The long-acting gonadotropin-releasing hormone analogues impaired the implantation rate*

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1 FERTILITY AND STERILITY Copyright c 1996 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. The long-acting gonadotropin-releasing hormone analogues impaired the implantation rate* Fabienne Devreker, M.D.t* Isabelle Govaerts, M.D.t Evelyne Bertrand, B.S. t Marc Van den Bergh, M.T.t Catherine Gervy, Dr.Sc~ Yvon Englert, Ph.D.t Free University of Brussels, Brussels, Belgium Objectives: To determine the efficacy and innocuousness of long-acting versus short-acting GnRH analogues (GnRH-a) in long protocol for in IVF-ET. Design: Prospective randomized study. Setting: The IVF unit at an academic hospital. Patients: One hundred couples admitted for their first IVF-ET attempt. Main Outcome Measures: Serum concentrations of LH, E 2, and P during the all cycles and duration of pituitary desensitization were assessed, as well as fertilization rate, embryo quality, and implantation and pregnancy rates. Results: Significantly more days (10.8 ± 1.8 versus 9.2 ± 1.7 days) of stimulation and more ampules of hmg (47 ± 22 versus 33 ± 16) were necessary to obtain similar numbers of embryos of quality with the long-acting GnRH-a. Implantation and delivery rates were significantly lower with the long-acting GnRH-a (32.8% versus 21.1%; 48.9% versus 29.1 %, respectively). Conclusions: As the long-acting GnRH-a might interfere with the luteal phase and embryo development, short-acting GnRH-a should be preferred for ovarian hyperstimulation in IVF- ET. Fertil Steril 1996;65:122-6 Key Words: Long-acting gonadotropin releasing-hormone analogue, implantation rates, IVF-ET Gonadotropin-releasing hormone analogues (GnRH-a) are used widely in association with hmg for controlled ovarian stimulation before IVF-ET cycles. Little work has been done comparing short-acting buserelin acetate with long-acting D-triptoreline in long protocol, and the results in terms of implantation and pregnancy rates are conflicting (1,2) (Demoulin A, abstract). Because one injection of a long-acting GnRH is more Received November 21,1994; revised and accepted August 17, * Presented at the X Congress of European Society of Human Reproduction and Embryology (ESHRE), Brussels, Belgium June t Department of Obstetrics and Gynecology, Fertility Unit, Erasme Hospital. * Supported by a grant from the Royal Society, London, United Kingdom. Reprint requests: Fabienne Devreker, M.D., Department of Obstetrics and Gynecology, Fertility Unit, Route de Lennik, 808, Brussels 1070, Belgium (FAX: ). ~ Department of Biological Medicine. 122 Devreker et al. Lower implantation rates with LA-Dtrp convenient for the patients than a daily administration of a short-acting one, it is important to know if the longacting D-triptoreline might have adverse effects during the luteal phase and the early pregnancy. To answer this question, this prospective randomized, cross-over, i and open study compared two GnRH-a in long protocol, for patients admitted for their first IVF attempts. MATERIALS AND METHODS After they had given their informed consent, patients starting their first IVF attempt were allocated prospectively and randomly among the following four groups for two cycles of treatment: group A, buserelin acetate and long-acting D-triptoreline; group B, long-acting D-triptoreline and buserelin acetate both with the analogues started during the luteal phase; group C, buserelin acetate and longacting D-triptoreline; and group D, long-acting D-triptoreline and buserelin acetate both with the analogues started on the first day of menstruation. Patients were allowed to start the treatment if P

2 was ~5 nglml (15.9 nmol/l) in the blood test performed on the day before. Women who were aged ~ 38 years, oocyte donors, and cycles requiring micromanipulation were excluded from this study. The mean age of the women was similar among the different groups (32.8 ::t 0.7 years), as was the mean duration of infertility (4.8 ::t 0.9 years). The different etiologies for the infertility also were distributed evenly. Buserelin acetate was administered by intranasal spray: 300 f,.lg/d three times and D triptoreline by one injection of3.75 mg 1M until complete pituitary desensitization was achieved (E2 < 50 pg/ml [< 183 pmol/lj), with no follicular growth observed by ultrasound (US). When ovarian cysts were observed (diameter > 20 mm) they were punctured after the pituitary desensitization before stimulation with hmg. Ovarian stimulation was started with two ampules per day of hmg, after which the dose was modified according to the ovarian response monitored by blood tests and vaginal US. Ovulation was induced by injection of 10,000 UI hcg. Routine IVF was performed as described previously (3). Forty-four to 48 hours after insemination the embryos obtained were scored under an inverted microscope. A numerical score was calculated on the basis of embryo morphology and cleavage rate (4): four points were given for an embryo with regular blastomeres and no anucleate fragments; three points were given for an embryo with irregular blastomeres, and one or two anucleate fragments; two or one points were given for an embryo with anucleate fragments over respectively ::s;~ or ~~, respectively, of the embryonic surface. Two more points were added if the embryo had reached the four-cell stage 44 hours after. After transfer of between two or three embryos of the highest score on day 2 after oocyte retrieval, luteal support was maintained by injections of 100 mg 1M oily P or with intravaginal pessaries (three times 200 mg/d of micronized P). The luteal phase was assessed through the E2:P ratio (5). Clinical pregnancy was defined either by a fetal sac at 5 weeks after oocyte retrieval by US or by villosity present in miscarriage material. An ongoing pregnancy was defined as one pregnancy that continues to develop after 20 weeks of gestation. The total number of fetal sacs divided by the total number of embryos transferred characterized the implantation rate. Statistical evaluation was effected by applying analysis of variance, X 2 test, or by the Fisher's exact probability test when necessary. The results were expressed as means ::t SEM. Only significant differences were reported. RESULTS One hundred couples underwent 133 cycles of treatment with 100 first cycles and 33 second cycles. Table 1 Comparison of Superovulation, Fertilization, and ET Mter Down-Regulation With Buserelin Acetate or Long-Acting D-Triptoreline No. of attempts No. of cycles cancelled* No. of cycles with pituitary desensitization> 15 days No. of ovarian cysts of stimulation:j: No. of hmg ampules E2 maximum No. of retrievals No. of oocytes/oocyte pick-up Fertilization rate (%) No. of embryos/oocyte pick-up Mean score embryos No. of ET per transfer Mean score ET Buserelin acetate (10) :':: :':: 16 2,556:':: :':: :':: :':: :':: :':: 0.9 * Values in parentheses are percentages. t p < :j:p < Cancelled cycles excluded. Long-acting D-triptoreline (9) 8t 2t 10.8 :':: 1.8:j: 47:':: 22:j: 2,808 :':: 1, :':: :':: :':: :':: :':: 1.1 Sixty-seven couples did not undergo a second treatment cycle either because they became pregnant during the first cycle or because they elected not to return for further treatment. The day of the cycle at which pituitary down-regulation was initiated, whether with buserelin acetate or long-acting D triptoreline, had no effect on the stimulation or IVF outcome. The number of ampules ofhmg (8.9 ::t 1.3 versus 9.7 ::t 2.1), days of stimulation (31 ::t 14 versus 35 ::t 18 days), and the maximum E2 levels (2,465 ::t 1,045 [9,046 ::t 3,835 pmol/l] versus 2,671 ::t 913 [9,803::t 3,351 pmolimlj) were similar between buserelin acetate started on day 22 or day 1. Similarly, the number of ampules of hmg (10.8 ::t 1.9 versus 10.7 ::t 1.6), days of stimulation (47 ::t 25 versus 47 ::t 21 days), and the maximum E 2 level (2,877 ::t 1,358 [10,559 ::t 4,984 pmoliml] versus 2,734 ::t 1,181 [10,034 ::t 4,334 pmolimlj) were similar between long-acting D-triptoreline started on day 22 or day 1. The implantation rates were similar with buserelin acetate started on day 22 or day 1 of the cycle (27% and 40%, not significant). However, the implantation rates with long-acting D-triptoreline, although similar when started on day 22 or day 1 (20.6% and 20.4%, respectively), were lower compared with buserelin acetate. Thus, the data for all the patients treated with buserelin acetate, irrespective of when they started treatment, were pooled, as were the data for patients treated with long-acting D-triptoreline (Table 1). The number of cancelled cycles because of poor ovarian response was similar between the two groups (Table 1). The duration of the pituitary de- Devreker et al. Lower implantation rates with LA-Dtrp 123

3 sensitization was significantly longer with buserelin acetate than with long-acting D-triptoreline (P < 0.05). More than 15 days were necessary for 28 cycles, with a maximum of 28 days for two of them. More ovarian cysts were also observed with bus erelin acetate (P < 0.05). Although pituitary desensitization was achieved more rapidly with the long-acting D-triptoreline, a significantly longer period of stimulation and a higher dose ofhmg was necessary to achieve similar levels of E2 and to obtain similar number of follicles, oocytes, and embryos as those obtain with buserelin acetate. The quality of these embryos also was similar between the two groups. The hormone profiles during the follicular phases are illustrated in Figure 1. In the group treated with buserelin acetate, the E2 level was significantly higher than with long-acting D-triptoreline 10 days after starting the analogue, 71 :±: 10 versus 38 :±: 3 pg/ml (260 :±: 37 versus 139 :±: 11 pmolll), P < On the day ofhcg injection, the E 2 level was significantly higher for patients with long-acting D-triptoreline than with buserelin acetate, 2,585 :±: 176 versus 2,169 :±: 122 pg/ml (9,461 :±: 644 versus 7,939 :±: 447 pmolll) (P < 0.05). Luteinizing hormone levels were higher throughout the stimulation with buserelin acetate compared with long-acting D-triptoreline, significantly 3 days (3.96 :±: 0.23 versus 3.15 :±: 0.05 IU/mL, P < 0.001) and 2 days (4.14 :±: 0.39 versus 3.11 :±: 0.04 IU/mL, P < 0.01) before the oocyte puncture. However, the levels ofp were similar with the two analogues. During the luteal phases, no differences could be found for the E 2:P ratio between the two groups. Although the embryos were similar both in number and quality, the implantation and the delivery rates were significantly lower with long-acting D triptoreline than with buserelin acetate (P < 0.02 and P < 0.05, respectively). The pregnancy rate (PR) by transfer (Table 2) tended also to be lower with the long-acting D-triptoreline (P < 0.053). When the results were pooled by cycle day, no difference in the implantation rates were observed between the patients treated on day 1 or on day 22 (30.3% versus 23.5%, respectively). DISCUSSION Several different GnRH-a are now available for pituitary desensitization before superovulation and by IVF. The long-acting forms are more convenient for the patients because usually one injection of the analogue is sufficient for one cycle of treatment. But this raises the problem of the persistent release of the GnRH-a in the circulation during the luteal phase and early pregnancy. All previous studies (1, 2,6) (Demoulin A, abstract) concur to say that with 124 Devreker et al. Lower implantation rates with LA-Dtrp UI/mL 4,5 4 3,5 2,5 1,5 0,5 pg/ml ng/ml 4,5 4 3,5 2,5 1,5 0, r----,-----r----~--~ o HCG HCG 1 (A) (B) Figure 1 Hormonal profiles during the follicular phase according to the administration of the GnRH-a buserelin acetate versus long-acting D-triptoreline. Mean ± SEM concentrations of (A) LH, (B) E 2, and (C) P. 0, long-acting D-triptoreline; 0, buserelin acetate. *p < 0.05; **p < 0.01; ***p < long-acting analogues the period of pituitary desensitization was shorter but that more days of stimulation and more ampules ofhmg were necessary compared with the short-acting forms. Despite this, the number of follicles punctured as well as the number and the quality of embryos available were similar between the cycles treated by the long- or short-act- (C)

4 Table 2 Pregnancy Outcome Mter Down-Regulation With Buserelin Acetate or Long-Acting D-Triptorelin No. of transfers No. of clinical pregnancies PRioocyte pick-up (%) PRitransfer (%) Delivery rate/transfer (%) Implantation rate (%) * p < t p < Buserelin acetate * 59.2* 49* 32.8t Long-acting D-triptorelin ing GnRH-a. This study here confirms that significantly more days of stimulation and more ampules ofhmg are required with the long-acting D-triptoreline to obtain a similar number of embryos of similar quality compared with the short-acting buserelin acetate. However, differences have been found regarding pregnancy and implantation rates. Two series (6) (Demoulin A, abstract) did not observe any differences between the two treatments for the PR or for the number of miscarriages. Zullo et al (Zullo F, Colacurci N, Perrone D, Galosso M, Armanek E, De Placido G, abstract) observed higher implantation and pregnancy rates (although not significantly), over a large number of patients, with the long-acting D-triptoreline compared with short-acting buserelin acetate. However, the low implantation rates observed in this study would have distorted the interpretation of the results. In contrast, Gonen et al (2) observed in his study a significantly lower pregnancy rate and a significantly higher number of miscarriages with the longacting D-triptoreline than with the short-acting buserelin acetate. In the present study, the implantation and the pregnancy rates were also significantly lower with the long-acting D-triptoreline (P < 0.03) than with buserelin acetate. However, the number of miscarriages was similar in the two groups. As the implantation rate is related to both embryo viability and the endometrium, the GnRH-a could interfere with the embryo quality, the endometrium receptivity, or both. Several hypotheses can be proposed to explain the differences observed in this study. The more pronounced inhibition of the ovarian function during the follicular phase due to the administration of long-acting analogues may have impaired both oocyte quality and the function of the corpus luteum. However, similar fertilization rates along with number and comparable quality of the embryos suggest that the oocyte quality was not impaired by the use of the long-acting analogue. On the other hand, the existence of GnRH receptors in the ovary has been demonstrated (7). Moreover, it has been proved (5,8) that analogues diffuse into the follicular fluid (FF) and that small amounts of buserelin acetate are still detectable in the FF 35 hours after the last intranasal administration (8). It can be supposed that this is also true for the longacting D-triptoreline, differing from buserelin acetate by the absence of the GLY 6 substitution. This difference in the structure ofthe two molecules could be responsible for differences in the pharmacokinetic and biologic activities (9). With long-acting GnRH-a, persistent amounts of molecules are released in the blood circulation far beyond the injection ofhcg and oocyte retrieval The follicular concentration of the D-triptoreline may be higher than with buserelin acetate, exerting a more significant inhibition on granulosa-cells during the follicular phase, resulting in the requirement of higher doses ofhmg for stimulation. Moreover, the presence of the GnRH-a in the FF during oocyte maturation could interfere with embryonic metabolism, impairing the potential of the future embryo to implant and giving rise to abnormalities that do not manifest themselves as morphological abnormalities of the embryo. Differences in metabolism and embryo quality recently have been shown after superovulation with different doses of hmg (10). These alterations of the embryonic metabolism could also explain the lower postsurvival and implantation rate achieved by the supernumerary frozen-thawed embryos obtained from cycles treated with GnRH-a (9, 11). Although previous studies (12, 13) of the inadvertent administration of short-acting GnRH-a during early pregnancy did not observe any specific teratogenicity or morphological abnormalities in the fetuses or neonates, the analogues were administrated after the period of implantation. However, one study (14) observed a very low rate of ongoing pregnancy. By their inhibition on the ovarian function, the long-acting analogue may impair the function of the corpus luteum. Gonadotropin-releasing hormone analogues have been shown to reduced the secretion of P during the early luteal phase by their inhibiting steroidogenesis in granulosa-cells (15) (Zullo F, abstract). A lower level ofp may prevent adequate maturation of the endometrium for implantation of the embryos. The administration of luteal support to patients undergoing both treatments may conceal differences in E 2:P ratio between the two groups (buserelin acetate and long-acting D-triptoreline). Alternatively, the persistent release of GnRH-a during the luteal phase may reduce the efficiency of the luteal support and decrease the implantation rate. It would appear, then, that both the degree of ovarian inhibition and the presence of the analogue through- Devreker et at. Lower implantation rates with LA Dtrp 125

5 out the oocyte maturation and the process of imp lantation may be responsible for the impaired implantation and pregnancy rates observed with the longacting D-triptoreline. Long-acting GnRH-a requiring a single injection certainly are more convenient for the patients, but they prolong the period of stimulation and increase the number of ampules, increasing the cost of one attempt compared with short-acting analogues. Moreover the implantation and pregnancy rates are impaired by the long acting GnRH-a. Embryos resulting from long-acting GnRH down-regulated cycles could be replaced, after cryopreservation or oocyte donation during cycles without GnRH-a. This would provide information on the effect of long-acting GnRH-a on embryo quality and uterine receptivity. Until these studies have been achieved, the short-acting analogues should be preferred for the treatment of ovarian stimulation in IVF. Acknowledgment. We thank Kate Hardy, Ph.D., for her help in writing this manuscript. D-Triptorelin was provided by Ipsen Co., Gent, Belgium. REFERENCES 1. Gianaroli L, Ferraretti AP, Feliciani E, Tabanelli C, Magli C, Fortini D. Prospective randomized study of protocols for medically assisted conception cycles. Hum Reprod 1994; 9: Gonen Y, Dirnfeld M, Goldman S, Koifman M, Abramovici H. The use of long-acting gonadotropin-releasing hormone agonist (GnRH-a; Decapeptyl) and gonadotropins versus short-acting GnRH-a (Buserelin) and gonadotropins before and during ovarian stimulation for in vitro fertilization (NF). J In Vitro Embryo Transf 1991;8: Englert Y, Van den Bergh M, Rodesch C, Bertrand E, Biramane J, Legreve A. Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Hum Reprod 1992; 7: Puissant F, Van Rysselberge M, Barlow P, Deweze J, Leroy F. Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod 1987;2: Smitz J, Ron-EI R, Tarlatzis BC. The use of gonadotrophin releasing hormone agonists for in vitro fertilization and other assisted procreation techniques: experience from three centres. Hum Reprod 1992;7: Porcu E, Dal Prato L, Seracchioli R, Fabbri R, Longhi M, Flamigni C. Comparison between depot and standard release triptoreline in in vitro fertilization: pituitary sensitivity, luteal function, pregnancy outcome, and perinatal results. Fertil Steril 1994;62: Latouche J, Crumeyrolle-Arias M, Jordan D, Kopp N, Augendre-Ferrante B, Cedard L, et al. GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study. Endocrinology 1989; 125: Loumaye E, Coen G, Pampfer S, Vankrieken L, Thomas K. Use of a gonadotropin-releasing hormone agonist during ovarian stimulation leads to significant concentrations of peptide in follicular fluids. Fertil Steril 1989; 52: Testart J, Lefevre B, Gougeon A. Effects of gonadotrophinreleasing hormone agonists (GnRHa) on follicle and oocyte quality. Hum Reprod 1993;8: Hardy K, Robinson FM, Paraschos T, Wicks R, Franks S, Winston RML. Normal development and metabolic activity of preimplantation embryos in vitro from patients with polycystic ovaries. Hum Reprod 1995;10: Oehninger S, Toner JP, Veeck LL, Brzyski RG, Acosta AA, Muasher SJ. Performance of cryopreserved pre-embryos obtained in in vitro fertilization cycles with or without a gonadotropin-releasing hormone agonist. Fertil Steril 1992;57: Wilshire GB, Emmi AM, Gagliardi CC, Weiss G. Gonadotrophin-releasing hormone agonist administration in early human pregnancy is associated with normal outcomes. Fertil Steril 1993;60: Young DC, Snabes MC, Poindexter AN. GnRH agonist exposure during the first trimester of pregnancy. Obstet Gynecol 1993;81: Isherwood PJ, Ibrahim ZMZ, Matson PL, Moroll DR, Burslem RW, Lieberman BA. Endocrine changes in women conceiving during treatment with an LHRH agonist. Hum Reprod 1990; 4: Testart J, Forman R, Belaisch-Allart J, Volante M, Hazout A, Strubb N, et al. Embryo quality and uterine receptivity in in-vitro fertilization cycles with or without agonists of gonadotrophin-releasing hormone. Hum Reprod 1989;4: Devreker et al. Lower implantation rates with LA-Dtrp