preimplantation genetics: a 25-year perspective
TRANSCRIPT
Preimplantation Genetics: A 25-Year Perspective
Alan H Handyside
School of Biosciences, University of Kent, Canterbury, The Bridge Centre, London and Illumina, Cambridge, UK
SINGLE CELL GENETICS AND GENOMICS1987-
PREIMPLANTATION GENETIC DIAGNOSIS1990-
PREIMPLANTATION GENETIC SCREENING1994-
COMMERCIAL PGD/PGS TESTS 2008-
First Live Births Following PGD of Sex-linked DiseaseBorn July 1990
Gender identification and selective transfer of unaffected female embryos in X-linked disease
Handyside et al. Nature (1990) 344, 768
• Single gene defectsDuchenne muscular dystrophyHaemophiliaAdrenoleukodystrophyCystic fibrosisSpinal muscular atrophyBeta-thalassaemiaSickle cell diseaseTay-Sachs diseaseGaucher diseaseBattens disease
• Late onsetHuntington’s diseaseEarly onset Alzheimer’s
Some PGD Indications Licensed by the Human Fertilisation and Embryology Authority (HFEA) in the UK to date
• Cancer predispositionsFamilial polyposis coliRetinoblastomaLi-Fraumeni syndrome
• Chromosome abnormalitiesAneuploidyTranslocationsStructural
• ‘Saviour siblings’HLA matching
Fiorentino et al. (2005) Eur J Hum Genet 13, 953
Combined PGD for Beta Thalassaemia and HLA matching
Single cell genomics
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
Preimplantation genetic diagnosis (PGD)
Genome-wide genotyping of 300,000 SNP markers in 24h on a beadarray
Natesan et al. (2014) Genetics in Medicine 16, 838
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
Preimplantation genetic screening (PGS)
• 456 clinics176,275 cycles51,294 live births65,179 babies born
• >1% of all births• Average success rates:
28.45% per cycle35.45% per transfer38% multiple pregnancyrate in women <35
US IVF/ICSI Live Birth Rates 2012 (CDC)
<35 35-37 38-40 41-42 43-44 >440
10203040506070
Live Birth Rates 2012
per cycleper ET
Maternal age
%
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 (%)
Live birth (%)
Miscarriage (%)
US CDC/SART data
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
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
• 13 previous failed IVF cycles
• 7/9 first polar bodies aneuploid
September 2, 2009
New IVF test–Array CGHProduces baby Oliver,offering hope to infertile
Polar Body Biopsy With Follow Upat Cleavage Stages on Day 3
Christopikou et al. (2013) Hum Reprod 28, 1426
Euploid
Array CGH on single blastomeres biopsied fromcleavage stage embryos
+16
Complex
24 chromosome copy number detection by blastocyst biopsy andreal time quantitative PCR
n = 15,169
Incidence of Euploid Human Blastocysts withMaternal Age
Modified from Franasiak et al. (2014) Fertil Steril 101, 656
Aneuploid and non-viable
30–34 35–37 38–40 41–420%
10%
20%
30%
40%
50%
60%
70%
80%
90%
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
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)
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
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
Array CGH NGS
Samples analysed by array CGH and NGS highly concordant
Fiorentino et al. (2014) Fertility and Sterility 101, 1375Fiorentino et al. (2014) Human Reprod 29, 2802
Trisomy 13
Trisomy 13
Trisomy 21
1
2
3
Increased ratio change as relationship between mapped fragment countsand copy number is linear
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
• 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
... the next 25 years
Rapid Advances in Single Cell Genomicsby 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
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
<35 35-37 38-40 41-42 43-44 >440
10203040506070
Live Birth Rates 2012
per cycleper ET
Maternal age
%
<35 35-37 38-40 41-42 43-44 >440
10203040506070
Live Birth Rates with PGS
per cycleper ET
Maternal age
%
<5% multiple pregnancies!