CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 3, April 2012
154
AFRICA
in the non-pregnant state and who had hypertension at the first
antenatal visit prior to 20 weeks’ gestation.
All clinical data were recorded on a structured form and
included the highest blood pressure measurement, level of
proteinuria, maternal age, parity, gestational age, mode of
delivery and neonatal outcomes.
Pre-partum blood samples were obtained from participants
who were in early labour. Postpartum central placental samples
were obtained soon after delivery of the babies.The blood samples
were centrifuged at 4°C at 3 000 rpm for 30 minutes and aliquots
of serum samples and placental tissue samples were stored at
–70°C until used. All participants with hypertension (chronic
hypertensives and pre-eclamptics) were on antihypertensive
medication, namely methyldopa.
The quantitative sandwich enzyme immunoassay technique
was performed on serum samples to analyse for the levels of
sFlt-1, VEGF and PlGF using Quantikine ELISA kits (R & D
Systems, Minneapolis, USA). All assays were performed in
duplicate according to the manufacturer’s instructions.
RNA was extracted from placental samples using a protocol
previously described,
15,16
and synthesis of cDNA was performed
using the Bio-Rad iScript cDNA synthesis kit according to
the manufacturer’s protocol (Bio-Rad Laboratories (Pty) Ltd).
Thereafter, real-time PCR was performed to determine the levels
of mRNA expressions of sFlt-1, VEGF, PlGF and AT1 using
standard methods.
16-18
Statistical analysis
All values are expressed as means
±
SEM (standard error of
mean). Statistical tests were performed using SPSS version
15.0 (SPSS Inc, Chicago, Illinois, USA). A
p
-value of
<
0.05
was considered statistically significant. The Kruskal-Wallis test
was done to assess for any overall significant differences across
the four groups within each variable in clinical data, as well as
between the groups for each mRNA gene expression. This was
followed by the Mann-Whitney test to determine which groups
displayed these differences.
One-way ANOVA was performed to determine if there were
significant differences in the serum concentrations of sFlt-1,
PlGF and VEGF, and the mRNA expression levels of sFlt-1,
VEGF, PlGF and the AT1 receptor among the groups. The Tukey-
Kramer multiple comparison test and the Mann-Whitney test
compared each group against the other and determined which
two groups displayed a significant difference (
p
<
0.05).
Results
Table 1 shows the demographic and clinical data including
neonatal outcomes of all patients. Except for changes in blood
pressure and proteinuria, there were no significant differences in
any of the parameters among the four groups.
The body mass of the neonates in the early-onset (2.83
±
0.38 kg) and late-onset (3.03
±
0.16 kg) pre-eclamptic groups
were lower than those of the normotensive (3.24
±
0.08 kg) and
chronic hypertensive (3.34
±
0.17 kg) groups. However, these
differences were not statistically significant (
p
>
0.05).
The serum sFlt-1 concentrations in the normotensive controls
(9 603
±
1 797 pg/ml) were significantly less than those in
the early-onset pre-eclamptic group (26 682
±
5 482 pg/
ml) (
p
<
0.05) (Fig. 1). The levels in the early- and late-onset
pre-eclamptic (16 069
±
4 305 pg/ml) groups were higher than
those in the chronic hypertensive group (8 811
±
2 008 pg/ml)
but these changes were not significantly different.
The serum concentrations of VEGF in all four groups were
below the detectable limit of the assay.
Fig. 2 shows that the
normotensive group (0.83
±
0.11 pg/ml) had significantly raised
serum PlGF levels compared to the early-onset pre-eclamptic
group (0.23
±
0.031 pg/ml) (
p
=
0.001). Furthermore, there
was a significant difference in the levels of PlGF between the
normotensive (0.83
±
0.11 pg/ml) and chronic hypertensive
(0.42
±
0.063 pg/ml) groups (
p
<
0.05) (Fig. 2). In addition, the
early-onset pre-eclamptic group had relatively lower serum PlGF
levels compared with the late-onset group (0.45
±
0.103 pg/ml).
The relative placental mRNA expression levels of sFlt-1,
VEGF, PlGF and AT1 were compared across the four groups and
were normalised to the housekeeping gene, GAPDH. Data are
expressed as fold changes.
TABLE 1. DEMOGRAPHIC DATAAND CLINICAL CHARACTERISTICS OFALL PATIENTS
Variable
Group
Normotensive controls
(n
=
29)
Chronic hypertension
(n
=
9)
Early-onset pre-eclampsia
(n
=
10)
Late-onset pre-eclampsia
(n
=
9)
Maternal age (years)
28.48
±
1.19
28.78
±
1.86
29.88
±
3.40
28.33
±
2.05
Parity
2
±
0.23
2
±
0.40
2
±
0.53
1
±
0.33
Gestational age (weeks)
38.46
±
0.26
(n
=
28)
38.44
±
0.47
36.38
±
1.72
37.67
±
0.73
Blood pressure (mmHg)
Systolic
118.59
±
1.79
139.67
±
3.16
152.75
±
6.39*
161.78
±
3.68*
#
Diastolic
72.24
±
1.57
91.33
±
2.74
94.63
±
5.79*
102.22
±
3.08*
#
HIV status (%)
Positive
44.83
44.44
25
66.66
Negative
51.72
55.55
62.5
33.33
Unknown
3.45
0
12.5
0
Mass of neonate (kg)
3.24
±
0.08
3.34
±
0.17
2.83
±
0.38
(n
=
9)
3.03
±
0.16
*Significant difference in comparison with normotensive control group (
p
<
0.05).
#
Significant difference in comparison with chronic hypertensive group (
p
< 0.05).
