Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos

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1 Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos Cristina Gutierrez-Mateo, Ph.D., a Pere Colls, Ph.D., a Jorge Sanchez-Garcıa, Ph.D., a Tomas Escudero, B.Sc., a Renata Prates, B.Sc., a Kelly Ketterson, B.Sc., a Dagan Wells, Ph.D., b and Santiago Munne, Ph.D. a a Reprogenetics, Livingston, New Jersey; and b Reprogenetics UK, Institute For Reproductive Sciences, Oxford Business Park, North, Oxford, United Kingdom Objective: To validate and determine the best array comparative genomic hybridization (acgh; array-cgh) protocols for preimplantation genetic screening (PGS). Design: Embryos had one cell removed as a biopsy specimen and analyzed by one of two array-cgh protocols. Abnormal embryos were reanalyzed by fluorescence in situ hybridization (FISH). Setting: Reference laboratory. Patient(s): Patients donating embryos or undergoing PGS. Intervention(s): Embryo biopsy, array-cgh, FISH reanalysis. Main Outcome Measure(s): Diagnosis, no result rate and error rate. Result(s): Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after array-cgh combined with single blastomere biopsy. Conclusion(s): Array-CGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, array- CGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Preimplantation genetic diagnosis, PGD, PGS, array-cgh, array-cgh, CGH, aneuploidy, embryos Chromosomal abnormalities are common in embryos from assisted reproductive technology (1 4), ranging from 60% abnormal embryos in women younger than 35 years to 80% in women 41 years and older (4). The majority of numerical chromosome abnormalities detected in cleavage-stage embryos are not compatible with implantation or birth (5 9), and their high frequency is likely to have a substantial effect on the success of assisted reproductive treatments. Preimplantation genetic screening (PGS) aims to provide a means of identifying potentially viable euploid embryos, which may have higher chances of producing a pregnancy, to be prioritized for transfer. Prior PGS strategies using fluorescence in situ hybridization (FISH) have involved the analysis of 5, 9, or 12 chromosomes (5 7). Techniques that comprehensively detect all instances of aneuploidy and assess each chromosome at multiple loci should reduce or minimize the risk of misdiagnosis. The first technique clinically applied for the comprehensive detection of aneuploidy Received May 17, 2010; revised August 25, 2010; accepted September 9, 2010; published online October 25, C.G-M. has nothing to disclose. P.C. has nothing to disclose. J.S-G. has nothing to disclose. T.E. has nothing to disclose. R.P. has nothing to disclose. K.K. has nothing to disclose. D.W. has nothing to disclose. S.M. has nothing to disclose. Reprint requests: Cristina Gutierrez-Mateo, Ph.D., 3 Regent Street, Suite 301, Livingston, NJ ( munne@reprogenetics.com). in day-3 embryos was comparative genomic hybridization (CGH) (8, 9). However, the standard CGH protocol is time consuming and incompatible with day-3 biopsy and replacement in a fresh cycle thus is currently applied in polar bodies (10, 11) or blastocyst biopsy specimens (12, 13) in combination with vitrification (14). Blastocyst biopsy combined with CGH and vitrification has been shown to significantly improve implantation rates, from 46.5% in controls to 72.2% in cycles with PGS, with 100% of blastocysts surviving biopsy, vitrification, and warming (13). However, many clinics are not yet proficient at blastocyst culture, biopsy, and vitrification, and many patients prefer to have a transfer in a fresh cycle. Thus, day-3 biopsy remains the PGS approach most frequently requested by IVF centers and patients. There are two techniques that can produce a comprehensive chromosome analysis of single cells and are compatible with day-3 biopsy and day-5 replacement in a fresh cycle. These techniques are microarray CGH (array-cgh or acgh) (15 17) and single nucleotide polymorphism (SNP) microarrays (18 20). Array-CGH is a technique based on the same principle as CGH, but the hybridization of test and reference DNA is shorter and does not take place on metaphase spreads, but on chips containing thousands of loci. Therefore, array-cgh may have the same advantages for PGS as those found by Schoolcraft et al. by using conventional CGH (13). Similarly, SNP arrays investigate thousands of SNPs across the genome, allowing the detection of chromosomal abnormalities and the diagnosis of inherited gene mutations by linkage /$36.00 Fertility and Sterility â Vol. 95, No. 3, March 1, doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 analysis. So far, array-cgh is used in cytogenetic analysis of prenatal and postnatal diagnosis (21, 22). In addition, the probes affixed to the microarray that detect an abnormality can also be used as FISH probes, allowing confirmatory analyses to be conducted. Because of the intrinsic and sometimes unpredictable problems with every technology, a new technique should be thoroughly validated, ideally against a different and more established method. Validating a new approach against the same technique might preclude the detection of these technically related flaws. The objective of this study is to validate two protocols of array- CGH by reanalyzing the diagnosed embryos using FISH for a standard set of 12 chromosomes plus any additional chromosomes suggested as being abnormal by the initial array-cgh analysis. This validation approach will then be compared to other studies using chromosome comprehensive analysis. MATERIALS AND METHODS Study Design Blastomeres were split into two experimental groups. Group 1 underwent amplification with GenomePlex (Sigma-Aldrich, Dorset, UK) and group 2 with SurePlex (BlueGnome, Cambridge, UK). The two groups were compared according to the number of cells with DNA amplification (no result rate) and the frequency of embryos misdiagnosed when comparing the PGS results obtained by array-cgh with the analysis by FISH of the remainder of the embryos (error rate). We considered an FISH-reanalyzed embryo as being erroneously classified by array-cgh when the embryo was initially classified as normal or abnormal, but found by FISH to be abnormal or normal, respectively. Material Two types of material were used for this study. One source was non-pgs embryos that were not suitable for replacement because of morphologic or developmental abnormalities. The second source of material was embryos from PGS cycles. All these embryos had a single cell biopsied and analyzed by array-cgh within hours. The rest of the embryo was fully disaggregated into single cells and reanalyzed with FISH to calculate the error rate in each experimental group. Patients signed an informed consent in accordance with an independent institutional review board protocol (WIRB, study number , approved on November 16, 2009). Array-CGH Biopsied cells were washed and collected into sterile PCR tubes. Several negative controls, consisting of 2 ml of Non-sticking Washing Buffer (NWB) taken from drops used for washing the biopsied cells, were placed in separate tubes. The samples were lysed, fragmented, and amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (Sigma- Aldrich) modified according to the protocol of Fiegler et al. (group 1) (23). Alternatively, single blastomeres were lysed and amplified using the SurePlex kit, according to the manufacturer s instructions (BlueGnome; group 2). One nanogram of genomic DNA and one reagent negative control (amplification mixture only) were also subjected to whole genome amplification. Amplified samples were processed according to the BlueGnome CytoChip protocol (available upon request at Scanned images were analyzed and quantified, and whole chromosomal copy number ratios were reported using the CytoChip algorithm fixed settings in BlueFuse Software (BlueGnome). Array-CGH Scoring Criteria Once a specific amplification was observed (i.e., low autosomal noise), autosomal profiles were analyzed for gain or loss of whole chromosomal ratios using a 3xSD assessment, > 0.3log 2 ratio call, or both. To pass hybridization quality controls, sex mismatched female samples had to show a consistent gain on chromosome X and a consistent loss of chromosome Y. Sex-matched male samples had to show consistently no change on either chromosome X or Y. Negative amplifications showed a consistent partial gain on chromosome X with no loss of chromosome Y. FISH Analysis of the Remaining Embryo The remaining cells of nontransferred embryos were fixed for FISH analysis to calculate the error rate. The fixed embryos were screened for chromosomes X, Y, 8, 13, 14, 15, 16, 17, 18, 20, 21, and 22 in three rounds of hybridization, as described previously (5). A fourth round of hybridization was performed to confirm any aneuploidy involving chromosomes indicated to be abnormal by array-cgh analysis that were not included in the three previous rounds. RESULTS Embryos Analyzed and Amplification Efficiency A total of 161 embryos were analyzed in group 1 and 654 embryos in group 2. After biopsy, all cells in group 1 and 634 in group 2 showed a nucleus. Of the embryos with a nucleated cell observed, failure of amplification occurred in 11.2% (18/161) of cells in group 1 and 3% (19/634) of group 2 (P<0.001; Table 1). Amplification was not detected in any of the negative controls (washing media or reagent negative controls) included in the preclinical validation or in the clinical cases. Error Rate per Embryo: Comparison between Array-CGH and FISH Results A total of 66 group 1 embryos and 54 group 2 embryos, previously diagnosed by array-cgh based on a single cell, were disaggregated, and the remaining cells were reanalyzed by FISH (Table 1). Of these, 9.1% (6/66) of group 1 embryos and 1.9% (1/54) of group 2 embryos were misdiagnosed. The discordances for these seven embryos are described in Table 2. Aneuploidy Detection, Chromosomes Involved, and Correlation with Maternal Age Noisy profile array-cgh results were obtained in 3.5% (5/143) of the amplified samples with method 1 and 2.5% (16/634) for method 2. After another analysis of six of the embryos classified as having noisy profiles, all were found to be chromosomally abnormal; therefore, the occurrence of noisy profiles was determined not to be caused by either protocol but by underlying problems with the biopsied cell, which most likely contained degraded DNA resulting from apoptosis. Combining groups 1 and 2, a total of 759 embryos had clear and well-defined array-cgh profiles (Fig. 1). Of those embryos, 35.4% TABLE 1 Comparison of array-cgh methods. Method 1 Method 2 Embryos included Embryos with nucleated cell biopsied Embryos without 18 (11.2%) 19 (3%) a amplification Embryos reanalyzed Embryos misdiagnosed 6 (9.1%) 1 (1.9%) b a P< b Not significant. 954 Gutierrez-Mateo et al. Validation of array-cgh for PGS Vol. 95, No. 3, March 1, 2011

3 TABLE 2 Embryos misdiagnosed. Embryo Method array-cgh result FISH result Reason misdiagnosis R ,XY, 3, 7, 8, 16, 17 46,XY (3)/44,XY, 21, 22 (1)/ 44,XY, 13, 21 (1)/44, X, 15 (1) False positive, technical artifact, mosaicism R ,XY, þ7, þ11 46,XY (4)/ 92,XXYY (1) False positive, technical artifact R ,XY 47,XY, þ22 (1) False negative, technical artifact, mosaicism R7014,3 1 45,XY, 22 46,XY (15) False positive, technical artifact R7029,5 1 43,XX, 9, 20, 21 46,XX (10) False positive, technical artifact R7029,6 1 44,XY, 17, 22 46,XY (10) False positive, technical artifact R7132,9 2 45,X 45,X (2)/46,XX (9) False positive, mosaicism Note: Cells analyzed by FISH are in parentheses. (269/759) were classified as euploid, and the rest were classified as abnormal, for a population of embryos with a mean SD age of years. As expected, chromosome abnormalities increased with maternal age, from 51% (126/245) in patients younger than 35 years, to 60% (125/208) in patients years old, and to 78% (239/306) in patients 40 years old or older (P<0.001). The chromosome abnormalities are summarized in Table 3. FISH analysis of 120 embryos revealed that 90 were abnormal, 40 were mosaic (44.4%), and 50 displayed the same abnormality in all cells analyzed. Array-CGH detected 1,615 aneuploid events involving all chromosomes 970 losses and 645 gains (Table 3). Of these, FISH would have detected 41.2% events with nine probes or 54.1% with 12 chromosomes, respectively. However, embryos with two or more abnormalities, of which FISH would detect at least one, would be scored as abnormal by FISH (Fig. 1C). Thus 9- or 12-chromosome FISH analysis would still detect 81% and 87% of the abnormal embryos detected by array-cgh, respectively. The chromosomes most frequently involved in aneuploidy were chromosomes 16, 22, 21, and 15. The chromosomes least involved in aneuploidy were chromosomes 4 and 6. Of the single aneuploid events detected, there were more loses than gains of chromosomes (95 single chromosome losses and 72 single chromosome gains; Table 3). Polyploidy Detection Array-CGH cannot detect some triploidy, such 69,XXX, and tetraploidy, such 92,XXXX or 92,XXYY, whereas it will detect as trisomic for gender triploidy 69,XXY, tetraploidy 92,XXXY, and 92,XYYY. In addition, any polyploidy not detectable by array- CGH that is also abnormal for aneuploidy will be classified as aneuploid. To determine how many of these pure polyploidies are missed by array-cgh, our database of embryos analyzed by FISH (using 9 12 probes) was queried. We found that 7.5% (6,898/92,018) embryos were polyploid or haploid. However, the majority of these embryos had other abnormalities detectable by CGH or array-cgh, and only 2.3% (2,136/91,073) of the embryos was homogeneously polyploid or haploid. In addition, of those homogeneously polyploid or haploid, those that had combinations of gonosomes that were different from NX or 0.5N (XY), where N is the ploidy, could be detected by array-cgh as abnormal and classified as aneuploid, such as 69,XXY. Only 1.7% (1,587/92,018) of embryos that were polyploid or haploid were not detectable by array-cgh (i.e., 69,XXX or 92,XXYY). Furthermore, 87% of these embryos were arrested or dysmorphic and consequently would have not been replaced. Homogeneously polyploid or haploid embryos that were not detectable by array-cgh and exhibited a good morphology accounted for approximately 0.2% of the embryos. Clinical Results Of the cycles performed with protocol 2 and for all with biopsy on day 3, clinical information past the first trimester was available on 104 cycles from 98 patients with average maternal age of 37.5 years. Of those, normal embryos were found for transfer in 76 cycles, which resulted in 44 pregnancies (42% per retrieval, 58% per transfer), with 40 ongoing or going to term (10% miscarriage rate). The implantation rate was 42% (53 sacs per 127 embryos replaced; average of 1.2 embryos replaced). At least 10 couples have given birth to healthy children. DISCUSSION Our results indicate that array-cgh will detect approximately 42% more abnormalities and 13% more abnormal embryos than the standard 12-probe FISH approach. The results are almost identical to those obtained by CGH (5, 10); this is due to a tendency of abnormalities for nontested chromosomes occurring in conjunction with abnormalities for chromosomes tested, which is in agreement with previous studies (10, 12, 24 26). The results also showed, as previously reported (27), that the chromosomes most involved in aneuploidy events at the cleavage stage are 16, 22, 21, and 15. Correlations among aneuploidy, maternal age, and an excess of monosomies over trisomies was also detected, which is in agreement with other studies (3, 27). The fact that these well-documented phenomena were observed provides a biologic validation of this new technique. The other form of validation was to use FISH to reanalyze nontransferred embryos after PGS with array-cgh. Because of the high rate of mosaicism in cleavage-stage embryos (i.e., 44% in the present study), which precludes the exact cytogenetic confirmation based on the analysis of other cells from the same embryo, we focused on clinical validation that is, if an embryo was classified as abnormal after PGS and the reanalysis was again abnormal, the result was confirmed, regardless of exact genetic confirmation. The error rate after array-cgh using method 1 was higher than using method 2 (9% and 1.9%, respectively). Both rates are well within the lower range reported in most PGS studies (28, 29), but method 1 clearly showed technical errors and method 2 showed only one error, Fertility and Sterility â 955

4 FIGURE 1 Example of blastomere array-cgh results. (A) Array profile showing an XX cell with loss of chromosome 18. (B) Female cell with gain of chromosome 4. (C) Male blastomere displaying double aneuploidy, gain of chromosome 19, and loss of chromosome 22. (D) Complex aneuploid male blastomere showing loss of chromosomes 10 and 16 and gain of chromosome 21. (E) Normal female profile. likely caused by mosaicism (Table 2). The lower frequency of single cells producing no amplification and the lower error rate obtained using method 2 make it our preferred amplification method for array-cgh analysis. Consequently, it is this method that we are now using clinically. To our knowledge, this is the first comprehensive chromosome screening technique that has been validated by comparing its results to the analysis of the remaining embryo analyzed by another technique (e.g., FISH). This method allowed the detection of intrinsic limitations of the technique. Other groups using 956 Gutierrez-Mateo et al. Validation of array-cgh for PGS Vol. 95, No. 3, March 1, 2011

5 TABLE 3 Chromosome abnormalities detected by array-cgh and reanalysis with FISH. n(%) Abnormalities found by array-cgh analysis (single cell) Single loss (i.e., monosomy) 95 (19.4) Single gain (i.e., trisomy) 72 (14.7) Two aneuploidies 121 (24.7) Complex aneuploid a 202 (41.2) Total 490 (100) Abnormalities found by FISH analysis (multiple cells) Embryos with homogeneous aneuploidy in all the cells Single monosomy 19 (21.1) Single trisomy 7 (7.8) Double aneuploidy 16 (17.8) Complex aneuploid a 7 (7.8) Haploid 0 (0) Polyploid and monosomy 1 (1.1) Mosaics embryos 100% abnormal 36 (40) 50-99% abnormal 2 (2.2) <50% abnormal 2 (2.2) Total embryos 90 (100) a Complex aneuploid: three or more abnormal chromosomes. SNP arrays have not yet performed this type of validation (19, 30). It is also important to note that, regardless of the high rate of mosaicism observed in cleavage stage embryos, the error rate with the current protocol is only 2%. Because most mosaics contain 100% abnormal cells as reported previously, regardless of which c is biopsied, the diagnosis of abnormal remains correct (28). In fact, the miscarriage rate in this study is only 10% for an average age of 37.5 years. The 2008 SART U.S. national summary (31) reports a miscarriage rate of 21.8% for patients years old, 18.1% for years old and 26.7% for years old. Array-CGH has minor limitations and some advantages compared to other techniques. First, in contrast to FISH (29), CGH and array- CGH allow the simultaneous enumeration of all chromosomes while evaluating multiple loci along the length of each chromosome. Another advantage over FISH is that CGH and arrays do not require cell fixation onto slides. Difficulties associated with fixation and technical ability might explain the variation in reported FISH error rates, ranging from 5% to 50% (28, 29, 32 35). Second, although standard CGH combined with blastocyst biopsy and vitrification has shown promising results (19), acgh and SNP arrays allow for day-3 biopsy and day-5 replacement. Third, CGH and array-cgh cannot detect some polyploidies, whereas FISH can. SNP arrays allow detection of polyploidy only in cases of three or more different chromosome complements. However, as shown in the Results section, only 0.2% of nonarrested embryos will be misdiagnosed owing to polyploidy. Fourth, unlike SNP arrays, FISH, array-cgh, and CGH cannot detect uniparental disomy. However, uniparental disomy is extremely rare. For example, uniparental disomy 15 is estimated to occur in 0.001% of newborns (OMIM). Fifth, the array-cgh amplification step produces enough DNA to allow the simultaneous detection of aneuploidy via array-cgh and gene defects via PCR based methods, whereas SNP arrays could do it in one step. Sixth, FISH, CGH, array-cgh, and SNP arrays can all be used to detect translocations. Seventh, in a comparison of SNP arrays, FISH and array-cgh do not require testing of DNA samples from the couple or the couple s relatives before the IVF cycle, and they can be scheduled on the day of biopsy based on the number of embryos available for biopsy. This difference is important because approximately 20% of cycles are cancelled owing to low numbers of embryos produced, and the cost of testing the parents for SNP array analysis may be wasted in those cases. Eighth, testing parental DNA for SNP arrays has been made into a virtue out of necessity. In lay terms, not all SNPs will return a signal, and each SNP returning a signal will have certain risk of giving an incorrect result. The data obtained by SNP arrays is thus dirty, requiring parental DNA support to make sense of it and reconstruct the sequence of potential SNPs in a chromosome based on the four potentially inherited types of chromosomes. Testing parental DNA, in return, offers the possibility to determine the origin of any extra or missing chromosome. However it is well known that approximately 95% of aneuploidies originate from errors in the maternal meiosis (36) and few from sperm meiotic errors (37). Some groups are making the mistake of assuming that mitotic aneuploidy has a parental origin. Although the chromosome gained or lost in the cell of a mosaic embryo has an origin, this chromosome was been gained or lost at random, and its parental origin has no biologic importance or therapeutic relevance (4, 28, 38). As such, we think that point seven trumps point eight. In conclusion, array-cgh is the only technique to provide a comprehensive chromosome count that has been fully validated by comparing it to another technique, thus detecting any shortcomings of array-cgh that are intrinsic to this technique (e.g., polyploidy). 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