Article Effect of cetrorelix dose on premature LH surge during ovarian stimulation

RBMOnline - Vol 16. No 6. 2008 772-777 Reproductive BioMedicine Online; www.rbmonline.com/article/3181 on web 18 April 2008 Article Effect of cetrorelix dose on premature LH surge during ovarian stimulation Yu-Hung Lin graduated from the School of Medicine, National Taiwan University in 1991. After completing his residency training at the National Taiwan University Hospital, he served as an attending physician at the Shin Kong Wu Ho-Su Memorial Hospital. He is also an assistant professor of Fu Jen Catholic University. His clinical interests include infertility and minimally invasive surgeries. His research interests are in assisted reproduction technology, especially ovarian stimulation and in-vitro maturation of oocytes. Dr Yu-Hung Lin YH Lin 1,2,3, KM Seow 1,2,4, HJ Chen 1,2, BC Hsieh 1,2, LW Huang 1,2, CR Tzeng 5, JL Hwang 1,5,6 1 Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, No. 95, Wen Chang Road, Shih Lin District, Taipei, Taiwan; 2 School of Medicine, Fu Jen Catholic University; 3 Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei; 4 Department of Obstetrics and Gynecology, Li Shin Hospital, Taoyuan; 5 Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan 6 Correspondence: Tel: +886 2 28332211, ext. 3879; Fax: +886 2 28389416; e-mail: m002179@ms.skh.org.tw Abstract Previous studies have shown that ovarian stimulation with clomiphene citrate (CC), human menopausal gonadotrophin (HMG), and multiple-dose gonadotrophin-releasing hormone (GnRH) antagonist is associated with a high rate of premature LH surge. This study assessed whether administration of the GnRH antagonist cetrorelix at an incremental dose or at a high dose (0.5mg) from the start could prevent premature LH surge. Couples with male factor or unexplained infertility who were going to undergo intrauterine insemination were randomized into two stimulation protocols. All women were stimulated with CC and HMG. In protocol A, cetrorelix was given at 0.25 mg per day when the leading follicles reached 14 mm, and increased to 0.5 mg when the leading follicles were 16 mm. With protocol B, cetrorelix was given at 0.5 mg per day when the leading follicles reached 14 mm. The primary outcome measure was the incidence of premature LH surge. Premature LH surge occurred in 21.6% of patients undergoing protocol A, and in 18.9% of patients undergoing protocol B. Cetrorelix at incremental dose or at 0.5 mg per day does not prevent premature LH surges associated with the CC/HMG/multiple-dose cetrorelix stimulation protocol. Keywords: cetrorelix, clomiphene citrate, GnRH antagonist, premature LH surge Introduction 772 Introducing gonadotrophin-releasing hormone (GnRH) antagonists offers a short and simple stimulation protocol for patients undergoing assisted reproduction treatment. Patients treated with GnRH antagonists require a significantly lower gonadotrophin dose, and the duration of stimulation is significantly shorter compared with patients receiving the GnRH agonist (GnRHa) long protocol (Al-Inany and Aboulghar, 2002). The incidences of premature LH surge are not different (Al-Inany and Aboulghar, 2002). To prevent multiple pregnancies and ovarian hyperstimulation syndrome, there has been a trend to use mild stimulation in recent years. One of the proposed protocols is combining clomiphene citrate (CC), gonadotrophin, and GnRH antagonist. Craft et al. (1999) used CC, FSH and multiple-dose cetrorelix acetate for poor responders and obtained favourable results. The incidence of premature LH surge was not mentioned in that study, but Engel et al. (2002a) showed that a CC/gonadotrophin/multiple-dose cetrorelix protocol was associated with a 21.5% rate of premature LH surge, which made it unacceptable as a stimulation protocol for IVF cycles. They postulated that increasing the daily dose of cetrorelix from 0.25 to 0.5 mg might decrease the incidence of premature LH surge, but the hypothesis remained untested. The purpose of this study was to assess if increasing the dose of cetrorelix can prevent the premature LH surge in the CC/human menopausal gonadotrophin (HMG)/multiple-dose cetrorelix protocol. Since CC and gonadotrophin, combined with 0.25 mg 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

cetrorelix, is associated with a high rate of premature LH surge, it was postulated that increasing the dose of cetrorelix at a later stage or from the beginning of its administration might prevent premature LH surge. Materials and methods Patients From January to October 2005, couples with male factor infertility or unexplained infertility who were going to undergo intrauterine insemination (IUI) were included in the study. The study was approved by the Institutional Review Board of the hospital. The women were aged 20 38 years, with regular cycles, day 3 FSH concentrations 12 miu/ml and a body mass index (BMI) between 18.5 and 24.9 kg/m 2. Patients with polycystic ovarian syndrome were excluded. The women were randomized by block randomization into two stimulation protocols. It was assumed that the rate of premature LH surge with the CC/ gonadotrophin/multiple-dose cetrorelix protocol was 21.5% (Engel et al., 2002a), and that increasing the dose of cetrorelix would reduce the incidence of premature LH surge by 50%. The sample size required would be 71 with each protocol, to give a test of significance of 0.05 and a power of 0.8 (Size version 2.00; WHO, Geneva, Switzerland). The primary outcome measure was the incidence of premature LH surge. All women were stimulated with CC and HMG. Clomiphene citrate (Clomid; Shionogi, Tokyo, Japan) at 100 mg per day was given from day 3 to day 7 of the cycle. Two ampoules of HMG (Pergonal; Serono, Geneva, Switzerland) were given on days 4, 6 and 8, and then every day from day 9. With protocol A, cetrorelix acetate (Cetrotide; Serono, Bari, Italy) was given at 0.25 mg per day when the leading follicles reached 14 mm. The dose was increased to 0.5 mg per day when the leading follicles were 16 mm. With protocol B, cetrorelix was given at 0.5 mg per day when the leading follicles reached 14 mm, and the dose was maintained until the day of human chorionic gonadotrophin (HCG) administration. When at least one follicle reached 18 mm, 5000 IU HCG (Pregnyl; NY Organon, Oss, The Netherlands) was injected, and IUI was performed 36 h later. If premature LH surge occurred, 5000 IU HCG was given immediately and IUI was performed the next day. Pregnancy was defined as fetal heart activity visible on ultrasound scan. An LH surge is defined as LH 10 miu/ml and progesterone 1.0 ng/ml (Olivennes et al., 2000; Engel et al., 2002a). A premature LH surge means that the LH surge occurs before the administration of HCG. Serum LH 10 miu/ml without an increase in progesterone concentration is defined as an LH rise (Olivennes et al., 2000). For the hormonal study, each woman had blood tests for oestradiol, progesterone and LH every morning from the time that a follicle reached 14 mm until HCG injection. LH was measured by immunometric assay using an Immulite kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The sensitivity and intra- and inter-assay coefficients of variation (CV) were 0.1 miu/ml, 6.5 and 7.1% respectively. Oestradiol and progesterone were measured by competitive immunoassay using an Immulite kit, with intra- and inter-assay CV of 6.3 and 6.4% for oestradiol, and 6.3 and 5.8% for progesterone respectively. Sensitivity was 15 pg/ml (55 pmol/l) for oestradiol and 0.2 ng/ml (0.6 nmol/l) for progesterone. Statistical analysis The results were expressed as mean ± SD, except for LH concentration, which was expressed as median and range. Statistical analysis was carried out using Statistics Package for Social Sciences software (Version 13.0; Chicago, IL, USA). The various parameters of patients with and without premature LH surge or rise were compared by independent t-test for parametric data or Mann Whitney U-test for non-parametric data. Multiple regression analysis was performed to explore the relationship between LH concentrations on the day of LH surge or HCG injection and age, ampoules of HMG used, and oestradiol and progesterone concentrations. Receiver operating characteristic (ROC) curve analysis was used to estimate the predictive power of the various hormone concentrations to predict the occurrence of premature LH surge. Predictive power was defined by the area under the ROC curve. Statistical significance was defined as P < 0.05. Results With each protocol, the study was discontinued after 37 cycles because of high rates of premature LH surge. The mean age of the 37 women stimulated with protocol A was 31.5 ± 3.3 years. Seven women were pregnant (18.9%), including two twin pregnancies and five singletons. Premature LH surge occurred in eight women (21.6%), and there were two clinical pregnancies (25.0%, both singleton). An LH rise occurred in two women (5.4%), and one ectopic pregnancy resulted (Table 1). Among the 27 women who did not develop premature LH surge or LH rise, five women became pregnant (18.5%). To identify the patient characteristics associated with premature LH surge or rise, the clinical outcome parameters of the 27 women without premature LH surge or rise were compared with those of the 10 women with premature LH surge or rise. The following parameters were similar between the two groups of women: mean age, ampoules of HMG used, days of initiation of cetrorelix and HCG injection, LH, progesterone and oestradiol concentrations at the start of cetrorelix, and endometrial thickness on the day of HCG injection. The hormonal changes on the final 4 days of stimulation are shown in Table 2. Oestradiol concentrations between the two groups of women were similar each day. Progesterone concentration on the day of HCG injection was higher in the women with premature LH surge or rise (1.6 ± 0.8 versus 0.9 ± 0.4, P = 0.011). Progesterone concentrations were also different on the day before HCG injection. Similarly, median LH concentrations were different on the day of and on the day before HCG injection. The changes in the median LH concentrations of the two groups of women are shown in Figure 1. On the day of HCG injection, there were more follicles >16 mm in women without premature LH surge or rise (4.1 ± 1.8 versus 2.9 ± 1.1, P = 0.038). In the 37 women stimulated with protocol B, the mean age was 32.5 ± 3.2 years. Eight women became pregnant (21.6%), including one twin pregnancy. Premature LH surge occurred in seven women (18.9%), and only one was pregnant (14.3%, singleton). No LH rise occurred in these women (Table 1). 773

Table 1. Patient characteristics and outcomes in the two stimulation protocols. Parameter Protocol A Protocol B (n = 37) (n = 37) Age (years) 31.5 ± 3.3 32.5 ± 3.2 Ampoules of HMG used 14.9 ± 4.8 14.4 ± 4.0 Day of HCG injection 12.7 ± 1.3 12.5 ± 1.1 Oestradiol on day of HCG injection (pg/ml) 2004.0 ± 912.2 2246.14 ± 1084.96 Median LH on day of HCG injection; range (miu/ml) 3.60; 0.70 25.2 3.20; 0.89 25.0 Progesterone on day of HCG injection (ng/ml) 1.06 ± 0.63 1.21 ± 0.80 No. of follicles >16 mm 3.8 ± 1.7 4.3 ± 2.4 Duration of cetrorelix injection (days) 3.5 ± 1.1 3.3 ± 1.1 0.25 mg 1.7 ± 0.7 0.5 mg 1.8 ± 0.9 No. of pregnancies (%) 7 (18.9) 8 (21.5) No. of premature LH surges (%) 8 (21.6) 7 (18.9) No. of pregnancies (%) 2 (25.0) 1 (14.3) No. of LH rises (%) 2 (5.4) 0 No. of pregnancies 0 0 All women were stimulated with CC and HMG. In protocol A, cetrorelix was given at 0.25 mg per day when the leading follicles reached 14 mm, and increased to 0.5 mg when the leading follicles were 16 mm. In protocol B, cetrorelix was given at 0.5 mg per day when the leading follicles reached 14 mm. Values are mean ± SD, unless otherwise stated. HCG = human chorionic gonadotrophin; HMG = human menopausal gonadotrophin. Table 2. Hormonal changes relative to day of human chorionic gonadotrophin (HCG) injection in women undergoing protocol A. Hormone 3 2 1 0 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Oestradiol (pg/ml) 835.6 ± 391.2 794.0 ± 426.6 1240.8 ± 566.4 1067.8 ± 524.3 1691.6 ± 961.5 1315.6 ± 521.4 2028.9 ± 932.6 1936.8 ± 899.3 Median LH; range (miu/ml) 5.8; 0.6 17.0 3.3; 1.0 6.0 3.8; 0.4 15.6 3.2; 1.2 4.9 3.4 a ; 0.6 8.4 4.7 a ; 2.7 9.0 2.8 b ; 0.7 9.5 13.3 b ; 10.4 25.2 Progesterone (ng/ml) 0.7 ± 0.3 0.4 ± 0.1 0.6 ± 0.2 0.5 ± 0.1 0.6 ± 0.3 c 0.8 ± 0.1 c 0.9 ± 0.4 d 1.6 ± 0.8 d Protocol A = following stimulation with CC and HMG, cetrorelix was given at 0.25 mg per day when the leading follicles reached 14 mm, and increased to 0.5 mg when the leading follicles were 16 mm. Group 1 = no premature LH surge or rise (n = 27); group 2 = premature LH surge or LH rise (n = 10). Values are mean ± SD, unless otherwise stated. Values with the same superscript letter are significantly different: a P = 0.018; b P < 0.001; c P = 0.034; d P = 0.011. 774 Among the 30 women who did not develop premature LH surge or LH rise, seven women became pregnant (23.3%). As with the women in protocol A, the mean ages, ampoules of HMG used, day of initiation of cetrorelix and HCG injection, LH, progesterone, and oestradiol concentrations at the start of cetrorelix, and endometrial thickness on the day of HCG injection were similar between the women with and without premature LH surge. The hormonal changes on the last 4 days of stimulation are shown in Table 3. Oestradiol and progesterone concentrations on the day of HCG injection and 3 days before did not differ between women with and without premature LH surge. Progesterone concentration on the day of HCG injection was apparently higher in women with premature surge than in women without premature LH surge (2.3 ± 0.6 versus 0.9 ± 0.6), but the difference was not significant. The LH concentration was higher in women with premature LH surge on the day before HCG injection. The changes in median LH concentrations in the two groups of women are shown in Figure 2. The numbers of follicles >16 mm were similar between the women with and without premature LH surge (3.3 ± 1.3 versus 4.6 ± 2.6). No cycle in either study was cancelled. Multiple regression analysis showed that with both protocols, LH concentrations on the day of HCG injection (in women without premature LH surge) or on the day of LH surge or rise were not associated with age, amount of HMG used, or oestradiol and progesterone concentrations.

LH (miu/ml) 20 15 10 5 No premature LH surge Premature LH surge 0 3 2 1 0 Figure 1. Median LH concentrations with protocol A. HCG = human chorionic gonadotrophin. *P < 0.05. Table 3. Hormonal changes relative to day of human chorionic gonadotrophin (HCG) injection in women undergoing protocol B. Hormone Oestradiol (pg/ml) Median LH; range (miu/ml) Progesterone (ng/ ml) 3 2 1 0 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 948.0 ± 566.2 4.5; 0.9 18.5 1086.0 ± 529.5 2.6; 1.1 10.1 1193.9 ± 619.2 3.1; 0.7 11.3 1467.1 ± 352.4 3.0; 1.6 4.1 1609.6 ± 782.0 2.6 a ; 0.7 17.9 2192.0 ± 648.9 4.9 a ; 3.4 5.6 2036.8 ± 3143.4 ± 868.1 1032.3 2.4 b ; 0.9 7.5 15.2 b ; 10.7 25.0 0.6 ± 0.2 0.6 ± 0.2 0.6 ± 0.2 0.7 ± 0.1 0.7 ± 0.3 1.0 ± 0.2 0.9 ± 0.6 2.3 ± 0.6 Protocol B = following stimulation with CC and HMG, cetrorelix was given at 0.5 mg per day when the leading follicles reached 14 mm. Group 1 = no premature LH surge (n = 30); group 2 = premature LH surge (n = 7). Values are mean ± SD, unless otherwise stated. Values with the same superscript letter are significantly different: a P = 0.001; b P < 0.001. LH (miu/ml) 20 15 10 5 No premature LH surge Premature LH surge 0 3 2 1 0 Figure 2. Median LH concentrations with protocol B. HCG = human chorionic gonadotrophin. *P < 0.05. 775

776 To see if hormonal parameters could predict the occurrence of premature LH surge, ROC curve analysis was used to analyse oestradiol, progesterone, and oestradiol/(progesterone 100) on the day of premature LH surge and 3 days before. It was not possible to predict premature LH surge with either protocol. Discussion Combining CC and HMG lowers the requirement for HMG and produces higher pregnancy and birth rates than HMG alone (Dickey et al., 1998). However, because of the LH surge, more cycles were cancelled with the CC/HMG protocol than with the GnRHa/HMG protocol (Dickey et al., 1998). An earlier study showed that the incidence of premature LH with the CC/HMG protocol might be up to 30% (Nisker et al., 1993). Because GnRH antagonist causes rapid suppression of LH, it is plausible to incorporate GnRH antagonist to prevent the undesirable LH surge. However, Engel et al. found CC and gonadotrophins, in conjunction with multiple-dose, 0.25 mg, cetrorelix, were associated with premature LH surges in 21.6% of patients (Engel et al., 2002a). Similarly, Tavaniotou et al. (2003) found CC/gonadotrophin/0.25 mg cetrorelix regimens were associated with 11.1 (sequential regimen) and 28.5% (concurrent regimen) risks of premature LH surge. In both studies, cetrorelix was initiated from stimulation day 6. Mansour et al. (2003) used a similar stimulation protocol, except 0.25 mg cetrorelix was initiated when the leading follicles reached 16 mm; premature LH surges occurred in 6.5% (2/31). The present study also showed that premature LH surge was common with the CC/ HMG/multiple-dose cetrorelix protocol. Premature LH surge occurred in 21.6% of cases with protocol A and in 18.9% of cases with protocol B. In contrast, the incidence of premature LH surge with gonadotrophins and multiple-dose GnRH antagonist protocol was <3% (Al-Inany and Aboulghar, 2002). This indicates that adding CC increases the occurrence of premature LH surge. The possible mechanism of the adverse effect of CC may be as follows. Administration of CC results in depletion of oestrogen receptors on the hypothalamus and pituitary. As a result, oestrogen-negative feedback is interrupted centrally and gonadotrophin secretion is increased. In the late follicular phase, because of the long half-life of CC, there continues to be depletion of oestrogen receptors centrally, and increased oestradiol concentration is not capable of negative feedback centrally (Casper and Mitwally, 2006). Moreover, there is evidence that CC heightens the sensitivity of the pituitary for GnRH and reduces the efficacy of subsequent GnRH antagonist (Emons et al., 1986; Engel et al., 2002b). Increasing the dose of GnRH antagonist may overcome the elevated risk of premature LH surge associated with the CC/gonadotrophin protocol. Engle et al. (2003) demonstrated in 10 patients that 3 mg cetrorelix effectively prevented premature LH surge in stimulation protocols combining clomiphene and gonadotrophins. Similarly, in a previous study, it was shown that CC and HMG, in combination with single-dose, 2.5 mg cetrorelix, successfully prevented the LH surge (Hwang et al., 2003; Lin et al., 2006). However, the present study proved that multiple-dose cetrorelix, either at incremental doses or at 0.5 mg per day, could not completely prevent the premature LH surge in a CC/HMG protocol. Increasing the dose of cetrorelix further may be able to prevent premature LH surge, but it will not be cost effective. Thus, achieving a total receptor blockade by administering a larger single-dose GnRH antagonist seems to be a more logical approach with the CC/gonadotrophin protocol. However, considering the relatively lower pregnancy rate with IUI, this protocol may not be cost effective in IUI cycles. Although a premature LH surge was common in the present study, on the day when LH surge occurred, the leading follicles were at least 16 mm, when the oocytes were mature or nearly mature. In the women who developed premature LH surge or rise with protocol A, on the day when LH surge or rise occurred, the mean number of follicles >16 mm was 2.9 (range: 1 5). In women stimulated with protocol B, the mean number of follicles >16 mm when premature LH surge occurred was 3.3 (range: 1 5). Therefore, the LH surge or rise in the present study was in fact not premature. This might explain why a favourable pregnancy rate could still be achieved even when IUI was performed in advance. To maximize the chance of success, timing of IUI should be closely related to ovulation (Allen et al., 1985). However, there is wide variation in the interval between LH surge and ovulation. A multi-centred study from the World Health Organization (WHO) found that ovulation occurred 24 56 h from the onset of LH surge and between 8 and 40 h after its peak (WHO, 1980). Garcia et al. (1981) reported that ovulation occurred after a mean of 27.3 h from the onset of LH surge. In IVF cycles, oocytes retrieved 36 38 h from the start of LH surge achieved good fertilization rates (Testart et al., 1981). Seibel et al. (1982) reported that oocytes might take up to 38 h to mature from the onset of the LH surge. Since spermatozoa can survive in vivo up to 72 h and oocytes can retain their fertility potential for 12 24 h, the timing of insemination does not have to be as strict as oocyte retrieval in IVF programmes. When HCG is given before the LH surge, there can be mis-timing of follicle maturity. If HCG is given after the LH surge, higher pregnancy rates can be achieved (Fuh et al., 1997). Similarly, Mitwally et al. (2004) reported that in IUI cycles, there was a trend for a higher pregnancy rate when HCG was given after LH surge in CC treatment, either alone or with FSH. These results may explain why a favourable outcome could still be obtained in women with premature LH surge in the present study. There seems to be no way to predict the occurrence of premature LH surge. The oestradiol, progesterone and LH concentrations at the start of cetrorelix were similar between women with and without premature LH surge or rise. ROC curve analysis also failed to demonstrate any cut-off values of hormonal parameters. With protocol A, the progesterone concentration began to rise 1 day before LH surge or rise, but this phenomenon could not be observed with protocol B. With both protocols, LH concentrations began to increase 1 day before LH surge or rise. Thus, it may be prudent to watch for a premature LH surge once the LH concentration begins to rise with the CC/HMG/ multiple-dose cetrorelix protocol. In conclusion, CC and HMG, combined with multiple-dose cetrorelix, was associated with a high rate of premature LH surge, even when cetrorelix was increased to 0.5 mg. However, a favourable outcome could still be achieved in the IUI

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