Concordance between different trophectoderm biopsy sites and the inner cell mass of chromosomal composition measured with a next-generation sequencing platform.

STUDY QUESTION: In PGS, does chromosomal constitution differ among trophectoderm (TE) biopsy sites and between them and the inner cell mass (ICM)? SUMMARY ANSWER: The ploidy concordance between ICM and TE was independent of whether the biopsy site in the TE was near to or far from the ICM. WHAT IS KNOWN ALREADY: TE biopsies are considered less harmful to developing embryos than blastomere biopsies. Removal of multi-cellular samples permits high-resolution next-generation sequencing (Veriseq NGS) to detect aneuploidy present in a minority of cells (mosaicism of diploid and aneuploid cells). However, the prevalence of ploidy discrepancies between different TE biopsy sites and the ICM, as well as confined mosaicism (aneuploidy only in a particular area), has not been established. STUDY DESIGN, SIZE, DURATION: Biopsies were taken from a site opposite to the ICM (TE1), near the ICM (TE2) and within the ICM of the same embryo in 33 donated blastocysts obtained from 12 volunteer patients. The samples were analyzed by the Veriseq NGS to assess ploidy concordance. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mean age of the patients was 34.4 years, and samples from all three biopsy sites were achieved in 29 frozen thawed blastocysts. The aneuploid percentage in each sample was interpreted by Veriseq NGS at the finest resolution involving the number of reads after filtering, sample overall noise score, and average quality/alignment scores according to the Veriseq quality control assessment. Ploidy concordance was then assessed between different TE fractions, and between the TE and ICM. MAIN RESULTS AND THE ROLE OF CHANCE: The euploid rates were similar in the TEs and ICM, and no preferential allocation of euploid lineage within a blastocyst was demonstrated. Whether the biopsy site in the TE was near to or far from the ICM, the chromosomal consistency rate was similar [TE1-to-ICM, 86.2% (25/29) versus TE2-to-ICM, 89.7% (26/29); P = 1.0], suggesting that the cells with different chromosomal components may spread randomly throughout the TE. The following two types of inconsistent PGS conclusions between TE and ICM due to confined mosaicism were observed: (i) euploid TE with mosaic ICM (3%) (1/29); and (ii) mosaic TE with euploid ICM (3%) (1/29) or with aneuploid ICM (7%) (2/29). Thus, the overall rate of confined mosaicism was 14% (4/29). LARGE SCALE DATA: N/A. LIMITATION, REASONS FOR CAUTION: The approach used in the present study was affected by biopsy manipulation limitations involving possible cell contamination and the technical challenge of comprehensive chromosomal screening (CCS) procedures. WIDER IMPLICATIONS OF THE FINDINGS: The rate of confined mosaicism in the blastocysts was estimated in this preliminary study, thus, specifying the incidence of biological sampling biases. The results also verified the random distribution of different cell lineages, and the representative value of a single biopsied sample from the TE. STUDY FUNDING AND CONFLICT OF INTEREST(S): No external funding was obtained; all the authors declare no conflicts of interest regarding this study.

[Progress in polyphosphate and related metabolizing enzymes].

Inorganic polyphosphate (Poly P) is a polymer consisting of ten to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds, which is abundantly found in all organisms and nature. Here the basic facts of PolyP are summarized: genes regulated by polyP, role in DNA uptake, motility of microorganism, function in stress response, the virulence of pathogens, as well as the proliferation of mammary cancer cells, blood coagulation, cell calcification and the modulation of mitochondrial activity. Enzymes with activities requiring polyP, such as endopolyphosphatase, glucokinase, NAD kinase, AMP phosphotransferase are outlined too. The structure and activity of enzymes regulating polyP level such as polyphosphate kinase and exopolyphosphatase are noted. A thorough analysis of the mycobacterium tuberculosis PPX protein homologs and their biochemical activity is presented.

High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq.

The Ion S5 (Thermo Fisher Scientific) and Miseq (Illumina) NGS systems are both widely used in the clinical laboratories conducting PGT-A. Each system employs discrepant library preparation steps, sequencing principles, and data processing algorithms. The automatic interpretation via Ion Reporter software (Thermo Fisher Scientific) and the manual interpretation via BlueFuse Multi software (Illumina) for chromosomal copy number variation (CNV) represent very different reporting approaches. Thus, it is intriguing to compare their ability of ploidy detection as PGT-A/NGS system. In the present study, four aneuploid cell lines were individually mixed with a diploid cell line at different aneuploid ratios of 0% (0:5), 10% (1:9), 20% (1:4), 40% (2:3), 50% (3:3), 60% (3:2), 80% (4:1) and 100% (5:0) to assess the sensitivity and specificity for whole chromosomal and segmental aneuploidy detection. The clinical biopsies of 107 blastocysts from 46 IVF/PGT-A cycles recruited between December 2019 and February 2020 were used to calculate the concordance. Initially, the pre-amplified products were divided into two aliquots for different library preparation procedures of each system. Applying the same calling criteria, automatic identification was achieved through the Ion Reporter, while well-trained technicians manually identified each sample through the BlueFuse Multi. The results displayed that both systems reliably distinguished chromosomal CNV of the mixtures with at least 10% aneuploidy from karyotypically normal samples ([Ion S5] whole-chromosomal duplication: 2.14 vs. 2.05, p value = 0.009, segmental deletion: 1.88 vs. 2.05, p value = 0.003; [Miseq] whole-chromosomal duplication: 2.12 vs. 2.03, p value = 0.047, segmental deletion: 1.82 vs. 2.03, p value = 0.002). The sensitivity and specificity were comparable between the Ion S5 and Miseq ([sensitivity] 93% vs. 90%, p = 0.78; [specificity] 100% vs. 100%, p value = 1.0). In the 107 clinical biopsies, three displayed chaotic patterns (2.8%), which could not be interpreted for the ploidy. The ploidy concordance was 99.04% (103/104) per embryo and 99.47% (2265/2277) per chromosome pair. Since their ability of detection were proven to be similar, the automatic identification in Ion S5 system presents comparatively faster and more standardized performance.

Identification of mosaic and segmental aneuploidies by next-generation sequencing in preimplantation genetic screening can improve clinical outcomes compared to array-comparative genomic hybridization.

BACKGROUND: Chromosomal mosaicism is observed as the presence of both euploid and aneuploid cells in a particular blastocyst. Recent studies have reported that the implantation rate of mosaic embryo transfer is remarkably lower than the euploid embryos. The superior capability of next-generation sequencing (NGS) to detect chromosomal mosaicism in preimplantation genetic screening (PGS) remains controversial, and several data displayed similar implantation and pregnancy rates using NGS or array comparative genomic hybridization (aCGH). RESULTS: In this study, the main inconsistency of aneuploidy detection and clinical performance between the NGS and aCGH were assessed. The phase I consisted of a parallel comparison in 182 blastocysts from 45 selected PGS patients for both the NGS and aCGH platforms. The phase II retrospectively compared the clinical outcomes of 90 patients with NGS-screened euploid embryo transfer to that of 129 patients with aCGH-screened euploid embryo transfer. The parallel comparison showed that the inconsistency of embryo euploidy was 11.8% (p = 0.01). Chromosomal mosaicism (10.7% with NGS vs. 3.9% with aCGH) and segmental aneuploidy (10.7% with NGS vs. 6.7% with aCGH) contributed to the discrepancy mainly. The chromosomally mosaic embryos (20%-50% of aneuploidy) and several embryos with segmental aneuploidy (≥10 Mbp) were hard to distinguish using the aCGH platform, but could be clearly identified using the NGS platform. After the first euploid embryo cryotransfer, the ß-HCG(+) rate and implantation rate significantly increased in the PGS/NGS patients (HCG[+] rate: 73.3% in PGS/NGS vs. 60.5% in PGS/aCGH, p = 0.048; implantation rate: 53.2% in PGS/NGS vs. 45.0% in PGS/aCGH, p = 0.043). The clinical and ongoing pregnancy rates appeared higher in the NGS group, but did not reached statistical significance. CONCLUSIONS: The results demonstrated that the NGS platform can identify embryos with chromosomal mosaicism and segmental aneuploidy more precisely than the aCGH platform, and the following clinical performance of NGS was more favorable.