Amir Ravhon, M.D., Ramon Aurell, M.D., Henrietta Lawrie, M.B., B.S., Raul Margara, M.D., and Robert M. L. Winston, F.R.C.O.G.

Transcription

1 FERTILITY AND STERILITY VOL. 73, NO. 2, FEBRUARY 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. The significance of delayed suppression using buserelin acetate and recombinant follicle-stimulating hormone in a long protocol in vitro fertilization program Amir Ravhon, M.D., Ramon Aurell, M.D., Henrietta Lawrie, M.B., B.S., Raul Margara, M.D., and Robert M. L. Winston, F.R.C.O.G. Wolfson Family Clinic and Flick Laboratories, Department of Reproductive Medicine and Science, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom Objective: To determine whether the time taken to achieve ovarian suppression has an impact on ovarian responsiveness and the outcome of IVF-ET. Design: Retrospective analysis. Setting: An assisted reproduction unit at a university center. Patient(s): Patients undergoing a long protocol of IVF-ET that included buserelin acetate therapy initiated on day 2 of the cycle and recombinant FSH. Intervention(s): Patients were divided into two groups according to the duration of buserelin acetate therapy required to achieve pituitary and ovarian suppression (group 1 2 weeks, n 172; group 2 3 weeks, n 337). Main Outcome Measure(s): Number of recombinant FSH ampules administered, duration of ovarian stimulation (days), ovarian response, and IVF outcome. Result(s): The patients in group 2 had lower mean E 2 levels after 5 days and 9 days of stimulation than the patients in group 1. The number of recombinant FSH ampules administered and the number of days of stimulation required were higher in group 2 than in group 1. These differences were prominent in the subgroups of older patients ( 36 years) and patients who had no evidence of polycystic ovaries on ultrasound examination. The number of oocytes retrieved and fertilized, the cancelation rate, and the pregnancy rate were similar in the two groups. Conclusion(s): Prolonged administration of a GnRH agonist to achieve suppression leads to a reduced ovarian response, particularly in women 36 years of age, but does not affect the success rate of IVF-ET. (Fertil Steril 2000;73: by American Society for Reproductive Medicine.) Key Words: Recombinant FSH, GnRH agonist, buserelin acetate, long protocol IVF, ovarian suppression Received April 7, 1999; revised and accepted August 25, Presented at the 15th Annual Meeting of the European Society of Human Reproduction and Embryology, Tours, France, June 27 30, Reprint requests: Amir Ravhon, M.D., Department of Obstetrics and Gynecology, Kaplan Medical Center, P.O.B. 1, Rehorot 76100, Israel (FAX: ; aravhon@bezeqint.net) /00/$20.00 PII S (99)00521-X The introduction of pituitary suppression with GnRH agonists (GnRH-a) in IVF treatment has led to improved pregnancy rates and fewer canceled cycles (1 3). The use of long protocol IVF, in which pituitary suppression is initiated during the early follicular phase or the midluteal phase, enables clinics to run more efficiently (4). The cycle also can be managed more easily if the day on which ovarian stimulation is started is coordinated and oocyte retrieval is deferred to avoid weekends and holidays (5 7). Delaying gonadotropin stimulation in patients in whom suppression of the pituitaryovarian axis already has been achieved by GnRH-a administration (defined by an E 2 level below a cutoff value of pg/ml (4, 6, 8) has not been found to affect ovarian responsiveness (6) and may even be associated with higher success rates (4). In contrast, in a number of studies (8 11), patients in whom GnRH-a administration was prolonged because of delayed suppression had lower pregnancy rates. This study was performed to determine whether the time taken to achieve ovarian suppression has an impact on ovarian responsiveness and IVF outcome in patients who begin receiving a GnRH-a on the second day of the menstrual cycle. 325

2 MATERIALS AND METHODS Five hundred nine IVF cycles were performed in 484 patients between August 1997 and March Pituitary desensitization was induced with buserelin acetate (Suprecur; Hoechst, Hounslow, United Kingdom) therapy started on the second day of the menstrual cycle. Buserelin acetate was administered as a nasal spray (0.3 mg tid) or as an SC injection (0.5 mg od) according to patient preference. After 2 weeks, the degree of ovarian suppression was assessed. Endometrial thickness was measured using transvaginal ultrasound equipment (Combison 311; Kretz Technic, Zipf, Austria) and blood samples were obtained to assess levels of E 2 and LH. When the E 2 level was 100 pmol/l, the LH level was 1 IU/L, and the endometrial thickness was 5 mm, the patient began receiving injections of recombinant FSH (rfsh, Puregon; Organon, Newhouse, United Kingdom). If these criteria were not met, the dosage of buserelin acetate was increased to 0.5 mg bid in patients receiving injections and to 0.3 mg five times a day in patients receiving the nasal spray. The dosage of buserelin acetate was reduced to the initial level in these patients once ovarian stimulation commenced. For patients who were undergoing their first IVF cycle, the starting dosage of rfsh was 100 IU/d for patients 32 years of age. For patients years of age, the starting dosage was 150 IU/d. Patients 36 years of age were started on 200 IU/d. Patients who had ultrasound findings consistent with polycystic ovaries (12) were started on half these dosages. For patients who had undergone a previous IVF cycle, the starting dosage of rfsh depended on the dosage required in the previous cycle. Plasma E 2 levels were measured 5 days after the initiation of rfsh therapy and then daily beginning on the eighth day. The first ultrasound scan was performed on day 9 and subsequent scans were performed every 2 or 3 days as required. The dosage of rfsh was adjusted from day 5 of stimulation according to the ovarian response. Human chorionic gonadotropin (10,000 IU, Profasi; Serono, Welwyn Garden City, United Kingdom) was given when 3 follicles had a mean diameter of 17 mm and the plasma E 2 level was 3,000 pmol/l. Egg collection was performed approximately 36 hours after hcg injection, and ET was done 2 3 days later. Two embryos were transferred unless there were adverse features. Adverse features included older patient age, poor embryo morphology, and multiple previous failed cycles. In those circumstances, three embryos were transferred. Progesterone was given for 12 days after ET and then the serum hcg level was measured. A clinical pregnancy was confirmed by transvaginal ultrasound scan 2 weeks later. Cycles were canceled if the ovarian response was poor or excessive. Statistical analysis included the use of the Mann-Whitney U test for comparison of means and the 2 test for proportions. P.05 was considered statistically significant. Analysis was performed using the STATA statistical package (Statacorp 1996, College Station, TX). The study was approved by our institution s research ethics committee. RESULTS The 509 cycles were divided retrospectively into two groups according to the length of time required to achieve suppression. consisted of 172 cycles (33.8%) in which ovarian suppression was achieved within 2 weeks; group 2 consisted of 337 cycles (66.2%) in which 3 weeks of GnRH-a therapy were required to achieve ovarian suppression. Patients in the two groups were similar in terms of age, number of previous IVF cycles, and indications for IVF. The results of ovarian stimulation and the outcomes of IVF-ET are presented in Table 1. To investigate whether patients were affected differently, the groups were subdivided according to patient age and the presence of polycystic ovaries on ultrasound examination. The results of ovarian stimulation and the outcome of IVF-ET in the younger patients ( 36 years) are presented in Table 2; those of the older patients ( 36 years) are presented in Table 3. The effect of the presence of polycystic ovaries is included in each table. DISCUSSION Women in whom buserelin acetate therapy was initiated on the second day of the cycle with long protocol IVF had a reduced ovarian response to rfsh when desensitization was attenuated. However, no differences were found in the number of oocytes retrieved or fertilized, or in the pregnancy rate between the two groups. The effect of attenuated pituitary desensitization was exaggerated in older patients without polycystic ovaries and was absent in younger patients with polycystic ovaries. Although this effect was not statistically significant in the group of older women with polycystic ovaries, this may be due to the relatively small number of patients in this group. Our results do not agree with those of Alvarez et al. (11), who found that the ovarian response was not affected by prolonged suppression ( 13 days); however, the pregnancy rate in these patients was reduced. Our results also contrast somewhat with those of Seifer et al. (8), who found that both the ovarian response and the pregnancy rate were reduced in patients in whom prolonged treatment was required to achieve ovarian suppression. The different results in these studies may be related to the treatment protocols used. Alvarez et al. (11) used luteal phase suppression, whereas we used early follicular phase suppression. The timing of the initiation of GnRH-a therapy influences the time course of ovarian suppression (13). More 326 Ravhon et al. 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3 TABLE 1 Ovarian response and IVF outcome in the two study groups according to the time required to achieve ovarian suppression. Variable (n 172) (n 337) P value Age (y) NS Buserelin acetate injected (%) Ampules of rfsh (50 IU each) No. of days of rfsh Day5E 2 level (pmol/l) Day9E 2 level (pmol/l) 2,124 2,101 1,436 1, E 2 level on day of hcg administration (pmol/l) 6,579 2,807 6,020 2,645 NS No. of oocyte retrievals NS No. of fertilized eggs NS Cancelation rate (%) NS No. of embryos replaced NS Pregnancy rate per eggs collected (%)* NS Note: Values are means SD unless otherwise indicated. NS not significant. had a suppression period of 2 weeks; group 2 had a suppression Ninety-five percent confidence interval 8.5% 7.7%. consistent and prompt ovarian suppression was achieved with midluteal phase initiation of GnRH-a therapy than with early follicular phase initiation (13). The initiation of GnRH-a therapy in the luteal phase compared with the follicular phase may have different effects on downregulation and therefore different groups of patients may require longer periods of treatment to achieve suppression. The increased dose of buserelin acetate required in our patients with delayed suppression also may have contributed to the reduced ovarian response to stimulation. The different results also may be related to the GnRH-a used. Alvarez et al. (11) and Seifer et al. (8) used leuprolide acetate, whereas we used buserelin acetate. Different patterns of follicular growth have been observed in humans with the use of different GnRH-a (14). In a study that compared the use of buserelin acetate, triptorelin, and leuprorelin during IVF cycles, the FSH level after 15 days of treatment and the E 2 level on the day of hcg administration were significantly lower when buserelin acetate was used (14). Testart et al. (15) postulated certain differences be- TABLE 2 Ovarian response and IVF outcome of patients 36 years old who had normal ovaries or polycystic ovaries in the two study groups. Patients with normal ovaries Patients with polycystic ovaries Variable (n 70) (n 129) P value (n 24) (n 34) P value Age (y) NS NS Buserelin acetate injected (%) NS NS Ampules of rfsh (50 IU each) NS No. of days of rfsh NS Day5E 2 level (pmol/l) NS Day9E 2 level (pmol/l) 2,262 2,301 1,625 1,444 NS 2,184 2,727 1,527 2,038 NS E 2 level on day of hcg administration (pmol/l) 6,704 2,875 6,523 2,591 NS 7,434 2,763 6,888 2,614 NS No. of oocyte retrievals NS NS No. of fertilized eggs NS NS No. of embryos replaced NS NS Pregnancy rate per eggs collected (%)* NS NS Note: Values are means SD unless otherwise indicated. NS not significant. had a suppression period of 2 weeks; group 2 had a suppression FERTILITY & STERILITY 327

4 TABLE 3 Ovarian response and IVF outcome of patients 36 years old who had normal ovaries or polycystic ovaries in the two study groups. Patients with normal ovaries Patients with polycystic ovaries Variable (n 64) (n 158) P value (n 14) (n 16) P value Age (y) NS NS Buserelin acetate injected (%) NS Ampules of rfsh (50 IU each) NS No. of days of rfsh Day5E 2 level (pmol/l) NS Day9E 2 level (pmol/l) 1,878 1,635 1,287 1, ,416 1,744 1, E 2 level on day of hcg administration (pmol/l) 6,062 2,777 5,408 2,559 NS 6,961 2,597 6,808 3,041 NS No. of oocyte retrievals NS NS No. of fertilized eggs NS NS No. of embryos replaced NS NS Pregnancy rate per eggs collected (%)* NS NS Note: Values are means SD unless otherwise indicated. NS not significant. had a suppression period of 2 weeks; group 2 had a suppression tween various GnRH-a in their action on follicular growth according to follicle size. Buserelin acetate has been proposed to have a strong antigonadotropin effect on small follicles. This effect of buserelin acetate was confirmed in the monkey (16). The fact that we adjusted the dosage of rfsh according to the ovarian response (17 19) in our protocol may explain why the success rate was similar in the two groups. By increasing the dosage of rfsh in response to suboptimal ovarian activity, folliculogenesis might be improved. Seifer et al. (8) did not specify the protocol they used for hmg administration; however, if they did not adjust the dosage of rfsh in response to a suboptimal ovarian response, this could explain the continued suboptimal ovarian response in some patients. We found a similar pregnancy rate in the two study groups, with a 95% confidence interval of 8.5% 7.7%. Although, theoretically, the pregnancy rate could be lower in cycles with delayed suppression, the difference is much smaller compared with the difference in pregnancy rates in the two other studies discussed (8, 11). In addition, the different results in the various studies (4, 6 11) may be due to the fact that two distinct groups of patients were described; the first underwent prolonged GnRH-a administration after achieving suppression (4, 6) and the second underwent prolonged GnRH-a administration to achieve desensitization (8 11). Once suppression is achieved, the duration of GnRH-a administration is not associated with an altered ovarian response (4, 6). Fabregues et al. (20) found that E 2 levels were lower in cycles with long-term suppression (4 months) than in those with short-term suppression. When a longer interval was required for suppression, the ovarian response and/or the pregnancy rate was reduced (8, 10, 11). Seifer et al. (8) suggested that the reduced ovarian response seen after prolonged GnRH-a treatment to achieve pituitary suppression may be due to an unspecified underlying dysfunction of the hypothalamic-pituitary-ovarian axis and not to the GnRH-a itself. The effect of buserelin acetate on the ovary may be direct or indirect. The activity of gonadotropins during GnRH-a treatment might be reduced as a result of the production and release of a deglycosylated form of FSH (21). These isoforms compete at the receptor level and thus act as endogenous antihormones. This may explain why the longer the duration of GnRH-a treatment and/or the higher the dose, the greater the need for FSH to achieve an adequate response. However, this phenomenon has been described in a single article (21) without subsequent confirmation. Given that the expression of GnRH receptor messenger RNA in human ovaries has been demonstrated, the ovary may be a target for GnRH action (22). Buserelin acetate may have a direct inhibitory effect on ovarian steroidogenesis with a deleterious effect on the ovary that is dose- and/or time-related (22). The recovery of E 2 secretion after longterm GnRH-a administration in women with polycystic ovary disease was found to be much slower than the recovery of FSH and LH secretion (23). One explanation for this finding could be an abnormal ovarian responsiveness to FSH after GnRH-a administration (23). Profound suppression of LH also may be important. 328 Ravhon et al. Aspects of delayed suppression in IVF Vol. 73, No. 2, February 2000

5 Fleming et al. (24) showed that profound suppression of LH during ovarian stimulation is correlated with lower E 2 concentrations. This may be exaggerated in cycles that are stimulated with rfsh because of a complete lack of exogenous LH. Our results demonstrate a statistically significant difference in E 2 levels between the two study groups on day 5 of rfsh administration. The difference in E 2 levels became smaller thereafter. This finding may be explained by a reduced ovarian response to rfsh stimulation in group 2 that was compensated for by the increased rfsh dose. However, a mechanism of delayed response in group 2 instead of (or combined with) a reduced response cannot be excluded. Because our treatment protocol included adjustment of the rfsh dose, we cannot determine whether the ovarian response to stimulation is reduced or only delayed. The confounding results reported in the literature do not help in resolving this issue (4, 6 11). We found that older patients were more affected by prolonged suppression, although it was not clear whether this was a cause or an effect. Simon et al. (25) found that a reduced GnRH-a dose during folliculogenesis had no effect on the hmg dose or success rate. However, Feldberg et al. (26) and Olivennes et al. (27) reported that patients with a poor response or with relatively high FSH levels tended to respond better to ovarian stimulation when the GnRH-a dose was reduced. This study demonstrated that the ovarian response to stimulation is reduced in women who require prolonged GnRH-a treatment to achieve suppression, and that this effect is seen mainly in older patients. A higher total gonadotropin dose compensates for this reduction. The effect of attenuated desensitization should be taken into consideration when clinical decisions are made about the initial gonadotropin dose and the ovarian response to stimulation. It also should be considered when data regarding a patient s previous ovarian response to stimulation are used in planning a subsequent cycle. References 1. Wildt L, Diedrich, K, van der Van H, Al Jassani S, Huebner H, Klassen R. Ovarian hyperstimulation for in-vitro fertilization controlled by GnRH agonist administered in combination with human menopausal gonadotropins. Hum Reprod 1986;1: Neveu S, Hedon B, Bringer J, Chinchole JM, Arnal F, Humeau C, et al. Ovarian stimulation by a combination of a gonadotropin-releasing hormone agonist and gonadotropins for in vitro fertilization. Fertil Steril 1987;47: Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a metaanalysis of randomized controlled trials. Fertil Steril 1992;58: Damario MA, Moomjy M, Tortoriello D, Moy F, Davis OK, Rosenwaks Z. Delay of gonadotropin stimulation in patients receiving gonadotropin-releasing hormone agonist (GnRH-a) therapy permits increased clinic efficiency and may enhance in vitro fertilization (IVF) pregnancy rates. Fertil Steril 1997;68: Dimitry ES, Bates SA, Oskarsson T, Margara R, Winston RML. Programming in vitro fertilization for a 5- or 3-day week. Fertil Steril 1991;55: Chang SY, Lee CL, Wang ML, Hu ML, Lai YM, Chang MY, et al. No detrimental effects in delaying initiation of gonadotropin administration after pituitary desensitization with gonadotropin-releasing hormone agonist. Fertil Steril 1993;59: Scott RT, Neal GS, Illions EH, Hayslip CA, Hofmann GE. The duration of leuprolide acetate administration prior to ovulation induction does not impact ovarian responsiveness to exogenous gonadotropins. Fertil Steril 1993;60: Seifer DB, Thornton KL, DeCherney AH, Lavy G. 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