Leptin in functional hypothalamic amenorrhoea

Silvia Andrico, Alessandro Gambera,1, Cristina Specchia, Carolina Pellegrini, Leopoldo Falsetti and Enrico Sartori

Department of Gynecological Endocrinology, University of Brescia, 25128 Brescia, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Leptin, body weight, body mass index (BMI) and other hormones in women with functional hypothalamic amenorrhoea (FHA) were investigated and the hypothesis proposed that energy imbalance is the predominant mechanism for leptin reduction in patients with FHA. METHODS: Eighty-eight women with FHA and 65 age- and weight-matched controls were divided into homogeneous groups on the basis of their BMI: women with different degrees of underweight (BMI 15–16, 17–18 kg/m2) and of normal weight (BMI 19–21, 22–24 kg/m2). Hormone and carrier protein assays were measured in all groups. RESULTS: In each BMI group of patients with FHA, gononadotrophins, prolactin, insulin, free tri-iodothyronine and leptin levels were significantly lower than those of the respective controls, whereas cortisol and insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) were higher. We found significant linear positive correlations between leptin and body weight, BMI, LH, peptide-C, insulin, IGF-1 values and negative correlations with cortisol and IGFBP-1. CONCLUSIONS: Leptin values in women with FHA are significantly lower than controls, even in the group of patients having normal body weight and BMI. Leptin profile is different between patients with FHA and controls: it is suggested that energy balance can interfere with the ratio of body weight/leptin and BMI/leptin in FHA.

Key words: body weight/energy balance/functional hypothalamic amenorrhoea/IGF-1/leptin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Leptin is a peptide, coded by the ob gene and secreted with circadian rhythm mainly by adipocytes (Laughlin and Yen, 1997Go). This protein is claimed to be a potent satiety factor that regulates adipose tissue mass, the neuroendocrine system and many other functions (Considine and Caro, 1996Go). It has been well documented that body weight and the percentage of fat tissue substantially influence reproductive function. In humans, leptin secretion is also regulated by energy balance (Kolaczynski et al., 1996aGo), insulin (Saad et al., 1998Go) and sexual endogenous (Wauters et al., 2000Go) and exogenous hormones. Some studies have suggested that estrogen administration could stimulate leptin secretion (Shimizu et al., 1997Go). Accordingly, it has been suggested that leptin is the hormonal factor that signals to the reproductive system concerning the nutritional status (Wauters et al., 2000Go). The presence of leptin receptors both at the hypothalamus–pituitary level (Couce et al., 1997Go) and in the ovary (Karlsson et al., 1997Go) strengthen the hypothesis regarding the role of leptin in reproduction.

There are leptin receptors in the hypothalamus and these are found in both the area associated with eating behaviour and energy balance and the area concerned with reproduction regulation (arcuate, ventro-medialis and paraventricular nucleus) (Couce et al., 1997Go). In the hypothalamus the target of leptin is neuropeptide-Y (NPY) and pro-opiomelanocortin (POMC), both involved in regulation of body weight and reproduction (Laughlin and Yen, 1997Go). The NPY gene expression is increased by insulin and glucocorticoids and reduced by leptin (Speroff et al., 1999aGo). Central administration of NPY or ß-endorphin increases the calorific intake and inhibits GnRH (Kalra and Kalra, 1996Go). Leptin feed-back on the paraventricular nucleus could inhibit the release of the corticotrophin-releasing hormone (CRH) (Heiman et al., 1997Go). Thus, an inadequate leptin production, as occurs in women with nutritional amenorrhoea, could induce hypercortisolism through an increase in CRH secretion and adrenergic activation (Heiman et al., 1997Go). Leptin could act directly on hypothalamic receptors or indirectly by affecting NPY or POMC (Laughlin and Yen, 1997Go).

Evidence indicates that psychological/metabolic/nutritional stress could be the basis of functional hypothalamic amenorrhoea (FHA) (Warren et al., 1999Go). Patients with FHA have a deficiency in GnRH pulsatile secretion. Based on the degree of GnRH suppression, FHA is clinically characterized by various menstrual irregularities (inadequate luteal phase, occasional anovulation or amenorrhoea), low or normal gonadotrophin levels and normal radiological imaging of the Sella Turcica bone (Speroff et al., 1999bGo). Usually, women with FHA have a reduced secretion of LH, FSH and PRL, but an increased secretion of cortisol, insulin-like growth factor binding protein-1 (IGFBP-1) and growth hormone (GH) (Berga et al., 1989Go, 1997Go; Laughlin and Yen, 1996Go). The increase of IGFBP-1 can inhibit the insulin-like growth factors (IGFs) activity in the hypothalamus (Laughlin and Yen, 1996Go). The calorific deprivation, fasting and weight loss are associated with an increased cortisol and nocturnal melatonin secretion and a decrease in LH pulse frequency and thyronine levels (Berga et al., 2001Go).

The aim of this study was to determine leptin profiles in a wide sample of women with FHA, stratified for different classes of body mass index (BMI) and to verify the correlations between leptin, body weight, BMI and the other hormones involved in this dysfunction. We hypothesize that energy imbalance is the predominant mechanism for leptin reduction in patients with FHA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eighty-eight women with FHA were admitted to the Department of Gynecological Endocrinology of the University of Brescia from January 1999 to March 2001. FHA was diagnosed by clinical (amenorrhoea for a period >=6 months) and hormonal findings (Table IGo) and the negative radiological evaluation of the Sella Turcica bone surrounding the pituitary.


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Table I. Endocrine profile of patients with FHA and controls.
 
On admission patients had a mean (± SD) age of 24.9 ± 5.6 years, a mean body weight of 54.4 ± 7.2 kg, a mean BMI of 20.0 ± 2.3 kg/m2 and presented secondary amenorrhoea (mean 13.2 months, range 6–48 months).

Based on hormone profile, no patient had polycystic ovarian syndrome, premature ovarian failure, hyperprolactinaemia, hypo/hyper adrenal activity, hypo/hyperthyroidism, anorexia nervosa, a history of drug abuse, or had used any medication in the 6 months preceding admission. Anorexia nervosa was excluded because no patient weighed <85% of their ideal body weight, and none responded to the Diagnostic and Statistical Manual of Mental Disorders (DSM IV) criteria (American Psychiatric Association, 1994Go).

All women underwent a complete history to individualize the possible causes of FHA, a clinical examination, haematochemical and hormone assays and a radiological evaluation of the Sella Turcica bone.

The control group consisted of 65 women with a mean age of 26.1 ± 4.6 years, a mean body weight of 56.5 ± 6.9 kg, a mean BMI of 20.8 ± 2.0 kg/m2 and with regular ovulatory menstrual cycles. Ovulatory cycles were documented by mid-luteal phase plasma progesterone levels >4 ng/ml. Patients with FHA and controls had similar mean (± SD) age, body weight and BMI. Hormonal assays were assessed in fasting conditions between 8:00 and 9:00 a.m. for 2 consecutive days, except for prolactin which was assayed in a supine position at 9:30 and 10:00 a.m. Reported hormonal values are the mean of two measurements. In the controls, samples were taken in the early follicular phase of the menstrual cycle (days 4–7 of the cycle), using the same procedure as for women with FHA.

Patients with FHA and controls underwent the following assays: LH, FSH, prolactin (PRL), 17ß-estradiol (E2), cortisol, peptide-C (Pep-C), insulin (I), insulin-like growth factor-1 (IGF-1), thyrotrophin (TSH), free tri-iodothyronine (FT3), free thyroxine (FT4) and leptin. Furthermore, insulin-like growth factor binding globulin-3 and -1 (IGFBP-3, IGFBP-1) were assessed.

Women with FHA and controls were divided on the basis of their BMI into four and three homogeneous groups respectively (BMI 15–16, 17–18, 19–21, 22–24 kg/m2), due to the lack of women with BMI <17 kg/m2 in the control group. Hormone levels were also divided on the basis of BMI.

The Institutional Review Board of the University of Brescia approved the project and all subjects provided informed consent.

Hormonal assays
Hormones were determined by an automated analyser with a chemioluminescent method using: Vitros EcI, Ortho-clinical Diagnostics, for LH, FSH, PRL and E2 (reagents by Ortho-clinical Diagnostics, Amersham, UK); Immulite Automated Analyzer 2000, Medical Systems, for cortisol, Pep-C and I (reagents by Diagnostic Products Corporation, Los Angeles, California, USA); Liaison, Byk-Gulden, for IGF-1 (reagents by Nichols Advantage, Nichols Institute Diagnostics, San Juan Capistrano, California, USA); Elexis, Roche, for TSH (electrochemioluminescent ECLIA); and Ria-mab semiautomated system, Cobra Packard, for FT3 and FT4 (reagents by Ortho-clinical Diagnostics, Amersham, UK).

The remaining hormones were tested using radioimmunoassay commercial kits as follows: IGFBP-1, Diagnostic System Laboratories, Webster, Texas, USA; IGFBP-3, Nichols Institute Diagnostics, San Juan Capistrano, California, USA; and leptin, DRG Diagnostics, Berlin, Germany.

The average intra- and inter-assay coefficients of variation were: 2.6 and 3.8% for LH, 7.1 and 7.2% for FSH, 8.1 and 8.7% for PRL, 7.1 and 7.8% for E2, 6.8 and 7.1% for cortisol, 4.8 and 5.8% for Pep-C, 4.6 and 5.3% for I, 5.2 and 5.7% for IGF-1, 4.6 and 6.0% for IGFBP-1, 3.8 and 6.3% for IGFBP-3, 5.0 and 5.5% for TSH, 3.1 and 4.2% for FT3, 3.0 and 3.8% for FT4, 3.0 and 4.7% for leptin respectively.

Statistical analysis
All values were expressed as mean ± SD. Student's unpaired t-test was used to compare the hormone levels of the women with FHA with those of the controls, and the analysis of variance to compare the hormone levels between the different groups of BMI within each population.

The Spearman test was used to verify the correlations between leptin and the other hormones. The relationship between leptin, body weight and BMI was determined by linear regression analysis after normalization of data. A value of P < 0.05 was considered statistically significant. The statistical package STATA (STATA Corp; 1999, Texas, USA) was used to perform the statistical analysis.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table IGo shows the basal endocrine profile of patients with FHA compared with normal women. Patients with FHA have significantly lower LH, FSH, PRL, E2, Pep-C, I, FT3 and leptin levels and higher cortisol and IGFBP-1 levels than controls. IGF-1, IGFBP-3, FT4 and TSH are not statistically different.

Anamnestic factors associated with the development of FHA are shown in Table IIGo. In the group `competitive sporting activity' four patients were professional cyclists, six were long-distance runners, one was a professional gymnast and one a ballet dancer.


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Table II. Anamnestic causes (88 patients)
 
Table IIIGo shows the leptin levels in the different groups of BMI in FHA and controls. Women with FHA had significantly lower leptin levels in each BMI group than controls. Analysis of variance shows that plasma leptin significantly increases with the increase of the body weight and the BMI in both FHA and controls. Furthermore, Figures 1 and 2GoGo show a strong linear positive correlation, in both FHA and controls, between leptin and body weight (r2 = 0.3142, P < 0.001 and r2 = 0.4940, P < 0.001 respectively) and between leptin and BMI (r2 = 0.3395, P < 0.001 and r2 = 0.7087, P < 0.001 respectively). However, the profile of leptin is significantly different in FHA and controls, as shown by the course of the straight lines (95% confidence intervals: –13.613, –3.929, 0.199 and 0.374 in FHA for body weight, versus –18.657, – 6.246, 0.319 and 0.537 in the controls; –16.655, –5.523, 0.620 and 1.168 in FHA for the BMI, versus –31.749, 19.621, 1.510 and 2.092 in the controls).


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Table III. Leptin levels in the different groups of BMI, in women with FHA and controls. (P within the table is 4 or 3 points ANOVA)
 


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Figure 1. Linear positive correlation between leptin and body weight in FHA and controls (linear regression analysis).

 


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Figure 2. Linear positive correlation between leptin and BMI in FHA and controls (linear regression analysis).

 
Table IVGo shows, for each group of BMI, the hormone values that were statistically significant between FHA and controls in the general endocrine profile. Hormone values of women with FHA, with BMI 15–16 kg/m2, were excluded due to the small number of the sample (n = 4) and the lack of a control group. This group of patients presented the following hormone values: LH = 2.0 ± 1.2 mIU/ml, FSH = 4.3 ± 1.0 mIU/ml, PRL = 6.8 ± 3.6 ng/ml, cortisol = 21.3 ± 3.8 µg/dl, Pep-C = 1.8 ± 0.5 µg/ml, I = 4.7 ± 1.5 µIU/ml, IGFBP-1 = 52.0 ± 7.4 ng/ml and FT3 = 2.6 ± 0.3 pg/ml.


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Table IV. Endocrine profile of patients with FHA and controls, divided according to BMI. (P-value within the Table is 3 points ANOVA)
 
In FHA, each group of BMI showed LH, FSH, PRL, I and FT3 levels significantly lower than those of the respective controls, whereas cortisol was significantly higher. After stratification for BMI groups, the Pep-C values of FHA were not statistically different from controls. IGFBP-1 was significantly higher in the groups with BMI of 17–18 and 19–21 kg/m2 than the controls. Furthermore, in women with FHA there is a significant (P < 0.001) and progressive increase of IGFBP-1, parallel to the reduction of BMI (Spearman's rho = –0.358; P < 0.001).

Leptin decrease is significantly correlated with a reduction of LH, Pep-C, I and IGF-1 levels, and with an increase of IGFBP-1 and cortisol levels. In particular, leptin is positively correlated with LH (r = 0.256, P < 0.01), Pep-C (r = 0.376, P < 0.001), I (r = 0.380, P > 0.001), and IGF-1 (r = 0.384, P < 0.001) levels, and negatively correlated with IGFBP-1 (r = –0.412, P < 0.001) and cortisol (r = –0.313, P < 0.003) levels.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our study demonstrated that, in women with FHA, leptin values are significantly lower than controls, even in the group of patients having normal body weight and BMI. Overall, leptin levels are directly and positively correlated with body weight and BMI. Nevertheless, leptin profiles are different between FHA and controls (Figures 1 and 2GoGo). Each increase of 1 kg/m2 in FHA corresponds to a leptin increase of 0.89 ng/ml, which is 50% lower than controls (1.8 ng/ml). Similarly, a difference of 1 kg in body weight corresponds to a leptin increase of 0.28 ng/ml in FHA and of 0.43 ng/ml in controls. Thus, it seems that other factors, such as energy balance and calorific intake, can greatly interfere with the ratio of body weight/leptin and BMI/leptin in FHA.

There is evidence that energy imbalance can regulate leptin secretion, independently from the body fat reserves (Caro et al., 1996Go). In fact, fasting in humans results in a dramatic decrease in leptin concentrations, but 1 day of overfeeding, which does not change body weight, results in a 40% increase in serum leptin (Kolaczynski et al., 1996bGo). The importance of the relationship between energy balance/BMI/leptin is underlined by the higher recovery rate in women with FHA, after improvement of the energy balance and body weight increases leptin secretion (Falsetti et al., 2002Go).

FHA is mainly caused by psychological stressors and energy imbalance, with or without weight loss, either due to a reduction of the calorific intake (improper diet), or due to an increased energy expenditure (e.g. athletes). Stress and metabolic challenge are known to induce a hypothalamic–pituitary–adrenal axis hyperfunction with a consequent increased secretion of CRH-adrenocoricotrophic hormone (ACTH)-cortisol (Dorn and Chrousos, 1997Go), an increase of the central dopaminergic tone with a reduction of PRL circulating levels (Genazzani et al., 1995Go) and a nocturnal melatonin hypersecretion (Kadva et al., 1998Go). Leptin reduction, independently of weight loss, is a typical feature of FHA and is correlated with the other hormones involved in this dysfunction. Our study showed significant positive correlations between leptin and LH, Pep-C, I, IGF-1 and negative correlations with IGFBP-1 and cortisol.

Leptin influences the pulsatile secretion of GnRH-LH and the CRH–ACTH–cortisol axis. In particular, leptin reduction may be able to directly induce an LH decrease. Recent evidence suggests that low energy intake suppresses LH pulsatility through leptin (Loucks and Heath, 1994aGo). Leptin antiserum administration suppresses LH pulsatility, which is reversed and prevented by leptin administration (Carro et al., 1997Go). Thus, in FHA, decreased serum leptin, due to the altered energy balance and/or BMI, may contribute to the reduction of GnRH-LH secretion, to anovulation and to menstrual irregularities.

Statistical analysis confirms the inverse correlation between leptin and cortisol levels. The inadequate leptin secretion, as observed in FHA, could increase cortisol secretion through the CRH and adrenergic activation (Macut et al., 1998Go). Furthermore hypoleptinaemia, increasing NPY levels, would stimulate a central release of ACTH and endogenous opiods, further activating cortisol secretion (Kalra and Kalra, 1996Go; Speroff et al., 1999aGo). Hypercortisolaemia could also contribute to hypoleptinaemia through a peripheral adrenergic inhibitory effect or through a central effect (Macut et al., 1998Go). Thus a vicious circle is set up between hypoleptinaemia and hypercortisolaemia. Furthermore, it is well known that cortisol per se is able to inhibit the GnRH-LH secretion (Saketos et al., 1993).

In accordance with other studies (Schwartz et al., 1997Go), we found a significant correlation between leptin, Pep-C and insulin. Increasing evidence supports the notion that insulin is a regulator of ob gene expression and is a leptin secretagogue (Saad et al., 1998Go). Leptin, in turn, in high concentration and for long exposition, suppresses insulin secretion by central action and direct action on pancreatic ß-cells (Kieffer and Habener, 2000Go). Since plasma leptin levels are directly proportional to body fat mass, the low Pep-C levels and the hypoinsulinaemia observed in patients with FHA could be related to the decrease of BMI and to the altered energy balance.

Several studies have demonstrated that, in FHA, IGFBP-1 is increased as a combined effect of negative energy balance, hypoinsulinaemia, hypoleptinaemia and hypercortisolaemia (Laughlin et al., 1998Go). The leptin-mediated increase of IGFBP-1 reduces the free and active fraction of IGF-1, and limits IGF-1 activity in the hypothalamus–pituitary axis. This can be another mechanism for suppression of GnRH secretion (Speroff et al., 1999bGo).

In agreement with some authors (Couzinet et al., 1999Go), we found lower FT3 plasma levels (P < 0.001) in FHA than in controls. Reduced FT3 and I levels are suggestive of a negative energy balance in FHA (Loucks and Heath, 1994bGo). The decrease in thyroid function of women with FHA could also be also attributed to leptin (Legradi et al., 1997Go), in fasting mice, treatment with leptin normalizes thyroid functionality (Ahima et al., 1996Go).

In conclusion, leptin provides an additional level of communication between adipose tissue, energy balance and the functional integrity of the reproductive axis in humans. Leptin may co-ordinate the neuroendocrine responses associated with energy depravation, with or without BMI/body weight variations so as not to waste metabolic fuels. Thus, leptin might have a key role in the onset and resolution of FHA.


    Notes
 
1 To whom correspondence should be addressed at: Via XXV Aprile, no.10, 24058, Romano di Lombardia (BG), Italy. E-mail: gynecol.endocrinol{at}libero.it Back


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Submitted on August 28, 2001; resubmitted on March 18, 2002; accepted on May 9, 2002.