Abstract

Introduction

Perinatal asphyxia is a leading cause of perinatal mortality in developing countries. According to the World Health Organization, in 2004, worldwide there were 133 million live births and 3.7 million neonatal deaths, 98% of which occurred in developing countries.[1] About 23% of neonatal deaths were due to perinatal asphyxia.[2] It is estimated that about 3% of live births in developing countries require neonatal resuscitation,[3] but even in major hospitals in some developing countries resuscitation measures may be inappropriate.[4] Perinatal asphyxia is a major cause for neonatal mortality and morbidity around the world especially in developing countries like India.[1] Oxygen deprivation and excess accumulation of CO₂ results in metabolic acidosis. This can alter the ionic exchange and cause defect in the liberation of adenosine triphosphate eventually leading to energy failure. Thus, cations accumulate inside the cell causing cytotoxic edema. The reduction of O₂ results in the generation of reactive oxygen species (ROS) which interact with nucleic acid and make an alteration in the structure and functioning of the genome.[2] Peripheral blood lymphocyte culture technique has been found to be useful in analyzing the morphology and aberrations of human chromosome. We studied the effect of therapeutic hypothermia (TH) on chromosomes with karyotyping.

Subjects and Methods

This study was a randomized, controlled trial among term babies with perinatal asphyxia. It was single-blinded and carried out at Tertiary Care Centre in India between March 2011 and June 2014. This trial was approved by Institute Ethical Committee and was registered in the Clinical Trial Registry of India (No: CTRI/2011/10/002094).

There were totally 42 babies were recruited in hypothermia and 43 in the control group. Babies whose gestational age was ≥37 weeks with umbilical cord blood or arterial blood (within the first postnatal hour) pH ≤7 or base deficit ≥16 meq with evidence of encephalopathy and with any two of the following criteria viz: (i) Apgar at 10 min <5; (ii) evidence of fetal distress; (iii) assisted ventilation for at least 10 min after birth; (iv) evidence of any organ dysfunction, were included in the study. Babies were excluded from the study if they were more than 6 h of age at the time of randomization, had major congenital abnormalities, did not establish spontaneous respiration by 20 min after birth or parents refused consent. After obtaining informed written consent from the parent of the newborn fulfilling the inclusion criteria were randomized into hypothermia or control group using a computer-generated sequence of random numbers. Allocation concealment was done using sealed envelopes. Neonatologist who was not involved in the study did random number generation, concealment, and assignment of intervention.

Babies in the hypothermia group were cooled by placing cloth-covered gel packs on the side of the chest, abdomen, back, head, and axilla to achieve target rectal temperature of 33–34°C. This temperature was maintained by continuous rectal and skin temperature monitoring. Vital parameters were recorded and at the end of 72 h, the cooling gel packs were removed one by one, and the radiant warmer was turned on to raise the body temperature by 0.25°C/h to reach the target temperature of 36.5°C. Cooling was stopped before 72 h if there was any complication. Both groups were treated with the same standard regimen, except for the hypothermia. Blood (1 ml) was collected after completion of TH (at 72 h) for karyotyping. Chromosomal analysis was done with IKAROS karyotyping system, metasystems, Germany, based on recommendations of International System of Human Cytogenetic Nomenclature.[5]

Data were analyzed using both descriptive and inferential statistics. The continuous parameters were expressed as mean with standard deviation and the distribution of categorical data as frequencies and percentages. The comparison of the distribution of categorical data between the groups was carried out by using Chi-square or Fishers exact test. The mean between groups was compared using independent student's t-test. All statistical analysis was carried out at 5% level of significance, and P < 0.05 was considered as significant. The statistical analysis was carried out using IBM SPSS version 19.0.

Results

Karyotyping was done in 42 neonates of hypothermia group and 43 of the control group. Karyotyping was done at 72 h of life to assess chromosomal aberration. The detailed analysis on the comparison of chromosomal aberration between the groups at 72 h is given in Table 1. At 72 h (after completion of hypothermia), chromosomal aberration was significantly higher in control than hypothermia group (P < 0.01). The median chromosomal aberration was lower in hypothermia (2 [0–5]) than control group (4 [1–7]). Chromatid breakage is the most common aberration seen among both hypothermia and control group, followed by dicentric chromosomes [Table 2 and Figure 1].

Table 1

Comparison of chromosomal aberration between the groups

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Table 2

Distribution of chromosomal aberration between cases and controls

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Figure 1

Different chromosomal aberrations. (a) Chromosome breaks, (b) chromatid loss, (c) dicentric chromosome, (d) ring chromosome

Comparison of chromosomal aberration in relation to severity of encephalopathy among hypothermia group is given in Table 3. Chromosomal aberration was significantly higher in severe encephalopathy group than moderate encephalopathy group. The median chromosomal aberration in moderate encephalopathy was 1 (0–3), and severe encephalopathy was 3 (1-6). In control group also, Chromosomal aberration was significantly higher in severe encephalopathy group than moderate encephalopathy group. The median chromosomal aberration in moderate encephalopathy was 2 (1–5), and severe encephalopathy was 5 (4–7) [Table 3].

Table 3

Chromosomal aberration in relation to severity of encephalopathy in hypothermia group at 72 h

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Discussion

To the best of our knowledge, this is probably first human randomized controlled trial to explore the effect of TH on chromosomal aberration [Figure 1]. We used gel packs to induce TH. Efficacy of gel packs in maintaining temperature has been proved in earlier studies.[6,7,8] Inclusion criteria used for our study were relatively similar to that used in the National Institute of Child Health and Human Development trial for cooling study.[9] In this study, we evaluated the effect TH on chromosomal aberration with karyotyping.

The DNA damage results in chromosomal aberration due to direct attack by ROS that is overproduced during postischemic reperfusion.[10,11] ROS readily crosses the nuclear membrane and therefore, reacts with DNA molecule resulting in loss of its purine and pyrimidine bases. This insult to DNA leads to the formation of apurinic and apyrimidinic sites (AP sites) which is expressed as single or double strand DNA breaks. The primary DNA damage caused by genotoxic effects of ROS interferes during cell division since the damaged bases block DNA replication process and thereby induce chromosomal aberration. In this scenario, karyotyping of chromosomes will serve as a sensitive indicator of cellular oxidative injury.[11,12,13,14] Study of chromosomal aberration has been proven as an effective method to assess the DNA damage. Some of the previous studies have shown higher chromosomal aberration in asphyxiated babies compared to normal neonates.[15,16]

In this study, maternal antenatal and intrapartum characters were similar in both groups. Chromosomal analysis showed higher number of aberrations in control group than hypothermia group. Our findings are in accordance with the findings of previous animal study,[11] in which the effect of TH was studied in a rat model with occlusion of the middle cerebral artery and reperfusion. The study showed that TH attenuates the strand breaks. With the above results, we conclude that TH reduces the DNA damage in the cases with perinatal asphyxia.

On subgroup analysis based on the severity of hypoxic-ischemic encephalopathy (HIE), chromosomal aberrations were found to be significantly higher in severe HIE than moderate HIE group among both hypothermia and control group. Previous studies have shown that outcome after hypothermic treatment was strongly influenced by the severity of HIE. TH is less effective in severe HIE than the moderate HIE cases.[8,9,17,18] It could be because of shorter latent phase, more secondary energy failure, and increased cortical gray matter neuronal death.[19] Thus, we can postulate that TH is effective in all HIE cases but more effective in moderate than severe HIE.

We conclude that the TH significantly reduces DNA damage in perinatal asphyxia. It could be an important mechanism by which TH works in HIE.

References