Supplementary Material for: Similarities and differences in diurnal salivary adrenal hormones in monozygotic twins with discordant birthweight

Introduction DHEAS is increased in formerly small-for-gestational-age singletons and in smaller twins compared to their normal-weight co-twin. Less is known concerning other adrenal hormones. We compared diurnal salivary profiles in monozygotic twins with intra-twin birthweight(bw)-differences to analyse the long-term impact of bw and persistent intra-twin auxological differences on various adrenal hormones. Methods 46 monozygotic pre-pubertal twin-pairs with bw-difference of <1SDS (concordant; n=29, 13 female) and ≥1SDS (discordant; n=17, 6 female) were recruited. At a mean age of 6.9 years (yrs) saliva samples were collected (7 am/waking, 1 pm, 6 pm, and 9 pm) and analysed with liquid chromatography-tandem mass spectrometry. Results Diurnal salivary concentrations showed significant intra-twin correlations in all twin-pairs for nearly all time-points: progesterone and androstenedione (4/4 time-points), 17-hydroxyprogesterone (2/4). However, in the discordant group mean progesterone differed significantly (p=0.018) between the smaller (3.27nmol/L, SD 3.87) and the larger twin (0.72nmol/L, SD 0.42) at 7 am and intra-twin differences were observed at 3/4 time-points. Regression analyses of intra-twin differences confirmed that actual hormonal parameters were explained mainly by the co-twin parameter. However, at 6 pm progesterone concentration of the smaller twin was explained by bw (Β -1.102; SE 0.563; p=0.05) and actual weight (Β 0.799; SE 0.327; p=0.019). Conclusion The significant correlations within monozygotic twin-pairs in all measured hormones at nearly all time-points suggest a strong genetic influence on the adrenal axis. Among the discordant twin pairs, we found significant variations in progesterone, suggesting additional long-lasting influence from intrauterine conditions on adrenal hormones with possible metabolic consequences. Word count 249