Vendrell, X. et al. New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos. Syst. Biol. Reprod. Med. 63, 162–178 (2017).

The authors developed a highly reliable assay for detecting aneuploidy in single blastomeres and samples of 5–10 trophectoderm cells. Their approach—a low-coverage whole genome sequencing method from WGA libraries plus an original algorithm and copy number viewer—depended on PicoPLEX technology to amplify nanogram amounts of gDNA from picogram-level inputs for their NGS protocol. They combined PicoPLEX's limited displacement preamplification with barcodes, followed by PCR amplification, and achieved both a high level of useful reads as well as full correlation of chromosome dose with samples processed using a CGH-BAC microarray approach. They found no differences in sequencing and mapping parameters between unamplified control DNA samples and WGA products from single blastomeres, indicating an unbiased coverage using PicoPLEX technology. Their assay enabled the accurate detection of segmental aneuploidy, with rearrangements of segments down to 1 Mb in length.

Vera-Rodríguez, M. et al. Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing. Fertil. Steril. 105, 1047–1055.e2 (2016).

The authors evaluated a commonly used NGS-based PGT-A technique for the detection of segmental and whole-chromosome aneuploidies from trophectoderm biopsies and compared these results to the typical aCGH approach. The overall concordance rate between NGS and aCGH was 99.8%, and 92.9% of segments detected by both NGS and aCGH were confirmed by FISH. PicoPLEX technology was used for whole genome amplification, followed by library preparation. The NGS approach detected segmental aneuploidies down to 10 Mb with the same efficiency as aCGH; furthermore, NGS detected several mosaic segmental aneuploidies that were not detected by aCGH. Additionally, the authors determined that most of the segmental aneuploidies resulted from mitotic errors leading to mosaicism, and this mosaic pattern was reliably detected by NGS.

Macaulay, I. C. et al. G&T-seq: Parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519–522 (2015).

The authors introduced a new technique, genome and transcriptome sequencing (G&T-seq), which enables a more in-depth characterization of single cells through simultaneous full-length RNA sequencing, whole-genome or targeted sequencing, and genome-wide copy number variation (CNV) detection. Their method involves bead-based isolation of polyA+ mRNA from gDNA, and then separate amplification, library preparation, and sequencing. PicoPLEX technology was chosen to amplify DNA for downstream CNV analysis. The authors produced several interesting and novel findings using their G&T-seq method: 1) chromosomal copy number in a single cell is corroborated by the expected changes in gene expression in that cell; 2) an aneuploidy event that occurs during a single cell division tracks with gene expression dosage; 3) within a single cell, there is high concordance of single nucleotide variation detected in gDNA and mRNA; and 4) a previously unknown fusion and the causative chromosomal rearrangement underlying the fusion were discovered.

Fuchs Weizman, N. et al. Towards improving embryo prioritization: parallel next generation sequencing of DNA and RNA from a single trophectoderm biopsy. Sci. Rep. 9, 2853 (2019).

The authors created a clinically applicable method, called preimplantation genetic testing for aneuploidy and transcriptome (PGT-AT), that prepares a biopsy of 4–6 trophectoderm cells for simultaneous DNA- and RNA-seq to determine the ploidy status and transcriptome profile. In this study, two gold-standard Takara Bio technologies were employed: SMART-Seq chemistry for cDNA library preparation from full-length mRNA and PicoPLEX chemistry for cell lysis, whole genome amplification, and library preparation. In addition to achieving 100% concordance of CNV detection between the standard PGT-A method and the PGT-AT method performed on the same blastocysts, they discovered that pathways regulating gene expression and energy metabolism were downregulated in the euploid samples. The authors expressed confidence that the transcriptome profiling aspect could be safely introduced into the current clinical PGT-A workflow, and doing so could greatly improve our embryo prioritization abilities.

Tortoriello, D. V., Dayal, M., Beyhan, Z., Yakut, T. & Keskintepe, L. Reanalysis of human blastocysts with different molecular genetic screening platforms reveals significant discordance in ploidy status. J. Assist. Reprod. Genet. 33, 1467–1471 (2016).

The authors reanalyzed blastocysts that returned abnormal PGT-A results to determine the reliability of different assays run by different labs. They examined the levels of concordance between the first and second rounds of trophectoderm biopsies analyzed by two different PGS labs using aCGH or SNP array (first biopsy/lab) and SNP array or NGS (second biopsy/lab). For NGS, WGA was performed with PicoPLEX technology. Chaotic results were obtained, including normal karyotype or gender-discrepant results upon second biopsy, and it could not be determined whether the lack of concordance was due to mosaicism, differences between platforms, or human error. The authors recommended regular, independent oversight verifying test performance and accuracy.

Ho, J. R. et al. Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos. Fertil. Steril. 110, 467–475.e2 (2018).

The authors examined cell-free DNA from spent embryo medium (SEM) to detect ploidy and sex and to determine whether assisted hatching and morphologic grade have an impact on cell-free DNA amount and predictive value. All samples (SEM, trophectoderm biopsies, and whole embryos) were subjected to WGA with PicoPLEX technology followed by NGS-based PGT-A. A cell-free DNA concentration of 63.2 ng/µl was enough to get an accurate ploidy diagnosis, and assisted hatching did not seem to affect cell-free DNA metrics. Based on low concordance rates, the authors conclude that cell-free DNA from spent medium is not ready to replace cellular DNA in PGT-A, but it is a promising tool for noninvasive screening.

Vera-Rodriguez, M. et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Obstet. Gynecol. Surv. 73, 355–356 (2018).

The authors examined the cell-free DNA content of SEM used to culture blastocysts in order to determine cell-free DNA amount and whether a chromosomal diagnosis would be concordant between trophectoderm biopsies and their corresponding culture media samples. NGS-based aneuploidy testing and SNP analysis to detect maternal DNA contamination were performed. PicoPLEX technology was used for double WGA of cell-free DNA in SEM, while FISH was used on the whole blastocysts to detect mosaicism. The cell-free DNA in the media afforded a diagnosis but was discordant with trophectoderm biopsies at a rate of 67%, which was likely due to maternal DNA contamination (92%). Mosaicism was present in most embryos, a possible explanation for the observation of different chromosomal alterations detected between trophectoderm samples and the cell-free DNA from the matching SEM samples.

Kuznyetsov, V. et al. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLoS One 13, e0197262 (2018).

The authors were the first to assess the suitability of SEM in combination with blastocoel fluid (BF) as a noninvasive PGT tool, and they introduced a new technique for noninvasive collection of BF. PicoPLEX technology was used for WGA of SEM, BF, trophectoderm biopsy, and whole blastocyst samples, followed by NGS-based PGT-A. Relatively high levels of concordance of PGT-A results were obtained for all samples. The authors not only demonstrated high concordance between the noninvasive and invasive samples, but they also remarked that the WGA method on combined SEM + BF samples showed superior amplification rates of 100% over previously reported amplification rates from SEM or BF alone.

Tšuiko, O. et al. Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass. Fertil. Steril. 109, 1127–1134.e1 (2018).

The authors were the first to use high-resolution NGS to compare samples from three compartments of the same blastocyst—blastocoel fluid (BF), trophectoderm (TE), and inner cell mass (ICM)—to determine whether cell-free DNA from BF represents the chromosomal status of the rest of the embryo. Using PicoPLEX technology, DNA from all samples was successfully amplified and then subjected to NGS-based PGT-A. The authors found low concordance (40%) between BF and TE or ICM samples, compared to 86% between TE and ICM. BF contained more mosaic aneuploidies and affected chromosomes than matching TE and ICM samples. They conclude that as a single source of genetic material, BF is not diagnostically acceptable with current protocols, and TE biopsy remains the most effective and safest way of determining karyotype.