1 Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, 2 Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, 3 Department of Obstetrics and Gynecology, E-Da Hospital/I-Shou University, Kaohsiung County, and 4 College of Medicine, Fu Jen Catholic University, Sinchuang City, Taipei County, Taiwan
5 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 111, Taiwan. Email: m001015{at}ms.skh.org.tw
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Abstract |
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Key words: cetrorelix acetate/Diane-35/HMG/GnRH antagonist/PCOS
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Introduction |
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Diane-35 (Schering, Berlin, Germany), an oral contraceptive pill (OCP) containing 2 mg of cyproterone acetate (CPA) plus 0.035 mg of ethinyl estradiol, has been widely used in treating the problems of seborrhoea and hirsutism in PCOS patients (Golland and Elstein, 1993; Prelevic et al., 1993
). It decreases LH production and subsequently androgen production through the inhibitory effect on the hypothalamus and pituitary gland. After three consecutive treatment cycles, serum concentrations of LH, testosterone and andostenedione returned to normal values, and sex hormone-binding globulin (SHBG) increased significantly in PCOS patients (Golland and Elstein, 1993
; Prelevic et al., 1993
).
GnRH antagonists inhibit gonadotrophin release within several hours through binding competitively to pituitary GnRH receptors (Felberbaum and Diedrich, 1998). Without a flare up effect being found with GnRH agonist, GnRH antagonist has been administered in the late follicular phase to prevent or interrupt the LH surge successfully during COS (Albano et al., 2000
; Olivennes et al., 2000
; Hwang et al., 2003
). If a similar application was made in PCOS patients undergoing IVF treatment, the early follicular phase high tonic LH secretion would probably still be present.
We proposed a stimulation protocol incorporating Diane-35 and GnRH antagonist in patients with PCOS who were to undergo IVF treatment. First of all, three consecutive cycles of Diane-35 pre-treatment were used in an attempt to decrease serum LH and androgens. One dose of a GnRH antagonist, cetrorelix acetate (Cetrotide; Serono, Geneva, Switzerland), was administered to augment the suppressive effect of Diane-35. Following this, cetrorelix acetate was administered concomitantly with exogenous gonadotrophin to prevent progressive tonic LH elevation in the early follicular phase and a premature LH surge in the late follicular phase. Part I of the study was an observational pilot study to evaluate the efficacy and hormonal changes associated with this protocol (Diane/cetrorelix protocol). Part II of the study was a prospective randomized controlled study to compare this protocol with the GnRH agonist long protocol. The hypothesis is that, by using the Diane/cetrorelix protocol, a similar fertilization, pregnancy and implantation rate could be achieved; and a similar serum LH and testosterone profile will be attained upon starting and during the HMG stimulation, compared with the GnRH agonist long protocol.
We tried to investigate (i) whether a similar fertilization, pregnancy and implantation rate could be achieved; and (ii) whether these two protocols attained a similar serum LH and testosterone profile upon starting and during gonadotrophin stimulation.
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Materials and methods |
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Study part I
From June 2002 to December 2002, 29 patients with PCOS were recruited from our infertility centre for this study. The age, duration of infertility and body mass index (BMI) were (mean±SD) 31.3±4.4 years old, 4.5±1.2 years and 22.9±2.9 kg/m2, respectively. Before entering the IVF programme, more than six cycles of ovulation induction with gonadotrophin had been done. The patients did not take any ovulation drugs or hormones for at least 3 months prior to the trial. The stimulation protocol is shown in Figure 1. In all patients, serum concentrations of FSH, LH, estradiol (E2), progesterone and testosterone were assessed on day 3 of induced or spontaneous menstruation. Diane-35 was prescribed as one tablet per day from day 5 of the cycle for 21 days. A total of three consecutive cycles were given. After pre-treatment with Diane-35, patients returned to the clinic for a hormonal profile examination, including FSH, LH, E2 and testosterone, in the morning of day 3 of the cycle. Ultrasonography was performed to exclude the presence of any ovarian cyst >10 mm. Cetrorelix acetate was then initiated with a single dose of 0.25 mg administered s.c. between 6.00 and 8.00 p.m. on day 3 to augment the suppressive effect of Diane-35. Patients returned to the clinic the following morning (day 4) for a repeat hormonal determination. From day 4 to day 9, cetrorelix acetate was reduced to 0.125 mg/day and ovarian stimulation was initiated with 150 IU of HMG (Pergonal; Serono, Geneva, Switzerland) every day. The dose of cetrorelix acetate was increased to 0.25 mg/day from day 10 until the day before HCG (Pregnyl; NY Organon, Oss, The Netherlands) injection, and the dose of HMG was adjusted according to the patient's response (Figure 1). Serum levels of LH, FSH and E2 were measured on days 3, 4, 10, the day of HCG injection and as indicated. HCG, 10 000 IU, was administered i.m. when at least two follicles reached 18 mm in diameter with adequate E2 response. Transvaginal oocyte retrieval (TVOR) was performed 36 h later. ICSI was performed for all patients because our previous experience (unpublished data) and that of Stadtmauer et al. (2001) suggested that a number of patients with PCOS had poor fertilization rates or unexpected fertilization failure with IVF. ICSI was performed according to the method described by Van Steirteghem et al. (1993)
. The potential risks of ICSI were thoroughly explained to the patients. Embryo transfer was performed 3 days after oocyte recovery. 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 of gestation.
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Study part II
During the period of January to December 2003, PCOS patients undergoing the first cycle of IVF treatment were randomized prospectively to receive one of the two ovarian stimulation protocols. Randomization was done by opening sealed envelopes containing computer-generated block randomization numbers with a block size of 10. The laboratory staff were blinded to the stimulation protocol. The ovarian stimulation protocol in the study group was the same as in part I of the study (Diane/cetrorelix protocol). The control protocol was a GnRH agonist long protocol. A GnRH agonist, buserelin acetate (Supremon; Hoechst, Frankfurt, Germany), 500 µg/day was administrated from day 3 of induced or spontaneous menstration. After 14 days of buserelin injection, patients returned to the clinic for blood tests and the ultrasound examination to ensure pituitary downregulation. The criteria of downregulation were serum E2 levels <50 pg/ml and the absence of ovarian cysts >10 mm in diameter. If the criteria were not met, a check-up was performed 3 days later. Buserelin was continued until the day of HCG injection, while the dosage was decreased to 250 µg/day at the beginning of HMG administration. HMG, at a dosage of 150 IU/day, was prescribed for 6 days beginning from the day of ensuing pituitary downregulation. Subsequent dosage was adjusted according to the follicular response as determined by serial ultrasound examination and serum E2 change. The timing of HCG injection, the method of oocyte retrieval, insemination, embryo culture, embryo transfer and luteal support were the same as in part I of the study. The primary outcome measures were fertilization, pregnancy and implantation rates. The secondary outcome measures were serum LH and testosterone status upon starting and during HMG administration, and the total days of injection.
Statistical analysis
We assumed 50% of patients received the Diane/cetrorelix protocol and 50% of patients received the GnRH agonist long protocol. The sample size required would be 25 in each group in the study to give a test of significance of 0.05 and a power of 0.8 (SAS; SAS Institute, Cary, NC), assuming a similar pregnancy rate of 0.4 between these two groups. Normality of continuous variables was assessed with the KolmogorovSmirnov test. A paired or unpaired t-test was used for the statistical analysis in continuous variable with normal distribution. The MannWhitney test was used for variables without normal distribution. Between-groups differences in non-continuous variables were assessed with the 2 method and the Yates correction, if needed. A P-value <0.05 was considered to be significant. Analysis was performed using the SPSS statistical package window 10.0 (SPSS Inc., Chicago, IL).
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Results |
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Hormonal profiles before and after Diane-35 pre-treatment, after the first dose of cetrorelix acetate injection (day 4), after 6 days concomitant treatment of 0.125 mg/day cetrorelix acetate and HMG (day 10) and the day of HCG injection are presented in Table I. As shown, PCOS patients had higher baseline LH and testosterone levels. After 3 months of pre-treatment with Diane-35, serum LH levels declined significantly to normal values (P<0.001). Before Diane-35 pre-treatment, 14 out of 29 patients had elevated serum LH levels (>10 mIU/ml) or an LH/FSH ratio >2. After OCP pre-treatment, only one patient still had abnormal serum LH levels that decreased to normal after a subsequent injection of cetrorelix acetate. The LH levels were suppressed to an even lower level after the first dose of 0.25 mg cetrorelix acetate (P<0.001). Seventeen of 29 patients had abnormal serum testosterone levels, and all of them returned to normal values after pre-treatment with Diane-35 (P<0.001). However, there was no further change after the first dose of cetrorelix acetate injection. Serum FSH and E2 levels did not change significantly after Diane-35 pre-treatment, but a significant decrease was noted after the first injection of cetrorelix acetate. FSH levels increased with the injections of HMG.
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Discussion |
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The fertilization, pregnancy and implantation rates of the two methods of ovarian stimulation were comparable. The serum LH levels during HMG stimulation were relatively constant in the Diane/cetrorelix group. There was a trend of progressive deeper serum LH suppression during the GnRH agonist long protocol. It could contribute to the higher amount of HMG used, the higher serum E2 on the day of HCG injection and three embryo transfer cancellations for fear of severe OHSS in this group of patients. Both protocols used in this study worked well with regard to prevention of a premature LH surge.
For non-PCOS patients undergoing IVF treatment, many reports have mentioned the application of a GnRH antagonist either by multiple dose daily administration or by a single dose injection in the late follicular phase to prevent a premature LH surge successfully, with satisfactory clinical results (Albano et al., 2000; Olivennes et al., 2000
; Hwang et al., 2003
). However, there are few reports regarding GnRH antagonist application during IVF cycles in PCOS patients. Craft et al. (1999)
reported seven patients with polycystic ovary (seven IVF cycles) using cetrorelix acetate in combination with clomiphene citrate and FSH. Cetrorelix acetate was administered daily until the leading follicle reached 14 mm in size. Three pregnancies were achieved from six completed cycles, with one ectopic pregnancy, one miscarriage and one live birth. There was one report retrospectively comparing 13 cycles of IVF/ICSI in PCOS patients using the leuprolide long protocol and 18 cycles using the ganirelix protocol. Ganirelix was given when the leading follicle reached 1314 mm. The pregnancy rate was comparable, while the peak E2 and total days of injections were significantly higher in the leuprolide long protocol (Abae et al., 2002
). The endocrine feature of the early follicular phase in PCOS patients treated by the traditional GnRH antagonist protocol is not well studied. It might be possible that high tonic LH hypersecretion is still present. Unsuppressed LH levels during the early follicular phase have been speculated to be related to a lower pregnancy rate consistently observed in the GnRH antagonist group during phase 3 comparative trials between agonist and antagonist (Kolibianakis et al., 2003
). Albano et al. (1997)
reported one premature LH surge in seven patients treated with 0.1 mg of cetrorelix acetate in the late follicular phase. Therefore, we thought that 0.125 mg/day would be enough to maintain low serum LH levels in the early follicular phase, which was shown to be between 2 and 3 mIU/ml in this study. The advantage is the decrease in patients' cost. On day 7 of HMG administration, cetrorelix acetate was increased to 0.25 mg/day in order to prevent a premature LH surge.
CPA, the progestational agent in Diane-35, possesses both anti-gonadotrophic and anti-androgenic properties that produce an inhibition of ovarian androgen production and a competitive inhibition of androgen receptors on the skin (Golland and Elstein, 1993; Prelevic et al., 1993
). In addition to the skin, androgen receptors have been reported to be present in the human ovary and endometrium (Horie et al., 1992
; Apparao et al., 2002
). Apparao et al. (2002)
have demonstrated an elevated endometrial androgen receptor expression in women with PCOS, which may reduce the endometrial receptivity. Favourable effects, in terms of three pregnancies in eight PCOS women, within 3 months after discontinuation of Diane-35 therapy have been reported (Prelevic et al., 1989
).
In summary, this study demonstrates that the incorporation of Diane-35 and cetrorelix acetate into the COS protocol for patients with PCOS undergoing IVF treatment could achieve a degree of pituitary suppression similar to that of the GnRH agonist long protocol at the start of HMG stimulation. The fertilization, pregnancy and implantation rate were similar to those of the GnRH agonist long protocol, with lower amounts of HMG used and lower serum E2 levels on the day of HCG injection. The total days of injections are fewer, while long-term Diane-35 administration is the major disadvantage. Further study is needed to clarify the minimal duration of Diane-35 pre-treatment.
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Submitted on June 10, 2003; accepted on May 10, 2004.
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