One of the major contentions about administering higher concentrations of oxygen while resuscitating a neonate in the delivery room is the risk of exuberant production of free oxygen radicals. This argument gains more importance in a premature neonate with physiologically immature antioxidants defense mechanism. A quest to address this contention became the key objective of this clinical trial – measuring 8-isoprostane levels after birth. The major observations of the current trial are as follows: a) no difference was observed in the 8-isoprostane values at 4 hours from birth between the room air and 100% oxygen groups, b) change in 8-isoprostane values from cord blood to 4 hours and then at 72 hours after birth was not significantly different between the study groups, c) no significant differences were observed in other important short term clinical outcomes such as mortality by discharge and abnormal neurological status by discharge even though the study was not powered to discriminate these outcomes and d) neonates in the room air group more frequently had treatment failure (defined as required chest compressions or heart rate remained < 60/minute or had apnea or poor respiratory efforts even after 60 seconds of PPV with the trial gas) and took longer time to establish regular respiratory efforts compared to 100% oxygen group.
Instead of a hard clinical primary outcome, we chose to study an oxidative stress associated biochemical marker to better understand the bio-causal mechanistic pathway when administering 100% oxygen instead of room air. Contrary to the often-quoted indirect markers of oxidative stress like superoxide dismutase, catalase, and reduced glutathione (GSH), we preferred to measure a direct marker of lipid peroxidation, plasma Isoprostane (28, 29). Isoprostane is superior to other biochemical markers and is considered as the best available biomarkers of oxidative stress and lipid peroxidation in vivo (29). This is due to its chemical stability, direct and more specific nature, its presence in detectable amounts in all normal tissues and biological fluids including plasma and ease to measure by a simple enzyme linked immunosorbent assay (29). Few studies have shown that Isoprostane levels increases robustly in preterm neonates following an oxidant injury and remain elevated to be detected for few days after birth (25, 30).
We arbitrarily chose 4 hours of postnatal age as the primary time point for measurement of Isoprostane as studies have shown that Isoprostane elevation starts at about 30 minutes after birth and stabilizes little later (28, 29). An animal experiment have reported that the peak levels of this marker will be achieved 4 hours after the hypoxemic insult (31). Apart from the 4 hours’ time point, we also measured the levels in cord blood as a surrogate of intrapartum hypoxic stress and repeated at 72 hours of life to study the change in Isoprostane levels postnatally, especially following resuscitation.
Our observations of lack of significant difference in Isoprostane levels over time, between the study groups and over time and between groups (Table 4) were in contrast with the previous studies done in term and preterm neonates(4–7, 32). Previous studies that have measured oxidative stress following such interventions has shown a decreased levels of oxidative stress markers in neonates resuscitated with room air compared to 100% oxygen (32). However, the biochemical markers used in those studies were different. A study by Vento et al comparing 30% versus 90% oxygen for resuscitation in DR in ≤28 weeks neonates have shown significantly higher GSSG/reduced glutathione at day 1 and 3 in those neonates who received high initial oxygen (90%). However, they reported no difference in the urinary Isoprostane metabolite levels between the groups on both the days (10). The same group studied the role of various biochemical markers of oxidative stress in moderate preterm infants and their role in the prolongation of hospitalization. The authors reported that at 7 days of life, Isofurans, products of lipid peroxidation with a substituted tetrahydrofuran ring, and not Isoprostane was significantly correlated with increased length of hospitalization (33).
Even though Isoprostanes are superior to many other biochemical indicators of lipid peroxidation, their formation seems to be impaired at elevated oxygen tensions, such as hyperoxia as part of resuscitation with 100% oxygen in delivery room (34). Fessel et al reported that this short coming can be overcome by measurement of Isofurans, which are not affected by high oxygen tension conditions and their formation is favored in such conditions (35). A combined measurement of both the Isoprostanes and isofurans may offer the best assessment of oxidative stress under all circumstances. Absence of significant difference in Isoprostanes in both the current trial and in the study by Vento et al may possibly be due to the above-described phenomenon and this needs further exploration in future studies (10).
We observed that those neonates resuscitated with room air had a higher incidence of treatment failure (required endotracheal intubation) in comparison to the 100% oxygen group. This contrasts with the beneficial effect observed in earlier trials which involved term neonates resuscitated with room air (4, 5, 32). Preterm neonates respond differently to hypoxemia by decreased respiratory efforts and delayed onset of regular respiration (22). A previous study involving preterm neonates (24–28 weeks’ gestation) reported shorter duration of oxygen, continuous positive airway pressure and mechanical ventilation in those resuscitated with 30% oxygen compared to 90% oxygen (36). However, this was not supported by current and few other studies (9, 37).
One of the limitations in the current study was our inability to assess the long-term neurodevelopmental outcome due to inadequate sample size for this outcome. The sample size for a composite outcome of death or abnormal neurodevelopmental outcome at 18–22 months of 35% for an effect size of 10% with α and β errors of 5 and 20% respectively would be 690 neonates which would require a multicentric participation. Second limitation was absence of comparison of different oxygen concentrations as the study site did not have blenders to mix air and oxygen. However, this offered us the opportunity to design and conduct a pragmatic clinical trial keeping in mind the infrastructural facilities in most of the DRs in India and other LMIC and further helped to effectively blind the intervention, which otherwise would have been much more difficult. The current study had two important strengths. Methodologically, this was the first blinded trial from a LMIC that compared two different concentrations of oxygen for DR resuscitation of preterm neonates. Blinding and allocation concealment were ensured by innovatively randomizing the trial cylinders (for the type of gas) and days in a week (for random allocation of trial cylinder). Secondly, as the protocol allowed us to defer consent in those mothers who arrived at the DR in an advanced labor, we could enroll many eligible pregnant women thereby increasing the generalizability of the results.
To conclude, we did not observe any difference in the levels of 8-isoprostane at 4 h and 72 h of life between those preterm neonates who received room air or 100% oxygen for delivery room resuscitation nor the change in Isoprostane levels from cord blood to 72 h of life was significantly different. Neonates resuscitated with room air had a higher incidence of need for endotracheal intubation in comparison to the 100% oxygen group and took longer time to establish regular respirations. Multi-centric studies from LMICs comparing different initial concentrations of oxygen for resuscitation of preterm neonates for long-term neurodevelopmental and neurosensory outcomes are required. Till then 21% oxygen should not be recommended as the initial concentration in preterm neonates.