In this prospective, randomized, double-blinded, placebo-controlled trial, we enrolled 78 newborns at > 36 weeks gestational age diagnosed HIE according to standard from pediatrics section of the Chinese Medical Association, which was similar to our previous RCT study protocol. The randomization codes were generated by computer and supplied in numbered, sealed envelopes. Participants were from hospitalized during May 1, 2014 to May 1, 2018 on Department of Neonatology, Children's Hospital of Fudan University. This study was approved by the Ethics Committee. Written informed consent was obtained from the parents.
We hypothesized that high doses of Epo (1000 U/kg per dose) given combined with TH will release the level of biomarkers associated with poor prognosis, decrease the brain injury in MRI, improve the long-term neurological outcome and be safe.
All participants met four inclusion criteria: (1) ≥ 36 weeks gestational age, ≥ 2500 g birth weight, within 6 hours of age; (2) pH < 7.00 or base deficit ≥ 15 mmol/L in cord or arterial blood within 60 minutes of birth; (3) perinatal emergency events (i.e. severe late fetal / abnormal deceleration, umbilical cord prolapse, rupture of the umbilical cord, rupture of the uterus) with 5 minute Apgar score < 5 or need for resuscitation (including endotracheal or mask ventilation) at 10 minutes after birth; (4) evidence of encephalopathy, such as seizures, disturbance of consciousness
(lethargy, stupor, or coma), abnormal reflexes.(11–13)
Additional exclusion criteria including: congenital metabolic disorder or anomaly; severe perinatal infectious; head trauma or skull fracture causing intracranial hemorrhage.
Withdrawal standards: (1) intractable pulmonary hypertension; (2) hypotension (MAP ≤ 30 mmHg) insensitive to treat; (3) severe arrhythmia; (4) platelet count < 50 × 109/L; (5) oliguria (urine volume < 0.5 mL/kg/h continuous for 24 hours and progressive aggravated azotemia); (6) severe infections with clinical and laboratory evidence; (7) scleroderma; (8) parents/legal guardians refuse to continue.
Within 6 hours after birth, participants began TH continuously for 72 hours. Epo group were given 1000U/kg per dose human recombinant erythropoietin (r-Hu-Epo)intravenously every other day for two weeks and the placebo group given an equal volume of physiological saline. The basal treatment and supportive care were identical between two groups: intravenous nutrition to meet the energy requirement; when seizures occurred, phenobarbital was used first at 20 mg/kg, with maintenance dose of 5 mg/kg.d ; dopamine, fentanyl or antibiotics were utilized when necessary.
Potential neurological biomarkers
Participants will have at least two blood samples (before TH and after TH) for potential neurological biomarkers serum level detecting. Reviewed previous study, we picked up several potential biomarkers which were common detected in clinical uses including: creatine kinase (CK), lactate dehydrogenase (LDH), Na+, K+, Mg2+ and Ca2+. CK, usually used in judging myocardial ischemic injury, have also been found that high level was associated with serious brain damage.(14) LDH, one of the important enzyme participating in anaerobic glycolysis and gluconeogenesis, have been demonstrated the level was significantly higher in the HIE group.(15) Besides, HI triggers the disruption of ionic homeostasis, characterizing by enhanced K+ efflux and Na+, Ca2+ influx, which has been considered as the most important alteration which eventually leads to cell death or injury. (16, 17) Evidences showed Mg2+ may keep cell membrane stabilization, inhibits free radical production and decreases secondary inflammation. (18, 19) The laboratory technician was blinded to the group distribution.
Neonatal brain MRI was performed within 14 days of age. The BG/W score system, obtained basing on combining damage region, with the first-echo T2-weighted sequence, was the useful score for predicting motor outcome at 3 and 12 months and cognitive outcome at 12 months. The score ranged from 0 to 4, and higher scores were given for more extensive damage. Detailed scoring criteria as below: 0 = normal; 1 = abnormal signal in basal nuclei (BG or thalamus); 2 = abnormal signal in cortex; 3 = abnormal signal in cortex and basal nuclei (BG or thalamus); 4 = abnormal signal in entire cortex and basal nuclei.(20) Two central reviewers after training were blinded to treatment allocation, independently scored brain MRI outcome.
Forty patients (40/78, 51%) succeeded in achieving 18-month follow up data. In the Epo group, three infants died after discharged from hospital at the age around three days to one month. In the placebo group, three patients died during their hospitalization (two of persistent pulmonary hypertension, one of multiple organ failure) and one died after left hospital for one week. The Bayley II developmental scale results showed that the Epo group were likely to perform better in MDI score (mean, 88 vs 81, P = .23) and PDI score (mean, 84 vs 78, P = .29) The totally adverse outcomes were trend to decline in the Epo group (35% vs 60%, P = .21) (Table 4).
Participants will have at least three blood samples (prior to TH, end of TH approximately 4d after birth and finished time of Epo/placebo transfusion). We detected serum concentrations of sodium, potassium, calcium, blood sugar, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), blood urea nitrogen (BUN), prothrombin time (PT), activated partial thromboplastin time (APTT) and serum total bilirubin. Complete blood counts (CBC) were collected through peripheral blood. Continuous noninvasive blood pressure monitoring and anal temperature data were collected. Hyponatremia is defined as serum sodium lower than 130 mmol/L; hypokalemia is defined as serum potassium lower than 3.5 mmol/L; hypocalcemia is defined as blood calcium lower than 1.8 mmol/L or free calcium lower than 0.9 mmol/L; hypoglycemia is defined as blood sugar lower than 2.6 mmol/L; hyperbilirubinemia is defined as serum total bilirubin over than 221umol/L; liver dysfunction is defined as level of AST over than 200 IU/L or ALT over than 100 IU/L; renal dysfunction is defined as level of Cr over than 88ummol/L or BUN over than 7.5 mmol/L; coagulation disorder is defined as PT delay over than 3 s or APTT delay over than 10 s; thrombocytopenia is defined as platelet lower than 100 000 per ul; polycythemia is defined as hematocrit over than 60%; hypotension is defined as mean arterial pressure (MAP) lower than 40 mmHg or need vasoactive drugs containing; hypothermia is defined as rectal temperature lower than 33.5℃ over 1 h; hyperthermia is defined as rectal temperature higher than 37.5℃ over 1 h. The laboratory technician was blinded to the study group.
For accurately analysis of treatment effect, intention-to-treat strategy was used. We analyzed the data by using SPSS software (SPSS Inc, Chicago, IL). χ 2 test or Fisher’s exact test (when the count was ≤ 5) were used when compared categorical variables. If continuous variables meet normal distribution, data were expressed as mean ± SD and counted in t test, otherwise median and inter-quartile range (IQR) were used and used Wilcoxon rank sum test for analyzing. Potential biomarkers comparison between two groups were used 2-sided T test or Mann-Whitney U test as appropriate. P values of < .05 (2-sided) were considered statistically significant.