The Impact of Holding During Therapeutic Hypothermia on Saliva Oxytocin and Cortisol Levels in Mother-Infant Pairs
Phase: NA
Conditions: Hypoxic-Ischemic Encephalopathy, Neonatal Encephalopathy
Sponsor: MaineHealth

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The Impact of Holding on Stress and Bonding in Mother-Infant Pairs during Therapeutic Hypothermia 
Research Protocol | IRB Number: 1183063  
Introduction: The inability to hold an infant being treated with therapeutic hypothermia in the neonatal 
intensive care unit has been subjectively reported by ours and other research groups as a significant source of 
stress for parents1-7. We aim to assess the impact of holding on endocrinological markers of stress and bonding. 
Specifically, we plan to collect salivary cortisol and oxytocin levels from infants undergoing therapeutic 
hypothermia and their mothers prior to and immediately after a 30-minute holding period. We anticipate 
elevated oxytocin and decreased cortisol levels as quantitative markers of increased bonding and stress 
reduction in the mother-infant dyad after holding. 
Background: Therapeutic hypothermia is a standard of care intervention that has been consistently shown to 
increase disability-free survival in term infants with neonatal encephalopathy8-13. Infants treated with 
therapeutic hypothermia often have central lines, are on continuous electroencephalogram (EEG) monitoring, 
and are intubated.  Due to concerns for dislodging equipment and potential for warming the infant, parents 
historically have not been permitted to hold their infants during therapeutic hypothermia. While therapeutic 
hypothermia has important neurological benefits to the newborn, prior research conducted with nurses and 
parents indicates that the experience is psychologically traumatic1,2,4,5,14. A parent’s inability to hold their infant 
during therapeutic hypothermia is often cited as a major impediment to parent-infant bonding2. 
For prematurely born infants, not treated with therapeutic hypothermia, skin-to-skin care has emerged as a 
therapeutic intervention that substantially improves bonding between mothers and infants15. It has also been 
shown to have an analgesic effect on infants16 and cortisol lowering effects17 on mothers and infants. Likewise, 
oxytocin, a hormone produced by the pituitary gland and associated with maternal-infant bonding and stress 
reduction, has been shown to increase in mothers while they hold their infants18. Previous research demonstrates 
correlation between salivary and serum oxytocin levels19. Skin-to-skin care has led to reduced salivary cortisol 
levels and increased salivary oxytocin levels in 28 premature infants and their parents.20  
Holding a newborn is a critical moment, described as the moment in which the new parent feels like a parent for 
the first time2.  Holding a baby is a necessary precursor to feeding a baby which is a fundamental maternal 
instinct.  Barriers to holding are perceived as barriers to feeding which can disrupt the maternal bond with an 
infant.  In prematurely born infants, the inability to feed a baby is associated with a higher level of maternal 
stress21.  High levels of stress matter because maternal depression and anxiety frequently occur in the NICU22 
and research on very preterm infants has shown alterations in language development and higher anxiety traits in 
children of mothers who expressed higher levels of stress23.  
Increased bonding and reduced stress may lead to improved neurodevelopmental outcomes for infants being 
treated with therapeutic hypothermia and lower rates of psychological trauma for mothers. We anticipate the 
sampling of salivary cortisol and oxytocin will quantitatively demonstrate an endocrinological biomarker for 
stress reduction and increased bonding for mother-infant dyads who are being treated with therapeutic 
hypothermia. The results of this study will support and promote protocolized holding interventions for infants 
during therapeutic hypothermia, in a similar vein as skin-to-skin care for the prematurely born patient 
population.  
Preliminary Data: A mixed methods study completed by our team demonstrated feasibility of holding 
medically stable infants for 30 minutes during therapeutic hypothermia (manuscript under review at Acta 
Paediatrica). There were no reported adverse events during holding including dislodging of central lines or EEG 

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leads or early termination of holding due to vital sign or other infant instability. A thin foam barrier was placed 
between the mother and infant to prevent unintentional rewarming.  The mean infant temperature prior to 
holding was 33.4ºC and at completion of holding the mean temperature was 33.5ºC (p=0.18).  There was no 
significant bradycardia (heart rate less than 80 bpm), hypotension (mean arterial pressure less than 40 mmHg) 
or oxygen desaturation (less than 93%) (Figure 1). Mothers and nurses responded to survey questions with 
overwhelming enthusiasm for the perceived benefits of holding. Nurses reported that holding during 
hypothermia seemed safe. Additionally, mothers responded to our survey with reports of lower stress levels and 
improved ability to bond with their baby after holding.  
 
Figure 1: A: Temperature by case; B: Heart rate by case; C: Mean arterial pressure by case; D: Oxygen 
saturation by case; bold horizontal lines are the mean for the 30 minute period of holding for each case. 
Hypothesis 
We hypothesize that measurable increases in salivary oxytocin levels, coinciding with the reported qualitative 
increased levels of bonding, will be observed after the holding period. We anticipate the reported stress 
reduction after holding to be quantified by measurable decreases in salivary cortisol levels. We hypothesize 
these hormone changes will be present in both the mother and the infant when compared to samples taken 
without the holding intervention.  
Primary outcome: To compare the change in the level of oxytocin in maternal saliva between two conditions; 
one assessing the difference between oxytocin levels before and immediately after a 30-minute period of 
holding during therapeutic hypothermia compared to the second condition in which pre and post-levels of 
salivary oxytocin surrounding a 30-minute period of “not holding” are obtained.  
Secondary outcomes: 

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1. To compare the change in the level of cortisol in maternal saliva between the same two conditions  
2. To compare the change in the level of oxytocin and cortisol in infant saliva between the same two conditions 
3. Comparison between the holding and “not holding” conditions of infant vital signs including temperature, 
heart rate, mean arterial blood pressure and oxygen saturation collected 30 minutes prior to holding, every 2 
minutes during, and 45 minutes after holding.  
4. Subjective maternal reports of the experience of holding the infant during hypothermia and responses to 
questions about stress level and bonding. 
5. Subjective nurse report about the experience of supporting holding. 
Significance 
The subjective evidence of psychological stress for mothers in the neonatal intensive care unit (NICU) has been 
well-reported in prior research. This is exceptionally challenging for mothers of infants undergoing therapeutic 
hypothermia, who historically have not been permitted to hold their infants due to concerns for dislodging 
equipment and potential infant re-warming. Our previous research has demonstrated feasibility of holding 
infants during therapeutic hypothermia. This study aims to elucidate quantitative data to support holding as a 
potential standard intervention during therapeutic hypothermia. We aim to measure the proposed benefits of 
holding by using endocrinological biomarkers.  Future research may use these biomarkers to noninvasively 
(saliva, not blood) assess the impact of stress and bonding on neurodevelopmental outcomes in infants treated 
with hypothermia, as well as a myriad of other clinically stressful scenarios in the NICU. 
Methods: 
Study Design: This is a single center, prospective randomized controlled crossover mixed methods study of the 
effect of a 30-minute holding intervention on salivary levels of cortisol and oxytocin in mothers and infants 
treated with therapeutic hypothermia. 
Power Calculation: The power calculation was performed using the data from the Vittner paper20 for our 
primary outcome, the maternal oxytocin response to holding.  The maternal oxytocin level was chosen as the 
primary outcome for several reasons. First, we are confident in the ability to obtain salivary samples from an 
adult and second because several of the mothers in the pilot study reported symptoms during or after holding 
that would be consistent with increased oxytocin levels such as uterine contractions and/or increased breast milk 
production. 
A sample size of 15 from a finite population of infants treated with hypothermia annually (N=40), will have 
80% power to detect a difference in means of 113 pg/ml, (a first condition µ=275 pg/ml and a second condition 
µ=162 pg/ml) assuming a conservative estimate for standard deviation of 175 pg/ml using a paired t-test with a 
0.05 two-sided level of significance. Of the 40 babies treated with therapeutic hypothermia each year, 25-33% 
are expected to meet exclusion criteria.  
Location: A convenience sample of infants undergoing therapeutic hypothermia and their mothers will be 
recruited from the neonatal intensive care unit at Maine Medical Center.  
Inclusion/exclusion criteria: Inclusion criteria will be; gestational age at birth of 35 weeks or greater, absence of 
clinical or electrographic seizures during the first 24 hours of therapeutic hypothermia, and designation as 
“clinically stable” by the attending neonatologist with the infant on room air, nasal cannula, continuous positive 

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airway pressure or intubated on conventional ventilator. Exclusion criteria will be use of inhaled nitric oxide for 
persistent pulmonary hypertension of the newborn, high frequency oscillator ventilation, presence of 
electrographic seizures, use of vasopressors or paralytic agents, presence of chest tubes, wound vacuums, or 
drains, and in utero opiate exposure.  
Informed consent: Dr. Alexa Craig is the neonatal neurologist involved in the clinical care of all infants at 
Maine Medical Center treated with therapeutic hypothermia. Within this patient population, Dr. Craig will 
identify potential patients to recruit for the study. Dr. Craig will describe the research study to mothers of 
infants who meet the inclusion criteria. Therapeutic hypothermia treatment takes 72 hours to complete (three 
days). Drs. Craig, Fox or Utley will wait 24 hours into therapeutic hypothermia to determine if the infant meets 
all inclusion criteria and does not meet any exclusion criteria. At this time, Drs. Craig,  Fox, Deerwester, or 
Utley will obtain a single signed informed consent from the mother on behalf of herself and on behalf of the 
infant. After randomization, a calendar of events will be created with mothers, NICU nurses and research 
personnel for the holding plan on day two and day three of hypothermia.  
Assisting mothers to hold their infants is within the scope of practice and routine clinical duties for NICU 
nurses, including babies born prematurely (up to 23 weeks) and intubated babies. A discussion will take place 
between the NICU nurse and research personnel prior to enrolling a patient into this study. Any concerns or 
discomfort can be addressed, and no nurse will be obliged to participate in the study. Should a nurse decline to 
assist with holding a patient in this study, another nurse volunteer may step in. The patient’s nurse will be 
expected to continue to care for the patient in all non-study related aspects. Nurses will go through an informed 
consent process before completing the questionnaire. There is no penalty for declining to complete the 
questionnaire.  
Randomization: Infants and mothers will be randomly assigned to the holding condition occurring on the 
second versus the third day of hypothermia. A random number generator will be used with those numbers being 
translated into a list of sequentially numbered opaque envelopes that will be opened by the principal 
investigator (PI) in order at the time of randomization following the informed consent process (Figure 1). 
Data Collection and Management:  
After obtaining consent and randomization, a plan will be made with the mother and a discussion will take place 
regarding the timing of events. Mothers will be assisted with holding their infants for a single 30-minute period 
using the holding protocol developed for the feasibility study (Appendix A). Holding will occur between 9:00 
am and 11:00am, to account for the diurnal variation of cortisol, on day two or day three of hypothermia 
treatment. Transitioning of the infant to the mother’s arm will require the assistance of the NICU nurse caring 
for the baby and one other nurse. For condition A, four samples of saliva will be obtained; two from the mother 
(pre and post holding) and two from the infant (pre and post holding). The first sample will be collected prior to 
holding, and the second sample immediately at the end of holding before the infant is placed back in the bed 
(condition A). For condition B, four additional samples of saliva will be obtained; two from the mother (pre and 
post “not holding”) and two from the infant (pre and post “not holding”).  

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All of the infants at Maine Medical Center treated with therapeutic hypothermia are recorded in the Maine 
Medical Center Neonatal Encephalopathy REDCap database (IRB NET976245-1). We will access the database 
for demographic and descriptive characteristics for the participants of this this study. Medical history will also 
be obtained from this database, including mothers’ obstetric history and infants’ medical history. This will be 
entered into a different REDCap database specific for this study.  
A ten-question survey will be given to Mothers after holding. An informed consent form with be obtained and a 
de-identified 15-question survey will be given to nurses immediately after assisting with holding. Infant data 
and maternal surveys will be stored in a binder in a locked office in the neonatal intensive care unit or at 55 
Spring Street, Scarborough, ME. The data will periodically be manually entered into the HIPPA compliant 
REDCap by co-investigators.  
Safety:  Infant vital signs will be continuously monitored during holding and “not holding” and recorded at the 
following intervals; 30 minutes prior to, every two minutes during, and 45 minutes after holding to monitor 
infant safety. Early termination of holding will occur if there is an adverse event as defined in the feasibility 
study.  This includes a change in infant temperature greater than 0.5°C above or below 33.5°C. This cutoff is 
used based on historical data at our institution in which the infant core temperature during therapeutic 
hypothermia normally varies by up to 0.5°C above or below 33.5°C. Accidental dislodgement of central lines or 
disruption of EEG leads during holding will also be considered adverse events. Early termination of holding 
will occur if there is clinical instability of infant heart rate, blood pressure or oxygen saturation present for 
greater than 2 recorded measurements; infant bradycardia defined as heart rate less than 80 beats per minute11, 
infant hypotension with mean arterial pressure (MAP) less than 40 mmHg11, oxygen saturation of less than 
93%24, or evidence of clinical instability as determined by the NICU nurse and/or attending neonatologist.  
Laboratory Studies: Saliva will be collected from mothers by passive drool method, the gold standard when 
collecting oral fluid for biological testing. Infant saliva will be obtained using a 1 mL syringe and suctioning 
saliva from the infants mouth until 0.1mL of volume is obtained. Research indicates that oxytocin increases in 
anticipation of breast-feeding and begins to normalize after 30 minutes25. Therefore, mothers will be asked to 
pump prior to the first saliva collection, which will allow prior anticipatory oxytocin increases to normalize. 
The collection will take place within the 30-minute window after pumping, prior to the rebound increase in 
oxytocin level. The mother will be asked not to eat, drink, smoke, or exercise for one hour before the study 

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session - all factors that confound salivary cortisol levels. Infants receiving therapeutic hypothermia are not fed 
by mouth so this issue will not impact infant subjects. The half-life of oxytocin has been shown to be 10-15 
minutes18. The half-life for cortisol has been shown to be one hour26. After collection, the saliva will be mixed 
with RNAprotect® Saliva Reagent with cOmplete™ protease inhibitor, to stabilize the sample. It will then be 
frozen immediately at -20°C, transferred to Dr. Jill Maron’s lab, and stored at -80°C, a temperature which has 
been shown to stabilize cortisol and oxytocin samples.20,27,28 An enzyme-linked immunosorbent assay (ELISA) 
will be used to analyze cortisol and oxytocin concentrations in saliva28 at Dr. Jill Maron’s lab at Tufts in 
Boston. A premade salivary cortisol ELISA from ENZO Life Sciences (ADI-901-071) and a premade oxytocin 
ELISA from ENZO (ADI-901-153A) will be used. Samples will be run in triplicate.  The cortisol and oxytocin 
levels will be tested in one batch at the completion of the study.  
The sensitivity of the cortisol assay is 56.72 pg/ml. The intra- and interassay coefficients of variation are 6.6% 
and 7.8% respectively. Crossreactivities for the cortisol ELISA include: Prednisolone (122.35%), 
Corticosterone (27.68%), 11-deoxycortisol (4.0%), Progesterone (3.64%), Prednisone (0.85%), Testosterone 
(0.12%) and less than <0.10% androstenedione, cortisone and estradiol. The sensitivity of oxytocin assay is 15 
pg/ml, without correcting for concentration. The intra- and interassay coefficients of variation are 10.2% and 
16.5% respectively for our anticipated oxytocin concentration (100-300 pg/ml11). The oxytocin ELISA is 
reported to cross react with the following substances: mesotocin (7%), Arg-vasotocin (7.5%), Ser ,Ile -Oxytocin 
(<0.2%), TRH (<0.2%), Somatostatin (<0.2%), Met-Enkephalin (<0.2%), VIP (<0.2%), Lys -Vasopressin 
(<0.2%), Arg -Vasopressin (<0.2%), α−ANP (<0.2%), Growth Hormone (<0.2%), Tocinoic acid (<0.2%), and 
Melanostatin (<0.2%). 
Data Analysis: Descriptive characteristics of infants and their mothers will be presented by percentages for 
categorical variables and by means and standard deviation (or non-parametric equivalent) for continuous 
variables. Salivary cortisol and oxytocin concentrations will be determined by comparing the optical density of 
the ELISA result to a control curve.  A paired t-test will be used to assess for significant differences in hormone 
levels pre and post-holding. Responses to surveys will be described using proportions of “agree”, “strongly 
agree”, “disagree” and “strongly disagree.” 
Potential Problems: Our previous research demonstrated feasibility of holding clinically stable infants for 30 
minutes during therapeutic hypothermia. Based on extensive experience of allowing mothers to hold critically 
ill premature infants, our NICU nurses developed and implemented a safe technique to transition hypothermic 
infants from the bed to their mother’s arms without adverse events. The thermal barrier successfully prevented 
rewarming of the infant.  
For this subsequent study on salivary biomarkers, we are confident in the ability to transition infants safely to 
holding, but acknowledge that problems could occur with saliva collection or storage, since these are the new 
components of the protocol. There is also the potential for difficulty in coordinating the timing of maternal 
pumping of breast milk and the collection of the first maternal “pre-holding” saliva specimen. We intend to 
address this with education and written instructions, as well as assistance from nursing staff. There may also be 
potential issues that arise around the availability and proper storage of dry ice. We will work with the Maine 
Medical Center patient laboratory to develop a specimen handling and storage protocol. The specimens, once 
collected, will be hand delivered to the lab for storage. 
 
Appendix A: Updated Safety Protocol 
 

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REASON FOR RESEARCH: 
Holding in normothermic infants has been shown to reduce stress hormones, decrease crying and irritability, 
regulate heart rate and breathing patterns, enhance mother-infant bonding, and provides more restful sleep 
patterns. This research project is aimed at understanding whether or not this holds true for hypothermic babies 
and their mothers. 
 
OUTCOME MEASURE: 
Maternal and Infant saliva will be collected before and after holding. Saliva will be tested for cortisol and 
oxytocin levels. We hypothesize that there will be a decrease in cortisol (marker of stress) and increase in 
oxytocin (marker of bonding) after the 30-minute holding period, when compared to samples obtained 30 
minutes apart without holding taking place.  
 
ELIGIBILITY CRITERIA: 
 Infant with gestational age ≥35 weeks treated with therapeutic hypothermia 
 Infant without seizures in the first 24 hours on EEG 
 Infants who are clinically stable either intubated, on bubble CPAP, nasal cannula or who have no respiratory 
support.  This criterion MUST be confirmed by the attending neonatologist. 
 Infants with all types of vascular access are eligible. 
o Use appropriate caution and attention to peripheral arterial lines and central lines (PICC, UAC, 
UVC, tunneled central catheters) 
 Informed consent for research study signed by mother who is at MMC. 
 
EXCLUSION CRITERIA: 
 High frequency oscillator ventilation 
 Use of inhaled nitric oxide 
 Persistent pulmonary hypertension of the newborn 
 Presence of seizures on EEG 
 Use of vasopressors or paralytic agents 
 Presence of chest tubes, wound vacuum or drains 
 Neonatal abstinence syndrome 
 
GENERAL INSTRUCTIONS: 
 Holding will occur exclusively as part of the research study. 
 May ONLY be performed by an RN in conjunction with the research team 
 Instruct the mothers to plan to stay in the NICU for a least one to two hours 
 Only the mother can hold the infant during the research project. 
 Instruct mother to take care of her own personal needs (i.e. bathroom) before holding.  The mother will be 
asked not to eat, drink, smoke, or exercise for one hour before the study session. The mother will be asked 
to pump breastmilk within 30 minutes prior to holding.  
 Encourage mothers to discuss their concerns regarding holding with their infant’s nurse.   
 
EQUIPMENT: 
1. Comfortable chair/recliner 
2. Research personnel will be present to assist the RN with transition of infant from Giraffe stand to mother’s 
arms, monitor vital signs, and handle saliva specimens.   
3. Pillows for support 

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4. Insulating barrier 
5. Four saliva specimen collection kits for infant  
6. Four saliva specimen collection kits for Mother 
 
PROCEDURE FOR HOLDING INTERVENTION:   
1. Assemble equipment 
2. Obtain saliva for Mother and Infant per kit collection protocol.  
3. Obtain initial set of vital signs (heart rate, respiratory rate, oxygen saturation, blood pressure and 
temperature). Record complete set of vitals 15 minutes into the 30-minute holding session.  Record another 
complete set of vitals after holding session is over.  Research team to record temperature and oxygen 
saturation every 2 minutes during holding.  Temperature will be recorded from the esophageal probe.   
4. Position reclining chair next to Giraffe stand.  
5. Have mother sit in recliner and place thermal layer over her chest and abdomen 
6. Transfer infant to mothers by placing hands under the cooling blanket in order to lift blanket and infant 
together 
7. Support infant’s head and lines immediately after transfer  
8. Assess need for increased oxygen by monitoring the oxygen saturation 
9. Offer pillows as necessary for additional support.  
10. If the infant shows signs/symptoms of not tolerating holding (i.e. drop in oxygen saturation below 90% or if 
temperature increases greater than 1°C) STOP HOLDING and return infant to Giraffe stand. Continue to 
monitor cardiorespiratory status of the infant for 30 minutes following holding. 
11. At the end of holding, prior to transferring infant back to Giraffe stand, obtain second saliva samples from 
mother and infant.  
 
PROCEDURE FOR NO-HOLDING INTERVENTION: 
1. Assemble equipment 
2. Obtain saliva for Mother and Infant per kit collection protocol.  
3. Obtain initial set of vital signs (heart rate, respiratory rate, oxygen saturation, blood pressure and 
temperature) 
4. After 30 minutes, obtain second saliva samples from mother and infant.  
 
 
DOCUMENTATION: 
1. RN will document in EPIC the three sets of vital signs taken before, during and after holding session (HR, 
RR, BP, T, O2) 
2. RN will document in EPIC note the length of holding time upon completion of session and any 
complications from holding 
 
 
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