HYPOVITAMINOSIS D DELETERIOUSLY AFFECT PREGNANCY OUTCOME: A PROPOSED ROLE FOR DAILY VITAMIN D SUPPLEMENTATION TO IMPROVE OUTCOME

1. Department of Obstetrics & Gynecology, Faculty of Medicine, Benha University. 2. Follow & Researcher of Medical Biochemistry, Hospital Lab., Clinical Pathology department, Faculty of Medicine, Benha University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Objectives: To estimate serum 25-hydroxy vitamin D (25OHD) levels in newly pregnant women and to evaluate its relation to development of gestational diabetes mellitus (GDM) and maternal and fetal outcome.
Patients & Methods: The study included 328 pregnant women, and at 6 th week gestational age (GA), women's clinical and obstetric data were collected and blood samples were obtained for estimation of baseline blood glucose (BG) and serum 25OHD levels. Studied women were divided into two equal groups: Group A included pregnant women received supplemental vitamin D3 in a daily dose of 1000 IU oral capsule starting since 6 th week GA till delivery. Group B included pregnant women who completed their pregnancy without vitamin D supplementation. All women underwent the 75g Oral Glucose Tolerance Test (75-OGTT) at the 20 th , 28 th and 36 th week for detection of GDM. All women were followed up till delivery and maternal and neonatal outcome. Results: Serum 25OHD levels were sufficient, insufficient and deficient in 42, 142 and 144 women, respectively. Forty-three women (13.1%) developed GDM and 62 women (18.9%) had emergency and 23 women had elective CS. Moreover, 48 neonates (14.6%) required NICU admission; 15 for respiratory-aid, 22 for phototherapy and 11 neonates for management of sepsis, but 4 neonates died. Women of group A showed significantly lower frequency of GDM with significantly lower BG levels, significantly lower frequency of CS especially emergency CS and significantly lower frequency of NICU admission. Conclusion: Low serum 25OHD levels are a widespread problem among pregnant women and are associated with higher frequency of GDM, CS delivery especially emergency ones and NICU admission rate. Supplemental vitamin D therapy did well for reducing the frequencies of these events. Supplemental daily dose of 1000 IU appears appropriate and convenient as it was taken in the form of oral capsules.

ISSN: 2320-5407
Int. J. Adv. Res. 5 (12), 1940-1950 1941 Introduction:-Vitamin D is an important secosteroid hormone in skeletal and non-skeletal systems (Yoon, 2017). After absorption, vitamin D is bound to a binding protein which carries it in blood stream to the liver where vitamin D is hydroxylated into 25(OH) vitamin D (calcidiol), (Lapillonne, 2010) and then to the kidneys, where it is hydroxylated by CYP27B1 to 1,25 dihydroxy-vitamin D (calcitriol), which is the biologically active form of vitamin D (Cho et al., 2013).
Vitamin D receptors (VDR) are present in many non-skeletal tissues, so vitamin D via the binding to VDR can produce a number of desired biological effects via different mechanisms and, therefore, contributes to the improvement of human health (Teegarden & Donkin, 2009).
However, accumulating evidence suggests that the metabolic pathways of vitamin D may play a key role in the development of gynecological/obstetric diseases (Colonese et al., 2015), such as polycystic ovary syndrome (Velija-Ašimi, 2014), endometriosis (Sayegh et al., 2014), and ovarian and even breast cancer (Goodwin et al., 2009). Moreover, the maternal-fetal unit is under the influence of vitamin D, as a breakdown in its homeostasis may underlie infertility, preeclampsia, and gestational diabetes mellitus (Colonese et al., 2015).
Gestational diabetes mellitus (GDM) arises in women developed insufficient β-cell compensation for the insulin resistance of late pregnancy (Retnakaran et al., 2016). GDM is characterized by rising incidence, fostered by the worldwide increase of pathological nutritional status from young age (Triunfo et al., 2017).
The mechanisms underlying both normal antepartum β-cell adaptation and its aberrancy in GDM are unclear (Retnakaran et al., 2016). However, high maternal blood glucose (BG) levels during pregnancy induces adverse intrauterine environment, which has a negative impact on health outcomes for both the mother and the offspring (Silva-Zolezzi et al., 2017). On the other side, maternal vitamin D status is independently associated with markers of physiological and pathological growth in term infants (Gernand et al., 2012).
According to that previously documented by Wahlberg et al., (2016), each of vitamin D deficiency and GDM has been associated with increased risk of adverse perinatal outcomes, but the consequences of simultaneous presence of both conditions during pregnancy needs to be evaluated; thus, the current study aimed to estimate serum 25-hydroxy vitamin D (25OHD) levels in newly pregnant women and to evaluate its relation to development of GDM and maternal and fetal outcome.

Patients & Methods:-
The present study was started since Jan 2015 to July 2017. The study protocol was approved by the Local Ethical Committee and only women signed a written fully informed consent were included in the study. All pregnant women who attended the Antenatal Outpatient Clinic, at Benha University hospital for assurance of diagnosis of being pregnant were eligible for study inclusion. At 6 th week gestational age (GA), women's demographic and anthropometric data and previous clinical and obstetric data were collected. Body mass index (BMI) was calculated using the formula: weight (kg)/ height (m 2 ) and patients were classified according to BMI using the World Health Organization ranges as underweight: BMI<18.5 kg/m 2 , normal weight: BMI=18.5-24.9 kg/m 2 , overweight: BMI=25-29.9 kg/m 2 and obese: BMI=≥30 kg/m 2 (14) . At meantime, a random blood samples were obtained under complete aseptic conditions from the antecubital vein and divided into two parts: 1. The first part was put in a tube containing sodium fluoride (2 mg sodium fluoride/ ml blood) to prevent glycolysis. Plasma was separated by centrifugation and used for estimation of fasting blood glucose (FBG) by glucose oxidase method (Tinder, 1969). Women with current DM, history of previous GDM or obesity-inducing endocrinopathy, evident manifestations of hypo-parathyroidism, thyrotocixosis, renal or hepatic diseases and women lost during the course of pregnancy were excluded from the study.
After fulfilling the inclusion and exclusion criteria, enrolled women were randomly divided using sealed envelopes, prepared by an assistant blinded about the study plan, target for grouping and results of laboratory investigation, into two equal groups: -Group A: included pregnant women assigned to received supplemental vitamin D3 according to Grant et al., (2014) in a daily oral dose of 1000 IU softgels to be taken with mail (Sunvite High Potency Vitamin D3 1000 IU; Puritan's Pride, Inc., Oakdale, NY, USA). Vitamin D supplementation started since 6 th week GA till delivery. -Group B: included pregnant women who will complete their pregnancy without vitamin D supplementation.
All enrolled women were asked to fast and attend the antenatal care unit at the 20 th , 28 th and 36 th week GA. At time of each visit, all women underwent the 75g Oral Glucose Tolerance Test (75-OGTT) which entails obtaining three blood samples an overnight fasting blood sample and then each woman was given an oral snake containing 75 gm glucose and another two blood samples were obtained at 1-hr and 2-hr postprandial (PP) for estimation of blood glucose. Women were categorized as having gestational diabetes mellitus (GDM) or not according to the criteria of the International Association of Diabetes and Pregnancy Groups (IADPSG, 2010) for abnormal OGTT, as follows: FBG ≥92 mg/dl, 1-h BG ≥180 mg/dl and 2-h BG ≥153 mg/dl. All women were followed up till delivery and maternal and neonatal outcome were reported.

Statistical analysis:-
Obtained data were presented as mean±SD, ranges, numbers and ratios. Results were analyzed using One-way ANOVA with post-hoc Tukey HSD test, and Chi-square test (X2 test). Statistical analysis was conducted using the SPSS (Version 15, 2006) for Windows statistical package. P value <0.05 was considered statistically significant.

Results:-
Throughout the duration of the study, 361 women were eligible for evaluation, 33 women were excluded and 328 women were included in the study (Fig. 1). Patients' demographic and clinical data and laboratory findings determined at time of 1 st antenatal visit showed non-significant (p>0.05) difference between both groups as shown in table 1.  (Fig. 2). Patients' distribution according to extent of 25-OHD sufficiency showed non-1943 significant (p>0.05) difference between studied groups among the main categories and categories of deficiency. Moreover, the differences between estimated levels showed non-significant (p>0.05) difference between patients of both groups (Table 2, Fig. 3). Throughout the observation period, 43 women developed gestational diabetes mellitus (GDM) for a frequency of 13.1%. Only, 12 of women received supplemental vitamin D therapy (Group A), while 31 of women did not receive supplemental vitamin D therapy (Group B) developed GDM with significantly (p=0.002) lower frequency of GDM among women of group A than group B. Time course development showed non-significantly (p>0.05) lower frequency of women developed GDM among women of group A compared to women of group B at 20 th and 36 th week GA, while the frequency was significantly (p=0.004) lower at 28 th week GA (Fig. 4). Concerning blood glucose levels estimated during 75-OGTT, at the 20 th week GA, FBG and 2-hr PP blood glucose levels were nonsignificantly (p>0.05) lower, while 1-hr PP blood glucose levels were significantly (p=0.046) lower in women of group A than women of group B. At both 28 th and 36 th week GA, all of the three blood samples showed significantly lower blood glucose levels in women of group A compared to women of group B (Table 3).   1946 Eighty-five women (25.9%) had cesarean section (CS); 34 in group A and 51 in group B with significantly (p=0.032) lower frequency of CS among women of group A than in group B. Distribution of women had CS showed significance difference between women of both groups categorized according to extent of sufficiency of vitamin D levels (p=0.042) and according to development of GDM (p=0.035) (Fig. 5). Sixty-two women (18.9%) had emergency CS; 21 in group A and 41 in group B with significantly (p=0.041) lower frequency of emergency CS among women of group A than in group B. Distribution of women had emergency CS showed significance difference between women of both groups categorized according to extent of sufficiency of vitamin D levels (p=0.047) and non-significantly (p=0.097) according to development of GDM (Table 4).  1947 Mean APGAR score determined at 1-min and 5-min was significantly higher in neonates of group A than those of group B. Differentially according to the extent of vitamin D sufficiency, mean APGAR score was significantly higher in neonates of group A than in neonates of group B. Mean APGAR score of women categorized according to estimated blood glucose levels was significantly higher in neonates of group A than in group B. Forty-eight neonates (14.6%) required NICU admission with significantly (p=0.029) lower frequency of NICU admission among neonates of group A compared to group B. Fifteen neonates required respiratory-aid and 22 required phototherapy management of hyperbilirubinemia with non-significantly higher frequencies among neonates of group B. Eleven neonates were NICU admitted for management of sepsis with significantly higher frequency among neonates of group B. Unfortunately, three neonates of group B and one of group A died with non-significant difference in favor of group A (Table 5).  The reported neonatal sequale of vitamin D status is in accordance with Weinert et al., (2016) who found newborns of women with vitamin D deficiency had higher incidences of ICU admission and of hypoglycemia. Ajmani et al., (2016) found that 9.5% of women with vitamin D deficiency delivered low-birth weight babies, 6% delivered premature babies and 8% of babies had APGAR score <7 at 5 min, and 6% of babies were admitted in NICU. Bartáková et al., (2017) detected higher risk for adverse perinatal outcomes in women with low vitamin D levels.
Pregnant women included in group A, who had received supplemental vitamin D therapy since 6 th week GA till delivery in a daily dose of 1000 IU, showed significantly lower frequency of developing GDM with significantly lower blood glucose levels throughout the observation period in comparison to pregnant women who did not receive supplemental vitamin D therapy (Group B). Moreover, supplemental vitamin D therapy improved maternal and fetal outcome of women of group A that was manifested as significant reduction for the need for CS especially emergency CS with significantly lower frequency of newborns that required NICU admission. Interestingly, newborns of women of group A showed significantly lower frequency of NICU admission for septic indication, a finding indicating improvement of immunity of both the mother and the fetus that reflected as decreased frequency of neonatal sepsis.
In line with the use of supplemental vitamin D during pregnancy, Wierzejska et al., (2017) documented that despite improvement of vitamin D levels in summer, it does not guarantee that supplementation throughout the whole year is inessential. Moreover, Kiely et al., (2017) reported that estimated vitamin D metabolites in umbilical cord blood emphasize the high risk of very low vitamin D status in infants born to un-supplemented mothers.
The provided daily dose of supplemental vitamin D goes in hand with that previously documented in literature; where Cooper et al., (2016) found supplementation of women with cholecalciferol 1000 IU/day during pregnancy is sufficient to ensure that most pregnant women are vitamin D replete, and it is safe. Ceccaldi et al., (2017) documented that despite national guidelines on vitamin D supplementation, the rates are currently insufficient and the recommended 100,000 IU single dose helps to limit deficiency in newborns, but it fails to cover infant's needs for optimal status. Yilmaz et al., (2017) who documented that in order to be able to prevent neonatal vitamin D deficiency, 1200 IU/day vitamin D was supplemented to mothers from the 12 th gestational week to 6 th month of the birth, In support of the fetal benefit of maternal vitamin D supplementation, De-Regil et al., (2016) reported that data from three trials involving 477 women suggest that vitamin D supplementation during pregnancy reduces the risk preterm birth and increases infant length and head circumference at birth compared to no intervention or placebo and Kilicaslan et al., (2017) found that height; head circumference and chest circumference of newborns born to mothers who had received vitamin D support were significantly higher compared to non-receivers.

Conclusion:-
Low serum 25OHD levels are a widespread problem among pregnant women and are associated with higher frequency of GDM, CS delivery especially emergency ones and NICU admission rate. Supplemental vitamin D therapy did well for reducing the frequencies of these events. Supplemental daily dose of 1000 IU appears appropriate and convenient as it was taken in the form of oral capsules. However, wider scale studies are recommended to assure the benefit of vitamin D supplementation for other pregnancy-induced complications.