1.Understanding Infertility New Delhi, India August 2011 Recent Advances inStimulation ProtocolsEsteves, 1

2. 1. Present an evolution perspective of gonadotropins2. GnRH analogues for controlled ovarian stimulation (COS)3. Protocols for COS: taking advantage of new productsEsteves, 2 3. Esteves, 3 4. Patients Doctors Industry 5. 600,000 120,000 Worldwide urine quantity (1000 litres)Number of donors 600120 1965 1975 1985 1990 19952000Donors from different regions around the worldEsteves, 6 6. Up to 65% of couples dropout from IVF without achievingpregnancy before they complete 3 cycles1-5 Reasons1,5,6Psychological burden 49%-26%Prognosis 40%-23%Oocyte retrieval52% Embryo transfer 29%Cost of treatment23%-0% Injections29%Relationship/divorce 15%-9% Physical pain 20%Physical burden7-6%Blood tests 14% 1. Olivius K t al, Fertil Steril 2004;81:258; 2. Land JA et al, Fertil Steril 1997; 68:278; 3. Schroder AK, et al, RBM Online 2004; 5:600; 4. Osmanangaoglu K et al, Hum Reprod 2002; 17:2655; 5. Rajkhowa M et al, Hum Reprod 2006; 21:358; 6. Brandes M et al, Hum Reprod 2009;24:3127; 7. Hammarberg K et al, Hum Reprod 2001; 16:374. 7. Typical Cycle (long protocol):Daily SC GnRH-a: x21FSH/LH: x10-15hCG: x1Progesterone: x14Blood tests: x4-7Number of sticks: 36-57 8. Gonadotropins: better today Milestones in the development of gonadotrophins20011940 1962 Full recombinant 2008First hCG1993 2000First Purified u-hMG gonadotrophinextracted from First highly purified First r-hLHr-hLH+r-FSH (Pergonal) and u- portfolio availablehuman urineFSH-only product launched combined hCG (Profasi) launched(Luveris)(Pergoveris)become available (Metrodin HP) 1949 1980s 19952001 2002 First hMG extractedFirst FSH-onlyFirst r-hFSH First r-hCG First filled-by-mass from urine poolsproduct launchedlaunched launched product launched (Metrodin)(GONAL-f)(Ovidrel/Ovitrelle) (GONAL-f FbM) Milestones in the development of r-hFSH 19801983 1985 1988 1992-subunit-subunit -FSH gene cloned andHuman FSH expressedFirst pregnancysequenced sequenced expressed in fibroblasts in CHO cells with r-hFSH1. Bassett et al. Reprod Biomed Online 2005;10:169177 CHO, Chinese hamster ovary2. Lunenfeld. Hum Reprod Update 2004;10:453467 9. CulturemediaBioreactor HarvestCell attachment and Concentration of proliferationsupernatantr-hFSH production and Chromatographic secretion purification stepsCollection of cell culture supernatantUltrasterile filtration medium containingCharacterization r-hFSHand full QC ofIn-process QC bulk r-hFSHEsteves, 10 10. Gonadotropins: better todayQuality SafetyKey ClinicalissuesEfficacyPatientconven- ienceEsteves, 11 11. From urinary to recombinantChoragonUrinary -hCG from different manufacturers FerringPregnylOrganon- hMG HP30% of impurities per vial with39 differentproteins identified (varied from batch to batch) Protein FSHimpuritiesLaboratoire De Spectometrie de MBO October/2009 van de Weijer et al. Reprod Biomed Online 2003;7:547557Esteves, 12 Kuwabara Y et al, J Reprod Med 2009; 54:459466 12. Impuritiescannot be associated with a better or worse outcomebut certainly are not needed for COHu-hMG HPMolecular (5 batches)r-hFSH weight (follitropinmarkers alfa)Esteves, 13 Merck Serono data on file 13. Australia 1996:Recognizes higherUK 2003: Metrodin HP standards of purity andwithdrawn safety of recombinants; unacceptable risks givenEncourages their use over that there are alternatives urinary, human derived.Esteves, 14 14. Purity Mean specificInjected(FSHFSH activity protein content) (IU/mg protein) per 75 IU(mcg)hMG < 5% ~100~750*hMG-HP < 70% 20002500~33*r-hFSH Follitropin beta700010,000 8.1*Follitropin alfa> 99%13,645 6.1Esteves, 15 1. Bassett et al. Reprod Biomed Online 2005;10:169177 15. Novel analitycal Conventional methodBioassay Physiochemical technique Highin vivovariability Minimal batch-to- (rat) batch variability (1.6%)1,2Urinary gonadotropins Follitropin beta Follitropin alfaEsteves, 16 1. Bassett et al. Reprod Biomed Online 2005;10:169177; 2. Driebergen et al. Curr Med Res Opin 2003;19:4146 16. Concept of Dose PrecisionClinical implicationsBatch variability Batch variability+20%, -25% 2%IURisk of OHSS27016.5 mcg225 (225 IU)170Poor response Bioassay Filled by MassUrinary and Follitropin betaFolitropin alfa 17. Patient Compliancesc rec FSH (pen device)sc rec LHsc hCG (prefilled syringe) 18. Evidence-based truth: Scientific truth: recFSH is more potent recFSH is purer 3.1 oocytes(Bosch, 2008)Non urine- extracted product 1.8 oocytes(MERIT, 2006)Recombinanttechnology 2.8 oocytes (Hompes, 2007)Esteves, 19 19. GnRH analogues in ARTLH surge prevention by GnRH agonistspyro (Glu) His Trp Ser Tyr Gly Leu Arg Pro Gly NH2Activation of the Regulation of Regulation of receptorGnRH receptor receptor affinity biological activity 20. GnRH analogues in ARTLH surge prevention by GnRH antagonists 12 3 pyro (Glu) His Trp Ser Tyr Gly Leu Arg Pro Gly NH2 Activation of the AntagonisticRegulation of Regulation of receptor GnRH receptoreffect receptor affinity biological activity 21. LH surge preventionGnRH antagonistsAgonistFollicularLH, FSH30 E2 , P 4Luteal20 2-4 weeks10Synchronized follicles 06 Half-life ~20h (Cetrorelix)Antagonist5 Suppress LH by 80% of4baseline levelsLH (IU/L)3 Antagonist210-6 0 6 12 1824 30 36 42 48 Hours 22. LH surge preventionGnRH antagonists vs agonistsPrevent OHSSCan be integratedby GnRH-aNo flare in spontaneous Single or multipleeffect withNo hormonal and OI cycles Antagonistdose GnRHadministrationpossible cystwithdrawalantagonist protocolformationGonadotropin administrationLess gona-Can excludedotropinsearly pregnancyFlare upPituitary effect suppressionGonadotropin administration Long GnRH agonist protocolLonger Agonist administration treatment Pre-treatment cycleTreatment cycle 23. Day 1 Rec-hCGFollicle (prefilled syringe) of rFSH13 mmor GnRH-a rec-FSH (fbm)Individualized rec-FSH dose112.5-450 UI(recLH supplementation)Vaginalmenses progesterone GnRH antagonist Day 2 or 30.25 mg/day (flexible) of mensesDay 1Rec-hCGDay 6(prefilled syringe) of rFSH of rFSHCyclerecFSH (fbm)Individualized rec-FSH doseday 21112.5-450 UI(rec-LH supplementation)Vaginal progesterone GnRH Agonist menses Day 2-5 of mensesEsteves, 24 24. Group A (hMG; N=299)Group B (HP-hMG; N=330)Group C (r-hFSH; N=236)Day Day 1 Day 6 of hCGCycleday 21 Gonadotropin rFSH/hMG Individualized dose112.5-450 UIVaginal progesterone Agonist (nasal spray): Nafarelin acetate (400 mcg/day; fixed)mensesEsteves, 25 Day 2-5 of menses 25. Outcome Measure HMG HP-hMGr-hFSHP-n=299N=330 n=236 valueTotal gonadotropin dose (IU)2,685 2,9032,268