Evaluation of reference ranges of four circulating sex steroids from dried blood spots in women aged 18-40 years

Capillary dried blood spot collection offers an excellent less invasive alternative to serum or plasma collection by venesection. This study aimed to generate reference ranges for four sex steroids in capillary whole blood samples collected from healthy premenopausal women as dried blood spots as a screening method to detect disorders of sex development. Using this method we evaluated variations according to age, body mass index, menstrual cycle, and hormonal contraceptive use. This cross-sectional, community-based study included 120 healthy premenopausal women aged 18-40 years, not pregnant or breastfeeding. Whole blood testosterone (T), androstenedione (A4), 17α hydroxyprogesterone (17OHP), and progesterone (P4) were measured by liquid chromatography-tandem mass spectrometry (LCMS) from dried filter paper blood spots. The median (95 % confidence limits) for T were 0.75 (0.28, 2.20) nmol/L, A4 2.69 (1.02, 6.03) nmol/L, 17OHP 1.62 (0.32, 8.52) nmol/L and P4 0.65 (0.18, 41.0) nmol/L. Age by 5-year interval was associated with a progressive reduction in A4 but not for other steroids. Menstrual variation was evident for 17OHP and P4 with increases in luteal phase with a small mid-cycle peak for A4 but no other consistent changes. All sex steroid concentrations were significantly lower in participants taking a systemic hormonal contraceptive (combined oral contraceptive or etonogestrel implant). None of the sex steroid concentrations varied significantly with body mass index. These data provide reference ranges for whole blood T, A4, 17OHP and P4 measured by LCMS from dried blood spots from healthy young adult premenopausal women. Small but significant variations were evident according to age, hormonal contraceptive use and menstrual cycle phase.