Efficacy and Safety of Enteral Recombinant Human Insulin in Preterm Infants: A Randomized Clinical Trial.

IMPORTANCE: Feeding intolerance is a common condition among preterm infants owing to immaturity of the gastrointestinal tract. Enteral insulin appears to promote intestinal maturation. The insulin concentration in human milk declines rapidly post partum and insulin is absent in formula; therefore, recombinant human (rh) insulin for enteral administration as a supplement to human milk and formula may reduce feeding intolerance in preterm infants. OBJECTIVE: To assess the efficacy and safety of 2 different dosages of rh insulin as a supplement to both human milk and preterm formula. DESIGN, SETTING, AND PARTICIPANTS: The FIT-04 multicenter, double-blind, placebo-controlled randomized clinical trial was conducted at 46 neonatal intensive care units throughout Europe, Israel, and the US. Preterm infants with a gestational age (GA) of 26 to 32 weeks and a birth weight of 500 g or more were enrolled between October 9, 2016, and April 25, 2018. Data were analyzed in January 2020. INTERVENTIONS: Preterm infants were randomly assigned to receive low-dose rh insulin (400-µIU/mL milk), high-dose rh insulin (2000-µIU/mL milk), or placebo for 28 days. MAIN OUTCOMES AND MEASURES: The primary outcome was time to achieve full enteral feeding (FEF) defined as an enteral intake of 150 mL/kg per day or more for 3 consecutive days. RESULTS: The final intention-to-treat analysis included 303 preterm infants (low-dose group: median [IQR] GA, 29.1 [28.1-30.4] weeks; 65 boys [59%]; median [IQR] birth weight, 1200 [976-1425] g; high-dose group: median [IQR] GA, 29.0 [27.7-30.5] weeks; 52 boys [55%]; median [IQR] birth weight, 1250 [1020-1445] g; placebo group: median [IQR] GA, 28.8 [27.6-30.4] weeks; 54 boys [55%]; median [IQR] birth weight, 1208 [1021-1430] g). The data safety monitoring board advised to discontinue the study early based on interim futility analysis (including the first 225 randomized infants), as the conditional power did not reach the prespecified threshold of 35% for both rh-insulin dosages. The study continued while the data safety monitoring board analyzed and discussed the data. In the final intention-to-treat analysis, the median (IQR) time to achieve FEF was significantly reduced in 94 infants receiving low-dose rh insulin (10.0 [7.0-21.8] days; P = .03) and in 82 infants receiving high-dose rh insulin (10.0 [6.0-15.0] days; P = .001) compared with 85 infants receiving placebo (14.0 [8.0-28.0] days). Compared with placebo, the difference in median (95% CI) time to FEF was 4.0 (1.0-8.0) days for the low-dose group and 4.0 (1.0-7.0) days for the high-dose group. Weight gain rates did not differ significantly between groups. Necrotizing enterocolitis (Bell stage 2 or 3) occurred in 7 of 108 infants (6%) in the low-dose group, 4 of 88 infants (5%) in the high-dose group, and 10 of 97 infants (10%) in the placebo group. None of the infants developed serum insulin antibodies. CONCLUSIONS AND RELEVANCE: Results of this randomized clinical trial revealed that enteral administration of 2 different rh-insulin dosages was safe and compared with placebo, significantly reduced time to FEF in preterm infants with a GA of 26 to 32 weeks. These findings support the use of rh insulin as a supplement to human milk and preterm formula. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02510560.

Thermoultrasonication, ultraviolet-C irradiation, and high-pressure processing: Novel techniques to preserve insulin in donor human milk.

BACKGROUND & AIMS: Donor human milk (DHM) is recommended as the first alternative for preterm infants if their mother's own milk is not available or if the quantity is not sufficient. The most commonly used technique to eliminate microbial contaminants in DHM is holder pasteurization (HoP). However, the heating process during HoP partially destroys milk bioactive factors such as insulin. Therefore, innovative techniques have been developed as alternatives to HoP. The objective of this study was to determine the effect of HoP, high-temperature-short-time (HTST), thermoultrasonication (TUS), ultraviolet-C irradiation (UV-C), and high-pressure processing (HPP) on the insulin concentration in DHM. METHODS: Milk samples from 28 non-diabetic mothers were collected. The milk samples were aliquoted and either left untreated or treated with HoP (62.5 °C; 30 min), HTST (72 °C; 15 s), TUS (60 W; 6 min), UV-C (4863 J/L), or HPP (500 MPa; 5 min). RESULTS: The mean insulin concentration in untreated milk was 79 ± 41 pmol/L. The mean insulin retention rate was 67% for HoP, 78% for HTST, 97% for TUS, 94% for UV-C, and 106% for HPP. The mean insulin concentration in milk treated with HoP was significantly lower compared to untreated milk (p = 0.01). CONCLUSION: TUS, UV-C, and HPP preserve insulin in DHM. The insulin concentration in DHM is affected to a larger extent by HoP than by HTST. These results indicate that TUS, UV-C, and HPP may serve as alternatives to HoP.

Insulin Concentration in Human Milk in the First Ten Days Postpartum: Course and Associated Factors.

BACKGROUND OBJECTIVES: Human milk (HM) is better tolerated than formula in preterm infants. Insulin, which is naturally present in HM but not in formula, has been suggested as a key factor for feeding tolerance, as it appears to stimulate intestinal maturation. Its precise concentrations during the early postnatal period, however, remains unknown. The objective of this study was to assess the natural timecourse of the HM insulin concentration during the first ten days postpartum. The effect of preterm delivery, maternal obesity, and diurnal rhythm were also assessed. METHOD: HM was collected from 31 non-diabetic mothers (21 preterm [gestational age (GA) < 37 weeks]; 10 at-term [GA ≥ 37 weeks]) on ≥ 4 time-points per day during the first five days, and once on the tenth day postpartum. RESULTS: The HM insulin concentration declined rapidly within the first three days postpartum (day 1: 516 [312-1058] pmol/L; day 3: 157 [87-299] pmol/L), after which the concentration remained relatively stable. The insulin concentrations were higher in HM from obese mothers than from non-obese mothers (P < 0.001). Preterm delivery did not significantly affect HM insulin concentrations when adjusted for maternal pre-pregnancy body mass index category (P = 0.270). Diurnal rhythm was characterized by an insulin concentration decline throughout the night (P = 0.001), followed by an increase in the morning (P = 0.001). CONCLUSION: The HM insulin concentration declines rapidly in the first three days postpartum, follows a diurnal rhythm, and is higher in obese mothers compared to non-obese mothers. HM insulin concentrations are not affected by preterm delivery.

Rapid quantification of insulin in human milk by immunoassay.

Human milk (HM) contains numerous non-nutritive bioactive factors, amongst which the peptide hormone insulin. HM insulin has been suggested to accelerate intestinal maturation, thereby promoting feeding tolerance. Therefore, recombinant human insulin for enteral administration has been developed which might serve as supplement to HM or formula for preterm infants. However, the natural course of the HM insulin concentration directly following delivery is unknown, which hampers the development of dosage schedules in clinical trials. The aim of this study was to validate a method for insulin determination in small volumes of HM, and to assess the stability of HM insulin. The results showed that the HM insulin concentration can be measured rapidly and reliably by using an automated immunoassay. In addition, HM insulin is stable at 4 °C for at least 72 h, at room temperature for a maximum of 12 h, at -20 °C for at least 2.5 years, and during at least five freeze-thaw cycles.

Simultaneous assessment of intestinal permeability and lactase activity in human-milk-fed preterm infants by sugar absorption test: Clinical implementation and analytical method.

BACKGROUND & AIMS: Experimental (nutritional) interventions in preterm infants frequently focus on intestinal maturation, as improving tolerance to enteral nutrition is a major goal. Intestinal permeability and lactase activity serve as markers for intestinal maturation. We aimed to develop a protocol for the simultaneous assessment of both markers in human-milk-fed preterm infants by a sugar absorption test. In addition, we developed a new gas chromatography-mass spectrometry (GC-MS) method for the analysis of lactulose, lactose, and mannitol in urine and milk collected during the sugar absorption test. METHODS: The sugar absorption test was performed on days 4, 7, and 14 postpartum in 12 preterm infants (gestational age of 26-32 weeks). Human milk was collected, pooled, and divided into equal portions to provide a stable lactose intake for 24 h. Urine was collected in the last 6 h of this 24 h period, after administration of a bolus test sugar solution. Samples were analyzed by GC-MS after derivatization by oxime formation combined with acetylation. RESULTS: The GC-MS method was validated and used for the accurate measurement of lactulose, lactose, and mannitol concentrations. The urinary lactulose/mannitol ratio declined with time, suggesting a decreased intestinal permeability. The urine-to-milk-lactulose/lactose ratio increased as a result of increased lactase activity with time. CONCLUSIONS: The developed protocol for simultaneous assessment of intestinal permeability and lactase activity can be used to monitor the effect of experimental (nutritional) interventions in human-milk-fed preterm infants. Urine and milk samples obtained during the sugar absorption test can be accurately analyzed by GC-MS.

Enteral Bioactive Factor Supplementation in Preterm Infants: A Systematic Review.

Feeding preterm infants with mother's own milk is associated with a reduction in postnatal complications and an improved neurocognitive outcome. Therefore, the bioactive factor composition of human milk has been used as a tool for the development of nutritional supplements with a potential prophylactic or therapeutic effect. The aim of this systematic review was to provide an overview on bioactive factors which have been studied as supplement to enteral nutrition in randomized controlled trials, and to provide an overview of ongoing trials. MEDLINE, EMBASE, CENTRAL, and clinical trial registers were searched. Studies on the antimicrobial protein lactoferrin were excluded as these were summarized very recently in three separate systematic reviews. Studies on vitamins D, K and iron were also excluded as they are already incorporated in most international guidelines. We identified 17 different bioactive factors, which were investigated in 26 studies. Despite the encouraging potential effects of several bioactive factors, more high-quality studies with a sufficient number of preterm infants are required before a certain factor may be implemented into clinical practice. Three large trials (n > 500) that investigate the effects of either enteral insulin or vitamin A are currently ongoing and could provide more definite answers on these specific supplements.

Holder-Pasteurized Human Donor Milk: How Long Can It Be Preserved?

OBJECTIVE: When own mother's milk falls short, pasteurized human donor milk is recommended as alternative feeding for preterm infants. Donor milk has to meet the highest safety standards, but its processing and storage is expensive. The recommended storage time of pasteurized donor milk is 3 months. The objective of the present study was to determine whether the frozen storage time of pasteurized donor milk can be extended beyond 3 months without compromising its safety and quality. METHODS: For this prospective observational study breast milk samples of 34 unique women, collected between November 2014 and June 2015, were provided by the Dutch Human Milk Bank. Samples were Holder pasteurized within 3 months after expression and stored at -20°C. Analysis of both bacterial growth (by inoculation of milk on a blood and a cysteine-, lactose-, and electrolyte-deficient agar) and fat, crude protein, carbohydrate and energy content of milk (analyzed by infrared spectroscopy) was done monthly during the first 6 months and every 2 months thereafter, up to 1 year postpasteurization. RESULTS: Thirty of 306 (9.8%) follow-up samples showed bacterial growth when cultured. None of the samples showed sequential contamination with the same strain up to 8 months of frozen storage. No significant decreases in macronutrients and energy content were observed over 8 months. CONCLUSION: Pasteurized human donor milk can be stored safely for 8 months at -20°C, without compromising its macronutrient and energy content. This longer storage time will reduce disposal of expired donor milk and subsequently reduce costs.

Effect of Donor Milk on Severe Infections and Mortality in Very Low-Birth-Weight Infants: The Early Nutrition Study Randomized Clinical Trial.

IMPORTANCE: Infections and necrotizing enterocolitis, major causes of mortality and morbidity in preterm infants, are reduced in infants fed their own mother's milk when compared with formula. When own mother's milk is not available, human donor milk is considered a good alternative, albeit an expensive one. However, most infants at modern neonatal intensive care units are predominantly fed with own mother's milk. The benefits of add-on donor milk over formula are not clear. OBJECTIVE: To determine whether providing donor milk instead of formula as supplemental feeding whenever own mother's milk is insufficiently available during the first 10 days of life reduces the incidence of serious infection, necrotizing enterocolitis, and mortality. DESIGN, SETTINGS, AND PARTICIPANTS: The Early Nutrition Study was a multicenter, double-blind randomized clinical trial in very low-birth-weight infants (birth weight <1500 g) admitted to 1 of 6 neonatal intensive care units in the Netherlands from March 30, 2012, through August 17, 2014. Intent-to-treat analysis was performed. INTERVENTIONS: Infants received pasteurized donor milk or preterm formula during the first 10 days of life if own mother's milk was not (sufficiently) available. MAIN OUTCOMES AND MEASURES: The primary end point was cumulative occurrence of serious infection (sepsis or meningitis), necrotizing enterocolitis, or mortality during the first 60 days of life. RESULTS: A total of 930 infants were screened for inclusion; 557 were excluded, resulting in 373 infants (183 receiving donor milk and 190 receiving formula) who were evaluated by intent-to-treat analysis (median birth weight, 1066 g; mean gestational age, 28.4 weeks). Own mother's milk comprised 89.1% and 84.5% of total mean intake during the intervention period for the donor milk and formula groups, respectively. The incidence of the combined outcome was not different (85 [44.7%] [formula] vs 77 [42.1%] [donor milk]; mean difference, 2.6%; 95% CI, -12.7% to 7.4%). The adjusted hazard ratio was 0.87 (95% CI, 0.63-1.19; P = .37). CONCLUSIONS AND RELEVANCE: In the current study, pasteurized donor milk and preterm formula as supplemental feeding during the first 10 days of life yielded similar short-term outcomes in very low-birth-weight infants regarding safety and efficacy when own mother's milk availability was insufficient. Future studies investigating longer duration of use of human donor milk on short-term and long-term outcomes are necessary. TRIAL REGISTRATION: trialregister.nl Identifier: NTR3225.