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

2. 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

3. 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

4. Participating clinics 25 clinics in 7 countries

5. 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

6. 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 +5 13-15 days after ET 5-6 weeks after ET 10-11 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

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

8. 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)

9. METHODOLOGY

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

11. 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%

12. 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

13. EMBRYO ASSESSMENT

14. 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: 2141–2148 2. 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. 1999. Pp 378–388

15. Endometrial assessment Thickness Triple-layer structure Echogenicity pattern

16. SUBJECT DISPOSITION

17. 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)

18. BASELINE PARAMETERS

19. Demographics and treatment history – ITT population DemographicsHP-hMG (N=374) rFSH (N=375) Age (years)30.8 ± 2.830.4 ± 2.6 Weight (kg)60.6 ± 6.859.9 ± 7.0 BMI (kg/m 2 )22.1 ± 1.921.9 ± 2.0 Duration of infertility (yrs)3.2 ± 1.83.1 ± 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

20. ENDOCRINE PROFILE

21. Endocrine Profile – Stimulation day 1 Endocrine profileHP-hMG (N=374) rFSH (N=375) FSH (IU/L)7.5 ± 2.37.4 ± 2.4 LH (IU/L)6.2 ± 2.36.2 ± 2.2 Estradiol (pmol/L)180 ± 106177 ± 100 Progesterone (nmol/L)2.2 ± 1.1 Total testosterone (nmol/L)1.6 ± 0.81.7 ± 0.8 Inhibin B (ng/L)87 ± 4085 ± 35 AMH (pmol/L)27 ± 1927 ± 20 ITT-populationData are mean ± SD

22. Endocrine profile – stimulation day 6 HP-hMG (N=374) rFSH (N=375) p value LH (IU/L)4.9 ± 5.05.5 ± 6.00.558 hCG (IU/L)1.7 ± 0.6-- Estradiol (pmol/L)2626 ± 14052973 ± 17020.003 Progesterone (nmol/L)2.2 ± 1.92.8 ± 10.80.025 Total testosterone (nmol/L)1.9 ± 0.9 0.169 Inhibin B (ng/L)604 ± 324722 ± 424<0.001 ITT-population Data are mean ± SD

23. 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

24. HP-hMG (N=374) rFSH (N=375) p value LH (IU/L)2.8 ± 2.82.1 ± 1.6<0.001 hCG (IU/L)2.1 ± 0.8-- Estradiol (pmol/L)8797 ± 60307022 ± 4945<0.001 Progesterone (nmol/L)3.1 ± 3.43.1 ± 3.30.630 Total testosterone (nmol/L)2.5 ± 1.22.1 ± 1.0<0.001 ITT-populationMean ± SD Endocrine profile – last stimulation day

25. 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

26. TREATMENT EFFICIENCY

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

28. Oocytes ITT-population with oocyte retrieval Protocol target 8 – 10 HP-hMG (N=362) rFSH (N=362) p value Oocytes retrieved 9.1 ± 5.210.7 ± 5.8<0.001

29. Exposure to gonadotrophins and GnRH antagonist HP-hMG (N=374) rFSH (N=375) p value Duration of gonadotrophin use (days) 8.8 ± 1.68.5 ± 1.30.077 Total gonadotrophin dose (IU) 1433 ± 3711353 ± 2960.009 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

30. Endometrial pattern – Day of embryo transfer HP-hMG (N=374) rFSH (N=375) p value Endometrial thickness (mm) 11.1 ± 2.111.1 ± 2.2- Triple-layer structure53%54%0.873 Echogenic pattern Hypoechogenic5%6% 0.983 Isoechogenic17% Hyperechogenic73% Not possible to evaluate 5%4% ITT-population

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

32. 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)

33. Pregnancy rates per started cycle PP-populationITT-population

34. p=0.95 p=<0.05 0 5 10 15 20 25 30 HP-hMGrFSH Ongoing pregnancy rate/cycle initiated (%) 29 16 29 30 Progesterone >4nmol/L Progesterone ≤4nmol/L Significantly lower ongoing pregnancy rate in rFSH patients with higher progesterone levels at the end of stimulation

35. Blastocyst quality and ongoing pregnancy rate Expansion and hatching status HP-hMG (N=304) rFSH (N=315) 4-5 46%41% 1-3 13%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

36. 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%

37. 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