Growth and tolerance of formula with lactoferrin in infants through one year of age: double-blind, randomized, controlled trial

Healthy 12- to 16-day-old infants were recruited at 24 clinical sites in the United States. Eligible infants were singleton births at 37–42 weeks gestational age with birth weight ≥ 2500 g and solely formula-fed at least 24 h prior to randomization. Exclusion criteria included history of underlying disease or congenital malformation likely to interfere with normal growth and development or participant evaluation; signs of acute infection including fever, diarrhea, or antibiotic use; feeding difficulties or formula intolerance; weight at randomization <98 % of birth weight; large for gestational age born from a mother diabetic at childbirth; and immunodeficiency.

In this multicenter, double-blind, randomized, controlled, parallel-group, prospective trial, participants were enrolled between July 2010 and January 2011. The study sponsor created a computer-generated, gender-stratified randomization schedule provided in sealed consecutively-numbered envelopes for each study site. Study formula was assigned by opening the next sequential envelope from the appropriate set at the study site. Study formulas, each designated by two unique codes known only to the sponsor, were dispensed to parents at each study visit prior to study completion or withdrawal. Participants were randomly assigned to receive one of three study formulas (isocaloric, 20 calories/fluid oz): a routine cow’s milk-based infant formula (Control; marketed Enfamil®, Mead Johnson Nutrition, Evansville, IN) or one of two investigational formulas with bLf (DMV International bv) at 0.6 g/L (LF-0.6) or 1.0 g/L (LF-1.0) from 14 days of age up to 365 days of age (Table 1). All study formulas provided DHA at 17 mg/100 kcal. ARA was provided at 34 mg/100 kcal in the Control and 25 mg/100 kcal in the investigational formulas. Each investigational formula also had 4 g/L (1:1 ratio) of a blend of PDX (Litesse® Two Polydextrose; Danisco) and GOS (Vivinal® GOS Galactooligosaccharide; Friesland Foods Domo).

Anthropometric measures (body weight, length, and head circumference) were recorded at 14, 30, 60, 90, 120, 180, 275, and 365 days of age. At study enrollment, parents completed a baseline recall of tolerance (fussiness and gassiness), and stool characteristics (frequency and consistency) and provided information on participant’s history of breastfeeding (0, ≤7, or >7 days prior to study enrollment), family history of allergy in one or more relatives (including biological mother, father, sibling, and/or half-sibling) and exposure to smoking in the home. At all subsequent study visits, information on enrollment in daycare, exposure to smoking in the home and/or daycare, and 24-h recall of formula intake (fluid oz/day), tolerance (fussiness and gassiness), and stool characteristics (frequency and consistency) was collected. Responses were scaled from 0–3 for amount of gas (none, slight amount, moderate amount, excessive amount); 0–4 for fussiness (not fussy, slightly fussy, moderately fussy, very fussy, extremely fussy); and 1–5 for stool consistency (hard, formed, soft, unformed or seedy, watery). The primary outcome was weight growth rate from 14–120 days of age. Secondary outcomes included anthropometrics, tolerance measures, and medically-confirmed adverse events through 365 days of age. Adverse events were coded according to specific event (e.g. otitis media, colic, etc.) and the body system involved including: Body as a Whole; Cardiovascular; Eye, Ears, Nose, and Throat; Endocrine; Gastrointestinal; Metabolic and Nutrition; Musculoskeletal; Nervous; Respiratory; Skin; and Urogenital. Participants received exclusive study formula feeding through 120 days of age. Participants who continued in the study through 365 days of age were considered to complete the study even if study formula consumption discontinued or decreased to fewer than 2 feedings/day after 275 days of age (approximately 9-months-old).

The sample size was chosen to detect a clinically relevant difference of 3 g/day in weight gain from 14–120 days of age (80 % power; one-tailed). Assuming a standard deviation of 6 g/day for male and 5 g/day for female participants, approximately 78 males and 55 females were needed to enroll in each group with the expectation that 51 male and 36 female participants per study group would complete the study through 120 days of age. Analysis of variance (ANOVA) was used to assess growth rates in four pre-specified time intervals: from 14 to 30, 60, 90, or 120 days of age, calculated for each participant by linear regression of weight on age. Mean weight growth rates by gender for each investigational formula group were compared with the Control using one-tailed tests as outlined in guidance provided by the American Academy of Pediatrics (AAP) Task Force on Clinical Testing of Infant Formulas [23]. For all secondary outcomes, overall comparisons for the three formula groups were two-tailed. Unadjusted pairwise comparisons were performed if the overall test was statistically significant. All tests were conducted at α = 0.05. Achieved weight, length, and head circumference; length and head circumference growth rates; formula intake; and stool frequency were analyzed by ANOVA. Stool consistency, fussiness, and gas were analyzed using the Cochran-Mantel-Haenszel (CMH) row mean score test. Incidence of adverse events as well as incidence of allergic manifestations, gastrointestinal infections, respiratory infections, or any infection-related adverse events were analyzed using Fisher’s exact test. All analyses were conducted using SAS version 9.2 (Cary, NC).

A total of 480 participants were enrolled and randomized (Control: 155; LF-0.6: 165; LF-1.0: 160). Participants who were randomized but consumed no study formula (Control: 1; LF-0.6: 1; LF-1.0: 2) were not included in subsequent analyses (Fig. 1). No differences in body weight, length, or head circumference were observed by gender among groups at study enrollment (Table 2). Birth anthropometric measures as well as gender, race, and ethnic distribution, history of breastfeeding, family history of allergy, and exposure to smoking in the home were also similar among groups (data not shown). No group differences from 30 to 365 days of age in daycare enrollment and exposure to smoking in the home and/or daycare were detected (data not shown). A total of 353 infants completed the study (Control: 110; LF-0.6: 127; LF-1.0: 116).

Growth rates were analyzed from 14–365 days of age. As outlined in guidance provided by the AAP Task Force on Clinical Testing of Infant Formulas, rate of weight gain (g/day) is used as the most important parameter in clinical evaluation of infant formulas with differences of >3 g/day over a 3–4 month period considered clinically significant [23]. Consequently, no statistically significant group differences by gender in the primary outcome, weight growth rate from day 14–120, were detected (Table 3). No statistically significant differences were observed for weight, length, or head circumference growth rates by gender for any measured age range among study groups with the exception of lower weight growth rate for females in the LF-1.0 compared to the Control group from day 14–60 (29.7 ± 0.9 vs 32.4 ± 1.0 g/day; P < 0.05). This small difference within a single measured age range at less than 3 g/day was not considered clinically significant. In addition, no other statistically significant differences were observed for mean achieved weight, length, or head circumference at any measured time point up to 365 days of age. Mean achieved weight for males (Fig. 2) and females (Fig. 3) plotted on the WHO weight-for-age standard growth chart [24, 25] fell approximately within the 25^th and 75^th percentiles at all study time points.

Parent-reported mean study formula intake (fl oz/day; data not shown) increased from day 30–120 for all groups by gender, indicating normal intake for LF-0.6 and LF-1.0 groups when compared to the Control for this time period (examples: females at day 30, 25.9–26.9 fl oz/day and day 120, 33.3–34.2 fl oz/day; males at day 30, 28.2–30.0 fl oz/day and day 120, 35.0–35.7 fl oz/day). Intake for female participants by group was similar at all time points assessed. Statistically significant group differences in intake were noted among males at days 180 (Control: 36.5 fl oz/day; LF-0.6: 31.9 fl /day; LF-1.0: 33.7 fl oz/day; Control vs LF-0.6, P < 0.05) and 275 only. However, by day 180, mean reported study formula intake began to decline in general for both male and female participants which could be expected as parents and caregivers likely begin to offer complementary foods to infants at approximately 4–6 months of age. Parent-reported gassiness and fussiness were similar among groups at all study time points (data not shown). Using 24-h recall, the amount of gas most commonly reported was “slight amount” or “moderate amount” up to 180 days of age and “none at all” or “slight amount” by 275 and 365 days of age. Fussiness was most often characterized as “slightly fussy” or “not at all fussy” in all groups. No significant group differences in mean (±SE) stool frequency (number/day) were detected at any time point assessed (Table 4). No group differences in mean (±SE) stool consistency (with categories corresponding to 1 = hard, 2 = formed, 3 = soft, 4 = unformed or seedy, 5 = watery; Table 4) were detected at baseline. Significant differences in stool consistency were detected between Control and investigational formula groups from day 30 through 180. By category, the primary differences at these study time points were more infants with a formed and fewer infants with an unformed or seedy stool consistency in the Control compared to LF-0.6 and LF-1.0 groups. The majority of infants in all groups from day 30–365 were reported to have a soft stool consistency. There were no significant differences among study formula groups by day 275, possibly reflecting the increased amount of complementary feeding in older children consuming less formula.

In the overall study population (all participants up to 365 days of age) no statistically significant group differences were detected for study formula discontinuation either related to study formula (Control: 18, 12 %; LF-0.6: 20, 12 %; LF-1.0: 17, 11 %) or not related to study formula (Control: 50, 32 %; LF-0.6: 42, 26 %; LF-1.0: 49, 31 %). Of the 55 participants with formula-related discontinuation, formula intolerance as determined by the study investigator was the most common reason (Control: 13; LF-0.6: 14; LF-1.0: 15) with fussiness (Control: 5; LF-0.6: 8; LF-1.0: 10) and gas (Control: 6; LF-0.6: 3; LF-1.0: 6) as the most common symptoms. Parental decision was the most common reason for discontinuation not related to study formula (Control: 13; LF-0.6: 7; LF-1.0: 19).

No group difference was detected in the number of participants for whom at least one medically-confirmed adverse event was reported (Control: 141, 92 %; LF-0.6: 154, 94 %; LF-1.0: 149; 94 %). There were no statistically significant group differences detected in the overall incidence of adverse events for the following systems: Body as a Whole; Cardiovascular; Eyes, Ear, Nose and Throat; Endocrine; Gastrointestinal (GI); Metabolic and Nutrition; Musculoskeletal; Nervous System; Respiratory; and Skin. Significantly fewer participants in the Control (6, 4 %) and LF-0.6 (7, 4 %) groups versus the LF-1.0 group (17, 11 %; P < 0.05) experienced Urogenital system events. Although no significant group differences were detected for specific adverse events in this system, the incidence of penile adhesion (Control: 2, 2 %; LF-0.6: 3, 3 %; LF-1.0: 8, 9 %), commonly associated with circumcision in young male infants, appeared to drive the overall statistical difference. Within the GI System, the medically-confirmed incidence of diarrhea, constipation, emesis, or gas was low with no significant group differences; however gastroesophageal (GE) reflux (Control: 27, 18 %; LF-0.6: 24, 15 %; LF-1.0: 41, 26 %) was significantly lower in the LF-0.6 versus the LF-1.0 group (P < 0.05). Within the Nervous System the incidence of macrocephaly (defined as head circumference >98^th reference percentile; Control: 3, 2 %; LF-0.6: 0; LF-1.0: 0; P < 0.05) was low, albeit statistically significant. No associated underlying health conditions were reported by study investigators for these participants and all completed the study. No group differences were detected in the incidence of allergy- or infection-related adverse events; however, this study was not powered to detect likely subtle differences in this population of healthy term infants. A total of 41 participants (Control: 14, 9 %; LF-0.6: 13, 8 %; LF-1.0: 14, 9 %) experienced serious adverse events (SAEs). With the exception of one SAE in the Control group in which the participant was diagnosed with likely cow’s milk protein intolerance, all SAEs were deemed unrelated to study formulas as assessed by study physicians.