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Department of Reproductive Medicine (A.H.B.), Leeds General Infirmary, Leeds, LS2 9NS United Kingdom; Erasmus Medical Center (A.G.M., B.C.F.), 3000 CA Rotterdam, The Netherlands; Catharina Ziekenhuis (B.C.S.), 5602 ZA Eindhoven, The Netherlands; University Hospital of Antwerp (M.A.R.), B-2650 Edegem, Belgium; Center for Reproductive Medicine (P.D.), Dutch-Speaking Brussels Free University, 1090 Brussels, Belgium; and NV Organon (M.J.S., B.M.M.), 5340 BH Oss, The Netherlands
Address all correspondence and requests for reprints to: Bernadette Mannaerts, M.Sc., Clinical Development Department, P.O. Box 20, 5340 BH Oss, The Netherlands. E-mail: b.mannaerts{at}organon.com.
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Abstract |
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After drug administration, the induced follicular response varied widely among subjects in each dose group. The percentage of subjects with a follicular response (at least one follicle 
10.0 mm) increased with the dose (P < 0.01) and was 10, 31, 70, 73, and 82% in the placebo and 7.5-, 15-, 30-, and 60-µg treatment groups, respectively. In responding subjects, the average maximum number of follicles was 4.0, 7.6, 13.4, and 20.0, respectively, which was reached at 6.5, 6.9, 6.6, and 8.2 d after a single dose of 7.5, 15, 30, and 60 µg FSH-CTP, respectively. The dose-response for the number of follicles was statistically significant within the dose range tested (P < 0.01). Peak serum inhibin-B levels were significantly correlated with serum estradiol (E2) levels (r = 0.84, P < 0.01), and peak concentrations of inhibin-B and E2 correlated with the number of follicles observed at the same time point (for both hormones; r = 0.47, P < 0.01). Overall per treatment group, serum E2 and inhibin B concentrations significantly increased only in the two highest FSH-CTP dose groups, reaching peak concentrations at d 3 in the 30-µg group and at d 5 in the 60-µg group. Thereafter these hormone values declined rapidly, returning to baseline within 1 wk after FSH-CTP administration.
In total, nine of the 55 treated subjects (16.4%) ovulated after drug administration: one subject in the placebo group, two subjects in the 7.5-µg group, three subjects in the 15-µg group, two in the 30-µg group, and one in the 60-µg group. Three subjects had monofollicular ovulation after placebo (n = 1) and a single dose of 15 µg FSH-CTP (n = 2). In two subjects with too many preovulatory follicles, (multiple) ovulation was prevented by GnRH antagonist administration.
Thus, a single low dose of long-acting FSH-CTP was able to induce one or more follicles to grow up to ovulatory sizes, but the anovulatory status was not reversed because the incidence of subsequent (mono)ovulations was low.
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Introduction |
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Alternatives to CC administration may be treatment with an FSH preparation that would mimic the CC-induced flare-up of FSH (7) without a concomitant rise in LH and would also lack any antiestrogenic effect. Of specific interest in this respect might be the novel long-acting FSH [FSH-carboxy terminal peptide (CTP), ORG 32489, corifollitropin alfa], which is a recombinant molecule constructed by coupling the carboxy terminal peptide of the ß-subunit of human chorionic gonadotropin to the FSH ß-subunit. Like recombinant wild-type FSH, FSH-CTP is expressed by a Chinese hamster ovary cell line that is able to produce glycoprotein hormones with carbohydrate side chains that closely resemble their native structure. FSH-CTP contains four N-linked carbohydrate chains (
52, 78, ß7, and ß24) and 4 O-linked carbohydrate chains at the carboxy terminal peptide (ß115, ß121, ß126, and ß132). These four O-linked carbohydrates, rather than the addition of the peptide, causes a 3- to 4-fold increased in vivo half-life, compared with wild-type recombinant (r)FSH (8). Phase I studies in men with hypogonadotropic hypogonadism (9) and female volunteers of reproductive age (10) have suggested that FSH-CTP treatment is safe and that the drug is nonimmunogenic in humans. After a single sc injection in women, peak levels are reached on average after 36 h, in comparison with 10 h for wild-type rFSH, and the terminal half-life is between 60 and 75 h (10). A recent study in in vitro fertilization patients indicated that FSH-CTP administered as a single dose of 120–240 µg is a potent inducer of multiple follicular growth for controlled ovarian stimulation (11). It is hypothesized that in anovulatory women, after a single low dose of FSH-CTP, the initial increase in FSH may stimulate several immature follicles, whereas the declining levels that will be reached 2–3 d after injection may prevent the smaller follicles from continuing their growth (12) and therefore lead to monofollicular growth and monoovulation.
The aim of the current study was to explore whether single low dosages of FSH-CPT induced ovulation in anovulatory and oligoovulatory women. The study was designed as a double-blind study and included placebo treatment because spontaneous ovulation may occur in oligomenorrheic women.
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Subjects and Methods |
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A total of 55 subjects presenting with oligomenorrhea (cycle length 41 d or longer) or amenorrhea (no menstrual period for more than 6 months) were randomized. All subjects had a wish to conceive and were at least 18 and at most 39 yr of age, with a body mass index 18 or greater and 32 kg/m2 or less. Subjects had normal serum FSH (<10 IU/liter), prolactin, and TSH concentrations and either a progestagen-induced withdrawal bleed or spontaneous menstrual bleeding. At screening, ultrasonography was performed to identify those patients with polycystic ovaries (at least 10 follicles < 8 mm in each ovary) and serum FSH, LH, and total testosterone to identify those subjects with normal FSH but increased LH and/or androgens. The study was approved by the local ethical committees of all five participating centers and is in agreement with the Declaration of Helsinki for Medical Research Involving Human Subjects.
Study design and assessments
In this double-blind, placebo-controlled study, subjects received a single injection of placebo or FSH-CPT (ORG 36286, corifollitropin alfa, NV Organon, Oss, The Netherlands). All 55 randomized subjects received a single injection of either placebo (n = 10), 7.5 µg FSH-CTP (n = 13), 15 µg FSH-CTP (n = 10), 30 µg FSH-CTP (n = 11), or 60 µg FSH-CTP (n = 11). The injection was given sc in the abdominal wall at 2 or 3 d after the onset of a spontaneous or progestagen-induced withdrawal bleed. Before injection and during the first week thereafter, ultrasound monitoring and blood sampling was performed every other day. Follicle sizes were reported as the mean of two measurements in two dimensions. One week after injection, subjects were monitored daily by ultrasonography, serum assessment of hormone concentrations, and urine collection to detect the preovulatory LH peak. If 10 d after injection, no follicles 12 mm or larger were observed or if follicles 12 mm or larger stopped growing (no increase in size and number for 4 d), monitoring was stopped and the cycle was discontinued. Ovulation was considered to have occurred with the observation of a positive urinary LH rise or a serum LH rise confirmed by a serum progesterone value of 15 nmol/liter or more assessed within 2 wk (usually 7 d after the moment of ovulation). Alternatively, ovulation after a urinary or serum LH rise could be confirmed by the rupture of the leading follicle(s) as observed by ultrasound. In case of hyperstimulation, endogenous gonadotropins could be suppressed and spontaneous ovulation prevented by giving daily injections of 2 mg of a GnRH antagonist (ganirelix, NV Organon).
Hormone assessments
Serum FSH-CTP concentrations were determined by enzyme immunoassay with a coefficient of variation (CV) less than 20% and a lowest detection limit of 0.079 ng/ml. Antibodies against FSH-CTP were assessed using a RIA based on the formation of an immune complex between a specific antibody and [125I]-labeled FSH-CTP. These samples were assessed with a CV less than 20% [the assays for FSH-CTP and antibodies against FSH-CTP were also described by Bouloux et al. (9)]. Serum FSH, LH, E2, and progesterone (P) levels were determined by time-resolved fluoroimmunoassay (AutoDELFIA, Wallac Oy, Finland) with a CV less than 20%. Lowest detection limits were 0.25 IU/liter, 0.6 IU/liter, 13.6 pg/ml, and 0.31 ng/ml, respectively. All of the aforementioned assays were assessed at NV Organon using validated assays.
Serum inhibin-B concentrations were determined by enzyme immunoassay (Oxford Bio-Innovation, Oxfordshire, UK) with a CV less than 20% and a lowest detection limit of 15.6 pg/ml. The inhibin-B concentrations were assessed by a central laboratory (AAI, Neu-Ulm, Germany) using a validated assay.
Pharmacokinetic evaluation
Serum FSH-CTP levels were analyzed in 41 subjects randomized to FSH-CTP treatment excluding four subjects having inconsistent or no values available for evaluation (one subject in the 15-µg group and three subjects in the 60-µg group). Serum FSH-CTP concentration-vs.-time data were pooled and analyzed using the nonlinear mixed effects modeling program NONMEM (University of California, San Francisco; GloboMax LLC, Hanover, MD). Serum FSH-CTP concentrations in time were best described by a one-compartment model with first-order absorption and a separate estimate for clearance after dosing with 7.5 µg. A fixed value for absorption rate of 0.102 h–1 was taken from a previous, dedicated PK trial with FSH-CTP because no information was available in this trial on the absorption phase of the pharmacokinetic profile. For this model both population (mean) and individual parameter estimates for total serum clearance (CL/F) and volume of distribution (V/F) were obtained. Individual estimates of exposure to FSH-CTP [area under the curve (AUC) and maximum concentration (Cmax)] were calculated from the individual parameter estimates for CL/F and V/F.
Statistical methods
The main objective of this explorative double-blind study was to examine whether a single dose of FSH-CTP was able to induce (mono)ovulation in anovulatory subjects. To illustrate the value of the planned sample size (n = 15 per treatment group), the precision for estimating the dose that yields ovulation after one treatment attempt was calculated. The method, as described by Patel (13), was applied assuming that the highest dose of 60 µg FSH-CTP would induce ovulation in 60% of all subjects and that the dose of 7.5 µg would induce ovulation in 10% of the subjects. The dose with a response of 40% was then to be estimated with a precision (SE) of approximately 1.8 µg. However, the study planned to include in total 75 subjects (15 subjects per treatment group), but the inclusion of new subjects was discontinued at 55 subjects because of the overall low success rate.
In addition to summary statistics presented as an intention-to-treat analysis (of all patients who were randomized in the study), the treatment effect on the maximum number of follicles and the time interval to reach these numbers was analyzed by using a linear regression model with FSH-CTP dose as covariate. The correlation between parameters like the maximum number of follicles, maximum inhibin-B concentrations, and E2 concentrations was assessed by the Pearson correlation test. The incidence of ovulations was analyzed by means of the Fischer’s exact test. The dose response on the incidence of follicular response was tested by means of the Cochran-Armitage test. A result was considered statistically significant if P < 0.05.
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Results |
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The five treatment groups, 55 subjects in total, were similar with respect to age, body height, body weight, and body mass index, and the overall means were 28.1 yr, 163.6 cm, 70.1 kg, and 26.1 kg/m2, respectively. The majority of subjects (76.4%) participating in this trial were Caucasian. Table 1
presents a summary of the gynecological and treatment history per treatment group. In total 43 (78.2%) subjects suffered from oligomenorrhea (cycle length 41 d), and 12 subjects (21.8%) suffered from amenorrhea (no bleeding > 6 months). For the majority of subjects (47 subjects, 85.5%), anovulation was the only cause of infertility. Overall, the mean duration of infertility was 2.5 (range 0.7–9.0) yr, and 33 subjects (60.0%) suffered from primary infertility. In total, 28 subjects (50.9%) had received previous CC treatment, and for 22 of these 28 subjects, ovulation was confirmed after at least one clomiphene treatment cycle. A total of 16 subjects (29.1%) had previous ovulation induction by means of gonadotropins. No relevant differences were found between the treatment groups.
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Pharmacokinetic evaluation
Mean serum FSH-CTP concentrations and derived pharmacokinetic parameters are presented in Table 2. The population FSH-CTP concentration-vs.-time curves for the four dose groups are presented in Fig. 1.
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Follicular response per treatment group
Table 3 presents for each treatment group the number of subjects without follicle growth (follicles < 8.0 mm) or with follicular growth that reached a certain maximum follicle size (divided into five size classes). A follicular response was defined as the development of at least one follicle of 10.0 mm.
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). The percentage of subjects with a follicular response (at least one follicle 10.0 mm) was, respectively, 10, 31, 70, 73, and 82% (P < 0.01, see Table 3
).
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After a single dose of 7.5 µg FSH-CTP (n = 13), a follicular response was noted in two subjects who ovulated at d 9 and 15, respectively. In six subjects initial follicular growth arrested at a maximum follicle size of 10.0–11.9 mm, and no follicular growth was noted in five subjects.
After a single dose of 15 µg FSH-CTP (n = 10), no follicular growth was observed in two subjects, initial follicular growth up to 11.9 mm was noted in three subjects, and in five subjects follicles 12.0 mm or larger were noted Three of the five responding subjects ovulated at 10, 12, and 19 d after FSH-CTP administration. For the other two subjects who did not ovulate, a maximum follicle size of 15.0–17.9 mm and 12.0–14.9 mm was reached at d 8 and 5–7, respectively.
After a single dose of 30 µg FSH-CTP (n = 11), one subject did not respond, five subjects showed initial follicle growth up to 11.9 mm, and five subjects had a follicular response resulting in follicles larger than 12.0 mm. In these five responders, follicle growth did not progress up to 18 mm; however, still two of these five subjects ovulated.
A single dose of 60 µg FSH-CTP (n = 11) was unable to induce a follicular response in two subjects. Follicles larger than 12.0 mm were observed in nine subjects; however, in six subjects the leading follicle(s) did not exceed a size of 14.9 mm and in four of these six subjects, a large cohort of small follicles between 8.0 and 11.9 mm was observed. Two of the three subjects with follicles exceeding 15.0 mm developed too many preovulatory follicles, namely eight and 10 follicles 15.0 mm or larger, respectively, and were treated for 1–2 wk with 2 mg GnRH antagonist to prevent multiple ovulation. One subject with two follicles 15.0 mm or larger ovulated 8 d after FSH-CTP administration.
Average follicular response after FSH-CTP treatment
Figure 2 shows the overall mean number of ovarian follicles observed just prior (d 1) to and after single administration of 7.5, 15, 30, and 60 µg FSH-CTP (intention-to-treat groups). Restricted to responders (Table 3), the average maximum number of follicles observed with a diameter of 8.0 mm or larger was 4.0, 7.6, 13.4, and 20.0 after administration of 7.5, 15, 30, and 60 µg FSH-CTP. Also the size of follicles increased with the dose of FSH-CTP. The dose response for the number of follicles was statistically within the tested dose range of 7.5–60 µg FSH-CTP (P < 0.01). Also, the size of follicles increased with the dose of FSH-CTP. The time interval to reach this maximum number of follicles was 6.5, 6.9, 6.6, and 8.2 d, respectively, and was not significantly longer with increasing doses of FSH-CTP.
Hormones
Serum concentrations of FSH, LH, E2, and inhibin-B measured during the first 10 d after drug administration are presented in Fig. 3. In contrast to serum FSH-CTP immunoactivity (Table 2), the total FSH immunoreactivity measured by Delfia increased only in the 30- and 60-µg dose group during the first days after FSH-CTP administration (Fig. 3A). Similarly, endogenous LH was only temporarily decreased in the two highest dose groups (Fig. 3B), but average values in each treatment group remained between 2 and 10 IU/liter during the first 10 d after drug administration. Overall serum E2 and inhibin-B levels did increase up to d 3 in the 30-µg group and up to d 5 in the 60-µg group (Fig. 3, C and D). At these time points, serum inhibin-B levels correlated well with serum E2 levels (r = 0.84, P < 0.01), and peak serum inhibin-B and E2 both correlated with the number of follicles measured at the same time points (for both parameters r = 0.47, P < 0.01). After peak concentrations, serum E2, and inhibin-B values rapidly declined, returning to baseline 1 wk after FSH-CTP administration, whereas according to ultrasound assessments, follicle growth continued for another 3 d. At all time points, serum P levels were similar between the treatment groups (data not shown).
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In total, nine of the 55 treated subjects (16.4%) ovulated after drug administration: one subject in the placebo group, two subjects in the 7.5-µg group, three subjects in the 15-µg group, two in the 30 µg group, and one in the 60-µg group (NS between the treatment groups). Subjects who ovulated suffered from oligomenorrhea with the exception of the placebo-treated subject who suffered from amenorrhea but ovulated in nine previous CC cycles. Ovulation was documented by either an urinary LH peak, a serum LH rise (>10 IU/liter), ultrasonography, and/or a midluteal P rise (Table 4). Three of the ovulating subjects had monofollicular cycles (no other follicles 15 mm at the day when the first follicle exceeded or was equal to 18 mm) after treatment with placebo (n = 1) or 15 µg FSH-CTP (n = 2). One subject who ovulated had two follicles larger than 18 mm, whereas four subjects ovulated after reaching one or two follicles of 15–17 mm. For one subject who became pregnant, ovulation was not documented because she was prematurely discontinued because of insufficient ovarian response.
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None of the subjects experienced a serious adverse event or ovarian hyperstimulation syndrome, and none of the subjects discontinued because of an adverse event. In total 29 of the 55 subjects experienced at least one adverse event of mild to moderate intensity, of which headache was the most frequently reported in all five treatment groups. No antibodies against FSH-CTP were detected. In general, FSH-CTP was well tolerated in terms of the assessed safety parameters, and no relevant differences between treatment groups were observed.
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Discussion |
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Evaluation of serum FSH-CTP levels has indicated that the bioavailability of FSH-CTP in this study with anovulatory women is very similar to that previous established in pituitary-suppressed female volunteers (10) and in in vitro fertilization patients treated in a flexible GnRH antagonist regimen (11). This implies that the low success rates in this study are not related to an exposure different from anticipated. Moreover, it also indicates that the pharmacokinetic properties of FSH-CTP are not influenced by the endocrine status of the treated subjects. Within the dose range of 15–60 µg FSH-CTP, peak levels of serum FSH-CTP levels increased in a linear dose-related manner, whereas the elimination half-life was independent of from the dose given. In the current study, the lowest dose of 7.5 µg FSH-CTP tended toward a longer terminal half-life than the 15- to 60-µg doses of FSH-CTP. This longer half-life was due to lower serum clearance (CL/F = 0.218 liter/h vs. 0.307 liter/h), whereas the volume of distribution was the same for all dose groups. Mean clearance values reported in other trials with FSH-CTP varied from 0.205 to 0.375 liter/h, and, therefore, the lower clearance of the 7.5-µg group is assumed to be a chance finding.
Although spontaneous ovulation appears to have occurred in one subject in the placebo group, this study demonstrates that FSH-CTP is capable of inducing follicular growth in anovulatory women in a dose-dependent manner because the percentage of responding subjects and the number and size of follicles increased with the dose of FSH-CTP administered. Accordingly, follicular growth arrested too early in the lowest dose group and too many preovulatory follicles developed in the highest dose group, indicating that the therapeutic window is very small.
In contrast to CC treatment, the administration of exogenous FSH does not result in a reduced negative feedback by endogenous estrogens. Rather the exogenous FSH induces an initial estrogen rise that enforces negative feedback. Thus, exogenous FSH is required until serum estradiol levels are sufficiently high to induce the natural (endogenous) LH surge. When subjects are treated with exogenous FSH, therefore, serum FSH levels should be sufficiently high to select and support the leading follicle up to ovulation (14). In the current study using long-acting FSH, the dilemma seems to be that too high a dose selects too many follicles, whereas a lower single dose is unable to support follicle growth up to ovulation; thus, the therapeutic window of appropriate FSH levels is small. Moreover, a distinct individual variability in sensitivity of the ovary to respond to FSH is another complicating factor. The ability to predict the individual FSH threshold (15) may represent a step forward by allowing for individual dose adjustments. Induction of ovulation with long-acting FSH may be feasible in anovulatory subjects if followed by a repeated low dose of FSH-CTP or daily FSH injections to support the leading follicle(s) up to ovulation.
After treatment with 30 or 60 µg FSH-CTP, initial rises of serum E2 and inhibin-B peaked before the maximum number or size of follicles was reached. Whereas these hormones decreased and returned to baseline levels, follicular growth continued for another 3 d. This discrepancy suggests that after treatment with long-acting FSH, the amount of FSH required to support follicular growth may be lower than the amount of FSH required to support hormonal production during follicular development.
Ovulation induction aims to achieve repeated unifollicular ovulation in anovulatory infertility, the most common cause of which is polycystic ovary syndrome. Strategies to induce ovulation include weight loss, oral antiestrogens (principally CC), parenteral gonadotropin therapy, and laparoscopic ovarian surgery. In addition, more recently there has been interest in the use of insulin-lowering agents (such as metformin and the thiazolidinediones) and the aromatase inhibitors (16). There have been no adequately powered randomized studies to determine which of these therapies provides the best overall chance of an ongoing pregnancy. Women with polycystic ovary syndrome are at risk of multiple follicle growth and hence multiple pregnancy and ovarian hyperstimulation syndrome. Because of this, ovulation induction has to be carefully monitored with serial ultrasound scans.
Today gonadotropin therapy is indicated (17, 18) only for anovulatory women who have been treated with antiestrogens if they have either failed to ovulate or they have a response to CC that is likely to reduce their chance of conception (e.g. persistent hypersecretion of LH). The main question addressed by the current feasibility study was whether a single low dose of FSH-CTP could replace first- and second-line treatment of anovulatory women, assuming that both CC-responders and CC-resistant patients could be successfully treated by this long-acting FSH. After injection, serum FSH-CTP initially rises up to peak levels at 1–2 d, and thereafter serum FSH-CTP slowly decreases. This overall profile mimics the FSH flare-up induced by CC treatment and the step-down approach in classical ovulation induction, although there are some essential differences. The CC-induced flare-up of FSH lasts usually from the first treatment day of CC up to the last treatment day of CC and only declines thereafter. With the step-down protocol, follicular recruitment is achieved using 150 or 225 IU daily for 3–4 d before decreasing the dose to 75 IU to maintain follicular development (19, 20, 21). Experimental studies have indicated that initiation of follicular growth requires a 10–30% increment in the dose of exogenous FSH, and the threshold changes with follicular growth due to an increased number of FSH receptors so that the concentration of FSH required to maintain growth is less than that required to initiate it (22).
It can be extremely difficult to predict the response to stimulation of women with polycystic ovaries; indeed, this is the greatest therapeutic challenge in all ovulation induction therapies, regardless as to whether daily or long-acting FSH is applied. The polycystic ovary is characteristically quiescent, at least when viewed by ultrasound, before often exhibiting an exuberant and, sometimes, explosive response to stimulation. It can be very challenging to stimulate the development of a single dominant follicle and whereas attempts have been made to predict a multifollicular response (23) by looking at midfollicular endocrine profiles and numbers of small follicles, it is harder to do so before commencing ovarian stimulation and hence determine the required starting dose of gonadotropin.
Multiple pregnancy is the main undesirable side effect of fertility therapy because of the increased rates of perinatal morbidity and mortality. Gonadotropin therapy should therefore be given in low doses to women with anovulatory infertility, and strict criteria should be employed before the administration of the ovulatory trigger. The underlying principle of all methods of ovulation induction for women with polycystic ovary syndrome must always be to use the lowest possible dose to achieve unifollicular ovulation.
Therefore, development of a long-acting FSH preparation by means of a single starting dose for all patients may not be feasible. Because each patient has her own threshold above which monofollicular growth may occur, patients may benefit when started in the first cycle with a very low dose of FSH-CTP and, if insufficient, step up to a higher dose in the next cycle. In addition, this small dose-finding study indicates that in the same cycle a second low dose of FSH-CTP or adjuvant rFSH may be required to induce ovulation Whether the development of long-acting FSH for monofollicular ovulation will be feasible needs additional research that may lead to a new strategy and treatment regimen including a single injection of long-acting FSH.
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Acknowledgments |
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Footnotes |
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Abbreviations: AUC, Area under the curve; CC, clomiphene citrate; CL/F, total serum clearance; Cmax, maximum concentration; CTP, carboxy terminal peptide; CV, coefficient of variation; E2, estradiol; P, progesterone; r, recombinant; V/F, volume of distribution.
Received April 7, 2004.
Accepted September 14, 2004.
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  N. S. Macklon, R. L. Stouffer, L. C. Giudice, and B. C. J. M. Fauser The Science behind 25 Years of Ovarian Stimulation for in Vitro Fertilization Endocr. Rev., April 1, 2006; 27(2): 170 - 207. [Abstract] [Full Text] [PDF]
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