Materials and Methods

This study is a prospective observational study of nulliparous women attending antenatal

outpatient department (OPD) and was performed over a period of two years starting from
February 2010 till January 2012 at a tertiary care hospital of armed forces, India. The local ethics
committee approved the study protocol.
The study included 306 nulliparous women attending antenatal OPD. The inclusion criteria were:
Antenatal cases registered before 14 weeks of gestation and who were willing to participate in
the study. The exclusion criteria were: 1. Multipara, 2. Pre-existing hypertension, 3. Connective
tissue disorder like systemic lupus erythematosus (SLE), 4. Anemia (Hb < 8 gm%), 5. Multiple
pregnancies.
Total 332 patients were given consent for the study, and by using exclusion criteria, 26 patients
were excluded from the study (anemia-16, pre-existing hypertension-05, multiple pregnancy-04,
SLE-01). Remaining 306 booked nulliparous patients meeting the inclusion criteria were studied.
Detailed history-taking and examination was done. Period of gestation (POG) was confirmed by
dates and correlated with ultrasonography (USG). In case of unsure dates, ultrasonographyexpected date of delivery (USG-EDD) of first trimester was taken to calculate POG. All 306
patients underwent serum lipid profile and ceruloplasmin levels estimation at 14-16 weeks POG
as sample 1 and at 18-20 weeks as sample 2. All cases were then followed up till the end of
pregnancy for development of pre-eclampsia.
Pre-eclampsia was defined as a rise in blood pressure combined with proteinuria after 20 weeks
gestation. Hypertension was defined as a sustained blood pressure (BP) reading of 140/90 on at
least two occasions 6 h apart in a previously normotensive woman. Blood pressure was recorded
in the right arm with a random zero sphygmomanometer; the reported values represented the
mean of two readings taken at one-minute intervals. Proteinuria was defined as a protein dipstick
measurement of 1+ on a midstream urine sample at least twice (24 h apart) or a 24-h urine
excretion 0.3 g protein in the absence of a urinary infection. Maternal fasting blood samples
were collected after an overnight fast of at least 8 h. All samples were analyzed for triglycerides
(TG), total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and
ceruloplasmin concentrations. Cholesterol and triglyceride concentrations were measured with
the enzymatic colorometric method (Cobas Integra 800, Roche, Germany). HDL cholesterol was
separated from LDL and very LDL cholesterol by a chemical precipitation technique and
ultrasound.[
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Destroy user interface control12] Ceruloplasmin was measured by automatable assay for
ceruloplasmin as feroxidase.[
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Destroy user interface control13] Body mass index was calculated as weight in kilograms
divided by the square of height in meters. The height and pre-gestational weight of each patient
were reported; if the patients did not know their weight or if it had been measured a long time
before conception, they were weighed during their initial visit. For socio-economic status,
monthly income was taken into consideration and divided in three groups namely poor (<
15,000), middle class ( 15,000-30,000), and rich (> 30,000).
The first value for the biochemical markers was done in all subjects at 14 to 16 weeks gestation.
The second value of biochemical markers at 18-20 weeks period of gestation in maternal blood
for oxidative stress for three cases cannot be performed as they had mid-trimester abortion.
Twenty-four patients developed pre-eclampsia, and they are taken as cases of interest, and others
who did not develop pre-eclampsia are taken as normals for statistical analysis. Baseline value of
the biochemical markers for statistical analysis has been taken at 14-16 weeks period of
gestation.

Statistical analysis
The collected data so obtained was statistically analyzed by SPSS statistical software using t test
for comparison of average values, Chi-square test for evaluation of qualitative variables. The
results were expressed as percent and mean with standard deviation. The P values of less than
0.05 was considered as statistically significant. Univariate analysis was carried out for all the
categorical variables using Chi square test and for quantitative variables using student t test at
14-16 weeks and at 18-20 weeks of POG. Logistic multivariate analysis was carried out to assess
the effect of demographic and oxidative stress parameters at 18-20 weeks simultaneously on the
dependent outcome variable of pre-eclampsia.
Findings of the study were compared with the other studies on biochemical markers of oxidative
stress as predictors of pre-eclampsia given in literature, and conclusion was drawn about the
prediction of pre-eclampsia in early gestation.
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Results
The univariate analysis [Table 1] revealed that there was no statistically significant difference
between the normals and pre-eclampsia cases at 14-16 week for all the oxidative stress
parameters.

Table 1
Comparison between normals and cases at 14-16 weeks period of gestation
The univariate analysis [Table 2] revealed that there was statistically significant difference
between the normals and pre-eclampsia cases at 18-20 week in cholesterol and ceruloplasmin
parameters.

Table 2
Comparison between normals and cases at 18-20 weeks period of gestation
The regression model revealed that only cholesterol was statistically significant parameter (P =
0.034, Odds ratio 1.034, 95% CI = 1.002-1.067). All the other variables that is age, BMI, socioeconomic status, triglyceride, HDL, LDL, ceruloplasmin was not significant as revealed in Table
3.

Table 3
Logistic multivariate analysis
Maternal and fetal characteristics are shown in table 4 and table 5 is showing the total number of
pre-eclampsia cases, which developed during different period of gestation. In this study,
incidence of development of pre-eclampsia was 8%. Two cases had early onset pre-eclampsia,
i.e., pre-eclampsia developed before 32 weeks period of gestation. Between 32-34 weeks period
of gestation, 5 cases developed pre-eclampsia, and 17 cases had pre-eclampsia after 34 weeks
period of gestation. Most of the pre-eclampsia cases (70%) developed after 34 completed weeks.
There was 1 case of eclampsia, which developed at 29 weeks period of gestation.

Table 4
Maternal and fetal characteristics

Table 5
Number of preeclampsia cases at different period of gestation
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Discussion
A potential role for oxidative stress in the pathophysiology of pre-eclampsia was first proposed
by Stark in 1993.[
The following popper user interface control may not be accessible. Tab to the next button to
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Destroy user interface control14] Raised oxidative stress markers glutathione peroxidase,
superoxide dismutase, and malondialdehyde in maternal serum of patient of pre-eclampsia
suggest that oxidative stress markers play a significant role in pathophysiology of pre-eclampsia.
[
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Destroy user interface control15] The incidence of pre-eclampsia in this study was 8% as most of
the patients were primigravidas, and it was comparable with other studies done at same local
population.[
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Destroy user interface control17]

Triglyceride-rich remnants are known to cause endothelial dysfunction, and because the
triglyceride content of intermediate dense lipoproteins (IDL) was positively correlated with
elevated blood pressure and proteinuria, triglyceride-rich remnant lipoproteins might contribute
to the pathophysiology of pre-eclampsia.[
The following popper user interface control may not be accessible. Tab to the next button to
revert the control to an accessible version.
Destroy user interface control18] In our study, triglyceride level was not found to be significant
between the normals and pre-eclampsia cases at 14-16 weeks and 18-20 weeks. However, Hubel,
et al., in 1996, found that triglycerides and fatty acid levels are elevated in patients with preeclampsia, and these changes antedate clinically evident disease by weeks to months,[
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Destroy user interface control9] which is not supported by our findings. The levels of HDL, LDL
were not found to be significant between the normals and pre-eclampsia cases at 14-16 weeks
and 18-20 weeks in our observation. Bainbridge and associates, in 2005,[
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Destroy user interface control19] stated increased levels of lipid peroxidase like
malondialdehyde and variety of pro-oxidants or potentiators of pro-oxidants, including iron,
trasferrin, and ferritin; blood lipids, including triglycerides, free fatty acids, and lipoproteins are
increased, and antioxidants, including ascorbic acid and vitamin E, are decreased in preeclampsia.
Cekmen, et al. concluded that in the pre-eclamptic group, plasma total triglyceride, low-density
lipoprotein cholesterol (LDL-C), malondialdehyde (MDA), and apolipoprotein B (apo-B) were
significantly increased, while plasma high-density lipoprotein cholesterol (HDL-C) was
significantly decreased compared to that of control group. There was no significant difference in
total cholesterol and apolipoprotein A1 concentrations,[
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Destroy user interface control20] which is contrary to our study, in which cholesterol is found to
be significantly increased in pre-eclamptic cases.
Total cholesterol concentrations were significantly higher in pre-eclamptic patients compared
with controls in the present study. Van den Elzen, et al. reported that first trimester serum total
cholesterol concentrations were significantly associated with the risk of pre-eclampsia, and that

those with such elevated levels of cholesterol experienced a 5-fold increase in the risk of preeclampsia after accounting for confounding factors like maternal body mass index and
gestational age.[
The following popper user interface control may not be accessible. Tab to the next button to
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Destroy user interface control21] Enquobahrie, et al. also found that there was a 3.60-fold
increase in the risk of pre-eclampsia among women with total cholesterol of >205 mg/dL before
16 weeks gestation.[
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The interactions among a disordered lipid profile, endothelial cells, and oxidative stress have
been hypothesized to be of major significance to the pathophysiology of pre-eclampsia by
several investigators.[
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Destroy user interface control9,
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Destroy user interface control23,
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Destroy user interface control24] High concentrations of circulating triglyceride-rich lipoproteins
may induce endothelial dysfunction through the generation of small dense LDL sub-fractions,
which have been found to be oxidized more readily than their larger counterparts. It was
indicated that small dense LDL fractions had a greater capacity to stimulate the thromboxane
synthesis by endothelial cells and an increase in intracellular calcium in vascular smooth muscle,
which might be relevant to vasospasm in pre-eclampsia.[
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Destroy user interface control25] Studies have shown this endothelial cell damage in the kidney
and placenta.[

The following popper user interface control may not be accessible. Tab to the next button to
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In the present study, there was no statistically significant difference between the normals and preeclampsia cases at 14-16 week for all the oxidative stress parameters, but there was statistically
significant difference between the normals and pre-eclampsia cases at 18-20 weeks in
ceruloplasmin parameter. This shows that ceruloplasmin level increases during the middle of
second trimester in patients who destined to develop pre-eclampsia. Aksoy, et al., in 2003,
support our findings as they also observed that the plasma antioxidant potential and transferrin
levels in pre-eclampsia patients were reduced and the malondialdehyde with ceruloplasmin
levels were increased as compared to the normotensive pregnant women.[
The following popper user interface control may not be accessible. Tab to the next button to
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Destroy user interface control26] Shamsi AZ and associates reported that serum level of
ceruloplasmin significantly lower in normal pregnancy than mild and severe pre-eclampsia.[
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The strengths of our study include the prospective design and the high follow-up rate. In
addition, the collection of blood early in pregnancy enabled us to show that increased levels of
serum cholesterol and ceruloplasmin concentrations precede the clinical manifestations of preeclampsia. We used logistic regression to adjust for a number of confounders. We also used
fasting blood samples to determine lipid concentrations. However, in studies of non-pregnant
individuals, investigators have found a strong correlation between fasting and post-prandial lipid
concentration measurements.[
The following popper user interface control may not be accessible. Tab to the next button to
revert the control to an accessible version.
Destroy user interface control28] In addition, almost all our patients had similar socio-economic
situations, education, and smoking status.
Several important limitations must be considered when interpreting the results of our study. First,
two measurement has resulted in some misclassification of maternal lipid profiles and
ceruloplasmin levels during pregnancy. Longitudinal studies with serial measurements of
maternal lipid and ceruloplasmin concentrations are needed to elucidate patterns of their levels,
the determinants of these changes, and the pathophysiologic consequences of such changes
during pregnancy. Second, in our study, only 24 patients had pre-eclampsia. Relatively small
number of pre-eclampsia patients hindered inferences from some of our analyzes. Third,

although we compared for many potential confounders, we cannot exclude the possibility of the
other confounding from unmeasured covariates. Finally, another limitation of the present study is
that it was done in only one medical center.
This study suggests that cholesterol and ceruloplasmin levels in second trimester at 18-20 weeks
POG can predict the development of pre-eclampsia. Information from this study may contribute
to the development and evaluation of behavioral and medical interventions aimed at reducing the
occurrence of pre-eclampsia.
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Footnotes
Source of Support: Nil.
Conflict of Interest: None declared.
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