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

2. 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.

3. 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.

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

5. 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.

8. Intracytoplasmic Sperm Injection (ICSI)

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

10. Blastomere Biopsy

16. 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

17. % 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

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

19. 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.

20. 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

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

22. 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.

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

24. 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

25. 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

26. 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

27. 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

28. 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

29. Maternal Age and Infertility
Society for Assisted Reproductive Technology 2011 (
♀ 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 to 5.3 over 42 yrs 29

30. 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.

31. Additional rounds: TEL and LSI
RCT Advanced Maternal Age yrs ( )
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 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

32. 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

33. 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 , CI [ ]
Ongoing implantation rate (%)
12/67 (22.1)
26/71 (36.6)
0.0112
OR , CI [ ]
* One-side Fisher´s test
Rubio et al., Fertil Steril 2013

34. CLINICAL RESULTS using CGH in different indicationsRM
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

35. Single Gene Disease Screening (Monogenic Diseases)

36. PCR
DNA
DOUBLE HELIX
CHROMOSOME

37. Single Gene Disease Screening

38. 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).

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

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

41. Work in progress: NGS
Illumina Sept 2013

42. Work in progress: NGS
Life Tech November 2013

43. 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.

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

45. 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.

46. Thank you