| Research Article |
Open Access |
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| Maternal Obesity and Placental Oxidative Stress in the First Trimester |
| Mark C. Alanis1*, Elizabeth M. Steadman1, Yefim Manevich2, Danyelle M. Townsend3 and Laura M. Goetzl1 |
| 1Department of Obstetrics and Gynecology, Medical University of South Carolina, 96 Jonathan Lucas Street, CSB 634, Charleston, South Carolina 29425, USA |
| 2Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, Charleston, South Carolina
29425, USA |
| 3Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, 280 Calhoun Street, Charleston, South Carolina 29425, USA |
| *Corresponding author: |
Mark C. Alanis
Department of Obstetrics and
Gynecology
Medical University of South Carolina, 96 Jonathan Lucas Street
CSB 634, Charleston, SC 29425, USA
Tel: (843) 792-1241
Fax: (843) 792-0533
E-mail: alanis@musc.edu |
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| Received June 20, 2012; Accepted August 06, 2012; Published August 12, 2012 |
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Citation: Alanis MC, Steadman EM, Manevich Y, Townsend DM, Goetzl LM (2012)
Maternal Obesity and Placental Oxidative Stress in the First Trimester. J Obes Wt
Loss Ther 2:143.
doi:10.4172/2165-7904.1000143 |
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| Copyright: © 2012 Alanis MC, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. |
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| Abstract |
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| Objective: Maternal obesity is associated with adverse pregnancy outcomes affected by placental dysfunction.
We sought to compare levels of placental oxidative stress between obese and lean women in the first trimester. |
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| Study Design: Obese and lean women matched 1:1 for baseline variables and gestational age were enrolled
between 8 and 13 weeks of gestation at the time of voluntary surgical abortion. The global cellular redox status was
determined by measuring the total protein thiol content in placental homogenates and serum (ThioGlo-1). |
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| Results: There were no differences in baseline variables between obese (n=22, median BMI 35.0) and lean
controls (n=22, median BMI 22.0). The median level of placental oxidative stress was 31% greater in the obese group
compared to the lean group (141.1 [117-156] vs. 203.7 [189-234] counts/sec/μg protein, respectively; p <0.001). A
similar but statistically insignificant difference was noted in the serum (12.2 [9-15] vs. 13.6 [12-23] counts/sec/μcg
protein; p=0.09). |
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| Conclusion: Maternal obesity is associated with placental oxidative stress in the first trimester. Oxidative stress
in the first trimester may reflect or contribute to impaired placentation and placental dysfunction in obese women. |
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| Keywords |
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| Obesity; Oxidative stress; Placenta; Redox; Protein
sulfhydryls; Thiols; ThioGlo-1 |
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| Introduction |
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| Pregnancy has been described as a state of oxidative stress due to
increased metabolic activity of the placenta and decreased antioxidant
capacity during normal pregnancy [1]. Rapidly dividing placental cells
produce large amounts of Reactive Oxygen Species (ROS), including
superoxide anion, as a byproduct of aerobic respiration by complexes
I and III of the mitochondrial electron transport chain [2]. NAD(P)H
oxidase (Nox) is the other main source of superoxide anion generation
in the placenta and is expressed in the cell membrane of the syncytial
layer, the vascular endothelium, and maternal granulocytes found at the
maternal-fetal interface [3,4]. Collectively, these observations suggest
that normal pregnancy is a state close to the limit at which oxidative
stress may become pathological. Decreased antioxidant capacity and
increased ROS are associated with placental dysfunction resulting in
preeclampsia, intrauterine fetal hypoxia and growth restriction, and
stillbirth [1-3,5-7]. |
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| Oxidative stress markers such as levels of plasma lipid peroxidation
and urinary F2 isoprostanes are known to be increased in obese, nonpregnant
women [8,9]. However, the relationship between maternal
obesity and placental oxidative stress is unclear. Maternal obesity
is associated with placental dysfunction, characterized clinically by
preeclampsia, second trimester spontaneous abortion, and stillbirth,
in a dose-dependent fashion with body mass index (BMI) [10-15].
More than 1 in 3 women in the United States are obese at the time of
conception, defined as a BMI equal to or greater than 30 kg/m2[16].
Thus, there is a broad interest among public health experts, physicians,
and researchers in elucidating mechanistically based interventions to
reduce the impact of maternal obesity on pregnancy outcome. The
aim of this study was to determine the effect of maternal obesity on
placental oxidative stress in the first trimester. |
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| Methods |
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| Subjects |
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| Obese (BMI ≥ 30) and lean (18.5 ≤ BMI <35) patients undergoing voluntary surgical abortion were recruited from a single reproductive
services clinic in this IRB approved study. Inclusion criteria included a
gestational age between 8(0/7) and 13(6/7) weeks and signed informed
consent. Exclusion criteria included co-existing diabetes mellitus, renal
disease, and use of antibiotics in the previous 6 weeks, use of any nonsteroidal
anti-inflammatory drug in the previous 24 hours, use of oral
or systemic steroids, or recent (<6 weeks) pelvic inflammatory disease.
BMI was determined by height and weight measured immediately
prior to the procedure. Obese subjects were enrolled consecutively,
and a matched lean controls was subsequently enrolled for each obese
subject. Matching was 1:1 by race/ethnicity, smoking status (yes/no
in pregnancy), and gestational age (± 3 d). All gestational ages were
confirmed by measurement of the crown-rump-length with ultrasound
prior to the procedure. |
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| Blood and placenta collection |
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| Blood samples were obtained at the time of intravenous access
and prior to initiation of the procedure. Blood samples were placed on
ice and allowed to clot for at least 30 minutes. Immediately following
dilation and curettage, products of conception were floated in ice-cold
phosphate buffered saline (PBS, pH 7.4). Placenta was identified by the
consistent frond-like appearance of villi (Figure 1). Placental specimens
were washed with ice-cold PBS then flash-frozen in liquid nitrogen.
The entire tissue collection process was completed in less than 20
minutes after dilation and curettage in all cases. Placental specimens were stored at -80° C until batched testing. Clotted blood samples were
centrifuged at 4,000 rpm for 20 minutes at 4° C and serum fractions
were kept for analyses. |
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Figure 1: Separation of gestational tissues and collection of placenta. |
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| Sample Preparation |
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| Frozen placental samples were manually homogenized in
lysis buffer containing 50 mMTris-HCl (pH 7.5), 2mM Ethylene
Diaminetetraacetic Acid (EDTA), 2mM Ethylene Glycol Tetraacetic
Acid (EGTA), 1% Triton X-100, 150 mMNaCl, 10 glycerol, and diluted
1:100 with Phenyl Methyl Sulfonyl Fluoride (PMSF). The lysis buffer
was supplemented with a proteinase inhibitor cocktail containing
4-(2-amnioethyl) benzenesulfonyl fluoride (AEBSF), pepstatinA, E-64,
bestatin, leupeptin, and aprotinin (Sigma-Aldrich, St. Louis, MO).
Samples were incubated in the lysis buffer with periodic vortexing at
40C for 15 minutes. The incubated samples were then centrifuged at
15,000 rpm for 15 minutes to precipitate non-soluble material. The
supernatant was collected and passed through a Biospin-6 (Bio-Rad
Laboratories, Inc., Hercules, CA) size-exclusion chromatography
mini-column and the protein concentration was determined using
the Bradford Assay (Bio-Rad Laboratories, In., Hercules, CA). The
resultant filtrate was diluted 1:100 with 20 mM PBS. |
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| Fluorescent detection of oxidative stress |
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| Oxidative stress was assessed through the measurement of
reduced protein thiol content using the sulfhydryl-specific maleimide
fluorescent dye, ThioGlo-1 (Calbiochem Inc., San Diego, CA) as
previously described [17-18]. The total reduced protein thiol content
is the reciprocal of the total oxidized protein content. The fluorescent
emission of ThioGlo-1-protein sulfhydryl adducts, therefore, is an
accurate measure of the global protein redox status [19]. Freshly
prepared placental homogenates and thawed serum were analyzed
using real-time kinetics mode of a QM-4 fluorometer (Photon
Technology International, Inc., Piscatway, NJ; Figure 2). Saturation
ThioGlo-1 fluorescence was normalized for the total protein content
of each sample. |
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Figure 2: Original representative traces of TG-1(5 µM) fluorescent detection
of protein-sulfhydryl in glutathione-free samples of placenta homogenate or
plasma in 20 mm PBS, pH=7.4 at 37°C. |
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| Statistical Analysis |
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| Continuous data were described as medians (IQR), and categorical
data were described by frequencies (column percents). The primary
outcome was ThioGlo-1 emission, normalized for protein content
(counts/sec/μg protein). Group comparisons between obese and
lean women were performed using Wilcoxon rank sum tests or Chisquare
tests as appropriate. Linear relationships between placental and
serum ThioGlo-1 emission and between gestational age and ThioGlo-1
emission were assessed by Spearman rank correlation coefficients.
P-values <0.05 were considered statistically significant for all tests (SAS
9.1.3 (Cary, NC)). |
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| Results |
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| A total of 44 subjects (22 matched pairs) were enrolled. There
were no differences in background or demographic variables (Table
1). Maternal obesity was associated with a 31% median increase in
placental oxidative stress compared to lean controls (Figure 3a). Thirtytwo
subjects (16 in each group) also had serum available for peripheral oxidative stress assessment. A similar, but non-significant increase in
serum oxidative stress was found (Figure 3b). There was no significant
correlation between placental and serum oxidative stress either within
groups (obese r=-0.11, p=0.67; lean r=-0.06, p=0.83) or overall (r=0.25,
p=0.17, Figure 4a). There was also no correlation between gestational
age and placental (r=-0.16, p=0.28) or serum (r=0.26, p=0.14) oxidative
stress (Figure 4b). Overall, the level of oxidative stress measured in the
placenta was far more pronounced than in the serum (Figure 3a, Figure
3b). |
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Table 1: Continuous variables presented as medians and interquartile range (IQR)
and analyzed by Wilcoxon rank sum tests. Categorical variable presented as
frequencies and column percents and analyzed by Chi-square tests. |
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Figure 3a: First trimester ThioGlo-1 emission (513 nm) in obese compared to
lean women. The top and bottom bars represent the full range of observations.
placental homogenates (n=44, medians [IQR]=203.7 [189-234] and 141.1
[117-156] for normal weight and obese subjects, respectively, p<0.001). |
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Figure 3b: Serum (n=32, medians=13.6 [12-23] and 12.2 [9-15] for normal
weight and obese subjects, respectively, p=0.09). |
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Figure 4a: Relationships between placental oxidative stress and peripheral
oxidative stress and gestational age. Spearman-rank correlation between
placental ThioGlo-1 emission (513 nm) and serum ThioGlo-1 emission (513
nm) (n=32, p=0.17). |
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Figure 4b: Spearman-rank correlation between placental ThioGlo-1 emission
(513 nm) and gestational age (n=44, overall p=0.26). |
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| Discussion |
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| Maternal obesity is an emerging public health concern of enormous
clinical impact and research interest. Our data shows for the first time
that maternal obesity is associated with placental oxidative stress in
the first trimester. This finding supports a first trimester origin for
the observed increased rate of placental dysfunction noted in obese
women later in pregnancy, although a direct relationship cannot
be established from this investigation [10-15]. Oxidative stress is
increasingly viewed as an upstream process resulting in inflammation
and cellular injury. Indeed, maternal obesity is associated with robust
placental inflammation at term Challier et al. [20] demonstrated that
mRNA expression of TNF-α and other pro-inflammatory cytokines are
elevated in placentas of obese women compared to lean women at term
[20]. These investigators found that an accumulation of activated CD14+
macrophages in the placenta was considered the primary source for
these pro-inflammatory cytokines [20]. Other investigators have found
that placental mitochondrial ROS production is stimulated by TNF-α
[21]. Therefore, increased obesity-related placental inflammatory
cytokines may promote further production of ROS and induce a feed
forward cycle of placental cellular damage [22]. |
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| Our finding that global placental redox status in obese women is
independent of gestational age was surprising. A common belief is
that placental oxidative stress occurs only after 10 weeks of gestation,
based on the observation that only after this time point does the fetal
circulation come into direct contact with the uterine spiral arteries
and the intervillous oxygen tension rises sharply (pO2=50 mmHg) [23,24]. Prior to this period, the fetal environment is very hypoxic
(pO2<20 mmHg) [24].The reason for independence of oxidative
stress for gestational age in obese women is unclear. There are two
potential explanations for this phenomenon. The first is hypoxiainduced
production of ROS from the placental mitochondrial electron
transport chain [2]. The second involves a priori increased NADPH
oxidase activity in the placenta of obese women. In fact, NADPH
oxidase appears to be the major contributor of ROS production in
non-pregnant obese women [8,25,26]. Our findings differ from those
earlier reported by Roberts et al. [27] who found no direct relationship
between lean, overweight, and obese BMI classes and oxidative stress
[27]. The disagreement between our two studies most likely reflect
that their study: 1) was limited to term gestations without pregnancy
complications; 2) contained a very limited number of subjects (7
lean, 5 overweight, and 8 obese), which may have limited the power
to detect increases in oxidative stress; 3) used quasi-quantifiable methods, such as western blot, to measure placental oxidative stress;
and 4) recruited both laboring and non-laboring women [27]. In
contrast, our study examines the induction of placental oxidative stress
at the critical time period following placentation. Further, our study
was not confounded by the increase in oxidative stress known to be
associated with parturition [17,28]. Finally, Roberts et al. [27] selected
for uncomplicated pregnancies [27], which may have reduced the
likelihood of detecting placental oxidative stress, while our sample is
unselected. Another advantage of our study is that our subjects were
matched for important baseline variables that could potentially affect
oxidative stress. Consistent with non-pregnant studies [8], the level of
oxidative stress measured in the serum was slightly increased in our
obese subjects. However, only a subset of subjects had serum available
for analysis, limiting our power to establish statistical significance. |
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| There are numerous advantages to the assessment of placental
oxidative stress as described and performed in our study. First, Hansen
et al. demonstrated the appropriateness of determination of the global
cellular sulfhydryl status as an indicator of oxidative stress [19]. Protein
cysteine residues (e.g. sulfhydryls) are considered to be redox switches
and mediate oxidative and nitro sative stress-induced signaling events
that are critical to cell fate. Rather than assaying a specific component
of the oxidative stress (e.g. antioxidant levels), which may fail to detect
oxidative stress, tissue assessment of protein redox status is highly
sensitive. Second, this direct measurement of the total protein redox
status is straightforward and reproducible. Third, samples can be stored
and assayed in batch, further reducing variability within the assay. |
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| We anticipate that our approach affords the opportunity to
identify obesity-related mechanisms of placental oxidative stress in the
first trimester. Further, our study implicates pre-pregnancy maternal
obesity as a potential first-trimester marker for selection of those whom
may be candidates for antioxidant trials for the prevention of adverse
pregnancy outcome. |
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