A randomised, double-blind, controlled trial to evaluate the effects of a nutritional product on brain integrity in preterm infants

Despite advances in perinatal care, extremely preterm infants still face
significant neurodevelopmental challenges, with over 50% of extremely preterm
infants exhibiting cognitive disabilities, behavioural problems and mild to
moderate motor impairments. These neurodevelopmental deficits place a major
burden on health care and society. Hence, there is an urgent need for
neuroprotective strategies to improve outcomes for these children.

During the third trimester of pregnancy, important processes of brain growth
and maturation take place with a rapid increase in both white and grey matter
volumes and subsequent cortical folding. Moreover, this phase is characterised
by rapid development of glial cells and neurons in the white matter. Extremely
preterm infants are thus exposed to extra uterine life in a period of critical
brain development, especially of white matter structures that render them
particularly susceptible to injury. Not surprisingly, white matter injury is
the most common pattern of brain injury in extremely preterm infants.

Perinatal infection has been recognised as an important risk factor for white
matter injury in preterm infants. In a recent study, Chau and colleagues showed
widespread abnormalities of microstructural and metabolic brain development, in
addition to white matter injury in preterm infants with postnatal infections.
Compared to preterm infants without infections, infected newborns demonstrated
lower FA values predominantly in the posterior white matter, increased average
diffusivity, indicating delayed myelination and maturation of the
oligodendrocyte lineage, and lower N-acteylaspartate/choline ratios -
reflecting impaired neuronal integrity and metabolism. The main pathogenetic
mechanism of white matter injury is considered to be inflammation, that may be
potentiated by co-existing ischemia.

The impact of nutrition on brain growth and cognition in preterm infants has
been increasingly appreciated. Furthermore, in recent years there has been an
increasing body of evidence that supplementation of probiotics may be
beneficial to preterm infants. Probiotics are micro-organisms that colonise the
gut and provide health benefits to the host through improved mucosal barrier
integrity, regulation of appropriate bacterial colonisation, and
immunomodulation. A recent Cochrane review including 2842 has demonstrated
that supplementation of enteral probiotics reduced the incidence of significant
NEC (Bell stage >= 2) and mortality, with a relative risk (RR) of 0.35 (95%
confidence interval (CI): 0.24; 0.52) for NEC and RR 0.40 (95% CI: 0.26; 0.60)
for all-cause mortality. In many level III neonatal intensive care units
(NICUs) across the globe, such as in Finland, Japan, Columbia, Denmark, Italy,
Germany, New-Zealand, and Australia, supplementation of probiotics has already
been integrated into routine clinical practice because of the presumed
beneficial effects outlined above.

The combination of probiotics and prebiotics is known to be synergistic, with
prebiotics enhancing the survival of endogenous probiotic organisms in the
host. The most widely studied prebiotics are galacto-oligosaccharide (GOS),
fructo-oligosaccharide (FOS), and inulin. Oligosaccharides represent an
important component of human milk and have been assigned important prebiotic,
antimicrobial, immunomodulatory and anti-inflammatory functions.
Oligosaccharides have the potential to improve the infant*s intestinal
microbiota by promoting growth of Bifidobacteria and Lactobacillus, that in
turn reduce the burden of potentially pathogenic micro-organisms in the gut.The
bifidogenic effect on the gut microbiota may support the immature immune system
by establishing an immunologic balance. The immune-modulating capacity of
prebiotic oligosaccharides is also likely to be microbiota-independent through
direct interaction with immune cells. Because of their presumed health
benefits, prebiotic oligosaccharides are supplemented to preterm formula as
part of routine clinical care. Meta-analyses of trial data have demonstrated
the safety of prebiotic supplementation, yet no convincing evidence was found
for a beneficial effect on serious neonatal infections. Therefore,
evidence-based recommendations of prebiotics as an isolated supplement, have so
far not been made.

Glutamine is the most abundant amino acid in the body. It is considered to be
an important fuel to rapidly dividing cells such as enterocytes and lymphocytes
and plays a substantial role in maintaining functional integrity of the gut.
Concomitantly, glutamine depletion leads to impaired functional integrity and
immune suppression. VLBW infants are at particular risk of glutamine depletion,
because they are primarily dependent on parenteral nutrition that contains
little to no glutamine. It is therefore postulated that glutamine-enriched
nutrition may improve immune function and intestinal integrity in these
vulnerable infants. Meta-analysis of trial data from five randomised controlled
trials showed a significantly lower incidence of invasive infections in preterm
infants who had received glutamine-supplemented enteral nutrition compared to
controls. No differences in the incidence of NEC or all-cause mortality were
found. Interestingly, a Dutch study (GEEF study) demonstrated long-term
benefits on brain development of early enteral glutamine supplementation. VLBW
infants who had received glutamine-enriched enteral nutrition in the neonatal
period showed larger brain volumes and improved white matter integrity at eight
years of age compared to controls. Differences in white matter volume and white
matter integrity of the hippocampus measured by FA were strongly associated
with the number of serious neonatal infections. Hence, these findings emphasise
the impact of neonatal infections on white matter integrity and support the
hypothesis that glutamine may improve brain development. To the best of our
knowledge, no studies have so far been conducted to assess the impact of
probiotics, or prebiotics on neonatal infections and/or inflammation in
relation to brain development. This is of particular interest because of the
postulated importance of inflammation and infection in the pathogenesis of
white matter injury and the well-known association between white matter injury
and subsequent neurodevelopmental impairment.

In conclusion, there is substantial evidence for the favourable effect of
probiotics on NEC and all-cause mortality in preterm infants. Enteral glutamine
supplementation has been shown to reduce the incidence of serious neonatal
infections. The benefits of oligosaccharides have not yet been established, as
literature shows only circumstantial evidence for a reduction of serious
infections in infants that received oligosaccharides as mono-therapy. All three
supplements have shown to be safe and no serious side effects have been
reported in large clinical trials. To date, there is no literature on the
combined effects of these nutritional supplements, or on their impact on
neonatal brain development. It is hypothesised that a combination of
probiotics, prebiotics, and glutamine may act synergistically. This hypothesis
is supported by animal studies in allergic mice, in which the combination of
L-glutamine, Bifidobacterium breve (B. breve), and scGOS/lcFOS was
demonstrated to exert a more beneficial effect on behaviour than the
combination of B. breve and scGOS/lcFOS alone. Therefore, we have decided to
combine these ingredients. Oligosaccharides promote survival of probiotics in
the host, and may improve the effect of probiotics on immunomodulation and
intestinal integrity, in addition to their intrinsic benefits on immune
function, host microbiota and mucosal barrier integrity. Glutamine may further
enhance these favourable effects. Subsequently, the combination of probiotics,
prebiotics and

>Primary objective:
During the intervention period (from randomisation until and including Term
Equivalent Age [TEA]):
To investigate the effect of the test product vs. the control product given to
preterm infants born at 24+0 to <30+0 weeks gestational age, on white matter
microstructure integrity (specifically: Fractional Anisotropy of the white
matter tracts, analysed using Tract-Based Spatial Statistics [TBSS]), as
assessed using magnetic resonance diffusion tensor imaging at TEA.


>Secondary objectives:
During the intervention period (from randomisation until and including TEA):
To investigate the effect of the test product vs. the control product, given to
preterm infants born at 24+0 to <30+0 weeks gestational age, on:
- White matter injury assessed on T2 and T1 weighted MR images at TEA
- Brain tissue volumes and cortical morphology assessed on T2 and T1 weighted
MR images at TEA
- Occurrence of serious neonatal infectious morbidity until TEA
- Development of immune function as measured by specific circulating
inflammatory markers until TEA (optional)

During the follow-up period (after TEA until 24 months corrected age):
To investigate the effect of the test product vs. the control product, given to
preterm infants born at 24+0 to <30+0 weeks gestational age, on:
- Neurodevelopmental outcome at 24 months corrected age as measured by Bayley
Scales of Infant and Toddler Development, Third Edition

>Safety and Tolerance objectives:
During the intervention period (from randomisation until and including TEA):
To investigate the effect of the test product vs. the control product given to
preterm infants born at 24+0 to <30+0 weeks gestational age, on:
- The occurrence of adverse events and serious adverse events
- Adequate growth
- Number of days of parenteral nutrition, time in days to achieve full
enteral nutrition (in the UMC Utrecht defined as 120 ml/kg/day for at least 1
day)
- Feeding intolerance
- Serum glutamine and glutamate concentrations (optional, in a subgroup)

During the follow-up period (after TEA until 24 months corrected age):
To investigate the effect of the test product vs. the control product, given to
preterm infants born at 24+0 to <30+0 weeks gestational age, on:
- The occurrence of adverse events and serious adverse events
- Growth velocity and anthropometric z-scores until two years corrected age

Double-blind, randomised, controlled, parallel-group, single-centre study

The intervention comprises a nutritional product that consists of two
components (part A and part B).
Test product group will receive:
- Part A: one daily dose of Bifidobacterium breve M-16V (3 x 109 cfu per day
for infants with birth weight >=1000 g; 1.5 x 109 cfu per day for preterm
infants with birth weight < 1000g until they reach enteral feeds of 50-60
ml/kg/day, then 3 x 109 cfu per day)
- Part B: L-glutamine (0.3g/kg/day) and short chain galacto-oligosaccharides
(scGOS)/long chain fructo-oligosaccharides (lcFOS) (9:1) (0.6g/kg/day)
supplemented to the regular enteral feed

Control product group will receive:
- Part A: One daily dose of carrier material
- Part B: Carrier material and casein and whey protein hydrolysates
supplemented to the regular enteral feed

Supplement
Based on previous research, it is believed that use of the test product may
lead to a reduction in the infectious and inflammatory burden experienced by
premature infants, with subsequent indirect and potentially direct benefits in
brain integrity/brain development.

The probiotic and overall prebiotic dosages lie within the range of the tested
dosages shown to be safe and well tolerated in premature infants. The
L-glutamine dosage has been extensively tested without reported adverse effects
in randomised controlled trials in premature neonates.

The only recognised possible adverse reaction associated with probiotic
administration is positive culture of the probiotic organism B. breve strain
M-16V from a normally sterile site. This is a very rare and unexpected event:
with B. breve M-16V it has never been previously reported, and with other B.
breve strains it has only been reported once (in an infant with an omphalocele
- a condition which is applied as an exclusion criterion in the current study).
The only recognised possible adverse reaction associated with prebiotic
administration is mild gastrointestinal discomfort (loose stools, constipation,
abdominal pain and/or flatulence). Gastrointestinal discomfort and feeding
tolerance will be closely monitored throughout the intervention period. (For
details regarding the above refer to the PIB).

MRI measurements
Potential risks of MRI scanning include noise-related hearing damage,
respiratory compromise and feeding problems in the first few hours after
scanning. The latter two could only be potentially present if sedation is
administered. To minimise the risk of noise-related hearing damage, appropriate
hearing protection is applied to the subject*s ears prior to scanning. Sedation
involves the potential risk of desaturation, apnea and feeding problems in the
first few hours after scanning, due to drowsiness. Although these side effects
are rarely reported, appropriate safety measures will be taken, according to
the local NICU protocol in case sedation is administered. Under the conditions
stated above, MRI scanning is considered a safe procedure. The benefit of
scanning at TEA is considered to outweigh the burden, because of the importance
for evaluation of the study objective and because even extremely preterm
infants tolerate MRI scanning well, if appropriate measures for hearing
protection and monitoring - if sedation is given - are taken.

Other study procedures, including sampling
There are no additional risks involved in other study procedures. Blood
sampling is considered to be a minimal burden, because it does not involve
extra handling of the subject. Samples are collected from indwelling arterial
catheters that have been inserted for clinical purposes or - in case the infant
does not have an arterial line - during routine sampling for clinical purposes.
Some sample material will be reserved for the purposes of the study. In case no
blood sampling is done for clinical purposes, blood samples will be omitted.
Collecting samples from the skin of the cheek, oral cavity, nasopharynx, and
stools is considered a minimal burden. Samples are obtained non-invasively and
will preferably be collected in combination with routine clinical sampling in
order to minimise handling of the subject and/or burden for the pregnant woman.
Moreover, timing of sampling during the intervention period will be dependent
on the infant*s clinical condition and stool pattern.