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Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore (M.Gu., D.R., M.Gi., R.S., L.S., R.A., A.L.), 00168 Rome, Italy; and OASI Institute for Researche (A.L.), 94018 Troina, Italy
Address all correspondence and requests for reprints to: Dr. M. Guido, Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy. E-mail: m.guido{at}rm.unicatt.it.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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As the exact basic etiology of PCOS remains unknown, continuing controversies and proposals arise about the preferential therapeutic strategy. In a large number of cases, the symptomatic therapy, according to the principal complaint of the patient, may represent the treatment of choice.
In this respect the combination of cyproterone acetate (CPA) and ethinyl estradiol (EE) has been largely used in past years to treat menstrual irregularities and androgenic clinical manifestations in PCOS women who were not interested in conceiving. The suppression of gonadotropin secretion and androgen production from ovaries and adrenal glands, the stimulation of SHBG production, along with the inhibition of peripheral androgen receptors are thought to account for the ability of these drugs to ameliorate hirsutism, acne, and alopecia in hyperandrogenic women with PCOS (2, 3).
A novel, low dose, estro-progestin combination, containing EE (30 µg) and drospirenone (DRSP; 3 mg; Yasmin, Berlex Laboratories, Wayne, NJ) has gained popularity beyond the contraceptive efficacy, thanks to the peculiar characteristics of its synthetic progestin (4). DRSP (6ß,7ß,15ß,16ß-dimethylene-3-oxo-17
-pregn-4-ene-21,17-carbolactone) is, indeed, an analog of the aldosterone antagonist spironolactone and possesses biochemical and pharmacological profiles closely related to those of endogenous progesterone (5). Previous studies demonstrated that adverse effects commonly associated with the use of other progestogens may be favorably reduced with an estro-progestinic drug containing DRSP (6).
The objective of the present study was to evaluate the effects of long-term administration of the EE/DRSP combination on the clinical and endocrino-metabolic features of women affected by PCOS.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The menstrual patterns were defined according to van Hooff et al. (9): regular cycles, length of cycle between 22 and 41 d; irregular cycles: oligomenorrhea, length of cycle between 42 and 180 d; polymenorrhea, length of cycle 21 d or less; amenorrhea, absence of menstruation for 180 d or more (two or more such irregularities during the past year).
A normal LH/FSH ratio was not considered an exclusion criterion (8). The presence of a late-onset adrenal enzyme defect was excluded by an ACTH test (250 µg, iv; Synachten, Ciba-Geigy, Basel, Switzerland) according to published criteria of New et al. (10). Significant liver (aspartate aminotransferase, alanine aminotransferase, total bilirubin, or alkaline phosphatase >2 times the upper limit of normal) or renal (serum creatinine, >1.8 ng/dl) impairment, neoplasm, cardiovascular disease, and unstable mental illness were considered exclusion criteria.
Because of the impact of body fat distribution on androgen levels and glucose metabolism (11, 12), waist to hip ratios were measured. Waist circumference was determined as the minimum value between the iliac crest and the lateral costal margin, whereas hip circumference was determined as the maximum value over the buttocks.
The ratio of testosterone x 100/SHBG was used to calculate the free androgen index (FAI).
Informed consent was obtained from each patient, and the study protocol was approved by our institutional review board.
At baseline, during the early follicular phase of a spontaneous or inducted (MAP, 10 mg/d for 7 d) menstrual cycle (d 3–7), the patients were hospitalized and underwent gynecological and medical examinations. All patients also underwent transvaginal ultrasonography. The grade of hirsutism was established using the Ferriman-Gallwey (F-G) score (13), in which hair growth in each of 11 androgen-sensitive zones is graded from 0 (none) to 4 (frankly virile). On the base of this method, four hirsutism levels were identified: score less than 8, no hirsutism; score of 8–16, low hirsutism; score of 17–25, moderate hirsutism; and score greater than 25, severe hirsutism. Patients using cosmetic measures were requested not to depilate for at least 1 month before each evaluation. We considered clinically meaningful the change from one level to a lower one.
After following a standard carbohydrate diet (300 g/d) for 3 d and fasting overnight for 10–12 h, blood samples were collected to perform the basal hormone assessment (testosterone, dehydroepiandrosterone sulfate, androstenedione, 17-hydroxyprogesterone, progesterone, FSH, LH, SHBG, prolactin, cortisol, estradiol, and IGF-I), serum lipid assays [triglycerides, total cholesterol, high and low density lipoproteins (HDL and LDL), very low density lipoprotein (VLDL), and nonesterified fatty acids (NEFA)], complete blood count, and hepatic and renal chemistries. Patients then underwent an oral glucose tolerance test (OGTT). On the following day, after another overnight fast, a euglycemic-hyperinsulinemic clamp was performed.
The OGTT was performed as follows. At 0900 h after overnight fasting, an indwelling catheter was inserted into the antecubital vein of one arm. Blood samples were collected basally, after ingestion of 75 g glucose in 150 ml water within 5 min, and at 30, 60, 90, 120, 180, and 240 min. Insulin, glucose, and C peptide were assayed in all samples.
The hyperinsulinemic-euglycemic clamp was performed after a 10-h overnight fast to estimate peripheral insulin sensitivity. At 0800 h, an iv catheter was placed in the antecubital vein for the infusion of glucose and insulin. Another catheter was placed in the dorsal vein of the controlateral hand for blood withdrawal and was warmed to 65 C with a warming box. A primed constant infusion of insulin (Actrapid HM, Novo Nordisk, Copenhagen, Denmark; 40 mIU/m2·min) was given (14). After reaching the steady state velocity for the insulin infusion within 10 min to achieve a steady state insulin level of approximately 100 µIU/ml (717 pmol/liter) during the clamp [range, 80–125 µIU/ml (574–897 pmol/liter)], a variable infusion of 20% glucose was begun via a separate infusion pump, and the rate was adjusted, on the basis of plasma glucose samples drawn every 5 min, to maintain plasma glucose between 79.3 and 89.9 mg/dl (4.40 and 4.99 mmol/liter). The plasma glucose level was determined by the glucose oxidase technique with a glucose analyzer (BD Biosciences, Palo Alto, CA). The glucose infusion rate during the last 60 min of a 2-h infusion was then taken as the estimate of peripheral insulin sensitivity and measured as M (milligrams per kilogram per minute). We preferred the use of this index as the measure of insulin sensitivity because the M/I ratio fails to narrow the range of individual sensitivity values (15). Plasma samples for glucose concentration were collected in tubes containing an inhibitor of glycolysis (sodium fluoride) to be analyzed within 5 h. Plasma samples for insulin and C peptide concentrations were placed in tubes standing in ice, centrifuged for 10 min at 1000 x g using a 4226 ALC Centrifuge (ALC, Milan, Italy), and remained frozen at –30 C until assayed.
The first day of the following menstruation, therapy with 30 µg EE and 3 mg DRSP (Yasmin, Berlex Laboratories) was started; the drug was taken from d 1–21 of the cycles, followed by a pill-free interval of 7 d, for 12 cycles. During the study, chronically stabilized therapies not interfering with the parameters under evaluation were permitted. The use of antidiabetic drugs was not allowed. Patients were recommended not to modify their usual diet. After three cycles of therapy, on the third day of the menstrual bleeding, patients returned to hospital for a transvaginal ultrasonographic examination for evaluation of the F-G score and basal hormone assessment. The patient returned to hospital at the sixth and 12th cycles and repeated the basal study.
On each visit, compliance with treatment was checked with a questionnaire about the side-effects and a subjective evaluation of the tolerability of the administered drug; the patients were also asked about incidental missed administrations, but all reported that they had correctly followed the scheduled treatment.
OGTT data were analyzed as the area under the curve after the glucose ingestion, calculated by the trapezoidal rule and expressed as picomoles per liter per 240 min for insulin and C peptide and as nanomoles per liter per 240 min for glucose.
Hepatic insulin extraction was estimated according to the following: difference between molar secretory areas of C peptide and insulin divided by molar secretory area of C peptide.
All hormone assays were performed with commercial RIA kits (Radim, Rome, Italy). The intra- and interassay coefficients of variation for all hormones were less than 8% and less than 15%, respectively. For each determination, all samples from the same patient were assayed simultaneously.
Plasma glucose concentrations were determined by the glucose oxidase technique with a glucose analyzer (Beckman Coulter, Fullerton, CA). Total cholesterol and triglyceride concentrations were determined by an enzymatic assay (Bristol, Paris, France). HDL concentrations were determined after precipitation of chylomicrons, VLDL, and LDL (Roche, Mannheim, Germany), and VLDL was separated (as the supernatant) from LDL and HDL by lipoprotein ultracentrifugations. A magnesium chloride/phosphotungstic acid technique was used to precipitate LDL from the bottom fraction after ultracentrifugation. NEFA were determined by an acryl-coenzyme A oxidase-based colorimetric method. All lipids assay were performed according to our standard laboratory procedures, as previously reported (11).
Statistical methods
All data are presented as the mean ± SD. Descriptive statistics, including the number of observations, mean, SD, minimum, and maximum were presented for continuous data at each visit. Distribution of the data were tested by the Kolmorov-Smirnov test to verify whether the samples follow a specified distribution. We found that the majority of the variables were not normally distributed.
On main efficacy variables, an ANOVA model for repeated measures was applied to describe the within-subjects time profile during the study. Within-group changes were tested with the Mann-Whitney U test.
For all analyses, P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The treatment was well tolerated, and correct intake of the drug was made by each of the remaining subjects. The most common adverse events either possibly or probably related to EE/DRSP were headache and breast pain, reported by two and three patients, respectively; no serious adverse events were related to EE/DRSP. Cycle control was acceptable in the entire studied group; the duration of withdrawal menstrual bleeding ranged from 4–7 d, and the intensity was referred to as normal by 80% of subjects, scarce by 13%, and abundant by 7%. No case of amenorrhea was observed. Breakthrough bleeding and/or spotting occurred in 20% of patients during the first cycle, and 13% of patients reported these disturbances during at least one of the following cycles.
Table 1
shows the impact of treatment with EE/DRSP on the grade of hirsutism in our PCOS patients. The efficacy of the treatment is documented by the ability of the EE/DRSP therapy to significantly decrease the mean F-G score within six cycles, with a significant trend of amelioration being maintained after 12 cycles of treatment (P < 0.01 for 6th and 12th cycles vs. baseline). At the end of the treatment, 10 patients experienced a change in the grade of hirsutism toward a lower level (see Patients and Methods), and four patients showed a reduction in F-G score, although it was not significant. Only one subject failed to respond to the treatment.
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shows the anthropometrical features of the studied patients at baseline and on each evaluation visit. The majority of the studied patients were normal weight (n = 12; body mass index range, 18.1–24.3), whereas 33% of them were obese (n = 6; body mass index range, 25.2–42). A slight trend toward s decrease in mean body weight was observed throughout the treatment period, with no significant differences between normal weight and obese subjects. At the end of the study, a weight gain of greater than 4 kg occurred only in one normal weight patient, whereas a weight loss greater than 4 kg was obtained in three patients (two obese and one normal weight). No significant change in body fat distribution emerged from the analysis of waist/hip ratio values.
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also depicts the variations in the endocrine features of the patients during treatment. A significant decrease in mean LH levels was observed from the third cycle of drug assumption. Ovarian and adrenal androgen plasmatic concentrations significantly decreased during treatment. At the same time, mean SHBG levels rose markedly, achieving by the 12th cycle an increase of 384% compared with baseline values. As a consequence of such modifications, a significant decline in FAI was observed after three, six, and 12 cycles of therapy compared with baseline. The treatment with EE/DRSP induced an increase in cortisol plasma concentrations, which attained statistical significance at the sixth and 12th cycles of therapy. The other hormonal parameters under evaluation did not change significantly during treatment.
The main metabolic features of the treated patients during the trial are shown in Fig. 1. None of the studied subjects exhibited impaired glucose tolerance at baseline; moreover, EE/DRSP treatment did not produce any deterioration in the glycemic and insulinemic responses to glucose load. Peripheral insulin sensitivity showed a trend toward a decline, although not statistically significant, in the first half of treatment period, followed by a significant rise in the last six cycles of therapy, so that values at the end of treatment were similar to those observed at baseline. An analog trend of modification was seen for hepatic insulin extraction.
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shows the changes in lipid profile during treatment. Serum triglyceride levels rose at the sixth cycle (83% increase vs. baseline values); however, no evidence of further increase occurred in the following six cycles of drug administration. A similar relative trend was seen for total cholesterol and LDL cholesterol serum concentrations after six cycles (31.4% and 34.2% increases, respectively); however, in the remaining six cycles of treatment, total cholesterol plasma levels did not change, whereas LDL cholesterol plasma concentrations tended to decrease (27% decrease). A parallel increment in serum HDL cholesterol levels was induced by the therapy. As a result of a balance between such modifications, the mean HDL/LDL and total cholesterol/LDL ratios remained unvaried throughout the study period (Fig. 3). A trend toward improvement, although devoid of statistical significance, was found in serum NEFA concentrations during treatment (data not shown).
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Discussion |
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DRSP is the unique progestin derived from 17-spironolactone and shares progesterone’s antiandrogenic and antimineralcorticoid properties, with no androgenic, estrogenic, glucocorticoid, or antiglucocorticoid activity (19, 20). The present study shows that the EE/DRSP pill administered for 12 consecutive cycles is able to significantly improve clinical manifestations of hyperandrogenism in patients with PCOS. This finding reflects the above-mentioned properties of DRSP, which is thought to act through different mechanisms. At a central level, beyond the ability of the EE/DRSP combination, similar to other estro-progestinic drugs, to inhibit gonadotropin release from the hypophysis (21), DRSP seems to possess a strong antigonadotropic activity per se; previous studies using animal models demonstrated that administration of this progestin alone resulted in a significant reduction of plasma LH levels (22, 23). Several lines of evidence documented augmented LH pulse amplitude and frequency and increased expression of LH receptors in the ovaries of women with PCOS, so that many investigators consider these alterations to be central in the pathogenesis of the syndrome or at least of the hyperandrogenism that frequently characterizes it (24, 25). In this respect, the significant decrease in serum LH observed in the present study appears particularly beneficial, as it was obtained in PCOS patients showing elevated pretreatment LH values. Our results are similar to those obtained by Falsetti et al. (26) using the combination EE and CPA; in patients suffering from PCOS, a significant decrease in plasma LH was obtained by these investigators after six treatment cycles, and a significant trend for improvement was maintained until the 12th cycle, with no further variations after 60 cycles of EE/CPA administration. The percent decrease in LH levels obtained with EE/CPA is comparable with that observed in our PCOS patients after treatment with EE/DRSP.
At the ovarian level, the combination of EE/DRSP has been demonstrated to directly reduce the production of testosterone and its precursors by an inhibition of the steroidogenic enzymatic activities (19). Consistent with these data, a significant reduction in plasma testosterone levels was achieved in our patients from the third cycle of therapy. However, a contemporaneous decrement in 17-hydroxyprogesterone and dehydroepiandrosterone sulfate occurred in the studied group, indicating the capacity of the DRSP to also act on the adrenal enzymatic chain. It is conceivable, in fact, that this feature resembles the capacity in other progestins, such as CPA, to directly act on the adrenal cortex (27).
In addition, DRSP has been documented to display antiandrogenic activity at the peripheral level by repression of androgen receptor-mediated transcription. In fact, previous studies, based on in vitro trans-activations assays, have demonstrated for DRSP competitive binding to the androgen receptor that is intrinsic to its molecular structure and dose dependent (20). The blockade of androgen receptors in the skin (sebaceous glands and hair follicles) represents an additional mechanism implicated in the positive effects of EE/DRSP on the cosmetically unacceptable signs of hyperandrogenism in PCOS women (acne, seborrhea, and hirsutism) (28).
The strong capacity to enhance SHBG production completes the wide antiandrogen spectrum of activity of DRSP. Although the reduced SHBG levels observed in a great percentage of PCOS patients are partially related to the higher incidence of obesity compared with that in the general population, a defect in the hepatic synthesis of this globulin has also been found in lean PCOS women and is believed to constitute an intrinsic feature of the syndrome. Our PCOS patients had baseline SHBG levels at the lower limit of the normal range. A very marked increase in this parameter and, in concert, a decrease in FAI occurred during treatment. These effects are probably related to the specific chemical properties of DRSP; unlike 19-nortestosterone derivatives (norethindrone and levonorgestrel), DRSP does not counteract the stimulatory effect of EE on SHBG synthesis and does not prevent androgen binding with it (5, 29). Furthermore, the magnitude of increase in plasma SHBG concentrations obtained in our PCOS patients (384%) is greater than those reported in literature after administration of 17-hydroxyprogesterone derivatives, such as CPA (2, 3). These actions may account for the marked and lasting amelioration of F-G scores obtained in our patients.
Concerning other hormonal parameters, an increase in plasma cortisol levels was observed during the treatment period. To our knowledge, previous studies using the EE/DRSP combination did not consider the changes induced by this therapy on the hypothalamus-pituitary-adrenal axis. However, several reports exist on the importance of the hippocampal mineralocorticoid receptor in regulation of the basal activity of the hypothalamus-pituitary-adrenal system. The blockade of this receptor obtained with the administration of spironolactone has been demonstrated to cause a significant increase in plasma cortisol, probably mediated by a rise in ACTH and ß-endorphin (30, 31). It is conceivable that the strict analogy between the molecular structures of spironolactone and DRSP could justify a similar mechanism of action on the structures of the central nervous system.
A positive profile of action with respect to body weight and blood pressure was seen for EE/DRSP in our patients. By activation of the renin-angiotensin-aldosterone system, EE may increase fluid volume in the body, thus producing weight gain and a rise in blood pressure in susceptible women (32). DRSP has a strong antimineralcorticoid activity and acts primarily as a potassium-sparing diuretic (33). Similar to the physiological effects of progesterone in the luteal phase of spontaneous cycles, DRSP is able to induce sodium excretion and a compensatory increase in renin secretion, plasma renin activity, angiotensin II, and plasma aldosterone, thus minimizing the estrogen-related water retention (34). These actions may explain the absence of any significant impact of the therapy on body weight in our PCOS patients and the lack of changes in blood pressure during treatment with EE/DRSP. Data from the present study are in line with the results obtained in normo-ovulatory women treated with EE/DRSP by other investigators, and as weight gain is commonly recognized as one of the main causes of estro-progestinic treatment interruption, our results confirm that an estro-progestinic containing DRSP may provide an advantage in terms of patient compliance (4, 35).
Beyond the effects on body weight and blood pressure, in past years the use of estro-progestinic drugs has been associated with an increased risk of cardiovascular disease because of their negative impact on lipid and glyco-insulinemic metabolism (36, 37). When a dose of EE less than 50 µg/d is used, these effects seem to be related to the progestin contained in the combination (38). More recently, an international multicenter study by the WHO concluded that the use of estro-progestinic drugs is related to an increased risk for myocardial infarction only in women with recognized cardiovascular risk factors (39). In this respect, although it is generally recognized that PCOS is associated with a higher incidence of diabetes mellitus and cardiovascular disease, it remains controversial whether this syndrome represents a condition at risk per se, or whether the combination of characteristic features of such disorders (obesity, hyperinsulinemia, and hyperlipidemia) may account for the high incidence of long-term health consequences (40). On this basis, the effect of treatment with EE/DRSP on carbohydrate and lipid metabolism was carefully examined. Interestingly, unlike other monophasic combined estro-progestinic, no impairment in glucose tolerance emerged from analysis of the glycemic response to OGTT during treatment in our PCOS patients. In addition, administration of EE/DRSP did not affect insulin secretion from the pancreas, which remained stable throughout the study. No significant variations in peripheral insulin sensitivity and clearance were suggested from the comparison between values at the end of therapy and at baseline.
Controversial data have been reported on the changes occurring in lipid profile under estro-progestin treatment, and the available results on the effect of DRSP on lipid assessment are not univocal (34, 35). In the present trial, although triglyceride levels rose significantly during treatment with EE/DRSP, they remained within the normal range in all of the PCOS subjects studied. For total and LDL cholesterol, a significant increase was observed at the sixth cycle of treatment; however, a slight trend toward improvement occurred in the following six cycles of therapy. Mean values of both parameters remained within the normal range during the 12 cycles of treatment. This result is in line with those obtained in previous studies using preparations containing 30 µg EE plus desogestrel, gestodene, or CPA (38, 41).
A significant increase in HDL cholesterol serum concentrations was induced by treatment in our PCOS women. As a consequence of these variations, the HDL/LDL ratio remained unchanged. Most studies to identify the atherogenic risk factors in the female population found that decreased HDL levels and HDL/LDL ratio together with a decline in the total cholesterol/LDL ratio are the parameters more closely related to an increased incidence of cardiovascular disease in the female population (42, 43). In this respect, the changes in lipid profile induced by EE/DRSP therapy along with the favorable effects of the drug on blood pressure, body weight, and insulin metabolism do not seem to negatively affect the epidemiologically determinant factors for coronary heart disease.
In conclusion, the antiandrogenic and antimineralcorticoid activities of DRSP resulted in remarkable benefits with regard to biochemical and clinical signs of hyperandrogenism, and this makes the EE/DRSP formulation a valid therapeutic option to treat PCOS women who are not interested in conceiving. Similar to the majority of the pilot studies, issues of both selection and information bias could be potentially operative. Nevertheless, data obtained from our patients are of interest and encourage further investigations.
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Footnotes |
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Received July 8, 2003.
Accepted March 9, 2004.
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, P < 0.01 vs. sixth cycle.
