Systemic immune markers and infection risk in preterm infants fed human milk fortified with bovine colostrum or conventional fortifier, a secondary analysis of the FortiColos trial

Abstract

Background
For very preterm infants, human milk is often fortified with formula products based on processed bovine milk. Intact bovine colostrum (BC), rich in anti-inflammatory milk factors, is considered an alternative. We investigated if BC affects anti-inflammatory/TH2 immunity and infection risk in very preterm infants.

Methods
For a secondary analysis of a multicenter, randomized controlled trial (NCT03537365), very preterm infants (26–31 weeks gestation, 23% small for gestational age, SGA) were randomized to receive BC (ColoDan, Biofiber, Denmark, n = 113) or conventional fortifier (PreNAN, Nestlé, Switzerland, n = 116). Infection was defined as antibiotic treatment for five or more consecutive days and 29 cytokines/chemokines were measured in plasma before and after start of fortification.

Results
In general, infection risk after start of fortification was associated with low gestational age, SGA status and antibiotics use prior to fortification. Adjusted for confounders, infants fortified with BC showed more infection episodes (20 vs 12%, P < 0.05) and higher cumulative infection risk (hazard ratio, HR 1.9, P = 0.06), particularly for SGA infants (HR 3.6, P < 0.05). Additionally, BC-fortified infants had higher levels of TH2-related cytokines/chemokines (IL-10, MDC, MCP4) and reduced levels of cytokines related to TH1/TH17-responses (IL-15, IL-17, GM-CSF). The differences were most pronounced in SGA infants, displaying higher levels of TH2-related IL-4, IL-6, and IL-13, and lower interferon-γ and IL-1α levels in the BC group.

Conclusion
Infants fortified with BC displayed a delayed shift from TH2- to TH1-biased systemic immunity, notably in SGA infants, possibly influenced by multiple confounding factors, alongside elevated antibiotic use, suggesting increased susceptibility to infection.

Full text

Vol.:(0123456789)
Infection
https://doi.org/10.1007/s15010-024-02280-3
RESEARCH
Systemic immune markers andinfection risk inpreterm infants fed
human milk fortified withbovine colostrum orconventional fortifier,
asecondary analysis oftheFortiColos trial
OleBæk1,2· TikMuk1· LiseAunsholt2,3· GitteZachariasen4,5,6· PerTorpSangild1,2,4,7· DucNinhNguyen1
Received: 22 January 2024 / Accepted: 21 April 2024
© The Author(s) 2024
Abstract
Background For very preterm infants, human milk is often fortified with formula products based on processed bovine milk.
Intact bovine colostrum (BC), rich in anti-inflammatory milk factors, is considered an alternative. We investigated if BC
affects anti-inflammatory/TH2 immunity and infection risk in very preterm infants.
Methods For a secondary analysis of a multicenter, randomized controlled trial (NCT03537365), very preterm infants
(26–31weeks gestation, 23% small for gestational age, SGA) were randomized to receive BC (ColoDan, Biofiber, Denmark,
n = 113) or conventional fortifier (PreNAN, Nestlé, Switzerland, n = 116). Infection was defined as antibiotic treatment for
five or more consecutive days and 29 cytokines/chemokines were measured in plasma before and after start of fortification.
Results In general, infection risk after start of fortification was associated with low gestational age, SGA status and antibi-
otics use prior to fortification. Adjusted for confounders, infants fortified with BC showed more infection episodes (20 vs
12%, P < 0.05) and higher cumulative infection risk (hazard ratio, HR 1.9, P = 0.06), particularly for SGA infants (HR 3.6,
P < 0.05). Additionally, BC-fortified infants had higher levels of TH2-related cytokines/chemokines (IL-10, MDC, MCP4)
and reduced levels of cytokines related to TH1/TH17-responses (IL-15, IL-17, GM-CSF). The differences were most pro-
nounced in SGA infants, displaying higher levels of TH2-related IL-4, IL-6, and IL-13, and lower interferon-γ and IL-1α
levels in the BC group.
Conclusion Infants fortified with BC displayed a delayed shift from TH2- to TH1-biased systemic immunity, notably in
SGA infants, possibly influenced by multiple confounding factors, alongside elevated antibiotic use, suggesting increased
susceptibility to infection.
Keywords Preterm infant· Infection· Anti-inflammatory· Fortification· Bovine colostrum· Immune development
Introduction
Very preterm infants (born before 32weeks of gestation)
show greater risk of infections in early life, with risks
increasing with the degree of immaturity, particularly in
those born small for gestational age (SGA) [1, 2]. These vul-
nerable infants are also at risk of post-natal growth failure,
partly due to co-morbidities and systemic infections, but also
because enteral feeding poses challenges, with risks of feed-
ing intolerance, dysregulated metabolism and necrotizing
* Per Torp Sangild
pts@sund.ku.dk
* Duc Ninh Nguyen
dnn@sund.ku.dk
1 Comparative Pediatrics andNutrition, Department
ofVeterinary andAnimal Sciences, Faculty ofHealth
andMedical Sciences, University ofCopenhagen,
Frederiksberg, Denmark
2 Department ofNeonatology, Rigshospitalet, Copenhagen,
Denmark
3 Department ofClinical Medicine, University ofCopenhagen,
Copenhagen, Denmark
4 Hans Christian Andersen Children’s Hospital, Odense
University Hospital, Odense, Denmark
5 University ofSouthern Denmark, Odense, Denmark
6 Open Patient Explorative Network, Odense University
Hospital, Odense, Denmark
7 Faculty ofTheology, University ofCopenhagen,
Copenhagen, Denmark
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O.Bæk et al.
enterocolitis (NEC) [3]. Mother’s own milk (MOM) is the
preferred milk diet for infants born preterm and reduces the
risk of early life infections compared with infant formula
[4–6]. MOM contains a multitude of bioactive compounds
such as antimicrobial peptides, immunomodulatory proteins,
commensal microorganisms, maternal leucocytes and prebi-
otics [7]. Unfortunately, MOM is often unavailable, or not
sufficient in amounts or nutritional composition sufficient
to nourish infants for optimal growth after preterm birth.
In many countries, donor human milk (DHM) is used as a
substitute for MOM. However, DHM is obtained from moth-
ers in late lactation, with relatively low level of nutritional
and bioactive proteins, partly degraded by heat pasteuriza-
tion [8]. Adequate growth can be attained when MOM and
DHM are fortified with nutrient fortifiers, often based on
bovine milk formula products [9]. Such fortifiers are highly
processed and proteins often pre-hydrolyzed to enhance
amino acid absorption and avoid constipation and allergic
reactions [10].
Recently, intact bovine colostrum (BC) has been proposed
as an alternative nutrient fortifier for preterm infants [11,
12]. Like human colostrum, BC is rich in protein and
milk bioactive and immune-modulating compounds, like
immunoglobulin G and A, lactoferrin, multiple growth
factors [13–16]. When gently dried, heat-pasteurized
and irradiated to obtain a near-sterile product suitable for
clinical use, bioactivity of BC proteins is largely preserved
[17, 18]. Based on our previous pre-clinical research on BC
in preterm pigs [19–23] and reports from clinical trials in
preterm infants [12, 24, 25], BC fortification may modulate
immune development and infection risk, potentially by
promoting a more anti-inflammatory immune phenotype.
A recent randomized controlled trial in very preterm
infants (n = 232) investigated if BC fortification could
adequately support infant growth during hospitalization,
compared with a conventional fortifier (CF) [26]. The
results showed similar body growth between groups, and BC
fortification improved bowel habits, as indicated by less use
of laxatives [26, 27]. However, in the per-protocol analysis
there was a tendency to more use of antibiotics in the BC
group and further investigation revealed that this tendency
was most prominent in infants born SGA. In this secondary
analysis of the data, we explore in detail the impact of BC
fortification on infection risk and development of systemic
immunity, based on levels of cytokines and chemokines
in plasma collected before and after start of fortification.
We hypothesize that an increased risk of infections in BC
fortified infants is accompanied by changes to the circulating
cytokine profile, possibly affected by SGA status.
Methods
Study design
The registered protocol (clinicaltrials.gov: NCT03537365)
has been published along with the main clinical finding
[26, 28]. Briefly, very preterm infants (26–31weeks of
gestation, n = 232, 23% SGA), from eight neonatal units
in two regions of Denmark, were randomized to receive
either BC (pasteurized, spray-dried and irradiated powder,
Biofiber, Denmark) or a conventional bovine-based
fortifier (CF, FM85 PreNAN, Nestlé, Switzerland). All
infants received an enteral diet of MOM and/or DHM and
fortification was initiated when enteral feeding volumes
reached 100–140mL/kg/day and blood urea nitrogen
was < 5 mmol/L. Fortification was continued until a
gestational age of 34 + 6weeks or discharge. The amount
of fortification followed local guidelines and international
recommendations, with added protein not exceeding
1.4g per 100mL of human milk [29]. Infants with major
congenital malformations, gastrointestinal surgery or
receiving infant formula before start of fortification were
excluded. Written informed consent was obtained from all
participants’ parents or guardians. In cases of withdrawn
informed consent, data collected up to that point in time
was used, if allowed by parents/guardians. Infants were
classified as SGA by a birthweight Z-score less than two
standard deviations for their gestational age, based on the
growth chart by Niklasson et al. [30].
Antibiotics use andinfection incidence
Use of all medications were registered during the trial,
both before and after start of fortification. In the current
analysis, the type and duration of intravenous antibiotic
medications were identified and calculated. In three
cases (1 CF/SGA and 2 BC/AGA), records on prescribed
medications were unavailable and these infants were
removed from further analyses. A list of the prescribed
antibiotics is shown in Supplementary TableS1.
An episode of infection was pre-defined in the study
protocol as five or more consecutive days on any type
of intravenous antibiotics, regardless of blood culture
or other biochemical findings [28]. As such, early-onset
infection was defined as five or more consecutive days
of antibiotics, starting within the first 48h after birth,
while late-onset infections would occur after 72h post-
partum. Two periods of antibiotic use, more than 24h
apart, were considered as inconsecutive. Clinical blood
samples, measuring C-reactive protein (CRP), blood gas
and bacterial cultures, were not collected consistently
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Systemic immune markers andinfection risk inpreterm infants fed human milk fortified with…
across the units in relation to suspected infections. Hence,
these confirmatory blood results were available only from
a subset of the infants with suspected infection.
Blood sampling andcytokine measurements
Blood samples were collected before fortification and at
approximately 1(7 ± 1days) and 2weeks (14 ± 2days) after
start. Blood was collected by capillary puncture in EDTA-
coated tubes, cooled down and centrifuged (2500 × g, 4°C,
10min) for plasma collection within 4h and stored at –20°C
until further analysis. Levels of 29 plasma pro- and anti-
inflammatory cytokines and chemokines related to TH1/TH2/
TH17 polarization were measured by multiplex, fluorescent
immunoassays, as per the manufacturer’s instructions
(V-plex proinflammatory cytokine/chemokine analysis
panels, Meso Scale Diagnostics, USA). Levels of markers
below the detection limits, but higher than blank values,
were set to half of the lowest standard for each individual
cytokine or chemokine.
Statistics
All statistics were performed using Stata 14 (StataCorp,
USA). Before the start of fortification, group differences
in antibiotic use and number of infectious episodes were
calculated using a Chi-square or student t-test, depending
on data type. After the start of fortification, the effect of
the intervention on incidence of infections were calculated
using a logistic regression model while the cumulative risk
of infection was calculated with a cox proportional hazard,
conditional risk set model, accounting for multiple infections
per infant [31]. Duration of antibiotic treatment and cytokine
levels were evaluated using a generalized linear model. In
models, sex, SGA status, gestational age, Apgar score and
antibiotics use prior to intervention was used as cofactors.
During recruitment it became evident that randomization
was unevenly distributed in the Eastern and Western regions
of Denmark, and feeding practices and probiotics use also
differed. For that reason, geographical region was also
added as a covariate [26]. Choice of covariates was based
on their perceived impact on later infection incidence and
chosen before analysis. Plasma cytokines and chemokines
were analyzed by a similar mixed effect model, with assay
plate number was as a random factor to control for any inter-
plate differences. If residuals of models could not conform
to normality, data was log transformed. Any data that could
not conform to normality was analyzed with an appropriate
non-parametric model. All models were performed first on
all infants, and then stratified according to SGA or AGA
status, using the same covariates. Due to the lower number
of SGA infants, incidence of infections was calculated with
a penalized logistic regression [32] and cumulative infection
risk using the log-rank test. P values less than 0.05 were
considered statistically significant, while values less than 0.1
were considered as a tendency to a significant effect.
Results
Infection risk andantibiotics use beforestart
offortification
Postnatal age at start of intervention was about 1day shorter
in the BC group (Table1, P < 0.05), however this difference
was not apparent for SGA infants (Table1). Other baseline
characteristics for infants did not differ between diet or
AGA/SGA groups, except that Apgar scores were lower
in BC-fortified infants (but only in AGA infants, P < 0.01,
Table1). Incidences of early- or late-onset infections, before
start of the intervention, did not differ between groups, with
or without AGA/SGA stratification, although a tendency
to fewer late-onset infections was observed in BC-fortified
AGA infants (6 vs 13%, P = 0.09, Table1). Before start
of intervention, about 50% of all the infants received
antibiotics, with similar distribution between early- and late-
onset infections, and between BC and CF groups, with or
without SGA (Table1, all P > 0.1). No infant had more than
one late-onset infection before start of fortification while two
infants had both an early and late onset infection (one from
each group). Similarly, the duration of antibiotic treatment
before start of intervention, did not differ between groups,
with/without AGA/SGA stratification (Table1, all P > 0.1).
Infection risk andantibiotic use afterstart
offortification
Following the start of fortification, 52 episodes of infection
were observed across 37 infants. Results of clinical blood
cultures or samples collected around the time of antibiotic
treatment are shown in Supplementary Table2. Blood
cultures were only performed in 50% (26/52) of reported
infection episodes and 65% (17/26) of these showed growth
of a possibly pathogenic bacteria, with no significant
differences in the culture positive rate or pathogen found
between the groups. Likewise in available samples CRP, pH
and lactate levels were similar between groups. Of the 52
reported episodes of infection, in 42 (14 CF, 28 BC) CRP
was > 10ug/mL, with/without positive blood culture, while
in four episodes (2 CF, 2 BC) CRP was < 10ug/mL and
blood cultures negative/not performed. In six cases (4 CF, 2
BC),) no CRP or blood culture results were available.
The overall risk of infection following fortification was
most strongly associated with gestational age (P < 0.001,
Supplementary FigureS1A), but not with postnatal age at
start of fortification (P > 0.1, Supplementary FigureS1B).
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O.Bæk et al.
Likewise, SGA status or antibiotic use before start of forti-
fication was associated with later infection (both P < 0.05,
Supplementary FigureS1 CS1C,D).
Across all infants, the incidence of at least one infectious
episode was higher in the BC group (Table2, 20 vs 12%,
P < 0.05). In SGA infants, the mean incidence of infection
was twice as high in BC-fortified SGA infants vs CF-forti-
fied SGA infants, but not significantly (Table2, 32 vs 15%,
P > 0.1). There was a tendency towards higher cumulative
infection risk among all BC-fortified infants (Cox model
HR: 1.9, CI95% 1.0–3.7, P = 0.06, Fig.1A), with no differ-
ence in risk among AGA infants (HR: 1.4, CI95% 0.6–3.3,
P > 0.1, Fig.1B). However, cumulative infection risk among
SGA infants was significantly higher in those receiving BC
fortification (risk ratio, RR: 3.3, CI95% 1.1–10.2, P < 0.05,
Fig.1C). In a sensitivity analysis, including only the 42
cases of infection with increased CRP and/or positive blood
culture, infection risk was significantly increased across all
BC-fortified infants (HR: 2.3, CI95% 1.1–4.3, P < 0.05, Sup-
plemental FigureS2A), as well as for BC-fortified AGA
infants (HR: 2.3, CI95% 1.0–5.4, P = 0.06, Supplemental
FigureS2B) and SGA infants (RR: 3.0, CI95% 1.0–9.2,
P < 0.05, Supplemental FigureS2C).
Across all infants, total length of antibiotic treatment
was longest in the BC group, although this is explained
by the higher incidence of infection as length of antibiot-
ics treatment did not differ among infants without infection
(Table2). Again, the effects were most pronounced for the
SGA subgroups (Table2). Importantly, the use of parenteral
nutrition, an additional risk factor for postnatal infection, did
not differ after start of fortification (Table2). Interestingly,
there was interaction between use of antibiotics prior to start
of fortification (for any duration of time), and later risk of
infection. Without antibiotics use, infants fortified with CF
Table 1 Baseline characteristics and infection risk before start of fortification
Baseline characteristics, risk of infections and use of intravenous antibiotics (AB) in very preterm infants randomized to be fortified with either
conventional fortifier (CF) or bovine colostrum (BC). Shown for all infants and stratified for birth weight status
AGA birthweight appropriate for gestational age, SGA small for gestational age, NS not significant
a Information on birth complications could not be obtained for all infants, see individual n numbers.
b Defined as more than 5 consecutive days on AB, starting within 48h of birth.
c Defined as more than 5 consecutive days on antibiotics, starting more than 48h of birth. Binomial data shown as percentage and continuous
data shown as means with corresponding standard deviation
All infants AGA SGA
CF
n = 116
BC
n = 113
P CF
n = 91
BC
n = 85
P CF
n = 25
BC
n = 28
P
Age at fortification, days 9.4 (3.7) 8.4 (2.8) < 0.05 9.6 (4.1) 8.5 (2.9) < 0.05 8.5 (2.9) 8.2 (2.7) NS
Gestational age at birth, days 200 (10) 201 (10) NS 200 (11) 202 (10) NS 202 (9) 200 (9) NS
Male sex 54% (63/116) 60% (68/113) NS 52% (47/91) 59% (50/85) NS 64% (16/25) 64% (18/28) NS
Birthweight, g 1167 (322) 1175 (333) NS 1242 (309) 1285 (289) NS 894 (198) 840 (211) NS
Cesarian section 74% (86/116) 70% (79/113) NS 67% (61/91) 61% (52/85) NS 100% (25/25) 96% (27/28) NS
Complications during birtha
Preeclampsia 21% (23/112) 23% (26/111) NS 10% (9/89) 14% (12/83) NS 61% (14/23) 50% (14/28) NS
Premature rupture of
membranes
21% (24/116) 20% (23/113) NS 24% (22/91) 26% (22/85) NS 8% (2/26) 4% (1/28) NS
Chorioamnionitis 3% (3/113) 4% (5/113) NS 2% (2/89) 6% (5/85) NS 4% (1/25) 0% (0/28) −
Placental abruption 6% (7/114) 12% (13/111) NS 8% (7/89) 15% (13/84) NS 0% (0/26) 0% (0/27) −
Mean APGAR score at 5min 9.3 (± 1.4) 8.7 (± 1.9) < 0.05 9.4 (± 1.2) 8.6 (± 2.1) < 0.01 8.8 (± 1.8) 9.3 (± 0.7) NS
APGAR score < 7 at 5min 7% (8/116) 11% (12/113) NS 5% (4/91) 14% (12/85) 0.03 17% (4/25) 0% (0/28) −
Infections and antibiotics use prior to intervention
Early onset infectionb10% (12/116) 12% (13/113) NS 9% (8/91) 8% (7/85) NS 15% (4/26) 21% (6/28) NS
Late onset infectionc13% (15/116) 8% (9/113) NS 13% (12/91) 6% (5/85) 0.09 12% (3/25) 14% (4/28) NS
Any AB 47% (55/116) 54% (61/113) NS 46% (42/91) 53% (45/85) NS 52% (13/25) 57% (16/28) NS
Total AB use, days 3.0 (± 4.2) 3.4 (± 5.0) NS 3.0 (± 4.3) 3.1 (± 4.9) NS 3.4 (± 4.0) 4.4 (± 5.4) NS
AB use during early onset
infections, days
8.8 (± 1.4) 11.2 (± 1.7) NS 8.8 (± 2.0) 10.7 (± 2.8) NS 9.0 (± 1.3) 11.7 (± 2.2) NS
AB use during late onset
infections, days
10.8 (± 1.0) 13.8 (± 1.5) NS 11.3 (± 1.3) 15.2 (± 2.3) NS 9.0 (± 1.0) 12.0 (± 2.1) NS
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Systemic immune markers andinfection risk inpreterm infants fed human milk fortified with…
showed lower infection risk later than the corresponding BC
infants, or infants receiving antibiotics before start of forti-
fication (Supplementary FigureS3 A−C).
Plasma cytokines beforeandafterstart
offortification
Before fortification no differences in plasma cytokines
were observed (Fig.2A−L). 1week after start of the
intervention, BC infants had significantly higher levels
of TH2 cytokine IL-10 (Fig.2A, P < 0.01, both AGA and
SGA) with a tendency to higher IL-4 levels (Fig.2B,
P = 0.07 for SGA infants). At the same time, BC-forti-
fied infants had lower levels of IL-5, IL-15, IL-17 and
granulocyte–macrophage colony-stimulating factor (GM-
CSF, Fig.2C−F, all P < 0.05). BC-fortified SGA infants
also tended to have lower levels of interferon-γ (IFN-γ,
Fig.2G, P = 0.07). The above differences between diet
were less pronounced at 2weeks after start of fortifi-
cation. However, differences persisted within the SGA
Table 2 Risk of infections and use of antibiotics and parenteral nutrition after start of fortification
Risk of infections and use of intravenous antibiotics (AB) and parenteral nutrition in very preterm infants fortified with either conventional
fortifier (CF) or bovine colostrum (BC). Shown for all infants and stratified for birth weight status
AGA birthweight appropriate-for-gestational age, SGA small-for-gestational age, NS not significant
a Infection defined as 5 or more consecutive days on antibiotics after start of fortification
b AB prescribed for less than 5days after start of fortification. Binomial data shown as percentage and continuous data shown as means with
corresponding standard deviation
All infants AGA SGA
CF
n = 116
BC
n = 113
P CF
n = 91
BC
n = 85
P CF
n = 25
BC
n = 28
P
Infectionsa
Incidence of infection 12% (14/116) 20% (23/113) 0.04 10% (10/91) 16% (14/85) NS 16% (4/25) 32% (9/28) NS
Multiple infections 29% (4/14) 26% (6/23) NS 40% (4/10) 14% (2/14) NS 0% (0/4) 44% (4/9) −
AB without infectionb9% (10/116) 7% (8/113) NS 10% (9/91) 6% (5/85) NS 4% (1/25) 10% (3/28) NS
Antibiotics
Total use, days 1.9 (± 5.4) 3.6 (± 9.5) 0.02 2.1 (± 5.9) 2.9 (± 1.0) NS 1.4 (± 3.0) 5.5 (± 8.5) 0.06
Without infection, days 0.3 (± 0.8) 0.3 (± 1.1) NS 0.3 (± 0.1) 0.2 (± 0.1) NS 0.1 (± 0.7) 0.7 (± 1.7) NS
With infection, days 13.9 (± 8.9) 16.2 (± 15.5) NS 16.3 (± 9.5) 16.5 (± 19.2) NS 8.0 (± 1.4) 15.8 (± 8.0) NS
Parenteral nutrition
Incidence 28% (32/116) 31% (35/113) NS 23% (21/91) 26% (22/85) NS 44% (11/25) 46% (13/28) NS
Duration, days 1.5 (± 1.5) 1.6 (± 1.5) NS 1.6 (± 1.7) 1.7 (± 1.2) NS 1.4 (± 1.3) 1.4 (± 1.8) NS
Fig. 1 Time to infectious episodes in infants fortified with bovine
colostrum (BC) or conventional fortifier (CF), shown as Kaplan
Meyer curves with results of corresponding Cox proportional hazard
models or log rank test shown as text. Shown for all infants (A) or
stratified by birth weight status (B AGA appropriate for gestational
age; C SGA small for gestational age). HR hazard ratio, RR risk ratio
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O.Bæk et al.
subgroup for IL-10, IL-15 and GM-CSF (Fig.2A,C,E).
In addition, levels of IL-6 and IL-13 were increased for
BC-fortified SGA infants (Fig.2H, I, both P < 0.01),
while the levels of GM-CSF and IL-1α were decreased
(P < 0.01). Interestingly, BC-supplemented AGA infants
showed lower IL-6 levels both 1 and 2weeks after start
of fortification (Fig.2H, P < 0.01), contrasting the above
effects in SGA infants.
To investigate whether cytokine levels and their
responses to fortification were influenced by the presence
or absence of suspected infection, a sensitivity analysis,
with removal of the 37 infants with infection, was
conducted. In this analysis, the majority of the previously
reported differences persisted. Specifically, SGA infants
fed with BC continued to show higher levels of IL-6,
IL-10, and IL-13 (all with P < 0.05), along with decreased
levels of GM-CSF, IL-1α, IL-1β, IL-5, and IL-17 (all
with P < 0.05) at 1 and/or 2weeks after the initiation of
fortification (data presented in Supplementary TableS3).
Plasma chemokines beforeandafterstart
offortification
The effects of BC fortification on circulating chemokine lev-
els were less obvious. BC-fortified infants showed higher
levels of monocyte chemoattractant protein 4 (MCP-4 or
CCL13) and IFN-γ inducible protein-10 (IP-10 or CXCL10)
1 and 2weeks after the start of fortification. This was driven
mainly by effects among AGA infants (Fig.3A, B, P < 0.01
and P < 0.05), with a similar trend observed for thymus and
activation regulated chemokine (TARC or CCL17, Fig.3C,
P = 0.06 and P = 0.07).
Discussion
Securing adequate enteral nutrition in preterm infants is
key to achieve optimal postnatal growth but the choice
of milk diet may also influence other aspects of health,
Fig. 2 Plasma cytokine levels in infants fortified with bovine colos-
trum (BC) or conventional fortifier (CF). Shown before start of for-
tification (Baseline) and 1 and 2weeks after start of fortification for
all infants or stratified by birth weight status (AGA, appropriate for
gestational age; SGA, small for gestational age). Shown as 95% per-
centile box plots for differences between BC and CF fortification, *:
P < 0.05, **: P < 0.01, while P values between 0.05 and 0.1 are shown
as text
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Systemic immune markers andinfection risk inpreterm infants fed human milk fortified with…
including systemic immunity. We found that the use of BC
as a fortifier to human milk (MOM, DHM or a mixture) was
associated with an increased use of intravenous antibiotics,
indicating an increased incidence of infections, especially
in infants born SGA, even after excluding infants without
positive blood cultures or high CRP levels. Fortification
with BC was also associated with a blood immune profile
that reflected higher levels of anti-inflammatory/TH2/TReg
cytokines (e.g. IL-10) and lower levels of pro-inflammatory/
TH1/TH17-related cytokines (e.g. IL-15, IL-17, GM-CSF),
most clearly after 1week of fortification. Again, this pattern
was most clear for SGA infants, where infants fortified with
BC also showed higher levels of TH2 cytokines (IL-4, IL-6,
IL-13) and a tendency to lower levels of key TH1 cytokines
(IFN-γ, IL-1α). These differences therefore indicate that the
immune profile of BC-fortified infants, especially those born
SGA, could be skewed towards a more anti-inflammatory/
TH2 driven immune phenotype. Importantly, these effects
persisted after excluding infants that experienced an
infectious episode after start of fortification, suggesting that
the observed effects were driven by the diet intervention and
not by inflammation associated with infections. However,
IL-5, a classical TH2 cytokine related to eosinophil
activation showed the opposite effect with lower levels
in BC-fortified infants. This could be explained by TReg
derived IL-10 which is known to suppress IL-5 production
at mucosal surfaces [33, 34]. Together with the observed
interaction of BC effects on infection risk with antibiotics
use prior to the start of fortification, this could indicate that
BC effects are mediated via the gut mucosal immune system
and/or the gut microbiota. We are currently investigating
stool samples collected during the trial to elucidate any
effects BC fortification and antibiotics had on gut microbial
composition.
The increased infection risk in BC infants was
unexpected, as previous use of BC supplementation in
humans and animals indicates protection against gut and
respiratory infections [11, 35–39]. However, these results
may not be valid for longer term supplementation of BC
to a very preterm infant, with an immature gastrointestinal
tract and immune system, beyond the first week of life.
In preterm pigs, exclusive or partial BC feeding just after
birth promotes gut maturation, prevents NEC, improves
bacterial clearance during systemic infections and increases
the number of circulating TReg cells, suggesting an impact
on systemic immunity [20, 22, 40, 41]. In our trial, the
incidence of NEC was low and equal in the two groups [26].
Likewise, administration of BC to adult mice reduced their
blood immune cell pro-inflammatory responses [42, 43].
Previously, three larger randomized and one pilot trial of
BC supplementation to preterm infants have been conducted
[12, 24, 25, 44]. In one of these trials, administration of BC
just after preterm birth, with no MOM feeding, reduced the
incidence of severe late-onset sepsis, defined as infection-
related organ dysfunction. However, BC had no impact on
overall infection risk or antibiotic use but was linked to
increased blood TReg cells in the weeks following preterm
birth (27). Our other recent study in China, supplementing
MOM with BC instead of formula in the first week after
birth, showed no effects on systemic infections. However
extensive use of antibiotics immediately following birth
in this trial may have clouded any positive or negative BC
effects (26). Likewise, BC used as a fortifier to human
milk for very preterm infants from 2–3weeks in China
showed no effect on infections, although almost all these
Fig. 3 Chemokine profiles in infants fortified with bovine colostrum
(BC) or conventional fortifier (CF). Shown before start of fortification
(Baseline) and 1 and 2weeks after start of fortification for all infants
and stratified by birth weight status (AGA, appropriate for gestational
age; SGA, small for gestational age). Shown as 95% percentile box
plots for differences between BC and CF fortification, *: P < 0.05,
while P values between 0.05 and 0.1 are shown as text
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

O.Bæk et al.
infants received antibiotics prior to start of fortification
(ClinicalTrials.gov NCT03822104, our unpublished results).
In the present study, we identified infections based on
antibiotics use and could not judge disease severity, but
increased IL-10 levels in BC infants may result from more
circulating TReg cells. The resulting anti-inflammatory/
TH2/TReg-biased immunity may predispose to systemic
infections later. For instance, very preterm infants diagnosed
with severe systemic infections show reduced capacity to
mount pro-inflammatory responses prior to infection onset
[45]. However, BC may lead to reduced pro-inflammatory
responses, with an impaired ability to fight infections,
depending on other variables, such as birth status (SGA/
AGA), and postnatal age. Likewise, the impact of the base
milk diet (DHM, MOM, or combinations) is unknown and
may act as an effect-modifier on BC fortification. Possibly,
anti-inflammatory effects of BC supplementation affect
preterm infants differently compared with term infants or
older children. Compared with term infants, preterm infants
already show diminished pro-inflammatory responses at
birth, while their ability to produce IL-10 is less affected or
even improved [45–48]. This immature immune phenotype
is further affected by fetal growth restriction, induced by
a variety of antenatal factors, as cord blood immune cells
from SGA and intrauterine growth restricted preterm infants
show an even lower capacity to produce pro-inflammatory
cytokines [49–51]. It is therefore plausible that BC
increase the infection susceptibility specifically in SGA
infants because they already show low pro-inflammatory
capacity. In our study, the infection risk was similar in
CF- and BC-fortified AGA infants, even if they showed
differences in plasma cytokine levels. Finally, it cannot be
excluded that anti-inflammatory effects of longer-term BC
supplementation negatively affect later systemic infections
in preterm infants, but positively affect immune responses
at mucosal surfaces (gut, lungs, skin) (35,36).
Our study has several limitations. Given the secondary
nature of the analysis, we cannot define the causal links
between plasma cytokine profiles and infection risk. Despite
randomization there were uneven distributions in some
factors between the BC and CF groups, including region
of birth and infection risk prior to start of fortification.
Although we included these factors as covariates in our
statistical models, residual confounding cannot be ruled
out. Especially since we find signs of interacting effects
of BC fortification with use of Antibiotics prior to start of
fortification. Finally, we were unable to control for any role
of MOM versus DHM (or mixed diets) on infection rates and
cytokine levels. It is also important to note that our trial was
not designed and powered to investigate effects of BC on
risk of infections but had a focus on safety and feasibility to
secure adequate growth rates [26]. Consequently, only a part
of the infants with suspected infections had blood sampled
for bacterial cultures and assessment of CRP levels and since
the trial was not blinded, we cannot exclude possible bias of
clinicians. Yet, we find it unlikely that clinicians would be
more likely to prescribe antibiotics to BC-fortified infants.
Our study provides novel insights into the immune-
modulating effects of BC fortification in very preterm infants
As adjusted for relevant confounders, we suggest that BC
fortification is associated with increased risk of infection,
especially in SGA infants, and with increased levels of
anti-inflammatory cytokines. The randomized controlled
trial design, coupled with comprehensive evaluations of
cytokine profiles and antibiotics use, strengthens the validity
of our findings and raise important questions regarding
the influence of continued supplementation with an anti-
inflammatory milk diet like BC beyond the immediate
neonatal period. Especially in already immune-suppressed
SGA infants, such diets may increase the risk of systemic
infections. Larger studies, controlling for age and weight at
birth, milk diet and antibiotics treatment prior to intervention
are required to elucidate these highly complex and possibly
interacting effects.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s15010- 024- 02280-3.
Author contributions GZ and PTS planned the original trial and LA
assisted in its execution. The secondary analysis was planned by OB
and DNN. Laboratory work and data analysis was performed by OB
and TM. OB wrote the first draft of the manuscript while PTS and DNN
held joint responsibility for the final contents and all authors read and
approved the final manuscript.
Funding Open access funding provided by Copenhagen University.
The FortiColos trial was funded by Innovation Fund Denmark (Grant
nr: 6150-00004B).
Data availability Anonymized raw data files can be made avaliable
upon reasonble request to the corresponding authors.
Declarations
Conflict of interest The University of Copenhagen holds a patent on
the use of bovine colostrum for pediatric patients. PTS is listed as a
sole inventor but has declined any share of potential revenue arising
from commercial exploitation of such a patent. The remaining authors
declare no conflict of interest.
Ethical approval This study adhered to the principles outlined in the
Declaration of Helsinki and was conducted in accordance with the ethi-
cal standards of the Danish National Center for Ethics. The trial was
approved by the Scientific Ethical Committee of the Region of South-
ern Denmark (S-20170095) and the Danish Data Protection Agency
(17/33672). An independent data safety monitoring board reviewed
trial data and safety during the enrolment period, incorporating pre-
liminary assessment of key outcomes and potential adverse events.
Written informed consent was obtained from all participants’ parents
or guardians before enrollment in the study.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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