Abstract:This is the first report analyzing distribution patterns of segmental aneuploidies in trophectoderm biopsy by NGS. We have demonstrated that NGS allows the detection of pure and mosaic segmental aneuploidies with the same efficiency as aCGH. The FISH analysis confirmed the existence of these events in the trophectoderm and the inner cell mass.
“…That is, no false-positive predictions of aneuploidy were made by either platform when standard criteria were applied.Fig. 2
a Sensitivity across three sets of analyses for each mixture level: qPCR default settings, VeriSeq default settings, and VeriSeq with criteria defined by Vera-Rodriguez, et al [14] (custom VeriSeq). Sensitivity is based on detecting trisomy of 13, 15, and 18, and monosomy of X ( n = 24 at each mixture level for each platform).…”
Section: Resultsmentioning
confidence: 99%
“…In contrast, when applying custom analysis criteria (not using automated calls from Bluefuse Multi software), as defined by Vera-Rodriguez et al [14], and although significantly improved sensitivity of detecting aneuploidy was observed from 17 to 66 % aneuploidy levels ( p < 0.05) (Fig. 2a), the gain in sensitivity resulted in a significant increase ( p < 0.001) in the rate of false positives.…”
Section: Resultsmentioning
confidence: 99%
“…One protocol involved the use of either SelectCCS (Foundation for Embryonic Competence Inc., Basking Ridge, New Jersey), a previously validated qPCR platform [2, 3, 16], or VeriSeq PGS (Illumina Inc., Santa Clara, CA), a commercially available method involving WGA and next-generation sequencing (NGS) on a MiSeq. Blinded computational prediction of aneuploidy was made with either (i) previously established criteria for qPCR [16], termed “default qPCR,” (ii) as recommended by the supplier utilizing the automatic aneuploidy calls made by Bluefuse Multi software (BlueFuse, Illumina Inc., version 4.2(20289)), termed “default VeriSeq,” or (iii) using previously defined [14] customized criteria for VeriSeq PGS (which examine changes in the median copy numbers and override automated calls made by Bluefuse Multi software), termed “custom VeriSeq”. It is important to note here that the default settings of the software used by these platforms in this study are not able to be manually altered and, any further tweaking of criteria must be done post-analysis with an independent algorithm.…”
Section: Methodsmentioning
confidence: 99%
“…Therefore, two platforms were tested using default settings, and one platform (NGS) was further investigated using additional published criteria. As previously defined [13], the custom VeriSeq analysis criteria predicted additional “mosaic” aneuploidies when median copy number values of the chromosomes were either between 1.2 and 1.8 or between 2.2 and 2.8 [14]. Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Some have argued that next-generation sequencing (NGS)-based strategies may provide an enhanced and unique opportunity to predict mosaicism within a trophectoderm biopsy [13], and one group recently published criteria for predicting mosaicism within a trophectoderm biopsy using intermediate values of copy number [14]. Nonetheless, there remains a lack of data regarding the actual capabilities and comparative performance of contemporary CCS platforms for predicting aneuploidy in a mosaic sample.…”
PurposeA subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy.MethodsFour cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms.ResultsWith the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy).ConclusionsBy demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.
“…That is, no false-positive predictions of aneuploidy were made by either platform when standard criteria were applied.Fig. 2
a Sensitivity across three sets of analyses for each mixture level: qPCR default settings, VeriSeq default settings, and VeriSeq with criteria defined by Vera-Rodriguez, et al [14] (custom VeriSeq). Sensitivity is based on detecting trisomy of 13, 15, and 18, and monosomy of X ( n = 24 at each mixture level for each platform).…”
Section: Resultsmentioning
confidence: 99%
“…In contrast, when applying custom analysis criteria (not using automated calls from Bluefuse Multi software), as defined by Vera-Rodriguez et al [14], and although significantly improved sensitivity of detecting aneuploidy was observed from 17 to 66 % aneuploidy levels ( p < 0.05) (Fig. 2a), the gain in sensitivity resulted in a significant increase ( p < 0.001) in the rate of false positives.…”
Section: Resultsmentioning
confidence: 99%
“…One protocol involved the use of either SelectCCS (Foundation for Embryonic Competence Inc., Basking Ridge, New Jersey), a previously validated qPCR platform [2, 3, 16], or VeriSeq PGS (Illumina Inc., Santa Clara, CA), a commercially available method involving WGA and next-generation sequencing (NGS) on a MiSeq. Blinded computational prediction of aneuploidy was made with either (i) previously established criteria for qPCR [16], termed “default qPCR,” (ii) as recommended by the supplier utilizing the automatic aneuploidy calls made by Bluefuse Multi software (BlueFuse, Illumina Inc., version 4.2(20289)), termed “default VeriSeq,” or (iii) using previously defined [14] customized criteria for VeriSeq PGS (which examine changes in the median copy numbers and override automated calls made by Bluefuse Multi software), termed “custom VeriSeq”. It is important to note here that the default settings of the software used by these platforms in this study are not able to be manually altered and, any further tweaking of criteria must be done post-analysis with an independent algorithm.…”
Section: Methodsmentioning
confidence: 99%
“…Therefore, two platforms were tested using default settings, and one platform (NGS) was further investigated using additional published criteria. As previously defined [13], the custom VeriSeq analysis criteria predicted additional “mosaic” aneuploidies when median copy number values of the chromosomes were either between 1.2 and 1.8 or between 2.2 and 2.8 [14]. Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Some have argued that next-generation sequencing (NGS)-based strategies may provide an enhanced and unique opportunity to predict mosaicism within a trophectoderm biopsy [13], and one group recently published criteria for predicting mosaicism within a trophectoderm biopsy using intermediate values of copy number [14]. Nonetheless, there remains a lack of data regarding the actual capabilities and comparative performance of contemporary CCS platforms for predicting aneuploidy in a mosaic sample.…”
PurposeA subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy.MethodsFour cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms.ResultsWith the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy).ConclusionsBy demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.
We aimed to use next generation sequencing (NGS) to investigate chromosomal abnormalities in blastocyst trophectoderm (TE) samples, and reproductive outcomes with the different types of chromosomal rearrangements (CR) and for each sex of CR carrier. A total of 1189 blastocyst TE samples were evaluated using NGS to detect chromosomal unbalanced translocations as well as aneuploidy, including blastocytes from 637 blastocysts from carriers of balanced CR and 552 blastocysts from carriers of normal chromosomes. The optimal embryos had lower chromosomal abnormality rates compared to the poor‐quality embryos. The experimental group had significantly reduced rates of normal embryos and euploidy, and higher rates of total abnormalities, aneuploidy and unbalanced chromosomal aberrations. Carriers of reciprocal translocations had a reduced rate of normal embryos and an increased percentage of embryos with total abnormalities and unbalanced chromosomal aberrations compared with carriers of Robertsonian translocations. Couples with female carriers of chromosomal abnormalities had significantly reduced rates of normal embryos and euploidy, and a higher percentage of embryos with total abnormalities, aneuploidy, and unbalanced chromosomal aberrations compared with couples of male carriers. Our preimplantation genetic testing (PGT) study identified higher rates of chromosomal abnormalities, including chromosomal unbalanced translocations and aneuploidy, in blastocysts from CR carriers, especially from the female carriers, in a Chinese population. The PGT cycles successfully improved clinical outcomes by increasing the fertilization rate and reducing the early spontaneous abortion rate compared with the in vitro fertilization and intracytoplasmic sperm injection cycles, especially for CR carriers.
The adoption of natural‐based fibers in place of inorganic reinforcements is an effective approach to reduce the environmental and economic impact of composite materials. In particular, hemp is an attractive solution due to its mechanical, physical, and growing properties. The present article deals with the manufacturing of thermoset hemp‐reinforced composite materials. In particular, the investigation moves into the production by resin transfer molding and by resin powder molding with the use of epoxy polymeric material. To describe the effects of the technological cycle onto the characteristic of realized products, different manufacturing parameters have been combined during the braiding of reinforcement and the polymerization of the final composite. Computed tomography, microscopical analysis, and tensile tests have been used to observe the main effects of the manufacturing process and mechanical properties of the materials. Furthermore, elastic moduli of the materials have been estimated by means of modified rule of mixture and Halpin‐Tsai models in order to verify their effectiveness in forecasting stiffness of the hemp‐reinforced composites in the early design phase. The article extends the existing knowledge base on hemp‐reinforced thermoset composites manufactured with different processes. Results also illustrate relations existing between error introduced by calculation models and the intrinsic variability in mechanical properties.
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