Cortisol assessment

Salivary cortisol concentration were analysed at the Centre for Child Research, Stockholm South General Hospital, Stockholm, Sweden, using the standardised CORT-CT2 radioimmunoassay kit (Cisbio Bioassays, Codolet, France) according to the manufacturer’s instructions. The detection interval ranged from concentrations of 1–100 nmol/L. Inter-assay and intra-assay variations were below 5%. Each sample was analysed twice and the mean value of these were used for analysis²². Salivary cortisol level was log-transformed in all analyses. As the microbiota analysis was performed in evening saliva samples, we only included morning salivary cortisol as anexposure in our regression analyses, as we wanted to reduce the risk of potential bias from a matrix effect. The saliva matrix refers to the overall saliva composition and all other components of a saliva sample than the analytes of interest, i.e water concentration and pH level, or presence of food or drug residues²⁴. Analysing both cortisol and microbiota from the same saliva samples may thereby introduce spurious associations due to sample confounding.