Human Milk Oligosaccharides, Gut Bifidobacterium, Vitamin D, and Infant Immunity

Human Milk Oligosaccharides in Breast Milk and Its Relation to Gut Bifidobacterium, Vitamin D and Immune Modulation in Infants

The goal of this observational study is to learn how certain natural sugar components in breast milk may be linked with vitamin D level, healthy gut bacteria, and immune markers in healthy breastfed infants. The study will include exclusively breastfeeding mothers and their healthy infants aged 1 to 2 months.

The main questions it aims to answer are:

• Is this natural sugar in breast milk linked with the baby's vitamin D level?
• Is this natural sugar in breast milk linked with healthy gut bacteria?
• Is this baby's vitamin D level linked with healthy gut bacteria?
• Is this natural sugar linked with immune factors in breast milk? Participants will answer health questions from questionnaire, undergo a brief physical examination, allow breast milk and a small baby blood sample to be collected, and provide an baby stool sample.

Researchers will test these samples in the laboratory and analyze the results to answer the research questions.

Subject recruitment was conducted during immunization activities at community health centers (Puskesmas) and integrated health posts (Posyandu). The subject collection technique used the quota sampling method, a non-probability subject selection technique carried out by determining the desired sample size (quota) from a population with predetermined criteria, and researchers collected sample units until the target quota was reached. Researchers provided an explanation of the research protocol and obtained informed consent from participants regarding their willingness to participate in the study. Participants who met the eligibility criteria (inclusion and exclusion criteria) were declared as research subjects. Next, a research questionnaire was completed, including recording the subject's identity (mother and baby), the mother's health and nutritional history, childbirth and breastfeeding history, observation of the baby's stool, history and risk factors for allergies, and a general physical examination.

Mature breast milk samples will be analyzed to determine maternal secretion status, HMO status, Bifidobacterium in breast milk, and the status of immune markers such as sCD14, TGF-beta, and IgA in breast milk. Stool samples will be analyzed to determine the infant's intestinal Bifidobacterium. Blood samples will also be analyzed to determine vitamin D levels. Statistical correlations between these parameters will be performed to determine the research objectives.

The collected data is grouped based on the purpose and type of data, then the appropriate statistical method is selected, namely:

1. Univariate analysis (Used to describe basic data characteristics, in the form of frequency, mean value, standard deviation, and range)

2. Bivariate analysis

   1. Comparison Test (To see differences between groups)

      • Chi-Square Test → If the variable is categorical (e.g., the relationship between maternal secretory status (Secretory vs. Non-Secretory) and delivery method (Vaginal vs. Cesarean section). An alternative test, the Fisher's Exact test, is performed if the basic assumptions of the Chi-Square are not met.
      • T-Test (Independent T-Test) → If you want to compare the means of two groups (e.g., the difference in vitamin D levels between male and female infants). An alternative test, the Mann-Whitney test, is used if the data is not normally distributed.
      • ANOVA Test → If you want to compare the means of more than two groups (e.g., the difference in vitamin D levels based on the timing of early breastfeeding initiation: <1 hour, 1-6 hours, >6 hours). An alternative test, the Kruskal-Wallis test, is used if the data is not normally distributed.

   2. Correlation Analysis (To see the relationship between two numeric variables)

      • Pearson Correlation → If the data is normally distributed.
      • Spearman Correlation → If the data is not normally distributed or is ordinal

Inclusion Criteria:

1. Maternal Subject :

   • Women aged at least 18 years who are physically healthy;
   • Have a healthy baby aged 1-2 months with a full-term gestational age (37-42 weeks) and a normal birth weight (2500-4000 grams);
   • Exclusively breastfeeding;
   • Agree to participate in this study by signing an informed consent form.

2. Infant Subjects :

   • All infants of mothers who meet the inclusion criteria

Exclusion Criteria:

1. Maternal Subject :

   • Currently participating in another clinical trial;
   • Having a condition such as a breast abscess or other breast pathology;
   • Currently taking medications for conditions that may affect breast milk, such as antibiotics;
   • Subjects who cannot comply with the study protocol

2. Infant Subjects:

   • Currently participating in another clinical trial;
   • Having a history of antimicrobial treatment (oral or parenteral antibiotics);
   • Subjects are unable to comply with the research protocol.