Preimplantation Genetic Screening

PGS-NGS 360°™

Genetic Laboratory

Innovators in Reproductive Genetics

PGS-NGS 360°™

PGS-NGS 360°™ significantly improves success rates of IVF program.

Results of IVF programs after PGS-NGS 360°™ using Next Generation Sequencing.

Primplantation genetic screening for aneuploidy using Next Generation Sequencing (NGS) is currently the most effective method for selecting genetically normal embryos for transfer.

Studies conducted by a team led by Prof. Krzysztof Łukaszuk1 have shown that using NGS to identify embryos free of any chromosomal changes can significantly increase success rates of IVF procedures. In a study of 45 patients, PGS-NGS 360°™ was carried out on biopsies from embryos in the 3rd day of development, and then transferred in a fresh cycle. The control sample consisted of 53 female patients selected according to age, AMH level, number of antral follicles and the duration of their infertility. After the PGS-NGS 360°™ was performed, a significant increase in the pregnancy rate (PR) and implantation rate (IR) was seen.

Clinical pregnancy Implantation

with PGS-NGS 360°™ without PGS-NGS 360°™

Reference: Łukaszuk K, Pukszta S, Wells D, et al. Routine use of next–generation sequencing for preimplantation genetic diagnosis of blastomeres obtained from embryos on day 3 in fresh in vitro fertilization cycles. Fertil Steril. 2015:1–6. doi:10.1016/j.fertnstert.2014.12.123.

PGS-NGS 360°™ is a test for aneuploidy screening of all 24 chromosomes.

84,4%61,5%

41,5%34,8%

PGS-NGS 360°™ (preimplantation genetic diagnosis using Next Generation Sequencing) employs state of the art human genome sequencing techniques (direct reading of genetic information) to examine embryos. It opens up new diagnostic possibilities.

In the past, embryos were selected mainly based on the basis of their morphology, The FISH and microarray methods were used for chromosome analysis. However, both techniques were limited in terms of range and accuracy. But with the PGS-NGS 360°™, all 24 chromosomes are analysed with unprecedented precision. This provides doctors with a unique opportunity to help couples exposed to increased risk of genetic abnormalities in their children.

Chromosomal aneuploidy (an abnormal number of chromosomes) is one of the main causes for the failure of IVF programs. Most embryos with aneuploidies are unable to nest in the uterus, and those that are implanted often miscarry in the first three months of the pregnancy1, 2. Preimplantation genetic diagnosis used to provide optimal selection of embryos for transfer, can greatly increases the chances of success of the procedure and the birth of a healthy child.

Age of woman over 35 Failure of IVF programs (although embryos of normal morphology were

transferred) Recurrent miscarriages Genetic defects diagnosed in previous pregnancies / birth of a child with

genetic abnormalities in the past A history of genetic defects in the family An elective single embryo transfer during in vitro program The intention of ruling out the presence of genetic defects in previously

frozen embryos Need to increase the chances of pregnancy in an IVF cycle with

donor cells Fear of childbirth with chromosomal defects

Analyses all autosomal and sex chromosomes Allows for diagnosis of the most common genetic defects, including:

Downs, Edwards, Patau, Turner, and Klinefelter’s syndrome, while still at the embryo stage

Increases the embryo implantation rate Reduces the risk of miscarriage Increases the number of healthy births Increases the efficiency of single embryo transfer by reducing

the number of multiple pregnancies

What is PGS-NGS 360°™?

What are the indications for a PGS-NGS 360°™?

Why is it worth doing the PGS-NGS 360°™?

Embryo biopsy

Completing biopsy report

Preparation of material for transport

Preparation of shipping document

Dispatch to INVICTA Genetic Laboratory

Results available within 7–14 days

• Sample is collected via biopsy of embryos at 3rd or 5/6th day of the culture.

• Blastomere or trophectoderm cells are used for tests.

• Until dispatch, the material sample must be stored in temp. –20°C.

• For transportation, 3 cartridges are used that are frozen to a temperature of –20°C. Cartridges should be placed around the box with a stand for the test tubes.

• During transport the material from biopsy must be kept cool – preferably frozen.

• The transport time depends on the place and time of shipment.

Cooperation step by step

This test is designed to detect aneuploidy and/or irregularities from Roberstonian translocation. This test does not detect partial aneuploidy (i.e. fragments of chromosomes) or chromosome mosaicism and structural chromosomal abnormalities (e.g. a fragments of chromosome deletions, inversions, duplications) or uniparental disomy, triploidy and tetraploidy.

Limitations

Biopsy

Transport

Result

1

2

3

Ready–to–use universalINVICTA PGD BIOPSY KIT™

Shipping address: INVICTA Genetic LaboratoryGdańsk Science and Technology ParkTrzy Lipy 3, 80–172 GdańskE–mail: info@invictagenetics.comTel.: +48 784 373 593

NGS methodology

Sample preparation

Libraries preparation with barcoding

Sequence preparation

Information read–out

Data analysis

The world’s first application in clinical use of NGS (Next Generation Sequencing) technology in preimplantation diagnosis.INVICTA Genetic Laboratory: August 2013

* Clinical pregnancy rate provided for both the IVF cycle and for transfer (transfers were made for all female patients) and defined as the detection of a heartbeat at the 6th week and the 1st–3rd day using ultrasound.

SpecificationRIF patients

with PGS-NGS 360°™RIF patients

without PGS-NGS 360°™

Number of IVF cycles with transfer 45 53

Number of embryo's biopsies 252 N/A

Number of transferred embryos 65 89

Average number of transferred embryos 1.4 1.7

Average female patient's age 34.0 34.4

Clinical pregnancy rate 84.4% (38/45) 41.5% (22/53)

Implantation rate 61.5% 34.8%

Table 1. The results of efficacy studies of IVF programs in patients with Repeated Implantation Failure (RIF) using the PGS-NGS 360°™.

NGS provides an accuracy of 99.999% (Q50 quality assessment by Phred Quality Scores – an indicator developed for evaluation of DNA sequence analysis methods).

99,999%is currently the most up–to–date method of analysing DNA information in the world. It ensures exceptionally precise, reliable and comprehensive result to determine the causes of miscarriage.

NGS Next Generation Sequencing

Literature1. Łukaszuk K, Pukszta S, Wells D, [et.al.]. Routine use of

next–generation sequencing for preimplantation genetic diagnosis of blastomeres obtained from embryos on day 3 in fresh in vitro fertilization cycles. J.FertilSteril. 2015 Jan 23.

2. Scott RT Jr, Ferry K, Su J, Tao X, Scott K, et al. (2012) Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, non–selection study. FertilSteril 97(4): 870–875.

3. Fiorentino F, Biricik A, Bono S, Spizzichino L, Cotroneo E, Cottone G, Kokocinski F, Michel CE. Development and validation of a next–generation sequencing–based protocol for 24–chromosome aneuploidy screening of embryos. FertilSteril. 2014 May;101(5):1375–82. doi: 10.1016/j.fertnstert.2014.01.051. Epub 2014 Mar 6.

4. Fiorentino F1, Bono S2, Biricik A2, Nuccitelli A2, Cotroneo E2, Cottone G2, Kokocinski F3, Michel CE3, Minasi MG4, Greco E4.Application of next–generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles. HumReprod. 2014 Dec;29(12):2802–13. doi: 10.1093/humrep/deu277. Epub 2014 Oct 21.

5. Anderson SH, Stankewicz–McKinney T, Glassner MJ, Hanshew K, Ketterson K, et al. (2013) Similar pregnancy and implantation rates following day 6 embryo transfer or frozen embryo transfer (FET) after blastocyst biopsy for preimplantation genetic screening (PGS). FertilSteril 100(3): S37.

6. Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, et al. (2012) Array CGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod Biomed Online 24(6): 614–620.

7. Balmir F, Hughes M, Jenkins J, Stelling JR (2013) A pilot study comparing fluorescence in situ hybridization (FISH) analysis in preimplantation genetic screening (PGS) to array comparative genomic hybridization (aCGH) technique. FertilSteril 100(3): S206.

8. Brezina PR, Brezina DS, Kearns WG (2012) Preimplantation genetic testing. BMJ 345:e5908.

9. Colls P, Coates A, Peters A, Acacio B, Roche M, et al. (2013) Preimplantation genetic diagnosis at blastocyst stage by array comparative genomic hybridization. Error rate determination. FertilSteril 100(3): S196.

10. Fishel S, Thornton S, Dowell K (2011) A new era of PGS for IVF – will it yield the anticipated improved efficiency? J Fertil In Vitro 1:1.

11. Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, et al. (2013) In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. FertilSteril 100(1): 100–7.e1.

12. Fragouli E, Wells D (2011) Aneuploidy in the human blastocyst. Cytogenet Genome Res 133(2–4): 149–159.

13. Fragouli E, Wells D (2012) Aneuploidy screening for embryo selection. SeminReprod Med 30(4): 289–301.

14. Handyside AH (2010) Preimplantation genetic diagnosis after 20 years. Reprod Biomed Online 21(3): 280–282.

15. Handyside AH (2013) 24–chromosome copy number analysis: a comparison of available technologies. FertilSteril 100(3): 595–602.

16. Harasim T, Roesemann M, Heinrich U, Wagner A, Schiller J, et al. (2013) Detection of genetic mosaicism during preimplantation genetic diagnosis (PGD). In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1).

17. Harton G, Braude P, Lashwood A, Schmutzler A, Traeger–Synodinos J, et al. (2011) ESHRE PGD consortium best practice guidelines for organization of a PGD centre for PGD/preimplantation genetic screening. Hum Reprod 26(1): 41–46.

18. Keltz MD, Vega M, Sirota I, Lederman M, Moshier EL, et al. (2013) Preimplantation Genetic Screening (PGS) with Comparative Genomic Hybridization (CGH) following day 3 single cell blastomere biopsy markedly improves IVF outcomes while lowering multiple pregnancies and miscarriages. J Assist Reprod Genet 10: 1333–1339.

19. Lee, H–L, Hodes–Wertz B, Alexis A, Lee T–L, McCulloh D, et al. (2013) Preimplantation genetic screening improves IVF success rate in women over 40. FertilSteril 100(3): S83.

20. Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, et al. (2012) Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation. ClinExpReprod Med 39(2): 52–57.

21. Lorwatthanasirikul J, Quangkananurug W, Rattanajitr T, Chanchamroen S, Sawakwongpra K (2013) Comprehensive chromosome screening with vitrified warmed single blastocyst transfer increases the pregnancy rate in advanced maternal age. In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1).

INVICTA Genetic Laboratory

Gdańsk Science and Technology ParkTrzy Lipy 3, 80–172 GdańskT: +48 58 58 58 804

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Customer Service

M: info@invictagenetics.comT: +48 784 373 593F: +48 58 746 30 02

Prof. Krzysztof Łukaszuk MD, Ph. D.Medical Directorof INVICTA Fertility Clinics

Bożena Maj M. Sc.Director of INVICTAMedical Laboratories

Sebastian Pukszta Ph.D.INVICTA Genetic LaboratoryDeputy Laboratory Managerfor Molecular Biology

Joanna Liss Ph.DDirector of INVICTAIVF Laboratory

Team