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

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          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.

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          Most cited references23

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          Delayed diagnosis and a lack of information associated with dissatisfaction in women with polycystic ovary syndrome

          Polycystic ovary syndrome (PCOS) is a complex, chronic, and under-recognized disorder. Diagnosis experience may have lasting effects on well-being and self-management.
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            Hyperandrogenemia Predicts Metabolic Phenotype in Polycystic Ovary Syndrome: The Utility of Serum Androstenedione

            Context: Polycystic ovary syndrome (PCOS) is a triad of anovulation, insulin resistance, and hyperandrogenism. Androgen excess may correlate with metabolic risk and PCOS consensus criteria define androgen excess on the basis of serum T. Here we studied the utility of the androgen precursor serum androstenedione (A) in conjunction with serum T for predicting metabolic dysfunction in PCOS. Patients and Methods: Eighty-six PCOS patients fulfilling Rotterdam diagnostic consensus criteria and 43 age- and body mass index-matched controls underwent measurement of serum androgens by tandem mass spectrometry and an oral glucose tolerance test with homeostatic model assessment of insulin resistance and insulin sensitivity index calculation. We analyzed 24-hour urine androgen excretion by gas chromatography/mass spectrometry. Results: PCOS patients had higher levels of serum androgens and urinary androgen metabolites than controls (all P < .001). Within the PCOS cohort, both serum A and T were positively correlated with the free androgen index (T × 100/SHBG) and total androgen metabolite excretion (all P < .001). All subjects with T above the normal reference range [high T (HT)] also had high A (HA/HT group, n = 56). However, the remaining 30 patients had normal T levels, either in the presence of HA (HA/NT; n = 20) or normal A (NA/NT; n = 10). The groups did not differ in age or BMI. The HA/HT and HA/NT groups had higher total androgen excretion than NA/NT (P < .01 and P < .05, respectively). Multiple linear regression showed a strong negative association between serum androstenedione and insulin sensitivity. The incidence of dysglycemia according to an oral glucose tolerance test increased with the severity of androgen phenotype (NA/NT, 0%; HA/NT, 14%; HA/HT, 25%, P = .03). Conclusion: Simultaneous measurement of serum T and A represents a useful tool for predicting metabolic risk in PCOS women. HA levels are a sensitive indicator of PCOS-related androgen excess.
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              The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome.

              Ovarian theca cells propagated from patients with polycystic ovary syndrome (PCOS) convert steroid precursors into T more efficiently than normal theca cells. To identify the basis for increased T production by PCOS theca cells, we examined type I-V 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isoform expression in long-term cultures of theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR analysis demonstrated that theca cells express type V 17 beta HSD a member of the aldo-keto reductase (AKR) superfamily (17 beta HSDV, AKR1C3), whereas expression of type I, II, and IV 17 beta HSD, which are members of the short-chain dehydrogenase/reductase superfamily, was limited to granulosa cells. Type III 17 beta HSD, the testicular isoform, was not detected in either granulosa or theca cells. Northern and real-time PCR analyses demonstrated that 17 beta HSDV transcripts were not significantly increased in PCOS theca cells compared with normal theca cells. RT-PCR analysis revealed that theca cells also express another AKR, 20 alpha HSD (AKR1C1). Both basal and forskolin-stimulated 20 alpha HSD mRNA levels were increased in PCOS theca cells compared with normal theca cells. However, 17 beta HSD enzyme activity per theca cell was not significantly increased in PCOS, suggesting that neither AKR1C3 nor AKR1C1 contributes to the formation of T in this condition. In contrast, 17 alpha-hydroxylase/C17,20 lyase and 3 beta HSD enzyme activities were elevated in PCOS theca cells, driving increased production of T precursors. These findings indicate that 1) increased T production in PCOS theca cells does not result from dysregulation of "androgenic" 17 beta HSD activity or altered expression of AKRs that may express 17 beta HSD activity; and 2) increased synthesis of T precursors is the primary factor driving enhanced T secretion in PCOS.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: ValidationRole: Writing – original draft
                Role: Data curationRole: Project administration
                Role: Data curation
                Role: InvestigationRole: Resources
                Role: Investigation
                Role: InvestigationRole: Resources
                Role: InvestigationRole: Methodology
                Role: Data curationRole: Investigation
                Role: Data curationRole: Investigation
                Role: Data curationRole: Investigation
                Role: Resources
                Role: Resources
                Role: InvestigationRole: Methodology
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: Project administrationRole: Resources
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 October 2018
                2018
                : 13
                : 10
                : e0203903
                Affiliations
                [1 ] Department of Nephrology and Hypertension and Department of BioMedical Research, Inselspital, University Hospital, University of Bern, Bern, Switzerland
                [2 ] Pediatric Endocrinology and Diabetology, Department of Pediatrics and Department of BioMedical Research, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
                [3 ] Department of Obstetrics and Gynecology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
                [4 ] Lindenhofspital, Bern, Switzerland
                [5 ] Department of Diabetes, Endocrinology, Clinical Nutrition and Metabolism, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
                [6 ] Nephrology Service, Department of Specialties of Internal Medicine, University Hospital of Geneva, Genève, Switzerland
                [7 ] Nephrology Service, University Hospital of Lausanne, Lausanne, Switzerland
                [8 ] Institute of Social and Preventive Medicine, University Hospital of Lausanne, Lausanne, Switzerland
                Universite Clermont Auvergne, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                ¶ Membership of the Swiss Kidney Project on Genes in Hypertension (SKIPOGH) Study Group is provided in the Acknowledgments.

                Author information
                http://orcid.org/0000-0002-4568-5504
                Article
                PONE-D-18-19531
                10.1371/journal.pone.0203903
                6181287
                30308019
                37f7d57c-8ab6-4d3c-b77d-9ea196caf9ed
                © 2018 Dhayat et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 July 2018
                : 29 August 2018
                Page count
                Figures: 3, Tables: 2, Pages: 15
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001711, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
                Award ID: 320030-146127
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001711, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
                Award ID: FN 33CM30-124087
                Award Recipient :
                This study was supported by two free project grants of the Swiss National Science Foundation ( www.snf.ch), namely grant numbers 320030-146127 and FN 33CM30-124087. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and Health Sciences
                Oncology
                Cancers and Neoplasms
                Gynecological Tumors
                Polycystic Ovary Syndrome
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Steroids
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Steroids
                Biology and Life Sciences
                Biochemistry
                Hormones
                Androgens
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Metabolites
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Urine
                Medicine and Health Sciences
                Anatomy
                Body Fluids
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                Biology and Life Sciences
                Physiology
                Body Fluids
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                Medicine and Health Sciences
                Physiology
                Body Fluids
                Urine
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzyme Chemistry
                Enzyme Metabolism
                Biology and Life Sciences
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                Hormones
                Steroid Hormones
                Biology and Life Sciences
                Physiology
                Physiological Processes
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                Medicine and Health Sciences
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                Physiological Processes
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