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The first clinical use of the next generation sequencing in preimplantation genetic diagnosis of Robertsonian translocation – case report.

Bialobrzeska D1,2, Lukaszuk K1,2,3,, Pukszta S 4, Liss J1, Ochman K.4

1 INVICTA Fertility and Reproductive Centre, Gdansk, Poland; 2 INVICTA Fertility and Reproductive Centre, Warsaw, Poland; 3 Department of Nursing, Medical University, Gdansk, Poland; 4 INVICTA, Molecular Biology Laboratory, Gdansk, Poland

IntroductionThe Robertsonian translocation carrier-state is connected with high risk during reproduction for unbalanced conceptuses with complete aneuploidy. The

standard multicolor FISH analysis for translocation chromosomes is not capable to eliminate risk connected with interchromosomal effect compared with

new screening methods detecting all chromosomes number abnormalities. Therefore, our aim was to determine the normal pregnancy outcome

following preimplantation genetic diagnosis (PGD) with use of next-generation sequencing (NGS) as a screening method for 24 chromosome aneuploidy

in case of Robertsonian translocation. This NGS-based PGS provides the first successful clinical application allowing to improve outcome through

comprehensive identification of euploid embryos from Robertsonian translocation carrier couple.

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Materials and methodsPatient(s): Couple (female 31, male 46) previously diagnosed as a male infertility with severe oligospermia. All the hormonal results were normal. Female

kariotype was normal. The male karyotype was abnormal with Robertsonian translocation 45,XY,der(14;15)(q10;q10).

IVF: The IVF program according to standard long protocol in Invicta Clinic started in January 2012.

PGD procedure: Day-3 embryos screening for chromosomal aneuploidy was performed in two consecutive IVF cycles firstly with FISH and then with NGS-

based protocol. In each IVF attempt three embryos were biopsied. The PGD for rob(14;15) was performed with FISH for translocation chromosomes and

search for aneuploidy with set of probes for five chromosomes. In the next IVF and PGD trial the genome material from single biopsy blastomere was

analysed with use of the Ion Torrent Personal Genome Machine (Life Tech). The short duration of procedures allowed fresh embryo transfer without need

for vitrification.

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ResultsThe embriological and PGD results for studied couple are

shown in Table 1. We analyzed 3 blastomeres biopsied from

3 embryos on day 3 of the culture in both cycles. PGS FISH

resulted in pregnancy miscarriaged on 10 week, whereas

PGS NGS resulted in healthy baby born.

Table 1. The embriological and PGD results for this couple.

PGD FISH programme

01.2012 PGD NGS programme

09.2013

Antral follicles count (AFC) 17 10

Stimulation duration (days) 9 10

HMG total dosage (IU) 2025 2250

Number cumulus oocyte complexes

12 15

MII 6 11

2PN 4 5

Number of biopsied embryos on day 3

3 3

Embryos PGD results

1: (14q D14S1419x2) (D15Z4x2) 2: (14q D14S1419x0) (D15Z4x1) 3: (14q D14S1419x3) (D15Z4x2)

1: normal, 46 X 2: unbalanced, 45 (-22) 3: unbalanced, 44, (-7, -11, -17, +21)

Total reads 1 embryo: 4 2 embryo: 1 3 embryo: 5

1 embryo: 51,810 2 embryo: 73,847 3 embryo: 70,246

Embryos transferred 1 1

4 weeks 2 days HCG level (mIU/mL)

94 322,3

Ongoing pregnancy miscarriage on 10 weeks healthy baby born

Result I. Normal:

nuc ish (14q D14S1419x2) (D15Z4x2)

Result II. Abnormal:

nuc ish (14q D14S1419x0) (D15Z4x1)

Result III. Abnormal:

nuc ish (14q D14S1419x2) (D15Z4x1)nuc ish (14q D14S1419x3) (D15Z4x2)

FIG 1. PGS FISH aneuploidy detection results.

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amo

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to

f si

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number of chromosome

Normal result: 46 XX Abnormal result: XX, -22

amo

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f si

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number of chromosome

Abnormal result: XX, -7, -11, -17, +21

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number of chromosome

FIG 2. PGS NGS aneuploidy detection results.

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ConclusionThe application of NGS to preimplantation genetic screening of embryos to predict chromosome copy number for

diagnosis of aneuploidy gives alternative to FISH and CGH-microarray new approach without limitations of those methods.

In case of Robertsonian tranclocation carriers it allows to evaluate simultaneously results of abnormal segregation of

translocation chromosomes both with interchromosomal effect products.

INVICTA Genetic Laboratoryul. Trzy Lipy 3, 80-172 Gdansk, Poland, T: +48 58 58 58 804, E: pgd@invicta.pl; W: www.invictagenetics.com

About INVICTA Genetic LaboratoryINVICTA is an experienced genetics laboratory (since 2000)

offering wide range Preimplantation Genetic Diagnosis testing using state of art Next Generation Sequencing (NGS) techniques.