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Article

Pathogenesis of preeclampsia:Implications of apoptotic markersand oxidative stress

OG Shaker1 and NAH Sadik2

Abstract

This study aimed to investigate the implication of some apoptotic and lipid peroxidation markers in preeclampsia(PE). A total of 25 women with PE and 25 age- and parity-matched normal pregnant women were enrolled in thisstudy. The malondialdehyde (MDA) level, caspase-9 activity and the percentage of DNA fragmentation were sig-nificantly higher in placental tissue of PE than in control women. The serum level of MDA was significantly elevatedin women with PE having delivery by cesarean section (CS) than in women with PE having vaginal delivery. In vitro

study demonstrated that the addition of 0.5 mM Fe

2

and 0.1 mM ascorbate caused increase in the production ofMDA level in placental tissue with PE than normal placentas, and vitamin E (100 mM) caused lower inhibition ofin vitro lipid peroxidation in placental tissue with PE when compared with normal tissue. The activity ofcaspase-9 and percentage of DNA fragmentation were associated with the severity of the PE and both could differ-entiate betweenPE and control womenwith 88% and 100% sensitivityand 96% and 100% specificity,respectively. Theactivities of caspase-8 and/or -9 were positively correlated with the maternal age but only caspase-8 was negativelycorrelated with neonatal birth weight and placental weight. In conclusion, the elevations of MDA, caspase-9 activityand the percentage of DNA fragmentation in the placentas of women with PE implicate the involvement of lipid per-oxidation and apoptosis in PE. The placenta represents a considerable source of the elevated circulating MDA in PE.

Keywords

Preeclampsia, apoptosis, lipid peroxidation, MDA, caspases, DNA fragmentation

Introduction

Preeclampsia (PE) is a syndrome defined by the new

onset of hypertension in the second half of pregnancy

that is generally, but not always, accompanied by pro-

teinuria. It is a multisystem disorder characterized by

uteroplacental and maternal endothelial dysfunction.

PE complicates 35% of all pregnancies and contin-

ues to be a major cause of morbidity and mortality

both for the mother and the infant. It is a significantly

greater problem than gestational hypertension alone,as the latter by definition does not affect maternal

organ systems. While in recent times the understand-

ing and management of this condition have improved,

the exact cause of PE has not been fully defined.1,2

Increased awareness of the long-term complications

of PE and its associations with vascular disease in

later life,35 both for the mother and the infant of

low-birth weight, is of major concern.6,7

Oxidative stress is described as an imbalance in the

production of reactive oxygen species and the ability

of antioxidant defenses to scavenge them. In PE and

some pathologic pregnancies, a heightened level of

oxidative stress is encountered.8 Lipid peroxide forma-

tion, a marker of oxidative stress, is increased during

pregnancy and PE. The lipid peroxide concentration

of microvillus membrane can be quantified by malon-

dialdehyde (MDA) level the most widely used lipid

peroxidation marker in syncytiotrophoblast plasma

membranes. The concentrations of MDA are highly

1Medical Biochemistry and Molecular Biology Department,

Faculty of Medicine, Cairo University, Cairo, Egypt2Biochemistry Department, Faculty of Pharmacy, Cairo University,

Cairo, Egypt

Corresponding author:

Nermin Abd EL-hamid Sadik, Biochemistry Department, Faculty

of Pharmacy, Cairo University, Kasr El-Eini Street, Cairo 11562,

Egypt.Email: [email protected]

Human and Experimental Toxicology

32(11) 11701178

The Author(s) 2013

Reprints and permission:

sagepub.co.uk/journalsPermissions.nav

DOI: 10.1177/0960327112472998

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correlated with those of isoprostane, another previously

described specific marker for oxidative stress in PE.9

Apoptotic DNA fragmentation is a key feature of

apoptosis, a type of programmed cell death. Apoptosis

is characterized by the activation of endogenous endo-

nucleases with subsequent cleavage of chromatin DNA

into internucleosomal fragments, which is roughlyequal to or multiples of 180 base pairs (360, 540, etc.).

DNA fragmentation is a secondary consequence, rather

than an integral cause, of apoptosis.10 Apoptosis in tar-

get cells can be evaluated by detecting the pattern of

DNA fragmentation by agarose gel electrophoresis.

Apoptosis can be initiated by toxicants, drugs or

perturbation of the cellular homeostasis in some dis-

ease conditions. Apoptosis has been implicated in the

pathogenesis of many disease states including cancer,

neurological disorders, autoimmune diseases and

aging.11

Its rate is thought to be increased in PE asa result of placental oxidative stress.1,12 Apoptosis

signaling events can roughly be grouped into four dif-

ferent phases: the premitochondrial initiation phase,

during which a cell receives the death signal at the

plasma membrane; the mitochondrial decision phase;

the postmitochondrial amplification phase, which leads

to activation of different caspases that act in concert to

propagate the death signal; the late (degradation)

phase, during which cellular proteins are proteolyti-

cally cleaved with the evidence of DNA fragmenta-

tion.13 Caspases comprise a structurally related group

of cysteine proteases that share a dominant specificity

for cleaving peptide bonds following Asp residues.14

They are roughly classified into initiator caspases, such

as caspase-8 and -9, and effector caspases, such as

caspase-3 and -7.13

Therefore, the aim of the present study was to inves-

tigate the changes in the activities of caspase-8 and -9,

percentage of DNA fragmentation and MDA level as

markers of apoptosis and oxidative stress in preeclamp-

tic women and the possible interplay between these

markers and the maternal risk factors and perinatal out-

come along with the response of the placental tissue toprooxidants (iron chloride (FeCl2) and low dose of

ascorbate) and the antioxidant a-tocopherol.

Patients and methods

A total of 25 preeclamptic primigravidas and 25

randomly selected age- and parity-matched normal

pregnant women, who attended the outpatient clinic

of the Gynaecology Department at Kasr El-Aini Hos-

pital, Cairo University, Cairo, Egypt, were enrolled in

this study. Informed consent was obtained from all

participants. The study protocol was approved by the

institute ethics committee and was conformed by the

ethical guidelines of the 1975 Helsinki Declaration.

The diagnosis of PE was made by strict criteria

according to Chesley.15 These criteria included onset

of hypertension during the third trimester (140/90 mmHg on two occasions), detectable urinary pro-

tein (1 by dipstick or 300 mg/24 h) and edema.

Women with a history of hypertension before

20 weeks gestation and patients whose pregnancies

were complicated by diabetes, peripheral vascular

disease, chronic renal disease, multifetal gestation or

major fetal anomalies were excluded from this study.

The PE group (group 1) was subdivided into two

subgroups: group 1(A), which included 12 patients

with mild PE, and group 1(B), which included 13

patients with severe PE. The criteria for severe PEwere determined by systolic blood pressure (SBP)

>160 mm Hg or diastolic blood pressure (DBP)

>110 mmHg on two separate occasions: >4 h apart

in the presence of repeated proteinuria ( or more)

and at least 4 h apart.16

Materials

Maternal venous blood samples (*5 ml) and human-

term placentas were collected immediately after

delivery from each woman. Placental samples were

taken away from areas with infarction or calcification.Placentas from cases with prolonged rupture of mem-

branes were avoided because of chorioamnionitis.

Small pieces of placental tissues were immediately

frozen at 80C till assays.

Laboratory assays

Estimation of lipid peroxides in serum and placental

tissues. Lipid peroxidation was estimated by measur-

ing thiobarbituric acid reactive substances mainly

MDA in placental tissues and serum according to the

method of Esterbauer and Cheeseman.17 The level ofMDA in the serum and the placental tissue samples

was expressed in micromoles per liter and nanomoles

per milligram protein, respectively. The protein con-

centration in tissue extracts was measured by the

method of Bradford.18

In vitro lipid peroxide formation. Placental tissues were

homogenized in four volumes of 0.15 M sodium

chloride (NaCl)10 mM sodium phosphate buffer,

pH 7.4, and filtered through a gauze. The filtrate was

Shaker and Sadik 1171

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diluted with 0.15 M potassium chloride (KCl)10 mM

Tris-HCl buffer, pH 7.4, so as to give a final protein

concentration of 1 mg/ml. According to the method

of Buege and Aust,19 formation of lipid peroxide in

relation to time (after 1, 2 and 3 h of incubation at

37C) was examined in placental tissue with PE com-

pared with the normal placenta. Applying the samemethod, lipid peroxidation response to the addition

of prooxidants (0.5 mM FeCl2, 0.1 mM ascorbate) and

antioxidant (100 mM a-tocopherol) was determined

after 2 h of incubation at 37C. For each placental

sample, a control was run simultaneously without

addition of prooxidants or antioxidants. The results

were expressed as percentage stimulation or percent-

age inhibition calculated as follows: % stimulation (or

%inhibition) (nM MDA of test sample/nM MDA of

its control) 100.

Assessment of apoptosis in placental tissues

Determination of activities of caspase-8 and -9. The

activities of both caspase-8 and -9 in placental tis-

sues were determined using Apo Target colori-

metric assay kits (BioSource Europe S.A., Nivelles,

Belgium) according to the manufacturers instruc-

tions. Each kit includes substrate and optimized buf-

fers. The substrate was composed of chromophore p-

nitroaniline and a synthetic tetrapeptide IETD (ILe-

Glu-Thr-Asp) or LEHD (Leu-Glu-His-Asp) in

caspase-8 or -9 kits, respectively. Upon cleavage of

the substrate by caspase-8 or -9, free p-nitroaniline

was liberated and quantified spectrophotometrically

at 405 nm.

Determination of percentage of DNA fragmentation. The

percentage of DNA fragmentation in placental tissues

was measured by diphenylamine (DPA) assay and

confirmed by agarose gel electrophoresis according

to the method of Perandones et al.20 Placentas were

mechanically dissociated in hypotonic lysis buffer.

The cell lysate was centrifuged at 13,000gfor 15 min,then the supernatant containing small DNA fragments

was separated immediately and half the supernatant

was used for gel electrophoresis. The other half as

well as the pellet containing large pieces of DNA

were used for the colorimetric determination by DPA

assay. Briefly, 200 ml perchloric acid (0.5 M) was

added to the pellet (reconstituted in 200 ml hypotonic

lysis buffer) containing native DNA and to the super-

natant containing fragmented DNA followed by the

addition of two volumes of a solution containing

0.088 M DPA, 98% v/v glacial acetic acid, 1.5% v/v

sulfuric acid and 0.5% acetaldehyde solution. The

samples were kept at 4C for 48 h. The color devel-

oped was then spectrophotometrically measured at

575 nm. The percentage of DNA fragmentation was

expressed by the formula:

Percent of DNA fragmentation absorbance ofsupernatant 100/2 (absorbance of supernatant

absorbance of pellet)

Measurement of the serum level of uric acid

The serum level of uric acid was determined by the

enzymatic colorimetric method21 using the kit pro-

vided by Randox Laboratories Ltd. (Crumlin, UK)

according to the manufacturers instructions. The

results were expressed as milligrams per deciliter.

Statistical analysis

The statistical data are reported as the mean + SD

and percentages when appropriate. Differences

between the two groups were evaluated with students

t test or chi square test, as appropriate (Table 1). A

comparison of the variables between more than two

groups was performed using a one-way analysis of

variance (ANOVA) test followed by Duncans test for

inter-group comparisons as in Table 2. The Pearson

correlation coefficient was used to determine correla-

tions between different variables (Table 3 and Fig-

ure 2). A receiver operating characteristic (ROC)analysis was used to determine the optimum cutoff

value for the studied markers (Table 4). The accuracy

is represented using the terms sensitivity and specifi-

city. ps < 0.05 were consideredstatistically signifi-

cant. All statistical calculations were performed

using the computer program SPSS (Statistical Pack-

age for the Social Science; SPSS Inc., Chicago, Illi-

nois, USA) version 15 for Microsoft Windows.

ResultsA total of 50 primigravidas were included in this study;

12 were with mild PE, 13 with severe PE and 25 age-

matched normotensive control women. The basic char-

acteristics of the patients and controls are shown in

Table 1. In Table 1, the mean admission SBP and DBP

blood pressures are significantly higher in the PE group

than the control women (p < 0.001, p < 0.01, respec-

tively). The gestational age, neonatal birth weight and

placental weight are lower in the women with PE com-

pared with the control group (p< 0.01).

1172 Human and Experimental Toxicology 32(11)

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With the exception of caspase-8 activity, the expres-

sion of apoptotic markers caspase-9, percentage of

DNA fragmentation (eachp < 0.001) and the lipid per-

oxidation product; serum (p < 0.001) and placental

MDA (p < 0.05) and the serum level of uric acid

(p < 0.05) were significantly higher in the PE group

than the control group.

According to the mode of delivery, the serum level

of MDA was shown to be higher in women with PE

having delivery by cesarean section (CS) than in

women with PE having vaginal delivery(mean + SD 1.4 + 0.37 vs. 1.03 + 0.33, respec-

tively) atp < 0.05. Also, the serum level of MDA was

higher in the women with PE having vaginal delivery

than the control women who had vaginal delivery

(mean+ SD 1.03+ 0.33 vs. 0.39 + 0.14, respec-

tively) atp < 0.001. Meanwhile, the mode of delivery

did not cause any statistically significant change in

the placental level of MDA (data not shown).

The results of the in vitro lipid peroxide forma-

tion showed higher levels of MDA formation in

placental tissue with PE than in normal placentas

when incubated at 37C for 1, 2 or 3 h (data not

shown). After 2 h of incubation period at 37C in the

presence and absence of prooxidants (Figure 1), the

percentage stimulation by 0.5 mM FeCl2 was signifi-

cantly higher in the placental tissue with PE

(mean 187.9+31.68%) than in the normal placenta(mean 139.8+ 25.6%) atp < 0.01. Furthermore, a

significant increase in percentage stimulation was

induced by 0.1 mM ascorbate in theplacentaltissue with

PE (mean 165.2 +50.06%) compared with the nor-

mal placenta (mean 130.18 + 32.06%) atp < 0.05.

Addition of 100mMa-tocopherol caused a significantly

lower percentage inhibition in the placental tissue with

PE (mean 80+ 12.77%) than in the normal placenta

(mean 52.27+10.57%) at p< 0.001.

Blood pressure, gestational age, neonatal birth

weight and placental weight as well as serum MDA,caspase-9 activity, percentage of DNA fragmentation

and serum level of uric acid (Table 2) were signifi-

cantly different in women with mild and severe PE

compared with the normal controls. Interestingly,

severe PE group did not differ significantly from the

mild group regarding neonatal birth weight, placental

weight and other biochemical measures (Table 2).

Correlation study among clinical data, pregnancy

outcomes and laboratory data (Table 3) revealed that,

whereas the maternal age was positively correlated

with the activities of caspase-8 (r 0.53,p < 0.05) and

caspase-9 (r 0.36,p < 0.05) in the PE group, it was

negatively correlated with the percentage of DNA frag-

mentation in the PE groups (r 0.33, r 0.43,

p < 0.05, respectively) and control (data not shown).

Caspase-8 was negatively correlated with neonatal birth

weight (r 0.56, p < 0.05) and placental

weight(r 0.33,p < 0.05); both neonatal birth weight

and placental weight correlated positively with each

other (r 0.35,p < 0.05). The serum level of MDA was

positively correlated with the serum level of uric acid

(Figure 2;r 0.8,p< 0.05) only in the PE group. The

placental MDA was positively correlated with thepercentage of DNA fragmentation (r 0.33,p < 0.05)

in the PE group but not in the control group. SBP was

positively correlated with maternal and negatively with

gestational age (r 0.6 and 0.33, respectively,

p< 0.05).

In the placental tissue with PE, ROC curve analysis

(Table 4) showed that the percentage of DNA frag-

mentation was the most sensitive (100%) and specific

(100%) followed by caspase-9 activity: 88% sensitiv-

ity and 96% specificity.

Table 1.Characteristics of the patient and control groups.

Control(n 25)

Preeclampsia(n 25) p value

Maternal age(years)

26 + 5.69 26.7 + 5.02 >0.05(1836) (1938)

Gestational age(weeks)

39.3 + 1.1 38.3 + 1.4

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Discussion

Despite intensive efforts to find mechanisms and mar-

kers that induce PE, no specific etiological factor has

been identified.12 Oxidative stress occurs when

hypoxic placental tissues are reoxygenated as a resultof defective remodeling of uterine spiral arteries in

PE.8 The product of attack by free radicals on polyun-

saturated fatty acids can be considered as a footprint

of free radical activity. Our results revealed higher

placental level of MDA in the preeclamptic group

compared with the control group.

It was reported previously that the mode of deliv-

ery affects the rate of lipid peroxidation in the placen-

tas of normal pregnancy because during vaginal

delivery, placental anoxia may occur as a result of

uterine contractions22 and lipid peroxides may be

formed from unsaturated lipids in the course of prosta-

glandin synthesis, which is needed for the initiation of

labor in spontaneous delivery.23 The current results sug-

gest that the situation is different in PE, as the placental

level of MDA did not significantly differ between

women having vaginal delivery (40% of our cases) and

those having delivery by CS. Meanwhile, there was an

increase in the serum level of MDA in women with

PE having delivery by CS (60% of our cases) over

women with PE having vaginal delivery. Therefore,

we suggested that the hyperoxic state that occurs during

delivery by CS may constitute considerable source for

the increased serum level of MDA in our preeclamptic

group. Zhang et al.24 and Khaw et al.25 reported that

Table 3. Correlation between clinical data, pregnancy outcome, and laboratory data in the preeclamptic group.

Mat.age

Gest.age SBP DBP

Birthwt

Placentalwt

SerumMDA

PlacentalMDA

Caspase-8

Caspase- 9

% DNAfragmentation

0.43 0.13 0.47 0.3 0.15 0.28 0.02 0.33 0.21 0.34

Caspase-9 0.36 0.04 0.11 0.18 0.25 0.25 0.07 0.02 0.28Caspase-8 0.53 0.15 0.24 0.17 0.56 0.33 0.14 0.12Placental MDA 0.24 0.34 0.25 0.02 0.06 0.10 0.27Serum MDA 0.19 0.10 0.11 0.04 0.18 0.10Placental weight 0.2 0.02 0.17 0.07 0.35Birth weight 0.32 0.29 0.41 0.05DBP 0.01 0.13 0.55SBP 0.60 0.33

MDA: malondialdehyde; SBP: systolic blood pressure; DBP: diastolic blood pressure.Bold characters indicate significant values at r +0.33 and p < 0.05. Gest., gestational; Mat., maternal.

Table 2. Comparison of different variables in the normal control, mild, and severe preeclamptic groups.

Control (n 25) Mild PE (n 12) Severe PE (n 13)

Maternal age (years) 26.1 + 5.7 25.4 + 3.9 28.0 + 5.7Gestational age (weeks) 39.3 + 1.1 38.4 + 1.4* 38.3 + 1.4*

SBP (mmHg) 117.6 + 11.3 156.7 + 16.7* 190.8 + 25.9*,#

DBP (mmHg) 78.0 + 7.6 100.4 + 8.1*

116.9 + 10.3*,#

Neonatal birth wt. (kg) 3.2 + 0.6 2.61 + 0.797* 2.36 + 0.68*

Placental wt (kg) 0.49 + 0.08 0.30 + 0.07* 0.29 + 0.06*

Serum MDA (nmol/l) 0.39 + 0.14 1.48 + 0.29* 1.28 + 0.47*

Placental MDA(nmol/mg protein) 0.12 + 0.01 0.15 + 0.01 0.14 + 0.01Caspase-8 (U/mg protein) 6.4 + 0.9 6.6 + 1.3 6.9 + 0.96Caspase-9 (U/mg protein) 5.8 + 1.2 10.9 + 2.9* 11.6 + 3.25*

% DNA fragmentation 21.6 + 4.5 53.2 + 14.6* 43.7 + 9.6*

Uric acid (mg/dl) 4.4 + 0.82 5.78 + 1.5* 6.37 + 1.7*

Data are represented as mean + SD.* and # indicate statistical significance from control and mild PE groups, respectively at p value < 0.05 using one way analysis of variance(ANOVA) test.MDA: malondialdehyde; SBP: systolic blood pressure; DBP: diastolic blood pressure.


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hyperoxia per se can generate free radicals via directmitochondrial electron transfer.

The current study revealed increase in the level of

uric acid in the preeclamptic group compared with the

control group. Increase in the serum uric acid level in

our PE group is in harmony with other reports. The

association of uric acid with PE has been known since

the late 1800s as reported by Bainbridge and

Roberts.26 Uric acid is a marker of oxidative stress,

tissue injury and renal dysfunction and its raised level

has been suggested to be associated with an almost

doubled risk of severe complications in PE including

perinatal death.27 Uric acid is the end product of pur-

ine metabolism and is synthesized by the enzyme

xanthine oxidase. Hypoxia, ischemia of the placenta

and cytokines such as interferon induce the expres-

sion of xanthine oxidase and therefore increase the

production of uric acid.28 Also, a significant positive

correlation between the serum levels of uric acid and

MDA levels was found in our preeclamptic group.

Taken together, our results suggest that the increase

in serum level of MDA in the preeclamptic patients

represents part of the disease process. However, a par-

tial contribution by the hyperoxic state that accompa-nied CS should also be considered.

To emphasize the role of placental tissue as a con-

siderable source of elevated circulating MDA in PE,

we measured the levels of MDA in PE and normal pla-

centas after 1, 2 and 3 h of incubation at 37C. The

results revealed time-dependent increase in the produc-

tion of MDA by placental tissue with PE compared

with the normal placental tissue (data not shown).

Additionally, our results demonstrated exaggerated

response (% stimulation) of placental tissues with PE

to the prooxidant effects of Fe2 (0.5 mM) and ascor-

bate (0.1 mM) when compared with the corresponding

normal tissue. Furthermore, the inhibition of lipoperox-

ide formation by a-tocopherol (100 mM) was signifi-

cantly lower in placental tissue with PE than in

normal placental tissue. This was attributed to higher

rate of peroxidation and probably due to lower basal

level of vitamin E in preeclamptic placentas.

Few studies reported the involvement of caspase-8and -9 in PE.1,2 Caspase-8 and -9 are mainly involved

in the initiation of apoptosis.13 Our study demon-

strates higher activity of only caspase-9 in

the preeclamptic than in the normal placentas. Addi-

tionally, ROC curve analysis revealed that both

caspase-8 and -9 had similar sensitivity (88%) but the

specificity of caspase-9 was much higher than that of

caspase-8 (96% versus 36%). Since it was reported

that the death receptor pathway of apoptosis was

mediated by caspase-8, while the mitochondrial

Figure 1. In vitro formation of lipid peroxides induced by 0.5 mM ferrous chloride or 0.1 mM ascorbate and the inhibitioninduced by 100 mmol a-tocopherol in normal (n 25) and preeclamptic (n 25) placental tissues. Incubation time 2 h.*,** and # indicate statistical significance from the corresponding controls at p < 0.05, 0.01 and 0.001, respectively.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 1 2 3 4 5 6 7

Serum uric acid (mg/dl)

SerumMDA(nmol/l)

r= 0.8

P< 0.05

Figure 2. Correlation between the serum levels of uricacid and MDA in the PE group.


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pathway of apoptosis was mediated by caspase-9,29

our results demonstrate the involvement of both the

caspases in the pathogenesis of PE, with the domina-

tion of mitochondrial caspase-9 signal transductionpathway.

The relationship between oxidative stress and

apoptosis was explained previously.2,12,30 In agree-

ment with these reports, the current study revealed a

positive correlation between the placental level of

MDA and percentage of DNA fragmentation, which

is a marker of the degradation (late) phase of apopto-

sis in PE. Meanwhile, the sensitivity and specificity of

percentage DNA fragmentation and caspase-9 were

much higher than those of placental MDA. This find-

ing may suggest that apoptosis is more dominant than

oxidative stress in the pathogenesis of PE.

In the current study, statistically significant differ-

ences were found upon comparing the groups of

severe and mild PE with the normal control group.

These differences included some of the investigated

clinical data (SBP, DBP and gestational age) as well

as some of the laboratory data (serum MDA, percent-

age DNA fragmentation, caspase-9 activity and serum

level of uric acid). The results also demonstrated a

decrease in the fetal birth weight in relation to the

severity of PE.

In the context of PE, our study is one of the fewreports that investigated the relationship between the

activities of caspase-8 and -9, percentage of DNA frag-

mentation and MDA and the maternal risk factors and

perinatal outcome. Pregnancy in older women was

reported to be associated with many confounding fac-

tors that end with increased incidence of maternal and

fetal complications.31 Research increasingly suggests

that changes in the levels of estrogen during aging may

be a risk factor for many diseases.32 Conflicting results

were obtained regarding the relationship between

maternal age and apoptosis. Yamada et al.33 reported

that placentas from older women overcome the age-

related hypofunction by increasing the proliferative

signals and reducing the apoptotic signals. Moreover,

Smith et al.34 reported the absence of any significant

correlation between the maternal age and the incidence

of apoptotic cells in normal placentas as demonstratedby microscopic examination.

Interestingly, our results revealed that the percent-

age of DNA fragmentation was negatively correlated

with the maternal age in the preeclamptic as well as

the control group. This may indicate that apoptosis

decreases with the advance of the maternal age

regardless of the presence or absence of PE. Mean-

while, the positive correlation between the activities

of caspase-8 and -9 with the maternal age in the pre-

eclamptic group and its absence in the normal control

group may indicate the presence of unidentified fac-tors in placentas of preeclamptic old women that abort

the apoptotic cascade before reaching its late stage.

So, further studies are recommended to elucidate the

precise mechanism by which maternal aging affects

the apoptotic and proliferative activity of placental

tissue with PE.

Considering gestational age, our results demon-

strated its decrease in the PE group compared with the

control group. This confirms the previously reported

correlation between PE and perinatal outcome,35

which can be attributed to the enhanced apoptotic pro-

cess in preeclamptic placental cells during late stages

of pregnancy as suggested by Wang et al.36 and loss of

the normal placental protection that decrease the lipid

peroxidation process with the advance of gestational

age as reported by Takehara et al.37 Our results sup-

port both mechanisms through (1) the negative

(though statistically insignificant) correlation

between the activity of both caspases (-8 and -9) and

gestational age, (2) the positive correlation between

placental MDA and gestational age in the PE group.

Apoptosis is a potentially important determinant of

placental size and function, which is crucial for growthand development of the fetus throughout gestation.38

Upon examining the relationship between apoptosis

and placental and fetal growth, our results demon-

strated a negative correlation between the activities

of each of caspase-8 and -9, and both weights in the

PE group. This result is consistent with the previously

reported correlation between apoptosis and intrauterine

fetal death in mice.38 Meanwhile, the absence of the

expected significant reciprocal relationship between

fetal birth weight and placental level of MDA may

Table 4. Sensitivity and specificity of the different labora-tory data in PE placentas as calculated by the ROC curve.

Variable Cutoff Sensitivity Specificity AUC

Caspase-9 activity(unit/mg protein)

7 88% 96% 0.99

% DNAfragmentation

29.9% 100% 100% 1

Placental MDA(nmol/mg protein)

0.14 76% 48% 0.63

Caspase-8 activity(unit/mg protein)

7.4 88% 36% 0.61

AUC: area under ROC curve; MDA: malondialdehyde.


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indicate that the fetus was protected from the maternal

oxidative stress but not from the apoptotic process.

Although sex difference and long-term perinatal out-

come are not the topics studied in our study, it is worth

mentioning that evidence suggests interplay of hormo-

nal modulation and genetically determined apoptotic

mechanisms, through which perinatal females may beafforded a level of protection againstperinatal insult that

is greater than that for perinatal males .39 Clinical find-

ings show that male infants are not only more vulnerable

to perinatal insult but they also suffer more long-term

cognitive deficits when compared with females with

comparable injury.39-41 This may be because the activa-

tion of the caspase-dependent apoptotic pathway in

females may afford greater protection, potentially due

to the actions of X-linked inhibitor of apoptosis (XIAP)

within this pathway.39,42,43

In conclusion, the elevations of MDA, caspase-9activity and the percentage of DNA fragmentation

in the placentas of women with PE implicate the

involvement of lipid peroxidation and apoptosis in

PE. The placenta represents a considerable source of

the elevated circulating MDA in PE. The negative

correlation of maternal age with the percentage of

DNA fragmentation and its positive correlation with

the activities of caspase-8 and -9 in placentas of

women with PE support the need for further studies.

Further studies are also recommended to test modula-

tion of apoptosis by drugs inhibiting caspases and/or

affecting the release of the mitochondrial cell death

effectors in a model of preeclamptic placentas.

Conflict of interest

The authors declared no conflicts of interest.

Funding

The present work was partially supported by the financial

assistance provided by Faculty of Medicine, Cairo Univer-

sity, Cairo, Egypt.

References

1. Hung TH, Skepper JN, Charnock-Jones DS and Burton

GJ. Hypoxia-reoxygenation: a potent inducer of apop-

totic changes in the human placenta and possible etio-

logical factor in preeclampsia. Circ Res 2002; 90:

12741281.

2. Aban M, Cinel L, Arslan M, Dilek U, Kaplanoglu M,

Arpaci R, et al. Expression of nuclear factor-Kappa

B and placental apoptosis in pregnancies complicated

with intrauterine growth restriction and preeclampsia:

an immunohistochemical study. Tohoku J Exp Med

2004; 204: 195202.

3. Bellamy L, Casas JP, Hingorani AD and Williams DJ.

Pre-eclampsia and risk of cardiovascular disease and

cancer in later life: systematic review and metaanaly-

sis. BMJ2007; 335: 974.

4. Magee LA and von Dadelszen P. Pre-eclampsia and

increased cardiovascular risk.BMJ2007; 335: 945946.

5. Smith GN, Walker MC, Liu A, Wen SW, Swansburg M,

Ramshaw H, et al. Pre-eclampsia new emerging team

(PE-NET). A history of preeclampsia identifies women

who have underlying cardiovascular risk factors. Am J

Obstet Gynecol2009; 200(1): 58.e158.e8.

6. Chan PY, Morris JM, Leslie GI, Kelly PJ and Gallery

ED. The long-term effects of prematurity and intrauter-

ine growth restriction on cardiovascular, renal, and

metabolic function.Int J Pediatr2010; 2010: 280402.

7. Pettit F and Brown MA. The management ofpre-eclampsia: what we think we know. Eur J Obstet

Gynecol Reprod Biol2012; 160(1): 612.

8. Myatt L and Cui X. Oxidative stress in the placenta.

Histochem Cell Biol2004; 122(4): 369382.

9. Walsh SW, Vaughan JE, Wang Y and Roberts LJ.

Placental isoprostane is significantly increased in pre-

eclampsia.FASEB J2000; 14(10): 12891296.

10. Wyllie AH. Glucocorticoid-induced thymocyte apop-

tosis is associated with endogenous endonuclease acti-

vation.Nature1980; 284(5756): 555556.

11. Favaloro B, Allocati N, Graziano V, Di Ilio C and De

Laurenzi V. Role of apoptosis in disease. Aging

(Albany NY) 2012; (5): 330349.

12. Bainbridge SA, Sidle EH and Smith GN. Direct pla-

cental effects of cigarette smoke protect women from

preeclampsia: the specific roles of carbon monoxide

and antioxidant systems in the placenta.Med Hypoth-

eses2005; 64: 1727.

13. Stegh AH and Peter ME. Apoptosis and caspases.

Cardiol Clin2001; 19: 1329.

14. Stennicke HR and Salvesen GS. Properties of the

caspases.Biochim Biophys Acta 1998; 1387: 1731.

15. Chesley LC. Diagnosis of preeclampsia.Obstet Gyne-col1985; 65(3): 423425.

16. Livingston JC, Park V, Barton JR, Elfering S, Haddad

E, Mabie WC, et al. Lack of association of severe pre-

eclampsia with maternal and fetal mutant alleles for

tumor necrosis factora and lymphotoxin a genes and

plasma tumor necrosis factor a levels. Am J Obstet

Gynecol2001; 184(6): 12731277.

17. Esterbauer H andCheeseman KH. Determination of alde-

hydic lipid peroxidation products: malondialdehyde and

4-hydroxynonenal.Meth Enzymol1990; 186: 407421.


7/24/2019 23515498

9/10

18. Bradford M. A rapid and sensitive method for the

quantitation of microgram quantities of protein utiliz-

ing the principle of protein-dye binding.Anal Biochem

1976; 72: 248254.

19. Buege JA and Aust SD. Microsomal lipid peroxida-

tion.Meth Enzymol1987; 52: 302310.

20. Perandones CE, Illera VA, Peckham D, Stunz LL and

Ashman RF. Regulation of apoptosis in vitro in mature

murine spleen T cells.J Immunol1993; 151:35213529.

21. Fossati P, Prencipe L and Berti G. Use of 3,5-dichlor-

o-2-hydroxybenzenesulfonic acid/4-aminophenazone

chromogenic system in direct enzymatic assay of uric

acid in serum and urine.Clin Chem1980; 26: 227231.

22. Diamant S, Kissilevitz R and Diamant Y. Lipid perox-

idation in human placental tissue: general properties

and influence of gestational age. Biol Reprod 1980;

23: 776781.

23. Robak J and Sobanska B. Relationship between lipidperoxidation and prostaglandin generation in rabbit

tissues.Biochem Pharmacol1976; 25: 22332236.

24. Zhang Z, Blake DR, Stevens CR, Kanczler JM, Win-

yard PG, Symons MC, et al. A reappraisal of xanthine

dehydrogenase and oxidase in hypoxic reperfusion

injury: The role of NADH as an electron donor. Free

Rad Res1998; 28: 151164.

25. Khaw KS, Wang CC, Nagn Kee WD, Pang CP and

Rogers MS. Effects of high inspired oxygen fraction

during elective cesarean section under spinal anesthe-

sia on maternal and fetal oxygenation and lipid perox-

idation.Br J Anaesth 2002; 88: 1823.

26. Bainbridge SA and Roberts JM. Uric acid as a pathogenic

factor in preeclampsia. Placenta 2008; 29(Suppl. A):

S67S72.

27. Koopmans CM, van Pampus MG, Groen H, Aarnoudse

JG, van den BergPP and Mol BW. Accuracyof serum uric

acid as a predictive test for maternal complications in

pre-eclampsia: bivariate meta-analysis and decision anal-

ysis.Eur J Obstet Gynecol Reprod Biol2009; 146: 814.

28. Martin AC and Brown MA. Could uric acid have a

pathogenic role in pre-eclampsia? Nat Rev Nephrol

2010; 6: 744748.29. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F,

Tomaselli KJ, et al. Two CD95 (APO-1/Fas) signaling

pathways.EMBO J1998; 17: 16751687.

30. Wiktor H, Kankofer M, Schmerold I, Dadak A,

Lopucki M and Niedermuller H. Oxidative DNA dam-

age in placentas from normal and pre-eclamptic preg-

nancies.Virchows Arch 2004; 445: 7478.

31. Jolly M, Sebire N, Harris J, Robinson S and Regan

L. The risks associated with pregnancy in women

aged 35 years or older. Hum Reprod 2000; 15:

24332437.

32. Petrovska S, Dejanova B and Jurisic V. Estrogens:

Mechanisms of neuroprotective effects.J Physiol Bio-

chem2012; 68(3): 455460.

33. Yamada Z, Kitagwa M, Takemura T and Hirokawa K.

Effect of maternal age on incidences of apoptotic and

proliferative cells in trophoblasts of full-term human

placenta.Mol Hum Reprod2001; 7: 11791185.

34. Smith SC, Leung TN and Baker PN. Apoptosis is a rare

event in the first trimester placental tissue. Am J Obstet

Gynecol2001; 183: 697699.

35. Al-MulhimAA, Abu-Heija A, Al-Jamma F andEl-Harith

EA. Preeclampsia: maternal risk factors and perinatal

outcome.Fetal Diagn Ther2003; 18: 275280.

36. Wang X, Yi S, Athayde N and Trudinger B. Endothe-lial cell apoptosis is induced by fetal plasma from preg-

nancy with umbilical placental vascular disease. Am J

Obstet Gynecol2002; 186: 557563.

37. Takehara Y, Yoshioka T and Sasaki J. Changes in the

levels of lipoperoxide and antioxidant factors in human

placenta during gestation. Acta Med Okayama 1990;

44(2): 103111.

38. Mu J, Kanzaki T, Si X, Tomimatsu T, Fukuda H, Shioji

M, et al. Apoptosis and related proteins in placenta of

intrauterine fetal death in prostaglandin F

receptor-deficient mice. Biol Reprod 2003; 68:

19681974.

39. Hill CA and Fitch RH. Sex differences in mechanisms

and outcome of neonatal hypoxia-ischemia in rodent

models: implications for sex-specific neuroprotection

in clinical neonatal practice. Neurol Res Int 2012;

2012: 867531.

40. Donders J and Hoffman NM. Gender differences in

learning and memory after pediatric traumatic brain

injury. Neuropsychology2002; 16(4): 491499.

41. Raz S, Debastos AK, Newman JB and Batton D.

Extreme prematurity and neuropsychological outcome

in the preschool years. J Int Neuropsychol Soc 2010;16(1): 169179.

42. Deveraux QL, Takahashi R, Salvesen GS and Reed JC.

X-linked IAP is a direct inhibitor of cell-death pro-

teases.Nature1997; 388(6639): 300304.

43. Carrel L and Willard HF. X-inactivation profile reveals

extensive variability in X-linked gene expression in

females.Nature2005; 434(7031): 400404.


7/24/2019 23515498

10/10

C o p y r i g h t o f H u m a n & E x p e r i m e n t a l T o x i c o l o g y i s t h e p r o p e r t y o f S a g e P u b l i c a t i o n s , L t d .

a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v w i t h o u t

t h e c o p y r i g h t h o l d e r ' s e x p r e s s w r i t t e n p e r m i s s i o n . H o w e v e r , u s e r s m a y p r i n t , d o w n l o a d , o r

e m a i l a r t i c l e s f o r i n d i v i d u a l u s e .

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