gestational diabetes mellitus ( gdm ) , glucose intolerance first diagnosed in pregnancy , has been a growing health problem among women in the last few decades ( 1 ) . about 14% of pregnancies in the us and 4.8% in iran were complicated with gdm ( 2 , 3 ) . screening for gdm is recommended at weeks 24 - 28 weeks of gestation , according to the iranian national screening program . women at high risk are the exception to this guideline , and for these patients , the oral glucose tolerance test ( ogtt ) is performed at first prenatal visit . risk factors for developing gdm include high maternal age , obesity , multiparity , previous history of gdm or complicated pregnancies , family history of type 2 diabetes , and asian ethnicity ( 4 - 6 ) . vitamin d ( 25-hydroxy vitamin d , or 25ohd ) is a fat - soluble vitamin with skeletal and non - skeletal functions . it affects glucose metabolism through decreasing insulin resistance and improving insulin secretion ( 7 ) . an inverse relationship between serum vitamin d levels and glucose metabolism in women with gdm has been reported in several observational studies ( 8 - 11 ) . hypovitaminosis d has also been associated with insulin resistance and reduced  cell function among individuals at risk of type 2 diabetes , including postpartum patients with a recent history of gdm ( 12 ) . a significant proportion of these patients develop isolated impaired fasting glucose ( ifg ) or impaired glucose tolerance ( igt ) , and 5% - 14% of these cases convert to persistent type 2 diabetes even shortly after delivery ( 13 , 14 , 15 ) . a recent study showed that post - parturition vitamin d supplementation may prevent the progression of insulin resistance in patients with a recent history of gdm ( 16 ) . however , to the best of our knowledge , the impact of prenatal supplementation on post - partum glucose metabolism has not yet been investigated . we therefore sought to explore the effect of vitamin d supplementation in women with gdm and hypovitaminosis d , both on post - partum glucose levels and insulin resistance , in a randomized clinical trial . we conducted a balanced ( 1:1 ) randomized clinical trial at a single gdm clinic in zanjan . for the purposes of the present study , we selected from the referred pregnant women with gdm only those who met our eligibility criteria , which included : maternal age > 16 years , singleton pregnancy , and gestational age between 12 and 32 weeks ( n = 117 ) . the gdm cases were referred from primary health centers affiliated with zanjan university of medical sciences , as well as private obstetric clinics throughout the city . diagnoses of gdm cases were made according to carpenter - coustan criteria or american diabetes association ( ada ) criteria , usually at weeks 24 - 28 of gestation , but always in the first prenatal visit for high risk patients . carpenter - coustan criteria were defined as fasting , 1h , 2h , and 3h plasma glucose ( pg ) levels of 95 , 180 , 155 , and 140 mg / dl after administration of 100 g ogtt , with or without a prior glucose challenge test ( gct , n = 99 ) ( 17 ) . ada criteria were defined as fasting , 1h , and 2h pg levels of 92 , 180 , and 153 mg / dl after one step 75 g ogtt ( n = 18 ) ( 18 ) . the ada criteria were employed for gdm diagnosis after the national gdm screening protocol was changed . those women with known type 1 or 2 diabetes before pregnancy , a history of hypertension , or untreated thyroid disorders were not included . we excluded those women using assisted reproductive techniques ( n = 1 ) or those with a history of high dose vitamin d consumption during the previous 3 months ( n = 1 ) . the selected women were randomly assigned to receive either vitamin d in the intervention group ( i ) or no supplementation in the control group ( c ) . the randomization was carried out by the statistician with a block size of four in a 2 : 2 ratio . the random allocation papers were concealed in sequentially - numbered envelopes . after obtaining informed consent from the patients , baseline characteristics , and medical and obstetrical history were recorded in their medical profile by the investigators . serum concentrations of 25ohd and calcium of those women in group i were measured immediately . however , the blood sample was stored at -80c for women in group c , and was assayed for 25ohd at the end of trial . we excluded the subjects with sufficient basal serum vitamin d in group i initially , and in group c at the end of trial . after excluding the patients with sufficient basal vitamin d , the remaining participants in group i were instructed to take 200,000 iu vitamin d3 for each of the first two days , and then 50,000 iu per week thereafter , up to 700,000 iu in total . those at week 28 of gestation or later were asked to take 100,000 iu weekly . compliance was self - reported by the patients , and was recorded in their medical profile . patients were allowed to take supplements prescribed by their obstetrician , including multivitamins with calcium and 400 iu vitamin d3 . all patients took the prescribed 700,000 iu vitamin d3 before delivery , and there were no cases in which vitamin d3 consumption was stopped . after taking 400,000 iu vitamin d3 , a random urinary calcium / creatinine ( ca / cr ) test was performed as a safety measure against vitamin d toxicity , which is the first sign of hypervitaminosis d ( 25ohd > 100 ng / ml ) ( 19 ) . vitamin d supplementation was stopped if the urinary ca / cr ( mg / dl / mg / dl ) level was  1 with associated hypervitaminosis d. both groups received the routine prenatal care for gdm . insulin was administered in the required cases and adjusted in weekly visits up to delivery . the primary outcomes were maternal fasting plasma glucose ( fpg ) , 2-h post 75 g glucose load plasma glucose ( 2-hplg ) , fasting serum insulin , homeostasis model assessment of insulin resistance ( homa - ir ) , hba1c , and serum 25ohd at 6 - 12 weeks after delivery . the secondary outcomes were maternal , including pre - eclampsia , pre - term labor , type of delivery , and neonatal data including abortion , stillbirth , birth weight , hypoglycemia , congenital anomalies , macrosomia , and hyperbilirubinemia that required hospital admission and phototherapy . six to twelve weeks after delivery , and after 12 hours overnight fasting , a 75 g ogtt was performed . after glucose measurement , the sera and whole blood were kept at -80c and 4c , respectively , for testing of lipids , insulin , and hba1c . serum 25ohd levels were determined by the elisa ( enzyme - linked immunosorbent assay ) method ( immunodiagnostic systems ltd . , tyne & wear , uk ) ; the intra - assay cv were 5.3% , 5.6% , and 6.7% , at 15.6 , 26.8 , and 66 ng / ml , respectively , and the inter - assay cv were 4.6% , 6.4% , and 8.7% , at 16.1 , 28.8 , and 52.8 ng / dl , respectively . insulin was measured by the ecl ( electrochemiluminescence ) method ( roche diagnostic co. , mannheim , germany ) ; the cv were 2.6% , 2.8% , and 2.5% , at 6.3 , 20.9 , and 747 u / ml , respectively . blood glucose measurement was carried out by the enzymatic ( god - glucose oxidase ) method ( pars azmun co. , tehran , iran ) , and hba1c measurement was performed with the enzymatic method ( sekisui medical co. , osaka , japan ) . hypovitaminosis d was defined by serum 25ohd levels < 30 ng / ml ( 75 nmol / l ) . vitamin d insufficiency and deficiency were defined as serum 25ohd levels of 20 - 30 ng / ml ( 50 - 75 nmol / l ) and < 20 ng / ml ( 50 nmol / l ) , respectively ( 20 ) . homa - ir was calculated with the formula 1 : to calculate the study sample size , we assumed the frequency of dysglycemia after delivery in the control group to be 35% ( 21 ) , the efficacy of intervention to be a 25% reduction of the frequency of dysglycemia , type i error to be equal to 0.05 for a study power of 80% , and a 10% drop out rate . dysglycemia was defined as the development of ifg , igt , or type 2 diabetes in subjects as measured by the post - partum tests . ifg was defined by fpg levels of 100 to 125 mg / dl , igt by 2-hplg levels of 140 to 199 mg / dl , and type 2 diabetes by fpg levels  126 or 2-hplg levels  200 mg / dl ( 18 ) . statistical analyses were carried out using spss for windows , version 11.5 ( spss inc . , the independent samples t - test was used for comparing the means of continuous variables between groups , and the chi - square test was used to compare the categorical variables between study groups . logistic regression analysis was performed to detect the independent risk factors of post - partum dysglycemia , including age , pre - pregnancy bmi , parity , history of type 2 diabetes in first degree relatives , history of previous gdm in the patient , and secondary serum 25ohd , as independent variables . one hundred and seventeen pregnant women were recruited to the investigation between june , 2012 and may , 2014 . ninety six women with gdm at weeks 12 - 32 of gestation were randomly assigned to either the intervention ( n = 48 ) or control group ( n = 48 ) . there was no significant difference between intervention and control groups in terms of age , pre - pregnancy bmi , gestational age at gdm diagnosis , and pg levels in ogtt . abbreviations : sd , standard deviation ; dm , diabetes mellitus ; ga , gestational age ; gdm , gestational diabetes mellitus ; fpg , fasting plasma glucose ; ogtt , oral glucose tolerance test . fisher s exact test p value . the initial serum 25ohd in group i was slightly lower than in group c ( 14.6  6.3 ng / ml vs. 17.7  6.1 ng / ml , p = 0.04 ) . insulin was prescribed for 38 ( 90% ) women in group i with a mean dose of 22.3  22 units / day at the end of pregnancy , compared with 35 ( 83% ) women in group c with a mean dose of 16.1  19 units / day . this difference was not statistically significant ( p = 0.33 , p = 0.17 ) . there was one subject in group i with urinary ca / cr  1 and with no associated hypervitaminosis d ( 36 ng / ml ) . maternal and neonatal outcomes were not significantly different between the groups ( table 2 ) . two cases of still birth were recorded : one occurred at week 25 of gestation in group c , and the second fetal loss was at week 39 of gestation in group i. the median follow up time in the study groups was 26 weeks , and occurred between the earliest eligible gestational age ( 12 weeks ) at recruitment and the last post - partum ogtt . fisher exact test p value . at 6 - 12 weeks post parturition , the mean serum level of 25ohd in group i ( 32.4  14.4 ng / ml ) was significantly higher than that in group c ( 19.3  9.6 ng / ml , p the high supplement dose dramatically raised the mean 25ohd in group i ( + 17.6  14.4 ng / ml , p < 0.001 ) , leading to a 20% deficiency rate in this group , compared to 58% in group c ( p = 0.001 ) . mean fasting pg , 2-hplg , and hba1c did not differ significantly between groups ( p = 0.12 , p = 0.56 , and p = 0.24 , respectively ) . among all patients , 13 ( 31% ) developed post - partum dysglycemia in each group , including 5 and 8 cases of ifg , 5 and 6 cases of igt , and 5 and 1 cases of type 2 diabetes in group i and c , respectively ( p = 0.36 , p = 0.74 , and p = 0.09 ) . homa - ir in group i was 2.0  1.3 , compared with 1.8  1.9 in group c. this difference was not significant ( p = 0.58 ) ( table 2 ) . in the logistic regression analysis , pre - gestational bmi , previous history of gdm , and a history of type 2 diabetes in first degree relatives were the significant independent risk factors for post - partum dysglycemia ( table 3 ) . to our knowledge , this is the first study to evaluate the effects of vitamin d supplementation during pregnancy on glucose metabolism shortly after delivery . our study demonstrates that high dose vitamin d supplementation during pregnancy in women with gdm and impairment of 25ohd concentration has no effect on post - partum plasma glucose and insulin resistance . however , supplementation caused an elevated 25ohd serum concentration (  20 ng / ml ) in 80% of women even two months after delivery . fifty eight percent of group c remained vitamin d deficient , which indicates that the usual multivitamin that is prescribed for a majority of pregnant women ( containing 400 iu vitamin d3 ) is not adequate to correct hypovitaminosis d. therefore , pregnant women need a higher dose of vitamin d than that typically consumed . several observational studies indicate that serum 25ohd has an inverse relationship with insulin resistance and incidence of gdm ( 22 , 23 ) , and that vitamin d deficiency is associated with type 2 diabetes ( 24 ) . in addition , animal studies have demonstrated that vitamin d deficiency causes insulin secretion defects ( 25 ) . however , there is scarce information about the effect of vitamin d supplementation on glucose metabolism , especially during pregnancy and after delivery . a recently published study by asemi et al . ( 26 ) revealed a positive effect of co - supplementation with vitamin d and calcium on insulin resistance during pregnancy . in this study , 28 participants in the intervention group were assigned to receive a total dose of 100,000 iu vitamin d3 and daily 1,000 mg calcium , and were compared with 25 participants in a placebo group . the fasting plasma glucose , insulin level , and homa - ir after six weeks of intervention decreased significantly compared to the control group . in that study , gdm diagnosis was based on 75-g glucose ogtt ( the new ada criteria ) , whereas in our study , 84% of the recruited patients were diagnosed by 100-g glucose ogtt , which detects severe cases of gdm ( 27 ) . study , patients requiring insulin therapy during pregnancy were excluded , whereas in our study , 90% of the patients in group i and 83% in group c required insulin for glycemic control . these facts indicate the higher severity of our cases , which might have affected the final results . other possible causes of the lack of effect of this intervention include the significantly higher weight gain in group i ( 8.9 kg ) in comparison to the weight gain in the control group 6.7 kg ( p = 0.04 ) . this could potentially affect the post - partum ogtt results , although the cause of this significant weight gain is not clear . although we can not ignore the likely effect of calcium synergism on the positive result in the asemi study , a study by de boer et al . ( 28 ) found that 1000 mg elemental calcium plus 400 iu vitamin d daily , in middle age and elderly women for a median of 7 years , did not reduce the incidence of diabetes and insulin resistance . in contrast with our study , in which all patients were evaluated for basal vitamin d insufficiency or deficiency , the proportion of the patients with initial hypovitaminosis was not clarified in the asemi study . in another investigation by yap et al . ( 29 ) on 179 women with gdm , plasma glucose level and homa - ir were compared in two groups receiving high dose ( 5000 iu daily ) and low dose ( 400 iu daily ) vitamin d supplementation . they found that high dose supplementation for a mean of 14 weeks during pregnancy was not beneficial to the patients plasma glucose level or insulin resistance during pregnancy , but that their babies benefitted from the supplementation with decreased neonatal hypovitaminosis d. the authors concluded that the commencement of vitamin d supplementation may have been too late to cause any observable effects on -cell function , and they suggested that earlier supplementation , even before pregnancy , might be effective . in our study , over a median interval of 26 weeks between commencement of vitamin d supplements and ogtt measurements , high dose vitamin d led to maternal and neonatal 25ohd level increases , but not to the predicted effects on glucose level and insulin resistance . this negative result was likely caused by inadequate doses of vitamin d3 , time limitations , or the lack of a causal relation between vitamin d and type 2 diabetes . as mentioned by yap , this result may indicate that it is actually dysglycemia that leads to defects in liver hydroxylation of vitamin d. vitamin d supplementation has diverse effects on the non - pregnant population . ( 30 ) reported that daily 4,000 iu vitamin d3 supplementation for 6 months ( 720,000 iu total ) significantly decreased insulin resistance in south asian women with vitamin d deficiency and insulin resistance . however , decreases in insulin resistance were not significant in the first 3 months , although resistance was reduced . the authors concluded that 6 months is an appropriate time interval for observing the presumed effects on insulin resistance . similarly , our patients received high dose vitamin d3 ( 700,000 iu ) with a median follow up time of 6.5 months in the intervention group . no effects on fasting plasma glucose and insulin resistance were observed in our study , and no trend in the reduction of insulin resistance was detected . thus , the effectiveness of longer periods of supplementation with vitamin d is doubtful . in another study of the non - pregnant population , and consistent with our results , davidson and colleagues found that a mean supplement dose of 88,865 iu vitamin d per week , in 56 subjects with hypovitaminosis d and pre - diabetes , had no effect on insulin resistance and plasma glucose levels at 3 , 6 , 9 , and 12 months after intervention , compared to 53 subjects in a placebo group ( 31 ) . in this study , application of a proper dose and duration of vitamin d supplementation led to a rapid increase in 25ohd , up to near 70 ng / ml , which was maintained for the duration of the study . the only limitation of this study appears to be the small sample size of the pre - diabetes population . however , it is doubtful that any significant difference would have been found with a larger sample size ( 32 ) . as previously discussed , a limited number of studies have evaluated the effect of vitamin d on glucose metabolism and insulin resistance , especially during and after pregnancy . consistent with our results , these studies have found that vitamin d is ineffective or has uncertain effects . the study was designed to detect any effects of vitamin d on insulin resistance by allowing for adequate time between the start of supplementation and the measurement of outcomes , and with the fewest number of patients lost to follow up . in addition , maternal and neonatal outcomes were assessed at delivery , and we administered a reasonable dose of vitamin d sufficient to achieve the necessary serum vitamin d levels in group i. however , our study also had several shortcomings . the sample size of the study was not sufficient to identify effects on maternal and neonatal outcomes , and a very large sample size would be necessary to detect these low - incident clinical outcomes . another shortcoming of the study is that none of the investigators or the patients were blinded to the intervention . we did not attempt to compare the daily calorie intake and physical activity between the two study groups . we did not adjust the supplementation dose of vitamin d3 based on the patient s weight in order to provide an appropriate volume of distribution of vitamin d in every patient , and the results would have been more reliable by calculating the proper dose for each individual . regarding the predicted prevalence of 35% , the detected incidence of dysglycemia ( 31% ) might have led to an underestimation of the impact of vitamin d in our study . however , it is unlikely that a greater sample size would change the direction of the hazard ratio , which was found to be 1.02 ( range : 0.98 - 1.06 ) . in spite of these limitations , this is currently the only study in the literature that evaluates the influence of vitamin d supplementation during pregnancy on postpartum dysglycemia . in conclusion , prenatal vitamin d supplementation in patients with gdm and hypovitaminosis d this increase persists for several weeks after delivery , but does not affect fasting glucose and insulin levels , insulin resistance , or clinical outcomes .
backgroundhypovitaminosis d has been associated with the development of gestational diabetes mellitus ( gdm ) in many observational studies.objectiveswe report the first study of the impact of prenatal vitamin d supplementation on postpartum dysglycemia in gdm patients in a randomized clinical trial.patients and methodswomen with gdm at 12 - 32 weeks of gestation were assigned randomly to either the intervention group ( in which serum 25-hydroxy vitamin d [ 25ohd ] levels were measured immediately , n = 48 ) or the control group ( in which the serum was stored and assayed at 6 - 12 weeks post - partum , n = 48 ) . 
 participants with initial serum 25ohd < 30 ng / ml in the intervention group were instructed to take a total of 700,000 iu vitamin d3 during pregnancy . 
 the primary outcomes were fasting plasma glucose ( fpg ) , insulin , 2-h post 75 g glucose load plasma glucose ( 2-hplg ) , homeostasis model assessment of insulin resistance ( homa - ir ) , hba1c , and 25 ohd at 6 - 12 weeks after delivery.resultsthe mean  sd of serum 25ohd in the intervention group raised dramatically from 14.6  6.3 to 32.4  14.4 ng / ml , whereas no significant change occurred in the control group ( from 17.7  6.1 to 19.3  9.6 ng / ml , p < 0.001 ) . 
 thirteen participants developed dysglycemia in each group . 
 mean fpg , 2-hplg , and homa - ir were not significantly different between the groups . 
 there was no significant difference between the groups for maternal and neonatal outcomes.conclusionsalthough the high vitamin d supplementation dose in the present study ( compared to the 400 iu / day dose usually recommended for pregnancy ) safely increases the serum 25ohd , in gdm cases , the higher dose does not affect the plasma glucose level or insulin resistance at short term follow - up after delivery .