POLYCYSTIC OVARIAN SYNDROME (PCOS) is a common

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1 /03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(4): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /jc The Biological Variation of Testosterone and Sex Hormone-Binding Globulin (SHBG) in Polycystic Ovarian Syndrome: Implications for SHBG as a Surrogate Marker of Insulin Resistance V. JAYAGOPAL, E. S. KILPATRICK, P. E. JENNINGS, D. A. HEPBURN, AND S. L. ATKIN Department of Medicine, University of Hull (V.J., P.E.J., D.A.H., S.L.A.), Department of Clinical Biochemistry and Immunology, Hull Royal Infirmary (E.S.K.), and York District General Hospital (P.E.J.), Hull, United Kingdom HU3 2RW This study was designed to assess the biological variability of total testosterone and SHBG in polycystic ovarian syndrome (PCOS) and to determine the use of SHBG as a surrogate marker of insulin resistance in PCOS. Fasting blood samples were collected at 4-d intervals on 10 consecutive occasions from 12 PCOS patients and 11 age- and weight-matched controls. Duplicate samples were analyzed for SHBG, testosterone, and insulin in a single batch, and insulin resistance was calculated by the homeostasis model assessment method (HOMA-IR). The PCOS group had higher testosterone (mean SD, vs nmol/liter; P 0.001), lower SHBG ( vs nmol/liter; P 0.001), and greater HOMA-IR POLYCYSTIC OVARIAN SYNDROME (PCOS) is a common endocrine disorder characterized by chronic anovulation and hyperandrogenism (1). The association between hyperinsulinemic insulin resistance and PCOS is well recognized (2) and may play a pathogenic role in the development of PCOS. Positive correlations have previously been observed between circulating concentrations of androgens and insulin in PCOS (3, 4). Hyperinsulinemia is known to suppress SHBG synthesis in the liver (5), and treatment with insulin-sensitizing agents, such as metformin and troglitazone, has been shown to cause a reduction in circulating androgen levels and an increase in SHBG concentration (6, 7). A previous study on the diurnal variation in hormones in women with PCOS reported no significant variation in SHBG concentration (8). However, there are no data on the intraindividual variability in testosterone in individuals with PCOS and the relationship between the variation in insulin resistance and the variation of SHBG in PCOS. We have recently shown that the biological variability in insulin resistance in individuals with PCOS is significantly higher than that in women without PCOS (9). Therefore, the aim of the present analysis was to quantify the biological variations in testosterone and SHBG in individuals with PCOS and to determine the implication of this variability when using diagnostic criteria and screening for the disorder. In addition, a comparison of the variation in insulin resistance with that Abbreviations: FAI, Free androgen index; HOMA, homeostasis model assessment method; HOMA-IR, insulin resistance calculated by the homeostasis model assessment method; IoI, index of individuality; PCOS, polycystic ovarian syndrome. ( vs U; P 0.001) than the controls. In contrast to HOMA-IR (1.09 vs U; P 0.001), the intraindividual variation in SHBG was lower in the PCOS group (mean, 3.4 vs. 6.3 nmol/liter; P 0.041). The index of individuality for SHBG and testosterone in PCOS was 0.49 and 0.69, respectively. This study shows that for patients with PCOS, SHBG is an integrated marker of insulin resistance that may be of use to identify insulin-resistant individuals for targeted treatment with insulin-sensitizing agents. However, SHBG and testosterone concentrations measured in isolation are inherently unsuitable for use as tests to detect hyperandrogenemia. (J Clin Endocrinol Metab 88: , 2003) in SHBG was undertaken to assess whether SHBG was a surrogate marker of insulin resistance in PCOS. Subjects Subjects and Methods Subjects were recruited initially for a study to assess the biological variation in insulin resistance in individuals with PCOS (9). Twelve overweight Caucasian women diagnosed with PCOS (median age, 28 yr; range, yr) and 11 weight-matched Caucasian women (controls) with regular menses (every d) and without PCOS (median age, 30 yr; range, yr), participated in the study. The diagnosis of PCOS was based on evidence of a history of oligomenorrhea and either hirsutism or acne together with hyperandrogenemia (free androgen index, 8; mean sd: PCOS, ; controls, ). Nonclassical 21-hydroxylase deficiency, hyperprolactinemia, and androgen-secreting tumors were excluded by appropriate tests before the diagnosis of PCOS was made (2). No subject was taking any medication at the start of the study or for the preceding 6 months, and there was no concurrent illness. All subjects were on an unrestricted diet and were instructed not to modify their usual eating patterns during the period of sampling. Fasting plasma glucose, age, body mass index, and current smoking status were determined. One subject with PCOS and 1 healthy subject smoked. They were asked to abstain from smoking overnight, and as the results from these 2 individuals were indistinguishable from those of the other subjects in both groups their data were included in the analysis. The body mass index in the PCOS group (mean sd, ) was nonsignificantly greater (P 0.151) than that in the control group ( ). Fasting venous blood was collected into serum gel tubes (BD Biosciences, Mountain View, CA) and a fluoride oxalate tube at the same time each day ( h) on 10 consecutive occasions at 4-d intervals. Samples were separated by centrifugation at 2000 g for 15 min at 4 C, and aliquots of the serum were stored at 20 C within 1 h of collection. Plasma glucose was analyzed in singleton within 4 h of collection. The serum samples were split before assay. All subjects gave their informed 1528

2 Jayagopal et al. Variation in Testosterone and SHBG in PCOS J Clin Endocrinol Metab, April 2003, 88(4): written consent before entering the study, which was approved by the Hull and East Riding local research ethics committee. Reagents Before analysis, all serum samples were thawed and thoroughly mixed. The duplicate samples (i.e. two per visit) were randomized and then analyzed in a single continuous batch using a single batch of reagents. Serum testosterone was measured on an Architect analyzer (Abbott Laboratories, Inc., Maidenhead, UK), SHBG and insulin were measured on the Immulite 2000 (Euro/DPC, Llanberis, UK) analyzer using the manufacturer s recommended protocol. The interassay coefficients of variation for total testosterone, SHBG, and insulin as determined from duplicate study samples were 10%, 8%, and 8%, respectively. The Architect analyzer uses chemiluminescent microparticle immunoassay technology that is based on a patented acridinium (nsulfonyl) carboxamide chemiluminescent assay. Results using this assay have been shown to demonstrate good agreement with a reference isotope dilution gas chromatography-mass spectrometry method (10). The analytical sensitivity of the insulin assay was 2 U/ml, and there was no stated cross-reactivity with proinsulin. Plasma glucose was measured using a Synchron LX 20 analyzer (Beckman-Coulter, High Wycombe, UK), using the manufacturer s recommended protocol. The coefficient of variation for this assay was 1.2% at a mean glucose value of 5.3 mmol/liter during the study period. Statistical analysis Statistical analysis was performed using SPSS for Windows NT, version 9.0 (SPSS, Inc., Chicago, IL). The insulin resistance was calculated using the homeostasis model assessment (HOMA) method (HOMA- IR (insulin glucose)/22.5) (11). Biovariability data were analyzed by calculating analytical, within-subject, and between-subject variances (sd A 2, sd I 2, and sd G 2, respectively) according to the methods described by Fraser and co-workers (12, 13). By this technique, analytical variance (sd A 2 ) was calculated from the difference between duplicate results for each specimen: sd A 2 d 2 /2N, where d is the difference between duplicates, and N is the number of paired results. The variance in the first set of duplicate results for each subject on the 10 assessment d was used to calculate the average biological intraindividual variance (sd I 2 ) by subtraction of sd A 2 from the observed dispersion (equal to sd I 2 sd A 2 ). Subtracting sd I 2 sd A 2 from the overall variance in the set of first results determined the interindividual variance (sd G 2 ). The intraindividual (sd i ) and interindividual (sd g ) variations were estimated as square roots of the respective variance component estimates. The reference change value or critical difference between two consecutive samples in an individual subject (i.e. the smallest percent change unlikely to be due to biological variability) was calculated using the formula 2.77(CV I ), where CV I is the within-subject biological coefficient of variation (12). The index of individuality (IoI) was derived from the ratio of intra- and interindividual variation (sd i /sd g ) (12, 14). When the IoI for a particular test is 0.6 or less, conventional population-based reference intervals are of limited value in the detection of unusual results for a particular individual. When the IoI is 1.4 or more, the variation in an individual will fit populations reference limits more closely, thus being suitable as a screening test. Results The baseline clinical and biochemical details of the subjects are shown in Table 1. The distribution of serum total testosterone and SHBG was Gaussian (by Kolmogorov-Smirnov) in both the PCOS group and controls. The distribution of HOMA-IR was found to be log Gaussian in the PCOS group and Gaussian in the control population. Figure 1 shows the mean and range of HOMA-IR and SHBG for the individuals in the two groups. The serum total testosterone was higher and SHBG was lower in the group with PCOS compared with the control group [mean sd, vs nmol/liter (P 0.001) and vs nmol/liter (P 0.001), TABLE 1. Baseline clinical and biochemical features of subjects Parameter PCOS (n 12) Controls (n 11) Age (yr) NS BMI (kg/m 2 ) NS Fasting glucose NS (mmol/liter) Fasting insulin ( U/ml) Testosterone (nmol/liter) SHBG (nmol/liter) Data is presented as mean SD. NS, Not significant. respectively]. The HOMA-IR in the PCOS group was significantly greater than that in the controls (mean sd, vs U; P 0.001). An inverse relationship was demonstrated between SHBG concentration and both serum insulin (r 0.24; P 0.001) and HOMA-IR (r 0.21; P 0.001). For serum total testosterone, in the control group the analytical variance contributed 1.5% of the total test variance, interindividual variance contributed 83.4%, and intraindividual variance contributed 15.1%. The IoI was 0.43, and the critical difference was 1.4 nmol/liter, i.e. 43% of the mean value in health. In the group with PCOS the analytical variance contributed 5.2% of the total test variance, interindividual variance contributed 64.1%, and intraindividual variance contributed 30.7%. The IoI was 0.69, and the critical difference was 1.3 nmol/liter, i.e. 33% of the mean value. For SHBG in the control group, the analytical variance contributed 1.0% of the total test variance, interindividual variance contributed 94.7%, and intraindividual variance contributed 4.3%. The IoI was 0.21, and the critical difference was 17.3 nmol/liter, i.e. 30% of the mean value in health. In the group with PCOS the analytical variance contributed 1.3% of the total test variance, interindividual variance contributed 79.3%, and intraindividual variance contributed 19.4%. The IoI was 0.49, and the critical difference was 9.5 nmol/liter, i.e. 37% of the mean value. Variation in SHBG and total testosterone expectedly resulted in variation in the calculated free androgen index [FAI (total testosterone/shbg) 100]. Figure 2 shows the mean and range of HOMA-IR and SHBG for the individuals in the two groups. An FAI of 8 or more was used as an indicator of hyperandroginemia in women, and all of the PCOS women included in the study had a FAI of 8 or more at baseline. In the PCOS group, 92.4% of the FAI values remained above the diagnostic threshold, and 7.6% of the values were below the diagnostic threshold of 8 during the period of sampling. In the control women, 67.9% of the values were below and 32.1% were above the diagnostic threshold of 8 during the period of sampling. The intraindividual variance in SHBG rose linearly with increasing SHBG concentrations (r 0.88; P ), and after accounting for analytical variation, the mean intraindividual variation of SHBG was substantially lower in the group with PCOS than in the control group (mean, 3.4 vs. 6.3 nmol/liter; P 0.041). In contrast, the mean intraindividual variations in serum insulin (mean, 15.7 vs. 2.3 U/ml; P ) and HOMA-IR (mean, 1.09 vs U; P 0.001) were significantly higher in the PCOS group compared with those in the controls. P

3 1530 J Clin Endocrinol Metab, April 2003, 88(4): Jayagopal et al. Variation in Testosterone and SHBG in PCOS FIG. 1. Means and range of values (unadjusted for analytical variation) for insulin resistance and SHBG in women with PCOS and controls. No significant difference was observed in the mean intraindividual variation for testosterone (mean, 0.46 vs nmol/liter; P 0.692) between the PCOS group and controls. Discussion The biological variation of total testosterone, SHBG concentration, and insulin resistance in women with PCOS demonstrated in this study shows that SHBG may be suitable for use as a surrogate marker of insulin resistance in this group. A low SHBG concentration is associated with adverse cardiovascular risk factors (15) and is considered an independent risk marker for the development of type 2 diabetes in women (16, 17). Correlations between low SHBG concentrations and hyperinsulinemia have previously been shown in

4 Jayagopal et al. Variation in Testosterone and SHBG in PCOS J Clin Endocrinol Metab, April 2003, 88(4): FIG. 2. Means and range of values (unadjusted for analytical variation) for total testosterone and FAI in women with PCOS and controls. both health and hyperinsulinemic disease states such as PCOS and type 2 diabetes (18 20). This strong association has prompted suggestions that a low level of SHBG could be used as a marker to identify individuals with insulin resistance (21, 22). In accord with previous studies we found an inverse relationship between hyperinsulinemia and SHBG concentration (20, 22, 23). In addition, it was found that although HOMA-IR was significantly higher and more variable in the group with PCOS, the converse was true for SHBG; the group with PCOS not only had lower mean SHBG concentrations, but also demonstrated significantly less intraindividual variation than the healthy controls. This indicates that in PCOS a low SHBG concentration is an integrated marker of insulin resistance and may reflect insulin resistance more consistently than HOMA-IR itself. However, the low index of individuality of SHBG caused by the relatively

5 1532 J Clin Endocrinol Metab, April 2003, 88(4): Jayagopal et al. Variation in Testosterone and SHBG in PCOS higher inter- to intraindividual variation seen in PCOS suggests that it is unsuitable as a screening marker for insulin resistance alone, but, rather, that it is a marker for insulin resistance only after the diagnosis of PCOS. Once the diagnosis of PCOS is made, then a low SHBG concentration may be useful to identify those individuals with PCOS who are insulin resistant for targeted treatment with insulin-sensitizing agents. Having established this potential therapeutic utility for SHBG measurement, the next step would be to determine the diagnostic index for low SHBG concentration and insulin resistance in PCOS, which is outside the scope of this study and will require data from a larger series. An inverse correlation between SHBG concentration and body weight has previously been described (24), but this was accounted for here, as both groups were matched for body mass index. The inherent limitation of a single serum total testosterone measurement as a tool to establish the presence of hyperandrogenemia and hence PCOS has been previously highlighted (25, 26) and is in part due to the significant interindividual variability even among women with other clinical and ultrasound features of PCOS. The intraindividual variability in serum testosterone in individuals with PCOS has not been previously studied and is presented here. As expected, the mean testosterone level was significantly higher in the group with PCOS than in controls. However, the intraindividual variation in total testosterone was not only similar in both groups, but was also less than the interindividual variation observed in both groups. This, therefore, means that the variation observed in serum testosterone in both health and PCOS is mostly due to differences between individuals rather than within each individual. The low index of individuality confirms that when used in isolation, total serum testosterone is unsuitable as a screening test to detect the presence of hyperandrogenemia. Total testosterone is not alone in being fundamentally limited as a potential screening tool because of a low IoI. Serum creatinine, for example, has an IoI of only 0.27, which contrasts with a newer marker of renal function, cystatin C, that has an IoI of 1.64 (27). This means the latter test is likely to be the more useful to screen subjects for reduced glomerular filtration rate using a population-based (rather than the subject s own) reference interval. The clearance and bioavailability of testosterone are affected by the concentration of SHBG (28), and variations in SHBG levels would therefore be expected to influence the variability in serum testosterone. This was indeed reflected in this study, where the total biological variation (sd i sd g ) in both testosterone and SHBG were found to be less in the group with PCOS than in the controls (i.e. when SHBG variability was less, the variability of testosterone was also reduced). There remains considerable debate about the accuracy (bias) of different total testosterone methods of measurements, especially when using automated platform analyzers such as the one used in this study. It is therefore reassuring that other studies have shown this method to compare well to reference techniques (10). Even if this were not the case, the findings of this study should not have been unduly affected, since both cases and controls used the same testosterone method. In conclusion, this study shows that in PCOS a low SHBG concentration reflects an elevation in IR and may be a useful marker to identify those individuals with PCOS who are insulin resistant and may therefore benefit from treatment with insulin-sensitizing agents. However, total serum testosterone and SHBG concentrations when used in isolation are inherently unsuitable for use as tests to detect the presence of hyperandrogenemia in both healthy subjects and women with PCOS. Acknowledgments Received April 8, Accepted January 7, Address all correspondence and requests for reprints to: Dr. V. Jayagopal, University of Hull, Michael White Centre for Diabetes and Endocrinology, Hull Royal Infirmary, Anlaby Road, Hull, United Kingdom HU3 2RW. v.jayagopal@hull.ac.uk. References 1. Franks S 1995 Polycystic ovary syndrome. N Engl J Med 333: Dunaif A 1997 Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. 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