Clinical trial NCT03593473
Conditions: Depression, Postpartum Anxiety Disorders Stress, Psychological
Keywords: Oxytocin Hypothalamic-pituitary-adrenal axis (HPA)
Sponsor: University of North Carolina, Chapel Hill
Start date: 2019-02-07; Completion date: 2023-07-25
Enrollment: 109 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Mothers who were enrolled in the Mood, Mother and Infant study will be eligible to participate in the 6-year follow-up maternal visit. At the time of this visit, mothers will be randomized to a single 24 IU dose of nasal oxytocin or placebo. Following administration of the study drug, women will participate in the Trier Social Stress Test (TSST), and blood samples will be collected to quantify HPA axis reactivity.

Detailed description:
The Mood, Mother and Infant Study is a prospective observational cohort study that began enrollment in May of 2013. Women were recruited in the 3rd trimester of pregnancy and followed prospectively through 12 months postpartum. Mother-infant pairs who completed the 12-month visit will be invited to participate in the current Psychobiology of Resilience in Maternal-Child Pairs follow-up study. The trial described here is a randomized controlled trial embedded within the Psychobiology of Resilience study.

Non-pregnant women will be randomized to either 24 IU of nasal oxytocin (OT) or placebo. The Investigational Drug Service (IDS) will use a random number generator to prepare a randomization table. Participants will be block randomized by risk status at enrollment in the Mood, Mother and Infant (MMI) study, as verified by structured clinical diagnostic interview (No history of depression or anxiety, Past depression or anxiety, current depression or anxiety). Both participants and study personnel will be blinded to allocation group. At the end of the protocol, participants will be asked which condition they believed they were in ('oxytocin,' 'control,' 'not sure') to ascertain success of blinding. Forty minutes after treatment, women will undergo the Trier Social Stress Test (TSST), comprised of a speech task and a math task; the TSST reliably induces large and consistent HPA and cardiovascular responses. The TSST is administered as follows: Pre-Task Instructions: (5 minutes) Subjects will be introduced to 3 people (the 'selection committee') and asked to assume the role of a job applicant. Anticipation Period: The subject prepares her speech for 3 minutes in the presence of the committee. Speech: The committee asks the subject to deliver her talk for 5 minutes while being video and audio-recorded. If the subject finishes early, the committee responds with prepared questions to ensure that she speaks for the entire 5 minutes. These questions are designed to be non-harassing but to create a feeling of lack of predictability/controllability (e.g., "Do you have any enemies?") Serial Subtraction (PASST): The committee will ask the subject to subtract the number 7 from 2000 as quickly and accurately as possible for 5 minutes. For each mistake, the committee says "Stop -- mistake -- start over at 2000." Stress Recovery: The subject sits quietly alone for 20 minutes.

Blood will be collected at baseline, during the speech and math tasks, and at 10 and 20 minutes of recovery, as HPA-axis responses are reliably found 10-30 minutes following the TSST. Evidence regarding optimal timing of stress testing is conflicting. Visits will be scheduled for 1 pm to increase likelihood of detecting a stress response unopposed by the circadian influence, based on the experience of investigators in our laboratory and published studies of postpartum women. These investigators have found menstrual cycle phase does not affect TSST results; therefore, the date of last menstrual period and hormone use will be recorded, but visits will not be scheduled based on cycle phase.

Arms: arm 1: Participants block randomized to Oxytocin intranasal spray by risk status at enrollment in the Mood, Mother and Infant (MMI) study and as verified by structured clinical diagnostic interview (no history of depression or anxiety, past depression or anxiety, current depression or anxiety). arm 2: Participants block randomized to placebo Intranasal spray with all equivalent ingredients except oxytocin. Blocks will be stratified by risk status at enrollment in the Mood, Mother and Infant (MMI) study and as verified by structured clinical diagnostic interview (no history of depression or anxiety, past depression or anxiety, current depression or anxiety).
Interventions: intervention 1: Intranasal Oxytocin intervention 2: Placebo
Intervention descriptions: intervention 1: Six intranasal sprays of oxytocin. Each insufflation delivers 4 IUs of oxytocin for a total oxytocin dosage of 24 IUs. intervention 2: Six intranasal sprays of placebo manufactured to mimic oxytocin nasal spray containing all equivalent ingredients except oxytocin.

Protocol documents:
Prot_SAP_001.pdf: 
Protocol 
 
 
Inhaled Oxytocin and HPA Axis Reactivity 
 
 
NCT number NCT03593473 
Document Date 01/19/2024 
 


Complete Title:  Inhaled Oxytocin and HPA Axis Reactivity  
Short Title: Inhaled Oxytocin and HPA Axis Reactivity 
 
Drug or Device Name(s): Inhaled Oxytocin 
FDA IND/IDE (if applicable): Exempt 
Sponsor: NICHD 
Protocol Date: January 19, 2024 
Amendment 1 Date: 
Amendment 2 Date:  
Amendment 3 Date: 
Amendment 4 Date:  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Mood, Mother and Child: The Psychobiology of Dyadic Resilience 
NICHD, R01HD093901 
 


 
 
2
 
 
 
Table of Contents 
 
1. 
Abbreviations and Definitions of Terms 3 
2. 
Protocol Synopsis 3 
3. 
Background and Rationale 5 
4. 
Investigational Plan 6 
5. 
Study Procedures 8 
6. 
Study Intervention (Device, Drug, or Other Intervention) 11 
7. 
Study Intervention Administration (If Applicable) 11 
8. 
Safety Management 12 
9. 
Data Collection and Management 12 
10. 
Recruitment Strategy 12 
11. 
Consent Process 12 
12. 
Plans for Publication 13 
13. 
References 13 
14. 
Appendix 14 
 
 
 


 
 
3
 
1. 
Abbreviations and Definitions of Terms 
Abbreviation 
Definition 
OT 
Oxytocin 
TSST 
Trier Social Stress Test 
CRT 
Cortisol 
ACTH 
Adrenocorticotropic hormone 
MMI 
Mood, Mother and Infant Study 
MMC 
Mood, Mother and Child Study 
 
2. 
Protocol Synopsis 
Study Title 
Inhaled Oxytocin and HPA Axis Reactivity 
Funder 
NICHD 
Clinical Phase 
n/a 
Study Rationale 
This is Randomized Parallel Assignment Quadruple masked protocol to test the 
effect of inhaled oxytocin vs. placebo on HPA axis reactivity during a standardized 
social stressor, the Trier social stress test. This work will address a substantial gap 
regarding the effect of OT on psychobiology in women and test the extent to which 
differences in OT response contribute to dyadic development. 
 
Study Objective(s) 
Primary  
The primary objective of this experiment is to test the extent to which oxytocin 
compared with placebo alters cortisol trajectory during the Trier Social Stress Test. 
 
Secondary 
The secondary objective of this experiment is to test the extent to which oxytocin 
compared with placebo alters other measures of stress reactivity during the Trier 
Social Stress Test. We will measure trajectory of ACTH, lagged association between 
ACTH and CRT, heart rate variability, and pre-ejection period during the Trier Social 
Stress Test. 
 
Other 
We will test the extent to which 1) maternal depression/anxiety and 2) OXTR 
genotype modifies the effect of oxytocin vs. placebo on measures of stress 
reactivity. 
 
Test Article(s) 
(If Applicable) 
Participants will receive six intranasal sprays of oxytocin or placebo manufactured to 
mimic oxytocin nasal spray containing all equivalent ingredients except oxytocin.  
Each insufflation delivers 4 IUs of oxytocin for a total oxytocin dosage of 24 IUs. 
 
Study Design 
 
Randomized Parallel Assignment Quadruple masked experiment 
Subject Population 
key criteria for 
Inclusion and 
Exclusion: 
This study will follow-up the existing Mood, Mother and Infant (MMI) prospective 
longitudinal cohort (R01HD073220), comprised of 222 mother-infant dyads who 
were recruited between May 2013 and April 2017 and completed the 12-month MMI 
visit. Inclusion Criteria for Inhaled Oxytocin and HPA Axis Reactivity, a sub study of 


 
 
4
the Psychobiology of Resilience in Mother-Child Pairs follow-up study: 1) 
Participated in the MMI study 2) Both mother and child willing and able to participate 
in the follow-up visits at 6 to 8 years postpartum. 3) Not pregnant, verified by urine 
pregnancy test on day of study visit. 
Number Of Subjects  
 
Our projected enrollment is 150 participants, recruited from the 222 individuals who 
enrolled in the MMI study.  
Study Duration 
Each subject’s participation in the Inhaled Oxytocin and HPA Axis Reactivity study is 
expected to last 2 hours. Participation in the Mood, Mother and Infant and Mood, 
Mother and Child study began with recruitment in the third trimester of pregnancy. 
Child follow-up has currently extended to about 8 years. 
 
Study Phases 
Screening 
Study Treatment 
Follow-Up   
 (1) Screening: Participants in the Mood, Mother, and Infant study are invited to 
participate in the Mood, Mother, and Child follow-up study.  Those who meet 
inclusion criteria are scheduled for a mother-only visit for the Inhaled Oxytocin and 
HPA Axis Reactivity sub study.  
(2) Intervention: Non-pregnant women will be randomized to either 24 IU of nasal 
OT or placebo prior to the Trier Social Stress Test. 
(3) Follow-up:  Effects of nasal OT will be measured during the study visit, which will 
last about 2 hours.  
Efficacy Evaluations 
This is not a treatment study; this is a translational study to test the effect of oxytocin 
on the HPA axis.  
Pharmacokinetic 
Evaluations 
NA 
Safety Evaluations 
Prior to and during a 90-minute period following intranasal OT or placebo, mothers 
will be monitored for vital signs (heart rate, blood pressure, ear temperature), 
vascular resistance and mood. 
Statistical And Analytic 
Plan 
We will use a general linear model to test the hypothesis that participants who are 
randomized to receive nasal oxytocin experience lower HPA reactivity, 
operationalized using CRT area under the curve (AUC) during the TSST, to evaluate 
this proposed causal pathway 
Data And Safety 
Monitoring Plan 
Study PI, Dr. Alison Stuebe, will provide medical oversight for the randomized trial of 
oxytocin vs. placebo. Any adverse events, as well as any significant deterioration in 
mood, health or wellbeing, will be reported directly to Dr. Stuebe (the PI). All 
moderate and severe adverse events will be reported within one week to the FDA, 
whether anticipated or unanticipated. 
 
 
 


 
 
5
3. 
Background and Rationale 
3.1. 
Introduction 
Mothers who were enrolled in the Mood, Mother and Infant study1 will be eligible to 
participate in the 6-year follow-up maternal visit. At the time of this visit, mothers will be 
randomized to a single 24 IU dose of nasal oxytocin or placebo. Following 
administration of the study drug, women will participate in the Trier Social Stress Test 
(TSST), and blood samples will be collected to quantify HPA axis reactivity.  
 
3.2. 
Name and Description of Investigational Product or Intervention  
Active drug: Six intranasal sprays of oxytocin. Each insufflation delivers 4 IUs of 
oxytocin for a total oxytocin dosage of 24 IUs. 
Placebo: Six intranasal sprays of placebo manufactured to mimic oxytocin nasal spray 
containing all equivalent ingredients except oxytocin. 
 
3.3. 
Non-Clinical and Clinical Study Findings  
Limited evidence suggests that exogenous OT has mixed effects on women with 
perinatal depression. Mah et al administered nasal OT and placebo in a within-subject 
cross-over study among women with PND. Nasal OT increased protective behavior 
towards the infant, but did not affect sensitive caregiving; moreover, with OT, women 
were more likely to report a harsh response to infant crying2-4. No studies to our 
knowledge have examined exogenous OT among mothers of older children. In the 
present proposal, we plan a randomized controlled trial of nasal OT vs. placebo among 
MMC mothers. This work is innovative, because few studies have tested the effect of 
nasal oxytocin on HPA axis reactivity, and the overwhelming majority of subjects 
studied have been male5,6: of 147 individuals in the extant literature who received OT or 
placebo prior to the TSST, only 24 were women. These studies found varying 
associations between OT and HPA axis activity, with moderation by factors such as 
concurrent social support7 emotional regulation abilities8, substances use9 and 
childhood trauma history22,10. These differential effects of OT on HPA axis may 
contribute to perinatal depression or anxiety. We will test the effect of nasal OT vs. 
placebo among 150 women with detailed, longitudinal data, including patterns of 
endogenous OT secretion during breastfeeding and maternal experience of trauma in 
her own childhood. 
3.4. 
Relevant Literature and Data 
Perinatal depressive symptoms are associated with dysregulation of the HPA axis. 
Jolley et al11 measured ACTH and cortisol during an exercise stressor at 6 and 12 
weeks postpartum and found expected correlations between ACTH and cortisol among 
euthymic women (N=13), but not among women with depressive symptoms (N=9). 
Heinrichs compared lactating mothers who breastfed vs. held their infants 30 minutes 
before the TSST and found that recent breastfeeding blunted cortisol, but not ACTH12. 
This blunting is thought to be mediated by oxytocin, and a growing literature implicates 
dysregulation of OT in PND. Findings in our laboratory using extracted samples 
demonstrate an inverse association between OT during breastfeeding and maternal 


 
 
6
symptoms of depression and anxiety at 2 months postpartum13. We further found that 
OT AUC differentially impacted HPA axis activity during the TSST in women with or 
without mood symptoms14. These results implicate OT and HPA axis dysregulation in 
the psychobiology of perinatal depression; in the proposed study, we will determine 
whether these differences persist at 6 to 8 years postpartum.  
3.5. 
Study Objectives 
3.5.1. Primary Objective 
The primary objective of this experiment is to test the extent to which oxytocin 
compared with placebo alters cortisol trajectory during the Trier Social Stress Test. 
 
3.5.2. Secondary Objective 
The secondary objective of this experiment is to test the extent to which oxytocin 
compared with placebo alters other measures of stress reactivity during the Trier Social 
Stress Test. We will measure trajectory of ACTH, lagged association between ACTH 
and CRT, heart rate variability, and pre-ejection period during the Trier Social Stress 
Test. 
 
3.5.3. Other objectives:  
We will test the extent to which maternal depression/anxiety modifies the effect of OT 
on CRT, ACTH, lagged association between ACTH and CRT, heart rate variability, and 
pre-ejection period during the Trier Social Stress Test. 
 
We will test the extent to which OXRT genotype modifies the effect of OT on CRT, 
ACTH, lagged association between ACTH and CRT, heart rate variability, and pre-
ejection period during the Trier Social Stress Test. 
4. 
Investigational Plan 
4.1. 
Study Design 
This is Randomized Parallel Assignment Quadruple masked experiment to test the 
effect of inhaled oxytocin vs. placebo on HPA axis reactivity during a standardized 
social stressor, the Trier social stress test. Mothers who were enrolled in the Mood, 
Mother and Infant study will be eligible to participate in the 6-year follow-up maternal 
visit. At the time of this visit, mothers will be randomized to a single 24 IU dose of nasal 
oxytocin or placebo. Following administration of the study drug, women will participate 
in the Trier Social Stress Test (TSST), and blood samples will be collected to quantify 
HPA axis reactivity during a 90-minute period following study drug administration.  
   
4.2. 
Allocation to Treatment Groups and Blinding (if applicable) 
Non-pregnant women will be randomized to either 24 IU of nasal OT or placebo. The 
Investigational Drug Service will use a random number generator to prepare a 
randomization table. Participants will receive a nasal insufflation bottle of oxytocin 
intranasal spray or placebo intranasal spray manufactured to mimic oxytocin nasal 
spray. 


 
 
7
 
4.3. 
Study Duration, Enrollment and Number of Subjects 
Each subject’s participation in the Inhaled Oxytocin and HPA Axis Reactivity study is 
expected to last 2 hours. Participation in the Mood, Mother and Infant1 and Mood, 
Mother and Child15 study began with recruitment in the third trimester of pregnancy, with 
ongoing follow-up through the child’s 6 to 8th birthday. 
 
4.4. 
Study Population 
This study will follow-up the existing Mood, Mother and Infant (MMI) prospective 
longitudinal cohort (R01HD073220)1, comprised of 222 mother-infant dyads who were 
recruited between May 2013 and April 2017 and completed the 12-month MMI visit. In 
the MMI study, 222 mothers ages 18-45 and their infants were enrolled. Participants 
were recruited from community clinics in the third trimester of pregnancy and continued 
to participate in the study through 12 months postpartum. At the 12-month visit, mothers 
were invited to continue to be followed via online surveys at 6-month intervals; more 
than 80% of women who have completed the MMI study to date have continued to 
participate. Enrolled participants in the MMI study met the following inclusion and 
exclusion criteria: 
 
4.4.1. MMI Inclusion Criteria: 
• Singleton pregnancy; 
• Intention to breastfeed (due to the centrality of breastfeeding to the oxytocin 
assessment); 
• Intention to remain within 40 miles of the University of North Carolina - Chapel 
Hill through infant's first birthday; 
• Ability to communicate in English. 
 
4.4.2. MMI Exclusion Criteria: 
• Maternal diagnosis of Axis I disorders other than unipolar depression or anxiety 
disorders. Women with a history of bipolar disorder were excluded, given their 
increased risk of postpartum psychosis. 
• Active substance abuse at enrollment in the 3rd trimester of pregnancy 
(Tobacco, alcohol, illicits); 
• Major congenital anomaly; 
• Chronic medication/medical condition contraindicated for breastfeeding; 
• Current use of tricyclic antidepressants, which alter cortisol and heart rate 
variability. 
At enrollment, all participants underwent a Structured Clinical Interview Non-Patient 
version (SCID-NP). 


 
 
8
4.5. 
 Inclusion Criteria for Inhaled Oxytocin and HPA Axis Reactivity 
This is a trial embedded within the Psychobiology of Resilience in Mother-Child Pairs 
follow-up study. Subjects were included who: 
• Participated in the MMI study 
• Both mother and child willing and able to participate in the follow-up visits at 6 to 
8 years postpartum.  
• Were not pregnant, verified by urine pregnancy test on day of study visit. 
5. 
Study Procedures 
The Mood, Mother and Infant Study is a prospective observational cohort study that 
began enrollment in May of 2013. Women were recruited in the 3rd trimester of 
pregnancy and followed prospectively through 12 months postpartum. Mother-infant 
pairs who completed the 12-month visit will be invited to participate in the current 
Psychobiology of Resilience in Maternal-Child Pairs follow-up study. The Inhaled 
Oxytocin and HPA Axis Reactivity trial described here is a randomized controlled trial 
embedded within the Psychobiology of Resilience study.   
5.1. 
Screening/Baseline Visit procedures 
When the study staff member speaks with the participant to ascertain interest in 
participating in the MMC visits, the staff member will screen the participant for any 
contraindications to inhaled oxytocin and review the risks and benefits of OT 
administration. Before the visit, the study staff member will email the participant the 
consent document. Prior to the visit, a study staff member who has completed 
DocuSign Part 11 training will speak with the participant by phone and review the full 
consent document. Following this discussion, the participant will sign an electronic 
consent. Once the participant has signed the electronic consent, Dr. Stuebe will review 
and sign the order for IDS. 
 
5.2. 
Intervention/Treatment procedure for study visit 
Non-pregnant women will be randomized to either 24 IU of nasal oxytocin (OT) or 
placebo. The Investigational Drug Service (IDS) will use a random number generator to 
prepare a randomization table. Participants will be block randomized by risk status at 
enrollment in the Mood, Mother and Infant (MMI) study, as verified by structured clinical 
diagnostic interview (No history of depression or anxiety, Past depression or anxiety, 
current depression or anxiety). Both participants and study personnel will be blinded to 
allocation group. At the end of the protocol, participants will be asked which condition 
they believed they were in ('oxytocin,' 'control,' 'not sure') to ascertain success of 
blinding. Forty minutes after treatment, women will undergo the Trier Social Stress Test 
(TSST), comprised of a speech task and a math task; the TSST reliably induces large 
and consistent HPA and cardiovascular responses16,17. The TSST is administered as 
follows: Pre-Task Instructions: (5 minutes) Subjects will be introduced to 3 people (the 
'selection committee') and asked to assume the role of a job applicant. Anticipation 
Period: The subject prepares her speech for 3 minutes in the presence of the 
committee. Speech: The committee asks the subject to deliver her talk for 5 minutes 
while being video and audio-recorded. If the subject finishes early, the committee 


 
 
9
responds with prepared questions to ensure that she speaks for the entire 5 minutes. 
These questions are designed to be non-harassing but to create a feeling of lack of 
predictability/controllability (e.g., "Do you have any enemies?") Serial Subtraction 
(PASST): The committee will ask the subject to subtract the number 7 from 2000 as 
quickly and accurately as possible for 5 minutes. For each mistake, the committee says 
"Stop -- mistake -- start over at 2000." Stress Recovery: The subject sits quietly alone 
for 20 minutes. 
5.3. 
Follow- up procedures : NA 
5.4. 
Unscheduled visits: NA  
5.5. 
Concomitant Medication documentation: NA 
5.6. 
Rescue medication administration ( if applicable): NA 
5.7. 
Subject Completion/ Withdrawal procedures 
All participants will also be reminded before starting the procedures that they would be 
free to withdraw at any time without penalty, and may contact our IRB review board or 
the Principal Investigator (numbers are provided) if she feels that rights regarding 
privacy or risk to health have been infringed. The right of the participants to withdraw 
from the study or to refuse any procedure will be made absolutely clear. The original 
consent form will be kept in a secured location in a locked office, and a copy will be 
provided to the participant. 
5.8. 
Screen failure procedures 
Some mothers who are eligible for Inhaled Oxytocin and HPA Axis Reactivity are 
expected to be pregnant at the time of the follow-up study. Because oxytocin can cause 
contractions, the Inhaled Oxytocin and HPA Axis Reactivity visit will not be scheduled 
while the mother is known to be pregnant, and will be deferred until 6 months 
postpartum. All mothers without a known pregnancy will be screened with a urine 
pregnancy test on arrival to the Mother Visit; if the pregnancy test is positive, the nasal 
OT and TSST protocol will not be performed. 
5.9. 
STUDY EVALUATIONS AND MEASUREMENTS 
Mobile Impedance Cardiographs (MindWare Tech Ltd, Gahanna, OH) will be used to 
measure cardiac rate, interbeat interval and pre-ejection period (PEP) in mothers and 
children. MindWare HRV software will be used to derive respiration and to calculate 
high frequency HRV and respiratory sinus arrhythmia from the IBI series as indices of 
parasympathetic activity. Respiratory rate will be used in all HRV analyses. MindWare 
Impedance Cardiography Analysis software will calculate cardiac time intervals (PEP, 
LVET), stroke volume and cardiac output. PEP will index sympathetic activation. 
 
Blood will be collected at baseline, during the speech and math tasks, and at 10 and 20 
minutes of recovery, as HPA-axis responses are reliably found 10-30 minutes following 
the TSST. Evidence regarding optimal timing of stress testing is conflicting. Visits will be 
scheduled for 1 pm to increase likelihood of detecting a stress response unopposed by 
the circadian influence, based on the experience of investigators in our laboratory and 
published studies of postpartum women. These investigators have found menstrual 


 
 
10
cycle phase does not affect TSST results; therefore visits will not be scheduled based 
on cycle phase.  
 
At baseline, 40 minutes after nasal OT, at the end of the TSST, and at the end of 
recovery, participants will complete a brief task assessment questionnaire, rating on a 
scale of 0-10 to what extent then felt 1) Stressed, Worried, Nervous, or Tense; 2) 
Happy, Relaxed, Comfortable, or Satisfied; 3) Irritated, Annoyed, ‘Pissed off,’ or 
Furious; 4) Depressed, Sad, Down, or Unhappy; and 5) Overwhelmed, Unable to 
Control Things, or Discouraged. 
 
At the end of the protocol, participants will be asked which condition they believed they 
were in ('oxytocin,' 'control,' 'not sure') to ascertain success of blinding. 
5.10. Efficacy Evaluation: NA 
5.11. Pharmacokinetic Evaluation: NA 
5.12. Safety Evaluations 
Prior to and during a 90-minute period following intranasal OT or placebo, mothers will 
be monitored for vital signs (heart rate, blood pressure, ear temperature), vascular 
resistance and mood.  
5.13. STATISTICAL CONSIDERATIONS 
 
5.13.1. 
Primary Endpoint 
The primary endpoint of this analysis is CRT trajectory during the TSST. 
 
5.13.2. 
Secondary Endpoint 
Our secondary end points comprise other measures of stress reactivity during the 
TSST, including trajectory of ACTH, lagged association between ACTH and CRT, heart 
rate variability, and pre-ejection period.  
 
5.13.3. 
Other endpoint 
In exploratory analyses, we will test the extent to which 1) maternal depression/anxiety 
and 2) OXTR genotype. 
5.14. Statistical Methods 
Our primary objective is to test the extent to which nasal oxytocin compared with 
placebo alters the trajectory of CRT during the TSST. We will use repeated measures 
analysis to quantify the effects of group (OT vs. placebo) on CRT trajectory during the 
TSST. We will first establish the shape of unconditional trajectory models for CRT using 
generalized linear mixed models with a random intercept and slopes to account for 
individual differences in CRT trajectories. We will test the fit of quadratic, cubic, and 
exponential effects of time as well as linear spline models. The most parsimonious 
unconditional trajectory model that fits the data well will be used for hypothesis testing. 
The intercept of the CRT trajectories will be placed at peak CRT. We will regress growth 
parameters (intercept and slopes) on treatment arm. A significant effect of treatment 
arm on the intercept will indicate a difference in peak CRT across groups, and a 


 
 
11
significant effect of treatment arm on slopes will indicate differences in the rate of 
recovery during the TSST. 
 
For secondary endpoints, we will similarly use repeated measures analysis to quantify 
the effects of group (OT vs. placebo), time, and group x time on each measure’s 
trajectory during the TSST. We will use repeated measures analysis with an interaction 
term to test the extent to which maternal depression/anxiety and OXTR genotype 
modifies the effect of oxytocin vs. placebo on measures of stress reactivity. 
5.15. Sample Size and Power 
Our projected sample size is 150 participants whose cortisol is assessed 8 times during 
the visit. Heinrichs et al (2001) assessed the effect of breastfeeding vs. holding a baby 
on cortisol levels among postpartum women undergoing the Trier Social Stress Test. 
Cortisol levels were similar prior to the social stressor, and then diverged, with values 
that were 1.03, 1.22, and 0.73 standard deviations higher among those who had held 
their baby compared with those who had breastfed. Because CRT growth parameters 
are individual-level parameters, power for detecting the effect of individual 
randomization to treatment arm on CRT trajectories can be simplified into an 
independent samples t-test. We used SAS 9.4 to estimate the minimum detectable 
difference for a two-sample t-test based on sample size, with 80% power and an alpha 
of 0.05. With only 30 participants per group, we are powered to detect a standardized 
effect of 0.74 standard deviations (Table). This effect is in line with the smallest 
plausible effect size in Heinrichs et al. 
 
Table: Minimum detectable difference by N per group 
N per Group 
30 
35 
40 
45 
50 
55 
60 
65 
70 
75 
Mean Diff 
0.74 
0.68 
0.63 
0.60 
0.57 
0.54 
0.52 
0.50 
0.48 
0.46 
 
5.16. Interim Analysis: NA 
6. 
Study Intervention (Device, Drug, or Other Intervention) 
The study drug and placebo are obtained from Apotheke Roter Ochsen (Schaffhausen, 
Switzerland). The study drug was shipped to the UNC Investigational Drug Service, 
which took responsibility for storage, distribution, drug return and destruction.  
7. 
Study Intervention Administration (If Applicable)  
Randomization: Non-pregnant women will be randomized to either 24 IU of nasal 
oxytocin (OT) or placebo. The Investigational Drug Service (IDS) will use a random 
number generator to prepare a randomization table. Participants will be block 
randomized by risk status at enrollment in the Mood, Mother and Infant (MMI) study, as 
verified by structured clinical diagnostic interview (No history of depression or anxiety, 
Past depression or anxiety, current depression or anxiety). 
Masking: Participants will receive a nasal insufflation bottle of oxytocin intranasal spray 
or placebo intranasal spray manufactured to mimic oxytocin nasal spray. A licensed 
provider will supervise administration of the study drug. Both participants and study 
personnel will be blinded to allocation group. At the end of the protocol, participants will 


 
 
12
be asked which condition they believed they were in ('oxytocin,' 'control,' 'not sure') to 
ascertain success of blinding. 
8. 
Safety Management 
Study PI, Dr. Alison Stuebe, will provide medical oversight for the randomized trial of 
oxytocin vs. placebo. In addition, a licensed provider will administer the study drug and 
be present for the Mother Visit. If a participant reports an unanticipated problem or 
adverse event, Dr. Stuebe will be notified immediately, and she will follow-up directly 
with the participant. If medical intervention is required, treatment allocation will be 
disclosed to the participant. The study coordinator will monitor AEs at study visits, 
tabulating events weekly. Any adverse events, as well as any significant deterioration in 
mood, health or wellbeing, will be reported directly to Dr. Stuebe (the PI). All moderate 
and severe adverse events will be reported within one week to the FDA, whether 
anticipated or unanticipated. 
9. 
Data Collection and Management 
Participant contact information required for scheduling study visits and phone visits will 
be maintained and shared by team members only as required. This information will be 
stored in locked filing cabinets in locked rooms and/or transmitted on secure computers 
only using masked study id's. All other data communicated to research team 
(biostatisticians, lab personnel) will be masked. 
10. 
Recruitment Strategy 
This study will follow-up the existing Mood, Mother and Infant (MMI) prospective 
longitudinal cohort (R01HD073220), comprised of 222 mother-infant dyads who were 
recruited between May 2013 and April 2017 and completed the 12-month MMI visit. In 
the MMI study, 222 mothers ages 18-45 and their infants were enrolled. Participants 
were recruited from community clinics in the third trimester of pregnancy and continued 
to participate in the study through 12 months postpartum. At the 12-month visit, mothers 
were invited to continue to be followed via online surveys at 6-month intervals; more 
than 80% of women who have completed the MMI study continued to participate in 
follow-up surveys. We will contact MMI participants and invite them to participate in the 
Mood, Mother, and Child study, of which Inhaled Oxytocin and HPA Axis Reactivity is a 
component. 
11. 
Consent Process 
Each participant has previously enrolled in the Mood, Mother, and Infant Study. As with 
the MMI study, each participant in the Mood, Mother, and Child Study will receive a 
verbal and written explanation of the purpose, procedure and potential hazards of this 
protocol. If a participant agrees to participate in the MMC study, they will receive a 
verbal and written explanation of the purpose, procedures and potential hazards of the 
protocol for both the mother and child. When the study staff member speaks with the 
participant to ascertain interest in participating in the MMC visits, the staff member will 
screen the participant for any contraindications to inhaled oxytocin and review the risks 
and benefits of OT administration. Before the visit, the study staff member will email the 
participant the consent document. Prior to the study visit, a study staff member who has 
completed DocuSign Part 11 training will speak with the participant by phone and 


 
 
13
review the full consent document. Following this discussion, the participant will sign an 
electronic consent. 
12. 
Plans for Publication 
We plan to submit our findings to a peer-reviewed publication, such as Biological 
Psychiatry 
13. 
References 
 
1. 
Stuebe AM, Meltzer-Brody S, Propper C, et al. The Mood, Mother, and Infant Study: 
Associations Between Maternal Mood in Pregnancy and Breastfeeding Outcome. Breastfeed Med. Oct 
2019;14(8):551-559. doi:10.1089/bfm.2019.0079 
2. 
Mah BL, Bakermans-Kranenburg MJ, Van IMH, Smith R. Oxytocin promotes protective behavior 
in depressed mothers: a pilot study with the enthusiastic stranger paradigm. Depress Anxiety. Feb 
2015;32(2):76-81. doi:10.1002/da.22245 
3. 
Mah BL, Van Ijzendoorn MH, Out D, Smith R, Bakermans-Kranenburg MJ. The Effects of 
Intranasal Oxytocin Administration on Sensitive Caregiving in Mothers with Postnatal Depression. Child 
psychiatry and human development. Apr 21 2016;doi:10.1007/s10578-016-0642-7 
4. 
Mah BL, Van Ijzendoorn MH, Smith R, Bakermans-Kranenburg MJ. Oxytocin in postnatally 
depressed mothers: Its influence on mood and expressed emotion. Progress in neuro-
psychopharmacology & biological psychiatry. Oct 17 2012;40C:267-272. 
doi:10.1016/j.pnpbp.2012.10.005 
5. 
Cardoso C, Kingdon D, Ellenbogen MA. A meta-analytic review of the impact of intranasal 
oxytocin administration on cortisol concentrations during laboratory tasks: moderation by method and 
mental health. Psychoneuroendocrinology. Nov 2014;49:161-70. doi:10.1016/j.psyneuen.2014.07.014 
6. 
Bakermans-Kranenburg MJ, van IJMH. Sniffing around oxytocin: review and meta-analyses of 
trials in healthy and clinical groups with implications for pharmacotherapy. Transl Psychiatry. 
2013;3:e258. doi:10.1038/tp.2013.34 
7. 
Heinrichs M, Baumgartner T, Kirschbaum C, Ehlert U. Social support and oxytocin interact to 
suppress cortisol and subjective responses to psychosocial stress. Biol Psychiatry. Dec 15 
2003;54(12):1389-98.  
8. 
Quirin M, Kuhl J, Dusing R. Oxytocin buffers cortisol responses to stress in individuals with 
impaired emotion regulation abilities. Psychoneuroendocrinology. Jul 2011;36(6):898-904. 
doi:10.1016/j.psyneuen.2010.12.005 
9. 
McRae-Clark AL, Baker NL, Maria MM, Brady KT. Effect of oxytocin on craving and stress 
response in marijuana-dependent individuals: a pilot study. Psychopharmacology (Berl). Aug 
2013;228(4):623-31. doi:10.1007/s00213-013-3062-4 
10. 
Simeon D, Bartz J, Hamilton H, et al. Oxytocin administration attenuates stress reactivity in 
borderline personality disorder: a pilot study. Psychoneuroendocrinology. Oct 2011;36(9):1418-21. 
doi:10.1016/j.psyneuen.2011.03.013 
11. 
Jolley SN, Elmore S, Barnard KE, Carr DB. Dysregulation of the hypothalamic-pituitary-adrenal 
axis in postpartum depression. Research Support, N.I.H., Extramural 
Research Support, Non-U.S. Gov't. Biological research for nursing. Jan 2007;8(3):210-22. 
doi:10.1177/1099800406294598 
12. 
Heinrichs M, Meinlschmidt G, Neumann I, et al. Effects of Suckling on Hypothalamic-Pituitary-
Adrenal Axis Responses to Psychosocial Stress in Postpartum Lactating Women. J Clin Endocrinol 
Metab. October 1, 2001 2001;86(10):4798-4804.  
13. 
Stuebe AM, Grewen K, Meltzer-Brody S. Association between maternal mood and oxytocin 
response to breastfeeding. Journal of Women's Health. Apr 2013;22(4):352-61. 
doi:10.1089/jwh.2012.3768 


 
 
14
14. 
Cox EQ, Stuebe A, Pearson B, Grewen K, Rubinow D, Meltzer-Brody S. Oxytocin and HPA 
stress axis reactivity in postpartum women. Psychoneuroendocrinology. Feb 25 2015;55:164-172. 
doi:10.1016/j.psyneuen.2015.02.009 
15. 
Mills-Koonce WR, Grewen K, O'Shea NG, et al. The Mood, Mother and Child Study: Protocol for 
a Prospective Longitudinal Study and Randomized Controlled Trial. JMIR Res Protoc. Oct 26 
2023;12:e51132. doi:10.2196/51132 
16. 
Kirschbaum C, Pirke KM, Hellhammer DH. The 'Trier Social Stress Test'--a tool for investigating 
psychobiological stress responses in a laboratory setting. Neuropsychobiology. 1993;28(1-2):76-81.  
17. 
Kudielka BM, Hellhammer DH, Kirschbaum C. Ten Years of Research with the Trier Social 
Stress Test -- Revisited. In: Harmon-Jones E, Winkielman P, eds. Social neuroscience : integrating 
biological and psychological explanations of social behavior. Guilford Press; 2007. 
14. 
Appendix 
 
Study visit schematic: Inhaled Oxytocin and HPA Axis Reactivity 
 
 

ICF_000.pdf: 
Informed Consent 
 
 
The Psychobiology of Resilience in Mother-child Pairs: Inhaled 
Oxytocin and HPA Axis Reactivity 
 
 
NCT number NCT03593473 
Document Date 12/08/2022 
 


12-2061 
Consent Addendum for Current Subjects 
Page 1 of 7 
University of North Carolina at Chapel Hill 
Consent to Participate in a Research Study 
Addendum to provide additional information to subject after original consent 
 
Consent Form Version Date: V7.12/08/22 
IRB Study # 12-2061 
Title of Study: MOOD, MOTHER AND INFANT (MMI): The psychobiology of impaired dyadic 
development 
Principal Investigator: Alison Stuebe, MD 
Principal Investigator Department: Obstetrics and Gynecology 
Principal Investigator Phone number: (919) 966-1601 
Principal Investigator Email Address: astuebe@med.unc.edu 
Funding Source and/or Sponsor: National Institutes of Health (NIH) 
 
Study Contact Telephone Number: (919) 843-8084 
Study Contact Email: bpearson@med.unc.edu 
 
_________________________________________________________________ 
 
The following information should be read as an addition to the original Consent form that you 
read and signed at the beginning of the study. Unless specifically stated otherwise in the 
following paragraphs, all information contained in that original Consent Form is still true and 
remains in effect. Your participation continues to be voluntary. You may choose not to 
participate, or may withdraw your consent to participate at any time, and for any reason, 
without jeopardizing your future care at this institution or your relationship with your study 
doctor.  
 
New or additional information 
MMI Phase 3 – Mood, Mother and Child (MMC) 
When your MMI child is between 5.5 and 8 years old, you will be invited to participate in Mood, 
Mother and Child (MMC). MMC involves two visits to the Mother-Child Behavior Lab at UNC 
(the same lab that you visited for your MMI lab visits). One visit will be a Mother-Only visit and 
the other will be a Mother-Child visit. After completion of the MMC visits you will be invited to 
complete post-MMC online surveys. 
Structured Psychiatric Interview 
As part of the MMC study, we will complete the SCID (Structured Psychiatric interview); the 
same assessment that was completed at your first MMI visit and several cognitive assessments.  
Online Surveys 
You will be asked to complete a set of online surveys, similar to the surveys you completed 
during the MMI study and during the MMI phase 2 online follow-up. 


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Consent Addendum for Current Subjects 
Page 2 of 7 
Mother-Only Visit (approximately 2½ - 3 hours) 
You will complete the re-consenting process (this document) during which we will review the 
MMC visit protocols in detail and you will have an opportunity to ask any questions and receive 
answers to your questions. Following re-consenting we will complete the following tasks: 
Urine Sample:  
• You will go to a private rest room to give a urine sample.   
• We will screen the urine sample immediately for pregnancy.  
o If the pregnancy screening is negative (you are NOT pregnant), your study visit 
will continue. 
o If the pregnancy screening is positive (you ARE pregnant), your Mother-Only 
visit will be rescheduled. 
Vital signs:  
• We will measure your height and weight.  
• We will apply sensors to your chest and a blood pressure cuff to your arm to measure 
your heart’s activity.  
• Your blood pressure and heart rate will be taken at various times during the visit.  
 
Blood samples:  
• A licensed practitioner on the study team will insert a soft catheter (IV) into a vein in 
your arm so they can take 8 small blood samples painlessly during the visit without 
having to insert a needle more than once.   
• A total of around 113 ml, or 7.64 tablespoons, of your blood will be collected. 
• We will use these blood samples to measure hormone level changes that happen in 
response to the nasal spray and tasks you complete during the visit.   
 
Nasal Spray:  
• At the end of the 10 minute rest period, the licensed practitioner will show you how to 
correctly breathe in the nasal spray.  
• You will breathe in 6 puffs of the spray (3 in each nostril).  
• Some participants will get oxytocin spray.  Other participants will get a harmless spray 
substance with no medical effects (placebo).  
• If you get oxytocin, you will get a dose of 24 international units (IU) of oxytocin. This 
dose is standard for testing the effects of oxytocin in medical studies.  It has been used 
in over 1500 research participants with rates of side effects that are the same as those 
reported by participants who received a placebo (a substance with no medical effects). 
• You will then sit quietly for another 40 minutes, while we measure your heart rate, 
respiration, and other bodily activity again. During this time, you will be asked to not use 
your cell phone or any social media (like email, text messages, Facebook). We will 
provide you with a selection of videos to watch, as well as coloring books.   


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Consent Addendum for Current Subjects 
Page 3 of 7 
• You will be asked to rate your mood and any physical discomfort before, during and 
after taking the dose of nasal spray. 
• At the end of the visit you will be asked whether or not you thought you received the 
“oxytocin”, the “placebo”, or “not sure”. 
 
Trier Social Stress Test:  
• At the end of the 40 minute rest period you will complete the Trier Social Stress Test, 
the same task you completed at your MMI 2-month visit including math and speech 
exercises. 
Following the Trier, you will have a 30 minute rest period. You will be asked to not use 
your cell phone or any social media (like email, text messages, Facebook) during this 
time. 
 
 
Mother-Child Visit (approximately 2 - 3 hours) 
During this visit, you and your child will be completing a series of tasks, some of which you will 
do together and some your child will do on their own. Sometimes a research assistant (RA) who 
is a member of the study team will be interacting with your child. The tasks that you complete 
together will help us understand the nature of the attachment between you and your child. The 
games that your child plays alone will help us understand your child’s memory development, 
attention skills, and responses to challenging tasks.  
We will also video record several of these tasks. The recorded portion of the visit will be used 
for several purposes:  to show the video for staff training, to make sure we are using our 
equipment correctly, to make sure the task is administered correctly, and for research 
purposes.  We will keep these videos indefinitely, but no names will be on the videos and they 
will be kept in locked offices.  
Vital signs:  
• We will measure you and your child’s height and weight. 
• We will apply sensors to your chest and your child’s chest and a blood pressure cuff to 
your arm to measure your heart’s activity.  
• Your blood pressure and heart rate will be taken at various times during the visit. 
  
Hair and Saliva samples:  
• We will take a cheek swab from your child (same as MMI). 
• We will collect hair samples from you and your child. 
 
75 minute Child Cognitive Tasks: 
Your child will complete the following computerized games: Working Memory Span Task, Pick 
the Picture Game, Silly Sounds Stroop, Spatial Conflict Task, Animal Go No-Go Task, and 


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Consent Addendum for Current Subjects 
Page 4 of 7 
Something’s the Same Game. The RA will provide your child with the opportunity to practice 
each game before starting and to take a break between games. All of these games are designed 
for children between the ages of 4 – 8 years old.  Your child will also be administered the 
Wechsler Preschool and Primary Scale of Intelligence (WPPSI), a standardized assessment of 
cognitive performance appropriate for children 2.5 to 8 years of age. The WPPSI uses child-
friendly, developmentally appropriate measures of verbal comprehension, visual spatial 
abilities, fluid reasoning, working memory, and processing speed. As with all study assessments, 
this assessment is for research purposes only and is not used for diagnostics or individual 
evaluation.  As such, all information obtained from assessments is retained for research use 
only and will not be made available to participants. 
 
45 minute Mother Cognitive tasks 
While your child is completing tasks in another room with the research assistant, you 
will complete a set of cognitive tasks that include computer-based assessments of 
executive functions (including memory, planning, and attention).  
 
5 minute Mother Speech Sample 
While your child is completing tasks in another room with the research assistant, you 
will be recorded talking about your child.  
 
20 minute Mother-Child tasks – Tower Construction Task, Slap Jack Card Game and Etch-a-
Sketch: 
 
Tower Construction Task 
Before the task begins you and your child will be shown a box that contains several 
small blocks of different shapes and sizes, as well as one large rectangular cube (a 
tower). The purpose of the task is to replicate the large tower out of the smaller blocks 
by using as many combinations as possible and before you start we will show you 
several examples. The RA will explain that you should allow your child to attempt the 
task first, and then to help however you see fit. 
 
Slap Jack Card Game 
Before the game begins you and your child will be given instructions on how to play the 
“slap jack” card game. You will be shown a deck of cards that is divided into two stacks. 
You and your child will take turns placing the cards face up in a single pile. When a jack 
is placed in the pile, the first one to slap the pile of cards gets to keep all of them. The 
object of the game is to get all of the cards. 
 
Etch-a-Sketch Maze 
You and your child will use an Etch-a-Sketch to complete a maze. You will control one 
knob and your child will control the other knob. The object of the task is to complete the 
maze together. 
 


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What are the possible risks or discomforts involved from being in this study? 
In addition to the risks described in your MMI consent, side effects from the oxytocin nasal 
spray are the only new potential risks. Oxytocin produces powerful contractions in the uterus of 
women who are giving birth and helps release milk during breastfeeding.  Oxytocin is most 
commonly given to start or increase contractions in the uterus of women who are giving birth.  
When oxytocin is given as a medication, rare bad effects have been reported including: 
• 
Severe allergic reactions (less than 1 in 1,000 people) 
• 
Raised blood pressure (less than 1 in 10,000 people) 
• 
Drop in blood pressure (less than 1 in 10 people) 
• 
Electrical abnormalities of the heart (less than 1 in 100 people) 
• 
Nausea or vomiting (less than 1 in 10 people) 
• 
Drop in sodium levels in blood which can cause confusion, seizures and even 
coma (less than 1 in 10,000 people) 
Much higher doses of oxytocin than have been given to postpartum women for the clinical 
purposes described above have been given to a much smaller number of people for research 
purposes (approximately several hundred to a few thousand). With the exception of one 55 
year old man with obsessive compulsive disorder who developed psychotic symptoms, no 
medical problems have been reported in these subjects. 
Protections: 
• 
We will ask you to immediately tell us if you get dizzy or nauseous, or if you have 
any pelvic pain or breast discharge.  
• 
A licensed practitioner will monitor your blood pressure and heart rate during 
the 80 minutes following your nasal spray dose. 
•        There are no known risks to a breastfeeding child from oxytocin administration; 
it may trigger a letdown. 
Risks to a pregnancy: If you are pregnant the Mother-Only visit will be cancelled. Oxytocin can 
cause contractions in the uterus that could cause harm to a pregnancy.   
Protections: A urine pregnancy test will be done at the start of the M-O visit, and you will not 
be given the nasal spray if this test is positive (you ARE pregnant). The cost of this test will be 
paid for by the research study. 
Will you and your child receive anything for being in this study? 
We will provide $100 compensation for completing the Mother-Only visit and $150 for 
completing the Mother-Child visit. As in MMI free parking will be provided. Subject 
compensation for MMC survey-only visit will be $25. 
What is a Certificate of Confidentiality? 
This research is covered by a Certificate of Confidentiality. With this Certificate, the researchers 
may not disclose or use information, documents or biospecimens that may identify you in any 


12-2061 
Consent Addendum for Current Subjects 
Page 6 of 7 
federal, state, or local civil, criminal, administrative, legislative, or other proceedings in the 
United States, for example, if there is a court subpoena, unless you have consented for this use. 
The Certificate cannot be used to refuse a request for information from personnel of a federal 
or state agency that is sponsoring the study for auditing or evaluation purposes or for 
information that must be disclosed in order to meet the requirements of the federal Food and 
Drug Administration (FDA). 
The Certificate of Confidentiality will not be used to prevent disclosure as required by federal, 
state, or local law, such as mandatory reporting requirements for child abuse or neglect, 
disabled adult abuse or neglect, communicable diseases, injuries caused by suspected criminal 
violence, cancer diagnosis or benign brain or central nervous system tumors or other 
mandatory reporting requirement under applicable law. The Certificate of Confidentiality will 
not be used if disclosure is for other scientific research, as allowed by federal regulations 
protecting research subjects or for any purpose you have consented to in this informed consent 
document. 
You should understand that a Certificate of Confidentiality does not prevent you from 
voluntarily releasing information about yourself or your involvement in this research. If an 
insurer, employer, or other person obtains your written consent to receive research 
information, then the researchers may not use the Certificate to withhold that information. 
 
 


12-2061 
Consent Addendum for Current Subjects 
Page 7 of 7 
Consent Form Version Date: V7.12/08/22 
IRB Study # 12-2061 
Title of Study: MOOD, MOTHER AND INFANT (MMI): The psychobiology of impaired dyadic 
development 
Principal Investigator: Alison Stuebe, MD 
Participant's Agreement: 
I have read the information provided above.  I have asked all the questions I have at this time.  I 
voluntarily agree to continue to participate in this research study.  
 
______________________________________________________ 
Signature of Research Participant 
 
____________________ 
Date 
 
______________________________________________________ 
Printed Name of Research Participant 
  
  
 
______________________________________________________ 
Signature of Research Team Member Obtaining Consent 
 
____________________ 
Date 
 
______________________________________________________ 
Printed Name of Research Team Member Obtaining Consent