This was a prospective comparative study conducted in the Department of Obstetrics and Gynaecology, AIMSR, Bathinda from January 2017 to October 2018 after taking approval from Research and Ethical Committee (Ref No.: AIMSR/MC/Estt/10/2k/16/3261). Sixty pregnant women attending the outpatient department and labour room, 30 each from normotensive and pre-eclamptic group were included in the study after applying inclusion and exclusion criteria. Inclusion criteria were maternal age of 20-40 years with gestational age of more than 20^th weeks of gestation. Whereas, women on vitamin D supplements and women having Diabetes Mellitus (DM), Chronic hypertension, Renal disease, Liver disease, Inflammatory or infective disorders, Severe anaemia, Preterm labor, History of smoking, History of tobacco chewing, Bone diseases, Current steroid therapy, Use of diuretics, beta blockers and Nonsteroidal Anti-Inflammatory Drug (NSAIDs), Multiple pregnancy, Hypoparathyroidism were excluded.

Sample size was calculated using the formula:

Where, Z=Static for 95% confidence interval (=1.96), S=Standard deviation, D=permissible error in mean (5%), Confidence interval=95%

According to a study conducted by, Sadin B et al., the standard deviation of Vitamin D level in pre-eclamptic women is 6.60 [8]. Now using the above formula which uses difference in mean and standard deviation the minimum sample came out to be 27 and therefore, 30 patients per group were selected for the study. The diagnosis of pre-eclampsia was confirmed with Systolic Blood Pressure (SBP) >140 mmHg and Diastolic Blood Pressure (DBP) >90 mmHg (measured after a period of rest of four hours, twice daily) and proteinuria (>300 mg protein/24 hour) after 20^th week of gestation. All the subjects who agreed to participate in the study gave their informed written consent. On enrollment, complete history was taken and clinical examination was done. Total vitamin D levels were measured quantitatively by immune-fluorescence assay test using Vitamin D kit on Tosho AIA 360 fully automatic hormone analyser.

Subjects were classified into four categories according to serum vitamin D levels [9]:

MS Excel 2010 software was used to analyse the data. Quantitative data were expressed as a mean±standard deviation. Categorical values were expressed as numbers (n) and percentages (%). Data were analysed with a 95% confidence interval and a p-value <0.05 was accepted as significant. Depending on the type of variables p-value was derived using chi-square and independent sample t-test.

Significant negative correlation was observed between Vitamin D and SBP/DBP in Pre-eclampsia group (p<0.05) while no significant correlation was observed between Vitamin D and SBP/DBP in Control group [Table/Fig-1]. Mean Vitamin D level was significantly lower in Pre-eclampsia group as compared to Control group (8.7±5.32 and 14.2±7.88 ng/mL, respectively) with p<0.05 which was statistically significant [Table/Fig-2]. Vitamin D level <5 ng/mL was associated with a 14.58 fold increase in the odds ratio of pre-eclampsia while Vitamin D level of 5-10 ng/mL was associated with a 11.42 fold increase in the odds ratio of pre-eclampsia [Table/Fig-3].

Pre-eclampsia of varying degrees of severity adds to number of admissions to the hospitals. In addition to the well known maternal risk factors such as hypertension, diabetes mellitus, antiphospholipid antibody syndrome and obesity, the role of genetic and immunological factors in the pathogenesis of pre-eclampsia has been postulated. The incidence of pre-eclampsia has been found to be lower in summers and higher in winters which suggest a possible role of Vitamin D and sunlight [10]. An inverse association of circulating vitamin D levels with blood pressure has been demonstrated and it has been shown that the treatment with vitamin D supplements reduces blood pressure [10]. Maternal vitamin D deficiency may predispose to a pro-inflammatory response, increased oxidative stress and lead to endothelial dysfunction that characterises pre-eclampsia. In addition, there is evidence showing that vitamin D affects the genes responsible for trophoblast invasion and angiogenesis critical for implantation, which might be an important factor in the pathophysiology of pre-eclampsia [11].

Studies have shown that Vitamin D improves angiogenesis and inhibits release of adhesion molecules from endothelial cells [12,13]. The recommendation of calcium supplementation for individuals with decreased calcium intake to prevent and treat pre-eclampsia by the World Health Organisation (WHO) has increased the popularity of calcium and vitamin D trials in particular thus considering them as “identifiable”, “modifiable” risk factors which will in turn, aid in the primary prevention by avoiding occurrence of the disease [14]. Various studies have shown the relationship between pre-eclampsia and vitamin D to be complex. They have shown a low vitamin D level in the second trimester to be an indicator of pre-eclampsia [15-17]. The cut-off level for vitamin D deficiency is acceptable at 20 ng/mL in the latest studies, although previously it was accepted at 15 ng/mL [18,19].

In the present study, serum vitamin D deficiency was universally present in both the groups but it was more severe in the pre-eclamptic group. Sadin B et al., carried out a study to assess the levels of 25(OH)D concentrations in women with pre-eclampsia and healthy women and found that 25(OH)D levels <10 ng/mL were associated with 15 fold increased risk of pre-eclampsia as compared to healthy controls [8]. Dhillon MK et al., also observed lower vitamin D levels in women with pre-eclampsia as compared to healthy women which are consistent with the present study [20]. Hypponen E et al., did a systematic review and meta-analysis and concluded that low maternal serum 25(OH)D levels lead to an increased risk of pre-eclampsia whereas its supplementation lowered this risk [21]. Further supporting the present results, Sahu M et al., evaluated serum vitamin D levels in normal pregnant females and pre-eclampsia or eclampsia individuals in the third trimester [10]. They found that 75% of the patients in hypertensive group (pre-eclampsia or eclampsia) have very severe vitamin D deficiency (<5 ng/mL) as compared to 25% of normotensive group. The mean serum vitamin D level was 9.06±5.20 ng/mL in pre-eclamptic group compared to 13.67±7.24 ng/mL in healthy pregnant group which was found to be statistically significant.

Bakacak M et al., also compared Vitamin D levels in 40 normotensives, 32 eclamptic and 83 pre-eclamptic pregnant women. The levels were found to be 23.7±5.93, 18.5±5.47 and 19.3±4.31 ng/mL in healthy normotensive women, eclamptic women and pre-eclamptic women, respectively. Thus, Vitamin D levels were found to be significantly lower in the eclamptic and pre-eclamptic groups compared to healthy pregnant women which were statistically significant (p <0.001) [22].

However, limitations of this study include the stage at which these changes begin to manifest as rising BP with proteinuria is yet to be recognised. Also, the lower limit of Vitamin D levels associated with increased risk of pre-eclampsia is still an area of investigation. Thus a larger study with longer follow-up including neonatal outcome is required for proper guidelines for serum Vitamin D levels in patients with pre-eclampsia and eclampsia.

The present study concludes that Vitamin D levels are lowered in hypertensive women when compared to normotensive pregnant women beyond 20 weeks of pregnancy. Hence, early detection of vitamin D deficiency during pregnancy may be helpful in predicting and preventing the occurrence of hypertensive disorders and its associated co-morbidities thus alleviating maternal morbidity and preterm birth outcomes. Moreover, it may decrease burden on healthcare setup especially in developing countries.