Identification of a Pool of miRNA to Improve Early Management of Perinatal Asphyxia and Hypoxic Ischemic Encephalopathy

Study Overview
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Brief Summary
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Hypoxic-ischemic encephalopathy is the most common cause of neurological damage in the neonatal period. It has an incidence of about 1.5-2.5% of livebirths in developed countries. It is associated with a high rate of mortality and morbidity. Major neurological outcomes such as cerebral palsy, mental retardation, learning disabilities, epilepsy occur in approximately 25% of survivors. The diagnostic and prognostic tools currently available for enrollment have limitations and additional reliable biomarkers are needed for all phases of clinical management. Sarnat staging has taken on a role in identifying those infants who may benefit from treatment of hypothermia, resulting in the need for neurological evaluation and staging within 6 hours of life. Therapeutic hypothermia is still the best therapeutic treatment. A new tool in neuroscience research is represented by micro-ribonucleic acid (microRNA) profiling. The presence of microRNAs in blood, urine and saliva and the ability to measure their levels non-invasively has opened new doors in the search for peripheral biomarkers for the diagnosis and prognosis of neurodegenerative diseases and also as possible pharmacological targets. The aim of the present study is to analyze a specific cluster of miRNAs selected from data obtained by macroarray (NGS Pannel) on the entire microRNAome in healthy newborns with normal cord arterial pH value (7.26-7.35) as control cases and in newborns with fetal metabolic acidosis with a pH threshold value lower than 7.12 of the blood gas analysis from cord arterial blood. This latter group will be further stratified into two groups, neonates who will practice therapeutic hypothermia according to current guidelines and a further group who will not practice therapeutic hypothermia. This study will make a further international contribution in evaluating and identifying the potential of microRNAs as diagnostic and prognostic biomarkers in perinatal asphyxia and hypoxic ischemic encephalopathy. Furthermore, the study aims to identify specific microRNA sequences as new possible markers to be used as an additional parameter for the enrollment of therapeutic hypothermia, especially in cases of mild hypoxic-ischemic encephalopathy.

Detailed Description
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Hypoxic-ischemic encephalopathy is the most common cause of neurological damage in the neonatal period. It has an incidence of about 1.5-2.5% of livebirths in developed countries. It is associated with a high rate of mortality and morbidity. About 1 million newborns worldwide die of hypoxic-ischemic encephalopathy. In the neonatal period die between 20 and 50% of asphyxiated infants who develop hypoxic-ischemic encephalopathy. Major neurological outcomes such as cerebral palsy, mental retardation, learning disabilities, epilepsy occur in approximately 25% of survivors. Therapeutic hypothermia is still the best therapeutic treatment. The diagnostic and prognostic tools currently available for enrollment have limitations and additional reliable biomarkers are needed for all phases of clinical management. One of the difficulties in interpretation lies in the identification of mild-grade hypoxic-ischemic encephalopathy which appears to be operator dependent, as well as the timing of the diagnosis. Sarnat staging has taken on a role in identifying those infants who may benefit from treatment of hypothermia, resulting in the need for neurological evaluation and staging within 6 hours of life. This is a relatively short time frame in which it is plausible to think that mild hypoxic-ischemic encephalopathy could become moderate at 6 h. In fact, some studies have retrospectively demonstrated the outcome of infants with mild hypoxic-ischemic encephalopathy defined between 1 and 6 hours of birth showing worse neurodevelopmental outcomes than the data reported in the pre-hypothermia literature. A new tool in neuroscience research is represented micro-ribonucleic acid (microRNA) profiling. Significant numbers of microRNAs have been observed outside cells including various body fluids. MicroRNAs have been detected in plasma, serum, milk, tears, saliva, urine, amniotic fluid, cerebrospinal fluid, and seminal fluid. Despite the instability of most RNA molecules in the extracellular environment, the presence and apparent stability of microRNAs has proven surprising. In particular, they were found to be very stable and resistant to RNases, freezing and pH variations. The presence of microRNAs in blood, urine and saliva and the ability to measure their levels non-invasively has opened new doors in the search for peripheral biomarkers for the diagnosis and prognosis of neurodegenerative diseases and also as possible pharmacological targets. In view of their usefulness, in recent years more and more different microRNAs have been analyzed as possible diagnostic and prognostic markers of perinatal asphyxia but specific sequences with high specificity and sensitivity have not yet been identified as markers of neonatal hypoxia and hypoxic ischemic encephalopathy with the need to perform further confirmatory studies. The aim of the present study is to analyze a specific cluster of miRNAs selected from data obtained by macroarray (NGS Pannel) on the entire microRNAome in healthy newborns with normal cord arterial pH value (7.26-7.35) as control cases and in newborns with fetal metabolic acidosis with a pH threshold value lower than 7.12 of the blood gas analysis from cord arterial blood. This latter group will be further stratified into two groups, neonates who will practice therapeutic hypothermia according to current guidelines and a further group who will not practice therapeutic hypothermia. This study will make a further international contribution in evaluating and identifying the potential of microRNAs as diagnostic and prognostic biomarkers in perinatal asphyxia and hypoxic ischemic encephalopathy. Furthermore, the study aims to identify specific microRNA sequences as new possible markers to be used as an additional parameter for the enrollment of therapeutic hypothermia, especially in cases of mild hypoxic-ischemic encephalopathy. Subsequently, this study will allow to evaluate their potential as new possible pharmacological targets in the pediatric field for hypoxic-ischemic encephalopathy in future preclinical studies as already reported in the literature in various preclinical studies, as therapeutic perspectives in ischemic stroke in adults.

Official Title
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Role of microRNAs as Diagnostic and Prognostic Biomarkers of Neonatal Perinatal Asphyxia and Hypoxic Ischemic Encephalopathy

Conditions
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Hypoxic-Ischemic Encephalopathy, Asphyxia Perinatal

Intervention / Treatment
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* Other: Metabolic acidosis
* Diagnostic Test: microRNA
* Other: Hypothermia


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: newborns with at least 35 weeks of gestational age body weight of at least 1800 g Exclusion Criteria: Withdrawal of informed consent

Ages Eligible for Study
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Minimum Age: 35 Weeks
Maximum Age: 42 Weeks

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Group A<br>15 healthy newborns with umbilical cord arterial pH between 7.26 and 7.35 | Diagnostic Test: microRNA<br>* Evaluation of microRNA levels<br>|
| Group B<br>15 newborns with metabolic acidosis at birth with umbilical cord arterial pH at birth < 7.12 and who do not practice therapeutic hypothermia due to lack of enrollment criteria | Other: Metabolic acidosis<br>* Metabolic acidosis at birth with umbilical cord arterial pH at birth < 7.12<br>Diagnostic Test: microRNA<br>* Evaluation of microRNA levels<br>|
| Group C<br>15 newborns with metabolic acidosis at birth with umbilical cord arterial pH at birth < 7.12 and practicing therapeutic hypothermia in accordance with current guidelines | Other: Metabolic acidosis<br>* Metabolic acidosis at birth with umbilical cord arterial pH at birth < 7.12<br>Diagnostic Test: microRNA<br>* Evaluation of microRNA levels<br>Other: Hypothermia<br>* Therapeutic induced hypothermia refers to a lowering of the central body temperature for therapeutic purposes<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Evaluation of microRNA levels | Quantitative characterization of microRNAs (ng/uL) in healthy neonates and neonates with metabolic acidosis at birth with or without therapeutic hypothermia and who developed hypoxic ischemic encephalopathy | 60 minutes of life | 
| Qualitative evaluation of microRNAs | Characterization of the type of microRNAs in healthy neonates and neonates with metabolic acidosis at birth with or without therapeutic hypothermia and who developed hypoxic ischemic encephalopathy | 60 minutes of life | 
| Evaluation of microRNA levels | Quantitative characterization of microRNAs (ng/uL) in healthy neonates and neonates with metabolic acidosis at birth with or without therapeutic hypothermia and who developed hypoxic ischemic encephalopathy | 3 hours of life | 
| Qualitative evaluation of microRNAs | Characterization of the type of microRNAs in healthy neonates and neonates with metabolic acidosis at birth with or without therapeutic hypothermia and who developed hypoxic ischemic encephalopathy | 3 hours of life | 
| Evaluation of microRNA levels | Quantitative characterization of microRNAs (ng/uL) in healthy neonates and neonates with metabolic acidosis at birth with or without therapeutic hypothermia and who developed hypoxic ischemic encephalopathy | 72 hours of life | 
| Qualitative evaluation of microRNAs | Characterization of the type of microRNAs in healthy neonates and neonates with metabolic acidosis at birth with or without therapeutic hypothermia and who developed hypoxic ischemic encephalopathy | 72 hours of life | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Predictive role of microRNAs | To assess the role of microRNAs as marker of perinatal asphyxia | 60 minutes of life | 
| Predictive role of microRNAs | To assess the role of microRNAs as markers of mild hypoxic ischemic encephalopathy | 60 minutes of life | 
| Predictive role of microRNAs | To assess the role of microRNAs as markers of perinatal asphyxia | 3 hours of life | 
| Predictive role of microRNAs | To assess the role of microRNAs as markers of mild hypoxic ischemic encephalopathy | 3 hours of life | 
| Predictive role of microRNAs | To assess the role of microRNAs as markers of perinatal asphyxia | 72 hours of life | 
| Predictive role of microRNAs | To assess the role of microRNAs as markers of mild hypoxic ischemic encephalopathy | 72 hours of life | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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newborn, microRNA, asphyxia, encephalopathy, hypothermia