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Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing
ORIGINAL ARTICLE: GENETICS
Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing María Vera-Rodríguez, M.Sc.,a Claude-Edouard Michel, Ph.D.,b Amparo Mercader, Ph.D.,c,d Alex J. Bladon, M.Phys., Ph.D.,b Lorena Rodrigo, Ph.D.,a,d Felix Kokocinski, Ph.D.,b Emilia Mateu, Ph.D.,a,d n, MD, Ph.D.,a,c,d,f and Carmen Rubio, Ph.D.a,d Nasser Al-Asmar, MSc,e David Blesa, PhD,a,d Carlos Simo a
Igenomix, Valencia, Spain; b Illumina, Cambridge, United Kingdom; c Instituto Universitario IVI, Valencia University; n Instituto Valenciano de Infertilidad (FIVI)/INCLIVA, Valencia, Spain; e IviGen, Miami, Florida; and Fundacio f Department of Obstetrics and Gynecology, School of Medicine, Stanford University, Stanford, California d
Objective: To evaluate the ability of next-generation sequencing (NGS) to detect pure and mosaic segmental aneuploidies in trophectoderm biopsies and to identify distribution patterns in whole blastocysts. Design: Validation study. Setting: Reference laboratory. Patient(s): Seventy couples with known karyotypes who had undergone preimplantation genetic screening with diagnoses at the blastocyst stage using array comparative genomic hybridization (aCGH). Intervention(s): None. Main Outcome Measure(s): Concordance rates for segmental and whole-chromosome aneuploidies determined between aCGH and NGS, and estimates of mosaicism levels of segmental aneuploidies in fixed blastocysts. Result(s): We used NGS with amplified DNA from trophectoderm biopsies in which segmental aneuploidies had been previously detected by array comparative genomic hybridization (aCGH). Single-cell fluorescent in situ hybridization (FISH) was then used as an independent form of analysis. The concordance rate between NGS and aCGH was 124 (98.4%) of 126 for the detection of segmental aneuploidies, and 48 (96.0%) of 50 for whole-chromosome aneuploidies. The overall concordance rate was 99.8% (2,276 of 2,280 chromosomes assessed). After FISH analyses with 41.4 24.3 cells per blastocyst, 26 (92.9%) of 28 segmentals detected by aCGH and NGS were confirmed. The FISH analysis did not detect the segmentals in two blastocysts, in which all cells analyzed were euploid. Conclusion(s): This is the first report analyzing distribution patterns of segmental aneuploidies in trophectoderm biopsy by NGS. We have demonstrated that NGS allows the detection of pure and mosaic segmental aneuploidies with the same efficiency as aCGH. The FISH analysis confirmed the existence of these events in the trophectoderm and the inner cell Use your smartphone mass. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Array comparative genomic hybridization (aCGH), blastocyst, mosaicism, nextand connect to the generation sequencing, segmental aneuploidy Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/verarodriguezm-segmental-aneuploidies-ngs/
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hromosomal abnormalities are found in over 50% of embryos involved in assisted reproduc-
tive techniques, including those in the blastocyst stage (1). Occasionally a small piece of chromosome is gained
Received September 15, 2015; revised and accepted December 16, 2015. M.V.-R. has nothing to disclose. C.-E.M. participated in the development, optimization, and validation of the VeriSeq PGS methodology and protocol. A.M. has nothing to disclose. A.J.B. participated in the development, optimization, and validation of the VeriSeq PGS methodology and protocol. L.R. has nothing to disclose. F.K. participated in the development, optimization, and validation of the VeriSeq PGS methodology and protocol. E.M. has nothing to disclose. N.A.-A. has nothing to disclose. D.B. has nothing to disclose. C.S. has nothing to disclose. C.R. has nothing to disclose. Reprint requests: María Vera-Rodríguez, M.Sc., Igenomix, Catedratico Agustin Escardino, 9, Paterna, Spain (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.12.022
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or lost during cell division, resulting in either segmental or partial aneuploidy. Segmental aneuploidies are inherited from a carrier of a balanced structural abnormality, or they can be generated de novo by a meiosis error during gametogenesis or by a mitosis error during embryo development. The frequency of de novo segmental aneuploidies varies between the stages of embryo development (2). The frequency of segmental aneuploidies during the cleavage stage
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ORIGINAL ARTICLE: GENETICS varies between studies, from as low as 3.9% (3), 5% (4), or 15% (2, 5), to as high as 70% (6) with no reported correlation with maternal age (2–5). The reported incidence of segmental aneuploidies at the blastocyst stage ranges from 6% to 7% (2, 7). Several methods currently allow for the study of segmental aneuploidies, including fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) arrays, and comparative genomic hybridization arrays (aCGH). The reported copy number resolution of comparative genomic hybridization arrays is approximately 20 Mb, although aCGH has been shown to detect segmental aneuploidies as small as 5 Mb and 6 Mb for trophectoderm and blastomeres, respectively (8). It is interesting that a recent study using cells lines to mimic different levels of mosaicism showed that aCGH allowed for the detection of a shift from normality when >25% aneuploid cells were present (9). This study supported a previous report in which aCGH from trophectoderm biopsies detected mosaicism in 95% of the embryos, when mosaicism levels were higher than 30% (10). Next-generation sequencing (NGS) was recently presented as an effective technique for the analysis of copy number variation in single cells (11). This methodology then proved to be useful for the detection of whole-chromosome aneuploidies and segmental aneuploidies in single blastomeres (12–16) and in trophectoderm cells (15–17). Next-generation sequencing has also been applied in clinical practice for the detection of segmental aneuploidies from translocations and inversion carriers, showing no significant differences in pregnancy rates when compared with trophectoderm cells screened by SNP arrays (18). Another study demonstrated that NGS screening of single blastomeres led to a higher pregnancy rate compared with a control group without preimplantation genetic screening (PGS) (19). A randomized clinical study comparing NGS and aCGH screening showed comparable pregnancy rates between the two techniques (20). Although NGS can offer a higher dynamic range to discriminate segmental aneuploidies with different patterns of mosaicism in comparison to aCGH, NGS has yet to be validated for the detection of aneuploidies with a mosaic pattern in trophectoderm biopsy. Here, we present a comparative study of the use of NGS and aCGH in the detection of whole-chromosome aneuploidies and segmental aneuploidies as small as 10 Mb with mosaic patterns.
MATERIALS AND METHODS Study Design Samples were collected from clinical PGS and preimplantation genetic diagnosis programs using aCGH at the blastocyst stage. In the period between March 2013 and May 2014, every sample containing at least one segmental aneuploidy was selected for this study. The project was approved by the institutional review board of the Instituto Valenciano Infertilidad (IVI) in Valencia, Spain. Whole-genome amplification (WGA) material was retrospectively collected for NGS analysis, and whole blastocysts, if available, for FISH analysis. Samples were assigned to the control group if the segmental abnormalities were inherited from reciprocal translocations carriers, or
to the study group if they came from couples with normal karyotypes, meaning that they were generated de novo during gametogenesis or embryo development. We collected samples from 12 couples with reciprocal translocations for the control group, with an average maternal age of 34.0 3.7 years old. In this group, all PGS indications were structural abnormality in the karyotype, as all embryos came from translocations carriers. In total, 22 blastocysts with 40 segmental aneuploidies and 11 wholechromosome aneuploidies were selected for this group (Fig. 1). Each aneuploidy was classified either as pure or mosaic according to the aCGH profile. The study group consisted of samples from 58 couples with an average maternal age of 36.7 4.7 years old. The PGS clinical indications were repetitive implantation failure (n ¼ 20), advanced maternal age (n ¼ 17), recurrent miscarriage (n ¼ 12), previous aneuploid pregnancy (n ¼ 5), male factor infertility (n ¼ 3), and abnormal sperm FISH (n ¼ 1). In some instances, more than one clinical indication was described for the same patient. In total, 66 blastocysts were selected with a total of 84 segmental aneuploidies and 37 whole-chromosome aneuploidies (see Fig. 1). Each aneuploidy was defined either as pure or mosaic. Finally, a subset of whole blastocysts still available at the time of the study (2–6 months later) underwent FISH analysis of individual cells. This analysis including blastocysts from both the control and study groups (see Fig. 1).
Embryo Biopsy and Vitrification Trophectoderm biopsy was performed on day 5 or day 6 as previously described elsewhere (21). After the biopsy, samples were washed in 5 mL of phosphate-buffered saline 1% polyvinylpyrrolidone buffer (Cell Signaling Technology) and transferred to a 0.2 mL polymerase chain reaction (PCR) tube under sterile conditions. The tubes were stored at 20 C until further analysis. When a chromosomally normal embryo transfer was planned for a deferred cycle, all embryos were immediately cryopreserved after the trophectoderm biopsy. The vitrification protocol was completed as previously described elsewhere (22).
Array Comparative Genome Hybridization We performed DNA extraction and WGA using the Sureplex Kit (Illumina), and the array protocol was performed according to the manufacturer's instructions (3). Data were analyzed using BlueFuse Multi software, version 3.2 (Illumina). Aneuploidies were classified as whole-chromosome when the majority of the length of the chromosome showed a copynumber change and was automatically identified by the software. Segmental aneuploidies were manually identified when only a fragment of the chromosome deviated from standard thresholds for euploidy. Taking into account empirical resolution levels described by the manufacturer, only segmental imbalances above 10 Mb were considered for the study. Because multiple cells were analyzed at the same time, we sought to differentiate between pure and mosaic aneuploidies. It has been reported that aCGH can detect 25% of aneuploid VOL. - NO. - / - 2016
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FIGURE 1
Study design for the analysis of concordance rates between next-generation sequencing (NGS) and array comparative genomic hybridization (aCGH). Number of samples and events included in the study, divided between the control group (samples from translocations carriers) and study group (samples from couples with normal karyotype). Number of events from both groups reanalyzed by fluorescent in situ hybridization (FISH) of individual blastocyst cells. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
cells (9). However, due to the limitations of our sample size, we used X-separation as a reference for experimental error, calculated as the absolute log2 value for the X chromosome. When the aneuploidy absolute log2 value was higher than half of the X-separation, it was labeled as pure because it was likely that all of the analyzed cells contained that aneuploidy. When the absolute aneuploidy log2 ratio was not higher than half of the X-separation, the event was labeled as mosaic because an unknown number of cells harbored the anomaly.
Next-generation Sequencing We performed NGS on WGA products amplified with the SurePlex DNA Amplification System (Illumina). The NGS libraries were prepared using a VeriSeq PGS-MiSeq kit (Illumina) from quantified WGA products. The resulting library pools were sequenced by synthesis on a MiSeq instrument (Illumina) using the VeriSeq PGS recipe. The WGA SurePlex PCR products of each biopsy and control were quantified using a fluorometry-based method (Qubit High Sensitivity dsDNA kit; Life Technologies). One nanogram of double-stranded DNA of each unpurified WGA product was tagged and fragmented by the VeriSeq PGS transposome, creating a population of fragmented nucleic acid molecules. A limited-cycle PCR reaction used the unique adapter sequences at the ends of the fragments to amplify the insert DNA and add index sequences (barcodes) on both ends of the DNA, enabling dual-indexed sequencing.
The libraries were purified using Solid Phase Reversible Immobilization paramagnetic bead-based technology (AMPure XP beads; Beckman Coulter). Purified libraries were normalized using the VeriSeq PGS bead-based sample normalization kit and pooled in accordance with the manufacturer's instructions. Dual index 36 base pair reads (1x36 DI) sequencing was performed according to the VeriSeq PGS recipe (Rev. O), using a MiSeq Reagent Kit v3-PGS (Illumina). MiSeq Reporter software (Illumina) was used to perform on-board secondary data analysis using the VeriSeq PGS workflow. BlueFuse Multi Software, version 4.2 (BFM 4.2; Illumina), was used to analyze sequencing data generated by the MiSeq instrument and to report results as previously described elsewhere (14). The NGS pattern detection and classification of aneuploidies was determined by copy number variation (CNV) values. Aneuploidies with CNV values lower than 1.20 or higher than 2.80 were labeled as pure; CNV values between 1.80 and 2.20 were considered euploid; and aneuploidies with CNV values between 1.2 and 1.80 or between 2.2 and 2.80 were considered mosaic. We adopted differences of 0.2 copies to differentiate between pure and mosaic aneuploidies as NGS was expected to have a higher dynamic range than aCGH.
Whole-blastocyst FISH Analysis Chromosomally abnormal blastocysts were fixed as described by Mir et al. (23). The slides were stored at 20 C before
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ORIGINAL ARTICLE: GENETICS analysis for a maximum of 2 weeks. The FISH analysis was performed using subtelomeric DNA probes (Abbott) for the region involved in the segmental aneuploidy according to the manufacturer's instructions. Nuclei with unclear or tetraploid signals were excluded from the study, and blastocysts with less than 10 informative cells by FISH were also excluded. Cells were considered informative when there was no overlap between them, they had a well-defined outline, and they showed signals of similar size separated by a minimal distance corresponding to twice the diameter of each of the cells. In total, 24 whole blastocysts were analyzed with an average number of 41.4 24.3 informative cells.
Data Analysis To detect differences in the labeling of whole-chromosome and partial aneuploidy between the two sequencing methods, statistical comparisons were performed using SPSS Statistics software, version 21 (IBM). A Pearson chi-square test was performed to compare frequencies between groups. To evaluate the similarity between the techniques, we calculated concordance rates. For the comparison between technique results, we calculated two type of concordance rates: the concordance rate per analyzed chromosome, where we considered the total number of chromosomes independently if they were called as euploid or aneuploidy (24 chromosomes per embryo); and the concordance rate per aneuploid chromosome, where we considered only the detected aneuploidies. In addition, each type of concordance rate was calculated according to the type of aneuploidies: all aneuploidies, whole-chromosome aneuploidies, or segmental aneuploidies. For FISH, only the concordance rate per aneuploid chromosome was calculated, as all analyzed chromosomes by FISH were segmental aneuploidies detected previously by aCGH and NGS.
RESULTS Concordance Rate Between NGS and aCGH The concordance rates of NGS and aCGH were calculated independently of the type of the aneuploidy detected. First, all chromosomes analyzed were taken into account (91 embryos 24 chromosomes ¼ 2,184 chromosomes). The NGS results were found to be nonconcordant with aCGH results for four chromosomes. In all cases, NGS detected them as aneuploid while aCGH classified them as euploid; this gave a final concordance rate between NGS and aCGH of 99.8% per chromosome analyzed (Fig. 2A). From the 176 detected aneuploidies, 172 were concordant between platforms; the concordance rate per aneuploid chromosome was 97.7% (see Fig. 2A).
Detection of Whole-chromosome Aneuploidies by NGS and aCGH From the 91 blastocysts with at least one segmental event detected by aCGH, 29 also showed whole-chromosome aneuploidies, resulting in a 30.5% coexistence rate between segmental and whole-chromosome events. In total, 48 whole-chromosome aneuploidies were identified, with
54.2% showing chromosome losses (n ¼ 26) and 45.8% showing chromosomal gains (n ¼ 22). We next differentiated between pure and mosaic patterns and found 44 aneuploidies with a pure pattern (91.7%) and 4 aneuploidies that were mosaic (9.1%). Of the pure whole-chromosome aneuploidies, all events were confirmed by NGS technology (Supplemental Fig. 1A, available online), although one of them showed a mosaic pattern by NGS (see ID:PA95, chromosome 22 in Supplemental Table 1, available online). In the mosaic group, all events detected by aCGH were also confirmed (see Supplemental Fig. 1B) and showed the same pattern with NGS. In addition, NGS detected two additional whole-chromosome aneuploidies with a mosaic pattern that were not originally detected by aCGH. Both were found in the same sample (see Supplemental Fig. 1C; Supplemental Table 1, see ID:PA63, chromosomes 12 and 16). In summary, the total concordance rate for the detection of whole-chromosome aneuploidies between aCGH and NGS per analyzed chromosome was 99.9% (2,182 of 2,184; see Fig. 2A). The concordance rate per aneuploid chromosome was 96.0% (48 of 50; see Fig. 2A).
Detection of Segmental Aneuploidies by NGS and aCGH Segmental aneuploidies (n ¼ 124) were more frequently discovered as losses (62.1%) than as gains (37.9%) of chromosomal fragments. We did not find a statistically significant difference between segmental and whole-chromosome aneuploidies. Segmental aneuploidies were differentiated between the control group (n ¼ 40; from reciprocal translocation carriers) and the study group (n ¼ 84; from normal karyotype couples). In the control group, all 40 events detected by aCGH were also detected by NGS (concordance rate per segmental aneuploidy detected in the control group: 100%; 40 of 40; see Fig. 2A) from the 600 chromosomes analyzed in total (concordance rate per analyzed chromosome: 100%, 600 of 600; see Fig. 2A). In the study group, aneuploidies were classified into pure (n ¼ 58) and mosaic (n ¼ 26) segmental events according to the aCGH pattern. In the pure group, all segmental aneuploidies were also identified by NGS (concordance rate per pure segmental aneuploidy detected in the study group: 100%, 58 of 58; see Fig. 2A), with 55 showing a pure pattern (Fig. 3A) and 3 a mosaic (see Fig. 3B; Supplemental Table 1, see ID:PA55 chromosome 20q, ID:PA66 chromosome 1q, ID:PA67, chromosome 9q). In the group of mosaic segmental aneuploidies detected by aCGH, 26 events were detected by NGS showing the same mosaic pattern (see Fig. 3C). In addition, NGS detected two new mosaic segmental events that were not identified by aCGH (see Fig. 3D; Supplemental Table 1, see ID:PA73 chromosome 8q, ID:PA90 chromosome 9p), giving a concordance rate of 92.9% (26 of 28; see Fig. 2A) for the detection of mosaic segmental aneuploidies in the study group. Unfortunately, full FISH blastocyst reanalysis was not available for these two samples. VOL. - NO. - / - 2016
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FIGURE 2
Concordance rates between next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and fluorescent in situ hybridization (FISH) for aneuploidy detection. (A) The concordance rates were calculated per analyzed chromosome, including euploid and aneuploidy chromosomes, and exclusively per aneuploid chromosome. Each type of concordance rate was calculated for all aneuploidies, for whole-chromosome aneuploidies and for segmental aneuploidies. Finally, the concordance rates for segmental aneuploidies were calculated for those events detected in the control group (translocations carriers) and the study group (normal karyotypes). (B) The concordance rates for those samples with FISH analyses were calculated per aneuploidy chromosome as all events analyzed were previously detected as segmental aneuploidies by aCGH and NGS. The concordance rates were also differentiated between the control and the study group, and between pure and mosaic events inside the study group. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
The total concordance rate for the detection of segmental aneuploidies between aCGH and NGS independently of the parental karyotype and the detected pattern was 98.4% (124 of 126) per aneuploidy detected and 99.9% (2,182 of 2,184) per analyzed chromosome (see Fig. 2A).
FISH Analysis to Determine Distribution Pattern of Segmental Aneuploidies Twenty-four chromosome spreads from full blastocysts were analyzed by FISH at the single-cell level; 6 blastocysts
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FIGURE 3
Comparison of segmental aneuploidy detection by array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS). (A) Both aCGH and NGS detected similarly a pure segmental aneuploidy for chromosome 6. (B) One segmental aneuploidy for chromosome 20 identified as pure by aCGH was detected by NGS as a mosaic pattern. (C) Array CGH and NGS detected an aneuploidy for chromosome 3 showing a mosaic pattern in both cases. (D) One mosaic segmental aneuploidy for chromosome 8 detected by NGS was not originally detected by aCGH. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
originated from patients with an altered karyotype (control group), and 18 blastocysts from patients with normal karyotype (study group). In total, we studied 28 segmental events from the control and study groups (Fig. 4). FISH analyses detected 10 events in blastocyst from the control group with 93% of the cells showing the segmental aneuploidy (concordance rate per detected segmental aneuploidy in the control group: 100%; 10 of 10; see Fig. 2B). All patterns, whether pure or mosaic, were consistent between the three technologies with the exception of one event that was detected as pure by aCGH and NGS whereas FISH detected a low degree mosaic with 25 (92.6%) of 27 aneuploid cells.
We analyzed 18 segmental events by FISH from the study group patients with normal karyotypes (see Fig. 4). In the pure group, nine out of the 10 segmental events detected by aCGH and NGS were also detected by FISH (concordance rate for pure events in the study group: 90%, 9 of 10; see Fig. 2B) but with a variable percentage of aneuploid cells, showing mainly mosaic patterns (n ¼ 7). There was one pure event (9q, 45.5 Mb) that was not detected in the 21 cells analyzed by FISH; this discrepancy may be a technical artifact. The percentage of aneuploid cells in the pure study group was 63%. In the mosaic events from the study group, FISH analyses detected 7 out 8 segmentals (see Fig. 4), which is a VOL. - NO. - / - 2016
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FIGURE 4
Segmental aneuploidies analyzed by array comparative genomic hybridization (aCGH), next-generation sequencing (NGS), and fluorescent in situ hybridization (FISH). Aneuploidies were identified as pure if they scored beyond the euploidy confidence range in aCGH and NGS, or if all cells analyzed by FISH were aneuploid. Aneuploidies were identified as mosaic if they showed an intermediate value within the confidence range, or, in FISH analysis, if aneuploid and euploid cells were detected in the same embryo. The color scale represents the percentage of aneuploid cells in the group of cells analyzed by FISH, with dark green indicating 100% aneuploidy, dark red indicating 0 aneuploidy, and intermediary colors indicating different levels of mosaicism (see Supplemental Fig. 2). Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
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ORIGINAL ARTICLE: GENETICS concordance rate per mosaic segmental event in the study group of 87.5% (7 of 8; see Fig. 2B). All seven showed a mosaic pattern, ranging from 11.7% to 64.7% of aneuploid cells. There was only one segmental event (þ5p, 54 Mb) that was not detected by FISH in the 29 cells analyzed. The final percentage of cells in the mosaic study group with segmental events detected was 24%. In total, the concordance rate per segmental aneuploidy in the study group, independent of the pattern, was 16 (88.9%) of 18 (see Fig. 2B). Finally, and regardless of the parental karyotypes of the blastocysts, the concordance rate between FISH, aCGH, and NGS for the detection of segmental aneuploidies was 26 (92.9%) of 28 (see Fig. 2B), with only two embryos showing discordant results (see Fig. 4).
DISCUSSION The evidence presented here supports the use of NGS for the detection of segmental aneuploidies in trophectoderm biopsies that are typically measured by aCGH. Although we focused our study on the detection of segmental aneuploidies, we also studied whole-chromosome aneuploidies in parallel. Our data support the use of NGS as a viable technique for detecting not only aneuploidies showing a clear, pure pattern, but also for analyzing samples with mosaic patterns. In fact, we found that mosaic events can be identified more easily due to the increased dynamic range of NGS data using BlueFuse Multi profiles. Additionally, we showed that FISH analysis of whole blastocysts could be used to confirm the presence and pattern of segmental aneuploidies. Our results suggest that NGS can successfully identify whole-chromosome and segmental aneuploidies previously detected by aCGH, offering, in addition, a higher dynamic range and a more cost-effective laboratory routine if there is a higher sample throughput. Previous studies have described a minimum size for the detection of imbalances by NGS of approximately 14 Mb (14, 15, 17), with only one recent report detecting imbalances of 1.8 Mb by NGS (24). Here, we report the detection of segmental aneuploidies as small as 10 Mb by NGS with the same efficiency as aCGH. It is interesting that we detected four additional aneuploidies by NGS: two whole-chromosome and two segmental aneuploidies, all of them exhibiting a mosaic pattern. Unfortunately, FISH could not be performed in these samples, and we could not conclude which technique reflected the most accurate chromosomal status of the blastocyst. The relevance of segmental aneuploidies in the development of the human embryo is often questioned in PGS programs. Here, we have shown that most of the segmental aneuploidies detected by NGS or aCGH were not technical artifacts, because they were also detected by FISH analysis. Even in the two cases in which the segmental aneuploidies were not confirmed by FISH, we do not know whether the discrepancy was due to a technical WGA artifact or to mosaicism in the blastocyst, which we found to be a frequent phenomenon in aneuploid human blastocysts. Furthermore, it has been described that the frequency of segmental aneuploidies in miscarriages is approximately 6% (25) and approximately 5 in 10,000 newborns (26). These numbers match the
described frequency of segmental aneuploidies in blastocysts (6% to 7%) (2, 7), implying that these types of abnormalities should be taken into consideration in clinical practice. Recently, it was reported that the detection of segmental aneuploidies in single cells is not optimal when the cells are in the S phase of the cell cycle (27). It is important to note that we used trophectoderm biopsy, increasing the probability of obtaining cells in the G0/G1 phase. Nevertheless, this phenomenon could underlie the two instances in which FISH did not detect the segmental aneuploidy detected by NGS and aCGH (7.1%). Additional studies are needed to determine the maximum mosaicism level compatible with the development of a healthy embryo. Array CGH has been shown to detect mosaicism degrees higher than 25% (9), which may explain why those events were detected as mosaic by FISH with more than 75% of aneuploid cells shown as pure by aCGH or NGS. The threshold to detect mosaicism in trophectoderm cells is a parameter that needs further discussion given that an increase in the sensitivity of the techniques described could result in a simultaneous decrease in the specificity, which could lead to a decrease in the detection of euploid embryos. Three main points must be addressed to optimize the identification of segmental aneuploidies. First, it is important to decrease the biological variability; this can be accomplished by using a common starting material for both techniques, such as the same amplified DNA. In some studies, different trophectoderm biopsies have been used to compare different methods of analysis (16). As such, it is difficult to know whether any discrepancies are due to technical or biological issues, such as mosaicism. Second, to better characterize the presence of segmental aneuploidies a third technique with a different molecular basis is recommended. For instance, when comparing aCGH and NGS, because they both share the same first WGA step, quantitative PCR or FISH can be used to confirm results and sort out any discrepancies. Currently, segmental aneuploidies can only be identified using FISH with specific probes. Third, it is necessary to characterize the parental karyotypes before the PGS cycle to avoid the mislabeling of very small segmental aneuploidies. We accomplished each of these using NGS and aCGH to assess segmental aneuploidy. Unfortunately, we could not analyze the blastocysts in which discrepancies were found between NGS and aCGH, because they were not available at the time of the study. Nevertheless, FISH analysis was extremely useful to confirm the existence of segmental events as well as the pure or mosaic patterns in the whole blastocyst. Although in our FISH study we did not differentiate between trophectoderm and inner cell mass, previous studies have shown concordant ploidy results between both types of cells (7,28–30); even when euploid or aneuploid mosaicism is present at the blastocyst stage, aneuploid cells are still found in both trophectoderm and inner cell mass cells (30). In conclusion, we have shown that FISH analysis revealed the existence of segmental aneuploidies in the majority of the blastocysts diagnosed, suggesting that most cases are not technical artifacts of aCGH or NGS. In addition, we demonstrated through the analysis of individual cells by FISH that most of the segmental aneuploidies originated from mitotic VOL. - NO. - / - 2016
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Fertility and Sterility® errors creating different degrees of mosaicism, and this pattern can be detected by NGS. Finally, we have validated NGS for segmental aneuploidies with a concordance rate per analyzed chromosome of 99.9 % and 98.4% of concordance rate per detected segmental event. Acknowledgments: The authors thank the clinicians and embryologists at the IVF units utilized in this study, with particular thanks to Pilar Campos for her assistance during blastocyst thawing, and our colleagues in Igenomix for all their helpful suggestions during data analysis.
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Taylor TH, Patrick JL, Gitlin SA, Crain JL, Wilson JM, Griffin DK. Blastocyst euploidy and implantation rates in a young (<35 years) and old (R35 years) presumed fertile and infertile patient population. Fertil Steril 2014;102: 1318–23. Fragouli E, Alfarawati S, Spath K, Jaroudi S, Sarasa J, Enciso M, et al. The origin and impact of embryonic aneuploidy. Hum Genet 2013;132:1001–13. Rodrigo L, Mateu E, Mercader A, Cobo AC, Peinado V, Milan M, et al. New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization. Biomed Res Int 2014;2014:517125. Voullaire L, Wilton L, McBain J, Callaghan T, Williamson R. Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure. Mol Hum Reprod 2002; 8:1035–41. Rabinowitz M, Ryan A, Gemelos G, Hill M, Baner J, Cinnioglu C, et al. Origins and rates of aneuploidy in human blastomeres. Fertil Steril 2012;97: 395–401. 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. Johnson DS, Cinnioglu C, Ross R, Filby A, Gemelos G, Hill M, et al. Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass. Mol Hum Reprod 2010;16:944–9. Colls P, Escudero T, Fischer J, Cekleniak NA, Ben-Ozer S, Meyer B, et al. Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos. Reprod Biomed Online 2012; 24:621–9. Mamas T, Gordon A, Brown A, Harper J, Sengupta S. Detection of aneuploidy by array comparative genomic hybridization using cell lines to mimic a mosaic trophectoderm biopsy. Fertil Steril 2012;97:943–7. Fragouli E, Alfarawati S, Daphnis DD, Goodall NN, Mania A, Griffiths T, et al. Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation. Hum Reprod 2011;26: 480–90. Zhang C, Zhang C, Chen S, Yin X, Pan X, Lin G, et al. A single cell level based method for copy number variation analysis by low coverage massively parallel sequencing. PLoS One 2013;8:e54236. Wang L, Wang X, Zhang J, Song Z, Wang S, Gao Y, et al. Detection of chromosomal aneuploidy in human preimplantation embryos by nextgeneration sequencing. Biol Reprod 2014;90:95. Wang L, Cram DS, Shen J, Wang X, Zhang J, Song Z, et al. Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos. Biol Reprod 2014;91:37. Fiorentino F, Biricik A, Bono S, Spizzichino L, Cotroneo E, Cottone G, et al. Development and validation of a next-generation sequencing-based proto-
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SUPPLEMENTAL FIGURE 1
Comparison of array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) whole-chromosome aneuploidy detection. (A) A pure, whole-chromosome aneuploidy detected by aCGH for chromosome 9 was also detected by NGS as pure. (B) A mosaic wholechromosome aneuploidy detected by aCGH for chromosome 18 showed a similar pattern using NGS. (C) NGS detected mosaic aneuploidies for chromosomes 12 and 16 that were not initially identified by aCGH. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
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SUPPLEMENTAL FIGURE 2
Fluorescent in situ hybridization (FISH) analysis for the detection of segmental aneuploidies in blastocyst cells. (A–C) FISH analysis of blastocyst in which a segmental loss (55.3 Mb) in the p arm of chromosome 1 was previously detected by array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) showing a mosaic pattern. We analyzed 34 cells successfully using Spectrum red probes specific to the p subtelomere region of chromosome 1 (Aquarius; Cytocell), with (A) 12 cells (23.5%) containing a single signal, (B) eight cells (35.3%) with two signals, and (C) 14 cells (41.2%) with three signals. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
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Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing María Vera-Rodríguez, M.Sc.,a Claude-Edouard Michel, Ph.D.,b Amparo Mercader, Ph.D.,c,d Alex J. Bladon, M.Phys., Ph.D.,b Lorena Rodrigo, Ph.D.,a,d Felix Kokocinski, Ph.D.,b Emilia Mateu, Ph.D.,a,d n, MD, Ph.D.,a,c,d,f and Carmen Rubio, Ph.D.a,d Nasser Al-Asmar, MSc,e David Blesa, PhD,a,d Carlos Simo a
Igenomix, Valencia, Spain; b Illumina, Cambridge, United Kingdom; c Instituto Universitario IVI, Valencia University; n Instituto Valenciano de Infertilidad (FIVI)/INCLIVA, Valencia, Spain; e IviGen, Miami, Florida; and Fundacio f Department of Obstetrics and Gynecology, School of Medicine, Stanford University, Stanford, California d
Objective: To evaluate the ability of next-generation sequencing (NGS) to detect pure and mosaic segmental aneuploidies in trophectoderm biopsies and to identify distribution patterns in whole blastocysts. Design: Validation study. Setting: Reference laboratory. Patient(s): Seventy couples with known karyotypes who had undergone preimplantation genetic screening with diagnoses at the blastocyst stage using array comparative genomic hybridization (aCGH). Intervention(s): None. Main Outcome Measure(s): Concordance rates for segmental and whole-chromosome aneuploidies determined between aCGH and NGS, and estimates of mosaicism levels of segmental aneuploidies in fixed blastocysts. Result(s): We used NGS with amplified DNA from trophectoderm biopsies in which segmental aneuploidies had been previously detected by array comparative genomic hybridization (aCGH). Single-cell fluorescent in situ hybridization (FISH) was then used as an independent form of analysis. The concordance rate between NGS and aCGH was 124 (98.4%) of 126 for the detection of segmental aneuploidies, and 48 (96.0%) of 50 for whole-chromosome aneuploidies. The overall concordance rate was 99.8% (2,276 of 2,280 chromosomes assessed). After FISH analyses with 41.4 24.3 cells per blastocyst, 26 (92.9%) of 28 segmentals detected by aCGH and NGS were confirmed. The FISH analysis did not detect the segmentals in two blastocysts, in which all cells analyzed were euploid. Conclusion(s): This is the first report analyzing distribution patterns of segmental aneuploidies in trophectoderm biopsy by NGS. We have demonstrated that NGS allows the detection of pure and mosaic segmental aneuploidies with the same efficiency as aCGH. The FISH analysis confirmed the existence of these events in the trophectoderm and the inner cell Use your smartphone mass. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Array comparative genomic hybridization (aCGH), blastocyst, mosaicism, nextand connect to the generation sequencing, segmental aneuploidy Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/verarodriguezm-segmental-aneuploidies-ngs/
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hromosomal abnormalities are found in over 50% of embryos involved in assisted reproduc-
tive techniques, including those in the blastocyst stage (1). Occasionally a small piece of chromosome is gained
Received September 15, 2015; revised and accepted December 16, 2015. M.V.-R. has nothing to disclose. C.-E.M. participated in the development, optimization, and validation of the VeriSeq PGS methodology and protocol. A.M. has nothing to disclose. A.J.B. participated in the development, optimization, and validation of the VeriSeq PGS methodology and protocol. L.R. has nothing to disclose. F.K. participated in the development, optimization, and validation of the VeriSeq PGS methodology and protocol. E.M. has nothing to disclose. N.A.-A. has nothing to disclose. D.B. has nothing to disclose. C.S. has nothing to disclose. C.R. has nothing to disclose. Reprint requests: María Vera-Rodríguez, M.Sc., Igenomix, Catedratico Agustin Escardino, 9, Paterna, Spain (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.12.022
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or lost during cell division, resulting in either segmental or partial aneuploidy. Segmental aneuploidies are inherited from a carrier of a balanced structural abnormality, or they can be generated de novo by a meiosis error during gametogenesis or by a mitosis error during embryo development. The frequency of de novo segmental aneuploidies varies between the stages of embryo development (2). The frequency of segmental aneuploidies during the cleavage stage
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ORIGINAL ARTICLE: GENETICS varies between studies, from as low as 3.9% (3), 5% (4), or 15% (2, 5), to as high as 70% (6) with no reported correlation with maternal age (2–5). The reported incidence of segmental aneuploidies at the blastocyst stage ranges from 6% to 7% (2, 7). Several methods currently allow for the study of segmental aneuploidies, including fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) arrays, and comparative genomic hybridization arrays (aCGH). The reported copy number resolution of comparative genomic hybridization arrays is approximately 20 Mb, although aCGH has been shown to detect segmental aneuploidies as small as 5 Mb and 6 Mb for trophectoderm and blastomeres, respectively (8). It is interesting that a recent study using cells lines to mimic different levels of mosaicism showed that aCGH allowed for the detection of a shift from normality when >25% aneuploid cells were present (9). This study supported a previous report in which aCGH from trophectoderm biopsies detected mosaicism in 95% of the embryos, when mosaicism levels were higher than 30% (10). Next-generation sequencing (NGS) was recently presented as an effective technique for the analysis of copy number variation in single cells (11). This methodology then proved to be useful for the detection of whole-chromosome aneuploidies and segmental aneuploidies in single blastomeres (12–16) and in trophectoderm cells (15–17). Next-generation sequencing has also been applied in clinical practice for the detection of segmental aneuploidies from translocations and inversion carriers, showing no significant differences in pregnancy rates when compared with trophectoderm cells screened by SNP arrays (18). Another study demonstrated that NGS screening of single blastomeres led to a higher pregnancy rate compared with a control group without preimplantation genetic screening (PGS) (19). A randomized clinical study comparing NGS and aCGH screening showed comparable pregnancy rates between the two techniques (20). Although NGS can offer a higher dynamic range to discriminate segmental aneuploidies with different patterns of mosaicism in comparison to aCGH, NGS has yet to be validated for the detection of aneuploidies with a mosaic pattern in trophectoderm biopsy. Here, we present a comparative study of the use of NGS and aCGH in the detection of whole-chromosome aneuploidies and segmental aneuploidies as small as 10 Mb with mosaic patterns.
MATERIALS AND METHODS Study Design Samples were collected from clinical PGS and preimplantation genetic diagnosis programs using aCGH at the blastocyst stage. In the period between March 2013 and May 2014, every sample containing at least one segmental aneuploidy was selected for this study. The project was approved by the institutional review board of the Instituto Valenciano Infertilidad (IVI) in Valencia, Spain. Whole-genome amplification (WGA) material was retrospectively collected for NGS analysis, and whole blastocysts, if available, for FISH analysis. Samples were assigned to the control group if the segmental abnormalities were inherited from reciprocal translocations carriers, or
to the study group if they came from couples with normal karyotypes, meaning that they were generated de novo during gametogenesis or embryo development. We collected samples from 12 couples with reciprocal translocations for the control group, with an average maternal age of 34.0 3.7 years old. In this group, all PGS indications were structural abnormality in the karyotype, as all embryos came from translocations carriers. In total, 22 blastocysts with 40 segmental aneuploidies and 11 wholechromosome aneuploidies were selected for this group (Fig. 1). Each aneuploidy was classified either as pure or mosaic according to the aCGH profile. The study group consisted of samples from 58 couples with an average maternal age of 36.7 4.7 years old. The PGS clinical indications were repetitive implantation failure (n ¼ 20), advanced maternal age (n ¼ 17), recurrent miscarriage (n ¼ 12), previous aneuploid pregnancy (n ¼ 5), male factor infertility (n ¼ 3), and abnormal sperm FISH (n ¼ 1). In some instances, more than one clinical indication was described for the same patient. In total, 66 blastocysts were selected with a total of 84 segmental aneuploidies and 37 whole-chromosome aneuploidies (see Fig. 1). Each aneuploidy was defined either as pure or mosaic. Finally, a subset of whole blastocysts still available at the time of the study (2–6 months later) underwent FISH analysis of individual cells. This analysis including blastocysts from both the control and study groups (see Fig. 1).
Embryo Biopsy and Vitrification Trophectoderm biopsy was performed on day 5 or day 6 as previously described elsewhere (21). After the biopsy, samples were washed in 5 mL of phosphate-buffered saline 1% polyvinylpyrrolidone buffer (Cell Signaling Technology) and transferred to a 0.2 mL polymerase chain reaction (PCR) tube under sterile conditions. The tubes were stored at 20 C until further analysis. When a chromosomally normal embryo transfer was planned for a deferred cycle, all embryos were immediately cryopreserved after the trophectoderm biopsy. The vitrification protocol was completed as previously described elsewhere (22).
Array Comparative Genome Hybridization We performed DNA extraction and WGA using the Sureplex Kit (Illumina), and the array protocol was performed according to the manufacturer's instructions (3). Data were analyzed using BlueFuse Multi software, version 3.2 (Illumina). Aneuploidies were classified as whole-chromosome when the majority of the length of the chromosome showed a copynumber change and was automatically identified by the software. Segmental aneuploidies were manually identified when only a fragment of the chromosome deviated from standard thresholds for euploidy. Taking into account empirical resolution levels described by the manufacturer, only segmental imbalances above 10 Mb were considered for the study. Because multiple cells were analyzed at the same time, we sought to differentiate between pure and mosaic aneuploidies. It has been reported that aCGH can detect 25% of aneuploid VOL. - NO. - / - 2016
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FIGURE 1
Study design for the analysis of concordance rates between next-generation sequencing (NGS) and array comparative genomic hybridization (aCGH). Number of samples and events included in the study, divided between the control group (samples from translocations carriers) and study group (samples from couples with normal karyotype). Number of events from both groups reanalyzed by fluorescent in situ hybridization (FISH) of individual blastocyst cells. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
cells (9). However, due to the limitations of our sample size, we used X-separation as a reference for experimental error, calculated as the absolute log2 value for the X chromosome. When the aneuploidy absolute log2 value was higher than half of the X-separation, it was labeled as pure because it was likely that all of the analyzed cells contained that aneuploidy. When the absolute aneuploidy log2 ratio was not higher than half of the X-separation, the event was labeled as mosaic because an unknown number of cells harbored the anomaly.
Next-generation Sequencing We performed NGS on WGA products amplified with the SurePlex DNA Amplification System (Illumina). The NGS libraries were prepared using a VeriSeq PGS-MiSeq kit (Illumina) from quantified WGA products. The resulting library pools were sequenced by synthesis on a MiSeq instrument (Illumina) using the VeriSeq PGS recipe. The WGA SurePlex PCR products of each biopsy and control were quantified using a fluorometry-based method (Qubit High Sensitivity dsDNA kit; Life Technologies). One nanogram of double-stranded DNA of each unpurified WGA product was tagged and fragmented by the VeriSeq PGS transposome, creating a population of fragmented nucleic acid molecules. A limited-cycle PCR reaction used the unique adapter sequences at the ends of the fragments to amplify the insert DNA and add index sequences (barcodes) on both ends of the DNA, enabling dual-indexed sequencing.
The libraries were purified using Solid Phase Reversible Immobilization paramagnetic bead-based technology (AMPure XP beads; Beckman Coulter). Purified libraries were normalized using the VeriSeq PGS bead-based sample normalization kit and pooled in accordance with the manufacturer's instructions. Dual index 36 base pair reads (1x36 DI) sequencing was performed according to the VeriSeq PGS recipe (Rev. O), using a MiSeq Reagent Kit v3-PGS (Illumina). MiSeq Reporter software (Illumina) was used to perform on-board secondary data analysis using the VeriSeq PGS workflow. BlueFuse Multi Software, version 4.2 (BFM 4.2; Illumina), was used to analyze sequencing data generated by the MiSeq instrument and to report results as previously described elsewhere (14). The NGS pattern detection and classification of aneuploidies was determined by copy number variation (CNV) values. Aneuploidies with CNV values lower than 1.20 or higher than 2.80 were labeled as pure; CNV values between 1.80 and 2.20 were considered euploid; and aneuploidies with CNV values between 1.2 and 1.80 or between 2.2 and 2.80 were considered mosaic. We adopted differences of 0.2 copies to differentiate between pure and mosaic aneuploidies as NGS was expected to have a higher dynamic range than aCGH.
Whole-blastocyst FISH Analysis Chromosomally abnormal blastocysts were fixed as described by Mir et al. (23). The slides were stored at 20 C before
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ORIGINAL ARTICLE: GENETICS analysis for a maximum of 2 weeks. The FISH analysis was performed using subtelomeric DNA probes (Abbott) for the region involved in the segmental aneuploidy according to the manufacturer's instructions. Nuclei with unclear or tetraploid signals were excluded from the study, and blastocysts with less than 10 informative cells by FISH were also excluded. Cells were considered informative when there was no overlap between them, they had a well-defined outline, and they showed signals of similar size separated by a minimal distance corresponding to twice the diameter of each of the cells. In total, 24 whole blastocysts were analyzed with an average number of 41.4 24.3 informative cells.
Data Analysis To detect differences in the labeling of whole-chromosome and partial aneuploidy between the two sequencing methods, statistical comparisons were performed using SPSS Statistics software, version 21 (IBM). A Pearson chi-square test was performed to compare frequencies between groups. To evaluate the similarity between the techniques, we calculated concordance rates. For the comparison between technique results, we calculated two type of concordance rates: the concordance rate per analyzed chromosome, where we considered the total number of chromosomes independently if they were called as euploid or aneuploidy (24 chromosomes per embryo); and the concordance rate per aneuploid chromosome, where we considered only the detected aneuploidies. In addition, each type of concordance rate was calculated according to the type of aneuploidies: all aneuploidies, whole-chromosome aneuploidies, or segmental aneuploidies. For FISH, only the concordance rate per aneuploid chromosome was calculated, as all analyzed chromosomes by FISH were segmental aneuploidies detected previously by aCGH and NGS.
RESULTS Concordance Rate Between NGS and aCGH The concordance rates of NGS and aCGH were calculated independently of the type of the aneuploidy detected. First, all chromosomes analyzed were taken into account (91 embryos 24 chromosomes ¼ 2,184 chromosomes). The NGS results were found to be nonconcordant with aCGH results for four chromosomes. In all cases, NGS detected them as aneuploid while aCGH classified them as euploid; this gave a final concordance rate between NGS and aCGH of 99.8% per chromosome analyzed (Fig. 2A). From the 176 detected aneuploidies, 172 were concordant between platforms; the concordance rate per aneuploid chromosome was 97.7% (see Fig. 2A).
Detection of Whole-chromosome Aneuploidies by NGS and aCGH From the 91 blastocysts with at least one segmental event detected by aCGH, 29 also showed whole-chromosome aneuploidies, resulting in a 30.5% coexistence rate between segmental and whole-chromosome events. In total, 48 whole-chromosome aneuploidies were identified, with
54.2% showing chromosome losses (n ¼ 26) and 45.8% showing chromosomal gains (n ¼ 22). We next differentiated between pure and mosaic patterns and found 44 aneuploidies with a pure pattern (91.7%) and 4 aneuploidies that were mosaic (9.1%). Of the pure whole-chromosome aneuploidies, all events were confirmed by NGS technology (Supplemental Fig. 1A, available online), although one of them showed a mosaic pattern by NGS (see ID:PA95, chromosome 22 in Supplemental Table 1, available online). In the mosaic group, all events detected by aCGH were also confirmed (see Supplemental Fig. 1B) and showed the same pattern with NGS. In addition, NGS detected two additional whole-chromosome aneuploidies with a mosaic pattern that were not originally detected by aCGH. Both were found in the same sample (see Supplemental Fig. 1C; Supplemental Table 1, see ID:PA63, chromosomes 12 and 16). In summary, the total concordance rate for the detection of whole-chromosome aneuploidies between aCGH and NGS per analyzed chromosome was 99.9% (2,182 of 2,184; see Fig. 2A). The concordance rate per aneuploid chromosome was 96.0% (48 of 50; see Fig. 2A).
Detection of Segmental Aneuploidies by NGS and aCGH Segmental aneuploidies (n ¼ 124) were more frequently discovered as losses (62.1%) than as gains (37.9%) of chromosomal fragments. We did not find a statistically significant difference between segmental and whole-chromosome aneuploidies. Segmental aneuploidies were differentiated between the control group (n ¼ 40; from reciprocal translocation carriers) and the study group (n ¼ 84; from normal karyotype couples). In the control group, all 40 events detected by aCGH were also detected by NGS (concordance rate per segmental aneuploidy detected in the control group: 100%; 40 of 40; see Fig. 2A) from the 600 chromosomes analyzed in total (concordance rate per analyzed chromosome: 100%, 600 of 600; see Fig. 2A). In the study group, aneuploidies were classified into pure (n ¼ 58) and mosaic (n ¼ 26) segmental events according to the aCGH pattern. In the pure group, all segmental aneuploidies were also identified by NGS (concordance rate per pure segmental aneuploidy detected in the study group: 100%, 58 of 58; see Fig. 2A), with 55 showing a pure pattern (Fig. 3A) and 3 a mosaic (see Fig. 3B; Supplemental Table 1, see ID:PA55 chromosome 20q, ID:PA66 chromosome 1q, ID:PA67, chromosome 9q). In the group of mosaic segmental aneuploidies detected by aCGH, 26 events were detected by NGS showing the same mosaic pattern (see Fig. 3C). In addition, NGS detected two new mosaic segmental events that were not identified by aCGH (see Fig. 3D; Supplemental Table 1, see ID:PA73 chromosome 8q, ID:PA90 chromosome 9p), giving a concordance rate of 92.9% (26 of 28; see Fig. 2A) for the detection of mosaic segmental aneuploidies in the study group. Unfortunately, full FISH blastocyst reanalysis was not available for these two samples. VOL. - NO. - / - 2016
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FIGURE 2
Concordance rates between next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and fluorescent in situ hybridization (FISH) for aneuploidy detection. (A) The concordance rates were calculated per analyzed chromosome, including euploid and aneuploidy chromosomes, and exclusively per aneuploid chromosome. Each type of concordance rate was calculated for all aneuploidies, for whole-chromosome aneuploidies and for segmental aneuploidies. Finally, the concordance rates for segmental aneuploidies were calculated for those events detected in the control group (translocations carriers) and the study group (normal karyotypes). (B) The concordance rates for those samples with FISH analyses were calculated per aneuploidy chromosome as all events analyzed were previously detected as segmental aneuploidies by aCGH and NGS. The concordance rates were also differentiated between the control and the study group, and between pure and mosaic events inside the study group. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
The total concordance rate for the detection of segmental aneuploidies between aCGH and NGS independently of the parental karyotype and the detected pattern was 98.4% (124 of 126) per aneuploidy detected and 99.9% (2,182 of 2,184) per analyzed chromosome (see Fig. 2A).
FISH Analysis to Determine Distribution Pattern of Segmental Aneuploidies Twenty-four chromosome spreads from full blastocysts were analyzed by FISH at the single-cell level; 6 blastocysts
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FIGURE 3
Comparison of segmental aneuploidy detection by array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS). (A) Both aCGH and NGS detected similarly a pure segmental aneuploidy for chromosome 6. (B) One segmental aneuploidy for chromosome 20 identified as pure by aCGH was detected by NGS as a mosaic pattern. (C) Array CGH and NGS detected an aneuploidy for chromosome 3 showing a mosaic pattern in both cases. (D) One mosaic segmental aneuploidy for chromosome 8 detected by NGS was not originally detected by aCGH. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
originated from patients with an altered karyotype (control group), and 18 blastocysts from patients with normal karyotype (study group). In total, we studied 28 segmental events from the control and study groups (Fig. 4). FISH analyses detected 10 events in blastocyst from the control group with 93% of the cells showing the segmental aneuploidy (concordance rate per detected segmental aneuploidy in the control group: 100%; 10 of 10; see Fig. 2B). All patterns, whether pure or mosaic, were consistent between the three technologies with the exception of one event that was detected as pure by aCGH and NGS whereas FISH detected a low degree mosaic with 25 (92.6%) of 27 aneuploid cells.
We analyzed 18 segmental events by FISH from the study group patients with normal karyotypes (see Fig. 4). In the pure group, nine out of the 10 segmental events detected by aCGH and NGS were also detected by FISH (concordance rate for pure events in the study group: 90%, 9 of 10; see Fig. 2B) but with a variable percentage of aneuploid cells, showing mainly mosaic patterns (n ¼ 7). There was one pure event (9q, 45.5 Mb) that was not detected in the 21 cells analyzed by FISH; this discrepancy may be a technical artifact. The percentage of aneuploid cells in the pure study group was 63%. In the mosaic events from the study group, FISH analyses detected 7 out 8 segmentals (see Fig. 4), which is a VOL. - NO. - / - 2016
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FIGURE 4
Segmental aneuploidies analyzed by array comparative genomic hybridization (aCGH), next-generation sequencing (NGS), and fluorescent in situ hybridization (FISH). Aneuploidies were identified as pure if they scored beyond the euploidy confidence range in aCGH and NGS, or if all cells analyzed by FISH were aneuploid. Aneuploidies were identified as mosaic if they showed an intermediate value within the confidence range, or, in FISH analysis, if aneuploid and euploid cells were detected in the same embryo. The color scale represents the percentage of aneuploid cells in the group of cells analyzed by FISH, with dark green indicating 100% aneuploidy, dark red indicating 0 aneuploidy, and intermediary colors indicating different levels of mosaicism (see Supplemental Fig. 2). Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
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ORIGINAL ARTICLE: GENETICS concordance rate per mosaic segmental event in the study group of 87.5% (7 of 8; see Fig. 2B). All seven showed a mosaic pattern, ranging from 11.7% to 64.7% of aneuploid cells. There was only one segmental event (þ5p, 54 Mb) that was not detected by FISH in the 29 cells analyzed. The final percentage of cells in the mosaic study group with segmental events detected was 24%. In total, the concordance rate per segmental aneuploidy in the study group, independent of the pattern, was 16 (88.9%) of 18 (see Fig. 2B). Finally, and regardless of the parental karyotypes of the blastocysts, the concordance rate between FISH, aCGH, and NGS for the detection of segmental aneuploidies was 26 (92.9%) of 28 (see Fig. 2B), with only two embryos showing discordant results (see Fig. 4).
DISCUSSION The evidence presented here supports the use of NGS for the detection of segmental aneuploidies in trophectoderm biopsies that are typically measured by aCGH. Although we focused our study on the detection of segmental aneuploidies, we also studied whole-chromosome aneuploidies in parallel. Our data support the use of NGS as a viable technique for detecting not only aneuploidies showing a clear, pure pattern, but also for analyzing samples with mosaic patterns. In fact, we found that mosaic events can be identified more easily due to the increased dynamic range of NGS data using BlueFuse Multi profiles. Additionally, we showed that FISH analysis of whole blastocysts could be used to confirm the presence and pattern of segmental aneuploidies. Our results suggest that NGS can successfully identify whole-chromosome and segmental aneuploidies previously detected by aCGH, offering, in addition, a higher dynamic range and a more cost-effective laboratory routine if there is a higher sample throughput. Previous studies have described a minimum size for the detection of imbalances by NGS of approximately 14 Mb (14, 15, 17), with only one recent report detecting imbalances of 1.8 Mb by NGS (24). Here, we report the detection of segmental aneuploidies as small as 10 Mb by NGS with the same efficiency as aCGH. It is interesting that we detected four additional aneuploidies by NGS: two whole-chromosome and two segmental aneuploidies, all of them exhibiting a mosaic pattern. Unfortunately, FISH could not be performed in these samples, and we could not conclude which technique reflected the most accurate chromosomal status of the blastocyst. The relevance of segmental aneuploidies in the development of the human embryo is often questioned in PGS programs. Here, we have shown that most of the segmental aneuploidies detected by NGS or aCGH were not technical artifacts, because they were also detected by FISH analysis. Even in the two cases in which the segmental aneuploidies were not confirmed by FISH, we do not know whether the discrepancy was due to a technical WGA artifact or to mosaicism in the blastocyst, which we found to be a frequent phenomenon in aneuploid human blastocysts. Furthermore, it has been described that the frequency of segmental aneuploidies in miscarriages is approximately 6% (25) and approximately 5 in 10,000 newborns (26). These numbers match the
described frequency of segmental aneuploidies in blastocysts (6% to 7%) (2, 7), implying that these types of abnormalities should be taken into consideration in clinical practice. Recently, it was reported that the detection of segmental aneuploidies in single cells is not optimal when the cells are in the S phase of the cell cycle (27). It is important to note that we used trophectoderm biopsy, increasing the probability of obtaining cells in the G0/G1 phase. Nevertheless, this phenomenon could underlie the two instances in which FISH did not detect the segmental aneuploidy detected by NGS and aCGH (7.1%). Additional studies are needed to determine the maximum mosaicism level compatible with the development of a healthy embryo. Array CGH has been shown to detect mosaicism degrees higher than 25% (9), which may explain why those events were detected as mosaic by FISH with more than 75% of aneuploid cells shown as pure by aCGH or NGS. The threshold to detect mosaicism in trophectoderm cells is a parameter that needs further discussion given that an increase in the sensitivity of the techniques described could result in a simultaneous decrease in the specificity, which could lead to a decrease in the detection of euploid embryos. Three main points must be addressed to optimize the identification of segmental aneuploidies. First, it is important to decrease the biological variability; this can be accomplished by using a common starting material for both techniques, such as the same amplified DNA. In some studies, different trophectoderm biopsies have been used to compare different methods of analysis (16). As such, it is difficult to know whether any discrepancies are due to technical or biological issues, such as mosaicism. Second, to better characterize the presence of segmental aneuploidies a third technique with a different molecular basis is recommended. For instance, when comparing aCGH and NGS, because they both share the same first WGA step, quantitative PCR or FISH can be used to confirm results and sort out any discrepancies. Currently, segmental aneuploidies can only be identified using FISH with specific probes. Third, it is necessary to characterize the parental karyotypes before the PGS cycle to avoid the mislabeling of very small segmental aneuploidies. We accomplished each of these using NGS and aCGH to assess segmental aneuploidy. Unfortunately, we could not analyze the blastocysts in which discrepancies were found between NGS and aCGH, because they were not available at the time of the study. Nevertheless, FISH analysis was extremely useful to confirm the existence of segmental events as well as the pure or mosaic patterns in the whole blastocyst. Although in our FISH study we did not differentiate between trophectoderm and inner cell mass, previous studies have shown concordant ploidy results between both types of cells (7,28–30); even when euploid or aneuploid mosaicism is present at the blastocyst stage, aneuploid cells are still found in both trophectoderm and inner cell mass cells (30). In conclusion, we have shown that FISH analysis revealed the existence of segmental aneuploidies in the majority of the blastocysts diagnosed, suggesting that most cases are not technical artifacts of aCGH or NGS. In addition, we demonstrated through the analysis of individual cells by FISH that most of the segmental aneuploidies originated from mitotic VOL. - NO. - / - 2016
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Fertility and Sterility® errors creating different degrees of mosaicism, and this pattern can be detected by NGS. Finally, we have validated NGS for segmental aneuploidies with a concordance rate per analyzed chromosome of 99.9 % and 98.4% of concordance rate per detected segmental event. Acknowledgments: The authors thank the clinicians and embryologists at the IVF units utilized in this study, with particular thanks to Pilar Campos for her assistance during blastocyst thawing, and our colleagues in Igenomix for all their helpful suggestions during data analysis.
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SUPPLEMENTAL FIGURE 1
Comparison of array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) whole-chromosome aneuploidy detection. (A) A pure, whole-chromosome aneuploidy detected by aCGH for chromosome 9 was also detected by NGS as pure. (B) A mosaic wholechromosome aneuploidy detected by aCGH for chromosome 18 showed a similar pattern using NGS. (C) NGS detected mosaic aneuploidies for chromosomes 12 and 16 that were not initially identified by aCGH. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
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SUPPLEMENTAL FIGURE 2
Fluorescent in situ hybridization (FISH) analysis for the detection of segmental aneuploidies in blastocyst cells. (A–C) FISH analysis of blastocyst in which a segmental loss (55.3 Mb) in the p arm of chromosome 1 was previously detected by array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) showing a mosaic pattern. We analyzed 34 cells successfully using Spectrum red probes specific to the p subtelomere region of chromosome 1 (Aquarius; Cytocell), with (A) 12 cells (23.5%) containing a single signal, (B) eight cells (35.3%) with two signals, and (C) 14 cells (41.2%) with three signals. Vera-Rodríguez. Detection of segmentals by NGS. Fertil Steril 2016.
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