Biology of Reproduction, 2022, 106(3), 397–407

https://doi.org/10.1093/biolre/ioab223
Advance access publication date 7 December 2021
Review

The impact of perfluoroalkyl substances on pregnancy,

birth outcomes, and offspring development: a review
of data from mouse models†
Zahra Aghaei, Katherine L. Steeves, Karl J. Jobst and Lindsay S. Cahill*
Department of Chemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada

Downloaded from https://academic.oup.com/biolreprod/article/106/3/397/6454968 by guest on 02 October 2023

*Correspondence: Department of Chemistry, Memorial University of Newfoundland, 283 Prince Philip Drive, St. John’s, NL A1B 3X7,
Canada. E-mail: lcahill@mun.ca
†
Grant Support: Funding for this work was provided by the Banting Research Foundation and the Natural Sciences and Engineering Research Council of
Canada

Abstract
Per- and polyfluoroalkyl substances (PFASs) such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are persistent in
the environment and bioaccumulate in wildlife and humans, potentially causing adverse health effects at all stages of life. Studies from human
pregnancy have shown that exposure to these contaminants are associated with placental dysfunction and fetal growth restriction; however,
studies in humans are confounded by genetic and environmental factors. Here, we synthesize the available results from mouse models of
pregnancy to show the causal effects of prenatal exposure to PFOA and PFOS on placental and fetal development and on neurocognitive
function and metabolic disorders in offspring. We also propose gaps in the present knowledge and provide suggestions for future research
studies.
Summary Sentence
Using mouse models of pregnancy, maternal exposure to perfluoroalkyl substances has been shown to have significant impact on placental
development, fetal growth, neurocognitive function, and risk for disease in both mother and offspring.
Keywords: fetal development, mouse, perfluoroalkyl substances, perfluorooctanesulfonic acid, perfluorooctanoic acid, pregnancy

Introduction
Per- and polyfluoroalkyl substances (PFASs) have been widely
used in industry and household products for more than
eight decades [1]. Many of these contaminants are thought to
persist in the environment and bioaccumulate in humans and
wildlife. The main routes of human exposure include
contaminated food, drinking water, and air inhalation [2–5].
While there are over 9000 known PFASs that could be
released into the envi- ronment [6, 7], only a few have been
identified, monitored, and tested for their biological
activity.
Available analytical techniques such as liquid chromatog-
raphy tandem mass spectrometry make it possible to repro-
ducibly detect low levels of PFASs in a variety of matrices
including blood and biological tissues [8–11]. A study in 2004
was the first to report that two PFASs, perfluorooctanoic
acid (PFOA) and perfluorooctanesulfonic acid (PFOS), were
detected in the blood of nonoccupationally exposed humans
in Canada [12]. This, combined with evidence of high con-
centrations of PFASs in the tissue of wildlife [13], prompted
concerns about the toxicity and potential health risks of
exposure to PFOA and PFOS, including during reproduction,
pregnancy, and neonatal development. Previous reviews have
assessed the impact of PFOA and PFOS exposure on birth
weights in humans and experimental animals and found a
negative association [14, 15]. A recent review by Blake and
Fenton described the association between PFOA and PFOS
exposure in humans and placental dysfunction and
increased susceptibility to chronic diseases later in life [16].
Despite growing evidence of exposure-related human health
effects, the ability to establish causality between environ-
mental exposure and adverse health outcomes in humans is
challenging. The use of experimental mice allows for greater
genetic and environmental control, ease of tissue collection,
and opportunities to evaluate the direct effect of pollutants
on behavior and cognitive function. Here, we focus on the
evidence from mouse models that maternal exposure to PFOA
and PFOS can impact reproduction, pregnancy, and fetal
development. We also summarize how PFOA and PFOS expo-
sure can affect postnatal development of murine offspring.
The Stockholm Convention environmental treaty restricted
the production and use of PFOA and PFOS. While the success
of this effort has led to a steady decline in blood concentration
of PFOA and PFOS in humans [17], these two legacy PFASs
remain in the environment and can continue to impact human
health. A better understanding of the health effects of PFOA
and PFOS will guide future studies to establish the impact of
replacement PFAS compounds on reproduction, pregnancy,
and fetal development. The search for this review was con-
ducted using PubMed and Web of Science databases up to
August 2021 using the following search terms: “perfluorooc-
tanoic acid” or “perfluorooctanesulfonic acid” and “mouse”
and “reproduction,” “pregnancy” or “development.” Full-
length English language articles were included if they inves-
tigated the effects of PFOA or PFOS on mouse reproduc-
tion, pregnancy, fetal development, or postnatal development.
There were no exclusion criteria.

Received: August 15, 2021. Revised: October 29, 2021. Accepted: December 2, 2021
© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please
e-mail: journals.permissions@oup.com
398 Impact of perfluoroalkyl substances on pregnancy, 2022, Vol. 106, No. 3
Common mouse strains to study pregnancy
Mice reproduce many of the genetic, molecular, and phys-
iologic processes of human pregnancy. For example, both
mice and humans have a hemochorial placenta with a similar
vascular and cellular structure [18]. This, in addition to being
efficient for research with low cost, rapid gestation and large
litter sizes, makes the mouse a widely used model for studying
placental development [19], pregnancy complications, and
fetal neurodevelopment [20]. Two of the most commonly used
mouse strains for pregnancy research are CD1 and C57BL/6
mice. CD1 mice are an outbred strain commonly used to
study healthy pregnancy and fetal development. C57BL/6
mice are an inbred strain that are often used because of
the ability to perform genetic manipulations. The vascular
morphology and development of the placenta are known to
be significantly different between CD1 and C57BL/6 mice
[21]. The two strains also show large differences in average
litter sizes (13 and 6 for CD1 and C57BL/6, respectively)
and in fetal weight at term [21, 22]. Knowing that several
pregnancy-related parameters are dependent on mouse strain,
we will report the mouse strain of each of the studies described
in this review. This review describes several studies using
Kunming mice, an outbred strain that first originated from
Swiss albino mice and are the most highly produced mouse
strain in China. Two other mouse strains that appeared most
often in our literature search were NMRI and BALB/c mice.
NMRI mice are an outbred strain that are used for toxicology
experiments, while BALB/c mice are an inbred strain that
find application in immunology. To maintain consistency
between studies, we will use the annotation of gestational
day (GD) where the morning a vaginal plug is detected
is denoted GD1. Full term in CD1 and C57BL/6 mice is
considered GD19 and mice are weaned at postnatal day 21
(PND 21).
Effects of PFOA and PFOS exposure
during pregnancy and early life
Effects on the placenta
The placenta is an essential organ for a successful mammalian
pregnancy [23]. It acts as the interface between the mother
and the fetus, providing the hormones and growth factors
necessary for fetal development and allowing the exchange
of oxygen, nutrients, and waste products [24]. Studies have
shown that many environmental pollutants can be transported
across the placenta, potentially impacting pregnancy outcome
[25]. The experimental details of the studies investigating
the effect of PFOA and PFOS exposure on placental and
fetal development are summarized in Table 1. In studies of
maternal exposure to PFOA and PFOS in mouse models
of pregnancy, the pollutants were found to be transferred
across the placental barrier. PFOA has been detected in dam
and pup serum, amniotic fluid, urine, milk, and mammary
tissue following just one day of exposure of PFOA late in
gestation to pregnant CD1 mice, confirming placental and
lactational transfer [10, 26]. In addition, PFOA and PFOS
were detected in maternal liver, placenta, and fetal liver after
prenatal exposure of CD1 mice [27, 28]. In a recent study,
images from dynamic positron emission tomography (PET)
showed placental uptake of PFOA and its transport to the
fetus after exposure of pregnant CD1 mice through tail vein
injection and oral gavage [29].
PFOA and PFOS can affect placental structure and
function; however, the direction of the effect is inconsistent.
While a study in Kunming mice showed that maternal expo-
sure to PFOA significantly decreased the placental weight
compared to controls [30], a study in CD1 mice found PFOA
exposure resulted in higher placental weights [31] and some
studies showed that exposure to PFOA and PFOS did not alter
placental weight [11, 32]. One common clinical measure of
the efficiency of the placenta is the fetoplacental weight ratio.
Studies on the effect of maternal exposure to PFOA and PFOS
on placental efficiency most often reported a reduction [31,
33, 34], with one study finding no effect [11]. A reduction in
the fetoplacental weight ratio indicates an inefficient placenta
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that fails to adapt its nutrient transfer capacity to meet the
needs of the rapidly growing fetus and is consistent with fetal
growth restriction [35]. Using an in vitro placental transport
assay, prenatal exposure to PFOS has been shown to impact
nutrient transport with a significant reduction in transport of
glucose and amino acid analogues across the maternal–fetal
interface [11].
Using histopathology, animal studies have reported patho-
logical findings in placental tissues following maternal expo-
sure to PFOA. Atrophy, necrosis, congestion, and a decrease
in the number of microvessels have been described for the
placental labyrinth in mice exposed to PFOA [30, 31, 33].
PFOA also results in fewer uterine natural killer cells and an
increase in cell apoptosis in the placenta [30].
Effects on fetal development/outcome and
physical maturation
The studies described above suggest placental function is com-
promised following maternal exposure to PFOA and PFOS.
Reduced placental function can lead to fetal growth restriction
or, in severe cases, stillbirth [36]. Babies born growth
restricted are at increased risk of neurological damage and
chronic dis- eases in adult life [37, 38]. The question of
whether exposure to environmental pollutants such as PFOA
and PFOS causes growth restriction has been the focus of
several studies over the past twenty years (Table 1).
There is no clear consensus in the literature about whether
exposure to PFOA and PFOS causes maternal toxicity, evi-
denced by a decrease in maternal weight gain. Maternal PFOS
exposure most often resulted in a decrease in maternal weight
gain in CD1 mice [34, 39, 40]; however, there was no effect
in two studies (one in CD1 [11] and one in C57BL/6/Bkl mice
[41]). On the other hand, maternal exposure to PFOA had
no effect on maternal weight gain in the majority of studies
(one in CD1 [26], two in 129 [32, 42], one in C57BL/6/Bkl
[41], and one in C57BL/6JxFVB mice [43]), with three studies
in CD1 mice reporting a reduced maternal weight gain [33,
44, 45]. When a decrease in maternal weight was reported,
it was usually accompanied by an increase in maternal liver
weight [39, 40, 44–47], indicating liver toxicity and possible
liver disease (see below).
While most studies found significant reduction in fetal
weights following maternal exposure to PFOA and PFOS in
CD1 [11, 31, 33, 34, 39, 40, 44, 45], C57BL/6JxFVB [43],
and Kunming mice [30], five studies reported no effect of
exposure on fetal weights in CD1 [26, 46], 129 [32, 42], and
C57BL/6/Bkl [41] mice. Survival rates also varied between
studies, with some reporting prenatal mortality [33, 34, 39,
45], demise shortly after birth [32, 40, 42, 44, 46], or no effect
Table 1. Developmental effects of exposure of pregnant mice to PFOA/PFOS

Z. Aghaei et al., 2022, Vol. 106, No. 3

Ref Mouse strain Exposure Dosage Number of live Maternal body Fetal body weight Maternal liver Placental weight FPWR Additional results
time (mg/kg/day) fetuses weight gain weight
[11] CD1 mice GD 5–18 1, 3 PFOS NR NS Reduced in 3 mg/kg Increased in 3 mg/kg NS NS Placental nutrient transport
was reduced in a
dose-dependent manner.
[26] CD1 mice GD 17 0.1, 1, 5 PFOA NS NS NS NS NR NR PFOA accumulates in maternal
and pup fluids.
[30] Kunming mice GD 1–13 2.5, 5, 10 PFOA NR NR Reduced NR Reduced in 5 and NR Interstitial edema of placenta
10 mg/kg from 5 mg/kg; increase in cell
apoptosis in placenta and
decrease in uterine natural
killer cells.
[31] CD1 mice GD 2–12 or 1, 5 PFOA NS NR Reduced in 5 mg/kg Increased from Increased in 5 mg/kg Reduced in Histopathological lesions in
2–18 at GD 18 1 mg/kg (GD 12) and at GD 18 5 mg/kg at placenta; atrophy, necrosis,
from 5 mg/kg (GD GD 18 and congestion in placental
18) labyrinth in 5 mg/kg (GD 18).
[32] Wild-type, PPARα-null GD 2–18 3 PFOA All born alive but NS NS Increased at GD 18 NS NR
and PPARα-humanized survival reduced in
mice (Sv/129 WT
background)
[33] CD1 mice GD 11–16 2, 10, 25 PFOA Reduced in a Reduced from GD13 Reduced in a NR Reduced Reduced Histopathological lesions and
dose-dependent dose-dependent cell necrosis in placenta from
manner manner 10 mg/kg.
[34] CD1 mice GD 11–16 0.5, 2, 8 PFOS Reduced in 8 mg/kg Reduced from GD14 Reduced in a NR Reduced in a Reduced Histopathological lesions and
dose-dependent dose-dependent cell necrosis in placenta in a
manner manner dose-dependent manner.
[39] CD1 mice GD 2–18 1, 5, 10, 15, 20 Reduced in 20 mg/kg Reduced in 20 mg/kg Reduced in 10 and Increased in a NR NR Observed birth defects
PFOS 15 mg/kg dose-dependent including cleft palate,
manner anasarca, ventricular septal
defect, enlargement of the right
atrium primarily in 15 and
20 mg/kg.
[40] CD1 mice GD 1–18 1, 10, 20 PFOS All born alive but Reduced n 20 mg/kg Reduced from Increased from NR NR Fetuses showed skeletal
survival reduced 10 mg/kg 10 mg/kg malformations, lung
from 10 mg/kg atelectasis, and dilatation of
intracranial blood vessels from
10 mg/kg.
[41] C57BL/6/Bkl mice GD 1–19 0.3 PFOS or NR NS NS NR NR NR
PFOA
[42] 129S1/SvlmJ wild-type GD 2–18 0.1, 0.3, 0.6, 1, Survival reduced in NS NS Increased in 1 mg/kg NR NR Delayed pubertal maturation
(WT) and PPARa 3, 5, 10, 20 WT neonates at in WT and 3 mg/kg from 1 mg/kg in WT.
knockout (KO) mice PFOA 0.6 mg/kg; full litter in KO
resorptions at

399
 5 mg/kg
[43] C57BL/6 J x FVB mice GD 1–19 0.003, 0.01, NS NS Reduced in a Increased in a NR NR
0.03, 0.1, 0.3, 1, dose-dependent dose-dependent
3 PFOA manner from PND 4 manner
[44] CD1 mice GD 1–18 1, 5, 10 PFOA All born alive but Reduced at 10 mg/kg Reduced in 5 and Increased in a NR NR Delayed bone formation in
survival reduced 10 mg/kg dose-dependent fetuses in 10 mg/kg.
from 5 mg/kg manner
[45] CD1 mice GD 2–18 1, 3, 5, 10, 20, Reduced in 20 mg/kg Reduced in 20 mg/kg Reduced in 20 mg/kg Increased in a NR NR Accelerated sexual maturation
40 dose-dependent was observed in male
PFOA manner offspring; slight pubertal delay
in female offspring in
40 mg/kg.

BW, body weight; FPWR, fetoplacental weight ratio; GD, gestational day; NR, not reported; NS, no significant difference
 Z. Aghaei et al., 2022, Vol. 106, 4

on survival [26, 31, 43]. In addition to effects on fetal

female mice showing no significant differences from controls
weight, birth defects have been reported following prenatal
[41, 54]. Muscle strength and motor coordination were also
PFOA and PFOS exposure including craniofacial
found to be dependent on sex, with only male C57Bl/6/Bkl-
abnormalities, delayed bone formation, abnormal bone
exposed mice showing deficits compared to controls [41].
mineral density, and cardiac defects [39, 44, 48, 49].
Both male and female CD1 mice were found to have delays in
Prenatal exposure to PFOS in CD1 mice resulted in severe
motor function following exposure to PFOS [56]. One study
lung collapse in neonates [40]. More- over, exposure to
investigated the response of mice to a nicotine challenge where
PFOA and PFOS throughout gestation and into adulthood in
the expected response from untreated mice is hyperactivity. In
BALB/c mice had a negative impact on lung development
contrast, following maternal exposure to PFOA or PFOS, the
[50].
NMRI mice were found to be hypoactive [55]. However, a
Many of these studies looked at multiple doses of PFOA
study in CD1 mice did not reproduce this result following
and PFOS and found the effects to be dose dependent. The
nicotine injection but instead found a hypoactive response
variability in dose and length of administration could explain
with a methamphetamine challenge [57]. It should also be
some of the inconsistency in the results. For example, a low

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noted that there are studies that found no differences in
dose (0.3 mg/kg/day) of PFOA or PFOS had no effect on
habituation in CD1 mice exposed to PFOA or PFOS prenatally
maternal weight gain or fetal weights in C57BL/6/Bkl mice
[56–58]. One explanation for this is that two of the studies
[41] and one day exposure of CD1 dams late in gestation
only looked at exposure during late gestation (GD 13–19) [56,
(GD17) also had no effect on maternal or fetal body weight
58] and one only looked at low doses of PFOA (≤1 mg/kg/day)
[26]. The consensus appears to be that at a significantly high
[57].
dose, exposure to PFOA and PFOS results in fetal growth
Exposure to PFOA and PFOS also impacts cellular and
restriction and, in some cases, impacts maternal health and
molecular mechanisms in the brain. Significant neurologi-
fetal survival. This is consistent with a 2014 meta-analysis
cal effects of PFOA were demonstrated in neonatal NMRI
using data from seven mouse studies, which concluded that
mice by increased levels of the proteins CaMKII, GAP-43,
exposure to PFOA in pregnant mice resulted in a decrease in
synaptophysin, and Tau in the hippocampus, all of which
birth weight of 0.023 g per a 1-mg/kg body weight (BW)/day
are important for neuronal growth and synaptogenesis [59].
increase in PFOA dose [15]. A 2017 meta-analysis by Negri
Quantitative PCR analyses of brain tissue from NMRI mice
et al. [14] reported on the toxicological effects of PFOA (12
exposed to PFOS early in life demonstrated reduced transcrip-
studies in mice) and PFOS (6 studies in mice) on fetal growth.
tion of dopamine receptor-D5 in the cortex and increased
They concluded that PFOA and PFOS were associated with
transcription of tyrosine hydroxylase in the hippocampus,
lower fetal weight when the maternal dose was greater than
showing that PFOS affects the developing cerebral dopamin-
5 mg/kg BW and 10 mg/kg BW, respectively. The authors
ergic system at the gene transcriptional level [60]. In another
cautioned about important differences that should be consid-
study, Kunming mice exposed to PFOA prenatally showed
ered when using mouse studies to draw conclusions about the
enhanced levels of nerve growth factor in sera and cortex cells
effect of maternal PFOA and PFOS exposure in humans. For
at PND 21 [61].
example, the effective serum concentrations in mice compared
The evidence from mouse models of pregnancy suggests
to those found in the general human population are 2–3 orders
prenatal exposure to PFOA and PFOS results in neurological
of magnitude higher [14]. Another important difference is that
impairments including effects on activity levels and motor
PFOA and PFOS are eliminated much faster from animals
function. Several studies reported reduced habituation fol-
than from humans [51]. These differences in elimination kinet-
lowing prenatal PFOA or PFOS exposure. Habituation, usu-
ics may be explained by several factors including differences
ally measured as reduced exploration of an open field, is
in renal tubular resorption and differences in expression of
a normal response in animals and is considered a simple
organic anion transporters [52] [53].
form of learning [62]. To translate the results to humans, we
Effects on brain development and behavior recommend further work investigating the effects of PFOA
The central nervous system can be negatively impacted by and PFOS exposure on more complex tests of learning and
maternal and early life exposure to environmental memory like the Barnes maze [63], Morris water maze [64],
pollutants [54]. Both PFOA and PFOS have been identified in or the fear conditioning task [65]. To our knowledge, none
the brain of offspring of C57BL/6/Bkl mice, with a four of these behavioral assays have been performed in mice
times higher accumulation of PFOS [41]. PFOA and PFOS are exposed prenatally to PFASs.
considered developmental neurotoxicants and the effects on
behavior have been extensively studied (Table 2). Effects on pregnancy hormones and the
Neurocognitive func- tion in mice is often evaluated using a reproductive and immune systems
battery of behavioral tests including mazes to study learning
A growing body of literature has explored the effects of
and memory, open field tests to study anxiety and exploratory
exposure to PFOA and PFOS on pregnancy hormones and
behavior (habituation), rotarod to study motor function, and
the reproductive system of both male and female mice. The
the hanging wire test to measure muscle strength.
effects of direct exposure to PFOA or PFOS in male mice
Mice exposed to PFOA or PFOS prenatally did not show
has shown reproductive toxicity [66], blood–testis barrier
abnormal levels of anxiety measured using the elevated plus
damage [67, 68], reduction in testosterone levels in the testis
maze [41, 55] or depression-like behavior determined by the
[69], and reduction in sperm quality [69]. Similarly, in female
forced swimming test [41]. In an open field test, prenatal
mice, exposure to PFOA and PFOS negatively impacted the
exposure to PFOA or PFOS resulted in reduced habituation
reproductive system. Peripubertal exposure to PFOA stunted
and abnormal activity levels in adult male NMRI mice [55],
mammary gland development and delayed vaginal opening in
male C57BL/6/Bkl mice [41], and male C57BL/6 J mice [54]
BALB/c and C57BL/6 mice [70]. Prenatal exposure to PFOA
compared to controls. This behavior was sex-specific, with
4 Impact of perfluoroalkyl substances on pregnancy, 2022, Vol. 106,

Table 2. Brain developmental and behavioral effects of exposure of mice prenatally to PFOA/PFOS

Ref Mouse strain Exposure time Dosage Motor activity Habituation Additional results
(mg/kg/day)
[41] C57BL/6/Bkl GD 1–19 0.3 PFOS or Reduced (male in PFOS Reduced Reduced muscle strength
mice PFOA group); increased (male in (PFOS, males)
PFOA group)
[54] C57BL/6 J mice GD 8 until PND 0.1 PFOA Increased (male) Reduced (male)
21
[55] NMRI male mice PND 10 0.75, 11.3 Reduced at the beginning Reduced (highest Hypoactivity following
PFOS;0.58, 8.70 and increased at the end dosed) nicotine challenge
PFOA (highest dosed)
[56] CD1 mice GD 13–19 6 PFOS NS NS Delay in neuromotor
maturation
[57] CD1 male mice GD 2–18 0.1, 0.3, 1 PFOA Increased in 1 mg/kg NS Hypoactivity following

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methamphetamine
challenge
[58] CD1 mice GD 13–19 6 PFOS NS NS

BW, body weight; GD, gestational day; NR, not reported; NS, no significant difference; PND, postnatal day

Table 3. Effects of prenatal exposure to PFOA/PFOS on the reproductive system

Ref Mouse strain Exposure time Dosage (mg/kg/day) Serum testosterone

Additional results
level
[72] Kunming mice GD 2–18 1, 5, 10, 20, 40
PFOA NR Increase in the number of apoptotic
[73] Kunming mice GD 2–8 1, 5, 10, 20, 40 uterine cells.
PFOA NR Increase in the number of apoptotic
uterine cells.
[74] Kunming mice GD 1–7 or 1–13 2.5, 5, 10 PFOA NR Reduced numbers and sizes of corpora
lutea; luteal function is inhibited via
ovarian oxidative stress and apoptosis;
reduced serum progesterone levels.
[76] CD1 mice GD 2–18, 9–18, 3, 5 PFOA NR Delayed mammary gland development.
8–18
[77] CD1 and C57BL/6 GD 2–18 0.01, 0.1, 0.3, NR Delayed mammary gland development.
mice 1 PFOA
[78] CD1 mice GD 2–18, 9–18, 5 PFOA NR Reduction in mammary gland epithelial
13–18 differentiation.
[79] CD1 mice GD 11–18 0.01, 0.1, 1 PFOA NR Stunted mammary epithelial growth.
[80] Kunming mice GD 1–17 5 PFOA Reduced Architectural damage and cell apoptosis
in testes.
[81] Kunming mice GD 2–18, 11–18 1, 2.5, 5 PFOA Reduced Architectural damage and decrease in the
number of Leydig cells in the testes.
[82] C57BL/6 mice GD 2–18 0.1, 1, 5 PFOS Reduced (male) Increased estradiol levels only in males.

BW, body weight; GD, gestational day; NR, not reported

in CD1 mice resulted in a significant increase in uterine weight

differentiation was reduced [78], and mammary epithelial
compared to controls and there were histopathologic changes
growth was significantly stunted [79]. Male Kunming mice
to the uterus, cervix and vagina in the exposed group [71].
were shown to have reduced testosterone levels and structural
In pregnant Kunming mice, PFOA exposure resulted in
damage and apoptosis in the testes after prenatal PFOA
apop- tosis of uterine cells, leading to slow embryo
exposure [80, 81]. At 8 weeks of age, significant reduction
development or decreased survival rates [72, 73]. Moreover, it
in testosterone levels was also reported in male C57BL/6 mice
was shown that exposure of pregnant Kunming mice and
(Shanghai Laboratory Animal Center) after prenatal exposure
CD1 female mice to PFOA and PFOS, respectively, reduced
to PFOS [82].
the numbers and sizes of corpora lutea and significantly
The developing immune system can also be affected by
decreased the serum progesterone levels [74, 75]. Other
prenatal exposure to PFOA and PFOS. Prenatal exposure
hormonal effects of PFASs can be seen on thyroid hormones
to PFOS had a significant impact on immunological func-
with serum T4 levels reduced in CD1 pregnant mice exposed
tion, with a decline in natural killer cell activity in 8 weeks
to PFOS during pregnancy [39]. Table 3 summarizes the
old male and female B6C3F1 offspring and a decrease in
studies on the effects of prenatal PFOA and PFOS exposure on
immunoglobulin M production in males but not in females
the reproductive system.
[83]. Male C57BL/6 offspring (Shanghai Laboratory Animal
In addition to the direct effect on the parents, maternal
Center) were found to be more sensitive than females to the
exposure to PFOA and PFOS has been shown to have adverse
immunotoxic effects of prenatal exposure to PFOS [82]. In
effects on pubertal development. Prenatal PFOA exposure
this study, there was a significant effect on lymphoid organ
is associated with delays in the development of mammary
weights and an imbalance in TH1/TH2-type cytokines with
glands in the female offspring of CD1 and C57BL/6 mice [76,
excess TH2 cytokines (IL-4) in 4 and 8 weeks old male mice
77]. In addition, in CD1 offspring mammary gland, epithelial
 4 Impact of perfluoroalkyl substances on pregnancy, 2022, Vol. 106,

but not in females. Similarly, exposure of pregnant C57BL/6 N

mice to PFOA showed no effect on lymphoid organ weights
Conclusions and future directions
or T cell–dependent antibody responses in female offspring
PFOA and PFOS have been detected in the placenta and are
(male offspring were not studied) [84].
known to cross the placental barrier into the fetus. Animal
studies of exposure to PFOA and PFOS during pregnancy
Effects on liver and metabolites have clearly demonstrated the detrimental effects of these
pollutants on maternal and fetal health. Exposure to PFOA
Mouse studies have shown that PFOA and PFOS exposure
and PFOS has wide-ranging effects in mice including mater-
during pregnancy results in increased maternal liver weight
nal liver dysfunction, fetal growth restriction, neurological
(Table 1) and histopathological findings that are consistent
impairments, and increased risk for chronic diseases in adult-
with liver toxicity and metabolic disorders including abnor-
hood such as liver disease and cancer. While the experimental
mal hepatic cells, increased lipid accumulation, and evidence
outcomes were generally the same for PFOA and PFOS,
of lesions [31, 43, 85]. Exposure of pregnant CD1 mice
there were some notable differences. Further research into
to PFOA also has a significant impact on liver enzymes

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the mechanism of action of these two pollutants may explain
resulting in lower serum albumin, increased aspartate amino-
these differences. After normalizing for the body surface area
transferase, increased sorbitol dehydrogenase, and lower total
between humans and mice [92], 0.1–5 mg/kg of PFOA/PFOS
serum protein when compared to controls [31]. Liver toxicity
equates to a human equivalent dose of 0.008–0.4 mg/kg for
following PFOA exposure may result from oxidative damage.
a 60-kg person. The human tolerable daily intake (TDI) for
A recent study in pregnant Kunming mice showed PFOA
PFOA and PFOS is 1.5 μg/kg and 150 ng/kg, respectively
exposure induced significant decreases in two antioxidant
[93]. Therefore, the dose range typically administered in mice
enzymes in liver tissue, superoxide dismutase and glutathione
to study the health effects of PFASs is much higher than the
peroxidase, that protect cells from damage by oxidative stress
human TDI. This underlies the urgency of studying exposures
[73]. The authors also found an increase in malondialdehyde,
to low concentrations using experimental assays that can
a marker for oxidative stress.
detect subtle differences following exposure as new PFASs are
Several studies show damage to the liver of the offspring,
identified in humans and wildlife. For example, metabolomics
evidenced by a significant increase in fetal liver weight fol-
approaches are highly sensitive and can detect subtle changes
lowing prenatal exposure to PFOA [41–43, 79, 86]. Table 4
in an organism’s biochemistry that may have a significant
summarizes the effects of prenatal exposure to PFOA and
impact on health. One major limitation of the available results
PFOS on the liver and metabolites. Consistent with liver cell
is that comparing data between studies is challenging because
damage, significant increases in the serum levels of
of the lack of consensus on dose (concentration and single
aspartate aminotransferase and alanine aminotransferase
vs. multiple dose), gestational window for administration,
were reported for Kunming and CD1 offspring after prenatal
and mouse strain. A goal for the reproductive toxicology
exposure to PFOA [27, 61, 86] and C57BL/6 offspring after
field would be for researchers using experimental animals
prenatal exposure to PFOS [28]. Exposure of pregnant
to adopt standardized guidelines such as those described by
CD1 mice to PFOA resulted in abnormal lipid metabolism, a
the International Council for Harmonisation (ICH) and the
hepatic inflammatory response, and histopathologic lesions
Organization for Economic Co-operation and Development
in the livers of the offspring [27, 87, 88]. A similar result
(OECD).
was found for PFOS exposure, with the offspring of CD1 and
The evidence from mouse models of pregnancy suggests
Kunming mice showing an inflammatory response and
prenatal PFOA and PFOS exposure has a significant impact
abnormal syn- thesis and metabolism of fatty acids and
on placental development. However, the effect of exposure
lipids in the liver [28, 85]. Lai et al. [28] also reported an
on placental vascular morphology and blood flow has yet to
activation of some liver cancer–promoting pathways,
be established. Ways to address these knowledge gaps are ex
suggesting prenatal PFOS exposure plays a role in liver
vivo microcomputed tomography imaging of the fetoplacental
cancer in offspring.
arterial tree [94] and the use of high-frequency ultrasound
Exposure to PFOA and PFOS is also associated with other
biomicroscopy [95] to quantify changes in umbilical artery
metabolite alterations such as changes in glucose metabolism
blood flow after exposure to PFOA or PFOS. Microcomputed
in dams and offspring. For instance, in CD1 mice, it was
tomography has been used to study the negative effects of
observed that prenatal exposure to PFOA led to an increase in
maternal exposure to polycyclic aromatic hydrocarbons on
serum concentration of insulin and leptin at 21 and 33 weeks
placental vascular structure [96], and high-frequency ultra-
of age [89]. A study of PFOS exposure in CD1 pregnant mice
sound has been used to investigate changes to placental blood
reported a significant increase in serum insulin levels at 3 and
flow in environmental exposure models of pregnancy compli-
9 weeks of age but only in male offspring [90]. Metabolomics,
cations such as in utero exposure to chronic hypoxia [97].
employing nuclear magnetic resonance or mass spectrometry
While PFOA and PFOS have been shown to accumulate in
to measure low molecular weight metabolites, is an excit-
the heart [98] and kidneys [99] of adult rodents, no study has
ing field that provides a unique “chemical fingerprint” of
investigated the effects of prenatal PFAS exposure on heart
the physiological state of an organism. Alterations in the
and kidney function in either the mother or her offspring.
profile of metabolites indicate abnormal cellular processes.
Another important consideration for future investigations is
How prenatal exposure to PFASs impacts metabolites has not
the importance of sex as a biological variable. Placental struc-
been fully explored. Reardon et al. showed that exposure to
ture and function and brain development are known to be sex-
PFOS early in life altered metabolite profiles in the rat brain
ually dimorphic. Several studies have reported significant sex
[91]. This approach has the potential to provide insight into
differences in outcomes following prenatal exposure to PFOA
the sublethal effects of these contaminants on biochemical
and PFOS; however, many studies only included one sex.
pathways during pregnancy and to identify serum biomarkers
To better understand how exposure to contaminants impact
of exposure to PFASs.
 Z. Aghaei et al., 2022, Vol. 106, 4
Table 4. Effects of prenatal exposure to PFOA/PFOS on liver and metabolites

Ref Mouse strain Exposure time Dosage (mg/kg/day) Fetal liver weight Additional results
[27] CD1 mice GD 13–18 0.05
PFOA NR Increased serum levels of aspartate
aminotransferase, alanine aminotransferase, and
alkaline phosphatase (male offspring)
Hepatic inflammatory response, abnormal lipid
metabolism, and lipid accumulation in
[28] CD1 mice GD 1–19 0.3 hepatocytes (male offspring)
PFOS NR Hepatic inflammatory response, abnormal
synthesis, and metabolism of fatty acids and
lipids in the fetal liver
Activation of liver cancer–promoting signaling
pathways in fetal liver
Increased serum levels of aspartate

Downloaded from https://academic.oup.com/biolreprod/article/106/3/397/6454968 by guest on 02 October 2023

aminotransferase and alanine aminotransferase
in offspring
[41] C57BL/6/Bkl mice GD 1–19 0.3 PFOS or PFOA Increased in PFOA group
[42] 129S1/SvlmJ GD 2–18 0.1, 0.3, 0.6, 1, 3, Increased from 0.1 mg/kg
wild-type (WT) and 5, in WT and 3 mg/kg in KO
PPARα knockout 10, 20 PFOA
(KO) mice
[43] C57BL/6 J x FVB Increased in a Nuclear dysmorphology of the hepatic cells
mice GD 1–19 0.003, 0.01, 0.03, dose-dependent manner Increased lipid accumulation (male offspring)
0.1, 0.3, 1, 3 PFOA
[61] Kunming mice GD1-PND21 5 NR Increased serum levels of aspartate
PFOA aminotransferase and alanine aminotransferase
at PND 21
[73] Kunming mice GD 2–8 1, 5, 10, 20, 40 NR Decreased superoxide dismutase and glutathione
PFOA peroxidase in liver in dose-dependent manner
Increased malondialdehyde from 10 mg/day
[79] CD1 mice GD 2–18 0.3, 1, 3 PFOA Increased at all doses for
GD 11–18 0.01, 0.1, 1 GD 2–18 and in 1 mg/kg
PFOA for GD 11–18
[85] Kunming mice GD 1–21 0.5, 5 NR Inflammatory response and abnormal synthesis
PFOS and metabolism of fatty acids and lipids in the
offspring liver
[86] Kunming mice GD 2–18 1, 2.5, 5, 10 PFOA Increased Increased serum levels of aspartate
aminotransferase and alanine aminotransferase
(female offspring)
Morphological changes in liver of female
offspring from 5 mg/kg
[87] CD1 mice GD 2–18 0.01, 0.1, 0.3, 1 NR Hepatic inflammatory response in the livers of
PFOA the female offspring at PND 21 and 91
Abnormal metabolism of fatty acids and lipids in
the female offspring liver
[89] CD1 mice GD 2–18 0.01, 0.1, 0.3, 1, 3, 5 NR Increased serum concentration of insulin and
PFOA leptin at 21 and 33 weeks of age in 0.01 and
0.1 mg/kg
[90] CD1 mice GD3-PND21 0.3, 3 NR Increased serum insulin levels at 3 and 9 weeks
PFOS of age in male offspring

BW, body weight; GD, gestational day; NR, not reported; NS, no significant difference; PND, postnatal day

birth outcomes and fetal development, we must include both

the frequency of placental abnormalities. Another common
sexes in the study design and include sex as a covariate in
replacement for PFOA and PFOS, perfluorobutanesulfonate
the analyses. Finally, to extend the findings of these mouse
(PFBS), has likewise shown negative impacts on development,
studies to humans, we need to make use of noninvasive
although only female CD1 offspring were studied [100]. This
experimental assays such as high-frequency ultrasound and
study showed a decrease in fetal body weight, a decrease in
magnetic resonance imaging.
thyroxine and an increase in thyroid-stimulating hormone,
The large body of evidence showing the negative impacts
and delayed pubertal development. Other alternatives are
of PFOA and PFOS has led to replacement chemicals being
PFAS polymers that are thought to be less harmful to human
implemented. Although there are few studies examining the
and environmental health; however, the production of these
developmental effects of these compounds, the results indicate
compounds creates nonpolymers and the polymers break
somewhat similar findings to studies of PFOA and PFOS.
down over time forming the same monomeric PFASs that
Hexafluoropropylene oxide dimer acid, often referred to as
are being replaced [101]. PFOA and PFOS can also be
GenX, is a common substitute for PFASs that has been shown
generated by (bio)transformation of other precursor PFASs.
to similarly increase maternal weight gain, maternal liver
For exam- ple, 8:2 fluorotelomer alcohol (FTOH) can be
weight, and placental weight in CD1 mice [31]. It was also
transformed into PFOA by both metabolism and
shown to reduce the fetoplacental weight ratio and increase
atmospheric oxidation [102]. Following maternal exposure
4 Impact of perfluoroalkyl substances on pregnancy, 2022, Vol. 106,

of CD1 mice to 8:2

 Z. Aghaei et al., 2022, Vol. 106,
FTOH, PFOA was detected in the fetuses and found to result
in an increase in fetal liver weight and in neural tube defects
[103]. The paucity of pregnancy research on recently used
PFASs such as GenX chemicals, short chain PFASs like PFBS
and FTOH, and other PFAS polymers presents a large knowl-
edge gap in this field. These compounds should be highly
considered for future investigations to determine their impacts
on mouse dams and the developing fetus. In addition, there
are over 9000 compounds that are structurally similar to
PFOA and PFOS but whose occurrence and toxicology are
unknown. Some of these substances can be complex mixtures
of homologues and therefore the actual number of unknown
PFASs could be orders of magnitude higher. There is an urgent
need to develop methods to enable the identification of undoc-
umented or unknown PFASs. One way to accomplish this is
the use of high-resolution mass spectrometry (HRMS) and
nontargeted analysis [104, 105]. The mouse promises to play
an important role in determining the potential health risks
during pregnancy following exposure to these new PFASs.
Conflict of interest
No conflicts of interest are declared by the authors.
Data availability statement
No new data were generated or analyzed in support of this
research.
Author contributions
ZA, KLS, and LSC drafted the manuscript; ZA, KLS, KJJ, and
LSC edited and revised the manuscript; ZA, KLS, KJJ, and
LSC approved the final version of the manuscript.
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