The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better.

STUDY QUESTION: How well can whole chromosome copy number analysis from a single trophectoderm (TE) biopsy predict true mosaicism configurations in human blastocysts? SUMMARY ANSWER: When a single TE biopsy is tested, wide mosaicism thresholds (i.e. 20-80% of aneuploid cells) increase false positive calls compared to more stringent ones (i.e. 30-70% of aneuploid cells) without improving true detection rate, while binary classification (aneuploid/euploid) provides the highest diagnostic accuracy. WHAT IS KNOWN ALREADY: Next-generation sequencing-based technologies for preimplantation genetic testing for aneuploidies (PGT-A) allow the identification of intermediate chromosome copy number alterations potentially associated with chromosomal mosaicism in TE biopsies. Most validation studies are based on models mimicking mosaicism, e.g. mixtures of cell lines, and cannot be applied to the clinical interpretation of TE biopsy specimens. STUDY DESIGN, SIZE, DURATION: The accuracy of different mosaicism diagnostic thresholds was assessed by comparing chromosome copy numbers in multiple samples from each blastocyst. Enrolled embryos were donated for research between June 2019 and September 2020. The Institutional Review Board at the Near East University approved the study (project: YDU/2019/70-849). Embryos showing euploid/aneuploid mosaicism (n = 53), uniform chromosomal alterations (single or multiple) (n = 25), or uniform euploidy (n = 39) in their clinical TE biopsy were disaggregated into five portions: the inner cell mass (ICM) and four TE segments. Collectively, 585 samples from 117 embryos were analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated blastocysts were warmed, allowed to re-expand, and disaggregated in TE portions and ICM. PGT-A analysis was performed using Ion ReproSeq PGS kit and Ion S5 sequencer (ThermoFisher). Sequencing data were blindly analysed with Ion Reporter software to estimate raw chromosome copy numbers. Intra-blastocyst comparison of copy number data was performed employing different thresholds commonly used for mosaicism detection. From copy number data, different case scenarios were created using more stringent (30-70%) or less stringent criteria (20-80%). Categorical variables were compared using the two-sample z test for proportions. MAIN RESULTS AND THE ROLE OF CHANCE: When all the five biopsies from the same embryo were analysed with 30-70% thresholds, only 8.4% (n = 14/166) of patterns abnormal in the original analysis revealed a true mosaic configuration, displaying evidence of reciprocal events (3.6%, n = 6/166) or confirmation in additional biopsies (4.8%, n = 8/166), while most mosaic results (87.3% of total predicted mosaic patterns) remained confined to a single TE specimen. Conversely, uniform whole chromosome aneuploidies (28.3% of total patterns, n = 47/166) were confirmed in all subsequent biopsies in 97.9% of cases (n = 46/47). When 20-80% thresholds were employed (instead of 30-70%), the overall mosaicism rate per biopsy increased from 20.2% (n = 114/565) to 40.2% (n = 227/565). However, the use of a wider threshold range did not contribute to the detection of additional true mosaic patterns, while significantly increasing false positive mosaic patterns from 57.8% to 79.5% (n = 96/166; 95% CI = 49.9-65.4 vs n = 271/341; 95% CI = 74.8-83.6, respectively) (P < 0.00001). Moreover, the shift of the aneuploid cut-off from 70% to 80% of aneuploid cells resulted in mosaicism overcalling in the high range (50-80% of aneuploid cells), impacting the accuracy of uniform aneuploid classification. Parametric analysis of thresholds, based on multifocal analysis, revealed that a binary classification scheme with a single cut-off at a 50% level provided the highest sensitivity and specificity rates. Further analysis on technical noise distribution at the chromosome level revealed a greater impact on smaller chromosomes. LIMITATIONS, REASONS FOR CAUTION: While enrolment of a population enriched in embryos showing intermediate chromosome copy numbers enhanced the evaluation of the mosaicism category compared with random sampling such study population selection is likely to lead to an overall underestimation of PGT-A accuracy compared to a general assessment of unselected clinical samples. This approach involved the analysis of aneuploidy chromosome copy number thresholds at the embryo level; future studies will need to evaluate these criteria in relation to clinical predictive values following embryo transfers for different PGT-A assays. Moreover, the study lacked genotyping-based confirmation analysis. Finally, aneuploid embryos with known meiotic partial deletion/duplication were not included. WIDER IMPLICATIONS OF THE FINDINGS: Current technologies can detect low-intermediate chromosome copy numbers in preimplantation embryos but their identification is poorly correlated with consistent propagation of the anomaly throughout the embryo or with negative clinical consequences when transferred. Therefore, when a single TE biopsy is analysed, diagnosis of chromosomal mosaicism should be evaluated carefully. Indeed, the use of wider mosaicism thresholds (i.e. 20-80%) should be avoided as it reduces the overall PGT-A diagnostic accuracy by increasing the risk of false positive mosaic classification and false negative aneuploid classification. From a clinical perspective, this approach has negative consequences for patients as it leads to the potential deselection of normal embryos for transfer. Moreover, a proportion of uniform aneuploid embryos may be inaccurately categorized as high-level mosaic, with a consequent negative outcome (i.e. miscarriage) when inadvertently selected for transfer. Clinical outcomes following PGT-A are maximized when a 50% threshold is employed as it offers the most accurate diagnostic approach. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by Igenomix. The authors not employed by Igenomix have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Molecular tools for the genomic assessment of oocyte's reproductive competence.

The most important factor associated with oocytes' developmental competence has been widely identified as the presence of chromosomal abnormalities. However, growing application of genome-wide sequencing (GS) in population diagnostics has enabled the identification of multifactorial genetic predispositions to sub-lethal pathologies, including those affecting IVF outcomes and reproductive fitness. Indeed, GS analysis in families with history of isolated infertility has recently led to the discovery of new genes and variants involved in specific human infertility endophenotypes that impact the availability and the functionality of female gametes by altering unique mechanisms necessary for oocyte maturation and early embryo development. Ongoing advancements in analytical and bioinformatic pipelines for the study of the genetic determinants of oocyte competence may provide the biological evidence required not only for improving the diagnosis of isolated female infertility but also for the development of novel preventive and therapeutic approaches for reproductive failure. Here, we provide an updated discussion and review of the progresses made in preconception genomic medicine in the identification of genetic factors associated with oocyte availability, function, and competence.

Technical factors to consider when developing an Expanded Carrier Screening platform.

PURPOSE OF REVIEW: Expanded Carrier Screening (ECS) is a genetic test able to detect carriers for a large number of autosomal recessive and X-linked diseases. Its clinical utilization is increasing but some technical aspects for its implementation are still controversial. RECENT FINDINGS: In the current literature, several aspects of ECS panel implementation have been addressed. One of the most relevant topics involves which genes/pathologies should be included in an optimized ECS panel and which variants should be reported. SUMMARY: Here, we review the best practice criteria to refine and improve clinical utility and validity of an ECS panel. The criteria for optimal ECS panel implementation include the severity of pathologies, the prevalence of diseases in general population and a definitive or strong gene/disease association. Moreover, we discuss the main complications associated with the reporting of Variant of Uncertain Significance and the need for periodic reassessment.

Application of nanotechnology to target and exploit tumour associated proteases.

Proteases are hydrolytic enzymes fundamental for a variety of physiological processes, but the loss of their regulation leads to aberrant functions that promote onset and progression of many diseases including cancer. Proteases have been implicated in almost every hallmark of cancer and whilst widely investigated for tumour therapy, clinical adoption of protease inhibitors as drugs remains a challenge due to issues such as off-target toxicity and inability to achieve therapeutic doses at the disease site. Now, nanotechnology-based solutions and strategies are emerging to circumvent these issues. In this review, preclinical advances in approaches to enhance the delivery of protease drugs and the exploitation of tumour-derived protease activities to promote targeting of nanomedicine formulations is examined. Whilst this field is still in its infancy, innovations to date suggest that nanomedicine approaches to protease targeting or inhibition may hold much therapeutic and diagnostic potential.

Development of an advanced nanoformulation for the intracellular delivery of a caspase-3 selective activity-based probe.

The ability to label active caspase-3 represents a useful pharmacodynamic strategy to determine the efficacy of anti-tumour drugs. Activity-based probes (ABPs) provide a method for the labelling of activated caspases and the recent development of hybrid combinatorial substrate libraries (HyCoSuL) has allowed for the generation of highly selective ABPs to discriminately label these proteases. Here using this approach, a novel caspase-3 selective ABP (CS1) has been developed and validated in apoptotic cells to selectively bind caspase-3 over the closely related caspase-7. However, a critical bottleneck for ABPs is their cell penetrance and therefore this cell-impermeable CS1 probe was subsequently formulated into PLGA-based nanoparticles (CS1-NPs). We demonstrate the ability of these particles to be taken up by the cells and facilitate intracellular delivery of the ABP to effectively label caspase 3 in response to apoptotic stimuli. This work forms the foundation of a novel approach for the labelling of caspase 3 and may have downstream utility to measure real time apoptosis in tumours and other organs.

USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells.

BACKGROUND: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs). METHODS: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed. RESULTS: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib. CONCLUSIONS: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

Clearance of intracellular Klebsiella pneumoniae infection using gentamicin-loaded nanoparticles.

Klebsiella pneumoniae is a foremost gram-negative pathogen that can induce life-threatening nosocomial pulmonary infections. Although it can be phagocytosed successfully by lung resident macrophages, this pathogen remains viable within vacuolar compartments, resulting in chronic infection and limiting therapeutic treatment with antibiotics. In this study, we aimed to generate and evaluate a cell-penetrant antibiotic poly(lactide-co-glycolide) (PLGA)-based formulation that could successfully treat intracellular K. pneumoniae infection. Screening of formulation conditions allowed the generation of high drug loaded nanoparticles through a water-in-oil-in-water approach. We demonstrated the therapeutic usefulness of these gentamicin-loaded nanoparticles (GNPs), showing their ability to improve survival and provide extended prophylactic protection towards K. pneumoniae using a Galleria mellonella infection model. We subsequently showed that the GNPs could be phagocytosed by K. pneumoniae infected macrophages, and significantly reduce the viability of the intracellular bacteria without further stimulation of pro-inflammatory or pro-apoptotic effects on the macrophages. Taken together, these results clearly show the potential to use antibiotic loaded NPs to treat intracellular K. pneumoniae infection, reducing bacterial viability without concomitant stimulation of inflammatory or pyroptotic pathways in the treated cells.