Abstract
Perinatal asphyxia is a severe medical condition resulting from oxygen deficiency (hypoxia) at the time of birth, causing worldwide approximately 680,000 newborn deaths every year. Better prediction of severity of damages including early biomarkers is highly demanded. Elevated levels of circulating cell-free DNA (cfDNA) in blood have been reported for a range of different diseases and conditions, including cancer and prematurity. The objective of this study was to validate methods for assessing cfDNA in blood and cerebrospinal fluid (CSF) and to explore temporal variations in a piglet model of neonatal hypoxia-reoxygenation. Different cfDNA extraction methods in combination with cfDNA detection systems were tested, including a fluorescent assay using SYBR Gold and a qRT-PCR-based technique. Newborn piglets (n = 55) were exposed to hypoxia-reoxygenation, hypoxia-reoxygenation and hypothermia, or were part of the sham-operated control group. Blood was sampled at baseline and at post-intervention, further at 30, 270, and 570 minutes after the end of hypoxia. Applying the fluorescent method, cfDNA concentration in piglets exposed to hypoxia (n = 32) increased from 36.8±27.6 ng/ml prior to hypoxia to a peak level of 61.5±54.9 ng/ml after the intervention and deceased to 32.3±19.1 ng/ml at 570 minutes of reoxygenation, whereas the group of sham-operated control animals (n = 11) revealed a balanced cfDNA profile. Animals exposed to hypoxia and additionally treated with hypothermia (n = 12) expressed a cfDNA concentration of 54.4±16.9 ng/ml at baseline, 39.2±26.9 ng/ml at the end of hypoxia, and of 41.1±34.2 ng/ml at 570 minutes post-intervention. Concentrations of cfDNA in the CSF of piglets exposed to hypoxia revealed at post-intervention higher levels in comparison to the controls. However, these observations were only tendencies and not significant. In a first methodological proof-of-principle study exploring cfDNA using a piglet model of hypoxia-reoxygenation variations in the temporal patterns suggest that cfDNA might be an early indicator for damages caused by perinatal asphyxia.
Highlights
Perinatal- or birth asphyxia is caused by insufficient oxygen dispersion to the fetus and/or neonate at the time of birth resulting in over 680,000 deaths of newborns every year [1]
The concentrations of cell-free DNA (cfDNA) in the newborn piglets used for our study were twice as high as in an adult pig, 20.1±1.1 ng/ml versus 37.3±18.3 ng/ml, respectively (S3 Fig)
We investigated variations in cfDNA concentrations in blood plasma and cerebrospinal fluid (CSF) in a piglet model of perinatal asphyxia
Summary
Perinatal- or birth asphyxia is caused by insufficient oxygen dispersion to the fetus and/or neonate at the time of birth resulting in over 680,000 deaths of newborns every year [1]. Reliable and early diagnostic markers for the severity and overall outcome of perinatal asphyxia could substantially improve intervention strategies and treatment. A number of clinical and experimental animal studies indicate that perinatal asphyxia provokes cellular energy failure, generation of reactive oxygen species (ROS), formation of oxidative stress, and damages of cell-structures [7,8,9,10]. Previous studies have shown that biomarkers of ROS and reactive nitrogen species (RNS) can be measured shortly after hypoxic-ischemic injury [11]. These reactions are expected to trigger necrosis, apoptosis, and autophagy resulting in organ failures, like the clinical picture of hypoxic-ischemic encephalopathy [12]. To estimate oxidative stress reactions and damages in perinatal medicine, many different biomarkers have been investigated with unsatisfying results, including isoprostanes, isofuranes, gluthathione GSH/GSSG ratio, 8-hydroxy-2-deoxyguanosine, non protein-bound iron, superoxide dismutase, malondialdehyde, and S100B [13, 14]
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