According to NIEHS, it is imperative to develop methods to assess the health effects associated with complex exposures in order to minimize their impact on the development of disease. NIEHS has held several meetings on mixtures, including the 2015 workshop on Statistical Approaches for Assessing Health Effects of Environmental Chemical Mixtures in Epidemiology Studies. Conclusions include the following. 1) An interdisciplinary perspective is needed, including insights from environmental epidemiology, statistics/mathematics, toxicology and exposure science. 2) Mixtures epidemiology has three key goals: a) identify components of a mixture contributing to the outcome; b) examine interactions between the components; c) construct summary measures of exposure where possible. 3) Different methods have different strengths and weaknesses that may be complementary. We propose to build upon three methods that performed well at the 2015 workshop: Bayesian kernel machine regression (BKMR), exposure space smoothing (ESS) and weighted quantile sum regression (WQS). We will develop two complementary methods: 1) BKMR/ESS. We will expand and combine aspects of BKMR and ESS into one method that primarily addresses the first two goals: variable selection and proposal are i) extension to binary health outcomes, the most common type of outcome data in epidemiology (the 2015 NIEHS workshop examined continuous outcomes); ii) variable selection interactions. Crucial aspects of our using the hierarchical structures observed for correlations between exposures; iii) incorporation of toxicological information. 2) Single index model: We will evaluate a generalization of WQS, the single index model (SIM). SIM non-parametrically estimates a one-dimensional smooth function of a weighted sum of exposures. The weighted sum represents a summary measure of exposure (one based on toxicological principles), a third goal of mixtures epidemiology. Following method development, we will test the methods using both synthetic and real world data sets, including the Environment And Reproductive Health (EARTH) cohort study. We will incorporate causal inference tools such as directed acyclic graphs (DAGs). For example, correlated exposures (co-exposures) are confounders under some DAGs and colliders or intermediate variables under others. This must be taken into account in both generation of synthetic data and proper interpretation of results. The specific aims of this project are as follows: Specific Aim 1: Combine features of BKMR and ESS to produce a method for analyzing epidemiologic data that incorporates toxicological information; can handle continuous, binary and repeated measures outcome data; select important exposure variables; flexibly model and examine interactions; adjust for confounders; is robust to influential points; Specific Aim 2: Evaluate the single index model (SIM) as a method for analyzing epidemiologic mixtures data and generating exposure summary measures; Specific Aim 3: Make benchmark synthetic data and method computer code publicly available.

Phthalates, a class of endocrine disrupting compounds (EDCs) used in plastics and personal care products, are ubiquitous environmental contaminants resulting in widespread human exposure. Epidemiologic data implicate paternal phthalates with adverse reproductive health including poor sperm quality, and more recently, with longer time to pregnancy ? the latter suggests a semen-derived effect. Traditionally, sperm have been considered vehicles only for the delivery of the paternal genome upon fertilization. However, compelling animal data demonstrate that environmental conditions in adulthood are embodied within sperm without altering the underlying DNA sequence, and in turn, these alterations affect offspring health and development. A biological pathway for this paternal contribution to reproductive success has been shown to include aberrations in sperm DNA methylation. To date, research in this area has been largely restricted to the effects of nutritional manipulations in animal models; therefore, relatively little is known about how adult exposures to toxicants affect epigenetic information in sperm ? especially in human populations. We propose that a key to understanding how adult exposures to phthalates impact reproductive health lies within sperm DNA methylation. This application capitalizes on the concurrent sample collection from the Sperm Environmental Epigenetics and Development Study (SEEDS; PI: Richard Pilsner) and the Environmental and Reproductive Health Study (EARTH; PI Russ Hauser), two independent epidemiologic studies investigating the link between paternal phthalate exposure and adverse reproductive health among in vitro fertilization (IVF) populations. Moreover, to address potential generalizability issues stemming from IVF cohorts, we also include in our replication aim, sperm methylation analyses from samples collected from the Longitudinal Investigation of Fertility and the Environment (LIFE; PI Germaine Buck Louis) study, a prospective cohort of couples representing the general population. For our first aim, genome-wide methylation will be analyzed via Illumina?s EPIC array on isolated sperm remaining after in-vitro fertilization (IVF) from SEEDS. Next, we will determine the relationships of sperm methylome and on the reproductive outcomes of embryo quality and probability of a live birth in SEEDS. Finally, after completion of these objectives in SEEDS, we will perform replication studies in sperm samples from EARTH and LIFE to determine the concordance of findings across the three cohorts. The proposed research is expected to uncover pathways linking paternal phthalate exposures with adverse reproductive outcomes via sperm DNA methylation. Characterization of potential intermediate pathways between the exposure and outcome continuum is of significant importance because it will inform avenues of translational research for the development of novel approaches to treat and prevent adverse reproductive health.

The etiology of male and female infertility and adverse pregnancy outcomes are not well understood, but likely arise as a complex interplay of environmental and lifestyle factors evident at the population level. It is well appreciated that environmental exposures are potential risk factors for infertility, with experimental animal studied linking several classes of chemicals to infertility and adverse pregnancy outcomes. These include phthalates, bisphenol A, and parabens, all of which have documented widespread general population exposure. To increase our understanding of potential human health risks of environmental chemicals, we need to consider exposure to complex mixtures of chemicals rather than focusing only on a single (or a few) chemicals at a time and in essence ignoring other simultaneous exposures. The NIEHS Strategic Plan (2012- 2017) identified mixtures research as highly relevant to many of the Goals, with Goal 4 focusing on 'combined exposures', specifically calling for research to determine human health risks from exposure to multiple chemicals. In the proposed study, motivated by our earlier findings of associations of individual chemicals with adverse reproductive and pregnancy outcomes, we will extend our aims to explore the impact of chemical mixtures on pregnancy outcomes. Furthermore, most epidemiologic studies focus on either male or female exposures with few studies assessing the joint contribution of both paternal and maternal exposure to a healthy pregnancy. Our proposed study is innovative because it will explore the joint impact of both paternal and maternal exposure to mixtures of environmental chemicals. In our preliminary data, we found associations of male and female exposure to phthalates, bisphenol A and parabens with adverse pregnancy outcomes. In the proposed study, we will extend recruitment to provide sufficient power to determine the joint effects of maternal-paternal exposure to chemical mixtures on the primary outcomes of implantation failure and live birth, and the secondary outcomes of chemical pregnancy (with no subsequent clinical pregnancy) and spontaneous abortion. Our study is cost-effective because we have chemical levels and clinical data available for over 300 couples; we will recruit 250 additional couples. We collect both pre- and peri-conception maternal and paternal measures of exposure to phthalates, BPA and parabens, and maternal measures throughout pregnancy. We propose to use in vitro fertilization (IVF) as a model to study the impact of mixtures of chemicals on early human development and pregnancy. IVF provides epidemiologists with novel and unique opportunities to study early development and pre-clinical pregnancy loss. These early endpoints are not observable in women conceiving naturally, but are of high importance as they represent the majority of failures of pregnancy among all women and are considered more sensitive to environmental chemicals than later pregnancy endpoints.

There is currently scientific, government and public concern regarding adverse effects from exposure to chemicals that may act through disruption of the endocrine system. Studies with laboratory and domestic animal species, and limited human studies, have shown that exposure to some environmental disrupting chemicals (EDCs) may negatively affect oocyte and embryo quality, therefore impacting fertility. In a cohort of IVF patients, we have shown a significant dose-response association between urinary BPA concentrations and decreased numbers of oocytes retrieved and fertilized, as well as decreased proportions of high quality embryos and blastocysts formed. Furthermore, in a recent study on in vitro maturation in human oocytes, we showed a dose response association between bisphenol A (BPA) and a decreased progression of oocytes to metaphase II (MII), an increased incidence of both spontaneous activation and degeneration and, of those oocytes that did mature, an increase in spindle abnormalities and chromosome alignment. As normal spindle organization and chromosome alignment in MII oocytes are critical for formation of a developmentally competent oocyte, any chemically-induced perturbations at this stage will adversely affect fertility. Of specific relevance to this application, the pathways and potential mechanisms by which EDCs alter meiotic progression to MII are currently unknown. Oocyte maturity and health are dependent upon a tightly coordinated communication network between the oocyte and follicular granulosa compartment. Innovative studies have proposed that such intercellular transmission within the follicle may involve secretion and uptake of microRNAs (miRNAs) carried in extracellular vesicles such as exosomes. Exosomes are present in human FF, which raises the possibility that miRNA exosome profiles are perturbed by endocrine disruptors. The objective of our proposed study is to determine if urinary levels of BPA and molar DEHP metabolites are associated with miRNA profile in FF-isolated exosomes and with oocyte maturation and day 3 embryo quality. We will also use mediation analysis to estimate the indirect effects of BPA and molar DEHP metabolites on oocyte maturation and embryo quality that are mediated through FF isolated exosomes miRNA expression. This concept, that miRNAs from FF exosomes may play a role in intra-follicular oocyte control is novel and may provide important insights for the understanding of the development of a mature oocyte and a competent embryo. We expect the results will provide the basis for future research investigating the associations among endocrine disrupting chemicals, ovarian function, oocyte development, embryo implantation and fetal health.

The causes of male and female infertility and adverse pregnancy outcomes are not well understood, but likely arise as a complex interplay of environmental and lifestyle factors evident at the population level. Etiologic factors may include exposure to a wide range of synthetic chemicals that have become ubiquitous and as such result in widespread general population exposure. The Centers for Disease Control and Prevention found that a majority of the U.S. population, including men and women of reproductive age, are exposed to the flame retardants known as polybrominated diphenyl ethers (PBDE). While PBDEs are the flame retardants that have received the most attention to date, their use is being phased out in the U.S. and other countries. Although PBDEs will continue to be a public health concern for decades to come due to their presence in existing consumer products and their high level of persistence, there is an urgent need to also consider other flame retardants that are increasingly being used as alternatives to PBDEs. Despite evidence from experimental studies that PBDEs and some of their alternatives are developmental and reproductive toxicants, there are limited human studies on the potential health risks of PBDEs and no human studies on their alternatives. We propose to leverage our ongoing prospective preconception cohort study (ES009718) by extending recruitment to sufficiently power the exploration of the effect of maternal and paternal exposure to FRs on pregnancy outcomes and to also explore the joint effects (maternal-paternal interactions) on pregnancy outcomes. The study is innovative because we have access to both pre- and peri-conception maternal and paternal measures of exposure and couple level clinical outcome data. We propose to use in vitro fertilization (IVF) as a model to study the impact of flame retardants on early human development and pregnancy. IVF provides epidemiologists with novel and unique opportunities to study early development and pre-clinical pregnancy loss. These early endpoints are not observable in women conceiving naturally, but are of high importance as they represent the majority of failures of pregnancy among all women and are considered more sensitive to environmental chemicals than later pregnancy endpoints. In the proposed study, we will measure not only PBDEs, but will apply new and novel biomarkers of exposure for their replacements, including components of Firemaster 550 and organophosphate flame retardants. The specific aims are to explore both the maternal and paternal effects of flame retardants, as well as their joint effects on clinical pregnancy outcomes. Clinical outcomes of interest include oocyte fertilization, implantation failure, pregnancy loss and live birth. As shown in our preliminary data, there is widespread exposure of our cohort to PBDEs and alternative flame retardants, measured using novel analytical methods developed by our research team. Furthermore, in our preliminary data, we report suggestive associations of flame retardants with reproductive outcomes that although intriguing were within small datasets and need to be expanded to the full cohort study through the proposed application.

Although the widespread use of modern synthetic chemicals dates back many decades, we have a limited understanding of their potential impact on clinical diseases and public health. Two classes of modern chemicals, phthalates and bisphenol-A (BPA), adversely affect development and reproductive function in experimental animals. Despite a majority of the U.S. general population being exposed to phthalates and BPA, there are very limited clinical and epidemiologic studies on their potential health risks, particularly in relation to fertility and pregnancy loss. In experimental studies, phthalates alter male reproductive tract development, decrease semen quality, and induce anovulatory infertility and pregnancy loss. BPA has been shown in experimental studies to lead to oocyte aneuploidy, pregnancy loss and adversely affect spermatogenesis. The proposed competing continuation is designed to determine the developmental and reproductive toxicity of phthalates and BPA in couples undergoing assisted reproductive technologies (ART), which can be used as a model for the assessment of the early stages of human development. The methods of gamete manipulation used in ART are now widely practiced and provide clinicians and epidemiologists with novel and unique opportunities to study infertility and early pregnancy loss. ART allows us to assess oocyte quality and fertilization rates, pre-implantation embryo development, and pre-clinical (early) pregnancy loss. These early developmental endpoints are not observable in women conceiving naturally, but are of high importance as they represent the majority of failures of pregnancy. Furthermore, experimental data show stage-specific differences in sensitivity to both phthalates and BPA, with early development endpoints being more sensitive to adverse affects than later pregnancy endpoints. The proposed study is a prospective preconception cohort study in which biological samples for phthalate and BPA exposure are collected at multiple time points, including preconception, periconception and throughout pregnancy. In addition, ovarian follicular fluid and amniotic fluid samples are collected to assess exposure in relevant target organs or in the fetal compartment, respectively. The specific aims include the determination of: 1) the relationship of urinary concentrations of BPA, mono-n-butyl (MnBP) and mono-(2-ethylhexyl) phthalate (MEHP), metabolites of di-n-butyl (DnBP) and di(2-ethylhexyl) phthalate (DEHP), respectively, with clinical measures of male fertility, including semen quality and sperm DNA damage; and 2) the relationship of pre- and periconception urinary concentrations of BPA, MnBP and MEHP with early developmental and pregnancy outcomes. Specific health endpoints of investigation include oocyte quality, fertilization rates, embryo quality, implantation failure and early pregnancy loss. Additional specific aims are to explore whether inter-individual phenotypic differences in DEHP metabolism modifies the association of urinary concentrations of MEHP with sperm DNA damage and adverse developmental and pregnancy endpoints.