Preimplantation Genetics Publications

Transcription

1 Preimplantation Genetics Publications An overview of recent publications featuring Illumina 24sure and karyomapping technology.

2 TABLE OF CONTENTS Introduction 3 Improving IVF Success Rate 5 Randomized Control Trials 6 Retrospective Studies 7 Advanced Maternal Age 9 Single Embryo Transfer 11 Accuracy and Reliability 12 Technology 13 Polar Body Biopsy 15 Cleavage Stage Biopsy 16 Trophectoderm Biopsy 17 Other Samples 19 Correlation Between Aneuploidy and Morphology 20 Early Embryo Development 21 Aneuploidy Rate 22 Origins of Aneuploidy 23 Mosaicism 25 24sure+: PGD for Translocation Carriers 26 Karyomapping 29 Bibliography 30 2

3 INTRODUCTION In vitro fertilization (IVF) success rates are low, with only around 1 in 3 cycles resulting in a healthy pregnancy 1. One of the major reasons for this is that many embryos are aneuploid 2 ; that is, they have an incorrect number of chromosomes. As most aneuploidies are maternally derived, the incidence increases with maternal age 3 (Figure 1). As such, IVF success rates begin to fall dramatically as a woman reaches her late 30s and early 40s. However, even in younger patients the aneuploidy rate can still be significant enough to have a severe impact on IVF success 4. In natural conceptions, aneuploid embryos would fail to implant or would miscarry so early on that they wouldn t even be noticed. But in IVF, when the embryo is carefully transferred into the uterus of a hopeful patient, these losses when so much time, money, and emotion have been invested can be devastating. Scientists developed preimplantation genetic screening (PGS) to increase IVF success. The groundbreaking 24sure technology delivers aneuploidy screening of embryos before transfer, so that only euploid embryos are transferred. All 24 chromosomes (22 autosomes plus X and Y) can be screened within a 24-hour period, starting from single cells. This fits comfortably into the time span permitted by the constraints of a fresh IVF cycle. Since its launch, many papers, conference presentations, and posters have shown the potential of the 24sure assay to improve IVF success rates, as well as to discover more about the early stages of embryo development. To date, over 350,000 clinical biopsies have been tested using 24sure technology and it is now a well-established technique in IVF labs across the globe. This booklet highlights some of the key papers that have been published using 24sure technology. It also provides some general background information for those interested in PGS and preimplantation genetic diagnosis (PGD). Figure 1: Aneuploidy Increases with Maternal Age 90.0% 100.0% 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% Implantation Aneuploidy Miscarriage 90.0% 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% Implant Aneuploidy Rate Miscarriage 0.0% % As maternal age increases, aneuploidy rate rises, and the number of euploid embryos decreases. 1 Human Fertilization and Embryology Authority: Latest UK IVF figures: 2010 and Available: pdf: Accessed 11 February Fragouli E, Wells D (2011) Aneuploidy in the human blastocyst. Cytogenet Genome Res 133(2 4): Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, et al. (2012) Array CGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod Biomed Online 24(6): Wells D, Delhanty JD (2000) Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod 6(11):

4 Reviews Handyside AH (2013) 24-chromosome copy number analysis: a comparison of available technologies. Fertil Steril 100(3): Brezina PR, Brezina DS, Kearns WG (2012) Preimplantation genetic testing. BMJ 345: e5908. Handyside AH (2012) Molecular origin of female meiotic aneuploidies. Biochim Biophys Acta 1822(12): Harton G, Braude P, Lashwood A, Schmutzler A, Traeger-Synodinos J, et al. (2011) ESHRE PGD consortium best practice guidelines for organization of a PGD centre for PGD/preimplantation genetic screening. Hum Reprod 26(1): Harton GL, De Rycke M, Fiorentino F, Moutou C, SenGupta S, et al. (2011) ESHRE PGD consortium best practice guidelines for amplificationbased PGD. Hum Reprod 26(1): Harton GL, Harper JC, Coonen E, Pehlivan T, Vesela K, et al. (2011) ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridization-based PGD. Hum Reprod 26(1): Harton GL, Magli MC, Lundin K, Montag M, Lemmen J, et al. (2011) ESHRE PGD consortium/embryology Special Interest Group best practice guidelines for polar body and embryo biopsy for pre-implantation genetic diagnosis/screening. Hum Reprod 26(1): Fishel S, Thornton S, Dowell K (2011) A new era of PGS for IVF will it yield the anticipated improved efficiency? J Fertiliz In Vitro 1:1. Handyside AH (2010) Preimplantation genetic diagnosis after 20 years. Reprod Biomed Online 21(3): Harper J, Coonen E, De Rycke M, Fiorentino F, Geraedts J, et al. (2010) What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD consortium steering committee. Hum Reprod 25(4): Geraedts J, Collins J, Gianaroli L, Goossens V, Handyside A (2010) What next for preimplantation genetic screening? A polar body approach! Hum Reprod 25(3): Fragouli E, Wells D (2012). Aneuploidy screening for embryo selection. Semin Reprod Med 30(4): Koehler U, Schoen U, Mayer V, Holinski-Feder E (2013) Preimplantation genetic diagnosis for monogenic disorders and chromosomal rearrangements the German perspective. Journal fur Reproduktionsmedizin und Endokrinologie 2009(2):

5 IMPROVING IVF SUCCESS RATE A growing number of researchers report that 24sure technology can dramatically increase the success rate of IVF. This body of evidence incorporates studies that have tested embryos at various stages of development and included patients of various ages, demonstrating that the 24sure assay can be applied in many scenarios. 5

6 RANDOMIZED CONTROL TRIALS The recent publication of two randomized control trials (RCTs) represent a landmark addition to the growing collection of evidence showing the benefits of 24sure technology. Researchers in both studies performed 24sure screening for a cohort of young, good prognosis patients undergoing IVF. The first study looked at fresh embryo transfer and showed an increase in ongoing pregnancy rate from 41.7% (control) to 69.1% (with 24sure screening). The second study used frozen embryo transfer and showed an increase in implantation rate from 33% to 65%. Additionally, there was a much lower miscarriage rate when embryos had been screened before transfer, with no miscarriages at all in the cohort where frozen embryo transfer was performed after 24sure screening. Yang Z, Liu J, Collins GS, Salem SA, Liu X, et al. (2012) Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with arraycgh for good prognosis patients: results from a randomised pilot study. Mol Cytogenet 5: 24. This paper focuses on increasing the use of single embryo transfer (SET) and improving the pregnancy rates associated with SET by using 24sure technology in good prognosis IVF patients (< 35, no prior miscarriage). In addition, this publication represents the first randomized study of its kind, and shows a significant increase in pregnancy rates using the 24sure assay compared with the control group (morphology scores only). Ongoing pregnancy rates at 20 weeks per cycle were 41.7% in the control group and 69.1% in the 24sure analysis group. Yang Z, Salem SA, Liu X, Kuang Y, Salem RD, et al. (2013) Selection of euploid blastocysts for cryopreservation with array comparative genomic hybridization (acgh) results in increased implantation rates in subsequent frozen and thawed embryo transfer cycles. Mol Cytogenet 6: 32. These data demonstrate the efficacy of 24sure technology when combined with frozen embryo transfer in good prognosis patients. This study shows that screening by array comparative genomic hybridization (acgh) before vitrification increases implantation rate per embryo (65% versus 33% in embryos screened by morphology only). The study also shows that acgh screening decreases miscarriage rates (no miscarriages occurred in the group screened using the 24sure assay), and reduces costs associated with embryo storage, as aneuploid embryos do not need to be stored. Meeting and conference abstracts Fiorentino F, Rienzi L, Bono S, Capalbo A, Spizzichino L, et al. (2013) Preimplantation genetic screening on day 3 embryos using array comparative genomic hybridization in patients with advanced maternal age: a prospective double blinded randomized controlled trial. Hum Reprod 28 (suppl 1): i49-i51. Harton G (2013) Results of randomized controlled studies using microarray technology. Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis. Reprod Biomed Online 6 (suppl 1). 6

7 RETROSPECTIVE STUDIES In addition to randomized control trials, there have been many retrospective and case-control studies comparing clinic data from patients who underwent PGS with those where embryos were selected based on morphology alone. These studies have consistently demonstrated the ability of 24sure technology to raise measures of IVF success, such as implantation rate and ongoing pregnancy rate, and to reduce negative outcomes such as miscarriage and multiple births. Keltz MD, Vega M, Sirota, I, Lederman M, Moshier EL, et al. (2013) Preimplantation Genetic Screening (PGS) with Comparative Genomic Hybridization (CGH) following day 3 single cell blastomere biopsy markedly improves IVF outcomes while lowering multiple pregnancies and miscarriages. J Assist Reprod Genet 10: This study looks at pregnancy outcomes in IVF patients who underwent aneuploidy screening using the 24sure platform and compares them to a control group. In both age groups (< 35 and 35), 24sure analysis resulted in a significantly higher implantation rate, clinical pregnancy rate, and ongoing pregnancy rate. Across all ages, implantation rate more than doubled compared to controls (52.63% versus 19.15%). These differences remained significant even though cycles with no euploid embryos to transfer were included. Fishel S, Craig A, Lynch C, Dowell K, Ndukwe, G, et al. (2011) In vitro assessment of 19,803 paired chromosomes and clinical outcome from first 150 cycles using array CGH of the first polar body for embryo selection and transfer. J Fertil In Vitro 1(1): 1 8. This paper by CARE Fertility Group presents their experience using 24sure technology on polar bodies since 2008, when they first assessed acgh aneuploidy screening and achieved a live birth following use of the technology. They conclude that acgh is proven to generate robust chromosome information and improve implantation rates in women with poor prognosis. Hodes-Wertz B, Grifo J, Ghadir S, Kaplan B, Laskin, CA, et al. (2012) Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos. Fertil Steril 98(3): This paper explores the possibility of aneuploid embryos being a significant factor in unexplained recurrent pregnancy loss. The study includes 287 cycles of couples with idiopathic pregnancy loss. Following euploid embryo selection with the 24sure assay, the study reports a 45% implantation rate, with 92% of those having ongoing pregnancies or deliveries at time of publication. The miscarriage rate was much reduced: 6.9% compared to 33.5% in a recurrent pregnancy loss (RPL) control group. Based on these results and previous studies, the authors suggest that selection of euploid embryos for transfer could be a huge benefit. Rubio C, Rodrigo L, Mir P, Mateu E, Peinado V, et al. (2013) Use of array comparative genomic hybridization (array-cgh) for embryo assessment: clinical results. Fertil Steril 99(4): This review paper reports the clinical outcome following array-based screening of all 24 chromosomes, showing improved pregnancy rates for different indications to a higher degree than fluorescence in situ hybridization (FISH). The authors show that most data regarding the controversy of day 3 biopsy comes from FISH cycles, and the utility of day 3 biopsy using array-based screening should be further validated through randomized control trials. Liu J, Wang W, Sun X, Liu L, Jin H, et al. (2012) DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic. Biol Reprod 87(6): 148. This paper reports that high proportions of human blastocysts produced in vitro from women of advanced maternal age are aneuploid (56.6%) and mosaic. The transfer of blastocysts screened with 24sure technology produced high pregnancy (70.2%) and implantation (62.5%) rates. It was established that errors can occur in any of the 23 pairs of chromosomes. 7

8 References Rechitsky S, Verlinsky O, Kuliev A (2013) PGD for cystic fibrosis patients and couples at risk of an additional genetic disorder combined with 24-chromosome aneuploidy testing. Reprod Biomed Online 26(5): Meeting and conference abstracts Breborowicz A, Vega M, Sauerbrun M, Sirota I, Stein D, et al. (2013) Euploid embryos following day 3 biopsy for comparative genomic hybridization (CGH) have excellent outcomes even with no advanced blastocysts. Fertil Steril 100(3): S201. Resetkova N, Tobler KJ, Kearns WG, Werner EF. (2013) In vitro fertilization (IVF) with 23-chromosome pair preimplantation genetic screening (PGS) is cost effective to achieve a live birth compared to IVF alone for recurrent pregnancy loss (RPL). Fertil Steril 100(3): S99-S100. Rubio C, Rodrigo L, Mateu E, Mercader A, Peinado V, et al. (2013) ArrayCGH versus FISH for PGS in recurrent miscarriage couples. Hum Reprod 28(suppl 1): i291-i311. 8

9 ADVANCED MATERNAL AGE IVF success rates decrease with maternal age, in large part due to the increase in aneuploidy 5. Therefore, 24sure technology can have a significant impact with those of advanced maternal age. By screening embryos before transfer and selecting only those that are euploid, ongoing pregnancy rates have been shown to remain static across age groups up to the age of around Clearly, in patients of more advanced maternal age, the chance of having no euploid embryos to transfer increases and this can be seen as a potential disadvantage to PGS. This chance depends on the number of blastocysts produced, as demonstrated by Ata et al. (2012) 7 using retrospective data from their clinic (Table 1). Clearly counseling must be provided to make sure that couples are informed of the possibility that no embryo will be available for transfer. While this would be a significant disappointment, these aneuploid embryos would not go on to develop into healthy children. Finding out at this stage that the IVF treatment is not going to work may be a better outcome rather than the distress that couples may experience when an embryo fails to implant, or worse, miscarries. In patients for whom euploid embryos do exist, the chance of achieving a successful pregnancy are substantially increased with the use of 24sure technology. This has been documented in many studies. Additionally, the cost of cryopreserving the remaining embryos would usually decrease as only those that are euploid would need to be stored 8. Harton GL, Munnè S, Surrey M, Grifo J, Kaplan B, et al. (2013) Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization. Fertil Steril 100(6): This study aimed to determine if identification of euploid embryos using 24sure technology overcame the decrease in implantation rates observed during IVF as maternal age increases. Embryos from patients undergoing PGS in fertility clinics across the U.S. were screened using the 24sure assay following biopsy on either day 3 or day 5/6. Despite observing the expected increase in aneuploidy with increasing maternal age, implantation rates and ongoing pregnancy rates were constant between age groups up to 42 years of age; additionally, pregnancy loss was significantly reduced. References Liu J, Wang W, Sun X, Liu L, Jin H, et al. (2012) DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic. Biol Reprod 87(6): Fragouli E, Wells D (2011) Aneuploidy in the human blastocyst. Cytogenet Genome Res 133(2 4): Harton GL, Munnè S, Surrey M, Grifo J, Kaplan B, et al. (2013) Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization. Fertil Steril 100(6): Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, et al. (2012) ArrayCGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod Biomed Online 24(6): Liu J, Sills ES, Yang Z, Salem SA, Rahil T, et al. (2012) Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation. Clin Exp Reprod Med 39(2):

10 Meeting and conference abstracts Barritt J, Hill D, Surrey M, Tormasi S, Welch C, et al. (2013) Estimated number of mature oocytes needed for fertility preservation patients based on the number of euploid blastocysts diagnosed following preimplantation genetic screening (PGS). Fertil Steril 100(3): S64. Lee, HL, Hodes-Wertz B, Alexis A, Lee TL, McCulloh D, et al. (2013) Preimplantation genetic screening improve IVF success rate in women over 40. Fertil Steril 100(3): S83. Mateu E, Rodrigo L, Milán M, Mercader A, Simón C, et al. (2013) Chromosomal abnormalities and clinical outcome in patients with advanced maternal age (AMA) using comprehensive chromosome screening (CCS). Fertil Steril 100(3): S130. Munné S, Mills E, Hill D, Wagner C, Grifo J, et al. (2013) Implantation and ongoing pregnancy rate with euploid embryo transfer is independent of maternal age. Fertil Steril 100(3): S131. Lorwatthanasirikul J, Quangkananurug W, Rattanajitr T, Chanchamroen S, Sawakwongpra K (2013) Comprehensive chromosome screening with vitrified warmed single blastocyst transfer increases the pregnancy rate in advanced maternal age. Abstracts of the 12th International Conference of Preimplantation Genetic Diagnosis, Reprod Biomed Online 6 (suppl 1). 10

11 SINGLE EMBRYO TRANSFER The risks of multiple births, both to mother and children, are well-documented. Babies born as multiples are significantly more likely to be admitted to the neonatal intensive care unit after birth than singletons, and are more likely to be premature and be of low birth weight 9. Despite these risks, it has traditionally been common practice to transfer more than one embryo during the IVF process to compensate for the low implantation rate usually observed. By transferring more than one embryo, the chance of the IVF cycle succeeding greatly increases. 24sure technology permits single embryo transfer as a viable option for many couples. By raising the implantation rate, PGS increases the chance of success when a single screened blastocyst is transferred so that it is equivalent to the chance of success with double transfer of unscreened embryos 10. This means that the problems of multiple births can be effectively eliminated without reducing the ongoing pregnancy rate. Many papers have demonstrated this possibility, and promote this practice to improve patient care and potentially reduce total long-term healthcare costs. Sills ES, Yang Z, Walsh DJ, Salem SA (2012) Comprehensive genetic assessment of the human embryo: can empiric application of microarray comparative genomic hybridization reduce multiple gestation rate by single fresh blastocyst transfer? Arch Gynecol Obstet 286(3): This review article contains similar themes to the Yang et al. (2012) paper regarding single embryo transfer. The authors conclude that it will soon be standard practice to determine which (single) embryo to transfer in an IVF cycle by assessing the chromosome complement by acgh. Schoolcraft WB, Katz-Jaffe MG (2013) Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age. Fertil Steril 100(3): This review paper outlines the advantages of single embryo transfer as opposed to double embryo transfer, and discusses how comprehensive chromosome screening (CCS) techniques (such as the 24sure assay) can enable this to occur without a reduction in IVF success rates. The authors suggest a strategy of trophectoderm biopsy, comprehensive chromosome screening, embryo vitrification, and frozen embryo transfer. In developing this strategy, the authors cite evidence that blastocyst biopsy is not detrimental to the embryo (whereas there is evidence that cleavage stage biopsy may be), that RCT evidence shows that chromosome screening improves success rates, and that frozen embryo transfer may also result in higher clinical pregnancy rates. Meeting and conference abstracts Hodes-Wertz B, Lee H-L, Adler A, Ampeloquio E, Clarke-Williams M, et al. (2013) Single euploid embryo transfer improves IVF pregnancy, miscarriage, and multiple gestation outcomes compared to national society for assisted reproductive technology database (SART) outcomes. Fertil Steril 100(3): S209. Morin, SJ, Melzer K, Grifo J, Munné S (2013) Single embryo transfer (SET) vs. double embryo transfer (DET) in preimplantation genetic screening (PGS) cycles: does size of euploid embryo cohort matter? Fertil Steril 100(3): S84. Sills, ES, Collins GS, Salem SA, Jones CA, Salem RD (2013) Array CGH and mandatory single fresh embryo transfer to normalize preterm delivery & multiple gestation outcomes with IVF: fiscal analysis of coverage expansion by a California state health exchange. Fertil Steril 100(3): S134. Whitney JB, Nugent N, Zozula S, Anderson RE, Schiewe MC (2013) Single embryo transfer (SET) yields equivalent clinical pregnancy rates and higher implantation rates compared to dual embryo transfer (DET) of euploid blastocysts. Fertil Steril 100(3): S Schoolcraft, WB, Katz-Jaffe MG. (2013) Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age. Fertil Steril 100(3): Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, et al. (2013) In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril 100(1): e1. 11

12 ACCURACY AND RELIABILITY Many studies demonstrate the accuracy and reliability of the 24sure assay in predicting aneuploidies in embryos. These studies have been carried out at all biopsy stages (polar body, blastomere, and blastocyst), (Table 2). For example, some studies have taken multiple biopsies at different time points and checked that results remain consistent, whereas others have used discarded embryos and analyzed and compared every cell separately. These studies are also useful in establishing how common mosaicism is, and at which stages of development it is most prevalent. Before the introduction of acgh, early attempts at PGS were carried out using FISH. This technique suffers from inherent problems when analyzing single cells, including difficulty interpreting signals that may be overlapping or split, and the inability to analyze all of the chromosomes. Currently, FISH is sometimes used to show that acgh provides a higher level of accuracy and can detect more abnormalities. 12

13 TECHNOLOGY 24sure technology is based on bacterial artificial chromosome (BAC) microarrays. These consist of a glass slide with thousands of single-stranded DNA probes bound to it. Each DNA probe hybridizes to a specific place on a specific chromosome. Patient DNA is fluorescently labeled and added to the glass slide. The probes bind to their specific targets in the patient DNA. Excess unbound DNA is washed off. A scanner scans the glass slide and detects how much fluorescence is present at each probe. The computer software uses the positional information for each probe to produce a chart, plotting the amount of fluorescence along each chromosome. Much less fluorescence in one area of a chromosome compared to other areas indicates a deletion. A greater amount of fluorescence at a certain point indicates a gain, such as a duplication. BAC microarray technology is sufficiently robust for applications such as the 24sure assay. It uses long probes, to which multiple fragments of sample DNA bind, which averages out the data for this region and reduces the effect of artifacts inherent in single cell samples. This produces a reliable test, with high accuracy and low failure rates. Handyside A (2013) 24-chromosome copy number analysis: a comparison of available technologies. Fertil Steril 100(3): This review article summarizes the key technologies that can be used for PGS. The advantages and disadvantages of each are discussed in the context of cost, scalability, biopsy stage, and whether they are appropriate as a screening test or a diagnostic test. acgh is widely discussed and is described as being reliable, accurate, and robust, which is why it is used extensively around the world. This paper builds on issues discussed previously as well as exploring newer technologies including karyomapping. Reviews Gresham D, Dunham MJ, Botstein D (2008) Comparing whole genomes using DNA microarrays. Nat Rev Genet 9(4): There are three stages of embryo development at which 24sure analysis can be carried out: polar body, cleavage stage (day 3), and blastocyst (day 5). The literature has many examples of successful application of the 24sure assay at all of these stages. There have also been studies that have compared all three to check for concordance. Each biopsy stage is associated with its own intrinsic advantages and disadvantages (Table 2), with no definitive answer as to which is best. Ultimately, the decision often depends on a number of factors individual to the lab itself such as lab workflow, experience in the different biopsy types, and local laws and regulations. 13

14 Table 2: Advantages and Disadvantages of 24sure Analysis at Each Stage of Embryo Development Polar body Advantages Doesn t involve removal of any material from the embryo itself May be the only legal option in some countries Provides sufficient time for analysis before transfer Is applicable to virtually all patients 8 Disadvantages Won t detect aneuploidies from paternal contribution Won t detect mitotic errors Requires large numbers of samples tested per cycle Cleavage stage (3 day) Is practiced most widely Tests both maternal and paternal contribution Provides sufficient time for analysis before transfer Is applicable for most patients Mosaicism is most common at this stage Viability of embryo may be affected in some cases Blastocyst (5 day) Has less mosaicism than at 3-day stage Tests more than one cell, reducing failure rate and improving the sensitivity in mosaics Tests both maternal and paternal contribution Has little or no impact on viability of embryo More time pressured in the context of a fresh transfer May not be appropriate for patients who are not expected to produce many blastocysts 14

15 POLAR BODY BIOPSY Most aneuploidies arise maternally in the oocyte and, therefore, can be detected at the earliest stages of embryo development through polar body testing. To do this, the 24sure assay analyzes both polar body 1 and polar body 2. The resulting copy number information is used to deduce the likely chromosome constitution of the egg itself. For example, a gain of a chromosome in the second polar body indicates an oocyte with a loss of the same chromosome. The technique of polar body biopsy has been well described in the literature and best practice guidelines drawn up by the European Society of Human Reproduction and Embryology (ESHRE) PGD consortium 11. The main advantage of polar body testing is that it is the biopsy method least likely to affect the embryo. Polar body cells do not form any part of the resulting embryo and are by-products of oocyte formation. Therefore, this method may be more ethically acceptable to the individual patient, or in some countries may be the only option available legally due to local regulations. It also provides the most time for analysis before embryo transfer. An inherent disadvantage in not testing the embryo itself is that any conclusions about the embryo have to be inferred, and are not definitive. Additionally, polar body testing will not pick up any aneuploidies due to other causes, such as paternal aneuploidies or mitotic errors. Nevertheless, many studies have used 24sure technology to show that the predictive value of this testing is high, and that it can improve IVF success rates significantly. Christopikou D, Tsorva E, Economou K, Shelley P, Davies S, et al. (2013) Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age. Hum Reprod 28(5): This paper shows that polar body biopsy and acgh analysis are efficient for accurate prediction of most aneuploidies in cleavage stage embryos. Almost all of the aneuploidies detected were associated with copy number changes in the polar bodies (93%) and all but one (98.5%) were predicted to be aneuploidy. References Geraedts J, Collins J, Gianaroli L, Goossens V, Handyside A, et al. (2010) What s next for preimplantation genetic screening? A polar body approach! Hum Reprod 25(3): Geraedts J, Montag M, Magli MC, Repping S, Handyside A, Staessen C, et al. (2011) Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results. Hum Reprod 26(11): Magli MC, Montag M, Köster M, Muzi L, Geraedts J, et al. (2011) Polar body arraycgh for prediction of the status of the corresponding oocyte. Part II: technical aspects. Hum Reprod 26(11): Fishel S, Craig A, Lynch C, Dowell K, Ndukwe G, et al. (2011) In vitro assessment of 19,803 paired chromosomes and clinical outcome from first 150 cycles using array CGH of the first polar body for embryo selection and transfer. J Fertil In Vitro 1(1): Harton GL, Magli MC, Lundin K, Montag M, Lemmen J, et al. (2011) ESHRE PGD Consortium/Embryology Special Interest Group-best practice guidelines for polar body and embryo biopsy. Hum Reprod 26(1):

16 CLEAVAGE STAGE BIOPSY At day three, the embryo is at cleavage stage. It consists of around eight cells. Biopsy at this stage involves the removal of just one (or at most two) cells. Traditionally, this is the most widely practiced biopsy method. A major advantage of testing at this stage is that paternal and mitotic aneuploidies will be detected with a comfortable amount of time available in which to complete testing before fresh embryo transfer. However, there is some evidence that biopsying at this stage may occasionally damage the embryo, and so biopsy requires a high level of expertise and experience to be performed safely. Additionally, embryos at this stage can be highly mosaic, so it is less certain that the single cell being tested is representative of the whole embryo. Despite this, the advantages in selecting euploid blastocysts outweigh the potential detrimental effects and biopsy at this stage still consistently produces significantly higher implantation and pregnancy rates than if biopsy were not performed at all. Keltz MD, Vega M, Sirota I, Lederman M, Moshier EL, et al. (2013) Preimplantation Genetic Screening (PGS) with Comparative Genomic Hybridization (CGH) following day 3 single cell blastomere biopsy markedly improves IVF outcomes while lowering multiple pregnancies and miscarriages. J Assist Reprod Genet 10: This study looks at pregnancy outcomes in IVF patients who underwent aneuploidy screening using the 24sure platform and compares them to a control group. In both age groups (< 35 and 35), 24sure analysis resulted in a significantly higher implantation rate, clinical pregnancy rate, and ongoing pregnancy rate. Across all ages, implantation rate more than doubled compared to controls (52.63% compared to 19.15%). These differences remained significant even though cycles with no euploid embryos to transfer were included. Rubio C, Rodrigo L, Mir P, Mateu E, Peinado V, et al. (2013) Use of array comparative genomic hybridization (array-cgh) for embryo assessment: clinical results. Fertil Steril 99(4): This review paper reports that the clinical outcome following array-based screening of all 24 chromosomes improved pregnancy rates for different indications to a higher degree than FISH. The authors find that most data regarding the controversy of day 3 biopsy comes from FISH cycles, and the utility of day 3 biopsy using array-based screening should be further validated through randomized control trials. Gutiérrez-Mateo C, Colls P, Sánchez-García J, Escudero T, Prates R, et al. (2011) Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos. Fertil Steril: 95(3): This study shows 24sure technology to be highly robust, specific, and able to provide results within 24 hours. In addition, the SurePlex DNA Amplification Kit used in the 24sure assay 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 acgh can detect 42% more abnormalities than the standard 12-probe FISH approach. Meeting and conference abstracts Mir P, Rodrigo L, Cervero A, Mercader A, Delgado A, et al. (2010) Validation of arraycgh on day-4 single blastomeres from day-3 embryos diagnosed as abnormal by FISH. In: Abstracts of the 26th Annual Meeting of ESHRE Human Reproduction 25 (suppl 1): i61-i63. Mir P, Rodrigo L, Mateu E, Peinado V, Milan-Sanchez M, et al. (2013) Re-analysis of whole day-5 embryos using the same arraycgh platform used for day-3 diagnosis showed a high confirmation rate. Hum Reprod 28 (suppl 1): i49 i51. 16

17 TROPHECTODERM BIOPSY At day 5, the embryo has formed a blastocyst consisting of an inner cell mass (which goes on to form the embryo), surrounded by the trophectoderm (a sphere of cells that form the supportive tissues such as the placenta). At this stage, a proportion of the mosaicism seen in cleavage-stage embryos has been eliminated 12,13. Biopsy at the blastocyst stage involves removal of a few of the trophectoderm cells. These do not go on to form the actual embryo, reducing the risk of the biopsy impacting embryo viability. Additionally, there are many more cells at this stage so more than one can be removed. Testing this small cluster of cells together increases the chance of detecting any mosaicism present at a level that could affect the embryo. As this is performed at day 5, there is still time for testing to occur before embryo transfer at day 6; albeit with more time pressure than at other stages. Alternatively, frozen transfer can take place, which may actually offer further improvements in success rate 14 and obstetric outcomes 15. A growing body of evidence is showing that trophectoderm biopsy is more advantageous than cleavage stage and polar body biopsy, with larger improvements in implantation rate being observed in comparative studies 16. Capalbo A, Wright G, Elliott T, Ubaldi FM, Rienzi L, et al. (2013) FISH reanalysis of inner cell mass and trophectoderm samples of previously array-cgh screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage. Hum Reprod 28(8): In this paper, embryos screened at the blastocyst stage using FISH on the inner cell mass and trophectoderm were analyzed using 24sure technology. Results show that the 24sure assay accurately predicts the ploidy of the embryo. Fragouli E, Alfarawati S, Daphnis DD, Goodall N-N, Mania A, et al. (2011) Cytogenetic analysis of human blastocysts with the use of FISH, CGH and acgh: scientific data and technical evaluation. Hum Reprod 26(2): 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 have previously been reported as a problem that can lead to misdiagnosis. The authors conclude that most mosaic blastocysts contained no normal cells. As such, trophectoderm acgh analysis is an appropriate tool for determining blastocyst aneuploidy. Mamas T, Gordon A, Brown A, Harper J, Sengupta S (2012) Detection of aneuploidy by array comparative genomic hybridization using cell lines to mimic a mosaic trophectoderm biopsy. Fertil Steril 97(4): This paper uses a cell culture system to imitate levels of mosaicism that may be present in an embryo. The level of aneuploidy was measured using 24sure technology in the mixed cell populations. It was determined that aneuploidy could be seen in cell populations where 25% of cells were aneuploid and confidently called where 50% of cells were aneuploid. 12. Mertzanidou A, Spits C, Nguyen HT, Van de Velde H, Sermon K (2013) Evolution of aneuploidy up to day 4 of human preimplantation development. Hum Reprod: 28(6): Evsikov S, Verlinsky Y (1998) Mosaicism in the inner cell mass of human blastocysts. Hum Reprod 13(11): Schoolcraft WB, Katz-Jaffe MG (2013) Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age. Fertil Steril 100(3): Maheshwar, A, Pandey S, Shetty A, Hamilton M, Bhattacharya S (2012) Obstetric and perinatal outcomes in singleton pregnancies resulting from the transfer of frozen thawed versus fresh embryos generated through in vitro fertilization treatment: a systematic review and meta-analysis. Fertil Steril 98(2): e Harton GL, Munné S, Surrey M, Grifo J, Kaplan B, McCulloh DH, et al. (2013) Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization. Fertil Steril 100(6):

18 Meeting and conference abstracts Anderson SH, Stankewicz-McKinney T, Glassner MJ, Hanshew K, Ketterson K, et al. (2013) Similar pregnancy and implantation rates following day 6 embryo transfer or frozen embryo transfer (FET) after blastocyst biopsy for preimplantation genetic screening (PGS). Fertil Steril 100(3): S37. Balmir F, Hughes M, Jenkins J, Stelling JR (2013) A pilot study comparing fluorescence in situ hybridization (FISH) analysis in preimplantation genetic screening (PGS) to array comparative genomic hybridization (acgh) technique. Fertil Steril: 100(3): S206. Colls P, Coates A, Peters A, Acacio B, Roche M, et al. (2013) Preimplantation genetic diagnosis at blastocyst stage by array comparative genomic hybridization. Error rate determination. Fertil Steril: 100(3): S196. Ribustello L, Escudero T, Margiatto E, Nagy P, Acacio BMD, et al. (2013) Differences between blastomere and blastocyst biopsies for preimplantation genetic diagnosis (PGD) using array comparative genomic hybridization (acgh). Fertil Steril 100(3): S130. Tobler KJ, Kaufmann R, Ross R, Dickey RP, Benner AT, et al. (2013) Preimplantation genetic screening (PGS) on day-5 blastocysts with a day-6 embryo transfer results in a clinical pregnancy rate of 71%. Fertil Steril 100(3): S198. Kearns WG (2013) PGS/PGD evaluation of 7059 embryos from 872 in vitro fertilization (IVF) cycles using 23-chromosome single nucleotide polymorphism (SNP) or 23-chromosome comparative genomic hybridization (acgh) microarrays. In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1). Harasim T, Roesemann M, Heinrich U, Wagner A, Schiller J, et al. (2013) Detection of genetic mosaicism during preimplantation genetic diagnosis (PGD). In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1). 18

19 OTHER SAMPLES One group used 24sure technology to show that embryonic DNA exists within the blastocoele fluid. This is sometimes removed before vitrification, and so would not involve any further handling of the embryo in order to obtain a sample for PGS. However, only limited evidence exists so far. More validation work is required before clinical use. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, et al. (2013) Genomic DNA in human blastocoele fluid. Reprod Biomed Online 26(6): This study explores the feasibility of using the DNA isolated from the blastocoele fluid of the blastocyst in a PGS test. Some clinics remove this fluid from the blastocyst cavity routinely, so it is essentially a by-product. This study uses real-time PCR to show that genomic DNA was present in about 90% of blastocoele fluid samples harvested during the vitrification procedure and successful amplification of specific genomic loci. In addition, whole-genome amplification of the isolated DNA was achieved and microarray comparative genomic hybridization was attempted to confirm the sex of embryos as well as to detect several aneuploidies. The authors conclude that further studies are needed to validate this approach and confirm that the accuracy is sufficient for diagnostic purposes. Meeting and conference abstracts Poli M, Jaroudi S, Sarasa J, Spath K, Child T, et al. (2013) The blastocoele fluid as a source of DNA for preimplantation genetic diagnosis and screening. Fertil Steril 100(3): S37. 19

20 CORRELATION BETWEEN ANEUPLOIDY AND MORPHOLOGY Traditionally, embryos were selected for transfer based on morphology alone. Characteristics such as how expanded the blastocyst is, the appearance of the inner cell mass, and hatching status were scored to give each embryo a final grade. These grades were used to select the best embryo for transfer, and further good quality embryos for freezing. Yet this technique is subjective and suffers from intrinsic inter-operator variation. Additionally, morphology is a poor predictor of embryo viability, and there is no guarantee that even the most morphologically perfect embryo will implant. With the use of 24sure technology, it soon became apparent that this was because aneuploidy rarely manifests morphologically. Even chromosomally chaotic embryos can appear morphologically normal, yet would clearly not produce a healthy pregnancy. In fact, around half of embryos with the best morphological scores are aneuploid 17. Recent innovations have made time-lapse imaging a possibility for embryo selection. Although this produces better predictive information than assessing the embryo at a single time point, studies indicate that it is still not indicative of aneuploidy, and PGS should still be carried out. This is entirely understandable when you consider that some aneuploidies (such as trisomy 21, trisomy 18, and trisomy 13) are compatible with live birth. Given this fact, there is little reason to suggest that they would cause obvious changes at such an early time point. Fragouli E, Alfarawati S, Spath K, Wells D (2014) Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos. Mol Hum Reprod 20(2): This study aimed to determine whether aneuploidy results in any detectable differences in embryo morphology. In total, 1,213 embryos were tested using the 24sure assay, and morphological characteristics of euploid and aneuploid embryos were compared at both the cleavage and blastocyst stages. Results show virtually no difference between the two groups at the cleavage stage. At the blastocyst stage, embryos with three or more aneuploidies tended to have poorer morphology and be less well developed; however, most clinically relevant abnormalities would be undetectable by morphology alone. Meeting and conference abstracts Melzer KE, Noyes N, Hodes-Wertz B, McCulloh D, Munné S, et al. (2013) How well do morphokinetic (MK) parameters and time-lapse microscopy (TLM) predict euploidy? A pilot study of TLM with trophectoderm (TE) biopsy with array comparative genomic hybridization (acgh). Fertil Steril 100(3): S209. Schneiderman A, Parks JC, Mitchell S, Stevens J, Schoolcraft WB, et al. (2013) Time lapse observation during embryo development differentiates blastocyst quality but not chromosome constitution or gender. Fertil Steril 100(3): S256. Cetinkaya M, Pirkevi C, Kumtepe Y, Beyazyurek C, Ekmekci CG, et al. (2013) Comparative kinetic evaluation of aneuploid and euploid embryos using time-lapse. In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1). Mazur P, Nagornyy V, Mykytenko D, Semeniuk L, Zukin V (2013) Time-lapse investigation of embryos with different types of aneuploidy. In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1). Olivares R, Lee H-L, Adler A, Grifo J, McCaffrey C (2013) Correlation between embryo development and morphology on blastocysts on day 5 with chromosome status analyzed by array comparative genomic hybridization (acgh) in patients younger than 37 years. In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1). 17. Alfarawat, S, Fragouli E, Colls P, Stevens J, Gutiérrez-Mateo C, et al. (2011) The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender. Fertil Steril 95(2):

21 EARLY EMBRYO DEVELOPMENT Before the introduction of 24sure technology, it was difficult to study aneuploidy in embryos. FISH was the primary method used, but it has many limitations, as described previously. 24sure technology facilitated a much more thorough investigation of the chromosomal constitution of the early embryo than was possible previously. This has enabled investigation of the origins of aneuploidy, as well as the frequency, and given us an insight into when, why, and how chromosomal abnormalities arise. For example, it was commonly thought that most aneuploidies arise through chromosomal non-disjunction. Yet, many independent studies have now shown that premature separation of sister chromatids is likely to be the most common mechanism. This finding was made possible by the ability to distinguish between single chromatids and whole chromosomes by analyzing the size of the log 2 ratio change in the acgh data. 24sure technology has also allowed more thorough investigation of mosaicism in the early embryo. Analysis of different stages of preimplantation embryo development has shown that mosaicism is common up to, and including, day 4 of development. By day 5, the number of mosaic embryos, and the level of mosaicism, is much reduced. The reason for this pattern has yet to be established, but this finding has clear implications for both PGD and PGS. 21

22 ANEUPLOIDY RATE It is well established that the incidence of aneuploidy rises with increasing maternal age. Further investigations have discovered that a significant proportion of embryos produced by younger patients is also aneuploid, with aneuploidy levels estimated at up to 50%, even for patients in the youngest age categories 18. Other studies have looked at whether there are increased aneuploidy levels in patients who, for example, are affected by a particular type of infertility, or have had a certain number of miscarriages, or whether aneuploidy rate correlates with the number of embryos produced. Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, Tan SL, et al. (2012) Array CGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod Biomed Online 24(6): This study analyzed existing 24sure data from Reprogenetics in North America. It provides up-to-date information on the frequency of aneuploidy across female ages for day 6 and day 3 biopsies. The authors conclude that the rate of aneuploidy is unrelated to the number of embryos generated per cycle. References Liu J, Wang W, Sun X, Liu L, Jin H, et al. (2012) DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic. Biol Reprod 87(6): 148. Meeting and conference abstracts Goldman KN, Hodes-Wertz B, McCulloh D, Grifo JA (2013) Euploidy is not associated with body mass index (BMI) in patients undergoing aneuploidy screening with trophectoderm (TE) biopsy and array comparative genomic hybridization (acgh). Fertil Steril 100(3): S80. Martinez E, Ariza M, Nogales M, Guillen A, Agudo D, et al. (2013) Rate of chromosomal chaotic embryos may be affected by the type of patient or clinical indication. In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1). Bronet F, Martinez E, Ariza M, Nogales M, Pareja S, et al. (2013) Embryo chaotic rate in thawed oocytes. In: Abstracts of the 12th International Conference on Preimplantation Genetic Diagnosis; Reprod Biomed Online 6 (suppl 1). 18. Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, Tan SL, et al. (2012) Array CGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod Biomed Online 24(6):

23 ORIGINS OF ANEUPLOIDY Until recently, it was thought that aneuploidies most commonly arise maternally through chromosomal non-disjunction during meiosis. Through careful analysis of 24sure data, it is possible to differentiate between the loss of a chromatid and the loss of a whole chromosome by looking at the degree to which the log 2 ratio shifts. As a result, it is becoming increasingly apparent that most aneuploidies arise through premature sister chromatid separation rather than chromosomal non-disjunction. Additionally, other studies have investigated the paternal incidence of aneuploidies. As 24sure technology is designed specifically for analysis of single cells, it can also be used to analyze the chromosomal constitution of sperm. This was demonstrated by a recent study that looked at the number of chromosomal abnormalities in men who had undergone chemotherapy compared to a control cohort. Capalbo A, Bono S, Spizzichino L, Biricik A, Baldi M, et al. (2013) Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum Reprod 28(2): This paper uses 24sure technology to analyze all PGS biopsy stages of an embryo (polar body, cleavage stage, and blastocyst) to review the incidence of aneuploidies at each time point. This approach enabled determination of the origin of aneuploidies, as well as calculation of the number of corrected aneuploidies and confirmation of the mechanism (e.g., trisomy rescue). This shows that the number of aneuploidies increased up to day 3 of embryo development, but then a proportion of these aneuploidies were corrected by the blastocyst stage. Additionally, it was demonstrated that maternally derived aneuploidies in this cohort of advanced maternal age patients were derived from chromatid missegregation at meiosis II. Gabriel AS, Thornhill AR, Ottolini CS, Gordon A, Brown A, et al. (2011) Array comparative genomic hybridisation on first polar bodies suggests that non-disjunction is not the predominant mechanism leading to aneuploidy in humans. J Med Genet 48(7): This article uses 24sure technology to address the question of whether aneuploidy arises from non-disjunction (whole chromosome segregation errors) or through precocious separation (sister chromatid segregation errors). The authors conclude that most aneuploidy arises from sister chromatid separation errors (92% compared to 8%), which is opposed to the accepted wisdom that non-disjunction is the primary mechanism leading to human aneuploidy. Handyside A, Montag M, Magli MC, Repping S, Harper J, et al. (2012). Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation. Eur J Hum Genet 20(7): This paper uses data compiled during the ESHRE PGS taskforce acgh proof-of-principle trial to determine when meiotic division errors occur. In 34 couples undergoing IVF, first and second polar bodies were examined for aneuploidy using 24sure technology. In cases where aneuploidy was predicted in the zygote, the corresponding zygote was also analyzed. Comparisons of aneuploidy in the three products of female meiosis enabled study of the incidence of aneuploidy in human oocytes at conception for the first time. It also identified in which meiotic division errors occurred and concluded that premature predivision of chromatids causes most errors in the first division. Patassini C, Garolla A, Bottacin A, Menegazzo M, Speltra E, et al. (2013) Molecular karyotyping of human single sperm by arraycomparative genomic hybridization. PLoS ONE 8(4): e This paper describes the first use of comprehensive chromosome screening on single sperm. It identifies differences in the number of abnormal sperm when comparing the chromosome complement from normal sperm with sperm isolated from men who had undergone chemotherapy. Additional validation of the technique was carried out through the analysis of single sperm from a carrier of a balanced translocation. 23