Role of Human Recombinant Erythropoietin ( rHuEPO ) in Perinatal Asphyxia-a randomized controlled trial

Background : Perinatal asphyxia is an insult to the fetus or newborn infant due to lack of oxygen (hypoxia) and/or a lack of perfusion (ischemia) to various organs, which will manifest as difficulty in establishing spontaneous respiration evident by delayed cry after birth, at least after one minute. World-wide, perinatal asphyxia accounts for about 900,000 deaths each year. In Bangladesh it is a major cause of neonatal death. A substantial proportion of the children that survive suffer late effects such as cerebral palsy and epilepsy. Objective : To determine the efficacy of erythropoietin in improving the neurological outcome of term neonates with perinatal asphyxia (HIE stage II and III). Materials and methods : A Randomized Controll Trial was carried out in the Neonatal ward and NICU of Dhaka Shishu Hospital from 1st April 2014 to 30th Sep 2015. A total 68 neonates with perinatal asphyxia (both HIE stage II and III) who fulfill the inclusion criteria were enrolled and randomly assigned to intervention group (n=35) and control group (n=33). Intervention group received rHuEPO 300500 U/kg/dose daily subcutaneously for 5 days within first 48 hours of birth along with the standard treatment protocol and control group received standard treatment protocol only. Results : Baseline clinical characteristics, USG of brain during hospital stay were almost similar in both groups. Statistically significant effect was noted in seizure control, tolerance of oral feeding, hospital stay and neurological outcome at 3 months of age (p=008). USG of brain at 3 months of age also improved significantly (p=0.027). Conclusion : This study demonstrates the effectiveness of early administration of rHuEPO to term neonates with moderate to severe asphyxia, beneficial effect on short term outcomes like seizure control, tolerance of oral feeding and neurological outcome at 3 months of age. A large multicenter study would be done for further evaluation of these findings.


Introduction
Perinatal asphyxia is an important cause of acute neurologic injury, occurring in 2 to 3 cases per 1000 term live births in developed countries, with a higher incidence in less developed countries including Bangladesh. 1 Acute neurologic injury i.e.Hypoxic-ischemic encephalopathy (HIE) is an important cause of death and disability in full-term infants.The incidence of moderate or severe hypoxicischemic encephalopathy has remained essentially unchanged over the past 20 years, at 1.5 to 2 per 1000 live births in the United States.
Primary energy failure Reperfusion 6-24 hours 24-40 hours Latent phase Secondary energy fai l ure "window of opportunity"

Original Article
Hypoxia leads to Phase 1 primary energy failure.
Phase 2, Laten Phase -A short time after reoxygenation, aerobic metabolism and cell functions are reestablished.However, as a result of a cascade of cellular mechanisms, 7,8 after this «latent phase» of 6-24 hours mitochondrial energy production again begins to fail.
Phase 3-Secondary energy failure lasts for 24-48 hours after the hypoxic event.The damage that occurs during this phase is considerable. 9uroprotective treatment targeting the «latent phase» may limit the secondary neuron damage due to perinatal asphyxia. 5ythropoietin (EPO) is a haematopoietic hormone that also has receptors in the brain, 10 having an anti-inflammatory effect, EPO can reduce brain damage after hypoxia through reduced nitric oxide (NO) production, 11 inhibited glutamate toxicity 12 and reduced lipid peroxidation. 13Neuronal anti-apoptotic mechanisms, angiogenesis and neurogenesis are also stimulated and modulated. 14ythropoietin (EPO) as a neuroprotective agent has been studied extensively both in animal and human.Erythropoietin and its receptors were up regulated following hypoxic injury and were correlated positively with outcomes.Studies have demonstrated anti apoptotic action in neurons following hypoxic injury where NO levels were reduced with the use EPO. 15me previous studies occur in Romania, Japan, China and USA proved that Erythropoietin is neuroprotective with minimal side effects but there is no clinical trial in Bangladesh.Our study the role of erythropoietin in improving the neurological outcome of term neonates with perinatal asphyxia is probably the first research work in Bangladesh.Therefore, this study will help us to evaluate the effectiveness of erythropoietin in perinatal asphyxia with HIE stage II and III.Baby with IUGR, age >48 hours, haemolytic disease, congenital malformation along with maternal eclampsia and diabetes were excluded from the study.

A Randomized Controlled
In this study period total 142 neonates were assessed for eligibility.Out of them 68 eligible neonates were enrolled according to inclusion criteria.These 68 asphyxiated neonates were randomly assigned to Intervention group, (Case,n=35) who received rHuEPO 300-500 U/kg/dose daily subcutaneously for 5 days within first 48 hours of birth along with the standard treatment protocol and Control group, (n=33) who received standard treatment according to the unit protocol for perinatal asphyxia.
The erythropoietin dose (500 IU/kg/dose) for 5 days was based on studies by Zhu et al 14 and Elmahdy et al. 15 The neonates were monitored for short term outcomes such as 1) seizure control 2) tolerance of oral feeding 3) duration of hospital stay 4) neurological assessment done at 1 and 3 months of age 5) USG of brain were done on admission and at 3 months of age.

Discussion
Few studies have been done worldwide on the neuroprotective effects of Epo on human infants with HIE.The first study by Zhu et al 16 in 2009 compared Epo to supportive care in infants with moderate-to-severe encephalopathy.The study concluded that EPO reduced the risk of disability for infants with moderate HIE, without side effects.
In this study baseline characteristics (neonate and mother) of both the groups were found similar which was consistent with the study of Pradeep KB et al. and Elmahdy H. 17,18 Outcome variables such as initiation of oral feeds, duration of seizure control, duration of hospital stay and neurological outcome at 1 and 3 months of age were based on the study conducted by Kumar P et al. 15 In our study we observed that the seizures lasted for a longer duration in control group (median of 66 hours) compared to intervention group (median of 39 hours).The p value was significant (p=0.011).This results consistent with the previous study done by Elmahdy et al 18 and Kumar P et al. 15 Reaching oral feeds (sucking) within 5-7 days was one of the short term neurological outcomes suggesting oromotor coordination in asphyxiated newborns study done by Bhat et al. 19 In our study to see this effect, babies were initiated on tube feeds after assessing the gut function (absence of nasogastric aspirates or abdomen distension) which was gradually increased as per the tolerance.
Once the babies were hemodynamically stable, they were assessed for oromotor coordination (suck and swallow) and started on cup & spoon feeds.This study shown significant improvement on initiation of oral feeding in treatment group than control group.This correlated with previous study done by Kumar P et al. 15 Mean duration of hospital stay in treatment and control group is 6.93±1.91 and 9.13±2.87,the difference is statistically significant.There is no significant difference in USG of brain findings in treatment and control group on admission (P>.05).
USG was performed on 49 follow up patients.Among them 23(88.5%)out of 26 in treatment group found normal findings in comparison to 14(60.9%)out of 23 in control group.The difference is statistically significant and correlated with previous study done by Bhat et al. 19 This study also showed that erythropoietin is neuroprotective that was reflected by fewer neonates with neurologic abnormalities in intervention group.Prognosis were assessed clinically by developmental assessment and neurosonogram at 3 months.These results were consistent with the previous studies done by Zhu et al, 16 where the neurological outcome were assessed at 18 months of age using Thompson neurological assessment and Elmahdy et al 18 where the neurological outcome were assessed at 6 month of age using Denver Developmental Screening Test.In our study due to time constraint the clinical developmental assessment was used for early assessment of neurological outcome at the end of 3 months.
Elmahdy et al 18 found normal EEG backgrounds, compared with control infants with HIE 10 vs 3 infants (P=0.010).In our study due to financial constraints small number of patient were able to perform EEG therefore we could not compare it statistically.
Erythropoietin had no effect in reducing the mortality rate, but the rate of disability was reduced.Neurological abnormality showed 56.5% in control group and 19.2 % in intervention group (p = .008)at 3 months.This is analogous to the results noted after head cooling, which reduced significantly the rate of disability. 20e role of Epo and its receptor in the brain has been widely studied in multiple preclinical models ranging from neonatal brain injury to adult injuries.Both Epo and Epo-analogs may prove useful for treating pathologies of the central nervous system.In neonates, the side effect profile of erythropoietin has been minimal.Treated infants have not shown common side effects seen in adults such as thrombosis and polycythemia. 21e study findings could have important implications on a child survival program in tertiary level hospital.

Conclusion
This study demonstrated the feasibility and potential efficacy of the use of human recombinant erythropoietin for term neonate with HIE.The use of rHuEPO was associated with promising clinical improvements, improved neuroimaging backgrounds and favorable neurodevelopmental outcomes at 3 months of age.
In this study we found that early administration of erythropoietin to neonate with moderate to severe HIE improves seizure control, early oral feeding, reduces hospital stay and better neurodevelopmental outcome at 3 months of age.
Trial was carried out in the Neonatal ward and NICU of Dhaka Shishu Hospital from 1 st April 2014 to 30 th Sep 2015.Neonates eligible for the study had (40.7%) in control group.Statistically the sex distribution between groups was not significant.Mean (±SD) birth weight of the case and control group of neonates were 2.83±0.23 (range 2.60-3.20)and 2.78±0.19(range 2.50-3.10)kg, respectively.The mean difference of the birth weight was not statistically significant.
parameter were monitored routinely.Clinical assessments including neurological status were done at admission and during hospital stay, the grade of HIE (moderate or severe), the type of respiratory support needed, the presence of seizures and time of initiation of oral feeding.Prognosis (Physical and neurological) were assessed clinically by developmental assessment (gross and fine motor examination, vision, hearing, cognition and assessment of muscle tone) and neurosonogram with 2 follow up at 1 and 3 months.Renal function test, Liver function test and EEG were done as required.The data was analyzed according to standard procedure.SPSS Win version 17 programs have been used for data analysis.Results of the findings was verified by doing standard test for significance like Unpaired student "t" test and Chi-Square (X 2 ) tests and finding out the P value.Statistically significant P value considered if P was <0.05.The Ethical Review Committee of BICH, Dhaka Shishu Hospital approved the study.Sex distribution shows that in case group male were 15 (53.6%) and 16 (59.3) in control group, female were 13 (46.4%) in case group and 11