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Impact of recombinant follicle-stimulating hormone and human menopausal gonadotropins on in vitro fertilization outcome
FERTILITY AND STERILITY威 VOL. 75, NO. 2, FEBRUARY 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Impact of recombinant follicle-stimulating hormone and human menopausal gonadotropins on in vitro fertilization outcome Erwin Strehler, M.D.,a Markus Abt, Ph.D.,b Imam El-Danasouri, Ph.D.,a Mariella De Santo, M.D.,c and Karl Sterzik, M.D.a Institute for Reproductive Medicine, Ulm, and Institute for Mathematics, University of Augsburg, Augsburg, Germany; and University of Chieti, Chieti, Italy
Objective: To investigate possible differences between using recombinant FSH (rFSH) and hMG for ovarian stimulation in IVF/intracytoplasmic sperm injection (ICSI) cycles. Design: Parallel group design. Prospective, randomized clinical study. Setting: A tertiary care infertility clinic. Patient(s): A total of 578 patients of our IVF/ICSI routine were recruited. Intervention(s): Treatment with hMG was used for 282 patients (282 cycles), whereas 296 patients (296 cycles) were treated with rFSH. The number of cycles leading to an embryo transfer were 248 and 259, respectively. Main Outcome Measure(s): Primary: clinical pregnancy rate. Secondary: treatment days, total dose of gonadotropin administered, number of oocytes retrieved, number of mature oocytes, and embryo quality. Result(s): Of the cycles with embryo transfer, the pregnancy rates were 30.1% and 32.3% in the rFSH and the hMG groups, respectively. This difference is not statistically significant (P⫽0.798). Treatment with rFSH resulted in a significantly higher number of recovered oocytes compared with the hMG group but was also associated with a higher number of ampoules needed to reach the criterion for hCG administration. No significant differences were found with regard to the number of mature oocytes, the number of treatment days, and the embryo quality. Conclusion(s): In terms of the clinical pregnancy rate, no significant differences between the two stimulation regimens can be stated. (Fertil Steril威 2001;75:332– 6. ©2001 by American Society for Reproductive Medicine.) Key Words: ICSI, IVF, generalized linear model, hMG, recombinant FSH Received February 8, 2000; revised and accepted August 3, 2000. Reprint requests: Erwin Strehler, M.D., Institute for Reproductive Medicine, Frauenstrasse 51, D-89073 Ulm, Germany. (FAX: ⫹49731-9665130; E-mail: [email protected]). a Institute for Reproductive Medicine, Ulm, Germany. b Institute for Mathematics, University of Augsburg, Augsburg, Germany. c Department of Gynecology and Obstetrics, University of Chieti, Chieti, Italy. 0015-0282/01/$20.00 PII S0015-0282(00)01696-4
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FSH and hMG are widely used for ovarian stimulation in IVF programs. The clinical benefits of recombinant gonadotropins as well as the role of LH in folliculogenesis are still subjects of controversy. It is questionable whether the presence of LH activity is necessary for adequate multiple-follicle development. Some authors postulate that exogenous LH administration may have an adverse effect on the pregnancy rate (1), whereas others cannot detect a detrimental effect of the exogenous LH-like activity contained in hMG (2). Several clinical trials compare the efficacy of recombinant FSH and highly purified urinary FSH in inducing superovulation in women
undergoing assisted reproductive techniques. Although some trials and a meta-analysis of hMG vs. urinary FSH in assisted reproduction have been published (3, 4), clinical results of comparative studies investigating recombinant FSH vs. hMG on a large number of patients are still lacking. The aim of our prospective, randomized study was to compare the treatment results of two different gonadotropins for IVF/ intracytoplasmic sperm injection (ICSI) using either recombinant FSH (rFSH) or hMG in our routine program. The main objective was to investigate whether possibly existing differences in the clinical pregnancy rates between the two gonadotropins are relevant in clinical practice.
MATERIALS AND METHODS Patients From January 1998 to June 1999, a total of 578 healthy women undergoing IVF or ICSI treatment were recruited into the study at the Institute of Reproductive Medicine in Ulm, Germany. The inclusion criteria were [1] age ⱕ40 years and [2] no more than four previous IVF or ICSI cycles. In order to obtain a study population that was comparable to a typical IVF population in a clinical setting, we did not apply any further patient selection. Using computerized randomization, the patients were assigned to one of the two treatment groups. The first group received rFSH (Gonal-F 75, Serono, Unterschleissheim, Germany), and the second group received hMG (Menogon, Ferring, Kiel, Germany). The gonadotropins were administered subcutaneously.
Ovarian Stimulation In all cases, pituitary was down-regulated with nafarelin acetate (Synarela, Heumann Pharma, Nuremberg, Germany) in a short protocol starting on the first cycle day and continuing until the day of hCG injection. Ovarian stimulation was performed with either two to six ampules of rFSH or with two to six ampules of hMG, beginning on cycle day 3. The dosage was individually adjusted and, in cases of previous treatment cycles, determined in accordance to previous response. In our IVF program, we used a simplified routine program with minimal monitoring and without estradiol measurement, as described by Wikland et al. (5). On day 8 of stimulation, follicular development was measured by transvaginal ultrasonography using the widest diameter in two planes (5). The daily dosage was adjusted depending on follicular growth. hCG (Pregnesin, 5,000 IU, Serono) was administered when at least one follicle reached a maximum size of 20 mm and two others, 16 mm in diameter.
Oocyte Retrieval, In Vitro Culture, and Embryo Transfer Transvaginal ultrasound-guided needle aspiration of follicular fluid was carried out 36 to 38 hours after hCG administration. Immediately after follicle puncture, the oocytes were retrieved, assessed, and fertilized in vitro. Sperm preparation and culture conditions did not differ in both groups. In cases of severe male subfertility, ICSI was performed as described in (6). Forty-eight hours after the conventional IVF or ICSI procedure, embryos were evaluated according to their appearance as type 1, 2, 3, or 4 (7). Up to three embryos were transferred into the uterine cavity on day 2 or 3 after oocyte retrieval. Luteal phase support was given by transvaginal progesterone administration (Utrogest, Kade, Berlin, Germany; 200 mg t.i.d.). Progesterone administration was initiated on the day after oocyte retrieval and continued until serum -hCG measurement 14 –16 days after transfer and, in cases of pregnancy, until gestation week 8. FERTILITY & STERILITY威
End Points The primary end point of the study was the clinical pregnancy rate after IVF or ICSI treatment. Clinical pregnancy was defined by the presence of a fetal sac at ultrasound examination 6 weeks after embryo transfer. Secondary variables included the number of treatment days, the total dose of gonadotropin administered, the number of oocytes retrieved, the number of mature oocytes in case of ICSI, and the embryo quality on the day of transfer.
Statistical Methods Generalized linear models (8) were used to analyze the primary as well as the secondary response variables. Besides the administered gonadotropin (rFSH or hMG), the models also contained the age of the patients, the number of embryos transferred, and the number of previous cycles as covariates. The importance of including covariates is twofold: it compensates for a possible imbalance between the two groups with respect to these variables and also increases the chance to detect small treatment effects which might otherwise go unnoticed. On the basis of residual plots and the deviance statistic (where appropriate), none of the fitted models was found to be inadequate. All statistical analyses were carried out using S-PLUS (9).
RESULTS A total of 578 patients was recruited for the study, resulting in 578 cycles. Only 1 cycle for each patient is used in the subsequent analyses to avoid bias from those patients who fail to conceive being reentered into the study. According to the randomization, 282 patients were treated with hMG, whereas rFSH was used for 296 patients. As Table 1 shows, there were no substantial differences between the two groups with respect to the diagnosis of infertility. Note that because both partners might have been diagnosed as infertile, the corresponding percentages in Table 1 add to a total larger than 100%. Twenty of the 282 hMG cycles were canceled before oocyte retrieval because of poor response (less than four mature follicles), in contrast to the case with 28 of the 296 rFSH cycles. In the rFSH group, ICSI was performed in 61.9%, and in the hMG group, in 63.7% of all cycles. Fertilization rates were comparable in both groups (rFSH: IVF 57.1%, ICSI 61.9%; hMG: IVF 54.4%, ICSI 62.9%). The numbers of cycles that finally led to an embryo transfer were 248 and 259 for the hMG and the rFSH group, respectively. Among these 507 cycles with embryo transfer, the two groups appear homogenous with respect to the number of embryos transferred and the age of the patients at study entry (see Table 1). As Table 1 also shows, the pools slightly differ in the number of previous cycles from earlier treatments. For the pregnancy rate, the analysis shows a positive correlation with the number of embryos transferred 333
TABLE 1 Profile and cycle characteristics of patients with hMG or rFSH. Variable No. (%) of patients started No. (%) of cycles with tubal disease No. (%) of cycles with endometriosis No. (%) with infertility due to male factor No. (%) with unknown cause of infertility No. (%) of cycles with oocyte retrieval No. (%) of conventional IVF cycles No. (%) ICSI cycles No. of cycles with embryo transfer Mean (⫾SD) no. of embryos transferred Mean (⫾SD) age of y Mean (⫾SD) no. of previous cycles
hMG group
rFSH group
P value
282 (100.0) 69 (24.4) 8 (2.8) 227 (80.5) 21 (7.4) 262 (92.9) 95 (36.3) 167 (63.7) 248 — 2.21 (⫾ 0.74) 31.78 (⫾ 4.62) 0.77 (⫾ 0.91)
296 (100.0) 68 (23.0) 13 (4.4) 235 (79.4) 15 (5.0) 268 (90.5) 102 (38.1) 166 (61.9) 259 — 2.28 (⫾ 0.73) 32.35 (⫾4.22) 1.15 (⫾ 0.93)
— 0.745 0.438 0.820 0.312 0.379 0.735 0.735 — 0.566 0.149 ⬍0.001
Note: All P values for percentages are based on results from Fisher’s exact test for the comparison of binomial probabilities. Strehler. Recombinant FSH and hMG on IVF outcome. Fertil Steril 2001.
(P⫽0.008) and a negative correlation with the number of previous cycles (P⬍0.001). The age of the patients appears to be of minor importance (P⫽0.188). From the generalized linear model, the ratio of the odds for pregnancy in the rFSH group divided by the odds for pregnancy in the hMG group is 1.055, with an associated 95% confidence interval ranging from 0.698 to 1.596. (The odds for pregnancy for each of the two groups are defined as the probability of pregnancy divided by the probability that no pregnancy resulted.) Note that an odds ratio of 1 would correspond to equal pregnancy rates. Because this value is included in the confidence interval, a statistically significant difference between the two gonadotropins with respect to the chance of achieving pregnancy cannot be established (P⫽0.798; Table 2). The confidence interval also indicates the range in which with 95% probability, a true difference in the odds for pregnancy between the two groups can be expected. For example, in the
rFSH group, the odds for pregnancy might be as much as 59.6% higher but also up to 30.2% lower than those in the hMG group. The range is rather wide and allows for the possibility of rFSH to be superior to hMG. More extensive studies might provide more information on whether a possibly existing difference is therapeutically relevant. The analysis also showed that the nonexistence of an overall difference between rFSH and hMG with respect to the pregnancy rate is not due to a cancellation of possibly opposite effects in the IVF and ICSI groups. For IVF and ICSI, the odds ratios are 1.085 (95% confidence interval [0.569,2.067]) and 1.027 (95% confidence interval [0.618, 1.705]), respectively. Also, the pregnancy rates between IVF and ICSI cannot be shown to be different (P⫽0.227). The secondary variables, as summarized in Table 2, will be discussed next. Embryo grading before transfer was an-
TABLE 2 IVF/ICSI program outcomes in cycles with embryo transfer. hMG group (n ⫽ 248)
rFSH group (n ⫽ 259)
80 14 32.3 68.17 9.11 (⫾ 2.13)
78 11 30.1 66.97 9.48 (⫾ 3.18)
20.21 (⫾ 7.27) 9.67 (⫾ 5.92) 7.48 (⫾ 4.55)
28.69 (⫾ 10.62) 12.29 (⫾ 7.80) 8.64 (⫾ 5.70)
Variable No. of patients with clinical pregnancy No. of patients with multiple pregnancy Clinical pregnancy rate per transfer (%) Embryos of grade 1 (%) Mean (⫾SD) no. of treatment days Total dose of gonadotropins used: mean (⫾SD) no. of ampules Mean (⫾SD) no. of oocytes retrieved Mean (⫾SD) no. of mature oocytes
P value — — 0.798 0.475 0.132 ⬍0.001 ⬍0.001 0.175
95% confidence interval — — [0.70, 1.60] [0.65, 1.22] [⫺1.25, 10.0] [33.94, 55.74] [17.67, 47.62] [⫺14.60, 137.12]
Note: The pregnancy rates and the embryo grading are reported in percentages, and the associated confidence intervals are for the odds ratio (rFSH/hMG). For all other variables, the confidence intervals indicate the possible excess (in %) of the mean of the variable in the rFSH group relative to hMG. Strehler. Recombinant FSH and hMG on IVF outcome. Fertil Steril 2001.
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alyzed in a similar way as the pregnancy rates above. Only very few embryos received grades 3 and 4 (⬍6% across both groups) and were therefore not considered for analysis. Embryos of grades 1 and 2 thus account for almost all embryos transferred and only embryos of grade 1 will be analyzed, as an analysis of embryos of grade 2 would necessarily lead to very similar conclusions with respect to significant differences among the two gonadotropins. In the hMG and rFSH groups, the respective overall proportions (taken across all embryos of all cycles in the respective group) of embryos of grade 1 were 68.1% and 67.0%. From the generalized linear model, the estimated odds-ratio becomes 0.89 (95% confidence interval from 0.65 to 1.22). A difference in embryo quality between the two gonadotropins cannot be established (P⫽0.475). Duration of stimulation was, on average, very similar in both groups (9.12 days in the hMG group vs. 9.47 days in the rFSH group, P⫽0.132). The mean cumulative amount of gonadotropins necessary to reach the criterion for hCG administration was significantly higher in the rFSH than in the hMG group (28.73 ampoules vs. 20.16 ampules, respectively, P⬍0.001). Treatment with rFSH led to a significantly higher number of recovered oocytes compared with the hMG group (P⬍0.001). On average, the number of retrieved oocytes under rFSH is expected to exceed the corresponding number for hMG by 31.8%. With 95% confidence, this excess is within the range of 17.7% and 47.6%. However, no difference between the two gonadotropins can be established when comparing the number of mature oocytes (P⫽0.175). The expected excess in the number of mature oocytes in the rFSH group compared with in the hMG group is about 42.3%, with the 95% confidence interval ranging from ⫺14.6% to 137.1%. On the basis of the observed data, it is therefore not possible to narrow down a possible treatment effect accurately. More extensive studies would have to be conducted.
DISCUSSION There is evidence that a moderate amount of LH activity may be advantageous for ovulation induction. LH is necessary to stimulate theca cell androgens that are subsequently transformed into estrogens in granulosa cells, which in turn exert positive effects on the oocyte development and on embryo maturation (2). Reduced follicular fluid estradiol levels may be responsible for a poor outcome of in vitro fertilization (10). On the other hand, during the time of follicle development and in the periovulatory phase, a relationship between increased LH levels and potential detrimental effects on embryo quality has been suggested (1). In a prospective, randomized comparison between highly purified urinary FSH and hMG, Westergaard et al. (4) could not recognize detrimental effects of the exogenous LH-like FERTILITY & STERILITY威
activity contained in hMG on the clinical outcome of IVF in GnRH agonist down-regulated cycles. Although the clinical results of IVF- and HP-FSH–treated normogonadotropic women were similar, the outcome indicates that exogenous LH does make a difference. The hMG-treated patients had a significantly lower risk of complete failure of fertilization and produced significantly more transferable pre-embryos than those treated with HP-FSH (4). Up to now, only one study had been performed comparing rFSH and hMG in infertile patients (n ⫽ 89) undergoing IVF without the use of a GnRH agonist (11). Although a higher number of oocytes was retrieved and a higher ongoing pregnancy rate was observed in the rFSH group, the differences did not reach statistical significance. A negative role of exogenous LH seemed unlikely in this study because LH concentrations on the day of hCG administration were comparable in both groups and because fertilization data from patients with LH concentrations ⬎10 IU/L did not show apparent detrimental effects (11). Because a comparison between rFSH and hMG on a large number of patients is still lacking, we performed a randomized, prospective study in a study population comparable to a general IVF population. Our primary end point was the clinical pregnancy rate obtained in patients treated with the two forms of gonadotropin stimulations in our IVF/ICSI program. The effects of the secondary end points as well as the age of the patients, number of cycles, and the influence of the two treatments IVF and ICSI on the pregnancy rates were investigated by generalized linear models. Our results show no significant advantage of rFSH in our clinical setting, which is in accordance with the above mentioned studies of Westergaard et al. (4) and Jansen et al. (11). The exogenous LH contained in menotropins seems not to lead to significantly lower pregnancy rates in hMG-treated patients. Treatment with rFSH resulted in a significantly higher number of recovered oocytes compared with the hMG group, but this advantage of FSH was associated with a higher number of ampoules needed to reach the criterion for hCG administration. Interpretation of this finding should be done with recognition that we did not use the same fixed daily drug dose for all patients and that we did not measure estradiol concentration on the day of hCG injection. Nevertheless, the results may be explained by the results of a recent study of Filicori et al. (12), who investigated the role of LH activity during ovarian hyperstimulation by supplementing highly purified (HP) FSH with low-dose hCG in GnRH agonist–suppressed women. Patients with FSH and hCG required a shorter stimulation time. They concluded that LH activity promotes folliculogenesis in synergy with FSH in the mid to late follicular phase and that low-dose hCG coadministration optimizes controlled ovarian hyperstimulation by enhancing FSH action and lowering HP-FSH requirements. In our study, a difference in embryo quality between the 335
two gonadotropins could not be established. In a retrospective review, Schoolcraft et al. (13) analyzed the influence of stimulation protocol on blastocyst transformation and transfer outcome. Patients were arbitrarily given various gonadotropin stimulations, containing pure FSH (n ⫽ 121) or FSH and LH (n ⫽ 53). Patients of the FSH/LH group received 50% hMG and 50% pure FSH throughout the entire stimulation protocol. The inclusion of LH in the stimulation protocol was associated with significant increase in the blastocyst implantation rate and the subsequent pregnancy rate. In a meta-analysis, Daya et al. (3) compared randomized trials of urinary FSH vs. hMG use in ovarian stimulation protocols, with or without GnRH agonists, in IVF treatment cycles. The authors (3) state that the use of urinary FSH produces better results than hMG and that an improvement in clinical pregnancy rates of ⬎50% can be expected in all three outcome denominators, namely, cycle start, oocyte retrieval, and embryo transfer. These findings could not be confirmed by the present study and the studies mentioned above (4, 11), because in none of them could a 50% higher pregnancy rate be achieved using FSH for ovarian stimulation in IVF treatment. Agrarwal et al. (14) reanalyzed in a meta-analysis the results of using FSH alone and hMG during IVF treatment, taking into account the different protocols of administration of GnRH analogs. The results suggested that in the long and short GnRH agonist protocol of IVF, FSH and hMG were equally effective in achieving clinical pregnancy rates per cycle (14). In summary, both stimulation regimens, rFSH and hMG, are comparable in their pregnancy rates in our clinical setting. We believe that the main advantage of rFSH, totally devoid of contaminants and nonspecific activities, is greater safety compared with hMG use, whereas hMG may be an alternative from the point of view of lowering the treatment costs for IVF. Concerning the pregnancy rates, a significant advantage of one of the two stimulation regimens cannot be
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stated. Maybe existing treatment differences can be shown in more extensive studies. References 1. Stanger J, Yovich JJ. Reduced in vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase. Br J Obstet Gynaecol 1985; 92:385–93. 2. Filicori M. The role of luteinizing hormone in folliculogenesis and ovulation induction. Fertil Steril 1999;71:405–14. 3. Daya S, Gunby J, Hughes E, Collins JA, Sagle MA. Follicle-stimulating hormone versus human menopausal gonadotropin for in vitro fertilization cycles: a meta-analysis. Fertil Steril 1995;64:347–54. 4. Westergaard LG, Erb K, Laursen S, Rasmussen PE, Rex S. The effect of human menopausal gonadotropin and highly purified, urine derived follicle stimulating hormone on the outcome of in-vitro fertilization in down-regulated normogonadotrophic women. Hum Reprod 1996;11: 1209 –13. 5. Wikland M, Borg J, Hamberger L, Svalander P. Simplification of IVF: minimal monitoring and the use of subcutaneous highly purified FSH administration for ovulation induction. Hum Reprod 1994;9:1430 – 6. 6. Palermo GD, Schlegel PN, Colombero LT, Zaninovic N, Moy F, Rosenwaus Z. Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum Reprod 1996;11:1023–9. 7. Staessen C, Camus M, Khan I, Smitz J, Van Waesberghe L, Wisanto A, Devroey P, Van Steirteghen AC. An 18-month survey of infertility treatment by in vitro fertilization, gamete and zygote intrafallopian transfer, and replacement of frozen-thawed embryos. J In Vitro Fertil Embryo Transfer 1989;6:22–9. 8. McCullagh P, Nelder JA. Generalized linear models. 2nd ed. London: Chapman and Hall, 1989. 9. Statistical Sciences. S-PLUS guide to statistical and mathematical analysis, version 3.3. Seattle, WA: StatSci, 1995. 10. Loumaye E, Engrand P, Howles CM, O’Dea L. Assessment of the role of serum luteinizing hormone and estradiol response to follicle-stimulating hormone on in vitro fertilization outcome. Fertil Steril 1997;67: 889 –99. 11. Jansen CAM, Van Os HC, Out HJ, Coelingh Bennink HJT. A prospective randomized clinical trial comparing recombinant follicle stimulating hormone (Puregon) and human menopausal gonadotrophins (Humegon) in non-down-regulated in-vitro fertilization patients. Hum Reprod 1998;13:2995–9. 12. Filicori M, Cognigni GE, Taraborrelli S, Spettoli D, Ciampaglia W, de Fatis CT, et al. Luteinizing hormone activity supplementation enhances follicle-stimulating hormone efficacy and improves ovulation induction outcome. J Clin Endocrinol Metab 1999;84:2659 – 63. 13. Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999;72:604 –9. 14. Agrarwal R, Holmes J, Jacobs HS. Follicle-stimulating hormone or human menopausal gonadotropin for ovarian stimulation in in vitro fertilization cycles: a meta-analysis. Fertil Steril 2000;73:338 – 43.
Vol. 75, No. 2, February 2001
Impact of recombinant follicle-stimulating hormone and human menopausal gonadotropins on in vitro fertilization outcome Erwin Strehler, M.D.,a Markus Abt, Ph.D.,b Imam El-Danasouri, Ph.D.,a Mariella De Santo, M.D.,c and Karl Sterzik, M.D.a Institute for Reproductive Medicine, Ulm, and Institute for Mathematics, University of Augsburg, Augsburg, Germany; and University of Chieti, Chieti, Italy
Objective: To investigate possible differences between using recombinant FSH (rFSH) and hMG for ovarian stimulation in IVF/intracytoplasmic sperm injection (ICSI) cycles. Design: Parallel group design. Prospective, randomized clinical study. Setting: A tertiary care infertility clinic. Patient(s): A total of 578 patients of our IVF/ICSI routine were recruited. Intervention(s): Treatment with hMG was used for 282 patients (282 cycles), whereas 296 patients (296 cycles) were treated with rFSH. The number of cycles leading to an embryo transfer were 248 and 259, respectively. Main Outcome Measure(s): Primary: clinical pregnancy rate. Secondary: treatment days, total dose of gonadotropin administered, number of oocytes retrieved, number of mature oocytes, and embryo quality. Result(s): Of the cycles with embryo transfer, the pregnancy rates were 30.1% and 32.3% in the rFSH and the hMG groups, respectively. This difference is not statistically significant (P⫽0.798). Treatment with rFSH resulted in a significantly higher number of recovered oocytes compared with the hMG group but was also associated with a higher number of ampoules needed to reach the criterion for hCG administration. No significant differences were found with regard to the number of mature oocytes, the number of treatment days, and the embryo quality. Conclusion(s): In terms of the clinical pregnancy rate, no significant differences between the two stimulation regimens can be stated. (Fertil Steril威 2001;75:332– 6. ©2001 by American Society for Reproductive Medicine.) Key Words: ICSI, IVF, generalized linear model, hMG, recombinant FSH Received February 8, 2000; revised and accepted August 3, 2000. Reprint requests: Erwin Strehler, M.D., Institute for Reproductive Medicine, Frauenstrasse 51, D-89073 Ulm, Germany. (FAX: ⫹49731-9665130; E-mail: [email protected]). a Institute for Reproductive Medicine, Ulm, Germany. b Institute for Mathematics, University of Augsburg, Augsburg, Germany. c Department of Gynecology and Obstetrics, University of Chieti, Chieti, Italy. 0015-0282/01/$20.00 PII S0015-0282(00)01696-4
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FSH and hMG are widely used for ovarian stimulation in IVF programs. The clinical benefits of recombinant gonadotropins as well as the role of LH in folliculogenesis are still subjects of controversy. It is questionable whether the presence of LH activity is necessary for adequate multiple-follicle development. Some authors postulate that exogenous LH administration may have an adverse effect on the pregnancy rate (1), whereas others cannot detect a detrimental effect of the exogenous LH-like activity contained in hMG (2). Several clinical trials compare the efficacy of recombinant FSH and highly purified urinary FSH in inducing superovulation in women
undergoing assisted reproductive techniques. Although some trials and a meta-analysis of hMG vs. urinary FSH in assisted reproduction have been published (3, 4), clinical results of comparative studies investigating recombinant FSH vs. hMG on a large number of patients are still lacking. The aim of our prospective, randomized study was to compare the treatment results of two different gonadotropins for IVF/ intracytoplasmic sperm injection (ICSI) using either recombinant FSH (rFSH) or hMG in our routine program. The main objective was to investigate whether possibly existing differences in the clinical pregnancy rates between the two gonadotropins are relevant in clinical practice.
MATERIALS AND METHODS Patients From January 1998 to June 1999, a total of 578 healthy women undergoing IVF or ICSI treatment were recruited into the study at the Institute of Reproductive Medicine in Ulm, Germany. The inclusion criteria were [1] age ⱕ40 years and [2] no more than four previous IVF or ICSI cycles. In order to obtain a study population that was comparable to a typical IVF population in a clinical setting, we did not apply any further patient selection. Using computerized randomization, the patients were assigned to one of the two treatment groups. The first group received rFSH (Gonal-F 75, Serono, Unterschleissheim, Germany), and the second group received hMG (Menogon, Ferring, Kiel, Germany). The gonadotropins were administered subcutaneously.
Ovarian Stimulation In all cases, pituitary was down-regulated with nafarelin acetate (Synarela, Heumann Pharma, Nuremberg, Germany) in a short protocol starting on the first cycle day and continuing until the day of hCG injection. Ovarian stimulation was performed with either two to six ampules of rFSH or with two to six ampules of hMG, beginning on cycle day 3. The dosage was individually adjusted and, in cases of previous treatment cycles, determined in accordance to previous response. In our IVF program, we used a simplified routine program with minimal monitoring and without estradiol measurement, as described by Wikland et al. (5). On day 8 of stimulation, follicular development was measured by transvaginal ultrasonography using the widest diameter in two planes (5). The daily dosage was adjusted depending on follicular growth. hCG (Pregnesin, 5,000 IU, Serono) was administered when at least one follicle reached a maximum size of 20 mm and two others, 16 mm in diameter.
Oocyte Retrieval, In Vitro Culture, and Embryo Transfer Transvaginal ultrasound-guided needle aspiration of follicular fluid was carried out 36 to 38 hours after hCG administration. Immediately after follicle puncture, the oocytes were retrieved, assessed, and fertilized in vitro. Sperm preparation and culture conditions did not differ in both groups. In cases of severe male subfertility, ICSI was performed as described in (6). Forty-eight hours after the conventional IVF or ICSI procedure, embryos were evaluated according to their appearance as type 1, 2, 3, or 4 (7). Up to three embryos were transferred into the uterine cavity on day 2 or 3 after oocyte retrieval. Luteal phase support was given by transvaginal progesterone administration (Utrogest, Kade, Berlin, Germany; 200 mg t.i.d.). Progesterone administration was initiated on the day after oocyte retrieval and continued until serum -hCG measurement 14 –16 days after transfer and, in cases of pregnancy, until gestation week 8. FERTILITY & STERILITY威
End Points The primary end point of the study was the clinical pregnancy rate after IVF or ICSI treatment. Clinical pregnancy was defined by the presence of a fetal sac at ultrasound examination 6 weeks after embryo transfer. Secondary variables included the number of treatment days, the total dose of gonadotropin administered, the number of oocytes retrieved, the number of mature oocytes in case of ICSI, and the embryo quality on the day of transfer.
Statistical Methods Generalized linear models (8) were used to analyze the primary as well as the secondary response variables. Besides the administered gonadotropin (rFSH or hMG), the models also contained the age of the patients, the number of embryos transferred, and the number of previous cycles as covariates. The importance of including covariates is twofold: it compensates for a possible imbalance between the two groups with respect to these variables and also increases the chance to detect small treatment effects which might otherwise go unnoticed. On the basis of residual plots and the deviance statistic (where appropriate), none of the fitted models was found to be inadequate. All statistical analyses were carried out using S-PLUS (9).
RESULTS A total of 578 patients was recruited for the study, resulting in 578 cycles. Only 1 cycle for each patient is used in the subsequent analyses to avoid bias from those patients who fail to conceive being reentered into the study. According to the randomization, 282 patients were treated with hMG, whereas rFSH was used for 296 patients. As Table 1 shows, there were no substantial differences between the two groups with respect to the diagnosis of infertility. Note that because both partners might have been diagnosed as infertile, the corresponding percentages in Table 1 add to a total larger than 100%. Twenty of the 282 hMG cycles were canceled before oocyte retrieval because of poor response (less than four mature follicles), in contrast to the case with 28 of the 296 rFSH cycles. In the rFSH group, ICSI was performed in 61.9%, and in the hMG group, in 63.7% of all cycles. Fertilization rates were comparable in both groups (rFSH: IVF 57.1%, ICSI 61.9%; hMG: IVF 54.4%, ICSI 62.9%). The numbers of cycles that finally led to an embryo transfer were 248 and 259 for the hMG and the rFSH group, respectively. Among these 507 cycles with embryo transfer, the two groups appear homogenous with respect to the number of embryos transferred and the age of the patients at study entry (see Table 1). As Table 1 also shows, the pools slightly differ in the number of previous cycles from earlier treatments. For the pregnancy rate, the analysis shows a positive correlation with the number of embryos transferred 333
TABLE 1 Profile and cycle characteristics of patients with hMG or rFSH. Variable No. (%) of patients started No. (%) of cycles with tubal disease No. (%) of cycles with endometriosis No. (%) with infertility due to male factor No. (%) with unknown cause of infertility No. (%) of cycles with oocyte retrieval No. (%) of conventional IVF cycles No. (%) ICSI cycles No. of cycles with embryo transfer Mean (⫾SD) no. of embryos transferred Mean (⫾SD) age of y Mean (⫾SD) no. of previous cycles
hMG group
rFSH group
P value
282 (100.0) 69 (24.4) 8 (2.8) 227 (80.5) 21 (7.4) 262 (92.9) 95 (36.3) 167 (63.7) 248 — 2.21 (⫾ 0.74) 31.78 (⫾ 4.62) 0.77 (⫾ 0.91)
296 (100.0) 68 (23.0) 13 (4.4) 235 (79.4) 15 (5.0) 268 (90.5) 102 (38.1) 166 (61.9) 259 — 2.28 (⫾ 0.73) 32.35 (⫾4.22) 1.15 (⫾ 0.93)
— 0.745 0.438 0.820 0.312 0.379 0.735 0.735 — 0.566 0.149 ⬍0.001
Note: All P values for percentages are based on results from Fisher’s exact test for the comparison of binomial probabilities. Strehler. Recombinant FSH and hMG on IVF outcome. Fertil Steril 2001.
(P⫽0.008) and a negative correlation with the number of previous cycles (P⬍0.001). The age of the patients appears to be of minor importance (P⫽0.188). From the generalized linear model, the ratio of the odds for pregnancy in the rFSH group divided by the odds for pregnancy in the hMG group is 1.055, with an associated 95% confidence interval ranging from 0.698 to 1.596. (The odds for pregnancy for each of the two groups are defined as the probability of pregnancy divided by the probability that no pregnancy resulted.) Note that an odds ratio of 1 would correspond to equal pregnancy rates. Because this value is included in the confidence interval, a statistically significant difference between the two gonadotropins with respect to the chance of achieving pregnancy cannot be established (P⫽0.798; Table 2). The confidence interval also indicates the range in which with 95% probability, a true difference in the odds for pregnancy between the two groups can be expected. For example, in the
rFSH group, the odds for pregnancy might be as much as 59.6% higher but also up to 30.2% lower than those in the hMG group. The range is rather wide and allows for the possibility of rFSH to be superior to hMG. More extensive studies might provide more information on whether a possibly existing difference is therapeutically relevant. The analysis also showed that the nonexistence of an overall difference between rFSH and hMG with respect to the pregnancy rate is not due to a cancellation of possibly opposite effects in the IVF and ICSI groups. For IVF and ICSI, the odds ratios are 1.085 (95% confidence interval [0.569,2.067]) and 1.027 (95% confidence interval [0.618, 1.705]), respectively. Also, the pregnancy rates between IVF and ICSI cannot be shown to be different (P⫽0.227). The secondary variables, as summarized in Table 2, will be discussed next. Embryo grading before transfer was an-
TABLE 2 IVF/ICSI program outcomes in cycles with embryo transfer. hMG group (n ⫽ 248)
rFSH group (n ⫽ 259)
80 14 32.3 68.17 9.11 (⫾ 2.13)
78 11 30.1 66.97 9.48 (⫾ 3.18)
20.21 (⫾ 7.27) 9.67 (⫾ 5.92) 7.48 (⫾ 4.55)
28.69 (⫾ 10.62) 12.29 (⫾ 7.80) 8.64 (⫾ 5.70)
Variable No. of patients with clinical pregnancy No. of patients with multiple pregnancy Clinical pregnancy rate per transfer (%) Embryos of grade 1 (%) Mean (⫾SD) no. of treatment days Total dose of gonadotropins used: mean (⫾SD) no. of ampules Mean (⫾SD) no. of oocytes retrieved Mean (⫾SD) no. of mature oocytes
P value — — 0.798 0.475 0.132 ⬍0.001 ⬍0.001 0.175
95% confidence interval — — [0.70, 1.60] [0.65, 1.22] [⫺1.25, 10.0] [33.94, 55.74] [17.67, 47.62] [⫺14.60, 137.12]
Note: The pregnancy rates and the embryo grading are reported in percentages, and the associated confidence intervals are for the odds ratio (rFSH/hMG). For all other variables, the confidence intervals indicate the possible excess (in %) of the mean of the variable in the rFSH group relative to hMG. Strehler. Recombinant FSH and hMG on IVF outcome. Fertil Steril 2001.
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alyzed in a similar way as the pregnancy rates above. Only very few embryos received grades 3 and 4 (⬍6% across both groups) and were therefore not considered for analysis. Embryos of grades 1 and 2 thus account for almost all embryos transferred and only embryos of grade 1 will be analyzed, as an analysis of embryos of grade 2 would necessarily lead to very similar conclusions with respect to significant differences among the two gonadotropins. In the hMG and rFSH groups, the respective overall proportions (taken across all embryos of all cycles in the respective group) of embryos of grade 1 were 68.1% and 67.0%. From the generalized linear model, the estimated odds-ratio becomes 0.89 (95% confidence interval from 0.65 to 1.22). A difference in embryo quality between the two gonadotropins cannot be established (P⫽0.475). Duration of stimulation was, on average, very similar in both groups (9.12 days in the hMG group vs. 9.47 days in the rFSH group, P⫽0.132). The mean cumulative amount of gonadotropins necessary to reach the criterion for hCG administration was significantly higher in the rFSH than in the hMG group (28.73 ampoules vs. 20.16 ampules, respectively, P⬍0.001). Treatment with rFSH led to a significantly higher number of recovered oocytes compared with the hMG group (P⬍0.001). On average, the number of retrieved oocytes under rFSH is expected to exceed the corresponding number for hMG by 31.8%. With 95% confidence, this excess is within the range of 17.7% and 47.6%. However, no difference between the two gonadotropins can be established when comparing the number of mature oocytes (P⫽0.175). The expected excess in the number of mature oocytes in the rFSH group compared with in the hMG group is about 42.3%, with the 95% confidence interval ranging from ⫺14.6% to 137.1%. On the basis of the observed data, it is therefore not possible to narrow down a possible treatment effect accurately. More extensive studies would have to be conducted.
DISCUSSION There is evidence that a moderate amount of LH activity may be advantageous for ovulation induction. LH is necessary to stimulate theca cell androgens that are subsequently transformed into estrogens in granulosa cells, which in turn exert positive effects on the oocyte development and on embryo maturation (2). Reduced follicular fluid estradiol levels may be responsible for a poor outcome of in vitro fertilization (10). On the other hand, during the time of follicle development and in the periovulatory phase, a relationship between increased LH levels and potential detrimental effects on embryo quality has been suggested (1). In a prospective, randomized comparison between highly purified urinary FSH and hMG, Westergaard et al. (4) could not recognize detrimental effects of the exogenous LH-like FERTILITY & STERILITY威
activity contained in hMG on the clinical outcome of IVF in GnRH agonist down-regulated cycles. Although the clinical results of IVF- and HP-FSH–treated normogonadotropic women were similar, the outcome indicates that exogenous LH does make a difference. The hMG-treated patients had a significantly lower risk of complete failure of fertilization and produced significantly more transferable pre-embryos than those treated with HP-FSH (4). Up to now, only one study had been performed comparing rFSH and hMG in infertile patients (n ⫽ 89) undergoing IVF without the use of a GnRH agonist (11). Although a higher number of oocytes was retrieved and a higher ongoing pregnancy rate was observed in the rFSH group, the differences did not reach statistical significance. A negative role of exogenous LH seemed unlikely in this study because LH concentrations on the day of hCG administration were comparable in both groups and because fertilization data from patients with LH concentrations ⬎10 IU/L did not show apparent detrimental effects (11). Because a comparison between rFSH and hMG on a large number of patients is still lacking, we performed a randomized, prospective study in a study population comparable to a general IVF population. Our primary end point was the clinical pregnancy rate obtained in patients treated with the two forms of gonadotropin stimulations in our IVF/ICSI program. The effects of the secondary end points as well as the age of the patients, number of cycles, and the influence of the two treatments IVF and ICSI on the pregnancy rates were investigated by generalized linear models. Our results show no significant advantage of rFSH in our clinical setting, which is in accordance with the above mentioned studies of Westergaard et al. (4) and Jansen et al. (11). The exogenous LH contained in menotropins seems not to lead to significantly lower pregnancy rates in hMG-treated patients. Treatment with rFSH resulted in a significantly higher number of recovered oocytes compared with the hMG group, but this advantage of FSH was associated with a higher number of ampoules needed to reach the criterion for hCG administration. Interpretation of this finding should be done with recognition that we did not use the same fixed daily drug dose for all patients and that we did not measure estradiol concentration on the day of hCG injection. Nevertheless, the results may be explained by the results of a recent study of Filicori et al. (12), who investigated the role of LH activity during ovarian hyperstimulation by supplementing highly purified (HP) FSH with low-dose hCG in GnRH agonist–suppressed women. Patients with FSH and hCG required a shorter stimulation time. They concluded that LH activity promotes folliculogenesis in synergy with FSH in the mid to late follicular phase and that low-dose hCG coadministration optimizes controlled ovarian hyperstimulation by enhancing FSH action and lowering HP-FSH requirements. In our study, a difference in embryo quality between the 335
two gonadotropins could not be established. In a retrospective review, Schoolcraft et al. (13) analyzed the influence of stimulation protocol on blastocyst transformation and transfer outcome. Patients were arbitrarily given various gonadotropin stimulations, containing pure FSH (n ⫽ 121) or FSH and LH (n ⫽ 53). Patients of the FSH/LH group received 50% hMG and 50% pure FSH throughout the entire stimulation protocol. The inclusion of LH in the stimulation protocol was associated with significant increase in the blastocyst implantation rate and the subsequent pregnancy rate. In a meta-analysis, Daya et al. (3) compared randomized trials of urinary FSH vs. hMG use in ovarian stimulation protocols, with or without GnRH agonists, in IVF treatment cycles. The authors (3) state that the use of urinary FSH produces better results than hMG and that an improvement in clinical pregnancy rates of ⬎50% can be expected in all three outcome denominators, namely, cycle start, oocyte retrieval, and embryo transfer. These findings could not be confirmed by the present study and the studies mentioned above (4, 11), because in none of them could a 50% higher pregnancy rate be achieved using FSH for ovarian stimulation in IVF treatment. Agrarwal et al. (14) reanalyzed in a meta-analysis the results of using FSH alone and hMG during IVF treatment, taking into account the different protocols of administration of GnRH analogs. The results suggested that in the long and short GnRH agonist protocol of IVF, FSH and hMG were equally effective in achieving clinical pregnancy rates per cycle (14). In summary, both stimulation regimens, rFSH and hMG, are comparable in their pregnancy rates in our clinical setting. We believe that the main advantage of rFSH, totally devoid of contaminants and nonspecific activities, is greater safety compared with hMG use, whereas hMG may be an alternative from the point of view of lowering the treatment costs for IVF. Concerning the pregnancy rates, a significant advantage of one of the two stimulation regimens cannot be
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stated. Maybe existing treatment differences can be shown in more extensive studies. References 1. Stanger J, Yovich JJ. Reduced in vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase. Br J Obstet Gynaecol 1985; 92:385–93. 2. Filicori M. The role of luteinizing hormone in folliculogenesis and ovulation induction. Fertil Steril 1999;71:405–14. 3. Daya S, Gunby J, Hughes E, Collins JA, Sagle MA. Follicle-stimulating hormone versus human menopausal gonadotropin for in vitro fertilization cycles: a meta-analysis. Fertil Steril 1995;64:347–54. 4. Westergaard LG, Erb K, Laursen S, Rasmussen PE, Rex S. The effect of human menopausal gonadotropin and highly purified, urine derived follicle stimulating hormone on the outcome of in-vitro fertilization in down-regulated normogonadotrophic women. Hum Reprod 1996;11: 1209 –13. 5. Wikland M, Borg J, Hamberger L, Svalander P. Simplification of IVF: minimal monitoring and the use of subcutaneous highly purified FSH administration for ovulation induction. Hum Reprod 1994;9:1430 – 6. 6. Palermo GD, Schlegel PN, Colombero LT, Zaninovic N, Moy F, Rosenwaus Z. Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum Reprod 1996;11:1023–9. 7. Staessen C, Camus M, Khan I, Smitz J, Van Waesberghe L, Wisanto A, Devroey P, Van Steirteghen AC. An 18-month survey of infertility treatment by in vitro fertilization, gamete and zygote intrafallopian transfer, and replacement of frozen-thawed embryos. J In Vitro Fertil Embryo Transfer 1989;6:22–9. 8. McCullagh P, Nelder JA. Generalized linear models. 2nd ed. London: Chapman and Hall, 1989. 9. Statistical Sciences. S-PLUS guide to statistical and mathematical analysis, version 3.3. Seattle, WA: StatSci, 1995. 10. Loumaye E, Engrand P, Howles CM, O’Dea L. Assessment of the role of serum luteinizing hormone and estradiol response to follicle-stimulating hormone on in vitro fertilization outcome. Fertil Steril 1997;67: 889 –99. 11. Jansen CAM, Van Os HC, Out HJ, Coelingh Bennink HJT. A prospective randomized clinical trial comparing recombinant follicle stimulating hormone (Puregon) and human menopausal gonadotrophins (Humegon) in non-down-regulated in-vitro fertilization patients. Hum Reprod 1998;13:2995–9. 12. Filicori M, Cognigni GE, Taraborrelli S, Spettoli D, Ciampaglia W, de Fatis CT, et al. Luteinizing hormone activity supplementation enhances follicle-stimulating hormone efficacy and improves ovulation induction outcome. J Clin Endocrinol Metab 1999;84:2659 – 63. 13. Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999;72:604 –9. 14. Agrarwal R, Holmes J, Jacobs HS. Follicle-stimulating hormone or human menopausal gonadotropin for ovarian stimulation in in vitro fertilization cycles: a meta-analysis. Fertil Steril 2000;73:338 – 43.
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