Preventive Effect of a Dietary Supplement With Two Probiotic Limosilactobacillus Reuteri Strains on Excessive Crying and Colic in Healthy Newborns.

Peak measured crying time at 6 (±1) weeks according to the prospectively kept baby day diary

The criteria for infant colic are:

• Recurrent and prolonged periods of infant crying, fussing, or irritability reported by caregivers that occur without obvious cause and cannot be prevented or resolved by caregivers. Fussing refers to intermittent distressed vocalization and refers to behavior that is not quite crying but not awake and content either.
• Caregiver reports infant has cried or fussed for 3 or more hours/day during 3 or more days in a given week.
• Total 24-hour crying plus fussing in the selected group of infants is confirmed to be 3 hours or more when measured by at least one, prospectively kept, 24-hour behavior diary.

Peak measured fussiness time at the age of 6 (±1) weeks according to the prospectively kept baby day diary.

Overall (estimated + measured) crying and/or fussiness time in the first 3 months (±1 week) of life.

Overall measured and/or estimated unconsolable crying time in the first 3 months (±1 week) of life

Parental stress as measured by the Parental Stress Scale (PSS) at 6 (±1) weeks and 3 months (±1 week) of both (if applicable) caregivers. Parents can indicate the degree to which they agree or disagree with the statements in this questionnaire, ranging from strongly disagree (1 point) to strongly agree (5 points). Total scores range from 18 points (low score signify a low level of stress) to 90 points (high score signify a high level of stress).

Infant and toddler quality of life questionnaire(ITQOL™) at 3 months (±1 week) and of 12 months (±2 weeks) age. ITQOL is scored on a scale from 0 to 100, where higher scores mean better health/quality of life.

Parental satisfaction scale measured by a 5-point Likert scale at 6 (±1) weeks and 3 months (±1 week). A scale where parents indicate how satisfied they are with their baby's current level of crying and fussing, with a minimum score of 2 and maximum score of 10. Parents who are very dissatisfied have a low score, while parents who are very satisfied have a high score.

Presence and degree of atopic dermatitis (AD, measured via SCORAD) at 3 months (±1 week) and 12 months (±2 weeks) of age. The score ranges from 0 to 103 and is used to determine the stages of AD severity. Where a score lower than 25 is seen as mild state, 25-50 is a moderate state and >50 is a severe state.

Assessed by the parents in a continuous daily bowel diary over the study period using the Brussels infant and toddler stool scale (BITSS). BITSS was validated as a reliable instrument to assess stools of non-toilet trained children There are 4 possible answers: A: Hard stool, B: Formed stool, C: Loose Stool and D: Watery stool, based on pictures.

assessed by the parents and day care centers in a continuous daily bowel diary over the study period using the BITSS

Fecal calprotectin at 6 (±1) weeks and 3 months (±1 week) of age

Morning saliva melatonin level at 6 (±1) weeks and 3 months (±1 week) of age

Number of (serious) adverse events throughout the study period. Parents are asked to provide detailed information on any adverse events their child may experience during the study. For each event, the diagnosis, sign, or symptom is written down, as well as the date the event started and ended. Parents should also describe the severity of the event, rating it as mild, moderate, or severe, and provide their assessment of its relationship to the study product, categorizing it as unrelated, unlikely, probable, or related. Moreover the outcome of the event should be specified, such as fatal, not resolved, resolved, resolved with sequelae, resolving, or unknown. Lastly, parents should report any actions taken regarding the study product, including whether the dose was unchanged, reduced, increased, temporarily suspended, or definitively withdrawn, or if there was any concomitant medication given to the child or no action was required.

Weight for age z score at each study visit

Stool pH at 6 (±1) weeks and 3 months (±1 week)

SCFA analysis at 6 (±1) weeks and 3 months (±1 week) of age. Concentrations of both straight-chain SCFA as branched-chain SCFA will be measured to be associated with the health status of the gut microbiota.

16S ribosomal RNA (rRNA) gene amplicon sequencing of skin microbiome (or shotgun metagenomics sequencing if the budget allows for this) at 6 weeks, 3 months and 12 months. To gain insight into the quantitative and qualitative abundance of microbial composition and diversity

16S ribosomal RNA (rRNA) gene amplicon sequencing of stool microbiome (or shotgun metagenomics sequencing if the budget allows for this) at 6 weeks, 3 months. To gain insight into the quantitative and qualitative abundance of microbial composition and diversity

Height for age Z-score at each study visit

Weight for height z score at each study visit