Human-derived Human Milk Fortifiers (H2MF), Gut Microbiota and Oxidative Stress in Premature Infants

Study Overview
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Brief Summary
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This is a randomized controlled trial of a human-derived human milk fortifier (H2MF) vs standard bovine-derived human milk fortifier (HMF) evaluating fecal microbiota and fecal and urinary biomarkers of oxidative stress in premature infants.

Detailed Description
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While breast milk provides complete nutrition for full term infants, supplementation with human milk fortifiers (HMF) is required to achieve optimal weight gain in very low birthweight (VLBW) preterm neonates. Traditionally, HMF have been derived from bovine milk. Bovine-based infant formula has been shown to cause dysbiosis of the infant gut microbiome (Azad et al 2013) and increased oxidative stress in preterm neonates (Friel et al 2011). Microbiome dysbiosis and oxidative stress have been implicated in numerous inflammatory conditions, including both acute (eg. necrotizing enterocolitis, NEC) and long-term (eg. asthma, metabolic syndrome) sequela of preterm birth (Torrazza et al 2013, Goulet et al 2015, Flora et al 2007, Perrone et al 2014). Recent studies show that new human-derived HMF (H2MF) are superior to standard bovine HMF for nourishing VLBW preterm infants and preventing NEC (Sullivan et al 2010, Cristofalo et al 2013). However, the biological basis for these clinical benefits is unknown, which limits our ability to inform and improve feeding strategies for VLBW preterm infants. This will be the first study to evaluate the impact of H2MF on gut microbiota and oxidative stress in preterm infants. Specific Objectives: To evaluate the effect of H2MF vs. HMF on gut microbiota composition in premature infants born <1250 gr between 26 and 30 weeks of gestational age. To evaluate the effect of H2MF vs. HMF on fecal and urinary biomarkers of oxidative stress in premature infants born <1250 gr between 26 and 30 weeks of gestational age.

Official Title
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The Impact of Human-derived Human Milk Fortifiers (H2MF) on Gut Microbiota Development and Oxidative Stress in Premature Infants

Conditions
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Very Low Birth Weight Baby, Premature Birth, Microbial Colonization, Oxidative Stress

Intervention / Treatment
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* Dietary Supplement: H2MF


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male or female infant with birth weight <1250 grams Gestational age between 26+0 to 30+0 weeks at birth Able to adhere to feeding protocol Parenteral nutrition must be started by day of life 2 Enteral feeding >80 ml/kg/d should be reached by day of life 14 Subject's parent(s)/legal guardian(s) has provided signed and dated informed consent and authorization to use protected health information, as required by national and local regulations. In the investigator's opinion, the subject's parent(s)/legal guardian(s) understands and is able to comply with protocol requirements, instructions, and protocol-stated restrictions, and is likely to complete the study as planned. Exclusion Criteria: Gestational age > 30+0 weeks at birth (to guarantee a minimum of 3 weeks H2MF treatment, since fortification ends at 33+0 AGA) Gestational age < 26+0 weeks at birth (to minimize baseline heterogeneity, since gestational age influences gut microbiota) Received antibiotics on the first day of specimen collection (to minimize baseline heterogeneity, since antibiotics influence gut microbiota) Note: all infants are expected to receive up to 48 hr antibiotic prophylaxis at birth according to standard NICU protocol; this criterion will exclude infants receiving extended courses of antibiotics. Received probiotics at any time (to minimize baseline heterogeneity, since probiotics influence gut microbiota) Unlikely to survive the study period Presence of clinically significant congenital heart disease or other major congenital malformation Presence prior to enrollment of intestinal perforation or stage 2 necrotizing enterocolitis (NEC) prior to tolerating fortified feeds

Ages Eligible for Study
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Maximum Age: 7 Days

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Using a randomized, controlled, un-blinded parallel design we will enroll 30 VLBW premature neonates (birth weight < 1250 grams, gestational age 26+0 to 30+0 weeks) from the NICU at the Health Sciences Centre Children's Hospital in Winnipeg, MB, Canada. Currently, H2MF is not routinely used in this population at this centre.
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| No Intervention: HMF (standard of care)<br>The HMF Control Group will receive the current standard feeding protocol of human milk fortified with bovine (cow) human milk fortifier (HMF) | |
| Experimental: H2MF<br>The H2MF Intervention Group will receive identical treatment with human-derived human milk fortifier (H2MF) replacing standard bovine HMF until the baby reaches an adjusted gestation age of 33 weeks; followed by a 5 day ween to standard HMF according to the manufacturer's recommendation. | Dietary Supplement: H2MF<br>* As described in the Experimental Arm description.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fecal microbiome composition at end of intervention | Relative abundance of operational taxonomic units (OTUs) determined by 16S rRNA Illumina sequencing | 33+0 weeks adjusted gestational age (end of intervention) | 
| Fecal microbiome diversity at end of intervention | Shannon diversity index of microbiota, determined by 16S rRNA Illumina sequencing | 33+0 weeks adjusted gestational age (end of intervention) | 
| Fecal microbiome community structure at end of intervention | Principal coordinate analysis using UniFrac distance matrices based on 16S rRNA Illumina sequencing | 33+0 weeks adjusted gestational age (end of intervention) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fecal microbiome at 1 week after intervention begins | Fecal microbiome composition, diversity and community structure from 16S rRNA Illumina Sequencing. | Study day 7 (1 week after intervention begins) | 
| Fecal microbiome at 2 weeks after intervention ends | Fecal microbiome composition, diversity and community structure from 16S rRNA Illumina Sequencing. | 35+0 weeks adjusted gestational age (2 weeks after intervention ends) | 
| Oxidative stress (urinary biomarkers) at end of intervention | F2-isoprostanes, 8-hydroxy deoxyguanine, and visfatin measured in urine. | 33+0 weeks adjusted gestational age (end of intervention) | 
| Oxidative stress (fecal calprotectin) at end of intervention | Calprotectin measured in feces | 33+0 weeks adjusted gestational age (end of intervention) | 
| Oxidative stress at 1 week after intervention begins | Fecal and urinary biomarkers of oxidative stress (fecal calprotectin, urinary F2-isoprostanes, urinary 8-hydroxy deoxyguanine, urinary visfatin). | Study day 7 (1 week after intervention begins) | 
| Oxidative stress at 2 weeks after intervention ends | Fecal and urinary biomarkers of oxidative stress (fecal calprotectin, urinary F2-isoprostanes, urinary 8-hydroxy deoxyguanine, urinary visfatin). | 35+0 weeks adjusted gestational age (2 weeks after intervention ends) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Microbiome