R E V I E W Open Access
Recent insights on indirect mechanisms in
developmental toxicity of nanomaterials
Battuja Batbajar Dugershaw
, Leonie Aengenheister
, Signe Schmidt Kjølner Hansen
Karin Sørig Hougaard
and Tina Buerki-Thurnherr
Background: Epidemiological and animal studies provide compelling indications that environmental and
engineered nanomaterials (NMs) pose a risk for pregnancy, fetal development and offspring health later in life.
Understanding the origin and mechanisms underlying NM-induced developmental toxicity will be a cornerstone in
the protection of sensitive populations and the design of safe and sustainable nanotechnology applications.
Main body: Direct toxicity originating from NMs crossing the placental barrier is frequently assumed to be the key
pathway in developmental toxicity. However, placental transfer of particles is often highly limited, and evidence is
growing that NMs can also indirectly interfere with fetal development. Here, we outline current knowledge on
potential indirect mechanisms in developmental toxicity of NMs.
Short conclusion: Until now, research on developmental toxicity has mainly focused on the biodistribution and
placental translocation of NMs to the fetus to delineate underlying processes. Systematic research addressing NM
impact on maternal and placental tissues as potential contributors to mechanistic pathways in developmental
toxicity is only slowly gathering momentum. So far, maternal and placental oxidative stress and inflammation,
activation of placental toll-like receptors (TLRs), impairment of placental growth and secretion of placental
hormones, and vascular factors have been suggested to mediate indirect developmental toxicity of NMs. Therefore,
NM effects on maternal and placental tissue function ought to be comprehensively evaluated in addition to
placental transfer in the design of future studies of developmental toxicity and risk assessment of NM exposure
Keywords: Nanomaterials, Developmental toxicity, Indirect toxicity pathways, Placental barrier, Pregnancy
Since the thalidomide scandal in the early 1960s, it has
become evident that the placenta does not provide a
tight barrier, and that fetuses are exceptionally suscep-
tible to potentially toxic substances compared to adults,
due to the phases of rapid growth, range of developmen-
tal events and often irreversible nature of the induced
changes . The first indications of developmental
toxicity of nanosized particles came from epidemio-
logical studies, showing association of particulate matter
(PM) exposure with adverse pregnancy outcomes such
as low birth weight, preterm birth and preeclampsia [2–4].
Recently, it has been confirmed that environmental black
carbon reaches the fetal side of the placenta in exposed
pregnant women . With the advent of nanotechnology,
novel NMs with unique properties can be industrially pro-
duced at large scales for application in food (reviewed in
[6,7]), cosmetics (reviewed in [7,8]), medicine (reviewed
in [9,10]) and high-technology products (reviewed in [10,
11]). These engineered NMs further contribute to human
exposure to nanosized particles, and due to their high
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* Correspondence: firstname.lastname@example.org
Laboratory for Particles-Biology Interactions, Empa, Swiss Federal
Laboratories for Materials Science and Technology, Empa, Lerchenfeldstrasse
5, 9014 St.Gallen, Switzerland
Full list of author information is available at the end of the article
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31
reactivity, pose additional health risks. However, investiga-
tions of the toxicological effects of engineered NMs, espe-
cially in vulnerable populations such as pregnant women
and their unborn children, have lagged behind the deve-
lopment of new applications. Importantly, to support safe-
by-design and sustainable use of NMs, it is imperative to
gain knowledge on the potential developmental toxicity of
NMs and to understand the mechanisms underlying such
In principle, NMs can affect fetal development
through two fundamentally different pathways: a direct
and an indirect pathway  (Fig. 1), that, however, are
not mutually exclusive. Direct developmental toxicity
may arise from particles in maternal blood that cross the
placental barrier [13–15] and directly damage fetal tis-
sues due to their high surface reactivity and propensity
to induce inflammation [16–18], reactive oxygen species
(ROS)  and hence oxidative stress reactions [20–22],
among others. Several FNMs are able to cross primary
biological tissue barriers (e.g., lung [23,24] and
gastrointestinal (GI) tract [24,25]) as well as the pla-
centa [26–29], even if translocation is usually rather
limited [30,31]. Direct effects on embryonic and fetal
tissues have been described for a variety of NMs in sev-
eral in vitro studies as well as across species, including
fish, chicken, and in vitro human stem cell (SC) models
(reviewed in ). However, findings from organisms
that lack a placenta or have a distinctly different placen-
tal structure might not directly correlate to the human
The potential for NMs to affect fetal development by
indirect pathways has been only marginally investigated
and understood. Here, the concept is that NMs can
interfere with fetal development in an indirect manner
without being in direct contact with fetal tissue (Fig. 1).
NMs deposited in primary maternal tissue barriers at
the point of entry following oral, inhalation, dermal or
intravenous (i.v.) exposure might induce oxidative stress
and subsequently inflammation, leading to the release of
inflammatory mediators and soluble signaling factors
that can reach the placenta and fetus to induce potential
toxic effects (maternal mediated developmental toxicity).
Alternatively, particles reaching the placenta can cause
similar responses in the placental tissue, compromising
Fig. 1 Scheme illustrating direct and indirect pathways of NM-mediated developmental toxicity
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 2 of 22
placental function and inducing the release of placental
signaling factors, which might impair embryo-fetal de-
velopment (placental mediated developmental toxicity).
The aim of this review is to (i) collect the current
knowledge base on the indirect developmental toxicity
of NMs, (ii) compile and describe already known signal-
ing pathways, (iii) propose novel candidate pathways and
(iv) suggest directions of future research needs.
Risks and opportunities of NMs in pregnancy
For a proper risk assessment of NMs, a central aspect is
to understand the exposure of pregnant women to NMs,
including all relevant routes of exposure . Due to the
use of NMs in many consumer, high-technology and
biomedical products, pregnant women could be exposed
to NMs via inhalation, absorption through damaged
skin, ingestion or injection (Fig. 1) (reviewed in [34,35]).
At production sites with applications of NMs, pregnant
women can be exposed to NMs by inhalation, since the
established protective legislation  does not come into
action until the employer is made aware of the preg-
nancy, most often not until after the first 4–6 weeks.
Even then, NM exposure might continue, as the regula-
tion does not specifically regulate NM relative to preg-
nancy [37,38]. Ingestion of NMs used as food additives,
in food packaging material or personal care products,
constitutes another realistic route of exposure during
pregnancy. For example, the white food colorant E171
consists of particulate titanium dioxide (TiO
approximately 17–35% of the particles being within the
nano-range (reviewed in [7,39,40]), and is present in
toothpaste and various food products such as beverages,
soups, cakes or candy in the European Union [41,42]. In
the United States, the dietary intake of TiO
to be 1–2 mg/kg body weight per day for children, and
0.2–0.7 mg/kg body weight per day for other age groups
[7,42]. Dermal uptake of NMs present in personal care
products, such as sunscreen, is expected to be minimal
since the intact skin forms a tight barrier for NMs
(reviewed in ). Finally, particles may be directly
injected into the body in case of medical application of
NMs (reviewed in [9,44,45]), but currently, nano-
medical therapies during pregnancy are still in the
investigational stage. For instance, King et al. demon-
strated the potential of iRGD (9-amino acid cyclic pep-
tide: CRGDKGPDC)-decorated liposomes loaded with
insulin-like growth factor (IGF)-2 for the treatment of
fetal growth restriction in mice . An oxytocin recep-
tor coated liposomal carrier loaded with the tocolytic
drug indomethacin substantially decreased preterm birth
rates in mice . Nevertheless, before clinical use in
pregnant women, not only the efficacy of the potential
treatment in humans but also the safety of the NMs dur-
ing pregnancy needs to be proven.
I.v. injection would make NMs readily systemically
available. In contrast, only a low fraction of air and food-
borne NMs would be expected to reach the systemic cir-
culation and become bioavailable for maternal, placental
and fetal tissues. Dermal exposure is expected to con-
tribute very little to the systemic burden [27,31]. Once
NMs have reached the systemic circulation, they can
distribute to maternal organs, including the placenta. As
a highly perfused organ, the placenta is extensively ex-
posed to circulating substances. Placental cells have been
described to take up nanosized particles from the blood
stream in experimental animals as well as the ex vivo
human placenta perfusion model (e.g. [48–51]). Studies
on placental translocation of NMs in rodents, in the hu-
man ex vivo and in in vitro placenta models have shown
that some types of NMs are retained in the maternal cir-
culation while others can pass the placenta (reviewed in
[26,52]). Placental transfer appears to partially correlate
withphysicochemical properties of NMs, in particular
particle size . However, other factors such as the ges-
tational stage or combined physico-chemical properties
can also affect placental translocation of NMs, making
this process difficult to predict . As an example, a re-
cent study demonstrated decreased fetal viability and
growth, when 13 nm zinc oxide (ZnO) NPs were orally
administered (7.2 mg/mouse) during organogenesis (ges-
tational day (GD)7–16) in mice. However, when ZnO
NP exposure occurred during the peri-implantation
period (GD1-GD10) no fetal toxicity, but a slight change
in placental weight, was observed .
For most routes of uptake (inhalation, ingestion and
injection), gestational NM exposure has been associated
with developmental toxicity for a variety of different
NMs (extensively reviewed in [36,55–58]).However, we
have yet to identify the underlying mechanisms and
which particle properties are of particular concern.
Organ systems of relevance for pathways of indirect
For sure, the placenta should be a key focus in any
mechanistic study on NM-mediated developmental tox-
icity due to its position at the interface between mother
and fetus and its numerous essential functions during
pregnancy. As a transient organ, the placenta starts
forming after implantation of the conceptus in the uter-
ine wall. It consists of tissues of maternal (decidua) and
fetal origin (amnion, chorion) [59,60]. Anatomically, the
maternal side of the placenta comprises the multinuclear
syncytiotrophoblast (ST) layer, which is supported by a
basal membrane, underlying cytotrophoblast cells, mes-
enchymal tissue and the microvascular endothelium of
the fetal small blood vessels (Fig. 2). This interface be-
tween the inner mucous membrane of the uterus (endo-
metrium) and the fetus defines the degree to which
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 3 of 22
maternally delivered substances reach the fetal tissue
. During pregnancy, the placenta undergoes dramatic
structural and functional changes to fulfill the evolving
needs of the developing fetus. During early pregnancy,
the placental barrier is relatively thick (20–30 μm) and
bilayered [62–64], but thins (2–4μm) , becomes
predominantly monolayered [62–64], and increases its
surface area tremendously (to approx. 12 m
the end of pregnancy to allow for efficient exchange of
nutrients and gases required to sustain rapid fetal
growth. Placental damage, disease or impairment of its
development or function are responsible for numerous
pregnancy complications, including preeclampsia ,
miscarriage [63,67] and intrauterine growth restriction
[63,67], and can likely impact offspring health later in
life . It should also be highlighted that the placenta
is the most species-specific organ among mammals and
shows remarkable differences in global structure, tissue
layer organization, trophoblast cell types [69,70] as well
as molecular features . Therefore, translation from
animal studies to the human situation should be done
with caution, and the use of physiologically relevant
placenta models is encouraged.
Also, maternal organs could mediate indirect develop-
mental toxicity of NMs. Here, a focus should be on
tissues at the port of entry that are in direct contact with
particles such as the lung, the skin or the GI tract upon
inhalation, dermal deposition or oral exposure, respect-
ively. Uptake and accumulation of NMs in these tissues
could affect organ functions locally, but effects may
spread to distant sites, including the placenta or the de-
veloping fetus, if particles interfere with essential signal-
ing pathways. This concept is nicely exemplified in a
recent study in mice, where systemic adverse effects (i.e.
increased retention of activated leukocytes, secondary
thrombocytosis, and pro-inflammatory responses in
secondary organs) were observed only upon inhalation
exposure to carbon NPs, but not after intra-arterial
injection of an equivalent dose of particles to bypass the
lung . The mechanism(s) underlying the observed
indirect systemic toxicity of carbon NPs appeared to
involve inflammatory responses of the lung tissue .
In addition to pro-inflammatory actions, NMs may also
Fig. 2 Scheme of the human placental barrier in early and late pregnancy. In the first trimester, the placental barrier consists of the
syncytiotrophoblast (ST), cytotrophoblasts (CT), basal lamina (BL) and the endothelial cells (E) of the fetal capillaries (FC). Other cell types in the
villous mesoderm include fibroblasts (F) and Hofbauer cells (HC). Various immune cells are also present in the maternal decidual tissue, including
dendritic cells (DC), macrophages (MP), uterine natural killer cells (uNK), T cells (TC) and B cells (BC). Extravillous trophoblasts (EVT) of the
anchoring villi invade the maternal spiral arteries (SA) and form a plug that prevents entry of maternal blood into the intervillous space, and
uterine glands (UG) provide histiotrophic nutrition. After the first trimester, the EVT plug is released and placental villi are now surrounded by
maternal blood. Towards the end of pregnancy, the placental barrier decreases in size by thinning of the ST layer and spreading of the CT layer,
and the FCs move towards the periphery of the floating villi
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 4 of 22
interfere with essential functions of the lung, skin or GI,
such as gas exchange, digestion, nutrient uptake, meta-
bolism or transport (Fig. 1). For instance, ZnO NPs can
reduce iron and nutrient uptake and transfer at the
intestinal barrier [73,74].
Evidence for indirect developmental toxicity
To gather an overview on potential indirect pathways in
developmental toxicity, we searched the open literature
for studies reporting adverse effects of NMs on gestation
and fetal development in the absence of detectable
materno-fetal particle transfer (Table 1). However, since
direct and indirect toxicity pathways may jointly contrib-
ute to developmental toxicity, we also included studies
that provide hints for indirect toxicity pathways even if
placental transfer of NMs was detected (Table 2) or un-
known (Table 3). The studies are briefly described below
alongside the provided evidence and forwarded hypoth-
eses for indirect mechanisms of toxicity.
Studies without detectable placental particle transfer
We identified a total of ten studies that reported devel-
opmental toxicity in the absence of detectable NM
translocation across the placental barrier (Table 1). Most
used pregnant mice as the experimental model, but a
few studies used in vitro cell culture systems for more
mechanistic studies. Gestational and litter parameters
were affected in four of the murine studies, including re-
duced survival rate of offspring from dams inhaling cop-
per oxide (CuO) (3.5 mg/m
at GD 3–19) ,
decreased fetal size and delayed neonatal growth from
cadmium oxide (CdO) NP inhalation (100 μg/m
at GD 4.5–16.5) , and increased fetal re-
sorption and malformations following maternal exposure
to SWCNTs by the oral (10 or 100 mg/kg body weight
at GD 9)  and i.v. route (10 ng to 30 μg/mouse at
GD 5.5) . Other studies described effects on placen-
tal structure and function, offspring lung development
and function and neurodevelopment. Regarding the pla-
centa, intratracheal instillation of TiO
and cerium diox-
) NPs (total 300 μg/mouse: 100 μg on GD 2.5,
9.5 and 16.5, respectively) decreased placental efficiency
, injection of CdO NPs altered placental weight 
and injection of SWCNTs induced vascular lesions and
increased placental level of ROS . Gestational NM
exposure can also affect maternal and fetal lungs as
exemplified by maternal lung inflammation induced by
inhalation of TiO
on GD 8–18)  or CuO
NPs (3.5 mg/m
on GD 3–19) , or long-lasting
impairment of lung development in the offspring result-
ing from maternal intratracheal instillation of TiO
NP . Neurodevelopmental abnormalities, like
reactive astrogliosis and increased DNA damage in the
fetal hippocampus, have been observed after injection of
cobalt-chrome (CoCr) NPs into pregnant mice on GD 9
(0.12 mg per mouse) . In a similar direction, both
maternal inhalation with 42 mg/m
(GD 8–18)  and
injection with 1000 μg/mouse (GD 9)  of TiO
caused behavioral deficits in the offspring.. Finally, im-
munomodulatory effects were reported upon CuO inhal-
ation . Importantly, NM translocation to the fetus
was addressed but not observed in these studies, there-
fore strongly supporting the presence of indirect toxicity
pathways. However, it is conceivable that a small
amount of particles might have crossed the placental
barrier, which were below the detection limit of the ap-
plied analytical techniques (i.e. ICP-MS, gAAS, TEM,
histological and micro-Raman analysis), as for example
suggested by Hougaard et al., 2010 . Moreover, for
soluble NPs (e.g. CuO), placental translocation of small
quantities of dissolved ions might also partially account
for developmental toxicity even in the absence of par-
ticle transfer. Nevertheless, the adverse effects upon
CuO inhalation in mice observed by Adamcakova-Dodd
et al.  were not associated with detectable increase in
fetal or maternal blood Cu levels. Proposed pathways for
indirect developmental toxicity included both placenta-
and maternally mediated secondary mechanisms. Mater-
nally mediated pathways comprised oxidative stress,
inflammatory, immune and endocrine responses [75,77,
78,83,84], whereas placental mediated pathways in-
volved oxidative stress, inflammation, placental insuffi-
ciency, release of mediators (e.g., ATP, IL-6) and
changes in placental transport of zinc, vitamin B12,
micronutrients or oxygen [76,79–88]
Studies with placental particle transfer
Several publications suggested a role for indirect devel-
opmental toxicity of NMs even if particles in some cases
were shown to cross the placental barrier and adverse
effects could have resulted from direct embryo-fetal
exposure (Table 2). The gestational and litter parameters
described in these studies include increased rate of mis-
carriage from quantum dot (QD) injection , smaller
fetuses and increased fetal resorption from silica dioxide
) and TiO
NP injection , growth retardation
from diesel exhaust particle (DEP) inhalation or
multi-walled carbon nanotube (MWCNT) injection 
and fetal organ damage from QD  or SWCNT/
MWCNT injection [94,95]. The maternal parameters
reported were decreased maternal body weight upon
NP injection (0.8 mg/mouse on GD 16
and 17)  and hepatocellular injury from QD injection
(100 mg/kg body weight on GD 17 in mice and 25 mg/
kg on GD 100 in monkeys) . Paul et al. observed
long-lasting impairment of lung development in off-
spring of pregnant mice intratracheally instilled with sil-
ver (Ag) NPs (total 300 μg/mouse: 100 μg on GD 2.5, 9.5
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 5 of 22
Table 1 Studies with evidences for indirect fetotoxicity pathways without placental transfer of NMs
NP size exposure/model application
and litter parameters)
developmental toxicity (other
hypothesis by authors on indirect
5–6 nm mouse i.v./ 100 or
in fetus or
no overt fetal
impact on postnatal
behavioral deficits relevant to ASD
and related neurodevelopmental
disorders in neonates
maternal mediated unknown
pathways due to absence of
particles in placenta and fetal
12.3 ± 0.1/ 22.4 ±
total 300 μg/
2.5, GD 9.5
and GD 16.5)
Ti and Ce
but not in
not evaluated long-lasting impairment of lung
development in offspring/
decreased placental efficiency
together with the presence of NPs
in the placenta/ no increase of
inflammatory mediators in amniotic
fluid, placenta or offspring lungs/
decreased pulmonary expression of
VEGF-αand MMP-9 at the fetal
stage (GD 17.5) and FGF-18 at the
alveolarization stage (postnatal day
probably involves placental
insufficiency secondary to the
presence of NPs in this organ with
ensuing down regulation of critical
mediators of lung development
without any amniotic fluid or fetal
lung inflammation/ not mediated
via fetal or maternal lung
20.6 ± 0.3 nm mouse inhalation/ 1
h/day to 42
in fetal liver
no impact on
gestational and litter
deficits/ persistent lung
inflammation in pregnant dams
dissolution and translocation of
contaminating metal ions/
placental transfer of inflammatory
cytokines released from NP-
exposed maternal lung tissue
CuO 16 nm mouse inhalation/
4 h/day/ GD
levels in pla-
survival rate of 7
week old pups
reduced/ no impact
on litter size, male/
female ratio, body
weight and lenght at
maternal pulmonary inflammation/
no histopathological changes of
immunomodulatory effects in
offspring (differential expression of
several Th1/Th2 or other immune
response genes in spleen)
changes in maternal inflammatory
and immune responses
CdO 11–15 nm mouse inhalation/
day or 230
for 2.5 h/ GD
but not in
fetus by gAAS
altered placental weight disruption in placental oxygen
transfer by Cd / decrease in
fetal length could be due to
alterations in the fetal and/or
maternal IGF system [80,81]/
changes in the placental transport
of zinc, vitamin B12, and other
micronutrients due to placental Cd
1–2 nm diameter
and 5–30 μm
mouse oral/ 10 mg/
kg or 100 mg/
kg/ GD 9
fetal liver and
resorption and fetal
at 10 mg/kg but not
none oxidative stress and inflammatory
response in placenta/maternal
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 6 of 22
Table 1 Studies with evidences for indirect fetotoxicity pathways without placental transfer of NMs (Continued)
NP size exposure/model application
and litter parameters)
developmental toxicity (other
hypothesis by authors on indirect
at higher dose
2.37 nm diameter,
0.85 μm length/
1.58 nm diameter,
0.76 μm length/
1.8 nm diameter,
0.37 μm length
mouse i.v./ 10 ng to
high percentage of
lowest effective dose
vascular lesions and increased ROS
in placenta/ increased ROS in
malformed fetuses/ no increased
ROS or evident morphological
alterations in maternal tissues
oxidative stress in placental tissue 
CoCr 29 nm BeWo Transwell
40 μg/ml/ 24
and Cr levels
in whole fetus
not applicable DNA damage to the fibroblasts
without significant cell death/
mechanism involving transmission
of purine nucleotides (e.g. ATP) and
intercellular signalling within the
placental barrier through connexin
gap junctions or hemichannels and
fetal damage mediated by
placental tissue via release of
mediators (e.g. ATP)
CoCr 29 nm Bewo Transwell
mono- or bilayers
with underlying BJ
40 μg/ml / 24
not applicable DNA damage to fibroblasts or Oct4-
hES cells only with BeWo double
indirect toxicity only across
(mice) placental barrier
mouse i.v./ 0.12 mg or
9.5 or 12.5
and Cr levels
in whole fetus
changes in neonatal
DNA damage in neonatal blood
and liver at GD 12.5 (placenta with
three layers established) but not at
GD 9.5 (nutrient exchange via
uterus and yolk sac)/ no
pathological changes in placenta
CoCr 29 nm Bewo Transwell
media transfer to
NPC or NPC-
40 μg/ml / 24
not applicable altered differentiation of human
NPC and DNA damage in the
derived neurons and astrocytes/
importance of autophagy and IL-6
release from placental tissue in NP-
induced DNA-damaging singalling/
NPs can cause developmental
neurotoxicity across placental bar-
riers/ astrocytes are key mediators
of this neurotoxicity/ fetal hippo-
campus is particularly affected in
exposure of the human placenta to
CoCr NPs could initiate a singalling
cascade that perturbs the
relationship between astrocytes
and neurons during
mouse i.v./ 0.12 mg /
ASD autism spectrum disorders, gAAS graphite furnace atomic absorption spectroscopy, FGF-18 fibroblast growth factor 18, GD gestation day, ICP-OES inductively coupled plasma optical emission spectrometry, IGF
insulin growth factor, i.v. intravenous, MMP-9 matrix metalloproteinase 9, NP nanoparticles, NPC neural progenitor cells, ROS reactive oxygen species, TEM transmission electron microscopy. VEGF-αvascular endothelial
growth factor α
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 7 of 22
Table 2 Studies with evidences for indirect fetotoxicity pathways with placental transfer of NMs
NP size Exposure/
placental transfer developmental toxicity
(gestational and litter
developmental toxicity (other
hypothesis by authors on indirect
60 nm mouse i.v./ 100 mg/
kg/ GD 17
levels in umbilical
cord and fetuses
no gestational or fetal
abnormalities or complications
no significant abnormalities in
maternal blood biomarkers,
histopathology or behavior
acute hepatocellular injury and
possible stress caused by the
injection did eventually
contribute to the high
miscarriage rate in macaques
macaques i.v./ 25 mg/
kg/ GD 100
Cd levels in fetal
organs by ICP-MS
increased rate of miscarriage no pathological changes in the
placenta or major organs of the
miscarried fetuses/ no
inflammatory response or injury
in maternal liver and lung tissues/
acute maternal hepatocellular
Si and TiO
70 nm and 35 nm mouse i.v./ 0.8 mg/
16 and 17
Si and TiO
liver and brain by
decrease of maternal body
weight at GD 17/18/ lower
uterine weights/ higher fetal
resorption rates/ smaller fetuses
Si NP induced structural and
functional abnormalities in
placenta (decreased sFlt-1)/ hep-
arin improved fetal weight and
sFlt-1 levels in Si NP exposed
adverse effects are linked to
structural and functional
abnormalities in the placenta/
activation of coagulation,
complement and oxidative stress
in the placenta
Ag 12.3/ 22.4 / 10.4
GD 2.5, GD
9.5 and GD
Ag in placenta
and fetal lung by
not evaluated long-lasting impairment of lung
development in offspring/
decreased placental efficiency
together with the presence of
NPs in the placenta/ no increase
of inflammatory mediators in
amniotic fluid, placenta or
offspring lungs/ decreased
pulmonary expression of VEGF-α
and MMP-9 at the fetal stage (GD
17.5) and FGF-18 at the alveolari-
zation stage (postnatal day 14.5)
probably involves placental
insufficiency secondary to the
presence of NPs in this organ
with ensuing down regulation of
critical mediators of lung
development without any
amniotic fluid or fetal lung
inflammation/ not mediated via
fetal or maternal lung
inflammation/ combination of
direct and indirect pathways
possible due to low placental
transfer of Ag
Ag 18–20 nm mouse inhalation/
1 or 4 h/day
to 640 μg/
/ GD 0.5–
Ag in maternal
and fetus by
TEM/ no particles
or ions detected
increased number of resorbed
reduced oestrogen plasma levels
(in 4 h/day exposures)/ increased
expression of pregnancy-relevant
inflammatory cytokines in the pla-
centas/ no major pathological
changes in the lung of the
mothers and only minor lesions
in maternal liver and kidney
adverse effects at least in part
related to the release of
inflammatory mediators by the
placenta/ reduction of circulating
oestrogen levels could indicate
an endocrine disrupting action of
2–15 or 5–15 nm ex vivo
40 or 75 μg/
ml / 6 h
low levels of Ag
NPs > 25 nm in
not applicable low translocation of Ag ions and
Ag NPs (below 0.02% of initial
dose)/ considerable uptake of Ag
NPs in placental tissue (4.2% of
initial dose for AgCOONa; 0.75%
low translocation but comparably
high accumulation of ionic Ag
and Ag NPs in placental tissue
may result in indirect placenta-
mediated developmental toxicity
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 8 of 22
Table 2 Studies with evidences for indirect fetotoxicity pathways with placental transfer of NMs (Continued)
NP size Exposure/
placental transfer developmental toxicity
(gestational and litter
developmental toxicity (other
hypothesis by authors on indirect
Diesel exhaust 69 nm rabbit inhalation/
2 h/day, 5
NP observed in
fetal blood by
growth retardation reduced placental efficiency/
reduced placental vascularization/
reduced plasma insulin and IGF1
concentrations/ in second
generation, fetal metabolism was
adverse effects on placental
structure and function and
reduced plasma IGF-1 may con-
tribute to the observed growth
retardation/ effects could be due
to either NP or contaminants
mouse i.v./ 20 mg/
NPs in placental
tissue and foetal
liver, lung and
poor embryo development/
fetal growth restriction/
embryonic death/ abortion/
reduced fetal weight/ fetal heart
and brain damage
decreased progesterone levels
and increased oestradiol levels in
serum/ decreased VEGF levels
and increased ROS amounts in
placental tissue/ number of
placental blood vessels decreased
fetal growth restriction due to
vascular reduction in the
placenta/ toxicity higher in first
time pregnancies as adaptations
in the placenta may occur/
oMWCNT affect secretion of
< 8 nm, 20–30 nm
or 50 nm
2000 nm length
i.v./ 2 mg/kg
or 5 mg/kg/
12.5 or 15.5/
all CNTs in
and fetal liver by
larger sized MWCNT restricted
the development of fetuses and
induced brain deformity (only at
GD 10.5 and only in p53−/−
fetuses)/ SWCNTs and smaller
sized MWCNTs showed no or
MWCNTs directly triggered p53-
dependent apoptosis and cell
cycle arrest in response to DNA
damage/ N-acetylcysteine (anti-
oxidant) pevented CNT-induced
nuclear DNA damage andreduce
brain development abnormalities
placenta mediated toxicity
thorugh interference with
FGF-18 fibroblast growth factor 18, GD gestation day, ICP-OES/MS inductively coupled plasma optical emission spectrometry/mass spectroscopy, IGF insulin growth factor, i.v. intravenous, MMP-9 matrix
metalloproteinase 9, NP nanoparticles, PAH polycyclic aromatic hydrocarbons, ROS reactive oxygen species, spICP-MS single particle ICP-MS, TEM transmission electron microscopy; VEGF-αvascular endothelial growth
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 9 of 22
Table 3 Studies with evidences for indirect fetotoxicity pathways with unknown placental transfer of NMs
NP size Exposure/
(gestational and litter
developmental toxicity (other parameters) hypothesis by authors on indirect
21 nm rat inhalation/
burden of 525 μg/
not evaluated increased placental vascular resistance and
impaired umbilical vascular reactivity
impaired fetoplacental vascular
reactivity/ altered placental reactivity
Si 70 nm mouse i.v. injection/ 0.025 or
0.04 mg/g/ GD 13–14
increased fetal resorption and
reduced fetal weight at 0.04
particle uptake in placenta/ 0.04 mg/ml:
abnormalities in placental structure and
reduced placental weight/ nanosilica
upregulated the inflammasome component
NLRP3 and induced placental inflammation
and ROS, resulting in pregnancy
complications/ pregnancy complications
were dependent on the balance between
an inflammatory cytokine (IL-1a) and an
anti-inflammatory cytokine (IL-10)/ compli-
cations were completely prevented by ei-
ther inhibition of ROS generation or forced
expression of IL-10
placental inflammation 
2 nm rat i.p./ 5, 10 or 20 mg/
kg/ GD 13
survival rate of fetuses/
reduction of fetal body length
and mass/ disturbed
ossification of limbs
placental tissue damage (decreased
placental weight, abnormal morphological
impeded embryogenesis due to the
placental damage rather than QD
penetration and accumulation in the
fetuses/ distinct developmental toxicity
effects than upon Cd
3 nm/ 10–20
0–25 μg/mL/ 24 h not applicable reduction of β-hCG secretion at sub-lethal
interference with hormone release 
5–7 nm first
10 nM and 1 μM/ 24
not applicable charge-dependent accumulation of
particles/ no major acute toxicity but
reduced secretion of β-hCG for charged
particles at the lower concentration
potentially hazardous influences of
charged dendritic polygylcerol particles
on early placental physiology by
reduction of β-hCG hormone levels
MWCNT 13 μm length mouse i.p or intratracheally/
2,3,4 or 5 mg/kg/ GD
fetal malformations/ increased
leucocyte and related
hemocyte number and
increased weight of spleen in
none inflammatory mechanism 
CB 14 nm mouse inhalation: 42 mg/m
1 h/day/ GD 8–18
instillation: 2.75, 13.5
or 67 μg/mouse/ GD
7, 10, 15 and 18
neither inhalation nor
instillation affected gestation
DNA strand breaks in maternal and
offspring liver after inhalation but not
instillation exposure/ persistent lung
inflammation in exposed mothers
translocation across lung, GI tract and
placenta expected to be very low for
highly insoluble CB; changes in
signalling cascades proposed e.g.
CB 14 nm mouse intratracheal
instillation/ 2.75, 13.5
or 67 μg/mouse/ GD
see (Jackson 2011) changes in the expression of several genes
and proteins associated with inflammation
in maternal lungs/ hepatic response in
responses in newborns secondary to
inflammation in dams
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 10 of 22
Table 3 Studies with evidences for indirect fetotoxicity pathways with unknown placental transfer of NMs (Continued)
NP size Exposure/
(gestational and litter
developmental toxicity (other parameters) hypothesis by authors on indirect
7, 10, 15 and 18 offspring at highest dose
instillation/ 50 μg/
mouse/ GD 14
not evaluated increased allergic susceptibility in offspring components of DEP (especially PAHs)
could mediate pro-allergic effects by in-
creased production of Th2 cytokines
(e.g., IL- 4), known to be important me-
diators of allergy and asthma
40 μm; 20
2D BeWo or
0–40 μg/mL/ 6 h, 24 h
or 48 h
not applicable particle uptake in BeWo cells/ no major
acute toxicity but reduced secretion of β-
hCG and transient reduction in barrier
interference with hormone release and
< 2.5 μm human ambient PM
exposures over the
from 5.54 to 29 μg/
not evaluated positive relationship between PM2.5
exposure during preconception and
pregnancy and intrauterine inflammation
intrauterine inflammation upon PM2.5
exposure in pregnancy may influence
subsequent fetal growth, development,
and health outcomes
<10μm human mean exposure levels
were 30.3 μg/m
and 39.9 μg/m
not evaluated short-term maternal PM10 exposure was
modestly associated with elevated maternal
CRP levels in early pregnancy and that
long-term maternal PM10 and NO
ure during pregnancy was associated with
elevated fetal CRP levels at delivery
exposure to air pollution during
pregnancy may lead to maternal and
fetal inflammatory responses
<10μm human mean exposure levels
were 30.3 μg/m
and 39.9 μg/m
not evaluated associations of PM10 and NO
with changes in fetal sFlt-1 and PlGF levels
at delivery/ higher PM10 and NO
sures were associated with lower placenta
weight/ air pollution exposure was not con-
sistently associated with other markers of
placental growth and function
maternal air pollution exposure may
influence markers of placental growth
BC black carbon, CB carbon black, DEP diesel exhaust particles, GD gestation day, ICP-OES inductively coupled plasma optical emission spectrometry, hCG human chorionic gonadotropin, HVMF human villous
mesencyhmal fibroblasts, IGF insulin growth factor, i.p. intraperitoneally, i.v: intravenous, NP nanoparticles, PAHs polycyclic aromatic hydrocarbons, PM particulate matter, ROS reactive oxygen species, TEM transmission
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 11 of 22
and 16.5, respectively) and suggested that the underlying
mechanisms may involve placental insufficiency with en-
suing down-regulation of critical mediators of lung de-
velopment . Other proposed placenta mediated
indirect pathways involve adverse effects of NMs on pla-
cental structure and function [90,92,93,95], the release
of placental inflammatory mediators , reduction in
placental vasculature [93,94] and activation of coagula-
tion, complement and oxidative stress in the placenta
 and disruption of endocrine signaling [91,93].
Studies with unknown placental particle transfer
In several studies, placental translocation was not
assessed, but the authors nevertheless postulated a role
for indirect pathways of developmental toxicity based on
observed interference of NMs with maternal organs or
placental function (Table 3). Most of these studies did
not evaluate gestational and litter parameters, but alter-
ations of these parameters have previously been de-
scribed following maternal exposure to TiO
PM [2–4] and DEPs . Injection of SiO
NPs in preg-
nant mice (0.025 or 0.04 mg/g body weight on GD 13
and 14) resulted in increased fetal resorption and re-
duced fetal weight, possibly through particle-induced in-
flammatory responses in the placental tissue . These
complications were entirely prevented by ROS inhibitors
or forced expression of IL-10 . Maternal or intra-
uterine inflammatory pathways were also proposed to
mediate developmental toxicity from exposure to air
pollution [49,104] carbon black (CB) [17,101] and
MWCNTs . Besides inflammatory pathways, inter-
ference with the placenta (structure, growth or function/
reactivity) has been suggested to constitute another in-
direct pathway for developmental toxicity of air
pollution particles , cadmium telluride (CdTe) QDs
NP  in vivo or graphene oxide (GO)
in vitro . For prenatal exposure to CB, TiO
CeO NPs (intratracheal instillation: 50 μg/mouse on GD
14), Fedulov et al. observed increased allergic suscepti-
bility in the offspring that was proposed to occur due to
NM-induced production of Th2 cytokines in maternal
Overall, for all of the three study categories (studies
investigating but not detecting placental transfer
(Table 1), studies detecting placental transfer (Table 2)
and studies with unknown placental transfer (Table 3)),
indications of potential indirect toxicity pathways medi-
ated by maternal and/or placental tissue have been identi-
fied. Moreover, considering that maternal and placental
tissues are probably exposed to NMs at earlier time points
and higher dose levels compared to the fetal compart-
ment, extending the focus from direct to indirect effects is
of key importance to advance our understanding of risks
associated with NM exposure during pregnancy.
Candidate pathways for indirect developmental toxicity
Developmental toxicity is mostly assessed in experimen-
tal animals and often centers on classical gestational and
litter parameters. In light of the growing evidence for
maternal and placenta mediated developmental toxicity
of NMs, it is crucial to perform more comprehensive
assessments of placental, maternal and fetal/offspring
tissue and organ functions. In this section, we will com-
pile and discuss the different indirect pathways for-
warded in the reviewed literature, to outline how NMs
may adversely affect developmental outcomes without
direct exposure of the fetus to NMs. Although we will
mostly describe individual indirect pathways, these are
likely interlinked and jointly contribute to adverse fetal
outcomes, potentially even in conjunction with direct
NM-induced oxidative stress and inflammatory responses
The placenta has a very high turnover of oxygen and
ROS are generated continuously, with the main source
being the mitochondrial respiratory chain. Overall, the
balance between oxidants and antioxidants is vital for
maintaining physiological homeostasis. During normal
pregnancy conditions, ROS are eliminated by the
corresponding and abundant production of antioxidants
by the feto-placental unit. If this redox balance is dis-
turbed pathological conditions may emerge . Sev-
eral types of NMs induce the generation of ROS, either
directly or via activation of oxidative enzymatic pathways
[106–109]. Excessive amounts of ROS may overwhelm
the capacity of the intrinsic antioxidants and result in a
condition of oxidative stress . ROS can damage
cells by interaction with lipids, proteins and DNA and
by induction of inflammation [108,111]. Placental in-
flammation is a well-established risk factor for preg-
nancy and fetal development . If NMs are taken up
by placental cells, the subsequent generation of ROS,
oxidative stress and inflammation has been hypothesized
to represent one indirect mechanistic pathway by which
NMs can interfere with placental development and
function, and hence, with fetal development [36,55].
Inhaled particles that deposit in the lung alveoli can
also locally induce ROS and inflammation. This will
often be accompanied by increased transcription of pro-
inflammatory genes and ultimately the production of
inflammatory mediators, such as cytokines and acute-
phase proteins that can become systemically available
[113,114]. It is increasingly being described that mater-
nal inflammation is a potent modulator of fetal develop-
ment and that the developing nervous system may be
especially sensitive. Maternal inflammation has been
proposed to constitute an immune challenge to the fetus
that could prime early alterations in the inflammatory
response systems and, in turn, disrupt development and
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 12 of 22
maturation of the central nervous system and enhance
sensitivity to additional stress factors . Maternal in-
flammation may not necessarily result in fetal inflamma-
tion, but the placenta may act as a sensor of maternal
inflammation and subsequently adapt to the inflamma-
tory environment and may act both as a target and a
producer of inflammatory mediators .
Shirasuna et al. (2015) elegantly aimed to investigate if
NPs induce pregnancy complications through placental
inflammation . Pregnant mice were injected i.v. with
0.04 mg/kg body weight of 70 nm silica particles on GD
13 and 14. This exposure increased fetal resorptions, in-
duced placental dysfunction, ROS generation and infil-
tration with neutrophil granulocytes (3-fold). Also,
placental protein levels of several inflammatory cyto-
kines were significantly increased (IL-1β, IL-6, TNF-α,
and CCL2). Pre-treatment with the antioxidant N-acetyl
cysteine (NAC) almost completely reversed the placental
and fetal effects of the injected NPs, reduced placental
ROS levels, cell infiltration and secretion of IL-1βand
IL-1α. Findings in specific knock-out mouse strains indi-
cated that the balance between the inflammatory cyto-
kine IL-1 and the anti-inflammatory IL-10 was pivotal
for induction of adverse effects. Therefore, the study was
repeated with forced expression of IL-10 by injection of
adeno-associated virus vectors encoding murine IL-10.
Again, the placental and fetal effects of the injected NPs
were reversed. Of note, inhibition of placental phagocyt-
osis and hence uptake of NPs significantly blocked IL-1β
and IL-1αsecretion, indicating that uptake of NPs into
the cells might has been involved in inducing inflamma-
tory pathways in placental cells .
The induction of placental ROS by NMs was also ad-
dressed by Qi et al. (2014) . Pregnant mice were
injected i.v. with 20 mg of oxidized (o-) MWCNTs/kg on
GD 17. Exposure increased the ROS levels in placentas,
but not in maternal plasma, indicating that the placenta
may respond more vigorously or faster to o-MWCNTs
than other maternal tissues. The observation of placental
implication in developmental toxicity has some resem-
blance to reports on the effects of SWCNTs, TiO
silica NPs [85,90].
Also, other studies have attempted to investigate the
degree to which oxidative stress contributes to develop-
mental effects by administering antioxidants alongside
the maternal exposure to NMs . Onoda and co-
workers investigated the protective effects of antioxi-
dants on the development of reactive astrogliosis in the
offspring that had been observed following maternal in-
tranasal instillation of CB NM (95 μg/kg body weight)
on GD 5 and 9 in several previous studies. N-acetyl cyst-
eine or ascorbic acid were administered intraperitoneally
to pregnant mice prior to CB instillation. N-acetyl cyst-
eine partly prevented, whereas ascorbic acid slightly
enhanced, astrogliosis in the offspring . Another
study investigated the developmental effects of
MWCNTs injected intravenously to pregnant p53+/−
mice (2 mg/kg or 5 mg/kg body weight on GD 10.5, 12.5
or 15.5 as a single or repeated dosis). MWCNTs in-
creased the incidence of brain defects in the offspring
and decreased offspring survival rate after birth. The
underlying mechanism seemed to involve MWCNTs dir-
ectly triggering p53-dependent apoptosis and cell cycle
arrest in response to DNA damage. Co-injection of an
antioxidant markedly decreased the number of fetuses
with brain defects, indicating that oxidative stress may
be implicated. In this study, MWCNTs were found to
distribute to the placenta and fetal liver but were not ob-
served in the fetal brain . Finally, intratracheal instil-
lation of 4–5 mg MWCNTs/kg to pregnant mice on GD
9 was found to induce fetal malformations and to signifi-
cantly increase maternal leukocyte counts in peripheral
blood. At a lower dose of 3 mg/kg, no abnormality oc-
curred. This suggests that maternal inflammation may
be contributing to fetal toxicity .
Overall, these findings offer evidence of the involve-
ment of oxidative stress in developmental toxicity of
NMs. It is, however, important to keep in mind that ob-
servation of protection by antioxidants does not specify
whether the effects occurred due to oxidative stress-
induced directly by particles or indirectly via other
mechanistic pathways. In some studies, particle exposure
also induced pregnancy complications, such as fetal
death, that could be associated with apoptosis and hence
generation of increased levels of ROS. Therefore, it is
not possible to deduct whether the increases in ROS
levels occurred due to particle exposure or pregnancy
NM interference with placental toll-like receptors
Several cell types express receptors for recognition of
pathogen-associated molecular patterns present on the
surface of microorganisms. Probably the best-described
group of pattern recognition receptors are the TLRs, a
group of evolutionary conserved transmembrane pro-
teins . Until now, 11 mammalian TLRs have been
defined. TLR 4 is crucial for response to lipopolysac-
charide (LPS) and, thereby, to gram-negative bacteria.
TLR 2 recognizes a broader array of molecular patterns
from bacteria and fungi. Ligand recognition by the TLRs
mostly results in the activation of the intracellular sig-
naling pathway of NFκB, ultimately increasing the pro-
duction of cytokines and antimicrobial factors .
The human placenta expresses all of the TLRs, varying
in a temporal and spatial manner . Activation of
trophoblast TLRs enhances cytokine expression, which
may be followed by significant recruitment of immune
cells (macrophages, NK cells) to the placenta. TLR-
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 13 of 22
activation is associated with negative pregnancy outcomes
(preterm labor, fetal loss and preeclampsia), but also plays
a role in long-term adverse outcomes in the offspring,
such as the function of the immune and central nervous
systems . Placental TLRs may, however, also be in-
volved in the protective effects hypothesized to occur in
case of “adequate”non-infectious microbial exposure as
proposed by the hygiene hypothesis .
Accumulating evidence indicate that TLRs might
recognize some NMs and activate similar pathways as
upon contact with LPS and bacteria [122,123]. Hence,
MWCNTs have been shown to induce DNA damage in
human lung epithelial cells due to the activation of TLR
9 and subsequent generation of nitric oxide (NO) .
Also, SWCNTs have been reported to provoke
chemokine secretion in macrophages via the TLR 2/4-
MyD88-NFκB signaling pathway . Interestingly,
when graphene oxide was tested in the same setup, no
such response was elicited, indicating that TLRs may
have a differential preference for subgroups of NMs
. In silico investigations show that the internal
hydrophobic pockets of TLR 4 might be able to bind
small-sized carbon nanostructures such as fullerenes and
CNTs . TLR 4 has, however, been shown to also
recognize non-carbonaceous NMs, such as iron and TiO
NPs, to promote inflammatory responses [127–129].
In the human placenta, TLR 2 and TLR 4 have been ob-
served to lack in the ST but to be expressed in villous and
extravillous trophoblasts, at least during early pregnancy
. This could indicate that, at this stage, the placenta
responds primarily to pathogen-associated molecular pat-
terns if the ligand has broken through the outer layer
. Therefore, NMs would need to be internalized by
the trophoblast for TLR activation. Interestingly, several
studies in the ex vivo human placenta model and experi-
mental animals report that nanosized particles accumulate
in placental tissue [130–132] and that particles can be
visualized in trophoblasts [90,91,133–135].
Activation of placental TLRs by NMs would implicate
the presence of NMs in maternal blood and their
uptake/penetration into the ST. Some TLRs do also
respond to endogenous molecules via so-called danger-
associated molecular patterns, including, but not
restricted to, ROS and proteins released from dead or
dying cells . Hence induction of ROS or inflamma-
tion by NMs in placental tissue, via direct or indirect
pathways, may indirectly activate TLRs.
NM interference with endocrine signaling
Endocrine signaling pathways are central in mediating
physiological and metabolic adaptations required for a
successful pregnancy and are orchestrated by the pla-
centa and the maternal endocrine organs (e.g., the pituit-
ary, thyroid and adrenal glands, and the ovaries) [136,
137]. First evidence that NMs can have endocrine-
disrupting activity came from studies in non-pregnant
individuals, where NMs have been reported to affect
levels of both female and male sex hormones in vitro
and in vivo (reviewed in [138,139]). For example, expos-
ure of female and male rats to nickel (Ni) NPs by gavage
resulted in altered hormone regulation (FSH and LH
levels were elevated and estradiol lowered in females
while testosterone and FSH levels were diminished in
males) and induced pathological changes in testes and
ovaries (reviewed in ). However, it largely remains
to be established if NMs might act as endocrine disrup-
tors during pregnancy and how this could potentially
affect pregnancy and offspring health later in life.
In pregnancy, one of the critical hormones secreted by
the human placenta is human chorionic gonadotropin
(hCG) . It supports the function of the corpus
luteum, a transient ovarian structure particularly import-
ant in the early gestational phase, which secretes ovarian
progesterone and estrogens to maintain a successful
pregnancy . hCG also regulates the formation of
the ST [142,143], modulates immune responses ,
ensures uterine quiescence , promotes angiogenesis
of the endometrial spiral arteries [143,144], and dilates
these vessels to enhance maternal blood flow . Due
to these various crucial functions of hCG, disturbances
in the tightly regulated levels of this hormone could,
therefore, increase the risk of adverse pregnancy out-
comes . A few in vitro studies using BeWo tropho-
blast monocultures , 3D placental co-culture
microtissues (BeWo cells/primary human villous mesen-
chymal fibroblasts)  or first trimester human placen-
tal explants  showed a significant reduction of hCG
release after exposure to GO, CdTe and CuO NPs or
dendritic polyglycerol NPs, respectively. This emphasizes
that disturbances in hCG release should be considered
in developmental toxicity studies.
Also, the steroid hormones estrogen (reviewed in
) and progesterone (reviewed in ) are indis-
pensable to maintain human pregnancy. Estrogens are
essential for vasodilation and local angiogenesis due to
their close interaction with angiogenic factors like vascu-
lar endothelial growth factor (VEGF) and placental
growth factor (PLGF) (reviewed in ). Dysregulation
of estrogen secretion could, therefore, play a major role
in the development of preeclampsia and other adverse
conditions during pregnancy. Progesterone is essential
for the reproductive process. Altered progesterone secre-
tion has been associated with miscarriage and preterm
birth . So far, only a few descriptive studies report-
ing NM effects on steroid hormone levels in pregnant
animals are available. Inhalation of Ag NPs decreased es-
trogen plasma levels in pregnant mice, but it was unclear
if the Ag NP exposure caused the increase in observed
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 14 of 22
fetal resorptions . In another study, serum levels of
progesterone decreased while estradiol levels increased
in pregnant mice injected with MWCNTs . Further-
more, VEGF levels and placental vascularization de-
creased and were linked with the reported growth
restriction in the offspring , indicating a potential
interconnection between endocrine and vascular
Other relevant placental and maternal hormones in-
clude human placental lactogen, the placental growth
factor prolactin, the neuropeptides serotonin, melatonin
and oxytocin (reviewed in [136,137,141,149,150]as
well as IGFs (reviewed in [150–153]). Inhalation of DEPs
in pregnant rabbits resulted in intra-uterine fetal growth
retardation that was accompanied by reduced placental
efficiency alongside a decrease in fetal plasma levels of
IGF-1 , a peptide hormone essential for the regula-
tion of feto-placental growth (reviewed in [150,153]).
However, it remains to be shown in how far the ob-
served changes in fetal IGF-1 levels are responsible for
the observed developmental toxicity. Moreover, further
clarification is needed if contaminants (e.g. metals or
polyaromatic hydrocarbons) associated with the particles
could be involved or even be responsible for the adverse
effects of DEPs on fetal development.
In summary, the few available studies indicate that
NMs might act as endocrine disruptors and interfere
with hormonal signaling in pregnancy. The underlying
mechanisms are not yet well understood and might in-
clude direct effects on hormone biosynthesis and secre-
tion, interference of NMs with hormone-receptor
binding on target cells or with downstream signaling
pathways (reviewed in ) as well as indirect effects,
e.g., via the induction of inflammatory processes, which
have been shown to cause endocrine imbalance ).
Overall, a better understanding of the possible interfer-
ence of NMs with the endocrine system during preg-
nancy is still needed and should include long-term
studies since endocrine responses often take time before
NM-triggered effects are manifested.
NM interference with vascular signaling and utero-placental
development and function
To accommodate the developing fetus, extensive vascu-
lar adaptations take place in the uterus and placenta.
Maternal blood volume increases around 45%  and
uterine placental blood flow increases ten-fold .
Maternal blood pressure usually decreases or remains
unchanged during pregnancy. This is mainly achieved
through a decreased uterine vascular resistance, which is
ultimately determined by a combination of an increase
in vessel diameter, a reduced vascular tone (vasodilation)
and an establishment of the placenta . To ensure
that vascular remodeling does neither harm the mother
nor the fetus, microcirculatory regulation of blood flow
is crucial [157,158]. Early in pregnancy, the uterine
spiral arteries are structurally converted from small
diameter arteries into low-resistance large diameter ves-
sels by interaction with the fetal placental extravillous
trophoblasts that invade the myometrium and the spiral
arteries . Some chemicals have been shown to
interfere with vascular remodeling and development of
the placenta, thereby impairing oxygen and nutrient de-
livery to the fetus and ultimately increasing the risk for
adverse pregnancy outcomes (reviewed in [160,161]).
Several epidemiological studies have found that compo-
nents of air pollution, including PM with 2.5 μm or less
in diameter (PM
were associated with increased risk
of pregnancy-induced hypertensive disorders .
For engineered NMs, studies in experimental animals
indicate that maternal NM exposure during gestation in-
terferes with maternal vascular reactivity and placental
vascular development and function. Exposure to NMs
has been associated with increased tone and contractility
of the uterine vasculature [162–166]. In some studies,
placentas from exposed pregnant rats were larger, while
their offspring were smaller compared to the control
group, indicative of effects downstream from the uterine
vascular impairment [163,166]. As an example, i.v. ad-
ministration of MWCNTs (100 μg/kg body weight) or
Ag NPs (200 μg/rat) to pregnant rats at GD 17–19 in-
creased the contractility of the uterine artery. The ma-
ternal mesenteric artery and thoracic aorta were
unaffected, suggesting that the extensively remodeled
and functionally dynamic uterine vasculature is more
vulnerable to particle exposure than vascular beds in the
adult organism [162,163]. For Ag NPs, the effects fur-
thermore depended on particle size (only for 10 nm but
not 100 nm Ag NPs) and surface modification (more
substantial effect of citrate compared to PVP-coated Ag
NPs) . Using the less invasive inhalation route of
NPs have been shown to attenuate both
the endothelium-dependent and -independent vessel
dilation at the uterine-arteriolar level in both non-
pregnant and pregnant rats [164,165]. This was accom-
panied by significantly increased plasma levels of the
pro-inflammatory factors IL-4 and IL-6 , indicating
that systemic inflammation could have been implicated
in the observed changes. Intratracheally instilled TiO
NPs induced a systemic Th2 inflammatory response,
which was mediated by group II innate lymphoid cells
(ILC2) in the lungs. At the same time, endothelium-
dependent dilation of the uterine radial arterioles was
impaired . IL-33 potently drives the production of
Th2-associated cytokines. Treatment with an anti-IL-33
antibody prior to the TiO
NP exposure attenuated the
upregulation of circulating IL-33 levels and improved
the endothelium-dependent dilation. Uterine microvascular
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 15 of 22
dysfunction may, therefore, arise via activation of ILC2 cells
in the lung and the subsequent systemic Th2-dependent
inflammation. The study was performed in non-pregnant
females, but the mechanisms ought to be also investigated
during pregnancy. In another study, inhalation of TiO
during pregnancy was found to augment vasoconstriction
of the uterine artery in response to Kisspeptin .
Kisspeptin is a potent vasoconstrictor and an essential re-
productive hormone, which is secreted by the placenta
during late pregnancy at 10,000-fold higher levels compared
to the non-pregnant state (reviewed in ). It is, there-
fore, possible that TiO
NP exposure perturbed the endo-
crine vascular axis via a kisspeptin-dependent mechanism
. Finally, a recent study investigated blood perfu-
sion in placentas from pregnant rats inhaling TiO
NPs. An endothelium-dependent increase in placental
vascular resistance, measured as decreased outflow
vein pressure, was observed. NO release plays a crucial
role in maintaining the low basal tone of the materno-
fetal circulation  and was therefore hypothesized
to be implicated in the signaling causing the decrease
in outflow pressure. Increased sensitivity to angiotensin
II, a potent vasoconstrictor implicated in preeclampsia
, was observed in both the placental and umbil-
ical artery , also identifying the renin-angiotensin
system as a perhaps crucial modulator of tissue tone.
Several studies observed abnormalities in placental de-
velopment and function following exposure to NMs.
Mice treated with 70 nm silica NPs (nSP70) i.v. (0.8 mg/
mouse on GD 16–17) failed to form spiral artery canals,
and blood flow in the fetal vascular sinuses decreased,
perhaps due to abnormalities in structure and length of
placental villi. The authors suggested that structural and
functional changes to the placenta might be a result of
the decreased levels of soluble Flt-1 , a potent anti-
angiogenic factor involved in placental vascularization
. Orally administrated TiO
NPs impaired placenta-
tion, possibly through the observed dysregulation of pla-
cental vascularization, proliferation and apoptosis .
Reductions were also observed for the expression of reg-
ulators for placental vascularization as well as the num-
ber of uterine natural killer (uNK) cells , a cell type
that plays important roles in remodeling of spiral arter-
ies, control of trophoblast invasion and placental devel-
opment [172–174]. Injection of QDs (10 or 20 mg/kg
body weight) on GD 13 was associated with placental
vascular anomalies, as exposed rats displayed a reduced
diameter of the labyrinth and basal zone and necrosis of
invasive trophoblasts when examined on GD20 . It is
possible that the reduced placental size observed after
QD treatment occurred due to the release of cadmium
ions, as cadmium is a recognized placental toxicant
. A recent mouse study observed (non-particulate)
cadmium to up-regulate several inflammatory cytokines
in the placenta through the Akt signaling pathway ,
so the mechanisms of developmental toxicity of QD
likely differ compared to that of other NMs. Vascular
effects of NMs could involve the uptake of particles by
extravillous trophoblasts resulting in impaired extra-
villous trophoblast invasion and vascular remodeling.
Hence, a recent in vitro study found uptake of platinum
NPs by autophagy to affect extravillous trophoblast func-
tions in exposed cells . Finally, there is also few
hints that some NMs might interfere with placental bar-
rier integrity and eventually transport. For instance, GO
NPs induced a transient decrease in the integrity of
BeWo trophoblast cells in vitro . Blum et al., 
suggested that developmental toxicity from CdO NP in-
halation in mice might result from the accumulation of
Cd ions and interference with transport in the placenta
since a previous epidemiological study reported that pla-
cental Cd can alter the transport of zinc, vitamin B12
and other micronutrients . However, conclusive evi-
dence for NM effects on placental transfer in vivo is still
lacking.In conclusion, it is becoming increasingly evident
that exposure to NMs can interfere with vascular and
placental development, structure and ultimately func-
tion, and, therefore, cause severe adverse effects on
embryo-fetal development and offspring health. The
underlying mechanisms, however, are still to be
NM interference with extracellular vesicle signaling
Cells release extracellular vesicles (EVs) as part of their
intercellular communication, with microvesicles (MVs)
and exosomes as two main subgroups (reviewed in
). EVs contain various molecules such as lipids, nu-
cleic acids and proteins, dependent on their donor cell
type and physiological state, and the stimuli that medi-
ated their formation and release . The cargo is con-
sidered as information that EVs shuttle from donor to
target cells, and even across internal barriers like the
blood-brain-barrier [179–181]. Upon contact with the
recipient cell, EVs have been shown to initiate different
processes depending on their cellular origins, such as an
immune response (exosomes originating from B lym-
phoblastoid or dendritic cells) [182,183] or the trans-
formation of healthy cells to tumor cells (MVs
originating from MDAMB231 breast carcinoma and U87
glioma cells) .
During pregnancy, EVs are indicated to be involved in
critical processes such as maternal-placental
vascularization  and regulation of the maternal im-
mune response . However, when pregnant women
suffer from certain diseases (e.g., preeclampsia and ges-
tational diabetes mellitus), serum EV levels, composition,
and function differ [187–192]. Even though it is still un-
clear, if the altered EV signaling is a contributing factor
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 16 of 22
or only a symptom of a pregnancy complication, these
studies indicate that EV levels and cargo reflect the
physiological state of pregnancy.
Since NMs and EVs are of comparable size and circu-
late in similar compartments, interactions between them
are considered possible, even though only few studies in-
vestigated this topic so far. In mice, intratracheal instilla-
tion of magnetic iron oxide NPs (MIONs with primary
size ~ 43 nm) increased secretion of exosomes in the al-
veolar region, which contained increased levels of iron,
albeit no MIONs could be imaged inside the vesicles by
transmission electron microscopy [193,194]. These exo-
somes were shown to initiate a systemic Th1-type im-
mune response via direct and indirect T cell activation
[193,194]. In another study, subtoxic concentrations of
ZnO (primary size ~ 10 nm) and TiO
NPs (primary size
of ~ 21 nm) did not significantly affect the characteristics
of exosomes released by primary human peripheral
blood mononuclear cells nor by monocyte-derived den-
dritic cells, such as size and number. Moreover, NPs
were not found attached to the outside or inside of the
exosomes . In contrast, 20 nm sized gold NPs were
taken up by human macrophages and found in released
exosomes afterwards . Interestingly, the only study
investigating effects of NMs on placental-derived EVs
demonstrated similar results. Human placental mesen-
chymal SCs released NP-loaded exosomes after exposure
to hollow gold NPs (primary size 40 nm). These vesicles
were further shown to exclusively migrate to the cell
type of origin in vitro .
These studies indicate that the release of EVs and their
characteristics can be altered by NM exposure, which
could systemically affect other tissues in the body. In
pregnancy, maternal exposure to NMs may lead to an
EV-mediated modulation of cell communication be-
tween maternal, placental and fetal tissues. Considering
that crucial processes during gestation are facilitated via
EVs, disturbances in EV signaling could jeopardize preg-
nancy, which calls for more extensive investigation of
potential influences of NMs on the EV system.
Conclusions and future perspectives
Indirect pathways probably play a major role in NM-
induced developmental toxicity. The placenta likely takes
a central position in mediation hereof due to its location
at the interface between mother and fetus and the many
essential functions it undertakes during pregnancy. An
increasing number of studies in experimental animals,
in vitro and ex vivo models highlight how NM may exert
effects indirectly via induction of maternal and placental
oxidative stress and inflammation, activation of placental
TLRs, impairment of placental growth, and secretion of
placental hormones and vascular factors. Potentially,
EVs could play a role in signaling between maternal and
fetal organs. The impact of NMs on maternal and pla-
cental tissues can ultimately result in pregnancy compli-
cations and long-term effects on offspring health, even
in the absence of or limited particle transfer across the
Our understanding of the involved mechanisms is,
however, still scarce, and this emphasizes the need for
more systematic studies. Comprehensive knowledge of
the mechanisms underlying indirect toxicity increases
the possibility of identifying triggers of effects and hence
to categorize NMs based on shared properties of various
materials. This will facilitate the risk assessment of hu-
man health effects, and furthermore, the application of a
safe-by-design approach in the design of new materials
. An important issue is here, whether the effects of
NP exposure depends on the existing state of inflamma-
tion in the mother, as e.g. asthma and obesity is associ-
ated with chronic low-grade inflammation . To our
knowledge, this aspect has only been marginally ad-
dressed. A study in pregnant mice indicated that LPS-
induced intrauterine inflammation can increase the
materno-fetal transfer of small AuNPs (3, 13 nm) after
i.v. injection at GD 17 . Identification of the key
processes in NM induced developmental toxicity will
further provide a basis for improvement of in vitro test
systems, which are suitable for the screening of a broad
range of NMs and allow for the identification of poten-
tial hazards to pregnancy. In addition, the application of
advanced in vitro models would reduce the use of
experimental animals .
Apart from identification of the mechanisms under-
lying developmental toxicity of NMs, the advancement
of relevant in vitro systems will profit from the identifi-
cation of the maternal and fetal organ systems that are
most prone to disruption by NMs. To include pathways
of indirect toxicity pathways, predictive developmental
toxicity assessment of NMs requires the interconnection
of multiple in vitro models such as the placenta, mater-
nal tissues and the embryo, either directly in co-cultures
[86,87,201], or indirectly via the transfer of conditioned
media . The potential of such approaches has been
nicely exemplified in some recent studies on the indirect
developmental toxicity of CoCr NPs, revealing DNA
damage to occur across the placental barrier in neurons
and astrocytes in the absence of NP transfer [86–88].
Recently, a novel microfluidic multitissue platform com-
bining the embryonic SC test and liver microtissues for
advanced embryotoxicity testing has been successfully
implemented . If the placental barrier could be in-
tegrated into such a platform, this might offer another
auspicious approach for future developmental toxicity
screening of NMs in a dynamic environment. However,
the combination of different models often requires com-
promises and substantial modification of the cultivation
Dugershaw et al. Particle and Fibre Toxicology (2020) 17:31 Page 17 of 22
conditions (e.g., culture medium, cultivation time), and
thus, the predictive value of the newly developed multi-
tissue models should be carefully validated. For instance,
the use of serum-free media is required for cultivation of
embryonic SCs or neural precursor cells to prevent their
differentiation, but this might affect the protein corona
of NMs, which could alter NM uptake and biological re-
sponses in the cells (reviewed in [203,204]). Another
challenge will be to distinguish between direct and indir-
ect toxicity mechanisms for NMs that cross the placental
barrier. Here, the comparison of different exposure con-
ditions (e.g., direct versus indirect exposure to NMs as
performed by Bhabra et al., (2009) ) or depletion of
conditioned medium from NMs by centrifugation may
allow to better understand the involvement of direct and
indirect pathways. Finally, to identify novel mechanisms
for indirect developmental toxicity mechanisms of NMs,
unbiased omics approaches (e.g., transcriptomics, prote-
omics, secretomics or epigenomic profiling) should be
explored to understand the array of molecular and func-
tional changes that result from NM exposure.
Ag: Silver; BC: B cell; BL: Basal lamina; CB: Carbon black; CdO: Cadmium oxide;
CdTe: Cadmium telluride; CeO2: Cerium dioxide; CoCr: Cobalt-chrome;
CuO: Copper oxide; DC: Dendritic cell; DEP: Diesel exhaust particle;
E: Endothelial; EVs: Extracellular vesicles; F: Fibroblasts; FC: Fetal capillary;
GD: Gestational day; GI: Gastrointestinal; GO: Graphene oxide; HC: Hofbauer
cell; hCG: Human chorionic gonadotropin; i.v.: intravenous; IGF: Insulin-like
growth factor; ILC2: Innate lymphoid cell; LPS: Lipopolysaccharide;
MIONs: Magnetic iron oxide NPs; MVs: Microvesicles; MWCNT: Multi-walled
carbon nanotube; NAC: N-acetyl cysteine; Ni: Nickel; NM: Nanomaterial;
NO: nitric oxide; NP: Nanoparticle; nSP70: 70 nm silica NPs; PLAP: Placental-
type alkaline phosphatase; PlGF: Placental growth factor; PM: Particulate
matter; QDs: Quantum dots; ROS: Reactive oxygen species; SC: Stem cell; sFlt-
1: Soluble Flt-1; SiO2: Silica dioxide; ST: Syncytiotrophoblast; SWCNT: Single-
walled carbon nanotube; TC : T cell; TiO2: Titanium dioxide; TLR: Toll-like
receptor; UG: Uterine gland; uNK: Uterine natural killer; VEGF: Vascular
endothelial growth factor; ZnO: Zinc oxide
All authors contributed to the design and concept of this article and drafted
the manuscript. All authors critically revised the manuscript. All authors read
and approved the final manuscript.
This research is supported by funding from the Swiss National Science
Foundation (grant no 31003A_179337). Karin Sørig Hougaard was supported
by the Danish Working Environment Research Fund (Danish Centre for
Nanosafety II) and Signe Schmidt Kjølner Hansen was supported by a grant
from the Independent Research Fund Denmark.
Availability of data and materials
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Laboratory for Particles-Biology Interactions, Empa, Swiss Federal
Laboratories for Materials Science and Technology, Empa, Lerchenfeldstrasse
5, 9014 St.Gallen, Switzerland.
National Research Centre for the Working
Environment, Copenhagen, Denmark.
Biotech Research and Innovation
Centre, University of Copenhagen, Copenhagen, Denmark.
Public Health, University of Copenhagen, Copenhagen, Denmark.
Received: 25 March 2020 Accepted: 14 June 2020
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