Vitamin D and Angiogenic Markers in Odense Child Cohort 1

Overall: The Odense Child Cohort Study (OCC) is a population-based cohort study, comprising pregnant women recruited between January 1st 2010 and December 31st 2012. All women who were pregnant in the municipality of Odense during this time were eligible for participation, and 6,707 women were approached directly with recruitment material.

The study complied with the Helsinki declaration and was approved by the Regional Scientific Ethical Committee for Southern Denmark, no. S-20090130. All participants gave informed consent. From a population base of 6,707 pregnant women, 2,874 (42.9%) enrolled in the OCC up to December 31st, 2012. The children will be followed until 18 years of age.

Serum samples were taken in early pregnancy (GA 2-25 weeks), late pregnancy (GA 26-30 weeks), maternal serum and cord blood at birth, and from the children along with general examinations at ages 3 months, 1 year, 3 years.

Questionnaires were completed by the families in early pregnancy, late pregnancy, after birth, and at the times of clinical examination of the children.

Register data on health variables were further available from the Danish registries.

Objective: The Vitamin D and Angiogenic Marker Study 1 examines the relationship between serum biomarkers 25(OH)D and soluble Fms-like kinase 1 (sFlt-1) and placental growth factor (PlGF) in pregnancy and risk of miscarriage as an adverse outcome in early pregnancy.

Method:

• 25(OH)D analysis: Serum was stored at -80º Celsius until analysis, which was performed by liquid chromatography mass spectrometry (LC-MS/MS). Triple deuterium marked 25(OH)vitamin D3 was added to serum samples as internal standard and deproteinized with ZnSO4 in methanol, centrifuged at 2750 g for 10 minutes, and 100 μl was injected on the TurboFlow column (Thermo Scientific) on the LC-MS/MS. The LC-MS/MS consisted of a Thermo Scientific TLX1 system connected to a Thermo Scientific Vantage TSQ. 25(OH)D2 and 25(OH)D3 were concentrated on a Thermo Scientific Cyclone P 50 x 1.0 mm column and back-flushed on the analytical column, Phenomenex Gemini C18 50 x 3.0 mm and eluted from the analytical column by a gradient. Mobile phases were A: 10 mM NH4Ac in water and B: 10 mM NH4Ac in methanol. Human serum was spiked with appropriate amounts of 25(OH)D2 and 25(OH)D3 in order to produce six point calibration curves (weighed 1/x2) and 3 levels of QC samples (low, mid, high). The method was calibrated against NIST standard 972.25 The C3 epimer of D3 was detected along with D3 and the two were not distinguishable from one another. Lowest detectable concentrations were 0.15 nM for both D2 and D3. Values of D2 and D3 were only considered if above 6.5 nM.
• Measurements for BRAHMS sFlt-1 and PIGF KRYPTOR assays were performed on the fully automated KRYPTOR compact Plus system (KRYPTOR PlGF and KRYPTOR sFlt-1; Thermo Fisher Scientific) according to the manufacturer's instructions. The BRAHMS sFlt-1 and PlGF KRYPTOR assays are homogeneous sandwich immunoassays based on the Trace technology24. The total duration of the assays are 9 minutes (sFlt-1) and 29 minutes (PlGF) and the sample volume is 9 µl, and 70 μl, respectively. The assays are calibrated with recombinant human sFlt-1 and PIGF and standardized against the Quantikine PIGF ELISA (R\&D Systems Europe Ltd, Abingdon, UK) and against the sFlt-1 Elecsys assay (Elecsys, Penzberg, Germany). According to the manufacturer's instructions for use, the KRYPTOR sFlt-1 assay covered a measuring range of 22-90,000 pg/ml. The limit of detection is 22 pg/ml and the limit of quantization (functional sensitivity) is 29 pg/ml. The intra- and inter-assay variations at an sFlt-1 concentration of 1540 pg/ml are 0.3% and 0.8%, respectively; at 2988 pg/ml 0.5% and 1.1%; and at 9666 pg/ml 0.5% and 1.0%, respectively. The KRYPTOR PlGF assay covers a measuring range of 3.6-7.000 pg/ml. The limit of detection is 3.6 pg/ml and the limit of quantitation is 6.9 pg/ml. The intra- and inter-assay variations at a PlGF concentration of 35 pg/ml are 4.6% and 7.3%, respectively; at 103 pg/ml 2.1% and 3.1%, and at 430 pg/ml 0.9% and 2.3%, respectively.
• Measurements for Elecsys sFlt-1 and PIGF assays were performed on the fully automated Elecsys system (Elecsys, Penzberg, Germany) according to the manufacturer's instructions. The Elecsys assays are sandwich immunoassays based on the electrochemiluminescence technology. The total duration of the assays is 18 minutes and the sample volume is 20 µl for sFlt-1 and 50 µl for PIGF. The assays are calibrated with recombinant human PIGF and sFlt-1 and standardized against the Quantikine PIGF and vascular endothelial growth factor-R1 ELISAs (R\&D Systems, Minneapolis, MN). According to the manufacturer's instructions for use, the Elecsys sFlt-1 assay covers a measuring range from 10-85,000 pg/ml, the Elecsys PIGF assay from 3-10,000 pg/ml. The limit of detection is 10 pg/ml (sFlt-1) and 3 pg/ml (PIGF). The limit of quantization is 15 pg/ml for sFlt-1 and 10 pg/ml for PIGF.

The Vitamin D and Angiogenic Marker Study 1:

Vitamin D and angiogenic markers in early pregnancy. Epidemiology and associations to early-pregnancy miscarriage

• In 1728 serum samples collected in early pregnancy, the levels of serum 25(OH)D were investigated by LC-MS/MS and levels of sFlt-1 and PlGF were determined by immunoassays in 1720 samples. Questionnaire and medical file information was used to determine the factors which were influential on 25(OH)D levels and sFlt/PlGF in early pregnancy by multiple linear regression and logistic regression.
• In 1675 serum samples collected before gestational age 22+0 weeks, analysis was performed to evaluate the predictive properties of sFlt-1 and PlGF levels as biomarkers of miscarriage (n=59).
• In 1684 serum samples collected before gestational age 22+0 weeks, the levels of 25(OH)D were compared between women who subsequently suffered spontaneous abortions (n=59) and the rest of the population, corrected for select covariates.