Complex genetic variation in nearly complete human genomes.

Diverse sets of complete human genomes are required to construct a pangenome reference and to understand the extent of complex structural variation. Here, we sequence 65 diverse human genomes and build 130 haplotype-resolved assemblies (130 Mbp median continuity), closing 92% of all previous assembly gaps1,2 and reaching telomere-to-telomere (T2T) status for 39% of the chromosomes. We highlight complete sequence continuity of complex loci, including the major histocompatibility complex (MHC), SMN1/SMN2, NBPF8, and AMY1/AMY2, and fully resolve 1,852 complex structural variants (SVs). In addition, we completely assemble and validate 1,246 human centromeres. We find up to 30-fold variation in α-satellite high-order repeat (HOR) array length and characterize the pattern of mobile element insertions into α-satellite HOR arrays. While most centromeres predict a single site of kinetochore attachment, epigenetic analysis suggests the presence of two hypomethylated regions for 7% of centromeres. Combining our data with the draft pangenome reference1 significantly enhances genotyping accuracy from short-read data, enabling whole-genome inference3 to a median quality value (QV) of 45. Using this approach, 26,115 SVs per sample are detected, substantially increasing the number of SVs now amenable to downstream disease association studies.

Implementation of a Quality Improvement Learning Collaborative to Support Implementation of the 2016 CDC Guideline for Prescribing Opioids for Chronic Pain: Case Study from Nurse-Led Clinics.

PURPOSE: The authors describe a case study of a quality improvement initiative to implement the 2016 CDC Guideline for Prescribing Opioids for Chronic Pain2 ("2016 CDC Guideline") into nurse-led primary care practices in central Appalachia. DESIGN: In this controlled pre-post quality improvement study, a policy change, an electronic health record form, and supporting education were implemented. Knowledge change and quality improvement metrics were measured before and after implementation. DATA SOURCES: The data comprised pre- and post-knowledge survey and quality improvement metrics from the electronic health record. RESULTS: After the implementation of the chronic pain intake form and supporting training and education, marked improvements in documentation and completion of the 2016 CDC Guideline and Tennessee Clinical Practice Guideline-concordant activities were observed, suggesting an increase in compliance with guidelines. CONCLUSIONS: Quality improvement efforts that focus on opioid management best practices may be effective at enhancing 2016 CDC Guideline-concordant care in clinics, including nurse-led ones. Similar strategies could be trialed to ensure the 2022 CDC Clinical Practice Guideline recommendations for opioid and pain management are adopted effectively. PRACTICE IMPLICATIONS: Interventions to improve opioid and pain management through quality improvement efforts require policy changes, clinician and patient education, and electronic record tools.

Mapping MAVE data for use in human genomics applications.

The large-scale experimental measures of variant functional assays submitted to MaveDB have the potential to provide key information for resolving variants of uncertain significance, but the reporting of results relative to assayed sequence hinders their downstream utility. The Atlas of Variant Effects Alliance mapped multiplexed assays of variant effect data to human reference sequences, creating a robust set of machine-readable homology mappings. This method processed approximately 2.5 million protein and genomic variants in MaveDB, successfully mapping 98.61% of examined variants and disseminating data to resources such as the UCSC Genome Browser and Ensembl Variant Effect Predictor.

Ecological momentary assessments of cognitive dysfunction and passive suicidal ideation among college students.

Cognitive dysfunction (CD), inclusive of specific cognitive content (e.g., hopelessness, unbearability) or impaired cognitive processes (e.g., attentional fixation on suicide, rumination), is a key risk factor for suicidal ideation (SI). This study aimed to evaluate multiple forms of CD using ecological momentary assessment (EMA) to determine the unique contributions of CD to concurrent and prospective SI. Fifty-five college students with a history of SI or non-suicidal self-injury completed EMA surveys measuring momentary CD and passive SI ("Wish to Die" [WTD], "Wish to Stay Alive" [WTL]) four times a day for 14 days (2149 total observations). Passive SI and CD variables showed notable within-person variability. Multiple CD variables were significant predictors of concurrent ideation when examined simultaneously in multilevel models with random intercepts and fixed slopes, and associations were stronger when participants were around others. Controlling for concurrent passive SI, between-person rumination was a significant predictor of prospective WTD, and both within-person unbearability and between-person hopelessness were each predictive of prospective WTL. These findings provide evidence for the roles of specific types of CD in conferring risk for passive SI and highlight potentially malleable factors that can be changed through targeted interventions.

Long-read sequencing and structural variant characterization in 1,019 samples from the 1000 Genomes Project.

Structural variants (SVs) contribute significantly to human genetic diversity and disease 1-4 . Previously, SVs have remained incompletely resolved by population genomics, with short-read sequencing facing limitations in capturing the whole spectrum of SVs at nucleotide resolution 5-7 . Here we leveraged nanopore sequencing 8 to construct an intermediate coverage resource of 1,019 long-read genomes sampled within 26 human populations from the 1000 Genomes Project. By integrating linear and graph-based approaches for SV analysis via pangenome graph-augmentation, we uncover 167,291 sequence-resolved SVs in these samples, considerably advancing SV characterization compared to population-wide short-read sequencing studies 3,4 . Our analysis details diverse SV classes-deletions, duplications, insertions, and inversions-at population-scale. LINE-1 and SVA retrotransposition activities frequently mediate transductions 9,10 of unique sequences, with both mobile element classes transducing sequences at either the 3'- or 5'-end, depending on the source element locus. Furthermore, analyses of SV breakpoint junctions suggest a continuum of homology-mediated rearrangement processes are integral to SV formation, and highlight evidence for SV recurrence involving repeat sequences. Our open-access dataset underscores the transformative impact of long-read sequencing in advancing the characterisation of polymorphic genomic architectures, and provides a resource for guiding variant prioritisation in future long-read sequencing-based disease studies.

Can serum progesterone concentration direct a fresh or freeze-all transfer strategy in the first in vitro fertilisation cycle?

PURPOSE: To examine the interaction between serum progesterone concentration on the trigger day and choice of freeze-all and fresh transfer strategies on live birth in an unselected population as well as in patients over 35 years old. METHODS: We performed a retrospective cohort study of 26,661 patients commencing their first IVF cycle in a large fertility centre between 2015 and 2019, including 4687 patients over 35 years old. We performed a multivariable fractional polynomial interaction analysis within a logistic regression model to investigate the interaction between serum progesterone concentration and the choice of freeze-all or fresh transfer strategy following the first transfer. RESULTS: 15,539 patients underwent a fresh embryo transfer and 11,122 underwent a freeze-all strategy in their first IVF cycle. The freeze-all group had a higher live birth rate compared to the fresh group (43.9% vs 40.3%). After adjusting for confounding factors, there was a positive interaction between serum progesterone concentrations and the choice of a freeze-all versus fresh embryo transfer on live birth (p for interaction 0.0001), with a larger magnitude of effect when progesterone concentration was higher. Such an interaction was also observed in patients over 35 years old (p for interaction 0.01), but the treatment effect curve over progesterone concentrations was almost flat. CONCLUSIONS: In an unselected population, frozen transfer is associated with greater chances of live birth, especially in patients with higher serum progesterone concentration. In patients over 35 years old, the benefit of a freeze-all policy appears small across all serum progesterone concentrations.

Storage trends, usage and disposition outcomes following egg freezing.

RESEARCH QUESTION: What happens to eggs after egg freezing? DESIGN: A retrospective cohort study was performed spanning 2012-2022. Data were obtained from seven assisted reproductive technology clinics in Victoria, Australia. Aggregated, de-identified data were collected on cycles that resulted in egg freezing and the following outcomes, including treatment involving thawed eggs and disposition outcomes of surplus eggs. RESULTS: The number of patients with eggs in storage grew rapidly from 144 in 2012 to 2015 in 2022. In 2022, 73% of patients had stored their eggs for <5 years, 25% for 5-10 years, and 2% for ≥10 years. Most thaw cycles (600/645, 93%) involved eggs that had been frozen for <5 years, of which 47% had been frozen for <6 months. Overall, the live birth rate per initiated thaw cycle was 12%. Across the study period, 2800 eggs from 286 patients were either discarded, donated or exported. Of the 128 patients who discarded their eggs, 32% had stored their eggs for <5 years, 32% for 5-10 years and 36% for >10 years. Of the 23 patients who donated their eggs to someone else, all but four had stored their eggs for <5 years. No eggs were donated to research over the study period. CONCLUSIONS: This study shows that very few patients have made the decision to use or relinquish their eggs. Strategies may be needed to address the prolonged storage of surplus eggs, and ensure that patients are supported to make decisions regarding the fate of their eggs which align with their preferences and values.

Mobilisation and analyses of publicly available SARS-CoV-2 data for pandemic responses.

The COVID-19 pandemic has seen large-scale pathogen genomic sequencing efforts, becoming part of the toolbox for surveillance and epidemic research. This resulted in an unprecedented level of data sharing to open repositories, which has actively supported the identification of SARS-CoV-2 structure, molecular interactions, mutations and variants, and facilitated vaccine development and drug reuse studies and design. The European COVID-19 Data Platform was launched to support this data sharing, and has resulted in the deposition of several million SARS-CoV-2 raw reads. In this paper we describe (1) open data sharing, (2) tools for submission, analysis, visualisation and data claiming (e.g. ORCiD), (3) the systematic analysis of these datasets, at scale via the SARS-CoV-2 Data Hubs as well as (4) lessons learnt. This paper describes a component of the Platform, the SARS-CoV-2 Data Hubs, which enable the extension and set up of infrastructure that we intend to use more widely in the future for pathogen surveillance and pandemic preparedness.

Implementation of a Colorectal Surgical Site Infection Prevention Bundle and Checklist: A Quality Improvement Project.

After noting an elevated surgical site infection rate in 2019 associated with colorectal surgeries, leaders at two Central Virginia health system hospitals convened an interdisciplinary team to audit current practices and research infection prevention strategies. After identifying a lack of standardization in care processes for colorectal surgery patients and reviewing the literature on colorectal bundles, the team created a bundle focusing on the use of antibiotics, chlorhexidine gluconate wipes or baths, separate closing instrument trays, nasal decolonization, bowel preparation, and maintaining patient normothermia. After synthesis and stakeholder input, the team implemented the colorectal bundle along with a checklist for all users to complete to ensure compliance and standardization of practice and for auditing purposes. Implementation results were positive: the total number of colorectal infections decreased from nine in 2020 to three in 2021. Education was critical to securing staff member engagement for successful implementation of and compliance with the bundle.

The complete sequence of a human Y chromosome.

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4,5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, showing the complete ampliconic structures of gene families TSPY, DAZ and RBMY; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a previous assembly of the CHM13 genome4 and mapped available population variation, clinical variants and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

DECIPHER: Improving Genetic Diagnosis Through Dynamic Integration of Genomic and Clinical Data.

DECIPHER (Database of Genomic Variation and Phenotype in Humans Using Ensembl Resources) shares candidate diagnostic variants and phenotypic data from patients with genetic disorders to facilitate research and improve the diagnosis, management, and therapy of rare diseases. The platform sits at the boundary between genomic research and the clinical community. DECIPHER aims to ensure that the most up-to-date data are made rapidly available within its interpretation interfaces to improve clinical care. Newly integrated cardiac case-control data that provide evidence of gene-disease associations and inform variant interpretation exemplify this mission. New research resources are presented in a format optimized for use by a broad range of professionals supporting the delivery of genomic medicine. The interfaces within DECIPHER integrate and contextualize variant and phenotypic data, helping to determine a robust clinico-molecular diagnosis for rare-disease patients, which combines both variant classification and clinical fit. DECIPHER supports discovery research, connecting individuals within the rare-disease community to pursue hypothesis-driven research.

SUsPECT: a pipeline for variant effect prediction based on custom long-read transcriptomes for improved clinical variant annotation.

Our incomplete knowledge of the human transcriptome impairs the detection of disease-causing variants, in particular if they affect transcripts only expressed under certain conditions. These transcripts are often lacking from reference transcript sets, such as Ensembl/GENCODE and RefSeq, and could be relevant for establishing genetic diagnoses. We present SUsPECT (Solving Unsolved Patient Exomes/gEnomes using Custom Transcriptomes), a pipeline based on the Ensembl Variant Effect Predictor (VEP) to predict variant impact on custom transcript sets, such as those generated by long-read RNA-sequencing, for downstream prioritization. Our pipeline predicts the functional consequence and likely deleteriousness scores for missense variants in the context of novel open reading frames predicted from any transcriptome. We demonstrate the utility of SUsPECT by uncovering potential mutational mechanisms of pathogenic variants in ClinVar that are not predicted to be pathogenic using the reference transcript annotation. In further support of SUsPECT's utility, we identified an enrichment of immune-related variants predicted to have a more severe molecular consequence when annotating with a newly generated transcriptome from stimulated immune cells instead of the reference transcriptome. Our pipeline outputs crucial information for further prioritization of potentially disease-causing variants for any disease and will become increasingly useful as more long-read RNA sequencing datasets become available.

Is online learning during the COVID-19 pandemic associated with increased burnout in medical learners?: A medical school's experience.

INTRODUCTION: The COVID-19 pandemic necessitated a shift to virtual curriculum delivery at Canadian medical schools. At the NOSM University, some learners transitioned to entirely online learning, while others continued in-person, in-clinic learning. This study aimed to show that medical learners who transitioned to exclusively online learning exhibited higher levels of burnout compared to their peers who continued in-person, clinical learning. Analysis of factors that protect against burnout including resilience, mindfulness, and self-compassion exhibited by online and in-person learners at NOSM University during this curriculum shift were also explored. METHODS: As part of a pilot wellness initiative, a cross-sectional online survey-based study of learner wellness was conducted at NOSM University during the 2020-2021 academic year. Seventy-four learners responded. The survey utilized the Maslach Burnout Inventory, the Brief Resilience Scale, Cognitive and Affective Mindfulness Scale-Revised, and the Self-Compassion Scale-Short Form. T-tests were utilized to compare these parameters in those who studied exclusively online and those who continued learning in-person in a clinical setting. RESULTS: Medical learners who engaged in online learning exhibited significantly higher levels of burnout when compared with learners who continued in-person learning in a clinical setting, despite scoring equally on protective factors such as resilience, mindfulness, and self-compassion. CONCLUSION: The results discussed in this paper suggest that the increased time spent in a virtual learning environment during the COVID-19 pandemic might be associated with burnout among exclusively online learners, as compared to learners who were educated in clinical, in-person settings. Further inquiry should investigate causality and any protective factors that could mitigate negative effects of the virtual learning environment.

Pelvic inflammatory disease and infertility.

BACKGROUND: Pelvic inflammatory disease (PID) is a major cause of morbidity and reproductive difficulty in women of childbearing age. OBJECTIVE: This article outlines the pathogenesis, clinical evaluation and management of PID with a focus on the management of long-term fertility-related sequelae. DISCUSSION: The clinical presentation of PID can be variable and clinicians need to have a low threshold for suspecting the diagnosis. Despite a good clinical response to antimicrobials, the risk of long-term complications is high. Therefore, a history of PID would warrant early review in couples planning conception for further evaluation and discussion of the various modalities available for treatment if spontaneous conception does not occur.

High anti-Müllerian hormone (AMH) is associated with increased risks of ectopic pregnancy in women undergoing fresh embryo transfer cycle, a cohort study.

BACKGROUND: Ectopic pregnancy is more common amongst assisted reproduction cycles and is a cause of significant maternal morbidity. Few predictive markers exist to help identify and modify risk of ectopic pregnancy in preparing for embryo transfer. The relationship between serum and AMH and ectopic pregnancy rate is unknown. METHODS: This was a retrospective cohort study investigating women who underwent fresh embryo transfer cycles from January 2017 to December 2019 in Peking University Third Hospital. The primary outcome was ectopic pregnancy. Restricted cubic splines with four knots for AMH concentration (0-3, 3-6, 6-12, 12-max) were used to map out the non-linear relationship between the predicted ectopic pregnancy rate and the serum AMH concentration. Log binomial regression was used to test the crude risk ratio (cRR) and the adjusted risk ratio (aRR) after adjustment for confounders with 95% confidence intervals (CI) to determine the difference across various groups. RESULTS: A total of 13,718 cycles in women undergoing fresh embryo transfer were eligible for analysis. The ectopic pregnancy rate was 1.3% per embryo transfer cycle initiated and 3.3% per pregnancy. Serum AMH concentrations were higher amongst women with ectopic pregnancy than in women with a confirmed intrauterine pregnancy or heterotopic pregnancy or who did not become pregnant (Mean levels: 4.0 ng/ml vs 3.2 ng/ml, 1.7 ng/ml, and 2.8 ng/ml). An AMH concentration of 7 ng/ml represented the best cut-off value to predict ectopic pregnancy. The ectopic pregnancy rate was 3.4% per cycle and 7.5% per pregnancy in women with AMH levels ≥ 7 ng/ml; and 1.2% per cycle and 2.9% per pregnancy in women with AMH levels < 7 ng/ml. Serum AMH concentration ≥ 7 ng/ml was associated with an increased risk of ectopic pregnancy in all fresh embryo transfer cycles started (aRR = 2.35 (1.45, 3.58)) as well in women who became pregnant (aRR = 2.23 (1.49, 3.33). CONCLUSIONS: Baseline AMH concentration ≥ 7 ng/ml is associated with an increased risk of ectopic pregnancy in fresh embryo transfer cycles.

ABCDEFGHI Systematic Approach to wound assessment and management.

ABSTRACT: The ABCDEFGHI approach introduces a systematic approach to wound care. It instructs the clinician to Ask pertinent questions, including those that may identify local and systemic Barriers to wound healing. After obtaining a thorough history, the clinician may proceed to Clean the wound and Do a physical examination, specifically looking for Exposed structures and Factors that will complicate the healing process. Good Healing strategies involving various dressings can then be implemented to promote healing. If necessary, a referral can be made to Involve specialists using various referral pathways. Information used to synthesize this approach was obtained through a review of national and international guidelines and Google Scholar, MEDLINE, and PubMed databases. The ABCDEFGHI approach to wound assessment and management is a simple and easy-to-follow guide that can be easily implemented into practice, thereby improving clinician confidence and competence in wound care.

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders.

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

Alternatives to in vitro fertilization.

There have been concerns on the potential overuse of in vitro fertilization (IVF) in view of the lack of evidence on effectiveness in certain populations, potential short and long-term safety risks, and economic considerations. On the other hand, the use of alternatives to IVF seems to be underappreciated in clinical practice as well as research. In this review, we summarized the up-to-date evidence on the effectiveness, safety as well as cost-effectiveness of different alternatives to IVF, including expectant management, intrauterine insemination, tubal flushing, in vitro maturation as well as intravaginal culture. We also discussed the trend of IVF use over the last decade and the available tiers of service because of intravaginal culture, and revisited the roles of different alternatives to IVF in modern reproductive medicine from both clinical and research perspectives.

Strong and reliable synaptic communication between pyramidal neurons in adult human cerebral cortex.

Synaptic transmission constitutes the primary mode of communication between neurons. It is extensively studied in rodent but not human neocortex. We characterized synaptic transmission between pyramidal neurons in layers 2 and 3 using neurosurgically resected human middle temporal gyrus (MTG, Brodmann area 21), which is part of the distributed language circuitry. We find that local connectivity is comparable with mouse layer 2/3 connections in the anatomical homologue (temporal association area), but synaptic connections in human are 3-fold stronger and more reliable (0% vs 25% failure rates, respectively). We developed a theoretical approach to quantify properties of spinous synapses showing that synaptic conductance and voltage change in human dendritic spines are 3-4-folds larger compared with mouse, leading to significant NMDA receptor activation in human unitary connections. This model prediction was validated experimentally by showing that NMDA receptor activation increases the amplitude and prolongs decay of unitary excitatory postsynaptic potentials in human but not in mouse connections. Since NMDA-dependent recurrent excitation facilitates persistent activity (supporting working memory), our data uncovers cortical microcircuit properties in human that may contribute to language processing in MTG.