Early cerebral hypoxia in extremely preterm infants and neurodevelopmental impairment at 2 year of age: A post hoc analysis of the SafeBoosC II trial.

BACKGROUND: The SafeBoosC II, randomised clinical trial, showed that the burden of cerebral hypoxia was reduced with the combination of near infrared spectroscopy and a treatment guideline in extremely preterm infants during the first 72 hours after birth. We have previously reported that a high burden of cerebral hypoxia was associated with cerebral haemorrhage and EEG suppression towards the end of the 72-hour intervention period, regardless of allocation. In this study we describe the associations between the burden of cerebral hypoxia and the 2-year outcome.
METHODS: Cerebral oxygenation was continuously monitored from 3 to 72 hours after birth in 166 extremely preterm infants. At 2 years of age 114 of 133 surviving children participated in the follow-up program: medical examination, Bayley II or III test and the parental Ages and Stages Questionnaire. The infants were classified according to the burden of hypoxia: within the first three quartiles (n = 86, low burden) or within in the 4th quartile (n = 28, high burden). All analyses were conducted post hoc.
RESULTS: There were no statistically significant differences between the quantitative assessments of neurodevelopment in the groups of infants with the low burden of cerebral hypoxia versus the group of infants with the high burden of cerebral hypoxia. The infants in the high hypoxia burden group had a higher-though again not statistically significant-rate of cerebral palsy (OR 2.14 (0.33-13.78)) and severe developmental impairment (OR 4.74 (0.74-30.49).
CONCLUSIONS: The burden of cerebral hypoxia was not significantly associated with impaired 2-year neurodevelopmental outcome in this post-hoc analysis of a feasibility trial.

Introduction

Being born extremely preterm is associated with a risk of death and later moderate- to severe neurodevelopmental impairment. Several studies have shown associations between cerebral hypoxia, as measured by cerebral near infrared spectroscopy (NIRS), and neurodevelopmental impairment [1-4]. NIRS-guided interventions have been shown to reduce the cerebral burden of hypoxia both in the first 15 minutes after birth in preterm infants [5] and during the first 72 hours of life in extremely preterm infants by [6]. NIRS-guided management, however, did not improve early biomarkers of brain injury [7] or outcome at 2 years [8]. A post-hoc analysis showed that a burden of cerebral hypoxia in the highest quartile was significantly associated with death before discharge, severe brain injury on cranial ultrasound (cUS), and reduced electrical brain activity compared to infants with the burden of cerebral hypoxia in the three lower quartiles in the SafeBoosC-II trial, regardless of group allocation [9]. In this post-hoc analysis we wish to explore the relationship between the burden of cerebral hypoxia during the first 72 hours of life and developmental outcome at two years corrected age in the extremely preterm infants included in the SafeBoosC-II trial while disregarding group allocation.

Patients and methods

The SafeBoosC II trial was a multicentre randomised trial with eight centres recruiting a total of 166 preterm infants within 3 hours after birth. The study was conducted between June 2012 and December 2013 [6]. Of these 133 infants were alive at discharge and 114 children were included in the follow up analysis. The burden of cerebral hypoxia did not differ between the infants who were followed up and the infants who were not [8]. The trial was registered at ClinicalTrial.gov, NCT01590316, the protocol is published [10] and available in full at http://www.safeboosc.eu.

The burden of cerebral tissue hypoxia

The infants were included in the SafeBoosC II study three hours after birth and regional cerebral oxygenation (rStO2) was continuously monitored by NIRS during until 72 hours after birth. The infants were randomised to the intervention or control group. Intervention group: the cerebral oxygenation level was visible and the target range was set at 55% to 85%. The clinicians were provided with a dedicated treatment guideline listing possible interventions if the cerebral oxygenation was out of range [11]. Control group: the cerebral oxygenation levels were recorded, but the clinicians were blinded to the results and the infants were given standard treatment and care, only. The primary outcome of the SafeBoosC II trial was the combined burden of hypo- and hyperoxia, calculated as time spent outside the target range of rStO2 predefined as 55–85%. The burden of cerebral hypoxia was calculated as the time spent below the target limits multiplied by the mean deviation from the lower limit during the first 72 hours of life, expressed as percentage hours (%hours. e.g. 10%hours represent one hour with 45% rStO2 or 2 hours with a 50% value of rStO2).

In this post-hoc analysis we wish to explore the relationship between the burden of cerebral hypoxia and developmental outcome at two years corrected age, regardless of trial group allocation.

Cranial ultrasound

cUS was performed at day 1, 4, 7, 14, and 35 and at term equivalent age. The cUS was categorised as no, mild/moderate or severe brain injury as previously described [12].

No brain injury: None of the findings below.

Mild/moderate brain injury: Grade 1–2 Intraventricular haemorrhage (IVH, including germinal layer haemorrhage), isolated ventriculomegaly with ventricular index

Severe brain injury: IVH III (ventricular index >p97 during the acute phase), post haemorrhagic ventricular dilatation, parenchymal/periventricular haemorrhagic infarction, unilateral porencephalic cysts, cystic periventricular leukomalacia (bilateral), cerebellar haemorrhage, cerebral atrophy at term age or stroke.

Neurodevelopmental evaluation

At 2 years corrected age, the participants were invited to a follow-up visit consisting of a medical examination and an assessment of their neurodevelopment with the Bayley II or III, and the parents were asked to fill in an Ages and Stages Questionnaire (ASQ) [8].

Medical examination: Basic growth measurements were collected. Vision and auditory functions were evaluated. If the child showed signs of cerebral palsy, the gross motor function was classified using the Gross Motor Function Classification System (GMFCS). The doctor performing the medical examination may not have been blind to the intervention.

Bayley Scales of Infant and Toddler Development, Second Edition (Bayley- II) or Third Edition (Bayley-III), depending on what version the centre was using at the time of the study. Bayley III is known to underestimate the developmental deficit when compared to Bayley II [13], we therefore calculated the predicted mental developmental index for the Bayley III edition, as previously described [14]. The psychologists conducting the tests were blinded to cerebral NIRS outcome and imaging results.

ASQ: A set of parental questionnaires covering the development of children aged from four months to five years. Each questionnaire contains five developmental domains, communications, gross motor skills, fine motor skills, problem-solving and personal-social skills. The parents returned the questionnaires by mail or handed them in on the day of medical examination. The parents were not blinded to intervention group nor imaging results.

Neurodevelopmental impairment was classified according to Classification of health status at 2 years as a perinatal outcome, British Association of Perinatal Medicine [15]. Severe neurodevelopmental disability if any of the following conditions was present: Cerebral palsy (CP) with a GMFCS score of 3–5; a cognitive function score below -3 standard deviations scores, Mental Development Index (MDI) below 55; hearing impairment with no useful hearing even with aids; no meaningful words or signs and blind or only able to perceive light or light reflecting objects. Moderate neurodevelopmental disability if any of the following conditions was present: CP with a GMFCS score of 1–2; a cognitive function score between -3 and -2 standard deviations scores, (MDI 55–70); hearing impairment, but useful hearing with aids; fewer than five words or signs and moderately impaired vision.

Statistics

The goal of the statistical analysis was to examine the association between neonatal cerebral hypoxia and neurodevelopmental outcome. For this purpose, the median and interquartile range was determined for the burden of cerebral hypoxia and the infants were divided in two groups according to a burden within the 4th quartile (highest burden of cerebral hypoxia) or below (lower 3 quartiles of burden of cerebral hypoxia). This dichotomisation was previous used in our group when comparing biomarkers [9] and chosen a priory for the present post-hoc data analysis.

We compared the baseline patient characteristics, the interventions conducted during the 72 hours NIRS monitoring, the neonatal complications, and the outcomes between the two groups, using the t-test and Chi-square test as appropriate. Thereafter we calculated the mean difference and the 95% confidence intervals for the ASQ score and Bayley II/III scores and the odds ratio and 95% confidence interval for adverse outcome in the group of infants with in the 4th quartile versus the children in the three lower quartiles.

If baseline patient characteristics, the interventions conducted during the 72 hours NIRS monitoring, or the neonatal morbidity rate (severe brain injury on cUS, necrotising enterocolitis, bronchopulmonary dysplasia or retinopathy of prematurity) differed significantly (p<0.05) and the differences between the developmental outcomes in the two groups were significant (p<0.05), additional multiple logistic and linear regression analyses was planned–as the basis for discussing the possibilities as regards causal paths as well as confounding.

None of the analyses reported here were specified in the SafeBoosC-II study protocol.

The statistics was performed using IBM SPSS Statistics for Windows Version 20.0 (Armonk, New York, USA).

Ethics

The SafeBoosC II trial was approved by each hospital’s research ethics committee: Ethikkommission, Medizinische Universität Graz, Austria; De videnskabetiske komiteer, Copenhagen, Denmark; Comité de Protection des Personnes Sud Est III–Groupement Hospitalier Edouard Herriot, Lyon, France; Clinical Research Ethics Committee, University College Cork, Ireland; Comitato de Etica, Drezione Scientifica, Fondazione IRCCSA Ca Granda, Ospetale Maggiore Plicliico, Milano, Italy; De Medische Ethische Toetsingescommissie, Universitair Medisch Centum, Utrecht, Netherland; Comité Etico de Investigación Clinica, Hospital Universitario La Paz, Madrid, Spain, and where required also by the competent authority responsible for medical devices (Austria, Denmark and France). Written informed parental consent was mandatory before inclusion. The trial was conducted according to the guidelines of the Declaration of Helsinki and the International Conference on Harmonisation good clinical practice.

Results

One-hundred sixty-six children were randomised to the SafeBoosC-II trial. Due missing data on cerebral oxygenation, death before discharge, death after discharge and loss to follow-up only data of 114 children were included in this post-hoc follow up analysis (). The children were classified into two groups according to the burden of cerebral hypoxia: children within the first three quartiles (n = 86, low burden) and children within in the 4th quartile (n = 28, high burden). The ranges of hypoxia in the 2 groups were: 0.1–78.3%hours in the low group and 78.7 to 803.9%hours in the high group.

The baseline patient characteristics () and treatment and major neonatal morbidities () were compared. There were significantly fewer boys in the high group, more children received vasopressors or inotropes. There were also more control group infants from the randomised trial in the high burden group versus low burden group.

The rates of major neonatal morbidities complication rates were similar ().

There were no statistically significant differences between the Bayley II or III sub- or total scores when comparing the group of infants form the high burden of cerebral hypoxia versus the low burden group (). The combined outcome of Bayley II and III for the 2 groups was mean (SD) 83.6 (17.6) for the children with a high burden of cerebral hypoxia versus 90.5 (16.9) for the children with the low burden of cerebral hypoxia (Cohen’s d = -0.40, 95% CI: -0.12 to 0.93, p = 0.13). There was no clear visible relation between developmental scores and the burden of cerebral hypoxia (). The ASQ-scores were similar in the two groups (). The rates of developmental impairment were higher in the high hypoxic burden group: cerebral palsy (OR 2.14 (0.33–13.78)) and severe developmental impairment (OR 4.74 (0.74–30.49) (), but these differences were not statistically significant (p = 0.59 and 0.11).

Burden of cerebral hypoxia and mental development index at 2 year.

Log-burden of hypoxia and Mental Development Index (MDI) combined of Bayley II and III at 2 year corrected age. The X-axis is a logarithmic scale (base2), i.e. one unit corresponds to a doubling of the hypoxic burden, and the limit between the two groups is at 78%hours. There is no suggestion of a different threshold of hypoxic burden, nor of a correlation across the scales.

Multiple regression was not done, since no statistically significant effects of cerebral hypoxia was found on bivariate analysis.

Discussion

In this post–hoc analysis of data collected as part of a randomised controlled phase II trial we found no statistically significant differences at two years of age between the children in the high hypoxic burden group compared to the low hypoxic burden group.

The SafeBoosC II was a randomised trial with the primary objective of investigating the possibility of reducing the combined burden of cerebral hyper- and hypoxia. As this showed possible, more infants from the control group ended up in the high burden group used in the present analysis. We focused solely on the hypoxic burden since the burden of cerebral hyperoxia was negligible and unaffected by the intervention [6].

Several publications report that low cerebral oxygenation is associated with developmental impairment of preterm infants. Among 67 infants born below 32 weeks of gestation, those within the lower (as well as higher) quartile of rStO2 in the neonatal period more often had impaired cognitive outcome at 2–3 year [4]. The burden of tissue hypoxia was defined as time with rStO2 below 50% and cerebral oxygenation was estimated from a 2-hour period of recording each day, while in the infants in the SafeBoosC II trial, the threshold was 55% and cerebral tissue oxygenation was continuously monitored from 3 hours after birth until 72 hours after birth [10]. In a cohort of 724 infants born at gestational age below 32 weeks and monitored with cerebral NIRS during the first 72 hours after birth, spending more than 20% of the time with rStO2 below 55% was associated with impaired cognitive outcome at 2 years corrected age (Odds Ratio 1.4 95% CI 1.1–1.7) [1]. The same group published that prolonged suboptimal rStO2 in preterm infants with patent ductus arteriosus was associated with reduced cerebral brain volume at term equivalent age [3]. They also identified that regardless of mean arterial blood pressure cerebral oxygenation below 50% for more than 10% of the time was related to adverse outcome at 18 months in 66 preterm infants born at gestational age below 32 weeks [2].

Several other studies have shown an association between cerebral oxygenation immediately after birth [16] or within the first 3 days [17] with adverse short-term outcome in preterm infants.

We previously reported that death, brain injury, and reduced brain electrical activity were more common in the high hypoxia burden group compared to the low burden group, i.e. precisely the groups that were also compared in the present analysis [9]. We were not able to demonstrate a similar association to neurodevelopmental deficits at two years of age. This may just be due to the limited statistical power, thus the confidence limits included clinical relevant associations. On the other hand, it is possible that the ‘true’ effects of cerebral hypoxia ‘wane’ with age due to the multifactorial aetiology of long-term outcome in preterm infants. In particular, it should be mentioned that severe brain injury as defined by cranial ultrasound imaging in the neonatal period is only a moderately strong predictor of moderate-to-severe neurodevelopmental deficit at two years of age or later.

Strength and limitations

The strengths are the use of data from a randomised controlled trial with prospective and well-defined data collection, as well as a pre-defined classification of the hypoxic exposure. In this way the problem with multiple results of exploratory, post-hoc analyses was minimised.

Our sample size was relatively small: 114 extremely preterm infants and the incidence of moderate-to-severe developmental impairment was relatively low (16 of 114 infants (14%). Thus, the statistical strength with this sample size is limited and the confidence limits are wide and include clinically relevant effect sizes. Furthermore, the original approach of the SafeBoosC-II trial, using a simple area-under-the curve to quantify the burden of hypoxia may be too simple for the purpose of associating with brain injury and neurodevelopmental consequences. For pulse oximetry, the duration of the individual episodes seems to be important in particular [18, 19]. Furthermore, more infants in the high hypoxia burden group received vasopressors or inotropes and red blood cell transfusions, so they likely were more sick than the infants in the low burden group, on the other hand this group included fewer boys who are at increased risk of neurodevelopmental impairment. Since the differences between the two groups in terms of neurodevelopmental impairment were not statistically significant, we abstained from additional analyses to adjust for potential confounding. In any case, causal inference would be difficult given the uncertain direction of effect among the neonatal variables. As a further weakness, the cerebral oxygenation reported by the two different devices used in the trial is not identical and using a single cut-off value may not be optimal [20-22].

Finally, the SafeBoosC II study allowed the use of either Bayley II or III and in order to compare the two editions the results from the Bayley III was converted into a predicted MDI [14]–as this procedure was conducted in both groups, it should not affect the result. However developmental delay as measured by Bayley at 2 years of age may not be the best predictor later cognitive outcomes [23].

Conclusion

The burden of cerebral tissue hypoxia was not statistically significantly associated with 2-year neurodevelopmental outcome in this post-hoc analysis of data from a randomised controlled trial, but the confidence limits were wide, and a clinically relevant association could not be excluded. A larger, appropriately powered randomised trial is needed to test the clinical value of cerebral oximetry in preterm infants.

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7 Oct 2020

PONE-D-20-27390

Early cerebral hypoxia in extremely preterm infants and neurodevelopmental impairment at 2 year of age: A Post hoc analysis of the SafeBoosC II trial

PLOS ONE

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Reviewer #1: Early cerebral hypoxia in extremely preterm infants and neurodevelopmental impairment at 2 years of age: A post hoc analysis of the SafeBoosc II trial (ReviewPLOSone-2020-1005)

This report is an exploratory post-hoc analysis of the SafeBoosC II trial to assess if there is a relationship between the burden of cerebral hypoxia and neurodevelopmental outcome at 2 years corrected age. The SafeBoosC II trial was a randomized study among 166 preterm infants to determine if continuous NIRS recording of cerebral oxygenation over the first 72 hours after birth combined with a protocol to maintain cerebral oxygenation between 55-85% reduces the time with cerebral hypoxia or hyperoxia. A series of publications have demonstrated that the intervention could reduce the time with cerebral hypoxia but outcome measures (biomarkers, EEG, imaging, neurodevelopment) have not be altered. The current manuscript builds on an earlier publication reporting that infants in the highest quartile of cerebral hypoxia exposure had more severe intracranial hemorrhage, altered EEG or death compared to infants in the lowest 3 quartiles.

Comments:

1) Methods: Although there have been multiple publications from this trial which provide details of the study, it would be helpful for readers not familiar with the trial to provide a brief description of the study within the methods section (more than currently provided).

2) Methods: Since the results for the CUS are reported, there should be a description of mild, moderate and severe brain injury.

3) Methods/neurodevelopmental assessments: Were examiners blinded to treatment intervention and other clinical data such as imaging results?

4) Methods: definition of neurodevelopmental impairment and CP belong in the methods and not the legend of a table.

5) Methods/statistics: The sentence "If baseline patient characteristics, the interventions conducted during the 72 hours of NIRS... is a run on sentence and is awkward. Although it is recognized that there was no adjustment for neonatal complications, it is not clear why one would consider doing any adjustment for neonatal complications since they may be in the causal path to the outcome at 2 years.

6) Methods/statistics: The two most important issues are as follows;

a. Death is not accounted for in the analyses. Shouldn't the primary outcome be death or a measure of neurodevelopment and a secondary outcome of neurodevelopment among survivors as presented? A value below the lowest measured values for cognitive and motor functions could be assigned to allow an assessment of death for this analysis.

b. There is no adjustment for baseline differences in characteristics among the lowest 3 quartiles and the highest quartile of infants (eg, p values < 0.2 or some other cut point for univariate analyses).

7) Results: the list of baseline characteristics is quite short. Is this the extent of data that is available? Is there information regarding maternal health (diabetes, hypertension), maternal education or sociodemographic status, placental problems (abruption, previa), mode of delivery, histological chorioamnionitis, intubation at birth?

8) Results: umbilical pH is not a treatment.

9) Results/treatment in the first 72 hours; is their information on pneumothorax and placement of a chest tube?

10) Results: the statement "neonatal complication rates were similar" needs more detail as to what complications are being referred to.

11) Results: "The rates of developmental impairment tended to be increased..." Reporting of trends can be problematic and sometimes misleading. It may be more objective to state that the point estimate for outcome x was in the direction of an increase in impairment but the confidence limits were very wide.

12) Discussion/1st paragraph: Discussing trends can be problematic. Consider a more objective statement that the wide confidence limits precludes the ability to assert that there is or is not an association between 2 year outcome and cerebral hypoxia.

13) Discussion: the statement "The fact that the difference in the cerebral outcomes waned with age underline the multifactorial influence in long term outcome..." is an important statement and could be viewed as the conclusion. The authors need to do a better job of integrating this possible conclusion with the limitation of their data. For example as the authors note, although the difference in Bayley scores is approximately a third of a standard deviation in favor of less cerebral hypoxia, the confidence limits for neurodevelopmental impairment are wide. This study is relatively small for 2 year outcome and as noted in the limitations may be under powered. Better integration of these comments would help the message. It seems like it is an open question.

14) Discussion: not performing adjustment of baseline characteristics because there were no differences in the outcome seems very unconventional.

15) Conclusion: This statement should reflect that this was an exploratory analysis. In addition stating that cerebral hypoxia was not statistically associated with 2 year outcome may be misconstrued as more definitive than it is. The wide confidence limits limit the ability to draw conclusions and further study is needed seems like a better way to summarize this interesting data.

Reviewer #2: - This manuscript should include a flow diagram showing how the children recruited to the overarching trial whittle down to the number included in this analysis.

- In the methods, a description of the various tools used for neurodevelopmental evaluation should be included.

- The statistical analysis requires a number of improvements

(1) the table of descriptive characteristics of should be separated from the table describing associations between the main exposure and outcomes. Descriptive characteristics should be summarised in terms of counts and percentages for categorical ones and either means and SDs or medians and IQRs or ranges, for continuous ones. Statistical tests comparing groups are not necessary at this stage, unless the authors have some a-priori hypotheses to test - otherwise these tests simply constitute data dredging/multiple testing (which would be difficult to adjust for in this underpowered study).

(2) a separate table presenting the associations between the main exposure and the various outcomes should be presented. For each outcome either counts (%) or means (SEs) should be presented for each group, followed by the effect estimate, its 95% confidence intervals and p-values. p-values should not be included for subgroup analyses, e.g. the various components of the neurodevelopmental outcome scores, as this also constitutes multiple testing.

Although the authors have previously described the main exposure by contrasting the lower three quartiles of burden of cerebral hypoxia to the uppermost, this approach collapses potentially heterogeneous individuals into fewer groups resulting in loss of information - this is particularly critical in this study where there are few individuals and a small number of outcome events. The authors should include sensitivity analyses where the main exposure is treated as a continuous outcome, to explore linear (or log-linear) trends and departures from linearity/log-linearity in the association between burden of hypoxia and each of the outcomes.

Additionally, more careful consideration should be made about how potential confounders are identified and controlled for. Only potential confounders which could plausibly herald hypoxia (and are not on a causal pathway in the association between the main exposure and each outcome) should be further explored for adjustment.

The methods, results and discussion should then be updated to reflect these changes.

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Reviewer #2: No

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25 Jan 2021

Georg M. Schmölzer

Academic Editor

PLOS ONE

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17 Mar 2021

PONE-D-20-27390R1

Early cerebral hypoxia in extremely preterm infants and neurodevelopmental impairment at 2 year of age: A Post hoc analysis of the SafeBoosC II trial

PLOS ONE

Dear Dr. Anne Mette Plomgaard ,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Major comments

I find this manuscript a bit more confusing since the last round of reviews. My understanding is that the authors are exploring the association between cerebral hypoxia (the main exposure) and 2-year outcomes including neurodevelopmental scores, growth, vision and auditory functions, cerebral palsy, etc.

The reason for my confusion is that the approach to the analysis is not clearly articulated and seems incorrect in various places, therefore I am unable to make any sense of the results.

In particular, it is confusing why comparisons such as that in Table 2 are presented: if the hypothesis is that burden of hypoxia might affect outcomes, then performing a statistical test to check whether there are differences in neonatal morbidities and treatments between the groups does not serve any purpose, especially when those morbidities and treatments could influence the outcomes of interest here i.e. these morbidities are on the causal pathway between the main exposure and the outcome. I feel that the authors have not clearly considered the potential relationships (and the directions of those potential relationships) between the factors explored, therefore some of the analyses conducted don't really serve a useful purpose.

Another bit of analysis that doesn't make sense is in Table 3. For example, odds ratios are presented for mental development index and ASQ scores - these are continuous outcome measures for which I would have expected to see mean difference in these measures between the two cerebral hypoxia groups. Presenting odds ratios implies that the authors have explored the effects of the neurodevelopment scores as exposures on hypoxia as the outcome - which is clearly the wrong direction of potential causal relationship between the factors, since the hypoxia was assessed 15 minutes after birth and the nourodevelopmental outcomes at 2 years; therefore the hypoxia is the exposure and the neurodevelopmental score the outcome in this relationship.

Here is what I would suggest the authors do to focus this analysis more clearly:

- make very clear in the methods what the main exposure and outcome are

- the table of descriptive characteristics should present the participant characteristics (already present in table 1), treatments received, and morbidities in the two exposure groups. There is no need to conduct statistical tests comparing these groups in terms of these factors unless some factors are identified which are associated with the main exposure and the main outcomes and not on the causal pathway between them - the problem with the current analysis is that some of these factors are potentially on the causal pathway (for example, the burden of cerebral hypoxia could influence treatment received or neontatal morbidity which could then influence outcomes). Being on the causal pathway means even if the factor is associated with exposure, it is inappropriate to consider adjusting for it in the analysis of the outcome.

- the table of outcomes should list the main outcomes, the means and standard errors of the continuous ones or the counts and proportions/percentages of the binary/categorical ones in each of the hypoxia groups, followed by the estimate of association e.g. mean difference, 95%CI and p-value for continuous outcomes and ORs or RRs with 95%CI and p-values (in that order) for binary outcomes. This analysis should be limited to the main outcomes only and not their components as this would result in multiple testing which there isn't enough power in this study to adjust for.

Minor comments

- the statement just above the 'statistics' section relating to figure 1 belongs to the results section.

- the denominators for each column in Table 2 are missing; there's only 'n=' at the top of each column.

- as advised previously, please report the standard errors (SEs) not standard deviations (SDs) for continuous outcomes in Table 3. SEs are preferred for inference while SDs are appropriately used for description in Table 1.

- also, please report the p-value after the effect estimate and its 95%CI in Table 3.

- the statistical methods say you compared the baseline patient characteristics; in fact, you presented the baseline characteristics, which is appropriate to do i.e. no comparison required, and then you compared the interventions, complications and outcomes between the two groups. Please clarify these in the methods.

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Reviewer #1: No

Reviewer #2: No

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14 Jun 2021

Response to reviewers is attached as one of the files.

Submitted filename: response_to_reviewers.docx

Click here for additional data file.

9 Jul 2021

PONE-D-20-27390R2

Early cerebral hypoxia in extremely preterm infants and neurodevelopmental impairment at 2 year of age: A Post hoc analysis of the SafeBoosC II trial

PLOS ONE

Dear Dr. Anne Mette Plomgaard ,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by August 31 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Georg M. Schmölzer

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: - the authors' response regarding the reporting of SDs instead of SEs in Table 3 is unsatisfactory. The authors say that "it is more conventional to give SDs to serve the interpretation of differences on a population scale" - this is incorrect. SDs are descriptive of a sample only. The equivalent statistic that applies to the population is the SE. Please see Altman & Bland's brief discussion of this at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255808/. Furthermore, their response that "the p-values also serve to support the interpretation as regards inference" is only partly correct, as p-values are used to make inference with regards to the null hypothesis only; on their own they do not give any information on the uncertainty about estimates of effect or association, which the SEs provide. It remains my recommendation that the SDs in Table 3 and in the text pertaining to the outcomes should be replaced with SEs as is the standard practice in reporting estimates of effect or association for inference.

- all tables need one more improvement: wherever proportions are reported (e.g. proportion of male sex in table 1), these need to be accompanied by counts i.e. in this example, the number of males. Similarly wherever counts are reported e.g. in table 3, the counts for cerebral palsy, these need to be accompanied by the proportions.

- there is still need for some improvement in how the study is described; for example, in the first paragraph on page 4, I needed several reads to understand that the sentence that begins with 'Control group,' was a section describing the control group.

- aspects of the reporting of results also still need improvement; wherever comparing differences between the hypoxia groups, this needs to be clear. For example, statements such as "there were no differences between the Bayley... scores" in the abstract and results sections need to be explicit that this comparison was between the hypoxia groups.

**********

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Reviewer #2: No

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29 Aug 2021

All comments has been addressed in response to reviewers

Submitted filename: responce to reviewer.docx

Click here for additional data file.

3 Jan 2022

Early cerebral hypoxia in extremely preterm infants and neurodevelopmental impairment at 2 year of age: A Post hoc analysis of the SafeBoosC II trial

PONE-D-20-27390R3

Dear Dr. Anne Mette Plomgaard ,

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Reviewers' comments:

13 Jan 2022

PONE-D-20-27390R3

Early cerebral hypoxia in extremely preterm infants and neurodevelopmental impairment at 2 year of age: A Post hoc analysis of the SafeBoosC II trial

Dear Dr. Plomgaard:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Table 1

Baseline patient characteristics.

	Burden of cerebral hypoxia
	Quartile 1 to 3	Quartile 4	p-value
	n = 86	n = 28
Gestational age (week), mean (SD)	26.6 (1.1)	26.7 (1.2)	0.72
Birth weight (gram), mean (SD)	878 (212)	892 (191)	0.71
Male sex, n (%)	46 (53)	8 (29)	0.04
Twins, n (%)	16 (19)	10 (36)	0.11
Antenatal steroids full course, n (%)	61 (71)	25 (89)	0.13
Prolonged rupture of membranes, n (%)	26 (30)	11 (41)	0.44
Maternal chorioamnionitis, n (%)	3 (4)	3 (11)	0.33
Umbilical pH, mean (SD)	7.31 (0.09)	7.31 (0.08)	0.69
APGAR score <5 points at 5 minutes, n (%)	13 (15)	7 (25)	0.36

Between group comparison was performed using the t-test and Chi-square test as appropriate.

Table 2

Treatment during the first 72 hours and major neonatal morbidities.

	Burden of cerebral hypoxia
	Quartile 1 to 3	Quartile 4	p-value
Treatment during the first 72h of life	n = 86	n = 28
Surfactant treatment, n (%)	62 (72)	19 (69)	0.67
Mechanical ventilation, n (%)	52 (60)	15 (54)	0.52
Persistent ductus arteriosus treatment, n (%)	10 (12)	2 (7)	0.73
Use of vasopressors/inotropes, n (%)	9 (11)	10 (37)	0.01
Any red blood cell transfusion, n (%)	20 (23)	10 (37)	0.16
Corticosteroids, n (%)	0 (0)	2 (7)	0.06
Intervention group, n (%)	55 (64)	10 (37)	0.01
Major neonatal morbidities
Severe brain injury on cranial ultrasound, n (%)	9 (10)	6 (21)	0.19
Necrotising enterocolitis, n (%)	8 (9)	4 (14)	0.49
Bronchopulmonary dysplasia, n (%)	46 (54)	14 (50)	0.71
Retinopathy of prematurity, n (%)	12 (14)	5 (18)	0.76

Between group comparison was performed using the t-test and Chi-square test as appropriate.

Table 3

Developmental outcome at 2 year of age.

	Burden of cerebral hypoxia
	Quartile 1 to 3	Quartile 4	p-value	Mean difference (95% CI)
Bayley II (n)	22	8
Age (months), mean (SE)	24.8 (0.3)	25.2 (0.3)
Psychomotor developmental index, mean (SE)	87.6 (3.0)	81.1 (3.9)
Mental developmental index, mean (SE)	93.5 (3.1)	85.3 (3.4)	0.18	-8.2 (-20.4 to +3.9)
Bayley III (n)	39	13
Age (months), mean (SE)	24.6 (0.6)	24.7 (0.5)
Cognitive index, mean (SE)	98.1 (2.6)	91.3 (4.8)
Language index, mean (SE)	95.4 (2.8)	91.6 (4.0)
Motor index, mean (SE)	95.4 (2.4)	93.1 (3.7)
Predicted mental developmental index, mean (SE)	88.6 (3.0)	82.5 (5.7)	0.35	-6.2 (-19.3 to +7.0)
Ages and Stages Questionnaire (ASQ) (n)	69	21
Age (months) mean (SE)	25.8 (0.5)	25.2 (0.4)
Gross motor score, mean (SE)	42.4 (2.0)	40.5 (3.5)
Fine motor score, mean (SE)	43.6 (2.0)	42.8 (2.4)
Communication score, mean (SE)	43.5 (2.0)	43.8 (3.3)
Personal social score, mean (SE)	42.5 (1.7)	42.4 (2.9)
Problem solving score, mean (SE)	43.8 (1.7)	41.4 (2.7)
Total ASQ score, mean (SE)	215.5 (7.4)	210.9 (12.4)	0.76	-4.6 (-31.5 to +23.3)
Medical examination (n)	63	21		Odds ratio (95% diff)
Head circumference (centimetre), mean (SE)	48.1 (0.2)	48.0 (0.4)
Weight (kg), mean (SE)	11.1 (0.2)	11.2 (0.3)
Height (centimetre), mean (SE)	85.2 (0.6)	85.5 (0.9)
Cerebral palsy, n (%)	3 (5)	2 (10)	0.59	2.14 (0.33–13.78)
Vision impairment, n (%)	2 (3)	2 (10)	0.28	3.21 (0.43–24.36)
Combined outcomes (n)	62	22
Moderate neurodevelopmental impairment, n (%)	8 (13)	3 (14)	0.93	1.07 (0.26–4.34)
Severe neurodevelopmental impairment, n (%)	2 (3)	3 (14)	0.11	4.74 (0.74–30.49)
Moderate or severe developmental impairment, n (%)	10 (16)	6 (27)	0.25	1.95 (0.61–6.02)

Between group comparison was performed using the t-test and Chi-square test as appropriate.