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24sure publications

First to deliver 24 chromosome aneuploidy screening in 12 hours 8019 24sure A5 1.4 (8019 S)_Layout 1 17/07/2012 15:12 Page 2

24sure is a ground breaking technology which delivers aneuploidy screening of 24 chromosomes, within a 12 hour period, starting from single cells. 24sure uses a reliable and proven amplification method that delivers representative amplification of genomic DNA. Amplified samples are then co-hybridised to 24sure or 24sure+ slides, BAC microarray platforms designed to enable robust and reliable calling of results. 24sure uses microarray based approaches to investigate whole chromosome copy number ploidy for pre-implantation genetic screening (PGS).

24 chromosome aneuploidy screening is rapidly evolving. This document contains a list of the key publications in the preimplantation genetic screening field and studies that use 24sure. Included in this compilation are articles concerned with validating the use of 24sure to screen all 24 chromosomes, articles that use the 24sure and 24sure+ platforms, general interest PGD/PGS articles such as the ESHRE PGD consortium best practice guidelines. Finally, a number of abstracts are included from conferences that have taken place in 2011 and to date in 2012.

Articles and abstracts included in this document were compiled in June 2012. For a more up to date list please visit: http://www.24suretest.com/literature

Key

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Version 1.4 June 2012

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Meeting abstracts – Fertility 2011

Application of aCGH for preimplantation genetic diagnosis of chromosome rearrangements and additional unrelated chromosome aneuploidy Colleen Lynch, Dr Lucy Jenner, Dr Simon Fishel, CARE Fertility, Nottingham, NG7 8PZ, UK

Trophectoderm biopsy and arrayCGH to detect unbalanced forms of t(9;15)(q22.2;q11.1) after failed FISH Colleen Lynch, Ellen Cater, Lucy Jenner, Simon Fishel, CARE Fertility, Nottingham, NG7 8PZ, UK

Multiple Factor PGD – Diagnosis of chromosome aneuploidy and translocation status, HLA type and Fanconi Anaemia Type A in a single blastomere Colleen Lynch, Kathryn Berrisford, Natalie Keown, Dr Lucy Jenner, Dr Anthony Brown, Dr Simon Fishel, Dr Mark Hughes, CARE Fertility, Nottingham, NG7 8PZ, UK

Comprehensive characterisation of chromosome abnormalities in human blastocysts Samer Alfarawati, Elpida Fragouli, Anastasia Mania, Tracey Griffiths, Tony Gordon, Danny Daphnis, Dagan Wells; Reprogenetics UK, Oxford, UK; Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, Oxford, UK

High implantation, pregnancy and birth rates after comprehensive chromosomal screening of blastocysts Dagan Wells, Samer Alfarawati, Elpida Fragouli, Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, UK

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Head office US office Singapore CPC4, Capital Park 823 Valencia Avenue Representative Office Fulbourn, Cambridge, CB21 5XE Half Moon Bay 3 Science Park Drive #02-12/25 United Kingdom CA 94019 The Franklin tel: +44 (0) 1223 885900 USA Science Park I fax: +44 (0) 1223 885949 toll free: 1-800-418-9656 118223 email: [email protected] tel: (650) 284 9732 Singapore web: www.24suretest.com fax: (814) 284 8650 tel: (65) 9831 0060 8019 24sure A5 1.4 (8019 S)_Layout 1 17/07/2012 15:12 Page 3

Contents

Validation of 24sure technology

Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with arrayCGH for good prognosis IVF patients: results from a randomised pilot study . .10

Validation of array comparative genome hybridisation for diagnosis of translocations in preimplantation human embryos ...... 11

Polar body arrayCGH for prediction of the status of the corresponding oocyte. Part I: clinical results ...... 13

Polar body arrayCGH for prediction of the status of the corresponding oocyte. Part II: technical aspects ...... 14

Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation ...... 15

Validation of microarray comparative genomic hybridisation for comprehensive chromosome analysis of embryos ...... 16

24sure – articles using preimplantation genetic screening

ArrayCGH analysis shows that aneuploidy is not related to the number of embryos generated ...... 17

Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation ...... 18

Detection of aneuploidy by array comparative genomic hybridisation using cell lines to mimic a mosaic trophectoderm biopsy ...... 19

Assessment of 19,803 paired chromosomes and clinical outcome from first 150 cycles using arrayCGH of the first polar body for embryo selection and transfer ...... 20

Array comparative genomic hybridisation on first polar bodies suggests that non-disjunction is not the predominant mechanism leading to aneuploidy in humans ...... 21

Live birth after polar body array comparative genomic hybridisation prediction of embryo ploidy – the future of IVF? ...... 21

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24sure+ – articles using translocation preimplantation genetic diagnosis

PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridisation ...... 22

Combined translocation and aneuploidy-screening after polar body biopsy and arrayCGH ...... 23

First births after preimplantation genetic diagnosis of structural chromosome abnormalities using comparative genomic hybridisation and microarray analysis ...... 24

PGD/PGS general interest

ESHRE PGD consortium best practice guidelines for organisation of a PGD centre for PGD/preimplantation genetic screening ...... 25

ESHRE PGD consortium best practice guidelines for amplification-based PGD ...... 25

ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridisation-based PGD ...... 25

ESHRE PGD Consortium/Embryology Special Interest Group – best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS) . . 25

A new era of PGS for IVF – will it yield the anticipated improved efficiency? ...... 26

Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes ...... 26

Preimplantation genetic diagnosis after 20 years ...... 26

What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium Steering Committee ...... 26

What next for preimplantation genetic screening? A polar body approach! ...... 27

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Contents

Meeting abstracts

PGDIS 2012

Oral presentations

[S3] Polar body biopsy and arrayCGH for aneuploidy in advanced maternal age ...... 28

[S14] Blastocyst biopsy for aneuploidy screening ...... 28

[S4] The use of polar bodies and cumulus cells to assess oocytes and select embryos . . . . . 28

[S13] PGD for aneuploidy and translocation chromosome imbalance by cleavage stage biopsy ...... 28

[O1] Polar body 1, polar body 2 and blastomere analysis in PGS treatment ...... 28

[S16] Karyomapping as a means of universal diagnosis and providing insight into early human development ...... 28

[S6] Polar body analysis by arrayCGH and follow up at cleavage stages ...... 29

[O2] Altered morphokinetic parameters of embryos identified as aneuploid by single cell arrayCGH analysis at the 8-cell stage ...... 29

[S11] Chromosomal mosaicism in the cleavage stage embryo revisited ...... 29

[O3] Aneuploidy screening reveals high incidence of abnormalities of chromosomes which are not involved in the rearrangements ...... 29

[S7] Blastocyst biopsy for aneuploidy screening ...... 29

[O8] Accuracy rates in biopsies performed on day 3 and day 5 embryos using CGHarray technology for PGS ...... 29

Posters

[P18] Preimplantation genetic diagnosis for translocation t(6;14)(q26;q31) using array comparative genomic hybridisation at the blastocyst stage ...... 29

[P8] First experiences with PGD after trophectoderm biopsy at Kinderwunsch Centrum Munich (KCM), Germany ...... 29

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[P51] Multiple Factor PGD – 4 case reports involving testing for up to 4 indications in a single blastomere ...... 29

[P35] First Hungarian results on aneuploidy patterns of day three embryos analysed by array comparative genome hybridisation ...... 30

[P48] Application of aCGH for pre-implantation genetic diagnosis of chromosome rearrangements and additional unrelated chromosome aneuploidy ...... 30

[P38] Birth after Preimplantation Genetic Diagnosis (PGD) using Microarray Comparative Genomic Hybridisation (MaCGH) for chromosome inversion (1) (p35q42) ...... 30

[P26] Increased pregnancy rates after trophectoderm biopsy for PGD of monogenic diseases and chromosomal aberrations ...... 30

[P27] Implementation of array-CGH technology in Preimplantation Genetic Diagnosis for rob(13;14)(q10;q10) Robertsonian translocations ...... 30

[P23] Detection of whole chromosome vs. chromatid errors and possible germinal mosaicism at various stages of oocyte maturation by array-CGH ...... 30

[P28] Checking on mosaicism in day 5-blastocysts – first results from a microarray based diagnostic setting ...... 30

[P29 Developmental potential of day 3 embryos diagnosed by arrayCGH analysis on the blastomere ...... 30

[P50] Aneuploidy testing of polar bodies by array comparative genomic hybridisation (arrayCGH) ...... 30 Alpha Conference

[PP38] First Hungarian results on aneuploidy patterns of day 3 embryos analysed by array comparative genome hybridisation ...... 31

[PP40] Identifying a suitable patient population for arrayCGH on first polar bodies to improve embryo selection and clinical outcomes ...... 31 The 1st Biomarker meeting in Reproductive Medicine: Emergence of a new field

A comparison of efficiency of aCGH and FISH PGS in patients with multiple IVF failure . . . . . 31

Use of Comparative Genomic Hybridisation (CGH) for embryo assessment: Clinical results . . 31

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Contents

Best of ASRM and ESHRE: where we are and where we are going?

Who is the most important for a successful outcome in ART? The clinician or the embryologist? ...... 32

Preimplantation Genetic Testing: Current Technology and Global Experience – When should it be done and by what technique(s)? The European view ...... 32 14th World Congress on Human Reproduction 2011

PGD for reciprocal translocations using a BAC Microarray ...... 32

Chromosomal mosaicism in embryos from young ART patients determined by Array Comparative Genomic Hybridisation (aCGH) ...... 33

PGS 2.0 – new genetic tests and complete cromosome assessment of human embryos . . . 34

Confirmation of FISH diagnosis and level of mosaicism for embryos clinically diagnosed with a single aneuploidy ...... 34

First clinical results using Microarray analysis for detection of aneuploidy in a PGD program . . 34 ASRM 2011

[O-76] Significant decrease in miscarriages after preimplantation genetic diagnosis (PGD) for recurrent pregnancy loss using array comparative genome hybridisation (arrayCGH) ...... 36

[O-83] 24 chromosome analysis of products of conception specimens by arrayCGH allows for more results than conventional karyotyping and allows for simultaneous maternal cell contamination analysis ...... 37

[O-88] Are patients undergoing PGD for chromosome rearrangements at increased risk of aneuploidy affecting chromosomes unrelated to their rearrangement (interchromosomal effect)? ...... 38

[O-183] Implantation of euploid blastocysts, assessed by array comparative genomic hybridisation (aCGH), in unstimulated cycles is not correlated with maternal age ...... 39

[O-198] Efficiency of preimplantation genetic screening (PGS) using arrayCGH compared to matched control IVF patient populations with and without day 3 PGS FISH ...... 40

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[O-202] Trophectoderm biopsy on day 5, 6 or 7 – does it matter? ...... 41

[O-205] Chromosome breakage in oocytes and embryos: assessment of frequency, origin and clinical relevance of genetic instability during preimplantation development ...... 42

[O-209] The progress of chromosome abnormalities from meiosis to the blastocyst stage . . . 43

[O-284] Embryo cohort size does not affect euploidy ...... 44

[O-371] Trophectoderm biopsy – age matters ...... 45

[P-396] PGD via array comparative genome hybridisation (aCGH) can be used for any translocation to simultaneously detect unbalanced embryos and aneuploidy ...... 47

[P-406] Chromosomal mosaicism in day 3 embryos from young, successful art patients as determined by array comparative genomic hybridisation (CGH) ...... 48

[P-473] Analysis of 4795 day 3 embryos by array comparative genome hybridisation (aCGH): aneuploidy patterns ...... 49 ASHG 2011

Mosaicism does not affect the accuracy of 24 chromosomes preimplantation genetic screening on cleavage stage embryos ...... 50 ESHG 2011

Optimising aneuploidy detection in the human preimplantation embryo using 24sure™ . . . . . 51 ESHRE 2011

[O-063] An altered pattern and high incidence of multiple meiotic errors in women of advanced maternal age undergoing IVF ...... 52

[O-250] PGD for translocations using array comparative genome hybridisation ...... 53

[P-407] Maternal meiosis 1 errors detected by array comparative genomic hybridisation of first polar bodies correlate with poor embryo quality ...... 54

[P-430] Clinical application of array comparative genomic hybridisation in preimplantation genetic diagnosis for reciprocal and robertsonian translocations ...... 55

[P-432] Successful clinical application of arrayCGH for 24 chromosome aneuploidy on day 3 preimplantation embryos: high efficiency and reliability ...... 56

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Contents

PCRS 2011

[O-6] Implantation and miscarriage rates following arrayCGH analysis at the cleavage and blastocyst stages ...... 58

[P-35] Reanalysis of monosomic embryos at the blastocyst stage following day 3 biopsy and array comparative genomic hybridisation (aCGH) ...... 59

[P-36] Aneuploidy patterns in 3143 day 3 embryos analysed by arrayCGH ...... 60

[P-42] Trophectoderm cells derived from blastocyst biopsy are suitable for arrayCGH analysis of 24 chromosomes ...... 61 GfH 2011

Preimplantation genetic diagnosis – arrayCGH of pluripotent trophoblast cells after trophectoderm biopsy ...... 62 Fertility 2011

Can first polar body arrayCGH results predict developmental potential ...... 62

Developmental potential of embryos derived from oocytes found to have a single aneuploidy in the first polar body ...... 62

Oocyte chromosome complement does not appear to be a factor in fertilisation – a comparison of first polar body analysis of fertilised and unfertilised metaphase II oocytes . . 62

Application of aCGH for preimplantation genetic diagnosis of chromosome rearrangements and additional unrelated chromosome aneuploidy ...... 63

Trophectoderm biopsy and arrayCGH to detect unbalanced forms of t(9;15)(q22.2;q11.1) after failed FISH ...... 63

Multiple Factor PGD – Diagnosis of chromosome aneuploidy and translocation status, HLA type and Fanconi Anaemia Type A in a single blastomere ...... 63

Comprehensive characterisation of chromosome abnormalities in human blastocysts ...... 63

High implantation, pregnancy and birth rates after comprehensive chromosomal screening of blastocysts ...... 63

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Validation of 24sure technology

This paper focuses on increasing the use of single embryo transfer (SET) and improving the pregnancy rates associated with SET by using 24sure in good prognosis IVF patients (<35, no prior miscarriage). In addition this publication represents the first randomised study of its kind, and shows a significant increase in pregnancy rates using 24sure compared with the control group (morphology scores only). On-going pregnancy rates at 20 weeks per cycle started, were 41.7% in the control group, 69.1% with 24sure analysis.

Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with arrayCGH for good prognosis IVF patients: results from a randomised pilot study Yang Z 1, Liu J2, Collins GS3, Salem SA1, Liu X2, Lyle SS1, Peck AC1, Sills ES1, Salem RD1. 1Division of Reproductive Endocrinology Research, Pacific Reproductive Center, Torrance, CA 90505, USA; 2IVF Division, Beijing Jia En De Yun Hospital, Beijing 100083, People’s Republic of China; 3Centre for Statistics in Medicine, Wolfson College Annexe, University of Oxford, Oxford, UK Molecular Cytogenetics 2012, 5:24

Background: Single embryo transfer (SET) remains underutilised as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by arrayCGH (aCGH) to provide this advantage and improve pregnancy rate from SET.

Methods: First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomised into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups.

Results: For patients in Group A (n = 55), 425 blastocysts were biopsied and analysed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies.

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Validation of 24sure

Conclusion: Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomised to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.

This study validates the use of arrayCGH to assess embryos for structural chromosomal abnormalities. They aim to determine the minimal resolution detectable, the error rate of detection in translocation cases, investigate mosaicism and determine the number of historical cases that could have been helped by arrayCGH.

Validation of array comparative genome hybridisation for diagnosis of translocations in preimplantation human embryos Colls P, Escudero T, Fischer J, Cekleniak NA, Ben-Ozer S, Meyer B, Damien M, Grifo JA, Hershlag A, Munné S. Reprogenetics, Livingston, NJ 07039, USA Reprod Biomed Online. 2012 Jun;24(6):621-9

Abstract Fluorescent in-situ hybridisation (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormalities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridisation (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were _6Mbp and _5Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by arrayCGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for arrayCGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by arrayCGH. This study validates arrayCGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. ArrayCGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy. Fluorescent in-situ hybridisation (FISH) has been used in preimplantation genetic diagnosis (PGD) for structural chromosome abnormalities. This approach has limitations such as the need for testing of carriers, poor fixation and no possibility for aneuploidy screening of all chromosomes. The use of array comparative genome hybridisation (CGH) can solve

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these limitations. ArrayCGH was used in 47 PGD cases for Robertsonian or reciprocal translocations. The smallest detectable fragment size was ~6Mbp for single blastomeres (day 3) and ~5Mbp for trophectoderm (day 5) biopsies. Translocation cases in which three or more translocated fragments were detectable by arrayCGH underwent PGD. Of those, a total of 54 non-transferred embryos analysed by arrayCGH were reanalysed by FISH. The error rate for arrayCGH was 1.9% (1/54). Of the 402 embryos analysed by arrayCGH, 81 embryos were normal or balanced, 92 were unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. We retrospectively reviewed 926 PGD cases for reciprocal translocations performed by FISH in order to identify those in which fewer than two fragments were less than _6Mbp. In conclusion, this study validates the use of arrayCGH in PGD for translocations showing a low error rate and shows that arrayCGH can identify all embryos with unbalanced reciprocal and Robertsonian translocations. ArrayCGH is a better approach than FISH or single-nucleotide polymorphism arrays because it allows simultaneous screening of all chromosomes for aneuploidy and does not require previous genetic testing of the parents.

This review article, contains similar themes to the recent Yang et al (2012 page 10) paper, tackling the topic of single embryo transfer. They conclude that it will soon be standard practice to determine which (single) embryo to transfer in an IVF cycle by assessing the chromosome complement and support the use of arrayCGH to do this.

Comprehensive genetic assessment of the human embryo: can empiric application of microarray comparative genomic hybridisation reduce multiple gestation rate by single fresh blastocyst transfer? Sills ES, Yang Z, Walsh DJ, Salem SA. Reproductive Research Division, Pacific Reproductive Center, Orange County, 10 Post, Irvine, CA, 92618, USA Arch Gynecol Obstet. 2012 Jun 8. [Epub ahead of print]

Purpose: The unacceptable multiple gestation rate currently associated with in vitro fertilization (IVF) would be substantially alleviated if the routine practice of transferring more than one embryo were reconsidered. While transferring a single embryo is an effective method to reduce the clinical problem of multiple gestation, rigid adherence to this approach has been criticised for negatively impacting clinical pregnancy success in IVF. In general, single embryo transfer is viewed cautiously by IVF patients although greater acceptance would result from a more effective embryo selection method.

Methods: Selection of one embryo for fresh transfer on the basis of chromosomal normalcy should achieve the dual objective of maintaining satisfactory clinical pregnancy rates and minimizing the multiple gestation problem, because embryo aneuploidy is a major contributing factor in implantation failure and miscarriage in IVF. The initial techniques for preimplantation genetic screening unfortunately lacked sufficient sensitivity and did not yield the expected results in IVF. However,

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Validation of 24sure

newer molecular genetic methods could be incorporated with standard IVF to bring the goal of single embryo transfer within reach.

Results: Aiming to make multiple embryo transfers obsolete and unnecessary, and recognizing that array comparative genomic hybridisation (aCGH) will typically require an additional 12 h of laboratory time to complete, we propose adopting aCGH for mainstream use in clinical IVF practice.

Conclusion: As aCGH technology continues to develop and becomes increasingly available at lower cost, it may soon be considered unusual for IVF laboratories to select a single embryo for fresh transfer without regard to its chromosomal competency. In this report, we provide a rationale supporting aCGH as the preferred methodology to provide a comprehensive genetic assessment of the single embryo before fresh transfer in IVF. The logistics and cost of integrating aCGH with IVF to enable fresh embryo transfer are also discussed.

Two papers were published following the ESHRE PGS taskforce proof of principle trial to determine the reliability of PGS by polar body biopsy.

Polar body arrayCGH for prediction of the status of the corresponding oocyte. Part I: clinical results Geraedts J, Montag M, Magli MC, Repping S, Handyside A, Staessen C, Harper J, Schmutzler A, Collins J, Goossens V, van der Ven H, Vesela K, Gianaroli L. Department of Genetics and Cell Biology, Research Institute GROW, Faculty of Health, Medicine and Life Sciences, Maastricht University, PO Box 5800, Maastricht, AZ 6202, The Netherlands Hum Reprod. 2011 Nov;26(11):3173-80. Epub 2011 Sep 9

Background: Several randomised controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridisation (arrayCGH) analysis.

Methods: In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilised by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by arrayCGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by arrayCGH for concordance analysis.

Results: Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1-15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%).

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With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by arrayCGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant.

Conclusions: This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by arrayCGH analysis of both PBs.

Polar body arrayCGH for prediction of the status of the corresponding oocyte. Part II: technical aspects Magli MC, Montag M, Köster M, Muzi L, Geraedts J, Collins J, Goossens V, Handyside AH, Harper J, Repping S, Schmutzler A, Vesela K, Gianaroli L; Department of Reproductive Medicine, SISMER, Via Mazzini 12, Bologna 40138, Italy Hum Reprod. 2011 Nov;26(11):3181-5. Epub 2011 Sep 9

Background: The purpose of this study was to assess the technical aspects related to polar body (PB) biopsy, which might have an influence on the results of the microarray comparative genomic hybridisation analysis. Furthermore, a comparison was made between two biopsy methods (mechanical and laser).

Methods: Biopsy of the first and second PB (PB1 and PB2) was performed by mechanical- or laser-assisted biopsy in two different IVF centres. PBs were separately amplified by whole genome amplification.

Results: The method of biopsy, mechanical or laser had no influence on the proportion of successfully biopsied oocytes. Especially, for the PB2, the timing of biopsy after ICSI was directly correlated to amplification efficiency.

Conclusions: Special care has to be taken with respect to the timing of biopsy of the PB2. Mechanical- and laser-assisted biopsy give the same performance in terms of diagnostic efficiency.

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Validation of 24sure

This paper investigates whether the use of cells from the trophectoderm of blastocyst stage embryos offers an optimal strategy for PGS. High rates of mosaicism in blastocysts has previously been reported as a problem that can lead to misdiagnosis. Here it is concluded that most mosaic blastocysts contained no normal cells, as such trophectoderm aCGH analysis is an appropriate tool for determining blastocyst anueploidy.

Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation Fragouli E, Alfarawati S, Daphnis DD, Goodall N, Mania A, Griffiths T, Gordon A, Wells D; Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, UK Human Reproduction. 2011 Feb;26(2):480-90. Epub 2010 Dec 8

Background: Recent studies have suggested that biopsy of several trophectoderm (TE) cells from blastocysts followed by comparative genomic hybridisation (CGH) analysis might represent an optimal strategy for aneuploidy detection, but few data on accuracy are available. The main question concerns the rate of mosaicism at the blastocyst stage, and to what extent this might cause misdiagnoses. We assessed blastocyst aneuploidy and mosaicism rates and evaluated the accuracy and efficiency of CGH and microarrayCGH (aCGH) for TE analysis.

Methods: A total of 52 blastocysts, from 20 couples, were biopsied and their chromosomes examined by CGH. The remaining cells were spread and tested by fluorescent in situ hybridisation (FISH). Of the 52 blastocysts, 20 underwent a second TE biopsy and were tested using aCGH.

Results: CGH and aCGH produced results for 98% of TE samples. 42.3% of blastocysts were uniformly euploid, 30% were uniformly aneuploid and 32.4% were mosaic. Of the mosaic embryos, 15.4% were found to be composed of a mixture of different aneuploid cell lines, while 17% contained both normal and aneuploid cells. Mosaic diploid–aneuploid blastocysts with >30% normal cells accounted for <6% of analysed embryos.

Conclusions: Comprehensive chromosome screening and follow-up assessment of large numbers of cells provided a unique insight into the cytogenetics of human blastocysts. Meiotic and post-zygotic errors leading to mosaicism were common. However, most mosaic blastocysts contained no normal cells. Hence, CGH or aCGH TE analysis is an accurate aneuploidy detection tool and may assist in identifying viable euploid embryos with higher implantation potential.

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This study shows 24sure to be highly robust, specific and able to provide results within 24 hours. In addition SurePlex is compared with an alternative DNA amplification method. A distinct advantage of SurePlex is evident through failed amplifications and misdiagnoses with the alternative method. The study also shows arrayCGH can detect 42% more abnormalities than the standard 12-probe FISH approach.

Validation of microarray comparative genomic hybridisation for comprehensive chromosome analysis of embryos Gutiérrez-Mateo C, Colls P, Sánchez-García J, Escudero T, Prates R, Ketterson K, Wells D, Munné S; Reprogenetics, Livingston, New Jersey, USA Fertil Steril. 2011 Mar 1;95(3):953-8. Epub 2010 Oct 25

Objectives: To validate and determine the best array-comparative genomic hybridisation (aCGH; arrayCGH) protocols for preimplantation genetic screening (PGS).

Design: Embryos had one cell removed as a biopsy specimen and analysed by one of two arrayCGH protocols. Abnormal embryos were reanalysed by fluorescence in situ hybridisation (FISH).

Setting: Reference laboratory.

Patients: Patients donating embryos or undergoing PGS.

Interventions: Embryo biopsy, arrayCGH, FISH reanalysis.

Main outcome measure(s): Diagnosis, no result rate and error rate.

Results: Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after arrayCGH combined with single blastomere biopsy.

Conclusions: ArrayCGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, arrayCGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary.

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24sure articles

24sure – articles using preimplantation genetic screening

This descriptive study was performed on existing 24sure data from Reprogenetics in North America. It provides up to date information on the frequency of aneuploidy across female ages for day 5 and day 3 biopsies. They conclude that the rate on aneuploidy is unrelated to the number of embryos generated per cycle.

ArrayCGH analysis shows that aneuploidy is not related to the number of embryos generated Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, Tan SL, Munné S. MUHC Reproductive Center, Div. of Reproductive Endocrinology and Infertility, Dept. of Obstetrics and Gynecology, McGill University, Montreal, Canada H3A 1A1; Assisted Reproduction Unit, Dept. Of Obstetrics and Gynecology, Uludag University, Gorukle, Bursa, 16059, Turkey Reprod Biomed Online. 2012 Jun;24(6):614-20 Abstract This study retrospectively analysed array comparative genomic hybridisation (CGH) results of 7,753 embryos from 990 patients to determine the frequency of embryonic euploidy and its relationship with the cohort size (i.e. the number of embryos available for biopsy and arrayCGH analysis). Linear regression analysis was performed to assess the effect of cohort size on euploidy rate adjusted for the effect of female age. While increasing female age was associated with a significant decrease in euploidy rate of day 3 and day 5 embryos (P<0.001 for both groups), cohort size was not significantly associated with euploidy rate. Logistic regression analysis was performed to assess the effect of cohort size, adjusted for maternal age, on the likelihood of having at least one euploid embryo available for transfer. The odds of having at least one euploid embryo in an assisted cycle was significantly decreased by increasing female age (P<0.01 for both day 3 and day 5 embryos) and was significantly increased by every additional embryo available for analysis (P<0.001 for both day 3 and day 5 embryos). Array comparative genomic hybridisation (CGH) enables assessment of the entire chromosomal component of human embryos, which was not possible with the formerly used preimplantation genetic diagnosis (PGD) technology. We analysed arrayCGH PGD results of 7,753 embryos from 990 women to determine the frequency of embryonic euploidy and its relationship with the number of embryos available for biopsy (hereafter called cohort size) in an assisted reproduction cycle. While increasing female age was associated with a significant decrease in euploidy rate of day 3 and day 5 embryos (P<0.001 for both groups), cohort size was not significantly associated with euploidy rate. Logistic regression analysis was performed to assess the effect of cohort size, adjusted for maternal age, on the likelihood of having at least one euploid embryo available for transfer. The odds of having at least one euploid embryo in an assisted cycle was significantly decreased by increasing female age (P<0.01 for both day 3 and day 5 embryos) and was significantly increased by every additional embryo available for analysis (P<0.001 for both day 3 and day 5 embryos). In conclusion, increasing the number of embryos available in a conventionally stimulated cycle increases the chance of having an euploid embryo available for transfer.

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This paper uses data compiled during the ESHRE PGS taskforce arrayCGH proof of principle trial. In 34 couples undergoing IVF, first and second polar bodies were examined for aneuploidy using 24sure, cases where aneuploidy was predicted in the zygote, the corresponding zygote was also analysed. Comparisons of aneuploidy in the three products of female meiosis enabled the incidence of aneuploidy in human oocytes at conception to be studied for the first time. It also identified in which meiotic division errors occured and concluded that most errors in the first division are caused by premature predivision of chomatids as previously shown by quantitative analysis (Gabriel et al, 2011).

Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation Handyside AH 1,9, Montag M 2,9, Magli MC 3,9, Repping S 4,9, Harper J 5,9, Schmutzler A 6,9, Vesela K 7,9, Gianaroli L 3,9 and Geraedts J 8,9. 1 London Bridge Fertility, London, UK; 2 Department of Gynecological Endocrinology and Reproductive Medicine, University of Heidelberg, Germany; 3 SISMER, Bologna, Italy; 4 Department of Obstetrics and Gynecology, University of Amsterdam, The Netherlands; 5 UCL Centre for PG&D, University College London, London, UK; 6 Center for Reproductive Medicine, Christian-Albrechts-University Kiel, Germany; 7 Sanatorium Repromeda, Brno, Czech Republic; 8 Department of Genetics and Cell Biology, Research Institute GROW, Maastricht University, The Netherlands. 9 The authors are members of the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Screening (PGS) Task Force. Eur J Hum Genet. advanced online publication Feb 8 2012

Abstract Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (arrayCGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes.

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24sure articles

This paper uses a cell culture system to imitate levels of mosaicism that may be present in an embryo. 24sure was used to measure the level of aneuploidy in the mixed cell populations. It was determined that anueploidy could be seen in cell populations where 25% of cells were aneuploid and confidently called where 50% of cells were aneuploid.

Detection of aneuploidy by array comparative genomic hybridisation using cell lines to mimic a mosaic trophectoderm biopsy Mamas T, Gordon A, Brown A, Harper J, Sengupta S. UCL Centre for PGD, Institute for Women's Health, University College London, London, UK Fertil Steril. 2012 Jan 24. [Epub ahead of print]

Objective: To examine the effect of mosaicism in the array comparative genomic hybridisation result during preimplantation genetic screening after blastocyst biopsy.

Intervention: Mixing of euploid and aneuploid cells to create mosaic trophectoderm and blastocyst models.

Main outcome measure: The level of aneuploidy in samples with different ratios of aneuploid cells was measured after array comparative genomic hybridisation.

Result: A shift from normality was present when the level of aneuploid cells in the sample was >25%. Aneuploidy could be confidently called when the level of aneuploid cells was >50%.

Conclusion: This study determined that aneuploidy in mosaic samples can be detected by array comparative genomic hybridisation and that the result may also indicate the proportion of the aneuploid cells present in the sample.

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This paper from CARE Fertility group, led by Simon Fishel, presents their experience of using 24sure on polar bodies since 2008 when they first assessed arrayCGH aneuploidy screening and achieved a live birth following use of the technology, see page 21 [Fishel et al p15 of March version]. They conclude that arrayCGH has proven to generate robust chromosome information and to generate improved implantation rates in women with very poor prognosis.

Assessment of 19,803 paired chromosomes and clinical outcome from first 150 cycles using arrayCGH of the first polar body for embryo selection and transfer Fishel S1, Craig A3, Lynch C1, Dowell K1, Ndukwe G1, Jenner L1, Cater E1, Brown A3, Gordon A3, Thornton S1, Campbell A2, Berrisford K1, Kellam L1, Sedler M2; 1CARE Nottingham, CARE Fertility Group, Nottingham NG8 6PZ, UK; 2CARE Manchester, CARE Fertility Group, Manchester M14 5QH, UK; 3BlueGnome Ltd, Cambridge CB22 5LD, UK J Fertiliz In Vitro 2011 1:101

Background: Meiosis 1 errors are believed to be the largest single cause of clinical embryo failure and early miscarriage. Following the failure of FISH technology and concerns over embryo mosaicism, our aim was to assess paired chromosome status and error rates, and predict oocyte aneuploidy using the metaphase 2 polar body with a novel arrayCGH platform as a means to select embryos for couples with multiple IVF failures.

Methods: The PB1 was removed from metaphase 2 oocytes using a laser-assisted breech of the zona pellucida to effect the biopsy. Reliable 23-paired chromosome analysis was obtained, and in time for fresh embryo transfer. It was a non- randomised investigation in patients seeking this technology as a result of previous multiples failures of IVF.

Results: 134 couples presented for 150 cycles of arrayCGH with a median age of 41.0. 861 polar bodies were evaluated, 67.4% and 32.6% were aneuploid and euploid, respectively. 19,803 paired chromosomes were analysed, 3.5% and 3.4% of chromosome errors resulted in either a gain or loss, respectively. There was a positive correlation between female age and aneuploidy, but no correlation with numbers oocytes harvested. 26% of cycles (n=39) failed to achieve embryo transfer (ET) as none of the oocytes were euploid. The live birth rate per ET and implantation was 24.1% and 27.7%, respectively, and 5.2% of chemical pregnancies (1.9% of transfers) resulted in a dizygotic multiple pregnancy.

Conclusion: ArrayCGH was proven to generate robust chromosome information. Chromosome segregation error rates were found to be inversely proportional to chromosome length and proportional to the G/C base content. Clinically, the transfer of a single embryo after PB 1 arrayCGH analysis appeared to generate improved implantation rates in women with very poor prognosis, whilst reducing the risk of a multiple pregnancy.

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24sure articles

This article addresses the question of whether aneuploidy arises from non-disjunction (whole chromosome segregation errors) or through precocious separation (sister chromatid segregation errors) with the use of 24sure. They conclude that most anueploidy arises from sister chromatid separation errors (92% vs 8%) which is opposed to the accepted wisdom that non-disjunction is the primary mechanism leading to human aneuploidy.

Array comparative genomic hybridisation on first polar bodies suggests that non-disjunction is not the predominant mechanism leading to aneuploidy in humans Gabriel AS, Thornhill AR, Ottolini CS, Gordon A, Brown AP, Taylor J, Bennett K, Handyside A, Griffin DK; School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK J Med Genet. 2011 Jul;48(7):433-7. Epub 2011 May 26

A case report of a 41 year old lady who previously had 13 cycles of failed IVF. This case uses 24sure with polar body biopsy to evaluate all 24 chromosomes copy number. Two of the nine eggs were euploid, these were selected for transfer to the uterus, despite appearing morphologically inferior to the other embryos, resulting in the birth of a normal healthy baby. It was the first reported case of a live birth after arrayCGH diagnosis of a polar body.

Live birth after polar body array comparative genomic hybridisation prediction of embryo ploidy – the future of IVF? Fishel S, Gordon A, Lynch C, Dowell K, Ndukwe G, Kelada E, Thornton S, Jenner L, Cater E, Brown A, Garcia-Bernardo J; CARE Fertility, John Webster House, 6 Lawrence Drive, Nottingham Business Park, Nottingham NG8 6PZ, UK Fertil Steril. 2010 Feb;93(3):1006.e7-1006.e10. Epub 2009 Nov 25

Conclusions: Selection of euploid eggs, as an objective parameter of subsequent embryo viability and with the opportunity to transfer embryos in the same cycle could maximise the opportunity for live birth after IVF even in cases with poor prognosis.

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24sure+ – articles using translocation preimplantation genetic diagnosis

This paper uses 24sure+ to detect translocations in day 3 embryos; balanced embryos were then selected for transfer on day 5 of the same cycle. Of 200 embryos analysed from 24 couples, 93.5% were successfully diagnosed. 16% of embryos were found to be normal/ balanced for all chromosomes, 17.1% had the translocation and normal for aneuploidy on other chromosomes, 39.6% had the translocation and anueploidy, 27.3% were normal or balanced for the expected translocation, but had aneuploidy on other chromosomes. Embryos suitable for transfer were identified in 17 cycles (60.7%). 12 couples achieved clinical pregnancy (70.6% per embryo transfer) with a total of 14 embryos implanted (63.6% per transferred embryo).

PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridisation Fiorentino F, Spizzichino L, Bono S, Biricik A, Kokkali G, Rienzi L, Ubaldi FM, Lammarrone E, Gordon A, Pantos K; GENOMA-Molecular Genetics Laboratory, Rome, Italy Hum Reprod. 2011 Jul;26(7):1925-35. Epub 2011 Apr 12

Background: Fluorescence in situ hybridisation (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements in preimplantation embryos but it is known to have several technical limitations. We describe the clinical application of a molecular-based assay, array comparative genomic hybridisation (arrayCGH), to simultaneously screen for unbalanced translocation derivatives and aneuploidy of all 24 chromosomes.

Methods: Cell biopsy was carried out on cleavage-stage embryos (Day 3). Single cells were first lysed and DNA amplified by whole-genome amplification (WGA). WGA products were then processed by arrayCGH using 24sure + arrays, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on Day 5 of the same cycle.

Results: Twenty-eight consecutive cycles of preimplantation genetic diagnosis were carried out for 24 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5%) embryos were successfully diagnosed. Embryos suitable for transfer were identified in 17 cycles (60.7%), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6% per embryo transfer), with a total of 14 embryos implanted (63.6% per transferred embryo). Three patients delivered three healthy babies, during writing, the other pregnancies (two twins and seven singletons) are ongoing beyond 20 weeks of gestation.

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24sure+ articles

Conclusions: The data obtained demonstrate that arrayCGH can detect chromosome imbalances in embryos, also providing the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. ArrayCGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity and reliability.

This German article uses 24sure to analyse polar bodies and clearly detects translocations (nice profiles).

Combined translocation and aneuploidy-screening after polar body biopsy and arrayCGH kombinierte translokations- und aneuploidieuntersuchungen nach polkörperbiopsie und array-comparative genomic hybridisation Montag M, Köster K, van der Ven K, Bohlen U, Bender F, van der Ven H; Department of Gynecological Endocrinology & Reproductive Medicine, University Clinics Bonn, Bonn, Germany J. Reproduktionsmed. Endokrinol 2010; 7 (6), 498-502

Abstract In females carrying a balanced chromosomal translocation biopsy of the 1st and 2nd polar body allows an indirect chromosomal investigation of the oocytes. It is possible to detect if the oocyte is unbalanced or balanced/normal in regard to the chromosomes which are involved in the translocation. In the present study polar body biopsy was carried out in 3 cases and the analysis was performed by array-Comparative Genomic Hybridisation (CGH). This allowed the simultaneous detection of numerical aberrations for all chromosomes as well as aberrations caused by the translocation chromosomes. In patient 1 (aged 33, Robertsonian translocation 45,XX,rob(13;14) (q10;q10)) 5 out of 12 oocytes were balanced/normal, 3 showed an unbalanced translocation and another 4 were aneuploid for other chromosomes. In patient 2 (36, reciprocal translocation 46,XX,t(11;19)(p10;p10)) 3 out of 11 oocytes were balanced/normal. The remaining 8 oocytes were unbalanced and 2 were additionally aneuploid for other chromosomes. In patient 3 (39; recirpocal translocation 46,XX,t(5;8)(q22;p11.2)) all 6 oocytes were chromosomally unbalanced and in each oocyte at least one other chromosome was aneuploid. In general, the unbalanced state was detectable in 1st as well as 2nd polar bodies. In patients 1 and 2 the transfer of 2 respectively 3 embryos (6A, 8A day 3; 2A, 2A, 4A day 2) resulted in two ongoing twin pregnancies. Polar body biopsy in combination with arrayCGH in female translocation patients allows a distinct chromosomal diagnosis of the oocyte. Furthermore, aneuploidies of chromosomes other than those involved in the translocation can be reliably detected.

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In this article conventional CGH and arrayCGH were applied to polar bodies, cleavage-stage embryos and blastocysts to assess the chromosomes of embryos from 16 patients known to be carriers of reciprocal or Robertsonian translocations or inversions. Results were obtained for 121 out of 132 samples (91.7%), 48.8% were found to carry abnormalities associated with the known rearrangement, sometimes together with other aneuploidies, 28.9% were balanced/normal for the rearrangement but contained other chromosomal aneuploidies, only 22.3% were chromosomally normal.

First births after preimplantation genetic diagnosis of structural chromosome abnormalities using comparative genomic hybridisation and microarray analysis Alfarawati S, Fragouli E, Colls P, Wells D; Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK Human Reprod. 2011 Jun;26(6):1560-74. Epub 2011 Mar 29

Background: Balanced chromosomal rearrangements represent one of the most frequent indications for preimplantation genetic diagnosis (PGD). Although fluorescence in situ hybridisation (FISH) has been successfully employed for diagnosis in such cases, this approach usually restricts assessment of the chromosomes involved in the rearrangement. Furthermore, with FISH-based strategies, it is sometimes necessary to create patient-specific protocols, increasing the waiting time and costs. In the current study, we explored the use of two comprehensive chromosome screening methods, conventional metaphase comparative genomic hybridisation (CGH) and microarrayCGH (aCGH), as alternatives for PGD of chromosome rearrangements.

Methods: The study included 16 patients who underwent 20 cycles of PGD for a variety of chromosome rearrangements (reciprocal or Robertsonian translocations or inversions). Testing was performed at various embryonic stages using CGH (9 cases) or aCGH (11 cases).

Results: Results were obtained for 121 out of 132 samples (91.7%). Of the diagnosed samples, 48.8% were found to carry abnormalities associated with the rearrangement, either alone or in combination with other chromosomal abnormalities. A further 28.9% of samples were normal/ balanced for the rearranged chromosomes, but affected by aneuploidy for other chromosomes. Only 22.3% of samples were chromosomally normal. Of the 15 patients who completed their treatment cycles, 5 became pregnant after one or two cycles resulting in four healthy births. The delivery rate per cycle was 21% (27% per embryo transfer).

Conclusions: This is the first study to describe the clinical application of comprehensive chromosome screening applied to polar bodies, blastomeres or trophectoderm cells from patients carrying inversions and translocations. Using these techniques, most patients requesting PGD for a chromosome rearrangement can be treated using a single protocol. Additionally, the detection of abnormalities affecting chromosomes unrelated to the rearrangement may assist in the selection of viable embryos for transfer.

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PGD/PGS general interest

Four articles containing ESHRE PGD guidelines were published in Hum Reprod. 2010 Oct 21. These articles offer an update since the previous guidelines were published in 2005 and many new technologies and evolution of existing techniques have occurred since this time. The new guidelines are separated into four documents including one outlining PGD centre organisation and three relating to the methods used: amplification-based testing, fluorescence in situ hybridisation (FISH)-based testing and polar body/embryo biopsy.

ESHRE PGD consortium best practice guidelines for organisation of a PGD centre for PGD/preimplantation genetic screening Harton G, Braude P, Lashwood A, Schmutzler A, Traeger-Synodinos J, Wilton L, Harper JC; European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium; Reprogenetics LLC, Livingston, New Jersey 07039, USA Hum Reprod. 2011 Jan;26(1):14-24. Epub 2010 Oct 21

ESHRE PGD consortium best practice guidelines for amplification-based PGD Harton GL, De Rycke M, Fiorentino F, Moutou C, SenGupta S, Traeger-Synodinos J, Harper JC; European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium; Reprogenetics LLC, Livingston, New Jersey 07039, USA Hum Reprod. 2011 Jan;26(1):33-40. Epub 2010 Oct 21

ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridisation-based PGD Harton GL, Harper JC, Coonen E, Pehlivan T, Vesela K, Wilton L; European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium; Reprogenetics LLC, Livingston, New Jersey 07039, USA Hum Reprod. 2011 Jan;26(1):25-32. Epub 2010 Oct 21

ESHRE PGD Consortium/Embryology Special Interest Group – best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS) Harton GL, Magli MC, Lundin K, Montag M, Lemmen J, Harper JC; European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium/Embryology Special Interest Group; Reprogenetics LLC, Livingston, New Jersey 07039, USA Hum Reprod. 2011 Jan;26(1):41-6. Epub 2010 Oct 21

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This recent review explains the emergence of PGS and the use of arrayCGH to assess aneuploidy. The different cell types that can be assayed are also discussed and the pros and cons of different techniques.

A new era of PGS for IVF – will it yield the anticipated improved efficiency? Simon Fishel, Simon Thornton, Ken Dowell; CARE Fertility Group, CARE Nottingham, John Webster House, 6, Lawrence Drive, Nottingham Business Park, Nottingham NG8 6PZ, UK Fishel et al. J Fertiliz In Vitro 2011, 1:1

This paper describes a method that uses SNP arrays for genome wide preimplantation genetic diagnosis which could remove the need to develop individual gene specific tests.

Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes Handyside AH, Harton GL, Mariani B, Thornhill AR, Affara N, Shaw MA, Griffin DK; London Bridge Fertility, Gynaecology and Genetics Centre, One St Thomas Street, London SE1 9RY, UK J Med Genet. 2010 Oct;47(10):651-8. Epub 2009 Oct 25

This commentary piece from Alan Handyside discusses the technical advances in PGD over the last 20 years. He addresses the issue of cost of IVF-PGD versus the cost of lifetime healthcare of an individual with a serious genetic disease.

Preimplantation genetic diagnosis after 20 years Alan H Handyside; London Bridge Fertility, Gynaecology and Genetics Centre, London, UK Reprod Biomed Online. 2010 Sep;21(3):280-2. Epub 2010 Jul 21

Discussion of prior PGS trial data and outline the setting up of ESHRE trial using polar body biopsy and arrayCGH.

What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium Steering Committee. Harper J, Coonen E, De Rycke M, Fiorentino F, Geraedts J, Goossens V, Harton G, Moutou C, Pehlivan Budak T, Renwick P, Sengupta S, Traeger-Synodinos J, Vesela K; UCL Centre for PG&D, Institute for Women's Health, University College London, 86-96 Chenies Mews, WC1E 6HX London, UK Hum Reprod. 2010 Apr;25(4):821-3

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PGD/PGS general interest

Abstract Since 2004, there have been 11 randomised controlled trials (RCTs) mainly for advanced maternal age (AMA), which have shown no benefit of performing preimplantation genetic screening (PGS). Ten of the RCTs have been performed at the cleavage stage and one at the blastocyst stage. It is probable that the high levels of chromosomal mosaicism at cleavage stages, which may result in the tested cell not being representative of the embryo, and the inability to examine all of the chromosomes using fluorescence in situ hybridisation, have contributed to the lack of positive outcome from the RCTs. We suggest that future RCTs should examine alternative biopsy timing (polar body and/or trophectoderm biopsy), and should apply technologies that allow more comprehensive testing to include all chromosomes (microarray-based testing) to determine if PGS shows an improvement in delivery rate. Currently there is no evidence that routine PGS is beneficial for patients with AMA and conclusive data (RCTs) on repeated miscarriage, implantation failure and severe male factor are missing. To evaluate benefits of PGS, an ESHRE trial has recently been started on patients with AMA using polar body biopsy and array-comparative genomic hybridisation, which should bring more information on this patient group in the near future.

Outline of the proof of principle study by the ESHRE PGS Task Force to determine whether chromosome analysis of first and second polar bodies by arrayCGH enable timely identification of the chromosomal status of an oocyte.

What next for preimplantation genetic screening? A polar body approach! Geraedts J, Collins J, Gianaroli L, Goossens V, Handyside A, Harper J, Montag M, Repping S, Schmutzler A; Department of Genetics and Cell Biology, Research Institute GROW, Faculty of Health, Medicine and Life Sciences, Maastricht University, PO Box 5800, 6202 AZ Maastricht, The Netherlands Hum Reprod. 2010 Mar;25(3):575-7

Abstract Screening of human preimplantation embryos for numerical chromosome abnormalities has been conducted mostly at the preimplantation stage using fluorescence in situ hybridisation. However, it is clear that preimplantation genetic screening (PGS) as it is currently practiced does not improve live birth rates. Therefore the ESHRE PGS Task Force has decided to start a proof of principle study with the aim of determining whether biopsy of the first and second polar body followed by subsequent analysis of the complete chromosome complement of these polar bodies using an array based technique enables a timely identification of the chromosomal status of an oocyte. If the principle of this approach can be proven, it is obvious that a multicentre randomised controlled trial should then be started to determine the clinical value of this technique. In this way the ESHRE PGS Task Force hopes to redirect preimplantation screening from the blind alley to the main road of assisted reproduction.

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Meeting abstracts PGDIS 2012

There were a number of oral presentations and posters that used 24sure at the 11th International Conference on Preimplantation Genetic Diagnosis (17 May 2012 - 19 May 2012) held in Bregenz. The abstracts from this meeting have been published in Reproductive biomedicine online (1 May 2012 volume 24, supplement 2) and the abstracts freely available. In total 25 abstracts using 24sure were presented, from 29 different institutions. To view the abstracts, please visit http://www.rbmojournal.com/supplements

Oral presentations

[S3] Polar body biopsy and arrayCGH for aneuploidy in advanced maternal age M. Montag – University Clinics Heidelberg, Germany

[S14] Blastocyst biopsy for aneuploidy screening D. Wells – Oxford, UK

[O4] Preimplantation Genetic Diagnosis (PGD) for chromosomal rearrangements (CR) using arrays of comparative genome hybridisation (aCGH). Risk assessment depending on the type of CR, the maternal age and the sex of the carrier A. Jimenez-Macedo, E. Garcia-Guixé, T. Escudero, P. Colls, S. Munné, C. Gimenez, M. Sandalinas – Reprogenetics, Barcelona, Spain and Livingstone, NJ, USA

[S4] The use of polar bodies and cumulus cells to assess oocytes and select embryos E. Fragouli – Reprogenetics, Oxford, UK

[S13] PGD for aneuploidy and translocation chromosome imbalance by cleavage stage biopsy F. Fiorentino – Genoma, Rome, Italy

[O1] Polar body 1, polar body 2 and blastomere analysis in PGS treatment S. Fishel, E. Cater, C. Lynch, L. Jenner, A. Campbell – Care Fertility, Manchester and Nottingham, UK

[S16] Karyomapping as a means of universal diagnosis and providing insight into early human development D.K. Griffin, A.R. Thornhill, A. Gordon, A.H. Handyside – University of Kent, The Bridge Centre, London and BlueGnome, Cambridge, UK

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Meeting abstracts – PGDIS 2012

[S6] Polar body analysis by arrayCGH and follow up at cleavage stages D. Christopikou – Embryogenesis, Athens, Greece

[O2] Altered morphokinetic parameters of embryos identified as aneuploid by single cell arrayCGH analysis at the 8-cell stage S. Davies, D. Christopikou, E. Tsorva, T. Karagianni, M. Mastrominas, A.H. Handyside – Embryogenesis, Athens, Greece and University of Leeds, UK

[S11] Chromosomal mosaicism in the cleavage stage embryo revisited L. Wilton – Melbourne IVF, Australia

[O3] Aneuploidy screening reveals high incidence of abnormalities of chromosomes which are not involved in the rearrangements C. Beyazyurek, C. Yapan, C.G. Ekmekci, M. Yesil, G. Altin, H.K. Yelke, S. Kahraman – Istanbul, Turkey

[S7] Blastocyst biopsy for aneuploidy screening S. Munné, P. Colls, G. Harton, E. Fragouli – Reprogenetics, Livingston, NJ, USA and Oxford, UK

[O8] Accuracy rates in biopsies performed on day 3 and day 5 embryos using CGHarray technology for PGS P. Mir, L. Rodrigo, E. Mateu, A. Mercader, L. Escrich, P. Buendía, A. Delgado, M.E. Poó, M. Vera, C. Simón, C. Rubio – Iviomics, Valencia, Spain

Posters

[P18] Preimplantation genetic diagnosis for translocation t(6;14)(q26;q31) using array comparative genomic hybridisation at the blastocyst stage Y.N. Su, C.C. Hung, M.Y. Fang, S.U. Chen – National Taiwan University, Taipei, Taiwan

[P8] First experiences with PGD after trophectoderm biopsy at Kinderwunsch Centrum Munich (KCM), Germany R. Suttner, D. Shakeshaft, U. Koehler, U. Schon, Th. Harasim, A. Wagner, E. Holinski-Feder, I. Rost, W. – Munich and Martinsried, Germany

[P51] Multiple Factor PGD – 4 case reports involving testing for up to 4 indications in a single blastomere L. Jenner, C. Lynch, E. Cater, K. Berrisford, N. Keown, K. Shaut, A. Brown, S. Fishel, M.R. Hughes – Care Fertility and Genesis Genetics, Nottingham, UK and Detroit, MI, USA

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[P35] First Hungarian results on aneuploidy patterns of day three embryos analysed by array comparative genome hybridisation A. Vereczkey, K. Szabó, B. Faragó, G. Tölgyesi, M. Csenki, É. Margittai, Zs. Kósa – Budapest, Hungary

[P48 Application of aCGH for pre-implantation genetic diagnosis of chromosome rearrangements and additional unrelated chromosome aneuploidy C. Lynch, N. Keown, H. Rouse, L. Jenner, S. Fishel, M.R. Hughes – Care Fertility and Genesis Genetics, Nottingham, UK and Detroit, MI, USA

[P38] Birth after Preimplantation Genetic Diagnosis (PGD) using Microarray Comparative Genomic Hybridisation (MaCGH) for chromosome inversion (1) (p35q42) C.S.S. Lee, S.Y. Low, W.Y. Leong – Alpha, Selangor, Malaysia

[P26] Increased pregnancy rates after trophectoderm biopsy for PGD of monogenic diseases and chromosomal aberrations U. Schön, U. Koehler, V. Mayer, A. Stegerer, K. Sendelbach, M. Locher, B. Paulmann, D. Shakeshaft, R. Suttner, B. Seifert, W. Wurfel, E. Holinski-Feder – Munich and Regensburg, Germany

[P27] Implementation of array-CGH technology in Preimplantation Genetic Diagnosis for rob(13;14)(q10;q10) Robertsonian translocations L. Xanthopoulou, H. Ghevaria, S. Cawood, P. Serhal, S.B. SenGupta, J.D.A. Delhanty – UCL, London, UK

[P23] Detection of whole chromosome vs. chromatid errors and possible germinal mosaicism at various stages of oocyte maturation by arrayCGH H. Ghevaria, T. Mamas, T. Sabhnani, P. Serhal, J.D.A. Delhanty – UCL, London, UK

[P28] Checking on mosaicism in day 5-blastocysts – first results from a microarray based diagnostic setting S. Bug, M. Stecher, J. Pricelius, B. Groß, U. Noss, C. Nevinny-Stickel-Hinzpeter – Synlab and Centre for Reproductive Medicine, Munich, Germany

[P29] Developmental potential of day 3 embryos diagnosed by arrayCGH analysis on the blastomere E. Cater, C. Lynch, L. Jenner, S. Fishel – Care Fertility, Nottinham, UK

[P50] Aneuploidy testing of polar bodies by array comparative genomic hybridisation (arrayCGH) Th. Harasim, A. Wagner, U. Heinrich, M. Gabert, S. Thoms, K. Fiedler, G. Krüsmann, R. Suttner, D. Shakeshaft, H.G. Klein, I. Rost, W. Würfel – Martinsried, Germany

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Meeting abstracts – Alpha Conference & Biomarker meeting

Alpha Conference

There were two posters that used 24sure at the Alpha Conference (26 – 29th April 2012) held in London. The abstracts from this meeting have been published in Reproductive biomedicine online (1 May 2012 volume 24, supplement 1) and are freely available at www.rbmojournal.com/supplements

[PP38] First Hungarian results on aneuploidy patterns of day 3 embryos analysed by array comparative genome hybridisation Attila Vereczkey, Mariann Csenki, Katalin Szabé, Bernadett Faragéo, Diaéna Debreceni, Éva Margittai, Zsolt Kósa – Versys Clinics, Hungary

[PP40] Identifying a suitable patient population for arrayCGH on first polar bodies to improve embryo selection and clinical outcomes Christian Ottolini, Christina Lappa, Darren Griffin, Michael Summers, Karen Sage, Shaun Rogers, Alan Handyside, Alan Thornhill – The Bridge Centre, London and University of Kent, UK

The 1st Biomarker meeting in Reproductive Medicine: Emergence of a new field

The first in this series of meetings that explore the novel technologies in reproductive medicine was held in Valencia, Spain (March 30-31, 2012). Two groups presented data from using 24sure technology, these talks are available to download from www.cytochip.com

A comparison of efficiency of aCGH and FISH PGS in patients with multiple IVF failure Dmytro O. Mykytenko; Valery V. Zukin – Clinic of reproductive medicine ‘NADIYA’, Ukraine

Use of Comparative Genomic Hybridisation (CGH) for embryo assessment: Clinical results Carmen Rubio – IVIOMICS, Valencia, Spain

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Best of ASRM and ESHRE: where we are and where we are going?

Two talks from the Best of ASRM and ESHRE meeting held in Cortina D’Ampezzo in Italy, (1-3 March 2012) used 24sure aneuploidy screening. These can be viewed on www.cytochip.com

Who is the most important for a successful outcome in ART? The clinician or the embryologist? Laura Rienzi, Senior Clinical Embryologist – GENERA Rome, Italy

Preimplantation Genetic Testing: Current Technology and Global Experience – When should it be done and by what technique(s)? The European view Prof. Dr. Joep Geraedts – Maastricht University, The Netherlands

14th World Congress on Human Reproduction 2011

There were a number of presentations using 24sure at this recent meeting held in Melbourne, Australia (30th November – 3rd December 2011). Three of the talks presented are available to view on www.cytochip.com

PGD for reciprocal translocations using a BAC Microarray Mirjana Martic, Anke Kohfahl, Peter Coleman, Sophie Falle, Sharyn Stock-Myer, Paisu Tang, Andrea Twomey, Leeanda Wilton, Preimplantation Genetics, Melbourne IVF

Aim: Fluorescent in situ hybridisation (FISH) has been used for many years to detect unbalanced segregants in the embryos of reciprocal translocation carriers in PGD programs. Individualised FISH tests can take months to develop and usually can only identify errors in the chromosomes involved in the translocation. Here we present the application of a specialised BAC array system to PGD for reciprocal translocation carriers.

Methods: Embryos diagnosed as unbalanced using PGD/FISH from two reciprocal translocation carriers, 46,XX,t(7;14)(q11.23;q11.2 and 46,XY,t(11;22)(q23;q11), were succumbed and separated into individual blastomeres. These were subjected to whole genome amplification and products labelled and hybridised onto 24sure+ BAC arrays (BlueGnome, UK) that have >15,000 clones targeted to the sub-telomere regions and specifically designed to detect imbalance of small segments.

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Meeting abstracts – 14th Word Congress on Human Reproduction

Results: 26 single blastomeres were amplified with 92% efficiency and results obtained on 100% of amplified cells. In each case the BAC array confirmed the segmental imbalance predicted by FISH. Importantly, this BAC array system was able to detect aneuploidy of other chromosomes not involved in the translocation. To date, one clinical case has been performed for a carrier of t(11;22)(q23;q11). Two embryos were available for testing and both found to be unbalanced for the translocation.

Conclusion: The 24sure+ BAC array system can reliably detect unbalanced chromosome segments as well as aneuploidy of any other chromosome in single blastomeres. This is an off-the-shelf test with no feasibility testing required and is simpler, faster and cheaper than FISH.

Chromosomal mosaicism in embryos from young ART patients determined by Array Comparative Genomic Hybridisation (aCGH) Leeanda Wilton, Andrea Twomey, Mirjana Martic, Anke Kohfahl, Paisu Tang, Preimplantation Genetics, Melbourne IVF

Aim: Chromosomal mosaicism, where different cells have a different chromosomal complement, reportedly affects approximately 60% of cleavage stage embryos. Of particular concern are diploid mosaic embryos which contain a mixture of diploid and aneuploid cells as they will confound preimplantation genetic screening (PGS) outcomes because the tested cell may not represent the embryo. The majority of this information is derived after analysis of less than 10 chromosomes and cells diagnosed as diploid may be aneuploid for chromosomes that were not tested for. This study reports the extent of mosaicism of all 24 chromosomes in cleavage stage embryos from young, successful ART patients.

Methods: 23 day 3 embryos were allowed to succumb, separated into 115 individual blastomeres (mean 5.0/embryo) and subjected to single cell arrayCGH using Sureplex whole genome amplification and 24sure BAC array analysis (Bluegnome, UK).

Results: Seven (30%) embryos were euploid in every cell, 5 (22%) had a consistent aneuploidy in every cell, which is presumed to be meiotic in origin, and 11 (57%) were mosaic. Of the mosaic embryos, 6 (26%) were aneuploid mosaic where every cell was aneuploid but with different aneuploidies in different cells. Five (22%) embryos were diploid mosaic with a mean of 31% aneuploid and 69% diploid cells per embryo.

Conclusion: This study shows that, even in good quality embryos from young ART patients, i) only a minority of embryos are euploid in every cell, ii) meiotic aneuploidy affects about 1 in 5 embryos, iii) diploid mosaicism affects only about 1 in 5 embryos and a minority of these blastomeres are aneuploid.

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PGS 2.0 – new genetic tests and complete chromosome assessment of human embryos Jacques Cohen, IVF-Online / Reprogenetics, Livingston, New Jersey, USA

Confirmation of FISH diagnosis and level of mosaicism for embryos clinically diagnosed with a single aneuploidy Peter Coleman, Melbourne IVF, Melbourne, Victoria, Australia

First clinical results using rapid Microarray analysis for aneuploidy detection in a PGD program Leeanda Wilton, Peter Coleman, Sophie Falle, Anke Kohfahl, Mirjana Martic, Paisu Tang, Sharyn Tock-Myer and Andrea Twomey, Preimplantation Genetics, Melbourne IVF

Aim: Metaphase comparative genomic hybridisation (mCGH) has demonstrated that many early human embryos harbour chromosomal aneuploidies that cannot be detected using fluorescent in situ hybridisation (FISH) for 8-10 chromosomes as practiced in most PGD laboratories. mCGH is unsuitable for routine clinical application and we have recently validated a microarray protocol that enables simple, rapid analysis of every chromosome in a single cell within 30 hours allowing euploid embryos to be transferred in the same cycle. Here we present the first clinical outcomes of arrayPGD for aneuploidy diagnosis.

Methods: Single blastomeres were biopsied from day 3 embryos and loaded into 4µl PBS. Cells were subjected to whole genome amplification, fluorochrome labelling and co-hybridisation with male reference DNA onto a 24sure 1Mb BAC array and analysed with Bluefuse software (BlueGnome, UK). Embryos diagnosed as euploid for all chromosomes were selected for transfer.

Results: Sixteen cycles of arrayPGD have been performed for indications including implantation failure, advanced maternal age and recurrent miscarriage. Results were obtained on 94% of cells and 30% were euploid. 31% of cells had only aneuploidies that would have been missed using FISH. Twelve embryo transfers have occurred with seven (58%) ongoing positive beta-hCG results. Four are now at fetal heart stage with one set of monozygotic twins. The clinical pregnancy rate per ET is 4/9 (44%) and implantation rate is 5/10 (50%).

Conclusion: This study shows that arrayPGD can be used clinically in PGD to perform a complete single-cell molecular karyotype within 30 hours. Euploid embryos can be transferred in the same cycle without the need for cryopreservation.

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Meeting abstracts – ASRM 2011

ASRM 2011

There were a large number of presentations and posters that used 24sure at the ASRM 2011 meeting held in Orlando in October 2011. This collection of work really shows the impact on outcomes that are coming from using aneuploidy screening. Abstracts from the meeting are published in Fertility and Sterility (vol 96(3) S1).

[O-74] FISH reanalysis of inner cell mass and trophectoderm samples of previously arrayCGH screened blastocysts reveals high accuracy of diagnosis and no sign of mosaicism or preferential allocation A. Capalbo, G. Wright, L. Themaat, T. Elliott, L. Rienzi, Z. P. Nagy GENERA, Reproductive Medicine, Rome, Italy; Reproductive Biology Associates, Atlanta, Georgia, USA

Objectives: To study the blastocyst genetic constitution (possible chromosomal mosaicism between ICM and TE and within TE), after isolating ICM by a novel method and to evaluate arrayCGH accuracy against FISH testing.

Design: Cross-sectional study.

Materials and methods: 32 blastocysts diagnosed as genetically abnormal by array-CGH were donated to research. ICM was isolated from the trophectoderm (TE) using a novel mechanical separation which was confirmed by KRT18 immunostaining. 24 of the 32 embryo had single or double aneuploidy and 8 aneusomic for unbalanced product of translocation or for long single arm deletions/duplications but with normal ploidy (by aCGH). After biopsy, individual samples were fixed by Carnoy method and analysed with conventional 9-chromosomes FISH and locus specific probes.

Results: We reanalysed 32 ICM and 96 TE samples (3 separate groups of cells from each TE of the same embryo). Looking at the single chromosome level, 28 out of 28 aneuploidies diagnosed by aCGH were confirmed by FISH reanalysis both in ICM and TE samples resulting in a analytical sensitivity of 100%(95%CI 90.2-100). For almost all embryos, the reported aneuploidies were constitutive. As regard to chromosomes diagnosed as disomic, only 2 out of 248 (specificity 99.1 95%CI 97.4-99.8) were misdiagnosed by aCGH, a trisomy 13 in an unbalanced embryo from reciprocal translocation carrier observed in a constitutive state and a mosaic nullisomy/monosomy 16 in a double aneuploid embryo. Consequently, only one out of 32 embryos was misdiagnosed as false negative. Only in one case we observed a confined trisomy 17 in all nuclei of a TE sample from a trisomic embryo for chromosome 13. In all other embryos ICM and TE samples were concordant and confirmed the original a-CGH diagnosis.

Conclusions: Our data show a very high analytical accuracy of aCGH to diagnose embryo chromosomal constitution. Concordance between ICM and TE was reported for all embryos analysed, showing no sign of mosaicism or preferential allocation at blastocyst stage.

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[O-76] Significant decrease in miscarriages after preimplantation genetic diagnosis (PGD) for recurrent pregnancy loss using array comparative genome hybridisation (arrayCGH) J. Grifo, S. Ghadir, B. Kaplan, C. A. Laskin, M. Glassner, S. Munne NYU Fertility Center, New York University, New York, NY, USA; ART Reproductive Center, Beverly Hills, California, USA; Fertility Centers of Illinois, Highland Park, Illinois, USA; LifeQuest Centre for Reproductive Medicine, Toronto, ON; Main Line Fertility and Reproductive Medicine, Bryn Mawr, Pennsylvania, USA; Reprogenetics, Livingston, New Jersey, USA

Objectives: ArrayCGH can analyse all chromosomes for aneuploidy and may further reduce the risk of miscarriage in patients suffering from recurrent pregnancy loss (RPL). The objective of this study was to determine any beneficial effects of PGD by arrayCGH for RPL patients in comparison to their expected loss rate and to PGD by FISH results.

Design: Multicenter retrospective study.

Materials and methods: The study included 177 cycles of couples with idiopathic RPL (defined as 3 or more miscarriages), average maternal age of 36.3 years, undergoing ART at multiple fertility centers. PGD was done using day 3 biopsy (n =143) or day 5 biopsy (n = 34), followed by analysis by aCGH. Each PGD patient was matched with their expected loss rate for RPL patients (Brigham et al. 1999). An ongoing pregnancy was defined as past second trimester.

Results: In total 1517 embryos were analysed, of which 33% were normal, 63% were abnormal and 4% none analyzable (no nucleus or no DNA amplification). Of the 176 cycles, pregnancy data was available for 154, of which 66 (43%) became pregnant with an implantation rate of 45% (95 sacs / 212 replaced embryos) and 62 cycles (40%) are ongoing past second trimester or delivered. We would expect a 34.9% miscarriage rate in this specific group of patients, but the miscarriage rate found was only 6% (4/66) (p<0.001).

Conclusions: Previous PGD results using FISH showed that the miscarriage rate in idiopathic RPL patients was significantly reduced from 26% to 10% in patients younger than 35, and from 39% to 13% in older patients. Current PGD results with aCGH indicate a significant decrease in the miscarriage rate of idiopathic RPL patients and high pregnancy rates for the current average maternal age of 36. These data suggest that aCGH further improves the results previously obtained by FISH. Furthermore, this confirms that idiopathic recurrent miscarriage is most caused by embryonic chromosome abnormalities.

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Meeting abstracts – ASRM 2011

[O-83] 24 chromsome analysis of products of conception specimens by arrayCGH allows for more results than conventional karyotyping and allows for simultaneous maternal cell contaminations analysis J. Sanchez, C. Sweet, P. Colls, B. Berger, D. Kenigsberg, G. Harton Reprogenetics, Livingston, New Jersey, USA; Specialists in Reproductive Medicine and Surgery, Fort Myers, Florida, USA; Boston IVF, Waltham, Massachusetts, USA; Long Island IVF, Melville, New York, USA

Objectives: To assess POC analysis by aCGH as compared to conventional karyotyping.

Design: A multi-center prospective study.

Materials and methods: Products of conception (POC) samples (n = 45) were collected following miscarriage and shipped overnight to our lab for further processing. At the time of the evacuation procedure, a cheek swab or blood sample was also collected to allow for fingerprinting analysis of maternal cell contamination in case of a molecular karyotype of 46,XX (normal female). Following removal of maternal tissue, DNA was extracted from the POC, fluorescently labeled and run on a bacteria artificial chromosome (BAC) array.

Results: All samples processed have resulted in a diagnosis (100%), which is an improvement over conventional karyotyping where approximately 40% of specimens end up with no result. Thirty three per cent of samples (15/45) were diagnosed as having maternal cell contamination, while 67% (30/45) of samples did not and were considered diagnostic. Sixty per cent (18/30) of the samples with a fetal diagnosis were determined to be chromosomally abnormal, including autosomal trisomies and sex chromosome anomalies. The other 40% (12/30) were diagnosed as normal following analysis of short tandem repeats (STRs) to rule out maternal cell contamination. Of those that were normal, 50% were male (6/12) and 50% were female (6/12). All results were returned within 24-72 hours of sample receipt.

Conclusions: Analysis of products of conception by aCGH allows for more thorough reporting of abnormalities, detection of maternal cell contamination and provides medical professionals and patients more information about pregnancy loss in a timely manner. Since maternal contamination was screened out, in contrast to routine POC karyotype analysis, 50% of normal miscarriages were male and 50% female. Analysis of POC by aCGH should be considered for patients that have a recognised miscarriage, although additional studies are strongly encouraged.

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[O-88] Are patients undergoing PGD for chromosome rearrangements at increased risk of aneuploidy affecting chromosomes unrelated to their rearrangement (interchromosomal effect)? S. Alfarawati, E. Fragouli, M. Konstantinidis, D. Wells Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, UK; Reprogenetics UK, Oxford, UK

Objectives: Balanced chromosomal rearrangements represent a relatively common form of genetic abnormality affecting 0.19% of newborns. Several studies have suggested that the presence of such rearrangements might interfere with normal chromosome segregation, increasing the chance of additional chromosome abnormalities occurring, known as an interchromosomal effect (ICE). This study aimed to investigate whether this phenomenon truly exists.

Design: Comprehensive cytogenetic evaluation of oocytes/embryos from carriers of chromosome rearrangements.

Materials and methods: 28 patients undergoing PGD for a wide variety of different chromosome rearrangements participated, with all chromosomes screened using microarray CGH (aCGH).

Results: Results were obtained for 180 samples, from patients with chromosome rearrangements (>7,000 chromosome segregations assessed). A robust control group was created for each patient by careful matching with multiple individuals of normal karyotype and identical age, undergoing routine preimplantation genetic screening (PGS) using aCGH. The control group consisted of 3,309 samples (analysis of >134,000 chromosomes). Overall, no evidence of ICE was observed. Subdivision of samples by class of rearrangement confirmed no ICE for Robertsonian or reciprocal translocations. However, a significant (p=0.04) increase in aneuploidy was observed associated with chromosome inversions.

Conclusions: Much evidence for and against the existence of an ICE has been presented, but controversy continues due to a lack of a definitive study. The investigation reported here offers the best chance of detecting an ICE if one exists, since the analysis was not limited to a small number of chromosomes as has been the case for all previous studies. Detailed analysis was unable to detect any elevated risk of chromosome malsegregation for carriers of Robertsonian or reciprocal translocations, conclusively demonstrating that no ICE exists for such patients. However, it remains possible that an ICE might exist in case of inversions.

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Meeting abstracts – ASRM 2011

[O-183] Implantation of euploid blastocysts, assessed by array comparative genomic hybridisation (aCGH), in unstimulated cycles is not correlated with maternal age G. Harton, M. Surrey, J. Grifo, B. Kaplan, P. Ahlering, J. Cohen Reprogenetics, Livingston, New Jersey, USA; ART Reproductive Center, Beverly Hills, California, USA; NYU Fertility Center, New York, NY; Fertility Centers of Illinois, Highland Park, Illinois, USA; Sher Institutes For Reproductive Medicine-St. Louis, Peoria, Illinois, USA

Objectives: To assess the relationship between maternal age and implantation after preimplantation genetic diagnosis with array comparative genomic hybridisation after biopsy on day3 or day5.

Design: A multi-center retrospective study.

Materials and methods: Patients from several IVF clinics had embryos biopsied on day 3 or 5. Analysis on day 3 involved 1-cell biopsy, aCGH and transfer of euploid embryos on day 5. Analysis of blastocysts involved trophectoderm biopsy on day 5/6 with aCGH, vitrification and transfer of euploid embryos in a hormone replacement cycle.

Results:

Day 3 biopsy vs. day 5 biopsy Day 3 biopsy Day 5 biopsy Age group Implantation Aneuploidy rate Age group Implantation Aneuploidy (+ sac) (+ sac) rate 30-34 51.6 (94/182) 50.5 (92/182) 30-34 85.3 (29/34) 41.2 (14/34) 35-39 38.1 (83/218) 65.1 (142/218) 35-39 63.5 (33/52) 48.1 (25/52) 40-42 31.2 (54/173) 77.5 (134/173) 40-42 76.3 (29/38) 60.5 (23/38)

Conclusions: Blastocyst biopsy produced better outcomes than day 3 biopsy across all ages. This may be due to less damage to the embryo during trophectoderm biopsy and/or better uterine receptivity. Implantation rates for euploid blastocysts were not significantly different between young and old patients, however, implantation rates from day 3 biopsy declined with advancing maternal age. Both techniques detect the same abnormalities with very low error rates, thus the decrease in implantation with age following biopsy at day 3 is unlikely a screening problem and suggests the involvement of other non-chromosomal factors. It is possible that the asynchrony between embryo development and the window of implantation is exacerbated in older patients due to higher doses of gonadatropins and longer stimulation times. Diagnosis of euploid blastocysts with cryopreservation and transfer in an unstimulated cycle may alleviate that effect. Mounting data suggests that blastocyst biopsy with 24 chromosome PGD may allow for single embryo transfer regardless of maternal age with excellent implantation and pregnancy rates.

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[O-198] Efficiency of preimplantation genetic screening (PGS) using arrayCGH compared to matched control IVF patient populations with and without day 3 PGS FISH A. Capalbo, G. Wright, T. Elliott, S. Slayden, D. Mitchell-Leef, Z. P. Nagy GENERA, Reproductive Medicine, Rome, Italy; Reproductive Biology Associates, Reproductive Medicine, Atlanta, Georgia, USA

Objectives: To evaluate if PGS with arrayCGH combined with blastocyst stage biopsy is a more efficient approach than the traditional FISH analysis combined with day 3 embryo biopsy or no biopsy (control).

Design: Retrospective matched control study.

Materials and methods: 30 patients had biopsy on blastocyst; (cryopreserved and ET in subsequent cycle) using aCGH (PGS/CGH group); 72 patients had PGS using FISH (PGS/FISH group; biopsy on day 3 with day 5 fresh ET) and 103 IVF patients having the same demographic were selected without PGS (Control; day 5 fresh ET). Clinical and laboratory data were analysed by One-way ANOVA (with Bonferroni post-hoc test) and Fisher’s exact tests with P<0.05.

Results: In the PGS/CGH, PGS/FISH and Control groups, ages of female patients were 37.4 (+/-2.9), 38.4 (+/-2.4) and 38.5 (+/-2.1) (NS); the mean number of zygotes obtained were 11.9 (+/-5.4), 11.3 (+/-4.9) and 10.0 (+/-4.4) (NS); the mean number of embryos biopsied were 8.3 (+/-3.5), 10.3 (+/-4.2) and 0 (P<0.05); and the mean number of embryos transferred were 1.6 (+/-0.9), 1.6 (+/-0.8) and 2.1 (+/-1.3) (P<0.05 between PGS/FISH and Control). Proportion of euploid embryos were significantly higher (P<0.05) in the PGS/CGH group (40.6%) compared to the PGS/FISH group (26.3%). Clinical pregnancy rate was significantly higher in the PGS/CGH group (60.9%, P<0.05) compared to PGS/FISH (25.4%) and not different to Control (41.2% p=0.057). Implantation rate was significantly higher (P<0.05) in the PGS/CGH group (45.9%) compared either to PGS/FISH (24.8%) or to Control (29.2%). Logistic regression analysis adjusted for female age and number of embryos transferred shown that day 5 CGH improved clinical pregnancy chance by 2.45 (95%CI 1.12 to 6.71) compared to control.

Conclusions: These results show that PGS using aCGH with blastocyst biopsy provides higher implantation and pregnancy rates than day 3/FISH or control and also confirms that day 3 biopsy with FISH does not improve outcomes.

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Meeting abstracts – ASRM 2011

[O-202] Trophectoderm biopsy on day 5, 6 or 7 – does it matter? M. P. Portmann, L. S. Morrison, S. M. Carney, C. F. Boylan, R. F. Feinberg, G. Kovalevsky Reproductive Associates of Delaware, Newark, Delaware, USA

Objectives: To determine if there are differences in aneuploidy rates between day 5, 6 and 7 blasts utilising trophectoderm biopsy and 24 chromosome microarray comparative genomic hybridisation (24-aCGH).

Design: Retrospective compilation of 24-aCGH blast biopsy results from patients undergoing PGS from Feb 2009 to Feb 2011.

Materials and methods: Candidates for IVF and PGS due to prior pregnancy losses, previous failed IVF and unexplained infertility were counseled prior to proceeding. Patients opting for PGS had trophectoderm biopsy of their embryos via laser excision of 2 to 10 cells on cycle days 5, 6, or 7. Following biopsy, blasts were vitrified and biopsies were sent for 24-aCGH.

Results: To date, 231 blasts were biopsied for PGS. Of these, 48.5% were euploid, 43.7% were aneuploid and 7.8% failed to produce a signal. A significant difference in euploidy rate was observed between day 5 and 6 and trending significance between day 5 and day 7. There was no difference in euploidy rate between day 6 and 7. Many aneuploid embryos displayed more than one anomaly.

Biopsy data Day 5 Day 6 Day 7 Total Number of blasts biopsied 136 85 10 231 Euploid status 77 (56.6%)1, 2 33 (38.8%)1 2 (20%)2 112 (48.5%) Aneuploid status 51 (37.5%) 44 (51.8%) 6 (60%) 101 (43.7%) Failed DNA amplification 8 (5.9%) 8 (9.4%) 2 (20%) 18 (7.8%) Monosomies 32 38 3 73 Trisomies 43 20 7 70 Male / female ratio 52% / 48% 58% / 42% 75% / 25% 55% / 45%

1 p=0.016; 2 p=0.050

Conclusions: These results suggest a higher percentage of day 5 blasts are more likely to be euploid than day 6 and 7. The much maligned morphometric indices for blast selection provide some benefit in choosing euploid blasts, but it is likely to be far from a one-to-one relationship. This data supports the notion that day 5 transfer is preferred over day 6 whenever possible. This study also questions the relevance of extended culture of embryos to day 7. The application of PGS and successful cryopreservation of euploid blasts may lead to a broader application of eSET by identification of a single penultimate embryo.

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[O-205] Chromosome breakage in oocytes and embryos: assessment of frequency, origin and clinical relevance of genetic instability during preimplantation development D. Wells, S. Alfarawati, K. Gardiner, P. Colls Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, Oxfordshire, UK; Reprogenetics UK, Oxford, Oxfordshire, UK; Reprogenetics LLC, Livingston, New Jersey, USA

Objectives: A high incidence of embryos containing losses and gains of chromosome fragments has recently been reported. This has raised concerns that abnormalities of this type, undetectable and overlooked by previous cytogenetic studies of embryos, could be extremely common in IVF cycles, increasing the risk of congenital abnormality, miscarriage and unsuccessful treatment cycles. However, the only published data is derived from a small number of embryos from a single clinic and thus require urgent verification. We aimed to determine the true incidence and origin of segmental chromosome anomalies, confirming whether or not they are a cause for clinical concern.

Design: Prospective study.

Materials and methods: Human oocytes and embryos were examined using a well-validated microarray, capable of detecting losses/gains of chromosomal material as small as 5Mb. Over 1,700 embryos were tested at the cleavage or blastocyst stages in addition to >400 oocytes. Samples were derived from 9 different clinics.

Results: Chromosome breakage affected 3.5% of oocytes, 20% of cleavage stage embryos and 6% of blastocysts. Breakage did not occur at random, with specific sites more frequently affected than expected by chance. In many cases one whole chromosome arm was lost while the other arm was duplicated, due to isochromosome formation. Some patients displayed a significant increase in chromosome breakage (P<0.05).

Conclusions: This is the first study to assess timing, location and frequency of de novo chromosome breakage in human oocytes and embryos. Most segmental errors arose at the cleavage stage, after which the incidence declined, indicating that abnormal cells/embryos are probably selected against. We conclude that segmental imbalance is likely to be clinically relevant, although its incidence is less than previous reports have suggested. The tendency for some patients to produce multiple affected embryos argues for procedure or patient-specific affects that warrant further investigation.

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Meeting abstracts – ASRM 2011

[O-209] The progress of chromosome abnormalities from meiosis to the blastocyst stage E. Fragouli, S. Alfarawati, M. Konstantinidis, S. Jaroudi, D. Wells; Reprogenetics UK, Oxford, Oxfordshire, UK; Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, Oxfordshire, UK

Objectives: Despite the clinical importance of aneuploidy, little is known concerning the ultimate fates of specific types of cytogenetic anomaly. This study aimed to shed light on the genesis, progression and survival of different forms of aneuploidy during transition from gamete to blastocyst. The most appropriate stage for preimplantation genetic screening (PGS) was also considered.

Design: Comprehensive cytogenetic evaluation of oocytes/embryos.

Materials and methods: A total of 2,055 samples (polar bodies, blastomeres and trophectoderm biopsies) derived from 319 couples were examined using microarrayCGH.

Results: Progression from day 0 to 5 was associated with an increase in the proportion of embryos that were normal or affected by a single chromosome anomaly (65% to 76%; P<0.001). Concurrently, a decrease in the incidence of embryos with >3 abnormal chromosomes was observed (14% to 4%; P<0.001). Although the trend from day 0 to day 5 was towards reduced abnormalities, a transient increase in aneuploidy was seen at the cleavage stage, the incidence of highly abnormal embryos doubling, matched by a decline in the proportion of normal embryos.

Conclusions: This study confirms that mitotic errors occur frequently at the cleavage stage, but that many of the affected embryos either arrest or succeed in eliminating their abnormal cells. No selection against aneuploidy was seen until after embryonic genome activation (day 3), but from that point most chromosome anomalies had a small negative effect on development. A few aneuploidies were unusual, showing neutral or even enhanced survival (chromosomes 15, 16, 22 and X), whereas others were highly detrimental (9, 10, 14, 19 and 20). Our data provides reassurance that PGS applied on day 0 or 5 is likely to be predictive of viability. The accuracy of day 3 PGS will depend on whether mosaic embryos are fundamentally flawed and succumb or whether they can successfully eliminate abnormal cells. This remains an open question.

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[O-284] Embryo cohort size does not affect euploidy B. Kaplan, H. Danzer, M. Glassner, M. Opsahl, J. Cohen, S. Munne Reprogenetics, Livingston, New Jersey, USA; Fertility Centers of Illinois, Highland Park, Highland Park, Illinois, USA; ART Reproductive Center, Beverly Hills, California, USA; Main Line Fertility and Reproductive Medicine, Bryn Mawr, PA; Northwest Center for Reproductive Sciences, Kirkland, Washington, USA

Objectives: Chromosome abnormalities cause the failure of a high proportion of embryos to implant or reach term. A correlation between follicular response and cohort size could indicate that ovarian reserve is correlated with egg or embryo quality and euploidy. The goal of this study was to determine the frequency of euploidy in cleaved embryos as well as blastocysts across maternal age groups according to the cohort size.

Design: Retrospective multicenter study

Materials and methods: Ovarian stimulation protocols varied and were clinic-specific. More than 60 clinics were included in this study. Preimplantation genetic diagnosis was performed by array Comparative Genomic Hybridisation (aCGH) following day 3 biopsy (649 cycles) or day 5 (blastocyst) biopsy (124 cycles). A total of 5435 day 3 embryos and 701 blastocysts were biopsied. Based on reanalysis of non-replaced embryos (n=221), the technical error rate of aCGH was 3% for day 3 and 0% for day 5 biopsied embryos.

Results:

% Euploid embryos according to maternal age and cohort size # embryos in cohort Egg donor <35 35-39 40-42 >42 Day 3 biopsy 1-4 50% 43% 18%* 17% 6% 5-7 40% 38% 26% 15% 5% 8-10 65% 35% 28% 15% 8% >10 47% 47% 32%* 16% 8% Day 5 biopsy 1-4 50% 55% 50% 36% 20% 5-7 70% 66% 44% 38% 23% 8-10 73% 64% 50% 36% n/a >10 58% 57% n/a n/a n/a

* p<0.05

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Meeting abstracts – ASRM 2011

Conclusions: The results do not support the notion that larger cohorts of embryos or aggressive stimulation regimes produce chromosomally inferior embryos. Furthermore, a smaller cohort of embryos reduces the absolute number of normal embryos available for fresh/frozen-thawed transfers while reducing the cumulative chances of pregnancy. Increasing the cohort size when possible, combined with comprehensive chromosome assessment and embryo selection followed by high yield freezing or vitrification procedures may optimise single embryo transfer and increase cumulative pregnancy rates.

[O-371] Trophectoderm biopsy – age matters M. P. Portmann, L. S. Morrison, S. M. Carney, C. F. Boylan, R. F. Feinberg, G. Kovalevsky Laboratory, Reproductive Associates of Delaware, Newark, Delaware, USA

Objectives: To determine the difference in aneuploidy rate among patients in different age groups utilizing trophectoderm biopsy and 24 chromosome microarray comparative genomic hybridisation (24-aCGH).

Design: Retrospective compilation of 24-aCGH blastocyst biopsy results from patients undergoing PGS between February 2009 and February 2011. Patients were stratified into 3 groups based on their age at the time of biopsy. The groups were as follows <35, 35-39 and >39.

Materials and methods: Patients considered potential candidates for IVF and PGS due to prior pregnancy losses, previous failed IVF attempts and unexplained infertility were counseled extensively prior to proceeding. Patients who opted to undergo PGS testing had trophectoderm biopsy of their embryos via laser excision of 2 to 10 cells post retrieval on day 5, 6, or 7. Following biopsy, blastocysts were vitrified individually, and the biopsed cells were sent for 24-aCGH.

Results: To date, a total of 231 blastocysts were biopsied for PGS. Of these, 48.5% were euploid, 43.7% aneuploid and 7.8% failed to produce a signal. A significant difference in the euploidy rate was observed between <35 and >39 age groups and between 35-39 and >39 age groups. There was no difference in euploidy rate between <35 and 35-39. Many aneuploid embryos displayed more than one anomaly, the frequency of which increased with age.

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Biopsy data <35 35-39 >39 Total Number of blasts biopsied 83 109 39 231 Euploid status 51 (61.4%)1 56 (51.4%)2 5 (12.8%)1, 2 112 (48.5%) Aneuploid status 26 (31.3%) 45 (41.3%) 30 (76.9%) 101 (43.7%) Failed DNA amplification 6 (7.2%) 8 (7.3%) 4 (10.3%) 18 (7.8%)

1p<0.01; 2p<0.01

Conclusions: Previous reports have suggested a relationship between age and aneuploidy rate. Utilising trophectoderm biopsy with microarray CGH, the long held notion of increasing aneuploidy rate with age was confirmed in this small study.

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Meeting abstracts – ASRM 2011

[P-396] PGD via array comparative genome hybridisation (aCGH) can be used for any translocation to simultaneously detect unbalanced embryos and aneuploidy P. Colls, T. Escudero, J. Fischer, N. Cekleniak, S. Munne Reprogenetics LLC, Livingston, New Jersey, USA; New York University, New York, NY; Institute for Reproductive Medicine & Science, Livingston, New Jersey, USA

Objectives: Determine the resolution of aCGH for translocations.Validate aCGH for translocations. Determine % of abnormal embryos that would had been missed by FISH.

Design: Prospective validation of the technique and retrospective comparison with FISH.

Materials and methods: Translocation carriers underwent PGD by aCGH using 24sure arrays (BlueGnome, UK). Abnormal embryos by aCGH were analysed by FISH for the translocation. Once the smallest size detectable by aCGH was determined, we checked all previous PGD cases for translocations, inversions and deletions performed via FISH to determine the % analyzable by aCGH.

Results: 19 PGD for reciprocal translocations and 15 for Robertsonian translocations were performed. Translocated fragments >4Mb could be detected. A total of 1326 cases of PGD for translocations, inversions and deletions analysed by FISH were reviewed. A total of 931 procedures were for PGD of reciprocal translocations. Of these, 196 had one four fragment <4 Mb, none had two fragments <4 Mb. All 351 Robertsonian translocation cases were analyzable by aCGH. All 35 inversion cases and 9 deletion cases had fragments large enough to be analysed by aCGH. Embryos unbalanced by aCGH were analysed by FISH and all (n=23) were found to be consistent with the aCGH diagnosis. Of the 34 PGD aCGH cases, 22.7% of embryos were normal or balanced, 19.9% were unbalanced but normal for the other chromosomes, 31.9% were unbalanced and aneuploid, and 23.1% were only aneuploid.

Conclusions: With PGD via FISH only few chromosomes can be tested simultaneously with a translocation. aCGH can detect any translocation analyzable by regular karyotyping (4Mb resolution) and any aneuploidy even if one of the four fragments is below aCGH resolution. The aCGH error rate are 0% for translocations, 2% for aneuploidy (Gutierrez-Mateo et al. 2011). 23% more abnormal embryos can be detected than using FISH with probes for the translocation only.

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[P-406] Chromosomal mosaicism in day 3 embryos from young, successful art patients as determined by array comparative genomic hybridisation (CGH) L. J. Wilton Preimplantation Genetics, Melbourne IVF, East Melbourne, Victoria, Australia

Objectives: Chromosomal mosaicism, where different cells have a different chromosomal complement, is reported to affect approximately 60% of cleavage stage embryos. Of particular concern are diploid mosaic embryos which contain a mixture of diploid and aneuploid cells as they will confound preimplantation genetic screening (PGS) outcomes because the tested cell may not represent the embryo. The majority of this information is derived after analysis of less than 10 chromosomes and cells diagnosed as diploid may be aneuploid for chromosomes that were not tested for. This study reports the extent of mosaicism of all 24 chromosomes in cleavage stage embryos from young, successful ART patients.

Design: ArrayCGH was used to determine the chromosomal complement of individual blastomeres from surplus, good quality, ICSI generated, day 3 embryos from 7 women who were less than 37 years old (mean 33 years) and had delivered a baby after ART treatment.

Materials and methods: 21 day 3 embryos were allowed to succumb (as required by local law), separated into 115 individual blastomeres (mean 5.5/embryo) and subjected to single cell arrayCGH using Sureplex whole genome amplification and 24sure BAC array analysis (BlueGnome, UK).

Results: Informative results on all chromosomes were obtained on 108 (94%) of cells. Five (24%) embryos were euploid in every cell, 4 (19%) had a consistent aneuploidy in every cell, which is presumed to be meiotic in origin, and 12 (57%) were mosaic. Of the mosaic embryos, 7 (33%) were aneuploid mosaic where every cell was aneuploid but with different aneuploidies in different cells. Five (24%) embryos were diploid mosaic with a mean of 33% aneuploid and 67% diploid cells per embryo.

Conclusions: This study shows that, even in good quality embryos from young ART patients, i) only a minority of embryos are diploid in every cell, ii) meiotic aneuploidy affects about 1 in 5 embryos, iii) diploid mosaicism affects only about 1 in 4 embryos and a minority of these blastomeres are aneuploid.

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Meeting abstracts – ASRM 2011

[P-473] Analysis of 4795 day 3 embryos by array comparative genome hybridisation (aCGH): aneuploidy patterns S. Munne, J. F. Sanchez-Garcia, R. Prates, S. Tormasi, G. Harton, C. Pere Reprogenetics, Livingston, New Jersey, USA

Objectives: To assess aneuploidy patterns in a large cohort of human embryos analysed by Preimplantation Genetic Diagnosis (PGD) using aCGH.

Design: Multicenter retrospective study.

Materials and methods: One cell was biopsied from day 3 embryos and analysed by aCGH (BlueGnome, UK). Because of the low error rate of this technique (2%, Gutierrez-Mateo et al. 2010) PGD results on day 3 embryos were assumed to be representative of the chromosome content of the whole embryo. Embryos with >2 chromosome abnormalities were considered ‘complex abnormal’.

Results: 4795 day 3 embryos were analyzable, of which 33% were normal, 29% complex abnormal, 14% double aneuploidies, 15% monosomies and 10% trisomies. Complex abnormal embryos significantly increased with advancing maternal age while normal embryos decreased, from 21% and 48% in patients <35 to 40% and 22% in patients 40 and older, respectively (p<0.001). There were more monosomy events (n = 1515, ave. age = 37.6) than trisomy events (n = 1071, ave. age = 38.2) with one third of that excess due to monosomy X (ave. age = 36.6). Normal embryos had an average maternal age of 35.8 years, and complex abnormal embryos of 38.5 years. The most common aneuploidies were for chromosomes 16, 22, X, 15, 21, 19 and 13.

Conclusions: This study confirms previous observations on FISH studies: 1) An excess of monosomies, 2) normal embryos had an average age lower than aneuploid ones, with the exception of monosomy X, and 3) most common aneuploidies involve chromosomes included in most FISH probe panels. However, many embryos classified by aCGH as complex abnormal must be true aneuploid and not mosaic, because they increase with maternal age. All abnormalities combined increase significantly with maternal age, with higher ages leading to more double and multiple aneuploidies. In addition, the excess of monosomies observed is not an artifact, their overall lower maternal age indicates the existence of a maternal-age independent mechanism, such as post-meiotic abnormalities.

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ASHG 2011

Anil Biricik from the Genoma Laboratory in Rome gave an interesting talk at the ICHG/ASHG 2011 meeting in Montreal presenting data using 24sure.

Mosaicism does not affect the accuracy of 24 chromosomes preimplantation genetic screening on cleavage stage embryos A. Biricik, L. Spizzichino, S. Bono, ‘GENOMA’ - Molecular Genetics Laboratory, Rome, Italy; F. Fiorentino, G. Kokkali, K. Pantos, Centre for Human Reproduction, Genesis Athens Hospital, Athens, Greece; L. Rienzi, F.M. Ubaldi, G.E.N.E.R.A Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; A. Gordon, BlueGnome Ltd, Cambridge CB22 5LD, UK

Introduction: Different randomised clinical trials (RCTs) have shown that preimplantation genetic screening (PGS), as it was practiced, has not provided the expected benefits. The possible explanation for this poor clinical performance has been mainly attributed to the embryonic chromosomal mosaicism, that is present on day 3 of development, which may decrease the chances of a live birth by prematurely labeling an embryo as abnormal. In this study we aimed to evaluate the accuracy of the 24-chromosomes PGS performed on cleavage stage embryos, in order to ascertain if the tested blastomeres were representative for the whole embryo.

Methods: Embryos biopsy was carried out at day 3. Single cell DNA was amplified by whole genome amplification (WGA) and processed by Array-CGH according to the 24sure protocol, BlueGnome. Euploid embryos were then selected for transfer on day 5 or day 6 of the same cycle. In order to verify the results obtained following day 3 PGS, chromosomally abnormal embryos that developed to blastocyst stage were re-biopsied on day 6 and reanalysed.

Results: 111 PGS cycles were carried out for 104 couples. The mean maternal age was 39.0 ±3.7 years. A total of 838 embryos were biopsied on day 3. Overall, 768 (91.6%) embryos were successfully diagnosed, 553 (72.0%) of which resulted aneuploid. Embryos suitable for transfer where identified in 75 cycles (67.6%). Following transfer of 124 embryos, 50 women (mean maternal age 38.1±3.2 years) had a clinical pregnancy (66.7% pregnancy rate/ET). A total of 63 embryos implanted (50.8% implantation rate/ET), for 60 of which heart beat was also detected. After the clinical cases, 218 nontransferred embryos from 64 PGS cycles were successfully reanalysed. Aneuploidy mosaicism was detected in 100/218 (45.9%) embryos. Despite high levels of mosaicism found, all day 3 aneuploid embryos followed-up were again diagnosed as abnormal after re-analysis on trofectoderm cells, confirming at the end the previous results regardless of the actual abnormal genotype.

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Meeting abstracts – ESHG 2011

Conclusions: Post-zygotic errors leading to mosaicism were common. However, mosaic embryos were confirmed as chromosomally abnormal after re-analysis at blastocyst stage. Although a larger follow-up study are required in order to confirm the above findings, arrayCGH analysis on single blastomeres has demonstrated an accurate aneuploidy detection tool and may assist in identifying abnormal embryos at cleavage stage.

ESHG 2011

Optimising aneuploidy detection in the human preimplantation embryo using 24sure™ Prof Alan Handyside

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age as women reach the menopause. Single cell arrayCGH using 24sure now allows analysis of copy number for all 24 chromosomes in polar bodies biopsied from human oocytes within 12-13 hours. Chromosome imbalance resulting from balanced structural chromosome abnormalities can also be detected with high resolution as well as de novo structural abnormalities and aneuploidies of paternal origin in cells biopsied from cleavage and blastocyst stage embryos. The optimal stage of biopsy for different indications will be discussed.

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ESHRE 2011

A number of abstracts and posters presented at the ESHRE 2011 conference in Stockholm used 24sure and 24sure related products. The abstract book is available for free download from the ESHRE website.

[O-063] An altered pattern and high incidence of multiple meiotic errors in women of advanced maternal age undergoing IVF A. H. Handyside1, J.P.M. Geraedts2, M. Montag3, M.C. Magli4, S. Repping5, C. Staessen6, J. Harper7, A. Schmutzler8, J. Collins9, V. Goossens10, H. van der Ven3, K. Vesela11, L. Gianaroli4. 1 London Bridge Fertility Gynaecology and Genetics Centre, Department of Preimplantation Genetics, London, UK; 2 Maastricht University, Department of Genetics and Cell Biology, Maastricht, The Netherlands; 3 University of Bonn, Department of Gynecological Endocrinology & Reproductive Medicine, Bonn, Germany; 4 SISMER, Department of Reproductive Medicine, Bologna, Italy; 5 University of Amsterdam, Department of Obstetrics and Gynecology, Amsterdam, The Netherlands; 6 University Hospital, Center for Medical Genetics, Brussels, Belgium; 7 University College London, UCL Centre for PG&D Institute for Women’s Health, London, UK; 8 Christian-Albrechts-University Kiel, Center for Reproductive Medicine, Kiel, Germany; 9 McMaster University, Department of Obstetrics & Gynecology, Hamilton, Canada; 10 ESHRE, Central Office, Grimbergen, Belgium; 11 Sanatorium Repromeda, Director, Brno, Czech Republic

Introduction: Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age as women reach the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that aneuploidy arises mainly in female meiosis and particularly in the first meiotic division. Here we have studied copy number gains and losses for all 24 chromosomes in the three products of female meiosis (first and second polar bodies and the fertilised zygote) in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (arrayCGH).

Materials and methods: Thirty four couples undergoing IVF consented to have polar body biopsy and aneuploidy testing by arrayCGH because of advanced maternal age (n = 31) or a maternal balanced translocation (n = 3). The average maternal age was 40 years (range 33-44). Following intracytoplasmic sperm microinjection, the first and second polar bodies were biopsied from all normally fertilised zygotes, collected in PBS, the whole genome amplified by PCR and the DNA labelled for arrayCGH (24sure, BlueGnome, UK). Each polar body was analysed for gains and losses and if there was one or more in either or both of them, the presumed aneuploid zygote was also tested.

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Meeting abstracts – ESHRE 2011

Results: Excluding structural chromosome abnormalities, the pattern of whole chromosome gains and losses in the two female meiotic divisions (meiosis I and II) was analysed in 105 presumed aneuploid zygotes from 34 cycles. In 227 out of 2376 chromosomes analysed (9.5%) maternal meiotic errors were detected, involving all chromosomes (except chromosome 3) though with an increased incidence for the smaller chromosomes. Of these errors, 125 (55%) occured in meiosis I and 102 (45%) in meiosis II. However, 48 meiosis I errors were compensated by gain or loss in meiosis II. Hence only 77 (43%) of maternal aneuploidies in the corresponding zygote originated in meiosis I. Almost all meiosis I errors had patterns consistent with premature predivision of chromatids and only 4 (3%) were caused by non-disjunction of bivalent chromosomes. Also 37 out of 64 (58%) zygotes with only maternal aneuploidies, had two or more maternal aneuploidies (range 2-7). Forty eight (2% per chromosome analysed) aneuploidies were identified only in the zygote and are presumed to be of paternal origin or a result of chromosome loss.

Conclusions: The pattern of meiotic errors revealed here by arrayCGH analysis of the products of female meiosis, mainly in women of advanced maternal age undergoing IVF, is significantly different from natural conception. For example, most pregnancies and live births affected by trisomy 21 (Downs syndrome), following natural conception, originate in female meiosis I. Of 16 chromosome 21 gains detected in zygotes here, only 4 originated in meiosis I, 9 in meiosis II and 3 were presumed to be of paternal origin. These differences in the etiology of meiotic aneuploidies in women of advanced maternal age and the high incidence of multiple meiotic errors may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes.

[O-250] PGD for translocations using array comparative genome hybridisation P. Colls, J. Fischer, T. Escudero, K. Ketterson, G. Harton, S. Munne; Reprogenetics, PGD Lab, Livingston, New Jersey, USA

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[P-407] Maternal meiosis 1 errors detected by array comparative genomic hybridisation of first polar bodies correlate with poor embryo quality C.S. Ottolini1, D.K. Griffin1, A.H. Handyside2, M.C. Summers3, A.R. Thornhill3; 1 University of Kent, Biosciences, Canterbury, UK; 2 University of Leeds, Biological Sciences, Leeds, UK; 3 The Bridge Centre, London, UK

Introduction: Aneuploidy of preimplantation embryos resulting from meiotic errors of the gametes is a major influence on IVF success rates. Studies using fluorescence in situ hybridisation (FISH) to detect up to 8 chromosomes have suggested a correlation between the chromosomal complement of the embryo and embryo quality. More recently, a weak correlation has been reported between chromosomal complement and competent blastocyst formation. However, the precise effects of maternal and paternal derived aneuploidy on embryo quality are not well understood. Preimplantation genetic diagnosis of aneuploidy (PGDA) using array comparative genomic hybridisation (aCGH) of the first polar body (PB1) of oocytes enables a determination of the maternal contribution to embryo aneuploidy for all 24 chromosomes. We compared the results of PB1 aCGH with embryo development and morphology to test the hypothesis that maternal meiotic I errors are correlated with embryo quality.

Materials and methods: All embryos analysed were from the first 25 consecutive IVF treatment cycles using PB1 PGDA at The Bridge Centre (UK). Patients were grouped by maternal age: Group 1) ≥ 40 years, Group 2) < 40 years. PB1 was biopsied directly following ICSI. Each PB1 was individually tubed and whole genome amplification (WGA) performed prior to 24 chromosome analysis by aCGH using ‘24sure’ microarray slides BlueGnome (UK) for all normally fertilised (2PN) oocytes. Embryos were graded at either cleavage or blastocyst stage of development. Cleavage stage embryos were graded following The Bridge Centre protocol and blastocyst stage embryos were scored using a modification of the Gardner blastocyst scoring system. The embryos were grouped by quality as either; good quality or poor quality following their suitability for cryopreservation as per The Bridge Centre protocol for cryopreservation of embryos. Comparisons between the two groups were made using t-tests with significance set at the p < 0.05 level.

Results: 143 PB1 were analysed with 134 (94%) returning aCGH results. Overall the mean maternal age was 39.4 years (± 6.0) and the mean number of 2PN embryos/patient was 5.8 (± 3.4). 52/134 (38.8%) embryos were of good quality and, of these, 38.5% (20/52) returned an aneuploid PB1 aCGH result. In the remaining poor quality embryos the aneuploidy rate was significantly higher at 65.9% (54/82)A total of 59 embryos was analysed from 12 patients in Group 1 with a mean age of 44.3 years (± 3.3). The mean number of 2PN embryos/patient was 5.1 (± 2.9). A significantly smaller proportion (p < 0.005) of good quality embryos (58.3% -14/24) had aneuploid results compared with poor quality embryos (97.1% - 34/35). A total of 75 embryos was analysed from 13 patients in Group 2 with a mean age of 34.9 years (± 3.8). The mean number of 2PN embryos/patient was 6.3 (± 3.7). A smaller proportion of good quality embryos (21.4% -6/28) had aneuploid results compared with poor quality embryos (42.6% - 20/47) but this difference was not significant (p = 0.06).

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Meeting abstracts – ESHRE 2011

Conclusions: In this relatively small data set, we observe a negative correlation between aneuploidy and embryo quality. In general, oocytes with maternal meiosis 1 errors subsequently developed into poorer quality embryos. This pattern was observed across both age groups although, in the current data set, the correlation is significant only in embryos from patients over 40 years. This is interesting considering we had no information on PB2 errors which are now thought to be at least as frequent as those in PB1. While these findings corroborate previous findings using FISH, it is difficult to determine causality since poor quality eggs probably predispose to meiotic errors and separately this may have an effect on embryo quality.

[P-430] Clinical application of array comparative genomic hybridisation in preimplantation genetic diagnosis for reciprocal and robertsonian translocations F. Fiorentino1, L. Spizzichino1, S. Bono1, A. Biricik1, G. Kokkali2, L. Rienzi3, F.M. Ubaldi3, E. Iammarrone1, A. Gordon4, K. Pantos2; 1 GENOMA – Molecular Genetics Laboratory, Preimplantation Genetic Diagnosis Section, Rome, Italy; 2 Genesis Athens Hospital, Centre for Human Reproduction, Athens, Greece; 3 G.E.N.E.R.A, Centre for Reproductive Medicine, Rome, Italy; 4 BlueGnome Ltd, Cambridge CB22 5LD, UK

Introduction: Fluorescence in-situ hybridisation (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements on preimplantation embryos. FISH is known to have several well-documented technical limitations which may lead to incorrect interpretation of the results and a potentially adverse outcome. The clinical application of a molecular-based assay, known as array comparative genomic hybridisation (arrayCGH), used to simultaneously screen for unbalanced translocation derivatives as well as aneuploidy of all 24 chromosomes is described in this study.

Materials and methods: This is a prospective longitudinal cohort study performed in order to evaluate the clinical efficiency of arrayCGH in a specific category of patients. Cell biopsy was carried out on cleavage stage embryos (day 3). Single cell were first lysed and DNA amplified by whole genome amplification (WGA). WGA products were then processed in 24 hours by arrayCGH using high density arrays 24sure +, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on day 5 of the same cycle. The chromosomally abnormal embryos that developed to blastocyst stage were reanalysed in order to confirm PGD results.

Results: Twenty-eight consecutive cycles of PGD were carried out for 23 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5%) embryos were successfully diagnosed, 16% (30/187) were normal or balanced, 17.1% (32/187) were unbalanced for the translocation and normal for aneuploidy, 39.6% (74/187) had aneuploidy and were unbalanced, 27.3% (51/187) were normal or balanced but showed aneuploidy of chromosomes not involved in the translocation. The re-analysis at blastyocyst stage were concordant for all embryos followed-up. Despite high levels of mosaicism, all day 3 aneuploid embryos were again diagnosed as abnormal after re-analysis on trofectoderm cells, confirming at the end the previous results regardless of the

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actual abnormal genotype. Embryos suitable for transfer were identified in 17 cycles (73.9%), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6% per ET), with a total of 14 embryos implanted (63.6% per transferred embryo). Six patients delivered 7 healthy babies, while the other pregnancies (1 twins and 5 singletons) are currently ongoing beyond 20 weeks of gestation.

Conclusions: The data obtained demonstrates that arrayCGH is able to detects chromosome imbalances on embryos. Unlike some alternative techniques, this approach does not need prior pre-clinical workup for validation of the PGD protocol, reducing the time required for initiation of treatment for translocation couples. It also provides the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. ArrayCGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity, and reliability.

[P-432] Successful clinical application of arrayCGH for 24 chromosome aneuploidy on day 3 preimplantation embryos: high efficiency and reliability L. Rodrigo, P. Mir, A. Cervero, E. Mateu, A. Mercader, C. Vidal, J. Giles, J. Remohi, A. Pellicer, J. Martin, C. Rubio, Institut Universitari – IVI Valencia, Valencia, Spain

Introduction: Array Comparative Genomic Hybridisation (aCGH) has emerged in the field of Preimplantation Genetic Screening (PGS) as a new technique to analyse aneuploidies from all the human chromosomes. The aim of this study was to describe the clinical outcome of day 3 single cell aCGH and the reliability of the technique by means of the re-analysis of day 5 embryos. Additionally, two cycles were performed in carriers of Robertsonian translocations to check the presence of interchromosomal effect.

Materials and methods: This study included 13 patients from our Preimplantation Genetic Diagnosis program in whom day 3 embryo biopsy was performed for different indications: 7 cycles in recurrent miscarriage (RM); 3 cycles in recurrent implantation failure (RIF); 1 cycle in severe male factor (SMF); and 2 cycles in carriers of Robertsonian translocations (RT). Mean female age was 37.6 ± 1.4 years (± SD). A single cell from each embryo underwent Whole Genome Amplification (WGA) using Sureplex (BlueGnome Ltd, Cambridge, UK). WGA products and genomic DNA used as control were labelled in Cy3 and Cy5 fluorophores respectively. Both labelled DNAs were co-hybridised in a 24sure array (BlueGnome Ltd, Cambridge, UK). After washing, slides were scanned using Innoscan 710 (Innopsys, Carbonne, France). Data were analysed by BlueFuse Multi software (BlueGnome Ltd, Cambridge, UK). We used a 24 hours aCGH protocol for single cell analysis. Transference of normal/balanced embryos was performed on day 5. Abnormal or arrested normal embryos after day 3 aCGH analysis were fixed on day 5, and all the cells were analysed by fluorescence in situ hybridisation (FISH) using probes for the affected chromosomes (Vysis Inc. Downers Grove, IL, USA).

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Meeting abstracts – ESHRE 2011

Results: A successful WGA was obtained in 68 out of 71 biopsied embryos (95.8%), and all the amplified products were labelled successfully. A total of 39 embryos were diagnosed as abnormal (57.4%). After 12 embryo transferences (92.3% cycles with embryo transfer), clinical pregnancy was observed in 8 patients (61.5% per cycle; 66.7% per transference), with 47.8% of implantation rate. In patients with RM, RIF and SMF the percentage of abnormal embryos was 57.4% (31/54); 20.4% (11/54) showed aneuploidies only for the chromosomes analysed in our routine FISH program (chromosomes 13, 15, 16, 17, 18, 21, 22, X, Y); 22.2% (12/54) also had aneuploidies for other chromosomes; and 14.8% (8/54) of the embryos had aneuploidies only for chromosomes different from the analysed in our FISH program. In patients with RT the percentage of abnormal embryos was 57.1% (8/14); 35.7% (5/14) showed abnormalities only for the chromosomes of the translocation; 21.4% (3/14) also had aneuploidies for other chromosomes; and none of the embryos (0/14) had aneuploidies only for chromosomes not implicated in the translocation. A total of 28 embryos were re-analysed on day 5 by FISH for the affected chromosomes on day 3, with a mean number of analysed cells per embryo of 28.5 ± 18.5 (± SD). The diagnosis of day 3 was confirmed in 26 of the embryos (92.9%), with 30.8% showing mosaicism for the chromosomes analysed. Of the two embryos not confirmed, one was diagnosed as normal on day 3 but as mosaic on day 5 (48 cells analysed with two cell lines: trisomy 15 (70%)/ diploid (30%)); and the other one was diagnosed with trisomy 15 on day 3 but as mosaic on day 5 (25 cells analysed with three cell lines: diploid (88%)/tetraploid (8%)/trisomy 13 and 21 (4%)).

Conclusions: PGS using aCGH is suitable on single blastomeres due to the high rate of successful amplification and reliable results. This protocol allows maintaining our routine of day 3 biopsy and day 5 embryo transfer. So far, no interchromosomal effect has been detected in the two couples with Robertsonian translocations.

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PCRS 2011

A number of abstracts and posters presented at the PCRS 2011 conference in Palm Springs used 24sure and 24sure related products. Abstracts from the meeting are published in Fertility and Sterility (vol 95(4) S1) and are free to view.

[O-6] Implantation and miscarriage rates following arrayCGH analysis at the cleavage and blastocyst stages G. Harton, M. Surrey, J. Grifo, B. Kaplan, S. Munne, Reprogenetics, Livingston, New Jersey, USA

Objectives: To assess the effect of maternal age on implantation after PGD with Comparative Genomic Hybridisation (CGH) or microarray CGH (aCGH) after biopsy at day 3 (cleavage) or day 5 (blastocyst) of embryo development.

Design: Retrospective multi-center comparative study.

Materials and methods: PGD Patients from various IVF clinics had embryos biopsied on day 3 or 5. Analysis on day 3 involved 1-cell biopsy, aCGH and transfer of euploid embryos on day 5. Analysis of blastocysts involved trophectoderm biopsy on day 5 or 6 followed by CGH, vitrification and transfer of euploid embryos in a later cycle.

Results:

Age group Implant (sac) Sacs lost Aneup rate Day 3 30-34 51.4 (70/136) 7.1 (5/70) 50.5 Day 5 30-34 85.7 (24/28) 8.3 (2/24) 40.4 P<0.005 NS P<0.005 Day 3 35-39 38.2 (63/165) 9.5 (6/63) 65.1 Day 5 35-39 63.8 (23/36) 4.3 (1/23) 49.2 P<0.01 NS P<0.005 Day 3 40-42 31.0 (40/121) 15.0 (6/40) 77.8 Day 5 40-42 77.7 (28/36) 7.1 (2/28) 61.1 P<0.001 NS P<0.001

Conclusions: There were fewer abnormal embryos when biopsied at the blastocyst stage compared with embryos biopsied at the cleavage stage, suggesting selection against aneuploidy during extended culture. However, blastocyst aneuploidy rates remained high and increased with advancing maternal age. Interestingly, implantation rates for euploid blastocysts were not significantly different

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Meeting abstracts – PCRS 2011

across all age groups, however, for day 3 biopsy, implantation rates declined with age. Since both techniques detect the same abnormalities, the decrease in implantation with age following biopsy at day 3 is unlikely to be a screening problem and suggests the involvement of non-chromosomal factors. The impact of biopsy on embryos at the cleavage stage versus the blastocyst stage is largely unknown. An age-related uterine receptivity problem may be magnified after controlled ovarian stimulation and alleviated by transfer of diagnosed blastocysts in unstimulated cycles. There are several limitations of this retrospective analysis; however the findings are intriguing enough to warrant further study.

[P-35] Reanalysis of monosomic embryos at the blastocyst stage following day 3 biopsy and array comparative genomic hybridisation (aCGH) P. Colls, S. Ghadir, H. Danzer, M. Surrey, D. Hill, S. Munne, Reprogenetics, Livingston, New Jersey, USA

Background: The diagnosis of ‘monosomy’ by FISH in some cases can be a technical artifact originated by overlapping of two signals, which can be avoided by a DNA ratio analysis test such as aCGH. In addition, based on the observations of Sandalinas et al. (2001) that monosomies did not reach the blastocyst stage, blastocyst embryos classified as monosomic by FISH on day 3 were sometimes replaced.

Objectives: To calculate the error rate of blastocyst embryos classified as monosomic on day 3 by aCGH, and based on that, provide guidance on whether to replace these embryos or not.

Materials and methods: Single blastomeres from day 3 embryos were analysed by aCGH (24sure, BlueGnome, UK). Embryos diagnosed as monosomic that reached the blastocyst stage were fixed for complete cell reanalysis by FISH using a 12 chromosomes panel (X,Y,8,13,14,15, 16,17,18, 21,22) plus any other chromosome previously diagnosed as abnormal by aCGH.

Results: 42 embryos diagnosed as monosomic for one or more chromosomes on day 3 by aCGH, with 50 monosomy events, reached blastocyst stage. The reanalysis by FISH of the whole embryos on day 5 showed confirmation of the ‘monosomy’ diagnosis in 40 (95.2%) embryos and 48 (96%) monosomy events. This compares with a concordance rate for all reanalysed embryos of 97.1% (132/136), a non-statistically significant difference. Thirty five (87.5%) of the 40 embryos correctly diagnosed as monosomic on day 3 showed a status of pure monosomy on day 5 and 5 (12.5%) showed a mosaicism of normal and monosomic cells.

Conclusions: The reanalysis by FISH on day 5 of embryos diagnosed as monosomic on day 3 by aCGH showed an error rate per embryo of 4.5% and of 4% per monosomy event, not different from the overall 2.9% error rate, or the published 2.5% (Gutierrez-Mateo et al. 2010). Finally, although earlier studies showed that pure monosomies were not able to reach the blastocyst stage (Sandalinas et al. 2001), the present study shows pure monosomy in 87.5% of day 5 monosomic

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embryos analysed, a change probably related to the improvement in culture media during the last decade. Based on the high likelihood that monosomies detected on day 3 are true monosomies, and that they can reach blastocyst stage, we do not recommend replacement of these embryos.

[P-36] Aneuploidy patterns in 3143 day 3 embryos analysed by arrayCGH S. Munne, P. Colls, J. Sanchez, R. Prates, S. Tormasi, N. Goodall, G. Harton, Reprogenetics, Livingston, New Jersey, USA

Background: Previous FISH studies with day 3 biopsy showed an excess of monosomies, a correlation between maternal age and percentage of normal embryos (lower maternal age= higher percentage of normal embryos), and no correlation between maternal age and mosaicism. These studies were performed with 9-12 chromosome probes and thus did not include more than half of the chromosome complement.

Objectives: To assess the types of chromosome abnormalities observed by arrayCGH and their relationship to maternal age.

Materials and methods: Single blastomeres from day 3 embryos were analysed by aCGH (24sure, BlueGnome, UK). This technique can analyse all chromosomes with a very low error rate, including errors caused by mosaicism, (2%, Gutierrez-Mateo et al. 2010) and thus we assume that day 3 PGD results are mostly representative of the rest of the embryo. An embryo with 3 or more chromosomes out of the 24 being abnormal was considered ‘complex abnormal’.

Results: Complex abnormal embryos and double aneuploidies significantly increased with advancing maternal age, while trisomies and monosomies did not. Normal embryos significantly decreased with advancing maternal age (48% in the <35 years of age group to 21% in the >39 years). We observed an excess of monosomy events (n = 1006) compared to trisomy events (n = 695) with one third of that excess being caused by monosomy X. There was a slightly higher average maternal age for aneuploidy events on chromosomes 13 to 22 (av. Age 38.4) than 1 to 12 (37.7), while monosomy X results showed an average maternal age of 36.4 years. Normal embryo results had an average maternal age of 35.9 years, compared to complex abnormal embryo results of 38.7 years. The most common aneuploidies were for chromosomes 16 (n = 204), 22 (157), X (125), 15 (119), 21 (107), and 19 (91).

Conclusions: Large studies using FISH (Munne et al. 2007) considered complex abnormalities akin to chaotic mosaics. However, by analysing all chromosomes with aCGH, many embryos classified by aCGH as complex abnormal must be true aneuploid, as seen by their increased maternal age. The percentage of abnormal embryos does increase significantly with maternal age, as does the percentage of double aneuploids and complex abnormals. The excess of monosomies observed is not an artifact, as described in a separate abstract by Colls et al.

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Meeting abstracts – PCRS 2011

[P-42] Trophectoderm cells derived from blastocyst biopsy are suitable for arrayCGH analysis of 24 chromosomes Z. Yang, S. Salem, S. S-Lyle, A. Bayrak, R. Salem, Pacific Reproductive Center, Torrance, California, USA

Background: Recent studies raise concern that use of blastomeres derived from cleavage stage embryo biopsy may compromise PGD results due to high incidence of mosacism. Use of trophectoderm cells derived from blastocyst biopsy may become the choice for chromosomal screening with arrayCGH.

Objectives: Our objective is to evaluate the efficacy of arrayCGH analysis of 24 chromosomes of trophectoderm cells derived from blastocyst biopsy using the 24sure arrayCGH (BlueGnome, Cambridge) for screening of all chromosomes.

Materials and methods: All embryos used in this study were donated for research by patients with informed consent. Embryos were biopsied at 8-cell and blastocyst stages. To obtain trophectoderm cells, some of the embryos were cultured to blastocyst stage and the trophectoderm cells were carefully removed from each blastocyst. The trophectoderm cells and blastomeres were washed in x PBS prior to loading into the PCR tube containing lysis buffer. Whole genome amplification was performed using the SurePlex kit. Sample and control DNA were labeled with Cy3 and Cy5 fluorophores. Labeled DNA was resuspended in dexsulphate hybridisation buffer and hybridised under cover slides. After washing, the hybridised 24sure slides were scanned using a laser scanner. The data was analysed using the BlueFuse software for whole chromosome loss or gain. Data of the trophectoderm cells of blastocysts was compared to that of the blastomeres of 8-cell stage embryos.

Results: Analysis of 24 chromosomes of trophectoderm cells from blastocysts and blastomeres from 8-cell embryos using the 24sure arrayCGH method were completed within 24 hours of sample receipt. All trophectoderm cells had more clear signals when compared with blastomeres of 8-cell embryos. The percentage of undiagnosed embryos was 0% for trophectoderm cells derived from blastocyst biopsy while 7.5% of blastomeres derived from 8-cell stage embryos were undiagnosed.

Conclusions: The above preliminary data suggests that trophectoderm cells derived from blastocyst biopsy are more suitable for the arrayCGH analysis of 24 chromosome using the 24sure methods within 24 hours. This finding makes it feasible to perform biopsy on day 5 and embryo transfer on day 6 with an in-house arrayCGH program or to freeze the blastocysts after biopsy for later transfer based on individual patients conditions.

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GfH 2011

This poster from MGZ in Munich uses 24sure and 24sure+ and was presented at the GfH meeting in Regensburg March 2011.

Preimplantation genetic diagnosis – arrayCGH of pluripotent trophoblast cells after trophectoderm biopsy U. Koehler, K. Sendelbach, B. Paulmann, U. Hehr, B. Seifert, E. Holinski-Feder, Medizinisch Genetisches Zentrum Munchen (MGZ), Munchen, Germany

Introduction: Preimplantation genetic diagnosis (PGD, PID) of pluripotent trophectoderm cells has been legalised by the German Supreme Court in July 2010 for families at high risk for severe genetic disorders. So far, investigations to detect maternal derived mutations or chromosomal translocations were carried out on polar bodies using PCR based techniques or fluorescent in situ hybridisation of selected chromosome probes. Thus only the maternal component of the fertilised oocyte could be tested. The court decision from July 2010 has changed this setting. Through the permission to test pluripotent trophoblast cells after trophectoderm biopsy at day 5 after fertilisation both the maternal and the paternal genetic component can be investigated.

Fertility 2011

A number of abstracts and posters presented at the Fertility 2011 conference in Dublin used 24sure and 24sure related products. They can be viewed on www.cytochip.com

Can first polar body arrayCGH results predict developmental potential Ellen Cater, Colleen Lynch, Kathryn Berrisford, Louise Kellam, Andrew Craig, Lucy Jenner, Simon Fishel, CARE Fertility, Nottingham, NG7 8PZ, UK

Developmental potential of embryos derived from oocytes found to have a single aneuploidy in the first polar body Ellen Cater, Colleen Lynch, Kathryn Berrisford, Louise Kellam, Andrew Craig, Lucy Jenner, Simon Fishel, CARE Fertility, Nottingham, NG7 8PZ, UK

Oocyte chromosome complement does not appear to be a factor in fertilisation – a comparison of first polar body analysis of fertilised and unfertilised metaphase II oocytes Colleen Lynch, Ellen Cater, Kathryn Berrisford, Louise Kellam, Dr Lucy Jenner, Andrew Craig, Dr Simon Fishel, CARE Fertility, Nottingham, NG7 8PZ, UK

62 24sure publications | BlueGnome