NUTRIPROTECT-Children Nutrition as a Protective or Health-risk Factor
Condition: Breastfeeding; Dietary Intake; Health; Body Composition; Intestinal Microbiota
Study type: Observational

Summary: 
      Despite the intensive worldwide research dedicated to the nutrition and faecal microbiota,
      the studies that follow the link between the early nutrition, initial intestinal microbiota,
      and development of children over a longer time period are limited. From 2010 to 2013, the
      investigators conducted a study "My-Milk" in Slovenia. From 185 participating pregnant women
      involved in the detailed part of the protocol (comprising of 4-days weighed dietary record,
      visits at clinic and biological sample collection), 152 participants completed the study at
      the child's first year of age. Five of the participants bore twins whose data was excluded
      from the research because infant's identity was not consistently followed throughout the
      research. In addition to studying the microbiota and fatty acid composition of breast milk
      and the initial development of intestinal microbiota of breastfed babies, the investigators
      also monitored the diet of expecting and nursing participants, concentration of vitamin D in
      serum and bone mineral density in participating mothers and children. The investigators
      performed anthropometric measurements of children during the first year of age. All singleton
      children who have completed My-Milk study (N= 147) will be invited in the follow-up study
      "My-Milk-2". The first step of the proposed study will be the evaluation of nutritional
      status of children aged 6 to 7 years in 2017/2018, including the assessment of the body
      composition (bone density, percentage of fat and muscle tissue), health indicators (blood
      pressure, blood cholesterol, frequency and severity of infections) and fecal microbiota. The
      obtained data of My-Milk and My-Milk-2 studies will enable the prospective cohort study to be
      carried out on the effects of early nutrition and various factors of mother and child during
      the first year of age on the health and developmental status of children aged 6- 7 years. The
      project results will significantly improve the progress in research areas of public health
      and nutrition. The interactions along the axis nutrition in early childhood, gut microbiota
      composition/ diversity and potential health problems in later life are still not fully
      explored. The investigators expect the results of the proposed study will contribute to the
      scientific development in this field in global prospective. The acquired information will
      also be an excellent basis for development of new dietary guidelines for children.
    

Detailed description: 
      Nutrition can act either as a risk or protective factor in the development of a series of
      non-communicable diseases. While such effects were established for the whole life cycle, the
      nutrition in early life may be particularly relevant for its long-term impact on later life
      health. The reliable scientific data on dietary intakes and nutritional status of the
      representing sample population serve as a basis to elucidate the role, the specific
      nutritional factors exert on human health. The scientific background of the proposed
      applicative research project is based on the fact that the studies, following the link
      between the early nutrition, initial intestinal microbiota, and development of children, over
      a longer time period, are limited. Therefore, the key scientific challenges addressed are
      focused on the effects of the early life nutrition on growth and health of participating
      children, and to investigate the relationship among various markers of health.

      While the work programme of this multidisciplinary applicative prospective study will be
      focused onto children, it shall be tightly linked to former "My-Milk" project (SRA J43606;
      finished in the year 2013), where the role of early nutrition in the development of breast
      fed children and their intestinal microbiota, during the first year of life, was
      investigated. This study will be conducted within the population of 6-7 years olds included
      in the first "My-Milk" study. The study will characterize the key modifying factors during
      participant's early life (defined through maternal variables (BMI before pregnancy, weight
      gain during pregnancy, nutrition during pregnancy and lactation, fatty acid composition of
      human milk, and vitamin D concentration in serum) and infant variables (duration of
      breastfeeding and antibiotic treatments, faecal microbiota at first month, anthropometrics
      and bone mineral density in the neonatal period)) on participant's nutritional status at the
      age 6-7 years. The current nutritional status will be defined by participant's dietary
      intakes, anthropometrics, body composition - bone density, percentage of fat and muscle
      tissue), health (blood pressure, blood cholesterol, frequency and severity of infections),
      and fecal microbiota.

      The study population invited to participate will include all singleton children of mothers
      who completed the detailed part of the My-Milk study at child's first year of age (N=147).
      Considering a very low drop-out rate in the first detailed part of the study My-Milk (33
      participating mothers out of 185, approximately 18 percent), investigators expect that at
      least 70-75 percentage of the invited participants will respond, meaning between 100 - 110
      children may get enrolled in the study. This population will represent 0,5 percentage of
      children born in 2011 (21947 children - statistical data for Slovenia).

      The following parameters will be measured and evaluated: the nutrition of children by using
      the standard dietary assessment methodology for children and adults/elderly population as
      provided in the European Food Safety Authority guidance (EFSA). Namely, the food consumption
      survey will be carried out on the individual level by repeated 24-h food diary and Computer
      Assisted Personal/Telephone Interview method (CAPI/CATI) and by supporting General
      Questionnaire and the Food Propensity Questionnaire (FPQ) according to PANCAKE - Pilot study
      for the Assessment of Nutrient intake and Food Consumption Among Kids in Europe. The 24-h
      structured diet recall interview is a quantitative research method used in the nutritional
      assessment. A trained dietitian investigator will ask a parent or caretaker to recall all
      foods and drinks that the participating child consumed during the previous 24 hours at two
      different time points during the study (non-consecutive days at least 8 - 20 days between the
      two records) to receive the proper information about the habitual intake of foods. From the
      data obtained by 24-h recall investigators intend to evaluate nutritional intakes of the
      population sub-groups. In addition, connections between the nutrition of children and body
      composition and health markers will be studied. FPQ will enable the estimation of certain
      relevant, infrequently consumed food groups. Data will be collected using validated
      questionnaires using computer -assisted data collection methods and processing. Evaluation of
      the dietary intake data will be performed by dietary software Open Platform for Clinical
      Nutrition (OPEN) by Jožef Stefan Institute (IJS). OPEN is a web-based application for dietary
      assessment and treatment as well as upgraded to support the 24h -recall/food diary method and
      FPQs in line with the EFSA pan-European food consumption survey, also known as the EU Menu,
      methodology. OPEN is an efficient, first Slovenian web-based tool for dietary assessment
      based on the local food items. OPEN allows the dietitian to enter the food and amounts for
      each participant. After food items, amounts and preparations are entered into the OPEN, the
      program creates an analysis with the breakdown of nutritional information.

      The assessment of growth, development, and health status in 6-7 years old participants will
      base on the conducted clinical examination and data obtained from the medical records.
      Clinical examination will include the assessment of clinical status, anthropometric
      measurements, and quantitative ultrasound measurements of the bone mineral density and
      measurement of blood pressure.

      The clinical examination will be performed at the University Children's Hospital and will be
      carried out by a specialist doctor of paediatrics and investigator biologist. It will include
      the assessment of clinical status, anthropometric measurements, and quantitative ultrasound
      measurements of bone mineral density. The bone mineral density will be measured at the right
      tibia using the (QUS) Sunlight Omnisense (Sunlight Medical Ltd, Tel Aviv, Israel) which is
      simple, painless and radiation free investigation and gives information about the bone
      density and micro architecture. The QUS measures the time taken by the ultrasound signal to
      travel between two transmitters and two receivers contained within the probe. These
      propagation times are used by a proprietary algorithm to determine the bone speed of sound
      (SOS), which is expressed as meters per second (m/s). The software will use the three most
      consistent measurements to compute the result. The results will be taken as SOS and Z score
      (units of standard deviations relative to the mean for age- and sex-matched population
      reference values). Unlike in children, in adults, measurements are taken on a radius. For
      anthropometry: body height [cm] and body mass [kg] will be measured with a certified medical
      scale barefooted and in a minimal clothing. The precision of a height measurement will be 0.1
      cm and of a mass measurements 0.1 kg. Skinfold thicknesses [mm] at six standard locations
      will be measured according to the standardised methodology with the precision of 0.1 mm by a
      calibrated certified skinfold calliper (Harpenden, England). Circumferences [cm] will be
      measured once on the trunk or on the right side of the body with a non-stretchable narrow
      measuring tape (fibre glass tape, China) with the precision of 0.1 cm. The estimates of body
      composition will be linked to the acquired nutritional data.

      Other information regarding the current health status (including blood cholesterol) and child
      development will be obtained from the medical documentation. For this purpose investigators
      will ask paediatricians for the access to the participants' medical records. The data on the
      eventual disruption of a regular physical development shall be analysed (the accelerated
      growth, attenuated growth, low body mass and BMI for overweight or obesity), and on the
      presence of allergies and emerging chronic diseases. The emphasis will also be laid on the
      infections, medication intakes (antibiotic treatments) and chronic diseases.

      The study of children fecal microbiota will base on molecular approaches such as quantitative
      real-time PCR (qPCR) and next-generation sequencing (NGS) sequencing of the 16 sedimentation
      coefficient (16S) subunit of ribosomal ribonucleic acid (rRNA) gene amplicons using Illumina
      technology. A single fecal sample of an apparently healthy child will be collected at the
      clinical examination. Priorly, the parents will be supplied with the coded sterile sampling
      containers with a spoon attached to the lid and with sampling instruction by post and e-mail.
      They will be asked to collect approximately 2 g (2 spoons) of participating child's faeces
      possibly on the planned day of visit at the Pediatric clinic or one day before. Samples will
      be kept refrigerated/cooled until the visit and will be immediately frozen at -20 ˚C upon the
      arrival to the clinic by the medical personnel. Within a month after being collected, -20 ˚C
      frozen fecal samples will be transferred to the laboratory and stored at -80 ˚C until the
      performance of microbial analyses. QPCR and 16S rRNA amplicon sequencing methods, also
      applied in the first part of My-Milk project, are suitable for the assessment of the
      biodiversity among total bacterial communities and composition of fecal microbiota, as well
      as, the evaluation of the quantitative fluctuations of specific target groups in the complex
      microbial ecosystems such as gastrointestinal tract.

      Data about the nutrition of 6-7-year old participants will be studied in connection with body
      composition (bone density, fat percentage, and muscle tissue), health markers (blood
      pressure, blood cholesterol), and fecal microbiota. After quality control of the sequenced
      reads, the faecal microbiome biodiversity and relative abundance of bacterial taxa will be
      gained and biodiversity measures such as the Chao1 score and the Shannon diversity index will
      be calculated.

      Afterwards, all data will be deposited in the central database together with those already
      obtained during the first part of My-Milk study. The appropriate statistical analysis will be
      applied in investigating the impact of early life exposures on later growth, nutritional
      status, health and faecal microbiota in participants at the current age of 6-7 years. The
      central database will allow (a) protected access to already collected information; (b)
      addition of new attributes and (c) export of data for the purposes of statistical analysis.
      Database will not contain any personal identifications (subjects will be coded by
      identification numbers only). The majority of the collected data from My-Milk study have
      already been converted into electronic form. Statistical methodology will comprise of the two
      steps: the first exploratory step, which consists of the data checking, graphical displays
      and reduction of higher dimensional space into lower dimensional space via multivariate
      statistical methods, such as principal component analysis and multivariate scaling; the
      second step will be devoted to statistical inference. The appropriate statistical tests will
      be used, regression models will be derived taking into account the research hypotheses. The
      power of the tests will be calculated and the models will be critically assessed by modern
      simulation techniques (e. g. bootstrap approach).
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        All singleton children and their mothers who have completed a detailed part of the My-Milk
        study 1 (147) will be invited in this prospective follow-up study.
      
Biospecimen description: 
      child's feces
    
Keywords: Nutritional Intake,; Child Growth and Development; Bone Density; Antibiotics