PGS-NGS 360 Preimplantation Genetic Screening

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1 Genetic Laboratory PGS-NGS 360 Preimplantation Genetic Screening Innovators in Reproductive Genetics

2 PGS-NGS 360 PGS-NGS 360 is a test for aneuploidy screening of all 24 chromosomes. Primplantation genetic screening for aneuploidy using Next Generation Sequencing (NGS) is currently the most effective method for selecting genetically normal embryos for transfer. PGS-NGS 360 significantly improves success rates of IVF program. Studies conducted by a team led by Prof. Krzysztof Łukaszuk 1 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. Results of IVF programs after PGS-NGS 360 using Next Generation Sequencing. 84,4% 61,5% 41,5% 34,8% 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: /j.fertnstert

3 What is PGS-NGS 360? 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 pregnancy 1, 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. Why is it worth doing the PGS-NGS 360? 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 are the indications for a PGS-NGS 360? 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

4 Cooperation step by step 1 Biopsy Embryo biopsy Completing biopsy report Sample is collected via biopsy of embryos at 3rd or 5/6th day of the culture. Blastomere or trophectoderm cells are used for tests. 2 Transport Preparation of material for transport Preparation of shipping document 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. Dispatch to INVICTA Genetic Laboratory During transport the material from biopsy must be kept cool preferably frozen. The transport time depends on the place and time of shipment. 3 Result Results available within 7 14 days Shipping address: INVICTA Genetic Laboratory Gdańsk Science and Technology Park Trzy Lipy 3, Gdańsk E mail: Tel.: Ready to use universal INVICTA PGD BIOPSY KIT NGS methodology Sample preparation Libraries preparation with barcoding Sequence preparation Information read out Data analysis Limitations 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.

5 The world s first application in clinical use of NGS (Next Generation Sequencing) technology in preimplantation diagnosis. INVICTA Genetic Laboratory: August 2013 Table 1. The results of efficacy studies of IVF programs in patients with Repeated Implantation Failure (RIF) using the PGS-NGS 360. Specification RIF patients with PGS-NGS 360 RIF patients without PGS-NGS 360 Number of IVF cycles with transfer Number of embryo's biopsies 252 N/A Number of transferred embryos Average number of transferred embryos Average female patient's age Clinical pregnancy rate 84.4% (38/45) 41.5% (22/53) Implantation rate 61.5% 34.8% * 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. NGS Next Generation Sequencing 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. 99,999% NGS provides an accuracy of % (Q50 quality assessment by Phred Quality Scores an indicator developed for evaluation of DNA sequence analysis methods).

6 Team Prof. Krzysztof Łukaszuk MD, Ph. D. Medical Director of INVICTA Fertility Clinics Bożena Maj M. Sc. Director of INVICTA Medical Laboratories Sebastian Pukszta Ph.D. INVICTA Genetic Laboratory Deputy Laboratory Manager for Molecular Biology Joanna Liss Ph.D Director of INVICTA IVF Laboratory Literature 1. 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 Jan 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): 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 May;101(5): doi: /j. fertnstert Epub 2014 Mar 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 Dec;29(12): doi: /humrep/ deu277. Epub 2014 Oct 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): S 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): 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): S 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). 8. Brezina PR, Brezina DS, Kearns WG (2012) Preimplantation genetic testing. BMJ 345:e 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): 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): S 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: 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): e Fragouli E, Wells D (2011) Aneuploidy in the human blastocyst. Cytogenet Genome Res 133(2 4): Fragouli E, Wells D (2012) Aneuploidy screening for embryo selection. SeminReprod Med 30(4): Handyside AH (2010) Preimplantation genetic diagnosis after 20 years. Reprod Biomed Online 21(3): Handyside AH (2013) 24 chromosome copy number analysis: a comparison of available technologies. FertilSteril 100(3): INV.10.MAR.PRO _EN INVICTA Genetic Laboratory Customer Service Gdańsk Science and Technology Park Trzy Lipy 3, Gdańsk T: M: T: F: 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: 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): S 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): 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).

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