1 Department of Gynaecology and Obstetrics, Academic Hospital Groningen, 2 Department of IVF, 3 Diagnostic Centre SSDZ and 4 Department of Internal Medicine and Endocrinology, Reinier de Graaf Groep, Delft-Voorburg, The Netherlands
5 To whom correspondence should be addressed at: Department of Internal Medicine and Endocrinology, Reinier de Graaf Groep, Fonteynenburghlaan 5, 2275 CX Voorburg, The Netherlands. e-mail: schweitz{at}rdgg.nl
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Abstract |
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Key words: cortisol/11-deoxycortisol/extra-adrenal factors
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Introduction |
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For overweight women, the first step in improving ovulation rates is to encourage weight reduction (Bates and Whitworth, 1982; Pasquali et al., 1989
). Notably, insulin-sensitizing agents can also promote ovulation, thereby reducing circulating androgen concentrations (Dunaif et al., 1996
; Ehrmann et al., 1997a
,b; Nestler et al., 1998
). Both elevated androgens as well as abnormal regulation of the hypothalamicpituitaryadrenal (HPA) axis and elevated cortisol clearance rates have previously been reported in obese women (Strain et al., 1980
, 1982). By contrast, women with Cushings syndrome commonly present with irregular menstrual bleedings, and ovarian morphology similar to that of PCOS (Kaltsas et al., 2000
).
In a previously reported study, several isolated or combined enzyme deficiencies were reported in a third of women with irregular cycles with a substantial number of subjects exhibiting a diminished 11-hydroxylase activity, suggestive for mild forms of congenital adrenal hyperplasia (CAH) (Eldar Geva et al., 1990
). In the current study, we analysed the calculated 11-deoxycortisol/cortisol ratios, indicative for 11
-hydroxylase activity, in women with normo-gonadotrophic, normo-estrogenic oligomenorrhoea and clomiphene citrate-resistant infertility, compared with lean and obese ovulatory controls. Infertility is commonly observed in obese women who frequently cope with clinical features of pseudo-hypercortisolism, even though their morning serum cortisol concentrations are normal or even low, but their 24 h free urinary cortisol excretion usually is normal or even high, meaning that although cortisol production in adipose tissue is high the clearance rate is also increased (Prelevic et al., 1993
).
In the present study we tested the hypothesis that changes in cortisol metabolism of infertile women are more related to extra-adrenal factors than to adrenocorticotrophic hormone (ACTH)-driven steroidogenesis. Our study objective was to determine 11-deoxycortisol/cortisol ratios before and after ACTH stimulation, thereby differentiating CAH (11-hydroxylase deficiency) from extra-adrenal factors.
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Materials and methods |
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The Medical Ethical Committee of Hospital Reinier de Graaf Groep approved the study. Both patients and controls gave their informed consent for participation in the study.
Methods
Hormone assessments were determined by commercially available assays. Hormones and steroid precursors were analysed in one assay, except for 17-hydroxypregnenolone (17OH-preg). The latter steroid was assayed in additional runs for reasons of validation. Three experienced laboratory technicians performed all assays. All precursors were assayed at baseline and at 30 min after an ACTH bolus i.v. [250 µg synthetic
124-ACTH (Cortrosyn®)].
Fasting insulin, reference values: <20 mIU/l (enzyme-linked immunoassay, EIA; IMX-Abbott Laboratories, USA), cortisol, reference values: <0.73 µmol/l between 0800 and 0900; total testosterone, reference values: <3.1 nmol/l before menopause. All these hormones were measured using EIA (Immulite; DPC, Los Angeles, CA, USA). The reference value for fasting glucose was <6.0 mmol/l.
According to the manufacturers instructions, blood samples assayed for 17OH-preg (ng/ml) were prepared with an ethyl acetate/hexane extraction and column chromatography. The final analysis was done with a solid-phase 3H radioimmunoassay (ICN Pharmaceuticals, Costa Mesa, CA, USA). The entire procedure had a maximal intra- and inter-assay coefficient of variation (CV) of 10.3 and 15.0% respectively. Mean ± SD values in 24 ovulatory controls before and after ACTH stimulation were 4.5 ± 4.7 and 9.7 ± 4.7 ng/ml. 17-Hydroxyprogesterone (17OH-prog; nmol/l) was determined with a solid-phase 125I radioimmunoassay (DPC) with maximal intra- and inter-assay CV of 5.6 and 5.7% respectively. Values in ovulatory controls were 6.2 ± 3.9 and 10.9 ± 3.7 nmol/l before and after ACTH stimulation respectively. 11-Deoxycortisol (ng/ml) was determined with a solid-phase 125I double antibody radioimmunoassay (ICN Pharmaceuticals) with a maximal intra- and inter-assay CV of 5.9 and 13.7% respectively. Values in ovulatory controls before and after ACTH stimulation were 2.8 ± 1.2 and 4.3 ± 1.4 ng/ml.
Steroid precursor-to-product ratios
Three enzyme activities were indirectly analysed by calculating precursor-to-product ratios; these were (i) 3-hydroxy-
5-steroid dehydrogenase (17OH-preg/17OH-prog), (ii) 21
-hydroxylase (17OH-prog/11-deoxycortisol) and (iii) 11
-hydroxylase (11-deoxycortisol/cortisol). Normal laboratory reference ranges of these ratios before and after the ACTH bolus were obtained from the control group, which consisted of both lean and obese ovulatory women. Ratios higher than the 90th percentile values in the controls were considered indicative of abnormal steroidogenesis. ACTH-stimulated ratios higher than the 90th percentile of values in the controls were diagnostic for late onset CAH. The reference values obtained from the current study were 1.2 (basal) and 11.1 (after ACTH) for 3
-hydroxy-
5-steroid dehydrogenase, 3.5 (basal) and 7.9 (after ACTH) for 21
-hydroxylase, and 8.3 (basal) and 18.1 (after ACTH) for 11
-hydroxylase.
Statistical analysis
Hormone and steroid levels from the lean ovulatory, obese ovulatory and infertile groups were compared using analysis of variance. Comparisons per group of basal and post-ACTH values were performed using paired Students t-tests. Correlations between groups and physical variables such as BMI, fasting glucose and insulin, testosterone, 11-deoxycortisol, cortisol and the calculated precursor-to-product ratio were analysed using stepwise regression analysis. P < 0.05 was considered statistically significant.
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Results |
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Twenty-three out of 26 infertile women (88%) had a low 11-deoxycortisol to cortisol conversion (11-deoxycortisol/cortisol), i.e. a baseline value >8.1 (reference value). Three of these women (BMI: 38.0, 35.0 and 44.1 kg/m2) had CAH (11-hydroxylase deficiency) exhibiting elevated ACTH-stimulated 11-deoxycortisol/cortisol ratios (35.7, 32.1 and 18.3) (reference value after ACTH: >18.1). From the infertile group, six women (23%) were lean (e.g. BMI <25 kg/m2) and 20 (77%) were considered to be obese (e.g. BMI >25 kg/m2). Of the lean women with infertility, three had mildly increased basal 11-deoxycortisol/cortisol ratios compared with the ratios in ovulatory women, 9.8, 14.6 and 14.8 (reference value: 8.3). None of these women had higher ACTH-stimulated deoxycortisol/cortisol ratios than the reference value of 18.1. All 20 infertile obese women had increased basal 11-deoxycortisol/cortisol ratios but only three of them had increased deoxycortisol/cortisol ratios after ACTH (18.3, 32.0 and 35.9).
As demonstrated in Table II, baseline and ACTH-stimulated morning cortisol concentrations were similar among the three groups. Baseline 11-deoxycortisol concentrations were elevated in the infertile group, and increased significantly after ACTH stimulation (Table I). Finally, basal 11-deoxycortisol/cortisol ratios were significantly higher in the infertile group. Although this ratio remained unchanged after ACTH stimulation, these levels were still significantly higher than those in either of the ovulatory control groups.
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Discussion |
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Clinical features of pseudohypercortisolism even with low morning cortisol concentrations have been reported in obese individuals (Björntorp and Rosmond, 2000). Based on the current observations, we questioned whether the abnormalities of cortisol metabolism that were observed should be ascribed to a mild form of CAH and/or to the concomitant state of an obesity-related metabolic disorder. Notably, it appeared that 11-deoxycortisol/cortisol ratios were highest in the infertile group.
In previous studies, it has been demonstrated that obesity is related to several changes in the HPA axis. Obese individuals sometimes have low serum cortisol concentrations despite having an increase in cortisol production (Strain et al., 1980, 1982). Subtle alterations in cortisol responses were described in obese individuals after corticotrophin-releasing factor and ACTH stimulation as well as after very low dose dexamethasone suppression (Kopelman et al., 1988
; Marin et al., 1992
; Pasquali et al., 1993
; Ljung et al., 1996
; Rosmond et al., 1998
). Finally, stress-related cortisol hyper-responders showed interactions with clinical features of the metabolic syndrome (central obesity, hypertension, insulin resistance, and dyslipidaemia; Rosmond et al., 1998
). Apart from these changes, obesity can also be related to an increase in cortisol production rate (Szenas and Pattee, 1959
; Migeon et al., 1963
; Prezio et al., 1964
; Zelissen et al., 1991
). Moreover, there is compelling evidence that cortisol is exposed to enzymatic transformation in peripheral target tissues, mostly in liver and visceral fat (Bujalska et al., 1997
). One cortisol conversion system includes the peripheral 11
-hydroxysteroid-dehydrogenases (11
-HSD), metabolizing cortisol to the less active cortisone (Type 2 enzymes) or back to cortisol (Type 1 enzymes) (Bujalska et al., 1997
; Seckl, 1997
). Non-obese individuals with hypertension may have subtle adrenal 11
-hydroxylase or peripheral 11
-HSD Type 2 deficiencies, resulting in mild mineralocorticoid excess. Obese individuals, displaying features of the metabolic syndrome, have evidence of an altered peripheral handling of glucocorticoids through a reinforcement of peripheral 11
-HSD, Type 1. This enzyme system enhances the local production of cortisol, particularly in visceral adipose tissues (Rask et al., 2001
, 2002). We speculate that the imbalance between cortisol and its precursor steroid (11-deoxycortisol), as observed in infertile women, mirrors an insufficient cortisol production in relation its high clearance rate. Local cortisol production in peripheral tissues (11
-HSD, Type 1 in visceral fat) may compensate this minute systemic low cortisol status.
The presence of defects in ovarian and adrenal steroidogenesis of women with infertility has been previously reported in the laboratory (Gilling-Smith et al., 1997) and in clinical studies (Gonzalez, 1997
; Rosenfield, 1999
). Notably, (patient) populations described in previous research are heterogeneous as a result of the inclusion of different clinical syndromes with infertility. In an effort to be consistent in the selection of patients, we only included women with normo-gonadotrophic normo-estrogenic oligomenorrhoea who were resistant against clomiphene citrate. It is widely believed that this infertile group embodies several different reproductive disorders. In daily practice, these women are commonly divided in two distinct subgroups: obese women with high fasting insulin concentrations and lean women with insulin levels which are adequately low (Meirow et al., 1995
). Convincing arguments have been provided to support the concept that PCOS includes a phenotypic spectrum of metabolic disorders. Firstly, insulin resistance is also observed in lean women with PCOS (Dunaif et al., 1989
). Secondly, lean and fat body mass calculations have shown that in lean women with PCOS, changes in fat distribution involved primarily an increase of visceral fat mass (Kirchengast and Huber, 2001
). Finally, we have observed changes in cortisol metabolism mainly due to extra-adrenal factors in lean as well as obese women with infertility.
In conclusion, abnormal baseline 11-deoxycortisol/cortisol conversions were found in 88% of women with clomiphene citrate-resistant normo-gonadotrophic normo-estrogenic oligomenorrhoea. This abnormality disappeared almost completely after ACTH administration, thus excluding overt CAH and suggesting an alternative operational pathway in non-adrenal tissues. Further studies are needed to compare the effects of interventions such as dietary programmes or insulin-sensitizing agents, to investigate cortisol metabolism, and ultimately to improve ovulation and pregnancy rates for these women.
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Acknowledgements |
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Submitted on July 5, 2002; resubmitted on September 19, 2002; accepted on November 5, 2002.