Hyperinsulinemia and circulating dehydroepiandrosterone sulfate in white and Mexican American women with polycystic ovary syndrome

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1 Hyperinsulinemia and circulating dehydroepiandrosterone sulfate in white and Mexican American women with polycystic ovary syndrome Robert P. Kauffman, M.D., a Vicki M. Baker, R.N., a Pamela DiMarino, B.S., a and V. Daniel Castracane, Ph.D. b a Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center School of Medicine, Amarillo, Texas; and b Foundation for Blood Research, Scarborough, Maine Objective: To determine whether Mexican American women with polycystic ovary syndrome (PCOS), a population more insulin resistant than white women with PCOS, demonstrate differences in adrenal androgen production. Design: Retrospective study. Setting: University gynecology clinic and research laboratory. Patient(s): One hundred eleven white women and 50 Mexican American women with PCOS based on the 2003 Rotterdam Consensus Statement. Intervention(s): Blood sampling, oral glucose tolerance testing, and ultrasonography. Main Outcome Measure(s): Serum total T, free T, DHEAS, and calculation of multiple insulin sensitivity indices after an oral glucose challenge. Result(s): Mexican American women with PCOS were significantly more insulin resistant than their white counterparts but had lower circulating levels of DHEAS, a reliable index of adrenal androgen production. Age and body mass index (BMI) were each inversely proportional to serum DHEAS, but no association was found between circulating insulin and serum DHEAS levels. Testosterone levels were similar between groups. Conclusion(s): The lower levels of DHEAS observed in the more insulin resistant Mexican American group with PCOS (compared to a similar group of white women living in the same locale) further corroborates the extent of phenotypic variability among specific PCOS populations. Hyperinsulinemia does not appear to significantly influence circulating adrenal androgen levels in PCOS. (Fertil Steril 2006;85: by American Society for Reproductive Medicine.) Key Words: Polycystic ovary syndrome, hyperandrogenism, DHEAS, insulin resistance, testosterone, adrenal gland, Mexican American women, ethnic differences Hyperandrogenism and ovulatory dysfunction are two of the cardinal findings in women with polycystic ovary syndrome (PCOS) (1). Between 20% 70% of women with PCOS demonstrate excess serum levels of DHEAS, a useful marker for adrenal androgen production in clinical practice given that the zona reticularis of the adrenal cortex is responsible for more than 95% of its production (2 4). Dehydroepiandrosterone and its sulfate ester DHEAS are the most abundant circulating steroid hormones in humans (5). Although they lack significant androgenicity, they function as precursors to the more potent androgen testosterone (T) (3, 5). Androstenedione (A) also tends to correlate with serum DHEAS levels (6). Unlike cortisol and aldosterone, DHEA and DHEAS concentrations slowly decline beginning in the third decade, and hence, only 20% 25% of women with PCOS Received June 1, 2005; revised and accepted September 24, Presented at the 53rd Annual Clinical Meeting of the American College of Obstetricians and Gynecologists, May 9, 2005, San Francisco, California. Reprint requests: Robert P. Kauffman, M.D., Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center School of Medicine, 1400 Coulter Drive, Amarillo, Texas (FAX: ; robert.kauffman@ttuhsc.edu). may have truly elevated DHEAS levels if age-adjusted normal values are considered (4, 6 8). The mechanisms regulating DHEA and DHEAS production remain obscure. In addition to aging, other factors are known or suspected to affect adrenal androgen production including obesity, low-density lipoprotein (LDL) production, ethnicity, gender, ovarian androgen production, menopausal status, insulin, and insulin-like growth factors (IGF) (2, 4, 9 16). Women with PCOS also appear to be hyperresponsive to ACTH, resulting in enhanced DHEA production (17, 18). The ACTH levels are similar among normal women and those with PCOS (19). Insulin has a direct stimulatory effect on ovarian androgen synthesis through stimulation of 17 -hydroxylase activity at the ovarian theca and variably attenuating 17,20-lyase responsiveness in women with PCOS (15, 20). Attempts to associate hyperinsulinemia and excess adrenal DHEAS production in normal ovulatory women have been inconsistent, demonstrating either no effect or a decline in adrenal androgen synthesis with direct insulin infusion or oral glucose loading-induced hyperinsulinemia (20 24). However, in women with PCOS, some studies have demonstrated an 1010 Fertility and Sterility Vol. 85, No. 4, April /06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 increase in abnormal androgen production proportional to circulating insulin levels (20, 23, 25, 26), while other investigators have not (27 29). Administration of exogenous insulin appears to induce adrenocortical androgen production in women with PCOS through stimulation of 17 -hydroxylase activity (30). A relationship between circulating insulin levels and hyperandrogenism in women with PCOS is further suggested by the ability of insulin sensitizing agents including metformin, troglitazone, rosiglitazone, and pioglitazone to reduce ovarian and adrenal androgen production (31 33). Nonetheless, diminution of adrenal androgen production in response to insulin sensitizing agents has not been universally reported (34, 35). We have previously demonstrated that Mexican American women with PCOS are more insulin resistant than a comparable group of white women (36). Based on available data, we hypothesized that Mexican American women with PCOS would demonstrate higher levels of circulating adrenal and ovarian androgens than a matched group of white women living in the same locale. In the present study, we investigated the relationship between hyperinsulinemia and serum ovarian and adrenal androgens in these two populations. MATERIALS AND METHODS Patient Selection All cases of PCOS seen at the reproductive medicine clinic at our institution between 1999 and 2004 were reviewed retrospectively. Polycystic ovary syndrome was defined according to the American Society for Reproductive Medicine (ASRM) and European Society of Human Reproduction and Embryology (ESRHE) consensus conference held in Rotterdam in 2003 (1). All women in the database had presented with one of the following complaints: [1] oligomenorrhea ( 6 cycles per year) or dysfunctional uterine bleeding, [2] hirsutism or other symptoms of hyperandrogenism, [3] infertility, or [4] a combination of these complaints. Hirsutism was defined when the modified Ferriman-Gallwey score was 6. Only patients identifying themselves as white or Mexican American between the ages of 14 and 44 years were selected from the database. Women identifying themselves as multiethnic or Hispanic, not of Mexican American origin, were excluded from further study. Patients identified with comorbid endocrinopathies such as 21-hydroxylase deficiency, untreated hypothyroidism, ovarian or adrenal neoplasms, Cushing s disease, diabetes mellitus, or impaired glucose tolerance were also eliminated from further consideration. Similarly, those using hormonal contraception, glucocorticoids, or insulin sensitizers within the preceding 3 months were also eliminated. A total of 161 women with PCOS (111 white and 50 Mexican American) were included in the final analysis. This study received approval by the Institutional Review Committee of Texas Tech University. Laboratory Evaluation Each study patient underwent serum measurements of total T, free T, DHEAS, 17-hydroxyprogesterone (17-OHP), TSH, PRL, and a 3-hour 100-g oral glucose tolerance test (OGTT) with glucose and insulin levels drawn at 0, 1, 2, and 3 hours. Testing was performed in the fasting state in the follicular phase. Further testing for other causes of hyperandrogenism (e.g., 24-hour urinary free cortisol, ACTH stimulation test, ultrasonography of the ovaries or adrenal glands) was performed as clinically indicated. Patient height and weight was recorded to calculate the body mass index (BMI) in kilograms per meter squared. Most (78%) patients underwent transvaginal ultrasonography (at the discretion of the treating physician.) In accordance with the ASRM/ESHRE criteria, ultrasonographic appearance of polycystic ovaries alone was insufficient to make the diagnosis of PCOS (1). The DHEAS, 17-OHP, TSH, PRL, and insulin samples were assayed using a chemiluminescent system (Immulite Diagnostic Products, Los Angeles, CA). Total T was assayed using a radioimmunoassay (Diagnostic Systems Laboratories, Webster, TX), and free T was measured using an analogue hormone RIA (Diagnostic Products Corporation) Glucose was assayed by the catalyzed glucose oxidase technique (Ortho Clinical Diagnostics, Rochester, NY). Normal Laboratory Values Normal values for free and total T, DHEAS, and 17-OHP were previously determined from a multiple year analysis of normal weight, ovulatory women without clinical evidence of hyperandrogenism. The DHEAS was considered elevated at serum levels 250 g/dl. The upper limit of normal for total T and free T in our laboratory is 0.6 ng/ml and 2.5 pg/ml, respectively. 17-hydroxyprogesterone at levels 2 ng/ml or post-acth stimulated values of 8 ng/ml were considered elevated. Mathematical Calculations Fasting glucose (in milligrams per deciliter) and insulin levels (in microunits per milliliter) from each subject were used to calculate the homeostatic model of insulin resistance (HOMA-IR) and quantitative insulin sensitivity check (QUICKI), both of which have demonstrated reasonable correlation against the hyperinsulinemic euglycemic clamp technique (37). The following equations were used to calculate these values: Fasting glucose Fasting insulin HOMA QUICKI log Fasting glucose log Fasting insulin Area under the curve for insulin (AUC insulin ) using the fasting insulin plus 1-, 2-, and 3-hour insulin levels (postin- Fertility and Sterility 1011

3 gestion of 100 g of a commercial glucola solution) was calculated for each study subject using the trapezoidal rule. Statistical Analysis Assessments for Gaussian distributions were performed using the D Agostino-Pearson test. Data obtained from the two ethnic groups were compared using either the Student s t-test for parametric data or the Mann-Whitney test for nonparametric data. Univariate linear regression was used to investigate the relationships between serum DHEAS and each index of insulin sensitivity individually. Serum DHEAS levels from each ethnic group were also compared against patient age and BMI, individually. Spearman s correlation coefficients (r s ) from the regression analyses were then compared to determine whether the relationships for each independent variable were similar between ethnic groups. An level 0.05 was considered statistically significant. Med- Calc 8.0 statistical software (Mariakerke, Belgium) was used for the statistical analysis. RESULTS The two ethnic groups were similar in age, BMI, fasting glucose, total T, free T, and 17-OHP values (Table 1). Each index of insulin resistance calculated in this study confirmed our previous finding that Mexican American women with PCOS were considerably more insulin resistant than their white counterparts (36). The median DHEAS level in white women with PCOS was higher than that in a comparable Mexican American group (median for white women with PCOS g/dl [95% confidence interval {CI} ] and for Mexican Americans g/dl [95% CI ]). The difference was statistically significant at P.009. When circulating DHEAS levels were assessed separately for each ethnic group using fasting insulin, HOMA-IR, QUICKI, AUC insulin, age, and BMI as the independent variables (Table 2), DHEAS levels were inversely proportional to age (r s 0.27, P.006 for white women and r s 0.38, P.01 for Mexican Americans) and BMI (r s 0.20, P.04 for white women and r s 0.34, P.02 for Mexican Americans). These relationships are demonstrated graphically in Figure 1. In contrast, no relationship between circulating DHEAS levels and any of the insulin sensitivity indices measured was observed in either ethnic group with PCOS (Table 2). When Spearman s r values derived from each study population were compared, the relationship between circulating DHEAS and each independent variable was similar (Table 2). DISCUSSION Because 95% of DHEAS production is adrenal in origin, it is a particularly suitable marker to assess adrenal androgen steroidogenesis in large populations of reproductive age women. There is relatively little data in the peer-reviewed literature addressing ethnic differences in PCOS. Carmina et al. (38) reported similar serum DHEAS levels in a study of Hispanic American (not necessarily Mexican American), Italian, and Japanese women with PCOS. In contrast to these findings, we detected significantly higher levels of DHEAS in white women with PCOS than in a similar age- and weight-matched group of Mexican American women with a mean difference of approximately 35 g/dl (24%). The Hispanic American group studied by Carmina and associates (38) demonstrated mean DHEAS levels that were twice that of our Mexican American group (approximately 300 g/dl in Carmina s population vs g/dl in our TABLE 1 Summary of clinical results. White (n 111) 95% CI Mexican American (n 50) 95% CI P Age BMI (kg/m 2 ) Fasting glucose (mg/dl) Fasting insulin ( U/mL) a HOMA-IR a QUICKI a AUC insulin ( g/ml min) 16,476 13,780 21,002 30,120 17,799 44, a DHEAS ( g/dl) a Total T (ng/ml) Free T (pg/ml) Note: Parametric data analyzed using the Student s t-test for independent samples and nonparametric data by the Mann-Whitney test. AUC area under the curve. a Statistical significance at P.05. Kauffman. Ethnicity, PCOS, and DHEAS. Fertil Steril Kauffman et al. Ethnicity, PCOS, and DHEAS Vol. 85, No. 4, April 2006

4 TABLE 2 Effect of independent variables on serum DHEAS concentrations. Independent variable (x-axis) Dependent variable (y-axis) Spearman s r (White) P Spearman s r (Mexican American) Within ethnic groups P z-statistic P Between ethnic groups HOMA-IR DHEAS QUICKI DHEAS Fasting insulin DHEAS AUC insulin DHEAS Age DHEAS a a BMI DHEAS a a Note: Spearman s r values calculated by univariate linear regression. AUC area under the curve. a Statistical significance at P.05. Kauffman. Ethnicity, PCOS, and DHEAS. Fertil Steril population of Mexican Americans) despite the fact that the two populations were relatively similar in age and obesity status. A more detailed comparison of weights between these two studies was not possible due to different systems of recording weight (percentage of ideal body weight vs. BMI). An explanation for this difference in serum DHEAS levels is not readily apparent, but variances in inclusion criteria, hormone assay techniques, or genetic differences between the two populations (Hispanic Americans vs. Mexican Americans) may explain these differences. Fasting insulin levels among the two studies were similar. In another comparison of two ethnic PCOS populations (white and African American patients living in Alabama), Kumar et al. (4) found no differences in mean DHEAS or fasting insulin concentrations. The mean BMI and ages of Kumar s study groups were similar to our study populations. For the same reasons noted previously, a direct comparison of mean DHEAS and insulin values between the two studies should be approached with caution. Regulation of adrenal androgen production is not well understood. Adrenal androgen production is regulated by the steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme (CYP11A1), 17 -hydroxylase/17,20 lyase (CYP17), and 3 -hydroxysteroid dehydrogenase (3 HSD). Differential expression of these enzymes or DHEAsulfotransferase activity may explain ethnic differences in circulating DHEAS levels. Intracellular signaling mechanisms or disparities in receptor sensitivities between the two ethnic populations offer another potential explanation for the disparity in circulating DHEAS found in our study populations (39). Increased production of ovarian androgen precursors elevates circulating DHEAS levels possibly by stimulating sulfotransferase activity (40), but this explanation alone is unlikely to explain the findings in this study as serum T levels were similar between ethnic groups. Differences in steroid elimination could also account for differences in adrenal androgen concentrations among different ethnic populations. The role of insulin in adrenal androgen steroidogenesis has not been fully elucidated. Although insulin, acting synergistically with LH, stimulates T production at ovarian theca cells, studies addressing insulin s role in adrenal androgen synthesis have been conflicting. Published studies have demonstrated diminished DHEAS production in the presence of hyperinsulinemia in normal women (20, 23, 24) and in one study of white women with PCOS (4). In hyperandrogenic women with PCOS, most (but not all) studies have shown a stimulatory effect by insulin on adrenal androgen production (20, 26, 41 43). Some investigators have reported that insulin modestly increases 17,20-lyase activity and subsequent DHEAS production in women with PCOS, whereas other researchers have reported the opposite (30, 44). Our study failed to demonstrate an association between DHEAS concentrations and circulating insulin in women with PCOS, which has been reported by other studies as well (4, 29, 45). Some (31, 46, 47) but not all (32, 33, 48) investigators have demonstrated significant decreases in adrenal androgen production with insulin sensitizing agents. Azziz, et al. (31, 49), in the largest of these studies (305 women with PCOS), reported diminished adrenal androgen concentrations with administration of troglitazone commensurate with lower insulin concentrations, but inhibition of 17 -hydroxylase and 3 HSD activity may explain lower adrenal androgens observed after troglitazone treatment. When serum DHEAS levels were analyzed against BMI, age, and insulin sensitivity indices by univariate linear regression in both arms of this study, only BMI and age demonstrated an inverse relationship with circulating DHEAS, similar to findings in other populations (3, 4, 7). However, the negative correlation between DHEAS and BMI has not been a uni- Fertility and Sterility 1013

5 FIGURE 1 Relationships between DHEAS and multiple independent variables studied. Univariate linear regression demonstrates significant inverse relationships between age and DHEAS levels (A and B) and body mass index (BMI) and DHEAS (C and D) in both whites and Mexican Americans with polycystic ovary syndrome (PCOS). No such correlation was observed when the homeostatic QUICKI test (E and F) and AUC insulin (G and H) were analyzed against DHEAS as the dependent variable. r s Spearman s correlation coefficient. Kauffman. Ethnicity, PCOS, and DHEAS. Fertil Steril Kauffman et al. Ethnicity, PCOS, and DHEAS Vol. 85, No. 4, April 2006

6 versal finding in all populations. In a small study (n 27) of African American women with PCOS, no association between DHEAS and BMI was detected (4), whereas in a population of Swedish women with PCOS, Holte and coworkers (50) found a positive association between BMI and DHEAS. Again, these discrepant findings suggest a wide array of phenotypic expression by genes associated with PCOS. Strengths of this study include limiting our Hispanic population specifically to Mexican Americans alone, a group known to be more insulin resistant than white women living in the southwestern United States (36). Greater insulin resistance observed in Mexican American populations probably reflects genetic influences from the indigenous Pima Indian population, the most insulin resistant population in the world (51). Our study also included larger subject numbers than most studies of DHEAS in other ethnic PCOS populations. A wide range of ages and BMIs were used in this study to test our hypothesis during the reproductive lifespan. Our population was significantly large enough to verify the inverse relationships between circulating DHEAS levels vs. age and BMI. Hence, if hyperinsulinemia had an appreciable effect on DHEAS production, it is likely that this relationship would have been detected. Elimination of women with diabetes mellitus or impaired glucose tolerance resulted in a more homologous population of women with PCOS and eliminated hyperglycemia as a confounding variable. Our findings may be difficult to compare with those from other studies of PCOS because of the different inclusion criteria used in studies of women with PCOS. Our application of the ASRM/ESHRE Rotterdam definition may have produced a population that is more heterologous than those using hyperandrogenism and ovulatory dysfunction only. Furthermore, serum measurements of DHEAS may not truly reflect adrenal DHEAS production, although DHEAS concentrations closely correlate with serum A and DHEA (6). The hyperinsulinemic euglycemic clamp technique is the contemporary gold standard in the quantification of insulin sensitivity, but is impractical to use in large populations because it is expensive, labor intensive, and requires multiple venipunctures. The insulin sensitivity indices used in this study have demonstrated good correlation against clamp techniques in some studies (37); however, Ciampelli and colleagues (52) established that AUC insulin measurements derived from an OGTT were superior to homeostatic assessments when compared against clamp techniques. In this study, AUC insulin results correlated well with HOMA-IR and QUICKI. The difference between DHEAS concentrations among the two PCOS populations suggests that ethnicity may need to be considered in addition to age, gender, and BMI when establishing normal values for DHEAS. It should be noted, however, that this study was not designed to assess differences in DHEAS production between healthy white and Mexican American women (without PCOS), the population on which normal circulating DHEAS ranges should be based. From a clinical viewpoint, whether higher insulin levels and lower DHEAS concentrations in the Mexican American population with PCOS might impact protocols for ovulation induction or future health care needs requires further investigation. In conclusion, Mexican American women with PCOS exhibit lower mean serum levels of DHEAS than age- and weight-matched white women, which appears to be largely independent of circulating insulin levels. 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