chromosomal abnormalities are an important cause of low implantation rate, pregnancy failure and birth defects in human embryos . The early miscarriage rate of natural pregnancy is about 15%-20%, the early miscarriage rate of in vitro fertilization and embryo transfer (IVF-ET) is about 25%, of which embryonic chromosomal abnormalities account for about 40-50%, and the incidence of neonatal chromosomal abnormalities is 0.5%-1%. For patients or their families with chromosomal abnormalities, monogenetic diseases, repeated spontaneous abortions and implant failures, after receiving genetic counseling, they may be advised to undergo embryo testing from multiple PGT platforms, such as PGT-APGT-M, PGT-APGT-SR, and even PGT-APGT-MPGT-SR, to avoid recurrent miscarriages of and genetic diseases in the offspring. 's current PGT-A/M/SR is performed by different technical platforms, with high cost, few materials, long time, and limited promotion.
PGT Dilemma: Multiple indications require multiple platforms, and clinical needs are difficult to meet
Preimplantation genetic testing (PGT) can detect chromosome number and structural abnormalities before embryos are implanted into the uterus, abandon abnormal embryos (single-gene abnormal embryos, chromosomal structural rearrangement embryos and aneuploid embryos), and select normal or non-disease-induced embryos to implant into the maternal uterus, which can effectively reduce the miscarriage rate, improve pregnancy rate, and reduce the birth of newborns with defective or severe genetic diseases.
PGT technology can effectively improve embryo implantation and pregnancy rate, reduce miscarriage rates, and prevent the birth of children with hereditary diseases. PGT includes three aspects of detection: PGT for aneuploidy (PGT-A), PGT for monogenic/single gene defects (PGT-M), and PGT for structural rearrangements (PGT-SR). A variety of molecular biology technologies, and molecular genetics, technologies can be used for chromosome genetic detection and analysis, including FISH (Fluorescence in situ hybridization) technology, PCR (Polymerase chain reaction) technology, aCGH (Array-based comparative genomic hybridization) technology, SNP-array (Single nucleotide polymorphism array) technology and NGS (Next Generation Sequencing) technology.
At present, the PGT integrated solutions mainly include Karyomapping, Haplarithmisis, OnePGT, MARSALA, MaReCs, Haploseek, integrated cPGT, triple expansion analysis, single gene deletion detection, non-invasive PGT, long-read sequencing, etc. (Table 1). OnePGT mainly uses the RRGS method to evaluate aneuploidy and structural rearrangements through read count-based methods and examines single-gene diseases by SNP-based haplotypes. Although triplose, haploid and UPD can be inferred from OnePGT data, the OnePGT software does not automatically provide this information; the Haplaseek platform is suitable for all clinical PGT applications (PGT-A/M/SR) and de novo mutations, and can be successfully applied to close couples with complete family member information; WGS-based integrated cPGT does not reduce sequencing coverage, but uses another strategy to reduce sequencing costs by reducing the sequencing depth of embryo samples to 4 times. It uses universal read count analysis for PGT-A, and at the same time uses haplotype for PGT-SR (including balanced translocation ) and PGT-M; another comprehensive PGT based on SNP array has been developed, and it also detects aneuploidy, CNV, single gene mutations and equilibrium translocations, but does not indicate the origin of CNV and chimera . Its PGT-A and CNV analysis relies on SNP allele frequency and Log-R ratio, while PGT-M and PGT-SR utilize linkage analysis. However, the SNP array is limited by its fixed number of probes.
Table 1 Integrated PGT solution
Data source: [1]丨Graphics: Biological Exploration Editorial Team
So far, no technology can use a set of reagents to complete the detection and analysis of three chromosomal abnormalities in an embryo sample in a workflow, and at the same time distinguish between normal embryos and translocation-carrying embryos of chromosome balance translocations . To overcome obstacles related to integrated PGT, application experts are seeking to combine PGT-A, PGT-M and PGT-SR in a single detection.
HaploPGT: Integrated PGT detection technology can achieve multiple indications and simultaneous detection
September 6, 2022, the joint research team of the National Human Stem Cell Engineering Research Center, CITIC Xiangya Reproduction and Genetic Hospital, Changsha Development and Carcinogenic International Science and Technology Cooperation Base, Beikang Medical, Central South University and Shanghai Jiaotong University published research results entitled "A novel multifunctional haplotyping-based preimplantation genetic testing for different genetic conditions" in Human Reproduction (Figure 1) [2]. study provides a multifunctional haplotype-based embryo preimplantation genetics detection platform for detecting different genetic conditions .
Figure 1 Research results (Photo source: [2])
This study collected 188 embryo samples from 43 families and all were tested using the HaploPGT platform. Most of the genetic abnormalities of the samples were determined in advance by different conventional PGT methods. There were 12 families carrying only structural rearrangements (115 embryos), of which 9 families received implantation, 5 families had normal ART birth results, 7 families only carried single-gene diseases (26 embryos), and 3 families carried both structural rearrangements and single-gene diseases (26 embryos). Twelve mononuclear fertilized eggs (1PN) samples and nine suspected triploid samples were collected from 21 families.
Results found that
1 80M genomic data can balance the cost and accuracy of HaploPGT
In 80 million reads (80M) genomic data, the window (1 million base pairs (Mb)) containing two or more informational single nucleotide polymorphisms (SNP) sites was 97.81%, and the genotyping error rate was stable at a low level (2.19%); in addition, informational single nucleotide polymorphisms were evenly distributed in the genome, establishing a whole genome haplotype (Figure 2). Therefore, 80M is chosen to balance the cost and accuracy in HaploPGT.
Figure 2 SNP distribution of different size sequencing data and 80M data haplotypes (Figure source: [2])
2 HaploPGT can identify abnormal embryos, and can even distinguish between parental sources
sequencing data has low coverage but high reading depth, and can accurately detect SNPs in the whole genome, thereby using the BAF of all SNPs to identify triploid, diploid and LOH. HaploPGT can identify abnormal embryos with triploid, full heterozygous and partial heterozygous deletion, and can even distinguish the parental source of copy number variation in mosaic and non-mosaic embryos (Figure 3).
Figure 3 Detection of triploPGT, parental diploid, haploid and LOH (Figure source: [2])
3 The available results of HaploPGT and PGT detection are 100% consistent
In addition, by retrospectively analyzing 188 embryo samples from 43 families, the available results of HaploPGT and PGT detection reference methods (including PGT-A, PGT-M, PGT-SR and PGT-HLA) are 100% consistent.
Figure 4 Distinguishing chromosomal rearrangements in embryos by haplotypes (Figure source: [2]
This study developed an integrated PGT detection platform named HaploPGT. HaploPGT performs copy number analysis, BAF analysis and haplotype analysis of RAD-seq data from blastocyst biopsy samples to perform PGT-A, PGT-SR, PGT-M and other extended applications. HaploPGT has the following advantages: (i) the amount of data required for WGS is reduced, and the number of effective SNPs that can be used to construct haplotypes is sufficient ; (ii) can be used to generalize Haplotype for parents, probands and embryos; (iii) sequencing data is very small, and the workflow is common. PGT-A, PGT-M and PGT-SR greatly reduce costs, materials, complexity and operating time ; (iv) can show its application in other functions, such as detecting 1PN, triploid, mosaic ratio of mosaic embryos or abnormal CNV of parent source . The above embryo conditions are all shown in a test, which can provide more information for doctors to choose better embryos for transplantation or scientific research.
Figure 5 Overview of HaploPGT (Photo source: [2]
HaploPGT is an efficient and cost-effective detection platform with high clinical value in detecting genetic status . This platform can promote the application of PGT in ART, improve pregnancy rate, and reduce the birth of children with genetic diseases.Although HaploPGT can bring many benefits, it still requires more family member information to infer parental haplotypes to identify balanced translocations and single gene mutations in the test embryo. In the case of PGT-SR, additional family members may be reference embryos with unbalanced translocations. PGT-M usually requires a proband. In both cases, genomic information from grandparents or parents’ siblings may contribute to theoretical haploid typing. Another limitation is that HaploPGT cannot distinguish between haploids and diploids produced by haploid replication, but it is able to identify partial loss of heterozygous properties in the embryonic genome. Furthermore, the locations and mutation sites of rearrangement breakpoints are complex, meaning that some genetic diseases may not be fully detected.
Written by | Wen Zhengze
Typeset | Feng Lixiao
End
Reference materials:
[1]Volozonoka L, Miskova A, Gailite L. Whole Genome Amplification in Preimplantation Genetic Testing in the Era of Massively Parallel Sequencing. Int J Mol Sci. 2022 Apr 27;23(9):4819. doi: 10.3390/ijms23094819. PMID: 35563216; PMCID: PMC9102663.
[2]Xie P, Hu X, Kong L, et al. A novel multifunctional haplotyping-based preimplantation genetic testing for different genetic conditions. Hum Reprod. 2022 Sep 6:deac190. doi: 10.1093/humrep/deac190. Epub ahead of print. PMID: 36066440.
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