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.

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.

Neonatal Gut and Immune Responses to ß-Casein Enriched Formula in Piglets.

BACKGROUND: ß-casein is the main casein constituent in human milk (HM) and a source of bioactive peptides for the developing gastrointestinal tract and immune system. Infant formulas contain less ß-casein than HM, but whether different concentrations of ß-casein affect tolerability and gut and immune maturation in newborns is unknown. OBJECTIVES: Using near-term piglets as a model for newborn infants, we investigated whether increasing the ß-casein fraction in bovine-based formula is clinically safe and may improve gut and immune maturation. METHODS: Three groups of near-term pigs (96% gestation) were fed formula with bovine casein and whey protein (ratio 40:60): 1) standard skim milk casein (BCN-standard, 35% ß-casein of total casein, n = 18); 2) ß-casein enrichment to HM concentrations (BCN-medium, 65%, n = 19); and 3) high ß-casein enrichment (BCN-high, 91%, n = 19). A reference group was fed 100% whey protein concentrate (WPC) as protein (WPC, n = 18). Intestinal and immune parameters were assessed before and after euthanasia on day 5. RESULTS: Clinical variables (mortality, activity, body growth, and diarrhea) were similar among the groups, and no differences in intestinal or biochemical parameters were observed between BCN-standard and BCN-medium pigs. However, pigs receiving high amounts of ß-casein (BCN-high) had lower small intestine weight and tended to have more intestinal complications (highest gut pathology score, permeability, and interleukin-8) than the other groups, particularly those receiving no casein (WPC pigs). Blood lymphocyte, thrombocyte, and reticulocyte counts were increased with higher ß-casein, whereas eosinophil counts were reduced. In vitro blood immune cell responses were similar among groups. CONCLUSIONS: ß-casein enrichment of bovine-based formula to HM concentrations is clinically safe, as judged from newborn, near-term pigs, whereas no additional benefits to gut maturation were observed. However, excessive ß-casein supplementation, beyond concentrations in HM, may potentially induce gut inflammation together with increased blood cell populations relative to natural ß-casein concentrations or pure whey-based formula.

Insulin-like growth factor-1 effects on kidney development in preterm piglets.

BACKGROUND: Preterm birth disrupts fetal kidney development, potentially leading to postnatal acute kidney injury. Preterm infants are deficient in insulin-like growth factor 1 (IGF-1), a growth factor that stimulates organ development. By utilizing a preterm pig model, this study investigated whether IGF-1 supplementation enhances preterm kidney maturation. METHODS: Cesarean-delivered preterm pigs were treated systemically IGF-1 or vehicle control for 5, 9 or 19 days after birth. Blood, urine, and kidney tissue were collected for biochemical, histological and gene expression analyses. Age-matched term-born pigs were sacrificed at similar postnatal ages and served as the reference group. RESULTS: Compared with term pigs, preterm pigs exhibited impaired kidney maturation, as indicated by analyses of renal morphology, histopathology, and inflammatory and injury markers. Supplementation with IGF-1 reduced signs of kidney immaturity, particularly in the first week of life, as indicated by improved morphology, upregulated expression of key developmental genes, reduced severity and incidence of microscopic lesions, and decreased levels of inflammatory and injury markers. No association was seen between the symptoms of necrotizing enterocolitis and kidney defects. CONCLUSION: Preterm birth in pigs impairs kidney maturation and exogenous IGF-1 treatment partially reverses this impairment. Early IGF-1 supplementation could support the development of preterm kidneys. IMPACT: Preterm birth may disrupt kidney development in newborns, potentially leading to morphological changes, injury, and inflammation. Preterm pigs have previously been used as models for preterm infants, but not for kidney development. IGF-1 supplementation promotes kidney maturation and alleviates renal impairments in the first week of life in preterm pigs. IGF-1 may hold potential as a supportive therapy for preterm infants sensitive to acute kidney injury.

Mineral supplementation for very preterm infants fed fortified human milk.

OBJECTIVES: The safety and feasibility of human milk fortification with bovine colostrum (BC) were investigated in very preterm infants (FortiColos trial, NCT03537365). The BC product contained lower calcium, phosphate, and iron levels compared to the conventional fortifier (CF). We tested whether fortification with BC plus extra phosphate was sufficient to support the infants' mineral status assessed by blood biochemistry. METHODS: In a randomised controlled trial (FortiColos, NCT03537365), mineral status was compared after fortification with BC versus CF. Blood calcium, phosphate, and haemoglobin were determined before and up to 3 weeks after the start of fortification (at the mean age of 8-9 days). The maximum supplemental doses of calcium, phosphate, and iron given were retrieved from patient medical records. Results were adjusted for gestational age, birth weight, and enteral nutrition with the mother's own milk and/or donor human milk. RESULTS: Blood values of calcium, phosphate, and haemoglobin were similar between groups. Infants in both groups required supplementation with calcium and phosphate, but infants fed BC required higher maximum doses of phosphate and calcium (p < 0.05) to maintain acceptable blood values. Regardless of fortification groups, the most immature (<29 weeks of gestation) and small for gestational age infants showed a higher risk for requiring additional phosphate (OR: 3.9, p < 0.001; OR: 2.14, p = 0.07, respectively). CONCLUSIONS: The use of BC as a fortifier for human milk requires additional phosphate and calcium relative to a CF. Regardless of the fortification product, the most immature and small infants require additional mineral supplementation.

Glucose-regulatory hormones and growth in very preterm infants fed fortified human milk.

BACKGROUND: Bovine colostrum (BC) contains a range of milk bioactive components, and it is unknown how human milk fortification with BC affects glucose-regulatory hormones in very preterm infants (VPIs). This study aimed to investigate the associations between hormone concentrations and fortification type, birth weight (appropriate/small for gestational age, AGA/SGA), milk intake, postnatal age, and body growth. METHODS: 225 VPIs were randomized to fortification with BC or conventional fortifier (CF). Plasma hormones were measured before, one and two weeks after start of fortification. ΔZ-scores from birth to 35 weeks postmenstrual age were calculated. RESULTS: Compared with CF, infants fortified with BC had higher plasma GLP-1, GIP, glucagon, and leptin concentrations after start of fortification. Prior to fortification, leptin concentrations were negatively associated with growth, while IGF-1 concentrations associated positively with growth during fortification. In AGA infants, hormone concentrations generally increased after one week of fortification. Relative to AGA infants, SGA infants showed reduced IGF-1 and leptin concentrations. CONCLUSION: Fortification with BC increased the plasma concentrations of several glucose-regulatory hormones. Concentrations of IGF-1 were positively, and leptin negatively, associated with growth. Glucose-regulatory hormone levels were affected by birth weight, milk intake and postnatal age, but not closely associated with growth in VPIs. IMPACT: Little is known about the variation in glucose-regulatory hormones in the early life of very preterm infants (VPIs). This study shows that the levels of glucose-regulatory hormones in plasma of VPIs are highly variable and modified by birth weight (appropriate or small for gestational age, AGA or SGA), the type of fortifier, enteral nutritional intake, and advancing postnatal age. The results confirm that IGF-1 levels are positively associated with early postnatal growth in VPIs, yet the levels of both IGF-1 and other glucose-regulatory hormones appeared to explain only a small part of the overall variation in growth rates.

Enteral plasma supports brain repair in newborn pigs after birth asphyxia.

Newborns exposed to birth asphyxia transiently experience deficient blood flow and a lack of oxygen, potentially inducing hypoxic-ischaemic encephalopathy and subsequent neurological damage. Immunomodulatory components in plasma may dampen these responses. Using caesarean-delivered pigs as a model, we hypothesized that dietary plasma supplementation improves brain outcomes in pigs exposed to birth asphyxia. Mild birth asphyxia was induced by temporary occlusion of the umbilical cord prior to caesarean delivery. Motor development was assessed in asphyxiated (ASP) and control (CON) piglets using neonatal arousal, physical activity and gait test parameters before euthanasia on Day 4. The ASP pigs exhibited increased plasma lactate at birth, deficient motor skills and increased glial fibrillary acidic protein levels in CSF and astrogliosis in the putamen. The expression of genes related to oxidative stress, inflammation and synaptic functions was transiently altered in the motor cortex and caudate nucleus. The number of apoptotic cells among CTIP2-positive neurons in the motor cortex and striatal medium spiny neurons was increased, and maturation of preoligodendrocytes in the internal capsule was delayed. Plasma supplementation improved gait performance in the beam test, attenuated neuronal apoptosis and affected gene expression related to neuroinflammation, neurotransmission and antioxidants (motor cortex, caudate). We present a new clinically relevant animal model of moderate birth asphyxia inducing structural and functional brain damage. The components in plasma that support brain repair remain to be identified but may represent a therapeutic potential for infants and animals after birth asphyxia.

A Gently Processed Skim Milk-Derived Whey Protein Concentrate for Infant Formula: Effects on Gut Development and Immunity in Preterm Pigs.

SCOPE: Processing of whey protein concentrate (WPC) for infant formulas may induce protein modifications with severe consequences for preterm newborn development. The study investigates how conventional WPC and a gently processed skim milk-derived WPC (SPC) affect gut and immune development after birth. METHODS AND RESULTS: Newborn, preterm pigs used as a model of preterm infants were fed formula containing WPC, SPC, extra heat-treated SPC (HT-SPC), or stored HT-SPC (HTS-SPC) for 5 days. SPC contained no protein aggregates and more native lactoferrin, and despite higher Maillard reaction product (MRP) formation, the clinical response and most gut and immune parameters are similar to WPC pigs. SPC feeding negatively impacts intestinal MRP accumulation, mucosa, and bacterial diversity. In contrast, circulating T-cells are decreased and oxidative stress- and inflammation-related genes are upregulated in WPC pigs. Protein aggregation and MRP formation increase in HTS-SPC, leading to reduced antibacterial activity, lactase/maltase ratio, circulating neutrophils, and cytotoxic T-cells besides increased gut MRP accumulation and expression of TNFAIP3. CONCLUSION: The gently processed SPC has more native protein, but higher MRP levels than WPC, resulting in similar tolerability but subclinical adverse gut effects in preterm pigs. Additional heat treatment and storage further induce MRP formation, gut inflammation, and intestinal mucosal damage.

Early parenteral nutrition is associated with improved growth in very low birth weight infants: a retrospective study.

OBJECTIVE: To assess the association between early initiation of parenteral nutrition (PN) and body growth in preterm infants with very low birth weight (VLBW). DESIGN: Causal inference analysis with confounders preselected by causal diagram based on the NeoNutriNet cohort containing data of infants born between 2011 and 2014 from 13 hospitals from 5 continents. PATIENTS: Neonates with birth weight ≤1500 g. INTERVENTIONS: PN initiated within the first day of life (early PN) versus within day 2-5 (delayed PN). MAIN OUTCOME MEASURES: The primary outcome was body weight z-scores at postmenstrual age (PMA) 36 weeks or early discharge or death, whichever comes first (WT z-score END). Secondary outcomes included WT z-scores at week 1 and 4 of life (WT z-scores CA1 and CA4), corresponding growth velocities (GVs), mortality and incidence of necrotising enterocolitis (NEC), and duration and episodes of antibiotic treatment. RESULTS: In total, 2151 infants were included in this study and 2008 infants were in the primary outcome analysis. Significant associations of early PN were found with WT z-score END (adjusted mean difference, 0.14 (95% CI 0.05 to 0.23)), CA4 (ß, 0.09 (0.04 to 0.14)) and CA1 (0.04 (0.01 to 0.08)), and GV PMA 36 weeks (1.02 (0.46 to 1.58)) and CA4 (1.03 (0.56 to 1.49), all p<0.001), but not with GV CA1 (p>0.05). No significant associations with mortality, incidence of NEC or antibiotic use was found (all p>0.05). CONCLUSIONS: For VLBW infants, PN initiated within the first day of life is associated with improved in-hospital growth.

Gut development following insulin-like growth factor-1 supplementation to preterm pigs.

BACKGROUND: Reduced insulin-like growth factor-1 (IGF-1) levels may contribute to impaired organ development in preterm infants. Using preterm pigs as a model, we hypothesized that IGF-1 supplementation improves health and gut development during the first three weeks of life. METHODS: First, clinical and organ endpoints were compared between artificially-reared, cesarean-delivered preterm pigs and vaginally-delivered, sow-reared term pigs at 5, 9 and 19 days. Next, preterm pigs were treated with recombinant human IGF-1 for 19 days (2.25 mg/kg/day, systemically). RESULTS: Relative to term pigs, preterm pigs had lower body weight, fat, bone contents, relative weights of liver and spleen and a longer and thinner intestine at 19 days. Preterm birth reduced intestinal villi heights and peptidase activities, but only at 5 and 9 days. In preterm pigs, IGF-1 reduced mortality primarily occurring from gastrointestinal complications and with a tendency towards salvaging smaller pigs. IGF-1 supplementation also increased spleen and kidney weights, small intestine length and maltase to lactase activity, reflecting gut maturation. CONCLUSION: Preterm birth affects body composition and gut maturation in the first 1-2 weeks, but differences are marginal thereafter. Supplemental IGF-1 may improve gut health in pigs and infants in the first few weeks after preterm birth. IMPACT: Insulin-like growth factor 1 (IGF-1) supplementation may improve gut health and development in prematurity, but whether the effects are sustained beyond the immediate postnatal period is unclear. In preterm pigs, the prematurity effects on IGF-1 and gut health deficiencies are most pronounced during the first week of life and diminishes thereafter. In preterm pigs, IGF-1 supplementation beyond the first week of life reduced mortality. The present study provides evidence of a sustained effect of IGF-1 supplementation on the gastrointestinal tract after the immediate postnatal period.

Ultra-High Temperature Treatment of Liquid Infant Formula, Systemic Immunity, and Kidney Development in Preterm Neonates.

SCOPE: Ready-to-feed liquid infant formulas (IFs) are increasingly being used for newborn preterm infants when human milk is unavailable. However, sterilization of liquid IFs by ultra-high temperature (UHT) introduces Maillard reaction products (MRPs) that may negatively affect systemic immune and kidney development. METHODS AND RESULTS: UHT-treated IF without and with prolonged storage (SUHT) are tested against pasteurized IF (PAST) in newborn preterm pigs as a model for preterm infants. After 5 days, blood leukocytes, markers of systemic immunity and inflammation, kidney structure and function are evaluated. No consistent differences between UHT and PAST pigs are observed. However, SUHT increases plasma TNFα and IL-6 and reduces neutrophils and in vitro response to LPS. In SUHT pigs, the immature kidneys show minor upregulation of gene expressions related to inflammation (RAGE, MPO, MMP9) and oxidative stress (CAT, GLO1), together with glomerular mesangial expansion and cell injury. The increased inflammatory status in SUHT pigs appears unrelated to systemic levels of MRPs. CONCLUSION: SUHT feeding may impair systemic immunity and affect kidney development in preterm newborns. The systemic effects may be induced by local gut inflammatory effects of MRPs. Optimal processing and length of storage are critical for UHT-treated liquid IFs for preterm infants.

Bovine colostrum to supplement the first feeding of very preterm infants: The PreColos randomized controlled trial.

BACKGROUND & AIMS: Gut immaturity leads to feeding difficulties in very preterm infants (<32 weeks gestation at birth). Maternal milk (MM) is the optimal diet but often absent or insufficient. We hypothesized that bovine colostrum (BC), rich in protein and bioactive components, improves enteral feeding progression, relative to preterm formula (PF), when supplemented to MM. Aim of the study is to determine whether BC supplementation to MM during the first 14 days of life shortens the time to full enteral feeding (120 mL/kg/d, TFF120). METHODS: This was a multicenter, randomized, controlled trial at seven hospitals in South China without access to human donor milk and with slow feeding progression. Infants were randomly assigned to receive BC or PF when MM was insufficient. Volume of BC was restricted by recommended protein intake (4-4.5 g/kg/d). Primary outcome was TFF120. Feeding intolerance, growth, morbidities and blood parameters were recorded to assess safety. RESULTS: A total of 350 infants were recruited. BC supplementation had no effect on TFF120 in intention-to-treat analysis [n (BC) = 171, n (PF) = 179; adjusted hazard ratio, aHR: 0.82 (95% CI: 0.64, 1.06); P = 0.13]. Body growth and morbidities did not differ, but more cases of periventricular leukomalacia were detected in the infants fed BC (5/155 vs. 0/181, P = 0.06). Blood chemistry and hematology data were similar between the intervention groups. CONCLUSIONS: BC supplementation during the first two weeks of life did not reduce TFF120 and had only marginal effects on clinical variables. Clinical effects of BC supplementation on very preterm infants in the first weeks of life may depend on feeding regimen and remaining milk diet. TRIAL REGISTRATION: http://www. CLINICALTRIALS: gov: NCT03085277.

Bovine colostrum as a fortifier to human milk in very preterm infants - A randomized controlled trial (FortiColos).

BACKGROUND: Human milk for very preterm infants need fortification for optimal growth and development but the optimal fortification product remains to be identified. AIMS: To investigate feasibility, safety and preliminary efficacy on growth and blood biochemistry when using intact bovine colostrum (BC) as a fortifier to human milk in very preterm infants. METHODS: In an open-label, multicenter, randomized controlled pilot trial (infants 26-31 weeks' gestation), mother's own milk or donor human milk was fortified with powdered BC (n = 115) or a conventional fortifier (CF, bovine-milk-based, n = 117) until 35 weeks' postmenstrual age. Fortifiers and additional micronutrients were added to human milk according to local guidelines to achieve optimal growth (additional protein up to +1.4 g protein/100 mL human milk). Anthropometry was recorded weekly. Clinical morbidities including necrotizing enterocolitis (NEC) and late-onset sepsis (LOS) were recorded. Clinical biochemistry included plasma amino acid (AA) levels to assess protein metabolic responses to the new fortifier. RESULTS: A total of 232 infants, gestational age (GA) 28.5 ± 1.4 (weeks + days), fulfilled inclusion criteria. Birthweight, GA and delta Z scores from birth to end of intervention on weight, length or head circumference did not differ between groups, nor between the subgroups of small for gestational age infants. Likewise, incidence of NEC (BC: 3/115 vs. CF: 5/117, p = 0.72, unadjusted values), LOS (BC: 23/113 vs. CF: 14/116, p = 0.08) and other morbidities did not differ. BC infants received more protein than CF infants (+10%, p < 0.05) and showed several elevated AA levels (+10-40%, p < 0.05). CONCLUSION: Infants fortified with BC showed similar growth but received more protein and showed a moderate increase in plasma AA-levels, compared with CF. Adjustments in protein composition and micronutrients in BC-based fortifiers may be required to fully suit the needs for very preterm infants.

Uncovering the gastrointestinal passage, intestinal epithelial cellular uptake, and AGO2 loading of milk miRNAs in neonates using xenomiRs as tracers.

BACKGROUND: Human breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiating enteral nutrition. However, this requires that relevant milk miRNA amounts survive the gastrointestinal (GI) passage, are taken up by cells, and become available to the RNA interference machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and GI passage. OBJECTIVES: Our goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine model in vivo to contribute to the discussion of whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals. METHODS: We performed cross-species profiling of miRNAs via deep sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells using adenovirus-type 5-mediated miRNA gene transfer. RESULTS: Mammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula nutrition in human preterm stool and 9 d after the onset of enteral feeding in intestinal cells (ICs) of preterm piglets. Thereafter, several xenomiRs accumulated in the ICs. Moreover, a few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in argonaute RISC catalytic component 2 (AGO2)-immunocomplexes from intestinal biopsies. CONCLUSIONS: Milk-derived miRNAs survived GI passage in human and porcine neonates. Bovine-specific miRNAs accumulated in ICs of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood concentrations of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in ICs. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract.

Insulin-Like Growth Factor-1 Supplementation Promotes Brain Maturation in Preterm Pigs.

Very preterm infants show low levels of insulin-like growth factor-1 (IGF-1), which is associated with postnatal growth restriction and poor neurologic outcomes. It remains unknown whether supplemental IGF-1 may stimulate neurodevelopment in preterm neonates. Using cesarean-delivered preterm pigs as a model of preterm infants, we investigated the effects of supplemental IGF-1 on motor function and on regional and cellular brain development. Pigs were treated with 2.25 mg/kg/d recombinant human IGF-1/IGF binding protein-3 complex from birth until day 5 or 9 before the collection of brain samples for quantitative immunohistochemistry (IHC), RNA sequencing, and quantitative PCR analyses. Brain protein synthesis was measured using in vivo labeling with [2H5] phenylalanine. We showed that the IGF-1 receptor was widely distributed in the brain and largely coexisted with immature neurons. Region-specific quantification of IHC labeling showed that IGF-1 treatment promoted neuronal differentiation, increased subcortical myelination, and attenuated synaptogenesis in a region-dependent and time-dependent manner. The expression levels of genes involved in neuronal and oligodendrocyte maturation, and angiogenic and transport functions were altered, reflecting enhanced brain maturation in response to IGF-1 treatment. Cerebellar protein synthesis was increased by 19% at day 5 and 14% at day 9 after IGF-1 treatment. Treatment had no effect on Iba1+ microglia or regional brain weights and did not affect motor development or the expression of genes related to IGF-1 signaling. In conclusion, the data show that supplemental IGF-1 promotes brain maturation in newborn preterm pigs. The results provide further support for IGF-1 supplementation therapy in the early postnatal period in preterm infants.

Neonatal prophylactic antibiotics after preterm birth affect plasma proteome and immune development in pigs.

BACKGROUND: Most preterm infants receive antibiotics to prevent serious infections shortly after birth. However, prolonged antibiotic treatment predisposes to gut dysbiosis and late-onset sepsis. Using preterm pigs as model, we hypothesized that neonatal prophylactic antibiotics impair systemic immune development beyond the days of antibiotic treatment. METHODS: Preterm pigs (90% gestation) were fed formula for 9 days, treated with sterile water (CON) or enteral antibiotics from day 1 to 4. On days 5 and 9, blood was collected for haematology, in vitro LPS stimulation, and plasma proteomics. RESULTS: Antibiotic treatment altered the abundance of 21 and 47 plasma proteins on days 5 and 9, representing 6.6% and 14.8% of the total annotated proteins, respectively. Most antibiotics-induced proteome changes related to complement cascade, neutrophil degranulation, and acute phase responses. Neutrophil and lymphocyte counts were higher in antibiotics-treated pigs on day 5 but did not change from days 5-9, in contrast to increasing cell counts in CON. The antibiotics treatment suppressed TNF-alpha and IL-10 responses to in vitro LPS challenge on day 5, 7 and 9. CONCLUSION: Few days of antibiotics treatment following preterm birth alter the plasma proteome and inhibit systemic immune development, even beyond the days of treatment. IMPACT: 1. Neonatal prophylactic antibiotics alter the plasma proteome and suppress systemic immune development in preterm pigs 2. The effects of prophylactic antibiotics last beyond the days of treatment. 3. Neonatal antibiotics treatment for compromised human newborns may predispose to longer-term risks of impaired immunity and infections.

Relationship of Early Anemia with Neurodevelopment and Brain Injury in Very Low Birth Weight Preterm Infants-A Prospective Cohort Study.

Anemia is associated with neurodevelopmental delays and brain injury in infants and toddlers, but whether early anemia has a similar effect in neonatal preterm infants is largely unknown. Thus, this study aimed to determine the relationship of early anemia with neurodevelopment and brain injury in very-low-birth-weight (VLBW) preterm infants within the neonatal period. A prospective cohort study including 110 VLBW preterm infants was conducted in Southern China from 2016 to 2018. All participants were followed from birth to 1 month corrected age. Early anemia is defined as hemoglobin of ≤145 g/L within the first week after birth. The non-anemic group (control group, N = 55) was 1:1 matched with the early anemia group (N = 55) according to birth weight and gestational age. Neurodevelopment at 1 month corrected age and brain injury within 1 month corrected age were measured by neonatal behavioral neurological assessments (NBNA) and cranial ultrasound, respectively. Compared to the control group, the early anemia group had a lower score in behavioral ability in the NBNA test [11 (10-12) vs. 10 (9.5-11), p = 0.033]. Early anemia was negatively associated with the NBNA total score (ß= -0.680, 95% CI: -1.300, -0.059), especially with the behavioral ability score (ß= -0.504, 95% CI: -0.941, -0.067) after adjusting for the confounders. However, no association between early anemia and brain injury was observed. In conclusion, in VLBW preterm infants, early anemia is negatively correlated with neurodevelopment, especially with behavioral ability.

A Randomized, Controlled Study to Investigate How Bovine Colostrum Fortification of Human Milk Affects Bowel Habits in Preterm Infants (FortiColos Study).

Background: Human milk does not meet the nutritional needs to support optimal growth of very preterm infants during the first weeks of life. Nutrient fortifiers are therefore added to human milk, though these products are suspected to increase gut dysmotility. The objective was to evaluate whether fortification with bovine colostrum (BC) improves bowel habits compared to a conventional fortifier (CF) in very preterm infants. Methods: In an unblinded, randomized study, 242 preterm infants (26−31 weeks of gestation) were randomized to receive BC (BC, Biofiber Damino, Gesten, Denmark) or CF (FM85 PreNAN, Nestlé, Vevey, Switzerland) as a fortifier. Stools (Amsterdam Stool Scale), bowel gas restlessness, stomach appearance score, volume, and frequency of gastric residuals were recorded before each meal until 35 weeks post-menstrual age. Results: As intake of fortifiers increased, stools became harder in both groups (p < 0.01) though less in BC infants (p < 0.05). The incidence of bowel gas restlessness increased with laxative treatments and days of fortification in both groups (p < 0.01), but laxatives were prescribed later in BC infants (p < 0.01). With advancing age, stomach appearance scores improved, but more so in BC infants (p < 0.01). Conclusions: Although there are limitations, a minimally processed, bioactive milk product such as BC induced similar or slightly improved bowel habits in preterm infants.

Early antibiotic use and incidence of necrotising enterocolitis in very preterm infants: a protocol for a UK based observational study using routinely recorded data.

INTRODUCTION: Necrotising enterocolitis (NEC) remains a major contributor to preterm mortality and morbidity. Prolonged duration of antibiotic therapy after delivery is associated with later NEC development but recent evidence suggests that absence of antibiotic treatment after delivery may also increase NEC risk. We will explore this controversy using a large pre-existing dataset of preterm infants in the UK. METHODS AND ANALYSIS: This is a retrospective cohort study using data from UK National Neonatal Research Database (NNRD) for infants born 1 January 2012 to 31 December 2020. Eligible infants will be <32 weeks gestation, alive on day 3. Primary outcome is development of severe NEC, compared in infants receiving early antibiotics (days 1-2 after birth) and those not. Subgroup analysis on duration of early antibiotic exposure will also occur. Secondary outcomes are: late onset sepsis, total antibiotic use, predischarge mortality, retinopathy of prematurity, intraventricular haemorrhage, bronchopulmonary dysplasia, focal intestinal perforation and any abdominal surgery. To address competing risks, incidence of death before day 7, 14 and 28 will be analysed. We will perform logistic regression and propensity score matched analyses. Statistical analyses will be guided by NEC risk factors, exposures and outcome presented in a causal diagram. These covariates include but are not limited to gestational age, birth weight, small for gestational age, sex, ethnicity, delivery mode, delivery without labour, Apgar score, early feeding and probiotic use. Sensitivity analyses of alternate NEC definitions, specific antibiotics and time of initiation will occur. ETHICS AND DISSEMINATION: We will use deidentified data from NNRD, which holds permissions for the original data, from which parents can opt out and seek study-specific research ethics approval. The results will help to determine optimal use of early antibiotics for very preterm infants. IMPLICATIONS: This data will help optimise early antibiotic use in preterm infants. TRIAL REGISTRATION NUMBER: ISRCTN55101779.

Ultra-High Temperature Treatment and Storage of Infant Formula Induces Dietary Protein Modifications, Gut Dysfunction, and Inflammation in Preterm Pigs.

SCOPE: Ready-to-feed liquid infant formula is increasingly used for preterm infants when human milk is unavailable. These formulas are sterilized by ultra-high temperature treatment, but heating and storage may reduce bioactivity and increase formation of Maillard reaction products with potential negative consequences for immature newborns. METHODS AND RESULTS: Using preterm pigs as a model for sensitive newborn infants, the study tests the intestinal responses of feeding experimental liquid formula within 5 days. A pasteurized formula (PAST) with the same nutrient composition but less protein modifications serves as control to ultra-high temperature-treated formula without (UHT) and with prolonged storage (SUHT). Relative to PAST, UHT contains lower levels of lactoferrin and IgG. Additional storage (40 °C, 60 days, SUHT) reduces antimicrobial capacity and increases non-reducible protein aggregates and Maillard reaction products (up to 13-fold). Pigs fed SUHT have more diarrhea and show signs of intestinal inflammation (necrotizing enterocolitis) compared with pigs fed PAST and UHT. These clinical effects are accompanied by accumulation of Maillard reaction products, protein cross-links, and inflammatory responses in the gut. CONCLUSION: The results demonstrate that feeding UHT infant formulas, particularly after prolonged storage, adversely affects gut maturation and function in preterm pigs used as a model of preterm infants.

Clinical outcome and gut development after insulin-like growth factor-1 supplementation to preterm pigs.

Background: Elevation of circulating insulin-like growth factor-1 (IGF-1) within normal physiological levels may alleviate several morbidities in preterm infants but safety and efficacy remain unclear. We hypothesized that IGF-1 supplementation during the first 1-2 weeks after preterm birth improves clinical outcomes and gut development, using preterm pigs as a model for infants. Methods: Preterm pigs were given vehicle or recombinant human IGF-1/binding protein-3 (rhIGF-1, 2.25 mg/kg/d) by subcutaneous injections for 8 days (Experiment 1, n = 34), or by systemic infusion for 4 days (Experiment 2, n = 19), before collection of blood and organs for analyses. Results: In both experiments, rhIGF-1 treatment increased plasma IGF-1 levels 3-4 fold, reaching the values reported for term suckling piglets. In Experiment 1, rhIGF-1 treatment increased spleen and intestinal weights without affecting clinical outcomes like growth, blood biochemistry (except increased sodium and gamma-glutamyltransferase levels), hematology (e.g., red and white blood cell populations), glucose homeostasis (e.g., basal and glucose-stimulated insulin and glucose levels) or systemic immunity variables (e.g., T cell subsets, neutrophil phagocytosis, LPS stimulation, bacterial translocation to bone marrow). The rhIGF-1 treatment increased gut protein synthesis (+11%, p < 0.05) and reduced the combined incidence of all-cause mortality and severe necrotizing enterocolitis (NEC, p < 0.05), but had limited effects on intestinal morphology, cell proliferation, cell apoptosis, brush-border enzyme activities, permeability and levels of cytokines (IL-1ß, IL-6, IL-8). In Experiment 2, rhIGF-1 treated pigs had reduced blood creatine kinase, creatinine, potassium and aspartate aminotransferase levels, with no effects on organ weights (except increased spleen weight), blood chemistry values, clinical variables or NEC. Conclusion: Physiological elevation of systemic IGF-1 levels for 8 days after preterm birth increased intestinal weight and protein synthesis, spleen weight and potential overall viability of pigs, without any apparent negative effects on recorded clinical parameters. The results add further preclinical support for safety and efficacy of supplemental IGF-1 to hospitalized very preterm infants.