A randomised controlled trial of 300 versus 225 IU recombinant FSH for ovarian stimulation in predicted normal responders by antral follicle count

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1 DOI: /j x Fertility and assisted reproduction A randomised controlled trial of 300 versus 225 IU recombinant FSH for ovarian stimulation in predicted normal responders by antral follicle count K Jayaprakasan, J Hopkisson, B Campbell, I Johnson, J Thornton, N Raine-Fenning Nottingham University Research and Treatment Unit in Reproduction (NURTURE), Division of Human Development, School of Clinical Sciences, University of Nottingham, Nottingham, UK Correspondence: Dr K Jayaprakasan, NURTURE, B Floor, East Block, Queen s Medical Centre, Nottingham, Nottinghamshire NG7 2UH, UK. k.jayaprakasan@nottingham.ac.uk Accepted 10 February Published Online 29 March Objective To test the hypothesis that among women predicted to have a normal ovarian response, ovarian stimulation using 300 IU follicle-stimulating hormone (FSH) results in the retrieval of more mature oocytes than 225 IU during in vitro fertilisation (IVF)/ intracytoplasmic sperm injection (ICSI) treatment. Design Prospective randomised controlled study. Setting University-based assisted conception unit. Population A cohort of 131 women predicted to have a normal ovarian response to gonadotrophin stimulation, based on antral follicle count. Methods Subjects undergoing their first cycle of IVF/ICSI were randomised to receive a fixed daily dose of 300 (experimental arm) or 225 IU (control arm) of recombinant FSH (Gonal-F). Main outcome measures Number of mature oocytes retrieved and live birth rates. Results The number (mean ± standard deviation) of mature oocytes retrieved (8.2 ± 5.0 versus 9.0 ± 4.8, for 300 and 225 IU, respectively; P = 0.34) was similar in each group. There were no differences between the 300- and 225 IU arms in live birth rates (31 versus 41%, respectively; P = 0.25), cycle cancellations resulting from insufficient ovarian response (0 versus 6.1%, respectively; P = 0.12), and prevalence of moderate (3.1 versus 3.0, respectively; P = 1.0) and severe (0 versus 1.5%, respectively; P = 1.0) ovarian hyperstimulation syndrome. Conclusions The use of a higher daily dose of 300 IU of recombinant FSH for ovarian stimulation does not improve the number of mature oocytes retrieved, or live birth rates, among women with a predicted normal response during conventional IVF/ICSI. Keywords Antral follicle count, IVF/ICSI outcome, ovarian response, predicted normal responders, randomised controlled trial, recombinant gonadotrophin. Please cite this paper as: Jayaprakasan K, Hopkisson J, Campbell B, Johnson I, Thornton J, Raine-Fenning N. A randomised controlled trial of 300 versus 225 IU recombinant FSH for ovarian stimulation in predicted normal responders by antral follicle count. BJOG 2010;117: Introduction The aim of ovarian hyperstimulation during conventional in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) treatment is to obtain sufficient potentially fertilisable, mature oocytes and embryos to allow selection and transfer of the best to achieve a healthy singleton pregnancy, and to leave a sufficient number of good-quality embryos to cryopreserve so as to optimise the total reproductive potential of the couple. 1 An ideal ovarian stimulation protocol should induce an intermediate response that is within the margins of safety, although pregnancies occur following both poor and exaggerated ovarian responses. Follicle-stimulating hormone (FSH) is the key hormone required to induce multifollicular development during controlled ovarian stimulation (COH). The optimum dose of FSH required to exceed the FSH threshold during the therapeutic FSH window, 2 in order to achieve an optimum ovarian response, has not yet been determined and, therefore, there is a wide variation in the dose of gonadotrophins that are used for ovarian stimulation between different assisted conception units. The knowledge of the ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology 853

2 Jayaprakasan et al. most effective daily dose of FSH in predicted intermediate responders will allow the development and assessment of treatment protocols in predicted poor and exaggerated responders, through adjustments in the dose or by the addition of luteinising hormone (LH). Several prospective, randomised controlled trials have compared different starting doses of recombinant FSH (rfsh) using gonadotrophin-releasing hormone (GnRH) agonist protocols in predicted intermediate responders. 3 9 The primary outcome measure in all of these studies was the number of oocytes retrieved. Although some reported an increased retrieval of oocytes with higher doses of FSH, these findings are by no means consistent. Whereas one group compared 225 with 150 IU of rfsh, 3 the others compared 100 and 200 IU, or 150 and 250 IU, of rfsh. Moreover, to date there have been no reported studies examining the effect of 300 IU of rfsh, which may offer an even greater improvement in ovarian response, and lower cancellation rates as a result. Although all of these studies were performed in predicted intermediate responders, this was determined on the participants age and/or basal FSH levels, which are not the most sensitive or specific markers of ovarian reserve. 10,11 Furthermore, according to the Rotterdam consensus, 12 a proportion of these participants would also have polycystic ovary syndrome (PCOS), which is a recognised risk factor for exaggerated ovarian response and poorer oocyte quality. 13,14 Recent data suggest that the total antral follicle count (AFC) is the single best predictor of ovarian response when compared with conventional markers, including age and basal FSH. 15,16 Selection of participants based on total AFC would ensure a more appropriate study group of intermediate responders, and this approach permits the use of a higher starting dose of gonadotrophin (300 IU), which may potentially allow the development of more follicles and the retrieval of more oocytes, with a subsequent increase in the cumulative pregnancy rate, without compromising the safety of the treatment. Recent work has suggested that a total AFC of seven or less identifies, with excellent sensitivity and specificity, women who are likely to have a poor response to ovarian stimulation. 17 In contrast, participants with a total AFC of 22 or more are likely to have PCOS, and are more prone to develop ovarian hyperstimulation syndrome (OHSS). 18 Therefore, in this study we considered women with a total AFC of 8 21 as being potential intermediate responders. The main objective of this study was to test the hypothesis that increasing the daily fixed dose of FSH from 225 to 300 IU in participants undergoing IVF/ICSI treatment using the GnRH agonist long protocol, and who are predicted to have a normal response to ovarian stimulation based on their total AFC, results in an improved ovarian response, as manifested by the retrieval of a higher number of mature oocytes without compromising safety. Methods Experimental design The study was designed as a prospective, randomised controlled trial. The participants were undergoing their first cycle of assisted reproduction treatment at Nottingham University Research and Treatment Unit in Reproduction (NURTURE), between September 2006 and March At screening, all participants under 39 years of age underwent a 3D transvaginal ultrasound assessment, and a blood test for the estimation of their basal FSH and LH levels. The transvaginal scan was performed by a single investigator (KJ) using a Voluson Expert 730Ô (General Electric Medical Systems, Zipf, Austria) and a 4D 5 9-MHz transvaginal transducer. Our ultrasound examination technique has been described in detail before, 19 but briefly comprised the acquisition of two volumetric data from each ovary, one with greyscale and the other with power Doppler information, which were stored for subsequent off-line analysis using the 4D view programme (v6.0; General Electric Medical Systems). Antral follicles measuring 2 10 mm in diameter were counted using the multiplanar view method, 17 and ovarian volume was calculated using the VOCAL technique, 19 in which the user manually defined the ovarian cortex in a series of image planes as the data set was rotated about 180 through 9 rotation steps. Quantification of power Doppler information within the resultant 3D ovarian model was performed using the histogram facility to generate three indices of ovarian vascularity: vascularity index (VI), flow index (FI) and vascularity flow index (VFI). Two measures of antral follicle number, ovarian volume and vascularity indices were performed, and the mean values were used for analysis. The AFC was expressed as the total value for both ovaries, and the ovarian volume and vascularity indices were expressed as the mean of the two ovaries. All measures were performed by a single observer (KJ), whose reliability was confirmed by mean intraclass correlation coefficients and 95% confidence intervals of ( ), ( ), ( ), ( ) and ( ), for the measurement of the number of antral follicles, ovarian volume and the vascularity indices VI, FI and VFI, respectively. The study was approved by the UK s National Health Service (NHS) research ethics committee, the Medicines and Healthcare products Regulatory Agency (MHRA), and the Nottingham University Hospital NHS Trust Research & Development (R&D) department. Informed, written consent was obtained prior to the enrolment of each participant. The trial was conducted in accordance with the 854 ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology

3 RCT of 300 versus 225 IU of rfsh ethical principles that have their origin in the Declaration of Helsinki, 1996, the principles of Good Clinical Practice, in accordance with the Medicines for Human Use Regulations, Statutory Instrument 2004, 1031 and its subsequent amendments, and the Department of Health Research Governance Framework for Health and Social Care, The research committee within the School of Human Development, University of Nottingham, monitored the data from the study regularly. Participant selection Participants below the age of 39 years were included if their basal FSH level was below 12 IU/l, if both ovaries were present, if their total AFC was 8 21, if there was no past history of ovarian surgery, if they had a regular, spontaneous menstrual cycle of days, and if their body mass index (BMI) was between 20 and 35. Participants were excluded following their baseline scan at the screening visit if they were found to have an ovarian cyst or a follicle measuring 20 mm or more in diameter, or other significant pelvic pathology, such as fibroids, a hydrosalpinx, an endometrioma, or a uterine anomaly. Participants with unilateral or bilateral polycystic ovaries, as defined by the Rotterdam criteria of the presence of 12 or more follicles measuring 2 9 mm in diameter, or an ovarian volume of more than 10 cm 3, were also excluded. Participants were also excluded if they were known to have PCOS or if their subfertility was related to, or associated with, any other recognised endocrine abnormalities, such as hyperprolactinaemia, thyroid dysfunction, and hyperandrogenism. Randomisation Once participants had been screened and were found to be eligible, they were randomised to receive 300 or 225 IU of rfsh (Gonal-F; Merck Serono Pharmaceuticals Ltd, Feltham, UK) by a computer-generated pseudorandom code using random permuted blocks of varying size created by the University of Nottingham Clinical Trials Support Unit (CTSU). Randomisation was stratified by age and BMI in order to avoid imbalance of these prognostic variables, which could potentially affect the outcome and distort the apparent treatment effect between the two treatment arms. The treatment allocation for each participant was accessed by means of a remote, internet-based randomisation system developed and maintained on a secure server by the University of Nottingham CTSU. Access to the sequence of treatment allocations was confined to an independent CTSU Data Manager, and allocation to treatment was in a 1:1 ratio. In accordance with the randomisation process, each participant was assigned a participant number once they were considered eligible for treatment. An identification label with the participant s unique number was placed on the front cover of the clinical file of all of the enrolled participants to facilitate their identification. All of the medications used in treatment of these participants were supplied through the pharmacy at Queen s Medical Centre, Nottingham University Hospitals NHS Trust, Nottingham, UK. Treatment protocol All participants underwent IVF/ICSI treatment using a standard long protocol. This involved down-regulation with GnRH agonists (500 lg/day of Buserelin; Suprefact Ò ; Aventis Pharma, Kent, UK; or 800 lg/day of Nafarelin; Synarel Ò ; Pharmacia, Milton Keynes, UK) started in the mid-luteal phase of the menstrual cycle 7 days prior to the earliest expected date of menstruation. Two weeks later, following confirmation of pituitary desensitisation, defined as an endometrial thickness of less than 5 mm and no ovarian activity evident on transvaginal ultrasound, in association with an estradiol level below 200 pmol/l, ovarian stimulation was commenced using either 300 or 225 IU of Gonal-F, dependent on their randomisation and group allocation. Participants were monitored for follicular recruitment and growth by serial transvaginal ultrasound and serum estradiol measurements from the fifth day of stimulation. Changes in the dose of Gonal-F were not allowed during the treatment. Human chorionic gonadotrophin (hcg; 6500 IU of Ovitrelle; Merck Serono Pharmaceuticals Ltd) was administered when there were three follicles measuring 18 mm or more in diameter, and oocyte retrieval was performed 36 hours later. Once this criterion was met, no delay in hcg administration was allowed. Participants who developed more than 20 follicles measuring more than 10 mm in diameter, in association with an estradiol level of pmol/l or more, were counselled regarding the increased risk of OHSS, 20,21 and their treatment was then cancelled. Participants that did not develop three or more follicles measuring 18 mm or more in diameter after 14 days of rfsh treatment also had their treatment cancelled or were converted to intrauterine insemination (IUI) treatment, dependent on other clinical factors, including tubal patency and semen analysis. Transvaginal ultrasound-guided oocyte retrieval was performed under sedation or general anaesthesia. All of the visible follicles measuring 10 mm or more in diameter were aspirated, and each follicle was flushed twice if an oocyte was not initially recovered. The embryologists were blinded to the gonadotrophins dose that the participants had received. The oocytes obtained were fertilised by IVF or ICSI treatment, dependent on previous seminal fluid analyses and the quality of the sperm obtained on the day of oocyte retrieval. For participants with partners considered to have normal seminal fluid parameters, IVF was performed by incubating the collected oocytes in groups of ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology 855

4 Jayaprakasan et al. five in a sperm suspension containing sperm/ml, and the meiotic stage of oocytes was assessed by the embryologists hours post-insemination. For participants receiving ICSI, the meiotic maturity was assessed after denudation, and only mature oocytes were injected with sperm following immobilisation. Oocytes were classified as mature or metaphase-2 oocytes (extruded polar body and absent germinal vesicle), metaphase-1 oocytes (absent polar body and absent germinal vesicle) and germinal vesicles (presence of a large nucleus or germinal vesicle). 22 Fertilisation as determined by the presence of two pronuclei (PN) was assessed at hours post-insemination. On day 2 or 3, based on the cleavage rate, the size, shape, symmetry and cytoplasmic appearance of the blastomeres, and the presence or absence of a nucleus, embryo quality was graded by the embryologists as grade 1, 2, 3 or Grade-1 and -2 embryos were considered to be of high quality. Whereas embryo transfer was performed with the two best available embryos, regardless of their grading, only top quality, grade-1 and -2 embryos were considered to be eligible for freezing, and this option was then offered to the couple. The protocols within the embryology laboratory were based on the standard operating procedures (SOPs) of the unit, and remained the same throughout the duration of the study. A maximum of two embryos were transferred into the uterus 2 or 3 days after oocyte retrieval. Single embryo transfer was discussed and offered to all participants, in line with our normal practice. Luteal support using progesterone pessaries (Cyclogest pessaries; Shire Pharmaceuticals Ltd, Basingstoke, Hants, UK) was provided from the day of embryo transfer, and the serum hcg level was measured 16 days later to determine the outcome. If the test was positive (hcg > 50 IU/l), a transvaginal ultrasound was arranged 2 weeks later to confirm the viability of the pregnancy (clinical pregnancy). A repeat ultrasound scan was performed at 12 weeks of gestation to ensure that the pregnancy remained viable (continuing pregnancy). All pregnant subjects were followed up to ascertain the eventual outcome of their pregnancies. Live birth is defined as a viable infant born after 24 weeks of gestation. Hormonal analysis Blood samples were collected from each participant into a 6-ml plain tube, and these samples were immediately centrifuged to separate the serum. The assays for FSH, LH, and estradiol were performed within 2 3 hours of venepuncture or at the start of the next day, in which case the serum was stored at )2 C. Follicle-stimulating hormone, LH, and estradiol levels were measured using the Microparticle Enzyme Immunoassay (MEIA) method on an AxSYM auto-analyser (AxSYM; Abbott Laboratories, Abbott Park, IL, USA). The lowest detection limit and the intra- and inter-assay coefficients of variation for FSH were 0.37 IU/l, <5%, and <5%, respectively. The lowest detection limit and the intra- and inter-assay coefficients of variation for LH were 0.5 IU/l, <7%, and <8%, respectively. The lowest detection limit and the intra- and inter-assay coefficients of variation for estradiol were 8 pmol/l, %, and %, respectively. Primary outcome The primary outcome was the number of mature oocytes retrieved during the first treatment cycle post-randomisation. Secondary outcomes included the number of follicles measuring more than 10 mm in diameter and more than 14 mm in diameter on the day of hcg administration, cycle cancellation because of poor ovarian response, the number of frozen embryos, the prevalence of moderate or severe OHSS, and the clinical and continuing pregnancy rates and live birth rates. Statistical analysis Power calculations revealed that a sample size of 64 participants per group (128 in total) was needed to detect a difference of 2.5 mature oocytes, with a standard deviation of five oocytes between the two treatment groups, with a power of 80%, and with a significance threshold of 5%. We estimated the sample size to detect a difference of 2.5 mature oocytes between the study group and the control group, but not to estimate the mean number of oocytes in each group. Considering the 60% fertilisation rate following IVF/ICSI and the 90% cleavage rate, a difference of 2.5 mature oocytes would have resulted in a difference of one embryo available for transfer between the two groups. The Statistical Package for the Social Sciences (spss v14.0; SPSS, Chicago, IL, USA) was used for statistical analysis. The distribution of the data was checked for normality by the application of the Kolmogorov Smirnov test. The demographic data and the outcome data of the two treatment groups were compared by a Student s t test or Mann Whitney U test for continuous variables, dependent on the statistical distribution of the data. The chi-square or Fisher s exact tests were used for comparing the dichotomous variables. Results The participant flow diagram is shown in Figure 1. The experimental and control groups had comparable clinical, endocrine and ultrasound markers of ovarian reserve, and causes and type of infertility (Table 1), suggesting that the confounding factors thought to influence the outcome of assisted reproduction treatment were matched. Compliance with the allocated treatment was good (Table 2). 856 ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology

5 RCT of 300 versus 225 IU of rfsh Total ART cycles during the study period (n = 598) Assessed for eligibility (n = 280) Eligibility criteria not met (n = 105) Excluded after scan (n = 34) (PCO (n = 30); follicle >20mm (n = 4)) Invited (n = 141) Declined to participate (n = 6) Randomised (n = 135) Allocated to 300 IU of rfsh (n = 67) Allocated to 225 IU of rfsh (n = 68) Excluded post-randomization (n = 2) due to pregnancy (1), personal reasons (1) Excluded post-randomization (n = 2) due to pregnancy (1), personal reasons (1) Ovarian stimulation started at 300 IU (n = 65) Ovarian stimulation started at 225 IU (n = 66) Egg retrieval cancelled due to exaggerated response (n = 1) Egg retrieval done (n = 64) Egg retrieval done (n = 62) Egg retrieval cancelled due to poor response (n = 4) Assessed for primary endpoint (number of mature oocytes retrieved) included cancelled cycle due to exaggerated response (n = 65) Assessed for primary endpoint (number of mature oocytes retrieved) included cancelled cycles due to inadequate response (n = 66) Figure 1. CONSORT flow diagram of the randomised trial. The primary and secondary outcomes are shown in Tables 3 and 4. There was no difference in the number of mature oocytes retrieved between the two treatment arms (Table 3). The results stratified according to absolute AFC are shown in Figure 2. This also revealed no difference in the number of mature oocytes retrieved between the two groups. The pregnancy rates and live birth rates per cycle started were comparable between the two treatment arms (Table 4). Discussion The data in this study indicate that COH using a higher dose of 300 IU of rfsh in predicted normal responders does not offer any advantage in terms of increasing the ovarian response, as assessed by the number of mature oocytes retrieved. This is the first prospective, randomised study to compare a fixed daily dose of 300 IU and 225 IU of rfsh in predicted normal responders during assisted reproduction treatment. It is also the first study to prospectively define normal responders according to their total AFC, which is currently one of the best conventional markers of ovarian reserve. The ovarian response in terms of the number of mature oocytes retrieved was similar between the two groups, even when the data were stratified according to the absolute AFC, which positively reflects the number of gonadotrophin-sensitive follicles within the ovaries. 24 The correlation between the AFC and the number of mature oocytes retrieved was similar in the treatment arms, suggesting that the use of daily gonadotrophin doses above 225 IU does not improve the ovarian response. These findings are in keeping with the scientific evidence and the concept of an FSH threshold and follicular sensitivity to FSH. Although the serum FSH levels should be above the FSH threshold level required for multifollicular development, the magnitude of the FSH rise above this threshold is less important. 2 In contrast, the duration of stimulation above the FSH ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology 857

6 Jayaprakasan et al. Table 1. Baseline characteristics of the study population Characteristic Mean ± SD (range) or n (%) rfsh dose group P 300 IU (n = 65) 225 IU (n = 66) Age (years) 34.2 ± 3.5 (25 38) 33.1 ± 3.7 (23 38) 0.08 Body mass index (kg/m 2 ) 24.6 ± 3.7 (20 35) 23.7 ± 2.9 (20 33) 0.21 Subjects with primary subfertility (%) 42 (65.6) 44 (66.7) 0.96 Cause of subfertility (%) Tubal factor 13 (20) 14 (21) 0.90 Male factor 21 (32) 30 (45) 0.14 Unexplained 22 (34) 17 (26) 0.28 Mixed 2 (3) 2 (3) 0.58 Endometriosis 7 (11) 3 (5) 0.17 Basal FSH (IU/l) 7.3 ± 1.8 ( ) 6.8 ± 1.8 ( ) 0.07 Basal LH (IU/l) 5.8 ± 3.1 ( ) 4.8 ± 1.8 ( ) 0.17 Basal estradiol (pmol/l) 154 ± 51 (42 307) 157 ± 63 (43 373) 0.84 Basal inhibin-b (pmol/l) 45 ± 28 (7 115) 55 ± 29 (7 164) 0.09 Basal AMH (ng/ml) 1.3 ± 0.7 ( ) 1.4 ± 0.8 ( ) 0.20 Total antral follicle count 14.1 ± 4.0 (8 21) 15.4 ± 3.9 (8 21) 0.07 Mean ovarian volume (cm 3 ) 6.3 ± 1.8 ( ) 6.9 ± 1.8 ( ) 0.07 Mean ovarian VI (%) 7.0 ± 4.4 ( ) 7.4 ± 3.7 (0.8 20) 0.60 Mean ovarian FI (0 100) 35.1 ± 7 ( ) 35.5 ± 6.4 ( ) 0.66 Mean ovarian VFI (0 100) 2.7 ± 1.9 ( ) 2.9 ± 1.7 ( ) 0.61 AMH, anti-müllerian hormone; FI, flow index; rfsh, recombinant follicle-stimulating hormone; LH, luteinising hormone; VFI, vascular flow index; VI, vascular index. Table 2. Compliance with allocated treatment and co-treatments Characteristic Mean ± SD (range) or n (%) rfsh dose group P 300 IU (n = 65) 225 IU (n = 66) Participants down-regulated with Buserelin (%) 44 (68) 45 (68) 0.95 Participants down-regulated with Nafarelin (%) 21 (32) 21 (32) 0.95 Total dose of gonadotrophins (IU) 3206 ± 403 ( ) 2481 ± 351 ( ) <0.001 Duration of stimulation (days) 10.7 ± 1.3 (8 13) 11 ± 1.6 (9 14) 0.61 Number of IVF cycle (%) 33 (51) 31 (47) 0.93 Number of ICSI cycle (%) 31 (48) 31 (47) 0.93 ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilisation; rfsh, recombinant follicle-stimulating hormone. threshold is the key determinant of the number of follicles that will develop, as the FSH-sensitive follicles continue to grow to the preovulatory stage, rather than undergoing atresia. 25 However, it is important to stress that these findings relate to participants predicted to have a normal response to gonadotrophins based on their AFC, and may not relate to participants predicted to have a poor or exaggerated response using the same assessment criteria. The strengths of our study are having predefined normal responders based on strict clinical, endocrine and ultrasound criteria, a predetermined sample size, and good treatment compliance. Nevertheless, we have not excluded the small effect of the increased daily doses on ovarian response as being a result of a small study population. The mean (95% CI) difference in the number of mature oocytes retrieved between the 300 and 225 IU arms in our study was 0.8 (from )0.9 to +2.4). There remains a 2.5% chance that the use of 300 IU of rfsh may have increased the number of mature oocytes by 2.4. However, any such effect in ovarian response in a minority of women is less likely to have any significant effect on improving pregnancy outcomes; conversely, the use of higher doses potentially 858 ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology

7 RCT of 300 versus 225 IU of rfsh Table 3. Ovarian stimulation characteristics Table 4. Embryological and pregnancy outcome characteristics Characteristic Mean (standard deviation) or n (%) rfsh dose group 300 IU (n = 65) 225 IU (n = 66) P Characteristic Mean (standard deviation) or n (%) rfsh dose group 300 IU (n = 65) 225 IU (n = 66) P Total number of mature 8.2 (5.0) 9.0 (4.8) 0.34 oocytes (primary outcome) Peak estradiol level (pmol/l) 8214 (7622) 7972 (4615) 0.83 Number of follicles >10 mm 15.0 (6.0) 17.2 (7.1) 0.07 in diameter on the day of hcg Number of follicles >14 mm 11.2 (5.9) 12.7 (6.5) 0.17 in diameter on the day of hcg Total number of follicles 13.0 (6.0) 14.3 (7.1) 0.27 aspirated Total number of oocytes 10.2 (5.9) 11.0 (5.4) 0.34 retrieved Egg maturity rate (%) 82 (18.1) 83 (13.7) 0.17 Cycle cancellations because 0 (0%) 4 (6.1%) 0.12 of an inadequate response (n; %) Cycle cancellations because 1 (1.5%) 0 (0%) 0.50 of an exaggerated response (n; %) Moderate OHSS (n; %) 2 (3.1%) 2 (3.0%) 1.0 Severe OHSS (n; %) 0 (0%) 1 (1.5%) 1.0 hcg, human chorionic gonadotrophin; OHSS, ovarian hyperstimulation syndrome; rfsh, recombinant follicle-stimulating hormone. increase drug costs and the risk of an exaggerated ovarian response. A dose-dependent increase in follicular recruitment and in the subsequent number of oocytes retrieved has been demonstrated in the other prospective, randomised study of follitropin a (Gonal-F), which compared daily doses of 150 and 225 IU. 3 This apparent better response did not result in an improved outcome, however, as the pregnancy rate, live birth rates, and number of embryos available for cryopreservation were comparable between the groups, although the study was not sufficiently powered to detect such differences. Our study has also shown a similar pregnancy outcome between the two treatment arms. The number of fertilisable oocytes retrieved is an important parameter, as it positively influences the number of embryos available for transfer and the number suitable for freezing, both of which influence the overall success of assisted reproduction treatment. 26 This was comparable in the two arms in our study, but was significantly different in the study of Yong et al. (2003). This suggests that any future work designed to examine the effect of the dose of FSH on IVF outcome should compare against 225 IU of Egg retrieval performed (%) 64 (98) 62 (94) 0.18 Failed fertilisation (%) 0 (0) 3 (4.5) 0.24 Fertilisation rate (%) 63 (26.0) 66 (22.9) 0.15 Failed cleavage (%) 3 (4.6) 1 (1.5) 0.62 Embryo transfer cancelled 0 1 (1.5) 1.0 because of the development of OHSS (%) Embryo transfer performed 61 (94) 57 (86) 0.15 (%) Number of embryos 1.8 (0.6) 1.7 (0.6) 0.98 transferred per cycle Number of embryos 0.6 (1.3) 1.0 (1.7) 0.33 frozen per cycle Positive pregnancy test (%) 33 (52) 33 (50) 0.93 Clinical pregnancy per cycle 28 (43) 30 (46) 0.78 started (%) 1st trimester miscarriage (loss 4 (14) 1 (3) 0.19 of clinical pregnancy) (%) Ectopic pregnancy (% of all 1 (3) 0 (0) 1.0 biochemical pregnancies) Ongoing pregnancy per cycle 23 (35) 29 (44) 0.32 started (%) Live birth rate per cycle started (%) 20 (31) 27 (41) 0.25 OHSS, ovarian hyperstimulation syndrome; rfsh, recombinant folliclestimulating hormone. FSH. Such studies should consider cumulative pregnancy rates or live birth rates per cycle of treatment as the primary outcome, and a large multicentre randomised controlled trial with a sample size of over 750 participants is needed to notice a difference of 10% in the outcome between the two groups, for a power of 80%. Whereas all the stimulation characteristics were similar between the two treatment groups, all four participants that had their treatment cancelled because of poor ovarian response were in the lower dose group. This difference was not statistically significant, but this may also relate to the sample size. The inclusion and exclusion criteria were set to define a study group of predicted normal responders, based on the AFC, age, and basal FSH. Although we measured anti-müllerian hormone (AMH) and inhibin-b for all the participants, and found similar levels between the two groups, we did not consider these markers for defining normal responders. However, none of these markers of ovarian reserve have been proven to be perfect in identifying all potential poor responders. 27 The variation in ovar- ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology 859

8 Jayaprakasan et al. Number of mature oocytes retrieved Number of mature oocytes retrieved 20 P = ± ± AFC (8 11) 20 P = ± ± AFC (15 18) Number of mature oocytes retrieved Number of mature oocytes retrieved 20 P = ± ± AFC (12 14) 20 P = ± ± AFC (19 21) Figure 2. Comparison of the mean number of mature oocytes retrieved in the two treatment groups (225 and 300 IU), categorised by each quartile of the total antral follicle count. Each box plot indicates the mean number of antral follicle count with standard deviation of the group, the values of which are shown above each box plot. ian sensitivity to gonadotrophins may also relate to follicular microcirculation, which influences the level of delivery of gonadotrophins, 28 or genetic factors such as the FSH receptor genotype. 29,30 The number of participants who developed moderate or severe OHSS was also similar in the two groups. Although the participants with polycystic ovaries, the most significant risk factor for the development of OHSS, were excluded in our study, a small proportion (5/131, 3.8%) still demonstrated an exaggerated ovarian response associated with moderate or severe OHSS, regardless of the dose of gonadotrophins. This may relate to an increased ovarian sensitivity to gonadotrophins, or may reflect the limitations in the predictive value of the tests used to estimate ovarian reserve and probable response used in this study. Whereas the AFC has been repeatedly shown to have a high sensitivity and specificity for the prediction of ovarian response, it is not a perfect test, and this may relate to the reliability of measurement technique or to the presence of small recruitable, gonadotrophin-responsive follicles that are not evident on ultrasound. In this study we used 3D ultrasound to count the total number of antral follicles, and this has been shown to offer a high degree of measurement reliability, 31 and so the latter is a more probable explanation. The use of serum AMH to individualise treatment strategies based on the predicted ovarian response may overcome this limitation, as has been demonstrated in a recent study 32. There are other randomised studies that have compared different doses of the other rfsh preparation that is currently in use, follitropin-b (Puregon). These have shown different and conflicting results. Some studies report an increased number of retrieved oocytes with a daily dose of 200 IU compared with 100 IU, 7 9 whereas others have not seen any significant difference between the same daily doses, 4,5 or between daily doses of 150 and 250 IU (Latin- American Puregon IVF Study Group 2001). 33 A recent randomised controlled trial reported a dose-dependent increase in the ovarian response with increasing fixed daily doses of rfsh (Gonal-F) from 75 up to IU (the number of oocytes retrieved increased from 8.3 to 12.7), but not with 225 IU (number of oocytes retrieved: 8.3) during conventional IVF using GnRH agonist in women aged below 35 years. 34 The starting dose in this study was individualised based on an algorithm derived according to four prognostic factors: age, FSH levels, BMI and AFC. The studies discussed above, examining both forms of recombinant FSH, suggest that a daily dose of 225 or IU represents the most effective way to ensure an appropriate response, balanced against the risk of an aberrant one, both in terms of poor and exaggerated responses. This dose may not be the appropriate starting dose or daily dose for participants predicted to have a higher chance of poor or exaggerated response to ovarian stimulation, and future studies should evaluate the effect of different protocols, 860 ª 2010 The Authors Journal compilation ª RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology

9 RCT of 300 versus 225 IU of rfsh including the recently evolving milder stimulation protocols, 35 to optimise IVF outcome in women. This study is limited by the fact that the AFC, a major determinant of the degree of ovarian response, was not stratified, unlike age and BMI, before randomisation. However, it is unlikely that this would have affected the results, as the AFCs in both the treatment arms were similar. Moreover, there was no difference in the ovarian response between the two treatment groups across each quartile of AFC (Figure 2). The ideal primary outcome should have been live births, but for this we need a much larger study population, which is only possible in a multicentre collaboration. Although we agree that the sample size is small in this study, but appropriate for the objectives stated, the similar ovarian response between the study arm and the control arm indicates that the use of a lower daily dose of 225 IU does not seem to adversely affect the IVF outcome. Conclusion Participants predicted to have a normal response to COH for assisted reproduction treatment have similar outcomes in terms of the number of mature oocytes retrieved, the number of embryos available for freezing, and the continuing pregnancy rates following fresh embryo transfer, regardless of whether a daily dose of 300 or 225 IU of recombinant FSH (Gonal-F) is used for ovarian stimulation. This study was not sufficiently powered to assess the effect of these different daily doses of FSH on pregnancy outcome, which requires multicentre collaboration. The data do suggest, however, that the use of a lower daily dose of 225 IU is effective, and does not seem to adversely affect the ovarian response. It is also potentially cost-effective as it is associated with the use of a lower total dose of gonadotrophins. Disclosure of interests None. Contribution to authorship All authors have substantially contributed to the conception and design of the study, recruitment, data collection or data analysis, interpretation, drafting the article or revising it critically for important intellectual content and final approval of the version to be published. Details of ethics approval Ethics approval was granted by Trent Multi-centre Research Ethics Committee on 16 August 2006: ref. 06/MRE04/33. Funding This study was supported by an investigational grant from Merck-Serono, Feltham, Middlesex, UK. Acknowledgements The authors would like to thank the University of Nottingham CTSU, particularly Daniel Simpkins, the CTSU data manager, and the clinical trials pharmacists Sheila Hodgson and Lisa Humphries at Queen s Medical Centre, University of Nottingham, for their assistance with setting up the web-based randomisation and for dispensing Gonal-F, respectively. j References 1 Arslan M, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S. Controlled ovarian hyperstimulation protocols for in vitro fertilization: two decades of experience after the birth of Elizabeth Carr. Fertil Steril 2005;84: Dorn C. FSH: what is the highest dose for ovarian stimulation that makes sense on an evidence-based level? Reprod Biomed Online 2005;11: Yong PY, Brett S, Baird DT, Thong KJ. A prospective randomized clinical trial comparing 150 IU and 225 IU of recombinant folliclestimulating hormone (Gonal-F*) in a fixed-dose regimen for controlled ovarian stimulation in in vitro fertilization treatment. 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