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Cell-free DNA (cfDNA) is a rich source of biomarkers for various pathophysiological conditions. Preanalytical variables, such as the library preparation protocol or sequencing platform, are major confounders of cfDNA analysis. We present DAGIP, a novel data correction method that builds on optimal transport theory and deep learning, which explicitly corrects for the effect of such preanalytical variables and can infer technical biases. Our method improves cancer detection and copy number alteration analysis by alleviating the sources of variation that are not of biological origin. It also enhances fragmentomic analysis of cfDNA. DAGIP allows the integration of cohorts from different studies.

STUDY QUESTION How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? SUMMARY ANSWER Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of ‘mosaic’ embryos. WHAT IS KNOWN ALREADY The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. STUDY DESIGN, SIZE, DURATION This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. PARTICIPANTS/MATERIALS, SETTING, METHODS The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. MAIN RESULTS AND THE ROLE OF CHANCE Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. LIMITATIONS, REASONS FOR CAUTION Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. WIDER IMPLICATIONS OF THE FINDINGS This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. STUDY FUNDING/COMPETING INTEREST(S) The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to ‘Methods for haplotyping single-cells’ and ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’, and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. DISCLAIMER This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation.  ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type.  Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE. 

Wolf–Hirschhorn syndrome is caused by anomalies of the short arm of chromosome 4. About 55% of cases are due to de novo terminal deletions, 40% from unbalanced translocations and 5% from other abnormalities. The facial phenotype is characterized by hypertelorism, protruding eyes, prominent glabella, broad nasal bridge and short philtrum. We used dense surface modelling and pattern recognition techniques to delineate the milder facial phenotype of individuals with a small terminal deletion (breakpoint within 4p16.3) compared to those with a large deletion (breakpoint more proximal than 4p16.3). Further, fine-grained facial analysis of several individuals with an atypical genotype and/or phenotype suggests that multiple genes contiguously contribute to the characteristic Wolf–Hirschhorn syndrome facial phenotype.

Since the introduction of noninvasive prenatal testing (NIPT)7 in prenatal diagnostics in 2011, incidental findings of an occult maternal malignancy following a \"falsepositive\" NIPT test have been reported repeatedly. [...]proper identification of cancer through routine NIPT screening would have a major effect on the management of these pregnant women. Because lymphomas are the most frequently detected tumor type before breast cancer following an aberrant NIPT outcome, whole-body MRI examination should be complemented with the analysis of hematological parameters (such as cell counts and cytology and clinical biochemistry) to search for the presence of a hematological malignancy that might not be detectable via MRI scanning. [...]vitamin B12 or folate deficiency or the presence of autoimmune diseases such as systemic lupus erythematosus might also lead to an abnormal NIPT result, although the profile of chromosomal imbalances found in these conditions might be discriminated from typical cancer-like CNAs.

Circulating cell-free DNA (cfDNA) fragments have characteristics that are specific to the cell types that release them. Current methods for cfDNA deconvolution typically use disease tailored marker selection in a limited number of bulk tissues or cell lines. Here, we utilize single cell transcriptome data as a comprehensive cellular reference set for disease-agnostic cfDNA cell-of-origin analysis. We correlate cfDNA-inferred nucleosome spacing with gene expression to rank the relative contribution of over 490 cell types to plasma cfDNA. In 744 healthy individuals and patients, we uncover cell type signatures in support of emerging disease paradigms in oncology and prenatal care. We train predictive models that can differentiate patients with colorectal cancer (84.7%), early-stage breast cancer (90.1%), multiple myeloma (AUC 95.0%), and preeclampsia (88.3%) from matched controls. Importantly, our approach performs well in ultra-low coverage cfDNA datasets and can be readily transferred to diverse clinical settings for the expansion of liquid biopsy. Deconvolution of cfDNA fragmentation benefits from cell type-specific reference data. Here, the authors create a disease agnostic cfDNA cell type of origin analysis and show it can successfully predict cell types of origin from plasma samples.

Background During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres. Results Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest. Conclusions Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF.

Structural variants (SVs) are important contributors to human disease. Their characterization remains however difficult due to their size and association with repetitive regions. Long-read sequencing (LRS) and optical genome mapping (OGM) can aid as their molecules span multiple kilobases and capture SVs in full. In this study, we selected six individuals who presented with unresolved SVs. We applied LRS onto all individuals and OGM to a subset of three complex cases. LRS detected and fully resolved the interrogated SV in all samples. This enabled a precise molecular diagnosis in two individuals. Overall, LRS identified 100% of the junctions at single-basepair level, providing valuable insights into their formation mechanisms without need for additional data sources. Application of OGM added straightforward variant phasing, aiding in the unravelment of complex rearrangements. These results highlight the potential of LRS and OGM as follow-up molecular tests for complete SV characterization. We show that they can assess clinically relevant structural variation at unprecedented resolution. Additionally, they detect (complex) cryptic rearrangements missed by conventional methods. This ultimately leads to an increased diagnostic yield, emphasizing their added benefit in a diagnostic setting. To aid their rapid adoption, we provide detailed laboratory and bioinformatics workflows in this manuscript.

Background A subset of developmental disorders (DD) is characterized by disease-specific genome-wide methylation changes. These episignatures inform on the underlying pathogenic mechanisms and can be used to assess the pathogenicity of genomic variants as well as confirm clinical diagnoses. Currently, the detection of these episignature requires the use of indirect methylation profiling methodologies. We hypothesized that long-read whole genome sequencing would not only enable the detection of single nucleotide variants and structural variants but also episignatures. Methods Genome-wide nanopore sequencing was performed in 40 controls and 20 patients with confirmed or suspected episignature-associated DD, representing 13 distinct diseases. Following genomic variant and methylome calling, hierarchical clustering and dimensional reduction were used to determine the compatibility with microarray-based episignatures. Subsequently, we developed a support vector machine (SVM) for the detection of each DD. Results Nanopore sequencing-based methylome patterns were concordant with microarray-based episignatures. Our SVM-based classifier identified the episignatures in 17/19 patients with a (likely) pathogenic variant and none of the controls. The remaining patients in which no episignature was identified were also classified as controls by a commercial microarray assay. In addition, we identified all underlying pathogenic single nucleotide and structural variants and showed haplotype-aware skewed X-inactivation evaluation directs clinical interpretation. Conclusion This proof-of-concept study demonstrates nanopore sequencing enables episignature detection. In addition, concurrent haplotyped genomic and epigenomic analyses leverage simultaneous detection of single nucleotide/structural variants, X-inactivation, and imprinting, consolidating a multi-step sequential process into a single diagnostic assay.

Purpose Noninvasive prenatal screening (NIPS) using genome sequencing also reveals maternal copy-number variations (CNVs). Those CNVs can be clinically actionable or harmful to the fetus if inherited. CNVs in the DMD gene potentially causing dystrophinopathies are among the most commonly observed maternal CNVs. We present our experience with maternal DMD gene CNVs detected by NIPS. Methods We analyzed the data of maternal CNVs detected in the DMD gene revealed by NIPS. Results Of 26,123 NIPS analyses, 16 maternal CNVs in the DMD gene were detected (1/1632 pregnant women). Variant classification regarding pathogenicity and phenotypic severity was based on public databases, segregation analysis in the family, and prediction of the effect on the reading frame. Ten CNVs were classified as pathogenic, four as benign, and two remained unclassified. Conclusion NIPS leverages CNV screening in the general population of pregnant women. We implemented a strategy for the interpretation and the return of maternal CNVs in the DMD gene detected by NIPS.

This Timeline article reviews the evolution of both prenatal and pre-implantation genetic testing, from their historic origins to ongoing development and implementation of tools for genome-wide single-cell and cell-free fetal DNA analysis. Future developments and some of the ethical issues that arise from these new technologies are also discussed. The past decade has seen the development of technologies that have revolutionized prenatal genetic testing; that is, genetic testing from conception until birth. Genome-wide single-cell arrays and high-throughput sequencing analyses are dramatically increasing our ability to detect embryonic and fetal genetic lesions, and have substantially improved embryo selection for in vitro fertilization (IVF). Moreover, both invasive and non-invasive mutation scanning of the genome are helping to identify the genetic causes of prenatal developmental disorders. These advances are changing clinical practice and pose novel challenges for genetic counselling and prenatal care.