Simultaneous Measurement of Thirteen Steroid Hormones in Women with Polycystic Ovary Syndrome and Control Women Using Liquid Chromatography-Tandem Mass Spectrometry
Background The measurement of adrenal and ovarian androgens in women with PCOS has been difficult based on poor specificity and sensitivity of assays in the female range. Methods Women with PCOS (NIH criteria; n = 52) and control subjects with 25–35 day menstrual cycles, no evidence of hyperandrogenism and matched for BMI (n = 42) underwent morning blood sampling. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to simultaneously measure 13 steroids from a single blood sample to measure adrenal and ovarian steroids. Androgen and progesterone results were compared in the same samples using RIA. Results Testosterone, androstenedione, progesterone and 17OH progesterone levels were higher when measured using RIA compared to LC-MS/MS, although the testosterone RIA demonstrated the best agreement with the LC-MS/MS using a Bland-Altman analysis. Results using LC-MS/MS demonstrated that the concentration of androgens and their precursors were higher in women with PCOS than controls [median (2.5, 97.5th %ile); 1607 (638, 3085) vs. 1143 (511, 4784) ng/dL; p = 0.03]. Women with PCOS had higher testosterone [49 (16, 125) vs. 24 (10, 59) ng/dL], androstenedione [203 (98, 476) vs. 106 (69, 223) ng/dL] and 17OH progesterone levels [80 (17, 176) vs. 44 (17, 142) ng/dL] compared to controls (all P<0.02), but no differences in serum concentrations of the adrenal steroids DHEAS, cortisol, corticosterone and their 11 deoxy precursors. Women with PCOS also had an increase in the product:precursor ratio for 3β-hydroxysteroid dehydrogenase [22% (6, 92) vs. 20% (4, 43); p = 0.009]. Conclusion LC-MS/MS was superior to RIA in measuring androstenedione, progesterone and 17OH progesterone levels, while testosterone measurements were better matched in the two assays. Androgen levels were higher in women with PCOS in the absence of a difference in adrenal-predominant steroids. These data support previous findings that the ovary is an important source for the androgen excess in women with PCOS.
References
[1]
Azziz R, Carmina E, Dewailly D, Amanti-Kandarakis E, Escobar-Morreale HF, et al. (2006) Criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess Society guideline. J Clin Endocrinol Metab 91: 4237–4245. doi: 10.1210/jc.2006-0178
[2]
Gilling-Smith C, Story H, Rogers V, Franks S (1997) Evidence for a primary abnormality of thecal cell steroidogenesis in the polycystic ovary syndrome. Clin Endocrinol (Oxf) 47: 93–99. doi: 10.1046/j.1365-2265.1997.2321049.x
[3]
Nelson VL, Legro RS, Strauss III JF, McAllister JM (1999) Augmented androgen roduction is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries. Mol Endocrinol 13: 946–957. doi: 10.1210/mend.13.6.0311
[4]
Kumar A, Woods KS, Bartolucci AA, Azziz R (2005) Prevalence of adrenal androgen excess in patients with the polycystic ovary syndrome (PCOS). Clin Endocrinol (Oxf) 62: 644–649. doi: 10.1111/j.1365-2265.2005.02256.x
[5]
Welt CK, Arason G, Gudmundsson JA, Adams JM, Palsdottir H, et al. (2006) Defining constant versus variable phenotypic features of women with polycystic ovary syndrome using different ethnic groups and populations. J Clin Endocrinol Metab 91: 4361–4368. doi: 10.1210/jc.2006-1191
[6]
Pasquali R, Stener-Victorin E, Yildiz BO, Duleba AJ, Hoeger K, et al. (2011) PCOS Forum: research in polycystic ovary syndrome today and tomorrow. Clin Endocrinol (Oxf) 74: 424–433. doi: 10.1111/j.1365-2265.2010.03956.x
[7]
Rosner W, Auchus RJ, Azziz R, Sluss PM, Raff H (2007) Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab 92: 405–413. doi: 10.1210/jc.2006-1864
[8]
Legro RS, Schlaff WD, Diamond MP, Coutifaris C, Casson PR, et al. (2010) Total testosterone assays in women with polycystic ovary syndrome: precision and correlation with hirsutism. J Clin Endocrinol Metab 95: 5305–5313. doi: 10.1210/jc.2010-1123
[9]
Janse F, Eijkemans MJ, Goverde AJ, Lentjes EG, Hoek A, et al. (2011) Assessment of androgen concentration in women: liquid chromatography-tandem mass spectrometry and extraction RIA show comparable results. Eur J Endocrinol 165: 925–933. doi: 10.1530/eje-11-0482
[10]
Vogeser M, Parhofer KG (2007) Liquid chromatography tandem-mass spectrometry (LC-MS/MS)–technique and applications in endocrinology. Exp Clin Endocrinol Diabetes 115: 559–570. doi: 10.1055/s-2007-981458
[11]
Jaque J, Macdonald H, Brueggmann D, Patel SK, Azen C, et al. (2013) Deficiencies in immunoassay methods used to monitor serum Estradiol levels during aromatase inhibitor treatment in postmenopausal breast cancer patients. SpringerPlus 2: 5. doi: 10.1186/2193-1801-2-5
[12]
Naessen T, Kushnir MM, Chaika A, Nosenko J, Mogilevkina I, et al. (2010) Steroid profiles in ovarian follicular fluid in women with and without polycystic ovary syndrome, analyzed by liquid chromatography-tandem mass spectrometry. Fertil Steril 94: 2228–2233. doi: 10.1016/j.fertnstert.2009.12.081
[13]
Taylor AE, McCourt B, Martin KA, Anderson EJ, Adams JM, et al. (1997) Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab 82: 2248–2256. doi: 10.1210/jcem.82.7.4105
[14]
Zawadzki JK, Dunaif A (1992) Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: Dunaif A, Givens JR, Haseltine FP, Merriam GR, editors. Polycystic Ovary Syndrome. Boston: Blackwell Scientific. 377–384.
[15]
Welt CK, Adams JM, Sluss PM, Hall JE (1999) Inhibin A and inhibin B responses to gonadotropin withdrawal depends on stage of follicle development. J Clin Endocrinol Metab 84: 2163–2169. doi: 10.1210/jcem.84.6.5757
[16]
Isaacs D, Altman DG, Tidmarsh CE, Valman HB, Webster AD (1983) Serum immunoglobulin concentrations in preschool children measured by laser nephelometry: reference ranges for IgG, IgA, IgM. J Clin Pathol 36: 1193–1196. doi: 10.1136/jcp.36.10.1193
[17]
Kasum M, Radakovic B, Simunic V, Oreskovic S (2013) Preovulatory progesterone rise during ovarian stimulation for IVF. Gynecol Endocrinol 29: 744–748. doi: 10.3109/09513590.2013.798280
[18]
Hamdine O, Broekmans FJ, Eijkemans MJ, Lambalk CB, Fauser BC, et al. (2013) Early initiation of gonadotropin-releasing hormone antagonist treatment results in a more stable endocrine milieu during the mid- and late-follicular phases: a randomized controlled trial comparing gonadotropin-releasing hormone antagonist initiation on cycle day 2 or 6. Fertil Steril 100: 867–874. doi: 10.1016/j.fertnstert.2013.05.031
[19]
Barnes RB, Rosenfield RL, Burstein S, Ehrmann DA (1989) Pituitary-ovarian responses to nafarelin testing in the polycystic ovary syndrome. N Engl J Med 320: 559–565. doi: 10.1056/nejm198903023200904
[20]
Rosencrantz MA, Coffler MS, Haggan A, Duke KB, Donohue MC, et al. (2011) Clinical evidence for predominance of delta-5 steroid production in women with polycystic ovary syndrome. J Clin Endocrinol Metab 96: 1106–1113. doi: 10.1210/jc.2010-2200
[21]
Simon JA (2002) Estrogen replacement therapy: effects on the endogenous androgen milieu. Fertil Steril 77 Suppl 4: S77–S82. doi: 10.1016/s0015-0282(02)02986-2
[22]
Kirschner MA, Jacobs JB (1971) Combined ovarian and adrenal vein catheterization to determine the site(s) of androgen overproduction in hirsute women. J Clin Endocrinol Metab 33: 199–209. doi: 10.1210/jcem-33-2-199
[23]
Wajchenberg BL, Achando SS, Okada H, Czeresnia CE, Peixoto S, et al. (1986) Determination of the source(s) of androgen overproduction in hirsutism associated with polycystic ovary syndrome by simultaneous adrenal and ovarian venous catheterization. Comparison with the dexamethasone suppression test. J Clin Endocrinol Metab 63: 1204–1210. doi: 10.1210/jcem-63-5-1204
[24]
Gilling-Smith C, Willis DS, Beard RW, Franks S (1994) Hypersecretion of androstenedione by isolated thecal cells from polycystic ovaries. J Clin Endocrinol Metab 79: 1158–1165. doi: 10.1210/jc.79.4.1158
[25]
Nelson VL, Qin KN, Rosenfield RL, Wood JR, Penning TM, et al. (2001) The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome. J Clin Endocrinol Metab 86: 5925–5933. doi: 10.1210/jcem.86.12.8088
[26]
Vassiliadi DA, Barber TM, Hughes BA, McCarthy MI, Wass JA, et al. (2009) Increased 5 alpha-reductase activity and adrenocortical drive in women with polycystic ovary syndrome. J Clin Endocrinol Metab 94: 3558–3566. doi: 10.1210/jc.2009-0837
[27]
Rodin A, Thakkar H, Taylor N, Clayton R (1994) Hyperandrogenism in polycystic ovary syndrome. Evidence of dysregulation of 11 beta-hydroxysteroid dehydrogenase. N Engl J Med 330: 460–465. doi: 10.1056/nejm199402173300703
[28]
Fanelli F, Belluomo I, Di Lallo VD, Cuomo G, De Iaso R, et al. (2011) Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults. Steroids 76: 244–253. doi: 10.1016/j.steroids.2010.11.005
