Ovarian stimulation by clomiphene citrate and hMG in combination with cetrorelix acetate for ICSI cycles

Jiann-Loung Hwang, Lee-Wen Huang, Bih-Chwen Hsieh, Yieh-Loong Tsai, Shih-Chia Huang, Chin-Yu Chen, Mei-Ling Hsieh, Pei-Hsin Chen and Yu-Hung Lin1

Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan 1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Shin-Kong Wu Ho-Su Memorial Hospital, No. 95, Wen Chang Road, Shih Lin District, Taipei, Taiwan. e-mail: m002179{at}ms.skh.org.tw


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The introduction of GnRH antagonists such as cetrorelix acetate has made possible the simplification of ovarian stimulation. However, the most effective protocol for their administration has not yet been clearly defined. METHODS: Forty women with male-factor infertility undergoing 40 ICSI cycles were included in the study. Clomiphene citrate at 100 mg a day was given from cycle day 3 through day 7. hMG at 150 IU was given on cycle days 4, 6 and 8, and was adjusted from day 9 according to the follicular and hormone responses. Cetrorelix acetate at 2.5 mg was administered when the leading follicle reached 14 mm. The remaining 0.5 mg was divided into two 0.25 mg injections for possible later use. Serum FSH, LH, estradiol and progesterone levels were measured daily from the day of cetrorelix acetate injection until hCG was given. RESULTS: Serum LH level was suppressed effectively for 4 days. Four patients (10%) needed one or two additional injections of 0.25 mg cetrorelix acetate. No premature LH surge was detected in any of the women treated. Sixteen women became pregnant (40%), of which 14 pregnancies (35%) were ongoing at the time of writing. CONCLUSIONS: This study demonstrates that this new protocol is feasible for couples with male-factor infertility undergoing ICSI.

Key words: cetrorelix acetate/clomiphene citrate/hMG/GnRH antagonist


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infertility affects ~15% of couples, and the male factor is the most common single cause of infertility (Irvine, 1998Go). Among infertile couples, the male factor is solely responsible in 20% and is contributory in 30–40% of cases (Thonneau et al., 1991Go). For couples with male factor infertility, intrauterine insemination (IUI), IVF, or even ICSI are usually necessary to achieve pregnancy. No matter which treatment strategy is used, controlled ovarian stimulation (COS) is usually used to increase oocyte numbers and pregnancy rates. In IVF/ICSI cycles, COS using clomiphene citrate (CC) and/or gonadotrophin has been replaced by gonadotrophin plus GnRH agonist because the former is associated with a 20–25% chance of a premature LH surge (Eibschitz et al., 1986Go). Among the different ovarian stimulation protocols currently available, the ‘long protocol’ is most commonly used and is generally considered the most effective (Biljan and Tan, 1998Go). However, the long protocol has the disadvantages of a longer treatment period and an increased requirement for gonadotrophins (Smitz et al., 1992Go).

Recently, there have been extensive studies on GnRH antagonists. GnRH antagonists bind to GnRH receptors and induce a fall in FSH and LH levels within hours. Their administration in the late follicular phase can effectively prevent or interrupt the LH surge. They do not cause a ‘flare-up’ as found with GnRH agonists, so prior desensitization for a period of some weeks is not necessary (Felberbaum and Diedrich, 1998Go). Two GnRH antagonists have been introduced, cetrorelix acetate and ganirelix (Antagon; NY Organon, Oss, The Netherlands). The development of GnRH antagonists makes ‘softer’ and simpler stimulation protocols possible. Felberbaum and Diedrich (1998Go) proposed that CC in combination with gonadotrophin and concomitant GnRH antagonist might be the way to a cheaper, more efficient and softer stimulation protocol. Craft et al. (1999Go) used daily administration of 0.25 mg cetrorelix acetate in conjunction with CC and FSH for 18 poor responders and found that this protocol produced favourable results. In comparison with previous IVF cycles with GnRH agonists, more oocytes were produced at a lower dose of gonadotrophin. Similar daily GnRH antagonist administration protocols were applied to 21 poor responders with three resulting clinical pregnancies (Nikolettos et al., 2001Go) and to 20 male-infertile couples with five resulting ongoing pregnancies (Tavaniotou et al., 2002Go).

Since most of the female partners in couples with male infertility factor have normal ovulatory function, it is not necessary for these women to undergo vigorous ovarian stimulation. In contrast to a study using multiple cetrorelix acetate injections (Tavaniotou et al., 2002Go), we evaluated the efficacy of CC and hMG in combination with single-dose cetrorelix acetate, which is supposed to be more patient friendly, for male-factor infertile couples undergoing ICSI. In a study using a single-dose cetrorelix acetate (at 3 mg)/hMG protocol, 9.6% of patients needed one or two additional doses at 0.25 mg to prevent the LH surge, which undoubtedly increased the cost (Olivennes et al., 2000Go). In order to reduce the patients’ economic burden, we reduced the dose of cetrorelix acetate to 2.5 mg, reserving the remaining 0.5 mg for possible later use. Its efficacy in suppressing the LH surge was tested.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This was a non-randomized, observational study to investigate the efficacy of a new protocol. Forty women aged 24–38 (31.9 ± 3.7) years who were scheduled to undergo ICSI to treat male infertility were enrolled in this study. The mean body weight was 54.1 ± 9.4 kg, and the mean body mass index (BMI) was 20.9 ± 2.9 kg/m2. The exclusion criteria included female age >38 years, a day 3 FSH level >15 mIU/ml, and an irregular menstrual cycle. All couples were required to sign an informed consent. The study was approved by the Ethics Committee of Shin Kong Wu Ho-Su Memorial Hospital. Among the 40 male patients, 36 had severe oligoasthenozoospermia with repeated failures of IUI, while the other four had obstructive azoospermia. Cryopreserved–thawed epididymal sperm obtained by percutaneous aspiration were used for the ICSI procedures, as in our previous study (Hwang et al., 2001Go). Seven patients had concomitant female factor infertility, including four with tubal factor and three with endometriosis. COS was carried out by starting clomiphene citrate (Clomid; Shionogi, Tokyo, Japan) at 100 mg per day from days 3–7 of the menstrual cycle. hMG (Pergonal; Serono, Geneva, Switzerland) at 150 IU was given on days 4, 6 and 8 of the cycle. From day 9, this was adjusted according to the follicular response until at least two follicles had reached 18 mm with an appropriate serum estradiol (E2) level. On cycle day 3, blood was drawn for analysis of serum E2, FSH, LH and progesterone as baseline data. Ultrasonography was carried out prior to COS. Daily ultrasonography from day 9 and blood sampling for E2, FSH, LH and progesterone were performed from the day of cetrorelix acetate injection (Cetrotide; ASTA Medica, Frankfurt, Germany) until administration of hCG (Pregnyl; NY Organon, Oss, The Netherlands). An LH surge is defined as an LH level >=10 mIU/ml with progesterone elevation (>=1.0 ng/ml). Serum LH >=10 mIU/ml without an increase in the progesterone level was defined as LH elevation (Olivennes et al., 2000Go). Serum FSH and LH were measured with an immunometric assay using an Immulite® kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The sensitivity for FSH was 0.1 mIU/ml. The intra- and inter-assay coefficients of variation (CV) were 7.7 and 7.9% respectively. For the LH assay, the sensitivity and intra- and inter-assays were 0.1 mIU/ml, 6.5, and 7.1% respectively. Estradiol and progesterone were measured by competitive immunoassay using an Immulite kit, with intra- and inter-assay CVs of 6.3 and 6.4% for estradiol, and 6.3 and 5.8% for progesterone respectively. Sensitivity was 15 pg/ml (55 pmol/l) for estradiol and 0.2 ng/ml (0.6 nmol/l) for progesterone.

The GnRH antagonist used was cetrorelix acetate in a 3 mg package. Prior to injection, 3 mg of lyophilized cetrorelix acetate was dissolved in 3 ml of injection-grade water to a final concentration of 1 mg/ml. An aliquot of 2.5 ml of the solution was withdrawn and injected s.c. into the abdominal wall. The rest of the cetrorelix acetate solution was separated into two 0.25 mg doses and kept at 4°C for possible later use. Cetrorelix acetate at 2.5 mg was given in the morning when the leading follicle had reached a size of 14 mm. If hCG was not going to be given 4 days after cetrorelix acetate injection, 0.25 mg cetrorelix acetate was given every day until the day of hCG injection. Oocyte retrieval was performed 36 h after hCG (10 000 IU) was given. ICSI was performed according to the method described by GoVan Steirteghem et al. (1993). Embryo transfer was performed on day 2 or 3. All patients received luteal phase support with 600 mg of vaginally administered micronized progesterone (Utrogestan; Laboratoires Piette International S.A., Brussels, Belgium) daily starting from the day after oocyte retrieval. Clinical pregnancy was defined as a visible fetal heart beat on ultrasonography at 7 weeks gestation. Values are expressed as the mean ± standard deviation (SD) unless otherwise indicated. Fisher’s exact test was used for statistical analysis. A value of P < 0.05 was considered as statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All patients underwent oocyte retrieval and embryo transfer (n = 40). Three patients needed one additional 0.25 mg cetrorelix acetate injection, and one needed two additional injections (4/40; 10%). Eighteen patients (45%) received a cetrorelix acetate injection on day 9; 14 (35%) on day 10; six (15%) on day 11; and two (5%) on day 12 of the cycle.

Hormonal profiles are shown in Table I. There was a statistically significant decrease in serum LH level 1 day after cetrorelix acetate injection (Table I). Serum values of LH, FSH, and E2, with respect to the day of cetrorelix acetate injection are presented in Figures 1, 2 and 3. It was noted that 2.5 mg cetrorelix acetate effectively suppressed the LH surge for 4 days in all patients (Table I, Figure 1). A premature LH surge did not occur in any patient. The serum FSH level was not suppressed as effectively as was LH (Table I, Figure 2). One patient experienced LH elevation on the day of cetrorelix acetate injection. The serum LH level dropped from 14.5 mIU/ml to 0.74 mIU/ml 1 day after cetrorelix acetate injection. Premature LH surge did not occur on the day of hCG injection and the woman became pregnant. Serum E2 steadily increased during the time of ovarian stimulation with a slow rising curve 1 day after cetrorelix acetate injection (Figure 3). There was a trend toward progesterone elevation near the day of hCG injection (Figure 4). On the day of hCG injection, 21 patients (52.5%) were found to have serum progesterone levels >=1.0 ng/ml. There were no significant differences in the day of oocyte retrieval, the number of metaphase II oocytes, the number of 2 pronuclei, the implantation rate, or the clinical pregnancy rate with regard to the progesterone level on the day of hCG injection (Table II). Statistically significant differences existed in the E2 and progesterone levels on the day of hCG injection and in the number of oocytes retrieved.


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Table I. Hormonal profile (mean ± SD) on day 3 of the cycle, day of cetrorelix acetate injection, one day after cetrorelix acetate injection, and the day of hCG injection
 


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Figure 1. Serum LH values relative to the day of cetrorelix injection. D = day.

 


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Figure 2. Serum FSH values relative to the day of cetrorelix injection. D = day.

 


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Figure 3. Serum E2 values relative to the day of cetrorelix injection. D = day.

 


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Figure 4. Serum progesterone values relative to the day of hCG injection D0. D = day.

 

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Table II. Comparison of clinical parameters (mean ± SD) in two groups of patients with regard to serum progesterone levels on the day of hCG injection
 
Table III shows the data of the clinical results including the day of oocyte retrieval, the number of ampoules of hMG used, the number of oocytes retrieved, the number of metaphase II oocytes, the number with 2 pronuclei, the number of embryos transferred, the implantation rates and the clinical and ongoing pregnancy rates. There were 16 clinical pregnancies (40%), of which two experienced intrauterine fetal death before 12 weeks gestation, so there were 14 ongoing pregnancies (35%) at the time of writing (11 singletons and three sets of twin pregnancies). No case suffered from severe ovarian hyperstimulation syndrome (OHSS).


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Table III. Patients and cycle characteristics
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates that 2.5 mg cetrorelix acetate, in combination with CC/hMG, successfully prevents the LH surge and is a feasible stimulation protocol for male factor infertility. Modern assisted reproductive technologies rely heavily on COS to increase the number of available oocytes and embryos and therefore the pregnancy rate. With the long protocol, women have to undergo multiple, prolonged drug injections with increasing incidence of side effects such as OHSS and at relatively higher costs (Felberbaum and Diedrich, 1998Go). Furthermore, as discussed by Edwards et al. (1996Go), such stimulation protocols may not be the optimal treatment for achieving the best implantation rates. Recently, there has been a trend toward ‘friendly IVF’ in order to decrease patients’ physical discomfort and economic burden. One way to simplify the stimulation protocol is with the help of a GnRH antagonist (Felberbaum and Diedrich, 1998Go).

The development of GnRH antagonists seems promising. A GnRH antagonist causes an immediate decrease in the LH level, thus allowing more flexible and shorter stimulation. Two protocols have been proposed for the use of GnRH antagonists in combination with hMG. In the multiple-dose or Lübeck protocol, 0.25 mg of antagonist is injected every day from day 5 or 6 of stimulation until the day of hCG administration (Diedrich et al., 1994Go; Felberbaum and Diedrich, 1998Go; Albano et al., 2000Go). In the single-dose or French protocol, 3 mg of antagonist should be administrated on stimulation day 7 to prevent a premature LH surge. If ovulation has not been triggered within 4 days of injection, 0.25 mg of antagonist is administered daily until hCG is given (Olivennes et al., 2000Go). Both protocols have been shown to be equally effective in suppressing the LH surge, and compare favourably with the long protocols with similar pregnancy rates, shorter stimulation periods, lower amounts of gonadotrophin used and decreased incidences of OHSS (Albano et al., 2000Go; Olivennes et al., 2000Go; European and Middle East Orgalutran Study Group, 2001). We prefer the single-dose protocol because it requires fewer injections. In one study, among the 126 patients receiving a single dose 3 mg cetrorelix acetate injection, nine patients (7.9%) needed one additional dose and two women (1.7%) received two additional doses of 0.25 mg cetrorelix acetate (Olivennes et al., 2000Go). In contrast, in the multiple-dose protocol, a mean of 5.7 injections of cetrorelix acetate was required (Albano et al., 2000Go).

In the Cochrane review of GnRH antagonists, it was found that GnRH antagonist was associated with shorter duration of ovarian stimulation and fewer ampoules of gonadotrophin were used, but there were no significant differences regarding prevention of a premature LH surge and severe OHSS compared with the GnRH agonist long protocol. Oocytes recovered were fewer, and there were significantly fewer clinical pregnancies in those treated with GnRH antagonist (Al-Inany and Aboulghar, 2002Go)

In a dose-finding study comparing a single dose of either 3 or 2 mg of cetrorelix acetate given on day 8 of the hMG stimulation cycle (hMG starting on day 2), no LH surge developed in the 3 mg group (n = 34); however, there was one LH surge (1/32 or 3%) in the 2 mg group (Olivennes et al., 1998Go). A single injection of 3 mg of cetrorelix acetate reliably prevented the LH surge for at least 4 days (Olivennes et al., 1998, 2000Go). Nowadays, cetrorelix acetate comes in two packages of either 0.25 or 3 mg. Since one of the drawbacks of cetrorelix acetate is its high cost, we have speculated on ways to decrease the cost of the stimulation protocol. We modified the protocol of single-dose cetrorelix acetate by giving only 2.5 mg and reserving the rest for possible later use after 4 days. We admit that this is against the manufacturer’s instruction, which requires that the solution be used immediately after reconstitution. This study showed that 2.5 mg of cetrorelix acetate could be as effective as 3 mg in suppressing the LH surge. Four patients (10%) in our study needed one or two additional 0.25 mg cetrorelix acetate injections 4 days after the initial injection. No patient required three additional injections, as in a previous study using 3 mg (Olivennes et al., 2000Go). In other words, our modification of giving only 2.5 mg cetrorelix acetate and reserving the rest for possible later use is an effective and economical method for most patients stimulated with CC/hMG. Tavaniotou et al. (2002Go) reported five ongoing pregnancies out of 20 patients with male infertility using multiple-dose cetrorelix acetate in combination with CC/hMG or CC/recombinant FSH. Our clinical result is comparable with that found in their report.

One of our patients had LH elevation on the day of cetrorelix acetate injection. In the study of Olivennes et al. (2000Go), 18 of 115 patients (15.7%) had LH elevation (LH >=10 IU/l) on the day of cetrorelix acetate administration. In common with the results of Olivennes et al. (2000Go), LH was suppressed within 24 h after injection of 2.5 mg cetrorelix acetate. The one patient in our study with LH elevation became pregnant, as did 4/18 patients in Olivennes (2000Go) study. These data are too small to draw any conclusions about the effects on the pregnancy rate when LH is elevated on the day of cetrorelix acetate injection. In both studies, 3 and 2.5 mg of cetrorelix acetate reduced the LH level within 24 h and effectively suppressed the LH surge for 4 days. There was no premature LH surge or LH elevation on the day of hCG injection in our patients. Because previous studies on GnRH antagonists were performed mostly on Caucasians, it cannot be ruled out that our results could be due to racial differences. It has been reported that serum GnRH antagonist levels have a linear inverse relationship with body weight, so smaller women may need lower doses of antagonists (Al-Inany and Aboulghar, 2002Go). However, the five studies included in the Cochrane review were restricted to women with normal BMI (18–30 kg/m2). It is reasonable to believe that our new protocol can also be applied to Caucasians. Further study is necessary to determine the minimal effective dose of cetrorelix acetate to prevent the LH surge in Caucasian populations.

We observed that a high percentage of patients (n = 21, 52.5%) had an elevated progesterone level (progesterone >=1.0 ng/ml) on the day of hCG injection. This incidence is much higher than that reported previously (Ubaldi et al., 1996Go). In that study, a subtle progesterone rise (progesterone >=1.1 ng/ml) was noted in 20.8% of patients (5/24) stimulated with hMG and multiple-dose cetrorelix acetate with no premature LH surge. The clinical results, including the number of oocytes retrieved, the number of mature oocytes, the number of fertilized oocytes, the implantation rates, and the pregnancy rates did not significantly differ between the two groups of patients with or without progesterone elevation (Table II). The higher percentage with a subtle progesterone rise might be due to the inclusion of CC in the stimulation protocol.

It has been reported that the combination of CC/hMG may be superior to either hMG or CC alone (Diamond et al., 1986Go), and may decrease hMG requirements compared with hMG alone (Dickey et al., 1993Go). With the addition of cetrorelix acetate, the potential for a LH surge in the CC/hMG protocol is negated. From this preliminary study, the pregnancy rate was also satisfactory.

Couples undergoing infertility treatments often experience relationship problems. For couples with male factor infertility, it seems unfair to make female partners go through the strenuous processes of ovarian stimulation and oocyte retrieval. We believe that the use of this simpler protocol will increase couples’ acceptance due to fewer injections, lower costs and potentially decreased marital conflict.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on August 6, 2002; accepted on September 30, 2002