Transcript Presentation

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
Risk of mosaicism
Accuracy
87.8%
95-98%
98%
Clinical results
30% PR/transfer
60% PR/ transfer
60-70% PR/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
6.0
Pregnancy rate /transfer
58.6
68.2
Implantación Rate
49.6
50.0
Pregnancy rate /cycle
47.0
48.4
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
Polarmiscarriages)
Body
Cleavage stage
biopsy
biopsy
 Severe male factor
Day-0
Day-1
Day-3
Blastocyst
biopsy
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
Results <24hours
Sample 1
Biopsy
Sample 2
Cell (s) loading
Amplification (~ 3 hrs) (98.3%)
Labelling (2 hrs)
Cy3
Cy5
DNA precipitation (~ 1 hrs)
24sure
BlueGnome
2684 clones
1Mb coverage
BlueFuse Multi
software
Hybridisation (5–12 hrs)
Washing (~ 1/2 hr)
Scanning
Euploid embryo
Loss of chromosome 8
Gain chromosome 21
Partial gain chromosome 2p
Complex aneuploidy
Chaotic pattern
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
No. of cycles
<35
35-37 38-40 41-42
>42
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%
Results: Incidence of chromosomal aneuploidies
♀ AGE (yrs)
38
39
40
41
42
43
44
45
46
P-value
No. of cycles
151
230
383
415
275
179
100
45
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
59.6
62.5
65.8
<0.0001
% Embryos with
partial aneuploidies
5.6
3.8
3.7
2.7
2.7
2.8
1.6
3.8
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
Pearson’s correlation (p<0.05)
RCT Advanced Maternal Age 41-44 yrs (2009-2011)
13, 16, 18, 21, 22
15, 17, X, Y
Additional rounds: TEL and LSI
Tel 16qx2
16
16
INITIAL DIAGNOSIS
Monosomy 16
FINAL DIAGNOSIS
Normal
Blastocyst
PGS
P-value
90
93
----
74 (82.2)
70 (75.3)
NS
----
69.2
----
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)*
P<0.0001
Live birth rate (%)
14/90 (15.5)
30/93 (32.3)*
P=0.0099
No. of PGS cycles
No. of transfers (%)
% Abnormal embryos
Mean embryos transferred (SD)
* 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
24 (96.0)
20 (90.9)
Ongoing PR/Transfer
29.2
75.0
Ongoing PR/Cycle
28.0
68.2
Miscarriage Rate
36.4
0
No. of embryo transfers (%)
RCT Repetitive Implantation Failure (2004- 2011)
Blastocyst
PGS
P-value
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
----
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]
No. of cycles
Mean embryos transferred (SD)
* One-side Fisher´s test
Rubio et al., Fertil Steril 2013
CLINICAL RESULTS using CGH in different indications
AMA
RM
RIF
PTP
MF
(≥38 yrs)
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
1.5 (0.5)
1.5 (0.6)
1.4 (0.5)
1.5 (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
No. of cycles
Mean embryos tr. (SD)
Single Gene Disease Screening
(Monogenic Diseases)
PCR
DNA
CHROMOSOME
DOUBLE
HELIX
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.
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