Abortion, Spontaneous Clinical Trial
Official title:
Vitamin D and Angiogenic Markers in Odense Child Cohort 1: A Prospective Cohort Study on Their Role in Early Pregnancy Adverse Outcome
The Vitamin D and Angiogenic Marker Study 1:
Vitamin D and angiogenic markers in early pregnancy. Epidemiology and associations to
early-pregnancy miscarriage
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.
;
Observational Model: Cohort, Time Perspective: Prospective
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