Urinary steroid profiling in women hints at a diagnostic signature of the polycystic ovary syndrome: A pilot study considering neglected steroid metabolites

Abstract

Background: Although the polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women with vast metabolic consequences, its etiology remains unknown and its diagnosis is still made by exclusion. This study aimed at characterizing a large number of urinary steroid hormone metabolites and enzyme activities in women with and without PCOS in order to test their value for diagnosing PCOS.

Methods: Comparative steroid profiling of 24h urine collections using an established in-house gas-chromatography mass spectrometry method. Data were collected mostly prospectively. Patients were recruited in university hospitals in Switzerland. Participants were 41 women diagnosed with PCOS according to the current criteria of the Androgen Excess and PCOS Society Task Force and 66 healthy controls. Steroid profiles of women with PCOS were compared to healthy controls for absolute metabolite excretion and for substrate to product conversion ratios. The AUC for over 1.5 million combinations of metabolites was calculated in order to maximize the diagnostic accuracy in patients with PCOS. Sensitivity, specificity, PPV, and NPV were indicated for the best combinations containing 2, 3 or 4 steroid metabolites.

Results: The best single discriminating steroid was androstanediol. The best combination to diagnose PCOS contained four of the forty measured metabolites, namely androstanediol, estriol, cortisol and 20βDHcortisone with AUC 0.961 (95% CI 0.926 to 0.995), sensitivity 90.2% (95% CI 76.9 to 97.3), specificity 90.8% (95% CI 81.0 to 96.5), PPV 86.0% (95% CI 72.1 to 94.7), and NPV 93.7% (95% CI 84.5 to 98.2).

Conclusion: PCOS shows a specific 24h urinary steroid profile, if neglected metabolites are included in the analysis and non-conventional data analysis applied. PCOS does not share a profile with hyperandrogenic forms of congenital adrenal hyperplasias due to single steroid enzyme deficiencies. Thus PCOS diagnosis by exclusion may no longer be warranted. Whether these findings also apply to spot urine and serum, remains to be tested as a next step towards routine clinical applicability.

Fig 1. Scheme of alterations in 24-hour urine steroid excretion and steroid enzyme activities in PCOS compared to controls adjusted for age and BMI in multivariable analyses.

Abbreviations: DHEA: dehydroepiandrosterone. An “OH” in enzyme names indicates a hydroxylase. An “OH” in steroid names indicates a hydroxyl group. DH: dehydro; TH: tetrahydro; HSD: hydroxysteroid dehydrogenase; POR: P450 oxidoreductase; Cyt b5: Cytochrome b5; 5α-R: 5α reductase; 5β-R: 5β reductase.

Fig 2. Diagnostic performance of urinary steroid metabolites in the prediction of PCOS.

ROC curves for different classifiers of urinary steroid metabolites are shown on the left side, the corresponding plots of sensitivity-specificity versus the classifier are shown in the middle, and corresponding contingency tables on the right hand side. Dashed lines around the ROC curves indicate the 95% CI of the sensitivity at the given specificity. The AUC and its 95% CI is indicated. The dashed vertical lines in the sensitivity-specificity versus classifier plots indicate the threshold where sensitivity and specificity are simultaneously maximized. The main diagnostic performance parameters corresponding to this threshold are indicated. A-C. Classifier androstanediol. D-F. Classifier: (androstanediol^1.5×20β-DH-cortisone)/(20β-DH-cortisone+[cortisol× log(estriol)]) represents the best combination of 4 steroid metabolites. Abbreviations: 5α3αdiol: androstanediol, F: cortisol, 20βDHE: 20β-DH-cortisone, PPV: positive predictive value, NPV: negative predictive value, log: natural logarithm.

Fig 3. Prospective evaluation of PCOS classifiers.

Black points scattered within the boxplots represent study participants. The ten not-filled point symbols between the boxplots represent urine profiles from suspected PCOS women, which were sent to our lab for excluding hyperandrogenism due to 21-hydroxylase deficiency, while the two black-filled point symbols represent urine profiles from women with genetically confirmed 21-hydroxylase deficiency. The dashed horizontal lines indicate the diagnostic thresholds of PCOS classifiers. A. 21-hydroxylase activity. A higher ratio of 17-OH-pregnanolone/TH-cortisone indicates a lower 21-hydroxylase activity. B. Classifier androstanediol. C. Classifier (androstanediol1.5×20β-DH-cortisone)/(20β-DH-cortisone+[cortisol× log(estriol)]).