European Journal of (2010) 162 611–615 ISSN 0804-4643

CLINICAL STUDY Defining hyperandrogenism in polycystic syndrome: measurement of and androstenedione by liquid chromatography–tandem mass spectrometry and analysis by receiver operator characteristic plots Julian H Barth1, Helen P Field1, Ephia Yasmin2 and Adam H Balen2 Departments of 1Clinical Biochemistry and 2Reproductive Medicine, Leeds General Infirmary, Leeds LS1 3EX, UK (Correspondence should be addressed to J H Barth; Email: [email protected])

Abstract Objective: Hyperandrogenism is one of the diagnostic criteria for the polycystic ovary syndrome (PCOS) despite no agreed definition of hyperandrogenism. In part, this is due to the quality of testosterone immu- noassays. We have developed liquid chromatography–tandem mass spectrometry methods for analysing testosterone and androstenedione (Ad) to study their reference ranges and diagnostic utility in PCOS. Design, setting and subjects: A consecutive series of 122 women attending a reproductive medicine clinic. Methods: Blood samples were taken during the early follicular phase for measurement of LH, FSH, oestradiol, Ad, testosterone and sex hormone-binding globulin (SHBG). Retrospective case note analysis was used to determine the clinical features and ultrasound findings. Results: The incidence of PCOS was 13.9%. The reference interval for testosterone was !1.8 nmol/l and for Ad was 1.4–7.4 nmol/l. There were significant differences in total testosterone (PZ0.001), Ad (P!0.05) and free index (FAI; P!0.0001) between the women with and without PCOS. Diagnostic performance using receiver operator characteristic plots showed area under the curve (AUC) for FAI 0.81, testosterone 0.75 and Ad 0.66. The AUC for the LH:FSH ratio was 0.72. Conclusions: Our analysis of a consecutive series of women attending a reproductive clinic has provided an appropriate series on which to construct reference ranges for key in women. Secondly, it has allowed us to conclude that early follicular serum testosterone measured using tandem mass spectrometry, FAI and the LH:FSH ratio are valuable laboratory tests in the diagnosis of PCOS.

European Journal of Endocrinology 162 611–615

Introduction performance and there was a hope that multiple assays would give more information. However, the develop- All guidelines and consensus statements on the ment of liquid chromatography–tandem mass spec- diagnosis of polycystic ovary syndrome (PCOS) use the trometry (LC–MS/MS) and its introduction into clinical terms clinical and biochemical hyperandrogenism in practice should obviate this need (3). It is for this reason their definitions. Moreover, the terms hyperandrogen- that we recently proposed that a single testosterone ism and hyperandrogenaemia are both used to describe measurement taken in the early follicular phase should increased concentrations of serum androgens. This be used for the diagnosis of PCOS (4). We now have used definition is unclear and creates a degree of uncertainty this assay technology to define the reference intervals of as shown by the majority of reported studies of women testosterone and Ad in women without PCOS and with PCOS, which have typically measured multiple assessed its diagnostic accuracy in women with PCOS. androgens such as total and free testosterone, andros- tenedione (Ad), dehydroepiandrosterone (DHAS), sex hormone-binding globulin (SHBG) and the free andro- gen index (FAI). This has occurred despite the concern Methods of the analytical accuracy of total testosterone measure- Subjects ment (1) and the variability of different formulae for assessing free testosterone (2). Moreover, there has been One hundred and twenty-two consecutive women little clarity over the definition of the limits of hormone attending the Reproductive Medicine Unit at the Leeds concentrations in ‘normal’ women. General Infirmary were identified from consecutive The dependence on multiple androgen measurements blood samples received in the endocrinology laboratory. may stem back to the days when RIA showed poor The blood samples were not taken during the clinic visit

q 2010 European Society of Endocrinology DOI: 10.1530/EJE-09-0741 Online version via www.eje-online.org

Downloaded from Bioscientifica.com at 09/25/2021 06:11:21AM via free access 612 J H Barth and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 162 but at a later time to coincide with days 1–5 of their was 9.34% at 0.79 nmol/l, 5.72% at 2.76 nmol/l and next menstrual cycle. The diagnoses of the patients were 4.4% at 5.88 nmol/l (6Z10). made retrospectively by examination of the case records Ad was measured in 100 ml serum by LC–MS/MS after (E Y). The following features were noted: menstrual extraction with 1 ml MTBE. Calibrators were prepared history; presence of ; hirsuties; body mass index by weighing out Ad (stated purity R98%, Sigma) on a (BMI); and early follicular phase hormones. A diagnosis six-place balance and dissolving in Analar grade ethanol of polycystic was based on the presence of 12 or (Hayman Ltd, Witham, UK) to give a stock solution of more follicles measuring 2–9 mm and/or ovarian 5 mmol/l. This stock solution was diluted in HPLC grade volume measuring O10 cm3 (Table 1). water (Fisher Scientific) to give a series of solutions that The ultrasound examinations were performed trans- were added to aqueous methanol, 1:1 solution by vaginally in the early follicular phase of the menstrual volume, to make seven calibrator solutions ranging cycle in menstruating women or after a withdrawal from 0.5 to 50 nmol/l. Calibrators were stored at 4 8C bleed in oligomenorrhoeic and amenorrhoeic women. before use. The response of the calibrators was linear Follicles were counted and the ovarian volumes were between 0.5 and 50 nmol/l. Serum samples, calibrators measured using the formula of the prolate ellipsoid or quality control samples (Lyphochek Immunoassay (0.5!length!width!thickness). In non-ellipsoid plus control levels 1, 2 and 3, Bio-Rad Laboratories) ovaries, the ovary was outlined manually in the scan were pipetted into washed disposable glass tubes and automated calculation applied (5). (12!75 mm). Internal standard (50 ml of 3 nmol/l The diagnosis of PCOS was based on the Rotterdam heptadeuterated Ad in HPLC grade water (d7Ad, ( Joint European Society for Human Reproduction supplied by C/D/N Isotopes Inc., Point-Claire, Quebec, and Endocrinology (ESHRE)/American Society for Canada)) was added to each tube, and the contents were Reproductive Medicine (ASRM)) consensus (6) criteria. mixed for 30 s. The Rotterdam criteria require two of the following Ad was extracted into 1 ml MTBE by vortex mixing three features for the diagnosis of PCOS: oligomenor- for 5 min. After the aqueous layer was frozen in a dry rhoea or amenorrhoea; clinical or biochemical evidence ice–methanol bath, the organic layer was decanted into of hyperandrogenism; polycystic ovaries by ultrasound a washed disposable glass tube (10!75 mm). Solvent examination as well as exclusion of other causes. was removed using a vacuum oven at 35–40 8C; then, However, in order to be able to develop hormonal data the dried residue was reconstituted in 100 ml aqueous on which to diagnose PCOS, the testosterone data were methanol (1:1 solution by volume). After 5 min at room not used to define subjects. temperature, the extracts were vortex mixed and then transferred to plastic 1.5 ml microcentrifuge tubes Hormone assays (Sarstedt, Leicester, UK). The autosampler plates were sealed as for the testosterone assay. The equipment, Testosterone was measured in 50 mlserumby HPLC reagents and LC column used for the Ad assay LC–MS/MS as previously described (3), after extraction were as described for the testosterone assay (3). Thirty with 1 ml HPLC grade methyl tert-butyl ether (MTBE, microlitres of the extract were injected into the column, supplied by Fisher Scientific, Loughborough, UK). The held at 40 8C. The initial mobile phase conditions were a transition 291O111 was used for dideuterated 50:50% mixture of HPLC grade water (solvent A) and testosterone. Recovery of testosterone was assessed HPLC grade methanol (solvent B), each containing using external quality assessment (UKNEQAS) samples ammonium acetate (2 mmol/l, SigmaUltra, Sigma) and and ranged from 101 to 105%. Inter-assay precision formic acid (1 ml/l, BDH Aristar grade, VDR,

Table 1 Patient characteristics.

Non-PCOS (nZ107) PCOS (nZ17)

Mean (S.D.) Median (IQR) Mean (S.D.) Median (IQR) P

Age (years) 33.2 (5.43) 33.0 (29.0–37.0) 31.35 (4.85) 31 (28.3–35.5) 0.168 BMI 24.26 (3.97) 24.00 (21.00–26.00) 27.04 (5.74) 26.60 (21.83–30.58) 0.081 Testosterone (nmol/l) 0.66 (0.40) 0.56 (0.40–0.80) 1.01 (0.51) 0.90 (0.62–1.17) 0.001 Androstenedione 3.56 (1.56) 3.25 (2.55–4.52) 4.32 (1.59) 3.80 (3.37–5.16) 0.034 (nmol/l) SHBG (nmol/l) 59.2 (26.5) 52.2 (41.6–73.2) 50.3 (29.2) 39.2 (29.5–66.2) 0.083 FAI 1.22 (0.83) 1.01 (0.66–1.56) 2.39 (1.23) 2.16 (1.46–3.12) !0.0001 Oestradiol (pmol/l) 188 (104) 163 (126–219) 222 (79) 218 (161–248) 0.042 LH (IU/l) 5.39 (4.89) 4.70 (3.6–6.0) 6.38 (3.7) 5.9 (4.03–8.10) 0.120 FSH (IU/l) 7.51 (6.87) 6.40 (5.2–7.8) 5.24 (1.63) 5.30 (4.27–5.93) 0.005 LH:FSH ratio 0.77 (0.34) 0.69 (0.53–0.98) 1.20 (0.66) 1.02 (0.81–1.54) !0.005

One hundred and twenty-two consecutive patients; 17 defined as having PCOS using the Rotterdam consensus criteria (see Methods section). Significance was tested using Mann–Whitney U test.

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Lutterworth, Leicestershire, UK). The flow rate was 0.6 ml/min. A linear gradient was run to 5% A:95% B over 5 min; then, the column was washed with 95% B at 1 ml/min and then reequilibrated with 50% A:50% B. Column eluate was diverted from the mass spectrometer during the first 2 min of the run and then later during the column washing stage. The retention time for Ad and d7Ad was 4.8 min, and the total run time was 7 min. The MS/MS settings in positive ion mode for Ad were capillary voltage 0.8 kV, cone voltage 25 V and collision energy 20 eV. The multiple reaction monitoring transitions used for Ad and d7Ad were m/z 287O97 and 294O100 respectively. Ion suppression was assessed by post-column infusion and occurred only between 0.8 and 1.2 min. The limit of quantification of Ad was 0.5 nmol/l using the definition of signal:noise ratio R10 and coefficient of variation (CV) %20%. The recovery of Ad ranged from 87 to 111%. Intra-assay imprecision for Ad in patient pools was 6.4% at 1.1 nmol/l, 4.3% at 2.3 nmol/l, 4.5% at Figure 1 ROC plot of testosterone, androstenedione, SHBG and 5.2 nmol/l and 5.7% at 10.3 nmol/l. Inter-assay FAI in 122 consecutive women with (17) and without (105) PCOS. imprecision for Ad in quality control material was 8.8% at 2.9 nmol/l, 7.3% at 8.7 nmol/l and 5.4% at Analysis by receiver operator characteristic (ROC) 21.5 nmol/l. Extracts were stable for 10 days at 4 8C. was used to determine the diagnostic performance of the Gonadotrophins and oestradiol (OE2) were measured androgens. The AUC for each androgen was: FAI 0.81, by chemiluminescence on an Advia Centaur (Siemens testosterone 0.75, Ad 0.66 and SHBG 0.63. There was a Medical Solutions, Camberley, UK) with typical significant difference between the AUC for FAI and between-batch CV: OE2 18% at 52 pmol/l, FSH 7.0% SHBG (P!0.005), but not between the other variables. at 6.8 IU/l, LH 6.0% at 2.5 IU/l. SHBG was measured by The AUC for LH:FSH ratio was 0.72 and LH 0.62 chemiluminescence on Siemens Immulite 2000 with (LH:FSH ratio O LH, P!0.01). CV 7.4% at 78 nmol/l. FAI was calculated using the formula: (testosterone/SHBG)!100. Discussion Statistical analysis There have been several attempts over the past years to define PCOS. Each of these definitions uses the term Statistical tests were performed using the Analyse-it hyperandrogenism and hyperandrogenaemia, yet no version 2.08 add-in package for Microsoft Excel (www. definition is given for these terms despite the enormous analyse-it.com). subjective and physiological variations (6–8).Wehave previously proposed that a single blood sample for serum testosterone should be taken in the morning during the early follicular phase as the diagnostic Results criterion for hyperandrogenaemia (4). In this study, we have evaluated the diagnostic utility of testosterone, Ad The incidence of PCOS in our consecutive cohort was and gonadotrophins in such early follicular samples 13.9% (17/122). The patient characteristics and using state of the art technology to determine the best endocrinology are tabulated in Table 1. There is a laboratory strategy to diagnose PCOS. significant difference in total testosterone and Ad The introduction of LC–MS/MS into routine practice concentrations and the FAI and LH:FSH ratio between has permitted precise and accurate measurements the women with and without PCOS (Fig. 1). of serum androgens for the first time. In particular, The distribution of all androgens was non-Gaussian it means that testosterone can be accurately measured using the Anderson–Darling A2 test. Data were log- in the range !5 nmol/l that is typically seen in women. transformed before calculations were performed. The Furthermore, it has been used to show that the reference intervals for women without PCOS were: inaccuracy of testosterone immunoassay is in part testosterone !1.8 nmol/l, Ad 1.4–7.4 nmol/l, SHBG duetocross-reactivitywithDHEAS(9, 10).This 22–129 nmol/l and FAI 0.30–3.36. There were trivial represents a significant proportion of the testosterone differences when the ten women with BMI O30 were measured with immunoassays and has made it excluded. necessary to define the upper reference value found in

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Table 2 Published studies of the incidence of polycystic ovary syndrome (PCOS).

Location Recruitment Number PCOS (%)

Leeds, UK Consecutive attenders at reproductive medicine clinic 122 13.9 US (21) Consecutive attenders at employment medical 400 (608 total cohort) 6.6 Lesbos (22) Open unspecified invitation 192 6.8 Madrid (23) Consecutive blood donors 154 6.5 Oxford (15) Open unspecified invitation 230 8a Mexican–Americans (11) Unselected consecutive subjects in MACAD study 156 13 (FH Ischaemic heart ) Madrid (24) Consecutive clinic attendees 113 28

All studies cited in this table (except this study) have used the NIH criteria for diagnosing PCOS rather than the Rotterdam (2003) criteria. aWhen these data were reanalysed using the Rotterdam criteria, the incidence of PCOS rose to 26% (see Discussion section). normal women. The situation for Ad is further beset by ratio has poor within-person reproducibility in women a lack of international standard preparations to with or without proven PCOS; secondly, Escobar- compare methods. The development of Ad methods by Morreale et al. (18) using consecutive series of women LC–MS/MS increases the chance of harmonisation showed that LH and FSH had practically no diagnostic between different laboratories. The use of LC–MS/MS utility, although samples were taken at random times to measure serum can then be used to within the menstrual cycle. These two studies contrast standardise a key diagnostic criterion on which to with our findings where LH/FSH is significantly elevated define the term ‘hyperandrogenism’ in the definition of in samples taken during the early follicular phase. This PCOS and make clinical decisions. may be due to different endocrine pathologies in slim We have chosen to study a consecutive series of and obese women with PCOS. In slim women, episodic women attending a Reproductive Medicine Unit rather pituitary hypersecretion of LH is the cause of hyperan- than a series of randomly recruited women. We believe drogenism, whereas in the obese women, elevated LH is that this removes recruitment bias, as the subjects dependent upon hyperinsulinaemia. This would be represent a group of women presenting with the supported by the studies of obese women with PCOS appropriate gynaecological and dermatological symp- who do have raised LH:FSH ratios (19, 20). toms that might lead to the consideration of PCOS as a ROC curves are used to assess sensitivity and diagnosis. This group would therefore appear to be ideal specificity across the dynamic range of an analytes for testing the diagnostic performance of testosterone concentration. However, these two variables are quite and Ad measured by LC–MS/MS. The case mix of dependent upon the case mix. Therefore, it is necessary consecutive patients attending a reproductive medicine to have a patient series that reflects the population clinic might have been expected to show a rather high under study and not to pick patients and controls in proportion of subjects with PCOS, but our population artificial proportions. ROC curves have been used to had an incidence of only 13.9%. This is greater than evaluate diagnostic tests in PCOS, but the only study we unselected women in previous studies but similar to the have identified that has used an appropriate series of Goodarzi et al. (11) study and significantly lower than a consecutive patients is that of Escobar-Morreale et al. study of obese women (see Table 2). Goodarzi et al. (17). They measured testosterone and SHBG using the studied women with a family history of ischaemic heart Immulite methods, and identified SHBG and FAI as the disease, which might be considered to have a high best assays with AUC 0.875 and 0.867. Their proportion of women with PCOS in view of the population had a similar BMI range to our population. relationship with ischaemic heart disease (12). Our analysis of a consecutive series of women However, it should be noted that all the studies cited attending a reproductive clinic has provided an in Table 2 including Goodarzi et al. used the National appropriate series on which to construct a reference Institute of Health (NIH) criteria for diagnosing PCOS range for androgens in women. Secondly, it has allowed (7), and studies comparing the Rotterdam and NIH us to conclude that early follicular serum testosterone criteria suggest that the former criteria diagnose PCOS measured using tandem MS, FAI and LH:FSH ratio are in about 1.5 times the number diagnosed with NIH valuable laboratory tests in the diagnosis of PCOS. criteria (13, 14). In the study of young women in Oxford, UK (15), the incidence of PCOS increased from 8 Declaration of interest to 26% when the Rotterdam criteria were used instead of the NIH (16). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. There is much debate in the literature regarding the diagnostic utility of LH and the LH:FSH ratio in PCOS. The Rotterdam consensus criteria proposed that LH had Funding no role in the diagnosis (6). This is supported by two This research did not receive any specific grant from any funding studies: firstly, Cho et al. (17) found that the LH:FSH agency in the public, commercial or not-for-profit sector.

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Author contribution statement 12 Shroff R, Kerchner A, Maifeld M, Van Beek EJ, Jagasia D & Dokras A. Young obese women with polycystic ovary syndrome J H Barth designed the study, H P Field developed the LC–MS/MS have evidence of early coronary atherosclerosis. Journal of Clinical methods and clinical information was collected by E Yasmin and A H Endocrinology and Metabolism 2007 92 4609–4614. Balen. All authors have reviewed and approved the final report. 13 Broekmans FJ, Knauff EA, Valkenburg O, Laven JS, Eijkemans MJ & Fauser BC. PCOS according to the Rotterdam consensus criteria: change in prevalence among WHO-II and association with metabolic factors. British Journal of Obstetrics and Gynaecology 2006 113 1210–1217. References 14 Hsu MI, Liou TH, Chou SY, Chang CY & Hsu CS. Diagnostic criteria for polycystic ovary syndrome in Taiwanese Chinese women: 1 Rosner W, Auchus RJ, Azziz R, Sluss PM & Raff H. Position comparison between Rotterdam 2003 and NIH 1990. 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