Hydrolyzed Protein Formula for Allergy Prevention in Preterm Infants: Follow-Up Analysis of a Randomized, Triple-Blind, Placebo-Controlled Study

Background: Allergic diseases are a major public health burden worldwide. Evidence suggests that early nutrition might play a key role in the future development of allergies and the use of hydrolyzed protein formulas have been proposed to prevent allergic disease, mainly in term infants with risk factors. Aim: To evaluate the preventive effect of a hydrolyzed protein formula vs. an intact protein formula on allergy development in preterm infants with or without risk factors. Methods: We performed a 3-year follow-up study of a previous triple-blind, placebo-controlled randomized trial. Evidence of atopic dermatitis, asthma and IgE-mediated food allergies were evaluated according to a validated parental questionnaire (Comprehensive Early Childhood Allergy Questionnaire). Food sensitization was also investigated by skin prick test at 3 years of chronological age. Results: Of the 30 subjects in the intact protein formula group and 30 in the extensively hydrolyzed formula group, respectively 18 and 16 completed the 3-year follow-up and entered the final analysis. No group differences in the incidence of atopic dermatitis, asthma, IgE-mediated food allergies, and food sensitization were found. Conclusion: Despite the small number of cases, extensively hydrolyzed protein formula seems to be ineffective in allergic diseases prevention in preterm neonates. Further adequately powered, randomized controlled trials evaluating hydrolyzed protein formula administration to prevent allergic diseases in preterm neonates are needed.


INTRODUCTION
Allergic rhinitis (AR), asthma, eczema, and food allergy are some of the most common pediatric chronic conditions worldwide and have a major impact on children health and quality of life (1).
Allergic diseases are genetically determined but also influenced by several factors such as environmental pollution, smoke, aeroallergens, and early feeding pattern (2). To date, a tailored approach seems to be the best strategy to hamper the so-called "atopic march" (3). In this perspective, standard operative procedures to prevent allergy have become a priority in managing public health and infant feeding is considered the most important modifiable factor that can be targeted (4).
World Health Organization (WHO) states that breast milk is the best source of nutrients for both term and preterm infants, and there is some evidence of its role in decreasing the risk of allergy development (5,6). Unfortunately human milk is not always available and the challenge for many pediatric societies remains to draw up standardized and definitive guidelines to recommend the most effective infant formula in allergy prevention (7).
More recently, hydrolyzed formulas (HF) have been proposed for prevention of allergy and many studies suggested the use of these formulas in formula-fed infants with a family history of allergic diseases (8).
The main differences between each HF are the degree and method of hydrolysis, with consequent different immunological, clinical, and nutritional effects: extensively hydrolyzed formulas (eHF) contain mostly peptides ≤3 kD, while partially hydrolyzed formulas (pHF) ≤5 kD (9).
Despite preterm infants could be at higher risk for food allergy because of their increased intestinal permeability and their possible higher food antigen uptake, they do not show higher incidence of allergic diseases when compared to term infants (10,11).
At the moment, human milk represents the best source of nutrients for preterm infants for its bioactive effect (12). On the contrary, there is limited evidence regarding nutritional preventive action against the future development of allergies in this vulnerable population (13). This paper describes the followup results of a previous published triple-blind, controlled, clinical trial, in preterm neonates fed with either intact protein formula or extensively hydrolyzed formula (14, 15).

METHODS
Study design, inclusion and exclusion criteria, randomization and study group allocation, and feeding protocol are thoroughly described in the previous articles (14, 15). To all participants, complementary feeding was recommended after the age of 4 months, without restriction of possible allergenic foods and with intact protein milk formulas in case of insufficient breast milk. To compare the allergypreventive effect of eHF vs. IPF, all infants were followed-up 6-monthly until 3 years of chronological age, for evidence of atopic dermatitis, asthma, and IgE-mediated food allergies according to a validated parental questionnaire (Comprehensive Early Childhood Allergy Questionnaire) (16). Food sensitization, based on positive skin prick tests at 3 years of chronological age, was also investigated.

Statistical Analysis
Participant characteristics at enrolment were compared by Student t-test (gestational age and birth weight) or chi-square test (gender, birth type, cesarean section). Outcomes such as evidence of atopic dermatitis, asthma, and IgE-mediated food allergies were analyzed by chi-square test. All participants who met study entrance criteria and completed the 3 years follow-up period were evaluated. A subset analysis was carried out to assess participants at high-risk for allergy. High-risk infants were defined as having at least one parent or a single first-degree relative with a history of allergic disease. All tests were conducted at α = 0.05. All analyses were conducted using IBM R SPSS R Statistics 23.

RESULTS
A total of 34/60 (56.6%) participants completed the 3-year followup study (IPF: 18; eHF: 16) and were included in the primary analysis. Dropouts occurred in 26 children due to protocol violation (3 patients) and voluntary withdrawal by parents during the follow-up period (23 patient) (Figure 1).
Infant characteristics at 3 years of chronological age were similar between groups ( Table 1). No difference in incidence of atopic dermatitis, asthma, IgEmediated food allergies and food sensitization (positive skin prick test) were detected (primary or subset analysis) between groups ( Table 2).

DISCUSSION
The results of our study indicated that eHFs did not provide any preventive effects of allergy in preterm infants. Despite the low number of patients and the inadequate sample size, our findings are in keeping with the most recent meta-analysis regarding HF effect on allergy prevention in term neonates. In 2015, Boyle et al. found no consistent evidence to support the use of HF formula for reducing risk of allergic diseases (17). Similarly, Osborn et al. in a 2018 Cochrane found no difference in allergic diseases such as asthma, eczema, rhinitis, and food allergy in infants fed with a HF compared to IPF (18).
Only another randomized, double-blind study was conducted in high-risk preterm infants by Szajewska et al. (19). They failed to demonstrate a decrease in the incidence of allergic diseases, yet highlighted only a preventive effect of eHF on atopic dermatitis (AD) during a short follow-up of 12 months.
Conversely, several studies have been conducted in the last decades to investigate the preventive role of HFs on allergy development in term infants ( Table 3).
A preventive effect of eHF both on food allergy and sensitization was first outlined in 1992, in the RCT conducted by Zeiger et al. on 288 high-risk infants (20).
This result, despite the lack of strong evidence, was later confirmed by Odelram et al. (24) and Oldaeus et al. (25). Mallet et al. in their study on 177 high-risk infants found a preventive effect on eczema linked to the early use of eHF, without any effect on asthma (21).
Similar results on eczema were found by Vandenplas et al., in a RCT on 58 high-risk infants (23).
Another RCT on 158 high-risk infants was conducted by Halken et al., who found a reduction of CMA in the first 6 months of life associated to the early use of eHFs (whey or casein) (22).
Subsequently these results were confirmed by the same authors in a larger study on 595 high-risk infants randomized to receive eHF-W, eHF-C, or pHF (26).
The German Infant Nutritional Intervention (GINI) study is to date the largest, spontaneous, quasi-randomized trial in which 2,252 children (with a family history of allergic diseases) were randomized to receive extensively hydrolyzed  Frontiers in Pediatrics | www.frontiersin.org casein formula (eHF-C), extensively hydrolyzed whey formula (eHF-W), partially hydrolyzed whey formula (pHF-W), or standard cow's milk formula, in order to evaluate the possible preventive role of these formulas in the development of allergic diseases during a long term follow-up (27,(29)(30)(31)(32). They found a protective effect of eHF-C and pHF-W against AD at 1 year follow-up and a significant reduction of AD at 7-10 years of age in children. However, no preventive effect against asthma or AR was shown. These results were confirmed in the 15-year followup study, where the authors reported also fewer diagnoses of AR and asthma in those children fed with hydrolysates (mainly eHF-C) in their early stages of life, as if the preventive effects of HF on these two pathologies had occurred later in time. Throughout the follow-up period eHF-W did not show any preventive effects toward allergic diseases and none of the HF had influence on IgE sensitization. Differently from GINI study, the Melbourne Atopic Cohort Study, conducted on 620 high-risk infants randomized to pHF-W, soy formula or cow's milk formula, showed neither significant difference in allergic outcomes (eczema and food reactions) in the first 2 years of life nor evidence of reduced risk of SPT reactivity and of lower risk for AR, eczema and asthma up to 6-7 years of age (28).
Furthermore, in an observational population-based study, Goldsmith et al. examined the possible association between the development of food allergy at 1 year of age and either duration of exclusive breastfeeding or use of pHF (33). They found that the incidence of food allergy was not reduced by either the duration of exclusive breastfeeding or by the use of pHF, suggesting that allergen avoidance may not be helpful in allergy prevention.
Pooling data on HF in meta-analyses is problematic due to the heterogeneity of HF products and the different sources of proteins, hence different HF should not be considered equivalent. For this reason, Szajewska et al. conducted a meta-analysis taking into consideration exclusively studies using a unique 100%-whey pHF. They found a preventive effect against all allergic diseases and eczema (34). Based on all data from the literature, use of pHF-W formula has been considered safe in healthy term neonates (35,36).
At present, the conclusions of guidelines and meta-analysis on the role of HF for prevention of allergic diseases differ in term of recommendations, outcomes, and target population. According to some pediatric Societies the use of pHF is still indicated in infants at high risk of allergy, when mother's milk is not available or is insufficient (37,38). Differently, other Societies, based on emerging evidence, changed their previous recommendations and no longer proposed HF for prevention of allergic diseases (39)(40)(41).
The aim of this paper was to analyse the effect of HF on allergy prevention in preterm infants using follow-up data of a previous randomized, triple-blind, placebo-controlled study. To our knowledge, this is the first long-term follow-up paper concerning allergy prevention with HF in preterm infants. We acknowledge a possible limitation of our literature search due to the search restriction for preterm infants. In fact, using Medical Subject Headings-Terms for preterm infants, we could have missed studies in which subgroup analysis of preterm infants have been carried out, but not mentioned in the title or abstract. However, in our view, publication bias could be negligible. The main limitations of the present study can be considered the underpowered number of preterm enrolled, the short period of eHF administration, the drop-out rate and the lack of quantitative diagnostic methods used to diagnose allergy other than a validate questionnaire and SPT.
To sum up, further well-designed large studies in preterm infants should be conducted to address the preventive effects of HF for allergic diseases and the nutritional non-inferiority of preterm HF compared to modern preterm IPF in these vulnerable population. Moreover, data on long term safety of HF in preterm infants and cost/benefit ratio analysis are needed.
Evidences of hypersensitivity have been described also as predisposing factor in patients with functional gastrointestinal diseases (42), whose high prevalence have been recently found in preterm newborns (43). Despite self-limited diseases, a preventive intervention for FGIDs, especially for high-risk population, might have important clinical, and socioeconomical effects (44,45). HFs have been investigated as dietary modification for management of these conditions with inconsistent evidences, despite some authors suggested a decreased incidence in infants fed with pHF (46,47).
Finally, bio-effective agents such as probiotic have been recently added to HF to enhance their putative role in allergic disease prevention (48) and should be evaluated since preterm infants could be considered a strategy population for their wellknown dysbiosis-related conditions (49,50).

CONCLUSION
To date many rigorous systematic reviews and meta-analyses evaluating term infants concluded that evidence is not robust to support the use of HF to prevent atopic diseases. Our data did not show any allergy preventive effect of eHF in a small cohort of preterm infants and highlighted the need of further large studies to better clarify the possible role of HF for allergy disease prevention in this population.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Policlinico di Bari Ethics Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
MB conceptualized and designed the study. MT and NLae assessed study participants and collected study data. MF performed statistical analyses. AD interpreted data and drafted the initial manuscript. GB and AZ performed literature search. MC, LP, SS, and RP revised the manuscript. NLaf coordinated and supervised all activities. All authors contributed to the intellectual content, reviewed and revised the manuscript, and approved the final version. All authors contributed to the article and approved the submitted version.