Frank Gonzalez, M.D.,* Lillie Chang, M.D., Theresa Horab, R.N.,* Frank Z. Stanczyk, Ph.D., Kent Crickard, M.D.,* and Rogerio A. Lobo, M.D.

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1 FERTILITY AND STERILITY VOL. 71, NO. 3, MARCH 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Adrenal dynamic responses to physiologic and pharmacologic adrenocorticotropic hormone stimulation before and after ovarian steroid modulation in women with polycystic ovary syndrome Frank Gonzalez, M.D.,* Lillie Chang, M.D., Theresa Horab, R.N.,* Frank Z. Stanczyk, Ph.D., Kent Crickard, M.D.,* and Rogerio A. Lobo, M.D. School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, and University of Southern California School of Medicine, Los Angeles, California Received June 17, 1998; revised and accepted October 8, Supported in part by a Research Competition Award from the University at Buffalo Research Foundation, Buffalo, New York. Presented at the 42nd Annual Meeting of The Society for Gynecologic Investigation, Chicago, Illinois, March 15 18, Reprint requests: Frank Gonzalez, M.D., Department of Gynecology and Obstetrics, Children s Hospital of Buffalo, 219 Bryant Street, Buffalo, New York (FAX: ). * Department of Gynecology and Obstetrics, School of Medicine and Biomedical Sciences, State University of New York at Buffalo. Department of Obstetrics and Gynecology, University of Southern California School of Medicine. Present address: Department of Obstetrics and Gynecology, College of Physicians and Surgeons of Columbia University, New York, New York /99/$20.00 PII S (98) Objective: To test the hypothesis that in women with polycystic ovary syndrome (PCOS), adrenal cytochrome P450c 17 activity is different after physiologic vs. pharmacologic ACTH stimulation and that ovarian activity promotes adrenal hyperactivity that is different after physiologic vs. pharmacologic ACTH stimulation. Design: Prospective controlled pilot study. Setting: Reproductive endocrinology unit of an academic medical center. Patient(s): Six women with PCOS who had adrenal hyperandrogenism were compared with four women with normal ovulation. Intervention(s): Adrenal dynamic blood sampling was performed before and after 6 months of GnRH agonist administration. Main Outcome Measure(s): Comparison of physiologic and pharmacologic ACTH-stimulated levels of progesterone, 17-hydroxyprogesterone, and androgens before and after ovarian steroid modulation. Result(s): In women with PCOS, exaggerated responses of androstenedione and 11 -hydroxyandrostenedione as well as elevated ratios of 17-hydroxyprogesterone to progesterone and of androstenedione to 17- hydroxyprogesterone after physiologic ACTH stimulation did not persist after GnRH-agonist administration. Three of the six women with PCOS had an increased response of androstenedione and a ratio of androstenedione to 17-hydroxyprogesterone that were 2 SD above the mean of those in the women with normal ovulation after pharmacologic ACTH stimulation; this finding persisted after GnRH-agonist administration. Conclusion(s): In women with PCOS, increases in adrenal androgen sensitivity after physiologic ACTH stimulation reflected in both arms of cytochrome P450c 17 activity may be influenced by ovarian activity. However, 17,20-lyase hyperactivity in a subset after pharmacologic ACTH stimulation may be an intrinsic adrenal disorder. (Fertil Steril 1999;71: by American Society for Reproductive Medicine.) Key Words: Polycystic ovary syndrome, adrenal dynamic testing, GnRH agonist, adrenal androgen sensitivity, 17,20-lyase hyperactivity Adrenal androgen excess in women with polycystic ovary syndrome (PCOS) has been attributed to adrenal androgen hyperresponsiveness to ACTH or to an intrinsic adrenal enzyme dysfunction (1). This adrenal androgen hyperresponsiveness has been observed after pharmacologic ACTH administration or physiologic ACTH release in response to corticotropin-releasing hormone administration (2 4). The latter phenomenon is an indicator of increased adrenal androgen sensitivity (1, 5) and also is more reflective of the in vivo circumstance because circulating levels of ACTH are normal in women with PCOS (6). Adrenal enzyme dysfunction characterized by a deficiency is a relatively rare phenomenon and is an entity that should be differentiated from PCOS (7 9). In contrast, enhanced adrenal enzymatic activity has been proposed to occur in association with PCOS. 439

2 Previous investigators (10) have reported a dysregulation of the ovarian cytochrome P450c 17 -hydroxylase 17,20- lyase enzyme complex (cytochrome P450c 17 ) characterized by an increase in the activity of 17 -hydroxylase relative to that of 17,20-lyase. Data supporting this abnormality (10) revealed a markedly increased 17-hydroxyprogesterone (17-OHP) response to ovarian dynamic testing. Dysregulation of the adrenal counterpart of this enzyme complex has been suggested as the cause of excess adrenal androgen secretion in women with PCOS (11). In a study by Azziz et al. (12), increases in the activity of either arm of adrenal cytochrome P450c 17 could not be confirmed in most of the hyperandrogenic women evaluated. In contrast, we have published data supporting the contention that some women with PCOS may have increased adrenal activity of only the 17,20-lyase arm of this enzyme complex (9). Contributing to this controversy is the fact that only pharmacologic doses of ACTH were used to evaluate this enzyme activity. Moreover, the use of physiologic doses of ACTH for this purpose to simulate the normal circulating levels of ACTH observed in vivo in women with PCOS (6) has never been attempted. There are data to suggest that ovarian activity also influences adrenal androgen excess in women with PCOS. Significant reductions in the basal levels of serum DHEAS and the post-acth responses of other androgens have been observed after GnRH agonist (GnRH-a) administration in women with PCOS who have elevated adrenal androgen levels but not in those who have normal adrenal androgen levels (2, 13 15). In addition, we have reported enhancement of adrenal 17,20-lyase activity by the administration of estrogen during GnRH-a of women with PCOS who have adrenal androgen excess (16). Thus, we designed a pilot study to test a two-part hypothesis. First, we proposed that adrenal cytochrome P450c 17 activity in women with PCOS is different when assessed by physiologic compared with pharmacologic ACTH stimulation. Second, we explored the hypothesis that ovarian activity may promote adrenal hyperactivity in women with PCOS that may be different in response to physiologic vs. pharmacologic ACTH stimulation. MATERIALS AND METHODS Subjects Six women with PCOS were selected for the study. They all had elevated basal levels of serum DHEAS as determined with a well-established RIA (17, 18) with a normal range for women of reproductive age between 70 g/dl and 300 g/dl. They all exhibited the classic features of oligomenorrhea and hirsutism and had no evidence of virilization or pelvic masses. The mean ( SEM) number of menses was per year and the mean ( SEM) Ferriman-Gallwey hirsutism score was All the women had withdrawal bleeding after progesterone administration. All the women had normal levels of prolactin, TSH, and 17-OHP. Four women with normal ovulation, two of whom previously had endometriosis diagnosed and one of whom had uterine myomas, were selected as the control group because they had regular menses and no evidence of hirsutism. These women were matched by age and weight with the women with PCOS. The mean ( SEM) age of the women with normal ovulation ( years) was not significantly different than that of the women with PCOS ( years). The mean ( SEM) body mass index of the women with normal ovulation was kg/m 2, whereas that of the women with PCOS was kg/m 2. All four control subjects demonstrated a biphasic BBT curve and a luteal phase progesterone level of 6 ng/ml. None of the subjects in either group had received any hormonal medication for at least 6 weeks before the study. This study was approved by our institutional review board, and written informed consent was obtained from each subject. Protocol All the study subjects received a GnRH-a (Lupron depot 7.5 mg IM; TAP Pharmaceutical Corp., Deerfield, IL) monthly at 8 AM for 6 months. The injections in subjects with PCOS were begun without regard to the patient s menstrual period and those in women with normal ovulation were begun on day 3 of the menstrual cycle. Each subject underwent a baseline evaluation before GnRH-a that included determination of hormone levels and assessment of adrenal dynamic responses after ACTH stimulation. In women with PCOS, blood samples were collected at 8 AM every 3 days ( 7, 4, and 1 days) and pooled to represent basal secretion. In women with normal ovulation, blood samples for basal secretion were collected at 8 AM on day 2 of the menstrual cycle. All the subjects received dexamethasone, 1 mg orally at 11 PM on the evening that basal blood drawing was completed and at 7 AM the next morning. The protocol for physiologic ACTH stimulation was as follows: Synthetic ACTH (1-24, Cortrosyn; Organon Pharmaceuticals, West Orange, NJ) was administered at a dose of 200 ng intravenously in two boluses at 9 AM and 11 AM, respectively. This was followed by a more conventional pharmacologic stimulation with 0.25 mg of synthetic ACTH (1-24) intravenously at 1 PM. Blood samples were collected through an indwelling catheter at 15, 0, 15, and 30 minutes after each physiologic ACTH bolus and at 15, 0, 60, and 120 minutes after the pharmacologic ACTH stimulation. At the completion of 6 months of GnRH-a administration, basal secretion and the adrenal dynamic responses to ACTH were reevaluated in a fashion identical to that of the pre protocol. 440 Gonzalez et al. Physiologic vs. pharmacologic ACTH in PCOS Vol. 71, No. 3, March 1999

3 TABLE 1 Mean basal steroid hormone levels of study subjects before and after GnRH agonist. Patient group Polycystic ovary syndrome Normal ovulation Hormone Before After Before After E 2 level (pg/ml) * * Testosterone level (ng/dl) * Androstenedione level (ng/ml) * * DHEAS level ( g/dl) OHA level (ng/ml) Note: All values are means SEM. 11 -OHA 11 -hydroxyandrostenedione. * P.05 (before GnRH agonist vs. after GnRH agonist in either group). P.05 (PCOS group vs. control group). Assays Luteinizing hormone, E 2, progesterone, 17-OHP, testosterone, androstenedione, DHEA, DHEAS, and 11 -hydroxyandrostenedione (11 -OHA) were measured with the use of validated RIAs, by methods described previously (17 20). The intra-assay and interassay coefficients of variation were 6% and 15%, respectively, for all assays. Data Analysis Hormonal measurements obtained from blood drawn after each physiologic ACTH bolus were averaged to represent the postphysiologic ACTH adrenal dynamic response. Adrenal androgen sensitivity was defined as the maximum values of DHEA, androstenedione, and 11 -OHA achieved after physiologic ACTH administration. The activities of 17 -hydroxylase and 17,20-lyase were represented by the mean ratios of the maximum values of 17-OHP to progesterone and of androstenedione to 17-OHP, respectively, after either physiologic or pharmacologic ACTH stimulation. Women with PCOS in whom the postpharmacologic ACTH maximum value of androstenedione and the ratio of the maximum values of androstenedione to 17-OHP were 2 SD above the mean of those in women with normal ovulation were considered to have 17,20-lyase hyperactivity (9). Only 4 steroid pairs and not those of the 5 pathway were used to evaluate enzyme activity based on previous data (16) demonstrating an estrogen effect on 17,20-lyase activity primarily through the 4 pathway. Statistical evaluation was performed with the use of Student s paired and unpaired t-tests where appropriate. Onetailed significance was considered sufficient when paired comparisons were assessed for the basal serum levels of E 2, testosterone, and androstenedione, because only declines in these hormones were anticipated in response to GnRH-a administration, as demonstrated previously (2, 21 23). However, two-tailed significance was used for all other comparisons. Data are expressed as means SEM. RESULTS Basal Hormonal Response The mean ( SEM) baseline LH concentration was significantly (P.01) higher in the women with PCOS ( miu/ml) than in the women with normal ovulation ( miu/ml). Table 1 depicts the mean basal steroid hormone values in both groups before and after GnRH-a. The women with PCOS exhibited significantly (P.05) higher baseline levels of E 2, testosterone, androstenedione, and DHEAS compared with those of the women with normal ovulation. In the women with normal ovulation, the mean levels of serum E 2, androstenedione, and testosterone were significantly (P.05) reduced, after GnRH-a, whereas those of serum DHEAS and 11 -OHA remained unaffected. In the women with PCOS, the mean levels of serum E 2 and androstenedione declined significantly (P.05) after GnRH-a. The mean serum testosterone concentration exhibited a moderate decline in women with PCOS and was not statistically significant. In both study groups, serum E 2 levels after GnRH-a declined to 33 pg/ml. Adrenal Dynamic Response Before GnRH-a Administration The physiologic ACTH responses of DHEA, androstenedione, and 11 -OHA before GnRH-a, representing baseline adrenal androgen sensitivity, are shown in Figure 1. Although there was no difference in the mean response of DHEA between the groups, the mean responses of androstenedione and 11 -OHA were twofold higher in the women with PCOS compared with the controls, which was statistically significant (P.05). The ratios of 17-OHP to progesterone and of androstenedione to 17-OHP in response to physiologic ACTH stimulation at the beginning of the study are depicted in Figure 2. Both these ratios were significantly (P.05) FERTILITY & STERILITY 441

4 FIGURE 1 Baseline postphysiologic ACTH responses (mean SEM) representing adrenal sensitivity for androstenedione (A; left axis) and 11 -hydroxyandrostenedione (11 -OHA; left axis) as well as DHEA (right axis) in women with normal ovulation ( ) and women with polycystic ovary syndrome (PCOS; u). * Statistically significant difference (P.05) between women with normal ovulation and women with PCOS. greater in the women with PCOS compared with the controls. As shown in Figure 3, there was no difference in the ratio of 17-OHP to progesterone between the groups in response to pharmacologic ACTH stimulation at the beginning of the study. However, the ratio of androstenedione to 17-OHP after pharmacologic ACTH stimulation was significantly (P.05) higher in the women with PCOS compared with the women with normal ovulation before GnRH-a administration. This higher pre ratio of androstenedione to FIGURE 2 Baseline postphysiologic ACTH ratios (mean SEM) of 17- hydroxyprogesterone (17-OHP) to progesterone (P) and of androstenedione (A) to 17-OHP in women with normal ovulation ( ) and women with polycystic ovary syndrome (PCOS, u). * Statistically significant difference (P.05) between women with normal ovulation and women with PCOS. 17-OHP in response to pharmacologic ACTH stimulation, reflecting increased 17,20-lyase activity, was attributed to three patients with PCOS (depicted by triangles in Fig. 3) who met the criteria for 17,20-lyase hyperactivity. Adrenal Dynamic Response After GnRH-a administration After 6 months of GnRH-a administration, all responses to physiologic ACTH stimulation were similar in the women with PCOS and the controls. For example, the previously elevated androstenedione and 11 -OHA responses in the women with PCOS ( ng/ml and ng/ml; respectively) were similar to those in the women with normal ovulation ( ng/ml and ng/ml, respectively). The ratios of 17-OHP to progesterone and of androstenedione to 17-OHP also were similar in the two groups after GnRH-a ( and , respectively, in the PCOS group and and , respectively, in the control group). In response to pharmacologic ACTH stimulation after GnRH-a administration, the ratio of 17-OHP to progesterone in the women with PCOS remained unchanged compared with the pre value, and the significantly (P.05) elevated ratio of androstenedione to 17-OHP ( ) persisted in this group compared with that of the control group ( ). All three of the women with PCOS who exhibited 17,20-lyase hyperactivity before GnRH-a continued to meet the criteria for this enzyme abnormality after GnRH-a. DISCUSSION The women with PCOS in our study exhibited baseline hormonal characteristics that concur with previously published findings for this syndrome (2, 9, 24). Declines in the basal serum levels of E 2, androstenedione, and testosterone in the women with PCOS after GnRH-a administration indicate a reduction in ovarian steroidogenesis that is consistent with the results of several previous studies (2, 13, 14, 16, 21, 22). The baseline increase in adrenal androgen sensitivity after the administration of a physiologic dose of ACTH in women with PCOS is similar to our previous findings with the use of corticotropin-releasing hormone administration (4, 16). To our knowledge, the significant baseline elevations in the activities of 17 -hydroxylase and 17,20-lyase after physiologic ACTH stimulation in these patients compared with the controls is a novel finding. In contrast, only the 17,20-lyase arm of cytochrome P450c 17 demonstrated a baseline increase in activity after pharmacologic ACTH administration in the women with PCOS compared with the controls. In this study, the 17,20-lyase hyperactivity was present in a subset (3 of 6) of these patients. This finding concurs with our initial description of this enzyme abnormality in a larger series of women with PCOS who had elevated adrenal an- 442 Gonzalez et al. Physiologic vs. pharmacologic ACTH in PCOS Vol. 71, No. 3, March 1999

5 FIGURE 3 Baseline postpharmacologic ACTH ratios ( ) of 17-hydroxyprogesterone (17-OHP) to progesterone (P) and of androstenedione (A) to 17-OHP as well as baseline maximum absolute values of A in women with normal ovulation and women with polycystic ovary syndrome (PCOS). The horizontal lines and error bars represent the mean SEM of values for each group and the boxes represent 2 SD above and below the mean of values in women with normal ovulation. Three women with PCOS ( ) met the criteria for 17,20-lyase hyperactivity. * Statistically significant difference (P.05) between women with normal ovulation and women with PCOS. drogen levels (9). Because the hallmark of cytochrome P450c 17 dysregulation as described for the ovary by previous investigators (10) is a relative increase in 17 hydroxylase activity compared with that of 17,20-lyase, our findings are at variance with the concept of adrenal cytochrome P450c 17 dysregulation in women with PCOS. The exaggerated adrenal responses to physiologic ACTH stimulation in our subjects with PCOS were observed to normalize after suppression of ovarian steroidogenesis. This was suggested by the lack of statistically significant differences between the groups in the responses of androstenedione and 11 -OHA as well as the ratios of 17-OHP to progesterone and of androstenedione to 17-OHP after physiologic ACTH stimulation at the completion of GnRH-a administration. Although the number of patients in our pilot study is too small to draw a definitive conclusion, the possibility that ovarian function may influence adrenal androgen sensitivity and adrenal cytochrome P450c 17 activity in women with PCOS cannot be excluded. Significant declines in adrenal androgen responses to standard doses of ACTH after GnRH-a have been shown previously in women with PCOS who have adrenal hyperandrogenism (13, 14). These data, coupled with those of our current study and our previous report (16), support the contention that estrogen in particular may be the ovarian steroid responsible for inducing increased adrenal androgen sensitivity and increased cytochrome P450c 17 activity in women with PCOS under physiologic circumstances. We found that the 17,20-lyase hyperactivity uncovered by pharmacologic ACTH stimulation in a subset of women with PCOS was not influenced by a reduction in ovarian steroids. This enzyme abnormality persisted in affected individuals during this form of adrenal dynamic testing at the end of GnRH-a. A recently discovered point mutation in cytochrome P believed to modify the phenotypic expression of PCOS has been shown to upregulate the transcription of cytochrome P (25). On the other hand, excessive serine phosphorylation of cytochrome P to increase cofactor binding has been proposed as a mechanism for promoting 17,20-lyase hyperactivity (26). Thus, 17,20-lyase hyperactivity probably is an intrinsic adrenal abnormality and may be the result of a genetic or regulatory defect that is evident in some, but not all, women with PCOS who have adrenal androgen excess. However, this adrenal enzyme defect does not appear to be modified by estrogen or other ovarian factors. In summary, we identified separate adrenal abnormalities in response to physiologic and pharmacologic ACTH stimulation in women with PCOS who had elevated adrenal androgen levels. Increased adrenal androgen sensitivity and an increase in the activities of both arms of cytochrome P450c 17 occurred after physiologic ACTH stimulation that may have resolved after GnRH-a induced modulation of ovarian steroids. In contrast, the 17,20-lyase hyperactivity that was evident in some patients after pharmacologic ACTH stimulation persisted after long-term GnRH-a. FERTILITY & STERILITY 443

6 In conclusion, we propose that in women with PCOS, physiologic ACTH stimulation may uncover acquired adrenal defects promoted by the ovary in the anovulatory state, whereas pharmacologic ACTH stimulation is necessary to expose a subtle intrinsic adrenal abnormality that appears to be independent of an ovarian influence. Acknowledgments: We thank TAP Pharmaceuticals, Deerfield, Illinois, for donating the Lupron depot used in this study. We also thank Carmen Todaro, M.D., Rosendo Intengan, M.D., Mario Reyes, M.D., Judith Ortman-Nabi, M.D., and Sheri Baczkowski, M.D., for their prompt patient referrals from their respective gynecologic practices in Buffalo, New York, and Miguel Rodriguez-Bigas, M.D., of the State University of New York at Buffalo for the thorough statistical evaluation. References 1. Gonzalez F. Adrenal involvement in polycystic ovary syndrome. Semin Reprod Endocrinol 1997;15: Gonzalez F, Hatala DA, Speroff L. 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Am J Obstet Gynecol 1975;121: Carey AH, Waterworth D, Patel K, White D, Little J, Novelli P, et al. Polycystic ovaries and male pattern baldness are associated with one allele of the steroid metabolism gene CYP17. Hum Mol Genet 1994; 3: Zhang L, Rodriguez H, Ohon S, Miller WL. Serine phosphorylation of human P450 c17 increases 17,20 lyase activity: implications for adrenarche and for the polycystic ovary syndrome. Proc Natl Acad Sci USA 1995;92: Gonzalez et al. Physiologic vs. pharmacologic ACTH in PCOS Vol. 71, No. 3, March 1999