Based on pilot data of 15 patients (not included in the study) it was found that the standard deviation (SD) of PIPP was 3.7. Taking SD as 3.7 and clinically significant difference as 2 on the PIPP score and considering 5% level of significance with a power of 80%, a sample size of 50 in each condition was required, Considering a 5% drop out rate the estimated sample size was 52. Balanced randomization of the participant neonates was done into the two groups using GraphPad software. Sealed opaque envelopes containing the randomization code were opened just before the procedure by the person who was responsible for giving oral sucrose and recording videos.

Newborns in the Intervention group received oral sucrose (Arbineo 24% w/v oral solution, dose: 1mL for 32-40 weeks, 2mL for >40 weeks) 2 minutes prior to echocardiography by a dropper, whereas newborns in the control group did not receive any medication/placebo. Investigators performing echocardiography were unaware of the status of the neonate. Echocardiography involved all regular views done during targeted neonatal echocardiography and did not involve removal of electrodes from the chest in any neonate. Echocardio-graphy in the present study was conducted by neonatal fellows, trained in targeted neonatal echocardiography. During echocardiography, saturation of oxygen (SPO2) and pulse rate monitoring was done using Philips IntelliVue MP 20 monitor, and video recording was performed using smart phones (Micromax A110Q, Sony Xperia SP-C5302 and Samsung Galaxy SIII). The videos were then transferred to a hard disc and erased from the recording instrument. The investigators performing the video analysis were blinded to the group allocation. Baseline recording of behavioral state, heart rate and SPO2 was done for 15 seconds prior to initiating echocardiography in both the groups. Gestational age was calculated using Naegel’s formula.

Premature infant pain profile (PIPP) scale was used for assessment of pain during echocardiography [16]. PIPP has good inter-rater reliability and can also be applied for term neonates [17]. No specific PIPP scores were done for different views. The lowest reading of SPO2, highest reading for heart rate and the maximum facial expression irrespective of time, during the procedure of echocardiography were utilized for scoring. Video analysis was done by three experienced researchers in a group, and PIPP scores were assigned to the videos after reaching a consensus. Monitoring of the neonate was done for one hour after the administration of sucrose to look for regurgitation or other side effects. Blood sugar was done after one hour to determine hyperglycemia. A week later, a follow up interview of the caretakers was done to determine if any complications like necrotizing enterocolitis (NEC), feed intolerance etc. had occurred.

Statistical analysis: Data was entered into Microsoft Excel. Analysis of the data was performed using SPSS 14.0. Independent sample t-test and descriptive statistics were used for analysis. Bonferroni’s correction was applied to determine the significance of various domains of PIPP score. There are seven domains in the PIPP score and the significance value was redefined by dividing the conventional P value of 0.05 by 7. Thus a P value < 0.007 was considered statistically significant.

Results

A total of 104 echocardiography examinations of 76 neonates were included in the study (52 examinations in each group) (Fig. 1). The baseline characteristics of both the groups were comparable (Table I). The mean (SD) time for which echocardiography was performed in the intervention group was 6.1 (3.05) minutes, and for the control group was 4.9 (2.18) minutes. (P=0.024).

TABLE I	Comparison of Basal Characteristics of the Groups

Characteristics	Intervention	Control group
	group  (n = 52)	(n = 52)
Gest. age, Mean (SD), wk	37.1 (2.3)	37.6 (2.4)
B. weight, Mean (SD), kg	2.1 (0.7)	2.2 (0.6)
SGA	36 (69.2%)	34 (65.4 %)
AGA	16 (30.7%)	18 (34.6 %)
Male gender	30 (57.6%)	26 (50 %)
Feeding characteristics
BF	23 (44.2%)	31 (59.6 %)
KS Feed	7 (13.4%)	7 (13.4 %)
RT Feed	22 (42.3%)	14 (26.9 %)
SpO2, Mean (SD)	96.3 (2.9)	97.2 (2.9)
Heart rate, Mean (SD)	147 ( 21)	138 (19)
SGA – small for gestational age, AGA – appropriate for gestational age, BF – breastfeeding, KS – katori spoon feeding, RT – ryle’s tube.

Fig. 1 Participant CONSORT flow diagram.

The mean (SD) PIPP score of intervention group was 5.2 (1.92) as compared to 7.4 (3.78) in control group (Fig. 2). The mean difference in the PIPP score of both the groups was 2.15 (P<0.001) (Table II). Thirty (57.7%) of the neonates belonging to the control group experienced pain while doing echocardiography (PIPP score ³7). Of these neonates, 24 (46.1%) experienced mild-moderate pain (PIPP score = 7-12), while others experienced severe pain (PIPP score >12). In the intervention (sucrose) group, 42 (80.8%) neonates experienced no pain as indicated by a PIPP score <7, 10 (19.2%) neonates experienced mild-moderate pain (PIPP score = 7-12). No neonate in the sucrose experienced severe pain (PIPP score >12). Four (7.6%) neonates spitted the sucrose solution after administration. No episode of hyperglycemia, necrotizing enterocolitis, or feed intolerance was reported after sucrose administration.

Fig. 2 Box plot showing mean PIPP score of both the groups.

TABLE II Details of the PIPP Score of the Groups

A total of 52 echocardiography examinations in each group were included in the study. Both of the study limbs were comparable in terms of their demographic profile. Mean (SD) PIPP score of intervention group was 5.25 (1.92) as compared to 7.40 (3.78) in control group. Forty six percent neonates experienced mild-moderate pain, and 12% neonates experienced severe pain in control group, while in intervention (sucrose) group, nineteen percent neonates experienced mild-moderate pain with no neonate experiencing severe pain.

The present study included neonates with established enteral feeding, and many neonates on respiratory support, having critical CHD or profound shock were excluded. Hence majority of neonates who needed frequent and longer echocardiography were excluded. Another aspect to be considered is the duration of action of sucrose, which varies from 2 to 5 minutes, extending unto 45 minutes [15]. This duration of action may sometimes be shorter than the duration of echocardiography, necessitating repeat dosing of sucrose. In the present study, no repeat dosing was given, as the personnel performing echocardiography were blinded and duration of echocardiography was short. Oral placebo was not utilized in the present study. Difference in the intervention time can have its independent influence on the PIPP score, but in spite of longer echocardiography time in intervention group they had lower PIPP scores, thereby showing effectiveness of oral sucrose.

In present study, we included neonates with gestational age of 32-42 weeks because in a study by Johnston, et al. [18,19], it was reported that preterm infants <31 weeks who had received >10 doses of sucrose per day in the first week of life had poor neurologic outcome. As shown in previous Cochrane analyses, there are wide variations in the inclusion criteria of various studies [6,8]. Probable cause for this difference in spectrum of patients enrolled might be the different clinical practices and socio-economic scenario world over.

It is evident that sucrose is effective in lowering mean PIPP score and the behavioral components of the PIPP score but has no/little effect on the physiological components of the PIPP scale (heart rate and SpO2 variability). A study by Slater, et al. [20] showed no significant effect of sucrose administered orally in reducing brain activity specific to pain, identified by EEG, in spite of significant change in PIPP score. These observations indicate that measuring pain is a complex process, and measuring only parts of the PIPP score may often be misleading and the whole score in total should be interpreted [21]. The present study also provides indirect evidence, as to why a scale which combines both the physiological and behavioral parameters should be used and not a scale which focuses only on one aspect, as there was no statistically and clinically significant difference in the mean change in heart rate and SPO2 during echocardiography between both the groups, but there was significant change in behavioral parameters. There was a clinically and statistically significant difference in PIPP score in intervention group 5.25 (1.92) vs. control group 7.4 (3.78), P<0.001. This was significant even after Bonferroni’s correction. This finding is similar to other studies [8, 22].

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