Pre-implantation Genetic Testing Pre-implantation Genetic Diagnosis Pre-implantation Genetic Testing Yacoub Khalaf MD, FRCOG Director of Assisted Conception Unit & Centre for Pre-implantation Genetic Diagnosis Guy’s & St Thomas’ Hospital and King’s College, London, UK Centre for Preimplantation Genetic Diagnosis 1

Pre-implantation Genetic Testing To diagnose/exclude embryos that are affected by serious genetic disorders (PGD). To screen IVF embryos to exclude aneuploid embryos from transfer with a view to improving chances of pregnancy (PGS).

PGD

Health legacy of genetic disorders Recurrent pregnancy loss Neonatal and childhood death Structural abnormality Mental disability Chronic disease Late onset disease Centre for Preimplantation Genetic Diagnosis

Reproductive options for those with serious genetic risk Reproductive roulette Gamete donation Adoption Remain childless Prenatal diagnosis and termination of pregnancy Centre for Preimplantation Genetic Diagnosis

Centre for Preimplantation Genetic Diagnosis There is another way! Pre-implantation Genetic Diagnosis Centre for Preimplantation Genetic Diagnosis

Pre-implantation Genetic Diagnosis Detection of genetic information in an embryo at risk of a specific genetic disease by examining a representative sample taken at a preimplantation stage of development What is PGT Centre for Preimplantation Genetic Diagnosis 7

Centre for Preimplantation Genetic Diagnosis The principle of PGD affected affected affected Transfer only unaffected embryos to the patient Centre for Preimplantation Genetic Diagnosis

Preimplantation Genetic Diagnosis 9

Centre for Preimplantation Genetic Diagnosis Tissues for Preimplantation Biopsy egg cleavage stage blastocyst Polar Body Blastomere Trophectoderm Centre for Preimplantation Genetic Diagnosis

Assisted Conception Unit Fertilisation in vitro Genetics Centre Fertilisation in vitro (IVF or ICSI) Accurate genetic diagnosis Appropriate Genetic Counselling Embryo biopsy Diagnosis by FISH PCR Transfer 2 unaffected embryos Steps to PGD

Types of genetic disorders Single Gene disorders (PCR) Spinal Muscular Atrophy Cystic Fibrosis Huntingdon’s Disease Sickle cell disease EB Chromosome rearrangements (Arrays) 64 Reciprocal translocations 14 Robertsonian 6 Inversions Sex Linked disorders e.g. OTC, Hunter’s, ALD etc Centre for Preimplantation Genetic Diagnosis

Diseases detectable by PCR on single cells Ornithine transcarbamylase deficiency X-linked hydrocephalus Crouzon syndrome Stickler syndrome Tuberous sclerosis Central core disease Gaucher's disease Hyperinsulinaemic hypoglycaemia Hunter's syndrome Agammaglobulinaemia Alzheimers disease HLA typing Cystic Fibrosis Sickle cell disease Haemophilia A and B Thalassaemia Fanconi Anaemia Spinal and bulbar muscular atrophy Retinitis Pigmentosa Alport Syndrome Phenylketonuria Tay Sachs disease Alpha 1 antitrypsin deficiency Myotonic Dystrophy Huntington's disease Adenine deaminase deficiency Fragile X syndrome Marfan disease Lesch Nyhan syndrome P53 mutations Epidermolysis bullosa Plakophilin deficiency Kennedy disease Long chain acyl CoA dehydrogenase deficiency Charcot Marie tooth disease type 1a, 2a Spinocerebellar ataxia type 7 Osteogenesis imperfecta type I and IV Duchenne muscular dystrophy Congenital adrenal hyperlasia

PGH/HLA

Outline Experience at GSTFT PGD Centre Compare data with PGD Consortium Data Factors affecting likelihood of conception Recent developments - Elective single blastocyst transfer - Freezing of PGD Blastocysts Centre for Preimplantation Genetic Diagnosis

Outline Experience at GSTFT PGD Centre Compare data with PGD Consortium Data Factors affecting likelihood of conception New developments - Elective single blastocyst transfer - Freezing of PGD Blastocysts Centre for Preimplantation Genetic Diagnosis

Number of PGD cycles started by year

UK PGD cycles HFEA 3 year aggregate data ACU, Guy’s Hospital UCH, London CARE, Nottingham The Bridge Centre, London Glasgow Royal Infirmary IVF Hammersmith, London Oxford Fertility Unit Edinburgh ACU ARGC, London 18 18

Main conditions in 2009-2013 2009 2010 2011 2012 2013 CF 35 28 39 29 HD 17 31 32 26 DMD 11 8 5 9 16 Fragile X 10 Hbopathy 6 4 MD 7 12 3

Type of chromosomal rearrangement

Cycle Outcome (%)

% transferable = 42% (2.6 embryos) 48% 51% 27%

1741 sequential cycles of PGD GSTT Centre for Pre-implantation Genetic Diagnosis (Sept 1997 - Dec 2013) Stimulated 921 123 697 1741 Cycles to OR 863 105 652 1620 Cycles to biopsy 820 100 597 1517 Cycles to ET 726(79%) 92(75%)437(63%) 1255(72%) Clinical Preg 304 33 168 505 Per egg collection 36% 31% 26% 31% Per Transfer 42% 36 % 39% 40% Rearrange X-linked Total Single gene Centre for Preimplantation Genetic Diagnosis

Outline Experience at GSTFT PGD Centre Compare data with PGD Consortium Data Factors affecting likelihood of conception New developments - Elective single blastocyst transfer - Freezing of PGD Blastocysts Centre for Pre-implantation Genetic Diagnosis

PGD outcome at GSTT (1997–2011) cf ESHRE PGD consortium (1997-2007) SGD XL FISH Chromosome Total Cycles to OR 561 (4733) 95 (1167) 495 (4253) 1151 (10153) Cycles to ET 480 (3727) 80 (880) 341 (2731) 901 (7338) Mean no. replaced 1.4 (1.9) 1.4 (1.8) 1.4 (1.7) CPR/OR 36% (22%) 33% (19%) 27% (17%) 32% (19%) CPR/ET 42% (29%) 39% (26%) 40% (27%) Centre for Preimplantation Genetic Diagnosis

Outline Experience at GSTFT PGD Centre Compare data with PGD Consortium Data Factors affecting likelihood of conception New developments - Elective single blastocyst transfer - Freezing of PGD Blastocysts Centre for Preimplantation Genetic Diagnosis

Factors affecting likelihood of conception in couples having PGD Condition being tested Female age No. of fertilised oocytes No. of embryos suitable for transfer Centre for Preimplantation Genetic Diagnosis

Clinical pregnancy rate and type of genetic condition Centre for Preimplantation Genetic Diagnosis

Effect of age on likelihood of pregnancy ≤38 years 3x more likely to get pregnant per cycle

No. of fertilised eggs and chance of transfer & pregnancy OR 2.4 P<0.05 % OR 2.9 P<0.003 Centre for Preimplantation Genetic Diagnosis

Clinical pregnancy rate and embryo availability Centre for Preimplantation Genetic Diagnosis

Clinical pregnancy rate and freezing Centre for Preimplantation Genetic Diagnosis

Centre for Preimplantation Genetic Diagnosis Adverse factors Age >38 years <8 fertilised oocytes <3 transferable embryos No freezing Centre for Preimplantation Genetic Diagnosis

Outline Experience at GSTFT PGD Centre Compare data with PGD Consortium Data Factors affecting likelihood of conception Recent developments - Elective single blastocyst transfer - Trophectoderm biopsy and freezing of PGD Blastocysts Centre for Preimplantation Genetic Diagnosis

PGS

Does screening for aneuploidy make a difference to ART outcome?

PGS Outcome – implantation Gianaroli 1997, Gianaroli 1999, Munne 1999, Pellicer 1999, Kahraman 2000, Obasaju 2001, Munne 2003, Rubio 2003, Pehlivan 2003, Oter 2004, Montag 2004, Wilding 2004, Platteau 2005, Rubio 2005 Non-randomised observational studies 38 38

Outcomes in Women Who Underwent Preimplantation Genetic Screening and in Controls Table 2. Outcomes in Women Who Underwent Preimplantation Genetic Screening and in Controls.

It’s not all about the technology – It’s the biology! We have to take a step back and realise that it isnt all about the technology – we need to look at the biology

10 whole chromosome mosaicism Structural mosaicism in 12 cells Chromosome complements of the blastomeres analysed by array-CGH ( in 9 young patients whose previous transfer resulted in live birth) Microarray analysis reveals abnormal chromosome complements in over 70% of 14 normally developing human embryos. Mertzanidou A, Wilton L, Cheng J, Spits C, Vanneste E, Moreau Y, Vermeesch J, Sermon K. Hum. Reprod. 2012 4 uniformly diploid; 10 whole chromosome mosaicism Structural mosaicism in 12 cells

ICM T1 T3 T2

ICM / trophoblast results may conflict Is this a sufficiently robust test to discard patients’ embryos

For an embryo selection technique to be adopted ethically Have clear established indications Repeatable and reliable in any reasonably competent professional hands A low false negative or false positive rate Must make a significant clinical difference shown in properly constructed clinical trials Cost effective from the patient / health provider perspective PGS using FISH has failed all of these tests.

Conclusions PGD is an important option that should be offered to couples at risk of having a child with serious genetic disease. PGS is appealing in theory but has so far failed to show improvement in the chances of pregnancy per started cycle and should not be offered for IVF patients.