Meeting Protein and Energy Requirements of Preterm Infants Receiving Human Milk

Abstract

Mother's own milk is universally recognized as the optimal source of nutrition for preterm infants, although most authorities agree a multi-nutrient fortifier must be added in order to support nutrient accretion at a rate comparable to in utero. Nevertheless, many preterm infants face a gap between achieved growth and what could have been achieved in utero. In this narrative review, we provide an overview on the macronutrient content in mother's own milk and donor milk and how this can be enhanced by the various available multi-nutrient fortifiers. We describe their general compositions and formulation, as well as several of their theoretical and practical advantages and drawbacks. In addition, differences between standardized fortification, or a more individualized approach like adjusted and targeted fortification are discussed. The optimal strategy however remains to be elucidated, and more experimental well-powered studies are therefore urgently needed. Until then, financial considerations and practical capabilities are likely to be the main drivers of local fortification strategies.

To facilitate nutrient accretion and growth in any individual, sufficient nutrition is of course required. Basically, this encompasses adequate amounts of protein of good quality, energy to finance the cost of protein synthesis, essential fatty acids, and all micronutrients including trace elements. Other requirements besides these nutrients are for example oxygen to facilitate energy generation, and anabolic hormones. The latter are, however, often suppressed in infants who are critically ill or inflamed, so that an anabolic state may be impossible to achieve.

Despite this basic knowledge, for many infants there is still a major gap between achieved growth in preterm infants admitted to a neonatal intensive care unit (NICU) and what would have been achieved on average if the fetus remained in utero. Although the incidence and severity of so-called extrauterine failure has decreased over the last decades, many infants at discharge have lost 1.2 standard deviations on average when compared to their birth standard deviation, as was shown in a recent report from 11 European countries [1]. In fact, 14% lost more than 2 standard deviations when compared to their birth z-score. On the other hand, there are also some reports typically from single centers that show this fall in the growth percentile can be prevented in the majority of preterm infants when paying meticulous attention to nutrition and growth [2].

In this narrative review, we will consider the various ways that the macronutrient intake in human milk-fed preterm infants can be improved. The discussion of adding specific bioactive factors (like for example human milk oligosaccharides, lactoferrin, vitamins, probiotics, enzymes, and hormones, etc.) is outside the scope of this review and can be found elsewhere [3]. All such factors may potentially interfere with nutrient availability, uptake, and utilization and therefore impact feeding tolerance and growth, either directly or indirectly. While there is a widely held belief that mother's own milk (MOM) from women delivering preterm contains more protein than from women that delivered at or close to term, there is actually only a clinically significant difference in the first few days after birth, as for example summarized in a systematic review on human milk nutrient content from a few years ago [4]. Furthermore, the protein content decreases rapidly after the first few weeks after which it stabilizes at around 1 gram per deciliter, both in human milk destined for either preterm or term infants. This finding was recently confirmed in the so-called “premature milk study,” in which over 1,900 milk samples from 225 women were analyzed [5]. Whereas the interquartile range on milk protein content ranged from 1.0 to 1.5 g/dL at around postpartum day 10, these amounts decreased to 0.8-1.2 g/dL 4 weeks after birth. Outside the interquartile ranges, there is a much wider variation in nutrient content, which makes assumptions on nutritional intake in daily practice very difficult, if milk is not regularly measured for macronutrient content in a standardized way. Partly because of this wide variation, multi-nutrient fortifiers have tended to err on what might be considered by some to be the safe or lower side, in order not to provide too much protein or other nutrients. On average, typical powdered multi-nutrient fortifiers add about 1.0-1.2 g of protein to each 100 mL milk. If we convert what this protein content translates into daily milk consumption, one can infer that at around postnatal day 10, a preterm infant receives on average around 3.5-4.3 g protein per kg/day, an amount that would meet most recommendations which currently advise around 3.5-4.5 g/kg/day. However, after 4 weeks of age, when protein content in expressed milk is lower, the interquartile range of protein intake has decreased to about 3.2-3.8 g/kg/day after standard fortification. This might not be enough for many infants admitted at our NICUs if nutrient accretion similar to the intrauterine rate is the objective.

Protein Requirements

There are various different ways that protein requirements for preterm infants could be defined. First of all, one can take a factorial or theoretical approach. This simply involves determination of the amount of protein deposited in newly formed tissue at a growth rate that is similar to intrauterine. In addition, the amount of oxidized amino acids together with some inefficiency factors need to be added; also known as the obligatory nitrogen losses [6]. All-in-all, approximately 4 g/kg/day of enteral protein is assumed to be required to achieve a desired growth rate of 15-20 g/kg/day. However, as previously mentioned, this also depends on the availability of all other macro- and micronutrients as well as an optimal clinical condition.

Defining protein requirements can also be based on experimental evidence by providing more or less protein or multi-nutrient fortifier to human milk and assessing subsequent growth and other clinical outcomes. While the availability of the many very recent Cochrane reviews on human milk fortification for preterm infants may appear promising at deriving a scientifically based answer [712], the results are a little underwhelming. In the review by Brown et al. [7] on multi-nutrient fortification on human milk in general, for example, results on almost 1,500 preterm infants included in 18 trials are summarized. The authors concluded there were some modest increases in growth rates if human milk was fortified, and the lack of adverse effects of fortification were seen to be reassuring. On the other hand, no long-term benefits were demonstrated, although not unsurprisingly as it was only rarely reported and thus significantly underpowered. Besides that, there were several other limitations, in that for example the range of added protein was most often low (0.4-1.0 g/100 mL milk) and relatively old studies were included often with suboptimal methodological design. Besides, many studies also included moderately preterm infants (i.e., those above 32 weeks' gestation), were performed in low- and middle-income countries, or formula powder was used as fortifier rather than a specific human milk multi-nutrient fortifier.

Similar issues together with a lack of high-quality studies were encountered in the other cited Cochrane reviews, which make drawing firm and meaningful conclusions challenging. This means there are still several uncertainties around the use of multi-nutrient fortification of MOM or donor milk [13]. However, several cohort studies show that poor growth is associated with poor neurode- velopmental outcome [14, 15], and that these relationships remain valid after statistical adjustment from confounders like sepsis or necrotizing enterocolitis (NEC) or many other background variables. Other cohort studies, however, were less convincing at demonstrating such relationships [16]. Unfortunately, no large, high-quality, experimental randomized controlled trials (RCTs) in human milk-fed preterm infants have been performed. Thus, convincing evidence that more nutrients and better growth result in better long-term outcomes is lacking. However, there is some evidence from a highly cited RCT on preterm infants who were completely or partially formula fed to complement insufficient MOM [17]. In this trial conducted in the 1980s, over 400 preterm infants were randomized to receive in the first 4 postnatal weeks, either human milk supplemented with regular term formula if human milk was insufficient or unavailable, or a newly designed preterm formula with more nutrients in case of insufficient MOM. Following several years of follow-up, the study consistently showed that those in the intervention group not only grew better, but had persistently better neurological outcomes, up to the age of 16 years [18].

Multi-Nutrient Fortifiers

Since it is very clear that unfortified human milk results in poor postnatal growth, it is advised by for example the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) [19], the European Milk Bank Association (EMBA) [20], and the American Academy of Pediatrics (AAP) [21] that for all preterm infants a multi-nutrient fortifier is added to human milk. Various types of multi-nutrient fortifiers are theoretically available to choose from, as also depicted in Figure 1. The main composition of several available multi-nutrient fortifiers is outlined in Table 1. Comparisons of powdered with liquid fortifiers are slightly complicated due to the volume of human milk that is replaced by the fortifier. As such, in Table 2, nutrient content after mixing the fortifier with preterm milk (with an average nutrient content) is calculated for easier comparisons. Practically, however, local availability of the different types or brands of fortifiers may be limited due to national legislation, registration, and allowance.



Multi-nutrient fortifiers typically contain currently at least 1 g of protein that is added per 100 mL of milk. The remainder is then made up of carbohydrates and sometimes lipids (vide infra) together with micronutrients, including electrolytes, vitamins, and several trace elements. The minerals phosphate and calcium, however, represent quantitatively the major contribution of the micronutrients and are essential in order to prevent metabolic bone deficiency of prematurity. In most of these fortifiers, the protein is of bovine milk origin. However, there is also a fortifier available that is produced from condensed donor human milk. Evaluation of the clinical advantage of a so-called human only diet requires large high-quality trials. Currently, only one RCT which studied this particular approach has been conducted so far [22], by comparing a standard multi-nutrient fortifier from bovine origin with a condensed human milk-based fortifier, and where both groups received donor milk in case of insufficient MOM availability. In this trial (n = 125), there were no differences in feeding intolerance as the primary outcome, nor were there differences in NEC stage ≥2, sepsis, or death, although there was less severe retinopathy of prematurity in the intervention group as a secondary outcome.



However, there are also significant challenges associated with the use of a condensed human milk-based fortifier. First of all, depending on the strength of applied fortification, 20-50% of MOM volume is displaced by current liquid hu-man milk-based fortifier formulations. This leads to a significant reduction in the intake of beneficial bioactive factors present in fresh MOM, but which are much less abundant in the fortifier which is vat-pasteurized. Furthermore, the costs of this type of fortifier are high, and there are a range of organizational, societal, and logistical issues that might preclude widespread roll out of this type of product [23]. Therefore, large-scale trials independent of commercial funding and influence will be needed before this approach should be recommended. One such trial is the Swedish N-forte RCT (ClinicalTrials.gov: NCT03797157) where 222 preterm infants are included, and the primary outcome is expected to be complete early 2022. However, in order to prove a reduction in NEC, sample sizes in trials need to be much higher to gain sufficient power; for example, at least 862 infants are required to demonstrate a reduction in NEC rates from 10 to 5%.

Multi-nutrient fortifiers from bovine origin come in two forms; either powdered or as a liquid additive to MOM or donor milk. Due to the fear of contamination of powdered products with Cronobacter sakazakii, liquid fortifiers are available, and especially in the United States widely used. Liquid fortifiers have the advantage of complete sterility, either by heat treatment or acidification, which forms another distinction between these types (Fig. 1). Another benefit of liquid fortifiers is easier mixing. Yet, the main downside of liquid fortifiers is their volume replacement of MOM, typically a reduction of about 16.7% (% v/v = 20/120; Table 1). Furthermore, acidified liquid fortifiers have been associated with metabolic acidosis and poorer growth [24].

Powdered multi-nutrient fortifiers may be subdivided in the traditional or newer generation fortifiers. In more recent fortifiers, the amount of carbohydrates is reduced and replaced by lipids, which even form the largest caloric contribution (Fig. 2). These lipids are mainly in the form of medium-chain triglycerides, although essential fatty acids as well as the long-chain poly-unsaturated fatty acids (LC-PUFAs) docosahexaenoic acid, and arachidonic acid may be added in small amounts. This may help prevent a postnatal fall in LC-PUFA concentrations, although more research is needed to demonstrate if this is truly beneficial.



In order to increase the amount of bioactive factors in human milk, there is currently a trial being conducted, which adds powdered bovine colostrum to human milk as a new type of multi-nutrient fortifier (ClinicalTrials.gov: NCT03537365). Whether this approach can help reducing neonatal morbidities in preterm neonates, is to be awaited.

Fortification Strategies

As stated above, despite the various Cochrane reviews that have appeared in the last 2 years on human milk fortification [7-12], the evidence base on how to op-timally fortify human milk remains relatively limited. On the other hand, there are also no consistent indications that adding fortifiers impairs gastrointestinal tolerance or other safety measures [25]. Once enteral feedings are tolerated after a few days of life, many clinicians choose to start to fortify MOM or donor milk, although the optimal timing (i.e., early or late introduction) in terms of growth or other clinical endpoints is understudied and thus unknown [8]. In addition, some clinicians and manufacturers prudently advocate to start with half-dose fortification as a precautionary measure, although scientifically, there is no reason to do so. Early and full-strength fortification not only increases macronutrient intake, but perhaps even more importantly, it may also aid to partially prevent or overcome early hypophosphatemia, which is frequently encountered in growth-restricted very preterm infants during the first week of life [26]. Similarly, on the other end, it is unknown when to stop multi-nutrient fortification: around discharge, several months after, or around a certain body weight. Simultaneously, practical issues become more important once the infant is partly or fully breast-fed, rather than fed with expressed milk.

During multi-nutrient fortification, various approaches may be employed as is also graphically displayed in Figure 3. Classically, multi-nutrient fortification is protocolized in a standardized fashion according to manufacturer's instruction, although sometimes it may be topped up in case of suboptimal growth rates. Alternatively, fortification may be more individualized by adjusting according to measured serum urea concentrations or milk may be fortified to meet targeted macronutrient content goals after analyzing the milk. These approaches are also acknowledged by EMBA for example [20], but the optimal strategy remains to be elucidated [9]. Until then, financial considerations and practical capabilities are likely to be the main drivers of local fortification strategies.

Regardless of the employed fortification strategy as outlined in Figure 3, the first step always encompasses addition of a powdered or liquid multi-nutrient Human Milk fortifier in amounts specified by the manufacturer (also depicted in Tables 1 and 2). In case donor milk is prescribed, some NICUs choose to add a standard ex- tramodular protein supplement with or without extra modular lipids. This may replete the lower protein content of expressed human milk, amounting to around 1.0 g/dL or even less, when donated several months after birth to a milk bank [4, 27]. Similarly, due to the multiple container changes before milk arrives from the donor to the infant, fat content decreases slightly due to adherence to each of the several bottle walls. In addition, during classic Holder pasteurization of milk, for example bile-salt stimulated lipase and lipoprotein lipase are degraded almost entirely, which could hamper lipid digestion and uptake [27]. As such, standard modular lipid addition in the form of long-chain or medium-chain triglycerides may be warranted when relatively large volumes of donor milk are consumed.
In case of suboptimal growth despite standardized fortification, several op-tions are available. One approach is to do nothing and accept poor growth, or one may increase total daily feeding volumes if tolerated gastrointestinally and by the current pulmonary and cardiac condition [28]. Modular protein with or without lipids may also be used as a top-on to promote growth as a more aggressive form of fortification. However, maintaining an appropriate protein to energy ratio is important to prevent oxidation of the majority of added proteins for energy production. We therefore recommended to monitor for high serum urea concentrations occasionally as a reflection of protein oxidation rather than anabolism, although safe and effective upper limits are unknown [29].



This latter approach partly resembles the so-called individualized adjustable fortification strategy, whereby default serum urea concentrations are measured, and fortification levels are either increased or decreased based on a relatively narrow bandwidth of serum urea concentrations. EMBA currently advises to maintain serum urea concentrations between 3.6 and 5.7 mmol/L (10-16 mg urea-N/dL, or 21-34 mg urea/dL) [20]. However, the evidence base for this tight range is very limited, and renal function or fluid status may make interpretations even more difficult. In addition, the recommended twice weekly urea measurements requiring blood are invasive and must also be considered.

Finally, a fully individualized approach when fortifying human milk, is the targeted approach [20]. This requires the analysis of the macronutrient compo-sition of pooled expressed MOM or donor milk several times per week ideally, and the addition of modular protein, carbohydrates, and lipids on top of a standard multi-nutrient fortifier, to reach predefined levels. Obviously, this requires an appropriately calibrated milk analysis device and a dedicated nutritional team. On the other hand, by doing so, growth rates may be promoted, and there are some data to show they also result in increased fat-free mass, as shown in a recent RCT [30].

Conclusion

MOM is universally recognized as the optimal source of nutrition for preterm infants, although most authorities agree it must be fortified to support nutrient accretion rates comparable to in utero. Despite this, the optimal strategy of multi-nutrient fortification remains unclear due to a limited evidence base from high-quality controlled trials. More experimental well-powered studies are therefore urgently needed.
Epilogue

A potential downside of fortifying MOM that is often ignored by doctors is the psychological effect of all supplementation, as some mothers may feel that their milk may not be good enough for their child. This may hamper the motivation to continue expressing or giving breast milk after discharge, especially in mothers with additional mental health issues such as feelings of failure or guilt associated with premature birth [31]. It may also impact on health professional beliefs and attitudes towards breast milk: if we need to add so many nutrients, why not switch to formula feeding? It is therefore important to continually emphasize the advantageous components and effects of MOM itself, which are not lost during fortification and are of major benefit to the preterm infant.

Conflict of Interest Statement

C.H.P.v.d.A. reports participating in scientific advisory boards and giving lectures in educational symposia for Nutricia Early Life Nutrition, Baxter, and Nestlé Nutritional Institute.

N.D.E. reports research grants paid to his institution from Prolacta Bioscience and Danone Early Life Nutrition; and reports lecture honoraria from Nestlé Nutrition Institute.

M.J.V. is board member of the Dutch neonatal parent organization (Care4Neo) and of the National Breastfeeding Council; and participates in the scientific advisory board of Neobiomics; and reports grants paid to her institution from Nutricia - Danone.

J.B.v.G. is member of the National Health Council and founder and director of the National Human Donor Milk Bank in the Netherlands.

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