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The use of arrays in preimplantation genetic diagnosis and screening
MODERN TRENDS Edward E. Wallach, M.D. Associate Editor
The use of arrays in preimplantation genetic diagnosis and screening Joyce C. Harper, Ph.D.,a and Gary Harton, B.S.b a Reader in Human Genetics and Embryology, University College London Centre for Preimplantation Genetics and Diagnosis, Institute for Women’s Health, University College London and Centre for Reproductive and Genetic Health, Institute for Women’s Health, University College London Hospital, London, United Kingdom; and b Preimplantation Genetic Diagnosis, Genetics & IVF Institute, Fairfax, Virginia
Background: In preimplantation genetic diagnosis (PGD), polymerase chain reaction has been used to detect monogenic disorders, and in PGD/preimplantation genetic screening (PGS), fluorescence in situ hybridization (FISH) has been used to analyze chromosomes. Ten randomized controlled trials (RCTs) using FISH-based PGS on cleavage-stage embryos and one on blastocyst-stage embryos have shown that PGS does not increase delivery rates. Is the failure of PGS due to a fundamental flaw in the idea, or are the techniques that are being used unable to overcome their own, inherent flaws? Array-based technology allows for analysis of all of the chromosomes. Two types of arrays are being developed for use in PGD; array comparative genomic hybridization (aCGH) and single nucleotide polymorphism–based (SNP) arrays. Each array can determine the number of chromosomes, however, SNP-based arrays can also be used to haplotype the sample. Objective(s): To describe aCGH and SNP array technology and make suggestions for the future use of arrays in PGD and PGS. Conclusion(s): If array-based testing is going to prove useful, three steps need to be taken: [1] Validation of the array platform on appropriate cell and tissue samples to allow for reliable testing, even at the single-cell level; [2] deciding which embryo stage is the best for biopsy: polar body, cleavage, or blastocyst stage; [3] performing RCTs to show improvement in delivery rates. If RCTs are able to show that array-based testing at the optimal stage for embryo biopsy increases delivery rates, this will be a major step forward for assisted reproductive technology patients around the world. (Fertil Steril 2010;94:1173–7. 2010 by American Society for Reproductive Medicine.) Key Words: PGD, PGS, CGH, array CGH, SNP arrays, mosaicism, embryo biopsy, ART
PREIMPLANTATION GENETIC DIAGNOSIS Preimplantation genetic diagnosis (PGD) for inherited disorders was first successfully applied in 1990, allowing couples carrying a genetic abnormality to have a normal child (1, 2). PGD makes use of routine IVF procedures to generate embryos in vitro. Three different embryo development stages have been described: polar bodies removed from the oocyte/zygote (polar body biopsy), blastomeres removed from cleavage-stage embryos (cleavage stage biopsy), or trophectoderm cells removed from the blastocyst (blastocyst biopsy). The sample removed from the oocyte/zygote or growing embryo is then tested using either polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH). PCRReceived November 11, 2009; revised April 20, 2010; accepted April 26, 2010; published online June 25, 2010. J.C.H. has nothing to disclose. G.H. has nothing to disclose. Dr. Harton’s present address: Reprogenetics, 3 Regent Street, Suite 301, Livingston, NJ 07039. Reprint requests: Joyce Harper, UCL Centre for PG & D, Institute for Women’s Health, University College London, 86-96 Chenies Mews, London, WC1E 6HX, England (FAX: 00-44-207-383-7429; E-mail: joyce.harper@ ucl.ac.uk).
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based techniques are used to diagnose specific genetic disease, while FISH-based techniques have been used to analyze chromosomes for patients carrying chromosome abnormalities or for embryo sexing for patients carrying X-linked diseases. The European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium has collected data on the use of PGD over the last 10 years including the diseases that have been diagnosed and the success of the procedure (3).
PREIMPLANTATION GENETIC SCREENING (PGS) PGS was developed to try and improve pregnancy rates in certain groups of patients undergoing IVF procedures owing to infertility. Reported indications for PGS include patients with advanced maternal age, repeated miscarriage, repeated implantation failure, and severe male factor infertility (3). PGS was first reported by Verlinsky et al. (4) and Munne et al. (5) in the analysis of polar bodies. Since these first reports, there have been numerous papers on the use of PGS; however, as with many new technologies brought into the IVF clinic, there has been little evidence to show that PGS increases delivery rates. There are now 11 randomized controlled trials (RCTs) applied to both good- (6–10) and
Fertility and Sterility Vol. 94, No. 4, September 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
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poor- (11–16) prognosis patients. None of these trials have shown that PGS improved the delivery rate compared with a control group, while some have actually shown that PGS decreased the delivery rate. All of these studies used FISH-based techniques to study 5–12 chromosomes, and almost all were applied to cleavage-stage embryos, except Jansen et al. (8), who performed trophectoderm biopsy. The vast majority of randomized and nonrandomized studies on PGS have been performed with biopsy at the cleavage stage, which may be part of the reason for the poor performance of PGS so far. Embryos at this stage of development show high levels of chromosome abnormality and mosaicism (17, 18), therefore, analysis of one cell from a cleavage-stage embryo may not be representative of the genetic status of the whole embryo. In addition, almost all of these studies have used FISH-based testing of a limited number of chromosomes. Interphase FISH was developed and optimized for use on samples with many hundreds of cells to count, allowing for natural variations in hybridization efficiency, signal overlap, and other technical issues to be washed out over the whole analysis of the sample. While a few cells may have an aberrant count for a particular FISH probe due to technical issues, the majority of the cells will have the actual count allowing for diagnosis of the sample. FISH was not designed to test a sample set of one cell, therefore technical limitations of the test are magnified and will lead to false-positive and false-negative results, which will further affect the ability of FISH-based testing in PGS. It is no wonder that the above RCTs using FISH-based testing on cleavage-stage embryos have shown no improvements in delivery rates. Now is the time to assess the timing of biopsy and to find new methods that are able to analyze all of the chromosomes. Biopsy at the polar body or blastocyst stage and new testing methods including metaphase comparative genomic hybridization (m-CGH), array-CGH (a-CGH), and single nucleotide polymorphism (SNP) arrays must be investigated and rigorously tested, ushering in a new stage in PGD and PGS.
COMPARATIVE GENOMIC HYBRIDIZATION Comparative genomic hybridization (CGH) is a technique that bridges the gap between molecular genetics and cytogenetics (19). In CGH, DNA from the test sample and DNA from a normal control DNA are amplified separately using a whole genome amplification (WGA) approach such as degenerate oligonucleotide primer PCR (20, 21), primer extension preamplification (PEP) PCR (22), or newer methods such as iPEP (23), multiple displacement amplification (MDA) (24, 25), or GenomePlex (26). WGA application on single cells for PGD have been reported both for clinical and research purposes (27–32). To date, there does not seem to be a universal WGA that can be used for any arraybased platform. For CGH, the amplified DNA is differentially labeled with one of two fluorochromes, for example, red for the test DNA and green for the control DNA. After labeling, both DNAs are mixed together in equal proportions and are allowed to compete to hybridize to either metaphase spreads from a normal male control cell line (m-CGH) or onto an array platform containing small pieces of chromosome (a-CGH) (Fig. 1). In m-CGH, specialized computer software analyzes the ratio of red-to-green fluorescence along the length of each metaphase chromosome in about six different metaphase sets and plots the ratios on an ideogram. Gains in red indicate that the test sample is deficient in that region or chromosome, and gains in green indicate that the test has extra copies of that region or chromosome. The process is time-consuming and technically challenging, taking up
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to 72 hours to perform the laboratory bench work, with additional time required for the detailed analysis of the data. Array-CGH works on the same principal as m-CGH but uses a chip of DNA (usually bacterial artificial chromosomes [BACs]) affixed to the microscope slide instead of metaphase chromosomes and has been applied to single cells and embryos (25, 33, 34). Each BAC clone corresponds to a specific part of a chromosome and computer software analyzes the ratio of red and green fluorescence on each spot that will correspond to a loss or gain of that region, similar to m-CGH. The analysis is fully automated, and the whole procedure can be performed within 24 hours. For day 3 biopsy, it is possible to perform the ET on day 5 of embryo development in a fresh cycle. However, for some groups, especially those performing blastocyst biopsy and/or transport PGD, it may be preferable to freeze the biopsied embryos while the diagnosis and analysis is performed. Clinically, m-CGH has been applied in PGS patients at the polar body stage (35), the cleavage stage (36), and the blastocyst stage (37, 38). Owing to the time-consuming nature of m-CGH, the studies on cleavage-stage embryos and blastocysts required cryopreservation of embryos to allow time for genetic testing. Schoolcraft et al. (37) performed embryo biopsy at the blastocyst stage, with vitrification of all biopsied embryos during genetic testing. Chromosomally normal embryos were warmed and transferred in a subsequent controlled cycle. In this study, 45 patients underwent testing as outlined above. The main outcome measure of the study, implantation rate, was significantly increased in the study population when compared with a control set of patients that were well matched for most clinical parameters; however, there was a high multiple pregnancy rate (70%) and no increase in delivery rates. This same group is currently undertaking a three-armed RCT to further elucidate the benefits of testing using blastocyst biopsy and mCGH for comprehensive chromosome screening (CCS). Since mCGH takes several days to perform, embryo freezing is required. In this new study, the patients will be randomized into one of three groups: no treatment (day 5 blastocyst biopsy); day 5 blastocyst biopsy with CCS and vitrification of embryos for transfer in a subsequent, nonstimulated cycle; and no biopsy or testing but vitrification of all blastocysts on day 5 and day 6 with subsequent transfer in a nonstimulated cycle. In addition, Schoolcraft and colleagues are working on a number of follow-up papers to further discuss their data and findings (personal communication). Array-CGH is just beginning to be applied clinically to patients (39). In this study, a total of 41 embryos were assessed with a-CGH from eight PGS cycles, with pregnancy achieved in five of six cycles that had an ET. While CGH-based tests have been applied more rapidly in the assisted reproductive technology (ART) world than other array-based tests, there are some shortfalls of using CGH-based technology. Array-CGH cannot detect polyploidies, such as triploidies, as there is no imbalance in the total DNA content. In addition, CGH tests cannot detect balanced translocations or inversions as the total amount of DNA in the sample is the same as in the control sample. These systems cannot detect changes in DNA sequences (point mutations, intragenic insertions or deletions, triplet repeat expansion, etc.) or gains or losses in regions of the genome not covered by the array.
SINGLE NUCLEOTIDE POLYMORPHISM ARRAYS Single nucleotide polymorphisms (SNP) are areas of the genome in which a single nucleotide in the sequence varies within the population. Most SNPs are biallelic; they exist in either of two Vol. 94, No. 4, September 2010
FIGURE 1 (A) Metaphase CGH and (B) array CGH. First the biopsied material undergoes WGA and the embryonic DNA is labeled in green fluorescence. A control sample is labeled in red fluorescence. The samples are then cohybridized onto either (A) a metaphase spread or (B) an array platform. In both cases, a computer analyzes the ratio of red-to-green fluorescence. Courtesy of Thalia Mamas and Leoni Xanthopoulou, University College London Centre for PG&D.
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forms and occur over the entire genome. SNP arrays can use this information to distinguish one person from another and can be drilled down further to distinguish one chromosome from another in any person. A typical SNP array will have hundreds of thousands of SNPs to assess and, with the correct software, can distinguish how many of each chromosome was inherited by an embryo using both quantitative information and/or analyzing the expected combinations of inherited chromosomes and comparing them with the actual outcome (40). SNP arrays are based mainly in molecular genetics and rely on a WGA step to amplify the single cell or small number of cells removed from a developing embryo (34, 41, 42). Newer methods of performing WGA like MDA and GenomePlex are used for most applications of SNP arrays. These WGA methods tend to allow for better overall coverage of the genome and are less inclined to preferentially amplify some parts of the genome while leaving others unamplified or under amplified. After amplification, the DNA is labeled with red and green fluorescent molecules with one version of the SNP in red and the other version of the SNP in green. This DNA is then assessed for intensity of signal and number of SNP calls and compared with a control population by powerful computer software to allow for diagnosis of inheritance. SNP arrays are just beginning to be used clinically in PGD, although no publications have appeared in the literature as of yet. A Fertility and Sterility
number of groups around the world are currently validating SNP arrays and analysis software for clinical use in PGD. It is expected that clinical data from SNP arrays will closely match the data from CGH testing for PGS applications. While they use different technology to analyze the genome, both systems are essentially answering the same question: how many of each chromosome is present in a sample? SNP-based arrays offer other options for testing that are not available on CGH-based systems. SNP-based arrays, owing to the data that are collected and the way they are analyzed, allow for simultaneous testing of specific genetic diseases and aneuploidy in each embryo. This will allow for the selective transfer of genetically and chromosomally normal embryos for patients undergoing IVF with PGD for monogenic diseases. Simple haplotyping of SNPs surrounding and embedded in disease-causing genes allows for selection of embryos that have not inherited the affected chromosome. In addition, simple haplotyping of various markers around the genome allow for analysis of live-born children to determine which embryo or embryos implanted in any given ART cycle. This last test may help IVF centers develop technology to assess an embryo’s implantation potential that can be added on to tests for genetic and chromosomal normalcy, allowing for increased implantation and delivery rates in IVF centers around the world. With most countries moving toward single ET, a set of tests that pinpoints the one best embryo for transfer would be a welcome addition.
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ETHICAL ISSUES FOR SNP ARRAYS SNP arrays, owing to the vast amount of information gathered from each test, do pose the risk of additional ethical issues. Most scientists and clinicians agree that testing for inheritance of monogenic diseases is acceptable, and PGD has been proven to be an effective method to test embryos. More debate surrounds the idea of aneuploidy screening (43, 44, 45) and testing for inheritance of predisposition to disease (i.e., BRCA1 screening in embryos). SNP arrays could allow for analysis of other, less certain, inheritance patterns for multifactorial diseases such as diabetes as well as autism or heart disease. These types of tests will require much more validation and will certainly generate even more debate. While the data may be available from SNP arrays, we may not know enough about these conditions to safely test for them.
CLINICAL ISSUES While both platforms listed above offer advantages over current technology, they both also come with a set of issues that need to be overcome before they can be placed into widespread clinical use. Beyond the need for proof in an RCT of the efficacy (increased delivery rate vs. not testing) of testing embryos for aneuploidy, there are practical issues that cannot be ignored. To perform CCS on day 5 or day 6 embryos, embryology laboratories must be proficient at blastocyst culture. New formulations of medium for embryo culture have allowed many embryology labs to move toward longer development in vitro with transfer of blastocysts on day 5 of development becoming more practical, but by no means is this routine in all IVF centers. Beyond culture to the blastocyst stage, blastocyst biopsy is performed clinically in a small number of labs around the world at this time, therefore most IVF centers will most likely not be able to offer biopsy at this stage of embryo development. Training on blastocyst culture and biopsy by experts will happen, but it will take some time to develop widescale use in IVF centers around the world. Analysis using chip-based platforms can be performed in 24 hours. If polar body or cleavage-stage biopsy is used, this should allow ample time to perform the diagnosis. However, if blastocyst biopsy and/or transport PGD is used, laboratories will need to be proficient at cryopreserving embryos immediately after biopsy. Two different methods of cryopreservation are widely used in embryology labs, slow cooling and vitrification. Both methods seem to offer advantages and disadvantages when compared, and no real best method has been decided on at this time. The dogma of assisted reproduction is that fresh transfer of embryos is always better than frozen, however, with current techniques, the gap may be closing. As referenced above, one planned RCT using metaphase CGH will include an arm that looks at cryopreservation of untested embryos to determine the effect (perhaps benefit) of this part of recent studies. It could be argued that some of the success of donor egg IVF comes from the fact that the embryos are being transferred into a uterus that has not seen exogenous FSH and has been prepared and monitored for uterine lining development. None of the issues discussed above are insurmountable, however, they do offer a number of hurdles that must be cleared before blastocyst biopsy and array-based testing can become routine in any IVF center around the world.
WHAT NEXT FOR PGS? There are three questions we need to ask before applying any type of array clinically for PGS:
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1. Which type of array would be the best for PGD/PGS and have the arrays been validated on embryonic samples, including polar bodies, single blastomeres, and trophectoderm cells? 2. At what stage should we do the biopsy for PGS? The answer to this question seems to be pointing away from cleavage stage and toward polar body or trophectoderm biopsy. 3. Has an RCT shown that this technology coupled with the biopsy stage statistically improves delivery rates?
ESHRE PGS TASK FORCE—PILOT AND RCT ESHRE is undertaking its first ever clinical trial—a two-stage study developed to assess the efficacy of PGS using polar body biopsy and analysis of 24 chromosomes using array technology (45). The first part of the study aims to determine whether the technique of polar body biopsy and analysis of 24 chromosomes by array CGH is feasible. Any abnormal oocytes will also be examined to see whether the polar body abnormalities can really be used to predict that the oocyte will be abnormal. If the proof of principle part of the study is successful, the second part of the study will involve a multicenter RCT involving at least six centers in different European Union countries to determine whether PGS gives an increased delivery rate in women of advanced maternal age. It was decided to run the trial on polar bodies as this will allow for a fresh transfer, the biopsy is less invasive, and hopefully the results will be more reliable than cleavage or blastocyst biopsy as mosaicism will not affect the results.
CONCLUSION The scientific basis of aneuploidy screening, removing chromosomally abnormal embryos from the cohort of embryos available after IVF, seems to be sound; however, proof of the concept is lacking (44). Recent studies (37) have renewed hope that new techniques (testing of 24 chromosomes instead of 8–12) and a fresh look at the moment of biopsy (moving away from cleavage stage where mosaicism is rampant) may finally show the usefulness of aneuploidy screening. It remains to be seen, in an RCT, whether PGS will improve delivery rates over traditional IVF with transfer of the bestlooking embryo. If the RCTs show that PGS using arrays improves delivery rates, this will be great news for IVF patients. The cost of arraybased testing, which at this time is higher than conventional methods, needs to be taken into account and will hopefully continue to drop as most new technologies do. Timing of embryo biopsy, as well as the ability to grow blastocysts and vitrify or freeze them after biopsy, are hurdles some IVF centers will struggle to overcome. There are concerns about the slippery slope that the SNP-based testing may bring, however, the promise of a test that will be able to assess a cohort of embryos and allow the choice of the one best embryo for transfer will be a huge leap forward for IVF centers around the world. It is hoped that other IVF/PGD centers will undertake rigorous RCTs to validate the use of PGS and only use techniques in clinical practice with appropriate evidence-based data. There is a lesson to be learned from the application of PGS in the early days, its clinical use, and the debate that has followed the RCTs published to date (44).
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REFERENCES 1. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;244:768–70. 2. Harper JC. Preimplantation genetic diagnosis. Cambridge: Cambridge University Press, 2009. 3. Goossens V, Harton G, Moutou C, TraegerSynodinos J, Van Rij M, Harper JC. ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786–810. 4. Verlinsky Y, Cieslak J, Freidine M, Ivakhnenko V, Wolf G, Kovalinskaya L, et al. Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod 1995;10:1923–7. 5. Munne S, Dailey T, Sultan KM, Grifo J, Cohen J. The use of first polar bodies for preimpantation diagnosis of aneuploidy. Hum Reprod 1995;10:1015–21. 6. Staessen C, Verpoest W, Donoso P, Haentjens P, Van der Elst J, Liebaers I, et al. Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer. Hum Reprod 2008;23:2818–25. 7. Meyer LR, Klipstein S, Hazlett WD, Nasta T, Mangan P, Karande VC. A prospective randomized controlled trial of preimplantation genetic screening in the ‘‘good prognosis’’ patients. Fertil Steril 2009;91:1731–8. 8. Jansen RPS, Bowman MC, de Boer KA, Leigh DA, Lieberman DB, McArthur SJ. What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy. Hum Reprod 2008;23:1476–8. 9. Mersereau JE, Pergament E, Zhang X, Milad MP. Preimplantation genetic screening to improve in vitro fertilization pregnancy rates: a prospective randomized controlled trial. Fertil Steril 2008;90:1287–8. 10. Blockeel C, Schutyser V, De Vos A, Verpoest W, De Vos M, Staessen C, et al. Prospectively randomised controlled trial of PGS in IVF/ICSI patients with poor implantation. Reprod Biomed Online 2008;17: 848–54. 11. Staessen C, Platteau P, Van Assche E, Michiels A, Tournaye H, Camus M, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod 2004;19:2849–58. 12. Stevens J, Wale P, Surrey ES, Schoolcraft WB, Gardner DK. Is aneuploidy screening for patients aged 35 or over beneficial? A prospective randomized trial. Fertil Steril 2004;82:S249–S249. 13. Debrock S, Melotte C, Spiessens C, Peeraer K, Vanneste E, Meeuwis L, et al. Preimplantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial. Fertil Steril 2010;93: 364–73. 14. Hardarson T, Hanson C, Lundin K, Hillensj€o T, Nilsson L, Stevic J, et al. Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomised controlled trial. Hum Reprod 2008;23:2806–12. 15. Mastenbroek S, Twisk M, van Echten-Arends J, Sikkema-Raddatz B, Korevaar JC, Verhoeve HR, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007;357:9–17.
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16. Schoolcraft WB, Katz-Jaffe MG, Stevens J, Rawlins M, Munne S. Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial. Fertil Steril 2009;92:57–62. 17. Harper JC, Coonen E, Handyside AH, et al. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn 1995;15:41–9. 18. Munne S, Sultan KM, Weier HU, et al. Assessment of numeric abnormalities of X, Y, 18, and 16 chromosomes in preimplantation human embryos before transfer. Am J Obstet Gynecol 1995;172: 1191–9. 19. Kallioniemi A, Kallioniemi OP, Sudan D. Comparative genomic hybridisation for molecular cytogenetic analysis of solid tumours. Science 1992;258:818–21. 20. Telenius H, Carter NP, Bebb CE, Nordenskj€old M, Ponder BA, Tunnacliffe A. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 1992;13:718–25. 21. Hu DG, Webb G, Hussey N. Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization. Mol Hum Reprod 2004;10: 283–9. 22. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A 1992;89:5847–51. 23. Dietmaier W, Hartmann A, Wallinger S, Heinmoller E, Kerner T, Endl E, et al. Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am J Pathol 1999;154:83–95. 24. Dean FB, Hosono S, Fang L, et al. Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci U S A 2002;99:5261–6. 25. LeCaignec C, Spits C, Sermon K, De Rycke M, Thienpont B, Debrock S, et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res 2006;34:e68. 26. Fiegler H, Geigl JB, Langer S, Rigler D, Porter K, Unger K, et al. High resolution arrayCGH analysis of single cells. Nucleic Acids Res 2007;35:e15. 27. Wilton L, Voullaire L, Sargeant P, Williamson R, McBain J. Preimplantation aneuploidy screening using comparative genomic hybridization of fluorescence in situ hybridization of embryos from patients with recurrent implantation failure. Fertil Steril 2003;80:860–8. 28. Hellani A, Coskun S, Tbakhi A, Al-Hassan S. Clinical application of multiple displacement amplification in preimplantation genetic diagnosis. Reprod Biomed Online 2005;10:376–80. 29. Renwick PJ, Lewis CM, Abbs S, Ogilvie CM. Determination of the genetic status of cleavagestage human embryos by microsatellite marker analysis following multiple displacement amplification. Prenat Diagn 2007;27:206–15. 30. Lledo B, Ten J, Galan FM, Bernabeu R. Preimplantation genetic diagnosis of Marfan syndrome using multiple displacement amplification. Fertil Steril 2006;86:949–55.
31. Burlet P, Frydman N, Gigarel N, et al. Multiple displacement amplification improves PGD for fragile X syndrome. Mol Hum Reprod 2006;12:647–52. 32. Ren Z, Zhou C, Xu Y, Deng J, Zeng H, Zeng Y. Mutation and haplotype analysis for Duchenne muscular dystrophy by single cell multiple displacement amplification. Mol Hum Reprod 2007;13:431–6. 33. Fiegler H, Redon R, Andrews D, Scott C, Andrews R, Carder C, et al. Accurate and reliable high-throughput detection of copy number variation in the human genome. Genome Res 2006;16:1566–74. 34. Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, et al. Chromosome instability is common in human cleavage-stage embryos. Nat Med 2009;15:577–83. 35. Wells D, Escudero T, Levy B, Hirschhorn K, Delhanty JD, Munne S. First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril 2002;78:543–9. 36. Wilton L, Williamson R, McBain J, Edgar D, Voullaire L. Birth of a healthy infant after preimplantation confirmation of euploidy by comparative genomic hybridization. N Engl J Med 2001;345:1537–41. 37. Schoolcraft WB, Fragouli E, Stevens J, Munne S, KatzJaffe MG, Wells D. Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril 2009. [Epub ahead of print]. 38. Hellani A, Abu-Amero K, Azouri J, El-Akoum S. Successful pregnancies after application of arraycomparative genomic hybridization in PGSaneuploidy screening. Reprod Biomed Online 2008;17:841–7. 39. Handyside AH, Harton G, Mariani B, Thornhill AR, Affara NA, Shaw MA, et al. Karyomapping: a universal method for genome crossovers between parental haplotypes wide analysis of genetic disease based on mapping. J Med Genet 2009. [Epub ahead of print]. 40. Treff NR, Su J, Mavrianos J, Bergh PA, Miller KA, Scott RT Jr. Accurate 23 chromosome aneuploidy screening in human blastomeres using single nucleotide polymorphism (SNP) microarrays. Fertil Steril 2007;86:S217. 41. Kearns WG, Pen R, Benner A, Kittai A, Widra E, Leach R. SNP microarray genetic analyses to determine 23-chromosome ploidy, structural chromosome aberrations and genome-wide scans to identify disease risks from a single embryonic cell. Fertil Steril 2008;90:S23. 42. Harper JC, Sermon K, Geraedts J, et al. What next for preimplantation genetic screening. Hum Reprod 2008;23:478–80. 43. Harper JC, Coonen E, De Rycke M, Geraedts J, Goosens V, Harton G, et al. What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium steering committee. Hum Reprod 2010;25:821–3. 44. Munne S, Gianaroli L, Tur-Kaspa I, Magli C, Sandalinas M, Grifo J, et al. Substandard application of preimplantation genetic screening may interfere with its clinical success. Fertil Steril 2007;88:781–4. 45. Geraedts J, Collins J, Devroey P, Gianaroli L, Goossens V, Handyside A, et al. What next for preimplantation genetic screening? A polar body approach!. Hum Reprod 2010;25(3):575–7.
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The use of arrays in preimplantation genetic diagnosis and screening Joyce C. Harper, Ph.D.,a and Gary Harton, B.S.b a Reader in Human Genetics and Embryology, University College London Centre for Preimplantation Genetics and Diagnosis, Institute for Women’s Health, University College London and Centre for Reproductive and Genetic Health, Institute for Women’s Health, University College London Hospital, London, United Kingdom; and b Preimplantation Genetic Diagnosis, Genetics & IVF Institute, Fairfax, Virginia
Background: In preimplantation genetic diagnosis (PGD), polymerase chain reaction has been used to detect monogenic disorders, and in PGD/preimplantation genetic screening (PGS), fluorescence in situ hybridization (FISH) has been used to analyze chromosomes. Ten randomized controlled trials (RCTs) using FISH-based PGS on cleavage-stage embryos and one on blastocyst-stage embryos have shown that PGS does not increase delivery rates. Is the failure of PGS due to a fundamental flaw in the idea, or are the techniques that are being used unable to overcome their own, inherent flaws? Array-based technology allows for analysis of all of the chromosomes. Two types of arrays are being developed for use in PGD; array comparative genomic hybridization (aCGH) and single nucleotide polymorphism–based (SNP) arrays. Each array can determine the number of chromosomes, however, SNP-based arrays can also be used to haplotype the sample. Objective(s): To describe aCGH and SNP array technology and make suggestions for the future use of arrays in PGD and PGS. Conclusion(s): If array-based testing is going to prove useful, three steps need to be taken: [1] Validation of the array platform on appropriate cell and tissue samples to allow for reliable testing, even at the single-cell level; [2] deciding which embryo stage is the best for biopsy: polar body, cleavage, or blastocyst stage; [3] performing RCTs to show improvement in delivery rates. If RCTs are able to show that array-based testing at the optimal stage for embryo biopsy increases delivery rates, this will be a major step forward for assisted reproductive technology patients around the world. (Fertil Steril 2010;94:1173–7. 2010 by American Society for Reproductive Medicine.) Key Words: PGD, PGS, CGH, array CGH, SNP arrays, mosaicism, embryo biopsy, ART
PREIMPLANTATION GENETIC DIAGNOSIS Preimplantation genetic diagnosis (PGD) for inherited disorders was first successfully applied in 1990, allowing couples carrying a genetic abnormality to have a normal child (1, 2). PGD makes use of routine IVF procedures to generate embryos in vitro. Three different embryo development stages have been described: polar bodies removed from the oocyte/zygote (polar body biopsy), blastomeres removed from cleavage-stage embryos (cleavage stage biopsy), or trophectoderm cells removed from the blastocyst (blastocyst biopsy). The sample removed from the oocyte/zygote or growing embryo is then tested using either polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH). PCRReceived November 11, 2009; revised April 20, 2010; accepted April 26, 2010; published online June 25, 2010. J.C.H. has nothing to disclose. G.H. has nothing to disclose. Dr. Harton’s present address: Reprogenetics, 3 Regent Street, Suite 301, Livingston, NJ 07039. Reprint requests: Joyce Harper, UCL Centre for PG & D, Institute for Women’s Health, University College London, 86-96 Chenies Mews, London, WC1E 6HX, England (FAX: 00-44-207-383-7429; E-mail: joyce.harper@ ucl.ac.uk).
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based techniques are used to diagnose specific genetic disease, while FISH-based techniques have been used to analyze chromosomes for patients carrying chromosome abnormalities or for embryo sexing for patients carrying X-linked diseases. The European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium has collected data on the use of PGD over the last 10 years including the diseases that have been diagnosed and the success of the procedure (3).
PREIMPLANTATION GENETIC SCREENING (PGS) PGS was developed to try and improve pregnancy rates in certain groups of patients undergoing IVF procedures owing to infertility. Reported indications for PGS include patients with advanced maternal age, repeated miscarriage, repeated implantation failure, and severe male factor infertility (3). PGS was first reported by Verlinsky et al. (4) and Munne et al. (5) in the analysis of polar bodies. Since these first reports, there have been numerous papers on the use of PGS; however, as with many new technologies brought into the IVF clinic, there has been little evidence to show that PGS increases delivery rates. There are now 11 randomized controlled trials (RCTs) applied to both good- (6–10) and
Fertility and Sterility Vol. 94, No. 4, September 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
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poor- (11–16) prognosis patients. None of these trials have shown that PGS improved the delivery rate compared with a control group, while some have actually shown that PGS decreased the delivery rate. All of these studies used FISH-based techniques to study 5–12 chromosomes, and almost all were applied to cleavage-stage embryos, except Jansen et al. (8), who performed trophectoderm biopsy. The vast majority of randomized and nonrandomized studies on PGS have been performed with biopsy at the cleavage stage, which may be part of the reason for the poor performance of PGS so far. Embryos at this stage of development show high levels of chromosome abnormality and mosaicism (17, 18), therefore, analysis of one cell from a cleavage-stage embryo may not be representative of the genetic status of the whole embryo. In addition, almost all of these studies have used FISH-based testing of a limited number of chromosomes. Interphase FISH was developed and optimized for use on samples with many hundreds of cells to count, allowing for natural variations in hybridization efficiency, signal overlap, and other technical issues to be washed out over the whole analysis of the sample. While a few cells may have an aberrant count for a particular FISH probe due to technical issues, the majority of the cells will have the actual count allowing for diagnosis of the sample. FISH was not designed to test a sample set of one cell, therefore technical limitations of the test are magnified and will lead to false-positive and false-negative results, which will further affect the ability of FISH-based testing in PGS. It is no wonder that the above RCTs using FISH-based testing on cleavage-stage embryos have shown no improvements in delivery rates. Now is the time to assess the timing of biopsy and to find new methods that are able to analyze all of the chromosomes. Biopsy at the polar body or blastocyst stage and new testing methods including metaphase comparative genomic hybridization (m-CGH), array-CGH (a-CGH), and single nucleotide polymorphism (SNP) arrays must be investigated and rigorously tested, ushering in a new stage in PGD and PGS.
COMPARATIVE GENOMIC HYBRIDIZATION Comparative genomic hybridization (CGH) is a technique that bridges the gap between molecular genetics and cytogenetics (19). In CGH, DNA from the test sample and DNA from a normal control DNA are amplified separately using a whole genome amplification (WGA) approach such as degenerate oligonucleotide primer PCR (20, 21), primer extension preamplification (PEP) PCR (22), or newer methods such as iPEP (23), multiple displacement amplification (MDA) (24, 25), or GenomePlex (26). WGA application on single cells for PGD have been reported both for clinical and research purposes (27–32). To date, there does not seem to be a universal WGA that can be used for any arraybased platform. For CGH, the amplified DNA is differentially labeled with one of two fluorochromes, for example, red for the test DNA and green for the control DNA. After labeling, both DNAs are mixed together in equal proportions and are allowed to compete to hybridize to either metaphase spreads from a normal male control cell line (m-CGH) or onto an array platform containing small pieces of chromosome (a-CGH) (Fig. 1). In m-CGH, specialized computer software analyzes the ratio of red-to-green fluorescence along the length of each metaphase chromosome in about six different metaphase sets and plots the ratios on an ideogram. Gains in red indicate that the test sample is deficient in that region or chromosome, and gains in green indicate that the test has extra copies of that region or chromosome. The process is time-consuming and technically challenging, taking up
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to 72 hours to perform the laboratory bench work, with additional time required for the detailed analysis of the data. Array-CGH works on the same principal as m-CGH but uses a chip of DNA (usually bacterial artificial chromosomes [BACs]) affixed to the microscope slide instead of metaphase chromosomes and has been applied to single cells and embryos (25, 33, 34). Each BAC clone corresponds to a specific part of a chromosome and computer software analyzes the ratio of red and green fluorescence on each spot that will correspond to a loss or gain of that region, similar to m-CGH. The analysis is fully automated, and the whole procedure can be performed within 24 hours. For day 3 biopsy, it is possible to perform the ET on day 5 of embryo development in a fresh cycle. However, for some groups, especially those performing blastocyst biopsy and/or transport PGD, it may be preferable to freeze the biopsied embryos while the diagnosis and analysis is performed. Clinically, m-CGH has been applied in PGS patients at the polar body stage (35), the cleavage stage (36), and the blastocyst stage (37, 38). Owing to the time-consuming nature of m-CGH, the studies on cleavage-stage embryos and blastocysts required cryopreservation of embryos to allow time for genetic testing. Schoolcraft et al. (37) performed embryo biopsy at the blastocyst stage, with vitrification of all biopsied embryos during genetic testing. Chromosomally normal embryos were warmed and transferred in a subsequent controlled cycle. In this study, 45 patients underwent testing as outlined above. The main outcome measure of the study, implantation rate, was significantly increased in the study population when compared with a control set of patients that were well matched for most clinical parameters; however, there was a high multiple pregnancy rate (70%) and no increase in delivery rates. This same group is currently undertaking a three-armed RCT to further elucidate the benefits of testing using blastocyst biopsy and mCGH for comprehensive chromosome screening (CCS). Since mCGH takes several days to perform, embryo freezing is required. In this new study, the patients will be randomized into one of three groups: no treatment (day 5 blastocyst biopsy); day 5 blastocyst biopsy with CCS and vitrification of embryos for transfer in a subsequent, nonstimulated cycle; and no biopsy or testing but vitrification of all blastocysts on day 5 and day 6 with subsequent transfer in a nonstimulated cycle. In addition, Schoolcraft and colleagues are working on a number of follow-up papers to further discuss their data and findings (personal communication). Array-CGH is just beginning to be applied clinically to patients (39). In this study, a total of 41 embryos were assessed with a-CGH from eight PGS cycles, with pregnancy achieved in five of six cycles that had an ET. While CGH-based tests have been applied more rapidly in the assisted reproductive technology (ART) world than other array-based tests, there are some shortfalls of using CGH-based technology. Array-CGH cannot detect polyploidies, such as triploidies, as there is no imbalance in the total DNA content. In addition, CGH tests cannot detect balanced translocations or inversions as the total amount of DNA in the sample is the same as in the control sample. These systems cannot detect changes in DNA sequences (point mutations, intragenic insertions or deletions, triplet repeat expansion, etc.) or gains or losses in regions of the genome not covered by the array.
SINGLE NUCLEOTIDE POLYMORPHISM ARRAYS Single nucleotide polymorphisms (SNP) are areas of the genome in which a single nucleotide in the sequence varies within the population. Most SNPs are biallelic; they exist in either of two Vol. 94, No. 4, September 2010
FIGURE 1 (A) Metaphase CGH and (B) array CGH. First the biopsied material undergoes WGA and the embryonic DNA is labeled in green fluorescence. A control sample is labeled in red fluorescence. The samples are then cohybridized onto either (A) a metaphase spread or (B) an array platform. In both cases, a computer analyzes the ratio of red-to-green fluorescence. Courtesy of Thalia Mamas and Leoni Xanthopoulou, University College London Centre for PG&D.
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forms and occur over the entire genome. SNP arrays can use this information to distinguish one person from another and can be drilled down further to distinguish one chromosome from another in any person. A typical SNP array will have hundreds of thousands of SNPs to assess and, with the correct software, can distinguish how many of each chromosome was inherited by an embryo using both quantitative information and/or analyzing the expected combinations of inherited chromosomes and comparing them with the actual outcome (40). SNP arrays are based mainly in molecular genetics and rely on a WGA step to amplify the single cell or small number of cells removed from a developing embryo (34, 41, 42). Newer methods of performing WGA like MDA and GenomePlex are used for most applications of SNP arrays. These WGA methods tend to allow for better overall coverage of the genome and are less inclined to preferentially amplify some parts of the genome while leaving others unamplified or under amplified. After amplification, the DNA is labeled with red and green fluorescent molecules with one version of the SNP in red and the other version of the SNP in green. This DNA is then assessed for intensity of signal and number of SNP calls and compared with a control population by powerful computer software to allow for diagnosis of inheritance. SNP arrays are just beginning to be used clinically in PGD, although no publications have appeared in the literature as of yet. A Fertility and Sterility
number of groups around the world are currently validating SNP arrays and analysis software for clinical use in PGD. It is expected that clinical data from SNP arrays will closely match the data from CGH testing for PGS applications. While they use different technology to analyze the genome, both systems are essentially answering the same question: how many of each chromosome is present in a sample? SNP-based arrays offer other options for testing that are not available on CGH-based systems. SNP-based arrays, owing to the data that are collected and the way they are analyzed, allow for simultaneous testing of specific genetic diseases and aneuploidy in each embryo. This will allow for the selective transfer of genetically and chromosomally normal embryos for patients undergoing IVF with PGD for monogenic diseases. Simple haplotyping of SNPs surrounding and embedded in disease-causing genes allows for selection of embryos that have not inherited the affected chromosome. In addition, simple haplotyping of various markers around the genome allow for analysis of live-born children to determine which embryo or embryos implanted in any given ART cycle. This last test may help IVF centers develop technology to assess an embryo’s implantation potential that can be added on to tests for genetic and chromosomal normalcy, allowing for increased implantation and delivery rates in IVF centers around the world. With most countries moving toward single ET, a set of tests that pinpoints the one best embryo for transfer would be a welcome addition.
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ETHICAL ISSUES FOR SNP ARRAYS SNP arrays, owing to the vast amount of information gathered from each test, do pose the risk of additional ethical issues. Most scientists and clinicians agree that testing for inheritance of monogenic diseases is acceptable, and PGD has been proven to be an effective method to test embryos. More debate surrounds the idea of aneuploidy screening (43, 44, 45) and testing for inheritance of predisposition to disease (i.e., BRCA1 screening in embryos). SNP arrays could allow for analysis of other, less certain, inheritance patterns for multifactorial diseases such as diabetes as well as autism or heart disease. These types of tests will require much more validation and will certainly generate even more debate. While the data may be available from SNP arrays, we may not know enough about these conditions to safely test for them.
CLINICAL ISSUES While both platforms listed above offer advantages over current technology, they both also come with a set of issues that need to be overcome before they can be placed into widespread clinical use. Beyond the need for proof in an RCT of the efficacy (increased delivery rate vs. not testing) of testing embryos for aneuploidy, there are practical issues that cannot be ignored. To perform CCS on day 5 or day 6 embryos, embryology laboratories must be proficient at blastocyst culture. New formulations of medium for embryo culture have allowed many embryology labs to move toward longer development in vitro with transfer of blastocysts on day 5 of development becoming more practical, but by no means is this routine in all IVF centers. Beyond culture to the blastocyst stage, blastocyst biopsy is performed clinically in a small number of labs around the world at this time, therefore most IVF centers will most likely not be able to offer biopsy at this stage of embryo development. Training on blastocyst culture and biopsy by experts will happen, but it will take some time to develop widescale use in IVF centers around the world. Analysis using chip-based platforms can be performed in 24 hours. If polar body or cleavage-stage biopsy is used, this should allow ample time to perform the diagnosis. However, if blastocyst biopsy and/or transport PGD is used, laboratories will need to be proficient at cryopreserving embryos immediately after biopsy. Two different methods of cryopreservation are widely used in embryology labs, slow cooling and vitrification. Both methods seem to offer advantages and disadvantages when compared, and no real best method has been decided on at this time. The dogma of assisted reproduction is that fresh transfer of embryos is always better than frozen, however, with current techniques, the gap may be closing. As referenced above, one planned RCT using metaphase CGH will include an arm that looks at cryopreservation of untested embryos to determine the effect (perhaps benefit) of this part of recent studies. It could be argued that some of the success of donor egg IVF comes from the fact that the embryos are being transferred into a uterus that has not seen exogenous FSH and has been prepared and monitored for uterine lining development. None of the issues discussed above are insurmountable, however, they do offer a number of hurdles that must be cleared before blastocyst biopsy and array-based testing can become routine in any IVF center around the world.
WHAT NEXT FOR PGS? There are three questions we need to ask before applying any type of array clinically for PGS:
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1. Which type of array would be the best for PGD/PGS and have the arrays been validated on embryonic samples, including polar bodies, single blastomeres, and trophectoderm cells? 2. At what stage should we do the biopsy for PGS? The answer to this question seems to be pointing away from cleavage stage and toward polar body or trophectoderm biopsy. 3. Has an RCT shown that this technology coupled with the biopsy stage statistically improves delivery rates?
ESHRE PGS TASK FORCE—PILOT AND RCT ESHRE is undertaking its first ever clinical trial—a two-stage study developed to assess the efficacy of PGS using polar body biopsy and analysis of 24 chromosomes using array technology (45). The first part of the study aims to determine whether the technique of polar body biopsy and analysis of 24 chromosomes by array CGH is feasible. Any abnormal oocytes will also be examined to see whether the polar body abnormalities can really be used to predict that the oocyte will be abnormal. If the proof of principle part of the study is successful, the second part of the study will involve a multicenter RCT involving at least six centers in different European Union countries to determine whether PGS gives an increased delivery rate in women of advanced maternal age. It was decided to run the trial on polar bodies as this will allow for a fresh transfer, the biopsy is less invasive, and hopefully the results will be more reliable than cleavage or blastocyst biopsy as mosaicism will not affect the results.
CONCLUSION The scientific basis of aneuploidy screening, removing chromosomally abnormal embryos from the cohort of embryos available after IVF, seems to be sound; however, proof of the concept is lacking (44). Recent studies (37) have renewed hope that new techniques (testing of 24 chromosomes instead of 8–12) and a fresh look at the moment of biopsy (moving away from cleavage stage where mosaicism is rampant) may finally show the usefulness of aneuploidy screening. It remains to be seen, in an RCT, whether PGS will improve delivery rates over traditional IVF with transfer of the bestlooking embryo. If the RCTs show that PGS using arrays improves delivery rates, this will be great news for IVF patients. The cost of arraybased testing, which at this time is higher than conventional methods, needs to be taken into account and will hopefully continue to drop as most new technologies do. Timing of embryo biopsy, as well as the ability to grow blastocysts and vitrify or freeze them after biopsy, are hurdles some IVF centers will struggle to overcome. There are concerns about the slippery slope that the SNP-based testing may bring, however, the promise of a test that will be able to assess a cohort of embryos and allow the choice of the one best embryo for transfer will be a huge leap forward for IVF centers around the world. It is hoped that other IVF/PGD centers will undertake rigorous RCTs to validate the use of PGS and only use techniques in clinical practice with appropriate evidence-based data. There is a lesson to be learned from the application of PGS in the early days, its clinical use, and the debate that has followed the RCTs published to date (44).
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REFERENCES 1. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;244:768–70. 2. Harper JC. Preimplantation genetic diagnosis. Cambridge: Cambridge University Press, 2009. 3. Goossens V, Harton G, Moutou C, TraegerSynodinos J, Van Rij M, Harper JC. ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786–810. 4. Verlinsky Y, Cieslak J, Freidine M, Ivakhnenko V, Wolf G, Kovalinskaya L, et al. Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod 1995;10:1923–7. 5. Munne S, Dailey T, Sultan KM, Grifo J, Cohen J. The use of first polar bodies for preimpantation diagnosis of aneuploidy. Hum Reprod 1995;10:1015–21. 6. Staessen C, Verpoest W, Donoso P, Haentjens P, Van der Elst J, Liebaers I, et al. Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer. Hum Reprod 2008;23:2818–25. 7. Meyer LR, Klipstein S, Hazlett WD, Nasta T, Mangan P, Karande VC. A prospective randomized controlled trial of preimplantation genetic screening in the ‘‘good prognosis’’ patients. Fertil Steril 2009;91:1731–8. 8. Jansen RPS, Bowman MC, de Boer KA, Leigh DA, Lieberman DB, McArthur SJ. What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy. Hum Reprod 2008;23:1476–8. 9. Mersereau JE, Pergament E, Zhang X, Milad MP. Preimplantation genetic screening to improve in vitro fertilization pregnancy rates: a prospective randomized controlled trial. Fertil Steril 2008;90:1287–8. 10. Blockeel C, Schutyser V, De Vos A, Verpoest W, De Vos M, Staessen C, et al. Prospectively randomised controlled trial of PGS in IVF/ICSI patients with poor implantation. Reprod Biomed Online 2008;17: 848–54. 11. Staessen C, Platteau P, Van Assche E, Michiels A, Tournaye H, Camus M, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod 2004;19:2849–58. 12. Stevens J, Wale P, Surrey ES, Schoolcraft WB, Gardner DK. Is aneuploidy screening for patients aged 35 or over beneficial? A prospective randomized trial. Fertil Steril 2004;82:S249–S249. 13. Debrock S, Melotte C, Spiessens C, Peeraer K, Vanneste E, Meeuwis L, et al. Preimplantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial. Fertil Steril 2010;93: 364–73. 14. Hardarson T, Hanson C, Lundin K, Hillensj€o T, Nilsson L, Stevic J, et al. Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomised controlled trial. Hum Reprod 2008;23:2806–12. 15. Mastenbroek S, Twisk M, van Echten-Arends J, Sikkema-Raddatz B, Korevaar JC, Verhoeve HR, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007;357:9–17.
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16. Schoolcraft WB, Katz-Jaffe MG, Stevens J, Rawlins M, Munne S. Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial. Fertil Steril 2009;92:57–62. 17. Harper JC, Coonen E, Handyside AH, et al. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn 1995;15:41–9. 18. Munne S, Sultan KM, Weier HU, et al. Assessment of numeric abnormalities of X, Y, 18, and 16 chromosomes in preimplantation human embryos before transfer. Am J Obstet Gynecol 1995;172: 1191–9. 19. Kallioniemi A, Kallioniemi OP, Sudan D. Comparative genomic hybridisation for molecular cytogenetic analysis of solid tumours. Science 1992;258:818–21. 20. Telenius H, Carter NP, Bebb CE, Nordenskj€old M, Ponder BA, Tunnacliffe A. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 1992;13:718–25. 21. Hu DG, Webb G, Hussey N. Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization. Mol Hum Reprod 2004;10: 283–9. 22. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A 1992;89:5847–51. 23. Dietmaier W, Hartmann A, Wallinger S, Heinmoller E, Kerner T, Endl E, et al. Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am J Pathol 1999;154:83–95. 24. Dean FB, Hosono S, Fang L, et al. Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci U S A 2002;99:5261–6. 25. LeCaignec C, Spits C, Sermon K, De Rycke M, Thienpont B, Debrock S, et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res 2006;34:e68. 26. Fiegler H, Geigl JB, Langer S, Rigler D, Porter K, Unger K, et al. High resolution arrayCGH analysis of single cells. Nucleic Acids Res 2007;35:e15. 27. Wilton L, Voullaire L, Sargeant P, Williamson R, McBain J. Preimplantation aneuploidy screening using comparative genomic hybridization of fluorescence in situ hybridization of embryos from patients with recurrent implantation failure. Fertil Steril 2003;80:860–8. 28. Hellani A, Coskun S, Tbakhi A, Al-Hassan S. Clinical application of multiple displacement amplification in preimplantation genetic diagnosis. Reprod Biomed Online 2005;10:376–80. 29. Renwick PJ, Lewis CM, Abbs S, Ogilvie CM. Determination of the genetic status of cleavagestage human embryos by microsatellite marker analysis following multiple displacement amplification. Prenat Diagn 2007;27:206–15. 30. Lledo B, Ten J, Galan FM, Bernabeu R. Preimplantation genetic diagnosis of Marfan syndrome using multiple displacement amplification. Fertil Steril 2006;86:949–55.
31. Burlet P, Frydman N, Gigarel N, et al. Multiple displacement amplification improves PGD for fragile X syndrome. Mol Hum Reprod 2006;12:647–52. 32. Ren Z, Zhou C, Xu Y, Deng J, Zeng H, Zeng Y. Mutation and haplotype analysis for Duchenne muscular dystrophy by single cell multiple displacement amplification. Mol Hum Reprod 2007;13:431–6. 33. Fiegler H, Redon R, Andrews D, Scott C, Andrews R, Carder C, et al. Accurate and reliable high-throughput detection of copy number variation in the human genome. Genome Res 2006;16:1566–74. 34. Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, et al. Chromosome instability is common in human cleavage-stage embryos. Nat Med 2009;15:577–83. 35. Wells D, Escudero T, Levy B, Hirschhorn K, Delhanty JD, Munne S. First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril 2002;78:543–9. 36. Wilton L, Williamson R, McBain J, Edgar D, Voullaire L. Birth of a healthy infant after preimplantation confirmation of euploidy by comparative genomic hybridization. N Engl J Med 2001;345:1537–41. 37. Schoolcraft WB, Fragouli E, Stevens J, Munne S, KatzJaffe MG, Wells D. Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril 2009. [Epub ahead of print]. 38. Hellani A, Abu-Amero K, Azouri J, El-Akoum S. Successful pregnancies after application of arraycomparative genomic hybridization in PGSaneuploidy screening. Reprod Biomed Online 2008;17:841–7. 39. Handyside AH, Harton G, Mariani B, Thornhill AR, Affara NA, Shaw MA, et al. Karyomapping: a universal method for genome crossovers between parental haplotypes wide analysis of genetic disease based on mapping. J Med Genet 2009. [Epub ahead of print]. 40. Treff NR, Su J, Mavrianos J, Bergh PA, Miller KA, Scott RT Jr. Accurate 23 chromosome aneuploidy screening in human blastomeres using single nucleotide polymorphism (SNP) microarrays. Fertil Steril 2007;86:S217. 41. Kearns WG, Pen R, Benner A, Kittai A, Widra E, Leach R. SNP microarray genetic analyses to determine 23-chromosome ploidy, structural chromosome aberrations and genome-wide scans to identify disease risks from a single embryonic cell. Fertil Steril 2008;90:S23. 42. Harper JC, Sermon K, Geraedts J, et al. What next for preimplantation genetic screening. Hum Reprod 2008;23:478–80. 43. Harper JC, Coonen E, De Rycke M, Geraedts J, Goosens V, Harton G, et al. What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium steering committee. Hum Reprod 2010;25:821–3. 44. Munne S, Gianaroli L, Tur-Kaspa I, Magli C, Sandalinas M, Grifo J, et al. Substandard application of preimplantation genetic screening may interfere with its clinical success. Fertil Steril 2007;88:781–4. 45. Geraedts J, Collins J, Devroey P, Gianaroli L, Goossens V, Handyside A, et al. What next for preimplantation genetic screening? A polar body approach!. Hum Reprod 2010;25(3):575–7.
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