Cortisol was assessed from saliva by the Salivette sampling device (Sarstedt, Numbrecht, Germany). The cortisol response to social stress was measured based on four cortisol samples. Sample 1 was taken just before the start of the GSST and reflects HPA axis activity when participants filled out a rating scale while sitting quietly. Sample 2 was collected directly after the end of the GSST and reflects HPA axis responses during the start of the GSST. Sample 3 reflects HPA axis activity around the end of the GSST. Sample 4 was collected 40 min after the end of the GSST and is considered to reflect post-stress activity of the HPA axis. Samples were brought to the laboratory of the University Medical Center in Groningen where they were stored at −20°C until analysis. The intra-assay coefficient of variation was 8.2% for concentrations of 1.5 nmol/l, 4.1% for concentrations of 15 nmol/l, and 5.4% for concentrations of 30 nmol/l. The inter-assay coefficients of variation were, respectively, 12.6, 5.6, and 6.0%. The detection border was 0.9 nmol/l. Missing cortisol samples (C1, n = 20, C2, n = 24, C3, n = 27, C4, n = 24) were due to detection failures in the lab (52%) or insufficient saliva in the tubes (48%). Missing values were imputed on the basis of group mean and standard deviation, where group is either ‘Boys’, ‘FC-girls’, or ‘OC-girls’, and the (standardized) mean of participant’s non-missing cortisol samples.
Heart rate (HR)
was recorded during and after the GSST in four blocks for several seconds: speech preparation (H1) (240 s), speech (H2) (360 s), mental arithmetic (H3) (360 s), and post test (H4) (300 s). A three-lead electrocardiogram was registered using 3M/RedDot–Ag/AgCl electrodes (type 2255, 3M Health Care, D-41453 Neuss, Germany), while the participant was sitting and breathing spontaneously. With a BIOPAC Amplifier-System (MP100), the signals were amplified and filtered before digitization at 250 samples/s. Dedicated software (PreCARSPAN), previously used in, e.g., Dietrich et al. (2006
) was used to check signal stationarity, to correct for artifacts, to detect R-peaks, and to calculate the IBI between two heart beats. IBI is inversely related to heart rate by the equation HR = 60000/IBI. HR was defined as the number of beats per minute (bpm). Blocks were considered invalid if they contained artifacts with duration of more than 5 s, if the total artifact duration was more than 10% of the registration, or if the block length was less than 100 s. Missing heart rate recordings (H1, n
= 6, H2, n
= 3, H3, n
= 3, H4, n
= 6) were due to recording failure (42%) or signal-analysis failure (58%). Missing values were imputed in a similar way as described above for cortisol levels.
Cortisol and heart rate response variables were computed to analyze if the GSST induced significant increases in cortisol and heart rate. We determined the peak response (indicated by either C2 or C3 for cortisol and H2 or H3 for heart rate) and subtracted the pretest measure (C1 or H1) from this peak. A difference larger than zero was considered a meaningful increase.
Use of OC was assessed at the day of the experiment, while type and name of the pill were asked as part of a questionnaire that was assessed previously, at school. In total, OCs were used by 93 girls, of whom 66 (70.2%) used a monophasic OC and four girls a tri-phasic Pill (both ethinylestradiol and progestagen based). Ten girls used the ‘Diane’ Pill, which contains only progestagen (cyproteronacetaat) and is mainly prescribed for the beneficial effects on acne vulgaris. Thirteen girls did not know what type of OC they used.
was assessed at the start of the experimental session, by means of the Dutch version of the short Profile of Mood Scale (POMS; Wald and Mellenbergh 1990
). The Depressed Mood scale includes eight items describing current mood (down, helpless, sad, lonely, unhappy, unworthy, melancholic, desperate), which could be rated on a five-point scale (1 = not at all, 2 = a little, 3 = partly, 4 = kind of, 5 = very much). Cronbach’s alpha was 0.87.