Comparación de diferentes concentraciones de proteínas en la leche materna enriquecida para promover el crecimiento y el desarrollo neurológico de los lactantes prematuros
Resumen
Antecedentes
Es posible que la leche materna por sí sola no proporcione las cantidades adecuadas de proteínas para satisfacer las necesidades de crecimiento de los lactantes prematuros, debido a las restricciones en la cantidad de líquido que pueden tolerar. Se ha convertido en una práctica habitual alimentar a los lactantes prematuros con leche materna enriquecida con proteínas y otros nutrientes, pero existe un debate sobre la concentración óptima de proteínas en los fortificantes disponibles en el mercado.
Objetivos
Comparar los efectos de diferentes concentraciones de proteínas en el fortificante de la leche materna con el que se alimenta a los lactantes prematuros, sobre el crecimiento y el desarrollo neurológico.
Métodos de búsqueda
Se utilizó la estrategia de búsqueda estándar del Grupo Cochrane de Neonatología (Cochrane Neonatal Group) para realizar búsquedas en CENTRAL (2019, número 8), Ovid MEDLINE y CINAHL el 15 de agosto de 2019. También se buscaron ensayos controlados aleatorizados (ECA) y cuasialeatorizados en bases de datos de ensayos clínicos y en las listas de referencias de artículos obtenidos.
Criterios de selección
Se incluyeron todos los ensayos aleatorizados, cuasialeatorizados y aleatorizados por conglomerados, publicados y no publicados, que compararon dos concentraciones diferentes de proteínas en el fortificante de la leche materna.
Se incluyeron lactantes prematuros (menos de 37 semanas de edad gestacional). Los participantes pueden haber sido alimentados exclusivamente con leche materna o haber recibido un suplemento de leche maternizada.
La concentración de proteínas se clasificó como baja (< 1 g de proteína/100 ml de leche materna extraída [LME]), moderada (≥ 1g a < 1,4 g de proteína/100 ml LME) o alta (≥ 1,4 g de proteína/100 ml LME). Se excluyeron los ensayos que compararon dos concentraciones de proteínas que se encontraban en la misma categoría.
Obtención y análisis de los datos
La obtención de los datos y los análisis se realizaron mediante los métodos estándar del Grupo Cochrane de Neonatología. Dos autores de la revisión evaluaron los ensayos de forma independiente. Los desenlaces primarios incluyeron el crecimiento, el desenlace del desarrollo neurológico y la mortalidad. Los datos se sintetizaron mediante las razones de riesgos (RR), las diferencias de riesgos y las diferencias de medias (DM), con los intervalos de confianza (IC) del 95%. Se utilizaron los criterios GRADE para evaluar la certeza de la evidencia.
Resultados principales
Se identificaron nueve ensayos que incluyeron a 861 lactantes. Hay un ensayo en espera de clasificación y nueve ensayos en curso. Los ensayos se realizaron principalmente en lactantes nacidos con < 32 semanas de edad gestacional o con < 1500 g de peso al nacer, o ambos. Todos utilizaron un fortificante derivado de la leche de vaca. Dos ensayos alimentaron a los lactantes exclusivamente con leche materna, tres ensayos administraron suplementos de alimentos con leche materna de donantes y cuatro ensayos administraron suplementos con leche maternizada para lactantes prematuros. En general los ensayos fueron pequeños, con un riesgo de sesgo bajo o incierto.
Concentración de proteínas alta versus moderada en el fortificante de la leche materna
Hubo evidencia de certeza moderada de que una alta concentración de proteínas probablemente aumentó la ganancia de peso en el hospital, en comparación con la concentración moderada del fortificante de la leche materna (DM 0,66 g/kg/día; IC del 95%: 0,51 a 0,82; ensayos = 6, participantes = 606). Hubo mucha incertidumbre con respecto al efecto de la concentración alta versus moderada de proteínas sobre el aumento de la talla (DM 0,01 cm/semana; IC del 95%: ‐0,01 a 0,03; ensayos = 5, participantes = 547; evidencia de certeza muy baja) y el aumento del perímetro cefálico (DM 0,00 cm/semana; IC del 95%: ‐0,01 a 0,02; ensayos = 5, participantes = 549; evidencia de certeza muy baja).
Solo un ensayo informó sobre la mortalidad neonatal, sin muertes en los grupos (participantes = 45).
Concentración de proteínas moderada versus baja del fortificante de la leche materna
Un fortificante con una concentración de proteínas moderada, en comparación con una concentración baja, puede aumentar la ganancia de peso (DMP 2,08 g/kg/día; IC del 95%: 0,38 a 3,77; ensayos = 2, participantes = 176; evidencia de certeza muy baja), con poco o ningún efecto sobre el aumento del perímetro cefálico (DMP 0,13 cm/semana; IC del 95%: 0,00 a 0,26; I² = 85%; ensayos = 3, participantes = 217; evidencia de certeza muy baja), pero la certeza de la evidencia es muy baja. Hubo evidencia de certeza baja de que una concentración moderada de proteínas puede aumentar la talla (DM 0,09 cm/semana; IC del 95%: 0,05 a 0,14; ensayos = 3, participantes = 217).
Solo un ensayo informó sobre la mortalidad y no encontró diferencias entre los grupos (RR 0,48; IC del 95%: 0,05 a 5,17; participantes = 112).
Ningún ensayo informó sobre el crecimiento a largo plazo ni sobre los desenlaces del desarrollo neurológico, incluida la parálisis cerebral y el retraso del desarrollo.
Conclusiones de los autores
Alimentar a los lactantes prematuros con un fortificante de la leche materna que contiene altas cantidades de proteínas (≥ 1,4 g/100 ml LME), en comparación con un fortificante que contiene una concentración moderada de proteínas (≥ 1 g a < 1,4 g/100 ml LME), da lugar a pequeños aumentos de peso durante el ingreso del lactante. También puede haber pequeños incrementos en el peso y la talla cuando los lactantes son alimentados con un fortificante que contiene una concentración de proteínas moderada, en comparación con una concentración baja (< 1 g de proteína/100 ml de LME). La certeza de esta evidencia es muy baja o moderada; por lo tanto, los resultados pueden cambiar cuando se disponga de los hallazgos de los estudios en curso. No hay evidencia suficiente para evaluar la repercusión de la concentración de proteínas sobre los efectos adversos o los desenlaces a largo plazo, como el desarrollo neurológico. Se necesitan más ensayos para determinar si los modestos aumentos de peso observados con los fortificantes con concentraciones de proteínas más altas se asocian con efectos beneficiosos o perjudiciales en el crecimiento y el desarrollo neurológico a largo plazo.
PICO
Resumen en términos sencillos
Comparación de diferentes concentraciones de proteínas en la leche materna enriquecida para promover el crecimiento y el desarrollo neurológico de los lactantes prematuros
Pregunta de la revisión
Entre los recién nacidos prematuros, ¿la cantidad de proteína utilizada para fortificar la alimentación con leche materna da lugar a alguna diferencia en los desenlaces de crecimiento y desarrollo neurológico (desarrollo del cerebro para mejorar el rendimiento o el funcionamiento [p.ej., la inteligencia, la capacidad de lectura, las habilidades sociales, la memoria, la atención o la capacidad de concentración])?
Antecedentes
La leche materna es la mejor forma de nutrición para los recién nacidos prematuros. Sin embargo, como los recién nacidos prematuros suelen tener dificultades para tolerar grandes cantidades de leche, es posible que no obtengan las cantidades recomendadas de proteínas solo de la leche materna. Se ha convertido en una práctica clínica habitual el enriquecimiento de la leche materna de los recién nacidos prematuros con nutrientes adicionales mediante un producto conocido como fortificante de la leche materna. Con el tiempo, la cantidad (concentración) de proteínas utilizada en los fortificantes de la leche materna ha aumentado, pero existe debate sobre cuál es la concentración óptima de proteínas del fortificante de la leche materna.
Características de los estudios
Esta revisión incluyó nueve ensayos con 861 recién nacidos. Seis ensayos compararon una concentración alta versus moderada de proteínas en el fortificante de la leche materna, y tres ensayos compararon una concentración moderada versus baja de proteínas. Los desenlaces principales fueron el crecimiento (p.ej., peso, talla, circunferencia de la cabeza), el desarrollo neurológico y la muerte. La información fue incompleta para todos los desenlaces, que en su mayoría tuvieron un riesgo de sesgo bajo o incierto. La búsqueda está actualizada hasta agosto de 2019.
Resultados clave
La alimentación de los recién nacidos prematuros con un fortificante de la leche materna que contiene una alta concentración de proteínas, en comparación con una concentración moderada de proteínas, dio lugar a pequeños aumentos de peso, pero no a un aumento de la talla o el crecimiento de la cabeza durante el ingreso en el hospital después del nacimiento. Hubo pequeños aumentos en la ganancia de peso y la talla en los recién nacidos alimentados con un fortificante de la leche materna que contenía concentraciones moderadas de proteínas, en comparación con las concentraciones bajas. No hubo un efecto claro de la concentración de proteínas sobre la muerte infantil durante la hospitalización inicial. Solo se dispone de datos limitados sobre otros desenlaces de salud, y esta evidencia indica que la cantidad de proteínas en el fortificante de la leche materna no afecta el riesgo de infecciones ni de problemas de alimentación o intestinales. No hubo datos disponibles en los ensayos sobre el crecimiento de los recién nacidos después del alta hospitalaria, ni sobre su desarrollo a largo plazo.
Conclusiones
Aunque hubo alguna evidencia de que el uso de leche materna enriquecida con una concentración de proteínas alta o moderada se asocia con pequeños aumentos de peso durante la estancia en el hospital, no hay datos sobre el impacto en el crecimiento después del ingreso en el hospital, ni en los desenlaces del desarrollo. Se necesitan más ensayos bien diseñados para determinar si la cantidad de proteína en la leche materna enriquecida se asocia con efectos beneficiosos o perjudiciales a largo plazo.
Certeza de la evidencia
La certeza de esta evidencia fue muy baja a moderada debido a los resultados inconsistentes que informaron algunos ensayos y al posible sesgo relacionado con la forma en la que se realizaron algunos ensayos.
Authors' conclusions
Summary of findings
| High compared to moderate protein concentration of human milk fortifier for promoting growth and neurological development in preterm infants | ||||||
| Patient or population: promoting growth and neurological development in preterm infants Setting: neonatal intensive care units in Australia, Belgium, France, Germany, Italy, Switzerland, UK, USA Intervention: addition of human milk fortifier Comparison: high (≥ 1.4 g protein/100 mL EBM) vs moderate (≥ 1 g to < 1.4 g protein/100 mL EBM) protein concentration of human milk fortifier | ||||||
| Outcomes | Anticipated absolute effects* (95% CI) | Relative effect | № of participants | Certainty of the evidence | Comments | |
|---|---|---|---|---|---|---|
| Risk with moderate protein concentration of human milk fortifier | Risk with high protein concentration of human milk fortifier | |||||
| Weight gain (g/kg/day) until the end of the intervention | Mean weight gain was 16.51 g/kg/day | MD 0.66 g/kg/day higher | — | 606 | ⊕⊕⊕⊝ | — |
| Length gain (cm/week) until the end of the intervention | Mean length gain was 1.11 cm/week | MD 0.01 cm/week higher | — | 547 | ⊕⊝⊝⊝ | — |
| Head circumference gain (cm/week) until the end of the intervention | Mean head circumference gain was 1.00 cm/week | MD 0 cm/week higher | — | 549 | ⊕⊝⊝⊝ | — |
| Cerebral palsy at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported cerebral palsy. |
| Developmental delay at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported developmental delay. |
| Mortality during intervention period | Preterm infants | Not estimable | 45 | — | — | |
| 0 per 1000 | 0 per 1000 | |||||
| *The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval; EBM: expressed breast milk; MD: mean difference; RCT: randomised controlled trial. | ||||||
| GRADE Working Group grades of evidence | ||||||
| aRisk of bias: three studies were rated low risk of bias overall, two studies provided insufficient details of methodology for assessment and one study was rated high risk of bias for blinding; therefore, the overall evidence was downgraded one level. | ||||||
| Moderate compared to low protein concentration of human milk fortifier for promoting growth and neurological development in preterm infants | ||||||
| Patient or population: preterm infants Setting: neonatal intensive care units in Canada, India, USA Intervention: addition of human milk fortifier Comparison: moderate (≥ 1 g to < 1.4 g protein/100 mL EBM) versus low (< 1 g protein/100 mL EBM) protein concentration of human milk fortifier | ||||||
| Outcomes | Anticipated absolute effects* (95% CI) | Relative effect | № of participants | Certainty of the evidence | Comments | |
|---|---|---|---|---|---|---|
| Risk with low protein concentration of human milk fortifier | Risk with moderate protein concentration of human milk fortifier | |||||
| Weight gain (g/kg/day) until the end of the intervention | Mean weight gain was 12.86 g/kg/day | MD 2.08 g/kg/day higher | — | 176 | ⊕⊝⊝⊝ | — |
| Length gain (cm/week) until the end of the intervention | Mean length gain was 0.77 cm/week | MD 0.09 cm/week higher | — | 217 | ⊕⊕⊝⊝ | — |
| Head circumference gain (cm/week) until the end of the intervention | Mean head circumference gain was 0.71 cm/week | MD 0.13 cm/week higher | — | 217 | ⊕⊝⊝⊝ | — |
| Cerebral palsy at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported cerebral palsy. |
| Developmental delay at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported developmental delay. |
| Mortality during the intervention period | Preterm infants | RR 0.48 | 112 | — | — | |
| 36 per 1000 | 17 per 1000 | |||||
| *The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval; EBM: expressed breast milk; MD: mean difference; RCT: randomised controlled trial; RR: risk ratio. | ||||||
| GRADE Working Group grades of evidence | ||||||
| aRisk of bias: one study was rated low risk of bias overall and the other study provided insufficient details of methodology for assessment; therefore, the overall evidence was downgraded one level. | ||||||
Background
Description of the condition
Human milk is the optimal nutrition for preterm infants because of its immunological properties, ease of digestion and absorption, and it has been associated with more favourable neurodevelopmental outcomes when compared with infant formula (AAP 2012). However, as preterm infants are often unable to tolerate large fluid volumes initially, feeding these infants with human milk alone (in usual volumes) may not provide adequate amounts of protein, energy and minerals to meet the high needs for growth (Arslanoglu 2019). Infants with insufficient nutrient intake are at increased risk of postnatal growth faltering and impaired neurodevelopment, which is particularly a problem for extremely low birth weight (< 1000 g) infants (Kumar 2017). To address the nutritional needs of preterm infants, it has become a common clinical practice to supplement or fortify breast milk with additional nutrients using commercially available human milk fortifier (Arslanoglu 2019; Brown 2020).
Description of the intervention
Protein is one of the main constituents of human milk fortifiers. Preterm infants require higher levels of protein intake as they grow rapidly (in line with fetal growth trajectories in the last trimester) and have higher nitrogen excretion in urine and faeces and via the skin than term infants (Hay 2010). However, protein needs cannot be considered in isolation since adequate energy must be supplied in order for the protein to be used for anabolic growth. Protein intake and protein energy ratio (grams of protein per 100 kcal) are the main determinants of growth in preterm infants (Koletzko 2014). Despite the routine use of commercial human milk fortifiers, there is evidence that many preterm infants do not meet their needs for protein intake and accrue substantial protein deficits during their hospital admission (Hay 2016; Moro 2015; Radmacher 2017). For this reason, clinical guidelines have changed to recommend an increased protein intake that is based on weight and gestation, ranging between 3.5 g/kg/day and 4.5 g/kg/day (ESPGHAN 2010; Koletzko 2014; Ziegler 2011), particularly in extremely preterm (less than 28 weeks' gestation) or extremely low birth weight infants where the recommended intake is 4 g/kg/day to 4.5 g/kg/day (ESPGHAN 2010). However, evidence about the impact of high intakes of protein on infant health outcomes is lacking. For example, one Cochrane Review of different protein concentrations in infant formula found a protein intake ≥ 3g/kg/day and < 4 g/kg/day was associated with improved weight gain in formula fed low birth weight infants, but there was insufficient evidence to evaluate the impact of formula containing ≥ 4g/kg/day protein or greater protein (Fenton 2020).
The protein in fortifiers is usually derived from bovine (cow) milk, predominantly whey protein, with some fortifiers using hydrolysed protein. The use of hydrolysed protein in formula may increase gastric emptying (Mihatsch 2002), and calcium absorption rate (Picaud 2001), without inducing the risk of feeding intolerance or necrotising enterocolitis (NEC) (Ng 2019). However, it is not known if these effects also apply to hydrolysed protein fortifier. More recently, fortifiers derived from human milk have become available, which allows for a diet that is exclusively human milk based. Bovine protein is thought to induce inflammation (Abdelhamid 2013), increase gut permeability (Taylor 2009), and may even cause death of intestinal cells (Penn 2012).
Regardless of the source and type of proteins used in fortifiers, there is considerable clinical practice variation in how and when fortifiers are administered. The usual regimen of a standard amount of fortifier added to human milk fails to account for individual and temporal variations in the protein concentration in maternal milk (Gidrewicz 2014), and the differing requirements of preterm infants of different gestational ages and weight (ESPGHAN 2010). Individualised regimens are now recommended by some groups (Arslanoglu 2019; ESPGHAN 2010), where either the amount of fortifier is titrated against blood urea nitrogen concentration or adjusted according to the level of nutrients in mother's milk. In addition, several trials have evaluated the timing of fortification, with evidence that starting fortification early rather than delaying is well tolerated but does not improve short term growth of infants (Godden 2019). These different fortification strategies (Fabrizio 2019), and timing of commencement of fortification (Thanigainathan 2019), are the focus of other Cochrane Reviews and consequently, are not addressed in this review.
How the intervention might work
Protein is an essential component of all cells in the body. Protein provides the amino acids required for adequate growth, particularly lean tissue and maturation of multiple organ systems. Growth failure is commonly reported in very low birth weight (< 1500 g) infants and this is thought to be related to inadequate protein intake (Arslanoglu 2019; Kumar 2017). Related Cochrane Reviews have shown that, when compared with unsupplemented human milk, both protein (Amissah 2018), and multi‐nutrient fortifier (Brown 2020), resulted in short term weight gain (protein: mean difference (MD) 3.82 g/kg/day, 95% confidence interval (CI) 2.94 to 4.70; multi‐nutrient fortifier: 1.76 g/kg/day, 95% CI 1.30 to 2.22).
However, increasing protein intake has potential adverse effects. These include metabolic acidosis and high serum levels of protein metabolites, such as urea and ammonia (Goldman 1969; Senterre 1983), high levels of some amino acids (Avery 1967), and increased risk of sepsis (Moltu 2013). However, results from these earlier studies may be due to the poor quality of protein (Hay 2010), and research has shown that adverse effects may be due to other nutritional components (e.g. minerals) (Rochow 2011). Amissah's Cochrane Review showed increased blood urea levels in the protein supplements groups (MD 0.95 mmol/L, 95% CI 0.81 to 1.00) (Amissah 2018). However, these remained within the normal range and may reflect adequate rather than excess protein intake. Nevertheless, there may be risks of feed intolerance and NEC associated with increased protein intake (Amissah 2018), therefore, the benefits of increased protein intake must be balanced with any potential adverse effects.
Why it is important to do this review
There remains considerable debate regarding the optimum protein concentration of fortifiers (Bertino 2017). When human milk fortifier was first introduced, the common practice was to add less than 1 g of protein per 100 mL of human milk due to safety and tolerance concerns. Since then, there has been some evidence of more favourable developmental outcomes with higher protein (with adequate energy intakes) (Coviello 2018; Stephens 2009). The concentration of protein added into human milk has gradually increased over time in many neonatal units, from around 1 g/100 mL of expressed breast milk (EBM), to greater than 1.4 g/100 mL of EBM. However, the upper limit of protein intake, where no further benefit is conferred or an adverse effect may occur (or both), has not been determined. There has been no systematic review assessing the effect of different levels of protein fortification of human milk on growth and safety in preterm infants. Although there is another review comparing human milk‐derived versus bovine milk‐derived human milk fortifier for mortality prevention and subsequent growth and neurodevelopment of preterm infants (Premkumar 2019), we will undertake subgroup analyses specifically comparing the effect of proteins derived from these different sources.
Objectives
To compare the effects of different protein concentrations in human milk fortifier, fed to preterm infants, on growth and neurodevelopment.
Methods
Criteria for considering studies for this review
Types of studies
We included all published and unpublished randomised trials, quasi‐randomised trials and cluster‐randomised trials comparing two or more different concentrations of protein in human milk fortifier.
Types of participants
Preterm infants (less than 37 weeks' gestational age) fed any human milk with added human milk fortifier. Infants may also be receiving parenteral nutrition when beginning fortification. Participants may have been exclusively fed human milk or be supplemented with formula.
Types of interventions
The intervention should have compared two or more different concentrations of protein added as a human milk fortifier to EBM. This may have included comparison of two different fortifier products or comparison of the same base fortifier product but with a protein supplement added to increase protein concentration. The fortifier could have been bovine (or other animal) milk based or human milk based and could have included other nutrients including fat and vitamins and minerals. We classified the protein concentrations of the fortifier as:
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low: less than 1 g protein/100 mL EBM;
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moderate: 1 g to less than 1.4 g protein/100 mL EBM; or
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high: 1.4 g, or greater, protein/100 mL EBM.
We excluded trials that compared two different protein concentrations that both fell within the same category (low, moderate or high). Infants in trial comparison groups should have received similar care.
Types of outcome measures
Primary outcomes
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Growth as assessed at birth (or as defined by study author) to discharge from hospital, at 12 months or 18 months (or both) or beyond corrected age expressed in absolute terms or relative to intrauterine or postnatal growth standard for the following:
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weight gain (g/kg/day or g/day);
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length gain (cm/week);
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head circumference gain (cm/week);
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measures of body composition (lean/fat mass);
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proportion of infants who were small for gestational age (SGA) (less than 10th percentile of intrauterine growth standards or post‐term growth charts as defined by study author).
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Neurodevelopmentaloutcomes
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Neurodevelopmental disability at 18 months' corrected age or greater defined as a neurological abnormality including any one of the following:
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cerebral palsy on clinical examination;
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developmental delay more than two standard deviations (SD) below population mean on a standardised test of development;
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blindness (visual acuity less than 6/60);
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deafness (any hearing impairment requiring amplification) at any time after term corrected.
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Mortality.
Secondary outcomes
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Safety measures, as reported by study authors, including:
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blood urea nitrogen (mmol/L);
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plasma amino acid levels (µmol/L);
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plasma pH levels;
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incidence of NEC (Bell's Stage II or greater);
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sepsis (as confirmed by blood culture).
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Tolerance as assessed by:
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episodes of interruption of feeds;
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days to reach full enteral feeds (enteral intake 150 mL/kg/day or greater) or as defined by study author.
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Length of hospital stay (days).
Search methods for identification of studies
We used the criteria and standard methods of Cochrane Neonatal (see the Cochrane Neonatal search strategy for specialised register; neonatal.cochrane.org/resources-review-authors). We searched for errata or retractions for included studies published in full text on PubMed (www.ncbi.nlm.nih.gov/pubmed).
Electronic searches
We conducted a comprehensive search including: Cochrane Central Register of Controlled Trials (CENTRAL 2019, Issue 8) in the Cochrane Library; Ovid MEDLINE(R) and Epub Ahead of Print, In‐Process & Other Non‐Indexed Citations, Daily and Versions(R) (1946 to 15 August 2019); and CINAHL (1981 to 15 August 2019). We included the search strategies for each database in Appendix 1. We applied no language restrictions.
We searched clinical trial registries for ongoing or recently completed trials. We searched The World Health Organization's International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en/), and the US National Library of Medicine's ClinicalTrials.gov (clinicaltrials.gov), via Cochrane CENTRAL. Additionally, we searched the ISRCTN Registry for any unique trials not found through the Cochrane CENTRAL search (www.isrctn.com/).
Searching other resources
We searched the reference lists of any articles selected for inclusion in this review to identify additional relevant articles.
Data collection and analysis
We used standard methods of Cochrane Neonatal to extract study information (Higgins 2011a).
Selection of studies
One review author (CG) screened titles and abstracts of all records and a second review author (split between JM, CC, AR) independently reviewed the titles and abstracts. One review author (CG) read the full texts and assessed each article for eligibility for inclusion based on the prespecified review criteria and another review author checked them (of JM, CC and AR). We resolved any disagreements by discussion, or when necessary with a third review author.
Data extraction and management
Two review authors (CG, JM) independently extracted data using a form based on the Cochrane Effective Practice and Organisation of Care Group data collection checklist (Cochrane EPOC 2017). Where there review authors were authors of included studies (Miller 2012; Reid 2018), another review author performed data extraction (CG).
We extracted the following characteristics from each included study:
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administrative details: study author(s); published or unpublished; year of publication; year in which study was conducted; presence of vested interest; details of other relevant papers cited;
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study: study design; type, duration and completeness of follow‐up (e.g. greater than 80%); country and location of study; informed consent; ethics approval;
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participants: sex, birth weight, gestational age, number;
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interventions: initiation, dose and duration of administration;
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outcomes as mentioned above under Types of outcome measures.
We resolved any disagreements by discussion. We collected information about ongoing studies identified by our search, when available, detailing the primary author, research question(s), methods and outcome measures, together with an estimate of the reporting date.
If the data reported were insufficient or incomplete, we contacted study investigators/authors for clarification. We used Cochrane's statistical software for data entry (Review Manager 2014). We replaced any standard error (SE) of the mean by the corresponding SD.
Assessment of risk of bias in included studies
Two review authors (CG, JM) independently assessed the risk of bias (low, high or unclear) of all included trials using the Cochrane 'Risk of bias' tool (Higgins 2011b), for the following domains.
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Sequence generation (selection bias).
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Allocation concealment (selection bias).
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Blinding of participants and personnel (performance bias).
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Blinding of outcome assessment (detection bias).
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Incomplete outcome data (attrition bias).
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Selective reporting (reporting bias).
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Any other bias.
We resolved disagreements by discussion or with a third review author. See Appendix 2 for a more detailed description of risk of bias for each domain.
Measures of treatment effect
We performed statistical analyses using Review Manager 5 (Review Manager 2014). We analysed treatment effects for categorical data using risk ratio (RR) and risk difference (RD). We calculated mean differences (MDs) between treatment groups where outcomes were measured in the same way for continuous data. Where outcomes were measured differently, we reported data as standardised mean differences (SMD). Where trials reported continuous data as median and interquartile range (IQR), we summarised data narratively in the results section. We report 95% confidence intervals (CIs) for all outcomes.
Unit of analysis issues
All included trials were individually randomised, thus the unit of analysis was the participating infant. All infants were considered only once in the analysis. If future updates of this review include cluster randomised trials we will analyse them using an estimate of the intracluster correlation coefficient (ICC) derived from the trial (if possible), or from a similar trial or from a study with a similar population as described in Section 16.3.6 of the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011a).
Dealing with missing data
Where outcome data were missing, we contacted the study investigators. We did not impute any missing data.
Assessment of heterogeneity
We examined heterogeneity among trials by inspecting the forest plots and quantifying the impact of heterogeneity using the I² statistic. The degree of heterogeneity was graded as: less than 50%, not serious, low heterogeneity; 50% to 75%: serious, substantial heterogeneity; more than 75%: very serious, considerable heterogeneity. Where we detected serious/very serious statistical heterogeneity (I² greater than 50%), we explored the possible causes (e.g. differences in study quality, participants, intervention regimens or outcome assessments).
Assessment of reporting biases
We assessed reporting bias by comparing the stated primary outcomes and secondary outcomes and reported outcomes, where study protocols were available, we compared these to full publications to determine the likelihood of reporting bias. We were unable to formally assess publication bias by generating funnel plots due to insufficient numbers of studies reporting the same outcome (fewer than 10).
Data synthesis
We performed meta‐analysis using Review Manager 5 (Review Manager 2014). We used a fixed‐effect model to combine data where it was reasonable to assume that studies were estimating the same underlying treatment effect. We used a random‐effects model to combine data where there was substantial level of heterogeneity. Where there was evidence of clinical heterogeneity, we attempted to explain this based on the different study characteristics and planned to perform subgroup analyses where there was sufficient data on subgroups.
Certainty of the evidence
We used the GRADE approach, as outlined in the GRADE Handbook to assess the certainty of evidence of the primary outcomes (Schünemann 2013).
Two review authors (CG, JM) independently assessed the certainty of the evidence for each of the primary outcomes. We considered evidence from randomised controlled trials (RCTs) as high certainty but downgraded the evidence one level for serious (or two levels for very serious) limitations based upon the following: design (risk of bias), consistency across studies, directness of the evidence, precision of estimates and presence of publication bias. We used the GRADEpro GDT Guideline Development Tool to create 'Summary of findings' tables to report the certainty of the evidence for the primary outcomes.
Subgroup analysis and investigation of heterogeneity
We planned to explore high statistical heterogeneity in the outcomes by visually inspecting the forest plots and by removing the outlying studies in the sensitivity analysis (Higgins 2011a). Where statistical heterogeneity was significant, we interpreted the results of the meta‐analyses accordingly; and downgraded the certainty of evidence in the 'Summary of findings' tables, according to the GRADE recommendations.
We planned to consider the following groups for subgroup analysis of the primary outcomes:
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less mature infants (defined as less than 1250 g birth weight or less than 28 weeks' gestation);
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source of protein (bovine, human, other animal);
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use of hydrolysed protein;
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the energy content of the two human milk fortifiers compared (isocaloric or non‐isocaloric).
Sensitivity analysis
We planned to conduct sensitivity analyses for primary outcomes to determine if the findings were affected by inclusion of only those trials considered to have used adequate methodology with a low risk of bias (selection and performance bias). We performed a sensitivity analysis for high versus moderate protein comparison including three trials (Maas 2017; Miller 2012; Reid 2018). We were unable to perform a sensitivity analysis for the moderate versus low protein comparison due to only one trial that met the standard to be included in such an analysis (Dogra 2017).
Results
Description of studies
Results of the search
See Figure 1 for the study flow diagram.
Study flow diagram.
We included nine trials (see Characteristics of included studies table), and excluded 28 full text reports (see Characteristics of excluded studies table).
There is one study awaiting classification due to lack of detail about the protein concentration of the fortifiers compared (see Characteristics of studies awaiting classification table). We identified nine ongoing trials (see Characteristics of ongoing studies table).
Included studies
The nine included trials were conducted in different countries/regions including USA, Europe, Australia and India (Dogra 2017; Kim 2015; Maas 2017; Miller 2012; Moya 2012; Porcelli 2000; Reid 2018; Rigo 2017; Sankaran 1996). In general, the included trials that were undertaken in the late 1990s compared low and moderate protein concentrations, whereas the more recent trials compared moderate to high protein concentrations, reflecting a shift in practice to higher protein fortification.
Participants
In total, 861 preterm infants participated in the included trials, most of whom were born at less than 32 weeks' gestation or with a birth weight less than 1500 g, or both, were appropriate weight for gestational age, and their mothers intended to provide breast milk during the study period. We excluded preterm infants with a congenital or chromosomal abnormality.
Intervention
Six trials compared moderate (≥ 1 g, < 1.4 g/100 mL EBM) versus high (≥ 1.4 g/100 mL EBM) protein concentrations (Kim 2015; Maas 2017; Miller 2012; Moya 2012; Reid 2018; Rigo 2017).
Three trials compared low (< 1 g/100 mL EBM) versus moderate protein concentrations ( ≥ 1 g, < 1.4 g/100 mL EBM) (Dogra 2017; Porcelli 2000; Sankaran 1996).
Human milk fortifiers used in these studies were mostly commercially available and all were bovine milk based, either in liquid or powder form. All fortifiers had additional nutrients, with varying amounts of fat, vitamins, minerals and trace elements. Three trials manipulated the concentration of other macronutrients to ensure the fortifiers were strictly isocaloric (Miller 2012; Reid 2018; Rigo 2017).
Where there were insufficient volumes of mother's own milk to meet enteral feeding requirements, three trials supplemented infants with donor human milk (Kim 2015; Moya 2012; Rigo 2017), and four trials supplemented with preterm infant formula (Dogra 2017; Maas 2017; Miller 2012; Reid 2018). Only two trials fed infants with mother's own milk exclusively (Porcelli 2000; Sankaran 1996).
Outcomes
All trials reported growth parameters until the end of the intervention, which was either the end of study period, or until hospital discharge or 40 weeks' postmenstrual age. The most common time period reported for growth was from when the infant achieved 80 mL/kg/day to 100 mL/kg/day feeding until hospital discharge or estimated delivery date. Other commonly reported outcomes included blood biochemistry (blood urea nitrogen and pH), adverse events (mortality, incidence of NEC and sepsis). No study reported neurodevelopmental outcomes of these infants.
Excluded studies
We excluded 28 studies (Arco 2002; Atchley 2019; Berseth 2004; Biasini 2012; Brumberg 2010; Coscia 2018; Costa‐Orvay 2011; Davies 1975; Ditzenberger 2013; ISRCTN27916681; Kanmaz 2013; Liang 2018; Loui 2004; Lucas 1996; McLeod 2016; Moltu 2013; Morlacchi 2016; Moro 1991; Moyer‐Mileur 1992; Polberger 1989; Quan 2020; Schanler 2018; Strommen 2017; Tan 2008; Thoene 2014; Thoene 2016; Thomaz 2014; Tracy 2015).
See Characteristics of excluded studies for details of exclusion.
Studies awaiting classification
We identified one study as potentially eligible for inclusion (Kashaki 2018). The study administered human milk fortifier without mixing with EBM, instead, as in‐between feeds four to five times a day. Both groups received human milk fortifiers, and the intervention group received an additional protein supplement of 0.6 g/kg/day to 0.8 g/kg/day mixed with EBM to achieve a higher level of intake. We are unable to determine the protein concentration consumed by the infants as grams per 100 mL fortified EBM, and are awaiting a response from the authors to clarify the protein dosage. See Characteristics of studies awaiting classification table.
Ongoing studies
We identified nine ongoing trials. See Characteristics of ongoing studies table.
Risk of bias in included studies
See 'Risk of bias' summary (Figure 2).
Risk of bias summary: review authors' judgements about each risk of bias item for each included study.
Allocation
Seven trials used computer generated randomisation schedules and were considered low risk of bias (Dogra 2017; Kim 2015; Maas 2017; Miller 2012; Reid 2018; Rigo 2017; Sankaran 1996); the remaining two trials provided insufficient information about randomisation, and were at unclear risk of bias (Moya 2012; Porcelli 2000).
Five trials were at low risk of bias for allocation concealment (Dogra 2017; Kim 2015; Maas 2017; Miller 2012; Reid 2018). Four trials were at unclear due to insufficient information (Moya 2012; Porcelli 2000; Rigo 2017; Sankaran 1996).
Blinding
Six trials were at low risk of performance and detection bias, as there were adequate methods described for blinding or personnel and outcome assessors (Dogra 2017; Maas 2017; Miller 2012; Reid 2018; Rigo 2017), or a clear rationale for use of single blinding (Porcelli 2000).
Moya 2012 reported that outcome assessors were blinded but it was unclear if carers were blinded; therefore, the trial was rated at unclear risk of bias.
Sankaran 1996 did not provide sufficient information for assessment regarding blinding and was at unclear risk of bias.
Kim 2015 reported the trial was unblinded and was, therefore, at high risk of bias.
Incomplete outcome data
Six trials were at low risk of attrition bias, as all infants were accounted for, and there was similar attrition rate between the comparison groups (Dogra 2017; Kim 2015; Maas 2017; Miller 2012; Reid 2018; Rigo 2017).
Three trials were at unclear risk of attrition bias due to insufficient information provided (Moya 2012; Porcelli 2000; Sankaran 1996). In Sankaran 1996, results from 41 infants (out of 60 infants originally enrolled) were available, whereas Porcelli 2000 reported most outcomes on a per protocol analysis. Moya 2012 reported significant attrition (27% overall, 23.6% for moderate protein group and 31.1% for high protein group) as the trial progressed, classifying the reasons as either 'fortifier related' or 'non‐fortifier related' but with no further details.
Selective reporting
There was insufficient evidence to judge whether there was reporting bias as we did not have access to trial protocols. As a result, all trials were considered unclear risk of reporting bias (Dogra 2017; Kim 2015; Maas 2017; Miller 2012; Moya 2012; Porcelli 2000; Reid 2018; Rigo 2017; Sankaran 1996).
Other potential sources of bias
Five trials were sponsored by the formula industry (Kim 2015; Moya 2012; Porcelli 2000; Rigo 2017; Sankaran 1996). For all these trials, there was no further information reported regarding the role of the sponsor in trial design and conduct, or data analysis and interpretation, or preparation and approval of the manuscripts; therefore, they were at unclear risk of bias. The remaining four trials specified that the human milk fortifier manufacturer donated products for trial but were not involved in any other way and were, therefore, at low risk of bias (Dogra 2017; Maas 2017; Miller 2012; Reid 2018).
Effects of interventions
See: Summary of findings 1 High compared to moderate protein concentration of human milk fortifier for promoting growth and neurological development in preterm infants; Summary of findings 2 Moderate compared to low protein concentration of human milk fortifier for promoting growth and neurological development in preterm infants
High (≥ 1.4 g/100 mL EBM) versus moderate (≥ 1 g, < 1.4 g/100 mL EBM) protein concentration
Primary outcomes
1. Growth outcomes
1.1 Weight gain
Six trials including 606 infants reported data for weight gain (Kim 2015; Maas 2017; Miller 2012; Moya 2012; Reid 2018; Rigo 2017). Meta‐analysis showed there was a higher rate of weight gain in infants fed high protein fortifier compared with moderate protein fortifier (MD 0.66 g/kg/day, 95% CI 0.51 to 0.82; I² = 21%; moderate certainty evidence; Analysis 1.1; Figure 3).
Forest plot of comparison: 1 High versus moderate protein concentration of human milk fortifier, outcome: 1.1 Weight gain (g/kg/day).
1.2 Length gain
Five trials including 547 infants reported length gain (Kim 2015; Miller 2012; Moya 2012; Reid 2018; Rigo 2017). Meta‐analysis showed no difference in length gain between infants fed a high protein fortifier compared with a moderate protein fortifier (MD 0.01 cm/week, 95% CI –0.01 to 0.03; I² = 53%; very low certainty evidence; Analysis 1.2; Figure 4).
Forest plot of comparison: 1 High versus moderate protein concentration of human milk fortifier, outcome: 1.2 Length gain (cm/week).
1.3 Head circumference gain
Five trials including 549 infants contributed data (Kim 2015; Miller 2012; Moya 2012; Reid 2018; Rigo 2017). Meta‐analysis showed there was no effect of protein concentration on head circumference gain (MD 0 cm/week, 95% CI –0.01 to 0.02; I² = 0%; very low certainty evidence; Analysis 1.3; Figure 5).
Forest plot of comparison: 1 High versus moderate protein concentration of human milk fortifier, outcome: 1.3 Head circumference gain (cm/week).
1.4 Measures of body composition
No trials reported body composition data.
1.5 Proportion of infants who were small for gestational age
Miller 2012 reported the proportion of infants classified SGA at the end of study period, based on weight, length and head circumference measurements (where available). For weight, 15/35 (43%) infants in the high and 17/35 (49%) in the moderate protein group were SGA (P = 0.84). For length, 21/49 (43%) infants in the high and 31/63 (49%) in the moderate protein group were SGA (P = 0.047). For head circumference, 8/19 (42%) infants in the high and 11/22 (50%) infants in the moderate protein group were SGA (P = 0.66).
2. Neurodevelopmental outcomes
No trials reported on neurodevelopmental outcomes including cerebral palsy, developmental delay, blindness and deafness.
3. Mortality
Maas 2017 reported no deaths in either the high or moderate protein groups.
Four other trials did not specifically report death, but all infants were accounted for in the trial follow‐up period (Kim 2015; Miller 2012; Reid 2018; Rigo 2017).
Secondary outcomes
1. Safety measures
1.1 Blood urea nitrogen
Three trials including 230 infants monitored blood urea nitrogen regularly during the study period (Kim 2015; Miller 2012; Reid 2018). The meta‐analysis was based on the last measurement taken before the trial concluded and showed that infants fed a high protein fortifier had a higher blood urea nitrogen concentration compared with infants fed a moderate protein fortifier (MD 1.25 mmol/L, 95% CI 1.19 to 1.32; I² = 0%; Analysis 1.6).
1.2 Plasma amino acid levels
No trials reported plasma amino acid levels.
1.3 Plasma pH levels
One study reported no difference in plasma pH levels between groups (mean 7.35, SD 0.03 for high protein group versus 7.35, SD 0.04 for moderate protein groups; P = 0.8) (Miller 2012).
1.4 Incidence of necrotising enterocolitis
Four trials including 326 infants reported on NEC (Kim 2015; Maas 2017; Miller 2012; Reid 2018). Meta‐analysis showed there was no difference in the risk of NEC between infants fed a higher versus lower protein fortifier (RR 0.78, 95% CI 0.27 to 2.27; I² = 0%; Analysis 1.8).
1.5 Sepsis
Four trials including 326 infants reported on sepsis (Kim 2015; Maas 2017; Miller 2012; Reid 2018). Meta‐analysis showed there was no difference in the risk of sepsis between infants fed a higher versus lower protein fortifier (RR 1.18, 95% CI 0.51 to 2.73; I² = 0%; Analysis 1.9).
2. Tolerance
2.1 Episodes of interruption of feeds
One trial reported a significantly higher rate of feed interruptions in the high protein group (n = 11, 35%) compared with moderate protein (n = 6, 20%) (P = 0.01) (Reid 2018).
2.2 Days to reach full enteral feed
Two trials including 162 infants reported days to reach full enteral feed (Miller 2012; Reid 2018). Meta‐analysis showed there was no difference in the number of days to reach full enteral feeds between infants fed a higher versus moderate protein fortifier (MD 0.26 days, 95% CI –1.78 to 2.31; I² = 0%; Analysis 1.11).
3. Length of hospital stay
One trial reported length of hospital stay and found no difference between groups (mean: 77 days (SD 21) in high protein group versus 73 days (SD 16) in moderate protein group; P = 0.06) (Miller 2012).
High (≥ 1.4 g/100 mL EBM) versus low (< 1 g/100 mL EBM) protein concentration
No trials compared a high versus low protein concentration fortifier.
Moderate (≥ 1 g, < 1.4 g/100 mL EBM) versus low (< 1 g/100 mL EBM) protein concentration
Primary outcomes
1. Growth outcomes
1.1 Weight gain
Two trials including 176 infants reported weight gain (Dogra 2017; Porcelli 2000). Meta‐analysis showed there was a higher rate of weight gain in infants fed a moderate protein fortifier compared with low protein fortifier (MD 2.08 g/kg/day, 95% CI 0.38 to 3.77; I² = 91%; very low certainty evidence; Analysis 2.1; Figure 6).
Forest plot of comparison: 2 Moderate versus low protein concentration of human milk fortifier, outcome: 2.1 Weight gain (g/kg/day).
1.2 Length gain
Three trials involving 217 infants reported length gain (Dogra 2017; Porcelli 2000; Sankaran 1996). Meta‐analysis showed there was a higher rate of length gain in infants fed a moderate protein fortifier compared with low protein fortifier (MD 0.09 cm/week, 95% CI 0.05 to 0.14; I² = 0%; low certainty evidence; Analysis 2.2; Figure 7).
Forest plot of comparison: 2 Moderate versus low protein concentration of human milk fortifier, outcome: 2.2 Length gain (cm/week).
1.3 Head circumference gain
Three trials including 217 infants reported head circumference gain (Dogra 2017; Porcelli 2000; Sankaran 1996). Meta‐analysis showed there was no difference in head circumference gain in infants fed a moderate protein fortifier versus low protein fortifier (MD 0.13 cm/week, 95% CI 0 to 0.26; I² = 85%; very low certainty evidence; Analysis 2.3; Figure 8).
Forest plot of comparison: 2 Moderate versus low protein concentration of human milk fortifier, outcome: 2.3 Head circumference gain (cm/week).
1.4 Measures of body composition
No trials reported data on body composition.
1.5 Proportion of infants who were small for gestational age
No trials reported data on SGA for weight, length or head circumference.
2. Neurodevelopmental outcomes
No trials reported on neurodevelopmental outcomes including cerebral palsy, developmental delay, blindness and deafness.
3. Mortality
One trial reported neonatal death, with no difference observed between groups (1/57 in moderate protein group versus 2/55 in low protein group; P = 1.00) (Dogra 2017).
Sankaran 1996 did not report death specifically, but all infants were accounted for in the trial follow‐up period.
Secondary outcomes
1. Safety measures
1.1 Blood urea nitrogen
Two trials including 165 infants reported blood urea nitrogen measurements either at the end of study (Porcelli 2000), or at hospital discharge (Dogra 2017). Meta‐analyses showed there was no difference in blood urea concentrations between infants fed a moderate protein fortifier versus low protein fortifier (MD 0.44 mmol/L, 95% CI –0.16 to 1.03; I² = 97%; Analysis 2.5).
1.2 Plasma amino acid levels
No trials reported data for plasma amino acid levels.
1.3 Plasma pH levels
No trials reported data for plasma pH levels.
1.4 Incidence of necrotising enterocolitis
One trial reported on NEC, and found no difference between groups (1/57 in moderate protein group versus 1/55 in low protein group; P = 1.00) (Dogra 2017).
1.5 Sepsis
One trial reported on sepsis, and found no differences between groups (1/57 in moderate protein group versus 2/55 in low protein group; P = 0.68) (Dogra 2017).
2. Tolerance
2.1 Episodes of interruption of feeds
No trials reported episodes of interruption of feeds.
2.2 Days to reach full enteral feed
One trial reported median days to reach full enteral feed, with no difference between groups (median 9 days (IQR 5 to 11); n = 57 in moderate protein group versus 8 days (IQR 5 to 11); n = 55 in low protein group; P = 0.37) (Dogra 2017).
3. Length of hospital stay
One trial reported length of hospital stay, with no difference between groups (median 29.2 days (IQR 22 to 45); n = 57 in moderate protein group versus 29.5 days (IQR 20 to 43); n = 55 in low protein group; P = 0.89) (Dogra 2017).
Subgroup analysis – birth weight < 1000 g
One trial reported growth outcomes among infants with a birth weight less than 1000 g fed a high versus moderate protein concentration fortifier; therefore, meta‐analyses could not be performed (Rigo 2017). Rigo 2017 showed no significant difference between the two groups in length gain (1.07 cm/week (SD 0.52); n = 19 in high protein group versus 1.27 cm/week (SD 0.52); n = 21 in low protein group; P = 0.563) or head circumference gain (1.04 cm/week (SD 0.34); n = 19 in high protein group versus 0.94 cm/week (SD 0.28); n = 21 in low protein group; P = 0.223).
Subgroup analysis – energy content
Three trials compared fortifiers that were designed to be strictly isocaloric (e.g. the same energy content in both groups) (Miller 2012; Reid 2018; Rigo 2017). However, in other trials, there were relatively small differences in the energy content of the two human milk fortifiers compared, especially when the fortifier was further diluted with EBM. Therefore, we also considered trials to be isocaloric when there was less than a 5% difference in the total energy content between comparison groups when the fortifiers were mixed with EBM (assuming 85 kcal/100 mL unless otherwise stated by the study authors). Based on this, seven trials compared fortifiers that were classified as isocaloric (Dogra 2017; Maas 2017; Miller 2012; Moya 2012; Porcelli 2000; Reid 2018; Rigo 2017), and two were non‐isocaloric (Kim 2015; Sankaran 1996).
High versus moderate protein concentration
In this comparison, five trials compared fortifiers that were isocaloric (Maas 2017; Miller 2012; Moya 2012; Reid 2018; Rigo 2017), and one compared non‐isocaloric fortifiers (Kim 2015). Meta‐analyses showed a significant subgroup effect for the outcome of length gain (Chi² = 4.14, degree of freedom (df) = 1, P = 0.04, I² = 75.8%; Analysis 3.2; Figure 9). In the subgroup of trials that compared isocaloric fortifiers, meta‐analysis showed there was a higher rate of length gain in infants fed a high versus moderate protein fortifier (MD 0.05 cm/week, 95% CI 0.01 to 0.09; I² = 30%; studies = 4; participants = 418; Analysis 3.2; Figure 9), whereas there was no difference in length gain in the one trial that compared fortifiers that were non‐isocaloric (MD 0.00 cm/week, 95% CI –0.02 to 0.02; participants = 129). There were no subgroup differences by energy content for the outcomes of weight gain (Analysis 3.1) or head circumference gain (Analysis 3.3).
Forest plot of comparison: 4 Subgroup analysis – calorie content, outcome: 4.2 Length gain (cm/week) – high versus moderate.
Moderate versus low protein concentration
In this comparison, two trials reporting on weight gain compared fortifiers that were isocaloric, thus subgroup analyses could not be performed (Analysis 3.4) (Dogra 2017; Porcelli 2000). There were no subgroup differences by energy content for the outcomes length gain (Analysis 3.5), or head circumference gain (Analysis 3.6).
Subgroup analysis – use of hydrolysed protein
We were unable to perform subgroup analysis using hydrolysed protein due to the lack of trials that used intact protein in both groups. Four trials compared fortifiers that both used hydrolysed protein, either extensively or partially hydrolysed (Maas 2017; Miller 2012; Reid 2018; Rigo 2017). Two trials specifically compared an intact and a hydrolysed protein human milk fortifier, making it difficult to separate out the effects of protein concentration from use of hydrolysed protein (Kim 2015; Moya 2012). Three studies did not report sufficient detail about the use of hydrolysed or intact protein (Dogra 2017; Porcelli 2000; Sankaran 1996).
Subgroup analysis – source of protein
We were unable to perform subgroup analysis by the source of protein as all fortifiers in the included trials were derived from bovine milk.
Sensitivity analysis
For the comparison of high versus moderate protein concentration, three trials were at low risk of bias and included in the sensitivity analysis (Maas 2017; Miller 2012; Reid 2018). All three reported weight gain, and meta‐analyses showed no difference between groups (MD 0.09 g/kg/day, 95% CI –0.52 to 0.70; I² = 0%; participants = 200; Analysis 4.1). Two of these trials reported length gain and head circumference gain, and meta‐analyses showed no difference in either outcome between comparison groups (length gain: MD 0.04 cm/week, 95% CI –0.01 to 0.08; I² = 38%; participants = 152; Analysis 4.2; head circumference gain: MD –0.01 cm/week, 95% CI –0.05 to 0.04; I² = 0%; participants = 152; Analysis 4.3) (Miller 2012; Reid 2018).
We were unable to perform sensitivity analysis for trials that compared the effect of moderate versus low protein concentration as there was only one trial considered low risk of bias for allocation concealment and randomisation (Dogra 2017).
Discussion
Summary of main results
We included nine trials involving 861 preterm infants. The available evidence suggests there is a small but statistically significant increase in weight gain among preterm infants fed a high protein fortifier when compared with a moderate protein fortifier. There was no overall effect on length gain; however, in the subgroup analysed on energy content, there were subgroup differences, with a significant increase in length gain identified only in trials that compared fortifiers with the same or similar energy content. Small increases in weight gain and length gain were also observed in infants fed a moderate protein fortifier compared with a low protein fortifier. The level of protein in the fortifier had no effect on head circumference gain.
No trials reported on measures of quality of growth (e.g. body composition), growth after hospital discharge or long term neurodevelopmental outcomes, and there were insufficient data to examine any effect on neonatal mortality.
Blood urea nitrogen concentrations were generally higher in infants fed a high or moderate protein concentration fortifier but all reported blood urea nitrogen values were within the normal range. There was insufficient evidence to examine the effect of protein concentration on other biochemical outcomes, including plasma pH and plasma amino acid concentrations. There was no evidence of an effect of protein concentration on any other clinical outcomes including sepsis, days to reach full enteral feeds, feed intolerance or NEC, or on length of stay of the initial neonatal admission.
Overall completeness and applicability of evidence
While the meta‐analyses showed statistically significant effects that suggested a higher protein fortifier is associated with increased growth rates, these findings should be interpreted and applied with caution as the effect sizes are small. Based on moderate certainty evidence, the use of a high protein fortifier rather than a moderate protein fortifier, over a 12‐week admission to the neonatal unit, would result in 55 g/kg difference in weight gain. The effects were greater in the moderate to low protein comparison, namely 175 g/kg difference in weight gain and 1.08 cm/kg difference in length gain; however, the certainty of the evidence for this comparison was low to very low. In addition, the relevance of the moderate versus low comparison to contemporary clinical practice is unclear as manufacturers of commercially available fortifiers have tended to increase their protein concentration in recent years.
For some growth outcomes, the meta‐analysis showed moderate to high levels of heterogeneity. Due to the small number of trials overall, there was limited ability to explore possible sources of heterogeneity in sensitivity and subgroup analyses. Nevertheless, for the high versus moderate protein comparison, the small improvements in weight gain seen overall were not present in meta‐analysis of the trials rated at low risk of bias, suggesting differences in trial design may be contributing to heterogeneity. Sensitivity analyses could not be undertaken for the moderate to low protein fortifier comparisons as only one trial was rated at low risk of bias. However, data reported in Porcelli 2000 were based on a per protocol analysis, which may explain the greater effect size observed and contribute to heterogeneity.
Participants in the included trials were broadly similar, mostly preterm infants born less than 32 weeks or of low birth weight, or both. We found no major differences in the subgroup analyses undertaken to explore the effect of different energy content. The exception was length gain, where a small but significant increase in length gain was observed in trials that compared isocaloric high versus moderate protein fortifiers, suggesting that additional energy may contribute to the higher length gain in infants fed a high protein fortifier. However, this should be interpreted with caution, as there was high heterogeneity in this subgroup, and very few trials included in this review compared a non‐isocaloric intervention. In addition, the clinical relevance of modest differences in length gain is unclear. Subgroup analyses based on source of protein (human versus bovine) and use of hydrolysed protein could not be undertaken.
It is possible that the high heterogeneity seen in the moderate versus low protein comparison may reflect different supplementary feeding practices, as some of the trials gave infants complementary feeds with infant formula whereas in others infants were exclusively fed mother's own milk. Trials in this comparison were also conducted almost two decades apart, therefore it is possible that other changes in clinical practice such as the introduction of probiotics into feeding regimens may also have contributed to heterogeneity. Four trials specifically excluded infants given probiotics or other interventions known to influence growth (e.g. steroids), or both (Kim 2015; Moya 2012; Porcelli 2000; Rigo 2017), whereas the extent of these practices was unclear in the remaining trials.
We found no trials that compared high protein versus low protein concentration. This is unlikely to be addressed in new trials, as current clinical practice has shifted towards higher protein fortification due to concerns about growth. This is reflected in the trials included in this review, where in general, trials comparing moderate to low protein concentrations fortifiers were the oldest, whereas more recent studies published since the 2010s compared higher to moderate concentrations. Notably, only one trial was undertaken in a low/middle income setting (Dogra 2017), thus the review findings may not be generalisable to clinical practice outside of high income settings where the burden of preterm birth lies.
The clinical significance of the small increases in growth rates during the neonatal admission is not clear, as none of the trials reported on long term growth or development of the preterm infants.
Quality of the evidence
The overall certainty of evidence of primary outcomes was moderate, low or very low (summary of findings Table 1; summary of findings Table 2). The methodological approach was mostly well described, and the very low certainty of evidence was mainly driven by considerable levels of heterogeneity and imprecision due to wide CI or CI that included zero, which includes an indicator of potential harmful effects. The certainty of evidence was further downgraded if the number of total participants included for the outcome was fewer than 400. We did not grade the overall certainty of evidence of secondary outcomes due to small numbers of trials and infrequent reporting of these outcomes.
Potential biases in the review process
Potential bias during study screening and selection and data extraction was minimised by at least two review authors working independently. However, there was an insufficient number of studies to assess reporting bias by generating funnel plots. As manufacturers of the fortifiers sponsored many of the trials in this review, it is possible that trials that did not find favourable results for one particular concentration of fortifier have not been published. We attempted to minimise the risk of publication bias by searching reference lists and clinical trial registries to identify trials that have not yet been published. However, it is possible that trials that did not report statistically significant differences have not been published and therefore not included in this review.
We classified trials into the low, moderate or high protein groups based on the protein concentration added to EBM. We did this to overcome difficulties ascertaining protein intake specifically from the fortifier, as trials often only report total protein intake based on the fortifier concentration plus an assumed protein concentration of EBM. This is problematic as there can be significant variation in the protein concentration of breast milk between mothers and by postnatal age (Gidrewicz 2014). Therefore, to minimise bias associated with misclassification of exposure to added protein, we compared the protein concentration of the fortifier as reported by the manufacturer (often reported as numbers of packs per volume of human milk).
The protein concentrations classified as low, moderate and high were designed to capture changes in fortification practices over time. When the review was first prepared in 2008, we considered fortifiers with less than 1 g of added protein per 100 mL EBM to be low and anything above this was considered high protein concentration, reflecting updated clinical guidelines recommending increased protein intake (e.g. ESPGHAN 2010). However, in more recently, several human milk fortifiers containing 1.4 g to 1.8 g added protein per 100 mL EBM have been developed and tested (e.g. see Maas 2017; Reid 2018; Rigo 2017). To permit examination of the impact of more recent changes in practice, in this version of the review we created another category by separating the high concentration group into two groups (moderate and high). Therefore, the classifications reflect a pragmatic approach and are not based on specific cut‐offs postulated to be associated with benefits or harms.
Agreements and disagreements with other studies or reviews
We found one systematic review and meta‐analysis that compared growth outcomes of preterm infants fed with human milk fortifier containing higher‐than‐standard versus standard (0.7 g to 1.1 g added protein/100 mL EBM) concentrations of protein (Liu 2015). The review included five studies (both trials and observational studies) involving 352 preterm infants and found improved weight gain (MD 1.77 g/kg/day, 95% CI 0.81 to 2.73; P = 0.0003; I² = 29%), length gain (MD 0.21 cm/week, 95% CI 0.12 to 0.29; P < 0.0001; I² = 0%) and head circumference gain (MD 0.19 cm/week, 95% CI 0.07 to 0.31; P = 0.002; I² = 56%) in infants fed a higher protein concentration fortifier. The reported effect sizes were generally higher than those reported in our review. This may reflect differences in the categorisation of fortifier concentration, with the review by Liu and colleagues grouping moderate and high protein concentration fortifiers in the same category. In addition, the "standard" fortifier category in that review included concentrations of protein that would now be considered low and thus their findings may have less relevance to current clinical practice.
The findings of our review are broadly consistent with other Cochrane Reviews examining the effects of protein for promoting growth in preterm infants, with short‐term weight gain reported in meta‐analyses of infants fed with higher versus lower protein concentration infant formula (Fenton 2020) or multi‐nutrient fortified versus unfortified human milk (Brown 2020), and among those fed with human milk supplemented with additional protein (Amissah 2018).
Study flow diagram.
Risk of bias summary: review authors' judgements about each risk of bias item for each included study.
Forest plot of comparison: 1 High versus moderate protein concentration of human milk fortifier, outcome: 1.1 Weight gain (g/kg/day).
Forest plot of comparison: 1 High versus moderate protein concentration of human milk fortifier, outcome: 1.2 Length gain (cm/week).
Forest plot of comparison: 1 High versus moderate protein concentration of human milk fortifier, outcome: 1.3 Head circumference gain (cm/week).
Forest plot of comparison: 2 Moderate versus low protein concentration of human milk fortifier, outcome: 2.1 Weight gain (g/kg/day).
Forest plot of comparison: 2 Moderate versus low protein concentration of human milk fortifier, outcome: 2.2 Length gain (cm/week).
Forest plot of comparison: 2 Moderate versus low protein concentration of human milk fortifier, outcome: 2.3 Head circumference gain (cm/week).
Forest plot of comparison: 4 Subgroup analysis – calorie content, outcome: 4.2 Length gain (cm/week) – high versus moderate.
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 1: Weight gain (g/kg/day)
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 2: Length gain (cm/week)
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 3: Head circumference gain (cm/week)
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 4: Proportion of infants who were small for gestational age
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 5: Mortality
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 6: Blood urea nitrogen (mmol/L)
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 7: Plasma pH levels
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 8: Incidence of necrotising enterocolitis
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 9: Sepsis
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 10: Episodes of interruption of feeds
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 11: Days to reach full enteral feeds
Comparison 1: High versus moderate protein concentration of human milk fortifier, Outcome 12: Length of hospital stay (days)
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 1: Weight gain (g/kg/day)
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 2: Length gain (cm/week)
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 3: Head circumference gain (cm/week)
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 4: Mortality
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 5: Blood urea nitrogen (mmol/L)
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 6: Incidence of necrotising enterocolitis
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 7: Sepsis
Comparison 2: Moderate versus low protein concentration of human milk fortifier, Outcome 8: Length of hospital stay (days)
Comparison 3: Subgroup analysis – calorie content, Outcome 1: Weight gain (g/kg/day) – high vs moderate
Comparison 3: Subgroup analysis – calorie content, Outcome 2: Length gain (cm/week) – high vs moderate
Comparison 3: Subgroup analysis – calorie content, Outcome 3: Head circumference gain (cm/week) – high vs moderate
Comparison 3: Subgroup analysis – calorie content, Outcome 4: Weight gain (g/kg/day) – moderate vs low
Comparison 3: Subgroup analysis – calorie content, Outcome 5: Length gain (cm/week) – moderate vs low
Comparison 3: Subgroup analysis – calorie content, Outcome 6: Head circumference gain (cm/week) – moderate vs low
Comparison 4: Sensitivity analysis, Outcome 1: Weight gain (g/kg/day) – high vs moderate
Comparison 4: Sensitivity analysis, Outcome 2: Length gain (cm/week) high vs moderate
Comparison 4: Sensitivity analysis, Outcome 3: Head circumference gain (cm/week) high vs moderate
| High compared to moderate protein concentration of human milk fortifier for promoting growth and neurological development in preterm infants | ||||||
| Patient or population: promoting growth and neurological development in preterm infants Setting: neonatal intensive care units in Australia, Belgium, France, Germany, Italy, Switzerland, UK, USA Intervention: addition of human milk fortifier Comparison: high (≥ 1.4 g protein/100 mL EBM) vs moderate (≥ 1 g to < 1.4 g protein/100 mL EBM) protein concentration of human milk fortifier | ||||||
| Outcomes | Anticipated absolute effects* (95% CI) | Relative effect | № of participants | Certainty of the evidence | Comments | |
|---|---|---|---|---|---|---|
| Risk with moderate protein concentration of human milk fortifier | Risk with high protein concentration of human milk fortifier | |||||
| Weight gain (g/kg/day) until the end of the intervention | Mean weight gain was 16.51 g/kg/day | MD 0.66 g/kg/day higher | — | 606 | ⊕⊕⊕⊝ | — |
| Length gain (cm/week) until the end of the intervention | Mean length gain was 1.11 cm/week | MD 0.01 cm/week higher | — | 547 | ⊕⊝⊝⊝ | — |
| Head circumference gain (cm/week) until the end of the intervention | Mean head circumference gain was 1.00 cm/week | MD 0 cm/week higher | — | 549 | ⊕⊝⊝⊝ | — |
| Cerebral palsy at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported cerebral palsy. |
| Developmental delay at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported developmental delay. |
| Mortality during intervention period | Preterm infants | Not estimable | 45 | — | — | |
| 0 per 1000 | 0 per 1000 | |||||
| *The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval; EBM: expressed breast milk; MD: mean difference; RCT: randomised controlled trial. | ||||||
| GRADE Working Group grades of evidence | ||||||
| aRisk of bias: three studies were rated low risk of bias overall, two studies provided insufficient details of methodology for assessment and one study was rated high risk of bias for blinding; therefore, the overall evidence was downgraded one level. | ||||||
| Moderate compared to low protein concentration of human milk fortifier for promoting growth and neurological development in preterm infants | ||||||
| Patient or population: preterm infants Setting: neonatal intensive care units in Canada, India, USA Intervention: addition of human milk fortifier Comparison: moderate (≥ 1 g to < 1.4 g protein/100 mL EBM) versus low (< 1 g protein/100 mL EBM) protein concentration of human milk fortifier | ||||||
| Outcomes | Anticipated absolute effects* (95% CI) | Relative effect | № of participants | Certainty of the evidence | Comments | |
|---|---|---|---|---|---|---|
| Risk with low protein concentration of human milk fortifier | Risk with moderate protein concentration of human milk fortifier | |||||
| Weight gain (g/kg/day) until the end of the intervention | Mean weight gain was 12.86 g/kg/day | MD 2.08 g/kg/day higher | — | 176 | ⊕⊝⊝⊝ | — |
| Length gain (cm/week) until the end of the intervention | Mean length gain was 0.77 cm/week | MD 0.09 cm/week higher | — | 217 | ⊕⊕⊝⊝ | — |
| Head circumference gain (cm/week) until the end of the intervention | Mean head circumference gain was 0.71 cm/week | MD 0.13 cm/week higher | — | 217 | ⊕⊝⊝⊝ | — |
| Cerebral palsy at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported cerebral palsy. |
| Developmental delay at ≥ 18 months | — | — | — | (0 studies) | — | None of the included studies reported developmental delay. |
| Mortality during the intervention period | Preterm infants | RR 0.48 | 112 | — | — | |
| 36 per 1000 | 17 per 1000 | |||||
| *The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval; EBM: expressed breast milk; MD: mean difference; RCT: randomised controlled trial; RR: risk ratio. | ||||||
| GRADE Working Group grades of evidence | ||||||
| aRisk of bias: one study was rated low risk of bias overall and the other study provided insufficient details of methodology for assessment; therefore, the overall evidence was downgraded one level. | ||||||
| Outcome or subgroup title | No. of studies | No. of participants | Statistical method | Effect size |
| 1.1 Weight gain (g/kg/day) Show forest plot | 6 | 606 | Mean Difference (IV, Fixed, 95% CI) | 0.66 [0.51, 0.82] |
| 1.2 Length gain (cm/week) Show forest plot | 5 | 547 | Mean Difference (IV, Fixed, 95% CI) | 0.01 [‐0.01, 0.03] |
| 1.3 Head circumference gain (cm/week) Show forest plot | 5 | 549 | Mean Difference (IV, Fixed, 95% CI) | 0.00 [‐0.01, 0.02] |
| 1.4 Proportion of infants who were small for gestational age Show forest plot | 1 | 92 | Risk Ratio (M‐H, Fixed, 95% CI) | 1.01 [0.57, 1.76] |
| 1.5 Mortality Show forest plot | 1 | 45 | Risk Ratio (M‐H, Fixed, 95% CI) | Not estimable |
| 1.6 Blood urea nitrogen (mmol/L) Show forest plot | 3 | 230 | Mean Difference (IV, Fixed, 95% CI) | 1.25 [1.19, 1.32] |
| 1.7 Plasma pH levels Show forest plot | 1 | 92 | Mean Difference (IV, Fixed, 95% CI) | 0.00 [‐0.01, 0.01] |
| 1.8 Incidence of necrotising enterocolitis Show forest plot | 4 | 326 | Risk Ratio (M‐H, Fixed, 95% CI) | 0.78 [0.27, 2.27] |
| 1.9 Sepsis Show forest plot | 4 | 326 | Risk Ratio (M‐H, Fixed, 95% CI) | 1.18 [0.51, 2.73] |
| 1.10 Episodes of interruption of feeds Show forest plot | 1 | 60 | Risk Ratio (M‐H, Fixed, 95% CI) | 1.72 [0.73, 4.04] |
| 1.11 Days to reach full enteral feeds Show forest plot | 2 | 162 | Mean Difference (IV, Fixed, 95% CI) | 0.26 [‐1.78, 2.31] |
| 1.12 Length of hospital stay (days) Show forest plot | 1 | 92 | Mean Difference (IV, Fixed, 95% CI) | 4.00 [‐3.71, 11.71] |
| Outcome or subgroup title | No. of studies | No. of participants | Statistical method | Effect size |
| 2.1 Weight gain (g/kg/day) Show forest plot | 2 | 176 | Mean Difference (IV, Random, 95% CI) | 2.08 [0.38, 3.77] |
| 2.2 Length gain (cm/week) Show forest plot | 3 | 217 | Mean Difference (IV, Fixed, 95% CI) | 0.09 [0.05, 0.14] |
| 2.3 Head circumference gain (cm/week) Show forest plot | 3 | 217 | Mean Difference (IV, Random, 95% CI) | 0.13 [‐0.00, 0.26] |
| 2.4 Mortality Show forest plot | 1 | 112 | Risk Ratio (M‐H, Fixed, 95% CI) | 0.48 [0.05, 5.17] |
| 2.5 Blood urea nitrogen (mmol/L) Show forest plot | 2 | 165 | Mean Difference (IV, Random, 95% CI) | 0.44 [‐0.16, 1.03] |
| 2.6 Incidence of necrotising enterocolitis Show forest plot | 1 | 112 | Risk Ratio (M‐H, Fixed, 95% CI) | 0.96 [0.06, 15.05] |
| 2.7 Sepsis Show forest plot | 1 | 112 | Risk Ratio (M‐H, Fixed, 95% CI) | 0.48 [0.05, 5.17] |
| 2.8 Length of hospital stay (days) Show forest plot | 1 | 41 | Mean Difference (IV, Fixed, 95% CI) | ‐3.00 [‐7.13, 1.13] |
| Outcome or subgroup title | No. of studies | No. of participants | Statistical method | Effect size |
| 3.1 Weight gain (g/kg/day) – high vs moderate Show forest plot | 6 | 606 | Mean Difference (IV, Fixed, 95% CI) | 0.66 [0.51, 0.82] |
| 3.1.1 Isocaloric | 5 | 477 | Mean Difference (IV, Fixed, 95% CI) | 0.32 [‐0.19, 0.83] |
| 3.1.2 Non‐isocaloric | 1 | 129 | Mean Difference (IV, Fixed, 95% CI) | 0.70 [0.54, 0.86] |
| 3.2 Length gain (cm/week) – high vs moderate Show forest plot | 5 | 547 | Mean Difference (IV, Fixed, 95% CI) | 0.01 [‐0.01, 0.03] |
| 3.2.1 Isocaloric | 4 | 418 | Mean Difference (IV, Fixed, 95% CI) | 0.05 [0.01, 0.09] |
| 3.2.2 Non‐isocaloric | 1 | 129 | Mean Difference (IV, Fixed, 95% CI) | 0.00 [‐0.02, 0.02] |
| 3.3 Head circumference gain (cm/week) – high vs moderate Show forest plot | 5 | 549 | Mean Difference (IV, Fixed, 95% CI) | 0.00 [‐0.01, 0.02] |
| 3.3.1 Isocaloric | 4 | 420 | Mean Difference (IV, Fixed, 95% CI) | 0.00 [‐0.03, 0.04] |
| 3.3.2 Non‐isocaloric | 1 | 129 | Mean Difference (IV, Fixed, 95% CI) | 0.00 [‐0.02, 0.02] |
| 3.4 Weight gain (g/kg/day) – moderate vs low Show forest plot | 2 | 176 | Mean Difference (IV, Fixed, 95% CI) | 2.52 [2.10, 2.94] |
| 3.4.1 Isocaloric | 2 | 176 | Mean Difference (IV, Fixed, 95% CI) | 2.52 [2.10, 2.94] |
| 3.4.2 Non‐isocaloric | 0 | 0 | Mean Difference (IV, Fixed, 95% CI) | Not estimable |
| 3.5 Length gain (cm/week) – moderate vs low Show forest plot | 3 | 217 | Mean Difference (IV, Fixed, 95% CI) | 0.09 [0.05, 0.14] |
| 3.5.1 Isocaloric | 2 | 176 | Mean Difference (IV, Fixed, 95% CI) | 0.09 [0.05, 0.14] |
| 3.5.2 Non‐isocaloric | 1 | 41 | Mean Difference (IV, Fixed, 95% CI) | 0.30 [‐1.89, 2.49] |
| 3.6 Head circumference gain (cm/week) – moderate vs low Show forest plot | 3 | 217 | Mean Difference (IV, Fixed, 95% CI) | 0.14 [0.10, 0.18] |
| 3.6.1 Isocaloric | 2 | 176 | Mean Difference (IV, Fixed, 95% CI) | 0.14 [0.10, 0.18] |
| 3.6.2 Non‐isocaloric | 1 | 41 | Mean Difference (IV, Fixed, 95% CI) | 0.30 [‐1.86, 2.46] |
| Outcome or subgroup title | No. of studies | No. of participants | Statistical method | Effect size |
| 4.1 Weight gain (g/kg/day) – high vs moderate Show forest plot | 3 | 200 | Mean Difference (IV, Fixed, 95% CI) | 0.09 [‐0.52, 0.70] |
| 4.2 Length gain (cm/week) high vs moderate Show forest plot | 2 | 152 | Mean Difference (IV, Fixed, 95% CI) | 0.04 [‐0.01, 0.08] |
| 4.3 Head circumference gain (cm/week) high vs moderate Show forest plot | 2 | 152 | Mean Difference (IV, Fixed, 95% CI) | ‐0.01 [‐0.05, 0.04] |
