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

引言
脱氢表雄酮硫酸盐（DHEAS）在既往为小于胎龄儿（small-for-gestational-age）的单胎新生儿，以及与正常体重同胞双生子相比体型更小的双生子中水平升高。目前针对其他肾上腺激素的相关研究仍较少。本研究针对存在双生子间出生体重（birthweight, bw）差异的同卵双生子（monozygotic twins），比较其唾液激素的昼夜分泌谱，以分析出生体重以及持续存在的双生子间生长学差异对多种肾上腺激素的长期影响。

方法
本研究纳入46对同卵青春前期双生子，根据双生子间出生体重差异分为两组：差异<1标准差评分（standard deviation score, SDS）的一致组（n=29，其中女性13名），以及差异≥1SDS的不一致组（n=17，其中女性6名）。在平均年龄6.9岁时收集唾液样本（采集时间为晨起7时、午后1时、傍晚6时及晚间9时），采用液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry）进行检测分析。

结果
几乎所有时间点的唾液激素昼夜浓度在所有双生子对中均呈现显著的双生子间相关性：孕酮（progesterone）与雄烯二酮（androstenedione）在全部4个时间点均存在相关性，17-羟孕酮（17-hydroxyprogesterone）在2/4个时间点存在相关性。但在不一致组中，晨起7时，体型更小的双生子的孕酮平均浓度（3.27nmol/L，标准差3.87）显著高于体型更大的双生子（0.72nmol/L，标准差0.42；p=0.018），且在3/4个时间点观察到双生子间激素差异。针对双生子间激素差异的回归分析显示，多数实际激素参数主要由同胞双生子的激素水平所解释。不过在傍晚6时，体型更小的双生子的孕酮浓度可由出生体重（β=-1.102；标准误=0.563；p=0.05）及实际体重（β=0.799；标准误=0.327；p=0.019）进行解释。

结论
所有检测激素在几乎所有时间点的同卵双生子对内部均存在显著相关性，提示肾上腺轴受较强的遗传调控影响。在不一致双生子对中，我们发现孕酮水平存在显著差异，这提示宫内环境对肾上腺激素存在额外的长期影响，并可能伴随相关代谢后果。

字数统计：249