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Rheumatology 2001; 40: 310-314
© 2001 British Society for Rheumatology

Assessment of pituitary gonadotropin release to gonadotropin releasing hormone/thyroid-stimulating hormone stimulation in women with systemic sclerosis

G. La Montagna, A. Baruffo, D. Pasquali1, A. Bellastella1, G. Tirri and A. A. Sinisi1

Institute of Clinical Medicine and Division of Rheumatology and
1 Institute of Endocrinology, Second Naples University, Naples, Italy


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To evaluate basal and dynamic levels of pituitary gonadotropin release in female systemic sclerosis (SSc) patients of childbearing age and in post-menopausal SSc patients.

Methods. We performed stimulation tests for gonadotropin-releasing hormone (GnRH) and thyroid-stimulating hormone (TRH) during the early follicular phase in 12 women of childbearing age [mean age (S.E.M.) 34.8 (2.4) yr] with SSc to determine serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin. Blood samples were also obtained from six post-menopausal women with SSc [mean age 46.8 (2.4) yr], after TRH stimulation; only serum prolactin concentration was determined, because elevated basal concentrations of FSH and LH were expected. Hormone concentrations were estimated by radioimmunoassay. Comparisons were made with healthy control women matched for age and reproductive status.

Results. In SSc patients of childbearing age, basal FSH, LH and oestradiol (E2) levels were not significantly different from those in controls, whereas basal prolactin concentration was significantly higher than in controls (P=0.0001). After the stimulation test, the peak concentrations of FSH (P=0.0001) and prolactin (P<0.0001) were significantly higher than in controls. The net integrated response curves [net area under the curve (AUC)] for FSH and LH did not differ significantly between SSc patients and controls. On the contrary, the net AUC for prolactin in response to TRH stimulation was significantly higher than in controls (P=0.001). In post-menopausal patients, basal E2, FSH, LH and prolactin levels were not significantly different between women with SSc and controls. However, after TRH stimulation, peak levels and net AUC for prolactin were not significantly higher in patients than those in controls. No significant correlations were found between basal and stimulated FSH, LH and prolactin levels and the severity of involvement of various organ systems. Multiple regression analysis showed that basal and stimulated prolactin concentrations were associated with skin sclerosis and peripheral vascular and lung involvement.

Conclusion. Our results suggest that subclinical primary hypogonadism can occur in SSc patients. They also confirm an alteration in the mechanism for prolactin secretion and release, which may not only contribute to further disturbance of the reproductive axis but may also have an influence on the disease.

KEY WORDS: Systemic sclerosis, Prolactin, Gonadotropins.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) is a connective tissue disease characterized by immunological abnormalities, vascular lesions and fibrosis of the skin and various internal organs [1]. Excessive collagen deposition in affected tissues is responsible for most of the clinical manifestations. It has been suggested [2, 3] that fertility is decreased in women with SSc, and that an increased incidence of problems in reproduction before and after the clinical onset of the disease can also occur [4, 5]. In addition, it has been hypothesized that fibrosis of the uterine cervix and genital organs may be the cause of infertility in SSc [6]. However, data regarding the role of the hypothalamic–pituitary–gonadal (HPG) axis are lacking. High serum levels of prolactin (PRL) have been reported recently in SSc patients [7, 8]. Such findings are important because raised PRL levels may cause dysregulation of the HPG axis and may play a role in the pathogenesis of the disease [9].

In this study we evaluated the basal and dynamic hormone levels of pituitary gonadotropin release in women with SSc of childbearing age and in post-menopausal SSc patients.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
Eighteen women attending the Division of Rheumatology and who satisfied the preliminary American College of Rheumatology (ACR) criteria for the classification of SSc [10] were studied. Their mean (S.E.M.) age at the time of the study was 38.8 (2.2) yr (range 19–54 yr) and disease duration 9.2 (1.5) yr (range 1–26 yr), disease onset being taken as the first appearance of Raynaud's phenomenon or other characteristic symptoms of SSc.

Twelve of the patients were of childbearing age [age 34.8 (2.4) yr, disease duration 7.7 (1.3) yr] and six were post-menopausal [age 46.8 (2.4) yr, interval since menopause 5.2 (2.4) yr, disease duration 12.2 (3.1) yr]. Eleven of the 18 women were married, and all were pregnant before the disease onset. Only two had a further pregnancy after disease onset. The ratio of total number of pregnancies:parous women was 1.36. Six of the women had had an abortion; five of them had 10 spontaneous abortions. Eight of these ten events were registered before disease onset. Seven of the SSc patients recalled oligomenorrhoea after disease onset.

According to the clinical criteria of LeRoy et al. [11], 16 of the women were classified as having limited cutaneous SSc and two as having diffuse cutaneous SSc. All patients had antinuclear antibodies detected by indirect immunofluorescence, using the Hep2 cell line as substrate; three patients showed the anticentromere pattern. Nine patients were positive for anti-topoisomerase I antibodies (anti-Scl 70), detected by enzyme-linked immunosorbent assay.

Patients were re-examined yearly. The investigations included routine laboratory tests to detect internal organ involvement, electrocardiogram, Doppler echocardiography, M- and B-mode echocardiography, capillaroscopy, pulmonary function tests, high-resolution computed tomography of the chest, oesophageal and/or gastrointestinal barium studies and the xylose absorption test.

At the time when blood samples were taken, a preliminary score on the SSc severity scale of Medsger et al. [12] was calculated for each of nine organs/systems [general, peripheral vascular, skin, joints/tendons, muscles, gastrointestinal (GI) tract, lungs, heart, kidneys]. The scores were as follows: 0, no documented involvement; 1, mild involvement; 2, moderate involvement; 3, severe involvement; 4, end-stage disease). Organ/system involvement was as follows: peripheral vascular, 94.4% of cases; skin, 100%; GI tract, 83.3%; lungs, 66.7%; heart, 33.3%. No involvement was recorded under the headings of general, joint/tendon, muscles and kidneys. The severity scores for the organ systems involved were as follows: peripheral vascular system, score 1 in three cases, 2 in two cases, 3 in 12 cases; skin, score 1 in 12 cases and 2 in six cases; GI tract, score 1 in 13 cases and 2 in two cases; lung, score 1 in eight cases and 3 in four cases; heart, score 1 in one case and 2 in five cases.

Sixteen of the 18 patients were taking disease-modifying drugs: five were taking D-penicillamine (300– 600 mg/day), five were taking griseofulvin (1000 mg/day) and six were taking griseofulvin plus hydroxychloroquine (1000 mg plus 400 mg/day respectively).

None of the patients was pregnant or breastfeeding, had renal failure or used medications known to affect the PRL level [13], including oral contraceptives.

Because of the frequent impairment of thyroid function in SSc [14, 15], all patients included in the study were required to have normal thyroid tests (normal levels of free thyroxine, free triiodothyronine and thyrotropin-stimulating hormone, and absence of antithyroid antibodies).

The control group consisted of 18 healthy women matched with the patients for age and reproductive status. Patients were informed about the nature of the study and gave verbal or written consent according to the Declaration of Helsinki.

Methods
Gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) stimulation tests were used to evaluate pituitary responsiveness with respect to follicle-stimulating hormone (FSH), luteinizing hormone (LH) and PRL. This was performed during the follicular phase in women of childbearing age and after a drug washout period of at least 3 days. Blood specimens were taken for FSH, LH and PRL determinations in the morning, after an overnight fast (basal condition). GnRH (100 µg) and TRH (200 µg) were then given i.v. and blood samples were taken at 20, 60, 90 and 120 min for FSH, LH and PRL determinations. In post-menopausal SSc women, blood samples were collected after stimulation with TRH to determine only serum PRL, because elevated basal levels of FSH and LH were expected in these women. In addition, blood samples were also taken from all subjects to determine the basal concentration of 17ß-oestradiol (E2). No further downstream hormones were evaluated. After centrifugation, serum was stored at -30°C until assayed. Concentrations of E2 (pg/ml), FSH (mIU/ml), LH (mIU/ml) and PRL (ng/ml) were determined by radioimmunoassay using a commercial kit manufactured by BioChem Immuno Systems Italia. The detection limits were 0.27 pg/ml for E2, 0.25 mIU/ml for FSH and LH, and 0.3 ng/ml for PRL.

Statistical analysis
Data were analysed using the Statistical Package for Social Science (SPSS) for Windows version 6.1 (SPSS Italia, Bologna, Italy), and are expressed as mean (S.E.M.). Peak hormonal responses were the highest responses reached during the stimulation test. The hormonal responses to the GnRH/TRH stimulation test were integrated over time and evaluated as net area under the response curve (net AUC) from 0 to 120 min considering delta values from baseline. The difference between the mean values of the basal hormonal concentrations and hormonal responses to stimulation tests in SSc patients and control subjects, subdivided according to menopausal status, was analysed with the non-parametric Mann–Whitney U-test. P<0.01 was considered significant. Multiple regression analysis (stepwise method) was used to explore the effects of disease duration and basal and stimulated FSH, LH and PRL concentrations on the SSc organ/system severity scale. Spearman rank correlation analysis was used to assess significance after Bonferroni correction to achieve an acceptable {alpha} risk. Using a very conservative approach, we required P<0.0027 for statistical significance.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Serum levels of FSH, LH and PRL
There was no difference in the mean FSH and LH levels between the 18 SSc patients [18.1 (3.6) and 17 (5.3) mIU/ml respectively] and the healthy controls [18.5 (5.4) and 12.4 (4.1) mIU/ml]. In SSc patients the mean level of PRL [14.2 (2.1) ng/ml] differed significantly from that of the controls [5.7 (0.69) ng/ml P=0.002].

SSc patients of childbearing age
In patients of childbearing age, E2 levels were not significantly different from those of the controls in the follicular phase [84.4 (11.5) and 65 (8.8) pg/ml respectively]; similarly, patients and controls did not differ significantly in basal FSH and LH concentrations (Table 1Go). On the contrary, basal PRL concentrations in the patients were significantly higher than in the controls (P=0.0001). Peak FSH and PRL concentrations after stimulation with GnRH and TRH were significantly higher in the patients than in the controls (P=0.0001 for both); peak LH levels of the patients were similar to those of the controls. The net integrated FSH and LH responses to GnRH/TRH stimulation (net AUC) in the patients were not significantly different from those in the control subjects. On the contrary, the net AUC for PRL was significantly higher in the women with SSc than in the controls (P=0.001) (Table 1Go).


View this table:
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  		TABLE 1. Basal and peak levels and net integrated response (net AUC) of gonadotropins after GnRH/TRH stimulation in female SSc patients of childbearing age and healthy control women [mean (S.E.M.)]

 

Post-menopausal SSc patients
In the post-menopausal patients, basal E2 levels were not significantly different from those of the controls [13.3 (1.7) and 10 (0.9) pg/ml respectively], and the same was true for basal concentrations of FSH, LH and PRL (Table 2Go). In the TRH stimulation test, the post-menopausal SSc patients had significantly higher values than controls for the PRL peak response (P=0.004) and the net AUC for PRL (P=0.0038) (Table 2Go).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Basal gonadotropin concentrations and basal, peak and net integrated responses (net AUC) of PRL after TRH stimulation in post-menopausal SSc patients and healthy control women [mean (S.E.M.)]

 

Correlation between hormonal levels, disease duration and severity
Scores on the SSc organ/system severity scale were not associated with disease duration or basal or stimulated FSH, LH and PRL concentrations after Bonferroni correction.

Multiple regression analysis was performed to assess the relative importance of some variables in SSc organ/system involvement. The statistical models included disease duration and basal and stimulated FSH, LH and PRL concentrations. General, muscle and kidney involvement were excluded by stepwise analysis; peripheral vascular involvement was significantly associated with basal PRL (ß=0.44, P=0.002); skin involvement was significantly associated with disease duration (ß=0.38, P=0.002) and peak PRL level (ß=0.40, P=0.001), whereas joint/tendon involvement was significantly associated with peak LH level (ß=0.45, P=0.001); GI tract involvement was significantly associated with basal (ß=1.10, P=0.000) and peak concentrations of FSH (ß=-1.3, P=0.000) and peak PRL concentration (ß=0.44, P=0.001). Lung involvement was significantly associated with basal PRL (ß=0.69, P=0.000) and peak LH concentrations (ß= 0.40, P=0.001). No relationship was demonstrated between heart involvement and the variables considered.


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
In this study we have demonstrated an increase in the basal level of PRL together with abnormalities in the responsiveness of FSH and PRL to GnRH and TRH in female SSc patients of childbearing age. On the contrary, basal FSH, LH and PRL levels were similar in post-menopausal patients and controls. This suggests that there may be underlying alterations in pre-menopausal SSc patients. In particular, the increased FSH release with normal follicular-phase E2 levels points to subclinical gonadal failure, which can also explain the functional oligomenorrhoea and the precocious post-menopausal status of some of our patients. Subclinical hypothyroidism, which is a common cause of increased PRL release in SSc [14, 15], was excluded by our patient selection criteria.

It has been shown that in autoimmune disorders, soluble factors, such as cytokines, released by immune cells may influence the release of hormones. It has been shown that secretion of PRL by the pituitary is affected by interleukin 2 [16]. Moreover, direct secretion of PRL by mononuclear cells from systemic lupus erythematosus patients has been demonstrated recently [17]. These data are consistent with the existence of bidirectional interaction between the neuroendocrine and immune systems [18] and can explain the raised PRL levels as a consequence of disease activity.

When the severity of illness was ranked according to a scale that considered general, skin and peripheral vascular involvement and changes in internal organ systems, it was shown that basal and stimulated PRL secretion was associated with skin severity grade and the involvement of the peripheral vascular system, the GI tract and the lungs.

In agreement with the association between PRL levels and the severity of involvement of some organ/systems, recent studies assign an immunoregulatory role to this hormone [9, 17, 19, 20]. Hyperprolactinaemia has been associated with spontaneous autoimmunity involving non-specific polyclonal B-cell activation [21], increased disease activity in human systemic lupus erythematosus [22] and murine lupus [23], and antigen-dependent T-cell development [24]. Recent findings in SSc patients [16] are consistent with the effects of PRL on the soluble interleukin 2 receptor, and are correlated with changes in total skin score [25]. Such findings support a role for T-lymphocyte-mediated immune activation in SSc.

However, immunomodulatory effects appear not to be limited to PRL. There is evidence suggesting that FSH and LH are also important, independently of gonadal steroids, in the pathogenesis of autoimmune diseases [26–28].

In our patients, hyperprolactinaemia was unrelated to therapy. Increased PRL concentrations have not been demonstrated during treatment with D-penicillamine [29] or chloroquine [30]. Griseofulvin is not mentioned among the drugs inducing an increased PRL concentration [13].

Although the small number of cases suggests caution, our results indicate that impairment of pituitary gonadotropin release occurs in SSc patients. Furthermore, an unusual primary subclinical hypogonadism appears to be associated with increased PRL secretion and release. The relationship between these findings and infertility in some of these patients needs to be investigated further, as does the role of hyperprolactinaemia in the pathogenesis of the disease.


   Notes
 
Correspondence to: G. La Montagna, Seconda Università di Napoli, Cattedra e Divisione di Reumatologia, Via S. Pansini 5, 80131 Napoli, Italy. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 22 February 2000; Accepted 2 October 2000


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