Cord Blood Test May Flag Neonatal Hypoxia Risk

Damian McNamara

January 08, 2019

MicroRNA in umbilical cord blood may signal risk for imminent hypoxic-ischemic encephalopathy (HIE) in neonates, offering potential advantages over current diagnostic methods, new research suggests.

In a study of 160 full-term infants, two specific microRNAs (miR-374a-5p and miR-376c-3p) distinguished both infants with perinatal asphyxia (PA) and infants with HIE from healthy neonates.

To increase the clinical application of the findings, the researchers also identified a third microRNA (mir-181b-5p) that may help determine which neonates would benefit from therapeutic hypothermia.

The need for a better measure of HIE following PA prompted the investigators to search for a test that is quantifiable, robust, and available early enough to guide decision making in the first few hours after birth.

"Apgar score is still our best current measure, but it is subjective," study investigator Deirdre M. Murray, MD, PhD, professor in the Department of Pediatrics and Child Health, University College Cork, Ireland, told Medscape Medical News.

"Clinicians find it hard to rely on the Apgar score assigned by someone else, especially from another center," she added.

The findings were published online December 28 in JAMA Neurology.

20% of Infants "Missed"

MicroRNAs can be measured noninvasively in umbilical cord blood. Identifying which of these tiny ribonucleic acids best informs a differential diagnosis could lead to rapid polymerase chain reaction–based assays in the clinical setting, the investigators note.

Research in microRNA is still quite novel; previous studies of miR-374a and miR-376c have focused on cancer. Neonatal HIE can cause long-term neurologic disability and death in newborns.

Current diagnostic strategies include Apgar scores, initial lactate measures, and base deficit, but these "individually have poor positive predictive values," the investigators write.

In addition, the Sarnat grading scale of HIE with electroencephalogram grading can be accurate when assessment is conducted 24 hours post partum — but that can be too late to start treatment.

"Although therapeutic hypothermia improves outcome, it is only effective if commenced within 6 hours of birth. During this narrow window, babies need to be identified, stabilized, and transferred to a cooling center," Murray said.

"We currently miss about 20% of infants who are not offered cooling, as they are not identified using clinical examination alone. No robust, quantifiable, early marker exists to aid clinical decisions," she added.

The current study builds on previous work from the same researchers. It demonstrated miRNA profiling in a human neonatal cohort and showed downregulation of cord blood miR-374a-5p in infants with HIE.

Discover and Verify

The investigators evaluated 90 full-term infants in Cork, Ireland, as part of the Biomarkers in Hypoxic-Ischemic Encephalopathy (BiHiVE1) study. In BiHiVE2, the discovery phase of the study, they enrolled 80 infants from May 2009 through June 2011 at centers in Cork and in Stockholm, Sweden. Of a total of 170 infants, 160 were included in the final analysis.

The PA group had biochemical or clinical signs of perinatal hypoxia but no clinical encephalopathy.

Murray and colleagues also conducted BiHIVE2, a second multicenter cohort study. They enrolled infants from March 2013 through June 2015 for this validation study.

The researchers collected umbilical cord blood immediately after delivery of the placenta and processed samples within 3 hours of delivery.

The microRNAs that were significantly altered across both cohorts (miR-376c, miR-374a, and miR-181b) regulate RNA metabolic processes, postsynaptic specialization, DNA binding, and more.

Both studies employed healthy control infants from uneventful deliveries whose neonatal examination results were normal and whose 5-minute Apgar scores were greater than 8.

In BiHiVE1, there were significant differences between the 16 infants in the mild HIE group, five in the moderate group, and six with severe HIE. For example, miR-181b was significantly reduced in the severe group compared with the mild group (median, 0.08; interquartile range [IQR], 0.04 - 0.21 vs 0.82; IQR, 0.36 - 1.38; P = .01).

Therapeutic Targets?

The investigators analyzed 12 microRNAs in BiHiVE1. From this panel, they discovered five microRNAs differentially expressed in affected infants: miR-374a, miR-376c, miR-410, miR-181b, and miR-199a.

They also considered miR-376a, which demonstrated a strong but not significant trend in differentiating PA from HIE, and miR-155, because it may play a role in immune response and microglial activation.

Ultimately, this process led to the three microRNAs of interest. Each was significantly reduced in the cord blood of affected infants in the validation study.

For example, miR-374a-5p was lower in infants diagnosed with HIE compared with the healthy control group (median relative quantification, 0.38; IQR, 0.17 - 0.77 vs 0.95; IQR, 0.68 - 1.19; P = .009).

In addition, miR-376c-3p was significantly decreased in infants with PA compared with healthy control infants (median, 0.42; IQR, 0.21 - 0.61 vs 0.90; IQR, 0.70 - 1.30; P = .004), and mir-181b-5p was decreased in infants eligible for therapeutic hypothermia (median, 0.27; IQR, 0.14 - 1.41 vs 1.18; IQR, 0.70 - 2.05; P = .02).

The latter finding indicates "that miRNAs may be useful in identifying those infants who should be offered therapeutic hypothermia," Murray said.

"In two separate cohorts of infants with hypoxic ischemic encephalopathy...we have shown that microRNA alterations are present in umbilical cord blood of infants with HIE," Murray said.

A negative predictive value of 99% from miR-181b, in particular, could make this miRNA a useful tool in the clinical setting because it is quantifiable and not subjective, the researchers note.

Now that some of the actors have been identified, future research could examine the roles that they play.

"We will try to learn more about the microRNAs involved and figure out whether they are just innocent bystanders or if they have a role to play in the pathogenesis of the hypoxic ischemic insult," Murray said. She added that cellular and small animal model studies are planned.

"MicroRNAs can be overexpressed or inhibited easily within a cell, so we are very excited about whether they could represent therapeutic targets in the future," she said.

In addition, "we will look at early postnatal samples to see if the effects remain significant at 1 hour post delivery" to potentially expand their clinical utility.

"Extreme Variability"

Commenting on the findings for Medscape Medical News, Sarka Pospisilova, PhD, head of the Center of Molecular Medicine at University Hospital Brno, Czech Republic, noted an increased or decreased expression of various miRNAs in hypoxic conditions has been described previously, for example, in various cancers.

"These microRNAs appear to be promising biomarkers also in fetal hypoxia. However, the problem is the extreme variability of miRNA expression," said Pospisilova, who was not involved in the study.

"The birth itself is a very complex and complicated process with a great impact on the expression of selected biological markers. Thus, in order to establish a reliable biomarker, it is necessary to perform a very extensive study involving hundreds of patients," she added.

The need to start therapeutic hypothermia within 6 hours of birth in infants at risk requires "perfect cooperation" between obstetricians and molecular biologists. In addition, molecular biology laboratories at maternity hospitals should be open 24 hours a day, 7 days a week, "which is far from the standard," she added.

"To conclude, new biomarkers selected in this study are very promising, but another study on a larger cohort is needed to confirm that these miRNAs are reliable biomarkers for clinical use," Pospisilova said.

She and her colleagues conducted a study, published in December, that supports the potential of another microRNA, miR-201, as a marker for acute fetal hypoxia.

The study was funded by the National Children's Research Center, the Health Research Board, a Science Foundation Research Center award, and a Science Foundation Ireland grant. The authors and Dr Pospisilova have disclosed no relevant financial relationships.

JAMA Neurol. Published online December 28, 2019. Abstract

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