Preimplantation Genetic Diagnosis M Rafati, MD, PhD

History In the late 1980s several pioneers and their groups began preimplantation genetic diagnosis (PGD) with the detection of single gene disorders 990The first successful PGD cycle was reported Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344:768– 70 by the mid-1990s there were still only 100 live born children resulting from PGD

History For most of the next decade, relatively few clinical cases were performed, and the field generally was considered ‘‘boutique’’ in nature By 2013 the mood has changed, PGD is no longer boutique

What is PGD? The procedure which involves the removal of one or more blastomeres to test for mutations in a specific gene sequence or chromosomal abnormalities before transferring

PGD indications An alternative to conventional PND for couples with a significant risk of transmitting a serious genetic disorder to their offspring Avoiding abortion (enormous physical and psycological burden) Presence of ethical concerns in aborting embryoes affected with specific disorders ( for example: hearing loss, skeletal disorders, …)

Clinical applications Carriers of mutations (Autosomal dominant disorders, Autosomal recessive disorders ) Female carriers of X- linked disorders HLA matching Carriers of a balanced chromosomal translocation, inversion or other structural rearrangements

PGD procedure Stimulation : controlled ovarian stimulation Retrieval : oocyte retrieval Fertilization : fertilization (standard IVF/ICSI) 2 pronuclei 4 cell embryo

Biopsy Methods In looking at the entire set of data, the most common method of zona breaching used was laser drilling 15 467/26 609 (58%), cleavage stage biopsy has predominated data collections I–X.

PGD procedure aspiration of blastomere biopsied blastomere blastomeres zona drilling aspiration of blastomere Courtesy of Paul Mitchell, ACS Suite, GRI

PGD procedure Methods of genetic analysis: PCR-based techniques FISH Hybridization based techniques FISH Array CGH

PGD procedure Embryo sexing 3 probe mix: male nucleus FISH chromosome X chromosome Y chromosome 18 male nucleus

PGD for chromosome abnormalities

PGD for chromosome abnormalities 4253 cycles of PGD for inherited chromosome Abnormalities In data I, only 40 cycles for chromosome abnormalities were performed and by data X, 729 cycles were reported.

Transfer Rate From 58 817 oocytes retrieved only 11% were suitable for transfer and, from 24 773 embryos that were successfully diagnosed, only 26% were suitable for transfer The low number of transferable embryos resulted in a low number of cycles that had a transfer (64%; 2731/4253), compared with 79% for single gene disorders (3727/4733)

PGD for chromosome abnormalities Relatively low pregnancy rate (17% per oocyte collection and 26% per transfer) reflects the low proportion of embryos considered to be chromosomally normal and available for transfer

Distribution of chromosome abnormalities

Robertsonian versus reciprocal There was a difference in outcome according to the type of translocation being tested. Robertsonian translocations showed a higher number of normal/balanced embryos available for transfer leading to a higher pregnancy rate compared with reciprocal translocations

Pregnancy Rate

Pregnancy Rate The relatively low pregnancy rate (17% per oocyte collection and 26% per transfer) reflects the low proportion of embryos considered to be chromosomally normal and available for transfer. Robertsonian translocations had a higher pregnancy rate compared with reciprocal translocations

PGD versus natural conception Live birth rate: IVF/PGD: 31-35% per cycle Natural conception/medical management: 55-74%

PGS When the genetic parents are known or presumed to be chromosomally normal and their embryoes are screened for aneuploidy

PGS indications Advanced maternal age History of recurrent early pregnancy loss Repeated IVF failure Severe male infertility Sex selection

PGS The application of PGS has become controversial in recent years after the publication of eleven randomized controlled trials that failed to demonstrate a benefit to pregnancy rates and live birth outcomes. Consequently, many centres reduced or stopped PGS treatment and this probably explains the drop in PGS cycles reported to the Consortium in data collection X. Possible reasons: the well-documented chromosomal mosaicism which exists in early human embryos Limited number of chromosomes studied

Array CGH, PGS All chromosomes are analysed in a single cell The ESHRE PGS task force has completed a pilot on the feasibility of using array-CGH and polar body biopsy and a multi-centre randomized controlled trial has been set up.

Does PGS improves pregnancy rates? Current evidences do not support that the use of PGS can increase live birth rates in: Women of advanced maternal age Patients with repeated implantation failure Couples receiving IVF/ICSI for male factor infertility

PGD for single gene diseases 4733 PGD cycles

The most common indications

Successful diagnosis rate An increasing trend in the proportion of embryos with a successful diagnosis following testing: Early data sets: 83% Data set X: 90% Landmark advances in DNA amplification over the past decade including the introduction of fluorescent PCR, whole genome amplification and multiplexed PCR amplification of loci PGD for single gene disorders have shown the highest pregnancy rate (23% per OR, 29% per ET) compared with all other indications

Social sexing Reported cycles: 671 cycles 197 positive hCG tests 143 cycles with a positive fetal heart beat Clinical pregnancy rate per transfer: 29%

Desired Gender Male: 66% The vast majority of the cases for social sexing originated from one centre in the USA where MicroSort sperm selection was available to the general public The reason of uneven distribution: Average sort purity is >90% for X-bearing sperm, compared with an average sort purity of just over 70% for Y-bearing sperm

Social sexing Social sexing remains illegal in most countries (i.e. Australia, China, Europe), however, preferential selection of one sex over another most likely occurs and is not reported Indirect social sexing through PGS

Pregnancies and Babies 6458 fetal sacs 5187 clinical pregnancies 744 pregnancy complications 4140 deliveries 5135 newborns (singletons: 3182, twins: 921, triplets: 37) PND in 2462 cases and postnatal investigation in 2049 cases

Pregnancies and Babies Till 2013: 50000 cycles 10000 babies

What about PGD babies? Among 4021 newborns: Major malformations: 84 (2%) Neonatal complications: 402 (10%) These cumulative data again confirm that pregnancies and babies born after PGD are similar to the pregnancies obtained and babies born after ICSI treatment

Misdiagnosis Complexity of misdiagnosis estimation Underestimation: many transferred embryos do not result in a pregnancy, some spontaneously abort Pregnancy termination in mistakenly predicted ones without confirmation Overestimation: natural conception

Misdiagnosis after FISH testing Among 15981 embryo transfer: 16 (0.1%) For chromosomal rearrangements: 0.1% For PGS: 0.08% For X-linked disease testing: 0.5% For social gender selection: 0.3%

Misdiagnosis after FISH testing The reasons: Technical limitations: overlapping FISH signals hybridization failure non-specific hybridization the difficulty of interpreting closely adjacent signals biological factors: Mosaicism

Misdiagnosis after PCR testing Allele dropout (ADO) and contamination are inherent pitfalls of single cell PCR and each can lead to an adverse misdiagnosis Misdiagnosis rate of 10/3727 (0.27%) after embryo transfer for single gene disorders Misdiagnosis rate of 3.6% in sex determination FISH-based analysis, is technically more robust than a simple PCR assay for sex determination

Next Generation Sequencing Based PGD

New insight Faded boundaries

NGS and PGD Advantages: Turn around time Simultanous screening for both aneuploidy and Single gene disorders. Patients do not always choose to test for both when doing PGD, but it is recommended because a normal genotyping result does not necessarily guarantee that the embryo is also euploid. For instance, embryo 5 in theWWScase was trisomic for chromosome 9 but genotyped as normal for the c.1167insA mutation in the FKTN gene Turn around time This procedure can be completed in less than 24 hours: Preamplification step: 2 hours Library preparation: 8 hours Template preparation: 6 hours Sequencing: 3 hours

Recommendation Before PGD is performed, genetic counseling must be provided to ensure that patients fully understand the risk of having an affected child, the impact of the disease on an affected child and the limitations of available options that may help to avoid the birth of an affected child.

Thanks for Your Attention Rafati M 64 5/22/13