Subjects and sampling

The present study was an observational study performed in the area of Antwerp (Belgium) between June 2009 and July 2010. The protocol was approved by the ethics committee of the hospital network of Antwerp (ZNA), Institutional Review Board-ZNA/OCMW Antwerpen. The trial is registered in the ISRCTN register: ISRCTN66704989.

One hundred and forty-three healthy pregnant women were recruited via gynecologists. Subjects were eligible for participation when they showed a normal course of pregnancy and a good physical and mental health status as determined by the medical history and general clinical examination according to the investigators’ judgment. Exclusion criteria were 1) Birth in water; 2) Participation in another clinical trial; 3) Alcohol consumption (>7 units/week); 4) Illegal drug use. Written informed consent was obtained before inclusion in the study. Subjects that fulfilled one or more of the following criteria during the study, were excluded from further participation: 1) premature delivery (before 37 weeks of pregnancy); 2) bacterial/viral infection within 2 weeks prior to delivery; 3) congenital malformation(s) in the subjects’ infant; 4) use of immune-modulatory drugs by subject and/or subjects’ infant prior to (4 weeks) delivery and during the study; 5) use of antibiotics between 2 weeks prior to delivery and 2 weeks after delivery, for any reason except for prophylactic use (e.g. cesarean section).

Upon enrolment (about 1–2 months before the expected delivery date) information with respect to the subjects’ demographics, relevant medical history, medication and nutritional supplement use were documented. Subjects were issued with questionnaires to register information on the diet, possible allergic/gastrointestinal symptoms, and medication to be completed at several time points during the study (before delivery, immediately after delivery, at 1/2/3/4 weeks after delivery, and at 2/3/4/5/6 months after delivery).

After delivery fecal samples of the neonates were collected at several time points (first defecation, 2 days after first defecation, 1 week after birth, 1/3/6 months after birth, 1 week after weaning started).

Participants were given a pre-weighed 15 ml fecal collection tube (Sarstedt AG & Co., Numbrecht, Germany) containing 2 ml of RNAlater (Thermo Fisher Scientific Inc., Waltham, MA), and an empty fecal collection tube. They placed a spoonful of fecal sample (approximately 0.5 g) into each collection tube immediately after defecation. The samples were kept at 4°C in a cooling box with refrigerants and sent to Yakult Honsha European Research Center for Microbiology ESV (Ghent, Belgium), within 72 h of collection.