ELIAS M. DAHDOUH, M.D., M.Sc. Head & Medical Director, ART-PGD Center Department of Obstetrics-Gynecology CHU Sainte-Justine, University of Montreal Associate Member, PROCREA Clinics Montreal 3 At the conclusion of this presentation, participants should be able to explain and discuss:  WHY PGD/PGS for RPL is performed?  WHEN to biopsy?  HOW to test?  WHAT are the clinical results? Elias M. Dahdouh, M.D., M.Sc. has nothing to disclose for this presentation  50% of RPL have no definitive cause  High rate of aneuploidies in RPL  High risk of subsequent Pregnancy Loss  Yet majority will achieve a spontaneous Live birth (>50%)  Therefore comparison: Treatment versus Expectant ! Shahine, Lathi. Sem Reprod Med 2014 “Any intervention for patients with RPL must be shown to provide a higher chance of live birth beyond no intervention” 7 PGD PGS Aim Identify genetically normal embryos Achieve a live birth Indication Monogenic disorder X-linked Chromosome abnormality HLA typing Gender Selection … AMA RIF RPL idiopathic Severe male factor Embryo Selection Fertility Often fertile Infertile Prenatal diagnosis Indicated Indicated for same risk factors as natural conceptions Harper et al. Hum Genet 2012 8 1. IVF ± ICSI 4. Embryo Transfer 2. Embryo Biopsy 3. Genetic Analysis 9  PGD: avoid unbalanced embryos  Avoids medical termination  Alternative to prenatal diagnosis  Genetic counselling/Multidisciplinary approach  Risks:  Infertility  Spontaneous abortions  BB with mental retardation and/or developmental delay Fiorentino F. Curr Opin Obstet Gynecol 2012 12  PGS: to increase clinical outcomes in IVF  Euploid ET  Invasive embryo selection  Aneuploidies are frequent in IVF cycles  High rate of embryonic aneuploidies (30%  80%)  Low IR (30%  6%) Munné S. Curr Genomics 2012 13 14 % aneuploidy % No euploid Franasiak et al. Fert Steril 2014 The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening 15 16 17 1st and 2nd Polar Body Characteristics  Simultaneous (8-12 h after ICSI) or sequential  Performed where embryo biopsy is considered illegal  Detects only maternal anomalies (aneuploidies)  30% of postmeiotic anomalies not detected Montag et al. Fertil Steril 2013 18 Day 3: 1 Blastomere Pros & Cons PROS Worldwide experience Enough time for fresh ET Suitable for many patients CONS Less DNA High rate of mosaicism Lower implantation? Harton et al. Hum Reprod 2011 19 Blastocyst: 3-10 trophectoderm Cells Pros & Cons PROS  More DNA: less no results  Less mosaicism: less error rate  No impact of embryo biopsy  Less embryos to test: lower cost  eSET possible  Frozen ET: better endometrial environment CONS  Not all embryos reach blasctocyst  Requires experience Schoolcraft et al. Fertil Steril 2010 20 21 Scott et al. Fertil Steril 2013 22 Polar Body (oocyte) D3 blastomere D5-6 trophectoderme Advantages 1- No effect on development 2- time for genetic test 3- excellent for maternal origin 4- avoid legal and ethical concerns 1- small number of cells 2- all indications 3- time for genetic test 1- few number to test 2- more cells: efficient +++ 3- all indications 4- less mosaïsme Drawbacks 1- high number tested 2- sequential biopsy 1- F+ ou F-: mosaïsme 1- blastocyst culture 2- may need vitrification 2- less implantation? 3- No information on mutations of paternal origin Xu K, Montag M. Hum Genet 2012 3- expertise 23 24  FISH: old technology  Comprehensive Chromosome Screening (CCS): new technology  aCGH  SNP microarray  NGS 25 FISH Technique  Hybridization of specific DNA probes marked with different colors  Detects translocations and partial aneuploidies 26 27  Complete 24-chromosome analysis (simultaneous testing for translocations and aneuploidies)  No pre-IVF validation required by parental DNA (aCGH)  Automated analysis, < 24 hours (aCGH)  Fresh ET still possible (if D3 or D5 biopsy + aCGH) Handyside A. Fertil Steril 2013 28 Munné S. Curr Genomics 2012 29 30  ONLY: Prospective & Retrospective Observational studies  No single study with expectant management as control group  No single randomized controlled trial (RCT) Franssen et al. Hum Reprod Uptodate 2011 32  Limited number of chromosomes tested  Technical problems: subjective, hybridization failure, signal overlap, and splitting  Negative impact of D3 biopsy on development  Negative effect with PGS-FISH on IVF program Mastenbroek et al. N Engl J Med 2007     Prospective, n = 28 (D3+aCGH) Cycles with ET: 60.7% Detection rate = 93.6% IR = 63.6% and PR = 70.6%  Prospective, n = 18 (Blastocyst+SNP)  Cycles with ET: 67%  IR = 45% and PR = 75% Fertil Steril. 2011 Tan et al. Hum Reprod 2013 37 38 39 Study Group N Age Blastocysts Platform Yang et al. 2012 55 31.2 8.3 aCGH Forman et al. 2013 89 35.1 5.8 qPCR Scott et al. 2013 72 32.2 7.1 qPCR 40 PGS Control P Yang et al 2012 70.9% 45.8% 0.017 Forman et al 2013 63.2% 51.7% 0.08 Scott et al 2013 79.8% 63.2% 0.01 41  Experience in extended embryo culture (%blastulation)  Experience in Blastocyst biopsy  Validated and tested CCS platform  Effective cryopreservation program (Frozen ET) Schoolcraft et al. Fertil Steril 2013  Level of evidence PGD-PGS for RPL is weak !  FISH on D3 biopsy is associated with low outcomes  CCS on blastocyst biopsy for RPL appears promising  CCS can be performed by aCGH, SNP microarray, or NGS  RCTs based on intention-to-treat are still needed (ideally expectant should be the control group)  PGD-PGS is invasive, carries financial burdens. Not for every patient, not for every clinic !!! 43 March 2015