Person:

Williams, Paige

Background: CYP19 and PPARγ are two genes expressed in the placental trophoblast that are important to placental function and are disrupted by phthalate exposure in other cell types. Measurement of the mRNA of these two genes in human placental tissue by quantitative real-time polymerase chain reaction (qPCR) offers a source of potential biomarkers for use in epidemiologic research. We report on methodologic challenges to be considered in study design. Methods: We anonymously collected 10 full-term placentas and, for each, sampled placental villi at 12 sites in the chorionic plate representing the inner (closer to the cord insertion site) and outer regions. Each sample was analyzed for the expression of two candidate genes, aromatase (CYP19) and peroxisome proliferator activated receptor protein gamma (PPARγ) and three potential internal controls: cyclophilin (CYC), 18S rRNA (18S), and total RNA. Between and within placenta variability was estimated using variance component analysis. Associations of expression levels with sampling characteristics were estimated using mixed effects models. Results: We identified large within-placenta variability in both transcripts (>90% of total variance) that was minimized to <20% of total variance by using 18S as an internal control and by modelling the means by inner and outer regions. 18S rRNA was the most appropriate internal control based on within and between placenta variability estimates and low correlations of 18S mRNA with target gene mRNA. Gene expression did not differ significantly by delivery method. We observed decreases in the expression of both transcripts over the 25 minute period after delivery (CYP19 p-value for trend = 0.009 and PPARγ (p-value for trend = 0.002). Using histologic methods, we confirmed that our samples were comprised predominantly of villous tissue of the fetal placenta with minimal contamination of maternally derived cell types. Conclusion: qPCR-derived biomarkers of placental CYP19 and PPARγ gene expression show high within-placental variability. Sampling scheme, selection of an appropriate internal control and the timing of sample collection relative to delivery can be optimized to minimize within-placenta and other sources of underlying, non-etiologic variability.

Background: Although urinary concentrations of phthalate metabolites are frequently used as biomarkers in epidemiologic studies, variability during pregnancy has not been characterized. Methods: We measured phthalate metabolite concentrations in spot urine samples collected from 246 pregnant Dominican and African-American women. Twenty-eight women had repeat urine samples collected over a 6-week period. We also analyzed 48-hr personal air samples (n = 96 women) and repeated indoor air samples (n = 32 homes) for five phthalate diesters. Mixed-effects models were fit to evaluate reproducibility via intraclass correlation coefficients (ICC). We evaluated the sensitivity and specificity of using a single specimen versus repeat samples to classify a woman’s exposure in the low or high category. Results: Phthalates were detected in 85–100% of air and urine samples. ICCs for the unadjusted urinary metabolite concentrations ranged from 0.30 for mono-ethyl phthalate to 0.66 for monobenzyl phthalate. For indoor air, ICCs ranged from 0.48 [di-2-ethylhexyl phthalate (DEHP)] to 0.83 [butylbenzyl phthalate (BBzP)]. Air levels of phthalate diesters correlated with their respective urinary metabolite concentrations for BBzP (r = 0.71), di-isobutyl phthalate (r = 0.44), and diethyl phthalate (DEP; r = 0.39). In women sampled late in pregnancy, specific gravity appeared to be more effective than creatinine in adjusting for urine dilution. Conclusions: Urinary concentrations of DEP and DEHP metabolites in pregnant women showed lower reproducibility than metabolites for di-n-butyl phthalate and BBzP. A single indoor air sample may be sufficient to characterize phthalate exposure in the home, whereas urinary phthalate biomarkers should be sampled longitudinally during pregnancy to minimize exposure misclassification.

Background: Although sources and routes of exposure to dioxins and polychlorinated biphenyls (PCBs) have been studied, information regarding exposure among children is limited. Breast-feeding and diet are two important contributors to early life exposure. To further understand other significant contributors to childhood exposure, we studied a cohort of children from a city with high environmental dioxin levels. Objectives: We investigated predictors of serum concentrations of polychlorinated dibenzo-p-dioxins (PCDDs)/polychlorinated dibenzofurans (PCDFs)/co-planar PCBs (C-PCBs), toxic equivalents (TEQs), and PCBs among 8- to 9-year-old boys in Chapaevsk, Russia. Methods: We used general linear regression models to explore associations of log10-transformed serum concentrations of PCDDs/PCDFs/C-PCBs, TEQs, and PCBs at study entry with anthropometric, demographic, geographic, and dietary factors in 482 boys in Chapaevsk, Russia. Results: The median (25th, 75th percentile) concentration for total 2005 TEQs was 21.1 pg/g lipid (14.4, 33.2). Boys who were older, consumed local foods, were breast-fed longer, and whose mothers were employed at the Khimprom chemical plant (where chlorinated chemicals were produced) or gardened locally had significantly higher serum dioxins and PCBs, whereas boys with higher body mass index or more educated parents had significantly lower serum dioxins and PCBs. Boys who lived less than 2 km from Khimprom had higher total TEQs (picograms per gram lipid) [adjusted mean = 30.6; 95% confidence interval (CI), 26.8–35.0] than boys who lived greater than 5 km away (adjusted mean = 18.8; 95% CI, 17.2–20.6). Conclusions: Our findings suggest that there are specific local sources of dioxin and PCB exposure among children in Chapaevsk including maternal gardening, consumption of locally grown food, and residential proximity to the Khimprom plant.

Background: Gestational phthalate and bisphenol A (BPA) exposure may increase the risk of adverse maternal/child health outcomes, but there are few data on the variability of urinary biomarkers before and during pregnancy.: Objective: We characterized the variability of urinary phthalate metabolite and BPA concentrations before and during pregnancy and the ability of a single spot urine sample to classify average gestational exposure. Methods: We collected 1,001 urine samples before and during pregnancy from 137 women who were partners in couples attending a Boston fertility clinic and who had a live birth. Women provided spot urine samples before (n ≥ 2) and during (n ≥ 2) pregnancy. We measured urinary concentrations of monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-iso-butyl phthalate, monobenzyl phthalate (MBzP), four metabolites of di-(2-ethylhexyl) phthalate (DEHP), and BPA. After adjusting for specific gravity, we characterized biomarker variability using intraclass correlation coefficients (ICCs) and conducted several surrogate category analyses to determine whether a single spot urine sample could adequately classify average gestational exposure. Results: Absolute concentrations of phthalate metabolites and BPA were similar before and during pregnancy. Variability was higher during pregnancy than before pregnancy for BPA and MBzP, but similar during and before pregnancy for MBP, MEP, and ΣDEHP. During pregnancy, MEP (ICC = 0.50) and MBP (ICC = 0.45) were less variable than BPA (ICC = 0.12), MBzP (ICC = 0.25), and ΣDEHP metabolites (ICC = 0.08). Surrogate analyses suggested that a single spot urine sample may reasonably classify MEP and MBP concentrations during pregnancy, but more than one sample may be necessary for MBzP, DEHP, and BPA. Conclusions: Urinary phthalate metabolites and BPA concentrations were variable before and during pregnancy, but the magnitude of variability was biomarker specific. A single spot urine sample adequately classified MBP and MEP concentrations during pregnancy. The present results may be related to unique features of the women studied, and replication in other pregnancy cohorts is recommended.:

Background: Phthalates can alter steroidogenesis and peroxisome proliferator–activated receptor gamma (PPARγ)–mediated transcription in rodent tissues. The placenta offers a rich source of biomarkers to study these relationships in humans. Objective: We evaluated whether gestational phthalate exposures in humans were associated with altered human placental steroidogenesis and trophoblast differentiation as measured by markers of mRNA transcription. Methods: We measured seven target genes in placentas collected from 54 Dominican and African-American women at delivery in New York City using quantitative real-time polymerase chain reaction (qPCR), normalized to 18S rRNA. qPCR results for the target genes were log-transformed, converted to Z-scores, and grouped into two functional pathways: steroidogenesis (aromatase, cholesterol side chain cleavage enzyme, 17β-hydroxysteroid dehydrogenase type 1, and cytochrome P450 1B1) and trophoblast differentiation (PPARγ, aryl hydrocarbon receptor, and human chorionic gonadotropin). Repeated measures models were used to evaluate the association of phthalate metabolites measured in third-trimester urine samples with each group of target genes, accounting for correlation among the genes within a pathway. Results: Higher urinary concentrations of five phthalate metabolites were associated with lower expression of the target genes reflecting trophoblast differentiation. Results were less consistent for genes in the steroidogenesis pathway and suggested a nonlinear dose–response pattern for some phthalate metabolites. Conclusions: We observed a significant association between prenatal exposure to phthalates and placental gene expression within two pathways. Further studies are warranted to understand the significance of this association with respect to fetal development and placental function.

Background: Toxicological studies and limited human studies have demonstrated associations between exposure to polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) and adverse developmental and reproductive health effects. Given that children may be particularly susceptible to reproductive and developmental effects of organochlorines, and the paucity of information available regarding childhood exposures to dioxins in particular, we undertook a pilot study to describe the distribution of, and identify potential predictors of exposure to, dioxin-like compounds and dioxins among adolescent boys in Chapaevsk, Russia. The pilot study was also designed to guide the development of a large prospective cohort study on the relationship of exposure to PCDDs, PCDFs, and PCBs with growth and pubertal development in peri-pubertal Chapaevsk boys. Methods: 221 boys age 14 to 17 participated in the pilot study. Each of the boys, with his mother, was asked to complete a nurse-administered detailed questionnaire on medical history, diet, and lifestyle. The diet questions were used to measure the current and lifetime consumption of locally grown or raised foods. Blood samples from 30 of these boys were sent to the Centers for Disease Control and Prevention (CDC) for analysis of dioxins, furans and PCBs. Results: The median (25th, 75th percentile) concentrations for total PCDDs, PCDFs and coplanar PCBs were 95.8 pg/g lipids (40.9, 144), 33.9 pg/g lipids (20.4, 61.8), and 120 pg/g lipids (77.6, 157), respectively. For WHO-TEQs, the median (25th, 75th percentile) for total PCDDs, PCDFs, and coplanar PCBs were 0.29 (0.1, 9.14), 7.98 (5.27, 12.3), and 7.39 (4.51, 11.9), respectively. Although TCDD was largely non-detectable, two boys had high TCDD levels (17.9 and 21.7 pg/g lipid). Higher serum levels of sum of dioxin-like compounds and sum of dioxin TEQs were positively associated with increased age, consumption of fish, local meats other than chicken, PCB 118, and inversely with weeks of gestation. Conclusion: The total TEQs among Chapaevsk adolescents were higher than most values previously reported in non-occupationally exposed populations of comparable or even older ages. Dietary consumption of local foods, as well as age and weeks of gestation, predicted dioxin exposure in this population.

Background: Epidemiologic studies suggest a temporal trend of earlier onset and longer duration of puberty, raising concerns regarding the potential impact of environmental factors on pubertal development. Lead exposure has been associated with delayed pubertal onset in girls; however, epidemiologic data in boys are limited. Methods: We used multivariable logistic regression models to explore the cross-sectional association of blood lead levels with growth and pubertal onset based on physician-assessed testicular volume (TV) and pubertal staging in 489 boys 8–9 years of age from Chapaevsk, Russia. We used multivariable linear regression models to assess associations of blood lead levels with somatic growth at the study entry visit. Results: The median (25th–75th percentile) blood lead level was 3 μg/dL (2–5 μg/dL). Height, weight, body mass index, birth weight, and gestational age were predictive of the onset of puberty as assessed either by TV (greater than 3 mL), genitalia stage (G2), or both. Blood lead level was inversely associated with height (p less than 0.001) and weight (p = 0.06) after adjustment for birth weight, gestational age, and age at examination. In multivariable adjusted analyses, boys with blood lead levels ≥ 5 μg/dL had 43% reduced odds of having entered G2 compared with those with lower levels (odds ratio = 0.57; 95% confidence interval, 0.34–0.95, p = 0.03). Conclusions: Relatively low environmental blood lead levels were associated with decreased growth and differences in pubertal onset in periadolescent Russian boys. Future analyses of this prospective cohort will address pubertal onset and progression in relation to lead and other environmental chemicals.

Previously, we reported evidence of inverse associations between exposure to some polychlorinated biphenyls (PCBs) and some phthalate monoesters in relation to semen parameters, specifically sperm motility. Because humans are exposed to both phthalates and PCBs and because experimental studies suggest that PCBs may interact with glucuronidative enzymes that are responsible for phthalate metabolism, we explored the potential interaction between phthalates and PCBs in relation to human semen quality. We studied 303 men who were partners in subfertile couples seeking infertility diagnosis from the andrology laboratory at Massachusetts General Hospital. Semen parameters were dichotomized based on World Health Organization reference values, and phthalate and PCB levels were dichotomized at their respective medians. After adjusting for age and abstinence time, for below reference sperm motility there was a greater than additive interaction between monobenzyl phthalate and PCB-153 [relative excess risk due to interaction (RERI) = 1.40; 95% confidence interval (CI), 0.41–3.22], sum of PCBs (RERI = 1.24; 95% CI, 0.15–2.94), and cytochrome P450 (CYP450)-inducing PCBs (RERI = 1.30; 95% CI, 0.21–3.06). For below-reference sperm motility, there was also a greater than additive interaction between monobutyl phthalate (MBP) and PCB-153 (RERI = 1.42; 95% CI, 0.09–3.76) and CYP450-inducing PCBs (RERI = 1.87; 95% CI, 0.56–4.52) and a suggestive interaction between MBP and sum of PCBs (RERI = 1.35; 95% CI, −0.11 to 3.48). In conclusion, because there are important risk assessment and public health implications of interactions between these two ubiquitous classes of compounds, further studies need to be conducted to confirm these results and identify potential mechanisms of interactions.

Background: Bisphenol A (BPA) is a synthetic chemical widely used in the production of polycarbonate plastic and epoxy resins found in numerous consumer products. In experimental animals, BPA increases embryo implantation failure and reduces litter size. Objective: We evaluated the association of urinary BPA concentrations with implantation failure among women undergoing in vitro fertilization (IVF). Methods: We used online solid phase extraction–high performance liquid chromatography–isotope dilution tandem mass spectrometry to measure urinary BPA concentrations in 137 women in a prospective cohort study among women undergoing IVF at the Massachusetts General Hospital Fertility Center in Boston, Massachusetts. We used logistic regression to evaluate the association of cycle-specific urinary BPA concentrations with implantation failure, accounting for correlation among multiple IVF cycles in the same woman using generalized estimating equations. Implantation failure was defined as a negative serum β-human chorionic gonadotropin test (β-hCG < 6 IU/L) 17 days after egg retrieval. Results: Among 137 women undergoing 180 IVF cycles, urinary BPA concentrations had a geometric mean (SD) of 1.53 (2.22) µg/L. Overall, 42% (n = 75) of the IVF cycles resulted in implantation failure. In adjusted models, there was an increased odds of implantation failure with higher quartiles of urinary BPA concentrations {odds ratio (OR) 1.02 [95% confidence interval (CI): 0.35, 2.95}, 1.60 (95% CI: 0.70, 3.78), and 2.11 (95% CI: 0.84, 5.31) for quartiles 2, 3, and 4, respectively, compared with the lowest quartile (p-trend = 0.06). Conclusion: There was a positive linear dose–response association between BPA urinary concentrations and implantation failure.

Background: Hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT), and dichlorodiphenyldichloroethylene (DDE) are persistent chlorinated pesticides with endocrine activity that may adversely affect the early stages of human reproduction. Objective: Our goal was to determine the association of serum levels of HCB, DDT, and DDE with implantation failure, chemical pregnancy, and spontaneous abortion in women undergoing in vitro fertilization (IVF) from 1994 to 2003. Methods: Levels of HCB and congeners of DDT and DDE were measured in serum collected during the follicular phase. Multivariable-adjusted statistical models accommodating multiple outcomes and multiple cycles per woman were used to estimate the relation between serum pesticide levels and IVF outcomes. Results: A total of 720 women with a mean (\pm) SD age 35.4 (\pm) 4.2 years at enrollment contributed 774 IVF cycles. All samples had detectable levels of HCB, DDT, and DDE, with median levels of 0.087 ng/g serum for HCB, 1.12 ng/g serum for total DDT, and 1.04 ng/g serum for p,p´-DDE. Compared with the lowest quartile (Q1) of HCB, the lipid- and multivariable-adjusted odds ratio (OR) for failed implantation was significantly elevated for those with higher HCB quartiles [Q2–Q4; adjusted ORs: for Q2, 1.71; 95% confidence interval (CI): 1.03, 2.82; for Q3, 2.30; 95% CI: 1.39, 3.81; for Q4, 2.32; 95% CI: 1.38, 3.90] and showed a significantly increasing trend (p = 0.001). No statistically significant associations were observed between DDT/DDE and IVF outcomes or between HCB and chemical pregnancy or spontaneous abortion. Conclusions: Serum HCB concentrations were on average lower than that of the general U.S. population and associated with failed implantation among women undergoing IVF.