Abstracts — Day Two

Young Investigators Career Mentoring Breakfast

Editor's Guide to Writing and Publishing Your Paper
Brooke Grindlinger, PhD, The New York Academy of Sciences, New York, NY

Publishing is critical to the scientific profession, yet training and guidance on the topic is very limited. Brooke Grindlinger, PhD, former Science Editor at the Journal of Clinical Investigation, will discuss what makes a good paper, strategies for selecting the appropriate journal, the review process, and how to navigate resubmissions.
 

Session IV: Decidualization and Placentation

Fate Specification during Human Pre-Implantation Development
Susan J. Fisher, PhD, University of California, San Francisco

The timing and mechanisms of fate specification during human embryogenesis are poorly understood. To gain insights, we studied the ultrastructure of human blastocyst-stage embryos. Electron microscopy showed an outer layer of mononuclear cytotrophoblasts that were connected by tight junctions. Inner cell mass components had disparate morphologies, varying with regard to nuclear/cytoplasmic ratio and chromatin packing. In contrast, the components of the mouse blastocyst inner cell mass had a more uniform appearance. To gain insights into the molecular correlates of the morphological differences among human embryonic precursors, we derived 9 human embryonic stem cell (hESC) lines from single blastomeres (B) of 8-cell embryos donated by one couple. Microarray analyses showed that the genetically related/identical B-hESCs had gene expression patterns that were different from blastocyst-derived hESCs. Hierarchical clustering suggested that individual cell lines established from blastomeres of different embryos were more closely related than lines derived from the same embryo. Immunolocalization, in 8-cell human embryos, of a group of genes that were differentially expressed among the B-hESC lines showed expression restricted to subsets of blastomeres. With varying rates of efficiency, embryoid bodies formed from the B-hESCs could be differentiated into CDX-2-positive trophoblasts and propagated in an undifferentiated state. Upon differentiation, they gave rise to multinucleated syncytiotrophoblasts that produced placental hormones and HLAG- positive invasive cytotrophoblasts. All the B-hESCs also formed derivatives of the three embryonic germ layers. These results suggested that B-hESCs have features of totipotency and human blastomeres at the 8-cell stage are heterogeneous.
 

Coauthors: Tamara Zdravkovic, PhD¹, Olga Genbacev, PhD¹, Nicholas Larocque, BA¹, Matthew Gormley, BS¹, Matthew Donne, BA¹, Michael McMaster, PhD¹, Kristopher Nazor, BS², Louise Laurent, MD, PhD², Ana Krtolica, PhD¹, Diana Valbuena, MD³, Carlos Simon, MD³, Jeanne Loring, PhD^2
1University of California San Francisco, San Francisco, CA
²The Scripps Research Institute, La Jolla, CA
³Instituto Universitario IVI, Valencia University, Valencia, Spain

Regulatory Pathways Controlling Hemochorial Placentation
Michael J. Soares, PhD, University of Kansas Medical Center, Kansas City, KS

The maternal-fetal interface is a dynamic site where uterine and placental structures cooperate to promote development of the fetus. The rat, mouse, and human each possess a hemochorial placenta. This type of placentation is characterized by erosion of the maternal uterine epithelium and vasculature permitting the direct flow of maternal nutrients to trophoblast. Defective hemochorial placentation, including impairments in uterine spiral artery remodeling, leads to pregnancy related disorders. The extravillous/invasive trophoblast lineage is of fundamental importance to development of the maternal-fetal interface and understanding diseases of placentation and pregnancy. Organization of rat and human placentation sites share common features, especially regarding trophoblast-directed remodeling of the uterine spiral arteries. Both species exhibit deep endovascular trophoblast invasion. We have established relevant in vitro and in vivo methodologies using the rat as an animal model to investigate the extravillous/invasive trophoblast lineage. Two pathways controlling the extravillous/invasive trophoblast lineage have been identified: i) hypoxia/hypoxia inducible factor and ii) phosphatidylinositol 3-kinase/AKT/FOSL1. Understanding molecular mechanisms underlying development of the extravillous/invasive trophoblast lineage is key to identifying developmentally sensitive events that are potentially susceptible to dysregulation; and represent opportunities to discover and evaluate approaches for the early detection and treatment of diseases of placentation. (Supported by NIH – HD20676)
 
Coauthors: Damayanti Chakraborty, MS, Stephen J. Renaud, PhD, Kaiyu Kubota, PhD, Pengli Bu, PhD, and M.A. Karim Rumi, MD, PhD, University of Kansas Medical Center, Kansas City, KS
 

Conversion of Umbilical Cord Mesenchyme to Trophoblast: A Glimpse at the Past
R Michael Roberts, PhD, Division of Animal Sciences, University of Missouri, Columbia, MO

An established model for generating cells of the trophoblast (TB) lineage involves treating human embryonic stem cells (ESC) with BMP4. For this conversion to favor TB it is essential to avoid the co-induction of mesoderm and endoderm by minimizing the effects of the growth factors, activin A and FGF2, which can be achieved by excluding their presence from the culture medium and adding inhibitors that block the MEK1/2 and SMAD2/3 signaling pathways. It would appear that when signaling networks supporting pluripotency of ESC become unsustainable and when specification towards mesoderm and endoderm is rendered inoperative, TB emerges as a major default state to pluripotency. Under these conditions, there is up-regulation of several transcription factors implicated in TB lineage emergence within 3 h of BMP4 exposure and, over a period of days and especially under a high O2 gas atmosphere, there is a gradual appearance of cell types carrying markers for differentiated TB cell types, including extravillous TB and syncytioTB. A similar BMP-response occurs with human induced pluripotent cells derived by driving the ectopic expression of reprogramming genes in mesenchymal fibroblasts from umbilical cord. Thus, it is possible to recreate TB from a past pregnancy. We are currently applying this procedure to umbilical cord cells from children born after normal pregnancies outcomes to cells from pregnancies complicated by early onset pre-eclampsia. The experiments are designed to determine whether there are abnormalities in cell invasiveness and other aspects of TB phenotype associated with pre-eclampsia.
 
Coauthors: Bhanu Prakash VL Telugu, DVM, PhD, Division of Animal Sciences, University of Missouri, Columbia, MO, Mitsuyoshi Amita, MD, PhD, Division of Animal Sciences, University of Missouri, Columbia, MO, Andrei Alexenko, PhD, Division of Animal Sciences, University of Missouri, Columbia, MO, Jessica Schlitt, BS, Department of, Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, MO, Danny Schust, MD, Department of, Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, MO, Laura C Schulz, PhD, Department of, Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, MO, Toshihiko Ezashi, PhD, Division of Animal Sciences, University of Missouri, Columbia, MO

 

Keynote Address

Epigenetics, Imprinting, and the Fetal Origins of Disease Susceptibility
Randy L. Jirtle, PhD, Duke University Medical Center, Durham, NC

Human epidemiological and animal experimental data indicate that the risk of developing adult onset diseases and neurological disorders is influenced by persistent adaptations to prenatal and early postnatal nutrition. A subset of epigenetically regulated targets that potentially link environmental exposures early in development to adult diseases are those with metastable epialleles. They have highly variable expression because of stochastic allelic changes in the epigenome rather than mutations in the genome. The viable yellow agouti (Avy) mouse harbors a metastable Agouti gene because of an upstream insertion of a transposable element. We have demonstrated in the Avy mouse that maternal dietary supplementation during pregnancy, with either methyl donors (i.e. folic acid, vitamin B12, choline and betaine) or genistein, alters coat color and decreases adult disease incidence in the offspring by increasing DNA methylation and altering histone marks at the Avy locus. Moreover, these nutritional supplements can counteract the negative effects on the epigenome caused by the endocrine disruptor, bisphenol A (BPA). We now have evidence that low doses of ionizing radiation also alter the epigenome at the Avy locus in a dose-response manner, and that these epigenetic alterations can be blocked by antioxidants, like vitamin E and vitamin C.

 

Session V: Genetic and Environmental Influences Affecting Fetal Programming In Utero

Programming of Glucose Tolerance
John R. G. Challis, PhD, McMaster University and University of Toronto, Canada

Fetal development can be modified either as an adjustment in response to adversity, including hypoxemia and nutritional compromise, or as a result of fetal exposure to excess glucocorticoid; where these adaptations serve to prepare the fetus for the anticipated postnatal environment. In cases where the prediction and the actual environment mismatch, these developmental modifications are maladaptive. In this regard, reduced growth in utero is associated with impaired glucose tolerance and non-insulin dependent diabetes in later life. Increased risk of metabolic compromise after early life adversity is embedded in developmental modifications of glucose regulation. Cohort studies have shown that famine-exposed individuals born after exposure in mid or late gestation demonstrate higher stimulated glucose levels; an effect that was greatest in individuals born as thin babies to mothers of low body weight. Experimental studies have highlighted novel regulatory pathways including modifications of the fetal hypothalamic-pituitary adrenal axis, adipo-insular axis, and epigenetic regulation of key regulatory genes that may explain altered glucose homeostasis. Similarly, fetal exposure to excess glucocorticoid in late pregnancy impairs pancreatic function and produces glucose intolerance in animals and humans. These data have implications for the long-term health of future generations since glucose intolerant females will likely have maternal hyperglycemica and/or diabetes during pregnancy thus perpetuating similar offspring outcomes. Although the mechanisms are still unclear, the early life environment clearly represents a critical window within which intervention strategies could be developed to reduce the burden of glucose intolerance and eventual diabetes.
 

Coauthor: Deborah M Sloboda, PhD, McMaster University and University of Toronto, Canada

Paternal Influence on Offspring Metabolism
Oliver J. Rando, MD, PhD, University of Massachusetts Medical School, Worcester, MA

Epigenetic information can be inherited through the mammalian germline, and represents a plausible transgenerational carrier of environmental information. To test whether transgenerational inheritance of environmental information occurs in mammals, we carried out an expression profiling screen for genes in mice that responded to paternal diet. Offspring of males fed a low protein diet exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis, and decreased levels of cholesterol esters, relative to the offspring of males fed a control diet. These results, in conjunction with recent human epidemiological data, indicate that parental diet can affect cholesterol and lipid metabolism in offspring, and define a model system to study environmental reprogramming of the heritable epigenome. I will also describe our ongoing efforts to understand the mechanistic basis for paternal transmission of environmental information.
 

Role for Placental Metabolic Pathways in Modulating Fetal Brain Development: Impact of Serotonin
Pat R. Levitt, PhD, University of Southern California

A role for fetal serotonin (5-hydroxytryptamine; 5-HT) in neurodevelopment has been suggested, with the importance of maternal-fetal interactions being a centerpiece of the hypothesis. Yet this concept had not been examined directly. Recent studies address the regulatory role of 5-HT in neural development, the source of extra-embryonic 5-HT to the fetal brain, and an unusual bioactivity of selective serotonin transport inhibitors (SSRIs) on axon growth. Several findings will be discussed including: 1) serotonin serves as a modulator of other axon guidance molecules, including the netrins. In utero electroporation studies that manipulate gene expression confirm the in vitro work, showing that altering 5-HT signaling in the forebrain is sufficient to change trajectory of growing axons; 2) demonstration of extraembryonic 5-HT in the forebrain but not the hindbrain of mouse fetal brain; 3) based on analyses of genetic mutants, a maternal source of 5-HT could not be verified; 4) an alternative source of 5-HT, the placenta, was examined first by demonstrating its synthetic capacity to produce 5-HT from the precursor tryptophan. Furthermore, a new ex vivo placentometer was developed and used to show direct synthesis of 5-HT and transport to the fetal vasculature. In contrast, 5-HT, when presented in the uterine vessel, failed to transport. These findings suggest a new, direct role for placental metabolic pathways in modulating fetal brain development, which has further implications for a variety developmental factors that may influence placental function, and which increase risk for human disease.
 
Coauthor: Alexandre Bonnin, PhD, Zilkha Neurogenetic Institute, Keck School of Medicine of University of Southern California, Los Angeles, CA
 

Mechanisms of Genetic Quality Control in the Germline
John Schimenti, PhD, Cornell University

The germline—from primordial germ cells (PGCs) through the eggs and sperm—are the carriers of the genetic material. High fidelity transmission of the genome, with minimal deleterious mutations, is of paramount importance not only to individual progeny but to the species. Diverse species have evolved checkpoint systemsto monitor the genome of all cell types for incipient mutations, and to either repair them or to trigger elimination of cells with irreparable or intolerable DNA damage levels. Due to the importance of maintaining the germline's genome, it follows that there are particularly sensitive quality control mechanisms to prevent transmission of gametes containing deleterious mutations. We have used genetic approaches to identify genes and mutations in mice that reveal a heightened sensitivity of germline cells to DNA replication-linked errors during fetal development (Mcm9 and Fancm), and to identify a critical component of the DNA damage checkpoint surveillance system that operates during meiosis (genes to be presented). The implications behind defects of these mechanisms for fertility and birth defects will be discussed.
 
Coauthors: Suzanne Hartford, PhD, Yunhai Luo, and Ewelina Bolcun-Filas, PhD, Cornell University, College of Veterinary Medicine
 

Maternal High-Fat Diet, Alcohol and Fetal Programming
Sarah F. Leibowitz, PhD, The Rockefeller University

Maternal consumption of a fat-rich diet or alcohol during pregnancy has been shown, in both human and preclinical studies, to increase the offspring's appetite for dietary fat and alcohol. The mechanisms underlying these phenomena have yet to be identified. Our recent studies in rodents demonstrate that in utero exposure to a high-fat diet (HFD), as compared to a balanced diet, stimulates the birth of excess neurons that express specific neurochemicals, such as the peptides galanin (GAL), orexin (OX), and opioid enkephalin (ENK), that are potent stimulants of both fat consumption and alcohol drinking. The density at birth of these peptide-expressing neurons in the hypothalamus is markedly increased. This phenomenon, which occurs in close association with higher blood lipids caused by the HFD, persists postnatally long after the diet is removed, suggesting that the neuronal changes at this early age are permanent. This is supported by evidence showing that a HFD during pregnancy simulates the proliferation of neuroepithelial and neuronal precursor cells of the embryonic hypothalamic third ventricle, the proliferation and differentiation of neurons, and the migration of these neurons toward hypothalamic areas where the peptides act. Remarkably, very similar results are obtained with maternal consumption of a low dose of alcohol, which also increases circulating lipids. Together with other disturbances in opioid and dopamine receptor systems in extra-hypothalamic areas, these neuronal changes associated with maternal hyperlipidemia may contribute to the increased appetite for fat and alcohol observed in the offspring. (Research supported by USPHS grants: DA 21518 and AA 12882)

 

Session VI: Pregnancy Disorders and Prematurity: Gene–Environment Interactions

Genetics and Genomics of Human Preterm Birth
Louis J. Muglia, PhD, Cincinnati Children's Hospital Medical Center

The regulation of the molecular pathways leading to term birth in humans, and how these normal timing mechanisms are disrupted to result preterm birth, is unknown. While model organisms have provided important insights into fetal development, the physiology of pregnancy maintenance and parturition in these same model systems has been difficult to extrapolate to the observed human physiology. Human genetics and genomics provide powerful non-biased approaches to identify key pathways involved in normal birth timing and the pathogenesis of preterm birth. We will provide evidence that 30-40% of the variation in human birth timing is contributed by genetic factors, and these genetic factors largely reside within the maternal genome. This evidence arises from population epidemiology, classic twin studies and segregation analysis in families. With this knowledge, we have performed genome wide association studies, linkage analysis, and most recently whole exome sequencing to reveal risk loci for preterm birth. To refine loci analyzed in the GWAS, we apply an evolutionary filter for rapidly evolving genes, testing the hypothesis that genes accelerated in their rate of evolution along the human lineage would harbor variants involved in birth timing due to unique constraints in human birth stemming from a large fetal head and a narrow maternal pelvis, needed to facilitate bipedalism. We find that genetic variants in FSHR, PLA2G4C, and IGF1R contribute to being born prematurely, and provide new targets for functional and therapeutic advances. Future studies will similarly analyze the fetal genome for contributions to preterm birth.
 
Coauthors: Jude J. McElroy, AB, Vanderbilt University School of Medicine, Nashville, TN, Courtney Gutman. MD, Vanderbilt University School of Medicine, Nashville, TN, Jevon Plunkett, PhD, Washington University School of Medicine, St. Louis, MO, Kari Teramo, MD, PhD, Helsinki University Central Hospital, Helsinki, Finland, Mikko Hallman, MD, University of Oulu, Oulu, Finland, Justin Fay, PhD, Washington University School of Medicine, St. Louis, MO
 

The Rebirth of Progesterone: Cervical Ultrasound and Progesterone to Prevent Preterm Birth
Roberto Romero, MD, Perinatal Research and Obstetrics, Intramural Division, NICHD, NIH, DHHS

Preterm birth is the leading cause of perinatal morbidity and mortality worldwide, with 13 million preterm births occurring annually. The prediction and prevention of preterm birth has been a major challenge in obstetrics. Preterm parturition is a syndrome caused by multiple etiologies—one of them is a suspension of progesterone action, which may lead to cervical shortening in the midtrimester and can be detected with ultrasound. A short cervix is a powerful predictor of spontaneous preterm delivery. Patients with a cervical length of 15mm or less have a 50% likelihood of having a preterm delivery at <33 weeks. Three interventions have been proposed to treat a short cervix: 1) progesterone; 2) cervical cerclage; or 3) a pessary. Several randomized clinical trials of vaginal progesterone have been conducted in patients with singleton gestations and a history of preterm birth, twin gestations and a short cervix. The administration of vaginal progesterone is associated with a reduction in the rate of early preterm birth, respiratory distress syndrome and neonatal morbidity. An individual patient meta-analysis suggests that cervical cerclage may be effective in women with a history of previous preterm birth and a cervix of <25mm. A recent randomized clinical trial suggests a role for a vaginal pessary in selected patients (Lancet, April 2012). This presentation will review the role of progesterone in pregnancy maintenance in the 1st, 2nd and 3rd trimesters, the mechanisms of action of this hormone, and the cost-effectiveness of a universal screening program including cervical sonography and the administration of progesterone to women with a short cervix.
 

Angiogenesis and Preeclampsia: Functional Characterization of Two Novel Secreted Gene Products Of Placental Origin (Sflt1 And Seng)
S. Anath Karumanchi, MD, Beth Israel Deaconess Medical Center

Imbalance of angiogenic growth factors in the maternal circulation contributes to the pathogenesis of preeclampsia. Soluble fms-like tyrosine kinase 1 (sFlt1), an endogenous anti-angiogenic protein that antagonizes vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) appears to be a central player in this paradigm. Overexpression of sFlt1 in pregnant rats produced hypertension, proteinuria and glomerular endotheliosis, the classical pathological renal lesion of preeclampsia. High serum sFlt1 and low serum free PlGF and free VEGF have been observed in preeclampsia. Abnormalities in these circulating angiogenic proteins are not only present during clinical preeclampsia, but also antedate clinical symptoms by several weeks. Another potential soluble factor secreted by the placenta that appears to be elevated in women with preeclampsia is soluble endoglin (sEng). Endoglin (Eng) is an angiogenic receptor expressed mainly on the surface of endothelial cells, but also by placental syncytiotrophoblast. Eng acts as a co-receptor for transforming growth factor-beta, a potent pro-angiogenic molecule) signaling in endothelial cells. Eng mRNA is up-regulated in the preeclamptic placenta. In addition, the extra-cellular region of endoglin referred to as sEng, is released in excess quantities into the circulation of preeclamptic patients. Furthermore, sEng appeared to exacerbate the vascular damage mediated by sFlt1 in pregnant rats resulting in severe preeclampsia-like illness including the development of thrombocytopenia and fetal growth restriction. Measurement of angiogenic markers in the serum/plasma may be particularly useful for the diagnosis and prediction of preterm preeclampsia. Methods aimed at interfering sFlt1 and sEng actions may be a novel therapeutic strategy in severe preterm preeclampsia.

 

Session VII: Environment, Hormone Action, and Pharmacological / Endocrine Disruptors

Disruption of Uterine Cytodifferentation by Developmental Exposures to Diethylstilbrestrol
Liang Ma, Division of Dermatology, Department of Medicine and Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO

Diethylstilbestrol (DES) is a synthetic estrogen that was widely prescribed to prevent miscarriage from 1940s to 1970s. During that time period, millions of women and their offspring were exposed to this compound. In utero exposure to DES causes reproductive tract malformations, affects fertility and increases the risk of clear cell carcinoma of the vagina and cervix in humans. In mice, perinatal DES exposure represses the expression of critical developmental regulators such as Hox and Wnt genes in the developing uterus. Using a well-established mouse DES model and global transcript profiling, we have systematically studied the underlying molecular mechanism of DES-induced uterine metaplasia. Analyses of the microarray results revealed many developmental control genes as well as anti-apoptotic proteins as DES targets. Genes involved in cellular metabolism accounted for the largest proportion of the DES-regulated genes. Interestingly, multiple components of the Peroxisome Proliferator-Activated Receptor gamma (PPARγ)-mediated adipogenic/lipid metabolism pathway, including PPARγ itself, are targets of DES in the neonatal uterus. TEM and Oil Red O staining further demonstrate a dramatic increase in lipid deposition in the uterine epithelial cells upon DES exposure. Our study for the first time implicates the adipogenic program as a downstream target of DES in the uterine epithelium, suggesting that DES and possibly other EDCs may activates similar genetic pathways in other estrogenresponsive tissues, such as the adipocytes, to cause obesity in adulthood.
 

Coauthors: Yan Yin, Congxing Lin, G. Michael Veith, Division of Dermatology, Department of Medicine and Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO

Making Eggs and Sperm: Environmental Effects on Gametogenesis
Patricia Hunt, PhD, School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA

Studies in rodents suggest that the environmental endocrine disruptor, bisphenol A (BPA) impacts the developing reproductive tract in males and females. BPA is present in a wide variety of consumer products and humans are exposed on a daily basis, raising concerns about the impact of BPA on human fertility. We have focused on the effect of BPA on game to genesis. In the female, BPA adversely affects three different stages of egg development: 1) the onset of oogenesis in the fetal ovary, 2) the formation of follicles in the perinatal ovary and, 3) the resumption and completion of the meiotic divisions in the adult. Effects on the fetal ovary are of particular concern since they have the potential to impact the entire cohort of eggs ovulated by the adult female. To determine if effects observed in rodents extend to primates, we conducted studies in the rhesus monkey and observed similar effects on the fetal ovary. In the male mouse, perinatal BPA exposure has been reported to reduce testis size and sperm counts. To determine if, as in the female, BPA exposure causes meiotic defects, we initiated studies of male mice exposed on days 1-12 postpartum. Our findings suggest that exposure during test is development is sufficient to permanently alter the process of spermatogenesis in the adult. Taken together, our studies suggest that BPA adversely impacts game to genesis and fertility in both sexes, and our findings in rhesus monkeys make it likely that these effects extend to humans.
 

Prenatal Developmental Toxicity Study Design for Pharmaceuticals
L. David Wise, PhD, Merck Research Laboratories

Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is described by the International Conference on Harmonization (ICH) Guidance S5(R2) document. This guidance replaces similar guidelines developed in various countries over the years following the thalidomide disaster. The studies that assess developmental hazard (i.e., Embryo-fetal developmental toxicity [EFD] studies) are generally conducted in rodents and rabbits under Good Laboratory Practice (GLP) conditions. The designs and types of data obtained from these studies (including dose range-finding studies) will be described. In addition, the timing of these studies in relation to clinical studies that enroll women of childbearing potential as outlined in S5(R2) will be discussed. Optional parameters that may be included in the studies will be discussed, as will study designs that combine assessments of fertility and developmental toxicity. The successful completion of such studies requires a substantial investment in training and resources, but results in the high-quality data needed to assess human safety.