Prenatal organophosphate pesticide exposure and reproductive hormones in cord blood in Shandong, China

International Journal of Hygiene and Environmental Health 225 (2020) 113479

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Prenatal organophosphate pesticide exposure and reproductive hormones in cord blood in Shandong, China

T

Kaili Qina, Yan Zhanga, Yiwen Wangb, Rong Shia, Rui Pana, Qian Yaoa, Ying Tiana,b,∗∗, Yu Gaoa,∗ a b

Department of Environmental Health, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China MOE-Shanghai Key Laboratory of Children's Environmental Health, Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Pregnant women Organophosphate pesticides Reproductive hormone Birth cohort

Background: Organophosphate pesticides (OPs) have been found to be associated with endocrine disorders, but limited research has been conducted to evaluate the relationship between maternal OP exposure and fetal reproductive hormone levels. In this study, we investigated the association between prenatal OP exposure and fetal reproductive hormones. Methods: A total of 306 healthy pregnant women were enrolled between September 2010 and February 2012. Pesticide exposure was assessed via the analysis of maternal urinary nonspecific metabolites of OPs (dialkylphosphate, DAP), and four reproductive hormones were measured in cord blood. Linear regression models and generalized linear models were used to estimate the associations between DAP metabolites and reproductive hormones, and further stratified by infant sex. Results: We found that concentrations of diethylphosphate (DEP) (β = −0.03; 95% CI: −0.07, −0.00) were inversely associated with estradiol (E2). Dimethylphosphate (DMP) (β = −0.08; 95% CI: −0.13, −0.03), diethylthiophosphate (DETP) (β = −0.08; 95% CI: −0.14, −0.01), and DAPs (β = −0.10; 95% CI: −0.17, −0.03) were inversely associated with testosterone (T) levels. DMP was inversely associated with follicle-stimulating hormone (FSH) levels (β = −0.03; 95% CI: −0.05, −0.01). DMP (β = −0.06; 95% CI: −0.10, −0.01) and DETP (β = −0.07; 95% CI: −0.13, −0.01) showed inverse associations with the testosterone/ estradiol (T/E2) ratio. Moreover, the magnitude of associations notably increased in higher quartiles of concentrations in a dose-response manner. After stratification by sex, these effects were mainly observed among female infants. Conclusion: Our findings suggest the potential impacts of prenatal OP exposure on fetal reproductive hormones, and that sex-related differences may exist.

1. Introduction Organophosphate pesticides (OPs) are extensively used in agriculture and households for pest control and now account for more than one-third of all pesticides used in China (Shu et al., 2016). The extensive use of OPs leads to continuous human exposure via contaminated food and household use (Katsikantami et al., 2019). Studies have demonstrated that OP exposure is widespread among susceptible populations, including pregnant women and fetuses (Ye et al., 2009; Barouki et al., 2012). Therefore, the potential health effects of prenatal OP exposure on fetuses could be an important public health issue and deserve more attention.

OPs have been identified as potential endocrine disrupting chemicals (EDCs) (Viswanath et al., 2010). Several recent animal studies have suggested that exposure to OPs is related to changes in reproductive hormones (Jeong et al., 2006; Yu et al., 2013; Geng et al., 2015; Ventura et al., 2016). For example, OP exposure in adult rats caused decreases in serum estradiol (E2), luteinizing hormone (LH) (Ventura et al., 2016), testosterone (T), and follicle-stimulating hormone (FSH) (Geng et al., 2015). In addition, two studies found that prenatal OP exposure in rats disrupted their offspring's reproductive hormonal profile (Yu et al., 2013; Jeong et al., 2006). Epidemiological studies in human populations have mainly focused on adult men. These studies have reported that OP exposure was associated with lower levels of T,

∗∗ Corresponding author. Department of Environmental Health, School of Public Health, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, 200025 Shanghai, China. ∗ Corresponding author. Department of Environmental Health, School of Public Health, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, 200025, Shanghai, China. E-mail addresses: [email protected] (Y. Tian), [email protected] (Y. Gao).

https://doi.org/10.1016/j.ijheh.2020.113479 Received 24 October 2019; Received in revised form 27 January 2020; Accepted 2 February 2020 1438-4639/ © 2020 Elsevier GmbH. All rights reserved.

International Journal of Hygiene and Environmental Health 225 (2020) 113479

K. Qin, et al.

samples for high and low concentrations) at an interval of once every 38 urine samples. The percent of relative standard deviation (%RSD) for DAP metabolites ranged from 0.5% to 6.3% for the within-run precision and from 3.0% to 17.8% for the between-run precision, as described elsewhere (Wang et al., 2017). The analytical laboratory responsible for the metabolite analysis did not participate in the G-EQUAS external quality assessment scheme. DAP metabolite levels in urine samples were adjusted to correct for variable urine dilutions using creatinine concentrations, which were measured by an automated chemistry analyzer (7100 Hitachi; Tokyo, Japan).

E2 (Meeker et al., 2006, 2008; Omoike et al., 2015) and FSH (BlancoMunoz et al., 2010), and with higher levels of LH and FSH (Melgarejo et al., 2015). However, no data exist from human studies on prenatal OP exposure and its effects on reproductive hormone levels in the offspring. Studies have reported that maternal-fetal transfer of OPs may occur (Whyatt et al., 2005; Abu-Qare and Abou-Donia, 2001), and fetuses are highly sensitive to hazardous environmental substances due to their rapid growth rates (Schug et al., 2011). The prenatal period is of great importance for the growth of fetuses, and reproductive issues among adults may have fetal origins (Barker et al., 1993; Lagiou et al., 2011; Crain et al., 2008; Juul et al., 2014). Therefore, based on a birth cohort from northern China, we studied the OP exposure levels in pregnant women by analyzing the urinary nonspecific metabolites of OPs (dialkylphosphate, DAP) and investigated whether prenatal OP exposure was associated with reproductive hormone measurements in cord blood.

2.3. Cord blood reproductive hormone measurement Cord blood samples were gathered immediately following delivery and centrifuged at 1500 rpm for 20 min to separate serum, which was collected into precleaned glass vials and stored at −80 °C. Serum samples were analyzed for four reproductive hormones, including E2, T, LH and FSH. Four hormones were separately detected using radioimmunoassay (RIA) kits (Beijing North Institute of Biotechnology, Beijing, China). In this study, assay sensitivities were 2 pg/mL for E2, 0.02 ng/mL for T, 1.0 mIU/mL for LH and 1.0 mIU/mL for FSH. The intra-assay and inter-assay coefficients of variation (CVs) of the four kits were < 10% and < 15%, respectively.

2. Methods 2.1. Participants This investigation was based on the Laizhou Wan Birth Cohort (LWBC) from the southern coastal area of Laizhou Wan (Bay) of the Bohai Sea, Shandong Province, northern China. Detailed descriptions of the study, including the population, information on recruitment, data collection, and contents of questionnaires, were provided previously (Chen et al., 2015; Han et al., 2018). Briefly, healthy pregnant women who were admitted to a local hospital for delivery were recruited from September 2010 to December 2013. The eligibility and exclusion criteria in this study are shown in Supplemental Table 1. In total, 773 mother-infant pairs met the eligibility criteria and participated in the study (baseline group). Of these 773 subjects, 467 women were excluded due to insufficient urine samples or cord blood samples for OP exposure or reproductive hormone measurements. Thus, 306 women who had measurements of both maternal urinary DAP metabolites and cord blood reproductive hormone levels were included in the present study (study population). The participating women were interviewed by specially trained nurses in the hospital using standardized questionnaires, which included demographic characteristics (maternal age, education level, address, household salary), lifestyle characteristics (alcohol use, cigarette smoking, dietary habits), environmental exposure (a nearby field or the use of pesticides during pregnancy), and reproductive and medical histories. This research was approved by the Medical Ethics Committee of Xinhua Hospital affiliated with Shanghai Jiao Tong University School of Medicine.

2.4. Statistical analysis Descriptive statistical analyses were performed for characteristics of the study population and concentrations of DAP metabolites and reproductive hormones. Because of the low detection rates of DMDTP and DEDTP (< 5%), they were excluded from further statistical analysis. Individual metabolite levels below the LOD were replaced by a value equal to the LOD divided by the square root of 2 (Hornung and Reed, 1990). Molar sums of DAP (DAPs) concentrations (nmol/L) were calculated by summing molar concentrations of DMP, DMTP, DEP, and DETP to provide summary measures of OP exposure. The formulas to convert each metabolite from its untransformed concentration (μg/L) to the corresponding molar concentration (nmol/L) has been reported elsewhere (DMP = concentration/0.126 μg/nmol; DMTP = concentration/0.142 μg/nmol; DEP = concentration/0.154 μg/nmol; DETP = concentration/0.170 μg/nmol) (Arcury et al., 2006). Potential associations between DAP metabolite concentrations and reproductive hormone levels were estimated via the multiple linear regression model. Concentrations of both DAP metabolites and reproductive hormones were log10 transformed to obtain normal distributions. In each model, the individual DAP metabolite level was the independent variable, the hormone was the dependent variable, and the standardized coefficient (β) and its 95% confidence interval (95% CI) were estimated. Changes in reproductive hormone concentrations associated with a 10-fold increase in individual urinary DAP metabolites were calculated as 10^β, where β is the coefficient from the corresponding multivariable regression model. DAP metabolites were categorized into concentration quartiles to examine the dose-response relationship using generalized linear model (GLM) with the lowest quartile as the reference group. To calculate a p-value for the trend, the median values of each metabolite within the quartiles of maternal urinary DAP concentrations were assigned (Greenland, 1995). The effect of OP exposure may differ by sex (Liu et al., 2016; Wang et al., 2012). Sex differences may exist in pesticide metabolism or the processes of repairing the damage caused by OPs (Arbuckle, 2006). To assess the effect modification of the relationship between DAP metabolites and fetal reproductive hormones by infant sex, all the models were stratified by infant sex. The inclusion of covariates was based on the literature and biological considerations (Blanco-Munoz et al., 2010; Dalsager et al., 2017; Araki et al., 2018). Covariates were also included in models if they were

2.2. Urinary OP exposure measurement Maternal midstream spot urine samples were collected during hospital admission before delivery. All samples were immediately aliquoted and stored at −80 °C until analysis. Six nonspecific DAP metabolites of OPs in maternal urine were analyzed: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The metabolites were measured using gas chromatography-mass spectrometry (GC-MS) (PE Clarus600s/MSD, USA). The limits of detection (LODs) for DAP metabolites were 0.18 μg/ L for DMP, 0.3 μg/L for DMTP and DMDTP, 0.06 μg/L for DEP and DETP, and 0.09 μg/L for DEDTP. Quality control (QC) samples consisted of urine blanks and urine spikes. The urine blanks were composed of pooled urine from eight healthy adult volunteers, and two kinds of urine spikes were prepared by adding predetermined high vs. low concentrations of six individual DAP metabolites to urine blanks separately. QC samples and urine samples were measured simultaneously. We measured one urine blank and four urine spikes (2 duplicate 2

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Table 1 Sociodemographic characteristics of the study population. Characteristic Continuous variables [Mean ± SD, (range)] Maternal age (years) Pre-pregnancy BMI (kg/m2) Gestational age (weeks) Categorical variables (n%) Maternal education (years) ≤9 (middle school) 10–12 (high school) ≥13 (greater than high school or college) Household monthly income (RMB, yuan) ≤3000 3000-5000 > 5000 Smoking during pregnancy No Lived with smoker Yes Parity 0 ≥1 Infant sex Male Female a b c

This study (n = 306)

Original cohort (n = 773)

p

28.43 ± 4.33 (18.00–41.00) 22.11 ± 3.53 (12.86–40.39) 39.38 ± 1.39 (30.00–43.00)

28.34 ± 4.50 (18.00–44.50) 21.93 ± 3.25 (12.86–40.39) 39.47 ± 1.39 (30.00–44.00)

0.779a 0.410a 0.377a

138 (45.1) 92 (30.1) 76 (24.8)

357 (46.2) 219 (28.3) 197 (25.5)

169 (55.2) 106 (34.7) 31 (10.1)

461 (59.6) 239 (30.9) 73 (9.5)

196 (64.1) 108 (35.3) 2 (0.6)

521 (67.4) 248 (32.1) 4 (0.5)

199 (65.0) 107 (35.0)

519 (67.1) 254 (32.9)

158 (51.6) 148 (48.4)

397 (51.4) 376 (48.6)

0.899b

0.212b

0.569c

0.828c

0.935c

Means were compared by T-test. Mean rank was compared by Mann-Whitney U test. Proportions were compared by Pearson chi-square test.

3672.98 pg/mL, 2.29 ng/mL, 15.59 mIU/mL, and 5.51 mIU/mL, respectively (Supplemental Table 2).

associated with any fetal reproductive hormones in our study (p < 0.15). Finally, the regression models included the following covariates: maternal age, pre-pregnancy BMI, maternal education, parity, household monthly income, and smoking during pregnancy. All statistical analyses were performed using SPSS 19.0 (IBM); p < 0.05 (two-tailed) was considered statistically significant.

3.3. OP exposure and reproductive hormones We analyzed the relationships between maternal DAP metabolite levels and reproductive hormone concentrations (Table 3), and further analyzed the dose-response relationship between reproductive hormone levels and quartiles of DAP metabolites (Fig. 1). After fully adjusting for the potential confounders, we found a significant inverse association between log-unit DEP concentrations and log-unit E2 levels (β = −0.03, 95% CI: −0.07, −0.00), indicating that a 10-fold increase in prenatal DEP concentrations was associated with a statistically significant 1.07 pg/mL decrease in E2 levels in cord blood. In the same way, for each 10-fold increase in DMP, DETP and DAPs concentrations, T levels were reduced by 1.20–1.26 ng/mL (DMP: β = −0.08, 95% CI: −0.13, −0.03; DETP: β = −0.08, 95% CI: −0.14, −0.01; DAPs: β = −0.10, 95% CI: −0.17, −0.03). For each 10-fold increase in DMP concentrations, FSH levels were reduced by 1.07 mIU/mL (β = −0.03, 95% CI: −0.05, −0.01). In addition, each 10-fold increase in DMP and DETP exposure was associated with a decrease in the T/E2 ratio of 1.15–1.17 (DMP: β = −0.06, 95% CI: −0.10, −0.01; DETP: β = −0.07, 95% CI: −0.13, −0.01). The results of categorical variables mostly showed dose-response relationships (p-trend < 0.05) and were consistent with those of continuous variables, except for DMP and FSH (p-trend = 0.072) and DETP and the T/E2 ratio (p-trend = 0.103). However, we observed additional dose-response relationships for DETP, DAPs and FSH (DEPT: p-trend = 0.040; DAPs: p-trend = 0.041), as well as DAPs and the E2/T ratio (p-trend = 0.036). There was no significant association between any DAP metabolite concentrations and LH levels in the linear regression models. After stratified by infant sex (Table 4), inverse associations were mainly observed among female infants. We found that concentrations of DMP (β = −0.05; 95% CI: −0.07, −0.02), DEP (β = −0.06; 95% CI: −0.09, −0.02), DETP (β = −0.04; 95% CI: −0.08, −0.00) and DAPs (β = −0.07; 95% CI: −0.11, −0.04) were inversely associated with E2; DMP (β = −0.12; 95% CI: −0.18, −0.06), DETP (β = −0.10; 95% CI: −0.18, −0.02) and DAPs (β = −0.13; 95% CI: −0.21,

3. Results 3.1. Population characteristics Table 1 describes the socio-demographic characteristics of this study population (n = 306) as well as those of the original cohort (n = 773). The average age of the pregnant women was 28.43 years (SD = 4.33), and the average pre-pregnancy BMI was 22.11 kg/m2 (SD = 3.53). A total of 54.9% of the women graduated from high school or above, and more than half of women (55.2%) lived in a household with a monthly income of less than RMB (¥) 3000 yuan. Two-thirds of the study population (65.0%) was composed of primipara women. Few women smoked, but one-third (35.3%) experienced passive smoking during pregnancy. Among the infants, 51.6% were male, and the average gestational age was 39.38 weeks (SD = 3.53). We found no substantial differences in sociodemographic characteristics between the baseline population (n = 773) and the study population (n = 306), indicating that the study population was generally representative of the original cohort. 3.2. Description of OP exposure and reproductive hormones The concentrations of DAP metabolites in maternal urine samples, both unadjusted and adjusted for creatinine, are shown in Table 2. The concentrations of DMDTP and DEDTP were not shown because of low detection rates (< 5%). The detection rate was 95.4% for DMP, 83.0% for DMTP, 98.4% for DEP, and 97.4% for DETP. The median level and range adjusted for creatinine was 22.00 (< LOD-480.36) μg/g creatinine for DMP, 2.17 (< LOD-106.34) μg/g creatinine for DMTP, 10.67 (< LOD-585.04) μg/g creatinine for DEP, and 1.82 (< LOD-102.24) μg/g creatinine for DETP. The median levels of E2, T, LH and FSH were 3

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Table 2 Concentrations of organophosphate pesticide (OP) metabolites in maternal urine (n = 306). Detection rate (%)

Not adjusted for creatinine (μg/L)

Adjusted for creatinine (μg/g)

Percentiles

DMP DMTP DEP DETP DAPsa a

95.4 83.0 98.4 97.4 –

Range

25th

50th

75th

3.69 0.43 2.33 0.31 71.20

10.23 0.76 5.02 0.76 149.46

19.54 1.87 10.98 2.41 332.18

Percentiles

< LOD-224.46 < LOD-74.57 < LOD-292.19 < LOD-48.33 4.28–2638.43

Range

25th

50th

75th

10.28 1.16 5.31 1.01 176.62

22.00 2.17 10.67 1.82 346.74

44.18 4.52 23.01 4.57 590.90

< LOD-480.36 < LOD-106.34 < LOD-585.04 < LOD-102.24 13.94–4641.53

The unit for DAPs (not adjusted for creatinine) is nmol/L; the unit for DAPs (adjusted for creatinine) is nmol/g Cre.

−0.05) were inversely associated with T; and DMP was inversely associated with FSH (β = −0.03; 95% CI: −0.05, −0.00) and the T/E2 ratio (β = −0.07; 95% CI: −0.13, −0.02) among females. In addition, the results of the categorical variables were similar to those of the continuous variables (Supplemental Figure 2). In male infants, concentrations of DMP were inversely associated with FSH levels (β = −0.04; 95% CI: −0.07, −0.01), and DETP showed an inverse association with the T/E2 ratio (β = −0.09; 95% CI: −0.17, −0.00), but no significant dose-response associations were observed (Supplemental Figure 1). There was no statistically significant association between any DAP metabolites and LH levels in either sex.

female offspring rats after in utero and postnatal exposure to chlorpyrifos-methyl (CPM), indicating that OP exposure could disrupt the reproductive hormonal profile of the offspring. Yu et al. (2013) observed decreased T and LH and increased E2 levels in male rats and increased T levels in female rats after utero exposure to OP mixtures. The results from animal studies were consistent with ours, showing that prenatal OP exposure may be related to reproductive hormone changes in newborns. Cord blood represents a mixture of hormones synthesized and secreted by the placenta and the fetus. Reproductive hormones, including E2, T, LH and FSH, are important for the growth of the gonads, such as pituitary, testicular and ovarian during fetus (Araki et al., 2018; Kuiri-Hänninen et al., 2014). Moreover, reproductive hormones also play pivotal roles for reproductive health in the long-term, including puberty development, growth and functioning of a broad range of tissues (Hart et al., 2016; Pepe et al., 2013; Kuijper et al., 2013). The fluctuations in reproductive hormones essential for fetal reproductive development may disturb fetal programming and potentially cause permanent damage (Costa, 2016). In the present study, we observed significant reductions in E2, T, FSH and the T/E2 ratio in association with DAP metabolites (DEP, DMP, DETP and DAPs) among all infants. OPs are neurotoxicants and may disturb the development of the neuro-endocrine axis. A possible mechanistic explanation for the reduction in FSH might be the inhibition of acetylcholine esterase (AChE) by OPs. The increase in ACh levels resulting from AChE activity inhibition may interfere with gonadotropin-releasing hormone (GnRH) release (Krsmanovic et al., 1998) and may have suppressed the synthesis and/or release of gonadotropins, such as FSH (Khokhar and Tyndale, 2012; Sarkar et al., 2000). T is produced by Leydig cells, which are regulated by a negative feedback loop, and the process is controlled by the hypothalamic pituitary gonadal (HPG) axis (Ivell et al., 2013). An animal study suggested that malathion exposure may cause oxidative damage to Leydig cells, thus reducing T levels (Silmen et al., 2014). On the other hand, studies have suggested that chlorpyrifos irreversibly inhibits CYP3A4, thus inhibiting the formation of 3-hydroxycarbofuran in human liver microsomes, resulting in activation of T metabolism and reduction of T levels (Usmani et al., 2004). T is produced in fetal adrenal and gonadal glands

4. Discussion In the present study, we found that prenatal OP exposure was inversely associated with T, FSH, and the T/E2 ratio. After stratified by sex, sex-related differences in the associations were observed. These results suggested that prenatal OP exposure may be related to reproductive endocrine disruption in the offspring and that this disruption might differ by sex. To the best of our knowledge, no data exist from human studies on the relationship between prenatal OP exposure and reproductive hormone levels in the offspring. Only a few epidemiological studies have estimated associations between reproductive hormones and exposure to OPs in adult men, suggesting that there are reproductive endocrinedisrupting effects of OP exposure in adult men. These studies reported inconsistent results (Omoike et al., 2015; Melgarejo et al., 2015; Blanco-Munoz et al., 2010). The conflicting results are probably due to different populations, which were recruited from infertility clinics, occupational populations or general populations, as well as due to the conduction of studies in different years and different countries. However, none of the populations studied were comparable to our population, which was composed of pregnant women and newborns. Animal studies have already reported that OP exposure could interfere with reproductive function among offspring (Jeong et al., 2006; Yu et al., 2013; Shin et al., 2015). For instance, Jeong et al. (2006) reported that serum levels of E2 and T were significantly lower in both male and

Table 3 Association of prenatal OP metabolite levels with reproductive hormone concentrations in cord blood among all infants. LgE2 β (95% CI) DMP DMTP DEP DETP DAPs

LgT a

−0.02 (−0.05, 0.01) 0.01 (−0.03, 0.04) −0.03 (-0.07, -0.00)* −0.01 (−0.04, 0.03) −0.04 (−0.08, 0.00)

LgLH

p

β (95% CI)

0.157 0.729 0.041 0.702 0.063

−0.08 (-0.13, -0.03)** −0.04 (−0.10, 0.03) −0.02 (−0.08, 0.04) −0.08 (-0.14, -0.01)* −0.10 (-0.17, -0.03)**

a

LgFSH

p

β (95% CI)

0.004 0.233 0.488 0.020 0.007

−0.02 (−0.08, 0.04) −0.02 (−0.09, 0.05) 0.04 (−0.03, 0.11) −0.05 (−0.12, 0.03) −0.00 (−0.09, 0.08)

a

Lg (T/E2)

p

β (95% CI)

0.540 0.560 0.248 0.213 0.914

−0.03 (-0.05, -0.01)** −0.00 (−0.02, 0.02) 0.01 (−0.01, 0.03) −0.01 (−0.03, 0.02) −0.02 (−0.05, 0.01)

a

p

β (95% CI)a

p

0.001 0.879 0.539 0.542 0.117

−0.06 (-0.10, -0.01)* −0.04 (−0.10, 0.01) 0.01 (−0.04, 0.07) −0.07 (-0.13, -0.01)* −0.06 (−0.12, 0.00)

0.015 0.113 0.629 0.015 0.057

* Significant at p < 0.05. ** Significant at p < 0.01. a Models adjusted for maternal age, pre-pregnancy BMI, maternal education, parity, household monthly income, and smoking during pregnancy. 4

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Table 4 Association of prenatal OP metabolite levels with reproductive hormone concentrations in cord blood among male and female infants. LgE2

LgT

Male DMP DMTP DEP DETP DAPs Female DMP DMTP DEP DETP DAPs

LgLH

β (95% CI)

p

β (95% CI)

0.01 (−0.04, 0.07) 0.04 (−0.01, 0.10) −0.01 (−0.07, 0.05) 0.03 (−0.03, 0.08) 0.01 (−0.06, 0.08)

0.647 0.141 0.676 0.402 0.810

−0.04 −0.01 −0.01 −0.06 −0.07

−0.05 (-0.07, -0.02)** −0.03 (−0.07, 0.01) −0.06 (-0.09, -0.02)** −0.04 (-0.08, -0.00)* −0.07 (-0.11, -0.04)**

0.001 0.098 0.001 0.002 0.000

−0.12 (-0.18, -0.06)** −0.07 (−0.15, 0.01) −0.03 (−0.11, 0.05) −0.10 (-0.18, -0.02)* −0.13 (-0.21, -0.05)**

a

a

(−0.13, (−0.11, (−0.11, (−0.16, (−0.19,

0.05) 0.08) 0.08) 0.04) 0.05)

LgFSH

p

β (95% CI)

p

β (95% CI)

0.351 0.792 0.788 0.229 0.237

0.03 (−0.07, 0.13) −0.01 (−0.12, 0.10) 0.03 (−0.07, 0.14) −0.06 (−0.17, 0.05) 0.00 (−0.13, 0.13)

0.531 0.843 0.537 0.313 0.993

0.000 0.106 0.409 0.021 0.002

−0.06 (−0.13, 0.02) −0.01 (−0.10, 0.09) 0.05 (−0.03, 0.14) −0.02 (−0.12, 0.07) −0.00 (−0.10, 0.10)

0.128 0.874 0.211 0.628 0.971

a

Lg (T/E2) p

β (95% CI)a

p

−0.04 (-0.07, -0.01)** −0.00 (−0.03, 0.03) 0.01 (−0.02, 0.04) −0.00 (−0.03, 0.03) −0.03 (−0.06, 0.01)

0.007 0.942 0.502 0.942 0.178

−0.06 (−0.13, 0.02) −0.06 (−0.14, 0.03) 0.00 (−0.08, 0.08) −0.09 (-0.17, -0.00)* −0.08 (−0.18, 0.02)

0.159 0.180 0.984 0.046 0.122

−0.03 (-0.05, -0.00)* −0.00 (−0.03, 0.03) 0.00 (−0.03, 0.03) −0.02 (−0.05, 0.02) −0.02 (−0.05, 0.02)

0.021 0.919 0.927 0.383 0.297

−0.07 (-0.13, -0.02)** −0.04 (−0.11, 0.04) 0.03 (−0.04, 0.09) −0.06 (−0.13, 0.02) −0.06 (−0.13, 0.02)

0.008 0.332 0.459 0.144 0.143

a

* Significant at p < 0.05. ** Significant at p < 0.01. a Models adjusted for maternal age, pre-pregnancy BMI, maternal education, parity, household monthly income, and smoking during pregnancy among male and female infants separately.

and < LOD in the NHANES population (U.S. CDC, 2019). The relatively high level of OP exposure in the current population may be attributable to the heavy use of OP pesticides and high residue in fruits and vegetables (Chen et al., 2009; Wang et al., 2012). Given the relatively high level of OP exposure among pregnant women, China urgently needs to conduct studies to evaluate the health effects of OP exposure on susceptible populations. However, there are some limitations in this study. First, we used single-point DAP metabolite measurements from spot urine samples to represent the level of OP exposure during the whole pregnancy. The measured DAP concentrations in a single spot urine sample might be less-representative biomarkers of OP chronic exposure, given the short half-lives (12–36 h) and high within-person variability in these compounds (Needham, 2005; Bradman et al., 2013; Spaan et al., 2015). However, previous studies have pointed out that a person's exposure level would be stable if his or her living environment, activities and habits were relatively stable and if levels of the contaminant in the microenvironments remained consistent over the course of months (Meeker et al., 2005). Nevertheless, repeated measurements of urine samples collected throughout pregnancy may provide a more accurate estimate of the average exposure during the whole gestational period. Second, although we adjusted for multiple confounders with potential effects on reproductive hormones, other possible covariates that may be associated with the outcome were unadjusted, such as co-exposure to other EDCs with reproductive endocrine disrupting activities. Third, although the measurement of DAP metabolites is the most current technique to estimate multiple OP exposures from different sources, it should be noted that the levels of DAP metabolites may reflect exposure not only to OP parent compounds but also to DAP metabolites in the environment and foods (Lu et al., 2005). Fourth, given the cross-sectional nature of the design, our findings only provide evidence of potential associations that warrant further evaluation. Fifth, we corrected DAP levels using creatinine levels, which may not accurately reflect urine dilution in pregnant women (MacPherson et al., 2018; Kuromoto et al., 2010), although many researchers continue to adjust DAP metabolites by urine creatinine in pregnant women (Endre et al., 2016; O’Brien et al., 2017). Nonetheless, we also excluded samples with urinary creatinine levels below 0.05 g/L (Eskenazi et al., 2004; Wolff et al., 2008) in the sensitivity analysis to validate our results, and this exclusion did not alter the observed associations (data not shown). Sixth, the results observed in our study have small effect differences. Until now, it has been difficult to predict the clinical significance of these numbers owing to the lack of available reference ranges for reproductive hormones in cord blood as well as the limited reported data. A balanced hormonal environment has been considered to be essential

in late gestation and is then delivered to the placenta and converted to E2 by aromatase (Kuijper et al., 2013). The decreasing levels of T may be responsible for the reduction in E2. The T/E2 ratio is considered an indicator of aromatase enzyme activity (Araki et al., 2018), and the decreased T/E2 ratio in this study indicated increased aromatase enzyme activity. A previous animal study showed that monocrotophos, one kind of OP, led to elevated gonadal aromatase (cyp19A1a) gene expression and increased the transcription of brain aromatase (cyp19A1b) (Zhang et al., 2013). Overall, we speculate that OPs could induce anti-androgenic effects on infants after prenatal exposure. Additional studies on alteration in reproductive hormones associated with OP exposure are required to test these hypotheses. Furthermore, we observed a sex-dependent association of prenatal OP exposure with the cord blood reproductive hormones; female infants were more vulnerable than males. Prenatal OP exposure was significantly inversely associated with E2 and T levels in cord blood only among female infants but not among male infants. However, limited evidence is available. Similar to our study, Wang et al. (2012) found an inverse association between DEP levels and duration of gestation among female infants but no association in males. Another follow-up study showed that prenatal occupational pesticide exposure might be related to earlier breast development, higher serum androstenedione levels and lower anti-Mullerian hormone (AMH) in girls (WohlfahrtVeje et al., 2012). The possible mechanisms remain unclear; however, sex-dependent fetal endocrine alterations could be induced by prenatal exposure to EDCs, and the placentas of fetuses of different sexes might possess different abilities to respond to environmental fluctuations (Rosenfeld, 2012). To our knowledge, this is the first study assessing the possible relationships between maternal OP exposure and cord blood reproductive hormone status among Chinese pregnant women. Women in our study were recruited from the general population, and our findings therefore should be reasonably generalizable to the overall population. Moreover, we measured urinary concentrations of metabolites of OPs among pregnant women, and these metabolites have been widely used as biomarkers of most OP pesticides and reflect the integration of multiple routes of exposure to OPs (Hardt and Angerer, 2000; Lu et al., 2005). The maternal median concentrations of major DAP metabolites (DMP and DEP) in our study were higher than those reported in developed countries (Supplemental Table 3). For DMP, the median concentration was 22.00 μg/g in the present study versus 2.20 μg/g in Japan (Hioki et al., 2019), 8.37 μg/g in Europe (Haug et al., 2018) and < LOD in the NHANES population (U.S. CDC, 2019). For DEP, the median concentration was 10.67 μg/g in the present study versus 4.80 μg/g in Japan (Hioki et al., 2019), 3.33 μg/g in Europe (Haug et al., 2018) 5

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Fig. 1. Adjusted regression coefficients [β (95% CI)] of the association of a change in lg (reproductive hormones) with increasing quartiles of DAP metabolites among all infants. The p -trend was calculated by assigning the median concentrations to all persons for each corresponding quartile. The results were based on generalized linear models that were adjusted for maternal age, pre-pregnancy BMI, maternal education, parity, household monthly income, and smoking during pregnancy. Q, quartile. CI, confidence interval * Significant at p < 0.05; ** Significant at p < 0.01.

with larger sample sizes are needed to confirm our findings.

for normal fetal development during pregnancy (Williams et al., 2001), and small effects that alter levels of reproductive hormones could have been associated with some pathologies, such as intrauterine growth restriction or preeclampsia (Kuijper et al., 2013). Given that maternal urine pesticide levels in the LWBC were much higher than those reported in developed countries, more studies on the effects of OP exposure are warranted. Finally, our results should be interpreted with caution because of the potential for multiple comparisons. However, we found that the association of DAP metabolites with concurrently decreased E2, T, and the T/E2 ratio is consistent with the dose-response relationships, supporting that our results may not be completely due to chance.

Funding This study was funded by National Key Research and Development Program of China (2016YFC1000203, 2017YFC1600500), National Natural Science Foundation of China (81773387, 81630085, 81602823, 81803185), Science and Technology Commission of Shanghai Municipality (17ZR1415800), Shanghai Municipal Health and Family Planning Commission (201840180). Declaration of competing interest The authors declare they have no actual or potential competing financial interests.

5. Conclusions In summary, the present study, based on the LWBC, suggested the potential impacts of prenatal OP exposure on fetal reproductive hormones. Sex-related differences might exist, with female infants being more vulnerable than males. However, more epidemiological studies

Acknowledgements We thank the Department of Environmental Health staff, students, 6

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participants, and hospital partners.

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