Preeclampsia is a pregnancy induced hypertensive disorder with an incidence of 2% - 8% of pregnant women worldwide  . It contributes to maternal/neonatal mortality and morbidity particularly in developing countries where there is poor antenatal care, illiteracy, lack of awareness and poverty  . As per American College of Obstetrics and Gynecology Task Force on Hypertension in Pregnancy, the diagnosis of preeclampsia requires blood pressure ≥ 140/90 mmHg with renal insufficiency, impaired liver function, hematological and neurological complications  . Preeclampsia can cause unfavorable pregnancy outcomes like fetal growth restriction with oligohydramnios, preterm birth, low birth weight, severe birth asphyxia, still birth or intrapartum death  . It is a disease of the placenta that results from insufficient trophoblast invasion leading to oxidative stress, inflammation and endothelial dysfunction  . As there is no curative treatment, except removal of the placenta, early diagnosis of this obstetric disorder by using simple tests could aid in management for improvement of pregnancy outcomes.
In first trimester of pregnancy, serum uric acid levels found to fall less than 3 mg/dL due to estrogen effect, expanded blood volume and increased glomerular filtration rate. The levels slowly increase as gestation proceeds up to 5 mg/dL by term  . However, women who are at risk for developing preeclampsia have elevated levels of uric acid as early as 10 weeks of gestation prior to the presence of proteinuria or hypertension  . Studies suggest that hyperuricemia is not only a prognostic indicator of maternal and fetal complications in preeclampsia but also correlates with the disease progression and contributes to its pathogenesis by virtue of its ability in promoting inflammation, oxidative stress and endothelial dysfunction in both placenta and maternal vasculature    .
Calcium not only helps in mineralization of fetal bone and teeth but also plays a crucial role as a second messenger in many signaling pathways in the placenta  . The intracellular calcium is also known to play a key role in blastocyst implantation, placental development and function  . Hence, alterations in maternal calcium homeostasis could be associated with impaired placental function that can contribute to preeclampsia and adverse fetal outcomes   .
Mean arterial pressure (MAP), a biophysical marker is known to be a better predictor for screening preeclampsia   . It represents average pressure in a patient’s arteries during one cardiac cycle and is considered as a better indicator of perfusion to vital organs than systolic blood pressure. MAP derived by calculation presented by Park H.J. et al.  . The formula describes doubling the diastolic blood pressure (DBP), addition to the systolic blood pressure (SBP) divided by 3 i.e., (MAP = SBP + 2(DBP)/3). Few studies reported increased MAP in prediction of preeclampsia during gestation period intervals.
Xanthine oxidase (XO) or xanthine oxido-reductase catalyzes the oxidation of hypoxanthine and xanthineto uric acid. The information available on the importance of serum XO activity, serum calcium and uric acid in all trimesters of pregnancy and preeclampsia followed by pregnancy outcome is limited. Therefore, in this study context, an attempt was made to consider these parameters as markers to know pregnancy complications and also to evaluate the possible association between serum uric acid, total calcium with XO activity, MAP and fetal outcome in normotensive pregnants and preeclamptic cases in a nested group.
2. Materials and Methods
The study was conducted in joint collaboration of Department of Biochemistry with Department of Obstetrics and Gynaecology in R. L. Jalappa Hospital and Research Centre, Kolar, Karnataka, between August 2017-May 2018. Clinically confirmed pregnant women at first trimester were enrolled after obtaining informed consent and followed till delivery. Ethical clearance for the study was obtained from Central ethics Committee bearing No. SDUAHER/KLR/R&D/47/ 2017-18.
2.2. Sample Collection
The study design was a nested case control. Three milliliters of blood was collected from anti-cubital vein under aseptic conditions using vacutainer from pregnants under non-fasting conditions in first (8 - 11 weeks) and second trimester (19 - 24 weeks) during their visit to the Department of Obstetrics and Gynaecology for regular antenatal check-up. Blood was allowed to retract, centrifuged at 3000 rpm at room temperature for 20 minutes to get clear serum and stored at −20˚C until analysis.
2.3. Inclusion Criteria
This study has 86 pregnant women of primigravida with singleton pregnancy visited for antenatal check-up in first trimester and second trimester of gestation in the age group of 20 - 35 years were included and followed until delivery to record fetal outcome.
2.4. Exclusion Criteria
Pregnant women with history of hypertension, liver disease and renal failure, hyperparathyroidism, cardiovascular or any vascular diseases were excluded from the study.
SBP and DBP were measured in pregnant women using a sphygmomanometer at first and second trimester of pregnancy. MAP was calculated by using the formula described by Katz 2004 which was adapted in research work of Park HJ et al. in 2015   .
The formula describes doubling the diastolic blood pressure (DBP), addition to the systolic blood pressure (SBP) divided by 3 i.e., (MAP = SBP+2(DBP)/3).
Serum total calcium was measured by Arsenazo III dye method, where calcium reacts with dye to produce purple colored complex that was measured in dry chemistry analyzer Vitros FS5.1 (Johnson and Johnson, USA).
Serum uric acid was measured by Uricase enzymatic method. Uric acid in the sample was catalyzed by uricase to produce allantoin, carbon dioxide and hydrogen peroxide. By the action of Peroxidase and in the presence of phenol-derivative,3,5-Dichloro-2-hydroxy-benzenesulfonic acid and 4-Aminoantipyrine, Hydrogen peroxide gives a red colored quinone complex that was measured in dry chemistry analyzer Vitros FS5.1 (Johnson and Johnson, USA).
The assay of serum Xanthine oxidase activity was carried out by colorimetric method as per the procedure supplied by Biovision, USA. XO oxidizes xanthine to hydrogen peroxide which reacts stoichiometrically with OxiRedTM Probe to generate pink color. The absorbance of the pink colour was measured at 570 nm.
4. Statistical Analysis
The obtained data was subjected for statistical analysis by using licensed version of SPSS 20. All variables were expressed as mean, ±SD. Data was checked for normality. Since the data was not normally distributed, the significance of difference in the mean of the measured parameters in first and second trimester of pregnancy was calculated by a non-parametric Wilcoxon rank sum test. Pearson’s Correlation (r) was applied to find the association between serum uric acid and total calcium with XO activity, MAP and fetal birth weight. Mann-Whitney unpaired test was used for comparison of non-normally distributed data between the preeclampsia and control group. p value less than 0.05 (p < 0.05) was considered to indicate statistical significance.
Eighty Six women were enrolled in the study and followed in their first and second trimester of pregnancy till delivery. Table 1 displays the mean ± SD of the demographic variables of the pregnant group (n = 86) in first and second trimester. The mean ± SD of the hematological and biochemical parameters in first and second trimester are shown in Table 2. The mean ± SD of the study parameters in the first and second trimester are shown in Table 3. The first and second trimester mean ± SD values of the study subjects for SBP (108.13 ± 8.74, 101.04 ± 10.40), DBP (72.20 ± 7.57, 67.09 ± 8.79), MAP (84.32 ± 6.71, 78.40 ± 8.53), serum uric acid (2.01 ± 0.85, 4.8 ± 1.93), serum total calcium (10.88 ± 1.97, 9.72 ± 2.04) and serum XO activity (11.96 ± 1.91, 14.05 ± 3.09) respectively. Data checked for normality. Since the data was not normally distributed, Wicoxon rank sum test was used to determine the mean difference between the two sets of observations. p value less than 0.05 was considered statistically significant.
Serum uric acid and calcium were correlated with MAP, XO activity and fetal birth weight in first and second trimesters of pregnancy using Pearson’s correlation, p value less than 0.05 considered statistically significant. The level of significance and correlation of the study parameters were tabulated Table 4. Graphical representation of positive and negative correlations is shown in Figure 1 & Figure 2.
In first trimester, a positive non-significant correlation was observed between uric acid and MAP (r = +0.116, p = 0.288). A positive non-significant correlation was observed between uricacid and serum XO activity (+0.170, p = 0.117). A negative correlation was observed between uric acid and fetal birth weight (r = −0.118, p = 0.279), calcium and XO activity (r = −0.105, p = 0.328) with no
Table 1. Demographic variables of the study group in first trimester and second trimester of pregnancy.
SD: Standard Deviation.
Table 2. Hematological and biochemical parameters of the study group in first and second trimester of pregnancy.
*p < 0.05―statistically significant by Wilcoxon rank sum test; SD: Standard Deviation.
Table 3. Showing mean, ±SD of the study parameters in first and second trimester of pregnancy.
*p < 0.05―statistically significant by Wilcoxon rank sum test; SBP: systolic blood pressure DBP: diastolic blood pressure MAP: mean arterial pressure FBW: fetal birth; weight XO: xanthine oxidase SD: Standard Deviation.
Table 4. Showing positive and negative correlations between serum uric acid and total calcium with mean arterial pressure and fetal birth weight in first and second trimesters of pregnancy.
*p < 0.05―statistically significant by Pearson’s correlation; MAP: mean arterial pressure, FBW: fetal birth weight, XO: xanthine oxidase.
significance. A significant negative correlation was observed between calcium and MAP(r = −0.288, p = 0.007).
In second trimester, a significant positive correlation was observed between uric acid and MAP (r = +0.246, p = 0.022). A significant positive correlation was also observed between calcium and MAP (r = + 0.326, p = 0.007). A significant negative correlation was observed between uric acid and fetal birth weight (r = −0.641, p = 0.000). A negative correlation was also observed between calcium and fetal birth weight(r = −0.316, p = 0.003) and between proteinuria and fetal birth weight (r = −0.514, p = 0.000) with significance. A significant positive correlation was observed between serum uric acid and xanthine oxidase activity in second trimester (r = +0.222, p = 0.040)
The mean ± SD of the demographic and biochemical parameters in preeclampsia and normotensive subjects are tabulated in Table 5. Mann-Whitney unpaired test was used for comparison of non-normally distributed data. P < 0.05 was considered statistically significant. Compared to the normotensive group, women who translated into preeclampsia showed statistical significance in the maternal age (25.04 ± 1.22 vs. 22.85 ± 0.69, p = 0.000). In the first trimester, there was no significant difference in the blood pressure (SBP, DBP and MAP) and calcium levels in both controls and preeclampsia group. First trimester uric acid levels (2.9 ± 0.88 vs. 1.93 ± 0.80, p = 0.005) and serum XO activity (13.57 ± 2.21 vs. 11.82 ± 1.83 p = 0.017) were significantly higher in preeclampsia when compared to the controls respectively. In the second trimester, the preeclampsia and control group showed significant difference in SBP (125.71 ± 5.34 vs.
Figure 1. Showing the graphical representation of correlations between the study parameters in first trimester of pregnancy. (a) Positive correlation between uric acid and mean arterial pressure (r = +0.116, p = 0.288); (b) Negative correlation between uric acid and fetal birth weight (r = −0.118, p = 0.279); (c) Negative correlation between calcium and mean arterial pressure (r = −0.288, 0.007).
Figure 2. Showing the graphical representation of correlations between the study parameters in second trimester of pregnancy. (a) Significant positive correlation between uric acid and MAP (r = 0.246, p = 0.022); (b) Significant negative correlation between uric acid and fetal birth weight (r = −0.641, p = 0.000); (c) Significant positive correlation between calcium and MAP (r = 0.326, p = 0.002); (d) Significant negative correlation between calcium and fetal birth weight (r = 0.361, p = 0.003).
98.86 ± 7.52, p = 0.000), DBP (88.57 ± 3.77 vs. 65.18 ± 6.17, p = 0.000), MAP (100.95 ± 2.52 vs. 76.41 ± 5.41, p = 0.000), serum uric acid (7.3 ± 2.19 vs. 4.6 ± 1.75, p = 0.003), XO activity (21.70 ± 3.50 vs. 13.37 ± 1.93, p = 0.000), Proteinuria (2 vs. 0, p = 0.000), delivery age in weeks (36.14 ± 1.06 vs. 38.96 ± 0.912, p = 0.000), fetal birth weight (2.21 ± 10.10 vs. 2.75 ± 0.25, p = 0.000) respectively.
Eighty-six normotensive pregnant women were followed from first trimester during their prenatal care visit until delivery. The samples were analysed in first and second trimesters for the quantification of serum uric acid, total calcium and also to identify their relationship with MAP, XO activity and fetal outcome. The results indicated seven cases developed preeclampsia which amounts to 8.1% from the study group (n = 86). The remaining seventy-nine pregnant
Table 5. Demographic characteristics and biochemical parameters normotensive and preeclampsia subjects in the study.
*p < 0.05 statistically significant by Mann-Whitney U test. SBP: systolic blood pressure DBP: diastolic blood pressure MAP: mean arterial pressure XO: xanthine oxidase.
women without pregnancy complications were studied as control group.
A significant decrease was observed in the mean of SBP, DBP and MAP from first trimester to second trimester of pregnancy. Study results are in line with the reports of Rebelo F. et al. They reported a decrease in SBP and DBP from first to the second trimester  . The possible explanation for the drop in blood pressure in the second trimester of pregnancy is due to cardiovascular adaptations including increased cardiac output, decreased vascular resistance and other metabolic changes  . Similar observations were also reported by studies conducted by HH Klein et al. and Sanghavi M. et al.   . In the second trimester, our study results showed increased blood pressure in women who developed preeclampsia. Many studies confirmed the presence of hypertension in second trimester in women who develop preeclampsia. Khalil A. et al. investigated the longitudinal changes in the uterine artery mean pulsatility index and MAP in women who developed preeclampsia and gestational hypertension and reported MAP increased from 12 weeks onwards in women who develop preeclampsia  . Tayyar A. et al. also reported MAP increases with gestational age in pregnancies that developed preeclampsia and also suggested assessment in the second and third trimesters aims to identify women at risk of developing preeclampsia  .
Uric acid is heterocyclic and an end product of purine metabolism; possess biological function as an antioxidant at lower concentrations. During early pregnancy uric acid concentration falls and starts rising towards third trimester. This initial drop is due touricosuric effect of estrogen, expanded blood volume and increased glomerular filtration rate  and the rise in late pregnancy may be secondary to increased fetal production, decreased binding to albumin and increased tubular reabsorption  . In the present study, a significant increase of uric acid levels from first trimester (2.01 ± 0.85) to second trimester of pregnancy (4.8 ± 1.93) was noticed. Our study observations are similar with that of the research work carried out by Powers R.W. et al. and Laughon S.K. et al.   . Out of 86 normotensive pregnant women, 7 developed preeclampsia. Serum uric acid levels were measured in control and preeclampsia group in both first and second trimester. The study results showed preeclampsia is associated with increased XO activity and hyperuricemia in first and second trimesters of pregnancy. Increased generation of uric acid is due to increased xanthine oxidase activity in ischaemic placenta, which contributes to the development of hypertension and renal damage in preeclampsia via endothelial dysfunction or renin activation  .
Studies have reported elevated uric acid levels in pregnancies complicated by preeclampsia and thus of good diagnostic and prognostic value for fetus     . Based on these observations, uric acid can be considered as a reliable and cost effective screening test for the early detection of preeclampsia. However there are other studies which have reported the measurement of uric acid levels is not a good predictor of preeclampsia or its complications   .
In the present study, uric acid positively correlated with MAP in the first trimester (r = +0.116, p = 0.288) and second trimester (r = +0.246, p = 0.022) of pregnancy. Study results are in agreement with the research work carried out by Zhou G et al. in 2018 where they reported Hyperuricemia and its association with elevated blood pressure during pregnancy  . Similarly, Laughon SK et al. in 2011 reported a linear association between elevated uric acid with later development of preeclampsia. He also reported uric acid not only predicts the development of preeclampsia but also contributes to the pathogenesis of the disorder  . However, de Jong CL et al. in 1999 also reported the same kind of observations  .
In our follow-up study, a negative correlation was observed between first trimester (r = −0.118, p = 0.279) and second trimester (r = −0.641, p = 0.000) uric acid and fetal birth weight. These results are similar with the studies conducted by Aparna Nair et al., Akahori Y. et al. and Yalamati P. et al. where they reported an association between uric acid and severity of preeclampsia with adverse fetal outcome    . These observations were experimentally evidenced by a set of in-vitro experiments that demonstrated elevated uric acid decreases endothelial cell proliferation and migration thus placental development  , impaired placental amino acid uptake,  trophoblast invasion and endothelial proliferation  , thus blocking fetal angiogenesis resulting in small for gestational age (SGA) infants  . However, a study by Williams KP et al. 2002 indicated contradictory results that uric acid is not a good prognostic indicator of the severity of the maternal or fetal complications  .
In this study, measured total calcium was significantly reduced in the second trimester (9.72 ± 2.04) in comparison to first trimester (10.88 ± 1.97). The results of the study are similar to the results obtained by Hanna B. et al. who reported reduced total calcium levels in second and third trimester compared to first trimester  . The probable reasons for reduced calcium levels in second trimester is due to increased nutritional demands of the growing fetus, expanded intravascular space, reduced albumin concentration, serum PTH is lower by 50% in pregnancy and increased excretion of calcium as a result of increased GFR  .
Negative correlation was also observed between serum calcium and MAP in first trimester of pregnancy (r = −0.288, p = 0.007). There are research evidences that documented an inverse relationship between intake of calcium in pregnancy and incidence of preeclampsia   . In 2011, WHO recommended calcium supplementation with 1.5 - 2.0 g per day in calcium deficient pregnant women for the prevention of pregnancy hypertensive disorders  . However, few contradictory studies also reported the non-beneficial aspect of calcium in prevention of preeclampsia    .
Unlike the first trimester, in second trimester, a strong positive correlation was observed between calcium and MAP with significance (r = +0.326, p = 0.007). The underlying explanation for the positive correlation might be functional role of calcium, where calcium not only brings about smooth muscle contraction but excess calcium can also cause an increase in peripheral vascular resistance leading to essential hypertension  . This biological property of hypercalcemia can be antagonized by using dihydropyridine/nifedipine that blocks influx of extracellular calcium on transmembrane of vascular smooth muscle cells which results in dilatation and reduces the hypertension in preeclampsia and preterm labor   .
There is limited information available on serum total calcium in relation with fetal birth weight. In the current study indicated significant negative correlation between serum total calcium levels and fetal birth weight (r = −0.361, p = 0.003). However, the effect of maternal calcium intake and its influence on infant growth remains unclear. The results of the study by Abalos E. et al. and Abdel-Aleem H. et al. also in agreement with current study where they reported calcium supplementation has no effect on fetal birth weight   . Thoughfew studies have shown calcium intake during pregnancy has a positive effect on the fetal birth weight   . The study results also showed significant negative correlation between Proteinuria and fetal birth weight (r = −0.514, p < 0.001). The results of this study are in agreement with Tayal et al. who reported severe proteinuria could also be an indicator of adverse fetal outcome  .
XO is a metalloenzyme contains iron and molybdenum as an integral component catalyzes the oxidation of xanthine/hypoxanthine into uric acid. Pre-eclampsia occurs only in the presence of placenta which depends on the decreased placental perfusion due to impaired remodeling of spiral arteries. Inadequacy of placental perfusion might result in hypoxic interface between maternal-fetus results in destruction of fetal tissue that can release XO, and substrates like xanthine/hypoxanthine, cytokines etc.
Measurement of XO level in all trimesters of pregnancy is required to understand if gradual increase of XO activity can act as a marker to denote the number of chances translated into pre-eclampsia. Therefore, in the present study understanding of pre-eclampsia at early stage is a good indication to decide suitable treatment strategies to prevent its onset and pathological changes.
Our study results prominently presents the XO activity of the normotensive group in first (13.57 mU/mL), second (21.70 mU/mL) trimesters and in preeclampsia in third trimester (53.60 mU/mL) of pregnancy as shown in Figure 3. Therefore, XO can be considered as cost effective parameter to know at early the later onset of preeclampsia along with uric acid and MAP. Uric acid was also positively correlated with XO activity (r = +0.170, p = 0.117) in first trimester and second trimester (r = +0.222, p = 0.040) of pregnancy.
Our study results also focussed on in finding relationship between elevated calcium levels and Proteinuria in preeclampsia when compared to control group
Figure 3. Showing XO activity in I, II trimesters of normal pregnancy and cases developed preeclampsia.
in second trimester. On successful follow up of the preeclampsia cases showed early gestational age of delivery and low fetal birth weight compared to control group. Supportive evidences to this observation reported by Aabidha P.M. et al. and Seyom E. et al. who showed that neonatal complications like prematurity, growth restriction and low birth weight are generally seen associated with preeclampsia   .
The limitation of the study is small sample size and non inclusion of other indicators denoting fetal outcome.
The nested case-control study on normotensive pregnant women evinced measurement of simple cost effective routine parameters like uric acid, calcium and mean arterial pressure serve as good indicators in early understanding of later onset of preeclampsia. First trimester screening is crucial over the second trimester to know the early onset of the disorder associated with the underlying placentation process. Besides, evaluation of xanthine oxidase activity in all trimesters of pregnancy is more informative and becomes newer aspect relating to placental ischemia. The preeclampsia group showed poor fetal outcome i.e., pre-term delivery and low fetal birth weight. Hence, measured biochemical and physical parameters facilitate their effectiveness in early assessment of preeclampsia.
We would like to thank the Authorities of Sri Devaraj Urs Academy of Higher Education and Research and Proteomics research laboratory for supporting this doctoral study.