Copenhagen, Denmark; 8 Holbæk Hospital, Holbæk, Denmark; 9 Hvidovre Hospital, Copenhagen, Denmark. doi: /humrep/dem317

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1 Human Reproduction Vol.23, No.2 pp , 2008 Advance Access publication on November 16, 2007 doi: /humrep/dem317 Recombinant LH supplementation to recombinant FSH during the final days of controlled ovarian stimulation for in vitro fertilization. A multicentre, prospective, randomized, controlled trial A. NyboeAndersen 1,10, P. Humaidan 2, G. Fried 3, J. Hausken 4, L. Antila 5, S. Bangsbøll 1, P.E. Rasmussen 6, S. Lindenberg 7, H. Ejdrup Bredkjaer 8 and H. Meinertz 9, The Nordic LH study group 9 1 The Fertility clinics 4017, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark; 2 Skive Hospital, Skive, Denmark; 3 Karolinska University Hospital, Stockholm, Sweden; 4 Haugesund Hospital, Haugesund, Norway; 5 The Family Federation of Finland, Turku, Finland; 6 Odense University Hospital, Odense, Denmark; 7 Herlev University Hospital, Copenhagen, Denmark; 8 Holbæk Hospital, Holbæk, Denmark; 9 Hvidovre Hospital, Copenhagen, Denmark 10 Correspondence address. Tel: þ /4976; Fax: þ ; anyboea@rh.hosp.dk BACKGROUND: The purpose of this multicentre, multinational trial was to study whether rlh supplementation to recombinant FSH (rfsh) during the late follicular phase increased pregnancy rates. METHODS: After downregulation with nafarelin, 526 women were randomized on Day 1 of stimulation to use either rfsh (Gonal-F) alone (n 5 261) or to continue after Day 6 of stimulation with both rfsh (Gonal-F) and rlh (Luveris) (n 5 265) from Day 6. The starting dose of rfsh was IU/day according to age below or above 35 years. RESULTS: Ongoing pregnancy rate at week was 28.7% after rfsh alone and 27.2% after rfsh 1 rlh. This showed no evidence of a difference. Administration of rlh significantly (P<0.001) increased serum LH. Ongoing pregnancy rates in patients with low LH levels (<33 percentile) on Days 1 and 6 of stimulation showed no difference between the group treated with rfsh only (23.9% low Day 1 LH; 22.1% low Day 6 LH) versus rfsh 1 rlh (25.0% low Day 1 LH; 28.9% low Day 6 LH). CONCLUSIONS: Supplementing rfsh with daily doses of IU of rlh during the second half of the follicular phase showed no evidence of increasing the ongoing pregnancy rates in the general population. (ClinicalTrials.gov, trial number: KF02-035/03). Introduction When the long agonist protocol is used for controlled ovarian stimulation (COS) before IVF or ICSI, the circulating LH levels are suppressed by the agonist, whh may additionally lower the biological activity of the secreted LH. During the course of stimulation, a further lowering of circulating LH is induced, due to the negative feedback of rising estrogens and non-steroidogenic factors during multifollicular development. The consequences are, that serum LH levels are low at the start of ovarian stimulation and even lower during the final days of follicular maturation (Westergaard et al., 2001). These low circulating LH levels have become a concern after the introduction of purified and later recombinant FSH (rfsh) for COS. Low endogenous LH and lack of exogenous LH support has been implicated in less high-quality embryos for cryopreservation (Fleming et al., 1998), lower pregnancy rates (Westergaard et al., 2001) and an increased miscarriage rate (Westergaard et al., 2000). Experience with rfsh treatment of the rare patients with hypogonadotrophic hypogonadism has accumulated unequivocal evidence that normal steroid secretion, ovulation and pregnancy rates needs LH support in addition to rfsh. This can be achieved in almost all patients by either supplementing the rfsh with doses of 75 IU/day of rlh (Burgues et al., 2001) or by treating the patients with human menopausal gonadotrophins (Shoham et al., 1991). In ovulatory women treated with COS for IVF or ICSI, the main clinical question is whether the degree of LH suppression is so pronounced that these patients, or a proportion of these patients, may benefit from co-administration of rlh in terms of higher pregnancy rates. A few randomized trials have investigated the role of rlh supplementation. Ferraretti et al. (2004) found an increased pregnancy rate in 54 hyporesponders after supplementation with rlh, and Lisi et al. (2005) found that 79 patients treated with combined rfsh and rlh had significantly higher pregnancy rates compared with a control # The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 427

2 NyboeAndersen et al. groups. Furthermore, Humaidan et al. (2004) found higher implantation rates in patients with high endogenous LH during stimulation. Additionally, low responders have been reported to have more oocytes retrieved after addition of rlh from Day 8 compared with those when the rfsh dose is increased (Placido et al., 2005). The largest randomized study so far, investigating the effects of supplementation with 150 IU of rlh from Day 6 of stimulation, showed a nonsignificant trend towards lower pregnancy rates in younger patients, but the opposite in patients.35 years of age (Marrs et al., 2003). Overall, the presently available randomized trials do not provide evidence of an increased pregnancy rate after rlh supplementation during the late follicular phase (Marrs et al., 2003; Fabreques et al., 2006; Tarlatzis et al., 2006). The purpose of the present study that was planned during late 2002 was to perform a superiority trial testing whether addition of rlh to rfsh from Day 6 of COS increased the ongoing pregnancy rate after IVF or ICSI using the long agonist stimulation protocol. Patients and methods The study was an investigator-initiated and -driven trial involving a total of 526 randomized patients, included from August 2003 to November The inclusion criteria were: indication for IVF or ICSI, first, second or third cycle, a regular menstrual cycle defined as bleeding intervals between 21 and 35 days, the appropriate mean daily dose of rfsh should be considered to be 225 IU/day, female with below 40 years of age and a basal serum FSH below 10 IU/l at cycle Days 2 5. The treatment schedule involved the long agonist protocol. Downregulation was initiated on Day 21 of the cycle, using nafarelin (Synarela, Pfizer) in 200 mg three times daily for at least 14 days. Following down-regulation, the nafarelin dose was decreased to 200 mg twice a day, and this dose was maintained until the day when rhcg (Ovitrelle, Serono) 250 mg s.c. was administered. Following confirmation of down-regulation, just prior to start of gonadotropin stimulation, patients were randomized to treatment with either rfsh (Gonal-F, Serono) alone throughout the stimulation period or to receive supplementation of the rfsh with rlh (Luveris, Serono) from Day 6 of stimulation. The randomization was done at this time-point in order to avoid any post-randomization withdrawals. The randomizations were done at each centre using the sealed envelope technique and blocks of 10 was used at each centre in order to maintain a balance between the number of patients within the two groups. The randomization envelopes were sequentially numbered. The block size was unknown to the participating centres, except at the centre of the first author. The starting dose of rfsh was 150 IU/day in all patients 35 years of age and 225 IU/day in all patients.35 years of age. The rfsh dose was fixed for the first six days, after which the dose could be individualized. On stimulation Day 1, the patients (n ¼ 526) were randomized to either continue after Day 6 with rfsh alone (n ¼ 261), or to receive a combination of rfsh and rlh (n ¼ 265) after Day 6. The dose of rlh was fixed at 75 IU/day in patients 35 years and 150 IU/day in patients.35 years of age. This differential dose regimen for women below or.35 years of age was based on unpublished data from a Serono database as well as previously published data (De Moustier et al., 2002). Additionally, it can be argued that when the sensitivity to FSH is decreased (in patients.35 years of 428 age) the same may be true for LH, and the traditioonal stimulation with human menopausal gonadotrophins has used a 50/50 distribution of FSH and LH. The criteria for administration of hcg, the oocyte retrieval, fertilization, embryo culture and transfer techniques were done according to local procedures. Luteal phase support was in the form of either Progestan (Organon) vaginal tablets or Crinone (Serono) vaginal gel. The primary end-point of the study was the number of ongoing pregnancies in week per initiated stimulation cycle in each of the two arms of the study. In order to simplify this investigator-driven study, data were only processed for randomized patients. As randomization occurred at the day when stimulation was started (Day 1) the number of randomized patients was the same as the number of rfsh-treated patients. Data have not been collected for eligible and/or included patients who were not subsequently randomized and started COS. The flow of patients is shown in Fig. 1 Serum FSH was determined at the local laboratory in each clinic. Blood samples for serum LH were sampled and stored locally and subsequently measured at a central laboratory. Serium LH measurements involved samples from Days 2 5 of the cycle where down-regulation was initiated, and on Days 1 and 6 of stimulation and on the day of hcg. All serum samples were stored at 2208C until assayed. LH was analysed at the Department of Clinical Chemistry, Odense University Hospital, Denmark, using an AutoDelfia hlh specific fluro-immunoassay. The sensitivity of the assay is,0.05 IU/l, with intra-assay variations,3.1% and inter-assay variations,9.4%. The LH analyses for each patient were done within the same assay. The study was approved by the Ethics Committee in each country and in each region within the country according to the local legal Figure 1: Flowchart of patients

3 rlh supplementation in the long agonist protocol for IVF practice. All participants gave written informed consent. The primary investigator was in Denmark, and the study was here approved by the regional Ethics Committee of Copenhagen and Frederiksberg Municipal, with the approval number KF, /03. A total of 22 centres contributed to the study. The national distribution of centres was: Denmark 10 centres (11 50 patients per centre), Finland 2 centres (5 and 26 patients per centre) Norway 4 centres (10 31 patients per centre) and Sweden 6 centres (4 27 patients per centre). Participants were enrolled by the doctor. The study nurse who gave instructions for medication also randomized the patient and handed out the study medication. Statistical analysis The sample size calculation was based on the assumption that the ongoing pregnancy rate in week in the control arm was This is equivalent to recent live pregnancy rates as published annually by the Danish Fertility Society. With this assumption 400 patients should be included in each arm to document a rise in pregnancy rates by in the Gonal-F þ Luveris arm. A total of 800 patients would give a power of 77% and a P-value of,0.05. All statistical analysis were performed using SAS software (SAS Institute Inc., NC, USA). All tests were carried out at the two-sided 0.05 significance levels. Data were analysed using all randomized patients in the group they were randomized to (intention-to-treat population, ITT). Summary statistics for quantitative data were number of values, mean and standard deviation. For categorical data, summary statistics were frequencies and proportions. For comparison purpose, Fishers s exact test was used for binary outcome. CMH (Cochrane Mantel Haenszel) test for general association was used for categorical outcome when there were more than one category to compare and ANOVA/ANCOVA models were used on the raw data for all continuous data. Results The study included 526 randomized patients and did therefore not reach the planned sample size. The reason was exclusively that a number of centres did not complete the number of patients that was planned for that specific centre. No interrim analysis was done. Table I shows demographic data. Table II shows that in all 526 randomized patients gonadotrophin stimulation resulted in a similar proportion of oocyte aspirations (rfsh alone n ¼ 246, 94.3%; rfsh þ rlh n ¼ 250, 94.3%) in the study and the control arm. The number with embryo transfer was also similar (rfsh alone n ¼ 224, 85.8%; rfsh þ rlh n ¼ 228, 86.0%) in the study and the control arm. Table II also indicates that there was no evidence of differences in relation to efficiency parameters like rfsh dose alterations from Day 6 onwards, total dose of rfsh and days of stimulation. Similarly, supplementation with rlh showed no evidence of influencing the number of follicles or the total number of oocytes collected (rfsh alone 9.5; rfsh þ rlh 8.8). In each arm, the mean number of embryos transferred was 1.6, and the average number of cryopreserved embryos per cycle did not differ significantly (rfsh, 1.2) and (rfsh þ rlh, 1.1). In relation to pregnancy rates, Table II shows that the clinical pregnancy rates per started cycle was 33.7% after rfsh alone Table I. Demographic and infertility characteristics, all patients. Gonal-F alone, N ¼ 261 Gonal-F and Luveris, N ¼ 265 Mean age (years, mean, SD) (3.98) (3.87) Women.35 years of age (n; %) 51 (19.5) 49 (18.5) Women 35 years of age (n; %) 210 (80.5) 216 (81.5) Duration of infertility 3.07 (1.88) 3.21 (1.92) (years, mean, SD) Cycle number, n (%) a (70.3) 168 (64.4) 2 48 (18.5) 65 (24.9) 3 29 (11.2) 28 (10.7) IVF in present cycle ICSI in present cycle Mixed or unspecified b Cause of infertility Tubal, n (%) 49 (18.8) 49 (18.5) Male, n (%) 127 (48.7) 131 (49.4) Anovulation, n (%) 5 (1.9) 6 (2.3) Endometriosis, n (%) 13 (5.0) 11 (4.2) Unexplained, n (%) 83 (31.8) 79 (29.8) Others, n (%) 4 (1.5) 9 (3.4) Combined, n (%) 19 (7.3) 23 (8.7) a cycle number missing in 2 and 4 in the two groups; b mixed is both IVF and ICSI in the same cycle. and 31.3% after rfsh þ rlh. The ongoing live intrauterine pregnancy rates at the seventh week showed no evidence of a difference (rfsh, 32.6%) and (rfsh þ rlh, 29.8%). The predefined primary end-point of the study, the ongoing live pregnancy rate at the 10 12th week of gestation was 28.7% after rfsh alone and 27.2% after rfsh þ rlh. This showed no evidence of a difference. The clinical abortion rates defined as the difference between the number of ongoing pregnancies at week and the number of clinical pregnancies at week 7 was 14.8% (13/88) after rfsh alone and 13.3% (11/83) after rfsh þ rlh. This difference is not significant. In the rfsh group, 81% of pregnancies were singleton and 19% twin gestations. In the rfsh þ rlh group, the corresponding figures were 75 and 25%, respectively, for singleton and twin gestations. Table III shows the results regarding the same parameters as in Table II in the stratum of women 35 years of age and Table IV shows the results in the group with.35 years of age. It is seen that there were no evidence of differences between the groups treated with rfsh alone versus those treated with rfsh þ rlh, in either of these age groups. Table V shows serum LH levels on Day 2 5 of the cycle where down-regulation was started, at Days 1 and 6 of stimulation and on the day of hcg administration. Mean serum LH showed the expected drop from around 5 IU/l on Day 2 5 to 1.7 IU/l on Day 1 of stimulation and to around 1.3 IU/l on Day 6. Serum LH increased marginally in the rfsh alone treated group to 1.6 on the day of hcg. Addition of rlh from Day 6 onwards increased serum LH in all patients to 2.25 IU/l on the day of hcg in the rfsh þ rlh-treated group. This is significantly (P, 0.001) higher than the serum LH of 1.6 IU/l in the rfsh alone treated group. Serum LH in the group with 35 years of age, who were treated with 75 IU of rlh per day, was 2.15 IU/l on the day of hcg. This is significantly (P, 0.001) higher that in the 429

4 NyboeAndersen et al. Table II. Days of treatment, doses of Gonal-F, number of follicles, oocytes, embryos and pregnancies in all patients (N ¼ 526). rfsh alone, N ¼ 261 RFSH þ rlh, N ¼ 265 P-values df Initiated cycles, n (%) 261 (100) 265 (100) Oocyte retrieval, n (%) Yes 246 (94.3) 250 (94.3) No 15 (5.7) 15 (5.7) Transfers, n (%) Yes 224 (85.8) 228 (86.0) No 37 (14.2) 37 (14.0) Patients with change dose of rfsh from Day 6, n (%)* a a Increase 134 (51.5) 129 (48.7) Unaltered 96 (36.9) 106 (40.0) b 2 Decrease 30 (11.5) 30 (11.3) Total dose in IU of rfsh, (mean, SD) (626.5) (757.6) c 522 Days of stimulation (day of hcg), (mean, SD) 11.2 (1.8) 11.3 (2.1) c 522 Follicles 10 mm (day of hcg), (mean, SD) 10.6 (4.9) 10.7 (6.0) c 491 Follicles 17 mm (day of hcg), (mean, SD) 4.6 (3.8) 4.3 (3.6) c 504 Diameter (mm) of leading follicle, (mean, SD) 18.8 (2.1) 18.7 (2.2) c 501 Oocytes aspirated, (mean, SD) 9.5 (4.8) 8.8 (4.8) c 494 Embryos transferred (mean, SD)** 1.6 (0.5) 1.6 (0.5) c 449 Embryos cryopreserved, (mean, SD) 1.2 (2.2) 1.1 (2.3) c 506 Intrauterine clinical pregnancy (at least one gestational sac), n (%) 88 (33.7) 83 (31.3) a Number of gestational sacs (n) Number of embryos transferred (n) Implantation rate (mean, SD) (sacs/embryos transferred)*** 0.29 (0.39) 0.28 (0.40) c 446 Number of gestational sacs with live fetus (n) Live implantation rate (mean, SD) (sacs with live fetus/embryos 0.27 (0.37) 0.26 (0.39) c 448 transferred)*** Number of patients with at least one live gestation at seventh week, n (%) [CI] d 85 (32.6) [26.9; 38.6] 79 (29.8) [24.4; 35.7] a Number of patients with live fetus, n (%) d One fetus 69 (81.2) 59 (74.7) b 1 Two fetuses 16 (18.8) 20 (25.3) Ongoing live gestation at 10 12th week, n (%) [CI] d 75 (28.7) [23.3; 34.6] 72 (27.2) [21.9; 33.0] a a Fisher s exact test; b CMH test for general association; c ANOVA/ANCOVA on the raw data; d on all pregnancy end-points the demoninator is started cycles (261 and 265, respectively). *one missing data (patient 381) in rfsh alone group; **calculated based on the subjects with at least one embryo transferred; ***three patients (60, 217, 493) having more gestational sacs than embryos transferred were excluded from the analysis. Table III. Days of treatment, doses of rfsh, number of follicles, oocytes, embryos and gnapregnancies in patients 35 years of age. rfsh alone, N ¼ 210 rfsh þ rlh, N ¼ 216 P-values df Initiated cycles, n (%) 210 (100) 216 (100) Oocyte retrieval, n (%) Yes 196 (93.3) 206 (95.4) No 14 (6.7) 10 (4.6) a Transfers, n (%) Yes 178 (84.8) 187 (86.6) No 32 (15.2) 29 (13.4) a Increase 112 (53.6) 107 (49.5) Patients with change dose of rfsh from Day 6, n (%)* Unaltered 75 (35.9) 86 (39.8) b 2 Decrease 22 (10.5) 23 (10.6) Total dose in IU of rfsh, (mean, SD) (606.4) (664.1) c 422 Days of stimulation (day of hcg), (mean, SD) 11.2(1.8) 11.2 (2.1) c 422 Follicles 10 mm (day of hcg), (mean, SD) 10.7 (4.9) 10.9 (5.9) c 399 Follicles 17 mm (day of hcg), (mean, SD) 4.6 (3.9) 4.2 (3.5) c 408 Diameter (mm) of leading follicle, (mean, SD) 18.8 (2.2) 18.5 (2.2) c 406 Oocytes aspirated, (mean, SD) 9.6 (5.0) 8.9 (4.5) c 400 Embryos transferred (mean, SD)** 1.6 (0.5) 1.6 (0.5) c 362 Embryos cryopreserved, (mean, SD) 1.2 (2.4) 1.1 (2.1) c 409 Intrauterine clinical pregnancy (at least one gestational sac), n (%) 70 (33.3) 69 (31.9) a Number of gestational sacs (n) Number of embryos transferred (n) Implantation rate (mean, SD) (sacs/embryos transferred)*** 0.28 (0.39) 0.28 (0.40) c 360 Number of gestational sacs with live fetus (n) Live implantation rate (mean, SD) (sacs with live fetus/embryos 0.27 (0.38) 0.27 (0.39) c 361 transferred)*** Number of patients with at least one live gestation at seventh week, n, (%) [CI] d 67 (31.9) [25.7; 38.7] 67 (31.0)[24.9; 37.6] a Number of patients with live fetus, n (%) d One fetus 54 (80.6) 50 (74.6) Two fetuses 13 (19.4) 17 (25.4) b 1 Ongoing live gestation at 10 12th week, n (%) [CI] d 58 (27.6) [21.7; 34.2] 64 (29.6) [23.6; 36.2] a a Fisher s exact test; b CMH test for general association; c ANOVA/ANCOVA on the raw data; d on all pregnancy end-points the demoninator is started cycles (210 and 216, respectively). *one missing data (patient 381) in Gonal-f alone group; **calculated based on the subjects with at least 1 embryo transferred; ***three patients (60, 217, 493) having more gestational sacs than embryos transferred were excluded from the analysis. 430

5 rlh supplementation in the long agonist protocol for IVF Table IV. Days of treatment, doses of rfsh, number of follicles, oocytes, embryos and pregnancies in patients.35 years of age. rfsh alone, N ¼ 51 rfsh þ rlh, N ¼ 49 P-values df Initiated cycles, n (%) 51 (100) 49 (100) Oocyte retrieval, n (%) Yes 50 (98.0) 44 (89.8) No 1 (2.0) 5 (10.2) a Transfers, n (%) Yes 46 (90.2) 41 (83.7) No 5 (9.8) 8 (16.3) a Increase 22 (43.1) 22 (44.9) Patients with change dose of rfsh from Day 6, n (%) Unaltered 21 (41.2) 20 (40.8) b 2 Decrease 8 (15.7) 7 (14.3) Total dose in IU of rfsh, (mean, SD) (478.9) (883.2) c 98 Days of stimulation (day of hcg), (mean, SD) 11.2 (1.5) 11.3 (2.2) c 98 Follicles 10 mm (day of hcg), (mean, SD) 10.3 (4.9) 9.9 (6.6) c 90 Follicles 17 mm (day of hcg), (mean, SD) 4.6 (3.7) 4.8 (4.0) c 94 Diameter (mm) of leading follicle, (mean, SD) 18.8 (1.6) 19.2 (2.4) c 93 Oocytes aspirated, (mean, SD) 9.3 (4.2) 8.5 (5.9) c 92 Embryos transferred (mean, SD)* 1.7 (0.5) 1.7 (0.5) c 85 Embryos cryopreserved, (mean, SD) 1.3 (1.7) 1.1 (3.1) c 95 Intrauterine clinical pregnancy (at least one gestational sac), n (%) 18 (35.3) 14 (28.6) a Number of gestational sacs (n) Number of embryos transferred (n) Implantation rate (mean, SD) (sacs/embryos transferred) 0.29 (0.402) 0.26 (0.41) c 84 Number of gestational sacs with live fetus (n) Live implantation rate (mean, SD) (sacs with live fetus/embryos 0.26 (0.36) 0.22 (0.37) c 85 transferred) Number of patients with at least one live gestation at seventh week, n (%) [CI] d 18 (35.3) [22.4; 49.9] 12 (24.5) [13.3; 38.9] a Number of patients with live fetus, n (%) d One fetus 15 (83.3) 9 (75.0) Two fetuses 3 (16.7) 3 (25.0) b 1 Ongoing live gestation at 12th week, n (%) [CI] d 17 (33.3) [20.8; 47.9] 8 (16.3) [7.3; 29.7] a a Fisher s exact test; b CMH test for general association; c ANOVA/ANCOVA on the raw data; d on all pregnancy end-points the demoninator is started cycles (51 and 49, respectively). *calculated based on the subjects with at least one embryo transferred. Table V. Serum LH levels before and during stimulation according to age groups. rfsh alone, N ¼ 261 RFSH þ rlh, N ¼ 265 P-values df All patients Serum LH (N ¼ 459) Day 2 5, previous cycle (mean, SD) 5.24 (2.85) 5.03 (2.47) a 457 Serum LH (N ¼ 507) Day 1 (the day of start of stimulation) (mean, SD) 1.74 (1.11) 1.74 (0.99) a 505 Serum LH (N ¼ 501) Day 6 (mean, SD) 1.38 (1.30) 1.23 (0.77) a 499 Serum LH (N ¼ 485) day of hcg (mean, SD) 1.57 (1.04) 2.25 (1.30),0.001 a 483 Patients 35 years Serum LH (N ¼ 381) Day 2 5, previous cycle (mean, SD) 5.22 (2.95) 5.04 (2.55) a 379 Serum LH (N ¼ 411) Day 1 (the day of start of stimulation) (mean, SD) 1.71 (0.83) 1.77 (1.01) a 409 Serum LH (N ¼ 403) Day 6 (mean, SD) 1.41 (1.41) 1.22 (0.80) a 401 Serum LH (N ¼ 392) day of hcg (mean, SD) 1.61 (1.11) 2.15 (1.26),0.001 a 390 Patients.35 years Serum LH (N ¼ 78) Day 2 5, previous cycle (mean, SD) 5.30 (2.35) 4.94 (2.00) a 76 Serum LH (N ¼ 96) Day 1 (the day of start of stimulation) (mean, SD) 1.84 (1.88) 1.58 (0.90) a 94 Serum LH (N ¼ 98) Day 6 (mean, SD) 1.26 (0.72) 1.24 (0.65) a 96 Serum LH (N ¼ 93) day of hcg (mean, SD) 1.38 (0.69) 2.73 (1.38),0.001 a 91 a ANOVA/ANCOVA on the raw data. rfsh-treated group (1.61 IU/l). In the group with.35 years of age treated with 150 IU of rlh per day serum LH was 2.73 IU/l, which is significantly (P, 0.001) higher that in the rfsh-treated group (1.38 IU/l). Patients treated with 150 IU of rlh had mean serum levels of 2.73 IU/l, which is significantly (P, 0.001) higher than the 2.15 IU/l of those treated with 75 IU of rlh per day. Intrauterine pregnancy rates at week in relation to LH levels has also been analysed. Irrespective of serum LH levels being below the 33th percentile, addition of rlh in the rfsh þ rlh group showed no evidence of increasing the pregnancy rates, although the sample size may be too small to detect smaller, but arguably clinically relevant differences. The pregnancy rates in patients with normal LH defined as those with a serum LH that was.33 the percentile on each specific day has also been analysed. Addition of rlh in the rfsh þ rlh group showed no evidence of altering the pregnancy rates. Additional serum LH threshold points have also been tested. In patients 431

6 NyboeAndersen et al. with Day 6 serum LH levels 0.5 IU/l, the ongoing pregnancy rate at week showed no evidence of differences after treatment with rfsh alone versus rfsh and rlh. Additionally, basal Days 2 5 LH levels in the cycle where down-regulation was started, was not related to the pregnancy rates, as these were similar in patients with low and high LH levels and this was the case irrespective of whether the patients were treated with rfsh alone or with rlh and rfsh. The number of recorded patients with ovarian hyperstimulation syndrome was 10 (3.8%) in the rfsh group versus 7 (2.6%) in the rfsh þ rlh group. Discussion This is a large randomized controlled trial including 526 patients, comparing the pregnancy rates after stimulation with rfsh versus a combination of rfsh and rlh during the second half of the stimulation phase of the long GnRH agonist protocol. Overall, the clinical pregnancy rate per started cycle was 33.7% after treatment with rfsh alone and 31.3% after treatment with rfsh and rlh. The primary predefined end-point of the study was the ongoing pregnancy rate in week and this was similar, i.e. 28.7% (rfsh alone) versus 27.2% (rfsh þ rlh). The study was designed to show superiority of rlh supplementation, but the planned sample size was not reached due to logistic reasons. The trend was that the higher ongoing pregnancy rates were found with rfsh alone, so with the reservation that the planned sample size was not reached, the overall conclusion of the study is that general supplementation with rlh to rfsh during the late follicular phase is not warranted in standard patients,40 years of age. Even though the planned sample size was not reached, the almost identical pregnancy rates supports this conclusion. These results are consistent with the findings in the publication by Marrs et al. (2003), and two recent papers (Fabregues et al., 2006; Tarlatzis et al., 2006) who also used the long agonist protocol. Marrs et al. (2003) conducted the other large study and randomized 431 patients who were downregulated with daily s.c. administration of leuprolide. The COS was with either 225 IU/day of rfsh or with rfsh and supplementation with rlh 150 IU/day from Day 6 of stimulation. Significantly more embryos were transferred in the rfsh þ rlh group, but the implantation rates showed no evidence of differences (27% rfsh alone versus 23% rfsh þ LH). Marrs et al. (2003) used the same age stratification as in the present study. In younger patients there was a trend towards lower implantation rates after addition of rlh, but in patients.35 years of age the implantation rates were higher, although this showed no evidence of a difference, in the group receiving supplementation with rlh from Day 6. In a recent study, Fabregues et al. (2006) used decapeptyl 0.1 mg s.c. daily and randomized 120 patients.35 years of age to either high dose rfsh alone or to supplementation with 150 u/day of rlh from Day 6 of stimulation. The pregnancy rates after transfer of and average of 2.5 embryos were similar, i.e. 42% (rfsh) and 40% (rfsh þ rlh). Tarlatzis et al. (2006) used buserelin 0.2 mg for down-regulation and 432 randomized 114 women to either rfsh alone or to rfsh and LH supplementation with 75 IU/day of rlh from the day when the leading follicle was 14 mm. Although there were differences in surrogate end-points like serum estradiol (E 2 ), which was higher after rlh supplementation, the pregnancy rates showed no evidence of a difference (23.7% after rfsh alone versus 16.4% after rfsh þ rlh). The study by Lisi et al. (2005) using down-regulation with triptorelin 0.1 mg, found an increased pregnancy rate after rlh supplementation in 79 patients treated with 75 IU/day. A recent meta-analysis of the four studies (Oliveira et al., 2007) indicated that rlh supplementation does not increase the clinical pregnancy rate, implantation rate or decrease the miscarriage rate. However, this meta-analysis of the four trials indicated that rlh supplementation in addition to increase serum E 2, may give 0.2 days shorter rfsh stimulation period and may reduce the number of rfsh units by around 50 IU. The present large study does not support any change in these efficiency parameters. In the short antagonist protocol, serum LH levels are also suppressed during the course of stimulation due to the negative feedback following multifollicular development. Additionally, circulating LH will be further suppressed after the antagonist is given. However, a recent meta-analysis concluded that supplementation with rlh after antagonist administration did not increase the pregnancy rates (Baruffi et al., 2007). As all studies both in agonist and antagonist protocols have used rlh supplementation during the final days of follicular maturation when the endogenous LH levels are the lowest, the evidence is now fairly substantial to conclude that doses of IU rlh during the late follicular phase does not, in general, increase the pregnancy rate compared with currently used standard protocols using rfsh. In our randomized trials, we followed the patients until an ongoing pregnancy in week The reason was that it has been suggested that low Day 6 serum LH levels are associated with an increased abortion rate (Westergaard et al., 2000) and it has been speculated whether or not rlh or treatment with LH/hCG containing preparations could reduce the early miscarriage rates (Westergaard et al., 2000). In our series, the clinical abortion rates defined as the difference between the number of clinical pregnancies at week 7 and the ongoing pregnancies at week was 17.1% after rfsh alone and 7.2% after rfsh þ rlh, but the difference was not significant. That rlh supplementation does not reduce the early miscarriage rate is confirmed in a meta-analysis (Oliveira et al., 2007). A number of variables should be considered before a final conclusion can be made regarding the lack of benefit of adding rlh to currently used protocols. First of all, we need trials where rlh has been given throughout the stimulation period. Earlier and recent trials have compared rfsh versus highly purified hmg (HP-hMG) (European Israeli Study Group, 2002; Nyboe Andersen et al., 2006), where the LH activity is mainly due to hcg and the drug has been given from stimulation Day 1. Even though a number of differences have been observed between HP-hMG and rfsh in relation to oocyte number and endocrinology, these trials showed no evidence of differences in ongoing pregnancy rates compared

7 rlh supplementation in the long agonist protocol for IVF with rfsh, although a trend was observed in both studies towards more ongoing pregnancies in the HP-hMG groups. However, in these trials the FSH was urinary derived rather than recombinant, so it remains to be determined whether rlh supplementation throughout the rfsh stimulation phase would be of benefit. Another variable to consider is the type of down-regulation. All trials using rlh have used nasal GnRH agonists as in the present study or daily s.c. injections of either buserelin 0.2 mg daily (Tarlatzis et al., 2006), leuproline 0.5 mg daily (Marrs et al., 2003) or triptorelin 0.1 mg daily (Fabregues et al., 2006). A more profound down-regulation using for instance depot preparation could influence results, but it should be noted that the available evidence from available GnRH agonist dose-finding study using triptorelin 15, 50 and 100 mg per day (Janssens et al., 2000), and comparative studies of different agonists (Parinaud et al., 1992) is that the dose or type of the agonist and thus the degree of downregulation is not related to clinical outcome. Whether rlh supplementation would be of benefit in the long agonist protocols using depot formulations is thus merely speculative. A third variable to consider is the dose of rlh. We have chosen the IU/day range, due to physiological and clinical considerations. Treating patients with hypogonadotrophic hypogonadism with ovulation induction, Burgues et al. (2001) have clearly documented that the far majority of the WHO type I patients obtain a good ovarian response, E 2 rise and pregnancy rates with a dose of 75 IU/day of rlh, and only a few patients need 150 IU/day. We did not measure the E 2 response to rlh supplementation, but the recent meta-analysis showed that treatment with these doses of rlh increased serum E 2 (Oliveiri et al., 2007). A number of non-interventional, observational studies have analysed associations between serum LH levels and pregnancy rates. Balasch et al. (2001) and Cabrera et al. (2005) found no association between early-, mid- and late follicular phase serum LH levels and the pregnancy rates in 144 and 157 patients, respectively. Penarrubia et al. (2003) similarly found no association between Day 7 LH levels and pregnancy rates in 214 patients, and Bjercke et al. (2005) showed than in an uncontrolled clinical setting, Day 1 serum LH had no predictive value in relation to pregnancy rates. Humaidan et al. (2002) who studied 207 patients found that both low and high mid-follicular phase serum LH was associated with poorer response and pregnancy rates, compared with normal LH levels. The pattern of observations from these studies does therefore not give a clear picture supporting a negative role of low LH levels during the long agonist cycle. In our study, the fairly large sample size allowed sub-analysis of those patients who, from a physiological point of view, were most likely to benefit from rlh supplemention, i.e. those patients with very low serum LH levels. Using the 33th percentile of the median as a threshold, patients with low serum LH levels at all time-points had higher ongoing pregnancy rates after rfsh and rlh, whereas the opposite was found in women with LH in the normal range. However, none of the differences were significant. The only finding providing some indirect evidence of a beneficial effect of rlh was the observation that patients treated with rfsh alone, had a significantly (P, 0.05) higher pregnancy rate when their serum LH at Day 6 and the day of hcg was normal, compared with when serum LH was low. This difference in pregnancy rates was eliminated, however, when patients were supplemented with rlh. This could indicate that some patients with low LH levels may benefit from rlh supplementation. From a quantitative point of view, however, such an effect may be small. In a recent review of existing literature, Kolibianakis et al. (2006) concluded that low endogenous LH levels would not be a sensible rationale for LH supplementation. The present study has not been powered for specific subgroup analysis. Such analysis with smaller sample sizes always has the risk of making significant findings by chance or not being able to demonstrate true differences. It thus remains speculative whether or not subgroups exist that may benefit from rlh supplementation in the doses and time-interval given here. We did a priori want to analyse for subgroups effects on both age and serum LH levels. Age was stratified for groups at up to 35 or above. Regarding serum LH, each assay is unique and pre-study determination of the most appropriate threshold between low and normal LH would be inappropriate. The 33th percentile was arbitrarily chosen, but other threshold points (like 0.5 IU/l) have been analysed without changing the findings. Being an investigator-driven and -conducted study, without the resource allocation normally following large clinical studies, the present study had some methodological limitations (Arce et al., 2005). First of all, it allowed for local clinical variability in relation to many post-randomization procedures, like FSH dose adjustments after Day 6 of stimulation and allowance for different types of luteal support. Additionally, recording of for instance embryo number and quality was limited to number of usable embryos that were either transferred or cryopreserved, and the data recording and handling only dealt with patients who were randomized and thus started stimulation. The study does not allow accounts of eligible patients and reasons for dropout before randomization. The strength of the study was its size and that we focused on a single well-defined and clinically relevant end-point: the ongoing pregnancy rate in the 10 12th week of gestation, with or without rlh supplementation. In conclusion, the results of the present study show that supplementation with rlh to rfsh from Day 6 of the stimulation phase showed no evidence of improving the ongoing pregnancy rates in general. In patients with low LH Day 6 there may be lower pregnancy rates after rfsh alone. However, it remains to be documented that supplementation with rlh increases the pregnancy rates, even in this subpopulation. Future clinical trials should focus on the effect of increasing the dose of rlh and extending the period of rlh supplementation both in an unselected population and in subgroups. Acknowledgements Serono Nordic provided Luveris (rlh), and funded the central measurements of serum LH. The statistical Unit, Serono International, Switzerland, did the statistical analysis. 433

8 NyboeAndersen et al. Appendix The Nordic LH Study Group Denmark A.N.A. and S.B., Rigshospitalet, Copenhagen; L. Bungum and P.H., Fertilitetsklinikken, Viborg/Skive Hospital, Skive; H.B. E., Holbæk Hospital, Holbæk; F. Hald, Braedstrup Hospital, Braedstrup; A. Lindhard, Fertilitetsklinikken, Roskilde Hospital, Roskilde; P. Lundström, Fertilitetsklinikken, Ballerup; H. M., Fertilitetsklinikken, Hvidovre Hospital, Hvidovre; S.L., Herlev Hospital, Herlev; K. Petersen, CICONIA Aarhus Privathospital, Højbjerg; P.E.R., Odense University Hospital, Odense. Finland L.A., The Family Federation of Finland (Väestöliitto Turun klinikka) FI Turku; H. Tinkanen, IVF-enhet, Tampere. Norway J.H., Fertilitetsseksjonen, Haugesunds Sykehus, Haugesund; M. Hentemann, Universitetssykehuset Nord-Norge, Tromsø; K. Kierulf, Ullevål Universitetssykehus, Oslo; A. Tandberg, Haukeland Sykehus, Bergen. Sweden G.F., Karolinska Universitets Hospital, Stockholm; B. Jablonowska, Universitetssjukhuset i Linköping, Linköping; P.-O. Karlström, Kvinnokliniken, Akademiska sjukhuset Uppsala; M. Lood, Kvinnokliniken, Universitetssjukhuset Örebro, Ørebro; L. Marsk, Sofiahemmet, Stockholm; S. Nilsson, Falu Lasarett, Falun. References Arce J-C, NyboeAndersen A, Collins J. Resolving methodological and clinical issues in the design of efficacy trials in assisted reproductive technologies: a mini-review. Hum Reprod 2005;20: Balasch J, Vidal E, Penarrubia J, Casamitjana R, Carmona F, Creus M, Fabregues F, Vanrell JA. Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in down-regulated women stimulated with recombinant FSH. Hum Reprod 2001;16: Baruffi JB, Mauri AL, Petersen CG, Felipe V, Martins AM, Cornicelli J, Cavanha M, Oliveira JB, Franco JG Jr. Recombinant luteinizing hormone supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-antagonist protocol: a meta-analysis. Reprod Biomed Online 2007;14: Bjercke S, Fedorcsak P, Åbyholm T, Storeng R, Ertzeid G, Oldereid N, Omland A, Tanbo T. IVF/ICSI outcome and serum LH concentration on day 1 of ovarian stimulation with recombinant FSH under pituitary suppression. Hum Reprod 2005;20: Burgues S, the Spanish Collaborative Group on Female Hypogonadotrophic Hypogonadism. The effectiveness and safety of recombinant human LH to support follicular development induced by recombinant human FSH in WHO group 1 anovulation: evidence from a multicentre study in Spain. Hum Reprod 2001;16: Cabrera RA, Stadtmauer L, Mayer JF, Gibbons WE, Oehninger S. Follicular phase serum levels of luteinizing hormone do not influence delivery rates in in vitro fertilization cycles down-regulated with a gonadotropin-releasing hormone agonist and stimulated with recombinant follicle-stimulated hormone. Fertil Steril 2005;83: De Moustier B, Brinsden P, Bungum L, Fisch B, Pinkstone S, Warne D, Loymaye E. The effects of combined treatment of recombinant FSH and LH in ratios 1:1, 2:1 and 3:1 in women aghed years undergoing IVF/ICSI treatment. Hum Reprod 2002;17:55. European Israeli Study Group on highly purified hmg versus rfsh. Efficacy and safety of highly purified menotropin versus recombinant FSH in in vitro fertilization/intracytoplasmatics sperm injection cycles: a randomised, comparative trial. Fertil Steril 2002;78: Fabregues F, Creus M, Penarrubia J, Manau D, Vanrell JA, Balasch J. 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Effects of recombinant LH supplementation in women undergoing assisted reproduction with GnRH agonist down-regulation and stimulation with recombinant FSH: an opening study. Reprod Biomed Online 2004;8: Janssens RMJ, Lambalk CB, Vermeiden JPW, Schats R, Bernards JM, Rekers-Mombarg LTM, Schoemaker J. Dose-finding study of triptorelin acetate for prevention of a premature LH surge in IVF: a prospective, randomised, double-blind, placebo-controlled study. Hum Reprod 2000;15: Kolibianakis EM, Collins J, Tarlatzis B, Papanikolaou ER, Devroey P. Are endogenous LH levels during ovarian stimulation for IVF using GnRH analogues associated with the probability of ongoing pregnancy? A systematic review. Hum Reprod Update 2006;12:3 12. Lisi F, Rinaldi L, Fishel S, Caserta D, Lisi R, Campell A. Evaluation of two doses of recombinant luteinizing hormone supplementatioin in an unselected group of women undergoing follicular stimulation for in vitro fertilization. Fertil Steril 2005;83: Marrs R, Meldrum D, Muasher S, Scvhoolcraft W, Ewerlin L, Kelly E. Randomized trial to compare the effect of recombinant human FSH (follitropin alfa) with opr without recombinant LH in women undergoing assisted reproduction treatment. RBM Online 2003;8: Nyboe Andersen A, Devroey P, Arce JC. Clinical outcome following stimulation with highly purified hmg or recombinant FSH in patients undergoing IVF: a randomised assessor-blind controlled trial. Hum Reprod 2006;21: Oliveira JBA, Mauri AL, Petersen CG, Martins AMC, Cornicelli J, Cavanha M, Pontes A, Baruffi RLR, Franco JG Jr. Recombinant luteinizing hormone supplementaion to recombinant follicle- stimulating hormone during induced ovarian stimulation in the GnRH-agonist porotocol: a meta-analysis. J Assist Reprod Genet 2007; DOI / s Penarrubia J, Fabregues F, Creus M, Manau D, Casmitjana R, Guimera M, Carmona F, Vanrell JA, Balasch J. LH serum levels during ovarian stimulation as predictors of ovarian response and assisted reproduction outcome in down-regulated women stimulated with recombinant FSH. Hum Reprod 2003;18: Placido GDe, Alviggi A, Perino A, Strina I, Lisi F, Fasolino A, Palo RDe, Ranieri A, Colacurci N, Mollo A, on behalf of the Italian Collaborative Group on Recombinant Human Luteinizing Hormone. Recombinant human LH supplementation versus recombinant human FSH (rfsh) step-up protocol during controlled ovarian stimulation in normogonadotrophic women with initial inadequate ovarian response to rfsh. A multicentre, prospective, randomized controlled trial. Hum Reprod 2005;20: Parinaud J, Oustry P, Perineau J, Rème JM, Monroziès X, Pontonnier G. Randomized trial of three luteinizing hormone-releasing hormone analogues used for ovarian stimulation in an in vitro fertilization program. Fertil Steril 1992;57: Shoham Z, Balen A, Patel A, Jacobs HS. 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