ABSTRACT
Although non-invasive pre-implantation genetic testing for aneuploidy (NIPGT-A) is potentially appropriate to assess chromosomal ploidy of the embryo, practical application of it in a routine IVF centre have not been started in the absence of a recommendation. Our objective in this study was to provide a comprehensive workflow for a clinically applicable strategy for NIPGT-A based on next-generation sequencing (NGS) technology with the corresponding bioinformatic pipeline. In a retrospective study, we performed NGS on spent blastocyst culture media of Day 3 embryos fertilised with intracytoplasmic sperm injection (ICSI) with quality score on morphology assessment using the blank culture media as background control. Chromosomal abnormalities were identified by an optimised bioinformatics pipeline applying copy number variation (CNV) detecting algorithm. In this study, we demonstrate a comprehensive workflow covering both wet- and dry-lab procedures supporting a clinically applicable strategy for NIPGT-A that can be carried out within 48 h, which is critical for the same-cycle blastocyst transfer. The described integrated approach of non-invasive evaluation of embryonic DNA content of the culture media can potentially supplement existing pre-implantation genetic screening methods.
Subject(s)
Aneuploidy , DNA Copy Number Variations , Embryo Culture Techniques/methods , Fertilization in Vitro/methods , Genetic Testing/methods , High-Throughput Nucleotide Sequencing/methods , Preimplantation Diagnosis/methods , Adult , Embryo Implantation , Female , Humans , Retrospective StudiesABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The aim of this study was to visualize the tumor propagation and surrounding mucosal field in radiography-based 3D model for advanced stage HNSCC and combine it with HPV genotyping and miRNA expression characterization of the visualized area. 25 patients with T1-3 clinical stage HNSCC were enrolled in mapping biopsy sampling. Biopsy samples were evaluated for HPV positivity and miR-21-5p, miR-143, miR-155, miR-221-5p expression in Digital Droplet PCR system. Significant miRNA expression differences of HPV positive tumor tissue biopsies were found for miR-21-5p, miR-143 and miR-221-5p compared to the HPV negative tumor biopsy series. Peritumoral mucosa showed patchy pattern alterations of miR-21-5p and miR-155 in HPV positive cases, while gradual change of miR-21-5p and miR-221-5p was seen in HPV negative tumors. In our study we found differences of the miRNA expression patterns among the HPV positive and negative tumorous tissues as well as the surrounding mucosal fields. The CT based 3D models of the cancer field and surrounding mucosal surface can be utilized to improve proper preoperative planning. Complex evaluation of HNSCC tissue organization field can elucidate the clinical and molecular differentiation of HPV positive and negative cases, and enhance effective organ saving therapeutic strategies.
Subject(s)
Imaging, Three-Dimensional/methods , MicroRNAs/genetics , Mouth Neoplasms/pathology , Mucous Membrane/pathology , Oropharyngeal Neoplasms/pathology , Papillomavirus Infections/complications , Tomography, X-Ray Computed/methods , Adolescent , Adult , Aged , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/diagnostic imaging , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/virology , Child , Child, Preschool , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Genomics/methods , Humans , Infant , Infant, Newborn , Male , Middle Aged , Mouth Neoplasms/diagnostic imaging , Mouth Neoplasms/genetics , Mouth Neoplasms/virology , Mucous Membrane/diagnostic imaging , Mucous Membrane/metabolism , Mucous Membrane/virology , Oropharyngeal Neoplasms/diagnostic imaging , Oropharyngeal Neoplasms/genetics , Oropharyngeal Neoplasms/virology , Papillomaviridae/isolation & purification , Papillomavirus Infections/virology , Prognosis , Survival Rate , Young AdultABSTRACT
Perchloric acid (PCA) precipitation is a well-known method for the separation of heavily glycosylated proteins and for reducing the masking effect of major serum proteins. The aim of this study is to characterize PCA-soluble serum proteins in healthy individuals and in patients with systemic inflammatory diseases, such as Crohn's disease and sepsis. A PCA precipitation protocol was prepared and adapted to the analytical methods. After PCA treatment of the serum, the soluble proteins in the supernatant were analyzed by SDS-PAGE and by microchip gel electrophoresis (MGE). Characteristic changes of the electrophoretic patterns of the PCA-soluble fractions were observed. Four characteristic bands (at â¼11, â¼65, â¼85, and â¼120 kDa) with varying intensity were detected by MGE. The proportion of the â¼65, â¼85, and â¼120 kDa bands were significantly higher in systemic inflammatory conditions than in healthy individuals (p < 0.001), and characteristic patterns were observed in patients with acute inflammation. The marked differences in the acid-soluble protein patterns, which were observed in patients with ongoing systemic inflammation, might be a good indicator of inflammation. The MGE analysis is a fast screening and quantification method for the detection of characteristic changes among acid-soluble serum proteins.
