Use of buserelin acetate in an in vitro fertilization program: a comparison with classical clomiphene citrate-human menopausal gonadotropin treatment

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1 FERTILITY AND STERILITY Copyright'" 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Use of buserelin acetate in an in vitro fertilization program: a comparison with classical clomiphene citrate-human menopausal gonadotropin treatment Bernard Lejeune, M.D., Ph.D. * Patricia Barlow, M.D. Francoise Puissant, M.D. Annick Delvigne, M.D. Michel Vanrysselberge, M.D. Fernand Leroy, M.D., Ph.D. IVF Clinic, Saint-Pierre Hospital, Free University of Brussels, Brussels, Belgium A comparison has been established retrospectively between clomiphene citrate-human menopausal gonadotropin (CC-hMG) and buserelin acetate-hmg treatments in in vitro fertilization trials performed over a 3-year period. The analysis of 466 CC-hMG and 319 buserelin acetate-hmg trials shows that buserelin acetate-hmg stimulation generates a greater ovarian response resulting in higher numbers of oocytes being retrieved ( versus ) and fertilized ( versus ). More embryos are thus obtained, allowing a wider choice for intrauterine replacement and cryopreservation. Mean embryonic vitality scores do not differ ( versus ), implying that the embryonic quality remains similar in both treatments. A premature demise of the corpus luteum occurs in a large proportion of bus ere lin acetate-hmg cycles. However, when suppletive progesterone treatment is given, there is a trend toward a better implantation rate per embryo, and a significantly higher ongoing pregnancy rate is observed in relation to buserelin acetate-hmg treatment (20%) as compared with CC-hMG cycles (14%). Fertil Steril54:475, 1990 Analogs of the gonadotropin-releasing hormone (GnRH-a) have been used in ovarian stimulation since The rationale for using these compounds is based on the observation that high basal levels or an untimed surge of the luteinizing hormone (LH) during the follicular phase, lead to premature luteinization and impairment of normal oocyte maturation. 4 Moreover, these modifications are associated with reduced fertilization rates,5 frequent fertilization failure,6 and a lower ongoing pregnancy rate after in vitro fertilization (IVF) and embryo transfer. 7 Previous studies dealing with small numbers of patients 8-10 highlighted the relevance of inhibiting gonadotropin secretion by GnRH -a, especially in cases showing abnormal LH Received December 13, 1989; revised and accepted May 17, * Reprint requests: Bernard Lejeune, M.D., Ph.D., IVF Clinic, Department of Gynecology and Obstetrics, Saint-Pierre Hospital, 322, rue Haute, 1000, Brussels, Belgium. levels during the follicular phase and in "poor responders.,,11-12 The aim of this study was to compare the efficiency of an easy-to-manage buserelin acetate-human menopausal gonadotropin (hmg) treatment with the classical clomiphene citrate (CC)-hMG treatment in clinical use. MATERIALS AND METHODS Our present study is based on a 3-year survey of GnRH-a (buserelin acetate) use in our IVF program and gives a retrospective comparison between the results obtained either with buserelin acetatehmg or with classical CC-hMG stimulation over the same period. From January 1986 to December 1988, 466 CC-hMG treatments and 319 buserelin acetate-hmg stimulations were performed. Infertile couples attending our IVF clinic were classified into four etiological categories: (1) pure tubal factors, (2) idiopathic cases, (3) male factors, and (4) mixed infertility. For each patient, trials were clas- Lejeune et al. Use of buserelin acetate in IVF 475

2 Figure 1 I I Ici. ldqmg/d I hmg IU~ a tcg IU t! Itt t t, E, (bid) t I!!, us (b/d) tutti LH (6x/d) I hcg IU ' hmg IU /d 4 x 200 ~g/d " ! t t t! t, E, us (hid) Schedules of ovarian stimulation and monitoring: (a) with clomiphene + hmg and (b) with buserelin acetate + hmg. E z, estradiol; US, ultrasound examination. sified according to their chronological rank, trial rank 1 being the first IVF attempt performed in the couple. Ovarian stimulation associated with pituitary desensitization was initially reserved for patients in whom abnormal LH secretion had been evidenced and for patients with "occult ovarian failure" or so-called "poor responders." For practical reasons, GnRH-a-hMG treatment was then extended to patients living far from the hospital and was eventually applied to all patients because its use was more convenient for both patients and medical staff. Patients in the buserelin acetatehmg group were thus treated on average at a later stage during the survey period than patients in the CC-hMG group. Patients treated with CC-hMG received 100 mg CC/d (Clomid; Merrel, Brussels, Belgium) from days 3 to 7 of the menstrual cycle and 150 to 300 IV hmg (Humegon; Organon, Oss, The Netherlands or Pergonal; Serono, Milano, Italy) from day 7 until follicular maturation was reached (Fig. 1). In patients treated with pituitary desensitization, buserelin acetate administration was started on the 1st day of menstruation. Two hundred micro- " grams were given intranasally four times a day at regular intervals until human chorionic gonadotropin (hcg) injection. Human menopausal gonadotropin (150 to 300 IV/d) was injected from day 5 until follicular maturation was reached (Fig. 1). Preliminary studies had established that the opti- b mal frequency for intranasal administration of buserelin acetate was 4 times a day at a dosage of 200 Ilg by spray. Indeed, with three applications per day, several cases of premature LH peak (8%) were still observed. With both treatments, estradiol (E2) and follicular size were monitored daily from day 7. Moreover, in CC-hMG cycles, the patients were admitted to the hospital from day 10 onward, and serum LH determinations were then performed every 4 hours with LH Coatria (Biomerieux, Charbonnieres les Bains, France) to detect the onset of an LH surge. By contrast, patients treated with buserelin acetate-hmg were hospitalized only the evening before oocyte pickup. Ten thousand IV hcg (Pregnyl; Organon) were injected when at least three follicles showed a mean diameter> 18 mm and with a serum E2 level> 300 pg.ml-1.follicle-1 > 16 mm or else as soon as a spontaneous LH surge was observed. Cycles were canceled if a LH peak appeared with a follicular diameter < 18 mm (untimed LH surge) and/or if the E2 level was <900 pg/ml, or if the number of follicles was <3 (insufficient ovarian response). Oocytes were retrieved by laparoscopy or by ultrasonically guided transvaginal puncture. Oocytes were inseminated after retrieval with a sperm suspension obtained by swim-up preparation at a concentration of approximately 50,000 motile spermatozoa/ml. Embryo culture was performed in Whittingham's T6 medium supplemented with 10% maternal serum. Embryo quality was assessed using the score developed by Puissant et al.13 Four points were allocated to embryos with clear, regular blastomeres without anucleate fragments, two points to embryos with anucleate fragments covering no more than l of the embryonic surface, and one point to embryos with anucleate fragments covering> l of the embryonic surface. Two points were added when embryos had reached stage IV within the first 48 hours. When available, up to three embryos were replaced 48 hours after insemination through transcervical route, the remaining embryos being frozen for possible replacement in a subsequent spontaneous cycle. Preliminary studies by our group14 and by others 9 have shown that major luteal phase defects are often observed after buserelin acetate administration. Luteal support thus seemed mandatory, and in almost all buserelin acetate-hmg trials with embryo replacement, the luteal phase was supported by a daily intramuscular injection of 100 mg pro- r! 476 Lejeune et al. Use of buserelin acetate in IVF Fertility and Sterility

3 Table 1 Characteristics of the Enrolled Cycles acetate- CC-hMG hmg Probability Enrolled cycles Canceled cycles 82 (15)a 28 (8) <0.001 Oocyte pickup Age 32.2 ± 4.2b 32.2 ± 4.3 b ' Indications Tubal 255 (55) 151 (47) Idiopathic 96 (21) 66 (21) Male 76 (16) 59 (18) Mixed 39 (8) 43 (14) Trial rank Rank (56) 145 (46) Rank (26) 78 (24) Rank 3 51 (11) 46 (14) <0.001 Rank (7) 50 (16) No. of days of ovarian stimulation 9.5 ± l.ob 11.9 ± 2.6b <0.001 No. of hmg ampullae (75 IV) 16.4 ± 9.0b 26.9 ± 13.2b <0.001 Mode of oocyte pickup Laparoscopy 168 (36) 12 (4) Echoguided 298 (64) 307 (96) <0.001 Luteal support Unsupported 362 (78) 71 (22) Supported 104 (22) 248 (78) <0.001 a Values in parentheses are percentages per pickup except the cancellation rate, which is per enrolled cycles. b Values are means ± SEM.,, not significant. gesterone (P) in oil from the replacement day onward. By contrast, CC-hMG cycles were only supplemented when an abnormal luteal phase had been evidenced either in the endocrinologic evaluation performed before IVF enrollment or during a previous IVF trial because only these patients have been shown to benefit from luteal support. 14 Serum E2 and P were determined on day 6, day 10, day 14, and day 17 after oocyte pickup using E2 and P Coatria radioimmunoassays (RIAs, Biomerieux). Human chorionic gonadotropin levels were measured with a highly specific monoclonal RIA (Hybritech, Liege, Belgium). Statistical analysis of the results was performed using X 2, Student's t-test or Mann-Whitney U-test as appropriate, with the SPSS V3.0 program (SPSS Inc., Chicago, IL) running on an IBM PS-2j50Z computer (International Business Machines, Brussels, Belgium). RESULTS Patient and treatment characteristics are given in Table 1. Age distribution and indications for IVF treatment were similar. A somewhat higher, but not significantly different, proportion of mixed in- dications (i.e., male plus tubal factors) was observed in the buserelin acetate group. In the distribution of trial ranks, a higher proportion of trials belonging to ranks over four (18% versus 8%) was evident in the buserelin acetate group (P < 0.001). The total amount of hmg given per treatment cycle was higher in the buserelin acetate group. The mean number of days of hmg administration required for adequate ovarian stimulation was increased (P < 0.001), and with the applied schedule of buserelin acetate treatment, oocyte pickup took place on average on day 15.7 (±3.1), compared with day 13.3 (±1.9) in CC-hMG cycles. The cancellation rate was significantly reduced (P < 0.001). Because in recent years, oocyte pickup has evolved from the laparoscopic to the echoguided approach, distribution of the oocyte retrieval mode differs between groups. Whereas 65% of pickups were echoguided in the CC-hMG group, this procedure was used in 96% of cases in the buserelin acetate-hmg group. Ovarian response was greater in the buserelin acetate group. The E2 peak was higher; the numbers of retrieved oocytes, of obtained zygotes, and of replaced and frozen embryos were all significantly increased. The fertilization rate was slightly but significantly elevated (Table 2). The drop in E 2levels occurring after the preovulatory peak was significantly greater in the buserelin acetate group, whereas P rose earlier and more sharply, thus reducing the E2:P ratio on the 2nd day of the luteal phase significantly (Table 3, Fig. 2). By contrast, in the late luteal phase of unsupplemented cycles, E2 and P were lower on day 10 in the buserelin acetate group (Fig. 2). Embryonic vitality scores, evaluated according to the method applied by Puissant et al.,13 did not show any significant difference between the groups Table 2 Ovarian Response in Relation to Stimulation Type Prob- CC-hMG acetate-hmg ability No E 2 peak (pgjml) 2,197 ± 978 a 3,054 ± 1,489 <0.001 No.oocytes 6.2 ± ± 5.2 <0.001 Fertilization rate (%) <0.01 No. fertilized oocytes 2.76 ± ± 3.84 <0.001 No. replaced embryos 1.8 ± l ± 1.2 <0.001 No. frozen embryos 0.4 ± ± 2.1 <0.003 Cycles with frozen embryos 53 (l1)b 60 (19)b <0.01 a Values are means ± SD. b Values in parentheses are percents. Lejeune et a1. Use of buserelin acetate in IVF 477

4 r! Table 3 Ovarian Hormone Levels in the Luteal Phase in Relation to Stimulation Type and P Supplementation a CC-hMG acetate-hmg Probability (E2 peak - E2 nadir)/e2 peak E 2:Pon day 2 Unsupplemented cycles Supplemented cycles E2 on day 10 (pregnancies excluded) (pg/ml) Unsupplemented cycles P on day 10 (pregnancies excluded) (ng/ml) Unsupplemented cycles a Values are means ± SD ± ± 0.10 < ± ± ± ± <0.01 < ± ±47 < ± ± 7.1 b, not significant. (Table 4). The gross implantation rate, calculated as the overall number of pregnancies per number of embryos replaced, was identical in both groups, but the "successful" implantation rate, calculated as the number of babies born per number of embryos replaced was slightly higher in the buserelin acetate group. It should be noted that among pregnant patients, no difference was observed between the groups in terms of successful implantation rate (Table 4). Data on the outcome of the trials are given in Table 5. Ongoing pregnancies were significantly more numerous in the buserelin acetate group, whereas the multiple pregnancy rate remained similar to that observed in the CC-hMG group. DISCUSSION Our first 40 trials treated with buserelin acetate were managed according to the long protocol developed by Fleming and Coutts. 2 They are not in- cluded in the present study. After a few months, we switched over to a shorter protocol in which buserelin acetate was given from the onset of menstruation. The reasons for abandoning the long protocol were: (1) the cost of the GnRH-a; (2) the problem of menstruation predictability in oligoanovulatory women; and (3) the potential interference with an incipient spontaneous pregnancy. Two cases of buserelin acetate administration during early pregnancy were indeed observed, fortunately without adverse effects on the outcome of these pregnancies. The management of cycles with buserelin acetate-hmg is simpler than with CC-hMG: no LH peak is expected so that no serial LH determinations are necessary. Only one LH determination per day was performed simultaneously with the daily E2 determinl;ltion and the echographic measurements. The hospitalization could therefore be reduced from 7 (in the CC-hMG group) to 2 days (in the buserelin acetate-hmg group) and oocyte E2 P9/ml /:,,. //.' Unsupplemenled cycles i ~ _---% EPR x ~ T 20! 10 P ng/ml fij--: /. 30,/! a CI-hMG.. 8-hMG / ':::-::: Cases supplemented with 100mg P 1M. ~. '. ~io'le : 0 -, b p ng/ml ''''1. CI-hMG 80 " I.. 8-hMG I I 60 i I,/ 'I. V'<..~ Figure 2 Evolution of estradiol (E2) and progesterone (P) levels during the luteal phase of nons upplemented (a) and supplemented (b) cycles with daily P treatment. EPR, E 2:P ratio; shaded areas, range of EPR values in normal, nonstimulated cycles on day 2. *, P < a, Cl-hMG, 13 cases; B-hMG, 6 cases. b, Cl-hMG, 21 cases; B-hMG, 146 cases. 478 Lejeune et al. Use of buserelin acetate in IVF Fertility and Sterility

5 Table 4 Embryonic Vitality Scores and Implantation Rates in Relation to Ovarian Stimulation Type a CC-hMG acetate-hmg Probability Mean embryonic score of all embryos obtained Mean embryonic score of replaced embryos Implantation rate (incipient pregnancies/no. of embryos replaced) Ongoing implantation rate (ongoing pregnancies/no. of embryos replaced) Ongoing implantation rate for pregnant patients only a Values are means ± SD. pickup could be programmed during office hours. Cancellation of a trial as a result of elevated basal LH levels or the premature onset of an LH peak was avoided, and only cycles with low E2 levels (8%) needed to be canceled, whereas 15% of the CC-hMG cycles were canceled for either premature LH rise or low E2 levels. In terms of ongoing pregnancies, ovarian stimulation performed with hmg during pituitary desensitization treatment with the GnRH-a buserelin acetate appears to yield better results than classical CC-hMG treatment for IVF. We observed a 20% ongoing pregnancy rate by oocyte pickup in the buserelin acetate-hmg group (25% per transfer) as compared with 14% in CC-hMG cycles (19% per transfer) (P < 0.04 for both comparisons, see Table 5). These results do not take into account the possible pregnancies obtained after replacement of the frozen embryos and are in agreement with those of other studies using different short or long treatment protocols with a GnRH_a Table 5 Outcome of IVF Trials in Function of Ovarian Stimulation Type Fertilization failures No. replacement Failures after replacement Biochemical pregnancies c Abortions Ectopic pregnancies Singleton pregnancies Twin pregnancies Triplets Ongoing pregnancies All failures (including biochemical, aborted, and ectopic pregnancies) CC-hMG acetate-hmg (n = 466) (n = 319) 114 (24)a 9 (2) 228 (49) 34 (7) 15 (3) 1 (0.2) 47 (10) 16 (3) 2 (0.4) 65 (14) 401 (86) a Values in parentheses are percents. b, not significant. C Transient hcg rise. 58 (18) 7 (2) 156 (49) 22 (7) 10 (3) 3 (1) 48 (15) 13 (4) 2 (0.6) 63 (20)} 256 (80) Probability b < ± ± 1.3 b 4.33 ± ± ± ± ± ± ± ± 0.20 b, not significant. A bias on the patients' age may be ruled out because its distribution was identical in both treatment groups. Although a somewhat higher number of mixed etiologies was observed in the buserelin acetate group, this small difference cannot be responsible for the better results of the buserelin acetate-treated cases because in this group results are worsened by both low fertilization rates arising from male shortcomings and a relatively poor prognosis associated with tubal antecedents. is The higher proportion of trials over rank 4 in the buserelin acetate group is explained by the fact that in the first period of bus ere lin acetate administration, patients having experienced several failures with the classical treatment were preferentially treated with this drug. However, this cannot be the cause of the better results observed in the buserelin acetate group as previous studies show a worse prognosis in relation to high rank trials.19 Similarly, methods of oocyte pickup were not found to influence the outcome of IVF in our care20 or in other IVF programs.21 On average, patients in the buserelin acetatehmg group were treated at a later stage of the survey period than patients in the CC-hMG group. However, 2 years before the beginning of the present study, our results in terms of ongoing pregnancy rates had reached a plateau of approximately 15% per oocyte pickup. Because no changes were introduced in the laboratory procedure during the study, it is very unlikely that any bias was linked to the treatment period. Contrary to data produced by others,17 ovarian response was significantly increased in the buserelin acetate-hmg group. Higher levels of E2 in the late follicular phase as well as higher numbers of retrieved and fertilized oocytes and of obtained, replaced, or frozen embryos may be related to the higher doses of hmg injected per day (2.2 ampules in buserelin acetate-hmg versus 1.8 in CC-hMG) or to the longer period of hmg administration Lejeune et al. Use of buserelin acetate in IVF 479

6 r, (11.9 days in buserelin acetate-hmg versus 9.5 in CC-hMG). This last feature can be partially explained by the absence of a premature onset of a LH peak in the buserelin acetate-hmg cases that would allow follicles to reach a more mature state before ovulation triggering. The improved fertilization rate is probably related to the increased maturity of the follicles because a premature LH peak associated with a reduced fertilization rate5,6 was prevented with the use of bus ere lin acetate. The mean vitality score of the replaced embryos as well as the mean score of the total population of embryos obtained are identical in both treatment groups (Table 4). This observation implies that the embryonic quality is not affected by the use of the GnRH-a during ovarian stimulation. Therefore, the higher ongoing pregnancy rate observed after buserelin acetate-hmg treatment does not seem to be related to an improvement in embryonic vitality. However, by increasing the number of embryos, the use of a GnRH -a widens the options for selection before replacement and freezing. Comparison between early luteal phases of CChMG and buserelin acetate-hmg cycles shows that the drop in E2 levels between the E2 peak 1 day before pickup and the E2 nadir observed 48 hours after pickup is more important in buserelin acetatehmg than in CC-hMG cycles. Moreover, whereas E2 is more sharply reduced, P levels are increased earlier in the luteal phase of buserelin acetatehmg cycles as compared with CC-hMG cases. This difference applies both to supplemented and unsupplemented cycles (Fig. 2). Therefore, the E2:P ratio at luteal day 2 was significantly lower when the GnRH-a was used. Low values of E 2:P ratio in the early luteal phase of IVF trials have been demonstrated to be associated with a significantly higher ongoing pregnancy rate by at least two independent groups,22,23 and ultrastructural characteristics of the endometrium are compatible with better implantation conditions when E 2:P is low.24 This might explain why a significantly better ongoing pregnancy rate was observed among buserelin acetate-hmg-treated cases and why, although not statistically significant (P = 0.10), the difference in implantation rates between groups shows the same trend (14% versus 11%). However, when considering all incipient pregnancies, there is no difference in the implantation rate between the CC-hMG and buserelin acetate-hmg groups (16% versus 17%). Similarly, the implantation rate is also identical when considering pregnant patients only (46% versus 44%). Jointly, these results suggest that, be- cause embryos are of a similar quality, nidation begins at comparable rates in the CC-hMG and buserelin acetate-hmg groups. When endometrial conditions are favorable, implantation is maintained at the same rate after both treatments. However, endometrial conditions would appear to be more frequently favorable in buserelin acetatehmg cycles. This hypothesis could also explain why the multiple pregnancy rate does not differ between the treatment groups (27.7% of the pregnancies in CC-hMG versus 24% in buserelin acetatehmg cycles). When endometrial conditions are favorable, the number of implanted embryos depends only on their quality, which appears to be the same with both treatments. By contrast, in the late luteal phase of un supplemented cycles (i.e., from day 10 to 14), E2 and Pare much lower in the buserelin acetate-hmg group, suggesting premature demise of the corpus luteum (CL). This observation confirms another report 9 and appears to be related to the extremely low levels of LH found during luteal phases of the buserelin acetate-hmg cycles. 14 Luteal support thus appeared mandatory after the first 40 trials with busere lin acetate, entailing a higher number of supplemented cycles in this group. Most of the studies dealing with CC-hMG cycles failed to establish any significant benefit related to luteal support.25 However, we were able to demonstrate a significant effect of such treatment in patients given CC-hMG and having experienced a short, hyperestrogenic luteal phase in previous trials. 14 This group represented no more than 20% of the patients. The beneficial effect of this luteal support in such cases appeared to be related, at least partially, to the improvement of the E 2:P ratio in the early luteal phase. As with buserelin acetate, the E2:P ratio is kept low without any luteal supplementation, the luteal support in this case is intended to prevent the shortening of the luteal phase that was observed in un supplemented buserelin acetate-hmg cycles. Therefore, whereas luteal support does not seem to be able to enhance the ongoing pregnancy rate in CC-hMG cycles of an unselected population, in the buserelin acetatehmg cases, it appears necessary to maintain adequate endometrial conditions until CL rescue occurs. In conclusion, as compared with CC-hMG treatment, the use of GnRH -a buserelin acetate during the follicular phase generates an increased ovarian response, a greater number of embryos for selection when replacing and freezing, an improved 480 Lejeune et al. Use of buserelin acetate in IVF Fertility and Sterility

7 early luteal function, but a premature demise of the CL. A higher rate of ongoing pregnancies is obtained when appropriate luteal support is given. acetate also makes the management of stimulated cycles much easier by preventing spontaneous LH peaks. REFERENCES 1. Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS: Induction of ovulation for in-vitro fertilization using buserelin and gonadotropins. Lancet 1:1284, Fleming R, Coutts JRT: Induction of multiple follicular growth in normally menstruating women with endogenous gonadotropin suppression. Fertil Steril 45:226, Neveu S, Hedon B, Bringer J, Chinchole J-M, Arnal F, Humeau C, Cristol P, Viala J -L: Ovarian stimulation by a combination of a gonadotropin-releasing hormone agonist and gonadotropins for in vitro fertilization. Fertil Steril4 7:639, Fleming R, Haxton MJ, Hamilton MPR, McCune GS, Black WP, Macnaughton MC, Coutts JR: Successful treatment of infertile women with oligomenorrhea using a combination of a luteinizing hormone releasing hormone agonist and exogenous gonadotrophins. Br J Obstet Gynaecol 92:369, Stanger JD, Yovich JL: Reduced in-vitro fertilization ofhuman oocytes from patients with raised basal luteinizing hormone levels during the follicular phase. Br J Obstet Gynaecol 92:385, Barlow P, Englert Y, Puissant F, Lejeune B, Delvigne A, Van Rysselberge M, Leroy F: Fertilization failure in IVF: why and what next? Hum Reprod 5:451, Lejeune B, Degueldre M, Camus M, Vekemans M, Opsomer L, Leroy F: In vitro fertilization and embryo transfer as related to luteinizing hormone rise or human chorionic gonadotropin administration. Fertil SteriI45:377, Wildt L, Diedrich K, Van der Ven H, Al Hasani S, Hubner H, Klasen R: Ovarian hyperstimulation for in vitro fertilization controlled by GnRH agonist administered in combination with human menopausal gonadotrophins. Hum Reprod 1:15, Smitz J, Devroey P, Braeckmans P, Camus M, Khan I, Staessen C, Van Waesberghe L, Wisanto A, Van Steirteghem AC: Management of failed cycles in an IVF/GIFT programme with the combination of a GnRH analog and HMG. Hum Reprod 2:309, Awadalla SG, Friedman CI, Chin NW, Dodds W, Park JM, Kim MH: Follicular stimulation for in vitro fertilization using pituitary suppression and human menopausal gonadotropins. Fertil Steril48:811, Serafini P, Stone B, Kerin J, Batzofin J, Quinn P, Marrs RP: An alternate approach to controlled ovarian stimulation in "poor responders": pretreatment with a gonadotropin-releasing hormone analog. Fertil Steril49:90, Howles CM, Macnamee MC, Edwards RG: Short term use of an LHRH agonist to treat poor responders entering an in-vitro fertilization programme. Hum Reprod 2:655, PuissantF, Van Rysselberge M, Barlow P, Deweze J, Leroy F: Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod 2:705, Lejeune B: Phase luteale et implantation en fecondation in vitro. Contracept Fertil Sex 16:825, Macnamee MC, Howles CM, Edwards RG, Taylor PJ, Elder KT: Short-term luteinizing-hormone-releasing-hormone agonist treatment: prospective trial of a novel ovarian stimulation regimen for in vitro fertilization. Fertil Steril 52:264, Cohen J, Junca AM, Debache C, Pez JP: Stimulation ovarienne pour fecondation in vitro avec Decapeptyl en protocole long. Contracept Fertil Sex 17:903, Chetkowski RJ, Kruse LR, Nass TE: Improved pregnancy outcome with the addition of leuprolide acetate to gonadotropins for in vitro fertilization. Fertil Steril 52:250, Van Rysselberge M, Puissant F, Barlow P, Lejeune B, Delvigne A, Leroy F: Fertility prognosis in IVF treatment of patients with cancelled cycles. Hum Reprod 4:663, Bouckaert A, Baeten S, de Cooman S, Thomas K, Loumaye E: In vitro fertilization: how many attempts before success? Hum Reprod 4:261, Van Rysselberge M, Lejeune B, Delvigne A, Leroy F: Comparaison des techniques de prelewement ovocytaire: laparoscopie ou ponction transvaginale echoguidee: deux ans d'experience. Contracept Fertil Sex 17:1101, Brinsmead M, Stanger J, Olivier M, Shumack J, Raymond S, Clark L: A randomized trial of laparoscopy and transvaginal ultrasound-directed oocyte pickup for in vitro fertilization. J In Vitro Fert Embryo Transfer 6:149, Gidley-Baird A; O'Neill C, Sinosich MJ, Porter RN, Pike IL, Saunders DM: Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen ratios in humans and mice. Fertil Steril45:69, Lejeune B, Camus M, Deschacht J, Leroy F: Differences in the luteal phases after failed or successful in vitro fertilization and embryo replacement. J In Vitro Fert Embryo Transfer 3:358, Dehou MF, Lejeune B, Arijs C, Leroy F: Endometrial morphology in stimulated in vitro fertilization cycles and after steroid replacement therapy in cases of primary ovarian failure. Fertil Steril 48:995, Daya S: Efficacy of progesterone support in the luteal phase following in vitro fertilization and embryo transfer: metaanalysis of clinical trials. Hum Reprod 3:731, 1988 Lejeune et al. Use of buserelin acetate in IVF 481