www.cogen2017.cme-congresses.com

Antonio Capalbo, PhD

Laboratory Director GENETYX, reproductive genetics laboratory, Italy

PGT responsible GENERA centers for reproductive medicine, Italy

Debate on PGS Technology: Targeted vs. Whole genome approach

Targeted qPCR

Discolsure

Stake shareholder of GENETYX S.R.L

Comprehensive Chromosome Testing Methods

SNP array aCGH NGS

WGA PCR

qPCR tNGS

• Lower reproducibility

• Preferential amplification;

• Incidental findings;

• Variants of unknown significance

• Costs and times;

• Simple workflow- low costs;

• Avoid Incidental findings;

• Allow CNV and SNV analysis

• Extensively validated;

ARRAYS and NGS ARE USEFUL BUT SLOW,

EXPENSIVE, AND LABOR INTENSIVE

qPCR based CCT: simple workflow, data analysis and

wide range of throughput capabilities

Lyse (20’) Multiplex PCR (90’) Load 384-well plates (5’) • 4 TaqMan CNV assays per chr • 96 TaqMan CNV assays x test

Real-time PCR (90’) Automatic

plates loader

Easy to be fully automatized

Faster, cheaper, and more flexible than

WGA!

• Biopsy to results < 4 hours;

• 20 minutes hands on;

• Single-tube (1-witnessing step)

• Low lab space and instalment costs

• 30 tests/day on each instrument.

• High scalability (2x2)

witness

Treff et al., 2012

∆CT(embryo) - ∆CT(normal) = ∆∆ CT

chromosome

co

py

nu

mb

er

Low resolution for chromosome diagnosis:

o The vast majority of abnormal embryos have trisomies or monosomies;

o Segmental aneuploidies presents at low frequencies in pregnancies

and poses many challenging for diagnosis and interpretation;

qPCR based CCT: avoid issues related to partial aneuploidies diagnosis and management

• Immediate report;

• No need of bioinformatics

team

• No need of expensive

informatics equipment

• No need of dedicated

servers

• Easy trouble-shooting

1. Treff NR, et al Development and validation of an accurate quantitative real-time polymerase chain reaction-based assay for human blastocyst comprehensive chromosomal aneuploidy screening. Fertil Steril. 2012

2. Capalbo A et al Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies. Eur J Hum Genet. 2014

3. Capalbo A et al Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies. Eur J Hum Genet. 2014

4. Franasiak JM et al Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure. J Assist Reprod Genet. 2014

5. Franasiak JM, et al The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil Steril. 2014

1. Scott RT Jr, et al Blastocyst biopsy with comprehensive chromosome screening and fresh embryotransfer significantly

increases in vitro fertilization implantation and deliveryrates: a randomized controlled trial. Fertil Steril. 2013 2. Forman EJ, et al In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril.

2013 3. Ubaldi FM et al., Reduction of multiple pregnancies in the advanced maternal age population after implementation

of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study. Human Reproduction 2015

4. Cimadomo et al., Failure mode and effects analysis of witnessing protocols for ensuring traceability during PGD/PGS cycles. RBM online 2016;

5. Capalbo et al., Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies. Human Reproduction 2016

6. Werner MD et al Clinically recognizable error rate after the transfer of comprehensive chromosomal screened euploid embryos is low. Fertil Steril. 2014

qPCR preclinical and clinical validation data

Cell Line Results: 98.9% (91/92) consistency with karyotypes

Human Embryo Results: 98.6% consistency with SNP arrays; Lower FP error rate compared to aCGH (7% aCGH vs 0.5% qPCR)

RCTs and large cohort studies: improvement of clinical outcomes: implantation; miscarriage rate; cumulative live-birth rate.

Failure and Effect Mode Analysis (FMEA) from biopsy to qPCR report

Reproducibility across IVF centers and embryologists

Clinically recognizable error rate ( 0.1% from over 3.000 pregnancies)

Default qPCR detects mosaic samples with

high sensitivity and maximal specificity

TaqMan CNVs assays:

Uniform whole chr aneuploidies;

Mosaicism;

Goodrich et al. JARG 2016

Chromosome CNVs

Default qPCR detects large segmental

aneuploidies in TE biopsies

TaqMan CNVs assays:

Uniform whole chr aneuploidies

Mosaicisms;

Large segmental;

Chromosome CNVs

Large de novo segmental aneuploidies (5/5)

are detected by default qPCR

(GENETYX-RMA data., in preparation)

Similar number of TE cells btw practitioners

Capalbo et al., HR 2016

Neal et al., Fert Steril 2017

Standard curve to derive the cell number of TE biopsies based on Ct values of qPCR

TaqMan CNVs assays:

o Uniform whole chr aneuploidies

o Mosaicisms;

o Large DEL/DUP;

o Cellularity;

Chromosome CNVs

Monitoring TE biopsy cellularity help in the

standardization of TE biopsy in IVF laboratory and

improves QC programme

5

1

0

15

20

Mean Number of biopsied TE cells

Capalbo et al., HR 2016

7 cells

(range 2-14)

Single nucleotide variants (SNVs) can be

accurately detected in parallel with CNVs testing

CNVs TaqMan assays:

• Whole chr aneuploidies;

• Mosaicisms;

• Large segmental;

• Cellularity

A

B

B

A

Chromosome CNVs Single Nucleotide Variants

Multiplex PCR

Add primers for SNVs

testing in parallel

Presence of Allele 1

Presence

of Allele 2

TaqMan Genotyping Assay

qPCR capabilities for multifactor genetic

testing of embryo biopsy

CNVs TaqMan assays:

• Whole chr aneuploidies;

• Mosaicisms;

• Large segmental;

• Cellularity of TE biopsies

TaqMan genotyping assays

• PGT-M

• PGT-mtDNA mutations

• DNA fingerprinting

• Contamination

• Ploidy analysis

A

B

B

A

Chromosome CNVs Single nucleotide Variants

1. Treff NR et al Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial DNA disorder. Fertil Steril. 2012; 2. Scott RT 3rd, et al Trophectoderm DNA fingerprinting by quantitative real-time PCR successfully distinguishes sibling human

embryos. J Assist Reprod Genet. 2014 3. Zimmerman RS et al., Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders

and comprehensive chromosomal aneuploidy screening without whole genome amplification. Fertil Steril 2016 4. Bettio et al., 2016. 45,X product of conception after preimplantation genetic diagnosis and euploid embryo transfer: evidence

of a spontaneous conception confirmed by DNA fingerprinting. Reprod Biol Endocrinol. 5. Capalbo et al., Abnormally fertilized oocytes can result in healthy live births: improved genetic technologies for preimplantation

genetic testing can be used to rescue viable embryos in IVF cycles .In press

Quantification of the allelic ratios of variable SNPs

to determine the embryonic ploidy

AAB or BBA AB A or B Genotype

Diploid Triploid Haploid

Capalbo et al., in press

Ploidy analysis from the same biopsy can be used

to rescue viable embryos from abnormally

fertilized oocytes

Prospective cohort study

(Jan 2015-Sept 2016) involving 556

women undergoing 719 PGT-A cycles

Three AFO-derived live-births were

achieved, one from a 1PN and two

from 3.1PN zygotes.

Capalbo et al., in press

Whenever a blastocyst was obtained

from an AFO, an independent set of

primers for 40 highly variable SNPs was

incorporated in the preamplification

reaction

qPCR capabilities for multifactor genetic

testing of embryo biopsy

CNVs TaqMan assays:

• Whole chr aneuploidies;

• Mosaicisms;

• Large segmental;

• Cellularity of TE biopsies

TaqMany genotyping

• PGT-M

• DNA fingerprinting

• Contamination

• Ploidy analysis

A

B

C

A

Chromosome CNVs Single Nucleotide Variants

mtDNA content

miRNAs analysis;

Epigenetic markers;

Proteins

Biomarkers of

reproductive potential

Capalbo et al., MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be

explored for human embryo reproductive competence assessment. Fert Ster 2016

Treff et al., Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos.

Hum Repr 2017

Marin et al., Comprehensive chromosome screening and gene expression analysis from the same biopsy in human

preimplantation embryos. Mol Hum Repr 2017

Summary and take home:

Lower costs per test

Lower tour-around time

No risks for incidental findings

Simple workflow (low expertise-low risk for mix-up;)

Simple bioinformatics analysis and data management in the clinical practice

Cost effective (low installation and personnel costs)

Wide range of throughput capabilities (from 2 to 30 samples/day)

Extensively validated (pre-clinical and RCTs)

Flexible (multifactor genetic testing from single TE biopsy)

Advantages of targeted based approaches:

Specific advantages of qPCR based PGT: