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Department of Obstetrics, Gynecology, Reproductive Medicine, and Public Health (N.C.-B.), Hospital Tenon, 75020 Paris, France; Equipe d’Accueil 1533 (N.C.-B., P.B.), Institut Fédératif de Recherche and University Pierre and Marie Curie, 75005 Paris, France; Department of Obstetrics and Gynecology (A.P.-K.), University of Liege, 4000 Liege, Belgium; and Endocrinology Unit (P.B.), Hospital Saint Antoine, 75012 Paris, France
Address all correspondence and requests for reprints to: Nathalie Chabbert-Buffet, Department of Obstetrics and Gynecology, Hospital Tenon, 4 Rue de la Chine, 75020 Paris, France. E-mail: nathalie.chabbert-buffet{at}tnn.aphp.fr.
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Abstract |
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Objective: We evaluated the effects of a new progesterone receptor modulator, VA2914, administered continuously for 3 months, on ovulation and endometrial maturation.
Design, Settings, and Patients: Forty-six normal women were included in a prospective, placebo-controlled, randomized trial, conducted in four referral centers.
Intervention: VA2914 (2.5, 5, or 10 mg/d) was administered continuously for 84 d. Pelvic ultrasound (treatment d 67 and 77), hormonal monitoring (FSH, LH, estradiol, and progesterone on treatment d 59, 63, 67, 70, 74, 77, 80, and 84), and endometrial biopsy (treatment d 77) were performed.
Main Outcome Measure: Ovulation inhibition was assessed by the absence of progesterone values above 3 ng/ml at any time during treatment month 3.
Results: Anovulation was observed in 81.8% women in the 5-mg group and 80% in the 10-mg group, and amenorrhea occurred in 81.2 and 90% of cases in the 5- and 10-mg groups. We did not detect any cases of endometrial hyperplasia despite estradiol levels that remained in the physiological follicular phase range throughout treatment cycle 3.
Conclusions: Continuous low-dose VA2914 can induce amenorrhea and inhibit ovulation without down-regulating estradiol levels or inducing endometrial hyperplasia in normal women. Long-term studies with a larger population are required to confirm the contraceptive efficacy of this regimen.
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Introduction |
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Progesterone receptor modulators (PRMs) with mild or potent antiprogestin activity are a class of progesterone receptor ligands that may exert contraceptive activity by different mechanisms, including blockade of ovulation and endometrial desynchronization (2, 3, 4). Mifepristone (5) was the first PRM to be used in women and has been evaluated for contraceptive use (6, 7, 8, 9). Low doses (up to 2 mg/d) cause a delay in ovulation and/or disturb the endometrial maturation without marked effects on follicular development (10). Its use is associated with good contraceptive efficacy although there is limited experience reported. Higher doses usually suppress follicular development (6, 11, 12). Finally, long-term use of RU 486 has been associated with increased endometrial thickness, in part due to excessive gland development without hyperplasia (13).
VA2914 is an orally active steroidal PRM initially developed by the National Institutes of Health (Contraceptive Development Branch) under the name of CDB-2914. VA2914 demonstrates potent progesterone antagonist activity in vitro and in vivo (14). However, its binding and antagonist potency with respect to the glucocorticoid receptor is significantly reduced, compared with that of mifepristone, indicating that VA2914 belongs to a new class of dissociated PRMs that have reduced antiglucorticoid activity (15, 16). The pharmacological effects of VA2914 have been examined in a variety of animal models, and the compound has been shown to exhibit antifertility activity during continuous low-dose administration (17, 18, 19). This compound is currently being evaluated for treatment of uterine leiomyoma and contraception.
We report the first prospective, randomized, controlled trial using VA2914 for ovulation inhibition. Three doses of VA2914 (2.5, 5, 10 mg/d) were administered continuously for 3 month and compared with placebo.
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Subjects and Methods |
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Women in good general health, aged 18–35 yr, were eligible for enrollment if they had regular menstrual cycles of 25–32 d in length, accepted the use of condoms, or were not sexually active. Exclusion criteria included pregnancy; use of oral contraception, intrauterine device, or other hormonal treatment (including herbal and homeopathic medication); use of agents known to induce hepatic P450, cardiovascular, hepatic, renal, or gastrointestinal disorder; metrorrhagia; cancer; depression; mental illness; epilepsy; migraines; and overweight or underweight women (body mass index < 16 or > 28 kg/m2).
Study design
This prospective, randomized, double-blind study was conducted at the Endocrine Unit of Hospital Saint Antoine and the Department of Obstetrics and Gynecology at Hospital Antoine Béclère (Clamart, France) as well as two clinical sites within the Department of Obstetrics and Gynecology at Liege University (Belgium) from October 2004 to April 2005.
The study was conducted in accordance with the guidelines of the Declaration of Helsinki, and the ethics committees of St. Antoine Hospital and Liege University approved the study protocol. Participants were recruited by community advertisements and primary care providers.
Due to the exploratory objectives of the study, the sample size was not based on statistical considerations but on usual practices in similar exploratory phase II studies. To obtain complete data from 10 women for each arm of the study, we set out to enroll 12 subjects per treatment group, thus a total inclusion of 48 subjects. Randomization was performed in blocks of four and stratified by study site to ensure even distribution of the treatment groups.
HRA Pharma (Paris, France) provided VA2914 (2.5, 5, and 10 mg) and placebo formulated in matching tablets in identical packaging, and funded all study-related expenses.
After having provided informed consent, participants were included in a pretreatment cycle designed to document normally ovulating spontaneous cycles and screen for exclusion criteria. Each participant received a home study card to complete daily with information on vaginal bleeding, side effects, treatment intake, concomitant treatments, and home urinary LH surge monitoring (Dectra Pharm, Strasbourg, France). Bleeding episodes were recorded as lighter than, similar to, or heavier than a normal period, with light bleeding within 2 d of an endometrial biopsy disregarded for analysis purposes.
Volunteers exhibiting an ovulatory baseline cycle (luteal phase P > 3 ng/ml on d 21) and normal transvaginal ultrasound (on d 21), normal laboratory exams, and negative urinary human chorionic gonadotropin were randomized. Treatment was started on the first or second day of menstrual bleeding after the baseline cycle and taken daily in a continuous fashion for 84 d. Participants were instructed to return their treatment packs at the end of the study to confirm compliance.
To determine the effects of the compound on cervical mucus, the Insler score (composite score of volume, elasticity, and Spinnbarkheit or ability to fern) was evaluated on treatment d 67 and compared with baseline (cycle d 11).
Endometrial biopsies were performed using a pipelle (CCD) and a cervical dilatator (Os Finder, Medsys SA, Gembloux, Belgium). Endometrial biopsies were scheduled 6–8 d after the LH surge during the baseline cycle as well as on treatment d 77 in the absence of a detectable urinary LH surge or 6–8 d after the LH surge between treatment d 57 and 84.
Pelvic ultrasounds were performed twice during treatment (d 67 and 77 or on the day of the endometrial biopsy) to determine ovarian size and structure, number and size of follicles, and endometrial thickness.
Blood samples were collected for the baseline cycle and treatment d 59 to determine biochemical and hematological markers as well as concentrations of serum cortisol, TSH, and prolactin. Blood samples were taken on d 3 and 21 of the baseline cycle and on treatment d 59, 63, 67, 70, 74, 77, 80, and 84 to measure serum estradiol (E2), FSH, LH, and progesterone (P). Concentrations of VA2914 and its principal metabolite (VA 3877A) were measured on treatment d 59 and 80. Samples were collected between 0800 and 0900 h before drug intake.
Hormonal analysis
Serum FSH and LH concentrations were measured by sensitive fluoroimmunometric assays (AutoDelphia; Wallac, Inc., Turku, Finland). FSH, within- and between-assay coefficients of variation (CVs) were 1.3 and 1.5%, respectively, at the level of 2 IU/liter. The detection limit was 0.01 IU/liter. For LH at the level of 0.3 IU/liter, CVs were 6.7 and 1.8%, respectively. The detection limit was 0.01 IU/liter.
E2 was measured by a direct RIA using Diasorin reagents (Anthony, France). The cross-reactivity of estrone and estriol was only 0.6%. CVs at the level of 50 pmol/liter were 3.6 and 10.4, respectively, and at the level of 260 pmol/liter, 2.9 and 6.8%, respectively.
P was measured by RIA using CIS Biointernational reagents (Gif sur Yvette, France). The only significant cross-reacting steroids were: deoxycorticosterone, 6.2%; 20
-dihydroprogesterone, 2.2%; and 6ß-dihydroprogesterone, 2.1%. CVs at the level of 10 ng/ml were 3.5 and 4.5, respectively, and at the level of 18.8 ng/ml, 4.4 and 4%, respectively.
Plasma concentrations of VA2914 and VA3877A, the compound’s main monodemethylated biologically active metabolite (16), were determined by Aster-Cephac (St. Benoît, France) using a fully validated bioanalytical method that couples HPLC with tandem mass spectroscopy (20).
Endometrial samples analysis
Endometrial biopsy samples were fixed in formalin and processed for routine light microscopy. Specimens were evaluated in a blinded fashion by two independent pathologists who dated by standard criteria (21, 22) and classified them as proliferative or early (d 17–19), mid- (d 20–22), or late (d 24–26) secretory according to the Noyes criteria (21, 22). When difficulties in biopsy dating were noted, unusual findings were written by pathologists in a specific comments section.
Data analysis and statistical methods
The primary outcome measure for this pilot trial was the absence of P levels above 3 ng/ml (23) at any time between treatment d 57 and 84 (absence of luteal function).
Secondary outcome measures included the following clinical and biological parameters measured after 84 d of treatment, analyzed in comparison with control cycles: cycle length, menstrual bleeding patterns, follicular and luteal phase levels of plasma E2 and P, diameter of the dominant follicle (ultrasound), endometrial maturation based on ultrasound (endometrial thickness and echo pattern), and biopsy.
Finally, this trial also aimed to characterize the steady-state pharmacokinetics of VA2914 on chronic administration.
Analyses of the treatment effect were performed for all randomized subjects that completed the study (84 d of treatment) for whom primary efficacy data were available (efficacy valuable population). Analyses were repeated for the per-protocol population after exclusion of major protocol deviations. Analyses of safety data were performed for all randomized and treated subjects that received at least one dose of study medication (safety valuable population).
A pair-wise comparison of the proportion of subjects without ovulation was performed between each of the three VA2914 treatment groups vs. placebo using a Fisher’s exact test, after a Hochberg procedure for multiplicity to maintain the overall type I error to 0.05. In addition, a Jonckheere-Terpstra trend test was used to evaluate whether the treatment effect was correlated with the dose of VA2914 administered. Comparison of the secondary outcome criteria and safety parameters were performed between treatment groups using a Fisher exact test for categorical variables and a Kruskall Wallis test for continuous variables. All statistical analyses were performed using two-tailed tests at a 5% significance level. Data management and statistical analyses was performed by Altizem (Nanterre, France) using Oracle Clintrial (SAS, Cary, NC).
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Results |
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Forty-six healthy women participated in the study, and the four groups were evenly balanced (Table 1
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Plasma levels of VA2914 and its metabolite VA 3877A increased in a dose-dependent manner as shown in Table 2.
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The threshold dose for inhibition of ovulation was 5 mg/d group (Table 3). A statistically significant dose-dependent trend was observed (P < 0.001).
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). FSH levels were normal (Fig. 1B). No statistical difference between groups was observed.
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). Persistently low levels of progesterone (i.e. < 3 ng/ml) were observed in most subjects in the 5- and 10-mg groups, although a temporary rise in LH levels was observed in four women (Fig. 1D). No statistically significant impairment of follicular growth was observed on ultrasound (US) scan. In fact, at least one growing follicle (diameter > 14 mm) was observed on treatment d 67 and 77 in 54.5% women in the 2.5-mg group, 63.6% women in the 5-mg group, and 60% women in the 10-mg group (P = ns vs. placebo for each treatment group). Ovarian cysts (>30 mm) occurred in one patient in the placebo group, one patient in the 2.5-mg/d group, four patients in the 5-mg/d group, and three patients in the 10-mg/d group. All cysts but one spontaneously resolved, whereas treatment was maintained. In one patient, who became pregnant shortly afterward, the cyst was still present at the end-of-study visit. It was subsequently diagnosed as an endometrioma during laparoscopy performed after delivery.
Cervical and endometrial effects
No significant modification of the Insler score was observed in treated women, compared with baseline (data not shown).
Amenorrhea rates and mean bleeding days per month for each treatment group are presented in Table 3. Bleeding days decreased with successive month of treatment, with amenorrhea in nearly all subjects in the 5- and 10-mg groups by treatment months 2 and 3. Breakthrough bleeding was reported in four women in the placebo group, four women in the 2.5-mg group, three women in the 5-mg group, and one woman in the 10-mg group.
Endometrial appearance on US did not show any polyps or thickening, compared with baseline, and there was no significant difference between treatment groups in terms of endometrial thickness (Table 3). In terms of echo pattern, a constant three-layer appearance was associated with inhibition of ovulation in treated women. This specific three-layer pattern included two hyperechogenic layers delineating a hypoestrogenic pattern, reminiscent of secretions in the uterine cavity.
Endometrial histology is summarized in Table 3. The degree of concordance between the two pathologists was 75%. The biopsies predominantly showed usual patterns of a secretory endometrium in the 2.5-, 5-, and 10-mg treatment groups, even in women with progesterone levels consistently less than 3 ng/ml throughout the treatment d 57–84 period. The glands exhibited cystic glandular dilatation for one patient in the 10-mg group and one in the 2.5-mg group (Fig. 2). Both of these individuals had persistently low levels of progesterone. Polyps were diagnosed in one woman at baseline and in one woman in the 2.5-mg group during treatment, but neither of them was detectable on US nor showed any atypia.
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Reported physical side effects are shown in Table 4. There were no blood pressure changes or weight gain.
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Two cases of hyperthyroidism were reported during the trial, both in the 5-mg group. Both women were subsequently found to have had a subclinical disease (abnormal TSH and thyroid antibodies) before enrollment.
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Discussion |
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. This PRM effect comes in contrast with what is commonly observed with progestins (24, 25, 26). Within the limits of our study, and in particular with the sampling protocol, transient increases in LH levels were observed in four women, in whom increases in P were absent (Fig. 1E). These LH increases may be inappropriate, in duration, kinetics, or timing in relation to follicular maturation, and are suggestive of an out-of-phase low LH response that has been associated with anovulation or luteinized unruptured follicle (27). The absence of a follicular response to an LH increase may also be related to a direct ovarian effect already described in the rat (28) as well as in women. In normal volunteers, mifepristone seems to act on FSH action, oocyte maturation, and granulosa cell function (29, 30, 31, 32). One of the key features of our study is the maintenance of follicular phase levels of E2 in treated women. This is indeed a significant advantage as far as clinical tolerance and bone fractures are concerned. This is in contrast to the consequences of using high-dose progestins (33).
The contraceptive effects of PRMs have been evaluated in several studies. The effects of mifepristone appear to be dose dependent: daily doses of 2–10 mg mifepristone suppress follicular development, block the LH surge, and delay ovulation (10, 34), with no pregnancies reported after 200 months in 50 women who received either 2 or 5 mg mifepristone daily as their only contraceptive method (8), whereas lower doses are usually inefficient (35). Administration of daily doses of 10–100 mg asoprisnil for 28 d (36) resulted in an increase in cycles length that was proportional to the dose of asoprisnil. In contrast with mifepristone, the effects on ovulation were inconsistent and lacked dose dependency. This is also in contrast with the results observed for VA2914.
Endometrial effects of PRMs are still a subject of contention because of the potential unopposed effect of persistent follicular phase levels of E2. Amenorrhea is induced in most studies, but histological data are controversial. In our study, bleeding patterns were excellent because 80 and 90% women were amenorrheic in the 5- and 10-mg/d groups, respectively, by treatment month 2. Very few days of spotting were reported by the participants. This may be a significant clinical advantage because once informed about the expected occurrence of amenorrhea during treatment, women may reach a high compliance rate (37).
The histological patterns were those of a normal cyclic endometrium and could be easily classified as proliferative, secretory, or atrophic endometrium, except for two subjects exhibiting cystic dilated glands. This aspect, which is specific to PRMs, has never been described before except during a trial with mifepristone (38) and very recently in a trial with asoprisnil (39). Secretory endometrium was also reported in women exhibiting low progesterone levels on the day of the endometrial biopsy, suggesting that the compound may also exert an agonistic action on the endometrium. The same aspects have been described with other PRMs [asoprisnil, the prototype of the so-called mesoprogestins (39) and mifepristone at high dose as reported by Gravanis et al. (40) in women with inactive ovaries]. The impact of such a finding on the contraceptive efficacy is probably mild because the main effect of PRMs is to block the LH surge. In most studies ovulation does not occur in 80–90% of volunteers. Finally, although anovulation does not occur in all patients, no pregnancy was observed in the Baird study (8), suggesting that the effects on the endometrium and the inhibition of ovulation are synergistic into producing efficient contraception. Obviously more studies are needed to assess this effect. Two women in the placebo group had proliferative endometrium, in both cases due to mistiming of their ovulation schedule, as is often the case in clinical trials. Samples without material obtained during treatment may be related to endometrial atrophy, defined as an endometrial thickness measured on US scan on the day of the endometrial biopsy less than 5 mm, according to the European Medicines Agency (41), for one subject in the placebo group, two subjects in the 2.5-mg group, and one subject in the 10-mg group. In other cases the lack of material may be related to operator failure. No specific patterns such as disordered architecture or abnormal vessels were present. Polyps without atypia were diagnosed on endometrial biopsies for the baseline cycle in one patient and in one patient from the 2.5-mg/d group, i.e. without any correlation with the dose of VA2914. There were no signs of endometrial hyperplasia in any of the specimens studied.
Very low doses of mifepristone (0.5–1 mg/d) induce a delay in endometrial maturation (42, 43, 44). The endometrium is inactive in women treated with low doses (2 or 5 mg) (7). These low doses of mifepristone are associated with a decrease in menstrual bleeding or even amenorrhea. Amenorrhea is probably related, at least in part, to the inhibition of ovulation (7, 8, 42). In long-term studies with mifepristone, endometrial hyperplasia has occasionally been observed. Simple endometrial hyperplasia was observed in 25% of women receiving 10 mg mifepristone daily for 6 months (13, 45) for the treatment of myoma. Administration of 1 mg mifepristone daily for 5 months was associated with increased endometrial thickness and dilated glands in 25 and 43% of the monophasic cycles, respectively (35). Mifepristone’s endometrial effects are incompletely understood but need to be compared with studies of the endometrium, using contraceptive methods that do not suppress E2 production in long-term studies. Endometrial samples in our study did not show the same appearance, but this may be related to the shorter treatment duration. Additionally the definition of a new histological classification may be useful to allow proper description of the aspects observed under PRM treatment. Such a modified classification has already been suggested (46) to allow a more accurate description of endometrial hyperplasia (47). In a recent expert meeting organized by the National Institutes of Health on the endometrial effects of PRMs, a panel of pathologists indicated that the current World Health Organization classification was not suitable to describe the non physiological secretory aspect observed during PRM treatment, including disordered architecture with desynchronization of glandular and stromal compartments, mixture of proliferative and secretory endometria with association of mitotic and apoptotic figures, mitotic and secretory aspects, and cystic glandular dilatation (McFarland, H., and D. Blithe, submitted for publication). In our study, nonphysiological secretory aspects were observed, with a discrepancy between a compact stroma and glands with morphological characteristics of an early secretory phase. Occasionally, rare mitoses could be observed in these glands.
General side effects were those usually observed with oral contraceptives. Pelvic pain was reported to be moderate or mild in this study. It was associated with ovarian cysts larger than 30 mm in two women. All of the cysts had disappeared on treatment d 77 except for one, subsequently diagnosed as endometriotic. Functional cysts have been described with progestins and in a study using continuous mifepristone 1 mg/d (35) for inhibition of ovulation. In this study recording incomplete inhibition of ovulation, cysts occurred in 22% women and may be due to the incomplete suppression of gonadotropins. No clear relationship between gonadotropin levels and the occurrence of cysts was identified.
Acne was reported by 18 and 25% of women in the 5- and 10-mg/d groups, respectively. Although the lesions were considered to be mild, this frequency is significant. None of the patients in the placebo or 2.5-mg/d group reported acne. Interestingly none of the patients had a significant increase in testosterone levels, although LH levels were not low as previously mentioned. In addition, the affinity of VA2914 for the androgen receptor is very low, compared with other androgen receptor ligands (relative binding affinity, 15%, compared with dihydrotestosterone; Blye, R., unpublished data).
In this preliminary study, we clearly demonstrate that the threshold dose for ovulation inhibition with continuous low-dose VA2914 is 5 mg/d. This dose induces amenorrhea in more than 80% of women after 2 months of treatment, whereas E2 levels remain in the physiological follicular phase range. Importantly, endometrial biopsies always showed an absence of hyperplasia, although longer studies are necessary. General and gynecological safety data were satisfactory. Further studies with long-term follow-up are required to evaluate the contraceptive potential of low doses of VA2914 and their endometrial impact. Because of their excellent tolerance and their capacity to inhibit ovulation without suppressing endogenous E2, PRMs are leading candidates for EE-free contraception.
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Acknowledgments |
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Footnotes |
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The study was registered (www.clinicaltrials.gov; ID NCT00456924).
Disclosure Summary: E.G., a member of the VA2914 Study Group, is an employee of HRA Pharma, the pharmaceutical company that is developing VA2914 and sponsored this study. None of the other authors has any commercial relationship with HRA Pharma besides the funding of the study.
First Published Online June 19, 2007
1 See Acknowledgments for members of the VA2914 Study Group. 
Abbreviations: COC, Combined oral contraceptive; CV, coefficient of variation; E2, estradiol; EE, ethinyl estradiol; P, progesterone; PRM, progesterone receptor modulator; US, ultrasound.
Received December 19, 2006.
Accepted June 13, 2007.
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