NTN-PW: normotensive pregnant women; BOS: biomarkers of oxidative
stress; AGMs: angiogenic growth mediators; O&G: obstetrics and
gynaecology; W1, wave 1 or visit 1, W2, wave 2 or visit 2; W3, wave 3 or
visit 3
A qualified consultant obstetrician/gynaecologist physically examined
all participants. PE was defined as systolic blood pressure (SBP)
/diastolic blood pressure (DBP) greater than or equal to 140/90mmHg with
visible proteinuria (≥1+ dipstick) or 24-hour proteinuria of ≥300mg/day
on two (2) occasions at least four (4) hours apart detected after 20
weeks gestation in previously normotensive pregnant women. Early-onset
PE (EO-PE) and late-onset PE (LO-PE) were defined as PE that occurred
before and at or after 34 weeks gestation, respectively (American
College of Obstetricians and Gynecologists & Task Force on Hypertension
in Pregnancy, 2013; Raymond & Peterson, 2011)
Laboratory assays
Serum, plasma and urine samples were obtained from all participants up
to a total of 3 visits or waves, at 10-week intervals throughout
gestation (median 17, 27, and 37 weeks). Samples were stored at -80oC (Thermo Scientific Ultra-Low Freezer) until the
biomarkers of OS and AGMs were analysed.
Following the manufacturer’s instructions, urinary and serum 8-OHdG were
analysed in duplicate using highly sensitive and competitive ELISA kits
(ab201734, Abcam, China). Serum concentrations of 8-OHdG were measured
immediately after sample collection to avoid autoxidation during long
storage. The inter-and intra- assay coefficients of variation (CV) were
3.5% and 4.5%, respectively. Urinary 8-OHdG concentrations were
normalised to creatinine (Cr) concentrations and recorded as ng/mg Cr.
Serum 8-epi-PGF2α was analysed in duplicate using competitive ELISA kits
from ELabscience, China (cat. log E-EL-0041). The intra-and-inter assay
coefficients of variation (CV) were 5.6% and 6.4%, respectively.
TAC reagents were obtained from Sigma-Aldrich (Hong Kong, China). Plasma
samples were thawed to measure TAC spectrophotometrically at 593 nm
using Mindray BA-88A, China. The estimation of TAC was based on the
Ferric Reducing Ability of Plasma (FRAP) and the protocol as described
by Benzie and Strain (1996). The absorbance was used to obtain the
concentrations after comparison to standard curves and recorded in
µmol/l.
AGMs including serum concentrations of VEGF-A, sFlt-1, PlGF, and sEng
were measured in duplicate using competitive Quantikine ELISA kits from
R&D System Inc. (Minneapolis, MN USA). Absorbance was measured at 450
nm wavelength using a microplate ELISA reader (Bio-Tek ELx808 microplate
reader, Hayward, CA, USA). The inter-and intra- assay coefficient of
variation obtained in our laboratory was 1.1 and 1.3 for VEGF-A, 1.5 and
3.8 for sFlt-1, 4.6 and 3.3 for PlGF and 2.8 and 5.2 for sEng,
respectively.
All laboratory assays were performed at the Molecular Medicine
Laboratory of the Kwame Nkrumah University of Science and Technology and
the Biochemistry and Immunology Department of the Komfo Anokye Teaching
Hospital, Ghana.
Statistical analyses
The normality of the data was tested using the Kolmogorov-Smirnov test.
Data were presented as median (interquartile ranges) for non-parametric
continuous variables and frequency (percentages) for categorical
variables. A Chi-square test was performed to test associations between
the proportions of variables among the study groups. Median comparisons
between more than two independent variables were performed using
Kruskal-Wallis one-way ANOVA followed by a Bonferroni posthoc multiple
comparison test and adjusted p-values were recorded. A receiver
operating characteristic (ROC) curve and area under the curve (AUC) were
generated to evaluate the diagnostic performance of the model. P< 0.05 was considered statistically significant. Data were
analysed using SPSS version 24 (IBM Corp, NY, USA), XLSTAT Premium
version 2018.1 and R version 3.4.3 (R core Team 2017).
Results
Unlike OHS groups, there was a statistically significant difference
between the median maternal ages between SHS pregnant group who
developed PE compared to those who did not (p <0.001).
There was a significantly increased SBP, DBP, sEng, sFlt-1, 8-epiPGF2α,
serum 8-OHdG, urinary 8-OHdG and combined ratios of sFlt-1/PlGF ratio,
8-epiPGF2alpha/PlGF ratio, 8-OHdG/PlGF ratio and sEng/PlGF ratio, and
correspondingly reduced PIGF, VEGF-A and TAC among PE groups compared to
NTN-PW group (p <0.001). Unlike the OHS groups, the
degree of imbalance in biomarkers of OS and AGMs was higher in SHS who
developed EO-PE followed by LO-PE compared to NTN-PW
(p <0.001). Although no statistical
significance was observed, the clinically significant difference
indicated by the high level of imbalances in favour of SHS rather than
the OHS group was observed in biomarkers of OS and AGMs. Meanwhile,
there was a significant difference in median SBP between SHS-associated
NTN-pregnancy and OHS- associated NTN-pregnancy (p =0.038)(Table 1) .
Overall, SBP, DBP, and biomarkers of OS and AGMs increased from baseline
to mid-pregnancy among SHS women who later developed PE and
NTN-pregnancies rather than OHS pregnant women who later developed PE
and NTN-pregnancies. At both early 2nd trimester
(10-20 weeks gestation, W1) and mid-pregnancy (21-31 weeks gestation,
W2), the median maternal serum levels of PlGF, VEGF-A, and plasma TAC
were significantly decreased whereas those of sEng, sFlt-1, 8-epiPGF2α,
8-OHdG, urinary 8-OHdG and the ratios: sFlt-1/PlGF, 8-epiPGF2α/PlGF,
8-OHdG/PlGF and sEng/PlGF were significantly increased among the SHS who
later developed EO-PE followed by LO-PE compared to NTN-PW
(p <0.001). Similar observations occurred among the OHS
group (p <0.001) even though the trend of imbalance was
higher among the SHS group. There was a clinically significant
difference between the SHS group and the OHS who later developed PE and
those who did not (Table 2).
Within each visit, there was no difference in gestational age across the
groups. Meanwhile, there was a significant difference in SBP and DBP at
both visit 1 or W1 and visit 2 or W2 across the study groups (p<0.05) (Table 2).
Compared to the individual biomarkers at visit 1 or W1 and visit 2 or
W2, the combined biomarkers of OS and AGMs, particularly the
mid-pregnancy (W2) 8-OHdG/PIGF ratio yielded the highest discriminating
power or AUC (0.93, p <0.001) (Figure 2c) with
the best sensitivity (85.6%), specificity (92.4%), positive predictive
value (PPV) (86.6%), negative predictive value (NPV) (85.2%), positive
likelihood ratio (LR+) (9.9) and negative likelihood ratio (LR-) (0.1)
at a cut-off value ≥0.80. At the cut-off value for 8-OHdG/PIGF ratio,
NTN-PW had 4.8-fold increased odds of developing PE (adjusted odds ratio
(aOR) =4.8 95%CI (1.5-11.5), p <0.001).
Except for W1 TAC levels, all the single and combined biomarkers of OS
and AGMs yielded a significant (all p <0.05)
discriminating power and adjusted odds ratios for predicting PE(Table S1) .