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Interleukins in Diagnosis of Perinatal Asphyxia: A Systematic Review

Hassan Boskabadi1*, Ali Moradi2 and Maryam Zakerihamidi3

1Department of Pediatrics, Neonatal Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2Orthopedic Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

3Department of Midwifery, Faculty of Medicine, Islamic Azad University, Tonekabon Branch, Tonekabon, Iran

*Corresponding Author:
Boskabadi H
Department of Pediatrics
Neonatal Research Centre
Faculty of Medicine
Mashhad University of Medical Sciences
Mashhad, Iran
Tel: +98 51 3841 3007
E-mail: [email protected]

Received date: June 02, 2017; Accepted date: June 15, 2017; Published date: June 23, 2017

Citation: Boskabadi H, Moradi A, Zakerihamidi M (2017) Interleukins in Diagnosis of Perinatal Asphyxia: A Systematic Review. Med Rep Case Stud 2: 138. doi: 10.4172/2572-5130.1000138

Copyright: © 2017 Boskabadi H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Abstract Background: Perinatal asphyxia is a serious perinatal problem that causes the death of infants all over the world each year. Biochemical markers including interleukins (IL) has been proposed for early diagnosis of asphyxia. This study has aimed to systematically review the significance of interleukin measurements in diagnosis of perinatal asphyxia. Materials and Methods: PubMed, Cochrane Library, Web of Science, Embase, and Scopus databases before 2017 were searched for the following keywords: asphyxia, neonatal, interleukin, and diagnosis. A total of 13 out of 300 searched papers were finally selected for evaluation. IL6 had been the most studied member among interleukin family (61.54% of the papers) with the most studies (23%) from Iran. interleukins under study were IL6 and IL-1β. Interleukins had been measured in ten studies by serum samples, two studies by samples of CSF (CSF) and one study by sample of umbilical cord blood. Briefing the available data was conducted on the studies having reported the relationship between neonatal asphyxia diagnosis and interleukins. The inclusion criteria were: studies on neonates; adequate information from the test results. Studies using markers other than interleukins to detect asphyxia, however; studies whose abstracts were only available were excluded. Results: Research on the issue suggests that IL6 >41 Pg/dl has the sensitivity of 84.88%, and the specificity of 85.43%, whereas IL-1ß >4.7 Pg/dl has the sensitivity of 78% and specificity of 83 %) in the diagnosis of neonatal asphyxia. Among diagnostic interleukins for neonatal asphyxia, combination of IL6 and IL-1ß had the highest sensitivity i.e. 92.9%. Conclusion: IL6 and IL-1β of serum samples are used in early diagnosis of perinatal asphyxia and are useful predictors for the outcomes of perinatal asphyxia and its intensity. In addition, simultaneous evaluation of IL-1β and IL6 can improve the sensitivity of the early diagnosis of perinatal asphyxia.

Keywords

Diagnosis; Interleukins; Perinatal asphyxia

Introduction

Perinatal asphyxia has been defined as the lack of oxygen which occurs either before, during, or after birth [1]. It is a significant cause of perinatal morbidity and mortality as well as neurological disabilities in the surviving babies. In addition to digestive, liver, metabolic, and hematologic disorders; respiratory distress syndrome; disseminated intravascular coagulation; subcutaneous fat necrosis; ischemic heart disease; adrenal hemorrhage; and acute tubular necrosis some of the affected newborns will suffer from hypoxic ischemic encephalopathy (HIE) [2,3].

Annually, 4 million babies are born with perinatal asphyxia; of whom, 800,000 die and the same number experience adverse clinical outcomes [4]. The mortality and morbidity rates among patients with moderate or severe HIE is very high. Half of patients with severe HIE will die, while almost all survivors suffer from neuro-developmental deficits, cerebral palsy, epilepsy, and learning disorders [5,6]. A previous two-year follow-up study has reported 26% mortality in asphyxic neonates, with 28% experiencing developmental delay [3].

Several pathophysiologic mechanisms of brain damage in neonates are linked to HIE. Early assessment of the severity of a HIE-induced acute brain injury can be very useful for the prevention or treatment decisions in such neonates [7]. Prediction of perinatal asphyxia is done using multiple assessments including the electronic monitoring of fetal heart rate during labor, cord or fetus pH measurements, meconiumstained amniotic fluid, Apgar score, the severity of HIE, PAB, blood markers (NRBC in umbilical cord blood), and multiple organs impairments. None of these factors alone are sufficient and combinations of parameters are clinically used for early diagnosis of perinatal asphyxia [8,9].

Recent studies have focused on the inflammatory cytokines such as IL-1ß, IL6, and IL8 for early diagnosis of brain damage. As interleukins are known as one of the early inflammatory responses to infections, they are potentially important in early diagnosis and hence proper management of both infectious and non-infectious conditions before the establishment of fulminant stage.

The inflammatory cytokines are involved in the biochemical pathways leading to ischemic-hypoxic injury [10,11]. Lymphocytes, monocytes, and macrophages secrete ILs in response to stimuli [12].

The role of inflammation in neonatal CNS injuries as well as the role of cytokines as mediators of injuries have recently been identified [13]. It is likely that the pathophysiology of perinatal asphyxia has a close relationship with the inflammatory mediators such as cytokines [14].

Many of these cytokines such as IL-1β, IL6, IL8, IL10, and IL12 increase during the inflammatory responses [15]. A study has shown that cytokines cause brain damage through direct injury to the white matter, weakening the germinal matrix endothelium, brain hemorrhage, and inflammatory reactions caused by microglia and astrocytes [16]. Although cytokines play a role in the regulation of cell apoptosis in CNS as well as leukocyte differentiation, proliferation, and infiltration, the precise role of pro-inflammatory cytokines such as IL6 as the main mediator in the development of brain damage is still unknown [17].

Although several studies have been conducted on the relationship between the ILs and infectious conditions, however, comparison of their value in confirmation or rule out of the diagnosis of infection has not been fully discussed yet. Identification of biochemical markers such as interleukin may be useful for early diagnosis of asphyxia. Early diagnosis of perinatal asphyxia helps to provide better health care and improved outcomes. Brain injury is also a common cause of sever morbidity with poor outcomes and high mortality during the perinatal period. The current systematic review was conducted aiming at the identification of neonatal perinatal asphyxia by IL levels.

Materials and Methods

Selection of ILs for the diagnosis of neonatal asphyxia

After an initial review of the literature, a list of interleukins was prepared to conduct a systematic review. The articles examining the role of interleukins in the diagnosis of neonatal asphyxia were addressed and studied. In this regard, articles containing interleukins such as interleukin-1, interleukin-6 or combinations of both were analyzed for the diagnosis of neonatal asphyxia.

Search strategy

PubMed, Cochrane Library, Web of Science, Embase, and Scopus databases were used for this systematic review. “Asphyxia”, “neonatal asphyxia”, “perinatal asphyxia”, “interleukin”, and “diagnosis” were the search keywords. All articles in English and Persian between and consisting 1997 and 2016 were searched.

Inclusion criteria

Articles with the following criteria were selected for this review: 1) Neonates as the study population; 2) Neonatal asphyxia as the specified study; 3) ILs must be examined for the diagnosis or prediction of neonatal asphyxia in umbilical cord blood, serum or CSF; and 4) Adequate information from the test results.

Exclusion criteria

Animal studies; articles that used other markers for the diagnosis of asphyxia rather than ILs; and articles with only abstract available were excluded from the study.

Data extraction and quality assessment of the articles

The full text articles were downloaded and the extracted data were collected in Microsoft Excel with the following titles: the authors’ name and surname, years of publication, method, study area, case group, control group, type of IL, location of sampling, time for measuring IL, sensitivity, specificity; positive predictive value, negative predictive value, and the results of the investigation.

Initially 300 studies were collected using EndNote software and the duplicate articles (n=115) were excluded from the review. According to the title and abstract 65 more articles were excluded. A total of 107 other articles were also removed due to incomplete data, unavailability of full text, animal studies, uncertainty of the study type and the target group. Finally, 13 related articles underwent further analysis (Figure 1).

medical-reports-case-studies-sensitivity

Figure 1: The sensitivity, specificity, positive, and negative predictive values (%) of IL6 in prediction of asphyxia* and its adverse outcomes.

The methodological qualities of the papers were determined using the quality control tool for diagnostic accuracy of the studies (QUADAS2) consisting of 14 questions and answers where 1, -1, and 0 were scored for "Yes", "No" and "Unknown", respectively. The maximum score was 14 [18].

Results

A total of 13 out of 300 articles with a total sample size of 1120 neonates were examined. Eight articles had discussed IL6 (61.54%), 2 studies (15.38%) had described about IL-1β, and 3 articles (23.08%) were related to the combination of both interleukins.

The number of the studies on ILs

Review of studies conducted from 1997 to 2016 showed that IL6 is the most frequently studied biomarker among interleukin family. Interleukins had been measured in 10 studies on serum samples (76.92%), 2 on CSF samples (15.39%) and 1 on umbilical cord blood samples (7.69%). All studies were prospective ones.

Heterogeneity of the articles

Studies on the relationship between IL and diagnosis of perinatal asphyxia were different in terms of inclusion criteria, sample size, sampling location, time of assessment, and the diagnostic value of interleukins. Only 5 studies have discussed the diagnostic value of interleukin (38.46%). Both IL6 and IL-1β cutoff values were 11.91-100 pg/ml and 3.35-6.7 pg/ml, respectively. The sensitivity and specificity as well as the positive and negative predictive values of interleukins were different in the articles under study.

IL6 (8 studies)

In a study on serum levels of IL6 at birth, 24 and 48 hours after delivery for 37 neonates with perinatal asphyxia and 45 healthy neonates serum concentrations of IL6 was reported as 43 times higher in asphyxiated neonates with HIE and 1.9 times higher in asphyxiated neonates without HIE compared to the healthy neonates.

Serum concentrations of IL6 were increased in neonates with asphyxia after birth and this was associated with the severity of encephalopathy and poor outcomes. The sensitivity, specificity, positive, and negative predictive value of IL6 in prediction of asphyxia and its adverse outcomes are shown in Figure 1. So following cerebrospinal system injury, IL6 plays an important role and its serum concentrations can be a useful predictor of HIE outcomes [1].

Another study comparing the IL6 in 50 neonates with noninfectious perinatal asphyxia with 113 healthy neonates at similar time points showed that the average concentrations of IL6 in neonates with HIE were 376 times higher than the healthy babies and 5.5 times higher in neonates with asphyxia without HIE. A critical relationship was also found between IL6 and the degree of HIE as well as neurodevelopmental outcomes at 2 years. Regardless of the outcomes, serum IL6 concentration was significantly lower in the first 24 and 48 hours after birth. Umbilical cord concentration of IL6 >100 pg/ml had the sensitivity and specificity of 70.8% and 80.9%, respectively, in predicting moderate to severe HIE. It was concluded that IL6 measurement in the umbilical cord blood of newborns with perinatal asphyxia may be useful in early detection of neonates with high risk of brain damage and adverse outcomes [19].

A study on the neurological markers (IL6) in perinatal asphyxia and its relationship with different stages of HIE was performed on 100 asphyxic and 100 healthy neonates with blood samplings in the first and third days of birth. The results showed that the average concentration of serum IL6 on the third day of birth of asphyxic neonates declined. There also was a negative relationship between IL6 concentrations in the first and third days of birth. The IL6 concentration had a reverse correlation with the HIE stages (Stages 1-3) within the 1st to the 3rd day of birth. In addition, there was a negative relationship between the first and third day of life in terms of IL6 concentrations in various stages of HIE. It was concluded that IL6 concentration increases after birth asphyxia and this increase is associated with the severity of HIE and poor outcomes [20].

In a study on the predictors of early-HIE in Egypt serum IL6 was measured in the first 12 hours of life in 27 perinatal asphyxic as well as 25 healthy neonates. The HIE group showed significantly higher IL6 levels compared to the controls. The IL6 levels were associated with the severity of HIE. It was suggested that assessment of IL6 level may be useful in early diagnosis of perinatal asphyxia [21].

IL6 measurements have also been performed in the CSF of full-term neonates with HIE. This prospective study was performed on 20 healthy newborns (no sepsis or meningitis; 1 and 2 min Apgar scores ≥9) and 15 neonates in the case group (with asphyxia and Apgar score of ≤4 in the 1st minute and ≤6 in the 5th minute, umbilical cord blood pH <7.20 or lactate >3 mmol/liter in the umbilical artery blood and need for artificial ventilation for at least 2 minutes after birth). The CSF samples were collected within the first 48 hours after birth to identify IL6. The results showed that the average IL6 in the case group (157.5 pg/ml) was significantly higher than the control group (4.1 pg/ml). The researchers concluded that IL6 levels in term neonates with HIE is higher than the control group [22].

A different study aiming at the evaluation of IL6 in CSF after perinatal asphyxia and its relationship with early and late nervous observations, was conducted on 20 infants, among whom, 3 cases had no HIE (stage 0) 5 infants were at stage 1, 6 infants at stage 2 and 6 infants at stage 3 of HIE. The IL6 concentration in CSF (from 8 to 90 hours of birth) in infants at stage 3 of HIE was higher than those at stage 0 to 2. Infants with brain damage and adverse outcomes showed higher CSF levels of IL6. The increase in CSF IL6 after perinatal asphyxia was related to the intensity of HIE, brain damage, and neurological outcomes. The researchers concluded that IL6 may be involved in hypoxic-ischemic brain damage [23,24].

A study on the clinical significance of serum IL6 in neonates with HIE was performed on 74 neonates with HIE along with 74 healthy neonates. An increase in the the inflammatory mediators was associated with the severity of the disease and positively correlated with prognosis. They reported high levels of IL6 in neonates with HIE. High concentrations of IL6 in infants with HIE suggests that these inflammatory mediators play an important role in the development and prognosis of the disease [23].

Serum concentrations of 50 asphyxic and 20 healthy neonates were measured on days 1, 3 and 7 after birth in a study on the relationship between IL6 and brain damage in perinatal asphyxia. The results showed that IL6 levels in asphyxic neonates reduced within a week after birth and reverted to the normal level on day 7 after birth; however, the IL6 levels were significantly lower in neonates with brain damage compared neonates with no brain injury. As a conclusion IL6 levels increase in neonatal asphyxia; hence, it can be involved in the pathophysiology of neonatal asphyxia [25].

IL-1β (2 articles)

Serum levels of IL-1β were studied in 38 neonates with noninfectious perinatal asphyxia (blood pH<7.2, low Apgar score, and fetal distress symptoms) and 41 healthy neonates (natural babies with no clinical signs of asphyxia during the 1st week after delivery) at birth as well as 24 and 48 hours after delivery. Serum levels of IL-1β in neonates with HIE were 5 times more than asphyxiated newborns without HIE and 6 times higher than healthy babies. Also a significant relationship was found between IL-1β and neonatal outcomes at discharge.

The sensitivity, specificity, positive, and negative predictive value of IL-1β in prediction of the occurrence of asphyxia and its adverse outcomes are shown in Figure 2.

medical-reports-case-studies-occurrence

Figure 2: The sensitivity, specificity, positive, and negative predictive values (%) of IL-1β in prediction of the occurrence of asphyxia* and its adverse outcomes.

The researchers concluded that the increase in serum levels of IL-1β in asphyxic neonates is a predictor of poor outcomes. In other words, serum IL-1β is a predictor for the severity of perinatal asphyxia and its short-term neural outcomes [26].

A different prospective study was conducted on the predictive value of plasma and CSF concentrations of IL-1β in the outcomes of 30 term neonates with HIE. Blood and CSF samples were collected within the first 24 hours after birth. Five babies died immediately after hypoxia.

The neurological examination and Denver Developmental Screening Test were performed at one year of age. Eleven neonates had normal and 14 had abnormal neurological findings or abnormal Denver Developmental Screening Test. The results indicated that the CSF concentration of IL-1β in unhealthy infants was significantly higher than healthy ones.

However, no significant difference was found in the plasma concentrations of IL-1β in two groups. Patients with CSF samples taken within 6 hours of hypoxia had higher levels of IL-1β compared to those with sampling after 6 hours of hypoxia. The researchers concluded that IL-1β level is correlated with CNS damage after hypoxia and can be a useful predictor for HIE [27].

Combination of IL6 and IL-1β (3 articles): Combination of IL6 and IL-1β at birth was studied in 38 infectious infants with perinatal asphyxia (pH<7.2, low Apgar score, and fetal distress symptoms) and 47 healthy infants.

Serum concentrations of IL6 and IL-1β were significantly higher in infants with perinatal asphyxia compared to the healthy ones (88.15 vs. 6.74 pb/ml for IL6 and 16.88 vs. 3.34 pb/ml for IL-1β). The sensitivity and specificity of IL6 and IL-1β are shown in Figure 3.

medical-reports-case-studies-diagnosis

Figure 3: The sensitivity and specificity (%) of IL6 and IL-1β in prediction of diagnosis of perinatal asphyxia [28].

The turning points for IL6 and IL-1β were 11.91 pb/ml and 3.35 pb/ml, respectively. The researchers concluded that simultaneous assessment of IL6 and IL-1β can improve the sensitivity and specificity for early diagnosis of perinatal asphyxia. In addition, the assessment of combination of IL-1β and IL6 was the best indicator for perinatal asphyxia [28].

In a different study on evaluation of the markers for early diagnosis of brain damage in the preterm low birth weight infants with perinatal asphyxia, 29 infants with perinatal asphyxia and 28 healthy infants were involved. Serum IL6 and IL-1β were measured in the first 24 hours and the 3rd and 7th days of birth. The study results showed that serum IL6 and IL-1β were significantly higher in infants with perinatal asphyxia in the first 24 hours after birth compared to the healthy infants. Neurologic evaluation of 14 infants with perinatal asphyxia and 12 healthy ones at 18 months of age revealed that 8 infants with perinatal asphyxia had abnormal findings that were associated with serum levels of IL6 and IL-1β in 24 hours after birth. The researchers concluded that increased levels of pro-inflammatory cytokines are the primary findings for the future neurological disorders in infants with asphyxia [29].

In an evaluation of the inflammatory cytokines in 55 healthy and 45 infants with HIE serum levels of IL6 and IL-1β were measured at birth, 24 and 72 hours after birth. A significant increase was found in IL6 and IL-1β in infants with HIE at three time points [30].

Combination of IL6 and IL-1ß had the highest average sensitivity (92.9%) and specificity (85.43%) among diagnostic interleukins in asphyxiated infants.

Discussion

Perinatal asphyxia is a common and serious problem which results in annually almost a million infant deaths in the world. Perinatal asphyxia may also have negative impacts on all main body organs [3]. HIE increases the quick expression of inflammatory cytokines of brain (IL1 and IL6) [31].

Cytokines, especially IL6 affect differentiation, growth and survival of cells in infants. Thus, infants’ brain is particularly sensitive to the cytokine concentrations [32]. IL6 level is one of the powerful predictors of HIE outcomes such as death and long-term neuro-developmental problems [19]. It appears as a considerable product among inflammatory cytokines in the pathogenesis of perinatal asphyxia [33]. IL6 is as an inflammatory mediator in brain injury and plays a central role in the inflammatory responses [34]. It is not clear whether IL6 has a devastating effect on neurons or healing effect after brain ischemic damage [35]. IL6 may be released as a protective response after hypoxic-ischemic brain damages. It acts as a cytokine with two pro-inflammatory and anti-inflammatory properties [36]. IL6 has been shown to play two roles in cerebral ischemia: as an inflammatory mediator during the acute phase; and as a neurotrophic mediator within the acute and long-term stages [37]. Higher levels of IL6 have been reported in infants with perinatal asphyxia and hypothermia [38].

IL6 is involved in the induction of acute reactions and control the inflammatory responses causing a reduction in pro-inflammatory cytokines and an increase in anti-inflammatory molecules during acute cerebral ischemia stage [39].

IL6 increases in CSF fluid in asphyxic infants and is related with the severity of HIE [24]. A direct relationship has been reported between the CSF levels of IL6 and TNF-α and neurological prognosis after acute cerebral ischemia in adults [40]. It has been shown that CSF levels of IL6 are significantly higher in infants with severe neurological observations compared to mild or moderate encephalopathy [24]. Increased IL6 in the CSF has been shown to be related with the severity of HIE, brain injury and neurological outcomes at 12 or 72 hours after perinatal asphyxia [24]. Serum concentrations of IL6 increased approximately 12 hours after birth [1]. A recent study reported an increase in serum levels of IL6 in infants with HIE [36]. Animal studies have shown an increase in the peak serum levels of IL6 in rats with HIE in 6 hours after creating HIE so that the concentration of interleukin returns to the base level after 20 hours [31]; However, researchers have not specified a certain turning point for IL6 which predicts long-term adverse outcomes. A significant relationship has been reported between the serum concentrations of IL6 and Sarnat encephalopathy grading [35]. Also, increased levels of IL6, IL11 and IL13 have been reported in dried blood samples of infants with cerebral palsy [41].

The results of an animal study showed that the serial injection of synthetic IL6 prevents learning disabilities and delays the loss of neurons [42]. In another study, the increase in serum IL6 was associated with poor outcomes or death in infants with perinatal asphyxia [43]. An increased IL6 in amniotic fluid and cord blood were associated with outcomes such as cerebral palsy and periventricular leukomalacia, respectively [44]. Serum IL6 had 86% positive predictive value and 100% specificity in predicting moderate to severe HIE [45].

Available studies have indicated that IL6<41 pg/dL had sensitivity and specificity of 88.84% and 85.43%, respectively, in the diagnosis of neonatal asphyxia. Therefore, the highest sensitivity and specificity are for the diagnosis of asphyxia related to IL6.

IL-1β in umbilical cord is a major bio-outcome for brain injury whose levels are significantly high in infants with HIE and predict severe HIE and adverse outcomes in 6 to 12 months of age [46]. IL-1β is an important cytokine which is often released by mononuclear cells and macrophages in response to infection and tissue injury and plays an important role in the metabolic disorders and organ failure caused by infection [47]. IL-1β transiently increases after hypoxia and reaches its peak within 6 hours [31].

Increased CSF concentrations of IL-1β are associated with neurologic outcomes after perinatal asphyxia. It could be concluded that IL-1β has neurotrophic and neuroprotective results. However, it is not clear whether IL-1β is involved in the destruction or repair of neurons after ischemic brain injuries [35]. CSF levels of TNF-α and IL-1β were higher in term infants with HIE who developed nervous disorders in one age [48]. Neuroprotective antagonist effects of IL-1β receptor against brain damages before or after exposure to hypoxia have already been reported [49]. The available studies showed that IL-1β <4.7 pg/dl had sensitivity and specificity of 78% and 83%, respectively, in the diagnosis of neonatal asphyxia. IL-1β and IL6 are significantly increased in birth asphyxia and the rate of increase is associated with the severity of encephalopathy. Simultaneous assessment of IL-1β and IL6 is the best indicator of perinatal asphyxia [28]. An association has been reported between the increase in proinflammatory cytokines and white matter brain damage or abnormal neurological outcomes. Increased TNF-α, IL-1β and IL6 in the amniotic fluid of preterm infants have shown a significant relationship with white matter damage [50].

IL-1β and IL6 levels have been reported to be significantly higher in infants with perinatal asphyxia compared to the control group in 24 hours after birth [29]. In another study, serum levels of IL6, IL8 and IL10 were higher in infants with severe asphyxia (death or poor outcome) than those with asphyxia but no poor outcome [43].

Serum concentrations of IL-1β and IL6 were significantly higher in infants with perinatal asphyxia than that in healthy infants. Simultaneous assessment of IL-1β and IL6 improved the sensitivity and specificity of early diagnosis of perinatal asphyxia. Assessment of combined IL-1β and IL6 was suggested as the best indicator of perinatal asphyxia [28].

Available studies indicated that simultaneous assessment of IL-1β and IL6 had the sensitivity of 93% and specificity of 43.5% in the diagnosis of neonatal asphyxia.

The current review is the only study that has examined the role of interleukin in the diagnosis of perinatal asphyxia. Limitations of this study include the lack of access to unpublished articles and reports, inappropriate and low quality reports, limited number of articles and infeasibility of accurate judgments about their effectiveness, lack of similar definitions for case groups, lack of cutoff point calculations, and lack of diagnostic value of IL in all the studies under review.

Conclusion

Serum and CSF concentrations of interleukins IL6 and IL-1β increased after asphyxia and the rate of increase was associated with the severity of asphyxia and adverse outcomes. Therefore, combination of IL6 and IL-1β can be used as a potential substantially powerful marker for early diagnosis of perinatal asphyxia. Further studies are required in order to identify more involved interleukins and standardize their cutoff values in early diagnosis of neonatal asphyxia.

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