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For identification of potentially surgically curable primary aldosteronism, guidelines recommend use of adrenal vein sampling (AVS) that requires selective catheterization of both adrenal veins as verified by using the cortisol-derived selectivity index. Unfortunately, bilaterally selective studies are not obtained under unstimulated conditions in a proportion of the cases ranging between 15% and 50% depending on the cutoff used. We therefore investigated whether 17-α-hydroxyprogesterone and androstenedione, which showed a higher step-up between adrenal vein and inferior vena cava blood than cortisol, can ascertain selectivity when cortisol failed to do so. We prospectively recruited 32 hypertensive patients with confirmed primary aldosteronism, who underwent bilaterally simultaneous sampling without cosyntropin stimulation and with the same predefined AVS protocol. All were consecutively selected because of a cortisol-based selectivity index <2.00 in at least one of the paired adrenal vein blood samples collected as per protocol. Results showed that the values of the selectivity index based on 17-α-hydroxyprogesterone and androstenedione were higher (P<0.01) on average by 1.6- and 12-fold, respectively, than those based on cortisol. With use of these steroids, we rescued 43% and 73% of the AVS, respectively, from being judged nonselective. Thus, in challenging patients with primary aldosteronism submitted to AVS use of 17-α-hydroxyprogesterone, and even more so of androstenedione, for ascertaining selectivity allows demonstration of correct catheter placement in a proportion of AVS studies better than cortisol. Thus, replacing cortisol measurement with these steroids, and particularly androstenedione, can improve the diagnostic yield of AVS.