Early Life Protein-enriched Human Milk Diets to Increase Lean Body Mass Accretion and Diversity of the Gut Microbiome in Extremely Preterm Infants: a Randomized Trial
Phase: NA
Conditions: Prematurity; Extreme, Feeding Disorder Neonatal, Breast Milk Expression, Growth Failure, Microbial Colonization
Sponsor: University of Alabama at Birmingham

1 
A single-center, masked, randomized, superiority trial of 
early life protein-enriched human milk diets to increase lean 
body mass accretion and diversity of the gut microbiome in 
extremely preterm infants 
 
 
 
 
 
 
 
Lead Study Investigator: 
Ariel A. Salas, MD, MSPH 
University of Alabama at Birmingham 
 
 
 
 
 
Version Date: 
3/23/2020 
 
 
 

2 
Contents 
 
Section 1. 
Abstract ............................................................................................................... 3 
Section 2. 
Statement of Problem ........................................................................................... 4 
2.1. 
Primary Hypothesis or Question ............................................................... 4 
2.2. 
Secondary Hypothesis or Questions (s) (if applicable) .............................. 4 
2.3. 
Background and Rationale ........................................................................ 4 
Section 3. 
Methods ............................................................................................................... 5 
3.1. 
Study Population ...................................................................................... 5 
3.1.1. Inclusion Criteria .......................................................................... 5 
3.1.2. Exclusion Criteria ......................................................................... 5 
3.2. 
Detailed Study Procedures ........................................................................ 5 
3.2.1. Screening ...................................................................................... 5 
3.2.2. Consent Procedures ...................................................................... 5 
3.2.3. Randomization Procedures............................................................ 5 
3.2.4. Study Intervention and Comparison .............................................. 6 
3.2.5. Blinding/Masking ......................................................................... 6 
3.2.6. Control or Monitoring of Co-interventions .................................... 6 
3.2.7. Primary Outcome .......................................................................... 6 
3.2.8. Secondary Outcomes .................................................................... 6 
3.2.9. Additional Safety Outcomes ......................................................... 7 
3.2.10. Compliance Monitoring ................................................................ 7 
3.2.11. Study Specimens .......................................................................... 7 
3.3. 
Potential Risks and Benefits to Subjects ................................................... 7 
Section 4. 
Analytical Plan .................................................................................................. 10 
4.1. 
Statistical Analysis Plan ......................................................................... 10 
4.2. 
Sample Size and Power Estimates .......................................................... 10 
4.3. 
Available Population .............................................................................. 11 
4.4. 
Projected Recruitment time .................................................................... 11 
4.5. 
Study Monitoring Plan ........................................................................... 11 
4.5.1. Reporting Adverse Events .......................................................... 11 
4.5.2. Data Monitoring Plan and Stopping Rules .................................. 11 
Section 5. 
References ......................................................................................................... 12 
 

3 
SECTION 1. ABSTRACT 
Study Hypothesis/Question  
The central hypothesis of the proposed work is that protein-enriched human milk diets during the 
first 2 weeks after birth increase fat-free mass(FFM)-for-age Z scores and promote maturation of 
the gut microbiome at term equivalent age in extremely preterm (EPT) infants. 
 
Study Design Type  
Parallel-group, masked randomized controlled trial in which study participants fed human milk 
will be randomly assigned in a 1:1 allocation ratio to receive either a protein-enriched human 
milk diet (intervention group) or a usual human milk diet (control group) within the first 96 
hours after birth.. 
 
Eligibility Criteria  
EPT infants with gestational age of 28 weeks or less admitted to the neonatal unit at the 
University of Alabama at Birmingham (UAB) Hospital will be included. Infants with major 
congenital anomalies and infants with a terminal illness in whom decisions to withhold or limit 
life support have been made will be excluded. 
 
Study Intervention/Methods  
Written informed consent will be obtained within the first 96 hours after birth to allow treatment 
allocation before or on the first day of enteral feeding via orogastric tube. Infants in the 
intervention group will receive expressed human milk or donor human milk on feeding day 1 
(within the first 96 hours after birth). On feeding day 2, a human milk-based product that 
increases protein content (Prolact®, Prolacta Bioscience, Inc. City of Industry, CA) will be 
added to human milk. This practice will continue until standard bovine-based human milk 
fortifiers are ordered. Infants in the control group will receive expressed human milk or donor 
human milk from feeding day 1. This practice will continue until standard bovine-based products 
are ordered. 
 
Primary Outcome  
The primary efficacy outcome will be FFM-for-age Z-score at 36 weeks of postmenstrual age 
(PMA). The primary microbiological outcome will be composition and diversity of the gut 
microbiome. 
 
Secondary Outcome(s)  
Secondary efficacy outcomes will include postnatal growth failure (PGF), FFM, %FFM, body fat 
(BF), and %BF at 36 weeks PMA, growth velocity rate (g/kg/d) between birth and 36 weeks 
PMA, and anthropometric measurements at 36 weeks PMA (weight, head circumference, and 
length). The primary safety outcomes will include intestinal perforation, NEC stage 2 or greater, 
culture-proven sepsis, and death.

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SECTION 2. STATEMENT OF PROBLEM 
2.1. 
PRIMARY HYPOTHESIS OR QUESTION 
 
Protein-enriched human milk diets during the first 2 weeks after birth increase FFM-for-
age Z scores at term equivalent age in extremely preterm (EPT) infants. 
 
2.2. 
SECONDARY HYPOTHESIS OR QUESTIONS (S) (IF APPLICABLE) 
 
Protein-enriched human milk diets during the first 2 weeks after birth promote maturation 
of the gut microbiome at term equivalent age in extremely preterm (EPT) infants. 
 
2.3. 
BACKGROUND AND RATIONALE 
 
Limited enteral nutrition aggravates the problem of cumulative nutritional deficits during 
the first 2 weeks after birth and increases the risk of postnatal growth failure in extremely 
preterm (EPT) infants born at 28 weeks of gestation or less. Postnatal growth failure occurs in 
approximately 6 of every 10 EPT infants by the time they reach 36 weeks of postmenstrual age 
(PMA). EPT infants with postnatal growth failure have a higher risk of adverse health outcomes, 
particularly when they have more fat mass (FM) gains than fat-free mass (FFM) gains. To restore 
cumulative nutritional deficits and prevent postnatal growth failure in EPT infants with limited 
enteral nutrition during the first 2 weeks after birth, most clinicians prescribe protein-enriched 
diets to promote catch-up growth only after full enteral nutrition is established. Emerging clinical 
evidence suggests that this approach is not effective to improve health outcomes in EPT infants.  
 
Likewise, increasing evidence from translational studies suggest that the practice of 
limiting enteral nutrition in early postnatal life shapes not only growth and FFM accretion, but 
also development of the gastrointestinal (GI) tract and composition of the gut microbiome. EPT 
infants unable to receive sufficient enteral nutrition during the first 2 weeks after birth need 
innovative early life dietary interventions. Not addressing this problem will perpetuate the 
practice of limiting enteral nutrition in EPT infants during a critical period of development in 
which human milk diets could influence development of the GI tract and help define composition 
of the gut microbiome. 

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SECTION 3. METHODS 
3.1. 
STUDY POPULATION 
 
Extremely preterm infants with gestational ages of 22 0/7 to 26 6/7 weeks of gestation 
admitted to the UAB hospital. This study population has been selected based on the frequency of 
feeding problems observed at these lower gestational ages and the increased risk of postnatal 
growth failure in this vulnerable population. 
 
3.1.1. Inclusion Criteria 
 
• Gestational age of 22 0/7 to 26 6/7 weeks of gestation 
• < 48 hours postnatal age 
 
3.1.2. Exclusion Criteria 
 
• Major congenital/chromosomal anomalies 
• Terminal illness requiring limited or withheld support 
 
3.2. 
DETAILED STUDY PROCEDURES 
 
3.2.1. Screening 
 
All 22 0/7 to 26 6/7 weeks of gestation infants admitted to the UAB neonatal unit will be 
screened to determine eligibility for the trial. To maximize the generalizability of our results, we 
will screen inborn and outborn infants in their first 96 hours to allow adequate time for informed 
consent, identify terminally ill infants, and exclude early deaths unrelated to enteral feeding. 
 
3.2.2. Consent Procedures 
 
Written informed consent will be obtained by the first 96 hours after birth to allow 
treatment allocation before or on the first day of enteral feeding. If a potential participant is 
identified, a member of the study will see the parents and/or mother in her room or the baby’s 
room and explain the study. The risks and benefits will be discussed with the parents and time 
will be given to them to ask questions. It will be made known to them that no treatment will be 
withheld from their infant if they participate in the study. Randomization will define study group 
assignment. 
 
3.2.3. Randomization Procedures  
 
Participants will be randomly assigned to one of the study groups following computer-
generated random-block sequences and with the use of numbered, opaque, sealed envelopes, 
which will be opened in sequential order only after informed consent is obtained. Twin infants 
will be randomized individually. 
 

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3.2.4. Study Intervention and Comparison  
 
Usual feeding practices in our neonatal unit include: 1) administration of enteral feeds as 
intermittent bolus gavage every 3 hours; 2) initiation of trophic feeds within the first 96 hours 
after birth with 20-25 ml/kg/d; 3) progression of enteral feeds with daily increments of 20-25 
ml/kg/d usually before postnatal day 7; 4) use of donor human milk as an alternative to mother’s 
own milk if the mother is not able to supply her own milk, and 5) addition of bovine-based 
products that increase protein content of human milk at approximately postnatal day 14 after full 
enteral nutrition is established (> 120 ml/kg/d) [Figure 4].  
 
Intervention group: Infants in the intervention group will receive expressed human milk 
or donor human milk on feeding day 1 (within the first 96 hours after birth). On feeding day 2, a 
human milk-based product that increases protein content (Prolact®, Prolacta Bioscience, Inc. 
City of Industry, CA) will be added to human milk. This practice will continue until standard 
bovine-based human milk fortifiers are ordered.  
 
Control group: Infants in the 
control group will receive 
expressed human milk or donor 
human milk from feeding day 1. 
This practice will continue until 
standard bovine-based products are 
ordered.  
 
3.2.5. Blinding/Masking 
 
Caregivers and primary outcome evaluators will be masked. Nutrition room staff not 
involved in patient care will be responsible for determining participant allocation to one of the 
supplementation groups by opening sequentially numbered sealed envelopes, dispensing feeding 
syringes with the allocated human milk diet (protein-enriched or usual), and masking caregivers 
administering the assigned dietary intervention. 
 
3.2.6. Control or Monitoring of Co-interventions 
 
This pragmatic trial will compare protein-enriched and usual human milk diets under 
normal clinical circumstances with no effort to strictly control interventions other than dietary 
intervention. Therefore, clinical care will be conducted at the clinician’s discretion. 
 
3.2.7. Primary Outcome 
 
• The primary efficacy outcome will be FFM-for-age Z-score at 36 weeks PMA. The 
primary microbiological outcome will be composition and diversity of the gut 
microbiome 
 
3.2.8. Secondary Outcomes  
 
Figure 4. Study Intervention 
 

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• PGF, FFM, %FFM, BF and %BF at 36 weeks PMA, growth velocity rate (g/kg/d) between 
birth and 36 weeks PMA, and anthropometric measures at 36 weeks PMA (weight, head 
circumference, and length).  
• Respiratory support at 36 weeks PMA 
• Bronchopulmonary dysplasia (BPD) at 36 weeks PMA 
 
3.2.9. Additional Safety Outcomes 
 
• Death 
• NEC stage 2 or 3 
• SIP 
• Culture-proven sepsis 
 
3.2.10. Compliance Monitoring  
 
Unlike many enteral feeding trials in EPT infants, including our preliminary trial, the 
primary intervention of this trial will be masked. Masking will reduce problems of compliance 
with the new intervention and reduce surveillance and ascertainment biases. 
 
3.2.11. Study Specimens 
 
We will obtain written informed consent from the parent(s) to collect stool samples 
weekly from birth to postnatal day 28 and a stool sample before hospital discharge. We will also 
obtain consent to collect a serum sample prior to hospital discharge to measure biomarkers of 
anabolism. 
 
3.3. 
POTENTIAL RISKS AND BENEFITS TO SUBJECTS 
 
This is non-exempt human subjects research. 
 
Risk to Human Subjects 
 
Human Subjects’ Involvement, Characteristics, and Design. In this clinical trial, 150 study 
participants will be randomly assigned in a 1:1 allocation ratio to receive either a protein-
enriched human milk diet (intervention group) or a usual human milk diet (control group) during 
the first 2 weeks after birth. Patients admitted to the UAB Neonatal Intensive Care Unit between 
July 2020 and December 2022 will be screened to determine eligibility for the trial. Our study 
population will reflect the epidemiology of prematurity in the state of Alabama, with 
approximately 51% being Black (African-American), 47% White (Non-Hispanic Caucasian), 
and the remaining 2% Hispanic/Other. This study population has been selected based on the 
frequency of postnatal growth problems observed in extremely preterm infants and the increased 
risk of adverse health outcomes in this vulnerable population.  
 
Study Procedures, Materials, and Potential Risks. For study participants, data will be collected 
from electronic medical records. Data containing identifying information will be available only 
to the PI and research personnel directly involved with this study. Information about the study 

8 
will be shared without individual identifiers. Data will be collected from medical records in 
accordance with the study protocol. This will include demographic data and other 
nutrition/feeding data. This protocol will be reviewed by the Institutional Review Board of the 
University of Alabama at Birmingham.   
 
Potential risks: The probability of risk for higher frequency of feeding interruptions in a patient 
that participates in this trial is not different than the probability of higher frequency of feeding 
interruptions in a patient that does not participate in the trial. Other theoretical risks of this study 
are related to clinical decompensation during assessment of infant body composition. They 
include increased risk of bradycardia or desaturations. Previous studies, including ours 
(unpublished data), have not reported an association between assessment of infant body 
composition and any of the above-mentioned risks. Similarly, studies of high-protein 
supplementation in preterm infants, primarily those fed formula did not report tolerance 
problems or increased risk of metabolic acidosis or high BUNs and it is unlikely that 
significantly adverse effects will be observed with excessive amounts of protein, particularly if 
the supplement is of high digestibility. Infants may experience some transient discomfort during 
the PeaPod® assessment which requires them to wear a tight-fitting cap but no other clothing or 
blanket. Therefore, there are no known risks associated with this trial except loss of 
confidentiality, as it involves data collection and imaging recording. This is one of the most 
common risks of participation in clinical research. Accordingly, our team has designed a strategy 
to protect participant confidentiality. All participants will be informed of study procedures and 
gauged for understanding of study tasks. In addition, study personnel will follow regulatory 
guidelines for obtaining informed consent and manage study data that includes personal 
information.  
 
Alternative treatments and procedures: Participants in the intervention and control groups will 
have full access to all available standard of care clinical services at our neonatal unit, and parents 
are permitted to withdraw or refuse participation at any time. Serious adverse events will be 
reported to the DSMB and to the principal investigator.    
 
Adequacy of Protection against Risks 
 
Informed Consent and Assent. After a potential study participant is identified, a member of the 
study will see the parents and/or mother in the baby’s room and explain the study. The risks and 
benefits will be discussed with the parents and time will be given to them to ask questions. It will 
be made known to them that no treatment will be withheld from their child if they participate in 
the study. The research team will attempt to obtain written consent after giving the parents a 
minimum of 24 hours to think about the study information and ask questions. Randomization 
will define study group assignment.  
 
Protection against Risks. Our team will make every effort to protect all participants’ confidential 
and private information in order to minimize possible study-associated risks. All findings related 
to any research will be available and provided to study participants in accordance with standard 
practices. We will also inform all participants that their participation is voluntary, and we will 
utilize study identification codes in place of personal identifiers on study materials. We will also 
employ storage and encryption techniques. All study personnel are required to renew Human 

9 
Subjects training biannually. No data will be accepted from or distributed to investigator or study 
staff if regulatory training is not current.  
 
Vulnerable Subjects.  This study population has been selected based on the frequency of 
postnatal growth problems observed in extremely preterm infants and the increased risk of 
adverse health outcomes in this vulnerable population. Because the probability of complications 
with protein-enriched human milk diets is not greater than the probability of complications with 
standard human milk diets, the category of children’s risk level is 1 – research not involving 
greater than minimal risk. The risks of harm anticipated in the proposed research are not greater 
than those ordinarily encountered during the assessment of feeding tolerance. 
 
Potential Benefits of the Proposed Research to Research Participants and Others 
 
Potential benefits of this study include safety and efficacy data on early life protein-enriched 
human milk diets as a dietary intervention to prevent nutritional deficits, reduce postnatal growth 
failure, and increase fat-free mass accretion in preterm infants.  Better quantitative and 
qualitative outcomes of growth may also be associated with improved long-term 
neurodevelopmental outcomes.   
 
Reductions of postnatal growth failure and improved nutritional parameters could improve long-
term neurodevelopmental outcomes of infants who survive to hospital discharge. There will be 
benefit to the medical community in providing additional information on infant body 
composition of preterm infants. 
 
Importance of the Knowledge to be Gained 
 
As the risk to individual participants is small and potential benefits are significant, the 
risk/benefit ratio is favorable. New knowledge on the effects of early diets on growth and the gut 
microbiome could measurably alter shift current practices and improve nutrition of preterm 
infants. 

10 
SECTION 4. ANALYTICAL PLAN 
4.1. 
STATISTICAL ANALYSIS PLAN 
 
All statistical analyses will use two-tail alpha to reject null hypotheses at 0.05, using R 
software. Continuous variables will be summarized as means ± standard deviations (SD) or as 
medians and interquartile ranges (IQRs). Categorical variable will be summarized as frequencies 
and proportions. Group differences will be evaluated using the T Test or Wilcoxon test for 
continuous variables and chi-square for categorical variables. The effect size of the primary 
outcome will be expressed as the mean difference with 95% confidence intervals (CIs). Risk 
ratios (RRs) with 95% CIs will be reported for categorical outcomes. All of the efficacy and 
safety outcomes of the trial will be analyzed with the intention-to-treat principle.  
 
To analyze our longitudinal microbiome data, we will use either negative binomial 
models or zero-inflated models. Since there is no explicit formula to calculate power and detect 
taxa based differences on neither negative binomial nor zero-inflated models, we performed 
extensive simulation studies to assess the statistical power needed to identify significant taxa 
with the proposed trial. We employed the function sim in the R package NBZIMM 
(https://github.com/nyiuab/NBZIMM) to simulate longitudinal microbiome count data, and then 
used negative binomial mixed models to analyze the simulated counts. For the proposed 
longitudinal microbiome study design (i.e. with 75 infants in the intervention group and 75 
infants in the control group that will have 3 to 6 stool samples collected over time), we will 
achieve ~80% power to detect ~ 2-fold effects. Since there is no explicit formula for analytically 
calculating power for detecting taxa based on neither negative binomial nor zero-inflated models, 
we performed extensive simulation studies to assess the statistical power to identify significant 
taxa with the proposed study design. We employed the function sim in the R package NBZIMM 
(https://github.com/nyiuab/NBZIMM) to simulate longitudinal microbiome count data, and then 
used negative binomial mixed models to analyze the simulated counts. To minimize possible bias 
and yield reasonable count values that are similar to real longitudinal microbiome data, we 
randomly generated the parameters (including the fixed fold-change effect, random effect, 
dispersion parameter, zero-inflation probability, etc.) in the model from reasonable ranges. For 
the proposed longitudinal microbiome study design (i.e. with 75 infants in the intervention group 
and 75 infants in the control group that will have 3 to 6 stool samples collected over time), we 
will achieve ~80% power to detect ~ 2-fold effects under a significance level of 5%.  
 
For clinical and microbiome outcomes, adjusted analyses will be performed with the 
following covariates: volume intake in ml/kg, proportion of human milk intake, exposure to 
antibiotics after birth (i.e., number of days receiving antibiotics), race, sex, gestational age, and 
maternal use of antibiotics. 
 
4.2. 
SAMPLE SIZE AND POWER ESTIMATES 
 
We used our own institutional data from a previous enteral feeding trial to calculate the 
sample size for this trial. To detect a 0.5-difference in FFM-for-age Z-scores between groups 
with SD of 1, 0.05 level of significance, and 80% power for a T-test that compares means from 

11 
two independent samples, we estimated that a sample size of 126 patients will be necessary in 
this superiority trial. Anticipating that approximately 20% of study participants will be lost to 
follow-up for assessment of the primary outcome at 36 weeks PMA, we will add 12 patients to 
each group and increase the sample size to 150. We will include a total of 75 patients in each 
group (n=150).  
 
4.3. 
AVAILABLE POPULATION 
 
The estimated UAB available population based on inclusion/exclusion criteria is 150 per 
year. 
 
4.4. 
PROJECTED RECRUITMENT TIME 
 
Assuming a consent rate of 60%, this trial will require 2.5 years for patient recruitment 
and 1 year for completion of microbiome analyses. Therefore, the time to study 
completion is 3.5 years. 
 
4.5. 
STUDY MONITORING PLAN 
 
4.5.1. Reporting Adverse Events 
 
Serious adverse events and suspected unexpected serious adverse reactions will be 
reported to the Data Safety and Monitoring Board (DSMB). 
 
4.5.2. Data Monitoring Plan and Stopping Rules 
 
Because not all neonatal units across the United States increase protein intake during the 
first 2 weeks after birth in extremely preterm infants, our DSMB will analyze all serious adverse 
events during the trial to determine whether they were result or consequence of participation in 
the trial. Unless modification or cessation of the protocol is recommended by the DSMB, the 
trial investigators will be unaware of the preliminary results described in these reports. Any 
provider or caregiver involved in the trial will be able to write to the DSMB to draw attention to 
any concern they may have about the possibility of harm arising from the intervention under 
investigation. The attending clinician may also withdraw the infant from the study if they 
consider this to be in the best interest of the infant’s health and well-being. 
 

12 
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