Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos

Reproductive BioMedicine Online (2012) 24, xxx– xxx

www.sciencedirect.com www.rbmonline.com

ARTICLE

Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos Pere Colls a,*, Tomas Escudero a, Jill Fischer a, Natalie A Cekleniak b, Snunit Ben-Ozer c, Bill Meyer d, Miguel Damien e, Jamie A Grifo f, ´a Avner Hershlag g, Santiago Munne a Reprogenetics, Livingston, NJ 07039, USA; b IRMS, Livingston, NJ 07039, USA; c ART Reproductive Center, Beverly Hills, CA 90210, USA; d Carolina Conceptions, Raleigh, NC 27607, USA; e East Coast Infertility and IVF, Little Silver, NJ 07739, USA; f New York University Fertility Center, New York, NY 10016, USA; g The Center for Human Reproduction, Manhasset, NY 11003, USA

* Corresponding author. E-mail address: [email protected] (P Colls). Pere Colls obtained his Bachelor’s degree in Biology in 1994 at Universitat Autonoma de Barcelona, Spain. At the same university, he started his research work in the field of human genetics and was awarded his PhD in 1998 with his thesis on cytogenetics of the human spermatozoon. He began his work in human reproduction in 1998 as embryologist and geneticist. In 2003 he moved to the USA to work in the field of preimplantation genetic diagnosis at Reprogenetics, New Jersey, USA, where he currently is laboratory director.

Abstract Fluorescent in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormal-

ities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridization (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were 6 Mbp and 5 Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by array CGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for array CGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by array CGH. This study validates array CGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: array CGH, PGD, translocations

1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2012.02.006

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Introduction Structural chromosome abnormalities occur with a frequency of 0.19% in the human population (Jacobs et al., 1974; Simpson and Bischoff, 2002) but this frequency may be as high as 5% among subfertile individuals (Frynes and Buggenhout, 1998; Stern et al., 1999). Reduced fertility in carriers of balanced translocations is due to increased risk of recurrent miscarriage and diminished ovarian reserve (Chen et al., 2005). Carriers also have an increased risk of producing chromosomally abnormal gametes, which, following fertilization, would generate embryos with the same defect (Munne ´ et al., 2000; Verlinsky et al., 2005) and, following implantation, offspring with unbalanced translocations (Chandley, 1998). The use of preimplantation genetic diagnosis (PGD) to detect unbalanced translocations in early embryos during IVF has proven to be successful in significantly reducing the rate of recurrent pregnancy loss as well as the risk of abnormal offspring in this group of patients (Fischer et al., 2010; Munne ´ et al., 1998a, 2000; Otani et al., 2006; Verlinsky et al., 2005). Since its first use in PGD for structural chromosome abnormalities (Conn et al., 1995, 1998; Munne ´ et al., 1998b), fluorescence in-situ hybridization (FISH) has been the technique most widely used, either in polar bodies via whole chromosome painting probes (Munne ´ et al., 1998c; Verlinsky and Evsikov, 1999) or in interphase nuclei using probes that bind to each of the chromosome fragments (Conn et al., 1998; Escudero et al., 2001; Munne ´ et al., 1998d, 2000) or breakpoint-spanning probes (Fung et al., 1998; Munne ´ et al., 2000; Weier et al., 1999). Despite their success, the accuracy of FISH-based methods can be compromised by split signals, cross-hybridization, chromosome polymorphisms, poor fixation quality or loss of micronuclei or chromosomes during fixation. Although different strategies such as the use of scoring criteria (Munne ´ et al., 1998e), improvement of the fixation technique (Velilla et al., 2002) or the use of extra probes (Colls et al., 2007) have been developed to address problematic areas, some general shortcomings still remain. The most important of these is the inability to simultaneously analyse chromosomes other than those involved in the structural chromosome rearrangement, especially in the case of advanced maternal age. Because of the decrease in FISH efficiency after each round of hybridization and time constraints, only a limited number of extra chromosomes can be analysed during FISH for structural chromosome abnormalities – usually those involved in spontaneous abortions (Fischer et al., 2010). Clinical outcome could be negatively affected if aneuploidy remains undetected. Unlike Robertsonian translocations, which do not require any previous work up since whole chromosomes are involved, reciprocal translocations require prior case preparation. This includes the selection of probes based on the chromosomes involved and the location of the translocation breakpoints, which allows for the detection of all possible unbalanced rearrangements in an interphase nucleus. This is followed by testing this combination of probes in lymphocyte metaphases of the translocation carrier (Munne ´ et al., 2000).

P Colls et al. Whole genome amplification approaches that have been successfully used for aneuploidy screening, including comparative genomic hybridization (CGH) (Fishel et al., 2010; Fragouli et al., 2010; Schoolcraft et al., 2010; Voullaire et al., 2002; Wells et al., 2002, 2008; Wilton et al., 2003) and microarray single-nucleotide polymorphisms (Treff et al., 2010) and microarray CGH (Gutie ´rrez-Mateo et al., 2011) have also been used for detection of reciprocal and Robertsonian translocations (Alfarawati et al., 2011; Fiorentino et al., 2010, 2011; Treff et al., 2011). These studies suggest that microarray methods can be used to simultaneously detect all numerical chromosome abnormalities as well as unbalanced translocations, within a time frame that is compatible with routine clinical schedule. This study describes the use of array CGH for diagnosis of reciprocal or Robertsonian translocations in human embryos and the reanalysis of the non-transferred embryos by FISH with probes designed specifically for each case to detect unbalanced translocations in interphase nuclei.

Materials and methods Study design This study had four aims: (i) to determine the minimal resolution size of array CGH; (ii) to determine the error rate for the technique in translocation cases; (iii) to determine the risk of misdiagnosis due to mosaicism; and (iv) to assess how many patients who have previously undergone PGD for translocations using older techniques could have been helped by array CGH taking into consideration the sizes of chromosome fragments and the minimal resolution of array CGH.

Resolution of array CGH Forty-seven cases in which one partner was a carrier of Robertsonian or reciprocal translocations were included in this analysis. The cases were referred to Reprogenetics by 28 collaborating IVF centres. The average maternal age was 33.4 ± 4.4 years (range 27–44 years); the chromosome rearrangements included 30 cases of reciprocal translocations and 17 cases of Robertsonian translocations (Table 1). To assess the feasibility of performing the analysis by array CGH, the structural chromosome abnormality was evaluated in detail for each case. The evaluation included the type of rearrangement, the chromosomes involved, location of the breakpoints and an estimation of the size of the chromosome fragments. To properly characterize a translocation, three of the four fragments must be detectable (Figure 1). Array CGH was performed either on day-3 (single blastomere biopsy) or day-5–6 (trophoectoderm biopsy) embryos produced by intracytoplasmic sperm injection. Embryo biopsy on day 3 was performed using conventional Tyrodes’s acid technique and a single blastomere was biopsied, while at blastocyst stage a laser ablation was used to remove a piece of trophoectoderm. Knowing the breakpoints and the size of each fragment in each case, this analysis allowed determination of the resolution in Mbp of the test by detection of, or failure to detect, gains or losses for the smallest fragment generated in each particular case as well as

Array CGH for translocations in preimplantation human embryos Table 1

623

Array comparative genome hybridization results from each case.

Karyotype

46,XY,t(2;11)(q21;q23) 46,XX,t(7;8)(q21.2;q22.3) 46,XY,t(5;9)(q33;p13) 46,XY,t(4;6)(q31.3;q16.2) 46,XY,t(3;8)(q25.3;q13) 46,XX,t(6;18)(q23.3;q21.1) 46,XX,t(6;13)(q23;q32) 46,XX,t(1;19)(q12;q13.1) 46,XX,t(1;2)(q42.1;q33) 46,XX,t(2;16)(q33;q22) 46,XX,t(2;8)(q21.1;q11.23) 46,XX,t(2;8)(q21.1;q11.23) 46,XX,t(3;13)(q21;q22) 46,XX,t(4;7)(q33;q21.2) 46,XX,t(7;13)(q21.2;q31) 46,XY,(11;17)(q13.1;q25.1) 46,XY,t(15;16)(q15;q22) 46,XY,t(2;6)(q36.1;p25.3) 46,XY,t(7;13)(q11.23;q32) 46,XY,t(8;13)(p11.2;q32) 46,XY,t(9;11)(q22;p13) 46,XX,t(9;12)(q22.3;q24.31) 46,XY,inv(7)(q22q36.1), t(10;11)(q22.3;p13) 45,XY,der(13;14)(q10;q10) 45,XX,der(13;14)(q10;q10) 45,XX,der(13;14)(q10;q10) 45,XX,der(13;15)(q10;q10) 45,XX,der(14;15)(q10;q10) 45,XX,der(14;21)(q10;q10) 45,XX,der(14;21)(q10;q10) 45,XY,der(13;14)((q10;q10) 45,XY,der(13;14)(q10;q10) 45,XY,der(13;14)(q10;q10) 45,XY,der(14;21)(q10;q10) 45,XY,der(14;22)(q10;q10) 45,XY,der(21;22)(q10;q10) 45,XY,der(21;22)(q10;q10) 46,XX,t(4;20)(q25;p11.2) 46,XY,t(16;22)(q12.1;q11.2) 46,XX,t(11;22)(q23.3;q11.2) 46,X,t(Y;9)(q11.21;p24) 46,XY,t(5;22)(p14;q12.2) 46,XY,t(8;15)(q24.3;q22.1) 46,XY,t(9;20)(p10;q10) 45,XX,der(13;14)(q10;q10) 45,XX,der(13;14)(q10;q10) 45,XX,der(13;21)(q10;q10) Total

Biopsy day

Smallest fragment Mbp)

Age (years)

No. of embryos

Array CGH finding

Biopsied

Normal or balanced

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

11 40 19 24 32 19 16 19 24 32 48 48 35 14 19 5 17 3 12 12 34 16 8

35 31 31 29 35 34 43 29 29 40 30 30 36 34 31 31 38 32 30 36 38 30 39

22 10 5 2 3 14 2 5 27 11 7 5 12 18 4 17 15 15 2 7 8 12 15

17 10 5 2 3 14 2 4 26 11 7 4 11 16 4 17 14 14 2 7 8 12 14

3 0 1 0 0 1 0 0 4 0 0 1 1 5 0 5 1 3 1 0 1 1 3

5 2 1 1 1 5 0 1 11 1 4 0 2 4 1 6 5 2 1 3 2 1 1

5 7 2 1 2 7 1 2 3 5 1 2 8 5 1 2 3 7 0 1 3 7 10

4 1 1 0 0 1 1 1 8 5 2 1 0 2 2 4 5 2 0 3 2 3 0

3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5

Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian Robertsonian 17 11 6 5 19 5 27 Robertsonian Robertsonian Robertsonian

37 30 30 35 28 31 29 38 30 34 31 33 40 40 29 28 34 28 42 38 35 44 27 29

4 15 5 13 2 15 6 2 3 16 4 14 7 10 5 1 12 11 9 16 5 6 4 9 432

3 11 5 13 2 14 4 2 2 16 4 14 6 7 5 1 12 10 9 14 5 6 4 9 402

3 6 0 2 1 4 1 0 1 2 1 5 3 4 3 1 1 1 0 3 1 0 2 5 81

0 3 2 1 0 1 0 0 1 1 0 4 1 0 2 0 6 3 2 3 2 0 0 0 92

0 0 1 4 0 6 1 0 0 0 0 2 1 2 0 0 2 5 4 2 0 4 1 3 123

0 2 2 6 1 3 2 2 0 13 3 3 1 1 0 0 3 1 3 6 2 2 1 1 106

detection of all possible unbalanced rearrangements. Simultaneous screening of all other chromosomes, apart from those involved in the structural rearrangement, allowed detection of aneuploidy in the same embryos.

Error rate of array CGH for translocations Error rate was determined through reanalysis of 54 non-transferred embryos using FISH probes that characterized each specific structural rearrangement. The embryos were fixed in whole on day 5, 6 or 7 of development as previously described (Sandalinas et al., 2001). The number of

Analysed

Unbalanced

Unbalanced and aneuploid

Aneuploid

analysable nuclei obtained varied depending on the stage of development, degradation level and quality of the fixation. To determine the error rate, FISH and array CGH results were compared. This validation approach was previously used to validate array CGH for aneuploidy (Gutie ´rrez-Mateo et al., 2011).

Error rate due to mosaicism Although array CGH could detect any unbalanced embryo if three out of four translocated fragments were detected, chaotic mosaicism, which refers to successive cell divisions

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P Colls et al. CARRIER

ALTERNATE

ALTERNATE

ADJACENT I

ADJACENT I

ADJACENT II

ADJACENT II

3:1

3:1

3:1

3:1

3:1

3:1

3:1

3:1

Figure 1 Profiles obtained by array comparative genome hybridization for three detectable fragments (blue, green and red) in a reciprocal translocation case. The fourth fragment (yellow) is not detectable. Each combination corresponds to a different meiotic segregation in the carrier and normal chromosomes from the normal partner.

with random distribution of chromosomes (Delhanty et al., 1997; Munne ´ et al., 2002), could create a situation in which an unbalanced gamete would contain only one unbalanced chromosome, which if it were below the resolution of the technique, would produce a misdiagnosis. To assess this possibility, a larger number of embryos would be needed for analysis. This study used a dataset of embryos previously analysed by array CGH from patients undergoing preimplantation genetic screening (not for translocations) and that were also reanalysed by 12 or more FISH probes. Some of these results have been previously published (Gutie ´rrez-Mateo et al., 2011) and others not. The purpose of this analysis was to determine the average number of abnormal cells detected in each of the mosaic types.

Specific utility of array CGH in diagnosis of translocations This study retrospectively surveyed 926 cases of PGD for reciprocal translocations by FISH, performed at Reprogenet-

ics from 1995 to 2011. Chromosome fragment sizes in each case were evaluated in order to estimate the number of cases for which array CGH could be used.

IRB approval The collaborating IVF centres that participated in the first component of the study obtained an IRB approval for the study from their respective institutions (NYU IRB 6902; IRB00002857). All patients were provided with counselling regarding PGD by array CGH and signed an informed consent to be included in the study. For the second and third parts of the study, IRB approval was not required, under common rule 45 CFR 46.101(b)(4) (Western IRB, Olympia, Washington). Exemption is granted to ‘research, involving the collection or study of existing data, documents, records, pathological specimens, if these sources are publicly available or if the information is recorded by the investigator in such manner that subjects cannot be identified, directly or through identifiers linked to subjects’.

Array CGH for translocations in preimplantation human embryos

Array CGH Biopsed cells were washed and collected into sterile PCR tubes. The samples and reference male DNAs were lysed, fragmented and amplified using the SurePlex whole genome amplification kit (BlueGnome, Cambridge, UK) according to the manufacturer’s instructions. Amplified DNA samples and reference male DNA were labelled with Cy3 and Cy5, respectively, using the BlueGnome fluorescent labelling system, according to the manufacturer’s instructions. Amplification and labelling protocols used in this study are available at www.cytochip.com. The platform used in this study was the 24Sure Cytochip (BlueGnome), which screens all 24 chromosomes for both gain and loss with a BAC pooling strategy together with software smoothing which enables robust results to be reported on the ploidy of the single cell on the basis of a single hybridization. Labelled samples and reference male DNAs were mixed and applied to a microarray and co-hybridized for a minimum of 3 h, after which they were washed in ·2 saline sodium citrate (SSC)/0.05% Tween 20 at room temperature for 10 min, followed by a wash in ·1 SSC at room temperature for 10 min and with ·0.1 SSC at 59C for 5 min and finally washed for 1 min at room temperature in the same solution. Microarray slides were dried in a centrifuge for 3 min and scanned with a laser scanner (InnoScan 710AL; Innopsys, Carbonne, France). Scanned images were analysed using BlueFuse Multi software (BlueGnome). Once a specific amplification was observed, autosomal profiles were analysed for gain or loss of whole or partial chromosomal ratios using a 3SD assessment, 0.3log2 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 consistently show no change on either chromosome X or Y (Gutie ´rrez-Mateo et al., 2011). Embryos were classified as ‘normal or balanced’ if the generated array CGH plot showed no gains or losses in any of the autosomes, ‘unbalanced’ if total or partial gains or losses were observed for the chromosomes involved in the chromosome rearrangements or ‘aneuploid’ if, being normal or balanced for the structural abnormality, gains or losses were observed for any other chromosome.

FISH reanalysis Non-transferred embryos previously analysed by array CGH were fixed and then reanalysed by FISH using a combination of probes (locus-specific identifiers, centromeric and subtelomeric) specially designed to properly characterize the structural rearrangements in interphase nuclei. For reciprocal translocations, a combination of probes was designed consisting of a probe above and a probe below the breakpoint for each of the chromosomes involved in the translocation. This approach yielded mainly combinations of four probes, each one binding to each of the four fragments. In cases where no probes were commercially available for one of the fragments, only three probes were used for the test. Probes were obtained from Abbot (Downers Grove, IL, USA). To minimize artefacts of fixation, only non-overlapping, good-quality nuclei were analysed. FISH signals were scored using criteria previously described

625

(Munne ´ et al., 1998e). Nuclei showing two signals for each probe were classified as ‘normal or balanced’, while any other combination was classified as ‘unbalanced’.

Results Array CGH resolution A total of 432 embryos from 47 cycles were biopsied for analysis of reciprocal and Robertsonian translocations using array CGH (Table 1). Of these, 354 embryos from 37 cycles were analysed on day 3 (single blastomeres) and 78 embryos from 10 cycles were analysed on day 5–6 (5–10 trophectoderm cells per embryo). In the day-3 group, 20 (5.6%) failed to amplify due to a lysed cell, DNA damage or absence of a nucleus in the biopsied cell. In an additional seven (2.0%) cases, the results were inconclusive because of chaotic array CGH profile attributable to DNA degradation. In the day-5–6 group, amplification failure occurred in three (3.8%) embryos and none had inconclusive results. Overall, results were obtained for 327 of 354 embryos and 75 of 78 embryos, meaning that the test efficiency rate was 92.4% and 96.2% for day 3 and day 5–6, respectively. In the reciprocal translocation group (30 cases), embryos were analysed from 29 different translocations. In 23 cases, the analysis was performed in day-3 single blastomeres while in the remaining seven cases it was carried out in trophoectoderm cells from day-5–6 blastocysts. Diagnosis was obtained for all the cases with a total of 280 embryos analysed, of which 41 (14.6%) were diagnosed as ‘normal or balanced’, 78 (27.9%) as ‘unbalanced’, 63 (22.5%) as ‘aneuploid’ and 98 (35.0%) as ‘unbalanced and aneuploid’. The size of the smallest fragment for each reciprocal translocation ranged from 3 Mbp to 48 Mbp. Gains or losses were detected for all fragments larger than 6 Mbp in all the embryos analysed. Five fragments were 6 Mbp or smaller, of which three (ranging from 5 Mbp to 6 Mbp) were detected in all the embryos analysed for that case, one (5 Mbp) was detected in seven of the 17 embryos (41%) analysed for the case and one (3 Mbp) was not detected in any of the embryos analysed for the case. None of the reciprocal translocations had two fragments smaller than 6 Mbp and thus unbalanced translocations were detectable in all embryos. In the Robertsonian translocation group (17 PGD procedures), a total of 122 embryos from seven different translocations were analysed. Diagnoses could be obtained for all the cases with a total of 40 embryos (32.8%) diagnosed as ‘normal or balanced’, 14 (11.5%) as ‘unbalanced’, 43 (35.2%) as ‘aneuploid’ and 25 (20.5%) as ‘unbalanced and aneuploid’.

Aneuploidy analysis Of 402 embryos analysed, 229 (57.0%) showed aneuploidy, whether balanced or unbalanced for the structural abnormality (Table 2). A total of 92 embryos (22.9%) were diagnosed as unbalanced only and normal for all the other chromosomes. In the reciprocal translocation group, a total of 28 of 280 (10.0%) embryos showed aneuploidy for chromosomes not

626 Table 2

P Colls et al. Array CGH results according to maternal age.

Age (years)

PGD cases

Embryos analysed

Unbalanced only

Aneuploid only

Unbalanced + aneuploid

Total aneuploid

<35 35–39 >39

29 12 6

250 111 41

63 (25.2) 25 (22.5) 4 (9.8)

63 (25.2) 30 (27.0) 13 (32.7)

68 (27.2) 38 (34.2) 17 (41.5)

131 (52.4) 68 (61.3) 30 (73.2)

Values are n or n (%). CGH = comparative genome hybridization; PGD = preimplantation genetic diagnosis.

involved in the translocation or any of the five additional chromosomes included in the FISH panel for aneuploidy (13, 16, 18, 21, 22). These, therefore, would have been missed if FISH had been used alone. In the Robertsonian translocation group, a total of 122 embryos were analysed, of which 10 (8.2%) showed aneuploidy for chromosomes not included in the standard 12-probe FISH panel (X, Y, 8, 13, 14, 15, 16, 17, 18, 21, 22). Overall, 38 (9.4%) of 402 embryos analysed presented one or more aneuploidies that would not have been detected with the use of standard FISH methods. In order to assess the usefulness of the simultaneous aneuploidy analysis, the PGD cycles were divided into three groups according to maternal age (Table 2). The first group (<35 years old) included 29 PGD cases with a total of 250 analysed embryos, the second (35–39 years old) included 12 cases with 111 analysed embryos and the third group (>39 years old) comprised six cases and 41 analysed embryos. The frequency of ‘unbalanced only’ embryos in the three groups was 25.2%, 22.5% and 9.8%, respectively. The significantly lower (P < 0.05) incidence of embryos unbalanced for the translocation but normal for the other chromosomes in the >39-years group was due to an increase in the incidence of aneuploidy for chromosomes not involved in the structural rearrangement due to advanced maternal age.

Table 3

Error rate for translocations Fifty-four embryos from seven array CGH reciprocal translocation cases were reanalysed by FISH using combinations of probes as described above (Table 3). A total of 268 cells were analysed. Of the 44 embryos previously diagnosed as ‘unbalanced’ by array CGH, 43 (97.7%) yielded the same diagnosis in the FISH reanalysis, while one (2.3%) was diagnosed as ‘normal or balanced’. All 10 embryos (100%) diagnosed as ‘normal or balanced’ with array CGH, whether or not they also showed aneuploidy, were also diagnosed as ‘normal or balanced’ by FISH. Based on these data, the incidence of false-positive and false-negative results were calculated as 2.3% and 0%, respectively, with a total error rate of 1.9%.

Average number of abnormal cells in mosaic embryos A total of 102 embryos classified as abnormal by array CGH (some from preimplantation genetic screening and some from PGD for translocations) were reanalysed entirely or partially by FISH for 12 chromosomes plus those chromosomes found to be abnormal by array CGH. Of these, 52 were homogeneously aneuploid and 45 were aneuploid mosaic or chaotic mosaic with either two or more aneuploid

Reanalysis by FISH of embryos analysed by array CGH.

Karyotype

PGD by array CGH Embryos analysed

46,XX,t(7;8)(q21.2;q22.3) 46,XY,t(5;9)(q33;p13) 46,XX,t(11;22)(q23.3;q11.2) 46,XY,t(2;6)(q36.1;p25.3) 46,XY,t(5;22)(p14;q12.2) 46,XY,t(9;11)(q22;p13) 46,XY,inv(7)(q22q36.1), t(10;11)(q22.3;p13) Total

Normal or balanceda

Reanalysis by FISH Unbalanced

Reanalysed

Normal or balanced array CGH Normal or balanced

Unbalanced array CGH

Unbalanced

Normal or balanced

Unbalanced

10 5 12 14 9 8 14

1 2 4 5 3 3 3

9 3 8 9 6 5 11

8 4 11 9 6 6 10

0 0 3 4 1 2 0

0 0 0 0 0 0 0

0 0 0 0 0 0 1

8 4 8 5 5 4 9

72

21

51

54

10

0

1

43

Values are n. The total error rate by array CGH was 1.90% (false positive 2.30%; false negative 0%). CGH = comparative genome hybridization; FISH = fluorescent in-situ hybridization. a Includes embryos that showed aneuploidy but were ‘normal or balanced’ for the structural abnormality.

Array CGH for translocations in preimplantation human embryos cell lines (in all 45 embryos, all cells were abnormal). Among the remaining five embryos, one had 18% abnormal cells, three had 66% abnormal cells and one had 90% abnormal cells. Overall, for the 102 abnormal embryos, the average number of abnormal cells was 97.3% (719/739). For the mosaic embryos, the average number of abnormal cells was 94.5% (343/363). Based on the study’s criteria, embryos with <25% abnormal cells were classified as errors, for a total false-positive error rate of 1%.

Screening potential of array CGH A total of 961 PGD cases that were performed by FISH between 1999 and 2010 were retrospectively reviewed; 926 of these were carriers of reciprocal translocations. The size of the smallest fragment of the rearrangement was determined for each case. A total of 203 (21.9%) cases had one fragment smaller than 6 Mbp and none had two or more fragments smaller than this size. Thus all translocations detectable by conventional karyotype analysis would have been analysable by array CGH.

Pregnancy outcome Overall, embryos were not transferred in 20.5% (9/44) of cycles due to lack of normal or balanced embryos available to transfer. A clinical pregnancy was achieved in 47.7% (21/44) of the cycles started and in 60.0% of those with transfer (21/35). In the Robertsonian translocation group, the pregnancy rates for cycles started and cycles with transfer were 71.4% (10/14) and 83.3% (10/12), respectively. In the reciprocal translocation group, the pregnancy rates for cycles started and cycles with transfer were 36.7% (11/30) and 47.8% (11/23), respectively. A successful pregnancy was defined as a delivery or an ongoing pregnancy in the third trimester. Successful pregnancy rates per cycles started were 64.3% and 33.3% for Robertsonian and reciprocal translocations, respectively. Successful pregnancy rates per cycles with transfer were 75% and 43.5%, respectively, for the mentioned groups. Overall, 90.5% (19/21) of the pregnancies were successful, with 90.0% (9/10) and 90.9% (10/11) for Robertsonian and reciprocal translocation groups, respectively. Loss rates were therefore 9.5%, 10.0% and 9.1%, respectively.

Discussion Array CGH has proven to be a powerful tool in PGD by successfully addressing most of the limitations that the FISH approach has presented, and therefore its use in aneuploidy screening has become routine in many laboratories (Gutie ´rrez-Mateo et al., 2011; Le Caignec et al., 2006; Wells et al., 2008). The test has been fully validated for that purpose showing a low 1.8% error rate (Gutie ´rrez-Mateo et al., 2011). Its use in PGD for structural chromosome abnormalities is even more advantageous since the technique not only overcomes limitations of FISH but it also eliminates the time-consuming work up necessary to prepare PGD cases for structural rearrangements. For diagnosis of translocations with array CGH, the only limiting factor is the resolution of the array.

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The present study, using the 24Sure aneuploidy array was able to detect fragments of 6 Mbp in single blastomeres and 5 Mbp in trophoectoderm cells, a resolution slightly lower than that found in other studies performed with high-density arrays (2.5–2.8 Mbp; Alfarawati et al., 2011; Fiorentino et al., 2011). For proper diagnosis, at least three of the four chromosome fragments must be detected. A possible exception is when all the cells are likely to be aneuploid and the embryo would not be considered for transfer on that basis alone, as it is the case in chaotic mosaicism (Gutie ´rrez-Mateo et al., 2011). The reanalysis of non-transferred embryos by FISH with probes specific for the structural chromosome abnormality indicates an error rate of 1.9%, which does not differ from that obtained in array CGH for aneuploidy (Gutie ´rrez-Mateo et al., 2011) and suggests that even though mosaicism is common, errors occur infrequently with this technique. No other microarray platform has been validated by reanalysis of non-transferred embryos with an alternative technique. This study surveyed a database of FISH results for translocation carriers and found that none of the 926 reciprocal translocation cases had two fragments smaller than the resolution limit of the array used for array CGH in this study, suggesting that array CGH could have been used for all of those cases. Since the limit of this array also coincides with the resolution limit of karyotyping (about 4 Mbp) it is possible that the population previously screened with FISH excluded patients with cryptic translocations (one in 8000 in the human population; Wise et al., 2009). For those patients, higher-resolution arrays may be needed (Fiorentino et al., 2011). Another important advantage of array CGH for diagnosis of structural chromosome abnormalities is that it allows simultaneous detection of aneuploidy for all 24 chromosomes without compromising the accuracy of the test. In this study, 26.4% of the embryos tested were normal or balanced for the structural rearrangement but they were aneuploid. Even with the use of the additional aneuploidy FISH panel for reciprocal translocations, which includes chromosomes 13, 16, 18, 21 and 22 and the use of the 12-chromosome FISH panel, including chromosomes X, Y, 8, 13, 14, 15, 16, 17, 18, 20, 21 and 22 for Robertsonian translocations, 9.4% of the aneuploid embryos would not have been detected. Those embryos could have been potentially transferred, increasing the risk of spontaneous abortions or implantation failure. As this study shows, in patients aged >30 years, the frequency of embryos with unbalanced structural abnormality but normal chromosome number is 9.8%. This is less than half that found in younger patients (22.5% for patients aged 35–39 years, and 25.2% in patients aged <35 years). Therefore, the use of array CGH for structural chromosome abnormalities is even more critical in older patients. In terms of pregnancy rates per cycle started as well as per cycle with transfer, these were superior to those obtained with FISH in previous studies (Fischer et al., 2010), overall and also for Robertsonian and reciprocal translocation groups. In conclusion, the use of array CGH for diagnosis of structural chromosome abnormalities in early human embryos is more efficient than FISH by increasing the pregnancy rate and more practical than single-nucleotide polymorphism

628 arrays since it can correctly detect unbalanced translocations. It is applicable to any translocation previously detected by karyotyping, allows simultaneous screening of all chromosomes for aneuploidy and does not require prior analysis of parental DNA. The technique has now been validated and shows an acceptable error rate.

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