Supplement ajog.

org

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

M ost adverse pregnancy outcomes

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

JULY 2016 American Journal of Obstetrics & Gynecology S1

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
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. ilekisj@mail.nih.gov, 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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org
the therapeutic agent to obtain the
desired effect. Will the therapeutic agent
be required to be administered contin-
uously or only for a short duration of
time? In the aforementioned FGR treat-
ment scenario, how long of a time period
is required to increase and maintain the
number of terminal villi? Once formed,
will the morphologic change remain
permanent, or will the induced
morphologic change regress if the stim-
ulus is not continued? Therefore, speci-
ficity, dosage, delivery, timing, and
length of exposure are some of the key
factors in the development of a success-
ful therapeutic agent.
Although much progress is being
made in understanding the molecular
pathways in the placenta that are
involved in the pathophysiologic condi-
tion of pregnancy-related disorders, a
significant gap exists in using this in-
formation for developing new drug
therapies to improve pregnancy
outcome. To address this concern, the
Eunice Kennedy Shriver National Insti-
tute of Child Health and Human
Development of the National Institutes
of Health sponsored a 2-day workshop
on March 5-6, 2015, titled “Placental
Origins of Adverse Pregnancy Out-
comes: Potential Molecular Targets,” to
discuss and reflect on placental drug
targeting to improve pregnancy out-
comes. The workshop brought together
leaders in the field to present and discuss
their particular area of research and
stimulate dialogue in the context of the
theme of the workshop. The goals of the
workshop were (1) to present the state of
the science with respect to the molecular
mechanisms that are involved in
placentation, (2) to identify potential
molecular pathways and developmental
time windows for targeting effective
“drug” interventions to avoid placenta-
tion defects in early pregnancy and
circumvent placental defects later in
pregnancy, and (3) to identify major
research gaps in our understanding of
placental molecular pathways that lead
to adverse pregnancy outcomes. This
article summarizes the proceedings of
that workshop. The overall objective of
the workshop was to stimulate the
research community to better apply the
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Obstetrics Supplement
TABLE 1
Workshop session themes and their respective topics
I. Review of placental development and function in the context of the molecular mechanisms
and pathways
Human trophoblast differentiation and placentation
Innate immune function of human trophoblast
Modeling trophoblast differentiation and placentation in the rat
Modeling trophoblast differentiation and placentation in the mouse
II. Potential “drug” targets of important placental molecular pathways throughout placental
development in relation to pregnancy disorders
Placental hypoxia as a therapeutic molecular target
Is fetal growth a feasible target for placental intervention?
Targeting oxidative/nitrative stress and mitochondrial dysfunction in placenta to relieve
adverse pregnancy outcomes
III. Identify potentially useful or experimental drugs to target important molecular pathways
Challenges and advantages of rescuing and repurposing
Effect of pregnancy on drug pharmacokinetics and pharmacodynamics
From bench to bedside: processes and pitfalls translating research findings into practice
paradigms
IV. Evolving technologies for placental specific “therapeutic/drug” delivery
Trophoblastic nanovesicles, microRNA, and their targets aberrant regulation of myometrial
contractility by maternal cell-free plasma RNA of placental origin: screening and therapeutic
implications
Maternally sequestered delivery systems for prevention of fetal drug exposure
Novel therapeutic interventions and delivery systems
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
knowledge that is obtained from the placenta and uterus is governed, in
laboratory bench for use at the bedside. large part, by the cytotrophoblast dif-
A summary of the research topic that was ferentiation pathway that enables
covered by each participant is presented invasion.20,21 With regard to the
along with their opinions on current and anatomic arrangement, placental cyto-
future opportunities and research gaps. trophoblasts emigrate from anchoring
The article is organized according to 4 villi and join cell columns that serve as
session themes. The session themes and conduits to the uterine wall (Figure 1).
their respective topics are listed in Within the uterus, the cytotrophoblasts
Table 1. Table 2 is a key to abbreviated invade nearly its entirety, normally
scientific terms that are used commonly stopping one-third of the way through
throughout the article. the muscle layer. Within the decidua,
interstitial cytotrophoblasts interact
Review of placental development and with specialized populations of maternal
function in the context of molecular immune cells that are allowed to enter
mechanisms and pathways this compartment. During invasion,
Human trophoblast differentiation the cells also remodel the uterine circu-
and placentation (Susan Fisher, lation, primarily by targeting the spiral
University of California San Francisco) arteries. They transform the walls of
Cytotrophoblast differentiation establishes these vessels. Endovascular cytotropho-
the anatomy of the human maternal-fetal blasts replace the endothelium and
interface. The complex cellular architec- intercalate within the smooth muscle
ture at the boundary between the cells of the tunica media. This process
JULY 2016 American Journal of Obstetrics & Gynecology S3
Supplement Obstetrics ajog.org

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.
CaO2 arterial oxygen content At a molecular level, cytotrophoblast
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-
trophoblasts undergo a stereotypical
DNA deoxyribonucleic acid
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-
entiation. Many investigators believe that
HLA human leukocyte antigen
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 cyto-
trophoblast 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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
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 cyto-
mRNA messenger RNA
trophoblasts from preeclampsia preg-
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 pre-
eclampsia.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,
NTU new therapeutic uses
there are no definitive answers to this
question. However, alternative explana- O2 oxygen
tions abound. For example, particular PAMP pathogen-associated molecular pattern
combinations of maternal natural killer
PaO2 partial pressure of arterial oxygen
(NK) cell expression of killer cell
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 pre-
eclampsia.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 hor-
mone, kiss-1 metastasis-suppressor 1, Prl prolactin
semaphoring 3B, and several pregnancy- PRR pattern recognition receptors
specific beta-1-glycoproteins) is nor- PTL/B preterm labor/birth
malized,35 which suggests that the defects
PTB preterm birth
are reversible and that pursuit of thera-
pies is warranted. Kþ 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/

JULY 2016 American Journal of Obstetrics & Gynecology S5

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Supplement Obstetrics ajog.org

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
bastic research into humans maternal blood,37 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 ap-
pears 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 under-
standing 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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org Obstetrics Supplement

cytotrophoblasts lacks a definitive

explanation. Therefore, it is very diffi- FIGURE 1
cult to devise targeted therapies for A schematic drawing of the maternal-fetal interface in human pregnancy
pregnancy disorders, from infertility to
preeclampsia, that are thought to have
an immune cause or component. Like-
wise, lack of knowledge impedes strate-
gies for dampening the maternal
immune response to infections during
pregnancy, which can lead to preterm
labor/birth. In cases of the latter syn-
drome with an unknown cause, thera-
pies lag because we do not understand
the pathways that normally trigger
normal labor and birth at the end of
pregnancy. Finally, preeclampsia ap-
pears 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)
Background. Placental trophoblast cells
can sense and respond to a variety
of infectious pathogen-associated mo-
lecular patterns that are expressed
by microbes, as well as noninfectious
host-derived damage-associated molec-
ular patterns (DAMPs) through their
expression of innate immune pattern
recognition receptors (PRRs).42,43
Depending on the trigger or receptor
that is activated, the trophoblast may
mount either a regulated protective
response that helps to maintain and
promote a healthy pregnancy or a
damaging response that might impact
pregnancy outcome adversely (Figure 3).
Moreover, expressions of some tropho-
blast PRRs are regulated gestationally,
which further impacts the placental
response.44
PRRs. Two major families of PRRs are the
Toll-like receptors (TLRs) and the Nod-
like receptors (NLRs). TLRs are trans-
membrane receptors, which allows for
the sensing of PAMPs or DAMPs either at
the cell surface or within endosomal
compartments.45 There are 10 human
and 12 murine TLRs, each with distinct
specificities.46 TLR4 recognizes Gram-
negative bacterial lipopolysaccharide.
TLR2, in cooperation with its coreceptors
TLR1, TLR6, or TLR10, recognizes
Gram-positive bacterial peptidoglycan
and lipoproteins. TLR3 senses viral
double-stranded RNA; TLR5 senses
bacterial flagellin. Mouse TLR7 and hu-
man TLR8 sense viral single-stranded
RNA, and TLR9 senses bacterial DNA.46
Four adapter proteins are involved in
TLR signaling: myelin and lymphocyte
protein 88 (MyD88), TIR-domaine
containing adapter-inducing interferon-
beta (TRIF), myelin and lymphocyte
protein (Mal), and TRIF-related adaptor
molecule.47,48 TLR2 and TLR4 signal
through MyD88/Mal. TLR4 can also
signal through TRIF/ TRIF-related
adaptor molecule. TLR3 signals through
TRIF; all other TLRs signal through
MyD88 alone.47,48 Downstream, TLR/
MyD88 signaling activates NFkB; TLR/
TRIF activates Tank-binding kinase-1
and interferon regulatory factor 3 that
leads to a type I interferon response
(Figure 4).48,49
NLRs are cytoplasmic-based PRRs
that provide an intracellular recognition
system for sensing microbe-associated
pathogen-associated molecular pattern
(PAMP) or as will be discussed, DAMPs.
NLRs can synergize with TLRs for a
greater response or provide a compen-
satory system for when TLR signaling
is absent or reduced.50,51 The NLR
proteins, Nod1 and Nod2, recognize
the peptidoglycan peptides Gram-
negative bacterial gamma-D-glutamyl-
meso-diaminopimelic acid (iE-DAP-)
and muramyl dipeptide (MDP) that is
expressed by all bacteria, respectively.
Both Nod1 and Nod2 signal through the
common adapter protein receptor-
interacting protein-like interacting
caspase-like apoptosis regulatory protein
kinase (RICK) to induce inflamma-
tion.52 Another NLR called NACHT,
leucine-rich repeat protein and the
NLR, Nalp3, are involved in mediating
the production of the proinflammatory
cytokine interleukin (IL)-1b.53,54
Because IL-1b has the potential to be
damaging, its regulation is tightly
controlled. Indeed, IL-1b is an important
mediator of preterm birth102-105 and
perinatal brain injury106-109 and can
trigger the production of other proin-
flammatory cytokines and chemokines
through the IL-1 receptor in a similar
manner to TLRs.55 Indeed, delivery
of a synthetic peptide to pregnant
mice that is a selective IL-1 receptor

JULY 2016 American Journal of Obstetrics & Gynecology S7

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Supplement Obstetrics ajog.org

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 inflam-
masome is the best characterized, there
are a number of inflammasomes: Nalp1/
apoptosis-associated speck-like protein
containing a CARD; absent in melanoma
2 protein A; NLR family, CARD domain
containing 4 protein; and interferon
gamma-inducible protein 16.112 Further-
more, Nod proteins can mediate IL-1b
production independent of the inflam-
masome. For example, Chlamydia tra-
chomatis 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 concentra-
tions, trigger mild proinflammatory
cytokines/chemokines (IL-8, IL-6,
monocyte chemoattractant protein-1
[MCP-1], and melanoma growth stim-
ulating activity, alpha [GRO-a]) re-
sponses 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
column formation and in the first cell layer of the column. aVb3 was up-regulated in the distal across gestation.63 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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org
preterm uterine contractility.100 High-
dose iE-DAP also induces inflammation
at the maternal-fetal interface and pre-
term birth.62 However, low-dose lipo-
polysaccharide can trigger preterm birth
in mice on a pathologic background,
such as an active viral infection67,68 or
IL-10 deficiency.69,70 Thus, although
TLRs and NLRs appear to be involved
in preterm birth in response to bacterial
components, in normal early pregnancy,
there appears to be some tolerance
towards certain triggers, such as lipo-
polysaccharide, that may always be pre-
sent at the maternal-fetal interface.71,72
Furthermore, although bacteria can
induce preterm birth,73,74 additional
signals, such as a virus, may further
sensitize the placenta to these bacterial
signals. 67,68
Gram-positive bacterial peptido-
glycan, which activates TLR2 in associ-
ation with TLR1, TLR6, or TLR10,
triggers a very different response in hu-
man first-trimester trophoblast cells
compared with term trophoblast. At
term, peptidoglycan activates these cells
to produce IL-8.75 However, in first-
trimester trophoblast cells, instead of
activating the classic inflammatory
cascade, peptidoglycan through TLR1,
TLR2, and TLR10 induces apoptosis and
down-regulates the cell’s basal cytokine/
chemokine expression. Moreover, this
apoptotic response is prevented by the
ectopic expression of TLR6, which is
absent in the first-trimester tropho-
blast.58,76-78 Similarly, delivery of pepti-
doglycan early in gestation induces
placental apoptosis without evidence of
inflammation,76 although delivery later
in gestation triggers preterm birth.79
Viral sensing by the trophoblast. The viral
sensors, TLR3 and TLR8, that recognize
viral double-stranded RNA and single-
stranded RNA, respectively, mediate a
rapid, robust chemokine/cytokine (IL-6,
IL-8), type I interferon, and antiviral
response in human first-trimester
trophoblast.80-82 Furthermore, inde-
pendent of TLR8, viral single-stranded
RNA also induces trophoblast expres-
sion of IL-1b, antiviral factors, and the
induction of apoptosis.82 This placental
single-stranded RNAeinduced response
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Obstetrics Supplement
FIGURE 3
Innate immune sensing by the trophoblast
Trophoblast cells sense infectious pathogen-associated molecular patterns that are expressed by
bacteria, viruses, fungi, and parasites through their expression of Toll-like receptors and Nod-like
receptors. Through these receptors, trophoblast cells also mount responses to noninfectious
host-derived damage-associated molecular patterns, such as uric acid, high mobility group B1,
glucose, and certain autoantibodies. Trophoblast expression of some Toll-like receptors and Nod-like
receptors are regulated across gestation and cell subtype. Depending on the trigger, receptor
activated, and type of signaling pathway used, the trophoblast may mount either a regulated pro-
tective response that helps to maintain and promote a healthy pregnancy or a damaging pathologic
response that might impact pregnancy outcome adversely.
PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns; HMGB1, high mobility group B1;
NLRs, Nod-like receptors; TLRs, Toll-like receptors.
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
is mirrored in vivo without triggering patients with preterm labor and preterm
preterm birth.82 In contrast, viral premature rupture of membranes,87 and
double-stranded RNA induces preterm in serum from pregnancies that are
birth through TLR379,83; other groups complicated by either preeclampsia or
have reported preeclampsia-like symp- reduced fetal movements.88,98 Term
toms in viral double-stranded RNA- trophoblast cells that are treated with
treated mice.84 HMGB1 secrete significantly increased
levels of IL-1b, IL-6, and monocyte
Trophoblast activation by DAMPs. In chemoattractant protein-1; however,
addition to understanding how the mechanistic studies are needed to show
trophoblast responds to PAMPs, the which receptors and signaling pathways
impact of DAMPs on trophoblast func- are activated by this DAMP.89 One type
tion has been investigated. DAMPs are of DAMP that has been characterized
host-derived factors that are either not extensively is the antiphospholipid
usually released from cells or tissues or, if antibody (aPL), which are autoanti-
present in the extracellular space, are bodies that specifically target the
normally at low levels. The DAMP, high- trophoblast by binding surface beta2
mobility group B1 (HMGB1) can be glycoprotein 1.90 These autoantibodies
released, passively from damaged cells or activate human first-trimester tropho-
actively in response to inflammatory blast TLR4 that results in inflammatory
triggers and can mediate inflammatory IL-8 and IL-1b secretion (Figure 4).91,92
responses via TLR2, TLR4, or receptor Downstream of TLR4, aPLs induce
for advanced glycation end products 85 endogenous uric acid, which is another
(Figure 4). HMGB1 levels are increased DAMP that specifically activates the
in the amniotic fluid from patients with Nalp3 inflammasome,93 which medi-
preterm birth or preterm premature tates proeIL-1b processing and secre-
rupture of membranes and intrauterine tion (Figure 5).92 In parallel, aPL via
infection,86 in fetal membranes from TLR4 induces the expression of the
JULY 2016 American Journal of Obstetrics & Gynecology S9
Supplement Obstetrics ajog.org

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 pre-
vented lipopolysaccharide-induced pre-
term uterine contractility in nonhuman
primates100 and knockout mice for TLR,
or associated adapter proteins are resis-
tant 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;
NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; PAMPs, pathogen-associated molecular patterns; PDG, pepti- acid may not activate112 and none
doglycan; ssRNA, single-stranded RNA; TBK, tank-binding kinase; TLR, Toll-like receptor; TRAF, tumor necrosis factor receptor-
associated factor; TRIF, tir-domainecontaining adapter-inducing interferon-b.
inflammasome-mediated pathways,113
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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org
endpoints and impact on pregnancy
outcome triggered by PAMPs and
DAMPs may be common, the upstream
mechanisms are often quite distinct.
These challenges for drug discovery and
applications highlight the need for a
greater understanding of the precise
molecular pathways that are involved in
placental sensing of infectious and
noninfectious triggers.
Modelling trophoblast differentiation
and placentation in the rat (Michael J.
Soares, University of Kansas)
Background. Fundamental to the estab-
lishment of pregnancy and formation of
the hemochorial placenta is remodeling
and restructuring of uterine spiral arteries
that allow for the flow of nutrients to the
placenta and ultimately to the fetus.114
Trophoblast cells play a central role in
this vascular remodeling process. Patho-
logic conditions that are associated with
trophoblast-directed uterine spiral artery
remodeling underlie some of the most
significant and challenging obstetric dis-
eases.114-116 There are several research
strategies for gaining insight into the
process of hemochorial placentation and
for the identification of potentially
vulnerable molecular mechanisms that
lead to disease. In this section, we address
the merits of animal models, especially
the rat, for placental research.
Placentation in the rat. The rat possesses
hemochorial placentation with deep
intrauterine trophoblast cell invasion
and trophoblast-directed uterine spiral
artery remodelling,117,118 which are fea-
tures shared with human placenta-
tion.114,119-121 Recognition of these
similarities spurred the establishment of
in vitro and in vivo research methods
using the rat as an animal model to
address mechanistic questions regarding
the development of the hemochorial
placenta and especially the role of inva-
sive trophoblast cells in the remodeling
of uterine spiral arteries. Rcho-1
trophoblast stem cells and blastocyst-
derived trophoblast stem cells are 2 rat
in vitro culture systems that have been
used extensively to investigate signaling
pathways and mechanisms that control
trophoblast cell differentiation.122,123
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Obstetrics Supplement
FIGURE 5
Nod-like receptor signaling
Nod-like receptors are cytoplasmic proteins that sense pathogen-associated molecular patterns.
Nod1 recognizes bacterial iE-DAP, and Nod2 senses bacterial muramyl dipeptide. Both Nod1 and
Nod2 signal through the adapter protein receptor-interacting protein-like interacting caspase-like
apoptosis regulatory protein kinase to induce nuclear factor kappa-light-chain-enhancer of acti-
vated B cells activation and subsequent cytokine/chemokine production. Nalp3 recruits ASC and
caspase-1 to form the inflammasome. Once the inflammasome has assembled, caspase-1 is
activated and processes pro-IL-1b into its active, secreted form. Refer to Table 2 for the key to
undefined abbreviations.
ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain [CARD]; DAMP, damage-associated molecular
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;
PAMPs, pathogen-associated molecular patterns; Pro-IL, precursor protein of interleukin; RICK, receptor-interacting protein-like
interacting caspase-like apoptosis regulatory protein kinase.
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
Regulatory factors identified in vitro observations illustrate the instructive
have been tested experimentally with the nature of oxygen delivery as signal-
use of an assortment of in vivo research driving decision-making within
strategies, which include transgenesis trophoblast stem/progenitor cell pop-
to monitor the invasive trophoblast ulations and ultimately affect hemo-
lineage,124 spontaneous mutant rat chorial placentation; however, it is
models that possess placental insuffi- important to appreciate that failures in
ciency118,125-128 and gain-of-function adaptive responses to hypoxia can lead to
and loss-of-function manipulations disruptions in placentation. Addition-
with the use of trophoblast-specific ally, hypoxia can also be a pathologic
lentiviral gene delivery and genome response to a failed placenta resulting in
editing.129-132 disease. Dissociating these potentially
A fundamental property of placenta- confounding outcomes of hypoxia is
tion is its plasticity, which is character- essential. Dissection of hypoxia-guided
ized by the acquisition of structural/ pathways within trophoblast stem/pro-
functional properties that permit adap- genitor cell populations offers an op-
tation to environmental challenges. portunity to identify molecular events in
Mechanisms underlying these adaptive the placentation process that could be
processes are an important feature of manipulated to improve and enhance
placentation and represent potential placental development and prevent the
therapeutic targets. Limiting oxygen de- spiraling consequences of a failed
livery to the placentation site at devel- placenta.
opmentally defined phases of gestation
effectively activates the acquisition of the Role of NK cells. Immune cells, especially
invasive trophoblast phenotype and NK cells, are active contributors to
promotes uterine spiral artery remodel- remodeling of uterine spiral arteries
ing124,133 that are adaptive responses during the establishment of pregnancy.
conserved in primates.134,135 These The involvement of NK cells in the
JULY 2016 American Journal of Obstetrics & Gynecology S11
Supplement Obstetrics ajog.org

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 pro-
cesses, 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 tropho-
blast cells, NK cells, and uterine spiral
arteries, a triad of key players in hemo-
chorial placentation.
A paradigm for investigating molec-
ular mechanisms that impact hemo-
chorial placentation with the use of
rodent trophoblast stem-cell models,
followed by the validation of the ob-
tained 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
(natural killer cell depleted) and killed on E13.5. Double immunofluorescence staining for A, B, experimentation represents a powerful
ANK61 (natural killer cell marker) and ACTA (smooth muscle marker) and C, D, 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
media (A, C); the asterisks identify blood vessels with intact tunica media (B). Scale bars ¼ 0.25 mm. the use of primary or immortalized hu-
NK, natural killer.
man trophoblast cell model systems and
Reprinted with permission from Chakraborty et al.133
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 op- in 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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org
importantly extends placental research
beyond description, classification,
in vitro analyses, and molecular pheno-
typing and permits a rational approach
for understanding the physiology of
placentation, the pathogenesis of
placental disease, and importantly the
identification and testing of potential
drug targets for treating placental
disease.
Modeling placental function and
pregnancy physiology in mice
(James C. Cross, University of Calgary)
Background. Understanding the molec-
ular, cellular, and physiologic functions
of the placenta in humans is limited to
expression studies in normal and path-
ologic human pregnancies and some
in vitro systems. Because of this, animal
models remain critical for investigation
of the basic biology and assessment of
biomarkers and treatments of pregnancy
complications. The mouse has been a
powerful model for understanding ani-
mal biology in the last 25 years with the
advent of transgenic and knockout
technologies. Hundreds of different gene
knockouts in mice have given molecular
insights into the development and
function of the placenta. Many of these
genes have human homologues that are
expressed in the placenta.144,145 How-
ever, before zeroing in on genes and cells,
if mice are to be truly useful for under-
standing human pregnancy complica-
tions, it is critical first to ask whether
mice and humans have similar physiol-
ogies of pregnancy. This is the starting
point for the use of mice to investigate
molecular mechanisms that give us
testable hypotheses to assess in human
studies.
Similar physiology of pregnancy in
humans and mice. Mice adapt to preg-
nancy with major changes in the
maternal cardiovascular, metabolic, and
immune systems. Similar to humans,
mice show increased cardiac output,
plasma volume, and a mid-gestation
drop in blood pressure.146,147 There are
also major changes in metabolism in
which the mother’s fat and muscle
become insulin resistant, requiring more
insulin to take up glucose, which helps to
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
shunt glucose to the fetus.148 To combat
insulin resistance, an increase in
pancreatic b cells and insulin synthesis
occurs.149-151 Gestational diabetes mel-
litus (GDM) occurs if there is inadequate
b-cell compensation.152,153 The most
important change in the immune system
during pregnancy is the appearance of
large numbers of uterine NK cells in the
decidua,154 first described in mice and
only later in humans. One difference
between mice and humans is that mice
are litter-bearing, whereas humans tend
to have singleton pregnancies; however,
what functional difference this has is not
clear, given the similarity of pregnancy
physiologic condition.
Trophoblast functions and pregnancy.
Most of the research on trophoblast cell
function in human pregnancy compli-
cations has focused on trophoblast cell
invasion and its association with spiral
artery remodeling. Cell ablation experi-
ments in mice showed that this is not just
an association and that trophoblast cell
association with spiral arteries is critical
for remodeling of those arteries,155
though uterine NK cells in the decidua
also play a role.156 Beyond just invasion
of spiral arteries, however, the human
placenta contains diverse extravillous
trophoblast subtypes,157 and mice have
diverse trophoblast cells in the junctional
zone that express complex patterns of
hormones.158
Several lines of evidence indicate that
the endocrine function of the placenta
modifies metabolism in the mother that
is necessary to promote fetal growth.
Scanning through microarray data in
the public domain indicates that the
human placenta expresses >80 different
hormones.159 Although a few are
placenta-specific hormones arising from
duplication of the GH 2 gene most are
from canonical hormone genes that are
expressed in the placenta, presumably
because of evolution of placenta-specific
promoter and/or enhancers. The hor-
mones include known regulators of
metabolism, blood cell production
and reproduction. Placental prolactin-
related hormones can promote prolifer-
ation of pancreatic b cells and insulin
synthesis.160 Glucose transporter-related
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
hormones,161 progesterone,162 resis-
tin,163 and leptin164 can promote insulin
resistance. Paradoxically, the placenta
also produces adiponectin165 that pro-
motes insulin sensitivity. It is interesting
that, although these hormones are nor-
mally expressed by the pituitary (pro-
lactin, GH), ovary (progesterone), and
fat (the “adipokines”: resistin, leptin,
adiponectin), the human placenta is a
major source during pregnancy, and
production from the maternal tissues is
down-regulated.
The mouse placenta expresses
approximately 40 protein hormone
genes.166 As with humans, prolactin-
and GH-like hormones, progesterone,
resistin, leptin, and adiponectin are
elevated during pregnancy. However, the
evolution of the system is slightly
different because the prolactin gene, and
not the GH gene, is duplicated in mice to
produce 22 placenta-specific mem-
bers167 with prolactin- and GH-like ac-
tivities. In addition, the placenta is not
the only source of the other metabolic
hormones. Progesterone is produced by
the ovary throughout pregnancy in
response to stimulation by prolactin-like
hormones from the placenta.168 Resis-
tin,169 leptin,170 and adiponectin171 are
produced by fat during pregnancy in
mice. Prolactin receptor signaling can
regulate adipokine expression,172-174
which suggests the possibility that,
although the mouse placenta is not
the direct source of all metabolic hor-
mones, unlike humans, it may still
orchestrate the network. The best
evidence in mice that placental hor-
mones regulate fetal growth is that of
the pleckstrin homology-like domain,
family A member 2 gene, which regulates
the fetal growth by influencing the
number of endocrine cells in the
placenta.175
Current opportunities. Despite the clear
evidence that placental hormones drive
fetal growth and regulate maternal
metabolism, it is curious that research
has not continued to understand their
roles in intrauterine growth restriction
(IUGR) and gestational diabetes mellitus
in humans. Placental hormones are
attractive targets from a diagnostic
S13
Supplement Obstetrics
FIGURE 7
Relationship of maternal
intervillous blood PO2 to fetal
umbilical venous PO2
Oxygen tension in the intervillous space of the
placenta is very low until the opening of the
spiral arteries to blood flow at approximately 10-
12 weeks of gestation. Light blue dots are in-
dividual data points that were obtained at 8-11
weeks gestational age. Medium blue dots are
data that were obtained from individual preg-
nancies at 11-16 weeks of gestation. Dark blue
dots are the mean of values that were obtained
in only a few women between 16-38 weeks of
gestation and have very wide confidence in-
tervals (>30 mm Hg). Red dots are umbilical
venous PO2. Note the tight relationship and
narrow diffusional gradient between intervillous
and fetal PO2 late in pregnancy. This Figure is a
composite of data obtained from various
references.182-185
Ilekis. Potential placental molecular therapeutic targets. Am J
Obstet Gynecol 2016.
standpoint because they can be
measured serially, and improvements in
multiplex immunoassays mean that
several hormones can be assessed at the
same time. Both hormone levels and
polymorphisms in the placenta GH-
related genes have been associated with
pregnancy complications in humans, 163
although the number of published
studies is limited and they have often
examined single hormones and not
made connections with anatomic
changes in the placenta.
Future opportunities. There is emerging
evidence from mouse studies that the
placental hormones are sensitive to
maternal nutrition and changes in their
levels likely reflect attempts to mitigate
the impact of poor nutrition on fetal
growth.176 Therefore, hormone levels
S14 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
should have good predictive value in
reflecting both stress to the pregnancy
and the robustness of the placenta’s
ability to mitigate the impact on the
fetus. In addition to diagnostic value, it is
easy to imagine therapeutic strategies in
which hormone supplementation or
blockade is used to treat pregnancy
complications.
Scientific gaps. The complexity of the
hormone network at play during preg-
nancy, both the number of hormones
and the systems of feedback and adap-
tation, will require the use of animal
models, particularly knockout and
transgenic mice, to understand them.
With the ability to study mouse physio-
logic condition, it is clear that, although
a mouse is not a human, we certainly can
learn from them.
Potential drug targets of important
placental pathways in relation to
pregnancy disorders
Placental hypoxia as a molecular target
(Stacy Zamudio, Hackensack
University Medical Center)
Background. Hypoxia is a pathologic
condition in which there is insufficient
oxygen to maintain normal physiologic
processes. However “hypoxia” is often
used imprecisely in the literature, inter-
changeably with some of its causes,
for example: hypoxemia-reduced partial
pressure of oxygen (PO2), anemia
(insufficient hemoglobin or hemoglo-
binopathies that alter oxygen binding/
release), or reduced environmental
oxygen availability (as in high-altitude
animals dwelling in burrows, diving
mammals). Hypoxia is usually assumed
to be present in any 1 of these conditions.
For example, a reduction in blood flow
to a specific organ or tissue, whether
acute or chronic, often is assumed to be a
hypoxic insult. However, hematologic
adaptations can compensate for lower
blood flow,177 and the volume and speed
with which blood travels, as well as
diffusional distances, affect tissue oxygen
delivery.178-182 Thus, in the absence of
direct measures of oxygenation in the
tissue, cell, or organ of interest, it is
difficult to tell whether hypoxia is
present.
ajog.org
Oxygen levels in the human placenta and
fetus. Normal oxygen levels within the
intervillous space of the placenta are
low early in gestation (approximately
20 mm Hg), can rise to as high as
80 mm Hg in the early second trimester,
and then decline progressively towards
term (Figure 7).183-186 Fetal umbilical
venous PO2 follows a similar pattern,
perhaps reaching as high as 40-50 mmHg
in the second trimester, but also
declining progressively towards term to
an average of w28 mmHg (<4% O2) at
term. Rising hemoglobin maintains
oxygen content. With normal values as
low as these, even small changes in fetal
PO2 can make a difference. Average
umbilical venous PO2 differs by only
3 mmHg in babies at >97% percentile
versus those at <3rd centile.187 Despite
difficulty in quantifying magnitude and
intensity of hypoxia, much less being able
to detect its onset, the bulk of evidence
has led to general acceptance that fetal
growth diseases often are associated with
placental hypoxia, specifically early-
onset preeclampsia, most nongenetic/
syndromic IUGR, preeclampsia with
IUGR, and some diabetes/GDM with
large for gestational age neonates.188-196
Figure 7 shows that what might be
considered abnormally low O2 levels in
the placenta will vary not only by gesta-
tional age, but also with location within
the placenta. The third-trimester
placenta is exposed to PO2s that can
range from <20 mm Hg when the
deoxygenated blood from the umbilical
artery flows back into the placenta to
>80 mm Hg when the maternal arterial
blood first enters the intervillous space.
This equates to 1-10% ambient O2 for
in vitro experimentation. Such data have
given rise to a convention in which 5-8%
O2 is used to mimic normoxic conditions
in the third trimester and <3% for
hypoxia.
Placenta hypoxia cannot be diagnosed
unambiguously because of the inherent
limitations of human experimentation
and the inaccessibility of the placenta
and fetus in vivo. Proxies are used
instead. For example, reduction in blood
flow severe enough to deprive tissues of
oxygen and glucose sufficient for meta-
bolic needs is assumed to be present
ajog.org
when elevated maternal/fetal Doppler
indices reflect increased impedance.
Elevated erythropoietin levels in
mothers and babies, nucleated red blood
cells in neonates, increased placental
expression of hypoxia-inducible-factor
(HIF), and its target gene products
have all been used to demonstrate
that placental hypoxia is present in
fetal growth diseases.192,197-209 Thus,
placental hypoxia is a target for thera-
peutic intervention, and unlike some of
the topics discussed at this workshop,
there have already been a number of
clinical trials designed to ameliorate or
prevent presumed consequences of
hypoxia such as inflammation210 and
oxidative stress.211-214
Chicken and egg, cause and effect. Of
critical importance is distinguishing be-
tween hypoxia per se and the hypoxia
response. The hypoxia response is evi-
dence that hypoxia is or has been present
(eg, up-regulation of HIF and HIF-
regulated genes, higher fetal hemoglo-
bin concentrations). The strength and
magnitude of the response may reflect
the intensity of the insult, but it is also
often adaptive in that it enables mecha-
nisms that increase the delivery of oxy-
gen to the fetus (as in metabolic
reprogramming or the development of
decreased vascular syncytial membrane
thickness). On the other hand, we must
be mindful of the possibility that failure
of an appropriate hypoxia response may
itself be part of the pathologic condition.
There is evidence of HIF dysregulation
and consequent over-expression without
adaptation in the more severe forms of
preeclampsia.207,208,215 In contrast, in
some severe, early-onset IUGR, there
often appears to be a lack of HIF-
mediated responses, when all evidence
suggests such response is needed.216-218
Studies at high altitude have revealed
the importance of being able to distin-
guish between adaptive hypoxia
response and pathologic condition, as
well as the subtlety with which O2 ten-
sion can exert an effect. Maternal partial
pressure of O2 in arterial blood (PaO2)
falls considerably at 2700-m elevation;
because of the sigmoid shape of the
O2 dissociation curve, PaO2 falls
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
precipitously at >3000 m. Even in
pregnancies with entirely normal out-
comes, physiologically lowered maternal
PaO2 results in a progressive slowing of
the third-trimester fetal growth trajec-
tory.219,220 This is despite significant
placental adaptation both structurally
(increased angiogenesis, decreased
vascular syncytial membrane thickness)
and metabolically.221-226 Altitude studies
in vivo showed that the human placenta
that is subjected to hypoxic stress engages
in a highly conserved process most
obviously seen in solid tumors.227-230
Metabolic reprogramming is a revers-
ible form of hypometabolism in which an
effective, largely mitochondrial-driven
switch from oxidative phosphorylation
to aerobic (ie, glycolytic) glucose con-
sumption by the trophoblast results in
increased cellular oxygen availability,
which is then available for diffusion to
the fetus.224,225 In essence, the mecha-
nism “spares” oxygen for the fetus, but at
the same time reduces fetal access to
glucose. This slows fetal growth but en-
sures that the fetus does not outgrow its
supply line. Even when placental meta-
bolic programming is active, a fetus with
greater umbilical venous O2 tension
consumes more O2.
How might hypoxia be ameliorated?
Therapeutic strategies must necessarily
depend on what is the pathologic barrier
to normal placental oxygen diffusion.
Four targets will be considered here:
blood O2 content, uteroplacental blood
flow, placental structure (angiogenesis),
and hypoxia-induced metabolic
reprogramming.
Raise arterial O2 content (CaO2) or
PaO2. In women who are anemic, the
obvious treatment would be increasing
maternal red cell mass and thereby
increasing CaO2. This is harder than it
sounds because women have less of a
response to erythropoietin than do
men,231 and the condition of up to 25% of
pregnant women who are treated with
erythropoietin does not respond.232 At
high altitude CaO2 is increased by raising
hemoglobin concentration. This preserves
maternal and fetal oxygen delivery176,233
despite an approximate 20% reduction
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
in uterine artery blood flow in mothers
and an even greater decrement in fetal
umbilical venous blood flow. Packed red
blood cell transfusion might work for
anemic mothers, but it is unlikely to be of
value where the barrier to O2 diffusion is
due to placental structural defects such as
the failed tertiary villous vascular devel-
opment (branching angiogenesis and
elongation/dilation of terminal capillary
loops) seen in idiopathic IUGR. Moreover,
there is a balance that must be maintained
between blood viscosity and O2 carrying
capacity (eg, the optimal hematocrit level
for preservation of O2 delivery in the brain
microcirculation is 15-40%).234 One
obvious method for increasing PaO2 is O2
supplementation. Sadly, a 2003 Cochrane
review found that, of 10 trials of O2 ther-
apy, only 3 were adequate for inclusion in
a metaanalysis. O2 compared with no O2
was given to mothers for improvement of
fetal growth. Rereview in 2009 changed
none of the Cochrane conclusions because
no additional trials were undertaken.235
The trials were poorly designed and
involved <100 women. However, 24-hr
supplemental O2 led to a decrease in the
perinatal mortality rate. The studies were
confounded in that the O2-treated group
was of greater gestational age at initiation
of treatment. Concerns over possible
response to “hyperoxia” such as the gen-
eration of free radicals, the inhibition of
potentially adaptive responses ongoing in
fetus and placenta have been raised as
objections to O2 therapy.
Blood flow. Deficits in uteroplacental or
fetal blood flow are associated strongly
with preeclampsia and IUGR as evi-
denced by Doppler ultrasound measures
of impedance to blood flow in the uter-
ine arteries and within the fetus. The
studies in which blood flow in human
pregnancy complications has been
measured quantitatively or semi-
quantitatively support this.236 Absent or
reversed end diastolic blood flow veloc-
ity in the umbilical arteries is a grave sign
and usually leads to emergent de-
livery.237 Elevated Doppler resistance
indices in the maternal uterine arteries
indicates that there is downstream
impedance to blood flow. This is
accompanied by morphologic evidence
S15
Supplement Obstetrics
of placental structural problems such as
a reduction in small arteries within the
tertiary stem villi,238 thickening of the
basal lamina, and erythrocyte congestion
in tertiary villous capillaries.239,240 As
with CaO2 or PaO2, how one might target
blood flow depends on what is the un-
derlying problem. Most of the blood
entering the intervillous space is carried
by the maternal uterine arteries. The
uterine arteries therefore are critical to
pregnancy success and a potential target.
In normal pregnancy, eccentric remod-
eling of the uterine arteries leads to
doubling of uterine artery diameter by
20 weeks of pregnancy and a further,
smaller increase, likely because of shear
stress, in the late third trimester.241
Eccentric remodeling is characterized
by changes in the composition of the
vessel wall that permits greater distensi-
bility. In addition, there is inhibition of
the myogenic response (the rise in vessel
tone with increasing pressure).242-244
In human and multiple experimental
animal models, endothelium-dependent
vasodilator response is increased mark-
edly in the uterine circulation during
pregnancy. In rodent models, depending
on what branch of the uterine artery
is investigated, nitric oxide contributes
30-80%, and endothelium-derived
hyperpolarizing effectors contribute
20-70% of the endothelium-dependent
vasodilator response of the pregnant
uterine arteries.245,246
Placental structure (eg, angiogenesis). In
idiopathic IUGR, the placenta frequently
has a dearth of branching angiogenesis
and poorly developed tertiary capillary
development, leading to reduced O2
diffusion capacity.178,216 Structural
placental defects in preeclampsia are less
clear. An excess of fibrinoid deposition
leading to, or as a result of, villous death
has been attributed to excess oxidative
stress or inflammation. In women with
preeclampsia, in general, placentas are
smaller (reduced weight and volume),
and the volume of functional tissue (pa-
renchyma) and villous surface area (area
of nutrient and gas exchange) are
reduced. 247 However, unlike IUGR, they
also have an increase in the fetal capillary
volumes and density (indicative of a
S16 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
hypoxia response). Late onset pre-
eclampsia (without fetal compromise)
shows no such changes and cannot be
statistically differentiated from normal
placentas.248 The interplay of many
angiogenic growth factors and of several
critical cell types (pericytes, endothelium,
trophoblasts) is involved in the normal
vasculogenesis of the placenta.223,249
Placental metabolic reprogramming. In
cancer biology, several recent unique
insights are relevant to the placenta and
consideration of molecular pathways
that might be amenable to therapeutic
intervention. Cancer cells have dysregu-
lated, Warburg-like glucose metabolism.
Energy production is abnormally
dependent on aerobic glycolysis; there is
increased fatty acid synthesis and
increased rates of glutamine meta-
bolism.250 In fact the term glutamine
addicted is now applied to many can-
cers.251 In cancer biology “glutamine
addicted” refers to an extension of
metabolic reprogramming in which
glutamine is required for essential amino
acid uptake to maintain activation of
mTOR. In many, if not all cancer cells,
glutamine is also the primary mito-
chondrial substrate. These metabolic
changes are linked to therapeutic resis-
tance in cancer treatment; hence, stra-
tegies are being developed to target this
altered cancer metabolism in conjunc-
tion with older treatments that were
designed to inhibit angiogenesis or
otherwise shrink the tumor.252,253 We
have demonstrated the same, evolu-
tionarily conserved mechanism of
metabolic reprogramming in the hyp-
oxic placenta that is associated with
stably elevated HIF-1alpha levels,
which initiates metabolic reprogram-
ming.205,224,225 However, in contrast to
cancer, the objective of a therapy that
targets metabolic reprogramming in
the placenta would be to sustain the
response, which leads to increased
intracellular oxygen levels and hence
more oxygen for diffusion to the fetus.
Current opportunities. There is wide-
spread acceptance that placental hypoxia,
acknowledging the already low
O2 environment that is normal, is
ajog.org
associated with pregnancy diseases.
However, before a placental molecular
intervention is attempted, there should be
a greater effort to examine relatively
noninvasive means of improving
oxygenation in the placenta. These should
include definitive studies of maternal O2
supplementation, dietary strategies
designed to increase maternal substrates
for nitric oxide, and other obviously more
benign strategies that target maternal
physiologic condition rather than
placental function.
Methods that might be used to pro-
vide additional means of carrying oxy-
gen in the circulation (eg, oxygen-filled
microbubbles) are under investiga-
tion.254-257 Moreover, the opportunity
exists currently to further test the effects
of supplemental O2; more modern
standards of clinical trials such as
establishing dose/response, effects of
treatment duration, minimum dosing
necessary surely should be attempted
as a simple, yet potentially, effective
intervention.
Beyond this are the effectors that are
responsible for altering vascular tone that
could be molecular targets, including
nitric oxide, by stimulating release via a
number of means that include the
administration of VEGF, PlGF, relaxin,
or stimulation of the reticular-activating
system, hemoxygenaseecarbon monox-
ide, large conductance Ca2þ- activated
potassium (Kþ) channels, and in-
creased dietary intact of arginine or
citrulline.258-260 For endothelium-derived
hyperpolarization, potential effectors
include Kþ C-type natriuretic peptide,
arachidonic acid derivatives, epox-
yeicosatrienoic acids and hydrogen
peroxide.
Three approaches have been or
currently are being investigated to in-
crease blood flow. The least invasive of
these involves dietary arginine (or
citrulline) supplementation, in theory,
to increase substrate for nitric oxide
production. A number of small trials led
to 1 randomized controlled clinical
trial.261 High-risk women (with a history
of preeclampsia in the previous preg-
nancy) had less than one-half the rate
of preeclampsia as women receiving
placebo. However, another trial in
ajog.org
women already diagnosed with pre-
eclampsia and treated acutely, rather
than throughout pregnancy, showed no
benefit.262 International recruitment for
a trial is ongoing for the treatment of
early-onset IUGR.263 This 6-year study
was based on several small studies
that showed that low-dose sildenafil
improved fetal growth and neonatal
survival.264 Another trial that is ongoing
and led by Dr Anna David in the United
Kingdom targets women with even
earlier IUGR, women whose IUGR is so
severe that the fetus normally would die
before viability.265 Treatment consists of
uterine artery catheterization and injec-
tion of adenovirally delivered VEGF-D.
Proof of concept studies in sheep
demonstrated that this treatment im-
proves uterine artery blood flow for
approximately1 month.266
Future opportunities. With respect to
angiogenesis, cancer therapies, especially
for solid tumors, provide some clues.
These therapies target the inhibition of
angiogenesis, although, in the placenta,
one might wish to stimulate organ-
specific angiogenesis. Effectors in this
molecular pathway provide a variety of
targets. Vascular endothelial-cadherin
and matrix metalloproteinases, for
example, loosen gap junctions in
the endothelium, which is required for
tip-cell formation, a prerequisite to
branching angiogenesis. However, tip
cell formation also requires coordinated
activity by VEGFR-2, various ligands for
Notch-1 receptor (such as Delta-like
ligand and jagged 1), neuropilin 1,
integrins, HIF-1a, and angiogenic
growth factors such as VEGF, fibroblast
growth factors, angiopoietins.
Metabolic reprogramming is HIF-
dependent, and, as indicated earlier,
HIF may be dysregulated in some
placental diseases. Targets other than
HIF that could be manipulated to sus-
tain the metabolic response to hypoxia
might include a variety of enzymes:
hexokinase, pyruvate kinase M2, lactate
dehydrogenase A, pyruvate dehydroge-
nase kinase, fatty acid synthase, and
glutaminase.
Inhibition of HIF-1a or its gene
products has been a primary focus in
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
the development of cancer drugs.
There are >400 drugs that specifically
target the HIF pathway, most of them
inhibitory. Many of them also target
specific, cancer-related mutations that
are related to the HIF pathway; hence, a
reverse-engineering approach could be
applied to existing drugs to see whether
they would be beneficial, as has been
done in human immunodeficiency vi-
rus.267,268 A placental strategy might
consist of controlled up-regulation of
HIF-1a, because it is known to be
elevated stably in hypoxic placentae
and likely contributes to the increased
angiogenesis that is a favorable adap-
tation when placental hypoxia is pre-
sent but not associated with a
disease.220
Scientific gaps. For further advances in
molecular targeting, greater information
is required in a number of areas. For
example, although there is a large
quantity of data on the role and effects of
vasodilators, the receptors for these
agents are understood poorly. Although
a targeted increase in receptors for va-
sodilators would be a possible thera-
peutic strategy, we clearly require more
knowledge of the broader effects of re-
ceptor modulation.
Similarly for the angiogenic growth
factors, what are the consequences if
some, but not other angiogenic growth
factors, are targeted in the placenta.
What might be the response to modu-
lating the angiogenic growth factor re-
ceptors rather than the growth factors
themselves? Might it be possible to
devise tissue and/or cell type selective
modulation that would bypass the
problems of the less-controlled distri-
bution of the ligands? Placental
vascular pericytes have been suggested
as a hematopoietic stem cell or
mesenchymal stem cell. They are
involved intimately in vessel stability
and angiogenesis.269-271 Pericytes can
be detached from their vascular niche
by angiopoietin-2, which would in-
crease the stimulus for branching
angiogenesis. Again we lack more
knowledge to determine whether these
supporting characters may be the key to
successful vascular therapies.
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
Is fetal growth a feasible target for
placental intervention? (Nicholas P.
Illsley, Hackensack University
Medical Center)
Background. Alterations in fetal nutrient
concentrations because of changes in
placental transport and/or metabolism
are associated with a variety of fetal
growth diseases. Deficits in oxygen are
associated with hypoxia, preeclampsia,
and IUGR. Deficits in glucose and/or
amino acids are associated with IUGR,
and an excess of glucose is associated
with macrosomia, obesity, and diabetes
mellitus. Alterations in placental
nutrient transporters are associated with
a variety of fetal growth diseases; the
glucose transporter 1 in hypoxia and
diabetes mellitus272,273 and amino acid
transporters in IUGR and diabetes mel-
litus.274,275 The simplest possibility in
these circumstances would be to reverse
these deleterious changes by manipula-
tion of maternal substrate levels. There
are situations in which action to reba-
lance abnormal nutrient concentrations
is capable of correcting growth prob-
lems: the restoration of normal maternal
glycemic status to prevent macrosomia
being an example. Overall, however, the
record with regard to maternal nutrient
supplementation is 1 of minimal effect
combined with potentially serious side-
effects.276
Another set of important questions
revolves around timing. Is there a win-
dow during which intervention is
possible, and perhaps more importantly,
how do we know when intervention is
necessary? In answer to the former,
intervention is clearly preferable before
structural and biochemical changes
make the growth restriction process
difficult to reverse, leading to the latter
question: how do we detect the initial
stages of growth restriction? The best
indicator we have currently that fetal
growth is deviating from normal is the
fetal growth trajectory derived from
serial ultrasound measurements. Given
that this requires multiple measure-
ments over weeks of gestation, by the
time we can verify growth restriction, the
fetus (and placenta) are already well
advanced through pathologic changes in
growth. This crude indicator signals a
S17
Supplement Obstetrics
process that is already well underway
and, at best, will allow for stabilization of
growth at a much lower percentile. It is
clear that, without a means to detect
the early stages of the processes that
contribute to growth restriction, inter-
vention will lag well behind. Our own
research into placental metabolic pro-
cesses has shown that the early stages
of growth reduction involve a switch
away from oxidative metabolism toward
an increased level of glycolytic meta-
bolism (placental metabolic reprogram-
ming).277 As a potential early indicator
of growth problems, the initiation of
placental metabolic reprogramming may
provide the crucial biomarkers needed to
detect the first stages of an altered
growth trajectory.
Current opportunities. Assuming that
combined biochemical/imaging methods
will eventually detect at-risk pregnancies,
what might we focus on as placental
targets for intervention? Is it possible to
see nutrient transporters as possible
molecular targets? The ones that concern
us here are those that transport the
essential, higher volume nutrients,
including glucose, amino acids and fatty
acids. Because the high volume glucose
transporters (ie, GLUT1 and GLUT3)
demonstrate unique polarized distribu-
tions between the maternal-facing
microvillous and fetal-facing basal sur-
faces,278,279 altering the balance of glucose
transporters within the syncytiotropho-
blast raises with it the question of the
selective polarized targeting that is needed
to achieve increased transport. The
polarized targeting question arises also
for amino acid transporters when the
asymmetric distribution acts to move
sufficient quantities and types of amino
acid against their gradient into the
fetus.280 In the absence of targeting in-
formation for these transporters, we need
to be assured that interventions will
provide the appropriate distribution be-
tween microvillous and basal faces
necessary to increase flux. Moreover, the
effects of increased transporter density,
although possibly increasing trans-
placental fluxes, will also pose other
problems. In the case of glucose, this is
likely to generate increased glycolytic
S18 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
metabolism and subsequent lactic
acidosis, as observed in glucose supple-
mentation interventions. In the case of
amino acids, the interconnected nature of
their transport and the broad substrate
specificity of amino acid transporters
suggest that alterations to an individual
transporter will have significant and
hard-to-predict ramifications for the
transport of other amino acids and for
placental amino acid metabolism. Simi-
larly, it is difficult to predict what the ef-
fect of altering the expression of placental
lipases, fatty acid transport, or fatty acid
binding proteins might be on processes
such as oxidative metabolism and the
transport of other nutrients. The essen-
tial, high-volume nutrients have a wide
range of metabolic fates, and they or their
metabolites play crucial roles in regu-
lating key metabolic pathways. Not sur-
prisingly therefore, changes in the
expression of individual nutrient trans-
porters are likely to be associated with
unwanted changes in placental and/or
fetal metabolism. The uncertainty
engendered by the alteration of individual
transporters makes them problematic
candidate molecular targets.
An alternative approach to targeting
individual transport or metabolic pro-
cesses is the generation of alterations in a
manner that combines multiple meta-
bolic and/or transport targets. In this
way, the transport and metabolic pro-
cesses that involve specific substrates are
modulated in an integrated fashion. We
already have detailed information on
many of these integrated pathways. They
form the traditional endocrine or growth
factor pathways that mediate regulation
of cellular function by extracellular fac-
tors. We know for example that insulin-
like growth factor I (IGF-1) regulates the
expression of placental glucose and
amino acid transporters281,282 and that it
is associated positively with fetal and
placental growth.283,284 Judicious
adjustment of the placental IGF-1
pathway might allow for an integrated
regulation of growth processes. Recent
research has shown that adenoviral-
mediated delivery of IGF-1 to the
placenta appears to stimulate glucose
and amino acid transporter expression
in vivo and to correct placental
ajog.org
insufficiency.285,286 Similar types of
growth factor-mediated regulation
might be possible via the signaling
pathways stimulated by IGF-2 or
placental GH. Perhaps the molecular
targets for these pathways might be
restricted to selected cell types by
altering receptor levels rather than
altering the concentration of the growth
factors themselves.
Future opportunities. Although the effec-
tors described earlier may be able to
stimulate integrated pathways for the
promotion of fetoplacental growth, the
signaling pathways that are activated in
this way may be too numerous or too
broadly based to avoid other less desir-
able effects. Growth factor signaling
pathways frequently have many trans-
duction pathways that branch off below
the growth factor receptor. Another
alternative is the targeting of metabolic or
signaling subnetworks that control mul-
tiple components but do not extend to all
of the endpoints for a signaling pathway.
For example mTOR is a serine kinase that
is the focus of a signaling pathway that
forms a nexus for multiple nutritional
and metabolic signals. MTOR affects
growth by modulating protein synthesis,
lipid synthesis, and energy metabolism,
primarily through phosphorylation of
the 4E-binding proteins that in turn have
marked effects on the production of the
proteins that control messenger RNA
(mRNA) translation.287 In the placenta,
in addition to effects on protein synthe-
sis, mTOR appears also to modulate
amino acid transporters by regulating the
membrane trafficking of transporter
protein.289 There is good evidence to
suggest that mTOR activity is propor-
tional to maternal body mass in-
dex288,289; however, in IUGR, syncytial
mTOR, while displaying increased
expression, shows decreased activ-
ity.288,290 This suggests that the mTOR
pathway may be a good potential regu-
latory point for growth-related inter-
vention. Is it possible, for example, to
target the mTOR system directly, below
the broadly regulatory phosphatidyli-
nositol-3-kinase/protein kinase B stage
(Figure 8)? Activation via agents, such as
the small experimental drugs MHY
ajog.org Obstetrics Supplement

1485291 or 3BDO,292 might confine the

effects to a smaller set of endpoints than FIGURE 8
the use of IGF-1. Will it be possible to Metabolic mechanistic target of rapamycin intervention points
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 inte-
grated, stimulation. This will require
detailed knowledge of the subsystems
that are involved and raises the question
of targeting and specificity.

Scientific gaps. Like substrate supple-

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

JULY 2016 American Journal of Obstetrics & Gynecology S19

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Supplement Obstetrics
FIGURE 9
Generation of oxidative and
nitrative stress
Superoxide generated from molecular oxygen by
membrane bound (myeloid differentiation pri-
mary response gene 88 oxidase) or cytosolic
(xanthine oxidase) enzymes or the mitochondrial
electron transport chain normally can be effec-
tively dismutated to hydrogen peroxide by su-
peroxide dismutase. Increased superoxide will
attack targets, which leads to oxidative stress.
With increasing generation of nitric oxide, nitric
oxide out competes superoxide dismutase for
superoxide and interacts to produce the more
powerful prooxidant peroxynitrite. Peroxynitrite
nitrates proteins at tyrosine residues and cova-
lently modifies function usually in a negative
manner. Superoxide dismutase is inactivated
when nitrated by prooxidant peroxynitrite, hence
leading to a negative feedback loop that leads to
oxidative and nitrative stress.
NO, nitric oxide; ONOOe, prooxidant peroxynitrite; SOD, super-
oxide dismutase.
Ilekis. Potential placental molecular therapeutic targets. Am J
Obstet Gynecol 2016.
superoxide, hydroxyl anion, hydrogen
peroxide, and the ability of various
antioxidant mechanisms to scavenge
them. Further, ROS can also interact
with reactive nitrogen species such as
nitric oxide to produce the powerful
prooxidant peroxynitrite, which, in turn,
can nitrate tyrosine residues in proteins,
such as superoxide dismutase300 (SOD;
Figure 9), causing nitrative stress, which
affects protein function, usually in a
negative manner.301 Not all reactive
oxygen and nitrogen species are equal in
potency, that being determined by their
cellular diffusion distance, defined as
the distance moved in aqueous solution
in 1 half-life. Hence, hydroxyl anion has
a diffusion distance of only 5 angstroms,
superoxide 0.4 mm, peroxynitrite 5 mm
S20 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
and nitric oxide 100 mm.302 Therefore, in
the placenta, the sites of action (intra- vs
extra- or transcellular) depends on
intracellular and cellular site of synthesis
in relation to placental structure such as
trophoblast thickness (5mm), stem
villous diameter (500-1500mm), and the
presence of antioxidant molecules to
scavenge them.303
Role of mitochondria. The major sources
of ROS are the mitochondrial electron
transport chain but also plasma mem-
brane enzymes, such as the nicotinamide
adenine dinucleotide phosphate oxi-
dases, cytosolic enzymes (including
xanthine oxidase, flavin enzymes, and
cytochrome p450s), lipoxygenases, and
cyclooxygenases.295 Although widely
acknowledged for their pathologic ef-
fects that include covalent modification
of proteins, lipids, and DNA, ROSs also
function as physiologic effectors, regu-
lating redox sensitive genes and pro-
teins.304 There are many methods
available to measure ROS. Because of
their ephemeral nature, direct measure-
ment of ROS is difficult; therefore,
measurement of effects that include lipid
peroxidation, covalent modification of
proteins, DNA damage and repair, levels
and activity of antioxidant molecules
and enzymes, and enzymes generating
ROS are used. There is no gold standard
measure of oxidative stress; rather one
should chose a measure related to the
area of interest.305
Mitochondria have many roles in
cellular function in addition to energy
production, which includes apoptosis,
steroid synthesis, calcium homeostasis,
and amino acid transport. Changes in
mitochondrial activity can be induced by
environmental factors such as nutrition,
hypoxia, aging, and obesity that impact
cellular survival and that are associated
with adverse pregnancy outcome. Mito-
chondria are the major source of ROS
under physiologic conditions because of
the release of high-energy electrons from
complexes I and III of the electron
transport chain; these electrons reduce
molecular O2 to superoxide, which
normally is scavenged by the mito-
chondrial SOD isoform, mitochondrial
antioxidant manganese SOD. However,
ajog.org
mitochondrial function itself can be
compromised by severe and/or pro-
longed oxidative stress via damage to
mitochondrial DNA, proteins, and
lipids. We recently have shown that
maternal adiposity leads to increased
oxidative stress in the placenta and that
this is associated with decreased
trophoblast mitochondrial respiration
measured in vitro using a Seahorse
extracellular flux analyzer (Seahorse
Bioscience, Copenhagen, Denmark).306
This dysfunction is further evidenced
by decreased mitochondria number,
expression of complexes I-V of the elec-
tronic transport chain, and decreased
placental adenosine triphosphate gener-
ation. In addition, these trophoblasts
appear metabolically inflexible because
they cannot switch to alternative energy
sources when placed on galactose to
prevent glycolysis.305
Current opportunities. Subsequent to
acquiring knowledge of increased
oxidative stress in pregnancies with
adverse outcomes, there have been many
trials of antioxidant therapy to try and
prevent these outcomes.307 All have
failed, which is an outcome variously
attributed to choice of antioxidant, time
and duration of treatment, heterogeneity
of patients studied, the fact that oxidative
stress may indeed not be part of the
pathophysiologic evidence, or the need
for targeted rather than global therapy.
This begs the question: what are the tar-
gets at the tissue and cellular levels?
Should antioxidants that are targeted to
placenta by use of nanoparticles or
liposomes be used rather than global
treatment? Cellular targets could include
antioxidant enzymes such as SOD,
glutathione peroxidase, thioredoxin
reductase and catalase, the enzymes that
synthesize ROS such as xanthine oxidase
and nicotinamide adenine dinucleotide
phosphate oxidases, extracellular anti-
oxidants (transferrin, ceruloplasmin,
uric acid, and bilirubin), intracellular
reducing elements (glutathione, coen-
zyme Q10, and cytochrome c oxidase)
and nutrients and supplements such as
vitamins C and E and melatonin. Because
mitochondria are the major source of
antioxidants, there has been growing
ajog.org
interest in mitochondrial targeting of
antioxidants by compounds such as the
mitochondria-targeted antioxidant drug,
MitoQ, where coenzyme Q10 is linked to
a lipophilic cation to allow adsorption
through the inner mitochondrial mem-
brane.308 Other approaches include the
use of selenium,309 which is found in the
active site of the selenoprotein gluta-
thione peroxidase, or of melatonin,
which functions as an antioxidant.310
Future opportunities. We recently have
shown that the hypoxamir, miRNA 210,
can inhibit mitochondrial respiration in
trophoblast cells,311 hence opening the
door to targeted therapies to alleviate
mitochondrial dysfunction and oxida-
tive stress in the placenta. The male fetus
is at greater risk of adverse pregnancy
outcome (eg, preterm birth and still-
birth) than is the female fetus, although
it is unclear whether this is because the
male places itself at risk by its desire for
greater growth or if the female adopts a
conservative strategy to ensure success-
ful delivery and propagation of the spe-
cies.312,313 Sexual dimorphism occurs in
placental gene expression,314 particularly
of genes that are involved in the in-
flammatory response, in response to
maternal adiposity and in diseases such
as preeclampsia. We found increased
expression of inflammatory and
apoptotic markers in placentas of male
fetuses from preeclamptic pregnancies
compared with female fetuses.315 Simi-
larly, we found differences in placental
expression of miRNA 210 and its mito-
chondrial target genes between male and
female fetuses of lean, overweight, and
obese women that were mediated by
differences in the transcription factor
NF-kB p50.305 Our current data there-
fore indicate that, in conditions such as
GDM, preeclampsia, and obesity, in-
flammatory pathways are activated in the
placenta in a sexually dimorphic manner
to regulate the production of reactive
O2 and nitrogen species that lead to
oxidative/nitrative stress and to mito-
chondrial dysfunction (Figure 10). This
results in placental dysfunction and
adverse pregnancy outcome and suggests
that targeting of inflammatory pathways
might be a useful approach to prevent
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
oxidative stress. Dietary manipulation
(eg, inclusion of omega 3 fatty acids
and vegetables), the use of antiin-
flammatories (eg, resveratrol) and
immune selective antiinflammatory de-
rivatives, or targeting strategies that
involve miRNA, siRNA, viral vectors,
nanoparticles, and biologics may prove
useful. These targeting strategies, how-
ever, may need to be different based on
the sex of the placenta.
Scientific gaps. Current gaps in the
knowledge include the absence of
proof of a cause-and-effect relationship
between oxidative stress and adverse
outcome. Perhaps an animal model of
induced oxidative stress with adverse
outcome would facilitate this. In addi-
tion, the tissue and cellular localization
and contribution of different sources of
oxidative stress (ie, including the gener-
ation and scavenging mechanisms of
ROS) must be studied comprehensively.
Further, the timing of the oxidative stress
insult, either early, mid, or late gestation
or continuously, on outcomes needs to
be considered when designing drug tar-
geting studies.
Identification of potentially useful
or experimental drugs to target
important
Challenges and advantages of rescuing
and repurposing (Christine Colvis,
National Center for Advancing
Translational Sciences)
Background. The most common reason
for an investigational drug to fail to make
it through the Food and Drug Adminis-
tration (FDA) approval for marketing is its
inability to demonstrate clinically mean-
ingful efficacy in the disease being studied.
At the stage of phase 2 trials, another
major reason for failure is simply because
of business strategy decisions that result in
the deprioritization of a drug.316,317 In
either case, there is no scientific reason
that the drug could not be pursued for an
indication other than that for which it was
originally being developed.
National Center for Advancing Trans-
lational Sciences (NCATS) new thera-
peutic uses program. The new therapeutic
uses program uses a novel approach to
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
FIGURE 10
Putative mechanism for the
involvement of inflammation-
mediated placental dysfunction
in fetal programming
The adverse inflammatory maternal environ-
ments of gestational diabetes mellitus, pre-
eclampsia, or obesity can generate increased
oxidative/nitrative stress and cause mitochon-
drial dysfunction in the placenta in a sexually
dimorphic manner. This disrupts placental
function and in turn may lead to alterations in
placental-mediated regulation of maternal
metabolism and fetal growth and differentiation
and hence result in fetal programming.
GDM, gestational diabetes mellitus.
Ilekis. Potential placental molecular therapeutic targets. Am J
Obstet Gynecol 2016.
provide academic researchers with ac-
cess to investigational drugs from phar-
maceutical companies.318 The program
leverages investments that have been
made by pharmaceutical companies in a
variety of drugs and biologics (agents)
but for which the company has often
times discontinued development. To
qualify for inclusion in the program, the
agent offered by the company has to have
been through at least a phase 1 clinical
trial, so that safety in humans has already
been assessed in at least 1 population. By
limiting the agents to only those that
have been tested in humans, the new
therapeutic uses program is able to move
projects quickly to phase 2a trials to test
the agents for efficacy in new indications.
Under the program, the pharmaceutical
companies provide agents (clinical sup-
ply including matched placebo and
preclinical material, if needed) and the
data that will be needed to file an inves-
tigational new drug application with the
FDA to study the agent for the new
S21
Supplement Obstetrics
indication. The National Institutes of
Health (NIH) provides the financial
support for phase 2a trials and, if needed,
phase 1b trials and/or preclinical studies.
The NIH and the pharmaceutical
companies are turning to the broader
research community to identify novel
uses for the agents. The value of this
strategy bore out when, as a pilot,
NCATS listed 58 agents provided by
companies in June 2012 and within
2 months received almost 160 pre-
applications with ideas of indications
that the agents might be used to treat.
Because of the limited time (12 months)
allowed under the program for preclin-
ical studies, pediatric trials were not
encouraged in the 2012 pilot. However,
in 2014, NCATS issued a new set of
solicitations or funding opportunity
announcements, and this time offered
an funding opportunity announcement
specifically for pediatric indications. The
companies, in turn, had identified those
agents that they believed could be
developed for a pediatric indication. In
general, it was expected that pediatric
trials would be proposed only when
there was no adult patient population
for the disease or for which the mecha-
nism of action of the drug might be
meaningful only if modulated at an
earlier stage in development than in
adulthood.
There are significant challenges when
trying to repurpose an investigational
drug for a pediatric indication. Unlike an
FDA-approved drug, these drugs often
have been studied in only dozens or
maybe a few hundred adults. So, side-
effects have been documented for
only a small number of individuals.
Furthermore, the metabolism of drugs
can be very different in younger
individuals than in adults. We face
similar challenges when we think about
exposing a pregnant woman to an
investigational drug. Effects of drug
exposure in pregnant women generally
will remain unknown until after the drug
has been on the market for years or even
decades.
Current opportunities. The NCATS
intramural program offers several op-
portunities for collaboration. Although
S22 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
funds are not offered, there are several
ways to access valuable resources and
expertise at no cost to our collaborators.
The intramural program that likely has
the most potential for repurposing drugs
for use in pregnant women is the
screening of the NCATS pharmaceutical
collection. Nearly 2750 small molecular
entities have been approved for clinical
use by the United States and in other
countries around the world. NCATS has
amassed a screening collection of 2500 of
these, along with approximately 1000
additional investigational compounds.
Because they have been approved for
clinical use, these drugs will have been
used in far more people than the inves-
tigational drugs that are used in the New
Therapeutic Uses program, thereby
providing a more comprehensive risk
profile for the drugs. Collaborators
generally approach NCATS with a high
throughput screening compatible assay
as a first step toward repurposing 1 of
these approved medications. Of course,
when a drug is first marketed, often, the
only data available on fetal effects
generally will come from preclinical
data. The FDA encourages the use of
pregnancy exposure registries as a first-
line strategy for gathering information
on drug exposure during pregnancy.
They have compiled a list of >60 regis-
tries to facilitate the gathering of valuable
data.319 Cross-referencing data from
such registries with findings from
in vitro assays that were used to screen
the NCATS pharmaceutical collection
may provide a strategy for repurposing
drugs to address placental health
indications.
Future opportunities. Repurposing both
investigational and approved drugs is a
strategy that is pursued currently in
small and large pharma and in
academia. The strategy is attractive to so
many because of the significant cost-
savings that result from the use of
drugs for which so much early devel-
opment has been completed already. In
the case of repurposing drugs for use in
pregnant women, it would be expected
that the longer a drug has been on the
market, the more information we would
have on exposure during pregnancy.
ajog.org
Unfortunately, the drugs that will fall in
this category will often be drugs for
which a generic is available, thereby
eliminating an enforceable use patent or
regulatory exclusivity. Currently, there
are no effective incentives to pursue a
new indication for a drug that is avail-
able as a generic, regardless of the indi-
cation, unless a unique formulation for
the drug is developed.320 Although this
is a barrier for all indications, it is
particularly unfortunate for pregnant
women, which is a population for which
the development of new drugs (investi-
gational drugs) is extremely unlikely.
Effect of pregnancy on drugs
pharmacokinetics and
pharmacodynamics (Maged M.
Costantine, MD, University of
Texas Medical Branch, Galveston)
Background. The use of medications
in pregnancy has been increasing pro-
gressively over the past 3-4 decades
(Figure 11).321-325 This is predominantly
because of changing in the de-
mographics of pregnant women,
because more women enter pregnancy at
a later age with increasing prevalence
of preexisting medical comorbidities
(such as diabetes mellitus, hypertension,
asthma, depression, and others) and
increased risk of obstetric complications
(eg, nausea and vomiting of pregnancy,
GDM, cholestasis of pregnancy, pre-
eclampsia.) that require pharmaco-
therapy.320-324 In a recent review, the
average number of medications this is
used by pregnant women is >4.320 This
increase in the use of medications is also
predominant in the first trimester, where
almost one-third of pregnant women use
at least 4 medications.320 This is con-
cerning because the first trimester is a
crucial period for organogenesis and
placental development that includes
trophoblast invasion, vascular remodel-
ing, and chorionic villi development; in
addition, many women are unaware of
their pregnancy status.320-324
Pregnancy physiologic changes and
their impact on medication pharmacoki-
netic and pharmacodynamics proper-
ties. Pregnancy is characterized by
significant changes in the anatomy
ajog.org Obstetrics Supplement

and physiology of almost all systems,

which affect medications’ pharmacoki-
netic/pharmacodynamics properties
(Figure 12). Pharmacokinetic generally
refers to “what the body does to the
drug” and usually is described using the
drug’s absorption, distribution, meta-
bolism, and elimination; pharmacody-
namics refers to “what the drug does to
the body” and usually influences the
drug’s efficacy 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
flow.327,331 Cardiac output is further
increased during labor, with additional
300-500 mL of blood reentering the
circulation with every contraction. Both
systemic and pulmonary vascular re-
sistances decrease by 20-30%. Blood
pressure also falls toward the end of
the first trimester and then rises again in
the third trimester to prepregnancy
levels. The increase in renal blood flow
leads to a parallel increase in the
glomerular filtration rate by approxi-
mately 40-65%.327,329,332 This can lead
to significant increase (20-60%) in the
elimination rates of renally cleared
medications, which lead to shorter half-
lives and risk of subtherapeutic concen-
trations. Examples of these medications
include lithium, ampicillin, cefuroxime,
cefazolin, piperacillin, atenalol, and
digoxin.326,329
During pregnancy, maternal blood
volume increases by 40-50%, and total
body water increases to almost 8.5 L by
the end of pregnancy. In addition,
maternal fat stores and fat mass increase
by almost 10-fold. The increase in blood
volume and water within the body
expands the volume of distribution of
hydrophilic substances.333 On the other
hand, the increase in fat mass leads to a
larger volume of distribution of lipo-
philic drugs, such as sedatives. For some
drugs, a larger volume of distribution
FIGURE 11
Medication use during pregnancy
Secular patterns of use of any medication that was restricted to the first trimester at any time during
the period of 1976-2008. Average number of medications and proportion of women taking 4
medications (n ¼ 25,313) is shown.
Reprinted with permission from Mitchell et al.321
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
could lead to decreased peak serum
concentration, which may necessitate a
higher initial and maintenance dose to
obtain therapeutic plasma concentra-
tions.329,334 Additionally, because of
the decrease in serum albumin during
pregnancy, highly protein bound com-
pounds (such as digoxin, midazolam,
and phenytoin) may display higher free
levels, which increases their peak
plasma concentrations.327,329 The hyper-
vascularity and edema of the upper res-
piratory mucosa theoretically may result
in an increased absorption of inhaled
agents such as corticosteroids.329,335 The
delayed gastric emptying and prolonged
small bowel transit time may alter the
bioavailability (decrease maximum con-
centration and time to maximum con-
centration) and thus decrease the efficacy
of oral drugs that are taken as a single
dose because a rapid onset of action is
desired.329,336
Drug metabolism is also altered in
pregnancy, in part, because of elevated
sex hormones. In general, drug meta-
bolism occurs through phase I
(oxidation, reduction, or hydrolysis)
and/or phase II metabolism (glucur-
onidation, acetylation, methylation, and
sulfation). Cytochrome (CYP) P450
represents a family of enzymes and is a
major route of drug metabolism that is
affected by pregnancy (activity increased
for CYP3A4, CYP2D6, CYP2C9,
CYP2A6, and decreased for CYP1A2,
CYP2C19, and CYP2D6 subtypes). For
example, the increase in activity and
abundance of CYP3A4 in pregnancy lead
to an increase in the clearance of its
many substrates, such as nifedipine,
carbamazepine, midazolam, saquinavir,
indinavir, lopinavir, ritonavir, and many
others.337-340
In summary, maternal physiologic
changes affect the pharmacokinetic/
pharmacodynamics of drugs through
changes in the drugs’ absorption,
increasing the volume of distribution,
protein binding, renal clearance, meta-
bolism, and placental biodisposition.
Any novel drug that is identified to act on
potential placental molecular targets
must be evaluated in the context of these

JULY 2016 American Journal of Obstetrics & Gynecology S23

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Supplement Obstetrics ajog.org

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 phar-
macokinetic and pharmacodynamics
throughout the pregnancy. Others rely
on primate animal models; however,
they are usually expensive and hard to
work with. Future opportunities in this
field include the development of novel
technologies to assess the placenta in
real-time and applying them to study
medication’s biodisposition in preg-
nancy. 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

Pregnancy induces major physiological changes that can affect the pharmacokinetic and pharma-
codynamic properties of medications.
CO, cardiac output; BP, blood pressure; HR, heart rate; NVP, nausea and vomiting of pregnancy; SVR, systemic vascular resistance.
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
changes so that appropriate dosing be
identified.
Current opportunities. Pregnant women
are still considered therapeutic orphans
because most current therapeutics were
never studied during pregnancy.341-343
In a recent review of the literature,
<2% of all pharmacokinetic studies
involved pregnant women.344 This is
concerning because the lack of data on
pregnancy-specific dosage of many
medications leads physicians to extrap-
olate drug dosage regimens from
nonpregnant subjects or men. This may
lead to over or under dosing and may
reduce efficacy and increase risk of
toxicity. In view of these gaps, the
opportunities to study the pharmacoki-
netic properties of many currently
used medications with potential impact
on placental development and use
in selective targeting of molecular
pathways are involved in the patho-
physiology of various obstetrical com-
plications. This will lead to more
effective treatment of pregnant women,
while reducing the risks of adverse
events.341 The new FDA rule regarding
drug classification represents a huge
current opportunity to encourage re-
search in this field.
Future opportunities. A major obstacle to
studying medications (current and
novel) in pregnancy is the lack of systems
never studied in pregnancy during
development, because pregnant women
were excluded from such studies.341-343
In addition, their safety, tolerability, ef-
ficacy, and dosing were extrapolated
from studies conducted in men or
nonpregnant women. This leads to un-
der or over dosing and affects efficacy
and toxicity of these medications. There
exists a large gap in information
regarding medications pharmacokinetic
properties in pregnancy and during
lactation. The effects of placental trans-
porters and biodisposition enzymes
on medication pharmacokinetic and
pharmacodynamics, and transplacental
transfer represent a large gap in our
scientific knowledge. Moreover, the
inability to study placental transport
expected after delivery with delivered
placentas widens this scientific gap.
From bench to bedside: processes and
pitfalls translating research findings
into practice paradigms (David M.
Haas, Indiana University)
Background. When Vannevar Bush wrote
“Science: the endless frontier” in 1945, it
inspired that science would provide
solutions to health problems. It further
described how investment in scientific
research could serve as a vehicle to

S24 American Journal of Obstetrics & Gynecology JULY 2016

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org
enhance the wealth and prosperity of the
nation.345 It is against that backdrop that
the public holds out hope for scientific
inquiry. This promise engenders a social
contract that funding of scientific in-
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 trans-
lational research spectrum and the dif-
ficulties with translating research into
practice, to articulate additional con-
cerns and difficulties with translational
research in pregnancy by highlighting
some translational successes in preg-
nancy research, and to propose some
mechanisms that are aimed at improving
the translation of findings into practice
and measuring research impact.
Translational research. The research
spectrum generally begins with basic
science findings that are then translated
to human and clinical research
(Figure 13). This is termed “T1” research
on the translational spectrum: the
translation of animal and basic science
research into humans.346 The “T2” step
in research translation is taking the
clinically relevant research findings and
translating them into practice guidelines
and having them become routine prac-
tice in health care.347,348 Some models of
the translational spectrum have included
more steps that can include the popula-
tion health impact and involvement of
consumers. All of the models serve to
help assess the success of translational
research at fulfilling the initial promise
to improve public health.
Globally, billions of dollars are spent
on biomedical, clinical, and health ser-
vices research, training of providers,
quality improvement initiatives, and
safety initiatives.347 However, it has been
demonstrated that, in the United States,
only 55% of patients receive the recom-
mended evidence-based care.349 Addi-
tionally, 20-30% of patients get care that
is not needed or could be potentially
harmful.350 This is a failure in translating
research findings into practice. Experts
have found that this can be due to system
failures. Grimshaw et al348 noted, “Sys-
tems fail to ensure that effective and
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Obstetrics Supplement
FIGURE 13
Translational research peaks and valleys
This figure illustrates the multiple-step model (5 hills and 4 valleys) of translating research into
practice and public health benefit and the many “valleys of death” that can be encountered along the
way. Depicted are the 5 phases of translational research separating 4 valleys: basic discovery
science to research involving humans (T1), from human studies to evidence-based guidelines (T2),
from guidelines to health practice (T3), and from health practices to population health programs (T4).
Reprinted with permission from Meslin et al.345
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
cost-effective programs, services, and practice, costs, limited resources,
drugs get to all those who need them commercialization issues, broad social
and providers fail to provide the level of policy implications, professional stan-
care they aspire to.” dards, governmental policy, and general
An example of this is seen in the fact science policy.344 There are many places
that in 2010, the NIH spent $31 billion where potential health-improving
on research. However, from 2006-2009, research findings can become derailed.
only 74 new drugs were approved by the The key is to overcome these difficulties
FDA, which was only one-half the and cross the translational valleys of
number approved during the previous death.
epoch.351 One interesting study looked
at 101 promising technologies that were Translational research in pregnancy.
published in the highest impact journals. Translational research in pregnancy
This report found that, in the 20 years holds additional potential pitfalls that
after publication, only 5 of these prom- generally flow from the concerns for the
ising technologies were licensed for impact of therapies on developing babies
clinical use, with only 1 angiotension- and potential long-term impacts that
converting enzyme inhibitor being used may be unrecognized for years after
regularly in clinical practice.352 The birth. It is for this reason that, for many
report found multiple steps where these years, pregnant women were actively
promising technologies failed, were lost, excluded from research. There are
refuted, or neglected along the trans- several examples in which translational
lational spectrum. It has also been found research failed to detect problems, lead-
that promising technologies take a long ing to major public health consequences.
time to get into routine practice, at times In 1956, thalidomide was introduced as a
up to 17 years.345 These areas of trans- “wonder drug” in Germany.353 It was
lational failure have been coined the purported to be a treatment for nausea
translational valleys of death.344 The and vomiting, insomnia, coughs, colds,
translational valleys of death can come and headaches. Early studies in mice and
from regulations that impede research or rats did not demonstrate congenital
JULY 2016 American Journal of Obstetrics & Gynecology S25
Supplement Obstetrics
anomalies; therefore, it was used as a
sedative and tranquilizer in early preg-
nancy. However, in 1961, reports sur-
faced of severe congenital anomalies that
included limb reduction defects that led
to its rapid withdrawal from the market.
This was a clear failure of T1 research.
Two notable examples of failure in T2
research in pregnancy have been seen
recently. The withdrawal from the mar-
ket of the combination drug Bendectin
was done because of lawsuit pressure
regarding congenital anomalies.354 The
drug was used by a large proportion of
pregnant women for nausea and vomit-
ing in early pregnancy. However, no re-
ports of defects were proved. Without an
alternative, women suffered. The rates
of hospitalization for severe dehydration
from hyperemesis gravidarum rose
dramatically. This was an example of
where research demonstrated safety and
practice patterns were established, but
fear and a lack of a supportive response
to the public caused the withdrawal of
an effective therapy, which led to a
negative public health impact. Even
more recently, the poor adoption rates of
the effective human papilloma virus
vaccines to prevent cervical cancer is
another example of success at the T1
stage, but unfortunately a failure at the
T2 stage.355 Despite proven efficacy and
safety and coverage by the Children’s
Immunization Program, vaccination
rates of the target populations remain
low.
However, there are some noteworthy
translational successes in pregnancy. The
development of Rhogam to prevent Rh
isoimmunization came about from a
confluence of observations along the
translational spectrum. After demon-
strating efficacy and safety in clinical
trials, it has been adopted universally as a
standard of care preventive therapy in
pregnancy, saving thousands of babies
every year.356,357 In similar ways, ante-
natal corticosteroids for accelerating
fetal lung maturity in threatened pre-
term birth have been a translational
success story in obstetrics.358 From
astute observations in sheep to landmark
clinical trials in humans and pivotal
metaanalyses, antenatal corticosteroids
are now used routinely on labor and
S26 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
delivery units and clearly have reduced
the rates of mortality in preterm
newborn infants.359-361 This was greatly
aided by NIH Consensus Conferences
highlighting the benefits and safety of
the therapy.362
How to improve translational success.
Many have proposed systems that are
aimed at crossing the translational val-
leys of death. One unifying theme is
aimed at getting stakeholder investment
in the translational process. Practicing
providers, the public, industry, and reg-
ulatory/governmental agencies must
have a stake in the translational processes
along the way if there is any hope of
translating important research findings
into practice aimed at improving the
public health.347,363-365 Otherwise, fail-
ure at T2 and beyond can occur
commonly. The formation of trans-
lational teams that involve the stake-
holders can ensure involvement from the
beginning and should involve the entire
translational spectrum.344,347 Effective
dissemination of the impact of trans-
lational research can inform those
providing funding and the public as to
the public good that is being gained.
Psychology of change research has
noted 3 stages of practice change:
awareness, acceptance, and adoption.366
Most translational research focuses on
the first 2 stages, whereas the reason that
most promising therapies fail to realize
their public health impact is the failure of
“adoption” into practice. A provider may
know that research shows a benefit and
accept that it might help the patients, but
there may be obstacles to getting it into a
routine practice pattern; thus, it is not
adopted. This is because “adoption”
takes different pathways in the brain
before these cognitive changes become
embedded in practice.365
A focus on stakeholders and align-
ment of incentives can help with the
adoption into practice. Dissemination of
the impact of research findings is crucial
to get stakeholder enthusiasm. For
instance, the Human Genome Project
turned a $4 billion government invest-
ment into $796 billion in economic
growth.367 Translational teams, like
those stimulated by the NIH Clinical and
ajog.org
Translational Science Awards, focus on
collaboration and development of net-
works of scientists, stakeholders, and
regulatory agencies in an effort to propel
translational science across the valleys of
death and into practice.368,369
Specific to pregnancy, there is a
tremendous need for public involve-
ment, education, and advocacy. The
focus of protection of mothers and in-
fants through research, instead of from
research, should be highlighted. Ethicists
and children’s advocacy groups should
also be engaged. The advent of individ-
ualized therapeutics and diagnostics
are forcing this conversation to accel-
erate.370 In addition, the utilization
of biorepositories can help stimulate
this translational process by focusing
on potential pooled resources and
collaborations.
In conclusion, bridging the trans-
lational research gap from bench to
bedside has many potential pitfalls.
Pregnancy translational research has had
well-documented missteps but several
important success stories as well.
Although research to cross the trans-
lational “valleys of death” is difficult,
using what is known about behavior
change will be important in ultimately
fulfilling the promise of scientific
research funding to improve public
health and the public good. Thinking
broadly about stakeholders and in-
volving these stakeholder teams in the
processes across the translational spec-
trum can help cross the valleys of death
and help take promising discoveries
from bench to bedside.
Current opportunities. Individualized
pharmacotherapy provides opportunities
currently to tie genomic and other
“-omic” technologies to ongoing clinical
trials. In addition, the development and
utilization of saved samples from past
trials and biorepositories provide a wealth
of opportunities for analyses using cutting
edge technologies to assay for biomarkers
that can serve as therapeutic monitoring
or individualized pharmacotherapy
model development aids for patients. The
focus on diagnostic and therapeutic
studies in human models and humans is a
clear priority moving forward.
ajog.org Obstetrics Supplement

Future opportunities. Clinical trials in the

future should have clear response bio- FIGURE 14
markers, pharmacokinetic/pharmaco- Schematic of extracellular microRNAs derived from human trophoblasts
dynamic/pharmacogenomic outcomes,
and harmonized efficacy outcome mea-
sures 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 diag-
nostic and therapeutic trials that
receive funding to have these types of
outcomes and measures will focus
investigators.

Scientific gaps. The plethora of basic sci-

ence data accumulating about placental
development and the interface between
mother and fetus must be linked clearly
to diagnostic and/or therapeutic needs.
In addition, because there is not perfect
animal model, there needs to be a way to
study these on human placental models
as often as possible. Another clear gap in
much of the therapeutic research in
pregnancy is a lack of long-term follow
up of the infant. Ensuring funding for
follow-up studies, including potential
“-omic” studies, of the offspring should
be a priority.
Evolving technologies for placental
specific therapeutic drugs
Trophoblastic nanovesicles, miRNA,
and their function (Yoel Sadovsky,
MD, Magee-Womens Research
Institute)
Background. Coordinated communica-
tion among cells and tissues is critical
for development, homeostatic func-
tion, and adaptation to change. The
trafficking of information between the
fetoplacental and maternal compart-
ments is essential for fetal develop-
ment, growth, and pregnancy health. It
also serves to reduce maternal-fetal
conflicts and to balance biologic re-
sources for the benefit of the 2 organ-
isms. In past years, it was believed that
communication between the fetopla-
cental unit and the mother was
executed by hormones and growth
factors, which are soluble in the blood
or are bound by protein carriers, and
serve as paracrine or endocrine signals
that control pregnancy health. It is now
MicroRNAs can be released from the trophoblast layer in different forms: microvesicle-enveloped
form; apoptotic bodyeenveloped form; nano-sized, exosome-encapsulated form; and RNA-
binding, protein-bound form. Exosomes are formed by budding in intraluminal vesicles to form
multivesicular bodies. Multivesicular bodies fuse with the plasma membrane and release their
intraluminal vesicles as exosomes into the extracellular space. In contrast, microvesicles are pro-
duced directly by budding and the detachment of membrane vesicles from the plasma membrane.
Apoptotic bodies (blebs) derive from cells that are undergoing apoptotic fragmentation and the
formation of membrane-enclosed vesicles.
miRNA, microRNA.
Reprinted with permission from Ouyang et al.395
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
clear that nucleic acids, and particularly It has become clear recently that, in
sRNA molecules, traffic among tissues addition to the effect of miRNAs on
and impact cell biology. intercellular gene regulatory networks,
miRNAs are also packaged within
MiRNAs and extracellular nanovesicles. A extracellular vesicles, where they can be
major class of these noncoding RNAs, trafficked to local and distant cell types
and 1 of the best studied, is the family of (Figure 14). Transport within extracel-
small regulatory RNAs called miRNA. lular vesicles may provide substantial
MiRNAs originally were described in the advantages, including stability, intravas-
nematode Caenorhabditis elegans and cular processing, and selective delivery
were later found in the genome of many to target cells.371-373 Diverse cell types
organisms, including humans. They (eg, hematopoietic cells, mast cells,
are single-strand RNA molecules of platelets, neurons) produce and release
20-24 nucleotides that characteristically extracellular vesicles, which can be
repress gene expression by guiding an found in the blood, urine, saliva, breast
RNA-induced silencing complex to a milk, amniotic fluid, ascites, cerebro-
target RNA, with subsequent attenuation spinal fluid, bile, lymph, tears, and
of gene expression by the inhibition semen.374-377 These vesicles are charac-
of mRNA translation and/or mRNA terized by their size, shape, and con-
degradation. tent.378 Importantly, when released to

JULY 2016 American Journal of Obstetrics & Gynecology S27

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Supplement Obstetrics ajog.org

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 charac-
terized 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 exo-
somes. Many studies on extracellular
vesicles in pregnancy have associated
a mixture of vesicles (exosomes,
microvesicles, apoptotic blebs) with
disorders of pregnancy, thus blurring
some the distinct properties of these
vesicles.398-401 Placental exosomes have
been implicated in several discrete
functions, such as immune modula-
tion,402-404 where exosomal cargo pro-
teins, 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-
Reprinted with permission from Delorme-Axford et al.417 tical to that of villous trophoblasts.396
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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org
transferred to other, nonplacental pri-
mary cells and cell lines by exposing
these cells to a medium that was pre-
conditioned by primary human tro-
phoblasts or to exosomes that are
produced in human trophoblasts
(Figure 15). We also found that this
effect was mediated, at least in
part, by exosome-packaged C19MC
miRNAs.393,396 Cells transfected with
a bacterial artificial chromosome that
harbors the C19MC locus or with
individual, highly expressed C19MC
miRNAs also became more resistant to
viral infections. Our data showed that
this effect of exosomal C19MC miRNAs
was mediated by the stimulation of
autophagy in recipient cells and that
pharmacologic or genomic inhibition of
autophagy diminished the conferral of
antiviral response to recipient cells.393
Although C19MC miRNAs likely act in
concert with other antiviral responses,
our data pointed to a unique and
transferrable antiviral response that is
mediated by human trophoblast-derived
exosomes.
Although the mechanisms, dynamics,
and targets of miRNA-containing
trophoblastic exosomes remain un-
known, our recent human and mouse
data suggest that trophoblastic C19MC
miRNAs are transferred primarily from
trophoblasts to maternal tissues. This
is plausible, because trophoblasts are
bathed directly in maternal blood yet
separated from the fetal blood by a
basement membrane and endothelial
cells. Together, these analyses shed new
light on miRNA-based communication
among the fetoplacental and maternal
compartments. Importantly, our data
suggest an extraordinary means of
nonhormonal communication between
the placenta and the maternal-fetal
compartments, which suggests a critical
role for miRNAs in pregnancy health.
Current opportunities. Although it is
clear that the plasma contains several
types of extracellular vesicles, their mo-
lecular and metabolic cargo are largely
unknown. Moreover, our understanding
of the function of these vesicles is rudi-
mentary. The formation of the placenta
during pregnancy adds a new source of
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
extracellular vesicles to the maternal
blood stream and possibly to the
fetal compartment. Whereas C19MC
miRNAs are packaged distinctly within
these placental vesicles, it is not clear
whether there are other, placenta-
specific, molecules and signals that are
packaged within vesicles. Importantly,
modern technology enables us to sepa-
rate the different types of vesicles and to
associate them with disorders of preg-
nancy that reflect placental function. It is
also possible that the state of pregnancy
changes the repertoire of extracellular
vesicles that are produced by maternal
tissues, thus adding to the landscape of
pregnancy-related extracellular vesicles.
This creates a new opportunity to catalog
these vesicles, assess their origin, con-
tent, and cellular targets and to define
the normal landscape of extracellular
vesicles (the “vesiculome”) throughout
human gestation.
Future opportunities. The development
of new tools to isolate extracellular
vesicles efficiently may allow us to
examine local and systemic targets of
trophoblastic extracellular vesicles and
whether their cargo faithfully informs
the health of trophoblasts. It may also
allow us to identify target cells and
interrogate the impact of pregnancy-
specific extracellular vesicles on these
cells in health and disease. Although
initial work has centered primarily on
maternal tissue targets, future research
may focus on intraplacental paracrine
targets and fetal targets of placental
extracellular vesicles. These opportu-
nities, coupled with recent de-
velopments in understanding of miRNA
function, will require the development
of innovative label-free technologies to
collect and sort extracellular vesicles,
which will shorten the time to clinical
translation and potentially offer a dra-
matic improvement in our ability to
assess placental health dynamically
throughout pregnancy using maternal
blood tests. Knowledge in this field may
also stimulate the development of
nanovesicles as carriers of noncoding
RNAs, drugs, and chemicals that selec-
tively target placental cells for thera-
peutic purposes.
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
Scientific gaps. The current and future
opportunities that were summarized
require us to bridge pertinent knowledge
gaps. We must understand the process of
cargo loading to diverse trophoblastic
vesicles and the potential selectivity of
this process. This should be followed by
analysis of the mechanisms used by
trophoblastic exosomes to enter target
cells selectively and impact their biologic
condition. The current state of perinatal
science raises additional questions:
Do surface and cargo proteins within
trophoblastic extracellular vesicles play a
role in this process? What determines
targeting by extracellular vesicles? Can
fetal vesicle-bound or free miRNAs and
other types of RNA molecules cross the
placenta into the maternal circulation
and inform on the health of fetal tissues?
How early in gestation can we find and
isolate placental extracellular vesicles
and RNA from maternal blood? The
answers to these and related questions
will greatly impact the field of placental
biology and its translation to pregnancy
diagnostics and therapy.
Aberrant regulation of myometrial
contractility by maternal cell-free
plasma (CFP) RNA of placental origin:
screening and therapeutic implications
(Carl P. Weiner, MD, University of
Kansas School of Medicine)
Background. The CFP transcriptome is
altered by numerous disorders, often
reflecting the stage of disease develop-
ment.420 Although the biologic roles of
these coding and noncoding RNAs are
unclear, they are not simply cellular
debris. Synthesized and released by a
range of cells that include human cho-
rionic villi, their biologic stability and
target tissue specificity are thought to
reflect their release within either exo-
somes or shedding vesicles or attached
to argonaute 2 proteins.421 Cell-to-cell
transfer and their ability to function
once transferred are shown in studies
that used mRNA/miRNA-loaded extra-
cellular vesicles and protein com-
plexes.422-424
Based predominantly on studies of
symptomatic women, spontaneous pre-
term birth (sPTB) is considered an
inflammatory process.428 We measured
S29
Supplement Obstetrics
both the mass restricted (MR) score and
IL-6 in 1004 consecutive amniotic fluid
samples at 16 weeks from healthy preg-
nancies with known outcomes (unpub-
lished data). Ten percent delivered at
<37 weeks gestation; 4% delivered at
<32 weeks gestation. Nineteen percent
had an MR score of 1; 2% had an MR
score of 3 or 4, which indicated signifi-
cant inflammation. However, there was
no relationship between either sPTB or
preterm premature rupture of mem-
branes and the MR score; 23 of 27
deliveries at <28 weeks gestation
occurred to women with an MR score of
0. IL-6 levels increased as the MR score
rose (0 ¼ 102 pg/mL; 3 ¼ 451 pg/mL,
and 4 ¼ 706 pg/mL) but was unrelated to
subsequent sPTB. Whatever the role of
inflammation in preterm or term birth,
it seemingly occurs after 20 weeks
gestation.
We proposed in 2011 that mRNA and
miRNA were candidate mechanisms for
the regulation of myometrial quiescence
activation (unpublished data) and have
been testing the hypothesis that CFP
RNAs enable communication between
mother and pregnancy and that, as a
direct result, changes in the maternal
CFP transcriptome predict both the
occurrence and mechanism of sPTB.
Methodologic refinements. In 2007, we
developed an extraction method that
yielded increased quantities of both CFP
nucleic acids and proteins in a single
process. The typical yield of total
RNA extracted is 18-35mg/mL plasma,
enabling comprehensive, transcriptome
wide study with the use of multiple
microarrays and/or next-generation
sequencing, plus the confirmatory po-
lymerase chain reaction studies all on the
same patient plasma sample.
To speed marker validation and
facilitate translation into the clinical
arena, we developed a series of tech-
niques that included economical, high-
throughput gene quantification that
allows for the running of panels of genes
to predict sPTB within a few hours.
The method allows us to run all
controls necessary for marker quantifi-
cation in a single polymerase chain
reaction well.
S30 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
The CFP transcriptome is altered in
women destined for sPTB by 16 weeks.
Using longitudinally obtained banked
plasma samples from women with
known pregnancy outcomes and
microarray/next-generation sequencing,
we identified and confirmed >90 novel
CFP RNAs in the plasma of women who
were destined for sPTB between 26-32
weeks gestation; some of the marker
expressions were altered as early as
16 weeks gestation. In a second cohort,
we found that several markers that were
abnormal at 16 weeks gestation were also
abnormal at 12 weeks gestation. We then
determined that 5 CFP markers were
known from studies of other cell systems
to interact with 7 myometrial gene/gene
products belonging to a previously
identified cluster of genes: the preterm
initiator set.425 Five of the 7 myometrial
genes were involved with the regulation
of intracellular calcium.
CFP RNAs originate from the pla-
centa. We next sought the anatomic
origin of the 5 CFP RNA markers; each
was dramatically overexpressed in the
placenta of women with <33 sPTB
weeks gestation, but not the placenta of
women delivered prematurely absent
labor and of women delivered at term in
labor.
CFP RNAs markers of sPTB can regulate
myometrial contractility. We then focused
on 1 particular CFP mRNA biomarker,
apolipoprotein, that, in silica, interacted
with the interferon gamma receptor,
which in turn interacted with 4 addi-
tional preterm initiator genes. Over-
expression of apolipoprotein in human
pregnant myometrial cells increased
both intracellular calcium flux and cell
contractility. The addition of interferon
gamma receptor siRNA blunted the
stimulatory effect of apolipoprotein,
which demonstrated that interferon
gamma receptor was at least 1 of the
targets of the apolipoprotein mRNA.
Current opportunities. These findings
support the often stated premise that
sPTB is a result of placental dysfunction.
Specific CFP RNAs are released from
the placenta of women who are destined
ajog.org
for sPTB at <33 weeks gestation by the
end of the first/beginning of the second
trimester that interfere with myometrial
quiescence, thus potentially creating
an environment that favors sPTB. The
early pregnancy time frame suggests
that effective therapy will require early
detection and initiation, which is
consistent with the general failure of
available tocolytic agents. It highlights
the preeminent importance of eluci-
dating the events that are involved in
human implantation and early placen-
tation. As such, the sPTB studies
described may serve an investigational
paradigm for the other great pregnancy
syndromes such as preeclampsia and
hypoxia that are associated FGR and
perinatal brain damage.
The first task along the path to un-
derstanding human placentation is
descriptive: the creation of an appro-
priately sized research medical center
network for sample processing (using
new and established biobanks) for the
rapid construction of an “omics” atlas of
“normal” from the time period covering
implantation through at least 16 weeks
gestation. Such an atlas would serve as a
basis for all future research and generate
a host of new targets for hypothesis-
based testing in vitro using systems
similar to those described earlier.
Future opportunities. Real-time assess-
ment of the human placenta all but re-
quires noninvasive methods that may
not exist presently or have not yet been
applied to the study of the placenta. So
much about human placental function is
shrouded by darkness that the areas for
investigation are almost unlimited, each
with the potential for making a contri-
bution. Yet, it is highly likely that much
placental dysfunction has its foundations
set by the early second trimester and that
it is here that new insights are needed
desperately. The application of newer
organ on chip technology could allow
the testing of potential interventions
that will be identified during atlas con-
struction.426 Real-time approaches will
require either new imaging modalities
or be “omics” in nature. As discrete
markers of placental function linked to
specific disease phenotypes emerge, it
ajog.org Obstetrics Supplement

may be possible to reduce the processing

from a minimum of a laboratory work FIGURE 16
day to less than a few hours, providing Translational steps for the development of nanoparticles for placental
near real-time feedback for nonimaging drug delivery
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 establish-
ment will be of modest impact. Future
efforts should build on the knowledge
derived from atlas construction to seek
targeted therapies that enhance implan-
tation and early placental development.

Nanoparticles for placental drug

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

JULY 2016 American Journal of Obstetrics & Gynecology S31

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Supplement Obstetrics
experimental models appear to be suit-
able for nanoparticle transport studies as
well. For example, the transplacental
transfer of polymeric nanoparticles has
been shown to be size-dependent in both
models, and limited transfer of silica
nanoparticles was demonstrated in both
BeWo cell and placental perfusion ex-
periments.435-437
Additional studies have shown that
gold nanoparticles do not cross the term
human placenta, that the transplacental
transport of poly-amidoamine den-
drimers is limited, and that liposome
transport across the placenta is size-
dependent.438-440 The observed size
dependence of nanoparticle transport
has been extended to demonstrate
increased drug transport across placental
trophoblast cells when the drug is
encapsulated in smaller polymeric
nanoparticles (diameter, 146 nm)
compared with larger nanoparticles
(diameter, 232 nm).441 Literature re-
ports of nanoparticles that have been
administered to pregnant rodents have
included titanium dioxide nanoparticles,
silica nanoparticles, zinc oxide nano-
particles, iron oxide nanoparticles, and
gold nanoparticles; results vary depend-
ing on particle size, surface charge, and
gestational age.442-446
Targeting strategies. Strategies for tar-
geted delivery of nanoparticles to the
placenta include the identification of
peptides by phage display.447,448 Re-
searchers have reported enhanced
transcellular transfer of T7 bacterio-
phages across BeWo cells with an
integrin-binding amino acid sequence
peptide. Harris et al used a similar
approach in pregnant mice and identi-
fied two peptides with increased
placental binding, designated as amino
acid sequence consisting of
CRGDKGPDC and KRK (unpublished
data).448 Kaitu’u-Lino et al449 designed
bacterial-derived nanocells that are
coated with antibodies to epidermal
growth factor receptor. The uptake of
these epidermal growth factor
receptoretargeted delivery vehicles in
ex vivo human placental explants was
greater than that of nontargeted nano-
cells. We have synthesized polymeric
S32 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
nanoparticles conjugated with folic acid
to take advantage of the relatively high
expression of folate receptors in the
placenta. This has resulted in greater
cellular uptake and transport of the
nanoparticles across BeWo cells.
Current opportunities. Immediate appli-
cations of these technologic advances
include fetal drug therapy and placental
therapy. Although medication during
pregnancy is most often prescribed to
treat maternal conditions, there are a
number of fetal diseases for which a
targeted, transplacental delivery strategy
potentially could attenuate the adverse
maternal side-effects that are associated
with current treatment modalities.
These include fetal cardiac arrhythmias,
congenital adrenal hyperplasia, and fetal
thyroid disorders.450 Targeted drug de-
livery to the placenta itself could also
improve therapeutic options for
malaria-infected placenta or prevent
mother-to-child transmission of HIV.451
Future opportunities. Nanotechnology
has also been suggested for placental
diagnostic applications.452 Opportu-
nities abound for the development of
nanoparticle-mediated biosensors and
imaging agents, and it is likely that many
recent advances in cancer diagnostics,
for example, can be utilized to monitor
the structure and function of the human
placenta.453 As progress towards these
goals continues, it is imperative that the
biocompatibility of such nanomaterials
is investigated thoroughly to prevent
unintended consequences of fetal expo-
sure to any potentially harmful imaging
agents. For example, gadolinium-based
magnetic resonance imaging contrast
agents are not recommended for use
during pregnancy because of concerns
for nephrogenic systemic fibrosis.454
Scientific gaps. Steps to translate the
preclinical promises of this technology
towards clinical trials and clinical prac-
tice will include scaling proof-of-
concept results from in vitro and small
animal studies to larger animal models
with placental structure and function
more similar to human placenta. For
example, recent progress has been made
ajog.org
to establish the pregnant baboon as a
nonhuman primate model for pharma-
cokinetic investigations.455-460 There is
also a need to refine the pool of potential
placental targeting moieties to minimize
off-target nanoparticle distribution.
Side-effects can be averted when the
disbursement of medication to off-target
sites is reduced. A necessary, but
exciting, task will be to determine the
magnitude of dose reductions that are
possible when targeted therapeutic
strategies are applied. Nanoparticle-
mediated delivery to the placenta offers
tremendous benefits for maternal-fetal
medicine.
Maternally sequestered delivery
systems for prevention of fetal drug
exposure (Gene Bidwell, University
of Mississippi Medical Center)
Background. Preeclampsia is a common
hypertensive disorder of pregnancy and
is one of the leading causes of maternal
and fetal morbidity and death. There is
currently no effective intervention for
preeclampsia short of induced delivery,
which makes it a leading cause of
premature birth. Improvements in
preeclampsia management have been
largely stifled because of deleterious
effects of proposed small-molecule
therapeutics on the developing fetus.
The objective of our studies was to
develop a drug delivery system capable of
stabilizing novel therapeutic agents in
the maternal circulation while protecting
them from entering the fetal circulation.
The use of a biopolymer drug carrier to
prevent drug transfer across the pla-
centa. The major focus of our research
group is to develop drug carriers capable
of preventing placental transfer and fetal
exposure to therapeutic agents. Protein
transport across the placental barrier is
highly regulated, and proteins that are
not substrates for active transporters
rarely cross into fetal circulation. To take
advantage of this, we are using a protein-
based biopolymer to fuse to therapeutic
agents and sequester them in the
maternal circulation (Figure 17). This
biopolymer, termed elastin-like poly-
peptide (ELP), is based on a repeating
sequence found in human elastin.461
ajog.org Obstetrics Supplement

ELP is an ideal drug carrier for several

reasons. Because of its origins in human FIGURE 17
elastin, it is not recognized as foreign by Model of the elastin-like polypeptide drug delivery vector
the immune system, does not induce an
inflammatory response or immunoge-
nicity, and is likely slowly broken down
into individual amino acids in vivo,
making it completely biocompatible.
Because the ELP sequence is encoded at
the DNA level, modification of the
polymer sequence (to tune the polymer
size, to add reactive sites for drug
conjugation, or to fuse targeting agents,
therapeutic peptides, or therapeutic
proteins) is a matter of simple molecular
biology.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

JULY 2016 American Journal of Obstetrics & Gynecology S33

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Supplement Obstetrics ajog.org

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 benefit of
increasing placental perfusion by
enhancing uterine or spiral artery blood
flow. We are also examining a related
strategy of supplementing PlGF by
administering an ELP-PlGF fusion pro-
tein. 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 administra-
tion 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
Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol 2016.
systemic macrophages and/or endothe-
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

Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
ajog.org
to free peptide, and prevents its transfer
across the placenta (unpublished data).
We hypothesize that a maternally
sequestered ELP-fused NF-kB inhibitor
will be effective as an antiinflammatory
agent and may slow the progression of
preeclampsia.
Current opportunities. The use of ELP or
related macromolecular carriers to
prevent fetal drug exposure represents a
promising new strategy for drug devel-
opment for disorders of pregnancy.
As described earlier, we are using
ELP fusions with therapeutic peptides
or proteins targeted to pathways of
importance in preeclampsia, but this
strategy is not limited to proteinaceous
therapeutics or to preeclampsia. ELP is
amenable to fusion with small molecule
drugs, and other projects in our labo-
ratory are examining the ability of
ELP to prevent transfer of known-toxic
or teratogenic drugs across the placenta,
thus potentially opening the door
for safe use of drugs that currently
are avoided in the pregnant patient
population.
Future opportunities. Our current strat-
egy is to use the ELP system to intervene
in late gestation, with therapeutics that
are targeted to pathways of currently
known importance in preeclampsia.
Future work will expand this strategy as
the list of potential drug targets con-
tinues to grow. The ELP system is
adapted easily for the delivery of nearly
any type of therapeutic, so expanding
this strategy to target newly identified
causative pathways is highly feasible. We
view the ELP system as a platform for
maternal sequestration of therapeutics,
and its attractive properties as a drug
carrier make it a promising platform
to be applied for delivery of novel
therapeutics.
Scientific gaps. Although we are focused
on treating symptoms of preeclampsia
during late gestation, the ideal scenario
would be to intervene early in pregnancy,
during trophoblast invasion and
placentation, to prevent the onset of
preeclampsia. However, to do that, we
need to gain a better understanding of
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
the causative factors that hamper proper
placental formation in preeclampsia,
and we need robust biomarkers to
identify patients who will experience the
disorder.
Another area that currently stands as a
hurdle to the development of therapeu-
tics for preeclampsia and other disorders
of pregnancy is the high level of risk
aversion when treating pregnant
mothers. This risk aversion is well justi-
fied given the vulnerability of the patient
population, and we have to address these
risks by doing all we can preclinically to
ensure the safety of developmental
therapeutics. One strategy for this is to
prevent fetal drug exposure using drug
carriers, as described earlier. In addition,
we must also strive to ensure that our
therapeutics is not adversely affecting
normal placental function. To address
these issues, we must use the best pre-
clinical models at our disposal to study
placental drug transport, fetal exposure,
and drug effects on normal placental
function, and we must follow offspring
in our preclinical models to insure
normal physical, mental, and physio-
logic development. Only after collecting
robust data in these areas should we
attempt to begin testing in human
pregnancies. Although we should pro-
ceed with caution and use every means at
our disposal to ensure safety, we should
not let fear of doing harm stifle innova-
tion in the development of diagnostics
and therapeutics for use in pregnant
patients.
Comment
Exciting research was presented at the
workshop in the area of placentation,
trophoblast migration, and spiral artery
remodeling. Research findings in these
areas will be critical for developing
future treatments and, ultimately, the
prevention of adverse pregnancy out-
comes of placental origin that include
preeclampsia and FGR. Knowledge of
the basic biology of trophoblast invasion
and spiral artery remodeling is critical to
understanding the cause of many preg-
nancy disorders; once the molecular
pathways that go awry are identified, it
will be possible to design therapeutics to
intervene in these pathways. However,
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
this goal also elucidates another major
gap in the field, the need for robust
biomarkers to predict the onset of the
disease early in pregnancy. Future
research in these areas will be critical for
filling this gap.
Data were also presented regarding
ongoing clinical efforts in the United
States and in Europe that are testing
novel interventions for preeclampsia and
FGR, including agents such as oral
arginine supplementation, sildenafil,
pravastatin, a virally delivered VEGF for
local administration into the uterine ar-
tery to improve blood flow and oxygen
supplementation therapy. Proposals
were also made to improve fetal health,
not by targeting maternal blood flow but
by enhancing nutrient transport to the
fetus by modulating glucose and amino
acid placental transporters. Strategies
were presented for inhibiting mito-
chondrial dysfunction and oxidative
stress to improve placental health; the
roles of inflammation on both tropho-
blast function and in the advanced stages
of preeclampsia were discussed. The
roles of miRNAs and placentally derived
exosomes were discussed in the context
of their use for diagnostics and as drug
targets. These diverse pathways illustrate
many opportunities for drug interven-
tion. Although a number of potential
drugs to target these pathways are avail-
able and have been shown to be relatively
safe, the therapeutic benefits of the ma-
jority of these drugs were never studied
in pregnancy. Hence, the safety and
pharmacokinetic profiles of these and
new therapeutics will need to be deter-
mined before their use in pregnancy. The
workshop discussed this aspect high-
lighting the unique pharmacokinetic
properties of pregnancy and the hurdles
and pitfalls of translating research find-
ings into practice. The workshop
concluded with discussions of drug de-
livery during pregnancy. The potential
for using nanoparticles or protein bio-
polymers during pregnancy was pre-
sented, either to prevent fetal drug
exposure by blocking placental transfer
or to encourage drug delivery to the fetus
for in utero therapy by attaching agents
that actively are transported by the
placenta. The use of macromolecular
S35
Supplement Obstetrics
carriers to prevent placental drug
transfer and fetal drug exposure repre-
sents an exciting new strategy for drug
development in pregnancy. Knowledge
that the fetus will not be exposed to the
therapeutic agent will open the door for
use of many drugs that currently are
avoided during pregnancy and will help
lessen the regulatory burden on drug
development for pregnancy-related
disorders.
The pregnant patient population
may be 1 of the most challenging co-
horts for which to develop drugs
because of concerns regarding drug ef-
fects on fetal outcome. Placental drug
transfer is a very real risk for many
small molecule drugs and even for
some actively transported biologics.
The potential for adverse fetal effects
not only represents a concern to preg-
nant mothers but also stifles drug
development because of risk aversion
from the pharmaceutical industry and
from regulatory bodies. For many
therapeutics, we simply do not know
the risk to pregnant mothers or to their
developing offspring because preg-
nancy is most commonly excluded
from clinical trials. Of course, inter-
vention during this vulnerable devel-
opmental period must be approached
with extreme care. We must use the best
preclinical models at our disposal to
maximize the chance of elucidating
adverse drug effects for novel test
agents. Also, the use of drug delivery
strategies to target the placenta specif-
ically and/or prevent placental drug
transfer and fetal drug exposure is an
exceptionally promising method of the
development of new and safe thera-
peutics for use in pregnant women.
In closing, a major theme that clearly
developed from the workshop was that
we, as a scientific community, need to
stop thinking of pregnant women as a
vulnerable patient population for which
drug development should be avoided
and begin to appreciate our opportunity
to improve both maternal and fetal
health. This strategy could not only have
an immediate benefit on pregnancy
outcome but also have a long-term
benefit on the future health of the in-
fant, because recent evidence has made it
S36 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
clear that the risks for many health out-
comes, which include the development
of cardiovascular diseases,482 metabolic
diseases,483 and cognitive developmental
disorders,484 just to name a few, are
strongly programmed by the in utero
environment. - 17. Van Rooij E, Kauppinen S. Development of
ACKNOWLEDGMENTS
We acknowledge Tamika Turner-Graydon for
her major contribution in the preparation and
editing of the manuscript and Rosalina Bray for
her excellent assistance in organizing the work-
shop meeting.
REFERENCES
1. Chaddha V, Viero S, Huppertz B, Kingdom J.
Developmental biology of the placenta and the
origins of placental insufficiency. Semin Fetal
Neonatal Med 2004;9:357-69.
2. Fisher SJ. The placental problem: linking
abnormal cytotrophoblast differentiation to the
maternal symptoms of preeclampsia. Reprod
Biol Endocrinol 2004;2:53.
3. Redline RW. Placental inflammation. Semin
Neonatol 2004;9:265-74.
4. Andraweera PH, Dekker GA, Roberts CT.
The vascular endothelial growth factor family in
adverse pregnancy outcomes. Hum Reprod
Update 2012;18:436-57.
5. Kingdom J, Huppertz B, Seaward G,
Kaufmann P. Development of the placental
villous tree and its consequences for fetal
growth. Eur J Obstet Gynecol Reprod Biol
2000;92:35-43.
6. Jansson T, Aye IL, Goberdhan DC. The
emerging role of mTORC1 signaling in placental
nutrient-sensing. Placenta 2012;33(suppl 2):
e23-9.
7. Perkins ND. Integrating cell-signalling path-
ways with NF-kappaB and IKK function. Nat Rev
Mol Cell Biol 2007;8:49-62.
8. Macintyre DA, Sykes L, Teoh TG,
Bennett PR. Prevention of preterm labour via the
modulation of inflammatory pathways. J Matern
Fetal Neonatal Med 2012;25(suppl 1):17-20.
9. The NCGC/NCATS Pharmaceutical Collec-
tion (vol 2015). Available at: http://tripod.nih.
gov/npc. Accessed: June 3, 2015.
10. PubChem. Available at: http://pubchem.
ncbi.nlm.nih.gov. Accessed: June 3, 2015.
11. The DrugBank database. Available at http://
www.drugbank.ca. Accessed: June 3, 2015.
12. KEGG DRUG. Available at http://www.
genome.jp/kegg/drug. Accessed: June 3, 2015.
13. GeneCards. Available at http://genecards.
org. Accessed: June 3, 2015.
14. Toporkiewicz M, Meissner J, Matusewicz L,
Czogalla A, Sikorski AF. Toward a magic or
imaginary bullet? Ligands for drug targeting to
cancer cells: principles, hopes, and challenges.
Int J Nanomedicine 2015;10:1399-414.
ajog.org
15. Silva AC, Lopes CM, Sousa Lobo JM,
Amaral MH. Nucleic acids delivery systems: a
challenge for pharmaceutical technologists. Curr
Drug Metab 2015;16:3-16.
16. Burnett JC, Rossi JJ. RNA-based thera-
peutics: current progress and future prospects.
Chem Biol 2012;19:60-71.
microRNA therapeutics is coming of age. EMBO
Mol Med 2014;6:851-64.
18. Svenson S, Prud’homme RE. Multifunc-
tional nanoparticles for drug delivery applica-
tions: imaging, targeting, and delivery. New
York: Springer-Verlag; 2012.
19. Vrachnis N, Kalampokas E, Sifakis S, et al.
Placental growth factor (PlGF): a key to opti-
mizing fetal growth. J Matern Fetal Neonatal
Med 2013;26:995-1002.
20. Maltepe E, Bakardjiev AI, Fisher SJ. The
placenta: transcriptional, epigenetic, and physi-
ological integration during development. J Clin
Invest 2010;120:1016-25.
21. Romero R, Dey SK, Fisher SJ. Preterm la-
bor: one syndrome, many causes. Science
2014;345:760-5.
22. Redman CW. Preeclampsia: a multi-stress
disorder. Rev Med Intern 2011;32(suppl 1):
S41-4.
23. Brosens IA, Robertson WB, Dixon HG. The
role of the spiral arteries in the pathogenesis of
preeclampsia. J Pathol 1970;101: Pvi.
24. Brosens I, Pijnenborg R, Vercruysse L,
Romero R. The “Great Obstetrical Syndromes”
are associated with disorders of deep placen-
tation. Am J Obstet Gynecol 2011;204:
193-201.
25. Fisher SJ. Why is placentation abnormal in
preeclampsia? Am J Obstet Gynecol
2015;213(suppl):S115-22.
26. Zhou Y, Damsky CH, Fisher SJ. Pre-
eclampsia is associated with failure of human
cytotrophoblasts to mimic a vascular adhesion
phenotype: one cause of defective endovascular
invasion in this syndrome? J Clin Invest 1997;99:
2152-64.
27. Zhou Y, McMaster M, Woo K, et al. Vascular
endothelial growth factor ligands and receptors
that regulate human cytotrophoblast survival
are dysregulated in severe preeclampsia and
hemolysis, elevated liver enzymes, and low
platelets syndrome. Am J Pathol 2002;160:
1405-23.
28. Clark DE, Smith SK, He Y, et al. A vascular
endothelial growth factor antagonist is produced
by the human placenta and released into the
maternal circulation. Biol Reprod 1998;59:
1540-8.
29. Maynard SE, Min JY, Merchan J, et al.
Excess placental soluble fms-like tyrosine kinase
1 (sFlt1) may contribute to endothelial dysfunc-
tion, hypertension, and proteinuria in pre-
eclampsia. J Clin Invest 2003;111:649-58.
30. Venkatesha S, Toporsian M, Lam C, et al.
Soluble endoglin contributes to the pathogen-
esis of preeclampsia. Nat Med 2006;12:642-9.
31. Levine RJ, Lam C, Qian C, et al. Soluble
endoglin and other circulating antiangiogenic
ajog.org
factors in preeclampsia. N Engl J Med
2006;355:992-1005.
32. Hirashima C, Ohkuchi A, Takahashi K,
Suzuki H, Matsubara S, Suzuki M. A novel three-
step approach for predicting the imminent onset
of preeclampsia within 4 weeks after blood
sampling at 19-31 weeks of gestation. Hyper-
tens Res 2014;37:519-25.
33. Chaiworapongsa T, Romero R, Whitten AE,
et al. The use of angiogenic biomarkers in
maternal blood to identify which SGA fetuses will
require a preterm delivery and mothers who will
develop pre-eclampsia. J Matern Fetal Neonatal
Med 2015:1-15.
34. Nakimuli A, Chazara O, Hiby SE, et al. A KIR
B centromeric region present in Africans but not
Europeans protects pregnant women from pre-
eclampsia. Proc Natl Acad Sci U S A 2015;112:
845-50.
35. Zhou Y, Gormley MJ, Hunkapiller NM, et al.
Reversal of gene dysregulation in cultured
cytotrophoblasts reveals possible causes of
preeclampsia. J Clin Invest 2013;123:
2862-72.
36. Berman J, Girardi G, Salmon JE. TNF-alpha
is a critical effector and a target for therapy in
antiphospholipid antibody-induced pregnancy
loss. J Immunol 2005;174:485-90.
37. Lo YM, Tein MS, Lau TK, et al. Quantitative
analysis of fetal DNA in maternal plasma and
serum: implications for noninvasive prenatal
diagnosis. Am J Hum Genet 1998;62:768-75.
38. Norton ME, Jacobsson B, Swamy GK, et al.
Cell-free DNA analysis for noninvasive exami-
nation of trisomy. N Engl J Med 2015;372:
1589-97.
39. Koh W, Pan W, Gawad C, et al. Noninvasive
in vivo monitoring of tissue-specific global gene
expression in humans. Proc Natl Acad Sci U S A
2014;111:7361-6.
40. Redman CW, Sargent IL. Circulating
microparticles in normal pregnancy and pre-
eclampsia. Placenta 2008;29(suppl A):S73-7.
41. Mincheva-Nilsson L, Baranov V. Placenta-
derived exosomes and syncytiotrophoblast
microparticles and their role in human repro-
duction: immune modulation for pregnancy
success. Am J Reprod Immunol 2014;72:
440-57.
42. Medzhitov R. Recognition of microorgan-
isms and activation of the immune response.
Nature 2007;449:819-26.
43. Jin C, Flavell RA. Innate sensors of pathogen
and stress: linking inflammation to obesity.
J Allergy Clin Immunol 2013;132:287-94.
44. Suman P, Gupta S. Immunology of Implan-
tation, Implantation Failure and Pregnancy. Part
A: Regulators of Early Invasion of Trophoblast
Cells. In: Chaouat G, Olivier S, Ledee N, eds.
Immunology of Pregnancy 2013: Bentham,
2013.
45. Kawai T, Akira S. The role of pattern-
recognition receptors in innate immunity:
update on Toll-like receptors. Nat Immunol
2010;11:373-84.
46. Akira S, Takeda K. Toll-like receptor signal-
ling. Nat Rev Immunol 2004;4:499-511.
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
47. Song DH, Lee JO. Sensing of microbial
molecular patterns by Toll-like receptors.
Immunol Rev 2012;250:216-29.
48. Kondo T, Kawai T, Akira S. Dissecting
negative regulation of Toll-like receptor
signaling. Trends Immunol 2012;33:449-58.
49. O’Neill LA. When signaling pathways collide:
positive and negative regulation of toll-like re-
ceptor signal transduction. Immunity 2008;29:
12-20.
50. Shaw MH, Reimer T, Kim YG, Nunez G.
NOD-like receptors (NLRs): bona fide intracel-
lular microbial sensors. Curr Opin Immunol
2008;20:377-82.
51. Franchi L, Warner N, Viani K, Nunez G.
Function of Nod-like receptors in microbial
recognition and host defense. Immunol Rev
2009;227:106-28.
52. Caruso R, Warner N, Inohara N, Nunez G.
NOD1 and NOD2: signaling, host defense,
and inflammatory disease. Immunity 2014;41:
898-908.
53. Jin C, Flavell RA. Molecular mechanism
of NLRP3 inflammasome activation. J Clin
Immunol 2010;30:628-31.
54. Martinon F, Tschopp J. Inflammatory cas-
pases and inflammasomes: master switches of
inflammation. Cell Death Differ 2007;14:10-22.
55. Cohen P. The TLR and IL-1 signalling
network at a glance. J Cell Sci 2014;127:
2383-90.
56. Nadeau-Vallee M, Quiniou C, Palacios J,
et al. Novel noncompetitive IL-1 receptor-biased
ligand prevents infection- and inflammation-
induced preterm birth. J Immunol 2015;195:
3402-15.
57. Netea MG, Simon A, Van De Veerdonk F,
Kullberg BJ, Van Der Meer JW, Joosten LA. IL-
1beta processing in host defense: beyond the
inflammasomes. PLoS Pathog 2010;6:
e1000661.
58. Abrahams VM, Bole-Aldo P, Kim YM, et al.
Divergent trophoblast responses to bacterial
products mediated by TLRs. J Immunol
2004;173:4286-96.
59. Abrahams VM, Visintin I, Aldo PB, Guller S,
Romero R, Mor G. A role for TLRs in the regu-
lation of immune cell migration by first trimester
trophoblast cells. J Immunol 2005;175:
8096-104.
60. Costello MJ, Joyce SK, Abrahams VM. NOD
protein expression and function in first trimester
trophoblast cells. Am J Reprod Immunol
2007;57:67-80.
61. Mulla MJ, Yu AG, Cardenas I, Guller S,
Panda B, Abrahams VM. Regulation of Nod1
and Nod2 in first trimester trophoblast cells. Am
J Reprod Immunol 2009;61:294-302.
62. Cardenas I, Mulla MJ, Myrtolli K, et al. Nod1
activation by bacterial iE-DAP induces maternal-
fetal inflammation and preterm labor. J Immunol
2011;187:980-6.
63. Ma Y, Kadner SS, Guller S. Differential ef-
fects of lipopolysaccharide and thrombin on
interleukin-8 expression in syncytiotrophoblasts
and endothelial cells: implications for fetal
survival. Ann N Y Acad Sci 2004;1034:236-44.
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
64. Elovitz MA, Mrinalini C. Animal models of
preterm birth. Trends Endocrinol Metab
2004;15:479-87.
65. Wang H, Hirsch E. Bacterially-induced pre-
term labor and regulation of prostaglandin-
metabolizing enzyme expression in mice: the
role of toll-like receptor 4. Biol Reprod 2003;69:
1957-63.
66. Elovitz MA, Wang Z, Chien EK, Rychlik DF,
Phillippe M. A new model for inflammation-
induced preterm birth: the role of platelet-
activating factor and Toll-like receptor-4. Am J
Pathol 2003;163:2103-11.
67. Cardenas I, Means RE, Aldo P, et al. Viral
infection of the placenta leads to fetal inflam-
mation and sensitization to bacterial products
predisposing to preterm labor. J Immunol
2010;185:1248-57.
68. Cardenas I, Mor G, Aldo P, et al. Placental
viral infection sensitizes to endotoxin-induced
pre-term labor: a double hit hypothesis. Am J
Reprod Immunol 2011;65:110-7.
69. Murphy SP, Fast LD, Hanna NN, Sharma S.
Uterine NK cells mediate inflammation-induced
fetal demise in IL-10-null mice. J Immunol
2005;175:4084-90.
70. Robertson SA, Skinner RJ, Care AS.
Essential role for IL-10 in resistance to
lipopolysaccharide-induced preterm labor in
mice. J Immunol 2006;177:4888-96.
71. Aagaard K, Ma J, Antony KM, Ganu R,
Petrosino J, Versalovic J. The placenta harbors a
unique microbiome. Sci Transl Med 2014;6:
237ra65.
72. Stout MJ, Conlon B, Landeau M, et al.
Identification of intracellular bacteria in the basal
plate of the human placenta in term and preterm
gestations. Am J Obstet Gynecol 2013;208:
226.e1-7.
73. Grigsby PL, Novy MJ, Sadowsky DW, et al.
Maternal azithromycin therapy for Ureaplasma
intraamniotic infection delays preterm delivery
and reduces fetal lung injury in a primate model.
Am J Obstet Gynecol 2012;207:475.e1-14.
74. Gravett MG, Adams KM, Sadowsky DW,
et al. Immunomodulators plus antibiotics delay
preterm delivery after experimental intraamniotic
infection in a nonhuman primate model. Am J
Obstet Gynecol 2007;197:518.e1-8.
75. Ma Y, Krikun G, Abrahams VM, Mor G,
Guller S. Cell type-specific expression and
function of toll-like receptors 2 and 4 in human
placenta: implications in fetal infection. Placenta
2007;28:1024-31.
76. Abrahams VM, Aldo PB, Murphy SP, et al.
TLR6 modulates first trimester trophoblast
responses to peptidoglycan. J Immunol
2008;180:6035-43.
77. Garg M, Potter JA, Abrahams VM. Identifi-
cation of microRNAs that regulate TLR2-
mediated trophoblast apoptosis and inhibition
of IL-6 mRNA. PLoS One 2013;8:e77249.
78. Mulla MJ, Myrtolli K, Tadesse S, et al. Cut-
ting-edge report: TLR10 plays a role in mediating
bacterial peptidoglycan-induced trophoblast
apoptosis. Am J Reprod Immunol 2013;69:
449-53.
S37
Supplement Obstetrics
79. Ilievski V, Lu SJ, Hirsch E. Activation of toll-
like receptors 2 or 3 and preterm delivery in the
mouse. Reprod Sci 2007;14:315-20.
80. Abrahams VM, Schaefer TM, Fahey JV, et al.
Expression and secretion of antiviral factors
by trophoblast cells following stimulation by
the TLR-3 agonist, Poly(I : C). Hum Reprod
2006;21:2432-9.
81. Aldo PB, Mulla MJ, Romero R, Mor G,
Abrahams VM. Viral ssRNA induces first
trimester trophoblast apoptosis through an in-
flammatory mechanism. Am J Reprod Immunol
2010;64:27-37.
82. Potter JA, Garg M, Girard S, Abrahams VM.
Viral single stranded RNA induces a trophoblast
pro-inflammatory and antiviral response in a
TLR8-dependent and -independent manner.
Biol Reprod 2015;92:17.
83. Koga K, Cardenas I, Aldo P, et al. Activation
of TLR3 in the trophoblast is associated with
preterm delivery. Am J Reprod Immunol
2009;61:196-212.
84. Chatterjee P, Weaver LE, Doersch KM, et al.
Placental Toll-like receptor 3 and Toll-like re-
ceptor 7/8 activation contributes to preeclamp-
sia in humans and mice. PLoS One 2012;7:
e41884.
85. Van Beijnum JR, Buurman WA,
Griffioen AW. Convergence and amplification of
toll-like receptor (TLR) and receptor for
advanced glycation end products (RAGE)
signaling pathways via high mobility group B1
(HMGB1). Angiogenesis 2008;11:91-9.
86. Romero R, Chaiworapongsa T, Alpay
Savasan Z, et al. Damage-associated molecular
patterns (DAMPs) in preterm labor with intact
membranes and preterm PROM: a study of the
alarmin HMGB1. J Matern Fetal Neonatal Med
2011;24:1444-55.
87. Bredeson S, Papaconstantinou J,
Deford JH, et al. HMGB1 promotes a
p38MAPK associated non-infectious inflamma-
tory response pathway in human fetal mem-
branes. PLoS One 2014;9:e113799.
88. Pradervand PA, Clerc S, Frantz J, et al. High
mobility group box 1 protein (HMGB-1): a
pathogenic role in preeclampsia? Placenta
2014;35:784-6.
89. Girard S Jr, Guller S, Sibley C, Abraham V.
Alarmins potently induce inflammation in
placental cells and impair trophoblast turnover.
Reprod Sci 2015;22:108A.
90. Meroni PL, Raschi E, Grossi C, et al. Ob-
stetric and vascular APS: same autoantibodies
but different diseases? Lupus 2012;21:
708-10.
91. Mulla MJ, Yu AG, Cardenas I, Guller S,
Panda B, Abrahams VM. Regulation of Nod1
and Nod2 in first trimester trophoblast cells. Am
J Reprod Immunol 2009;61:294-302.
92. Mulla MJ, Salmon JE, Chamley LW, et al.
A role for uric acid and the Nalp3 inflammasome
in antiphospholipid antibody-induced IL-1beta
production by human first trimester trophoblast.
PLoS One 2013;8:e65237.
93. Mulla MJ, Myrtolli K, Potter J, et al. Uric acid
induces trophoblast IL-1beta production via the
S38 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
inflammasome: implications for the pathogen-
esis of preeclampsia. Am J Reprod Immunol
2011;65:542-8.
94. Gysler SM. MiR-146a Regulates Anti-
phospholipid Antibody-Induced IL-8 Secretion
by Human Trophoblast Cells. Reprod Sci
2014;21:116A.
95. Gysler SM, Mulla MJ, Brosens JJ, et al.
Antiphospholipid antibody-induced miR-146a
activates TLR8 in trophoblast and is inhibited by
vitamin D. Reprod Sci 2015;22:71A.
96. Han CS, Herrin MA, Pitruzzello MC, et al.
Glucose and metformin modulate human first
trimester trophoblast function: a model and
potential therapy for diabetes-associated ute-
roplacental insufficiency. Am J Reprod Immunol
2015;73:362-71.
97. Koopmans CM, Van Pampus MG, Groen H,
Aarnoudse JG, Van Den Berg PP, Mol BW.
Accuracy of serum uric acid as a predictive test
for maternal complications in pre-eclampsia:
bivariate meta-analysis and decision analysis.
Eur J Obstet Gynecol Reprod Biol 2009;146:
8-14.
98. Girard S, Heazell AE, Derricott H, et al.
Circulating cytokines and alarmins associated
with placental inflammation in high-risk preg-
nancies. Am J Reprod Immunol 2014;72:
422-34.
99. Schmella MJ, Clifton RG, Althouse AD,
Roberts JM. Uric acid determination in gesta-
tional hypertension: is it as effective a delineator
of risk as proteinuria in high-risk women?
Reprod Sci 2015;22:1212-9.
100. Adams Waldorf KM, Persing D, Novy MJ,
Sadowsky DW, Gravett MG. Pretreatment
with toll-like receptor 4 antagonist inhibits
lipopolysaccharide-induced preterm uterine
contractility, cytokines, and prostaglandins in
rhesus monkeys. Reprod Sci 2008;15:121-7.
101. Filipovich Y, Lu SJ, Akira S, Hirsch E. The
adaptor protein MyD88 is essential for E coli-
induced preterm delivery in mice. Am J Obstet
Gynecol 2009;200:93.e1-8.
102. Gotsch F, Romero R, Chaiworapongsa T,
et al. Evidence of the involvement of caspase-1
under physiologic and pathologic cellular stress
during human pregnancy: a link between the
inflammasome and parturition. J Matern Fetal
Neonatal Med 2008;21:605-16.
103. Christiaens I, Zaragoza DB, Guilbert L,
Robertson SA, Mitchell BF, Olson DM. Inflam-
matory processes in preterm and term parturi-
tion. J Reprod Immunol 2008;79:50-7.
104. Kemp MW, Saito M, Newnham JP,
Nitsos I, Okamura K, Kallapur SG. Preterm birth,
infection, and inflammation advances from the
study of animal models. Reprod Sci 2010;17:
619-28.
105. Adams Waldorf KM, Rubens CE,
Gravett MG. Use of nonhuman primate models
to investigate mechanisms of infection-
associated preterm birth. BJOG 2011;118:
136-44.
106. Breen K, Brown A, Burd I, Chai J,
Friedman A, Elovitz MA. TLR-4edependent
and -independent mechanisms of fetal brain
ajog.org
injury in the setting of preterm birth. Reprod Sci
2012;19:839-50.
107. Burd I, Bentz AI, Chai J, et al. Inflammation-
induced preterm birth alters neuronal
morphology in the mouse fetal brain. J Neurosci
Res 2010;88:1872-81.
108. Girard S, Tremblay L, Lepage M, Sebire G.
IL-1 receptor antagonist protects against
placental and neurodevelopmental defects
induced by maternal inflammation. J Immunol
2010;184:3997-4005.
109. Leitner K, Al Shammary M, McLane M,
Johnston MV, Elovitz MA, Burd I. IL-1 receptor
blockade prevents fetal cortical brain injury but
not preterm birth in a mouse model of
inflammation-induced preterm birth and peri-
natal brain injury. Am J Reprod Immunol
2014;71:418-26.
110. Romero R, Tartakovsky B. The natural
interleukin-1 receptor antagonist prevents
interleukin-1-induced preterm delivery in mice.
Am J Obstet Gynecol 1992;167:1041-5.
111. Rosenzweig JM, Lei J, Burd I. Interleukin-1
receptor blockade in perinatal brain injury. Front
Pediatr 2014;2:108.
112. Dowling JK, O’Neill LA. Biochemical
regulation of the inflammasome. Crit Rev Bio-
chem Mol Biol 2012;47:424-43.
113. Kavathas PB, Boeras CM, Mulla MJ,
Abrahams VM. Nod1, but not the ASC inflam-
masome, contributes to induction of IL-1beta
secretion in human trophoblasts after sensing
of Chlamydia trachomatis. Mucosal Immunol
2013;6:235-43.
114. Pijnenborg R, Vercruysse L, Hanssens M.
The uterine spiral arteries in human pregnancy:
facts and controversies. Placenta 2006;27:
939-58.
115. Kaufmann P, Black S, Huppertz B. Endo-
vascular trophoblast invasion: implications for
the pathogenesis of intrauterine growth retar-
dation and preeclampsia. Biol Reprod 2003;69:
1-7.
116. Wallace AE, Fraser R, Cartwright JE.
Extravillous trophoblast and decidual natural
killer cells: a remodelling partnership. Hum
Reprod Update 2012;18:458-71.
117. Ain R, Canham LN, Soares MJ. Gestation
stage-dependent intrauterine trophoblast cell
invasion in the rat and mouse: novel endocrine
phenotype and regulation. Dev Biol 2003;260:
176-90.
118. Konno T, Rempel LA, Arroyo JA,
Soares MJ. Pregnancy in the brown Norway rat:
a model for investigating the genetics of
placentation. Biol Reprod 2007;76:709-18.
119. Pijnenborg R, Vercruysse L. Animal
models of deep trophoblast invasion. New York:
Cambridge University Press; 2010:127-39.
120. Soares MJ, Chakraborty D, Karim
Rumi MA, Konno T, Renaud SJ. Rat placenta-
tion: an experimental model for investigating the
hemochorial maternal-fetal interface. Placenta
2012;33:233-43.
121. Soares MJ, Chakraborty D, Kubota K,
Renaud SJ, Rumi MA. Adaptive mechanisms
controlling uterine spiral artery remodeling
ajog.org
during the establishment of pregnancy. Int J Dev
Biol 2014;58:247-59.
122. Faria TN, Soares MJ. Trophoblast cell dif-
ferentiation: establishment, characterization,
and modulation of a rat trophoblast cell line
expressing members of the placental prolactin
family. Endocrinology 1991;129:2895-906.
123. Asanoma K, Rumi MA, Kent LN, et al.
FGF4-dependent stem cells derived from rat
blastocysts differentiate along the trophoblast
lineage. Dev Biol 2011;351:110-9.
124. Rosario GX, Konno T, Soares MJ. Maternal
hypoxia activates endovascular trophoblast cell
invasion. Dev Biol 2008;314:362-75.
125. Konno T, Graham AR, Rempel LA, et al.
Subfertility linked to combined luteal insuffi-
ciency and uterine progesterone resistance.
Endocrinology 2010;151:4537-50.
126. Konno T, Rempel LA, Rumi MA, et al.
Chromosome-substituted rat strains provide
insights into the genetics of placentation. Physiol
Genomics 2011;43:930-41.
127. Goyal R, Yellon SM, Longo LD, Mata-
Greenwood E. Placental gene expression in a rat
‘model’ of placental insufficiency. Placenta
2010;31:568-75.
128. Goyal R, Zhang L, Blood AB, et al. Char-
acterization of an animal model of pregnancy-
induced vitamin D deficiency due to metabolic
gene dysregulation. Am J Physiol Endocrinol
Metab 2014;306:E256-66.
129. Lee DS, Rumi MA, Konno T, Soares MJ.
In vivo genetic manipulation of the rat tropho-
blast cell lineage using lentiviral vector delivery.
Genesis 2009;47:433-9.
130. Renaud SJ, Karim Rumi MA,
Soares MJ. Review: genetic manipulation of
the rodent placenta. Placenta 2011;32(suppl
2):S130-5.
131. Kent LN, Rumi MA, Kubota K, Lee DS,
Soares MJ. FOSL1 is integral to establishing the
maternal-fetal interface. Mol Cell Biol 2011;31:
4801-13.
132. Rumi MA, Dhakal P, Kubota K, et al.
Generation of Esr1-knockout rats using zinc
finger nuclease-mediated genome editing.
Endocrinology 2014;155:1991-9.
133. Chakraborty D, Rumi MA, Konno T,
Soares MJ. Natural killer cells direct hemochorial
placentation by regulating hypoxia-inducible
factor dependent trophoblast lineage de-
cisions. Proc Natl Acad Sci U S A 2011;108:
16295-300.
134. Zhou Y, Chiu K, Brescia RJ, et al.
Increased depth of trophoblast invasion after
chronic constriction of the lower aorta in rhesus
monkeys. Am J Obstet Gynecol 1993;169:
224-9.
135. Kadyrov M, Schmitz C, Black S,
Kaufmann P, Huppertz B. Pre-eclampsia and
maternal anaemia display reduced apoptosis
and opposite invasive phenotypes of extravillous
trophoblast. Placenta 2003;24:540-8.
136. Zhang J, Chen Z, Smith GN, Croy BA.
Natural killer cell-triggered vascular trans-
formation: maternal care before birth? Cell Mol
Immunol 2011;8:1-11.
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
137. Rossant J, Cross JC. Placental develop-
ment: lessons from mouse mutants. Nat Rev
Genet 2001;2:538-48.
138. Hou Z, Romero R, Uddin M, Than NG,
Wildman DE. Adaptive history of single copy
genes highly expressed in the term human
placenta. Genomics 2009;93:33-41.
139. Maltepe E, Fisher SJ. Placenta: the
forgotten organ. Annu Rev Cell Dev Biol
2015;31:523-52.
140. Cox B, Kotlyar M, Evangelou AI, et al.
Comparative systems biology of human and
mouse as a tool to guide the modeling of human
placental pathology. Mol Syst Biol 2009;5:279.
141. Kent LN, Konno T, Soares MJ. Phospha-
tidylinositol 3 kinase modulation of trophoblast
cell differentiation. BMC Dev Biol 2010;10:97.
142. Renaud SJ, Kubota K, Rumi MA,
Soares MJ. The FOS transcription factor family
differentially controls trophoblast migration and
invasion. J Biol Chem 2014;289:5025-39.
143. Genbacev O, Lamb JD, Prakobphol A,
Donne M, McMaster MT, Fisher SJ. Human
trophoblast progenitors: where do they reside?
Semin Reprod Med 2013;31:56-61.
144. Rossant J, Cross JC. Placental develop-
ment: lessons from mouse mutants. Nat Rev
Genet 2001;2:538-48.
145. Cross JC, Baczyk D, Dobric N, et al.
Genes, development and evolution of the
placenta. Placenta 2003;24:123-30.
146. Davisson RL, Hoffmann DS, Butz GM,
et al. Discovery of a spontaneous genetic mouse
model of preeclampsia. Hypertension 2002;39:
337-42.
147. Wong AY, Kulandavelu S, Whiteley KJ,
Qu D, Langille BL, Adamson SL. Maternal car-
diovascular changes during pregnancy and
postpartum in mice. Am J Physiol Heart Circ
Physiol 2002;282:H918-25.
148. Freemark M. Placental hormones and the
control of fetal growth. J Clin Endocrinol Metab
2010;95:2054-7.
149. Brelje TC, Parsons JA, Sorenson RL.
Regulation of islet beta-cell proliferation by pro-
lactin in rat islets. Diabetes 1994;43:263-73.
150. Parsons JA, Brelje TC, Sorenson RL.
Adaptation of islets of Langerhans to pregnancy:
increased islet cell proliferation and insulin
secretion correlates with the onset of placental
lactogen secretion. Endocrinology 1992;130:
1459-66.
151. Weinhaus AJ, Stout LE, Sorenson RL.
Glucokinase, hexokinase, glucose transporter 2,
and glucose metabolism in islets during preg-
nancy and prolactin-treated islets in vitro:
mechanisms for long term up-regulation of
islets. Endocrinology 1996;137:1640-9.
152. Devlieger R, Casteels K, Van Assche FA.
Reduced adaptation of the pancreatic B cells
during pregnancy is the major causal factor for
gestational diabetes: current knowledge and
metabolic effects on the offspring. Acta Obstet
Gynecol Scand 2008;87:1266-70.
153. Zhang H, Zhang J, Pope CF, et al. Gesta-
tional diabetes mellitus resulting from impaired
beta-cell compensation in the absence of
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
FoxM1, a novel downstream effector of
placental lactogen. Diabetes 2010;59:143-52.
154. Guimond MJ, Wang B, Fujita J,
Terhorst C, Croy BA. Pregnancy-associated
uterine granulated metrial gland cells in mutant
and transgenic mice. Am J Reprod Immunol
1996;35:501-9.
155. Hu D, Cross JC. Ablation of Tpbpa-
positive trophoblast precursors leads to
defects in maternal spiral artery remodeling
in the mouse placenta. Dev Biol 2011;358:
231-9.
156. Ashkar AA, Di Santo JP, Croy BA. Inter-
feron gamma contributes to initiation of uterine
vascular modification, decidual integrity, and
uterine natural killer cell maturation during
normal murine pregnancy. J Exp Med 2000;192:
259-70.
157. Benirschke K, Burton G, Baergen R. Early
development of the human placenta. pathology
of the human placenta, 6th ed. New York:
Springer; 2012:41-54.
158. Simmons DG, Rawn S, Davies A,
Hughes M, Cross JC. Spatial and temporal
expression of the 23 murine Prolactin/Placental
Lactogen-related genes is not associated with
their position in the locus. BMC Genomics
2008;9:352.
159. Gene Expression Omnibus (GEO) Data-
sets. Available at: http://www.ncbi.nlm.nih.gov/
gds/. Accessed: June 3, 2015.
160. Brelje TC, Scharp DW, Lacy PE, et al. Ef-
fect of homologous placental lactogens, pro-
lactins, and growth hormones on islet B-cell
division and insulin secretion in rat, mouse, and
human islets: implication for placental lactogen
regulation of islet function during pregnancy.
Endocrinology 1993;132:879-87.
161. Barbour LA, Shao J, Qiao L, et al. Human
placental growth hormone causes severe insulin
resistance in transgenic mice. Am J Obstet
Gynecol 2002;186:512-7.
162. Branisteanu DD, Mathieu C. Progesterone
in gestational diabetes mellitus: guilty or not
guilty? Trends Endocrinol Metab 2003;14:54-6.
163. Fasshauer M, Bluher M, Stumvoll M. Adi-
pokines in gestational diabetes. Lancet Diabetes
Endocrinol 2014;2:488-99.
164. Newbern D, Freemark M. Placental hor-
mones and the control of maternal metabolism
and fetal growth. Curr Opin Endocrinol Diabetes
Obes 2011;18:409-16.
165. Chen J, Tan B, Karteris E, et al. Secretion
of adiponectin by human placenta: differential
modulation of adiponectin and its receptors by
cytokines. Diabetologia 2006;49:1292-302.
166. Knox K, Leuenberger D, Penn AA,
Baker JC. Global hormone profiling of murine
placenta reveals Secretin expression. Placenta
2011;32:811-6.
167. Wiemers DO, Shao LJ, Ain R, Dai G,
Soares MJ. The mouse prolactin gene family
locus. Endocrinology 2003;144:313-25.
168. Galosy SS, Talamantes F. Luteotropic
actions of placental lactogens at midpregnancy
in the mouse. Endocrinology 1995;136:
3993-4003.
S39
Supplement Obstetrics
169. Chen D, Dong M, Fang Q, He J, Wang Z,
Yang X. Alterations of serum resistin in normal
pregnancy and pre-eclampsia. Clin Sci (Lond)
2005;108:81-4.
170. Tomimatsu T, Yamaguchi M, Murakami T,
et al. Increase of mouse leptin production by
adipose tissue after midpregnancy: gestational
profile of serum leptin concentration. Biochem
Biophys Res Commun 1997;240:213-5.
171. Combs TP, Berg AH, Rajala MW, et al.
Sexual differentiation, pregnancy, calorie
restriction, and aging affect the adipocyte-
specific secretory protein adiponectin. Dia-
betes 2003;52:268-76.
172. Ling C, Kindblom J, Wennbo H, Billig H.
Increased resistin expression in the adipose
tissue of male prolactin transgenic mice and in
male mice with elevated androgen levels. FEBS
Lett 2001;507:147-50.
173. Viengchareun S, Bouzinba-Segard H,
Laigneau JP, et al. Prolactin potentiates insulin-
stimulated leptin expression and release from
differentiated brown adipocytes. J Mol Endo-
crinol 2004;33:679-91.
174. Asai-Sato M, Okamoto M, Endo M, et al.
Hypoadiponectinemia in lean lactating
women: Prolactin inhibits adiponectin secretion
from human adipocytes. Endocr J 2006;53:
555-62.
175. Tunster SJ, Creeth HD, John RM. The
imprinted Phlda2 gene modulates a major
endocrine compartment of the placenta to
regulate placental demands for maternal re-
sources. Dev Biol 2016;409:251-60.
176. Chen PY, Ganguly A, Rubbi L, et al. Intra-
uterine calorie restriction affects placental DNA
methylation and gene expression. Physiol
Genom 2013;45:565-76.
177. Postigo L, Heredia G, Illsley NP, et al.
Where the O2 goes to: preservation of human
fetal oxygen delivery and consumption at high
altitude. J Physiol 2009;587:693-708.
178. Faber JJ. Review of flow limited transfer
in the placenta. Int J Obstet Anesth 1995;4:
230-7.
179. Ansari T, Fenlon S, Pasha S, et al.
Morphometric assessment of the oxygen
diffusion conductance in placentae from preg-
nancies complicated by intra-uterine growth
restriction. Placenta 2003;24:618-26.
180. Mayhew TM. Scaling placental oxygen
diffusion to birthweight: studies on placentae
from low- and high-altitude pregnancies. J Anat
1991;175:187-94.
181. Mayhew TM, Joy CF, Haas JD. Structure-
function correlation in the human placenta: the
morphometric diffusing capacity for oxygen at
full term. J Anat 1984;139:691-708.
182. Reshetnikova OS, Burton GJ,
Milovanov AP. Effects of hypobaric hypoxia on
the fetoplacental unit: the morphometric
diffusing capacity of the villous membrane at
high altitude. Am J Obstet Gynecol 1994;171:
1560-5.
183. Jauniaux E, Watson AL, Hempstock J,
Bao YP, Skepper JN, Burton GJ. Onset of
maternal arterial blood flow and placental
S40 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
oxidative stress: a possible factor in human early
pregnancy failure. Am J Pathol 2000;157:
2111-22.
184. Jauniaux E, Watson A, Burton G. Evalua-
tion of respiratory gases and acid-base gradi-
ents in human fetal fluids and uteroplacental
tissue between 7 and 16 weeks’ gestation. Am J
Obstet Gynecol 2001;184:998-1003.
185. Rodesch F, Simon P, Donner C,
Jauniaux E. Oxygen measurements in endo-
metrial and trophoblastic tissues during early
pregnancy. Obstet Gynecol 1992;80:283-5.
186. Soothill PW, Nicolaides KH, Rodeck CH,
Campbell S. Effect of gestational age on fetal
and intervillous blood gas and acid-base values
in human pregnancy. Fetal Ther 1986;1:
168-75.
187. Lackman F, Capewell V, Gagnon R,
Richardson B. Fetal umbilical cord oxygen
values and birth to placental weight ratio in
relation to size at birth. Am J Obstet Gynecol
2001;185:674-82.
188. Kuzmina IY, Hubina-Vakulik GI, Burton GJ.
Placental morphometry and Doppler flow
velocimetry in cases of chronic human fetal
hypoxia. Eur J Obstet Gynecol Reprod Biol
2005;120:139-45.
189. Rajakumar A, Whitelock KA, Weissfeld LA,
Daftary AR, Markovic N, Conrad KP. Selective
overexpression of the hypoxia-inducible tran-
scription factor, HIF-2alpha, in placentas from
women with preeclampsia. Biol Reprod
2001;64:499-506.
190. Rampersad R, Nelson DM. Trophoblast
biology, responses to hypoxia and placental
dysfunction in preeclampsia. Front Biosci
2007;12:2447-56.
191. Soleymanlou N, Jurisica I, Nevo O, et al.
Molecular evidence of placental hypoxia in pre-
eclampsia. J Clin Endocrinol Metab 2005;90:
4299-308.
192. Tal R. The role of hypoxia and hypoxia-
inducible factor-1alpha in preeclampsia patho-
genesis. Biol Reprod 2012;87:134.
193. Yinon Y, Nevo O, Xu J, et al. Severe intra-
uterine growth restriction pregnancies have
increased placental endoglin levels: hypoxic
regulation via transforming growth factor-beta 3.
Am J Pathol 2008;172:77-85.
194. Taricco E, Radaelli T, Rossi G, et al. Effects
of gestational diabetes on fetal oxygen and
glucose levels in vivo. BJOG 2009;116:
1729-35.
195. Escobar J, Teramo K, Stefanovic V, et al.
Amniotic fluid oxidative and nitrosative stress
biomarkers correlate with fetal chronic hypoxia in
diabetic pregnancies. Neonatology 2013;103:
193-8.
196. Hytinantti TK, Koistinen HA, Teramo K,
Karonen SL, Koivisto VA, Andersson S.
Increased fetal leptin in type I diabetes mellitus
pregnancies complicated by chronic hypoxia.
Diabetologia 2000;43:709-13.
197. Todros T, Sciarrone A, Piccoli E,
Guiot C, Kaufmann P, Kingdom J. Umbilical
Doppler waveforms and placental villous
angiogenesis in pregnancies complicated by
ajog.org
fetal growth restriction. Obstet Gynecol
1999;93:499-503.
198. Ayuk PT, Theophanous D, D’Souza SW,
Sibley CP, Glazier JD. L-arginine transport
by the microvillus plasma membrane of the
syncytiotrophoblast from human placenta in
relation to nitric oxide production: effects of
gestation, preeclampsia, and intrauterine
growth restriction. J Clin Endocrinol Metab
2002;87:747-51.
199. Ferrazzi E, Rigano S, Bozzo M, et al.
Umbilical vein blood flow in growth-restricted
fetuses. Ultrasound Obstet Gynecol 2000;16:
432-8.
200. Aardema MW, Oosterhof H, Timmer A,
Van Rooy I, Aarnoudse JG. Uterine artery
Doppler flow and uteroplacental vascular pa-
thology in normal pregnancies and pregnancies
complicated by pre-eclampsia and small for
gestational age fetuses. Placenta 2001;22:
405-11.
201. Yu CK, Smith GC, Papageorghiou AT,
Cacho AM, Nicolaides KH. An integrated model
for the prediction of pre-eclampsia using
maternal factors and uterine artery Doppler
velocimetry in unselected low-risk women. Am J
Obstet Gynecol 2006;195:330.
202. Rigano S, Bozzo M, Ferrazzi E, Bellotti M,
Battaglia FC, Galan HL. Early and persistent
reduction in umbilical vein blood flow in the
growth-restricted fetus: a longitudinal study. Am
J Obstet Gynecol 2001;185:834-8.
203. Rigano S, Bozzo M, Padoan A, et al. Small
size-specific umbilical vein diameter in severe
growth restricted fetuses that die in utero. Prenat
Diagn 2008;28:908-13.
204. Bellotti M, Pennati G, De Gasperi C,
Bozzo M, Battaglia FC, Ferrazzi E. Simultaneous
measurements of umbilical venous, fetal
hepatic, and ductus venosus blood flow in
growth-restricted human fetuses. Am J Obstet
Gynecol 2004;190:1347-58.
205. Widness JA, Susa JB, Garcia JF, et al.
Increased erythropoiesis and elevated erythro-
poietin in infants born to diabetic mothers and in
hyperinsulinemic rhesus fetuses. J Clin Invest
1981;67:637-42.
206. Zamudio S, Wu Y, Ietta F, et al. Human
placental hypoxia-inducible factor-1alpha
expression correlates with clinical outcomes in
chronic hypoxia in vivo. Am J Pathol 2007;170:
2171-9.
207. Nevo O, Soleymanlou N, Wu Y, et al.
Increased expression of sFlt-1 in in vivo and
in vitro models of human placental hypoxia is
mediated by HIF-1. Am J Physiol Regul Integr
Comp Physiol 2006;291:R1085-93.
208. Rajakumar A, Doty K, Daftary A, Harger G,
Conrad KP. Impaired oxygen-dependent
reduction of HIF-1alpha and -2alpha proteins in
pre-eclamptic placentae. Placenta 2003;24:
199-208.
209. Rajakumar A, Brandon HM, Daftary A,
Ness R, Conrad KP. Evidence for the functional
activity of hypoxia-inducible transcription factors
overexpressed in preeclamptic placentae.
Placenta 2004;25:763-9.
ajog.org
210. Eltzschig HK, Carmeliet P. Hypoxia
and inflammation. N Engl J Med 2011;364:
656-65.
211. Combined antioxidant and preeclampsia
prediction studies (CAPPS). Available at:
https://clinicaltrials.gov/ct2/show/NCT00135707?
term¼NCT00135707&rank¼1. Accessed May 1,
2015.
212. Effect of low dose aspirin on birth-
weight in twins: the GAP Trial. Available at:
https://clinicaltrials.gov/ct2/show/NCT02280031?
term¼NCT02280031&rank¼1. Accessed May 1,
2015.
213. Pravastatin for prevention of preeclampsia.
Available at: https://clinicaltrials.gov/ct2/show/
NCT01717586?term¼NCT01717586&rank¼1.
Accessed May 1, 2015.
214. RCT of antioxidant therapy to pre-
vent preeclampsia in Brazil. Available at:
https://clinicaltrials.gov/ct2/show/NCT00097110?
term¼NCT00097110&rank¼1. Accessed May 1,
2015.
215. Caniggia I, Winter J, Lye SJ, Post M. Ox-
ygen and placental development during the first
trimester: implications for the pathophysiology of
pre-eclampsia. Placenta 2000;21(suppl A):
S25-30.
216. Rajakumar A, Jeyabalan A, Markovic N,
Ness R, Gilmour C, Conrad KP. Placental HIF-1
alpha, HIF-2 alpha, membrane and soluble
VEGF receptor-1 proteins are not increased in
normotensive pregnancies complicated by late-
onset intrauterine growth restriction. Am J
Physiol Regul Integr Comp Physiol 2007;293:
R766-74.
217. Kingdom JC, Kaufmann P. Oxygen and
placental vascular development. Adv Exp Med
Biol 1999;474:259-75.
218. Kingdom JC, Kaufmann P. Oxygen and
placental villous development: origins of fetal
hypoxia. Placenta 1997;18:613-21. discussion
23-6.
219. Unger C, Weiser JK, McCullough RE,
Keefer S, Moore LG. Altitude, low birth weight,
and infant mortality in Colorado. JAMA
1988;259:3427-32.
220. Krampl E, Lees C, Bland JM, Espinoza
Dorado J, Moscoso G, Campbell S. Fetal
biometry at 4300 m compared to sea level in
Peru. Ultrasound Obstet Gynecol 2000;16:
9-18.
221. Zamudio S. The placenta at high altitude.
High Alt Med Biol 2003;4:171-91.
222. Espinoza J, Sebire NJ, McAuliffe F,
Krampl E, Nicolaides KH. Placental villus
morphology in relation to maternal hypoxia at
high altitude. Placenta 2001;22:606-8.
223. Mayhew TM. Thinning of the intervascular
tissue layers of the human placenta is an adap-
tive response to passive diffusion in vivo and
may help to predict the origins of fetal hypoxia.
Eur J Obstet Gynecol Reprod Biol 1998;81:
101-9.
224. Mayhew TM, Charnock-Jones DS,
Kaufmann P. Aspects of human fetoplacental
vasculogenesis and angiogenesis: III, changes in
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
complicated pregnancies. Placenta 2004;25:
127-39.
225. Illsley NP, Caniggia I, Zamudio S. Placental
metabolic reprogramming: do changes in
the mix of energy-generating substrates
modulate fetal growth? Int J Dev Biol 2010;54:
409-19.
226. Zamudio S, Torricos T, Fik E, et al. Hypo-
glycemia and the origin of hypoxia-induced
reduction in human fetal growth. PLoS One
2010;5:e8551.
227. Lu H, Forbes RA, Verma A. Hypoxia-
inducible factor 1 activation by aerobic glycolysis
implicates the Warburg effect in carcinogenesis.
J Biol Chem 2002;277:23111-5.
228. Semenza GL. Oxygen-dependent regula-
tion of mitochondrial respiration by hypoxia-
inducible factor 1. Biochem J 2007;405:1-9.
229. Lisy K, Peet DJ. Turn me on: regulating HIF
transcriptional activity. Cell Death Differ 2008;15:
642-9.
230. Aragones J, Fraisl P, Baes M, Carmeliet P.
Oxygen sensors at the crossroad of metabolism.
Cell Metab 2009;9:11-22.
231. Wilber RL, Stray-Gundersen J, Levine BD.
Effect of hypoxic “dose” on physiological re-
sponses and sea-level performance. Med Sci
Sports Exerc 2007;39:1590-9.
232. Sifakis S, Angelakis E, Vardaki E,
Koumantaki Y, Matalliotakis I, Koumantakis E.
Erythropoietin in the treatment of iron deficiency
anemia during pregnancy. Gynecol Obstet
Invest 2001;51:150-6.
233. Zamudio S, Postigo L, Illsley NP, et al.
Maternal oxygen delivery is not related to alti-
tude- and ancestry-associated differences in
human fetal growth. J Physiol 2007;582:
883-95.
234. Hudetz AG, Wood JD, Biswal BB, Krolo I,
Kampine JP. Effect of hemodilution on RBC
velocity, supply rate, and hematocrit in the
cerebral capillary network. J Appl Physiol
1999;87:505-9.
235. Say L, Gulmezoglu AM, Hofmeyr GJ.
Maternal oxygen administration for suspected
impaired fetal growth. Cochrane Database Syst
Rev 2003:CD000137.
236. Zamudio S. High-altitude hypoxia and
preeclampsia. Front Biosci 2007;12:2967-77.
237. Galan HL, Ferrazzi E, Hobbins JC. Intra-
uterine growth restriction (IUGR): biometric and
Doppler assessment. Prenat Diagn 2002;22:
331-7.
238. Giles WB, Trudinger BJ, Baird PJ. Fetal
umbilical artery flow velocity waveforms and
placental resistance: pathological correlation.
BJOG 1985;92:31-8.
239. Macara L, Kingdom JC, Kohnen G,
Bowman AW, Greer IA, Kaufmann P. Elabora-
tion of stem villous vessels in growth restricted
pregnancies with abnormal umbilical artery
Doppler waveforms. BJOG 1995;102:807-12.
240. Kreczy A, Fusi L, Wigglesworth JS. Cor-
relation between umbilical arterial flow and
placental morphology. Int J Gynecol Pathol
1995;14:306-9.
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
241. Palmer SK, Zamudio S, Coffin C, Parker S,
Stamm E, Moore LG. Quantitative estimation of
human uterine artery blood flow and pelvic blood
flow redistribution in pregnancy. Obstet Gynecol
1992;80:1000-6.
242. Mandala M, Osol G. Physiological remod-
elling of the maternal uterine circulation during
pregnancy. Basic Clin Pharmacol Toxicol
2012;110:12-8.
243. Osol G, Mandala M. Maternal uterine
vascular remodeling during pregnancy. Physi-
ology (Bethesda) 2009;24:58-71.
244. Osol G, Cipolla M. Interaction of myogenic
and adrenergic mechanisms in isolated, pres-
surized uterine radial arteries from late-pregnant
and nonpregnant rats. Am J Obstet Gynecol
1993;168:697-705.
245. Senadheera S, Bertrand PP, Grayson TH,
Leader L, Murphy TV, Sandow SL.
Pregnancy-induced remodelling and enhanced
endothelium-derived hyperpolarization-type
vasodilator activity in rat uterine radial artery:
transient receptor potential vanilloid type 4
channels, caveolae and myoendothelial gap
junctions. J Anat 2013;223:677-86.
246. Luksha L, Nisell H, Luksha N, Kublickas M,
Hultenby K, Kublickiene K. Endothelium-derived
hyperpolarizing factor in preeclampsia:
heterogeneous contribution, mechanisms, and
morphological prerequisites. Am J Physiol Regul
Integr Comp Physiol 2008;294:R510-9.
247. Boyd PA, Scott A. Quantitative structural
studies on human placentas associated with
pre-eclampsia, essential hypertension and in-
trauterine growth retardation. BJOG 1985;92:
714-21.
248. Teasdale F. Histomorphometry of the
human placenta in maternal preeclampsia. Am
J Obstet Gynecol 1985;152:25-31.
249. Geva E, Ginzinger DG, Zaloudek CJ,
Moore DH, Byrne A, Jaffe RB. Human placental
vascular development: vasculogenic and
angiogenic (branching and nonbranching)
transformation is regulated by vascular endo-
thelial growth factor-A, angiopoietin-1, and
angiopoietin-2. J Clin Endocrinol Metab
2002;87:4213-24.
250. Ratnikov B, Aza-Blanc P, Ronai ZA,
Smith JW, Osterman AL, Scott DA. Glutamate
and asparagine cataplerosis underlie glutamine
addiction in melanoma. Oncotarget 2015;6:
7379-89.
251. Wise DR, Thompson CB. Glutamine
addiction: a new therapeutic target in cancer.
Trends Biochem Sci 2010;35:427-33.
252. Medvetz D, Priolo C, Henske EP. Thera-
peutic targeting of cellular metabolism in cells
with hyperactive mTORC1: a paradigm shift. Mol
Cancer Res 2015;13:3-8.
253. Vivanco I. Targeting molecular addictions
in cancer. Br J Cancer 2014;111:2033-8.
254. Legband ND, Feshitan JA, Borden MA,
Terry BS. Evaluation of peritoneal microbubble
oxygenation therapy in a rabbit model of hyp-
oxemia. IEEE Trans Biomed Eng 2015;62:
1376-82.
S41
Supplement Obstetrics
255. Matsuki N, Ishikawa T, Ichiba S, Shiba N,
Ujike Y, Yamaguchi T. Oxygen supersaturated
fluid using fine micro/nanobubbles. Int J Nano-
medicine 2014;9:4495-505.
256. Matsuki N, Ichiba S, Ishikawa T, et al.
Blood oxygenation using microbubble suspen-
sions. Eur Biophys J 2012;41:571-8.
257. Bisazza A, Giustetto P, Rolfo A, et al.
Microbubble-mediated oxygen delivery to hyp-
oxic tissues as a new therapeutic device. Conf
Proc IEEE Eng Med Biol Soc 2008;2008:
2067-70.
258. Hammond S, Mathewson AM, Baker PN,
Mayhew TM, Dunn WR. Gap junctions and
hydrogen peroxide are involved in endothelium-
derived hyperpolarising responses to bradykinin
in omental arteries and veins isolated from
pregnant women. Eur J Pharmacol 2011;668:
225-32.
259. Vodstrcil LA, Tare M, Novak J, et al. Relaxin
mediates uterine artery compliance during
pregnancy and increases uterine blood flow.
FASEB J 2012;26:4035-44.
260. Morton JS, Davidge ST. Arterial
endothelium-derived hyperpolarization: poten-
tial role in pregnancy adaptations and compli-
cations. J Cardiovasc Pharmacol 2013;61:
197-203.
261. Vadillo-Ortega F, Perichart-Perera O,
Espino S, et al. Effect of supplementation during
pregnancy with L-arginine and antioxidant vita-
mins in medical food on pre-eclampsia in high
risk population: randomised controlled trial. BMJ
2011;342:d2901.
262. Staff AC, Berge L, Haugen G, Lorentzen B,
Mikkelsen B, Henriksen T. Dietary supplemen-
tation with L-arginine or placebo in women with
pre-eclampsia. Acta Obstet Gynecol Scand
2004;83:103-7.
263. Ganzevoort W, Alfirevic Z, Von
Dadelszen P, et al. STRIDER: Sildenafil Therapy
In Dismal prognosis Early-onset intrauterine
growth Restriction: a protocol for a systematic
review with individual participant data and
aggregate data meta-analysis and trial sequen-
tial analysis. Syst Rev 2014;3:23.
264. Von Dadelszen P, Dwinnell S, Magee LA,
et al. Sildenafil citrate therapy for severe early-
onset intrauterine growth restriction. BJOG
2011;118:624-8.
265. Everrest DA. Developing a therapy for fetal
growth restriction: a 6 year prospective study to
define the clinical and biological characteristic of
pregnancies affected by severe early onset fetal
growth restriction. London: University College;
2015.
266. David AL, Torondel B, Zachary I, et al.
Local delivery of VEGF adenovirus to the uterine
artery increases vasorelaxation and uterine
blood flow in the pregnant sheep. Gene Ther
2008;15:1344-50.
267. Hanauske-Abel HM, Popowicz AM. The
HAG mechanism: a molecular rationale for the
therapeutic application of iron chelators in hu-
man diseases involving the 2-oxoacid utilizing
dioxygenases. Curr Med Chem 2003;10:
1005-19.
S42 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
268. Hoque M, Hanauske-Abel HM,
Palumbo P, et al. Inhibition of HIV-1 gene
expression by ciclopirox and deferiprone, drugs
that prevent hypusination of eukaryotic initiation
factor 5A. Retrovirology 2009;6:90.
269. Zhang EG, Burton GJ, Smith SK, Char-
nock-Jones DS. Placental vessel adaptation
during gestation and to high altitude: changes in
diameter and perivascular cell coverage.
Placenta 2002;23:751-62.
270. Charnock-Jones DS. Soluble flt-1 and the
angiopoietins in the development and regulation
of placental vasculature. J Anat 2002;200:
607-15.
271. Shih SC, Ju M, Liu N, Mo JR, Ney JJ,
Smith LE. Transforming growth factor beta1
induction of vascular endothelial growth factor
receptor 1: mechanism of pericyte-induced
vascular survival in vivo. Proc Natl Acad Sci U
S A 2003;100:15859-64.
272. Zamudio S, Baumann MU, Illsley NP. Ef-
fects of chronic hypoxia in vivo on the expression
of human placental glucose transporters.
Placenta 2006;27:49-55.
273. Gaither K, Quraishi AN, Illsley NP. Diabetes
alters the expression and activity of the human
placental GLUT1 glucose transporter. J Clin
Endocrinol Metab 1999;84:695-701.
274. Glazier JD, Cetin I, Perugino G, et al.
Association between the activity of the system A
amino acid transporter in the microvillus plasma
membrane of the human placenta and severity
of fetal compromise in intrauterine growth re-
striction. Pediatr Res 1997;42:514-9.
275. Jansson T, Ekstrand Y, Bjorn C,
Wennergren M, Powell TL. Alterations in the
activity of placental amino acid transporters in
pregnancies complicated by diabetes. Diabetes
2002;51:2214-9.
276. Harding JE, Owens JA, Robinson JS.
Should we try to supplement the growth
retarded fetus? A cautionary tale. BJOG
1992;99:707-9.
277. Zamudio S, Torricos T, Fik E, et al. Hypo-
glycemia and the origin of hypoxia-induced
reduction in human fetal growth. PLoS One
2010;5:e8551.
278. Jansson T, Wennergren M, Illsley NP.
Glucose transporter protein expression in
human placenta throughout gestation and in
intrauterine growth retardation. J Clin Endocrinol
Metab 1993;77:1554-62.
279. Brown K, Heller DS, Zamudio S, Illsley NP.
Glucose transporter 3 (GLUT3) protein expres-
sion in human placenta across gestation.
Placenta 2011;32:1041-9.
280. Cleal JK, Lewis RM. The mechanisms and
regulation of placental amino acid transport to
the human foetus. J Neuroendocrinol 2008;20:
419-26.
281. Baumann M, Schneider H, Malek A, et al.
Regulation of human trophoblast GLUT1
glucose transporter by insulin-like growth factor I
(IGF-I). PLoS One 2014;9:e106037.
282. Roos S, Lagerlof O, Wennergren M,
Powell TL, Jansson T. Regulation of amino acid
transporters by glucose and growth factors in
ajog.org
cultured primary human trophoblast cells is
mediated by mTOR signaling. Am J Physiol Cell
Physiol 2009;297:C723-31.
283. Giudice LC, Martina NA, Crystal RA,
Tazuke S, Druzin M. Insulin-like growth factor
binding protein-1 at the maternal-fetal interface
and insulin-like growth factor-I, insulin-like
growth factor-II, and insulin-like growth factor
binding protein-1 in the circulation of women
with severe preeclampsia. Am J Obstet Gynecol
1997;176:751-8.
284. Sferruzzi-Perri AN, Owens JA, Pringle KG,
Roberts CT. The neglected role of insulin-like
growth factors in the maternal circulation
regulating fetal growth. J Physiol 2011;589:
7-20.
285. Jones HN, Crombleholme T, Habli M.
Adenoviral-mediated placental gene transfer
of IGF-1 corrects placental insufficiency via
enhanced placental glucose transport mecha-
nisms. PLoS One 2013;8:e74632.
286. Jones H, Crombleholme T, Habli M.
Regulation of amino acid transporters by
adenoviral-mediated human insulin-like growth
factor-1 in a mouse model of placental insuffi-
ciency in vivo and the human trophoblast line
BeWo in vitro. Placenta 2014;35:132-8.
287. Laplante M, Sabatini DM. mTOR signaling
in growth control and disease. Cell 2012;149:
274-93.
288. Jansson N, Rosario FJ, Gaccioli F, et al.
Activation of placental mTOR signaling and
amino acid transporters in obese women giving
birth to large babies. J Clin Endocrinol Metab
2013;98:105-13.
289. Aiko Y, Askew DJ, Aramaki S, et al.
Differential levels of amino acid transporters
System L and ASCT2, and the mTOR protein in
placenta of preeclampsia and IUGR. BMC
Pregnancy Childbirth 2014;14:181.
290. Roos S, Jansson N, Palmberg I, Saljo K,
Powell TL, Jansson T. Mammalian target of
rapamycin in the human placenta regulates
leucine transport and is down-regulated in
restricted fetal growth. J Physiol 2007;582:
449-59.
291. Choi YJ, Park YJ, Park JY, et al. Inhibitory
effect of mTOR activator MHY1485 on auto-
phagy: suppression of lysosomal fusion. PLoS
One 2012;7:e43418.
292. Peng N, Meng N, Wang S, et al. An acti-
vator of mTOR inhibits oxLDL-induced auto-
phagy and apoptosis in vascular endothelial cells
and restricts atherosclerosis in apolipoprotein
E(-)/(-) mice. Sci Rep 2014;4:5519.
293. Rosario FJ, Kanai Y, Powell TL, Jansson T.
Mammalian target of rapamycin signalling
modulates amino acid uptake by regulating
transporter cell surface abundance in primary
human trophoblast cells. J Physiol 2013;591:
609-25.
294. Andrzejewski S, Gravel SP, Pollak M,
St-Pierre J. Metformin directly acts on mito-
chondria to alter cellular bioenergetics. Cancer
Metab 2014;2:12.
295. Illsley NP, Caniggia I, Zamudio S. Placental
metabolic reprogramming: do changes in the
ajog.org
mix of energy-generating substrates modulate
fetal growth? Int J Dev Biol 2010;54:409-19.
296. Myatt L, Cui X. Oxidative stress in the
placenta. Histochem Cell Biol 2004;122:
369-82.
297. Menon R. Oxidative stress damage as a
detrimental factor in preterm birth pathology.
Front Immunol 2014;5:567.
298. Abuja PM, Albertini R. Methods for moni-
toring oxidative stress, lipid peroxidation and
oxidation resistance of lipoproteins. Clin Chim
Acta 2001;306:1-17.
299. Roberts VH, Smith J, McLea SA,
Heizer AB, Richardson JL, Myatt L. Effect of
increasing maternal body mass index on oxida-
tive and nitrative stress in the human placenta.
Placenta 2009;30:169-75.
300. Macmillan-Crow LA, Crow JP, Kerby JD,
Beckman JS, Thompson JA. Nitration and
inactivation of manganese superoxide dismut-
ase in chronic rejection of human renal allografts.
Proc Natl Acad Sci U S A 1996;93:11853-8.
301. Ischiropoulos H. Biological tyrosine nitra-
tion: a pathophysiological function of nitric oxide
and reactive oxygen species. Arch Biochem
Biophys 1998;356:1-11.
302. Pacher P, Beckman JS, Liaudet L. Nitric
oxide and peroxynitrite in health and disease.
Physiol Rev 2007;87:315-424.
303. Myatt L. Review: reactive oxygen and
nitrogen species and functional adaptation of
the placenta. Placenta 2010;31(suppl):S66-9.
304. Sen CK, Packer L. Antioxidant and redox
regulation of gene transcription. FASEB J
1996;10:709-20.
305. Halliwell B, Whiteman M. Measuring reac-
tive species and oxidative damage in vivo and
in cell culture: how should you do it and what do
the results mean? Br J Pharmacol 2004;142:
231-55.
306. Muralimanoharan S, Guo C, Myatt L,
Maloyan A. Sexual dimorphism in miR-210
expression and mitochondrial dysfunction in
the placenta with maternal obesity. Int J Obes
(Lond) 2015;39:1274-81.
307. Roberts JM, Myatt L, Spong CY, et al. Vi-
tamins C and E to prevent complications of
pregnancy-associated hypertension. N Engl J
Med 2010;362:1282-91.
308. Kelso GF, Porteous CM, Coulter CV, et al.
Selective targeting of a redox-active ubiquinone
to mitochondria within cells: antioxidant and
antiapoptotic properties. J Biol Chem 2001;276:
4588-96.
309. Khera A, Vanderlelie JJ, Perkins AV. Sele-
nium supplementation protects trophoblast cells
from mitochondrial oxidative stress. Placenta
2013;34:594-8.
310. Reiter RJ, Tan DX, Mayo JC, Sainz RM,
Leon J, Czarnocki Z. Melatonin as an antioxi-
dant: biochemical mechanisms and patho-
physiological implications in humans. Acta
Biochim Pol 2003;50:1129-46.
311. Muralimanoharan S, Maloyan A, Mele J,
Guo C, Myatt LG, Myatt L. MIR-210 modulates
mitochondrial respiration in placenta with pre-
eclampsia. Placenta 2012;33:816-23.
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
312. Vatten LJ, Skjaerven R. Offspring sex and
pregnancy outcome by length of gestation. Early
Hum Dev 2004;76:47-54.
313. Clifton VL. Review: sex and the human
placenta: mediating differential strategies of fetal
growth and survival. Placenta 2010;31(suppl):
S33-9.
314. Sood R, Zehnder JL, Druzin ML, Brown PO.
Gene expression patterns in human placenta.
Proc Natl Acad Sci U S A 2006;103:5478-83.
315. Muralimanoharan S, Maloyan A, Myatt L.
Evidence of sexual dimorphism in the placental
function with severe preeclampsia. Placenta
2013;34:1183-9.
316. Arrowsmith J, Miller P. Trial watch: phase II
and phase III attrition rates 2011-2012. Nat Rev
Drug Discov 2013;12:569.
317. Mullard A. Drug repurposing programmes
get lift off. Nat Rev Drug Discov 2012;11:505-6.
318. The National Center for Advancing Trans-
lational Sciences (NCATS). Available at: http://
www.ncats.nih.gov/ntu. Accessed May 1, 2015.
319. Reviewer guidance: evaluating the risks of
drug exposure in human pregnancies. Available
at: http://www.fda.gov/downloads/Science
Research/SpecialTopics/WomensHealthResearch/
UCM133359.pdf. Accessed May 1, 2015.
320. Shineman DW, Alam J, Anderson M, et al.
Overcoming obstacles to repurposing for
neurodegenerative disease. Ann Clin Transl
Neurol 2014;1:512-8.
321. Mitchell AA, Gilboa SM, Werler MM,
Kelley KE, Louik C, Hernandez-Diaz S. Medica-
tion use during pregnancy, with particular focus
on prescription drugs: 1976-2008. Am J Obstet
Gynecol 2011;205:51.e1-8.
322. Lupattelli A, Spigset O, Twigg MJ, et al.
Medication use in pregnancy: a cross-sectional,
multinational web-based study. BMJ Open
2014;4.
323. Andrade SE, Raebel MA, Morse AN, et al.
Use of prescription medications with a potential
for fetal harm among pregnant women. Phar-
macoepidemiol Drug Saf 2006;15:546-54.
324. Andrade SE, Gurwitz JH, Davis RL, et al.
Prescription drug use in pregnancy. Am J Obstet
Gynecol 2004;191:398-407.
325. Andrade SE, Raebel MA, Brown J, et al.
Use of antidepressant medications during
pregnancy: a multisite study. Am J Obstet
Gynecol 2008;198:194.e1-5.
326. Anderson GD. Pregnancy-induced
changes in pharmacokinetics: a mechanistic-
based approach. Clin Pharmacokinet 2005;44:
989-1008.
327. Anger GJ, Piquette-Miller M. Pharmacoki-
netic studies in pregnant women. Clin Pharma-
col Ther 2008;83:184-7.
328. Costantine MM. Physiologic and pharma-
cokinetic changes in pregnancy. Front Phar-
macol 2014;5:65.
329. Briggs G, Freeman R, Yaffe S. Drugs in
pregnancy and lactation: a reference guide to
fetal and neonatal risk. Philadelphia: Lippincott,
Williams and Wilkins; 2012.
330. Pacheco LD, MM C, Hankins GD.
Physiologic changes during pregnancy. In:
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
Mattison DR, ed. Clinical pharmacology during
pregnancy. London: Elsevier; 2013. pp. 5-14.
331. Robson SC, Hunter S, Boys RJ, Dunlop W.
Serial study of factors influencing changes in
cardiac output during human pregnancy. Am J
Physiol 1989;256:H1060-5.
332. Clark SL, Cotton DB, Lee W, et al. Central
hemodynamic assessment of normal term
pregnancy. Am J Obstet Gynecol 1989;161:
1439-42.
333. Hytten FE, Paintin DB. Increase in plasma
volume during normal pregnancy. J Obstet
Gynaecol Br Emp 1963;70:402-7.
334. Frederiksen MC. Physiologic changes in
pregnancy and their effect on drug disposition.
Semin Perinatol 2001;25:120-3.
335. Elkus R, Popovich J Jr. Respiratory phys-
iology in pregnancy. Clin Chest Med 1992;13:
555-65.
336. Parry E, Shields R, Turnbull AC. Transit
time in the small intestine in pregnancy. J Obstet
Gynaecol Br Commonw 1970;77:900-1.
337. Evans WE, Relling MV. Pharmacoge-
nomics: translating functional genomics into
rational therapeutics. Science 1999;286:
487-91.
338. Hellden A, Madadi P. Pregnancy and
pharmacogenomics in the context of drug
metabolism and response. Pharmacogenomics
2013;14:1779-91.
339. Ke AB, Rostami-Hodjegan A, Zhao P,
Unadkat JD. Pharmacometrics in pregnancy: an
unmet need. Annu Rev Pharmacol Toxicol
2014;54:53-69.
340. Weier N, He SM, Li XT, Wang LL, Zhou SF.
Placental drug disposition and its clinical impli-
cations. Curr Drug Metab 2008;9:106-21.
341. Koren G. Sex dependent pharmacoki-
netics and bioequivalence: time for a change.
J Popul Ther Clin Pharmacol 2013;20:
e358-61.
342. Shields KE, Lyerly AD. Exclusion of preg-
nant women from industry-sponsored clinical
trials. Obstet Gynecol 2013;122:1077-81.
343. Schonfeld T, Schmid KK, Brown JS,
Amoura NJ, Gordon B. A pregnancy testing
policy for women enrolled in clinical trials. IRB
2013;35:9-15.
344. McCormack SA, Best BM. Obstetric
pharmacokinetic dosing studies are urgently
needed. Front Pediatr 2014;2:9.
345. Meslin EM, Blasimme A, Cambon-
Thomsen A. Mapping the translational science
policy ‘valley of death.’ Clin Transl Med 2013;
2:14.
346. Morris ZS, Wooding S, Grant J. The
answer is 17 years, what is the question: un-
derstanding time lags in translational research.
J R Soc Med 2011;104:510-20.
347. Lenfant C. Shattuck lecture: clinical
research to clinical practice: lost in translation?
N Engl J Med 2003;349:868-74.
348. Grimshaw JM, Eccles MP, Lavis JN,
Hill SJ, Squires JE. Knowledge translation of
research findings. Implement Sci 2012;7:50.
349. McGlynn EA, Asch SM, Adams J, et al. The
quality of health care delivered to adults in the
S43
Supplement Obstetrics
United States. N Engl J Med 2003;348:
2635-45.
350. Schuster MA, McGlynn EA, Brook RH.
How good is the quality of health care in the
United States? 1998. Milbank Q 2005;83:
843-95.
351. Roberts SF, Fischhoff MA, Sakowski SA,
Feldman EL. Perspective: transforming science
into medicine: how clinician-scientists can build
bridges across research’s “valley of death”.
Acad Med 2012;87:266-70.
352. Contopoulos-Ioannidis DG, Ntzani E,
Ioannidis JP. Translation of highly promising
basic science research into clinical applications.
Am J Med 2003;114:477-84.
353. Botting J. The history of thalidomide. Drug
News Perspect 2002;15:604-11.
354. Kutcher JS, Engle A, Firth J, Lamm SH.
Bendectin and birth defects: II, ecological ana-
lyses. Birth Defects Res A Clin Mol Teratol
2003;67:88-97.
355. Palmer AK, Harris AL, Jacobson RM.
Human papillomavirus vaccination: a case study
in translational science. Clin Transl Sci 2014;7:
420-4.
356. Mittendorf R, Williams MA. Rho(D) immu-
noglobulin (RhoGAM): how it came into being.
Obstet Gynecol 1991;77:301-3.
357. Hamilton EG. Rho(D) immunoglobulin
(RhoGAM): how it came into being. Obstet
Gynecol 1991;77:957-8.
358. Crowley P. Prophylactic corticosteroids for
preterm birth. Cochrane Database Syst Rev
2000:CD000065.
359. Liggins GC. Premature delivery of foetal
lambs infused with glucocorticoids. J Endocrinol
1969;45:515-23.
360. Liggins GC, Howie RN. A controlled trial of
antepartum glucocorticoid treatment for pre-
vention of the respiratory distress syndrome in
premature infants. Pediatrics 1972;50:515-25.
361. American College of Obstetricians and
Gynecologists. ACOG practice bulletin no. 127:
management of preterm labor. Obstet Gynecol
2012;119:1308-17.
362. Effect of corticosteroids for fetal maturation
on perinatal outcomes. NIH Consensus Devel-
opment Panel on the Effect of Corticosteroids for
Fetal Maturation on Perinatal Outcomes. JAMA
1995;273:413-8.
363. Bradley EH, Schlesinger M, Webster TR,
Baker D, Inouye SK. Translating research into
clinical practice: making change happen. J Am
Geriatr Soc 2004;52:1875-82.
364. Bradley EH, Webster TR, Baker D, et al.
Translating research into practice: speeding the
adoption of innovative health care programs.
Issue Brief (Commonw Fund) 2004;724:1-12.
365. Kahn K, Ryan G, Beckett M, et al. Bridging
the gap between basic science and clinical
practice: a role for community clinicians. Imple-
ment Sci 2011;6:34.
366. Green LA, Seifert CM. Translation of
research into practice: why we can’t “just do it”.
J Am Board Fam Pract 2005;18:541-5.
367. National Institutes of Health, IMPACT: our
economy. Available at: http://www.nih.gov/
S44 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
about/impact/economy.htm. Accessed: April
6, 2015.
368. Liu G, Chen G, Sinoway LI, Berg A.
Assessing the impact of the NIH CTSA program
on institutionally sponsored clinical trials. Clin
Transl Sci 2013;6:196-200.
369. The Clinical and Translational Science
Award (CTSA). Available at: https://ctsacentral.
org/. Accessed June 1, 2015.
370. Haas DM. Pharmacogenetics and individ-
ualizing drug treatment during pregnancy.
Pharmacogenomics 2014;15:69-78.
371. Arroyo JD, Chevillet JR, Kroh EM, et al.
Argonaute2 complexes carry a population of
circulating microRNAs independent of vesicles
in human plasma. Proc Natl Acad Sci U S A
2011;108:5003-8.
372. Valadi H, Ekstrom K, Bossios A,
Sjostrand M, Lee JJ, Lotvall JO. Exosome-
mediated transfer of mRNAs and microRNAs is
a novel mechanism of genetic exchange be-
tween cells. Nat Cell Biol 2007;9:654-9.
373. O’Loughlin AJ, Woffindale CA, Wood MJ.
Exosomes and the emerging field of exosome-
based gene therapy. Curr Gene Ther 2012;12:
262-74.
374. Thery C, Ostrowski M, Segura E. Mem-
brane vesicles as conveyors of immune re-
sponses. Nat Rev Immunol 2009;9:581-93.
375. Simons M, Raposo G. Exosomes: vesicu-
lar carriers for intercellular communication. Curr
Opin Cell Biol 2009;21:575-81.
376. Raposo G, Stoorvogel W. Extracellular
vesicles: exosomes, microvesicles, and friends.
J Cell Biol 2013;200:373-83.
377. Raposo G, Nijman HW, Stoorvogel W,
et al. B lymphocytes secrete antigen-presenting
vesicles. J Exp Med 1996;183:1161-72.
378. Akers JC, Gonda D, Kim R, Carter BS,
Chen CC. Biogenesis of extracellular vesicles
(EV): exosomes, microvesicles, retrovirus-like
vesicles, and apoptotic bodies. J Neurooncol
2013;113:1-11.
379. Pitt JM, Charrier M, Viaud S, et al. Dendritic
cell-derived exosomes as immunotherapies in
the fight against cancer. J Immunol 2014;193:
1006-11.
380. Robbins PD, Morelli AE. Regulation of im-
mune responses by extracellular vesicles. Nat
Rev Immunol 2014;14:195-208.
381. Fais S, Logozzi M, Lugini L, et al. Exo-
somes: the ideal nanovectors for biodelivery.
Biol Chem 2013;394:1-15.
382. Tan A, Rajadas J, Seifalian AM. Exosomes
as nano-theranostic delivery platforms for
gene therapy. Adv Drug Deliv Rev 2013;65:
357-67.
383. Mevorach D, Mascarenhas JO,
Gershov D, Elkon KB. Complement-dependent
clearance of apoptotic cells by human macro-
phages. J Exp Med 1998;188:2313-20.
384. Taylor RC, Cullen SP, Martin SJ.
Apoptosis: controlled demolition at the cellular
level. Nat Rev Mol Cell Biol 2008;9:231-41.
385. Heijnen HF, Schiel AE, Fijnheer R,
Geuze HJ, Sixma JJ. Activated platelets release
two types of membrane vesicles: microvesicles
ajog.org
by surface shedding and exosomes derived
from exocytosis of multivesicular bodies and
alpha-granules. Blood 1999;94:3791-9.
386. Cocucci E, Racchetti G, Meldolesi J.
Shedding microvesicles: artefacts no more.
Trends Cell Biol 2009;19:43-51.
387. Leventis PA, Grinstein S. The distribution
and function of phosphatidylserine in cellular
membranes. Annu Rev Biophys 2010;39:
407-27.
388. Hugel B, Martinez MC, Kunzelmann C,
Freyssinet JM. Membrane microparticles: two
sides of the coin. Physiology (Bethesda)
2005;20:22-7.
389. Meckes DG Jr, Raab-Traub N. Micro-
vesicles and viral infection. J Virol 2011;85:
12844-54.
390. Pols MS, Klumperman J. Trafficking and
function of the tetraspanin CD63. Exp Cell Res
2009;315:1584-92.
391. Hemler ME. Tetraspanin functions and
associated microdomains. Nat Rev Mol Cell Biol
2005;6:801-11.
392. Fomina AF, Deerinck TJ, Ellisman MH,
Cahalan MD. Regulation of membrane traf-
ficking and subcellular organization of endocytic
compartments revealed with FM1-43 in resting
and activated human T cells. Exp Cell Res
2003;291:150-66.
393. Bobrie A, Colombo M, Raposo G, Thery C.
Exosome secretion: molecular mechanisms and
roles in immune responses. Traffic 2011;12:
1659-68.
394. Delorme-Axford E, Donker RB, Mouillet JF,
et al. Human placental trophoblasts confer viral
resistance to recipient cells. Proc Natl Acad Sci
U S A 2013;110:12048-53.
395. Ouyang Y, Mouillet JF, Coyne CB,
Sadovsky Y. Review: placenta-specific micro-
RNAs in exosomes: good things come in nano-
packages. Placenta 2014;35(suppl):S69-73.
396. Luo SS, Ishibashi O, Ishikawa G, et al.
Human villous trophoblasts express and secrete
placenta-specific microRNAs into maternal cir-
culation via exosomes. Biol Reprod 2009;81:
717-29.
397. Donker RB, Mouillet JF, Chu T, et al. The
expression profile of C19MC microRNAs in pri-
mary human trophoblast cells and exosomes.
Mol Hum Reprod 2012;18:417-24.
398. Aharon A, Brenner B. Placenta-derived
microparticles. Thromb Res 2013;131(suppl 1):
S22-4.
399. Mayhew TM. Turnover of human villous
trophoblast in normal pregnancy: what do we
know and what do we need to know? Placenta
2014;35:229-40.
400. Ishihara N, Matsuo H, Murakoshi H,
Laoag-Fernandez JB, Samoto T, Maruo T.
Increased apoptosis in the syncytiotropho-
blast in human term placentas complicated
by either preeclampsia or intrauterine growth
retardation. Am J Obstet Gynecol 2002;186:
158-66.
401. Salomon C, Kobayashi M, Ashman K,
Sobrevia L, Mitchell MD, Rice GE. Hypoxia-
induced changes in the bioactivity of
ajog.org
cytotrophoblast-derived exosomes. PLoS One
2013;8:e79636.
402. Taylor DD, Akyol S, Gercel-Taylor C.
Pregnancy-associated exosomes and their
modulation of T cell signaling. J Immunol
2006;176:1534-42.
403. Kshirsagar SK, Alam SM, Jasti S, et al.
Immunomodulatory molecules are released
from the first trimester and term placenta via
exosomes. Placenta 2012;33:982-90.
404. Sabapatha A, Gercel-Taylor C, Taylor DD.
Specific isolation of placenta-derived exosomes
from the circulation of pregnant women and their
immunoregulatory consequences. Am J Reprod
Immunol 2006;56:345-55.
405. Jeschke U, Hutter S, Heublein S, et al.
Expression and function of galectins in the
endometrium and at the human feto-maternal
interface. Placenta 2013;34:863-72.
406. Alegre E, Rebmann V, LemaoulT J, et al.
In vivo identification of an HLA-G complex as
ubiquitinated protein circulating in exosomes.
Eur J Immunol 2013;43:1933-9.
407. Mincheva-Nilsson L, Baranov V. The role of
placental exosomes in reproduction. Am J
Reprod Immunol 2010;63:520-33.
408. Gross JC, Chaudhary V, Bartscherer K,
Boutros M. Active Wnt proteins are secreted on
exosomes. Nat Cell Biol 2012;14:1036-45.
409. Atay S, Gercel-Taylor C, Suttles J, Mor G,
Taylor DD. Trophoblast-derived exosomes
mediate monocyte recruitment and differentia-
tion. Am J Reprod Immunol 2011;65:65-77.
410. Subra C, Grand D, Laulagnier K, et al.
Exosomes account for vesicle-mediated trans-
cellular transport of activatable phospholipases
and prostaglandins. J Lipid Res 2010;51:
2105-20.
411. Esser J, Gehrmann U, D’Alexandri FL, et al.
Exosomes from human macrophages and den-
dritic cells contain enzymes for leukotriene
biosynthesis and promote granulocyte migration.
J Allergy Clin Immunol 2010;126:1032-40.e1-4.
412. Record M. Intercellular communication by
exosomes in placenta: a possible role in cell
fusion? Placenta 2014;35:297-302.
413. Sarker S, Scholz-Romero K, Perez A, et al.
Placenta-derived exosomes continuously in-
crease in maternal circulation over the first
trimester of pregnancy. J Transl Med 2014;12:
204.
414. Bentwich I, Avniel A, Karov Y, et al. Iden-
tification of hundreds of conserved and non-
conserved human microRNAs. Nat Genet
2005;37:766-70.
415. Zhang R, Wang YQ, Su B. Molecular evo-
lution of a primate-specific microRNA family. Mol
Biol Evol 2008;25:1493-502.
416. Bortolin-Cavaille ML, Dance M, Weber M,
Cavaille J. C19MC microRNAs are processed
from introns of large Pol-II, non-protein-coding
transcripts. Nucleic Acids Res 2009;37:
3464-73.
417. Delorme-Axford E, Bayer A, Sadovsky Y,
Coyne CB. Autophagy as a mechanism of anti-
viral defense at the maternal-fetal interface.
Autophagy 2013;9:2173-4.
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
418. Bayer A, Delorme-Axford E, Sleigher C,
et al. Human trophoblasts confer resistance to
viruses implicated in perinatal infection. Am J
Obstet Gynecol 2015;212:71.e1-8.
419. Mouillet JF, Ouyang Y, Bayer A, Coyne CB,
Sadovsky Y. The role of trophoblastic micro-
RNAs in placental viral infection. Int J Dev Biol
2014;58:281-9.
420. Wang K, Huang C, Nice EC. Proteomics,
genomics and transcriptomics: their emerging
roles in the discovery and validation of colorectal
cancer biomarkers. Expert Rev Proteomics
2014;11:179-205.
421. Choi DS, Kim DK, Kim YK, Gho YS.
Proteomics, transcriptomics and lipidomics
of exosomes and ectosomes. Proteomics
2013;13:1554-71.
422. Montecalvo A, Larregina AT, Shufesky WJ,
et al. Mechanism of transfer of functional
microRNAs between mouse dendritic cells via
exosomes. Blood 2012;119:756-66.
423. Bartlett DW, Davis ME. Physicochemical
and biological characterization of targeted,
nucleic acid-containing nanoparticles. Bio-
conjug Chem 2007;18:456-68.
424. Bryniarski K, Ptak W, Martin E, et al. Free
extracellular miRNA functionally targets cells by
transfecting exosomes from their companion
cells. PLoS One 2015;10:e0122991.
425. Weiner CP, Mason CW, Dong Y,
Buhimschi IA, Swaan PW, Buhimschi CS. Human
effector/initiator gene sets that regulate myo-
metrial contractility during term and preterm la-
bor. Am J Obstet Gynecol 2010;202:474.e1-20.
426. Lee JS, Romero R, Han YM, et al.
Placenta-on-a-chip: a novel platform to study
the biology of the human placenta. J Matern
Fetal Neonatal Med 2016;29:1046-54.
427. Duncan R, Gaspar R. Nanomedicine(s)
under the microscope. Mol Pharm 2011;8:
2101-41.
428. Kabanov AV. Polymer genomics: an
insight into pharmacology and toxicology of
nanomedicines. Adv Drug Deliv Rev 2006;58:
1597-621.
429. Rytting E, Nguyen J, Wang X, Kissel T.
Biodegradable polymeric nanocarriers for pul-
monary drug delivery. Expert Opin Drug Deliv
2008;5:629-39.
430. Peer D, Karp JM, Hong S, Farokhzad OC,
Margalit R, Langer R. Nanocarriers as an
emerging platform for cancer therapy. Nat
Nanotechnol 2007;2:751-60.
431. Rytting E, Bur M, Cartier R, et al. In vitro
and in vivo performance of biocompatible
negatively-charged salbutamol-loaded nano-
particles. J Control Release 2010;141:101-7.
432. Cai C, Bakowsky U, Rytting E,
Schaper AK, Kissel T. Charged nanoparticles as
protein delivery systems: a feasibility study using
lysozyme as model protein. Eur J Pharm Bio-
pharm 2008;69:31-42.
433. Poulsen MS, Rytting E, Mose T,
Knudsen LE. Modeling placental transport:
correlation of in vitro BeWo cell permeability and
ex vivo human placental perfusion. Toxicol
In Vitro 2009;23:1380-6.
JULY 2016 American Journal of Obstetrics & Gynecology
Obstetrics Supplement
434. Li H, Van Ravenzwaay B, Rietjens IM,
Louisse J. Assessment of an in vitro transport
model using BeWo b30 cells to predict placental
transfer of compounds. Arch Toxicol 2013;87:
1661-9.
435. Cartwright L, Poulsen MS, Nielsen HM,
et al. In vitro placental model optimization for
nanoparticle transport studies. Int J Nano-
medicine 2012;7:497-510.
436. Wick P, Malek A, Manser P, et al. Barrier
capacity of human placenta for nanosized
materials. Environ Health Perspect 2010;118:
432-6.
437. Poulsen MS, Mose T, Maroun LL,
Mathiesen L, Knudsen LE, Rytting E. Kinetics of
silica nanoparticles in the human placenta.
Nanotoxicology 2015;9:79-86.
438. Myllynen PK, Loughran MJ, Howard CV,
Sormunen R, Walsh AA, Vahakangas KH.
Kinetics of gold nanoparticles in the human
placenta. Reprod Toxicol 2008;26:130-7.
439. Menjoge AR, Rinderknecht AL, Navath RS,
et al. Transfer of PAMAM dendrimers across
human placenta: prospects of its use as drug
carrier during pregnancy. J Control Release
2011;150:326-38.
440. Bajoria R, Contractor SF. Effect of the size
of liposomes on the transfer and uptake of car-
boxyfluorescein by the perfused human term
placenta. J Pharm Pharmacol 1997;49:675-81.
441. Ali H, Kalashnikova I, White MA,
Sherman M, Rytting E. Preparation, character-
ization, and transport of dexamethasone-loaded
polymeric nanoparticles across a human
placental in vitro model. Int J Pharm 2013;454:
149-57.
442. Yamashita K, Yoshioka Y, Higashisaka K,
et al. Silica and titanium dioxide nanoparticles
cause pregnancy complications in mice. Nat
Nanotechnol 2011;6:321-8.
443. Hong JS, Park MK, Kim MS, et al. Prenatal
development toxicity study of zinc oxide
nanoparticles in rats. Int J Nanomedicine
2014;9(suppl 2):159-71.
444. Di Bona KR, Xu Y, Ramirez PA, et al. Sur-
face charge and dosage dependent potential
developmental toxicity and biodistribution of iron
oxide nanoparticles in pregnant CD-1 mice.
Reprod Toxicol 2014;50:36-42.
445. Tsyganova NA, Khairullin RM,
Terentyuk GS, et al. Penetration of pegylated
gold nanoparticles through rat placental barrier.
Bull Exp Biol Med 2014;157:383-5.
446. Rattanapinyopituk K, Shimada A, Morita T,
et al. Demonstration of the clathrin- and
caveolin-mediated endocytosis at the maternal-
fetal barrier in mouse placenta after intravenous
administration of gold nanoparticles. J Vet Med
Sci 2014;76:377-87.
447. Basha S, Vaidhyanathan S, Pauletti GM.
Selection of peptide ligands for human placental
transcytosis systems using in vitro phage
display. Methods Mol Biol 2011;716:141-56.
448. Wu CH, Liu IJ, Lu RM, Wu HC. Advance-
ment and applications of peptide phage display
technology in biomedical science. J Biomed Sci
2016;23:8.
S45
Supplement Obstetrics
449. Kaitu’u-lino TJ, Pattison S, Ye L, et al.
Targeted nanoparticle delivery of doxorubicin
into placental tissues to treat ectopic pregnan-
cies. Endocrinology 2013;154:911-9.
450. Rytting E, Ahmed MS. Fetal drug therapy.
In: Mattison DR, ed. Clinical pharmacology during
pregnancy. Oxford, UK: Academic Press; 2013.
451. Reed MD, Mattison DR. Treating the
placenta: an evolving therapeutic concept.
Oxford, UK: Academic Press; 2013.
452. Khlebtsov N, Bogatyrev V, Dykman L, et al.
Analytical and theranostic applications of
gold nanoparticles and multifunctional nano-
composites. Theranostics 2013;3:167-80.
453. Engelberth SA, Hempel N, Bergkvist M.
Development of nanoscale approaches for
ovarian cancer therapeutics and diagnostics.
Crit Rev Oncog 2014;19:281-315.
454. Webb JA, Thomsen HS. Gadolinium
contrast media during pregnancy and lactation.
Acta Radiol 2013;54:599-600.
455. Rytting E, Wang X, Vernikovskaya DI, et al.
Metabolism and disposition of bupropion in
pregnant baboons (Papio cynocephalus). Drug
Metab Dispos 2014;42:1773-9.
456. Wang X, Paul JA, Nanovskaya TN,
Hankins GD, Ahmed MS. Quantitative determi-
nation of telavancin in pregnant baboon plasma
by solid-phase extraction and LC-ESI-MS.
J Pharm Biomed Anal 2014;98:107-12.
457. Wang X, Abdelrahman DR, Fokina VM,
Hankins GD, Ahmed MS, Nanovskaya TN.
Metabolism of bupropion by baboon hepatic
and placental microsomes. Biochem Pharmacol
2011;82:295-303.
458. Yan R, Nanovskaya TN, Zharikova OL,
Mattison DR, Hankins GD, Ahmed MS. Meta-
bolism of 17alpha-hydroxyprogesterone cap-
roate by hepatic and placental microsomes of
human and baboons. Biochem Pharmacol
2008;75:1848-57.
459. Zharikova OL, Ravindran S,
Nanovskaya TN, Hill RA, Hankins GD,
Ahmed MS. Kinetics of glyburide metabolism by
hepatic and placental microsomes of human and
baboon. Biochem Pharmacol 2007;73:2012-9.
460. Ravindran S, Zharikova OL, Hill RA,
Nanovskaya TN, Hankins GD, Ahmed MS. Iden-
tification of glyburide metabolites formed by he-
patic and placental microsomes of humans and
baboons. Biochem Pharmacol 2006;72:1730-7.
461. Urry DW, Long MM, Cox BA, Ohnishi T,
Mitchell LW, Jacobs M. The synthetic poly-
pentapeptide of elastin coacervates and forms
S46 American Journal of Obstetrics & Gynecology JULY 2016
Downloaded for rachmad sammulia (iwandrake@yahoo.co.id) at ClinicalKey Global Guest Users from ClinicalKey.com by Elsevier on May 17, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
filamentous aggregates. Biochim Biophys Acta
1974;371:597-602.
462. Bidwell GL 3rd, Raucher D. Application of
thermally responsive polypeptides directed
against c-Myc transcriptional function for
cancer therapy. Mol Cancer Ther 2005;4:
1076-85.
463. George EM, Liu H, Robinson GG, Mahdi F,
Perkins E, Bidwell GL 3rd. Growth factor purifi-
cation and delivery systems (PADS) for thera-
peutic angiogenesis. Vasc Cell 2015;7:1.
464. Meyer DE, Chilkoti A. Genetically encoded
synthesis of protein-based polymers with
precisely specified molecular weight and
sequence by recursive directional ligation: ex-
amples from the elastin-like polypeptide system.
Biomacromolecules 2002;3:357-67.
465. Daniell H, Guda C, McPherson DT,
Zhang X, Xu J, Urry DW. Hyper expression of a
synthetic protein-based polymer gene. Methods
Mol Biol 1997;63:359-71.
466. Urry D, Urry C, Luan T, et al. Temperature
of polypeptide inverse temperature transition
depends on mean residue hydrophobicity. J Am
Chem Soc 1991;113:4346-8.
467. George EM, Liu H, Robinson GG,
Bidwell GL. A polypeptide drug carrier for
maternal delivery and prevention of fetal expo-
sure. J Drug Target 2014;22:935-47.
468. Brosens JJ, Pijnenborg R, Brosens IA. The
myometrial junctional zone spiral arteries in
normal and abnormal pregnancies: a review of
the literature. Am J Obstet Gynecol 2002;187:
1416-23.
469. Lyall F, Robson SC, Bulmer JN. Spiral ar-
tery remodeling and trophoblast invasion in
preeclampsia and fetal growth restriction: rela-
tionship to clinical outcome. Hypertension
2013;62:1046-54.
470. Burke SD, Karumanchi SA. Spiral artery
remodeling in preeclampsia revisited. Hyper-
tension 2013;62:1013-4.
471. Palei AC, Spradley FT, Warrington JP,
George EM, Granger JP. Pathophysiology of
hypertension in pre-eclampsia: a lesson in inte-
grative physiology. Acta Physiol (Oxf) 2013;208:
224-33.
472. Warrington JP, George EM, Palei AC,
Spradley FT, Granger JP. Recent advances
in the understanding of the pathophysiology
of preeclampsia. Hypertension 2013;62:
666-73.
473. Bidwell GL 3rd, George EM. Maternally
sequestered therapeutic polypeptides: a new
ajog.org
approach for the management of preeclampsia.
Front Pharmacol 2014;5:201.
474. Maynard SE, Min JY, Merchan J, et al.
Excess placental soluble fms-like tyrosine
kinase 1 (sFlt1) may contribute to endothelial
dysfunction, hypertension, and proteinuria in
preeclampsia. J Clin Invest 2003;111:649-58.
475. Maynard SE, Venkatesha S, Thadhani R,
Karumanchi SA. Soluble Fms-like tyrosine
kinase 1 and endothelial dysfunction in the
pathogenesis of preeclampsia. Pediatr Res
2005;57:1R-7R.
476. Sawano A, Takahashi T, Yamaguchi S,
Aonuma M, Shibuya M. Flt-1 but not KDR/Flk-1
tyrosine kinase is a receptor for placenta growth
factor, which is related to vascular endothelial
growth factor. Cell Growth Differ 1996;7:213-21.
477. Gerber HP, McMurtrey A, Kowalski J, et al.
Vascular endothelial growth factor regulates
endothelial cell survival through the phosphati-
dylinositol 3’-kinase/Akt signal transduction
pathway: requirement for Flk-1/KDR activation.
J Biol Chem 1998;273:30336-43.
478. Rusterholz C, Hahn S, Holzgreve W. Role
of placentally produced inflammatory and regu-
latory cytokines in pregnancy and the etiology of
preeclampsia. Semin Immunopathol 2007;29:
151-62.
479. Kupferminc MJ, Peaceman AM, Wigton TR,
Rehnberg KA, Socol ML. Tumor necrosis factor-
alpha is elevated in plasma and amniotic fluid of
patients with severe preeclampsia. Am J Obstet
Gynecol 1994;170:1752-9.
480. Vince GS, Starkey PM, Austgulen R,
Kwiatkowski D, Redman CW. Interleukin-6,
tumour necrosis factor and soluble tumour ne-
crosis factor receptors in women with pre-
eclampsia. BJOG 1995;102:20-5.
481. Kim YM, Romero R, Oh SY, et al. Toll-like
receptor 4: a potential link between “danger
signals,” the innate immune system, and pre-
eclampsia? Am J Obstet Gynecol 2005;193:
921-7.
482. Demicheva E, Crispi F. Long-term follow-up
of intrauterine growth restriction: cardiovascular
disorders. Fetal Diagn Ther 2014;36:143-53.
483. Galjaard S, Devlieger R, Van Assche FA.
Fetal growth and developmental programming.
J Perinat Med 2013;41:101-5.
484. Tuovinen S, Eriksson JG, Kajantie E,
Raikkonen K. Maternal hypertensive pregnancy
disorders and cognitive functioning of the
offspring: a systematic review. J Am Soc
Hypertens 2014;8:832-47.e1.