The goal of this observational study is to demonstrate the presence of neurotrophic factors in the breast milk of mothers with 18-month-old children and the persistence of these factors in breast milk after the age of 2 years. The main questions it aims to answer are:

• Does the breast milk of mothers with 18-month-old babies contain Brain-Derived Neurotrophic Factor (BDNF), S100B protein and glial cell line-derived neurotrophic factor (GDNF)?
• Does the breast milk continue to contain BDNF, s100B protein and GDNF and-after 24 months?

Participants will be asked to give breastmilk at 3 separate times. Breast milk samples will be collected from mothers at 18th-month, 24th-month and ≥25 months of their babies. A total of 75 breast milk samples obtained from 25 mothers with their consent will be analyzed.

Growth factors are among the bioactive compounds found in breast milk and have effects on the growth and maturation of the baby's organs and systems. The completion of each organ maturation occurs at different times. Although the development of the nervous system reaches 60% of adults at the age of 2 years, it continues after the age of 2. Brain-Derived Neurotrophic Factor (BDNF), S100B protein, and glial cell line-derived neurotrophic factor (GDNF) are the neurotrophic growth factors found in breast milk and play a critical role in the development of the nervous system, the maintenance of neuronal functionality and the proliferation of neurons . They play a role during brain development, particularly in differentiating neuronal and glial cells. In addition, it is effective in synaptic plasticity and has important roles in the mature nervous system. The presence of neurotrophic factors in breast milk under the age of one has been demonstrated. However, the presence of these factors in breast milk has not been evaluated at 2 years and above. In this study, investigators aimed to demonstrate the presence of neurotrophic factors in the breast milk of mothers with 18-month-old children and the persistence of these factors in breast milk after the age of 2 years.

Study Design:

The study was conducted at Istanbul University, Istanbul Medical Faculty, Department of Pediatrics, Department of Social Pediatrics, Well Child Outpatient Clinic. All mothers participating in the study are informed in detail about the study and the importance of breast milk. According to Turkish Demographic and Health Survey (TDHS) 2018 data, the rate of breastfeeding is 66% at 12 months, 50% at 18 months, and 34% at 24 months. Considering the decrease according to age, it was assumed that the breastfeeding rate would be 18% after the age of 2. Considering the TDHS 2018 data of mothers, assuming that 18% of mothers breastfeed their babies after the age of 2, with a 95% confidence level and an acceptable sampling error of 0.15, the sample size calculation formula was used to estimate the population ratio, and the sample size was calculated for each mother. It is planned to have at least 25 mothers during the milk collection period (for each mother 18., 24., ≥ 25 months), with a total of 75 breast milk.

Breast milk collections and analysis:

1. Breast milk will be collected at 3 separate times at 18th-month, 24th-month and ≥25 months of their babies.
2. Using the manual milking method, 5 ml of expressed breast milk will be collected in Eppendorf tubes and transported in a polystyrene box containing ice (at 4°C) to the laboratory within 30 minutes. All breast milk samples will be stored at -20°C under laboratory conditions until the analysis.
3. All collected breast milk samples will be prepared for analysis by gradual thawing (breast milk will be taken to +4 C degrees one day before analysis, room temperature on the day of analysis) at the 12th month of the study. The neurotrophic factors (BDNF, S100B, GDNF) will be analyzed with ELISA (Elabsicence; United States) method.

Inclusion Criteria:

• Mothers who applied for the 18th-month follow-up of their exclusively breastfed baby for the first 6 months agreed to participate in the study.

Exclusion Criteria:

• Mothers of infants who were not exclusively breastfed in the first six months
• Mothers with infection, metabolic disease, central nervous system, breast disease or pregnancy
• Mothers of infants with infection, metabolic disease, malnutrition and central nervous system disease
• Mothers who stop breastfeeding before 25 months

Mothers will included

On the basis of the inclusion and exclusion criteria 25 mothers will be included in this study

The decision will be made after the permission of the authors and authors' institution