Maternal Serum 25-Hydroxy Vitamin D Concentrations and Calcium Levels in Preeclamptic Women, Sindh, Pakistan

Objectives: To analyse the levels of serum 25-hydroxyvitamin D 25(OH) Dand calcium concentrations in patients with pre-eclampsia, Hyderabad, Sindh, Pakistan. Methodology: Cross sectional study was conducted at the Gynaecology and Obstetrics Original Research Article Memon et al.; JPRI, 33(52B): 216-221, 2021; Article no.JPRI.77521 217 Department, LUHS, Hyderabad during the period of January 2020 to June 2020. Total 150 pregnant women were selected according to predesign Proforma and divided into two groups. 50 females were normal normotensive pregnant women as a control group, 100 were pre-eclamptic patients as case group. Obstetric history, clinical data was gathered and then 5ml of blood sample was collected from each patient. The blood samples were taken for the analysis of 25hydroxyvitamin D or 25(OH)D and calcium levels. 25(OH)D were performed on chemiluminescence, While calcium concentration of Microlab 300. Data was analysed by using SPSS version 23. Student t-test was used for analysed of continuous variables. Results: The maternal serum vitamin D and calcium levels were significantly decreased as compared to patient with preeclampsia and normotensive healthy pregnant ladies. The blood pressure also declined in case group with P<0.001.Multinomial logistic regression showed significant odd ratio of Vitamin-D and Calcium deficiency in preeclampsia. Conclusion: The present study reveals that 25-hydroxyvitamin D, and Calcium levels declined significantly in preeclamptic women, and mighty be biomarker of this life threating disease.


INTRODUCTION
Pre-eclampsia (PE) is one of the common complication of pregnancy. PE is characterized by development of hypertension, proteinuria after 20th week of pregnancy with previously normal pregnancy [1,2]. The metabolic demand is increases during the progressive period of pregnancy which is helpful for the better growth of foetus [3]. Proper nutrition throughout pregnancy is very important for mother and healthy foetus [4]. Vitamin Dis a lipophilic vitamin. It is existing in two forms, Vitamin D3 and Vitamin D2, with similar structures. Vitamin D3 also known as cholecalciferol contributes to 95% of the Vitamin-D levels in the human circulation [5]. Vitamin-D deficiency effects all age groups. Its classical function is on bone and calcium haemostasis but it is also pluripotent regulator of many biological functions [4]. It is vitamin effects on billions of world population and the prevalence of Vitamin-D in Pakistan is higher approximately 53.3% [6]. Vitamin-D paly vital role during pregnancy. Its deficiency not only effects on mother health but also impact on infant health. During pregnancy Vitamin-D deficiency results in complications like pre-eclampsia, gestational diabetes, intrauterine growth restriction and pre term birth is globally appreciated [7]. Vitamin D receptor (VDR) present in every tissue controlling by many genes. The placental components, maternal decidua and foetal trophoblast including syncytiotrophoblast express the CYP27B1 [8]. The CYP27B1 is responsible to produce 25(OH)2D and then converted to 1,25(OH)2D. The concentration of 25(OH)2D is increased in placenta dduring the pregnancy [9]. Vitamin-D and its other related compound localized in these tissues the placenta is one of the prominent tissues among these barrier sites. During first trimester of pregnancy initially maternal serum 25(OH)2D levels increased significantly however, some studies on animal and human models indicated that this is due to presence of VDR in the placenta and exerts its functions on specific tissue at the maternal-fetal interface [10]. These receptors act as autocrine. Studies suggest that vitamin D is also responsible for the placental transport of calcium [11]. Serum calcium play very important role in regulation of blood pressure by stimulating the parathyroid hormone (PTH) and release of renin results in inducing vasoconstriction. Calcium might also have an indirect effect on smooth muscle [12]. The aim of this study was to analysed and compared the maternal serum 25(OH)2D and calcium levels in in pre-eclamptic and normotensive females.
The objective of this study is to estimate the levels of serum 25-hydroxyvitamin D and calcium concentrations during preeclamptic Hyderabad women, Sindh.

Setting
The study was conducted in Gynaecology and Obstetrics Department, Liaquat University of Medical & Health Sciences, Jamshoro with collaboration of Pathology Department LUMHS Jamshoro / Hyderabad during the period of January 2020 to June 2020.

Sample Size
The sample size was calculated according to Biostatistics Rao software. Pre-eclampsia was 5% by using the proportion of 95% confidence interval with 5% margin of error, the sample size was stand to be n=70. Total 150 subjects were recruited and divided into two groups. Group A: n = 50 Healthy pregnant women with normal B.P after 20 th week of gestation and Group B: n = 100 patient with Pre-eclampsia after 20 th week of gestation. after taking history, anthropometric parameters, collecting the blood sample from all participants of the study and also taking the consent on pre-designed proforma.

Inclusion Criteria
Healthy pregnant women after 20 th week of gestation within 20-35 years of age and pregnant women with pre-eclampsia after 20 th week of gestation within 20-35 years of age.

Exclusion Criteria
Pregnant women below 20 or above 35 years, multiple Pregnancy, known hypertensive, diabetic, cardiac and renal, liver disease, smokers and any use of drug.

Blood Parameters
3 ml of Blood was collected from each participant by veno puncture into EDTA tubes under aseptic measures. The blood was centrifuged at 3500 rpm for 10 min, fractionated and transferred to eppendorf cups then stored at −20°C till required for analysis. Before the analysis, sample was first allowed to attain room temperature.The blood samples were taken for the analysis of 25(OH)D and calcium levels. 25(OH)D were performed on chemiluminescence, While calcium concentration of Microlab 300.

Statistical Analysis Procedure
The data was entered in Microsoft Excel and analyzed on SPSS (Statistical package for Social Sciences) Version 23. Independent unpaired student t-test for comparison between cases and control was used for continuous variable. Results were presented as mean and standard deviation.

RESULTS
The present study included 150 pregnant females, 33.4% were normal healthy pregnant whereas 66.6% were patients with preeclampsia. The Mean ± SD of maternal age of control and cases was 26.7 ±4.34 year versus 27.43±3.86 year. The p-value of gestational age, parity and gravida of control and cases were insignificant ( Table 1). The systolic, diastolic blood pressure was increased in cases group as compared to control. 25-hydroxyvitamin D and serum calcium was decreased in preeclamptic patients as compared to control with highly significant difference of (p< 0.001), shown in Table 1.
The predictor of severe and mild preeclampsia observed by Multinomial logistic regression. The odd ratio of maternal serum 25(OH)2D deficiency and hypocalcaemia in severe and mild patients of preeclampsia shown significant difference ( Table 2).

DISCUSSION
This is immense health related issue in pregnancy worldwide. Maternal serum 25(OH)2D deficiency during pregnancy is associated with adverse outcomes [13]. Lamminpaa et al., [14] revealed that advance maternal age exhibited pre-eclampsia. one more study of Ghana found that maternal age is also a major risk factors of pre-eclampsia [15]. Fondjo LA et al., [16] found that pre-eclampsia is commonly occurred in low gestational age group as compared to normotensive group with her gestational age with (p <0.001).We found similar results in present study. Preeclampsia is more common in developing country [1]. Nutritional deficiency is one of the most important cause and exerted adverse effects on health of mother as well as fetus [17]. The Achkar M et al., [13] reported that low Vitamin-D concentration is linked with high risks of preeclampsia, infertility and gestational diabetes mellitus. 25(OH)2D deficiency also associated with primary onset of preeclampsia with foetal growth retardation. Many researchers reported in their observational studies that significant association has been found between declined levels of maternal serum 25(OH)D results in enhance risk of preeclampsia [18]. We found similar results in present as mention in above studies. The odd ratio of mild and sever preeclampsia is approximately (1.97 and 1.78).
In contrast to other case control studies reported that who developed preeclampsia having low levels of maternal serum 25(OH)D concentration as compare to normal pregnant ladies [19]. The results were constituent with present study.
Calcium is regulator of PTH, and it is associated with 25(OH)2D. Many studies have focused on correlation among calcium, 25(OH)2D and PTH with Preeclampsia [20]. In our study the calcium concentration is significantly declined. Hypocalcaemiacan be mighty be a risk factor of preeclampsia as a life-threatening disease. Gabbay A et al., [21]. revealed that complications of low level of calcium such as secondary hyperparathyroidismmay play a vital role in the aetiology of preeclampsia.
Udenze IC and co-workers [22] reported that calcium concentration was low in preeclamptic women as compared to normal subjects. On another case control study revealed association of hypocalcaemia and preeclampsia [23]. In present study we found similar results.

CONCLUSION
The 25-hydroxyvitamin D and calcium play a vital role in pregnancy process of conception, implantation and development of placenta. The 25-hydroxyvitamin D and calciumdeficiency are major causative factors of Preeclampsia.

RECOMMENDATION
The vitamin D receptors and its histopathology should be in consideration for future studies. Calcium is also very important element further studies regarding Parathyroid hormone should be done.

CONSENT
As per international standard or university standard, respondents' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
The study was conducted strictly under the ethical rules after the approval from Ethical Review Committee of LUMHS Jamshoro.

ACKNOWLEDGEMENT
We would to like to thanks all authors and laboratory staff who cooperate with us during proceed of present study.

COMPETING INTERESTS
Authors have declared that no competing interests exist.