Preimplantation Genetic Diagnosis (PGD) in Medicine Dr. Hazem Al-Rumaih – FRCOG , MD Consultant OBGYN & reproductive Medicine

Introduction The past 100 yrs have given birth to the most profound changes in society, medicine & technology the world have ever witnessed. Genetics is one such field that have enjoyed a meteoric rise during this time.

History of Genetics Progressing from Mendelian genetics in the 1950s to the discovery of DNA in the 1960s with the consequent discoveries of genetic (hereditary) aetiotogy of many diseases, to the ability to diagnose genetic defects of embryos before birth in the 1980s. In 1990 the first PGD test done worldwide in the UK. Reaching the ability to sequence the whole human genome in 2002.

The Consequences This magnificent development in genetics have shifted medicine completely from only diagnosing to preventing hereditary disorders.

What is PGD / PGS It is genetic testing done to cell(s) extracted from day 3 / 5 embryos in the lab before transfer to the uterus as part of IVF treatment. This is done to diagnose or screen for genetic status &/or disorders.

Intracytoplasmic Sperm Injection (ICSI)

d2 Dobson et al, 2004; R Reijo Pera,2010

Blastomere Biopsy

Comparison of embryo-biopsy strategies Polar Body (PB) Cleavage (D3) Blastocyst (D5) Blastocyst rate 50% oocytes 80% D3 biopsied embryos Not all D5 embryos can be biopsied Cost Highest cost (PB1 and PB2 analyzed separately) Moderate cost Lowest cost (only evolutive blastocysts) Timing Up to 2-3 days 24 hours 24 hours / vitrification Informativity 90% 98-99% 99% Mosaicism No mosaicism / only maternal information Risk of mosaicism Accuracy 87.8% 95-98% 98% Clinical results 30% PR/transfer 60% PR/ transfer 60-70% PR/transfer

% complex aneuploidies % partial aneuploidies Pregnancy rate /transfer D3 vs D5/D6 biopsy VS. Day 3 (<38yrs) Blastocyst (<38yrs) Nº de cycles 479 31 Mean age 34.7 33.3 % cycles with ET 80.2 71.0 % abnormal embryos 66.3 49.0 % caotic embryos 14.8 1.6 % complex aneuploidies 19.1 12.6 % partial aneuploidies 6.0 Pregnancy rate /transfer 58.6 68.2 Implantación Rate 49.6 50.0 Pregnancy rate /cycle 47.0 48.4 On top we can see what we call a complex aneuploidy and at the bottom the results of a blastomere with a chaotic chromosome pattern. 17

Aim of PGD Offers couples at risk the chance to have an unaffected (desired) child , without facing termination of pregnancy.

Uses of PGD Diagnose & avoid embryos with chromosomal aberrations. Diagnose & avoid single gene disorders. Diagnose & avoid X-linked diseases (specific embryo gender). Human leucocyte antigen (HLA) typing for stem cell transplant for an affected offspring. First & second polar body testing can be done to study maternal genetic contribution.

Preimplantation Genetic Screening (PGS) Comprehensive Chromosome Screening (CCS) Advanced maternal age (≥38 yrs; >40 yrs) Implantation failure (≥ 3 IVF attempts) Recurrent miscarriage (≥ 2 miscarriages) Severe male factor Polar Body biopsy Day-3 Cleavage stage biopsy Blastocyst biopsy Day-0 Day-1 Day-5

Professional Pre Requisites Parents counseled & accepted. ≥ 5 grade A or 1 embryos. Both IVF & genetic labs are trained & prepared to do PGD.

PGD Techniques FISH (Fluorescent In Situ Hydridization) : for cytogenetic diagnosis of 5-7 chromosomes: (13,16,18,21,22,X &Y). Now obsolete. PCR (Polymerase Chain Reaction): for molecular diagnosis of specific diseases. More recent & more accurate: CGH-Array (Comparative Genomic Hydridization): 24 chromosones are tested.

FISH in Detection of Chromosomal Abnormality Triploid Chromosome 13 and normal other chromosomes

CCS with CGH arrays: 24-chromosomes screening Cy3 Cy5 24sure BlueGnome 2684 clones 1Mb coverage BlueFuse Multi software Biopsy Cell (s) loading Amplification (~ 3 hrs) (98.3%) Labelling (2 hrs) DNA precipitation (~ 1 hrs) Hybridisation (5–12 hrs) Washing (~ 1/2 hr) Results <24hours Scanning Sample 1 Sample 2 A single cell from each embryo underwent Whole Genome Amplification (WGA) with Sureplex DNA Amplification System Amplification efficiency was 98.3%. WGA products and control DNAs used were labeled with Cy3 and Cy5 fluorophores, and co-hybridized (>4 hours) in the 24sure platform. After washing, slides were scanned and analyzed by BlueFuse Multi software. 24

Euploid embryo Loss of chromosome 8 Different profiles of abnormalities were observed in the analysis of the embryos. In this slide we can see the results of the analysis of an euploid embryo and an embryo with loss of chromosome 8. 25

Partial gain chromosome 2p These are the images of the analysis of an embryo with a gain of chromosome 21 and an embryo with a partial aneuploidy for chromosome 2. 26

Chaotic pattern Complex aneuploidy On top we can see what we call a complex aneuploidy and at the bottom the results of a blastomere with a chaotic chromosome pattern. 27

Ethical & Legal Issues Couple’s Informed consent. Should not be done on social grounds. Should be done on professional grounds only. Should be provided only by well trained teams

Maternal Age and Infertility Society for Assisted Reproductive Technology 2011 (www.sart.org) ♀ AGE <35 35-37 38-40 41-42 >42 No. of cycles 39,721 19,930 20,130 10,277 6,033 Percentage of cancellations 6.4 9.5 12.7 16.3 20.7 Pregnancy rate/retrieval 46.2 38.5 29.3 19.5 9.1 Implantation rate 36.0 27.3 17.5 9.4 4.0 Delivery rate/ retrieval 42.9 35.2 24.8 14.5 5.3 ≈38% In the annual registry of 2011 of the Society for Assisted Reproductive Technology in USA, we can see: The high percentage of IVF cycles with own oocytes that were performed in women over 38 years age (up to 38%). How delivery rates per retrieval decreases after 37 years, from 35.2 to 5.3 over 42 yrs 29

Results: Incidence of chromosomal aneuploidies Pearson’s correlation (p<0.05) ♀ AGE (yrs) 38 39 40 41 42 43 44 45 46 P-value No. of cycles 151 230 383 415 275 179 100 30 ---- % Chromosomal abnormal embryos 74.0 75.6 79.0 85.8 88.2 95.0 95.7 94.2 91.1 0.001 % Embryos with complex aneuploidies 26.6 29.2 32.8 40.4 54.8 59.6 62.5 65.8 <0.0001 % Embryos with partial aneuploidies 5.6 3.8 3.7 2.7 2.8 1.6 0.0 0.015 % Embryos with chaotic pattern 16.1 11.8 15.0 16.3 15.8 13.7 18.1 17.3 20.0 0.045 Finally, the percentage of embryos with chaotic pattern was almost constant for different ages except for women of 46 years. Possibly, this significant increase could be attributed to the low number of embryos included in this age group.

Additional rounds: TEL and LSI RCT Advanced Maternal Age 41-44 yrs (2009-2011) 13, 16, 18, 21, 22 15, 17, X, Y Additional rounds: TEL and LSI 16 INITIAL DIAGNOSIS Monosomy 16 Tel 16qx2 FINAL DIAGNOSIS Normal Blastocyst PGS P-value No. of PGS cycles 90 93 ---- No. of transfers (%) 74 (82.2) 70 (75.3) NS % Abnormal embryos 69.2 Mean embryos transferred (SD) 2.8 (0.8)* 1.6 (0.6) P<0.0001 Ongoing PR/transfer (%) 14/74 (18.9) 30/70 (42.8)* P=0.0021 Ongoing implantation rate (%) 20/152 (13.1) 40/114 (35.1)* Live birth rate (%) 14/90 (15.5) 30/93 (32.3)* P=0.0099 However, the limitations of the FISH, embryo biopsy and culture conditions could have compromised the results of these studies. In fact, we carried out an RCT in women between 41-44 years, with at least 5 MII oocytes from 1 or 2 stimulated cycles. We performed day-3 embryo biopsies with day-5 embryo transfer and FISH analysis for 9 chromosomes and we observed a significant increase in live birth rates in the PGS group compared to day-5 blastocyst transfer without PGS. * Two-sides Fisher´s test Rubio et al., FS 2013

Day-3 RCT using CGH arrays (May 2012- Sept 2013) VS. ≥ 5 MII FM (<2 mill spz/ml) No PGS PGS No. of patients informed 34 35 No. of cycles performed 25 22 No. of embryo transfers (%) 24 (96.0) 20 (90.9) Ongoing PR/Transfer 29.2 75.0 Ongoing PR/Cycle 28.0 68.2 Miscarriage Rate 36.4 On top we can see what we call a complex aneuploidy and at the bottom the results of a blastomere with a chaotic chromosome pattern. 32

RCT Repetitive Implantation Failure (2004- 2011) Blastocyst PGS P-value No. of cycles 43 48 ---- Mean Age (SD) 35.3±2.9 35.2±3.5 No. of transfers (%) 36 (83.7) 43 (89.6) % Abnormal embryos 57.3 Mean embryos transferred (SD) 1.9±0.7 1.7±0.6 Ongoing PR/transfer (%) 12/36 (33.3) 23/43 (53.5) 0.0579 Ongoing PR/retrieval (%) 12/43 (27.9) 23/48 (47.9) 0-0402 OR 0.04218, CI [0.1755-1.009] Ongoing implantation rate (%) 12/67 (22.1) 26/71 (36.6) 0.0112 OR 0.03776, CI [0.1715-0.8317] * One-side Fisher´s test Rubio et al., Fertil Steril 2013

CLINICAL RESULTS using CGH in different indications RM RIF PTP MF AMA (≥38 yrs) No. of cycles 204 187 33 116 1808 Mean Age (SD) 35.9 (2.7) 36.5 (2.5) 36.8 (2.4) 34.8 (3.2) 41.5 (2.1) Mean embryos analyzed (SD) 5.5 (3.1) 5.9 (3.0) 5.7 (3.8) 6.8 (3.7) 4.6 (2.6) % abnormal embryos 68.2 67.7 71.5 65.4 85.7 % embryo transfers 76.0 79.1 81.8 83.6 40.7 Mean embryos tr. (SD) 1.5 (0.5) 1.5 (0.6) 1.4 (0.5) 1.3 (0.7) PR /transfer 58.1 57.4 44.4 62.9 50.3 Implantation rate 47.9 50.9 36.4 54.2 46.4 Miscarriage rate 13.3 4.7 16.6 3.3 6.5

Single Gene Disease Screening (Monogenic Diseases)

PCR DNA DOUBLE HELIX CHROMOSOME

Single Gene Disease Screening

Single Gene Disease Screening Mutation report and requisition form. 5 ml blood (EDTA tube) or buccal swab. Primer development (3 weeks common and 6 weeks for rare mutation). Day 5 biopsy for Single Gene disease plus Aneuploidy. Report 2 days after receiving the samples, i.e. freeze embryos & transfer later (FET).

Children Follow up PGD Children follow up showed similar outcome to IVF / ICSI without PGD.

Next Generation Sequencing (NGS) Near Future of PDG Next Generation Sequencing (NGS) Rapid Accurate More efficient Scan for more genes

Work in progress: NGS Illumina Sept 2013

Work in progress: NGS Life Tech November 2013

Conclusions PGD is currently easier & faster to do, cheaper, more comprehensive & accurate than before. It is now an integral part of ART. More advances in the technology is coming soon. It should be use within clear regulations. It has a great potential in reducing incidence of chromosomal & genetic disorders.

Future of Genetics Hopefully in the near future we will wetness the use of knowledge & technologies in genetics to repair or correct genetic defects.

Thanks Finally I would like to thank Prof. Carlos Simon Scientific Director of IVI & Igenomix Valencia , Spain For allowing me to share some of his results in this lecture.

Thank you