July 2015 is the 25th anniversary of the first births following preimplantation genetic diagnosis (PGD) world-wide. In this presentation, landmark developments are briefly reviewed and future developments are outlined which I believe will revolutionise the practice of IVF.

1. Preimplantation Genetics:
A 25-Year Perspective
Alan H Handyside
School of Biosciences, University of Kent, Canterbury, The
Bridge Centre, London and Illumina, Cambridge, UK
ahandyside@illumina.com

2. SINGLE CELL GENETICS AND GENOMICS
1987-
PREIMPLANTATION GENETIC DIAGNOSIS
1990-
PREIMPLANTATION GENETIC SCREENING
1994-
COMMERCIAL PGD/PGS TESTS
2008-

3. First Live Births Following PGD of Sex-linked Disease
Born July 1990
Gender identification and
selective transfer of
unaffected female
embryos in X-linked
disease
Handyside et al. Nature (1990) 344, 768

4. • Single gene defects
Duchenne muscular dystrophy
Haemophilia
Adrenoleukodystrophy
Cystic fibrosis
Spinal muscular atrophy
Beta-thalassaemia
Sickle cell disease
Tay-Sachs disease
Gaucher disease
Battens disease
• Late onset
Huntington’s disease
Early onset Alzheimer’s
Some PGD Indications Licensed by the Human Fertilisation
and Embryology Authority (HFEA) in the UK to date
• Cancer predispositions
Familial polyposis coli
Retinoblastoma
Li-Fraumeni syndrome
• Chromosome abnormalities
Aneuploidy
Translocations
Structural
• ‘Saviour siblings’
HLA matching

5. Fiorentino et al. (2005) Eur J Hum Genet 13, 953
Combined PGD for Beta Thalassaemia and HLA matching

6. Single cell genomics

7. Hou et al (2012) Cell 148, 873
2004
2012
2014
Handyside et al (2004) MHR 10, 767
Hellani et al (2004) MHR 10, 847
Hou et al (2014) Cell 155, 1492

8. Preimplantation genetic diagnosis
(PGD)

9. Genome-wide
genotyping of
300,000 SNP
markers in 24h
on a beadarray

10. Natesan et al. (2014) Genetics in Medicine 16, 838

11. Child
Chr 11 (0 – 135,007Kb)(a)
(b)
Embryo 1
Embryo 2
Embryo 3
Embryo 3
HBB
HBB
Chr 11p15.4 (3,047 – 7,451Kb)
Karyomapping provides thousands of informative
markers across each chromosome in a single
universal assay for single gene defects
PGD of Beta Thalassaemia by Karyomapping
Paternal chromosome
Maternal chromosome
Beta globin gene on chr 11
Paternal SNP markers
Maternal SNP markers

12. Preimplantation genetic screening
(PGS)

13. • 456 clinics
176,275 cycles
51,294 live births
65,179 babies born
• >1% of all births
• Average success rates:
28.45% per cycle
35.45% per transfer
38% multiple pregnancy
rate in women <35
US IVF/ICSI Live Birth Rates 2012 (CDC)
0
10
20
30
40
50
60
70
<35 35-37 38-40 41-42 43-44 >44
%
Maternal age
Live Birth Rates 2012
per cycle
per ET

14. 0
10
20
30
40
50
60
70
80
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
%
Maternal age
Oocyte aneuploidy and maternal age
Aneuploidy (%)
Livebirth (%)
Miscarriage (%)
US CDC/SART data

15. Genome-wide 24 chromosome screening:
Revolutionising IVF practice
• Across all ages, about 50% of human preimplantation embryos are
chromosomally abnormal and non-viable at later stages
• Main cause of IVF implantation failure and pregnancy loss
• Most chromosome copy number abnormalities (aneuploidy) arise in female
meiosis and are inherited in oocytes
• Maternal meiotic errors increase exponentially with age in women in their
30’s
• All women (including younger women) are affected though the relationship
with age varies
• Many oocytes in women in their 40’s have multiple aneuploidies
• Chromosome abnormalities also arise through abnormal fertilisation,
syngamy, mitotic divisions following fertilisation and paternal meiotic errors

16. Applications of 24 chromosome screening
• Elective single blastocyst transfer in young, good prognosis
patients
• Embryo selection in women in their mid/late 30’s
• Prognostic information for women in their 40’s to reduce
repeated treatment cycles with very low chance of pregnancy
and live birth
• Specific indications in high risk couples eg severe male factor
infertility and repeat pregnancy loss
• ?All eggs and embryos as routine to prevent transfer of non-
viable genetically abnormal embryos
• First live birth following polar body biopsy and array CGH in
2009
• To date, an estimated 500,000 embryos have been screened by
array CGH alone in approx 200 labs and clinics world-wide

17. • 13 previous failed IVF cycles
• 7/9 first polar bodies aneuploid
September 2, 2009
New IVF test–Array CGH
Produces baby Oliver,
offering hope to infertile

18. Polar Body Biopsy With Follow Up
at Cleavage Stages on Day 3
Christopikou et al. (2013) Hum Reprod 28, 1426

19. Euploid
Array CGH on single blastomeres biopsied from
cleavage stage embryos
+16
Complex

20. 24 chromosome copy
number detection by
blastocyst biopsy and
real time quantitative
PCR
n = 15,169
Incidence of Euploid Human Blastocysts with
Maternal Age
Modified from Franasiak et al. (2014) Fertil Steril 101, 656
Aneuploid and non-viable

21. 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
30–34 35–37 38–40 41–42
Implant (Sac) Aneuploidy Rate Sacs Lost
Blastocyst Biopsy (Day 5) and PGS by Array CGH:
Reduced maternal age effect on CPR per transfer
Harton et al (2013) Fert Stert 100, 1695

22. RCTs of 24 chromosome copy number screening
Array Comparative Genomic Hybridisation (array CGH)
(1) Yang et al (2012) Mol Cyto 5, 24
• Good prognosis patients (n=103), maternal age < 35 yr
(average 31 yr)
• Blastocyst biopsy on day 5 and array CGH vs morphological
selection for single blastocyst transfer on day 6
• 191/425 (44.9%) blastocysts aneuploid
• Ongoing pregnancy rate 69.1% vs 41.7% (p = 0.009)
• No twins
CPR per oocyte retrieval and 0% multiple
pregnancies in young, good prognosis patients with
elective single embryo transfer (eSET)

23. 36 nucleotides
1 2
24 chromosome copy number analysis
by low cost, low read depth (0.1x)
next generation sequencing (NGS) and
mapped fragment counting

24. Chr.1
Chr.2
Chr.3
Chr.4
Chr.5
Chr.6
Chr.7
Chr.8
Chr.9
Chr.10
Chr.11
Chr.12
Chr.13
Chr.14
Chr.15
Chr.16
Chr.17
Chr.18
Chr.19
Chr.20
Chr.21
Chr.22
Chr.X
Chr.Y
Euploid Control Embryo sample
Trisomy 22
Counts of sequenced fragments mapping to selected regions of
each chromosome: linear relationship with copy number

25. Array CGH NGS
Samples analysed by array CGH and NGS highly concordant
Fiorentino et al. (2014) Fertility and Sterility 101, 1375
Fiorentino et al. (2014) Human Reprod 29, 2802

26. Trisomy 13
Trisomy
13
Trisomy
21
1
2
3
Increased ratio change as
relationship between
mapped fragment counts
and copy number is linear

27. 24 chromosome copy number analysis by NGS
• WGA using the same PCR-library based protocol
• Sample to report in 12h
• Almost 100% concordance with array CGH
• Similar resolution to array CGH
• Linear relationship between displacement and copy number
• Low read depth (0.1x) allows multiplexing of up to 24 samples
reducing cost (currently equivalent to array CGH)
• Multiplexing up to 96 samples available soon
• First birth reported in 2013
• Possibility of testing for other features eg mitochondrial DNA

28. • Women ages 25-40 years with at least 2 blastocysts on Day 5
• Trophectoderm biopsy on Day 5 and PGS by NGS
• Comparator arm – standard morphological assessment
• All embryos will undergo vitrification prior to single embryo
transfer
• Pregnancy status will be followed up to 20 weeks gestation
• 600 patients with embryos transferred
• For consenting patients at 10+ weeks gestation, a maternal
blood sample will be drawn for non-invasive prenatal
aneuploidy testing
Multi-center, international randomized controlled trial (RCT)
of blastocyst biopsy and PGS by NGS with eSET (STAR)
www. clinicaltrials.com NCT02268786 started August 2014

29. ... the next 25 years

30. Rapid Advances in Single Cell Genomics
by Next Generation Sequencing
• High throughput and reduced cost
• Equipment combining high throughput NGS and
microarray scanning for high resolution
cytogenetics
• Simplified protocols and lab automation
• Targeted NGS-based strategies for chromosome
copy number and sequence level testing for
combined PGS/PGD

31. Revolution in Assisted Conception:
The Power of Genomics and NGS
• Increased patient screening for karyotype
abnormalities and pathological copy number
variants, carrier status of thousands of serious
single gene disorders and fertility related genes for
advanced diagnostics
• Routine genetic screening of all human eggs and
embryos using high throughput, low cost NGS for
combined testing of genetic imbalance and when
necessary PGD of inherited single gene defects

32. 0
10
20
30
40
50
60
70
<35 35-37 38-40 41-42 43-44 >44
%
Maternal age
Live Birth Rates 2012
per cycle
per ET
0
10
20
30
40
50
60
70
<35 35-37 38-40 41-42 43-44 >44
%
Maternal age
Live Birth Rates with PGS
per cycle
per ET
<5% multiple pregnancies!