Nordic Study on Human Milk Fortification in Extremely Preterm Infants: a Randomized Controlled Trial
Phase: PHASE2
Conditions: Necrotizing Enterocolitis, Sepsis, Mortality
Sponsor: Thomas Abrahamsson, MD, PhD

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
1 
STUDY TITLE: 
Nordic study on human milk fortification in extremely preterm infants: a 
randomized controlled trial 
 
ACRONYM: 
N-forte 
 
PROTOCOL No: 
2020/vers.4 
 
DATE OF PROTOCOL 
2020-03-25 
 
STUDY PRODUCT 
Human milk-based human milk fortifier (H2 MF)  
 
PRINCIPAL 
Associate Professor Thomas Abrahamsson, MD, PhD, Neonatologist. 
INVESTIGATOR 
 
INVESTIGATORS 
Professor Magnus Domellöf. MD, PhD, Neonatologist 
 
Associate Professor Anders Elfvin, MD, PhD, Neonatologist 
 
Associate Professor Fredrik Ahlsson, MD, PhD, Neonatologist 
 
Associate Professor Lars Naver, MD, PhD, Neonatologist 
  
 
 
CO-INVESTIGATORS 
Dr Ingela Danielsson, MD, Neonatologist 
 
Georg Bach Jensen, MD, Pediatrician 
 
STUDY COORDINATOR Gisela Hellgren, Fravil Clinical Consulting, Sweden 
 
SPONSOR 
Thomas Abrahamsson, MD, PhD 
H.K.H Kronprinsessan Victorias Barn- och ungdomssjukhus, plan 15  
Universitetssjukhuset 
Region Östergötland  
581 85 Linköping  
tel: 010 – 103 00 00 (vx) 
mob: 0709 – 56 68 15 
 
email: thomas.abrahamson@regionostergotland.se 
 
 
 
 

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Thomas Abrahamsson          
 
 
 
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Table of contents 
 
 
Table of contents ............................................................................................................................................................. 2 
1 
Summary ................................................................................................................................................................. 6 
1.1 
 Rationale ................................................................................................................................................................. 6 
1.2 
 Hypothesis ............................................................................................................................................................... 6 
1.3  
Objective .................................................................................................................................................................. 6 
1.4  
Study design ............................................................................................................................................................. 7 
2 
Flow chart ................................................................................................................................................................ 8 
3 
Background ............................................................................................................................................................. 9 
4.  
Objectives .............................................................................................................................................................. 11 
4.1 
Hypothesis .............................................................................................................................................................. 11 
5. 
 Research questions ............................................................................................................................................... 12 
5.1  
Primary research questions .................................................................................................................................... 12 
5.2  
Secondary research questions ................................................................................................................................ 12 
5.3  
Efficacy variables .................................................................................................................................................... 12 
5.3.1 
Primary variable ................................................................................................................................................. 12 
5.3.2 
Secondary variables ........................................................................................................................................... 12 
5.3.2.1 
Food intolerance and growth ..................................................................................................................................... 12 
5.3.2.2 
Clinical variables for morbidity ................................................................................................................................... 13 
5.3.2.3 
Laboratory variables ................................................................................................................................................... 15 
5.3.2.4 
Health economics ....................................................................................................................................................... 16 
5.3.3 
Associated predictor variables (covariates) ....................................................................................................... 16 
6  
Study design .......................................................................................................................................................... 17 
6.1  
Design .................................................................................................................................................................... 17 
6.2  
Selection of study design ........................................................................................................................................ 18 
6.3  
Selection of treatment ............................................................................................................................................ 18 
7.  
Study population ................................................................................................................................................... 19 
7.1  
Sample size ............................................................................................................................................................. 19 
7.2 
Inclusion criteria ..................................................................................................................................................... 20 
7.3  
Exclusion criteria .................................................................................................................................................... 20 
7.4 
 Withdrawal criteria ............................................................................................................................................... 21 
7.5 
Study termination .................................................................................................................................................. 21 
8  
Study period .......................................................................................................................................................... 22 
9  
Methods ................................................................................................................................................................ 22 
9.1 
Practical arrangements .......................................................................................................................................... 22 
9.1.1 
Inclusion and randomisation ..................................................................................................................... 22 
9.1.2 
Twins ............................................................................................................................................................ 23 
9.1.3 
Investigator file ............................................................................................................................................ 23 
9.1.4 
Start of the intervention .............................................................................................................................. 23 
9.1.5 
Prescription, preparation and administration of the study product ...................................................... 24 
9.1.6 
Case report form (CRF) and Nutrium ...................................................................................................... 25 
9.1.7 
Samples at 7 ± 2 days of age ................................................................................................................... 25 
9.1.8 
Samples at 14 ± 2 days of age ................................................................................................................. 25 

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9.1.9 
Samples at 21 ± 2 days of age ................................................................................................................. 25 
9.1.10 
Samples at 28 ± 2 days of age ................................................................................................................. 26 
9.1.11 
The end of the study intervention, gw 34+0 ............................................................................................ 26 
9.1.12 
Transfer to another neonatal unit ............................................................................................................. 27 
9.1.13 
Sample at gestational week 33+0-33+6 .................................................................................................. 27 
9.1.14 
Magnetic resonance imaging (MRI) ......................................................................................................... 27 
9.1.15 
The end of the neonatal period ..................................................................................................................... 27 
9.1.16 
Follow up at 2 years of age ............................................................................................................................ 28 
9.1.17 
Follow up at 5.5 years of age ......................................................................................................................... 28 
9.1.18 
Deaths ........................................................................................................................................................... 28 
9.2 
Assessment of efficacy ........................................................................................................................................... 28 
9.2.1 
Clinical variables ......................................................................................................................................... 28 
9.2.2 
Sample collection ........................................................................................................................................ 28 
Blood samples ................................................................................................................................................................. 28 
Faecal samples ................................................................................................................................................................ 29 
Urine samples ................................................................................................................................................................. 29 
Breast milk samples ........................................................................................................................................................ 29 
9.3 
Assessment of safety .............................................................................................................................................. 29 
9.3.1 
Adverse events ........................................................................................................................................... 29 
10. 
Concomitant therapy ......................................................................................................................................... 29 
11. 
Concomitant diseases ........................................................................................................................................ 29 
12. 
Study requirements ........................................................................................................................................... 29 
12.1 
Study product ......................................................................................................................................................... 29 
12.2 
Dose, administration, storage ................................................................................................................................ 30 
12.2.1 
Active study group ......................................................................................................................................... 30 
12.2.2 
Control study group ....................................................................................................................................... 30 
12.3 
Storage ................................................................................................................................................................... 30 
12.4 
Study product accountability ................................................................................................................................. 30 
12.5 
Randomisation ....................................................................................................................................................... 30 
13. 
Adverse Events .................................................................................................................................................. 31 
13.1 
Definitions .............................................................................................................................................................. 31 
13.1.1 
Non-serious adverse events ..................................................................................................................... 31 
13.1.2 
Serious adverse events ............................................................................................................................. 31 
13.1.3 
Suspected unexpected serious adverse reaction (SUSAR) ................................................................. 32 
13.2 
Reporting serious adverse events ........................................................................................................................... 32 
13.2.1  
Reporting SAE ............................................................................................................................................. 32 
13.2.2 
Reporting lethal cases and SUSAR ......................................................................................................... 32 
13.3 
Interim analysis ...................................................................................................................................................... 32 
13.4 
Data and safety monitoring board (DSMB) ............................................................................................................ 33 
14 
Data collection ....................................................................................................................................................... 33 
14.1 
Case Report Form (CRF) .......................................................................................................................................... 33 
14.2 
Data source ............................................................................................................................................................ 34 
15 
Data quality assurance ........................................................................................................................................... 34 
15.1. Study monitoring ........................................................................................................................................................ 34 
15.2. Study monitoring ........................................................................................................................................................ 34 
16 
Ethics ..................................................................................................................................................................... 34 

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17. 
Informed consent .............................................................................................................................................. 35 
18. 
Approval from research ethical board and other authorities .............................................................................. 36 
19. 
Study discontinuation ........................................................................................................................................ 36 
20. 
Statistical methods ............................................................................................................................................ 36 
21. 
Financing and insurance .................................................................................................................................... 37 
22. 
Final study report .............................................................................................................................................. 37 
23. 
Retention of records .......................................................................................................................................... 37 
24.  
Confidentiality and publication .......................................................................................................................... 38 
25. 
References ......................................................................................................................................................... 38 
25.  
Appendix ........................................................................................................................................................... 41 
Appendix 1 –Agreement between sponsor and investigator .............................................................................................. 41 
Appendix 2 –List of contributing neonatal units and their contact persons ........................................................................ 42 
Appendix 3 –Agreement with the head of the clinic (this was included in the ethical application) .................................... 43 
Appendix 4 – Data and safety monitoring board (DSMB) ................................................................................................... 44 
Appendix 5 –Reporting of SUSAR ........................................................................................................................................ 45 
Appendix 6 –Study information to the parents ................................................................................................................... 46 
Appendix 7 –Informed consent form ................................................................................................................................... 49 
Appendix 8 –Case report form ............................................................................................................................................. 50 
Appendix 9 –Prescription and preparation of the study product ........................................................................................ 51 
Appendix 10 – Transition to discharge feeding regimen ..................................................................................................... 54 
Appendix 11 –Classification of level of care ........................................................................................................................ 56 
Appendix 12 –Blood sample: instructions ........................................................................................................................... 57 
Appendix 13 –Stool sample: instructions ............................................................................................................................ 58 
Appendix 14 –Urine sample: instructions ............................................................................................................................ 59 
Appendix 15 –Breast milk sample: instructions ................................................................................................................... 60 
Appendix 16 –Nutritional content of H2 MF and Prolact CR ................................................................................................ 61 
Appendix 17 –Product description and safety information ................................................................................................. 62 
 
 
 

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List of abbreviations: 
 
AE= Adverse Event 
BPD=Bronchopulmonary dysplasia 
CRF= Case Report Form 
CRO=Contract research organisation 
CPAP=Continuous positive airway pressure 
DSMB=Data and Safety Monitoring Board 
EHM=Exclusively human milk 
ELBW=Extremely low birth weight 
ELGA=Extremely low gestational age 
GCP= Good Clinical Practice  
ICH= International Conference on Harmonisation 
IVH= Intraventricular haemorrhage 
ITT= Intention-to-treat 
NEC=Necrotizing enterocolitis 
NICU=Neonatal Intensive Care Unit 
PDA=Patent ductus arteriosus 
PPA= Per Protocol analysis 
PPROM=Preterm premature rupture of the membrane 
PVL= Periventricular leucomalacia 
REB=Research Ethic Board  
SAE= Serious Adverse Event 
SCFA=Short Chain Fatty Acids 
SIP=Spontaneous intestinal perforation 
SUSAR= Suspected Unexpected Serious Adverse Reaction 

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1 
Summary 
1.1 
 Rationale 
Although the care of premature infants has improved dramatically during the last decades, still about 30% of the 
extremely low gestational age (ELGA born < gestational week 28+0) infants die – and severe infections and 
necrotizing enterocolitis (NEC) are common causes of death.  There are clear links between nutrition and the risk 
of NEC, infection, neurological sequelae and mortality. A common cause of poor nutrition in this patient group is 
poor enteral feed tolerance due to immaturity and dysfunction of gastrointestinal tract – and thus ELGA infants 
usually require parenteral feeding though intravenous catheters that increase the risk of sepsis.  Nutritional 
problems and their consequences are a major problem in neonatal care. 
 
However, choice of diet has a major impact on morbidity and clinical course. Preterm infants receiving human 
milk have better feeding tolerance and lower incidence of NEC than those fed cow milk based preterm formula. 
Fortunately, because of the widespread availability of donor milk banks in Nordic countries, ELGA infants are 
generally receiving a base diet of human milk without the need for cow’s milk-based formulas. Despite this, the 
incidence of NEC in ELGA infants is still as high as 12% according to the Swedish National Quality Register 
(www.snq.se). We hypothesise that a key reason for this is that even fully human milk fed ELGA infants have a 
significant intake of cow’s milk protein.  This is because human milk alone does not fully meet the nutritional 
requirements in ELGA infants, but needs to be supplemented with a nutrient fortifier – and such fortifiers 
available in Nordic Countries are based on cow’s milk. Indeed, because breast milk protein levels are so low in 
relation to the needs of ELGA infants, such fortifiers often provide more cow’s milk protein than the infant 
receives from an all-human milk diet.  
 
H2MF® (Prolacta Bioscience, CA, USA) is a human milk-based nutrient fortifier for preterm infants. Previous 
trials evaluating Prolacta fortifiers in extremely preterm infants receiving exclusive human milk diet suggest that 
exclusive human milk diet, devoid of cow milk-containing products, is associated with lower mortality and 
morbidity in these infants without compromising growth. Thus, such an exclusive human milk diet has been 
reported to reduce the incidence of NEC, sepsis, bronchopulmonary dysplasia (BPD) and retinopathy of the 
prematurity (ROP). No side effects have been reported. Still, there is no well powered trial in ELGA infants 
comparing a human milk-based fortifier with bovine protein-based fortifier in which both study groups receive an 
all-human breast milk-based diet. Such a trial is needed to provide evidence that a human milk-based fortifier is 
superior to bovine-based one in ELBW infants also in centres with 100% coverage of breast milk donor banks. 
Moreover, due to the introduction of active intervention for infants also born before gestational week 25 during 
the last decade, a trial run in Nordic centres will have a predominance of infants born in gestational week 23-25, a 
patient population that could be expected to gain the most of a diet free from bovine protein. A significant positive 
result of this trial could easily be implemented in the NICUs, since Prolact H2MF® already exists and has been 
used extensively - by around 1 in 4 level 3 NICUs in the US. 
1.2 
 Hypothesis 
Extremely preterm infants exclusively fed with human breast milk (mother´s own milk and/or donor milk) 
supplemented with the human milk-based nutrient fortifier will have a lower incidence of severe complications: 
NEC, sepsis and mortality (as a composite) than ELGA infants supplemented with a bovine protein-based one.  
1.3  
Objective 
To evaluate if diet supplementation with the human milk-based nutrient fortifier H2MF® reduces severe 
complications - NEC, sepsis, mortality - and improves enteral feeding tolerance as compared with bovine protein-
based nutrient fortifier in extremely preterm infants receiving exclusive human breast milk (own mother´s milk 

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and/or donor milk). Possible mechanisms underlying the effect of H2MF® will be analysed in blood, urine and 
stool samples. The health economic impact of an implementation will also be evaluated. 
1.4  
Study design 
This is a randomised controlled multi-centre trial comparing the effect of diet supplementation of a human breast 
milk-based nutrient fortifier (H2MF®) with standard bovine protein-based nutrient fortifier in 222 extremely 
preterm infants (born before gestational week 28+0) exclusively fed with human breast milk (own mother´s milk 
and/or donor milk). The infants will be randomised to receive either the human breast-milk based H2MF® or the 
standard bovine protein-based nutrient fortifier when oral feeds have reached <100 ml/kg/day. If fortification with 
extra enteral lipids is needed during the intervention period, the infants receiving H2MF® will be supplemented 
with the human milk-based Prolact CR®, while the infants receiving standard bovine protein-based fortification 
will be supplemented with the standard lipid products used at the unit. The study subject will be enrolled at level 
III NICUs. Only infants with a home clinic with the logistics to maintain the intervention until gestational week 
34+0 will be included.  
 
The randomised intervention, stratified by centre, will continue until the target gestational week 34+0. The infant 
must not be fed with formula during the intervention period. The allocation will be concealed before inclusion, but 
after randomisation the study is not blinded. It would not be possible to prescribe the fortifier and prepare of the 
breast milk in a blinded fashion, since the fortifiers are not exactly equal in nutrient content and also look 
different. Instead the assessment of several of the outcomes will be made blinded, such as the assessment of X-ray 
images in NEC cases. 
 
The enrolled infants are characterised with clinical data including growth, feeding intolerance, use of enteral and 
parenteral nutrition, treatment, antibiotics and complications collected daily in a study specific case report form 
from birth until discharge from the hospital (not longer than gestational week 44+0). A follow up focusing on 
neurological development, growth and feeding problems will be performed at 2 years of age (corrected) and 5.5 
years of age.  
Since it is often difficult to distinguish between the diagnoses of NEC and sepsis, and their clinical consequences, 
our primary endpoint of the intervention is the composite variable NEC, sepsis and mortality. Secondary 
endpoints are feeding intolerance and other severe complication such as BPD, ROP and neurological impairment. 
Stool, urine and blood samples are also collected for microbiology, metabolomic and immunology analysis in 
order to study underlying mechanisms. Health economic analyses will be made to evaluate the costs and benefits 
of an introduction of human milk-based fortifier in NICUs in the Nordic countries. 
 
Analyses will be conducted using an intention to treat approach. An evaluation will be performed when 20 infants 
have been included to evaluate feasibility and make it possible to adjust the protocol for the remaining part of the 
study. Safety analyses will be performed by an independent data and safety monitoring board (DSMB) when 50, 
100 and 150 infants have been included. A sample size re-estimation will be made by an independent statistician 
when 150 infants have been included. Thus, the definitive sample size might be increased (never decreased) based 
on this interim analysis. The study can be terminated before 222 infants have been enrolled based on a decision of 
the sponsor and the DSMB, if the primary outcome is significantly lower (with a significance level <0.001) in the 
H2MF® than in the standard fortification group in the interim analysis made after 150 infants have completed the 
neonatal period.  The study subject will be enrolled at level III NICUs in the Nordic Countries. All study subjects 
will be followed during the neonatal period until discharge (not longer than gestational week 44+0) and also be 
included in a follow up at 2 and 5.5 years of age based on the national follow up program for extremely preterm 
infants.  
 

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2 
Flow chart 
Time point 
Inclusion    
 
 
 
Inter- 
vention 
ends 
 
Neonatal 
follow up 
ends 
Follow up 
at age of 
 2y         5.5y 
Day of life 
<100 
ml/kg/d 
7 d  
± 2 d  
14 d  
± 2 d  
21 d  
± 2 d 
28 d  
± 2 d 
 
 
 
 
 
Gestational week 
 
 
 
 
 
-w34+0 
w36+0 
-w44+0 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
Incl.-/exclusion criteria 
X 
 
 
 
 
 
 
 
 
 
Background history 
X 
 
 
 
 
 
 
 
 
 
Randomisation 
X 
 
 
 
 
 
 
 
 
 
Intervention 
||========================================|| 
 
 
 
 
Feeding intolerance 
||========================================|| 
 
 
 
 
NEC, sepsis, mortality 
 
||==================================================|| 
 
 
Neurological impairment* 
 
 
X 
X 
Antibiotics and 
concomitant medication  
X 
X 
X 
X 
X 
w34+0 
X 
X 
 
 
Weight** 
 
X  
X  
X  
X  
w34+0 
X 
X 
X 
X 
Height/head circumf.** 
 
7d 
14d 
21d 
28d 
w34+0 
X 
X 
X 
X 
Blood sample (if available) 
 
X 
X 
 
X 
w33+0-6 
 
 
 
 
Urine sample (if available) 
 
X 
X 
X 
X 
w33+0-6 
 
 
 
 
Stool sample (if available) 
 
X 
X 
X 
X 
w33+0-6 
 
 
 
 
Breast milk  (if available) 
 
X 
X 
X 
X 
w33+0-6 
 
 
 
 
Adverse Events  
X 
X 
X 
X 
X 
X 
X 
X 
 
 
*=Including examination by paediatrician and psychologist (incl. Bayley III test)  
**= In the table it is only notified when measurements must be made in the trial (weight is often measured every day). 

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3 
Background 
Although the care of premature infants has improved dramatically during the last decades, the extremely low birth 
weight (ELBW, birth weight <1000g) preterm infant remains a major challenge for neonatal intensive care.  
About 30% of these babies die 1 and the survivors may have a complicated clinical course with major morbidity 
and a significant risk of impaired later outcome.  
Nutrition is increasingly recognised as a key factor in the survival, clinical course and later outcome of high-risk 
preterm infants; and of particular relevance to proposed project is the broad evidence for beneficial roles of human 
milk in neonatal care, underpinning current recommendations for its use 2.  
 
Extremely preterm infants are usually fed parenterally while enteral feed tolerance develops, though even when 
feeds are tolerated, these infants are too immature to be fed orally and require tube feeding. Human milk, fed by 
tube, is generally expressed milk from the infant’s own mother. However, in Nordic countries, donor breast milk is 
usually available when mothers provide no or insufficient breast milk. A growing literature supports the clinical 
value of using human milk for feeding preterm infants, in particular for the smallest and most vulnerable premature 
infants. Proposed benefits of human milk for such infants are considered as follows. 
 
Reduction of necrotising enterocolitis (NEC) 
NEC remains a significant problem in neonatal care with an incidence in some centres in the US in ELGA infants 
of close to 20% 3 and around 12% in Sweden (www.snq.se), where the peak incidence is 16% for infants born at 
gestational week 24. NEC frequently results in death (around 20-30%) or the requirement for surgical resection of 
necrotic gut which may be sufficiently extensive to necessitate long term parenteral nutrition in hospital and 
subsequently at home. It is increasingly recognised that NEC is a major adverse factor for subsequent lower IQ, 
motor impairment, visual impairment and cerebral palsy 4. The health economic cost of NEC is large 5,6. 
A number of earlier observational studies including those by Lucas and Cole 7 in 1990, El-Mohandes et al in 1997 
8 were followed by a meta-analysis by Boyd et al in 2007, concluding that human milk feeding was associated with 
a reduced incidence of NEC 9. More extensive recent evidence comes from two randomised trials 10,11 and 8 
observational studies 3,12 13 in the US, collectively involving over 4000 preterm infants under 1250g birth weight. 
These studies, show on average over 70% reduction in NEC with the use of an exclusive human milk diet achieved 
by feeding human-milk based products. 
 
Reduction in neonatal sepsis 
Neonatal sepsis remains a life-threatening and common problem in neonatal care. The incidence is as high as 35-
40% in ELGA infants (www.snq.se). Growing evidence links sepsis to the mode of feeding.  Thus, early 
observational studies by Naraynan et al 14-16 and Lucas et al 17 showed human milk was associated with less sepsis. 
Further evidence comes from a randomised trial of breast milk fortification in which Lucas et al showed that those 
who received a cow’s milk-based breast milk fortifier had a significant increase in the combined outcome of NEC 
or proven sepsis. Each 10% increase in feed containing cow´s milk was associated with an 18% increase in sepsis 
18. Abrams et al also showed a dose related reduction in proven sepsis with increasing human milk usage 19.  Hair 
et al showed in a four centre, 1600 preterm infant observational study that introduction of an exclusive human milk 
diet was followed by a highly significant reduction in sepsis from 30% to 19% 3. 
 
Feed tolerance and reduced need for parenteral nutrition 
Poor feed tolerance is a significant and pervasive problem in the management of ELGA infants that poses the risk 
of under-nutrition, which not only undesirably prolongs he use of parental nutrition, but also may compromise 
optimal neurodevelopment 20. Several studies have shown that use of human milk is associated with better feed 
tolerance, reduced time to full enteral feeds and reduced need for parenteral nutrition 11,12. Parenteral nutrition in 

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10 
ELGA infants, though necessary from a nutritional perspective, is associated with potential clinical hazards, 
including systemic sepsis related to long lines required. 
 
 
Long-term impact of human milk in neonatal care  
As preterm infants are being followed increasingly throughout childhood and into adult life, long-term effects of 
breast milk in neonatal care are emerging. A beneficial effect of prior randomisation to an exclusive human milk 
diet has been shown in preterm-born 16-year old children. They had favourably reduced LDL/HDL cholesterol 
ratio, diastolic blood pressure, insulin resistance and metabolic tendency to fatness - key risk factors for 
cardiovascular disease 21-24. Larger bone mass in adolescence 25, higher cognitive performance in childhood 26,27 
and increased brain size and white matter in adolescence 28 have also been reported in those fed human milk as 
neonates. A recent observational study showed a reduction in all classes of retinopathy of prematurity in ELBW 
infants from 9% with cow’s milk products in the diet, down to 5% on an exclusive human milk diet 3. Moreover, 
preterm-born adults aged 25-28 years who were fed exclusive human milk in infancy have more normal sized 
cardiac ventricles and stroke volumes, whereas these were markedly reduced in those fed exclusively formula in 
the neonatal period 29.  
 
Nutritional inadequacy of breast milk for preterms: implications 
It is well recognised that despite the health benefits of human milk considered above, it does not meet the nutritional 
needs of ELBW infants whose rate of somatic and brain growth far exceeds that in full term neonates. In order to 
meet the nutritional needs of enterally fed preterm infants additional products are therefore required and these are 
generally cows-milk based. For infants whose mothers provide no or inadequate volumes of expressed breast milk 
and where there is no access to milk bank facilities, these infants require a cow’s milk based preterm formula. But 
even in Nordic countries where a 100% human milk base diet is achievable through the use of donor milk alongside 
mother’s milk, all the breast milk used needs to be fortified – again with a cows-milk based fortifier. 
  
Whilst cow’s milk products are used in modern neonatal care to provide adequate nutrition – for instance to fuel 
brain growth – current evidence, outlined above, indicates that cow’s milk products may have detrimental effects 
when compared to a human milk diet. 
 
The development of lacto-engineering 
The possibility of meeting nutritional needs of preterm infants whilst avoiding use of cow’s milk products was first 
explored in 1980 by Lucas et al 30, who showed it was possible to develop a prototype formula for premature infants 
made only from human milk components. This was achieved through adding freeze-dried human milk powder and 
human milk cream (as an energy source) to whole human milk.  
 
More recently, Prolacta Bioscience (CA, USA) has developed a series of human milk preparations that provide 
neonatologists with the first commercially available lacto-engineered products – preterm formulas and fortifiers - 
made from 100% human milk that have carefully controlled amounts of protein, carbohydrates and fat, as well as 
minerals and vitamins.  
 
Pasteurized human milk products that are made from donor human milk have the theoretical potential for 
transmitting infectious diseases such as HIV or hepatitis. Prolacta Bioscience incorporates a number of steps to 
reduce the possibility of infectious disease transmission (please see appendix 17). These include careful donor 
screening, blood testing of the donor, and testing of the milk pre and post pasteurization by culture and PCR. 
Nevertheless, whilst the transmission of disease remains a remote possibility there are no reports of any disease 
transmission or any other adverse events to date, despite the products under study here have been used in more than 
100,000 babies in around 400 centres across the United States and a few in Canada and Austria  

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11 
 
Clinical studies on human milk-based products 
Two randomised trials have examined the impact of these human milk-based formulations. The first, by Sullivan et 
al examined in 207 infants the impact of replacing cow’s milk based preterm formulas and fortifiers with the human 
milk equivalents to produce an exclusively human (EHM) based diet milk 10. Those fed the EHM diet showed a 
major reduction of NEC rate from 15.9% to 5.4% and a much larger reduction in NEC surgery rate from 11.6% to 
only 1.4%. In the second, smaller trial on the rare group of babies whose mothers provided no breast milk, 
randomisation was to a cow’s milk based preterm formula as sole enteral feed versus exclusive human milk based 
products 11. In the EHM group NEC surgery rate was zero compared to 17% in the preterm formula group. A study 
combining these two trials further showed a favourable impact of an exclusive human milk diet on sepsis risk and 
importantly a significant drop in death rate from 8% in those exposed to cow’s milk products to only 2% in those 
on an EHM diet 19. 
 
Since those trials, 8 investigator-led studies have examined during the period 2006-2014 the clinical impact of 
introducing an EHM diet compared to baseline morbidity 3,12 13. Collectively these studies on around 4000 subjects 
principally under 1250g birth weight showed a 75% reduction in NEC. The studies provided further evidence for a 
major adverse impact of cow’s milk based products on sepsis, mortality and retinopathy of prematurity, the latter 
now shown in 3 independent studies 3,31,32. 
 
In conclusion 
A growing literature supports the use of human milk-based products in clinical practise. The previous trials in the 
US, however, do not distinguish between the effects of replacing a cow’s milk based preterm formula with a human 
milk-based equivalent and those replacing a cow’s milk-based human milk fortifier with a human milk based one – 
in the main trial done in US 10 both were replaced. Thus, there is still no trial in ELBW infants comparing human 
milk-based nutrient fortifier with bovine protein-based one in which both study groups receive exclusive human 
breast milk. Moreover, due to the introduction of active interventions for infants also born before gestational week 
25 during the last decade, a trial run in Nordic centres will have a predominance of infants born in gestational week 
23-25, a patient population that could be expected to gain the most of a diet free from bovine protein. A significant 
positive result of this trial could easily be implemented in the NICUs, since the human milk-based nutrient fortifiers 
already exist as a product on the market and have been used and tested extensively. 
4.  
Objectives 
The primary objective is to evaluate if the supplementation of a human milk-based nutrient fortifier H2MF® 
reduces the severe complications of NEC, sepsis and mortality as compared with bovine protein-based nutrient 
fortifier in extremely preterm infants receiving exclusive human breast milk (own mother´s milk and/or donor 
milk). Secondary objectives are to evaluate if H2MF® supplementation improves enteral feeding tolerance and 
reduces other severe complications such as BPD, ROP and neurological impairment. Possible mechanisms 
underlying the effect of H2MF® will be analysed in blood, stool, urine and breast milk samples. Health economic 
analyses will be made to evaluate the costs and benefits of an introduction of human milk-based fortifier in 
NICUs in the Nordic countries.  
 
 
4.1 
Hypothesis 
The primary hypothesis is that extremely preterm infants exclusively fed with human breast milk (mother’s own 
milk and/or donor milk) will have a lower combined incidence of severe complications - NEC, sepsis and 

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12 
mortality – when supplemented with human milk-based nutrient fortifier H2MF® as compared to supplementation 
with a bovine protein-based one’.  
 
5. 
 Research questions 
5.1  
Primary research questions 
Does supplementation with the human milk-based nutrient fortifier H2MF® reduce NEC, culture-proven 
sepsis and mortality in extremely preterm infants? 
5.2  
Secondary research questions 
• Does supplementation with the human milk-based nutrient fortifier H2MF® reduce the composite of NEC 
and culture-proven sepsis in extremely preterm infants? 
• Does supplementation with the human milk-based nutrient fortifier reduce feeding intolerance in 
extremely preterm infants? 
• Does supplementation with the human milk-based nutrient fortifier improve growth and the neurological 
development in extremely preterm infants? 
• Does supplementation with the human milk-based nutrient fortifier reduce other severe complications such 
as BPD, ROP in extremely preterm infants? 
• Does supplementation with human milk-based nutrient fortifier reduce the days of intensive care needed 
and the length of stay at the hospital? 
• Does the use of human milk-based nutrient fortifier reduce total cost of neonatal care according to health 
economic analyses? 
• What are the immunological, microbiological and metabolic mechanisms underlying the effect of a human 
milk-based nutrient fortifier in extremely preterm infants? 
• What are the immunological, microbiological and metabolic mechanisms underlying feeding intolerance, 
growth restriction, neurological impairment, NEC, sepsis and bronchopulmonary dysplasia?  
5.3  
Efficacy variables 
5.3.1 
Primary variable 
 
The composite of NEC, culture-proven sepsis and mortality: An infant should have had any of these 
diagnoses to fulfil the criterion. Please see the definition of NEC and sepsis below. 
. 
5.3.2 
Secondary variables 
5.3.2.1 
Food intolerance and growth 
1. Time to reach full enteral feeds: the day of life the infant has received at least 150 mL/kg enteral feeds. 
2. Feeding interruption:  number of days feedings held for ≥12 hours OR feeds reduced by >50% (ml/kg/d) 
not due to a clinical procedure or transitioning to the breast. 
3. Parenteral nutrition: number of days of parental amino acid and/or lipid infusion. Only days when the 
enteral feed <150mL/kg/day should be included. 
4. Gastric aspirates: ≥100% pre-feed volume (2 hours feeding volume if continuous feeding). Lower limit=2 
ml/kg. 
5. Stool frequency: number of stools per day.  

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6. Time to regain birth weight: first day of three success days when the infant weight is greater than birth 
weight. Indicate 0 days, if the infant never has a weight below birth weight. 
7. Weight, height and head circumference at 7, 14, 21 and 28 days, the end of intervention (≤ 34+0), 
gestational week 36+0, at discharge from neonatal ward (or at gestational week 44+0, whatever comes 
first) and at 2 years of age (corrected) and 5.5 years of age (uncorrected). Standard deviation score (based 
on Niklasson 33) will be used in the calculations.  
 
5.3.2.2 
Clinical variables for morbidity 
-Neonatal period: 
1. Mortality 
 
2. Necrotising enterocolitis (NEC): Bell´s stage II-III 34. Stage II requires clinical signs of NEC with 
abdominal symptoms and radiological findings such as intramural and/or portal gas. Stage III requires the 
same findings as stage II in addition to heavily affected clinical condition (chock, need of respirator, 
mortality) and/or surgery. If surgery confirms NEC, there is no need of positive radiological findings for 
diagnosis. Radiological assessment will be made blinded by an independent radiologist. The final decision 
will be confirmed by a blinded consensus panel review consisting of the investigators. The age (in days) of 
onset will be noted. If the NEC diagnosis is confirmed, this diagnosis will replace any eventual sepsis 
diagnosis during the duration of the NEC episode.  
 
3. The composite of NEC and culture-proven sepsis: An infant should have had any of these two diagnoses 
to fulfil the criterion. Please see the definition of NEC above and sepsis below. 
 
4. Spontaneous intestinal perforation (SIP: intestinal perforation without signs of intramural and/or portal gas 
and no signs of inflammation at surgery.  
 
If there is an intestinal perforation it is important that the investigator rules out whether it is NEC or SIP, 
since the underlying mechanism probably differs. 
 
Although NEC criteria is not fulfilled, e.g. because the independent radiologist do not agree that the 
radiology findings are enough for the diagnosis, the clinical findings and laboratory results might fulfil 
the criteria for sepsis (please see below). 
 
5. Abdominal surgery 
 
6. Culture-proven sepsis: Positive blood- and/or urine- and/or CSF culture, clinical deterioration and 
laboratory inflammatory response* 
 
*Laboratory inflammatory response (one of the following is needed) 35: 
1. LPK < 5 or > 20 (x109 cells/L) 
2. TPK < 100 (x109 cells/L) 
3. CRP > 15 mg/L 
 
Culture proven sepsis should further be classified into early (<72 hours postpartum) or late (>72 hours 
postpartum) onset. Also, the age (in days) at onset will be noted. 
 

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The infant shall not get a sepsis diagnosis if it has got a NEC diagnosis during the same period 
 
Suspected sepsis, not culture-proven: clinical deterioration and laboratory inflammatory response* but 
negative blood culture. Age at onset will be noted.  
 
7. Pneumonia: Pathological X-ray confirmed by an independent radiologist, need of increased respiratory 
support/oxygen and laboratory inflammatory response* Age at onset will be noted. 
 
8. Bronchopulmonary dysplasia (BPD): Need of extra oxygen, high flow nasal cannula, CPAP or ventilator 
at gestational week 36+0 36 
9. Retinopathy of the prematurity (ROP): diagnosed by an independent ophthalmologist according to 
international classification. 37 Classified into stage I-V. The diagnosis is set after gestational week 42+0. 
10. Intraventricular haemorrhage (IVH): classified into grade I-IV according to Papile 38. Assessed by 
independent radiologist if the onset was after the intervention started. Age at onset will be noted. 
11. Periventricular leukomalacia (PVL). Criteria according to de Vries 39 assessed by an independent 
radiologist. 
12. Number of days with intensive care: need of respirator or CPAP until discharge (not later than gestational 
week 44+0). 
13. Length of stay at the hospital: gestational week and day at discharge (not later than gestational week 
44+0).  
14. Length of need of feeding tube: gestational week and day when the infant does not need it anymore (not 
later than gestational week 44+0). 
 
2-year follow up 
1. Weight, height and head circumference. 
2. Need of feeding tube after discharge from the hospital at the neonatal period (not later than gestational 
week 44+0)  
3. Extra nutritional support after discharge from the hospital at the neonatal period (not later than gestational 
week 44+0)  
4. Neurocognitive development: Bayleys III 40 
5. Cerebral palsy 
6. Epilepsy 
7. Squint and/or impaired vision 
8. Impaired hearing 
9. Need of extra oxygen and/or ventilatory support after discharge from the hospital at the neonatal period 
(not later than gestational week 44)  
10. Wheeze and/or asthma 
11. Severe infections after discharge from the neonatal unit (not later than gestational week 44). 
12. Mortality: including cause of death. 
13. Level of education of the parents 
14. Family status 
15. Day-care 
 
5.5 year follow up 
1. Weight, height and head circumference. 
2. Neurocognitive development: Wechsler Preschool and Primary Scale of Intelligence IV (WPPSI-IV TM) 
and Movement ABC-2 
3. Cerebral palsy 

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4. Epilepsy 
5. Squint and/or impaired vision 
6. Impaired hearing 
7. Wheeze and/or asthma 
8. Mortality: including cause of death. 
 
Data from the 2 and 5.5 follow up will be obtained from the Swedish neonatal quality register (www. 
snq.se 
 
5.3.2.3 
Laboratory variables 
Separate protocol for each laboratory analysis will be created. Time points of collection of samples are displayed 
in the Flow chart (paragraph 2).  
1. Microbiology analyses: Bacterial DNA will be extracted from stool and breast milk and samples analysed 
with high-throughput next generation sequencing methods. Shotgun metagenomics will be performed to 
sequence the whole bacterial genome in order to analyse bacterial functional genes. Specific focus will be 
on genes for degradation of oligosaccharides, formation of short chain fatty acids (SFCA), vitamin 
production, intestinal barrier function and virulence factors. Because of the prospective design the analyses 
could be performed in samples collected before the onset of an outcome parameter- thereby providing 
evidence related to a cause-and-effect relationship. Gut bacteria metabolites in faeces such as SCFA will 
be assessed with gas chromatography and be related to the microbiology analyses and the clinical 
outcome. 
 
2. Immunological analyses in blood samples: Blood samples are collected in EDTA tubes for plasma and 
separate pre-prepared tubes for masscytometry of blood cells (CyTOF). The plasma and cells will be 
stored at -70°C. The purpose of the immunological analyses is not only to compare the active and control 
group to assess the effect on the immune system of human milk-based and bovine-based nutrient fortifier, 
respectively, but also to map the immune system in this extreme group of patients and to identify 
immunological markers associated with specific diseases such as NEC and BPD.  
 
1. T helper subsets (TH1, Th2, TH17, Treg), T cells subsets associated with the intestinal mucosa (gd-T 
cells, MAIT cells) and neutrophils will be assessed using masscytometry.  
2. Levels of Anti- and proinflammatory cytokines (e.g. IL-10. IL-6, TNF-a) and chemokines (e.g. 
CXCL11, CCL18) as well as immune modulatory enzymes such as  Indoleamine 2,3-dioxygenase 
(IDO) are high enough in plasma to be measured even in these small infants. To use the plasma as 
efficiently as possible, it will be analysed using multiplex assays.  
 
3. Growth factors and neurotransmitters in blood samples: The levels of growth factors such as IGF-1 and the 
associated IGFBP-3 will be analysed. Besides being important for growth, these factors are also important 
in the pathogenesis of several of the complications (ROP, BPD) under study in this project. 
Neurotransmitters, which affect gut motility and neurological development, have been associated with the 
gut microbiota and will also be analysed. Markers for CNS damage such as Neurofilament light protein 
will be analysed in plasma.  
 
4. Lipidomic analyses will be performed in plasma samples and erythrocyte membranes to assess the effects 
of the different feeding regimens on lipid profiles 
 

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5. Metabolomic analyses will be performed in urine samples. Urine is collected using cotton wool (first urine 
after 8 am), centrifuged and divided to 2-3 aliquots of about 1 mL of urine and stored at -70°C. Low 
molecular weight metabolites (metabolome), intermediate or products of metabolism in human cells and 
gut bacteria, will be analysed with different techniques such as proton nuclear magnetic resonance 
spectroscopy (NMR), liquid chromatography (LC) and mass spectroscopy couple to gas chromatography 
(GC-MC). These methods allow us to prospectively identify potential biomarkers of malnutrition in ELGA 
infants and potential metabolic mechanism underlying the effect of human milk-based nutrient fortifiers.  
 
6. Proteomic analyses in breast milk samples after fortification from the mothers but also from the donor 
milk that the infant receives at the day of sample collection (at 7, 14, 21 and 28 days and w33 of life). Also 
human milk oligosaccharides will be measured in the breast milk samples. The protein profile in breast 
milk samples and in H2 MF and the cow milk protein-based fortifier will be related to feeding tolerance, 
NEC and other complications during the neonatal period in order to identify specific proteins or patterns of 
proteins important for the outcomes 
 
7. Vitamins and micronutrients will be analysed in a sample of infants with standard methods to evaluate if 
the different fortification may lead to different levels in blood. 
 
 
5.3.2.4 
Health economics 
 
The intervention is costly but may prevent important complications such as NEC, sepsis and food intolerance and 
may thereby reduce the overall cost of neonatal care. We will use level of care to calculate health care costs. 
1. The number of days at each level of care (as defined in Appendix 11) will be recorded until discharge from 
the hospital (not longer than gestational week 44+0). 
2. The cost will be calculated by multiplying the number of days at each level of care by the average cost. 
 
5.3.3 
Associated predictor variables (covariates) 
 
1. Gender 
2. Caesarean section 
3. Multiple pregnancies. 
4. Birth weight and height. 
5. Small for gestational age (SGA): Birth weight <2 SD. 
6. Maternal smoking during pregnancy. 
7. Preeclampsia: diagnosis by the responsible obstetrician. 
8. Chorioamnionitis:  diagnosis by the responsible obstetrician. 
9. Preterm premature rupture of the membranes (PPROM). Rupture > 1 hour before contractions started. 
10. Antenatal antibiotics: pertain the period of the mother´s actual attendance at the hospital. 
11. Antenatal corticosteroids: the mother should have received at least 12 mg Betametasone. The 
corticosteroid prophylaxis is considered completed if the mother has received 2 doses at least 24 hours 
before delivery.  
12. Born at level 1-2 NICUs. 
13. Apgar score. 
14. Surfactant-administration. 

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15. Intubation. 
16. Infant respiratory distress syndrome (IRDS): Verified by X-ray 
17. Respirator duration. 
18. Patent ductus arteriosus (PDA): requiring medical or surgical treatment.   
19. Antibiotics: name, treatment period and number of days. 
20. Probiotics: name, treatment period and number of days 
21. Opioids: name, treatment period and number of days 
22. Gastric acid inhibitors: name, treatment period and number of days 
23. Day of life when the supplementation of the study product was started 
24. The amount of enteral feeds per day that the infants received when the supplementation of the study 
product was started 
25. Number of days the infants has not received the study product 
26. Number of days with intravenous line 
27. Number of days with insulin treatment (and treatment period) 
28. Number of days and doses of postnatal corticosteroids 
29. Number of days with inotropic drugs 
30. Feeding regime: continuous or bolus. 
31. Amount of nutrient protein fortifier per day 
32. Amount of fat supplement per day 
33. Total amount of protein, fat, carbohydrate, energy and micronutrient per day. 
34. The relative amount of donor breast milk per day 
35. Amount of extra enteral lipid  
36. Breastfeeding: exclusive, partial.  
6  
Study design 
Birth 
Inclusion 
Randomisation          H2MF® 
 
 
 
 
 
Standard cow milk-based nutrient fortifier 
 
 
6.1  
Design 
This is a randomised controlled multi-centre trial comparing diet supplementation with human breast milk-based 
nutrient fortifier H2MF® and standard bovine protein-based nutrient fortifier in extremely preterm infants (born 
before gestational week 28+0) exclusively fed with human breast milk (own mother´s milk and/or donor milk). 
The infants will be randomised to receive either the human breast-milk based H2MF® or the standard bovine 
protein-based nutrient fortifier before oral feeds have reached 100 ml/kg/day. If fortification with extra enteral 
lipids is needed during the intervention period, the infants receiving H2MF® will be supplemented with the human 
milk-based Prolact CR®, while the infants receiving standard bovine protein-based fortification will be 
supplemented with the standard lipid products used at the unit. The study subjects will be enrolled at level III 
NICUs. Only infants with a home clinic with the logistics to maintain the intervention until gestational week 34+0 
will be included. 
 
<100 mL/kg/day 

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The intervention will continue until gestational week 34+0, but terminates earlier if the infant is transferred to a 
clinic without the logistics needed for the intervention, or if the clinic terminates the supplementation with donor 
breast milk earlier than gestational week 34+0 and the infant is not fed completely with mother´s own milk. After 
the intervention has ended there should be a transition period when the fortification of the breast milk during a 5-
day period gradually goes from 100% of H2 MF to 100% of standard bovine protein-based fortifier, if protein 
fortification still is needed. The infant must not be fed with formula during the intervention period. Thus, the 
interventions terminate when the infant receives the first dose of formula. A specific transition protocol is required 
in order to gradually wean the infant off the exclusive human milk diet. Please see Appendix 10. 
 
Randomisation will be stratified by inclusion centre, gestational week (22+0-24+6 and 25+0-27+6), gender and 
parity. The allocation will be concealed before inclusion, but after randomisation the study is not blinded 
 
The enrolled infants are characterized with clinical data including growth, feeding intolerance, use of enteral and 
parenteral nutrition, treatment, antibiotics and complications collected daily in a study specific case report form 
from birth until discharge from the hospital (not longer than gestational week 44+0). A follow up focusing on 
neurological development, growth and feeding problems will be performed at 2 and 5.5 years of age (corrected). 
Stool, urine, blood and breast milk samples are also collected for microbiology, metabolomic, proteomic and 
immunology analysis in order to study underlying mechanisms. Biopsies from the intestine will be collected if 
acute surgery is performed. Health economic analyses will be done to evaluate the costs and benefits of 
introducing a human milk-based fortifier into Nordic countries.  
6.2  
Selection of study design 
There is no trial in extremely preterm infants comparing a human milk-based breast milk fortifier with bovine 
protein-based breast milk fortifier in which both study groups receive exclusive human breast milk. Such a trial is 
needed to provide evidence that a human milk-based fortifier is superior to a bovine-based one in extremely 
preterm infants also in centres with 100% coverage of breast milk donor banks. Moreover, due to the introduction 
of active intervention for infants also born before gestational week 25 during the last decade, a trial run in Nordic 
centres will have a predominance of infants born in gestational week 23-25, a patient population that could be 
expected to gain the most of a diet free from bovine protein. The trial will be prospective randomised controlled to 
achieve the highest level of evidence. However, it will not be blinded. It would not be possible to prescribe the 
fortifier and prepare the breast milk in a blinded fashion, since the fortifiers are not exactly equal in nutrient 
content and also look different. Instead the assessment of several of the outcomes will be made blinded, such as 
the radiological assessment of NEC. The trial will also have multi-centre design in order to have an inclusion rate 
high enough but also to evaluate feasibility and generalisation in different NICUs. Since it is often difficult to 
distinguish between the diagnoses of NEC and sepsis, and the clinical consequences of NEC and sepsis are equal, 
our primary endpoint of the intervention is the composite variable of NEC, sepsis and mortality. 
6.3  
Selection of treatment  
Preterm infants receiving human milk have better feeding tolerance and lower incidence of NEC than those fed 
cow milk based preterm formula 9. Thanks to a high coverage of donor milk banks, extremely preterm infants are 
exclusively fed with human breast milk in the Nordic countries. Despite this, the incidence of NEC in extremely 
preterm infants is still as high as 12% according to the Swedish National Quality Register (www.snq.se). In 
extremely preterm infants, breast milk has to be supplemented with a multi-nutrient fortifier in order to provide 
extra protein and micronutrients needed to achieve adequate growth and body composition. However, the 
fortifiers used in the Nordic Countries are bovine protein-based. Thus, even when the base diet is 100% human 
milk, extremely preterm infants receive substantial intakes of cow’s milk protein – indeed often a higher intake 
than from the human milk diet since breast milk contains such a low protein level for the needs of extremely 

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preterm infants.  
 
H2MF® is one of a range of human milk-based milk fortifier with different protein contents (but with identical 
contents of micronutrients) designed for preterm infants by Prolacta Bioscience, CA, USA. H2MF® was selected 
for this trial since the protein level (at 2.7 g/100ml) was closest to that commonly provided using cow’s milk- 
based fortifiers available in Nordic countries. Previous trials evaluating this human milk-based fortifier in 
extremely preterm infants receiving exclusive human milk diet, suggest that exclusive human milk diet, devoid of 
cow milk-containing products, is associated with lower mortality and morbidity in these infants without 
compromising growth 3,10,11. Thus, such an exclusive human milk diet has been reported to reduce the incidence of 
NEC, sepsis, BPD and ROP. No side effects have been reported. However, there is no trial in extremely preterm 
infants comparing a human milk-based fortifier with bovine protein-based fortifier in which both study groups 
receive exclusive human milk base diet. Such a trial is needed to provide evidence that a human milk-based 
fortifier is superior to bovine-based one in extremely preterm infants also in centres with 100% coverage of breast 
milk donor banks. A significant positive result of this trial could easily be implemented in the NICUs, given the 
existence of the product and the extensive prior experience of its usage in well over 200 level 3 neonatal units, 
notably in the USA. The manufacturer recommends the use of the human milk-based Prolact CR® as the fat 
supplement to infants receiving H2MF®. Therefore, if an infant in the H2MF® group needs extra enteral fat during 
the intervention period, it will be supplemented with Prolact CR®.  An infant receiving standard bovine protein-
based fortification will be supplemented with the standard lipid products used at the unit. 
7.  
Study population 
7.1  
Sample size 
The incidence of the primary outcome, the composite of NEC, culture-proven sepsis and mortality, was 47.4% 
(1153/2428) in extremely preterm infants in 2007-2014 according to the Swedish Neonatal Quality Register 
(www.snq.se). Only infants surviving more than 3 days were included in this estimation since the inclusion will 
take place after 3 days of life. The NEC incidence (incl. NEC cases that also had a late onset sepsis episode) was 
12.6% (305/2428). The sepsis incidence (without NEC) was 29.7% (720/2428), and the mortality incidence 
(without sepsis and NEC) was 5.3% (128/2428). The reduction of sepsis in the exclusive human milk fed group 
using Prolacta human milk-based products in a previous trial was 37.2% 3. With a similar effect on sepsis there 
would be a reduction to 20.9% (507/2428) in the H2MF® group in our proposed trial (incl. 42 sepsis cases from 
the NEC with sepsis group above because as we estimate a 60% reduction of NEC, please see below). Since there 
is not any previous trial with 100% coverage of breast milk it is difficult to estimate the effect on NEC from 
previous trials. However, a conservative estimation would be a 60% reduction of the NEC incidence to 5.4 % 
(131/2428), as compared to US studies cited above where the fall in NEC was around 75%. Since many deaths 
may reflect undiagnosed NEC and sepsis episodes, mortality is estimated to be reduced by 25% to 4.0% 
(96/2428). Thus, the incidence of the composite outcome is estimated to be reduced to 28.0% (680/2428).  With at 
least 101 infants in each group a reduction from 47% in the control to 28% in the active group would be detected 
at a 5% level of significance and with 80% power using a large sample Gaussian approximation to the comparison 
of two binomial proportions (using a pooled estimate of variance). Thus, the aim would be to enrol a total of 222 
extremely premature infants in the trial to allow for an approximate 10% dropout rate.  
 
However, because there are uncertainties in the effect size presupposed here, the study will be conditioned power 
in the following sense. An evaluation of the overall rate of the primary clinical endpoint of NEC/sepsis/death will 
be made prior to the formal analysis of that aspect of the study data in order to determine whether the trial sample 
size should be re-evaluated and increased in order to continue study enrolment. This evaluation will be based on 
the methodology suggested by Gould et al. 41. Let σ be the pooled rate of this study endpoint in the two arms, i.e. 

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σ=(pcontrol+phuman fortifier group)/2, where p is the incidence of the primary study endpoint in the respective groups. Let 
σ* be the same value, but based on some assumed rates for these rates from the beginning of the trial. Then the 
following sample size multiplier is determined: 
 
f = [σ(1-σ)]/σ*(1-σ*) 
 
If f is greater than 1, then the new sample size, n1, is determined as f x n, where n is the original sample size (per 
group) of 101. If f is less than one, then the study may be analysed at that point for the primary clinical endpoints 
NEC/sepsis/death. Note that an independent statistician not associated with the study conduct will perform this 
conditional analysis. An independent statistician will make a sample size re-estimation when 150 infants have 
been included. Thus, the definitive sample size might be increased (never decreased) based on this interim 
analysis.  
 
Theoretically, if the effect is very low, the calculation according to Gould will suggest a very high number of 
infants needed. Thus, there is a need of a clinically relevant upper limit of the inclusion. With a conservative 
estimation of a 50% NEC reduction in the H2MF® group, a reduction of the composite outcome from 47% in the 
control and 31% in the active would be detected with at a 5% level and 80% power with 145 infants in each 
group. Based on that estimation, the upper limit of included infants would be 322 to allow for an approximate 
10% dropout rate. 
 
An estimation how many infants that would be needed for the composite of culture-proven sepsis and NEC has 
also been made, since this secondary outcome could be of special interest, as it is not affected by mortality rates. 
The incidence of this outcome was 42.2% (1045/2428) and could be estimated to be reduced to 26.2% (638 /2428) 
based on the background data described above. With at least 142 infants in each group, a reduction from 42% in 
the control to 26% in the active group would be detected at a 5% level of significance and with 80% power. Thus, 
with a dropout rate of 10% in total 312 infants would be needed for this outcome. A similar evaluation according 
to Gould et al. as described above will also be made for this outcome after 150 infants have completed the study 
in order to determine whether the trial sample size should be re-evaluated and increased. 
 
7.2 
Inclusion criteria 
• Gestational age at birth 22+0-27+6: based on prenatal ultrasonography. 
• Enteral feeds < 100 mL/kg/day at the day of randomisation. 
• Written informed consent from the legal guardians of the infant.  
• The home clinic of the infant has the logistics of maintaining the intervention until 
gestational week 34+0  
7.3  
Exclusion criteria 
• Lethal or complicated malformation known at the time of inclusion 
• Chromosomal anomalies known at the time of inclusion 
• No realistic hope for survival at the time of inclusion 
• Gastrointestinal malformation known at the time of inclusion 
• Abdominal surgery before the time of inclusion 
• Participation in another intervention trial aiming at having an effect on growth, nutrition, 
feeding intolerance or severe complications such as NEC and sepsis 
• Infants having nutrient fortifier or formula prior to randomisation 

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7.4 
 Withdrawal criteria 
Subjects may be discontinued from the study for the following reasons: 
• If it is in the best interest of the subject, based on the responsible physician’s discretion. 
• The parents/legal guardian, cancel the informed consent agreement. 
 
The reasons for exclusion of a subject should be stated in the Case Report Form (CRF). Discontinued subjects 
should always be included in the safety and tolerance analysis. 
 
Parents have the right to discontinue participation in the study at any time. Although the infant for any reason - 
physician’s or parents' decision - stops receiving H2MF®, he or she will still continue to be included in the study 
for clinical data collection, unless the parents oppose that. Thus, the infant will be included in the intention-to treat 
analyses as long as CRF protocol is completed to gestational week 36+0. That also applies to situations when the 
intervention ends because the infant has received formula or bovine-based nutrient fortifier. Note that parents do 
not have to give any reasons why to cancel the study. If parents wish to avoid sampling, they will be asked if the 
infant can remain in the study even if the sampling is omitted. 
 
If the stated reason for discontinuation is an adverse event, the principal investigator is to actively participate in all 
actions until the problem is resolved or the situation has become stable. 
 
The infant will not be excluded from the study if H2MF® has not been supplemented due to poor study 
compliance. Instead, a per protocol analysis will be made based on the length of the supplementation (please see 
statistics in section 20). The parents will be asked if their infant is included in another trial, both at inclusion and 
the end of the trial. The infant can be included in another trial as long as the other trial does not influence the 
infants’ nutrition, growth, feeding tolerance and risk of NEC. 
 
The attending physician or investigator has the right to withdraw the infant from the trial at any time on the 
assessment made that continued participation would be a serious health risk. 
 
A transfer to a clinic that does not participate in the study is not a reason for withdrawal. The situation is handled 
according to 9.1.12. 
 
7.5 
Study termination 
Please see the section 7.1 regarding study population. By ethical reasons, the study can be terminated prematurely 
based on a decision of the sponsor and the DSMB, if the primary outcome is significantly lower (with a 
significance level <0.001) in the H2MF® than in the standard fortification group in the interim analysis made after 
150 infants have completed the neonatal period. The study can also terminate prematurely if any severe 
complication is significantly more common in the H2MF® than in the standard fortification group in any interim 
analysis included in the safety analyses in the trial, please see section 13.3 and 13.4. 
 

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8  
Study period 
In total 222 extremely preterm infants are planned to be included. 270 extremely preterm infants are born in the 
catchment area of Gothenburg, Linköping, Stockholm, Uppsala and Umeå every year. Since not all available 
infants could be expected to be enrolled, the inclusion time is supposed to be approximately two-three years.  
9  
Methods 
9.1 
Practical arrangements 
9.1.1 Inclusion and randomisation 
The study subject will only be enrolled at level III NICUs. The infants should be randomised 1:1 to receive either 
the human breast-milk based H2MF® or the standard bovine protein-based nutrient fortifier before oral feeds have 
reached 100 ml/kg/day. The written informed consent could preferably be obtained from the parents well before 
the oral feeds have reached 70 ml/kg/day in infants that are expected to fulfil the inclusion criteria, since they 
should have time enough to give a well-thought-out consent. Thus, in practice, the informed consent can be 
obtained already at the second and third day of life but not after the oral feeds have passed 100 ml/kg/day.  
 
Randomisation, however, can only be done when all the inclusion criteria are fulfilled, and there are no exclusion 
criteria.  
 
Procedures: 
• Control that the infant fulfils all the inclusion criteria, please see section 7.2 
• Control that the infant does not have any exclusion criteria, please see section 7.3 
• Clear oral information to the parents, use an interpreter if needed, and give them the written information 
(Appendix 6). 
• Clear information to the parents that the participation is optional, and that they can withdraw their infant´s 
participation at any time without explaining why. Also inform about the basics in EU´s General Data 
Protection regulation (GDPR).  
• All eligible infants should be added in the Screening log anonymously. Thus, all infants at an inclusion 
site that were born before gestational week 28+0 and survived during the first 3 days should be included in 
the Screening log. That means that also infants that by accident reached enteral feeds >100 mL/kg/day 
before there was an opportunity to include them should be included in the Screening log. Also, eligible 
infants belonging to the region but born and treated outside the inclusion site should be included in the 
Screening log. 
• The informed consent form should be signed by legal guardians (appendix 7). If there is only one legal 
guardian, an informed consent from this person is sufficient. The attending physician shall note this in the 
medical record of the infant. 
• The information can be given via telephone, if one of the parents cannot attend at the information at the 
hospital. This parent has to sign the informed consent form and send to the hospital before inclusion. 
• The informed consent form should also be signed by a physician. It does not have to be done at the same 
date as the parents but it must be before the randomisation. 
• Randomisation is made according to section 12.5. The legal guardians are informed what study group their 
infant belongs to. The allocation and the study identification number (ID) should also be indicated in the 
Identification list and the CRF immediately.  
• All infants included in the study should be added in the Identification list, which include the infants’ 
personal number (“personnummer”) and the patient ID. Inclusion sites have separate series of patient 
identification number: Umeå starts with 01001, Uppsala with 02001, Linköping with 03001, Göteborg 

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with 04001, Örebro (Uppsala´s catchment area) with 5001, Solna (Stockholm´s catchment area) with 6001 
and Huddinge (Stockholm´s catchment area) with 7001. New cases are added consecutively at each site. 
Study group allocation is also added in the identification list. This list and the Screening log should be 
kept safe in the Investigator file at each study centre.  
• The intervention is started as described in section 9.1.4 
• A CRF (Appendix 8) is prepared and background data is filled in. The day the child was born is 
considered as day 0. Day 1 starts at moon (24.00/0.00).  
• The study nurse at the study site should be informed. 
• The tasks of the study nurse: 
o Inclusion and exclusion criteria are controlled. 
o Check that the Screening log and the Identification list are completed. 
o Note in the medical records:  
”Patienten deltar i den kliniska prövningen N-Forte. Barnets PatientID/StudieID är:_________. 
Studiegrupp:__________ ( skriv in H2MF eller standardberikning) 
H2MF är en humanbaserad bröstmjölksberikning och effekten av denna är det som testas i studien. 
Interventionen pågår från inklusionsdag__________ till gestationsvecka 34+0. 
Ansvarig prövare på denna studieort är:_______________________. 
Ansvarig prövare för hela studien är Thomas Abrahamsson, Region Östergötland. 
Föräldrar har lämnat informerat samtycke, datum___________.” 
o Write the Patient-ID in medical files of the infant; both in the medical records and the Cardex (if 
there is one at the study site) 
o Label all study material of the included infant with the Study ID. 
o The informed consent is stored in investigator file. 
o Give a copy of the signed informed consent form to the parents/legal guardians. 
o Prepare a study file for the infants.  
 
9.1.2 Twins 
Twins and larger multiple births will be allowed into the study. Randomisation will be stratified by parity to avoid 
imbalanced randomisation of multiple births. Twins will be randomised together. Thus, they will get the patient 
identification number and also the same nutritional protocol. Twin number one will get the suffix A and number 2 
the suffix B (e.g. 101 A and 101 B). There will also be stratification for gender. Because of limitations of 
randomisation/stratification procedure, a twin pair will be considered having the same gender when the 
randomisation is made. Thus, enter the gender of the first-born twin in the program (please see paragraph 12.5) 
 
9.1.3 Investigator file  
An investigator file will be kept locked at each study site. It should include the study protocol (including all 
appendices), parental information, informed consent form, CRF, copy of the ethical approval, screening log and 
identification list. 
 
9.1.4 Start of the intervention 
The intervention starts before the enteral feeds have reached 100 mL/kg/day, the parents have given their written 
informed consent (please see 9.1.1) and randomisation is completed (please see 12.5). Prescription and 
administration of the study product is described in 9.1.5. 
 

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9.1.5 Prescription, preparation and administration of the study product 
A specific protocol of preparing H2MF® is described in Appendix 17. The standard bovine protein-based milk 
fortifier used at the specific site will be prepared according the recommendation of manufacturer. Each centre will 
have a co-ordinator associated to the milk kitchen where the study product is prepared.  
It is essential that the clinician and/or the dietician that prescribe the enteral feeding knows in which study group 
the individual patient belongs to.  
 
1. The attending physician prescribes the enteral nutrition every day during the NICU stay, including the 
source of breast milk (own mother’s or donor milk), total volume and the level of fortification (normal, 
half normal or no fortification), based on which intervention group the infant is allocated to. The study is 
not blinded for the attending physician. 
 
a. The level of fortification is prescribed stepwise according local guidelines in order to achieve 
appropriate protein intake, see Appendix 9. 
b. The daily level of fortification for each infant is primarily based on protein intake and the aim is 
4.0-4.5 g/kg/day, even though a lower intake is acceptable when the infant is approaching term age 
and is growing adequately. 
c. Macronutrient analyses of own mother’s milk are performed weekly using an infrared breast milk 
analyser. Breast milk analyses of donor breast milk are performed once for each batch. 
d. The daily prescription is based on the analysed protein content in the breast milk which the infant 
receives. Charts for fortification levels for each study product and for different ranges of breast 
milk protein content are found in Appendix 9. 
e. Daily prescriptions are individualized, taking clinical condition, fluid balance and parenteral 
nutrition into consideration 
f. Daily prescriptions should be done in order to ensure that intakes of all nutrients are within 
recommended ranges. The Nutrium software (Nutrium AB, Umeå, Sweden) which is provided to 
all study sites has included the study products in its algorithms. Support from the level III sites will 
be given to physicians and dietitians if needed for the nutritional calculations.  
g. Additional vitamins (Multivitamin Unimedic, typical dose 7 drops/kg [0.25 mL/kg] for infants < 2 
kg) always need to be prescribed to infants who receive the H2MF product. 
h. When less than full dose fortification is given, additional micronutrient supplements (especially 
calcium and phosphorous supplements) are usually needed. 
i. Fat supplements can be considered if energy intake is low and growth is suboptimal. The infants 
receiving H2MF® will be supplemented with the human milk-based Prolact CR®, while the infants 
receiving standard bovine protein-based fortification will be supplemented with the standard lipid 
products used at the unit. 
 
 
2. The preparation of fortified milk is performed in the NICU milk kitchen by trained staff according to the 
manufacturer’s instructions, the prescribed dose and the mixing table in Appendix 9 and the investigator 
file. The milk is prepared in advance for each meal during the next 24-hour period. Fortified milk bottles 
are marked with patient name and/or ID and date and kept refrigerated until use. The staff in the milk 
kitchen are not blinded to the intervention. 
 
3. The milk is fed to each infant by the attending nurse according to local guidelines.  

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.  
9.1.6 Case report form (CRF) and Nutrium 
The CRF is established at randomisation. The study nurse fills in all the background factors from pregnancy and 
birth in collaboration with the investigators of the centre. The CRF will cover the period in the neonatal ward 
from birth, i.e. also before inclusion, until discharge from the hospital/neonatal home care (but not longer than 
GW 44+0). The day the child was born is considered as day 0. Day 1 starts at moon (0.00 am).. Regarding the 
transfer to another clinic, please see 9.1.11. Upon discharge home the CRF should be completed including 
evaluation of clinical diagnoses. The CRF should be controlled and signed by any of the investigator at the actual 
site. The original CRF shall be submitted to the sponsor/PI after been monitored by the study monitor. One of the 
copies should be submitted to the data entrance company. One copy of the CRF should always be stored at the 
home clinic.  
 
All data on nutrition and other fluids will be entered daily in the Nutrium software (Nutrium AB, Umeå, Sweden) 
which is provided to all study sites. Support will be given to physicians and dietitians if needed for the nutritional 
for this entrance of data. All nutritional data will be entered from birth to gestational week 34+0, when the 
intervention ends.  
The data of infants included in the N-forte trial will be transferred to the database after the study has been 
completed. 
 
 
9.1.7 Samples at 7 ± 2 days of age 
Note adverse events and possible antibiotic treatment since the previous registration in the CRF. The actual 
weight, length and head circumference at 7 days of age is noted in the CRF.  
Stool, urine, blood and breast milk samples are collected by the nurse according to the sampling instructions 
(Appendices 12-15). Breast milk samples are only collected if the mother has an excess of it. It is the breast milk 
that infants receive at day 7 that should be collected. Blood samples are only collected when routine blood 
sampling is made. Note in the CRF that the samples have been collected.  
 
Label the tubes CLEARLY with the study specific adhesive label, study identification number type of samples 
(stool, urine),  “Day 7” and the date of the sampling. WITHOUT IDENTIFICATION NUMBER AND DATE 
THE SAMPLE IS USELESS! 
 
9.1.8 Samples at 14 ± 2 days of age 
Note adverse events and possible antibiotic treatment since the previous registration in the CRF. Actual weight, 
length and head circumference at 14 days of age is noted in the CRF. 
Stool, urine, blood and breast milk samples are collected by the nurse according to the sampling instructions 
(Appendices 12-15). Breast milk samples are only collected if the mother has an excess of it. It is the breast milk 
that infants receive at day 14 that should be collected. Blood samples are only collected when routine blood 
sampling is made. Note in the CRF that the samples have been collected.  
 
Label the tubes CLEARLY with the study specific adhesive label, study identification number, type of samples 
(blood, stool, breast milk, urine), “Day 14” and the date of the sampling. WITHOUT IDENTIFICATION 
NUMBER AND DATE THE SAMPLE IS USELESS! 
 
9.1.9 Samples at 21 ± 2 days of age 
Note adverse events and possible antibiotic treatment since the previous registration in the CRF. Actual weight, 
length and head circumference at 21 days of age is noted in the CRF. 

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Stool, urine and breast milk samples are collected by the nurse according to the sampling instructions (Appendices 
13-15).  Breast milk samples are only collected if the mother has an excess of it. It is the breast milk that infants 
receive at day 21 that should be collected. Note in the CRF that the samples have been collected.  
 
Label the tubes CLEARLY with the study specific adhesive label, patient identification number, type of samples 
(stool, urine),  “Day 21” and the date of the sampling. WITHOUT IDENTIFICATION NUMBER AND DATE 
THE SAMPLE IS USELESS! 
 
9.1.10 Samples at 28 ± 2 days of age 
Please, see sample collection at 14 ± 2 days of age (9.1.8).  
 
Label the tubes CLEARLY with the study specific adhesive label, patient identification number, type of samples 
(stool, urine),  “Day 28” and the date of the sampling. WITHOUT IDENTIFICATION NUMBER AND DATE 
THE SAMPLE IS USELESS! 
 
9.1.11 The end of the study intervention, gw 34+0 
The intervention ends at gestational week 34+0, but can end earlier (please see 9.1.12). Note in the CRF the actual 
date the intervention ends. Actual weight, length and head circumference is noted in the CRF. Samples shall be 
collected before intervention ends! Please, see 9.1.13 
 
Infants that has completed the study to gestational week 34+0: 
1. The H2 MF group: 
After the intervention has ended there should be a transition period when the fortification of the breast 
milk during a 5-day period gradually goes from 100% of H2 MF to 100% of standard bovine protein-based 
fortifier, if protein fortification still is needed (please see Appendix 10). Still it is the staff in the milk 
kitchen that prepares the breast milk by using the transition protocol. 
A transition protocol must also be applied if formula is going to be introduced directly after the 
intervention period ((please see Appendix 10). If the infant has received Prolact CR as fat supplement it 
should end after the transition period is over. The standard lipid supplement will then be used if the if the 
child still need extra lipids.  
2.  The standard bovine protein-based fortifier group:  
The protein fortification continues according the routine of the clinic.  
 
Infants that end the study intervention before gestational week 34+0 
The most common reason for early discontinuation is that the infants are going to be transferred to a study 
site without the logistics for maintaining the intervention.  
 
1. The H2 MF group: 
A transition period is needed, when the fortification of the breast milk during a 5-day period gradually 
goes from 100% of H2 MF to 100% of standard bovine protein-based fortifier, if protein fortification still 
is needed (please see Appendix 10). The staff in the milk kitchen at the new site will prepare the breast 
milk by using the transition protocol. It is crucial that the transition protocol (Appendix 10), the product 
description (Appendix 17) and H2 MF much enough for the transition period, follow the infant to the new 
site. The investigator should ensure that the attending physician at the new site get information about the 
study at the day of transferal. 
 
2. The standard bovine protein-based fortifier group:  
The protein fortification continues according the routine of the clinic.  

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9.1.12 Transfer to another neonatal unit 
Take copy of the CRF. Keep the original in the study folder of the infant and send a copy to the receiving clinic. 
Basically, only infants with a home clinic that will be able to continue with the study product will be enrolled in 
the trial. However, if the receiving unit does not have the logistics to continue with the products until gestational 
week 34+0, a transition protocol shall be applied as described in section 9.1.11. The attending nurse and physician 
report that the infant is included in the study, that the transition protocol has to be followed and how to fill in the 
CRF.  
 
Thus, the aim is to continue with the intervention at the new clinic until gestational week 34+0. The responsible 
physician and nurse should report to the receiving unit in which study group the infants belong to. Also, the 
investigator and the study nurse and/or the staff at the milk kitchen shall confirm with the receiving unit that they 
have got the information. It might be a time gap of 1-3 days before this information can be forwarded (at 
weekends etc.). While waiting for this information, the infant must not get any bovine protein-based fortifier or 
formula. If the staff at the new clinic do not want to wait with the fortification, they can use H2 MF. However, 
when the staffs have got the information about the study group, the infant should start with the right study 
product. The dietician at the original clinic will be a resource helping smaller neonatal units calculating the degree 
of fortification. In most cases the fortification does not have to change during the short time period that is left until 
the infant has reached gestational week 34+0. 
 
The study nurse will contact the new clinic as soon as possible to help them with the CRF, sampling etc. In 
neonatal units that belong to the catchment area of Gothenburg, Linköping, Stockholm, Uppsala and Umeå, there 
will be one principal investigator and nurse that will co-ordinate procedures at their site. The principal investigator 
will be responsible for completing and signing the CRF when the neonatal follow up period has ended at 
discharge from the hospital. The fulfilled part of the original CRF shall be submitted to the sponsor/PI after been 
monitored by the study monitor. One of the copies of this part should be submitted to the data entrance company. 
One of these copies should follow the infant to the next study site. One copy of the CRF should always be stored 
at the home clinic. The non-used (empty) part of the original CRF should follow the infant to the next study site 
 
9.1.13 Sample at gestational week 33+0-33+6  
Please, see sample collection at 14 ± 2 days of age (9.1.8). The sample shall be collected before the interventions 
ends gw 34+0. Topical anaesthesia can be used before the blood sample is collected and a larger volume can be 
taken, 2.5 mL 
 
Label the tubes CLEARLY with the study specific adhesive label, patient identification number, type of samples 
(stool, urine),  “GW 36” and the date of the sampling. WITHOUT IDENTIFICATION NUMBER AND DATE 
THE SAMPLE IS USELESS! 
 
9.1.14  Magnetic resonance imaging (MRI) 
Applicable in centers that have MRI as routine examination when the infants have reached full term (gw 37+0-
42+0). Data from MRI will be filled in a specific protocol and added to the study file of the infant. 
 
9.1.15 The end of the neonatal period 
The follow up of the neonatal period ends at discharge from the hospital and/or the neonatal home care but not 
later than gestational week 44+0. The CRF should be completed, controlled and signed by any of the investigator 
at the actual site. Note that the result of the ROP examination shall be entered in CRF after gestational week 42+0. 

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Also, data on weight, length and head circumference from gestational week 36+0 +/2 days and from the day of 
discharge (or gestational week 44+0, whatever comes first) should be entered in the CRF. 
 
9.1.16  Follow up at 2 years of age 
A follow up focusing on neurological development, growth and feeding problems will be performed at 2 years (± 
3 months) of age (corrected). The follow up will be identical with the national follow-up program for extremely 
preterm infants that have been implemented in all paediatric clinics in Sweden (www.neo.barnlakarforeningen). In 
Sweden, all data from the national 2-year follow-up is entered in the Swedish Neonatal Quality Register (SNQ). 
Data in the SNQ will be used for analyses in the study. The 2-year follow-up includes a questionnaire that covers 
the period from the discharge from the hospital until 2 years of age. Cognitive function is tested with Bayley’s test 
III by a psychologist and neurological disability by an experienced paediatrician and/or physiotherapist.  Nurses 
coordinate the follow-up and meet with the family, measures weight, height and head circumference and complete 
a questionnaire about respiratory diseases, allergic diseases, feeding issues and socioeconomic factors.  
 
9.1.17 Follow up at 5.5 years of age 
A follow up focusing on neurological development, growth and feeding problems will be performed at 5.5 years 
(± 3 months) of age. The follow up will be identical with the national follow-up program for extremely preterm 
infants that have been implemented in all paediatric clinics in Sweden (www.neo.barnlakarforeningen). In 
Sweden, all data from the national 5.5-year follow-up is entered in the Swedish Neonatal Quality Register (SNQ). 
Data in the SNQ will be used for analyses in the study. Cognitive function is tested with WPPSI-IV by a 
psychologist and neurological disability by an experienced paediatrician and/or physiotherapist. A physiotherapist 
will also perform the ABC Movement test. Nurses coordinate the follow-up and meet with the family, measures 
weight, height and head circumference and complete a questionnaire about respiratory diseases, allergic diseases, 
feeding issues and socioeconomic factors.  
 
9.1.18 Deaths 
The sponsor shall be notified, if an infant dies before discharge from the neonatal ward. The information shall 
include study group and the cause of death. The CRF shall be filled in as completely as possible. That also 
includes diagnoses that would otherwise be filled in at discharge. 
 
9.2 
Assessment of efficacy  
9.2.1 Clinical variables  
Definitions, please see variables in section 5. Data entered in the CRF and Nutrium (9.1.6) 
9.2.2 Sample collection 
It is optional for each study site to collect samples. The study site must have the logistics to take care of the 
samples and store them. Sufficient statistical power is usually reached with fewer cases in laboratory than clinical 
outcomes.  
 
Blood samples 
Blood samples are collected in EDTA tubes for plasma and separate pre-prepared tubes for masscytometry of 
blood cells (CyTOF) 1, 2 and 4 weeks (± 2 days) of age and at gestational week 33+0-33+6. The plasma and cells 
will be stored at -70°C.  
 

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Faecal samples 
Faecal samples are collected at 1, 2, 3 and 4 weeks (± 2 days) of age and at gestational week 33+0-33+6  i sterile 
tubes. The sample is taken with a special spatula from the diaper. It is stored immediately in -20º C or -70º C. As 
soon as possible they should be transferred frozen to a -70º C freezer. PUT AS MUCH STOOL AS POSSIBLE 
IN THE TUBE (aim at 1 mL/”1cm”). It is an advantage if you use 2-3 tubes. 
 
Urine samples 
Urine is collected 1, 2, 3 and 4 weeks (± 2 days) of age and at gestational week 33+0-33+6  using cotton wool 
(first urine after 8.00 am), centrifuged and divided into 2-3 aliquots of about 1 mL of urine (placed in 1.5 ml 
sterile Eppendorf tube).  The urine sample is stored in a freezer and picked up by the lab personnel to be stored at 
-70 ° C. 
 
Breast milk samples 
Breast milk samples are only collected if the mother has an excess of it. The sample is collected from the breast 
milk that the infant will be fed with at the actual day (after fortification): at 1, 2, 3 and 4 weeks (± 2 days) of age 
and at gestational 33+0-33+6 in sterile 10 ml tubes. The sample is stored in -20ºC. As soon as possible they 
should be transferred frozen to a -70º C freezer.  
 
9.3 Assessment of safety 
 
9.3.1 Adverse events  
Adverse events will be recorded in the CRF and in some cases they will be reported on as soon as they are 
detected, which is described in detail in section 13.  
10. 
Concomitant therapy 
Besides the supplementation of the breast milk fortifier and the samples collected in the trial, the participating 
infants will not be treated differently compared to infants outside the trial. No drug is contraindicated during the 
course of the study. Antibiotics and other relevant drugs are recorded in the CRF. 
11. 
Concomitant diseases 
All diseases/adverse events that the infant has before inclusion shall be recorded in the CRF since they might 
confound the results. All diseases or symptoms that the infant has during the study shall be recorded as adverse 
events in the CRF. Please see section 13. 
12. 
Study requirements 
12.1 
Study product 
A description of the product (H2MF®), how it is stored, and how it is prepared is given in Appendix 17. In 
addition, the safety steps incorporated into the collection of the milk and in the production of the preparation also 
are indicated in that appendix. 
 

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12.2 Dose, administration, storage 
Whether the infant has received the study products (breast milk fortifier according to 12.2.1 and 12.2.2), will be 
registered in Nutrium every day from the start to the end of the intervention period. 
 
12.2.1 Active study group 
A description of the products (H2MF® and Prolact CR®), how they are stored, and how they are prepared is given 
in Appendix 17. Please, also see 9.1.5. 
 
12.2.2 Control study group 
The standard bovine protein-based milk fortifier used at the specific site will be prepared according the 
recommendation of manufacturer. Please, also see 9.1.5. 
12.3 Storage 
Storage of the H2MF® and Prolact CR® is at -20º C. The expiration of the specific batch(es) provided are 
indicated on the bottles. 
12.4 Study product accountability 
Prolacta Bioscience, CA, USA, manufactures the active products. Details of product manufacture, 
distribution, stability, storage conditions and disposal of unused material are at Appendix 17. The standard 
bovine protein-based milk fortifier used at the specific site will be prepared according the recommendation of 
manufacturer. 
 
Each study centre will be expected to record the use of investigational and standard care product in Nutrium. 
12.5 Randomisation 
This is a randomised trial. Randomisation will be based on the following stratification variables: primary 
enrolment site, gestational week (22+0-24+6 or 25+0-27+6), singleton/twin and gender. Because there are 4 
stratification variables and not a particularly large study, an adaptive randomisation scheme will be used based on 
the method of minimisation. This will include a biased-coin randomisation scheme as needed in the adaptive 
scheme. 42 A specific computer program will be used to accomplish this scheme and the program will be carefully 
validated prior to incorporation in the study. The allocation will be concealed before inclusion, but after 
randomisation the study is not blinded.  
 
When the infant fulfils the requirement of inclusion (please see section 9.1.1), a web-based randomisation service 
centre will be used:  Randomize.net (Interrand Inc., Ottawa, Ontario, Canada). The patient identification number 
will be the consecutive number in the identification list at the study site, while the randomisation service centre 
will provide to which study group the infant shall be allocated. It is the patient identification number that should 
be used in the CRF and on samples etc.  The allocation should be indicated in the identification list and also in the 
CRF immediately. To confirm eligibility inclusion and exclusion criteria will be checked for, including signed 
parental consent, before randomisation is done. 
 
Twins will be stratified for as indicated above. They will be randomised together. Thus, they will get the same 
randomisation number and also the same nutritional protocol. Twin number one will get the suffix A and number 
2 the suffix B (e.g. 101 A and 101 B). There will also be stratification for gender. Because of limitations of 
randomisation/stratification procedure, a twin pair will be considered having the same gender when the 
randomisation is made. Thus, enter the gender of the first-born twin in the program.  

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All study material of the included infant should be labelled with the patient identification number. The patient 
identification number will be five-digit and will start with 01001 in Umeå 02001 in Uppsala, 03001 in Linköping, 
04001 in Göteborg, 5001 in Örebro, 6001 in Solna and 7001 in Huddinge.  
 
13. 
Adverse Events  
The morbidity of premature infants with extremely low birth weight is very high. Thus, a high incidence of 
serious and non-serious adverse events (SAE) unrelated to the study product could be expected in the participating 
infants. Also, mortality rate is high; please see background figures in section 3. Typical serious conditions 
affecting premature infants are listed below (13.1.2). In principle, none of these are unexpected. 
 
As a consequence, special consideration must be given as to how the SAE is reported and followed up. 
 
The use of H2MF® is thoroughly studied and there no suggestions that H2MF® will increase the incidence of AE, 
SAE, or death. Rather, previous research supports that feeding problems, NEC, sepsis and mortality is lower 
among infants receiving H2MF® (please see section 3).  
13.1 Definitions 
13.1.1 Non-serious adverse events 
Non-serious adverse is reported in the CRF and will be analysed after all infants have completed the neonatal 
period. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
13.1.2 Serious adverse events 
 
Expected severe adverse events (SAE 

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13.1.3 Suspected unexpected serious adverse reaction (SUSAR) 
A high incidence of serious adverse events (SAE) unrelated to the study product could be expected in the 
participating infants. In principle, none of these are unexpected. However, if there is a suspected unexpected 
serious adverse reaction (SUSAR), it should be reporting according to 13.2.2. Whether an infant has got a SUSAR 
or not is decided by the investigator or the attending physician at the study site. 
13.2 
Reporting serious adverse events 
 
13.2.1  
Reporting SAE 
As a consequence of what is discussed above, SAE does not have to be reported immediately to the principal 
investigator or sponsor. Instead, interim safety analyses will be made according to a specific schedule - see 13.3 – 
to detect a potential increased incidence of SAE.  
 
Thus, all SAE will be recorded in the CRF. In principle, they will be the same as the clinical morbidity variables 
addressed in paragraph 5. 
 
13.2.2 
Reporting lethal cases and SUSAR 
Investigator or the attending physicians at the study site are required to report SUSARs to the sponsor within 24 
hours. A report form must be completed (Appendix 5) and promptly sent by mail or email to the sponsor. Phone 
number and address to the sponsor can be found at the research protocol first page and on the report form. 
 
The sponsor is required to report SUSARs to the manufacturer, Prolacta Bioscience, and the Data and safety 
monitoring board (DSMB). Deaths are expected to occur, and the sponsors are not required to report these. The 
DSMB will, however, be contacted if there is an unexpected high mortality among in the participants.  
13.3 
Interim analysis  
Interim analysis will be carried out for safety analyses, please see section 13.4. Also, it will be made for the final 
sample size calculation, please see 7.1. The interim analyses will be made by an independent statistician. 
 
• Severe infection such as pneumonia, sepsis or meningitis 
• CMV infection, upper respiratory viral infection 
• NEC, SIP and /or need of abdominal surgery 
• Respiratory distress syndrome and BPD 
• Intracranial bleeding, periventricular leucomalacia or hydrocephalus 
• Lung bleeding 
• Pneumothorax, pleural effusion 
• Pulmonary hypertension 
• PDA 
• Retinopathy of prematurity (ROP) 
• Death 
 
These events do not have to be reported immediately (please see 13.2.1). 
 

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13.4 
Data and safety monitoring board (DSMB) 
An independent DSMB (Appendix 4), consisting of at least three members with at least one specialist in 
Neonatology and one Biostatistician, will have regular meetings to make interim safety analyses. Such an interim 
analysis shall be made after 50, 100 and 150 completed CRFs until discharge from the hospital. If any of the SAE 
is significantly more common (p<0.05) in the active than the control group, the DSMB will bring in all medical 
data on infants affected by this specific SAE. The DSMB will then assess the causality between the use of H2MF® 
and the specific SAE in the affected infants. Based on this analysis the DSMB will decide if the trial can continue 
or not after consultation with the co-ordinating principal investigator and the sponsor. The DSMB may decide that 
it is ethically correct to pursue if the positive effects of the active intervention outweigh the SAE. 
 
In case of a very strong effect of the active treatment, the study can be terminated prematurely based on a decision 
of the sponsor and the DSMB, if the primary outcome is significantly lower (with a significance level <0.001) in 
the H2MF® than in the standard fortification group in the interim analysis made after 150 infants have been 
included 43,44. If the significant level is >0.001 the study enrolment will continue (please see section 20).  
14 Data collection 
This is discussed in detail in section 14.1. In principle, all data from the neonatal period in the CRF originate from 
medical records of the patient. All materials will be labelled with the patient identification number. To avoid 
mixing of the participating infants (e.g. if they taking care of in the same room), the medical file, bed etc. will also 
be labelled with the patient identification number. Data from completed CRFs will be entered into the database 
continuously. A copy is sent to the study monitor. A study database will be created by a CRO and the data will 
subsequently be forwarded to the sponsor and the co-ordinating principal investigator. The entrance of data to the 
database will also be made by a CRO. Data from the 2 and 5.5 years follow up originate from the Swedish quality 
register. 
 
All data on nutrition and other fluids will be entered daily in the Nutrium software (Nutrium AB, Umeå, Sweden) 
which is provided to all study sites. All nutritional data will be entered from birth to gestational week 34+0, when 
the intervention ends.  
The data of infants included in the N-forte trial will be transferred to the database after the study has been 
completed. 
14.1 Case Report Form (CRF) 
The CRF should be handled in accordance with instructions from the sponsor. All CRF should be filled out 
completely by examining personnel or the investigator/study nurse. The CRF are reviewed, signed, and dated by 
the investigator. 
 
The CRF is established at randomisation. The study nurse fills in all the background factors from the 
pregnancy and birth in collaboration with the investigators of the centre. The study nurse regularly checks 
the CRF to the patient medical record and completes the missing data. The CRF is kept together with 
medical file during the neonatal period and the infant´s study file after discharge from the clinic. In 
principle, all data collected in the study will be added in the CRF. Regarding the transfer to another clinic, 
please see 9.1.12. Upon discharge the CRF should be completed including evaluation of clinical 
diagnoses. The original CRF shall be submitted to the sponsor/PI after been monitored by the study 
monitor. One of the copies should be submitted to the data entrance company. One copy of the CRF 
should always be stored at the home clinic.  A study database will be created by a CRO and the data will 
subsequently be forwarded to the sponsor and the co-ordinating principal investigator.  

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If a question in the CRF cannot be answered, this box shall be barred. When making changes or corrections, cross 
out the original entry with a single line, and initial and date the change.  Do not erase, overwrite or use correction 
fluid or tape on the original.  
. 
If the CRF is sent by mail, it should be sent as a certified mail (“rekommenderat brev”)  
 
 
14.2 Data source 
In principle, all data in the CRF originate from data that also can be found in the medical records. Nutritional data 
on each case will transferred from Nutrium (Nutrium AB, Umeå, Sweden)  to the sponsor in the end of the trial 
and added to the database. Likewise, the data from the 2- and 5.5-years follow up in the quality register SNQ 
(www.snq.se) will be transferred to the sponsor and added to the study database. There is a list of the data sources 
in the investigator file. 
15 Data quality assurance 
15.1. Study monitoring 
Site visits will be conducted by an authorized representative of the sponsor to inspect study data, subjects’ 
medical records, and CRF in accordance with current Good Clinical Practices (GCP) and the respective local and 
national government regulations, internal SOP and guidelines. A monitor assigned by the CRO will conduct 
regular investigational centre visits for the purpose of study monitoring. The investigator will facilitate monitoring 
of the study and permit authorized representatives of the sponsor and international or national health authorities to 
inspect facilities and records relevant to this study. CRF data will be monitored during each visit according to the 
Study Monitoring Plan. All source data, subjects file, lab sheet etc. must be kept on file in accordance with GCP 
guidelines, i.e. for 10 years.  
 
15.2. Study monitoring 
CRO will employ appropriate quality control measures in critical processes in data management (including data 
handling and data transfers) to ensure that data reported from the study are true and reliable. 
 
 
16 Ethics 
Research including ELBW infants is crucial in order to achieve knowledge of possible causes underlying the 
development of severe complication and finding preventive strategies in this age group. A growing literature 
supports the use of human milk-based protein fortifier in clinical practise (please see background, section 3). 
Intervention studies are needed to refute or confirm the suggested effect H2MF®. We do not perceive any major 
health hazard with the study design. H2MF® is considered to be safe and could conceivably be used for simple, 
safe and effective prevention of severe complication in ELBW infants. Safety will also be monitored by a study 
specific DSMB.  
 

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Autonomy is very limited for this patient population. To adhere to ethical principles is therefore very important. 
Parents are the deputies of their child. A written informed consent is obtained from both parents prior to 
enrolment. Parents have the right to discontinue participation in the study at any time. 
 
Blood sampling can cause pain and also affect the haemoglobin levels. Therefore, sampling is co-ordinated with 
other blood samples and only small amounts are collected at each time. Confidentiality is important and therefore 
only the patient identification number will be used to identify the infant in the study protocol and the test tubes. 
 
Infants or their family have no specific benefit of participating in the trial besides to the possible positive effect of 
H2MF® in the active group. Besides the supplementation of the breast milk fortifier and the samples collected in 
the trial, the participating infants will not be treated differently compared to infants outside the trial. The potential 
benefit for future ELBW infant, however, could be substantial. Finding an intervention that reduces NEC, sepsis 
and mortality would have a major impact of the well-being in this patient population. It would also a beneficial 
effect on health economics. Only randomised clinical trials will give sufficient evidence for a general 
recommendation of a new treatment.  
 
The study is made in collaboration with the company Prolacta Bioscience producing H2MF®. The company also 
helps with randomisation and packaging. Without the aid from the company this trial would be difficult to 
conduct. The study, however is investigator-initiated, and the results are entirely owned by the principle 
investigator. None of the investigators have a financial interest in Prolacta Bioscience. Only excess breast milk 
produced by the donating mother that is beyond the consumptive needs of her nursing child are accepted. The 
donating mother signs informed consent form stating the rights and responsibilities of the donating mother as well 
as the payment for the provided breast milk. 
 
The potential benefits are considered to outweigh the discomfort to the infants and their families. The study is 
conducted according to ICH/GCP guidelines and was approved by the regional ethical review board at Linköping 
University (Dnr 2018/193-31). 
17. 
Informed consent 
The information letter and the informed consent form can be found in Appendix 7.  
 
If possible, the information can be given to the parents in several steps. Brief oral information can be given to the 
parents even before the birth if the child is expected to meet the inclusion criteria, please see 9.1.1. 
 
An oral and written informed consent could preferably be obtained from the parents well before the oral feeds 
have reached 70 ml/kg/day in infants that are expected to fulfil the inclusion criteria, since they should have time 
enough to give a well-thought-out consent. Thus, in practice, the informed consent can be obtained already at the 
second and third day of life but not after the oral feeds have passed 100 ml/kg/day.  
 
Once all the information has been provided, both parents/legal guardians will sign the inform consent form. If 
there is only one legal guardian, an informed consent from this person is sufficient. The attending physician shall 
note this in the medical record of the infant. If twins are enrolled, the parents/legal guardians shall sign an 
informed consent form for each twin. The original will be stored in the investigator file A copy is given to the 
parents.  

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18. 
Approval from research ethical board and other authorities  
The application to the regional ethical review board at Linköping University has been approved before the start of 
the trial, (Dnr. 2018/193-31). A copy of the approved ethical application will be stored in the Investigator file at 
each study site. Blood, stool, urine and breast milk samples are included in the biobank (516) at Children and 
Women centre at the University Hospital in Linköping, Region Östergötland. There is a multi-centre agreement 
allowing the samples to be stored in the all centre in the trial. Application to the Medical Product Agency is not 
applicable since H2MF® and standard fortifiers in this trial are considered as diet supplement and not classified as 
a drug but regarded as Food for Special Medical Purposes (FSMP). The regulation for FSMP is covered in 
SLVFS 2000:15. No application to the Swedish Food Agency is needed for clinical trials. 
19. 
Study discontinuation 
The sponsor may at any time terminate the study. In that case, the sponsor shall immediately inform all 
investigators. Furthermore, the sponsor should inform to the regional ethical review board at Linköping 
University and Prolacta Bioscience.  
20. 
Statistical methods 
With at least 101 infants in each group a reduction of the composite NEC, sepsis and mortality incidence of 47% 
in the control group (www.snq.se) to 28% in the active group could be detected at a two-sided 5% level of 
significance and with 80% power. To allow for a potential 10% dropout rate, a total of 222 patients will be 
enrolled (please see 7.1). 
The following is an outline of the statistical approaches to be used. A more detailed evaluation will be 
incorporated into a Statistical Analysis Plan. 
The primary basis for all analyses of the clinical outcome will use the intent-to-treat paradigm. This means that in 
this approach all randomised participants are included in the analysis regardless of their disposition in the study 
(including whether they actually adhered to the randomized nutritional protocol). In addition, a per protocol 
analysis will be conducted that will only include patients who appropriately complete the intervention. A further 
stratification in per protocol analysis will be if the onset of an outcome was before or after the start of the 
intervention. All analyses will be conducted at a two-sided 5% significance level. No adjustment will be made for 
multiple analyses for the clinical outcome. 
Descriptive statistics will be computed for all quantitative outcome variables: mean ± standard deviation and 
median ± interquartile range. For qualitative (categorical) outcomes, proportions and/or percentages will be 
computed. The comparison of baseline variables such as gestational age, birth weight, gender and ethnicity/race 
will be based on the Wilcoxon rank-sum test for quantitative data and the chi-square test for homogeneity for 
categorical data. 
The primary outcome variable is the occurrence of either (at least one event) NEC or sepsis and/or whether the 
infant dies. Since this is a binary variable, the two groups will be compared using the chi-square test for 
homogeneity. The CRF will be completed also in infants who end the intervention before gestational week 34+0. 
Decisive for the inclusion of the infant is that all episodes of the primary outcomes - NEC, sepsis and mortality- 
are filled in the CRF until the end of the neonatal follow-up at gestational week 36+0. The only reason that the 
infant is excluded from the intent-to-treat analysis is that parents deny the entry of these data into the CRF. A 
secondary evaluation of the primary endpoint will employ a multivariate adjustment model using logistic 
regression that will include appropriately selected covariates, e.g. birth weight, gestational age, gender, race and 

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37 
the use of antenatal steroids. This will allow for the demonstration of the sturdiness of the primary analysis in the 
face of these adjustment variables.  
There are a number of secondary outcome variables. Quantitative variables such as growth (weight velocity 
calculated as g/kg/day and change in z-score, length velocity calculated as cm/week and change in z-score and head 
circumference growth rate calculated as cm/week and change in z-score) will be compared between the study groups 
using the Wilcoxon rank-sum test. Any other non-censored quantitative variable will be evaluated in the same 
fashion. Right-censored time-to-event data (e.g. NICU or hospitalization time) will be evaluated by the method of 
Kaplan and Meier and compared between the groups using the log-rank test. In addition, secondary analyses will 
involve the use of various covariates that will be incorporated into the evaluation through the use of regression 
models (multiple linear, logistic, or Cox proportional-hazards depending on the type of data) and the covariates will 
include variables such as birth weight, gestational age, AGA versus SGA and other clinical variables to be decided 
upon prior to the initiation of data analysis. Qualitative variables such as the occurrence of BPD, ROP and IVH and 
the composite culture-proven sepsis and NEC will be compared between the groups using the chi-square test for 
homogeneity. Finally, number of days using TPN will be evaluated using the method of Ghandehari et al. 45 
 
A separate analysis of the cost data will be performed and this will use standard econometric methods to be defined. 
The statistical analyses of the laboratory data will also be defined in separate protocol for each analysis. 
 
The study can be terminated prematurely because the primary outcome is significantly lower (with a significance 
level <0.001) in the H2MF® than in the standard fortification group in the interim analysis made after 150 infants 
have completed the neonatal period (please see section 7.5) based on the Haybittle-Peto group sequential stopping 
boundary 43 44.  
 
21. 
Financing and insurance 
The study is investigator-initiated. The study, however, is made in collaboration with the company Prolacta 
Bioscience, CA, USA.  The company will provide H2MF® and Prolact CR® and also support randomisation and 
packaging. Prolacta Bioscience will also cover the cost for study nurses and monitoring. A financial agreement 
between the sponsor, each participating hospital and Prolacta Bioscience will be negotiated in accordance with 
national guidelines. 
 
Participating patients will be insured via the patient injury act. 
22. 
Final study report 
The co-ordinating principal investigator is responsible of the final study report. The results of the primary research 
question will be published in international scientific peer-reviewed journals. They will also be reported at 
international congresses and national meetings for physicians and nurses. The public will be informed through 
press releases and media.  
23. 
Retention of records 
Records and documents pertaining to the conduct of this study, including CRF, source documents, consent forms, 
laboratory test results, and the investigator must retain medication inventory records, for at least 10 years 
according with GCP guidelines.  

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24.  
Confidentiality and publication 
The Sponsor owns all the information obtained in the trial, and the information obtained by this study is 
confidential. Disclosure to third parties other than those noted below is prohibited. With the subject’s permission, 
medical information may be given to his or her physician or other appropriate medical personnel in accordance 
with national regulations. Data generated by this study must be available for inspection upon request by 
representatives of the international, national and local health authorities, and the REB for each study site. 
 
The data file with patient information must be encrypted and protected with a password. 
 
The lawfulness of the processing of personal data in the study is based upon Article 6 (e) in the General Data 
Protection Regulation (GDPR) which states that the “processing is necessary for the performance of a task carried 
out in the public interest”. Patient data must not be transferred to anyone without the approval by the co-
ordinating principal investigator. In order to lawfully process special category data (also called sensitive data) a 
controller must identify a lawful basis under Article 6 and a separate condition for processing special category 
data under Article 9 in the General Data Protection Regulation. The separate condition that the N-Forte study uses 
to process special category data is article 9 (2) (j) in the General Data Protection Regulation which states that 
“processing is necessary for archiving purposes in the public interest, scientific or historical research purposes or 
statistical purposes in accordance with Article 89 (1) based on Union or Member State law which shall be 
proportionate to the aim pursued, respect the essence of the right to data protection and provide for suitable and 
specific measures to safeguard the fundamental rights and the interests of the data subject”. 
 
Before a CRF is transferred to a new hospital, the physician in charge has to make a review for the disclosure of 
patient data to the new hospital according to Chapter 25 Paragraph 1 of the Public access to secrecy act. It should 
be manifestly evident that the information may be disclosed without the patient or a person closely related to the 
patient being harmed. The same kind of review must be undertaken before the Nutrium data is transferred to the 
datafile in the end of the study by the investigator at the actual site. 
 
The information obtained during this study may be made available to other researchers who are conducting 
similar studies and to international or national medical authority, with due respect to the scientific priority of the 
investigation and after consulting the co-ordinating Principal Investigator.  
The intention is to publish the results in an international peer-reviewed journal. A copy of any manuscript 
(including meeting abstracts) reporting the primary outcomes in the study will be provided to Prolacta Bioscience 
for review and comments at least 30 days prior the expected date of submission to the intended publisher.  
 
 
The trial is registered at ClinicalTrials.gov (NCT03797157) 
25. 
References 
 
1. 
EXPRESS. One-year survival of extremely preterm infants after active perinatal care in Sweden. 
JAMA 2009; 301(21): 2225-33. 
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Sullivan S, Schanler RJ, Kim JH, et al. An exclusively human milk-based diet is associated with a 
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Cristofalo EA, Schanler RJ, Blanco CL, et al. Randomized Trial of Exclusive Human Milk versus 
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Assad M, Elliott MJ, Abraham JH. Decreased cost and improved feeding tolerance in VLBW infants 
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Lucas A. Feeding Low Birthweight Infants. Roberton NRC, Textbook of Neonatology 2nd edition 
Churchill Livingstone 1992: 279-88  
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Lucas A, Fewtrell MS, Morley R, et al. Randomized outcome trial of human milk fortification and 
developmental outcome in preterm infants. Am J Clin Nutr 1996; 64(2): 142-51. 
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Abrams SA, Schanler RJ, Lee ML, Rechtman DJ. Greater mortality and morbidity in extremely 
preterm infants fed a diet containing cow milk protein products. Breastfeed Med 2014; 9(6): 281-5. 
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Lucas A, Morley R, Cole TJ. Randomised trial of early diet in preterm babies and later intelligence 
quotient. BMJ 1998; 317(7171): 1481-7. 
21. 
Singhal A, Cole TJ, Lucas A. Early nutrition in preterm infants and later blood pressure: two cohorts 
after randomised trials. Lancet 2001; 357(9254): 413-9. 
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Singhal A, Farooqi IS, O'Rahilly S, Cole TJ, Fewtrell M, Lucas A. Early nutrition and leptin 
concentrations in later life. Am J Clin Nutr 2002; 75(6): 993-9. 
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Singhal A, Fewtrell M, Cole TJ, Lucas A. Low nutrient intake and early growth for later insulin 
resistance in adolescents born preterm. Lancet 2003; 361(9363): 1089-97. 
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Singhal A, Cole TJ, Fewtrell M, Lucas A. Breastmilk feeding and lipoprotein profile in adolescents 
born preterm: follow-up of a prospective randomised study. Lancet 2004; 363(9421): 1571-8. 
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Fewtrell MS, Williams JE, Singhal A, Murgatroyd PR, Fuller N, Lucas A. Early diet and peak bone 
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26. 
Lucas A, Morley R, Cole TJ, Lister G, Leeson-Payne C. Breast milk and subsequent intelligence 
quotient in children born preterm. Lancet 1992; 339(8788): 261-4. 
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Anderson JW, Johnstone BM, Remley DT. Breast-feeding and cognitive development: a meta-
analysis. Am J Clin Nutr 1999; 70(4): 525-35. 
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Isaacs EB, Fischl BR, Quinn BT, Chong WK, Gadian DG, Lucas A. Impact of breast milk on 
intelligence quotient, brain size, and white matter development. Pediatr Res 2010; 67(4): 357-62. 
29. 
Lewandowski AJ, Lamata P, Francis JM, et al. Breast Milk Consumption in Preterm Neonates and 
Cardiac Shape in Adulthood. Pediatrics 2016; 138(1). 
30. 
Lucas A, Lucas PJ, Chavin SI, Lyster RL, Baum JD. A human milk formula. Early Hum Dev 1980; 
4(1): 15-21. 
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Zhou J, Shukla VV, John D, Chen C. Human Milk Feeding as a Protective Factor for Retinopathy of 
Prematurity: A Meta-analysis. Pediatrics 2015; 136(6): e1576-86. 
32. 
Manzoni P, Stolfi I, Pedicino R, et al. Human milk feeding prevents retinopathy of prematurity 
(ROP) in preterm VLBW neonates. Early Hum Dev 2013; 89 Suppl 1: S64-8. 
33. 
Niklasson A, Albertsson-Wikland K. Continuous growth reference from 24th week of gestation to 
24 months by gender. BMC Pediatr 2008; 8: 8. 
34. 
Bell MJ, Ternberg JL, Feigin RD, et al. Neonatal necrotizing enterocolitis. Therapeutic decisions 
based upon clinical staging. Ann Surg 1978; 187(1): 1-7. 
35. 
Gastmeier P, Geffers C, Schwab F, Fitzner J, Obladen M, Ruden H. Development of a surveillance 
system for nosocomial infections: the component for neonatal intensive care units in Germany. J Hosp Infect 
2004; 57(2): 126-31. 
36. 
Jobe AH, Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit Care Med 2001; 163(7): 
1723-9. 
37. 
Prematurity TICftCoRo. The International Classification of Retinopathy of Prematurity revisited. 
Arch Ophthalmol 2005; 123(7): 991-9. 
38. 
Papile LA, Burstein J, Burstein R, Koffler H. Incidence and evolution of subependymal and 
intraventricular hemorrhage: a study of infants with birth weights less than 1,500 gm. J Pediatr 1978; 92(4): 529-
34. 
39. 
de Vries LS, Eken P, Dubowitz LM. The spectrum of leukomalacia using cranial ultrasound. Behav 
Brain Res 1992; 49(1): 1-6. 
40. 
Bayley N. Bayley Scales of Infant and Toddler Development. 3rd ed, San Antonio 2006: TX: 
Harcourt Assessment Inc. 
41. 
Gould AL. Interim analyses for monitoring clinical trials that do not materially affect the type I error 
rate. Stat Med 1992; 11(1): 55-66. 
42. 
Pocock SJ, Simon R. Sequential treatment assignment with balancing for prognostic factors in the 
controlled clinical trial. Biometrics 1975; 31(1): 103-15. 
43. 
Pantiadosi S. Clinical trials: a methodological perspective, 2nd ed, p 384-386. John Wiley & sons, 
New Jersey, 2005. . 
44. 
Haybittle JL. Repeated assessment of results in clinical trials of cancer treatment. Br J Radiol 1971; 
44(526): 793-7. 
45. 
Ghandehari H, Lee ML, Rechtman DJ, Group HMS. An exclusive human milk-based diet in 
extremely premature infants reduces the probability of remaining on total parenteral nutrition: a reanalysis of the 
data. BMC Res Notes 2012; 5: 188. 
 

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 25.  
Appendix 
 
Appendix 1 –Agreement between sponsor and investigator 
 
PROTOCOL SIGNATURE PAGE 
 
By signing below, the Investigator agrees to adhere to the protocol as outlined and agrees that any changes to the 
protocol must be approved by the sponsor prior to seeking approval from the Research ethical board. 
This study will be conducted in accordance with current ICH regulations, the Declaration of Helsinki, and local 
ethical and legal requirements. 
 
Sponsor´s Signature:  ________________________________________ 
 
Printed Name:  ________________________________________________ 
 
Date:  _______________________________________________________ 
 
 
Co-ordinating principal Investigator’s Signature:  ________________________________________ 
 
Printed Name:  ________________________________________________ 
 
Date:  _______________________________________________________ 
 
Investigator’s Signature:  ________________________________________ 
 
Printed Name:  ________________________________________________ 
 
Date:  _______________________________________________________ 
 
Clinical Director Signature:_____________________________________________ 
(verksamhetschef) 
 
Printed Name: 
 
 
 
 
 
Date:________________________________________________________ 
  
Please submit a signed original of this page to the sponsor and add copy to the investigator file at the study site. 
 
 
 

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Appendix 2 –List of contributing neonatal units and their contact persons 
 
Neo-IVA, H.K.H Kronprinsessan Victorias Barn- och ungdomssjukhus i Linköping  
 
Principal investigator: 
Thomas Abrahamsson, Neonatolog, Överläkare, (principal investigator) 
thomas.abrahamsson@regionostergotland.se 
 
Address: 
H.K.H Kronprinsessan Victorias Barn- och ungdomssjukhus, plan 15 
Universitetssjukhuset i Linköping 
581 85 Linköping 
Tel: 010-1030000 vx 
 
 
 

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Appendix 3 –Agreement with the head of the clinic (this was included in the ethical 
application) 
 
 
Intyg om att erforderliga resurser finns för att garantera forskningspersonernas 
säkerhet. 
 
Härmed intygas att erforderliga ekonomiska, strukturella och personella resurser finns tillgängliga på denna klinik 
för att garantera forskningspersonernas säkerhet vid genomförandet av projektet ”Nordic study on human milk 
fortification in extremely preterm infants (N-Forte)” 
 
  
Verksamhetschefs signatur                                                                Datum 
 
Namnförtydligande 
 
Center/klinik 
 
 
 

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Appendix 4 – Data and safety monitoring board (DSMB) 
 
The member of the DSMB 
 
Baldvin Jonsson, Docent, Överläkare, Astrid Lindgrens Barnsjukhus, Stockholm (chairman). 
Baldvin.jonsson@karolinska.se 
 
Mats Hammar, Professor, Överläkare, Kvinnokliniken, Universitetssjukhuset i Linköping. 
mats.hammar@liu.se 
 
Mats Fredrikson, biostatistician, Forum Östergötland, Universitetssjukhuset i Linköping. 
mats.fredrikson@liu.se 
 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
45 
Appendix 5 –Reporting of SUSAR 
 
Investigator or the attending physicians at the study site are required to report SUSARs to the Co-ordinating 
Principal investigator within 24 hours. This report form must also be completed and promptly sent by mail or 
email to the principal investigator.  
 
 
Name of the patient 
Patient personal number 
Reserve number (if appl) 
Patient identification number 
 
SUSAR: 
 
 
 
Date 
Time 
Where? 
 
 
 
Send to: 
 
Co-ordinating Principal investigator: 
Thomas Abrahamsson,  
H.K.H Kronprinsessan Victorias Barn- och ungdomssjukhus, plan 15 
Universitetssjukhuset 
581 85 LINKÖPING 
010 – 103 00 00 (vx) 
mob: +46-70956 68 15 
 
email: thomas.abrahamson@regionostergotland.se 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
46 
Appendix 6 –Study information to the parents  
Vill ni delta i N-Forte studien, där vi jämför två olika 
proteinberikningar av bröstmjölk?  
 
Bästa föräldrar! 
Hur kan tillmatningen av för tidigt födda barn förbättras? Detta vill vi undersöka i en forskningsstudie där vi 
jämför två olika näringsberikningar av bröstmjölken.  Den ena är baserad på komjölk och den andra på human 
bröstmjölk. Syftet med studien är att se om risken för att få blodförgiftning och inflammation i tarmen kan minska 
om det nödvändiga proteintillskottet som för tidigt födda barn behöver kommer från donerad bröstmjölk istället 
för komjölk.  
 
Varför vill vi göra den här studien? 
Ett barn som föds extremt för tidigt är mycket känsligt och kan drabbas av allvarliga komplikationer under 
vårdtiden. Den medicinska utvecklingen har gått framåt och gjort att de flesta av dessa barn ändå överlever och 
klarar sig bra.  
 
Barnens tillmatning och tillväxt är en viktig utmaning. Ju bättre vi lyckas med detta, desto bättre blir barnets hälsa 
och utveckling – både på kort och på lång sikt. Målet är att ge dessa små barn förutsättningar att växa lika bra som 
de skulle ha gjort inuti livmodern. Vi vet att bröstmjölk är den bästa födan, även för prematura barn. Därför ger vi 
alltid någon milliliter mjölk redan första dygnet och ökar sedan mängden successivt. Efter ett antal veckor kan 
barnet helt och hållet matas med bröstmjölk och behöver inte dropp. I första hand ges mammans egna utpumpade 
mjölk. Om den inte räcker till ger vi bröstmjölk som har donerats till en bröstmjölksbank, så att barnet inte 
behöver få vanlig bröstmjölksersättning.  
 
Barn som föds extremt för tidigt har ett större näringsbehov än barn som föds efter vanlig graviditetslängd. Därför 
tillsätts rutinmässigt extra berikning (som innehåller protein, mineraler och vitaminer) till bröstmjölken för att de 
skall växa så bra som möjligt. Den berikning som används på sjukhusen idag är gjorda av komjölk eftersom det 
inte har funnits några alternativ.  
 
 
Det har nu utvecklats en berikning om kommer från human bröstmjölk, d.v.s. bröstmjölk som donerats av andra 
mammor och det finns en del forskningsresultat som tyder på att denna ”humana” bröstmjölksberikning kan ha 
positiva hälsoeffekter. Vi vill nu undersöka om risken för infektion och svår tarminflammation (NEC) minskar om 
de får denna berikning istället för den traditionella berikningen som gjorts av komjölk.  
 
Vilka kan vara med i studien? 
Alla barn som är födda i graviditetsvecka 22+0 till 27+6 som inte har någon allvarlig bukåkomma, medfödd 
sjukdom eller missbildning. Barnet skall inte fått något proteintillskott i bröstmjölken innan det går med i studien.  
 
Hur går studien till? 
Barnet skall inkluderas i studien innan vi börjar ge extra berikning i bröstmjölken, vilket oftast brukar ske vid 4-
21 dagars ålder, och det följs sedan hela vårdtiden under nyföddhetsperioden. Vi kommer även att göra en 
uppföljning när ert barn är två år gammalt samt vid 5,5 års ålder. Dessa uppföljningar görs i samband med den 
ordinarie uppföljningen som görs på alla för tidigt födda barn.  
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
47 
De barn som deltar i studien delas in i två grupper. I ena gruppen kommer barnen att få en berikning som baseras 
på donerad bröstmjölk (H2MF®) fram till graviditetsvecka 34+0. Om barnet behöver extra energi kommer ett 
fettillskott (ProlactCR®) som också är baserat på bröstmjölk att ges till barnen i den gruppen. I den andra gruppen 
kommer barnen få det berikning baserat på komjölk som är standard på avdelningen. Vid behov av extra fett 
kommer de få det fettillskott som är standard på avdelningen. Barnen kommer att lottas slumpmässigt till en av de 
två grupperna så att hälften av barnen får traditionell berikning och hälften får H2MF®. På så sätt kan vi jämföra 
om den nya berikningen är bättre än den nuvarande standardbehandlingen. Barnen i de båda grupperna kommer i 
övrigt att behandlas på precis samma sätt.  
 
Under vårdtiden kommer uppgifter om barnets tillmatning, tillväxt, behandlingar och symptom samt uppgifter om 
mamman som är viktiga för barnets hälsa samlas in från journalen och nutritionsprogram för att kunna tolka 
resultaten i studien rätt. Prover kommer att tas för att undersöka vilka mekanismer som kan ligga bakom 
förbättrad tillväxt, tarmfunktion och infektionsförsvar. Blodprov kommer att tas på barnet vid fyra tillfällen under 
vårdtiden i samband med att blodprov ändå tas av andra skäl. Dessutom kommer prov att tas på utpumpad 
bröstmjölk från mamma samt prov på avföring och urin från barnet. Dessa prover kommer att frysas in för 
analyser i framtiden. De kommer först ingå i vårdgivarens biobank och sedan flyttas till Barn- och 
kvinnocentrums biobank 516 i Region Östergötland, Linköping. Ni kan när som helst begära att dessa prover 
förstörs utan att ange orsak. 
 
Förutom provtagningen och studieprodukten kommer barnen inte att behandlas annorlunda än barn som inte deltar 
i studien.  
 
Är det farligt? 
Det nya kosttillskottet (H2MF®) och fettillskottet (ProlactCR®) har testats i flera studier på extremt för tidigt födda 
barn tidigare. Det finns inga rapporter om förekomst av ett ökat antal infektioner eller andra allvarliga 
biverkningar. För säkerhets skull kommer vi ändå kontrollera barnen noga.  
 
Kan man lämna studien? 
Ett deltagande i studien är helt frivilligt och om ni samtycker till deltagande men sedan ändrar er kan ni när som 
helst ta ert barn ur studien. Ni behöver inte berätta varför. Vi kommer i så fall att fråga er om vi även efter 
avbrytandet får registrera uppgifter om barnets tillväxt och hälsotillstånd. Om ni väljer att avbryta ert barns 
studiedeltagande kommer det inte att påverka ert barns fortsatta vård. Ingen ersättning utgår för deltagande i 
studien. Patientförsäkringen gäller för ert barn precis som för all vård.  
 
Vilka frågor vill vi få svar på? 
• Kan H2MF® minska förekomsten av allvarliga sjukdomar såsom blodförgiftning och inflammation i 
tarmen jämfört med komjölksbaserad proteinberikning? 
• Hur lång tid tar det innan barnet klarar av att få fulla mål med bröstmjölk? 
• Hur påverkas barnets tillväxt och utveckling? 
• Vilka faktorer kan ligga bakom effekten av H2MF®? 
• Vilka andra mekanismer ligger bakom allvarliga komplikationer, dålig tillväxt och mag- och tarmfunktion  
 
Vad gör vi med resultatet? 
Studiens resultat kommer att redovisas i internationella vetenskapliga tidskrifter. Om det visar sig att H2MF® 
minskar risken för komplikationer för det för tidigt födda barnet, är avsikten att kunna erbjuda denna berikning till 
alla för tidigt födda barn. Ni kommer att få information om resultaten via brev. 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
48 
Behandling av personuppgifter 
Uppgifterna tas ifrån patientjournalen och används bara för att besvara de frågor som vi har beskrivit ovan. De 
hanteras enligt EU´s dataskyddsförordning. Den lagliga grunden för hanteringen är dataskyddförordningens stöd 
för behandling av uppgifter för forskningsändamål. All data behandlas gruppvis och inga obehöriga kan ta del av 
dem. De måste sparas i en databas i minst 10 år och måste sparas längre om de vetenskapliga analyserna 
fortfarande pågår. Region Östergötland, Region Uppsala, Region Västerbotten, Västra Götalandsregionen och 
Stockholms läns landsting har ett gemensamt personuppgiftsansvar för all data i den sparade datafilen. Vid behov 
kan ni kontakta dataskyddsombudet i Region Östergötland på adressen Region Östergötland, Dataskyddsombud, 
581 91 Linköping, eller via e-post dataskyddsombud@regionostergotland.se. På studieprotokoll, provrör och i 
databasen anges inte namn eller personnummer utan bara en särskild kod som är unik för ert barn. För att vara 
säker på att alla uppgifter rapporteras in på ett korrekt sätt kommer en kvalitetsgranskning att göras. En oberoende 
person kan då komma att jämföra de formulär som fylls i med barnets journal. Röntgenbilder kan även komma att 
skickas för en extra bedömning till röntgenspecialister i annat landsting. I forskningssyfte kan forskare från andra 
universitet i Sverige och utomlands ta del av data i databasen. Det gäller även det USA-baserade företaget 
Prolacta som producerar kosttillskottet H2MF®. Ni har rätt att få ta del av det särskilda avtal som krävs för att data 
skall överföras till ett företag i USA. Det kanadensiska företaget Interrand får även enstaka uppgifter som behövs 
när de hjälper oss med lottningen till vilken grupp barnet skall höra (födelseort, om barnet föddes före 
graviditetsvecka 25+0, könstillhörighet, och om det är en tvilling eller inte). Inte heller till Interrand skickas namn 
eller personnummer. Alla personer som tar del av data har tystnadsplikt. Alla resultat som publiceras i 
vetenskapliga tidskrifter kommer presenteras på gruppnivå så inget enstaka barn kan identifieras. Ni har rätt att 
kostnadsfritt få ta del av de uppgifter om er som hanteras i studien och vid behov få eventuella fel rättade. För att 
säkerställa studieresultatets kvalitet kan, enligt gällande lagar och förordningar, data insamlade i forskningsstudier 
inte alltid raderas på er begäran. Eventuella klagomål på hanteringen av personuppgifterna kan ges till 
Datainspektionen 
 
Om Ni har några ytterligare frågor eller vill ha mer information, tag då gärna kontakt med någon av oss. 
 
 
 
För projektet, 
 
 
[ Namn och titel ansvarig läkare] 
 
 
[Namn och titel ansvarig ssk ] 
 
 
 
[Arbetsplats/klinik ] 
 
 
[Arbetsplats/klinik ] 
[ Tel nr ]  
 
 
[ Tel nr ] 
[E-mail ] 
 
 
[E-mail ] 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
49 
Appendix 7 –Informed consent form 
Samtycke till deltagande 
 
Undertecknande målsmän samtycker härmed till att vårt barn deltar i N-Forte studien, som undersöker om det 
bröstmjölksbaserade proteintillskottet H2MF® minskar risken för svåra komplikationer och förbättrar uppfödning 
och tillväxt hos extremt för tidigt födda barn jämfört med komjölksbaserat proteintillskott. Vårt barn kommer att 
antingen få H2MF® eller det komjölksbaserade proteintillskott som är standard på avdelningen. 
 
Vi har tagit del av den skriftliga informationen. Vi har haft möjlighet att ställa frågor och vi har fått 
tillfredställande svar.  
Vi godkänner att en utomstående person kan komma att granska studiehandlingarna och jämföra med uppgifter i 
patientjournalen. 
 
Angående lagring av biologiska prover 
Vi har informerats och samtycker till att biologiska prover (blod, bröstmjölk, urin och avföring) får sparas 
nedfrysta i biobank för framtida vetenskapligt bruk inom det område som detta projekt avser. Vi kan när som helst 
begära att dessa prover förstörs utan att ange orsak. 
 
Vi är medvetna om att deltagandet är helt frivilligt och att vi har möjlighet att när som helst avbryta studien utan 
att ange något skäl. 
 
…………………………………………… 
…………………………………………… 
Barnets personnummer 
Barnets efternamn och ev förnamn 
…………………………………………… 
…………………………………………… 
Namnteckning vårdnadshavare 1 
Namnteckning vårdnadshavare 2 
…………………………………………… 
Ort och datum 
…………………………………………… 
Ort och datum 
…………………………………………… 
…………………………………………… 
Textat namn  
Textat namn  
…………………………………………… 
……………………………………………. 
Telefon hem                           mobil 
Telefon hem                           mobil 
 
…………………………………………. 
…………………………………………. 
Ansvarig läkare (namnteckning och 
namnförtydligande) 
 
 
 
…………………………………………… 
Ort och datum 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
50 
 
Appendix 8 –Case report form 
 
In a separate file 
 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
51 
Appendix 9 –Prescription and preparation of the study product 
 
Dose of fortification in order to achieve protein intake 4.0-4.5 g/kg/d: 
Nutriprem 
 
 
PreNAN HMF  
 
 
H2MF 
 
 
Daily prescriptions should be done in order to ensure that intakes of all nutrients are within recommended ranges. 
The table above only give a overview how to prescribe the different fortifiers. The Nutrium software (Nutrium 
AB, Umeå, Sweden) which is provided to all study sites has included the study products in its algorithms. Support 
will be given to physicians and dietitians if needed for the nutritional calculations. It is not mandatory to use 
Nutrium for the prescription of the fortifier. Thus, some study sites will use locally developed tables instead of 
Nutrium to achieve the wanted protein level. For protein content in milk, true protein concentrations measured by 
the breast milk analyser are used. 
 
Total daily volume of enteral feeding should not exceed 170 ml/kg/d when the breast milk has been fortified with 
a full dose of H2MF, since the intakes of Calcium and Phosphorous otherwise will exceed recommendations. 
 
Observe that additional vitamins (Multivitamin Unimedic) always needs to be prescribed to infants who receive 
H2MF®. However, in most cased there is no need of extra Calcium and Phosphorous if the milk is fortified with 
H2MF®, please see investigator file for instructions. H2MF® contain little iron. Thus, extra iron supplement is 
often needed in this study group. 
 
Breast milk
Feed
Nutriprem
protein content volume
Dose
g/100 mL
ml/kg/d
0.9-1.0
200
Full
1.1-1.3
180
Full
1.4-1.7
160
Full
1.8-2.0
160
75%
2.1-2.2
160
Half
Breast milk
Feed
PreNAN HMF
protein content volume
Dose
g/100 mL
ml/kg/d
0.9-1.0
180
Full
1.1-1.4
160
Full
1.5-1.7
160
75%
1.8-2.1
160
Half
Breast milk
Feed
H2MF
protein contentvolume
Dose
g/100 mL
ml/kg/d
0.9-1.0
170
Full
1.1-1.6
160
Full
1.7-2.0
160
75%
2.1-2.3
160
Half

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
52 
How to prepare the different doses: 
 
Four levels of fortification  
This is the standard protocol for most sites in the study*/ 
 
Full dose fortification  
H2MF : 30 ml + 70 ml breast milk (42.9 ml added to 100 ml breast milk) 
Prenan HMF (”FM85”): 4 g per 100 ml breast milk 
Nutriprem: 2 sachets per 100 ml breast milk 
 
75% fortification 
H2MF: 22.5 ml + 77.5 ml breast milk (29 ml added to 100 ml breast milk) 
Prenan HMF: 3 g per 100 ml breast milk 
Nutriprem 1½ sachet per 100 ml breast milk 
 
Half dose fortification 
H2MF: 15 ml + 85 ml breast milk (17.6 ml added to 100 ml breast milk) 
Prenan HMF: 2 g per 100 ml breast milk 
Nutriprem: 1 sachet per 100 ml breast milk 
 
Quarter dose fortification (rarely used) 
H2MF: 7.5 ml + 92.5 ml breast milk (8.1 ml added to 100 ml breast milk) 
Prenan HMF: 1 g per 100 ml breast milk 
Nutriprem: ½ sachet per 100 ml breast milk 
 
*/ The NICU in Uppsala fortifies with H2MF according to this protocol, but they have chosen to continue with 
their previous guidelines of standard fortification with 15 levels of Prenan HMF, from full dose to 7% 
fortification: 4.2g, 3.9g, 3.6g, 3.3g, 3.1g, 2.8g, 2.5g, 2.2g, 1.9g, 1.7g, 1.4g, 1.1g, 0.8g, 0.6g and 0.3g. 
 
 
Eight levels of fortification 
The protocol used in the Stockholm region 
 
Full dose fortification 
H2MF : 30 ml + 70 ml breast milk (42.9 ml added to 100 ml breast milk)  
Prenan HMF (”FM85”): 4 g per 100 ml breast milk 
 
87,5% fortification 
H2MF: 26.3 ml + 73.7 ml (35.6 ml added to 100 ml breast milk) 
Prenan HMF: 3,5 g per 100 ml breast milk 
 
75% fortification 
H2MF: 22.5 ml + 77.5 ml breast milk (29 ml added to 100 ml breast milk)  
Prenan HMF: 3 g per 100 ml bröstmjölk  
 
62,5% fortification 
H2MF: 18.8 ml + 81.2 ml breast milk (23.1 ml added to 100 ml breast milk)  
Prenan HMF: 2,5 g per 100 ml breast milk 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
53 
 
Half dose fortification 
H2MF: 15 ml + 85 ml breast milk (17.6 ml added to 100 ml breast milk)  
Prenan HMF: 2 g per 100 ml breast milk 
 
37,5% fortification 
H2MF: 11,3 ml + 88,7 ml breast milk (12,7 ml added to 100 ml breast milk)  
Prenan HMF: 1,5 g per 100 ml breast milk 
 
25% fortification (unusual) 
H2MF: 7.5 ml + 92.5 ml breast milk (8.1 ml added to 100 ml breast milk)  
Prenan HMF: 1 g per 100 ml breast milk 
 
12,5% fortification (unusual)  
H2MF: 3,8 ml + 96,2 ml breast milk 3,9 ml added to 100 ml b breast milk)  
Prenan HMF: 0,5 g per 100 ml breast milk 
 
 
How to start 
The dose should be based on individual analysis of the breast milk (mother´s own milk or donor milk) when such 
analysis has been done. If the supplementation of the breast milk fortification starts before the first analysis has 
been done, a half dose fortification is recommended during the first 1-3 days in both study groups.  
 
To assist in calculating the individual nutritional needs the computer-aided nutrition calculation program Nutrium 
will be used. In Nutrium the H2MF fortification should be added as indicated in the above. I.e. for full dose 
fortification add 42,9 ml H2MF to 100 ml breast milk, for 75% fortification add 29 ml H2MF to 100 ml breastmilk 
etc. 
 
Fat supplement 
Fat supplements can be considered if energy intake is low.  The supplement should be based on the nutritional 
calculations with Nutrium. The H2MF group should only get human milk-based fat supplement, ProlactCR®. This 
product will be supplied by Prolacta Bioscience during the whole intervention period for free. After the 
intervention has ended, i.e. after gestational week 34+0, Prolact CR shall be exchanged to standard fat 
supplement. A specific transition period is not needed for this. The standard group will get the fat supplement that 
is standard at the unit. 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
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54 
Appendix 10 – Transition to discharge feeding regimen 
Needed for infants in the H2MF arm when weaning starts at gestational week 34+0. Also, needed for infants in 
both arms if weaning starts before gestational week 34+0 at a centre without the logistics for maintaining 
intervention. 
 
1. Transition protocol in infants that will continue with 100% breast milk after the transition from H2 
MF. The amount of the fortification (none, half or full supplementation) will be calculated as 
described in section 9.1.5 and Appendix 9.   
 
H2 MF transition protocol: in infants that will continue with 100% breast milk  
 
Feeding 
Schedule 
 
8 times/day 
Feedings: 
Last day of all 
Human milk  
Transition Day 
1 
 
BMd/BMe+  
Standard 
fortifier/ HUM 
fortifier 
Transition Day 
2 
 
BMd/BMe+  
Standard 
fortifier/ HUM 
fortifier 
Transition Day 
3 
 
BMd/BMe+  
Standard 
fortifier/ HUM 
fortifier 
Transition Day 
4 
 
BMd/BMe+  
100% Standard 
fortifier 
1 
BMd/BMe+ HUM 
fortifier  
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
2 
BMd/BMe+ HUM 
fortifier 
BMd/BMe+ 
HUM fortifier 
DBM/EBM+ 
HUM Fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
3 
BMd/BMe+ HUM 
fortifier 
BMd/BMe+ 
HUM fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
4 
BMd/BMe+ HUM 
fortifier 
BMd/BMe+ 
HUM fortifier 
DBM/EBM+ 
HUM Fortifier 
DBM/EBM+ 
HUM Fortifier 
BMd/BMe+  
Standard 
fortifier 
5 
BMd/BMe+ HUM 
fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
6 
BMd/BMe+ HUM 
fortifier 
BMd/BMe+ 
HUM fortifier 
DBM/EBM+ 
HUM Fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
7 
BMd/BMe+ HUM 
fortifier 
BMd/BMe+ 
HUM fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
BMd/BMe+  
Standard 
fortifier 
8 
BMd/BMe+ HUM 
fortifier 
BMd/BMe+ 
HUM fortifier 
DBM/EBM+ 
HUM Fortifier 
DBM/EBM+ 
HUM Fortifier 
BMd/BMe+  
Standard 
fortifier 
Footnote: BMd=donor breast milk; BMe=mother´s own expressed breast milk: HUM fortifier= H2 MF; Standard fortifier= 
bovine protein-based fortifier (Nutriprem, FM85 etc.) 
 
2. Transition protocol is not required in infants that will continue with 100% breast milk in bovine 
protein-based fortifier group.  
 
In practice, the staff in breast milk kitchen will not change anything but only provide the fortified milk  
 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
55 
3. Transition protocol in infants that will continue with formula (exclusively or partially) after the 
transition from H2 MF.  
 
H2 MF transition Protocol: in infants that will continue with formula (partial or exclusive)  
 
Feeding 
Schedule 
 
8 times/day 
Feedings: 
Last Day of all 
Human milk  
Transition Day 
1 
 
BMd/BMe/ 
Formula+  
HUM fortifier 
Transition Day 
2 
 
BMd/BMe/ 
Formula+  
HUM fortifier 
Transition Day 
3 
 
BMd/BMe/ 
Formula+  
HUM fortifier 
Transition Day 4 
 
Formula 
100%  
Standard fortifier 
1 
BMd/BMe+ 
HUM fortifier  
Formula 
Formula 
Formula 
*Formula/BMe 
2 
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
Formula 
*Formula/BMe 
3 
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
Formula 
Formula 
*Formula/BMe 
4 
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
*Formula/BMe 
5 
BMd/BMe+ 
HUM fortifier  
Formula 
Formula 
Formula 
*Formula/BMe 
6 
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
Formula 
*Formula/BMe 
7 
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
Formula 
Formula 
*Formula/BMe 
8 
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
BMd/BMe+ 
HUM fortifier  
*Formula/BMe 
Footnote: BMd=donor breast milk; BMe=mother´s own expressed breast milk: HUM fortifier= H2 MF; Standard fortifier= 
bovine protein-based fortifier (Nutriprem, FM85 etc.). Formula=the standard formula at the actual site. 
 
*If the infant is partially fed with breast milk and the breast milk still are going to be fortified with protein 
fortifier, one or feeding with formula will be replaced with breast milk+ standard fortification. 
 
4. Transition protocol is not required in infants that will continue formula in bovine protein-based 
fortifier group.  
 
The staff in the milk kitchen will follow the clinical guidelines at the neonatal unit. 
 
 
 
 
 

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56 
Appendix 11 –Classification of level of care 
 
0 
Infants who are not discharged but spend the whole day at home (cared for by home 
care team or only phone contact with neonatal unit) are cared for by home care team 
or who are on ”leave” 
  
1 
Infants who do not require continuous monitoring by health care staff. 
  
Example: 
Infant stays with parents in family room without continuous monitoring. 
  
2 
Infants who require continuous monitoring by staff and/or monitoring equipment 
  
Example: 
Treatment with low flow oxygen cannula 
Peripheral venous line 
  
3 
Low level intensive care: Infants who require continuous monitoring and CPAP 
treatment or central venous access. 
  
  
Example: 
Treatment with CPAP 
Central venous lines for nutrition 
Post-operative monitoring without need for respirator 
  
4 
Medium level intensive care: Infants who require continuous monitoring by intensive 
care staff 
  
Example: 
Stable on respirator treatment 
  
5 
High level intensive care: Infants who require continuous monitoring by more than 
one intensive care staff 
  
Example: 
Unstable infant on respirator treatment (respirator and need of NO and/or inotropic 
support) 
 
 
 
 
 

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57 
Appendix 12 –Blood sample: instructions 
 
Provrör och märkning: 
Provrör finns i särskild låda ipå neonatalavdelningen (valfritt att lägga till mer exakt beskrivning på studieorten). 
Det har särskild klisteretiketter för varje enskilt prov uppdelat på blod- avförings- urin- respektive 
bröstmjölksprov samt barnets ålder vid provtagningen. 
 
Blodprov: 
 
Dag 7, 14, och dag 28 provet 
Dessa prover skall tas vid 7±2, 14±2, respektive 28±2 dagars ålder. Använd rör avsedda för dag 7, 14, och 28. 
 
Blodprov i vecka 33 
Detta prov skall tas i gestationsvecka 33+0-33+6 
Ta två EDTA-rör i denna vecka så att vi får mer provmaterial. Ta dock fortfarande bara ett CyTOF-rör. 
 
Hantering av blodprover: 
1. Välj rör och klisteretikett med studienamn och ”Blod/CyTOF Dag 7”, ” Blod/CyTOF Dag 14”, ” 
Blod/CyTOF Dag 28” respektive ”B Blod/CyTOF gv 33”, respektive ”Blod/EDTA Dag 7”, ” Blod/EDTA 
Dag 14”, ” Blod/EDTA Dag 28” respektive ”B Blod/EDTA gv 33”. Särskilda etiketter finns även för 
frysrören efter centrifugeringen och separationen plasma och celler från EDTA-röret, vg se nedan. Man 
behöver endast fylla i barnets studie-ID nummer och provtagningsdatum. 
2. Prov tas i två olika rör: 
a. CyTOF/masscytometri: Ca 100 mikroliter (0.1 ml) blod i rör med fixeringslösning för 
masscytometri. 
b. EDTA-rör: Ca 500 mikroliter (0.5 ml) blod i EDTA-rör (kapillärrör) 
 
3. Provet för masscytometri blandas genom att vändas 5 – 6 ggr, låt stå i rumstemperatur 10 min och fryses 
sedan i -70°C, men kan korttidsförvaras i -20°C.  
4. Provet i EDTA-röret blandas genom att vändas 10 ggr.  Röret får stå i rumstemperatur högst 2 timmar och 
sedan måste det ha tagits om hand och frysts in enligt nedan. 
5. EDTA-röret centrifugeras 15 minuter, 1500g 
6. Från EDTA röret sugs plasma ut med pipett och sätts i ett 0.5 ml mikroör och detta centrifugeras en gång 
till i 15 minuter i 1500g. Plasman pipetteras över till nya mikrorör, gärna 2-3 rör om det går (minst 100 
mikroliter/rör). Märk frysröret med klisteretikett ”Plasma/EDTA dag X” och skriv studie-ID nummer och 
datum. 
7. EDTA-röret med kvarvarande celler skall också sparas i frys. Pipettera över cellerna till 0,5 ml mikrorör. 
Märk frysröret med klisteretikett ”Celler/EDTA dag X” och skriv studie-ID nummer och datum. 
8. Proverna kan korttidsförvaras i -20 °C innan långtidsförvaring i -70 ° 
9. Skriv in i CRF att prov har tagits och om något avvek från ordinarie rutiner. 
 
Detta är grundinstruktionen men enskilda studieorter kan justera instruktionen beroende på hur man har kommit 
överens med sitt lokala lab.  Exempelvis kommer lab och hämtar provet före centrifugering på vissa studieorter. 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
58 
Appendix 13 –Stool sample: instructions 
 
Provrör finns i särskild låda på neonatalavdelningen (valfritt att lägga till mer exakt beskrivning på studieorten). 
Det finns särskilda klisteretiketter för varje enskilt prov uppdelat på blod- avförings- urin- respektive 
bröstmjölksprov samt barnets ålder vid provtagningen. 
 
 
Avföringsprov: 
 
Avföringsprovet tas med spatel som sitter i locket i provröret. Det går bra att ta provet från blöjan. Ta 
så mycket som möjligt, d.v.s. hela rör. Det går bra att ta flera prov under dygnet, särskilt om det blir 
lite prov i de första rören. Sträva efter att ta 2-3 provrör. 
 
1. Avföringsprover skall tas vid 7±2, 14±2, 21±2 respektive 28±2 dagars ålder samt i gestationsvecka 33 
(33+0-33+6).  
2. Ta 2 rör per tillfälle. Sätt provet i frysfack, -20°C, i ”sköljen” i väntan på att de hämtas av labpersonal.  
3. Välj rör och klisteretikett med studienamn ”Feces dag 7”, ”Feces dag 14”, ”Feces dag 21”,”Feces dag 28” 
respektive ”Feces gv 33”. Ni behöver endast fylla i barnets studie-ID nummer och provtagningsdatum. 
4. Om föräldrarna tagit v33 provet hemma tar de med sig detta fryst (”iskubpåsar” i plastpåse). Provet sätts i 
frysfack i ”sköljen” i väntan på att de hämtas av labpersonal. 
5. Ange i CRF att provet har tagits och hur många rör totalt som sparats. 
 
 
Detta är grundinstruktionen men enskilda studieorter kan justera instruktionen beroende på hur man har kommit 
överens med sitt lokala lab.   
 

Protocol No: 2020/vers.4      2020-03-25            
 
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59 
 
Appendix 14 –Urine sample: instructions 
 
Provrör och märkning: 
 
Provrör finns i särskild låda på neonatalavdelningen (valfritt att lägga till mer exakt beskrivning på studieorten). 
Det finns särskilda klisteretiketter för varje enskilt prov uppdelat på blod- avförings- urin- respektive 
bröstmjölksprov samt barnets ålder vid provtagningen. 
 
Urinprov: 
 
Ett särskilt sterilt rör för urinprov skall användas (salivette). Detta rör har en övre kammare med ett litet hål i 
botten. I denna övre kammare finns en torr bomullsrulle (eller steril kompress som klippts till) som man sätter i 
barnets blöja. Den suger upp urinen. När den är blöt sätter man tillbaka den i röret och centrifugerar ut urinen som 
kommer ner i botten på röret genom hålet. Helst skall det första provet efter kl 8.00 timmar som inte 
kontaminerats av avföring användas. Det skall in i frys så snabbt som möjligt. Det är ok att ta flera rör (vid olika 
tillfällen), särskilt om man fick liten urinmängd i första provet. 
 
Som alternativ till bomullsrulle kan steril kompress användas. Den rullas ihop så att den passar i övre facket i 
röret. 
 
 
Hantering av urinprover: 
 
1. Urinprover skall tas vid 7±2, 14±2, 21±2 respektive 28±2 dagars ålder samt i gestationsvecka 33 (33+0-
33+6).  
2. Välj rör och klisteretikett med studienamn ” Urin dag 7”, ”Urin Dag 14”, Urin Dag 21”, ”Urin Dag 28” 
respektive ”Urin gv 33”. Ni behöver endast fylla i barnets studie-ID nummer och provtagningsdatum. 
3. Ta ur bomullsrulle och sätt i blöja efter 8.00. Om provet kontamineras med avföring får man göra om det 
med ny bomullsrulle/steril kompress. Som alternativ till bomullsrulle kan steril kompress användas. Den 
rullas ihop så att den passar i övre facket i röret. 
4. Ta hand om bomullsrullen så fort som möjligt när det blivit blöt så att den inte ligger länge i blöjan.  
5. Sätt bomullsrulle i det specifika röret. Centrifugera i minst 1500g i 2-5 min. Det skall centrifugeras inom 1 
timme för att urinprovet skall komma i frys så fort som möjligt. Urinen hälls/pipetteras över i frysrör. 
6. Provröret sätts i kyl omgående och efter centrifugeringen sätts provet i frys, -20°C eller ´-70°C  
7. Skriv in i CRF att provets tagits och hur många rör som sparats. 
 
Detta är grundinstruktionen men enskilda studieorter kan justera instruktionen beroende på hur man har kommit 
överens med sitt lokala lab. 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
60 
Appendix 15 –Breast milk sample: instructions 
 
Provrör och märkning: 
 
Provrör finns i särskild låda på neonatalavdelningen (valfritt att lägga till mer exakt beskrivning på studieorten). 
Det finns särskilda klisteretiketter för varje enskilt prov uppdelat på blod- avförings- urin- respektive 
bröstmjölksprov samt barnets ålder vid provtagningen. 
 
Bröstmjölksprov: 
 
Bröstmjölksprovet tas av den bröstmjölk som barnet skall få den aktuella dagen. Om det handlar om dag 14 
provet så tas alltså provet från den bröstmjölk som gjorts i ordning för dag 14. Provet skall tas efter berikning 
gjorts (eller om berikning ej påbörjad ännu tas provet av den bröstmjölk som barnet ska få). Ta så mycket som 
möjligt, 10 ml om möjligt, d.v.s. hela rör (men högst till 1 cm från rörkanten eftersom vätskan expanderar vid 
frysning).  
 
OBS, bröstmjölksprov vid 7, 14, 21 och 28 dagar skall bara tas om det finns ett överskott av bröstmjölk från 
mamma, d.v.s. om mammans sparade mjölk räcker till barnets aktuella dygnsbehov. Vid gestationsvecka 33-
0-36+6 tas provet även om mammans mjölk inte räcker till full tillmatning. 
 
Om barnet vid den aktuella tidpunkten får helt eller delvis donatorsmjölk så tas provet från denna mjölk 
istället (efter berikning). Markera i CRF om det var donators eller mammas egna mjölk som provet togs från.  
 
Eftersom vi skall ta berikad bröstmjölk måste mjölkköket bereda 10 ml mer bröstmjölk än barnet behöver den 
aktuella dagen. Meddela mjölkköket detta senast dagen före provtagning. 
 
1. Bröstmjölksprover skall tas dag vid 7±2, 14±2, 21±2 respektive 28±2 dagars ålder samt i gestationsvecka 
33+0-33+6 
2. Bröstmjölksprovet ska tas efter ev. tillsatt berikning! Skaka flaskan med mjölken direkt innan provet tas.  
3. Ta 1 rör per tillfälle. Sätt provet i frysfack, -20°C, i ”sköljen” i väntan på att de hämtas av labpersonal.  
4. Välj rör och klisteretikett med studienamn BM dag 7”, ”BM dag 14”, ”BM dag 21”, ”BM dag 28” 
respektive ”BM gv 33. Ni behöver endast fylla i barnets studie-ID nummer och provtagningsdatum. 
5. Skriv in i CRF att provet har tagits. 
 
Detta är grundinstruktionen men enskilda studieorter kan justera instruktionen beroende på hur man har kommit 
överens med sitt lokala lab.  
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
Thomas Abrahamsson          
 
 
 
61 
Appendix 16 –Nutritional content of H2 MF and Prolact CR 
 
Nutrient 
Units 
Prolact H2MF 
Prolact CR (Cream) 
 
 
 
 
 
 
 
 

Protocol No: 2020/vers.4      2020-03-25            
 
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62 
Appendix 17 –Product description and safety information 
 
Prolact+6 H2MF Concentrated Human Milk Fortifier 
(Human, Pasteurized) 
Each mL of Prolact+6 H2MF contains 1.41 kcal and 
0.06 grams of protein. 
Store at: -20°C or colder until ready to thaw for 
preparation and use. 
Available frozen in 125mL bottles containing 30mL of product. 
 
 
Under no circumstances should Prolact+6 H2MF be 
defrosted or warmed in a microwave. 
Remove bottle from the freezer and label with date 
and time. Thaw product using any of the 
following methods: 
1) Refrigeration: (2°C to 8°C) Place unopened bottle 
in refrigerator. Once thawed, must be administered 
within 48 hours. Do not refreeze, keep refrigerated. 
2) Rapid Thawing: Place bottle under lukewarm 
running water, or place in a water bath. Do not 
submerge top of bottle. Warm only until product is 
thawed. Continued warming, or exposure to high 
temperatures, could result in undesirable changes to 
the product. Wipe outside of bottle with appropriate 
disinfectant to reduce the risk of contamination. Once 
thawed, keep refrigerated, do not refreeze. Product 
must be administered within 48 hours of thawing. 
 
 
Always maintain aseptic technique when preparing and 
handling human milk. **DO NOT ADD WATER** 
Each bottle of Prolact+6 H2MF contains 30mL of fortifier 
and must be mixed with human milk according to 
Appendix 9. Once Prolact+6 H2MF has been properly 
thawed (see above), remove cap from bottle. Add human 
milk (expressed breast milk or banked milk) into the 
Prolact+6 H2MF bottle. Swirl the bottle gently to 
complete the mixing process. 
Once completed, the product is ready for use. Refrigerate 
(2°C to 8°C) until needed. Product must be administered 
according to the Prolacta thawing guidelines (see above), 
or according to hospital policy regarding human milk - 
whichever timeframe is shorter. 
Prolact+6 H2MF is 100% human milk. Abrupt transitioning 
from this product to non-human derived nutrition in the 
infant’s diet could result in feeding intolerance or GI 
complications.* 
All donors are screened for HIV 1 & 2, HTLV I & II, 
HBV, HCV and syphilis. 
All donor milk undergoes drug screening for 
amphetamine, benzodiazepine, cocaine, marijuana 
(THC), methamphetamine, opiates, nicotine, 
oxycodone, oxymorphone and their principle 
metabolites. 
Donor identity matching is performed on donated 
breast milk using DNA fingerprinting. 
Pasteurization has repeatedly demonstrated a 105 
reduction of pathogenic viruses such as HIV, HBV and 
HCV. It has been shown to kill pathogenic bacteria such 
as E. coli, S. aureus and Klebsiella species at levels of 
1015 or higher, the precise amount varying by strain and 
technique. 
HIV, also a marker virus for CMV, has been repeatedly 
demonstrated to inactivate during pasteurization. 
Pooled human breast milk is tested by PCR for the 
presence of HIV-1, HBV and HCV. 
 
 
Human milk, and contains less than 2% of one or more 
of the following: calcium glycerophosphate, calcium 
gluconate, sodium citrate, calcium chloride, calcium 
carbonate, potassium citrate, magnesium phosphate, zinc 
sulfate and cupric sulfate. 
 
 
 
Visit us at www.prolacta.com or call 1(888) PROLACT 
#1 for Customer Service 
Manufactured  by: Prolacta 
Bioscience, Inc. City of 
Industry, CA 91746 
PRODUCT DESCRIPTION 
SAFETY INFORMATION 
Prolact+6 H2MF® 
Human Milk Fortifier (Human, Pasteurized) 
30mL 
DIRECTIONS FOR THAWING 
INGREDIENTS 
PREPARATION INSTRUCTIONS 
FOR MORE INFORMATION