In animal models phthalates and stress can alter fetal androgens and disrupt androgen-sensitive endpoints. We will examine associations between exposure to phthalates and psychosocial stress at multiple time points in early childhood development and androgen-sensitive reproductive, anthropometric, and neurodevelopmental endpoints. In The Infant Development and the Environment Study (TIDES) we confirmed that first trimester phthalate exposure is significantly and inversely associated with boy's anogenital distance (AGD) and that prenatal stress can modify this association. These novel findings suggest hypotheses that can be addressed with further follow-up of the TIDES cohort during middle childhood. We propose to extend our methodology in several novel directions in order to achieve our overarching goal of identifying the effects of exposure to phthalates and stress on androgen-sensitive development. Children born to TIDES mothers will be invited to two study visits between 3 and 6 years of age. At the 1st visit we will measure height, weight, skin fold thickness, and head circumference, and collect a sample of the child's urine for phthalate measurement. We will assess the child's attention, hyperactivity, social responsiveness, and play behavior by parental report. At the 2nd visit we will repeat these assessments and measure the length of the 2nd and 4th digits and their ratio as well as measuring the child's verbal ability and visuospatial reasoning by direct assessment. At each visit mothers will complete instruments designed to measure perceived stress, anxiety and depression, job stress and stressful life events. We will create a novel index that transforms the binary classification of se into a gender continuum score and examine its association with pre and postnatal exposure to phthalates and psychosocial stress. We hypothesize that physical measurements (AGD, digit ratio, head circumference) will be sensitive to prenatal exposure, while neurodevelopmental endpoints may be sensitive to both prenatal and postnatal exposure. We hypothesize that AGD will correlate with anthropometric outcomes as well as one or more neurodevelopmental endpoints such as play behavior as our preliminary data suggest. This study could have several implications for public health and environmental policy. Finding robust associations between developmental endpoints and phthalate exposure at current low levels would suggest the need for further reducing exposure. If newborn AGD predicts dimorphic development in multiple domains this would strengthen its importance as a marker of altered androgen-related development. If maternal stress modifies associations between phthalates and dimorphic development, as we have seen with AGD, this would lend support to efforts to reduce stress-related exposures during pregnancy. Any one of these findings from this large, robust, multi- center prospective study would support the hypothesis that a common androgen-dependent mechanism underlies these associations. Our findings could impact future research and policy because our study exposures are ubiquitous and many of our study endpoints, or their correlates, are early predictors of lifelong health.

The aim of this application is to test the role of prenatal sex steroid hormones as the link between first trimester phthalate (synthetic endocrine disrupting chemical) exposure and newborn anogenital distance (AGD). Primary genitourinary development and programming occurs in the first 5-18 weeks of human gestation when the developing fetus is extremely sensitive to changes in sex steroid hormone concentrations that direct genital differentiation and development. AGD develops during this programming window and is a hormone sensitive reproductive toxicity endpoint commonly used in animal studies. Changes in AGD are associated with clinically significant newborn reproductive tract abnormalities and reduced fertility and abnormal semen parameters later in life. The general population is widely exposed to phthalates which can affect hormone concentrations and function during gestation leading to adverse hormone-mediated outcomes in animal and humans, including changes in AGD. The Prenatal Environmental Exposure and Reproductive Hormone Concentrations (PERCH) study will use data and samples from The Infant Development and the Environment Study to determine the relationship between phthalate exposures and sex steroid hormone concentrations in early pregnancy. It will also examine the relationship between early pregnancy sex steroid hormones and AGD in male and female infants. PERCH will be the first study to examine phthalate exposures during the fetal genitourinary programming window in relation to sex steroid hormone concentrations and newborn AGD. Results have important implications for understanding the biological pathway in which phthalates may impact reproductive development as well as for policy surrounding phthalate exposures in vulnerable populations.

In specific areas of industrialized nations, the rising incidence of hypospadias and cryptorchidism from 1950- 2000 parallels the production and use of some phthalates, anti-androgenic synthetic chemicals used as plasticizers, that have been found in the urine of over 90% of the general population in the United States. Genetic and environmental factors play a role in the development of abnormal development of male reproductive outcomes. Polymorphisms in several genes including AR, SRD5A2, INSL3/LGR8, ESR1, ESR2, ATF3, CTGF, and CYR61 are associated with adverse male reproductive development. Prenatal exposure to phthalates leads to hypospadias, cryptorchidism, and shortened anogenital distance (AGD) and smaller penile width (PW) in animal studies. Our hypothesis is that single nucleotide and repeat polymorphisms in genes involved in hormone mediated male genital development are associated with shortened AGD and ano-scrotal distance (ASD: a similar measure to AGD in animals) and smaller PW; and this relationship is modified by prenatal phthalate exposure. To address this hypothesis, the proposed GEAR (Genetics and the Environment Associated with Male Reproduction) study will genotype candidate genes and repeat polymorphism length in 400 male infants recruited into a prospective NIEHS-funded cohort study, TIDES (The Infant Development and Environment Study), and in whom AGD, ASD, and PW and maternal pregnancy urinary phthalate concentrations are being measured. We will obtain and genotype buccal swab/FTA card DNA samples from infants during already scheduled birth exams in TIDES. We will first examine the association between genetic variation in AR, ESR1, ESR2, SRD5A2, ATF3, INSL3/LGR8, CTGF, CYR61 and shortened AGD and ASD, and smaller PW; then proceed to test whether this association is modified by first trimester urine phthalate exposures. By taking advantage of a prospective parent study design, GEAR is cost effective and novel by allowing for assessment of genetic variation and phthalate exposures in male reproductive development. If a relationship exists between genetic polymorphisms associated with male reproductive abnormalities, phthalate exposure, and novel clinical phenotypes AGD, ASD, and PW, these findings would: 1) enhance understanding of the relationship between genotype and novel clinical phenotypes of early male genital development, and 2) be among the first studies to examine gene/phthalate interactions in male reproductive development, and 3) contribute to future research that uses genotype, environmental exposure, and phenotype to predict future male reproductive health.

The role of the placenta in the relationship of maternal and fetal exposure to endocrine disrupting chemicals (EDC), and the long-term effects on the health of the child is unknown. Yet knowledge of the role of the placenta can increase temporal and spatial precision in estimating health risks to the fetus. A class of EDCs called phthalates, are detected in all pregnant women in the U.S. Potential roles for the placenta in phthalate toxicity include transfer of phthalate metabolites to fetal circulation, phthalate-induced damage to placental development and function (nutrient transfer, invasion of the myometrium, oxygen regulation, immune function, parturition signaling), or hormonal mediation of fetal programming. Based on findings from a pilot study of placental gene expression and maternal phthalate exposure, we hypothesized that phthalates disrupt trophoblast differentiation (GCM1, PPARG, AHR, FATP, CGA). This would consequently alter placental function over the course of the pregnancy and possibly explain correlations between prenatal exposure and outcomes at birth. In the K99 phase, we conducted experimentation to test this hypothesis using two primary placental cell models of the undifferentiated and differentiated primary trophoblasts, dosing cells with the same concentrations of mono-n-butyl phthalate (MnBP) measured in prenatal urine. We found that a) the transcriptome response of the cells to MnBP differed by the sex of the fetus; b) MnBP at 150-200 nM increased the secretion of a placenta-specific hormone called human chorionic gonadotropin (hCG); c) and the effects of MnBP on the trophoblast overall were pro-differentiation and pro-invasiveness. Based on these insights, in the R00 phase we focused on the sex-specific molecular response of the placenta and the potential for placental mediation of fetal programming. In Aim 1, we experimentally confirmed the sex-specific effects of phthalates on trophoblast gene and protein expression, and specifically on hCG synthesis and secretion. In Aim 2, we translated our experimental findings back to an epidemiologic context in collaboration with a birth cohort study (TIDES) and by sampling maternal and placental tissue from first trimester pregnancy terminations (e3pad). In conclusion, we have confirmed the sex-specific effects of phthalates on the placental transcriptome, hCG transcription, translation and secretion in vitro and in human pregnancy. Secondly, we have demonstrated using observational data that the first trimester placenta may mediate the effects of phthalates on fetal sex differentiation by way of placental hormonal disruption. Thirdly, we have generated a bank of placental tissue samples from the TIDES cohort that can be used to further probe hypotheses regarding the fetal origins of endocrine disruption.

Environmental chemicals may contribute to increases in male reproductive disorders that have been noted in industrial nations. While much of this discussion has revolved around environmental estrogens, the most compelling links between impaired male reproductive function and environmental agents have been demonstrated for anti-androgens, and phthalates, in particular. In rodents, diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) induce a spectrum of male genital tract disorders termed the "phthalate syndrome". Anogenital distance (AGD), the distance from anus to genitals, is a defining marker of this syndrome, which also includes small testes size and incomplete testicular descent. We found higher concentrations of metabolites of DEHP and DBP in prenatal urine to be associated with shorter AGD in male infants. DEHP metabolites were also negatively correlated with penile width (PW) and incomplete testicular descent. Pregnancy cohort: A primary aim of The Infant Development and Environment Study (TIDES) is to examine genital landmarks in male infants in relation to prenatal DEHP and DBP exposure. To address this aim, we will recruit 1,000 women in the first trimester of pregnancy from four centers (Seattle, WA, Minneapolis, MN, San Francisco, CA and Rochester, NY) and store a urine sample in each trimester from all women. We will examine DEHP and DBP metabolite concentrations in first trimester urine samples in women who give birth to males, and store urine from female births for future study. We will measure metabolite concentration in urine samples from each trimester for 50 per cent of mothers of males to estimate metabolite variability across pregnancy. Birth cohort: The second aim of TIDES is to obtain population-based norms for AGD, PW, testicular volume (TV) and testicular location (TL) in male newborns, specific for birthweight and gestational age. We will examine 1,000 newborn males (including 500 males born to members of the pregnancy cohort and an additional sample of 500 boys from the newborn nursery) using precise methods and measures standardized across clinical centers with strict quality control procedures. Follow-up cohort: To examine the stability of birth measurements, we will repeat the birth exam at 6 and 12 months in males born to women in the pregnancy cohort. We will also pilot novel methods for conducting morphometry of the anogenital region from standardized digital images obtained by camera and ultrasound. Consequences: This research will provide standard methods for easily measuring AGD, PW, and TL (and perhaps TV) and norms for these genital landmarks obtained in a diverse, population-based, sample of male infants, which may be suitable for use in pediatric practice. Associations between phthalate metabolites and the androgen-sensitive measures we are examining could play an important role in public health policy given the ubiquitous nature of these exposures and the clinical importance of appropriate androgen stimulation during fetal development.