1 Department Obstetrics and Gynecology, University of Study of Siena, 2 Department Obstetrics and Gynecology, University of Study of Catania and 3 Centro Florence, Firenze, Italy
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Abstract |
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Key words: clomiphene/IGF-I/IGFBP-1/insulin/PCOS
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Introduction |
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IGF-I seems to have an overall negative effect on normal folliculogenesis and ovulation (Barbieri et al., 1986; Cara and Rosenfield, 1988
), favouring the production and accumulation of androgens in the ovary. Although plasma concentrations of total IGF-I are not reported to be significantly higher than those in women without PCOS (Homburg et al., 1992
), the bioavailability of IGF-I may be raised by a decrease in IGFBP-1 plasma concentrations. A reduction in IGFBP-1 concentrations with respect to normal women has been demonstrated in women with PCOS (Suikkari et al., 1989
). These lower concentrations of IGFBP-1 seem to be a consequence of hyperinsulinaemia, a recognized feature of PCOS (Suikkari et al., 1988
). It was recently demonstrated that PCOS is associated with elevated serum concentrations of free IGF-I measured with a new direct immunoradiometric assay (Van Dessel et al., 1999
).
The aim of the present study was to evaluate the effect of clomiphene on plasma concentrations of IGF-I and IGFBP-1 and on insulin-resistance associated with PCOS.
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Materials and methods |
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All women were normoprolactinaemic, normotensive and without evidence of any other serious medical disorder. All had normal thyroid function.
Clomiphene citrate was administered at a dose of 100 mg/day to all women from day 5 to day 9 of the spontaneous or medroxyprogesterone acetate (MAP)-induced menstrual cycle. Blood sampling and a 2 h oral glucose loading test (75 g) were performed the day before (day 4, 08.0010.00) and after the course of clomiphene (day 10, 08.0010.00).
The study was approved by the institutional review board of Siena University. Written informed consent was obtained from the patients.
Assays
Plasma concentrations of LH, FSH, oestradiol, testosterone, free testosterone and 17-hydroxyprogesterone (17-OHP) were measured by double antibody radioimmunoassay using Radim kits (Rome, Italy) for LH and FSH, Sorin kits (Saluggia, Italy) for testosterone, DPC kits (Los Angeles, CA, USA) for 17-OHP and free testosterone, and Biodata Kits (Rome, Italy) for oestradiol. Total IGF-I and IGFBP-1 were measured by immunoradiometric assay (IRMA) using DSL Kits (Webster, TX, USA). The samples were assayed in duplicate at two dilutions. All samples of a given subject were assayed together. Quality control pools at low, medium and high hormone concentrations were included in each assay. The detection limits of the assays were 0.20IU/l for LH, 0.18IU/l for FSH, 2.06ng/ml for IGF-I, 0.33 ng/ml for IGFBP-1, 18 pmol/l for oestradiol, 0.52 pmol/l for free testosterone, 277 pmol/l for testosterone and 0.21 nmol/l for 17-OHP.
Intra- and inter-assay variations were 7.8 and 8.2% for LH, 6.2 and 6.5% for FSH, 3.2 and 3.4% for free testosterone, 3.9 and 3.8% for IGF-I, 4.6 and 3.6% for IGFBP-1, 3.4 and 4.6% for testosterone, 4 and 4.8% for 17-OHP and 4.2 and 4.9% for oestradiol.
Statistical analysis
All values are mean ± SD. Areas under the insulin curves (AUC) were calculated by the trapezoidal method. Basal hormone concentrations were compared using Student's two-tailed t-test for paired data. Differences were considered significant for P < 0.05. Relationships between variables were sought by Pearson product-moment correlations (Glantz, 1988).
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Results |
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Discussion |
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Although oral glucose tolerance test provides a convenient, readily available means of classifying individuals into normal, mild to moderate and severe insulin resistant, euglycaemic clamp is considered the gold standard in the assessment of insulin action (Del Prato, 1999).
The fasting glucose:insulin ratio has been reported to correlate with dynamic testing of insulin in PCOS (Legro et al., 1998). In the current study patients were their own controls and the comparison was made between before and after treatment. Using a highly sensitive measure of insulin sensitivity such as glucose:insulin ratio makes unlikely the risk of missing real changes in insulin concentrations.
Several reports have focused on the changes of serum IGF-I and IGFBP-1 during the ovulatory cycle (Jesionowska et al., 1990; Van Dessel et al., 1996
) concluding that circulating concentrations of IGF-I and IGFBP-1 are not menstrual cycle dependent, hence observed modifications in these polypeptides are direct effects of clomiphene administration and not the result of having an ovulatory cycle.
The increase in IGFBP-1 could be responsible for the reduction in bioavailability of IGF-I in the ovary, as demonstrated by the great reduction in the IGF-I:IGFBP-1 ratio. IGF-I may contribute to ovarian hyperandrogenaemia in PCOS by autocrine and paracrine mechanisms. IGF-I has been shown to stimulate oestrogen production by granulosa cells (Erickson et al., 1990) and to act synergistically with FSH and LH in controlling granulosa cell aromatase concentrations (Garzo and Dorrington, 1984
; Erickson et al., 1989
). It has also been reported to act synergistically with LH to stimulate androgen production (Cara and Rosenfield, 1988
; Bergh et al., 1993
), probably via its receptors on thecal cells (Bergh et al., 1993
). In PCOS, plasma IGF-I concentrations are within the normal range whereas serum IGFBP-1 concentrations are reported to be significantly lower than in normal women and inversely correlated with serum concentrations of insulin (Suikkari et al., 1989
; Homburg et al., 1992
). This leads to an increased IGF-I:IGFBP-1 ratio and an increase in bioavailability of IGF-I. This hypothesis is confirmed by the recent demonstration of elevated serum free IGF-I in women with PCOS (Van Dessel et al., 1999
). Both high concentrations of insulin and IGF-I could amplify the effects of LH on granulosa cells, inducing terminal differentiation and leading to anovulation.
The reduced IGF-I:IGFBP-1 ratio after clomiphene administration could therefore play a role in the induction of ovulation by this drug. IGFBP-1 is a hepatic product, the synthesis of which is inhibited by insulin (Suikkari et al., 1988). IGFBP-1 synthesis also takes place in ovarian granulosa cells and endometrium (Koistinen et al., 1986
), and in both sites IGFBP-1 synthesis is inhibited by insulin.
On the basis of the present results, the increase in IGFBP-1 cannot be explained by a reduction in insulin resistance, since no changes in the insulin curve were observed in response to oral glucose loading after clomiphene therapy. The increase in plasma concentrations of IGFBP-1 may therefore be a direct effect of clomiphene.
Other hypotheses show that IGF-I directly suppresses secretion of IGFBP-1 by granulosa cells (Dor et al., 1992), decidua (Thraikill et al., 1990
) and HEPG2 cells (Conover and Lee, 1990
) and thus a primary reduction in IGF-I could itself be responsible for the increase in IGFBP-1. It is therefore possible that clomiphene causes an increase in IGFBP-1 by reducing IGF-I.
However, a direct effect of clomiphene cannot be excluded, since this drug, being an oestrogen receptor probe, may theoretically act on all tissues that express receptors for oestrogens. In fact, about 30 years ago, Schultz et al. (1968) showed that [14C]clomiphene accumulates in oestrogen target tissues, such as the pituitary, hypothalamus, ovary, liver, uterus and adrenal gland. Since the number of clomiphene non-responders is so small in the current study, it is not possible to verify existing data on the predicitivity of the response to clomiphene in terms of insulin resistance and IGFBP-1 (Tiitinen et al., 1993).
IGF-I has been shown to contribute to carbohydrate economy. Availability of recombinant IGF-I has made possible investigations on the regulatory relations among IGF-I and insulin. IGF-I suppresses insulin secretion and reduces fasting glucose, even under euglycaemic conditions (Bondy et al., 1994); however, this effect is for supraphysiological doses of recombinant IGF-I. In the current study, fasting glucose did not change and IGF-I concentrations were in the normal range, hence it is unlikely that a reduction in bioavailable IGF-I for a few days might lead to changes in insulin concentrations.
It is difficult to evaluate the real contribution of the reduction in the IGF-I:IGFBP-1 ratio to induction of ovulation by clomiphene. It was recently shown that the insulin-sensitizing drug metformin reduces the IGF-I:IGFBP-1 ratio in PCOS (De Leo et al., 2000). This drug has recently been proposed to treat anovulation in PCOS and is reported to improve menstrual cycles (Velazquez et al., 1997
) and the response to clomiphene (Nestler et al., 1998
) and to normalize ovarian response to gonadotrophins (De Leo et al., 1999
). Hence the reduction in the IGF-I:IGFBP-1 ratio seems to be a fundamental element of initiation of ovulatory cycles in PCOS.
In conclusion, the present results show that clomiphene does not cause changes in insulin resistance associated with PCOS but reduces plasma concentrations of IGF-I and increases those of IGFBP-1, with a consequent marked reduction in the IGF-I:IGFBP-1 ratio. Reduction of this ratio could play a basic role in clomiphene initiated ovulation, presumably by modifying the hyperandrogenic intrafollicular milieu recognized in PCOS. It can therefore be stated that clomiphene-induced ovulation in PCOS is the result of an action of the drug not only on the hypothalamus, pituitary and ovary, but also peripherally, as shown by changes in plasma concentrations of peptides of prevalently hepatic origin.
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Notes |
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References |
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Submitted on April 27, 2000; accepted on July 31, 2000.