Department of Obstetrics and Gynecology, University of Siena, Viale Bracci, 53100 Siena, Italy
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Abstract |
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Key words: ACTH/adrenal androgens/amenorrhoea/cortisol/diabetes mellitus
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Introduction |
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It has been postulated that diabetic amenorrhoeic women have increased dopaminergic tonus that could play a role in the pathogenesis of amenorrhoea (Djursing et al., 1983, 1984
). Berga et al. (1991) found increased dopaminergic tonus and accelerated LH pulsatility after administration of a dopamine antagonist in women with functional hypothalamic amenorrhoea. They also reported a variety of neuroendocrine aberrations in patients with functional hypothalamic amenorrhoea (Berga et al., 1989
). In these patients, increased activity of the hypothalamicpituitaryadrenal axis, demonstrated by increased cortisol concentrations, may play a primary role in the pathogenesis of amenorrhoea (Berga et al., 1997
). The aim of this study was to investigate the hypothalamicpituitaryovary and hypothalamicpituitaryadrenal axis in young amenorrhoeic women with insulin-dependent diabetes in good metabolic control.
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Materials and methods |
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Procedures
The women were admitted to the Gynecological Endocrinology Hospital Centre of Siena University 2 h before blood sampling was to begin. Eumenorrhoeic patients were investigated in midfollicular phase. All patients were administered conventional regular and intermediate acting insulin (s.c. injections twice daily). An indwelling catheter was inserted in the antecubital vein and saline solution was infused slowly to keep the vein patent. Blood samples were taken every 30 min from 08001000 h, 12001400 h, 16001800 h, 20002200 h, 24000200 h and 04000600 h. Sleeping was permitted only from 2300 to 0700 h. At 0800 h next day, all patients did the combined releasing hormone test simultaneously [100 µg corticotrophin releasing hormone (CRH; Nova Biochem, Zurich, Switzerland), 100 µg gonadotrophin releasing hormone (GnRH; Biochem Immunosystems, Milan, Italy), 200 µg thyrotrophin releasing hormone (TRH; Biochem Immunosystems)]. Blood samples were taken at 20, 10, 0, 15, 30, 45, 60, 75, 90, 105 and 120 min. A portion of the blood was immediately placed in EDTA-treated plastic tubes. The next day, i.v. metoclopramide (10 mg; Plasil; Lepetit, Frosinone, Italy) was administered. Blood samples were taken at 15, 0, 15, 30, 45 and 60 min.
Hormone assays
Plasma follicle stimulating hormone (FSH), LH, oestradiol, thyroid stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), prolactin, cortisol, dihydroepiandrosterone sulphate (DHEAS), androstenedione, 17-hydroxyprogesterone (17-OHP), sex hormone-binding globulin (SHBG), testosterone, free testosterone, free triiodothyronine and free thyroxine concentrations were assayed by double-antibody radioimmunoassay using commercial kits from Radim (Rome, Italy) for FSH, LH, cortisol, DHEAS, androstenedione and TSH, from Sorin (Saluggia-VC, Italy) for oestradiol and testosterone, from Biodata (Rome, Italy) for prolactin, from DPC (Los Angeles, CA, USA) for ACTH, SHBG, 17-OHP and free testosterone and from Immunotech (Marseille, France) for free thyroxine and free triiodothyronine. Samples were assayed in duplicate at two dilutions. Samples from a given subject were analysed for each hormone in the same assay to avoid inter-assay variation. Quality control pools at low, normal and high LH, FSH, oestradiol, prolactin, TSH, ACTH, testosterone, free testosterone, free triiodothyronine, free thyroxine, androstenedione, 17-OHP, SHBG, DHEAS and cortisol concentrations were present in each assay. The detection limit of the assay was 0.20 IU/l for LH, 0.18 IU/l for FSH, 18 pmol/l for oestradiol, 2.4 nmol/l for cortisol, 277 pmol/l for testosterone, 0.5 pmol/l for free testosterone, 1.7 pmol/l for ACTH, 0.2 mIU/l for TSH, 0.05 µmol/l for DHEAS, 0.21 nmol/l for 17-OHP, 0.2 nmol/l for SHBG, 104 pmol/ for androstenedione, 0.4 pmol/l for free triiodothyronine, 0.4 nmol/l for free thyroxine and 0.3 µg/l for prolactin. Intra- and interassay variations were 7.8 and 8.2% for LH, 6.2 and 6.5% for FSH, 4 and 4.8% for 17-OHP, 4.9 and 7.2% for DHEAS, 5.6 and 6.4% for androstenedione, 4.2 and 4.9% for oestradiol, 4.8 and 6% for cortisol, 3.4 and 4.6% for testosterone, 4.6 and 4.7% for free testosterone, 4.9 and 6.4% for ACTH, 6.1 and 8% for SHBG, 3.1 and 2.5% for TSH, 5.15 and 5.5% for free triiodothyronine, 3.9 and 3.3% for free thyroxine and 3.4 and 1.6% for prolactin.
Statistical analysis
The results are expressed as means and SD. The total integrated hormonal responses to GnRH, TRH, CRH and metoclopramide were calculated by the trapezoidal method and expressed as the area under the concentrationtime curve (AUC). Non-Gaussian-distributed variables were logarithmically transformed before analysis. For clarity the non-log-transformed data are presented in the tables and figures. Analysis of variance was performed to detect time-related differences. To compare the differences between the groups peak values (the maximum rise above baseline values) and the areas under curve were compared using Student's t-test. Statistical significance was taken for P < 0.05.
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Results |
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TRH test
There were no differences in total, incremental and AUC responses of TSH to TRH between the two groups. TRH administration was followed by a lower incremental response of prolactin in AD than in ED (140 ± 35 versus 185 ± 43 µg/l; P < 0.05). The AUC of prolactin was significantly smaller in AD than in ED (75 ± 18 versus 103 ± 28 µg/l.time; P < 0.05) (data not shown).
Metoclopramide test
This drug was followed by a larger total and incremental response of prolactin in ED than in AD (P < 0.05). In the latter group, the AUC was significantly smaller than in ED (P < 0.05) (Figure 1).
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Discussion |
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In the present study two groups of women with insulin-dependent diabetes were compared; there were no differences in duration of diabetes, metabolic control and BMI. As in certain other studies, no differences in the response of pituitary gonadotrophins to GnRH were found. However, there is disagreement in the literature, some authors reporting an enhanced and others an inhibited response of LH to GnRH. Administration of TRH was followed by a normal and similar response of TSH in both groups of patients. Basal plasma concentrations of prolactin were significantly lower in the amenorrhoeic group than in the eumenorrhoeic one, and the TRH response expressed as maximum increment and AUC was lower in the amenorrhoeic women and similar to that observed in women with functional hypothalamic amenorrhoea (Berga et al., 1989).
Administration of metoclopramide was followed by lower pituitary secretion of prolactin in the amenorrhoeic group than in the eumenorrhoeic one, in line with the results of other studies (Djursing et al., 1983). This finding of reduced prolactin response to antidopaminergic central drug is explained by an increase in dopaminergic tonus. In diabetic women, administration of metoclopramide causes an increase in gonadotrophins (Djursing et al., 1985
). Similar results were also seen (Berga et al., 1991
) in non-diabetic women with functional hypothalamic amenorrhoea. In one study performed in men with poorly controlled diabetes (Iranmesh et al., 1990
) a reduction in prolactin pulse amplitude has been reported, suggesting increased dopaminergic tonus. In women an increment of dopaminergic tonus could be partly responsible for amenorrhoea due to inhibition of GnRH secretion.
To our knowledge, there is no data in the literature on the activity of the hypothalamicpituitaryadrenal axis in amenorrhoeic women with insulin-dependent diabetes. Administration of CRH caused a lower increase in ACTH concentrations in AD women and the AUC was significantly less in amenorrhoeic patients. The maximum increment and AUC of cortisol to CRH were significantly lower in the amenorrhoeic group. The 24 h mean concentrations of cortisol were signficantly higher in the amenorrhoeic group than in the eumenorrhoeic one. Significant differences in plasma concentrations of cortisol between 2400 and 1000 h were also found between the two groups while significant differences were not found during the day.
Taken as a whole, these findings indicate activation of the hypothalamicpituitaryadrenal axis in diabetic amenorrhoeic patients. In a previous study it has been demonstrated (Wurzburger et al., 1990) that diabetics showed significant reductions in cortisol concentrations as metabolic control improved. The present results suggest that for a given metabolic status, amenorrhoeic patients have amplified cortisol concentrations and hyperactivity of the hypothalamicpituitaryadrenal axis. Similar results have been reported in non-diabetic women with functional hypothalamic amenorrhoea (Berga et al., 1997
). Other studies have reported a reduction in the response of cortisol to CRH (Biller et al., 1990
; Nappi et al., 1993
) in women with functional hypothalamic amenorrhoea with respect to eumenorrhoeic women. It is well known that hyperactivity of the hypothalamicpituitaryadrenal axis inhibits the hypothalamicpituitaryovarian axis, causing amenorrhoea. CRH is one of the main mediators of stress-induced inhibition of gonadotrophin secretion (Rivier et al., 1986
). In fact, central administration of CRH decreases LH secretion in rats and monkeys (Ono et al., 1984
; Olster et al., 1987). This reduction seems to be unrelated to increased adrenal steroid secretion, since the reduction in LH after CRH infusion in adrenalectomized monkeys is reported to be similar to that in normal monkeys (Xiao et al., 1989
). Diabetes mellitus is associated with low concentrations of insulin-like growth factor-I (IGF-I) and high concentrations of IGF binding protein-1 (IGFBP-1), a protein down-regulated by insulin (Hanaire-Broutin et al., 1996
). Even intensified s.c. insulin therapy does not normalize IGF-I plasma concentrations. It has been demonstrated that insulin, IGF-I and IGF-II play a role in modulating gonadotrophin-mediated folliculogenesis and steroidogenesis. Both insulin and IGF-I receptors are present in the human ovary (Poretsky and Kalin, 1987
). An autocrine function of IGF-I is suggested by its ability to amplify the FSH induction of LH receptors, progesterone sythesis and aromatase activity (Erickson et al., 1989
; Adashi et al., 1990
). Recently, a decrease in ovarian cytochrome P450c17
activity has been shown after reduction of insulin secretion by metformin in polycystic ovary syndrome (Nestler and Jakubowicz, 1996
).
It is also difficult to exclude the possibility that diabetes itself affects adrenal function. Evidence of adrenal hyperfunction has been reported in uncomplicated diabetes mellitus (Coiro et al., 1995). Dissociation of cortisol and DHEAS secretion has been found in children with insulin-dependent diabetes (Radetti et al., 1994
) and it has been proposed that insulin has a direct effect on the biosynthetic pathway of adrenal steroids (Ghizzoni et al., 1993
). It is therefore possible that insulin-dependent diabetes involves mild chronic hypercortisolism, probably by paracrine mechanisms, and this condition may affect metabolic control. Subsequent stress-induced activation of the hypothalamicpituitaryadrenal axis would enhance hypothalamic secretion of CRH which directly, and perhaps also indirectly through an increase in dopaminergic tonus, may inhibit GnRH secretion, leading to hypogonadotropic amenorrhoea.
In conclusion, the identification of the amenorrhoea associated with insulin-dependent diabetes with good metabolic control as a form of functional hypothalamic amenorrhoea is of considerable interest, because functional hypothalamic amenorrhoea is, by definition, `a theoretically reversible form of ovulatory impairment' requiring psychological and pharmacological management.
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Notes |
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References |
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Submitted on May 11, 1998; accepted on October 16, 1998.