Recombinant Human Luteinizing Hormone to Trigger ...

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OBJECTIVE: To evaluate recombinant human luteiniz- ing hormone (r-hLH) versus urine-derived human cho- rionic gonadotropin (u-hCG) to trigger ovulation in.

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Recombinant Human Luteinizing Hormone to Trigger Ovulation Randomized, Controlled, Dose-finding Pilot Study in Ovulation Induction Roger A. Pierson, M.S., Ph.D., Olufemi A. Olatunbosun, M.D., Donna R. Chizen, M.D., Helen Saunders, B.Sc. (Hons), Ernest Loumaye, M.D., Ph.D., and Beatrice De Moustier, M.D.

OBJECTIVE: To evaluate recombinant human luteinizment. All patients in the r-hLH 2,750 (13/13), 5,500 (12/ ing hormone (r-hLH) versus urine-derived human cho12), 11,000 IU (13/13), and u-hCG 5,000 IU (12/ rionic gonadotropin (u-hCG) to trigger ovulation in 12) groups ovulated; 3/5 patients in the r-hLH 825 women (aged 20–40 years) IU and 2/12 in the r-hLH with WHO Group II anovu22,000 IU group failed to latory infertility undergoing … the minimal effective dose of ovulate (p = 0.105 between ovulation induction (OI) evaluable groups). The mean r-hLH to trigger ovulation in with recombinant human ratio of ruptured follicles/ OI is 2,750 IU. follicle-stimulating hormone follicle ≥ 15 mm was 1.1 (r-hFSH) (150 IU/day start(p = 0.675 between groups). ing dose). The monofollicular ovulation STUDY DESIGN: For this Phase II, open-label, doserate was 15/60 (25%). Two cases of ovarian hyperstimufinding pilot study, patients were randomized to doses of lation syndrome were reported. 825, 2,750, 5,500, 11,000, or 22,000 IU r-hLH or u-hCG CONCLUSION: This open-label, pilot study (conducted (5,000 IU). Primary endpoints were ovulation and ratio in 1999–2001) suggests that the minimal effective dose of ruptured follicles/follicle ≥ 15 mm (day of r-hLH/ of r-hLH to trigger ovulation in women with WHO u-hCG administration). Secondary endpoints included Group II anovulatory infertility undergoing OI with monofollicular ovulation and clinical pregnancy rates. r-hFSH (150 IU starting dose) was 2,750 IU. (J Reprod RESULTS: All 67 randomized patients completed treatMed 2014;59:355–366) From the Department of Obstetrics, Gynecology and Reproductive Sciences, College of Medicine, University of Saskatchewan, Saskatchewan, Canada; Merck Serono S.A.–Geneva, Switzerland; and PregLem SA, Geneva, Switzerland. Data published as ASRM (American Society for Reproductive Medicine) 2002 abstract: Fertil Steril 2002;78(Suppl 1):S56. Address correspondence to: Roger A. Pierson, M.S., Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W8 ([email protected]). Financial Disclosure: This study was funded by Merck Serono S.A.–Geneva, Switzerland. Study monitoring and statistical analysis were performed by Merck Serono S.A.–Geneva, Switzerland. Dr. Pierson received a travel grant from Merck Serono S.A.–Geneva, Switzerland, to present the data at an international meeting. He is the President and CSO of Synergyne, Inc., in addition to being majority stockholder. He currently consults for ADE Therapeutics, Inc., Ferring Pharmaceuticals AS, Mitomics, Inc., Pelican MRI, Inc., and Pharmalytics, Inc. His past consultancies include Agile Therapeutics, Bioniche Life Sciences, Janssen-Ortho/R.W. Johnson Pharmaceutical Research Institute, Stem Cell Therapeutics, and Wyeth Pharmaceuticals. He is also a Scientific Advisory Board Member of ADE Therapeutics, Saskatoon, Saskatchewan, Canada. Ms. Saunders and Dr. De Moustier were employees of Merck Serono S.A.–Geneva, Switzerland, at the time of this study.

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Keywords: anovulatory infertility, human chorionic gonadotropin, infertility techniques, ovarian hyperstimulation syndrome, ovulation induction, recombinant human luteinizing hormone. The natural surge of luteinizing hormone (LH) is a key event in the menstrual cycle and triggers a physiologic cascade resulting in follicle rupture and expulsion of the oocyte at ovulation.1 The amplitude, duration, and appropriate timing of this surge are essential for successful ovulation. A surrogate LH surge must be provided when the feedback mechanisms that stimulate an endogenous LH surge are impaired.1 Historically, human chorionic gonadotropin (hCG) from urine was used to mimic the endogenous LH surge. hCG activates the same receptor as LH, and urine-derived hCG (u-hCG) was easy to source.2 However, there are important differences in the pharmacokinetic and pharmacodynamic profiles of the two molecules. The natural LH surge lasts for approximately 2 days,3 whereas serum hCG levels remain above baseline for ≤ 10 days following administration of 5,000 or 10,000 IU u-hCG.3-6 Compared with LH, hCG has a severalfold higher affinity for the human LH receptor,7 a slower rate of internalization,8 and more prolonged intracellular bioactivity.9 Currently, gonadotropins are used for ovulation induction (OI) in women with WHO Group II anovulatory infertility who have failed to conceive using clomiphene citrate.10 Other alternatives to clomiphene citrate for these patients have been investigated: for example, the aromatase inhibitor letrozole.11,12 Conventional gonadotropin OI therapy involves daily administration of folliclestimulating hormone (FSH) to recruit follicles, ideally resulting in the physiologic selection and preferential maturation of a single dominant follicle. A single injection of hCG is given when the dominant follicle reaches the optimal diameter to induce ovulation and luteogenesis. However, OI frequently results in multifollicular development, which is associated with risks of multiple ovulation, multiple pregnancy, and ovarian hyperstimulation syndrome (OHSS).13,14 Ovulation of a single dominant follicle is a key goal in fertility treatment cycles.15 Strategies to avoid excessive follicular development during OI include, but are not limited to, the use of low-dose and chronic low-dose FSH protocols.15-19 The lowdose approaches are associated with lower rates of

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multiple pregnancy and OHSS as compared with conventional treatment protocols.16-18 However, some cycles still must be canceled during the follicular phase to avoid the development of OHSS. Administration of low doses (225–450 IU) of recombinant human LH (r-hLH) during the late follicular phase may facilitate selective follicular growth and encourage monofollicular development.20 In addition, in assisted reproductive technology (ART) cycles, a single dose of r-hLH (5,000, 15,000, or 30,000 IU) was shown to induce final follicular maturation and early luteinization.2 Furthermore, r-hLH was as effective as u-hCG, 5,000 IU, in terms of oocyte recovery, fertilization, and embryo development.2 Importantly, the half-life of LH21 is shorter than that of hCG.4 Therefore, it is reasonable to hypothesize that the use of r-hLH to trigger ovulation could limit the number of follicles that ultimately rupture. Indeed, it has been suggested that the use of r-hLH to trigger ovulation could improve the safety of OI protocols by reducing the incidence of OHSS.22 Currently, r-hLH is approved for use in association with an FSH preparation for the stimulation of follicular development in adult women with severe LH and FSH deficiency (in clinical trials, these patients were defined by an endogenous serum LH level < 1.2 IU/L).23 The present study evaluated the efficacy of different doses of r-hLH versus u-hCG to induce ovulation during OI and investigated the minimal effective dose of r-hLH required. Materials and Methods This randomized, open-label, single-center, dosefinding, Phase II pilot study (Clinicaltrials.gov identifier NCT 01735422, Protocol #21321) was conducted in Canada in accordance with the Declaration of Helsinki, ICH Good Clinical Practice Guidelines, and the Canadian Tri-Council Policy Statement on the Ethical Conduct of Research Involving Humans. Written informed consent was obtained from all participants prior to study start. The primary objectives of the study were to assess the efficacy of r-hLH as compared with u-hCG in inducing ovulation in women undergoing ovarian stimulation with recombinant human FSH (rhFSH) and to estimate a minimal effective dose of r-hLH in this indication. Women aged 20–40 years who had WHO Group II anovulatory infertility and were eligible for OI with gonadotropins for in vivo conception were recruited from a tertiary care center. Key inclusion criteria included a body mass

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index (BMI) of 18–35 kg/m2, a normal uterine cavity, at least 1 patent fallopian tube, early follicular phase serum FSH level of < 12 IU/L (cycle day 2–4 or anytime if not menstruating regularly), and a serum prolactin level of < 800 mIU/L. A male partner with an analysis (performed within the previous 6 months) of semen considered acceptable for intrauterine insemination (IUI) (20 million motile sperm per raw semen ejaculate and/or 5 million motile sperm following preparation for IUI swimup preparation) was an original requirement for participation, but, following a protocol amendment, the use of donor sperm was permitted. Key exclusion criteria included a persistent ovarian cyst of > 20 mm, severe endometriosis (American Society for Reproductive Medicine classification Stage III or IV), ovarian endometrioma, pelvic inflammatory disease, known allergy or hypersensitivity to human gonadotropin preparations, abnormal undiagnosed gynecological bleeding, clinically significant systemic disease, or any other medical condition that may have affected the absorption, distribution, metabolism, or excretion of the study drug. Patients with thyroid-stimulating hormone levels at screening that indicated thyroid disease were also excluded. Interventions Patients received pretreatment with an oral contraceptive pill (Ortho-Cyclen 35, Ortho-McNeil Pharmaceutical, Inc., Raritan, New Jersey). Following a negative pregnancy test patients received r-hFSH (follitropin alfa, GONAL-f, Merck Serono S.A.– Geneva, Switzerland) administered subcutaneously. Daily r-hFSH treatment was initiated on cycle days 3–5 and continued for up to 28 days. A starting daily dose of 150 IU r-hFSH was used (as per the center’s standard practice at the time of the study). The r-hFSH dose could be altered according to ovarian response (reduced to 75 IU/day, increased to 225 IU/day). The ovarian response was monitored closely using transvaginal ultrasound (TVUS) and serum estradiol (E2) levels. TVUS examination was performed every second day from cycle day 6 and then daily when the dominant follicle reached 16 mm in diameter. Follicular development was considered adequate when ≥ 1 follicle with a mean diameter ≥ 18 mm and not more than 5 follicles ≥ 15 mm were observed and serum E2 levels were < 9,000 pmol/L. Patients were randomly assigned to 1 of 6 treatment groups: 825 IU (37.5 μg), 2,750 IU (125 μg),

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5,500 IU (250 μg), 11,000 IU (500 μg), or 22,000 IU (1 mg) of r-hLH (Luveris, Merck Serono S.A., Geneva, Switzerland) or u-hCG (5,000 IU). Randomization was performed using a computergenerated randomization list (SAS software v6.12 for Windows using the PROC PLAN procedure). Patients and investigators were aware of treatment allocation at the time of r-hLH/u-hCG administration. Patients received a single subcutaneous injection of study medicine administered by a healthcare professional. Ovulation was determined by daily TVUS monitoring of the ovaries for signs of follicular rupture for up to 4 days and on days 6–9 following r-hLH/ u-hCG administration.24 IUI was performed twice, approximately 36 and 60 hours after r-hLH/u-hCG administration (or earlier if at least 1 ruptured follicle was observed on TVUS performed 1 day after r-hLH/u-hCG administration). Luteal phase support (United States Pharmacopeia progesterone [P4], 200 mg, twice daily, delivered via a locally produced wax-based intravaginal pessary) was initiated 2 days after r-hLH/u-hCG administration. Serum E2 levels were measured locally (prior to study start, during r-hFSH treatment, and on the day of r-hLH/u-hCG administration) and at a central laboratory (day of r-hLH/u-hCG administration and 1, 2, and 6–9 days thereafter). A retrospective analysis was conducted of FSH, LH/hCG, P4, androstenedione, total renin, and vascular endothelial growth factor (VEGF) in serum collected on the day of r-hLH/u-hCG administration and 1, 2, and 6–9 days thereafter. Endometrial thickness was measured clinically as the distance from the anterior stratum basalis–– myometrial junction to the posterior stratum basalis––myometrial junction in the midsagittal plane. The sagittal plane of section that represented the largest dimension of the endometrium between 5 mm and 10 mm from the fundus was used as a standard location for measurements. Endometrial thickness was assessed on r-hLH/u-hCG days 0, 1, 2, 3, 4, and 6–9. Biochemical pregnancy was detected by a serum β-hCG test performed 15–20 days after the administration of r-hLH/u-hCG. A positive result (> 10 mIU/mL) was confirmed by a second test the following week. Clinical pregnancy was determined by TVUS visualization of an intrauterine gestational sac (with or without heart activity) 35–42 days after r-hLH/u-hCG administration. Each patient underwent only 1 study cycle. Pa-

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tients with no ovarian response, an excessive response to r-hFSH, premature luteinization, WHO grade 3 or 4 adverse event (AE), or an AE justifying treatment cessation were withdrawn from the study. Treatment Group Protocol Amendment. The r-hLH 825 IU treatment group was added following a protocol amendment. An ongoing review of primary efficacy data showed that the 6 patients already randomized to the 2,750 IU r-hLH dose group had all ovulated; therefore, the protocol amendment was required to enable evaluation of the minimum effective dose of r-hLH. At this point, half of the patients planned had been assigned to 1 of the 5 original treatment groups. Rapid recruitment to the new treatment group was required; therefore, weighted randomization was implemented. Six of the next 16 patients were assigned to the new treatment group. Outcomes Primary efficacy endpoints were ovulation, defined as follicle rupture 1–4 days after r-hLH/u-hCG administration, and the ratio of ruptured follicles per follicle ≥ 15 mm on the day of r-hLH/u-hCG administration. Additional post-hoc analyses were performed to provide supportive data, including an analysis of the number of intermediate-sized follicles (11–14 mm) and the ratio of ruptured follicles per follicle ≥ 11 mm on the day of r-hLH/u-hCG administration. Secondary efficacy endpoints included (1) the proportion of patients who achieved monofollicular ovulation, (2) the number of biochemical and clinical pregnancies, and (3) serum E2, P4, androstenedione, total renin and VEGF levels. AEs were recorded during the study and for up to 30 days after the administration of r-hCG/u-hCG. Data Analysis Sample Size. No formal dose-group sample-size calculations were performed. The sample size for this pilot study was determined by clinical judgment alone, as no prior efficacy data were available to support this estimation. Furthermore, at the time of study initiation, pilot data were required to investigate the use of LH in this indication of ovulation triggering; therefore, this study could provide valuable initial data, and, if a positive trend in outcome was seen, larger trials with greater statistical power could be designed. Following the treatment-

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group protocol amendment, it was planned to have 6 patients in the r-hLH 825 IU group and 12 patients in each of the other treatment groups. Any group in which 3 treatment failures occurred would be discontinued, as the small population would prevent valid statistical comparisons of efficacy. Patient Populations. The intent-to-treat population included all randomized patients and was used to evaluate the primary efficacy endpoint of ovulation. All other efficacy analyses were performed on the per-protocol population (defined as all randomized patients who completed the study without a major protocol deviation and who ovulated). The safety population was defined as all patients who received at least 1 dose of study drug. Change to Planned Analysis. Three of 5 patients randomized to the 825 IU dose group failed to ovulate. Therefore, recruitment to the 825 IU dose group was discontinued, and this group of patients was excluded from the per-protocol population. Statistics. The endpoints included binary, categorical, and continuous outcomes. Binary data were analyzed using two-sided Fisher’s exact tests (continuity corrected χ2). The exact Cochran–Armitage trend test was used to assess the dose-response relationship (to evaluate whether success increased with the dose of r-hLH). Categorical data were analyzed using the Cochran–Mantel–Haenszel method. Continuous efficacy endpoints were analyzed using analysis of variance, or analysis of covariance if a continuous covariate was also included. Polynomial contrasts were used to assess the dose-response relationship to define the dose of rhLH that led to the best response. All statistical tests were performed using a twosided significance level of 0.05 (SAS v6.12). Efficacy data are presented as means with standard deviations (SDs) unless stated otherwise. Results Patient Characteristics and Disposition The study was conducted between December 1999 and July 2001. In total, 67 patients (mean [SD] age, 33.1 [4.1] years) were randomized to receive r-hLH or u-hCG to trigger ovulation (Figure 1). There were 2 patients (both in the r-hLH 11,000 IU group) who had polycystic ovarian disease. All 67 patients completed treatment and underwent IUI. Patients in the r-hLH 2,750 IU group had a lower mean BMI than

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Figure 1 Patient disposition. aAs only 2/5 patients in the r-hLH 825 IU group ovulated, this group was excluded from the per-protocol population. ITT = intent to treat, PP = per protocol, r-hFSH = recombinant human follicle-stimulating hormone, r-hLH = recombinant human luteinizing hormone, u-hCG = urine-derived human chorionic gonadotropin.

those in the other treatment groups (p = 0.024) (Table I). No other significant differences in baseline demographic or fertility characteristics were found between treatment groups. The mean (SD) duration of r-hFSH stimulation was 11.2 (3.1) days (Table II); however, the duration of stimulation differed among treatment groups (p = 0.045). Overall, the mean (SD) total FSH dose requirement was 22.1 (7.9) 75 IU ampoules (Table II), and there was no difference among treatment groups (p = 0.242). The number of follicles ≥ 11 mm prior to ovulation triggering was higher in the r-hLH 2,750 IU group than in any of the other r-hLH groups (Table III); for example, the mean number of those follicles was 7.5 in the r-hLH 2,750 IU group versus 5.3 in the r-hLH 22,000 IU group (p = 0.060). The mean number of intermediate-sized follicles (11–14 mm) was higher in the 2,750 IU group than in all other groups combined (4.4 vs. 2.5, p = 0.014). Efficacy Ovulation was detected in 62/67 (93%) patients

(Figure 2). Three of the 5 (60%) patients in the r-hLH 825 IU group and 2/12 (17%) patients in the r-hLH 22,000 IU group failed to ovulate. No significant differences in ovulation rates were observed among evaluable treatment groups (p = 0.105). The mean ratio of ruptured follicles (on day 4 following r-hLH/u-hCG administration) per follicle ≥ 15 mm (on the day of r-hLH/u-hCG administration) was 1.1 (median 1.0; minimum 0.2, maximum 2.3); across the treatment groups the mean ratios ranged from 1.0–1.2; no differences among groups (p = 0.675) and no relationships with r-hLH dose were found. The ratio of ruptured follicles per follicle ≥ 15 mm was > 2 for 1 patient in the r-hLH 11,000 IU group. The mean ratio of ruptured follicles per follicle ≥ 11 mm was 0.7 and differed among groups (p = 0.033). The lowest mean ratio of ruptured follicles was observed in the r-hLH 2,750 IU group (0.5 vs. 0.7 in the other r-hLH groups and 0.9 in the u-hCG group). Lower doses of r-hLH were associated with a lower proportion of ruptured follicles (Figure 3); furthermore, the dose of r-hLH administered and

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Table IA Patients’ Baseline Demographic Characteristics (Intent-to-Treat Population) r-hLH Characteristic Age (years) Mean (SD) Median Range BMI (kg/m2) Mean (SD) Median Range Ethnicity, n (%) White Black/Asian/other

u-hCG

825 IU n=5

2,750 IU n = 13

5,500 IU n = 12

11,000 IU n = 13

22,000 IU n = 12

5,000 IU n = 12

Total n = 67

31.6 (3.4) 34.0 27.0–34.0

32.1 (4.2) 31.0 26.0–39.0

35.2 (3.1) 35.0 30.0–39.0

32.8 (3.9) 32.0 28.0–40.0

33.7 (4.3) 34.5 26.0–39.0

32.6 (4.8) 33.5 25.0–40.0

33.1 (4.1) 34.0 25.0–40.0

26.0 (5.3) 24.8 21.0–35.0

24.1 (3.5)a 23.6 20.4–32.4

25.9 (4.1) 24.5 21.4–33.2

26.9 (5.3) 27.0 19.1–34.9

29.0 (4.1) 29.4 23.3–35.0

27.4 (3.9) 26.5 21.8–34.9

26.6 (4.4) 25.8 19.1–35.0

5 (100.0) 0 (0.0)

11 (84.6) 2 (15.4)

12 (100.0) 0 (0.0)

12 (92.3) 1 (7.7)

7 (58.3) 5 (41.7)

12 (100.0) 0 (0.0)

59 (88.1) 8 (11.9)

ap = 0.024.

Table IB Fertility Characteristics of Patients (Intent-to-Treat Population) r-hLH Characteristic Type of infertility, n (%) Primary Secondary Duration of infertility (years) Mean (SD) Cause of infertility,a n (%) Male factor Endometriosis Unexplained Type of previous pregnancy reported, n (%) Spontaneous Ovulation induction

u-hCG

825 IU n=5

2,750 IU n = 13

5,500 IU n = 12

11,000 IU n = 13

22,000 IU n = 12

5,000 IU n = 12

Total n = 67

4 (80.0) 1 (20.0)

6 (46.2) 7 (53.8)

7 (58.3) 5 (41.7)

6 (46.2) 7 (53.8)

5 (41.7) 7 (58.3)

6 (50.0) 6 (50.0)

34 (50.7) 33 (49.3)

5.0 (2.5)

3.9 (1.9)

4.5 (2.2)

4.4 (2.4)

4.6 (2.4)

4.5 (1.4)

4.4 (2.1)

2 (40.0) 1 (20.0) 2 (40.0)

-2 (15.4) 7 (53.8)

2 (16.7) 3 (25.0) 5 (41.7)

2 (15.4) 1 (7.7) 7 (53.8)

2 (16.7) 2 (16.7) 6 (50.0)

5 (41.7) 3 (25.0) 2 (16.7)

13 (19.4) 12 (17.9) 29 (43.3)

1 (20.0) 1 (20.0)

5 (38.5) 3 (23.1)

3 (25.0) 3 (25.0)

4 (30.8) 4 (30.8)

6 (50.0) 2 (16.7)

5 (41.7) 1 (8.3)

24 (35.8) 14 (20.9)

aThe top 3 reasons for the overall population are reported; for some patients, infertility was attributed to more than one cause. All patients were classified as having WHO Group II anovulatory infertility.

the number of follicles ≥ 11 mm that ruptured were highly correlated (p = 0.003). On days 1, 2, 3, and 4, and 6–9 days following r-hLH/u-hCG administration, the mean (SD) number of ruptured follicles was 0.2 (0.6), 0.6 (1.3), 3.2 (2.0), 3.3 (2.0), and 3.4 (2.0). Thus, the majority of follicles ruptured > 36 hours after r-hLH/u-hCG administration. No differences among treatment groups were identified for the mean total number of ruptured follicles (p > 0.513). Monofollicular and bifollicular ovulation rates are shown in Figure 4. Overall, 15 patients (25%) achieved monofollicular ovulation, and 24 patients (40%) achieved either monofollicular or bifollicular ovulation. There were no significant differences in the proportion of patients who achieved mono-

follicular or bifollicular ovulation in each treatment group (p > 0.122), and no trends were observed across treatment groups (p > 0.834). Overall, 17/60 (28%) patients had a biochemical pregnancy, and 16/60 (27%) achieved a clinical pregnancy (Figure 4). There were no differences among treatment groups in the proportion of patients who achieved a biochemical pregnancy (p = 0.456) or clinical pregnancy (p = 0.655); no trends for either outcome were observed (p > 0.548). There was 1 biochemical miscarriage and one extrauterine pregnancy in the r-hLH 11,000 IU group, and 1 clinical miscarriage in the 2,750 IU group. Mean (SD) endometrial thicknesses (mm) were 9.9 (2.2), 9.7 (2.2), 10.0 (2.3), 10.2 (2.2), 10.3 (2.4), and 9.6 (3.0) on days 0, 1, 2, 3, 4, and 6–9 after r-hLH/

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Table II Duration and Total Dose of Recombinant Human Follicle-Stimulating Hormone Treatment (Intent-to-Treat Population) r-hLH Characteristic Duration, days Mean (SD) Median (range) Range 75 IU ampoules required, n Mean (SD) Median Range

825 IU n=5

2,750 IU n = 13

5,500 IU n = 12

11,000 IU n = 13

22,000 IU n = 12

5,000 IU n = 12

Total n = 67

8.8 (1.9) 8.0 7.0–12.0

10.7 (2.4) 10.0 8.0–17.0

11.5 (2.5) 11.5 8.0–16.0

13.4 (4.6) 11.0 9.0–24.0

11.1 (2.2) 11.0 8.0–16.0

10.3 (2.4) 11.0 6.0–14.0

11.2 (3.1) 11.0 6.0–24.0

17.4 (4.3) 16.0 14.0–25.0

20.2 (5.7) 20.0 13.5–30.0

24.3 (6.9) 23.0 16.0–39.0

25.7 (12.9) 20.0 10.0–59.0

21.0 (5.8) 19.5 14.0–35.0

20.9 (5.2) 22.0 12.0–29.0

22.1 (7.9) 20.0 10.0–59.0

Safety

u-hCG administration, respectively. The differences among treatment groups were of borderline significance on day 3 following r-hLH/u-hCG administration (p = 0.069); no significant differences were found on any other days (p > 0.281). Serum E2, P4 and androstenedione levels at 1, 2, and 6–9 days after treatment with r-hLH or u-hCG are presented in Table IV. No differences among treatment groups were observed in mean serum renin levels on any of the days assessed (p > 0.079) (Table IV), although a trend was found 6–9 days after r-hLH/u-hCG administration (p = 0.012). At this time point, higher doses of r-hLH were associated with higher mean serum total renin levels. Mean serum renin levels in the 5,000 IU u-hCG group were higher than in any r-hLH dose groups on days 6–9. No differences between treatment groups were found in mean serum VEGF levels on days 1, 2, or 6–9 after r-hLH/u-hCG administration (p > 0.611), and no trends were identified (p > 0.354).

Table III

u-hCG

Most patients (58/62; 94%) reported no local reactions to injections. Overall, 17/67 patients (25%) reported at least 1 AE (total of 29 AEs) after r-hLH/ u-hCG administration (plus 30 days). During this period all AEs were mild or moderate in severity. Two cases of OHSS were reported after r-hLH/ u-hCG administration (plus 30 days): 1 in the r-hLH 2,750 IU group (mild OHSS) and 1 in the r-hLH 11,000 IU group (moderate OHSS); both occurred before confirmation of pregnancy and were considered to be probably related to treatment. Both patients had elevated serum E2 levels (10,040 pmol/L and 9,150 pmol/L, respectively) and a high number of follicles ≥ 11 mm (11 and 8, respectively) on r-hLH/u-hCG day 0. Three (3/60; 5%) multiple pregnancies occurred: 1 twin pregnancy each in the r-hLH 5,500 IU and u-hCG 5,000 IU groups and a quadruple pregnancy in the r-hLH 2,750 IU group; this patient had 11 follicles ≥ 11 mm (and no dominant follicles > 15 mm)

Number of Follicles Prior to Ovulation Triggering (Intent-to-Treat Population) r-hLH

No. of follicles ≥ 15 mm Mean (SD) Median Range ≥ 11 mm Mean (SD) Median Range ap = 0.060

vs. r-hLH 20,000 IU group.

u-hCG

825 IU n=5

2,750 IU n = 13

5,500 IU n = 12

11,000 IU n = 13

22,000 IU n = 12

5,000 IU n = 12

Total n = 67

3.6 (1.3) 3.0 2.0–5.0

3.1 (1.4) 4.0 1.0–5.0

3.6 (1.1) 3.5 2.0–5.0

3.2 (1.3) 3.0 1.0–6.0

2.3 (1.3) 2.0 1.0–5.0

2.8 (1.3) 3.0 1.0–5.0

3.0 (1.3) 3.0 1.0–6.0

6.2 (3.5) 6.0 3.0–12.0

7.5 (3.6)a 8.0 1.0–14.0

6.3 (2.5) 6.5 2.0–10.0

6.0 (3.3) 7.0 1.0–11.0

5.3 (4.4) 3.5 1.0–13.0

4.1 (2.5) 4.0 1.0–8.0

5.9 (3.4) 6.0 1.0–14.0

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Table IV Serum Hormone Levels 1, 2, and 6–9 Days After Treatment with r-hLH or u-hCG (Per-Protocol Population) r-hLH Characteristic E2, pmol/L Day 1 Day 2a Days 6–9b P4, nmol/L Day 1 Day 2 Days 6–9c Androstenedione, nmol/L Day 1 Day 2a Days 6–9c Total renin, IU/mL Day 1 Day 2 Days 6–9 VEGF, pg/L Day 1 Day 2 Days 6–9

u-hCG

2,750 IU n = 13

5,500 IU n = 12

11,000 IU n = 13

22,000 IU n = 10

5,000 IU n = 12

Total n = 60

5627 (5585.8) 3096 (2806.0) 249 (153.9)

2933 (1651.2) 1357 (1153.3) 335 (313.8)

3885 (2512.5) 1672 (916.4) 524 (713.4)

3666 (3511.3) 1284 (1070.2) 579 (573.9)

2653 (1958.6) 1285 (1262.2) 1331 (1250.2)

3789 (3448.9) 1775 (1733.3) 597 (787.4)

13.5 (15.8) 18.1 (24.2) 41.1 (23.8)

8.5 (6.6) 16.4 (19.0) 43.2 (20.0)

9.0 (4.5) 16.5 (11.2) 53.7 (21.0)

10.0 (12.1) 14.5 (16.3) 63.1 (42.8)

6.7 (3.2) 10.7 (7.8) 97.7 (41.7)

9.6 (9.6) 15.3 (16.4) 58.6 (35.8)

11.0 (5.5) 11.3 (6.2) 4.4 (1.9)

7.6 (2.0) 6.5 (2.5) 3.7 (1.1)

9.8 (5.9) 8.7 (4.4) 4.3 (1.8)

8.2 (4.2) 6.8 (2.5) 4.3 (2.4)

9.0 (2.3) 7.7 (2.7) 7.9 (3.1)

9.2 (4.4) 8.3 (4.3) 4.9 (2.6)

427 (233.3) 636 (329.6) 234 (70.8)

294 (146.9) 482 (219.2) 258 (118.2)

442 (290.9) 726 (408.8) 270 (166.8)

380 (169.2) 701 (368.8) 272 (72.6)

287 (108.3) 638 (252.9) 363 (113.8)

368 (208.0) 636 (324.1) 279 (120.5)

231 (208.9) 230 (180.8) 235 (210.7)

262 (186.0) 256 (173.8) 232 (178.3)

198 (95.9) 199 (99.4) 201 (122.9)

295 (163.0) 292 (149.8) 304 (146.4)

249 (115.1) 253 (115.7) 267 (108.8)

244 (157.3) 243 (145.4) 245 (157.0)

All values are mean (SD). Between treatment groups: ap = 0.033, bp = 0.004, cp < 0.001.

on the day of r-hLH administration and subsequently underwent a selective reduction procedure.

Two serious AEs were reported (in the r-hLH 11,000 IU dose group and considered to be unrelat-

Figure 2 Cumulative ovulation rate 4 days after r-hLH/u-hCG administration (intent-to-treat population).

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Figure 3 Ratio of ruptured follicles on day 4, following r-hLH or u-hCG administration, per follicle ≥ 11 mm prior to ovulation triggering (perprotocol population). Treatment distributions exclude the patient group receiving 825 IU r-hLH.

ed to r-hLH): 1 ectopic pregnancy in a 33-year-old woman, which resolved with medical management (methotrexate), and 1 premature labor in a 29-yearold woman who went into premature labor at 26 weeks’ gestation; steroids were administered, and the infant was delivered the following day. The mother made a good recovery, but (in accordance with the study remit) no further information on the infant was available. Discussion This dose-finding pilot study evaluated the efficacy of 5 doses of r-hLH (825, 2,750, 5,500, 11,000, and 22,000 IU) versus u-hCG (at 5,000 IU) to trigger ovulation in women with WHO Group II anovulatory infertility, and thus estimated the minimal effective dose of r-hLH to trigger ovulation in OI. Ovulation occurred in the majority (93%) of patients. All patients in the r-hLH 2,750, 5,500, and 11,000 IU and u-hCG 5,000 IU groups ovulated, whereas only 2/5 patients in the 825 IU r-hLH group ovulated. This finding suggests that the minimal effective dose of r-hLH to trigger ovulation in OI is 2,750 IU. Monofollicular ovulation is a key treatment goal for ovarian stimulation protocols.15 It has been suggested previously that the use of hCG, with its long

half-life, to induce final follicular maturation may increase the number of preovulatory follicles present in the ovaries.25 Here, we expected that the optimum dose of r-hLH would generally lead to monofollicular rupture, regardless of the number of follicles present. However, only 25% of patients (15/60) in this study achieved monofollicular ovulation. Furthermore, we were surprised by the similar rates of monofollicular ovulation (23–40%) found in the r-hLH 2,750, 11,000, and 22,000 IU, and u-hCG treatment groups. The mean ratio of ruptured follicles per follicle ≥ 15 mm prior to ovulation triggering was 1.1 and was unrelated to the dose of r-hLH. Interestingly, the proportion of ruptured follicles per follicle ≥ 15 mm was > 2 for 1 patient, indicating that more than twice as many follicles ruptured as were visible on the day of r-hLH/u-hCG administration. This finding could be interpreted to mean that large follicles (≥ 15 mm) are poor indicators of the population of follicles that will rupture. It is possible that the intermediate-sized follicle (11–14 mm) population could provide a better indicator of the number of follicles that will ultimately rupture. A smaller proportion of follicles per follicle ≥ 11 mm ruptured in the r-hLH 2,750 IU group versus

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Figure 4 Rates of (A) monofollicular ovulation and monofollicular or bifollicular ovulation and (B) biochemical and clinical pregnancy by treatment group (per-protocol population).

the other r-hLH and u-hCG groups (p = 0.033). We believe that r-hLH at 2,750 IU may limit the number of intermediate- and large-sized follicles that rupture while still triggering the ovulation of at least 1 follicle. In this study, good clinical pregnancy rates (25– 39%) were obtained following administration of r-hLH 2,750–11,000 IU to trigger ovulation, and these are equivalent to or exceed those reported using r-hCG or u-hCG in OI (22–29%).26 The use of r-hLH to stimulate final follicular maturation in ART has also been investigated.2,27 Pooled data from these 2 studies showed that r-hLH at doses of

5,000–30,000 IU resulted in a clinical pregnancy rate of 25%.28 As this was a dose-finding pilot study, the conclusions that can be made regarding safety data are limited. The incidence of OHSS was low, with only 2 patients (1 in the r-hLH 2,750 IU group and 1 in the r-hLH 11,000 IU group) experiencing OHSS. The data on these 2 patients were interpreted to suggest an excessive response to OI therapy. The methodology reported here reflects the standard practice at the time that this study was conducted (1999–2001). However, the large number of follicles observed following r-hFSH treatment in

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the present study suggests that the protocol resulted in an ovarian response greater than that of a modern OI cycle. A high starting dose of 150 IU rhFSH was used as per the center’s standard practice in 1999–2001 (and could be increased to 225 IU in patients who had a reduced ovarian response). Such a high starting dose is no longer recommended in women with polycystic ovary syndrome (PCOS).29 Importantly, the criteria for triggering ovulation (1 follicle ≥ 18 mm but no more than 5 follicles ≥ 15 mm) were liberal by modern standards. Indeed, current recommendations to minimize the risk of complications in women with PCOS are to withhold hCG if there are > 2 follicles ≥ 16 mm, or > 1 follicle ≥ 16 mm plus 2 follicles ≥ 14 mm.29 The dose of u-hCG used in this study (5,000 IU) again reflects the standard clinical practice at the time, which was to use a dose of between 5,000 and 10,000 IU. Also, details of the ultrasound performed in our clinic at the time of the study have been reported previously.24,30-32 In addition, semen analysis techniques were nonstandardized at the time that this study was conducted. However, the high doses of r-hFSH administered and the liberal criteria for ovulation triggering limit detailed comparisons of our data with more recent work. In addition, over the last decade ART has become more readily available and now results in good clinical pregnancy rates.33,34 We believe that, in current real-life clinical practice, a substantial subset of the patients recruited to this study would undergo first-line therapy with ART rather than OI.29 Furthermore, we acknowledge the limitations of this pilot study, such as the open-label design, the lack of a sample size calculation and the use of a relatively small sample size for the evaluation of 6 randomized treatment groups. In summary, this pilot study indicated that the minimal effective dose of r-hLH to trigger ovulation in women with WHO Group II anovulatory infertility undergoing OI with r-hFSH may be 2,750 IU (125 μg). The use of r-hLH 2,750 IU to trigger ovulation limited the number of intermediate- and largesized follicles that ruptured (as compared with uhCG), while triggering ovulation of ≥ 1 follicle in all patients. Furthermore, r-hLH 2,750 IU resulted in a good clinical pregnancy rate of 31%. However, our data do not indicate a clinical benefit (in terms of monofollicular or bifollicular ovulation, multiple pregnancy, or OHSS rates) for any dose of r-hLH versus hCG and, owing to the high starting dose of

r-hFSH, do not apply to modern OI protocols in women with PCOS. Further studies of r-hLH in this indication, for example, using modern OI protocols, may prove to be valuable. Acknowledgments The authors thank John Deptuch (Women’s Health Imaging Research Laboratory, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Saskatchewan College of Medicine, Canada) for computer programming and database management, and Angela Baerwald (Department of Obstetrics, Gynecology and Reproductive Sciences, University of Saskatchewan College of Medicine, Canada) for ultrasonography assistance. The authors appreciate the valuable contributions from Heather Harris (Clinical Research Associate) and Lionel Pidoux (Medical Research Associate), who are both former employees of Merck Serono S.A.– Geneva, Switzerland. The authors also thank Alyson Bexfield, Hannah Wills, and Catherine Kidd of Caudex Medical (supported by Merck Serono S.A.–Geneva, Switzerland) for their assistance in the preparation of this manuscript. References 1. Shoham Z, Schacter M, Loumaye E, et al: The luteinizing hormone surge––the final stage in ovulation induction: Modern aspects of ovulation triggering. Fertil Steril 1995;64: 237-251 2. European Recombinant LH Study Group: Recombinant human luteinizing hormone is as effective as, but safer than, urinary human chorionic gonadotropin in inducing final follicular maturation and ovulation in in vitro fertilization procedures: Results of a multicenter double-blind study. J Clin Endocrinol Metab 2001;86:2607-2618 3. Hoff JD, Quigley ME, Yen SS: Hormonal dynamics at midcycle: A reevaluation. J Clin Endocrinol Metab 1983;57:792796 4. Weissman A, Lurie S, Zalel Y, et al: Human chorionic gonadotropin: Pharmacokinetics of subcutaneous administration. Gynecol Endocrinol 1996;10:273-276 5. Trinchard-Lugan I, Khan A, Porchet HC, et al: Pharmacokinetics and pharmacodynamics of recombinant human chorionic gonadotrophin in healthy male and female volunteers. Reprod Biomed Online 2002;4:106-115 6. Damewood MD, Shen W, Zacur HA, et al: Disappearance of exogenously administered human chorionic gonadotropin. Fertil Steril 1989;52:398-400 7. Galet C, Ascoli M: The differential binding affinities of the luteinizing hormone (LH)/choriogonadotropin receptor for LH and choriogonadotropin are dictated by different extracellular domain residues. Mol Endocrinol 2005;19:1263-1276 8. Mock EJ, Papkoff H, Niswender GD: Internalization of ovine luteinizing hormone/human chorionic gonadotropin recombinants: Differential effects of the alpha- and beta-

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subunits. Endocrinology 1983;113:265-269 9. Strickland TW, Puett D: Contribution of subunits to the function of luteinizing hormone/human chorionic gonadotropin recombinants. Endocrinology 1981;109:1933-1942 10. Messinis IE: Ovulation induction: A mini review. Hum Reprod 2005;20:2688-2697 11. Roy KK, Baruah J, Singla S, et al: A prospective randomized trial comparing the efficacy of Letrozole and Clomiphene citrate in induction of ovulation in polycystic ovarian syndrome. J Hum Reprod Sci 2012;5:20-25

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21-27 22. Emperaire JC, Edwards RG: Time to revolutionize the triggering of ovulation. Reprod Biomed Online 2004;9:480-483 23. Luveris 75 IU SPC: Luveris 75 IU Summary of Product Characteristics (UK). Available from: http://www.medicines.org. uk/emc/medicine/8289/SPC/Luveris+75+IU/. Accessed Oct 2, 2012 24. Hanna MD, Chizen DR, Pierson RA: Characteristics of follicular evacuation during human ovulation. Ultrasound Obstet Gynecol 1994;4:488-493

12. Polyzos NP, Mauri D, Tzioras S: Letrozole in ovulation induction: Time to make decisions. Hum Reprod Update 2009; 15:263-264

25. Edwards RG, Steptoe PC: Induction of follicular growth, ovulation and luteinization in the human ovary. J Reprod Fertil Suppl 1975;121-163

13. Balen AH, Braat DD, West C, et al: Cumulative conception and live birth rates after the treatment of anovulatory infertility: Safety and efficacy of ovulation induction in 200 patients. Hum Reprod 1994;9:1563-1570

26. International Recombinant Human Chorionic Gonadotropin Study Group: Induction of ovulation in World Health Organization group II anovulatory women undergoing follicular stimulation with recombinant human follicle-stimulating hormone: A comparison of recombinant human chorionic gonadotropin (rhCG) and urinary hCG. Fertil Steril 2001;75: 1111-1118

14. Levene MI, Wild J, Steer P: Higher multiple births and the modern management of infertility in Britain. The British Association of Perinatal Medicine. Br J Obstet Gynaecol 1992; 99:607-613 15. ESHRE Capri Workshop Group: Mono-ovulatory cycles: A key goal in profertility programmes. Hum Reprod Update 2003;9:263-274 16. Hedon B, Hugues JN, Emperaire JC, et al: A comparative prospective study of a chronic low dose versus a conventional ovulation stimulation regimen using recombinant human follicle stimulating hormone in anovulatory infertile women. Hum Reprod 1998;13:2688-2692 17. Homburg R, Levy T, Ben-Rafael Z: A comparative prospective study of conventional regimen with chronic low-dose administration of follicle-stimulating hormone for anovulation associated with polycystic ovary syndrome. Fertil Steril 1995;63:729-733 18. Homburg R, Howles CM: Low-dose FSH therapy for anovulatory infertility associated with polycystic ovary syndrome: Rationale, results, reflections and refinements. Hum Reprod Update 1999;5:493-499 19. White DM, Polson DW, Kiddy D, et al: Induction of ovulation with low-dose gonadotropins in polycystic ovary syndrome: An analysis of 109 pregnancies in 225 women. J Clin Endocrinol Metab 1996;81:3821-3824 20. Loumaye E, Engrand P, Shoham Z, et al: Clinical evidence for an LH ‘ceiling’ effect induced by administration of recombinant human LH during the late follicular phase of stimulated cycles in World Health Organization type I and type II anovulation. Hum Reprod 2003;18:314-322 21. Diczfalusy E, Harlin J: Clinical-pharmacological studies on human menopausal gonadotrophin. Hum Reprod 1988;3:

27. Manau D, Fabregues F, Arroyo V, et al: Hemodynamic changes induced by urinary human chorionic gonadotropin and recombinant luteinizing hormone used for inducing final follicular maturation and luteinization. Fertil Steril 2002;78:1261-1267 28. Al-Inany H, Aboulghar MA, Mansour RT, et al: Recombinant versus urinary gonadotrophins for triggering ovulation in assisted conception. Hum Reprod 2005;20:2061-2073 29. Thessaloniki ESHRE-ASRM-Sponsored PCOS Consensus Workshop Group: Consensus on infertility treatment related to polycystic ovary syndrome. Hum Reprod 2008;23:462-477 30. Pierson RA, Hanna MD: Ultrasonographic morphology of the follicle wall during human ovulation. Ultrasound Int 1999;5:67-78 31. Pierson RA, Hanna MD: Ultrasonographic morphology of the human preovulatory follicle wall. Ultrasound Int 1999; 5:4-13 32. Pierson RA, Martinuk SD, Chizen DR, et al: Ultrasonographic Visualization of Human Ovulation. From Ovulation to Implantation. In Proceedings of the 7th Reinier de Graaf Symposium, Maastricht, the Netherlands. Edited by JHL Evers, MJ Heineman. Amsterdam, Elsevier Science Publishers (Biomedical Division), 1990, pp 73-79 33. Yli-Kuha AN, Gissler M, Luoto R, et al: Success of infertility treatments in Finland in the period 1992-2005. Eur J Obstet Gynecol Reprod Biol 2009;144:54-58 34. Gunby J, Bissonnette F, Librach C, et al: Assisted reproductive technologies (ART) in Canada: 2007 results from the Canadian ART Register. Fertil Steril 2011;95:542-547

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