Androgen Responses to ACTH Infusion among Individual Women with Polycystic Ovary Syndrome

Abstract

Objective

To compare androgen responses during ACTH infusion among women with PCOS and normal women.

Design

Cross-sectional study.

Setting

Research center at an academic medical center.

Participants

Women with PCOS (n=13) and normal controls (n=15).

Interventions

Blood samples were obtained frequently during a 6-hour dose-response ACTH infusion.

Main Outcome Measures

Comparison of basal and stimulated levels of 17-OHP, androgens, and cortisol during ACTH infusion with those following hCG injection within individual subjects.

Results

In women with PCOS increased 17-OHP, A4, and DHEA responses during ACTH infusion were comparable to those observed in normal controls. The magnitude of responses was highly variable among PCOS women. Within individual women with PCOS adrenal responses to ACTH and ovarian responses to hCG were significantly correlated. Cortisol responses to ACTH were similar in PCOS and normal controls.

Conclusion

Within individual women with PCOS, enhanced androgen responses to ACTH are accompanied by comparable androgen responsiveness to hCG. These findings suggest that dysregulated steroidogenesis leading to hyperandrogenemia in this disorder is likely present in both adrenal and ovarian tissues.

Introduction

One of the hallmark features of polycystic ovary syndrome (PCOS) is excess androgen production. It has been well established that the primary source of androgen overproduction in women with PCOS is the ovary(1–3) while contribution from the adrenal has varied from 20–50% of cases(4–7). Moreover, adrenal androgen production has not been particularly associated with ovarian androgen excess in this disorder. Recently, it was demonstrated that androgen responses to gonadotropin stimulation were exaggerated in some PCOS women whereas in others androgen responses were similar to that of normal women(8). Of note, androstenedione and DHEA responses to ACTH stimulation did not distinguish between exaggerated and normal responder PCOS women. By comparison, Ehrmann et al. reported that in hyperandrogenic women with exaggerated ovarian androgen responses to gonadotropin stimulation, 57% had functional adrenal hyperandrogenism based on ACTH-dependent 17-ketosteroid excess whereas 43% had normal responses(9, 10). Conversely, in hyperandrogenic women with normal gonadotropin stimulated androgen responses, 59% had hyperresponsiveness to ACTH and 41% exhibited normal responses. These findings underscore variable androgen production by the adrenal much like that reported for ovarian androgen production in women with PCOS. Moreover, that ovarian hyperandrogenemia may arise from an inherent defect of theca cell steroidogenesis incriminates similar dysfunction of adrenal androgen production in this disorder. To examine whether excess androgen production by the ovary is associated with altered androgen production by the adrenal within individuals, we employed a 6-hour dose-response ACTH infusion in PCOS and normal women who had previously undergone hCG stimulation as reported previously(11).

Subjects and Methods

Subjects

There were 13 women with PCOS and 15 women with regular menstrual cycles recruited for the study. All PCOS individuals were oligo- or amenorrheic and demonstrated either biochemical or clinical evidence of hyperandrogenism. All study participants underwent 3D pelvic ultrasound. Patients with PCOS demonstrated evidence of bilaterally enlarged ovaries with more than 12 antral follicles per ovary. Circulating TSH and prolactin levels were normal and not significantly different between the two groups. Congenital adrenal hyperplasia was excluded based on a basal serum 17-OHP of less than 2 ng/ml. No participant received any hormone medication or metformin within two months of study enrollment. The study was approved by the Human Research Protection Program at the University of California, San Diego, and written informed consent was obtained for each individual prior to participation.

Procedures

Subjects were admitted to the Clinical and Translational Research Institute (CTRI) at the University of California, San Diego on the day of hCG stimulation. Normal subjects were studied during the midfollicular phase (cycle days 5–8), while PCOS patients were anovulatory and studied on a random day. The 17-OHP responses to r-hCG in 13 women with PCOS and 14 normal controls in this study have been previously reported(11). Briefly, each subject received intravenous (iv) administration of recombinant hCG (r-hCG), 25 μg. Blood samples were collected prior to and 24 hr following r-hCG injection.

Adrenal stimulation was performed in a subsequent month in the same patients. All study participants were instructed to begin fasting the midnight before the planned study day, and received 1 mg dexamethasone both at 11 pm the night before and at 7 am the morning of the study. On the day of study, an infusion of ACTH was initiated at 8 am with a starting rate of 0.1 μg/hr, and increased at hourly intervals (0.25, 1, 2.5, 10, and 25 μg/hr, respectively) over a 6 hour period. Baseline serum was obtained and subsequent blood sampling was performed every 30 minutes for the duration of the infusion.

For all portions of the study, none of the PCOS subjects experienced recent ovulation as evidenced by absence of recent menstrual bleeding for 2 months before study and serum progesterone (P₄) less than 1.0 ng/ml at in baseline sample.

Assays

Serum concentrations of LH and FSH were measured by radioimmunoassay (RIA) with intra-and inter-assay coefficients of variation (CV) of 5.4% and 8.0%, respectively, for LH and 3.0% and 4.6%, respectively, for FSH (Diagnostic Products Corp., Los Angeles, CA). Serum concentrations of estradiol (E₂), A4, T, and dehydroepiandrosterone (DHEA) were measured by well-established RIA with intra-assay CV less than 7%. Serum levels of 17-OHP, P₄, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEA-S) were measured by RIA with intra-assay CV less than 7% (Diagnostic Systems Laboratories, Inc., Webster, TX). Serum P₄, 17-hydroxyprogesterone (17-OHP), and dehydroepiandrosterone sulfate (DHEAS) were measure by RIA (Diagnostic Systems Laboratories, Inc., Webster, TX) with an intra-assay CV less than 7%. The detection limit for T, A4, DHEA, and 17-OHP were 3.4 pg, 10.4 pg, 50 pg, and 25 pg, respectively.

Statistics

For continuous data, normal distribution was determined visually using normal quantile plots. For cases where normal distribution was still in question, the Shapiro-Wilk test was used with a W<0.05 establishing non-normal distribution. For normally distributed continuous data, a two-sided Student’s t-test was used to establish statistical significance between two groups. For non-normally distributed continuous data, Wilcoxon ranked sums were used to establish statistical significance between two groups. To account for BMI, analysis of covariance was performed.

In order to compare the cumulative steroid response to ACTH infusion, the Riemann Sums method was used to approximate the area under the curve (AUC). Given baseline differences for steroid level amongst control and PCOS participants, we calculated the delta AUC (∆AUC) by subtracting the baseline from all Riemann Sum measurements.

In order to determine if there was an association between previously characterized ovarian theca cell responses to hCG and adrenal responses to ACTH infusion, Pearson correlations and p-values were obtained for comparison between continuous variables.

Results

Clinical features and basal hormone levels in PCOS women and normal controls

There was no difference in mean (±SE) age between PCOS and normal women. There was a trend towards greater BMI among women with PCOS but it did not reach statistical significance (p=0.06). As shown in Table 1, elevated circulating LH, A4, T and 17-OHP levels in PCOS women were significantly higher compared to those observed for normal controls. Levels of serum FSH, DHEA, DHEA-S, E₂ and cortisol were similar between groups. These comparisons did not change after adjusting for BMI.

Table 1.

Mean (± SD) clinical and basal hormone values for PCOS and normal women

Measure	PCOS (n=13)	Control (n=15)	P-value
Age (yrs)	26.1 ± 1.3	27.2 ± 1.3	0.60
BMI	31.3 ± 1.4	26.6 ± 1.7	0.06
LH (mIU/mL)	10.0 ± 1.0	4.1 ± 2.5	<0.001
FSH (mIU/mL)	5.7 ± 0.3	5.2 ±1.8	0.46
17-OHP (ng/mL)	0.9 ± 0.1	0.5 ± 0.2	0.001
A4 (ng/mL)	1.4 ± 0.1	0.9 ± 0.3	<0.001
T (ng/mL)	0.4 ± 0.03	0.19 ± 0.08	<0.001
DHEA (ng/mL)	1.9 ± 0.2	1.8 ± 1.0	0.51
DHEAS (ng/mL)	2933 ± 516	2145 ± 1780	0.30
E2 (pg/mL)	58 ± 4	83 ± 66	0.26
Cortisol (μg/dL)	2.5 ± 0.3	2.8 ± 2.3	0.71

To convert to SI units multiply by the following conversion factor: 17-OHP (3.03); A4 (3.49); T (3.47); DHEA (3.47): DHEA-S (0.0027); E₂ (3.67)

17-OHP and androgen responses to hCG in PCOS women and normal controls

The 17-OHP response to hCG in PCOS women, as measured by percent change above baseline, was significantly greater (p<0.03) compared to that observed in normal women. However, after adjusting for BMI this difference as well as those for A4, DHEA, and T following hCG were not significant between groups.

17-OHP and androgen responses to ACTH in normal controls and women with PCOS

During ACTH infusion, increased serum levels of 17-OHP and DHEA were observed for both PCOS and normal women compared to subtle rises of A4 and T for each group (Figure 1). These incremental changes of 17-OHP, DHEA, A4 and T between groups were not statistically significant.

Figure 1.

Mean (± SE) serum 17-OHP, DHEA, A4, and T levels during 6 hr ACTH step-wise, dose-response infusion in women with PCOS (filled circles) and normal controls (open circles). ACTH doses: 0.1, 0.25, 1.0, 2.5, 10, and 25 μg/hr.

Comparison of steroid hormone responses between ovarian (hCG) and adrenal (ACTH) stimulation

Within individual women with PCOS, the 17-OHP response to ACTH infusion, as measured by the net change of area under the curve (Δ AUC), was significantly correlated with the incremental 17-OHP response to hCG (R²=0.38; P=0.03). A corresponding association was also evident for ACTH-stimulated DHEA (P=0.04) and A4 (P=0.003) responses (Figure 2). Serum T responses during ACTH and after hCG were not correlated in PCOS women. In the normal control group, no correlations were observed between ACTH and hCG stimulated 17-OHP and androgen responses.

Figure 2.

Correlation of hormone responses to ACTH infusion with 24 hr responses to r-hCG for DHEA (upper panels), A4 (middle panels) and 17-OHP (lower panels) in individual control (left) and PCOS (right) subjects. The response to ACTH was determined by area under the curve above baseline during 6 hr infusion. The response to hCG was determined by the net change from baseline.

Discussion

The results of this study have demonstrated that in women with PCOS androgen dose-responsiveness during ACTH infusion was similar to that observed in normal controls. However, androgen responses among PCOS women were highly variable and, notably, correlated significantly with ovarian androgen production following hCG stimulation.

Our findings are consistent with previous published reports that showed in PCOS and normal women minimal and non-significant changes of 17-OHP and adrenal androgens following acute injection of ACTH(8, 12, 13). In contrast, Lachelin et al observed increases of 17-OHP and DHEA following a 2 hour infusion of ACTH in women with PCOS that were significantly greater than those noted for normal controls(13).

Among women with PCOS 17-OHP, A4 and DHEA responses during ACTH infusion were highly variable. In some subjects adrenal androgen responses were similar to those observed in normal women whereas in others, enhanced androgen production was obvious. The differing magnitudes of response suggest that adrenal androgen production is not the same in all women with PCOS and may relate to the inconsistent prevalence of increased serum DHEA-S levels reported in this disorder(4–6, 14). Notably, in our study mean levels of DHEA-S were greater PCOS women compared to normal women although a wide range of values likely precluded statistical significance.

The notion that dysregulated CYP17, 17-hyddroxylase, and 17–20-lyase activities may be present in both the ovary and adrenal of women with PCOS has been suggested by Ehrmann et al. (10). In a prospective study of 40 hyperandrogenic women with hirsutism or irregular menstrual bleeding that underwent both ovarian and adrenal stimulation with GnRH agonist (nafarelin, 100 μg, sq) and ACTH (10 μg/m², iv) 23 individuals exhibited functional ovarian hyperandrogenism (10). Of these, 13 (57%) had abnormal 17-OHP responses to ACTH indicative of functional adrenal hyperandrogenism. These results tended to imply co-existent abnormalities of ovarian and adrenal androgen overproduction in women with PCOS.

However, there have been few efforts to examine corresponding androgen responses to gonadotropin and ACTH stimulation within individual women with PCOS. In the current study, we detected positive correlations between ovarian and adrenal stimulated responses for 17-OHP, DHEA, and A4 in women with PCOS that suggested a common dysregulation of steroid biosynthesis involving CYP17 activities in both these tissues. Consequently, it follows that individual PCOS women with the highest DHEA and A4 responses to hCG stimulation also demonstrated highest responses during ACTH infusion. This commonality of dysregulated CYP17 activities underlying androgen excess of the ovary and adrenal in PCOS has been suggested by others as well(10, 15).

Additionally, other alterations of steroid synthesis may contribute to androgen overproduction in women with PCOS. Studies conducted with human PCOS theca cells demonstrated increased expression of CYP11A and CYP17 mRNA as well as enzyme activities for 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase on a per cell basis that suggested altered steroidogenesis in this disorder may involve multiple steps(16). In hyperandrogenic women 17-hydroxylase and 17, 20-lyase activities were examined by comparing precursor/product ratios before and after ACTH stimulation. There was no association between the estimated enzyme activities and circulating adrenal androgen levels which led the investigators to propose that a generalized alteration of adrenocortical biosynthesis existed in these women(14). While our results may be attributed to an inherent dysregulation of CYP17 activities in both ovarian and adrenal steroidogenic pathways, alterations of other enzyme activities may also exist. Overall, our findings suggest that in women with PCOS an inherent theca cell defect may reflect a general dysregulation of androgen steroidogenesis.

Our study did not assess insulin secretion in women with PCOS which has been shown, in vitro and in vivo, to enhance gonadotropin-stimulated androgen production in hyperandrogenic women with PCOS(17–19). Evidence to show an effect of insulin on adrenal androgen production is limited. It has been reported that insulin augmented ACTH stimulated A4 production in bovine adrenal tissue(20). However, in minced human adrenal tissue insulin failed to show a consistent effect on androgen production(21). Further studies using a human adrenocortical cell line, co-incubation with insulin was not associated with increases of T and DHEAS(22). In contrast, there is clinical evidence in humans to suggest that hyperinsulinemia or hyperglycemia may decrease adrenal androgen levels(23, 24). Our subjects with PCOS had higher BMI than that of normal controls that would suggest greater hyperinsulinemia in these women. Despite this consideration adrenal androgen responses to ACTH were not different between PCOS and normal women.

In summary, we have shown that within individuals with PCOS enhanced androgen responses to hCG are accompanied by comparable androgen responsiveness to ACTH. These findings suggest that dysregulated steroidogenesis leading to hyperandrogenemia is likely present in both the adrenal and ovary.

Capsule.

Among individual women with polycystic ovary syndrome androgen responses to ACTH infusion are variable and positively correlated with those following hCG injection.