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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 1-6

Current status and recent advances in preimplantation genetic testing for structural rearrangements

1 Department of Genetics, Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2 Department of Genetics, Shanghai Ji Ai Genetics and IVF Institute; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China

Date of Submission04-Jan-2020
Date of Decision13-Feb-2020
Date of Acceptance04-Mar-2020
Date of Web Publication2-Apr-2020

Correspondence Address:
Cong-Jian Xu
Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.281855

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Preimplantation genetic testing (PGT) is an early form of prenatal genetic diagnosis, which can identify the abnormal embryos cultured in vitro, allow only transfer of genetically normal embryos, and improve the pregnancy rate. In recent years, the rapid development of microarrays and next-generation sequencing (NGS) technologies has remarkably accelerated the clinical application of PGT. In particular, a variety of detection methods have emerged and achieved significant progress in PGT for structural rearrangements (PGT-SR). The detection-related abilities of these methods range from the detection of limited chromosome aneuploidy to comprehensive chromosome screening of the whole genome to differentiation of embryos with normal or balanced translocation/inversion karyotypes. In this study, we reviewed PGT-SR-related detection techniques to provide a better reference for clinical application and research. We have also discussed the potential development of novel techniques in the future.

Keywords: Chromosome Aneuploidy, Inversion Karyotype, Preimplantation Genetic Testing, Structural Chromosomal Rearrangement, Structural Rearrangement, Translocation Karyotype

How to cite this article:
Zhang S, Lei CX, Sun XX, Xu CJ. Current status and recent advances in preimplantation genetic testing for structural rearrangements. Reprod Dev Med 2020;4:1-6

How to cite this URL:
Zhang S, Lei CX, Sun XX, Xu CJ. Current status and recent advances in preimplantation genetic testing for structural rearrangements. Reprod Dev Med [serial online] 2020 [cited 2021 Jan 20];4:1-6. Available from: https://www.repdevmed.org/text.asp?2020/4/1/1/281855

  Introduction Top

Preimplantation genetic testing (PGT) has already been introduced since 1990 by selecting female embryos to prevent the birth of male patients affected with X-linked mental retardation and adrenoleukodystrophy. Initial experiments with animals as early as 1890 and those that followed in the middle and later parts of the 20th century paved the way for assisted reproductive technology and PGT.[1],[2][3] PGT can be divided into three types according to patients' indication: aneuploidy (PGT-A), monogenic disorders (PGT-M), and structural rearrangements (PGT-SR).[4] Among these, patients who undergo PGT-SR are primarily carriers of balanced chromosomal translocations and inversions. Because these carriers have no numerical gain or loss of genetic material, most are phenotypically considered normal. However, these carriers may face fertility problems, mainly as a result of producing high amounts of unbalanced gametes; this is associated with infertility, recurrent spontaneous abortion, and pregnancies involving congenital abnormalities. During the pachytene stage of prophase I, the translocated chromosomes and the two corresponding normal chromosomes form trivalent or quadrivalent structures, which allow the pairing of homologous regions and increase reproductive risk due to the complex segregation patterns. Transferring diploid embryos through in vitro fertilization combined with PGT-SR is the primary solution for these patients,[5],[6] and PGT-SR can improve the clinical pregnancy rate by 45%–70% in patients with translocations.[7],[8],[9] With the development of microarray and next-generation sequencing (NGS), PGT-SR can detect anomalies ranging from limited chromosome aneuploidy to comprehensive chromosome screening (CCS) of the whole genome and to effectively identify embryos with structural chromosomal abnormalities. The currently available techniques can effectively distinguish embryos that carry a normal karyotype from those with a balanced translocation karyotype, thereby preventing the birth of offspring who will face the same fertility complications as their parents.

  Key Techniques for Chromosome Aneuploidy Detection Top

At present, the primary techniques for detecting chromosome aneuploidy are fluorescence in situ hybridization (FISH), quantitative real-time polymerase chain reaction (qPCR), microarrays (including comparative genomic hybridization [array CGH] and single-nucleotide polymorphism array [SNP array]), and NGS. FISH was the first technique to be used for detecting chromosome aneuploidy, but technological limitations only allow for analysis of a limited number of chromosomes.[10] QPCR was developed later, and in recent years, microarrays and NGS have been used for CCS of biopsied embryos.[11],[12],[13],[14] Herein, we provide a brief introduction to each of these techniques.

In situ hybridization

In FISH, fluorescently labeled probes are used to hybridize with target complementary nucleic acid sequences in situ and DNA is qualitatively analyzed. It was the first technique used for preimplantation identification of chromosomal abnormalities,[15],[16] and this technique significantly improves the clinical pregnancy rate and reduces the risk of miscarriage for couples with reciprocal and Robertsonian translocations.[17],[18] However, conflicting results have been reported by Mastenbroek et al.,[19] who divided 408 women aged 35–41 years into a PGT (n = 206) or control group (n = 202) and used FISH to detect cleavage-stage embryos. Notably, the PGT group had lower pregnancy and live birth rates than the control group, raising questions about the ability of FISH to detect aneuploidy in blastomeres. The main limitation of FISH is low throughput. Due to the limited number of probes that can be used in each test, ≥2 rounds of hybridization are needed to analyze sufficient numbers of chromosomes. In addition, multiround hybridization of a single cell increases the error rate, i.e., FISH can only be practically used to detect aneuploidy in a limited number of chromosomes. Besides, overlapping fluorescence signals and signal division may affect the accuracy of results and lead to misdiagnosis.[10],[20]

Quantitative real-time polymerase chain reaction

qPCR is based on traditional polymerase chain reaction (PCR),[21],[22] in which the amplification products of a specific region of the chromosome are quantitatively analyzed in real time. By comparing the results with the amplification curves of reference genes, chromosome copy numbers can be identified before implantation. Treff et al.[21] determined the accuracy of qPCR to be 97.6% (41/42) and 98.6% (70/71) for the detection of 42 cell samples with confirmed karyotypes and 71 embryos, respectively. In addition, only 4 h was required to obtain the detection data. Thus, qPCR has been shown to have a high accuracy rate and can rapidly identify aneuploidy in all 23 pairs of chromosomes.[22] However, qPCR can only detect relatively large copy number variations (CNVs), and it cannot detect some microdeletions/microduplications or mosaics.[23] Moreover, qPCR can only amplify specific sequences and allelic dropout (ADO) is inevitable, with an incidence of 5%–20%.[24] Therefore, qPCR has not been widely applied in clinical practice.

Array comparative genomic hybridization

Array CGH, which was created by Kallioniemi in 1992,[25] extends the detection region covered by FISH probes to the whole genome and detects deletions or duplications of genetic material by comparing sample DNA with control (normal) DNA. The main principle of this procedure is to label sample and control DNA with fluorescent Cy5 and Cy3 dyes, respectively, and hybridize these on a DNA chip after mixing them in equal proportions. Then, the chip is scanned and the ratio of the two signals is determined. For a given genomic region, a 1:1 ratio indicates a normal copy number, whereas increased Cy3 signal indicates a trisomy and increased Cy5 signal indicates monosomy.[26] Fiorentino et al.[8] first reported the application of array CGH for translocation carriers in cleavage-stage embryos. Among 28 embryos tested, 93% were successfully categorized, with a successful implantation rate of 64% in each cycle.[8] Colls et al.[9] detected 432 embryos from carriers of reciprocal and Robertsonian translocations using array CGH, in which 354 were D3 blastomeres and 78 were blastocysts. Further, 7.6% of the blastomeres failed to detect for the absence of nucleus in the biopsied embryo or DNA degradation, and the accuracy of diagnosis was 92.4%. In addition, 3.8% of the blastocysts showed failed DNA amplification, with a diagnostic accuracy of 96.2%. This study demonstrated the potential of array CGH to perform CCS. To date, array CGH has been successfully applied in the detection of polar bodies,[27] cleavage-stage embryos,[28] and trophectoderm cells.[29] However, array CGH has been reported to have a higher misdiagnosis rate than qPCR.[23]

Single-nucleotide polymorphism array

SNP array involves the use of high-density microarray chips with a large number of SNP loci. Its principle is similar to that of array CGH.[30] A large number of high-density SNP sequence probes on the chip are hybridized with the sample genomic DNA to obtain the allele types of each locus. By analyzing the signal intensity ratios of allele heterozygotes, duplication and deletion can be detected in small CNVs with higher resolution than can be achieved using FISH or qPCR.[31] Due to its unique advantages, SNP array has been widely performed for PGT in recent years. Tan et al.[32] performed SNP array to screen embryos from carriers of reciprocal and Robertsonian translocations; the pregnancy and abortion rates after embryo transfer were 69.4% versus 73.8% and 12% versus 11.1%, respectively. What's more, SNP array can be performed to detect triploid and homologous uniparental disomy (UPD) and analyze the parental origin of duplicated chromosomal regions.[33]

Next-generation sequencing

NGS, also known as high-throughput sequencing, has considerable advantages over traditional Sanger sequencing in the processing of large-scale samples. NGS can simultaneously process hundreds of thousands to millions of DNA molecules. Apart from being a high-throughput technique, its advantages include low cost and high resolution.[34] NGS can detect a wide variety of variations including single-nucleotide mutation,[35] structural chromosomal variation,[36] and aneuploidy.[14] Fiorentino et al.[37] evaluated the accuracy of NGS with 18 single cells and 190 whole-genome amplification products. They detected a total of 4,992 chromosomes, of which 402 showed CNVs. The accuracy of detection was 99.98%, which is comparable with the results of array CGH. Another study reported no significant differences in embryo implantation rate, pregnancy rate, and abortion rate after testing with NGS or SNP array between carriers of reciprocal and Robertsonian translocations.[38] Moreover, NGS has been proven to detect 20% of mosaics.[38],[39] Due to the high cost and long time required for NGS to detect SNP loci, NGS has limited utility in the detection of the parental origin of abnormal chromosomes, UPD, and triploid embryos.

  Main Techniques for the Detection of Structural Chromosomal Rearrangement Top

Using microarrays and NGS, CCS can be performed on all 23 pairs of chromosomes in embryos. However, these techniques cannot distinguish embryos with a normal karyotype from those with structural chromosomal rearrangements, i.e., 50% of newborns could be carriers of chromosomal abnormalities and encounter the same infertility complications as their parents. Moreover, when reciprocal translocation involves the X chromosome, the phenotype of an embryo with balanced translocation is unpredictable due to random inactivation of the X chromosome during early embryonic development.[40] With the development of molecular genetics techniques, the detection of structural chromosomal rearrangements can help distinguish between embryos with abnormal and normal chromosomes. Currently, researches have reported several techniques used to distinguish embryos with normal or balanced translocation karyotypes such as NGS following microdissecting junction region (MicroSeq), mapping allele with resolved carrier state (MaReCS), preimplantation genetic haplotyping (PGH) method, and mate-pair sequencing.


MicroSeq-PGD combines chromosome microdissection and NGS. In the preclinical phase, chromosome microdissection is performed at breakpoints in cultures of peripheral blood of reciprocal translocation carriers, followed by PCR amplification of the microdissected DNA. Then, rearrangement breakpoints and adjacent SNPs are characterized by NGS. Junction-spanning PCR and Sanger sequencing are further performed to identify the precise location of breakpoints. In the clinical phase of embryo analysis, CCS is performed, and WGA products are amplified with junction-spanning-specific primers via PCR, with genomic DNA of translocation carriers serving as the positive control and genomic DNA from healthy donors serving as the negative control. Only embryos identified to be positive in the junction-spanning PCR analysis and/or those positive for informative SNPs were predicted to be carriers, whereas those that were identified to be negative in the junction-spanning PCR analysis and/or those negative for informative SNPs were predicted to be normal. The combination of direct amplification and linkage analysis improves the accuracy of detection. In 2013, Jancuskova et al.[41] reported that the combination of chromosome microdissection and NGS could be used to accurately identify the breakpoints of reciprocal translocations at individual base pair level in leukemia patients. Hu et al.[42] first developed MicroSeq-PGD in 2016 to identify precise translocation breakpoints and distinguish between embryos with normal and balanced karyotypes, which can also help accurately locate chromosomal breakpoints. However, microdissection requires specialized equipment, complicated sample preparation, and professional personnel to operate.[42]

Mapping allele with resolved carrier state

Translocation breakpoints in embryos with chromosomal imbalance are first identified by locating CNVs in unbalanced embryos through multiple annealing and looping-based amplification cycle and NGS. SNP markers located within 1 Mb of the detected breakpoints are used to identify the translocation-carrying allele. After its identification in embryos with chromosomal imbalance, the same groups of SNP markers are used to identify whether a chromosomally balanced embryo carries the translocation. In this way, a translocation-free chromosomally balanced embryo can be selected for implantation. In 2017, Xu et al.[43] successfully established a new technique, called MaReCS, and applied it in clinical practice. MaReCS enabled chromosome ploidy screening and resolution of the translocation carrier status in the same embryo. The limitation of MaReCS is that each translocation carrier requires personalized design.

Mate-pair sequencing and polymerase chain reaction breakpoint analysis

NGS mainly involves single-end sequencing and paired-end/mate-paired (PE/MP) sequencing.[44] Single-end sequencing involves sequencing each fragment after randomly interrupting the genome, and PE/MP sequencing is also known as bidirectional sequencing. The difference between PE and MP lies in library construction. In PE, the two ends of the DNA fragment are directly connected to short-length fragments to perform bidirectional sequencing. In MP, the DNA fragment is inserted to yield a ring to obtain the end information of the inserted fragment, and the length is relatively long.

Wang et al. designed and verified the accuracy of detection using combined MP sequencing and PCR breakpoint analysis to distinguish between embryos with normal and balanced translocation karyotypes.[45] The technique first enabled the analysis of the genomic DNA extracted from the peripheral blood of translocation carriers using MP sequencing. Next, the breakpoint sequences containing two related translocation chromosomes were identified and primers based on the upstream and downstream sequences of the breakpoint were designed. After biopsy, euploid embryos were identified using sequencing, and WGA products from these embryos were further tested by PCR using breakpoint and nonbreakpoint spanning primers to distinguish carrier and noncarrier embryos. Wang et al.[45] successfully identified translocation breakpoints in 9 (82%) of 11 patients, allowing for the preferential selection of noncarrier embryos for transplantation. This indicates that use of high-throughput deep sequencing to find translocation breakpoints can be used in PGT cycles of reciprocal translocation carriers. In the same year, Aristidou et al.[46] also reported that in apparently balanced translocations, 9 patients with balanced translocation were mapped in four families using whole-genome MP sequencing. Then, the breakpoint was verified by Sanger sequencing.[46] However, even if the sequencing depth was high, approximately 18% of breakpoints cannot be successfully identified, and when the original DNA template was restricted by ADO,[47] the embryo may be misdiagnosed.

Preimplantation genetic haplotyping technology

PGH method is based on whole-genome haplotype linkage analysis, patients were enrolled and genome-wide SNP genotyping is performed on a couple with structural chromosomal abnormality and one carrier's parent or unbalanced embryos or tissues from miscarriage, or offspring. Then, informative SNPs are defined and the analysis model is established. Haplotypes, including breakpoint regions, whole chromosomes involved in the translocation, and the corresponding homologous chromosomes are established with these informative SNPs in the couple, reference, and embryos. Afterward, embryo testing is conducted, in which molecular karyotypes and haplotypes are simultaneously established with this model in each embryo. SNP allele frequencies are used to identify embryos without chromosomal CNVs, and haplotypes are used to distinguish between balanced and structurally normal embryos. Zhang et al.[48] analyzed 68 blastocysts from 11 translocation families, of which 42 were unbalanced or aneuploidy and the other 26 were balanced or normal chromosomes. In the second trimester of successful pregnancy, the results of PGH were consistent with amniotic fluid karyotypes, verifying the accuracy of PGH. In 2019, Zhang et al.[49] reported a highly efficient approach for PGH, the larger number of SNP loci used further improved the accuracy of the analysis. Because haplotype of any region or chromosome in the genome could be established, PGH can be applied for any type of translocation. Treff et al.[50] successfully distinguished a balanced translocation carrier embryo from a truly normal embryo using SNP genotyping using an unbalanced embryo as a reference. In 2018, Wang et al.[51] also successfully distinguished embryos with normal karyotypes from those with Robertsonian karyotypes using PGH-based SNP array.

Long-read sequencing and breakpoint analysis

Long-read sequencing, also known as single-molecule sequencing, is characterized by ultra-long reads.[52] It has unique advantages for the study of structural chromosomal variations and has become a hot technique in recent genome research. In 2018, Hu et al.[53] and Chow et al.[54] successively applied Oxford Nanopore sequencing in PGT. First, DNA was extracted from the peripheral blood of reciprocal translocation carriers, and long-read sequencing was performed to obtain the sequence across breakpoints. Sanger sequencing was then performed to identify the precise location of breakpoints. Verifying the precise location of breakpoints by Sanger sequencing was critical for the successful design of PCR primers. Embryo DNA was directly amplified using breakpoint-spanning PCR. If an amplification product was found, the embryo was diagnosed as a balanced translocation carrier. In addition, Zhang et al.[55] detected the embryos of chromosomal inversion carriers combining long-read sequencing and breakpoint haplotype linkage analysis. However, it still cannot accurately identify the location of breakpoints for some chromosomal rearrangements in complex regions of the genome such as the regions with highly repetitive sequences.

  Future Challenges and Perspectives Top

Regarding PGT-SR, the reviewed techniques can be performed to detect chromosome aneuploidy and structural chromosomal variations in embryos. Preferential selection and transfer of diploid embryos with normal karyotypes not only improves the pregnancy rate but also helps avoid genetic transmission of chromosomal rearrangements. With the improvement in eugenic conscience, many couples express a strong desire to pursue more careful screening and transfer embryos without translocation. Techniques that cannot detect structural chromosomal rearrangement will gradually be eliminated, and the selection of an accurate and effective detection technique will be challenging. Several reviewed techniques can detect structural chromosomal rearrangement in embryos, among which PGH has significant advantages and has the potential to become the first option for PGT-SR due to its simplicity, efficiency, and accuracy. Along with PGT-SR, PGT also includes PGT-A and PGT-M. However, according to the needs of specific individuals, different methods/preparations are needed, which is not convenient in routine clinical practice. Therefore, there is a need to integrate these different indications simultaneously on one test platform. Because PGH technology is based on whole-genome SNP genotyping, linkage analysis of SNP loci in and around certain pathogenic genes can also be conducted as long as SNP data are available, in addition to the detection of chromosome aneuploidy and structural chromosomal abnormalities. Herein, we propose the concept of “PGH-One” that can perform PGT-A, PGT-M, and PGT-SR simultaneously in one test and may eventually become the standard practice in the development of PGT.

Financial support and sponsorship

The research was supported by the Science and Technology Innovation Action Plan Program of Shanghai (18411953800) and Shanghai Municipal Health Commission (20194Y0002).

Conflicts of interest

There are no conflicts of interest.

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