John Wilcox, M.D., a,e Daniel Potter, M.D., b,e Marva Moore, Ph.D., c Lee Ferrande, M.S., c and Eduardo Kelly, M.D., M.B.A. d

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1 Prospective, randomized trial comparing cetrorelix and ganirelix in a programmed, flexible protocol for premature luteinizing hormone surge prevention in assisted reproductive technologies John Wilcox, M.D., a,e Daniel Potter, M.D., b,e Marva Moore, Ph.D., c Lee Ferrande, M.S., c and Eduardo Kelly, M.D., M.B.A. d a Huntington Reproductive Center, Pasadena, California; b Huntington Reproductive Center, Fullerton, California; and c Reproductive Health Clinical Development Unit and d Reproductive Health, Serono, Inc., Rockland, Massachusetts; e on behalf of the CAP IV Investigator Group Objective: To compare the safety and efficacy of single-dose cetrorelix (3 mg) and daily ganirelix (0.25 mg) in the inhibition of premature LH surge in women undergoing cycle-programmed ovarian stimulation before Assisted Reproductive Technology (ART). Design: Prospective, open-label, randomized, comparative study. Setting: Sixteen ART centers in the United States. Patient(s): One hundred eighty-five infertile patients undergoing ART. Intervention(s): Single injection of cetrorelix (3 mg SC) or daily dose of ganirelix (0.25 mg SC) was administered when the lead follicle was 14 mm. Daily cetrorelix (0.25 mg) was administered if the criteria for hcg administration were not met 4 days after receiving 3 mg of cetrorelix. Main Outcome Measure(s): Percentage of patients who did not have a premature LH surge, defined as LH 10 IU/L on the day of hcg administration. The IVF and embryo transfer (ET) outcomes were assessed. Result(s): No patient in either treatment group had a premature LH surge. There were no statistically significant differences between treatments for any IVF/intracytoplasmic sperm injection (ICSI) or ET outcomes, including pregnancy rate (PR). However, cetrorelix required significantly fewer injections than ganirelix. Similar safety profiles were observed. Conclusion(s): Cetrorelix and ganirelix effectively prevented LH surges in oral contraceptive (OC) pill-programmed, flexible protocols, with similar safety profiles and PRs; however, cetrorelix required significantly fewer injections, increasing patient convenience. (Fertil Steril 2005;84: by American Society for Reproductive Medicine.) Key Words: Antagonist, cetrorelix, ganirelix, oral contraceptives programming, assisted reproductive technology, inhibition of premature LH surge, follitropin alfa In women undergoing infertility therapy with gonadotropins, the stimulation of more than one ovarian follicle may increase E 2 production above normal levels and often will induce a surge of LH before adequate follicular maturation (1). The premature surge induces luteinization of immature follicles resulting in developmental arrest of oocytes and IVF cycle cancellation (2 4). Gonadotropin-releasing hormone agonists and antagonists are currently used to suppress endogenous LH secretion during ovarian stimulation. Received December 2, 2004; revised and accepted March 16, Supported by Serono, Inc., Rockland, Massachusetts. Presented in part at the 60th Annual Meeting of the American Society for Reproductive Medicine (ASRM), Philadelphia, Pennsylvania, October Reprint requests: Eduardo Kelly, M.D., M.B.A. Serono, Inc., Rockland, Massachusettes (FAX: ; eduardo.kelly@ serono.com). The GnRH agonists are long-acting modifications of the native hormone and, after an initial augmented pituitary response (flare effect), pituitary gonadotropin secretion is suppressed (down-regulation). This suppressive effect avoids premature LH surges, and consequently reduces the incidence of cancelled cycles. The GnRH agonists have been applied successfully in ART procedures leading to a significant increase in pregnancy rate (PR) per initiated IVF cycle (5, 6). However, pituitary desensitization may take 2 3 weeks to occur, and thus increases treatment duration (7). The GnRH antagonists lack an initial flare effect and therefore, suppress gonadotropin secretion in a few hours rather than the days to weeks required for an agonist (7). The ability of GnRH antagonists to inhibit gonadotropin secretion rapidly without exerting transient stimulation makes them an alternative to agonists in most situations where gonadotropin suppression is indicated (7). Thus, the use of GnRH antagonists in an IVF protocol in the usual clinical 108 Fertility and Sterility Vol. 84, No. 1, July /05/$30.00 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 settings inhibits LH surges more efficiently without potentially adverse pituitary stimulation. Cetrorelix for injection (Cetrotide; Serono, Inc., Rockland, MA) and ganirelix (Ganirelix Acetate Injection [previously Antagon Injection]; Organon, Inc., West Orange, NJ) are GnRH antagonists approved by the U.S. Food and Drug Administration (FDA) for the inhibition of premature LH surge. The efficacy and safety of a single-dose cetrorelix regimen (3 mg) and a multiple-dose cetrorelix regimen (daily 0.25 mg) in the prevention of a premature LH surge have been demonstrated in numerous clinical trials (8 16). These clinical trials also have evaluated the dosing regimens in different treatment protocols (e.g., starting cetrorelix administration by cycle day, serum E 2 level, or lead follicle size). A tailored approach to cetrorelix administration has also been evaluated by Ludwig et al. (17). Olivennes et al. (18) reported the results of two large Phase IIIb clinical trials. The results from these clinical trials strongly suggest that the efficacy and safety of the cetrorelix single-dose regimen were similar to that of the cetrorelix multiple-dose regimen, while requiring only one injection in 73% of patients. Ganirelix is available only as a multiple-dose regimen (daily 0.25 mg), and has been shown to be safe and effective in the prevention of premature LH surge (19 22). In the four studies cited, ganirelix was administered on day 6 of stimulation and continued until the day of hcg administration. However, a recent study by Mochtar et al. (23) assessed an individualized ganirelix protocol. Oral contraceptives (OC) are commonly used in ART programs for cycle programming, which allows centers to plan and predict ovum retrieval to more efficiently schedule workload. Few publications have addressed outcomes of GnRH antagonist cycles in combination with OCs; however, based on these few studies, one can conclude that OC pretreatment can be used with GnRH antagonists to program ovum retrieval (24, 25) and that utilization of OC pretreatment may yield a more synchronized follicular cohort (26). In a recent literature review of GnRH antagonists, Griesinger et al. (27) noted that cetrorelix and ganirelix had not been directly compared in a clinical trial. Therefore, our goal was to perform a prospective randomized clinical trial with single-dose cetrorelix (3 mg) or ganirelix daily dosing (0.25 mg), comparing the percentage of patients with a premature LH surge (LH 10 IU/L) and IVF and ET outcomes with antagonists administered when the lead follicle was 14 mm. MATERIALS AND METHODS Study Design The study was designed as a Phase IV, prospective, openlabel, comparative, randomized clinical trial with 16 ART centers participating in the United States. A single dose of cetrorelix (3 mg) was compared with daily ganirelix (0.25 mg) in the inhibition of a premature LH surge (LH 10 IU/L on day of hcg administration) in women undergoing ovarian stimulation before ART. Other efficacy end points included the number of oocytes obtained, number of embryos obtained and transferred, oocyte/embryo quality, implantation rates, and PRs. Safety was assessed by the incidence and severity of adverse events, including ovarian hyperstimulation syndrome (OHSS). The antagonist treatment regimens were administered as flexible protocols in this clinical trial, as antagonist dosing began when the lead follicle was 14 mm. The ART protocols applied in this trial were consistent with established medical practice. During the pretreatment period of the trial, OCs were used for cycle programming. Patient Population Study patients were premenopausal infertile women desiring pregnancy whose physician recommended ART. Eligible patients included women aged years with regular menstrual cycles (25 35 days in length), screening follicular phase FSH levels within normal limits, and a transvaginal ultrasound revealing no clinically significant abnormal findings. Patients with any contraindication to pregnancy or carrying pregnancy to term, documented American Society for Reproductive Medicine (ASRM) grade III or IV endometriosis, prior history of severe OHSS, poor response in a previous ART cycle ( 3 oocytes retrieved), or 3 prior, initiated, consecutive ART cycles without a clinical pregnancy were ineligible for this study. The study was approved by an appropriately constituted Institutional Review Board at all study centers and all patients provided written informed consent before enrolling in the study. Study Procedures Pretreatment with daily OC dosing (Ortho-Cept 28 tablets; 21 tablets containing 0.15 mg of desogestrel and 0.03 mg of ethinyl E 2 ; Ortho-McNeil Pharmaceutical, Inc., Raritan, NJ) began on the first day of menses onset. Follitropin alfa multidose (recombinant hfsh; Gonal-f Multi-Dose; Serono, Inc.) was administered at a dose of 225 IU SC daily beginning 5 days after the last OC. The dose of follitropin alfa could be increased or decreased ( IU) beginning on stimulation day 6, and subsequently adjusted every 2 days based on individual patient response. Patients were randomly assigned to one of two treatment groups within a center. Group A was assigned to a single dose of cetrorelix for injection (3 mg; Cetrotide; Serono, Inc.). Group B was assigned to a daily dose of ganirelix (0.25 mg; Antagon Injection [name changed to Ganirelix Acetate Injection in 2004 after conclusion of the study]; Organon, Inc.). Randomization occurred in a 1:1 ratio stratified by centers. Patients were randomized Fertility and Sterility 109

3 using an Internet-based randomization system integrated in the electronic case report form. Patients randomized to group A (cetrorelix treatment) received a single dose of 3 mg SC of cetrorelix when the lead follicle reached 14 mm, as measured by ultrasound. If hcg administration criteria were not met within 4 days of receiving 3 mg of cetrorelix, a daily dose of 0.25 mg SC of cetrorelix was administered up to and including the day of hcg administration. Patients randomized to ganirelix treatment (group B) were administered a daily dose of 0.25 mg SC of ganirelix when the lead follicle reached 14 mm and continued up to and including the day of hcg administration. In addition to treatment with follitropin alfa multidose, both treatment groups were administered a daily dose of 75 IU of hmg 75 IU (Pergonal; Serono, Inc.) starting on the day of antagonist administration. Gonadotropin treatment continued until hcg administration criteria were met. The daily dose of gonadotropins (recombinant hfsh and hmg combined) did not exceed 450 IU. However, if a patient was suspected of being at excessive risk of OHSS, the dosage was reduced or discontinued. Coasting, defined as discontinuation of gonadotropin treatment and delayed administration of recombinant hcg for 2 or more days, was not permitted. Lyophilized recombinant hcg (250 g SC; Ovidrel; Serono, Inc.) was administered within 36 hours after the last dose of gonadotropin, when the patient had at least one follicle 18 mm, at least two other follicles 16 mm, an acceptable serum E 2 level according to the center s standard practice (approximately 150 pg/ml per mature follicle), and no evidence of a condition that satisfied a criterion for discontinuation. The LH level for the primary efficacy end point was measured on the day of, but before, recombinant hcg administration. Oocytes were retrieved vaginally under ultrasound monitoring in the morning approximately hours after hcg administration. Oocytes were categorized as mature if the coronal cells were still apposed to the egg but the cumulus had expanded into a fluffy mass. Oocytes were then fertilized in vitro using IVF or intracytoplasmic sperm injection (ICSI) according to each center s practice. Embryos were replaced 2 3 days after oocyte retrieval (i.e., days 4 5 after hcg administration) or 5 6 days after retrieval (days 7 8 after hcg administration) if transferred at the blastocyst stage, according to the center s standard procedure. Before transfer, embryos were scored for cell number and quality as judged by morphological appearance. Grade A was defined as embryos that showed evenly sized blastomeres of near spherical appearance with moderate refractility (i.e., not very dark) and with intact zona. Grade B was defined as embryos that had more uneven or irregular-shaped blastomeres, with mild variation in refractility, and no more than 10% fragmentation of blastomeres. No more than three embryos or two blastocysts were to be replaced per protocol. One or two doses of vaginal micronized progesterone (Crinone 8%; Serono, Inc.) were administered daily for luteal phase progesterone support. Luteal phase support started on the day of or the day after ovum pick-up. Administration continued either up to menstruation or, if the patient became pregnant, for at least 30 days after pregnancy was confirmed by laboratory evidence. Statistical Methods The difference in the primary end point, the percent of patients who did not have a premature LH surge during controlled ovarian stimulation for ART, for the treatment groups was assessed using Fisher s exact test. The efficacy and safety analyses included all patients who received at least one injection of either cetrorelix or ganirelix (intention-to-treat population). The criterion for declaring cetrorelix treatment noninferior to ganirelix treatment specified that the one-sided 97.5% lower confidence bound for the percent of patients treated with Cetrotide who did not have a premature LH surge during controlled ovarian stimulation for ART minus the percent of patients treated with ganirelix who did not have a premature LH surge during controlled ovarian stimulation for ART must be greater than 10% (sample size was calculated based on 80% power). Continuous secondary end points were analyzed by analysis of variance (ANOVA) with effects for treatment, center, and, where appropriate, their interaction. For nominal-scaled categorical end points, the Cochran-Mantel-Haenszel general association test was used to analyze the association between the end point and treatment taking into account center differences. The difference in the percentage of patients with grade A embryos transferred for the treatment groups was assessed using Fisher s exact test. All statistical analyses were performed using version 6.12 of the Statistical Analysis System (SAS Institute Inc., Cary, NC). RESULTS Disposition of Patients Of the 185 patients who enrolled, 180 patients received OCs and 177 patients received recombinant hfsh. A total of 176 patients were randomly assigned to receive cetrorelix (n 87) or ganirelix (n 89) at 16 participating study centers. The distribution of patients treated in each center and by treatment group is provided in Table 1. After randomization, 87 patients received cetrorelix and 88 patients received ganirelix (overall N 175), as one patient was randomized to receive ganirelix but did not receive the study drug due to lack of ovarian response to stimulation treatment. Ovum pick-up and fertilization were performed for all patients who received hcg (87 patients per treatment group, as one ganirelix-treated patient did not receive hcg due to excessive risk of OHSS; overall N 174). Embryo transfer (ET) was performed for 86 patients who received cetrorelix and 82 patients who received ganirelix. 110 Wilcox et al. Cetrorelix and ganirelix in IVF/ICSI Vol. 84, No. 1, July 2005

4 TABLE 1 Patients treated by center and treatment group. Center Cetrorelix (n 87) n(%) Ganirelix (n 88) n(%) (5.7) 6 (6.8) (5.7) 5 (5.7) (5.7) 5 (5.7) (6.9) 6 (6.8) (6.9) 6 (6.8) (4.6) 3 (3.4) (5.7) 5 (5.7) (5.7) 6 (6.8) (5.7) 5 (5.7) (6.9) 7 (8.0) (4.6) 5 (5.7) (5.7) 5 (5.7) (5.7) 4 (4.5) (6.9) 6 (6.8) (6.9) 5 (5.7) (10.3) 9 (10.2) Overall, five patients withdrew from the study during the pretreatment period. Of the 87 patients who were randomly assigned to cetrorelix, 86 patients (98.9%) completed the study, whereas 82 (93.2%) of the 88 patients who received ganirelix completed the study. Fewer patients withdrew from cetrorelix treatment (1.1%) than ganirelix treatment (6.8%). The one patient (1.1%) who withdrew from the study during cetrorelix treatment withdrew due to an adverse event (moderate OHSS). However, in the ganirelix group, two patients (2.3%) withdrew due to an adverse event (moderate OHSS and mild OHSS, respectively), one patient (1.1%) withdrew due to excessive risk of OHSS, and three patients (3.4%) withdrew because they had no ET due to arrested embryo development. Patient Demographics The median age of the 175 treated patients was 32 years (range years). The median body mass index (BMI) was 23.6 kg/m 2 (range kg/m 2 ). The majority of the patients were white (79.4%), whereas 2.9% were black, 5.7% were Asian, and 12.0% were reported as other. Both treatment groups showed a similar distribution for all demographic characteristics except for race, where the difference between the treatment groups was statistically significant (P.012) (Table 2). The last menses was spontaneous for the majority of patients (98.9%; 173/175). Primary infertility was reported by 42.9% (75/175) of patients, whereas secondary infertility was reported by 57.1% (100/175). The median duration of infertility was 36 months. The differences between the treatment groups were not statistically significant for any of these baseline parameters. For the type of infertility, however, the difference between the treatment groups was statistically significant (P.043) (Table 2). Although the history of male factor infertility was higher in the ganirelix group TABLE 2 Patient characteristics. Cetrorelix (n 87) Ganirelix (n 88) P value Age (y) 32 (26, 38) 32 (23, 39).451 a BMI (kg/m 2 ) 23.5 (18.3, 37.6) 23.8 (17.3, 35.1).897 a Race (%).012 b White 73 (83.9) 66 (75.0) African American 4 (4.6) 1 (1.1) Asian 6 (6.9) 4 (4.5) Other 4 (4.6) 17 (19.3) Type of infertility (%).043 b Female infertility only 33 (37.9) 25 (28.4) Male infertility only 21 (24.1) 37 (42.0) Female and male infertility 20 (23.0) 19 (21.6) Unexplained 13 (14.9) 7 (8.0) Note: Median (min, max). a P value from an ANOVA on ranked data with effects for treatment and center. b P value from Cochran-Mantel-Haenszel general association. Fertility and Sterility 111

5 TABLE 3 Comparison of patient stimulation outcomes in patients treated with cetrorelix or ganirelix. Cetrorelix (n 87) Ganirelix (n 88) P value Patients who received hcg Follicles 14 mm 9.0 (3.0, 27.0) 11.0 (4.0, 25.0).255 a Serum E 2 (pg/ml) 1,660.0 (530.0, 5,421.0) 2,240.0 (330.0, 5,980.0).005 a Serum E 2 (pg/ml)/follicle 14 mm (48.3, 547.5) (41.3, 631.4).080 a Oocytes retrieved 12.0 (2.0, 47.0) 13.0 (3.0, 34.0).299 a Oocytes intact 11.0 (2.0, 47.0) 13.0 (3.0, 34.0).238 a Mature oocytes 6.0 (0.0, 37.0) 7.0 (1.0, 22.0).581 a Metaphase II oocytes 8.0 (0.0, 40.0) 9.0 (2.0, 30.0).469 a 2PN oocytes 6.0 (1.0, 28.0) 8.0 (1.0, 23.0).707 b 2PN oocytes (% of inseminated) 67.6 (12.5, 100.0) 66.7 (13.3, 100.0).502 b Note: Median (min, max). a P value from an ANOVA on ranked data with effects for treatment and center. b P value from an ANOVA on ranked data with effects for treatment, center, and their interaction. (42.0%; 37/88) than in the cetrorelix group (24.1%; 21/87), the ganirelix group also had a greater percentage of ICSI-only cases than in the cetrorelix group, 63.2% (55/87) and 55.2% (48/87), respectively. However, this difference in ICSI-only cases was not statistically significant. There were also no statistically significant differences between the treatment groups in fertilization results or any of the oocyte/embryo quality data (Tables 3 and 4). Ovarian cysts 25 mm were observed in 3 (3.4%) of 87 patients in the cetrorelix group and 9 (10.2%) of 88 patients in the ganirelix group during the prestudy visit, but this difference was not statistically significant. A similar prestudy FSH level was observed for both treatment groups. For the overall patient population (N 175), the FSH level ranged from 1.2 to 12.0 IU/L, with a median of 5.9 IU/L. All patients had a prestudy FSH level within the normal limit for the early follicular phase as per the trial center s local laboratory. Prevention of Premature LH Surge No patient in either the cetrorelix or the ganirelix treatment group had a premature LH surge (LH level 10 miu/ml on the day of hcg administration) (Table 5). In the cetrorelix and ganirelix groups combined, 97.1% (169/174) of patients had an LH level 5 IU/L on the day of hcg administration. The remaining five patients, two (2.3%) in the cetrorelix group and three (3.4%) in the ganirelix group, had a serum LH level between 5 and 10 IU/L. The median LH level was 1.3 IU/L for each of the treatment groups; however, the range was smaller for the cetrorelix group (range IU/L) compared with the ganirelix group (range IU/L). Exposure to Treatment Regimens The median cetrorelix dose was 3 mg (range 3 4 mg), and the median ganirelix dose was 1 mg (range mg). The median number of cetrorelix injections (1 injection) was less than the median number of ganirelix injections (4 injections) (Table 6). Most women in the cetrorelix group (66.7%) received only a single 3 mg dose of cetrorelix. Of those patients requiring supplemental cetrorelix (0.25 mg), 89.7% (26/29) needed only one additional dose. Only one patient required more than two additional doses; this patient received four injections of 0.25 mg of cetrorelix. Two patients received the 0.25 mg injection less than 96 hours after the 3 mg cetrorelix injection. However, 62.5% (55/88) of patients who received ganirelix required four or more injections of 0.25 mg of ganirelix. The extent of exposure to Ortho-Cept 28, Gonal-f Multi- Dose, Pergonal, Ovidrel, and Crinone 8% was comparable between treatment groups (Table 6). Furthermore, there was no statistically significant difference in the duration of gonadotropin therapy, total recombinant hfsh and hmg administered, total Gonal-f administered, or total Pergonal administered between the treatment groups. Patient Stimulation Outcomes Table 3 summarizes the findings for follicle number, serum E 2 level, and oocyte number and quality. The median num- 112 Wilcox et al. Cetrorelix and ganirelix in IVF/ICSI Vol. 84, No. 1, July 2005

6 TABLE 4 Comparison of embryo/blastocyst transfer results in patients treated with cetrorelix or ganirelix. Cetrorelix (n 87) Ganirelix (n 88) P value Patients with day 2/day 3 embryos obtained Embryos obtained 5.0 (1.0, 23.0) 5.5 (1.0, 23.0).505 a Patients with blastocysts obtained Blastocysts obtained 5.0 (2.0, 10.0) 6.0 (3.0, 10.0).912 b Patients with day 2/day 3 embryos transferred Embryos transferred 3.0 (1.0, 4.0) 3.0 (1.0, 3.0).156 a Grade A embryos transferred 1.0 (0.0, 4.0) 1.0 (0.0, 3.0).256 b Grade B embryos transferred 1.0 (0.0, 4.0) 1.0 (0.0, 3.0).429 b Patients with blastocysts transferred Blastocysts transferred 2.0 (1.0, 3.0) 2.0 (1.0, 2.0).306 b Patients with embryos cryopreserved Embryos cryopreserved 4.5 (1.0, 11.0) 5.0 (1.0, 15.0).791 b Patients with blastocysts cryopreserved Blastocysts cryopreserved 2.0 (1.0, 7.0) 2.0 (1.0, 5.0).286 b Implantation rate (%) c b Note: Median (min, max). a P value from an ANOVA on ranked data with effects for treatment, center, and their interaction. b P value from an ANOVA on ranked data with effects for treatment and center. c Mean SD; implantation rate for each patient with embryos/blastocysts transferred was defined as 100% [(number of fetal sacs)/(number of embryos transferred number of blastocysts transferred)]. ber of follicles 14 mm was 9 for the cetrorelix group and 11 for the ganirelix group on the day of hcg administration. The difference between the treatment groups was not statistically significant. There was a statistically significant difference between the treatment groups in the serum E 2 levels on the day of hcg (P.005). The median E 2 level was higher in the ganirelix group (2,240.0 pg/ml) as compared to the cetrorelix group (1,660.0 pg/ml). However, the E 2 level per follicle 14 mm for the treatment groups was not significantly different (179.0 pg/ml in the cetrorelix group and pg/ml in the ganirelix group, P.080). Overall, there were no statistically significant differences between the treatment groups in the number or quality of oocytes. TABLE 5 Inhibition of premature LH surge, day of hcg administration. Patients with LH 5 IU/L (%) Patients with LH 5 IU/L and 10 IU/L (%) Patients with LH 10 IU/L (%) Cetrorelix (n 87) Ganirelix (n 87) 85 (97.7) 84 (96.6) 2 (2.3) 3 (3.4) 0 0 Embryo/Blastocyst Transfer Results Embryo/blastocyst transfer results are summarized in Table 4. Embryos were obtained from 81.6% of patients in the cetrorelix group (median of 5 embryos) and 75.9% of patients in the ganirelix group (median of 5.5 embryos) who underwent ovum pick-up. Embryos were transferred for 98.6% of patients in the cetrorelix group and 98.5% of patients in the ganirelix group who had embryos obtained. For both treatment groups, a median of three embryos was transferred. Embryo transfer deviated from the protocol for two patients in the cetrorelix group, as four embryos were transferred. Both deviations were due to incorrectly following the protocol. Although similar percentages of patients in each treatment group had embryos transferred, a greater percentage of cetrorelix patients had grade A embryos transferred Fertility and Sterility 113

7 TABLE 6 Exposure to treatment regimens. Cetrorelix (n 87) Ganirelix (n 88) a P value No. of Ortho Cept 28 tablets 20 (14.0, 30.0) 21 (12.0, 30.0) Gonadotropin stimulation b (days) 9.0 (6.0, 13.0) 9.0 (7.0, 14.0).688 c Total dose of gonadotropins b (IU) 2,175.0 (975.0, 4,725.0) 2,100.0 (1,350.0, 4,875.0).392 c Gonal-f Multi-dose (IU) 2,025 (675.0, 4,425.0) 1,875 (1,200.0, 4,650.0).574 c Pergonal (IU) 225 (75.0, 825.0) 225 (75.0, 600.0).173 c No. of antagonist injections 1.0 (1.0, 5.0) 4.0 (1.0, 6.0).001 c Ovidrel (mcg) Crinone 8% (total number of times applied) 31 (6.0, 88.0) 31 (1.0, 82.0) Note: Median (min, max). a In the ganirelix group, 87 patients received Ovidrel and 86 patients received Crinone 8%. b r-hfsh hmg combined. c P value from an ANOVA on ranked data with effects for treatment and center. (67.1%; 47/70) as compared to ganirelix patients (55.4%; 36/65). However, this difference was not statistically significant (P.215; Fisher s exact test), and a median of one grade A embryo was transferred for both treatment groups. Blastocysts were obtained from 16 patients (18.4%) in the cetrorelix group and 17 patients (19.5%) in the ganirelix group. All patients in both treatment groups with blastocysts obtained had a median of two blastocysts transferred. Blastocyst transfer deviated from the protocol for two patients in the cetrorelix group, as three blastocysts were transferred. One deviation was due to poor quality of the blastocysts, and the other was due to incorrectly following the protocol. Embryos were cryopreserved for 26 patients in the cetrorelix group and 29 patients in the ganirelix group. The median number of embryos cryopreserved was 4.5 embryos for the cetrorelix group and 5 embryos for the ganirelix group. Blastocysts were cryopreserved for 11 patients in the cetrorelix group and 13 patients in the ganirelix group. A median of two blastocysts was cryopreserved for the cetrorelix and the ganirelix groups. Overall, there were no statistically significant differences between the treatment groups in the number of embryos or blastocysts obtained, transferred, or cryopreserved. Pregnancy Rates Pregnancy rates for all patients and for those patients who had embryo/blastocyst transfer are summarized in Table 7. The overall PR was 51.7% (45/87) for the cetrorelix group and 48.9% (43/88) for the ganirelix group, with clinical PRs of 46.0% (40/87) and 43.2% (38/88), respectively. The overall difference between biochemical PR and clinical PR was the same for both treatment groups (5.7%). For patients with embryos/blastocysts transferred, TABLE 7 Comparison of pregnancy rates in patients treated with cetrorelix or ganirelix. Cetrorelix (n 87) Ganirelix (n 88) P value Pregnancy rate (%) 51.7 (45/87) 48.9 (43/88) a Pregnancy rate with transfer (%) 52.3 (45/86) 52.4 (43/82) a Clinical pregnancy rate (%) 46.0 (40/87) 43.2 (38/88) a Clinical pregnancy rate with transfer (%) 46.5 (40/86) 46.3 (38/82) a a P value from Cochran-Mantel-Haenszel general association test. 114 Wilcox et al. Cetrorelix and ganirelix in IVF/ICSI Vol. 84, No. 1, July 2005

8 the overall PR was 52.3% (45/86) for the cetrorelix group and 52.4% (43/82) for the ganirelix group, with clinical PRs of 46.5% (40/86) and 46.3% (38/82), respectively. Pregnancy was achieved by only one of the four patients who deviated from the protocol by having four embryos or three blastocysts transferred. Given the small number of patients in each center, formal statistical tests regarding a difference in the rates within a center for the two treatments are not appropriate. Safety Treatment-emergent adverse events were reported by 30 (34.5%) of 87 patients in the cetrorelix group and 25 (28.4%) of 88 patients in the ganirelix group. The most common treatment-emergent adverse event for both treatment groups was abdominal distension, which occurred in 10 (11.5%) of 87 patients in the cetrorelix group and in 11 (12.5%) of 88 patients in the ganirelix group. The percentage of patients who experienced OHSS was also similar for the cetrorelix group (8.0%; 7/87) and ganirelix group (6.8%; 6/88). Ectopic pregnancy occurred in four patients (three patients in the cetrorelix group and one patient in the ganirelix group). Ten patients reported reactions at the site of antagonist injection (six patients in the cetrorelix group and four patients in the ganirelix group). These reactions were all mild, with the exception of a moderate injection site irritation reported by a patient in the cetrorelix group. All other individual adverse events occurred in three or fewer patients in each treatment group. Three severe treatment-emergent events (injection site stinging, ectopic pregnancy, and urinary retention) were reported in the cetrorelix group. The severe injection site stinging occurred at the site of Ovidrel administration. In the ganirelix group, four severe events (OHSS, adnexa uteri pain, headache, and influenza), and one very severe event (ectopic pregnancy) were reported. For both treatment groups, all other treatment-emergent adverse events were categorized as mild or moderate in nature. Overall, mild or moderate OHSS occurred in 12 patients, and 1 severe case occurred in the ganirelix group resulting in hospitalization. In both treatment groups, only a small number of treatment-emergent adverse events were considered by the investigator to be probably or possibly related to study treatment, all of which were injection site related. Seven patients reported serious side effects (SAEs) during the study, five patients in the cetrorelix group and two patients in the ganirelix group. Of the five patients who reported SAEs in the cetrorelix group, three of these events occurred during the post-treatment period of the trial (two ectopic pregnancies and myasthenia gravis). Three SAEs required hospitalization of the patient, two in the cetrorelix group (severe myasthenia gravis and postprocedural hemorrhage) and one in the ganirelix group (OHSS). All seven SAEs were considered to be unlikely related or unrelated to study drug treatment. Overall, a similar safety profile was observed for cetrorelix and ganirelix. DISCUSSION Cetrorelix for injection (Cetrotide; Serono, Inc.) and ganirelix (Ganirelix Acetate Injection [previously Antagon Injection]; Organon, Inc.) are GnRH antagonists that are approved by the U.S. FDA for the inhibition of premature LH surge and are commercially available in the United States. This Phase IV study comparing a single dose of 3 mg of cetrorelix to a daily dosing of 0.25 mg of ganirelix in a cycle programmed, flexible protocol was the first clinical trial to directly compare 3 mg of cetrorelix and 0.25 mg of ganirelix. Premature LH surge, defined as LH level 10 miu/ml on the day of hcg administration, was not observed in any patient in either antagonist treatment group. For the antagonist treatment groups combined, almost all of the patients (97.1%) achieved an LH level 5 IU/L on the day of hcg administration. Of note was that the median LH level on day of hcg administration was 1.3 IU/L for each of the treatment groups; however, the range was smaller for the cetrorelix group (range IU/L) than the ganirelix group (range IU/L). Pang et al. (28) observed a similar median LH value in a stimulation protocol using recombinant FSH in a pen device and ganirelix (median 1.0 IU/L; range IU/L). Although LH levels of 10 IU/L or greater were not observed in our study, Pang et al. (28) observed two patients with an LH level 10 miu/ml (ranging from 20 to 21 miu/ml) after initiation of ganirelix treatment. Previously, there has been some controversy that has attributed potential clinical outcomes to differences in the pharmacokinetic profiles of these antagonists. However, our study, which is the first to report data from a direct comparison in a randomized clinical trial, showed no difference in the ability of these antagonists to prevent an LH surge, or in IVF outcomes or PRs. Significantly fewer injections of cetrorelix (median of 1 injection) were administered per patient as compared with ganirelix (median of 4 injections). Most women in the cetrorelix group (66.7%) received only a single 3-mg dose of cetrorelix, which is similar to the results from a large Phase IIIb clinical trial (n 541) by Olivennes et al. (18) that reported the cetrorelix single-dose regimen required only one injection in 73% of patients. In the present study, 89.7% of those patients requiring supplemental cetrorelix (0.25 mg) needed only one additional dose, and only one patient required more than two additional doses. However, most of the patients in the ganirelix group (62.5%) required four or more injections of 0.25 mg of ganirelix. The extent of exposure to OCs, gonadotropins, hcg, and P gel was comparable Fertility and Sterility 115

9 between treatment groups. The duration of gonadotropin therapy was also comparable between groups, and the duration was similar to other antagonist studies (14, 18, 20, 22). After treatment with cetrorelix or ganirelix, the difference in serum E 2 levels between the treatment groups was statistically significant; however, there was no statistically significant difference between the treatment groups in the number of follicles 14 mm on the day of hcg administration or in the E 2 level per follicle 14 mm. An explanation for the difference in serum E 2 levels is not readily available. Because serum E 2 levels are evidence of the composite E 2 product of all functioning follicles, one might expect to find fewer total follicles or mature follicles in the cetrorelix group. This was not the case, however, as the total number of oocytes retrieved was similar in both treatment groups. Similarly, profound LH suppression, which can be associated with inadequate ovarian steroid production, does not serve as a ready explanation for our observations. To counter any potential detrimental effects of LH suppression, all patients received supplemental LH in the form of hmg. Furthermore, LH levels on the day of hcg administration were similar in both groups. Also, a recent study by Sauer et al. (25) demonstrated that there was no benefit in adding LH to the ovulation stimulation protocol for normal gonadotropic women. Oocyte competence was not affected by the lower E 2 levels in the cetrorelix group, as the number of intact oocytes, mature oocytes, or metaphase II oocytes was not different for the treatments. Furthermore, there was no statistical difference between the treatment groups in the number of oocytes inseminated. For both antagonist treatments, approximately two-thirds of the oocytes inseminated per patient reached the 2PN stage. Embryos were obtained from 81.6% of cetrorelix patients and 75.9% of ganirelix patients who underwent ovum pick-up. There was no statistically significant difference in the percentage of patients with grade A embryos transferred in either treatment group. Blastocysts were obtained from similar percentages of cetrorelix and ganirelix patients. The numbers of embryos or blastocysts obtained, transferred, or cryopreserved were also comparable for the two antagonist treatments. Mean implantation rates were not statistically different between groups (25.8% in the cetrorelix group and 30.1% in the ganirelix group). Moreover, there were no statistically significant differences in the overall PR between the cetrorelix and ganirelix groups (52.3% vs. 52.4%) or for clinical pregnancies (46.5% vs. 46.3%). Interestingly, these PRs were higher in the present study than rates noted in previous cetrorelix or ganirelix Phase III/IIIb studies (14, 18, 20 22). However, previous studies used a fixed protocol for timing initiation of GnRH antagonist administration, whereas the present trial used a flexible protocol that included OC pretreatment and hmg added to recombinant hfsh during stimulation. Two of these studies (18, 21) had similar starting doses (225 IU) to the present study with similar total gonadotropins administered, whereas the other studies had a lower starting dose (150 IU) and used lower total gonadotropins. Two studies have evaluated fixed vs. flexible antagonist protocols. In a study by Ludwig et al. (17), estimation of PRs was not possible because there were too few patients to reliably calculate the rates. However, in the other study (23), clinical PRs were 33.0% and 22.7% for the fixed protocol and flexible protocol, respectively. These rates were also lower than those observed in the present study. The most common treatment-emergent adverse event for both treatment groups was abdominal distension. Similar rates of OHSS and ectopic pregnancy were noted for cetrorelix and ganirelix patients. For both treatment groups combined, mild or moderate OHSS occurred in 12 patients, and 1 severe case occurred in the ganirelix group resulting in hospitalization. However, OHSS, including abdominal distension, is considered an intrinsic risk of the stimulation procedure with gonadotropins. The majority of adverse events for both treatment groups were categorized as mild or moderate in nature. Overall, a similar safety profile was observed for both cetrorelix and ganirelix, and was also comparable with the currently known safety profiles of these antagonists. In conclusion, cetrorelix and ganirelix were effective in preventing LH surges when used in an OCprogrammed, flexible protocol. These antagonists had a similar safety profile and PR; however, cetrorelix required significantly fewer injections, increasing patient convenience. Acknowledgments: The authors thank the study investigators and their staff for their participation in this study. We also thank Emily Wiehe, R.N., lead Clinical Research Associate, Serono, Inc. for overseeing details related to investigator sites and patient enrollment and thank Sherri DeGiorgio, Senior Data Analyst, Serono, Inc., for supervising all aspects of data management. The expert writing assistance and editorial guidance of Barbara Johansson and Joan Schertz, Serono, Inc., is greatly appreciated. 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