1 University Women's Hospital of Basel, Schanzenstrasse 49, CH-4031 Basel, 2 Hormone Laboratory of the University of Basel, Switzerland and 3 Institute of Reproductive Medicine, University of Münster, Domagkstrasse 11, D-48129 Münster, Germany
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Abstract |
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Key words:
17-hydroxyprogesterone/adrenal cortex/granulosa/progesterone/theca interna
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Introduction |
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Despite the evident role of progesterone in the process of ovulation, its source during this period of the menstrual cycle has not been defined. The enzyme responsible for the synthesis of progesterone, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), is expressed in the adrenal cortex, the theca interna and, at least in some species, also in the granulosa of the Graafian follicle (Dupont et al., 1990). While some authors argue that the adrenals contribute significantly to preovulatory progesterone (Eldar-Geva et al., 1998
), others consider the ovary as the main source (Fanchin et al., 1997
; Urman et al., 1999
). In addition to these uncertainties, nothing is known about the regulatory signals that induce the rise of progesterone at the end of follicular development.
This communication summarizes the results of three prospective experiments, which were carried out to determine the origin and regulation of follicular phase progesterone secretion in regularly menstruating women. Two sets of experiments were performed during desensitization with a long-acting GnRH agonist in patients treated with IVF or ICSI, because this treatment effectively reduces the contribution of ovarian steroidogenesis. The third experiment was performed in normally menstruating volunteers during their untreated, natural cycle and, subsequently, during intake of a contraceptive pill.
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Material and methods |
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Additionally, in 11 women a GnRH-test, consisting of a bolus injection of 100 µg of GnRH (Ferring), and in 18 women an ACTH test, consisting of a bolus i.v. injection with 250 µg ACTH (Synacthen; Ferring), were performed to evaluate the activity of the pituitaryovarian and of the pituitaryadrenal axis respectively during desensitization with triptoreline acetate. The LH response in the GnRH challenge test is considered optimal for the assessment of pituitary desensitization during treatment with GnRH agonists (Scheele et al., 1996). Both the GnRH and the ACTH tests were performed before the start of ovarian stimulation with gonadotrophins. All serum samples were stored frozen at 20°C until assay.
Second study: suppression of follicular phase progesterone serum levels with DXM
DXM was used to suppress the adrenal production of progesterone and to observe the ovarian contribution to the progesterone levels during the follicular phase. Patients with regular menstrual cycles and a normal ovarian reserve (as assessed by day 3 cycle FSH level <9 IU/l), but suffering from tubal infertility or male immunological infertility, were recruited prior to their first treatment with IVF. All participants had normal, ovulatory menstrual cycles and none was suffering from the PCOS or any apparent endocrine abnormality of the ovaries or adrenals. The serum progesterone concentration was determined in three serum samples, taken every 10 min, during suppression of endogenous gonadotrophin secretion, which was achieved with triptoreline acetate 23 weeks earlier (Decapeptyl Retard; Ferring). Patients with all three basal serum progesterone concentrations uniformly >2 nmol/l during suppression of the ovaries were treated with DXM (1 mg daily, taken orally) to suppress the secretion of adrenal progesterone throughout ovarian stimulation with gonadotrophins. One control group of patients with three subsequent serum progesterone concentrations >2 nmol/l was not treated with DXM during ovarian stimulation. The patients were randomized based on their birth month (those with an even birth month were treated with DXM, and those with an uneven birth month were not). Patients with three subsequent serum concentrations of progesterone <2 nmol/l and treated with IVF for the first time during the same period constituted a further control group. Each couple was included into the study only once. The serum samples were taken prior to ovarian stimulation and on the day of ovulation induction with HCG (Profasi; Serono, Zug, Switzerland). These serum samples were stored frozen at 20°C until assay. The clinical data of the patients participating in this study and the results of treatment with DXM are summarized in Table II.
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All ACTH tests were performed during early morning (8.00 10.00 a.m.) and the nine participants were permitted to rest, lying throughout the procedure. Bolus i.v. injections of ACTH (250 µg bolus i.v.) were given on day 3 of an untreated menstrual cycle. Fasting serum samples were taken during morning hours 30 and 0 min prior to, and 30 and 60 min after, bolus administration of ACTH. The second ACTH test was performed during the preovulatory rise of progesterone, which was determined by measuring daily the early morning (e.g. 7.009.00 a.m.) serum progesterone concentration starting on day 12 of the untreated menstrual cycle. An additional ACTH test was performed on day 3 of the next menstrual cycle, after which the participants started taking 50 µg ethinyl estradiol for 7 days followed by 50 µg ethinyl estradiol and 0.125 mg desogestrel for an additional 15 days (Ovidol; Nourypharma). A fourth ACTH test was performed after 7 days of treatment with 50 µg ethinyl estradiol daily, before taking desogestrel. The serum samples were stored frozen at 20°C until assay.
Hormone concentration measurements
All hormone concentrations were measured with commercially available assay kits. Measuring the progesterone levels reliably during the follicular phase of the menstrual cycle warrants assay systems with high accuracy at the lower end of the standard curve. For the progesterone measurements, two sensitive assay systems were used: SR1 (ImmunoChem, Freiburg, Germany) and Elecsys (Roche Diagnostics, Basel, Switzerland), both with an analytical sensitivity of 0.48 nmol/l and a functional sensitivity of 1.43 nmol/l, i.e. the lowest concentration that can be measured reproducibly with an inter-assay coefficient of variation of 20%. During initial testing, the results of the SR1 enzyme-linked immunosorbent assay (ELISA) kit for the measurement of low progesterone concentrations were compared with a commerically available radioimmunoassay (RIA; Biermann, Bad Nauheim, Germany) and the correlation was found to be highly significant (0.811, P < 0.00001). The inter-assay and intra-assay coefficients of variations for the progesterone, estradiol and LH measurements were below 10.1, 5.7 and 9.0% respectively, determined with control solutions with concentrations set at 5 nmol/l, 196 pmol/l and 15 IU/l respectively. The concentration of androstenedione was measured with an RIA from Diagnostic Systems Laboratories I7nc. (DSL, Webster, TX, USA). The concentration of 17ß-hydroxyprogesterone was measured with a RIA from Diagnostic Products Corporation (DPC, Los Angeles, CA, USA). For androstenedione and 17-hydroxyprogesterone, the inter- and intra-assay coefficients of variation were below 7.1 and 8.4% respectively, determined with low concentration solutions at 3.3 and 2.6 nmol/l respectively.
Statistical analysis
Data were analysed with MannWhitney U-test, Kruskal Wallis or 2-test as appropriate using the Statgraphics statistical software package (Manugistics Inc., Rockville, MA, USA). The data were presented by the mean values together with their 95% confidence interval in all instances. The level of statistical significance was set at 5%.
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Results |
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DXM effectively lowered the serum progesterone concentration during follicular development in patients with initially elevated progesterone concentrations, but the pregnancy rate among the patients treated with DXM was not different from those with elevated basal serum levels of progesterone left untreated and of patients with low basal serum levels of progesterone. The serum concentration of progesterone on the day of HCG administrationin patients left untreated with DXM despite an elevated initial progesterone serum concentrationrose significantly higher than that of patients treated with DXM (P < 0.001).
Effect of ACTH on adrenocortical steroid secretion during the natural menstrual cycle and during intake of an oral contraceptive
The higher rise of progesterone levels in patients lacking suppression of adrenocortical steroidogenesis by DXM suggests a significant adrenal contribution during the late follicular phase. Therefore, the origin of preovulatory progesterone was further tested using ACTH to stimulate adrenal steroidogenesis. Nine participants with normal, untreated menstrual cycles were recruited for this study. The results of two participants had to be discarded, because ovulation occurred prior to the second ACTH test. The results of the seven remaining participants were summarized in Figure 3. Whereas the serum concentrations of progesterone and 17
-hydroxyprogesterone rose significantly 30 and 60 min after i.v. administration of ACTH, the changes observed for the androstenedione concentrations before and after administration of ACTH were statistically not significant (P < 0.05 and P < 0.001 for progesterone, P < 0.001 for 17
-hydroxyprogesterone). The basal concentrations of progesterone and 17
-hydroxyprogesterone prior to the administration of ACTH were higher in the late follicular phase as compared with the early follicular phase. However, differences of the basal values of 17
-hydroxyprogesterone and progesterone were significant only in comparison with those after 7 days of treatment with ethinyl estradiol (P < 0.02 and < 0.01 respectively). The changes in the concentrations of progesterone and 17
-hydroxyprogesterone provoked by ACTH during the late follicular phase were not different from those during the early follicular phase. The rise of the progesterone concentration induced by ACTH was significantly lower during intake of ethinyl estradiol than in all other tests performed (P < 0.05).
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Discussion |
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The adrenal cortex may also be stipulated as an important source of late follicular phase progesterone (Eldar-Geva et al., 1998). LH receptors have been detected in the human adrenal cortex (Pabon et al., 1996
), suggesting an involvement of this organ in the process of ovulation. Conversely, in PCOS patients, elevated levels of progesterone and 17
-hydroxyprogesterone originate in the ovaries rather than in the adrenals (Lachelin et al., 1979
; Chetkowski et al., 1984
; Azziz et al., 1990
). The present communication describes the contribution of the adrenal cortex to circulating progesterone during the follicular phase of women with regular ovulatory menstrual cycles.
The present data clearly establish the adrenals to be the main secretory source of circulating progesterone during the early follicular phase. This is demonstrated by the rapid rise of progesterone after administration of ACTH during suppression of endogenous gonadotrophin secretion with triptoreline acetate. ACTH stimulates the conversion of cholesterol to pregnenolone in the adrenal cortex (Simpson and Waterman, 1988), which is rapidly converted to progesterone, 17
-hydroxyprogesterone and androstenedione by the enzymes 3ß-HSD and 17
-hydroxylase/17,20-lyase (cytochrome P450c17) respectively. Although long-term administration of ACTH seems to disrupt ovarian steroidogenesis (Viveiros and Liptrap, 2000
), the short-term administration used in this study may not have affected any of the ovarian functions.
During the late follicular phase, the main source of circulating progesterone shifts towards the ovaries, as demonstrated by the lack of suppression by DXM towards the end of follicular development. The activity of the adrenals is not influenced by the cyclic activity of the ovaries, as demonstrated by the similar response of both progesterone and 17-hydroxyprogesterone secretion induced by ACTH in both the early and late follicular phase of the natural menstrual cycle.
However, an important aspect of the present study revealed a major contribution of the adrenal cortex to circulating progesterone levels during the preovulatory phase. The high progesterone concentration of certain patients rose significantly during the late follicular phase as compared with those patients with consistently low levels of progesterone. There appears to be an individual setpoint, which determines the rate of progesterone secretion both in the theca interna and in the adrenal cortex, in both the early follicular phase and the preovulatory phase. The ovaries mediate the contribution of the adrenal cortex. This is demonstrated by the suppressive effect of ethinyl estradiol on both the basal and the ACTH-stimulated concentrations of progesterone and 17-hydroxyprogesterone. A similar finding was presented previously by Lobo et al. who found a significantly reduced output of adrenal androgens after ACTH stimulation in ovarectomized women as compared with regularly ovulating women (Lobo et al., 1982
). Although a direct stimulatory effect of conjugated estrogens on adrenal steroidogenesis was demonstrated in that study, our data suggest rather a suppressive effect of ethinyl estradiol on the adrenal reactivity to ACTH.
Orally administered ethinyl estradiol induces a rise of transcortin, which not only binds cortisol but progesterone as well (Rosner, 1991). The effects of ethinyl estradiol in this study could be explained by the reduced availability of progesterone due to increased binding to transcortin. However, both assay systems measured total serum progesterone: bound and unbound. Therefore, our experimental results suggest a reduced adrenocortical secretion of progesterone in the presence of suppressed ovarian function.
The presence of an endocrine crosstalk between the ovaries and the adrenal cortex in the hormonal regulation of the menstrual cycle has been suggested both experimentally and clinically in the past. Transgenic mice deficient of the inhibin -subunit all develop gonadal tumours (Matzuk et al., 1994
; Kananen et al., 1995
). In these animals, adrenocortical tumours will develop after gonadectomy and the shift of tumorigenesis from the gonads to the adrenal cortex is mediated by LH (Rilianawati et al., 1998
). Other examples of adrenocorticalovarian crosstalk is found in PCOS, as ovarian wedge resection results in reduced reactivity of the progesterone and 17
-hydroxyprogesterone concentrations after ACTH administration (Wu et al., 2000
), and in ovarectomized women, in whom adrenal androgenesis is suppressed as compared with regularly ovulating controls (Lobo et al., 1982
).
The present findings clearly demonstrate that the adrenals constitute the main source of circulating progesterone during early follicular development, whereas the ovaries provide most of the circulating progesterone during the late follicular phase. Our data also demonstrate that adrenal steroidogenesis is influenced by ovarian action. The adrenocorticalovarian crosstalk may be similar to that between the granulosa and the theca interna, in which thecal progesterone synthesis is stimulated by the granulosa (Makris and Ryan, 1977; Kotsuji et al., 1990
; Yada et al., 1999
). The molecular nature of the endocrine and paracrine mediators between the granulosa, the theca interna and the adrenal cortex remains to be determined.
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Acknowledgements |
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Notes |
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References |
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Submitted on June 12, 2001; resubmitted on October 24, 2001; accepted on November 20, 2001.