Development of new comprehensive

Transcription

1 Development and validation of an accurate quantitative real-time polymerase chain reaction based assay for human blastocyst comprehensive chromosomal aneuploidy screening Nathan R. Treff, Ph.D., a,b Xin Tao, M.Sc., a Kathleen M. Ferry, B.Sc., a Jing Su, M.Sc., a Deanne Taylor, Ph.D., a and Richard T. Scott Jr., M.D., H.C.L.D. a,b a Reproductive Medicine Associates of New Jersey, Morristown; and b Division of Reproductive Endocrinology, Department of Obstetrics, Gynecology, and Reproductive Science, University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, New Brunswick, New Jersey Objective: To develop and validate a quantitative real-time polymerase chain reaction (qpcr) based method for blastocyst trophectoderm comprehensive chromosome screening (CCS) of aneuploidy. Design: Prospective, randomized, and blinded. Setting: Academic center for reproductive medicine. Patient(s): Nine cell lines were obtained from a commercial cell line repository, and 71 discarded human blastocysts were obtained from 24 IVF patients that underwent preimplantation genetic screening. Intervention(s): None. Main Outcome Measure(s): Consistency of qpcr diagnosis of aneuploidy compared with either conventional karyotyping of cell lines or microarraybased diagnoses of human blastocysts. Result(s): Samples from nine cell lines with well characterized karyotypes were diagnosed by qpcr with 97.6% (41/42) consistency. After applying a minimum threshold for concurrence, 100% consistency was achieved. Developmentally normal blastocysts designated as aneuploid or arrested blastocysts designated as euploid by single-nucleotide polymorphism microarray analyses were assigned identical 24 chromosome diagnoses by qpcr in 98.6% of cases (70/71). Overall euploidy (n ¼ 37) and aneuploidy (n ¼ 34) were assigned with 100% consistency. Data was obtained for both sample types in 4 hours. Conclusion(s): These data demonstrate the first qpcr technology capable of accurate aneuploidy screening of all 24 chromosomes in 4 hours. This methodology provides an opportunity to evaluate trophectoderm biopsies with subsequent fresh euploid blastocyst transfer. Randomized controlled trials to investigate the clinical efficacy of qpcr-based CCS are currently underway. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Quantitative PCR, real-time PCR, aneuploidy screening, comprehensive chromosome screening Development of new comprehensive aneuploidy screening methodologies has renewed efforts to use preimplantation genetic analysis to enhance embryo selection, increase implantation rates, reduce the time to pregnancy, reduce multiple gestations, and reduce the incidence of miscarriage for couples with infertility. A variety of methodologies for comprehensive DNA quantificatation exist and include the incorporation of array Received November 30, 2011; revised January 4, 2012; accepted January 24, 2012; published online February 18, N.R.T. has nothing to disclose. X.T. has nothing to disclose. K.M.F. has nothing to disclose. J.S. has nothing to disclose. D.T. has nothing to disclose. R.T.S. has nothing to disclose. Reprint requests: Nathan R. Treff, Ph.D., 111 Madison Ave, Suite 100, Morristown, New Jersey ( ntreff@rmanj.com). Fertility and Sterility Vol. 97, No. 4, April /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert comparative genomic hybridization, metaphase comparative genomic hybridization, and single-nucleotide polymorphism (SNP) microarray technologies (1). In addition to variations in the array-based methods of probing preimplantation-stage DNA, a variety of methods exist for the initial whole genome amplification and have been shown to provide variable accuracy for aneuploidy screening and genotyping applications (2). The stage of development at which to perform aneuploidy screening also varies, including polar VOL. 97 NO. 4 / APRIL

2 bodies from the oocyte and zygote, blastomeres from the cleavage-stage embryo, and trophectoderm from the blastocyst (3). Although there are advantages and disadvantages to each of these components of aneuploidy screening, the amount of time required to perform comprehensive chromosome screening (CCS) by any of these methodologies typically exceeds 12 hours. Although this is less critical when applied to polar bodies or blastomeres, analysis of aneuploidy in the trophectoderm would likely require cryopreservation of the blastocyst to provide sufficient time for analysis while also preserving the appropriate synchrony between embryo and endometrial development (4). Although methods of blastocyst cryopreservation have improved, some risk remains, and many patients prefer to avoid the additional expense and time associated with a frozen embryo transfer cycle. To routinely perform comprehensive aneuploidy screening of the blastocyst without cryopreservation, more rapid methodologies need to be developed. Indeed, a more rapid method of preimplantation-stage DNA analysis, polymerase chain reaction (PCR), has been in clinical practice to manage patients with risk of transmitting monogenic disorders for >20 years (5). However, the ability to reliably diagnose aneuploidy of all 24 chromosomes with the use of PCR has not been established. Because of the initial success with PCR-based preimplantation genetic diagnosis (PGD), many improvements in the application of PCR have been made, including the incorporation of multiplexing, nesting, and fluorescence detection (6). Particularly noteworthy is the development of quantitative real-time PCR (qpcr) (7). Although qpcr has typically been applied to gene expression studies where relative transcript quantities are determined, there is also potential to evaluate the quantity of DNA in a given sample (8). Nevertheless, the ability of qpcr to quantify chromosomes in limited numbers of cells has yet to be demonstrated. The present study characterizes the accuracy of a novel method of 24 chromosome quantificatation in limited starting material as a preclinical step toward the application of a rapid CCS method in the diagnosis of human embryonic aneuploidy at the blastocyst stage of development. MATERIALS AND METHODS Experimental Design This study was conducted in two phases with emphasis on evaluating the technical variation of qpcr-based 24-chromosome copy number assignments by avoiding analysis of samples with potential biologic variation. In phase I, only cell lines with little to no evidence of mosaicism (biologic variation) for the previously well characterized whole-chromosome aneuploidies were used. In a similar attempt to avoid the impact of mosaicism in embryos in phase 2, only blastocysts with two consistent SNP microarray based diagnoses were reevaluated by qpcr. Randomized and blinded evaluation of consistency of qpcr with cell line karyotypes and embryo SNP microarray diagnoses were used as a measure of accuracy. Phase 1: Cell Lines Nine established and stable cell lines (fibroblasts and lymphocytes) were purchased from the Coriell Cell Repository (Camden, NJ) and cultured as recommended by the supplier. Included were GM09286 (47,XY,þ9), GM02948 (47,XY,þ13), GM04610 (47,XX,þ8[75]/46,XX,þ8,dic(14;21)(14qter > 14p 13::21p13 > 21qter)[25]), GM04435 (48,XY,þ16,þ21[45]/ 47,XY,þ21[5]), GM00323 (46,XY), AG16777 (47,XX,þ21 [21]/47,XX,þ21,t(21;22)(q22;q13)[29]), AG16778 (46,XX), AG16782 (46,XY), and GM01454 (47,XY,þ12[48]/47,XY, þ12,add(13)(q34)[52]). Earlier studies have indicated that the typical trophectoderm biopsy contains about five cells (9). To model this in evaluating cell lines, 5-cell samples were prepared by placement of five individual cells into a PCR tube under a dissecting microscope, as previously described (10). Lymphocyte lines were prepared directly and fibroblast lines after trypsin EDTA treatment. Seven 5-cell samples from cell line GM00323 were used to serve as a reference dataset to interpret results from 42 randomized and blinded 5-cell test samples (GM00323, n ¼ 10; GM09286, n ¼ 4; AG16777, n ¼ 5; AG16778, n ¼ 3; AG16782; n ¼ 3; GM01454, n ¼ 5; GM02948, n ¼ 5; GM04610, n ¼ 5; and GM04435, n ¼ 2), as described subsequently. Randomization was performed using Microsoft Excel to avoid potential bias from sequential analysis of multiple samples from the same cell line. The identification of the origins of each sample was blinded by using decoded sample names created in Microsoft Excel. The amount of time to complete the procedure was recorded for each sample. Phase 2: Embryos Seventy-one embryos were included in this study. All embryos had two consistent SNP microarray based aneuploidy screening results of trophectoderm biopsies (from days 5 and 6), as previously described (9, 11, 12). Thirty-seven of the 71 embryos included in this study had arrested by day 6 and were subsequently found to be euploid by SNP microarray analysis. The remaining 34, despite developmental normalcy on day 6, were found to possess aneuploidy by SNP microarray analysis. A third biopsy of each of the 71 day 6 embryos was randomized, blinded, and evaluated by qpcr with the same seven 5-cell reference sample set used in the cell line study described above. Again, randomization and blinding was performed in Microsoft Excel to avoid interpretation bias. The amount of time to complete the procedure was recorded for each embryo. qpcr Cell line 5-cell samples and embryo biopsies were processed by alkaline lysis as previously described (13). Multiplex amplification of 96 loci (four for each chromosome, as previously described [14]) was performed with the use of TaqMan Copy Number Assays and TaqMan Preamplification Master Mix as recommended by the supplier (Applied Biosystems), and in a 50-mL reaction volume for 18 cycles using an Applied Biosystems 2720 thermocycler. Real-time PCR was performed in quadruplicate for each of the individual 96 loci using TaqMan Gene Expression Master Mix (Applied Biosystems), a 5-mL reaction volume, a 384-well plate, and a 7900 HT sequence detection system, as recommended by the supplier (Applied Biosystems). A unique method of the standard delta delta threshold cycle (ΔΔC t ) method of relative quantitation (15) 820 VOL. 97 NO. 4 / APRIL 2012

3 Fertility and Sterility was applied. First, a chromosome-specific ΔC t was calculated from the average C t of the 16 reactions targeting a specific chromosome (four replicates of four loci) minus the average C t of all of the 336 reactions targeting all of the remaining autosomes (four replicates of four loci of 21 remaining autosomes). The same process was used to individually determine the ΔC t for each of the 24 chromosomes in the test sample. Each chromosome-specific ΔC T was then normalized to the average chromosome specific ΔC t values derived from the same evaluation of seven normal male (GM00323) 5-cell samples (reference set). The resulting chromosome-specific ΔΔC t values were used to calculate fold change by considering the ΔΔC t values as the negative exponent of 2, as previously described (15). All autosome fold changes were then multiplied by 2, whereas the sex chromosome fold changes were used as is, to determine the 24-chromosome copy number in each sample. This methodology was designed to specifically identify whole-chromosome but not segmental aneuploidy. FIGURE 1 Statistics Sample specific concurrence. To evaluate the utility of a previously established strategy for identifying poorquality data independent of knowing its accuracy (12), the overall concurrence was calculated for each sample. In this analysis, it is first assumed that the qpcr assay can assess only whole-chromosome aneuploidy, such that the four copy number assignments within each chromosome should always agree. Therefore, the standard deviation of the four measurements of copy number for each chromosome was calculated. The standard deviations of each of the 24 chromosomes were then averaged for each sample. Outliers (nonconcurrent samples) were defined as samples found outside an interquartile range of 1.5 from the overall distribution of average sample-specific standard deviations for each sample type as determined with the use of Analyse-It software for Microsoft Excel. Means and variations of the rates of concurrence in cell lines and embryos were evaluated for significance with a Student t test and an F test, respectively. Consistency of diagnosis. Consistency of the cell line 5-cell samples qpcr-based 24-chromosome copy number predictions with the cell lines karyotype (previously established by the Coriell Cell Repository by conventional karyotyping) was evaluated at the level of individual chromosome copy numbers and for the entire 24 chromosomes of each sample tested. Consistency of embryo qpcr-based 24-chromosome copy number assignments with previously established SNP microarray based diagnoses was also evaluated at the level of individual chromosome copy numbers for the entire 24 chromosomes of each sample tested and for the overall diagnosis of aneuploidy or euploidy. Results were evaluated with and without the application of a threshold of concurrence as described above. RESULTS Phase 1: Cell Lines Forty-two randomized blinded samples were evaluated for 24-chromosome copy number and compared for consistency Examples of qpcr-based 24-chromosome copy number results from 5-cell samples derived from nine cell lines with previously well characterized karyotypes. with the cell lines karyotype previously determined by conventional g-banding at the commercial provider s laboratory. Examples of qpcr results for 5-cell samples from the cell lines are shown in Figure 1. One of the samples (GM00323; 46,XY) produced a false positive trisomy 18, giving an overall consistency of chromosome copy number assignment of 99.90% (1,007/1,008) and an overall 24-chromosome diagnosis consistency of 97.6% (41/42). There were no false negative VOL. 97 NO. 4 / APRIL

4 FIGURE 2 FIGURE 3 Box-whisker plots representing the distribution of average 24- chromosome four-loci copy number standard deviations for each of the 42 cell line samples and 71 blastocyst biopsies. For each sample type, one outlier was identified, including the only cell line sample with an inconsistent qpcr diagnosis. diagnoses for aneuploid chromosomes or inaccurate predictions of gender. Analysis of concurrence identified the only discordant cell line sample as the only outlier (i.e., nonconcurrent; Fig. 2). Therefore, by applying a threshold of concurrence, the cell line study resulted in 97.6% reliability of obtaining a diagnosis and a 100% level of consistency of chromosome-specific (n ¼ 984) and 24-chromosome copy number (n ¼ 41) assignments. The amount of time taken to complete the procedure for each sample was 4 hours. Phase 2: Embryos Seventy-one embryos with consistent SNP microarray based 24 chromosome aneuploidy screening results from 2 biopsies were rebiopsied, randomized, and blinded for analysis of consistency of qpcr-based diagnoses. These were selected to reduce the risk of mosaicism. Examples of embryo biopsy qpcr results are shown in Figure 3, and the details of karyotype predictions are included in Supplemental Table 1 (available online at In one embryo, consistently diagnosed as 45,XY, 13, 14,þ18 by SNP microarray analysis of two biopsies, qpcr failed to detect monosomy 14. All of the remaining chromosomes for all of the remaining samples were consistent between qpcr and microarray, giving an overall chromosome-specific consistency of 99.94% (1,703/ 1,704) and an overall 24-chromosome diagnosis consistency of 98.6% (70/71). There were no false positive aneuploid chromosomes observed or inaccurate predictions of sex. The overall rate of concurrence in cell lines from phase 1 was equivalent to the rate of concurrence in embryos in phase 2 (P¼.96). The variation in concurrence rates within cell lines and embryos was also equivalent (P¼.34). Analysis of concurrence identified only one embryo sample as an outlier (i.e., nonconcurrent; Fig. 2). However, this was not the sample with the false negative monosomy 14, and therefore the Examples of (gray) single-nucleotide polymorphism microarray and (white) qpcr-based 24-chromosome copy number results from blastocyst-stage embryo biopsies. consistency of the embryo results was the same with or without applying a threshold for concurrence. Because the only false negative aneuploidy diagnosis occurred in an embryo with other consistently diagnosed aneuploidies (monosomy 13 and trisomy 18), the overall qpcr-based diagnosis of aneuploidy or euploidy was 100% consistent with SNP 822 VOL. 97 NO. 4 / APRIL 2012

5 Fertility and Sterility microarray based predictions. The amount of time taken to complete the procedure for each sample was 4 hours. DISCUSSION Results of the present study have demonstrated the validity of a new 4-hour method for CCS in human blastocysts. The technical accuracy was measured in two phases. The first phase involved the use of cell lines with previously well characterized karyotypes. Although it is possible for biologic variation of cell line karyotypes to exist as a result of extended culture (16, 17) or from unidentified low-level mosaicism in the original sample used to create the cell line, the potential impact of these biologic artifacts can be avoided by the use of early passages of cell lines that show little to no evidence of mosaicism by conventional karyotyping. With this strategy we demonstrated a consistency of qpcr-based CCS of concurrent 5-cell samples of 100%. To evaluate a more relevant tissue type, the second phase of the study involved the evaluation of discarded human embryos. Because the presence of mosaicism in embryos as a result of postzygotic mitotic aneuploidy development represents a well documented phenomenon that could contribute to biologic variation in blastocysts (11, 18), we selected embryos which specifically demonstrated consistent SNP microarray diagnoses from 2 biopsies. This approach may help reduce the impact of mosaicism and biologic variation on evaluating the technical accuracy of new methods such as qpcr. Indeed, analysis of these well controlled blastocysts by qpcr demonstrated 98.6% 24- chromosome consistency with the highly validated method of SNP microarray based aneuploidy screening (12). Importantly, all SNP microarray based euploid embryos were diagnosed as euploid and all SNP microarray based aneuploid embryos as aneuploid by qpcr (100% laboratory diagnostic consistency). Furthermore, because trophectoderm biopsies may not all possess five cells as modeled in phase I, the results of evaluating actual trophectoderm biopsies in phase II provides additional evidence of validity to samples with variable and potentially fewer numbers of cells. Another important observation regarding the performance of this qpcr methodology was the equivalent levels of concurrence measured in cell lines and embryos (Fig. 2). It has been suggested that PGD-based assays typically perform differently on different cell types (i.e., lymphocytes, fibroblasts, and embryonic cells) (19). Given the high degree of similarity in performance between cell lines and embryos in the present study, qpcr-based aneuploidy screening appears to be a robust methodology independent of the cell type. This may be in part due to the use of locus-specific multiplex PCR rather than whole-genome amplification for the initial processing of the sample. It is also possible that the use of trophectoderm biopsies, which may possess more than five cells, provided an advantage compared with the use of five lymphocytes or fibroblasts for providing consistent copy number assignments across each chromosome. The same advantage might be expected when comparing concurrence of trophectoderm with either blastomeres or polar bodies where less template DNA is present. Although this method was not applied to blastomeres or polar bodies (single cells), it is theoretically possible. In addition, this methodology could also be applicable to evaluating segmental aneuploidies associated with inheritance of unbalanced translocations by simply adding specific assays targeting positions on either side of the breakpoints of the chromosomes involved. Finally, one important challenge that should be considered is the need to process multiple embryos in parallel. Although this certainly involves an additional expense (a limitation on its own), the procurement of multiple thermal cyclers and the use of standard laboratory automation solutions can be used to completely circumvent this challenge. In conclusion, with these measures of accuracy in place and the fact that this protocol can be accomplished within 4 hours of receiving a biopsy, this qpcr-based methodology provides the first opportunity for same-day trophectoderm biopsy 24-chromosome aneuploidy screening and fresh blastocyst transfer. Given the level of consistency with an established method of aneuploidy screening that has also demonstrated excellent predictive value for clinical outcome (20), this qpcr method can now be justifiably evaluated for clinical efficacy in a randomized controlled trial (RCT). Indeed, preliminary RCT results of 24-chromosome aneuploidy screening with qpcr on trophectoderm biopsies and subsequent fresh euploid blastocyst transfer indicate a significant increase in the success of IVF (21). REFERENCES 1. Harper JC, Harton G. The use of arrays in preimplantation genetic diagnosis and screening. Fertil Steril 2010;94: Treff NR, Su J, Tao X, Northrop LE, Scott RT Jr. Single-cell whole-genome amplification technique impacts the accuracy of SNP microarray-based genotyping and copy number analyses. Mol Hum Reprod 2011;17: Delhanty JD. Is the polar body approach best for pre-implantation genetic screening? Placenta 2011;32(Suppl 3):S van Voorhis BJ, Dokras A. Delayed blastocyst transfer: is the window shutting? Fertil Steril 2008;89: Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344: Harton GL, De Rycke M, Fiorentino F, Moutou C, SenGupta S, Traeger- Synodinos J, et al. ESHRE PGD consortium best practice guidelines for amplification-based PGD. Hum Reprod 2010;26: Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 1993;11: D Haene B, Vandesompele J, Hellemans J. Accurate and objective copy number profiling using real-time quantitative PCR. Methods 2010;50: Schoolcraft WB, Treff NR, Stevens JM, Ferry K, Katz-Jaffe M, Scott RT Jr. Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based comprehensive chromosome screening in infertile patients. Fertil Steril 2011;96: Treff NR, Su J, Tao X, Miller KA, Levy B, Scott RT Jr. A novel single-cell DNA fingerprinting method successfully distinguishes sibling human embryos. Fertil Steril 2009;94: Northrop LE, Treff NR, Levy B, Scott RT Jr. SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts. Mol Hum Reprod 2010;16: Treff NR, Su J, Tao X, Levy B, Scott RT Jr. Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays. Fertil Steril 2010;94: Cui XF, Li HH, Goradia TM, Lange K, Kasasian HH Jr, Galas D, et al. Single-sperm typing: determination of genetic distance between the G gamma-globin and VOL. 97 NO. 4 / APRIL

6 parathyroid hormone loci by using the polymerase chain reaction and allelespecific oligomers. Proc Natl Acad Sci U S A 1989;86: Treff NR, Tao X, Su J, Lonczak A, Northrop LE, Ruiz A, et al. Tracking embryo implantation using cell-free fetal DNA enriched from maternal circulation at 9 weeks gestation. Mol Hum Reprod 2011;17: Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc 2008;3: Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, et al. Genomic alterations in cultured human embryonic stem cells. Nat Genet 2005;37: Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 2004;22: Fragouli E, Wells D. Aneuploidy in the human blastocyst. Cytogenet Genome Res Glentis S, SenGupta S, Thornhill A, Wang R, Craft I, Harper JC. Molecular comparison of single cell MDA products derived from different cell types. Reprod Biomed online 2009;19: Scott RT Jr, Ferry K, Su J, Tao X, Scott K, Treff NR. Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, nonselection study. Fertil Steril doi: /j.fertnstert Scott RT Jr, Tao X, Taylor D, Ferry K, Treff N. A prospective randomized controlled trial demonstrating significantly increased clinical pregnancy rates following 24 chromosome aneuploidy screening: biopsy and analysis on day 5 with fresh transfer. Fertil Steril 2010;94:S VOL. 97 NO. 4 / APRIL 2012

7 Fertility and Sterility SUPPLEMENTAL TABLE 1 Results of SNP microarray and randomized blinded qpcr analysis of 24-chromosome aneuploidy screening in 71 blastocysts. Patient no. Embryo no. SNP microarray result 1 SNP microarray result 2 qpcr result ,XY 46,XY 46,XY ,XX, 8 45,XX, 8 45,XX, ,XX 46,XX 46,XX ,XY 46,XY 46,XY ,XX, 3 45,XX, 3 45,XX, ,XY 46,XY 46,XY ,XX 46,XX 46,XX ,XY 46,XY 46,XY ,XX 46,XX 46,XX ,XX,þ14 47,XX,þ14 47,XX,þ ,XX, 16, 17 44,XX, 16, 17 44,XX, 16, ,XX 46,XX 46,XX ,XX,þ11, 17 46,XX,þ11, 17 46,XX,þ11, ,XY,þ16 47,XY,þ16 47,XY,þ ,XX 46,XX 46,XX ,XX, 10 45,XX, 10 45,XX, ,XX 46,XX 46,XX ,XY,þ15 47,XY,þ15 47,XY,þ ,XY, 9 45,XY, 9 45,XY, ,XX,þ13 47,XX,þ13 47,XX,þ ,XX 46,XX 46,XX ,XX 46,XX 46,XX ,XY 46,XY 46,XY ,XX, 9, 22 44,XX, 9, 22 44,XX, 9, ,XY, 7 45,XY, 7 45,XY, ,XX,þ22 47,XX,þ22 47,XX,þ ,XY 46,XY 46,XY ,XY, 16 45,XY, 16 45,XY, ,XX,þ2, 15,þ19 47,XX,þ2, 15,þ19 47,XX,þ2, 15,þ ,XX 46,XX 46,XX ,XX, 15 45,XX, 15 45,XX, ,OY 45,OY 45,OY ,XY,þ1 47,XY,þ1 47,XY,þ ,XY 46,XY 46,XY ,XX, 14 45,XX, 14 45,XX, ,XY, 21, 22 44,XY, 21, 22 44,XY, 21, ,XY 46,XY 46,XY ,XX, 15,þ16, 22 45,XX, 15,þ16, 22 45,XX, 15,þ16, ,XX 46,XX 46,XX ,XY 46,XY 46,XY ,XX 46,XX 46,XX ,XY 46,XY 46,XY ,XY 46,XY 46,XY ,XY 46,XY 46,XY ,XX 46,XX 46,XX ,XX,þ7 47,XX,þ7 47,XX,þ ,XX 46,XX 46,XX ,XX, 4, 16, 22 43,XX, 4, 16, 22 43,XX, 4, 16, ,XY 46,XY 46,XY ,XY, 16 45,XY, 16 45,XY, ,XX,þ9 47,XX,þ9 47,XX,þ ,XY,þ7, 10, 22 45,XY,þ7, 10, 22 45,XY,þ7, 10, ,XX, 1,þ6,þ7,þ9,þ17 49,XX, 1,þ6,þ7,þ9,þ17 49,XX, 1,þ6,þ7,þ9,þ ,XX,þ16 47,XX,þ16 47,XX,þ ,XY, 13, 14,þ18 45,XY, 13, 14,þ18 46,XY, 13,þ18 a ,XX 46,XX 46,XX ,XY, 6,þ19, 21 45,XY, 6,þ19, 21 45,XY, 6,þ19, ,XY,þ18,þ22 48,XY,þ18,þ22 48,XY,þ18,þ ,XY,þ20 47,XY,þ20 47,XY,þ ,XX, 16 45,XX, 16 45,XX, ,XY, 12 45,XY, 12 45,XY, ,XY 46,XY 46,XY ,XX 46,XX 46,XX ,XY 46,XY 46,XY ,XY 46,XY 46,XY ,XX 46,XX 46,XX ,XY 46,XY 46,XY VOL. 97 NO. 4 / APRIL e1

8 SUPPLEMENTAL TABLE 1 Continued. Patient no. Embryo no. SNP microarray result 1 SNP microarray result 2 qpcr result ,XX 46,XX 46,XX ,XX 46,XX 46,XX ,XX 46,XX 46,XX ,XY 46,XY 46,XY a Inconsistent qpcr result. 824.e2 VOL. 97 NO. 4 / APRIL 2012