Kirbaş et al. BMC Pediatrics(2024) 24:568
https://doi.org/10.1186/s12887-024-05033-1

BMC Pediatrics

RESEARCH

Open Access

Effect of kangaroo mother care and white
noise on physiological-stress parametersin heel lancing: randomized controlled study
Zila Özlem Kirbaş1 , Elif OdabaşI Aktaş2* , Bülent Bayraktar3

and Hava Özkan4

Abstract
Background Newborns are exposed to varying degrees of stressful interventions due to procedures such as heel
lancing used in routine metabolic screenings. It is an examination of the effects of white noise and kangaroo care onsome physiological parameters and stress markers (cortisol and glucose-regulated protein 78-GRP78) in heel lancing
in newborns.
Methods Randomized controlled study was conducted at a gynecology service of a hospital between January and
September 2023. 90 babies were divided into three groups: 30 babies in the Kangaroo Care Group (KCG), 30 babies
in the White Music Group (WMG), and 30 babies in the Control Group (CG). All babies were randomly divided into
groups. Stress parameters were measured by saliva collection method and physiological parameters by saturation
device.
Results A statistically significant difference was determined between the total crying time, pulse and saturation
values according​​
to the groups (p < 0.001; p = 0.001). A statistically significant difference was determined between themean values of​​ cortisol and GRP78 measurements according to group and time interaction (p < 0.001). KCG was more
effective in reducing total crying time and stabilizing pulse, saturation, salivary cortisol, GRP-78 values compared to
WNG and CG.
Conclusion It was concluded that white noise and kangaroo care help reduce newborns’ stress in the case of heel
lancing.
Practical implications The practice of kangaroo care and the use of white noise methods may assist healthcare
professionals as supportive methods in stress management during invasive procedures.
Trial Registration NCT06278441, registered on 19/02/2024.
Keywords Newborn, Glucose-regulated protein 78 (GRP78), Cortisol, Kangaroo care, White noise, Physiologicalparameters*Correspondence:
Elif OdabaşI Aktaş
elifaktas@bayburt.edu.tr
1
Faculty of Health Sciences, Department of Nursing, Bayburt University,
Bayburt, Türkiye

2

Faculty of Health Sciences, Department of Midwifery Bayburt, Bayburt
University, Bayburt, Türkiye
3
Faculty of Health Sciences, Department of Physiotherapy andRehabilitation, Bayburt University, Bayburt, Türkiye
4
Faculty of Health Sciences, Department of Midwifery, Ataturk University,
Erzurum, Türkiye© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Kirbaş et al. BMC Pediatrics(2024) 24:568

Introduction
Heel lancing from the newborn has an important role in
the early diagnosis of diseases that can result in mental
and developmental delay and even death within the scopeof the newborn screening program. It is one of the basic
preventive health services in nursing and midwifery practices carried out to save lives. However, newborns, who
are often exposed to environmental stressors in the postnatal period, may experience pain and stress due to heel
lancing, which is an invasive procedure [1]. For this reason, pharmacological and non-pharmacological methods
reduce pain and stress in newborns during heel lancing. Non-pharmacological methods such as swaddling,massage, touching, white noise, and kangaroo care area few of them. The calming effect of white noise applied
to newborns in clinics and intensive care units has been
supported by studies with its positive effects, such as
feeding, starting breastfeeding, facilitating the transition
to sleep, providing pain control, reducing blood pressure
and stress, and reducing the length of hospital stay [2, 3].
Another non-pharmacological method used in interventions is kangaroo care. It has been observed that newborns who receive kangaroo care receive breast milk forlonger, are less stressed, and are more comfortable. It has
been proven that kangaroo care in newborns positively
affects physiological and behavioral parameters during
stressful invasive procedures and is effective on heart rateand crying duration [4].
In assessing the stress level in newborns, in addition to
behavioral scales, it is possible to evaluate cortisol andglucose-regulated protein 78 (GRP78) levels in biological fluids such as blood and saliva. Cortisol is a hormone
released from the adrenal cortex in response to stress.
Taking samples to measure cortisol concentration insaliva is simple, non-invasive and stress-free [5]. Endoplasmic reticulum (ER) is an organelle that performs
various functions such as lipid and protein synthesis,
folding, and calcium homeostasis. Glucose-regulated
protein 78 has a molecular weight of 78 kDa, known as
HSP5, an endoplasmic reticulum stress marker [6–8].
Unfolded proteins, which tend to form protein clusters
and accumulate hydrophobic amino acid residues within
the protein, are associated with many diseases, especially cancer, diabetes, neurodegenerative and metabolic
diseases. GRP78 plays a critical role in maintaining cell
survival in stressful situations, such as decreased calcium in the ER and accumulation of non-glycosylated
proteins [9, 10]. In the literature research, no studies
examined the effects of white noise and kangaroo careon some stress markers (cortisol, GRP78) in newborns
from whom heel lancing. Most research investigates theeffect of kangaroo care and white noise music on relieving neonatal stress and pain [11–14]; however, very few
studies compare the effectiveness of these methods andPage 2 of 10

their effect on the stress parameters cortisol. In fact, no
study has been found evaluating the effectiveness of thesemethods regarding GRP78 [15–17]. We believe that withthis study, healthcare professionals who are knowledgeable about the most effective methods for relieving stressduring invasive procedures performed on newborns will
provide better care. The current study is the first andserves as a reference. In this context, this study aimed
to examine the effects of white noise and kangaroo careduring heel lancing in newborns on some physiologicalparameters (pulse, saturation, crying duration) and stressmarkers (cortisol and GRP78).

Methods
Study design

This study, designed as a randomized controlled trial, wasconducted in the gynecology department of a hospital in
Türkiye between January and September 2023. In Türkiye, metabolic screenings for newborns are performed
within the first 36–72 h after birth in hospitals, before
discharge, and again on the 5th day at Family HealthCenters [18]. We selected our sample group from thehospital setting to ensure accurate monitoring of physiological parameters and to reduce sample loss. Five neonatal nurses and three pediatricians staff the hospital’s
gynecology department. This trial is registered with ClinicalTrials.gov (NCT06278441, 19/02/2024).
Participants

The study population was selected from the obstetrics
and gynecology service of the hospital, meeting specificinclusion criteria: gestational age of 37 weeks or more,birth weight over 2500 g, Apgar score of 8 or higher atboth the 1st and 5th minutes, and no anatomical, physiological, or hearing-related congenital or acquired diseases. Infants on sedative medications and those withsevere neonatal diseases were excluded. Based on reference study values, the minimum sample size required
was calculated to be 57, with 95% confidence (1-α), 95%
test power (1-β), and an effect size of 57, f = 0.476 [19].
To account for potential case losses, the biostatistician
recommended increasing the sample size to 90 infants.
These 90 newborns were then randomly assigned into
three groups: White Noise Group (WNG), Kangaroo
Care Group (KCG), and Control Group (CG), with 30
infants in each group. Randomization was conductedusing a computer program (http://www.randomizer.org),
and initial participants were assigned by a lottery method(WNG (1), KCG (2), CG (3)). Subsequent assignments
were made based on the random numbers generated(Fig. 1: CONSORT Flow Diagram).

Kirbaş et al. BMC Pediatrics(2024) 24:568

Page 3 of 10

Fig. 1 Flow chart showing the recruitment of patients for the study

Data collection tools

Data were collected using a ‘Mother/Baby Information
Form’, ‘Physiological and Stress Biomarkers Parameter
Follow-up Form’, ‘Portable speaker’, ‘Decibel measurement device (Benetech GM1351/TURKIYE)’, ‘Orhan
Osman’s Colic album - Don’t Let Your Baby Cry 2’.
‘Mother/Baby Information Form’ collected data on
various demographics and health indicators. It included
questions regarding the mother’s age, educational level,
number of children, and mode of delivery. For the newborns, it gathered information on Apgar scores, gestational age, gender, birth weight, and length.
‘Physiological and Stress Biomarkers Parameter Follow-up Form’ was designed by the researchers to trackphysiological parameters (respiratory rate, oxygen saturation, and heart rate) and crying duration before, during,
and after the procedure. Stress biomarkers (cortisol and
GRP78) were measured using saliva samples collected
before and after the procedure. Physiological parameters

were recorded one minute before the heel stick (when theinfant was calm and stable), during the procedure, andone minute afterward. Oxygen saturation and heart rate
were measured at the right wrist with a pulse oximeter,
which is a commonly used non-invasive device in clinicalsettings (ST03694 Console Type Pulse Oximeter Device
Nellcor Bedside). Crying duration during and after theprocedure was timed with a stopwatch by the researcher.
The stopwatch was started when the procedure beganand stopped when the baby ceased crying. Saliva samples
were collected using a Salimetrics SalivaBio Infant’s Swab
(SIS), placed under the newborn’s tongue and around themouth for 60–90 s, and then stored in a container. The
form’s content was reviewed and refined based on feedback from three pediatric nursing experts.
‘Speaker’, “JBL” brand speaker was used for white noisefor the newborns in the white noise group before, during
and after the heel sting procedure.

Kirbaş et al. BMC Pediatrics(2024) 24:568‘Sound Decibel Meter’ was used to set the sound levelon the white noise recorder below 50 dB [3, 20]. A BENETECH GM1351 model sound level meter was used tomeasure noise levels, which is a compact and accurate
device suitable for this type of measurement. It is a Benetech brand, easy-to-use, sound level meter and decibel
meter that can be attached to a tripod if desired via thescrew hole on the bottom.
The song ‘Don’t Let Your Baby Cry 2’ by Orhan Osman,featured in his album Colic, was utilized in this study.
This track is included in previous literature and is partof the Colic album released by ON Music Productions.
Orhan Osman created the album Colic by integrating
the sounds of the womb from Dr. Harvey Karp’s album
The Happiest Baby with his own musical compositions. It
should be added that this album falls under the categoryof white noise, which is known for its calming effects on
infants [2, 21].
Interventions
Control Group (CG)The heel lancing for the babies was conducted in a specially equipped intervention room. The room was
well-lit, temperature-controlled, and equipped with allnecessary medical supplies. A neonatal nurse was present
to ensure the procedure was performed safely. The safetyof the newborns was ensured throughout the procedureby continuously monitoring their vital signs and having
a trained neonatal nurse present at all times. Heel lancing was performed by a single experienced nurse without
support from other health professionals. The newborn
was positioned comfortably on a padded stretcher before,
during, and after the heel lancing. Heel blood samplingof newborns was done in the same intervention room
to maintain a consistent and controlled environment. A
saturation probe was attached to the right wrist of eachbaby to monitor its pulse and oxygen saturation levels before, during, and after the procedure. This group
did not receive any intervention prior to the procedure.
Saliva samples were collected immediately before theheel lance and 20 min afterward. To ensure reliability,
the same nurse from the clinic performed all heel lance
procedures. All interventions were carried out on the
newborns between 7:00 and 10:00 in the morning to alignwith their natural circadian rhythms, which can influence
stress and pain responses. Additionally, this time frame
ensured the availability of necessary medical staff andminimized potential disturbances from routine hospital
activities.
Kangaroo Care Group (KCG)

In the Kangaroo Care Group (KCG), each participant
received kangaroo care during the blood collection procedure. The baby was dressed only in underwear andPage 4 of 10

a diaper. Following the guidelines of the World HealthOrganization (WHO) for kangaroo care [22], the motherunbuttoned her shirt and placed the baby on her barechest. Throughout the blood collection, the baby wasallowed to move their arms and legs freely. The mothersupported the baby with one hand on their back and theother on their bottom. The ambient room temperature
was carefully maintained between 24 and 26 degrees Celsius for comfort. The mother reclined in a semi-fowler
position with her back propped by a pillow, ensuring thebaby faced her to facilitate bonding. The baby remained
wrapped in a blanket. Kangaroo care was started 5 min
before the heel prick and continued throughout theprocedure, ending 30 min after the procedure was completed. During this time, a saturation probe was affixed
to the baby’s right wrist to monitor pulse and oxygen
saturation levels before, during, and after the blood collection. Crying durations were meticulously recorded
using a stopwatch throughout the entire procedure. For
all groups, the stopwatch was started at the moment
the heel lancing began and stopped when the newborn
stopped crying. Saliva samples were collected immediately before the heel lancing and again 20 min afterward
for subsequent analysis.
White Noise Group (WNG)

After the newborns in this group were brought to theintervention room, white noise at 50 dB was played
through a speaker positioned 1 m away. The sound level
was calibrated to 50 dB using a decibel meter. Heel lancing was performed once the newborns had been exposedto the white noise and had calmed down. Physiological parameters of the newborns were monitored andrecorded before, during, and after the heel lancing procedure. Crying times were tracked with a stopwatch
and recorded on a follow-up form by the researchers.
The stopwatch was started at the moment the heel prickbegan and stopped when the newborn stopped crying,
not when the procedure was completed. Saliva samples
were collected just before and 20 min after the procedure
to measure the peak levels of stress biomarkers, as previous research indicates that these levels typically peakaround 20 min post-stressor [23].
Saliva collection and measurement

To collect saliva samples, the research team used Salimetrics® SalivaBio Infant Swabs (Carlsbad, CA). They held
one end of the swab and placed the other end against the
newborn’s buccal mucosa or sublingually to absorb saliva.
Immediately after collection, the swabs were placed intolabeled storage tubes [24]. The collected saliva samples
were then transferred to Eppendorf tubes and centrifuged at 1500 g for 15 min in a refrigerated centrifuge(NF 1200R, NUVE, Ankara, TURKIYE) in the laboratory.

Kirbaş et al. BMC Pediatrics(2024) 24:568

The samples were subsequently stored at -30 °C until
the analyses for cortisol hormone and GRP78 were performed. For the measurements, human-specific commercial kits were used: cortisol levels were measured using
BT LAB kits (Cat.NoE1003Hu, CHINA), and GRP78 levels were measured using ELISA kits from SinoGeneClon(Product code: SG-20252, CHINA).
Ethical considerations

Bayburt University Ethics Committee approved the study(Decision no: (2022/334) and it is registered into ClinicalTrials.com (NCT06278441). In addition, administrative
approval was obtained from the Provincial Health Directorate of the place where the study was conducted. An
informed consent form was signed after being informed
about the purpose and confidentiality of the dataobtained. Because neonates were minor human participants, informed consent was obtained from a parent and/or legal guardian in our study. Neonates of parents and/or legal guardians who did not give written consent were
not included in the study. The study protocol adheresto the ethical guidelines outlined in the Declaration of
Helsinki.
Data analysis

Data were analyzed in IBM SPSS V23. Compliance withnormal distribution was examined with skewness andkurtosis coefficients (± 2) [25]. Group and time used generalized Linear Models to compare data complying withnormal distribution, and multiple comparisons were analyzed with the Tukey test. Group and time used a Robust
ANOVA test to compare data that did not comply withnormal distribution, and multiple comparisons wereanalyzed with Bonferroni Correction. Analysis results
were presented as frequency (percentage) for categoricalvariables, mean ± standard deviation, and median (minimum–maximum) for quantitative variables. The significance value was taken as p < 0.05.

Results
A comparison of parameters by groups is given in detailin Table 1. A statistically significant difference exists
between the median values ​​of total crying time according
to groups (p < 0.001). CG’s total crying time median was
2.53, KCG 0.49, and WNG 1.17. No statistically significant difference exists between the distributions of otherparameters according to groups (p > 0.05, Table 1).
Mean pulse rate was 131 before the procedure, 156
during the procedure, 132 after the procedure; meansaturation was 96 before the procedure, 90 during theprocedure, 93 after the procedure. It is given in detail in
Tables 2 and 3. A statistically significant difference wasobtained between the median values ​​of pulse measurements according to groups (p < 0.001). The median CG

Page 5 of 10

pulse measurement was 138, KCG 132, and WNG 143.
KCG differed from other groups. A statistically significant difference was obtained between the median values​​of pulse measurements according to time (p < 0.001). The
median heart rate measurement before the procedure
was 131; during the procedure, it was 156; and after theprocedure, it was 132. The procedure orders differedfrom other times (Table 2).
A statistically significant difference was obtained
between the median values of​​ pulse measurements
according to the group and time interaction (p < 0.001).
While the highest median value of 175 was obtained during the CG procedure, the lowest median value of 126.5
was obtained after the CG procedure (Table 2).
A statistically significant difference was obtained
between the median values ​​of saturation measurements
according to groups (p = 0.001). The median saturation
measurement of KG was 93, KCG was 94, and WNG was
91. All groups differed from each other. A statisticallysignificant difference was obtained between the medianvalues ​​of saturation measurements according to time(p < 0.001). The median saturation measurement before
the procedure was 96; during the procedure, it was 90;
and after the procedure, it was 93. All times it differedfrom each other (Table 3).
A statistically significant difference was obtained
between the median values ​​of saturation measurements
according to the group and time interaction (p < 0.001).
While the highest median value of 96 was obtained forboth KCG and WNG before the procedure, the lowest
median value of 90 was obtained for both CG and WNG
during the procedure (Table 3).
There is a statistically significant difference between
the mean values ​​of cortisol measurement according to
the groups. The difference was obtained (p < 0.001). The
mean of CG cortisol measurement was 2.88, 2.38 in the
KCG group, and 2.56 in the WNG. CG differed fromother groups. A statistically significant difference wasobtained between the average values of​​ cortisol measurements according to time (p = 0.049). While the averagecortisol measurement before the procedure was 2.68, it
was accepted as 2.53 after the procedure (Table 4).
A statistically significant difference was obtained
between the mean values of​​ cortisol measurements
according to the interaction of group and time (p < 0.001).
The highest mean value of 3.09 was obtained after the CG
procedure. In contrast, the lowest mean value of 2.08 was
obtained after the KCG procedure (Table 4).
A statistically significant difference was obtained
between the mean values of​​ GRP78 measurement according to the groups (p < 0.001). The mean of CG GRP78
measurement was 0.50, 0.42 in the KCG group, and 0.44
in the WNG group. All groups differed from each other.
A statistically significant difference was obtained between

Kirbaş et al. BMC Pediatrics(2024) 24:568

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Table 1 Comparison of parameters by groups

MOTHER
Educational Status
Literate
Primary School
Middle School
High School
University and above
Number of children
1
2
3
4
5
Delivery Type
Vaginal birth
Cesarean birth
Mother’s Age
BABYBaby genderGirlBoy
Birth Weight (kg)Birth Height (cm)
Total Crying Time

Total

Test Statistics

p

0 (0)
2 (6,7)9 (30)
10 (33,3)
9 (30)

3 (3,3)
7 (7,8)26 (28,9)27 (30)27 (30)4.659

0.832*

10 (33,3)
14 (46,7)
3 (10)
2 (6,7)
1 (3,3)9 (30)
12 (40)6 (20)
3 (10)0 (0)29 (32,2)
35 (38,9)15 (16,7)
10 (11,1)
1 (1,1)5.603

0.729*

19 (63,3)
11 (36,7)
30,83 ± 5,82
29,5 (21–42)

14 (46,7)
16 (53,3)
30,33 ± 6,19
29,5 (21–42)

17 (56,7)
13 (43,3)
29,8 ± 6,21
28 (21–42)

50 (55,6)
40 (44,4)
30,32 ± 6,02
29 (21–42)

1.710

0.425**

0.217

0.805‡

21 (70)9 (30)
3324,67 ± 418,91
3305 (2495–4130)
50,13 ± 1,74
50 (48–54)
2,53 ± 0,96
2,53 (1,03–4,13)a15 (50)15 (50)3333,5 ± 388,08
3240 (2760–4050)
49,7 ± 1,1850 (48–52)
0,59 ± 0,30,49 (0,28 − 1,3)b

17 (56,7)
13 (43,3)
3227,33 ± 249,58
3245 (2890–4050)
50,2 ± 1,45
50 (48–53)
1,21 ± 0,37
1,17 (0,48 − 2,21)c53 (58,9)37 (41,1)
3295,17 ± 359,01
3250 (2495–4130)
50,01 ± 1,47
50 (48–54)
1,44 ± 1,02
1,16 (0,28 − 4,13)

2.570

0.277**

0.931

0.628⁑

1.02

0.365‡

64.546< 0.001⁑

Control Group

Kangaroo Care GroupWhite Noise Music Group

1 (3,3)3 (10)
11(36,7)
7 (23,3)
8 (26,7)

2 (6,7)
2 (6,7)6 (20)
10 (33,3)
10 (33,3)

10 (33,3)
9 (30)
6 (20)
5 (16,7)0 (0)*Fisher-Freeman-Halton test; **Pearson ki kare test ‡ One-Way Variance Analysis; ⁑Kruskal Wallis H test; a−c: There is no difference between groups with the same
letter; Mean ± Standard Deviation; Median (Minimum - Maximum)

the average values of​​ GRP78 measurements according totime (p < 0.001). While the mean GRP78 measurement
before the procedure was 0.47, it was obtained as 0.44
after the procedure (Table 5).
A statistically significant difference was obtained
between the mean values of​​ GRP78 measurements
according to the interaction of group and time (p < 0.001).
The lowest average value of 0.37 was obtained after the
KCG procedure, while the highest average value of 0.52
was obtained after the CG procedure (Table 5).

Discussion
In the study where we examined the effects of white noise
and kangaroo care during heel lancing on some physiological parameters and stress markers of newborns, there
was a significant difference in crying times between KCG
WNG and CG. Crying time was highest in CG and lowest in KCG. Considering these results, kangaroo care andwhite noise calm the newborn and reduce crying times
during heel lancing. In the study of Karakoç and Türker
[11], it was reported that white noise reduced crying

duration and pain. In another study, KCG was found tohave less crying duration than CG [12]. Crying is themost obvious reaction of newborns during painful andstressful procedures [11]. Studies conducted with healthy
newborns found that those who received kangaroo carecried less than those who did not [26, 27]. In anotherstudy conducted with newborns, less crying time wasfound in heel lancing in the mother’s lap compared tocontrol and swaddling positions [28]. Together with ourstudy, all these results were obtained from non-pharmacological methods. Studies show that white noise andkangaroo care reduce newborns’ pain, stress, and crying
times.
In the study, we observed that all groups showed anincrease in pulse values during the procedure, with thehighest increase noted in the CG, followed by WNG and
KCG, respectively. However, KCG exhibited less decreasein saturation during the procedure, with rapid increases
observed after the procedure compared to CG and WNG.
Our findings indicate that KCG potentially enhances cardiovascular and respiratory stability in infants, whichKirbaş et al. BMC Pediatrics(2024) 24:568

Page 7 of 10

Table 2 Descriptive statistics and multiple comparison results ofheart rate measurements by group and timeTime
Beforeprocedure
133.41 ± 11,98
131 (112–170)

Duringprocedure
157.43 ± 16,52
156 (126–194)

After
procedure
134.19 ± 12,89
132 (112–171) Total

175(138–194)C

126.5(112–161)A

Kangaroo Care 128.5
Group(112–170)A

141(126–172)BD

128(112–171)A

White Noise
Music Group

130.5(116–145)A

162(142–170)E

142(128–154)D

Total

131
(112–170)a156(126–194)b

132(112–171)a

Mean ± SD
Median(Min-Max)Group
Control Group 133.5(120–169)AB

Group
Time
Group*Time

Test statics*
21.4
102.3
59

138
(112–
194)a
132
(112–
172)b
143
(116–
170)a
138
(112–
194)

p
< 0.001< 0.001< 0.001a−b: There is no difference between groups with the same letter; A−E: There isno difference between interactions with the same letter; Median (Minimum Maximum); *Robust ANOVA; the median method was used as the comparison
methodTable 3 Descriptive statistics and multiple comparison results ofsaturation measurements by group and timeTime
Beforeprocedure
95.37 ± 2,42
96 (90–99)

Duringprocedure
89.98 ± 2,4
90 (80–95)

Afterprocedure
93.28 ± 2,38
93 (89–98)

95 (91–99)AB 90 (80–95)CD94 (91–97)A

96 (92–99)B

91,5 (87–95)E95 (93–98)ABWhite Noise Music 96 (90–99)AB 90 (87–93)CGroup

91 (89–94)DEMean ± SD
Median(Min-Max)Group
Control Group

Kangaroo Care
Group

Total

96 (90–99)a

Group
Time
Group*Time

Test statistics*
6.92
55.16
58.63

a−c

90 (80–95)b

93 (89–98)c

Table 4 Comparison of cortisol measurements by group and
time

Group
Control Group
Kangaroo Care GroupWhite Noise Music Group
Total
Group
Time
Group*Time

Total

Time
Beforeprocedure

After
procedure

2,66 ± 0,52B
2,68 ± 0,49B
2,71 ± 0,54AB
2,68 ± 0,51
F
13,84
3,93
15,54

3,09 ± 0,63A
2,08 ± 0,42C
2,40 ± 0,51BC
2,53 ± 0,67p
< 0,001
0,049< 0,001

2,88 ± 0,61a
2,38 ± 0,54b
2,56 ± 0,54b
2,6 ± 0,6
PES
0,137
0,022
0,152

a−b

: There is no difference between groups with the same letter; A−C: There isno difference between interactions with the same letter; Mean ± Standard
Deviation; F: Variance Analysis test statistic; R2=%26,49; Adjusted R2=%24,38;
PES: Partial Eta Squared

Table 5 Comparison of GRP78 measurements by group and
time

Group
Control Group
Kangaroo Care GroupWhite Noise Music Group
Total
Group
Time
Group*Time

Total

Time
Beforeprocedure

After
procedure

0,47 ± 0,07B
0,47 ± 0,05B
0,47 ± 0,05B
0,47 ± 0,06
F
28,08
15,58
23,06

0,52 ± 0,07A
0,37 ± 0,05D
0,42 ± 0,05C
0,44 ± 0,09p
< 0,001< 0,001< 0,001

0,50 ± 0,08a
0,42 ± 0,07b
0,44 ± 0,06c
0,45 ± 0,08
PES
0,244
0,082
0,210

a−c

Total

93
(80–
99)a
94
(87–
99)b
91
(87–
99)c
93
(80–
99)

p
0.001
< 0.001< 0.001: There is no difference between groups with the same letter; A−E: There isno difference between interactions with the same letter; Mean ± Standard
Deviation; Median (Minimum - Maximum); *Robust ANOVA; the median method
was used as the comparison method: There is no difference between groups with the same letter; A−D: Thereis no difference between interactions with the same letter; Mean ± Standart
Deviation; F: Variance Analysis test statistic; R2=%40,38; Adjusted R2=%38,67;
PES: Partial Eta Squaredaligns with existing literature suggesting that kangaroocare reduces adverse stress effects [13, 26]. This may be
attributed to mechanisms such as increased maternalinfant bonding, regulation of autonomic nervous system
activity, and improved thermal regulation, all contributing to physiological stability [29].
In the study of Çaksak [21], when white noise, fetal
position, and both applications were evaluated together
during the procedure, it was found that using both white
noise and fetal position together had the most significant calming effect on newborns. Karakoç and Türker
[11] found that white noise significantly reduced heart
rate and stabilized oxygen saturation levels in infants
and white noise exposure during painful procedures
like heel lancing resulted in lower heart rate variability
and quicker recovery to baseline levels. These findings
suggest that white noise can effectively mitigate stressresponses in newborns by promoting autonomic stability
and enhancing overall physiological resilience. Gokulu etKirbaş et al. BMC Pediatrics(2024) 24:568

al. [14] found a statistically significant difference in pulse
and saturation rates in healthy newborns before and afterheel lancing. Kostandy and Ludington-Hoe [27] reported
that kangaroo care caused a decrease in pulse and saturation rates during heel lancing in two-day-old healthy
newborns. As a result, while our study differs from otherstudies in terms of physiological parameters, it is parallel
to some. Considering the differences in working environments, newborns appear to have different physiologicalresponses to pain and stress during the procedure.
In our study, a significant difference was obtained insalivary cortisol measurements according to the timeinteraction of the groups. After the procedure, the highest cortisol values ​​were detected in CG, while the lowest
value was seen in KCG. A study evaluating salivary cortisol concentrations for procedural pain monitoring in
newborns found that salivary cortisol levels increased
after blood collection [30]. Stoye et al. [15] reported
in their study that cortisol levels in preterm newborns
before vaccination were higher than morning cortisol
levels and that this would cause a source of stress in thepreterm newborn. In the context of our study, this finding suggests that newborns may experience heightened
stress responses prior to medical procedures, such as heel
lancing. Elevated cortisol levels indicate that the infants
were already in a state of heightened arousal and stressbefore the procedure, which could potentially amplify
their physiological responses during the heel lancing.
This highlights the importance of interventions like kangaroo care and white noise in mitigating stress and stabilizing physiological parameters. By providing a calming
environment and reducing stress, these interventions
may help to lower cortisol levels and improve overalloutcomes for newborns undergoing painful procedures.
Gao et al. [16] examined the effectiveness and safety of
combining sucrose with massage, music, non-nutritive
sucking, and gentle human touch in the treatment of preterm infants with recurrent procedural pain. The study
concluded that combined interventions remained effective and safe in reducing recurrent procedural pain andreduced cortisol levels at discharge. It also showed that itsupports early neurobehavioral development in preterm
infants. Although our study parallels the studies in theliterature, we can say that KC, one of the interventions,is more effective in reducing stress than the other intervention. The ‘developmental origins of health and disease’
hypothesis proposes that environmental exposures earlyin life, during critical periods for development, can influence health throughout life [31]. However, a single study
has never assessed HPA axis activity in newborns due tothe high individual variability of salivary cortisol concentrations. We suggest that it should be based not on value
but on repeated measurements. Our study showed that
painful procedures performed on newborns increase thePage 8 of 10

stress level. It was observed that this increase was mainly
in CG, not KCG or WNG, after the procedure.
GRP78 is an endoplasmic reticulum stress marker, anessential endoplasmic reticulum chaperone, and a vital
regulator of the unfolded protein response. ER homeostasis is critical in maintaining cellular functions suchas protein synthesis, folding, and secretion. Disruption of ER homeostasis causes ER stress and triggers theunfolded protein response, which can lead to apoptosis
in cells under constant stress, especially cancer, diabetes,and many metabolic and neurodegenerative diseases. In
addition to playing an essential role in early embryonicdevelopment, GRP78 plays a role in age-related diseases[6, 7]. In our current study, the average saliva GRP78 levelin newborn groups was the lowest in the Kangaroo Caregroup and the highest in the control group. No studies on
GRP78 as a stress marker in newborns have been foundin the literature. Our current study is the first and serves
as a reference in this sense. There are several different
studies in the literature evaluating GRP78 [32, 33].
Strengths and limitations

The first limitation of the study is that the study was conducted in a single health center, and the results obtained
cannot be generalized to this sample group. Anotherlimitation is that no intervention was applied to the CG
to evaluate the effectiveness of the study. Finally, oneof our limitations is that not all researchers could beblinded due to the nature of the study. Specifically, theresearchers who administered the interventions (kangaroo care and white noise) could not be blinded because
they were directly involved in providing the interventions. However, the researchers who recorded the physiological parameters and analyzed the data were blinded
to the group assignments to minimize bias. This limitation means that there may be some degree of observerbias in the administration of the interventions, although
efforts were made to ensure objective data collection andanalysis.
Implications for practiceKangaroo care and white noise music techniques are
effective, simple and safe methods to reduce stress during the heel lancing procedure of newborns. These interventions provide a good way for healthcare professionals
to use during the heel lancing procedure. Among theseinterventions, kangaroo care application has a more positive effect on newborn physiological parameters, cortisol, and GRP78 levels compared to white noise. In ourstudy, we found that white noise also had positive effects
on crying time, pulse and saturation levels, and cortisol
and GRP78 levels of newborns throughout heel lancing.
However, kangaroo care was more effective in stabilizingthese parameters and reducing stress in newborns. This

Kirbaş et al. BMC Pediatrics(2024) 24:568

conclusion aligns with our findings that both interventions are beneficial, but kangaroo care provides greater
overall physiological and biochemical stability.

Conclusion
As a result, white noise and kangaroo care during heel
lancing had significant effects on various physiologicalparameters in newborns. Both interventions decreased
crying duration and pulse levels while helping to maintain higher saturation levels. Specifically, kangaroo care
was more effective in reducing cortisol and GRP78 levels compared to white noise. These findings suggest thatwhile both interventions are beneficial in mitigatingstress and stabilizing physiological parameters, kangaroocare provides a greater overall reduction in stress indicators and improved physiological stability in newborns.
In examining the stress response in newborns, cortisol
and GRP78 levels can be assessed, given their potential
use as stress biomarkers and their importance for a better understanding of neonatal stress physiology. Additionally, due to the scarcity of available information andthe fact that this study is the first study conducted innewborns, further experimental and prospective studies
are needed to determine the clinical significance of thefindings.
Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12887-024-05033-1.
Supplementary Material 1
Supplementary Material 2
Author contributions
ZOK Conceptualization; Investigation; Writing - original draft; Funding
acquisition; Methodology; Validation; Writing - review & editing; Visualization;
Software; Formal analysis; Project administration. EOA Supervision; Resources;
Writing - review & editing; Writing - original draft; Data curation. BB Software;
Formal analysis; Project administration; Data curation; Supervision; Resources.
HO Writing - original draft; Writing - review & editing.
Funding
This research received no external funding.
Data availability
The data sets used for this study are available from the corresponding author
on reasonable request.

Declarations
Ethical considerations
Bayburt University Ethics Committee approved the study (Decision no:(2022/334) and it is registered into ClinicalTrials.com (NCT06278441). In
addition, administrative approval was obtained from the Provincial HealthDirectorate of the place where the study was conducted. An informed
consent form was signed after being informed about the purpose andconfidentiality of the data obtained. Because neonates were minor humanparticipants, informed consent was obtained from a parent and/or legal
guardian in our study. Neonates of parents and/or legal guardians who did

Page 9 of 10

not give written consent were not included in the study. The study protocol
adheres to the ethical guidelines outlined in the Declaration of Helsinki.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Conflict of interest
No conflict of interest is declared by the authors.
Received: 16 May 2024 / Accepted: 26 August 2024

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