Polyphenols and pregnancy (1)

...........................................................................................................................
The effects of dietary polyphenols
on reproductive health and early
development†
Christina Ly1,2,*
, Julien Yockell-Lelie`vre2, Zachary M. Ferraro3,
John T. Arnason4, Jonathan Ferrier1,2,4,5, and Andreé Gruslin1,2,3
1
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 2
Chronic Disease Program,
Ottawa Hospital Research Institute, Ottawa, ON, Canada K1Y 4E9 3
Division of Maternal-Fetal Medicine, The Ottawa Hospital, Ottawa, ON,
Canada K1H 8L6 4
Centre for Research in Biotechnology and Biopharmaceuticals, University of Ottawa, Ottawa, ON, Canada K1N 6N5
5
Bruker BioSpin Corp., Billerica, MA 01821, USA
*Correspondence address. Tel: +1-613-218-1210; E-mail: cly032@uottawa.ca
Submitted on May 1, 2014; resubmitted on September 30, 2014; accepted on October 16, 2014
table of contents
† Introduction
† Methods
† Classification and dietary sources of polyphenols
† Polyphenol pharmacokinetics and bioavailability
Absorption, metabolism and elimination
Bioavailability
† Molecular targets of polyphenols: an overview of their potential beneficial effects
Polyphenols and oxidative stress
Polyphenols and inflammation
Polyphenols and AGEs
† Potential hazardous effects of polyphenols
Fertility and sexual development
Fetal health
Bioavailability of substrates
† Dietary intake of polyphenols during pregnancy
† Human studies and translational potential
† Conclusion and recommendations for future research
background: Emerging evidence from clinical and epidemiological studies suggests that dietary polyphenols play an important role in the
prevention of chronic diseases, including cancer, cardiovascular disease, diabetes and neurodegenerative disorders. Although these beneficial
health claims are supported by experimental data for many subpopulation groups, some studies purport that excessive polyphenol consumption
may have negative health effects in other subpopulations. The ever-growing interest and public awareness surrounding the potential benefits of
natural health products and polyphenols, in addition to their widespread availability and accessibility through nutritional supplements and fortified
foods, has led to increased consumption throughout gestation. Therefore, understanding the implications of polyphenol intake on obstetrical
health outcomes is of utmost importance with respect to safe consumption during pregnancy.
methods: Using relevant keywords, a literature search was performed to gather information regarding polyphenol pharmacology and the
molecular mechanisms by which polyphenols exert their biological effects. The primary focus of this paper is to understand the relevance of
these findings in the context of reproductive physiology and medicine.
†
This manuscript is dedicated to the memory of our co-author Andree Gruslin who passed away in 2014.
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Human Reproduction Update, Vol.0, No.0 pp. 1–21, 2014
doi:10.1093/humupd/dmu058
Human Reproduction Update Advance Access published November 5, 2014
atFloridaAtlanticUniversityonNovember18,2014http://humupd.oxfordjournals.org/Downloadedfrom

results: Evidence from both in vitro experiments and in vivo studies using animals and humans demonstrates that polyphenols regulate key
targetsrelatedtooxidativestress,inflammationandadvancedglycationendproducts.Althoughthemajorityofthesestudieshavebeenconducted
in the context of chronic diseases, such as cancer and diabetes, several of the key targets influenced by polyphenols are also related to a variety of
obstetrical complications, including pre-eclampsia, intrauterine growth restriction and preterm birth. Polyphenols have also been shown to influ-
ence fertility and sexual development, fetal health and the bioavailability of nutrients.
conclusions: Further research leading to a thorough understanding of the physiological roles and potential clinical value that polyphenol
consumption may play in pregnancy is urgently needed to help inform patient safety.
Key words: polyphenols / reproduction / pregnancy / molecular targets / beneficial and adverse effects
Introduction
Polyphenols (also known as phenolics) are the most abundant dietary
antioxidants and are common constituents of many plant food
sources, including fruits, vegetables, seeds, nuts, chocolate, wine,
coffee and tea. Natural polyphenols have garnered significant interest
within the scientific community and public media. This spotlight has
mainly resulted from emerging evidence which supports a role for poly-
phenols in the prevention of degenerative diseases, particularly cancer,
cardiovascular disease, diabetes and neurodegenerative disorders (Scal-
bert et al., 2005a, b). As well, an assumption by some members of the
general public is that if a natural health product is made of natural sub-
stances, then it should be safe to consume (Ipsos-Reid, 2010). As a
result, this has created a real interest from the general population to in-
crease their intake of polyphenols through a variety of sources. These
sources include nutraceutical foods (e.g. bran, flax and hemp harts), heri-
tage varieties of foods (e.g. purple potatoes), foods and drinks fortified
with nutraceutical extracts (e.g. pomegranate, grape and cranberry), as
wellas concentrated and diverse sourcesof polyphenolics in dietary sup-
plements (USA), natural health products (Canada), complementary and
alternative medicines (Australia), phytomedicines (EU) and traditional
Chinese medicines (Asia). Consequently, these sources are frequently
consumed at conception and throughout gestation. Despite the benefi-
cial effects observed in many human subpopulations, evidence from ex-
perimental studies raise concerns regarding the potential hazards that
excessive polyphenol consumption may have on health (Chavarro
et al., 2008; Zielinsky et al., 2010; Jacobsen et al., 2014). One of the
most at-risk groups may be pregnant women and their fetuses. There-
fore, understanding the influence of maternal consumption of these
widely available and used agents on reproductive health is imperative.
This article reviews polyphenol pharmacology and summarizes their
possible beneficial and/or adverse effects on reproductive health and
pregnancy.
Methods
A literature search was performed using the National Center for Biotechnol-
ogy Information (NCBI) PubMed database. The years covered by the search
dated from 1972 to 2014 and no language restrictions applied. Relevant key-
words (e.g. polyphenols, pharmacokinetics, pregnancy and fertility) were
entered inthesearchtogatherinformationregardingpolyphenol pharmacol-
ogy and the molecular mechanisms by which polyphenols exert their
biological effects. The primary focus of this paper is to understand the
relevance of these findings in the context of reproductive physiology and
medicine.
Classification and dietary sources
of polyphenols
According to the Quideau definition, the term ‘polyphenol’ is used to
definecompounds exclusively derivedfrom theshikimate/phenylpropa-
noid and/or the polyketide pathway, featuring more than one phenolic
unit and deprived of nitrogen-based functionalities (Quideau et al.,
2011). Simply, polyphenols may be considered plant-derived and/or
synthetic compounds containing one or more phenol structural units.
Most polyphenols are glycosylated and may be linked with other
phenols, or conjugated with glucuronic acid, galacturonic acid, or gluta-
thione, etc., after metabolism in the body (Tsao, 2010). The bioactivity
of polyphenols is as diverse as their many phytochemical structures
(Cody, 1988; Fig. 1). As such, polyphenols are classified into major
groups such as phenolic acids, stilbenes, lignans and flavonoids, which
can be sub-categorized as flavanols, flavonols, flavones, isoflavones, fla-
vanones, anthocyanins and proanthocyanidins. Phenolic acids and flavo-
noids arethe mostabundant dietary polyphenols; accounting for roughly
one- and two-thirds of the total sources, respectively (Han et al., 2007).
Although the content of various polyphenols present in food sources
varies, the general distribution and approximate quantities of these com-
pounds in common food items have been summarized in Tables I and II.
Since several thousand naturally occurring polyphenols have been iden-
tified, this review will focus on those most abundant in the human diet
and with the greatest documentation in the literature.
Polyphenol pharmacokinetics
and bioavailability
Although thehealth benefits of polyphenols appear generally to be dose-
dependent,themostabundantpolyphenolsinthehumandietarenotne-
cessarily the most bioactive. The bioactivity of each polyphenol depends
on the level of its activity (e.g. antioxidant capacity) and the extent to
which it is absorbed, distributed and metabolized within, and eliminated
from the body (i.e. its pharmacokinetics). Researchers have investigated
polyphenol pharmacokinetics in adult subjects by measuring plasma and
urine concentrations of known metabolites following single-dose admin-
istration of the pure compound or food/beverage of interest (Scalbert
and Williamson, 2000; Manach et al. 2004, 2005). There is wide variabil-
ity in the kinetics and bioavailability for different polyphenols and some
information regardingthe fate of these compounds remains unclear. Fur-
thermore, due to extensive metabolism by the intestine and liver, the
metabolites found in the circulation, urine and target organs often
differ from the parent compound (Manach et al., 2004); this adds
2 Ly et al.

another level of complexity when studying biological activity in vitro and
in animal models. Therefore, understanding polyphenol kinetics and
bioavailability is critical for understanding the health effects of these
compounds.
Absorption, metabolism and elimination
The physicochemical properties of polyphenols, including molecular
weight and extent of glycosylation and esteriﬁcation, are major determi-
nants of intestinal absorption (Scalbert et al., 2002). Higher molecular
weight polyphenols areless likely to be absorbed in the gut, as are antho-
cyanins which carry a positive charge (De´prez et al., 2001). As a general
rule, polyphenols in the form of esters and glycosides are absorbed less
rapidlyandlessefﬁcientlythanaglycones(compoundsremainingafterhy-
drolysis of phenolic glycosides and esters) and glucosides (glycosides
derived from glucose) (Olthof et al., 2001; Manach et al., 2004). This is
because glycosylated polyphenols are hydrophilic, thus unable to pas-
sively diffuse through the gut wall until they are hydrolyzed (Scalbert
and Williamson, 2000; Crespy et al., 2002; Ne´meth et al., 2003).
However, active transport mechanisms have also been shown in vitro
Figure 1 Chemical structures of selected polyphenols.
Polyphenols and reproduction 3

to carry phenolic glycosides through the intestinal cell wall in the rat
jejunum (Ader et al., 1996). Similarly, absorption of polyphenols
through theplacenta is believedtoinvolve selective transporter mechan-
isms(Unadkatetal.,2004;Chuetal.,2006);althoughtheidentityofthese
transporters remains to be elucidated.
Polyphenols are extensively metabolized by both Phase I and II
enzymesofxenobioticmetabolismwhenpassingthroughthesmallintes-
tine and again in the liver following first-pass clearance via the portal vein
(Donovan et al., 2001; Fisher et al., 2001; Wu et al., 2002). Phase I reac-
tions are primarily carried out by a superfamily of isozymes known as
cytochrome P450-dependent mixed-function oxidases (CYPs), which
make the molecule more polar and are important to facilitate Phase II
conjugation reactions that lead to excretion (Foster et al., 2005).
These reactions are highly efficient as evidenced by the absence or
traceamounts of freeaglycones in circulation afterpolyphenolconsump-
tion (Bell et al., 2000). The identification of conjugated metabolites has
only been investigated for a few polyphenols and the data regarding
the types of conjugates circulating in the human plasma is limited. Never-
theless, it is known that these metabolites are not free in the blood, but
rather extensively bound to plasma proteins, primarily albumin (Boulton
et al., 1998), and that the binding affinity of these metabolites to albumin
dependsontheirchemicalstructure(Dangleset al.,2001).However,the
degree of bindingto albuminand theeffectsthis hasonmetabolite rate of
clearance and biological activity remains unclear (Manach et al., 2004).
Phase I and II enzymes have been identified and are also well character-
izedintheplacentafortheirroleindrugdetoxification(Symeetal.,2004);
althoughtheirinvivointeraction withpolyphenolshasnotbeenreported.
Nonetheless,invitroassaysandinvivostudiesnotfocusedontheplacenta
have clearly shown that polyphenols can have complex effects on drug
metabolism through the activation and inhibition of CYP and Phase II
enzyme activity (Anger et al., 2005; Foster et al., 2005; Li et al., 2006;
Kimura et al., 2010). Ultimately, the effects ofpolyphenols ondrugmetab-
olism in the placenta may be similar, but should be investigated directly.
Following Phase I and II biotransformation, weakly conjugated poly-
phenols re-enter circulation, whereas extensively conjugated polyphe-
nols are excreted in the bile and enter the large intestine. The
microflora hydrolyze glycosides into aglycones and then metabolizes
the aglycones into different aromatic acids, which are well absorbed
across the colonic barrier (Scheline, 1991; Knaup et al., 2007). These
metabolic pathways are well established in animals, but data are still
limited in humans. As such, future research should further identify and
quantify microbial metabolites in humans and investigate any differences
in polyphenol metabolism amongst individuals depending on differences
in their microflora composition and diet. This is of particular importance
in the case of active metabolites (i.e. products of metabolism with bio-
logical activity) since they may have a physiological effect (Kim et al.,
1998). Identification of metabolites unique to the degradation of poly-
phenols may be useful biomarkers of phenolic intake and help research-
ers determine the biological activity of specific polyphenol-derived
conjugates present in vivo.
The elimination profile for each polyphenol is different according to
the nature of the compound, as demonstrated in animal studies
(Crespy et al., 2003). After ingestion, most dietary phenolic metabolites
are rapidly excreted in either urine or bile depending on size and degree
of conjugation (Manach et al., 2004). Generally, the extent of urinary ex-
cretion is proportional to the maximum concentration of metabolites in
the plasma. However, there are some exceptions, as demonstrated for
anthocyanins, where urinary excretion percentages are very low relative
totheplasmaconcentrations(Wu et al.,2002). Thismaybe explained by
higherbiliaryexcretionorextensivemetabolismtocurrentlyunidentified
metabolites or unstable compounds. Metabolites excreted in the bile
and in the intestinal lumen may also undergo bacterial-catalysed
.............................................................................................................................................................................................
Table I Major dietary polyphenols and their general distribution in foods.
Group Subgroup Examples Major food sources
Phenolic acids Benzoic acids Gallic acid Tea leaves21
p-Hydroxybenzoic acid Red fruit (e.g. strawberries and raspberries), onions17
Cinnamic acids Caffeic acid Virtually all fruit12
p-Coumaric acid Cereal grains20
Flavonoids Flavanols Epigallocatechin gallate Green and black tea11
Epicatechin Most fruits, chocolate2
Flavonols Kaempferol, quercetin Onions, broccoli, blueberries6,7,8,9
Anthocyanins Cyanin glucoside Highly pigmented fruit5
Flavones Apigenin, chrysin, luteolin Parsley, celery6,7,9
Isoflavones Daidzein, genistein Soya and its processed products3,16
Flavanones Naringenin Grapefruit14
Hesperetin Oranges14
Stilbenes Resveratrol Red wine, red grape juice15,22
Lignans Secoisolariciresinol Flaxseed13
Sesamin Sesame seed19
Others Chlorogenic acid Most fruit, coffee4
Curcumin Turmeric1
Rutin Citrus fruits10
Silibinin Milk thistle seeds18
1
Aggarwal et al. (2007), 2
Arts et al. (2000a, b), 3
Cassidy et al. (2000), 4
Clifford (1999), 5
Clifford (2000a, b), 6
Crozier et al. (1997), 7
Herrmann (1976), 8
Hollman and Arts (2000), 9
Justesen
et al. (1998), 10
Karimi et al. (2012), 11
Khan and Mukhtar (2007), 12
Manach et al. (2004), 13
Mazur (1998), 14
Mouly et al. (1994), 15
Prasad (2012), 16
Reinli and Block (1996), 17
Shahidi and
Naczk (1995), 18
Siegel and Stebbing (2013), 19
Smeds et al. (2012), 20
Sosulski et al. (1982), 21
Toma´s-Barberan and Clifford (2000), 22
Vitrac et al. (2002).
4 Ly et al.

.................................................. ................................................................................................................................................................
..........................................................................................................................................................................................................................................................
Table II Phenolic acid and ﬂavonoid content of selected foods (milligrams/100 g of fresh weight or 100 ml of liquids).
Source Phenolic acids Flavonoids
Benzoic acids Cinnamic acids Flavanols Flavonols Anthocyanins Flavones Isoﬂavones Flavanones
Fruit
Blueberry 0.3–0.7g
200–220p
1–7c
3–16p
25–500p
0.8h
n.d. 0.00n
Grapefruit 50–100u
0.1h
0.3h
; 40–100u
0.00x
1.4h
n.d. 26.5h
; 160u
Orange 80–100u
0.00c
40–50u
0.00x
0.7h
n.d. 2000u
Raspberry 6–10p
2–3p
3.2–48c
n.d. 23–995i
n.d. n.d. n.d.
Strawberry 2–9p
1–3p
0.6–12.5c
1.5g
; 1.9h
15–75p
; 78.5–385i
0.00–0.03n
n.d. 1.8h
Vegetables
Broccoli 15q
0.00n
0.4h
; 4–10p
0.00x
; 6h
0.8h
n.d. 0.00n
Celery 1.3q
0.00n
0.22n
; 3.5h
0.00x
1.3h
; 2–14p
; 50n
n.d. 0.00n
Parsley 6.2q
n.d. 15m
n.d. 24–184p
; 216m
n.d. 0.00m
Onion 0.79q
0.08n
; 0.1h
7.6–19.8h
; 35–120p
0.00–9.5x
0.00–0.40n
n.d. 0.00n
Cereal grains
Barley 45–130e
239k
n.d. n.d. n.d. n.d. n.d.
Rice 20–38e
1.6–260l
Beverages
Black tea 3.2–3.6o
n.d. 114.30n
4.05n
n.d. 0.00n
n.d. n.d.
Coffee n.d. n.d. 0.08d
0.10j
n.d. 0.00j
n.d. n.d.
Green tea 0.8–1.2o
n.d. 51.03–324.20t
2.81–4.77n
n.d. n.d. n.d. n.d.
Red grape juice n.d. n.d. 0.00r
0.69r
0.49r
n.d. n.d. n.d.
Red wine 2.2–3.4v
0.47–1.1v
11.08–18.36v
0.77–2.11v
19.27–152.98s
0.04–0.17v
n.d. 2.4a
Other
Dark chocolate n.d. n.d. 53.49–108.6f
n.d. n.d. n.d. n.d. n.d.
Soy beans 73q
37.41w
1.26b
n.d. 0.00b
20–90p
n.d.
Tofu n.d. n.d. n.d. 1.19b
n.d. 0.00b
8–70p
n.d.
n.d., indicates that the value has not been determined.
a
Achilli et al. (1993), b
Arai et al. (2000), c
Arts et al. (2000a), d
Arts et al. (2000b), e
Dykes and Rooney (2007), f
Gu et al. (2006), g
Schuster and Herrmann (1985), h
Harnly et al. (2006), i
Heinonen et al. (1998), j
Hertog et al (1993), k
Holtekjølen et al.
(2006), l
Huang and Ng (2012), m
Justesen and Knuthsen (2001), n
Justesen et al. (1998), o
Lin et al. (1998a, b), p
Manach et al. (2004), q
Mattila and Hellstro¨m (2007), r
Mullen et al. (2007), s
Nyman and Kumpulainen (2001), t
Price and Spitzer (1993),
u
Ramful et al. (2011), v
Rodriguez-Delgado et al. (2002), w
Sakakibara et al. (2003), x
Wu et al. (2006).
Polyphenolsandreproduction5
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hydrolysis via b-glucuronidases, which are able to release free aglycones
fromconjugatedmetabolites.Asaresult,aglyconescanbereabsorbedin
both the intestine and the colon and undergo enterohepatic recycling. In
this case, first-pass metabolism and disposition does not result in com-
plete elimination of the substance, but rather significantly increases the
elimination half-life (Wu et al., 2011).
Bioavailability
Postprandial plasma concentrations of polyphenols, primarily present as
conjugated metabolites, vary greatly depending on the chemical charac-
teristics of the polyphenol and the food source. In humans, maximum
plasma concentrations of flavonoids rarely exceed 1 mM, but have
beenreportedtorangebetween0.1and5 mM(ScalbertandWilliamson,
2000). Unless the polyphenol is absorbed only after metabolism by the
colon, peak concentrationsarecommonly reached 1–2 h afteringestion
and then rapidly decline (Scalbert and Williamson, 2000). Thus, main-
taining high concentrations in the plasma requires repeated and consist-
ent intake of the polyphenol (van het Hof et al., 1999; Moon et al., 2000;
Warden et al., 2001).
Bioavailability refers to the amount of phenolic compounds that enter
the circulation upon ingestion. However, what is more physiologically
relevant is the amount of polyphenol that reaches the target tissue and
is subsequently able to elicit a change in intracellular response. Some
studies have reported the concentrations of polyphenols in human
tissues, but these data is limited to only a few polyphenols and select
tissue types, mainly prostate and breast tissues (Hong et al., 2002;
Maubach et al., 2003; Henning et al., 2006). In these studies, polyphenol
concentrations in the tissues vary widely between participants and do
not directly correlate with plasma concentrations. This finding suggests
that caution should be taken when using plasma concentrations as
accurate biomarkers of exposure and intracellular activity within the
target tissue.
Pharmacokinetic studies in rat maternal plasma and fetuses have only
been performed for a few substances, including green tea catechins (Chu
et al., 2006) and grape seed flavanols (Arola-Arnal et al., 2013). In the
study conducted by Chu et al. (2006), dams at 15.5 days of gestation
were fed with 166 mg green tea extract tablet (considered moderate
dosage) containing various catechins, including epicatechin and epigallo-
catechin gallate (EGCG). At several time points after administration,
blood samples were collected and placental and fetal tissues were
obtained. Results showed that maternal plasma concentrations of cate-
chins were 10 times higher than in placenta and 50–100 times higher
than in the fetus. Levels of epicatechin were highest in the plasma while
the levels of EGCG were highest in the placenta and fetus. This suggests
thatepicatechiniswellabsorbedanddistributedinthemother,butnotin
the conceptus. The opposite phenomenon is true for EGCG, suggesting
that EGCG is selectively absorbed and retained by the fetus (Chu et al.,
2006). Arola-Arnal et al. (2013) reported that flavanols and their meta-
bolites were widely distributed in both pregnant and non-pregnant rat
plasma and tissues. Conjugated forms of flavanols were more abundant
intheliversofnon-pregnantratscomparedwithpregnantrats,suggesting
that flavanol metabolism is less active during pregnancy. Furthermore,
flavanol metabolites were abundant in the placenta and detected at
low levels in the fetus and amniotic fluid. Overall, this suggests that
these compounds are able to cross the placental barrier and therefore,
may have biological effects on the offspring.
Molecular targets of polyphenols:
an overview of their potential
beneficial effects
Although the molecular mechanisms of action of polyphenols have been
extensively characterized in systems such as cancer, diabetes and cardio-
vascular disease (Vauzour et al., 2010; Bahadoran et al., 2013), their
effect on pregnancy-related complications is a new and emerging field
of research. The health benefits of polyphenols have been traditionally
attributed to their antioxidant properties. However, more recent evi-
dence suggests that polyphenols can also attenuate inflammation and
inhibit the formation of advanced glycation end products (AGEs).
These mechanistic pathways are summarized in Table III and help
explain the beneficial effects of polyphenols demonstrated in other
systems. As such, the implications of polyphenols and the effects they
have on reproductive health will be discussed here.
Polyphenols and oxidative stress
Reactive oxygen species (ROS) and antioxidant enzyme systems are im-
portant components of many reproductive processes, including ovarian
follicular development, ovulation, fertilization, endometrium receptivity
and shedding, placentation, embryonic development and implantation
(Al-Gubory et al., 2010). Oxidative stress reflects an imbalance
between the generation of ROS/free radicals (e.g. superoxide radical,
hydroxyl radical and hydrogen peroxide) and antioxidant defences
[e.g. copper–zinc superoxide dismutase (SOD) and manganese SOD]
which can result in damage to DNA, proteins and lipids (Sugino et al.,
2007). During early pregnancy, there is a natural increase in ROS gener-
ation caused by the high metabolic rate of the placenta (Al-Gubory et al.,
2010). Consequently,the uterus, embryoand feto-placental unit require
adequate defence mechanisms to protect themselves against oxidative
damage. Theseadaptations areconsideredkeyeventsfora healthypreg-
nancy.Therefore,sufficientantioxidantcapacitycouldpreventorattenu-
ate the severity of those disorders induced by oxidative stress, such as
pre-eclampsia (PE), intrauterine growth restriction (IUGR), preterm
labour and miscarriage (Burton and Jauniaux, 2004; Myatt and Cui,
2004).
Polyphenols are able to directly scavenge free radicals and inhibit
metal-mediated free radical formation (Frei et al., 1989; Jovanavic
etal.,1996;Brownetal.,1998;FreiandHigdon,2003).Theconsumption
of polyphenol-rich foods and beverages has been shown to increase
plasma antioxidant capacity in humans (Prior et al., 2007) and decrease
oxidative stress in vivo and in vitro in human placenta and human placental
trophoblasts, respectively (Chen et al., 2012). Compared with endogen-
ousantioxidants, theimportance ofdietaryantioxidants invivoas oxidant
scavengersisconsideredtobeminorduetotheirlowerreductionpoten-
tials and bioavailability (Frei and Higdon, 2003). Instead, polyphenols are
believed to have a greater role in the prevention of oxidative stress
through indirect mechanisms, summarized by Frei and Higdon (2003)
to include: (i) inhibition of redox-sensitive transcription factors [e.g.
nuclear factor-kB (NF-kB)] (Siddiqui et al., 2008); (ii) down-regulation
of pro-oxidant enzymes [e.g. inducible nitric oxide synthase (iNOS)
and cyclooxygenase (COX)-2] (Chan et al., 1997; Lin and Lin, 1997);
and (iii) induction of Phase II enzymes [e.g. glutathione S-transferase
(GST)] (Khan et al., 1992; Lin et al., 1998a, b).
6 Ly et al.

Many of these pathways have been shown to play key roles in the
pathophysiologyofadversepregnancyoutcomes.Forinstance,immuno-
histochemical (IHC) analysis conducted by Vaughan and Walsh (2012)
showed that pre-eclamptic placenta displayed almost a 10-fold increase
in the p65 subunit of NF-kB localized mainly in the cyto- and syncytiotro-
phoblasts compared with healthy controls. Many of the gene products
stimulated by NF-kB [e.g. corticotropin-releasing hormone, tumour ne-
crosis factor alpha (TNF-a), and interleukin 1beta (IL-1b)] are also ele-
vated, suggesting that increased NF-kB signalling is implicated in the
pathogenesis of PE (Goksu et al., 2012). Moreover, IHC analysis of pre-
eclamptic placenta demonstrated a significantly elevated expression in-
tensity of iNOS in trophoblast cells (Schiessl et al., 2005) which is
known to lead to increased production of NO-derived oxidants
capable of damaging DNA and proteins. Furthermore, expression of
the pro-oxidant enzyme COX-2 was shown to be increased in placental
syncytiotrophoblasts (Goksu et al., 2012) and neutrophils (Bachawaty
et al., 2010). Lastly, placental levels of GST are reduced in PE (Zusterzeel
et al., 1999) which is of importance as GST is a major detoxifying enzyme
that neutralizes the reactivity of electrophiles and therefore, prevents
electrophile-mediated DNA and protein damage.
The antioxidant activity of polyphenols has also been demonstrated in
animal models of oxidativestress.Administration of tea polyphenols was
reported to attenuate experimentally induced decreases in antioxidant
enzyme activities, including infection-associated reduction of SOD
(Guleria et al., 2002) and ethanol-associated reduction of glutathione
peroxidase (Skrzydlewska et al., 2002a, b) activities. Although the
levels of these enzymes have been shown to be lower in pre-eclamptic
placentaltissuescomparedwithgestationalage-matchedcontrolplacen-
tae from non-pre-eclamptic pregnancies (Vanderlelie et al., 2005), the
preventive effects of polyphenol consumption on antioxidant enzymatic
activityduringpregnancyhasyettobeexplored.Inaddition,studiesusing
animal models of atherosclerosis have demonstrated that tea and tea
polyphenol administration increases the resistance of lipoproteins to
ex vivo oxidation and decreases the rate of low-density lipoprotein
(LDL) oxidation ex vivo (Anderson et al., 1998; Kasaoka et al., 2002;
Yokozawaetal., 2002). Similarly, assessmentofthiobarbituric acidreact-
ive substances, an indicator of lipid peroxidation, in plasma and tissue
samplesofanimalmodelsofcancerandatherosclerosisuponpolyphenol
consumptionsupportstheantioxidantcapabilitiesofplantpolyphenolsin
vivo (Matsumoto et al., 1996; Hayek et al., 1997; Tijburg et al., 1997). Of
.............................................................................................................................................................................................
Table III Molecular targets of polyphenols.
Target Biological effect
Oxidative stress
Free radicals Neutralize free radicals and free radical formation4,13,14,20
Redox-sensitive transcriptionfactors(e.g.NF-kB,AP-1) Prevent transcription factor binding to DNA26,29,36
Pro-oxidant enzymes (e.g. iNOS, COX-2) Down-regulate gene expression and enzyme activity7
Phase II enzymes (e.g. GST, GP, catalase, SOD) Activate enzyme activity22,25,26
Lipoproteins Attenuate the rate of LDL oxidation ex vivo1,21,42
Lipids Decrease lipid peroxidation17,28,38
Inflammation
COX-1/COX-2 Inhibit gene expression and enzyme activity; prevent COX-mediated PG synthesis19,23,40,41,43
LOX Inhibit enzyme activity10,24,32
PLA2 Selective inhibition of PLA2 isoforms27,39
iNOS Down-regulate transcription and translation; inhibit NO production9,16,37
NF-kB Inhibit activation and downstream signalling (e.g. production of cytokines)3,15,33
PPAR Activate receptor11,18,44
AGE–RAGE pathway
Reactive carbonyl species Scavenge intermediate products in AGE formation process which inhibits AGE production and
cross-link formation2,6,30,34
IKK Inhibit IKK activity; prevent NF-kB binding to DNA; attenuate AGE-mediated production of
TNF-a8,31
NADPH oxidase Reduce mRNA and protein expression12,35
RAGE Reduce protein expression5
NF-kB, nuclear factor-kB; AP-1, activator protein 1; iNOS, inducible nitric oxide synthase; COX-1, COX-2, cyclooxygenase-1, 2; GST, glutathione S-transferase; GP, glutathione
peroxidase; SOD, superoxide dismutase; LDL, low-density lipoprotein; PG, prostaglandin; LOX, lipoxygenase; PLA2, phospholipase A2; NO, nitric oxide; PPAR, peroxisome
proliferator-activated receptor; AGE, advanced glycation end product; IKK, IkB kinase; TNF-a, tumor necrosis factor alpha; NADPH, nicotinamide adenine dinucleotide phosphate;
RAGE, receptor for AGE.
1
Anderson et al. (1998), 2
Babu et al. (2006), 3
Bharrhan et al. (2012), 4
Brown et al. (1998), 5
Burckhardt et al. (2008), 6
Cervantes-Laurean et al. (2006), 7
Chan et al. (1997), 8
Chandler et al.
(2010), 9
Chenetal. (2001),10
Chi etal.(2001),11
Danesi etal. (2009),12
Da´valosetal. (2009),13
Freietal. (1989),14
Freiand Higdon (2003),15
Giorgi etal. (2012),16
Ha¨ma¨laïnen etal. (2007),
17
Hayeketal. (1997),18
Jacobetal. (2007),19
Jangand Pezzuto(1999),20
Jovanavicetal. (1996),21
Kasaoka etal.(2002),22
Khanetal. (1992),23
Landolfi etal. (1984),24
Laughtonetal. (1991),
25
Linetal. (1998a,b), 26
Lin andLin (1997),27
Lindahl andTagesson (1993),28
Matsumotoetal. (1996),29
McCarty (1998),30
Pengetal. (2008),31
Rasheedetal. (2009),32
Reddyetal. (1991),
33
Romier et al. (2008), 34
Sajithlal et al. (1998), 35
Sarr et al. (2006), 36
Siddiqui et al. (2008), 37
Soliman and Mazzio (1998), 38
Tijburg et al. (1997), 39
Tsao et al. (2012), 40
Williams et al. (1999),
41
Yasukawa et al. (1998), 42
Yokozawa et al. (2002), 43
Yoshimoto et al. (1983), 44
Zoechling et al. (2011).

great interest, however, is that increased plasma levels of LDL oxidation
and lipid peroxidation are associated with fetal growth restriction and PE
(Kharb, 2000; Sańchez-Vera et al., 2005; Qiu et al., 2006; Karowicz-
Bilinska et al., 2007).
In theory, sufficient maternal antioxidant status before and during
pregnancy may help prevent and/or manage adverse mechanisms intim-
ately relatedtopoor reproductiveoutcomesandthatarealsoassociated
with poor dietary habits and oxidative stress. However, results from
several clinical trials that have studied the use of antioxidant supplemen-
tation, specifically vitamin C and E, as a therapy to improve pregnancy
outcome have been unsuccessful. Briefly, vitamin C and E therapy
aimed at reducing the risk of PE in women at high risk or low/moderate
risk for PE was not effective (Spinnato et al., 2007; Roberts et al., 2010).
Instead, women supplemented with these vitamins were at increased
risk for developing gestational hypertension and premature rupture of
membranes (Conde-Agudelo et al., 2011). High-dose vitamin C and E
supplementationforwomenatriskofPEhasalsobeenshowntoincrease
the rate of babies born with a low birthweight (Poston et al., 2006). The
unsuccessful use of vitamin C and E supplements maybe partly explained
by an imbalanced administration of vitamins and/or trace elements
(Al-Guboryetal.,2010).AsdescribedbytheEUROFEDAproject (Euro-
pean Research on the Functional Effect of Dietary Antioxidants), no
single antioxidant is more essential than another, thus preferentially
selecting a specific antioxidant supplement may not be justified (Astley
and Lindsay, 2002; Al-Gubory et al., 2010). Furthermore, at higher
doses similar to those found in supplements, evidence suggests that vita-
mins C and E act as pro-oxidants (Rietjens et al., 2002; Poston et al.,
2006) which may explain the adverse effects seen with their usage. An
alternative approach to prevent adverse pregnancy and birth outcomes
associated with oxidative stress is through nutritional intervention by
using phytonutrients from fruits and vegetables that are nutritionally
balanced and rich in multiple antioxidant vitamins and essential trace
elements (Polidori et al., 2009; Al-Gubory et al., 2010). However,
more research on the requirements of maternal antioxidant micronutri-
ents for normal fetal growth and development is required and limited
at present.
Polyphenols and inflammation
Inflammation is required to promote healing and is an immunological
defence mechanism by which tissues respond to an insult. Inflammation
is characterized by the up-regulation of proinflammatory chemokines,
cytokines and other inflammatory mediators. Ovulation, menstruation,
implantation and parturition are all inflammatory processes. As such,
physiologic inflammatory responses are crucial to reproductive
success. In general, there are three immunological phases of a healthy
pregnancy which coincide with the first, second and third trimesters.
Briefly, the first and third trimesters are proinflammatory phases due
to the insults caused by blastocyst implantation and parturition, respect-
ively. Conversely, the second trimester represents a predominant anti-
inflammatory state since the maternal and feto-placental immune
systemsareatequilibrium(Moretal.,2011).Topreparefortheimmuno-
logical events during pregnancy, the human decidua contains a high
number of immune cells, including macrophages, dendritic cells, mast
cellsand naturalkillercells(Bulmeretal., 1988;King etal., 1997;Zenclus-
sen, 2005; Mor et al., 2006, 2011). These immune cells secrete proin-
flammatory agents to regulate trophoblast development and function
during the first trimester (Mor et al., 2011) and stimulate the production
ofuterineactivationproteinsduringthethirdtrimester(Christiaensetal.,
2008).Althoughdepletionofthesesignallingmoleculeshasseriousimpli-
cations for placental development, implantation and decidualization
(Manaseki and Searle, 1989; Greenwood et al., 2000; Hanna et al.,
2006), an exaggerated inflammatory response is also a mechanism for
disease in preterm labour, PE and other obstetrical complications
(Romero et al., 2007).
Greater intake of polyphenol-rich foods has been associated with
decreased incidence of chronic inflammatory diseases in many subpopu-
lations (Yoon and Baek, 2005). Also, several anti-inflammatory drugs, in-
cluding Aspirinw
and Merivaw
, have been derived from or are based on
phenolic compounds (Cragg et al., 1997; Belcaro et al., 2010; Fu¨rst and
Zu¨ndorf, 2014). Polyphenols are reported to exert their anti-
inflammatory effects through a variety of molecular targets which can
be divided into two pathways: the arachidonic acid (AA)-dependent
pathway and the AA-independent pathway. COX, lipoxygenase
(LOX) and phospholipase A2 (PLA2) are inflammatory mediators
included in the AA-dependent pathway. Activation of these proteins
leads to the release of AA (a starting point for the general inflammatory
response) and promotes the release of proinflammatory molecules
(Nijveldt et al., 2001). Conversely, NOS, NF-kB and peroxisome
proliferator-activated receptor (PPAR) promote inflammation through
AA-independent pathways.
Many polyphenols, including resveratrol and EGCG, have been shown
topreventprostaglandin (PG)synthesisbyinhibitingCOX-1andCOX-2
at the transcriptional and enzyme level (Yoshimoto et al., 1983; Landolfi
etal.,1984;Yasukawaetal.,1998;JangandPezzuto,1999;Williamsetal.,
1999). PGs areautocrine and paracrine lipid mediators that mediate cer-
vicalripening, stimulateuterinecontractions andmodulate hemodynam-
ic changes. Generally, increased production of stimulatory PGs is
involved in the mechanism leading to preterm labour (Ivanisevic´ et al.,
2001). Similarly, an increase in vasocontricting, platelet-aggregating
PGs is demonstrated in PE (Friedman, 1988). Despite the physiologically
relevant effects that polyphenols have on PG production, their use for
the clinical management of preterm parturition or PE has never been
investigated.
KaempferolandquercetinwereshowntoinhibitLOX(Laughtonetal.,
1991; Reddy et al., 1991; Chi et al., 2001). Normally, LOX activation sti-
mulates eicosanoid production which leads to increased myometrial
contractility (Bennett et al., 1987; Smith et al., 2001). Women with
preterm labour were noted to have increased concentrations of LOX
metabolites in their amniotic fluid, suggesting that these AA-derived
metabolites may play a role in the aetiology of preterm birth (Romero
et al., 1989). Interestingly, when the COX pathway is blocked by select-
ive flavonoids, the LOX pathway continues to produce mediators of in-
flammation (Moroney et al., 1988). In such cases, the production of
leukotrienes and other proinflammatory cytokines (via LOX activation)
mayevenbeaccelerated.Therefore,polyphenols,suchascurcumin,that
can inhibit both the COX and LOX pathways are desirable for treating
inflammation (Fiorucci et al., 2001; Hong et al., 2004; Yoon and Baek,
2005).
Evidence from in vitrostudies suggests that polyphenols exert selective
inhibition of various PLA2 isoforms. For instance, quercetin is a strong in-
hibitor of Group II secretory-PLA2, (s-PLA2), but a very weak inhibitor of
Group I s-PLA2 in plasma from septic shock patients (Lindahl and Tages-
son,1993). Furthermore,prophylacticadministrationofpolyphenol-rich
8 Ly et al.

grapeextractwasshown toattenuateendotoxin-induced s-PLA2 activity
in rats (Tsao et al., 2012), although the activities of specific s-PLA2 groups
were not discussed. In patients with PE, decidual, placental and plasma
levels of PLA2 are elevated (Jendryczko et al., 1989; Lim et al., 1995;
Staff et al., 2003) and plasma levels correlate with the severity of the
disease (Lim et al., 1995). As such, it may be useful to investigate thera-
peutic agents that can decrease levels of PLA2, as seen with polyphenols,
in the context of PE. Interestingly, not all PLA2 isoforms are associated
with increased inflammation. Group V s-PLA2 has been identified to
have a novel anti-inflammatory role in immune complex-mediated arth-
ritis (Boilard et al., 2010), but its interaction with polyphenols has not
been reported.
Ininflammatorydiseases,NOisproducedin greateramountsand acts
as a proinflammatory mediator. Placentae obtained from pregnancies
complicated by IUGR and fetal hypoxia displayed increased NO produc-
tion compared with controls (Tikvica et al., 2008). Moreover, exposure
of endothelial cells to pre-eclamptic plasma was found to stimulate NOS
activityandincreaseNOproduction(Bakeretal.,1995).Inregardstothe
AA-independent pathways, flavonoids, including quercetin and apigenin,
were found to inhibit the production of NO by down-regulating iNOS
transcription and translation in LPS/cytokine-induced cell models of in-
flammation (Soliman and Mazzio, 1998; Chen et al., 2001; Ha¨ma¨laïnen
et al., 2007). Flavonoids also inhibit the production of proinflammatory
cytokines and chemokines, including TNF-a, IL-1b and monocyte
chemoattractant protein-1 (Sato et al., 1997; Wadsworth and Koop,
1999; Nair et al., 2006; Sharma et al., 2007). These effects are likely
mediated through NF-kB, an important regulator of many proinflamma-
torygenes and found to be activein many proinflammatoryconditions.In
vitro studies using mononuclear cells from pre-eclamptic women have
shown that endogenous NF-kB activation and TNF-a and IL-1b
release are elevated compared with non-pregnant women and normo-
tensive pregnant women (Giorgi et al., 2012). However, when the cells
were treated with a silibinin, a main component of the flavonolignan
extract silymarin from milk thistle, levels of NF-kB and cytokines
TNF-aandIL-1bwerereduced(Giorgietal.,2012).Althoughthemech-
anism by which this extract exerts its anti-inflammatory activity is
unknown, in a human intestinal cell line (Caco-2), polyphenols could
inhibit NF-kB by preventing its inhibitor, IkB-a, from being deactivated
by phosphorylation (Romier et al., 2008). Moreover, Bharrhan et al.
(2012) found that polyphenolic compounds down-regulate the levels
of p50, a NF-kB subunit, in rat liver nuclear extracts, which would
further inhibit downstream signalling of NF-kB.
Polyphenols are also able to activate PPARs. PPARs are a group of
nuclear receptors activated by many factors, including PGs and leuko-
trienes. When activated, they act as transcription factors and regulate
processes such as cellular differentiation, apoptosis, lipid metabolism,
peroxisome proliferation and inflammatory responses. During preg-
nancy, PPAR signalling is known to regulate trophoblast invasion and dif-
ferentiation (Schaiff et al., 2000), placentation (Barak et al., 1999) and
maternal metabolism (Waite et al., 2000). Aberrant regulation of the
PPAR system is associated with complicated pregnancy-related condi-
tions, including PE, IUGR and preterm birth (Wieser et al., 2008). Evi-
dence from animal knockout studies and in vitro work suggests that
PPAR activation inhibits the expression of proinflammatory cytokines
anddirectsthedifferentiationofimmunecellstowardsanti-inflammatory
phenotypes (Devchand et al., 1996; Jiang et al., 1998; Martin, 2010).
Many dietary polyphenols have been described as direct agonists of
PPAR. For instance, phenolic compounds found in turmeric, red wine
and green tea, have all been reported to have anti-inflammatory roles
acting chiefly through PPAR activation (Jacob et al., 2007; Danesi et al.,
2009; Zoechling et al., 2011). In addition, polyphenols may up-regulate
the expression of other PPAR agonists, including paraoxonase-1
(Khateeb et al., 2010), furthercontributing to an anti-inflammatorystate.
Non-steroidal anti-inflammatory drugs are commonly prescribed to
treat fever, pain and inflammation. However, their use during pregnancy
has been associated with increased risks of embryo-fetal and neonatal
adverse outcomes (Antonucci et al., 2012). Consequently, future re-
searchneedstohighlightandevaluatemoreeffectivemedicinalstrategies
with feweradverse effects. Although the anti-inflammatory properties of
polyphenols makethese compoundsattractivetherapeuticcandidates in
various inflammatory-mediated diseases, more information regarding
the effects of polyphenols in the context of pregnancy-related pathology
is required. Further understanding of the mechanisms by which polyphe-
nolsexert theiranti-inflammatoryeffects aswellas informationregarding
dose and duration of treatment will be useful for future drug and/or
nutraceutical development.
Polyphenols and AGEs
AGEsareaheterogeneousgroupofcompoundsformednon-enzymatically
between carbonylgroups ofreducing sugars and amino groups of proteins,
lipidsandnucleicacids(BaynesandMonnier,1989;Fig.2).AGEproduction
occurs over a period of months and is part of the natural aging process.
However, their formation in vitro is accelerated by high glucose levels, or
in the presence of oxidative stress (Miyata et al., 1997) which may explain
why the levels of AGEs are more pronounced in diseases, such as PE and
diabetes, where oxidative stress and/or high glucose plays a role. AGEs
are believed to contribute to disease development by: (i) forming cross-
links with one another; and (ii) activating the AGE receptor (RAGE), a
member of the immunoglobulin superfamily of cell surface molecules.
Cross-link formation disrupts the physicochemical properties of a tissue
by increasing the stiffness of the protein matrix and preventing the
normal turnover and degradation of matrix proteins, such as collagen and
elastin, by proteolysis (Monnier et al., 1996; Singh et al., 2001). On the
other hand, AGE–RAGE interaction mediates cellular injury by triggering
a wide range of signalling events that modify the action of hormones, cyto-
kines and chemokines and ROS. Key targets of AGE–RAGE signalling
include NF-kB and nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase (Schmidt et al., 1994, 2000; Goldin et al., 2006).
Serum levels of AGEs in pre-eclamptic women have been reported to
be significantly higher than those in healthy non-pregnant women or
healthy pregnant women (Chekir et al., 2006). However, other studies
have reported contradictory results where serum AGE levels were not
elevated in PE, but other RAGE ligands, including HMGB1 and
S100A12, were (Harsem et al., 2008; Naruse et al., 2012). These dis-
crepancies may be explained by the heterogeneous nature of the
disease and sample size and population differences between these
studies. Nevertheless, there appears to be a general consensus in the lit-
erature that the AGE–RAGE system is altered in PE. Pre-eclamptic pla-
centae show significantly higher levels of AGE and RAGE than normal
placentae, as detected by IHC and western blot analyses, and these
findings positively correlate with the levels of lipid and DNA oxidation
in the pre-eclamptic samples (Chekir et al., 2006). Immunostaining of
myometrial and omentum tissues taken from non-pregnant, healthy

pregnant and pre-eclamptic women showed that RAGE protein levels
are elevated in both the myometrial and omentum vasculature during
pregnancy and more so in PE (Cooke et al., 2003).
Several plants rich in phenolic compounds, including lowbush blue-
berry (Vaccinium angustifolium Ait.), have been shown to inhibit the for-
mation of bovine serum albumin-modiﬁed AGEs in vitro (Peng et al.,
2008; McIntyre et al., 2009; Ferrier et al., 2012). Vaccinium angustifolium
has been used as atraditional medicine for millennia and its potent inhibi-
tory effect on AGE formation may help explain why it is an effective
natural health product for diabetes treatment in Canada (Martineau
et al., 2006). More recently, in vitro studies have shown that extracts
from this plant increase trophoblast migration and invasion (Ly et al.,
2013, 2014); two important cell functions required for normal placental
development and spiral artery remodelling. Furthermore, evidence in-
cluding that from placental bed biopsies suggests that abnormal tropho-
blast invasion and spiral artery remodelling play an important role in the
aetiology of PE (Brosens et al., 1972, 1977). Since the mechanism by
which the blueberry extract exerts its effects is still unknown, it would
be interesting to investigate if AGEs play a role in trophoblast migration
and invasion and therefore, determine if the effects seen with the
extract are through an AGE-dependent path. Furthermore, other in
vitro models using collagen as a substrate have demonstrated that rutin
and its metabolites inhibit the formation of AGE biomarkers, including
pentosidine and N1
-carboxymethyl-lysine adducts (Cervantes-Laurean
et al., 2006). Similarly, in vivo studies using diabetic rat models have
reported that oral consumption of green tea extracts and curcumin
reduces the formation of AGEs and the cross-linking of collagen (Sajithlal
et al., 1998; Babu et al., 2006).
Additionally, polyphenols are known inhibitors of AGE-mediated sig-
nalling cascades. Studies using murine microglia demonstrated thatsome
plant-derived polyphenols are able to attenuate AGE-induced NO and
TNF-a production in a dose-dependent manner (Chandler et al.,
Figure 2 AGE formation and AGE-mediated activation of NF-kB. (1) AGEs are formed non-enzymatically (Maillard reaction) between carbonyl groups
of reducing sugars (e.g. glucose) and amino groups of proteins, lipids and nucleic acids. The early and intermediate stages of the Maillard reaction lead to the
reversible formation of intermediate products (e.g. Schiff bases and Amadori products), after which classic rearrangement leads to the irreversible gener-
ationofAGEs (2)and cross-linkingof proteins(3). (4)Receptorfor AGE(RAGE) consistsof three extracellular domains, atransmembranehelix and ashort
cytoplasmic tail. Activation of RAGE by AGEs generates ROS through a membrane-associated enzyme, NAPDH oxidase. (5) Increased ROS production
stimulates NF-kB translocation into the nucleus and activation of NF-kB-mediated transcription. (6) Soluble RAGE (sRAGE) is an endogenous RAGE an-
tagonist found in human circulation. It is composed of only the extracellular domain of RAGE and is primarily generated through alternative splicing. sRAGE
acts as ‘decoy’ by binding RAGE ligands and preventing them from reaching RAGE.
10 Ly et al.

2010). According to Chandler et al. (2010), five compounds/plant
extracts were examined and apigenin was found to be the most potent
and did not affect cell viability at the concentrations tested. This study
did not investigate the mechanism of action; however, the authors
hypothesized, based on previous work, that the inhibitory effects are
likely mediated by NF-kB. Rasheed et al. (2009) were able to show
thatEGCG,agreenteapolyphenol,inhibitsAGE-inducedTNF-aproduc-
tionin humanchondrocytespartly by preventing the DNA-binding activity
of NF-kB. Green tea catechins also attenuate intermittent hypoxia-
induced increases in NADPH oxidase and RAGE expression in
Sprague–Dawley rats (Burckhardt et al., 2008). NADPH oxidase is a
membrane-associated enzyme responsible for the production of super-
oxide anions in phagocytic and vascular cells. Red grape juice, red wine
and pure polyphenols were able to reduce NADPH oxidase subunit ex-
pression at the transcriptional and protein level in human neutrophils
andmononuclearcells(Da´valosetal.,2009).Similarresultswereobserved
in hypertensive rats given red wine polyphenols in their drinking water.
Consumption of red wine polyphenols prevented angiotensin II-induced
hypertension and endothelial dysfunction in male rats (Sarr et al., 2006).
Moreover, a significant inhibitory effect on vascular ROS production and
NADPH oxidase expression was seen in the treatment group (Sarr
et al., 2006). Interestingly, hypertension and endothelial dysfunction are
two phenomena also seen in PE, thus investigating the role of polyphenols
in this context may warrant further investigation.
Although polyphenols represent an exogenous therapeutic approach
to delay AGE- and RAGE-mediated diseases, the body has endogenous
mechanisms dedicated to regulating homeostasis of this system. Studies
conducted in vivo and in vitro provide evidence that RAGE signalling can
be antagonized by soluble RAGE (sRAGE), an endogenous RAGE antag-
onistgenerated byeitheralternative splicing of RAGE mRNA orcleavage
of the extracellular domain of RAGE (Stern et al., 2002; Raucci et al.,
2008). sRAGE has the same binding specificity as RAGE and may act
as a ‘decoy’ by binding RAGE ligands (e.g. AGEs) and preventing them
from reaching membrane-bound RAGE, thus inhibiting the intracellular
effect. The clinical application of this work was noted by Germanova´
et al. (2010) who reported elevated maternal serum sRAGE levels in
the third trimester of women with PE and gestational hypertension.
Additionally, Oliver et al. (2011) expanded these findings by demon-
strating that maternal serum sRAGE levels were elevated in women
with severe PE, but not chronic hypertension, as early as 20 weeks of
gestation. This time point is typically recognized as the earliest diagnostic
cut-off point for this disease which suggests that in PE, the RAGE system
is active at an early gestational age and sRAGE may have a protective
function before a patient presents any noticeable clinical symptoms
(Oliver et al., 2011). Furthermore, treatment of placental explants
with xanthine/xanthine oxidase, an inducer of oxidative stress, stimu-
lated the release of sRAGE; potentially a compensatory mechanism
against tissue damage (Oliver et al., 2011). However, higher levels of
sRAGE may not be enough to account for the damage induced by the
AGE–RAGE system, especially if the levels of RAGE ligands exceed
sRAGE scavenging abilities. By measuring the ratio of sRAGE to AGEs,
Yu et al. (2012) demonstrated that the sRAGE scavenger capacity is
lower in women with Type I diabetes mellitus that subsequently devel-
oped PE versus those who did not. In this case, polyphenols may be a
useful therapeutic tool to attenuate RAGE activity in disease. Unfortu-
nately, the effects of polyphenols on sRAGE expression during preg-
nancy are still unknown.
Potential hazardous effects
of polyphenols
The beneficial effects of polyphenols, mainly demonstrated in experi-
mental studies, are encouraging. However, prior to initiating human
intervention trials there is a need to examine the potential adverse
effects of polyphenols during conception and pregnancy. The influence
ofpolyphenolconsumption onmale andfemale fertilityandsexualdevel-
opment, fetal health and the bioavailability of substrates are summarized
in Table IV and will be discussed below.
Fertility and sexual development
Oocyte quality is affected by the intrafollicular microenvironment.
During normal embryonic development, programmed cell death or
apoptosisfunctionstoremoveabnormalorredundantcellsinpreimplan-
tation embryos, contributing to the formation of organs and the embryo
itself (Brill et al., 1999). This process does not occur prior to the blasto-
cyststageinmouseembryos(Byrneetal.,1999).Instead,inductionofcell
death during oocyte maturation and early embryogenesis leads to devel-
opmental injury(Chen and Chan, 2012). In vitrostudies suggest thatpoly-
phenols may have a negative impact on female reproductive health. For
instance, curcumin, the predominant dietary pigment in turmeric, has
beenshowntopromotemouseoocyteapoptosiswhichleadstoasignifi-
cant reduction in the rate of oocyte maturation, fertilization and in vitro
embryonic development (Chen and Chan, 2012). Another study also
noted that curcumin induces apoptosis and developmental injury in
mouse blastocysts (Chen et al., 2010). Moreover, Chen and Chan
(2012) demonstrated using a mouse model that dietary consumption
of curcumin decreased the number of implantations and surviving
fetuses, decreased fetal weight and increased the number of resorption
sites. Similarly, Murphy et al. (2012) reported that parenteral administra-
tion of curcumin decreased folliculogenesis and hastened the onset of
puberty in female mice. Neonatal treatment with genistein, an isoflavo-
noid with estrogenic activity from soya products, has been shown to
lead to multi-oocyte follicles in mice (Jefferson et al., 2002). These
types of follicles are known to have reduced fertility rates during IVF
(Iguchi et al., 1990). Overall, these adverse effects are important to con-
sider and justify further investigations to understand the effects of poly-
phenols on female fertility and sexual development.
In males, treatment with curcumin reduced seminal vesicle weights,
but did not alter testes weights (Murphy et al., 2012). Other studies
suggest that curcumin reduces the motility and viability of human and
murine sperm (Rithaporn et al., 2003; Ashok and Meenakshi, 2004)
which results in failure of IVF (Naz, 2011). On the contrary, the
adverse effect of EGCG on sperm motility is not significant, but this poly-
phenol has been shown to have cytogenetic effects on mouse spermato-
zoainvitro(KusakabeandKamiguchi,2004).Uponinjectionintooocytes,
a significant proportion of spermatozoa treated with EGCG displayed
pronuclear arrest, degenerated sperm chromatin mass and structural
chromosome aberrations (Kusakabe and Kamiguchi, 2004). Furukawa
et al. (2003) proposed that at high concentrations, as used in this
study, EGCG is a pro-oxidant and Kusakabe and Kamiguchi (2004) sug-
gested that this leads to the deterioration of sperm plasma membrane.
Furthermore, dietary exposure of pregnant dams to genistein resulted
in aberrant or delayed spermatogenesis in the seminiferous tubules of
male pups (Delclos et al., 2001). In general, the possible adverse

effects of polyphenols on male reproduction require careful consider-
ation and further investigation, particularly in human studies.
Moststudiesonpolyphenolsandtheireffectsonfertilityandsexualde-
velopment have used animal models, thus data from human studies is
scarce. However, research on isoflavones and fertility in both men and
women has been identified in the literature. Isoflavones are phytoestro-
gens with chemical structures that closely resemble 17-b-estradiol and
therefore, have the potential to bind to both membrane and nuclear es-
trogen receptors, exert estrogenic activity and alter reproductive func-
tion (Vitale et al., 2013). A cross-sectional study by Jacobsen et al.
(2014) reported that North American Adventist women aged 30–50
years old with high isoflavone intake (≥40 mg/day) had a higher inci-
dence of nulliparity and nulligravidity compared with women with low
isoflavoneintake(,10 mg/day).Otherstudieshaveeasedtheconcerns
regarding the potential negative effects of isoflavone consumption on
female fertility by reporting that isoflavone intake is not associated
with sporadic anovulation (Filiberto et al., 2013) and that higher
urinary isoflavone levels may be associated with a shorter time to
pregnancy among couples who are attempting to conceive (Mumford
et al., 2014). Contrasting findings are also evident in studies examining
the effects of isoflavones on male fertility. For instance, studies report
that higher intake of soy foods and soy isoflavones is associated with
lower sperm concentration (Chavarro et al., 2008) and decreased
serum levels of dihydrotestosterone (Dillingham et al., 2005).
However, evidence from other studies suggests that isoflavone intake
does not adversely affect semen quality parameters, including sperm
concentration and sperm motility and morphology in healthy males
(Mitchell et al., 2001; Beaton et al., 2010). Genistein has also been
shown to accelerate capacitation and acrosome loss in human and
mouse sperm, although human gametes appear to be more sensitive
(Fraser et al., 2006). Thus, despite the many reported benefits of poly-
phenoladministration,datahighlighting thepotential hazardsofpolyphe-
nols, the variation of results between heterogeneous studies, and the
possibility of species-specific susceptibility stresses the need for
caution and further study in humans prior to implementing recommen-
dations for clinical practice.
.............................................................................................................................................................................................
Table IV Potential harmful effects of polyphenols on reproductive health and early development.
Field Polyphenol Experimental model Biological effect
Fertility and sexual
development
Curcumin Female mice Promote oocyte and blastocyst apoptosis4,5
Decrease number of implantations and surviving fetuses5
Increase number of resorption sites5
No effect on placental weight5
Reduce fetal weight5
Decrease folliculogenesis and hasten the onset of puberty16
Male mice Reduce seminal vesicle weight16
No effect on testes weight16
Human and murine sperm in vitro Reduce motility and viability of sperm2,17
Genistein Female mice Increase number of multi-oocyte follicles11
Female rats Alter spermatogenesis in seminiferous tubules of male pups6
Isoflavones Cross-sectional study in
non-pregnant women
Higher incidence of nulliparity and nulligravidity (estimated
intake ≥40 mg/day)10
Male partners in subfertile
couples
Inverse association between soy food intake and sperm
concentration3
Healthy men (20–40 years old) Decrease serum levels of dihydrotestosterone7
Human and murine sperm in vitro Accelerate capacitation and acrosome loss
(human sperm more sensitive)8
EGCG Murine sperm in vitro No effect on sperm motility14
Chromosomal abnormalities14
Fetal health Not specific (estimated total intake
.75th percentile)
Prospective analysis in pregnant
women
Increaseductalvelocitiesandright-to-leftventricularratiosin
exposed fetuses9,18
Bioavailability of
nutrients
Red wine and green tea Caco-2 Increase OC uptake15
Isoxanthohumol, Xanthohumol BeWo Reduce thiamine uptake (in chronic treatment)12
Epicatechin, Isoxanthohumol Reduce folic acid uptake (in acute treatment)13
Quercetin, isoxanthohumol,
xanthohumol
Increase folic acid uptake (in chronic treatment)13
Chrysin, EGCG, Quercetin,
Resveratrol, Xanthohumol
Reduce glucose uptake (in acute treatment)1
Catechin, Epicatechin, Rutin Increase glucose uptake (in acute treatment)1
Myricetin, Rutin Increase glucose uptake (in chronic treatment)1
EGCG, epigallocatechin gallate; OC, organic cation.
1
Arau´jo et al. (2008), 2
Ashok and Meenakshi (2004), 3
Chavarro et al. (2008), 4
Chen et al. (2010), 5
Chen and Chan (2012), 6
Delclos et al. (2001), 7
Dillingham et al. (2005), 8
Fraser et al.
(2006), 9
Galaõ et al. (2010), 10
Jacobsen et al. (2014), 11
Jefferson et al. (2002), 12
Keating et al. (2006), 13
Keating et al. (2008), 14
Kusakabe and Kamiguchi (2004), 15
Monteiro et al. (2005),
16
Murphy et al. (2012), 17
Rithaporn et al. (2003), 18
Zielinsky et al. (2010).
12 Ly et al.

Fetal health
Maternal intake of polyphenol-rich foods and beverages during the third
trimester has been associated with fetal ductal constriction (Zielinsky
et al., 2010), a risk factor for neonatal pulmonary hypertension (Levin
et al., 1979). In a prospective study conducted by Zielinsky et al.
(2010), measurements of fetal ductal flow dynamics were compared
between fetuses exposed to high levels of polyphenols (i.e. estimated
daily maternal consumption above 1089 mg) and low levels of polyphe-
nols (i.e. unexposed fetuses; estimated daily maternal consumption
below 127 mg). Results indicated that fetuses exposed to polyphenol-
rich foods had higher ductal velocities and right-to-left ventricular
ratios than unexposed fetuses; however, these parameters were still
within the normal range (Galaõ et al., 2010). Although maternal restric-
tion of polyphenol-rich foods was reported to reverse the effect on
ductal constriction (Zielinsky et al., 2012), whether this finding warrants
changes in perinatal diet remains to be determined, but certainly should
be thoroughly investigated before recommendations are made.
Bioavailability of substrates
Polyphenols are known to target the intestine and therefore, can affect
intestinal absorption of nutrients, drugs and other exogenous com-
pounds (i.e. xenobiotics). Similarly, polyphenols that are absorbed
from the gastrointestinal system into the maternal circulation can
target the placenta and affect placental transport of nutrients and
other bioactive substances (Martel et al., 2010). Polyphenols have
been reported to affect the bioavailability of various substrates, including
organic cations (OCs), thiamine, folic acid (FA) and glucose.
OCs possess net charges at physiological pH. Some examples include
various drugs (e.g. antihistamines, antacids and antihypertensives), vita-
mins (e.g. thiamin and riboflavin), amino acids and bioactive amines (e.g.
catecholamines, serotonin and histamine) (Zhang et al., 1998).
1-Methyl-4-phenylpyridinium (MPP+
) is widely used as a model for
OC intestinal uptake studies because it is not metabolized in vivo and is
efficiently taken up by intestinal epithelium (Martel et al, 2000; Martel
et al., 2010). Red wine has been shown to increase 3
H-MPP+
uptake in
Caco-2cellsinadose-dependentmanner(Monteiroetal.,2005).Incon-
trast, white wine caused a slight decrease in MPP+
uptake. Since both of
these wines had approximately the same amount of ethanol, Monteiro
et al. (2005) concluded that the differences in their effects were most
likely attributed to non-alcoholic components such as polyphenols.
Green tea has also been shown to increase MMP+
uptake in Caco-2
cells more so than black tea, which may be explained by differences in
their EGCG content (Monteiro et al., 2005).
Thiamine is a complex water-soluble B vitamin (vitamin B1) that is
required during pregnancy for normal fetal growth and development.
Therefore, understanding the regulation of thiamine transport across
the placenta is important. Keating et al. (2006) examined the short-
and long-term effects of different phenolics on [3
H] thiamine uptake in
BeWo cells, a human syncytiotrophoblast cell line. In the short-term
study, none of the 10 compounds tested influenced thiamine transport.
Long-termtreatmentwiththeprenylatedchalconesxanthohumoloriso-
xanthohumol, which are commonly found in beer, significantly reduced
thiamineuptakebyBeWocells.Thiseffectwasnotmediatedthroughdif-
ferential mRNA expression of the thiamine transporters, ThTr-1 and
ThTr-2, or the human serotonin transporter, both of which have been
previously reported to be involved in thiamine uptake in BeWo cells
(Keating et al., 2006). To further elucidate the mechanism by which
this effect occurs, future studies should examine the protein levels of
these transporters following treatment and quantify other transporters
known to carry thiamine across the placenta (e.g. amphiphilic solute fa-
cilitator family).
FA is a member of the large family of B vitamins and its derivatives are
required for a variety of cellular functions, including nucleic acid synthesis
and amino acid metabolism (Martel et al., 2010). Folate is the naturally
occurring form of the vitamin and is especially important during preg-
nancy for preventing fetal neural tube defects (Lucock, 2000). One Japa-
nese study noted that circulating levels of folate appear to be lower in
healthy pregnant women who consume high levels (i.e. greater than
the 75th percentile of participants) of green or oolong tea compared
with healthy pregnant women who do not consume high levels of
these beverages (Shiraishi et al., 2010). However, recent datafromCola-
pinto et al. (2011) showed that the vast majority of Canadian women in
child bearing age are receiving excessively high levels of folate through
supplementation and food. Therefore, folate deficiency does not seem
to be an issue in Canada.
In vitro studies using BeWo cells have shown that acute treatment with
the polyphenols epicatechin or isoxanthohumol reduced FA uptake
(Keatingetal.,2008).Conversely,xanthohumol,quercetinorlowercon-
centrations of isoxanthohumol increased FA uptake (Keating et al.,
2008). Polyphenols are believed to affect FA transport in BeWo cells
through direct interaction with FA transporters rather than influencing
transporter expression (Keating et al., 2008). Since the BeWo cell line
only acts as a simple model for a more complex biological system,
caution should be taken when interpreting these results. For instance,
the apparent differences in acute and chronic exposure of polyphenols
in vitro may not necessarily be reflective of what is seen in vivo, thus
further studies using villous explants or animal models would be interest-
ing to pursue.
Glucoseisthemainenergysubstrateformetabolismandgrowthofthe
feto-placental unit (Martel et al., 2010). Since the fetus cannot synthesize
the amount of glucose required for optimal development, it must obtain
glucose from the maternal circulation. Therefore, placental transport of
glucose is a major determinant of fetal health. Glucose transport is
mediated by members of the GLUT family of transporters; GLUT1
being the predominant transporter in the placenta (Barros et al., 1995;
Hahn et al., 1995). Short-term treatment of BeWo cells with resveratrol,
EGCG, quercetin, chrysin and xanthohumol reduced glucose uptake
while rutin, catechin and epicatechin increased glucose uptake in these
cells (Arau´jo et al., 2008). Chronic treatment with rutin and myricetin
increased glucose uptake in this model. However, whether polyphenols
when taken together with other phenolics or whole foods have similar
effects in humans is still unknown.
Dietary intake of polyphenols
during pregnancy
Polyphenol consumption variesgreatly between individuals and cultures.
An epidemiological study in southern Germany reported that the
average phenolic acid intake of men and women was 222 mg/day
within a large range from as low as 5 to 983 mg/day (Radtke et al.,
1998). Individuals who drink more than two cups of coffee per day can
easily consume 0.5–1 g of phenolic acids per day, as a 200 ml cup of

coffee contains 20–675 mg of the phenolic acid chlorogenic acid
(Clifford, 2000a, b). The estimated mean flavonoid intake for men and
women (non-pregnant) in the USA, captured through the nationally rep-
resentative National Health and Nutrition and Exercise Examination
Survey (NHANES), is roughly 190 mg/day (Chun et al., 2007);
however, the average polyphenol intake obtained from one 24 h recall
may be an underestimate.
As the use of nutritional supplements continues to grow in popularity,
the concentration of polyphenols found within these capsules and
powders should be considered when determining total phenolic
intake. Individuals who take supplements are estimated to consume
100 times more polyphenols than the common intakes in a Western
diet (Mennen et al., 2005), highlighting the importance of monitoring
the source of polyphenol ingestion. To assess the possible beneficial
and harmful effects of polyphenols, validated methods are being devel-
oped to quantify the concentration of these compounds in dietary sup-
plements (Harris et al., 2007; Colson et al., 2010; Hicks et al., 2012)
and food sources. However, adequately powered studies with large
sample sizes are needed to properly correlate polyphenol intake and
healthoutcome. The useof biochemical markerstomeasurepolyphenol
intake during pregnancy is subject to interpretation errors caused by in-
dividualdifferencesinabsorptionandmetabolism,geneticsandmetabol-
ic changes during pregnancy. Food frequency questionnaires (FFQ) have
well-documented limitations, but arethe mostcommon methodused to
evaluate dietary intake patterns given the low cost and ease of adminis-
tration (Archer et al., 2013; Schoeller et al., 2013). A recent study con-
ducted by Vian et al. (2013) was the first to test the reproducibility and
validity of a FFQ to quantify total ingestion of polyphenols for 120 preg-
nant women in Brazil. The average daily intake of total polyphenols esti-
mated by the FFQ was roughly 1 g, and this FFQ showed high
reproducibility and validity for the quantification of total polyphenol
consumption.
Studies that provide more precise individual data concerning intake of
specific classes of polyphenols during pregnancy are required and will
further our understanding of their potential impact on reproductive
health. Similarly, continuing to expand on food composition data
through the use of publicly accessible and open access databases, such
as ‘Phenol-Explorer’, will provide comprehensive data on polyphenol
content in foods and therefore, assist with identifying potential hazards
of consuming excess polyphenol-rich foods. Although the current
methods for measuring polyphenol content in foods and dietary
supplements (e.g. oxygen radical absorbance capacity assay and Folin–
Ciocalteu method) is accurate (Prior et al., 2005), developing a single
standardized assay would be beneficial to compare foods or nutritional
supplements. Lastly, obtaining accurate information with regards to ma-
ternal consumption of nutritional supplements high in polyphenols, such
as ginger, cranberry and raspberry herbal medicines (Kennedy et al.,
2013), will be useful for risk assessment and help guide clinical and re-
searchefforts.Asthesestudiesareintheirinfancy,considerablymorere-
search effort is needed in this area.
Human studies and translational
potential
The increasing interest and public awareness surrounding the potential
health benefits of polyphenol consumption, as well as the widespread
availability and accessibility of polyphenols through the use of nutritional
supplements and fortified foods, has prompted extensive research
focused on the biological effects of these compounds in regards to
chronic disease prevention and health maintenance. However, these
studies have included mostly cell and animal data, with minimal human
investigations. In fact, much less human data are available on the
effects of polyphenol consumption during pregnancy.
Nordeng and Havnen (2005) interviewed a total of 400 post-partum
women in Norway and found that 36% of the women reported herbal
medicine use during their pregnancy. Moreover, both women who had
used herbal medicines during pregnancy and those who did not, had a
positive attitude towards the consumption of polyphenol-rich supple-
ments (Nordeng and Havnen, 2005). In a different study conducted in
Italy, 700 pregnant women were interviewed and 27% of these
women reported that they consumed herbal supplements every day
for at least 3 months (Facchinetti et al., 2012). Similar findings have
been documented in a more recent multinational study in which nearly
30% of the 9500 women interviewed reported the use of herbal medi-
cines (Kennedyet al., 2013). Overall, the use of supplements rich in poly-
phenols appearsto be relatively high, thusidentifying theherbal products
used by pregnant women and understanding the potential benefits or
harm is needed.
In chronic diseases, including cancer, cardiovascular disease and dia-
betes, the consumption of polyphenol-rich foods and beverages has
been reported to have antioxidant and anti-inflammatory effects, such
as increasing the plasma antioxidant capacity in humans (Prior et al.,
2007) and decreasing the incidence of chronic inflammatory diseases
in many subpopulations (Yoon and Baek, 2005). To our knowledge,
there have been no studies to date examining the relationship between
polyphenols and the incidence of pregnancy-related complications asso-
ciated with oxidativestress and inflammation. However, Facchinetti et al.
(2012) reported that women who consumed almond oil, a herbal sup-
plement rich in polyphenols (Mandalari et al., 2010), on a regular basis
had a higher incidence of preterm birth. Most of the human studies
related to polyphenols and reproductive health focus on the effects of
isoflavone consumption on male and female fertility, and there appears
to be no clear consensus in this field (Mitchell et al., 2001; Dillingham
et al., 2005; Chavarro et al., 2008; Beaton et al., 2010; Filiberto et al.,
2013; Jacobsen et al., 2014; Mumford et al., 2014). Other studies have
reported that maternal intake of polyphenol-rich foods and beverages
during pregnancy may have adverse effects on fetal health (Zielinsky
et al., 2010, 2012); however, this topic is also controversial and
remains to be reconciled in the current literature.
Overall, studies examining the biological effects of polyphenol con-
sumption on human reproductive health are limited and inconclusive.
Based on the evidence accumulated from in vitro studies and animal
models, as well as human studies in other contexts, some may initially
believe that polyphenols have potential health benefits on human repro-
duction. On the otherhand, investigators who have studied theeffects of
polyphenols on fertility, sexual development and fetal health, have high-
lighted significant health concerns that should be considered prior to
conducting clinical trials and implementing recommendations for clinical
practice. The findings from these animal studies are difficult to extrapo-
late to humans due to a variety of species-related differences, including
inter- and intra-species variation in digestion, absorption, and metabol-
ism of polyphenols, and concentration and composition of the experi-
mental treatment. Therefore, further studies in humans are required
14 Ly et al.

and should employ large cohorts, with adequate powerand sample sizes
to detect changes in the primary outcome.
Conclusion and
recommendations for
future research
Both positive and negative effects have been associated with the con-
sumption of polyphenol-rich foods and beverages in human studies, as
well as with the treatment of individual phenolic compounds in experi-
mental in vitro and in vivo models. The mechanisms responsible for
these effects have only recently started to be elucidated, especially in
the context of reproductive health and pregnancy. As such, we must
remain critical particularly for at-risk populations, such as pregnant
women, when drawing conclusions regarding the potential health bene-
fits or adverse effects of polyphenols.
Successful advancement in this field of research will require the devel-
opment of extensive food composition tables for polyphenols and stan-
dardizedmethodsforexecutingexperimentalprocedures.Thiswillallow
researchers to conduct thorough observational epidemiological studies
and grant confidence when comparing results in the literature. Since the
active compound responsible for the biological effect may not be the
native polyphenol found in food, further studies are required to charac-
terize the activity of the metabolites rather than simply the native com-
pounds which are currently the most often tested agents in in vitro
studies. Finally, identifying the normal physiological range of polyphenols
andtheirmetabolitesinadulttissues andfetaltissues isofutmostimport-
ance if scientists aim to determine if the effects achieved from a certain
dose in an experimental study are physiologically relevant. Determining
the clinical relevance of results obtained from animal and in vitro studies is
difficult as these studies are conducted at doses which may exceed
normal physiologic concentrations. Even if concentrations are deemed
‘low’ in the fetus, we cannot disregard their potential biological activities
as the effective concentration in the fetus might be much lower than in an
adult.Collectively,alloftheseaspectsmustbeconsideredinthedesignof
future experimental studies, irrespective of whether they are aimed at
evaluating beneficial or adverse effects of polyphenols.
Acknowledgements
The authors thank Dr Tony Durst and Dr Ammar Saleem for their com-
ments on the figures.
Authors’ roles
C.L., J.Y.L., Z.M.F., J.T.A., J.F. and A.G. made substantial contributions to
the conception and design of the manuscript. C.L., Z.M.F., J.T.A, J.F. and
A.G. were involved in the acquisition of data. C.L., J.Y.L., Z.M.F., J.T.A.,
J.F. and A.G. played a role in the analysis and interpretation of data. C.L.
drafted the manuscript and J.Y.L., Z.M.F., J.T.A., J.F. and A.G. critically
revised the manuscript. C.L., J.Y.L., Z.M.F., J.T.A., J.F. and A.G. contrib-
uted to the final approval of the version to be published.
Funding
The following funds were used to support the authors during the prep-
aration of the manuscript: Queen Elizabeth II Graduate Scholarship in
Science and Technology (C.L.); Department of Cellular and Molecular
Medicine, University of Ottawa (C.L.); Division of Maternal-Fetal Medi-
cine, The Ottawa Hospital (Z.M.F., A.G.); Canadian Institutes of Health
Research (CIHR) Postdoctoral Fellowship (Z.M.F.); Mitacs Elevate Post-
doctoral Fellowship (J.F.); Natural Sciences and Engineering Research
Council Discovery Grant (J.T.A.); The Ottawa Hospital Academic
Medical Organization (J.Y.L., A.G.).
Conflict of interest
The authors declared no conflict of interests.
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