Timur G ü rgan MD Dean and Professor Yeni Yüzyıl University Faculty of Medicine

1. Highly Glycosylated FSH (HG-FSH): Available Clinical Results and Investigational Issues Timur Gürgan MD Dean and Professor Yeni Yüzyıl University Faculty of Medicine Dept. Of Obsterics and Gynecology Istanbul,Turkey

2. Is Dialogue Important ? • Dialogue in between couple and clinician • Dialogue in between clinician and embyologist • Dialogue in between cumulus complex and oocyte • Dialogue in between oocyte and sperm • Dialogue in between embryo and endometrium • Dialogue in between fetus and mother • Dialogue in between mother and obstetrician SINGLE HEALTHY CHILD

4. Structure of FSH FSH is a complex mix of molecules (isoforms) Four Asn-glycosylation sites: Alpha = Asn52 Asn78 Beta = Asn7 Asn24

5. Oligosaccharide chains structure Sialic Acid Galactose N-Acetyl-Glucosamine Mannose áglycosylation = ásialic acid = áacidity

6. Low sialic acid content High sialic acid content Basic Isoforms Acidic Isoforms - + Human FSH isoforms according to the point of isoelectricity (pI) pI 6 pI 5 pI 4 pI 3

7. SummaryWhat are FSH isoforms? • FSH is a family of molecules carrying different amounts of sugars (glycosylation) • Due to their content in sialic acid, more glycosylated molecules are more acidic • Acidity is used in analytical tests • Natural FSH is a wide mix of the different molecules

8. Oligosaccaride composition of industrial FSH products 0-sialic (%) 1-sialic (%) 2-sialic (%) 3-sialic (%) 4-sialic (%) Fostimon (IBSA) IBSA data on file 2 5 36 41 16 Bravelle (Ferring) Wolfenson 05 <5 2 57 36 <5 Puregon (Organon) De Leeuw 96 43 13 33 9 2 Gonal F (Serono) Driebergen 03 8 30 47 12 3

9. Fostimon Gonal-F FSH isoforms according to Iso Electro Focusing From Lispi et al., RBMonline 2006

10. BASIC ISOFORMS ACIDIC ISOFORMS Chromatofocusing, Fostimon vs Gonal-F FSH isoforms were separated by chromatofocusing and the amount of protein contained in each peak was measured by anti-FSH Ab. Results are given as percentages of the total FSH recovered, means of three different experiments. Data on file IBSA laboratories.

11. SummaryPhysiological role of FSH isoforms

12. Pattern of FSH glycosylation during the follicular cycle HG FSH LG FSH Imbalance for HG FSH during the recruitment phase, the opposite at time of ovulation

13. Sensitive already at 1.5 mg/ml, ceiling already at 5 mg/ml Less acidic (scarce glycosylation) Unselective: All follicles are stimulated Acidic (high glycosylation) Sensitive only at 40 mg/ml, ceiling as high as 100 mg/ml Selective: Only “good” follicles are stimulated Antral follicular threshold for FSH glycoforms (Nayudu et al., RBMonline 2002) Selective recruitment with acidic isoforms

14. Progressive increase of more biopotent, acidic forms with advancing age Compensative to the general ovarian resistance More precisely, reaction to the pathologic shortening of the early follicular phase Predominance of the very acidic forms in the recruitment phase i.e. luteal-follicular transition and early follicular phases Better selective for good quality follicles Longer (physiolocical) pre-antral phase SummaryNatural pattern of FSH isoform secretion Throughout the reproductive life In the follicular cycle FSH varies over time according to well defined patterns

15. Stimulation cycle withhighly glycosylated FSH (Fostimon) • Different pattern of follicle growth • Less follicles recruited • Slower growth rate in pre-antral phase • Fast development once entering the antral phase • Minor loss of follicles during maturation • Reduced number of small follicles at time of hCG (<OHSS) • Possibility for improved outcomes • Better quality of oocytes/embryos • Higher implantation rates • Improved outcome from frozen embryos

16. Company sponsored Multicentre Non-inferiority design to assess efficacy and tolerabiltiy Regulatory purposes EU study USA study Fostimon, Pivotal studies Aim: does Fostimon qualify as a suitable alternative to already existing drugs?

17. Fostimon, Pivotal studies EU Study: Antoine JMet al. ASRM 2006 - Poster presentation USA study: Baker VL et al. Fertility & Sterility 2009

18. Pivotal studies, Conclusions In good prognosis patients with a fixed starting dose of gonadotrophin, the authors found no difference between hFSH and rFSH with respect to: • Mean number of oocytes retrieved • Clinical pregnancy rate • Live birth rate • Safety and tolerability The results from the pivotal studies showed totally overlapping results

19. Published studies comparing Fostimon (HG FSH) to rFSH 6 Studies, 1349 cycles

20. Details: Drug consumption Global trend for a better efficiency

21. Details: Mature oocytes Likely no or just minor differences

22. Details: Fertilisation rate Trend for an improved fertilisability

23. Details: Number of embryos No differences

24. Details: Top quality embryos Strong trend for an improved embryo quality

25. Details: Implantation rates Slight trend for improved implantation

26. Details: Clinical pregnancy rates Possible trend for improved pregnancy rates

27. Stimulation cycle withhighly glycosylated FSH (Fostimon) • Different pattern of follicle growth • Less follicles recruited • Slower growth rate in pre-antral phase • Fast development once entering the antral phase • Minor loss of follicles during maturation • Reduced number of small follicles at time of hCG (<OHSS) • Possibility for improved outcomes • Better quality of oocytes/embryos • Higher implantation rates • Improved outcome from frozen embryos

28. Examples of cohorts hCG & pick-up Recruitment (pre-antral) Maturation (antral) Fertilisation Implantation Pregnancy Phase Fostimon rFSH Days [Estradiol]

29. Pre-antral phase Priming the oocyte for a correct reproductive function • Stimulation with a HG FSH should result in a more selective recruitment and in a slower (physiological) antral phase • A proper pre-antral phase is mandatory for good quality oocytes • DNA imprinting • Genetic synthesis • Proteic synthesis

30. triggering Day 6 Standard long down-regulation Stimulation Fostimon r-FSH r-FSH r-FSH Fostimon Fostimon hCG 5’000-10’000 IU 225 IU Ovarian stimulation protocols based on FSH glycosylation pattern: impact on oocyte quality and clinical outcome.Selman HA et al., Fertility & Sterility, 2009 Group A, n=63 Group B, n=65 Group C, n=60

31. Selman HA et al. 2009, Clinical outcomes

32. Investigational questions • Does the pattern of activation of the oocyte genoma really vary according to the (FSH) stimulation regimen? • Are cumulus cells as well sensitive to alternative stimulation types? A new study for a new approach: “Effect of FSH stimulation Regimens on The Pattern of mRNA expression in human Cumulus Cells” Benkhalifa M, Menezo Y, Demirol A, Gurgan T

33. Least acidic (Puregon) vs Sequential (Fostimon + Puregon) vs Acidic (Fostimon) Day 6 triggering Stimulation Standard long Down regulation Fostimon Puregon Fostimon or Puregon hCG, 10’000 IU 225 IU In this study we investigate the effect on the whole transcriptome of cumulus cells following follicular stimulation with regimens differing in their FSH isoforms composition

34. Effects of FSH stimulation regimens on the pattern of mRNA expression in human cumulus cells PreliminaryClinical data *:1 abortion

35. Conclusions I • FSH isoforms are likely to play an important phisiological role • The available FSH drug products differ each other in terms of isoform content • Fostimon contains more of HG FSH • Clinical data available so far showed interesting outcomes from the sequential use of differently glycosylated FSHs • Fostimon gives, more or less, equivalent response as sequence Fostimon+ Puregon better than Puregon alone

36. Conclusion II • Different CHO (controlled hyperstimulation) treatmentsinducedifferentresponses in CC biochemicalpathways • Our data on cumulustranscriptomeprofilingshowedthatthegroup of genesinvolved in thebiologicalfunctionandmolecularprocess can be expresseddifferently on relationwiththeused FSH glycoformsMorestudiesneedto be done ( mainlyproteomics) toeluciatethecontinueddialoguebetweentheoocytesanditssomaticenvironmenttoreachits final maturationandcompetency at ovulation.

37. Investigational AddressesWhy to use highly glycosylated FSH? • To improve the recruitment process • More selective, for better quality • Less of small follicles … OHSS? • To improve the quality of the oocytes/embryos • Better primed oocytes (pre-antral phase) • To increase the efficiency • Lower drug consumption • Reproductive ageing … • PCOS … • Male indications …

38. Why to use highly glycosylated FSH? • To improve the recruitment process • More selective, for better quality • Less of small follicles … OHSS? • To improve the quality of the oocytes/embryos • Better primed oocytes (pre-antral phase) • To increase the efficiency • Lower drug consumption • Reproductive ageing … • PCOS … • Male indications …

39. Y.Menezo M. Benkhalifa Shili Ons Jilani Nej MontJean Debbie Unilabs, Paris, Geneve ATL R&D. Reproductive Biology & Genetics Laboratory. Paris France A.Demirol , T.Gurgan & theembryologyTeam Women’sHealthClinic. IVF & Genetics Centre. Ankara. Turkey We Would Like to thank IBSA for the financial Support And Dr Maurizio Dattilo

40. Cluster Analysis Analysisper cluster shows modification in mRNAs expression in CC, according to the differenttreatments Itgives a too global response , to be handledwithcare But all FSH are not givingthesameresponse !

41. By analysing transcriptome profile of cumulus cells from three protocols,we concluded that there are 147 genes involved in the stimulation processes

42. Green colour means down regulated Red colour means upregulated

43. Biological processes Involved processes: • mRNA transcription • Protein targeting and localization • Endocytosis • Intracellular signaling cascade • Nucleoside, nucleotide and nucleic acid metabolism • mRNA transcription regulation • Ion transport • Whenwewantedtoanalysebiologicalprocessesdeeplyweobservedinvolvedprocesseswereprimarlyforcellcyclesqualityandcellcyclecommunication (oocyte &CC) No significant variations in biological process

44. Biological Processes Even if the pathways are not significantlyaffected , Fostimon & PuregonisratherdownregulatingwhereasPuregononlyisupregulating

45. Molecular Function Involved process : Deacetylase Homeobox transcription factor Acting binding cytoskeletal protein G-Protein Reductase Oxidoreductase Molecular pathways involved the pathways of mRNA transcription,metabolic pathways and antiappoptotic processes to reduce ROS No significant variations in molecular function

46. Molecular Function FostimonisratherdownregulatingwhereasPuregonisupregulating

47. Bioactivity of Fostimon’s isoform pools Restricted glycofractions of Fostimon were separated by chromatofocusing, collected in sub-pools and tested for bioactivity according to Steelman & Pohley. Data on file IBSA laboratories.

48. What does it mean? • 27% of the molecules give double of the average activity • i.e. this 27% of the molecules within a 75 IU vial give 40.5 IU • i.e. 54% of the activity is given as an FSH type that does almost not occur in other FSH products