Title: Serum Calcium and Its association with Preeclampsia

Abstract
Background: Preeclampsia is one of the major cause of maternal and fetal morbidity and
mortality. It is seen in 2% to 8% of all pregnancies and its pathogenesis is still obscure.
Objective: To evaluate the association of serum calcium concentration with preeclampsia.
Methods: This case control study was carried out in the department of obstetrics and
Gynecology of BSMMU, Dhaka, Bangladesh from January 2016 to December 2016. A total
number of 120 women were enrolled in this study and they were classified as mild
Preeclampsia (n=40) considered as Group A, severe preeclampsia (n=40) considered as
Group B and normal pregnant women without preeclampsia taken as controls considered as
Group C. Blood samples were collected and serum calcium was estimated by Arsenazol-III.
Statistical analyses of the results were obtained by using window based computer software
devised with Statistical Packages for Social Sciences (SPSS-20). Results: The mean serum
calcium concentration was found 8.2±0.2mg /dl in-Group-A, 7.6 ± 0.3 mg/dl in Group-B and
9.0±0.5 mg/dl in Group-C (p=0.001). Low serum calcium was found 53.7% and 12.5% in
case and control respectively. The difference was statistically significant (p=<0.05) between
case and control groups having OR=8.14 with 95% CI (2.67 - 26.47) %. Conclusion: This
study revealed that that hypocalcaemia and its concentration varies with the severity of the
disease process.
Key words: Serum calcium concentration, severity,, Preeclampsia, normal pregnancy &
proteinuria.

INTRODUCTION

Preeclampsia is a hypertensive disorder in pregnancy which is characterized by the

occurrence of new onset of persistent hypertension with new onset of proteinuria or in
absence of proteinuria presence of new onset end organ damage, usually after 20 weeks of
gestation (ACOG, 2013). Preeclampsia may be mild or may cause maternal mortality and
serious immediate morbidity like eclampsia, abruptio placenta, stroke, cerebral & pulmonary
edema, renal & hepatic failure or disseminated intravascular coagulation and late morbidity
like hypertensive disorder, renal disease. It also causes fetal and neonatal consequences such
as intrauterine growth restriction, stillbirth, severe iatrogenic prematurity due to delivery for
maternal indication as the only known treatment of preeclampsia is delivery, after which
symptoms usually resolved (Peters and Flack, 2004, Ota et al. 2014). As preeclampsia is the
3rd common cause of maternal death in the world. In spite of several-decade studies on
preeclampsia, the cause is still unknown (Bahadoran et al. 2010).

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Pre-eclampsia, is a multifactorial disorder that is associated with maternal and perinatal
morbidity and mortality worldwide, as well as with neurological disorders, liver disease and
abnormal renal function (Kiondo et al. 2014). The incidence in South-East Asia and North
African regions 1.51% and 1.56%, respectively (Abalos et al. 2014) but higher in South
Africa i.e., 1.8% to 7.1% (Osungbade and Ige, 2011). Around 40,000 women, mostly from
developing countries, die each year due to preeclampsia or eclampsia. Preeclampsia alone is
estimated to account for about 40% to 60% of maternal deaths in developing countries
(Lakew et al. 2013). The prevalence of preeclampsia in developing countries ranges from
1.8% to 16.7% (Lakew et al. 2013). World Health Organization (2014); despite active
research for many years, the etiology of this disorder remains unknown, although
contributory factors including obesity, diabetes, calcium deficiency, older maternal age and
job stress have been observed and studied.

Calcium is the micronutrient that has been best studied in relationship to preeclampsia.
Several epidemiological studies in developing nations indicate an association between
reduced calcium intake and preeclampsia. These observations led to the hypothesis that the
incidence of preeclampsia can be reduced in populations of low calcium intake by calcium
supplementation (Roberts et al. 2003).

An inverse relationship between calcium intake and hypertensive disorders of pregnancy was
first described in 1980 based on the observation that Mayan Indians in Guatemala had a low
incidence of preeclampsia as their intake of calcium was high due to the traditional method of
soaking corn in lime before cooking (Belizan & Villa, 1980). Mahomed et al. (2000) reported
a low intake of calcium during pregnancy in many different parts of the world such as Asia,
Latin America, and Africa as well as developed countries such as Canada, USA and the UK.
They reported a very low intake of calcium in India (250 mg/d).

Calcium plays an important role in muscle contraction and regulation of water balance in
cells. Modification of plasma calcium concentration leads to the alteration of blood pressure.
The lowering of serum calcium and the increase of cellular calcium can cause an elevation of
blood pressure in preeclamptic mothers. The increase of cellular calcium concentration when
serum calcium went lower led to constriction of smooth muscles in blood vessels and
increase of vascular resistance (Sukonpan & Phupong, 2005).

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Preeclampsia is the second leading cause of maternal death in Bangladesh. About 20% of all
maternal death occur due to preeclampsia/eclampsia in every year in our country (streatfield
et al. 2010) patients. Several studies revealed significant association between lower serum
calcium concentration and preeclampsia. Serum calcium concentration measurement is
relatively easy, minimally invasive test and not very much costly. Therefore, considering the
above mentioned facts, the present study was designed for evaluation of serum calcium
concentration in pregnant women with preeclampsia and comparing them with those of the
control group to verify the possible association between hypocalcaemia and PE. The
detection of association of hypocalcaemia with preeclampsia will help us to predict such a
serious condition early so that preventive measures can be taken.

MATERIALS

In this case control study, 120 women having gestational age within >20 weeks to 40 weeks
were included and distributed into three groups. Forty pregnant women with mild pre-
eclampsia were selected as group A, 40 pregnant women with severer pre-eclampsia as group
B and 40 normal healthy pregnant women as group C according to the selection criteria who
were attending in outpatient and inpatient department of Obstetrics and Gynaecology in
outpatient department (OPD) and inpatient department (IPD) of Bangabondhu Sheikh Mujib
Medical University (BSMMU), Dhaka, Bangladesh during the period from January 2016 to
December 2016. Pregnant women who were clinically diagnosed as preeclampsia during their
second half of pregnancy (> 20 weeks to 40 weeks) attending the outpatient department
(OPD) and inpatient department (IPD) of BSMMU as well as fulfilled the selection criteria
were selected as case. Age, parity and gestational age matched apparently healthy
normotensive pregnant women in their second half of pregnancy (> 20 weeks to 40 weeks)
attending the OPD and IPD of BSMMU as well as fulfilled the selection criteria were
selected as control. The exclusion criteria include patient with diabetes, renal disease, patient
with cardiovascular disease, patients with chronic hypertension and patient with hemorrhagic
disorders. After selection of the study subject according to inclusion criteria and objectives,
nature, purpose and potential risk of all procedures used for the study were explained in
details and written informed consent from pre-eclampsia and normal healthy pregnant women
were taken. A detailed general, systematic, obstetric examination and medical history were
taken. After selecting cases and controls, with all aseptic precaution 05ml antecubital venous
blood sample was collected from each subject for measurement of serum calcium. Blood was
immediately transferred into a clean dry test tube and sent to Biochemistry laboratory of
BSMMU. All these information were collected in a pre-designed structured data collection
sheets.
Blood pressure (BP):
As a baseline investigation, after 10 minutes rest, BP was measured on the both arms in a
sitting posture following all standard procedure. Blood pressure measured again after 5
minutes interval and the average were used for the analysis.
Measurement of serum calcium concentration:

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Serum calcium was estimated by Arsenazol-III. It is recommended that each laboratory
determine its own reference range based upon its particular locale and population
characteristics. Normal serum calcium 8.5 – 10.5 mg/dl.
Estimation of urinary protein:
Proteinuria was measured by dipstick method. Test for protein in urine by multiple reagent
strip (dipstick) as follows: Trace = 0.1 gm/L; 1+ = 0.3 gm/L; 2+ = 1.0 gm/L; 3+ = 3.0 gm/L;
4+ = 10.0 gm/L (Haugen et al. 2006).
Statistical analysis: The statistical analysis of the results were obtained by using window
based computer software devised with Statistical Packages for Social Sciences (SPSS-20).
Numerical parameters were expressed by mean and standard deviation. Mean comparisons
between two groups were done by Student’s t-test. Mean comparisons among three groups
were done by ANOVA test. For categorical variables, distributions were expressed by
frequency and their percentages. Chi square tests were done to see the significance of
differences between cases and controls. Strength of association was determined by estimating
odds ratios (OR) and their 95% confidence intervals (CI). The relationship between two
variables, pearson’s correlation coefficient (r) and spearman’s correlation coefficient (r s) was
done. Probability of <0.05 was considered as statistically significant.

Results

The mean age was found 27.6±4.4 years in group A and 28.9±4.6 years in group B and
26.5±4.9 years in group C. The mean gestational age was 33.3±4.1 weeks in group A,
34.2±3.6 weeks in group B and 34.0±3.7 weeks in group C. More than three fourth (87.5%)
subjects were Nulliparous in group A, 32(80.0%) in group B and 33(82.5%) in group C. The
difference were not statistically significant (p>0.05) between two groups. The mean
systolic BP was found 147.3±4.5 mmHg in group-A, 182.5±15.3 mmHg in group-B and
109.8±8.9 mmHg in group-C. Diastolic BP was found 94.1±4.2 mmHg in group-A,
119.0±7.8 mmHg in group-B and 72.0±7.9 mmHg in group-C. The difference was
statistically significant (p<0.05) among three groups. Proteinuria ++ was more common
19(47.5%) in group A and 13 (32.5%) in group B. The difference was statistically significant
(p<0.05) between two groups.

Table I: Result of serum calcium concentration in study subjects

Serum calcium(mg/dl) Case Control OR 95 CI% p Value
(n=80) (n=40)
n % n %
Low <8.5 mg 43 53.7 5 12.5
8.14 (2.67 – 26.47) 0.001s
Norma(8.5-10.5)mg 37 46.3 35 87.5
p-value reached from Chi square test
Chi value 18.90 with d.f 1

Table II: Comparison of mean serum calcium in case and control groups
Group A Group B Group C p value
(n=40) (n=40) (n=40)
Mean±SD Mean±SD Mean±SD
Serum calcium (mg/dl) 8.2±0.2 7.6±0.3 9.0±0.5 0.001s
p-value reached from ANOVA test

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F value:155.79 with d.f (2,117)

Severe preeclampsia
Mild preeclampsia

Figure 1: Serum calcium concentrations were measured by mg/dl and severity of

preeclampsia was ranked as mild PE = 1 and severe PE = 2. Scatter diagram showing
Spearman’s correlation coefficient was rs=-0.869 (p=0.001) showed a significant negative
correlation of serum calcium concentration with severity of preeclampsia.

Figure 2: Systolic blood pressure (SBP) of 80 cases of preeclampsia were expressed as

mmHg and serum calcium concentrations were measured by mg/dl. Scatter diagram
showing Pearson’s correlation coefficient was r=-0.746 (p=0.001) which indicate a

5
significant negative correlation between serum calcium concentration and systolic blood
pressure

Figure 3: Diastolic blood pressure (DBP) of 80 cases of preeclampsia were expressed as

mmHg and serum calcium concentrations were measured by mg/dl. Scatter diagram showing
Pearson’s correlation coefficient was r=-0.688 (p=0.001) which indicate a significant negative
correlation between serum calcium concentration and diastolic blood pressure.

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 ++
+

++

Figure 4: Proteinuria of 80 cases of preeclampsia were expressed as plus (+, ++, +++)
and serum calcium concentration were measured by mg/dl. Scatter diagram showing
Spearman’s correlation coefficient was rs=-0.232 (p=0.039) which indicate a negative
correlation between serum calcium concentration and proteinuria.

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DISCUSSION

Pre-eclampsia is one of the most common and potentially life-threatening complications of

pregnancy. Pre-eclampsia shares conventional risk factors with cardiovascular disease, such

as obesity, dyslipidaemia, hypertension, and insulin resistance (Acikgoz et al. 2013). The

common basis for these disorders is the presence of endothelial dysfunction. One pathway of

endothelial dysfunction is related to alterations in the plasma levels of lipids and

apolipoproteins as sources of lipoperoxidation and oxidative stress (Alvarez et al. 1996).

In this present study it was observed that the mean age distribution of three groups were
almost alike, no statistical significant (p>0.05) difference was observed among three groups.
Similarly, Nnodim et al. (2017) found the mean age was 22.51±3.42 years in preeclampsia
group and 23.55±3.11 years control group. Olusanya et al. (2015) observed the mean age was
found 26.5±6 years in Preeclampsia/Eclampsia group and 27.4±6 years in control group. The
difference were not statistically significant (p>0.05) between two groups in the above
mentioned studies, which are comparable with the present study. On the other hand Ibraheem
and Obiade, (2013) observed the mean age was 33.15±6.23 years in mild preeclampsia group
and 32.7±4.18 years in severe preeclampsia group and 32.95±4.86 years in normal pregnant
group, which is higher with the current study. Similarly Ositadinma et al. (2015) also found
higher mean age in their case control study.

Regarding the gestational age the mean difference was not statistically not significant
(p>0.05) among three groups. Similarly, Ositadinma et al. (2015) observed the mean
gestational age was 33.48±4.9 weeks in preeclampsia group and 34.33±4.61 weeks in control
group. The difference was not statistically significant (p>0.05) between two groups. But in
another study Nnodim et al. (2017) found the mean gestational age was significantly lower in
pre-eclampsia subjects (p<0.05), where they found 35.26±3.29 weeks in pre-eclampsia group
and 38.84±4.01 weeks control group.

Nulliparity almost triples the risk for pre-eclampsia (Lee et al. 2000). Olusanya et al., (2015)
observed more than half (54.8%) patients were nulliparous in pre-eclampsia group, 55.1%
were in control group. Ephraim et al., (2014) found 50.0% patients were nulliparous in
control group and 60.0% in PE group in respect of parity. Women with pre-eclampsia are
twice as likely to be nulliparous as women without pre-eclampsia (Coonrod et al. 1995). To
identify important outcomes such as mortality, and as the failure to identify and act on known
risk factors at booking contributes to deaths from pre-eclampsia (Confidential Enquiries into
Maternal Deaths, 2001), it is important to define risk at the beginning of pregnancy (Duckitt
and Harrington, 2005).
In this present study it was observed that the mean systolic and diastolic blood pressure
were significantly (p<0.05) higher in severe preeclampsia group. Kanagal et al., (2014) found
the mean systolic blood pressure was 155.50±12.18 mmHg in preeclampsia group and
108±6.50 mmHg in control group, which is consistent with the current study. Similarly,
Ibraheem and Obiade, (2013) mentioned that the systolic blood pressure of severe
preeclamptic women were more than mild preeclamptic women. Kanagal et al., (2014) found
the mean diastolic blood pressure was found 108.18±10.89 mmHg in preeclampsia group and
68.69±8.19 mmHg in control group. The diastolic blood pressure was significantly higher in
preeclampsia compared to controls, which is comparable with the current study. Similarly, in
another study Akinloye et al., (2010) obtained from their study showed mean diastolic blood
pressure of 93.0±2.9 mmHg in preeclamptic patients and and 73.3±6.6 mmHg in control
subjects, which also similar with the current study.

In this present study, it was observed that proteinuria ++ was more common 47.5% in group
A and 32.5% in group B. The difference was statistically significant (p<0.05) between two
groups. Munde et al., (2014) showed the degree of proteinuria was persistant 1+ on dipstick
in mild and 2+ or 3+ in severe preeclamptic cases. The above findings are consistent with the
current study. In our country Begum et al. (2011) found 81.0% of the cases had severe
proteinuria (urine albumin +++) and the rest 19.0% had moderate proteinuria (urine
albumin ++), which is higher with the current study.

In this study, it was found that low serum calcium level (<8.5mg/dl) 8.14 times significantly
increased to developed preeclampsia with 95% CI 2.67 – 26.47%. On the other hand the
mean serum calcium was found 8.2±0.2 mg/dl in group-A (mild preeclampsia), 7.6±0.3 mg/dl
in group-B (sever preeclampsia) and 9.0±0.5 mg/dl in group-C (normal pregnancy). The
mean serum calcium was significantly (p<0.05) lower in sever preeclampsia, compared to
mild preeclampsia and normal pregnant women. Baruah, (2015) obtained in his study that
serum calcium levels were significantly lower in preeclampsia group in comparison to those
of control group (p<0.05). Kanagal et al., (2014) obtained in their study that the serum
calcium concentration was significantly lower in the preeclamptic cases compared to the
normotensive controls, where the mean serum calcium concentration were 7.84±0.87 mg/dl
and 8.97± 0.69 mg/dl, (p<0.05) respectively, which support with the present study. In India
Sharma, (2014) observed the mean serum calcium was 7.20±0.44 mg/dl in normal pregnancy
women, 7.13±0.26 mg/dl in mild preeclampsia women and 6.89±0.31 mg/dl in severe
preeclampsia women. The serum calcium concentration in severe preeclamptic pregnant
women is significantly lower than that in normal pregnant women and mild preeclamptic
women. Simialrly, Suryono et al., (2016) done a study in Indonsia and observed the mean
serum calcium level was 7.97±0.34 mg/dl in preeclamptic group and 8.82±0.44 mg/dl in
normal pregnancy group. Many investigators Adewolu, (2013), Ibraheem & Obiade, (2013)
and Sukonpan and Phupong, (2005) showed serum calcium level in severe preeclamptic
women had significantly lower than normal pregnant women and mild preeclamptic women.
The above findings are closely resembled with the current study. The present study result was
contradictory to some studies that the mean serum calcium levels in preeclampsia were not
different from normal pregnancy like Amirabi et al. (2015), Villanueva et al. (2001), Magri et
al. (2003). Dietary calcium deficiency has been proposed as a possible cause of preeclampsia
by some authors. Levine et al., (1997) in a prospective study in an American population
showed that calcium supplementation during pregnancy did not prevent preeclampsia in
healthy nulliparous women. Conversely, Crowther et al., (1999) in Australia and Herrera et al.
(1998) in Colombia reported in their own studies lowering of blood pressure with calcium
supplementation in primigravid women, therefore role of calcium deficiency in
etiopathogenesis of preeclampsia is yet to be universally proven.

There were significant negative Spearman’s correlation was found between serum calcium
concentration with severity of preeclampsia in this study. Similarly, Ibraheem and Obiade,
(2013) mentioned in their study that the serum total calcium level and corrected total calcium
level were lower in the preeclamptic women and as compared to healthy control group and
even when compared between mild and severe preeclampsia group. Similarly, punthumapol
and kittichotpanich, (2008) showed that serum calcium in severe preeclamptic women was
lower than normal pregnant women and mild preeclamptic women.

There was a significant negative Pearson’s correlation was found between and serum calcium
concentration with systolic and diastolic blood pressure. Similarly, Suryono et al., (2016)
obtained in their study that there is a significant correlation between blood calcium levels
with systolic blood pressure (p<0.001), with a negative correlation coefficient of 0.62 which
indicates a very strong relationship between the decrease in blood calcium levels and increase
in systolic blood pressure, which is consistent with the current study. The authors reported
that there is a significant correlation between blood calcium level with diastolic blood
pressure (p<0.05), with a negative correlation coefficient of 0.65, which indicates a very
strong correlation between the decrease in blood calcium levels and the increase in diastolic
blood pressure. There was also a significant negative Spearman’s correlation was found
between serum calcium concentration with Proteinuria in this present study.
LIMITATION OF THE STUDY
The study was conducted with small sample size and done in a selected hospital from Dhaka
city, which may not be adequate to represents the total population. For the convenience of the
enrollment most of the time we enrolled participants from outpatient department which
restrict us to measure some laboratory test which preferred to do 24 hourly (e.g. urinary
protein). Due to limited resources we were unable to compare preeclampsia with other severe
feature as it need some Biochemical evaluation with some clinical sign. The sample was
taken purposively, so there may be a chance of bias which can influence the result.

CONCLUSION
This study finding revealed that low serum calcium concentration is associated with
preeclampsia. The mean serum calcium concentration is significantly lower in preeclampsia
cases compare to their normal counterpart. Within preeclampsia groups, serum calcium
concentration shows a negative correlation with severity of preeclampsia. However, there is a
negative association with both systolic and diastolic blood pressure with serum calcium
concentration. Similarly, there is also a significant negative association between proteinuria
with serum calcium concentration in preeclampsia cases.

RECOMMENDATIONS
This study suggests, serum calcium concentration is significantly associated with
preeclampsia and its severity and have negative association with blood pressure. Further
study with larger sample size in multiple centers may strengthen the outcome of this study
result. It may give more information if association of serum calcium would be seen with
maternal and fetal outcome in preeclampsia cases.

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