EFFECT OF VITAMIN D SUPPLEMENTATION ON MATERNAL GLUCOSE CONCENTRATIONS DURING PREGNANCY: The MO-VITD study Submitted in partial fulfilment of the regulation for the Award of the Degree of Master of Science in Human Nutrition This research project has been prepared by the instructions to the authors of British Journal of Nutrition Statement of Student Contribution This MSc project involved analysis of previously collected data as part of the MOTVID (Association between Maternal Body Weight and Vitamin D Status) undertaken at NICHE. This study was a double-blind randomised intervention study which aimed to assess the effects of supplementation (10µg/d vs 20µg/d) of vitamin D3 on maternal vitamin D status throughout pregnancy from 12 weeks gestation to delivery. I was involved with the analysis of the data using various statistical methodologies to compare the effects of supplementation of 10µg/d vs 20µg/d of vitamin D3 throughout pregnancy on maternal glucose concentration. Abstract Gestational diabetes is a frequently occurring pregnancy complication marked by high blood sugar levels. Several factors, including vitamin D deficiency, obesity, age, and a sedentary lifestyle, contribute to its prevalence, which currently stands at 14.7% worldwide. Research has looked into the impact of vitamin D on maternal glucose levels, but results have been inconsistent. This study examined the effects of daily vitamin D supplementation (20μg) on maternal glucose levels from week 12 of gestation until delivery, compared to a control group receiving 10μg, while also analyzing the influence of maternal BMI. The study involved 240 non-diabetic pregnant women, including those of normal weight, overweight, and obese, and blood samples were taken at various stages of pregnancy. Ultimately, no significant differences were found between the control and treatment groups, though overweight women with insufficient vitamin D levels showed higher glucose levels at baseline, and a higher maternal BMI was linked to higher glucose levels at week 28. These findings suggest that a higher dose of vitamin D may be necessary for pregnant women with higher BMIs to improve glucose levels. Keywords: Vitamin D supplementation, maternal glucose concentration, pregnancy, BMI, maternal obesity Word count: 250 INTRODUCTION Vitamin D also known as 25[OH]D2 is a fat-soluble vitamin which supports the nervous, musculoskeletal, and immune systems (Bouillon 2007). It plays a major role in calcium absorption, metabolism, bone health, hormone regulation, immunological modulation, and cellular proliferation, among other non-classical functions. Vitamin D is obtained via sunlight, diet, and dietary supplements, but despite these various sources, its deficiency is a common problem worldwide (Natasja et.al. 2011). In Europe and the United States, it is believed that between 40 and 100 per cent of the older population is found to be vitamin D deficient (Holick et.al. 2007). Vitamin D insufficiency is widespread among people of different ages and in a variety of places with the Middle East shown to have dangerously low levels of vitamin D (Benachi et.al. 2013). Numerous studies in both the northern and southern hemispheres have shown that many women have low levels of vitamin D both before and during pregnancy (Alfaham et.al. 1995; Sachan et.al. 2005; Gale et.al. 2008) with a higher prevalence of insufficiency among pregnant women, particularly those with dark skin complexions (Bowyer et.al. 2009), overweight and obesity (Alhomaid et.al. 2021). During pregnancy, vitamin D metabolism is altered to meet the physiological demands of both mother and foetus and increases approximately 2-fold during pregnancy (Hollis et.al. 2011). The incidence of vitamin D deficiency during pregnancy is influenced by seasonal variation, with a higher occurrence observed in the winter months as opposed to the summer months (Nicolaidou et.al. 2006; O'Riordan et.al.2008). Sloka (2009) has demonstrated that variations in latitude have a significant impact on vitamin D concentrations in the majority of pregnant women. Supplementation of vitamin D during pregnancy and lactation does not have a separate reference value according to current dietary reference values [MM9] and there has been a lack of scientific evidence on the amount of vitamin D that is sufficient in the population of pregnant women regardless of the additional risk factor such as obesity (Alhomaid et.al. 2021) which affects 26% of women of reproductive age and 1 in every 5 pregnant women in the United Kingdom (Heslehurst et.al 2010). However, there are variations in the recommended dose for supplementation ranging from 200 to 400 IU/d (5 to 10 µg/d) (Palacios et.al. 2016; Godel, 2007). The recommended daily supplementation dose of vitamin D is 10µg in the United Kingdom (SACN, 2016) and 15µg in the United States (IoM, 2010) for pregnant and lactating women. Inadequate levels of vitamin D in the mother during early pregnancy have been associated with an increased likelihood of complications such as gestational diabetes mellitus (GDM), [MM10] pre-eclampsia, infections, caesarean section, and foetal growth limitation (Kaludjerovic et.al. 2010; Scholl et.al. 2012). According to a meta-analysis by Aghajafari et.al. (2013), and Wei et.al. (2013) involving 31 and 24 observational studies respectively indicate that low vitamin D levels were associated with an increased risk of developing gestational diabetes. [MM11] The global prevalence of GDM is reportedly 14.7% based on the diagnostic criteria of the International Association of Diabetes and Pregnancy Study Group (IADPSG) identifying women with overt and undiagnosed diabetes by testing for fasting glucose, HbA1c and random blood glucose (Saeedi, et.al. 2021). GDM as defined by the World Health Organization (WHO, 2013) is any degree of glucose intolerance recognized during pregnancy, mostly diagnosed during the second and third trimester of pregnancy. The incidence of this condition is experiencing a global significant surge with pre-pregnancy overweight, obesity, high gestational weight gain, advanced maternal age, family history of type 2 diabetes (Ben‐ Haroush et.al. 2004; Casagrande et.al. 2018) and vitamin D deficiency (Holick 2007; Parikh et.al. 2004) contributing to its increase prevalence within the general population (Ferrara et.al. 2004) with approximately 7–14% increase in US pregnant women (Jovanovic et.al. 2001; Burris et.al. 2014). [MM12] Gestational diabetes mellitus has been recognized as a substantial risk factor for many adverse health outcomes in pregnant women and the developing foetus. It has been determined to increase the risk of having a caesarean section (Metzger et.al. 2009), gestational hypertensive disorders (Bryson et.al. 2003), premature birth (Hedderson et.al. 2003), and macrosomia (Yang et.al. 2018). During late pregnancy, serum concentration of 25-hydroxyvitamin D and fasting plasma glucose levels are inversely related (Clifton‐Bligh et.al. 2008) as reported in a recent meta-analysis of observational studies showing an association between poor vitamin D status and glucose level with a twofold increase in gestational diabetes mellitus risk (OR 1.85, 95% CI 1.47–2.33) (Corcoy et.al. 2018). The aetiology of gestational diabetes mellitus continues to be a subject of ongoing research (Harlev et.al. 2010), with more attention being given to the role of vitamin D insufficiency as a potential contributing factor (Dror. 2011). Vitamin D directly impacts glucose metabolism by stimulating insulin secretion via the vitamin D receptor on the pancreatic beta-cell thereby reducing insulin resistance through the vitamin D receptor in the muscle and liver (Li et.al. 2018; Holick et.al. 2007; Tehrani et.al. 2017). The association between obesity and gestational diabetes mellitus as well as vitamin D insufficiency has been extensively studied (Chu et.al. 2007; Solomon et.al .1997; Holick 2007; Cheng et.al. 2010), however, there is still uncertainty regarding the role of vitamin D deficiency in the development of gestational diabetes mellitus, particularly in the context of overweight and obesity. This study aimed to examine the effects of supplementation of 10μg and 20μg vitamin D daily on maternal glucose concentrations and to examine the influence of overweight and obesity on this relationship. METHODS The study at hand utilized previously collected samples and data from the MO-VITD trial which stands for Association between Maternal Body Weight and Vitamin D Status. The MOTVID trial was a double-blind randomized vitamin D intervention study that studied the effects of 10µg/d vs 20µg/d vitamin D supplementation from 12-week gestation to delivery on maternal and fetal vitamin D status, as detailed in the Alhomaid et.al. 2021 publication. The study included 240 pregnant women who were recruited during their first antenatal clinics in Northern Ireland, primarily at Altnagelvin Area Hospital, within Western Health and Social Care Trust (WHSCT). The women were equally distributed across three BMI categories: normal weight (BMI 25kg/m2), overweight (BMI 25–29.9kg/m2), and obesity (BMI ≥30kg/m2). Recruitment was done during the 12th gestational week (GW), and inclusion criteria were applied, including being at least 18 years old, having a BMI of 18.5kg/m2, and being free of any current pregnancy-related complications. Multiple pregnancies, involvement in another research study, previous history of complications including gastrointestinal, renal, hepatic, or vascular disorders, IVF (in-vitro fertilization) treatment, and pregnancies with neural tube defects were among the categories of participants who were excluded from the study. Participants who were already taking vitamin D supplements were instructed to solely take the study supplement going forward. During the study, pregnant participants were randomly assigned to two groups. The first group received 10µg/d of vitamin D3 from the 12th week of gestation until delivery, while the second group received 20µg/d of vitamin D3 during the same period. The participants were divided into BMI categories before they were randomized. A total of 118 pregnant women in the 10µg group were given two tablets each day. These tablets included one multivitamin tablet containing 10µg vitamin D and a placebo tablet that did not contain any vitamin D. In contrast, a total of 121 pregnant women in the 20µg group also received two tablets each day. These tablets included one multivitamin tablet containing 10µg vitamin D and another tablet containing 10µg vitamin D. The multivitamins were supplied by Vitabiotics Ltd., while the placebo was provided by Sona Nutrition. Both the vitamin D and placebo tablets were identical in size, colour, shape, and texture. The tablets were distributed in two groups: batch 1 from 12GW to 28GW, and batch 2 from 28GW to 40GW. As part of the study, the researchers contacted the participants twice to ensure they were following the guidelines. The first check was conducted between the 12th and 28th week, and the second check was done between the 28th and 34th week. The participants' compliance was deemed satisfactory if they had consumed more than 75% of the supplements given to them. Maternal anthropometry, which includes measurements of weight, height, and BMI, as well as body composition, was taken by a trained researcher in a private clinic setting during the first antenatal visit at 12 weeks gestational age (GW). The measurements were repeated at 28GW and 36GW. All participants were asked to complete a health and lifestyle questionnaire at the 12GW visit to gather information about age, medication and supplementation use, smoking habits, sun exposure, and sun holidays. At 28GW, participants were also asked to complete a food frequency questionnaire to assess their vitamin D intake from food. Non-fasting blood samples were collected from participants at 12GW, 28GW, and 36GW, as well as from the umbilical cord after delivery. The samples were analyzed for total serum glucose concentrations. A total of 20 mL of blood was collected, which included 2 x 8-mL serum tubes and 1 x 4-mL plasma tube. The samples were kept chilled and processed within 3 hours of collection. Plasma and serum aliquots were stored at −80°C until batch analysis. Total serum 25(OH)D concentrations analysis The study used stored serum samples to analyze the concentration of vitamin D. The LCMS/MS method was used to measure total serum 25(OH)D concentrations, which includes 25(OH)D2 and 25(OH)D3. The measurement was carried out using a commercially available kit from AB SCIEX, Chromsystems Instruments and Chemicals GmbH, and MassChrom 25OH-Vitamin D3/D2 (plate number 1), as reported by Alhomaid et al. in 2021. To analyze the concentration of plasma glucose, a standard hexokinase method was used. First, plasma was deproteinized using barium hydroxide and zinc sulfate. The clear supernatant was then subjected to the reaction. The glucose in the sample reacted with adenosine triphosphate (ATP) with the help of the HK enzyme to form glucose-6-phosphate. Glucose-6-phosphate was then acted upon by glucose-6-phosphate dehydrogenase (G6PD) in the presence of NADP+ or NAD+ to form NADPH or NADH and 6-phosphogluconate. The amount of NADPH or NADH generated was measured by recording the absorbance at 340nm. This reading is proportional to the glucose concentration in the sample. The method was reported by Ambade et al. in 1998 and Dickson et al. in 2019. Glucose analysis was carried out on the Clinical Chemistry analyzer at NICHE Ulster University. Statistical analysis The statistical analysis was conducted using the SPSS (Statistical Package for Social Sciences software) version 29 for Windows. The data was presented as mean and standard deviation, and normality was assessed using the Kolmogorov-Smirnov test. Maternal characteristics at baseline were evaluated using an independent sample t-test. To compare the effect of intervention on glucose concentration at 12GW, 28GW, 36GW, and in the umbilical cord after delivery across all BMI groups, adjusted for baseline glucose concentration, a general linear model was used. Correlation analysis was performed to examine the strength and direction of glucose changes, and regression analysis was done to determine if intervention was a predictor of glucose changes. Additionally, an independent sample t-test was conducted to determine if there were differences in glucose concentration of pregnant women who entered the study with either sufficient (≥50mmol/L) 25[OH]D concentration or insufficient (<50mmol/L) 25[OH]D concentration throughout pregnancy. Results were considered significant when P values < 0.05 in all analyses. RESULTS Figure 1 presents a flow diagram illustrating the recruitment process. A total of 240 pregnant women completed the baseline visit at 12 weeks gestation, with 119 in the 10 μg/d control group and 121 in the 20 μg/d intervention group. Samples were collected from all participants except for one who withdrew from the 10 μg/d control group. A total of 74 participants dropped out of the study after the baseline visit, with 37 in each group. There were no differences in the rate of dropout between BMI groups. Of the 166 participants who completed the 28-week gestation visit, 158 supplied blood samples. Of these, 78 were in the 10 μg/d group and 80 in the 20 μg/d group. Similarly, 166 participants completed the 36-week gestation visit, of which 153 provided blood samples. The 10 μg/d control group and 20 μg/d intervention group demonstrated great compliance with the intervention, with 92.4% and 91.7%, respectively. Moreover, participants did not report any adverse events during the study. Table 1 shows the maternal baseline characteristics of the total 239 pregnant women who were included in this analysis, with 118 in the 10 µg/d control group and 121 in the 20 µg/d intervention group. The results indicate that there was no significant difference in maternal characteristics between the control and intervention groups at baseline (12 weeks gestation). Table 2 presents the mean maternal glucose concentration at different time points during pregnancy. There were no statistically significant differences in maternal glucose concentration between the 10 µg/d control and 20 µg/d intervention groups at baseline (4.69 ± 0.77 mmol/L compared with 4.79 ± 0.80 mmol/L, p = 0.331). This trend was also observed at 28 and 36 weeks gestation, with no significant difference between the two groups. Additionally, the results were adjusted for confounding factors such as baseline vitamin D at 28 and 36 weeks gestation. Table 3 illustrates the differences in glucose concentration between the 10 µg/d and 20 µg/d vitamin D intervention group and BMI groups at different time points during pregnancy. The results indicate no significant difference at 12 and 36 weeks gestation (p > 0.05). However, at 28 weeks gestation, women with obesity in the 20 µg/d intervention group had a significantly higher glucose concentration (6.50 ± 1.92) than those who were normal weight and overweight (5.02 ± 1.16 and 5.44 ± 1.62, respectively), p = 0.008. No significant difference was observed in maternal glucose concentration within BMI groups in the 10 µg/d group. Regression analysis was conducted to determine whether vitamin D status at each time point during pregnancy was a predictor of glucose concentrations. The results indicate that vitamin D was not a determinant of glucose concentrations (p > 0.05) at baseline (12 weeks gestation) and 36 weeks gestation. BMI did not influence glucose concentration. However, at 28 weeks gestation, a higher BMI was predictive of increased glucose concentration (β = 0.268, p = 0.001) (Table 4). Table 5 shows the differences in glucose concentration of pregnant women who entered the study with either insufficient (<50 mmol 25(OH)D) or sufficient (≥50 mmol 25(OH)D) vitamin D concentration. The results indicate that at 28 and 36 weeks gestation, there was no difference in glucose concentration across all BMI groups regardless of vitamin D concentrations at baseline. At 12 weeks gestation, pregnant women who had insufficient 25(OH)D levels and were overweight had a higher glucose concentration than those with sufficient 25(OH)D levels (p = 0.028). However, no significant difference in glucose concentration was observed in women who were normal weight and with obesity. The study also found that glucose concentration across all BMI groups in this cohort falls within the normal range when compared with the normal clinical glucose concentration, which ranges between 3.9 mmol/L and 5.6 mmol/L (WHO, 2021). Moreover, 62 umbilical cord blood samples were collected, with 31 in the 10 µg/d group and 31 in the 20 µg/d group. The results indicate no significant differences in glucose concentration of infants born to mothers in the 10 µg/d control group compared with the 20 µg/d intervention group. Cord glucose concentration was not significantly different in any of the maternal BMI groups (normal weight, overweight, obesity), p > 0.05. 12 Gestational Week N= 240 10µg/d control group n= 121 20µg/d intervention group n= 118 10µg/d control group dropout n= 37 Withdraw without reason n= 20 Pregnancy-related sickness n= 13 Stopped taking supplements n= 2 Preterm delivery n= 1 Discontinued and requested data not used n=1 20µg/d intervention group dropout n= 37 Withdraw without reason n= 14 Pregnancy-related sickness n= 12 Stopped taking supplements n= 8 Miscarriage n= 3 Follow up 1 28 Gestational Weeks n= 166 20µg/d n= 84 Number of samples obtained n=4a Samples available for analysis n=80 10µg/d n= 82 Number of samples obtained n=4a Samples available for analysis n=78 Follow up 2 36 Gestational Weeks n= 166 10µg/d n= 82 Number of samples obtained n=9b Samples available for analysis n=73 20µg/d n= 84 Number of samples obtained n=4b Samples available for analysis n=80 Umbilical cord samples at birth N=62 10µg/d n= 31 20µg/d n= 31 Figure 1 Flow diagram of recruitment. aReason for no samples obtained: pregnancy sickness (n=2 in 10µg/d group , n=2 in 20µg/d group), unable to attend appointment (n=2 in 10µg/d group, n=2 in 20µg/d group). bReason for no samples obtained: pregnancy sickness (n=1 in 20µg/d group), unable to attend appointment (n=5 in 10µg/d group, n=2 in 20µg/d group). preterm delivery (n= 4 in 10µg/d group, n=2 in 20µg/d group) TABLE 1 Maternal characteristics at baseline (12GW) in the 10µg control group and 20µg intervention group of vitamin D supplementation Variables Intervention groups 10µg/d Mean±SD N=118 20µg/d Mean±SD N=121 Age (y) Weight (kg) Height (m) BP systolic (mm/hg) BP diastolic (mm/hg) BMI (kg/m2 ) Normal weight(n,%) Overweight(n,%) Obese(n,%) Education Secondary school(n,%) 3rd level education (n,%) 29.65±5.06 74.71±15.91 1.63±0.60 119.50±11.36 71.61±8.56 28.08±5.73 39(33.1) 39(33.1) 40(33.9) 38(32.8) 78(67.2) 29.49±5.48 74.41±15.91 1.63±0.63 120.31±11.98 72.61±9.32 27.78±5.35 41(33.9) 40(33.1) 40(33.1) 37(31.6) 80(68.4) Data were presented as mean±SD or n(%). Differences in the intervention groups were assessed by independent sample t-test, P<0.05 was considered significant. There were no significant differences in any of the maternal characteristics at baseline between the 10µg/d control and 20µg/d intervention groups. BMI=Body Mass Index BP= Blood Pressure TABLE 2 Maternal glucose concentration by BMI category in the 10µg/d control and 20µg/d intervention groups during pregnancy and in the umbilical cord at birth Glucose (mmol/l) Baseline (12GW) 10µg 20µg P N=118 N=121 28 GW 10µg 20µg P N=78 N=80 36 GW 10µg 20µg P N=73 N=80 Umbilical cord 10µg 20µg P N=31 N=31 All Normal weight (n=80) Overweight (n=79) Obese (n=80) 4.69±0.77 4.79±0.80 0.331 5.27±1.36 5.69±1.66 0.081 4.80±0.98 5.12±1.27 0.080 3.00±1.83 3.09±1.52 0.808 4.57±0.79 4.67±0.96 0.607 4.92±0.89 5.20±1.16 0.329 4.56±0.96 4.98±1.25 0.118 3.35±2.18 3.72±1.36 0.651 4.72±0.73 4.91±0.76 0.274 5.22±1.18 5.44±1.62 0.576 5.02±0.93 5.46±1.47 0.237 3.12±1.66 2.45±1.74 0.456 4.79±0.78 4.81±0.64 0.925 5.61±1.76 6.50±1.92 0.080 4.80±1.00 4.91±1.00 0.955 2 .56±1.65 2.90±1.23 0.671 Data were presented as Mean±SD. Differences in glucose concentration between the 10ug/d control and 20ug/d intervention groups were assessed using a general linear model. There were no differences in maternal glucose concentration at the different time points during pregnancy across all BMI groups p>0.05. GW= Gestational Weeks , BMI= Body Mass Index Table 3 Differences in glucose concentration between the 10µg/d control and 20µg/d vitamin D intervention groups and BMI groups at different time points during pregnancy. Glucose (mmol/l) Baseline (12GW) 10µg 20µg P 28GW) 10µg 20µg P 36GW 10µg 20µg P Umbilical cord 10µg 20µg P BMI Groups (Kg/m2) Normal weight overweight Obese P value 4.57±0.79a 4.67±0.96 a 0.607 4.92±0.89a 5.02±1.16a 0.333 4.56±0.96a 4.98±1.25a 0.187 3.35±2.18a 3.72± 1.36a 0.617 4.72±0.73a 4.91±0.76a 0.207 5.22±1.18a 5.44±1.62a 0.592 5.02±0.93a 5.46±1.47a 0.196 3.12±1.66a 2.45±1.74a 0.403 4.79±0.78a 4.81±0.64a 0.925 5.62±1.76a 6.50±1.92b 0.084 4.80±1.00a 4.91±1.00a 0.715 2.56±1.65a 2.90±1.23a 0.634 0.417 0.402 0.175 0.008 0.277 0.228 0.606 0.129 Results presented as Mean±SD. Data was assessed using independent sample t-test and One-way ANOVA accordingly to determine if there were differences in glucose concentration between intervention groups and BMI groups, mean that do not share subscript differ by p<0.05 between BMI groups according to Turkey’s Honestly Significant Differences. BMI= Body Mass Index, GW=Gestational Weeks TABLE 4 Regression analysis shows the prediction of glucose concentrations at different time points during pregnancy. Baseline (12GW) 28 GW 36 GW βPβPβP Umbilical cord βP Vitamin D at each time point BMI Group -0.039 0.184 0.20 0.806 -0.026 0.760 0.288 0.507 0.088 0.565 0.268 0.001 0.038 0.647 -0.214 0.104 Data were presented as Standardized coefficient of beta (β) and p-value. Regression analysis was used to assess if vitamin D status at each time point was a determinant of glucose changes at each time point during pregnancy. Results were considered significant if p< 0.05. BMI= Body Mass Index, GW=Gestational Weeks TABLE 5 Differences in glucose concentration according to baseline (12GW) vitamin D status across all BMI groups at each time point during pregnancy. Baseline (12GW) 28GW 36GW Mean±SD P value Mean±SD P value Mean±SD P value All Insufficient (<50mmol) 4.78±0.77 5.63±1.59 5.12±1.31 0.500 0.335 0.264 Sufficient (≥50mmol) 4.71±0.79 5.38±1.49 4.90±1.06 Normal Weight Insufficient (<50mmol) 4.60±0.80 5.40±1.18 4.94±1.21 0.758 0.245 0.616 Sufficient (≥50mmol) 4.66±0.91 4.96±1.00 4.76±1.16 Overweight Insufficient (<50mmol) 5.01±0.84 5.58±1.73 5.57±1.64 0.028 0.384 0.240 Sufficient (≥50mmol) 4.64±0.60 5.18±1.17 5.08±0.95 Obese Insufficient (<50mmol) 4.69±0.62 5.78±1.70 4.82±0.99 0.264 0.416 0.894 Sufficient (≥50mmol) 4.87±0.78 6.20±2.02 4.88±1.03 The data was analyzed using an independent sample t-test. The results indicate that there were no significant differences in glucose concentration between 28 and 36 weeks of gestational age across all BMI. However, at 12 weeks, there were significant differences in the mean glucose concentration of overweight women (p=0.028). It is noteworthy that p-values less than 0.05 are considered statistically significant. DISCUSSION The findings of the study suggest that there were no significant differences in glucose concentration between the control group (who received 10µg/d vitamin D) and the intervention group (who received 20µg/d vitamin D) during pregnancy. This was even after adjusting for baseline vitamin D level and BMI in a group of healthy pregnant women. However, significant differences were noted within BMI groups. Women with obesity in the intervention group had a higher glucose concentration at 28GW than women who were normal weight and overweight. Similarly, women who were overweight at baseline and had insufficient vitamin D levels had a higher glucose concentration. Increased BMI was also found to be a significant predictor of an increase in maternal glucose concentration at 28GW. Previous intervention studies have evaluated the effect of vitamin D supplementation during pregnancy on glucose metabolism. However, none of these studies found significant differences in glucose concentration following maternal vitamin D supplementation. Yap et al. in a cohort of 209 pregnant women supplemented 125µg/d of vitamin D commencing at 14GW and Hossain et al. in a cohort of 200 pregnant women supplementing 100µg/d of vitamin D commencing at 20GW reported that supplementation did not lead to an improvement in maternal glucose concentration compared to the control group receiving a daily supplemental dose of 10µg/d of vitamin D3 in pregnancy. Soheilykhah et al. tested three different doses of vitamin D supplementation (10µg/d, 100µg/d and 1250µg monthly) on maternal glucose metabolism in healthy pregnant women. However, no significant difference was observed in fasting blood glucose when comparing the control group (10µg/d) to the intervention groups. These findings align with the results of this present study which shows no significant differences in maternal glucose concentration between the control group (10µg/d) and the 20µg/d vitamin D supplementation intervention group. Despite the high dose of supplementation in these studies compared to the present study, no differences were established in blood glucose during pregnancy. It could be suggested that a very high dose of vitamin D supplements might be needed for differences to be established in blood glucose during pregnancy. Despite several studies that show no significant differences similar to the result of the present study, investigational studies have established significant differences in maternal glucose metabolism and the risk of GDM following vitamin D supplementation therefore contradicting this current study (Mojibian et.al. 2015; Zhang et.al. 2016; Asemi et.al. 2013). Mojibian et.al. (2015), administered[MM17] a dosage of 1250µg of vitamin D in a cohort of 500 pregnant women every two weeks and showed a decreased occurrence of GDM compared to the control group with a daily dosage of 10µg of vitamin D starting from 12GW to delivery (P=0.01 at 95% Confidence interval). Zhang et.al. (2016) supplemented with 1250µg vitamin D every 2 weeks in a cohort of 133 pregnant women and Asemi et.al. (2013) supplemented with 1250µg vitamin D twice (once at baseline ≥26week gestation and a second dose at day 21 of the intervention) in a total of 54 pregnant women during 6weeks of the study found that vitamin D supplementation significantly improves insulin resistance (P<0.05) and decreases fasting plasma glucose concentrations (p <0.001) respectively. The differences observed in these studies could be a result of the high dose (1250µg) of vitamin D supplement together with the fact that these studies were carried out in a population of pregnant women who were at high risk of developing diabetes and participants with GDM. Results from intervention studies evaluating the effect of vitamin D supplements on glucose level and the risk of developing GDM remain inconsistent, while some studies find no association some others show an association particularly in those with pre-existing risk factors for diabetes and those who already have been diagnosed with GDM. Therefore pregnant women who are at high risk of diabetes may respond well to vitamin D supplementation than those who are healthy or at a very low risk for developing gestational diabetes. This inconsistency in findings may be attributed to variations in the measurement of vitamin D level, supplementation doses, duration of intervention, presence of underlying health condition, sample size, disparities in population characteristics including ethnicity, gestational age at sample collection, geographic location, and diagnostic criteria for GDM. The differences in results from studies may also be explained by the lack of evidence surrounding the [MM18] potential mechanisms of glucose response to the improvement in vitamin D status. Vitamin D enhances pancreatic insulin secretion, via beta cell vitamin D receptor, thus lowering circulating glucose levels (Chiu et.al. 2004; Norman et.al. 1980), vitamin D, via its receptor, stimulates the expression of insulin receptors, which stimulates insulin sensitivity and thus increases glucose transport inside the cells (Vaidya et.al. 2012), as well as adjusting the balance between extracellular and intracellular calcium pools which are essential to intracellular insulin-mediated mechanisms of insulin-sensitive tissues, also resulting in glucose transportation (Draznin et.al. 1988). Results from this current study also found that at baseline (12GW) pregnant women who were overweight and had insufficient 25(OH)D level had a higher glucose concentration than those with sufficient 25(OH)D level and at 28GW women with obesity have a significantly higher glucose concentration than those who were normal weight or overweight indicating a high level of plasma glucose in those who are obese. Several clinical investigations have identified a relationship between vitamin D, glucose hemostasis and body weight. The result of Shao et.al (2020) shows that vitamin D deficiency was associated with increased fasting blood glucose concentrations among women who are overweight or with obesity. Loy et.al (2015) also demonstrated in a multi-ethnic Asian cohort that maternal 25(OH)D insufficiency (<30mmol/L) was associated with higher fasting blood glucose. The link between vitamin D and BMI can be explained by the fact that vitamin D is a fat-soluble vitamin and may be less bioavailable in individuals who are overweight or obese due to its sequestration in adipose tissue and increased levels of vitamin D binding protein (Rizzo et.al. 2019), as a result, decreased maternal vitamin D availability during pregnancy may be one of the potential mechanism contributing to increase glucose concentration and subsequently increasing the risk of developing GDM in overweight and obese pregnant women. The result from the MO-VITD study also establishes that individuals particularly overweight and those with obesity with a low level of 25(OH)D (<50mmol) at the start of pregnancy might not achieve sufficient levels of vitamin D with the recommended dosage (10µg) alone, with further increased risk in women with obesity (Alhomaid et.al. 2021). During pregnancy, the increased production of steroid hormones and insulin resistance can lead to the development of diabetes. Inflammatory cytokines from adipose tissue and the placenta can also contribute to insulin resistance and gestational diabetes mellitus. Deficiency of vitamin D in early pregnancy, particularly in women with obesity, can further increase the risk. A study showed that women with insufficient vitamin D at 12 weeks of pregnancy had higher glucose concentrations at 28 weeks. Increased vitamin D supplementation is crucial, especially for overweight and obese individuals. Vitamin D was not found to be a predictor of glucose changes in the study's cohort, possibly because all participants received vitamin D supplements. However, increased BMI was found to be a significant predictor of increased glucose concentration at 28 weeks. Maternal overweight and obesity are significant risk factors for gestational diabetes mellitus, which is mostly diagnosed during the second trimester. The study's strengths include measuring vitamin D levels and glucose concentration throughout gestation, using the gold standard measurement for Vitamin D, and considering early maternal BMI. Compliance with the intervention was also high. Limitations include being a secondary analysis of participants in a non-GDM-focused trial, using non-fasting blood glucose, a low supplementation dose, and a small sample size. CONCLUSION This study did not find any differences in the concentration of maternal glucose at any point during pregnancy between the intervention groups. However, it is shown that insufficient vitamin D levels during early pregnancy and increased BMI can affect glucose concentration during pregnancy. Further studies are required to better understand the association between maternal vitamin D insufficiency, glucose concentration, and gestational diabetes mellitus, considering maternal overweight and obesity. These studies should assess the influence of vitamin D status on glucose concentration as well as the biological pathways involved. They should also investigate the most effective and safe dose of vitamin D during pregnancy that could improve health outcomes, optimal timing to start supplementation, and treatment interval in a well-designed randomized control trial employing a large sample size. Additionally, public health initiatives should be put in place in hospitals and maternity clinics to emphasize the importance of vitamin D supplements during pregnancy for both mother and cord. It is important to highlight the significance of a healthy BMI and encourage vitamin D intake for women of reproductive age to avoid being deficient during pregnancy. 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Effect of various doses of vitamin D supplementation on pregnant women with gestational diabetes mellitus: a randomized controlled trial. Experimental and therapeutic medicine, 12(3), 1889-1895. Zhu Y, Li L, Li, P (2022). Vitamin D in gestational diabetes: A broadened frontier. Clinica Chimica Acta. [MM1]It would be more informative to note the direction of the association on glucose, negative or positive. [MM2]This is written unclearly - you would be better to note 10 vs 20ug, rather than the way it is currently stated. [MM3]Poor terminology [MM4]BMI ranges are not necessary to note in the abstract. [MM5]This statement is much too long. [MM6]Not previously abbreviated [MM7]High compared with what? This is unclear. [MM8]Beta value should also be included here. [MM9]This statement is poorly written and needs clarity. It is also very long with too many components. It would be better to split it into a few statements for clarity of thought. [MM10]Use this abbreviation from this point forward. [MM11]This should be a separate statement [MM12]Too long. [MM13]Spelling error [MM14]Subheading needed [MM15]You are not reporting these data so you don’t need this information in the methods [MM16]You need to then tell the reader about your thoughts and interpretation of these findings. Interpretation currently missing. [MM17]You need to relate this to the MOVITD study - have you got some thoughts on the dose used and how this may have impacted on your lack of associations? [MM18]Same as previous comments - you need to relate this to the MOVITD study. This should be a discussion of YOUR findings. You are providing relevant literature but you are not discussing your thoughts and interpretations. Currently, this discussion section is still too generic and reads like a review of other literature, rather than a discussion of MOVITD findings. [MM19]Not sure what this means. The study was powered to detect differences in maternal vitamin D status.