1 Serono International, Reproductive Health Clinical Development Unit, Geneva, Switzerland, 2 Department of Obstetrics and Gynecology, Kaplan Medical Center, Rehovot, Israel and 3 Department of Obstetrics and Gynaecology, University of Edinburgh Centre for Reproductive Biology, Edinburgh, UK
4 To whom correspondence should be addressed at: Serono International, 15bis Chemin des Mines, CH-1202 Geneva, Switzerland
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Abstract |
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Key words: anovulation/follicular growth/recombinant FSH/recombinant LH
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Introduction |
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Although follicular growth can be induced by FSH in the total absence of LH, the resulting follicles have developmental deficiencies such as abnormally low production of estradiol (E2) and an inability to luteinize and rupture in response to an hCG stimulus [Couzinet et al., 1988; Glasier et al., 1988
; Shoham et al., 1991b
; Shoham et al., 1993
; Schoot et al., 1994
; European Recombinant Human LH Study Group, 1998; Loumaye and ODea, 2002
; Hemsey et al., 2001
]. Optimal follicular development is therefore also dependent on a minimal exposure to LH or LH threshold. Extensive clinical testing in patients suffering from severe deficiency in LH and FSH has demonstrated that serum LH levels of
1.2 IU/l are necessary to provide adequate LH support to FSH-induced follicular development (Hemsey et al., 2001
; Loumaye and ODea, 2002
) when endogenous LH secretion is absent. Sufficient LH supply can be delivered by a daily injection of 75 IU rLH (European Recombinant Human LH Study Group, 1998).
Pre-clinical evidence has also accumulated suggesting that developing follicles have finite requirements for exposure to LH, beyond which normal maturation ceases (Hillier, 1994). This has given rise to the concept of an LH ceiling, which defines an upper limit of stimulation. The physiological relevance of the ability of LH to arrest follicular growth is potentially 2-fold. The most obvious is the response to the mid-cycle LH surge. Within hours after the onset of the surge, granulosa cell mitosis stops, the oocyte resumes meiosis and cumulus oophorus cells undergo functional and morphological changes in advance of ovulation (Shoham et al., 1995
). The pathological relevance of this effect has been demonstrated in patients undergoing IVF, who had undergone an attenuated LH surge prior to the time of oocyte recovery. This leads to low fertilization rate and reduced embryo viability (Howles et al., 1986
; Howles, 1990
; Loumaye, 1990
).
A second possible physiological role for LH is through its contribution to follicle dominance and mono-ovulation. During the early follicular phase, multiple follicles are present that are able potentially to develop into pre-ovulatory follicles under the influence of peri-menstrual increase in circulating FSH. The dominance of only one such follicle is attributed to the declining levels of FSH during the follicular phase, which falls below the threshold of secondary follicles. In addition to this, the rising serum LH level during the late follicular phase could contribute to this selection process by inducing secondary follicle growth arrest. If effective, this LH property could be exploited clinically to promote mono-ovulation when inducing ovulation in anovulatory women. However, no clinical data are available to support such a role for LH.
Ovulation induction with gonadotrophins is the first line therapy for anovulatory patients who have failed to ovulate or conceive after clomiphene citrate treatment. These treatments are extremely effective in both World Health Organization (WHO) group II/polycystic ovary syndrome (PCOS) patients and in WHO group I/hypogonadotrophic hypogonadal patients (WHO, 1973; Balen et al., 1994). However, a significant adverse outcome of these treatments is multiple follicular development resulting in multiple ovulation and multiple pregnancy (Levene et al., 1992
). Soft protocols have been developed to minimize this response such as chronic low dose FSH, in which the dose of FSH is gently titrated upwards, attempting to cross the FSH threshold level for a single dominant follicle (Seibel et al., 1984
; Buvat et al., 1989
; Hamilton-Fairley et al., 1991
; Meldrum, 1991
; Shoham et al., 1991a
; Homburg and Howles, 1999
; Marci et al., 2001
). In the step-down FSH protocol the process is reversed to take similar advantage of differences in FSH thresholds between the dominant follicle and secondary follicles (Fauser and Van Heusden, 1997
; van Santbrink and Fauser, 1997
). These regimens of administration lead to a mono-ovulation rate of
65% and a reduction in multiple pregnancy rates from 25 to
10% (White et al., 1996
; Homburg and Howles, 1999
).
The objective of the studies reported below was to test in a clinical setting the hypothesis that over-dosing with rLH during the late follicular phase would provide a means of suppressing the development of secondary follicles and promoting final maturation and ovulation of a single pre-ovulatory follicle.
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Materials and methods |
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Study A (WHO I) design
This was a double-blind, placebo-controlled, randomized, parallel group, pilot study, carried out in pre-menopausal women aged 1839 years with a clinical history of hypogonadotrophic hypogonadism (WHO group I type anovulation) who had stopped any treatment with gonadotrophins or steroid replacement therapy 1 month before screening, with a negative progesterone challenge test during screening and hormonal values within prescribed limits within 6 months of study entry, i.e. serum FSH <5.0 IU/l, LH <1.2 IU/l, TSH <6.5 µIU/ml, free testosterone between 11 and 24 pmol/l, testosterone <3.5 nmol/l and prolactin <520 mIU/l.
Patients were to have no clinically significant abnormal haematology, clinical chemistry or urinalysis finding in the 6 months before the study and a body mass index (BMI) between 18.4 (10th percentile for age 18 years) and 31.4 kg/m2 (90th percentile for age 38 years).
In the initial open phase of treatment, patients received treatment with both rFSH (Gonal-F®; Serono, Aubonne, Switzerland) (starting dose 112.5 IU/day; if necessary this could be increased at 7 day intervals to a maximum dose of 262.5 IU/day) and rLH (Luveris®, fixed dose of 225 IU/day; Serono). The open phase could continue for up to 28 days. When at least one follicle reached a mean diameter of 1013 mm, patients were randomized to one of three blinded treatments: (i) continued treatment with both drugs (rFSH/rLH group), (ii) rLH alone (placebo substituted for rFSH; rLH placebo group) or (iii) rFSH alone (placebo substituted for rLH; rFSH placebo group). rLH was given at the same fixed dose as in the open phase (i.e. 225 IU/day) and rFSH at the dose the patient was receiving at the end of the open phase. The blinded phase could continue for up to 7 days.
When one follicle reached a mean diameter of 18 mm and there were no more than two other follicles
11 mm in diameter, 10 000 IU urinary hCG was administered s.c.
The trial was designed to recruit 24 women who were anovulatory and wishing to conceive. A total of 24 patients was enrolled. Twenty of these 24 patients were qualified to start the blinded phase: six patients received rFSH alone, six patients received rLH alone and eight patients received both rFSH and rLH
Study B (WHO II) design
Study B was a double-blind, placebo-controlled, randomized, parallel group, pilot study, carried out in pre-menopausal women aged 1839 wishing to conceive. They were required to be infertile due to ovulatory dysfunction (WHO group II anovulation). To be eligible the patients must have been stimulated with FSH for ovulation induction and have started the FSH treatment within 5 days of spontaneous or induced menstruation. In addition, they were required to be euthyroid, and to have a BMI of <35 kg/m2.
Patients were recruited from a population undergoing routine ovulation induction with FSH in each of the participating clinical units. The distinctive eligibility criteria were a hyper-response to FSH treatment defined as the presence of 4 follicles that were
8 mm and <13 mm in diameter, no larger follicles and an endometrial thickness of
8 mm. If a patient satisfied these eligibility criteria, she was informed about the study on the same day and was asked if she wanted to participate. If affirmative, she was asked to sign the informed consent form. She then received a unique study number. Her FSH treatment was stopped and she was given the study drug allocated to this identification number randomizing her to one of three blinded treatments, i.e. (i) a daily s.c. injection of rLH (Luveris) at a dose of 225 IU/day s.c. or (ii) rLH at a dose of 450 IU/day s.c. or (iii) placebo. The rLH treatment phase was
7 days. The 7-day rLH/placebo treatment period could also be interrupted before completion if obvious regression of all follicles was recorded or if the patient was at risk of developing ovarian hyperstimulation syndrome. According to the protocol, when the follicular response was judged to be adequate (at least one follicle
18 mm and a total number of follicles
11 mm was
3), ovulation was to be triggered by one single s.c. injection of 5000 IU of hCG. No luteal support was administered.
The trial was originally designed to recruit 36 women who were anovulatory and wishing to conceive. Of these 36 patients, 12 were to be allocated to each of the three treatment arms. However, due to the slow patient recruitment and study drug shelf-life limitation, a total of 17 patients was enrolled. Of these, five patients were allocated to and received placebo, four patients were allocated to and received rLH 225 IU/day and eight were allocated to and received rLH 450 IU/day.
Selecting the LH doses
Mean serum LH levels are quite variable in the WHO group II PCOS anovulation population. Using an immunoradiometric assay (IRMA) assay (LH MAIA clone), in a population of 180 WHO group II anovulatory patients stimulated with FSH only (urinary human FSH or rFSH), the mean basal level of LH was 7.4 IU/l (SD 5.7; range 0.557). On the day of hCG administration, in 387 cycles, the mean serum LH level was 7.7 IU/l (SD 8.4, range 0.561) (Serono study GF 5642: data on file). If an average increase of 50% of the mean serum LH level is desired in this population, a dose of rLH resulting in a mean LH Cmax after each daily injection of 3.5 IU/l should be used. According to the pharmacokinetic characteristics of rLH, this daily dose is 450 IU rLH s.c. For study A it was decided to investigate a dose of 225 IU rLH/day; and for study B, to investigate two doses of rLH, i.e. 225 and 450 IU/day.
Randomization and drug used
Blinded vials of rLH/placebo were packed individually for each patient. rLH (Luveris) was supplied in vials containing 75 IU of LH and 47.75 mg of sucrose, phosphate buffer and Tween 20 in a lyophilized form. Matching placebo vials containing only sucrose, phosphate buffer and Tween 20 were also supplied. The rLH or placebo treatment assigned to each patient was determined according to a computer-generated randomization list stratified by centre. When a patient had signed the informed consent form and had been found eligible for the study, she received a unique patient identification number in sequential, chronological order.
Monitoring
Monitoring was primarily done by vaginal ultrasonography. The examinations were performed prior to the administration of rLH/placebo and at 12 day intervals (as clinically indicated) during LH treatment. All follicles with a mean diameter (= the mean of the two longest perpendicular diameters) >10 mm (i.e. 11 mm) were recorded according to their size.
Blood sampling was taken for retrospective analyses of E2, progesterone, androstenedione, LH and FSH prior to the administration of rLH/placebo and at each occasion the patient came to the centre for ultrasound. For study A, blood samples were collected just before starting rLH and rFSH stimulation (S1), on the day of randomization to rFSH placebo, rFSH and rLH or rLH placebo and on the day of hCG administration (or the last day of gonadotrophin treatment if no hCG was administered). For study B, blood samples were collected just before the day of randomization to placebo, rLH 225 IU or rLH 450 IU and on a regular basis up to the day of hCG administration (or the last day of gonadotrophin treatment if no hCG was administered). The luteal phase was assessed and monitored by measuring progesterone between days 6 and 9 post-hCG, and the day of spontaneous menstruation was recorded. Investigating the safety of the rLH for potential antibodies, one pre- and one post-treatment sample was assayed in a central laboratory.
Statistical methods
These were pilot studies, which were not designed on statistical grounds. Results are reported as mean ± SEM, median and range, unless otherwise specified. The statistical methods used were directly related to the nature of the variable analysed, i.e. Fishers exact test, analysis of variance, and analysis of covariance. P < 0.05 was considered to be statistically significant.
Hormone assays
All assays were performed in a central laboratory, SCL Bioscience Services, Bourn Hall Clinic, Cambridge, UK. Serum FSH and serum LH were measured using a validated, commercially available IRMA method (MAIAClone assay). The limit of quantification for serum LH and serum FSH was 1.0 IU/l. Serum E2, progesterone and androstenedione were measured using validated, commercially available immunoassays.
Data collection
The study was performed and monitored according to the sponsors Standard Operating Procedures for clinical trials, and data were collected on case report forms. The case report forms were completed by the study co-ordinator(s), checked and signed by the responsible principal investigator, and then checked against the source documents and verified for consistency prior to retrieval from the centre.
A double data entry was done using CLIMED (Simed, Paris, France) as data entry screens. After comparison of the two entries, computerized checks were carried out by the database monitor and statistician, and data inconsistencies thus detected were queried and then corrected in the database according to the investigators answers.
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Results |
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Patients distribution and demographic characteristics are summarized in Table II. Study B follicular development status at time of enrolment in the study was high, with an average number of follicles 8 mm equal to 10.6 ± 1.4 follicles per patient, confirming the compliance with the patient selection criteria for over-response. The mean ± SEM number of follicles was 14.0 ± 3.0, 9.8 ± 3.3 and 8.9 ± 1.4 in the placebo group, the 225 IU LH group and the 450 IU LH group respectively.
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Serum FSH, LH, E2, progesterone and androstenedione levels are summarized in Table III. As expected, serum FSH remained constant in patients who continued FSH administration whereas it dropped sharply in patients who stopped FSH administration. Serum LH remained low and most often below the limit of quantification (1 IU/l) in all three groups. Serum E2 did not further increase in the FSH-alone group, declined in the rLH-alone group and increased in the rFSH/rLH group. Serum progesterone remained low in all three groups during rLH administration. It is noteworthy that one patient developed a spontaneous LH surge and luteinized in the rFSH/rLH group. Androstenedione increased in all three groups. Two pregnancies occurred, both in the rFSH/rLH group.
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Study B efficacy results
Seventeen patients started the blinded treatment. Ten of those 17 received hCG and seven were cancelled prior to hCG administration for the following reasons: one patient for excessive response in the placebo group; two for follicular regression in the 225 IU rLH group and three for follicular regression and one excessive response in the 450 IU rLH group. rLH/placebo treatment lasted on average 4.5 ± 0.5 days with no significant difference between treatment groups.
At the end of this treatment phase, in 5/12 patients from the LH groups no follicular growth was recorded on ultrasound, resulting in the cancellation of hCG administration (two in the 225 IU/day LH group, and three in the 450 IU LH group). This contrasts with no patient (0/5) presenting similar lack of follicular growth in the placebo group. In addition, 1/5 patients had excessive follicular development in the placebo group and 1/12 in the rLH treatment group. The distribution of number of follicles by diameter per patient is presented in Figure 2.
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Mean serum FSH was elevated on the day of starting the rLH/placebo treatment. A significant decrease was recorded after stopping FSH administration in all three groups (Table IV). Serum LH levels were also elevated on the day of starting the treatment phase and remained elevated during rLH/placebo treatment. No statistically significant difference in serum LH was recorded between the placebo group and the rLH groups. Serum E2 was higher in the placebo group than in the rLH treatment groups. This is consistent with the higher number of follicles at baseline in the placebo group. Both mean and median serum E2 levels in the placebo group indicate that E2 secretion was maintained during placebo administration. Contrasting with this pattern, in the rLH treatment groups, median serum E2 levels underwent a large fall. Serum P was low at baseline in all three groups and remained low, including patients receiving rLH. In patients receiving hCG, mid-luteal phase serum E2 remained elevated whereas serum progesterone very significantly increased, consistent with proper luteinization. Two pregnancies were confirmed, both in the placebo group.
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Discussion |
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These two studies have in common the administration of rLH alone during the late follicular phase. As a control, the study conducted in WHO group II anovulatory patients (study B) had a placebo group in which no gonadotrophin was administered. The study conducted in WHO group I anovulatory patients (study A) did not have a placebo-only group, since the absence of endogenous gonadotrophin would have led to rapid follicular regression.
By protocol, all patients who entered these studies had some growing follicles, but none >13 mm in diameter. The assessment of follicular growth with ultrasound during the study showed a similar and consistent pattern of response to rLH in both studies. (i) Administration of rLH alone prevented follicles from growing, and of reaching a late antral stage in a significant number of patients (see Figures 1 and 2). A total of 9/26 patients had follicular growth arrest in the rLH-alone groups contrasting with 0/11 in the control groups (i.e. rFSH alone in study A, and placebo alone in study B) (Fishers exact P = 0.036). This illustrates the capability of rLH to arrest follicular growth at the dose used, in one-third of this patient population. (ii) In patients with continuing follicular growth, an additional effect of LH was noted. More patients had only one dominant follicle (defined as a follicle with a diameter 14 mm) in the rLH groups (8/17) than in the control groups (2/11) (Fishers exact P = 0.226). This observation suggests that LH may facilitate selective follicular growth.
This effect of rLH is different from the concept of coasting (stopping FSH administration) (Dhont et al., 1998). In study A, no coasting was performed in the control group and the comparison is with FSH-only treatment. In study B, coasting was performed in the control group but this was not associated with either follicular growth arrest or clear-cut emergence of a dominant follicle.
The hormone profiles during treatments with rLH were also informative. Stopping rFSH administration led, as expected, to a consistent decline in serum FSH levels in both populations. Within a few days, serum FSH levels dropped to 25% of initial levels in WHO group I patients. This is compatible with the rFSH terminal half-life of
35 h (Porchet et al., 1994
). By contrast, in WHO group II patients, serum FSH decline was limited to 50% of initial levels, reflecting the contribution of endogenous FSH in this population.
In terms of serum LH levels, at screening, prior to any LH administration, most patients in study A had a value below the limit of quantification (1.0 IU/l). The mean value was 1.1 IU/l and the median value was 1.0 IU/l. During the open phase when 225 IU rLH was administered daily, a minimal increase in serum LH level was recorded with a mean value of 1.4 IU/l and a median value of 1.0 IU/l. This is compatible with an rLH terminal half-life of 1012 h, and a minimal accumulation after repeated administration (Le Cotonnec et al., 1998). In this population, stopping LH administration led to a return of serum LH mean levels to 1 IU/l, while continuing with 225 IU/day resulted in a slight increase with a median value of 1.3 and 1.4 IU/l (see Table III). This observation indicates that the impact on follicular development summarized above is obtained with quite minor variations in serum LH levels. This also concurs with a previous observation that the threshold dose of 75 IU rLH does lead to a very significant pharmacodynamic effect without measurable variations in serum LH levels (European Recombinant Human LH Study Group, 1988). In study B, prior to any LH administration, serum LH levels were significant with a mean value of 5.8 ± 1.2 IU/l, which is compatible with WHO II PCO patients. In patients receiving 450 IU/day of rLH, the mean and median serum LH levels were increased by
40%. However, this was not statistically significant. In the other groups, a less predictable sequence of serum LH levels was recorded. Large individual value variations due to fluctuations of endogenous LH as well as lack of strict timing imposed for sampling the patients may account for this observation.
In study A, the E2 pattern was consistent with previous reports in which stimulation was performed in hypogonadotrophic hypogonadism with FSH only or with an association of FSH and LH (Couzinet et al., 1988; European Recombinant Human LH Study Group, 1998). FSH alone only maintained levels recorded at the end of the open phase treatment. In patients treated with rFSH and rLH, serum E2 continued to increase up to the day of hCG administration. In patients receiving rLH only, a sharp drop in E2 was recorded. Thus, in this group, E2 production parallels the follicular growth arrest documented with ultrasound. In WHO group II anovulation or assisted reproduction patients, E2 levels are known to drop when coasting is performed in ovulatory patients stimulated with FSH (Dhont et al., 1998
). In study B, placebo treatment was not associated with significant changes in serum E2 levels. Administration of rLH alone led to further increases in serum E2 levels as previously reported by Sullivan et al. (1999
). However, some patients with follicle regression presented a sharp decline in serum E2 levels, as illustrated in the median values (Table IV). From these two studies, it can be concluded that LH alone can support E2 secretion as long as follicular growth arrest does not occur and as long as some FSH is present, whether exogenous (study A) or endogenous (study B) in origin.
Progesterone levels remained low (<6 nmol/l) in all patients in both studies, including patients who received 450 IU rLH per day (the only exception was one patient who underwent a premature LH surge with full luteinization before hCG administration). Thus doses of rLH up to 225 IU/day given alone or in association with rFSH, or up to 450 IU/day given alone, do not trigger luteinization. Importantly, this observation also suggests that the follicular growth arrest induced by LH at this stage of follicular development is distinct from the peri-ovulatory luteinization phenomenon.
Finally, some patients treated with rLH alone did not show a normal luteinization response when exposed to hCG, contrasting with patients treated with rFSH and rLH in study A or patients treated with placebo only in study B, all of whom attained serum progesterone >25 nmol/l during mid-luteal phase.
The impact of LH on oocyte fertilizability and capacity to lead to a viable embryo cannot be directly assessed from these studies in which conception was intended to occur in vivo. Two pregnancies were recorded in study A and both were in the rFSH + rLH group. Two pregnancies were recorded in study B, and both were in the placebo group. Although the groups were small and establishing a pregnancy depends on numerous other parameters, this suggests that administering LH without FSH may be too detrimental for oocyte quality, including the dominant follicle. In other words, a specific balance between LH and FSH may be required.
In conclusion, these two studies provide preliminary clinical evidence for the existence of a development-related LH ceiling during the late pre-ovulatory follicular maturation, which, when breached, results in a spectrum of effects ranging from complete follicular growth arrest, to selective follicle growth arrest, to impaired ability to luteinize. Stopping exogenous FSH administration completely appears to be too detrimental, especially in WHO group I patients. Maintaining a low FSH supply during the administration of LH results in increased E2 levels with pregnancies, promoting development of a single dominant follicle in 45% of cases. Although no premature luteinization was recorded using LH in these patients, the possible impact of such treatment on oocyte quality remains unknown and requires further evaluation. Thus the results of this pilot study suggest a potential clinical benefit of the usage of rLH in ovarian stimulation regimes to promote mono-ovulation.
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FOOTNOTES |
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References |
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Submitted on July 7, 2002; accepted on October 18, 2002.