A total of 50 subjects (54% male) were enrolled. Mean age was 28.9±14.5 days (mean±SD) at enrollment. Most subjects’ caregivers rated them as ‘very’ fussy (76%), and the remaining were ‘extremely’ fussy (24%) at enrollment. The majority of subjects were African American (56%) and were receiving an intact protein-based infant formula at the time of enrollment (84%). Subject demographics are presented in Table 1.

Nine subjects did not complete the study per protocol. Three subjects were lost to follow-up, one subject moved, two subjects discontinued study formula and began consuming non-study formula during the study, and two subjects withdrew because of an adverse event (one with hard stools, rash, GERD; one with bronchiolitis). One subject was enrolled while using a thickened formula, and it was subsequently determined that the subject’s caregiver had started using the thickened formula without consulting her doctor, thus leading the site investigator to approve subject enrollment. This subject completed the study but was not included in the PP analysis as inclusion/exclusion criteria had not been met.

Baseline IGSQ for the intention-to-treat (ITT) population was 34.9±10.0 (mean ±SD; PP: 34.1 ±10.0). After 3 weeks of study formula use, the IGSQ score had a mean decrease of 12.7 points down to 22.1±7.5 (p<0.001; Figure 1). The per protocol (PP) population was similar with a mean decrease of 12.7 points down to 21.4±7.0 (p<0.001).

The baseline fussiness score was 3.2±0.4 in the ITT population. Compared to baseline, the fussiness score was significantly decreased after the first, second, and third feedings and after 24 hours (p<0.001 for all time points), ranging from 1.2 to 1.4. Similar results were seen with the PP population. No subjects worsened in their fussiness score from baseline to any of the time points assessed.

Caregivers’ satisfaction with the study formula was high. In the ITT population, 93% of caregivers stated their infants were comfortable on the formula, and 95% reported being satisfied with the study formula. All caregivers stated their infants liked the study formula. As expected, satisfaction was greater in the PP population, with 98% of caregivers stating their infants were comfortable on the formula, and all caregivers stating they were satisfied with the study formula.

Twelve subjects reported 17 adverse events throughout the study. Of these, one adverse event was reported as having a probable relationship to study formula. This was hard stools reported by one subject who withdrew from the study. All other adverse events were not related to study formula. There were no serious adverse events.

This was a single-arm study of healthy, exclusively formula-fed infants with caregiver-perceived intolerance. All subjects were switched from their usual formula to a commercially available formula with a partially hydrolyzed whey protein base, reduced lactose, 2-fucosyllactose and L. reuteri. IGSQ scores significantly decreased after three weeks of study formula feeding. In addition, fussiness as assessed by parents after the first three feedings of study formula and again 24 hours after enrollment, improved in nearly all subjects.

The use of the IGSQ tool in assessment of the subjects included in this study was advantageous for several reasons. The simplicity and broad assessment of GI symptoms is a major strength of the tool. The IGSQ is the only known tool to assess GI burden in infants to date that is simple to use, reliable and validated. To be enrolled in the study, subjects caregivers had to perceive their infants as being fussy , with no strict definitions given. This fussiness could have been attributed to any one of several factors commonly seen in infants, and the IGSQ inquires about five different domains including stooling, spit-up, flatulence, fussiness, and crying. Therefore changes in the overall combined score can be reflective of differences in any singular domain, or more likely, in any combination thereof. This tool has been validated and used in several infant formula studies 18192021. It is administered in a standardized way by trained personnel but answered by caregivers, providing an objective score for comparing an infant s GI symptoms. The intent of this research tool is to assess feeding tolerance through inquiries on GI distress and identification of non-normative patterns 18, which can translate into real-world situations of caregiver-perceived fussiness.

In this study, over 90% of caregivers documented improvements in fussiness as soon as after the first feeding, and this improvement in fussiness from baseline was maintained for the first three feedings as well as at 24 hours where 96% of caregivers reported an improvement. Importantly, the objective IGSQ scores which reflect gastrointestinal symptoms over the previous week were significantly decreased from baseline to three weeks after feeding of study formula. This indicates improvements in feeding tolerance occurred over the three-week period, remaining consistent with the reduction of fussiness perceived over the first 24 hours. From previous work, an IGSQ score of greater than 30 may be indicative of clinically meaningful digestive distress, and a score of 23 points has been observed for healthy infants18. In this single-arm study, subjects were enrolled with a mean IGSQ score of 34.9 points (indicating clinically meaningful GI distress) which then decreased after three weeks to a range associated with healthy infants without GI distress (score of 22.1).

Several attributes of the study formula may have contributed to the improvements recorded by caregivers. The protein base of the study formula was partially hydrolyzed whey which has been shown to empty rapidly from the stomach 222324, thereby potentially reducing the likelihood for spit-up and also producing softer stools when compared to intact cow s milk protein-based infant formula 25. L. reuteri strengthens the mucosal barrier, improves gastric emptying time, and balances the microbiota 91415. L. reuteri has been shown to reduce spit-up frequency in infants with regurgitation and reduce crying time in colicky infants 1526272829. The reduced lactose content in the study formula could also help in reduction in gas for any subjects who may have had a lactose sensitivity, though this is rare in infants.

The major limitation of this study was that it was a single-arm study. All subjects were switched to the same formula, and caregivers were aware this was not a randomized, controlled study. The placebo effect or a desire to please study staff on the 24 hour phone call could have played a role in caregiver-perceived fussiness. However, improvements were seen in fussiness in nearly all subjects (>90%) after the first feeding and after 24 hours (>95%) and post-hoc power calculations assuming a placebo effect for improved proportion ranging from 0.5 to 0.7 demonstrated over 80% power to detect the observed improved proportion for both the first feeding and the 24-hour telephone call in the ITT and PP populations.