Fetal Programming and Environmental Exposures: Implications for Prenatal Care and Pre-Term Birth

Abstracts — Day One

Keynote Address

Challenges for Reproductive Toxicology Posed By Unpredicted Effects on Fetuses Of Very Low-Doses Of Estrogenic Endocrine Disrupting Chemicals
Frederick S. vom Saal, PhD, University of Missouri–Columbia

The factors that lead to the disruption of normal fetal development are of great concern. For example, there is evidence that the incidence of prematurity and intrauterine growth restriction (IUGR) is increasing. Significant attention has been devoted to the impact on fetal health of maternal malnutrition in experimental animals (for example, severe protein restriction during pregnancy), although severe malnutrition is not the basis for the increase in prematurity or IUGR in developed countries. There has been less attention paid to the impact of maternal exposure to environmental chemicals on fetal growth and other fetal abnormalities. The chemicals of greatest concern with regard to the disruption of the normal programming of genes that occurs during critical periods in cell differentiation are chemicals that have been found to disrupt hormone action, which are referred to as endocrine disrupting chemicals (EDCs). There is experimental evidence that disruption of the programming of gene expression associated with changes in the epigenome is related to diseases that are expressed at various times in postnatal life (the Developmental Origins of Adult Health and Disease, DOHaD, hypothesis). The concern is that waiting for conclusive evidence from prospective epidemiological studies will lead to generations being exposed to chemicals shown to cause harm in experimental animals. A large number of animal studies show that EDCs disrupt fetal development at very low doses that are below the no adverse effect level (NoAEL) based on the traditional approach currently used in chemical risk assessments by the US-FDA and US-EPA as well as European regulatory agencies (Vandenberg et al, 2012). This traditional approach consists of testing very high doses using very insensitive outcomes to predict effects at the much lower levels of environmental chemicals to which humans are chronically exposed. This approach is flawed in that the core assumption is that all doseresponse relationships are monotonic, and thus effects at high doses predict effects at low doses, which is a false assumption for endogenous hormones and EDCs. In addition, the current approach by regulatory agencies in the initial (Tier 1) screen for the hazards posed by EDCs does not include exposure during development, and thus does not recognize that fetuses, infants and children are not little adults. Specific examples of adverse effects due to exposure to EDCs with estrogenic activity from epidemiological studies and experimental animal research include disruption of metabolic systems involved in obesity, type 2 diabetes and abnormalities in reproductive organs and behavior.
 

Session I: Genetic / Epigenetic Programming of Pre-implantation Development

Networks of RNA Binding Proteins Regulate Maternal mRNA Translation Essential for Oocyte Maturation and Early Embryo Development
Marco Conti, MD, University of California, San Francisco

In the final stages of oocyte maturation, completion of meiotic division is required to generate a haploid genome as well as assemble the molecular machinery responsible for nuclear reprogramming and activation of transcription in the zygote and early embryo. All of these functions rely on a program of translation of maternal mRNAs in the virtual absence of transcription. To define the pattern of protein expression for this critical window in development and to understand the mechanisms governing translational control of the oocyte-to-zygote transition, we have used a genome-wide approach to identify maternal mRNAsrecruited to the polysome fraction and, therefore, actively translated. This analysis has revealed that many of the cellular components necessary for cell cycle progression are finely regulated at the level of translation. Moreover, components that will be used during nuclear reprogramming and early embryo development are synthesized earlier during oocyte maturation and accumulate in the cytoplasm. To investigate the mechanisms underlying selective mRNA translation, the 3′ untranslated region of mRNAs undergoing comparable patterns of recruitment were investigated. This analysis demonstrated that consensus nucleotide sequences recognized by known RNA binding proteins are enriched in mRNAs that are recruited to the polysome during maturation. Knockdown and gain-of-function experiments strongly indicate that a network of RNA binding proteins is coordinating translation or repression of individual maternal mRNAs. This genome-wide approach provides a blueprint of protein expression critical for the oocyte-to-embryo transition.
 
Coauthors: Jing Chen, Matthew Cook, Chih-Jen Lin, Andrej Susor, and Jeong Su Oh, University of California, San Francisco.
 

Maternal Obesity, Oocyte Quality, and Reproductive Outcomes
Kelle H. Moley, MD, Washington University in St. Louis School of Medicine

Approximately 60% of women desiring pregnancy are obese by BMI criterion, with incidence rising exponentially over the last 15 yrs. Unfortunately, maternal obesity is associated with poor outcomes, including increased rates of infertility, pregnancy loss, developmental delay and neurological deficits. From current studies it is not clear if the origin of these problems is attributable to oocyte or embryo development, impaired uterine environment, or a combination of factors. The impact of abnormal metabolic environment on oocyte quality and pregnancy outcomes was initially seen in diabetic mouse models. In animal models of diabetes, oocytes are smaller, and show impaired maturation and increased granulosa cell apoptosis. These findings have been linked to poor reproductive outcomes including growth restriction and congenital anomalies. Similarly in murine models of obesity, exposure to an abnormal endocrine environment impacts the oocyte, the embryo, and pregnancy outcomes. Oocytes from obese mice are significantly smaller show delayed meiotic maturation, and exhibit increased follicular apoptosis. Mice fed a high fat diet have impaired ovulation, fertilization, embryonic development and growth restriction. The inciting metabolic factor is not clear, however, abnormal levels of free fatty acids in follicular fluid as well as sera have been associated with poor oocyte quality and decreased chance of pregnancy in patients undergoing IVF, respectively. Alternatively, these fetal changes, like growth restriction and anomalies, may be due to a combination of oocyte and early preimplantation exposure to free fatty acids as shown in animal models.
 
One proposed mechanism for compromised oocyte quality and poor reproductive outcomes in obese females includes altered mitochondrial activity at the oocyte stage. Abnormal mitochondria structure and function in oocytes from type 1 diabetic mice are associated with poor fertilization rates and abnormal embryo development. It is apparent that both obesity and diabetes influence mitochondrial activity. In obese mice, uneven mitochondrial distribution and altered mitochondrial DNA copy number are linked to impaired embryonic development from the zygote to blastocyst stage. Diabetic mice have increased abnormalities in mitochondria morphology, distribution, and mtDNA copy number. These mice also exhibit spindle defects/chromosome misalignment; defects associated with mitochondrial metabolism and changes in endocrine surroundings. In the unpublished data presented in this talk, we will show that maternal obesity adversely affects oocyte mitochondria and spindle formation, and that this isolated early preimplantation insult will negatively impact embryo outcomes when transferred into nonobese control recipient mice.
 

Trophoblast Cell Subtypes Orchestrating the Development of the Maternal–Fetal Interface
James Charles Cross, DVM, PhD, University of Calgary, Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Canada

The placenta is essential for growth and survival of the fetus during gestation because of both its ability to transport nutrients and oxygen to the fetus but also by producing hormones that promote maternal adaptations to pregnancy. Using the mouse as a model system, my group has identified key regulatory pathways that underlie the development of the placenta and described a complex array of differentiated trophoblast cell types that line the maternal blood spaces in the placenta. In the last few years, we and others have investigated the extent to which the normal development and functions of the placenta can adapt to alterations in the intrauterine environment. Results indicate that: 1) the ability of the placenta to adapt by expansion is limited by the fact that multipotent trophoblast stem cells do not persist beyond embryonic day (E) 8.0; 2) Tissue oxygen levels regulate patterns of trophoblast differentiation, influencing the balance of cell types that form; 3) Prolactin-related hormones from the placenta (the Placental Lactogens) protect the mother against diabetes during pregnancy in part by promoting an increase in the number of her pancreatic beta cells; 4) Expression levels of Placental Lactogens follow feeding patterns in the mother; 5) Specialized cells in the placenta, called glycogen trophoblast cells, can accumulate glucose and they localize both around spiral arteries which bring maternal blood into the placenta as well as small channels that lead maternal blood out of the placenta and into uterine veins. Glycogen content increases during gestational diabetes perhaps buffering against effects of hyperglycemia on the fetus. Together, these results indicate that placenta development and function can be modified by the environment in ways that benefit not just the fetus but also the mother.
 

Session II: Embryo–Uterine Cross-Talk

Paracrine Signals Regulating Embryo Implantation and Uterine Decidualization
Francesco J. DeMayo, PhD, Baylor College of Medicine, Houston

The ability of the uterus to support embryo implantation and pregnancy requires ovarian steroid hormone activation of a network of paracrine signaling between the endometrial epithelium and stroma cells. The identification of the regulatory networks governing uterine physiology has been facilitated by using the mouse as a model and exploiting gene expression and ablation approaches. One critical regulator of uterine physiology is Progesterone. Progesterone signaling through its cognate receptor, the progesterone receptor, PR, regulates uterine development, embryo implantation and the post-implantation support of embryo implantation. Ablation of the PR results in the inability of the uterus to support pregnancy. The PR is expressed in both epithelial and stromal compartments of the endometrium. In order to further define the role of PR action in the uterus, PR was ablated, specifically in the uterine epithelium. This analysis demonstrated that the epithelium is critical in the regulation of the signaling cascades required to activate the pathways that support embryo development and pregnancy. One signaling pathway component regulated by PR is morphogen Indian hedgehog, Ihh. PR acting in the uterine epithelium directly regulates the expression of Ihh in the preimplantation uterine epithelium. Once expressed, Ihh signals to endometrial stroma cells to express, Bone morphogenic protein (BMP), Epidermal Growth Factor (EGF) and Wnt. This in turn activates the signaling pathway to coordinate endometrial stroma, the proliferation, vascularization, and differentiation preparing the stroma of the endometrium for the support of embryo implantation. Ihh and COUP-TFII in the uterus demonstrated that these molecules regulate the window of receptivity in the uterus by coordinating endometrial stroma, proliferation, vascularization and endometrial Estrogen Receptor sensitivity in the uterine epithelium. Understanding the interactions of transcription factors and signaling pathways in the uterus will help determine what processes are required for normal pregnancy and aid in the identification of processes that are altered in infertility and early fetal loss or preterm delivery.
 
Specialized Cooperative Centers in Reproduction and Infertility Research, U54HD0077495 (F.J.D.) and NIH Grant RO1-CA77530 and the Susan G. Komen Award BCTR0503763 (J.P.L.)
 
Coauthor: John P. Lydon, Baylor College of Medicine, Houston.
 

Nuclear Receptor Regulation of Gestation
Bruce Murphy, PhD, Université de Montréal

Nuclear receptors interact with specific regulatory sequences of DNA to modulate gene transcription. The orphan nuclear receptor, liver receptor homolog-1 (Nr5a2) is expressed in the ovary, exclusively in granulosa and luteal cells. Germline deletion in the mouse is lethal during early embryogenesis, requiring tissue-specific disruption of the gene. A Cre/lox strategy employing Cre recombinase driven by the progesterone receptor (genotype PgrCre/⁺/Nr5a2^f/f) deleted Nr5a2 expression from the corpus luteum beginning 4 h after ovulation and completed by 24 h. The effect was abrogation of progesterone synthesis and gestational failure. Embryos were normal and, with progesterone replacement, implanted, but progesterone did not rescue pregnancy. By PCR and immunohistochemistry, Nr5a2 was shown to be expressed the uterine stroma of wild type mice at dpc 3-5, and ablated by dpc 5 in PgrCre/⁺/Nr5a2^f/f mice. Its absence from the uterine stroma resulted in embryo crowding, decidualization failure, and compromised gestation. Embryos displayed substantially reduced size and most pregnancies terminated spontaneously by day 18 of gestation. The ovarian and uterine Nr5a2 deletion resulted in deficiencies of expression of several genes, including Star, Cyp11a1, Hoxa10 and Wnt 4a, among others. Complementary investigations in which Nr5a2 mRNA abundance was reduced in primary cultures of human endometrial stromal cells by siRNA demonstrated a similar abrogation of decidualization, as indicated by cell morphology and reduced marker gene expression. We conclude that NR5A2 plays multiple, non-overlapping roles in reproductive processes, as it is essential for luteal function and placental formation. (Supported by CIHR OGP 10118 to B. Murphy)
 

Immune Surveillance of the Maternal–Fetal Interface: Implications for Fetal Loss and Preterm Birth
Adrian Erlebacher, New York University School of Medicine and New York University Cancer Institute

Successful pregnancy requires delicate control over the maternal immune cells that reside at the maternal/fetal interface. On the one hand, immune cell-mediated inflammation at the maternal/fetal interface is likely to be a major instigator of preterm birth, while immunogenic recognition of fetal/placental antigens by professional antigen presenting cells runs the risk of inducing T cell responses that might directly induce fetal demise. On the other hand, inadequate immune surveillance of the maternal/fetal interface might be expected to increase the risk of ascending infection, or the risk that such infection progresses to chorioamnionitis. We will discuss recent our recent work on the molecular and cellular pathways that regulate immune cell trafficking and homeostasis within the pregnant mouse uterus. Our results demonstrate how the developmental properties of the decidua, i.e. the specialized endometrial stromal tissue that directly encases the implanted embryo, minimize the tissue density of dendritic cells and prevent these cells from surveying the placenta for antigens. We will also discuss how the decidua regulates macrophage population dynamics to minimize macrophage densities. Since dendritic cells are key instigators of immunogenic T cell responses while macrophages are major promoters of tissue inflammation, these results have major implications for the immunology of pregnancy its complications.
 
Coauthors: Elisa Tagliani, Mary K. Collins, and Patrice Nancy, New York University School of Medicine.
 

Session III: Young Investigator Presentations

Asynchronous Peri-implantation Events Characterize a Mouse Model of Preeclampsia, bph/5
Jenny Sones, DVM, Cornell University, Ithaca, NY

Preeclampsia (PE), a pregnancy-specific disorder characterized by a sudden late gestational rise in blood pressure and urinary protein levels, is one of the leading causes of perinatal and maternal morbidity and mortality. Our lab discovered an inbred mouse strain (BPH/5) that spontaneously develops these cardinal signs of PE during pregnancy, along with early placental defects, decreased litter size, and low-birth-weight pups. Although abnormal placentation in early gestation is thought to be responsible for the cascade of events leading to the clinical syndrome of PE, the precise mechanisms are still not clear. Proper placental development begins with synchronous implantation of an activated blastocyst into the receptive uterus. We hypothesized that defects in the implantation process would be observed in the BPH/5 model of PE. BPH/5 mice exhibit a delay in timing of implantation as well as embryo clustering. BPH/5 pregnant females also show aberrant ovarian hormone profiles leading up to and during implantation, and BPH/5 embryos demonstrate abnormal maturation kinetics as compared to C57 controls. Gene expression patterns of the implantation signaling molecules Lif, Hb-egf, and Cox-2 are dysregulated in BPH/5 implantation sites and suggest delayed uterine receptivity and decidualization. Taken together, our data suggest that BPH/5 mice have dysregulated implantation, which is associated with both delayed blastocyst development and inadequate uterine receptivity. The role of asynchronous embryo-uterine cross-talk in the pathogenesis of PE in BPH/5 is the subject of ongoing investigations.
 

Coauthors: Yi Zhou, PhD,¹ Ashley Woods, PhD,¹ Emilie Williamson,¹ Ethan Green,¹ Catherine Isroff,¹ Robin L Davisson, PhD,^1,2
1Cornell University, Ithaca, NY
²Weill Cornell Medical College, New York, NY

Heightened Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling Provokes Premature Decidual Senescence and Preterm Birth
Jeeyeon Cha, Division of Reproductive Sciences, Cincinnati Children's Hospital Research Center, Cincinnati, OH, USA

p53, a tumor suppressor encoded by Trp53, is strongly associated with cancer and has diverse physiological functions. However, its role in reproduction is poorly understood. Female mice harboring a uterine deletion of Trp53 (p53^d/d) show normal ovulation, fertilization and implantation; but post-implantation decidual cells (day 8 of pregnancy) show increased terminal differentiation and senescenceassociated growth restriction. Interestingly, p53^d/d females show increased incidence of preterm birth without decreases in serum progesterone levels, mimicking aspects of human parturition. This condition is associated with increased levels of uterine Cox2 and PGF2_α. Preterm birth is rescued in these mice by an oral gavage of Cox2 inhibitor late in pregnancy, suggesting that PGF2_α generated by Cox2 is involved. More strikingly, we now have evidence that decidual senescence in p53^d/d females is reflected in increased mTORC1 signaling and a low dose of mTORC1 inhibitor rapamycin given early in pregnancy attenuates the senescence phenotype and rescues preterm birth. Collectively, this study shows for the first time that progressive decidual senescence approaching term birth is a normal phenomenon, and that premature decidual senescence early in pregnancy leads to preterm birth in mice. This study constitutes an unanticipated role of mTORC1 in preterm birth upstream of Cox2, suggesting that the mTORC1-Cox2 signaling axis is critical in timing of birth since targeting either of these players rescues preterm birth. These preclinical studies may help to elucidate the mechanism of human birth and develop new strategies to combat this global problem.
 

Coauthors: Yasushi Hirota², Takiko Daikoku¹, Sudhansu K. Dey^1
1Division of Reproductive Sciences, Cincinnati Children's Hospital Research Center, Cincinnati, OH, USA
²Department of Obstetrics and Gynecology, University of Tokyo, Tokyo, Japan

Extraembryonic Hematopoietic Niches: Cross-Talk Between Non-Hematopoietic and Hematopoietic Populations in Human Placenta and Chorion
Alicia Bárcena, PhD, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research Center for Reproductive Sciences, Department of Obstetrics, Gynecology & Reproductive Sciences University of California, San Francisco, San Francisco, CA

Hematopoietic progenitors and hematopoietic stem cells (HSCs) expressing CD34 and CD45 antigens are present in the placenta and chorion from 5 to 40 weeks of gestation. However, fully functional HSCs — defined by their ability to produce long-term and multilineage reconstitution when transplanted into immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice — only manifest in these extraembryonic tissues from the 15th week of gestation onward. Therefore, extraembryonic progenitors/HSCs become functionally mature when their density declines from a peak at 5-8 weeks. The switch that extraembryonic HSCs undergo at 15th week may reflect the immature nature of these cells earlier in ontogeny and could be due to signals delivered by their niche. Immunolocalization experiments of CD34⁺⁺CD45⁺ cells show that these cells reside in the vicinity of trophoblast progenitors cells (TBPCs) and interact with stromal cells in the chorion, and with endothelial cells in the placenta. To study the contribution of the niche’s different components on regulating hematopoesis, we performed co-culture experiments in the presence of mesenchymal stromal cell lines derived from placenta and chorion, and TBPCs derived from chorion. We conclude that both mesenchymal stromal cells and TBPCs regulate the hematopoietic potential of extraembryonic and intraembryonic HSCs. In addition, we investigated the expression of chemokine receptors, cytokine receptors and adhesion molecules on extraembryonic HSCs to assess whether the expression of these molecules changes during development. We have thus established a new culture system that could be applied to investigate the cells and molecules involved in the funtional regulation of HSCs during development.
 

Coauthors: Marcus O. Muench, Mirhan Kapidzic, Nicholas Larocque, Susan J. Fisher, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research Center for Reproductive Sciences, Department of Obstetrics, Gynecology & Reproductive Sciences University of California, San Francisco, San Francisco, CA

Effects of Bisphenol a Exposure on Genomic Imprinting in Mouse
Martha Susiarjo, PhD, Center of Excellence in Environmental Toxicology and Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

Exposure to the common compound bisphenol A (BPA) is linked to developmental abnormalities in model organisms and humans. The molecular mechanisms, however, are unclear and elucidating these pathways will provide insight into the windows of vulnerability and preventive measures. Recently, BPA has been shown to alter DNA methylation in rodents, proposing that epigenetic mechanisms may be relevant. We investigated effects of exposure onimprinted genes in the mouse. Imprinted genes are subject to differential DNA methylation and they regulate fetal, placental and postnatal development. Perturbed imprinting is linked to diseases including the Beckwith Wiedemann, Prader Williand Angelman Syndromes. We exposed pregnant mice to BPA and analyzed their offspring a tE9.5. Using real time PCR, we found that exposure disrupted expression of genes relevant to imprinting diseases, including the Snrpn, Ube3a, Igf2, Kcnq1ot1 and Cdkn1c genes. Furthermore, we observed tissue-specific effects with most genes affected in the placenta. Our bisulfite sequencing and pyrosequencing data demonstrated that gene transcription changes were associated with altered methylation at differentially methylated regions. Additionally, Luminometric Methylation Assay studies showed that exposure reduced global methylation in the placenta, but not the embryo. Histology revealed that BPA-exposed placentas were larger with expanded junctional zone but smaller labyrinth, and had increased accumulation of red blood cells. As the placenta influences embryonic growth and development, our data suggest that BPA-induced perturbed genomic imprinting candisrupt fetal and postnatal health. In the future, we will conduct genome wide methylation and expression studies to link altered epigenetic signatures to expression changes.
 
Research supported by University of Pennsylvania Center of Excellence in Environmental Toxicology 2008 Pilot Project Funding P30 ES013508 and NIEHS F32ES019416.
 

Coauthors: Isaac Sasson, Department of Obstetric and Gynecology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; Clementina Mesaros, Center of Excellence in Environmental Toxicology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; Marisa Bartolomei, Center of Excellence in Environmental Toxicology and Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA