A controlled study of gonadotropin-releasing hormone agonist (buserelin acetate*) for folliculogenesis in routine in vitro fertilization patients

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1 FERTILITY AND STERILITY Copyright" 1991 The American Fertility Society Vol. 56, No. 3, September 1991 Printed on acid-free paper in U.S.A. A controlled study of gonadotropin-releasing hormone agonist (buserelin acetate*) for folliculogenesis in routine in vitro fertilization patients David W. Polson, M.D.t Vivien MacLachlan, B.Sc. Jennifer A. Krapez, B.Sc. Carl Wood, M.B. David L. Healy, Ph.D. Monash IVF Programme, Infertility Medical Centre, Epworth Hospita~ Richmond, Victoria, Australia Study Objective: To determine if gonadotropin-releasing hormone agonist (GnRH-a) and gonadotropin therapy could improve folliculogenesis and pregnancy rates (PRs) in women with a previously satisfactory response to clomiphene citrate and human menopausal gonadotropin (hmg). Design: Randomized prospective study. Setting: Assisted reproduction clinic. Patients: One hundred fifty-seven women were randomized to receive either hmg alone or the GnRH-a buserelin acetate 600 p.gfd or buserelin acetate 1,200 p.gfd plus hmg. Results: Compared with hmg alone, pretreatment with buserelin acetate significantly increased the PR per cycle started by preventing a premature luteinizing hormone rise and thereby reducing the number of abandoned cycles. There was, however, no difference between the number of follicles aspirated, oocytes obtained, or fertilization rates between groups. Furthermore, agonist therapy significantly increased both the dose of hmg required and the duration of stimulation. Conclusion: The routine use of GnRH -a in in vitro fertilization programs must be questioned. Fertil Steril 56:509, 1991 The use of long-acting gonadotropin-releasing hormone agonists (GnRH-a) has been recently reported in women undergoing controlled hyperstimulation for in vitro fertilization (IVF) programs. 1-5 Reports suggest improvements in follicular recruitment, oocyte number and quality, fertilization rates, number of embryos transferred and pregnancy rates (PRs). In addition, there is a reported reduction in the number of abandoned cycles because of prevention of an inappropriate rise in the serum luteinizing hormone (LH) or progesterone (P) concentration. 6 This has led to the suggestion that GnRH -a be used in all IVF-gamete intrafallopian transfer (GIFT) treatment cycles. 4 5 Received December 27, 1989; revised and accepted May 9, * Buserelin acetate, Suprefact; Hoechst, Melbourne, Victoria, Australia. t Reprint requests: David Polson, M.D., Department of Obstetrics and Gynaecology, Withington Hospital, Nell Lane, West Didsbury, Manchester M20 8LR, United Kingdom. Vol. 56, No. 3, September 1991 Unfortunately, these conclusions have been drawn from poorly controlled and nonrandomized studies with patients being specifically allocated to GnRHa therapy Most of these reports do confirm improved folliculogenesis in women who fail to respond satisfactorily to treatment with clomiphene citrate (CC) and human menopausal gonadotropin (hmg). 7-9 There is, however, little evidence for a similar improvement in women who respond satisfactorily to CC-hMG. Furthermore, there have been no dose-response studies to determine the dose of agonist required. We have therefore performed a randomized study to: (1) compare folliculogenesis and pregnancy outcome in women with a previously normal response to CC-hMG after randomization to receive either hmg alone, buserelin acetate 600 f.l.g/d plus hmg, or buserelin acetate 1,200 f.l.g/d plus hmg; and (2) determine any differences in patient response to either of these two doses of buserelin acetate. Polson et al. GnRH-a in routine IVF patients 509 I

2 Patients MATERIALS AND METHODS One hundred fifty-seven apparently endocrinenormal IVF patients with tubal (n = 76) or idiopathic (n = 81) infertility entered the study between September 1988 and February Couples with idiopathic infertility had failed to conceive after at least 18 months regular unprotected intercourse despite ovulatory cycles, a normal semen analysis as defined by the World Health Organization classification and a normal pelvic appearance at laparoscopy. All patients were.::;;37 years old and had a previously satisfactory response after our standard regimen of CC (Clomid; Merrell Dow, Sydney, New South Wales, Australia) 100 mg days 3 to 7 and hmg (Pergonal; Serono, Melbourne, Victoria, Australia) 150 IU from day 4. 10' 11 This was defined by a serum estradiol (E2) concentration > 3,600 pmol/l, more than three follicles > 17 mm in diameter noted on ultrasound (US) before the administration of human chorionic gonadotrophin (hcg) and the recovery of more than one oocyte. Patients were randomized by simple randomization using a single sequence of random numbers. The randomization was centralized but was not s~ratified, and blocking was not used. The sample s1ze based on 80% power and a level of significance of 5% was 150 patients. Patients were randomized to receive hmg or intranasal buserelin acetate (Suprefact; Hoechst, Melbourne, Victoria, Australia) 600 J.Lg/d (buserelin acetate 600) or 1,200 J.Lg/d (buserelin acetate 1,200) plus hmg. Fifty women received hmg (26 with tubal infertility; 24 with idiopathic infertility), 51 received buserelin acetate 600 (25 tubal, 26 idiopathic), and 56 received buserelin acetate 1,200 (25 tubal, 31 idiopathic). Fifty-six cycles were initiated in each of the hmg and buserelin acetate 600 groups and 64 cycles in the buserelin acetate 1,200 group. The mean age (range) in each group was similar; 31.3 years (23 to 36), 32.5 years (26 to 37), and 32.7 years (26 to 37) respectively. Methods Women assigned to treatment with buserelin acetate started on day 3 of the cycle after measurements of serum E2, P, and LH concentrations. 11,12 One or two doses of buserelin acetate 150 J.Lg were administered intranasally at regular intervals four time~ ~er d~y. For the buserelin acetate 600 group, admtmstratwn was to one nostril only (4 X 150 J.Lg) and for the buserelin acetate 1,200 group to both nostrils (4 X 300 J.Lg). Initially, weekly blood tests were taken to assess pituitary and ovarian suppression. When the serum E2 concentrations were <180 pmol/l on two consecutive daily blood samples, treatment with hmg was started. Women assigned to receive hmg alone commenced on day 3 of the cycle. The hmg alone and buserelin acetate-hmg groups received the same initial dosage of 300 IU, 300 IU, 150 IU, and 150 IU hmg for the 1st 4 days. On the 4th day of treatment, and daily thereafter, serum E2, LH, and P concentrations were measured. The dosage of hmg was adjusted according to the rise in serum E2 concentration and follicular developmen~ as noted by vaginal pelvic ultrasonography, as prevwusly describedy Cycles were abandoned if there was an unsatisfactory follicular response and low serum E2 concentrations, despite a high daily dose of hmg {up to 675 IU). Human chorionic gonadotropin, 5,000 IU, was administered when there were at least three follicles > 17 mm in diameter and the serum E2 concentration was >3,600 pmoljl. Buserelin acetate was then discontinued. Depending on the indication for infertility, either gamete intrafallopian transfer (GIFT), pronuclear stage transfer, or an IVF procedure was performed 36 hours later, as previously described. 10,13 For IVF, embryo transfer (ET) took place 48 to 72 hours after US-guided vaginal oocyte recovery.14 The luteal phase was supported in all three groups. For the 1st 2 months of the study, 1,000 IU ofhcg was administered on days 3, 6, and 9, but after two cases of moderate hyperstimulation requiring hospitalization, this dose was reduced to 500 IU on days 6 and 9. Serum concentrations of hcg, E2, and P were measured 14 days after oocyte recovery and any pregnancies confirmed by ultrasonography 14 to 21 days later. Only clinical pregnancies were recorded (i.e., presence of gestational sac with or without fetal complex, or ectopic pregnancy). If an LH surge occurred in the presence of three or more follicles and satisfactory serum E2 concentrations (>3,600 pmol/l), oocyte recovery was performed 36 hours from the onset of the surge. If there was an LH surge without these criteria being met, the cycle was abandoned. Statistical Analysis Statistical analysis was performed using Wilcoxon's rank sum test, Fisher's exact probability test d 2 ' an the X test as nonparametric methods to ex- 510 Polson et al. GnRH-a in routine IVF patients Fertility and Sterility

3 amine relationships between groups. Probability value < 0.05 was considered significant. Down Regulation Phase RESULTS There was a similar time taken in both the buserelin acetate 600 and buserelin acetate 1,200 groups to achieve suppression of serum E 2 concentrations (buserelin acetate 600, 22 days [6 to 41]; buserelin acetate 1,200, 23 days [7 to 30]; median [range]). Serum LH and P concentrations were usually low (i.e., <10 IU/L and <3 nmol/l, respectively) within 2 weeks of commencing buserelin acetate, with the delay in E 2 suppression because of ovarian stimulation with the increase in endogenous gonadotropin release on commencing agonist therapy. Thirty-five of 56 buserelin acetate 600 cycles (62%) and 45 of 64 buserelin acetate 1,200 cycles (70%) showed an initial rise in serum E 2 concentrations to above 400 pmol/l. In both groups, the elevation was similar (buserelin acetate 600 1,325 pmol/l [425 to 6,050], buserelin acetate 1,200 1,465 pmol/l [410 to 5,800]; median [range]). This stimulation was not influenced by baseline serum LH or E 2 concentrations, which were similar for both groups. On the initial US examination, 61% of these women had one or more follicular cysts > 20 mm in diameter, which were presumably unruptured follicular cysts associated with ovarian stimulation because in 44 of these 49 cases there had been a rise in serum E 2 concentration. These cysts did not interfere with follicular growth during stimulation with hmg and were aspirated at the time of oocyte recovery. Stimulation Phase Table 1 summarises the number of completed cycles in each group and the indication for abandoning treatment. Women receiving hmg alone were significantly more likely to be discharged, usually because of a premature LH surge. Only 7 of the 120 (6%) buserelin acetate cycles were abandoned, and none were because of an LH surge. In four patients there was poor follicular development, two had elevated serum progesterone concentrations, and one woman stopped sniffing for social reasons. Both doses of buserelin acetate were associated with a significant increase in the duration and dosage of hmg required when compared with the hmg only group. Within the buserelin acetate group, women Table 1 Treatment Groups No. of women No. of cycles No. of abandoned cycles Serum E 2 < 1,600 pmol/l Premature LH surge Serum P > 7 nmol/l Self-discharge Buserelin acetate Buserelin acetate 1, HMG " 2 13 Buserelin acetate 600 and buserelin acetate 1,200 groups are significantly different from hmg group (P < 0.01). receiving 600 J.tg/d had a significantly shorter stimulation time and required less hmg than those receiving 1,200 J.tg/d. In neither group was there any correlation between the time taken to achieve suppression of serum E 2 concentrations and the subsequent stimulation phase with hmg, i.e., a long suppression period was not associated with the need for higher doses of hmg. Furthermore, follicular stimulation on commencing buserelin acetate did not influence the response to hmg therapy. The peak serum E 2 concentration before administration of hcg before oocyte recovery was significantly lower in both buserelin acetate groups compared with the hmg group (see Table 2). There was no relationship between the low E 2 concentration and the duration or dose of buserelin acetate therapy. Oocyte Recovery and Fertilization Rates A similar median number of follicles was aspirated in each group-9, 9, and 8 in the buserelin acetate 600, buserelin acetate 1,200, and hmg groups, respectively, with a similar range of between 2 and 29 follicles. There was also a similar median number of oocytes obtained-5 in the buserelin acetate 600 group (range 0 to 23), 6 in the buserelin acetate 1,200 group (O to 13), and 5 in the hmg group (O to 21). There was no significant difference in the fertilization rates between any of the groups (56.5%, 63.4%, and 65.1% for buserelin acetate 600, buserelin acetate 1,200, and hmg, respectively). Therefore, in this randomized study buserelin acetate did not improve the number of oocytes obtained compared with hmg alone, nor were the fertilization rates higher, suggesting similar oocyte quality. It was interesting to note that similar follicle and oocyte numbers were obtained from buserelin acetate cycles, despite a significantly lower peak serum E 2 concentration. Vol. 56, No.3, September 1991 Polson et al. GnRH-a in routine IVF patients 511

4 Table 2 Characteristics of the Three Groups Buserelin acetate 600 (n =52) Buserelin acetate 1,200 (n = 61) HMG (n = 41) Duration of hmg (d) Total dose of hmg (no. of ampules 75 IU FSH/75 IU LH) Peak serum E 2 concentration (pmol/l) (16 to 66)c 5,505 (355 to 13,210) 11a 30 (13 to 77)a 19 (11 to 35)b 5,870 (3,260 to 13,210) 7,340 (3,255 to 13,210)d a Buserelin acetate 600 significantly different from buserelin acetate 1,200 group (P < 0.05). b Buserelin acetate 600 and buserelin acetate 1,200 groups significantly different from hmg group (P < 0.001). c Values are medians with ranges in parentheses. d Buserelin acetate 600 and buserelin acetate 1,200 groups significantly different from hmg group (P < 0.01). There was a positive correlation between the E 2 level before oocyte recovery and the number of oocytes obtained for both groups: buserelin acetate (r = 0.32, P < 0.01; Spearman rank correlation) and hmg only (r = 0.38, P < 0.05). Outcome of Therapy Of the 52 cycles in women receiving buserelin acetate 600 resulting in oocyte recovery, there were 9 cycles without oocyte or ET because of a failure of fertilization, and in one cycle no oocytes were obtained. There were 31 cycles of IVF-ET, 10 GIFT, and 1 pronuclear stage transfer cycle. For women receiving buserelin acetate 1,200, no oocytes were obtained in 3 cycles and an additional 7 had no ET because of either a failure of fertilization (5 cycles) or abnormal embryonic development (2 cycles). This resulted in 51 completed cycles (35 IVF-ET, 14 GIFT, and 2 pronuclear stage transfer). In the hmg group, 5 of the 41 cycles did not proceed to oocyte/ ET because of no oocytes being obtained (3 cycles) or a failure of fertilization (2 cycles), leading to 26 IVF-ET and 10 GIFT cycles. Because GIFT and IVF have different PRs, which are dependent on the number of oocytes and embryos transferred, the pregnancy outcome for each group is classified accordingly (see Table 3). The pronuclear stage transfer cycles are included with the IVF results. Only clinical pregnancies are reported as defined earlier. For IVF-ET and pronuclear stage transfer cycles, the buserelin acetate 600 and buserelin acetate 1,200 groups had higher rates of 3 to 4 ETs than the hmg group (75% and 78% versus 58%). Despite this, there was a similar PR per ET for all groups. For GIFT cycles, there were no pregnancies in women receiving hmg only. This was significantly lower (P < 0.05; Fisher's test) than the PR in the buserelin acetate 1,200 group, although the numbers in each group were small. Considering both IVF and GIFT cycles and the various stimulation regimens, there was no difference in the overall clinical PR per transfer between the groups. However, if clinical pregnancies per treatment cycle commenced were compared, hmg stimulation achieved a significantly lower PR than women receiving buserelin acetate 1,200 (P < 0.05; Fisher's test). There was no difference in the PRs between the buserelin acetate 600 and buserelin acetate 1,200 groups. Sixteen of the 20 pregnancies with buserelin acetate had a peak serum E 2 concentration between 4,400 and 9,250 pmol/l with 1 < 3,600 pmol/l and 3 > 9,250 pmol/l. We have previously demonstrated that E 2 concentrations between 5,000 and 10,000 pmol/l in CC-hMG stimulation cycles are optimal for conception. 15 These similar results with buserelin acetate support the suggestion that the peak serum E 2 concentrations are important for the establishment and maintenance of a pregnancy. Table 3 Clinical PRs in Women Receiving Buserelin Acetate 600, Buserelin Acetate 1,200, or HMG Buserelin Buserelin acetate 600 acetate 1,200 HMG IVF 1 to 2 ET 0/8 1/8 1/11 3 to 4 ET 6/24 4/29 2/15 Total 6/32 5/37 3/26 (18.8)a (13.5) (11.5) GIFT 1 to 3 oocytes 0/2 0/0 0/2 4 to 6 oocytes 3/8 6/14 0/8 Total 3/10 6/14 0/10 (30) (42.8) (O)b IVF and GIFT clinical 9/42 11/51 3/36 PR/transfer (21.4) (21.6) (8.3) Clinical PR/cycle started 9/56 11/64 3/56 (16.1) (17.2) (5.4)b a Values in parentheses are percents. b Buserelin acetate 1,200 significantly different from hmg-only group (P < 0.05). 512 Polson et al. GnRH-a in routine IVF patients Fertility and Sterility

5 DISCUSSION Reports of improved follicular recruitment, oocyte quality, and PRs in women who fail to respond to CC-hMG stimulation by the use of GnRH-a have led some groups to suggest their routine use for all women undergoing ovarian stimulation for IVF-ET f GIFT. 4 5 Our results with buserelin acetate-hmg do confirm a higher PR per treatment cycle because of a significant reduction in the number of abandoned cycles rather than any improvement in folliculogenesis. Twenty-seven percent of the hmg cycles were abandoned. In all but two patients, this was because of an inappropriate LH surge at the time of a low follicle number and low serum E2 concentrations. No buserelin acetate cycles were canceled because of an LH surge, although two cycles were abandoned because of elevation of the serum P concentration. One of these cases was probably because of poor patient compliance and the second may have been related to the presence of a functional ovarian cyst (50 X 40mm) noted on her first US scan. Elevated serum P concentrations in the late follicular phase are associated with a poor IVF outcome, in contrast to a rise only in the follicular phase which does not influence PR Gonadotropin-releasing hormone agonists therapy clearly reduced the likelihood of a patient being discharged from treatment before oocyte recovery by prevention of an unwanted LH surge. Buserelin acetate ensured a higher percentage of patients proceeded to oocyte retrieval. We did not find buserelin acetate increased the number of oocytes obtained nor improved the quality of oocytes as suggested by their fertilization rate when compared with hmg alone. The same number of oocytes was obtained with a significantly lower serum E2 concentration. The reason for this is unclear but may reflect a direct action of buserelin acetate on follicle-stimulating hormone (FSH) action at the granulosa cell level or a reduction in FSH receptors on granulosa cells. It was not dependent on the duration of buserelin acetate therapy. However, serum E2 concentrations may be important for establishing pregnancy and Yamashita and associates15 have recently suggested serum E2 concentrations between 5,000 and 10,000 pmol/l were significantly more likely to result in pregnancy regardless of the number of embryos transferred. This may be because of improved endometrial receptivity, which has been suggested with GnRH-a therapy.18 The PR per ET was similar between the buserelin acetate and the hmg-only groups. Gamete intra- fallopian transfer treatment was more successful overall, although it remains unclear why there were no pregnancies in the hmg-only group. This may reflect a higher percentage of immature oocytes as previously reported with hmg stimulation19 or simply the relatively small numbers of patients in this study. The higher PR observed in the buserelin acetate group was at the expense of a significantly longer stimulation period and much higher hmg requirements. This may reflect the absence of endogenous gonadotropin release, although another theoretical consideration may be a reduction in growth hormone (GH) release from the pituitary. Recent results from in vitro studies have confirmed that GH has a facilitatory action on FSH-induced granulosa cell activity, acting either directly on granulosa cells20 or mediated through increased production of insulinlike growth factors In vivo work has also suggested that GH may play a role in folliculogenesis. 23 In addition to the significantly higher cost of hmg when used with buserelin acetate in this study, there is also the additional cost of the GnRH -a. We could find very little data on dose-response testing of GnRH-a in clinical IVF. This study shows that a dose of 600 f.lg/ d of buserelin acetate achieved satisfactory pituitary suppression and that higher doses are unnecessary. We would conclude that buserelin acetate-hmg appears to offer an advantage to hmg alone for IVF superovulation by the elimination of unwanted LH surges. Randomized trials of CC-hMG and buserelin acetate-hmg in endocrine-normal IVF patients undergoing their first or subsequent IVF cycle should now be considered. For GnRH -a to have a place in the routine management of IVF subjects, clinical advantages or improved ongoing PRs will need to be proven. For women in whom there is a premature LH surge or a failure of adequate folliculogenesis with CC-hMG, we would recommend the use ofbuserelin acetate 600 f.lg/ d to achieve ovarian suppression before hmg stimulation. Acknowledgments. The authors gratefully thank the clinical, secretarial, and laboratory staff of the Infertility Medical Centre for their assistance and Takanori Yamashita, M.D., for statistical advice. REFERENCES 1. Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS: Induction of ovulation for in-vitro fertilisation using buserelin and gonadotropins. Lancet 2:1284, 1984 Vol. 56, No.3, September 1991 Polson et al. GnRH-a in routine IVF patients 513

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