Ovarian and adrenal steroid production: regulatory role of LH/HCG

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1 Human Reproduction Vol.17, No.3 pp , 2002 Ovarian and adrenal steroid production: regulatory role of LH/HCG T.Piltonen 1, R.Koivunen 1, L.Morin-Papunen 1, A.Ruokonen 2, I.T.Huhtaniemi 3 and J.S.Tapanainen 1,4 Departments of 1 Obstetrics and Gynaecology, and 2 Clinical Chemistry, Oulu University Hospital, Oulu and 3 Department of Physiology, University of Turku, Turku, Finland 4 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Oulu University Hospital, PO Box 5000, FIN University of Oulu, Finland. juha.tapanainen@oulu.fi BACKGROUND: The contribution of the adrenal glands to the total circulating steroid pool in women is not well known. There is evidence that human adrenals express the LH receptor gene and that LH may affect adrenal androgen secretion. METHODS: HCG stimulation tests (a single dose of 5000 IU i.m.) were performed in women at reproductive age (group 1, n 6, age years) before and after treatment with a GnRH agonist for 3 weeks, and in oophorectomized post-menopausal women (group 2, n 6, years) during and after estrogen replacement therapy (ERT). RESULTS: HCG did not stimulate the secretion of cortisol, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) in group 2. In contrast, in group 1, the basal concentrations of serum 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone and estradiol (E 2 ) were stimulated significantly (17-OHP 105%, androstenedione 31%, testosterone 20%, E 2 136%) by HCG, and the treatment with GnRH agonist decreased the responses. The basal serum concentrations of these steroids were significantly lower in oophorectomized women (17-OHP 57%, androstenedione 46%, testosterone 25%), and HCG did not increase these levels. It can be approximated that the ovarian contribution to the circulating levels of 17-OHP, androstenedione and testosterone is 25 30%, and that the adrenals are the primary source of cortisol, DHEA and DHEAS. CONCLUSION: LH/HCG does not have a major role in the regulation of adrenal steroid synthesis in endocrinologically healthy women. Key words: adrenal glands/hcg/lh/ovary/steroids Introduction The role of adrenal androgen secretion, including its regulation and contribution to the total circulating steroid pool in women, is still largely undefined. It is known that adrenocorticotrophic hormone (ACTH) is the principal stimulator of glucocorticoid production and it also has a role in adrenal androgen secretion (Parker, 1991). However, it is unlikely that ACTH exclusively regulates adrenal androgen production since several studies have reported dissociation between cortisol production and adrenal androgens (McKenna and Cunningham, 1991; Parker, 1991; McKenna et al., 1997). The presence of receptors for HCG (Pabon et al., 1996; Mircescu et al., 2000; Lacroix et al., 2001) insulin, insulin-like growth factor 1 (IGF-1) (Penhoat et al., 1988; Pillion et al., 1989) and prolactin (Glasow et al., 1996) in the adrenal cortex raises the possibility of their involvement in the control of adrenal steroidogenesis. In the human, increased LH secretion occurs in polycystic ovarian syndrome (PCOS) and in infertility, often concomitant with adrenocortical dysfunction. In a study by Pabon et al. it was shown that human adrenals express the luteinizing hor- mone receptor (LHR) gene in zona fasciculata and reticularis (Pabon et al., 1996). There is also circumstantial evidence that LH may affect adrenal androgen secretion (Lacroix et al., 1999, 2001). This is supported by studies showing decreased serum dehydroepiandrosterone sulphate (DHEAS) levels during GnRH agonist treatment in women with PCOS (Azziz et al., 1998; Bayhan et al., 2000; De Leo et al., 2000). In addition, in a recent study on transgenic female mice with chronically elevated serum LH concentrations, infertility, and aberrant ovarian tumours, it was found that the adrenal glucocorticoid production was increased when compared with control mice (Kero et al., 2000). The observation was explained by ectopic LH-induced LHR expression in the adrenal gland. In conclusion, LH may play a role in the regulation of physiological adrenal androgen secretion, as well as in some pathological conditions such as PCOS where hyperandrogenism is associated with chronically elevated levels of LH (Martikainen et al., 1996; Morin-Papunen et al., 2000). To explore the importance of adrenals in female androgen secretion, and especially the possible role of LH in its regulation, we stimulated women at reproductive age with HCG before 620 European Society of Human Reproduction and Embryology

2 Role of LH/HCG in steroid production and during GnRH agonist treatment. In addition, to exclude the contribution of the ovaries to the androgen pool, oophorectomized post-menopausal women were also studied. Materials and methods Subjects Two groups of subjects were recruited for the study. Group 1 consisted of six women with moderate/severe endometriosis [age years, body mass index (BMI) ] and group 2 of six post-menopausal women (age years, BMI ) who underwent oophorectomy and hysterectomy. In group 1, the diagnosis of endometriosis was based on laparoscopy, and since none of the patients desired to become pregnant, GnRH treatment was recommended. Besides endometriosis, the subjects were otherwise healthy and had regular menstruation. In group 2, the indication of oophorectomy and hysterectomy was endometrial hyperplasia in one subject, benign ovarian cysts in four subjects and ovarian serous cystadenoma in one subject. All of them were on estrogen replacement therapy (ERT, oral or transdermal estradiol). Two subjects had anti-hypertensive medication, one had medication for bronchial asthma, and one for depression. A woman with simvastain treatment for hyperlipidaemia forgot to inform the research team about her medication. Her androgen levels did not differ from those of other subjects, and therefore she was included in the study. Informed written consent was obtained from each subject and the study was approved by the Ethics Committee of the Oulu University Hospital, Oulu, Finland. HCG test In group 1, the initial HCG test was performed 2 6 days after a spontaneous menstrual bleeding. After the i.m. injection of 5000 IU HCG (Pregnyl 5000IU; Organon, Oss, The Netherlands) between 0700 and 0900 h, fasting blood samples for 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone, E 2, DHEA, DHEAS and cortisol measurements were collected at 0, 24, 48, 72 and 96 h. Thereafter, GnRH agonist treatment (Enanton, Depot 3, 75 mg; Laboratoires Cassenne Osnay, Cerny-Pentoise Cedex, France) was started on day of the menstrual cycle, and HCG test was repeated 3 weeks after the agonist injection. In group 2, the first HCG test was performed between 0700 and 0900 h any time during ERT and the test was repeated 4 weeks after cessation of ERT. The blood samples were collected as described above. Assays The serum concentration of testosterone was analysed by using an automated chemiluminescence system (Ciba Corning ACS-180, Medfield, MA, USA). The serum concentrations of LH and FSH were analysed by fluoroimmunoassays (Wallac Inc. Ltd, Turku, Finland) and radioimmunoassays (RIA) were used for 17-OHP, androstenedione, DHEA, DHEAS (Diagnostic Products Corporation, Los Angeles, CA, USA), E 2 and cortisol (Orion Diagnostica, Oulunsalo, Finland), following the instructions of the manufacturers. Areas under curve (AUC) for the 17-OHP, androstenedione, testosterone, E 2, DHEA, DHEAS, cortisol responses were calculated by the trapezoidal method. The intra- and interassay coefficients of variation were 4.9 and 6.5% for LH, 3.8 and 4.3% for FSH, 5.0 and 5.4% for 17-OHP, 5.0 and 8.6% for androstenedione, 4.0 and 5.6% for testosterone, and 5.7 and 6.4% for E 2, 6.5 and 7.9% for DHEA, 5.3 and 7.0% for DHEAS and 4.0 and 4.3% for cortisol. Statistics To compare the absolute maximal serum levels of the hormones to the basal levels, paired sample t-tests were used for normally distributed variables and Wilcoxon signed rank test was used for variables with skewed distribution. The same tests were also used for comparison of the AUC. The limit of statistical significance was set at P Results Group 1 (women years) The maximal responses and changes of AUC of all hormones measured in the HCG test are shown in Tables I and III. 17-OHP Serum concentration of 17-OHP reached a maximum at 24 h after HCG and decreased thereafter up to 96 h. The basal concentrations of 17-OHP decreased by 27% after the GnRH agonist treatment, but the response pattern remained unchanged (Figure 1 and Table III). Androstenedione Androstenedione increased gradually until 96 h after HCG. The basal level of androstenedione and AUC decreased during GnRH agonist treatment and the stimulation pattern to HCG was similar to that seen before GnRH agonist (Figure 1 and Table III). Testosterone Serum concentrations of testosterone were increased by 20% at h after HCG and returned to the starting level at 96 h. GnRH agonist decreased the basal level of serum testosterone by 38%, and in contrast to the response pattern before GnRH agonist, testosterone increased after HCG up to 96 h (Figure 2 and Table III). E 2 Serum E 2 increased significantly at 24 h after HCG (P 0.03), and decreased temporarily at 48 h before returning to the 24 h level (P 0.03). GnRH agonist treatment decreased the basal level of E 2 and AUC by 70%, however, HCG stimulated its levels two-fold and the stimulation pattern remained similar to that observed in the first HCG test (Figure 2 and Table III). DHEA, DHEAS, cortisol The serum levels of cortisol, DHEA and DHEAS did not change after HCG or GnRH agonist (Table I). Group 2 (post-menopausal women) E 2 The basal concentration of E 2 decreased in oophorectomized women from to after cessation of ERT. The HCG test had no influence on E 2 concentration (Tables II and III). 17-OHP androstenedione, testosterone, DHEA, DHEAS, cortisol No significant changes were observed in the concentrations of 17-OHP, androstenedione, testosterone, DHEA, DHEAS and cortisol in HCG test during or after cessation of ERT (Tables II and III). Group 1 compared with group 2 The effects of GnRH agonist treatment and oophorectomy on basal steroid concentrations are summarized in Table IV. 621

3 T.Piltonen et al. Table I. HCG test of subjects aged years before and during GnRH treatment Hormone HCG test before GnRH agonist HCG test during GnRH agonist Basal Maximum Change (%) P Basal Maximum Change (%) P LH (IU/l) FSH (IU/l) OHP (nmol/l) Androstenedione (nmol/l) NS NS Testosterone (nmol/l) NS E 2 (nmol/l) DHEA (nmol/l) NS NS DHEAS (µmol/l) NS NS Cortisol (µmol/l) NS NS DHEA dehydroepiandrosterone; DHEAS dehydroepiandrosterone sulphate; 17-OHP 17-hydroxyprogesterone. Figure 1. Responses of serum 17-hydroxyprogesterone (17-OHP) and androstenedione to a single dose of HCG (5000 IU) in women aged years and in oophorectomized post-menopausal women. *P 0.05 compared with the basal level. ERT estrogen replacement therapy; GnRH-a GnRH agonist. Figure 2. Responses of serum testosterone and estradiol to a single dose of HCG (5000 IU). *P 0.05 compared with the basal level. 622 Discussion The results of HCG test performed in women of fertile age and in oophorectomized post-menopausal women show that LH/HCG does not have a major role in the regulation of adrenal steroid synthesis in endocrinologically healthy women, under the experimental conditions used. In women aged years, HCG stimulated the serum concentrations of 17-OHP, androstenedione, testosterone and E 2 significantly and the treatment with GnRH agonist decreased the responses, reflecting ovarian contribution to synthesis of these steroids. This was also supported by the observation that the basal concentrations were significantly lower in oophorectomized women (17-OHP 57%, androstenedione 46%, testosterone 25%), and HCG did not increase their serum levels. Although the basal serum concentrations of ovarian steroids, especially those of E 2, decreased during GnRH agonist treatment, the relative response to HCG did not change markedly. This demonstrates that the immediate ovarian response to gonadotrophic stimulation persists during pituitary downregulation, and as well known, estrogen secretion is primarily of ovarian origin. In contrast to the ovarian steroids, a single dose of HCG did not stimulate the secretion of steroids previously thought to be primarily of adrenal origin, i.e. DHEA, DHEAS and cortisol. Despite these in-vivo results, it has been previously reported that upon short-term stimulation, the incubation of guinea pig adrenal cells with HCG for 2 h stimulated cortisol and androstenedione secretion significantly, and that ACTH enhanced the responses (O Connell et al., 1994). These observations indicate that HCG/LH could play a role in modulating adrenal steroidogenesis in-vitro conditions. This is also supported by occasional earlier observations; for example, during pregnancy, plasma and urinary free cortisol levels are increased even though ACTH levels are low, suggesting a possible role of HCG in the regulation of adrenal steroid secretion (Fotherby, 1984). In fact this has been demonstrated in in-vitro studies on human fetal adrenal cells (Serón-Ferré et al., 1978). Furthermore, during adrenarche, adrenal androgen production begins to increase and reaches adult levels without concomitant increase in ACTH (Apter et al., 1979). The purity of HCG

4 Role of LH/HCG in steroid production Table II. HCG test of oophorectomized subjects aged years Hormone HCG test during ERT HCG test after ERT Basal Maximum Change (%) P Basal Maximum Change (%) P LH (IU/l) FSH (IU/l) OHP (nmol/l) NS NS Androstenedione (nmol/) NS NS Testosterone (nmol/l) NS NS E 2 (nmol/l) NS NS DHEA (nmol/l) NS NS DHEAS (µmol/) NS NS Cortisol (µmol/) NS NS ERT estrogen replacement therapy. Table III. HCG response in groups 1 and 2 Hormone Group 1 Group 2 before GnRH during GnRH during ERT after ERT agonist AUC agonist AUC Change (%) P AUC AUC Change (%) P 17-OHP (nmol/l) NS Androstenedione (nmol/) NS Testosterone (nmol/l) NS E 2 (nmol/l) DHEA (nmol/l) NS NS DHEAS (µmol/l) NS NS Cortisol (µmol/) NS NS Table IV. Percentual change of serum basal steroid and gonadotrophin levels during GnRH agonist treatment and after oophorectomy compared with subjects aged years Hormone GnRH agonist (%) Oophorectomy a (%) LH FSH OHP Androstenedione Testosterone E DHEA DHEAS 9 47 Cortisol a Change of ageing has not been taken into account. may have varied between studies, which could explain some of the differences between in-vivo and in-vitro experiments. The time of exposure to LH/HCG may also be important. Women with chronic anovulation have been reported to have elevated levels of LH and DHEAS but normal levels of ACTH (Hoffman et al., 1984). Transgenic (TG) mice with constitutive high LH secretion provide a useful model for studying disorders with chronically elevated serum LH and adrenal androgen production (Kero et al., 2000). TG female mice exhibiting chronically elevated serum LH levels have been shown to have increased corticosterone production. Furthermore, LHR mrna expression was detected in the adrenal glands, and they responded to HCG stimulation with significant increase in corticosterone production, and the response was greater when HCG was combined with ACTH. Interestingly, oophorectomized TG animals had normal corticosterone levels, suggesting a possible role of gonadal factors directly or indirectly in the regulation of adrenal steroidogenesis. Similarly, in the present study, the post-menopausal oophorectomized subjects had high serum LH levels and decreased DHEA and DHEAS levels compared with women in group 1. However, because the serum levels of these steroids decrease with age (Orentreich et al., 1984; Laughil and Barrett-Connor, 2000), the contribution of oophorectomy alone cannot be confirmed. The first HCG test in group 2 was performed during ERT, to avoid the chronic effect of high endogenous gonadotrophin levels and consequent possible down-regulation of LHR. However, since the gonadotrophin levels in post-menopausal women were also high during ERT it is possible that the study design was not optimal for prevention of the possible complete down-regulation of LHR. Alternatively, it is also possible that oophorectomy had eliminated some important ovarian factors, for instance estrogens, that would be required for maintaining 623

5 T.Piltonen et al. adrenal LHR as suggested based on the observations in gonadectomized TG mice (Kero et al., 2000). In the same study, they implied that E 2 could play a part in adrenal androgen production by up-regulating prolactin secretion. Since prolactin is an important up-regulator of LHR expression (Huhtaniemi and Catt, 1981; Gåfvels et al., 1992; Pakarinen et al., 1994), this hormone could be the ovary-dependent factor that enhances the LH responsiveness of the adrenal gland. The role of estrogens is emphasized by studies showing that E 2 enhances adrenal sensitivity to ACTH in women with PCOS (Ditkoff et al., 1995; Carmina et al., 1999), although in normal post-menopausal women this effect has not been observed (Slayden et al., 1998). In conclusion, it can be approximated that the ovarian contribution for the synthesis of 17-OHP, androstenedione and testosterone is 25 30%, and that the adrenals are the primary source of cortisol, DHEA and DHEAS. 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