Article Routine use of r-hfsh follitropin alfa filled-bymass for follicular development for IVF: a large multicentre observational study in the UK

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1 RBMOnline - Vol 9. No Reproductive BioMedicine Online; on web 12 October 2004 Article Routine use of r-hfsh follitropin alfa filled-bymass for follicular development for IVF: a large multicentre observational study in the UK Dr Amir Lass Dr Amir Lass was born in Israel in After 5 years military service as an officer in the navy, he obtained his MD degree from the Hebrew University Jerusalem in 1987, and completed the residency in obstetrics and gynaecology at Meir Hospital affiliated to Tel Aviv University in In order to specialize in reproductive medicine he took a post as research fellow at the IVF unit, Hammersmith Hospital, London, UK from , and then as consultant at Bourn Hall clinic from During that period he studied for and was awarded the MBA degree in London. Amir joined Serono in 2000 as Corporate Medical Director Reproductive Health, responsible for a few phase II III clinical trials and since April 2001 he has been Serono s Regional Medical Director Northern Europe. His research interests include ovarian reserve and ageing and he has published extensively on this topic and others. Amir lives in London, is married and has six children. A Lass 1,3, E McVeigh 2 on behalf of the UK Gonal-f FbM PMS Group 4 1 Serono Ltd, Feltham, Middlesex, London, 2 Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford, UK 3 Correspondence: Bedfont Cross, Stanwell Road, Feltham, Middlesex TW14 8NX, UK. Tel: ; Fax: ; amir.lass@serono.com 4 Participating centres are listed at the end of the article Abstract The technical advance of recombinant biotechnology to manufacture FSH has resulted in a biochemically pure FSH preparation with high batch-to-batch consistency. The protein content of recombinant human FSH (r-hfsh) can be reliably quantified in mass units by size exclusion high performance liquid chromatography (SE HPLC) enabling r-hfsh follitropin alfa (Gonal-F ) to be filled and released in the vial on the basis of mass with dose variability of only ±2% [filled-by-mass (FbM)]. Observational studies have merit in addressing the effectiveness of a new product in routine clinical practice. This non-interventional study assessed r-hfsh follitropin alfa FbM for the multifollicular stimulation of patients undergoing assisted reproduction. A total of 1427 patients were recruited in 21 centres in the UK and the mean age was 34.3 years (±4.48, range 18 48). Of those who started treatment, 1388 (97.3%) received human chorionic gonadotrophin and 1330 (93.2%) underwent oocyte retrieval. A total of 1213 (85%) patients had embryo transfer and in the majority of recorded cases, most embryos replaced were graded as 1 or 2 (74.6%). The ongoing clinical pregnancy rate was 29.2% per cycle and 34.4% per embryo transfer. The routine use of r-hfsh follitropin alfa FbM in normal clinical practice in IVF has been demonstrated to be effective, safe and well received by patients. Keywords: assisted reproduction, filled-by-mass, follitropin alfa, FSH, gonadotrophins 604 Introduction FSH is a complex heterodimeric glycoprotein used therapeutically to stimulate follicular development in the ovary. Current manufactured gonadotrophin preparations for therapeutic use are quantified by the Steelman Pohley rat invivo bioassay (Steelman and Pohley, 1953). Briefly, immature female rats (21 22 days old) pretreated with human chorionic gonadotrophin (HCG) are injected with a production batch of exogenous FSH once daily for 3 days and the ovaries weighed following autopsy. A direct linear relationship has been shown between ovarian weight gain and FSH administered (Steelman and Pohley, 1953) and the FSH bioactivity is measured in terms of international units (IU). However, this technique has inherent limitations due to the very nature of measurement of rat ovarian weight gain and results in a variability of 10 20% (Steelman and Pohley, 1953). A vial that nominally contains 75 IU of FSH on the basis of an initial assay meets the % (i.e IU) acceptance range and the % (i.e IU) fiducial limits. However, in reality the vial may contain anything from 48 IU to 117 IU FSH (Driebergen and Baer, 2003). The technical advance of recombinant biotechnology to manufacture FSH has resulted in a biochemically pure FSH preparation with high batch-to-batch consistency (Driebergen

2 and Baer, 2003). The degree of glycosylation of the gonadotrophin molecule can be specified and quantified using methods such as isoelectric focusing (IEF) (Siebold, 1996) and glycan mapping (Gervais et al., 2003). The protein content of recombinant human FSH (r-hfsh) can be reliably quantified in mass units by size exclusion high performance liquid chromatography (SE HPLC). These recent developments have enabled r-hfsh follitropin alfa (Gonal-F ) to be filled and released in vials on the basis of mass. As there is a constant relationship between FSH mass (μg) and bioactivity (Driebergen et al., 2002), the filled-by-mass (FbM) vial delivers a dose with variability of only ±2%. Recent studies (Neuspiller et al., 2002; Hugues et al., 2003; Balasch et al., 2004) have shown the r-hfsh follitropin alfa FbM to be bioequivalent to r-hfsh filled-by-bioassay. However, in the study by Hugues et al., the FbM batches appeared to deliver a more consistent therapeutic effect than the fill-by-bioassay batches. There was a smaller difference between the mean resultant oocytes retrieved (P = 0.039) and clinical pregnancy rate (P = 0.001) for FbM than for filled-bybioassay batches, implying a more consistent ovarian response with r-hfsh follitropin alfa FbM. To date, studies published using the new FbM preparation have been randomized controlled trials (RCT) with small patient numbers and by the very nature of design, select patient groups. Whilst RCT are the gold standard for the basis of strategy and consensus, observational studies or postmarketing surveillance (PMS) have a place in assessing the consequences of these treatments (Barlow, 2004). There is merit in addressing the effectiveness of a new product in routine clinical practice and in an unbiased population, so that the clinician can address product functionality in his or her clinic. For this reason, the largest units in the UK were invited to participate in a non-interventional observational study of r-hfsh follitropin alfa FbM for the multifollicular stimulation of patients prior to IVF/intracytoplasmic sperm injection (ICSI). In this way, r-hfsh follitropin alfa FbM could be assessed in a practical setting on a large scale encompassing all patient groups and treatment practices. The aim of this large, multicentre, prospective noninterventional study is to assess the safety and effectiveness of r-hfsh follitropin alfa FbM in a normal clinical practice across the UK over a period of 6 months immediately following its introduction to the UK. Materials and methods The new formulation of r-hfsh follitropin alfa FbM (Gonal- F FbM; Serono Ltd, Feltham, Middlesex, UK) has been available in the UK since May Twenty-one IVF centres throughout the UK participated in this non-interventional study between May and December 2003 (of 30 units which have been approached). The aim was to recruit a total of about 1500 patients, which was approximately 10% of the national total of IVF cycles performed in this period. Centres were asked to recruit around 60 consecutive patients presenting for an IVF cycle in whom r-hfsh follitropin alfa was suitable. Each patient was eligible only once for the PMS. There were no pre-selection criteria or randomization procedures. Each patient provided written informed consent to their IVF treatment cycle using consent forms specific to each unit. Each patient was free to stop treatment at any time. Treatment for follicular stimulation followed each unit s normal practice; controlled LH surge was employed in all units either by antagonist or agonist. r-hfsh follitropin alfa was available as freeze-dried powder plus solvent for solution, in a multidose vial of either 450 or 1050 IU or monodose vial of 75 IU (Serono Ltd, Feltham, Middlesex, UK) and administered according to the summary of product characteristics (SPC) for marketed products. Patients were shown how to selfadminister the injections using the monodose or multidose vial by a trained nurse. Follicular tracking, HCG administration and luteal support were performed according to each unit s own practice. Data were collected on a specified form and comprised of demographics, stimulation performance, clinical outcomes, embryology and nurse assessment on the ease of use of the multidose vial. Patient anonymity was maintained at all times. Statistical methods As this was a non-comparative, observational study, the evaluation of r-hfsh follitropin alfa FbM effectiveness was made on the basis of descriptive statistics. Safety evaluation was based on the cancellation rates due to risk of ovarian hyperstimulation syndrome (OHSS), multiple pregnancy rates and OHSS incidence. Where appropriate, certain data were presented as mean values ± SD. As with most PMS studies, a degree of missing data is inevitable. Where missing data occurred, the data was assumed to be missing at random and summaries and percentages were based on data available. For this reason, the number of cases reported for each variable might be less than the total number of patients assessed. Results Patient disposition and demographics A total of 1427 patients were recruited from 21 IVF centres. The average recruitment rate was 68 patients per centre (range ). The mean age of the patients was 34.3 years (SD ±4.48, range 18 48) with an average history of infertility of 4.24 years (SD ±2.80, 0 22). Figure 1 illustrates the number of previous treatment cycles patients had received prior to commencing the study. As there were no pre-selection criteria, a significant proportion (9.2%) had received more than three previous treatment cycles. A total of 843 (60.7%) patients (number available = 1388) had suffered from primary infertility. The causes of infertility are described in Table 1. For those patients in whom FSH concentrations in the 6 months prior to treatment were recorded (n = 750), the mean FSH concentration was 7.2 IU/l (SD ±2.55, ). A mean LH concentration of 4.8 IU/l prior to treatment was recorded for 449 patients. Stimulation results Table 2 details the treatments used during the cycle. Most patients received daily doses of gonadotrophin-releasing hormone (GnRH) agonists (84.5%). Of the 1427 patients who started treatment, 1388 (97.3%) received HCG and

3 Figure 1. Treatment cycle number. Table 1. Cause of infertility. Cause Number of patients (%) Tubal factor 237 (16.6) Male factor 482 (33.8) Unexplained infertility 217 (15.2) Endometriosis 74 (5.2) Anovulation 75 (5.3) More than one cause 252 (17.7) Other (not specified) 60 (4.2) No reason specified 30 (2.1) Total 1427 (100) Table 2. Proportion of patients using analogues and luteal phase support medications. Product Details No. of patients No. of patients (% of total; undergoing embryo total = 1427) transfer (% of total; total = 1213) GnRH agonist Daily 1208 (86) Long-acting 92 (6.6) GnRH 105 (7.5) antagonist Luteal phase Progesterone a 911 (87.2) support Progesterone b 70 (6.7) Progesterone c 11 (1.1) Progesterone d 3 (0.3) HCG 50 (4.7) Data not available 22 (1.5) 168 (13.8) a,b,c Commercial products from Serono: Cyclogest, Crinone gel, Gestone, respectively. d Product not specified. 606

4 (93.2%) underwent oocyte retrieval. Ninety-seven cycles (6.7%) were abandoned, mainly due to poor response to stimulation and 17 patients (1.2% of the total patients recruited) had a cycle cancelled due to risk of OHSS (Table 3). The stimulation results are described in Table 4. An average of 5.6 (SD ±4.6) follicles 14 mm and 4.0 (SD ±3.0) 18 mm were observed on the day of HCG. The mean number of oocytes retrieved was 10.3 (SD ±6.2). A total of 1213 (85%) patients had embryo transfer (mean 2.0 embryos per transfer). One hundred and seventeen patients who underwent oocyte retrieval did not have embryo transfer and the reasons are listed in Table 5. In the majority of recorded cases, two embryos (945, 78.0%) were put back. A single embryo transfer was performed in 103 cycles (8.5%) and in 163 cycles three embryos were transferred (13.4%). Most of the transferred embryos were graded as 1 or 2 (74.6%). The majority of patients (63.8%) received Cyclogest (Alfarma Ltd, UK; progesterone 200 or 400 mg vaginal or rectal suppository) for luteal support (Table 2). Of the 1427 patients treated, 478 had a positive HCG (33.5% per cycle, 39.4% per embryo transfer). At the time of writing, 417 patients (29.2% per cycle, 34.4% per embryo transfer) had an ongoing pregnancy >12 weeks. Eighty-three patients (19.9%) had a multiple pregnancy but only one of these was a triplet pregnancy. r-hfsh follitropin alfa FbM appears to be safe and well tolerated. Only 83 cases of OHSS (5.8% of patients) were reported, the majority being mild or moderate in nature (75 incidences, 5.3% of patients). Five cases of severe OHSS (0.4% of patients) occurred and only two were reported as requiring hospitalization with full recovery. All other cases resolved following routine management by the clinic. Table 3. Patient disposition during the course of the study. Numbers in parentheses are percentages. No. of patients Total no. of patients recruited 1427 No. of patients receiving HCG 1388 (97.3) No. of patients undergoing OPU 1330 (93.2) No.undergoing embryo transfer 1213 (85.0) Total no. with positive HCG 478 (33.5) Spontaneous abortion 60 Extrauterine pregnancy 1 Total with a pregnancy ongoing 417 (29.2) >12 weeks Ongoing clinical pregnancy rate per 34.4 embryo transfer, % Total no. of multiple pregnancies 83 (19.9) Two sacs 82 Three sacs 1 Total no. reporting OHSS 83 Mild 51 (3.5) Moderate 24 (1.7) Severe 5 (0.4) Unspecified 3 (0.2) Values in parentheses are percentages. Table 4. Stimulation profile of IVF cycle. Stimulation profile No. of patients Data as entered on with available data form (mean ± SD) Total r-hfsh follitropin alfa FbM dose (IU) ± 1136 Days of stimulation ± 2.3 Oestradiol concentration (pmol/ml) ± 4233 No. of follicles 14 mm on day of HCG ± 4.6 No. of follicles 16 mm on day of HCG ± 3.2 No. of follicles 18 mm on day of HCG ± 3.0 No. of oocytes retrieved ± 6.2 No. of available embryos ±

5 608 Table 5. Reasons for not performing embryo transfer following oocyte retrieval. Cause No oocytes recovered 6 Failure to fertilize/cleavage or poor 69 quality embryos Spermatozoa not available 2 Poor endometrium 2 Freezing embryos due to risk of OHSS 20 Freezing before chemotherapy 3 Unspecified/other 15 Total 117 Nurses trained most patients to handle the multidose injections and of those patients asked, 97.6% (1318) viewed the dosing flexibility with the multidose as an advantageous and simplified method. Discussion No. of cases The data from this study represent a large snapshot of the IVF patient population treated in the UK during the latter half of All patient groups were included and routine clinical practice observed. r-hfsh follitropin alfa FbM was demonstrated to be effective and safe in normal clinical practice. A high number of oocytes retrieved (10.3 ± 6.2), high quality embryos (70.8% graded 1 or 2) and high pregnancy rate (clinical pregnancy rate per cycle 29.2%) with a low multiple rate were achieved, which stand up favourably against published data (Bergh et al., 1997; Daya and Gunby, 1999; Bhattacharya et al., 2001; Katzorke et al., 2001) and current published results from the UK Human Fertilisation and Embryology Authority (HFEA) national registry (21.8%; HFEA, 2004). It is readily accepted that this study has some limitations. It was an observational and non-comparative assessment. As previously stated, small studies have been performed comparing r-hfsh follitropin alfa FbM to r-hfsh follitropin alfa fill-by-bioassay and have shown them to be bioequivalent, so it was felt that there was little additional merit in repeating this assessment on a larger scale. This study covered a variety of treatment protocols in various units; the only true constant being the use of r-hfsh follitropin alfa FbM for stimulation. In addition, the study was a PMS study conducted primarily by the clinicians such that monitoring of data collection was minimal. There is no information on which patients have been selected to enrolment; however, because the request was for a consecutive patients on r-hfsh, it can be assumed that there was no or limited bias in selecting patients. Missing data were inevitable, as were some data inconsistencies, so the analysis could only be based on what was actually entered on the database from the record forms. Therefore, some of the proportions calculated vary from variable to variable and the results must be viewed in this context. That said, the sample size assessed is still large by IVF study standards and generates some interesting results in terms of effectiveness considering the variability of the patient group. The age range was wide (34.3 years, range 18 48) and previous multiple failure cycles were included (9.2% more than three previous cycles): a group of patients usually excluded from RCT. The aim of this study was to assess r-hfsh follitropin alfa FbM in routine clinical practice and the results have shown that even with a wide range of patient types, results are comparable to published data performed in RCT (Bergh et al., 1997; Daya and Gunby, 1999). The advantage of filled-by-mass technology is the greater control over dose of FSH delivered and associated improved consistency of clinical response. It is well established that patients responses to ovarian stimulation are variable, impacting on clinical pregnancy rates as they are directly related to quantity and quality of embryos obtained and replaced, both prognostic factors for conception (Templeton and Morris, 1998). Interestingly, the overall pregnancy rate was also calculated on the basis of age. It was found that in patients aged less than 35 years, 37.5% had a pregnancy (316 out of 842). This is a favourable result from a multicentre study using different treatment protocols. In addition, the overall pregnancy rate in patients aged years was 27.7% and 19.1% in those aged over 40. It is well established that pregnancy rates following IVF treatment decline after the age of 35 years and again after 40 (Segal and Casper, 1990, Kenny, 1994, Dor et al., 1996, Tan et al., 1992), and may be related to declining embryo quality (Minaretzis et al., 1998) amongst other factors. However, the pregnancy rates achieved in this study in the older patients suggest that embryo quality is not the overriding factor and may not be greatly compromised; patients aged over 40 for example had a clinical pregnancy rate of 12.1%, a miscarriage rate of 6.9%. The embryo data collected suggested no differences in grading or quality of those embryos transferred between age groups, at least from morphological aspect. The low rate of serious adverse events, only five cases of severe OHSS, all of them resolved without complication, demonstrated the safety profile of the new formulation, which may reflect the consistency and accuracy of the delivered dose. By removing the variability of dose administered, the clinician is therefore better able to predict and manage patient treatment, particularly in the outlier patient groups. Now that this study has demonstrated the effectiveness of r-hfsh follitropin alfa FbM in routine practice, the logical step forward might be to assess and tailor treatment protocols in these problem patients. The data in this study have also provided an interesting observation on current practice of IVF treatment protocols in the UK, which was not an aim of the original study design. Due to its size, which reflects ~10% of the cycles performed in the UK in this period, and that there were no recommendations or limitation on choice of ovarian stimulation protocol, it can be assumed that the findings reflect accurately the normal current practice in the UK. The resulting data indicate that more than 90% of patients treated received GnRH agonists, presumably because these facilitate cycle programming and are efficacious (Loumaye, 1990; Hughes et al., 1992). The use of GnRH antagonists was limited to 10% of cycles; data on resulting pregnancy rates (Ludwig et al., 2001; Al-Inany and Aboulghar, 2003) and reduced cycle control are likely to be contributing factors in the lack of confidence in this new preparation. Half of the units screen for FSH concentrations

6 prior to treatment and a third screen for baseline LH. There are no data as to whether the results of the screening played any role in the decision to exclude patients from treatment, but it is a possible outcome as the highest recorded concentration was 20 miu/ml. One can assume that patients with basal FSH concentrations higher than 20 miu/ml have been excluded from treatment. The recent National Institute for Clinical excellence (NICE) guideline on infertility treatment in the UK discusses the merit of ovarian reserve tests including the basal FSH test in length and summarizes that tests of ovarian reserve do not currently have the necessary sensitivity or specificity for general application (NICE, 2004). In the majority of cycles (78%), only two embryos were transferred. The study was conducted in the immediate period before the ruling of the HFEA that (i) women aged under 40 at the time of transfer receive no more than two embryos in any one cycle, regardless of the procedure used; (ii) women aged 40 or over at the time of transfer receive no more than three embryos in any one cycle, regardless of the procedure used (HFEA, Code of Practice, 2004). However, the recently published data from the HFEA showed that in 47.6% of cycles, two embryos have been electively replaced. It is therefore encouraging to note the positive trend to reduce the number of embryos transferred whilst maintaining high clinical pregnancy rates and it is worth noting that only a single triplet pregnancy has been reported in this study. Of those patients who were recorded as undergoing embryo transfer and for whom information was supplied regarding type of luteal phase support, 95.2% received progesterone either vaginally (93.9%) or intramuscularly (1.3%). As the literature associates improved pregnancy rates with luteal support (Soliman et al. 1994), this is not surprising. However, vaginal pessaries were most commonly used despite some controversy in the literature suggesting a better clinical outcome with intramuscular injection (Pritts et al., 2002). The ease of use of pessaries is the probable reason for this choice. Patient convenience is a factor in the determination of treatment protocols. The introduction of a multidose vial for r-hfsh follitropin alfa FbM was seen to be advantageous for the patient and nurse in terms of ease of administration, as it simplified the reconstitution procedure that is otherwise required with the monodose vial. These findings add weight to the value of post-marketing studies that act as a pharmaco-epidemiological investigation into current practice. Whilst the shortcomings of this study have been discussed, the design is important in validating outcomes orientated research (de Mey, 2000) and in this case, the clinical pregnancy rate in routine practice following r-hfsh follitropin alfa FbM administration that could not be gleaned from RCT data. Moreover, recently published commentary on the value of observational non-randomized studies concluded that they provide a useful method for generating and testing hypothesis, often providing equivalent results to randomized trials (Concato and Horwitz, 2004). In conclusion, this observational PMS study has demonstrated r-hfsh follitropin alfa FbM to be effective and safe in routine practice use. Acknowledgements The authors would like to thank Dr Susan Bogle of Aysgarth Statistics for the statistical analysis of data and Mrs Hilary Hurst for assisting in writing the manuscript. Participating Centres In the UK, the Gonal-f PMS Group included: Assisted Reproduction Unit, Aberdeen Maternity Hospital; Bridge Fertility Clinic, London; Assisted Conception Unit, Chelsea and Westminster Hospital, London; Fertility Unit, Chelsfield Park Hospital, Orpington; Centre for Reproductive Medicine, London; Assisted Conception Unit, Guy s and St Thomas Hospital, London; Assisted Conception Unit, Hammersmith Hospital, London; Harley Street Fertility Centre, London; Essex Fertility Centre; The Oxford Fertility Unit, John Radcliffe Hospital, Oxford; Assisted Conception Unit, King s College Hospital, London; Assisted Conception Unit, Lister Hospital, London; Assisted Conception Unit, Ninewells Hospital, Dundee; Midland Fertility Services, Aldridge; Nuffield Hospital; Woking Fertility and Reproductive Endocrine Unit; Royal Infirmary Hospital, Edinburgh; Sheffield Fertility Centre (CARE); Three Shires Hospital, Northampton (CARE); Department of Obstetrics and Gynaecology, Queen Mary s Hospital, Sidcup; Centre for Reproductive Medicine and Surgery St Bartholomew s Hospital, London; CARU, University Hospital of Wales, Cardiff, UK References Al-Inany H, Aboulghar M 2001 Gonadotrophin-releasing hormone antagonists for assisted conception. 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