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Vitamin D deficiency in gestational diabetes mellitus and the role of the placenta

Published:August 16, 2013DOI:https://doi.org/10.1016/j.ajog.2013.08.015

      Objective

      The aim of this study was to evaluate the relationships between maternal vitamin D levels and gestational diabetes mellitus (GDM) and differences in the placental production of vitamin D receptor (VDR), CYP24A, and CYP27B1.

      Study Design

      Forty normal pregnant women and 20 women with GDM were included in this study. Serum levels of 25-hydroxyvitamin D (25[OH]D) were measured with enzyme-linked immunosorbent assay. The expression and production of VDR, CYP27B1, and CYP24A1 in the placenta were evaluated with real time–polymerase chain reaction and Western blot, respectively.

      Results

      We found that 27.5% of normal pregnant women and 85% of women with GDM had vitamin D deficiency, with serum 25(OH)D levels <20 ng/mL. Serum levels of 25(OH)D were lower in women with GDM than normal pregnant women (P < .01). The production of CYP24A1 protein and messenger RNA expression was significantly higher in placental tissue from patients with GDM than in those from normal pregnancies; however, the production of CYP27B1 and VDR protein and messenger RNA expression were not different between 2 the groups.

      Conclusion

      In this study, vitamin D deficiency was associated with GDM. Given that 25(OH)D is hydroxylated by CYP27B1 to the bioactive 1,25(OH)2D form, and CYP24A1 catabolizes both 25(OH)D and 1,25(OH)2D to the inactive metabolites, respectively, our data indicate that the elevated activity of CYP24A1 in the placenta may play a key role in the development of vitamin D deficiency in GDM.

      Key words

      Vitamin D is a secosteroid hormone that is well-known for its role in maintaining calcium homeostasis and promoting bone mineralization. Moreover, vitamin D has classic skeletal and nonclassic effects that include blood sugar control.
      • Holick M.F.
      Vitamin D deficiency.
      During pregnancy, vitamin D is also essential for maternal health and the prevention of adverse outcome. Vitamin D levels increase progressively from the first trimester and are increased by 100% during the third trimester relative to the nonpregnant state.
      • Cross N.A.
      • Hillman L.S.
      • Allen S.H.
      • Krause G.F.
      • Vieira N.E.
      Calcium homeostasis and bone metabolism during pregnancy, lactation, and postweaning: a longitudinal study.
      • Ritchie L.D.
      • Fung E.B.
      • Halloran B.P.
      • et al.
      A longitudinal study of calcium homeostasis during human pregnancy and lactation and after resumption of menses.
      The vitamin D metabolic pathway involves multiple enzymatic reactions. Vitamin D is metabolized in the liver to the form 25-(OH)D, which is used to determine a patient's vitamin D status: 25(OH)D is metabolized in the kidneys by CYP27B1 to its active form, 1,25-(OH)2D; 1,25-(OH)2D induces the expression of CYP24A1, which catabolizes both 25-(OH)D and 1,25-(OH)2D into biologically inactive, water-soluble calcitroic acid. The biologic functions of vitamin D are exerted through the interaction of 1,25-(OH)2D with a single vitamin D receptor (VDR) in the cell nucleus.
      • Holick M.F.
      Vitamin D deficiency.
      • Shin J.S.
      • Choi M.Y.
      • Longtine M.S.
      • Nelson D.M.
      Vitamin D effects on pregnancy and the placenta.
      The human placenta is known to express all of the components that are required for vitamin D signaling, which include VDR, CYP27B1, and CYP24A1.
      • Shin J.S.
      • Choi M.Y.
      • Longtine M.S.
      • Nelson D.M.
      Vitamin D effects on pregnancy and the placenta.
      Moreover, the placenta plays an important role of increasing vitamin D level
      • Delvin E.E.
      • Arabian A.
      Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from human term decidua.
      • Weisman Y.
      • Harell A.
      • Edelstein S.
      • David M.
      • Spirer Z.
      • Golander A.
      1 Alpha, 25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta.
      by an increase in the expression of CYP2B1 and the epigenetic down-regulation of CYP24A1.
      • Saffery R.
      • Ellis J.
      • Morley R.
      A convergent model for placental dysfunction encompassing combined sub-optimal one-carbon donor and vitamin D bioavailability.
      Gestational diabetes mellitus (GDM) is a type of glucose intolerance that first manifests in pregnancy and affects 3-8% of all pregnancies.
      • Metzger B.E.
      • Coustan D.R.
      Summary and recommendations of the Fourth International Workshop-Conference on Gestational Diabetes Mellitus: the Organizing Committee.
      GDM has serious adverse maternal and fetal outcomes. Currently, strategies for the prevention of GDM include diet and exercise, but these have limited effectiveness.
      • Oostdam N.
      • van Poppel M.N.
      • Wouters M.G.
      • van Mechelen W.
      Interventions for preventing gestational diabetes mellitus: a systematic review and meta-analysis.
      There is increasing interest in the relationship between vitamin D and GDM. Several studies have reported lower vitamin D levels in women with GDM,
      • Maghbooli Z.
      • Hossein-Nezhad A.
      • Karimi F.
      • Shafaei A.R.
      • Larijani B.
      Correlation between vitamin D3 deficiency and insulin resistance in pregnancy.
      • Clifton-Bligh R.J.
      • McElduff P.
      • McElduff A.
      Maternal vitamin D deficiency, ethnicity and gestational diabetes.
      and other studies have demonstrated lower levels at 16 weeks gestation among women who experienced the development of GDM
      • Zhang C.
      • Qiu C.
      • Hu F.B.
      • et al.
      Maternal plasma 25-hydroxyvitamin D concentrations and the risk for gestational diabetes mellitus.
      ; however, these results are controversial.
      • Baker A.M.
      • Haeri S.
      • Camargo Jr., C.A.
      • Stuebe A.M.
      • Boggess K.A.
      First-trimester maternal vitamin D status and risk for gestational diabetes (GDM) a nested case-control study.
      • Makgoba M.
      • Nelson S.M.
      • Savvidou M.
      • Messow C.M.
      • Nicolaides K.
      • Sattar N.
      First-trimester circulating 25-hydroxyvitamin D levels and development of gestational diabetes mellitus.
      Moreover, although the placenta plays an important role in vitamin D metabolism during pregnancy and several interdependent risk factors that regulate vitamin D metabolism at the fetomaternal interface increase the risk of placental dysfunction that causes adverse pregnancy outcome,
      • Saffery R.
      • Ellis J.
      • Morley R.
      A convergent model for placental dysfunction encompassing combined sub-optimal one-carbon donor and vitamin D bioavailability.
      to the best of our knowledge, placental vitamin D metabolism and the contribution of the placenta to altered vitamin D levels in GDM are unknown.

      Materials and Methods

      Participants and sample collection

      A case-control study was performed at Korea University, Department of Obstetrics and Gynecology from January 2011 to May 2011. We consecutively enrolled 20 Korean pregnant women with GDM and 40 age- and gestational age-matched normal Korean pregnant women as the control group. Gestational age at delivery was calculated according to the last menstrual period and was confirmed by ultrasonic examination during the first trimester. All women had singleton pregnancies without identified fetal anomalies at term (range, 37–42 gestational weeks). None of these women had any history of hypertension, preeclampsia, pre-GDM, or any other significant endocrine disorder. All participants gave written informed consent for participation in the study, which was approved by the clinical research ethics committee.
      All pregnant women were screened for GDM at 24-28 weeks of gestation with a 50-g oral glucose challenge test. Women with glucose levels of ≥140 mg/dL underwent a standard 100-g, 3-hour oral glucose tolerance test. The diagnosis of GDM was based on the criteria of Carpenter and Coustan
      • Carpenter M.W.
      • Coustan D.R.
      Criteria for screening tests for gestational diabetes.
      by which at least 2 of 4 of the following diagnostic criteria were met: fasting plasma glucose, ≥95 mg/dL; and 1-, 2-, and 3-hour glucose levels of ≥180 mg/dL, ≥155 mg/dL, ≥140 mg/dL, respectively.
      Maternal blood samples were taken from a cannulated vein immediately before delivery, and all blood samples were centrifuged (1500g at 4°C for 25 minutes). Placental tissues from the central cotyledon were also obtained immediately after delivery. Serum aliquots and placental tissues were immediately stored at –80°C until analysis.
      Body mass index (BMI) was calculated for each participant at the time of admission for delivery as weight in kilograms divided by height in square meters. General information including maternal age, parity, birthweight, neonatal gender, and delivery mode were obtained from patient medical records.

      Measurement of serum 25(OH)D

      Levels of 25(OH)D in serum were measured with a commercially available enzyme-immunoassay kit for 25(OH)D (Immundiagnostik AG, Bensheim, Germany) according to the manufacturers' instructions. The results were normalized to 25(OH)D levels and expressed in nanograms per milliliters. Vitamin D deficiency was defined as a 25(OH)D serum level of <20 ng/mL.
      American College of Obstetricians and Gynecologists
      ACOG Committee Opinion, No. 495: Vitamin D: screening and supplementation during pregnancy.

      Total RNA isolation and complementary DNA sample preparation

      RNA extraction and purification were conducted with the RNeasy mini kit (Qiagen, Valencia, CA) as described in the manufacturer's protocol. The concentration of RNA was measured with a spectrophotometer (DU530; Beckman Instruments Inc, Fullerton, CA). The total RNA sample (1 μg/sample) was used in the SuperScriptTM III First-Strand Synthesis System for reverse transcriptase–PCR kit (Invitrogen, Milan, Italy) at the 20 μL scale to generate complementary DNA. RNA was reverse transcribed under the following conditions: 25 mmol/L MgCl2, 10 mmol/L dNTP mix, 10 × RT buffer, 0.1 mol/L DTT, 200 U SuperScriptTM III (Invitrogen), 40 U RNaseOut, and 50 μmol/L oligo d(T) primers in a final volume of 20 μL. The reaction was run at 65°C for 5 minutes and 50°C for 50 minutes. The enzyme was then heat inactivated at 85°C for 5 minutes, and a 4-μL sample of the reaction products was used for real-time PCR reactions.

      Quantitative real-time PCR analysis

      Real-time PCR was used to quantify VDR, CYP24A1, and CYP27B1 gene expression. This expression was normalized using the GAPDH housekeeping gene as an endogenous reference. The primers and probes were designed for VDR, CYP24A1, and CYP27B1 using Primer Express (version 2.0; Applied Biosystems, Foster City, CA). VDR, CYP24A1, and CYP27B1 messenger RNA (mRNA) levels were quantified with the use of TaqMan real-time PCR with an ABI 7700 system (Applied Biosystems). Gene-specific probes and primer pairs for VDR (Assays-on-Demand; Hs01045840_m1, Applied Biosystems), CYP24A1 (Hs00167999_m1), and CYP27B1 (Hs00168017_m1) were used. For each probe/primer set, a standard curve was generated and confirmed a linear increase with increasing amounts of complementary DNA. The amplification conditions were 2 minutes at 50°C, 10 minutes at 95°C, and a 2-step cycle of 95°C for 15 seconds and 60°C for 60 seconds, for a total of 40 cycles.

      Western blot analysis

      Total tissue lysates were prepared by homogenization. The tissues were maintained in buffer that contained 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 10% glycerol, 1% Triton X-100, and a mixture of protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate [iNtRON Biotechnology, Sungnam, Korea]). Concentrations of the extracted proteins were measured according to the Bradford method (Bio-Rad Laboratories, Hercules, CA). Equal amounts of total protein (10 μg) were resolved on 12% sodium dodecylsulfate–polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane (HybondTM-P; Amersham Biosciences, Piscataway, NJ). After they were blocked overnight (Tris-buffered saline, 0.1% Tween 20) at 4°C, the membranes were incubated with primary antibodies for human VDR (1:1000; Abcam Inc, Cambridge, MA), CYP24A1 (1:1000, Bioworld Technology, Inc, St. Louis Park, MN) and CYP27B1 (1:1000, Santa Cruz Biotechnology, Inc, Santa Cruz, CA). After incubation, the blots were washed (Tris-buffered saline, 0.1% Tween 20) and incubated with secondary antibodies linked to horseradish peroxidase (1:2000; Bio-Rad Laboratories). Immunoreactive proteins were visualized by chemiluminescence with the SuperSignal West Dura Extended Duration Substrate (Pierce Chemical Co, Rockford, IL), and signals were detected on X-ray film (Agfa-Gevaert, Mortsel, Belgium). Signals in the linear range of the film were digitized and densitometry was performed using Quantity One (Bio-Rad Laboratories).

      Immunohistochemistry and immunofluorescence staining

      Immunohistochemical staining was used to localize and compare the distribution of CYP24A1. Tissue sections (5 μm) were deparaffinized and then rehydrated and blocked with 3% H2O2 in methanol for 30 minutes followed by blocking (normal serum, 1.5%; Vector Laboratories, Burlingame, CA). Antibodies that were reactive to human CYP24A1 (1:100, Bioworld Technology Inc) were used for 1 hour. After the primary antibodies were applied, the slides were incubated overnight at room temperature. Secondary antibodies were used for detection (Vector Laboratories). All samples were counterstained with Mayer's hematoxylin before being mounted with Immuno Mount (Lab Vision, Fremont, CA). Immunofluorescence staining was used to localize and compare the distribution of VDR and CYP27B1. Tissue sections (5 μm) were fixed with 4% paraformaldehyde for 10 minutes, followed by blocking solution (phosphate-buffered saline solution that contained 3% bovine serum albumin and 0.02%Triton X-100) for 30 minutes, and anti-VDR (1:100; Abcam Inc) and CYP27B1 (1:100, Santa Cruz Biotechnology Inc) antibodies were added for 1 hour at room temperature. After being washed, the cells were exposed to Alexa-488–conjugated anti-rabbit immunoglobulin G (1:500; Invitrogen) or anti-goat immunoglobulin G (1:500; Santa Cruz Biotechnology Inc) for 30 min at room temperature, and their nuclei counterstained with DAPI (1:5000, Invitrogen) for immunofluorescence. Confocal microscopy (Olympus BX61-32FA1-S08 microscope with fluorescence equipment; Olympus, Tokyo, Japan) was used for morphologic evaluation.

      Statistical analysis

      Data are expressed as the mean ± SD for continuous variables and as a percentage for categoric variables. Student t tests were used to assess statistical significance between normally distributed continuous variables. Otherwise, nonparametric Mann-Whitney U tests were used. Categoric variables were compared with the use of the χ2 test. A post-hoc power analysis with an alpha of .05 showed that a sample size of this study provides sufficient power (>99.9%) to identify a difference in vitamin D level between 2 groups. A model of multivariate logistic regression analysis was used to evaluate the risk of GDM according to the presence of vitamin D deficiency. Spearman rank correlation coefficients were used to evaluate associations between mRNA levels in placenta and serum 25(OH)D levels. Results were considered statistically significant when the probability values were < .05 (2-sided). Statistical analyses were performed using SPSS software (version 12.0; SPSS Inc, Chicago, IL).

      Results

      Clinical and demographic characteristics of study participants

      Table 1 shows the basic characteristics of pregnant women with GDM and normal pregnant women. There were no differences in age, gestational age at delivery, BMI, parity, birthweight, neonatal gender, delivery mode, or total calcium level between the 2 groups.
      Table 1Clinical and biochemical characteristics of the study participants
      VariablePregnant womenP value
      Without GDM (n = 40)With GDM (n = 20)
      Age, y32.68 ± 3.8533.45 ± 3.76.460
      Gestational age at delivery, wk38.79 ± 1.1438.41 ± 1.13.813
      Body mass index, kg/m226.53 ± 4.1228.37 ± 3.70.089
      Parity, n0.60 ± 0.780.50 ± 0.61.588
      Birthweight, kg3.20 ± 0.513.36 ± 0.42.053
      Male gender, %42.535.0.576
      Total calcium level, mg/dL8.99 ± 0.309.02 ± 0.43.815
      Vitamin D level, mg/dL34.52 ± 19.2711.65 ± 9.15< .001
      Vitamin D deficiency, %27.585.0< .001
      GDM, gestational diabetes mellitus.
      Cho. Vitamin D deficiency and GDM. Am J Obstet Gynecol 2013.

      Vitamin D deficiency in study participants

      Overall, 46.7% of participants reported vitamin D deficiency, which was defined as a 25(OH)D serum level of <20 mg/dL. Pregnant women with GDM had a lower serum vitamin D level and a higher prevalence of vitamin D deficiency compared with normal pregnant women (Table 1).
      The multivariate-adjusted odds ratios for GDM are shown in Table 2. Pregnant women with vitamin D deficiency had a 45.73-fold increased risk of having GDM after adjustment for age, BMI, parity, birthweight, neonatal gender, and total calcium level.
      Table 2The risk of gestational diabetes mellitus
      VariableAdjusted odds ratio (95% CI)
      Age, y0.92 (0.74–1.15)
      Body mass index, kg/m21.26 (1.02–1.56)
      Parity, n0.73 (0.21–2.51)
      Birthweight, kg4.97(0.73–33.59)
      Male gender, %0.67 (0.12–3.87)
      Total calcium level, mg/dL1.76 (0.20–15.27)
      Vitamin D deficiency, %30.78 (4.65–203.90)
      Vitamin D deficiency defined as 25(OH)D serum levels <20 ng/mL. The model is adjusted for the variables in the Table.
      CI, confidence interval.
      Cho. Vitamin D deficiency and GDM. Am J Obstet Gynecol 2013.

      Expression and production of placental VDR, CYP27B1, and CYP24A1

      Real time–PCR was used to determine the expression of VDR, CYP27B1, and CYP24A1 gene transcripts in the placenta. Densitometry analysis revealed that, in relation to the reference gene GAPDH, CYP24A1 gene expression in placental tissues from patients with GDM was increased significantly compared with normal term placental tissue. However, no differences in VDR and CYP27B1 expression between the 2 groups were found (Figure 1).
      Figure thumbnail gr1
      Figure 1Real time–polymerase chain reaction analysis of messenger RNA expression in placental tissue
      A, Vitamin D receptor (VDR); B, CYP24A1; C, CYP27B1. The asterisk denotes a probability value of < .05.
      GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
      Cho. Vitamin D deficiency and GDM. Am J Obstet Gynecol 2013.
      Western blot analysis was conducted to assess the production of VDR, CYP27B1, and CYP24A1 protein in placental tissues. Quantification of CYP24A1 production by densitometry revealed that the CYP24A1 protein level was increased significantly in placentas from participants with GDM when compared with normal term placental tissue. However, there were no differences in protein production of VDR and CYP27B1 between the 2 groups (Figure 2).
      Figure thumbnail gr2
      Figure 2Western blot analysis of protein expression in placental tissue
      A, Representative Western blot analysis of protein production in placental tissue and densitometric analysis of B, vitamin D receptor (VDR), C, CYP24A1 (the asterisk denotes a probability value of < .05), and D, CYP27B1 in placental tissue.
      GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
      Cho. Vitamin D deficiency and GDM. Am J Obstet Gynecol 2013.
      Immunohistochemical staining was performed for CYP24A1 tissue localization. Weak nuclear production of CYP24A1 was identified in the cytotrophoblasts and syncytiotrophoblasts. Immunostaining for CYP24A1 was increased in placental tissues from patients with GDM (Figure 3, B) compared with normal term placental tissue (Figure 3, A). VDR and CYP27B1 were visualized by immunofluorescence for tissue localization (Figure 4), which were localized to the cytoplasm of trophoblastic cells. No significant differences in the localization of VDR and CYP27B1 proteins were identified between the 2 groups.
      Figure thumbnail gr3
      Figure 3Immunohistochemistry for CYP24A1
      A, Normal placental tissue and B, placental tissue with gestational diabetes mellitus. The arrows show the immunostaining cells. (Original magnification, ×50; scale bar, 50 μm.)
      Cho. Vitamin D deficiency and GDM. Am J Obstet Gynecol 2013.
      Figure thumbnail gr4
      Figure 4Immunofluorescence
      A, B, Vitamin D receptor and C, D, CYP27B1 in A, C, normal placental tissue and B, D, placental tissue with gestational diabetes mellitus. (Original magnification, ×200.)
      Cho. Vitamin D deficiency and GDM. Am J Obstet Gynecol 2013.

      The association between vitamin D level and the expression of placental VDR, CYP27B1, and CYP24A1

      There was a significant negative correlation between serum 25(OH)D levels and mRNA expressions of CYP24A1 in placenta (Figure 5, B). However, no correlation between serum 25(OH)D levels and mRNA expressions of VDR (Figure 5, A) and CYP27B1 (Figure 5, C) in placenta were found.
      Figure thumbnail gr5
      Figure 5Correlation between serum 25(OH)D levels and messenger RNA levels in placenta
      A, Vitamin D receptor (VDR), B, CYP24A1, and C, CYP27B1. Statistical analysis by Spearman's rank test. Rho represents the correlation coefficient.
      GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
      Cho. Vitamin D deficiency and GDM. Am J Obstet Gynecol 2013.

      Comment

      In this study, vitamin D level was lower in pregnant women with GDM compared with normal pregnant women. In addition, vitamin D deficiency was associated with an increased risk of GDM, which is consistent with results from other studies.
      • Maghbooli Z.
      • Hossein-Nezhad A.
      • Karimi F.
      • Shafaei A.R.
      • Larijani B.
      Correlation between vitamin D3 deficiency and insulin resistance in pregnancy.
      • Clifton-Bligh R.J.
      • McElduff P.
      • McElduff A.
      Maternal vitamin D deficiency, ethnicity and gestational diabetes.
      • Zhang C.
      • Qiu C.
      • Hu F.B.
      • et al.
      Maternal plasma 25-hydroxyvitamin D concentrations and the risk for gestational diabetes mellitus.
      However, several studies have reported no association between vitamin D deficiency and GDM.
      • Baker A.M.
      • Haeri S.
      • Camargo Jr., C.A.
      • Stuebe A.M.
      • Boggess K.A.
      First-trimester maternal vitamin D status and risk for gestational diabetes (GDM) a nested case-control study.
      • Makgoba M.
      • Nelson S.M.
      • Savvidou M.
      • Messow C.M.
      • Nicolaides K.
      • Sattar N.
      First-trimester circulating 25-hydroxyvitamin D levels and development of gestational diabetes mellitus.
      This inconsistency between different studies, in part, could be due to variations in sampling time to measure vitamin D level. Serum vitamin D level increases 50-100% over the nonpregnant state through the second trimester and increases by 100% during the third trimester.
      • Cross N.A.
      • Hillman L.S.
      • Allen S.H.
      • Krause G.F.
      • Vieira N.E.
      Calcium homeostasis and bone metabolism during pregnancy, lactation, and postweaning: a longitudinal study.
      • Ritchie L.D.
      • Fung E.B.
      • Halloran B.P.
      • et al.
      A longitudinal study of calcium homeostasis during human pregnancy and lactation and after resumption of menses.
      Based on the findings that vitamin D is produced by the placenta
      • Delvin E.E.
      • Arabian A.
      Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from human term decidua.
      • Weisman Y.
      • Harell A.
      • Edelstein S.
      • David M.
      • Spirer Z.
      • Golander A.
      1 Alpha, 25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta.
      and the fact that an increase in placental mass occurs with gestation, it is reasonable to speculate that placental production of vitamin D is the principal cause for the increase in vitamin D levels in late pregnancy. Vitamin D level was measured at term in this study, and other studies reported lower vitamin D levels in pregnant women with GDM at the time of GDM screening (24-29 weeks' gestation).
      • Maghbooli Z.
      • Hossein-Nezhad A.
      • Karimi F.
      • Shafaei A.R.
      • Larijani B.
      Correlation between vitamin D3 deficiency and insulin resistance in pregnancy.
      • Clifton-Bligh R.J.
      • McElduff P.
      • McElduff A.
      Maternal vitamin D deficiency, ethnicity and gestational diabetes.
      A single study has demonstrated lower vitamin D levels at 16 weeks' gestation before the diagnosis of GDM.
      • Zhang C.
      • Qiu C.
      • Hu F.B.
      • et al.
      Maternal plasma 25-hydroxyvitamin D concentrations and the risk for gestational diabetes mellitus.
      Otherwise, studies that reported no association measured and assessed vitamin D levels early in pregnancy (11-14 weeks' gestation)
      • Baker A.M.
      • Haeri S.
      • Camargo Jr., C.A.
      • Stuebe A.M.
      • Boggess K.A.
      First-trimester maternal vitamin D status and risk for gestational diabetes (GDM) a nested case-control study.
      • Makgoba M.
      • Nelson S.M.
      • Savvidou M.
      • Messow C.M.
      • Nicolaides K.
      • Sattar N.
      First-trimester circulating 25-hydroxyvitamin D levels and development of gestational diabetes mellitus.
      ; therefore, these studies may have measured vitamin D levels at a stage too early to reflect the placental production of vitamin D and its effects on vitamin D levels. The association between vitamin D deficiency and GDM may be different according to gestational age, especially between levels at early and late gestation. Further studies are needed to evaluate longitudinal changes in vitamin D level over whole gestational periods and their associations with GDM. Moreover, differences of other factors that include ethics, criteria of GDM, and definition of vitamin D deficiency may also have contributed to the discrepancies between studies.
      To the best of our knowledge, this study is the first to report the role of the placenta in vitamin D metabolism in GDM. We found that the expression and production of CYP24A1 was increased in placental tissues from patients with GDM compared with normal placental tissues and that serum vitamin D level was correlated negatively with the expression of CYP24A1 in placenta. It is well established that CYP24A1 is correlated inversely with vitamin D status in target tissues for vitamin D.
      • St-Arnaud R.
      • Arabian A.
      • Travers R.
      • et al.
      Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.
      • Ly L.H.
      • Zhao X.Y.
      • Holloway L.
      • Feldman D.
      Liarozole acts synergistically with 1alpha,25-dihydroxyvitamin D3 to inhibit growth of DU 145 human prostate cancer cells by blocking 24-hydroxylase activity.
      • Anderson P.H.
      • O'Loughlin P.D.
      • May B.K.
      • Morris H.A.
      Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction.
      • Mimori K.
      • Tanaka Y.
      • Yoshinaga K.
      • et al.
      Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer.
      Therefore, given that the placenta has an important role for the increase of vitamin D during pregnancy,
      • Delvin E.E.
      • Arabian A.
      Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from human term decidua.
      • Weisman Y.
      • Harell A.
      • Edelstein S.
      • David M.
      • Spirer Z.
      • Golander A.
      1 Alpha, 25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta.
      increased placental expression and production of CYP24A1 may be responsible for the low level of vitamin D that is observed in GDM. Interestingly, it has also been reported that vitamin D levels were lower in women with preeclampsia
      • Baker A.M.
      • Haeri S.
      • Camargo Jr., C.A.
      • Espinola J.A.
      • Stuebe A.M.
      A nested case-control study of midgestation vitamin D deficiency and risk of severe preeclampsia.
      and that the expressions of CYP24A1 were elevated in preeclamptic placentas.
      • Ma R.
      • Gu Y.
      • Zhao S.
      • Sun J.
      • Groome L.J.
      • Wang Y.
      Expressions of vitamin D metabolic components VDBP, CYP2R1, CYP27B1, CYP24A1, and VDR in placentas from normal and preeclamptic pregnancies.
      Similarly, increased activity of CYP24A1 has been associated with other diseases in humans. The expression of CYP24A1 was shown to be increased in renal tissue from patients with diabetic nephropathy
      • Helvig C.F.
      • Cuerrier D.
      • Hosfield C.M.
      • et al.
      Dysregulation of renal vitamin D metabolism in the uremic rat.
      and chronic kidney disease,
      • Zehnder D.
      • Quinkler M.
      • Eardley K.S.
      • et al.
      Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation.
      which may be a significant mechanism that contributes to vitamin D deficiency and resistance to vitamin D therapy in chronic kidney disease.
      • Zehnder D.
      • Quinkler M.
      • Eardley K.S.
      • et al.
      Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation.
      CYP24A1 is also overexpressed in numerous human tumors, including breast, colon, prostate, esophagus, and lung
      • Mimori K.
      • Tanaka Y.
      • Yoshinaga K.
      • et al.
      Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer.
      • Anderson M.G.
      • Nakane M.
      • Ruan X.
      • Kroeger P.E.
      • Wu-Wong J.R.
      Expression of VDR and CYP24A1 mRNA in human tumors.
      ; here, it attenuates vitamin D–mediated growth inhibition.
      Despite significant public health efforts to ensure that all pregnant women achieve 25(OH)D intake of 10 mg/day (400 IU/day),

      Your health in pregnancy. In: Department of Health, editor. The pregnancy book 2009. London: Department of Health; 2009:24-39.

      there appears to have been little positive impact on the prevalence of vitamin D, which, in part, may be due to inadequate vitamin D supplementation doses in pregnancy.
      • Hollis B.W.
      • Wagner C.L.
      Assessment of dietary vitamin D requirements during pregnancy and lactation.
      In addition, increased placental activity of CYP24A1 observed in this study can also account for the resistance to vitamin D therapy in pregnancy.
      The important issue of whether low levels of vitamin D or vitamin D deficiency by the increased placental activity of CYP24A1 are causal of GDM or a consequence of the disease process is not clear. Several studies demonstrated specific receptors for vitamin D in pancreatic β cells
      • Roth J.
      • Bonner-Weir S.
      • Norman A.W.
      • Orci L.
      Immunocytochemistry of vitamin D-dependent calcium binding protein in chick pancreas: exclusive localization.
      and a role for vitamin D in the secretion of insulin.
      • Kadowaki S.
      • Norman A.W.
      Dietary vitamin D is essential for normal insulin secretion from the perfused rat pancreas.
      • Norman A.W.
      • Frankel J.B.
      • Heldt A.M.
      • Grodsky G.M.
      Vitamin D deficiency inhibits pancreatic secretion of insulin.
      It also has been reported that vitamin D deficiency is associated with insulin resistance and type 2 DM.
      • Chiu K.C.
      • Chu A.
      • Go V.L.
      • Saad M.F.
      Hypovitaminosis D is associated with insulin resistance and beta cell dysfunction.
      • Scragg R.
      • Holdaway I.
      • Singh V.
      • Metcalf P.
      • Baker J.
      • Dryson E.
      Serum 25-hydroxyvitamin D3 levels decreased in impaired glucose tolerance and diabetes mellitus.
      Moreover, in this study, maternal obesity with increased BMI was associated with increased risk of GDM after we controlled for several factors. Because obesity is associated with placental dysfunction by maternal hyperleptinemia
      • Farley D.M.
      • Choi J.
      • Dudley D.J.
      • et al.
      Placental amino acid transport and placental leptin resistance in pregnancies complicated by maternal obesity.
      and, moreover, because the placental tissue of women with GDM typically displays structural and functional abnormalities,
      • Alonso A.
      • Del Rey C.G.
      • Navarro A.
      • Tolivia J.
      • González C.G.
      Effects of gestational diabetes mellitus on proteins implicated in insulin signaling in human placenta.
      vitamin D deficiency by the increased placental activity of CYP24A1 through placental dysfunction may be attributed to the development of GDM. Otherwise, the vitamin D deficiency by increased placental activity of CYP24A1 that was observed in our study may be a consequence of factors that are associated with GDM. For example, tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is 1 of the key cytokines that are involved in the development of insulin resistance and type 2 diabetes mellitus.
      • Chen H.L.
      • Yang Y.P.
      • Hu X.L.
      • Yelavarthi K.K.
      • Fishback J.L.
      • Hunt J.S.
      Tumor necrosis factor alpha mRNA and protein are present in human placental and uterine cells at early and late stages of gestation.
      • Sjöholm A.
      • Nyström T.
      Inflammation and the etiology of type 2 diabetes.
      Although the exact mechanism is unknown, this relationship may be related to the association of TNF-α and inhibition of insulin signaling and insulin-regulated glucose uptake.
      • Mimori K.
      • Tanaka Y.
      • Yoshinaga K.
      • et al.
      Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer.
      During pregnancy, TNF-α is synthesized and secreted by adipose tissue as well as the placenta,
      • Chen H.L.
      • Yang Y.P.
      • Hu X.L.
      • Yelavarthi K.K.
      • Fishback J.L.
      • Hunt J.S.
      Tumor necrosis factor alpha mRNA and protein are present in human placental and uterine cells at early and late stages of gestation.
      and its level increases in late pregnancy, which causes the insulin resistance that is observed in pregnancy.
      • Mimori K.
      • Tanaka Y.
      • Yoshinaga K.
      • et al.
      Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer.
      Serum TNF-α levels and placental production of this cytokine are both augmented in GDM compared with control subjects.
      • Kirwan J.P.
      • Hauguel-De Mouzon S.
      • Lepercq J.
      • et al.
      TNF-alpha is a predictor of insulin resistance in human pregnancy.
      • Winkler G.
      • Cseh K.
      • Baranyi E.
      • et al.
      Tumor necrosis factor system in insulin resistance in gestational diabetes.
      Recently, Díaz et al
      • Díaz L.
      • Noyola-Martínez N.
      • Barrera D.
      • et al.
      Calcitriol inhibits TNF-alpha-induced inflammatory cytokines in human trophoblasts.
      reported that TNF-α caused a 2- to 5-fold increase in CYP24A1 gene expression in cytotrophoblasts. Therefore, increased TNF-α production in patients with GDM may cause the low vitamin D status through the activation of CYP24A1. Further studies are needed to clarify the underlying mechanisms that link vitamin D deficiency and GDM.
      Among women with GDM, there was a difference in vitamin D level between women with medical nutrition therapy (n = 11) and those with insulin therapy (n = 9). Moreover, there was no difference in vitamin D level between women with adequate glycemic control (n = 17) and those with poor glycemic control (n = 3; data not shown).
      Several limitations should be kept in mind with the interpretation of our findings. First, it was not possible to establish a cause-and-effect relationship between vitamin D deficiency and GDM, as mentioned earlier, because of the cross-sectional nature of this study. Second, we did not survey the data regarding the patient's supplemental vitamin D intake. Third, placental vitamin D and CYP24A1 may play an important role in vitamin D metabolism in normal and complicated pregnancies, including GDM. However, we could not determine the exact amount of vitamin D that was secreted from the placenta into the maternal circulation and the degree to which activated CYP24A1 contributed to the low level of vitamin D that was observed in GDM. Further studies are required to clarify these issues.
      In conclusion, women with GDM were shown to have a lower level of vitamin D compared with normal control subjects, and the placental activity of CYP24A1 was increased in GDM compared with normal control placental tissues. These observations provide an alternative to the placental-derived vitamin D metabolism paradigm to explain vitamin D deficiency in GDM. The regulation of CYP24A1 may provide an important target for physiologic interventions that are designed to reduce the risk of adverse pregnancy outcomes related to vitamin D deficiency.

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