The optimal choice of gonadotrophin in GnRH antagonist protocols Prof Dr P Devroey

Recent trends in ART practice Increasing use of GnRH antagonists with lower doses of gonadotrophin Increasing use of ICSI over the last decade Increasing use of single embryo transfer Increasing use of embryo culture to blastocyst stage Increasing use of vitrification instead of slow-freezing

Why the MEGASET trial? MEGASET compares HP-hMG (MENOPUR ® ) with rFSH (PUREGON ® ) in a setting that addresses these recent trends in ART practice Randomised, assessor-blind, parallel groups, multi- centre trial to demonstrate non-inferiority of HP- hMG compared to rFSH with respect to ongoing pregnancy rates

Participating clinics 25 clinics in 7 countries

Key design features Women 18–34 years BMI 18–24.9 kg/m 2 GnRH antagonist No programming 150 IU starting dose ICSI Blastocyst culture Single blastocyst transfer on Day 5 2 weeks luteal support Vitrification Replacement of a single warmed blastocyst in a natural cycle

rhCG 250 μg OR 6 HP-hMG or rFSH 1 3 follicles ≥ 17mm ET 1 blastocyst Oocyte/embryo/ blastocyst evaluation β-hCG 150 IU x 5 days Adjustment by 75 IU; minimum 4 days on dose Clin. P Progesterone 3x200 mg Progesterone 3x200 mg GnRH antagonist 0.25 mg OR days after ET 5-6 weeks after ET weeks after ET Ong. P Trial design Post-trial follow-up FER 1 blastocyst natural cycle Ongoing pregnancy Ongoing pregnancy No ongoing pregnancy No ongoing pregnancy Ongoing pregnancy Ongoing pregnancy No ongoing pregnancy No ongoing pregnancy Pregnancy outcome and neonatal health follow-up Pregnancy outcome and neonatal health follow-up Pregnancy outcome and neonatal health follow-up Pregnancy outcome and neonatal health follow-up

Investigations: All patients Endocrine profile Follicular development Ovarian response Endometrial profile Pregnancy rates Cumulus mass appearance Oocyte maturation, fertilisation Embryo quality Blastocyst quality

Additional investigations: Subgroups of patients Early-mid follicular phase endocrine profile Intrafollicular endocrine profile Uterine contractility Modelling of follicles Modelling of endometrium Gene expression in cumulus cells (mechanical dissection and enzymatic denudation)

METHODOLOGY

Primary endpoint of the study Ongoing pregnancy rates beyond 10–11 weeks after ET in a fresh cycle

Power calculation Estimated ongoing pregnancy rate of 30% was derived from previous studies on single blastocyst transfer Non-inferiority margin was set at –10% (absolute) At least 660 cycles was required to achieve a study power of 80%

Analysis of data Modified Intention-to-treat (ITT) analysis –All subjects who have been randomised and exposed to at least one dose of investigational medicinal product were analysed according to the actual treatment Per protocol analysis –All subjects from the modified ITT, except those who are excluded because of a major protocol deviation were analysed

EMBRYO ASSESSMENT

Embryo morphology assessment and grading Local embryologists only; no central evaluation Interobserver agreement and intraobserver reproducibility were validated in the MERiT trial showing good–excellent agreement on overall embryo morphology assessment and grading 1 Embryos were graded according to the Gardner and Schoolcraft classification system 2 1. Arce et al. Hum Reprod 2006; 21: 2141– Gardner and Schoolcraft. In: Towards reproductive certainty (Eds Jansen & Mortimer). The plenary proceedings of the 11 th world congress on in vitro fertilization and human reproductive genetics. The Parthenon Publishing Group Pp 378–388

Endometrial assessment Thickness Triple-layer structure Echogenicity pattern

SUBJECT DISPOSITION

Consort diagram Screened (N=810) Randomised and exposed (n=749) Oocyte retrieval N=362 rFSH (ITT; N=375) Embryo transfer N=305 β-hCG visit N=305 Ongoing pregnancy visit N=116 Ongoing pregnancy visit N=107 β-hCG visit N=316 Embryo transfer N=316 Oocyte retrieval N=362 HP-hMG (ITT; N=374)

BASELINE PARAMETERS

Demographics and treatment history – ITT population DemographicsHP-hMG (N=374) rFSH (N=375) Age (years)30.8 ± ± 2.6 Weight (kg)60.6 ± ± 7.0 BMI (kg/m 2 )22.1 ± ± 2.0 Duration of infertility (yrs)3.2 ± ± 1.7 Treatment historyHP-hMG (N=374) rFSH (N=375) 1 st or 2 nd COS cycle ever95% Previous IUI cycles, total49%52% Previous IUI cycles, with gonadotrophins 29%31% Unexplained 38% Primary reason of infertility Mild male factor 62% Unexplained 40% Mild male factor 60% rFSH HP-hMG

ENDOCRINE PROFILE

Endocrine Profile – Stimulation day 1 Endocrine profileHP-hMG (N=374) rFSH (N=375) FSH (IU/L)7.5 ± ± 2.4 LH (IU/L)6.2 ± ± 2.2 Estradiol (pmol/L)180 ± ± 100 Progesterone (nmol/L)2.2 ± 1.1 Total testosterone (nmol/L)1.6 ± ± 0.8 Inhibin B (ng/L)87 ± 4085 ± 35 AMH (pmol/L)27 ± 1927 ± 20 ITT-populationData are mean ± SD

Endocrine profile – stimulation day 6 HP-hMG (N=374) rFSH (N=375) p value LH (IU/L)4.9 ± ± hCG (IU/L)1.7 ± Estradiol (pmol/L)2626 ± ± Progesterone (nmol/L)2.2 ± ± Total testosterone (nmol/L)1.9 ± Inhibin B (ng/L)604 ± ± 424<0.001 ITT-population Data are mean ± SD

Early-mid follicular phase: LH HP-hMG (N=49) rFSH (N=50) Day 1 → Day 2 -22%-26% Day 2 → Day 4 -42%-47% Day 1 → Day 4 -60%-61% Day 4 → Day 6 10%24% Day 1 → Day 6 -50%-57% Change over time Median values ITT-population / early-mid follicular phase sub-group HP-hMG rFSH

HP-hMG (N=374) rFSH (N=375) p value LH (IU/L)2.8 ± ± 1.6<0.001 hCG (IU/L)2.1 ± Estradiol (pmol/L)8797 ± ± 4945<0.001 Progesterone (nmol/L)3.1 ± ± Total testosterone (nmol/L)2.5 ± ± 1.0<0.001 ITT-populationMean ± SD Endocrine profile – last stimulation day

HP-hMG (N=374) rFSH (N=375) Premature luteinization * -LH ≥ 10 IU/L -Progesterone ≥ 1 ng/mL (3.18 nmol/L) 5.9%6.1% ITT-population * Both LH and progesterone criteria to be met at the same visit (ie. Stimulation Day 6 or Last Stimulation Day) Premature luteinization

TREATMENT EFFICIENCY

Stimulation Day 6 Last Stimulation Day ITT-populationMean data p<0.05 Follicular development HP-hMG rFSH HP-hMG rFSH

Oocytes ITT-population with oocyte retrieval Protocol target 8 – 10 HP-hMG (N=362) rFSH (N=362) p value Oocytes retrieved 9.1 ± ± 5.8<0.001

Exposure to gonadotrophins and GnRH antagonist HP-hMG (N=374) rFSH (N=375) p value Duration of gonadotrophin use (days) 8.8 ± ± Total gonadotrophin dose (IU) 1433 ± ± Dose on Day 6 Decreased 1%2% Maintained 67%73% Increased 31%25% ITT-population Percentages may not add to 100% due to rounding off

Endometrial pattern – Day of embryo transfer HP-hMG (N=374) rFSH (N=375) p value Endometrial thickness (mm) 11.1 ± ± 2.2- Triple-layer structure53%54%0.873 Echogenic pattern Hypoechogenic5%6% Isoechogenic17% Hyperechogenic73% Not possible to evaluate 5%4% ITT-population

Availability of blastocysts on the day of ET ITT-populationHP-hMGrFSH Subjects with blastocysts82%85% Subjects with frozen blastocysts55%58%

Non-inferiority was demonstrated for both PP- and ITT-populations, as the lower limit of the 95% confidence interval was above the pre- established non-inferiority margin of -10% Ongoing pregnancy rate per started cycle: Primary endpoint HP-hMGrFSH HP-hMG – rFSH Difference (95% CI) PP30.0%27.0%3.0% (-3.8; 9.8) ITT28.9%26.7%2.2% (-4.2; 8.6)

Pregnancy rates per started cycle PP-populationITT-population

p=0.95 p=< HP-hMGrFSH Ongoing pregnancy rate/cycle initiated (%) Progesterone >4nmol/L Progesterone ≤4nmol/L Significantly lower ongoing pregnancy rate in rFSH patients with higher progesterone levels at the end of stimulation

Blastocyst quality and ongoing pregnancy rate Expansion and hatching status HP-hMG (N=304) rFSH (N=315) %41% %14% Ongoing pregnancy rate by quality of transferred blastocyst ITT-population with blastocysts on Day 5 ITT-population with embryo transfer Subjects according to their highest blastocyst quality Blastocyst expansion and hatching statu s

Pregnancy loss Biochemical pregnancy N=14 Ectopic pregnancy N=1 Intrauterine pregnancy without heart beat N=12 Abortion N=7 Biochemical pregnancy N=18 Ectopic pregnancy N=1 Intrauterine pregnancy without heart beat N=10 Abortion N=8 HP-hMG 37/374 = 10% rFSH 34/375 = 9%

Conclusions Primary endpoint of MEGASET study was achieved Largest multicentre, multinational RCT of HP-hMG vs rFSH addressing new trends in ART in a robust, high quality innovative trial with ICSI Demonstrates single blastocyst transfer is effective with mild stimulation and lower number of oocytes Reinforces the importance of progesterone during the late follicular phase –Higher pregnancy rate with HP-hMG than rFSH when progesterone >4 nmol/L