Fetal Inflammatory Response Syndrome and Cerebral Oxygenation During Immediate Postnatal Transition in Preterm Neonates

Introduction: Fetal inflammatory response syndrome (FIRS), defined as elevated umbilical cord blood interleukin-6 (IL-6) values > 11 pg/ml, is associated with an increased risk of neonatal morbidity and mortality. The primary aim of the present study was to evaluate a potential influence of FIRS on cerebral oxygen saturation (crSO2) and fractional tissue oxygen extraction (cFTOE) during immediate postnatal transition in preterm neonates. The secondary aim was to analyze the potential influence of FIRS on cerebral injury and mortality. Methods: Secondary outcome parameters of prospective observational studies were analyzed. Preterm neonates with measured IL-6 values from umbilical cord blood and cerebral near-infrared spectroscopy (NIRS) measurements during immediate transition after birth were included. Preterm neonates with FIRS (FIRS group) were matched 1:1 for gestational age (± 1 week) to preterm neonates without FIRS (non-FIRS group). crSO2, cFTOE, arterial oxygen saturation (SpO2), heart rate (HR), and fraction of inspired oxygen (FiO2) were compared between both groups. In addition, cerebral injury and mortality were compared between both groups. Results: A total of 46 preterm neonates were included. Twenty-three neonates in the FIRS group [median gestational age 32.1 (IQR 30.3–33.0) weeks; median IL-6 19.7 (IQR 12.2–37.0) pg/ml] were compared to 23 neonates in the non-FIRS group [gestational age: 32.0 (30.4–33.1) weeks; IL-6: 5.4 (3.0–6.7) pg/ml]. cFTOE showed significantly lower values within the first 4 min and a trend toward lower values in minute 5 after birth in the FIRS group. There were no significant differences in crSO2 within the first 15 min after birth between the two groups. SpO2 was significantly lower in minutes 5 and 6 and HR was significantly lower in minutes 2 and 4 after birth in the FIRS group compared to the non-FIRS group. Survival without cerebral injury was similar in both groups. Conclusion: In preterm neonates with FIRS the crSO2 was similar despite significantly lower cFTOE values during the first minutes after birth. This observation may be a result of compromised oxygen consumption and delivery in the first minutes after birth in neonates with FIRS.

Introduction: Fetal inflammatory response syndrome (FIRS), defined as elevated umbilical cord blood interleukin-6 (IL-6) values > 11 pg/ml, is associated with an increased risk of neonatal morbidity and mortality. The primary aim of the present study was to evaluate a potential influence of FIRS on cerebral oxygen saturation (crSO2) and fractional tissue oxygen extraction (cFTOE) during immediate postnatal transition in preterm neonates. The secondary aim was to analyze the potential influence of FIRS on cerebral injury and mortality.
Methods: Secondary outcome parameters of prospective observational studies were analyzed. Preterm neonates with measured IL-6 values from umbilical cord blood and cerebral near-infrared spectroscopy (NIRS) measurements during immediate transition after birth were included. Preterm neonates with FIRS (FIRS group) were matched 1:1 for gestational age (± 1 week) to preterm neonates without FIRS (non-FIRS group). crSO2, cFTOE, arterial oxygen saturation (SpO2), heart rate (HR), and fraction of inspired oxygen (FiO2) were compared between both groups. In addition, cerebral injury and mortality were compared between both groups.
Results: A total of 46 preterm neonates were included. Twenty-three neonates in the FIRS group [median gestational age 32.1 (IQR 30.3-33.0) weeks; median IL-6 19.7 (IQR 12.2-37.0) pg/ml] were compared to 23 neonates in the non-FIRS group [gestational age: 32.0 (30.4-33.1) weeks; IL-6: 5.4 (3.0-6.7) pg/ml]. cFTOE showed significantly lower values within the first 4 min and a trend toward lower values in minute 5 after birth in the FIRS group. There were no significant differences in crSO2 within the first 15 min after birth between the two groups. SpO2 was significantly lower in minutes 5 and 6 and HR was significantly lower in minutes 2 and 4 after birth in the FIRS group compared to the non-FIRS group. Survival without cerebral injury was similar in both groups.
In FIRS, pro-and anti-inflammatory cytokine release leads to oxidative stress resulting in cerebral cell damage (11). IL-6 is already known to be a risk factor of white matter injury (12). This raises the question if FIRS is associated with a compromised cerebral tissue oxygen saturation (crSO2) in neonates, aggravating adverse effects and cerebral injury.
Near-infrared spectroscopy (NIRS) enables non-invasive, continuous measurement of crSO2 and cerebral tissue fractional oxygen extraction (cFTOE) (13)(14)(15). Rallis et al. (16) observed in septic neonates a decrease in cerebral oxygenation (measured with NIRS) over the first seven days after birth. Cerebral NIRS monitoring is well established in the delivery room during the immediate transition period from intra-to extrauterine life (17)(18)(19)(20). However, so far there are no data about the effect of FIRS on crSO2 during the immediate transition after birth in preterm neonates.
The primary aim of the present study was to evaluate whether there is an association between FIRS and crSO2/cFTOE in preterm neonates during the first 15 min after birth. We hypothesized that in preterm neonates with FIRS, crSO2 values are lower and cFTOE is higher due to compromised perfusion. In addition, our secondary aim was to analyze cerebral injury and mortality until term-equivalent age or before discharge in neonates with and without FIRS.

Design
In the present study secondary outcome parameters of prospective observational studies, conducted between May 2010 and November 2019 at the Division of Neonatology, Medical University of Graz, Austria, were analyzed. All studies were approved by the Regional Committee on Biomedical Research Ethics (EC numbers: 19/291 ex 07/08, 23/403 ex 10/11, 27-465 ex 14/15, 30-450 ex 17/18) and written parental consent was obtained before study inclusion.

Patients
We included preterm neonates, in whom umbilical cord blood IL-6 values and crSO2 were measured during the first 15 min after birth. Exclusion criteria were major congenital anomalies. The preterm neonates were stratified into two groups according to their IL-6 values: neonates with IL-6 ≤ 11 pg/ml were assigned to the non-FIRS group, and those with IL-6 > 11 pg/ml to the FIRS group (1).

NIRS and Routine Monitoring
An INVOS 5100C Cerebral/Somatic Oximeter Monitor (Covidien, Massachusetts, U.S.A.) with a neonatal transducer was used for crSO2 measurements. After birth the cord was clamped according routine after at least 30 s. Preterm neonates were placed on the resuscitation table under an overhead heater immediately after birth. The NIRS transducer was applied on the left fronto-parietal head in each neonate immediately after birth without disturbing routine medical care. The sensor was secured with a gauze bandage. crSO2 measurements were conducted during the first 15 min after birth. The sample rate (period) of NIRS measurements was 0.13 Hz (8 s).
Arterial oxygen saturation (SpO2) and heart rate (HR) were measured with the IntelliVue MP30 monitor (Philips, The Netherlands). The transducer was placed on the right hand/wrist. crSO2, SpO2 and HR were recorded continuously during the first 15 min after birth and stored every second in a multichannel system (alpha-trace digital MM, B.E.S.T. Medical Systems, Austria) for subsequent analyses. cFTOE was calculated for each minute: (SpO2-crSO2)/SpO2. Body temperature was measured in minute 15 after birth using a rectal probe. Blood pressure was measured by a pneumatic cuff applied to the right upper arm with the IntelliVue MP30 monitor (Philips, The Netherlands) in minutes 5, 10, and 15 after birth. Afterwards, the mean of the three mean arterial blood pressure (MABP) values was calculated. Further, fraction of inspired oxygen (FiO2) was recorded and the need for respiratory support (continuous positive airway pressure (CPAP) or intubation) was documented.

IL-6, Procalcitonin and C-Reactive Protein
IL-6 was measured in umbilical cord blood plasma taken immediately after birth. The analysis of IL-6 was performed using the Endogen Interleukin-6 ELISA (Endogen Inc., Massachusetts, U.S.A.) according to the standard procedure. In addition, procalcitonin (PCT) levels were analyzed from the same umbilical cord blood sample. C-reactive protein (CRP) was determined twice after birth: within 24-48 h and 48-72 h after birth.

Cerebral Injury and Mortality
Cerebral injuries were evaluated by routine cerebral ultrasound examinations carried out in all preterm neonates on the first, fourth, and eighth day after birth and at term age or before discharge, depending on what came first. We recorded any grade of IVH and PVL, and death.

Groups Matching
Preterm neonates in the non-FIRS group were matched for gestational age ± 1 week to those of the FIRS group. The matching ratio was 1:1.

Statistical Analysis
Data are presented as mean and standard deviation or median and interquartile range (IQR) for continuous data and absolute and relative frequency for categorical data, respectively. Baseline differences between groups were analyzed using

RESULTS
Two-hundred sixty-eight preterm neonates were included in the prospective observational studies. Ninety-nine preterm neonates, with IL-6 values available and crSO2 measurements within the first 15 min after birth, fulfilled the inclusion criteria. Sixty-one neonates showed IL-6 values ≤ 11 pg/ml (non-FIRS group) and 38 neonates had IL-6 values > 11 pg/ml (FIRS group). Twentythree neonates in each group, matched for gestational age, were finally analyzed (Figure 1). Demographic data are presented in Table 1.
Twenty (87%) preterm neonates in the FIRS group and 21 (91%) preterm neonates in the non-FIRS group needed respiratory support and/or supplemental oxygen (CPAP) within the first 15 min after birth. Four (17%) neonates in the FIRS group and one (4%) neonate in the non-FIRS group were intubated within the first 15 min after birth. None of the neonates needed cardiopulmonary resuscitation.

NIRS Monitoring
The courses of crSO2 and cFTOE during the first 15 min after birth are demonstrated in Tables 2, 3 and Figures 2, 3. There were no significant differences in crSO2 between the two groups. In the FIRS group, cFTOE was significantly lower in minutes 2, 3, and 4, and showed a trend toward a lower value in minute 5 after birth compared to the non-FIRS group.

Routine Monitoring
SpO2, HR, and FiO2 during the first 15 min after birth are demonstrated in Figures 4-6 and Supplementary Tables 1-3. SpO2 was significantly lower in minutes 5 and 6 after birth in the FIRS group. Afterwards, there were no significant differences between the two groups in SpO2. HR was significantly lower in the FIRS group in minutes 2 and 4 after birth. There were no significant differences in FiO2 between both groups within the first 15 min after birth.

Cerebral Injury and Mortality
At term-equivalent age or before discharge no significant differences in cerebral injury or mortality were observed. Twenty (87%) preterm neonates in the FIRS group and 22 (96%) neonates in the non-FIRS group survived without cerebral injury. In the FIRS group one neonate had an IVH Grade I, one neonate an IVH Grade III and one neonate a PVL Grade I. The IL6 values of these neonates were 19.7, 686.2, and 959.9 pg/ml, respectively. In the non-FIRS group one neonate had a PVL Grade I (IL-6 value 3.0 pg/ml). One (4%) preterm neonate of the FIRS group died (IL-6 value 959.9 pg/ml).

DISCUSSION
To our knowledge, this is the first study to evaluate the potential influence of FIRS on cerebral oxygenation measured with NIRS in preterm neonates during the first 15 min after birth. Preterm neonates with FIRS showed significantly lower cFTOE values in the first 4 min after birth compared to neonates in the non-FIRS group.
One would also expect significantly higher crSO2 values when cFTOE is significantly lower. However, in the present study we did not observe significant differences in crSO2 despite significant differences in cFTOE between the groups. This suggests that in the FIRS group not only oxygen consumption but also oxygen delivery was lower, resulting in a similar crSO2 in both groups.
Reduced oxygen consumption during inflammation and sepsis can be explained by mitochondrial dysfunction with impaired energy production (21). There are three possible mechanism for this mitochondrial dysfunction: (i) dysfunction is secondary due to tissue hypoxia, (ii) impairment of oxygen utilization due to cytokines, and (iii) active mitochondrial measure of survival strategy resembling stunning or hibernation. In the present study the first explanation should not play an important role since tissue oxygenation was even higher in neonates with FIRS group.
Cerebral oxygen delivery is a function of Hb concentration, arterial oxygen content and cerebral blood flow depending on cardiac output and vascular resistance. SpO2 representing oxygen content was similar in both groups in the first 4 min. In neonates, cardiac output is strongly dependent on HR. Thus, HR determines strongly cerebral blood flow, depending on vascular resistance (22). HR was significantly lower in the first minutes in the FIRS group compared to the non-FIRS group in the present study. Therefore, it can be assumed that the observed lower HR in the FIRS group resulted in a reduction of cerebral blood flow and thus in a reduction of cerebral oxygen delivery. Cerebral oxygenation and cerebral perfusion might also be influenced immediately after birth by open shunts (persistent ductus arteriosus and persistent foramen ovale) (23). Influence of cardiac output and possible shunts via persistent ductus arteriosus and persistent foramen ovale on the present findings cannot be ruled out completely, since in the included studies no echocardiography was performed. In addition an elevation of IL-6 causes stress due to inflammation, under regulation of neural and endocrine response (24). In unprepared preterm neonates, it is assumed that the production of IL-6 causes stress induced by adrenal gland cells (25). cFTOE measured with NIRS reflects oxygen saturation in veins (70-80%), capillaries (5-10%) and arteries (15-25%) (26). If FIRS resulted in a centralization of the circulation, this may have changed the ratio of the arterial, capillary and venous compartments. As a consequence this resulted in a local decrease of oxygen consumption, because of a higher proportion of arterial blood vessels (relative to reduced number of capillary vessels within the measurement compartments), resulting in higher values of crSO2 and consecutively in lower cFTOE values (27,28). However, we do not have detailed information about the behavior of cerebral blood flow to define exact changes in cerebral perfusion to prove the underlying mechanism of the observed differences between groups (13,14,29). Rallis et al. (16) measured cerebral oxygenation with NIRS in neonates on the first, third, and seventh day of sepsis. No differences in crSO2 between neonates with sepsis and those without sepsis were found on the first and third day, but crSO2 was severely compromised on the seventh day of sepsis (16). In the present study, we found differences between the FIRS and the non-FIRS group already within the first 5 min after birth, with lower cFTOE values in the FIRS group but no significant differences in crSO2. This inconsistency between the studies can be explained by the different time point of measurement as well as by differences in circulation and ventilation between the first minutes after birth and the first week after birth.
In the present study, there were no differences between the two groups in PCT, determined from umbilical cord blood, and in CRP values, taken on the first and on the second day after birth. Therefore, we assume that FIRS is not always linked with a neonatal infection. Ebenebe et al. (23) investigated factors associated with elevated umbilical cord blood IL-6 values in neonates without infection. They demonstrated that neonates can cope with perinatal stress or intrauterine inflammation (elevated IL-6 values) without developing any clinical signs of inflammation or infection. In detail, out of 471 neonates with no clinical signs of infection within 72 h, 139 neonates showed IL-6 greater 11 pg/ml (25).
In the present study, no significant differences in cerebral injury or mortality at term-equivalent age or before discharge were observed between the two groups, whereby the overall number of cerebral injury and death in both groups was low.
Ozalkaya et al. (30) described that umbilical cord blood IL-6 concentrations > 37.7, > 26.7, and > 17.5 pg/ml predicted death, RDSand multi-organ failure, respectively. In addition, preterm neonates with a gestational age of > 32 weeks and a birth weight > 1,500 g have a lower risk for the development of cerebral injury, like IVH or PVL when compared to neonates born below 30 weeks of gestation (31)(32)(33)(34). In our patients, the median gestational age and birth weight were > 32 weeks and > 1,500g, respectively. Further, the majority of IL-6 values in the FIRS group were relatively low. Therefore, we suppose that the published association between FIRS and adverse neonatal outcome (3,30) may occur more often in preterm neonates with a lower gestational age and birth weight and higher IL-6 values.

Strengths and Limitations
The strength of the present study is the matching of the FIRS and non-FIRS group, rendering them comparable in terms of demographic parameters. However, due to matching the number of analyzed preterm neonates in both groups became rather small. Furthermore, the majority of IL-6 values in the FIRS group (median IL-6 19.7 pg/ml) was relatively low, although they fulfilled the evidence-based definition of FIRS (1). However, significant differences in cFTOE values between the two groups were observed.

CONCLUSION
In preterm neonates with FIRS, crSO2 was similar despite significantly lower cFTOE values during the first minutes after birth compared to neonates without FIRS. This observation may be a result of compromised oxygen consumption and delivery in the first minutes after birth in neonates with FIRS.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Committee on Biomedical Research Ethics, Medicial University of Graz, Auenbruggerplatz 2, 8036 Graz, Austria. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
CH, AA, and GP: substantially contributed to the conception and design of the work. CH, MB, NB-S, BS, LM, AA, BU, and GP: analysis and interpretation of data, drafting the work and revising it critically, final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

ACKNOWLEDGMENTS
We would like to express our gratitude to the parents for giving permission to investigate their infants and to the team of midwives, nurses, laboratory staff, and physicians involved in their care. We also want to thank Evelyn Ziehenberger for her assistance in completing this study and her valuable technical support during performance of study measurements.