Medires Publishers - Article

Abstract

Background: Perinatal asphyxia remains a significant cause of morbidity and mortality globally, representing the 3rd most common cause of neonatal death after pre-term births and severe infections. Brain injury following severe asphyxia insult usually end up with Hypoxic Ischemic Encephalopathy associated with elaboration of biomarkers of neuronal injury one of which is S100β. The aim of this study was to determine the serum levels of S100β protein in asphyxiated and non-asphyxiated neonates aged 0-6hrs.

Materials and Methods: comparative, cross-sectional study of 100 subjects (50 subjects with neonatal asphyxia, and 50 controls) over a seven-month period to determine the serum levels of S100β, using ELISA kit, in asphyxiated and non-asphyxiated newborns delivered at the University of Nigeria Teaching Hospital (UNTH), Ituku-Ozalla, Enugu.  Data was collected and analyzed using Statistical Package for Social Sciences SPSS version 20. Significant levels were assumed at p-values less than 0.05.

Results: The mean cord blood S100β levels (213pg/ml) of asphyxiated babies was higher than the mean (45.68pg/ml) obtained in non-asphyxiated babies(p=0.000).

Conclusion: Asphyxiated neonates have significantly higher S100β protein level than non-asphyxiated neonates.

Introduction

The neonatal period is a very turbulent time for all babies. With the transition from the uterine to the external environment, a number of physiological adjustments occur.1,2 Notably in-utero, there is appreciable decrease in the partial pressure of carbon dioxide.3 Adequate buffering mechanisms prevent significant fluctuations in maternal arterial pH.3 Optimal foetal metabolism gives rise to acids which are neutralized to maintain extracellular pH within a critical range. Foetal hypoxia is seen in pathologies that limit maternal oxygenation, maternal perfusion of placenta or return of oxygenated blood from the placenta to the fetus. Metabolism of substrates then continues along the anaerobic pathway with the release of such organic acids as lactic acid. Foetal distress and poor APGAR score would give rise to metabolic acidosis.3 Therefore, small fluctuations in pH may cause significant derangement in foetal function.

Perinatal asphyxia is a condition characterized by an impairment in the exchange of respiratory gases (oxygen and carbon dioxide) giving rise to hypoxaemia and hypercapnia, accompanied by metabolic acidosis in the period immediately before, during, or after the birth process.4 The basic mechanism involved in perinatal asphyxia ranges from an interruption in blood flow and gaseous exchange in the foetus during the prenatal period to an inability of the newborn to commence and maintain adequate respiration following delivery.5

Despite improvement, in perinatal care in the past years, asphyxia is still a significant cause of morbidity and mortality in newborns. Worldwide, it ranks as the third most common cause of neonatal death after preterm birth and severe neonatal infections.6 Perinatal asphyxia is recorded to be the fifth commonest cause of under-five mortality worldwide7  and accounts for approximately 23% of all newborn deaths.7 In 2010, the World Health Organization noted that between 4 and 9 million newborns suffered  from birth asphyxia with an estimated 1.2 million deaths and about a similar number  developed severe neurological deficits.7 In the United States and other advanced countries, the incidence of perinatal asphyxia is 1 to 4 cases per 1000 births.7  Developing countries, on the other hand, have recorded high rates of birth asphyxia ranging from 4.6/1000 live births in Cape Town to 7 to 26/1000 live births in Nigeria.7 Although many neonates with perinatal asphyxia usually recover quickly and proceed  to live normal lives, some go on to have hypoxic-ischaemic organ dysfunction involving the heart, lung, kidney and commonly the brain. A large proportion, however, die from hypoxic ischaemic encephalopathy due to cortical lesions in the brain.7

Hypoxic Ischaemic Encephalopathy (HIE) is a brain dysfunction or injury caused by a reduction in blood flow (ischemia) and supply of oxygen to the brain (hypoxia).8 The injury to the brain and other essential organs results when the precipitating event is of an adequate severity.8 Robertson et al defined HIE as “an acute non-static encephalopathy caused by intrapartum or late antepartum brain hypoxia and ischemia.” 9  The prevalence of HIE ranges from 1.5 to 2.5/1000 live births in developed countries.10 Given the higher frequency of avoidable complications of labour and the limited availability of specialized care during delivery in developing countries, intra-partum causes of perinatal asphyxia constitute a large percentage of cases, in contrast to antepartum causes documented in advanced countries.11  Where perinatal asphyxia occurs with progression to hypoxic ischemic encephalopathy, there is deranged cellular metabolism, followed by irreversible neuronal damage and consequent death.12 The resultant manifestations range from hyper-alertness and weak reflexes as indicated in Sarnat stages 1and 2 diseases to seizures, stupor and coma as seen  in Sarnat stage 3 disease.12  The complications of hypoxic ischaemic encephalopathy  are associated with unfavourable  motor, sensory, cognitive and behavioural outcomes. Moreover, HIE is a modifiable condition making it important to ascertain its presence or absence.

Basically, the aim in postnatal therapeutic treatment of perinatal asphyxia is to delay secondary neuronal injury.  After birth and resuscitation, the neonatal brain undergoes a period of partial recovery which is followed by a latent phase of about 1-6hours. In moderate to severe encephalopathy, the brain deteriorates to a phase of secondary injury characterized by complete mitochondrial energy production failure, cytotoxic edema, cell death and clinical deterioration. This takes place about 6-15hours after the hypoxic-ischemic event occured.13 This latent phase of 1-6 hours allows a therapeutic window during which the institution of therapy may prevent secondary injury.

Neuro-protective treatments are measures employed to prevent the progression of primary neuronal injury to secondary injury.  They reduce neuronal injury and improve the outcome of asphyxiated babies at risk of hypoxic ischaemic encephalopathy.  Most commonly applied is the therapeutic hypothermia.14 Most recent meta-analysis documented the efficacy of therapeutic hypothermia in term infants with moderate to severe encephalopathy especially when administered within 3-6 hours of birth.

Jacobs et al,15 in a Cochrane Review in 2013, noted that therapeutic hypothermia improves neurodevelopmental outcomes in patients with moderate-severe encephalopathy. Guillet et al16 noted that infants who were cooled within 3hours of birth had better neurologic outcomes compared with those whose cooling commenced between 3-6hours. The role of other emerging neo-adjuvant therapies like erythropoietin, osteopontin,17 melatonin,18 prophylactic barbiturates,19 antioxidants such as Nitric oxide, anti-inflammatory therapy, in improving neuro developmental outcome of asphyxiated newborns are still being evaluated.

The current standard of care of HIE is to optimize newborns ≥35weeks gestation with moderate-severe hypoxic ischemic encephalopathy by administering hypothermia as soon as possible after the hypoxic event occurs between 1-6hours.16   Guillet et al20 indicated that cooling babies before 3hours of age achieves the best neuroprotective results. Also in the TOBY (TOtal Body hYpothermia) study, the induction of hypothermia yielded best result in children who were treated within the 4hours post-partum.21   The use of the hypothermia protocol requires that prompt assessment of the severity of HIE as well as serial clinical assessments during the first 6 hours of commencing treatment be done. This could be feasible in centres in developed world, but may not be promptly instituted in low and middle-income countries where the therapeutic time-window for administering beneficial cooling may have been exceeded due to dearth of objective protocol in management. This may be due to delayed hospital admissions, prolonged/obstructed labour and lack of neonatal transport facilities. By the time the evidence of moderate to severe encephalopathy became clinically obvious, the latent period during which neuroprotective measures should have been instituted would have elapsed. Even in standard centres where detailed neurological examination of asphyxiated newborn using the criteria defined by Sarnat and Sarnat during 1st 48 hours of life is done, as recommended in treatment protocol, it may be defective in instances where newborns are either sedated or restless. In the above instance, a progression from Sarnat stage 1 to 2 diseases may be picked up later than the proposed time window.

Clinical examination, brain magnetic resonance imaging (MRI) and electroencephalography (EEG) are tools used to detect brain injury. Their major limitation is the inability to detect early neuronal brain injury in asphyxiated babies.22 The clinical signs characteristic of brain injury  evolve over  six hours to days as the effects of  the initial insult progress.22 Brain MRI scans, on the other hand, cannot be relied upon in the first 24-hours as indicators of brain injury can be delineated on second or third day when structural changes in the brain are obvious.23 Also in our environment it is difficult to move critically ill neonates for imaging to centers where equipment are available due to logistic reasons.  Electroencephalography shows electrical changes in the brain only after an extensive significant neuronal injury has occurred and is thus mainly used to predict outcomes.22 Furthermore, the equipment and interpretative expertise may not be readily available in most neonatal units in the country.

As a result of the irreversible nature of brain neuronal injury, the narrow time window for neuro-protective interventions and the limitation of currently available tools for assessing neuronal injury, it is necessary to identify early biomarkers which can predict the risk of neuronal injury even before it overtly occurs. This would identify those who would benefit from early intervention.

A biomarker is a measurable indicator or biological index that is objectively quantified and assessed as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapy.24 Various biomarkers of HIE have been proposed. These include S100β protein, Neuron specific enolase, Glial fibrillary acidic protein, Ubiquitin Carboxyl-terminal Hydrolase (UCH-L1), Creatine Kinase.25 Many of these substances are elaborated  by different cell types in the brain as soon as hypoxic-ischemic insult occurs.25 Of these biomarkers, S100β  has been noted by many workers25,26,27 to be very useful in diagnosing brain injury following neuronal insult as may occur in perinatal asphyxia.27 Furthermore, its level increases with  severity of asphyxia. It is, thus, important to find out the relationship between the status of S100β in asphyxiated newborns and their progression to encephalopathy. This will assist the clinician in early screening of infants for brain injury as well as monitoring disease progression with the aim of instituting time sensitive neuro-protective interventions.  The findings will add to the awareness and clinical utilization of S100β in the identification of neonates who may develop hypoxic ischemic encephalopathy following asphyxia. It will also form a template on which further studies can be conducted on the clinical problem.

Materials and Methods

Study Site and Design

The study was carried out in the Obstetric Unit of the University of Nigeria Teaching Hospital (UNTH), Ituku-Ozalla, Enugu. It was cross sectional done over a period of 7 months (between  April 2019 and October 2019). The study subjects were newborns from birth to six hours of life who had perinatal asphyxia and were admitted into the Newborn Special Care Unit of UNTH. They were full term neonates which, when defined by WHO, were live births delivered between 37 completed weeks and 42 weeks of gestation. The controls were non-asphyxiated newborns matched for age and sex who were delivered in UNTH and were being nursed either in the labour ward following vaginal delivery or the postnatal ward after caesarian section.

Sample Size Determination

The sample size was calculated using the formula114

N is sample size

Where Zα/2 is the critical value of the Normal distribution at α/2. For a confidence level of 95%, the critical value is 1.96,

Zβ is the critical value of the Normal distribution at β. For a power of 90%, the critical value is 2.33,

σ is the population standard deviation which is 0.044111, and σ2 is the variance

ꝺ   is the standard error and placed at 0.04.

Calculation of sample size based on S100β

The standard deviation of S100β for controls in a previous study105 was 0.044.

Substituting these figures into the formula:

Thus, 45 babies with asphyxia and 45 controls matched for sex and age were recruited into the study. Considering a dropout rate of 10%, 5 additional babies were recruited to each arm to make up for either possible incomplete data or late withdrawals. This brought the sample size to 50 subjects and 50 controls. Therefore, fifty subjects were recruited from each arm of the study population.

Sampling Method

The researcher was informed by the Obstetrics ward nurses of impending deliveries whether vaginal or caesarean. Once the researcher was informed, the mothers were identified, approached and the reason for the study explained to them. Consent was obtained from all mothers not later than first stage of labour. Where the need arose, the researcher assisted in the resuscitation of the babies. Relevant data such as age, place of domicile (urban or rural) were collected from the mother. Other data included occupation of the parents and highest educational qualification which were used in assigning the social class to the child using the method proposed by Oyedeji. (Appendix VII). The classes were grouped into upper (I &II), middle (III), low (IV &V). The first day of the last menstrual period was obtained to estimate gestational age of the baby.  Further information obtained included the use of tobacco as well as other maternal illnesses such as diabetes and hypertension. This and other relevant data were entered into the proforma for the subject. Babies who did not cry after birth and met the inclusion criteria for subjects were enrolled into the study. The APGAR score of the term babies was assessed by the researcher while in the labour ward. APGAR scores 0 to 3, and 4 to 6 were considered to be severe and moderate asphyxia respectively while term babies who had APGAR scores of 7-10 and matched for sex were enrolled as controls. 

Before delivery, four Howard Kelly forceps for double clamping of the umbilical cord were included with the surgical instruments to be used for the delivery process. These were previously sterilized by the hospital Central Sterile Supplies Department (CSSD).  Two Howard Kelly forceps labelled ‘a’ and ‘b’ were used to clamp the umbilical cord at the maternal end. Two other Howard Kelly forceps labelled ‘c’ and ‘d’ were used to clamp the umbilical cord at the subject’s end, about 20cm from the initial clamps ‘a’ and ‘b’ (Appendix VIII). With the baby kept at the same vertical height as the placenta without milking, the cord was then severed using operative scissors in between clamps ‘a’ and ‘b’ and clamps ‘c’ and ‘d’. This left a resultant independent length of umbilical cord with clamps ‘b’ and ‘c’ at each end each time with some amount of cord blood trapped for sampling.  This was placed into a small storage container designated for the study and kept at room temperature.  In a double clamped section of umbilical cord, blood could stay at room temperature for up to 60 minutes without clotting. Following this, the section in between the clamped portion of the cord was removed from the storage box. Subsequently, the Howard Kelly forceps on one end of the section of the cord was removed. The umbilical arteries were identified as the smallest of the three visible vessels, and which had a non-collapsible lumen. A size 21-guage needle with syringe was used to draw blood from the artery. A needle with the syringe attached was placed at 30 degree to the artery and pierced to avoid going through the back of the vessel into the vein. About 1ml of blood was withdrawn each time and placed into Na citrate bottle for arterial blood pH estimation.

The umbilical arterial blood pH estimation was done by the researcher within 30mins of collection using an iSTAT machine (Abbott i-STAT MN:300. Abbott point of care Inc. II USA).  Using a pipette, a drop of blood was withdrawn from the Na citrate bottle, inserted into the istat cartridge and loaded on the istat machine. The result of the pH was obtained within two minutes and documented.

Once the baby was delivered and assessed to have moderate-severe perinatal asphyxia (APGAR score < 6 in 5minutes and arterial blood pH of less than 7.25), blood sample was collected through a peripheral vein using 24G intravenous cannula within six hours. The blood was collected in a new sterile plain sample bottle and allowed to stand in a sample bottle rack a2qfor 15 to 20minutes to enable the serum to separate. Thereafter the blood sample was centrifuged (CSN-80, Medfield Equipment and Scientific Ltd, England) at 3000rpm for 15 minutes in the side laboratory of Newborn Special Care Unit to separate the serum.  The serum obtained was carefully extracted and transferred to another sterile plain sample bottle using a pipette. The sample was labeled and stored in a freezer in the hematology laboratory. Samples were transported in ice packed containers to the laboratory where S100β is assayed using ELISA kit under the guidance of a Consultant Chemical pathologist.

Peripheral venous blood sample of non-asphyxiated controls was also collected for S100β estimation using the same procedure.

Following stabilization, detailed general and systemic examinations were carried out for both subjects and controls. Those with obvious congenital anomalies were excluded. The occipito-frontal circumference, weight and length measurements were documented.

Birth weight: The weight of each newborn was measured using a weighing scale. (SECA infant weighing scale, model 725,). The scale was calibrated on each day using a known weight. The scale was standardized at each weighing by ensuring it returns to the zero mark. To eliminate parallax error, readings of measurement were obtained from a position directly in front of the scale. Neonates were weighed with all clothing and diapers removed. Birth weight was measured to the nearest 0.1kg. Birth weight was low if it is less than 2500g, normal if it is between 2500g and less than 4000g and macrosomic if greater than 4000.

Length: The length was measured using a length mat Seca 210 with a measuring range of 10-99cm.  Each baby was placed supine on the measuring mat with the head in contact with the headboard. The trunk and pelvis were aligned on the mat and the baby’s legs were held gently but firmly over the knees to keep the legs extended by an assistant. The toes pointed directly upward with the soles of the feet perpendicular to the horizontal back piece of the measuring device. The footboard was moved firmly against the sole of the feet and the measurement was read to the nearest 0.1cm.

Occipito-frontal circumference(OFC): OFC was measured using a standard inelastic measuring tape. The circumference of the head was taken from the occiput to the most anterior aspect of the frontal bone which is the glabella, 2cm above the supraorbital ridge with the examiner standing behind the patient.

Also the Modified Ballard Score was applied to verify the gestational age of the subjects. This was then cross-checked against the obtained gestational age from the maternal last menstrual period. Where the gestational age could not be ascertained from the LMP, the estimated gestational age with the Ballard scoring was used.

HIE was assessed using the method proposed by Sarnat and Sarnat (Appendix VI).

Ethical Approval

Ethical approval was obtained from the Health Research and Ethics Committee of UNTH.

Consent

Details of the study were explained to the parents of each baby. Neonates were enrolled if consent was obtained from their parents.

Data Analysis

The data was entered into Microsoft Excel spread sheet and data cleaning was done to ensure that they were properly entered into the spreadsheet. The data obtained was analyzed using IBM Statistical Package for Social Sciences (SPSS) Version 20 statistical software program. Descriptive statistics such as frequencies and percentages were used to summarize categorical variables. Kolmogorov Sminorv test of normality was done to check if the continuous variables (Mean S100β and pH) deviated from normal distribution. Parametric statistical test was employed for the pH since it follows normal distribution while, non-parametric statistics was used for S100β due to non-normality in distribution.

Results

One hundred and five babies were recruited for the study. However, four subjects and one control were dropped as their blood samples were lysed and not suitable for analysis. Fifty subjects and fifty controls were eventually enrolled into the study. Of each of those, 44(88%) were males and 6(12%) were females giving a male to female ratio of 7.3:1. Majority (74%) of the subjects were in social classes 4 and 5 while most of the controls (90%) were in social classes 3 and 4.  Twenty-four(48%) of the subjects and 23(46%) of the controls were in social class 4.

The mode of delivery of the study participants is presented in Table I. A greater number of the subjects (58%) were delivered by emergency caesarean section while vaginal delivery was the commonest (74%) mode of delivery for the control group (p=0.002).

The range of the weight of the subjects was 2.4 to 4.7kg (mean 3.67± 0.61kg). The range of the weight of the controls was 3.00 to 5.30kg (mean 3.87± 0.38 kg). 

Table II shows the distribution of subjects according to severity of asphyxia using APGAR scores. Thirty out of 50(60%) subjects had moderate asphyxia with APGAR scores 4 to 6 while 20 out of 50(40%) had severe asphyxia with APGAR 0 to 3.

Thirty-three subjects (66%) had pH within the mild asphyxia range. Nine subjects (18%) had their pH in the moderate category while 8(16%) had their pH in the severe category as indicated in Table III below.

Serum S100β levels of subjects ranged from 28 to 1188pg/ml (mean is 213.02± 281.43pg/ml) while serum S100β levels of controls ranged from 4 to 69pg/ml (mean 45.68± 14.41 pg.ml). The difference in mean values of the two groups was statistically significant (p <0.001).

Among the control group, 47(94%) had S100β levels within safe limits while three (6%) had values within the range at risk of brain injury. None of the controls had serum S100β levels at the pathological level. Among the subjects, 11(22%) were within safe limit, 4(8%) had their S100β at risk while 34(68%) subjects were within the pathological limit. This is presented in Table IV.

Table I: Sociodemographic characteristics and mode of delivery of study population

 *Significant P<0.05

Table II: Distribution of subjects according to degrees of asphyxia using APGAR

Table III: Distribution of subjects according to degree of Asphyxia defined by pH

Table IV: Categorization of S100β Serum values among study population

Fisher’s exact Chi-square: 64.889; p=<0.001*

*Significant P<0.05

Discussion

A total of fifty asphyxiated newborns with their fifty non-asphyxiated sex matched control group were studied. More males met the inclusion criteria for this study. Though male dominance in this study cannot be explained, it has been documented that males are more prone to neonatal asphyxia than their female counterparts.28

Twenty-nine (58%) out of the 50 subjects were delivered by emergency caesarean section as against 13(26%) of the controls. The dominant mode of delivery in the subjects was caesarean section due to the fact that the subjects had foetal distress. This is similar to findings documented by Martins et al.29

With APGAR scoring, subjects were categorized into two groups – moderate and severe asphyxia. But with umbilical cord blood pH, same subjects were categorized into mild, moderate and severe asphyxia. APGAR scoring, being subjective, may have allowed a number of subjects to be categorized as moderate when identified as mild asphyxia using pH categorization.  Also some subjects that were found to have severe asphyxia were noted to have moderate asphyxia using pH criteria.  This reflects the possible excessive categorization of babies into having perinatal asphyxia using a subjective parameter in assessing perinatal asphyxia as with the APGAR scoring system. Categorization of asphyxia using pH reflects the metabolic effect of hypoxic injury and as such may give a more objective assessment of injury due to asphyxia.

In this study the range of S100β in subjects (28-1188pg/ml) was significantly higher than in controls(4-69pg/ml). The mean serum S100β level was also significantly higher in asphyxiated babies(213pg/ml) compared with 45.68pg/ml found with non-asphyxiated babies. This difference is attributable to the metabolic events which occur in asphyxiated neonates following hypoxic injury which leads to neuronal injury with consequent brain damage. These events lead to the release of S100β from damaged cerebral astrocytes following ischaemic brain injury.  Earlier studies30,31,32   have also shown increases in serum S100β levels in asphyxiated babies when compared with those without asphyxia.

Subjects with values between 66-75pg/ml fell within the indeterminate group and so are described in this study as ‘at risk’ of HIE as their brain injury may not have been severe enough to cause a rise in serum S100β to pathological limits. The finding of this group is not clear though it calls for closer monitoring of such newborns for an early initiation of neuroprotective interventions when the need arises. Such timely initiation may avert attendant impaired neurodevelopment, over time, which they may be prone to if not timely monitored.

The mean serum S100β level of 213pg/ml found in this study amongst newborns with moderate-severe HIE is above the value of 120pg/ml reported as normal by Martin et al29 in Brazil. It is known that HIE is associated with glial cell damage from where these biomarkers are elaborated. The raised levels of S100β further supports the observation that brain injury increases the levels of the biomarkers in asphyxiated newborns. The different cut-off values of S100β observed in this study compared to others could be attributed to different assay techniques (ELISA) used. However, most studies supported the fact that raised serum S100β levels above cut-off values predicted brain injury irrespective of the assay method used. 

The fact that the number of study participants with raised S100β is higher in asphyxiated newborns than non-asphyxiated groups corroborates the finding that asphyxia is associated with neuronal injury with subsequent elaboration of S100β biomarker from the glial cells of the brain.  This observation is buttressed in this study where about 7 out of every 10 asphyxiated newborns have serum S100β levels above normal values with most attaining pathological limits. Though this finding may not seem significant on face value, its true burden becomes obvious when viewed in the context of high rate of asphyxiated deliveries in Nigeria. From this study, approximately 76% of asphyxiated babies have greater likelihood of raised S100β than their non-asphyxiated counterparts using a cut-off value of S100β greater than 75pg/ml. It is known that values above pathologic limits are associated with consequent significant impairment in neurodevelopment.25,26 implying a tendency to brain injury with increasing serum levels of S100β.

Conclusion

The mean serum S100β levels of asphyxiated term newborns was significantly higher than the mean serum S100β levels of non-asphyxiated newborns. There is no significant difference between the serum S100β levels of asphyxiated term newborns delivered by caesarean section and those delivered per vagina. No association was found between serum S100β levels and gender differences.

Recommendations

The recommendations based on the findings of this study are as follows:

• Serum S100β may be adopted to be part of routine examination of newborn babies
• Using the determined cut off points, serum S100β can be used to predict brain injury in asphyxiated newborns

Competing Interests

The authors have declared that no competing interests exists.

Author’s Contribution

Principal researcher, Dr Maduka designed the work, collected the data and wrote up the article.

References

1. Leone, T. A., & Finer, N. N. (2006). Fetal adaptation at birth. Current Paediatrics, 16(4), 269-274.
   View at Publisher | View at Google Scholar
2. Duncan, H. P. (1999). Physiology of fetal adaptation at birth. Current Paediatrics, 9(2), 118-122.
   View at Publisher | View at Google Scholar
3. Omo-Aghoja, L. (2014). Maternal and fetal acid-base chemistry: a major determinant of perinatal outcome. Annals of medical and health sciences research, 4(1), 8-17.
   View at Publisher | View at Google Scholar
4. Haider, B. A., & Bhutta, Z. A. (2006). Birth asphyxia in developing countries: current status and public health implications. Current problems in pediatric and adolescent health care, 5(36), 178-188.
   View at Publisher | View at Google Scholar
5. Ezechukwu, C. C., Ugochukwu, E. F., Egbuonu, I., & Chukwuka, J. O. (2004). Risk factors for neonatal mortality in a regional tertiary hospital in Nigeria. Nigerian Journal of clinical practice, 7(2), 50-52.
   View at Publisher | View at Google Scholar
6. Antonucci, R., Porcella, A., & Pilloni, M. D. (2014). Perinatal asphyxia in the term newborn. Journal of Pediatric and Neonatal Individualized Medicine (JPNIM), 3(2), e030269-e030269.
   View at Publisher | View at Google Scholar
7. Onyearugha, C. N., & Ugboma, H. A. A. (2010). Severe birth asphyxia: risk factors as seen in a tertiary institution in the niger delta area of nigeria. Continental Journal of Tropical Medicine, 4, 11.
   View at Publisher | View at Google Scholar
8. Airede, A. I. (1991). Birth asphyxia and hypoxic-ischaemic encephalopathy: incidence and severity. Annals of tropical paediatrics, 11(4), 331-335.
   View at Publisher | View at Google Scholar
9. Robertson, C. M., & Perlman, M. (2006). Follow-up of the term infant after hypoxic-ischemic encephalopathy. Paediatrics & child health, 11(5), 278-282.
   View at Publisher | View at Google Scholar
10. Kurinczuk, J. J., White-Koning, M., & Badawi, N. (2010). Epidemiology of neonatal encephalopathy and hypoxic–ischaemic encephalopathy. Early human development, 86(6), 329-338.
    View at Publisher | View at Google Scholar
11. Aslam, H. M., Saleem, S., Afzal, R., Iqbal, U., Saleem, S. M., Shaikh, M. W. A., & Shahid, N. (2014). Risk factors of birth asphyxia. Italian journal of pediatrics, 40, 1-9.
    View at Publisher | View at Google Scholar
12. Massaro, A. N., Tsuchida, T., Kadom, N., El-Dib, M., Glass, P., Baumgart, S., & Chang, T. (2012). aEEG evolution during therapeutic hypothermia and prediction of NICU outcome in encephalopathic neonates. Neonatology, 102(3), 197-202.
    View at Publisher | View at Google Scholar
13. Chalak, L. F., Sánchez, P. J., Adams-Huet, B., Laptook, A. R., Heyne, R. J., & Rosenfeld, C. R. (2014). Biomarkers for severity of neonatal hypoxic-ischemic encephalopathy and outcomes in newborns receiving hypothermia therapy. The Journal of pediatrics, 164(3), 468-474.
    View at Publisher | View at Google Scholar
14. Maoulainine, F. M. R., Elbaz, M., Elfaiq, S., Boufrioua, G., Elalouani, F. Z., Barkane, M., & El Idrissi Slitine, N. (2017). Therapeutic hypothermia in asphyxiated neonates: Experience from neonatal intensive care unit of University Hospital of Marrakech. International journal of pediatrics, 2017(1), 3674140.
    View at Publisher | View at Google Scholar
15. Jacobs SE, Bergs M, Hunt R,Tarnow-Mordi WO, Inder TE, Davis PG. et al. Cooling for Newborns with Hypoxic Ischemic Encephalopathy. The Cochrane Database of Syst Rev. 2013;(1):CD003311. Published 2013 Jan 31
    View at Publisher | View at Google Scholar
16. Guillet, R., Edwards, A. D., Thoresen, M., Ferriero, D. M., Gluckman, P. D., Whitelaw, A., & Gunn, A. J. (2012). Seven-to eight-year follow-up of the CoolCap trial of head cooling for neonatal encephalopathy. Pediatric research, 71(2), 205-209.
    View at Publisher | View at Google Scholar
17. O'Regan, A., & Berman, J. S. (2000). Osteopontin: a key cytokine in cell‐mediated and granulomatous inflammation. International journal of experimental pathology, 81(6), 373-390.
    View at Publisher | View at Google Scholar
18. Alonso-Alconada, D., Álvarez, A., Arteaga, O., Martínez-Ibargüen, A., & Hilario, E. (2013). Neuroprotective effect of melatonin: a novel therapy against perinatal hypoxia-ischemia. International journal of molecular sciences, 14(5), 9379-9395.
    View at Publisher | View at Google Scholar
19. Hall, R. T., Hall, F. K., & Daily, D. K. (1998). High-dose phenobarbital therapy in term newborn infants with severe perinatal asphyxia: a randomized, prospective study with three-year follow-up. The Journal of pediatrics, 132(2), 345-348.
    View at Publisher | View at Google Scholar
20. Thoresen, M., Tooley, J., Liu, X., Jary, S., Fleming, P., Luyt, K., ... & Sabir, H. (2013). Time is brain: starting therapeutic hypothermia within three hours after birth improves motor outcome in asphyxiated newborns. Neonatology, 104(3), 228-233.
    View at Publisher | View at Google Scholar
21. Azzopardi, D., Brocklehurst, P., Edwards, D., Halliday, H., Levene, M., Thoresen, M., ... & TOBY Study Group. (2008). The TOBY Study. Whole body hypothermia for the treatment of perinatal asphyxial encephalopathy: a randomised controlled trial. BMC pediatrics, 8, 1-12.
    View at Publisher | View at Google Scholar
22. Ling, X. B., & Sylvester, K. G. (2011). Proteomics and biomarkers in neonatology. NeoReviews, 12(10), e585-e591.
    View at Publisher | View at Google Scholar
23. Steinlin, M., Dirr, R., Martin, E., Boesch, C., Largo, R. H., Fanconi, S., & Boltshauser, E. (1991). MRI following severe perinatal asphyxia: preliminary experience. Pediatric neurology, 7(3), 164-170.
    View at Publisher | View at Google Scholar
24. Biomarker Definitions Working Group, Biomarkers and surrogate endpoints; preferred definitions and conceptual framework, Clin Pharmacol and Thers 2001;69:89-95
    View at Publisher | View at Google Scholar
25. Nagdyman, N., Kömen, W., Ko, H. K., Müller, C., & Obladen, M. (2001). Early biochemical indicators of hypoxic-ischemic encephalopathy after birth asphyxia. Pediatric research, 49(4), 502-506.
    View at Publisher | View at Google Scholar
26. Qian, J., Zhou, D., & Wang, Y. W. (2009). Umbilical artery blood S100β protein: a tool for the early identification of neonatal hypoxic-ischemic encephalopathy. European journal of pediatrics, 168, 71-77.
    View at Publisher | View at Google Scholar
27. Elsisy, A. A., El-Lahoney, D. M., El-Meshad, G. M., Helwa, M. A., & Barseem, N. F. (2017). Umbilical cord S100B protein in neonatal hypoxic-ischemic encephalopathy. Menoufia Medical Journal, 30(2), 588-594.
    View at Publisher | View at Google Scholar
28. Hill, C. A., & Fitch, R. H. (2012). Sex differences in mechanisms and outcome of neonatal hypoxia‐ischemia in rodent models: implications for sex‐specific neuroprotection in clinical neonatal practice. Neurology research international, 2012(1), 867531.
    View at Publisher | View at Google Scholar
29. Martins, R. O., Rotta, N. T., Portela, L. V., & Souza, D. O. (2006). S100B protein related neonatal hypoxia. Arquivos de Neuro-psiquiatria, 64, 24-29.
    View at Publisher | View at Google Scholar
30. Nagdyman, N., Kömen, W., Ko, H. K., Müller, C., & Obladen, M. (2001). Early biochemical indicators of hypoxic-ischemic encephalopathy after birth asphyxia. Pediatric research, 49(4), 502-506.
    View at Publisher | View at Google Scholar
31. Sofijanova, A., Piperkova, K., & Jordanova, O. (2012). S100B Protein in Serum as a Prognostic Marker for Brain Injury in Term Newborn Infants with Hypoxic Ischemic Encephalopathy-New Strategy for Early Brain Damage. Maced J Med Sci, 5(4), 416-422.
    View at Publisher | View at Google Scholar
32. Zaigham, M., Lundberg, F., & Olofsson, P. (2017). Protein S100B in umbilical cord blood as a potential biomarker of hypoxic-ischemic encephalopathy in asphyxiated newborns. Early human development, 112, 48-53.
    View at Publisher | View at Google Scholar