1. The Women’s Clinic 婦產科中心 Current Criticism on IVM Treatment Dr. Milton Leong MDCM DSc (McGill) Director, IVF Center, Hong Kong Sanatorium & Hospital Specialist in Reproductive Medicine Adjunct Professor, OBS-GYN, McGill University 1

2. The Women’s Clinic 婦產科中心 Current Criticism of IVM Lower fertilization rate Lower pregnancy & implantation rates Higher pregnancy loss Inadequate neonatal and postnatal data Possible genetic and epigenetic changes

3. The Women’s Clinic 婦產科中心 Current Criticism of IVM Inconsistent pregnancy rates Inconsistent clinical details regarding timing, endometrium, EC techniques Inconsistent laboratory details regarding EC, culture system and culture techniques Inertia from IVF success vs “experimentation”

4. The Women’s Clinic 婦產科中心 Current Status of IVM Like IVF 25 years ago, IVM is a treatment tool waiting to be universally applicable. At the moment, there is some sort of alchemy bias. There are also unresolved issues, like IVF then, that needs to be clarified.

5. The Women’s Clinic 婦產科中心 Current Status of IVM IVM is a CONCEPT IVM is a laboratory technique, not a stand alone treatment modality IVM is a method of preparing oocytes so IVF can be carried out IVF/M have its advantages over conventional IVF in some patients IVM can enable IVF to be performed when otherwise it is impossible

6. The Women’s Clinic 婦產科中心 Current Status of IVM Similar to IVF in 80’s, issues regarding IVM: Technical - timing of EC aspiration technique drugs to use Laboratory - culture system Outcome - now there is IVF to compare Safety - baby registry long term genetic changes

7. The Women’s Clinic 婦產科中心 Progress in IVM  Edwards 1965 : 1st in vitro matured oocyte  Veeck 1983 : 1st IVM pregnancy from an ovum derived from a  stimulated cycle  Cha et al. 1991 : the first pregnancy from in-vitro matured oocytes derived from a caesarean section donor  Trounson et al. 1994: IVM in women with PCOS  improvements in culture condition and transfer techniques have demonstrated that IVM is an effective treatment for women with PCO or PCOS.  In general, clinical pregnancy and implantation rates for infertile women with PCO or PCOS have reached approximately 30-35% and 10-15%, respectively, (Chian et al., 2004). 

8. The Women’s Clinic 婦產科中心 Advantages of IVM No/minimal stimulation: less OHSS less long term effect safety factor – cancer patients Flexible start time, no preparation Cancer patients no theoretical and actual risk (especially breast ca) can treat anytime

9. The Women’s Clinic 婦產科中心 Indications for IVFM treatment Polycystic ovaries (PCO) or polycystic ovary syndrome (PCOS) (primary); Hyper responders for gonadotropin stimulation (alternative); Delayed responders for gonadotropin stimulation (alternative); 9

10. The Women’s Clinic 婦產科中心 PCOS and IVF Pregnancies were comparable to non- PCOS patients Lowered fertilization rate Increased miscarriage rate FSH requirement leads to under or over response Higher cancellation rate Much higher chance of OHSS

11. The Women’s Clinic 婦產科中心 PCOS, OHSS and IVF Risk up 5-30 fold (6-30%) Estradiol >3000pg/ml # Follicles >20

12. The Women’s Clinic 婦產科中心 OHSS in PCOS Undergoing IVF Preventive Measures: Pre-treat with laparoscopic drilling Use GnRH-ant then GnRH-a to trigger Metformin (as short as 28 days) No Transfer, Cryopreservation Early Aspiration of Follicles - 2 operations Conversion to IVM IVM with and/or without stimulation

13. The Women’s Clinic 婦產科中心 GnRHa v.s. HCG in IVF-IVF used in PCOS 300 ųg Buserelin vs 10000 unit HCG Maturation ratesame(±50%) Fertilization ratesame85% Pregnancy ratesame41% OHSS rate< 1% A. Fukuda et al, Fertility and Sterility 2008 13

14. The Women’s Clinic 婦產科中心 IVM/IVF vs IVF for PCOS Child TJ, et al,2002

15. The Women’s Clinic 婦產科中心 IVM-IVF in POS 210 cycles 1883 oocytes56% maturation 83% fertilization Pregnancy rate31% fresh 32% frozen-thawed 50/56 pregnancies delivered Miscarriage11% A. Fukuda et al, Fertility & Sterility 2008 15

16. The Women’s Clinic 婦產科中心 McGill IVM results by age group (own oocytes only – patients with PCOS) Age(years) Implantation rate per embryo Clinical pregnancy rate per cycle started Live birth rate per cycle started <3514.4%34.8%20.0% 35-375.4%20.0%20.0% 38-405.0%20.0%10.0% 16

17. The Women’s Clinic 婦產科中心 Obstetric and perinatal outcomes of the IVM pregnancies

18. The Women’s Clinic 婦產科中心 Updated IVM success rates Clinical pregnancy rate 30-35% per ET; Implantation rate 9-15% per ET; More than 1,000 healthy live births; 18

19. The Women’s Clinic 婦產科中心 Pregnancy Outcome in IVM Malformation: –Cha, Fertil. Steril. 2005 5,3% major malformation rate Later neuromotor development: –Soderstrom-Anttila, Hum. Reprod. 2006 ))) Minor developmental delay at first year ))) No Difference in the second year

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21. The Women’s Clinic 婦產科中心 Outcome of IVM, IVF, ICSI and normal pregnancies obstetrical and perinatal outcome of 432 babies (55 IVM, 217 IVF, 160 ICSI) compared with 1,296 age-matched spontaneous pregnancies (controls) delivered at a single hospital (MUHC) Buckett et al. Obstet Gynecol 2007; 110:885-91

22. The Women’s Clinic 婦產科中心 Perinatal outcome IVMIVFICSIControlsp-value Twin pregnancy rate12.0%16.0%14.0%1.3%p<0.001 Triplet pregnancy rate4.0%2.0%3.0%0p<0.001 Mean birthweight (g)2,8122,8262,8013,289p<0.001 Mean gestational age (wks)37 3639p<0.001 Mean Apgar scores at 1 min8888n/s Mean Apgar scores at 5 min9999n/s Mean cord pH7.297.30 7.29n/s

23. The Women’s Clinic 婦產科中心 Congenital abnormalities following IVM (n=55) Major malformations2 ompalocele small ventricuoloseptal defect 1111 Minor malformations3 patent ductus arteriosus1 congenital hip dislocation2

24. The Women’s Clinic 婦產科中心 Relative risk for any congenital abnormality compared with controls RR95% CI IVM1.190.35 – 3.25 IVF1.010.52 – 1.90 ICSI1.410.72 – 2.68

25. The Women’s Clinic 婦產科中心 Pregnancy Outcome in IVM Mikkelsen et al. (2005) ----- 47 IVM babies –2 twins –1 NT Normal karyotype –2 preterm deliveries –1 stillbirth (42 weeks) –1 chromozomal abnormality

26. The Women’s Clinic 婦產科中心 Worldwide IVM babies born (2007) Korea455 Taiwan20 Colombia7 Canada131 Finland52 Turkey8 China58 Japan51 Vietnam42 Hong Kong18 Denmark34 Italy56 UK8 Total930

27. The Women’s Clinic 婦產科中心 Drawbacks in IVM – Lower development competence – Lower blastocysts formation – Lower implantation rate – Lower pregnancy rate – Higher miscarriage rate But, where is the beef? 27

28. The Women’s Clinic 婦產科中心 Development Competence of Oocytes 1.Nuclear maturation – progression to Meta phase II stage. 2.Cytoplasmic maturation – coordinated arrangement of proteins, organelles. Gosden R et al, Bioessays 1997 Nogueira D et al, Fertil Steril 2007 28

29. The Women’s Clinic 婦產科中心 Development Competence of Oocytes 1.IVM results in asynchronous nuclear vs. cytoplasmic maturation 2.IVM (mouse oocytes) showed different spindle morphology when assessed with immuncytologic staining. Combelles CM et al, Human Reprod 2002 Sanfins A et al, Biol Reprod 2003 29

30. The Women’s Clinic 婦產科中心 Results of the spindle and chromosome analysis in both experimental groups. No.(%) of oocytes with Oocyte GroupSpindle configurationChromosome configuration NormalAbnormalNormalAbnormal Oocytes matured in vitro27(56.3)21(43.7) a 32(66.7) 16(33.3) a (n = 48) Oocytes matured in vivo19(86.4)3 (13.6)20(90.9) 2(9.1) (n = 22) a P<.05 vs. oocytes matured In vivo. Li. Spindle and chromosome configurations of human oocytes. Fertil Steril 2008 30

31. The Women’s Clinic 婦產科中心 Does size matter – In vivo matured oocytes > immature. – Maturation rate (IVM) related to diameter of oocytes. – Zona thickness, total oocytes + zona diameter has no correlation. Cavilla JL et al, Human Reprod 2008 31

32. The Women’s Clinic 婦產科中心 Outcome of IVM – In mouse, in vitro vs. in vivo maturation. – In vitro: a) established optimized culture fluid b) no amino acid culture fluid –Comparing life span, and variety of physiological and psychological tests. –No difference between in vitro or in vivo life span or behavior. –In culture fluid a) reduction in cardiac output and pulse rate. Eppig JJ et al, Human Reprod 2009 32

33. The Women’s Clinic 婦產科中心 Outcome of IVM 21 children IVM vs. non IVM, age 2-5 PE – neurological examination and developmental (Bayley Scales) Findings: Normal Karyotype, Normal PE Mean Mental Developmental Index Score 92.7% vs. 97.2%, Mean Psychomotor Development Index Scores 96.7% vs. 96.2%. Conclusion: No developmental delay in infancy and early childhood Shu-chi M et al, Early Hum Dev 2006 33

34. The Women’s Clinic 婦產科中心 Outcome in IVM 46 children assessed at 6, 12, 24 months Muenchener Funktionelle Entwicklungs Diagnostik and Bayley Scales 6 mthsNormal 12 mths19% IVM expressed minor developmental problem 1/43 had optic glioma 24 mthsNormal development Suikkari M et al, Human Repro 2006 34

35. The Women’s Clinic 婦產科中心 Outcome of IVM IVM vs. IVF vs. ICSI N = 1581IVMIVFICSI Biochemical loss17.5%17%18%ND Clinical miscarriage rate 25.3%15.7%12.6%SD Ectopic preg.1%2.3%1.8%ND Late foetal loss1%2.7%2.9%ND PCOS24.5%22.2%ND 35 Buckett WM et al, Fertil Steril 2008

36. The Women’s Clinic 婦產科中心 IVM outcome High miscarriage rate may be due to other causes (endometrium?) because cytogenetic and DNA fragmentation analysis of the aborted embryos were not different from routine IVF. Benkhalifa Meta, Reprod Biomed Online 2009 36

37. The Women’s Clinic 婦產科中心 Genetics in IVM Methylation of the KCNQ1OT1 gene in vitro matured oocytes methylate DNA the same as GV and mature genes. At 28 hours of culture, however, GV and MI oocytes from natural cycles significantly methylate more than those from stimulated cycles. ?Hyperstimulation recruits too young follicles Khoueiry R, J Med Genet 2008 37

38. The Women’s Clinic 婦產科中心 Genetics in IVM No difference in meiotic spindle organization, chromosome alignment and aneuploidy (mouse oocytes) Xu L et al, Syst Biol Reprod Med. 2008 38

39. The Women’s Clinic 婦產科中心 Effect of IVM on Genes 39

40. The Women’s Clinic 婦產科中心 Genes and IVM Rhesus Monkey oocytes IVM vs. VVM Only 56 mRNA’s differentially expressed. These are related to cell homeostasis, cell-cell interactions, cell adhesion, mRNA stability and translation. Over expression of maternal genes. IVM oocytes can be very close to VVM oocytes but interruption of normal oocyte-somatic cell interaction may disturb full developmental competence. Lee YS et al, Physiol Genomics 2008 40

41. The Women’s Clinic 婦產科中心 Genes and IVM IVM, then examine GV, MI, MII oocytes, using H19. Differentially methylated region GV Unmethylated MIArrested – altered methylation pattern MII15/20 normal unmethylated pattern 5/20 methylated pattern Risk of epigenetic changes so safety of IVM should be assessed. Borghol N et al, Genomics 2006 41

42. The Women’s Clinic 婦產科中心 Genes in IVM Although in vivo maturated an in vitro maturated oocytes has very similar gene expression, the major difference is in persistence of immature genes. IVM oocytes differs in gene expression related to cytoplasmic function and cellular storage and homeostasis. Well D et al, Am J Obstet Gynecol. 2008 42

43. The Women’s Clinic 婦產科中心 IVM successes – PCOS patients – Natural cycle IVF/M – Birth after vitrification – Repeated successful pregnancies in same patients – Birth after preservation of oocytes for cancer patients McGill Reproductive Centre 43

44. The Women’s Clinic 婦產科中心 IVM is efficient infertility treatment, especially for patients with PCO/PCOS; IVM provides an alternative for some patients during ovarian stimulation cycles. Conclusions 44

45. The Women’s Clinic 婦產科中心 Conclusions IVM simplifies treatment, reduces costs and eliminates OHSS IVM successful in women with high AFC hCG increases final number of MII oocytes and rate of maturation IVM may be helpful in women with repeated poor embryo quality in previous IVF cycles for no obvious reason, or repeated poor responders to ovarian stimulation

46. The Women’s Clinic 婦產科中心 Conclusions IVM produces CPR/C of 35%, and up to 48% in selected cases, in women up to 35. obstetric and perinatal outcomes of IVM pregnancies comparable with IVF and ICSI IVM may be useful for oocyte donation or PGD IVM may offer a chance for fertility preservation to young women with cancer and undergoing cytotoxic treatment. IVM may not replace standard IVF but appears to play increasingly important role in ART

47. The Women’s Clinic 婦產科中心 Current Status of IVM An established treatment for PCO/PCOS In experienced hands closing on IVF Needs registry outcome and follow up Some animal data regarding genetic changes May be epigenetic changes All can be solved by proper practice and follow up and large data collection as in REGISTRY