Placental Origins of Adverse Pregnancy Outcomes: Potential Molecular Targets

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OBSTETRICS 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 Tsilou, MD; Susan Fisher, PhD; Vikki M. Abrahams, PhD; Michael J. Soares, PhD; James C. Cross, PhD; Stacy Zamudio, PhD; Nicholas P. Illsley, DPhil; 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

ost adverse pregnancy outcomes M can trace their origin to the Although much progress is being made in understanding the molecular pathways in the placenta. Preeclampsia and fetal growth placenta that are involved in the pathophysiology of pregnancy-related disorders, a restriction (FGR) are disorders that are significant gap exists in the utilization of this information for the development of new drug rooted in defects of early placental therapies to improve pregnancy outcome. On March 5-6, 2015, the Eunice Kennedy development.1,2 These defects include Shriver National Institute of Child Health and Human Development of the National In- poor trophoblast uterine invasion, stitutes of Health sponsored a 2-day workshop titled Placental Origins of Adverse impaired transformation of the uterine Pregnancy Outcomes: Potential Molecular Targets to begin to address this gap. Particular spiral arteries to high capacity and low emphasis was given to the identification of important molecular pathways that could serve impedance vessels, and/or abnormalities as drug targets and the advantages and disadvantages of targeting these particular in the development of chorionic villi. A pathways. This article is a summary of the proceedings of that workshop. A broad number number of poor pregnancy outcomes are of topics were covered that ranged from basic placental biology to clinical trials. This associated with placental inflammation included research in the basic biology of placentation, such as trophoblast migration and because of infectious or noninfectious spiral artery remodeling, and trophoblast sensing and response to infectious and causes and include early pregnancy loss, noninfectious agents. Research findings in these areas will be critical for the formulation stillbirth, and FGR.3 Significant progress of the development of future treatments and the development of therapies for the pre- is being made in understanding the vention of a number of pregnancy disorders of placental origin that include preeclampsia, molecular basis of these disorders to fetal growth restriction, and uterine inflammation. Research was also presented that begin contemplating targeting the mo- summarized ongoing clinical efforts in the United States and in Europe that has tested lecular pathways that are involved in novel interventions for preeclampsia and fetal growth restriction, including agents such as their pathophysiologic condition. oral arginine supplementation, sildenafil, pravastatin, gene therapy with virally delivered Several potential targets could be envi- vascular endothelial growth factor, and oxygen supplementation therapy. Strategies were sioned readily. In the case of pre- also proposed to improve fetal growth by the enhancement of nutrient transport to the eclampsia, an altered balance of fetus by modulation of their placental transporters and the targeting of placental mito- circulating angiogenic/antiangiogenic chondrial dysfunction and oxidative stress to improve placental health. The roles of factors that are derived from the placenta microRNAs and placental-derived exosomes, as well as messenger RNAs, were also are believed to responsible for the sys- discussed in the context of their use for diagnostics and as drug targets. The workshop temic vascular dysfunction that is discussed the aspect of safety and pharmacokinetic profiles of potential existing and new observed in preeclampsia.4 These factors therapeutics that will need to be determined, especially in the context of the unique include an increase in the antiangiogenic pharmacokinetic properties of pregnancy and the hurdles and pitfalls of the translation of proteins such as soluble fms-like tyro- research findings into practice. The workshop also discussed novel methods of drug sine kinase 1 (sFlt-1) and soluble endo- delivery and targeting during pregnancy with the use of macromolecular carriers, such as glin, whose pathways can serve as targets nanoparticles and biopolymers, to minimize placental drug transfer and hence fetal drug for inhibition, or a decrease in the exposure. In closing, a major theme that developed from the workshop was that the proangiogenic proteins such as placental scientific community must change their thinking of the pregnant woman and her fetus as a growth factor (PlGF), whose pathway vulnerable patient population for which drug development should be avoided, but rather can serve as a target for stimulation. In be thought of as a deprived population in need of more effective therapeutic interventions. the case of FGR, the stimulation of the Key words: PlGF pathway could also be targeted as a drugs, placenta, pregnancy, therapeutics, trial

JULY 2016 American Journal of Obstetrics & Gynecology S1 Supplement Obstetrics ajog.org means to increase the number of termi- aptamers), and nucleic acid therapies chance of a successful therapeutic agent. nal villi and thus increase the available (eg, DNA gene therapy and small RNAs These include that the ideal therapeutic surface area for the improvement of (sRNAs), such as microRNAs, (miR- agent should be highly specific to a key nutrient transfer between the maternal NAs) and silencing RNAs (siRNAs).14-17 step in the targeted pathway and that it blood and the growing fetus.5 Another A major obstacle in introducing acts as far down stream as possible to potential treatment to increase nutrient novel pharmaceutical interventions to produce the desired effect, thus mini- transfer to the malnourished fetus is the improve pregnancy outcomes is based mizing unfavorable upstream-mediated stimulation of the mammalian target of on the general fear of inflicting potential cascading events. Furthermore, the rapamycin (mTOR) pathway as a means harm, particularly to the fetus, that may ideal therapeutic should avoid or mini- to increase nutrient transporters.6 In the result in either short- or long-term mize maternal and fetal systemic effects. case of placental inflammation, the nu- deleterious effects. Understandably, a Thus, selectively targeting the placenta clear factor kappa-light-chain-enhancer very cautious direction is taken, and and optimizing the dosage would be of activated B cells (NF-kB) pathway, most studies involve either the evalua- important considerations. In this regard, which is a major pathway that is involved tion of off-label drugs with a very safe placental homing molecules coupled to a in mediating the inflammatory response, history or dietary supplementation for delivery system that contains the thera- could be targeted to decrease placental use in pregnancy. Although extreme peutic agent (such as nanoparticles, inflammation.7,8 A number of drugs to caution is warranted, the current chal- synthetic peptides, liposomes, exo- target these pathways and many others lenge is to overcome the overbearing somes) and cell-specific DNA expression already exist in the market place or are reticence of doing harm that unduly vectors show exciting promise to elimi- available at the experimental stage. A hinders the development and testing of nate or minimize any deleterious collat- listing of these drugs can be obtained new and novel approaches to improve eral effects for either the mother or easily through a number of accessible pregnancy outcomes. The first step is to fetus.18 The timing of the delivery of the databases.9-13 In addition, a promising test potential drug therapies for their therapeutic agent is also another pipeline of novel therapeutics are on the safety and efficacy in animal models, important consideration because the horizon that include natural or synthetic which then, in turn, can lead to human placenta is a developing organ with antibodies, synthetic small binding studies. A number of important factors certain pathways that take critical roles at molecules (eg, peptides and nucleic acid need to be considered to improve the different developmental stages. Thus, the modulation of a particular molecular From the Pregnancy and Perinatology Branch (Dr Ilekis) and the Obstetric and Pediatric pathway at an inappropriate time win- Pharmacology and Therapeutics Branch (Dr Tsilou), Eunice Kennedy Shriver National Institute of dow may result in deleterious effects by Child Health and Human Development (NICHD), National Institutes of Health, Department of Health interfering with the normal develop- and Human Services, Bethesda, MD; Department of Obstetrics, Gynecology, and Reproductive mental trajectory. For example, villous Sciences, University of California San Francisco, San Francisco, CA (Dr Fisher); Obstetrics, maturation undergoes an orderly devel- Gynecology and Reproductive Sciences, Yale School of Medicine; New Haven, CT (Dr Abrahams); Comparative Biology and Experimental Medicine, University of Calgary Health Sciences Centre, opmental process that is orchestrated by Calgary, Alberta, Canada (Dr Cross); Institute of Reproductive Health and Regenerative Medicine and the angiogenic factors vascular endo- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas thelial growth factor (VEGF) and City, KS (Dr Soares); Department of Obstetrics and Gynecology, Hackensack University Medical PlGF.1,5,19 VEGF is involved in early Center, Hackensack, NJ (Dr Zamudio); Department of Obstetrics and Gynecology, Hackensack villous formation and drives primary 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 and secondary branching angiogenesis. Program, National Center for Advancing Translational Sciences, National Institutes of Health, This is followed by nonbranching Bethesda, MD (Dr Colvis); Department of Obstetrics and Gynecology, University of Texas Medical angiogenesis and the formation of the Branch, Galveston, TX (Dr Costantine); Department of Obstetrics and Gynecology Indiana University, tertiary terminal villi, principally under Indianapolis, IN (Dr Haas); Magee-Womens Research Institute, Pittsburgh, PA (Dr Sadovsky); the control of PlGF. Primary and sec- 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 ondary branching angiogenesis generally Neurology, University of Mississippi Medical Center, Jackson, MS (Dr Bidwell). is complete by approximately 20 weeks Received Aug. 3, 2015; revised Feb. 11, 2016; accepted March 1, 2016. of gestation, after which tertiary termi- Comments and views of the author(s) do not necessarily represent the views of the NICHD. nal villi formation predominates and continues to term.1 Thus, in a hypo- 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. thetical situation for the treatment of The 2-day workshop was held in North Bethesda, MD, March 5-6, 2015. FGR, stimulating the PlGF pathway too early (ie, before the adequate completion This supplement was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, for the benefit of the American public. of primary and secondary branching Corresponding authors: John V. Ilekis, PhD and Ekaterini Tsilou, MD. [email protected], tsiloue@ angiogenesis) conceivably could result in mail.nih.gov malformation of normal villous struc- 0002-9378/$36.00 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ajog.2016.03.001 ture and function. Another factor to consider is the required exposure time to

S2 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement the therapeutic agent to obtain the desired effect. Will the therapeutic agent TABLE 1 be required to be administered contin- Workshop session themes and their respective topics uously or only for a short duration of I. Review of placental development and function in the context of the molecular mechanisms time? In the aforementioned FGR treat- and pathways ment scenario, how long of a time period Human trophoblast differentiation and placentation is required to increase and maintain the Innate immune function of human trophoblast number of terminal villi? Once formed, Modeling trophoblast differentiation and placentation in the rat will the morphologic change remain permanent, or will the induced Modeling trophoblast differentiation and placentation in the mouse morphologic change regress if the stim- II. Potential “drug” targets of important placental molecular pathways throughout placental ulus is not continued? Therefore, speci- development in relation to pregnancy disorders fi city, dosage, delivery, timing, and Placental hypoxia as a therapeutic molecular target length of exposure are some of the key factors in the development of a success- Is fetal growth a feasible target for placental intervention? ful therapeutic agent. Targeting oxidative/nitrative stress and mitochondrial dysfunction in placenta to relieve Although much progress is being adverse pregnancy outcomes made in understanding the molecular III. Identify potentially useful or experimental drugs to target important molecular pathways pathways in the placenta that are Challenges and advantages of rescuing and repurposing involved in the pathophysiologic condition of pregnancy-related disorders, a Effect of pregnancy on drug pharmacokinetics and pharmacodynamics significant gap exists in using this in- From bench to bedside: processes and pitfalls translating research findings into practice formation for developing new drug paradigms therapies to improve pregnancy IV. Evolving technologies for placental specific “therapeutic/drug” delivery outcome. To address this concern, the Trophoblastic nanovesicles, microRNA, and their targets aberrant regulation of myometrial Eunice Kennedy Shriver National Insti- contractility by maternal cell-free plasma RNA of placental origin: screening and therapeutic tute of Child Health and Human implications Development of the National Institutes Maternally sequestered delivery systems for prevention of fetal drug exposure of Health sponsored a 2-day workshop on March 5-6, 2015, titled “Placental Novel therapeutic interventions and delivery systems Origins of Adverse Pregnancy Out- Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. comes: Potential Molecular Targets,” to discuss and reflect on placental drug targeting to improve pregnancy out- knowledge that is obtained from the placenta and uterus is governed, in comes. The workshop brought together laboratory bench for use at the bedside. large part, by the cytotrophoblast dif- leaders in the field to present and discuss A summary of the research topic that was ferentiation pathway that enables their particular area of research and covered by each participant is presented invasion.20,21 With regard to the stimulate dialogue in the context of the along with their opinions on current and anatomic arrangement, placental cyto- theme of the workshop. The goals of the future opportunities and research gaps. trophoblasts emigrate from anchoring workshop were (1) to present the state of The article is organized according to 4 villi and join cell columns that serve as the science with respect to the molecular session themes. The session themes and conduits to the uterine wall (Figure 1). mechanisms that are involved in their respective topics are listed in Within the uterus, the cytotrophoblasts placentation, (2) to identify potential Table 1. Table 2 is a key to abbreviated invade nearly its entirety, normally molecular pathways and developmental scientific terms that are used commonly stopping one-third of the way through time windows for targeting effective throughout the article. the muscle layer. Within the decidua, “drug” interventions to avoid placenta- interstitial cytotrophoblasts interact tion defects in early pregnancy and Review of placental development and with specialized populations of maternal circumvent placental defects later in function in the context of molecular immune cells that are allowed to enter pregnancy, and (3) to identify major mechanisms and pathways this compartment. During invasion, research gaps in our understanding of Human trophoblast differentiation the cells also remodel the uterine circu- placental molecular pathways that lead and placentation (Susan Fisher, lation, primarily by targeting the spiral to adverse pregnancy outcomes. This University of California San Francisco) arteries. They transform the walls of article summarizes the proceedings of Cytotrophoblast differentiation establishes these vessels. Endovascular cytotropho- that workshop. The overall objective of the anatomy of the human maternal-fetal blasts replace the endothelium and the workshop was to stimulate the interface. The complex cellular architec- intercalate within the smooth muscle research community to better apply the ture at the boundary between the cells of the tunica media. This process

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converts the originally low-capacitance/ TABLE 2 high-resistance uterine arteries into Abbreviations for commonly used scientific terms [in file but NOT high-capacitance/low-resistance chan- EDITED] nels that perfuse the surface of the Abbreviation Description placenta, which is comprised of multi- AB apoptotic bleb nucleated syncytiotrophoblast, which is a transport epithelium. Thus, they can aPL antiphospholipid antibodies respond to the ever-increasing demands APOA4 apolipoprotein a-iv of the offspring for maternal blood over C19MC chromosome 19 miRNA cluster the course of pregnancy. At a molecular level, cytotrophoblast CaO2 arterial oxygen content invasion of the uterus is as remarkable as CARD caspase activation and recruitment domain the unique behaviors that the cells þþ Ca calcium exhibit. The progenitors, which are CFP cell-free plasma attached to the trophoblast basement CO cardiac output membrane of the chorionic villi, express an adhesion molecule repertoire that is CTB cytotrophoblast typical of epithelial cells (eg, epithelial- CYP cytochrome P cadherin and integrin a6/b4). As they DAMP damage associated molecular pattern enter the columns, the emigrating cyto- DNA deoxyribonucleic acid trophoblasts undergo a stereotypical transformation. They down-regulate dsRNA double-stranded ribonucleic acid those that are typical of an epithelial EDH endothelium derived hyperpolarizing monolayer and up-regulate receptors EGFR epidermal growth factor receptor that enable invasion (eg, aV family members; Figure 2), vascular endothelial ELP elastin-like polypeptide cadherin and integrin a1/b1. Remark- EPO erythropoietin ably, the end result of this transformation FDA Food and Drug Administration is vascular mimicry in which cyto- FGR fetal growth restriction; also known as IUGR trophoblasts of epithelial origin express a broad repertoire of adhesion molecules, FOA funding opportunity announcement growth factors, ephrin receptors and GDM gestatoinal diabetes mellitus their cognate ligands (ephrins), and GLUT glucose transporter notch family members that typically are GPx glutathione peroxidase associated with endothelium and the muscular tunica media of vessels. GRO-a melanoma growth stimulating activity, alpha HIF hypoxia inducible factor Preeclampsia is associated with abnormal HIV human immunodeficiency virus cytotrophoblast invasion and differ- HLA human leukocyte antigen entiation. Many investigators believe that preeclampsia (the sudden onset of HMGB1 high mobility group B1 maternal high blood pressure, protein- iE-DAP gamma-D-glutamyl-meso-diaminopimelic acid uria, and edema) occurs in 2 stages.22 IFNGR interferon-gamma receptor The first stage involves shallow cytotrophoblast invasion of the uterus, which IGF insulin-like growth factor was first described by Brosens et al.23,24 IL interleukin Failed transformation of spiral arteries IL interferon appears to be critical and leads to IRAK interleukin-1 receptor associated kinase hypoperfusion of the placenta and oxidative stress.25 The second stage in- IRF interferon regulatory factor cludes overactive maternal immune IUGR intrauterine growth restriction (also known as FGR) responses. Although these pathways are LPS lipopolysaccharide associated most commonly with pre- m meters eclampsia, similar diseases have been described in a subset of preterm labor/ Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. (continued) birth cases.21 The causes are under

S4 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement intense investigation. Severe cases of preeclampsia are associated with failed TABLE 2 cytotrophoblast transformation into Abbreviations for commonly used scientific terms [in file but NOT vascular-like cells coincident with EDITED] (continued) shallow uterine invasion.26 For example, Abbreviation Description placental cells that enter the uterine wall mmHg millimeters mercury fail to down-regulate epithelial cadherin MAL myelin and lymphocyte and to up-regulate vascular epithelial cadherin. They also misexpress a broad MCP-1 monocyte chemoattractant protein-1 array of angiogenic and/or vasculogenic MDP muramyl dipeptide factors. These include VEGF family miRNA micro-ribonucleic acid members. For example, invasive cytotrophoblasts from preeclampsia preg- mRNA messenger RNA nancies fail to stain with anti-VEGF A, MR mass restricted which their normal counterparts express mTOR mammalian target of rapamycin in abundance. In addition, they release MV microvesicle higher amounts of soluble VEGFR1 (sFlt-1),27 as do syncytiotrophoblasts.28 MVB multivesicular body Increasing circulating levels of sFlt-1 NADPH myeloid differentiation primary response gene 88 and other angiogenic factors (such as NCATS National Center for Advancing Translational Sciences endoglin) cause a preeclampsia-like NF-kB nuclear factor kappa beta syndrome in animal models.29,30 Thus, there has been a great deal of interest in NGS next generation sequencing whether or not circulating levels of NIH National Institutes of Health molecules that could have negative ef- NK natural killer fects on the maternal vasculature can be NLR nod-like receptor used to predict and/or diagnose preeclampsia.31-33 NO nitric oxide Is abnormal placental production of Nod nucleotide oligomerization domain protein angiogenic/vasculogenic factors a cause NRP1 neuropilin-1 or consequence of preeclampsia? As yet, there are no definitive answers to this NTU new therapeutic uses question. However, alternative explana- O2 oxygen tions abound. For example, particular PAMP pathogen-associated molecular pattern combinations of maternal natural killer PaO partial pressure of arterial oxygen (NK) cell expression of killer cell 2 immunoglobulin-like receptors that PD pharmacodynamic recognize the certain major histocom- PDG peptidoglycan patibility complex molecule, human PE preeclampsia leucocyte antigen C, on invading cyto- PK pharmacokinetic trophoblasts increase the risk of preeclampsia.34 Surprisingly, a recent study PI3K/Akt phosphatidylinositol-3-kinase/protein kinase B showed that, on isolation from pre- PlGF placental growth factor eclampsia placentas, cytotrophoblast PO2 partial pressure of oxygen gene expression (eg, growth hormone PPROM preterm premature rupture of membranes [GH] 2, corticotrophin-releasing hormone, kiss-1 metastasis-suppressor 1, Prl prolactin semaphoring 3B, and several pregnancy- PRR pattern recognition receptors fi speci c beta-1-glycoproteins) is nor- PTL/B preterm labor/birth malized,35 which suggests that the defects are reversible and that pursuit of thera- PTB preterm birth þ pies is warranted. K potassium RICK receptor-interacting protein-like interacting caspase- Current opportunities. As compared with like apoptosis regulatory protein kinase other medical conditions, very little Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. (continued) attention has been paid to therapeutic/

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does not cross the placenta, could be TABLE 2 evaluated in women who have a high Abbreviations for commonly used scientific terms [in file but NOT risk of pregnancy loss because of the EDITED] (continued) effects of this cytokine (eg, inflammatory Abbreviation Description and thrombotic placental lesions) in RNA ribonucleic acid the setting of autoimmune disorders such as antiphospholipid antibody syn- ROS reactive oxygen species drome. 36 sFlt-1 soluble fms-like tyrosine kinase 1 (also known as soluble VEGFR1) Future opportunities. Until recently, it SOD superoxide dismutase was thought that placental interactions sPTB spontaneous preterm birth with the mother occurred at a cellular level (eg, invasive cytotrophoblasts and STB syncytiotrophoblast maternal immune cells within the uter- sRNA small ribonucleic acid ine wall) or involved soluble proteins siRNA silencing ribonucleic acid (eg, human chorionic gonadotropin). ssRNA single-stranded ribonucleic acid However, this paradigm is shifting rapidly. Free fetal DNA, which circulates in T1 translational spectrum 1, translation of animal and 37 bastic research into humans maternal blood, is being used as a noninvasive means of prenatal genetic T2 translational spectrum 2, translation of clinical diagnoses.38 It is possible that circulating research findings to practice cell-free RNA could be used as a com- TBK-1 tank-binding kinase 1 plementary method and/or as a means of TLR toll-like receptor gaining additional information.39 Also, TNFa tumor necrosis factor; alpha like many cancer cells, the placenta appears to release a complex repertoire of TRAF TNF receptor associated factor extracellular vesicles the cargo of which TRAM trif-related adaptor molecule could have major effects on numerous TRIF tir-domain-containing adapter-inducing interferon-b maternal cells, tissues, and organs.40,41 TS trophoblast stem Thus, obtaining an in-depth understanding of the types and content of uNK uterine natural killer placental extracellular vesicles will in- VEGF vascular endothelial growth factor crease our understanding of their func- VEGFR VEGF receptor tions. For example, it would be interesting to determine how their contents and tar- Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. gets change over the course of gestation and the impact of the common pregnancy complications, which include pre- pharmacologic interventions for the antibodies, small molecules) that target eclampsia and preterm labor/birth, on great obstetric syndromes. In this particular vulnerabilities (eg, vascular the normal trajectory. Ultimately, this context, pregnancy complications are and/or immune functions) could be important information could lead to the equivalent of “orphan” diseases, not formulated as derivatives that prevent several types of clinical applications (eg, because they are rare conditions but syncytiotrophoblast transport, thus extracellular vesicles could be used to because there is very little monetary reducing the risk of untoward embry- infer important aspects of placental incentive for taking on the risk that onic/fetal events. As a first step, this functions). Other possibilities include treating pregnant women entails. How- general strategy could be tried with therapies that target extracellular vesicles ever, there are compelling reasons to agents that are already used to treat or take advantage of this system of inter- shift this paradigm. Most of the common pregnant women (eg, tumor necrosis cellular communication for drug delivery. diseases that derail human pregnancy factorealpha inhibitors that work, in affect the placenta. Many involve either part, by blocking the activation of Scientific gaps in relation to drug targe- fetal or maternal cells that reside within endothelial and immune cells that this ting. A myriad of questions remain the uterine wall. Thus, it is likely that cytokine produces. For example, certo- to be answered about mechanisms effective therapies could be designed to lizumab (a pegylated fragment antigen- that are central to the success of target these cells without crossing the binding fragment of a humanized normal pregnancy and go awry in preg- placenta and reaching the embryo/fetus. monoclonal antibody that inhibits nancy complications. For example, For example, many kinds of drugs (eg, tumor necrosis factorealpha), which maternal tolerance of hemiallogeneic

S6 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement cytotrophoblasts lacks a definitive FIGURE 1 explanation. Therefore, it is very diffi- A schematic drawing of the maternal-fetal interface in human pregnancy cult to devise targeted therapies for pregnancy disorders, from infertility to preeclampsia, that are thought to have an immune cause or component. Like- wise, 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 pla- Mononuclear placental cytotrophoblasts invade the uterine wall and its resident vasculature (right centa and the mother. The development panel). During this process, they transform spiral arteries into wide-bore vessels that perfuse the of drugs that intercept or redirect these placenta. Its tree-like chorionic villi are covered by multinucleated syncytiotrophoblasts, which signals will require a molecular dissec- transport a variety of substances to and from the fetus, enabling normal fetal growth. tion of their components. Reprinted with permission from Romero et al.21 Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. Innate immune function of human trophoblast (Vikki M. Abrahams, Yale University) negative bacterial lipopolysaccharide. NLRs can synergize with TLRs for a Background. Placental trophoblast cells TLR2, in cooperation with its coreceptors greater response or provide a compen- can sense and respond to a variety TLR1, TLR6, or TLR10, recognizes satory system for when TLR signaling of infectious pathogen-associated mo- Gram-positive bacterial peptidoglycan is absent or reduced.50,51 The NLR lecular patterns that are expressed and lipoproteins. TLR3 senses viral proteins, Nod1 and Nod2, recognize by microbes, as well as noninfectious double-stranded RNA; TLR5 senses the peptidoglycan peptides Gram- host-derived damage-associated molec- bacterial flagellin. Mouse TLR7 and hu- negative bacterial gamma-D-glutamyl- ular patterns (DAMPs) through their man TLR8 sense viral single-stranded meso-diaminopimelic acid (iE-DAP-) expression of innate immune pattern RNA, and TLR9 senses bacterial DNA.46 and muramyl dipeptide (MDP) that is recognition receptors (PRRs).42,43 Four adapter proteins are involved in expressed by all bacteria, respectively. Depending on the trigger or receptor TLR signaling: myelin and lymphocyte Both Nod1 and Nod2 signal through the that is activated, the trophoblast may protein 88 (MyD88), TIR-domaine common adapter protein receptor- mount either a regulated protective containing adapter-inducing interferon- interacting protein-like interacting response that helps to maintain and beta (TRIF), myelin and lymphocyte caspase-like apoptosis regulatory protein promote a healthy pregnancy or a protein (Mal), and TRIF-related adaptor kinase (RICK) to induce inflamma- damaging response that might impact molecule.47,48 TLR2 and TLR4 signal tion.52 Another NLR called NACHT, pregnancy outcome adversely (Figure 3). through MyD88/Mal. TLR4 can also leucine-rich repeat protein and the Moreover, expressions of some tropho- signal through TRIF/ TRIF-related NLR, Nalp3, are involved in mediating blast PRRs are regulated gestationally, adaptor molecule. TLR3 signals through the production of the proinflammatory which further impacts the placental TRIF; all other TLRs signal through cytokine interleukin (IL)-1b.53,54 response.44 MyD88 alone.47,48 Downstream, TLR/ Because IL-1b has the potential to be MyD88 signaling activates NFkB; TLR/ damaging, its regulation is tightly PRRs. Two major families of PRRs are the TRIF activates Tank-binding kinase-1 controlled. Indeed, IL-1b is an important Toll-like receptors (TLRs) and the Nod- and interferon regulatory factor 3 that mediator of preterm birth102-105 and like receptors (NLRs). TLRs are trans- leads to a type I interferon response perinatal brain injury106-109 and can membrane receptors, which allows for (Figure 4).48,49 trigger the production of other proin- the sensing of PAMPs or DAMPs either at NLRs are cytoplasmic-based PRRs flammatory cytokines and chemokines the cell surface or within endosomal that provide an intracellular recognition through the IL-1 receptor in a similar compartments.45 There are 10 human system for sensing microbe-associated manner to TLRs.55 Indeed, delivery and 12 murine TLRs, each with distinct pathogen-associated molecular pattern of a synthetic peptide to pregnant specificities.46 TLR4 recognizes Gram- (PAMP) or as will be discussed, DAMPs. mice that is a selective IL-1 receptor

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cleaved into its active secreted form.57 FIGURE 2 This second step (signal 2) is mediated Phenotypic transformation of cytotrophoblast during uterine invasion 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 proeIL-1b into its secreted form (Figure 5).57 Although the Nalp3 inflammasome 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- more, Nod proteins can mediate IL-1b production independent of the inflammasome. For example, Chlamydia trachomatis infection of human trophoblast induces IL-1b via Nod 1.113

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- Cytotrophoblasts switch their expression of integrin aVb family members as they invade the uterine cytiotrophoblast cells can generate a wall. Sections of the maternal-fetal interface at various weeks of gestation (18-22) were double strong inflammatory response to lower stained with anti-cytokeratin to mark A, C, E, G, cytotrophoblasts and B, D, F, H, anti-aVb5, anti- lipopolysaccharide doses. Thus, there aVb6, or anti-aVb3. aVb5 was detected on cytotrophoblasts in floating (data not shown) and are differential sensitivities of tropho- anchoring villi, but not in other locations. aVb6 was detected on villous cytotrophoblasts at sites of blast cells to bacterial components 63 column formation and in the first cell layer of the column. aVb3 was up-regulated in the distal across gestation. This dose dependency portions of the columns and on endovascular cytotrophoblasts that lined the maternal blood vessels. and role for TLRs and NLRs is AV, anchoring villi; b3, anti-aVb3; b5, anti-aVb5; b6, anti-aVb6; BV, blood vessels; CK, anti-cytokeratin; EC, endothelial cell. reflected in vivo. In pregnant mice, Reprinted with permission from Zhou et al.134 high-dose lipopolysaccharide induces Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. placental and uterine inflammation and subsequent preterm birth.64 TLR4 deficient mice are protected against modulator delays IL-1b delays and The first step requires induction of bacterial and lipopolysaccharide- lipopolysaccharide-induced preterm proeIL-1b expression. This is triggered induced preterm birth,65,66 and block- birth.56 Unlike most other cytokines, IL- through signals like TLRs (signal 1). ing TLR4 in nonhuman primates 1b production involves a 2-step process. Once expressed, proeIL-1b can be prevents lipopolysaccharide-induced

S8 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement preterm uterine contractility.100 High- FIGURE 3 dose iE-DAP also induces inflammation Innate immune sensing by the trophoblast at the maternal-fetal interface and preterm birth.62 However, low-dose lipopolysaccharide 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 lipopolysaccharide, that may always be present at the maternal-fetal interface.71,72 Trophoblast cells sense infectious pathogen-associated molecular patterns that are expressed by Furthermore, although bacteria can bacteria, viruses, fungi, and parasites through their expression of Toll-like receptors and Nod-like induce preterm birth,73,74 additional receptors. Through these receptors, trophoblast cells also mount responses to noninfectious signals, such as a virus, may further host-derived damage-associated molecular patterns, such as uric acid, high mobility group B1, sensitize the placenta to these bacterial glucose, and certain autoantibodies. Trophoblast expression of some Toll-like receptors and Nod-like signals. 67,68 receptors are regulated across gestation and cell subtype. Depending on the trigger, receptor Gram-positive bacterial peptido- activated, and type of signaling pathway used, the trophoblast may mount either a regulated pro- glycan, which activates TLR2 in associ- tective response that helps to maintain and promote a healthy pregnancy or a damaging pathologic ation with TLR1, TLR6, or TLR10, response that might impact pregnancy outcome adversely. triggers a very different response in hu- PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns; HMGB1, high mobility group B1; man first-trimester trophoblast cells NLRs, Nod-like receptors; TLRs, Toll-like receptors. compared with term trophoblast. At Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. term, peptidoglycan activates these cells to produce IL-8.75 However, in first- trimester trophoblast cells, instead of is mirrored in vivo without triggering patients with preterm labor and preterm activating the classic inflammatory preterm birth.82 In contrast, viral premature rupture of membranes,87 and cascade, peptidoglycan through TLR1, double-stranded RNA induces preterm in serum from pregnancies that are TLR2, and TLR10 induces apoptosis and birth through TLR379,83; other groups complicated by either preeclampsia or down-regulates the cell’s basal cytokine/ have reported preeclampsia-like symp- reduced fetal movements.88,98 Term chemokine expression. Moreover, this toms in viral double-stranded RNA- trophoblast cells that are treated with apoptotic response is prevented by the treated mice.84 HMGB1 secrete significantly increased ectopic expression of TLR6, which is levels of IL-1b, IL-6, and monocyte absent in the first-trimester tropho- Trophoblast activation by DAMPs. In chemoattractant protein-1; however, blast.58,76-78 Similarly, delivery of pepti- addition to understanding how the mechanistic studies are needed to show doglycan early in gestation induces trophoblast responds to PAMPs, the which receptors and signaling pathways placental apoptosis without evidence of impact of DAMPs on trophoblast func- are activated by this DAMP.89 One type inflammation,76 although delivery later tion has been investigated. DAMPs are of DAMP that has been characterized in gestation triggers preterm birth.79 host-derived factors that are either not extensively is the antiphospholipid usually released from cells or tissues or, if antibody (aPL), which are autoanti- Viral sensing by the trophoblast. The viral present in the extracellular space, are bodies that specifically target the sensors, TLR3 and TLR8, that recognize normally at low levels. The DAMP, high- trophoblast by binding surface beta2 viral double-stranded RNA and single- mobility group B1 (HMGB1) can be glycoprotein 1.90 These autoantibodies stranded RNA, respectively, mediate a released, passively from damaged cells or activate human first-trimester tropho- rapid, robust chemokine/cytokine (IL-6, actively in response to inflammatory blast TLR4 that results in inflammatory IL-8), type I interferon, and antiviral triggers and can mediate inflammatory IL-8 and IL-1b secretion (Figure 4).91,92 response in human first-trimester responses via TLR2, TLR4, or receptor Downstream of TLR4, aPLs induce trophoblast.80-82 Furthermore, inde- for advanced glycation end products 85 endogenous uric acid, which is another pendent of TLR8, viral single-stranded (Figure 4). HMGB1 levels are increased DAMP that specifically activates the RNA also induces trophoblast expres- in the amniotic fluid from patients with Nalp3 inflammasome,93 which medi- sion of IL-1b, antiviral factors, and the preterm birth or preterm premature tates proeIL-1b processing and secre- induction of apoptosis.82 This placental rupture of membranes and intrauterine tion (Figure 5).92 In parallel, aPL via single-stranded RNAeinduced response infection,86 in fetal membranes from TLR4 induces the expression of the

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this mechanism is not involved only in FIGURE 4 microbial-induced inﬂammation, but Toll-like receptor signaling 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 preterm 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-1b is a mediator of preterm birth102-105 and fetal brain injury,106-109 studies have focused on using IL-1 receptor an- tagonists or selective IL-1 receptor modulators to prevent these adverse outcomes.110,111 Preventing the upstream Toll-like receptors are transmembrane receptors that mediate the sensing of pathogen-associated induction of IL-1b by inhibiting placental molecular patterns expressed by microorganisms. Toll-like receptor 2, in cooperation with its cor- inflammasome activity may also serve as a eceptors Toll-like receptors 1, 6, or 10, recognizes Gram-positive bacterial peptidoglycan. Toll-like potential target for the prevention of receptor 4 recognizes Gram-negative bacterial lipopolysaccharide. Toll-like receptors 3 and 7/8 adverse pregnancy outcomes. 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 dinucleotideerich DNA Future opportunities. Because uric acid regions. Four adapter proteins are involved in Toll-like receptor signaling: MyD88, TRIF, Mal, and mediates placental inflammasome func- TRAM that trigger downstream pathways that lead to either nuclear factor kappa-light-chain- tion,92,93,96 currently available drugs that enhancer of activated B cells activation and subsequent cytokine/chemokine production or IRF-3/ inhibit xanthine oxidase, such as allo- IRF-7 activation that leads to a type I interferon response. Some Toll-like receptors also sense purinol or febuxostat, may provide host-derived damage-associated molecular patterns. Toll-like receptors 2 and 4 can sense high potential avenues to explore. However, mobility group B1 protein; Toll-like receptor 4 can be activated by antiphospholipid antibodies. Refer this is only 1 mechanism by which IL-1b to Table 2 for the key to undefined abbreviations. production can arise; there are a number 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 of other inflammasomes that uric regulatory factor; LPS, lipopolysaccharide; Mal, myelin and lymphocyte; MyD88, myeloid differentiation primary response protein 88; 112 e NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; PAMPs, pathogen-associated molecular patterns; PDG, pepti- acid may not activate and non doglycan; ssRNA, single-stranded RNA; TBK, tank-binding kinase; TLR, Toll-like receptor; TRAF, tumor necrosis factor receptor- fl 113 associated factor; TRIF, tir-domainecontaining adapter-inducing interferon-b. in ammasome-mediated pathways, Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. all of which could be possible targets. Similarly, as we expand our knowledge about the role of miRNAs in the medi- miRNA, miR-146a-3p, which drives IL-8 associated with elevated uric acid and is ation and regulation of placental func- secretion by activating TLR8.94,95 Thus, dependent on the activation of the Nalp3 tion and PRR activity, we can begin to aPL, through TLR4, induces endogenous inflammasome.96 Elevated serum uric consider these as potential therapeutic secondary messengers that subsequently acid has been associated with high-risk targets. activate other trophoblast innate im- pregnancies, such as those complicated mune PRRs. Hyperglycemic levels of by preeclampsia, gestational hyperten- Scientific gaps. Together, the studies dis- glucose have similar effects on these cells, sion, cases of reduced fetal movements, cussed herein demonstrate that the which suggests that overt pregestational or obstetric aPL syndrome.92,97-99 Thus, placenta is immunologically functional, diabetes mellitus may impact placental rather than simply correlating levels with with the trophoblast able to generate inflammation and function early in disease, uric acid can act as a direct specific and diverse innate immune-like pregnancy. Excess glucose induces a mediator of trophoblast inflammasome responses through their expression of a proinflammatory cytokine/chemokine activation and placental inflammation,93 range of PRRs. However, the type of profile (IL-1b, IL-6, IL-8, GRO-a, which suggests that it may play a path- response is highly dependent on the regulated on activation normal T cell ologic role. Furthermore, that those stimuli, the receptors expressed and expressed and secreted, chemokine, and host-derived noninfectious triggers activated, the downstream signaling granulocyte colony stimulating factor). (such as uric acid, glucose and aPL) can pathways involved, and the timing of Moreover, the IL-1b response is activate the inflammasome indicates that gestation. Indeed, although many of the

S10 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement endpoints and impact on pregnancy FIGURE 5 outcome triggered by PAMPs and Nod-like receptor signaling 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) Nod-like receptors are cytoplasmic proteins that sense pathogen-associated molecular patterns. Background. Fundamental to the estab- Nod1 recognizes bacterial iE-DAP, and Nod2 senses bacterial muramyl dipeptide. Both Nod1 and lishment of pregnancy and formation of Nod2 signal through the adapter protein receptor-interacting protein-like interacting caspase-like the hemochorial placenta is remodeling apoptosis regulatory protein kinase to induce nuclear factor kappa-light-chain-enhancer of acti- and restructuring of uterine spiral arteries vated B cells activation and subsequent cytokine/chemokine production. Nalp3 recruits ASC and fl that allow for the ow of nutrients to the caspase-1 to form the inflammasome. Once the inflammasome has assembled, caspase-1 is 114 placenta and ultimately to the fetus. activated and processes pro-IL-1b into its active, secreted form. Refer to Table 2 for the key to Trophoblast cells play a central role in undefined abbreviations. this vascular remodeling process. Patho- ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain [CARD]; DAMP, damage-associated molecular logic conditions that are associated with pattern; IL, interleukin; MDP, muramyl dipeptide; Nalp, protein complex consisting of Nalp3, apoptosis-associated speck-like protein that contains a [CARD] and caspase-1; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRs, Nod-like receptors; trophoblast-directed uterine spiral artery PAMPs, pathogen-associated molecular patterns; Pro-IL, precursor protein of interleukin; RICK, receptor-interacting protein-like remodeling underlie some of the most interacting caspase-like apoptosis regulatory protein kinase. significant and challenging obstetric dis- Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. eases.114-116 There are several research strategies for gaining insight into the process of hemochorial placentation and Regulatory factors identified in vitro observations illustrate the instructive for the identification of potentially have been tested experimentally with the nature of oxygen delivery as signal- vulnerable molecular mechanisms that use of an assortment of in vivo research driving decision-making within lead to disease. In this section, we address strategies, which include transgenesis trophoblast stem/progenitor cell pop- the merits of animal models, especially to monitor the invasive trophoblast ulations and ultimately affect hemo- the rat, for placental research. lineage,124 spontaneous mutant rat chorial placentation; however, it is models that possess placental insuffi- important to appreciate that failures in Placentation in the rat. The rat possesses ciency118,125-128 and gain-of-function adaptive responses to hypoxia can lead to hemochorial placentation with deep and loss-of-function manipulations disruptions in placentation. Addition- intrauterine trophoblast cell invasion with the use of trophoblast-specific ally, hypoxia can also be a pathologic and trophoblast-directed uterine spiral lentiviral gene delivery and genome response to a failed placenta resulting in artery remodelling,117,118 which are fea- editing.129-132 disease. Dissociating these potentially tures shared with human placenta- A fundamental property of placenta- confounding outcomes of hypoxia is tion.114,119-121 Recognition of these tion is its plasticity, which is character- essential. Dissection of hypoxia-guided similarities spurred the establishment of ized by the acquisition of structural/ pathways within trophoblast stem/proin vitro and in vivo research methods functional properties that permit adap- genitor cell populations offers an op- using the rat as an animal model to tation to environmental challenges. portunity to identify molecular events in address mechanistic questions regarding Mechanisms underlying these adaptive the placentation process that could be the development of the hemochorial processes are an important feature of manipulated to improve and enhance placenta and especially the role of inva- placentation and represent potential placental development and prevent the sive trophoblast cells in the remodeling therapeutic targets. Limiting oxygen de- spiraling consequences of a failed of uterine spiral arteries. Rcho-1 livery to the placentation site at devel- placenta. trophoblast stem cells and blastocyst- opmentally defined phases of gestation derived trophoblast stem cells are 2 rat effectively activates the acquisition of the Role of NK cells. Immune cells, especially in vitro culture systems that have been invasive trophoblast phenotype and NK cells, are active contributors to used extensively to investigate signaling promotes uterine spiral artery remodel- remodeling of uterine spiral arteries pathways and mechanisms that control ing124,133 that are adaptive responses during the establishment of pregnancy. trophoblast cell differentiation.122,123 conserved in primates.134,135 These The involvement of NK cells in the

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in structure, function, and especially FIGURE 6 molecular mechanisms that regulate the Natural killer cells and endovascular trophoblast cells contribute to placentation process; when the time and uterine spiral artery remodeling effort are taken to investigate these processes, it has been demonstrated that there is considerable merit for animal models in placental research.120,137-140

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 trophoblast cells, NK cells, and uterine spiral arteries, a triad of key players in hemochorial placentation. A paradigm for investigating molecular mechanisms that impact hemochorial 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 A-D , Rats were treated on E4.5 and E9.5 with normal rabbit serum (control) or anti-asialo GM1 then proceeding to in vivo rodent A, B, (natural killer cell depleted) and killed on E13.5. Double immunofluorescence staining for experimentation represents a powerful C, D, ANK61 (natural killer cell marker) and ACTA (smooth muscle marker) and cytokeratin and research approach.131,141,142 Evaluation ACTA2. The asterisks demarcate blood vessels that possess interruptions (arrowheads) in the tunica of conservation generally is limited to A, C B ¼ media ( ); the asterisks identify blood vessels with intact tunica media ( ). Scale bars 0.25 mm. the use of primary or immortalized hu- NK, natural killer. man trophoblast cell model systems and 133 Reprinted with permission from Chakraborty et al. expression analysis in human placental Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. tissues. A human trophoblast stem-cell culture system functionally equivalent to rodent trophoblast stem cells would hemochorial placentation process has supporting a conserved role for uterine be optimal for studying the regulation of been investigated effectively in the rat NK-cell actions within the human differentiation, especially the identifica- (Figure 6). NK cells directly regulate placentation site.136 Modulation of tion of conserved processes. Some uterine spiral artery growth and pro- uterine NK-cell behavior represents progress has been made in the develop- gression towards the developing another potential target for modulating ment of this important experimental placenta. These critical events impact the placentation process. in vitro tool.143 The recent availability of oxygen delivery to the placentation site genome editing strategies to generate and trophoblast differentiation.133 Fail- Current opportunities. Animal models are mutations in rodents should lead to the ures in NK cells result in attenuated important tools to understand human establishment of new animal models for uterine spiral artery development, local disease. Animal models provide the opin vivo testing of conserved molecular hypoxia at the placentation site, and portunity to study biologic phenomena mechanisms that control hemochorial premature and exaggerated activation not easily studied in the human. placentation. These in vivo approaches of endovascular invasive trophoblast Although no model is ideal, each provides should include targeting the activities of cells that lead to extensive uterine spiral useful insight relevant to the human specific trophoblast and immune cell artery remodeling. Thus, NK cells con- condition. The rat is a particularly populations in order to identify molec- trol oxygen delivery to the developing important experimental tool for investi- ular pathways that could serve as sites for placenta and regulate the timing gating regulatory processes that control therapeutic intervention. and extent of endovascular invasive hemochorial placentation. Species dif- trophoblast cell differentiation and ferences in placental organization and Scientific gaps. The use of relevant and trophoblast-directed uterine spiral ar- gene expression patterns are evident, but appropriate animal models, including tery remodeling. Evidence also exists there are also underlying commonalities rodents, to test hypotheses in vivo most

S12 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement importantly extends placental research shunt glucose to the fetus.148 To combat hormones,161 progesterone,162 resis- beyond description, classification, insulin resistance, an increase in tin,163 and leptin164 can promote insulin in vitro analyses, and molecular pheno- pancreatic b cells and insulin synthesis resistance. Paradoxically, the placenta typing and permits a rational approach occurs.149-151 Gestational diabetes mel- also produces adiponectin165 that pro- for understanding the physiology of litus (GDM) occurs if there is inadequate motes insulin sensitivity. It is interesting placentation, the pathogenesis of b-cell compensation.152,153 The most that, although these hormones are nor- placental disease, and importantly the important change in the immune system mally expressed by the pituitary (pro- identification and testing of potential during pregnancy is the appearance of lactin, GH), ovary (progesterone), and drug targets for treating placental large numbers of uterine NK cells in the fat (the “adipokines”: resistin, leptin, disease. decidua,154 first described in mice and adiponectin), the human placenta is a only later in humans. One difference major source during pregnancy, and Modeling placental function and between mice and humans is that mice production from the maternal tissues is pregnancy physiology in mice are litter-bearing, whereas humans tend down-regulated. (James C. Cross, University of Calgary) to have singleton pregnancies; however, The mouse placenta expresses Background. Understanding the molec- what functional difference this has is not approximately 40 protein hormone ular, cellular, and physiologic functions clear, given the similarity of pregnancy genes.166 As with humans, prolactin- of the placenta in humans is limited to physiologic condition. and GH-like hormones, progesterone, expression studies in normal and path- resistin, leptin, and adiponectin are ologic human pregnancies and some Trophoblast functions and pregnancy. elevated during pregnancy. However, the in vitro systems. Because of this, animal Most of the research on trophoblast cell evolution of the system is slightly models remain critical for investigation function in human pregnancy compli- different because the prolactin gene, and of the basic biology and assessment of cations has focused on trophoblast cell not the GH gene, is duplicated in mice to biomarkers and treatments of pregnancy invasion and its association with spiral produce 22 placenta-specific mem- complications. The mouse has been a artery remodeling. Cell ablation experi- bers167 with prolactin- and GH-like ac- powerful model for understanding ani- ments in mice showed that this is not just tivities. In addition, the placenta is not mal biology in the last 25 years with the an association and that trophoblast cell the only source of the other metabolic advent of transgenic and knockout association with spiral arteries is critical hormones. Progesterone is produced by technologies. Hundreds of different gene for remodeling of those arteries,155 the ovary throughout pregnancy in knockouts in mice have given molecular though uterine NK cells in the decidua response to stimulation by prolactin-like insights into the development and also play a role.156 Beyond just invasion hormones from the placenta.168 Resis- function of the placenta. Many of these of spiral arteries, however, the human tin,169 leptin,170 and adiponectin171 are genes have human homologues that are placenta contains diverse extravillous produced by fat during pregnancy in expressed in the placenta.144,145 How- trophoblast subtypes,157 and mice have mice. Prolactin receptor signaling can ever, before zeroing in on genes and cells, diverse trophoblast cells in the junctional regulate adipokine expression,172-174 if mice are to be truly useful for under- zone that express complex patterns of which suggests the possibility that, standing human pregnancy complica- hormones.158 although the mouse placenta is not tions, it is critical first to ask whether Several lines of evidence indicate that the direct source of all metabolic hor- mice and humans have similar physiol- the endocrine function of the placenta mones, unlike humans, it may still ogies of pregnancy. This is the starting modifies metabolism in the mother that orchestrate the network. The best point for the use of mice to investigate is necessary to promote fetal growth. evidence in mice that placental hor- molecular mechanisms that give us Scanning through microarray data in mones regulate fetal growth is that of testable hypotheses to assess in human the public domain indicates that the the pleckstrin homology-like domain, studies. human placenta expresses >80 different family A member 2 gene, which regulates hormones.159 Although a few are the fetal growth by influencing the Similar physiology of pregnancy in placenta-specific hormones arising from number of endocrine cells in the humans and mice. Mice adapt to preg- duplication of the GH 2 gene most are placenta.175 nancy with major changes in the from canonical hormone genes that are maternal cardiovascular, metabolic, and expressed in the placenta, presumably Current opportunities. Despite the clear immune systems. Similar to humans, because of evolution of placenta-specific evidence that placental hormones drive mice show increased cardiac output, promoter and/or enhancers. The hor- fetal growth and regulate maternal plasma volume, and a mid-gestation mones include known regulators of metabolism, it is curious that research drop in blood pressure.146,147 There are metabolism, blood cell production has not continued to understand their also major changes in metabolism in and reproduction. Placental prolactin- roles in intrauterine growth restriction which the mother’s fat and muscle related hormones can promote prolifer- (IUGR) and gestational diabetes mellitus become insulin resistant, requiring more ation of pancreatic b cells and insulin in humans. Placental hormones are insulin to take up glucose, which helps to synthesis.160 Glucose transporter-related attractive targets from a diagnostic

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should have good predictive value in Oxygen levels in the human placenta and FIGURE 7 reflecting both stress to the pregnancy fetus. Normal oxygen levels within the Relationship of maternal and the robustness of the placenta’s intervillous space of the placenta are intervillous blood PO to fetal 2 ability to mitigate the impact on the low early in gestation (approximately umbilical venous PO 2 fetus. In addition to diagnostic value, it is 20 mm Hg), can rise to as high as easy to imagine therapeutic strategies in 80 mm Hg in the early second trimester, which hormone supplementation or and then decline progressively towards blockade is used to treat pregnancy term (Figure 7).183-186 Fetal umbilical complications. venous PO2 follows a similar pattern, perhaps reaching as high as 40-50 mmHg Scientific gaps. The complexity of the in the second trimester, but also hormone network at play during preg- declining progressively towards term to nancy, both the number of hormones an average of w28 mmHg (<4% O2)at and the systems of feedback and adap- term. Rising hemoglobin maintains Oxygen tension in the intervillous space of the tation, will require the use of animal oxygen content. With normal values as placenta is very low until the opening of the models, particularly knockout and low as these, even small changes in fetal spiral arteries to blood flow at approximately 10- transgenic mice, to understand them. PO2 can make a difference. Average 12 weeks of gestation. Light blue dots are in- With the ability to study mouse physio- umbilical venous PO2 differs by only dividual data points that were obtained at 8-11 logic condition, it is clear that, although 3 mmHg in babies at >97% percentile weeks gestational age. Medium blue dots are a mouse is not a human, we certainly can versus those at <3rd centile.187 Despite data that were obtained from individual preg- learn from them. difficulty in quantifying magnitude and nancies at 11-16 weeks of gestation. Dark blue intensity of hypoxia, much less being able dots are the mean of values that were obtained Potential drug targets of important to detect its onset, the bulk of evidence in only a few women between 16-38 weeks of placental pathways in relation to has led to general acceptance that fetal gestation and have very wide confidence in- pregnancy disorders growth diseases often are associated with tervals (>30 mm Hg). Red dots are umbilical Placental hypoxia as a molecular target placental hypoxia, specifically early- venous PO2. Note the tight relationship and (Stacy Zamudio, Hackensack onset preeclampsia, most nongenetic/ narrow diffusional gradient between intervillous University Medical Center) syndromic IUGR, preeclampsia with and fetal PO2 late in pregnancy. This Figure is a Background. Hypoxia is a pathologic IUGR, and some diabetes/GDM with composite of data obtained from various condition in which there is insufficient large for gestational age neonates.188-196 references.182-185 oxygen to maintain normal physiologic Figure 7 shows that what might be Ilekis. Potential placental molecular therapeutic targets. Am J processes. However “hypoxia” is often considered abnormally low O2 levels in Obstet Gynecol 2016. used imprecisely in the literature, inter- the placenta will vary not only by gesta- changeably with some of its causes, tional age, but also with location within for example: hypoxemia-reduced partial the placenta. The third-trimester standpoint because they can be pressure of oxygen (PO2), anemia placenta is exposed to PO2s that can measured serially, and improvements in (insufficient hemoglobin or hemoglo- range from <20 mm Hg when the multiplex immunoassays mean that binopathies that alter oxygen binding/ deoxygenated blood from the umbilical several hormones can be assessed at the release), or reduced environmental artery flows back into the placenta to same time. Both hormone levels and oxygen availability (as in high-altitude >80 mm Hg when the maternal arterial polymorphisms in the placenta GH- animals dwelling in burrows, diving blood first enters the intervillous space. related genes have been associated with mammals). Hypoxia is usually assumed This equates to 1-10% ambient O2 for pregnancy complications in humans, 163 to be present in any 1 of these conditions. in vitro experimentation. Such data have although the number of published For example, a reduction in blood flow given rise to a convention in which 5-8% studies is limited and they have often to a specific organ or tissue, whether O2 is used to mimic normoxic conditions examined single hormones and not acute or chronic, often is assumed to be a in the third trimester and <3% for made connections with anatomic hypoxic insult. However, hematologic hypoxia. changes in the placenta. adaptations can compensate for lower Placenta hypoxia cannot be diagnosed blood flow,177 and the volume and speed unambiguously because of the inherent Future opportunities. There is emerging with which blood travels, as well as limitations of human experimentation evidence from mouse studies that the diffusional distances, affect tissue oxygen and the inaccessibility of the placenta placental hormones are sensitive to delivery.178-182 Thus, in the absence of and fetus in vivo. Proxies are used maternal nutrition and changes in their direct measures of oxygenation in the instead. For example, reduction in blood levels likely reflect attempts to mitigate tissue, cell, or organ of interest, it is flow severe enough to deprive tissues of the impact of poor nutrition on fetal difficult to tell whether hypoxia is oxygen and glucose sufficient for meta- growth.176 Therefore, hormone levels present. bolic needs is assumed to be present

S14 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement when elevated maternal/fetal Doppler precipitously at >3000 m. Even in in uterine artery blood flow in mothers indices reflect increased impedance. pregnancies with entirely normal out- and an even greater decrement in fetal Elevated erythropoietin levels in comes, physiologically lowered maternal umbilical venous blood flow. Packed red mothers and babies, nucleated red blood PaO2 results in a progressive slowing of blood cell transfusion might work for cells in neonates, increased placental the third-trimester fetal growth trajec- anemicmothers,butitisunlikelytobeof 219,220 expression of hypoxia-inducible-factor tory. This is despite significant valuewherethebarriertoO2 diffusion is (HIF), and its target gene products placental adaptation both structurally due to placental structural defects such as have all been used to demonstrate (increased angiogenesis, decreased the failed tertiary villous vascular devel- that placental hypoxia is present in vascular syncytial membrane thickness) opment (branching angiogenesis and fetal growth diseases.192,197-209 Thus, and metabolically.221-226 Altitude studies elongation/dilation of terminal capillary placental hypoxia is a target for thera- in vivo showed that the human placenta loops) seen in idiopathic IUGR. Moreover, peutic intervention, and unlike some of that is subjected to hypoxic stress engages thereisabalancethatmustbemaintained the topics discussed at this workshop, in a highly conserved process most between blood viscosity and O2 carrying there have already been a number of obviously seen in solid tumors.227-230 capacity (eg, the optimal hematocrit level clinical trials designed to ameliorate or Metabolic reprogramming is a revers- for preservation of O2 delivery in the brain prevent presumed consequences of ible form of hypometabolism in which an microcirculation is 15-40%).234 One 210 hypoxia such as inflammation and effective, largely mitochondrial-driven obvious method for increasing PaO2 is O2 oxidative stress.211-214 switch from oxidative phosphorylation supplementation. Sadly, a 2003 Cochrane to aerobic (ie, glycolytic) glucose con- review found that, of 10 trials of O2 ther- Chicken and egg, cause and effect. Of sumption by the trophoblast results in apy, only 3 were adequate for inclusion in critical importance is distinguishing be- increased cellular oxygen availability, a metaanalysis. O2 compared with no O2 tween hypoxia per se and the hypoxia which is then available for diffusion to was given to mothers for improvement of response. The hypoxia response is evi- the fetus.224,225 In essence, the mecha- fetal growth. Rereview in 2009 changed dence that hypoxia is or has been present nism “spares” oxygen for the fetus, but at none of the Cochrane conclusions because (eg, up-regulation of HIF and HIF- the same time reduces fetal access to no additional trials were undertaken.235 regulated genes, higher fetal hemoglo- glucose. This slows fetal growth but en- The trials were poorly designed and bin concentrations). The strength and sures that the fetus does not outgrow its involved <100 women. However, 24-hr magnitude of the response may reflect supply line. Even when placental meta- supplemental O2 led to a decrease in the the intensity of the insult, but it is also bolic programming is active, a fetus with perinatal mortality rate. The studies were often adaptive in that it enables mecha- greater umbilical venous O2 tension confounded in that the O2-treated group nisms that increase the delivery of oxy- consumes more O2. was of greater gestational age at initiation gen to the fetus (as in metabolic of treatment. Concerns over possible reprogramming or the development of How might hypoxia be ameliorated? response to “hyperoxia” such as the gen- decreased vascular syncytial membrane Therapeutic strategies must necessarily eration of free radicals, the inhibition of thickness). On the other hand, we must depend on what is the pathologic barrier potentially adaptive responses ongoing in be mindful of the possibility that failure to normal placental oxygen diffusion. fetus and placenta have been raised as of an appropriate hypoxia response may Four targets will be considered here: objections to O2 therapy. itself be part of the pathologic condition. blood O2 content, uteroplacental blood There is evidence of HIF dysregulation flow, placental structure (angiogenesis), Blood flow. Deficits in uteroplacental or and consequent over-expression without and hypoxia-induced metabolic fetal blood flow are associated strongly adaptation in the more severe forms of reprogramming. with preeclampsia and IUGR as evi- preeclampsia.207,208,215 In contrast, in denced by Doppler ultrasound measures some severe, early-onset IUGR, there Raise arterial O2 content (CaO2) or of impedance to blood flow in the uter- often appears to be a lack of HIF- PaO2. In women who are anemic, the ine arteries and within the fetus. The mediated responses, when all evidence obvious treatment would be increasing studies in which blood flow in human suggests such response is needed.216-218 maternal red cell mass and thereby pregnancy complications has been Studies at high altitude have revealed increasing CaO2.Thisisharderthanit measured quantitatively or semi- the importance of being able to distin- sounds because women have less of a quantitatively support this.236 Absent or guish between adaptive hypoxia response to erythropoietin than do reversed end diastolic blood flow veloc- response and pathologic condition, as men,231 andtheconditionofupto25%of ity in the umbilical arteries is a grave sign well as the subtlety with which O2 ten- pregnant women who are treated with and usually leads to emergent de- sion can exert an effect. Maternal partial erythropoietin does not respond.232 At livery.237 Elevated Doppler resistance pressure of O2 in arterial blood (PaO2) high altitude CaO2 is increased by raising indices in the maternal uterine arteries falls considerably at 2700-m elevation; hemoglobin concentration. This preserves indicates that there is downstream because of the sigmoid shape of the maternal and fetal oxygen delivery176,233 impedance to blood flow. This is O2 dissociation curve, PaO2 falls despite an approximate 20% reduction accompanied by morphologic evidence

JULY 2016 American Journal of Obstetrics & Gynecology S15 Supplement Obstetrics ajog.org of placental structural problems such as hypoxia response). Late onset pre- associated with pregnancy diseases. a reduction in small arteries within the eclampsia (without fetal compromise) However, before a placental molecular tertiary stem villi,238 thickening of the shows no such changes and cannot be intervention is attempted, there should be basal lamina, and erythrocyte congestion statistically differentiated from normal a greater effort to examine relatively in tertiary villous capillaries.239,240 As placentas.248 The interplay of many noninvasive means of improving with CaO2 or PaO2, how one might target angiogenic growth factors and of several oxygenationintheplacenta.Theseshould blood flow depends on what is the un- critical cell types (pericytes, endothelium, include definitive studies of maternal O2 derlying problem. Most of the blood trophoblasts) is involved in the normal supplementation, dietary strategies entering the intervillous space is carried vasculogenesis of the placenta.223,249 designed to increase maternal substrates by the maternal uterine arteries. The for nitric oxide, and other obviously more uterine arteries therefore are critical to Placental metabolic reprogramming. In benign strategies that target maternal pregnancy success and a potential target. cancer biology, several recent unique physiologic condition rather than In normal pregnancy, eccentric remod- insights are relevant to the placenta and placental function. eling of the uterine arteries leads to consideration of molecular pathways Methods that might be used to pro- doubling of uterine artery diameter by that might be amenable to therapeutic vide additional means of carrying oxy- 20 weeks of pregnancy and a further, intervention. Cancer cells have dysregu- gen in the circulation (eg, oxygen-filled smaller increase, likely because of shear lated, Warburg-like glucose metabolism. microbubbles) are under investiga- stress, in the late third trimester.241 Energy production is abnormally tion.254-257 Moreover, the opportunity Eccentric remodeling is characterized dependent on aerobic glycolysis; there is exists currently to further test the effects by changes in the composition of the increased fatty acid synthesis and of supplemental O2; more modern vessel wall that permits greater distensi- increased rates of glutamine meta- standards of clinical trials such as bility. In addition, there is inhibition of bolism.250 In fact the term glutamine establishing dose/response, effects of the myogenic response (the rise in vessel addicted is now applied to many can- treatment duration, minimum dosing tone with increasing pressure).242-244 cers.251 In cancer biology “glutamine necessary surely should be attempted In human and multiple experimental addicted” refers to an extension of as a simple, yet potentially, effective animal models, endothelium-dependent metabolic reprogramming in which intervention. vasodilator response is increased mark- glutamine is required for essential amino Beyond this are the effectors that are edly in the uterine circulation during acid uptake to maintain activation of responsible for altering vascular tone that pregnancy. In rodent models, depending mTOR. In many, if not all cancer cells, could be molecular targets, including on what branch of the uterine artery glutamine is also the primary mito- nitric oxide, by stimulating release via a is investigated, nitric oxide contributes chondrial substrate. These metabolic number of means that include the 30-80%, and endothelium-derived changes are linked to therapeutic resis- administration of VEGF, PlGF, relaxin, hyperpolarizing effectors contribute tance in cancer treatment; hence, stra- or stimulation of the reticular-activating 20-70% of the endothelium-dependent tegies are being developed to target this system, hemoxygenaseecarbon monox- þ vasodilator response of the pregnant altered cancer metabolism in conjunc- ide, large conductance Ca2 -activated þ uterine arteries.245,246 tion with older treatments that were potassium (K ) channels, and in- designed to inhibit angiogenesis or creased dietary intact of arginine or Placental structure (eg, angiogenesis). In otherwise shrink the tumor.252,253 We citrulline.258-260 For endothelium-derived idiopathic IUGR, the placenta frequently have demonstrated the same, evolu- hyperpolarization, potential effectors þ has a dearth of branching angiogenesis tionarily conserved mechanism of include K C-type natriuretic peptide, and poorly developed tertiary capillary metabolic reprogramming in the hyp- arachidonic acid derivatives, epox- development, leading to reduced O2 oxic placenta that is associated with yeicosatrienoic acids and hydrogen diffusion capacity.178,216 Structural stably elevated HIF-1alpha levels, peroxide. placental defects in preeclampsia are less which initiates metabolic reprogram- Three approaches have been or clear. An excess of fibrinoid deposition ming.205,224,225 However, in contrast to currently are being investigated to in- leading to, or as a result of, villous death cancer, the objective of a therapy that crease blood flow. The least invasive of has been attributed to excess oxidative targets metabolic reprogramming in these involves dietary arginine (or stress or inflammation. In women with the placenta would be to sustain the citrulline) supplementation, in theory, preeclampsia, in general, placentas are response, which leads to increased to increase substrate for nitric oxide smaller (reduced weight and volume), intracellular oxygen levels and hence production. A number of small trials led and the volume of functional tissue (pa- more oxygen for diffusion to the fetus. to 1 randomized controlled clinical renchyma) and villous surface area (area trial.261 High-risk women (with a history of nutrient and gas exchange) are Current opportunities. There is wide- of preeclampsia in the previous preg- reduced. 247 However, unlike IUGR, they spread acceptance that placental hypoxia, nancy) had less than one-half the rate also have an increase in the fetal capillary acknowledging the already low of preeclampsia as women receiving volumes and density (indicative of a O2 environment that is normal, is placebo. However, another trial in

S16 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement women already diagnosed with pre- the development of cancer drugs. Is fetal growth a feasible target for eclampsia and treated acutely, rather There are >400 drugs that specifically placental intervention? (Nicholas P. than throughout pregnancy, showed no target the HIF pathway, most of them Illsley, Hackensack University benefit.262 International recruitment for inhibitory. Many of them also target Medical Center) a trial is ongoing for the treatment of specific, cancer-related mutations that Background. Alterations in fetal nutrient early-onset IUGR.263 This 6-year study are related to the HIF pathway; hence, a concentrations because of changes in was based on several small studies reverse-engineering approach could be placental transport and/or metabolism that showed that low-dose sildenafil applied to existing drugs to see whether are associated with a variety of fetal improved fetal growth and neonatal they would be beneficial, as has been growth diseases. Deficits in oxygen are survival.264 Another trial that is ongoing done in human immunodeficiency vi- associated with hypoxia, preeclampsia, and led by Dr Anna David in the United rus.267,268 A placental strategy might and IUGR. Deficits in glucose and/or Kingdom targets women with even consist of controlled up-regulation of amino acids are associated with IUGR, earlier IUGR, women whose IUGR is so HIF-1a, because it is known to be and an excess of glucose is associated severe that the fetus normally would die elevated stably in hypoxic placentae with macrosomia, obesity, and diabetes before viability.265 Treatment consists of and likely contributes to the increased mellitus. Alterations in placental uterine artery catheterization and injec- angiogenesis that is a favorable adap- nutrient transporters are associated with tion of adenovirally delivered VEGF-D. tation when placental hypoxia is pre- a variety of fetal growth diseases; the Proof of concept studies in sheep sent but not associated with a glucose transporter 1 in hypoxia and demonstrated that this treatment im- disease.220 diabetes mellitus272,273 and amino acid proves uterine artery blood flow for transporters in IUGR and diabetes mel- approximately1 month.266 Scientific gaps. For further advances in litus.274,275 The simplest possibility in molecular targeting, greater information these circumstances would be to reverse Future opportunities. With respect to is required in a number of areas. For these deleterious changes by manipula- angiogenesis, cancer therapies, especially example, although there is a large tion of maternal substrate levels. There for solid tumors, provide some clues. quantity of data on the role and effects of are situations in which action to reba- These therapies target the inhibition of vasodilators, the receptors for these lance abnormal nutrient concentrations angiogenesis, although, in the placenta, agents are understood poorly. Although is capable of correcting growth prob- one might wish to stimulate organ- a targeted increase in receptors for va- lems: the restoration of normal maternal specific angiogenesis. Effectors in this sodilators would be a possible thera- glycemic status to prevent macrosomia molecular pathway provide a variety of peutic strategy, we clearly require more being an example. Overall, however, the targets. Vascular endothelial-cadherin knowledge of the broader effects of re- record with regard to maternal nutrient and matrix metalloproteinases, for ceptor modulation. supplementation is 1 of minimal effect example, loosen gap junctions in Similarly for the angiogenic growth combined with potentially serious side- the endothelium, which is required for factors, what are the consequences if effects.276 tip-cell formation, a prerequisite to some, but not other angiogenic growth Another set of important questions branching angiogenesis. However, tip factors, are targeted in the placenta. revolves around timing. Is there a win- cell formation also requires coordinated What might be the response to modu- dow during which intervention is activity by VEGFR-2, various ligands for lating the angiogenic growth factor re- possible, and perhaps more importantly, Notch-1 receptor (such as Delta-like ceptors rather than the growth factors how do we know when intervention is ligand and jagged 1), neuropilin 1, themselves? Might it be possible to necessary? In answer to the former, integrins, HIF-1a, and angiogenic devise tissue and/or cell type selective intervention is clearly preferable before growth factors such as VEGF, fibroblast modulation that would bypass the structural and biochemical changes growth factors, angiopoietins. problems of the less-controlled distri- make the growth restriction process Metabolic reprogramming is HIF- bution of the ligands? Placental difficult to reverse, leading to the latter dependent, and, as indicated earlier, vascular pericytes have been suggested question: how do we detect the initial HIF may be dysregulated in some as a hematopoietic stem cell or stages of growth restriction? The best placental diseases. Targets other than mesenchymal stem cell. They are indicator we have currently that fetal HIF that could be manipulated to sus- involved intimately in vessel stability growth is deviating from normal is the tain the metabolic response to hypoxia and angiogenesis.269-271 Pericytes can fetal growth trajectory derived from might include a variety of enzymes: be detached from their vascular niche serial ultrasound measurements. Given hexokinase, pyruvate kinase M2, lactate by angiopoietin-2, which would in- that this requires multiple measure- dehydrogenase A, pyruvate dehydroge- crease the stimulus for branching ments over weeks of gestation, by the nase kinase, fatty acid synthase, and angiogenesis. Again we lack more time we can verify growth restriction, the glutaminase. knowledge to determine whether these fetus (and placenta) are already well Inhibition of HIF-1a or its gene supporting characters may be the key to advanced through pathologic changes in products has been a primary focus in successful vascular therapies. growth. This crude indicator signals a

JULY 2016 American Journal of Obstetrics & Gynecology S17 Supplement Obstetrics ajog.org process that is already well underway metabolism and subsequent lactic insufficiency.285,286 Similar types of and, at best, will allow for stabilization of acidosis, as observed in glucose supple- growth factor-mediated regulation growth at a much lower percentile. It is mentation interventions. In the case of might be possible via the signaling clear that, without a means to detect amino acids, the interconnected nature of pathways stimulated by IGF-2 or the early stages of the processes that their transport and the broad substrate placental GH. Perhaps the molecular contribute to growth restriction, inter- specificity of amino acid transporters targets for these pathways might be vention will lag well behind. Our own suggest that alterations to an individual restricted to selected cell types by research into placental metabolic pro- transporter will have significant and altering receptor levels rather than cesses has shown that the early stages hard-to-predict ramifications for the altering the concentration of the growth of growth reduction involve a switch transport of other amino acids and for factors themselves. away from oxidative metabolism toward placental amino acid metabolism. Simi- an increased level of glycolytic meta- larly, it is difficult to predict what the ef- Future opportunities. Although the effec- bolism (placental metabolic reprogram- fect of altering the expression of placental tors described earlier may be able to ming).277 As a potential early indicator lipases, fatty acid transport, or fatty acid stimulate integrated pathways for the of growth problems, the initiation of binding proteins might be on processes promotion of fetoplacental growth, the placental metabolic reprogramming may such as oxidative metabolism and the signaling pathways that are activated in provide the crucial biomarkers needed to transport of other nutrients. The essen- this way may be too numerous or too detect the first stages of an altered tial, high-volume nutrients have a wide broadly based to avoid other less desir- growth trajectory. range of metabolic fates, and they or their able effects. Growth factor signaling metabolites play crucial roles in regu- pathways frequently have many trans- Current opportunities. Assuming that lating key metabolic pathways. Not sur- duction pathways that branch off below combined biochemical/imaging methods prisingly therefore, changes in the the growth factor receptor. Another will eventually detect at-risk pregnancies, expression of individual nutrient trans- alternative is the targeting of metabolic or what might we focus on as placental porters are likely to be associated with signaling subnetworks that control mul- targets for intervention? Is it possible to unwanted changes in placental and/or tiple components but do not extend to all see nutrient transporters as possible fetal metabolism. The uncertainty of the endpoints for a signaling pathway. molecular targets? The ones that concern engendered by the alteration of individual For example mTOR is a serine kinase that us here are those that transport the transporters makes them problematic is the focus of a signaling pathway that essential, higher volume nutrients, candidate molecular targets. forms a nexus for multiple nutritional including glucose, amino acids and fatty An alternative approach to targeting and metabolic signals. MTOR affects acids. Because the high volume glucose individual transport or metabolic pro- growth by modulating protein synthesis, transporters (ie, GLUT1 and GLUT3) cesses is the generation of alterations in a lipid synthesis, and energy metabolism, demonstrate unique polarized distribu- manner that combines multiple meta- primarily through phosphorylation of tions between the maternal-facing bolic and/or transport targets. In this the 4E-binding proteins that in turn have microvillous and fetal-facing basal sur- way, the transport and metabolic pro- marked effects on the production of the faces,278,279 altering the balance of glucose cesses that involve specific substrates are proteins that control messenger RNA transporters within the syncytiotropho- modulated in an integrated fashion. We (mRNA) translation.287 In the placenta, blast raises with it the question of the already have detailed information on in addition to effects on protein synthe- selective polarized targeting that is needed many of these integrated pathways. They sis, mTOR appears also to modulate to achieve increased transport. The form the traditional endocrine or growth amino acid transporters by regulating the polarized targeting question arises also factor pathways that mediate regulation membrane trafficking of transporter for amino acid transporters when the of cellular function by extracellular fac- protein.289 There is good evidence to asymmetric distribution acts to move tors. We know for example that insulin- suggest that mTOR activity is propor- sufficient quantities and types of amino like growth factor I (IGF-1) regulates the tional to maternal body mass in- acid against their gradient into the expression of placental glucose and dex288,289; however, in IUGR, syncytial fetus.280 In the absence of targeting in- amino acid transporters281,282 and that it mTOR, while displaying increased formation for these transporters, we need is associated positively with fetal and expression, shows decreased activ- to be assured that interventions will placental growth.283,284 Judicious ity.288,290 This suggests that the mTOR provide the appropriate distribution be- adjustment of the placental IGF-1 pathway may be a good potential regu- tween microvillous and basal faces pathway might allow for an integrated latory point for growth-related inter- necessary to increase flux. Moreover, the regulation of growth processes. Recent vention. Is it possible, for example, to effects of increased transporter density, research has shown that adenoviral- target the mTOR system directly, below although possibly increasing trans- mediated delivery of IGF-1 to the the broadly regulatory phosphatidyli- placental fluxes, will also pose other placenta appears to stimulate glucose nositol-3-kinase/protein kinase B stage problems. In the case of glucose, this is and amino acid transporter expression (Figure 8)? Activation via agents, such as likely to generate increased glycolytic in vivo and to correct placental the small experimental drugs MHY

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1485291 or 3BDO,292 might confine the FIGURE 8 effects to a smaller set of endpoints than Metabolic mechanistic target of rapamycin intervention points 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?293 Another possibility is suggested by the inhibitory actions of metformin on oxidative metabolism.294 Might it be possible to initiate or extend placental metabolic reprogramming effects through artificial stimulation of placental oxygen sparing and consequent increased glycolytic metabolism?295 Ac- tion via subsystems may 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 suc- Potential intervention points (open arrows) for modulation of placental metabolism. These range from cessful. This requires not only detailed extracellular endocrine regulation by insulin-like growth factor I through effects on mechanistic target knowledge of the pathways involved but of rapamycin by activators such as 3BDO (3-benzyl-5-[(2-nitrophenoxy) methyl]-dihydrofuran-2[3H]- also understanding of the lateral or one) or MHY 1485 (4,6-dimorpholino-N-[4-nitrophenyl]-1,3,5-triazin-2-amine) to points in the related pathways that may be affected to metabolic subsystems regulated by mechanistic target of rapamycin. The question marks indicate prevent generation of off-target effects. possible stages in the mTOR pathway that can be targeted. Whether stimulation via a growth fac- IGF-I, insulin-like growth factor I; mTOR, mechanistic target of rapamycin. tor pathway or an intracellular subsystem, Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. 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 pathologic events and the means to stress and that this is further increased these actions are under development. monitor growth and the ameliorative ef- in first, second, and third trimesters Viral or homing-peptide directed nano- fects of targeted interventions is an aspect in pregnancies that result in adverse particles carrying directly active agents or of this process that is just as important as outcomes such as preeclampsia and vectors with trophoblast-specificpro- the intervention itself. diabetes mellitus,296 preterm birth,297 moters may provide the means to over- stillbirth,298 and in those pregnancies come questions of targeting and Targeting oxidative /nitrative stress complicated by maternal obesity.299 specificity. But just as importantly, timing and mitochondrial dysfunction in However, a cause-and-effect relation- is everything! Unless it is possible to placenta to relieve adverse pregnancy ship between increased oxidative stress detect changes in nutrient transport or outcomes (Leslie Myatt, University of and adverse pregnancy outcomes still growth early enough to intervene, modi- Texas Health Science Center San remains to be proved definitively. fication of nutrient transport may exac- Antonio) Oxidative stress is defined as an imbal- erbate rather than resolve problems. Background. It has long been recognized ance between the production of reac- Research to determine the onset of that pregnancy is a state of oxidative tive oxygen species (ROS), including

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and nitric oxide 100 mm.302 Therefore, in mitochondrial function itself can be FIGURE 9 the placenta, the sites of action (intra- vs compromised by severe and/or pro- Generation of oxidative and extra- or transcellular) depends on longed oxidative stress via damage to nitrative stress intracellular and cellular site of synthesis mitochondrial DNA, proteins, and in relation to placental structure such as lipids. We recently have shown that trophoblast thickness (5mm), stem maternal adiposity leads to increased villous diameter (500-1500mm), and the oxidative stress in the placenta and that presence of antioxidant molecules to this is associated with decreased scavenge them.303 trophoblast mitochondrial respiration measured in vitro using a Seahorse Role of mitochondria. The major sources extracellular flux analyzer (Seahorse of ROS are the mitochondrial electron Bioscience, Copenhagen, Denmark).306 transport chain but also plasma mem- This dysfunction is further evidenced Superoxide generated from molecular oxygen by brane enzymes, such as the nicotinamide by decreased mitochondria number, membrane bound (myeloid differentiation pri- adenine dinucleotide phosphate oxi- expression of complexes I-V of the elec- mary response gene 88 oxidase) or cytosolic dases, cytosolic enzymes (including tronic transport chain, and decreased fl (xanthine oxidase) enzymes or the mitochondrial xanthine oxidase, avin enzymes, and placental adenosine triphosphate gener- electron transport chain normally can be effec- cytochrome p450s), lipoxygenases, and ation. In addition, these trophoblasts 295 fl tively dismutated to hydrogen peroxide by su- cyclooxygenases. Although widely appear metabolically in exible because peroxide dismutase. Increased superoxide will acknowledged for their pathologic ef- they cannot switch to alternative energy fects that include covalent modification sources when placed on galactose to attack targets, which leads to oxidative stress. 305 With increasing generation of nitric oxide, nitric of proteins, lipids, and DNA, ROSs also prevent glycolysis. oxide out competes superoxide dismutase for function as physiologic effectors, regulating redox sensitive genes and pro- Current opportunities. Subsequent to superoxide and interacts to produce the more 304 powerful prooxidant peroxynitrite. Peroxynitrite teins. There are many methods acquiring knowledge of increased nitrates proteins at tyrosine residues and cova- available to measure ROS. Because of oxidative stress in pregnancies with lently modifies function usually in a negative their ephemeral nature, direct measure- adverse outcomes, there have been many ment of ROS is difficult; therefore, trials of antioxidant therapy to try and manner. Superoxide dismutase is inactivated 307 when nitrated by prooxidant peroxynitrite, hence measurement of effects that include lipid prevent these outcomes. All have fi leading to a negative feedback loop that leads to peroxidation, covalent modi cation of failed, which is an outcome variously oxidative and nitrative stress. proteins, DNA damage and repair, levels attributed to choice of antioxidant, time NO, nitric oxide; ONOOe, prooxidant peroxynitrite; SOD, super- and activity of antioxidant molecules and duration of treatment, heterogeneity oxide dismutase. and enzymes, and enzymes generating of patients studied, the fact that oxidative Ilekis. Potential placental molecular therapeutic targets. Am J ROS are used. There is no gold standard stress may indeed not be part of the Obstet Gynecol 2016. measure of oxidative stress; rather one pathophysiologic evidence, or the need should chose a measure related to the for targeted rather than global therapy. area of interest.305 This begs the question: what are the tar- superoxide, hydroxyl anion, hydrogen Mitochondria have many roles in gets at the tissue and cellular levels? peroxide, and the ability of various cellular function in addition to energy Should antioxidants that are targeted to antioxidant mechanisms to scavenge production, which includes apoptosis, placenta by use of nanoparticles or them. Further, ROS can also interact steroid synthesis, calcium homeostasis, liposomes be used rather than global with reactive nitrogen species such as and amino acid transport. Changes in treatment? Cellular targets could include nitric oxide to produce the powerful mitochondrial activity can be induced by antioxidant enzymes such as SOD, prooxidant peroxynitrite, which, in turn, environmental factors such as nutrition, glutathione peroxidase, thioredoxin can nitrate tyrosine residues in proteins, hypoxia, aging, and obesity that impact reductase and catalase, the enzymes that such as superoxide dismutase300 (SOD; cellular survival and that are associated synthesize ROS such as xanthine oxidase Figure 9), causing nitrative stress, which with adverse pregnancy outcome. Mito- and nicotinamide adenine dinucleotide affects protein function, usually in a chondria are the major source of ROS phosphate oxidases, extracellular anti- negative manner.301 Not all reactive under physiologic conditions because of oxidants (transferrin, ceruloplasmin, oxygen and nitrogen species are equal in the release of high-energy electrons from uric acid, and bilirubin), intracellular potency, that being determined by their complexes I and III of the electron reducing elements (glutathione, coen- cellular diffusion distance, defined as transport chain; these electrons reduce zyme Q10, and cytochrome c oxidase) the distance moved in aqueous solution molecular O2 to superoxide, which and nutrients and supplements such as in 1 half-life. Hence, hydroxyl anion has normally is scavenged by the mito- vitamins C and E and melatonin. Because a diffusion distance of only 5 angstroms, chondrial SOD isoform, mitochondrial mitochondria are the major source of superoxide 0.4 mm, peroxynitrite 5 mm antioxidant manganese SOD. However, antioxidants, there has been growing

S20 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement interest in mitochondrial targeting of oxidative stress. Dietary manipulation FIGURE 10 antioxidants by compounds such as the (eg, inclusion of omega 3 fatty acids Putative mechanism for the mitochondria-targeted antioxidant drug, and vegetables), the use of antiin- involvement of inflammation- MitoQ, where coenzyme Q10 is linked to flammatories (eg, resveratrol) and mediated placental dysfunction a lipophilic cation to allow adsorption immune selective antiinflammatory de- in fetal programming through the inner mitochondrial mem- rivatives, or targeting strategies that brane.308 Other approaches include the involve miRNA, siRNA, viral vectors, use of selenium,309 which is found in the nanoparticles, and biologics may prove active site of the selenoprotein gluta- useful. These targeting strategies, how- thione peroxidase, or of melatonin, ever, may need to be different based on which functions as an antioxidant.310 the sex of the placenta.

Future opportunities. We recently have Scientific gaps. Current gaps in the shown that the hypoxamir, miRNA 210, knowledge include the absence of can inhibit mitochondrial respiration in proof of a cause-and-effect relationship trophoblast cells,311 hence opening the between oxidative stress and adverse door to targeted therapies to alleviate outcome. Perhaps an animal model of The adverse inflammatory maternal environ- mitochondrial dysfunction and oxida- induced oxidative stress with adverse ments of gestational diabetes mellitus, pre- tive stress in the placenta. The male fetus outcome would facilitate this. In addi- eclampsia, or obesity can generate increased is at greater risk of adverse pregnancy tion, the tissue and cellular localization oxidative/nitrative stress and cause mitochon- outcome (eg, preterm birth and still- and contribution of different sources of drial dysfunction in the placenta in a sexually birth) than is the female fetus, although oxidative stress (ie, including the gener- dimorphic manner. This disrupts placental it is unclear whether this is because the ation and scavenging mechanisms of function and in turn may lead to alterations in male places itself at risk by its desire for ROS) must be studied comprehensively. placental-mediated regulation of maternal greater growth or if the female adopts a Further, the timing of the oxidative stress metabolism and fetal growth and differentiation conservative strategy to ensure success- insult, either early, mid, or late gestation and hence result in fetal programming. ful delivery and propagation of the spe- or continuously, on outcomes needs to GDM, gestational diabetes mellitus. cies.312,313 Sexual dimorphism occurs in be considered when designing drug tar- Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. placental gene expression,314 particularly geting studies. of genes that are involved in the inflammatory response, in response to Identification of potentially useful maternal adiposity and in diseases such or experimental drugs to target provide academic researchers with ac- as preeclampsia. We found increased important cess to investigational drugs from phar- expression of inflammatory and Challenges and advantages of rescuing maceutical companies.318 The program apoptotic markers in placentas of male and repurposing (Christine Colvis, leverages investments that have been fetuses from preeclamptic pregnancies National Center for Advancing made by pharmaceutical companies in a compared with female fetuses.315 Simi- Translational Sciences) variety of drugs and biologics (agents) larly, we found differences in placental Background. The most common reason but for which the company has often expression of miRNA 210 and its mito- for an investigational drug to fail to make times discontinued development. To chondrial target genes between male and it through the Food and Drug Adminis- qualify for inclusion in the program, the female fetuses of lean, overweight, and tration (FDA) approval for marketing is its agent offered by the company has to have obese women that were mediated by inability to demonstrate clinically mean- been through at least a phase 1 clinical differences in the transcription factor ingful efficacy in the disease being studied. trial, so that safety in humans has already NF-kB p50.305 Our current data there- At the stage of phase 2 trials, another been assessed in at least 1 population. By fore indicate that, in conditions such as major reason for failure is simply because limiting the agents to only those that GDM, preeclampsia, and obesity, in- of business strategy decisions that result in have been tested in humans, the new flammatory pathways are activated in the the deprioritization of a drug.316,317 In therapeutic uses program is able to move placenta in a sexually dimorphic manner either case, there is no scientificreason projects quickly to phase 2a trials to test to regulate the production of reactive that the drug could not be pursued for an the agents for efficacy in new indications. O2 and nitrogen species that lead to indication other than that for which it was Under the program, the pharmaceutical oxidative/nitrative stress and to mito- originally being developed. companies provide agents (clinical sup- chondrial dysfunction (Figure 10). This ply including matched placebo and results in placental dysfunction and National Center for Advancing Trans- preclinical material, if needed) and the adverse pregnancy outcome and suggests lational Sciences (NCATS) new thera- data that will be needed to file an inves- that targeting of inflammatory pathways peutic uses program. The new therapeutic tigational new drug application with the might be a useful approach to prevent uses program uses a novel approach to FDA to study the agent for the new

JULY 2016 American Journal of Obstetrics & Gynecology S21 Supplement Obstetrics ajog.org indication. The National Institutes of funds are not offered, there are several Unfortunately, the drugs that will fall in Health (NIH) provides the financial ways to access valuable resources and this category will often be drugs for support for phase 2a trials and, if needed, expertise at no cost to our collaborators. which a generic is available, thereby phase 1b trials and/or preclinical studies. The intramural program that likely has eliminating an enforceable use patent or The NIH and the pharmaceutical the most potential for repurposing drugs regulatory exclusivity. Currently, there companies are turning to the broader for use in pregnant women is the are no effective incentives to pursue a research community to identify novel screening of the NCATS pharmaceutical new indication for a drug that is avail- uses for the agents. The value of this collection. Nearly 2750 small molecular able as a generic, regardless of the indi- strategy bore out when, as a pilot, entities have been approved for clinical cation, unless a unique formulation for NCATS listed 58 agents provided by use by the United States and in other the drug is developed.320 Although this companies in June 2012 and within countries around the world. NCATS has is a barrier for all indications, it is 2 months received almost 160 pre- amassed a screening collection of 2500 of particularly unfortunate for pregnant applications with ideas of indications these, along with approximately 1000 women, which is a population for which that the agents might be used to treat. additional investigational compounds. the development of new drugs (investi- Because of the limited time (12 months) Because they have been approved for gational drugs) is extremely unlikely. allowed under the program for preclin- clinical use, these drugs will have been ical studies, pediatric trials were not used in far more people than the inves- Effect of pregnancy on drugs encouraged in the 2012 pilot. However, tigational drugs that are used in the New pharmacokinetics and in 2014, NCATS issued a new set of Therapeutic Uses program, thereby pharmacodynamics (Maged M. solicitations or funding opportunity providing a more comprehensive risk Costantine, MD, University of announcements, and this time offered profile for the drugs. Collaborators Texas Medical Branch, Galveston) an funding opportunity announcement generally approach NCATS with a high Background. The use of medications specifically for pediatric indications. The throughput screening compatible assay in pregnancy has been increasing pro- companies, in turn, had identified those as a first step toward repurposing 1 of gressively over the past 3-4 decades agents that they believed could be these approved medications. Of course, (Figure 11).321-325 This is predominantly developed for a pediatric indication. In when a drug is first marketed, often, the because of changing in the de- general, it was expected that pediatric only data available on fetal effects mographics of pregnant women, trials would be proposed only when generally will come from preclinical because more women enter pregnancy at there was no adult patient population data. The FDA encourages the use of a later age with increasing prevalence for the disease or for which the mecha- pregnancy exposure registries as a first- of preexisting medical comorbidities nism of action of the drug might be line strategy for gathering information (such as diabetes mellitus, hypertension, meaningful only if modulated at an on drug exposure during pregnancy. asthma, depression, and others) and earlier stage in development than in They have compiled a list of >60 regis- increased risk of obstetric complications adulthood. tries to facilitate the gathering of valuable (eg, nausea and vomiting of pregnancy, There are significant challenges when data.319 Cross-referencing data from GDM, cholestasis of pregnancy, pre- trying to repurpose an investigational such registries with findings from eclampsia.) that require pharmaco- drug for a pediatric indication. Unlike an in vitro assays that were used to screen therapy.320-324 In a recent review, the FDA-approved drug, these drugs often the NCATS pharmaceutical collection average number of medications this is have been studied in only dozens or may provide a strategy for repurposing used by pregnant women is >4.320 This maybe a few hundred adults. So, side- drugs to address placental health increase in the use of medications is also effects have been documented for indications. predominant in the first trimester, where only a small number of individuals. almost one-third of pregnant women use Furthermore, the metabolism of drugs Future opportunities. Repurposing both at least 4 medications.320 This is con- can be very different in younger investigational and approved drugs is a cerning because the first trimester is a individuals than in adults. We face strategy that is pursued currently in crucial period for organogenesis and similar challenges when we think about small and large pharma and in placental development that includes exposing a pregnant woman to an academia. The strategy is attractive to so trophoblast invasion, vascular remodel- investigational drug. Effects of drug many because of the significant cost- ing, and chorionic villi development; in exposure in pregnant women generally savings that result from the use of addition, many women are unaware of will remain unknown until after the drug drugs for which so much early devel- their pregnancy status.320-324 has been on the market for years or even opment has been completed already. In decades. the case of repurposing drugs for use in Pregnancy physiologic changes and pregnant women, it would be expected their impact on medication pharmacoki- Current opportunities. The NCATS that the longer a drug has been on the netic and pharmacodynamics proper- intramural program offers several op- market, the more information we would ties. Pregnancy is characterized by portunities for collaboration. Although have on exposure during pregnancy. significant changes in the anatomy

S22 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement and physiology of almost all systems, FIGURE 11 which affect medications’ pharmacoki- Medication use during pregnancy 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; pharmacodynamics refers to “what the drug does to the body” and usually influences the drug’sefficacy and safety.326-330 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 Secular patterns of use of any medication that was restricted to the first trimester at any time during flow.327,331 Cardiac output is further the period of 1976-2008. Average number of medications and proportion of women taking 4 increased during labor, with additional medications (n ¼ 25,313) is shown. 300-500 mL of blood reentering the Reprinted with permission from Mitchell et al.321 circulation with every contraction. Both Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. systemic and pulmonary vascular re- sistances decrease by 20-30%. Blood pressure also falls toward the end of the first trimester and then rises again in could lead to decreased peak serum (oxidation, reduction, or hydrolysis) the third trimester to prepregnancy concentration, which may necessitate a and/or phase II metabolism (glucur- levels. The increase in renal blood flow higher initial and maintenance dose to onidation, acetylation, methylation, and leads to a parallel increase in the obtain therapeutic plasma concentra- sulfation). Cytochrome (CYP) P450 glomerular filtration rate by approxi- tions.329,334 Additionally, because of represents a family of enzymes and is a mately 40-65%.327,329,332 This can lead the decrease in serum albumin during major route of drug metabolism that is to significant increase (20-60%) in the pregnancy, highly protein bound com- affected by pregnancy (activity increased elimination rates of renally cleared pounds (such as digoxin, midazolam, for CYP3A4, CYP2D6, CYP2C9, medications, which lead to shorter half- and phenytoin) may display higher free CYP2A6, and decreased for CYP1A2, lives and risk of subtherapeutic concen- levels, which increases their peak CYP2C19, and CYP2D6 subtypes). For trations. Examples of these medications plasma concentrations.327,329 The hyper- example, the increase in activity and include lithium, ampicillin, cefuroxime, vascularity and edema of the upper res- abundance of CYP3A4 in pregnancy lead cefazolin, piperacillin, atenalol, and piratory mucosa theoretically may result to an increase in the clearance of its digoxin.326,329 in an increased absorption of inhaled many substrates, such as nifedipine, During pregnancy, maternal blood agents such as corticosteroids.329,335 The carbamazepine, midazolam, saquinavir, volume increases by 40-50%, and total delayed gastric emptying and prolonged indinavir, lopinavir, ritonavir, and many body water increases to almost 8.5 L by small bowel transit time may alter the others.337-340 the end of pregnancy. In addition, bioavailability (decrease maximum con- In summary, maternal physiologic maternal fat stores and fat mass increase centration and time to maximum con- changes affect the pharmacokinetic/ by almost 10-fold. The increase in blood centration) and thus decrease the efficacy pharmacodynamics of drugs through volume and water within the body of oral drugs that are taken as a single changes in the drugs’ absorption, expands the volume of distribution of dose because a rapid onset of action is increasing the volume of distribution, hydrophilic substances.333 On the other desired.329,336 protein binding, renal clearance, meta- hand, the increase in fat mass leads to a Drug metabolism is also altered in bolism, and placental biodisposition. larger volume of distribution of lipo- pregnancy, in part, because of elevated Any novel drug that is identified to act on philic drugs, such as sedatives. For some sex hormones. In general, drug meta- potential placental molecular targets drugs, a larger volume of distribution bolism occurs through phase I must be evaluated in the context of these

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that allow researchers to determine the FIGURE 12 safety and efﬁcacy of these medications. Factors that affect drug pharmacokinetics and pharmacodynamics in Some researchers rely on in vitro pregnancy placental transfusion models; however, these placentas typically are collected after delivery, usually at term, and may not represent the changes in drug pharmacokinetic 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.341-343 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 Pregnancy induces major physiological changes that can affect the pharmacokinetic and pharma- and toxicity of these medications. There codynamic properties of medications. exists a large gap in information CO, cardiac output; BP, blood pressure; HR, heart rate; NVP, nausea and vomiting of pregnancy; SVR, systemic vascular resistance. regarding medications pharmacokinetic Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. properties in pregnancy and during lactation. The effects of placental transporters and biodisposition enzymes changes so that appropriate dosing be opportunities to study the pharmacoki- on medication pharmacokinetic and identified. netic properties of many currently pharmacodynamics, and transplacental used medications with potential impact transfer represent a large gap in our Current opportunities. Pregnant women on placental development and use scientific knowledge. Moreover, the are still considered therapeutic orphans in selective targeting of molecular inability to study placental transport because most current therapeutics were pathways are involved in the patho- expected after delivery with delivered never studied during pregnancy.341-343 physiology of various obstetrical com- placentas widens this scientific gap. In a recent review of the literature, plications. This will lead to more <2% of all pharmacokinetic studies effective treatment of pregnant women, From bench to bedside: processes and involved pregnant women.344 This is while reducing the risks of adverse pitfalls translating research findings concerning because the lack of data on events.341 The new FDA rule regarding into practice paradigms (David M. pregnancy-specific dosage of many drug classification represents a huge Haas, Indiana University) medications leads physicians to extrap- current opportunity to encourage re- Background. When Vannevar Bush wrote olate drug dosage regimens from search in this field. “Science: the endless frontier” in 1945, it nonpregnant subjects or men. This may inspired that science would provide lead to over or under dosing and may Future opportunities. A major obstacle to solutions to health problems. It further reduce efficacy and increase risk of studying medications (current and described how investment in scientific toxicity. In view of these gaps, the novel) in pregnancy is the lack of systems research could serve as a vehicle to

S24 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement enhance the wealth and prosperity of the FIGURE 13 nation.345 It is against that backdrop that Translational research peaks and valleys the public holds out hope for scientific inquiry. This promise engenders a social contract that funding of scientificin- quiry 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 translational 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 This figure illustrates the multiple-step model (5 hills and 4 valleys) of translating research into mechanisms that are aimed at improving practice and public health benefit and the many “valleys of death” that can be encountered along the the translation of findings into practice way. Depicted are the 5 phases of translational research separating 4 valleys: basic discovery and measuring research impact. science to research involving humans (T1), from human studies to evidence-based guidelines (T2), from guidelines to health practice (T3), and from health practices to population health programs (T4). Translational research. The research Reprinted with permission from Meslin et al.345 spectrum generally begins with basic Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. science findings that are then translated to human and clinical research (Figure 13). This is termed “T1” research on the translational spectrum: the cost-effective programs, services, and practice, costs, limited resources, translation of animal and basic science drugs get to all those who need them commercialization issues, broad social research into humans.346 The “T2” step and providers fail to provide the level of policy implications, professional stan- in research translation is taking the care they aspire to.” dards, governmental policy, and general clinically relevant research findings and An example of this is seen in the fact science policy.344 There are many places translating them into practice guidelines that in 2010, the NIH spent $31 billion where potential health-improving and having them become routine prac- on research. However, from 2006-2009, research findings can become derailed. tice in health care.347,348 Some models of only 74 new drugs were approved by the The key is to overcome these difficulties the translational spectrum have included FDA, which was only one-half the and cross the translational valleys of more steps that can include the popula- number approved during the previous death. tion health impact and involvement of epoch.351 One interesting study looked consumers. All of the models serve to at 101 promising technologies that were Translational research in pregnancy. help assess the success of translational published in the highest impact journals. Translational research in pregnancy research at fulfilling the initial promise This report found that, in the 20 years holds additional potential pitfalls that to improve public health. after publication, only 5 of these prom- generally flow from the concerns for the Globally, billions of dollars are spent ising technologies were licensed for impact of therapies on developing babies on biomedical, clinical, and health ser- clinical use, with only 1 angiotension- and potential long-term impacts that vices research, training of providers, converting enzyme inhibitor being used may be unrecognized for years after quality improvement initiatives, and regularly in clinical practice.352 The birth. It is for this reason that, for many safety initiatives.347 However, it has been report found multiple steps where these years, pregnant women were actively demonstrated that, in the United States, promising technologies failed, were lost, excluded from research. There are only 55% of patients receive the recom- refuted, or neglected along the trans- several examples in which translational mended evidence-based care.349 Addi- lational spectrum. It has also been found research failed to detect problems, lead- tionally, 20-30% of patients get care that that promising technologies take a long ing to major public health consequences. is not needed or could be potentially time to get into routine practice, at times In 1956, thalidomide was introduced as a harmful.350 This is a failure in translating up to 17 years.345 These areas of trans- “wonder drug” in Germany.353 It was research findings into practice. Experts lational failure have been coined the purported to be a treatment for nausea have found that this can be due to system translational valleys of death.344 The and vomiting, insomnia, coughs, colds, failures. Grimshaw et al348 noted, “Sys- translational valleys of death can come and headaches. Early studies in mice and tems fail to ensure that effective and from regulations that impede research or rats did not demonstrate congenital

JULY 2016 American Journal of Obstetrics & Gynecology S25 Supplement Obstetrics ajog.org anomalies; therefore, it was used as a delivery units and clearly have reduced Translational Science Awards, focus on sedative and tranquilizer in early preg- the rates of mortality in preterm collaboration and development of net- nancy. However, in 1961, reports sur- newborn infants.359-361 This was greatly works of scientists, stakeholders, and faced of severe congenital anomalies that aided by NIH Consensus Conferences regulatory agencies in an effort to propel included limb reduction defects that led highlighting the benefits and safety of translational science across the valleys of to its rapid withdrawal from the market. the therapy.362 death and into practice.368,369 This was a clear failure of T1 research. Specific to pregnancy, there is a Two notable examples of failure in T2 How to improve translational success. tremendous need for public involve- research in pregnancy have been seen Many have proposed systems that are ment, education, and advocacy. The recently. The withdrawal from the mar- aimed at crossing the translational val- focus of protection of mothers and in- ket of the combination drug Bendectin leys of death. One unifying theme is fants through research, instead of from was done because of lawsuit pressure aimed at getting stakeholder investment research, should be highlighted. Ethicists regarding congenital anomalies.354 The in the translational process. Practicing and children’s advocacy groups should drug was used by a large proportion of providers, the public, industry, and reg- also be engaged. The advent of individ- pregnant women for nausea and vomit- ulatory/governmental agencies must ualized therapeutics and diagnostics ing in early pregnancy. However, no re- have a stake in the translational processes are forcing this conversation to accel- ports of defects were proved. Without an along the way if there is any hope of erate.370 In addition, the utilization alternative, women suffered. The rates translating important research findings of biorepositories can help stimulate of hospitalization for severe dehydration into practice aimed at improving the this translational process by focusing from hyperemesis gravidarum rose public health.347,363-365 Otherwise, fail- on potential pooled resources and dramatically. This was an example of ure at T2 and beyond can occur collaborations. where research demonstrated safety and commonly. The formation of trans- In conclusion, bridging the trans- practice patterns were established, but lational teams that involve the stake- lational research gap from bench to fear and a lack of a supportive response holders can ensure involvement from the bedside has many potential pitfalls. to the public caused the withdrawal of beginning and should involve the entire Pregnancy translational research has had an effective therapy, which led to a translational spectrum.344,347 Effective well-documented missteps but several negative public health impact. Even dissemination of the impact of trans- important success stories as well. more recently, the poor adoption rates of lational research can inform those Although research to cross the trans- the effective human papilloma virus providing funding and the public as to lational “valleys of death” is difficult, vaccines to prevent cervical cancer is the public good that is being gained. using what is known about behavior another example of success at the T1 Psychology of change research has change will be important in ultimately stage, but unfortunately a failure at the noted 3 stages of practice change: fulfilling the promise of scientific T2 stage.355 Despite proven efficacy and awareness, acceptance, and adoption.366 research funding to improve public safety and coverage by the Children’s Most translational research focuses on health and the public good. Thinking Immunization Program, vaccination the first 2 stages, whereas the reason that broadly about stakeholders and in- rates of the target populations remain most promising therapies fail to realize volving these stakeholder teams in the low. their public health impact is the failure of processes across the translational spec- However, there are some noteworthy “adoption” into practice. A provider may trum can help cross the valleys of death translational successes in pregnancy. The know that research shows a benefit and and help take promising discoveries development of Rhogam to prevent Rh accept that it might help the patients, but from bench to bedside. isoimmunization came about from a there may be obstacles to getting it into a confluence of observations along the routine practice pattern; thus, it is not Current opportunities. Individualized translational spectrum. After demon- adopted. This is because “adoption” pharmacotherapy provides opportunities strating efficacy and safety in clinical takes different pathways in the brain currently to tie genomic and other trials, it has been adopted universally as a before these cognitive changes become “-omic” technologies to ongoing clinical standard of care preventive therapy in embedded in practice.365 trials. In addition, the development and pregnancy, saving thousands of babies A focus on stakeholders and align- utilization of saved samples from past every year.356,357 In similar ways, ante- ment of incentives can help with the trials and biorepositories provide a wealth natal corticosteroids for accelerating adoption into practice. Dissemination of of opportunities for analyses using cutting fetal lung maturity in threatened pre- the impact of research findings is crucial edge technologies to assay for biomarkers term birth have been a translational to get stakeholder enthusiasm. For that can serve as therapeutic monitoring success story in obstetrics.358 From instance, the Human Genome Project or individualized pharmacotherapy astute observations in sheep to landmark turned a $4 billion government invest- model development aids for patients. The clinical trials in humans and pivotal ment into $796 billion in economic focus on diagnostic and therapeutic metaanalyses, antenatal corticosteroids growth.367 Translational teams, like studies in human models and humans is a are now used routinely on labor and those stimulated by the NIH Clinical and clear priority moving forward.

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Future opportunities. Clinical trials in the FIGURE 14 future should have clear response bio- Schematic of extracellular microRNAs derived from human trophoblasts markers, pharmacokinetic/pharmacodynamic/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 MicroRNAs can be released from the trophoblast layer in different forms: microvesicle-enveloped as often as possible. Another clear gap in form; apoptotic bodyeenveloped form; nano-sized, exosome-encapsulated form; and RNA- much of the therapeutic research in binding, protein-bound form. Exosomes are formed by budding in intraluminal vesicles to form pregnancy is a lack of long-term follow multivesicular bodies. Multivesicular bodies fuse with the plasma membrane and release their up of the infant. Ensuring funding for intraluminal vesicles as exosomes into the extracellular space. In contrast, microvesicles are pro- follow-up studies, including potential duced directly by budding and the detachment of membrane vesicles from the plasma membrane. “ ” -omic studies, of the offspring should Apoptotic bodies (blebs) derive from cells that are undergoing apoptotic fragmentation and the be a priority. formation of membrane-enclosed vesicles. miRNA, microRNA. Evolving technologies for placental Reprinted with permission from Ouyang et al.395 specific therapeutic drugs Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. Trophoblastic nanovesicles, miRNA, and their function (Yoel Sadovsky, MD, Magee-Womens Research Institute) clear that nucleic acids, and particularly It has become clear recently that, in Background. Coordinated communica- sRNA molecules, traffic among tissues addition to the effect of miRNAs on tion among cells and tissues is critical and impact cell biology. intercellular gene regulatory networks, for development, homeostatic func- miRNAs are also packaged within tion, and adaptation to change. The MiRNAs and extracellular nanovesicles. A extracellular vesicles, where they can be trafficking of information between the major class of these noncoding RNAs, trafficked to local and distant cell types fetoplacental and maternal compart- and 1 of the best studied, is the family of (Figure 14). Transport within extracel- ments is essential for fetal develop- small regulatory RNAs called miRNA. lular vesicles may provide substantial ment, growth, and pregnancy health. It MiRNAs originally were described in the advantages, including stability, intravas- also serves to reduce maternal-fetal nematode Caenorhabditis elegans and cular processing, and selective delivery conflicts and to balance biologic re- were later found in the genome of many to target cells.371-373 Diverse cell types sources for the benefitofthe2organ- organisms, including humans. They (eg, hematopoietic cells, mast cells, isms. In past years, it was believed that are single-strand RNA molecules of platelets, neurons) produce and release communication between the fetopla- 20-24 nucleotides that characteristically extracellular vesicles, which can be cental unit and the mother was repress gene expression by guiding an found in the blood, urine, saliva, breast executed by hormones and growth RNA-induced silencing complex to a milk, amniotic fluid, ascites, cerebro- factors, which are soluble in the blood target RNA, with subsequent attenuation spinal fluid, bile, lymph, tears, and or are bound by protein carriers, and of gene expression by the inhibition semen.374-377 These vesicles are charac- serve as paracrine or endocrine signals of mRNA translation and/or mRNA terized by their size, shape, and con- that control pregnancy health. It is now degradation. tent.378 Importantly, when released to

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extracellular vesicles, exosomes have FIGURE 15 been studied extensively in recent years Schematic depicts the exosome-mediated induction of viral resistance because of their ability to modulate target cell physiology, immune function, and carcinogenesis.393 Exosomes also harbor miRNAs as part of their cargo.371,394,365 We and others recently have characterized the trophoblastic miRNA landscape and the potential role of trophoblastic miRNA in fetoplacental to maternal communication.393-397 Whereas exosomes are found in the circulation of pregnant women, little is known about trophoblastic exosomes. 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.398-401 Placental exosomes have been implicated in several discrete functions, such as immune modulation,402-404 where exosomal cargo proteins, such as galectin-3405 and human leucocyte antigen-G,406 may perform an Chromosome 19 microRNA clusters were transferred to recipient cells. Primary trophoblast cells immune modulatory function. 407 Other release exosomes that contain chromosome 19 microRNA clusters, which are taken up by recipient exosomal cargo molecules, such as Wnt/ cells, thereby mediating chromosome 19 microRNA clusteredependent autophagy. Incoming viral b-catenin, fibronectin, and prostaglan- particles (red) are likely trafficked in endocytic vesicles from the endosomal pathway into preexisting dins, may play a role in supporting ho- autophagosomes, which then fuse with lysosomes to form autolysosomes, as a mechanism to meostasis throughout pregnancy.408-413 degrade these virus-containing vesicles. The gray arrows indicate the stepwise trafficking of We have shown that the miRNA trophoblast exosomes and viral particles to a recipient cell. landscape within the cargo of human AL, autolysosomes; APs, autophagosomes; C19, chromosome 19 microRNA clusters; EXO, exosomes. trophoblastic exosomes is nearly iden- 396 Reprinted with permission from Delorme-Axford et al.417 tical to that of villous trophoblasts. Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. The most abundant trophoblastic miRNA species are those derived from the chromosome 19 miRNA cluster the extracellular space, these vesicles may and fission of the plasma membrane, (C19MC),396,414 which is the largest target local and distant cell types, and where microdomains and associated miRNA cluster in the human genome, release nonhormonal signals that control protein cargo are enveloped for direct spanning approximately100 kb of physiologic condition, determine disease cellular exit.385-389 Exosomes (40-150 nm genomic DNA and harboring 46 intronic risk, and even harbor therapeutic po- in diameter) originate from the intracel- miRNA genes that express 58 miRNA tential.379-382 The major types of extra- lular endosomal network through serial species.396,413,415,416 Importantly, we cellular vesicles include apoptotic blebs, processes that are initiated by the found that, in comparison with non- microvesicles, and exosomes, each pro- enrichment of endosomal membrane trophoblastic cells, primary human tro- duced by different pathways. Apoptotic complexes by tetraspanins and other phoblasts are resistant to infection by blebs (500-4000 nm in diameter) are proteins.390,391 This is followed by diverse types of DNA and RNA viruses, formed during partial or complete cell budding of intraluminal vesicles within which include clinically relevant species death and disintegration and contain multivesicular bodies (MVBs) and sub- such as herpes simplex virus type 1, cell organelles and cytoplasmic proteins sequent movement to the plasma mem- rubella, HIV, and cytomegalovirus, but that are subsequently cleared from the branes, where multivesicular bodies fuse not the nonviral pathogens Listeria circulation by macrophages.377,383,384 with the cell membrane to release monocytogenes and Toxoplasma gon- Microvesicles (100-1000 nm in diam- actively intraluminal vesicles as extra- dii.393,417-419 A remarkable finding was eter) originate from outward budding cellular exosomes.377,392 Among all that the viral resistance could be

S28 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement transferred to other, nonplacental pri- extracellular vesicles to the maternal Scientific gaps. The current and future mary cells and cell lines by exposing blood stream and possibly to the opportunities that were summarized these cells to a medium that was pre- fetal compartment. Whereas C19MC require us to bridge pertinent knowledge conditioned by primary human tro- miRNAs are packaged distinctly within gaps. We must understand the process of phoblasts or to exosomes that are these placental vesicles, it is not clear cargo loading to diverse trophoblastic produced in human trophoblasts whether there are other, placenta- vesicles and the potential selectivity of (Figure 15). We also found that this specific, molecules and signals that are this process. This should be followed by effect was mediated, at least in packaged within vesicles. Importantly, analysis of the mechanisms used by part, by exosome-packaged C19MC modern technology enables us to sepa- trophoblastic exosomes to enter target miRNAs.393,396 Cells transfected with rate the different types of vesicles and to cells selectively and impact their biologic a bacterial artificial chromosome that associate them with disorders of preg- condition. The current state of perinatal harbors the C19MC locus or with nancy that reflect placental function. It is science raises additional questions: individual, highly expressed C19MC also possible that the state of pregnancy Do surface and cargo proteins within miRNAs also became more resistant to changes the repertoire of extracellular trophoblastic extracellular vesicles play a viral infections. Our data showed that vesicles that are produced by maternal role in this process? What determines this effect of exosomal C19MC miRNAs tissues, thus adding to the landscape of targeting by extracellular vesicles? Can was mediated by the stimulation of pregnancy-related extracellular vesicles. fetal vesicle-bound or free miRNAs and autophagy in recipient cells and that This creates a new opportunity to catalog other types of RNA molecules cross the pharmacologic or genomic inhibition of these vesicles, assess their origin, con- placenta into the maternal circulation autophagy diminished the conferral of tent, and cellular targets and to define and inform on the health of fetal tissues? antiviral response to recipient cells.393 the normal landscape of extracellular How early in gestation can we find and Although C19MC miRNAs likely act in vesicles (the “vesiculome”) throughout isolate placental extracellular vesicles concert with other antiviral responses, human gestation. and RNA from maternal blood? The our data pointed to a unique and answers to these and related questions transferrable antiviral response that is Future opportunities. The development will greatly impact the field of placental mediated by human trophoblast-derived of new tools to isolate extracellular biology and its translation to pregnancy exosomes. vesicles efficiently may allow us to diagnostics and therapy. Although the mechanisms, dynamics, examine local and systemic targets of and targets of miRNA-containing trophoblastic extracellular vesicles and Aberrant regulation of myometrial trophoblastic exosomes remain un- whether their cargo faithfully informs contractility by maternal cell-free known, our recent human and mouse the health of trophoblasts. It may also plasma (CFP) RNA of placental origin: data suggest that trophoblastic C19MC allow us to identify target cells and screening and therapeutic implications miRNAs are transferred primarily from interrogate the impact of pregnancy- (Carl P. Weiner, MD, University of trophoblasts to maternal tissues. This specific extracellular vesicles on these Kansas School of Medicine) is plausible, because trophoblasts are cells in health and disease. Although Background. The CFP transcriptome is bathed directly in maternal blood yet initial work has centered primarily on altered by numerous disorders, often separated from the fetal blood by a maternal tissue targets, future research reflecting the stage of disease develop- basement membrane and endothelial may focus on intraplacental paracrine ment.420 Although the biologic roles of cells. Together, these analyses shed new targets and fetal targets of placental these coding and noncoding RNAs are light on miRNA-based communication extracellular vesicles. These opportu- unclear, they are not simply cellular among the fetoplacental and maternal nities, coupled with recent de- debris. Synthesized and released by a compartments. Importantly, our data velopments in understanding of miRNA range of cells that include human cho- suggest an extraordinary means of function, will require the development rionic villi, their biologic stability and nonhormonal communication between of innovative label-free technologies to target tissue specificity are thought to the placenta and the maternal-fetal collect and sort extracellular vesicles, reflect their release within either exo- compartments, which suggests a critical which will shorten the time to clinical somes or shedding vesicles or attached role for miRNAs in pregnancy health. translation and potentially offer a dra- to argonaute 2 proteins.421 Cell-to-cell matic improvement in our ability to transfer and their ability to function Current opportunities. Although it is assess placental health dynamically once transferred are shown in studies clear that the plasma contains several throughout pregnancy using maternal that used mRNA/miRNA-loaded extra- types of extracellular vesicles, their mo- blood tests. Knowledge in this field may cellular vesicles and protein com- lecular and metabolic cargo are largely also stimulate the development of plexes.422-424 unknown. Moreover, our understanding nanovesicles as carriers of noncoding Based predominantly on studies of of the function of these vesicles is rudi- RNAs, drugs, and chemicals that selec- symptomatic women, spontaneous pre- mentary. The formation of the placenta tively target placental cells for thera- term birth (sPTB) is considered an during pregnancy adds a new source of peutic purposes. inflammatory process.428 We measured

JULY 2016 American Journal of Obstetrics & Gynecology S29 Supplement Obstetrics ajog.org both the mass restricted (MR) score and The CFP transcriptome is altered in for sPTB at <33 weeks gestation by the IL-6 in 1004 consecutive amniotic fluid women destined for sPTB by 16 weeks. end of the first/beginning of the second samples at 16 weeks from healthy preg- Using longitudinally obtained banked trimester that interfere with myometrial nancies with known outcomes (unpub- plasma samples from women with quiescence, thus potentially creating lished data). Ten percent delivered at known pregnancy outcomes and an environment that favors sPTB. The <37 weeks gestation; 4% delivered at microarray/next-generation sequencing, early pregnancy time frame suggests <32 weeks gestation. Nineteen percent we identified and confirmed >90 novel that effective therapy will require early had an MR score of 1; 2% had an MR CFP RNAs in the plasma of women who detection and initiation, which is score of 3 or 4, which indicated signifi- were destined for sPTB between 26-32 consistent with the general failure of cant inflammation. However, there was weeks gestation; some of the marker available tocolytic agents. It highlights no relationship between either sPTB or expressions were altered as early as the preeminent importance of eluci- preterm premature rupture of mem- 16 weeks gestation. In a second cohort, dating the events that are involved in branes and the MR score; 23 of 27 we found that several markers that were human implantation and early placen- deliveries at <28 weeks gestation abnormal at 16 weeks gestation were also tation. As such, the sPTB studies occurred to women with an MR score of abnormal at 12 weeks gestation. We then described may serve an investigational 0. IL-6 levels increased as the MR score determined that 5 CFP markers were paradigm for the other great pregnancy rose (0 ¼ 102 pg/mL; 3 ¼ 451 pg/mL, known from studies of other cell systems syndromes such as preeclampsia and and 4 ¼ 706 pg/mL) but was unrelated to to interact with 7 myometrial gene/gene hypoxia that are associated FGR and subsequent sPTB. Whatever the role of products belonging to a previously perinatal brain damage. inflammation in preterm or term birth, identified cluster of genes: the preterm The first task along the path to unit seemingly occurs after 20 weeks initiator set.425 Five of the 7 myometrial derstanding human placentation is gestation. genes were involved with the regulation descriptive: the creation of an appro- We proposed in 2011 that mRNA and of intracellular calcium. priately sized research medical center miRNA were candidate mechanisms for network for sample processing (using the regulation of myometrial quiescence CFP RNAs originate from the pla- new and established biobanks) for the activation (unpublished data) and have centa. We next sought the anatomic rapid construction of an “omics” atlas of been testing the hypothesis that CFP origin of the 5 CFP RNA markers; each “normal” from the time period covering RNAs enable communication between was dramatically overexpressed in the implantation through at least 16 weeks mother and pregnancy and that, as a placenta of women with <33 sPTB gestation. Such an atlas would serve as a direct result, changes in the maternal weeks gestation, but not the placenta of basis for all future research and generate CFP transcriptome predict both the women delivered prematurely absent a host of new targets for hypothesis- occurrence and mechanism of sPTB. labor and of women delivered at term in based testing in vitro using systems labor. similar to those described earlier. Methodologic refinements. In 2007, we developed an extraction method that CFP RNAs markers of sPTB can regulate Future opportunities. Real-time assess- yielded increased quantities of both CFP myometrial contractility. We then focused ment of the human placenta all but re- nucleic acids and proteins in a single on 1 particular CFP mRNA biomarker, quires noninvasive methods that may process. The typical yield of total apolipoprotein, that, in silica, interacted not exist presently or have not yet been RNA extracted is 18-35mg/mL plasma, with the interferon gamma receptor, applied to the study of the placenta. So enabling comprehensive, transcriptome which in turn interacted with 4 addi- much about human placental function is wide study with the use of multiple tional preterm initiator genes. Over- shrouded by darkness that the areas for microarrays and/or next-generation expression of apolipoprotein in human investigation are almost unlimited, each sequencing, plus the confirmatory po- pregnant myometrial cells increased with the potential for making a contri- lymerase chain reaction studies all on the both intracellular calcium flux and cell bution. Yet, it is highly likely that much same patient plasma sample. contractility. The addition of interferon placental dysfunction has its foundations To speed marker validation and gamma receptor siRNA blunted the set by the early second trimester and that facilitate translation into the clinical stimulatory effect of apolipoprotein, it is here that new insights are needed arena, we developed a series of tech- which demonstrated that interferon desperately. The application of newer niques that included economical, high- gamma receptor was at least 1 of the organ on chip technology could allow throughput gene quantification that targets of the apolipoprotein mRNA. the testing of potential interventions allows for the running of panels of genes that will be identified during atlas con- to predict sPTB within a few hours. Current opportunities. These findings struction.426 Real-time approaches will The method allows us to run all support the often stated premise that require either new imaging modalities controls necessary for marker quantifi- sPTB is a result of placental dysfunction. or be “omics” in nature. As discrete cation in a single polymerase chain Specific CFP RNAs are released from markers of placental function linked to reaction well. the placenta of women who are destined specific disease phenotypes emerge, it

S30 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement may be possible to reduce the processing FIGURE 16 from a minimum of a laboratory work Translational steps for the development of nanoparticles for placental day to less than a few hours, providing drug delivery 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 Nanoparticles may contain therapeutic, diagnostic, and/or targeting components. In vitro, ex vivo, delivery (Erik Rytting, University of and in vivo models will be used to investigate proof of concept and safety before clinical trials. Texas Medical Branch, Galveston) Nanomedicine. The field of nano- Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. medicine includes nanoparticles for medical diagnostics, nanoparticles for therapeutic delivery, and particles play- assessments of particle size, zeta poten- developed in our laboratory are based on ing both roles, referred to as theranostic tial (surface charge), encapsulation effi- lactic acid polymer. When this polymer nanoparticles (Figure 16).427 Types of ciency, and drug release kinetics. Particle breaks down within the body, the so- nanoparticles that are used in therapeu- size and surface charge can have a called degradation product is lactic tic delivery include liposomes, solid lipid significant effect on both particle distri- acid, a naturally endogenous compound. nanoparticles, polymeric nanoparticles, bution and cellular uptake. Encapsula- polymeric micelles, dendrimers, and tion efficiency represents the mass of Transport of nanoparticles across the pla- polymeric DNA or siRNA complexes drug actually encapsulated within a centa. To predict placental drug and called polyplexes.428 Advantages of nanoformulation as a percentage of the nanoparticle transport, our laboratory nanoparticles for drug delivery include intended drug loading. The drug release focuses primarily on 2 experimental improved bioavailability, protection of profile will demonstrate whether sus- models: the BeWo b30 human tropho- therapeutic payload, controlled release, tained delivery of the drug from the blast cell line and the dually perfused and increased drug targeting effi- nanoparticles can be anticipated, which human placental lobule. Our compara- ciency.429 Targeting ligands such as an- can reduce dosing frequencies. It has tive study demonstrated excellent cor- tibodies, peptides, aptamers, or small been demonstrated that drugs and pro- relation between the in vitro BeWo cell molecules can be conjugated or adsor- teins are still pharmacologically and line model and the ex vivo perfused bed onto the surface of nanoparticles to biologically active after their release from human placenta model in the prediction promote the accumulation of the parti- nanoparticles.431,432 of the transplacental transport of 4 cles in specific regions or tissues that are Biocompatibility is an important model compounds.433 A separate study facilitated by binding of the targeting consideration in the development of then expanded the comparison with ligands to a particular receptor.430 nanoparticles for therapeutic delivery. placental transport data in the literature The nanomaterial excipients themselves and reported a strong correlation (cor- Nanoparticle characterization and bio- must not elicit toxic responses such relation coefficient ¼ 0.95) between the compatibility. Characterization of drug- as inflammation or oxidative stress. in vitro relative transfer rate and the loaded nanoparticles typically includes Many of the polymeric nanoparticles ex vivo transfer index.434 Both

JULY 2016 American Journal of Obstetrics & Gynecology S31 Supplement Obstetrics ajog.org experimental models appear to be suit- nanoparticles conjugated with folic acid to establish the pregnant baboon as a able for nanoparticle transport studies as to take advantage of the relatively high nonhuman primate model for pharma- well. For example, the transplacental expression of folate receptors in the cokinetic investigations.455-460 There is transfer of polymeric nanoparticles has placenta. This has resulted in greater also a need to refine the pool of potential been shown to be size-dependent in both cellular uptake and transport of the placental targeting moieties to minimize models, and limited transfer of silica nanoparticles across BeWo cells. off-target nanoparticle distribution. nanoparticles was demonstrated in both Side-effects can be averted when the BeWo cell and placental perfusion ex- Current opportunities. Immediate appli- disbursement of medication to off-target periments.435-437 cations of these technologic advances sites is reduced. A necessary, but Additional studies have shown that include fetal drug therapy and placental exciting, task will be to determine the gold nanoparticles do not cross the term therapy. Although medication during magnitude of dose reductions that are human placenta, that the transplacental pregnancy is most often prescribed to possible when targeted therapeutic transport of poly-amidoamine den- treat maternal conditions, there are a strategies are applied. Nanoparticle- drimers is limited, and that liposome number of fetal diseases for which a mediated delivery to the placenta offers transport across the placenta is size- targeted, transplacental delivery strategy tremendous benefits for maternal-fetal dependent.438-440 The observed size potentially could attenuate the adverse medicine. dependence of nanoparticle transport maternal side-effects that are associated has been extended to demonstrate with current treatment modalities. Maternally sequestered delivery increased drug transport across placental These include fetal cardiac arrhythmias, systems for prevention of fetal drug trophoblast cells when the drug is congenital adrenal hyperplasia, and fetal exposure (Gene Bidwell, University encapsulated in smaller polymeric thyroid disorders.450 Targeted drug de- of Mississippi Medical Center) nanoparticles (diameter, 146 nm) livery to the placenta itself could also Background. Preeclampsia is a common compared with larger nanoparticles improve therapeutic options for hypertensive disorder of pregnancy and (diameter, 232 nm).441 Literature re- malaria-infected placenta or prevent is one of the leading causes of maternal ports of nanoparticles that have been mother-to-child transmission of HIV.451 and fetal morbidity and death. There is administered to pregnant rodents have currently no effective intervention for included titanium dioxide nanoparticles, Future opportunities. Nanotechnology preeclampsia short of induced delivery, silica nanoparticles, zinc oxide nano- has also been suggested for placental which makes it a leading cause of particles, iron oxide nanoparticles, and diagnostic applications.452 Opportu- premature birth. Improvements in gold nanoparticles; results vary depend- nities abound for the development of preeclampsia management have been ing on particle size, surface charge, and nanoparticle-mediated biosensors and largely stifled because of deleterious gestational age.442-446 imaging agents, and it is likely that many effects of proposed small-molecule recent advances in cancer diagnostics, therapeutics on the developing fetus. Targeting strategies. Strategies for tar- for example, can be utilized to monitor The objective of our studies was to geted delivery of nanoparticles to the the structure and function of the human develop a drug delivery system capable of placenta include the identification of placenta.453 As progress towards these stabilizing novel therapeutic agents in peptides by phage display.447,448 Re- goals continues, it is imperative that the the maternal circulation while protecting searchers have reported enhanced biocompatibility of such nanomaterials them from entering the fetal circulation. transcellular transfer of T7 bacterio- is investigated thoroughly to prevent phages across BeWo cells with an unintended consequences of fetal expo- The use of a biopolymer drug carrier to integrin-binding amino acid sequence sure to any potentially harmful imaging prevent drug transfer across the pla- peptide. Harris et al used a similar agents. For example, gadolinium-based centa. The major focus of our research approach in pregnant mice and identi- magnetic resonance imaging contrast group is to develop drug carriers capable fied two peptides with increased agents are not recommended for use of preventing placental transfer and fetal placental binding, designated as amino during pregnancy because of concerns exposure to therapeutic agents. Protein acid sequence consisting of for nephrogenic systemic fibrosis.454 transport across the placental barrier is CRGDKGPDC and KRK (unpublished highly regulated, and proteins that are data).448 Kaitu’u-Lino et al449 designed Scientific gaps. Steps to translate the not substrates for active transporters bacterial-derived nanocells that are preclinical promises of this technology rarely cross into fetal circulation. To take coated with antibodies to epidermal towards clinical trials and clinical prac- advantage of this, we are using a protein- growth factor receptor. The uptake of tice will include scaling proof-of- based biopolymer to fuse to therapeutic these epidermal growth factor concept results from in vitro and small agents and sequester them in the receptoretargeted delivery vehicles in animal studies to larger animal models maternal circulation (Figure 17). This ex vivo human placental explants was with placental structure and function biopolymer, termed elastin-like poly- greater than that of nontargeted nano- more similar to human placenta. For peptide (ELP), is based on a repeating cells. We have synthesized polymeric example, recent progress has been made sequence found in human elastin.461

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ELP is an ideal drug carrier for several FIGURE 17 reasons. Because of its origins in human Model of the elastin-like polypeptide drug delivery vector elastin, it is not recognized as foreign by the immune system, does not induce an inflammatory response or immunogenicity, 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.462-464 Also, because it is protein- based, ELP can be produced by recom- binant expression in bacterial or eukaryotic systems,459,460,465 and it is easily purified by a nonchromatographic procedure called inverse transition cycling.460,466 This makes scale-up to Elastin-like polypeptide is a biocompatible protein polymer that is used to protect attached cargo production levels that is required for from degradation, rapid renal clearance, and immunogenicity to prevent the transfer of cargo across therapeutic development very feasible. the placenta. A, Elastin-like polypeptide is modified with targeting agents or cell penetrating peptides Finally and most importantly, because of to enhance organ specificity or to mediate target cell uptake. Elastin-like polypeptide is also modified its large size, ELPs can protect small with cargo therapeutic peptides, therapeutic proteins, or with reactive sites for covalent attachment peptide or drug cargo from rapid of small molecule drugs. B, Placental transfer of elastin-like polypeptide. Quantitative fluorescence degradation and extend their plasma analysis revealed that the elastin-like polypeptide carrier accumulated highly in the placenta (red and half-life and bioavailability. yellow) but did not penetrate into the fetal circulation after bolus injection or chronic infusion in a rat Our strategy involves the use of a core pregnancy model. ELP biopolymer as a drug carrier; this Reprinted with permission from George et al.467 biopolymer is modified at its amino Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. 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 spiral artery remodeling during early approach is to target the pathways that recent study, we examined the pharma- placental development.468-470 Because lead to hypertension, impaired renal cokinetics and biodistribution of ELPs in the molecular basis for this failed function, neurologic complications, and a rat pregnancy model. The ELP carrier remodeling is understood poorly and other components of the preeclampsia accumulated at very high levels in the because a robust biomarker to predict syndrome in hopes of delaying the need maternal kidney, liver, and placenta after patients who will become preeclamptic for induced delivery for as long as systemic intravenous infusion, but little is lacking, it is not yet practical to possible; we aim to do so in a manner to no ELP crossed into the fetus after design targeted therapeutics to correct that protects the fetus from exposure to either a bolus administration or 5 days of spiral artery remodeling and prevent the therapeutic agents. continuous infusion (Figure 17, B).467 preeclampsia. However, preeclampsia The molecular pathways that lead to This study indicates that the ELP drug symptoms do not present until later in the symptom precipitation of symptoms carrier may be a powerful means of pregnancy, and, unlike the initiating in preeclampsia are a consequence of sequestering fused therapeutics to the factors in spiral artery remodeling, the placental hypoperfusion and hypoxia. maternal circulation and preventing fetal molecular pathways that lead to symp- In response, the hypoxic placenta re- drug exposure. tom precipitation are much better un- leases a multitude of factors that derstood. Therefore, now is the optimal affect the mother at nearly all organ Mechanisms of symptom onset in pre- time to develop therapeutics that are systems.473 Briefly, the major players in eclampsia and potential therapeutic targeted to the progression of pre- the progression of the preeclampsia interventions. Preeclampsia is a disease of eclampsia with the goal of extending syndrome can be divided into 3 groups, placental insufficiency that is rooted in a pregnancies and improving fetal out- antiangiogenic factors, proinflammatory poorly understood process of impaired comes.471,472 The overall goal of our factors, and inducers of ROS

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preeclampsia.475 This makes sFlt-1 one FIGURE 18 of the major targets for preeclampsia Model for the role of antiangiogenic factors and inflammatory cytokines drug development. One strategy we are in preeclampsia. using is to supplement VEGF levels by administering an exogenously prepared ELP-VEGF fusion protein.470 We have previously found that VEGF signaling activity is maintained after ELP fusion,460 and our goal with this strategy is to restore the angiogenic balance that was lost when sFlt-1 production was increased. In addition to the reduction of systemic endothelial dysfunction, this strategy might have the added benefitof 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-1,476 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 VEGFR2,477 supplementation of PlGF may be less prone to side-effects compared with the direct administration of VEGF. Other major drivers of preeclampsia The ischemic placenta is known to be a source of the vascular endothelial growth factor antagonist symptoms include the chronic systemic protein sFlt-1 and many proinflammatory cytokines. These factors cooperate with 1 another to inflammatory response (exacerbated produce systemic inflammation and endothelial dysfunction in the mother, which is manifested by placental production of inflamma- clinically in multiple organ systems. Illustrated are our proposed strategies for interfering with these tory cytokines).478 The inflammatory processes by either supplementing vascular endothelial growth factor or placental growth factor response in preeclampsia is known to be levels by the administration of exogenous elastin-like polypeptide-fused proteins or by giving elastin- mediated by tumor necrosis factor-a,479 like polypeptideestabilized inhibitors of NF-kB. proinflammatory interleukins,480 and ELP, elastin-like polypeptide; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; sFlt-1, soluble fms-like tyrosine agonists of TLR signaling481 that is pro- kinase; VEGF, vascular endothelial growth factor. duced in the placenta and possibly by Adapted with permission from Bidwell and George.473 systemic macrophages and/or endothe- Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016. lial cells. A common mediator of all these inflammatory signaling pathways is the transcription factor NF-kB. Another (Figure 18).470 These factors induce sFlt-1. SFlt-1 levels were found to be strategy that we are exploring is the ex- endothelial dysfunction and systemic elevated dramatically in patients with amination of NF-kB as a potential drug inflammation in systemic vascular beds preeclampsia with a corresponding target for preeclampsia. As a first of the maternal circulation that ulti- reduction in the plasma levels of its attempt, we have generated an ELP- mately lead to the clinical manifestations ligands-VEGF and PlGF.474 VEGF is delivered peptide inhibitor of NF-kB of preeclampsia. known to be important in endothelial signaling. The ELP fusion protects this One of the major proteins produced cell health, and its sequestration by sFlt-1 peptide from rapid renal clearance after by the hypoxic placenta thought to drive binding is hypothesized to lead to many intravenous administration, enhances its maternal preeclampsia symptoms is of the renal and vascular effects of placental deposition over 30-fold relative

S34 American Journal of Obstetrics & Gynecology JULY 2016 ajog.org Obstetrics Supplement to free peptide, and prevents its transfer the causative factors that hamper proper this goal also elucidates another major across the placenta (unpublished data). placental formation in preeclampsia, gap in the field, the need for robust We hypothesize that a maternally and we need robust biomarkers to biomarkers to predict the onset of the sequestered ELP-fused NF-kB inhibitor identify patients who will experience the disease early in pregnancy. Future will be effective as an antiinflammatory disorder. research in these areas will be critical for agent and may slow the progression of Another area that currently stands as a filling this gap. preeclampsia. hurdle to the development of therapeu- Data were also presented regarding tics for preeclampsia and other disorders ongoing clinical efforts in the United Current opportunities. The use of ELP or of pregnancy is the high level of risk States and in Europe that are testing related macromolecular carriers to aversion when treating pregnant novel interventions for preeclampsia and prevent fetal drug exposure represents a mothers. This risk aversion is well justi- FGR, including agents such as oral promising new strategy for drug devel- fied given the vulnerability of the patient arginine supplementation, sildenafil, opment for disorders of pregnancy. population, and we have to address these pravastatin, a virally delivered VEGF for As described earlier, we are using risks by doing all we can preclinically to local administration into the uterine ar- ELP fusions with therapeutic peptides ensure the safety of developmental tery to improve blood flow and oxygen or proteins targeted to pathways of therapeutics. One strategy for this is to supplementation therapy. Proposals importance in preeclampsia, but this prevent fetal drug exposure using drug were also made to improve fetal health, strategy is not limited to proteinaceous carriers, as described earlier. In addition, not by targeting maternal blood flow but therapeutics or to preeclampsia. ELP is we must also strive to ensure that our by enhancing nutrient transport to the amenable to fusion with small molecule therapeutics is not adversely affecting fetus by modulating glucose and amino drugs, and other projects in our labo- normal placental function. To address acid placental transporters. Strategies ratory are examining the ability of these issues, we must use the best pre- were presented for inhibiting mito- ELP to prevent transfer of known-toxic clinical models at our disposal to study chondrial dysfunction and oxidative or teratogenic drugs across the placenta, placental drug transport, fetal exposure, stress to improve placental health; the thus potentially opening the door and drug effects on normal placental roles of inflammation on both tropho- for safe use of drugs that currently function, and we must follow offspring blast function and in the advanced stages are avoided in the pregnant patient in our preclinical models to insure of preeclampsia were discussed. The population. normal physical, mental, and physio- roles of miRNAs and placentally derived logic development. Only after collecting exosomes were discussed in the context Future opportunities. Our current strat- robust data in these areas should we of their use for diagnostics and as drug egy is to use the ELP system to intervene attempt to begin testing in human targets. These diverse pathways illustrate in late gestation, with therapeutics that pregnancies. Although we should pro- many opportunities for drug interven- are targeted to pathways of currently ceed with caution and use every means at tion. Although a number of potential known importance in preeclampsia. our disposal to ensure safety, we should drugs to target these pathways are avail- Future work will expand this strategy as not let fear of doing harm stifle innova- able and have been shown to be relatively the list of potential drug targets con- tion in the development of diagnostics safe, the therapeutic benefits of the ma- tinues to grow. The ELP system is and therapeutics for use in pregnant jority of these drugs were never studied adapted easily for the delivery of nearly patients. in pregnancy. Hence, the safety and any type of therapeutic, so expanding pharmacokinetic profiles of these and this strategy to target newly identified Comment new therapeutics will need to be deter- causative pathways is highly feasible. We Exciting research was presented at the mined before their use in pregnancy. The view the ELP system as a platform for workshop in the area of placentation, workshop discussed this aspect high- maternal sequestration of therapeutics, trophoblast migration, and spiral artery lighting the unique pharmacokinetic and its attractive properties as a drug remodeling. Research findings in these properties of pregnancy and the hurdles carrier make it a promising platform areas will be critical for developing and pitfalls of translating research find- to be applied for delivery of novel future treatments and, ultimately, the ings into practice. The workshop therapeutics. prevention of adverse pregnancy out- concluded with discussions of drug de- comes of placental origin that include livery during pregnancy. The potential Scientific gaps. Although we are focused preeclampsia and FGR. Knowledge of for using nanoparticles or protein bio- on treating symptoms of preeclampsia the basic biology of trophoblast invasion polymers during pregnancy was pre- during late gestation, the ideal scenario and spiral artery remodeling is critical to sented, either to prevent fetal drug would be to intervene early in pregnancy, understanding the cause of many preg- exposure by blocking placental transfer during trophoblast invasion and nancy disorders; once the molecular or to encourage drug delivery to the fetus placentation, to prevent the onset of pathways that go awry are identified, it for in utero therapy by attaching agents preeclampsia. However, to do that, we will be possible to design therapeutics to that actively are transported by the need to gain a better understanding of intervene in these pathways. However, placenta. The use of macromolecular

JULY 2016 American Journal of Obstetrics & Gynecology S35 Supplement Obstetrics ajog.org carriers to prevent placental drug clear that the risks for many health out- 15. Silva AC, Lopes CM, Sousa Lobo JM, transfer and fetal drug exposure repre- comes, which include the development Amaral MH. Nucleic acids delivery systems: a challenge for pharmaceutical technologists. Curr sents an exciting new strategy for drug of cardiovascular diseases,482 metabolic 483 Drug Metab 2015;16:3-16. development in pregnancy. Knowledge diseases, and cognitive developmental 16. Burnett JC, Rossi JJ. RNA-based thera- 484 that the fetus will not be exposed to the disorders, just to name a few, are peutics: current progress and future prospects. therapeutic agent will open the door for strongly programmed by the in utero Chem Biol 2012;19:60-71. use of many drugs that currently are environment. - 17. Van Rooij E, Kauppinen S. Development of microRNA therapeutics is coming of age. EMBO avoided during pregnancy and will help Mol Med 2014;6:851-64. lessen the regulatory burden on drug ACKNOWLEDGMENTS 18. Svenson S, Prud’homme RE. Multifunc- development for pregnancy-related tional nanoparticles for drug delivery applica- We acknowledge Tamika Turner-Graydon for tions: imaging, targeting, and delivery. New disorders. her major contribution in the preparation and York: Springer-Verlag; 2012. The pregnant patient population editing of the manuscript and Rosalina Bray for 19. Vrachnis N, Kalampokas E, Sifakis S, et al. her excellent assistance in organizing the work- maybe1ofthemostchallengingco- Placental growth factor (PlGF): a key to opti- shop meeting. horts for which to develop drugs mizing fetal growth. J Matern Fetal Neonatal because of concerns regarding drug ef- Med 2013;26:995-1002. fects on fetal outcome. Placental drug 20. Maltepe E, Bakardjiev AI, Fisher SJ. 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