2. Outcome Measurement for Assisted Reproductive Technology DAVID L. KEEFE, M.D. Tufts New England Medical Center, Boston, Massachusetts Laboratory for Reproductive Medicine, Marine Biological Laboratory, Woods Hole, MA Brown University, and Women & Infants Hospital, Providence, RI

3. Overview of Presentation Introduction to ART procedures Study population –How factor in study populations for ART studies –How should IVF/ICSI/Donor Egg be factored in? Study Design –Efficacy measures: Primary and secondary endpoints –How should success be defined? –Safety endpoint measures A look into the future of ART outcome measurement

4. Assisted Reproductive Technologies In Vitro Fertilization/Embryo Transfer (IVF-ET), w/ or w/o ICSI Gamete Intrafallopian Tranfer (GIFT) Zygote Intrafallopian Transfer (ZIFT) Tubal Embryo Transfer (TET) Controlled Ovarian Hyperstimulation (COH) w/ Intrauterine Inseminations

5. IVF Steps Ovarian down-regulation w/ OCP, GnRH agonist or antagonist Controlled ovarian hyperstimulation with gonadotropins; U/S, E2 monitoring Trigger maturation with hCG Retrieval Fertilization by IVF or ICSI Culture embryos Transfer embryos w/ or w/o hatching Luteal support

6. IVF- Clinical Processes Asse ss sper m qual ity and cou nt Wash sample Sperm collection Egg equilibration As ses sm en t of fer tili zat io n IncubateIncubate Wash/re move excess sperm Assess & Transfer

7. IVF- Laboratory Processes Eggs retrieved Eggs stripped and cleaned Assess sperm quality and count Wash sample Sperm collection Egg equilibration Fertilization- IVF or ICSI Assess fertilization Incubate Wash/remove excess sperm Assess & Transfer Embryos

8. Controlled Ovarian Hyperstimulation Regimens for Assisted Reproductive Technology GnRH Antagonist Protocols GnRH Agonist Protocols 225 IU per day (150 IU Europe) Individualized Dosing of FSH/HMG 250  g per day antagonist Individualized Dosing of FSH/HMG GnRHa 1.0 mg per day up to 21 days 0.5 mg per day of GnRHa 225 IU per day (150 IU Europe) Day 6 of FSH/HMG Day of hCG Day 1 of FSH/HMG Day 6 of FSH/HMG Day of hCG 7 – 8 days after estimated ovulation Down regulation Day 2 or 3 of menses Day 1 FSH/HMG OCP

9. Overview of Presentation Introduction to ART procedures Study population –How factor in study populations for ART studies –How should IVF/ICSI/Donor Egg be factored in? Study Design –Efficacy measures: Primary and secondary endpoints –How should success be defined? –Safety endpoint measures A look into the future of ART outcome measurement

10. Differences in Study Populations be Factored Into ART Studies IVF/ICSI/Donor Egg patients differ in underlying disease Differ in rate of egg dysfunction IVF>ICSI>Egg donor Egg dysfunction (a.k.a. ovarian reserve, age, etc.) best predictor of outcome (can determine log-order differences in pregnancy rates among groups of patients) Studies should control for study population differences through inclusion/exclusion criteria, case-control or stratification

11. Overview of Presentation Introduction to ART procedures Study population –How factor in study populations for ART studies –How should IVF/ICSI/Donor Egg be factored in? Study Design –Efficacy measures: Primary and secondary endpoints –How should success be defined? –Safety endpoint measures A look into the future of ART outcome measurement

12. Outcome Measures for ART Deliveries/initiated cycles- the gold standard Surrogate clinical outcomes –Ongoing viable pregnancy (+FH) –Clinical pregnancy rate (+FH) –Biochemical pregnancy rate Surrogate biologic outcomes –Number of follicles –Peak E2 –Number eggs aspirated –Fertilization rate –Embryo cleavage and morphology rates

13. Outcome Measures for ART- Deliveries/Initiated Cycles The gold standard Large power needed Expensive Difficult-to-measure, but important patient differences have greater impact than drug therapy on this outcome

14. Outcome Measures for ART- Surrogate Clinical Outcomes Close to gold standard Less power needed Clinically important outcome May miss clinically-important differences, e.g. miscarriage rates Contaminated by clinic practices, e.g. cancellation policies

15. Outcome Measures for ART- Surrogate Biologic Outcomes Far from gold standard Much less power needed May not reflect clinically important outcome, e.g. young women with low response to COH still have excellent outcomes; subtle differences in drug potency on egg yield and E2 can be managed by altering dosing

16. How Should Success be Defined? Superiority to comparator (placebo;active control) Equivalence to active comparator Non-inferiority to active comparator Success should be defined not only according to pregnancy rate or its surrogate, but also according to convenience and discomfort level

17. Success Should be Defined Based on Equivalence or Non-Inferiority to Comparator Superiority to comparator (placebo;active control)- not necessary for new drug to prove useful for patient care Equivalence or Non-inferior drugs would: –Spur competition in market –Allow multiple options affecting convenience/comfort, which differ according to patient preference, e.g. vaginal vs. IM route for progesterone therapy

18. Example- Antagonist improves convenience w/o improving pregnancy outcome (also, vag. prog, s.q. gts.) Antagonist Agonist vs. * Based on median duration of use. North American Ganirelix study.

19. Safety Endpoints Ovarian hyperstimulation syndrome Miscarriage rate Multiple pregnancy rate Ectopic pregnancy rate

20. Safety Endpoints- Ovarian Hyperstimulation syndrome Life-threatening Risk sets upper limit on COH Risk may be modified by lowering peak estradiol e.g. aromatase inhibors, LH

21. Safety Endpoints-Miscarriage Rate Common (15-70%) Affected by patient-specific factors (e.g. age, ovarian reserve) May be influenced by all stages of ART, e.g. stimulation regimens, luteal phase support, culture media, etc.

22. Multiple Gestations and ART Common (15-50%) Major obstetric, pediatric and public health concern (prematurity, C.P., C/S rate, preeclampsia, gestational diabetes) Affected by patient-specific factors (e.g. age, ovarian reserve) Affected by (elusive ) clinician practices, e.g. number of viable embryos transferred Monozygotic twinning also should be considered, since is related to COH, increased in ART and causes significant morbidity (twin-twin tx) Should imprinting abnormalities (Beckwith- Wiedemann, Angelmann Sydromes, PIH) be considered an ART risk (DeBaun et al, AJHG, 2001)?

23. Overview of Presentation Introduction to ART procedures Study population –How factor in study populations for ART studies –How should IVF/ICSI/Donor Egg be factored in? Study Design –Efficacy measures: Primary and secondary endpoints –How should success be defined? –Safety endpoint measures A look into the future of ART outcome measurement

24. The Future of IVF Outcome Measurement Multicenter network to facilitate RCT’s Greater racial and ethnic diversity in clinical studies to ensure generalizability of data, as mandates increase access of working and middle class Americans to ART Improve biological surrogate outcomes

25. The Future of IVF Outcome Measurement- Improving Biological Surrogate Outcomes Aneuploidy ubiquitous and related to ART failure, through increased embryo apoptosis, implantation failure and miscarriage Thus, may provide a meaningful biologic surrogate outcome Safety problems with IVF stem from attempts to overcome egg aneuploidy through COH, e.g. OHSS and multiple gestations May be increased by COH (e.g. by short-cutting normal selection process, altering follicular environment) New technologies to dx aneuploidy e.g. CGH, SKY May be able to dx predisposition to aneuploidy

26. Aneuploid Embryos Can Develop Normally Until Day 5 of Life! Day 1 Day 5 Day 4Day 3 Day 2 Development of Embryo with Trisomy 21, determined by PGD on day 3, with develoment to normal- appearing blastocyst

27. Preimplantation Genetic Diagnosis (PGD) Can Improve Implantation Rate Identification of chromosomes X,Y,13,18,21,15,16,22 Implantation Rate PGD24.2% Controls12.4% (p<0.001) Gianaroli et al F+S, 1999

28. Preimplantation Genetic Diagnosis (PGD) Predicts IVF Outcome Age >37 > 2 failed cycles of IVF 216 couples 3 groups, depending on # normal embryos available after PGD 0 normal1 normal>1 normal #patients272655 #embryos114118322 #transfers81448 Births/patient4%15%31% Ferraretti, et al World Congress IVF, 2002

29. Preimplantation Genetic Diagnosis (PGD) in Patients with Repeated Miscarriages 76% of embryos from patients with Recurrent Pregnancy Loss have aneuploidy Pellicer, et al F+S 71:1033, 1999

30. Gt.s’ Play Key Role in Meiosis Metaphase II (MII) Metaphase I (MI) FSH then LH (Gonal F, Repronex, Follistim, Bravelle, then hCG) Immature Oocytes w/I Follicles

31. Eggs From Older Women Have Abnormal Spindles Age (years) % Abnormal Spindles 20-35 17 40-45 79 Battaglia et al, Hum Reprod. 1996;11:2217.

32. Spindle Function Imaged by Polscope

33. Eggs With Normal Spindles Develop Better

34. Telomere Shortening Explains Effects of Age on Aneuploidy: Late exit from the Production Line (Henderson and Edwards, 1968) The effects of low levels of MtDNA deletions (Keefe, 1995) Spindle abnormalities (Battaglia,1997) Reactive oxygen species (Tarin, 1998) Increased embryo arrest Increased embryo death