Article pubs.acs.org/est
Levels of Phthalate Metabolites in Urine of Pregnant Women and Risk of Clinical Pregnancy Loss Di Mu,†,‡ Fumei Gao,†,‡,§ Zhanlan Fan,‡ Huan Shen,*,§ Hui Peng,‡ and Jianying Hu*,‡ ‡
Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, People’s Republic of China § Reproductive Medical Center, Peking University People’s Hospital, Peking University, Beijing 100044, People’s Republic of China S Supporting Information *
ABSTRACT: Toxicological studies have shown that phthalate esters (PAEs), a class of widely used and environmentally prevalent chemicals, can increase the abortion rate in animals, but epidemiological evidence is scarce. This study aimed to explore the relationship between the urinary concentration of phthalate metabolites and the risk of clinical pregnancy loss. A total of 132 women who underwent clinical pregnancy loss (cases) and 172 healthy pregnant women (controls) were recruited from Beijing, China. Eight phthalate metabolites in urine were determined by ultraperformance liquid chromatography tandem mass spectrometry (UPLC−MS/MS). Five phthalate metabolites, monomethyl phthalate (MMP), monoethyl phthalate (MEP), monoisobutyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), and mono(2-ethlyhexyl) phthalate (MEHP), were detected in at least 95% of the urine samples, with the highest median concentration of 51.0 μg/ g of creatinine for MnBP of all participants. The differences in urinary concentrations of phthalate metabolites between cases and controls were evaluated using the Mann−Whitney U test. The concentrations of MEP (median of 18.7 μg/g of creatinine), MiBP (23.3 μg/g of creatinine), and MnBP (58.2 μg/g of creatinine) detected in the cases were significantly higher than those (15.7 μg/g of creatinine for MEP, 19.4 μg/g of creatinine for MiBP, and 43.9 μg/g of creatinine for MnBP) in the controls (p < 0.05). Increasing risks of clinical pregnancy loss were observed from the first to fourth quartiles of the MEP, MiBP, and MnBP concentrations (p < 0.05 for trend). We concluded that exposure to MEP, MiBP, and MnBP was associated with an increased risk of clinical pregnancy loss.
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INTRODUCTION Phthalates are a class of synthetic chemicals widely used in industrial and personal care products, such as paint, glue, lubricant solvents, polyvinyl chloride (PVC), food packing, and cosmetics.1−3 Because of their extensive use and non-covalent conjugation with the products, phthalates can be easily released and distributed into the surrounding environment.4 Phthalates have been found in indoor dust, foodstuffs, indoor and outdoor air, and water in various countries.5−7 It has been proven that humans are continuously exposed to these chemicals through ingestion, inhalation, dermal absorption, and contact with medical devices, and consequently, phthalate metabolites have been detected in urine in nearly 100% of individuals from various countries.8−10 It has been demonstrated that long-term exposure to low concentrations of phthalates could cause adverse effects, such as hepatocarcinogenicity and endocrine disruption, in laboratory animals. Several epidemiological studies suggested that exposure to phthalates was associated with key features of metabolic syndrome [increased body mass index (BMI), abdominal adiposity, and insulin resistance],11−13 although the results may be controversial.14 A special concern about © XXXX American Chemical Society
phthalates relates to their potential toxicity toward male reproductive development, including testicular atrophy, hypospadias, cryptorchidism, shorter anogenital distance (AGD), and malformation of epididymis, vas deferens, and seminal vesicles.15−18 Adverse female reproductive and developmental outcomes have been observed in several animal studies where the exposure of specific phthalates [diethyl phthalate (DEP), di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP), and diisobutyl phthalate (DiBP)] has significantly decreased embryo survival, increased the incidence of resorptions, reduced the number and size of litters, and increased the abortion rate in rats.19−22 The association between phthalate exposure and adverse effects on female reproductive function, such as female puberty, endometriosis, and lower birth weight, have also been observed in epidemiological studies.23−25 It has been reported that chronic occupational exposure to high levels of phthalates has been Received: May 29, 2015 Revised: August 3, 2015 Accepted: August 6, 2015
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DOI: 10.1021/acs.est.5b02617 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Chemical Analyses. In this study, a total of 304 samples were analyzed, and the concentrations of eight phthalate metabolites, including monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-n-butyl phthalate (MnBP), monoisobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), mono-n-octyl phthalate (MnOP), monoisononyl phthalate (MiNP), and MEHP, were determined. The eight phthalate metabolites (>99% purity) were purchased from AccuStandard (New Haven, CT). MEP−13C4, MnBP−13C4, MBzP−13C4, and MEHP−13C4 were purchased from Cambridge Isotope Laboratories (Andover, MA). β-Glucuronidase from Helix pomatia were purchased from Sigma-Aldrich (St. Louis, MO). The Oasis HLB (150 mg, 6 mL) cartridges were purchased from Waters (Milford, MA). The information on the solvent used in this study was presented in the Supporting Information In this study, the chemical procedure is according to the previous paper, with some modification.33 After 10 ng of MEP−13C4, MnBP−13C4, MBzP−13C4, and MEHP−13C4 was added, 1 mL of urine sample was adjusted to pH 6.5 and then treated with 20 μL of β-glucuronidase enzyme. Samples were then incubated at 37 °C for 120 min and then loaded on Oasis HLB cartridges, which were preconditioned with 5 mL of MeOH and 2 mL of Milli-Q water. After the cartridges were washed with 2 mL of distilled water, they were dried under a flow of nitrogen. A 4 mL mixture of MTBE/MeOH (9:1, v/v) was added to elute phthalate metabolites. The extract solution was dried under nitrogen gas and reconstituted in 1 mL of MeOH for analysis of ultraperformance liquid chromatography tandem mass spectrometry (UPLC−MS/MS, Waters, Milford, MA). The detailed information on mobile phases, ultraperformance liquid chromatography (UPLC) gradient conditions, mass spectrometry (MS) parameters, and multiple reaction monitoring (MRM) conditions is shown in the Supporting Information. For each batch of 20 samples analyzed, 2 procedural blanks were processed. Identification of the target analytes was accomplished by comparing the retention time (within 2%) and the ratio (within 20%) of the two selected precursor ionproduced ion transitions to those of standards. Recoveries were estimated by analysis of urine (n = 6) spiked with two different levels (20 μg/L for MiBP and MnBP, 15 μg/L for MEHP, 5 μg/L for MMP, MBzP, MnOP and MiNP, 60 μg/L for MiBP and MnBP, 45 μg/L for MEHP, and 15 μg/L for MMP, MBzP, MnOP and MiNP). The recoveries for all target chemicals ranged from 75.7 to 90.1%, and limits of detection (LODs) and limits of quantitation (LOQs) were 0.1−0.27 and 0.3−0.7 μg/ L, respectively (Table S2 of the Supporting Information). The creatinine concentration in urine was measured using an enzymatic reaction on a Roche Hitachi chemistry analyzer (Roche Hitachi, Basel, Switzerland), and the volume of the urine sample was 0.3 mL. Ethical Approval and Informed Consent. This study was approved by the Ethics Committee of Peking University People’s Hospital (2011-33). All participants were informed of the design and signed an informed consent. Statistical Analysis. Data analysis was performed with SPSS, version 16.0. Concentrations of phthalate metabolites in urine were not normally distributed; therefore, the median with range (P5 and P95) was used to describe their distributions. The comparison of concentrations between the cases and the controls were performed by the Mann−Whitney U test. Differences in concentrations of phthalate metabolites between
linked with decreased rates of pregnancy and higher rates of miscarriage in female factory workers.26 A higher urinary concentration of mono(2-ethlyhexyl) phthalate (MEHP) has been associated with biochemical pregnancy losses, which were identified on the basis of the human chorionic gonadotropin (hCG) elevation after menstrual bleeding.27 However, the relationship between the exposure of phthalates and the risk of clinical pregnancy loss, the most common adverse pregnancy outcome in humans,28 has not been examined in the nonoccupational population. This study aimed to monitor of phthalate metabolites in urine of pregnant women and explore the associations between phthalate exposure levels and clinical pregnancy loss. The results will be helpful to better understand the impacts of phthalate esters (PAEs) on the female reproduction function.
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MATERIALS AND METHODS Study Population and Urine Collection. Subjects were recruited women from the Department of Obstetrics and Gynecology of Peking University People’s Hospital in Beijing, China, from September 2011 to June 2014. A total of 399 women underwent clinical pregnancy loss, and 311 pregnant women were invited to participate in this study. Trans-vaginal ultrasound examination was used to check the development condition of embryos by experienced doctors. According to the diagnosis of clinical pregnancy loss, when women meet any of the criteria: (1) the mean sac diameter of their gestation sac is greater than 20 mm without a visible embryo, (2) the length of their embryo is more than 6 mm with invisible activity, or (3) repeat trans-vaginal ultrasound showing an absence of cardiac activity in embryos or fetus, the women were confirmed as clinical pregnancy loss. When pregnant women were found to have a fetal bud body with a heartbeat, they were invited to participate in this study as controls. Same selection criteria were adopted for exclusion of cases and controls. Gestation period (less than 20 weeks) and maternal age (20−45 years) are required. On the basis of a physical examination and self-report questionnaire, volunteers with recurrent miscarriages, history of infertility, reproductive tract abnormalities, gynecological inflammation, fever during early pregnancy, and husband in bad health were also excluded from the study. Finally, 174 cases remained, of which 132 of them completed the questionnaire and provided urine samples, and 172 controls completed the questionnaire and provided urine samples in the present study. The details of selection procedures were shown in the Supporting Information. According to several previous studies,29,30 the increase in maternal age affects the chances of miscarriage, especially for women over 35 years old. Cigarette smoking and the use of alcohol can also increase the risk of adverse pregnancy outcomes.31,32 Information of maternal age, alcohol consumption, and smoking are required. Each subject completed a face-to-face questionnaire that included information regarding demographics (age, BMI, nationality, and week of gestation), current smoking or drinking status, household income, lifestyle factors, and whether they were engaged in plastic-related occupations, such as beautician or hairdresser, under the guide of trained investigators. Urine samples were collected using a glass beaker in the morning of the fourth day after trans-vaginal ultrasound examination of the pregnancy and immediately transferred and stored in 8 mL brown glass bottles (pretreated at 500 °C for 6 h) and kept at −80 °C until analysis. B
DOI: 10.1021/acs.est.5b02617 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology cases and controls were evaluated using the Mann−Whitney U test. Demographic data suspected to influence the female risk of clinical pregnancy loss [age (continuous), BMI (continuous), weeks of gestation (≤5 weeks, 5−13 weeks, or 13−20 weeks, categorical), household income (low, medium, or high, categorical), smoking status (smokers or non-smokers, categorical), alcohol consumption (categorical), and occupation (yes or no, categorical)] were compared. t tests were performed for continuous data (age and BMI), and χ2 tests were performed for categorical data (weeks of gestation, household income, smoking status, alcohol consumption, and occupation). To explore the associations between the risk of clinical pregnancy loss and the concentrations of phthalate metabolites, we categorized the data by the quartiles of concentrations of the phthalate metabolites according to the controls, and the reference group is the lowest urinary concentration quartile of the controls. Urinary concentration quartiles (1−4) of the controls were used as the cutoff values in dose−response analysis. The risk of clinical pregnancy loss associated with urinary concentrations of phthalate metabolites was estimated by the odds ratio (OR) with 95% confidence internal (CI). The adjusted-OR was calculated using logistic regression and adjusted by confounders (age, BMI, weeks of gestation, household income, current smoking and drinking status, and occupation). Results of OR were calculated separately according to different kinds of phthalate metabolites. Trends in the OR of clinical pregnancy loss with increasing urinary phthalate metabolite levels were determined by increasing phthalate metabolite quartiles as an ordinal variable. A twotailed p value of LOD
median
P5
P95
percent > LOD
median
P5
P95
pb
MMP MEP MiBP MnBP MBzP MEHP
98.5 99.2 100 99.2 26.6 100
8.58 57.3 40.8 85.8 0.61 20.6
0.40 3.75 8.04 10.1
