Validation of a next-generation sequencing–based protocol for 24-chromosome aneuploidy screening of blastocysts

Huang, Jin; Yan, Liying; Lü, Sijia; Zhao, Nan; Xie, X. Sunney

doi:10.1016/j.fertnstert.2016.01.040

Cited by 46 publications

(29 citation statements)

References 26 publications

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“…Then, the amplified genome of each sample was sequenced at an approximate 0.04× genome depth using the Illumina HiSeq 2500 platform. Our previous study validated MALBAC sequencing as a satisfactory method for 24chromosome aneuploidy screening of cleavage-stage embryos and blastocysts [11,12]. In this study, MALBAC sequencing showed that 98.04% of blastocysts (50 of 51) had MALBAC sequencing results that were concordant with the aCGH diagnosis.…”

Section: Discussionsupporting

confidence: 61%

“…Using an Illumina HiSeq 2500 platform, the amplified genome of each biopsied cell was sequenced at an approximate 0.04× genome depth. Therefore, we sequenced a total of approximately 40 million bases, obtaining an average genome coverage of 3% for each single cell [11,12].…”

Section: Methodsmentioning

confidence: 99%

“…colleagues [3] evaluated semiconductor-based NGS for genetic analysis of human embryos. Huang [11,12] validated multiple annealing and looping-based amplification cycle (MALBAC) sequencing for 24-chromosome aneuploidy screening of cleavage-stage embryos and blastocysts.…”

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confidence: 99%

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Re-analysis of aneuploidy blastocysts with an inner cell mass and different regional trophectoderm cells

Huang

Yan

Lü

et al. 2017

J Assist Reprod Genet

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Section: Discussionsupporting

confidence: 61%

Section: Methodsmentioning

confidence: 99%

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Re-analysis of aneuploidy blastocysts with an inner cell mass and different regional trophectoderm cells

Huang

Yan

Lü

et al. 2017

J Assist Reprod Genet

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“…1). The MALBAC-NGS protocol has been previously validated in performing PGS with cleavage-stage and blastocyst-stage biopsies (29)(30)(31), and is increasingly used for single-gene PGD combined with chromosomal PGS (30,32). Similarly, we performed MALBAC-NGS on all 24 chromosomes from the corresponding D5 whole embryos, which we used as the gold standard to evaluate the chromosome screening results from the culture media.…”

Section: Significancementioning

confidence: 99%

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization

Fang

Chen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

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“…The complete workflow is typical of this kind of test: sample preparation, library generation, barcoding, sequencing, and data analysis. In the context of single blastomere sequencing, we offer advantages in comparison with previous validation schemes [Fiorentino et al 2014a;Wells et al 2014;Kung et al 2015;Huang et al 2016]. These advantages include simultaneous DNA amplification and library preparation in a single step, read lengths of 150 nucleotides, the use of paired-ends sequencing, and the design of specific algorithm/viewer for data analysis.…”

Section: Discussionmentioning

confidence: 99%

New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos

Vendrell

Fernández‐Pedrosa

Triviño

et al. 2017

Systems Biology in Reproductive Medicine

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Novel next-generation sequencing procedures have rapidly emerged into the preimplantation genetic screening framework. This work presents the design and validation of a new low-coverage whole-genome sequencing assay for aneuploidy detection in single blastomeres and trophectodermal samples from preimplantation embryos. The validation ensures analytical sensitivity, specificity, robustness, precision, limit of detection, resolution, and reproducibility. Specific parameters to measure the performance are defined, and the results are compared with a standardized array-based method to stablish the concordance. From the single cell genomics point of view, the main novelties are the length of reads of the libraries (150 nucleotides) together with a paired-end strategy and the design of an original algorithm and copy number viewer. A total of 129 samples were included in six experimental runs using a MiSeq Illumina platform. Samples included: single amniocytes, single blastomeres (cleavage-stage embryos), trophectoderm samples (blastocyst), and diluted DNA. Sensitivity and specificity were calculated per chromosome yielding 96% and 99%, respectively. The percentage of concordant samples was 98.2% and all of the aneuploid samples were confirmed. In conclusion, the validation yields highly reliable and reproducible results, representing an accurate and cost-effective strategy for the routine detection of aneuploidy in human embryos.

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Validation of a next-generation sequencing–based protocol for 24-chromosome aneuploidy screening of blastocysts

Abstract: MALBAC-NGS provides concordant chromosomal results when compared with aCGH and SNP array in blastocysts with chromosomal abnormalities.

Cited by 46 publications

References 26 publications

Re-analysis of aneuploidy blastocysts with an inner cell mass and different regional trophectoderm cells

Re-analysis of aneuploidy blastocysts with an inner cell mass and different regional trophectoderm cells

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization

New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos

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