Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Academic research paper on "Biological sciences"

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Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development

John V. Ilekis, PhD; Ekaterini Tsiiou, MD; Susan Fisher, PhD; Vikki M. Abrahams, PhD; Michael J. Soares, PhD; James C. Cross, PhD; Stacy Zamudio, PhD; Nicholas P. Illsley, DPhii; Leslie Myatt, PhD; Christine Colvis, PhD; Maged M. Costantine, MD; David M. Haas, MD; Yoel Sadovsky, MD; Carl Weiner, MD; Erik Rytting, PhD; Gene Bidwell, PhD

Most adverse pregnancy outcomes can trace their origin to the placenta. Preeclampsia and fetal growth restriction (FGR) are disorders that are rooted in defects of early placental development.1,2 These defects include poor trophoblast uterine invasion, impaired transformation of the uterine spiral arteries to high capacity and low impedance vessels, and/or abnormalities in the development of chorionic villi. A number of poor pregnancy outcomes are associated with placental inflammation because of infectious or noninfectious causes and include early pregnancy loss, stillbirth, and FGR.3 Significant progress is being made in understanding the molecular basis of these disorders to begin contemplating targeting the molecular pathways that are involved in their pathophysiologic condition. Several potential targets could be envisioned readily. In the case of pre-eclampsia, an altered balance of circulating angiogenic/antiangiogenic factors that are derived from the placenta are believed to responsible for the systemic vascular dysfunction that is observed in preeclampsia.4 These factors include an increase in the antiangiogenic proteins such as soluble fms-like tyro-sine kinase 1 (sFlt-1) and soluble endo-glin, whose pathways can serve as targets for inhibition, or a decrease in the proangiogenic proteins such as placental growth factor (PlGF), whose pathway can serve as a target for stimulation. In the case of FGR, the stimulation of the PlGF pathway could also be targeted as a

Although much progress is being made in understanding the molecular pathways in the placenta that are involved in the pathophysiology of pregnancy-related disorders, a significant gap exists in the utilization of this information for the development of new drug therapies to improve pregnancy outcome. On March 5-6, 2015, the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health sponsored a 2-day workshop titled Placental Origins of Adverse Pregnancy Outcomes: Potential Molecular Targets to begin to address this gap. Particular emphasis was given tothe identification of important molecular pathways that could serve as drug targets and the advantages and disadvantages of targeting these particular pathways. This article is a summary of the proceedings of that workshop. A broad number of topics were covered that ranged from basic placental biology to clinical trials. This included research in the basic biology of placentation, such as trophoblast migration and spiral artery remodeling, and trophoblast sensing and response to infectious and noninfectious agents. Research findings in these areas will be critical for the formulation of the development of future treatments and the development of therapies for the prevention of a number of pregnancy disorders of placental origin that include preeclampsia, fetal growth restriction, and uterine inflammation. Research was also presented that summarized ongoing clinical efforts in the United States and in Europe that has tested novel interventions for preeclampsia and fetal growth restriction, including agents such as oral arginine supplementation, sildenafil, pravastatin, gene therapy with virally delivered vascular endothelial growth factor, and oxygen supplementation therapy. Strategies were also proposed to improve fetal growth by the enhancement of nutrient transport to the fetus by modulation of their placental transporters and the targeting of placental mitochondrial dysfunction and oxidative stress to improve placental health. The roles of microRNAs and placental-derived exosomes, as well as messenger RNAs, were also discussed in the context of their use for diagnostics and as drug targets. The workshop discussed the aspect of safety and pharmacokinetic profiles of potential existing and new therapeutics that will need to be determined, especially in the context of the unique pharmacokinetic properties of pregnancy and the hurdles and pitfalls of the translation of research findings into practice. The workshop also discussed novel methods of drug delivery and targeting during pregnancy with the use of macromolecular carriers, such as nanoparticles and biopolymers, to minimize placental drug transfer and hence fetal drug exposure. In closing, a major theme that developed from the workshop was that the scientific community must change their thinking of the pregnant woman and her fetus as a vulnerable patient population for which drug development should be avoided, but rather be thought of as a deprived population in need of more effective therapeutic interventions.

Key words: drugs, placenta, pregnancy, therapeutics, trial

means to increase the number of terminal villi and thus increase the available surface area for the improvement of nutrient transfer between the maternal blood and the growing fetus.5 Another potential treatment to increase nutrient transfer to the malnourished fetus is the stimulation of the mammalian target of rapamycin (mTOR) pathway as a means to increase nutrient transporters.6 In the case of placental inflammation, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway, which is a major pathway that is involved in mediating the inflammatory response, could be targeted to decrease placental inflammation.7'8 A number of drugs to target these pathways and many others already exist in the market place or are available at the experimental stage. A listing of these drugs can be obtained easily through a number of accessible databases.9-13 In addition, a promising pipeline of novel therapeutics are on the horizon that include natural or synthetic antibodies, synthetic small binding molecules (eg, peptides and nucleic acid aptamers), and nucleic acid therapies

(eg, DNA gene therapy and small RNAs (sRNAs), such as microRNAs, (miR-NAs) and silencing RNAs (siRNAs).14-17 A major obstacle in introducing novel pharmaceutical interventions to improve pregnancy outcomes is based on the general fear of inflicting potential harm, particularly to the fetus, that may result in either short- or long-term deleterious effects. Understandably, a very cautious direction is taken, and most studies involve either the evaluation of off-label drugs with a very safe history or dietary supplementation for use in pregnancy. Although extreme caution is warranted, the current challenge is to overcome the overbearing reticence of doing harm that unduly hinders the development and testing of new and novel approaches to improve pregnancy outcomes. The first step is to test potential drug therapies for their safety and efficacy in animal models, which then, in turn, can lead to human studies. A number of important factors need to be considered to improve the chance of a successful therapeutic agent. These include that the ideal therapeutic

From the Pregnancy and Perinatology Branch (Dr Ilekis) and the Obstetric and Pediatric Pharmacology and Therapeutics Branch (Dr Tsilou), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Department of Health and Human Services, Bethesda, MD; Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA (Dr Fisher); Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine; New Haven, CT (Dr Abrahams); Comparative Biology and Experimental Medicine, University of Calgary Health Sciences Centre, Calgary, Alberta, Canada (Dr Cross); Institute of Reproductive Health and Regenerative Medicine and Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS (Dr Soares); Department of Obstetrics and Gynecology, Hackensack University Medical Center, Hackensack, NJ (Dr Zamudio); Department of Obstetrics and Gynecology, Hackensack University Medical Center, Hackensack, NJ (Dr Illsley); Center for Pregnancy and Newborn Research, University of Texas Health Science Center, San Antonio, TX (Dr Myatt); Therapeutics Discovery Program, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD (Dr Colvis); Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX (Dr Costantine); Department of Obstetrics and Gynecology Indiana University, Indianapolis, IN (Dr Haas); Magee-Womens Research Institute, Pittsburgh, PA (Dr Sadovsky); University of Kansas Medical Center, Kansas City, KS (Dr Weiner); Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX (Dr Rytting); Department of Neurology, University of Mississippi Medical Center, Jackson, MS (Dr Bidwell).

Received Aug. 3, 2015; revised Feb. 11, 2016; accepted March 1, 2016.

Comments and views of the author(s) do not necessarily represent the views of the NICHD.

G.B. is the owner of Leflore Technologies, LLC, a private company working to develop biopolymer-delivered therapeutics. All other authors report no conflict of interest.

The 2-day workshop was held in North Bethesda, MD, March 5-6, 2015.

Corresponding authors: John V. Ilekis, PhD and Ekaterini Tsilou, MD. ilekisj@mail.nih.gov, tsiloue@

mail.nih.gov

0002-9378 • Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). • http://dx.doi.org/10.10167j.ajog.2016.03.001

agent should be highly specific to a key step in the targeted pathway and that it acts as far down stream as possible to produce the desired effect, thus minimizing unfavorable upstream-mediated cascading events. Furthermore, the ideal therapeutic should avoid or minimize maternal and fetal systemic effects. Thus, selectively targeting the placenta and optimizing the dosage would be important considerations. In this regard, placental homing molecules coupled to a delivery system that contains the therapeutic agent (such as nanoparticles, synthetic peptides, liposomes, exo-somes) and cell-specific DNA expression vectors show exciting promise to eliminate or minimize any deleterious collateral effects for either the mother or fetus.18 The timing of the delivery of the therapeutic agent is also another important consideration because the placenta is a developing organ with certain pathways that take critical roles at different developmental stages. Thus, the modulation of a particular molecular pathway at an inappropriate time window may result in deleterious effects by interfering with the normal developmental trajectory. For example, villous maturation undergoes an orderly developmental process that is orchestrated by the angiogenic factors vascular endo-thelial growth factor (VEGF) and PlGF.1,5,19 VEGF is involved in early villous formation and drives primary and secondary branching angiogenesis. This is followed by nonbranching angiogenesis and the formation of the tertiary terminal villi, principally under the control of PlGF. Primary and secondary branching angiogenesis generally is complete by approximately 20 weeks of gestation, after which tertiary terminal villi formation predominates and continues to term.1 Thus, in a hypothetical situation for the treatment of FGR, stimulating the PlGF pathway too early (ie, before the adequate completion of primary and secondary branching angiogenesis) conceivably could result in malformation of normal villous structure and function. Another factor to consider is the required exposure time to the therapeutic agent to obtain the desired effect. Will the therapeutic agent

be required to be administered continuously or only for a short duration of time? In the aforementioned FGR treatment scenario, how long of a time period is required to increase and maintain the number of terminal villi? Once formed, will the morphologic change remain permanent, or will the induced morphologic change regress if the stimulus is not continued? Therefore, specificity, dosage, delivery, timing, and length of exposure are some of the key factors in the development of a successful therapeutic agent.

Although much progress is being made in understanding the molecular pathways in the placenta that are involved in the pathophysiologic condition of pregnancy-related disorders, a significant gap exists in using this information for developing new drug therapies to improve pregnancy outcome. To address this concern, the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health sponsored a 2-day workshop on March 5-6, 2015, titled "Placental Origins of Adverse Pregnancy Outcomes: Potential Molecular Targets," to discuss and reflect on placental drug targeting to improve pregnancy outcomes. The workshop brought together leaders in the field to present and discuss their particular area of research and stimulate dialogue in the context of the theme of the workshop. The goals of the workshop were (1) to present the state of the science with respect to the molecular mechanisms that are involved in placentation, (2) to identify potential molecular pathways and developmental time windows for targeting effective "drug" interventions to avoid placenta-tion defects in early pregnancy and circumvent placental defects later in pregnancy, and (3) to identify major research gaps in our understanding of placental molecular pathways that lead to adverse pregnancy outcomes. This article summarizes the proceedings of that workshop. The overall objective of the workshop was to stimulate the research community to better apply the knowledge that is obtained from the laboratory bench for use at the bedside.

TABLE 1

Workshop session themes and their respective topics

I. Review of placental development and function in the context of the molecular mechanisms and pathways

Human trophoblast differentiation and placentation Innate immune function of human trophoblast Modeling trophoblast differentiation and placentation in the rat Modeling trophoblast differentiation and placentation in the mouse

II. Potential "drug" targets of important placental molecular pathways throughout placental development in relation to pregnancy disorders

Placental hypoxia as a therapeutic molecular target

Is fetal growth a feasible target for placental intervention?

Targeting oxidative/nitrative stress and mitochondrial dysfunction in placenta to relieve adverse pregnancy outcomes

III. Identify potentially useful or experimental drugs to target important molecular pathways Challenges and advantages of rescuing and repurposing

Effect of pregnancy on drug pharmacokinetics and pharmacodynamics

From bench to bedside: processes and pitfalls translating research findings into practice paradigms

IV. Evolving technologies for placental specific "therapeutic/drug" delivery

Trophoblastic nanovesicles, microRNA, and their targets aberrant regulation of myometrial contractility by maternal cell-free plasma RNAof placental origin: screening and therapeutic implications

Maternally sequestered delivery systems for prevention of fetal drug exposure Novel therapeutic interventions and delivery systems

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

A summary of the research topic that was covered by each participant is presented along with their opinions on current and future opportunities and research gaps. The article is organized according to 4 session themes. The session themes and their respective topics are listed in Table 1. Table 2 is a key to abbreviated scientific terms that are used commonly throughout the article.

Review of placental development and function in the context of molecular mechanisms and pathways

Human trophoblast differentiation and placentation (Susan Fisher, University of California San Francisco)

Cytotrophoblast differentiation establishes the anatomy of the human maternal-fetal interface. The complex cellular architecture at the boundary between the placenta and uterus is governed, in large part, by the cytotrophoblast

differentiation pathway that enables invasion.20'21 With regard to the anatomic arrangement, placental cyto-trophoblasts emigrate from anchoring villi and join cell columns that serve as conduits to the uterine wall (Figure 1). Within the uterus, the cytotrophoblasts invade nearly its entirety, normally stopping one-third of the way through the muscle layer. Within the decidua, interstitial cytotrophoblasts interact with specialized populations of maternal immune cells that are allowed to enter this compartment. During invasion, the cells also remodel the uterine circulation, primarily by targeting the spiral arteries. They transform the walls of these vessels. Endovascular cytotropho-blasts replace the endothelium and intercalate within the smooth muscle cells of the tunica media. This process converts the originally low-capacitance/ high-resistance uterine arteries into

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TABLE 2

Abbreviations for commonly used scientific terms [in file but NOT EDITED]

Abbreviation Description

AB apoptotic bleb

aPL antiphospholipid antibodies

APOA4 apolipoprotein a-iv

C19MC chromosome 19 miRNA cluster

CaO2 arterial oxygen content

CARD caspase activation and recruitment domain

Ca++ calcium

CFP cell-free plasma

CO cardiac output

CTB cytotrophoblast

CYP cytochrome P

DAMP damage associated molecular pattern

DNA deoxyribonucleic acid

dsRNA double-stranded ribonucleic acid

EDH endothelium derived hyperpolarizing

EGFR epidermal growth factor receptor

ELP elastin-like polypeptide

EPO erythropoietin

FDA Food and Drug Administration

FGR fetal growth restriction; also known as IUGR

FOA funding opportunity announcement

GDM gestatoinal diabetes mellitus

GLUT glucose transporter

GPx glutathione peroxidase

GRO-a melanoma growth stimulating activity, alpha

HIF hypoxia inducible factor

HIV human immunodeficiency virus

HLA human leukocyte antigen

HMGB1 high mobility group B1

iE-DAP gamma-D-glutamyl-meso-diaminopimelic acid

IFNGR interferon-gamma receptor

IGF insulin-like growth factor

IL interleukin

IL interferon

IRAK interleukin-1 receptor associated kinase

IRF interferon regulatory factor

IUGR intrauterine growth restriction (also known as FGR)

LPS lipopolysaccharide

m meters

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

(continued)

high-capacitance/low-resistance channels that perfuse the surface of the placenta, which is comprised of multi-nucleated syncytiotrophoblast, which is a transport epithelium. Thus, they can respond to the ever-increasing demands of the offspring for maternal blood over the course of pregnancy.

At a molecular level, cytotrophoblast invasion of the uterus is as remarkable as the unique behaviors that the cells exhibit. The progenitors, which are attached to the trophoblast basement membrane of the chorionic villi, express an adhesion molecule repertoire that is typical of epithelial cells (eg, epithelial-cadherin and integrin a6/b4). As they enter the columns, the emigrating cyto-trophoblasts undergo a stereotypical transformation. They down-regulate those that are typical of an epithelial monolayer and up-regulate receptors that enable invasion (eg, aV family members; Figure 2), vascular endothelial [F2] cadherin and integrin a 1/^1. Remarkably, the end result of this transformation is vascular mimicry in which cytotrophoblasts of epithelial origin express a broad repertoire of adhesion molecules, growth factors, ephrin receptors and their cognate ligands (eph-rins), and notch family members that typically are associated with endothe-lium and the muscular tunica media of vessels.

Preeclampsia is associated with abnormal cytotrophoblast invasion and differentiation. Many investigators believe that preeclampsia (the sudden onset of maternal high blood pressure, protein-uria, and edema) occurs in 2 stages.22 The first stage involves shallow cytotrophoblast invasion of the uterus, which was first described by Brosens et al.23,24 Failed transformation of spiral arteries appears to be critical and leads to hypoperfusion of the placenta and oxidative stress.25 The second stage includes overactive maternal immune responses. Although these pathways are associated most commonly with pre-eclampsia, similar diseases have been described in a subset of preterm labor/ birth cases.21 The causes are under intense investigation. Severe cases of

preeclampsia are associated with failed cytotrophoblast transformation into vascular-like cells coincident with shallow uterine invasion.26 For example, placental cells that enter the uterine wall fail to down-regulate epithelial cadherin and to up-regulate vascular epithelial cadherin. They also misexpress a broad array of angiogenic and/or vasculogenic factors. These include VEGF family members. For example, invasive cyto-trophoblasts from preeclampsia pregnancies fail to stain with anti-VEGF A, which their normal counterparts express in abundance. In addition, they release higher amounts of soluble VEGFR1 (sFlt-1),27 as do syncytiotrophoblasts.28 Increasing circulating levels of sFlt-1 and other angiogenic factors (such as endoglin) cause a preeclampsia-like syndrome in animal models.29,30 Thus, there has been a great deal of interest in whether or not circulating levels of molecules that could have negative effects on the maternal vasculature can be used to predict and/or diagnose pre-

eclampsia.

Is abnormal placental production of angiogenic/vasculogenic factors a cause or consequence of preeclampsia? As yet, there are no definitive answers to this question. However, alternative explanations abound. For example, particular combinations of maternal natural killer (NK) cell expression of killer cell immunoglobulin-like receptors that recognize the certain major histocom-patibility complex molecule, human leucocyte antigen C, on invading cyto-trophoblasts increase the risk of pre-eclampsia.34 Surprisingly, a recent study showed that, on isolation from pre-eclampsia placentas, cytotrophoblast gene expression (eg, growth hormone [GH] 2, corticotrophin-releasing hormone, kiss-1 metastasis-suppressor 1, semaphoring 3B, and several pregnancy-specific beta-1-glycoproteins) is nor-malized,35 which suggests that the defects are reversible and that pursuit of therapies is warranted.

Current opportunities. As compared with other medical conditions, very little attention has been paid to therapeutic/ pharmacologic interventions for the

TABLE 2 Abbreviations for commonly used scientific terms [in file but NOT EDITED] (continued)

Abbreviation Description

mmHg millimeters mercury

MAL myelin and lymphocyte

MCP-1 monocyte chemoattractant protein-1

MDP muramyl dipeptide

miRNA micro-ribonucleic acid

mRNA messenger RNA

MR mass restricted

mTOR mammalian target of rapamycin

MV microvesicle

MVB multivesicular body

NADPH myeloid differentiation primary response gene 88

NCATS National Center for Advancing Translational Sciences

NF-kB nuclear factor kappa beta

NGS next generation sequencing

NIH National Institutes of Health

NK natural killer

NLR nod-like receptor

NO nitric oxide

Nod nucleotide oligomerization domain protein

NRP1 neuropilin-1

NTU new therapeutic uses

O2 oxygen

PAMP pathogen-associated molecular pattern

PaO2 partial pressure of arterial oxygen

PD pharmacodynamic

PDG peptidoglycan

PE preeclampsia

PK pharmacokinetic

PI3K/Akt phosphatidylinositol-3-kinase/protein kinase B

PIGF placental growth factor

PO2 partial pressure of oxygen

PPROM preterm premature rupture of membranes

Prl prolactin

PRR pattern recognition receptors

PTL/B preterm labor/birth

PTB preterm birth

K+ potassium

RICK receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. (continued)

TABLE 2

Abbreviations for commonly used scientific terms [in file but NOT EDITED]

(continued)

Abbreviation Description

RNA ribonucleic acid

ROS reactive oxygen species

sFlt-1 soluble fms-like tyrosine kinase 1 (also known as soluble VEGFR1)

SOD superoxide dismutase

sPTB spontaneous preterm birth

STB syncytiotrophoblast

sRNA small ribonucleic acid

siRNA silencing ribonucleic acid

ssRNA single-stranded ribonucleic acid

T1 translational spectrum 1, translation of animal and bastic research into humans

T2 translational spectrum 2, translation of clinical research findings to practice

TBK-1 tank-binding kinase 1

TLR toll-like receptor

TNFa tumor necrosis factor; alpha

TRAF TNF receptor associated factor

TRAM trif-related adaptor molecule

TRIF tir-domain-containing adapter-inducing interferon-b

TS trophoblast stem

uNK uterine natural killer

VEGF vascular endothelial growth factor

VEGFR VEGF receptor

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

great obstetric syndromes. In this context, pregnancy complications are the equivalent of "orphan" diseases, not because they are rare conditions but because there is very little monetary incentive for taking on the risk that treating pregnant women entails. However, there are compelling reasons to shift this paradigm. Most ofthe common diseases that derail human pregnancy affect the placenta. Many involve either fetal or maternal cells that reside within the uterine wall. Thus, it is likely that effective therapies could be designed to target these cells without crossing the placenta and reaching the embryo/fetus. For example, many kinds of drugs (eg, antibodies, small molecules) that target

particular vulnerabilities (eg, vascular and/or immune functions) could be formulated as derivatives that prevent syncytiotrophoblast transport, thus reducing the risk of untoward embryonic/fetal events. As a first step, this general strategy could be tried with agents that are already used to treat pregnant women (eg, tumor necrosis factor—alpha inhibitors that work, in part, by blocking the activation of endothelial and immune cells that this cytokine produces. For example, certo-lizumab (a pegylated fragment antigen-binding fragment of a humanized monoclonal antibody that inhibits tumor necrosis factor—alpha), which does not cross the placenta, could be

evaluated in women who have a high risk of pregnancy loss because of the effects of this cytokine (eg, inflammatory and thrombotic placental lesions) in the setting of autoimmune disorders such as antiphospholipid antibody syndrome. 36

Future opportunities. Until recently, it was thought that placental interactions with the mother occurred at a cellular level (eg, invasive cytotrophoblasts and maternal immune cells within the uterine wall) or involved soluble proteins (eg, human chorionic gonadotropin). However, this paradigm is shifting rapidly. Free fetal DNA, which circulates in maternal blood,37 is being used as a noninvasive means of prenatal genetic diagnoses.38 It is possible that circulating cell-free RNA could be used as a complementary method and/or as a means of gaining additional information.39 Also, like many cancer cells, the placenta appears to release a complex repertoire of extracellular vesicles the cargo of which could have major effects on numerous maternal cells, tissues, and organs.40'41 Thus, obtaining an in-depth understanding of the types and content of placental extracellular vesicles will increase our understanding of their functions. For example, it would be interesting to determine how their contents and targets change over the course of gestation and the impact of the common pregnancy complications, which include preeclampsia and preterm labor/ birth, on the normal trajectory. Ultimately, this important information could lead to several types of clinical applications (eg, extracellular vesicles could be used to infer important aspects of placental functions). Other possibilities include therapies that target extracellular vesicles or take advantage of this system of intercellular communication for drug delivery.

Scientific gaps in relation to drug targeting. A myriad of questions remain to be answered about mechanisms that are central to the success of normal pregnancy and go awry in pregnancy complications. For example, maternal tolerance of hemiallogeneic

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cytotrophoblasts lacks a definitive explanation. Therefore, it is very difficult to devise targeted therapies for pregnancy disorders, from infertility to preeclampsia, that are thought to have an immune cause or component. Likewise, lack of knowledge impedes strategies for dampening the maternal immune response to infections during pregnancy, which can lead to preterm labor/birth. In cases of the latter syndrome with an unknown cause, therapies lag because we do not understand the pathways that normally trigger normal labor and birth at the end of pregnancy. Finally, preeclampsia appears to arise because of profound miscommunication between the placenta and the mother. The development of drugs that intercept or redirect these signals will require a molecular dissection of their components.

Innate immune function of human trophoblast (Vikki M. Abrahams, Yale University)

Background. Placental trophoblast cells can sense and respond to a variety of infectious pathogen-associated molecular patterns that are expressed by microbes, as well as noninfectious host-derived damage-associated molecular patterns (DAMPs) through their expression of innate immune pattern recognition receptors (PRRs).42,43 Depending on the trigger or receptor that is activated, the trophoblast may mount either a regulated protective response that helps to maintain and promote a healthy pregnancy or a damaging response that might impact pregnancy outcome adversely (Figure 3). Moreover, expressions of some tropho-blast PRRs are regulated gestationally, which further impacts the placental

response.44

PRRs. Two major families of PRRs are the Toll-like receptors (TLRs) and the Nodlike receptors (NLRs). TLRs are transmembrane receptors, which allows for the sensing of PAMPs or DAMPs either at the cell surface or within endosomal compartments.45 There are 10 human and 12 murine TLRs, each with distinct specificities.46 TLR4 recognizes Gram-

FIGURE 1

A schematic drawing of the maternal-fetal interface in human pregnancy

Mononuclear placental cytotrophoblasts invade the uterine wall and its resident vasculature (right panel). During this process, they transform spiral arteries into wide-bore vessels that perfuse the placenta. Its tree-like chorionic villi are covered by multinucleated syncytiotrophoblasts, which transport a variety of substances to and from the fetus, enabling normal fetal growth.

Reprinted with permission from Romero et al.21

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

negative bacterial lipopolysaccharide. TLR2, in cooperation with its coreceptors TLR1, TLR6, or TLR10, recognizes Gram-positive bacterial peptidoglycan and lipoproteins. TLR3 senses viral double-stranded RNA; TLR5 senses bacterial flagellin. Mouse TLR7 and human TLR8 sense viral single-stranded RNA, and TLR9 senses bacterial DNA.46 Four adapter proteins are involved in TLR signaling: myelin and lymphocyte protein 88 (MyD88), TIR-domain-containing adapter-inducing interferonbeta (TRIF), myelin and lymphocyte protein (Mal), and TRIF-related adaptor molecule.47,48 TLR2 and TLR4 signal through MyD88/Mal. TLR4 can also signal through TRIF/ TRIF-related adaptor molecule. TLR3 signals through TRIF; all other TLRs signal through MyD88 alone.47,48 Downstream, TLR/ MyD88 signaling activates NFkB; TLR/ TRIF activates Tank-binding kinase-1 and interferon regulatory factor 3 that leads to a type I interferon response

(Figure 4).48,49

NLRs are cytoplasmic-based PRRs that provide an intracellular recognition system for sensing microbe-associated pathogen-associated molecular pattern (PAMP) or as will be discussed, DAMPs.

NLRs can synergize with TLRs for a greater response or provide a compensatory system for when TLR signaling is absent or reduced.50,51 The NLR proteins, Nod1 and Nod2, recognize the peptidoglycan peptides Gramnegative bacterial gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP-) and muramyl dipeptide (MDP) that is expressed by all bacteria, respectively. Both Nod1 and Nod2 signal through the common adapter protein receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK) to induce inflamma-tion.52 Another NLR called NACHT, leucine-rich repeat protein and the NLR, Nalp3, are involved in mediating the production of the proinflammatory cytokine interleukin (IL)-1^.53,54 Because IL-1b has the potential to be damaging, its regulation is tightly controlled. Indeed, IL-1b is an important mediator of preterm birth and

perinatal brain injury106-109 and canQ4 trigger the production of other proin- [F4] flammatory cytokines and chemokines through the IL-1 receptor in a similar manner to TLRs.55 Indeed, delivery of a synthetic peptide to pregnant mice that is a selective IL-1 receptor

FIGURE 2

Phenotypic transformation of cytotrophoblast during uterine invasion

È i AV V •

B ß5

fc > /jj V/ 7 - Skfil

Cytotrophoblasts switch their expression of integrin aVj3 family members as they invade the uterine wall. Sections of the maternal-fetal interface at various weeks of gestation (18-22) were double stained with anti-cytokeratin to mark A, C, E, G, cytotrophoblasts and B, D, F, H, anti-aV^5, anti-aV06, or anti-aV^3. aV@5 was detected on cytotrophoblasts in floating (data not shown) and anchoring villi, but not in other locations. aVffi was detected on villous cytotrophoblasts at sites of column formation and in the first cell layer of the column. aV@3 was up-regulated in the distal portions of the columns and on endovascular cytotrophoblasts that lined the maternal blood vessels.

AV, anchoring villi; ¡3, anti-aVb3; ¡5, anti-aVb5; ¡6, anti-aVb6; BV, blood vessels; CK, anti-cytokeratin; EC, endothelial cell. Reprinted with permission from Zhou et al.134

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

modulator delays IL-1b delays and lipopolysaccharide-induced preterm birth.56 Unlike most other cytokines, IL-1b production involves a 2-step process.

The first step requires induction of pro—IL-1 b expression. This is triggered through signals like TLRs (signal 1). Once expressed, pro—IL-1b can be

cleaved into its active secreted form.57 This second step (signal 2) is mediated typically by the Nalp3 inflammasome a protein complex consisting of Nalp3, apoptosis-associated speck-like protein that contains a caspase recruitment domain (CARD) and caspase-1.53 Once the inflammasome has assembled, caspase-1 is activated and processes pro—IL-1b into its secreted form (Figure 5).57 Although the Nalp3 inflam- [F5] masome is the best characterized, there are a number of inflammasomes: Nalp1/ apoptosis-associated speck-like protein containing a CARD; absent in melanoma 2 protein A; NLR family, CARD domain containing 4 protein; and interferon gamma-inducible protein 16.112 Further-Q5 more, Nod proteins can mediate IL-1b production independent of the inflam-masome. For example, Chlamydia trachomatis infection of human trophoblast induces IL-1 b via Nod 1.113 Q6

Trophoblast sensing of bacteria. Bacterial components such lipopolysaccharide, iE-DAP, and MDP, at high concentrations, trigger mild proinflammatory cytokines/chemokines (IL-8, IL-6, monocyte chemoattractant protein-1 [MCP-1], and melanoma growth stimulating activity, alpha [GRO-a]) responses in first trimester trophoblast cells through TLR4, Nod1, and Nod2, respectively, although lower, more physiological doses, are unable to induce this trophoblast inflammation.58-61 Third trimester trophoblast cells lack Nod2 and thus responses to MDP are altered.62 Similarly, third trimester syn-cytiotrophoblast cells can generate a strong inflammatory response to lower lipopolysaccharide doses. Thus, there are differential sensitivities of tropho-blast cells to bacterial components across gestation.63 This dose dependency and role for TLRs and NLRs is reflected in vivo. In pregnant mice, high-dose lipopolysaccharide induces placental and uterine inflammation and subsequent preterm birth.64 TLR4 deficient mice are protected against bacterial and lipopolysaccharide-induced preterm birth,65,66 and blocking TLR4 in nonhuman primates prevents lipopolysaccharide-induced

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Q7 preterm uterine contractility. Highdose iE-DAP also induces inflammation at the maternal-fetal interface and pre-term birth.62 However, low-dose lipo-polysaccharide can trigger preterm birth in mice on a pathologic background, such as an active viral infection67,68 or IL-10 deficiency.69,70 Thus, although TLRs and NLRs appear to be involved in preterm birth in response to bacterial components, in normal early pregnancy, there appears to be some tolerance towards certain triggers, such as lipo-polysaccharide, that may always be present at the maternal-fetal interface.71,72 Furthermore, although bacteria can induce preterm birth,73,74 additional signals, such as a virus, may further sensitize the placenta to these bacterial

signals. 67,68

Gram-positive bacterial peptido-glycan, which activates TLR2 in association with TLR1, TLR6, or TLR10, triggers a very different response in human first-trimester trophoblast cells compared with term trophoblast. At term, peptidoglycan activates these cells to produce IL-8.75 However, in firsttrimester trophoblast cells, instead of activating the classic inflammatory cascade, peptidoglycan through TLR1, TLR2, and TLR10 induces apoptosis and down-regulates the cell's basal cytokine/ chemokine expression. Moreover, this apoptotic response is prevented by the ectopic expression of TLR6, which is absent in the first-trimester tropho-blast.58,76-78 Similarly, delivery of pepti-doglycan early in gestation induces placental apoptosis without evidence of inflammation,76 although delivery later in gestation triggers preterm birth.79

Viral sensing by the trophoblast. The viral sensors, TLR3 and TLR8, that recognize viral double-stranded RNA and single-stranded RNA, respectively, mediate a rapid, robust chemokine/cytokine (IL-6, IL-8), type I interferon, and antiviral response in human first-trimester trophoblast.80-82 Furthermore, independent of TLR8, viral single-stranded RNA also induces trophoblast expression of IL-1b, antiviral factors, and the induction of apoptosis.82 This placental

FIGURE 3

Innate immune sensing by the trophoblast

Trophoblast cells sense infectious pathogen-associated molecular patterns that are expressed by bacteria, viruses, fungi, and parasites through their expression of Toll-like receptors and Nod-like receptors. Through these receptors, trophoblast cells also mount responses to noninfectious host-derived damage-associated molecular patterns, such as uric acid, high mobility group B1, glucose, and certain autoantibodies. Trophoblast expression of some Toll-like receptors and Nod-like receptors are regulated across gestation and cell subtype. Depending on the trigger, receptor activated, and type of signaling pathway used, the trophoblast may mount either a regulated protective response that helps to maintain and promote a healthy pregnancy or a damaging pathologic response that might impact pregnancy outcome adversely.

PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns; HMGB1, high mobility group B1; NLRs, Nod-like receptors; TLRs, Toll-like receptors.

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

is mirrored in vivo without triggering preterm birth.82 In contrast, viral double-stranded RNA induces preterm birth through TLR379,83; other groups have reported preeclampsia-like symptoms in viral double-stranded RNA-treated mice.84

Trophoblast activation by DAMPs. In addition to understanding how the trophoblast responds to PAMPs, the impact of DAMPs on trophoblast function has been investigated. DAMPs are host-derived factors that are either not usually released from cells or tissues or, if present in the extracellular space, are normally at low levels. The DAMP, high-mobility group B1 (HMGB1) can be released, passively from damaged cells or actively in response to inflammatory triggers and can mediate inflammatory responses via TLR2, TLR4, or receptor for advanced glycation end products 85 (Figure 4). HMGB1 levels are increased in the amniotic fluid from patients with preterm birth or preterm premature rupture of membranes and intrauterine

single-stranded RNA—induced response infection, in fetal membranes from

patients with preterm labor and preterm premature rupture of membranes,87 and in serum from pregnancies that are complicated by either preeclampsia or reduced fetal movements.88,98 TermQ8 trophoblast cells that are treated with HMGB1 secrete significantly increased levels of IL-1b, IL-6, and monocyte chemoattractant protein-1; however, mechanistic studies are needed to show which receptors and signaling pathways are activated by this DAMP.89 One type of DAMP that has been characterized extensively is the antiphospholipid antibody (aPL), which are autoanti-bodies that specifically target the trophoblast by binding surface beta2 glycoprotein 1.90 These autoantibodies activate human first-trimester tropho-blast TLR4 that results in inflammatory IL-8 and IL-1b secretion (Figure 4).91,92 Downstream of TLR4, aPLs induce endogenous uric acid, which is another DAMP that specifically activates the Nalp3 inflammasome,93 which meditates pro-IL-1b processing and secretion (Figure 5).92 In parallel, aPL via TLR4 induces the expression of the

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Toll-like receptor signaling

Toll-like receptors are transmembrane receptors that mediate the sensing of pathogen-associated molecular patterns expressed by microorganisms. Toll-like receptor 2, in cooperation with its cor-eceptors Toll-like receptors 1, 6, or 10, recognizes Gram-positive bacterial peptidoglycan. Toll-like receptor 4 recognizes Gram-negative bacterial lipopolysaccharide. Toll-like receptors 3 and 7/8 sense viral double-stranded RNA and single-stranded RNA, respectively. Toll-like receptor 5 senses bacterial flagellin, and Toll-like receptor 9 senses bacterial cytosine-guanine dinucleotide—rich DNA regions. Four adapter proteins are involved in Toll-like receptor signaling: MyD88, TRIF, Mal, and TRAM that trigger downstream pathways that lead to either nuclear factor kappa-light-chain-enhancer of activated B cells activation and subsequent cytokine/chemokine production or IRF-3/ IRF-7 activation that leads to a type I interferon response. Some Toll-like receptors also sense host-derived damage-associated molecular patterns. Toll-like receptors 2 and 4 can sense high mobility group B1 protein; Toll-like receptor 4 can be activated by antiphospholipid antibodies. Refer to Table 2 for the key to undefined abbreviations.

aPLs, antiphospholipid antibodies; CpG, cytosine-guanine dinucleotide; DAMPs, damage-associated molecular patterns; dsRNA, double-stranded RNA; HMGV1, high mobility group B1; IFNs, interferon; IRAK, interleukin-1 receptor-associated kinase; IRF, interferon regulatory factor; LPS, lipopolysaccharide; Mal, myelin and lymphocyte; MyD, ••••; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; PAMPs, pathogen-associated molecular patterns; PDG, peptidoglycan; ssRNA, single-stranded RNA; TBK, tank-binding kinase; TLR, Toll-like receptor; TRAF, tumor necrosis factor receptor-associated factor; TRIF, tir-domain—containing adapter-inducing interferon^.

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

miRNA, miR-146a-3p, which drives IL-8 secretion by activating TLR8.94'95 Thus, aPL, through TLR4, induces endogenous secondary messengers that subsequently activate other trophoblast innate immune PRRs. Hyperglycemic levels of glucose have similar effects on these cells, which suggests that overt pregestational diabetes mellitus may impact placental inflammation and function early in pregnancy. Excess glucose induces a proinflammatory cytokine/chemokine profile (IL-10, IL-6, IL-8, GRO-a, regulated on activation normal T cell expressed and secreted, chemokine, and granulocyte colony stimulating factor). Moreover, the IL-^ response is

associated with elevated uric acid and is dependent on the activation of the Nalp3 inflammasome.96 Elevated serum uric acid has been associated with high-risk pregnancies, such as those complicated by preeclampsia, gestational hypertension, cases of reduced fetal movements,

or obstetric aPL syndrome.92,97-99 Thus,

rather than simplycorrelating levels with disease, uric acid can act as a direct mediator of trophoblast inflammasome activation and placental inflammation,93 which suggests that it may play a pathologic role. Furthermore, that those host-derived noninfectious triggers (such as uric acid, glucose and aPL) can activate the inflammasome indicates that

this mechanism is not involved only in microbial-induced inflammation, but also in sterile-induced inflammation.

Current opportunities. Inhibiting PRR activation to prevent infection-associated preterm birth has been considered. A TLR4 antagonist prevented lipopolysaccharide-induced pre-term uterine contractility in nonhuman primates100 and knockout mice for TLR, or associated adapter proteins are resistant to microbial and PAMP-induced preterm birth.83'101 Similarly, because IL-1$ is a mediator of preterm birth102-105 and fetal brain injury,106-109 studies have focused on using IL-1 receptor antagonists or selective IL-1 receptor modulators to prevent these adverse outcomes.110,111 Preventing the upstream induction of IL- lS by inhibiting placental inflammasome activity may also serve as a potential target for the prevention of adverse pregnancy outcomes.

Future opportunities. Because uric acid mediates placental inflammasome func-tion,92,93,96 currently available drugs that inhibit xanthine oxidase, such as allo-purinol or febuxostat, may provide potential avenues to explore. However, this is only 1 mechanism by which production can arise; there are a number of other inflammasomes that uric acid may not activate112 and non— inflammasome-mediated pathways,113 all of which could be possible targets. Similarly, as we expand our knowledge about the role of miRNAs in the mediation and regulation of placental function and PRR activity, we can begin to consider these as potential therapeutic targets.

Scientific gaps. Together, the studies discussed herein demonstrate that the placenta is immunologically functional, with the trophoblast able to generate specific and diverse innate immune-like responses through their expression of a range of PRRs. However, the type of response is highly dependent on the stimuli, the receptors expressed and activated, the downstream signaling pathways involved, and the timing of gestation. Indeed, although many of the

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endpoints and impact on pregnancy outcome triggered by PAMPs and DAMPs may be common, the upstream mechanisms are often quite distinct. These challenges for drug discovery and applications highlight the need for a greater understanding of the precise molecular pathways that are involved in placental sensing of infectious and noninfectious triggers.

Modelling trophoblast differentiation and placentation in the rat (Michael J. Soares, University of Kansas)

Background. Fundamental to the establishment of pregnancy and formation of the hemochorial placenta is remodeling and restructuring of uterine spiral arteries that allow for the flow of nutrients to the placenta and ultimately to the fetus.114 Trophoblast cells play a central role in this vascular remodeling process. Pathologic conditions that are associated with trophoblast-directed uterine spiral artery remodeling underlie some of the most significant and challenging obstetric diseases.114-116 There are several research strategies for gaining insight into the process of hemochorial placen-tation and for the identification of potentially vulnerable molecular mechanisms that lead to disease. In this section, we address the merits of animal models, especially the rat, for placental research.

Placentation in the rat. The rat possesses hemochorial placentation with deep intrauterine trophoblast cell invasion and trophoblast-directed uterine spiral artery remodelling,117'118 which are features shared with human placenta-tion.114'119-121 Recognition of these similarities spurred the establishment of in vitro and in vivo research methods using the rat as an animal model to address mechanistic questions regarding the development of the hemochorial placenta and especially the role of invasive trophoblast cells in the remodeling of uterine spiral arteries. Rcho-1 trophoblast stem cells and blastocyst-derived trophoblast stem cells are 2 rat in vitro culture systems that have been used extensively to investigate signaling pathways and mechanisms that control

FIGURE 5

Nod-like receptor signaling

Nod-like receptors are cytoplasmic proteins that sense pathogen-associated molecular patterns. Nod1 recognizes bacterial iE-DAP, and Nod2 senses bacterial muramyl dipeptide. Both Nod1 and Nod2 signal through the adapter protein receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase to induce nuclear factor kappa-light-chain-enhancer of activated B cells activation and subsequent cytokine/chemokine production. Nalp3 recruits ASC and caspase-1 to form the inflammasome. Once the inflammasome has assembled, caspase-1 is activated and processes pro-IL-1 b into its active, secreted form. Refer to Table 2 for the key to undefined abbreviations.

ASC, DAMP, damage-associated molecular pattern; IL, interleukin; MDP, muramyl dipeptide; Nalp, ••••; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRs, Nod-like receptors; PAMPs, pathogen-associated molecular patterns; Pro-IL, ••••; RICK, receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase. Q38

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

122 123

trophoblast cell differentiation. ' Regulatory factors identified in vitro have been tested experimentally with the use of an assortment of in vivo research strategies, which include transgenesis to monitor the invasive trophoblast

lineage, spontaneous mutant rat models that possess placental insufficiency118'125-128 and gain-of-function and loss-of-function manipulations with the use of trophoblast-specific len-tiviral gene delivery and genome edit-ing.129-132

A fundamental property of placenta-tion is its plasticity, which is characterized by the acquisition of structural/ functional properties that permit adaptation to environmental challenges. Mechanisms underlying these adaptive processes are an important feature of placentation and represent potential therapeutic targets. Limiting oxygen delivery to the placentation site at devel-opmentally defined phases of gestation effectively activates the acquisition of the invasive trophoblast phenotype and promotes uterine spiral artery remodel-ing124'133 that are adaptive responses conserved in primates.134'135 These

observations illustrate the instructive nature of oxygen delivery as signal-driving decision-making within trophoblast stem/progenitor cell populations and ultimately affect hemo-chorial placentation; however, it is important to appreciate that failures in adaptive responses to hypoxia can lead to disruptions in placentation. Additionally, hypoxia can also be a pathologic response to a failed placenta resulting in disease. Dissociating these potentially confounding outcomes of hypoxia is essential. Dissection of hypoxia-guided pathways within trophoblast stem/progenitor cell populations offers an opportunity to identify molecular events in the placentation process that could be manipulated to improve and enhance placental development and prevent the spiraling consequences of a failed placenta.

Role ofNK cells. Immune cells, especially NK cells, are active contributors to remodeling of uterine spiral arteries during the establishment of pregnancy. The involvement of NK cells in the hemochorial placentation process has

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Natural killer cells and endovascular trophoblast cells contribute to uterine spiral artery remodeling

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A-D, Rats were treated on E4.5 and E9.5 with normal rabbit serum (control) or anti-asialo GM1 (natural killer cell depleted) and killed on E13.5. Double immunofluorescence staining for A, B, ANK61 (natural killer cell marker) and ACTA (smooth muscle marker) and C, D, cytokeratin and ACTA2. The asterisks demarcate blood vessels that possess interruptions (arrowheads) in the tunica media (A, C); the asterisks identify blood vessels with intact tunica media (B). Scale bars = 0.25 mm.

NK, natural killer.

Reprinted with permission from Chakraborty et al.133

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

been investigated effectively in the rat (Figure 6). NK cells directly regulate uterine spiral artery growth and progression towards the developing placenta. These critical events impact oxygen delivery to the placentation site and trophoblast differentiation.133 Failures in NK cells result in attenuated uterine spiral artery development, local hypoxia at the placentation site, and premature and exaggerated activation of endovascular invasive trophoblast cells that lead to extensive uterine spiral artery remodeling. Thus, NK cells control oxygen delivery to the developing placenta and regulate the timing and extent of endovascular invasive trophoblast cell differentiation and trophoblast-directed uterine spiral artery remodeling. Evidence also exists supporting a conserved role for uterine

and especially molecular mechanisms that regulate the placentation process; when the time and effort are taken to investigate these processes, it has been demonstrated that there is considerable merit for animal models in placental research

120,137-141

NK-cell actions within the human placentation site.136 Modulation of uterine NK-cell behavior represents another potential target for modulating the placentation process.

Current opportunities. Animal models are important tools to understand human disease. Animal models provide the opportunity to study biologic phenomena not easily studied in the human. Although no model is ideal, each provides useful insight relevant to the human condition. The rat is a particularly important experimental tool for investigating regulatory processes that control hemochorial placentation. Species differences in placental organization and gene expression patterns are evident, but there are also underlying commonalities in structure, function,

Future opportunities. Maximal benefits for animal models in placental research will be achieved when efforts are directed to regulatory events and mechanisms that are conserved in the human. The rat has been used effectively to investigate regulatory events that involve tropho-blast cells, NK cells, and uterine spiral arteries, a triad of key players in hemo-chorial placentation.

A paradigm for investigating molecular mechanisms that impact hemo-chorial placentation with the use of rodent trophoblast stem-cell models, followed by the validation of the obtained results in human placenta and human trophoblast cell models, and then proceeding to in vivo rodent experimentation represents a powerful research approach.131,142,143 Evaluation of conservation generally is limited to the use of primary or immortalized human trophoblast cell model systems and expression analysis in human placental tissues. A human trophoblast stem-cell culture system functionally equivalent to rodent trophoblast stem cells would be optimal for studying the regulation of differentiation, especially the identification of conserved processes. Some progress has been made in the development of this important experimental in vitro tool.144 The recent availability of genome editing strategies to generate mutations in rodents should lead to the establishment of new animal models for in vivo testing of conserved molecular mechanisms that control hemochorial placentation. These in vivo approaches should include targeting the activities of specific trophoblast and immune cell populations in order to identify molecular pathways that could serve as sites for therapeutic intervention.

Scientific gaps. The use of relevant and appropriate animal models, including rodents, to test hypotheses in vivo most

importantly extends placental research beyond description, classification, in vitro analyses, and molecular pheno-typing and permits a rational approach for understanding the physiology of placentation, the pathogenesis of placental disease, and importantly the identification and testing of potential drug targets for treating placental disease.

Modeling placental function and pregnancy physiology in mice (James C. Cross, University of Calgary)

Background. Understanding the molecular, cellular, and physiologic functions of the placenta in humans is limited to expression studies in normal and pathologic human pregnancies and some in vitro systems. Because of this, animal models remain critical for investigation of the basic biology and assessment of biomarkers and treatments of pregnancy complications. The mouse has been a powerful model for understanding animal biology in the last 25 years with the advent of transgenic and knockout technologies. Hundreds of different gene knockouts in mice have given molecular insights into the development and function of the placenta. Many of these genes have human homologues that are expressed in the placenta.145,146 However, before zeroing in on genes and cells, if mice are to be truly useful for understanding human pregnancy complications, it is critical first to ask whether mice and humans have similar physiologies of pregnancy. This is the starting point for the use of mice to investigate molecular mechanisms that give us testable hypotheses to assess in human studies.

Similar physiology of pregnancy in humans and mice. Mice adapt to pregnancy with major changes in the maternal cardiovascular, metabolic, and immune systems. Similar to humans, mice show increased cardiac output, plasma volume, and a mid-gestation drop in blood pressure.147,148 There are also major changes in metabolism in which the mother's fat and muscle become insulin resistant, requiring more insulin to take up glucose, which helps to

shunt glucose to the fetus.149 To combat insulin resistance, an increase in pancreatic b cells and insulin synthesis occurs.150-152 Gestational diabetes mel-litus (GDM) occurs if there is inadequate b-cell compensation.153,154 The most important change in the immune system during pregnancy is the appearance of large numbers of uterine NK cells in the decidua,155 first described in mice and only later in humans. One difference between mice and humans is that mice are litter-bearing, whereas humans tend to have singleton pregnancies; however, what functional difference this has is not clear, given the similarity of pregnancy physiologic condition.

Trophoblast functions and pregnancy. Most of the research on trophoblast cell function in human pregnancy complications has focused on trophoblast cell invasion and its association with spiral artery remodeling. Cell ablation experiments in mice showed that this is not just an association and that trophoblast cell association with spiral arteries is critical for remodeling of those arteries,156 though uterine NK cells in the decidua also play a role.157 Beyond just invasion of spiral arteries, however, the human placenta contains diverse extravillous trophoblast subtypes,158 and mice have diverse trophoblast cells in the junctional zone that express complex patterns of

hormones.159

Several lines of evidence indicate that the endocrine function of the placenta modifies metabolism in the mother that is necessary to promote fetal growth. Scanning through microarray data in the public domain indicates that the human placenta expresses >80 different hormones.160 Although a few are placenta-specific hormones arising from duplication of the GH 2 genem most are from canonical hormone genes that are expressed in the placenta, presumably because of evolution of placenta-specific promoter and/or enhancers. The hormones include known regulators of metabolism, blood cell production and reproduction. Placental prolactin-related hormones can promote proliferation of pancreatic b cells and insulin synthesis.161 Glucose transporter-related

hormones,162 progesterone,163

resis-

ting64 and leptin165 can promote insulin resistance. Paradoxically, the placenta also produces adiponectin166 that promotes insulin sensitivity. It is interesting that, although these hormones are normally expressed by the pituitary (prolactin, GH), ovary (progesterone), and fat (the "adipokines": resistin, leptin, adiponectin), the human placenta is a major source during pregnancy, and production from the maternal tissues is down-regulated.

The mouse placenta expresses approximately 40 protein hormone genes.167 As with humans, prolactin-and GH-like hormones, progesterone, resistin, leptin, and adiponectin are elevated during pregnancy. However, the evolution of the system is slightly different because the prolactin gene, and not the GH gene, is duplicated in mice to produce 22 placenta-specific mem-bers168 with prolactin- and GH-like activities. In addition, the placenta is not the only source of the other metabolic hormones. Progesterone is produced by the ovary throughout pregnancy in response to stimulation by prolactin-like hormones from the placenta.169 Resis-tin,170 leptin,171 and adiponectin172 are produced by fat during pregnancy in mice. Prolactin receptor signaling can

regulate adipokine expression,173-175

which suggests the possibility that, although the mouse placenta is not the direct source of all metabolic hormones, unlike humans, it may still orchestrate the network. The best evidence in mice that placental hormones regulate fetal growth is that of the pleckstrin homology-like domain, family A member 2 gene, which regulates the fetal growth by influencing the number of endocrine cells in the placenta.176 Q9

Current opportunities. Despite the clear evidence that placental hormones drive fetal growth and regulate maternal metabolism, it is curious that research has not continued to understand their roles in intrauterine growth restriction (IUGR) and gestational diabetes mellitus in humans. Placental hormones are attractive targets from a diagnostic

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FIGURE 7

Relationship of maternal intervillous blood PO2 to fetal umbilical venous PO2

Gestational age (weeks)

Oxygen tension in the intervillous space of the placenta is very low until the opening of the spiral arteries to blood flow at approximately 1012 weeks of gestation. Light blue dots are individual data points that were obtained at 8-11 weeks gestational age. Medium blue dots are data that were obtained from individual pregnancies at 11-16 weeks of gestation. Dark blue dots are the mean of values that were obtained in only a few women between 16-38 weeks of gestation and have very wide confidence intervals (>30 mm Hg). Red dots are umbilical venous PO2. Note the tight relationship and narrow diffusional gradient between intervillous and fetal PO2 late in pregnancy. This Figure is a composite of data obtained from various references.184-187

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

standpoint because they can be measured serially, and improvements in multiplex immunoassays mean that several hormones can be assessed at the same time. Both hormone levels and polymorphisms in the placenta GH-related genes have been associated with pregnancy complications in humans, 164 although the number of published studies is limited and they have often examined single hormones and not made connections with anatomic changes in the placenta.

Future opportunities. There is emerging evidence from mouse studies that the placental hormones are sensitive to maternal nutrition and changes in their levels likely reflect attempts to mitigate the impact of poor nutrition on fetal growth.177 Therefore, hormone levels

should have good predictive value in reflecting both stress to the pregnancy and the robustness of the placenta's ability to mitigate the impact on the fetus. In addition to diagnostic value, it is easy to imagine therapeutic strategies in which hormone supplementation or blockade is used to treat pregnancy complications.

Scientific gaps. The complexity of the hormone network at play during pregnancy, both the number of hormones and the systems of feedback and adaptation, will require the use of animal models, particularly knockout and transgenic mice, to understand them. With the ability to study mouse physiologic condition, it is clear that, although a mouse is not a human, we certainly can learn from them.

Potential drug targets of important placental pathways in relation to pregnancy disorders

Placental hypoxia as a molecular target (Stacy Zamudio, Hackensack University Medical Center)

Background. Hypoxia is a pathologic condition in which there is insufficient oxygen to maintain normal physiologic processes. However "hypoxia" is often used imprecisely in the literature, interchangeably with some of its causes, for example: hypoxemia-reduced partial pressure of oxygen (PO2), anemia (insufficient hemoglobin or hemoglo-binopathies that alter oxygen binding/ release), or reduced environmental oxygen availability (as in high-altitude animals dwelling in burrows, diving mammals). Hypoxia is usually assumed to be present in any 1 of these conditions. For example, a reduction in blood flow to a specific organ or tissue, whether acute or chronic, often is assumed to be a hypoxic insult. However, hematologic adaptations can compensate for lower blood flow,178 and the volume and speed with which blood travels, as well as diffusional distances, affect tissue oxygen delivery.179-183 Thus, in the absence of direct measures of oxygenation in the tissue, cell, or organ of interest, it is difficult to tell whether hypoxia is present.

Oxygen levels in the human placenta and fetus. Normal oxygen levels within the intervillous space of the placenta are low early in gestation (approximately 20 mm Hg), can rise to as high as 80 mm Hg in the early second trimester, and then decline progressively towards term (Figure 7).184-187 Fetal umbilical [F7 venous PO2 follows a similar pattern, perhaps reaching as.188 Despite difficultyQ10 in quantifying magnitude and intensity of hypoxia, much less being able to detect its onset, the bulk of evidence has led to general acceptance that fetal growth diseases often are associated with placental hypoxia, specifically early-onset pre-eclampsia, most nongenetic/syndromic IUGR, preeclampsia with IUGR, and some diabetes/GDM with large for

gestational age neonates.

Figure 7

shows that what might be considered abnormally low O2 levels in the placenta will vary not only by gestational age, but also with location within the placenta. The third-trimester placenta is exposed to PO2s that can range from <20 mm Hg when the deoxygenated blood from the umbilical artery flows back into the placenta to >80 mm Hg when the maternal arterial blood first enters the intervillous space. This equates to 1-10% ambient O2 for in vitro experimentation. Such data have given rise to a convention in which 5-8% O2 is used to mimic normoxic conditions in the third trimester and <3% for hypoxia.

Placenta hypoxia cannot be diagnosed unambiguously because of the inherent limitations of human experimentation and the inaccessibility of the placenta and fetus in vivo. Proxies are used instead. For example, reduction in blood flow severe enough to deprive tissues of oxygen and glucose sufficient for metabolic needs is assumed to be present when elevated maternal/fetal Doppler indices reflect increased impedance. Elevated erythropoietin levels in mothers and babies, nucleated red blood cells in neonates, increased placental expression of hypoxia-inducible-factor (HIF), and its target gene products have all been used to demonstrate that placental hypoxia is present in

fetal growth diseases.193,198-210 Thus,

placental hypoxia is a target for

therapeutic intervention, and unlike some of the topics discussed at this workshop, there have already been a number of clinical trials designed to ameliorate or prevent presumed consequences of hypoxia such as inflamma-tion211 and oxidative stress.212-215

Chicken and egg, cause and effect. Of critical importance is distinguishing between hypoxia per se and the hypoxia response. The hypoxia response is evidence that hypoxia is or has been present (eg, up-regulation of HIF and HIF-regulated genes, higher fetal hemoglobin concentrations). The strength and magnitude of the response may reflect the intensity of the insult, but it is also often adaptive in that it enables mechanisms that increase the delivery of oxygen to the fetus (as in metabolic reprogramming or the development of decreased vascular syncytial membrane thickness). On the other hand, we must be mindful of the possibility that failure of an appropriate hypoxia response may itself be part of the pathologic condition. There is evidence of HIF dysregulation and consequent over-expression without adaptation in the more severe forms of preeclampsia.208,209,216 In contrast, in some severe, early-onset IUGR, there often appears to be a lack of HIF-mediated responses, when all evidence suggests such response is needed.217-219 Studies at high altitude have revealed the importance of being able to distinguish between adaptive hypoxia response and pathologic condition, as well as the subtlety with which O2 tension can exert an effect. Maternal partial pressure of O2 in arterial blood (PaO2) falls considerably at >2700-m elevation; because of the sigmoid shape of the O2 dissociation curve, PaO2 falls precipitously at >3000 m. Even in pregnancies with entirely normal outcomes, physiologically lowered maternal PaO2 results in a progressive slowing of the third-trimester fetal growth trajectory.220,221 This is despite significant placental adaptation both structurally (increased angiogenesis, decreased vascular syncy-tial membrane thickness) and metabol-ically.222-227 Altitude studies in vivo showed that the human placenta that is

subjected to hypoxic stress engages in a highly conserved process most obviously seen in solid tumors.228-231 Metabolic reprogramming is a reversible form of hypometabolism in which an effective, largely mitochondrial-driven switch from oxidative phosphorylation to aerobic (ie, glycolytic) glucose consumption by the trophoblast results in increased cellular oxygen availability, which is then available for diffusion to the fetus.225,226 In essence, the mechanism "spares" oxygen for the fetus, but at the same time reduces fetal access to glucose. This slows fetal growth but ensures that the fetus does not outgrow its supply line. Even when placental metabolic programming is active, a fetus with greater umbilical venous O2 tension consumes more O2.

How might hypoxia be ameliorated? Therapeutic strategies must necessarily depend on what is the pathologic barrier to normal placental oxygen diffusion. Four targets will be considered here: blood O2 content, uteroplacental blood flow, placental structure (angiogenesis), and hypoxia-induced metabolic reprogramming.

Raise arterial O2 content (CaO2) or PaO2. In women who are anemic, the obvious treatment would be increasing maternal red cell mass and thereby increasing CaO2. This is harder than it sounds because women have less of a response to erythropoietin than do men,232 and the condition of up to 25% of pregnant women who are treated with erythropoietin does not respond.233 At high altitude CaO2 is increased by raising hemoglobin concentration. This preserves maternal and fetal oxygen delivery177,234 despite an approximate 20% reduction in uterine artery blood flow in mothers and an even greater decrement in fetal umbilical venous blood flow. Packed red blood cell transfusion might work for anemic mothers, but it is unlikely to be of value where the barrier to O2 diffusion is due to placental structural defects such as the failed tertiary villous vascular development (branching angiogenesis and elongation/dilation of terminal capillary loops) seen in idiopathic IUGR. Moreover, there is a balance that must be maintained

between blood viscosity and O2 carrying capacity (eg, the optimal hematocrit level for preservation of O2 delivery in the brain microcirculation is 15-40%).235 One obvious method for increasing PaO2 is O2 supplementation. Sadly, a 2003 Cochrane review found that, of 10 trials of O2 therapy, only 3 were adequate for inclusion in a metaanalysis. O2 compared with no O2 was given to mothers for improvement of fetal growth. Rereview in 2009 changed none ofthe Cochrane conclusions because no additional trials were undertaken.236 The trials were poorly designed and involved <100 women. However, 24-hr supplemental O2 led to a decrease in the perinatal mortality rate. The studies were confounded in that the O2-treated group was of greater gestational age at initiation of treatment. Concerns over possible response to "hyperoxia" such as the generation of free radicals, the inhibition of potentially adaptive responses ongoing in fetus and placenta have been raised as objections to O2 therapy.

Blood flow. Deficits in uteroplacental or fetal blood flow are associated strongly with preeclampsia and IUGR as evidenced by Doppler ultrasound measures of impedance to blood flow in the uterine arteries and within the fetus. The studies in which blood flow in human pregnancy complications has been measured quantitatively or semi-quantitatively support this.237 Absent or reversed end diastolic blood flow velocity in the umbilical arteries is a grave sign and usually leads to emergent delivery.238 Elevated Doppler resistance indices in the maternal uterine arteries indicates that there is downstream impedance to blood flow. This is accompanied by morphologic evidence of placental structural problems such as a reduction in small arteries within the tertiary stem villi,239 thickening of the basal lamina, and erythrocyte congestion in tertiary villous capillaries.240,241 As with CaO2 or PaO2, how one might target blood flow depends on what is the underlying problem. Most of the blood entering the intervillous space is carried by the maternal uterine arteries. The uterine arteries therefore are critical to pregnancy success and a potential target.

In normal pregnancy, eccentric remodeling of the uterine arteries leads to doubling of uterine artery diameter by 20 weeks of pregnancy and a further, smaller increase, likely because of shear stress, in the late third trimester.242 Eccentric remodeling is characterized by changes in the composition of the vessel wall that permits greater distensi-bility. In addition, there is inhibition of the myogenic response (the rise in vessel

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tone with increasing pressure).

In human and multiple experimental animal models, endothelium-dependent vasodilator response is increased markedly in the uterine circulation during pregnancy. In rodent models, depending on what branch of the uterine artery is investigated, nitric oxide contributes 30-80%, and endothelium-derived hyperpolarizing effectors contribute 20-70% of the endothelium-dependent vasodilator response of the pregnant

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uterine arteries.

Placental structure (eg, angiogenesis). In idiopathic IUGR, the placenta frequently has a dearth of branching angiogenesis and poorly developed tertiary capillary development, leading to reduced O2 diffusion capacity.179'217 Structural placental defects in preeclampsia are less clear. An excess of fibrinoid deposition leading to, or as a result of, villous death has been attributed to excess oxidative stress or inflammation. In women with preeclampsia, in general, placentas are smaller (reduced weight and volume), and the volume of functional tissue (parenchyma) and villous surface area (area of nutrient and gas exchange) are reduced. 248 However, unlike IUGR, they also have an increase in the fetal capillary volumes and density (indicative of a hypoxia response). Late onset pre-eclampsia (without fetal compromise) shows no such changes and cannot be statistically differentiated from normal placentas.249 The interplay of many angiogenic growth factors and of several critical cell types (pericytes, endothelium, trophoblasts) is involved in the normal

vasculogenesis of the placenta.224,250

Placental metabolic reprogramming. In cancer biology, several recent unique

insights are relevant to the placenta and consideration of molecular pathways that might be amenable to therapeutic intervention. Cancer cells have dysregu-lated, Warburg-like glucose metabolism. Energy production is abnormally dependent on aerobic glycolysis; there is increased fatty acid synthesis and increased rates of glutamine meta-bolism.251 In fact the term glutamine addicted is now applied to many can-cers.252 In cancer biology "glutamine addicted" refers to an extension of metabolic reprogramming in which glutamine is required for essential amino acid uptake to maintain activation of mTOR. In many, if not all cancer cells, glutamine is also the primary mito-chondrial substrate. These metabolic changes are linked to therapeutic resistance in cancer treatment; hence, strategies are being developed to target this altered cancer metabolism in conjunction with older treatments that were designed to inhibit angiogenesis or otherwise shrink the tumor.253,254 We have demonstrated the same, evolu-tionarily conserved mechanism of metabolic reprogramming in the hypoxic placenta that is associated with stably elevated HIF-1alpha levels, which initiates metabolic reprogram-

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ming. However, in contrast to

cancer, the objective of a therapy that targets metabolic reprogramming in the placenta would be to sustain the response, which leads to increased intracellular oxygen levels and hence more oxygen for diffusion to the fetus.

Current opportunities. There is widespread acceptance that placental hypoxia, acknowledging the already low O2 environment that is normal, is associated with pregnancy diseases. However, before a placental molecular intervention is attempted, there should be a greater effort to examine relatively noninvasive means of improving oxygenation in the placenta. These should include definitive studies of maternal O2 supplementation, dietary strategies designed to increase maternal substrates for nitric oxide, and other obviously more benign strategies that target maternal physiologic condition rather than placental function.

Methods that might be used to provide additional means of carrying oxygen in the circulation (eg, oxygen-filled microbubbles) are under investiga-tion.255-258 Moreover, the opportunity exists currently to further test the effects of supplemental O2; more modern standards of clinical trials such as establishing dose/response, effects of treatment duration, minimum dosing necessary surely should be attempted as a simple, yet potentially, effective intervention.

Beyond this are the effectors that are responsible for altering vascular tone that could be molecular targets, including nitric oxide, by stimulating release via a number of means that include the administration of VEGF, PlGF, relaxin, or stimulation of the reticular-activating system, hemoxygenase—carbon monoxide, large conductance Ca2+- activated potassium (K+) channels, and increased dietary intact of arginine orQ11 citrulline.259-261 For endothelium-derived hyperpolarization, potential effectors include K+ C-type natriuretic peptide, arachidonic acid derivatives, epox-yeicosatrienoic acids and hydrogen peroxide.

Three approaches have been or currently are being investigated to increase blood flow. The least invasive of these involves dietary arginine (or citrulline) supplementation, in theory, to increase substrate for nitric oxide production. A number of small trials led to 1 randomized controlled clinical trial.262 High-risk women (with a history of preeclampsia in the previous pregnancy) had less than one-half the rate of preeclampsia as women receiving placebo. However, another trial in women already diagnosed with pre-eclampsia and treated acutely, rather than throughout pregnancy, showed no benefit.263 International recruitment for a trial is ongoing for the treatment of early-onset IUGR.264 This 6-year study was based on several small studies that showed that low-dose sildenafil improved fetal growth and neonatal survival.265 Another trial that is ongoing and led by Dr Anna David in the United Kingdom targets women with even earlier IUGR, women whose IUGR is so

severe that the fetus normally would die before viability.266 Treatment consists of uterine artery catheterization and injection of adenovirally delivered VEGF-D. Proof of concept studies in sheep demonstrated that this treatment improves uterine artery blood flow for approximately! month. 267

Future opportunities. With respect to angiogenesis, cancer therapies, especially for solid tumors, provide some clues. These therapies target the inhibition of angiogenesis, although, in the placenta, one might wish to stimulate organ-specific angiogenesis. Effectors in this molecular pathway provide a variety of targets. Vascular endothelial-cadherin and matrix metalloproteinases, for example, loosen gap junctions in the endothelium, which is required for tip-cell formation, a prerequisite to branching angiogenesis. However, tip cell formation also requires coordinated activity by VEGFR-2, various ligands for Notch-1 receptor (such as Delta-like ligand and jagged 1), neuropilin 1, integrins, HIF-1a, and angiogenic growth factors such as VEGF, fibroblast growth factors, angiopoietins.

Metabolic reprogramming is HIF-dependent, and, as indicated earlier, HIF may be dysregulated in some placental diseases. Targets other than HIF that could be manipulated to sustain the metabolic response to hypoxia might include a varietyofenzymes: hexokinase, pyruvate kinase M2, lactate dehydroge-nase A, pyruvate dehydrogenase kinase, fatty acid synthase, and glutaminase.

Inhibition of HIF-1a or its gene products has been a primary focus in the development of cancer drugs. There are >400 drugs that specifically target the HIF pathway, most of them inhibitory. Many of them also target specific, cancer-related mutations that are related to the HIF pathway; hence, a reverseengineering approach could be applied to existing drugs to see whether they would be beneficial, as has been done in

human immunodeficiency virus.268,269

A placental strategy might consist of controlled up-regulation of HIF-1a, because it is known to be elevated stably in hypoxic placentae and likely

contributes to the increased angiogenesis that is a favorable adaptation when placental hypoxia is present but not associated with a disease.221

Scientific gaps. For further advances in molecular targeting, greater information is required in a number of areas. For example, although there is a large quantity of data on the role and effects of vasodilators, the receptors for these agents are understood poorly. Although a targeted increase in receptors for vasodilators would be a possible therapeutic strategy, we clearly require more knowledge of the broader effects of receptor modulation.

Similarly for the angiogenic growth factors, what are the consequences if some, but not other angiogenic growth factors, are targeted in the placenta. What might be the response to modulating the angiogenic growth factor receptors rather than the growth factors themselves? Might it be possible to devise tissue and/or cell type selective modulation that would bypass the problems of the less-controlled distribution of the ligands? Placental vascular pericytes have been suggested as a he-matopoietic stem cell or mesenchymal stem cell. They are involved intimately in

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vessel stability and angiogenesis. Pericytes can be detached from their vascular niche by angiopoietin-2, which would increase the stimulus for branching angiogenesis. Again we lack more knowledge to determine whether these supporting characters may be the key to successful vascular therapies.

Is fetal growth a feasible target for placental intervention? (Nicholas P. Illsley, Hackensack University Medical Center)

Background. Alterations in fetal nutrient concentrations because of changes in placental transport and/or metabolism are associated with a variety of fetal growth diseases. Deficits in oxygen are associated with hypoxia, preeclampsia, and IUGR. Deficits in glucose and/or amino acids are associated with IUGR, and an excess of glucose is associated with macrosomia, obesity, and diabetes mellitus. Alterations in placental

nutrient transporters are associated with a variety of fetal growth diseases; the glucose transporter 1 in hypoxia and diabetes mellitus273,274 and amino acid transporters in IUGR and diabetes mel-litus.275,276 The simplest possibility in these circumstances would be to reverse these deleterious changes by manipulation of maternal substrate levels. There are situations in which action to rebalance abnormal nutrient concentrations is capable of correcting growth problems: the restoration of normal maternal glycemic status to prevent macrosomia being an example. Overall, however, the record with regard to maternal nutrient supplementation is 1 of minimal effect combined with potentially serious side-effects.277

Another set of important questions revolves around timing. Is there a window during which intervention is possible, and perhaps more importantly, how do we know when intervention is necessary? In answer to the former, intervention is clearly preferable before structural and biochemical changes make the growth restriction process difficult to reverse, leading to the latter question: how do we detect the initial stages of growth restriction? The best indicator we have currently that fetal growth is deviating from normal is the fetal growth trajectory derived from serial ultrasound measurements. Given that this requires multiple measurements over weeks of gestation, by the time we can verify growth restriction, the fetus (and placenta) are already well advanced through pathologic changes in growth. This crude indicator signals a process that is already well underway and, at best, will allow for stabilization of growth at a much lower percentile. It is clear that, without a means to detect the early stages of the processes that contribute to growth restriction, intervention will lag well behind. Our own research into placental metabolic processes has shown that the early stages of growth reduction involve a switch away from oxidative metabolism toward an increased level of glycolytic metabolism (placental metabolic reprogram-ming).278 As a potential early indicator of growth problems, the initiation of

placental metabolic reprogramming may provide the crucial biomarkers needed to detect the first stages of an altered growth trajectory.

Current opportunities. Assuming that combined biochemical/imaging methods will eventually detect at-risk pregnancies, what might we focus on as placental targets for intervention? Is it possible to see nutrient transporters as possible molecular targets? The ones that concern us here are those that transport the essential, higher volume nutrients, including glucose, amino acids and fatty acids. Because the high volume glucose transporters (ie, GLUT1 and GLUT3) demonstrate unique polarized distributions between the maternal-facing microvillous and fetal-facing basal sur-faces,279,280 altering the balance of glucose transporters within the syncytiotropho-blast raises with it the question of the selective polarized targeting that is needed to achieve increased transport. The polarized targeting question arises also for amino acid transporters when the asymmetric distribution acts to move sufficient quantities and types of amino acid against their gradient into the fetus.281 In the absence of targeting information for these transporters, we need to be assured that interventions will provide the appropriate distribution between microvillous and basal faces necessary to increase flux. Moreover, the effects of increased transporter density, although possibly increasing trans-placental fluxes, will also pose other problems. In the case of glucose, this is likely to generate increased glycolytic metabolism and subsequent lactic acidosis, as observed in glucose supplementation interventions. In the case of amino acids, the interconnected nature of their transport and the broad substrate specificity of amino acid transporters suggest that alterations to an individual transporter will have significant and hard-to-predict ramifications for the transport of other amino acids and for placental amino acid metabolism. Similarly, it is difficult to predict what the effect of altering the expression of placental lipases, fatty acid transport, or fatty acid binding proteins might be on processes

such as oxidative metabolism and the transport of other nutrients. The essential, high-volume nutrients have a wide range of metabolic fates, and they or their metabolites play crucial roles in regulating key metabolic pathways. Not surprisingly therefore, changes in the expression of individual nutrient transporters are likely to be associated with unwanted changes in placental and/or fetal metabolism. The uncertainty engendered by the alteration ofindividual transporters makes them problematic candidate molecular targets.

An alternative approach to targeting individual transport or metabolic processes is the generation ofalterations in a manner that combines multiple metabolic and/or transport targets. In this way, the transport and metabolic processes that involve specific substrates are modulated in an integrated fashion. We already have detailed information on many of these integrated pathways. They form the traditional endocrine or growth factor pathways that mediate regulation of cellular function by extracellular factors. We know for example that insulinlike growth factor I (IGF-1) regulates the expression of placental glucose and amino acid transporters282,283 and that it is associated positively with fetal and placental growth.284,285 Judicious adjustment of the placental IGF-1 pathway might allow for an integrated regulation of growth processes. Recent research has shown that adenoviral-mediated delivery of IGF-1 to the placenta appears to stimulate glucose and amino acid transporter expression in vivo and to correct placental insuffi-

ciency.286,287 Similar types of growth

factor-mediated regulation might be possible via the signaling pathways stimulated by IGF-2 or placental GH. Perhaps the molecular targets for these pathways might be restricted to selected cell types by altering receptor levels rather than altering the concentration of the growth factors themselves.

Future opportunities. Although the effectors described earlier may be able to stimulate integrated pathways for the promotion of fetoplacental growth, the signaling pathways that are activated in

this way may be too numerous or too broadly based to avoid other less desirable effects. Growth factor signaling pathways frequently have many trans-duction pathways that branch off below the growth factor receptor. Another alternative is the targeting of metabolic or signaling subnetworks that control multiple components but do not extend to all of the endpoints for a signaling pathway. For example mTOR is a serine kinase that is the focus of a signaling pathway that forms a nexus for multiple nutritional and metabolic signals. MTOR affects growth by modulating protein synthesis, lipid synthesis, and energy metabolism, primarily through phosphorylation of the 4E-binding proteins that in turn have marked effects on the production of the proteins that control messenger RNA (mRNA) translation.288 In the placenta, in addition to effects on protein synthesis, mTOR appears also to modulate amino acid transporters by regulating the membrane trafficking of transporter pro-tein.289 There is good evidence to suggest that mTOR activity is proportional to maternal body mass index290,291; however, in IUGR, syncytial mTOR, while displaying increased expression, shows decreased activity.290,292 This suggests that the mTOR pathway may be a good potential regulatory point for growth-related intervention. Is it possible, for example, to target the mTOR system directly, below the broadly regulatory phosphatidylinositol-3-kinase/protein kinase B stage (Figure 8)? Activation via agents, such as the small experimental drugs MHY 1485293 or 3BDO,294 might confine the effects to a smaller set of endpoints than the use of IGF-1. Will it be possible to selectively target functions below the level of mTOR, for example modulating amino acid transporter expression via changes in microtubule organization?295 Another possibility is suggested by the inhibitory actions of metformin on oxidative metabolism.296 Might it be possible to initiate or extend placental metabolic reprogramming effects through artificial stimulation of placental oxygen sparing and consequent increased glycolytic meta-bolism?297 Action via subsystems may

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avoid the lateral signaling effects of classic growth factors while providing for confined, but integrated, stimulation. This will require detailed knowledge of the subsystems that are involved and raises the question of targeting and specificity.

Scientific gaps. Like substrate supplementation, alteration of an individual transporter appears likely to distort the transport and metabolism of other substrates and metabolites. Modulation of systems that integrate multiple aspects related to nutrient transfer, whether by endocrine or intracellular subsystem approaches, is likely to be more successful. This requires not only detailed knowledge of the pathways involved but also understanding of the lateral or related pathways that may be affected to prevent generation of off-target effects.

Whether stimulation via a growth factor pathway or an intracellular subsystem, a prerequisite is to restrict the site of action to the trophoblast. Renewed attention is required to the delivery vehicles for agents of interest. Some of the vehicles for these actions are under development. Viral or homing-peptide directed nanoparticles carrying directly active agents or vectors with trophoblast-specific promoters may provide the means to overcome questions of targeting and specificity. But just as importantly, timing is everything! Unless it is possible to detect changes in nutrient transport or growth early enough to intervene, modification of nutrient transport may exacerbate rather than resolve problems. Research to determine the onset of pathologic events and the means to monitor growth and the ameliorative effects of targeted interventions is an aspect of this process that is just as important as the intervention itself.

Targeting oxidative /nitrative stress and mitochondrial dysfunction in placenta to relieve adverse pregnancy outcomes (Leslie Myatt, University of Texas Health Science Center San Antonio)

Background. It has long been recognized that pregnancy is a state of oxidative

FIGURE 8

Metabolic mechanistic target of rapamycin intervention points

Potential intervention points {open arrowS) for modulation of placental metabolism. These range from extracellular endocrine regulation by insulin-like growth factor I through effects on mechanistic target of rapamycin by activators such as 3BDO (3-benzyl-5-[(2-nitrophenoxy) methyl]-dihydrofuran-2[3H]-one) or MHY 1485 (4,6-dimorpholino-N-[4-nitrophenyl]-1,3,5-triazin-2-amine) to points in the

metabolic subsystems regulated by mechanistic target of rapamycin. The question marks indicate ••••.

IGF-I, insulin-like growth factor I; mTOR, mechanistic target of rapamycin.

Hekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

stress and that this is further increased in first, second, and third trimesters in pregnancies that result in adverse outcomes such as preeclampsia and diabetes mellitus,298 preterm birth,299 stillbirth,300 and in those pregnancies complicated by maternal obesity.301 However, a cause-and-effect relationship between increased oxidative stress and adverse pregnancy outcomes still remains to be proved definitively. Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS), including superoxide, hydroxyl anion, hydrogen

peroxide, and the ability of various antioxidant mechanisms to scavenge them. Further, ROS can also interact with reactive nitrogen species such as nitric oxide to produce the powerful prooxidant peroxynitrite, which, in turn, can nitrate tyrosine residues in proteins, such as superoxide dismutase302 (SOD;

Figure 9), causing nitrative stress, which affects protein function, usually in a negative manner.303 Not all reactive oxygen and nitrogen species are equal in potency, that being determined by their cellular diffusion distance, defined as the distance moved in aqueous solution

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FIGURE 9

Generation of oxidative and nitrative stress

Nitrative stress

(protein nitration)

Superoxide generated from molecular oxygen by membrane bound (myeloid differentiation primary response gene 88 oxidase) or cytosolic (xanthine oxidase) enzymes or the mitochondrial electron transport chain normally can be effectively dismutated to hydrogen peroxide by superoxide dismutase. Increased superoxide will attack targets, which leads to oxidative stress. With increasing generation of nitric oxide, nitric oxide out competes superoxide dismutase for superoxide and interacts to produce the more powerful prooxidant peroxynitrite. Peroxynitrite nitrates proteins at tyrosine residues and cova-lently modifies function usually in a negative manner. Superoxide dismutase is inactivated when nitrated by prooxidant peroxynitrite, hence leading to a negative feedback loop that leads to oxidative and nitrative stress.

NO, nitric oxide; ONOO-, prooxidant peroxynitrite; SOD, superoxide dismutase.

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

in 1 half-life. Hence, hydroxyl anion has

a diffusion distance of only 5 angstroms,

superoxide 0.4 mm, peroxynitrite 5 mm

and nitric oxide 100 mm.304 Therefore, in the placenta, the sites of action (intra- vs

extra- or transcellular) depends on intracellular and cellular site of synthesis in relation to placental structure such as trophoblast thickness (5mm), stem villous diameter (500-1500mm), and the presence of antioxidant molecules to

scavenge them.305

Role of mitochondria. The major sources

of ROS are the mitochondrial electron transport chain but also plasma membrane enzymes, such as the nicotinamide adenine dinucleotide phosphate oxidases, cytosolic enzymes (including

xanthine oxidase, flavin enzymes, and cytochrome p450s), lipoxygenases, and cyclooxygenases.297 Although widely acknowledged for their pathologic effects that include covalent modification of proteins, lipids, and DNA, ROSs also function as physiologic effectors, regulating redox sensitive genes and pro-teins.306 There are many methods available to measure ROS. Because of their ephemeral nature, direct measurement of ROS is difficult; therefore, measurement of effects that include lipid peroxidation, covalent modification of proteins, DNA damage and repair, levels and activity of antioxidant molecules and enzymes, and enzymes generating ROS are used. There is no gold standard measure of oxidative stress; rather one should chose a measure related to the area of interest.307

Mitochondria have many roles in cellular function in addition to energy production, which includes apoptosis, steroid synthesis, calcium homeostasis, and amino acid transport. Changes in mitochondrial activity can be induced by environmental factors such as nutrition, hypoxia, aging, and obesity that impact cellular survival and that are associated with adverse pregnancy outcome. Mitochondria are the major source of ROS under physiologic conditions because of the release of high-energy electrons from complexes I and III of the electron transport chain; these electrons reduce molecular O2 to superoxide, which normally is scavenged by the mito-chondrial SOD isoform, mitochondrial antioxidant manganese SOD. However, mitochondrial function itself can be compromised by severe and/or prolonged oxidative stress via damage to mitochondrial DNA, proteins, and lipids. We recently have shown that maternal adiposity leads to increased oxidative stress in the placenta and that this is associated with decreased trophoblast mitochondrial respiration measured in vitro using a Seahorse extracellular flux analyzer (Seahorse Bioscience, Copenhagen, Denmark).308 This dysfunction is further evidenced by decreased mitochondria number, expression of complexes I-V of the electronic transport chain, and decreased

placental adenosine triphosphate generation. In addition, these trophoblasts appear metabolically inflexible because they cannot switch to alternative energy sources when placed on galactose to prevent glycolysis.307

Current opportunities. Subsequent to acquiring knowledge of increased oxidative stress in pregnancies with adverse outcomes, there have been many trials of antioxidant therapy to try and prevent these outcomes.309 All have failed, which is an outcome variously attributed to choice of antioxidant, time and duration of treatment, heterogeneity of patients studied, the fact that oxidative stress may indeed not be part of the pathophysiologic evidence, or the need for targeted rather than global therapy. This begs the question: what are the targets at the tissue and cellular levels? Should antioxidants that are targeted to placenta by use of nanoparticles or liposomes be used rather than global treatment? Cellular targets could include antioxidant enzymes such as SOD, glutathione peroxidase, thioredoxin reductase and catalase, the enzymes that synthesize ROS such as xanthine oxidase and nicotinamide adenine dinucleotide phosphate oxidases, extracellular anti-oxidants (transferrin, ceruloplasmin, uric acid, and bilirubin), intracellular reducing elements (glutathione, coen-zyme Q10, and cytochrome c oxidase) and nutrients and supplements such as vitamins C and E and melatonin. Because mitochondria are the major source of antioxidants, there has been growing interest in mitochondrial targeting of antioxidants by compounds such as the mitochondria-targeted anti-oxidant drug, MitoQ, where coenzyme Q10 is linked to a lipophilic cation to allow adsorption through the inner mitochondrial membrane.310 Other approaches include the use of selenium,311 which is found in the active site of the selenoprotein glutathione peroxidase, or of melatonin, which functions as an antioxidant.312 Q12

Future opportunities. We recently have shown that the hypoxamir, miRNA 210,Q13 can inhibit mitochondrial respiration in

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trophoblast cells, hence opening the door to targeted therapies to alleviate mitochondrial dysfunction and oxida-tive stress in the placenta. The male fetus is at greater risk of adverse pregnancy outcome (eg, preterm birth and stillbirth) than is the female fetus, although it is unclear whether this is because the male places itself at risk by its desire for greater growth or if the female adopts a conservative strategy to ensure successful delivery and propagation of the species.314'315 Sexual dimorphism occurs in placental gene expression,316 particularly of genes that are involved in the inflammatory response, in response to maternal adiposity and in diseases such as preeclampsia. We found increased expression of inflammatory and apoptotic markers in placentas of male fetuses from preeclamptic pregnancies compared with female fetuses.317 Similarly, we found differences in placental expression of miRNA 210 and its mito-chondrial target genes between male and female fetuses of lean, overweight, and obese women that were mediated by differences in the transcription factor NF-kB p50.307 Our current data therefore indicate that, in conditions such as GDM, preeclampsia, and obesity, inflammatory pathways are activated in the placenta in a sexually dimorphic manner to regulate the production of reactive O2 and nitrogen species that lead to oxidative/nitrative stress and to mito-227F10]chondrial dysfunction (Figure 10). This 2277 results in placental dysfunction and adverse pregnancy outcome and suggests that targeting of inflammatory pathways might be a useful approach to prevent oxidative stress. Dietary manipulation (eg, inclusion of omega 3 fatty acids and vegetables), the use of antiin-flammatories (eg, resveratrol) and immune selective antiinflammatory derivatives, or targeting strategies that involve miRNA, siRNA, viral vectors, nanoparticles, and biologics may prove useful. These targeting strategies, however, may need to be different based on the sex of the placenta.

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Scientific gaps. Current gaps in the knowledge include the absence of proof of a cause-and-effect relationship

between oxidative stress and adverse outcome. Perhaps an animal model of induced oxidative stress with adverse outcome would facilitate this. In addition, the tissue and cellular localization and contribution of different sources of oxidative stress (ie, including the generation and scavenging mechanisms of ROS) must be studied comprehensively. Further, the timing of the oxidative stress insult, either early, mid, or late gestation or continuously, on outcomes needs to be considered when designing drug targeting studies.

Identification of potentially useful or experimental drugs to target important

Challenges and advantages of rescuing and repurposing (Christine Colvis, National Center for Advancing Translational Sciences) Background. The most common reason for an investigational drug to fail to make it through the Food and Drug Administration (FDA) approval for marketing is its inability to demonstrate clinically meaningful efficacy in the disease being studied. At the stage of phase 2 trials, another major reason for failure is simply because of business strategy decisions that result in the deprioritization of a drug.318,319 In either case, there is no scientific reason that the drug could not be pursued for an indication other than that for which it was originally being developed.

National Center for Advancing Trans-lational Sciences (NCATS) new therapeutic uses program. The new therapeutic uses program uses a novel approach to provide academic researchers with access to investigational drugs from pharmaceutical companies.320 The program leverages investments that have been made by pharmaceutical companies in a variety of drugs and biologics (agents) but for which the company has often times discontinued development. To qualify for inclusion in the program, the agent offered by the company has to have been through at least a phase 1 clinical trial, so that safety in humans has already been assessed in at least 1 population. By limiting the agents to only those that

FIGURE 10

Putative mechanism for the involvement of inflammation-mediated placental dysfunction in fetal programming

Pregnancy Outcome (Programming)

The adverse inflammatory maternal environments of gestational diabetes mellitus, pre-eclampsia, or obesity can generate increased oxidative/nitrative stress and cause mitochon-drial dysfunction in the placenta in a sexually dimorphic manner. This disrupts placental function and in turn may lead to alterations in placental-mediated regulation of maternal metabolism and fetal growth and differentiation and hence result in fetal programming.

GDM, gestational diabetes mellitus.

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

have been tested in humans, the new therapeutic uses program is able to move projects quickly to phase 2a trials to test the agents for efficacy in new indications. Under the program, the pharmaceutical

companies provide agents (clinical supply including matched placebo and preclinical material, if needed) and the

data that will be needed to file an inves-tigational new drug application with the

FDA to study the agent for the new indication. The National Institutes of Health (NIH) provides the financial

support for phase 2a trials and, ifneeded, phase 1b trials and/or preclinical studies.

The NIH and the pharmaceutical

companies are turning to the broader research community to identify novel uses for the agents. The value of this strategy bore out when, as a pilot, NCATS listed 58 agents provided by companies in June 2012 and within 2 months received almost 160 pre-applications with ideas of indications that the agents might be used to treat. Because of the limited time (12 months)

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allowed under the program for preclin-ical studies, pediatric trials were not encouraged in the 2012 pilot. However, in 2014, NCATS issued a new set of solicitations or funding opportunity announcements, and this time offered an funding opportunity announcement specifically for pediatric indications. The companies, in turn, had identified those agents that they believed could be developed for a pediatric indication. In general, it was expected that pediatric trials would be proposed only when there was no adult patient population for the disease or for which the mechanism of action of the drug might be meaningful only if modulated at an earlier stage in development than in adulthood.

There are significant challenges when trying to repurpose an investigational drug for a pediatric indication. Unlike an FDA-approved drug, these drugs often have been studied in only dozens or maybe a few hundred adults. So, side-effects have been documented for only a small number of individuals. Furthermore, the metabolism of drugs can be very different in younger individuals than in adults. We face similar challenges when we think about exposing a pregnant woman to an investigational drug. Effects of drug exposure in pregnant women generally will remain unknown until after the drug has been on the market for years or even decades.

Current opportunities. The NCATS intramural program offers several opportunities for collaboration. Although funds are not offered, there are several ways to access valuable resources and expertise at no cost to our collaborators. The intramural program that likely has the most potential for repurposing drugs for use in pregnant women is the screening of the NCATS pharmaceutical collection. Nearly 2750 small molecular entities have been approved for clinical use by the United States and in other countries around the world. NCATS has amassed a screening collection of2500 of these, along with approximately 1000 additional investigational compounds. Because they have been approved for

clinical use, these drugs will have been used in far more people than the inves-tigational drugs that are used in the New Therapeutic Uses program, thereby providing a more comprehensive risk profile for the drugs. Collaborators generally approach NCATS with a high throughput screening compatible assay as a first step toward repurposing 1 of these approved medications. Of course, when a drug is first marketed, often, the only data available on fetal effects generally will come from preclinical data. The FDA encourages the use of pregnancy exposure registries as a firstline strategy for gathering information on drug exposure during pregnancy. They have compiled a list of >60 registries to facilitate the gathering ofvaluable data.321 Cross-referencing data from such registries with findings from in vitro assays that were used to screen the NCATS pharmaceutical collection may provide a strategy for repurposing drugs to address placental health indications.

Future opportunities. Repurposing both investigational and approved drugs is a strategy that is pursued currently in small and large pharma and in academia. The strategy is attractive to so many because of the significant cost-savings that result from the use of drugs for which so much early development has been completed already. In the case of repurposing drugs for use in pregnant women, it would be expected that the longer a drug has been on the market, the more information we would have on exposure during pregnancy. Unfortunately, the drugs that will fall in this category will often be drugs for which a generic is available, thereby eliminating an enforceable use patent or regulatory exclusivity. Currently, there are no effective incentives to pursue a new indication for a drug that is available as a generic, regardless of the indication, unless a unique formulation for the drug is developed.322 Although this is a barrier for all indications, it is particularly unfortunate for pregnant women, which is a population for which the development of new drugs (investigational drugs) is extremely unlikely.

Effect of pregnancy on drugs pharmacokinetics and pharmacodynamics (Maged M. Costantine, MD, University of Texas Medical Branch, Galveston) Background. The use of medications in pregnancy has been increasing progressively over the past 3-4 decades (Figure 11).323-327 This is predominantly because of changing in the demographics of pregnant women, because more women enter pregnancy at a later age with increasing prevalence of preexisting medical comorbidities (such as diabetes mellitus, hypertension, asthma, depression, and others) and increased risk of obstetric complications (eg, nausea and vomiting of pregnancy, GDM, cholestasis of pregnancy, pre-eclampsia.) that require pharmacotherapy.322-326 In a recent review, the average number of medications this is used by pregnant women is >4. This increase in the use of medications is also predominant in the first trimester, where almost one-third of pregnant women use at least 4 medications.322 This is concerning because the first trimester is a crucial period for organogenesis and placental development that includes trophoblast invasion, vascular remodeling, and chorionic villi development; in addition, many women are unaware of

322-326

their pregnancy status.

Pregnancy physiologic changes and their impact on medication pharmacokinetic and pharmacodynamics properties. Pregnancy is characterized by significant changes in the anatomy and physiology of almost all systems, which affect medications' pharmacoki-netic/pharmacodynamics properties (Figure 12). Pharmacokinetic generally refers to "what the body does to the drug" and usually is described using the drug's absorption, distribution, metabolism, and elimination; pharmacody-namics refers to "what the drug does to the body" and usually influences the drug's efficacy and safety.328-332 Cardiac output increases by 30-50% because of both increase in heart rate and stroke volume. Most of the increase occurs early in pregnancy (75% by the end of the first trimester). The increase in

cardiac output is preferential in that uterine blood flow increases 10-fold (17% of total cardiac output compared with 2% prepregnancy), and renal blood flow increases 50%; there are minimal alterations to the liver and brain blood flow. 329,333 Cardiac output is further increased during labor, with additional 300-500 mL of blood reentering the circulation with every contraction. Both systemic and pulmonary vascular resistances decrease by 20-30%. Blood pressure also falls toward the end of the first trimester and then rises again in the third trimester to prepregnancy levels. The increase in renal blood flow leads to a parallel increase in the glomerular filtration rate by approximately 40-65%. 329,331,334 This can lead to significant increase (20-60%) in the elimination rates of renally cleared medications, which lead to shorter half-lives and risk of subtherapeutic concentrations. Examples of these medications include lithium, ampicillin, cefuroxime, cefazolin, piperacillin, atenalol, and digoxin. 328,331

During pregnancy, maternal blood volume increases by 40-50%, and total body water increases to almost 8.5 L by the end of pregnancy. In addition, maternal fat stores and fat mass increase by almost 10-fold. The increase in blood volume and water within the body expands the volume of distribution of hydrophilic substances.335 On the other hand, the increase in fat mass leads to a larger volume of distribution of lipophilic drugs, such as sedatives. For some drugs, a larger volume of distribution could lead to decreased peak serum concentration, which may necessitate a higher initial and maintenance dose to obtain therapeutic plasma concentra-tions.331,336 Additionally, because of the decrease in serum albumin during pregnancy, highly protein bound compounds (such as digoxin, midazolam, and phenytoin) may display higher free levels, which increases their peak

plasma concentrations.329,331 The hyper-

vascularity and edema of the upper respiratory mucosa theoretically may result in an increased absorption of inhaled agents such as corticosteroids.331,337 The delayed gastric emptying and prolonged

FIGURE 11

Medication use during pregnancy

Secular patterns of use of any medication that was restricted to the first trimester at any time during the period of 1976-2008. Average number of medications and proportion of women taking >4 medications (n = 25,313) is shown.

Reprinted with permission from Mitchell et al.323

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

small bowel transit time may alter the bioavailability (decrease maximum concentration and time to maximum concentration) and thus decrease the efficacy of oral drugs that are taken as a single dose because a rapid onset of action is

desired. 331,338

Drug metabolism is also altered in pregnancy, in part, because of elevated sex hormones. In general, drug metabolism occurs through phase I (oxidation, reduction, or hydrolysis) and/or phase II metabolism (glucuronidation, acetylation, methylation, and sulfation). Cytochrome (CYP) P450 represents a family of enzymes and is a major route of drug metabolism that is affected by pregnancy (activity increased for CYP3A4, CYP2D6, CYP2C9, CYP2A6, and decreased for CYP1A2, CYP2C19, and CYP2D6 subtypes). For example, the increase in activity and abundance of CYP3A4 in pregnancy lead to an increase in the clearance of its many substrates, such as nifedipine, carba-mazepine, midazolam, saquinavir, in-dinavir, lopinavir, ritonavir, and many

others.339-342

In summary, maternal physiologic changes affect the pharmacokinetic/ pharmacodynamics of drugs through changes in the drugs' absorption, increasing the volume of distribution, protein binding, renal clearance, metabolism, and placental biodisposition. Any novel drug that is identified to act on potential placental molecular targets must be evaluated in the context of these changes so that appropriate dosing be identified.

Current opportunities. Pregnant women are still considered therapeutic orphans because most current therapeutics were never studied during pregnancy.343-345 In a recent review of the literature, <2% of all pharmacokinetic studies involved pregnant women.346 This is concerning because the lack of data on pregnancy-specific dosage of many medications leads physicians to extrapolate drug dosage regimens from nonpregnant subjects or men. This may lead to over or under dosing and may reduce efficacy and increase risk of toxicity. In view of these gaps, the

FIGURE 12

Factors that affect drug pharmacokinetics and pharmacodynamics in pregnancy

CO, cardiac output; BP, blood pressure; HR, heart rate; NVP, nausea and vomiting of pregnancy; SVR, systemic vascular resistance. Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

opportunities to study the pharmacoki-netic properties of many currently used medications with potential impact on placental development and use in selective targeting of molecular pathways are involved in the patho-physiology of various obstetrical complications. This will lead to more effective treatment of pregnant women, while reducing the risks of adverse events.343 The new FDA rule regarding drug classification represents a huge current opportunity to encourage research in this field.

Future opportunities. A major obstacle to studying medications (current and novel) in pregnancy is the lack ofsystems that allow researchers to determine the

safety and efficacy of these medications. Some researchers rely on in vitro placental transfusion models; however, these placentas typically are collected after delivery, usually at term, and may not represent the changes in drug phar-macokinetic and pharmacodynamics throughout the pregnancy. Others rely on primate animal models; however, they are usually expensive and hard to work with. Future opportunities in this field include the development of novel technologies to assess the placenta in real-time and applying them to study medication's biodisposition in pregnancy. The use of novel ultrasound techniques or other novel technologies to tag drugs and then be able to image the placenta and fetus to look for their

distribution would be a great future opportunity.

Scientific gaps. Most therapeutics were never studied in pregnancy during development, because pregnant women were excluded from such studies.343-345 In addition, their safety, tolerability, efficacy, and dosing were extrapolated from studies conducted in men or nonpregnant women. This leads to under or over dosing and affects efficacy and toxicity of these medications. There exists a large gap in information regarding medications pharmacokinetic properties in pregnancy and during lactation. The effects of placental transporters and biodisposition enzymes on medication pharmacokinetic and pharmacodynamics, and transplacental transfer represent a large gap in our scientific knowledge. Moreover, the inability to study placental transport expected after delivery with delivered placentas widens this scientific gap.

From bench to bedside: processes and pitfalls translating research findings into practice paradigms (David M. Haas, Indiana University)

Background. When Vannevar Bush wrote "Science: the endless frontier" in 1945, it inspired that science would provide solutions to health problems. It further described how investment in scientific research could serve as a vehicle to enhance the wealth and prosperity of the nation.347 It is against that backdrop that the public holds out hope for scientific inquiry. This promise engenders a social contract that funding of scientific inquiry would be translated into better health that can elevate the national good on many levels.

The objectives of this portion of the workshop were to describe the trans-lational research spectrum and the difficulties with translating research into practice, to articulate additional concerns and difficulties with translational research in pregnancy by highlighting some translational successes in pregnancy research, and to propose some mechanisms that are aimed at improving the translation of findings into practice and measuring research impact.

Translational research. The research spectrum generally begins with basic science findings that are then translated to human and clinical research (Figure 13). This is termed "T1" research on the translational spectrum: the translation of animal and basic science research into humans.348 The "T2" step in research translation is taking the clinically relevant research findings and translating them into practice guidelines and having them become routine practice in health care.349,350 Some models of the translational spectrum have included more steps that can include the population health impact and involvement of consumers. All of the models serve to help assess the success of translational research at fulfilling the initial promise to improve public health.

Globally, billions of dollars are spent on biomedical, clinical, and health services research, training of providers, quality improvement initiatives, and safety initiatives.349 However, it has been demonstrated that, in the United States, only 55% of patients receive the recommended evidence-based care.351 Additionally, 20-30% of patients get care that is not needed or could be potentially harmful.352 This is a failure in translating research findings into practice. Experts have found that this can be due to system failures. Grimshaw et al350 noted, "Systems fail to ensure that effective and cost-effective programs, services, and drugs get to all those who need them and providers fail to provide the level of care they aspire to."

An example of this is seen in the fact that in 2010, the NIH spent $31 billion on research. However, from 2006-2009, only 74 new drugs were approved by the FDA, which was only one-half the number approved during the previous epoch.353 One interesting study looked at 101 promising technologies that were published in the highest impact journals. This report found that, in the 20 years after publication, only 5 of these promising technologies were licensed for clinical use, with only 1 angiotension-converting enzyme inhibitor being used regularly in clinical practice.354 The report found multiple steps where these promising technologies failed, were lost,

FIGURE 13

Translational research peaks and valleys

This figure illustrates the multiple-step model (5 hills and 4 valleys) of translating research into practice and public health benefit and the many "valleys of death" that can be encountered along the way. Depicted are the 5 phases of translational research separating 4 valleys: basic discovery science to research involving humans (T1), from human studies to evidence-based guidelines (72), from guidelines to health practice (T3), and from health practices to population health programs (T4).

Reprinted with permission from Meslin et al.347

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

refuted, or neglected along the trans-lational spectrum. It has also been found that promising technologies take a long time to get into routine practice, at times up to 17 years. 347 These areas of trans-lational failure have been coined the translational valleys of death.346 The translational valleys of death can come from regulations that impede research or practice, costs, limited resources, commercialization issues, broad social policy implications, professional standards, governmental policy, and general science policy. 346 There are many places where potential health-improving research findings can become derailed. The key is to overcome these difficulties and cross the translational valleys of death.

Translational research in pregnancy. Translational research in pregnancy holds additional potential pitfalls that generally flow from the concerns for the impact of therapies on developing babies and potential long-term impacts that may be unrecognized for years after birth. It is for this reason that, for many years, pregnant women were actively excluded from research. There are

several examples in which translational research failed to detect problems, leading to major public health consequences. In 1956, thalidomide was introduced as a "wonder drug" in Germany.355 It was purported to be a treatment for nausea and vomiting, insomnia, coughs, colds, and headaches. Early studies in mice and rats did not demonstrate congenital anomalies; therefore, it was used as a sedative and tranquilizer in early pregnancy. However, in 1961, reports surfaced of severe congenital anomalies that included limb reduction defects that led to its rapid withdrawal from the market. This was a clear failure of T1 research.

Two notable examples of failure in T2 research in pregnancy have been seen recently. The withdrawal from the market of the combination drug Bendectin was done because of lawsuit pressure regarding congenital anomalies.356 The drug was used by a large proportion of pregnant women for nausea and vomiting in early pregnancy. However, no reports of defects were proved. Without an alternative, women suffered. The rates of hospitalization for severe dehydration from hyperemesis gravidarum rose dramatically. This was an example of

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where research demonstrated safety and practice patterns were established, but fear and a lack of a supportive response to the public caused the withdrawal of an effective therapy, which led to a negative public health impact. Even more recently, the poor adoption rates of the effective human papilloma virus vaccines to prevent cervical cancer is another example of success at the T1 stage, but unfortunately a failure at the T2 stage .357 Despite proven efficacy and safety and coverage by the Children's Immunization Program, vaccination rates of the target populations remain low.

However, there are some noteworthy translational successes in pregnancy. The development of Rhogam to prevent Rh isoimmunization came about from a confluence of observations along the translational spectrum. After demonstrating efficacy and safety in clinical trials, it has been adopted universally as a standard of care preventive therapy in pregnancy, saving thousands of babies every year.358'359 In similar ways, antenatal corticosteroids for accelerating fetal lung maturity in threatened pre-term birth have been a translational success story in obstetrics.360 From astute observations in sheep to landmark clinical trials in humans and pivotal metaanalyses, antenatal corticosteroids are now used routinely on labor and delivery units and clearly have reduced the rates of mortality in preterm newborn infants.361-363 This was greatly aided by NIH Consensus Conferences highlighting the benefits and safety of

the therapy. 364

How to improve translational success. Many have proposed systems that are aimed at crossing the translational valleys of death. One unifying theme is aimed at getting stakeholder investment in the translational process. Practicing providers, the public, industry, and regulatory/governmental agencies must have a stake in the translational processes along the way if there is any hope of translating important research findings into practice aimed at improving the

public health 349,365-367 Otherwise, failure at T2 and beyond can occur

commonly. The formation of trans-lational teams that involve the stakeholders can ensure involvement from the beginning and should involve the entire translational spectrum. 346,349 Effective dissemination of the impact of trans-lational research can inform those providing funding and the public as to the public good that is being gained.

Psychology of change research has noted 3 stages of practice change: awareness, acceptance, and adoption.368 Most translational research focuses on the first 2 stages, whereas the reason that most promising therapies fail to realize their public health impact is the failure of "adoption" into practice. A provider may know that research shows a benefit and accept that it might help the patients, but there may be obstacles to getting it into a routine practice pattern; thus, it is not adopted. This is because "adoption" takes different pathways in the brain before these cognitive changes become embedded in practice. 367

A focus on stakeholders and alignment of incentives can help with the adoption into practice. Dissemination of the impact of research findings is crucial to get stakeholder enthusiasm. For instance, the Human Genome Project turned a $4 billion government investment into $796 billion in economic growth.369 Translational teams, like those stimulated bythe NIH Clinical and Translational Science Awards, focus on collaboration and development of networks of scientists, stakeholders, and regulatory agencies in an effort to propel translational science across the valleys of

death and into practice.370,371

Specific to pregnancy, there is a tremendous need for public involvement, education, and advocacy. The focus of protection of mothers and infants through research, instead of from research, should be highlighted. Ethicists and children's advocacy groups should also be engaged. The advent of individualized therapeutics and diagnostics are forcing this conversation to accel-erate.372 In addition, the utilization of biorepositories can help stimulate this translational process by focusing on potential pooled resources and collaborations.

In conclusion, bridging the trans-lational research gap from bench to bedside has many potential pitfalls. Pregnancy translational research has had well-documented missteps but several important success stories as well. Although research to cross the trans-lational "valleys of death" is difficult, using what is known about behavior change will be important in ultimately fulfilling the promise of scientific research funding to improve public health and the public good. Thinking broadly about stakeholders and involving these stakeholder teams in the processes across the translational spectrum can help cross the valleys of death and help take promising discoveries from bench to bedside.

Current opportunities. Individualized pharmacotherapy provides opportunities currently to tie genomic and other "-omic" technologies to ongoing clinical trials. In addition, the development and utilization of saved samples from past trials and biorepositories provide a wealth of opportunities for analyses using cutting edge technologies to assay for biomarkers that can serve as therapeutic monitoring or individualized pharma-cotherapy model development aids for patients. The focus on diagnostic and therapeutic studies in human models and humans is a clear priority moving forward.

Future opportunities. Clinical trials in the future should have clear response bio-markers, pharmacokinetic/pharmaco-dynamic/pharmacogenomic outcomes, and harmonized efficacy outcome measures in this field. This will help evaluate molecular targets more robustly with fewer trials and may help us evaluate promising technologies in a more cost-effective manner. Requiring all diagnostic and therapeutic trials that receive funding to have these types of outcomes and measures will focus investigators.

Scientific gaps. The plethora of basic science data accumulating about placental development and the interface between mother and fetus must be linked clearly

to diagnostic and/or therapeutic needs. In addition, because there is not perfect animal model, there needs to be a way to study these on human placental models as often as possible. Another clear gap in much of the therapeutic research in pregnancy is a lack of long-term follow up of the infant. Ensuring funding for follow-up studies, including potential "-omic" studies, of the offspring should be a priority.

Evolving technologies for placental specific therapeutic drugs

Trophoblastic nanovesicles, miRNA, and their function (Yoel Sadovsky, MD, Magee-Womens Research Institute)

Background. Coordinated communication among cells and tissues is critical for development, homeostatic function, and adaptation to change. The trafficking of information between the fetoplacental and maternal compartments is essential for fetal development, growth, and pregnancy health. It also serves to reduce maternal-fetal conflicts and to balance biologic resources for the benefit of the 2 organisms. In past years, it was believed that communication between the feto-placental unit and the mother was executed by hormones and growth factors, which are soluble in the blood or are bound by protein carriers, and serve as paracrine or endocrine signals that control pregnancy health. It is now clear that nucleic acids, and particularly sRNA molecules, traffic among tissues and impact cell biology.

MiRNAs and extracellular nanovesicles. A major class of these noncoding RNAs, and 1 of the best studied, is the family of small regulatory RNAs called miRNA. MiRNAs originally were described in the nematode Caenorhabditis elegans and were later found in the genome of many organisms, including humans. They are single-strand RNA molecules of 20-24 nucleotides that characteristically repress gene expression by guiding an RNA-induced silencing complex to a target RNA, with subsequent attenuation of gene expression by the inhibition of mRNA translation and/or mRNA degradation.

FIGURE 14

Schematic of extracellular microRNAs derived from human trophoblasts

MicroRNAs can be released from the trophoblast layer in different forms: microvesicle-enveloped form; apoptotic body—enveloped form; nano-sized, exosome-encapsulated form; and RNA-binding, protein-bound form. Exosomes are formed by budding in intraluminal vesicles to form multivesicular bodies. Multivesicular bodies fuse with the plasma membrane and release their intraluminal vesicles as exosomes into the extracellular space. In contrast, microvesicles are produced directly by budding and the detachment of membrane vesicles from the plasma membrane. Apoptotic bodies (blebs) derive from cells that are undergoing apoptotic fragmentation and the formation of membrane-enclosed vesicles.

miRNA, microRNA.

Reprinted with permission from Ouyang et al.396

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

It has become clear recently that, in addition to the effect of miRNAs on intercellular gene regulatory networks, miRNAs are also packaged within extracellular vesicles, where they can be trafficked to local and distant cell types (Figure 14). Transport within extracellular vesicles may provide substantial advantages, including stability, intravas-cular processing, and selective delivery to target cells.373-375 Diverse cell types (eg, hematopoietic cells, mast cells, platelets, neurons) produce and release extracellular vesicles, which can be found in the blood, urine, saliva, breast milk, amniotic fluid, ascites, cerebro-spinal fluid, bile, lymph, tears, and semen.376-379 These vesicles are characterized by their size, shape, and con-tent.380 Importantly, when released to

the extracellular space, these vesicles may target local and distant cell types, and release nonhormonal signals that control physiologic condition, determine disease risk, and even harbor therapeutic po-tential.381-384 The major types of extracellular vesicles include apoptotic blebs, microvesicles, and exosomes, each produced by different pathways. Apoptotic blebs (500-4000 nm in diameter) are formed during partial or complete cell death and disintegration and contain cell organelles and cytoplasmic proteins that are subsequently cleared from the

circulation by macrophages.379,385,386

Microvesicles (100-1000 nm in diameter) originate from outward budding and fission of the plasma membrane, where microdomains and associated protein cargo are enveloped for direct

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Schematic depicts the exosome-mediated induction of viral resistance

Chromosome 19 microRNA clusters were transferred to recipient cells. Primary trophoblast cells release exosomes that contain chromosome 19 microRNA clusters, which are taken up by recipient cells, thereby mediating chromosome 19 microRNA cluster—dependent autophagy. Incoming viral particles (red) are likely trafficked in endocytic vesicles from the endosomal pathway into preexisting autophagosomes, which then fuse with lysosomes to form autolysosomes, as a mechanism to degrade these virus-containing vesicles. The gray arrows indicate ••••.

AL, autolysosomes; APs, autophagosomes; C19, chromosome 19 microRNA clusters; EXO, exosomes. Reprinted with permission from Delorme-Axford et al.420

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

387 391

cellular exit. - Exosomes (40-150 nm

in diameter) originate from the intracel-

lular endosomal network through serial

processes that are initiated by the

enrichment of endosomal membrane

complexes by tetraspanins and other proteins. ' This is followed by budding of intraluminal vesicles within multivesicular bodies (MVBs) and sub-

sequent movement to the plasma membranes, where multivesicular bodies fuse

with the cell membrane to release

actively intraluminal vesicles as extra-

cellular exosomes.379,394 Among all

extracellular vesicles, exosomes have been studied extensively in recent years because of their ability to modulate target cell physiology, immune function,

and carcinogenesis. Exosomes also harbor miRNAs as part of their

373,396,367

cargo.

We and others recently have characterized the trophoblastic miRNA landscape and the potential role of trophoblastic miRNA in fetoplacental to maternal communication.395-399 Whereas exosomes are found in the circulation of pregnant women, little is known about trophoblastic exo-somes. Many studies on extracellular vesicles in pregnancy have associated a mixture of vesicles (exosomes, microvesicles, apoptotic blebs) with disorders of pregnancy, thus blurring some the distinct properties of these vesicles.400-404 Placental exosomes have

been implicated in several discrete functions, such as immune modulation,405-407 where exosomal cargo proteins, such as galectin-3408 and human leucocyte antigen-G,409 may perform an immune modulatory function. 410 Other exosomal cargo molecules, such as Wnt/ b-catenin, fibronectin, and prostaglan-dins, may play a role in supporting ho-meostasis throughout pregnancy.411-416 We have shown that the miRNA landscape within the cargo of human trophoblastic exosomes is nearly identical to that of villous trophoblasts.398 The most abundant trophoblastic miRNA species are those derived from the chromosome 19 miRNA cluster (C19MC), 398,417 which is the largest miRNA cluster in the human genome, spanning approximately100 kb of genomic DNA and harboring 46 intronic miRNA genes that express 58 miRNA

species.398,416,418,419 Importantly, we

found that, in comparison with non-trophoblastic cells, primary human tro-phoblasts are resistant to infection by diverse types of DNA and RNA viruses, which include clinically relevant species such as herpes simplex virus type 1, rubella, HIV, and cytomegalovirus, but not the nonviral pathogens Listeria

monocytogenes and Toxoplasma gon-dii.395,420-422 A remarkable finding was

that the viral resistance could be transferred to other, nonplacental primary cells and cell lines by exposing these cells to a medium that was preconditioned by primary human trophoblasts or to exo-somes that are produced in human trophoblasts (Figure 15). We also found [F1, that this effect was mediated, at least in Q16 part, by exosome-packaged C19MC miRNAs. 395>398 Cells transfected with a bacterial artificial chromosome that harbors the C19MC locus or with individual, highly expressed C19MC miR-NAs also became more resistant to viral infections. Our data showed that this effect of exosomal C19MC miRNAs was mediated by the stimulation of auto-phagy in recipient cells and that phar-macologic or genomic inhibition of autophagy diminished the conferral of antiviral response to recipient cells.395 Although C19MC miRNAs likely act in concert with other antiviral responses,

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our data pointed to a unique and trans-ferrable antiviral response that is mediated by human trophoblast-derived exosomes.

Although the mechanisms, dynamics, and targets of miRNA-containing trophoblastic exosomes remain unknown, our recent human and mouse data suggest that trophoblastic C19MC miRNAs are transferred primarily from trophoblasts to maternal tissues. This is plausible, because trophoblasts are bathed directly in maternal blood yet separated from the fetal blood by a basement membrane and endothelial cells. Together, these analyses shed new light on miRNA-based communication among the fetoplacental and maternal compartments. Importantly, our data suggest an extraordinary means of nonhormonal communication between the placenta and the maternal-fetal compartments, which suggests a critical role for miRNAs in pregnancy health.

Current opportunities. Although it is clear that the plasma contains several types of extracellular vesicles, their molecular and metabolic cargo are largely unknown. Moreover, our understanding of the function of these vesicles is rudimentary. The formation of the placenta during pregnancy adds a new source of extracellular vesicles to the maternal blood stream and possibly to the fetal compartment. Whereas C19MC miRNAs are packaged distinctly within these placental vesicles, it is not clear whether there are other, placenta-specific, molecules and signals that are packaged within vesicles. Importantly, modern technology enables us to separate the different types of vesicles and to associate them with disorders of pregnancy that reflect placental function. It is also possible that the state of pregnancy changes the repertoire of extracellular vesicles that are produced by maternal tissues, thus adding to the landscape of pregnancy-related extracellular vesicles. This creates a new opportunity to catalog these vesicles, assess their origin, content, and cellular targets and to define the normal landscape of extracellular vesicles (the "vesiculome") throughout human gestation.

Future opportunities. The development of new tools to isolate extracellular vesicles efficiently may allow us to examine local and systemic targets of tropho-blastic extracellular vesicles and whether their cargo faithfully informs the health of trophoblasts. It may also allow us to identify target cells and interrogate the impact of pregnancy-specific extracellular vesicles on these cells in health and disease. Although initial work has centered primarily on maternal tissue targets, future research may focus on intraplacental paracrine targets and fetal targets of placental extracellular vesicles. These opportunities, coupled with recent developments in understanding of miRNA function, will require the development of innovative label-free technologies to collect and sort extracellular vesicles, which will shorten the time to clinical translation and potentially offer a dramatic improvement in our ability to assess placental health dynamically throughout pregnancy using maternal blood tests. Knowledge in this field may also stimulate the development of nanovesicles as carriers of noncoding RNAs, drugs, and chemicals that selectively target placental cells for therapeutic purposes.

Scientific gaps. The current and future opportunities that were summarized require us to bridge pertinent knowledge gaps. We must understand the process of cargo loading to diverse trophoblastic vesicles and the potential selectivity of this process. This should be followed by analysis of the mechanisms used by trophoblastic exosomes to enter target cells selectivelyand impact their biologic condition. The current state of perinatal science raises additional questions: Do surface and cargo proteins within trophoblastic extracellular vesicles play a role in this process? What determines targeting by extracellular vesicles? Can fetal vesicle-bound or free miRNAs and other types of RNA molecules cross the placenta into the maternal circulation and inform on the health of fetal tissues? How early in gestation can we find and isolate placental extracellular vesicles and RNA from maternal blood? The answers to these and related questions

will greatly impact the field of placental biology and its translation to pregnancy diagnostics and therapy.

Aberrant regulation of myometrial contractility by maternal cell-free plasma (CFP) RNA of placental origin: screening and therapeutic implications (Carl P. Weiner, MD, University of Kansas School of Medicine) Background. The CFP transcriptome is altered by numerous disorders, often reflecting the stage of disease development.423 Although the biologic roles of these coding and noncoding RNAs are unclear, they are not simply cellular debris. Synthesized and released by a range of cells that include human cho-rionic villi, their biologic stability and target tissue specificity are thought to reflect their release within either exo-somes or shedding vesicles or attached to argonaute 2 proteins.424 Cell-to-cell transfer and their ability to function once transferred are shown in studies that used mRNA/miRNA-loaded extracellular vesicles and protein com-

plexes.425-427

Based predominantly on studies of symptomatic women, spontaneous pre-term birth (sPTB) is considered an inflammatory process.428 We measured both the mass restricted (MR) score and IL-6 in 1004 consecutive amniotic fluid samples at 16 weeks from healthy pregnancies with known outcomes (unpublished data). Ten percent delivered at <37 weeks gestation; 4% delivered at <32 weeks gestation. Nineteen percent had an MR score of >1; 2% had an MR score of 3 or 4, which indicated significant inflammation. However, there was no relationship between either sPTB or preterm premature rupture of membranes and the MR score; 23 of 27 deliveries at <28 weeks gestation occurred to women with an MR score of 0. IL-6 levels increased as the MR score rose (0 = 102 pg/mL; 3 = 451 pg/mL, and 4 = 706 pg/mL) but was unrelated to subsequent sPTB. Whatever the role of inflammation in preterm or term birth, it seemingly occurs after 20 weeks gestation.

We proposed in 2011 that mRNA and miRNA were candidate mechanisms for

the regulation of myometrial quiescence activation (unpublished data) and have been testing the hypothesis that CFP RNAs enable communication between mother and pregnancy and that, as a direct result, changes in the maternal CFP transcriptome predict both the occurrence and mechanism of sPTB.

Methodologic refinements. In 2007, we developed an extraction method that yielded increased quantities of both CFP nucleic acids and proteins in a single process. The typical yield of total RNA extracted is 18-35mg/mL plasma, enabling comprehensive, transcriptome wide study with the use of multiple microarrays and/or next-generation sequencing, plus the confirmatory po-lymerase chain reaction studies all on the same patient plasma sample.

To speed marker validation and facilitate translation into the clinical arena, we developed a series of techniques that included economical, high-throughput gene quantification that allows for the running of panels of genes to predict sPTB within a few hours. The method allows us to run all controls necessary for marker quantification in a single polymerase chain reaction well.

The CFP transcriptome is altered in women destined for sPTB by 16 weeks. Using longitudinally obtained banked plasma samples from women with known pregnancy outcomes and microarray/next-generation sequencing, we identified and confirmed >90 novel CFP RNAs in the plasma of women who were destined for sPTB between 26-32 weeks gestation; some of the marker expressions were altered as early as 16 weeks gestation. In a second cohort, we found that several markers that were abnormal at 16 weeks gestation were also abnormal at 12 weeks gestation. We then determined that 5 CFP markers were known from studies of other cell systems to interact with 7 myometrial gene/gene products belonging to a previously identified cluster of genes: the preterm initiator set.429 Five of the 7 myometrial genes were involved with the regulation of intracellular calcium.

CFP RNAs originate from the placenta. We next sought the anatomic origin of the 5 CFP RNA markers; each was dramatically overexpressed in the placenta of women with <33 sPTB weeks gestation, but not the placenta of women delivered prematurely absent labor and of women delivered at term in labor.

CFP RNAs markers of sPTB can regulate myometrial contractility. We then focused on 1 particular CFP mRNA biomarker, apolipoprotein, that, in silica, interacted with the interferon gamma receptor, which in turn interacted with 4 additional preterm initiator genes. Overexpression of apolipoprotein in human pregnant myometrial cells increased both intracellular calcium flux and cell contractility. The addition of interferon gamma receptor siRNA blunted the stimulatory effect of apolipoprotein, which demonstrated that interferon gamma receptor was at least 1 of the targets of the apolipoprotein mRNA.

Current opportunities. These findings support the often stated premise that sPTB is a result of placental dysfunction. Specific CFP RNAs are released from the placenta of women who are destined for sPTB at <33 weeks gestation by the end of the first/beginning of the second trimester that interfere with myometrial quiescence, thus potentially creating an environment that favors sPTB. The early pregnancy time frame suggests that effective therapy will require early detection and initiation, which is consistent with the general failure of available tocolytic agents. It highlights the preeminent importance of elucidating the events that are involved in human implantation and early placen-tation. As such, the sPTB studies described may serve an investigational paradigm for the other great pregnancy syndromes such as preeclampsia and hypoxia that are associated FGR and perinatal brain damage.

The first task along the path to understanding human placentation is descriptive: the creation of an appropriately sized research medical center network for sample processing (using

new and established biobanks) for the rapid construction of an "omics" atlas of "normal" from the time period covering implantation through at least 16 weeks gestation. Such an atlas would serve as a basis for all future research and generate a host of new targets for hypothesis-based testing in vitro using systems similar to those described earlier.

Future opportunities. Real-time assessment of the human placenta all but requires noninvasive methods that may not exist presently or have not yet been applied to the study of the placenta. So much about human placental function is shrouded by darkness that the areas for investigation are almost unlimited, each with the potential for making a contribution. Yet, it is highly likely that much placental dysfunction has its foundations set by the early second trimester and that it is here that new insights are needed desperately. The application of newer organ on chip technology could allow the testing of potential interventions that will be identified during atlas con-struction.430 Real-time approaches will require either new imaging modalities or be "omics" in nature. As discrete markers of placental function linked to specific disease phenotypes emerge, it may be possible to reduce the processing from a minimum of a laboratory work day to less than a few hours, providing near real-time feedback for nonimaging modalities.

Scientific gaps. There are presently no targeted pharmacologic therapies for placental abnormalities. The fact that a placental-derived CFP RNA that is unique to women who are destined for sPTB at <33 weeks gestation modulates a myometrial gene that is associated with sPTB and that its inhibition with siRNA reverses the up-regulation demonstrates that therapeutic targets can be identified with current transcriptomic approaches. It is likely that attempts to address placental dysfunction after its establishment will be of modest impact. Future efforts should build on the knowledge derived from atlas construction to seek targeted therapies that enhance implantation and early placental development.

Nanoparticles for placental drug delivery (Erik Rytting, University of Texas Medical Branch, Galveston)

Nanomedicine. The field of nano-medicine includes nanoparticles for medical diagnostics, nanoparticles for therapeutic delivery, and particles playing both roles, referred to as theranostic nanoparticles (Figure 16).431 Types of nanoparticles that are used in therapeutic delivery include liposomes, solid lipid nanoparticles, polymeric nanoparticles, polymeric micelles, dendrimers, and polymeric DNA or siRNA complexes called polyplexes.432 Advantages of nanoparticles for drug delivery include improved bioavailability, protection of therapeutic payload, controlled release, and increased drug targeting effi-ciency.433 Targeting ligands such as antibodies, peptides, aptamers, or small molecules can be conjugated or adsorbed onto the surface of nanoparticles to promote the accumulation of the particles in specific regions or tissues that are facilitated by binding of the targeting ligands to a particular receptor.434

Nanoparticle characterization and bio-compatibility. Characterization of drug-loaded nanoparticles typically includes assessments of particle size, zeta potential (surface charge), encapsulation efficiency, and drug release kinetics. Particle size and surface charge can have a significant effect on both particle distribution and cellular uptake. Encapsulation efficiency represents the mass of drug actually encapsulated within a nanoformulation as a percentage of the intended drug loading. The drug release profile will demonstrate whether sustained delivery of the drug from the nanoparticles can be anticipated, which can reduce dosing frequencies. It has been demonstrated that drugs and proteins are still pharmacologically and

biologicallyactive after their release from nanoparticles.435,436

Biocompatibility is an important consideration in the development of nanoparticles for therapeutic delivery. The nanomaterial excipients themselves must not elicit toxic responses such as inflammation or oxidative stress. Many of the polymeric nanoparticles

FIGURE 16

Translational steps for the development of nanoparticles for placental drug delivery

In Vivo

Nanoparticles may contain therapeutic, diagnostic, and/or targeting components. In vitro, ex vivo, and in vivo models will be used to investigate proof of concept and safety before clinical trials.

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

developed in our laboratory are based on lactic acid polymer. When this polymer breaks down within the body, the so-called degradation product is lactic acid, a naturallyendogenous compound.

Transport of nanoparticles across the placenta. To predict placental drug and nanoparticle transport, our laboratory focuses primarily on 2 experimental models: the BeWo b30 human tropho-blast cell line and the dually perfused human placental lobule. Our comparative study demonstrated excellent correlation between the in vitro BeWo cell line model and the ex vivo perfused human placenta model in the prediction of the transplacental transport of 4 model compounds.437 A separate study then expanded the comparison with placental transport data in the literature and reported a strong correlation (correlation coefficient = 0.95) between the in vitro relative transfer rate and the

experimental models appear to be suitable for nanoparticle transport studies as well. For example, the transplacental transfer of polymeric nanoparticles has been shown to be size-dependent in both models, and limited transfer of silica nanoparticles was demonstrated in both BeWo cell and placental perfusion ex-

439-441

periments. 441

Additional studies have shown that gold nanoparticles do not cross the term human placenta, that the transplacental transport of poly-amidoamine den-drimers is limited, and that liposome transport across the placenta is size-dependent.442-444 The observed size dependence of nanoparticle transport has been extended to demonstrate increased drug transport across placental trophoblast cells when the drug is encapsulated in smaller polymeric nanoparticles (diameter, 146 nm) compared with larger nanoparticles (diameter, 232 nm).445 Literature re-

ex vivo transfer index.438 Both ports of nanoparticles that have been

administered to pregnant rodents have included titanium dioxide nanoparticles, silica nanoparticles, zinc oxide nano-particles, iron oxide nanoparticles, and gold nanoparticles; results vary depending on particle size, surface charge, and

446-450

gestational age.

Targeting strategies. Strategies for targeted delivery of nanoparticles to the placenta include the identification of peptides by phage display.451 Researchers have reported enhanced transcellular transfer of T7 bacteriophages across BeWo cells with an integrin-binding amino acid sequence peptide (unpub-Q18 lished data). Harris et al used a similar approach in pregnant mice and identified two peptides with increased placental binding, designated as amino acid sequence consisting of CRGDKGPDC and KRK (unpublished Q19 data).452 Kaitu'u-Lino et al453 designed bacterial-derived nanocells that are coated with antibodies to epidermal growth factor receptor. The uptake of these epidermal growth factor receptor—targeted delivery vehicles in ex vivo human placental explants was greater than that of nontargeted nano-cells. We have synthesized polymeric nanoparticles conjugated with folic acid to take advantage of the relatively high expression of folate receptors in the placenta. This has resulted in greater cellular uptake and transport of the nanoparticles across BeWo cells.

Current opportunities. Immediate applications of these technologic advances include fetal drug therapy and placental therapy. Although medication during pregnancy is most often prescribed to treat maternal conditions, there are a number of fetal diseases for which a targeted, transplacental delivery strategy potentially could attenuate the adverse maternal side-effects that are associated with current treatment modalities. These include fetal cardiac arrhythmias, congenital adrenal hyperplasia, and fetal thyroid disorders.454 Targeted drug delivery to the placenta itself could also improve therapeutic options for malaria-infected placenta or prevent mother-to-child transmission of HIV.455

Future opportunities. Nanotechnology has also been suggested for placental diagnostic applications.456 Opportunities abound for the development of nanoparticle-mediated biosensors and imaging agents, and it is likely that many recent advances in cancer diagnostics, for example, can be utilized to monitor the structure and function of the human placenta.457 As progress towards these goals continues, it is imperative that the biocompatibility of such nanomaterials is investigated thoroughly to prevent unintended consequences of fetal exposure to any potentially harmful imaging agents. For example, gadolinium-based magnetic resonance imaging contrast agents are not recommended for use during pregnancy because of concerns for nephrogenic systemic fibrosis.458

Scientific gaps. Steps to translate the preclinical promises of this technology towards clinical trials and clinical practice will include scaling proof-of-concept results from in vitro and small animal studies to larger animal models with placental structure and function more similar to human placenta. For example, recent progress has been made to establish the pregnant baboon as a nonhuman primate model for pharmacokinetic investigations.459-464 There is also a need to refine the pool of potential placental targeting moieties to minimize off-target nanoparticle distribution. Side-effects can be averted when the disbursement of medication to off-target sites is reduced. A necessary, but exciting, task will be to determine the magnitude of dose reductions that are possible when targeted therapeutic strategies are applied. Nanoparticle-mediated delivery to the placenta offers tremendous benefits for maternal-fetal medicine.

Maternally sequestered delivery systems for prevention of fetal drug exposure (Gene Bidwell, University of Mississippi Medical Center)

Background. Preeclampsia is a common hypertensive disorder of pregnancy and is one of the leading causes of maternal and fetal morbidity and death. There is currently no effective intervention for

preeclampsia short of induced delivery, which makes it a leading cause of premature birth. Improvements in preeclampsia management have been largely stifled because of deleterious effects of proposed small-molecule therapeutics on the developing fetus. The objective of our studies was to develop a drug delivery system capable of stabilizing novel therapeutic agents in the maternal circulation while protecting them from entering the fetal circulation.

The use of a biopolymer drug carrier to prevent drug transfer across the placenta. The major focus of our research group is to develop drug carriers capable of preventing placental transfer and fetal exposure to therapeutic agents. Protein transport across the placental barrier is highly regulated, and proteins that are not substrates for active transporters rarely cross into fetal circulation. To take advantage of this, we are using a protein-based biopolymer to fuse to therapeutic agents and sequester them in the maternal circulation (Figure 17). This biopolymer, termed elastin-like polypeptide (ELP), is based on a repeating sequence found in human elastin.465 ELP is an ideal drug carrier for several reasons. Because of its origins in human elastin, it is not recognized as foreign by the immune system, does not induce an inflammatory response or immunoge-nicity, and is likely slowly broken down into individual amino acids in vivo, making it completely biocompatible. Because the ELP sequence is encoded at the DNA level, modification of the polymer sequence (to tune the polymer size, to add reactive sites for drug conjugation, or to fuse targeting agents, therapeutic peptides, or therapeutic proteins) is a matter of simple molecular biology.466-468 Also, because it is protein-based, ELP can be produced by recombinant expression in bacterial or eukaryotic systems,463'464'469 and it is easily purified by a nonchromatographic procedure called inverse transition cycling.464,470 This makes scale-up to production levels that is required for therapeutic development very feasible. Finally and most importantly, because of its large size, ELPs can protect small

peptide or drug cargo from rapid degradation and extend their plasma half-life and bioavailability.

Our strategy involves the use of a core ELP biopolymer as a drug carrier; this biopolymer is modified at its amino and/or carboxy-termini with targeting agents, cell-penetrating agents, peptide or protein therapeutics, or reactive sites for drug attachment (Figure 17, A). In a recent study, we examined the pharmacokinetics and biodistribution of ELPs in a rat pregnancy model. The ELP carrier accumulated at very high levels in the maternal kidney, liver, and placenta after systemic intravenous infusion, but little to no ELP crossed into the fetus after either a bolus administration or 5 days of continuous infusion (Figure 17, B).471 This study indicates that the ELP drug carrier may be a powerful means of sequestering fused therapeutics to the maternal circulation and preventing fetal drug exposure.

Mechanisms of symptom onset in pre-eclampsia and potential therapeutic interventions. Preeclampsia is a disease of placental insufficiency that is rooted in a poorly understood process of impaired spiral artery remodeling during early placental development.472-474 Because the molecular basis for this failed remodeling is understood poorly and because a robust biomarker to predict patients who will become preeclamptic is lacking, it is not yet practical to design targeted therapeutics to correct spiral artery remodeling and prevent preeclampsia. However, preeclampsia symptoms do not present until later in pregnancy, and, unlike the initiating factors in spiral artery remodeling, the molecular pathways that lead to symptom precipitation are much better understood. Therefore, now is the optimal time to develop therapeutics that are targeted to the progression of pre-eclampsia with the goal of extending pregnancies and improving fetal out-comes.475'476 The overall goal of our approach is to target the pathways that lead to hypertension, impaired renal function, neurologic complications, and other components of the preeclampsia syndrome in hopes of delaying the need

FIGURE 17

Model of the elastin-like polypeptide drug delivery vector

Organ Targeting Peptide or

Cell Penetrating Peptide

Elastin-like Polypeptide

Therapeutic Protein, Therapeutic Peptide, or

Drug Binding Domain

B ft Mr . ™ .-yJlK M

gj 9 g

Elastin-like polypeptide is a biocompatible protein polymer that is used to protect attached cargo from degradation, rapid renal clearance, and immunogenicity to prevent the transfer of cargo across the placenta. A, Elastin-like polypeptide is modified with targeting agents or cell penetrating peptides Q42 to enhance organ specificity or to mediate target cell uptake. Elastin-like polypeptide is also modified with cargo therapeutic peptides, therapeutic proteins, or with reactive sites for covalent attachment of small molecule drugs. B, Placental transfer of elastin-like polypeptide. Quantitative fluorescence analysis revealed that the elastin-like polypeptide carrier accumulated highly in the placenta (redand yellow) but did not penetrate into the fetal circulation after bolus injection or chronic infusion in a rat pregnancy model.

Reprinted with permission from George et al.471

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

for induced delivery for as long as possible; we aim to do so in a manner that protects the fetus from exposure to the therapeutic agents.

The molecular pathways that lead to the symptom precipitation of symptoms in preeclampsia are a consequence of placental hypoperfusion and hypoxia. In response, the hypoxic placenta releases a multitude of factors that affect the mother at nearly all organ systems.477 Briefly, the major players in the progression of the preeclampsia syndrome can be divided into 3 groups, antiangiogenic factors, proinflammatory factors, and inducers of ROS (Figure 18).474 These factors induce endothelial dysfunction and systemic inflammation in systemic vascular beds of the maternal circulation that ultimately lead to the clinical manifestations of preeclampsia.

One of the major proteins produced by the hypoxic placenta thought to drive maternal preeclampsia symptoms is sFlt-1. SFlt-1 levels were found to be elevated dramatically in patients with preeclampsia with a corresponding reduction in the plasma levels of its ligands-VEGF and PlGF.478 VEGF is known to be important in endothelial cell health, and its sequestration by sFlt-1 binding is hypothesized to lead to many of the renal and vascular effects of pre-eclampsia.479 This makes sFlt-1 one of the major targets for preeclampsia drug development. One strategy we are using is to supplement VEGF levels by administering an exogenously prepared ELP-VEGF fusion protein.474 We have previously found that VEGF signaling activity is maintained after ELP fusion,464 and our goal with this strategy is to restore the angiogenic balance that

Review Obstetrics

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3701 > <=8

3702 c

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FIGURE 18

Model for the role of antiangiogenic factors and inflammatory cytokines in preeclampsia.

The ischemic placenta is known to be a source of the vascular endothelial growth factor antagonist protein sFlt-1 and many proinflammatory cytokines. These factors cooperate with 1 another to produce systemic inflammation and endothelial dysfunction in the mother, which is manifested clinically in multiple organ systems. Illustrated are our proposed strategies for interfering with these processes by either supplementing vascular endothelial growth factor or placental growth factor levels by the administration of exogenous elastin-like polypeptide-fused proteins or by giving elastin-like polypeptide—stabilized inhibitors of NF-kB.

ELP, elastin-like polypeptide; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; sFlt-1, soluble fms-like tyrosine kinase; VEGF, vascular endothelial growth factor.

Adapted with permission from Bidwell and George.477

Hekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.

was lost when sFlt-1 production was increased. In addition to the reduction of systemic endothelial dysfunction, this strategy might have the added benefit of increasing placental perfusion by enhancing uterine or spiral artery blood flow. We are also examining a related strategy of supplementing PlGF by administering an ELP-PlGF fusion

protein. This has the potential to be safer and easier to dose strategy than direct VEGF administration. The hypothesis is that the exogenous ELP-PlGF will bind sFlt-1 as well as the full length cell surface receptor Flt-1,480 thereby displacing VEGF and making it available to bind the more active receptor VEGFR2. Because sFlt-1 has no known signaling function

and VEGFR1 is much less active than VEGFR2,481 supplementation of PlGF may be less prone to side-effects compared with the direct administration of VEGF.

Other major drivers of preeclampsia symptoms include the chronic systemic inflammatory response (exacerbated by placental production of inflammatory cytokines).482 The inflammatory response in preeclampsia is known to be mediated by tumor necrosis factor-a,483 proinflammatory interleukins,484 and agonists of TLR signaling485 that is produced in the placenta and possibly by systemic macrophages and/or endothelial cells. A common mediator of all these inflammatory signaling pathways is the transcription factor NF-kB. Another strategy that we are exploring is the examination of NF-kB as a potential drug target for preeclampsia. As a first attempt, we have generated an ELP-delivered peptide inhibitor of NF-kB signaling. The ELP fusion protects this peptide from rapid renal clearance after intravenous administration, enhances its placental deposition over 30-fold relative to free peptide, and prevents its transfer across the placenta (unpublished data). We hypothesize that a maternally sequestered ELP-fused NF-kB inhibitor will be effective as an antiinflammatory agent and may slow the progression of preeclampsia.

Current opportunities. The use of ELP or related macromolecular carriers to prevent fetal drug exposure represents a promising new strategy for drug development for disorders of pregnancy. As described earlier, we are using ELP fusions with therapeutic peptides or proteins targeted to pathways of importance in preeclampsia, but this strategy is not limited to proteinaceous therapeutics or to preeclampsia. ELP is amenable to fusion with small molecule drugs, and other projects in our laboratory are examining the ability of ELP to prevent transfer of known-toxic or teratogenic drugs across the placenta, thus potentially opening the door for safe use of drugs that currently are avoided in the pregnant patient population.

Future opportunities. Our current strategy is to use the ELP system to intervene in late gestation, with therapeutics that are targeted to pathways of currently known importance in preeclampsia. Future work will expand this strategy as the list of potential drug targets continues to grow. The ELP system is adapted easily for the delivery of nearly any type of therapeutic, so expanding this strategy to target newly identified causative pathways is highly feasible. We view the ELP system as a platform for maternal sequestration of therapeutics, and its attractive properties as a drug carrier make it a promising platform to be applied for delivery of novel therapeutics.

Scientific gaps. Although we are focused on treating symptoms of preeclampsia during late gestation, the ideal scenario would be to intervene early in pregnancy, during trophoblast invasion and placentation, to prevent the onset of preeclampsia. However, to do that, we need to gain a better understanding of the causative factors that hamper proper placental formation in preeclampsia, and we need robust biomarkers to identify patients who will experience the disorder.

Another area that currently stands as a hurdle to the development of therapeutics for preeclampsia and other disorders of pregnancy is the high level of risk aversion when treating pregnant mothers. This risk aversion is well justified given the vulnerability of the patient population, and we have to address these risks by doing all we can preclinically to ensure the safety of developmental therapeutics. One strategy for this is to prevent fetal drug exposure using drug carriers, as described earlier. In addition, we must also strive to ensure that our therapeutics is not adversely affecting normal placental function. To address these issues, we must use the best pre-clinical models at our disposal to study placental drug transport, fetal exposure, and drug effects on normal placental function, and we must follow offspring in our preclinical models to insure normal physical, mental, and physiologic development. Only after collecting

robust data in these areas should we attempt to begin testing in human pregnancies. Although we should proceed with caution and use every means at our disposal to ensure safety, we should not let fear of doing harm stifle innovation in the development of diagnostics and therapeutics for use in pregnant patients.

Comment

Exciting research was presented at the workshop in the area of placentation, trophoblast migration, and spiral artery remodeling. Research findings in these areas will be critical for developing future treatments and, ultimately, the prevention of adverse pregnancy outcomes of placental origin that include preeclampsia and FGR. Knowledge of the basic biology of trophoblast invasion and spiral artery remodeling is critical to understanding the cause of many pregnancy disorders; once the molecular pathways that go awry are identified, it will be possible to design therapeutics to intervene in these pathways. However, this goal also elucidates another major gap in the field, the need for robust biomarkers to predict the onset of the disease early in pregnancy. Future research in these areas will be critical for filling this gap.

Data were also presented regarding ongoing clinical efforts in the United States and in Europe that are testing novel interventions for preeclampsia and FGR, including agents such as oral arginine supplementation, sildenafil, pravastatin, a virally delivered VEGF for local administration into the uterine artery to improve blood flow and oxygen supplementation therapy. Proposals were also made to improve fetal health, not by targeting maternal blood flow but by enhancing nutrient transport to the fetus by modulating glucose and amino acid placental transporters. Strategies were presented for inhibiting mito-chondrial dysfunction and oxidative stress to improve placental health; the roles of inflammation on both tropho-blast function and in the advanced stages of preeclampsia were discussed. The roles of miRNAs and placentally derived exosomes were discussed in the context

of their use for diagnostics and as drug targets. These diverse pathways illustrate many opportunities for drug intervention. Although a number of potential drugs to target these pathways are available and have been shown to be relatively safe, the therapeutic benefits of the majority of these drugs were never studied in pregnancy. Hence, the safety and pharmacokinetic profiles of these and new therapeutics will need to be determined before their use in pregnancy. The workshop discussed this aspect highlighting the unique pharmacokinetic properties of pregnancy and the hurdles and pitfalls of translating research findings into practice. The workshop concluded with discussions of drug delivery during pregnancy. The potential for using nanoparticles or protein biopolymers during pregnancy was presented, either to prevent fetal drug exposure by blocking placental transfer or to encourage drug delivery to the fetus for in utero therapy by attaching agents that actively are transported by the placenta. The use of macromolecular carriers to prevent placental drug transfer and fetal drug exposure represents an exciting new strategy for drug development in pregnancy. Knowledge that the fetus will not be exposed to the therapeutic agent will open the door for use of many drugs that currently are avoided during pregnancy and will help lessen the regulatory burden on drug development for pregnancy-related disorders.

The pregnant patient population may be 1 of the most challenging cohorts for which to develop drugs because of concerns regarding drug effects on fetal outcome. Placental drug transfer is a very real risk for many small molecule drugs and even for some actively transported biologics. The potential for adverse fetal effects not only represents a concern to pregnant mothers but also stifles drug development because of risk aversion from the pharmaceutical industry and from regulatory bodies. For many therapeutics, we simply do not know the risk to pregnant mothers or to their developing offspring because pregnancy is most commonly excluded from clinical trials. Of course, intervention during this vulnerable

developmental period must be approached with extreme care. We must use the best preclinical models at our disposal to maximize the chance of elucidating adverse drug effects for novel test agents. Also, the use of drug delivery strategies to target the placenta specifically and/or prevent placental drug transfer and fetal drug exposure is an exceptionally promising method of the development of new and safe therapeutics for use in pregnant women.

In closing, a major theme that clearly developed from the workshop was that we, as a scientific community, need to stop thinking of pregnant women as a vulnerable patient population for which drug development should be avoided and begin to appreciate our opportunity to improve both maternal and fetal health. This strategy could not only have an immediate benefit on pregnancy outcome but also have a long-term benefit on the future health of the infant, because recent evidence has made it clear that the risks for many health outcomes, which include the development of cardiovascular diseases,486 metabolic diseases,487 and cognitive developmental disorders,488 just to name a few, are strongly programmed by the in utero environment. ■

ACKNOWLEDGMENTS

We acknowledge Tamika Turner-Graydon for her major contribution in the preparation and editing of the manuscript and Rosalina Bray for her excellent assistance in organizing the workshop meeting.

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