Salivary Cortisol as Stress Marker in Newborns Subjected to Double Weighing With Body Weight Loss of 3±1,5% at 24 hr From Birth
Conditions: Cortisol Overproduction
Sponsor: Fondazione Poliambulanza Istituto Ospedaliero

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
  
 
 
Title Form 
 
 
“Salivary cortisol as stress marker in newborns subjected to double 
weighing with physiological body weight” 
 
 
Date: 13/01/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
 
 
 
PILOT STUDY PROTOCOL 
 
Salivary cortisol as stress marker in newborns subjected to double weighing with body 
weight loss of 3±1,5% at 24 hr from birth   
 
  
 
Principal Investigator 
 
Dr. Giuseppe De Bernardo, responsible S. S. Neonatal Intensive Care Unit 
Department of mother and child’s Health 
Poliambulanza Foundation, Brescia 
 
Co - principal investigator 
 
Dr.ssa Laura Linetti 
Neonatology Coordinator 
Poliambulanza Foundation, Brescia 
 
Statistic 
Maurizio Giordano 
University of Naples Federico II, School of Medicine 
 
 
 
 
 
 

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
  
 
INDEX 
 
1. Introduction 
2. Objective 
3. Study Location 
4. Population 
5. Inclusion Criteria 
6. Exclusion Criteria 
7. Procedure 
8. Primary Outcome 
9. Secondary Outcome 
10. Sample Size 
11. Randomization 
12. Reference 
 
 
 
 
 
 
 
 
 
 
 
 
1. Introduction 
 

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
 
 
 
After the phase of early adaptation, newborn must learn quickly to live out of the mother 
uterus. In this phase of life, physiological mechanisms are activated (1).  Newborn loss body 
weight because loss liquid by respiration, feces, urine and not adequate feeding (2). Body 
weight loss after birth is physiological when is lower than 10% but is also important the 
evaluation of body weight daily trend that must be lower than 5% (3,4). If newborn shows 
physiological body weight loss, no treatment will be necessary and rooming-in can continue 
together with exclusively breastfeeding (5,6)  
Furthermore, if newborn lose more than 10% of body weight during 48hr of life or more 
than 5% of body weight in a day, a dehydration state will occur that will influence negatively 
the physiological cardio-circulatory and kidney adaptation. In these cases, is important 
execute controls and a specific therapy (7). When body weight loss is between 7%-10%, 
usually in Neonatology wards it is used double weighing before and after breastfeeding (8). 
This practice evaluates colostrum quantity that is assumed from infant during feeding and 
the necessity of milk formula integration to prevent an excessive body weight loss. Diuresis 
is also monitored, and parents are informed on the benefits of breastfeeding.  
If body weight loss is greater than 10%, diuresis is monitored weighing the diaper and 
haematological examinations are performed. In this case, it is counselled an artificial milk 
integration after breastfeeding. Discharge can be influenced from the results of the controls 
and must be executed for the security of the newborn (9).  
Health care providers must not be underestimated the possible allergies to formula milk and 
the mothers that give up breastfeeding after the use of formula milk.  The newborn should 
receive breastfeeding from the delivery room (10,11). It is good practice, when the milk 
secretion is enough, to attach the infant to a single breast for each feeding and let it suck for 
15-20 minutes (12). Quantity of milk that newborn assume for each feeding is object of 
careful evaluation. Double weighing consists in weighing newborn before and after feeding: 
the body weight difference is the quantity of milk assumed. In the first day of life, newborn 
should suck 10 gr of colostrum or milk for each feeding. Then starting from the second day 
of life, the quantity of milk assumed must be increased of 10 gr/die for the first week.  

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
  
 
According to scientific revisions, salivary cortisol is a valid method for measuring 
adrenocortical activity in newborns because it is non-invasive in fact the measurement of 
salivary cortisol has been used as a stress marker in newborns since 1992 (13,14). The 
mechanism by which cortisol induces stress, concerns the hypothalamic-pituitary-adrenal 
axis. The hypothalamus produces the corticotropin-releasing hormone, which stimulates the 
production of adrenocorticotropin by hypophysis. Adrenocorticotropin induces the secretion 
of glucocorticoids such as cortisol by stimulating the cortex of the adrenal gland. An 
appropriate release of cortisol due to stress factor is vital for surviving and the loss of this 
mechanism increase the risk of morbidity and mortality for newborns. However, prolonged 
exposure to a high level of cortisol may increase the risk of cognitive and behavioural 
disorders, hypertension, hyperlipidaemia, insulin resistance, immune deficiency and 
hippocampal impairment (15,16). The separation from the mother, the exposure to painful 
procedures but also routine interventions such as physical examination, diaper changes and 
bathing increase the salivary cortisol level of the newborn compared to the basal state (17). 
The measurement of salivary cortisol in the newborn therefore has the aim of understanding 
and preventing those stressful conditions that may have long-term side effects. For this 
reason, the lose weight in the first days and double weighing should be a stress practice for 
the newborn. 
 
2. Objective 
Evaluation of salivary cortisol level as stress marker in newborns with body weight loss 
of 3±1,5% at 24 hr from birth subjected to double weighing or to body weight control at 
36 hr and 48 hr of life. Furthermore, evaluation of body weight trend between newborns 
subjected to double weighing or to body weight control at 36 hr and 48 hr of life.    
 
 
3. Study Location 
U.O. Neonatology, Department of mother and child’s health, Poliambulanza Foundation, 
Brescia.  

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
 
 
 
4. Population 
Group A (Case): Newborns subjected to double weighing before and after breastfeeding, 
with body weight loss >3±1,5% at 24 hrs or  6% at 48 hrs from birth  
Group B (Control): Newborns with body weight loss >3±1,5% at 24 hrs and 6 % at 48 hrs 
from birth and subjected to weight control at 36 hr and 48 hr of life 
 
5. Inclusion Criteria   
● Gestational age: 37-42 wks 
● Body Weight: >2500 g 
● Body weight loss >3±1,5% at 24 hr from birth 
● First-born 
● Eutocic delivery 
● Mother BMI:19-24  
● Glycaemia >50 mg/dl 
 
6. Exclusion criteria 
● Mother disease 
● Admission in NICU 
● Neonatal pathologies  
● Utilization of human milk 
● Body weight loss > 10% 
 
7. Procedure 
Body weight of newborns will be collected at birth(T0), at 24 hr of life (T1) and at 48 hr of life 
(T2) by Eura Mod.AS/1 OMIP, Milano.  
 
Group A will be subjected to double weighing, before and after the next 6 breastfeeding. 
Breastfeeding will be carried out each 4 hr.  

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
  
 
Group B will be subjected to monitoring of body weight only at 24, 36 hr and 48 hr of life.  
Saliva sample of the newborns will be collected in oral cavity at 48 hr of life. Samples 
collected will be storage at -80 C for up to 4 months and analysed by analysis laboratory of 
Poliambulanza Foundation.  
 
 
Measurement 
Weight scale used to obtain body weight is Eura Mod.AS/1 OMIP, Milano. 
Data collected will be reported on a database by Microsoft Office Excel 2016. 
SalivaBio Infant’s Swab (SIS) is the kit used to collect salivary cortisol.  
Statistical analysis will be carried out by IBM SPSS Statistics for Windows, v.25 
(Armonk,NY:IBM Corp.) 
 
8.Primary Outcome 
Evaluation of salivary cortisol levels at 48 hr of life after the practice of double weighing to 
analyse stress levels of the newborns 
 
H0: There is not difference in salivary cortisol level between group A and B. 
 H1: Salivary cortisol level in group A is greater than group B.  
 
9.Secondary Outcome 
Evaluation of body weight increase between group A and B.  
H0: There is not difference in the body weight increase between group A and B 
H1: Group A shows a greater body weight increase than group B 
 
 
10.Sample Size 
Primary outcome will be evaluated by independent two samples t-test. Sample size will be 
computed setting: 

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
 
 
 
Alfa error: 0.05 
Beta error: 0.90 
Effect size: 0.8  
Sample size will be of 56 patients (28 group A + 28 group B) 
 
 
 
Secondary outcome will be analysed by independent two samples t-test. Trend of body 
weight will be computed evaluating percentage increase from birth to 48 hr of life.   
 
11.Randomization 
Patients will be enrolled to group A or B by statistical software.  
12. Reference 
1) Janet M. Rennie, “Textbook of Neonatology” Elsevier Health Sciences, 
London, 2012. 
2)  Rodríguez G, Ventura P, Samper M P, Moreno L, Sarría A, Pérez-González J M. 

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
  
 
Changes in body composition during the initial hours of life in breast-fed 
healthy term newborns. Biol Neonate. 2000;77(1):12–6 
3) Thulier D. Weighing the facts: a systematic review of expected patterns of 
weight loss in full-term, breastfed infants. J Hum Lact. 2016;32:28–34  
4) Noel-Weiss J, Courant G, Woodend AK. Physiological weight loss in the 
breastfed neonate: a systematic review. Open Med 2008;2(4):e99-e110. 
5) Victora, C.G.; Bahl, R.; Barros, A.J.D.; França, G.V.A.; Horton, S.; Krasevec, J.; 
Murch, S.; Sankar, M.J.; Walker, N.; Rollins, N.C.; et al. Breastfeeding in the 
21st century: Epidemiology, mechanisms, and lifelong effect. Lancet 2016, 
387, 475–490. 
6) Horta, B.L.; Victora, C.G. Long-Term Effects of Breastfeeding: A Systematic 
Review; World Health Organization: Geneva, Switzerland, 2013, pp. 1–68. 
7) Flaherman VJ, Kuzniewicz MW, Li S, Walsh E, McCulloch CE, Newman TB. 
First-day weight loss predicts eventual weight nadir for breastfeeding 
newborns. Arch Dis Child Fetal Neonatal Ed. 2013;98:488–492 
8) G. Bertini, R. Breschi, C. Dani,” Physiological weight loss chart helps to identify 
high-risk infants who need breastfeeding support”, Acta Pædiatrica 2015 
104, pp. 1024–1027. 
9) Ferrández-González M, Bosch-Giménez V, López-Lozano J, Moreno-López N, 
Palazón-Bru A, Cortés-Castell E. Weight loss thresholds to detect early 
hypernatremia in newborns. J Pediatr (Rio J). 2018 Jul 18. pii: S0021-
7557(18).  
10) Righard L, Alade MO. Effect of delivery room routines on success of first 
breast-feed. Lancet. 1990 Nov 3;336(8723):1105-7. 
11) Jang GJ, Kim SH, Jeong KS. [Effect of postpartum breast-feeding support by 
nurse on the breast-feeding prevalence]. Taehan Kanho Hakhoe Chi. 2008 
Feb;38(1):172-9. Korean. 
12) Woolridge MW, Baum JD, Drewett RF. Individual patterns of milk intake 
during breast-feeding. Early Hum Dev. 1982 Dec 6;7(3):265-72. 

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
 
 
 
13) Cignacco, E.; Denhaerynck, K.; Nelle, M.; Buhrer, C.; Engberg, S. Variability in 
pain response to a non-pharmacological intervention across repeated routine 
pain exposure in preterm infants: A feasibility study. Acta Paediatr. 2009, 98, 
842–846 
14) De Bernardo G, Riccitelli M, Giordano M, Proietti F, Sordino D, Longini M, 
Buonocore G, Perrone S. Rooming-in Reduces Salivary Cortisol Level of 
Newborn. Mediators Inflamm. 2018 Mar 8;2018:2845352 
15) Levine, S. Developmental determinants of sensitivity and resistance to stress. 
Psychoneuroendocrinology 2005, 30, 939–946 
16) Mörelius E, Theodorsson E, Nelson N. Salivary cortisol and mood and pain 
profiles during skin-to-skin care for an unselected group of mothers and 
infants in neonatal intensive care. Pediatrics. 2005 Nov;116(5):1105-13. 
17) Gunnar MR, Talge NM, Herrera A. Stressor paradigms in developmental 
studies: what does and does not work to produce mean increases in salivary 
cortisol. Psychoneuroendocrinology. 2009 Aug;34(7):953-67 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
PROTOCOLLO di STUDIO PILOTA - CORTISOLO 
Versione 3.0 del 30/07/2018 
  
 
 
 
SIGNATURE OF APPROVALS 
 
 
 
 
_______________________                                _______________________ 
       SIGNATURE                                                                               DATE 
 
 
_______________________                                _______________________ 
       SIGNATURE                                                                               DATE 
 
 
_______________________                                _______________________ 
       SIGNATURE                                                                               DATE 
 
13-01-2019
13-01-2019
13-01-2019