Preterm Formula or Donor Breast Milk to Make up Any Shortfall in Mother's Own Milk
Phase: NA
Conditions: Adiposity, Insulin Resistance, Metabolomic Profile, microRNA Profile
Sponsor: Imperial College London

PREMFOOD Protocol 04/03/14 
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Study Protocol 
 
 
Short title 
 
PREMFOOD: PREterM Formula Or Donor breast milk for preterm babies 
 
Long Title 
 
A pilot randomised controlled trial of fortified Human Donor Milk, unfortified Human Donor 
Milk or Preterm Formula in preterm babies, to make up any shortfall in the volume of 
Mother’s Own Milk.  
 
 
 
 
 
 
Clinical Trials.gov NCT: 01686477 
 
Research Ethics Approval No: 12/LO/1391 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PREMFOOD Protocol 04/03/14 
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Contents 
 
1. Protocol summary 
 
2. Background 
2.1 
Summary 
2.2 
The knowledge gap 
2.2.1 
Human milk versus preterm formula: NEC 
2.2.2 
Human milk versus preterm formula: metabolic consequences 
2.2.3 
MicroRNA profiling and metabolic disease 
2.2.4 
Human 
milk 
versus 
preterm 
formula: 
neurodevelopmental 
consequences 
2.2.5 
Use of bovine fortifier for human milk  
 
 
2.3 
Bridging the gap 
 
2.4 
Importance to the NHS 
 
3. Aims and Hypothesis  
 
4. Methods 
 
4.1 
Design 
 
4.2 
Study Flowchart 
 
4.3 
Patient population 
 
   4.3.1 
Inclusion Criteria 
 
   4.3.2 
Exclusion Criteria 
 
4.4 
Screening and Enrolment 
 
4.5 
Explanation and informed consent for the study 
 
4.6 
Randomisation 
 
4.7 
Patient withdrawal 
 
    4.7.1 
Temporary discontinuation of feeds 
 
    4.7.2 
Withdrawal of infant from trial procedures and incomplete follow up 
      4.8. 
Trial interventions 
 
    4.8.1 
Description 
 
    4.8.2 
Feeding schedule 
 
    4.8.3 
Pasteurised Human Donor Milk 
 
    4.8.4 
Fortified Human Milk 
 
    4.8.5 
Unfortified Human Milk 
 
    4.8.6 
Preterm formula 
 
    4.8.7 
Assumed intakes 
 
4.9 
Trial observations, tests, and investigations 
 
   4.9.1 
Electronic data capture 
 
   4.9.2 
Case report form 
        4.9.3 
Randomisation 
 
   4.9.4 
Baseline evaluation 
 
   4.9.5 
Daily evaluation 
        4.9.6 
Weekly evaluation 
        4.9.7 
Pre-discharge evaluation 
 
   4.9.8 
Magnetic Resonance Investigations (MRI and MRS) 
    
   4.9.9 
Scan visits 
 
   4.9.10 
Feeding data 
 
   4.9.11 
Schedule of investigations 
 
 
 
4.10 
Assessment of feasibility 
 
   4.10.1 
Trial design peer review 
 
   4.10.2 
Trial acceptability to parents 
 
   4.10.3 
Trial design feedback from other sources 
 
   4.10.4 
Recruitment to target 
 
   4.10.5 
Trial requirement for HDM from NHS Human Milk Banks 
 
   4.10.6 
Electronic outcome data capture 

PREMFOOD Protocol 04/03/14 
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4.11 
Statistical considerations 
 
   4.11.1 
Sample size 
 
   4.11.2 
Statistical analysis 
 
4.12 
Timeline 
 
5. Regulatory and Ethical considerations 
 
 5.1 
Regulatory framework 
 
 5.2 
Research ethics approval and monitoring 
 
 5.3 
Adverse events 
 
 5.4 
Indemnity 
 
 5.5 
Sponsor 
 
 5.6 
Funding 
 
 5.7 
Audit 
 
 5.8 
Study Management 
 
 5.9 
Publication policy 
 
 5.4 
End of trial 
 
6. References 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PREMFOOD Protocol 04/03/14 
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1.Protocol summary 
 
 
Title 
A pilot randomised controlled trial of fortified Human Donor Milk 
(HDM), unfortified HDM, or Preterm Formula (PTF) in preterm 
babies, to make up any shortfall in the volume of Mother’s Own Milk 
(MOM) 
Study Centre 
Chelsea and Westminster Hospital Neonatal Unit 
Study 
Objectives 
1. To assess the feasibility of a multicentre trial addressing short 
term (Necrotising Enterocolitis and Bloodstream Infection) 
and long term (growth and metabolic) outcomes 
2. To address the null hypothesis: there are no differences in 
total body adiposity, as measured by whole body MRI, 
between any groups of infants randomised to receive 
unfortified MOM+/-HDM, fortified MOM+/-HDM or MOM+/-
PTF 
3. To assess the macronutrient content of HDM given to each 
infant in the HDM arms by analysis of HDM samples 
Study Design 
Single centre unblinded randomised controlled trial 
Study 
Population 
Inclusion Criteria 
• 
Preterm infants born between 25+0 to 31+6 weeks 
gestational age 
 
Exclusion Criteria 
• 
Major congenital or life threatening abnormalities or 
congenital abnormalities that preclude early milk feeding 
• 
Inability to randomise infant within 48 hours 
Intervention 
Either  
1. HDM, fortified with standard commercial multicomponent 
breast milk fortifier, once enteral intake of 150ml/kg/d 
reached, to supplement MOM 
Or 
2. Unfortified HDM to supplement MOM 
Comparator 
PTF to supplement MOM 
Target number 
of patients 
84 (28 in each group) 
Randomisation 
• 
Using minimisation 
• 
Stratifying factors are: gestational age (25-27+6 w and 28-
31+6 w), birth weight z score (<-1.28, >-1.28) and sex 
Primary 
outcomes 
• 
Total body adiposity, as measured by whole body MRI, at 
term equivalent age 
Secondary 
outcomes 
• 
Total body adiposity, as measured by whole body MRI at term 
plus 6 weeks corrected gestational age 
• 
Regional adiposity, as measured by whole body MRI 
• 
Non adipose tissue, as measured by whole body MRI 
• 
Anthropometry (weight, length, and head circumference) 
• 
Intrahepatocellular 
Lipid, 
as 
measured 
by 
magnetic 
resonance spectroscopy 
• 
Blood pressure 
• 
Stool and urine metabolomic profile 
• 
MicroRNA profile 
• 
Fasting blood Quantitative Insulin Sensitivity Check Index at 
term equivalent age 
Duration 
of 
study 
Recruitment: 18 months 
End of trial: Up to 24 months 
 

PREMFOOD Protocol 04/03/14 
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2. Background 
 
2.1 Summary 
 
Mother’s Own Milk (MOM) is believed to confer many benefits for preterm babies. However, 
on average, mothers giving birth preterm are able to provide less than half their baby’s 
milk requirements. In these circumstances standard clinical practice is to make up the 
shortfall in MOM with either pasteurised Human Donor Milk (HDM) or Preterm Formula 
(PTF). Pasteurisation reduces or destroys many biologically active components; human 
milk, unlike PTF, is very variable in composition and despite fortification, there are 
concerns that preterm macronutrient requirements are not met with HDM. Doctors who 
use HDM to supplement insufficient MOM, do so primarily in the hope that despite 
pasteurisation of HDM, this strategy will reduce two feared conditions, bloodstream 
infection and necrotising enterocolitis, a serious inflammatory disease involving bacterial 
invasion of the bowel. The mortality from these conditions is high and survivors are at 
substantially increased risk of neurodisability. Metabolic advantages of human milk for 
preterm infants may also exist. Follow-up of preterm babies recruited to landmark 
nutritional trials in the early 1980s suggest positive effects of human milk on insulin 
sensitivity, blood pressure, and other metabolic outcomes. Doctors who prefer PTF to 
supplement insufficient MOM, believe this will benefit growth, including brain growth, and 
long-term neurodevelopment. Which of these two approaches is more efficacious and 
effective is unknown.  
 
In order to address this crucial question, central to preterm newborn care, a multicentre 
UK-wide study randomising 4000 preterm babies would be necessary to achieve sufficient 
power to evaluate the impact on the short-term outcomes necrotising enterocolitis and 
bloodstream infection, and establish cohorts with power to address long-term growth and 
metabolic outcomes.  
 
The objective of this PhD proposal is to conduct a pilot study to inform an application to 
the NIHR or other major funding body to conduct a randomised controlled trial designed 
to address cardinal short-term (necrotising enterocolitis, bloodstream infection) and 
longer-term outcomes (growth, obesity, insulin resistance), that would require the 
recruitment of approximately 4000 babies across sites throughout the UK. This pilot trial 
will not involve any novel intervention, it will merely randomise babies to three existing, 
widely used, standard feeding practices. 
 
 
2.2 The knowledge gap 
 
 
2.2.1 Human milk versus preterm formula: Necrotising Enterocolitis 
 
Human milk not only provides nutrition, but is rich in biologically active molecules with 
advantages to infant host defence.  Large nutritional trials of MOM versus PTF in the late 
1980’s showed that infants who received MOM were 5-10 times less likely to develop 
necrotising enterocolitis (NEC) than infants fed with PTF, where both MOM and PTF were 
sole diet (1). A meta-analysis of seven trials in the 1970’s and 1980’s each comparing 
HDM with PTF for preterm infants (2), suggested that HDM given as a sole diet is 
associated with a lower risk of NEC compared with formula. However, in these trials, the 
total sample size was small, the methodological quality poor, and their nutritional 
protocols did not evaluate the currently widely used bovine human milk fortifiers, or 
contemporary preterm formulas. A more recent Cochrane meta-analysis has failed to 
show any such protection against NEC with HDM compared to PTF when used to 
supplement MOM (3). Furthermore, only one trial has ever compared bovine fortified 

PREMFOOD Protocol 04/03/14 
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HDM with PTF, in which no significant differences in mortality or NEC was found (4). 
These uncertainties are reflected in the varying attitudes of staff, parents and countries 
to HDM (5). The lack of good evidence of efficacy of HDM led the National Institute for 
Clinical Excellence to refrain from advising on clinical indications in their guidance “Donor 
breast milk banks: the operation of donor milk bank services” (NICE 2010).  
 
 
2.2.2  Human milk versus preterm formula: metabolic consequences 
 
The association between low birth weight and later adult metabolic disease such as type- 
2 diabetes is now well established (6,7). Epidemiological studies show an association 
between low birth weight and decreased lean mass, increased total adiposity, and 
increased central adiposity in adults (8) ,a body composition which has consistently been 
linked with later insulin resistance (9).  Third trimester and early postnatal growth 
appears to have a major impact on this later adult metabolic phenotype.  Longitudinal 
growth data indicate that those born small for gestational age but then show catch up 
growth, specifically from birth to 3 months, are most at risk of later insulin resistance in 
young adulthood (10) 
 
Prematurity, regardless of fetal growth, has been associated with later insulin resistance 
in adults (11). Using whole body MRI, our group has shown that the preterm born adult 
phenotype is characterised by increased total and abdominal adiposity compared to term 
born controls (12). As early as at term equivalent age we have shown that there is 
aberrant deposition of adipose tissue, with an excess of intra-abdominal adipose tissue, 
and reduced lean body mass in preterm infants in comparison to their term-born 
counterparts, with illness severity being the major determinant of adiposity (13).  
Although the neonatal morbidities associated with preterm birth may well contribute to 
long term metabolic health, mounting evidence highlights the impact of early growth 
velocity during postnatal third trimester growth. 
 
Follow-up to early adulthood of preterm babies recruited to landmark nutritional trials in 
the early 1980s by Lucas and colleagues showed lower insulin resistance at adolescence 
in preterm infants randomised to receive standard formula compared to a growth 
promoting enriched formula (14) , those infants with the greatest growth velocity in the 
first 2 weeks of life having the greatest insulin resistance at adolescence.  Consistent 
with these findings, Roteveel et al have recently shown that preterm infants who had 
highest early postnatal growth velocities had highest insulin resistance as young adults 
regardless of prior fetal growth (15). 
 
In parallel trials Lucas and colleagues showed positive effects of human milk on insulin 
sensitivity, blood pressure, and other metabolic outcomes (16-19). Whether the 
observed metabolic advantages with human milk are due to its unique bioactive 
properties, or result from a protective slower early growth as compared to preterm 
formula, remain unclear (20).  This pilot trial will begin to address this question by 
comparing fortified and unfortified HDM with PTF. 
 
Increased adiposity could be a plausible mechanism by which manipulation of early 
postnatal nutrition may predispose to poor metabolic health in preterm born adults.  
Nutritional differences are key causal candidates for poor metabolic health: the diet of 
the preterm neonates in intensive care is high in fat and carbohydrate and low in 
protein. This is in marked contrast to third trimester intrauterine diet which is high in 
amino acids, moderate in glucose and low in lipid (21).  But within this preterm diet, 
human milk may attenuate this adiposity: an observational study of an indirect 
measurement of body composition using dual energy x-ray absorptiometry (22) has 
shown increased fat mass in preterm babies at discharge when PTF was used compared 
to fortified HDM, in supplementing MOM. Using dual energy x-ray absorptiometry in a 
cohort of preterm infants recruited just before discharge, Cooke et al (23) showed that 

PREMFOOD Protocol 04/03/14 
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infants randomised to receive PTF or term formula had increased total non-fat and fat 
mass but not percentage fat mass, at 3,6, and 12 months corrected age, compared to 
breast fed preterm controls. In contrast to the pattern of adiposity that we observed, 
changes in fat mass were explained by increased fat accretion in the legs rather than 
centrally. However, no studies to date have examined the impact of feeding preterm 
infants from birth, on changes in adiposity quantity and distribution, at discharge and 
post-discharge. 
 
Emergence of insulin resistance has been shown to parallel that of central adiposity at 
age 2-4yrs in SGA infants who show catch up growth (24). Insulin resistance in this 
growth pattern correlates with growth velocity immediately after birth (10) and has been 
shown as early as 1yr of age, in a cohort of SGA infants who gained one birth centile in 
weight in the first year (25). The “gold standard” for measuring insulin resistance is the 
euglycaemic hyperinsulinaemic insulin-glucose clamp technique which cannot be used 
during infancy, but a surrogate marker of insulin resistance, a  logarithmic 
transformation of the fasting insulin and glucose concentration (Quantitative Insulin-
sensitivity Check Index, QUICKI (26) has been validated in children and adults, and has 
been measured in preterm infants shortly after birth in a handful of studies (27,28).  Yet 
as far as we know there are no prospective studies assessing direct measurements of 
adipose tissue distribution with growth and markers of insulin resistance in the first few 
weeks and months of life.  
 
Our group has also shown increased intrahepatocellular lipid (IHCL) content in preterm 
infants at term compared to healthy term infants (29). Raised IHCL is closely associated 
with abdominal adiposity (30), obesity, and dyslipidaemia. Recent work by Nobili et al 
(31), showed that breast feeding  had a protective effect, in a dose dependent manor, 
on progression of non-alcoholic fatty liver disease in a  sample of children diagnosed with 
the disease in a paediatric liver unit. Although there are several possible determinants of 
raised IHCL in the preterm infant, the impact of human milk warrants further 
investigation. 
 
Metabolic “profiles” can now be measured using small volumes of biological tissues and 
fluids, such as dried blood spots, urine and stool. Analysis is carried out using high 
throughput nuclear magnetic resonance (NMR) spectroscopy, which provides the 
opportunity to analyse a large number of metabolites simultaneously and therefore offers 
extensive scope to study metabolic responses in relation to normal development, disease, 
physiological variation, nutritional intake and medications (32). A small number of reports 
describe metabolic profiling in newborns and demonstrate the feasibility of this technique 
in detecting differences in metabolic responses to preterm birth (33). 
 
Yet there remains no randomised controlled enteral nutritional trial in preterm infants 
from birth to discharge and beyond with direct measures of body composition, QUICKI, 
IHCL or metabolomic profiling as the outcome. Furthermore, no long term follow up of 
such early preterm body composition, IHCL and metabolomic phenotypes exist, making 
any nutritional mechanistic pathways to poor adult metabolic health difficult to define. 
 
 
2.2.3 MicroRNA profiling and metabolic disease 
 
MicroRNAs (miRNAs) are small 22–25-nt-long noncoding RNA molecules that negatively 
regulate translation of target messenger RNAs. miRNAs normally bind to their target 
messenger RNA (mRNA) leading to translation inhibition and/or mRNA degradation (34). 
Over 500 miRNAs have been found in the human genome (35), and it has been 
estimated that they could regulate 74–92% of all protein coding mRNAs (36). By 
affecting gene regulation, miRNAs are likely to be involved in most biological processes, 
and aberrant expression of miRNAs has been implicated in numerous disease states 
including insulin resistance and type 2 diabetes. Several studies have identified 

PREMFOOD Protocol 04/03/14 
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dysregulation of several miRNAs in animal and human type 2 diabetes, in insulin 
sensitive tissues such as adipose tissue (37). The discovery of circulating miRNAs has 
highlighted their potential as both endocrine signalling molecules and disease markers 
(38). MicroRNA expression has been implicated in the complex interplay between genetic 
susceptibility to disease and environmental factors.  We aim to undertake the novel 
exploration into the impact of early nutrition on microRNA expression and correlate this 
with body composition, by global microRNA profiling at the time of discharge. 
 
 
2.2.4 Human milk versus preterm formula: neurodevelopmental consequences 
 
Accumulating evidence shows that poor early postnatal growth is associated with 
impaired neurodevelopment in preterm infants (39-42). Suboptimal nutritional intake 
and subsequent poor growth including brain growth has lasting consequences in this 
sensitive period. Follow-up studies showed that at 7 years of age, preterm infants 
randomised to receive standard formula from birth to discharge, demonstrated 
neurocognitive impairment with a significant reduction in IQ compared with infants 
randomised to receive enriched formula (39).  Despite slower growth in breast milk fed 
preterm infants, there is a growing body of evidence which suggests that neurocognitive 
outcome is better. Long-term studies at 8 years of age through adolescence suggest that 
intelligence test results and white matter and total brain volumes are greater in subjects 
who had received human milk as infants in the neonatal unit.(39,43). Extremely preterm 
infants receiving the greatest proportion of human milk in the neonatal unit had 
significantly greater scores for mental, motor, and behaviour scores at ages 18 months 
and 30 months. (44,45) These data remain significant after adjustment for confounding 
factors, such as maternal age, education, marital status, race, and infant morbidities. 
 
Evidence for neurodevelopmental advantage in the use of HDM used as sole diet or to 
supplement MOM, compared to PTF however, is less clear. In a meta-analysis of donor 
breast milk versus preterm formula for preterm infants, only one trial compared 
neurodevelopment as the outcome, and there were no significant differences in 
neurodevelopmental status at 9 or 18 months either used as sole diet or to supplement 
MOM (3). Indeed, the use of HDM, even with fortification, is consistently associated with 
slower growth compared to PTF (3, 4). The question of whether HDM has similar 
cognitive advantages as MOM, despite pasteurisation, and slower associated growth 
velocity compared to PTF, is worthy of further investigation. 
 
 
2.2.5 Use of bovine fortifier for human milk  
 
A Cochrane review (46) has shown that bovine multicomponent fortification of human 
milk in comparison to the unfortified human milk, improved short-term growth, but had 
no long-term advantage in terms of either growth or neurodevelopment, nor any clear 
effect on bone mineral content, and was not associated with clinically significant adverse 
effects.  Fortification of human milk for preterm infants is now widely practiced, yet there 
exists widespread variation in its indication, method of initiation and indication for 
stopping (5). This reflects the lack of good evidence for optimal use.   
 
Comparisons between fortified HM and PTF indicate that despite fortification, human milk 
fed preterm infants continue to grow slower than PTF fed infants (47, 48). Accumulating 
evidence suggests that a continuing protein deficit is primarily responsible for slow 
preterm growth (47,49) and there is growing concern that preterm protein requirements 
to maintain intrauterine growth rates and overcome catabolic deficits, is not being met 
(50). Recent ESPGHAN (2010) (51) guidelines have reflected this, recommending up to 
4.5g/kg/d of protein for ELBW infants. Although a sole diet of preterm formula could 
match this intake, the assessment of protein intake from human milk is inherently 
difficult. The protein concentration of human milk decreases with gestational age and 

PREMFOOD Protocol 04/03/14 
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duration of lactation but also varies unpredictably from sample to sample (52, 53).  
Furthermore, HDM is likely to have an even lower protein concentration than that from 
MOM as it is more likely to come from mothers of term infants. However, there is limited 
data on the macronutrient content of HDM, and we propose to analyse the macronutrient 
content of all HDM that each infant in the study receives, thus providing an accurate as 
possible nutritional intake for each interventional arm.  
 
 
 
2.3 Bridging the gap 
 
 
To begin to address these questions this feasibility pilot trial will assess growth and key 
candidate markers of long term metabolic health such as body composition, blood 
pressure, hepatic lipid content, QUICKI, metabolomic profile, in preterm infants 
randomised to receive PTF, fortified or unfortified HDM when there is a shortfall of MOM.  
An estimation of macronutrient content of HDM will be made by analysis of all HDM given 
to each infant in the HDM arm. To our best knowledge through wide professional and 
academic contacts, and searches of international registers, no trials similar to that which 
we propose are being undertaken in the UK or elsewhere.  
 
 
 
2.4 Importance to the NHS 
 
 
Infant nutrition studies involving randomisation, testing causality and unravelling 
biological mechanisms are rare. Yet infant nutrition is a cardinal candidate effector of long-
term health. Current practice in feeding the preterm infant invariably involves the use of 
fortified Human Donor Milk or Preterm Formula. There are however no national standards 
or guidelines for enteral feeding of extremely preterm infants, and clinical practice is very 
variable.   
 
Advocates of pasteurised HDM believe this is as efficacious as MOM in reducing two of the 
most feared conditions in newborn medicine, necrotising enterocolitis and bloodstream 
infection, and in improving long-term metabolic health. These result in major health 
services utilisation costs and a life-long burden of impairments on survivors.  
 
An adequately powered trial is required to address these outcomes but poses two major 
hurdles, recruitment of large numbers of babies, and long-term follow-up. The 
organisation of NHS neonatal specialist care and the establishment of the Neonatal Data 
Analysis Unit that holds and archives national neonatal data at Chelsea & Westminster 
Hospital, and has received permission for the National Information Governance Board to 
link this to Hospital Episode statistics data, offers unique international opportunity to 
conduct this trial in the UK and achieve long term follow-up at low cost.  Clarification of 
efficacy will advance clinical care, and enable economic evaluation and rational 
development of Human Milk Banking.  
 
 
 
 
 
 
 
 
 
 

PREMFOOD Protocol 04/03/14 
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3. Aims and Hypothesis 
 
Specific aims of this pilot trial are to:   
 
A:   
Assess feasibility 
1) test the acceptability of the trial design with parents and clinicians  
2) test that recruitment to target is achievable, estimate consent and dropout 
rates 
3) estimate the trial requirement for HDM from NHS Human Milk Banks 
4) test that clinical outcome data can be retrieved from operational NHS electronic 
records, thus minimising the burden of data capture and facilitating the use of 
linked NHS records to achieve long-term follow-up at low cost. 
 
B:  
Obtain data on the magnitude of effect at term equivalent age and again at term 
plus 6 weeks of age, on key mechanistic outcomes:  
 
1) the primary outcome of total body adiposity, addressing the null hypothesis: 
there are no differences in total body adiposity, as measured by whole body 
MRI, between infants randomised to receive fortified MOM+/-HDM, unfortified 
MOM+/-HDM, or MOM+/-PTF. 
2) the secondary outcomes of  
• 
Regional adiposity, as measured by whole body MRI 
• 
Non adipose tissue, as measured by whole body MRI 
• 
Total brain volume, as measured by MRI 
• 
Anthropometry (weight, length, and head circumference) 
• 
IHCL, as measured by magnetic resonance spectroscopy 
• 
blood pressure 
• 
Stool and urine metabolomic profile 
• 
Blood Quantitative Insulin Sensitivity Check Index (QUICKI) – at discharge 
only between infants randomised to receive fortified MOM+/-HDM, unfortified 
MOM+/- HDM or MOM+/-PTF. 
 
C:  
By using HDM with known macronutrient content, we aim to correlate 
macronutrient intake of each intervention with the above outcomes. 
 
 
4. Methods 
 
 
4.1 Design 
 
This will be a pilot single centre randomised controlled trial. There will be no novel 
interventions, merely randomisation of infants to three existing, widely used, standard 
feeding practices. For the purposes of clarity, this study will be in two parts. The first part 
will involve an opt out of feeding randomisation. Eligible preterm infants will be randomised 
by 48 hours to receive unfortified Mother’s Own Milk (MOM) +/- Human Donor Milk (HDM), 
fortified MOM +/- HDM, or MOM +/- Preterm Formula (PTF), where HDM or PTF are used 
to make up any shortfall in MOM. Anthropometric and blood pressure outcomes will be 
determined from routinely collected clinical data.  The second part will involve informed 
consent for collection and analysis of urine, stool, and blood samples and magnetic 
resonance imaging scans. The flowchart illustrates exactly what is involved if an infant is 
in the study. 
 
 
 
 

PREMFOOD Protocol 04/03/14 
11 
 
 
4.2 Study Flowchart 
 
Please see page 12 
 
 
4.3 Patient Population:  
 
Preterm infants born between 25+0 and 31+6 weeks gestational age. 
 
4.3.1 Inclusion Criteria 
• 
Preterm infants whose mothers are resident within the north west neonatal 
network,  born between 25+0 to 31+6 weeks gestational age 
 
4.3.2 Exclusion Criteria 
• 
Major congenital or life threatening abnormalities or congenital abnormalities 
that preclude early milk feeding 
• 
Inability to randomise infant within 48 hours 
• 
Infants less than 25+0 weeks or more than 31+6 weeks 
 
 
4.4 Screening and enrolment 
 
For part one of the study, we plan to randomise all eligible infants, unless explicitly 
requested by the parents for their baby not to take part.  Where possible parents will be 
approached before delivery, and the study explained to them eg for mothers at risk of 
premature delivery.  In all cases, the study will be explained to the parents by one of the 
clinical team or study investigators shortly after birth, and a written parent information 
leaflet (PIL pt 1) will be given to the parents. This process will be documented in the 
infant’s medical notes by the person explaining the study, with a confirmation sticker. 
Randomisation to one of the three feeding arms will take place between 24-48hrs of age.  
 
For part two of the study, parents will be approached by one of the clinical team or the 
study investigators after 48hrs of age, and part two of the study will be explained to them, 
with a written parent information leaflet (PIL pt 2) given to them.  At a convenient time 
for the parents, informed consent will then be sought for part 2 of the study. All infants 
will be recruited from the Chelsea and Westminster Hospital Neonatal Unit. 
 
 
4.5 Explanation and informed consent for the study 
 
Explanation and informed consent requires individual discussion with the infant’s parents 
about the nature of the research to be conducted in a language that is easy to comprehend. 
The parents will have the study verbally explained to them and also be given a written 
information sheet about the study.  The parents will have sufficient time to think about 
the study and ask any further questions regarding it.  It will be stressed to parents that 
their babies will follow one of three, widely used, standard feeding practices, and there 
will be no new treatments or interventions. The parents will also understand that refusal 
to participate in the study will not affect the quality of subsequent medical care and if they 
do consent to participate they may withdraw at any point without affecting their infant’s 
care. 
 
Before any trial-related procedures may be performed, informed consent must be obtained 
from the infant’s parents by the investigator by means of a signed declaration.  The 
investigator must record in the case report form to confirm that informed consent was 
obtained and store the original of the signed declaration of consent in the patient’s notes. 
A copy will be given to the infant’s parents and a copy filed in the investigator file.  

PREMFOOD Protocol 04/03/14 
12 
 
 
 
 
 
 
 
 
 
 
 
 
Written (PIL) and verbal information about the study given to  
 
 
 
 
 
 
 
 
 
 
 
parents shortly after birth, and where possible, before birth:                                      
Birth  
 
 
 
 
 
 
 
 
 
Informed that randomisation to feeding practice will take place  
 
 
 
 
 
 
 
 
 
 
 
at 24-48h unless parents opt out of study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
48h 
 
 
 
 
 
 
 
 
 
 
 
 
 
Written informed consent at a 
convenient time for collection and 
analysis of urine, stool, and blood 
samples, and MRI scans. Weight, 
length,  head circumference, blood 
pressure as part of standard care
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
25+0-31+6 
Randomisation of 
feeding  practice 
Opt out to randomisation of 
feeding  practice 
Fortified MOM+/-HDM 
Unfortified MOM+/-HDM 
MOM+/-PTF 
Feeding at the 
discretion of 
the attending 
clinician, not 
part of study 
 
 
Feeding according to maternal/clinician plan 
Discharge; MRI and MRS 
scan, stool and urine samples 
if consented for scans 
Discharge, exit study 
if not consented for 
Second MRI and MRS 
scan; stool and urine 
4.1 PREMFOOD Flowchart  
Baseline stool and urine samples if consented 
Discharge stool, urine, and blood samples if consented 
Pre-discharge urine, stool, blood 
samples collected if consented. Given 
feeding diary at discharge if consented 
for scans. Weight, length, head 
circumference, and urine and stool 
collected at both scan visits. Note baby 
may still be inpatient at first (and 
second) scan visit. 
Term/near term corrected 
35 weeks 
Term plus 6 weeks corrected 

PREMFOOD Protocol 04/03/14 
13 
 
 
 
4.5 Explanation and informed consent for the study 
 
Explanation and informed consent requires individual discussion with the infant’s parents 
about the nature of the research to be conducted in a language that is easy to comprehend. 
The parents will have the study verbally explained to them and also be given a written 
information sheet about the study.  The parents will have sufficient time to think about 
the study and ask any further questions regarding it.  It will be stressed to parents that 
their babies will follow one of three, widely used, standard feeding practices, and there 
will be no new treatments or interventions. The parents will also understand that refusal 
to participate in the study will not affect the quality of subsequent medical care and if they 
do consent to participate they may withdraw at any point without affecting their infant’s 
care. 
 
Before any trial-related procedures may be performed, informed consent must be obtained 
from the infant’s parents by the investigator by means of a signed declaration.  The 
investigator must record in the case report form to confirm that informed consent was 
obtained and store the original of the signed declaration of consent in the patient’s notes. 
A copy will be given to the infant’s parents and a copy filed in the investigator file.  
 
 
 
4.6 Randomisation 
 
Randomisation will be generated centrally using a computer program, with minimisation 
by infant sex, gestational age (25-27+6 wks, 28-31+6 wks) birth-weight Z score (<-1.28, 
>-1.28) Randomisation will take place between 24-48hrs of age. Infants will be 
randomised to receive either HDM or PTF to make up any shortfall in Mother’s Own Milk 
(MOM) until 34+6 weeks postmenstrual age. Blinding of the intervention is not practicable 
because of the distinctive appearances of human milk and formula, and the method of 
administration to preterm babies by syringe and nasogastric tube. At 35 weeks, the infant 
will be close to discharge, and feeding will be directly from the breast or formula by bottle 
in accordance with maternal choice.  
 
 
 
4.7 Withdrawals and protocol violations 
 
4.7.1 Temporary discontinuation of feeds/alternative feeds 
 
Infants in the trial may develop feed intolerance or suspected/confirmed necrotising 
enterocolitis (NEC) requiring the cessation of enteral feeds. The period of no feeding may 
vary from a few hours to a number of days.  Once feeding is re-established, the infant will 
continue with the allocated interventional arm. However, there may be circumstances in 
which continuation of the allocated feed is deemed inappropriate by the clinical team. 
Examples would include feed intolerance and the use of hydrolysed or elemental feeds. 
Infants would remain in the study and included in the “intention-to-treat analysis.” 
 
 
 
 
 
 
 
 
4.7.2 Withdrawal of infant from trial procedures and incomplete follow-up 

PREMFOOD Protocol 04/03/14 
14 
 
 
It is possible that parents may choose to withdraw their infant from trial procedures 
resulting in incomplete patient follow up and failure to capture outcome data.  Likewise, if 
an infant dies before discharge or is still on the neonatal unit and considered too unstable 
at term equivalent age or term plus 6 weeks, they will not have their MRI scan. 
 
 
 
4.8 Trial intervention 
 
4.8.1 Description 
 
The interventions will be fortified, or unfortified pasteurised Human Donor Milk (HDM) with 
the comparator Preterm Formula (PTF) to make up any shortfall in Mother’s Own Milk 
(MOM). 
 
4.8.2 Feeding schedule 
 
The trial is designed to reflect current clinical practice. Preterm infants will be introduced 
to parenteral nutrition and enteral feeds within the first 24 hours of birth.  Milk volumes 
will be increased up to 200ml/kg/day in accordance with a standard protocol modified if 
necessary in accordance with infant tolerance; parenteral intake simultaneously 
decreased.  For infants randomised to fortified MOM± HDM, fortification will begin when a 
total enteral intake of 100ml/kg/day is reached and continued to 34+6 weeks 
postmenstrual age.  At this point feeding will progress in accordance with maternal choice. 
 
 
4.8.3 Pasteurised Human Donor Milk 
 
The Chelsea and Westminster Hospital neonatal unit obtains all HDM from the Countess of 
Chester Human Milk Bank. The milk bank follows the recommendations for donor 
screening, pasteurisation, milk collection and storage published by NICE 2010 (54).  All 
HDM supplied has been analysed for macronutrient content using infrared spectrometry 
done at the Countess of Chester Human Milk Bank. All bottles will be labelled with their 
macronutrient content and daily intakes recorded on the clinical case report form. If an 
infant is allocated to the HDM intervention, then the instruction only to use HDM to 
supplement MOM will be made on the infants feeding plan. 
 
 
4.8.4 Fortified Human Milk 
 
For infants randomised to receive fortified HDM to supplement MOM, all milk will be 
fortified once a total enteral intake of 150ml/kg/d is reached.  This will be with a 
multicomponent bovine breast milk fortifier (Cow and Gate Nutriprem Breast Milk 
Fortifier).  The amount used will be in accordance with current practice, namely two 
sachets per 100ml. 
 
 
4.8.5 Unfortified Human Milk 
 
For infants randomised to receive unfortified human milk the instruction to only use 
unfortified HDM to supplement unfortified MOM will be made on the infants feeding plan.  
For these infants, the situation may arise where the growth of the infants is deemed 
suboptimal by the clinical team. Therefore, weight gain will be assessed two weeks after 
a milk volume of 120ml/kg/d has been reached. At this point, if the infant has crossed 
down 3 centile bandwidths from birthweight centile bandwidth, or shows continued 
downward crossing if less than the 0.4th centile, using birthweight centile charts, then the 

PREMFOOD Protocol 04/03/14 
15 
 
infant will discontinue the feeding intervention. There may be other causes of poor weight 
gain than inadequate enteral nutrition. These include for example, respiratory distress 
syndrome, infection, chronic lung disease, gastro-oesophageal reflux disease, anaemia, 
patent ductus arteriosus, or underlying congenital anomaly. Once these causes have been 
excluded or corrected, and the weight loss has been confirmed from at least two weights 
over a 72 hour period, fortification will be commenced.  Infants will then receive fortified 
HDM, and undergo all outcome measures. Analysis will be as per protocol, and also for 
categories of overall intake (see statistical analysis). 
 
This three centile drop cut off value is based on results of a longitudinal preterm growth 
reference by Cole et al (submitted). This is a study of routinely collected growth data (co-
ordinated by the Neonatal Data Analysis Unit) from 113 neonatal units across the UK from 
2006-2011. A growth reference for weight, length, and head circumference in preterm 
infants less than 32 weeks from birth to term/near term was constructed, stratified for 
birth gestation week, and compared to currently used birthweight centile charts. In this 
comparison, average growth curves for all infants by week of birth gestation, crossed down 
2-3 birthweight centile bandwidths over a period of 3-4 weeks from birth, before becoming 
parallel to the birthweight centile bandwidth curves.  Time taken to reach full milk feeds 
will vary depending on feeding tolerance, but typically will take from a few days to a couple 
of weeks. A period of two weeks after full milk feeding (120ml/kg/d and above) has been 
reached, is therefore expected to coincide with no further crossing down in birthweight 
centile bandwidth curves.  
 
 
4.8.6  Preterm formula 
 
For infants randomised to receive PTF, Nutriprem 1 will be used (Cow and Gate Nutriprem 
1) as this is in standard use in the Chelsea and Westminster neonatal unit. If an infant is 
allocated to the PTF intervention, then the instruction only to use PTF to supplement MOM 
will be made on the infants feeding plan. 
 
 
4.8.7 Assumed intakes 
 
For comparison purposes the following table illustrates the macronutrient daily intakes for 
a 1kg baby on 180ml/kg/d for different milks, based on mean macronutrient content from 
a large number of samples (415 sequential samples from 273 donors) of banked donor 
milk (55) and the widely accepted values taken from the mean of a number of studies of 
milk from mothers of preterm infants (56). This is compared with the ESPGHAN 2010 
recommended intakes (51). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 1. Assumed intakes compared with recommendations 

PREMFOOD Protocol 04/03/14 
16 
 
 
180ml 
of 
unfortified 
Mother’s 
Own 
Milk 
(MOM) 
180ml 
of 
unfortified 
Donor 
milk  
180ml of 
fortified 
MOM 
180ml 
of 
fortified 
Donor 
milk 
180ml 
of 
Nutriprem 
1 
ESPGHAN 2010 
Recommendation 
unit/kg/d 
Protein (g) 
2.7 
2.2 
4.7 
4.4 
4.7 
4.0-4.5 * 
3.5-4.0** 
Carbohydrate (g) 
12.4 
14.0 
17.3 
18.9 
15.1 
11.6-13.2 
Fat (g) 
6.3 
5.8 
6.3 
5.8 
7.0 
4.8-6.6 
Energy (kcal) 
117 
117 
144 
144 
144 
110-135 
Protein/Energy 
ratio (g/100kcal) 
2.3 
1.9 
3.3 
3.1 
3.3 
3.6-4.1* 
3.2-3.6** 
 
*For infants <1kg 
** For infants 1-1.8kg 
  
 
4.9 Trial observations, tests and investigations 
 
 
4.9.1 Electronic data capture 
 
The intention is to minimise the burden of data collection by utilising the National Neonatal 
Database; mortality, necrotising enterocolitis, blood-stream infection, growth, breast 
feeding at discharge and health resource utilisation (length of stay; intensive, high 
dependency and special care days, parenteral nutrition use) will be evaluated to discharge 
in all randomised babies; these data will be retrieved from the National Neonatal Research 
Database; this is created at the Neonatal Data Analysis Unit from electronic records 
captured at the point of care on all admissions to neonatal units nationally; the Neonatal 
Data Analysis Unit is based at Chelsea & Westminster Hospital and led by Professor Modi. 
Data will be retrieved electronically from the Neonatal Data Analysis Unit except that 
stated otherwise below. 
 
 
4.9.2 Case report form 
 
Randomisation data and certain clinical data will be collected on a case report form which 
will be stored securely at the neonatal data analysis unit.  Only the research team will 
have access to the case report form. 
 
 
4.9.3 Randomisation 
 
• 
Study number 
• 
Date and time of birth 
• 
Gender 
• 
Confirmation of eligibility (full eligibility check) 
• 
Date of parental consent 
• 
Name of person taking consent 
• 
Gestational age (in weeks and days) 
• 
Recruiting hospital 
• 
Allocated trial intervention (HDM or PTF) 
 
 
 
 
4.9.4 Baseline evaluation 

PREMFOOD Protocol 04/03/14 
17 
 
 
• 
Birth weight (kg) 
• 
Birth length (cm) 
• 
Head circumference (cm) 
• 
Ethnicity (NHS ethnicity) 
• 
Mother’s date of birth 
• 
Mother’s ethnicity 
• 
Mother’s height and weight (collected on case report form) 
• 
Father’s ethnicity 
• 
Father’s height and weight (collected on case report form) 
• 
Mode of delivery 
• 
Use of antenatal steroids 
• 
Diastolic blood pressure 
• 
Systolic blood pressure 
• 
Medical conditions and perinatal medications 
• 
Metabolomic profile (urine and stool) after informed consent 
 
 
 
4.9.5 Daily evaluations 
 
Performed daily until discharge from NICU 
• 
Record nutritional intake (parenteral nutrition, daily volume of MOM, daily volume 
of trial feed – all collected on case report form) 
• 
Record level of care (BAPM 2001 criteria) 
• 
Record if infant is nil by mouth and reason for this (collected on case report form) 
• 
Record of withdrawal information (collected on case report form) 
• 
Weight (when on parenteral nutrition) 
 
 
 
4.9.6 Weekly evaluations 
 
Performed weekly until discharge form NICU 
• 
Weight (when not on PN) 
• 
Length 
• 
Head circumference 
• 
Diastolic blood pressure 
• 
Systolic blood pressure 
• 
Record any episode of suspected or confirmed NEC 
• 
Record any episode of blood stream infection (blood culture positive) 
 
 
4.9.7 Pre-discharge evaluations 
 
• 
Metabolomic profile (urine and stool) 
Performed with routine weekly bloods as close as possible to discharge and when on 3hrly 
feeds: 
• 
Blood fasting (pre feeding) insulin and glucose (QUICKI), each 0.5ml volume. 
• 
Blood for microRNA profiling 0.5ml in volume. 
 
 
4.9.8 Magnetic Resonance Investigations (MRI and MRS) 
 
Infants will undergo two magnetic resonance investigations, according to a well-defined 
and well established protocol (57-59).  The first investigation will be as soon as possible 
after discharge. To investigate whether early feeding impacts on body composition 

PREMFOOD Protocol 04/03/14 
18 
 
changes which persist beyond discharge, a second scan at around a corrected gestational 
age of term plus 6 weeks will be performed. This protocol has been used for over a decade 
in a large number of preterm and term infants.  The MRI measurements are carried out 
during normal sleep without the need for sedation. All the MRI measurements (brain 
volume, body composition, and hepatic MRS) take a total of about 20-25 minutes.  Both 
quantity and distribution of adipose and non-adipose tissue will be assessed. The infants 
will be monitored with pulse oximetry and a trained neonatal doctor will be present 
throughout the scan.  Parents are invited to be present in the console room. 
 
 
4.9.9 Scan visits 
 
Data will be collected at each scan visit on the following: 
• 
Weight 
• 
Length 
• 
Head circumference 
• 
Feeding data (see below) 
• 
Urine and stool for metabolomic profile 
 
Urine samples (1 ml, from cotton wool placed in the nappy), and stool specimens (from 
the nappy) will be collected immediately after recruitment and at both scan appointments. 
All samples will be analysed at Imperial College using high throughput techniques 
employing in vitro NMR. This aspect of the proposed research will be supervised by 
Professor Elaine Holmes, a leading international authority in the field. 
 
 
 
4.9.10 Feeding data 
 
At each scan visit, data will be collected on breast/expressed breast milk and formula 
feeding from end of feeding intervention to time of scan. Breast feeding will be defined 
according to the criteria recommended by Labbock et al (60) and subsequently adopted 
by the World Health Organisation.  Feeding will be defined as: 
1. Full breast/expressed breast milk feeding (including exclusive and almost 
exclusive) 
2. Partial - High breast/expressed breast milk feeding (>80%) 
3. Partial - Medium breast/expressed breast milk feeding (20-80%) 
4. Partial - Low breast/expressed breast feeding (<20%) 
5. Full formula feeding (including token breast/expressed breast milk feeding) 
To avoid recall bias, parents will be given a simple feed diary to complete during the study 
period. Furthermore date of first formula feed, when first predominantly formula fed and 
when exclusively formula fed will be recorded.  Parents will be asked about method of 
feeding at both scan points and at all contact points throughout the study period (eg 
telephone contact to arrange appointment for MR studies).   
 
 
 
 
 
 
 
 
 
4.9.11 Schedule of investigations 

PREMFOOD Protocol 04/03/14 
19 
 
 
Table 2. Schedule of investigations 
Evaluation 
Baseline 
Daily 
Weekly 
Pre-
discharge 
Scan 1 
Scan 2 
Informed 
Consent 
+ 
 
 
 
 
 
Eligibility 
+ 
 
 
 
 
 
Randomisation + 
 
 
 
 
 
Weight 
# 
#* 
#** 
 
+ 
+ 
Length 
# 
 
# 
 
+ 
+ 
Head 
circumference 
# 
 
# 
 
+ 
+ 
Blood pressure # 
 
# 
 
 
 
Nutritional 
intake/feeding 
data 
+ 
+ 
+ 
 
+ 
+ 
Efficacy 
 
 
 
 
 
 
Whole 
body 
MRI, MRS 
 
 
 
 
+ 
+ 
Metabolomic 
Profile 
 
 
 
 
 
 
Urine 
and 
stool sample 
+ 
 
 
+ 
+ 
+ 
QUICKI 
 
 
 
 
 
 
Fasting blood 
insulin 
and 
glucose 
 
 
 
+ 
 
 
microRNA 
 
 
 
 
 
 
Blood 
 
 
 
+ 
 
 
Key 
+Research evaluation #Routine care evaluation *When on parenteral nutrition 
**When not on parenteral nutrition 
 
 
 
 
4.10 Assessment of feasibility 
 
 
4.10.1 Trial design peer review 
 
The trial protocol has had extensive peer review from the Neonatal Clinical Studies Group 
at the National Institute of Health Research Medicines for Children Research Network, on 
two separate occasions. This process has involved trial redesign and further review, 
resulting in favourable support for this feasibility trial. 
 
 
4.10.2 Trial acceptability to parents 
 
Trial design has also been reviewed at the parent research forum in the ‘Medicines for 
neonates’ programme – a National Institute of Health Research funded Research 
Programme, led by Professor Neena Modi. This programme includes several interrelated 
projects, one of which is ‘Data sharing in Neonatal Services’: understanding parents’ 
attitudes towards the use of NHS data for research purposes in the context of neonatal 
services. The parent research forum involved parents who have previously had preterm 

PREMFOOD Protocol 04/03/14 
20 
 
babies in the neonatal unit, and their feedback has proved invaluable in the redesign of 
the protocol, parent information leaflet, and consent form.  
 
 
4.10.3 Trial design feedback from other sources 
 
Trial design has also been reviewed by experts in the field by the neonatal nutrition club, 
and peer review from grant, and training fellowship applications. Human donor milk 
banks and the charity BLISS (Breastfeeding and Lactation Information Support Source) 
have also been involved in the trial design. 
 
 
4.10.4 Recruitment to target  
 
This feasibility study will test if recruitment to target is achievable; a conservative estimate 
is that 50% of eligible preterm infants will be recruited; the intention is to demonstrate 
that 2-4 eligible babies per month will be recruited (this would enable recruitment of 4000 
babies from 40 centres over 25-50 months). 
 
 
4.10.5 Trial requirement for HDM from NHS Human Milk Banks  
 
There are 17 human milk banks in the UK. A recent survey of human milk bank service 
provision in the UK (61) showed that the annual median number of donor mothers to the 
milk bank was 48, and the annual median number of litres of donor milk pasteurised by 
each milk bank was 206. It is anticipated that the provision of HDM from the Countess of 
Chester Human milk bank will match the demand for the purposes of this pilot trial. This 
Human milk bank currently provides all HDM for Chelsea and Westminster Hospital 
Neonatal Unit.  This study will provide an estimate of trial annual requirement of HDM from 
NHS human milk banks from data collected on HDM volume requirements of each infant 
in the HDM feeding arm. This estimate could then be compared to the current service 
provision of UK human milk banks. 
 
 
4.10.6 Electronic outcome data capture 
 
Test that outcome data can be retrieved from operational NHS electronic records, thus 
minimising the burden of data capture and facilitating the use of linked NHS records to 
achieve long-term follow-up at low cost; data will be captured as detailed above in all 
randomised babies to discharge. The National Neonatal Research Database is created at 
the Neonatal Data Analysis Unit from electronic records captured at the point of care on 
all admissions to neonatal units nationally; the Neonatal Data Analysis Unit is based at 
Chelsea & Westminster Hospital and led by Professor Modi.  
 
 
4.11 Statistical considerations 
 
4.11.1 Sample size 
 
Statistical considerations have been overseen by the Neonatal Data Analysis Unit 
Statistician Ms Shalini Santhakumaran. This study is primarily a feasibility pilot trial and 
has been designed as such.  It is unknown what constitutes a clinically relevant 
difference in the primary outcome total body adiposity, as measured by whole body MRI, 
following HDM and PTF supplemented MOM in preterm infants from birth to discharge.  
Indeed, one of the aims of the study is to obtain data on the magnitude of effect on total 
body adiposity.  However, an estimate of a clinically relevant difference has been made 
from one previous study using a different method of body composition measurement.  In 

PREMFOOD Protocol 04/03/14 
21 
 
an observational study, Pieltain et al (22) investigated change in body composition, 
measured using dual energy x-ray absorptiometry at birth and again at discharge, in 
preterm infants fed with fortified HDM or PTF to supplement MOM. Whole body 
composition was similar in the two groups at the initial measurement, but differed 
significantly at the time of the second measurement. They calculated that at the 5% 
significance level and 80% power, the minimal detectable difference in fat mass would 
be 86g or 96cc between groups for a total sample size of 40 (20 in each group).  
 
Using our group’s database of preterm infants at term age equivalent (July 2012), the 
mean (standard deviation) of directly measured total body adiposity, using whole body 
MRI is 0.677(0.243)l. Although the clinically relevant difference may be less than this, 
we have based our sample size on detecting a one standard deviation difference. A 
sample size of 22 infants in each group would enable us to detect a 0.243l difference in 
adiposity between groups with just over 80% power and at 5% significance level.  
Assuming 10% mortality prior to term and 10% drop out rate, 28 infants will be 
recruited in to each of the three groups or until 22 infants in each group has had their 
MRI and MRS at term age equivalent, a total of 66 scans. Thus, over a recruitment 
period of 18 months, with an estimated recruitment rate of 2-4 infants per month it is 
realised that the study will likely be underpowered for the outcome of total body 
adiposity. This sample size is based on single centre recruitment. In the eventuality of 
one or more centres being involved in the study, the following table has been 
constructed to demonstrate sample sizes and powers required for detecting a range of 
possible clinically significant differences in total body adiposity, for which the Bonferroni 
correction has been used for multiple testing. 
 
 
 
 
 
 
Table 3. Power to detect possible clinically relevant differences in adiposity for different 
sample sizes; AT=adipose tissue; l=litre 
 
Total 
n 
Power (%) to 
detect 0.096(l) diff 
in AT 
Power (%) to detect 
0.243(l) (1 sd) diff 
in AT 
Difference (l) in AT at 
80% power 
60 
12.8
78.1
0.249
66 
14.1
82.4
0.237
75 
16.1
87.5
0.222
90 
19.6
93.1
0.203
96 
21
94.7
0.197
105 
23.1
96.4
0.188
120 
26.8
98.1
0.176
135 
30.4
99.1
0.166
150 
34
99.6
0.157
165 
37.6
99.8
0.15
180 
41.2
99.9
0.144
210 
48
100
0.133
225 
51.3
 
0.129
240 
54.5
 
0.125
 
 
 

PREMFOOD Protocol 04/03/14 
22 
 
4.11.2 Statistical analysis 
 
Outcomes will be analysed both for by intention to treat and as per protocol using multiple 
linear regression with adjustment for covariates where appropriate. Baseline covariates 
such as gender, gestational age, and birthweight z score, will be controlled for by 
randomisation stratification, while those such as ethnicity will be adjusted for. To address 
the null hypothesis differences between groups will be tested using ANOVA, with 
examination of the 3 pairwise differences for magnitude of differences. For the purposes 
of modelling randomised babies will be assigned to categories of overall intake 
(predominantly MOM, predominantly PTF, predominantly HDM). Results will be presented 
as estimates and 95% confidence intervals for differences in means.  
 
 
4.12 Timeline 
 
00-06 months: development of protocol, submission for research ethics approval, 
submission of PhD proposal  
06-09 months: establishment of trial centres 
09-27 months: recruitment 
09-21 months: evaluation of design 
27-30 months: delivery of research protocol completed for final recruits 
30-36 months: final analyses and write-up 
 
 
 
 
5. Regulatory and ethical considerations 
 
 
5.1 Regulatory framework 
 
This study is a randomised trial of fortified or unfortified HDM versus PTF to supplement 
MOM. The fortifier used is multicomponent breast milk fortifier which is classified as a food 
supplement for special medical purposes and is not an investigational medicinal product, 
and as such is not subject to the Medicines for Human Use (Clinical Trials) Regulations 
2004. This study is registered with the clinicaltrials.gov online registry and results 
database. 
 
 
5.2 Ethical approval and monitoring 
 
The study protocol, patient information sheet and consent form will be submitted to the 
Research Ethics Committee for approval and subsequently to the R&D department of 
Chelsea and Westminster Hospital for site specific approval.  As part of the ethics approval 
process, this study has been extensively peer reviewed by reviewers as outlined in the 
peer review process by the Joint Research Compliance Office, Imperial College London. As 
part of the PhD, the progression of the study and PhD will be assessed by the Progress 
Review Panel, department of medicine, Imperial College London. 
 
 
 
 
 
 
 
 
5.3 Adverse Events 

PREMFOOD Protocol 04/03/14 
23 
 
 
As this study is simply comparing standard feeding practices, there will be no expected 
adverse events related to the study.  However, we are required by the sponsor to include 
the following information:  
 
ADVERSE EVENTS  
DEFINITIONS  
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study 
subject.  
Serious Adverse Event (SAE): any untoward and unexpected medical occurrence or 
effect that:  
? Results in death  
? Is life-threatening – refers to an event in which the subject was at risk of death at 
the time of the event; it does not refer to an event which hypothetically might have 
caused death if it were more severe  
- Requires hospitalisation, or prolongation of existing inpatients’ hospitalisation  
? Results in persistent or significant disability or incapacity  
? Is a congenital anomaly or birth defect  
Medical judgement should be exercised in deciding whether an AE is serious in other 
situations. Important AEs that are not immediately life-threatening or do not result in 
death or hospitalisation but may jeopardise the subject or may require intervention to 
prevent one of the other outcomes listed in the definition above, should also be 
considered serious.  
 
REPORTING PROCEDURES  
All adverse events should be reported. Depending on the nature of the event the 
reporting procedures below should be followed. Any questions concerning adverse event 
reporting should be directed to the Chief Investigator in the first instance.  
 
Non serious AEs  
All such events, whether expected or not, should be recorded.  
Serious AEs  
An SAE form should be completed and faxed to the Chief Investigator within 24 hours. 
However, relapse and death due to conditions associated with prematurity and 
hospitalisations for elective treatment of a pre-existing condition do not need reporting 
as SAEs.  
All SAEs should be reported to the Fulham Research and Ethics Committee where in the 
opinion of the Chief Investigator, the event was:  
? ‘related’, ie resulted from the administration of any of the research procedures; and  
? ‘unexpected’, ie an event that is not listed in the protocol as an expected occurrence  
Reports of related and unexpected SAEs should be submitted within 15 days of the Chief 
Investigator becoming aware of the event, using the NRES SAE form for non-IMP 
studies. The Chief Investigator must also notify the Sponsor of all SAEs.  
Local investigators should report any SAEs as required by their Local Research Ethics 
Committee, Sponsor and/or Research & Development Office.  
 
Contact details for reporting SAEs  
Fax: 0161 625 7299 attention Shehnaz Ishaq 
Please send SAE forms to:  
Fulham REC Coordinator 
4 Minshull Street 
Manchester M1 3DZ 
Tel: 0161 625 7821(Mon to Fri 09.00 – 17.00)  
 
 
 
5.4 Indemnity 

PREMFOOD Protocol 04/03/14 
24 
 
 
Imperial College London holds negligent harm and non-negligent harm insurance policies 
which apply to this study/ Imperial College Healthcare NHS Trust holds standard NHS 
Hospital Indemnity and insurance cover with NHS Litigation Authority for NHS Trusts in 
England, which apply to this study  
 
5.5 Sponsor 
 
Imperial College London will act as the main Sponsor for this study. Delegated 
responsibilities will be assigned to the NHS trusts taking part in this study.  
 
5.6 Funding 
 
This study will be funded through CI core budget while grant funding is being sought. 
 
5.7 Audit 
 
The study may be subject to inspection and audit by Imperial College London under their 
remit as sponsor and other regulatory bodies to ensure adherence to GCP and the NHS 
Research Governance Framework for Health and Social Care (2nd edition).  
 
5.8 Study Management 
 
The day to day management of the study will be co-ordinated through the section of 
Neonatal Medicine, Imperial College London. 
 
5.9 Publication Policy 
 
Results of the study will be disseminated by publication in peer reviewed scientific journals, 
internal report, and conference presentation. All parents of participants will be sent a 
report of the study findings in the form of a letter. This will be sent to them once all data 
have been collected, analysed and the findings have been written up. A copy of the main 
publication will be provided to all study investigators. Investigators will be encouraged to 
share results with participants' parents as well. 
 
5.10 End of Trial 
 
The end of trial will be declared when the last infant recruited completes the last follow up 
visit ie MRI scan at term plus 6 weeks of age. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6. References 
 

PREMFOOD Protocol 04/03/14 
25 
 
 
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336:1519-1523 
 
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infants: systematic review and meta-analysis. Arch Dis Child Fetal Neonatal Ed 
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3. Quigley M, Henderson G, Anthony MY, McGuire W. Formula milk versus donor 
breast milk for feeding preterm or low birth weight infants. Cochrane Database of 
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4. Schanler RJ, Lau C, Hurst NM, O’Brian Smith E. Randomized trial of donor human 
milk versus preterm formula as substitutes for mothers’ own milk in the feeding 
of extremely premature infants. Pediatrics 2005;116:400-406 
 
5. Kingenberg C, Embleton N, Jacobs SE, O’Connell LAF, Kuschel CA. Enteral feeding 
practices in very preterm infants: an international survey. Arch Dis Child Fetal 
Neonatal Ed 2012;97:F56-F61 
 
6. Barker DJ et al, 1989. Growth in utero, blood pressure in childhood and adult life, 
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7. Eriksson JG et al. 2002. Effects of size at birth and childhood growth on the insulin 
resistance syndrome in elderly individuals. Diabetologia 45:342-348 
8. Kensara OA et al. 2005. Fetal programming of body composition: relation between 
birth weight and body composition measured with dual-energy X-ray absorptiometry 
and anthropometric methods in older Englishmen. Am J Clin Nutr 82:980-7 
9. Saenger et al. 2007. Small for Gestational age: Short stature and beyond. Endocr Rev 
28(2):219-251 
10. Fabricus-Bjerre S et al. 2011. Impact of Birth Weight and Early Infant Weight Gain on 
Insulin Resistance and Associated Cardiovascular Risk Factors in Adolescence PLoS 
ONE 6(6): e20595 
11. Hofman et al. 2004. Premature birth and later insulin resistance. N Engl J Med 
351:2179-86 
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