Unraveling molecular pathways responsible for regulation of early embryonic development is crucial for our understanding of female infertility. Maternal determinants that control the transition from oocyte to embryo are crucial molecules that govern developmental competence of the newly conceived zygote. We describe a series of defects that are triggered by a disruption of maternal lethal effect gene, Nlrp5. Previous studies have shown that Nlrp5 hypomorph embryos fail to develop beyond the two-cell stage. Despite its importance in preimplantation development, the mechanism by which the embryo arrest occurs remains unclear. We confirmed that Nlrp5 mutant and wild-type females possess comparable ovarian germ pool and follicular recruitment rates. However, ovulated oocytes lacking Nlrp5 have abnormal mitochondrial localization and increased activity in order to sustain physiological ATP content. This results in an accumulation of reactive oxygen species and increased cellular stress causing mitochondrial depletion. Compromised cellular state is also accompanied by increased expression of cell death inducer Bax and depletion of cytochrome c. However, neither genetic deletion (Bax/Nlrp5 double knockout) nor mimetic interference (BH4 domain or Bax inhibitory peptide) were sufficient to alleviate embryo demise caused by depletion of Nlrp5. We therefore conclude that lack of Nlrp5 in oocytes triggers premature activation of the mitochondrial pool, causing mitochondrial damage that cannot be rescued by inactivation of Bax.

This paper presents a cellular force measurement technique that allows for mechanical characterization of mouse oocytes during microinjection (i.e., in situ) without requiring a separate characterization process. The technique employs an elastic cell holding device and a sub-pixel computer vision tracking algorithm to resolve cellular forces in real time with a nanonewton force measurement resolution (2 nN at 30 Hz). Mechanical properties (i.e., stiffness) of both healthy and defective mouse oocytes are characterized. The experimental results suggest that the in situ obtained force-deformation data are useful for distinguishing healthy mouse oocytes from those with aging-induced cellular defects, promising an approach for oocyte quality assessment during microinjection. Biomembrane and cytoskeleton structures of the healthy and defective oocytes are also investigated in an attempt to correlate the measured subtle mechanical difference to cellular structure changes.

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

Background: Liquid biopsy (LB) can detect actionable genomic alterations in plasma circulating tumor circulating tumor DNA beyond tissue testing (TT) alone in advanced non-small cell lung cancer (NSCLC) patients. We estimated the cost-effectiveness of adding LB to TT in the Canadian healthcare system. Methods: A cost-effectiveness analysis was conducted using a decision analytic Markov model from the Canadian public payer (Ontario) perspective and a 2-year time horizon in patients with treatment-naïve stage IV non-squamous NSCLC and ⩽10 pack-year smoking history. LB was performed using the comprehensive genomic profiling Guardant360™ assay. Standard of care TT for each participating institution was performed. Costs and outcomes of molecular testing by LB + TT were compared to TT alone. Transition probabilities were calculated from the VALUE trial (NCT03576937). Sensitivity analyses were undertaken to assess uncertainty in the model. Results: Use of LB + TT identified actionable alterations in more patients, 68.5 versus 52.7% with TT alone. Use of the LB + TT strategy resulted in an incremental cost savings of $3065 CAD per patient (95% CI, 2195–3945) and a gain in quality-adjusted life-years of 0.02 (95% CI, 0.01–0.02) versus TT alone. More patients received chemo-immunotherapy based on TT with higher overall costs, whereas more patients received targeted therapy based on LB + TT with net cost savings. Major drivers of cost-effectiveness were drug acquisition costs and prevalence of actionable alterations. Conclusion: The addition of LB to TT as initial molecular testing of clinically selected patients with advanced NSCLC did not increase system costs and led to more patients receiving appropriate targeted therapy.

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After reviewing the literature, nurses at the bedside seeking answers to clinical questions may find their inquiries remain unanswered. This article describes the yearlong Research Fellows Program in which candidates, funded for 12 hours per month of research release time, answered formal research questions in a curriculum designed to provide the skills to complete their study. Five have completed their studies; 1 has received a grant to continue. Two are in process of submitting manuscripts to journals.

Introduction: Molecular profiling of tumor tissue is the gold standard for treatment decision-making in advanced non-small cell lung cancer (NSCLC). Results may be delayed or unavailable due to insufficient tissue, prolonged wait times for biopsy, pathology assessment and testing. We piloted the use of plasma testing in the initial diagnostic workup for patients with suspected advanced lung cancer. Methods: Patients with ⩽15 pack-year smoking history and suspected advanced lung cancer referred to the lung cancer rapid diagnostic program underwent plasma circulating-tumor DNA testing using a DNA-based mutation panel. Tissue testing was performed per standard of care, including comprehensive next-generation sequencing (NGS). The primary endpoint was time from diagnostic program referral to cancer treatment in stage IV NSCLC patients (Cohort A) compared to a contemporary cohort not enrolled in the study (Cohort B) and an historical pre-COVID cohort referred to the program between 2018 and 2019 (Cohort C). Results: From January to June 2021, 20 patients were enrolled in Cohort A; median age was 70.5 years (range 33–87), 70% were female, 55% Caucasian, 85% never smokers, and 75% were diagnosed with NSCLC. Seven had actionable alterations detected in plasma or tissue (4/7 concordant). Fusions, not tested in plasma, were identified by immunohistochemistry for three patients. Mean result turnaround time was 17.8 days for plasma NGS and 23.6 days for tissue ( p = 0.10). Mean time from referral to treatment initiation was significantly shorter in cohort A at 32.6 days (SD 13.1) versus 62.2 days (SD 31.2) in cohort B and 61.5 days (SD 29.1) in cohort C, both p < 0.0001. Conclusion: Liquid biopsy in the initial diagnostic workup of patients with suspected advanced NSCLC can lead to faster molecular results and shorten time to treatment even with smaller DNA panels. An expansion study using comprehensive NGS plasma testing with faster turnaround time is ongoing (NCT04862924).

Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments. We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro. Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts. Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl‐x transcript. Cell death activation in human blastocysts was accompanied by changes in Bcl‐x splicing, favoring production of Bcl‐xS, an activator of cell death. Expression of Bcl‐xS was detected in a subset of human blastocysts that show particular clustering in dying and/or dead cells. Altering the Bcl‐xL/Bcl‐xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl‐xS, with concomitant downregulation of Bcl‐xL, compromised developmental potential and committed a subset of cells to undergoing cell death. This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content. In addition, altered Bcl‐x splicing in favor of Bcl‐xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development. Thus, we conclude that inappropriate culture conditions affect Bcl‐x isoform expression, contributing to compromised preimplantation embryo development.