Recombinant LH supplementation to recombinant FSH during the final days of controlled ovarian stimulation for in vitro fertilization. A multicentre, prospective, randomized, controlled trial

Abstract

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 down-regulation with nafarelin, 526 women were randomized on Day 1 of stimulation to use either rFSH (Gonal-F) alone (n = 261) or to continue after Day 6 of stimulation with both rFSH (Gonal-F) and rLH (Luveris) (n = 265) from Day 6. The starting dose of rFSH was 150–225 IU/day according to age below or above 35 years.

RESULTS

Ongoing pregnancy rate at week 10–12 was 28.7% after rFSH alone and 27.2% after rFSH + 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 + rLH (25.0% low Day 1 LH; 28.9% low Day 6 LH).

CONCLUSIONS

Supplementing rFSH with daily doses of 75–150 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 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 non-significant 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 2004. 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. Down-regulation was initiated on Day 21 of the cycle, using nafarelin (Synarela, Pfizer) in 200 µg three times daily for at least 14 days. Following down-regulation, the nafarelin dose was decreased to 200 µg twice a day, and this dose was maintained until the day when rhCG (Ovitrelle, Serono) 250 µg 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 = 526) were randomized to either continue after Day 6 with rFSH alone (n = 261), or to receive a combination of rFSH and rLH (n = 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 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 10–12 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

Figure 1:

Flowchart of patients

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 −20°C 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 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, 02-035/03.

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 10–12 in the control arm was 0.25. 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 0.09–0.34 in the Gonal-F + 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 = 246, 94.3%; rFSH + rLH n = 250, 94.3%) in the study and the control arm. The number with embryo transfer was also similar (rFSH alone n = 224, 85.8%; rFSH + rLH n = 228, 86.0%) in the study and the control arm.

Table I.

Demographic and infertility characteristics, all patients.

	Gonal-F alone, N = 261	Gonal-F and Luveris, N = 265
Mean age (years, mean, SD)	31.80 (3.98)	31.72 (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 (years, mean, SD)	3.07 (1.88)	3.21 (1.92)
Cycle number, n (%)a	259	261
1	182 (70.3)	168 (64.4)
2	48 (18.5)	65 (24.9)
3	29 (11.2)	28 (10.7)
IVF in present cycle	127	125
ICSI in present cycle	117	115
Mixed or unspecifiedb	17	25
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)

^acycle number missing in 2 and 4 in the two groups; ^bmixed is both IVF and ICSI in the same cycle.

Table II.

Days of treatment, doses of Gonal-F, number of follicles, oocytes, embryos and pregnancies in all patients (N = 526).

		rFSH alone, N = 261	RFSH + rLH, N = 265	P-values	df
Initiated cycles, n (%)		261 (100)	265 (100)
Oocyte retrieval, n (%)	Yes	246 (94.3)	250 (94.3)	1.000a
	No	15 (5.7)	15 (5.7)
Transfers, n (%)	Yes	224 (85.8)	228 (86.0)	1.000a
	No	37 (14.2)	37 (14.0)
Patients with change dose of rFSH from Day 6, n (%)*	Increase	134 (51.5)	129 (48.7)
	Unaltered	96 (36.9)	106 (40.0)	0.763b	2
	Decrease	30 (11.5)	30 (11.3)
Total dose in IU of rFSH, (mean, SD)		2131.6 (626.5)	2139.3 (757.6)	0.898c	522
Days of stimulation (day of hCG), (mean, SD)		11.2 (1.8)	11.3 (2.1)	0.910c	522
Follicles ≥10 mm (day of hCG), (mean, SD)		10.6 (4.9)	10.7 (6.0)	0.874c	491
Follicles ≥17 mm (day of hCG), (mean, SD)		4.6 (3.8)	4.3 (3.6)	0.460c	504
Diameter (mm) of leading follicle, (mean, SD)		18.8 (2.1)	18.7 (2.2)	0.581c	501
Oocytes aspirated, (mean, SD)		9.5 (4.8)	8.8 (4.8)	0.129c	494
Embryos transferred (mean, SD)**		1.6 (0.5)	1.6 (0.5)	0.833c	449
Embryos cryopreserved, (mean, SD)		1.2 (2.2)	1.1 (2.3)	0.544c	506
Intrauterine ‘clinical’ pregnancy (at least one gestational sac), n (%)		88 (33.7)	83 (31.3)	0.577a
Number of gestational sacs (n)		110	107
Number of embryos transferred (n)		366	372
Implantation rate (mean, SD) (sacs/embryos transferred)***		0.29 (0.39)	0.28 (0.40)	0.775c	446
Number of gestational sacs with live fetus (n)		101	97
Live implantation rate (mean, SD) (sacs with live fetus/embryos transferred)***		0.27 (0.37)	0.26 (0.39)	0.864c	448
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]	0.511a
Number of patients with live fetus, n (%)d	One fetus	69 (81.2)	59 (74.7)	0.317b	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]	0.699a

^aFisher's exact test; ^bCMH test for general association; ^cANOVA/ANCOVA on the raw data; ^don 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 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 + 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 + 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 and 31.3% after rFSH + rLH. The ongoing live intrauterine pregnancy rates at the seventh week showed no evidence of a difference (rFSH, 32.6%) and (rFSH + rLH, 29.8%). The pre-defined 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 + rLH. This showed no evidence of a difference. The clinical abortion rates defined as the difference between the number of ongoing pregnancies at week 10–12 and the number of clinical pregnancies at week 7 was 14.8% (13/88) after rFSH alone and 13.3% (11/83) after rFSH + rLH. This difference is not significant.

In the rFSH group, 81% of pregnancies were singleton and 19% twin gestations. In the rFSH + 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 + rLH, in either of these age groups.

Table III.

Days of treatment, doses of rFSH, number of follicles, oocytes, embryos and gnapregnancies in patients ≤35 years of age.

		rFSH alone, N = 210	rFSH + rLH, N = 216	P-values	df
Initiated cycles, n (%)		210 (100)	216 (100)
Oocyte retrieval, n (%)	Yes	196 (93.3)	206 (95.4)	0.405a
	No	14 (6.7)	10 (4.6)
Transfers, n (%)	Yes	178 (84.8)	187 (86.6)	0.678a
	No	32 (15.2)	29 (13.4)
Patients with change dose of rFSH from Day 6, n (%)*	Increase	112 (53.6)	107 (49.5)	0.680b	2
	Unaltered	75 (35.9)	86 (39.8)
	Decrease	22 (10.5)	23 (10.6)
Total dose in IU of rFSH, (mean, SD)		2018.2 (606.4)	2010.8 (664.1)	0.904c	422
Days of stimulation (day of hCG), (mean, SD)		11.2(1.8)	11.2 (2.1)	0.989c	422
Follicles ≥10 mm (day of hCG), (mean, SD)		10.7 (4.9)	10.9 (5.9)	0.736c	399
Follicles ≥17 mm (day of hCG), (mean, SD)		4.6 (3.9)	4.2 (3.5)	0.360c	408
Diameter (mm) of leading follicle, (mean, SD)		18.8 (2.2)	18.5 (2.2)	0.296c	406
Oocytes aspirated, (mean, SD)		9.6 (5.0)	8.9 (4.5)	0.175c	400
Embryos transferred (mean, SD)**		1.6 (0.5)	1.6 (0.5)	0.895c	362
Embryos cryopreserved, (mean, SD)		1.2 (2.4)	1.1 (2.1)	0.627c	409
Intrauterine ‘clinical’ pregnancy (at least one gestational sac), n (%)		70 (33.3)	69 (31.9)	0.836a
Number of gestational sacs (n)		86	89
Number of embryos transferred (n)		288	303
Implantation rate (mean, SD) (sacs/embryos transferred)***		0.28 (0.39)	0.28 (0.40)	0.885c	360
Number of gestational sacs with live fetus (n)		80	83
Live implantation rate (mean, SD) (sacs with live fetus/embryos transferred)***		0.27 (0.38)	0.27 (0.39)	0.972c	361
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]	0.917a
Number of patients with live fetus, n (%)d	One fetus	54 (80.6)	50 (74.6)	0.409b	1
	Two fetuses	13 (19.4)	17 (25.4)
Ongoing live gestation at 10–12th week, n (%) [CI]d		58 (27.6) [21.7; 34.2]	64 (29.6) [23.6; 36.2]	0.669a

^aFisher's exact test; ^bCMH test for general association; ^cANOVA/ANCOVA on the raw data; ^don 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.

Table IV.

Days of treatment, doses of rFSH, number of follicles, oocytes, embryos and pregnancies in patients >35 years of age.

		rFSH alone, N = 51	rFSH + rLH, N = 49	P-values	df
Initiated cycles, n (%)		51 (100)	49 (100)
Oocyte retrieval, n (%)	Yes	50 (98.0)	44 (89.8)	0.108a
No	1 (2.0)	5 (10.2)
Transfers, n (%)	Yes	46 (90.2)	41 (83.7)	0.384a
	No	5 (9.8)	8 (16.3)
Patients with change dose of rFSH from Day 6, n (%)	Increase	22 (43.1)	22 (44.9)	0.975b	2
	Unaltered	21 (41.2)	20 (40.8)
	Decrease	8 (15.7)	7 (14.3)
Total dose in IU of rFSH, (mean, SD)		2596.3 (478.9)	2703.6 (883.2)	0.450c	98
Days of stimulation (day of hCG), (mean, SD)		11.2 (1.5)	11.3 (2.2)	0.809c	98
Follicles ≥10 mm (day of hCG), (mean, SD)		10.3 (4.9)	9.9 (6.6)	0.736c	90
Follicles ≥17 mm (day of hCG), (mean, SD)		4.6 (3.7)	4.8 (4.0)	0.836c	94
Diameter (mm) of leading follicle, (mean, SD)		18.8 (1.6)	19.2 (2.4)	0.304c	93
Oocytes aspirated, (mean, SD)		9.3 (4.2)	8.5 (5.9)	0.471c	92
Embryos transferred (mean, SD)*		1.7 (0.5)	1.7 (0.5)	0.900c	85
Embryos cryopreserved, (mean, SD)		1.3 (1.7)	1.1 (3.1)	0.703c	95
Intrauterine ‘clinical’ pregnancy (at least one gestational sac), n (%)		18 (35.3)	14 (28.6)	0.524a
Number of gestational sacs (n)		24	18
Number of embryos transferred (n)		78	69
Implantation rate (mean, SD) (sacs/embryos transferred)		0.29 (0.402)	0.26 (0.41)	0.724c	84
Number of gestational sacs with live fetus (n)		21	14
Live implantation rate (mean, SD) (sacs with live fetus/embryos transferred)		0.26 (0.36)	0.22 (0.37)	0.600c	85
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]	0.279a
Number of patients with live fetus, n (%)d	One fetus	15 (83.3)	9 (75.0)	0.583b	1
	Two fetuses	3 (16.7)	3 (25.0)
Ongoing live gestation at 12th week, n (%) [CI]d		17 (33.3) [20.8; 47.9]	8 (16.3) [7.3; 29.7]	0.065a

^aFisher's exact test; ^bCMH test for general association; ^cANOVA/ANCOVA on the raw data; ^don 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 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 + 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 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.

Table V.

Serum LH levels before and during stimulation according to age groups.

	rFSH alone, N = 261	RFSH + rLH, N = 265	P-values	df
All patients
Serum LH (N = 459) Day 2–5, previous cycle (mean, SD)	5.24 (2.85)	5.03 (2.47)	0.402a	457
Serum LH (N = 507) Day 1 (the day of start of stimulation) (mean, SD)	1.74 (1.11)	1.74 (0.99)	0.974a	505
Serum LH (N = 501) Day 6 (mean, SD)	1.38 (1.30)	1.23 (0.77)	0.114a	499
Serum LH (N = 485) day of hCG (mean, SD)	1.57 (1.04)	2.25 (1.30)	<0.001a	483
Patients ≤35 years
Serum LH (N = 381) Day 2–5, previous cycle (mean, SD)	5.22 (2.95)	5.04 (2.55)	0.525a	379
Serum LH (N = 411) Day 1 (the day of start of stimulation) (mean, SD)	1.71 (0.83)	1.77 (1.01)	0.544a	409
Serum LH (N = 403) Day 6 (mean, SD)	1.41 (1.41)	1.22 (0.80)	0.107a	401
Serum LH (N = 392) day of hCG (mean, SD)	1.61 (1.11)	2.15 (1.26)	<0.001a	390
Patients >35 years
Serum LH (N = 78) Day 2–5, previous cycle (mean, SD)	5.30 (2.35)	4.94 (2.00)	0.481a	76
Serum LH (N = 96) Day 1 (the day of start of stimulation) (mean, SD)	1.84 (1.88)	1.58 (0.90)	0.400a	94
Serum LH (N = 98) Day 6 (mean, SD)	1.26 (0.72)	1.24 (0.65)	0.860a	96
Serum LH (N = 93) day of hCG (mean, SD)	1.38 (0.69)	2.73 (1.38)	<0.001a	91

^aANOVA/ANCOVA on the raw data.

Intrauterine pregnancy rates at week 10–12 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 + 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 + rLH group showed no evidence of altering the pregnancy rates. Additional serum LH threshold points have also been tested. In patients with Day 6 serum LH levels ≤0.5 IU/l, the ongoing pregnancy rate at week 10–12 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 + 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 pre-defined end-point of the study was the ongoing pregnancy rate in week 10–12 and this was similar, i.e. 28.7% (rFSH alone) versus 27.2% (rFSH + 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 down-regulated 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 + rLH group, but the implantation rates showed no evidence of differences (27% rFSH alone versus 23% rFSH + 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 + rLH). Tarlatzis et al. (2006) used buserelin 0.2 mg for down-regulation and 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 (E₂), 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 + 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 E₂, 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 75–150 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 10–12. 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 10–12 was 17.1% after rFSH alone and 7.2% after rFSH + 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 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 µg 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 down-regulation 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 75–150 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, E₂ 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 E₂ response to rLH supplementation, but the recent meta-analysis showed that treatment with these doses of rLH increased serum E₂ (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.

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-20100 Turku; H. Tinkanen, IVF-enhet, 33520 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.