)

24Sure™ V3 aCGH (Illumina)

NGS Ion Torrent™ PGM (Life Technologies)

Workflow

16-24h

Up to 28 embryos/microarray pack

$$$$

Workflow

15h

Up to 34 embryos/run in a 318™ chip loaded

$$

A 24-chromosome aneuploidy detection and unbalanced translocations determination of 100 embryos in PGS/PGD by means of NGS

European Society of Human Reproduction and Embryology 31st Annual Meeting (Lisbon, Portugal)

PENACHO V1, GONZÁLEZ-REIG S1, ALCARAZ LA1, RAMOS B2, ROGEL S2, HORCAJADAS JA3, AIZPURUA J2 1 BIOARRAY S.L. (ELCHE, SPAIN); 2 IVFSPAIN (ALICANTE, SPAIN); 3 PABLO DE OLAVIDE UNIVERSITY (SEVILLE, SPAIN)

o PGS has been designed for the 24-chromosome aneuploidy screening. PGD consists in the detection of point mutations by sequencing a selected gene, but also can be used for HLA matching, inherited predisposition to cancer, late on-set disorders and chromosome translocations. Both pre-conception analysis are required to select euploid and/or mutation-free embryos for implantation, increasing pregnancy success rates in IVF.

o Regarding to the 24-chromosome screening, technical limitations showed by FISH and aCGH can be overcome by genome-wide DNA sequencing in terms of NGS data providing an alternative approach for detecting Copy Number Variations (CNV)

INTRODUCTION

Thirty-five patients (mean age 39.1±4.13 years, range 32-52) were recruited for PGS validation by NGS. Also, one couple performed PGD for chromosome translocation; male was diagnosed as a carrier of a balanced translocation [46,XY,t(1;16)(q42.1;p13.11)] in peripheral blood by Q-banding.

Human oocyte fertilization by ICSI-IVF. Trophectoderm biopsy on day 5 of embryo culture (blastocyst stage). Frozen-thawed ET cycles.

Double-blinded genetic testing with both NGS and aCGH of 100 whole genome amplified DNA samples (WGA) amplified by PicoPlex® technique from trophectoderm cells.

CNV analysis was performed with BlueFuse Multi Software (aCGH) and Ion Reporter™ Software 4.4 (NGS), which determined the ploidy status with less than 0.01X read coverage.

METHODOLOGY

Evaluation in terms of Preimplantation Genetic Screening (PGS) for aneuploidy detection and Preimplantation Genetic Diagnosis (PGD) for unbalanced translocations, how the next-generation sequencing (NGS) Ion-Torrent™ PGM platform along with low-pass whole genome sequencing, provides the ability to assess many biopsied trophectoderm cells in order to confirm which euploid embryos were ready-to-transfer in an IVF cycle.

OBJECTIVES ?

PGS/PGD FOR ANEUPLOIDY AND TRANSLOCATION DETECTION - RESULTS

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VALIDATION BY NGS vs. aCGH - RESULTS

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TECHNICAL AND CLINICAL OUTCOMES - RESULTS

PGD

Both aneuploidy detection in PGS cycles and a precise identification of unbalanced translocations in PGD cycles were assessed by NGS Ion-Torrent™ PGM platform.

This NGS strategy is rapid, highly accurate, robust, sensitive, easily reproducible, and cost-effective in contrast to well-established 24Sure™ V3 aCGH (array-based comparative genomic hybridization)

CONCLUSIONS

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722 OOCYTES INJECTED IN 40 IVF CYCLES

587 OOCYTES FERTILIZED

(Range 4-32 per cycle)

287 TWO PRONUCLEI EMBRYOS BIOPSIED

100 BLASTOCYSTS

SELECTED

37 EUPLOID (20 males; 17 females)

63 ANEUPLOID

10 SINGLE TRISOMIES

15.87%

6 DOUBLE OR COMPLEX TRISOMIES

9.52%

19 SINGLE MONOSOMIES

30.16%

5 DOUBLE OR COMPLEX MONOSOMIES

7.94%

11 FULL GAIN + LOSSES

17.46%

12 PARTIAL IMBALANCES 19.05%

3 UNBALANCED TRANSLOCATIONS Paternal origin

4.76%

29 PATIENTS ACHIEVED EUPLOID EMBRYOS

Median euploid embryos/cycle: 3±2.39 (range 0-10)

Source: http://slodive.com

• NO technical failures • NO mistakes in gender determination • NO false positives and negatives

Higher accurate screening of two different imbalances by NGS

98/100 embryos (95%CI: 92.6%-99.7%)

2398/2400 chromosomes (95%CI: 99.8%-100%)

100% SPECIFICITY

100% SENSIBILITY

98.9% CONCORDANCE aCGH vs. NGS

Euploid ET (to date) (median ET/cycle: 1.35±0.49) 46

IVF cycles with transfer (to date) (18 SET, 12 DET) 30/40 (75%)

Women with transfer (to date) 28/35 (80%)

Clinical pregnancies (to date) 22/28 (78.57%)

Ongoing pregnancies (to date) 7/19 (36.84%)

Babies delivered (to the date) 12/19 (63.16%)

Women with advanced age (≥38) 23 (65.71%) (Mean age 41.5±2.7 years, range 38-52)

Women with polycystic ovary syndrome 9 (25.71%) (Mean age 35.7±3.1 years, range 32-43)

Women with recurrent miscarriages (≥2) 7 (20%) (Mean age 36.4±3.4 years, range 32-41)

Women with low IVF stimulation response 4 (11.43%) (Mean age 39.3±2.9 years, range 35-41)

Couples with oocytes donation 11 (31.43%) (Mean age 40.1±5.0 years, range 32-46)

Couples with sperm donation 6 (17.14%) (Mean age 39.8±1.5 years, range 38-41)

…Some clinical data

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PGS

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