Targeted qpcr. Debate on PGS Technology: Targeted vs. Whole genome approach. Discolsure Stake shareholder of GENETYX S.R.L

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Antonio Capalbo, PhD Laboratory Director GENETYX, reproductive genetics laboratory, Italy PGT responsible GENERA centers for reproductive medicine, Italy

1 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

2 Comprehensive Chromosome Testing Methods WGA PCR ARRAYS and NGS ARE USEFUL BUT SLOW, EXPENSIVE, AND LABOR INTENSIVE SNP array acgh NGS Lower reproducibility Preferential amplification; Incidental findings; Variants of unknown significance Costs and times; qpcr tngs Simple workflow- low costs; Avoid Incidental findings; Allow CNV and SNV analysis Extensively validated;

3 qpcr based CCT: simple workflow, data analysis and wide range of throughput capabilities Easy to be fully automatized witness Treff et al., 2012 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 Biopsy to results < 4 hours; 20 minutes hands on; Faster, cheaper, and more flexible than WGA! Single-tube (1-witnessing step) Low lab space and instalment costs 30 tests/day on each instrument. High scalability (2x2) C T(embryo) - C T(normal) = C T

trouble-shooting Low resolution for chromosome diagnosis: o o The vast majority of abnormal embryos have trisomies or

4 copy number 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 chromosome Easy trouble-shooting Low resolution for chromosome diagnosis: o o The vast majority of abnormal embryos have trisomies or monosomies; Segmental aneuploidies presents at low frequencies in pregnancies and poses many challenging for diagnosis and interpretation;

5 qpcr preclinical and clinical validation data 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 Capalbo Cell Line A et al Results: Comparison of array 98.9% comparative (91/92) genomic consistency hybridization with and quantitative karyotypes real-time PCR-based aneuploidy screening of blastocyst biopsies. Eur J Hum Genet 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 Franasiak Human JM Embryo et al Aneuploidy Results: across individual 98.6% consistency chromosomes at with the embryonic SNP arrays; level in trophectoderm biopsies: changes with patient age and chromosome structure. J Assist Reprod Genet Franasiak Lower FP JM, error et al The rate nature compared of aneuploidy to with acgh increasing (7% age acgh of the vs female 0.5% partner: qpcr) a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil Steril 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 Forman RCTs and EJ, et al large In vitro cohort fertilization studies: with single euploid improvement blastocyst transfer: of clinical a randomized outcomes: controlled trial. Fertil Steril Ubaldi FM et implantation; al., Reduction of multiple miscarriage pregnancies in the rate; advanced cumulative maternal age live-birth population after rate. implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study. Human Reproduction 2015 Failure and Effect Mode Analysis (FMEA) from biopsy to qpcr report 4. Cimadomo et al., Failure mode and effects analysis of witnessing protocols for ensuring traceability during PGD/PGS cycles. RBM online 2016; Reproducibility across IVF centers and embryologists 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 Clinically recognizable error rate ( 0.1% from over pregnancies) 6. Werner MD et al Clinically recognizable error rate after the transfer of comprehensive chromosomal screened euploid embryos is low. Fertil Steril. 2014

6 Default qpcr detects mosaic samples with high sensitivity and maximal specificity Chromosome CNVs TaqMan CNVs assays: Uniform whole chr aneuploidies; Mosaicism; Goodrich et al. JARG 2016

7 Default qpcr detects large segmental aneuploidies in TE biopsies Chromosome CNVs Large de novo segmental aneuploidies (5/5) are detected by default qpcr (GENETYX-RMA data., in preparation) TaqMan CNVs assays: Uniform whole chr aneuploidies Mosaicisms; Large segmental;

8 Similar number of TE cells btw practitioners Chromosome CNVs 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; Capalbo et al., HR 2016 Neal et al., Fert Steril 2017

9 Monitoring TE biopsy cellularity help in the standardization of TE biopsy in IVF laboratory and improves QC programme Mean Number of biopsied TE cells 7 cells (range 2-14) Capalbo et al., HR 2016

10 Single nucleotide variants (SNVs) can be accurately detected in parallel with CNVs testing Chromosome CNVs Single Nucleotide Variants A B B A CNVs TaqMan assays: Whole chr aneuploidies; Mosaicisms; Large segmental; Cellularity Add primers for SNVs testing in parallel Multiplex PCR TaqMan Genotyping Assay Presence of Allele 1 Presence of Allele 2

11 qpcr capabilities for multifactor genetic testing of embryo biopsy Chromosome CNVs Single nucleotide Variants A B B A 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 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 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 Bettio et al., ,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

12 Quantification of the allelic ratios of variable SNPs to determine the embryonic ploidy Diploid Triploid Haploid Genotype AB AAB or BBA A or B Capalbo et al., in press

13 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 Whenever a blastocyst was obtained from an AFO, an independent set of primers for 40 highly variable SNPs was incorporated in the preamplification reaction Three AFO-derived live-births were achieved, one from a 1PN and two from 3.1PN zygotes. Capalbo et al., in press

qpcr capabilities for multifactor genetic testing of embryo biopsy Chromosome CNVs Single Nucleotide Variants A C Biomarkers of reproductive potential B A CNVs TaqMan assays: Whole chr aneuploidies;

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.

14 qpcr capabilities for multifactor genetic testing of embryo biopsy Chromosome CNVs Single Nucleotide Variants A C Biomarkers of reproductive potential B A CNVs TaqMan assays: Whole chr aneuploidies; Mosaicisms; Large segmental; Cellularity of TE biopsies TaqMany genotyping PGT-M DNA fingerprinting Contamination Ploidy analysis mtdna content mirnas analysis; Epigenetic markers; Proteins 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: Advantages of targeted based approaches: Lower costs per test Lower tour-around time No risks for incidental findings Specific advantages of qpcr based PGT: Simple workflow

15 Summary and take home: Advantages of targeted based approaches: Lower costs per test Lower tour-around time No risks for incidental findings Specific advantages of qpcr based PGT: 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)

SNP array-based analyses of unbalanced embryos as a reference to distinguish between balanced translocation carrier and normal blastocysts

J Assist Reprod Genet (2016) 33:1115 1119 DOI 10.1007/s10815-016-0734-0 TECHNOLOGICAL INNOVATIONS SNP array-based analyses of unbalanced embryos as a reference to distinguish between balanced translocation