1 Department of Endocrinology, PANAGIA Hospital, Thessaloniki, Departments of 2 Biological Chemistry and 3 Obstetrics and Gynaecology, University of Ioannina and 4 Department of Obstetrics and Gynaecology, University of Thessalia, Larissa, Greece
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Abstract |
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Key words: growth hormone/insulin/insulin-like growth factor-I/polycystic ovarian syndrome/thyrotrophin releasing hormone
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Introduction |
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Materials and methods |
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Eighteen healthy, normally menstruating women, aged 2129 years, were used as controls. They were studied in the early follicular phase, whereas the women with PCOS were studied on day 4 following a spontaneous or a progesterone-induced menstrual bleeding. The study was approved by the Local Ethical Committee and informed consent was obtained from all subjects. Patients were instructed to eat meals containing at least 150 g of carbohydrates per day for 3 days prior to the study. On the day of the study, subjects were instructed to report after an overnight fast and underwent a 75 g oral glucose tolerance test (OGTT). Venous blood samples for glucose and insulin measurement were obtained before and at 30 min intervals for the first 3 h after glucose administration. Two days later, a TRH test was performed in the morning after an overnight fast. Blood samples in relation to a bolus i.v. injection of TRH (time 0) were obtained at 15, 0, 15, 30, 60 and 90 min. GH was measured in these blood samples. The dose of TRH was 200 µg (Relefact; Hoechst AG, Frankfurt (M), Germany). In the blood samples taken at 15 and 0 min, IGF-I, oestradiol, free testosterone, 4-androstenedione, prolactin and free fatty acids (FFA) were also measured. All blood samples were immediately centrifuged at 1000 g for 10 min at 4°C. Supernatant serum was aspirated, aliquotted and stored at 20°C until assayed.
Hormone assays
Insulin was measured by radioimmunoassay using a CIS bioInternational Kit (Gif-sur-Yvete, France). The results are expressed as µIU/ml. GH was measured by immunoradiometric assay (IRMA) using a Sorin Biomedica Kit (Dia Sorin, Salugia, Italy). The results are expressed as ng/ml. Free testosterone was measured by radioimmunoassay using a Coat-A-Count Kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The results are expressed as pg/ml. 4-androstenedione was measured by radioimmunoassay using a Radim Kit (Pomezia, Rome, Italy). The results are expressed as ng/ml. Prolactin was measured by radioimmunoassay using a Medgenix Diagnostics Kit (Fleurus, Belgium). The results are expressed as ng/ml. The lower limits of detection for insulin, GH, free testosterone,
4-androstenedione and prolactin were 3.6 µIU/ml, 0.15 ng/ml, 0.15 pg/ml, 0.1 ng/ml and 0.35 ng/ml respectively, while inter-assay and intra-assay coefficients of variation were 6.9 and 6.4%, 7.5 and 6.1%, 8.1 and 7.2%, 7.6 and 5.1% and 7.1 and 6.4% respectively. Glucose concentrations were determined by the glucose oxidase technique. A colorimetric assay kit (Sigma Chemical Co., St Louis, MO, USA) was used for glucose oxidase measurement. An enzymatic method was used for determination of serum FFA (Wako Chemicals Gmbh, Neuss, Germany). The intra- and inter-assay coefficients of variation for this assay were 2.9 and 3.7% respectively, while the lower limit of detection was 0.1 mmol/l. IGF-I concentrations were measured by RIA following acid-ethanol extraction. Kits were purchased from Nichols Institute, San Juan Capistrano, CA, USA. The inter- and intra-assay coefficients of variation were 5.8% and 4.4% respectively and the results are expressed as ng/ml. The lower limit of detection of IGF-I was 0.06 ng/ml. Serum oestradiol was measured using a competitive immunoassay based on enhanced luminescence. Kits were purchased from Amersham (Amerlite Estradiol-60 assay; Amersham Pharmacia Biotech UK Ltd, Little Chalfont, Bucks, UK). The results are expressed as pg/ml. The lower limit of detection for oestradiol was 50 pg/ml, while inter-assay and intra-assay coefficients of variation were 9.3 and 8.5% respectively.
Statistical analysis
The GH release in response to TRH administration and insulin levels after OGTT were estimated by computing the area under the curve (AUC), using the trapezoidal rule. The mean of the values at 15 and 0 min was used as baseline value. The GH peak concentration in response to TRH was defined as the highest concentration reached in each individual and the response was expressed as the difference from the baseline value (max). Women with PCOS who had
max GH responses higher than the mean +2 SD of the
max GH responses observed in normal controls were defined as over-responders. Where data were normally distributed, unpaired t-test was used for the comparison of the results. Correlations between variables were assessed by linear regression analysis. Statistical significance was considered for P < 0.05. For some parameters, data were not normally distributed and in this case, non-parametric tests such as MannWhitney U-test and Wilcoxon rank sum test were also applied.
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Results |
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Discussion |
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A modulatory influence of FT and insulin levels on the secretory balance of hypothalamic neurons regulating GH secretion cannot be excluded. In a previous study (Anapliotou et al., 1989), a paradoxical increase of GH secretion was observed after TRH stimulation in 48.4% of the studied PCOS patients, but the correlations between obesity or insulin and
max GH were not investigated. It is of interest that in women with PCOS obesity and hyperinsulinaemia negatively influence GH secretion and that these two parameters may have an additive deleterious action on GH secretions (Villa et al., 1999
). It is known that insulin suppresses basal and GHRH stimulated GH secretion from rat anterior pituitary cells in culture (Yamashita and Melmed, 1986
) and that testosterone stimulates somatostatin release (Devesa et al., 1992
). Another possibility is that hyperandrogenaemia and hyperinsulinaemia reduced the acute stores of releasable pituitary GH pool in the normal responders group, which had significantly higher insulin and free testosterone concentrations than the over-responders. In favour of this assumption is also the negative correlation between
max GH and basal insulin concentrations that was found in the normal responders. On the other hand, it is rather unlikely that GH secretion was influenced by BMI, IGF-I or oestradiol concentrations, because their values were similar between the two PCOS subgroups. Moreover, dopaminergic deficiency (Quigley et al., 1981
), which has been postulated to exist in PCOS, could not account for the oversecretion of GH in the over-responders, since it would be also be expected to have an effect on basal prolactin concentrations, which were not different in both subgroups of PCOS patients. Furthermore, earlier reports have demonstrated that free fatty acids are able to block GH response to a number of stimuli (Imaki et al., 1985
). Because the two PCOS subgroups had similar free fatty acid concentrations, this mechanism must be excluded.
As PCOS is a heterogeneous disorder, it may be impossible to isolate a single factor that alone could explain the paradoxical action of TRH on GH secretion. Rather, several independent abnormalities, acting in concert, could contribute to this paradoxical GH effect.
In conclusion, the present study demonstrates enhanced GH response to TRH administration in a subgroup of women with PCOS. The results of this study could have clinical implications since they demonstrate a new pharmacological stimulus which, it is speculated, could be used as a test of GH secreting capacity to single out those women who may benefit from additive GH, following the establishment of a correlation between the response of GH secretion and gonadotrophin requirements (Menashe et al., 1993)
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Notes |
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References |
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Submitted on February 17, 1999; accepted on July 27, 1999.