Articles Follitropin-alfa for ovarian stimulation during assisted reproduction treatment: a national collaborative study

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1 RBMOnline - Vol 3. No Reproductive BioMedicine Online webpaper 2001/102 on web 30 August 2001 Articles Follitropin-alfa for ovarian stimulation during assisted reproduction treatment: a national collaborative study Thomas Katzorke is Director of the Centre for Reproductive Medicine in Essen, Germany. Born in 1948, he obtained his MD in 1976 and subsequent qualification in obstetrics and gynaecology in 1981, both from the University of Essen. In 1977 he took up a post-doctoral fellowship under Professor AV Schally at Tulane University and Veterans Administration Hospital in New Orleans, USA. A further qualification was obtained in endocrinology and reproductive medicine in Current clinical research interests include studies with recombinant FSH, cryobiology, and assisted reproduction. He has in excess of 50 publications to his name. Dr Thomas Katzorke Thomas Katzorke 1 *, Hugo C. Verhoeven 2 *, Jutta Blechschmidt 3, Manfred Köhler 4 and Eduardo Kelly 5 1 Centre for Reproductive Medicine, Essen, Germany; 2 Centre for Reproductive Medicine, Düsseldorf, Germany; 3 Serono Pharma GmbH, Unterschleissheim, Germany; 4 Pharma-Biometrics-Consulting, Freiburg, Germany; 5 Correspondence: Clinical Development Unit, Serono Inc., 700 Longwater Drive, Norwell, MA USA. Tel ; Fax ; eduardo.kelly@serono.com *on behalf of the Gonal-F Study Group (see Appendix). Financial support provided by Serono Pharma GmbH, Unterschleissheim, Germany. Abstract This prospective, observational study investigated the efficacy and tolerability of follitropin-alfa for ovarian stimulation in routine clinical practice. The study involved a large, unselected population of women. Stimulation parameters and treatment outcomes were documented for 767 patients who underwent a total of 1098 cycles of follitropin-alfa according to the clinic s standard protocol. Stimulation lasted a median of 11 days and used a cumulative dosage of 28 ampoules of 75 IU follitropinalfa. Pituitary down-regulation was used in 81.2% of cycles, predominantly by the long protocol. The desired ovarian development was achieved in 99.5% of cycles and embryo transfer took place in 86.9% of cycles, with an average of 2.5 embryos transferred. The clinical pregnancy rate was 39.5% and the miscarriage rate was 12.2%. Ovarian hyperstimulation syndrome was observed in only 12 cycles (1.1%). In conclusion, follitropin-alfa is effective and well tolerated for ovarian stimulation during routine clinical practice. Keywords: follitropin-alfa, multiple follicular development, observational study, r-hfsh 98 Introduction Follitropin-alfa (Gonal-F ; Ares-Serono) is a recombinant human follicle stimulating hormone (r-hfsh) which is expressed by Chinese hamster ovary cells (Howles, 1996; Recombinant Human FSH Product Development Group, 1998), and it is the first biotechnology product to be developed for the treatment of infertility. Although urine-derived FSH (uhfsh) preparations have been established for more than 30 years for the induction of ovulation in women undergoing assisted reproduction techniques, they have a number of disadvantages, including contamination with luteinizing hormone (LH) and other urinary proteins, low specific activity and variable isoform content between batches (Guidice et al., 1994; Howles and Wikland, 1999). By contrast, the biotechnology processes used to produce follitropin-alfa ensure that it is highly pure biochemically (>99% FSH) with high specific activity (~ IU/mg protein), and that its isoform content is consistent between batches (Howles, 1996; Recombinant Human FSH Product Development Group, 1998). The high purity of r-hfsh provides good tolerability and low immunogenicity when injected subcutaneously, making it suitable for use in women with a history of adverse reactions to urinary FSH (Redfearn et al., 1995; Albano et al., 1996). Studies of in-vivo activity using the rat ovary bioassay (Steelman and Pohley, 1953) have shown that the biological activity of follitropin-alfa is equivalent to that of urinary human FSH (u-hfsh) (Recombinant Human FSH Product Development Group, 1998). In addition, a number of

2 comparative studies in women undergoing assisted reproduction treatment have shown that follitropin-alfa is more effective than u-hfsh for ovulation induction (Bergh et al., 1997; Frydman et al., 1998; Khalaf et al., 1998). For example, two randomized, double-blind studies which have compared follitropin-alfa with highly purified u-hfsh (u-hfsh HP) found that the women treated with follitropin-alfa required a shorter stimulation time and received a lower cumulative dose of FSH than those treated with u-hfsh HP, but that the number of oocytes recovered was significantly higher with the recombinant product (Bergh et al., 1997; Frydman et al., 1998). One of these studies also found that the number of embryos transferred was significantly higher in the woman who received follitropin-alfa (Frydman et al., 1998). A second r-hfsh preparation, follitropin-β, has also been developed. It is almost indistinguishable structurally and biochemically from follitropin-alfa, except for some minor differences in the percentage of oxidised/degraded isoforms, and the two preparations produce identical ovarian stimulation characteristics when administered on an equivalent IU basis (Brinsden et al., 2000). However, there is some evidence that follitropin-β is less well tolerated than follitropin-alfa, with a greater proportion of patients reporting pain and/or stinging after subcutaneous injection of follitropin-β than with follitropin-alfa (Brinsden et al., 2000). This paper presents the findings of a prospective, observational study with follitropin-alfa which was performed in Germany. While German law does not permit a randomized, comparative trial design for post-marketing surveillance studies, observational studies are required by law in Germany to collect further information about the safety and tolerability of drugs after they have been approved for use in clinical practice. A key feature of such studies is that they should not have any inclusion or exclusion criteria, thereby allowing the assessment of the product in a wide range of patients compared with the highly selected populations which are enrolled in phase II and III clinical trials. This study, therefore, presents data on the efficacy and safety of follitropin-alfa during routine use in a large patient population and so provides information about its use in real life rather than under the tightly controlled conditions of a clinical trial. Materials and methods Study design The open, prospective, observational study was performed between June 1996 and December 1997 at 44 centres for Reproductive Medicine in Germany in accordance with the requirements of the German Drug Law. Women undergoing multifollicular stimulation in preparation for an assisted reproduction treatment, such as in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), and who were considered by their physician to be appropriate candidates for treatment with subcutaneous injections of follitropin-alfa, were managed according to the clinic s regular protocols for pituitary down-regulation and ovarian stimulation. Up to three cycles of treatment with follitropin-alfa were documented for each woman. All data were collected and processed anonymously. In addition to demographic data, the reason for infertility, the duration of infertility, and any previous interventions were documented for each patient. The data collected from each treatment cycle included the duration of stimulation, the total dose of follitropin-alfa, the protocol for pituitary down-regulation, follicle size on the day of ovulation induction, number of oocytes retrieved, levels of LH and oestradiol on the day of induction, the number of clinical pregnancies, the number of miscarriages, and the nature and severity of any adverse reactions. Utilization of IVF or ICSI techniques were not differentiated as separate procedures. A clinical pregnancy was defined as the visualization of a gestational sac by transvaginal sonography. Statistical analyses All data were evaluated using SAS, Version 6.12 (SAS Institute, Cary, NC, USA).A stepwise logistical regression was performed to identify those factors, such as age of patient, duration of infertility, reason for infertility, type of pituitary down-regulation, cumulative r-hfsh dose, days of stimulation, cycle number and oestradiol per oocyte, which had a statistically significant influence on the outcome clinical pregnancy. These variables were included with a selection criterion of 15%. Factors with P < 0.05 were considered to be statistically significant. Logistical regressions were also performed to investigate the influence of the number of embryos transferred on the outcome clinical pregnancy, and to identify the influence of factors such as age and the number of embryos transferred on the outcome multiple births. Finally, a linear regression was performed to identify which of the above factors were related to the cumulative dose of r- hfsh. Results Patient demographics During the 18-month period of the study, treatment was documented for 767 women who underwent a total of 1098 cycles of treatment with follitropin-alfa. The median age of the women was 31 years, and the majority (70.3%) had primary Table 1. Patient demographics. Median no. % Number of patients 767 Age (years) a 31.0 ( ) Body mass index (kg/m 2 ) a ( ) Type of infertility Primary Secondary Duration of infertility (years) a 4.0 ( ) Cause of infertility b Tubal factor Andrological factor Polycystic ovary Idiopathic Other Patients with previous treatment Duration of previous treatment (years) a 2.0 ( ) a Values are median (range). b More than one cause was recorded. 99

3 infertility (Table 1). Andrological factors were the cause of infertility in 55.5% of patients, while tubal factors and idiopathic infertility accounted for 33.8% and 11.6% of patients respectively. The average duration of infertility was 4 years and just over half (59.1%) of the women had a history of previous treatment for infertility, with a median duration of 2 years. Treatment and outcomes For most of the women included in the study (525, 68.4%), a single cycle of treatment with follitropin-alfa was documented, while two and three cycles of treatment were documented for 153 (20%) and 89 (11.5%) women respectively. Thus, the analyses presented in this paper are based upon 767 first cycles (which accounted for 69.9% of all cycles documented), 242 second cycles (22% of cycles) and 89 third cycles (8.1% of cycles). Pituitary desensitization with a gonadotrophin-releasing hormone analogue (GnRHa) was performed in 891 cycles (81.2%) cycles, and in the majority of cases (70.9%) it was administered according to the long protocol (Figure 1). However, down-regulation was not used in 177 cycles (16.1%), and data were absent for 30 cycles (2.7%). The median duration of stimulation across all treatment cycles was 11 days, and an average of 28 ampoules of 75 IU r-hfsh were used per cycle (Table 2). Women who did not have pituitary down-regulation required fewer ampoules (average 20.5 per cycle) than the women with down-regulated cycles (average 30 ampoules per cycle). The desired follicular development was achieved in 1092 (99.5%) cycles. Ovulation was induced with human chorionic gonadotrophin (HCG) in 1082 cycles, at a dose of IU in the majority (78.3%) of cases. There was a median of nine follicles 10 mm diameter on the day of induction, and an overall average of eight oocytes was retrieved per cycle, although fewer oocytes were retrieved following non-down-regulated cycles (average five per cycle) Table 2. Summary of stimulation characteristics. Number of cycles (%) 1098 (100) Cycles with ovulation induction (%) 1092 (99.5) Days of stimulation a 11 ( ) Number of 75 IU ampoules a 28 ( ) Follicles 10 mm on day of induction a 9 (1-54) Oestradiol on day of induction (pg/ml) a (5.8 13,318) LH on day of induction (miu/ml) a 1.10 ( ) Oocytes obtained a 8 (1-55) Oestradiol per oocyte (pg/ml) a ( ) a Values are median (range). than following down-regulated cycles (average eight per cycle) (Table 2). Plasma oestradiol and LH concentrations were measured on the day of ovulation induction in 919 and 631 cycles respectively. The median oestradiol value prior to HCG administration was 1475 pg/ml ( pmol/l) for all cycles (Table 2), and was higher in cycles with pituitary down-regulation ( pg/ml) than in those without down-regulation (983.0 pg/ml). The average ratio of oestradiol per oocyte obtained was pg/ml (Table 2). Median plasma LH concentrations on the day of induction were 3.2 miu/ml for non-down-regulated cycles, compared with 1.0 miu/ml for down-regulated cycles. Embryos were transferred in 954 (86.9%) cycles (Table 3). Between one and three embryos were transferred per cycle, with an average of 2.5 embryos per cycle. The luteal phase was assisted in 936 (98.1%) of the cycles in which embryos were transferred, by administration of progesterone in 376 cycles (40.2%), by HCG in 134 cycles (14.3%) and by both progesterone and HCG in 426 cycles (45.5%). A total of 434 clinical pregnancies was achieved, representing a pregnancy rate of 39.5% per cycle, and there were 53 miscarriages (12.2% per cycle) (Table 3). Younger age was associated with a greater probability of becoming pregnant, with overall pregnancy rates declining from 43.7% in women under 30 years of age to 14.9 in women aged over 40 years. The incidence of multiple births was low, occurring in only 66 (15.2%) clinical pregnancies (Table 3). Twins were reported in 49 pregnancies, triplets in 16 pregnancies, and there was one case of quadruplets in a woman in whom three embryos had been transferred during a previous cycle of treatment. 100 Figure 1. Patients using pituitary down-regulation protocols. Down-regulation of pituitary function appeared to be associated with a better outcome than non-down-regulated cycles. The pregnancy rate was 43.4% for women who received GnRHa in a long protocol compared with 34.5% for women without pituitary down-regulation (Table 4). The miscarriage rate also appeared to be slightly lower amongst women with down-regulated cycles (11.6%) than in those without GnRHa treatment (13.1%). Safety and tolerability Follitropin-alfa was generally well tolerated, and adverse events were reported in only 19 (1.7%) cycles. Ovarian hyperstimulation syndrome (OHSS) was observed in 12 cycles (1.1%), and was rated as severe in six (0.5%) and as moderate in another six (0.5%) patients. Ten women were hospitalized

4 Table 3. Embryo transfer and pregnancy. No. % Cycles with embryo transfer Number of embryos transferred (cycles) Number of clinical pregnancies Total 434 a 39.5 < 30 years (n = 359) b years (n = 449) years (n = 226) > 40 years (n = 47) Number of miscarriages 53 - Miscarriage rate per clinical pregnancy Pregnancies with multiple births Twins Triplets Quadruplets a Pregnancy rate per cycle; b Number of cycles for women within each age group Table 4. The effect of pituitary down-regulation on pregnancy and miscarriage. No. No. pregnancies (%) miscarriages (%) Without GnRHa (n = 177) 61 (34.5) 8 (13.1) With GnRHa (n = 891) 361 (40.5) 42 (11.6) Long protocol (n = 632) 274 (43.4) 0 Short protocol (n = 194) 72 (37.1) 0 Ultra-short protocol (n = 30) 7 (23.3) 0 Table 5. Factors with a significant influence on clinical pregnancy outcome by stepwise logistical regression. Factor Odds ratio P-value Cycle number <0.001 Down-regulation by long protocol <0.01 Age Oestradiol/oocyte and ovulation was induced with a GnRHa; two other women had the treatment cycle terminated. A stepwise logistical regression identified the plasma oestradiol concentration and pregnancy as risk factors for developing OHSS. In 10 out of 12 cycles (83%) patients were pregnant. Local injection-site reactions were reported in only seven cycles (0.6%): burning was reported in six cycles (four mild, two severe) and pain in one cycle (mild). More than half of the patients (61.9%) self-injected follitropin-alfa, and this method of administration was rated as either good or very good by 19.3% and 39.4% of patients respectively. Factors influencing the outcome of clinical pregnancy A stepwise logistical regression identified age, cycle number, down-regulation by the long protocol and oestradiol per oocyte as the factors which had a statistically significant influence on the outcome clinical pregnancy for all cycles recorded during the study (Table 5). Cycle number (odds ratio (OR) 0.524) was the factor with the greatest influence on clinical pregnancy in the present study, and the probability of clinical pregnancy decreased on average by 47.6% for repeat cycles relative to the first cycle of follitropin-alfa stimulation. Pituitary downregulation by the long protocol (OR 0.59) also had a significant influence on outcome, with the probability of clinical pregnancy decreasing by 41% in relative terms in those women who were not down-regulated according to the long protocol. Increasing age (OR 0.953) was associated with a poorer outcome, and the probability of becoming pregnant decreased by 4.7% on average for each additional year of life. Finally, the highest pregnancy rates were associated with an oestradiol per oocyte value of <500 pmol/ml (140 pg/ml; OR 0.999). A logistical regression to investigate the influence of the number of embryos transferred on the outcome clinical pregnancy was performed for the sub-population of patients with embryo transfer. This confirmed that the number of embryos transferred had a significant effect on the probability of occurrence of pregnancy (P < 0.001). Thus, the pregnancy rate increased from 16.4% after transfer of one embryo to 37.1% and 51.9% after transfer of two and three embryos respectively. The dose of follitropin-alfa had no effect on the outcome of r-hfsh treatment. However, linear regression showed that it was positively correlated with both age (r = 0.89, P < 0.001) and with cycle number (r = 0.62, P < 0.001). Thus, older women received a higher dose of follitropin-alfa than younger women, and women undergoing a second or third cycle received higher doses than those having their first cycle of treatment. In addition, down-regulation was a borderline variable (P = 0.074), with an apparent but not significant trend towards lower doses of follitropin-alfa in patients without GnRHa treatment. Prognostic factors for clinical pregnancy and multiple births The probabilities of clinical pregnancy and the risk of multiple births were calculated for the variables age and number of embryos transferred. This showed that younger age and higher numbers of embryos transferred were associated with a higher probability of pregnancy. Thus, women under 30 years of age who had three embryos transferred had the greatest probability of pregnancy (58.7%) (Table 6). However, they also had the greatest risk of multiple births (26.9%). The probability of pregnancy decreased with age, to 40.6% in women aged over 40 years with three embryos transferred, as did the risk of multiple births. Discussion This multicentre, observational study of 1098 cycles of follitropin-alfa treatment for ovulation induction in women undergoing IVF procedures has demonstrated that it is an effective and well tolerated agent when used for this purpose in 101

5 102 Table 6. Age and number of embryos transferred as prognostic factors for pregnancy and multiple births. Age group Embryos Probability Risk of (years) transferred of pregnancy (%) multiple births (%) < > routine clinical practice. The desired ovarian development was achieved in 99.5% of cycles, and the overall mean clinical pregnancy rate was 39.5%. Adverse events were reported during only 19 cycles: there were 12 cases of OHSS and seven reports of local injection-site reactions. An important aspect of this study is that the data were obtained during the use of follitropin-alfa in an unselected group of patients. The only requirement of a woman to be included in the present study was that she was considered by her physician to be a candidate for r-hfsh treatment, and that she had no specific contra-indications for follitropin-alfa. This explains the large range of values for some of the variables seen in the present study, compared with those that might be expected in a controlled clinical study with extensive inclusion and exclusion criteria. Although many post-marketing observational studies of drugs investigate both their efficacy and safety, few include any objective measures of efficacy, relying instead upon global ratings by the patient and/or physician (Hasford and Lamprecht,1998). A key feature of the present study is, therefore, the inclusion of a number of objective measures of efficacy, including the number of oocytes retrieved, the number of embryos transferred and the number of clinical pregnancies. Prospective observational studies also permit the identification of factors which may influence the outcome of treatment in general practice. Thus, the present study found that age <30 years, pituitary down-regulation according to the long protocol, oestradiol/oocyte values <500 pmol/ml on the day of ovulation induction, and first cycle of ovarian stimulation were all factors which increased the probability of clinical pregnancy. These observations are all consistent with the results of previous studies. For example, the odds of becoming pregnant decreased by 4.7% for each additional year of life in the present study, and the overall pregnancy rate declined from 43.7% among women under 30 years of age to 14.9% in women aged over 40 years. Similarly, a study of the factors that influenced the probability of pregnancy following IVF, which was performed at a single centre in the UK, found that high success rates (54% cumulative conception rate over five cycles) were achieved in women up to 34 years of age, after which they began to decline, before dropping dramatically in women over 40 years of age (20.2% cumulative conception rate over five cycles) (Tan et al., 1992). As in the present study, which found that treatment cycle was the most important prognostic factor for clinical pregnancy, the UK study (Tan et al., 1992) also found that the probability of pregnancy declined with each successive cycle of treatment. This is probably due to the fact that women with a better overall fertility status are more likely to become pregnant during the initial treatment cycle, whereas those women with a poorer fertility status are more likely to require additional treatment cycles to increase their chance of becoming pregnant. It should be noted, however, that this phenomenon should not be confused with the individual pregnancy rate; even if this remains the same in the first cycle, the global pregnancy rate may decline with subsequent cycles (Hershlag et al., 1991). Thus, in the present study, it is probable that the proportion of women with a poorer prognosis was increased in the groups which required a second or third cycle of treatment, thereby reducing the pregnancy rates for these cycles. Hormonal factors are also important in determining the success of assisted reproduction techniques. A recent meta-analysis has shown that down-regulation according to the long protocol is associated with a greater likelihood of pregnancy than other protocols (Daya, 1997), and the present study also found that women who received GnRHa according to the long protocol had a higher pregnancy rate than women who had no downregulation. In addition, the oestradiol/oocyte ratio on the day of ovulation induction was identified as a prognostic factor for the outcome of clinical pregnancy, with a higher probability of pregnancy in women who had an oestradiol/oocyte ratio <500 pmol/ml (140 pg/ml). This is consistent with the findings of a previous study, in which Loumaye et al. (1997) also found that an oestradiol/oocyte ratio >140 pg/ml was associated with poorer pregnancy rates than lower values. Studies by Simon et al., (1995) and Pellicer et al., (1996) have also suggested that there is an association between high levels of oestradiol secretion and implantation failure. Comparative studies of r-hfsh and u-hfsh have suggested that the cumulative dose of the recombinant product which is required to provide adequate follicular stimulation is ~30% less than that of u-hfsh (Bergh et al., 1997; Frydman et al., 1998; Khalaf et al., 1998). The average of 28 ampoules of 75 IU follitropin-alfa per cycle which was used in the present study is in accordance with the doses used in previous investigations of r-hfsh treatment. No treatment emergent adverse events were reported in the unselected patient population that was the subject of the present study that had not previously been observed in the controlled clinical studies, providing further evidence of the good tolerability of follitropin-alfa. Furthermore, the incidence of OHSS was low, occurring in fewer than 2% of cycles, and only six cases of severe OHSS were reported. As the overall clinical pregnancy rate of 39.5% per initiated cycle is significantly higher than the mean pregnancy rate reported by the German National IVF registry (24-25% per embryo transfer), a number of factors may address this apparent discrepancy. Our series consisted of 1098 cycles, approximately 3.4% of the cycles reported in the German National IVF registry during the same period (June 1996 December 1997). Whereas the cycles reported to the registry represent all cycles, regardless of gonadotropin stimulation

6 regimen, the current report addresses only those cycles utilising follitropin-alfa. Additionally, selection bias may be a confounding factor as physicians selected patient candidates deemed appropriate for treatment with follitropin-alfa. Support for this observation are noted in the high percentage (69.9%) of first cycles represented by this study. (Furthermore the probability of clinical pregnancy decreased on average by 47.6% for repeat cycles relative to the first cycle of follitropinalfa stimulation.) The national registry does not differentiate between outcomes of first versus subsequent treatment cycles, therefore it is not possible to definitively attribute the contribution of the first cycle benefit to our results. The percent of cycles reaching ovulation induction (98.5% of initiated cycles) in this study is the same as the national registry rate (99.9% in 1997). Similar to the national registry report, three embryos were transferred in vast majority of cycles in this study (50% and 66.2%, respectively), reflecting the preference of patients to transfer the maximum number of embryos in compliance with the German Embryo Protection Law. In conclusion, the results of this prospective, observational study in an unselected population of women undergoing IVF procedures have shown that follitropin-alfa is an effective and well tolerated product for ovulation induction in routine clinical practice. In addition, it appears to provide a high probability of pregnancy with one cycle of treatment while permitting the use of a lower total dose of FSH than with u-hfsh preparations, and thereby may confer a lower risk of OHSS. Furthermore, follitropin-alfa may be injected subcutaneously, thus providing the possibility of self-administration and therefore greater convenience for the patient. References Albano C, Smitz J, Camus M et al Pregnancy and birth in an in-vitro fertilisation cycle after controlled ovarian stimulation in a woman with a history of allergic reaction to human menopausal gonadotrophins. Human Reproduction 11, Bergh C, Howles CM, Borg K et al Recombinant human follicle stimulating hormone (r-hfsh; Gonal-F) versus highly purified urinary FSH (Metrodin HP): results of a randomized comparative study in women undergoing assisted reproductive techniques. Human Reproduction 12, Brinsden P, Akagbosu F, Gibbons LM et al A comparison of the efficacy and tolerability of two recombinant follicle-stimulating hormone preparations in patients undergoing in vitro fertilizationembryo transfer. Fertility and Sterility 73, 114. Daya S 1997 Optimal protocol for gonadotrophin releasing hormone agonist use in ovarian stimulation. Proceedings of the 10th World Congress on In-Vitro-Fertilisation and Assisted Reproduction, Vancouver, Frydman R, Avril C, Camier B et al A double-blind, randomised study comparing the efficacy of recombinant follicle stimulating hormone (r-hfsh/gonal-f) and highly purified FSH (u-hfsh HP/Metrodin HP) in inducing superovulation in women undergoing assisted reproductive techniques (ART) (abstract O- 185). Human Reproduction 13, 94. Giudice E, Crisci C, Eshkol A et al Composition of commercial gonadotrophin preparations extracted from human postmenopausal urine: characterization of non-gonadotrophin proteins. Human Reproduction 9, Hasford J, Lamprecht T 1998 Company observational post-marketing studies: drug risk assessment and drug research in special populations - a study-based analysis. European Journal of Clinical Pharmacology 53, Hershlag A, Kaplan EH, Randall AL et al Heterogeneity in patient populations explains the difference in in vitro fertilisation programs. Fertility and Sterility 56, Howles CM 1996 Genetic engineering of human FSH (Gonal-F). Human Reproduction Update 2, Howles CM, Wikland M 1999 The use of recombinant human FSH in in vitro fertilisation. In Shohan Z, Howles CM, Jacobs HS (eds) Female infertility therapy: Current practice. Martin Dunitz, London, UK, Khalaf Y, Taylor A, Pettigrew R et al The relative clinical efficacy of recombinant human follicle stimulating hormone and the highly purified urinary FSH preparation (abstract P-955). Human Reproduction 13, 191. Loumaye E, Engrand P, Howles CM et al Assessment of the role of serum luteinizing hormone and estradiol response to follicle-stimulating hormone on in vitro fertilisation treatment outcome. Fertility and Sterility 67, Pellicer A, Valbuena D, Cano F et al Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period. Fertility and Sterility 65, Recombinant Human FSH Product Development Group 1998 Recombinant follicle stimulating hormone: development of the first biotechnology product for the treatment of infertility. Human Reproduction Update 4, Redfearn A, Hughes EG, O Connor M et al Delayed-type hypersensitivity to human gonadotrophin: case report. Fertility and Sterility 64, Simon C, Cano F, Valbuena D et al Clinical evidence for a detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients. Human Reproduction 10, Steelman SL, Pohley FM 1953 Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotrophin. Endocrinology 53, Tan SL, Royston P, Campbell S et al Cumulative conception and live birth rates after in vitro fertilisation. Lancet 339, Appendix The Gonal-F Study Group (Name, location and patient enrolment) Dr. med. F. Abu Hmeidan, Leipzig - 24; Prof. Dr. med. H. Alexander, Leipzig - 10; Dr. med. T. Bäckert, Tübingen - 11; Dr. med. I. Baron, Bielefeld - 22; Dr. med. L. Belkein, Münster - 17; Prof. Dr. med. F.D. Berg, München - 14; Dr. med. M. Bloechle, Berlin - 18; Dr. med. P. Böhm, Göttingen - 1; Dr. med. K. Brandenburg, Kiel - 47; Dr. med. C. Dorn, Bonn - 10; Dr. med. K.-U. Ebner, Deggendorf - 1; Dr. med. R Felberbaum, Lübeck - 13; Dr. med. A Haase, DRK Berlin - 1; Dr. med. M. Heeder, Oldenburg - 10; Dr. med. P Kaltwaßer, Halle - 26; Dr. med. T Katzorke, Essen - 94; Dr. med. C. Keck, Freiburg - 4; Dr. med. P. Klare, Berlin - 2; Dr. med. S. Klein, Hannover - 20; Prof. Dr. med. J. Kleinstein, Magdeburg - 14; Dr. med. B.T. Krause, Greifswald - 4; Dr. med. W. Maletz-Kehry, Heidelberg - 5; Dr. med. F. Nawroth, Neubrandenburg - 11; Dr. med. U. Noss, München - 10; Dr. med. Ö. Özdemir, Köln - 69; Prof. Dr. med. A.E. Schindler, Essen - 10; Dr. med. K. Scholz, Köln - 63; Dr. med. T. Schreiner, Berlin - 11; Dr. med. M.J. Seeler, Hamburg - 1; Dr. med. D. Seehaus, München, Klinikum Großhadern - 15; Prof. Dr. med. B. Seifert, Regensburg - 6; PD Dr. med. E.R. Siebzehnrübl, Erlangen - 16; Dr. med. M. Smolka, München, Frauenklinik Dr. Krüsmann - 2; Dr. med. G. Sonntag, Köln - 2; PD Dr. med. W. Starker, Jena - 16; Dr. med. T. Steck, Würzburg - 1; Prof. Dr. med. K. Sterzik, Ulm - 11; Dr. med. P. Sydow, Berlin - 11; Dr. med. A. Ularik, Münster - 11; Dr. med. H.C. Verhoeven, Düsseldorf - 80; Dr. med. W. von der Burg, Osnabrück - 12; Dr. med. A. von Stutterheim, Bremen - 20; Dr. med. G. Wilke, Hildesheim - 12; Dr. med. T. Wilken, Hannover