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) 2 D form, and CYP24A1 catabolizes both 25(OH)D and 1,25(OH) 2 D 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.
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. 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.
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) 2 D; 1,25-(OH) 2 D induces the expression of CYP24A1, which catabolizes both 25-(OH)D and 1,25-(OH) 2 D into biologically inactive, water-soluble calcitroic acid. The biologic functions of vitamin D are exerted through the interaction of 1,25-(OH) 2 D with a single vitamin D receptor (VDR) in the cell nucleus. The human placenta is known to express all of the components that are required for vitamin D signaling, which include VDR, CYP27B1, and CYP24A1. Moreover, the placenta plays an important role of increasing vitamin D level by an increase in the expression of CYP2B1 and the epigenetic down-regulation of CYP24A1.
Gestational diabetes mellitus (GDM) is a type of glucose intolerance that first manifests in pregnancy and affects 3-8% of all pregnancies. GDM has serious adverse maternal and fetal outcomes. Currently, strategies for the prevention of GDM include diet and exercise, but these have limited effectiveness. There is increasing interest in the relationship between vitamin D and GDM. Several studies have reported lower vitamin D levels in women with GDM, and other studies have demonstrated lower levels at 16 weeks gestation among women who experienced the development of GDM ; however, these results are controversial. 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, 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 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 (1500 g 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.
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 MgCl 2 , 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% H 2 O 2 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 χ 2 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.
| Variable | Pregnant women | P value | |
|---|---|---|---|
| Without GDM (n = 40) | With GDM (n = 20) | ||
| Age, y | 32.68 ± 3.85 | 33.45 ± 3.76 | .460 |
| Gestational age at delivery, wk | 38.79 ± 1.14 | 38.41 ± 1.13 | .813 |
| Body mass index, kg/m 2 | 26.53 ± 4.12 | 28.37 ± 3.70 | .089 |
| Parity, n | 0.60 ± 0.78 | 0.50 ± 0.61 | .588 |
| Birthweight, kg | 3.20 ± 0.51 | 3.36 ± 0.42 | .053 |
| Male gender, % | 42.5 | 35.0 | .576 |
| Total calcium level, mg/dL | 8.99 ± 0.30 | 9.02 ± 0.43 | .815 |
| Vitamin D level, mg/dL | 34.52 ± 19.27 | 11.65 ± 9.15 | < .001 |
| Vitamin D deficiency, % | 27.5 | 85.0 | < .001 |
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.
| Variable | Adjusted odds ratio (95% CI) |
|---|---|
| Age, y | 0.92 (0.74–1.15) |
| Body mass index, kg/m 2 | 1.26 (1.02–1.56) |
| Parity, n | 0.73 (0.21–2.51) |
| Birthweight, kg | 4.97(0.73–33.59) |
| Male gender, % | 0.67 (0.12–3.87) |
| Total calcium level, mg/dL | 1.76 (0.20–15.27) |
| Vitamin D deficiency, % | 30.78 (4.65–203.90) |
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 ).
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 ).
