Pyrethroid insecticide exposure and semen quality of young Japanese men

Reproductive Toxicology 43 (2014) 38–44

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Pyrethroid insecticide exposure and semen quality of young Japanese men Kanako Imai a , Jun Yoshinaga a,∗ , Mitsuha Yoshikane a , Hiroaki Shiraishi b , Makiko Naka Mieno c , Miki Yoshiike d , Shiari Nozawa d , Teruaki Iwamoto d,e a

Department of Environmental Studies, University of Tokyo, Kashiwa, Chiba 277-8563, Japan National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8563, Japan c Centre for Information, Jichi Medical University, Shimotsuke, Tchigi 329-0498, Japan d Department of Urology, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan e Center for Infertility and IVF, International University of Health and Welfare Hospital, Nasushiobara, Tochigi 329-2763, Japan b

a r t i c l e

i n f o

Article history: Received 12 June 2013 Received in revised form 17 October 2013 Accepted 24 October 2013 Available online 1 November 2013 Keywords: Pyrethroid insecticide Urinary 3-PBA Semen quality University student Multiple regression analysis

a b s t r a c t The present study aimed at assessing the relationship between exposure to pyrethroid insecticides and semen quality in 323 university students recruited in a population-based manner in Metropolitan Tokyo. Urinary concentrations of pyrethroid insecticide metabolite, 3-phenoxybenzoic acid (3-PBA), were measured by LC/MS/MS and semen parameters were measured by following internationally harmonized protocols. Median urinary 3-PBA concentration was 0.641 ng/mL (specific gravity-adjusted, n = 322). Median values of semen volume, sperm concentration, motility, total number of sperm, and total number of motile sperm were 2.5 mL, 56 × 106 /mL, 61%, 141 × 106 , and 82 × 106 , respectively. Urinary concentration of 3-PBA was not selected as significant in multiple regression models indicating, in contrast to previous findings, that environmental exposure to pyrethroid insecticides did not affect semen quality. This inconsistency may be related to exposure to different pyrethroid insecticides and/or levels of exposure as well as to survey design (hospital- vs population-based subject recruitment). © 2013 Elsevier Inc. All rights reserved.

1. Introduction Synthetic pyrethroid insecticides, such as permethrin, cypermethrin or fenvalerate, are used worldwide in residences and for agricultural purposes. This group of insecticides is favored because of its high insect to mammal acute toxicity ratio and it is replacing organophosphorus pesticides in many areas of applications. In Japan, in particular, synthetic pyrethroids are the most common active ingredient of insecticides used in domestic residences. The Ministry of the Environment of Japan estimated the nonagricultural (residential and municipal) use of permethrin alone to be 23 × 103 kg in 2010 [1]. Accordingly, human exposure to pyrethroid insecticides is already widespread in many countries as has been indicated by the frequent detection of their metabolites in human urine [2]. Pyrethroid insecticides can be ingested by the consumption of vegetables and fruits with residues on their surfaces and by the inhalation/ingestion of material from the indoor environment where insecticides have been applied for pest control. Although the acute toxicity of synthetic pyrethroids to mammals is considered low, concerns have been growing about their

∗ Corresponding author. Tel.: +81 4 7136 4716; fax: +81 4 7136 4716. E-mail address: [email protected] (J. Yoshinaga). 0890-6238/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2013.10.010

chronic toxicities to humans. Reproductive effects are one of such concerns. Decreased spermatogenesis was observed in adult mammals (rats and goats) administered with some pyrethroid insecticides [3–7]. This was assumed to be connected with the decreased testosterone biosynthesis that had been observed in adult male rats and mice exposed to pyrethroid insecticides in early life [8,9] and in maturity [3,4,10]. A handful of human studies have reported that pyrethroid insecticides exposure negatively impact semen quality. Tan et al. [11] reported poorer semen quality among fenvalerate-exposed workers than among a non-exposed reference population. More recently, for non-occupationally exposed male subjects, poorer semen quality was associated with higher urinary concentrations of 3-phenoxybenzoic acid (3-PBA), a biomarker of exposure to pyrethroid insecticides [12–15], although one study found a weak relationship that lacked statistical significance [16]. After intake pyrethroid insecticides are metabolized with a short biological halflife (e.g., 7.5 h for deltamethrin [17]). 3-PBA is a common metabolite of several pyrethroid insecticides and is excreted in urine making urinary levels a good indicator of recent exposure. Higher urinary 3PBA levels were also associated with altered reproductive hormone levels in male subjects; for example, a positive association between 3-PBA and serum luteinizing hormone concentrations [18,19] and a negative association between urinary metabolite concentrations

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and serum inhibin B, testosterone and the free androgen index were observed [19]. It appears that male reproductive effects seen in human studies have been consistent with animal studies despite the fact that exposure (dose) levels in these studies are considerably different. Except for one study of a small number of men recruited in a community setting [12], these prior human studies have involved subjects who were infertile for unexplained reasons [13,14] or who were partners in subfertile couples recruited in fertility clinics [15,16]. Limitations in sample size and generalizability could have affected the results of these prior studies, and studies further evaluating pyrethroid exposures and male reproductive impacts are needed in men recruited from the healthy general population. In the present study, level of exposure to pyrethroid insecticides and semen quality were examined in male subjects recruited from a population-based manner to determine if the deleterious effect on semen quality of exposure to pyrethroid insecticides in subjects associated with fertility problems and recruited in a hospital-based manner was reproduced.

2. Subjects and methods 2.1. Subjects The present subject population was a sub-population of a larger cross-sectional, multi-center study population for the establishment of reference semen quality in Japan [20]. Inclusion criteria for each male individual were (1) he was a university student, (2) he was 18–24 years old, and (3) his mother and he were born in Japan. Subject recruitment was carried out twice at the Kawasaki center (May 1999–May 2000 and April 2002–May 2003), located in the western part of Tokyo metropolitan area; the present subjects were from the latter recruitment period. The subjects voluntarily applied for inclusion in the study after they had read the leaflet which explained in detail the purpose and procedure of the study, and which had been distributed to universities in the Kawasaki region. In total 2034 leaflets were distributed and 323 subjects were included. They did not have any apparent problems with their general health, including their reproductive health, and thus were considered healthy. The subjects gave written informed consent after the purpose and procedures of the study were again explained to them at the time of participation. The study was approved by the Ethical Committee of St. Marianna University School of Medicine.

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Table 1 Demographic and anthropometric characteristics of the subjects (n = 322). Unit Age Height Body weight BMI Abstinence period before semen sampling Testicular volume (L) (R) Varicocelea (L) (R) a

Mean ± SD ± ± ± ± ±

years cm kg kg/m2 hours

20.2 173 64.3 21.5 78.1

1.5 5 9.0 2.5 32.3

mL mL % %

20.9 ± 4.3 21.5 ± 4.3 28.2 1.9

Range 18–24 160–188 48–120 16.5–37.9 37–351 12–30 12–30

Percentage of the subjects who had a varicocele: L, left side; R, right side.

were routinely and rigorously cross-validated between the urologists. The subjects were asked to complete a self-administered questionnaire that asked for lifestyle information such as smoking and alcohol consumption data and the frequency of consumption of selected foods (fish, dairy products, vegetable, fruits, etc.) during the period of normal life before the sampling visit, and were asked to submit it at the visit. Frequency of consumption of each food item was coded from 1 to 6, corresponding to: not eaten, 1–3/month, 1/week, 2–3/week, 1/day, and >2/day. Table 1 shows demographic and anthropometric characteristics of the study subjects. 2.3. Semen analysis Analysis of semen was carried out in accordance with the procedures described in detail in Iwamoto et al. [20]. Briefly, each semen sample, kept at 37 ◦ C since receiving, was first liquefied and its volume measured with a volumetric device. Sperm motility was assessed by observation under a microscope (×400) and categorized into 4 classes (A to D) according to WHO guidelines [21]. Duplicated measurements were done. The number of sperm in an aliquot of well-mixed semen was counted on a Burker-Turk hemocytometer after dilution with a solution containing 5% (w/v) NaHCO3 and 0.4% (w/v) formaldehyde. Based on these measurements, semen volume (mL/ejaculation), sperm concentration (number/mL), motility ([A + B + C]/[A + B + C + D] × 100%), and number of total and motile sperm per ejaculation were calculated. Quality control of semen analysis in this laboratory was rigorously assessed by cross-validation with external laboratories [20].

2.2. Sample collection and physical examination 2.4. Urinalysis Detailed information on the subjects and the procedures for semen sampling and physical examination of the subjects have been given in Iwamoto et al. [20]. Only a brief summary of that information is given here. The subjects were requested to visit the study center in Kawasaki for the sampling of semen and urine, and for physical examination. Twenty five to thirty subjects were invited in each month of the study period (April 2002–March 2003) so that sample collection was evenly distributed throughout that time. Semen sampling was carried out by masturbation in a private room at the study center. Prior to sample collection the subject was instructed to abstain from sexual activity for at least 48 h. Urine samples were obtained during the visit by the subject himself using a paper cup. The urine in the cup was dispensed into plastic tubes by study staff and then frozen at −80 ◦ C until analysis. Genitalia were examined during the visit by one of three urologists working at the study center. Testis size was measured with an orchidometer and the presence or absence of varicoceles was evaluated for both right and left testes. The measurement and evaluation

Urine samples collected at the time of semen sampling were analyzed for 3-PBA and specific gravity (SG) was determined. Analysis of 3-PBA was carried out with a method based on previous studies [22,23] with modifications which included, for example, acid hydrolysis in place of enzymatic hydrolysis for 3PBA deconjugation. Briefly, to 1 mL of urine was added 0.1 mL of 100 ng/g deuterium-labeled 3-PBA (Cambridge Isotope Laboratories Inc., USA) as an internal standard, and 0.5 mL of 6 mol/L HCl, and the solution heated at 100 ◦ C for 2 h for hydrolysis of conjugates. After adjustment to pH5 with 5 mol/L NaOH solution and the addition of 0.6 mol/L acetate buffer, the sample was loaded onto a solid-phase extraction cartridge (Oasis HLB, Waters, USA) that had been conditioned prior to use. The cartridge was then washed with 5% (v/v) aqueous methanol and was eluted with 100% methanol. The methanol was removed and the sample reduced to dryness in a gentle stream of N2 . The residue was re-dissolved in 0.1 mL 30% (v/v) aqueous acetonitrile and

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Table 2 Summary statistics of semen parameters of the subjects (n = 322).

Semen volumea Sperm concentration Motility Total number of sperm Total number of motile sperm

Unit

Min

25%tile

50%tile

75%tile

Max

Mean (SD)

mL ×106 /mL % ×106 /ejaculation ×106 /ejaculation

0.3 1.5 10 1.3 0.5

1.7 28 52 69 39

2.5 56 61 141 82

3.4 87 70 234 143

8.2 536 88 2787 724

2.7 (1.3) 70 (60) 60 (14) 176 (204) 103 (90)

a Semen volume was measured volumetrically; therefore the values in this table may underestimate by 0.4 mL the weight-based semen volume measurement carried out according to WHO guidelines. See text for details.

a 2 ␮L aliquot was injected into a liquid chromatograph (Agilent 1100 Series; Agilent Technologies, Tokyo, Japan) coupled to a tandem mass spectrometer (Quattro Ultima, Micromass, UK). The column used was a BETASIL C18 (1.0 mm × 100 mm, Thermo Scientific, USA) and the mobile phase was 30% acetonitrile/0.5% acetic acid with a 0.2 mL/min flow rate. A calibration curve was established for 0–40 ng/mL. Detection limit of urinary 3-PBA was 0.08 ng/mL. In every batch of urine samples undergoing pretreatment, a procedural blank and an in-house control urine sample were included for internal analytical quality control. The recovery of added deuterium-labeled 3-PBA was 74 ± 17% (n = 322) and that of added unlabeled 3-PBA was 97 ± 1% (n = 3). The mean and standard deviation of repeated measurements of in-house control urine was 1.28 ± 0.08 ng/mL (n = 18). Thus urine 3-PBA analysis was reliable in terms of trueness and precision. The specific gravity of urine samples was measured with a hand-held refractometer (Elma, Tokyo, Japan), and urinary 3-PBA concentrations were adjusted to SG = 1.020 according to the following equation: Cc = Cm ×

1.020 − 1 SG − 1

2.5. Statistical analysis The values of semen parameters (except for sperm motility) and of urinary 3-PBA concentration were not regarded as being normally distributed, and therefore cubic root transformation for semen parameters [20] and log-transformation for 3-PBA were carried out for statistical analysis. For sperm motility, crude values were used because their distribution was considered to be normal. The relationships between these parameters and those between these parameters and demographic, anthropometric and questionnaire data were examined by t-tests, analyses of variance (ANOVA) and correlation analyses. Multiple regression analysis was carried out using one of the semen parameters as dependent variable and urinary 3-PBA concentration as independent variable. Other variables which had statistically significant associations with the dependent variable in bivariate analysis were included in the model as independent variables. For food consumption data derived from the questionnaires, consumption frequency of a given food was categorized into two groups and included in the model in a 0/1 (−/+) manner. The criteria for dividing the subjects into two groups were determined in order to make the number of subjects in the two groups as similar as possible. Stepwise variable selection (Pin = 0.05 and Pout = 0.1) was employed for multiple regression analysis. The analysis was run on subjects with a value for any one of the semen parameters below the WHO lower reference limit (LRL) to see if exposure to pyrethroid insecticides affects those subjects with potential male-factor reproductive problems differently from those with normal semen quality. All statistical analyses were carried out using SPSS for Windows ver. 19.

3. Results 3.1. Subjects As shown in Table 1, mean age of the present subjects was 20.2 years and the mean height (173 ± 5 cm), weight (64.3 ± 9.0 kg) and BMI (21.5 ± 2.5) were in agreement with the national averages for 20–29 years age grade (171.8 ± 6.5 cm, 66.5 ± 11.5 kg and 22.5 ± 3.6, respectively) in 2004 [24]. 3.2. Semen parameters In Table 2, quartile concentrations of semen parameters of the subjects with minimum and maximum values are shown because of their skewed distributions. Sperm motility was the only parameter that could be assumed to follow a normal distribution. There were 25, 23, 38, and 52 subjects whose semen volume, motility, sperm concentration and TNS were below the 2010 WHO LRL [25]. The semen volume measurement in the present study was volume-based and was found to underestimate the semen quantity by 0.4 mL when compared with the weight-based method described in the WHO manual [19]. This should be taken into account when the semen volumes presented in Table 2 are considered. Therefore, the criterion for normal semen volume was set at 1.1 mL in this study in order to divide the subjects into two groups. There were 57 subjects who had at least one semen parameter below the WHO LRL. 3.3. Urinary 3-PBA concentration 3-PBA was detected in 294 of 322 samples of urine (91.3%). Note that urine sample was missing for one subject and total number of urine analyzed was 322. Average concentrations of 3-PBA in the urine of subjects are shown in Table 3. Arithmetic and geometric means, and median values for unadjusted and SG-adjusted concentrations are shown to facilitate comparison with literature values. 3.4. Bivariate analysis Bivariate associations between urinary 3-PBA concentrations and biological variables (age and BMI), seasonal factors, and smoking and dietary habits were sought. A significant variation in urinary 3-PBA concentration with the season of urine sampling was found (ANOVA, p < 0.001, Supplemental Fig. 1); the mean concentration of 3-PBA was significantly higher in urine sampled Table 3 Urinary 3-PBA concentration of subjects (ng/mL).

Unadjusted SG-adjusteda a

Mean (SD)

Geometric mean (SD)

Median (min–max)

1.22 ± 1.64 1.01 ± 1.32

0.679 (3.26) 0.588 (3.12)

0.808 (<0.08–13.2) 0.641 (<0.07–12.0)

Adjusted to specific gravity 1.020.

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in summer (June–August, n = 82) than those sampled in winter (December–February, n = 84) (Tukey HSD test, p < 0.001) and in spring (March–May, n = 84) (p < 0.01). The mean concentration of 3-PBA in urine sampled in autumn (September–November, n = 72) was significantly higher than that sampled in winter (p < 0.05). Significant variation due to consumption frequency of beef and vegetables was also found (ANOVA, p < 0.05). However, no linear relationship was apparent, i.e., increasing urinary 3-PBA concentration with increasing consumption frequency was not observed for either food. There was no association of other factors with urinary 3-PBA concentrations. Bivariate analyses between semen parameters and biological variables (age, BMI, testis size, and presence of varicoceles), seasonal factors, and smoking, and dietary habits revealed a significantly negative correlation between age and sperm concentration (Spearman’s correlation coefficient, r = −0.207, p < 0.001), between age and total number of sperm (r = −0.137, p < 0.05), and between age and total number of motile sperm (r = −0.118, p < 0.05). Abstinence period (in hours) significantly positively correlated with semen volume (r = 0.212, p < 0.001), sperm concentration (r = 0.276, p < 0.001), total sperm counts (r = 0.397, p < 0.001) and motile sperm counts (r = 0.311, p < 0.001). Testis size significantly correlated with sperm concentration (r = 0.246, p < 0.001), total number of sperm (r = 0.249, p < 0.001), and total number of motile sperm (r = 0.234, p < 0.001). Subjects with varicoceles had significantly lower motility (t = 3.162, p < 0.01). Marginally significant variation in motility due to the frequency of consumption of cheese (ANOVA, p = 0.05), and in semen volume and motile sperm count due to the frequency of consumption of soy products was found. Total number of sperm and total number of motile sperm varied significantly with the frequency of consumption of non-oily white fish. All of these relationships showed a tendency for increasing semen quality with increasing frequency of consumption of the various foods. Preliminary bivariate tests between semen parameters and urinary 3-PBA revealed no significant difference in urinary 3-PBA concentrations between subjects with a semen parameter below the 2010 WHO LRL (n = 23–52 depending on the parameter) and those with a value greater than the LRL (t-test, p > 0.05). There were 57 subjects who had at least one semen parameter below the WHO LRL; again there was no difference in urinary 3-PBA concentration between these 57 subjects and those of the others.

3.5. Multiple regression analysis Possible associations between any one of the semen parameters and urinary concentration of 3-PBA was analyzed using linear multiple regression analysis by including age, BMI, season of semen sampling, abstinence period, presence of a varicocele, testis size, frequency of consumption of cheese, soy and non-oily white fish flesh as covariates and by taking the bivariate analytical results into consideration. Subjects were reorganized into just 2 categories, namely, high and low frequency of consumption, for cheese, soy products and non-oily white fish with similar numbers of subjects in each group. As shown in Table 4, urinary 3-PBA concentration was not selected as a significant independent variable for any of the semen parameters. Instead, biological factors, dietary habits and season of semen sampling, some of which were found to be significant in the bivariate analyses, were also found to be significant in the multivariate models. When the analysis was run on subjects who had at least one semen parameter below the WHO LRL, urinary 3PBA was not selected as significant (data not shown). Note that the criterion for semen volume in this study was 1.1 mL as has been explained.

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4. Discussion 4.1. Pyrethroid insecticides exposure of the subjects The average urinary concentration of 3-PBA of the present subjects (Table 3) was slightly higher than the levels reported to date for Japanese populations; Ueyama et al. [26] reported geometric mean urinary 3-PBA concentrations (unadjusted) to be 0.38 and 0.29 ng/mL for occupationally exposed elderly farmers (n = 87) and non-occupationally exposed elderly subjects from the general population (n = 448), respectively. Our previous studies (Toshima et al. [15]) found the SG-adjusted geometric mean concentration for male partners (n = 42) of subfertile couples to be 0.547 ng/mL. There has been no substantial change in the domestic production of permethrin, a major pyrethroid insecticide, since 2003 when urine sampling for this study was carried out [27]. Note that urine sampling was done in 2005 and in 2010 for the Ueyama et al. and Toshima et al. studies, respectively. It is unlikely that a nation-wide decrease in the level of exposure level has occurred and the reason for the higher 3-PBA concentrations in urine of the present subjects is not clear. Significant variation in urinary 3-PBA concentration with season of urine sampling was found; 3-PBA concentrations in urine samples collected in summer and autumn were higher than those collected in winter (Supplemental Fig. 1). A similar finding was obtained in a recent Chinese study [28]. Seasonal variation in the exposure of the Japanese population to pyrethroid insecticides was indicated. Increased use of agricultural insecticides in summer and autumn is to be expected, and this would result in increased intake of pyrethroid insecticides through the diet. Increased non-dietary exposure (e.g., through inhalation, ingestion of house dust or dermal contact) to pyrethroid insecticides applied in the outdoor and indoor environment in summer and autumn may also contribute to this observed variation. However, in this study, there was no apparent trend of increasing urinary 3-PBA concentrations with increasing frequency of consumption of vegetables and fruit, in contrast to previous studies [29–31]. This suggested that pyrethroid insecticides from non-dietary sources contributed more to the exposure of the present subjects than did pyrethroid insecticides in the diet. 4.2. Semen quality of the subjects The subjects were a sub-population of a larger cross-sectional population recruited at 4 study centers in Japan [20]. The median semen parameters of the subjects (Table 2) were similar to those of the entire population (n = 1559) for which median values were 2.7 mL, 59 × 106 /mL, 69%, and 159 × 106 for semen volume, sperm concentration, motility and total number of sperm, respectively [20]. Iwamoto et al. [20] concluded that the semen quality of the 1559 young Japanese men was higher than that reported for populations in northern European countries. Although the subjects of the present study were apparently healthy at the time of recruitment, it was found that 57 of 322 (18%) had at least one semen parameter value below the 2010 WHO LRL. 4.3. Pyrethroid insecticides exposure and semen quality The multiple regression analyses did not find urinary 3-PBA concentration to be a significant variable in explaining variation in any of the semen parameters examined in the present study (Table 4). Thus the negative association between urinary 3-PBA concentration and semen parameters found in previous studies was not reproduced in the present population-based study. Xia et al. [13] found a significantly elevated probability of the concentration of sperm in the semen of Chinese males, infertile for unexplained

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K. Imai et al. / Reproductive Toxicology 43 (2014) 38–44

Table 4 Results of multiple regression analysis for semen parameters as dependent variable.

Age Abstinence period Presence of varicocelea Testis size Summerb Soyc Cheesed

Semen volume

Motility

ˇ

ˇ

0.195

0.181

p <0.001

p

−0.125 −0.186

0.024 0.001

0.137

0.013

0.170

0.002

0.001

Sperm concentration

Total number of sperm

Number of motile sperm

ˇ

p

ˇ

p

ˇ

p

−0.199 0.271

<0.001 <0.001

−0.119 0.381

0.018 <0.001

0.256

<0.001

0.273

<0.001

0.338 −0.118 0.237

<0.001 0.023 <0.001

0.115

0.022

0.130

0.012

Standardized partial correlation coefficients (ˇ) with p-values are given only for significant independent variables. Others were not selected as significant. Urinary 3-PBA and BMI were also included as independent variables but were not selected as significant for any of the dependent variables. a Varicocele present regardless of left or right, 1; absent, 0. b Season of semen sampling. Summer, 1; other season, 0. c Consumption frequency >2–3 times/week, 1 (n = 200); <1 time/week, 0 (n = 119). d Consumption frequency >1 time/week, 1 (n = 92); 2–3 times/month, 0 (n = 227).

reasons, having less than the WHO standard (<40 × 106 /mL) for the highest urine 3-PBA concentration quartile (>1.501 ␮g/g cre., corresponding to approximately >1.90 ng/mL unadjusted concentration). Ji et al. [14] found a significant negative linear association between urinary 3-PBA concentration and sperm concentration in semen in male subjects recruited in the same program as Xia et al. [13] and in an overlapping recruitment period. Toshima et al. [15] found a negative linear relationship between urinary 3-PBA concentration and motility among Japanese male partners of subfertile couples. Meeker et al. [16], however, did not find a significant association between urinary 3-PBA concentration and semen quality among men recruited at an infertility clinic in the USA, but they did find significant associations between urinary concentrations of trans-3-2,2-dichlorovinyl-cyclopropene-carboxylic acid (transDCCA), another metabolite of pyrethroid insecticides, and semen quality [16]. Several points need to be considered in attempting to explain the inconsistency between the results of the present study and previous Chinese studies [13,14] (Toshima et al. [15] was not included in the following comparison because it was a pilot study with a small sample size (n = 42).). First, the present study subjects were recruited in a populationbased manner while those in the Chinese studies were male factor infertility patients recruited in a hospital. Possibly individuals with male factor infertility responded differently from healthy subjects to reproductive toxicants, though any mechanism underlying this assumption cannot be proposed at this time. It must be noted, however, that 3-PBA was not selected as significant in multiple regression analyses using subjects who had at least one semen parameter below the 2010 WHO LRL in the current study (n = 57), indicating the absence of a negative relationship between pyrethroid exposure and semen quality among subjects with possible reproductive problems. Second, since 3-PBA is a non-specific metabolite of several pyrethroid insecticides, it is possible that subjects in different populations might be exposed to different pyrethroid insecticides that may have different toxicities. Although there have been no reports of animal studies comparing the effects of different pyrethroid insecticides on male reproductive functions, an in vitro study demonstrated different sex hormone receptor activities for different pyrethroid insecticides [32], thus indicating that different pyrethroid insecticides have different reproductive effects. The subjects in the Chinese studies might have been exposed to pyrethroid insecticides with more potent effects on semen parameters than were the present subjects. Inclusion of other metabolites with some specificity to parent pyrethroid insecticides, such as cis-DCCA and trans-DCCA, cis3-(2,2-dibromovinyl)-2,2-dimethyl-cyclopropane-carboxylic acid

(DBCA) or 4-fluoro-3-phenoxy-benzoic acid (4-F3-PBA) [33] as biomarkers of exposure may be warranted in future studies. Indeed, Meeker et al. [16] found a significant negative association between trans-DCCA and semen quality for those subjects whose urinary 3-PBA concentrations were not associated with semen quality. Third, exposure levels to pyrethroid insecticides of the present subjects were slightly lower than those in Chinese studies and this might contribute to the difference in results. In the Xia et al. [13] study, only those subjects in the highest urinary 3-PBA quartile range (approximately >1.9 ng/mL unadjusted concentration) had significantly more risk of being below the WHO standard for sperm concentration (<20 × 106 /mL) when compared with the subjects in the lowest quartile range. Thus, the threshold level of exposure to pyrethroid insecticides for deleterious effects on human semen quality might be around this urinary 3-PBA level. Thirty-five of 322 subjects (11%) in the present study had >1.9 ng/mL 3-PBA. It must be noted, however, that the exposure levels in the present study and in the Chinese studies were much less than administered doses in previous experiments with rodents; deleterious effects on semen parameters and circulating hormone levels were observed in rodents at administered dose levels of >10 mg/kg/day. A urinary concentration of 1 ng/mL 3-PBA roughly corresponds to an intake of parent pyrethroid insecticides at a level of 0.06 ␮g/kg/day if we assume that (1) most of the intake of the pyrethroid insecticides is excreted within 24 h (biological half-life of the pyrethroid insecticide deltamethrin was 7.5 h [17]), (2) daily urine volume is 2 L, (3) the typical molecular weight of pyrethroid insecticides is 400 (3-PBA is 214.2) and (4) the body weight of an adult is 60 kg. [The calculation: 1 ng/mL × 2000 mL/day/214.2 × 400/60 kg = 60 ng/kg/day.] If semen parameters are affected by exposure to pyrethroid insecticides at resulting urine concentrations of 3-PBA of around 1–2 ng/mL, humans would be more sensitive than rodents to pyrethroid insecticides by a factor of approximately 105 .

4.4. Biological factors and dietary habits responsible for variations in semen quality The multiple regression analyses extracted age, abstinence period, testis size, and the presence of varicoceles as significant variables to explain semen quality. The ˇ values for age and varicocele presence were negative and those for abstinence period and testis size were positive, except for the abstinence period for sperm motility where a negative value was found (Table 4). Of the biological variables selected as significant here, only abstinence period has been known to have an association with semen quality in previous studies (e.g. [34]). The observed associations between semen

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quality and other biological factors have not been generally recognized or are a matter of controversy [35–38]. In addition to some biological factors, the frequencies of consumption of cheese and soy products were selected as significant for some parameters in the multiple regression analyses (Table 4). The deleterious effect of soy food products, which are rich in isoflavones, naturally occurring estrogenic compounds, on semen quality has been reported in some studies [15,39] while no effect has been reported in others [40]. The present result, i.e., higher semen quality in frequent consumers of soy products, was not consistent with any of the results of the previous studies. As mentioned above, it is still uncertain as to which biological factors and dietary habits, as well as other factors [38], are involved as determinants of semen quality in men; therefore, it is important for future studies of the association between chemical exposure and semen quality to include these biological and dietary variables as covariates in the survey design to control for their possible involvement. 4.5. Strength and limitation of the present study The strength of the present study was that a moderate number of apparently healthy university students (number of urine samples analyzed: 322) was included, making the results more generalizable to the general public than those of previous studies involving subjects recruited at infertility clinics. However, it must also be recognized that the present study results are not fully generalizable to Japanese general population because the subjects were all university students living in a limited geographical area. Another strength of the current study is the inclusion of potential biological and lifestyle factors and dietary habits that may influence semen quality in multivariate models as far as was possible. The study indicated for the first time that exposure to pyrethroid insecticides at current environmental a level does not have deleterious effects on the semen quality of the general male population. The limitation of the present study was that the level of exposure to pyrethroid insecticides of the subjects was assessed by the measurement of 3-PBA concentrations in single spot urine samples. Although this was a common practice of assessing exposure in previous epidemiologic studies, it has some potential disadvantages. Since the biological half-life of pyrethroid insecticides is generally short, 3-PBA levels in spot urine reflects only very recent exposure to pyrethroid insecticides while, in contrast, spermatogenesis is a much longer process. This time-lag between assessments of exposure and effect might have obscured any potential association. In addition, as stated above, 3-PBA is a non-specific metabolite of several pyrethroid insecticides; therefore 3-PBA might not be an appropriate biomarker for all individual pyrethroid insecticides that could have reproductive effects of different potencies. Thus, an exposure assessment approach that considers specific pyrethroid insecticides individually and covers a longer period of time would be warranted for future studies. 5. Conclusion The present study indicated that exposure to pyrethroid insecticides at environmental levels in Japan did not affect the semen parameters (semen volume, sperm concentration, motility, total number of sperm, and total number of motile sperm) of apparently healthy university students. This result was in contrast to those of previous epidemiologic studies carried out on infertile patients in China and the USA. Since pyrethroid insecticides exposure is widespread among the general public in many countries and male reproductive toxicities of pyrethroids have been demonstrated in

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experimental animals, it is critically important to characterize their potential male reproductive toxicity in humans. Further human studies are warranted to this end.

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.reprotox. 2013.10.010.

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