Multisensory Early Oral Administration of Human Milk in Preterm Infants to Attenuate Early Life Toxic Stress on Epigenetic Modifications and Dysbiosis: Randomized Controlled Trial Pilot Study
Phase: NA
Conditions: Infant Development, Stress
Sponsor: Loyola University

Version Date: 05/07/2024 
1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Protocol Title: 
Multisensory Early Oral Administration of Human Milk in Preterm Infants  
to Attenuate Early Life Toxic Stress on Epigenetic Modifications and Dysbiosis:  
Randomized Controlled Trial Pilot Study 
 
Version Date: 05/07/2024 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Version Date: 05/07/2024 
2 
 
Table of Content 
I. 
Research Team .......................................................................................................................................... 3 
II. 
Abstract ...................................................................................................................................................... 4 
III. Background ................................................................................................................................................ 5 
IV. Study Aims ................................................................................................................................................. 6 
V. Administrative Organization ........................................................................................................................ 6 
VI. Study Design .............................................................................................................................................. 6 
VIII. Safety Monitoring Plan ............................................................................................................................. 13 
IX. Data Management and Analysis Plan ....................................................................................................... 16 
X. Study Timeline .......................................................................................................................................... 16 
XIII. Literature Cited ......................................................................................................................................... 16 
 
 
 

Version Date: 05/07/2024 
3 
 
I. 
Research Team 
Name and Credentials 
Job Tittle 
Affiliation 
Role on the  
Research Team
Thao Griffith, PhD, RN 
Assistant Professor 
Loyola University Chicago 
Principal Investigator 
Linda Janusek, PhD, RN, 
FAAN 
Professor Emerita 
Loyola University Chicago 
Consultant 
Rosemary White-Traut, PhD, 
RN, FAAN* 
Director  
Children’s Wisconsin, 
Nursing Research 
Consultant 
Sachin Amin, MD 
NICU Director 
Loyola University Medical 
Center 
Site Principal Investigator 
Cara Joyce, PhD 
Director 
Loyola University Chicago, 
Biostatistics Core, Clinical 
Research Office 
Biostatistician 
Talar Markossian, PhD, MPH 
Associate Professor 
Loyola University Chicago 
Co-investigator 
Julia Puchi, BSN, RN 
Research Nurse 
Loyola University Chicago 
Research Nurse 
Brianna Avery, BS 
Research Assistant 
Loyola University Chicago 
Research Assistant 
Jennifer Grunwaldt, MS, 
CCC-SLP 
Speech Therapist 
Loyola University Medical 
Center 
Co-investigator 
George Chlipala, PhD 
Associate Director 
University of Illinois at 
Chicago, Research 
Informatics Core 
Consultant 
Mark Maienschein-Cline, PhD
Director 
University of Illinois at 
Chicago, Research 
Informatics Core 
Consultant 
Stefan Green, PhD 
Director 
Rush University Medical 
Center, Core Lab Services 
Consultant 
Barb O’Rourke, RN* 
Registered Nurse 
Blank Children’s Hospital 
Synapse Care Solutions 
Reckitt-Mead Johnson 
Consultant 
Kristine Fuller, OTR/L, CNT, 
CLC* 
 
Occupational Therapist 
 
Caring Essentials 
Collaborative LLC 
Infant Feeding Strategies 
LLC 
Consultant 
*Rosemary White-Traut, Barb O’Rourke, and Kristy Fuller will not receive any PHI or other data from the study. Their 
role is to provide consultation on the protocol and logistics.  
 
 
 

Version Date: 05/07/2024 
4 
 
II. 
Abstract 
 
More than 60,000 infants are born between 22 to 32 weeks gestation age (very or extremely premature) 
annually in the US. Approximately 11% of them develop comorbidities, i.e., oral feeding difficulty, failure to 
thrive, delayed neurodevelopment, and maladapted parent-infant interaction. As part of the Neonatal Intensive 
Care Unit (NICU) hospitalization, very and extremely preterm infants inevitably endure early life toxic stress, 
e.g., parental separation, painful procedures, sleep fragmentation, harmful hospital organisms, and antibiotic 
therapy, without adequate protective buffers. Early life toxic stress results in adverse epigenetic modifications 
of glucocorticoid-related genes (DNA methylation of NR3C1 and HSD11B2) and dysbiosis (altered gut 
microbiome), both impairing neurodevelopment. These adversities further exacerbate the risk of comorbidities 
and inappropriate brain development during sensitive periods of neuroplasticity. Evidence suggests adverse 
epigenetic modifications and dysbiosis may set a life-long trajectory of risk for chronic health conditions, i.e., 
mental health disorders, cardiometabolic and immunologic diseases. Hence, it is a clinical and scientific priority 
to test an early NICU intervention to attenuate stress-related adverse epigenetic modifications and dysbiosis. 
Prior research has paved the way for a potential early NICU intervention as follows. Human milk influences the 
structure and relative abundance of healthy gut bacteria, i.e., short-chain fatty acid (SCFA)- and lactate-
producing bacteria (e.g., Bifidobacterium and Lactobacillus), and neurodevelopment. Maternal nurturing, e.g., 
licking and grooming (in rodents), and breastfeeding and touch (in humans), promotes neurodevelopment, 
reduces stress, and reverses stress-related epigenetic modifications. Building on what is known, the 
multisensory early oral administration of human milk (M-MILK) intervention by nurses or parents is designed to 
provide an enjoyable and nurturing experience for infants, through a safe and consistent infant-guided 
provision of human milk droplets, given orally as early as 22 weeks postmenstrual age. M-MILK is 
implemented after every hands-on care and during the beginning of a full gavage feeding. The objective of our 
planned NIH R01 randomized controlled trial is to examine the efficacy of M-MILK from birth to 2 months 
corrected age, and the mechanisms whereby this may occur. We propose the M-MILK pilot study (N = 20): a 2-
group, 10 per group, parallel design, and longitudinal randomized controlled trial in preterm infants who are 
born between 22 to 33 weeks gestational age. M-MILK will begin on day 3 of life. We will measure outcomes 
using validated methodologies: maternal postpartum depressive symptoms via the Edinburgh Postnatal 
Depression Scale, parent stress via the Parental Stressor Scale: NICU, parent discharge readiness via the 
Screening and Multi-disciplinary Risk Assessment Scale & Parent Activation Scale, neurodevelopment via the 
Neurobehavioral Assessment of the Preterm Infants and Ages & Stages Questionnaire-3, oral feeding skills via 
the Early Feeding Skills Assessment and NeoEAT, parent-infant interaction during feeding via the Nursing 
Child Assessment Satellite Training–Feeding Scale and Postpartum Bonding Questionnaire, DNA methylation 
of NR3C1 and HSD11B2 via buccal samples, and gut microbiome via fecal samples. Outcomes will be 
measured at baseline, 34 weeks postmenstrual age, discharge, and 2 months corrected age. The aims of this 
pilot study are to (1) determine the feasibility and acceptability of the M-MILK intervention by nurses and 
parents, (2) determine the feasibility of recruitment and retention, (3) obtain variability data for sample size 
estimation, and (4) evaluate the charges and costs of the NICU hospitalization and 2 months CA care between 
the M-MILK and control groups. M-MILK will be considered feasible if M-MILK is implemented by nurses or 
parents after every hands-on care and during the beginning of a full gavage feeding at least 90% of the time. 
The M-MILK acceptability by nurses and parents will be determined via M-MILK Acceptability Questionnaire. 
Recruitment and retention will be considered feasible if at least 50% of infants approached for participation 
enrolled, 90% of infants completed all inpatient data collection, and at least 50% of infants completed post-
discharge follow-ups. This pilot study will inform our R01 application regarding the feasibility and acceptability 
of M-MILK and sample size estimation. 
 
 
 

Version Date: 05/07/2024 
5 
 
III. 
Background  
 
More than 60,000 infants are born between 22 to 32 weeks gestation age (very or extremely premature) 
annually in the US.1 Approximately 11% of them develop comorbidities, i.e., oral feeding difficulty, failure to 
thrive, delayed neurodevelopment, and maladapted parent-infant interaction.2-7 As part of the Neonatal 
Intensive Care Unit (NICU) hospitalization, very and extremely preterm infants inevitably endure early life toxic 
stress, e.g., parental separation, painful procedures, sleep fragmentation, harmful hospital organisms, and 
antibiotic therapy.7-9 We define early life toxic stress in the NICU as excessive and prolonged stressful stimuli 
without buffer of a consistent, supportive, and protective parental relationship.7,10 Early life toxic stress results 
in adverse epigenetic modifications of glucocorticoid-related genes (DNA methylation of NR3C1 and 
HSD11B2)11-13 and dysbiosis (altered gut microbiome),14-17 both impairing neurodevelopment.11,13-15,18 These 
adversities further exacerbate the risk of comorbidities and inappropriate brain development during sensitive 
periods of neuroplasticity.7,19 Evidence suggests adverse epigenetic modifications and dysbiosis may set a life-
long trajectory of risk for chronic health conditions, i.e., mental health disorders, cardiometabolic and 
immunologic diseases.8,19-21 Hence, it is a clinical and scientific priority to test an early NICU intervention to 
attenuate stress-related adverse epigenetic modifications and dysbiosis. Prior research has paved the way for 
a potential early NICU intervention as follows. Human milk influences the structure and relative abundance of 
the healthy gut bacteria, i.e., short-chain fatty acid (SCFA)- and lactate-producing bacteria (e.g., 
Bifidobacterium and Lactobacillus), and neurodevelopment.12,22,23 Maternal nurturing, e.g., licking and 
grooming (in rodents) and breastfeeding and touch (in humans), promotes neurodevelopment, reduces stress, 
and reverses stress-related epigenetic modifications.13,21,24-26 Building on what is known, the multisensory early 
oral administration of human milk (M-MILK) intervention by nurses or parents is designed to provide an 
enjoyable and nurturing experience for infants, through a safe and consistent infant-guided provision of human 
milk droplets, given orally as early as 22 weeks postmenstrual age.27,28 M-MILK is implemented after every 
hands-on care and during the beginning of a full gavage feeding.28 The objective of our planned NIH R01 
randomized controlled trial is to examine the efficacy of M-MILK from birth to 2 months corrected age, and the 
mechanisms whereby this may occur.  
 
Initial evidence from prior studies is promising, however, the study designs lack rigor and comprehensiveness. 
In these studies, early administration of mother’s own milk or donor’s milk shortly after birth supported oral 
mechanical stimulation and subsequent maturation of suckling and swallowing skills.28,29 It is evidenced by less 
episodes of feeding intolerance and shorter duration to full oral feeding.28,29 Researchers also found that the 
intervention offered immunoprotection as evidenced by lower pathogenic microbacterial colonization of the 
gastrointestinal tract and incidence of ventilator-associated pneumonia.29 The intervention reduced duration of 
oxygen support, length of stay, and cost of care.27-30 However, the potential efficacy of M-MILK to attenuate the 
effects of early life toxic stress on epigenetic modifications and gut microbiome is unknown. This gap leads to a 
missed opportunity for individualized neonatal care and early parental involvement in the NICU which could 
protect very and extremely preterm infants against early life toxic stress. There is an urgent need to address 
this knowledge gap and determine the efficacy of M-MILK. 
 
To inform our planned R01 application, we propose the M-MILK pilot study (N = 12): a 2-group, 6 per group, 
parallel design, and longitudinal randomized controlled trial in infants who are born between 22 to 33 weeks 
gestational age. We will compare the outcomes between the M-MILK and attention control groups. M-MILK will 
begin on day 3 of life. We will measure outcomes using validated methodologies, e.g., maternal postpartum 
depressive symptoms via the Edinburgh Postnatal Depression Scale, parent stress via the Parental Stressor 
Scale: NICU, parent discharge readiness via the Screening and Multi-disciplinary Risk Assessment Scale & 
Parent Activation Scale, neurodevelopment via the Neurobehavioral Assessment of the Preterm Infants and 
Ages & Stages Questionnaire-3, oral feeding skills via the Early Feeding Skills Assessment and NeoEAT, 
parent-infant interaction during feeding via the Nursing Child Assessment Satellite Training–Feeding Scale and 
Postpartum Bonding Questionnaire, DNA methylation of NR3C1 and HSD11B2 via buccal samples, and gut 
microbiome via fecal samples. Outcomes will be measured at baseline, 34 weeks postmenstrual age, 
discharge, and 2 months corrected age. The aims of this pilot study are to (1) determine the feasibility and 
acceptability of the M-MILK intervention by nurses and parents, (2) determine the feasibility of recruitment and 

Version Date: 05/07/2024 
6 
 
retention, (3) obtain variability data for sample size estimation, and (4) evaluate the charges and costs of the 
NICU hospitalization and 2 months CA care between the M-MILK and control groups.  
 
This innovative pilot study will be the first to examine M-MILK on a cumulative scale of clinical and molecular 
outcomes of infants and parents. The findings will yield novel insights regarding an early NICU intervention that 
is clinically safe, effective, and scalable. By promoting epigenetic protection and healthy gut microbiome, M-
MILK has significant potential to improve future health outcomes and reduce the medical and financial burdens 
across the lifespan for very and extremely preterm infants. 
 
IV. 
Study Aims 
 
The aims of this pilot study are to (1) determine the feasibility and acceptability of the M-MILK intervention by 
nurses and parents, (2) determine the feasibility of recruitment and retention, (3) obtain variability data for 
sample size estimation, and (4) evaluate the charges and costs of the NICU hospitalization and 2 months CA 
care between the M-MILK and control groups. M-MILK will be considered feasible if M-MILK is implemented by 
nurses or parents after every hands-on care and during the beginning of a full gavage feeding at least 90% of 
the time. The M-MILK acceptability by nurses and parents will be determined via M-MILK Acceptability 
Questionnaire. Recruitment and retention will be considered feasible if at least 50% of infants approached for 
participation enrolled, 90% of infants completed all inpatient data collection, and at least 50% of infants 
completed post-discharge follow-ups. 
 
V. 
Administrative Organization 
 
Participants will be enrolled in the Neonatal Intensive Care Unit (NICU) at Loyola University Medical Center 
(LUMC) (Maywood, IL). The inpatient components of the study protocol will be conducted in the NICU at 
LUMC. The post-discharge follow-up components of the study protocol will be conducted via phone/zoom 
interview. Data storage and management will be carried out via REDCap (version 10.4.0, Vanderbilt University) 
and a secured research network drive, hosted by the Loyola University Chicago (Maywood, IL).  
 
Upon sample collection, buccal swab and fecal samples will be labeled only with study ID numbers and 
collection date, and stored in a locked cabinet in a locked laboratory at Loyola University Chicago (LUC) 
Center for Translational Research and Education (CTRE). All samples will be stored at room temperature. 
Quarterly, the buccal swab samples and fecal samples will be securely transported in a biospecimen transfer 
container (Saf T Pak, Austell, GA) via car to Rush University Medical Center Genomics and Microbiome Core 
Facility (Rush) (Chicago, IL) for processing and analyses. A Service Agreement between LUC and Rush will be 
submitted through LUC Research Portal. 
 
VI. 
Study Design 
 
a. Design 
The M-MILK pilot study (N = 12) is a 2-group, 6 per group, parallel design, and longitudinal randomized 
controlled trial. We will recruit preterm infants who are born between 22 to 33 weeks gestational age. We will 
compare the outcomes between the M-MILK and attention control groups. Infants will be enrolled into the study 
as soon as possible after they are admitted to the NICU. If infants are in the M-MILK group, M-MILK will begin 
on day 3 of life after every hands-on care and during the beginning of a full gavage feeding. We will measure 
outcomes using validated methodologies at baseline (enrollment), 34 weeks PMA, discharge, and 2 months 
CA. 
 
b. Setting 
The M-MILK pilot study will be conducted in a level III NICU at LUMC (Maywood, IL). The hospital provides 
care to families with diverse racial, ethnic, and socioeconomic backgrounds. The nursing staff are 
predominantly registered nurses and advanced nurse practitioners, and the physicians are board certified 

Version Date: 05/07/2024 
7 
 
neonatologists. Infants are fed using mother’s own milk, donor milk, and/or formula. However, we will only 
include infants who receive mother’s own milk or donor’s milk.  
 
c. Sample 
We will enroll 12 preterm infants (6 per group). Anticipated a 40% attrition rate, we expect the final sample size 
to be 12 preterm infants. Estimates of average values and variability of continuous measures are acceptable at 
this sample size using the “rule of 12” as recommended.31  
 
VII. 
Study Procedures 
 
a. Subject Selection Procedures 
 
i. 
Inclusion/Exclusion Criteria 
 
Inclusion criteria 
 
Born between 22 to 33 weeks gestational age 
 
Mother is pumping and infant is receiving mother’s own milk and/or donor’s milk to be in 
the intervention group. 
 
Exclusion criteria 
 
Congenital abnormalities in the digestive, e.g., clef lip and palate, omphalocele 
 
Necrotizing enterocolitis  
 
Congenital heart disease that requires surgery 
 
ii. 
Screening procedures 
 
After infants are admitted to the NICU, we will review their electronic medical records to screen for eligible 
infants according to the inclusion and exclusion criteria. Once an infant is enrolled into the study, the screening 
period continues until NICU discharge. The infant will be classified as screen failed and excluded if he/she 
develops any of the exclusion criteria. 
 
iii. 
Recruitment Procedures 
Parents of eligible infants will be approached during their post-partum hospitalization or in the NICU at LUMC. 
A research team member will provide details regarding the study purpose, procedures, and answer questions. 
Parents will be provided with a study brochure. If parents request additional time to make their decision, we will 
request a phone number and ask permission to follow-up. If parents agree to have their infant participate, a 
written informed consent and authorization to use and disclose the PHI will be obtained. Parents will receive a 
copy of their signed informed consent and authorization to use and disclose the PHI. A copy of the informed 
consent form will be uploaded to the infant’s electronic medical record. The original signed consent will be 
stored in a locked cabinet at LUC CTRE (Maywood, IL). Infant’s participation in the study will end after the 
phone/Zoom interview at 2 months CA. We will provide a $50 incentive e-gift card at NICU discharge and a 
$50 incentive e-gift card at the completion of the post-discharge follow-up, a total of $100 e-gift card. 
Premature infants, by virtue of their age, are unable to give consent or assent, and therefore, will not give 
assent or participate in the informed consent process.  
 
iv. 
Randomization Procedures 
 
Randomization is controlled by using two computer-generated lists of random numbers that are corresponded 
to each group: Group 1 (M-MILK) and Group 2 (attention control), balanced so that 1/2 of the infants are in 
each group. The lists are retained. The random numbers will be printed in order on small cards stated as 
Group 1 or Group 2. The cards will be placed in envelopes and placed in order in a box. Prior to approaching 
parents for recruitment, we will open the first envelope in the box to obtain the group assignment. We will 
request parents to not disclose their group assignment with other parents in the NICU. The research team 

Version Date: 05/07/2024 
8 
 
members, staff nurses, and parents cannot be blinded to the treatment condition. The research team members 
need to know which intervention to provide. Staff nurses provide direct care to infants. The two groups are 
described in the consent form as required by LUC Institutional Review Board.  
 
v. 
Study Intervention 
 
1. MMILK Intervention 
The M-MILK group will have access to the educational materials regarding preterm infant care such as Your 
Guide to Neonatal Intensive Care Unit, Breastfeeding in the NICU, and M-MILK Toolkit video. We will provide 
parents with two scent hearts to wear and bring back to place near the infant’s face. Parents will perform 
routine care such as taking temperature, bathing, changing diapers, reading, feeding, holding hands, as well as 
M-MILK when appropriate. Parents will receive an automatic daily text in the morning with a link to report their 
daily activities with infants when they visit the NICU.  
 
The M-MILK Intervention Protocol is a modified and improved version of previous protocols. This M-MILK 
protocol is carefully designed by a team of neonatal experts, with a strong focus on developmentally 
appropriate, trauma informed care, and family friendly language. M-MILK is implemented at day 3 of life even if 
the infant requires respiratory support and/or mechanical ventilation. M-MILK is implemented after every 
hands-on care and during the beginning of a full gavage feeding. Infants receive M-MILK as droplets via a 1-ml 
syringe. In our NICU, oral feeding attempts are initiated at approximately 33 to 34 weeks PMA and based on 
the infant’s pre-feeding cues. M-MILK is not offered with oral feeding attempts since the feeding itself is 
considered a positive experience. We will use either mother’s own milk and or donor’s milk depending on 
availability. Infants may receive up to 1 mL of milk each time based on their cues and responses. The 1 mL 
volume intake is included as part of their oral caloric intake. M-MILK is provided by the infant’s nurse or 
parents. Parents are encouraged and supported to provide M-MILK when appropriate with the nurse’s 
supervision.  
 
Intervention Training and Fidelity. Delivery of M-MILK by nurses or parents introduces potential threats to 
intervention fidelity. We will take the following strategies for training and improving intervention fidelity. 1) In-
person 1-hour M-MILK training sessions for all nurses, led by the PI and the M-MILK Expert Consultant, held in 
groups quarterly and individually as needed. The M-MILK training sessions will cover the M-MILK Toolkit. An 
acceptable attendance rate during the first quarter is 90%. 2) M-MILK Toolkit is available for nurses to view on 
the M-MILK study website and LUMC HealthStream platform for reference and continue education. 3) Nurses 
will document infant’s responses to M-MILK; and research team will review M-MILK documentation by nurses 
daily to ensure accuracy and completion. 4) In-person 30-minute M-MILK training sessions for parents in the 
M-MILK group, within 1 week of enrollment, provided by a Research Nurse. The M-MILK training sessions will 
cover the M-MILK Toolkit. The M-MILK training sessions for parents are followed by in-person observation and 
hands-on return demonstration, and questions/answers with a Research Nurse. An acceptable attendance rate 
for the parent M-MILK training sessions is 90%. 6) M-MILK Toolkit is available on the M-MILK study website for 
reference and continuing education. 7) Access to the M-MILK content on the website is restricted to nurses 
and parents in the M-MILK group, thus there will be 2 different access codes for each group. 8) We will request 
parents not to share the M-MILK content with other parents in the NICU. 9) Parents will document the M-MILK 
activities and infant’s responses via the daily activity link; acceptable completion rate for parent documentation 
is 85%. 10) Research Nurse will conduct an in-person audit of 10% of the M-MILK administrations by nurses or 
parents quarterly using M-MILK Intervention Fidelity Checklist; acceptable reliability rate is 90%. 11) The 
Research Nurse who is responsible for studying main outcome evaluations will be blinded to the infant’s group 
assignment. 
 
2. Attention Control Group 
For infants in the attention control group, standard of care will be provided, including hands-on care by their 
nurses, diapering, swaddling, feeding, etc. After assessment and initial cares are completed, infants are tucked 
and nested with boundaries to support comfort, security, and flexion. The infants’ hands are placed by their 
faces to encourage self-soothing, mouthing, and sucking. We will provide parents with two scent hearts to wear 

Version Date: 05/07/2024 
9 
 
and bring back to place near the infant’s face. Parents perform routine care such as taking temperature, 
bathing, changing diapers, reading, feeding, and holding hands. Parents will have access to educational 
materials regarding preterm infant care such as Your Guide to Neonatal Intensive Care Unit and Breastfeeding 
in the NICU. Parents will receive an automatic daily text in the morning with a link to report their daily activities 
with infants when they visit the NICU. Parents in the attention control group will not have access to the M-MILK 
content. 
vi. 
Study Assessments and Activities 
 
1. Measures 
 
Table 1. Concepts, Measures, and Operational Definitions 
Concepts 
Operational Definitions 
Data Source 
& Time 
Parent 
characteristics 
Parent characteristics will include both mother and father as applicable. We will collect 
race/ethnicity, education, age at delivery, household income, number of children, number of 
children under 24 months, and social determinants of health (i.e., physical activity, financial 
resource strain, housing stability, transportation needs, food insecurity, stress, social 
connections, intimate partner violence, and alcohol use) 
T0 
 
EPIC & self-
report 
Maternal postpartum 
depressive 
symptoms 
Maternal postpartum depressive symptoms are assessed by the Edinburgh Postnatal 
Depression Scale (EPDS).32 The EPDS consists of 10 short statements. A mother checks off 
one of four possible answers that is closest to how she has felt during the past week. 
Responses are scored 0, 1, 2 and 3 based on the seriousness of the symptom. Items 3, 5 to 
10 are reverse scored (i.e., 3, 2, 1, and 0). The total score is calculated by adding together 
the scores for each of the 10 items. Mothers scoring above 12 or 13 are likely to be suffering 
from depression and should seek medical attention. The pooled sensitivity and specificity of 
the EPDS were 0.81 and 0.87, respectively, with summary receiver operating characteristic 
curve of 0.90.33   
T0, T3, T4 
 
EPDS 
Parent stress 
We will also measure parent stress associated with their infant’s NICU hospitalization via the 
Parental Stressor Scale: NICU (PSS: NICU).34 The PSS:NICU measures parental stress after 
preterm birth and admission of their neonate to NICU. It is a self-report questionnaire, which 
can be rated on a five-point Likert-Scale: 1 = not at all stressful: the experience did not cause 
you to feel upset, tense, or anxious, 2 = a little stressful, 3 = moderately stressful, 4 = very 
stressful, and 5 = extremely stressful. Items describing situations that have not been 
experienced by the parents can also be answered with “not applicable.” The PSS: NICU has 
good concurrent and predictive validity and is internally consistent with Cronbach alpha 
ranging from 0.73 to 0.94.35 
T3 
 
PSS: NICU & 
Self-report 
Parent discharge 
readiness 
Parent discharge readiness will be evaluated by the Screening and Multi-disciplinary Risk 
Assessment Scale & Parent Activation Scale (PDR). The scale has 44 items and includes 
five sections: self-efficacy, social support, outcomes expectations and intent, knowledge, and 
perception of risk. The content validity of the scale is supported.36 
T3 
 
PDR & Self-
report 
 
Postpartum Bonding 
Questionnaire 
Parent-infant bonding will be evaluated by the Postpartum Bonding Questionnaire (PBQ).  
The PBQ comprises four subscales, i.e., general bonding disorders (12 items; 1, 2, 6, 7, 8, 9, 
10, 12, 13, 15, 16, 17), severe mother-infant relationship disorders (7 items; 3, 4, 5, 11, 14, 
21, 23), infant-focused anxiety (4 items; 19, 20, 22, 25), and risk of abuse (2 items; 18, 24). 
The total cumulative score, ranging from 0 to 125, is also used to screen for general bonding 
disorders (cut-off score ≥26), and severe bonding disturbances (cut-off score ≥40). 
The items are scored on a 6-point Likert scale (0-Always to 5-Never). Parents scoring ≥ 26, 
(indicating risk of general bonding disorders) or ≥ 40 (indicating risk of severe bonding 
disturbances) should seek medical attention. Parents scoring “rarely”, “sometimes”, “quite 
often”, “very often”, or “always” for item 18 “I have done harmful things to my baby” or item 24 
“I feel like hurting my baby” indicates risk of child abuse. In this case, it is our responsibility to 
report it to the state’s child protective agency. If the infant is in immediate danger, the 
research staff will be instructed to inform the parents of their immediate concerns for the 
infant’s safety, report the incident using a cellular phone, and call 911 until appropriate 
officials arrive. The PI will be informed of the family’s condition and always be available by 
phone or in person for consultation and instruction. As part of the training of our research 
team, the team will review content related to reporting of child neglect and abuse. 
T1, T3, T4 
 
PBQ & Self-
report 
Pumping and 
breastfeeding  
Mothers will report the following: pumping initiation date, pumping cessation date, pumping at 
discharge (yes/no), pumping at 2 months CA (yes/no), breastfeeding initiation date, 
breastfeeding cessation date, breastfeeding at discharge (yes/no), & breastfeeding at 2 
months CA (yes/no). 
Ongoing 
 
Self-report 

Version Date: 05/07/2024 
10 
 
Family daily NICU 
activities 
Parents will report the following: (1) How many hours were you able to visit your baby today? 
(2) What activities did you do with your baby while you were visiting? The answer choices for 
question (2) for parents in the M-MILK group are: M-MILK, skin-to-skin, feeding, taking 
temperature, bathing, changing diaper, reading, holding hands. 
Ongoing 
 
Self-report 
Infant characteristics 
race/ethnicity, & sex, GA at birth, birthweight, 5-minute Apgar score, small for gestational age 
(yes/no), intra-uterine growth restriction (yes/no), PROM (yes/no), medical diagnoses & 
therapies, daily weight gain, PMA at discharge, duration of time from initiation of oral feeding 
to discharge, & duration of NICU hospitalization; daily oral feeding intake, daily type of milk 
(mother’s own milk, donor’s milk, or formula), duration of tube feeding, duration of time from 
initiation of oral feeding to full oral feeding, & PMA at initiation & full oral feeding. 
Ongoing 
 
EPIC 
Infant medical 
severity 
Infant medical severity will be measured via the Neonatal Medical Index Classification (NMI), 
range 1-5, 1 as absence of serious medical problems & 5 as the most serious 
complications.37
T3 
 
EPIC 
Early life stress 
Early life stress will be measured daily by the modified Neonatal Infant Stressor Scale 
(NISS)38 using data from the EPIC during the entire NICU hospitalization. The Neonatal 
Infant Stressor Scale (NISS):38 summary scores, 47 acute interventions & 23 chronic 
conditions, weighted from 2-5, 2 as ‘a little stressful’ & 5 as ‘extremely stressful’.39 Predictive 
& construct validity were established, i.e., correlations between higher stress scores & brain 
structural changes,40 & poorer neurobehaviors.38,41  
Ongoing 
 
EPIC 
Neurodevelopment 
 
Neurodevelopment will be evaluated via the Neurobehavioral Assessment of the Preterm 
Infants (NAPI) at discharge.42-45 The PI will train a Research Nurse to criterion of ≥ 90% 
agreement. A Research Nurse (who is blinded to the infant’s group assignment) will conduct, 
rate and video record the NAPI assessment. A secondary trained evaluator will code 20% of 
the videos to assure continued reliability. The Neurobehavioral Assessment of the Preterm 
Infants (NAPI, 73 items, 32-37 weeks PMA): summary scores, normed referenced means, 
standard deviation, 7 clusters: scarf sign, motor development & vigor, popliteal angle, 
alertness and orientation, irritability, quality of cry, & percent sleep ratings.42-45 Construct 
validity, face validity, & sensitivity of the NAPI were described using an index of medical 
complications & general movements.43,44,46 Inter-rater reliability coefficient was 0.67 to 0.97.47 
T3 
 
NAPI 
Neurodevelopment will also be evaluated via the Ages and Stage Questionnaire (ASQ-3), 
completed by parents at 2 months CA.48 The ASQ-3 is a developmental screening tool that 
pinpoints developmental progress in children between the ages of one month to years. It is a 
validated and standardized screening tool based on parental report that reliably assesses 
five key developmental domains: communication, fine and gross motor, problem solving, and 
personal-social skills. The ASQ-3) has strong concurrent validity (r = 0.85), 2-week test-retest 
reliability (r = 0.75-0.82), interobserver reliability (r = 0.43-0.69), and internal consistency 
(α = 0.51-0.87).48 The ASQ-3 provides cutoff scores to indicate possible delay as follows: 
communication, gross motor, fine motor, problem solving, and personal-social. The AAP 
reports the ASQ-3 sensitivity range to be 0.70 to 0.90 and specificity to be 0.76 to 0.91.49 The 
five scored domains include communication, gross motor, fine motor, problem-solving, and 
personal–social. Scores of 10, 5, and 0 are applied, respectively, to caregiver responses of 
“yes,” “sometimes,” and “not yet” to 30 items. The scores are tabulated for each domain and 
are further categorized into a color-coded chart based on score interpretation. “Above cutoff” 
is considered typical development and is defined as any score in the white area (higher than 
1 SD below the mean); scores within the gray area indicate the “monitoring zone” in which 
the child should be observed and another screening may be desirable in a few months (1–2 
SD below the mean); and scores within the black area are “below cutoff” and indicate the 
child may be at risk for developmental delays and should be referred for further assessment 
(2 SD below mean).50 
T4 
 
ASQ-3 
Oral feeding skill 
development 
Oral feeding skill development will be evaluated using the Early Feeding Skill Assessment 
(EFS) at oral feeding initiation and discharge.51 The PI will train a Research Nurse to criterion 
of ≥ 90% agreement. A Research Nurse (who is blinded to the infant’s group assignment) will 
video record the feeding and rate the EFS. A secondary trained evaluator will code 20% of 
the videos to assure continued reliability. EFS has 22 items (32-50 weeks PMA), summary 
scores, 5 subscales: respiratory regulation, oral-motor functioning, swallowing coordination, 
engagement, & physiologic stability.51 Interrater reliability & construct validity have been 
established, i.e., Cronbach  = 0.81, correlations among EFS scores, GA & PMA. 
T2, T3 
 
EFS 
Oral feeding skill development will also be evaluated using the NeoEAT at 2-month CA.52,53 
There are 2 versions of the NeoEAT: bottle-feeding and breast-feeding and will be offered 
accordingly. The NeoEAT will be completed via during the home visit by a caregiver who is 
familiar with the infant’s eating. Neonatal Eating Assessment Tool– Bottle-feeding (NeoEAT– 
Bottle-feeding, 64 items, less than 7 months old, parent-report): summary scores, 5 
subscales: regulation, energy & physiologic stability, gastrointestinal tract function, sensory 
responsiveness, & compelling symptoms of problematic feeding.53 Internal consistency 
reliability (α = 0.92) and test-retest reliability (r = 0.90; P < .001) were excellent. Construct 
T4 
 
NeoEAT 

Version Date: 05/07/2024 
11 
 
validity was established with the I-GERQ-R (r = 0.74; P < .001) and IGSQ (r = 0.64; P < 
.001). Healthy infants scored lower on the NeoEAT – Bottle-feeding than infants with feeding 
problems (P < .001), supporting known-groups validity. Neonatal Eating Assessment Tool – 
Breast-feeding (NeoEAT – Breast-feeding, 62 items, less than 7 months old, parent-report): 
summary scores, 7 subscales:  regulation, energy & physiologic stability, oral-pharyngo-
esophageal function, gastroesophageal function, gastrointestinal function, feeding efficiency 
& sensory responsiveness, & compelling symptoms of problematic feeding.52 Internal 
consistency reliability (Cronbach's α = .92) was excellent. Test-retest reliability (r = 0.91) was 
high. Concurrent validity was established with the Infant Gastroesophageal Reflux 
Questionnaire (r = 0.69) and Infant Gastrointestinal Symptoms Questionnaire (r = 0.62). The 
NeoEAT-Breastfeeding total score and all subscale scores were higher in infants with feeding 
problems than in typically feeding infants (p < .001), supporting known-groups validity. 
Parent-Infant 
Interaction during 
Feeding 
The Nursing Child Assessment Satellite Training–Feeding Scale (NCAST-Feeding) will be 
used to assess behaviors between infants and mothers at oral feeding initiation, NICU 
discharge, and 2 months CA. The PI will train a Research Nurse to criterion of ≥ 90% 
agreement. A Research Nurse will video record the feeding interactions. A Research Nurse 
(who is blinded to the infant’s group assignment) will code the feeding interactions from the 
videos. A secondary trained evaluator will code 20% of the videos to assure continued 
reliability. The Feeding Scale (76 items) consists of six subscales: (a) maternal-
sensitivity to cues; (b) maternal response to distress; (c) maternal social-emotional 
growth-fostering; (d) maternal cognitive growth fostering; (e) infant clarity of cues; and 
(f) infant responsiveness to caregiver.54-59 Parent infant interaction will be video 
recorded. The NCAST has been widely used in parenting research and has well established 
reliability and validity for term and preterm infants. For preterm infants, the child subscales 
are significantly correlated to later IQ. Internal consistency is high for the total score (α = 
0.86), the mother total score (α = 0.83) and for the infant total score (α =.73). Concurrent 
validity is documented using the Teaching Scale and the Home Observation of the 
Environment (HOME). Construct validity is evidenced by the subscales’ ability to identify 
clinical problems. For example, if the child has been premature the child's score is low, while 
if the parent has a problem such as low education the mother's score is low.  
T2, T3 
 
NCAST-
Feeding 
DNA methylation of 
NR3C1 & HSD11B2 
We will evaluate DNA methylation in buccal cell DNA collected at baseline, 34 weeks PMA, 
NICU discharge, and 2 months CA, as an index of early life toxic stress. A trained research 
team member will be collecting, transporting, and storing the samples. We will collect two 
buccal swab samples from each infant at each visit using Isohelix DNA Buccal Swabs (Boca 
Scientific, Westwood, MA) before feeding to avoid contamination. The buccal swab samples 
will immediately be placed in a storage tube with a DNA stablizer Dri-Capsule (Boca 
Scientific, Westwood, MA). The samples collected will then be transported to a laboratory at 
the Loyola University Chicago Center for Translational Research and Education (Maywood, 
IL). All samples will be stored at room temperature. Quarterly, the buccal swab samples will 
be securely transported in a biospecimen transfer container (Saf T Pak, Austell, GA) via car 
to Rush University Medical Center Genomics and Microbiome Core Facility (Chicago, IL) for 
processing and analyses. DNA will be extracted from buccal swab samples using the 
Maxwell® RSC Buccal Swab DNA Kit (Promega, Maddison, WI) implemented on an 
automated Promega RSC device. The DNAm status for both the NR3C1 exon 1F and 
HSD11B2 promoter regions will be assessed using quantitative bisulfite amplicon sequencing 
using an Illumina MiSeq sequencer. Preparation of the bisulfite-converted DNA for 
sequencing will be accomplished with primers for NR3C1 and for HSD11B2, as described,11 
employing a two-stage polymerase chain reaction protocol.60 Using the Illumina MiSeq 
sequencer, we will be able to sequence the entire targeted region, including the 13 NR3C1 
CpG sites in the 1F region and the 4 HSD11B2 CpG sites. These sites are associated with 
neuroendocrine system regulation, including the HPA axis, and cortisol reactivity in rodent 
and human studies.21,61-63 Bisulfite conversion controls will be included with each sequencing 
run and will also be assessed for non-CpG ‘C’s. Samples with conversion rates below 93% 
will be re-processed. Libraries will be prepared in technical triplicate from each bisulfite 
converted DNA template, and if the replicates differ by > 10%, the sample analysis will be 
repeated. Unmethylated CpG sites will be converted to UpG (TpG upon PCR amplification) 
by bisulfite treatment. The percent methylation at any nucleotide coordinate will be 
determined by the ratio of C residues (derived from 5-methylcytosine) to C+T residues 
(derived from 5-methylcytosine plus cytosine, respectively) at any position within the NR3C1 
and HSD11B2 genomic DNA sequence. In addition, the methylation status of each read will 
be assessed individually by counting the number of methylated CpG sites observed within 
that read and summed to assess average methylation status in each amplicon. The percent 
DNAm at the NR3C1 and HSD11B2 promoters and at each CpG site will be quantified using 
CLC genomics workbench (Qiagen) software package. DNAm data will be analyzed as a 
T0, T1, T3, 
T4 
 
Buccal 
samples 

Version Date: 05/07/2024 
12 
 
continuous measure based on total percent of methylation for each NR3C1 and HSD11B2 
promoter region as well as for each individual CpG site. 
Gut Microbiome 
Infant fecal samples will be collected by a bedside nurse in the NICU at baseline, 34 weeks 
PMA, NICU discharge. For the 2 months CA sample, parents will receive a fecal collection kit 
with written instructions to collect and send samples to our lab via USPS postal services. 
Fecal samples from each infant will be collected using sterile, disposable spatulas during 
diaper changes and then placed into a sterile specimen container with microbial DNA 
stabilizer solution for 60 days at room temperature. The samples collected will be transported 
to a laboratory at the Loyola University Chicago Center for Translational Research and 
Education (Maywood, IL). All samples will be stored at room temperature. Monthly, the 
buccal swab samples will be securely transported in a biospecimen transfer container (Saf T 
Pak, Austell, GA) via car to Rush University Medical Center Genomics and Microbiome Core 
Facility (Chicago, IL) for processing and analyses. Genomic DNA will be extracted from fecal 
material using a Maxwell RSC48 autM-MILKted extraction device (Promega) and employing 
a Maxwell RSC Fecal Microbiome DNA kit, as described previously.64 Samples will be 
subject to physical shearing (i.e., bead-beating) using a TissueLyser II device (Qiagen) prior 
to extraction. Genomic DNA will be prepared for sequencing using a two-stage amplicon 
sequencing workflow65 and employing primers 515F-modified and 806r-modified.66 
Sequencing will be performed a MiniSeq sequencer (Illumina)64 with paired-end 2x154 base 
sequencing. Basic processing of sequence data, including merging of forward and reverse 
reads, quality and primer trimming, chimera removal, and selection and annotation of 
amplicon sequence variants (ASVs) will be performed.64 Alpha diversity indices (e.g., 
Shannon index) will be calculated on rarefied datasets67 in R using the vegan library 
package.68 Pairwise comparisons of alpha diversity indices will be performed using the non-
parametric Mann–Whitney test. Beta diversity analyses will be performed on pairwise Bray–
Curtis indices calculated in R using the metaMDSdist function in the vegan library v2.5-6. 
The dissimilarity indices will be modelled and tested for significance with the sample 
covariates using PERMANOVA.69 Differential abundance analyses of individual taxa between 
groups will be performed using the software package DESeq2, generating an FDR q-value.70 
DESeq2 has been shown to be appropriate for differential abundance comparisons in studies 
with small sample size groups (< 20) or unbalanced design.71 To identify taxa that most 
strongly explain between-group differences, a Linear discriminant analysis Effect Size 
(LEfSe) analysis will performed.71  
T0, T1, T3, 
T4 
 
Fecal 
samples 
Charges and costs 
of care  
Healthcare use and associated charges and costs of the NICU hospitalization and 2 months 
CA care will be extracted from LUMC’s electronic health and billing records. For infants 
receiving care outside LUMC 2 months CA, we will ask the parents about the extent and 
types of healthcare received outside LUMC. We will evaluate healthcare use, costs, and 
charges between the M-MILK and control groups. We will also describe the extent and types 
of healthcare use 2 months CA for infants receiving care outside LUMC at discharge. In the 
R01 trial, we will use activity-based costing approach to calculate the time and resources 
costs of delivering the M-MILK intervention by nurses and parents (which may not be fully 
reflected in the health system billing records), and query NICU hospitalization and 2 months 
CA healthcare use, charges, and costs to calculate total benefits and costs of M-MILK 
Intervention vs. control group.   
T3 & T4 
M-MILK 
Acceptability 
Questionnaire 
The M-MILK Acceptability Questionnaire will be completed by parents at discharge.  
The nurses will complete the M-MILK Acceptability Questionnaire at the end of the entire 
study.  
T3 
 
M-MILK 
Acceptability 
Questionnaire 
Note. EPIC = electronic medical record; GA = gestational age; PMA = postmenstrual age; CA = corrected age;  
Ongoing = throughout hospitalization from enrollment to discharge; T0 = baseline; T1 = 34 weeks PMA; T2 = oral feeding initiation; T3 
= discharge; T4 = 2 months CA 
 
ii. 
Procedures 
Figure 2. After informed consent is obtained, infants will be randomized into: Group 1 (M-MILK) and Group 2 
(attention control). All data collection will be conducted in the same manner and at the planned time points for 
both groups. For all infants, we will collect infant characteristics, NISS, and family NICU activities daily ongoing 
from EPIC and self-report. All parents will receive an automatic daily text in the morning with a link to report 
their daily activities with infants when they come to the NICU. All parents are signing up for daily texts for up to 
20 weeks to complete daily activities report every day during their participation. All parents will also receive a 
weekly phone call from a research nurse from enrollment until discharge. M-MILK is primarily implemented by 
nurses or parents. At baseline (T0), we will conduct the EPDS assessment, and collect parent characteristics, 

Version Date: 05/07/2024 
13 
 
buccal swab and fecal samples. For infants in the M-MILK group, the clinical team will place an order in EPIC 
for M-MILK to be provided starting on day 3 of life according to the M-MILK protocol. We will also schedule an 
in-person 30-minute M-MILK training session with parents in the M-MILK group, within 1 week of enrollment, 
provided by a research nurse. Parents in the M-MILK group are encouraged and supported to provide M-MILK 
when appropriate with the nurse’s supervision. At 34 weeks PMA (T1), we will collect PBQ, buccal swab and 
fecal samples. At oral feeding initiation (T2), we will collect the EFS, and NCAST-Feeding video recording. 
Day of initiation of oral feeding is defined as the first of at least two consecutive days when the infant is able to 
orally consume ≥ 10% of their prescribed feedings. Infants will be fed by nurses, speech therapist, or PI. We 
will implement the same feeding protocol, e.g., side-lying and swaddling during feeding. During the week of 
planned discharge (T3), we will conduct EPDS, PSS: NICU, PDR, PBQ, Pumping and Breastfeeding, NMI, 
NAPI, and EFS assessments. We will collect NCAST-Feeding video recording, buccal swab and fecal 
samples. For the NCAST-Feeding video recording, both parents and infants will be recorded. Parents will also 
complete the M-MILK Acceptability Questionnaire at T3. For EFS and NAPI assessments, we will video record 
the assessments, when possible, to obtain videos for inter-rater reliability. At 2 months CA (T4), we will send 
parents the fecal and buccal sample collection kits via USPS mail service. We will also conduct a phone/Zoom 
interview to complete the EPDS, PBQ, ASQ-3, and NeoEAT assessments. During the phone/Zoom interview, 
we will also provide parents instructions on how to collect buccal and fecal samples and securely send them 
back to us.  
 
VIII. 
Safety Monitoring Plan 
 
a. Potential Risk 
IRB oversight and approval will be based on 45CFR 46 HHS criteria.  We anticipate our study will be 
determined to be no more than “minimal risk”. The M-MILK intervention’s safety has been demonstrated in 
infants as young as 22 weeks GA. According to the M-MILK protocol, nurses or parents will carefully observe 
infants’ cues and responses during the M-MILK intervention and stop the administration if infants do not 
engage or exhibit any signs of distress. Thus, there is minimal risk associated with this protocol. Every effort 
will be made to minimize the risks. There may be risks associated with administration of M-MILK and collecting 
buccal swab samples including gagging and minor discomfort. Of note, these risks are no more expected risks 
to infants than what they might experience during a typical day, routine oral care, or feeding. Nurses will 
document infants’ responses or any unexpected and serious adverse events to M-MILK intervention in the M-
MILK Documentation paper sheet. A research nurse will review the documentation and report to PI if there are 
any unexpected and serious adverse events. For the NAPI assessment, we will perform maneuvers such as 
partially removing swaddle blanket, gentle stretching/bending of arms and legs, gently picking up, and 
stimulating vision and hearing with soft rattle sounds and talking voices. These maneuvers may pose minimal 
risks such as feeling cold, and mild discomfort due to stretching/bending/picking up, or noises. Of note, these 
risks are no more expected risks to infants than what they might experience during a typical day, routine care, 
or physical exam. Additionally, this research also carries a minimal risk of loss of data confidentiality/security. 
We also recognize that there are unique risks related to loss of confidentiality regarding a participant’s 
epigenetic and microbiome data; however, we consider this risk to be rare.  
 
b. Adequacy of Protection Against Risks 
 
i. 
Informed Consent and Assent 
The potential risks will be presented to the parents prior to enrollment. If parents are interested in having their 
preterm infants participate in the study, written informed consent will be obtained. Alternatively, the potential 
preterm infant will receive standard care if the parents choose to not enroll in the study. Premature infants, by 
virtue of their age cannot give consent or assent and therefore will not give assent or participate in the 
informed consent process.  
 
ii. 
Protections Against Risk 
 
1. Risks Related to Study Procedure 

Version Date: 05/07/2024 
14 
 
During the study, if an infant becomes clinically unstable and/or develops new diagnosis, and no longer meet 
the inclusion criteria, we will remove the infant from the research protocol. The M-MILK intervention’s safety 
has been demonstrated in infants as young as 22 weeks GA. According to the M-MILK protocol, nurses or 
parents will carefully observe infants’ cues and responses during the M-MILK intervention and stop the 
administration if infants do not engage or exhibit any signs of distress. When collecting the buccal swab 
samples and administering NAPI, we will make every effort to be as gentle as possible and omit any item(s) 
that may be causing discomfort to infants. If parents report a score of 12 or above on the EPDS, we will make 
referrals to the NICU Social Worker. If parents report “yes, quite often” or “sometimes” to the question “the 
thought of harming myself has occurred to me” on the EPDS, we will refer the parent to the local Emergency 
Department immediately. For the PBQ, parents scoring ≥ 26, (indicating risk of general bonding disorders) or ≥ 
40 (indicating risk of severe bonding disturbances) should seek medical attention. For the PBQ, parents 
scoring “rarely”, “sometimes”, “quite often”, “very often”, or “always” for item 18 “I have done harmful things to 
my baby” or item 24 “I feel like hurting my baby” indicates risk of child abuse. In this case, it is our responsibility 
to report it to the state’s child protective agency. If the infant is in immediate danger, the research staff will be 
instructed to inform the parents of their immediate concerns for the infant’s safety, report the incident using a 
cellular phone, and call 911 until appropriate officials arrive. The PI will be informed of the family’s condition 
and always be available by phone or in person for consultation and instruction. As part of the training of our 
research team, the team will review content related to reporting of child neglect and abuse. 
 
2. Risks related to Data Confidentiality 
Risks associated with loss of electronic data confidentiality/security will be mitigated through the utilization of 
secure electronic database (REDCap) and research network drive process to ensure that only authorized 
research personnel have access to the research data. The original signed informed consent documents will be 
kept in a locked cabinet at LUC CTRE. All infants will be assigned a study ID number. All of the data will be 
identified only by a study ID number. All discussions of specific research participants in our research team 
meetings will use this number rather than names. Electronic data will be encrypted with a password known and 
viewed only to the research staff. The password will be changed every six months.  
 
The M-MILK Documentation paper sheets will have the infant’s name on it and is available at the infant’s 
bedside for nurses to document M-MILK. The completed sheets will be collected daily by a NICU charge nurse 
and stored in a locked cabinet on the unit. A research team member will pick up the completed sheets daily, 
and stored them in a locked cabinet in at LUC CTRE (Maywood, IL) for data entry. Upon completion of data 
entry into REDCap, the completed sheets will be shredded immediately. 
 
Buccal swab and fecal samples will be labeled only with study ID numbers and collection date, and stored in a 
locked cabinet in a locked laboratory at LUC CTRE (Maywood, IL). We will securely transport buccal swab and 
fecal samples to Rush (Chicago, IL) using biosafety transfer containers and via car.  
 
Video recordings will be downloaded and stored on a secure research network drive, hosted by LUC. Infant’s 
and parent’s face will be visible in the videos as we need to observe their interactions and infant’s feeding 
skills. However, we will not include any identifiers such as name or date in the videos. The video files will be 
labeled only with study ID numbers and recorded date. 
 
De-identifiable data will be shared with University of Illinois at Chicago Research Informatics Core. All data; 
e.g., raw sequence data, processed data and sample mapping; will be transmitted between systems via 
encrypted transport protocols, i.e. HTTPS or SFTP. All desktop and laptop computers are protected by strong 
password authentication with whole disk encryption enabled. Remote servers are protected by network 
firewalls, intrusion detection software, and either bastions or two factor authentication for any remote logins. 
Any passwords on the remote systems are subject to strong password requirements, i.e., minimum password 
length, combinations of different character types (capital letters, lowercase letters, numbers, special 
characters), and are not typical English words.  
 

Version Date: 05/07/2024 
15 
 
Biospecimens are materials that come from infants that may include blood, tissue, urine, bone marrow, saliva, 
cells, etc. In this study, we will collect cheek tissue and stool. Most biospecimens contain DNA. We will not use 
biospecimens collected as a part of this study for whole genome sequencing. However, we will use 
biospecimens collected as a part of this study to sequence a short portion of the genes of interest that starts 
gene transcription found at the beginning of the genes. The de-identified sequence data will be submitted to 
online government data repositories per National Institute of Health requirements. All the sources of the 
sample are de-identified and infants’ individual identity will not be revealed.  
 
De-identified data, photos and/or selected brief segments of videos from this study may be shared with others 
for research/educational purposes in reports, scientific papers, research/educational meetings, and/or 
research/educational websites. De-identified data, photos and/or selected brief segments of videos from this 
study may also be shared on research/educational social media platforms. We will remove or code any 
personal identifiable information that could identify participants before data are shared with other researchers 
to ensure that no one will be able to identify participants from the information we share. De-identified data, the 
photos and/or selected brief segments of videos will be stored indefinitely. Informed consents and video 
recordings will be securely shredded/deleted after all related data are published or up to 5 years. Buccal swab 
and fecal samples will be stored and securely destroyed after all related data are published or up to 5 years.  
 
If any data are lost or stolen, the PI will immediately notify the LUC Information Services, at 773-508-4487 and 
will notify the LUC IRB of any unusual occurrence.   
 
The results of this research study may be published in a journal for the purpose of advancing medical 
knowledge. You will not be identified by name or by any other identifying information in any publication or 
report about this research. When we prepare our reports or publications, we will summarize the results of the 
research in a manner that will not reveal infants’ identity. In addition, no names will be used in any report of this 
study. We may want to use photos and/or brief segments of the videos to illustrate infants’ feeding skills and 
behaviors in reports, scientific papers, or educational meetings. We will obtain parents' written permission for 
use of evaluation photos and videos for educational purposes. 
 
3. Report of child neglect and abuse to the appropriate authority 
Researchers and professionals who work with families with children under the age of 18 are responsible for the 
welfare of children. For the PBQ, parents scoring “rarely”, “sometimes”, “quite often”, “very often”, or “always” 
for item 18 “I have done harmful things to my baby” or item 24 “I feel like hurting my baby” indicates risk of 
child abuse. If the research team suspects an infant has been physically or emotionally abused or neglected, 
following the guidelines set by the state during study evaluations, it is our responsibility to report it to the state’s 
child protective agency. If the infant is in immediate danger, the research staff will be instructed to inform the 
parents of their immediate concerns for the infant’s safety, report the incident using a cellular phone, and call 
911 until appropriate officials arrive. The PI will be informed of the family’s condition and always be available 
by phone or in person for consultation and instruction. As part of the training of our research team, the team 
will review content related to reporting of child neglect and abuse. 
 
iii. 
Vulnerable Subjects 
This research study is intended to evaluate an intervention for a specific group of preterm infants, those born 
between 22 to 33 weeks GA. It is essential that infants under one year of age are included. Older children are 
excluded because the study is evaluating preterm infants only. Our research team has expertise for working 
with preterm infants. For example, Dr. Griffith has more than 12 years of experience working with preterm 
infants and their families. Dr. White-Traut has more than 10 years of clinical experience and 40 years of 
research experience conducting numerous research projects, including H-HOPE intervention with neonates 
and their mothers. The infants will be cared for in the NICU as part of their hospitalization. The team will follow 
the U.S. Department of Health and Human Services regulatory requirements for parental permission and child 
assent for research involving children. Preterm infants, by virtue of their age cannot give consent or assent and 
therefore will not give assent or participate in the informed consent process.  
 

Version Date: 05/07/2024 
16 
 
IX. 
Data Management and Analysis Plan 
 
Strict confidentiality of data files will be maintained. Data management will be facilitated using REDCap. SAS 
(v 9.4) will be used for all statistical procedures and analyses. Levels of missingness, variable distributions, 
and outliers will be examined throughout data collection for quality assurance as well as prior to statistical 
modeling. We will calculate descriptive statistics for all variables of interest to describe the variability of study 
variables, and conduct sample size estimation for the planned R01 application. Comparisons will be made 
using an intent-to-treat approach with participants analyzed in the groups to which they were randomized. 
Feasibility will be assessed with respect to enrollment, adherence, acceptability, and safety. We will calculate 
the percentage of infants who (1) were eligible, approached, consented, enrolled, and randomized over the 
recruitment period, (2) were retained for the duration of the study, (3) completed all inpatient data collection, 
and (4) completed post-discharge follow-ups. To determine the adherence of M-MILK, we will calculate the 
daily numbers of completed M-MILK and the daily numbers of expected M-MILK implementation (numbers of 
hands-on care episodes before full gavage feedings) to obtain the daily percentage of M-MILK completion. We 
will summarize findings from M-MILK Acceptability Questionnaire by nurses and parents. We will also 
summarize findings from M-MILK EPIC charting regarding infant’s responses and any adverse events to 
monitor for the safety of M-MILK.  
 
X. 
Study Timeline 
 
Table 2. The proposed pilot study will require 1 year to complete. During the first 3 months, we will hire and 
train the team members. We anticipate enrolling 2 infants per month for 9 months. An additional 3 months will 
be required to complete the intervention and collect study data. Data entry and analyses will be ongoing. We 
will spend 6 months to complete data analyses. An additional 6 months will be needed to prepare the final 
report and disseminations. 
 
 
Table 2. Study Timeline 
 
Activity/Month 
Month 1-3 
Month 3-12 
Month 12-15 
Month 15-21 
Month 21-27 
Preparation & Staff Training 
X 
 
 
 
 
Recruitment & Enrollment  
 
X 
 
 
 
Data Collection 
 
X 
X 
 
 
Data Entry & Analyses 
 
X 
X 
X 
 
Final Reports & Dissemination 
 
 
 
X 
X 
 
XIII. 
Literature Cited 
 
1. 
Barfield WD. Public Health Implications of Very Preterm Birth. Clin Perinatol. Sep 2018;45(3):565-577. 
2. 
Pickler RH, Wetzel PA, Meinzen-Derr J, Tubbs-Cooley HL, Moore M. Patterned feeding experience for 
preterm infants: study protocol for a randomized controlled trial. Trials. Jun 4 2015;16:255. 
3. 
Griffith T, White-Traut R, Janusek LW. A Behavioral Epigenetics Model to Predict Oral Feeding Skills in 
Preterm Infants. Adv Neonatal Care. Oct 2020;20(5):392-400. 
4. 
Moschino L, Duci M, Fascetti Leon F, et al. Optimizing Nutritional Strategies to Prevent Necrotizing 
Enterocolitis and Growth Failure after Bowel Resection. Nutrients. Jan 24 2021;13(2). 
5. 
Tang MN, Adolphe S, Rogers SR, Frank DA. Failure to Thrive or Growth Faltering: Medical, 
Developmental/Behavioral, Nutritional, and Social Dimensions. Pediatr Rev. Nov 2021;42(11):590-603. 
6. 
Goh GL, Lim CSE, Sultana R, De La Puerta R, Rajadurai VS, Yeo KT. Risk Factors for Mortality From 
Late-Onset Sepsis Among Preterm Very-Low-Birthweight Infants: A Single-Center Cohort Study From 
Singapore. Front Pediatr. 2021;9:801955. 
7. 
Bergman NJ. Birth Practices: Maternal-Neonate Separation as a Source of Toxic Stress. Birth Defects 
Res. Sep 1 2019;111(15):1087-1109. 
8. 
de Magalhães-Barbosa MC, Prata-Barbosa A, da Cunha A. Toxic Stress, Epigenetics and Child 
Development. J Pediatr (Rio J). Mar-Apr 2022;98 Suppl 1(Suppl 1):S13-s18. 
9. 
National Association of Neonatal Nurses. Trauma-Informed Care in the NICU. 2022. 

Version Date: 05/07/2024 
17 
 
10. 
Malin K, Gondwe KW, Fial AV, et al. A scoping review of epigenetic alterations in premature infants 
secondary to early toxic stress exposure. Nursing Research. Accepted 2022. 
11. 
Lester BM, Marsit CJ, Giarraputo J, Hawes K, LaGasse LL, Padbury JF. Neurobehavior Related to 
Epigenetic Differences in Preterm Infants. Epigenomics. Oct 2015;7(7):1123-1136. 
12. 
Zhao T, Griffith T, Zhang Y, et al. Early-life factors associated with neurobehavioral outcomes in 
preterm infants during NICU hospitalization. Pediatr Res. Mar 25 2022. 
13. 
Provenzi L, Guida E, Montirosso R. Preterm Behavioral Epigenetics: A Systematic Review. Neurosci 
Biobehav Rev. Jan 2018;84:262-271. 
14. 
Cowan CSM, Stylianakis AA, Richardson R. Early-life stress, microbiota, and brain development: 
probiotics reverse the effects of maternal separation on neural circuits underpinning fear expression 
and extinction in infant rats. Dev Cogn Neurosci. Jun 2019;37:100627. 
15. 
D'Agata AL, Wu J, Welandawe MKV, Dutra SVO, Kane B, Groer MW. Effects of early life NICU stress 
on the developing gut microbiome. Dev Psychobiol. Jul 2019;61(5):650-660. 
16. 
Groer MW, Miller EM, D'Agata A, et al. Contributors to Dysbiosis in Very-Low-Birth-Weight Infants. J 
Obstet Gynecol Neonatal Nurs. May 2020;49(3):232-242. 
17. 
Henderickx JGE, Zwittink RD, van Lingen RA, Knol J, Belzer C. The Preterm Gut Microbiota: An 
Inconspicuous Challenge in Nutritional Neonatal Care. Front Cell Infect Microbiol. 2019;9:85. 
18. 
Liu X, Cong X, Li G, Maas K, Chen K. Multivariate log-contrast regression with sub-compositional 
predictors: Testing the association between preterm infants' gut microbiome and neurobehavioral 
outcomes. Stat Med. Feb 10 2022;41(3):580-594. 
19. 
Lu J, Claud EC. Connection Between Gut Microbiome and Brain Development in Preterm Infants. Dev 
Psychobiol. Jul 2019;61(5):739-751. 
20. 
Janusek LW, Tell D, Gaylord-Harden N, Mathews HL. Relationship of Childhood Adversity and 
Neighborhood Violence to a Proinflammatory Phenotype in Emerging Adult African American Men: An 
Epigenetic Link. Brain Behav Immun. Feb 2017;60:126-135. 
21. 
Janusek LW, Tell D, Mathews HL. Epigenetic Perpetuation of the Impact of Early Life Stress on 
Behavior. Curr Opin Behav Sci. 2019;28C:1-7. 
22. 
Cong X, Judge M, Xu W, et al. Influence of Feeding Type on Gut Microbiome Development in 
Hospitalized Preterm Infants. Nurs Res. Mar/Apr 2017;66(2):123-133. 
23. 
Xu W, Judge MP, Maas K, et al. Systematic Review of the Effect of Enteral Feeding on Gut Microbiota 
in Preterm Infants. J Obstet Gynecol Neonatal Nurs. May 2018;47(3):451-463. 
24. 
Lester BM, Conradt E, LaGasse LL, Tronick EZ, Padbury JF, Marsit CJ. Epigenetic Programming by 
Maternal Behavior in the Human Infant. Pediatrics. Oct 2018;142(4). 
25. 
Provenzi L, Brambilla M, Scotto di Minico G, Montirosso R, Borgatti R. Maternal Caregiving and DNA 
Methylation in Human Infants and Children: Systematic Review. Genes Brain Behav. Mar 
2020;19(3):e12616. 
26. 
Álvarez MJ, Fernández D, Gómez-Salgado J, Rodríguez-González D, Rosón M, Lapeña S. The Effects 
of Massage Therapy in Hospitalized Preterm Neonates: A Systematic Review. Int J Nurs Stud. Apr 
2017;69:119-136. 
27. 
Wetzel CM, Davis L, Grohler N, et al. A Quality Improvement Project to Improve the Use of Mother's 
Own Milk (MOM) With Precision Oropharyngeal Therapy. Adv Neonatal Care. Apr 2020;20(2):E19-e30. 
28. 
O'Rouke B, Fuller K, Parker LA, Moore TA. Provision of Positive Oral Experiences for Premature 
Infants by Offering Milk Drops: A Clinical Practice Change Initiative Adv Neonatal Care. 2022. 
29. 
Abd-Elgawad M, Eldegla H, Khashaba M, Nasef N. Oropharyngeal Administration of Mother's Milk Prior 
to Gavage Feeding in Preterm Infants: A Pilot Randomized Control Trial. JPEN J Parenter Enteral Nutr. 
Jan 2020;44(1):92-104. 
30. 
Muelbert M, Lin L, Bloomfield FH, Harding JE. Exposure to the Smell and Taste of Milk to Accelerate 
Feeding in Preterm Infants. Cochrane Database Syst Rev. Jul 16 2019;7(7):Cd013038. 
31. 
Moore CG, Carter RE, Nietert PJ, Stewart PW. Recommendations for planning pilot studies in clinical 
and translational research. Clin Transl Sci. Oct 2011;4(5):332-337. 
32. 
Cox JL, Holden JM, Sagovsky R. Detection of postnatal depression. Development of the 10-item 
Edinburgh Postnatal Depression Scale. The British journal of psychiatry : the journal of mental science. 
Jun 1987;150:782-786. 

Version Date: 05/07/2024 
18 
 
33. 
Park SH, Kim JI. Predictive validity of the Edinburgh postnatal depression scale and other tools for 
screening depression in pregnant and postpartum women: a systematic review and meta-analysis. Arch 
Gynecol Obstet. May 2023;307(5):1331-1345. 
34. 
Miles MS, Funk SG, Carlson J. Parental Stressor Scale: neonatal intensive care unit. Nurs Res. May-
Jun 1993;42(3):148-152. 
35. 
Kubicka Z, Zahr E, Clark P, Williams D, Berbert L, Arzuaga B. Use of an internet camera system in the 
neonatal intensive care unit: parental and nursing perspectives and its effects on stress. J Perinatol. 
Aug 2021;41(8):2048-2056. 
36. 
Fischer SL. Modification and Evaluation of the Late-preterm Screening and Multidisciplinary Risk 
Assessment and Parent Activation Scale. South Dakota, USA: South Dakota State University; 2013. 
37. 
Korner AF, Stevenson DK, Kraemer HC, et al. Prediction of the Development of Low Birth Weight 
Preterm Infants by a New Neonatal Medical Index. J Dev Behav Pediatr. Apr 1993;14(2):106-111. 
38. 
Cong X, Wu J, Vittner D, et al. The impact of cumulative pain/stress on neurobehavioral development of 
preterm infants in the NICU. Early Hum Dev. May 2017;108:9-16. 
39. 
Newnham CA, Inder TE, Milgrom J. Measuring Preterm Cumulative Stressors Within the NICU: The 
Neonatal Infant Stressor Scale. Early Hum Dev. Sep 2009;85(9):549-555. 
40. 
Smith GC, Gutovich J, Smyser C, et al. Neonatal Intensive Care Unit Stress is Associated with Brain 
Development in Preterm Infants. Ann Neurol. Oct 2011;70(4):541-549. 
41. 
Gorzilio DM, Garrido E, Gaspardo CM, Martinez FE, Linhares MB. Neurobehavioral Development Prior 
to Term-Age of Preterm Infants and Acute Stressful Events During Neonatal Hospitalization. Early Hum 
Dev. Dec 2015;91(12):769-775. 
42. 
Gabriel P, Formiga C, Linhares M. Early neurobehavioral development of preterm infants. Psicologia. 
2013;26(1):202-211. 
43. 
Constantinou JC, Adamson-Macedo EN, Mirmiran M, Ariagno RL, Fleisher BE. Neurobehavioral 
assessment predicts differential outcome between VLBW and ELBW preterm infants. J Perinatol. Dec 
2005;25(12):788-793. 
44. 
Snider L, Tremblay S, Limperopoulos C, Majnemer A, Filion F, Johnston C. Construct validity of the 
Neurobehavioral Assessment of Preterm Infants. Physical & occupational therapy in pediatrics. 
2005;25(3):81-95. 
45. 
Korner A, Brown J, Thom V, Constantinou J. Neurobehavioral Assessment of the Preterm Infant 
revised. 2nd ed. Van Nuys: Child Development Media, Inc. ; 2000. 
46. 
Korner AF, Constantinou J, Dimiceli S, Brown BW, Jr., Thom VA. Establishing the reliability and 
developmental validity of a neurobehavioral assessment for preterm infants: a methodological process. 
Child development. Oct 1991;62(5):1200-1208. 
47. 
Korner AF, Kraemer HC, Reade EP, Forrest T, Dimiceli S, Thom VA. A methodological approach to 
developing an assessment procedure for testing the neurobehavioral maturity of preterm infants. Child 
development. Dec 1987;58(6):1478-1487. 
48. 
Shuffrey LC, Firestein MR, Kyle MH, et al. Association of Birth During the COVID-19 Pandemic With 
Neurodevelopmental Status at 6 Months in Infants With and Without In Utero Exposure to Maternal 
SARS-CoV-2 Infection. JAMA Pediatr. Jun 1 2022;176(6):e215563. 
49. 
Lipkin PH, Macias MM. Promoting Optimal Development: Identifying Infants and Young Children With 
Developmental Disorders Through Developmental Surveillance and Screening. Pediatrics. Jan 
2020;145(1). 
50. 
Agarwal PK, Xie H, Sathyapalan Rema AS, et al. Evaluation of the Ages and Stages Questionnaire 
(ASQ 3) As a Developmental Screener at 9, 18, and 24 Months. Early Hum Dev. Aug 
2020;147:105081. 
51. 
Thoyre SM, Pados BF, Shaker CS, Fuller K, Park J. Psychometric Properties of the Early Feeding 
Skills Assessment Tool. Adv Neonatal Care. Oct 2018;18(5):E13-E23. 
52. 
Pados BF, Thoyre SM, Estrem HH, Park J, McComish C. Factor Structure and Psychometric Properties 
of the Neonatal Eating Assessment Tool-Breastfeeding. J Obstet Gynecol Neonatal Nurs. May 
2018;47(3):396-414. 

Version Date: 05/07/2024 
19 
 
53. 
Pados BF, Thoyre SM, Estrem HH, Park J, McComish C. Factor Structure and Psychometric Properties 
of the Neonatal Eating Assessment Tool-Bottle-Feeding (NeoEAT-Bottle-Feeding). Adv Neonatal Care. 
Jun 2018;18(3):232-242. 
54. 
Arianas EA, Rankin KM, Norr KF, White-Traut RC. Maternal weight status and responsiveness to 
preterm infant behavioral cues during feeding. BMC Pregnancy Childbirth. Apr 11 2017;17(1):113. 
55. 
Farel AM, Freeman VA, Keenan NL, Huber CJ. Interaction between high-risk infants and their mothers: 
the NCAST as an assessment tool. Res Nurs Health. Apr 1991;14(2):109-118. 
56. 
Jameson M, Fehringer K, Neu M. Comparison of two tools to assess dyad feeding interaction in infants 
with gastroesophageal reflux disease. J Spec Pediatr Nurs. Jan 2018;23(1). 
57. 
Letourneau NL, Tryphonopoulos PD, Novick J, Hart JM, Giesbrecht G, Oxford ML. Nursing Child 
Assessment Satellite Training Parent-Child Interaction Scales: Comparing American and Canadian 
Normative and High-Risk Samples. J Pediatr Nurs. May-Jun 2018;40:47-57. 
58. 
White-Traut R, Norr KF, Fabiyi C, Rankin KM, Li Z, Liu L. Mother-infant interaction improves with a 
developmental intervention for mother-preterm infant dyads. Infant Behav Dev. Dec 2013;36(4):694-
706. 
59. 
Whitfield KC, Ventura AK. Exploration of Responsive Feeding During Breastfeeding Versus Bottle 
Feeding of Human Milk: A Within-Subject Pilot Study. Breastfeed Med. Sep 2019;14(7):482-486. 
60. 
McFadden VC, Shalaby RE, Iram S, et al. Hepatic Deficiency of the Pioneer Transcription Factor FoxA 
Restricts Hepatitis B Virus Biosynthesis by the Developmental Regulation of Viral DNA Methylation. 
PLOS Pathog. Feb 2017;13(2):e1006239. 
61. 
Lester BM, Marsit CJ. Epigenetic Mechanisms in the Placenta Related to Infant Neurodevelopment. 
Epigenomics. Mar 1 2018;10(3):321-333. 
62. 
Casavant SG, Cong X, Moore J, Starkweather A. Associations between preterm infant stress, 
epigenetic alteration, telomere length and neurodevelopmental outcomes: A systematic review. Early 
Hum Dev. Apr 2019;131:63-74. 
63. 
Vaiserman AM, Koliada AK. Early-life adversity and long-term neurobehavioral outcomes: epigenome 
as a bridge? Hum Genomics. Dec 16 2017;11(1):34. 
64. 
Hooper MJ, LeWitt TM, Pang Y, et al. Gut dysbiosis in cutaneous T-cell lymphoma is characterized by 
shifts in relative abundances of specific bacterial taxa and decreased diversity in more advanced 
disease. J Eur Acad Dermatol Venereol. Sep 2022;36(9):1552-1563. 
65. 
Naqib A, Poggi S, Wang W, Hyde M, Kunstman K, Green SJ. Making and Sequencing Heavily 
Multiplexed, High-Throughput 16S Ribosomal RNA Gene Amplicon Libraries Using a Flexible, Two-
Stage PCR Protocol. Methods Mol Biol. 2018;1783:149-169. 
66. 
Walters W, Hyde ER, Berg-Lyons D, et al. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and 
Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys. 
mSystems. Jan-Feb 2016;1(1). 
67. 
Gihring TM, Green SJ, Schadt CW. Massively parallel rRNA gene sequencing exacerbates the 
potential for biased community diversity comparisons due to variable library sizes. Environ Microbiol. 
Feb 2012;14(2):285-290. 
68. 
R Package. Vegan: community ecology [computer program]. Version; 2018. 
69. 
Jackson A, Engen PA, Forsyth CB, et al. Intestinal Barrier Dysfunction in the Absence of Systemic 
Inflammation Fails to Exacerbate Motor Dysfunction and Brain Pathology in a Mouse Model of 
Parkinson's Disease. Front Neurol. 2022;13:882628. 
70. 
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data 
with DESeq2. Genome Biol. 2014;15(12):550. 
71. 
Weiss S, Xu ZZ, Peddada S, et al. Normalization and microbial differential abundance strategies 
depend upon data characteristics. Microbiome. Mar 3 2017;5(1):27.