The Role of Human Milk in Development of Breast Fed Child's Intestinal Microbiota

The main goal of the proposed study is to establish the link among mothers' nutrition, human milk LCPUFAs composition and microbiota and their potential influence on child's gut microbiota development. Since the information about the mothers´ diet, about the microbiota and fatty acids composition of human milk, about the faecal microbiota of infants and about the possible mechanisms of eventual transfer of microorganisms from mother to child will be needed to establish these connections. In addition, the influence of vitamine D levels in maternal blood on the bone mineral density in neonates will be determined in the first year of life. The dietary fatty acid composition of plasma lipids in the blood of pregnant women will be assessed.

Mothers and their respective infants will be recruited in the maternity hospitals in three Slovenian regions: Ljubljana, Murska Sobota and Izola. Pregnant women will be invited to participate in the study by informative leaflets during their first visits in the health care centres or gynaecological clinics. All pregnant women who will express interest to participate and fulfilling the participation criteria will be invited to the introductory meeting where they will be informed about the study and will get during 1-hour course, instructions about the writing of 4-day weighed dietary protocol. The meetings will be carried out in the selected regional maternity hospitals. All volunteers willing to participate in the study will get the possibility to get our opinion about their diet during the pregnancy as well as later, during lactation period. The volunteers pregnant women will be also asked to precisely register every probiotic food product or food supplement which they will eat during the last three months of pregnancy and during 4 weeks after delivery.

Informed consent:

The study will be described to pregnant women by principal investigator and/or co-investigators. Following the explanation, the interested pregnant women will be given a written description of the study and the approved informed consent to sign. One copy of the signed document will be provided to the mother and one copy will be retained in the records of the clinical study.

The mothers and respective infants fulfilling the participation criteria for further research will receive written instructions and especially designed health care diary for follow up child's first year health and performance.

The mother will give the second or the third day after the delivery, under the supervision of medical staff, the sample of colostrum. If available, the sample of infant´ meconium will be taken at the same time. Both samples will be subjected to the analyses of microbiota. Samples of mature human milk will be taken under the supervision of medical staff at the first visit of the paediatrician, i.e. 4 weeks after delivery (for determination of fatty acids and microbiota), and again between 12th and 14th week after delivery. Mothers will be asked to bring with also a sample of child's faeces. Cooperation between researchers and respective paediatricians in the health care centres responsible for the infants involved in the study will be also established in order to get insight in the basic data about the development and health of infants.

All infants will be followed up from birth to the age of 12 months according to: a) growth, b) feeding regime, c) development d) bone mineral density and e) health. During their first 12 months of life examinations will take part on six occasions:

• Immediately after birth and during the first three days of life
• At first month (age 28 to 38 days)
• At third month (age 80 to 100 days)
• At sixth month (age 110 to 130 days)
• At ninth month (age 260 to 280 days)
• At twelfth month of age (age 355 to 375 days).

For following growth, development and health standard paediatrics' protocols will be used. Feeding regime will be annotated as:

• exclusively breast feed (BF)
• partially BF (BF + formula)
• only formula. Data will be gathered from maternity department medical charts, from regular paediatric health care visits at the dispensaries and from especially designed health care diary each mother will keep until the end of her child's first year.

The researchers will be responsible for the collecting and keeping records of data about all volunteers involved, of their diaries and of data obtained from paediatricians from health care centres.

Long-chain polyunsaturated fatty acids (LCP), especially docosahexaenoic acid (DHA, C22:6n-3) play an important role for the optimal development and maturation of the nervous system. Adequate supply of LCP, especially of DHA, plays an important role during the period of accelerated growth and differentiation of fetal nervous system during the last trimester of pregnancy and in the first year of life. Recently new Consensus recommendations on dietary fat intake in pregnancy and lactation were issued. We aim to investigate whether dietary intake of Slovenian pregnant and lactating women is in accordance with those new recommendations as well as if their dietary intake is healthy.

Special attention will be given to consumption of LCP and probiotic fermented dairy products. The consumption of probiotic food supplements will be registered as well.

Pregnant women (n=300) from three regions of Slovenia, i.e. Ljubljana with surrounding area (n=100), Izola with surrounding area (n=100) and Murska Sobota with surrounding area (n=100), will be included in the study.

Diet will be examined by the use of 4-day weighed dietary protocol (4DP) which will be done twice:

1. during the pregnancy: in the last third of pregnancy (between 27th and 28th week of pregnancy)
2. during the breast-feeding: 4 weeks after delivery

Objectives:

• to investigate weather nutrition of pregnant and lactating Slovenian women is in accordance with the Central European dietary guidelines
• to investigate whether nutrition of pregnant and lactating Slovenian is in accordance with the recent consensus recommendations on dietary fat intake during pregnancy and lactation
• to evaluate the frequencies of consumption of probiotic products (data will be important for screening the breast milk for the presence of commercially important probiotics)

During the study mothers will continue with their ordinary diet. For all participating women, a lecture and one-hour course with practical examples and demonstrations by the use of a balance and examples of foods, about how to write a 4-day weighed dietary protocol (4DP) will be organised. After the evaluation of diaries, the mothers will also have a possibility to ask for an expert opinion about their diet during the pregnancy as well as during lactation.

At the University Medical Centre of Ljubljana, Division of Paediatrics, the collected data will be evaluated by the help of computer program Prodi 5.2 Expert plus (Stuttgart, Germany). Energy will be expressed as a daily intake in kcal and MJ. Nutrient intakes will be expressed in (micro) grams/day, while carbohydrate, protein and fat intakes will also be expressed as a percentage of contribution to the daily energy intake. The intake of sea fish and the fatty acids, especially of DHA as well as the intake of probiotic products will be also precisely evaluated.

The nutrition of pregnant and breast-feeding mothers will be compared with the Central European reference intakes, D-A-CH (D-German, A-Austrian; CH-Swiss) Reference Values for Nutrient Intake which are official in Slovenia since 2004. The intake of fats, especially of DHA, will be compared with the recent consensus recommendations on fat intake during pregnancy and lactation. As the D-A-CH Reference Values do not define the maximum amount of daily sodium and sugar intake, we will compare the sodium and sugar intake with the joint WHO/FAO recommendations (population nutrient intake goal for sodium: < 2 g/day and for free sugars: < 10 % of daily energy intake). The proportion of n-3 LCP in plasma lipids of pregnant women will be compared to their diet. The blood obtained from the pregnant women will be analysed for the content of D vitamine and fatty acids composition of plasma lipids. The purpose of these additional test will be

• to establish whether the consumption of marine fish as well as supplements containing fish oil or unicellular organisms with n-3 LCP (EPA and DHA) (capsules, syrups), before and during pregnancy affects the increase of n-3 LCP in plasma lipids, and
• to assess the influence of dietary factors on bone mineral density in women, and the influence of the levels of vitamin D in the blood of mothers to the bone mineral density in neonates.

Among pregnant women included in the study of diet of pregnant and breast-feeding mothers in Slovenia, 40 mothers from each of regions will be randomly selected, after evaluating of inclusion and exclusion criteria, for the investigation of fatty-acid composition and microbial composition of human milk as well as analysis of faecal microbiota of infants.

Objectives:

• the assessment of fatty acids composition of human milk, with emphasis on LCPUFAs
• the investigation of the correlation between dietary DHA intake (4DP) and its content (wt. %) in mature human milk
• the assessment of microbial diversity of human colostrum and milk microbiota, with emphasis on bifidobacteria and lactic acid bacteria - potential probiotics
• the assessment of the prevalence of bacteriocin producing lactic acid bacteria in the natural microbiota of human colostrum and milk
• the assessment of microbial diversity of child's meconium and faeces
• comparison of milk and child's faecal microbiota

Statistic evaluation of final results will be performed by independent statistician from the Institute of Biostatistics and Medical informatics which has been already included in the preparation of the protocols of clinical studies (WP1).