Background: Metabolic imprinting through early childhood nutrition seems to play an important role in the aetiology of obesity. Overweight at age two and later in life is associated with excessive weight gain as early as three months of age. Breastfeeding in the first year of life appears to be protective against obesity development.

Objective: of the "Josef Ressel Centre for Early Life Metabolic Programming of Dispositions of Obesity" is to identify maternal and infant predictors of metabolic risk of obesity. The main considerations of modifiable factors are early infant nutrition, 24-hours-drinking-volume, the velocity of infant weight gain, in relation to infant fat mass and fat free mass, to biomarker and to the nutritional status of the mother. A second focus is put on maternal feeding style, infant eating behaviour and the identification of satiety cues.

Multi-Study design: a monocentric prospective longitudinal cohort of 100 healthy, non-obese, non-smoking pregnant mothers and their term, normal birthweight, singleton babies. Mothers and exclusively breastfed versus exclusively formula fed children (at 16 weeks) will be examined at 36 weeks of pregnancy, 4 - 8 - 12 and 16 weeks of life, follow-up at 1 and 2 years of life.

Methods: four weighing protocols between 4th and 16th week of life, feeding diary, anthropometric data measurement of mother and child, child fat mass index by air displacement plethysmograph. Macronutrient and energy content of the breast milk will be analysed by MIRIS™.

Well-defined biomarkers of oxidative stress and inflammation, lipid profile, adipokines, insulin, as well as micro- und macronutrients will be analysed as meaningful indicators regarding the development of obesity and/or the metabolic syndrome in newborns. Samples, such as plasma, urine, saliva, and stool of the mothers and children will be examined with High Performance Liquid Chromatography, High Performance Gas Chromatography, Mass Spectrometry, Enzyme-Linked Immunosorbent Assay (ELISA) and more. Also questionnaires for the evaluation of the maternal milk feeding style are used as well as the Baby and Child Eating and Behaviour Questionnaire at 16 weeks, 1 and 2 years.

A semi-automatic recognition of infants' satiety cues during feeding will be performed. The recording environment includes video cameras and microphones, a pulse oximeter, etc. All signals are synchronously recorded with the aid of the hardware and software infrastructure.

Nutrition related diseases such as obesity are considered as the main health problems in the 21st century. Overweight and obesity are significant risk factors regarding the development of cardiovascular diseases, strokes, diabetes mellitus, and other non-communicable diseases. In the "Josef Ressel Centre for the Investigation of Early Life Metabolic Programming regarding Dispositions of Obesity" possible predictors towards obesity and their relationship to the volume uptake of formula are to be explored multi-dimensionally. During the first part of the project, the volume study, including a total of 66 mother-infant pairs at the 16th week of life, the overfeeding of infants will be investigated. Overfeeding of infants can be regarded in the context of the amount of food consumed, the ingredients, or in the context of satiety perception of babies by their mother. A closer inspection of the drinking volume of breast-fed infants in comparison to formula-fed ones could give an indication of how much and in what frequency infants ingest food and how the drinking volume affects the weight gain and body fat percentage. To do so, applicable anthropometric data will be collected and the measurement of body fat from mothers and infants will be carried out. Additionally, a semi-automatic detection of satiety signals during feeding will be performed. The identification of satiety signs and the reaction of the mother to those signals, in addition with the feeding style, are related to the weight of the infant and its risk of becoming obese later in life. During the second part of the research project, it will be emphasized whether a coherence exists between early infant nutrition and the manifestation of biomarkers of oxidative stress and inflammation. Thus, detailed analyses regarding macro- and micronutrients, adipokines and biomarkers of oxidative stress of mother milk and biological specimen from mothers and infants will be performed Furthermore, the microbiome of the gut of mothers and infants will be investigated. Results from bioanalytical experiments are intended to sup-port the interpretation of results from the volume study to perceive a deep understanding of the metabolic programming phenomenon of infants at the molecular level.

The five main areas of investigation are:

1. 24-hours-drinking-volume: Background: beyond the quality of nutrition during neonatal life, also the quantity in terms of overfeeding plays a critical role in the development of obesity. Objective: to derive evidence on the drinking volume of exclusively breastfed versus exclusively formula fed children, applying strictly the WHO definitions of exclusivity. Secondary objective is the integration of body composition and growth velocity, which may contribute to insights on critical time slots for programming overweight. Methods: weighing protocol, feeding diary, anthropometric data measurement of mother and child, child fat mass index by air displacement plethysmograph PeaPod™ and BodPod™ of the company Cosmed™. The macronutrient and energy content of the breast milk will be also analyzed by MIRIS™.

2. Nutritional Status of Mother and Child:

   Background: evidence suggests that programming may be induced by even mild intrauterine poor nutrition and may confer an increased risk of obesity, without neonates exhibiting birth weights below or above what is considered to be normal. Objectives: both maternal over- and undernutrition, seen as excess energy intake and/or deficiencies of micro- and macronutrients during (late) pregnancy, may increase infant fat mass. Methods: maternal food frequency questionnaire is adapted for pregnancy, an infant food frequency questionnaire is developed for the time span up to two years.

3. Maternal Feeding Style and Child Eating Behaviour:

   Background: a strong maternal control of eating habits is associated with obesity in childhood. Objectives: a prospective investigation of associations between infant eating behaviour and weight gain to gather evidence on the time of establishment and the causal role of infant eating behaviours in weight gain and BMI at two years. Methods: also questionnaires for the evaluation of the maternal milk feeding style are used as well as the Baby and Child Eating and Behaviour Questionnaire at 16 weeks, 1 and at 2 years.

4. Infant Satiety Cues:

   Background: there is an increasing probability that bottle feeding habits, like encouraging the infants to drink the whole content of the bottle, develops overfeeding habits and causes a lower feeling of satiety. Objective: the identification of satiety cues may directly contribute to an objective method to assess satiety. Methods: a semi-automatic recognition of infants' satiety cues during feeding will be performed. The recording environment includes video cameras and microphones, a pulse oximeter, etc. All signals are synchronously recorded with the aid of the hardware and software infrastructure (Noldus-Observer- XT™, Noldus-Media-Recorder™).

5. Biomarker:

Background and Objectives: well-defined biomarkers of oxidative stress and inflammation, lipid profile, ghrelin, leptin, adipokines insulin, as well as micro- und macronutrients will be analysed as meaningful indicators regarding the development of obesity and/or the metabolic syndrome in newborns. Methods: samples, such as plasma, urine, saliva, and stool of the mothers and children will be examined with High Performance Liquid Chromatography, High Performance Gas Chromatography, Mass Spectrometry, Enzyme-Linked Immunosorbent Assay (ELISA) and more.

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Inclusion Criteria:

1. Pregnancy or mother in puerperium
2. Delivery of baby at 37+0 to 41+6 gestational age
3. Legal age (18 years old), age limit 50 years
4. Written consent of test person after having been informed
5. BMI ≥ 18.5 kg/m² to < 30 kg/m²
6. Negative result of oGTT (oral glucose tolerance test) during pregnancy
7. Birth weight of baby 2.5-4.5 kg
8. Non-smoker since knowledge of pregnancy
9. Very good knowledge of German language and Caucasian
10. Exclusive breastfeeding or exclusive formula feeding at MR 2 (4th week of life)

Exclusion Criteria:

1. Birth before 37+0 week of pregnancy (WoP) or after the 41+6 WoP

2. Multiple pregnancy

3. Children with serious congenital malformations of

   • Nervous system
   • Mouth, throat, neck
   • Circulation system
   • Respiratory tract
   • Gastrointestinal tract
   • Urogenital tract
   • Chromosomal aberrations

4. Diseases or hospitalization or intensive medical care of child during neonatal period

5. Hereditary metabolic diseases of child

   • Hereditary disorders of fat metabolism (MCHAD: Medium Chain Acyl-CoA Dehydrogenase Deficiency, LCHAD: Long Chain Acyl-CoA Dehydrogenase Deficiency, VLCHAD: Very Long Chain Acyl-CoA Dehydrogenase Deficiency)
   • Hereditary disorders of amino acid metabolism (PKU: Phenylketonuria)
   • Hereditary disorders of carbohydrate metabolism (Glycogenosis, Galactosemia, Hereditary fructose intolerance, Diabetes mellitus Type 1)

6. Drug (tobacco) abuse

7. Mental illnesses that have to be treated with medicaments

8. Metabolic or autoimmune diseases of mother

9. Complications at birth (blood loss > 1000 ml or eclampsia)

10. Pre-conceptional diabetes (type 1 or 2)

11. Celiac disease and/or wheat protein allergy of mother

12. Breast surgery and/or hypomastia

13. Mixed feeding at MR 2 (4th week of life)

pregnant women and young (breastfeeding) mothers. Babys of both sex

term (37-41 gestational age), singleton healthy babies with normal birth weight (2.5-4.5 kg) from healthy, non-smoking, non-diabetic mothers with a normal pre-pregnancy BMI (18.5-30). Pregnant women, who plan to breastfeed will be selected from a primary care clinic (STGKK Graz) and young non-breastfeeding mothers will be selected at the maternity ward of the university hospital of Graz.