1 Department of Obstetrics & Gynecology, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, NY, 2 Department of Obstetrics and Gynecology, State University of New York at Buffalo School of Medicine & Biomedical Sciences, Buffalo, NY and 3 Department of Obstetrics & Gynecology, University of Southern California School of Medicine, Los Angeles, CA, USA
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Abstract |
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Key words: adrenal androgens/IGF-I/insulin/oestrogen/PCOS
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Introduction |
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Materials and methods |
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Protocol and measurements
During the mid-follicular phase of the cycle, between days 5 and 8, at 89 a.m. after an overnight fasting, a blood sample was obtained for measurements of oestradiol, unbound oestradiol, androstenedione, dehydroepiandrosterone sulphate (DHEA-S), insulin, IGF-I, IGF-II, IGFBP-3 and IGFBP-1. The following day, patients and controls underwent an adrenocorticotrophic hormone (ACTH) stimulation test (ACTH 117, 0.25 mg i.v. with blood samples at 0, 60, 120 and 180 min). Dexamethasone (1 mg) was administered orally the night before the ACTH test (at 11 p.m.). After ACTH, blood samples were obtained for 17-OH pregnenolone (17-OHPe), 17-OH progesterone (17-OHP), androstenedione, DHEA and DHEA-S. After ACTH, steroid ratios were calculated as approximate estimates of various enzymatic activities.
Serum hormone concentrations were quantified by well-established methods which were validated previously in our laboratory. All steroids were measured by specific radioimmunoassay (RIA) after extraction using previously described methods (Lobo and Goebelsmann, 1981; Hoffman et al., 1984
). Non-sex hormone-binding globulin (SHBG)-bound oestradiol was measured by adding 500 c.p.m. of tritiated oestradiol to 0.3 ml of serum prior to precipitation with ammonium sulphate. The percentage of tritiated oestradiol in the supernatant was calculated, as previously reported (Lobo et al., 1981
). Insulin and IGFBP-3 were quantified by use of direct RIA kits (Diagnostic Systems Laboratories, Inc., Webster, TX, USA). IGF-I and IGF-II were measured by RIA using material provided by Diagnostic Systems Laboratories. Both assays were performed after an acid ethanol extraction step. IGFBP-1 was measured by an immunoreactive assay using a kit provided by Diagnostic Systems Laboratories. The methods for the assay of IGF-I, IGFBP-3, IGFBP-1 have been previously described (Carmina et al., 1995
). There was no cross reactivity of the IGF-II antiserum, IGF-I, IGF binding proteins (1, 2, 3, 4, 5) insulin or growth hormone. The sensitivity of the IGF-II assay was 12 ng/ml. The interassay coefficient of variation ranged from 4.3 to 7.2%, while the interassay coefficient of variation ranged from 6.3 to 10.4%. In all other assays, intraassay and interassay coefficients of variation did not exceed 6 and 15% respectively.
Statistics
Analysis of the data was performed using Student's t-test for comparisons between the two groups: PCOS and controls. Correlations were analysed by Pearson product moment correlations and stepwise multivariate linear regression analysis with forward selection. P < 0.05 was considered significant. Results are expressed as mean ± SE.
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Results |
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Table II depicts the adrenal androgen responses to ACTH. In PCOS, patients exhibited an exaggerated response (P < 0.01) to all steroids measured.
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Both total and unbound oestradiol correlated significantly with basal DHEA-S but showed a negative correlation with the androstenedione response to ACTH (Table III). Moreover, total and unbound oestradiol correlated negatively with the ratio of
A/
DHEA and positively with the ratio (
DHEA/
17-OHP) (Table IV
), suggesting a relative increase in 17,20 lyase activity and reduction in 3ß-dehydroxysteroid dehydrogenase activity favouring the formation of
5 steroids.
IGF-II did not show any correlation with adrenal androgen secretion.
Thirteen women with PCOS were hyperinsulinaemic (mean insulin 21.3 ± 2 µIU/ml) with values >12 µIU/ml, which was the upper 95% confidence value of the controls. Twelve women with PCOS were normoinsulinaemic (mean insulin 7.1 ± 0.7 µIU/ml, range 3.110 µIU/ml). The two groups were significantly different in terms of BMI (hyperinsulinaemic PCOS: 32.6 ± 2, normoinsulinaemic PCOS: 23.4 ± 1, P < 0.01). As shown in Table V, the hyperinsulinaemic patients with PCOS had significantly (P < 0.05) higher ratios of
A/
17-OHP and
A/
17-OHPe compared to the normoinsulinaemic group.
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Multivariate analysis showed that BMI did not influence the correlations of unbound oestradiol and IGF-I with adrenal androgens. In spite of a strong influence of BMI on serum insulin, BMI did not affect the negative correlation of insulin with 17-OHP, or the positive correlation with the
A/
17-OHP ratio. Correcting for BMI, the correlation of insulin with
17-OHP was r = 0.43 (P < 0.05) and with
A/
17-OHP was r = 0.40 (P < 0.05). Multivariate analyses did not show any influence of insulin on the correlation between oestradiol and unbound oestradiol with adrenal androgens.
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Discussion |
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Novel implications of findings in this study are that these extra-adrenal hormones correlated differently with various adrenal androgens suggesting that these factors may induce AH by different mechanisms. Serum IGF-1 correlated with basal DHEA-S, but not with adrenal androgen responses to ACTH while insulin did not correlate with basal adrenal androgens but showed a positive correlation with the A/
17-OHP ratio and the
A/
17-OHPe ratio after ACTH stimulation. In evaluating steroid ratios in blood after ACTH, only the effect on true enzyme activity was estimated. Nevertheless, these data suggested an important influence of insulin on adrenal
4 A production. These findings were further enhanced by our findings in the subgroup of hyperinsulinaemic patients with PCOS whose responses were different from the normoinsulinaemic group. In this study, single time point measurements of fasting insulin for correlative analyses were used. Clearly, fasting insulin alone is inadequate to characterize the existence of insulin-resistance in these women.
Oestrogen (oestradiol and unbound oestradiol) on the other hand, correlated with both basal DHEA-S and adrenal androgen responses to ACTH (positively with the ratio of DHEA/
17-OHPe and negatively with the ratio of
A/
DHEA). This suggests a positive influence of oestrogen on the
5 pathway, predominantly resulting in an enhancement of DHEA/DHEA-S production.
In this study, BMI was similar in patients and controls, and multivariate analyses were carried out. It was shown that BMI did not influence the correlations observed.
In PCOS, it has been shown that the ovarian theca hypersecretes androstenedione (Gilling-Smith et al., 1994). Also, a genetic defect in serine phosphorylation in PCOS may lead to enhanced 17,20 lyase activity in both the ovary and adrenal (Zhang et al., 1995
). While hypersecretion of adrenal androgen in PCOS is heterogeneous, in ~50% of patients a more isolated increase in either the
4 or
5 pathway may be exhibited (Carmina et al., 1992
; Carmina 1997a
). Accordingly, it might be postulated that the more dominant the influence of one factor (oestradiol versus insulin), the more different may be the pattern of AH (
5 versus
4 steroids). Since in this study it was found that only serum IGF-I correlated positively with DHEA-S, this influence may be artifactual or may involve other factors not studied here such as peripheral or adrenal sulphatase activity.
It is interesting to observe that while the data regarding the influence of oestrogen are consistent with observations previously made using different experimental approaches (Lobo et al., 1982; Gonzalez et al., 1991
; Ditkoff et al., 1995
), and in-vitro data showing the influence of oestrogen of adrenal cell cultures, the data for insulin are not as consistent. It has been previously reported that insulin administration reduces 17,20 lyase activity in both normal (Nestler et al., 1992
) and hyperandrogenic (Moghetti et al., 1996) women. However, it has also been suggested that insulin may in fact increase 17,20 lyase activity in PCOS (Rosenfield, 1996
). It has also been reported that A responses to ACTH are increased in hyperinsulinaemic patients with PCOS (Lanzone et al., 1992
). It is possible that this discrepancy may be explained by the extent of insulin resistance. In PCOS, hyperinsulinaemia may be secondary to tissue insulin, which may include the adrenals as a site of this relative resistance. Interestingly, diet and other factors (such as diazoxide) which reduce serum insulin do not change adrenal androgen concentrations in PCOS (Nestler and Jakubowicz, 1989; Holte et al., 1994
) while insulin sensitizing agents and troglitazone, which decrease serum insulin by improving tissue sensitivity have been shown to reduce adrenal androgen concentrations (Dunaif et al., 1996
; Nestler et al., 1997). It should also be noted that the reduction in insulin also results in an increase in SHBG. As a consequence of this decrease, serum unbound oestradiol (Lobo and Carmina, 1997
) may be reduced and contribute to a reduction in AH.
This study was conducted to evaluate several factors by multivariate analyses. This approach was necessary because of the heterogeneous nature of AH and of the various factors influencing it in patients with PCOS. Thus, the results have to be viewed with the problems inherent in performing correlative analyses. In addition, use of steroid ratios to estimate true enzyme activities also should be interpreted with some caution. Nevertheless, it has been shown that several extra-adrenal hormones, specifically oestrogen and insulin, correlate with the presence of AH in PCOS and that these hormones may influence adrenal androgen pathways differently, thus explaining the heterogeneous presentation of AH in PCOS.
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Notes |
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References |
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Submitted on July 13, 1998; accepted on October 2, 1998.