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Original Articles: Hormones and Reproductive Health |
Geneva, Switzerland
Address correspondence and requests for reprints to: E. Loumaye, M.D., Ph.D., Serono International, 15 bis Chemin des Mines, CH-1202 Geneva, Switzerland.
Abstract
In a prospective, comparative, dose-finding study, the minimal effective dose of recombinant human LH (rhLH) required to induce final follicular maturation and early luteinization in patients undergoing in vitro fertilization and embryo transfer was determined. In addition, the efficacy and safety of rhLH were compared with urinary human CG (u-hCG). A total of 259 infertile women, aged 18–39 yr, were enrolled in the study. After pituitary desensitization using a GnRH agonist, rhFSH was administered for ovarian stimulation. Patients then received either rhLH or u-hCG to achieve final follicular maturation. The doses of rhLH administered were 5,000, 15,000, 30,000, or 15,000 + 10,000 IU (second injection administered 3 days after the first injection; 129 patients), and those of u-hCG were consistently 5,000 IU (121 patients). Ovum pick-up was performed 34–38 h after rhLH or u-hCG injection. After fertilization in vitro, up to three embryos were replaced in the uterine cavity. The numbers of oocytes retrieved after u-hCG or rhLH administration were not significantly different between the four different doses of rhLH, when compared with each corresponding u-hCG group, nor when compared with the pool of all u-hCG groups. Similarly, there were no statistically significant differences in: the number of oocytes retrieved per follicle with a diameter of over 10 mm on the day of u-hCG or rhLH administration; the number of patients with at least one oocyte retrieved; oocyte nuclear maturity; oocyte potential for fertilization; the number of embryos; the number of total, biochemical, and clinical pregnancies; and the embryo implantation rate. However, in many of these parameters, the lowest dose of rhLH seemed suboptimal when compared with the higher dose. In terms of safety, rhLH was well tolerated at a dose of up to 30,000 IU. Moderate ovarian hyperstimulation syndrome (OHSS) was reported in 12.4% of patients who received u-hCG and 12.0% of patients who received two injections of rhLH. No moderate or severe OHSS was reported in patients who received a single dose of rhLH up to 30,000 IU. The results show that a single dose of rhLH is effective in inducing final follicular maturation and early luteinization in in vitro fertilization and embryo transfer patients and is comparable with 5,000 IU u-hCG. A single dose of rhLH results in a highly significant reduction in OHSS compared with hCG. The dose of rhLH giving the highest efficacy to safety ratio was between 15,000 and 30,000 IU.
THE MIDCYCLE GONADOTROPIN surge is a major event in the dynamics of ovulation. Rapidly increasing levels of LH induce a number of key changes in both oocytes and follicular cells, which further modify the steroid and protein micro- and macroenvironment. These physiologic changes have a prominent role in the normal maturation of oocytes, the process of ovulation, and subsequent fertilization and implantation (1). Because of the inconsistency of the spontaneous LH surges during controlled ovarian stimulation in any of its forms, and due to the increasing use of GnRH agonist (GnRH-a) in in vitro fertilization (IVF) programs, human CG (hCG) has been adopted uniformly by all successful ovarian stimulation programs to effect the final triggering of oocyte maturation and ovulation.
hCG has been used as a surrogate LH surge because of the degree of
homology between the two hormones. Both LH and hCG are complex
heterodimeric glycoproteins with a molecular weight of 
30K
for recombinant human LH (rhLH) and 40K for hCG. They both have
identical 
-subunits and a high cystine content. Most importantly,
they have the same natural function—to cause luteinization and support
lutein cells. The major differences between the two hormones include
the sequence of the ß-subunit, the regulation of the secretion of the
two hormones, and the pharmacokinetics of clearance of hCG as opposed
to LH (2, 3).
hCG has a slower plasma metabolic clearance, which consists of a rapid phase in the first 5–9 h following im administration and a slower phase in the 1–1.3 days after administration. After 36 h, the calculated half-life of hCG was 2.32 days, as compared with LH, for which estimates have ranged from 1 h (3) to 3–5 h (4, 5). By day 10 after administration, less than 10% of the originally administered hCG was measurable (6). However, the long serum half-life of hCG is likely to be an undesirable characteristic in clinical practice. Residual hCG may be mistaken for early detection of de novo synthesis of hCG by a newly implanted pregnancy. High-serum hCG concentrations may result in a sustained luteotrophic effect ending with the development of multiple corpora lutea and supraphysiologic levels of estradiol (E2) and progesterone (P4). Excessive levels of circulating E2 have been implicated in the relatively high rates of implantation failure and early pregnancy loss in ovarian stimulation programs (7, 8). High serum concentrations of hCG can also lead to sustained high-level stimulation of corpora lutea, which may lead to ovarian hyperstimulation syndrome (OHSS), an important complication of gonadotropin therapy (9).
Until recently, there was no other biologic preparation that was as
effective as hCG in triggering the final stage of ovulation. Recent
developments using genetic engineering technologies and
posttranscriptional biosynthesis have led to the production of rhLH,
which has been available for use in clinical trials for several years
(10). The rhLH produced in vitro is purified
and further formulated to yield a pharmaceutical preparation with very
high specific immunoactivity and bioactivity. A cross-over
pharmacokinetic study has been performed in nonhuman primates to assess
and compare pituitary, urinary, and rhLH (11). After iv
administration of pituitary and urinary-derived LH, and rhLH, mean
concentration-time curves were parallel. The mean areas under the curve
(AUC) for concentration by time after dose curves were similar, after
correction for immunologic differences in dose. The mean clearance
estimates and volume of distribution at steady state, distribution, and
terminal half-lives were similar for all three types of LH.
Furthermore, the results of studies in human volunteers show that rhLH
and urinary-derived hLH have similar pharmacokinetic characteristics;
both preparations presented an iv distribution half-life of 1.2 h
and a terminal half-life of 10 h (12, 13, 14). rhLH total
body clearance was 2 L/h, with less than 5% of the dose being excreted
renally. The steady state volume of distribution was approximately 8 L
(13). Furthermore, it has been demonstrated that
coadministration of rhLH and rhFSH (follitropin ) does not modify
their respective pharmacokinetic characteristics (13).
Finally, low doses of rhLH have been shown to promote ovarian
steroidogenesis when follicular growth is induced with rhFSH in
hypogonadotropic hypogonadal women (15).
The aims of this study were to determine the minimal effective dose of rhLH required to induce final follicular maturation and early luteinization in patients undergoing IVF-embryo transfer (ET) and to compare the efficacy and safety of rhLH with those of the active control, urinary-derived hCG (u-hCG), looking at the possibility that rhLH, if proved efficient, would replace the well known u-hCG. A case report has been published suggesting the feasibility of rhLH in this indication (16). Here, we report the first extensive assessment of rhLH in IVF.
Materials and Methods
Study design
This prospective, randomized, double-blind, double-placebo study was designed to assess the safety and the minimal effective dose of rhLH required to induce final follicular maturation and early luteinization in patients undergoing IVF-ET and to compare the safety and efficacy of rhLH with those of u-hCG in this patient population. The different rhLH dosages were investigated in arms conducted sequentially, each following dose based on the results of the previous arm. The primary efficacy end point was the number of oocytes retrieved in the different study arms. Secondary efficacy end points were follicular and oocyte development, number of embryos, implantation rate, pregnancy rates, number of cryopreserved embryos and their fate, and the course of different hormone levels.
The study protocol was approved by the Institutional Review Board or Ethics Committee of all clinical centers before screening the first patient. Written informed consent was given before study entry, with the understanding that consent could be withdrawn by the patient at any time without prejudice.
Starting doses
Based on findings by Abdalla et al. (17) the dose of u-hCG was chosen to be 5,000 IU im in all control groups. The initial dose of rhLH was chosen to be 15,000 IU based on pharmacokinetic data of LH and a study by Chandrasekher et al. (18) comparing the effectiveness of human pituitary LH and u-hCG in ovulation induction in rhesus monkeys.
Selection of subsequent doses of rhLH
The decision to stop a study group and select the next rhLH dose was to be made based on the data from 25 patients treated with rhLH and 25 treated with u-hCG. The patients’ E2 and P4 serum levels assessed just before rhLH or u-hCG administration, on days 1 and 2 after rhLH or u-hCG injection, and the ovum pick-up (OPU) and IVF results were reported to the study center on an ongoing basis. The data were gathered and presented to the special group of investigators for review. The reviewers looked at blinded data and decided on the next dose of rhLH to be administered by considering major differences between the two treatments (rhLH and u-hCG). If the dose of 15,000 IU rhLH was judged to be as effective as 5,000 IU u-hCG, then the next dose of rhLH was to be lower (e.g. 5,000 or 10,000 IU). If the dose of 15,000 IU rhLH was judged to be less effective than 5,000 IU u-hCG, then the next dose of rhLH was to be higher (e.g. 20,000 or 25,000 IU).
Doses of rhLH tested
As a result of the study design and rhLH dose assignment described above, the rhLH doses tested during this study were: study arm 1, 15,000 IU rhLH vs. 5,000 IU u-hCG; study arm 2, 5,000 IU rhLH vs. 5,000 IU u-hCG; study arm 3, 30,000 IU rhLH vs. 5,000 IU u-hCG; study arm 4, 15,000 + 10,000 IU rhLH (the second dose of 10,000 IU was given 3 days after the first dose, after the OPU procedure) vs. 5,000 IU u-hCG.
Treatment protocol
Pituitary down-regulation. Commercially available GnRH-a (Suprefact, buserelin; Hoechst, Frankfurt, Germany) was self-administered sc into the thigh at a dose of 200 µg/day, starting in the midluteal phase (for a minimum of 10 days) and continuing until 24 h before the administration of rhLH or u-hCG. Pituitary desensitization was confirmed by a pelvic ultrasound (US) scan and measurement of plasma E2 levels (i.e. no evidence of ovarian activity on US examination and E2 <150 pmol/L or <40 pg/mL). If the patient was not down-regulated after 10 days of treatment, administration of GnRH-a was allowed to continue for another 15 days. If down-regulation was not achieved, the patient was removed from the study.
Stimulation of follicular growth
Treatment with rhFSH (Gonal-F; Serono, Geneva, Switzerland) was started after at least 10 days of GnRH-a therapy when pituitary desensitization had been achieved. rhFSH was administered once daily as a sc injection in the abdomen. The ovarian response was monitored by US and measurement of plasma E2 levels according to locally prevailing protocols in the different centers, and the dose was adjusted accordingly. The maximum dose allowed was 450 IU/day. The dose was reduced or discontinued if the patient was at risk of developing OHSS. US was performed at least once between day 10 and day 25 of pituitary suppression, on the day of rhLH or u-hCG administration (day 0), and at least once between days 6 and 9. LH, P4, E2, hCG, inhibin, testosterone, and androstenedione were determined once between days 10 and 25 of pituitary suppression (except for hCG), on the day of rhLH or u-hCG administration (day 0), and on days 1–3, day 6 or 7, and day 8 or 9. In addition, E2 was also determined on all days the patient came in for US examination. Anti-FSH and anti-LH antibodies were determined on the first day of rhFSH treatment, and total renin was determined on the day of rhLH or u-hCG administration. Serum hCG was also determined on day 15 and days 18 or 19.
Administration of study medication
Triggering of final follicular maturation with rhLH or u-hCG was performed in the evening, within 24 h of the last rhFSH and GnRH-a administration, when all of the following criteria had been met: the largest follicle had reached a mean diameter of at least 18 mm, at least one other follicle had a mean diameter of 16 mm, and serum E2 levels were within an acceptable range for the number of follicles present according to locally prevailing protocols in the different centers. Patients participating in treatment arms 1, 2, and 3 received an im injection of u-hCG (5,000 IU or placebo) in the buttock and a sc injection of rhLH (either 5,000 IU, 15,000 IU, 30,000 IU, or placebo) in the abdomen. Patients entering the fourth treatment arm received a single im injection of u-hCG (5,000 IU or placebo) and two sc injections of rhLH. The first rhLH injection (15,000 IU or placebo) was given on the same day as hCG, and the second injection (10,000 IU or placebo) was administered 3 days later.
Oocytes were retrieved by regular follicle aspiration 34–38 h after rhLH or u-hCG injection. Up to three embryos were replaced in the uterine cavity on day 2 or 3 after OPU.
Luteal phase support
Natural P4 (e.g. 200 mg micronized P4 three times a day) was administered by the vaginal route as luteal phase support, starting after oocyte collection. P4 treatment was continued up to menstruation or for at least the first 3 weeks of pregnancy if the patient became pregnant.
Early luteinization was assessed by measurement of serum P4 levels. At the midluteal phase, careful abdominal US assessment was performed to record any signs of OHSS. The patient was then followed up, and the outcome (pregnancy or menstruation) was recorded.
Selection of patients
Patients had to fulfill the following criteria: be a premenopausal woman; be between 18 and 39 yr old; have a body mass index (BMI) of 32 or less; have a menstrual cycle lasting between 21 and 35 days; have serum hormone levels of FSH 12 IU/L or less, PRL 1040 mIU/L or less, and TSH within the normal range of 0.3–4.1 mIU/L; and show normal results in pretreatment hematology, clinical chemistry, or urinalysis parameters. All patients had to be infertile due to at least one of the following causes and must have justified IVF treatment: tubal factor, mild endometriosis (American Fertility Society classification stage I or II), unexplained (with a history of at least 3 yr of infertility, and a postcoital test showing at least one forward progressive sperm per high power field), male factor (based on the investigator’s judgment, but only if an oocyte fertilization rate of more than 50% had been observed during a previous IVF attempt after regular insemination, or if donor sperm was used), severe male factor (based on the investigator’s judgment, but only if intracytoplasmic sperm injection was performed).
Patients had to have both ovaries and have undergone no more than three previous assisted reproductive technology cycles. In addition to these criteria, patients had to have had no treatment with clomiphene citrate or gonadotropins for at least 1 month before screening, and a normal uterine cavity confirmed by hysteroscopy, or hysterosalpingography or a US scan performed within the past 5 yr.
Method of assigning patients to study treatment
Eligible patients who achieved pituitary desensitization within 10–25 days of GnRH-a treatment were randomized by allocation of a unique patient identification number in sequential, chronological order before starting rhFSH therapy. The randomization scheme was prepared by a computer using Proc PLAN in SAS version 6.12 (SAS Institute Inc., Cary NC). Patients were randomly assigned to rhLH or u-hCG treatment according to balanced blocks of four subjects. rhLH/placebo vials and u-hCG/placebo ampoules were labeled with unique patient identification numbers. On the day of rhLH or u-hCG injection, the patient was given the rhLH or u-hCG medication carrying the identification number received when rhFSH treatment was started. The identification numbers of patients discontinued from the study were not reallocated.
Blinding
This was a prospective, double-blind, double-placebo study in which rhLH (5,000 IU, 15,000 IU, 30,000 IU, 15,000 + 10,000 IU or placebo) or u-hCG (5,000 IU or placebo) had to be administered as separate injections when satisfactory follicular development was achieved. The investigators and the patients were blinded to the treatment for the duration of the study. The dose of u-hCG was fixed, and the dose of rhLH varied depending on the treatment arm that was ongoing at the time of study. Three vials of 10,000 IU rhLH or corresponding placebo and one ampoule of 500 IU u-hCG or corresponding placebo were provided for each patient to keep the study blinded and to enable modification of the rhLH dose at any time.
Safety evaluation
Safety was assessed through: monitoring of all adverse events that occurred during the study, clinical assessment of local adverse reactions to injections, a questionnaire on clinical symptoms associated with OHSS at the time of the rhLH or u-hCG injection and at midluteal phase, midluteal phase US of the ovaries and abdomen, assessment of serum total renin levels, monitoring of any pathologic changes in routine laboratory values, and testing for anti-LH and anti-FSH antibodies.
Criteria used to define moderate OHSS
The criteria used to define moderate OHSS were: at least one of the following clinical symptoms—abdominal distension, abdominal pain, nausea, vomiting, diarrhea or dyspnea lasting for at least 3 days after rhLH or u-hCG injection; diameter of the ovaries (maximum of the left and right ovaries) on days rhLH or u-hCG 6 and 7 greater than 5 cm; and ascites on days rhLH or u-hCG 6 and 7. In addition, the E2 level measured on the day of rhLH or u-hCG injection was used as a predictive factor: in each treatment group, patients were classified based on an E2 cut-off value of 3000 pg/mL as well as on the number of follicles observed just before administration of rhLH or u-hCG, with a cut-off value of 20 follicles.
Serum hormone assays
Hormone analyses were performed with validated, commercially available immunoassay kits at Serono Clinical Laboratories (Cambridge, UK), which is a certified United Kingdom laboratory that conforms to the principles of Good Laboratory Practice. This same central facility also assayed antibodies to the study drug (rhFSH and rhLH) using immunoassays developed and validated by Serono. Serono Clinical Laboratories used the following assays methods and defined and applied the following limits of quantification (LOQ) throughout the study: LH, Serono MAIACLONE (now Biochem ImmunoSystems, Rome, Italy) immunoradiometric assay (IRMA; LOQ, 1.0 IU/L); hCG, Serono MAIACLONE IRMA (LOQ, 2.0 IU/L); FSH, Serono MAIACLONE IRMA (LOQ, 1.0 IU/L); PRL, Serono MAIACLONE IRMA (LOQ, 40 mIU/L); TSH, Kodak Clinical Diagnostics (Cardiff, UK) IRMA (LOQ, 0.5 µIU/mL); E2, Diagnostic Products (Los Angeles, CA) Count-a-coat RIA (LOQ, 100 pmol/L); P4, Diagnostic Products Count-a-coat RIA (LOQ, 1.0 nmol/L); renin, Nichols Institute Diagnostics (San Juan Capistrano, CA) IRMA (LOQ, 7 mIU/L).
Statistical evaluation
ANOVA models were used to detect a group or treatment effect. Tests of normality and equality of variance of the residuals were applied for validation of the model. Estimates, P values, and 95% confidence intervals (CIs) of the difference between groups and/or treatments were computed. For efficacy parameters for which several measurements were obtained on the same patient (i.e. oocyte nuclear maturity, grading of embryos), a generalized mixed linear model was applied. For proportions (i.e. pregnancies), a logistic regression model was used. Log odds ratios, P values, and 95% CIs of the log of the odds ratio between groups and treatments were computed. Laboratory and safety measures, unless otherwise noted, were analyzed using the same statistical tests.
Results
This double-blind, randomized, parallel-group clinical trial was
performed simultaneously in 22 centers in nine countries. A total of
259 patients were enrolled and randomized, and, of these, 258 entered
the clinical phase of the study. Of the 258 patients who underwent
ovarian stimulation treatment, 250 received rhLH (129 patients) or
u-hCG (121 patients). Nine patients did not receive rhLH or u-hCG
treatment: one was discontinued before starting rhFSH treatment because
she was found to be pregnant, five presented a risk of OHSS, and three
failed to develop a follicle with a mean diameter of at least 17 mm.
These patients were equally distributed over the different rhLH groups
as well as over their control groups. Of the 250 patients who received
rhLH or u-hCG treatment, 231 had at least one embryo transferred and
243 completed the midluteal phase assessment. The luteal phase had to
be monitored in all patients who received rhLH or u-hCG treatment,
whether or not ET was performed. Patient recruitment into the different
treatment groups is summarized in Table 1
.
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Demographic characteristics did not differ between the rhLH and
u-hCG treatment groups (Table 2). The
mean (± SD) age of the patients was
31.8 ± 3.6 yr, and the mean BMI was 22.6 ± 3.2
kg/m2. All patients had negative pregnancy tests.
The cause and duration of infertility, as well as the history of
previous assisted reproductive technique (ART) and non-ART pregnancies,
were similar between the treatment groups. At baseline, the mean serum
FSH level was 7.1 ± 1.9 IU/L, the mean serum PRL level was
268.8 ± 142.1 mIU/L, the mean serum TSH level was 1.47 ±
0.75 mIU/L, and uterine and ovarian size were comparable between the
treatment groups.
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The above information confirms that patients treated in this study corresponded well to the targeted population of regular IVF patients and that the different treatment groups were comparable at baseline.
Serum LH levels
Serum LH values on rhLH (day 0) were comparable between the
treatment groups, and a clear-cut increase above baseline in serum LH
levels was recorded in patients receiving a dose of rhLH. In addition,
there was a clear relationship between the dose of rhLH injected and
serum LH levels measured (Fig. 1), with
the highest levels recorded on day 1 in patients treated with 30,000 IU
(mean, 93 IU/L) and the lowest in those treated with 5,000 IU (mean,
23.28 IU/L). The dose-response relationship seemed to be linear between
doses of 5,000 IU and 15,000 IU rhLH, the latter showing a mean serum
LH level around three times higher than the former at days 1, 2, and 3.
This was not the case for the 30,000 IU dose, for which mean serum LH
levels at days 1, 2, and 3 were around 1.5 times higher than those
measured after a dose of 15,000 IU (Fig. 1).
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Exposure to treatment and study drugs
Patient exposure to treatment with GnRH-a and rhFSH is presented
in Table 3. The mean number of days of
GnRH-a therapy ranged from 23.9 ± 3.9 (15,000 + 10,000 IU
rhLH group) to 27.9 ± 5.1 days (15,000 IU u-hCG group).
When considering each group separately, the mean duration of GnRH-a
therapy was comparable between rhLH and u-hCG. The mean number of days
of rhFSH treatment ranged from 8.6 (15,000 IU rhLH) to 10.2 days
(30,000 IU rhLH).
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This treatment led to comparable follicular development in each study
group. At baseline, before rhLH/u-hCG administration, the number of
follicles was comparable as reported in Table 4. Moreover, serum
E2 was 7,925 pmol/L, 9,490 pmol/L, 9,045 pmol/L,
5,982 pmol/L, and 7,938 pmol/L in patients who received 5,000 IU rhLH,
15,000 IU rhLH, 30,000 IU rhLH, 15,000 + 10,000 IU rhLH, and 5,000 IU
u-hCG, respectively.
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IVF data from days u-hCG/rhLH 0–3 for patients in the 15,000 + 10,000 IU group were pooled with those from the 15,000 IU group, because the second rhLH dose of 10,000 IU was administered only 3 days after the first one. Embryo implantation data collected from day u-hCG/rhLH 2 onward and hormone levels from days u-hCG/rhLH 6–9 were compared between u-hCG (5,000 IU) and the four rhLH groups (5,000, 15,000, 30,000, and 15,000 + 10,000 IU), because, at this stage, patients in the 15,000 + 10,000 IU group had received the second injection of rhLH.
Follicular development and oocytes retrieved
The primary end point of this treatment protocol was to compare
the number of oocytes retrieved. Table 4
shows the mean number of
oocytes retrieved and the mean number of follicles with a diameter
greater than 10 mm at the time of rhLH or u-hCG administration.
Overall, the number of oocytes retrieved at OPU per patient ranged from
0–40. Two patients had no oocytes retrieved at OPU despite
satisfactory follicular development (one in the 30,000 IU rhLH group
and one in the 15,000 IU rhLH group). The serum LH values of these
patients were checked retrospectively and found to be comparable with
those of other patients in the same treatment group. The mean number of
oocytes retrieved per group varied from 10.23 (5,000 IU rhLH) to 12.62
(30,000 IU rhLH). The estimates of the difference between rhLH and
u-hCG treatments within each group did not reach statistical
significance (ANOVA P values = 0.3203, 0.9584, and
0.3362 for 5,000 IU, 15,000 IU pooled, and 30,000 IU, respectively)
(Fig. 2).
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To adjust for the number of follicles with a diameter greater than 10 mm observed before administration of rhLH or u-hCG, the ratio between the number of oocytes retrieved and the number of such follicles was calculated. This ratio ranged from 0.73–0.87, with no significant difference between the different groups.
Oocyte nuclear maturity
Nuclear maturity was observed in 785 oocytes. The majority of
oocytes were in metaphase II (83.1% overall; Table 4) with a maximum
proportion in the 15,000 IU pooled rhLH group (90.8%) and a minimum in
the 30,000 IU rhLH group (57.6%). The distribution of nuclear maturity
was comparable between rhLH and u-hCG and did not show any significant
differences. The test of linearity of the difference between rhLH and
u-hCG was not significant (P = 0.1838). However, when
comparing each rhLH dose with the mean proportions for all u-hCG
patients, the distribution of nuclear maturity was statistically
different between 30,000 IU rhLH and u-hCG (P = 0.014),
showing more oocytes at germinal vesicle (22.6% vs. 8.6%)
and metaphase I stages (18.9% vs. 3.5%) for patients
treated with 30,000 IU rhLH than for those treated with u-hCG.
Embryos
The mean total number of embryos varied from a minimum of 5.42
(5,000 IU rhLH) to a maximum of 7.67 (30,000 IU rhLH; Table 4). Within
each of the three groups, the mean total number of embryos were
comparable and showed no significant difference between rhLH and u-hCG
(ANOVA P = 0.1480, 0.7219, and 0.3339 for 5,000 IU,
15,000 IU pooled, and 30,000 IU, respectively). The test of linearity
for the difference between rhLH and u-hCG was not significant
(P = 0.0983).
Only embryos transferred during the study treatment cycle were
considered in this analysis. A total of 231 patients had at least one
embryo transferred. It was recommended in the protocol that no more
than three embryos be replaced; however, up to five embryos were
replaced in a few patients. The mean number of embryos transferred
varied from 2.39 (5,000 IU rhLH) to 2.78 (30,000 IU rhLH; Table 4).
Within each of the three groups, the mean numbers of embryos
transferred were comparable and showed no significant difference
between rhLH and u-hCG (ANOVA P = 0.5736, 0.5960, and
0.5771 for 5,000 IU, 15,000 IU pooled, and 30,000 IU, respectively).
The test of linearity of the difference between rhLH and u-hCG was not
significant (P = 0.4310).
Implantation rate
Due to the relatively small number of patients in each study
group, some variability was recorded in embryo implantation and
pregnancy rates. The mean implantation rate by group and treatment
varied from a minimum of 3% (u-hCG group of the 30,000 IU rhLH arm) to
a maximum of 19% (15,000 + 10,000 IU rhLH group). In each of the four
groups, the mean implantation rate was not significantly different
between rhLH and u-hCG (ANOVA P = 0.1413, 0.5024,
0.2803, and 0.7786 for 5,000 IU, 15,000 IU, 30,000 IU, and 15,000 +
10,000 IU, respectively; Table 4). In patients treated with rhLH, the
lowest and highest implantation rates were found in the 5,000 IU group
(6%) and the 15,000 + 10,000 IU group (19%). However, the test of
linearity of the difference between rhLH and u-hCG was not significant
(P = 0.1373).
Pregnancies
A total of 55 pregnancies (22.0%) were recorded for 250 patients; 41 (16.4%) were clinical pregnancies, and 30 (12.0%) resulted in delivery. Due to the relatively small numbers of patients, pregnancy outcome must be interpreted with caution. The proportion of patients achieving pregnancies was comparable between rhLH and u-hCG and was not significantly different for any of the four groups. Similar results were obtained when comparing each rhLH dose with the mean proportion of all u-hCG live births. Thirty patients delivered at least one baby, of whom 17 had singleton pregnancies and 13 twin pregnancies (43 births in total). The proportion of patients who delivered at least one baby was comparable between rhLH and u-hCG and was not significantly different for any of the four groups. The test of linearity within the four groups comparing rhLH and u-hCG was, however, of borderline significance (P = 0.0606). The highest proportion of patients with at least one live birth was found in the 15,000 + 10,000 IU rhLH group, in which 20% of patients had at least one live birth. By contrast, only 5% of the patients who received 5,000 IU rhLH had at least one live birth.
Cryopreserved embryos
At 20 of the 22 centers, 107 patients had at least one embryo
cryopreserved. The mean number of cryopreserved embryos varied by group
and treatment, from a minimum of 4.42 (5,000 IU rhLH group) to a
maximum of 9.89 (u-hCG group of 15,000 + 10,000 IU rhLH arm; Table 4).
The mean numbers of cryopreserved embryos from patients who received
5,000 IU rhLH (4.42 ± 2.65) or 15,000 + 10,000 IU rhLH
(5.75 ± 2.49) were significantly lower than those from
patients treated with u-hCG (6.81 ± 3.67 and 9.89
± 3.22; ANOVA P = 0.0325 and 0.0102,
respectively). On the other hand, the mean number of cryopreserved
embryos from patients who received 15,000 IU rhLH (7.93 ±
4.18) was significantly higher than the number from patients treated
with u-hCG (4.90 ± 3.24) (ANOVA P =
0.0088). When comparing each rhLH dose with the mean of all u-hCG
patients (6.25 ± 3.75), only patients who received 5,000 IU
rhLH had a significantly lower mean number of cryopreserved embryos
than patients treated with u-hCG (ANOVA P =
0.0497).
Cryopreserved ET
Seventeen of the 22 centers reported data on cryopreserved ET.
Of the 66 patients who had at least one cryopreserved ET, 17 had a
pregnancy (25.8%), 13 had a clinical pregnancy (19.7%), and 7 had at
least one delivery (10.6%). The number of transfers and pregnancies
for each study group are presented in Table 4. Because the numbers of
patients were small (from zero to five depending on the group and
treatment received), no statistical comparison was performed on these
data. It is, however, noteworthy that the majority of pregnancies were
recorded after transfer of embryos resulting from rhLH treatment. Five
patients had single pregnancies (in the rhLH 15,000 IU, u-hCG group of
15,000 IU rhLH arm, and rhLH 30,000 IU), one patient had a twin
pregnancy (rhLH 15,000 IU group), and one patient had a triplet
pregnancy (rhLH 5,000 IU group).
Serum P4 levels
Serum P4 profiles were evaluated from
days 0–3 and then on days 6–7 and 8–9 after rhLH or u-hCG
administration. Interpretation of these data requires consideration
that from day 2 (after blood sampling) onward natural
P4 was administered vaginally to all patients for
luteal support. Serum P4 levels at baseline (day
0) were comparable between the treatments (means ranged from 2.92–4.84
nmol/L). From days 1–3 after administration and in each of the four
groups, the increase in mean serum P4 levels was
comparable between rhLH and u-hCG and did not show any significant
difference. Similar results were obtained when comparing each rhLH dose
with the mean of all u-hCG patients. At study days 6–7 and 8–9, the
mean serum P4 levels measured in patients who
received 5,000 IU, 15,000 IU, or 30,000 IU rhLH were significantly
lower than those measured in patients treated with u-hCG (ANCOVA
P < 0.001 for each dose). The mean serum
P4 levels for patients who received 15,000 +
10,000 IU rhLH were not statistically different from those measured in
u-hCG patients (days 6 and 7, 242.7 vs. 279.0 nmol/L for
rhLH and u-hCG, respectively, P = 0.5147; days 8 and 9,
52.1 vs. 89.8 nmol/L for rhLH and u-hCG, respectively,
P = 0.1032) (Fig. 3).
|
Serum E2 levels at baseline (day 0) were
comparable between the treatments: means ranged from 5,982–9,490
pmol/L. From days 1–3 after administration, mean serum
E2 levels were comparable between rhLH and u-hCG
in each of the four groups and did not show any significant difference.
At study days 6–7 and 8–9, the mean serum E2
levels measured in patients who received 5,000 IU, 15,000 IU, and
30,000 IU rhLH were significantly lower than those in u-hCG-treated
patients (ANCOVA P < 0.001 for each dose). The mean
serum E2 levels of patients treated with 15,000 +
10,000 IU rhLH did not differ statistically from those of u-hCG-treated
patients (days 6 and 7, 3,079 vs. 4,960 pmol/L for rhLH and
u-hCG, respectively, P = 0.0822; days 8 and 9, 824
vs. 1,405 pmol/L for rhLH and u-hCG, respectively,
P = 0.1972) (Fig. 3).
Summary of adverse events
A total of 329 adverse events were reported by 148 patients
(59.2%). Adverse events were recorded from the start of GnRH-a
treatment until the final visit. A total of 158 (48.0%) of these
events occurred in 71 patients treated with rhLH (55.0%), and 171
(52.0%) occurred in 77 patients treated with u-hCG (63.6%). Of these
329 adverse events, 189 started before rhLH or u-hCG administration
(57.4%), and, of these, 126 resolved before rhLH or u-hCG injection
(36.1%). A total of 140 adverse events were experienced after rhLH or
u-hCG administration (42.6%): 65 (46.4%) occurred in 44 patients who
received rhLH treatment, and 75 (53.6%) occurred in 46 patients
treated with u-hCG. Table 5 lists the 13
nonserious adverse events that were reported at least five times and
their incidence in the five different treatment modalities. There were
no statistically significant differences between the treatment
arms.
|
OHSS
The proportion of patients presenting with moderate OHSS (as
defined above), independent of the number of follicles or the
E2 level, was highly statistically related to the
treatment received (exact P = 0.0004, Cochran-Armitage
trend test), with the higher incidence in patients treated with 15,000
+ 10,000 IU rhLH (12.0%) or 5,000 IU u-hCG (12.4%). In addition, the
proportion of patients who did not present any of the three criteria
for moderate OHSS was higher for the lower doses of rhLH than for the
15,000 + 10,000 IU rhLH or 5,000 IU u-hCG treatments (48.7%, 28.2%,
23.1%, 20.0%, and 17.4%, respectively, for rhLH 5,000 IU, 15,000 IU,
30,000 IU, or 15,000 + 10,000 IU rhLH and 5,000 IU u-hCG; exact
P = 0.0003, Cochran-Armitage trend test). Similar
results were obtained when taking into account the number of follicles
with a diameter greater than 10 mm or the E2
level on the day of rhLH or u-hCG injection (Table 6).
|
Because elevated serum renin levels have been reported in patients developing OHSS, serum total renin levels were measured in serum samples taken on the day of rhLH or u-hCG injection (before drug administration) and on day 6 or 7 after administration. The results show that total serum renin levels at baseline (day rhLH or u-hCG 0) were comparable between treatments: means ranged from 274.4–344.6 µIU/mL. The estimated differences at days 6 and 7 between rhLH and u-hCG for the four groups (-286.0 µIU/mL, -201.3 µIU/mL, -267.3 µIU/mL, and -5.3 µIU/mL) followed a dose-response relationship that was statistically significant (P = 0.0324).
Because elevated serum E2 on the day of hCG administration is a recognized risk factor for OHSS, the relationship between serum E2 level on the day of rhLH or u-hCG administration and serum renin levels in the midluteal phase was investigated.
The Pearson coefficient of correlation was highly statistically
significant for the 15,000 + 10,000 IU rhLH dose (positive correlation
= 0.737, P < 0.001) and the 5,000 IU u-hCG
treatment (all groups pooled, positive correlation = 0.437,
P < 0.001) only.
For the other rhLH doses, the correlation between these two parameters
was not significant (5,000 IU rhLH, = 0.196, P
= 0.244), slightly significant (15,000 IU rhLH, = 0.339,
P = 0.037), or of borderline significance (30,000 IU
rhLH, = 0.349, P = 0.094).
Discussion
This study has shown that the incidence of OHSS is significantly smaller when using a single dose of rhLH for induction of final maturation of follicles and oocytes in women treated for IVF, compared with the use of 5,000 IU u-hCG or two doses of rhLH. This finding is independent of the dose of rhLH, as long as it is administered in a single dose. The data presented in this study also demonstrate the efficacy of the first preparation of pure LH available for clinical use as a surrogate LH surge in regular IVF-ET patients. Treatment with 5,000 IU, 15,000 IU, 30,000 IU or 15,000 + 10,000 IU rhLH promotes final follicular maturation and luteinization. Therefore, this drug is promising in the struggle for restricting the potentially dangerous side effects of hyperstimulation of the ovaries, be it for IVF or for ovulation induction.
The efficacy and safety of hCG as a surrogate LH surge are well established. However, the occurrence of OHSS, of multiple pregnancies, and of a relatively low implantation rate after human menopausal gonadotropin-FSH/hCG therapy has drawn the interest of clinicians to the putative contribution of the prolonged activity of hCG to these adverse outcomes. A study published by Abdalla et al. (17) comparing single doses of 2,000, 5,000, and 10,000 IU u-hCG indicated that 2,000 IU u-hCG is not as effective as 5,000 IU u-hCG at inducing the final stages of ovulation, whereas 5,000 IU and 10,000 IU u-hCG are equally effective. The lesser efficacy of 2,000 IU was demonstrated by a significant increase in the proportion of patients in whom oocytes were not recovered during OPU and a reduction in the mean number of oocytes in patients who had at least one oocyte retrieved. Although the authors are aware of the fact that in many centers a dose of 10,000 IU hCG is routinely used, this observation formed the basis of this study’s primary efficacy end point and the u-hCG dose selected for the control group (5,000 IU u-hCG).
The relationship between the use of hCG and the occurrence of OHSS has long been recognized. OHSS rarely occurs when hCG is withheld, and multiple administrations of hCG (e.g. when hCG is used for luteal support) increases the incidence of OHSS. Also, when hCG is produced endogenously, as is the case during pregnancy, the course of OHSS is generally more prolonged and more severe (19).
Substitution of the hCG administration by a single dose of GnRH or GnRH-a has been advocated also to prevent OHSS (20, 21). It is, therefore, possible that OHSS is induced as well as supported by prolonged stimulation of the corpora lutea. Therefore, it is not surprising that also in this study the difference between single and multiple doses of rhLH translated into a higher incidence of OHSS. Apparently, the initial dose of LH at midcycle is less important, because even a dose of 30,000 IU rhLH did not induce one single case of moderate OHSS, whereas a smaller dose of 25,000 IU, given in two administrations, 3 days apart, did induce OHSS in the same frequency as 5,000 IU u-hCG.
The fact that single doses of rhLH have a short effect is further substantiated by the performance of rhLH in relation to the main characteristics of a natural LH surge as reported in the literature (1, 22). The natural surge lasts for about 2 days (49 ± 9 h) and is composed of an ascending phase (around 14 h), a plateau (around 14 h), and a descending phase (around 20 h). LH serum levels, when measured by RIA, are about 10–20 times the basal LH levels. The surge profile obtained after a single injection of 5,000 IU hCG is very different from that of the natural LH surge. The main differences are the total duration (the u-hCG surge can last up to 120 h) and the length of the descending phase. The profiles of the rhLH surges in this study are much closer to that of the natural surge: the duration of the surge in the 5,000 IU, 15,000 IU, and 30,000 IU rhLH groups was about 60 h.
Chandrasekher et al. (18) compared rhLH with u-hCG and pituitary hLH as an ovulatory stimulus in rhesus monkeys before IVF. These investigators found that a single injection of 2,500 IU rhLH was as effective as 1,000 IU u-hCG in inducing oocyte maturation, oocyte fertilizability, and luteinization of granulosa cells. The data suggested that using a conversion factor of 2.5, a dose of 12,500 IU rhLH would be as effective as 5,000 IU u-hCG in humans. Another study (unpublished data) looked at the spontaneous LH surges in seven healthy female volunteers with normal menstrual cycles after a single injection of either 250 µg GnRH-a (buserelin) or 5,000 IU u-hCG administered when the dominant follicle reached a diameter of 17 mm. It was found that the mean Cmax of the spontaneous LH surge was 47 IU/L (95% CI, 28–65 IU/L) and the mean AUC was 1,019 IU h/L (95% CI, 718–1,320 IU h/L). Using rhLH pharmacokinetic characteristics defined in Phase I studies (12, 13, 14), it was calculated that, to obtain a Cmax and an AUC larger than the corresponding mean values observed in spontaneous surges in 95% of cases, single sc injections of between 14,000 IU and 25,000 IU rhLH should be used.
Taking into account the findings of Abdalla et al. (17) and the above conversion factor, it was estimated that 10,000 IU rhLH would be less effective than 5,000 IU u-hCG and that 25,000 IU rhLH would be more effective than 5,000 IU u-hCG. Based on the above data, the first dose of rhLH chosen to be administered as a single sc injection was 15,000 IU.
The primary efficacy end point was the number of oocytes retrieved in relation to the dose of rhLH used and compared with that number after u-hCG. The results showed no statistically significant differences between the 5,000 IU, 15,000 IU, 30,000 IU, and 15,000 + 10,000 IU doses of rhLH compared with 5,000 IU u-hCG. The data showed a dose-effect relationship, the linearity of which, however, was not statistically significant.
Results for the secondary efficacy end points also showed no statistically significant differences. However, again there was a consistent trend toward lower numbers of oocytes, lower numbers of embryos, lower embryo implantation rates, lower numbers of clinical pregnancies, and lower live birth rates in patients treated with lower doses of rhLH. On one hand, this means that at least from a clinical point there is some evidence that the higher doses give better results; on the other hand, it may be that the optimal dose, as far as efficacy is concerned (number of oocytes retrieved vs. incidence of OHSS) has not yet been reached by the single dose of 30,000 IU rhLH. The low percentage of metaphase II oocytes in the rhLH 30,000 IU group is unexpected and unexplained.
The fact that serum E2 and P4 levels on days 6–7 and 8–9 in patients who received 5,000 IU, 15,000 IU, or 30,000 IU rhLH were statistically significantly lower than in patients treated with u-hCG and that mean serum P4 levels in patients who received 15,000 + 10,000 IU rhLH were not significantly different from those in u-hCG-treated patients, once more, shows that indeed the stimulation of the corpora lutea in the single dose rhLH-administered patients is less compared with the u-hCG and double dose rhLH-stimulated patients.
The rise in serum total renin levels from day 0 to days 6–7 and 8–9 showed a dose-effect relationship with rhLH. Serum E2 also correlated borderline with serum renin concentrations. The best correlations for both hormones were found with the double dose rhLH and with u-hCG. As shown in this study, OHSS was also preferentially found in the patients treated with the double dose rhLH and u-hCG. It might, therefore, well be that the elevated renin level is E2 dependent rather than dependent on the dose of rhLH or u-hCG.
In terms of safety, this study shows that rhLH is well tolerated at a dose of up to 30,000 IU. Reported adverse effects are similar to those usually reported during stimulation cycles with FSH and hCG. Finally, beyond the benefit of physiological half-life of rhLH documented in this study, the use of LH produced by recombinant technology also frees us from urinary supply difficulties and the possible spectra of communicable diseases related to extraction and pooling of biological materials obtained from large numbers of donors.
Based on this study, the recommended dose of rhLH is a single administration of at least 15,000 IU (750 µg rhLH) or 30,000 IU (1,500 µg rhLH). This dose will provide an efficacy at least equivalent to that of the reference treatment of 5,000 IU u-hCG and a safety superior to that of u-hCG, particularly with respect to the incidence of OHSS.
Acknowledgments
We thank Z. Shoham and J. Schoemaker for writing and M. O’Brien for reviewing the manuscript. We are also grateful to P. Cox, A. Kok, S. Lancaster, M. Peden, S. Rabut-Navarre, S. Rotem, M. Sauvage, G. Thomeczek, and G. Ursicino for help in the conception, conduct, and analysis of this study; and P. Fonjallaz and N. Waridel for editorial support.
Footnotes
1 Supported by Serono International (Geneva, Switzerland). This work
is dedicated to the memory of Prof. M. Hull. 
2 The following people from Serono Reproductive Health Clinical
Development Unit contributed to the conception, conduct, and analysis
of the study: P. Engrand, M. Arguinzoniz, A. Piazzi, S. Bologna, and
E. Loumaye. The following clinical investigators participated in
the conception and conduct of the study: P. Devroey, M.D., J. Smith,
M.D. (Belgium); A. N. Andersen, M.D. (Denmark); R. Frydman, M.D.,
B. Hedon, M.D., J. N. Hugues, M.D., R. Roulier,
M.D., J. Salat-Baroux, M.D. (France); R. Homburg, M.D., Z. Shoham,
M.D., A. Barash, M.D. (Israel); R. Palermo, M.D., C. Flamigni, M.D., E.
Porcu, M.D. (Italy); J. Schoemaker, M.D., E. R. Te Velde, M.D., D.
Braat, M.D. (The Netherlands); L. Hamberger, M.D. (Sweden); M. Germond,
M.D., B. Imthurn, M.D. (Switzerland); D. H. Barlow, M.D., P.
Brinsden, M.D., R. Fleming, M.D., M. Hull, M.D., G. Lockwood, M.D., A.
Templeton, M.D., R. Yates, M.D. (United Kingdom).
Received March 11, 2000.
Revised December 5, 2000.
Revised January 22, 2001.
Accepted January 29, 2001.
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