in rats [24].hus, these endocrine-disrupting APs may compromise male fer-ility not only through sperm number deterioration but also otherusceptible factors in relation to male reproductive competencempairment. This explains the lack of relationship between 4-n-P exposure and idiopathic male infertility with abnormal semenarameters while the association between 4-n-OP exposure and

diopathic male infertility was observed in all cases and controls inhe present study.

Recently, there is a growing concern about the effect of mixedxposure to contaminants on male infertility [21]. Previous paperlso indicated that mixture contained a combination of the short-hain alkylphenolethoxylates in addition to 4-NP and 4-t-OP hadigher estrogenic activity than NP only in fathead minnows [44].hen combining these APs exposure together, significant corre-

ations were found between idiopathic male infertility and suminear chain APs, sum OPs and sum APs exposure which reflectxposure levels from total APs (Fig. 1 and Tables S2 and S3, Sup-lementary material), suggesting potential male reproductive toxicffect caused by exposure to co-existent APs in day-to-day situa-ion. Additionally, although the three APs selected for the currenttudy are the commonly monitored urinary analytes to reflect APsxposure [7–11], they may only represent part of environmentalPs. On the other hand, due to isomeric heterogeneity of 4-NP, thereould be potent estrogenic isomer(s) that was not detected in thistudy. Collectively, these data emphasize that the male reproduc-ion risk caused by mixed exposure to APs in daily life should beaken into account.

Although there is evidence that exposure to BPA might beelated to impairment of male reproduction in animal and in vitro

tudies [1], the epidemiologic results of relationships betweenPA exposure and male reproductive function are still scarce andonflicting [32,33]. In this study there is no evidence for the asso-iation between BPA exposure and male infertility, which agrees

rials 250– 251 (2013) 115– 121

with the study in the US fertile population in which the authors didnot find relationship between BPA exposure and semen parameters[33].

In this study, we focused on a wide range of phenols whichare widely used endocrine disrupting chemicals, and studied theirassociation with male reproductive health, which may have greatpublic health significance. Strengths of the present study alsoinclude its sample size and the use of internal exposure estimation.

Our study had some limitations. One limitation of present studyis low detectable rate of urinary phenols in the general population.However, we conducted the statistical analysis according to pre-vious report, which has been proven to be useful in exploring theassociation between exposure to chemical with low detectable rateand health outcomes [38]. Moreover, in order to validate the find-ings, we conducted sensitivity analyses and compared the crudeand adjusted results, and found that major results in this studywere unchanged, suggesting that the results of this study are robust.Another limitation of this study is its case–control study naturewhich restricts our ability to make conclusions regarding causalrelationships. Future studies are required to confirm whether APsexposure increases the risk of idiopathic male infertility. Third, inthis study, we only determined three commonly monitored uri-nary APs [7–11]. Given the association found between APs exposureand idiopathic male infertility, a more comprehensive measure ofurinary APs should be conducted.

In conclusion, we explored the relationships between exposureto various environmental phenols and idiopathic male infertilitybased on a large sample size. We found significant link between APs(4-t-OP, 4-n-OP and 4-n-NP) exposure and idiopathic male infertil-ity. These findings provide the first evidence suggesting adverseeffects of APs on male reproductive health in humans, and war-rant further investigation in independent replication and follow-upstudies.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

We thank Ms. Rencheng Zhao for the chemical analysis. Wethank Dr. Jianling Bo and Dr. Honggang Yi for their assistance in sta-tistical analysis. This study was supported by National 973 Program(2009CB941703); Key Project of National Natural Science Founda-tion of China (No. 30930079); National Natural Science Foundationof China (No. 81072328); The Key Project of MOE (No. 211063); Pri-ority Academic Program Development of Jiangsu Higher EducationInstitutions (PAPD).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2013.01.061.

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