Review

Molecular Targets of Aspirin and Prevention of

Preeclampsia and Their Potential Association with
Circulating Extracellular Vesicles during Pregnancy
Suchismita Dutta 1, Sathish Kumar 2, Jon Hyett 3 and Carlos Salomon 1,4,5,*
1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical
Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane,
QLD 4029, Australia
2 Departments of Comparative Biosciences and Obstetrics and Gynecology, University of Wisconsin,

Madison, WI 53792, USA

3 Royal Prince Alfred Hospital Sydney, University of Sydney, Camperdown, NSW 2050, Australia

4 Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Ochsner Clinic Foundation,

New Orleans, LA 70124, USA

5 Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, University of Concepción,

Concepción, Region Bio-Bio 4070386, Chile

* Correspondence: [email protected]; Tel.: +61-7-3346-5500; Fax: +61-7-3346-5509

Received: 19 May 2019; Accepted: 26 August 2019; Published: 5 September 2019

Abstract: Uncomplicated healthy pregnancy is the outcome of successful fertilization, implantation

of embryos, trophoblast development and adequate placentation. Any deviation in these cascades
of events may lead to complicated pregnancies such as preeclampsia (PE). The current incidence of
PE is 2–8% in all pregnancies worldwide, leading to high maternal as well as perinatal mortality
and morbidity rates. A number of randomized controlled clinical trials observed the association
between low dose aspirin (LDA) treatment in early gestational age and significant reduction of early
onset of PE in high-risk pregnant women. However, a substantial knowledge gap exists in
identifying the particular mechanism of action of aspirin on placental function. It is already
established that the placental-derived exosomes (PdE) are present in the maternal circulation from
6 weeks of gestation, and exosomes contain bioactive molecules such as proteins, lipids and RNA
that are a “fingerprint” of their originating cells. Interestingly, levels of exosomes are higher in PE
compared to normal pregnancies, and changes in the level of PdE during the first trimester may be
used to classify women at risk for developing PE. The aim of this review is to discuss the
mechanisms of action of LDA on placental and maternal physiological systems including the role
of PdE in these phenomena. This review article will contribute to the in-depth understanding of
LDA-induced PE prevention.

Keywords: pregnancy; placentation; preeclampsia; low dose aspirin; exosomes

1. Introduction
Pregnancy is an important event that leads to significant changes in maternal physiology.
Successful pregnancy requires involvement of a series of processes commencing from fertilization to
establishment of placental and maternal vascular connection with the fetus in correct order. Adequate
placentation is one of the prerequisites for maintaining a normal healthy pregnancy. New insights
into the placentation process involve migration, invasion, adherence, proliferation and differentiation
of the placental principal cellular component, i.e., extravillous trophoblasts (EVTs), followed by their
interaction with the pre-decasualized maternal uterine blood vessels, glands and lymphatics [1].
Int. J. Mol. Sci. 2019, 20, 4370; doi:10.3390/ijms20184370 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2019, 20, 4370 2 of 25

Placentation further evolves by digestion of the extracellular matrix where the EVTs tolerate
surrounding maternal circulatory oxidative stress and the effects of soluble cytokines [1].
Nonetheless, the allogenic EVTs also interact with maternal decidual immune cells to provide
immune competence [2]. Any deviation in these events may lead to pathological pregnancies, i.e.,
preeclampsia (PE). The broad concept of PE pathophysiology includes defective trophoblast invasion
and inadequate uterine spiral arterial remodeling in the first trimester that follows with reduced
uteroplacental perfusion [3]. This subsequently leads to poorly perfused and stressed placental
syncytiotrophoblasts that release a range of mediators causing endothelial dysfunction and PE
clinical manifestations [3]. Moreover, such abnormal placentation leads to the secretion of abnormal
levels of anti-angiogenic and inflammatory proteins that enter the systemic maternal circulation and
impair maternal systemic vascular function, resulting in the clinical manifestations of PE. Since PE
and its clinical symptoms rapidly abate after delivery (removal of the placenta), the placenta must
play a central or initiating role in this pregnancy disorder.
Novel pharmacological interventions for the prevention of PE have not been developed for
many years, as the complex pathophysiology, diversified clinical presentation of the disease and
difficulties associated with conducting drug discovery research in pregnant women have hampered
their development. Low dose aspirin (LDA) is considered to be the most effective prophylactic
therapy for reducing disease prevalence in women at high risk for developing early-onset PE. The
use of LDA in pregnant women is generally considered to be safe as it does not affect the pregnant
mothers and/or their unborn fetuses inadvertently. It has been suggested that the principal
mechanism of action by which LDA exerts its effect is via the inhibition of thromboxane production
that leads to the inhibition of platelet aggregation. Additionally, LDA has a direct positive effect on
the villous trophoblasts [4]. However, recent evidence suggests that LDA prevents the development
of PE by promoting trophoblast invasion and migration into the uterine arteries, interfering with
cytokine production and stimulating the production of proangiogenic protein placental growth factor
(PlGF); thereby, inhibiting apoptosis and premature uterine arterial remodeling [5].
Recent meta-analysis suggest that LDA (>100 mg/day) in early gestation (before 16 weeks) is
beneficial in preventing common pregnancy complications; i.e., PE, fetal growth restriction, preterm
birth [6–9], suggesting that aspirin may have effect on implantation and early placentation [10]. Low-
dose aspirin has been utilized for many years to prevent PE [11–13]. A recent individual patient data
meta-analysis observed that LDA can reduce the risk of PE development by 10% and small for
gestational age (SGA) births by 24% [14] without posing a major safety risk to mothers or fetuses
other than placental abruption in some cases [15]. Other studies reported that low dose aspirin is
generally well tolerated within both preconception and early pregnancy periods [16].
In normal healthy pregnancy, placental syncytiotrophoblast release extracellular vesicles (EVs)
including exosomes into the maternal bloodstream that contain some information (i.e., micro RNA,
mRNA, proteins) to convey from the originating cells to their distant target cells such as maternal
immune cells in order to adapt to the pregnancy associated physiological changes [17]. This EV
release is further increased from the preeclamptic placenta due to oxidative stress, causing
widespread systemic endothelial dysfunction, giving rise to maternal hypertension, feto-placental
circulatory compromise and damaging various maternal organs [17]. Some recently published
reports have suggested that LDA could influence platelet derived EV release; however, the effect of
LDA on the regulation of placental EV release is not known. Therefore, in this review, we will discuss
the potential mechanisms of action of aspirin in the context of PE prevention and the potential role
of extracellular vesicles released from the placenta in this phenomenon (Figure 1).
Int. J. Mol. Sci. 2019, 20, 4370 3 of 25

Figure 1. Diagrammatic representation of preeclampsia (PE) development pathogenesis and

mechanism of prevention by low dose aspirin (LDA). In PE, syncytiotrophoblast-derived extracellular
vesicles (EVs), including exosomes, are released into the maternal circulation in increased amounts
due to inadequate placental vascular remodeling. These EVs activate the vascular endothelial cells,
leukocytes and platelets and cause dysfunction. LDA prevents the development of PE by
reducingendothelial cell dysfunction. The proposed mechanism that was investigated; acetylsalicylic
acid, the crude form of aspirin, modulates trophoblast derived exosome release and changes their
proteomic and microRNA contents.

2. Physiology of Pregnancy
Pregnancy induces a number of alterations to maternal physiology for maintaining the correct
course of pregnancy and it involves a cascade of processes commencing from fertilization to the
establishment of feto-maternal communication and cross-talk mediated via the placenta.
Preimplantation conditions, vascularisation, invasion of the embryonic cells to the maternal uterine
wall and oxidative stress are the essential regulators in the function of pregnancy events [18]. Prior
to implantation, there is a postovulatory surge of circulatory progesterone level that inhibits the
proliferation of estrogen-dependent uterine epithelium and induces secretory transformation of
uterine glands. In the early stages of development, i.e., ~day 6 of fertilization, the microvilli of the
blastocyst interact with the pinopodes of the uterine endometrial luminal epithelium in order to
establish apposition, which becomes stable through the increased adherence of the trophectoderm
and the uterine luminal epithelium. During this interaction, a range of molecules are secreted from
the immune activated cells including mucin, selectin, integrin and cadherin [18,19]. Shortly thereafter,
invasion begins and trophectoderm penetrates the uterine epithelium, invading the wall of the
uterine arteries where they interact with the cells of the maternal circulatory immune system and
Int. J. Mol. Sci. 2019, 20, 4370 4 of 25

mediate the remodeling of the uterine spiral arteries that supply the placenta. This is followed by
deportation of aggregates containing transcriptive materials.
There is direct evidence that platelets are involved in the placentation process. During
placentation, trophoblasts invade the decidual stroma including the uterine glands and migrate into
the maternal uterine spiral arteries replacing the vascular smooth muscle cells to remodel the arteries
as low resistance, large caliber vessels [20]. This process ensures adequate placental perfusion by the
remodeling of maternal uterine vessels. Histological examination shows deposition of maternal
platelets in the trophoblast aggregates formed in the uterine spiral arteries. A number of studies
discovered that these platelets are activated, releasing soluble factors enhancing the invasive capacity
of trophoblast cells [21]. The trophoblasts produce a range of vasoconstrictors and vasodialators that
are in balance to maintain the placental blood flow for proper fetal development during pregnancy
[22].
During pregnancy, the viscosity and coagulability of maternal blood upsurges due to the
increase in pro-coagulant agents, such as plasminogen activator inhibitor-1, fibrinogen, factor VII,
VIII, von Willebrand factor and to the reduced fibrinolysis. The hypercoagulable state is attributed
to the activation of platelets [23]. Maternal serum biochemical markers of pregnancy, namely alpha
fetoprotein (AFP), human chorionic gonadotrophin (hCG), unconjugated estriol [24] and inhibin-A
[25], are produced and found in higher concentration in the maternal peripheral circulation due to
the implantation and placentation processes of pregnancy.

3. Pathogenesis of Preeclampsia
Preeclampsia (PE) is defined as new onset of hypertension after 20 weeks’ gestation with renal,
hepatic, hematologic, neurological, pulmonary or fetal involvement. Physical signs of preeclampsia
are hypertension, proteinuria, renal insufficiency, hemolysis, reduced platelet count and/or increased
platelet activation [26]. It is a serious complication of pregnancy affecting ~7.6% pregnancies globally
and is associated with high morbidity and mortality in affected mothers and children [27]. It is a
lifelong disorder with increased risks of neonatal and child morbidity and mortality including health
risks in adulthood [27]. Pregnancy induced hypertension is one of the most prevalent risk factors for
the development of PE. The consequences of PE include intrauterine fetal growth restriction (IUGR)
and preterm birth. [28].
In PE, there is widespread systemic endothelial dysfunction that leads to hypertension and
concomitant proteinuria [29]. Clinical risk factors for developing PE assessed before 16 weeks of
gestation include prior history of hypertension, chronic hypertension, pre-gestational diabetes, pre-
pregnancy BMI > 30 and use of assisted reproductive technology [30]. The most commonly used
screening test for early prediction of PE involves analysis of maternal characteristics, maternal mean
arterial pressure, uterine arterial Doppler pulsatility index and serum biochemistry (PaPP-A and/or
PlGF). This test is performed at 11–13 weeks of gestation [31]. The present management of patients
with PE depends on symptom severity. Currently, some drugs are available to treat mild to severe
PE (e.g., methyldopa, hydralazine, magnesium sulphate) [32]. However, the best treatment currently
available for PE is delivery of the newborn and placenta as all the signs and symptoms of PE abolish
when the placenta is separated from the mother.
In early-onset PE, there is defective implantation and placentation due to inadequate
extravillous trophoblast invasion and partial failure of uterine arterial remodeling, resulting in high
resistance and low capacitance vascular supply to the placenta and fetus [33]; however, this
phenomenon is not evident in late-onset PE. Some research studies showed that during normal
healthy pregnancy, the invasive trophoblast cells replace the smooth muscle and elastic lamina of the
maternal uterine vessels, causing dilation and funneling at the vessel mouth and facilitating further
migration of trophoblasts. In the absence of conversion of the maternal uterine vessels, there is
retention of smooth muscle cells contributing to increased resistance to maternal blood flow.
Nonetheless, maternal blood enters into the intervillous space as a turbulent jet that increases the risk
of spontaneous vasoconstriction and ischemia-reperfusion injury, generating oxidative stress within
the maternal circulation [34]. This in turn gives rise to placental villous infarcts, constriction of spiral
Int. J. Mol. Sci. 2019, 20, 4370 5 of 25

arteries due to the mural hypertrophy and fibrin deposition, leading to the abnormal ultrasound
indices and biochemical markers seen in the maternal circulation. This failure in vascular dilation has
a direct impact on placental blood flow and is the primary determinant of pregnancy pathology [34].
In addition to the general concept of PE pathogenesis, where there is defective EVT invasion and
uterine arterial remodeling, there is also an imbalance of angiogenic and antiangiogenic factors. These
factors include vascular endothelial growth factor (VEGF), soluble endoglin, soluble fms-like tyrosine
kinase-1 receptors (sFlt-1) and placental growth factor (PlGF). Abnormal production of these factors
is closely associated with PE and intrauterine growth restriction [35]. At the end of the first trimester
of pregnancy, the extravillous trophoblasts (EVT) invade the uterine spiral arteries and replace the
vascular smooth muscle and the endothelium to remodel the arteries, which lead to the formation of
low resistance and high capacitance vessels that facilitates increased placental perfusion. When there
is perturbation of this process, there is reduced placental perfusion causing placental stress where
platelets aggregate and accumulate in the partially damaged placenta [36]. In PE, there are
interactions between maternal characteristics and risk factors and placental pathophysiological
factors leading to a vicious cycle of maternal inflammation, vascular dysfunction and the activation
of pro-coagulation pathways [33].
Current research on PE is focused on the role of extracellular vesicles released from the placenta
[33]. Following placentation, the residual syncytiotrophoblastic material generated from placental
shedding or by placental microparticles releases various vessel constricting factors that cause
systemic endothelial dysfunction [37,38]. These microparticles contain a set of proteins including
some pro-inflammatory and pro-coagulatory molecules that contribute to the development of PE [39–
41] A recent article on PE stated that there was interaction between fetal Human Leukocyte Antigen-
C (HLA-C) molecule and maternal natural killer cells’ killer-cell immunoglobulin-like receptor (KIR)
in severe PE; these molecules are carried by the EVs released from the placenta and maternal
circulatory cells [42]. Inadequate placentation causes the development of pregnancy-induced
hypertension (PIH) and preeclampsia (PE) [43,44], leading to focal regions of hypoxia that are
responsible for modifying the production of growth factors, cytokines [45], lipid peroxides [46] and
prostaglandins by placental trophoblasts [45]. Elevated placental levels of inflammatory cytokines,
such as tumor necrosis factor-α, interleukin (IL)-1α, IL-1β and IL-6, are generally considered
unfavorable to pregnancy [47]. Moreover, clinical studies have shown changes in the levels of
cytokines and prostaglandins in women with PE [48,49]. Maternal circulatory neutrophils are
activated in pregnancy and further activated in PE, which are the source of oxidative stress by
generating reactive oxygen species such as hydrogen peroxide and superoxide anion and these
molecules cause damage to the proteins, lipids and nucleic acids [50]. Neutrophil activation is
initiated in the intervillous space by increased secretion of lipid peroxides by the placenta, which is
abnormally increased in PE. This stimulates phospholipase A2 and cyclooxygenase enzymes to
increase the production of thromboxane. Thromboxane is implicated in monocyte activation
responses and plays role in mediating tumor necrosis factor alpha (TNF-α) production by neutrophils
in response to oxidative stress [50].
In PE, the production of thromboxane A2 and prostaglandin I2 is altered with excessive
accumulation of THXA2 metabolite in the maternal systemic circulation [51,52]. This results to the
increased activation and aggregation of platelets and vasoconstriction causing impaired placental
perfusion and oxidative stress [53–57]. The platelet count is reduced in PE due to platelet activation
and aggregation under the effect of elevated levels of ThXA2 Synthase [28]. In addition [36], PE
contributes to some biochemical changes in maternal circulatory system, such as, increase in
phosphodiesterase-5 [58], thromboxane synthase [28] and an elevated hCG level [59]. Nonetheless,
immunological changes also take place in PE. There is rise in anti β2 - glycoprotein I antibodies that
are related with aberrant implantation [60]. many predictive biomarkers for PE have been described
including placental biomarkers (PAPP-A, PLGF, s-FLT-1, placental protein 13 (PP 13)), Free HbF,
Alpha 1 Macroglobulin and Uterine Artery Doppler Pulsatility Index [61] [62]. Not all studies have
consistently shown value of these markers, for example changes in PP13 have not been consistently
Int. J. Mol. Sci. 2019, 20, 4370 6 of 25

replicated [63]. Measurements of total cell free DNA and fetal fraction in maternal plasma at 11–13
and 20–24 weeks are not predictive of PE [64].

4. Pharmacology of Aspirin and Basis for Its Use in PE

The chemical name of aspirin is acetylsalicylic acid (ASA) [65–69]. It is a nonsteroidal anti-
inflammatory drug (NSAID). It is typically used in two dose regimens—high dose (600 mg) and low
dose (60–150 mg). It has anti-inflammatory, analgesic, antipyretic and antiplatelet effects [70]. The
endothelial dysfunction in PE involves increased lipid peroxidation, which activates COX and
inhibits prostacyclin synthase, thus inducing rapid imbalance in the TXA2/prostacyclin (PGI2) ratio
in favor of TXA2 [51]. TXA2 favors systemic vasoconstriction, and increasing platelet aggregation
and adhesion, which is compensated in this context by the vasodilator effect of prostacyclins, levels
of which drop sharply. This imbalance is present from 13 weeks of gestation in high-risk PE patients
[71]. LDA treatment for 2 weeks reverses TXA2/PGI2 imbalance by inhibiting THXA2 production
[72,73]. Some studies observed that LDA can reduce the release of sFLT-1 from trophoblast cells and
induce the production of vascular endothelial growth factor thereby promoting angiogenesis [74].
LDA also modulates cytokine production, reduces apoptosis and alters cell aggregation and fusion
thereby improving defective trophoblast implantation [5]. LDA improves EVT migration and
invasion into the maternal uterine spiral arteries and reduces placental cell apoptosis [75]. PE is
associated with some augmented anti-angiogenic, oxidative and pro-inflammatory markers, as well
as increasing human polymorphonuclear neutrophil (PMN)-endothelial cell adhesion [76]. LDA
reduces the circulatory levels of these factors and improves the cytokine profile [5]. LDA causes
retardation in leukocyte-endothelial cell adhesion and interaction and thus it prevents the endothelial
cell dysfunction in PE [76]. Several reports have suggested that few biomarkers can be identified in
maternal blood to be monitored for assessing treatment response after initiation of LDA treatment in
pregnant women at high risk for preeclampsia; i.e., placental growth factor, placental protein 13,
alpha fetoprotein [77].
The mechanism of action of aspirin involves a cascade of events. Aspirin irreversibly acetylates
the platelet enzyme cyclooxygenase (COX), modifying the production of different prostaglandins
and also acts as an analgesic, anti-inflammatory agent. There are three isoforms of COX enzyme upon
which aspirin acts; the sources of these enzymes are mainly platelets, but they are also found in other
immune cells namely leukocytes, monocytes and macrophages. Aspirin inhibits COX-1 irreversibly
and COX-2 reversibly to a lesser extent. The resultant inhibition of COX-dependent generation of
thromboxane A2 prevents platelet aggregation. This effect is maintained for the entire platelet
lifespan of 8–9 days [78].

5. Low Dose Aspirin (LDA) and Pregnancy

Low dose aspirin reduces the mortality and morbidity in pregnant women at high risk for PE
[79–82]. National guidelines typically suggest that women considered to be at high risk of developing
pre-eclampsia should be treated with prophylactic low dose aspirin to reduce the prevalence of
disease, although there are differences in how “high risk” is defined (NICE guidelines and ACOG
recommendations (2017)). Acetylsalicylic Acid (ASA) is considered a highly attractive
pharmacological agent to use in pregnancy for the prevention of maternal and perinatal mortality
and morbidity worldwide due to its low cost, widespread availability, ease of administration and
safety profile [83]. Aspirin is listed as a US Food and Drug Administration (FDA) category C drug
during the first and second trimester and a category D drug in the third trimester of pregnancy [70].
Although some recent evidence has suggested that aspirin can affect the fetus adversely causing
congenital anomaly, the FDA has assigned this drug as pregnancy category C, and treatment is
relatively safe [84]. Although aspirin can cross the placenta, it is safe in low doses [85].
Low dose aspirin is a very effective treatment. Meta-analysis of a series of >30 randomized
controlled trials have shown that low dose aspirin prophylaxis (any dose, any gestation) reduces the
incidence of PE by 10% [11,12,14,86]. If analysis is restricted to assessment of outcomes for PE leading
Int. J. Mol. Sci. 2019, 20, 4370 7 of 25

to delivery before 34 weeks in women who commence aspirin <16 weeks gestation and have a higher
dose (>100 mg/day), then the data show a 90% reduction in early PE [87]. Aspirin also appears to be
effective at reducing the prevalence of intrauterine growth restriction (IUGR); once again, this meta-
analysis shows that treatment is more effective if a higher dose (>100 mg/day) is given and treatment
is started before 16 weeks [88]. Other meta-analyses have also shown that low dose aspirin may be
effective in preventing spontaneous preterm birth [6–9]. Other studies have demonstrated that low
dose aspirin is generally well tolerated in both preconception and early pregnancy periods [16].
To date, several studies have attempted to assess the beneficial effects of aspirin treatment in
gestational hypertensive disorders, in particular PE. In spite of different conflicting results on the
effects of aspirin in pregnancy, one study found that aspirin administered early i.e., from the eighth
week of gestation has in fact a positive effect on the pregnancy outcome without the manifestation of
teratogenicity or fetotoxicity [29]. Recent studies on PE found that in high risk pregnancies, any
preventative treatment should be aimed at or before 16 gestational weeks to be effective as
placentation and uterine spiral arterial remodeling is completed by 20 gestational weeks [11].
Additionally, to prevent perinatal death and to improve perinatal outcomes, low dose aspirin should
be prescribed before 16 gestational weeks [89]. Cost benefit analysis in a US based research study
showed that aspirin prophylaxis through pregnancy would reduce morbidity and mortality, leading
to a reduction in health care costs [90].
Following the preparation of a systematic review, the US Preventive Service Task Force
recommended the use of low-dose aspirin (81 mg/d) as preventive medication after 12 weeks of
gestation in women who are at high risk for PE [91–93]. The US Preventive Service Task Force also
found that LDA prophylaxis in early pregnancy does not increase the chances of placental abruption,
postpartum hemorrhage, fetal intracranial hemorrhage or perinatal mortality [94].
Other authors have suggested that the dose and timing of aspirin prophylaxis is also important.
Ayala et al., 2013, identified that (i) 100 mg/d ASA should be the recommended minimum dose for
prevention of complications in pregnancy; (ii) ingestion of low-dose ASA should be started at ≤16
weeks of gestation and (iii) low-dose ASA should be ingested at bedtime, not during the morning.
Aspirin prescribed in this way significantly regulates ambulatory blood pressure (BP) and reduces
the incidence of PE, gestational hypertension, preterm delivery and intrauterine growth restriction
(IUGR) [95].
Other agents have been used for prophylaxis against PE in high risk women, either alone or in
combination with LDA. There is a significant body of literature investigating whether low molecular
weight (LMWH) or unfractionated heparin can reduce rates of PE, preterm birth, perinatal mortality
and small for gestational age babies when prescribed to high risk women [96,97]. Heparin is safe from
a fetal perspective and does not cross the placental barrier due to its high molecular weight [85,98].
While some observational studies that have combined the use of aspirin and LMWH show significant
reduction in rates of PE in very high risk groups [99,100], an individual patient meta-analysis did not
show significant benefit to this intervention [101]. Calcium (1 g/day) has also been widely
investigated and appears to be particularly useful in low and middle income settings where dietary
calcium intake is poor [102]. Vitamin C, D, E [103–105], fish oil/omega 3, statins [35], L-arginine [106]
and antihypertensive drugs such as calcium channel blockers [107] have also been investigated,
although there is a paucity of randomized controlled trial-based data for these investigations. The
most significant ongoing research issues are to establish why aspirin is less effective in some groups
of women; for example, those that have chronic hypertension and to determine whether additional
agents can impact rates of term pre-eclampsia, which are not as significantly reduced using aspirin
therapy.
A table on recent studies involving aspirin and pregnancy has been presented in the Table 1.
Int. J. Mol. Sci. 2019, 20, 4370 8 of 25

Table 1. Recent Studies on Aspirin and Pregnancy.

Study Design Mode of Treatment Outcome of Study Reference

Low dose aspirin (LDA) and/or Low Improved pregnancy outcomes (Less PE and IUGR
Randomized controlled trial (RCT) [108]
Molecular Weight Heparin (LMWH) incidence)
Reduced incidence of unexplained recurrent
Prospective case-control study LDA and LMWH in first trimester [109]
spontaneous abortion
Database searching for RCTs involving LDA and
LDA or Placebo LDA reduces PE risk [110]
placebo in PE
LDA at 100 mg/day <16 gestational
Literature searching on LDA and PE Reduced PE incidence due to LDA prophylaxis [111]
weeks
LDA prophylaxis in at risk patients to develop PE have
higher advantages compared to negligible
Systematic literature searches about aspirin and PE LDA [112]
disadvantages i.e., feto-maternal bleeding, aspirin
resistance etc.
Systematic review and an individual participant data Antiplatelet aspirin therapy in early
10–15% reduction in the risk of PE [113]
meta-analysis pregnancy
LDA at <16 weeks, there was a significant reduction and
A systematic review and meta-analysis of randomized 50–150 mg/day aspirin or no treatment
a dose-response effect for the prevention of [87]
controlled trials at <16 or >16 gestational weeks
preeclampsia
A systematic review and meta-analysis through
LDA or placebo at <16 or >16 <16 weeks, significant reduction of PE.
electronic database searches (PubMed, Cochrane, [11]
gestational weeks >16 weeks, negligible impact on PE and related disorders
Embase).
LDA initiated at ≤ 16 weeks of gestation is associated
RCTs that evaluated the prophylactic
Databases searching involving keywords ‘aspirin’ and with a greater reduction of perinatal death and other
use of LDA (50–150 mg/day) during [114]
‘pregnancy’ adverse perinatal outcomes than when initiated at >16
pregnancy were included.
weeks.
Antiplatelet agents were associated with a significant
One or more antiplatelet agents (e.g.,
Meta-analysis of individual patient data recruited to 31 10% reduction in the relative risk of both PE (p = 0·004)
LDA or dipyridamole) versus a [86]
RCTs of PE primary prevention. and preterm birth before 34 weeks' gestation (p = 0·011)
placebo or no antiplatelet agent.
compared to control cases.
150 mg/day of aspirin was used to
Women at high risk for preterm PE were recruited to
reduce the incidence of aspirin LDA reduced the incidence of preterm PE [115]
RCTs
resistance and maximize the effect.
Int. J. Mol. Sci. 2019, 20, 4370 9 of 25

A planned secondary analysis of the Effects of Aspirin

Daily LDA (81 mg, n = 615) or placebo
in Gestation and Reproduction (EAGeR) trial, a Preconception LDA appears to be well tolerated by
(n = 613) and were followed for up to
multicenter, block-randomized, double-blind, placebo- women trying to conceive, women who become [16]
six menstrual cycles or through
controlled trial investigating the effects of LDA on the pregnant, and by their fetuses and neonates.
gestation if they became pregnant.
incidence of live birth.
Studies with smaller sample sizes are more likely to be
Chronological, cumulative meta-analyses of two biased against the null hypothesis. As such, cumulative
Antioxidant or Low Dose
recently published meta-analyses of RCTs examining meta-analysis is an effective tool in predicting potential [116]
Acetylsalicylic Acid (LDAA) therapy
the effects of antioxidant or LDA on the rates of PE. bias against the null hypothesis and the need for
additional studies.
The use of ASA may be associated with an increased risk
Prospective cohort study involving 533 pregnant
LDAA and LMWH of developing a sub-chorionic hematoma (SCH) during [117]
women in their first trimester
the first trimester.
Combined LMWH and aspirin treatment started before
Multicentre RCTs involving 32 women with a previous 12 weeks gestation in a subsequent pregnancy did not
The intervention was daily LMWH
delivery <34 weeks gestation with HD and/or SGA and show reduction of onset of recurrent HD either <34 [118]
with aspirin or aspirin alone.
aPLA were included before 12 weeks gestation. weeks gestation or irrespective of gestational age,
compared with aspirin alone.
Daily administration of LDA (81
Prospective randomized, placebo-controlled, double- mg/day) initiated between 6 and 13
PTB, PE, SGA, perinatal mortality were reduced. [119]
blinded, multinational clinical trial weeks of pregnancy and continued
upto 36 weeks.
Prospective RCTs Preconception LDA daily It is not associated with reduction of pregnancy loss [120]
Some of them received 150 mg/day
Primary outcome was delivery with PE before 37 weeks
Multicenter, double blind, placebo-controlled trial aspirin and some of them received
of gestation. Treatment with aspirin reduced the [121]
involving women at high risk for preterm PE placebo at 11–14 gestational weeks
incidence of preterm preeclampsia.
until 36 weeks of gestation
Int. J. Mol. Sci. 2019, 20, 4370 10 of 25

6. Effects of LDA on Placental and Maternal Body System Function

To date, a number of studies have attempted to elucidate the role of aspirin in the prevention of
adverse pregnancy outcomes. However, the particular function of LDA in preventing PE and other
pregnancy-induced hypertension is not clearly understood. Some in-vitro studies found that there is
no specific effect of LDA or LMWH on BeWo choriocarcinoma cells when treated with forskolin
except cell fusion due to the placental protein level 13 increase [122]. Some studies reported that
thromboxane has been found to be involved with vasoconstriction leading to placental ischemia,
thrombosis and platelet aggregation [123]. Other research studies reported that aspirin can negatively
act on COX2 enzyme, thereby inhibiting thromboxane A2 production from arachidonic acid
[124,125]. Interestingly, there are also data suggesting that aspirin can reduce the release of
thromboxane from the trophoblasts [22].
Low-dose aspirin, which selectively inhibits TXA2 production, is used to prevent high-risk PE
[28]. Low-dose aspirin, a common antiplatelet agent, usually restores prostacyclin and thromboxane
levels that prevent vasoconstriction, and therefore, has been targeted as an intervention to reduce PE
in at-risk women [124,126]. LDA increases the production of prostaglandin I2 by blocking the
synthesis of thromboxane A2 [73]. This PGI2 increases vasodilatation and prevent thromboxane
mediated damage [127]. Some studies have shown that TXA2 analogues cause hypertension in
pregnancy and TXAS depletion prevents hypertension and IUGR [128]. Urine specimens of PE
women show the presence of thromboxane B2, which is the metabolite of thromboxane A2 and LDA
shifts the balance between THXA2 and PGI2 favoring the production of PGI2 that increases the blood
flow to the placenta [129].
In normal, healthy pregnancies, uterine spiral arterial remodeling occurs at around 8 weeks of
gestation and is complete by 16–20 weeks [130]. However, in PE, placentation is inadequate and
under stress due to impaired uterine spiral arterial remodeling [131]. Some randomized controlled
clinical trials observed that LDA is associated with improvement in uterine arterial pulsatility index
when started in the first trimester of pregnancy [132,133]. Another study observed that low dose
aspirin reduces the UtA Doppler pulsatility index, indicating improved blood flow [134].
In-vitro studies found an association of LDA treated trophoblast cells and an improvement in
cytokine profile that prevents trophoblast apoptosis and promotes angiogenesis by increasing the
production of placental growth factor (PlGF) [75]. Another similar study by Panagodage, et al.
identified a number of factors that are involved in preeclampsia prevention with low dose aspirin
(LDA) treatment. The authors observed that placental growth factor is significantly decreased in
preeclamptic women’s sera compared to normotensive women’s sera; LDA increases trophoblast
secretion of PlGF and restores abnormal cytokine (Activated Leukocyte cell adhesion molecule
ALCAM, CXCL-16 and ErbB3) production by trophoblasts in PE [5]. Soluble fms-like tyrosine kinase-
1 (sFLT1) is an antiangiogenic factor and its expression is increased in preeclamptic placentas and in
cytotrophoblast exposed to hypoxia. Aspirin inhibits the production of sFLT1 in CTBs and this effect
is mediated by the inhibition of COX-1 [74].
Preeclampsia is associated with some augmented anti-angiogenic, oxidative and pro-
inflammatory markers, as well as increasing human polymorphonuclear neutrophil (PMN)-
endothelial cell adhesion. This cell adhesion is reduced when human PMN are incubated with ATL
(aspirin triggered lipoxin A4) [76]. This aspirin triggered lipoxin is similar to endogenously produced
lipoxins but the duration of action is prolonged [135]. ATL acts as an anti-inflammatory agent; it
promotes angiogenesis and causes immunosuppression and it also blocks the generation of reactive
oxygen species in the endothelial cells, inhibits chemotaxis of polymorphonuclear neutrophil and the
leukocyte-endothelial interaction [136–140] causes nuclear factor kappa B activation [137,141] and
secretion of tumor necrosis factor alpha (TNF-α) in activated T cells [142]. Additionally, ATL can
increase nitric oxide synthesis where the heme oxygenase-1 enzyme is also involved [143] and this
effect is responsible for resolving inflammation [143]. Heme oxygenase enzyme-1 degrades heme to
generate bilirubin, carbon monoxide and iron, exerting their anti-oxidant, antiapoptotic and
cytoprotective actions [144]. Additionally, another recently conducted study identified that aspirin
Int. J. Mol. Sci. 2019, 20, 4370 11 of 25

prevents TNF-alpha-induced endothelial cell dysfunction by regulating the NF-kappa B-dependent

miR-155/eNOS pathway in preeclampsia [145].
The pathophysiology of PE also involves the genetic expression of the STOX1 transcription
factor by extravillous trophoblasts that modulate trophoblast proliferation [146] [147]. The STOX1
gene is overexpressed in human placental extravillous trophoblasts and is associated with PE
pathogenesis [147–149]. Founds et al. [150] showed, in transcriptomic analysis, that STOX1 is
overexpressed during the first trimester of pregnancies that had a preeclamptic outcome. Other
studies have performed functional assays to determine the function of the STOX1 gene; using an in-
vivo mouse model, this gene was found to cause severe gestational hypertension, proteinuria, an
increased circulatory level of antiangiogenic factors and histological alterations in the kidney as well
as the placenta [151]. These researchers also demonstrated that low dose aspirin improved maternal
PE-like symptoms [152]. LDA improves uterine perfusion and favourably affects aspects of
reproduction [153]. In addition, empirical introduction of LDA during in vitro fertilization (IVF)
treatment improves the quality of oocytes and embyros [154]. Low dose aspirin and heparin in
combination improve the live birth rate in IVF for unexplained implantation failure [155]. Low-dose
aspirin effectively improves perifollicular artery blood flow and enhances oocyte quality and clinical
pregnancy rates [156].

7. Complications of LDA for Fetuses and Mothers

A systematic evidence review by the US Preventive Services Task Force (USPSTF) identified no
adverse impact on the mother or offspring during the perinatal period following aspirin use for
prevention of preeclampsia [157] including no documented adverse effect on neonatal platelets [158].
However, some studies have identified adverse effects with the antenatal and perinatal use of aspirin,
albeit taken at a higher dose. Potential risks associated with aspirin therapy during the third trimester
include premature closure of the ductus arteriosus and hemorrhagic complications [159],
subchorionic hematoma if administered in first trimester of pregnancy [117], fetal loss [160],
endocrine disturbances in the human fetal testis and interference in the testicular descent [161],
childhood asthma [162] and fetal complications [110]. Some research studies observed that high doses
of aspirin may affect fertility, increases the risk of miscarriages and may cause fetal cryptorchidism
[163–165]. Additionally, LDA therapy in the late gestational age has on rare occasion been reported
to cause renal injury, cardiovascular abnormality such as closure of the ductus arteriosus, necrotizing
enterocolitis and intracranial hemorrhage in the fetus as well as reduced breast milk supply in the
mother, likely due to the inhibition of cyclooxygenase enzyme pathways [164]. The common adverse
effects of aspirin in adults are significantly associated with gastrointestinal or cerebral bleeding
episodes [166]. Given the risks of aspirin therapy, it is better to reserve treatment for women deemed
high-risk of deep placentation related disorders rather than to prescribe it universally.

8. Predictive Biomarkers for Preeclampsia Cases Treated with Low Dose Aspirin
Few biomarkers have been identified in maternal blood as candidates for monitoring treatment
response after initiation of low dose aspirin treatment in pregnant women at high risk for
preeclampsia:
(i) Maternal serum concentrations of placental growth factor (PlGF) level are generally low in
preeclampsia.
(ii) Low-dose aspirin reduces adverse pregnancy outcome such as PE and delivery before 34 weeks
of gestation in pregnant women with unexplained elevated levels of alpha-fetoprotein (AFP)
[167,168].
(iii) Normotension in the first trimester is associated with reduced risk of PE [169].
(iv) In a randomized controlled clinical trial conducted by Asemi Z. et al., low dose aspirin (80 mg)
was administered with calcium supplementation (500 mg) in pregnant women who were at risk
for PE. The treatment was continued for nine consecutive weeks before measuring high
sensitivity C-reactive protein (hs-CRP), total antioxidant capacity (TAC), total glutathione (GSH)
Int. J. Mol. Sci. 2019, 20, 4370 12 of 25

in plasma and serum glucose and insulin level. The study showed a significant difference in
serum hs-CRP level and increased levels of plasma TAC and total GSH in pregnant women at
risk for preeclampsia as compared to those that took placebo (did not receive any treatment),
but serum insulin levels were not affected at all [170].

9. Extracellular Vesicles (EVs)

Extracellular vesicles (EVs) are mediators that can modify the function of target cells by
transferring proteins and genomic materials to other cells; thus, EVs have an active participation in
cell-to-cell communication [171]. EVs shred from a variety of cells and have a number of important
physiological as well as pathological functions as they are capable of trafficking and transfecting the
genetic material from cell to cell. The biogenesis and contents of EVs predominantly depends on the
originating cell type and their surrounding microenvironment [172]. Several studies using electron
microscopy analysis to characterize the morphology of EVs demonstrate that are spherical with lipid
bilayer membrane [173,174]. The correct classification of EVs still a manner of debate and the majority
of the information in the literature classify them according to their size and the different biogenesis
pathways. Typically, EVs are categorized as exosomes (~40–100 nm), microvesicles (~100–1,000 nm)
and apoptotic bodies (~1,000–5,000 nm) based on their size and origin. Microvesicles and apoptotic
bodies are formed directly via budding of the plasma membrane, whereas exosomes are produced
via an endocytic pathway [174]. Distinction between different EVs subgroups is difficult, due to the
minimal physical and morphological differences, to the lack of specific markers, and to the fact that
the same cellular source may dynamically produce different class of EVs in response to different
conditions [175]. Recently, the international society of extracellular vesicles has recommended
classifying the vesicles according to the their size in small EVs (<100–200 nm) and medium/large EVs
(>200nm), or density (low, middle, high, with each range will defined) or their biochemical
composition (e.g., CD63+ve) [176]. Currently, there is no single method allowing for accurate
characterization and discrimination of the different EVs classes [175]. EVs can be ordinarily isolated
from different biological fluids using the differential and buoyant density centrifugation methods
followed by ultrafiltration/size exclusion chromatography or flow cytometry or precipitation using
polymers or antibodies to enrich the pure EVs population [177]. EVs play an important role in cell-
to-cell communication and influence a variety of cellular functions, including cytokine production
modulation, cell proliferation, apoptosis and metabolism, by transferring their protein, lipid or
messenger RNA and micro RNA molecules [173,178]. EVs can be isolated both biological fluids (e.g.,
plasma/serum, urine, cerebrospinal fluid, saliva, etc.) and in vitro from cell-conditioned media.
Moreover, as EVs are natural carriers of bioactive molecules, different research studies are addressing
the therapeutic potential of EVs due to their specific genetic material packaging capabilities [175].
Several groups have identified EVs in maternal biofluids during normal and complication of
pregnancies and the potential role of EV during pregnancy have been reviewed in details by our
group previously [179–182].

10. Extracellular Vesicles/Exosomes in Normal and Preeclamptic Pregnancies

Different research studies utilized a variety of experimental models (i.e., biological fluids,
primary placental trophoblasts, trophoblast cell line, placental explant, placental perfusate etc.) to
isolate different subpopulations (exosomes, microvesicles) of EVs and studied their role in the context
of healthy as well as in pathological pregnancies. Small and large EVs originating from the placenta
have been identified in maternal plasma. Concentrations of both total and placenta-derived exosomes
present in maternal circulation increase across gestation [183] and are higher in complicated
pregnancies (such as those affected by PE) as compared to normal pregnancies [184,185].
Interestingly, the global miRNA profile within small vesicles such as exosomes differs between
normal and PE pregnancies across gestation and it is likely that PE is not only associated with changes
in the circulating levels of exosomes, but also in their miRNA content [186]. Recently, Biro et al.
identified that hsa-miR-210 level increased in the circulating exosomes isolated from PE pregnancies
Int. J. Mol. Sci. 2019, 20, 4370 13 of 25

[187]. Poor placentation is associated with hypoxia and oxidative stress, which are features of PE and
affects the invasion of extravillous trophoblast (EVT) and the uterine spiral arterial remodeling.
Truong et al. studied whether low oxygen tension alters exosome release and the exosomal miRNA
profile from HTR-8/SVneo cell line and examined their interaction with endothelial cells [188]. HTR-
8/SVneo cells are commonly used as a model for EVT cells, although they are not ideal, as they contain
a heterogenous population of trophoblast and stromal cells [189]. In this study, low oxygen tension
to exosomes from EVTs cultured under normoxic conditions. Moreover, a specific set of miRNAs
within exosomes from EVTs cultured under hypoxia were identified, and these miRNAs are present
in circulating exosomes at early gestation from women who develop PE later in pregnancy. This data
suggests that aberrant extracellular vesicle signaling is one of the common factors in the development
of PE. In normal healthy pregnancy, syncytiotrophoblast derived EVs release into the maternal blood
stream where they act upon their target endothelial cells and circulating immune cells [33,190–192].
Placental EVs carry different proteins, lipids and nucleic acids that play a crucial role in feto-maternal
communication to maintain pregnancy [193]. Interestingly, concentrations of large EVs gradually
increase through pregnancy irrespective of their origin [186] and these EVs convey pro-inflammatory
and pro-thrombotic antigens that might contribute to the hypercoagulable state observed in the last
trimester of pregnancy [186]. Chang et al. identified that high levels of preeclamptic exosomes contain
abundant sFlt-1 and sEng that can induce vascular dysfunction as these proteins were captured by
vascular endothelial cells [194]. Tannetta et al. investigated the level of expression of placental protein
13 in syncytiotrophoblast derived extracellular vesicles (STBEVs) isolated from PE and normal
pregnancy placental perfusate and found it was low in PE placenta [195]. Tong et al. described a novel
mechanism by which placental EVs can attenuate PE pathogenesis in the presence of
antiphospholipid antibody (aPL), which can induce the synthesis of toll-like receptors on placental
EVs to increase the level of expression of mitochondrial DNA in these vesicles [196]. Thus, placenta-
derived EVs are involved in gene regulation, placental homeostasis and cellular function that overall
reflect the placental-maternal crosstalk [197]. Placental exosomes were also observed in fetal blood
and their concentration correlated with fetal growth [198]. The concentration of placental exosomes
in the fetal circulation was higher than that found in the maternal circulation and was also higher in
pregnancies affected by PE [199]. Interestingly, not only the concentration of circulation exosomes in
PE is different compared with normal pregnancies, and specific changes in the protein cargo of
exosomes in PE have been identified [200].
Another recent study measured the level of different biomarkers including copeptin, annexin V
and placental growth factor in maternal serum derived microparticles at 10–14 gestational weeks in
women with PE and compared with that of normal healthy pregnancy [201]. Interestingly, the levels
of nitric oxide synthase enzyme in the STBEVs were lower in STBEVs from PE compared to normal
pregnancies [202]. In this regard, in a similar study, the levels of the protein neprilysin were increased
in EVs of PE placenta [203].
Kohli et al. identified a novel pathway by which the placental EVs interact and causes release of
EVs from endothelial cells and platelets that further activate the inflammasome in the trophoblast
resulting in the development of PE [204]. The role of EVs in relation to PE pathophysiology including
their different contents has been summarized in Table 2.
Int. J. Mol. Sci. 2019, 20, 4370 14 of 25

Table 2. Updated Research Studies on EVs in PE Pathophysiology.

Gestational Pregnancy
EVs Sample Type Isolation Method Biological Process/Results Reference
Age Condition
Maternal Blood Stream and other Body Fluids
Trophoblast derived Plasma and HTR-8 cell
Membrane affinity spin This micro RNA is responsible for PE
exosomal micro RNA (has- culture conditioned Third trimester Normal and PE [187]
column method pathogenesis
miR-210) media
Third trimester
Exosomes Plasma (before cesarean Commercial kit (ExoQuick) Normal and PE Vascular dysfunction [194]
section)
Placental mesenchymal
During
stem cells culture Ultracentrifugation Normal Pregnancy High level of exosomal miRNA-136, 494,
Exosomal micro RNAs cesarean section [205]
conditioned media and followed by Real Time PCR (NP) and PE 495 in PE
delivery
peripheral blood
Healthy non- Phosphorylation of renal tubular sodium
Urinary Exosomal proteins Urine After 20 weeks Centrifugation pregnant, Normal transporter proteins that enhance sodium [206]
pregnancy, PE reabsorption in PE compared to NP
Human umbilical cord Flow cytometry based
Effect on placental tissue morphology and
Exosomes mesenchymal stem cells After delivery detection of MSC surface PE [207,208]
angiogenesis in rat PE placenta
(MSC) markers
Placental syncytiotrophoblast Following
Normal and PE Lower level of placental protein 13 was
derived extracellular vesicles Placental perfusate cesarean section Centrifugation [195]
pregnancy found in STBEVs of PE placenta
(STBEVs) delivery
Presence of antiphospholipid antibody
Cultured human
Placental extracellular First trimester Sequential centrifugation increases the level of mitochondrial DNA
placental villi explant Normal pregnancy [196]
vesicles placenta and ultracentrifugation in the placental EVs and increases the risk
and Maternal serum
to develop PE
Serum copeptin, annexin V were higher
Microparticles Maternal serum 10–14 weeks Centrifugation Normal, PE, IUGR [201]
and placental growth factor was low in PE
Melatonin is secreted from placental
Placental explant and First trimester explant that reduce PE sera induced
Macovesicles/placental debris Centrifugation PE [209]
maternal serum (8–10 weeks) production of endothelial cell activating
placental EVs
First trimester
Transthyretin is increased in amount and
Nanovesicles Placenta and term Differential centrifugation PE [210]
incorporated in placental nanovesicles
placenta
EVs were stained for PE and Nephrin protein was packaged in
EVs Urine Maternal urine annexin, nephrin and Normotensive increased amount in urinary EVs of PE [211]
podocin proteins pregnant women women
Int. J. Mol. Sci. 2019, 20, 4370 15 of 25

Syncytiotrophoblast derived Placental perfusion and Gestational age Less nitric oxide synthase in STBEVs of PE
Differential centrifugation Normal and PE [202]
extracellular vesicles (STBEV) maternal plasma matched women
First and Endothelial dysfunction in severe early
EVs Placental explant second Sequential centrifugation Normal and PE onset PE is via soluble angiogenic factors, [212]
trimester not by EVs
Differential centrifugation,
First, second The concentration of exosomes is higher
ultracentrifugation
Exosomes Maternal plasma and third Normal and PE and miRNA content is different in PE [184]
followed by density
trimester compared to normal pregnancy
gradient centrifugation
Increase MP shedding from PE placenta;
At term (>37 Uncomplicated and upregulation of caveolin-1 and
Microparticles Placental trophoblasts Two-step centrifugation [213]
weeks) preeclamptic downregulation of eNOS in these MPs
which is modulated by vitamin-D
Endothelial cells and Inflammasome activation in placental
EVs Not mentioned Differential centrifugation Normal and PE [204]
Platelets trophoblasts results in PE development
Fetal Circulation
Differential Centrifugation No difference in concentration of exosomes
Exosomes Umbilical cord blood At delivery + Density gradient Normal in term, small for gestational age, fetal [198]
centrifugation growth restricted neonates
MPs were identified by size
and annexin V fluorescein MP levels is higher compared to maternal
Microparticle (MPs) Umbilical cord blood At delivery Normal and PE [199]
isothiocyanate (FITC) blood in PE
labelling
Differential centrifugation Altered protein expression profile that are
Exosomes Umbilical cord blood At delivery Normal and PE [200]
+ Filtration involved with PE etiology
Int. J. Mol. Sci. 2019, 20, 4370 16 of 25

A number of drugs that can be used to treat PE appear to modulate EV expression. Some studies
also addressed the mode of action of different antihypertensives, including thiazide diuretics that are
used to treat the hypertension in PE. Hu et al. identified some changes in the sodium transporters in
the renal tubule that were incorporated in the urinary exosomes isolated from PE women [206].
Another very interesting study by Chamley L. et al. identified melatonin as an effective agent that
can reduce the endothelial cell activating placental EVs release in PE [209]. In a similar study,
transthyretin which is the thyroxin binding protein, was found in aggregated form and packaged in
the small placental EVs in PE [210]. Xu et al. identified potential molecular mechanisms by which
vitamin-D can reduce oxidative-stress induced PE [213]. Among the different therapeutic agents, the
efficacy of aspirin was evaluated due to its availability and cost-effectiveness. However, there is lack
of understanding in the mechanism of action of aspirin in the context of EV secretion regulation.

11. Effects of LDA on Exosomal Secretion

Up until now, there has been very limited evidence on the potential effect of aspirin on the
release and content of EVs. Goetzl E.J. et al., discovered that in the presence of some coagulation
factors (e.g., thrombin/ collagen) induces changes in the plasmatic levels of platelet-derived exosomes
and their protein content (i.e., α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-
mobility group box 1 (HMGB1)) [214]. Incubation of normal platelets with aspirin significantly
inhibits arachidonic acid (AA)-induced platelet reactivity, EV formation and pro-coagulant activity
[215]. Interestingly, aspirin therapy can significantly reduce microparticle (MP) shredding from
erythrocytes, monocytes and vascular smooth muscle cells, reversing the effects of diabetes-induced
stress on these cells [216]. Other studies have identified that aspirin changes the miRNA profile and
EV release from platelet [217]. Syncytiotropholast-derived extracellular vesicles that are placental
alkaline phosphatase (PLAP) positive inhibit the aggregation of platelets that were treated with
aspirin [218]. Tannetta et al. observed that STBEV that are placental alkaline phosphatase (PLAP)
positive inhibit the aggregation of platelets that were treated with aspirin [218]. On the contrary,
platelets were activated and thrombus formation was increased by the STBEV isolated from
preeclamptic placentas. Another study observed the effect of anticoagulant therapy (treatment with
either unfractionated heparin (UFH) or low molecular weight heparin (LMWH), and/or LDA) on cell
derived microparticles and outcome of pregnancy [219]. These findings indicate that placenta-
derived extracellular vesicles may provide understanding in their potential role in low dose aspirin
induced placental functions. Although there is significant evidence for dysregulation of both
concentrations and bioactivity of circulating placental EVs in PE compared to normal pregnancies,
no studies have described the potential effect of aspirin on EVs released from placental cells and their
bioactivity.

12. Summary
Several studies focused on EVs (mainly small EVs called exosomes) highlighting their
extraordinary characteristics as natural carriers of bioactive molecules, which can be used as
biomarker for several pathological conditions including PE. These vesicles are unique in terms of
their cell trafficking and transfecting capabilities. These nanovesicles are released from almost all
types of cells into different human body fluids. Their release and contents are dependent on the
microenvironment where the cells are exposed and the origin of the cells [172]. Therefore, EVs can be
used as a diagnostic tool as well as prognostic marker for several pathologies, and we have proposed
that the analysis of placental vesicles in maternal plasma can function as a liquid biopsy to establish
placental function during pregnancy. During pregnancy, placenta and other cells, such as platelets
and immune cells, secrete exosomes into the maternal circulation; this process is exaggerated in
pathological pregnancies (i.e., PE, PIH, IUGR) in an attempt to modulate the pathology [43,190,191].
Very few studies [218,219] have been conducted to identify the particular mechanism of action that
exosomes can produce on the placenta and overall maternal physiological system when treated with
low dose aspirin and other antithrombotic medication. An avenue is open to explore the placental
Int. J. Mol. Sci. 2019, 20, 4370 17 of 25

and other cell derived exosomal functions in placental dysfunctional disorders when treated with
antithrombotic medications including low dose aspirin. A better understanding of these processes
may lead to the development of novel prognostic markers utilizing placenta specific exosomes or for
monitoring the response to aspirin treatment for placental pathologies.
Funding: Dr. Carlos Salomon is supported by The Lions Medical Research Foundation, National Health and
Medical Research Council (NHMRC; 1114013) and the Fondo Nacional de Desarrollo Científico y Tecnológico
(FONDECYT 1170809). Suchismita Dutta received the Australian postgraduate award scholarship.

Conflicts of Interest: The authors declare no conflict of interest.

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