Human milk (HM) is the optimal food source for the nutrition, growth, and development of newborns. The protein fraction of HM plays a crucial role in the healthy development of infants. HM contains a wide variety of minor whey proteins and peptides with important bioactive functions, many of which are still unknown. Proteomics allows for the study of biological samples with inherently complex protein mixtures. Proteins are essential for the development of living organisms, both in quantitative and qualitative terms. The combination of proteomic techniques currently enables the study of protein variability and minor peptides in HM across different lactation stages and allows for differential quantification according to gestational age and birth weight. However, studies on the human milk serum proteome during these stages are limited. The aim is to explore the minor whey proteins and peptides in human milk through a longitudinal analysis of five groups of breastfeeding mothers (with 30 extremely low birth weight newborns, 30 very low birth weight newborns, 30 low birth weight newborns, 30 adequate birth weight newborns, and 30 high birth weight newborns). Gestational age will also be considered to ensure homogeneous group distribution according to this condition. HM samples will be collected from each mother during three lactation periods after birth: within the first 48 hours (colostrum), at 5-14 days (transitional milk), and at 100-120 days (mature milk) for the five birth weight groups. In these neonatal/infant groups, minor proteins from whey fraction and peptides will be separated, quantified, and identified using label-free proteomic techniques. This study aims to expand our understanding of the minor proteins and peptides in human milk and their bioactive roles in neonatal health. By examining these components across different birth weight groups and lactation stages, the research will offer insights into how protein and peptide profiles vary by gestational age and birth weight, potentially influencing neonatal development. The findings from this proteomic analysis could not only demonstrate the complexity of human milk composition but also contribute to targeted nutritional support for preterm or low-birth-weight infants, customizing protein supplementation in HM banks and therefore enhancing their growth and developmental outcomes.
Healthy mothers
With monitored pregnancies within the care area of the Reina Sofía University Hospital in Córdoba
With newborns expected to be exclusively breastfed until 4 months
Mothers whose newborns have any of the following conditions: congenital malformation, chromosomal abnormality, hypoxia-ischemia, gastroschisis, polycythemia, hypoglycemia, sepsis, blood incompatibility
Pathological pregnancy, pregnancy by in vitro fertilization, or multiple pregnancies
With no plan to exclusively breastfeed until 4 months
Under medical treatment
Five groups of breastfeeding mothers: Group 1 (n=30) - with neonates of extremely low birth weight; Group 2 (n=30) - with neonates of very low birth weight; Group 3 (n=30) - with neonates of low birth weight; Group 4 (n=30) - with neonates of adequate birth weight; Group 5 (n=30) - with neonates of high birth weight. Additionally, gestational age will be considered to achieve a homogeneous distribution of the groups according to this condition.
Cohort
Prospective
Not listed
Minor whey proteins: Lysozyme
Concentration of lysozyme in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
Minor whey proteins: Lactoferrin
Concentration of lactoferrin in mg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
Minor whey proteins: Bile salt-dependent lipase (BSDL)
Concentration of bile salt-dependent lipase (BSDL) in µg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
Minor whey proteins: Lactoperoxidase
Concentration of lactoperoxidase in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).
Muscular ecography
Muscle thickness will be measured using a Phillips HD ultrasound equipment, with a high-frequency linear probe (7.5 MHz - 15 MHz), in B-mode. The thickness (expressed in mm) of the right quadriceps muscle and subcutaneous tissue will be measured, taken at the midpoint between the greater trochanter and tibial plateau: one anteroposterior in longitudinal section, another anteroposterior in transverse plane, and another perpendicular to the latter in the same plane.
Transfontanellar ecography
Brain structure and anatomy will be measured using a convex probe with a frequency range of (3.5 - 7 MHz) in B-mode. Longitudinal and transverse sections will be measured, and the thickness of the frontal cortex and the maximum thickness of the corpus callosum will be measured in millimeters (mm).
General information - Mothers
Mothers will complete a general questionnaire to gather information on sociodemographic factors, pregnancy, breastfeeding, medical conditions or complications, medications, and lifestyle habits
General information - Newborns
A general questionnaire will be completed regarding newborn characteristics, including gestational age, birth weight and length, sex, feeding type, and any pre- and/or postnatal complications or infections.
Dietary intake and eating habits
Food Consumption Frequency Questionnaire (CFCA) at the beginning of the study will be conducted, along with a 3-day food diary during the three stages of breastfeeding.
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Maimónides Biomedical Research Institute of Córdoba
Lead sponsor
Hospital Universitario Reina Sofia de Cordoba
Collaborator
Universidad de Granada
Collaborator
Universidad de Córdoba
Collaborator
