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BRIEF REPORT |
Department of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093
Address all correspondence and requests for reprints to: R. Jeffrey Chang, M.D., Department of Reproductive Medicine, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, California 92093-0633. E-mail: rjchang{at}ucsd.edu.
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Abstract |
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Main Objective: This study was conducted to determine whether blockade of androgen action in PCOS women by administration of the antiandrogen flutamide would alter E2 responses to rhFSH.
Design: We conducted a prospective cohort study.
Subjects and Setting: We studied 11 women with PCOS at an institutional general clinical research center.
Intervention: On study d 1, each subject received 150 IU rhFSH iv. Frequent blood samples were obtained over 24 h. After completion of rhFSH stimulation, each subject was treated with flutamide, 125 mg, twice daily, for 6 wk. Thereafter, the rhFSH stimulation test was repeated.
Main Outcome Measures: Baseline and stimulated E2 levels before and after treatment were assayed.
Results: Mean baseline and maximally stimulated E2, integrated E2 response, and fold change in E2 were not different before and after treatment. Levels of testosterone, androstenedione, progesterone, 17-hydroxyprogesterone, estrone, and SHBG before and after treatment were unchanged. Baseline dehydroepiandrosterone sulfate levels declined significantly after flutamide therapy.
Conclusion: These findings indicate that in women with PCOS, the E2 hyperresponsiveness to FSH may not be attributable to increased circulating androgens.
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Introduction |
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Ovarian hyperstimulation is also associated with marked increases of serum estradiol (E2) levels, which reflects the number and steroidogenic capacity of the stimulated follicle cohort. In PCOS women, heightened granulosa cell (GC) responsiveness to FSH stimulation compared with that of normal cells has been demonstrated in both in vitro and in vivo studies. Cultured GCs from PCOS follicles have been shown to exhibit greater sensitivity to FSH compared with that observed in normal cells (1). Correspondingly, we recently showed that in PCOS women, FSH-E2 dose responses were higher than those found in normal women (2).
In GCs obtained from follicles of unstimulated anovulatory PCOS ovaries, binding of radiolabeled FSH was significantly higher compared with that detected in cells from size-matched follicles of normal women (3). In vitro studies conducted in rodent and nonhuman primate ovaries have demonstrated that androgen exposure increases the number of FSH receptors in GCs as well as stimulates early stages of follicular development (4, 5, 6, 7). Consistent with these observations, ovaries of female to male transsexuals receiving high-dose androgen therapy were histologically similar to polycystic ovaries with increased FSH receptor message in GCs (8, 9, 10).
Based on the results of these studies, exaggerated GC responsiveness to FSH stimulation in PCOS women may be a consequence of excessive ovarian androgen production. To determine whether androgen may influence ovarian responses to gonadotropin administration, we examined FSH-stimulated E2 production in PCOS and normal women before and after androgen blockade with an antiandrogen, flutamide.
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Subjects and Methods |
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Thirteen women with PCOS were recruited for this study. Each subject had oligomenorrhea or amenorrhea and clinical or biochemical evidence of hyperandrogenism. Late-onset congenital adrenal hyperplasia was excluded by a serum 17-hydroxyprogesterone (17-OHP) level of less than 3 ng/ml (9.1 nmol/liter). TSH and prolactin levels were also normal in all subjects. None of the subjects received any hormonal medication or insulin-sensitizing agent for at least 3 months before study. Exclusion criteria were desire for pregnancy, abnormal liver function tests, or history of liver disease/alcoholism and diabetes. Eleven subjects completed the study. One subject was excluded from the final analysis because of recent ovulation. The mean age of PCOS subjects was 28.6 ± 4.6 yr, and mean body mass index was 32 ± 5.2 kg/m2. The study was approved by the Human Research Protection Program at the University of California, San Diego, and written informed consent was obtained from each subject before study.
Procedures
Each subject was admitted to the General Clinical Research Center at University of California, San Diego, on the morning of testing, on two separate occasions. On each occasion after baseline sampling, recombinant human FSH (rhFSH) was administered as an iv injection at a dose of 150 IU. The rhFSH was provided by Serono Laboratories, Inc. (Norwell, MA). None of the subjects had experienced recent ovulation as indicated by a lack of previous menstrual bleeding and retrospective analysis of baseline serum progesterone (P4) levels. Blood samples were drawn through an indwelling iv catheter at time –1, –2, 0, 2, 4, 6, 8, 10, 12, 16, 18, and 24 h. Sera were collected and stored at –4 C until assayed. Upon completion of the first rhFSH-stimulation test, each subject received flutamide orally at a dose of 125 mg twice daily for a period of 6–8 wk. After flutamide treatment, each subject returned to the General Clinical Research Center, and the second rhFSH stimulation test was performed. In each subject, blood samples were obtained 4 wk after starting flutamide and at the end of treatment to monitor liver function.
Assays
Serum LH and FSH concentrations were measured by RIA with intra- and interassay coefficients of variation, respectively, of 5.4 and 8% for LH and 3.0 and 4.6% for FSH (Diagnostic Products Corp., Los Angeles, CA). Serum concentrations of estrone (E1), E2, androstenedione (A4), testosterone (T), dehydroepiandrosterone sulfate (DHEAS), 17-OHP, SHBG, and P4 were measured by well-established RIA with intraassay coefficients of variation less than 7% (Diagnostic Systems Laboratories, Webster, TX).
Statistical analysis
Baseline values before and after treatment were compared using paired t tests. The E2 responses to FSH before and after treatment were analyzed as maximal concentration, absolute maximal change from baseline, fold change from baseline, and area under the curve (AUC). Values of A4, T, DHEAS, 17-OHP, SHBG, P4, and E1 were also compared using paired t tests. Data are expressed as means ± SEM.
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Results |
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There were no differences in the mean basal levels of T, A, 17-OHP, E1, and SHBG before and after treatment with flutamide. In contrast, the circulating concentration of DHEAS before treatment, 5.17 ± 0.98 ng/ml (0.014 ± 0.003 µmol/liter), became statistically significantly lower (P < 0.02) after flutamide therapy, 4.17 ± 0.97 ng/ml (0.011 ± 0.003 µmol/liter).
E2 responses to rhFSH
The mean basal serum E2 concentration was 92 pg/ml (337 pmol/liter) before treatment and 71 pg/ml (260 pmol/liter) after treatment. This difference was not statistically significant. Maximally stimulated E2 levels and the mean fold changes between groups before and after treatment were similar and not statistically different as shown in Table 1
. Similarly, the AUC between groups did not differ. Examination of the 24-h response patterns failed to demonstrate temporal differences in rhFSH-stimulated E2 production as a consequence of treatment (Fig. 1).
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Three subjects experienced transient elevations in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) during treatment. One month after the conclusion of study, liver function was reassessed, and in each subject, serum AST and ALT had returned to normal. Interestingly, the subjects with transient increases in liver transaminases were obese and were also being treated with antidepressive medications.
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Discussion |
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In vitro, androgens have been shown to enhance GC estrogen production as well as influence ovarian follicle development by increasing GC mitosis, facilitating transition of preantral to antral follicles, and up-regulating aromatase activity (6, 11, 12). All of these effects may be mediated by androgen-induced FSH receptor expression on GCs (4, 5, 6, 13, 14). Studies in nonhuman primates treated with either T or dihydrotestosterone have demonstrated a dose- and time-dependent increase in the number of preantral and antral follicles as well as increased FSH receptor mRNA expression in GCs (4, 5). That the androgen receptor colocalized with the FSH receptor suggested a direct effect on GCs (4).
In female-to-male transsexual patients treated with high-dose T for 6–12 wk before bilateral salpingo-oophorectomy, histological analysis revealed that the ovaries were morphologically similar to PCOS ovaries (8, 9, 10). Notably, there were increased numbers of antral follicles, theca cell hyperplasia, and greater FSH receptor expression on GCs compared with normal ovaries. Extrapolation of these data to GC function in PCOS suggests that the increased androgenic milieu may contribute to the higher antral follicle count and GC hyperresponsiveness to FSH seen in women with PCOS.
Despite this preceding evidence, the findings of our current study revealed that androgen blockade had essentially no effect on E2 responses to FSH, implying a lack of influence by circulating androgens in PCOS women. The lack of effect may be attributed to several factors.
One possible reason may be an inadequate dose and/or duration of treatment with flutamide. However, previous studies have shown that flutamide at doses of 250 mg/d or lower produces sufficient antiandrogenic effects to decrease hirsutism and improve ovulatory function (15, 16). In these studies, the duration of treatment was 12–18 months. In our study, DHEAS levels were significantly lowered after treatment with flutamide, which is consistent with previously reported responses to flutamide and indicated the clinical effectiveness of the dose employed (17).
Another consideration was whether E2 responsiveness to a single injection of FSH appropriately reflected the clinical consequence of multiple FSH administration. Although our FSH stimulation protocol has not been correlated to ovarian responses during ovulation induction, we have previously reported that the amount of FSH, 150 IU, used in the current study revealed GC hyperresponsiveness in PCOS women compared with E2 responses in normal women receiving the same dose (2). Collectively, our findings suggest that androgen does not influence GC responses to FSH in PCOS women. However, assessment of androgen blockade on ovarian responses during controlled ovarian hyperstimulation would be necessary to determine whether any effect on the risk of ovarian hyperstimulation syndrome exists in women with PCOS.
Alternatively, androgen may be a relatively weak effector of FSH receptor expression in GCs compared with other factors such as estrogens (18). In vitro, estrogen has been shown to up-regulate FSH receptor message in cultured GCs, thus providing a mechanism for greater FSH-stimulated E2 responses from diethylstilbestrol-treated GCs compared with untreated cells (19).
Three subjects developed transient mild elevations in serum AST and ALT levels during treatment, which had not been previously reported at the dose of flutamide employed in the current investigation (15, 16, 20). Whether PCOS subjects with obesity are susceptible to flutamide hepatotoxicity is unknown. Nevertheless, we believe that in obese PCOS women, flutamide should be used with caution because of a relatively high incidence of asymptomatic fatty liver in this disorder (21).
In conclusion, we have determined that short-term flutamide treatment in women with PCOS was not associated with alterations in E2 production after FSH stimulation. Whether the degree of androgen blockade in this study was sufficient to detect a role for androgen on ovarian follicle responsiveness is not clear. Currently, additional clinical studies are being conducted to determine whether androgen may influence GC function in women with PCOS.
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Footnotes |
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First Published Online June 27, 2006
Abbreviations: A4, Androstenedione; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the curve; DHEAS, dehydroepiandrosterone sulfate; E1, estrone; E2, estradiol; GC, granulosa cell; 17-OHP, 17-hydroxyprogesterone; P4, progesterone; PCOS, polycystic ovary syndrome; rhFSH, recombinant human FSH; T, testosterone.
Received April 6, 2006.
Accepted June 21, 2006.
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