1. Preimplantation Genetic Diagnosis
M Rafati, MD, PhD

2. 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

3. 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

4. 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

5. 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, …)

6. 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

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

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

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

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

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

12. PGD for chromosome abnormalities

13. 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.

14. Transfer Rate
From oocytes retrieved only 11% were suitable for transfer and, from 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)

15. 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

16. Distribution of chromosome abnormalities

17. 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

18. Pregnancy Rate

19. 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

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

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

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

23. 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

24. 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.

25. 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

26. PGD for single gene diseases
4733 PGD cycles

27. The most common indications

28. 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

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

30. 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

31. 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

32. 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

33. Pregnancies and Babies
Till 2013:
50000 cycles
10000 babies

34. 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

35. 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

36. Misdiagnosis after FISH testing
Among 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%

37. 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

38. 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

39. Next Generation Sequencing Based PGD

40. New insight Faded boundaries

41. 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

42. 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.

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