Overview Preimplantation Genetic Screening - (PGS)
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Overview Preimplantation Genetic Screening
(PGS)
Rosy Volpi
Sr Marketing Specialist
Padova 18 Gennaio 2016
© 2013 Illumina, Inc. All rights reserved.
Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium,
iSelect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks
of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.Dati di Prevalenza sull’Infertilità
L’infertilità è un fenomeno più comune di quel che si pensa
Key Statistics from the National Survey of Family Growth (data are for 2006-2010 ). http://www.cdc.gov/nchs/fastats/fertile.htm
2
ESHRE: http://www.eshre.eu/Guidelines-and-Legal/ART-fact-sheet.aspxIVF: una rivoluzione 3 World In-vitro Fertilization (IVF) Market Opportunities and Forecsasts, 2014-2021 (http://www.bigmarketresearch.com/IVF-in-vitro-fertilization-service-market)
IVF Overview 4
IVF: percentuali di successo basse
Percentuale di nati vivi, per ciclo in UK
In 20 anni,
25%
successo.
5 HFEA Fertility Trends & Figures 2011. http://www.hfea.gov.uk/docs/HFEA_Fertility_Trends_and_Figures_2011_-_Annual_Register_Report.pdfL’Aneuploidia è una delle principali cause di
fallimento della IVF
Dopo i 42 anni di età la frequenza di
aneuploidie nell’embrione è pari al
82%
Franasiak, M. J., et al. (2014). The nature of aneuploidy with increasing age of the female partner: a review of 15169 consecutive TE biopsies evaluated with CCS Fertility & Sterility.
6 101(3).Metodo Tradizionale di selezione degli
embrioni
Contestant A Contestant B Contestant C Contestant D
Aneuploid Aneuploid Aneuploid
Selezione del migliore embrione in base alla morfologia
1. Mayo Clinic In Vitro Fertilization Website –Blastocyst. http://www.mayoclinic.com/health/medical/IM04680/. Accessed Mar 25, 2013.
7 2. UNC Dep. Of OB/GYN Blog http://uncobgyn.blogspot.com/2011/08/should-cystic-fibrosis-carrier.html#!/2011/08/should-cystic-fibrosis-carrier.html
3. Texas Department of State Health Services. http://www.dshs.state.tx.us/newborn/expandparent.shtmPGS è…
Screening del corretto numero cromosomi in un embrione
prima dell’impianto al fine di selezionare e trasferire solo
embrioni euploidi.
Ulteriori embrioni euploidi possono essere congelati per utilizzi
futuri
8Perchè PGS?
Rileva le aneuploidie negli embrioni derivanti da IVF
Le aneuploidie sono presenti in tutte le età e aumentano con l’età
della madre1
La maggior parte degli embrioni aneuploidi falliscono l’impianto
tramite aborto spontaneo
Permette il trasferimento solo di embrioni euploidi
I Benefici
Aumento del successo degli impianti e percentuale di gravidanze
portate a termine2
Riduzione del numero di aborti spontanei2
Mitiga gli effetti dovuti all’età materna3
Permette il trasferimento di un singolo embrione, riducendo I rischi
associate a gravidanze con gestazioni multiple4
1. Franasiak, M. J., et al. (2014) The nature of aneuploidy with increasing age of the female partner: a review of 15169 consecutive TE biopsies evaluated with CCS Fert Steril 101(3): 656-663.
2. Yang, Z., et al. (2012) In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fert Steril 100(1): 100-107.
9 3. Harton, L. G., et al. (2013) Diminished effect of maternal age on implantation after Preimplantation Genetic Diagnosis with array comparative genomic hybridization. Fert Steril 100(6): 1695-1703..
4. Forman, R., et al. (2013) In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial Fert Sterili100(1): 100-107.PGS Mitiga gli Effetti dovuti all’età Materna
Siginificatività: pAumento della percentuale di successo nell’IVF
69.1%
Analisi
41.7% Morfologica
+
Solo analisi
PGS
Morfologica
Group A Group B
Cycles with fresh transfer 48 (100 %) 55 (100 %)
Clinical Pregnancy 22 (45.8 %) 39 (70.9 %)
Ongoing Pregnancy 20 (41.7 %) 38 (69.1 %)
Spontaneous Abortions 2 (9.1 %) 1 (2.6 %)
Yang Z, Liu J, Collins GS, Salem SA, Liu X, et al (2012) In vitro fertilization with single euploid blastocyst transfer: a randomized
11 controlled trial. Fert Steril 100(1): 100-107Riduzione del numero di gestazioni multiple
Pazienti di età inferiore ai 43 anni
Group A (Study Group) = trasferimento di un singolo embrione o di una blastocisti
euploide
Group B (Control Group) = trasferimento di embrioni doppi o blastocisti non testate
Group A Group B
No. di Pazienti 89 86
Età 34.5 35.1
Clinical PR 69% 81%
Ongoing PR 61% 65%
Multiples 0 48%
Forman et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial Fertility & Sterility 2013 Jul;100(1):100-7.
12Evoluzione delle tecniche di PGS
2014
aCGH
2008 2012 NGS
PCR 2010
FISH
1991-1995 qPCR SNP
arrays
2-12 chromosomes 24 chromosomes 24 chromosomes,
translocations.
13 For Research Use Only. Not for use in diagnostic procedures.Perchè NGS per PGS?
► I ricercatori in ambito PGS sono costantemente alla ricerca di tecnologie per rendere la
PGS una metodica sempre più efficiente ed accurata
► NGS offre la possibilità di migliorare la rilevazione di aneuplodie negli embrioni in
paragone ai metodi in uso
► Vantaggi della tecnologia NGS:
► Riduzione dei costi
► Aumento della precisione
► Analisi parallela e personalizzata di più embrioni in una singola corsa
(multiplexing)
► Aumento del range dinamico e della sensibilità di quantificazioneNGS-based PGS
Sequences are compared to the
reference human genome
Each region of the
genome sequenced
multiple times
Embryonic DNA is amplified
(WGA)
Millions of short sequences
producedembryonic
Amplified
DNA Fragmentation
into a Library of smaller
fragments (100-200 bp)Sample Multiplexing (Barcoding)
Specific barcode Libraries from each Barcode sequences Each set of reads is
sequences are sample are pooled are used to aligned to the
attached to DNA and sequenced in differentiate reads reference sequence
fragments parallel from each sampleAnalisi dei dati
Trisomia 22
Sequenze derivanti da ciascun cromosoma vengono analizzate tramite BlueFuse
software quindi comparate con un reference di controlloCaratteristiche NGS per PGS Approccio di sequenziamento massivo– almeno 25 milioni di reads Analisi in multiplex, fino a 24 campioni per run 800K a 1M reads per campione 36nt read length Le Reads vengono mappate e ragruppate in bins (median size 1 Mbp) Conta del numero di reads per bin Algoritmi per correggere bias tecnici e dovute alla presenza di GC Normalizzazione all’interno del campione, Numero di read all’interno di ogni bin è proporzionale al copy number – Una trisomia cromosomica avrà 1.5x più read rispetto ad una disomia cromosomica
Introducing the VeriSeq® PGS Solution
June, 2014 (MiSeq)
MiSeq
24 samples per run
NextSeq
Sample to result in approximately 12 hours 96 samples per run
Visualize both 24Sure and VeriSeq PGS data with BlueFuseVeriSeq PGS NGS solution for PGS MiSeq with 24 samples per run; NextSeq 96 samples per run “Sample to report” within 12hours – as per 24sure Simple substitution for 24sure arrays with NGS platform – Start with SurePlex – End with BlueFuse Multi analysis and reporting Cost per sample similar to 24sure Resolution comparable to 24sure No reference required
NGS Workflow and Timing
VeriSeq PGS
Massively parallel sequencing approach – 25 million reads per MiSeq run
Multiplex up to 24 samples per run by using indexing
800K to 1M reads per sample
36nt read length
Reads are mapped and grouped into bins (median size 1 Mbp)
Count number of reads per bin
Algorithms to correct for technical and GC biases
Normalisation within sample, assuming median bin count across all autosomes
corresponds to copy number 2
Number of fragments from each bin is proportional to its copy number
– A trisomy chromosome will have 1.5x more counts than a disomy
chromosomeValidation of VeriSeq
► Phase 1: large preclinical validation study on single
cells to determine the accuracy of the NGS-based 24-
aneuploidy screening protocol (Fiorentino et al., 2014)
► Phase 2: prospective clinical trial, performed on a
cohort of 55 consecutive PGS cycles, involving a double
blinded parallel evaluation of embryos at blastocyst
stage with both NGS and array-CGH techniques
(Fiorentino et al., submitted)
► The study aims to outline the potential for routine
clinical use of the NGS methodology for comprehensive
aneuploidy screening of preimplantation embryos
Fiorentino et al. Development and validation of a next-generation sequencing-based protocol
for 24-chromosome aneuploidy screening of embryos. Fertil Steril. 2014 May;101(5):1375-1382.VeriSeq Validation-Phase 1
► Step 1: blinded evaluation of karyotypically defined chromosomally abnormal single cells
derived from cultured amniotic fluid samples or products of conception (POC)
► Step 2: retrospective blinded assessment of WGA products from 68 clinical PGS cycles
performed by array-CGH on single blastomeres biopsied from cleavage stage embryos
► Consistency of NGS results was evaluated with:
► Previously established cytogenetic karyotypes (single cell lines, Step 1)
► Array-CGH–based diagnoses (WGA products, Step 2)
► At the level of individual chromosome copy numbers and for the overall
diagnosis of aneuploidy or euploidy
► Discordant samples were re-evaluated by a third methodology (QF-PCR)What Does Illumina Offer for PGS?
24sure VeriSeq PGS
Array comparative genome
Technology Next-generation sequencing
hybridization
• Well established in the market • Lower cost per sample
• Lower capital expenditure • More scalable
Advantages • More flexible at low throughput • Does not need a reference
• Broader dynamic range
• Allows menu expansion
Possible
Not scalable Higher capital cost
Disadvantages
25 For Research Use Only. Not for use in diagnostic procedures.VeriSeq PGS vs 24sure
Washing Scanning BlueFuse
Sample Prep Labelling Hybridisation
Analysis
Sample to report in ~ 12 hours
Sample BlueFuse
Prep Library Prep Sequencing Analysis
Sample to report in ~ 12 hours
26
For Research Use Only. Not for use in diagnostic procedures.VeriSeq PGS vs 24sure Profiles
24Sure
VeriSeq PGS
27 For Research Use Only. Not for use in diagnostic procedures.VeriSeq PGS vs 24sure Profiles
24Sure
VeriSeq PGS
28 For Research Use Only. Not for use in diagnostic procedures.Poorer Quality Amplification Products
24Sure
VeriSeq PGS
29 For Research Use Only. Not for use in diagnostic procedures.Pubblicazioni chiave NGS in PGS
“Given the high level of consistency with an ‘’NGS has demonstrated a reliable
established methodology, NGS has methodology,
demonstrated a robust high-throughput with the potential to improve chromosomal
methodology ready for clinical application diagnosis on embryos especially in terms of
in reproductive medicine.” high-throughput, automation and ability to
detect aneuploidy.’’
63.8% clinical
pregnancy
rate
30 For Research Use Only. Not for use in diagnostic procedures.Conclusioni da Fiorentino et al.
“Given the high level of consistency with an
established methodology, such as array-CGH, NGS
has demonstrated a robust high-throughput
methodology ready for clinical application in
reproductive medicine, with potential advantages of
reduced costs and enhanced precision.”
Fiorentino et al. Development and validation of a next-generation sequencing-based protocol
for 24-chromosome aneuploidy screening of embryos. Fertil Steril. 2014 May;101(5):1375-1382.Pubblicazioni & Abstracts
Pubblicazioni
Zheng et al. (2015) Application of NGS for 24-chromosome aneuploidy screening of human
preimplantation embryos. Molecular Cytogenetics, 8:38.
Abstracts CONCLUSIONI: “NGS has demonstrated a robust high-throughput
methodology ready for extensive clinical application in reproductive
medicine, with potential advantages of reduced costs and enhanced
precision”
32 For Research Use Only. Not for use in diagnostic procedures.Pubblicazioni
Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts
N Eng J Med 2015. 373;21 DOI: 10.1056/NEJMc1500421
Authors: Ermanno Greco, Maria Giulia Minasi & Francesco Fiorentino.
Identificazione di mosaicismi e trasferimento di questi embrioni a donne che non
presentano embrioni euploidi
6 su 18 donne hanno portato a termine la gravidanza con bimbi sani
“Transfer of mosaic embryos with purportedly ‘viable’ aneuploidies should
be considered with extreme caution”
33Collateral – Lead Generation
Peer Reviewed Papers PGS Publication Booklet PGS email blast
PGS Brochure
PGS landing page (overview of PGS)
34 For Research Use Only. Not for use in diagnostic procedures.Collateral – Product Positioning
VeriSeq PGS Data
Sheet
PGS Web page and
VeriSeq PGS Product Page
www.illumina.com/PGS
VeriSeq PGS Product
Information Sheet
35 For Research Use Only. Not for use in diagnostic procedures.PGD (Preimplantation Genetic Diagnosis)
PGD viene utilizzata per lo screening di embrioni quando vi è il rischio di severi disordini genetici
ereditati dai genitori.
Autosomal Recessive Model
Metodi tradizionali si basano sull’utilizzo di STR adiacenti a specifici loci coinvolti nella malattia
Oggi viene utilizzato preferenzialmente il Kariomapping
36Karyomapping Against
Traditional STR Analysis
Traditional STR analysis Karyomapping
STR – multiallelic; variation in repeat
Markers SNP – biallelic; variation in base
length
Number of markers Usually between 10 to 30 for each Over 280K SNPs providing genome
required locus wide coverage
Limited to a single locus in each set of Able to screen multiple loci in
Coverage
STR markers parallel
Location Requires knowledge of gene location Requires knowledge of gene location
Typically 3 to 6 months to work up and
Workup Off-the-shelf solution
validate multiple STR markers
Customised set of primers for each
Workflow Standard workflow for all cases
case
Linkage analysis Manual Assisted by BlueFuse Multi
Aneuploidy is not currently offered,
Aneuploidy screening is not part of
Aneuploidy but monosomies can be observed
analysis
easilyGrazie! 38
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