»My-Milk-2« Children nutrition as a protective or health-risk factor

The research "My-Milk-2" is a continuation of the study performed in the period from 2010 to 2013 in the framework of a research project entitled "The role of human milk in development of breast-fed child's intestinal microbiota” (ARRS J4-3606) - "My-milk". A total of 294 pregnant Slovene women were enrolled. All of them participated in the study of dietary habits, while a subset of 185 women was enrolled in an in-depth study in which we were monitoring the diet of pregnant women and nursing mothers and studied the microbiota and the fatty acid composition of breast milk and the initial development of the intestinal microbiota of breast-fed babies. In addition, we measured the concentration of vitamin D in the maternal serum, the mineral bone density of mothers and their children, and performed anthropometric measurements of mothers and their children up to the age of one year.

Important findings of the »My-Milk« study

  • the reported dietary intake of pregnant and lactating women was inadequate, with insufficient intake of folate, vitamin D, iodine, iron, zinc, docosahexaenoic acid, and dietary fibre, and excessive intake of salt, free sugar, and alcohol;
  • the mass gain and BMI at the age of 1 year were significantly higher in infants with shorter duration of breastfeeding;
  • mothers with high serum levels of 25-hydroxyvitamin D tended to have new-borns with higher bone mineral density at birth (p=0.001);
  • faecal microbiota (FM) of partially breast-fed infants was different from the faecal microbiota of exclusively breast-fed children;
  • infant early phenotypes - higher weight and lower ponderal index at first month, were associated with lower proportion of the bacterial group Bacteroides-Prevotella;
  • major change observed in the FM over the first 3 months, common to most infants, was an increase in the proportion of bifidobacteria; the infant FM diversity did not increase during the first three months of life.

Purpose and the objectives of the »My-Milk-2« study

Diet can be either a risk or a protective factor for the development of a series of non-communicable diseases, while nutrition in the early life and its long-term impact is of particular interest and importance. A strong link between mother’s diet and lifestyle and a risk of developing various chronic diseases in her offspring such as obesity, atopic disorders, asthma, and others was demonstrated in various studies (Sausenthaler et al., 2011; Netting, 2014; Grieger et al., 2016). Javaid et al. (2006) reported that children of mothers with low vitamin D status during late pregnancy had reduced bone mineral density (BMD) at the age of 9 years. Beside nutrition, the latest research suggests crucial involvement of the gut microbiota (GM) in infant growth. For example, research has shown the negative impact of poorly formed, 'immature' gut microbial consortia in 6-month-old children on their growth and nutritional status, mineral bone density and metabolism (Blanton et al., 2016). The challenge for many research groups remains the question, whether early differences in the composition of the GM in children are associated with metabolic or systemic problems later in life. Despite the intensive worldwide research dedicated to the nutrition and faecal microbiota, the studies that follow the link among the early nutrition, initial intestinal microbiota, and development of children over a longer time period are limited.

The results obtained in the “My-Milk” study are an excellent basis for the implementation of the prospective cohort study on the effects of early nutrition and various maternal and child factors during the first year of life on health and developmental status of children who will reach the age of 6 – 7 years in 2017/2018.

Study objectives

  • O1: To evaluate the nutritional status (dietary intake, anthropometrics, body composition (bone density, % of fat and muscle tissue)), health markers (blood pressure, blood cholesterol, frequency and severity of infections) and faecal microbiota composition in children at age of 6-7 years;
  • O2: To investigate the impact of early life exposure on later growth, body composition, nutritional status, health, and faecal microbiota composition in children aged 6-7 years. Early life exposures would be defined by maternal factors (BMI before pregnancy, weight gain during pregnancy, nutrition during pregnancy and lactation, fatty acid composition of human milk, and concentration of vit D in serum) and infant factors (duration of breastfeeding, antibiotic treatments, faecal microbiota composition at 3 month of age, anthropometrics and bone mineral density in the neonatal period).

Description of the »My-Milk-2« study«

Study population

ll children (mothers) who have completed a detailed part of My-Milk study (N=162) will be invited in this follow-up study, since only term (≥ 37 weeks of pregnancy with normal birth weight), healthy and breast-fed children had been included. A child will be included after a written consent of the parent to agree with the inclusion the child in the study.

Description of the work

The parents (children) who will respond to the invitation to participate will be invited to a preparatory meeting where the project leader and researchers will briefly present the most important results of the "My-Milk" study, followed by a more detailed presentation of the "follow-up" survey. Parents willing to include their children in a "follow-up" study will receive a document with a description of the research and a written consent of cooperation for signature.

Children included in the “My-Milk-2” study will be recorded by the codes they received in the My-Milk study. Data from both studies will be stored in the central database. The database will not contain identifiable personal information. The records will be encrypted with a locally generated identification number.

What and how will be measured - methods

The standard dietary assessment methodology for children population as provided in the EFSA guidance (EFSA, 2014) will be used. The food consumption survey will be carried out at an individual level by the repeated 24-h food diary and Computer Assisted Personal/Telephone Interview method with the support of the General Questionnaire and the FPQ according to PANCAKE - Pilot study for the Assessment of Nutrient intake and food Consumption Among Kids in Europe (Ocké et all, 2012). Evaluation of the dietary intake data will be performed by the Open Platform for Clinical Nutrition (OPEN, Jožef Stefan Institute IJS - OPEN is a web-based application for dietary assessment and treatment upgraded to support the h -recall/food diary method and FPQs in line with the EFSA »EU Menu« methodology.

Growth, development, and health status of children aged 6-7 years will be assessed based on the clinical examination and the data obtained from medical records. Clinical examination, carried out at the University Children’s Hospital, UMCL by the specialist doctor of paediatrics and nurses, will include assessment of the clinical status, anthropometric measurements, measurement of the bone mineral density, and measurement of the blood pressure. The bone mineral density will be measured using the quantitative ultrasound (Omnisense 7000P) which is a simple, painless, radiation free investigation that gives information about the bone density and the micro architecture.

Study of children faecal microbiota will be based on the molecular approach. Single faecal sample of an apparently healthy child will be collected at the first clinical examination. Parents will be supplied with sterile sampling containers with a spoon attached to the lid, provided with a code and sampling instruction. They will be asked to collect approximately 2 g (2 spoons) of child's faeces possibly on the planned day of visit at the Pediatric clinic or one day before. The samples will be kept refrigerated/cooled until the visit and will be immediately frozen upon the arrival to the clinic by the medical personnel.


The study, approved by the Republic of Slovenia National Medical Ethics Committee (NMEC) of the Ministry of Health, will be conducted in accordance with the Declaration of Helsinki on ethical principles for medical research involving human subjects. The parents of children invited to join the survey will be informed about the possibility to interrupt the collaboration of their child in the research at any time without giving any reason.

Identification numbers will be assigned to participants and used across the project, to avoid the use of personal data in data transfer and analyses. Access to personal data will be restricted. All data on the subjects and their health status will be protected as part of confidential medical documentation in accordance with all the legally required principles of personal data protection.

Significance of the research for the science and society

The project results will significantly contribute to the progress in research in the areas of public health and nutrition since the interactions within the axis nutrition in early childhood, gut microbiota composition and diversity, and potential health problems in later life are still not fully explored. It should be noted that this study will gain a lot of new data on dietary habits, the basic features of the intestinal microbiota, health status and the occurrence of certain diseases in Slovenian children. We believe that the results of our research will contribute to the development of more suitable nutritional interventions for prevention of certain chronic non-communicable diseases and their complications, including overweight and obesity. Moreover, the acquired data and knowledge will be an excellent basis for developing new dietary guidelines for the population of children.