Fetal Programming and Environmental Exposures: Implications for Prenatal Care and Preterm Birth

Resources

The impact of diet on fetal development

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Drugs and consumer products: Adverse effects

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New models for studying tropoblast development

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Genbacev O, Donne M, Kapidzic M, et al. Establishment of human trophoblast progenitor cell lines from the chorion. Stem Cells. 2011;29(9):1427-36.

Kent LN, Rumi MA, Kubota K, Lee DS, Soares MJ. FOSL1 is integral to establishing the maternal-fetal interface. Mol. Cell Biol. 2011;31(23):4801-13.

Lee DS, Rumi MA, Konno T, Soares MJ. In vivo genetic manipulation of the rat trophoblast cell lineage using lentiviral vector delivery. Genesis. 2009;47(7):433-9.

Marchand M, Horcajadas JA, Esteban FJ, McElroy SL, Fisher SJ, Giudice LC. Transcriptomic signature of trophoblast differentiation in a human embryonic stem cell model. Biol. Reprod. 2011;84(6):1258-71.

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Rosario GX, Konno T, Soares MJ. Maternal hypoxia activates endovascular trophoblast cell invasion. Dev. Biol. 2008;314(2):362-75.

Schulz LC, Ezashi T, Das P, Westfall SD, Livingston KA, Roberts RM. Human embryonic stem cells as models for trophoblast differentiation. Placenta. 2008;29 Suppl A:S10-6.

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Pre-term birth: Risks and treatments

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Bonnin A, Levitt P. Fetal, maternal, and placental sources of serotonin and new implications for developmental programming of the brain. Neurosci. 2011;197:1-7.

Cahill AG, Odibo AO, Caughey AB, et al. Universal cervical length screening and treatment with vaginal progesterone to prevent preterm birth: a decision and economic analysis. Am. J. Obstet. Gynecol. 2010;202(6):548 e1-8.

Deneris ES, Wyler SC. Serotonergic transcriptional networks and potential importance to mental health. Nat. Neurosci. 2012;15(4):519-27.

Fonseca EB, Celik E, Parra M, Singh M, Nicolaides KH. Progesterone and the risk of preterm birth among women with a short cervix. N. Engl. J. Med. 2007;357(5):462-9.

Gross GA, Imamura T, Luedke C, et al. Opposing actions of prostaglandins and oxytocin determine the onset of murine labor. Proc. Natl. Acad. Sci. USA. 1998;95(20):11875-9.

Handel MA, Schimenti JC. Genetics of mammalian meiosis: regulation, dynamics, and impact on fertility. Nat. Rev. Genet. 2010;11(2):124-36.

Hassan SS, Romero R, Vidyadhari D, et al. Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial. Ultrasound Obstet. Gynecol. 2011;38(1):18-31.

Hendricks TJ, Fyodorov DV, Wegman LJ, et al. Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior. Neuron. 2003;37(2):233-47.

Karumanchi SA, Stillman IE. In vivo rat model of preeclampsia. Methods Mol. Med. 2006;122:393-9.

Parikh SM, Karumanchi SA. Putting pressure on pre-eclampsia. Nat. Med. 2008;14(8):810-2.

Plunkett J, Doniger S, Orabona G, et al. An evolutionary genomic approach to identify genes involved in human birth timing. PLoS Genet. 2011;7(4):e1001365.

Rana S, Powe CE, Salahuddin S, et al. Angiogenic factors and the risk of adverse outcomes in women with suspected preeclampsia. Circulation. 2012;125(7):911-9.

Ratajczak CK, Fay JC, Muglia LJ. Preventing preterm birth: the past limitations and new potential of animal models. Dis. Model Mech. 2010;3(7-8):407-14.

Romero R, Nicolaides K, Conde-Agudelo A, et al. Vaginal progesterone in women with an asymptomatic sonographic short cervix in the midtrimester decreases preterm delivery and neonatal morbidity: a systematic review and metaanalysis of individual patient data. Am. J. Obstet. Gynecol. 2012;206(2):124 e1-19.

Sasaki H, Matsui Y. Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nat. Rev. Genet. 2008;9(2):129-40.

Suh EK, Yang A, Kettenbach A, et al. p63 protects the female germ line during meiotic arrest. Nature. 2006;444(7119):624-8.

Thadhani R, Kisner T, Hagmann H, et al. Pilot study of extracorporeal removal of soluble fms-like tyrosine kinase 1 in preeclampsia. Circulation. 2012;124(8):940-50.

Pre-implantation development

Chen J, Melton C, Suh N, et al. Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition. Genes Dev. 2011;25(7):755-66.

Freemark M, Avril I, Fleenor D, et al. Targeted deletion of the PRL receptor: effects on islet development, insulin production, and glucose tolerance. Endocrinology. 2002;143(4):1378-85.

Hu D, Cross JC. Development and function of trophoblast giant cells in the rodent placenta. Int. J. Dev. Biol. 2010;54(2-3):341-54.

Huang C, Snider F, Cross JC. Prolactin receptor is required for normal glucose homeostasis and modulation of beta-cell mass during pregnancy. Endocrinology. 2009;150(4):1618-26.

Hunkapiller NM, Gasperowicz M, Kapidzic M, et al. A role for Notch signaling in trophoblast endovascular invasion and in the pathogenesis of pre-eclampsia. Development. 2011 July;138(14):2987-98.

Jungheim ES, Moley KH. Current knowledge of obesity's effects in the pre- and periconceptional periods and avenues for future research. Am. J. Obstet. Gyneco. 2010;203(6):525-30.

Jungheim ES, Schoeller EL, Marquard KL, Louden ED, Schaffer JE, Moley KH. Diet-induced obesity model: abnormal oocytes and persistent growth abnormalities in the offspring. Endocrinology. 2010;151(8):4039-46.

Kang MK, Han SJ. Post-transcriptional and post-translational regulation during mouse oocyte maturation. BMB Rep. 2011;44(3):147-57.

Mantovani A. Hazard identification and risk assessment of endocrine disrupting chemicals with regard to developmental effects. Toxicology. 2002;181-182:367-70.

Oh JS, Susor A, Conti M. Protein tyrosine kinase Wee1B is essential for metaphase II exit in mouse oocytes. Science. 2011;332(6028):462-5.

Vom Saal FS, Myers JP. Bisphenol A and risk of metabolic disorders. JAMA. 2008;300(11):1353-5.

Vom Saal FS, Nagel SC, Coe BL, Angle BM, Taylor JA. The estrogenic endocrine disrupting chemical bisphenol A (BPA) and obesity. Mol. Cell Endocrinol. 2012;354(1-2):74-84.

Wang Q, Frolova AI, Purcell S, et al. Mitochondrial dysfunction and apoptosis in cumulus cells of type I diabetic mice. PLoS One. 2010;5(12):e15901.

Yu Z, Ji P, Cao J, et al. Dazl promotes germ cell differentiation from embryonic stem cells. J. Mol. Cell Biol. 2009;1(2):93-103.

Zoeller RT, Brown TR, Doan LL, et al. Endocrine-disrupting chemicals and public health protection: a statement of principles from the endocrine society. Endocrinology. 2012. [Epub ahead of print].

Embryo–uterine cross talk

Chaouat G, Petitbarat M, Dubanchet S, Rahmati M, Ledee N. Tolerance to the foetal allograft? Am. J. Reprod. Immunol. 2010;63(6):624-36.

Duggavathi R, Volle DH, Mataki C, et al. Liver receptor homolog 1 is essential for ovulation. Genes Dev. 2008;22(14):1871-6.

Erlebacher A. Immune surveillance of the maternal/fetal interface: controversies and implications. Trends Endocrinol. Metab. 2010;21(7):428-34.

Erlebacher A. Why isn't the fetus rejected? Curr. Opin. Immunol. 2001;13(5):590-3.

Han SJ, Hawkins SM, Begum K, et al. A new isoform of steroid receptor coactivator-1 is crucial for pathogenic progression of endometriosis. Nat. Med. 2012. [Epub ahead of print].

Lefevre P, Campos DB, Murphy BD. Talk to me: the embryo dictates gene expression by the endometrium. Endocrinology 2007;148(9):4170-2.

Lefevre PL, Murphy BD. Differential gene expression in the uterus and blastocyst during the reactivation of embryo development in a model of delayed implantation. Methods Mol. Biol. 2009;550:11-61.

Murphy BD. Revisiting reproduction: What a difference a gene makes. Nat. Med. 2010;16(5):527-9.

Nancy P, Tagliani E, Tay CS, Asp P, Levy DE, Erlebacher A. Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface. Science. 2012;336(6086):1317-21.

Rubel CA, Lanz RB, Kommagani R, Franco HL, Lydon JP, Demayo FJ. Research resource: genome-wide profiling of progesterone receptor binding in the mouse uterus. Mol. Endocrinol. 2012. [Epub ahead of print].

Spencer TE, Bazer FW. Biology of progesterone action during pregnancy recognition and maintenance of pregnancy. Front Biosci. 2002;7:d1879-d1898.

Wetendorf M, Demayo FJ. The progesterone receptor regulates implantation, decidualization, and glandular development via a complex paracrine signaling network. Mol. Cell Endocrinol. 2012;357(1-2):108-18.

Young investigators

Barcena A, Kapidzic M, Muench MO, et al. The human placenta is a hematopoietic organ during the embryonic and fetal periods of development. Dev. Biol. 2009;327(1):24-33.

Barcena A, Muench MO, Kapidzic M, Gormley M, Goldfien GA, Fisher SJ. Human placenta and chorion: potential additional sources of hematopoietic stem cells for transplantation. Transfusion. 2011;51 Suppl 4:94S-105S.

Campagnoli C, Fisk N, Overton T, Bennett P, Watts T, Roberts I. Circulating hematopoietic progenitor cells in first trimester fetal blood. Blood. 2000;95(6):1967-72.

Chakrabarty A, Tranguch S, Daikoku T, Jensen K, Furneaux H, Dey SK. MicroRNA regulation of cyclooxygenase-2 during embryo implantation. Proc. Natl. Acad. Sci. USA. 2007;104(38):15144-9.

Choufani S, Shapiro JS, Susiarjo M, et al. A novel approach identifies new differentially methylated regions (DMRs) associated with imprinted genes. Genome Res. 2011;21(3):465-76.

Davisson RL, Hoffmann DS, Butz GM, et al. Discovery of a spontaneous genetic mouse model of preeclampsia. Hypertension. 2002;39(2 Pt 2):337-42.

Hirota Y, Cha J, Yoshie M, Daikoku T, Dey SK. Heightened uterine mammalian target of rapamycin complex 1 (mTORC1) signaling provokes preterm birth in mice. Proc. Natl. Acad. Sci. USA. 2011;108(44):18073-8.

Hirota Y, Daikoku T, Tranguch S, Xie H, Bradshaw HB, Dey SK. Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice. J. Clin. Invest. 2010;120(3):803-15.

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Maynard SE, Min JY, Merchan J, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Invest. 2003;111(5):649-58.

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Sponsors

Presented by

Academy Friends

Abcam Inc.

Quartzy

Watson Pharmaceuticals, Inc.

Weill Cornell Medical College — The Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine

Grant Support

Burroughs Wellcome Fund

Supported in part by March of Dimes Foundation Grant No. 4-FY12-545.

Funding for this conference was made possible (in part) by grant number 1R13ES021699-01 from the National Institute of Environmental Health Sciences (NIEHS) and National Institute on Drug Abuse (NIDA). The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention by trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

Keynote Speaker:
Frederick S. vom Saal, University of Missouri–Columbia

Speakers:
Marco Conti, University of California, San Francisco
Kelle H. Moley, Washington University School of Medicine
James Charles Cross, University of Calgary

Highlights

• Low doses of endocrine-modifying chemicals that are prevalent in the environment can negatively affect fetal development.
• Networks of RNA-binding maternal proteins are crucial for oocyte maturation and "set the stage" for fertilization.
• Obesity, insulin resistance, and hyperglycemia can cause irreversible damage to oocytes and may lead to a block in embryogenesis.
• Prolactin hormones expressed by the placenta may help protect mothers from developing gestational diabetes.

Introduction

Fetal development is a complex process that relies on numerous environmental inputs from uterine tissue, the placenta, the maternal blood supply, and other sources. Prior to fertilization, oocytes and sperm are subject to chemical and metabolic influences within the respective parent. Although these interactions drive the proper sequence of developmental events and provide nourishment and protection, they can also result in exposure to toxic agents or other damaging conditions.

In addition, a growing body of research has suggested that the intrauterine environment contributes to more than the formation of an infant or even a mature individual—processes that generally fall under the umbrella of 'development.' This environment is also thought to ‘program’ the individual for susceptibility to adult disease, which may occur long after the developmental phase is complete. Given these findings, investigators interested in health at all stages of life are taking note of research into the effects of environmental exposures at the earliest timepoints of development.

Industrial chemicals and endocrine interference

The first day of the Fetal Programming and Environmental Exposures conference began with a keynote address by Frederick vom Saal from the University of Missouri–Columbia, who explained that low-level exposure to compounds that modify steroid hormones in utero can have negative effects on fetal development. vom Saal presented data suggesting that environmental chemicals such as bisphenol A (BPA), an organic compound commonly used in the plastics industry, mimic the action of estrogen hormones. He suggested that exposure to low doses of chemicals that affect the endocrine system may promote the development of diseases such as breast cancer, diabetes, and even developmental disorders like ADHD.

vom Saal expressed concern that toxicological risk-assessment methods employing conventional monotonic dose responses are inadequate. Endocrine-modifying chemicals such as BPA are capable of binding estrogen receptors at doses in the low nanomolar range, yet risk assessments frequently test compounds at much higher concentrations and make assumptions about the effect of low-level exposures in vivo. vom Saal argued that estrogen-like molecules can behave very differently depending on the level of exposure. As an example, he cited the anti-estrogen tamoxifen, which prevents the proliferation of breast cancer cells at pharmacological doses but can actually promote cell proliferation at lower doses. When testing potential endocrine-modifying chemicals, effects at both low and high doses should be considered. Risk assessment for fetal impacts must also acknowledge that low doses of such chemicals can affect a critical time in development when the fetus lacks a fully developed immune system, liver (to detoxify these compounds), and blood–brain barrier. vom Saal's lecture set the tone for the rest of the conference by addressing the sensitivity of fetal development and highlighting the public health concerns raised by the possibility of toxicity from agents in the environment such as industrial chemicals.

Chemical risk-assessment assumes a monotonic dose response relationship and makes assumptions about low-dose effects. (Image courtesy of Frederick vom Saal)

Embryonic development: Dazl, oocytes, and prolactins

The next presenter was Marco Conti from the Center for Reproductive Sciences at the University of California, San Francisco. Conti spoke about networks of RNA-binding proteins that regulate oocyte and embryonic development; specifically, the mechanism by which newly synthesized proteins "set the stage" for the developing embryo during a critical period—between oocyte maturation and activation of the embryonic genome—when gene transcription does not occur. Conti discovered a gene called Dazl (Deleted in azoospermia-like), which encodes a maternal protein that positively regulates RNA translation in the maturing oocyte before embryogenesis occurs. Due to its epigenetic effects on DNA and chromatin structure, Dazl also regulates meiosis in the zygote: Conti's research showed that the absence of Dazl prevents meiosis by disrupting microtubule organization and spindle formation. Thus, Conti's study highlights the role of epigenetic regulation, mediated by maternal proteins like Dazl, in embryogenesis.

As the conference progressed, participants learned how obesity and other metabolic disorders alter embryonic development in mammals. Kelle H. Moley from Washington University School of Medicine presented her work with a mouse model of diabetes. Oocytes from diabetic mice had alterations in cell structure and morphology, and mitochondria in these oocytes were not distributed properly, resulting in decreased levels of ATP, the cell's primary energy source. Based on these findings, Moley argued that obesity and diabetes antagonize the early stages of pregnancy.

The session concluded with a presentation from James Charles Cross from the University of Calgary. Cross examined the various types of cells that line the placenta–fetus interface and found that several types of trophoblast cells line the canal leading to the umbilical cord; the fetus relies on these cells for transportation of maternal blood and nutrients. Specialized placental cells express a family of genes called prolactins, hormones required for insulin production during pregnancy. Cross argued that prolactin hormones protect female mice from developing gestational diabetes during pregnancy, further illustrating the importance of the specialized cells that bridge the gap between mother and fetus.

Speakers:
Francesco J. DeMayo, Baylor College of Medicine
Bruce Murphy, Université de Montréal
Adrian Erlebacher, New York University Langone Medical Center

Highlights

• Epidermal growth factor receptors regulate the process of decidualization by controlling progesterone action.
• The NR5A2 gene encodes an orphan receptor that regulates a network of signaling molecules required for fertility.
• An epigenetic mechanism may be responsible for suppressing T-cell migration into the placenta, which could otherwise result in rejection of the developing fetus.

Nuclear receptors and immune surveillance

The second session began with a presentation by Francesco DeMayo from Baylor College of Medicine. DeMayo's group is investigating the role progesterone receptors play in embryo implantation and decidualization. A complex signaling cascade occurs between the epithelium and the stroma before implantation. DeMayo found that activation of the progesterone receptor in the epithelium sets off this cascade by regulating expression of the morphogen Indian Hedghog (Ihh). His group is also seeking to understand the mechanism of decidualization, the process by which progesterone transforms the endometrium during pregnancy. DeMayo spoke about the effects of the EGFR family of receptor tyrosine kinases—otherwise famous for their oncogenic effects in breast cancer—on decidualization. His group found that progesterone action is regulated by EGFR family members; in fact, pharmacological inhibitors of EGFR and HER2 prevent decidualization, suggesting that these inhibitors might one day be used to treat endometriosis. DeMayo's group is undertaking genome-wide studies to identify which genes are targets of the progesterone receptor and to further define how uterine biology is dependent on this hormone. Preliminary findings indicate the importance of genes involved in the circadian cycle and of the GATA2 gene, which encodes a transcription factor that tunes progesterone sensitivity in the epithelium.

Continuing on the topic of nuclear receptor signaling, Bruce Murphy from the Université de Montréal spoke about a family of genes called orphan nuclear receptors, which are similar to estrogen and progesterone receptors but for which no activating hormone has yet been identified. The NR5A2 gene encodes an orphan receptor that is expressed in granulosa and luteal cells in the ovary and the uterine stroma. Murphy and colleagues investigated this orphan receptor using tissue-specific knockout mice with the NR5A2 gene deleted in progesterone-receptive tissues only. His group discovered that NR5A2 deletion causes problems early in gestation by inhibiting progesterone synthesis. Knockout mice treated with progesterone exhibited gestational failure later, due to defects in the uterus. Transplanting wild-type ovaries into knockout mice did not correct the abnormality: embryos failed to develop normally and gestation failed. Gene-expression experiments suggest that the NR5A2 gene plays an important role in decidualization by regulating networks of signaling molecules and transcription factors that are required for fertility. While much attention has been directed towards understanding the action of well-established hormone receptors, these results indicate the importance of orphan nuclear receptors in regulating reproductive processes.

The final part of this session ventured into the field of immunology. Adrian Erlebacher from New York University Langone Medical Center began by asking a provocative question: Why does the immune system fail to reject a fetus? After all, the fetus is a foreign entity that, as such, should be rejected by the adaptive immune response. To investigate this, Erlebacher re-activated memory T cells in a transgenic mouse model to specifically migrate to, and attack at, the fetal–placental interface. His team found that T cells cannot cross into the decidua, suggesting that there is a molecular barrier preventing infiltration; they discovered that an innate epigenetic mechanism represses certain chemokine genes that are required for T-cell migration. Chemokines are hormones for immune cells that signal T cells to migrate into areas of infection; nature has created its own barrier to protect the developing fetus, preventing T cells from infiltrating the placenta. Erlebacher speculated that insufficient epigenetic silencing of chemokine genes would result in fetal/placental "rejection" or preterm birth secondary to deciduitis. Conversely, excessive silencing would result in an inability to combat decidual infection and lead to preterm birth secondary to chorioamnionitis or to stillbirth.

Memory T cells that have not been re-activated by an antigen are shown on the left (blue). Memory T cells re-stimulated with an antigen are activated (pink) but cannot infiltrate the decidual stroma cells due to epigenetic silencing of chemokines CXCL9 and CXCL10. (Image courtesy of Adrian Erlebacher)

Keynote Speaker:
Margaret Ann Miller, National Center for Toxicological Research, FDA

Speakers:
John R. G. Challis, University of Toronto
Oliver J. Rando, University of Massachusetts Medical School
Sarah F. Leibowitz, The Rockefeller University
Pat R. Levitt, University of Southern California
Liang Ma, Washington University School of Medicine
Patricia Hunt, Washington State University
L. David Wise, Merck

Highlights

• Fetal development is influenced by environmental factors that can have lasting impacts over a lifetime.
• Maternal and paternal diets can have significant metabolic and behavioral effects: high consumption of fat and alcohol during pregnancy can promote obesity in offspring.
• Studies show consumer products containing DES and BPA are detrimental to reproductive health.
• The FDA is developing improved toxicology evaluation and labeling to highlight drug actions during pregnancy.

Keynote—FDA evaluation of drugs used during pregnancy

The second day of the conference highlighted the impact of diet and drugs on fetal programming and presented current research into the causes and prevention of preterm birth. The keynote was given by Margaret Ann Miller from the National Center for Toxicological Research at the FDA, who presented recent work to improve the toxicological evaluation of drugs for use during pregnancy. To improve drug labeling for pregnant women, the FDA has broadened the scope of information and has transitioned from using an overly simplistic category system (reflecting risk, risk/benefit, or no data) to including descriptive text that addresses developmental abnormalities, maternal safety (i.e., maternal health outside of the pregnancy), recommended timing of exposure, and dosing during pregnancy, which has been lacking for many drugs.

It is considered unethical to enroll pregnant women in clinical trials unless the trials are testing a treatment specific to pregnancy; however, there is a need for better data. One means to identify specific effects during pregnancy is to extrapolate data from animal studies to explicate likely human outcomes. Another is to draw conclusions from clinical data that are already available. To the latter end, the FDA is building pregnancy exposure registries to collect data on broad margins of safety and pregnancy outcomes and to guide further studies. The goal is to enroll pregnant women who are currently taking drugs for conditions unconnected with their pregnancy; for example, allergy medications or antiepileptics. The FDA has also initiated pharmacokinetic studies to aid in determining appropriate dosing of medicines in pregnant women. Permission to conduct these studies was only granted when it was clear that there was minimal risk to the fetus and that the study would not alter the treatment of the women. These studies have been transferred to the NICHD. It is hoped that new labeling regulations and information from well-designed studies will lead to better recommendations for drug safety.

Impact of diet on fetal development and future health outcomes

Parental diet can impact fetal development: the challenge is to understand its effects and to pinpoint periods of gestation during which it is most influential on health outcomes. John R. G. Challis from the University of Toronto discussed a study that enrolled participants who were fetuses during a five month period in the Dutch winter famine when daily calorie rations fell to 400 to 800 calories per day; following participants from age 50 to 58, the study found, compared to baseline, numerous changes that impact disease, including decreased glucose tolerance and elevated insulin concentrations. Further analyses revealed epigenetic alterations including decreased DNA methylation of the IGF2 gene. These findings highlight the significant effect that maternal nutrition during gestation has on health later in life.

Data showing that paternal diet also impacts offspring metabolism were presented by Oliver J. Rando from the University of Massachusetts Medical School. His group is developing a mouse epigenetic pipeline to determine whether dietary information is carried by sperm. Using sperm from males with varied diets to fertilize eggs in vitro, researchers derive embryonic stem cells from developing blastocysts for further in vitro studies or implant these blastocysts into pseudo-pregnant females for in vivo studies. This strategy eliminates other factors that could potentially impact offspring metabolism, such as seminal fluid peptides, and in theory enables researchers to study only the epigenetic impact of the sperm in an otherwise genetically homogenous population. Thus far, analysis of the genetic and epigenetic landscape of sperm tissue has revealed modest cytosine methylation changes at putative gene enhancer regions, which could impact gene expression. Although methylation patterns generally clustered better with the generation of mice than with their diet, the interferon zeta cluster displayed a 10% to 15% decrease in methylation with a low protein diet. Research is ongoing to discover the implications of these results and to further refine the technique used.

Sarah Leibowitz from The Rockefeller University focused on the positive feedback loop between maternal diet during gestation and the dietary habits of offspring over their lifetime. In one study in rats, a high fat diet and high alcohol consumption were both correlated with increased production of hypothalamic peptides in offspring, which drive an appetite for a high fat diet. Prenatal exposure to a high fat diet increased the genesis of peptide-expressing neurons in the hypothalamus of developing rats at embryonic day (E) 11.5 and offspring exhibited a higher density of these neurons after birth. This higher density persisted even in the absence of a high fat diet over their lifetime and resulted in a range of behavioral and metabolic effects, such as a higher food intake and a preference for fatty foods. This study underscores the significant impact of maternal diet during pregnancy on the dietary habits of offspring.

Pat Levitt from the University of Southern California continued the discussion of metabolic pathways and fetal development, focusing on the role played by the placenta. There is a plethora of evidence supporting the hypothesis that maternal serotonin (5HT) impacts fetal brain development by directly influencing serotonin levels in the developing brain. Levitt showed that 5HT produced by the placenta contributes to serotonin levels in the developing brain: in Pet-1 knockout mice 5HT levels in the forebrain remained normal until embryonic day (E) 16.5, despite the lack of 5HT neurons. Demonstrating that the placenta is capable of producing 5HT until E18.5, Levitt's data shed light on the importance of this extra-embryonic source of a neurotransmitter for fetal development.

Serotonin levels in the embryonic mouse forebrain are regulated by serotonin synthesis in the placenta until E16 and can be affected by a range of environmental factors during gestation, such as maternal stress. (Image courtesy of Pat Levitt)

Investigating drugs and consumer products

There are numerous consumer products on the market for which safety data are lacking. The FDA, academic institutions, and non-governmental organizations are conducting studies to address the safety of these compounds for fetal development and postnatal health. Liang Ma from Washington University School of Medicine described his research into the mechanism of action of diethylstilbestrol (DES), a synthetic estrogen that was widely used as an anti-miscarriage drug between 1940 and 1970 but was later shown to induce reproductive tract malformations and other defects. Ma's data show that DES represses key developmental genes such as Hoxa10, causes cell death, reduces cell proliferation in the uterine epithelium, induces accumulation of lipid droplets, and promotes up-regulation of the glucose pathway. Ma predicts that the metabolic defects caused by DES exposure could also be present in other estrogen-responsive tissues, such as fatty tissue, and thus may contribute to obesity later in life.

Patricia Hunt from Washington State University discussed reproductive tract abnormalities induced by bisphenol A (BPA), an environmental endocrine disrupter found in many consumer products. Hunt's investigation of BPA was prompted by an observation that chromosomal abnormalities occur in mice that consume water from BPA bottles. Treating mice with BPA at fetal and perinatal stages increased chromosomal recombination, increased multioocyte follicle formation in females, and affected spermatogenesis in males. Similar effects were detected in experiments in rhesus monkeys. Future studies will analyze the effect of BPA on other organs, including the brain.

Chromosomal abnormalities such as synaptic defects and increased level of recombination are detected with increasing doses of BPA administration. (Image courtesy of Patricia Hunt)

L. David Wise from Merck described the procedure for designing toxicity studies for pharmaceutical drugs. He explained guidelines issued by the International Conference on Harmonization (ICH) that outline a study-design standard set by the U.S., E.U., and Japan for examining the effects of drugs on several stages of prenatal and postnatal development. These guidelines cover studies on fertility and early development (from fertilization until implantation), embryo–fetal development (from implantation until just before birth), prenatal and postnatal development (from implantation until weaning), and juvenile toxicity (from birth until after sexual maturity). The embryo–fetal study design, although labor intensive, provides the most suitable data for determining drug risk and efficacy information for women of childbearing age.

Speakers:
Susan J. Fisher, University of California, San Francisco
Michael J. Soares, Kansas University Medical Center
R. Michael Roberts, University of Missouri–Columbia
Louis J. Muglia, Cincinnati Children's Hospital Medical Center
Roberto Romero, National Institute of Child Health & Human Development, NIH
S. Ananth Karumanchi, Beth Israel Deaconess Medical Center and Harvard Medical School
John Schimenti, Cornell University

Highlights

• Researchers are developing new screening and treatment methods to prevent preterm birth and preeclampsia through identification of genetic and environmental risk factors.
• Current research is investigating the epigenetic and genetic regulators that contribute to preterm birth, focusing on trophoblast development.
• Human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines are useful models for studying early stages of embryonic development.

New models for studying trophoblast development

Trophoblast development and invasion are essential steps in placentation, establishing a fetal connection to maternal blood flow. Species differences in this developmental process have highlighted the need to establish better model systems to study human trophoblast invasion. Susan J. Fisher from the University of California, San Francisco described a new method to derive human embryonic stem cell (hESC) lines from single blastomeres from 8-cell embryos rather than from whole blastocysts. hESCs are a powerful model for understanding human development, but lines that are currently available have been established after weeks of in vitro development and thus exhibit heterogeneity in terms of gene expression and differentiation capacity. Fisher's new hESC lines appear to be more primitive and display a slightly different gene expression profile, with fewer genes expressed than previously established hESC lines (these new cell lines are awaiting NIH registration). Experiments are underway to differentiate these lines into trophoblast cells for studying human trophoblast development.

Michael J. Soares from Kansas University Medical Center described several models for studying trophoblast invasion. Soares investigated the molecular pathways that regulate trophoblast invasion using both transgenic rats, a model system with an invasive trophoblast lineage similar to that of humans, and blastocyst-derived trophoblast stem cell lines. Both knock-down of the gene Fosl1 and hypoxia resulted in decreased trophoblast invasion in rats. Fosl1 encodes a transcription factor that is a component of the PI3K/Akt signaling pathway, which is important in promoting cell growth and survival. Hypoxia induced the expression of several genes, including the histone demethylase Kdm3A, which regulates metalloprotease MMP12 expression. Members of the MMP family are key regulators of cell adhesion and invasion.

R. Michael Roberts from the University of Missouri–Columbia continued the discussion of trophoblast invasion and development. His results differed from those reported in a paper by Roger Pedersen's group, published in Cell Stem Cell in 2011, which showed that hESCs treated with the growth factor BMP4 differentiate along the mesodermal lineage. Roberts' data show, instead, that treating hESCs with BMP4 switches on expression of trophoblast markers and progesterone. The Matrigel invasion assay revealed increased trophoblast invasion, especially in cells grown in a high oxygen environment (20%). Roberts suggested that differences between these results could be due to the different media and substrates that were used by the two groups. His group has now derived 14 human-induced pluripotent stem cell (hiPSC) lines from fetal umbilical cord tissue (obtained from cases of preeclampsia, a condition that can develop during pregnancy and can result in the death of baby and/or mother from seizures and organ failure). Roberts' team plan to use the hiPSC lines to investigate possible genetic and cellular defects related to preeclampsia.

Preterm birth and birth defects: Identifying risks and developing treatments

Louis J. Muglia from Cincinnati Children's Hospital Medical Center shifted the focus to discuss factors impacting preterm delivery. Inactivation of Cyclooxygenase 1 (Cox1)—a known factor implicated in human birth that is required for prostaglandin synthesis—delayed birth in mice; however, implantation of Cox1-deficient blastocysts into the uterus of normal females resulted in normal duration of labor, suggesting that maternal Cox1 expressed in the uterine wall is sufficient for normal labor. Muglia utilizes comparative and whole-genome studies to identify gene variants that may contribute to preterm labor; this could allow for prenatal identification of at-risk individuals and help researchers to develop new therapies for treatment.

Roberto Romero from the National Institute of Child Health and Human Development at the NIH expanded on the topic of preterm birth. He presented data from several randomized clinical trials that studied vaginal progesterone levels in pregnant women at risk for preterm delivery, focusing on women with a short cervix—a prominent risk factor. His results reveal consistent and significant reductions in the rate of preterm births in women treated with progesterone. Romero discussed the value of universal screening programs to identify women with a short cervical size in order to combat preterm births by administering progesterone treatment.

S. Ananth Karumanchi from Beth Israel Deaconess Medical Center and Harvard Medical School talked about several angiogenic growth factors that contribute to preeclampsia development in women with abnormal levels of angiogenic factors in circulation. He described several methods that aim to restore vascular endothelial growth factor (VEGF), the depletion of which contributes to preeclampsia symptoms like hypertension and proteinuria. He also discussed strategies to treat preeclampsia by depleting proteins that interfere with critical angiogenic factors, such as VEGF, to shift the balance of factors towards "proangiogenesis."

John Schimenti from Cornell University presented work using forward and reverse genetic approaches to identify genes and pathways that impact germ cell survival. Germline mutations, if not corrected or eliminated by the DNA damage response (DDR) pathway, can result in birth defects, reduced fitness, and sterility. Intact cell-death machinery facilitates the elimination of germ cells that fail to be corrected by the DDR. Targeted deletion of either Mcm9 or Fancm, two DDR pathway genes that are needed for correcting mutations, resulted in germ-cell depletion in the ovaries and testes of mice. Deletion of the p53 gene, which triggers cell death in response to DNA damage, resulted in germ-cell depletion in Mcm9-knockout mice but not in Fancm-knockout mice. Analysis of a number of other knockouts revealed that germ cell depletion in response to DNA damage can occur via p53-dependent, as well as p53-independent and Check2-independent mechanisms. This provides evidence that several molecular pathways are at work to protect against the maintenance of germ cells carrying mutations that could result in embryos at risk for birth defects and sterility.

Mcm9 protects against DNA damage and germ-cell depletion. Germ cells (green cells) are reduced in the mouse testis in the absence of Mcm9. (Image courtesy of John Schimenti)

Speakers:
Jenny Sones, Cornell University
Jeeyeon Cha, Cincinnati Children's Hospital Medical Center
Alicia Bárcena, University of California, San Francisco
Martha Susiarjo, University of Pennsylvania

Highlights

• Preeclampsia is associated with inappropriate hormone dynamics and abnormal expression of the genes LIF and COX2.
• Celecoxib and low doses of rapamycin both reverse spontaneous preterm birth in mouse models.
• Embryogenesis requires a specific type of hematopoietic stem cell to repopulate the embryonic niche with new blood cells.
• Bisphenol A may cause genetic disorders by preventing the normal function of genomic imprinting.

Graduate students and postdoctoral fellows presented their research projects in fetal programming in a series of presentations. The first talk was given by Jenny Sones, a graduate student (and veterinarian by training) at Cornell University. Sones presented her work on a mouse model called BPH/5, an inbred strain that spontaneously develops symptoms similar to human preeclampsia, including late-gestational hypertension, proteinuria, renal glomerular lesions, and endothelial dysfunction. She discovered that the model mice had inappropriate hormone dynamics, which led to abnormal expression of two important genes that regulate implantation, LIF and COX2.

Jeeyeon Cha, a graduate student at Cincinnati Children's Hospital Medical Center, also spoke about abnormal COX2 expression in a mouse model of preterm birth. Cha and colleagues showed that an important enzymatic complex called mTORC1 is up-regulated in decidual cells to induce their senescence—premature exit from the cell cycle. They hypothesize that preterm birth is characterized by senescent decidual cells that have undergone premature terminal differentiation and found that preterm birth is reversed by the COX2 inhibitor celecoxib or by a low dose of rapamycin, which is commonly used to inhibit the mTORC1 activity. This work could one day facilitate novel preventative treatment strategies for women at risk for preterm birth.

Cellular senescence and premature differentiation of decidual cells may lead to defective decidualization and preterm birth. (The above scheme is adapted from JCI 120(4):1004-1015, 2010.)

Alicia Bárcena, a postdoctoral fellow at the University of California, San Francisco, presented her work on hematopoietic cells in the placenta and the chorion. She discovered that a certain type of hematopoietic stem cell exhibiting high expression of a primitive cell surface marker is capable of re-populating the extraembryonic niche with diverse blood cells; these stem cells also communicate with endothelial cells in the vasculature as well as trophoblasts in the chorion. This work will further our understanding of how primitive blood development occurs early in embryogenesis.

Martha Susiarjo, a postdoctoral fellow at the University of Pennsylvania, presented her work on the effects of Bisphenol A (BPA) in the mouse placenta. Susiarjo's talk revisited the role of BPA in altering epigenetic regulation and causing developmental defects and other diseases. She hypothesized that BPA prevents methylation of certain genes, leading to a loss of genomic imprinting (which silences particular maternal genes), and found evidence of DNA hypomethylation, an epigenetic event that prevents normal gene silencing. Future studies will determine whether BPA directly causes maternal imprinting and which genes are responsible for generating developmental defects.