Improved clinical utility of preimplantation genetic testing through the integration of ploidy and common pathogenic microdeletions analyses.

STUDY QUESTION: Can chromosomal abnormalities beyond copy-number aneuploidies (i.e. ploidy level and microdeletions (MDs)) be detected using a preimplantation genetic testing (PGT) platform? SUMMARY ANSWER: The proposed integrated approach accurately assesses ploidy level and the most common pathogenic microdeletions causative of genomic disorders, expanding the clinical utility of PGT. WHAT IS KNOWN ALREADY: Standard methodologies employed in preimplantation genetic testing for aneuploidy (PGT-A) identify chromosomal aneuploidies but cannot determine ploidy level nor the presence of recurrent pathogenic MDs responsible for genomic disorders. Transferring embryos carrying these abnormalities can result in miscarriage, molar pregnancy, and intellectual disabilities and developmental delay in offspring. The development of a testing strategy that integrates their assessment can resolve current limitations and add valuable information regarding the genetic constitution of embryos, which is not evaluated in PGT providing new level of clinical utility and valuable knowledge for further understanding of the genomic causes of implantation failure and early pregnancy loss. To the best of our knowledge, MDs have never been studied in preimplantation human embryos up to date. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort analysis including blastocyst biopsies collected between February 2018 and November 2021 at multiple collaborating IVF clinics from prospective parents of European ancestry below the age of 45, using autologous gametes and undergoing ICSI for all oocytes. Ploidy level determination was validated using 164 embryonic samples of known ploidy status (147 diploids, 9 triploids, and 8 haploids). Detection of nine common MD syndromes (-4p=Wolf-Hirschhorn, -8q=Langer-Giedion, -1p=1p36 deletion, -22q=DiGeorge, -5p=Cri-du-Chat, -15q=Prader-Willi/Angelman, -11q=Jacobsen, -17p=Smith-Magenis) was developed and tested using 28 positive controls and 97 negative controls. Later, the methodology was blindly applied in the analysis of: (i) 100 two pronuclei (2PN)-derived blastocysts that were previously defined as uniformly euploid by standard PGT-A; (ii) 99 euploid embryos whose transfer resulted in pregnancy loss. PARTICIPANTS/MATERIALS, SETTING, METHODS: The methodology is based on targeted next-generation sequencing of selected polymorphisms across the genome and enriched within critical regions of included MD syndromes. Sequencing data (i.e. allelic frequencies) were analyzed by a probabilistic model which estimated the likelihood of ploidy level and MD presence, accounting for both sequencing noise and population genetics patterns (i.e. linkage disequilibrium, LD, correlations) observed in 2504 whole-genome sequencing data from the 1000 Genome Project database. Analysis of phased parental haplotypes obtained by single-nucleotide polymorphism (SNP)-array genotyping was performed to confirm the presence of MD. MAIN RESULTS AND THE ROLE OF CHANCE: In the analytical validation phase, this strategy showed extremely high accuracy both in ploidy classification (100%, CI: 98.1-100%) and in the identification of six out of eight MDs (99.2%, CI: 98.5-99.8%). To improve MD detection based on loss of heterozygosity (LOH), common haploblocks were analyzed based on haplotype frequency and LOH occurrence in a reference population, thus developing two further mathematical models. As a result, chr1p36 and chr4p16.3 regions were excluded from MD identification due to their poor reliability, whilst a clinical workflow which incorporated parental DNA information was developed to enhance the identification of MDs. During the clinical application phase, one case of triploidy was detected among 2PN-derived blastocysts (i) and one pathogenic MD (-22q11.21) was retrospectively identified among the biopsy specimens of transferred embryos that resulted in miscarriage (ii). For the latter case, family-based analysis revealed the same MD in different sibling embryos (n = 2/5) from non-carrier parents, suggesting the presence of germline mosaicism in the female partner. When embryos are selected for transfer based on their genetic constitution, this strategy can identify embryos with ploidy abnormalities and/or MDs beyond aneuploidies, with an estimated incidence of 1.5% (n = 3/202, 95% CI: 0.5-4.5%) among euploid embryos. LIMITATIONS, REASONS FOR CAUTION: Epidemiological studies will be required to accurately assess the incidence of ploidy alterations and MDs in preimplantation embryos and particularly in euploid miscarriages. Despite the high accuracy of the assay developed, the use of parental DNA to support diagnostic calling can further increase the precision of the assay. WIDER IMPLICATIONS OF THE FINDINGS: This novel assay significantly expands the clinical utility of PGT-A by integrating the most common pathogenic MDs (both de novo and inherited ones) responsible for genomic disorders, which are usually evaluated at a later stage through invasive prenatal testing. From a basic research standpoint, this approach will help to elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of otherwise euploid embryos. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. S.C., M.F., F.C., P.Z., I.P., L.G., C.P., M.P., D.B., J.J.-A., D.B.-J., J.M.-V., and C.R. are employees of Igenomix and C.S. is the head of the scientific board of Igenomix. A.C. and L.P. are employees of JUNO GENETICS. Igenomix and JUNO GENETICS are companies providing reproductive genetic services. TRIAL REGISTRATION NUMBER: N/A.

Clinical validity and utility of preconception expanded carrier screening for the management of reproductive genetic risk in IVF and general population.

STUDY QUESTION: What is the clinical validity and utility of preconception Expanded Carrier Screening (ECS) application on the management of prospective parents? SUMMARY ANSWER: The high detection rate of at-risk couples (ARCs) and the high proportion opting for IVF/preimplantation genetic testing (PGT) treatment demonstrate the clinical utility of ECS in the preconception space in IVF and general population. WHAT IS KNOWN ALREADY: About 2-4% of couples are at risk of conceiving a child with an autosomal recessive or X-linked genetic disorder. In recent years, the increasing cost-effectiveness of genetic diagnostic techniques has allowed the creation of ECS panels for the simultaneous detection of multiple recessive disorders. Comprehensive preconception genetic screening holds the potential to significantly improve couple's genetic risk assessment and reproductive planning to avoid detectable inheritable genetic offspring. STUDY DESIGN, SIZE, DURATION: A total of 3877 individuals without a family history of genetic conditions were analyzed between January 2017 and January 2020. Of the enrolled individuals, 1212 were gamete donors and 2665 were patients planning on conceiving from both the IVF and the natural conception group. From the non-donor cohort, 1133 were analyzed as individual patients, while the remaining ones were analyzed as couples, for a total of 766 couples. PARTICIPANTS/MATERIALS, SETTING, METHODS: A focused ECS panel was developed following American College of Obstetrics and Gynecology ACOG-recommended criteria (prevalence, carrier rate, severity), including highly penetrant severe childhood conditions. Couples were defined at-risk when both partners carried an autosomal recessive pathogenic/likely pathogenic variant (PLP) on the same gene or when the woman was a carrier of an X-linked PLP variant. ARC detection rate defined the clinical validity of the ECS approach. Clinical utility was evaluated by monitoring ARCs reproductive decision making. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 402 individuals (10.4%) showed PLP for at least one of the genes tested. Among the 766 couples tested, 173 showed one carrier partner (22.6%), whereas 20 couples (2.6%) were found to be at increased risk. Interestingly, one ARC was identified as a result of cascade testing in the extended family of an individual carrying a pathogenic variant on the Survival Of Motor Neuron 1SMN1 gene. Of the identified ARCs, 5 (0.7%) were at risk for cystic fibrosis, 5 (0.7%) for fragile X syndrome, 4 (0.5%) for spinal muscular atrophy, 4 (0.5%) for Beta-Thalassemia/Sickle Cell Anemia, 1 (0.1%) for Smith-Lemli-Opitz Syndrome and 1 (0.1%) for Duchenne/Becker Dystrophy. Fifteen ARCs were successfully followed up from both the IVF and the natural conception groups. All of these (15/15) modified their reproductive planning by undergoing ART with Preimplantation Genetic Testing for Monogenic disease and Aneuploidies (PGT-M and PGT-A). To date, 6/15 (40%) couples completed their PGT cycle with euploid/unaffected embryos achieving a pregnancy after embryo transfer and three of them have already had an unaffected baby. LIMITATIONS, REASONS FOR CAUTION: The use of a limited panel of core gene-disease pairs represents a limitation on the research perspective as it can underestimate the rate of detectable carriers and ARCs in this cohort of prospective parents. Expanding the scope of ECS to a larger panel of conditions is becoming increasingly feasible, thanks to a persistent technological evolution and progressive cataloging of gene-disease associations. WIDER IMPLICATIONS OF THE FINDINGS: These results highlight the potential clinical validity and utility of ECS in reducing the risk of a pregnancy affected by a detectable inheritable genetic condition. The steady reduction in the costs of genetic analyses enables the expansion of monogenic testing/screening applications at the preimplantation stage, thus, providing valid decisional support and reproductive autonomy to patients, particularly in the context of IVF. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. A.C., M.F., S.C., M.P., L.G., and C.P. are employees of Igenomix Italy. C.S. is the head of the scientific board of Igenomix. TRIAL REGISTRATION NUMBER: N/A.

Isotopic biomonitoring of N pollution in rivers embedded in complex human landscapes.

The dynamic and hierarchical structure of rivers, together with disruption of the natural river continuum by human activities, makes it difficult to identify and locate sources of nutrient pollution affecting receiving waters and observe its dispersion, thus impairing monitoring efforts. The identification of reliable indicators of anthropogenic nitrogen inputs in catchments is therefore key to achieving effective management of polluted rivers. We tested the capacity of N isotopic signatures (δ15N) of epilithon and snails to provide useful indications of organic and inorganic anthropogenic N inputs in three Mediterranean rivers differing in terms of surrounding land use and physicochemical conditions. We used a combined approach based on (i) analysis of nutrient concentrations in water, (ii) CORINE land cover classification and drainage patterns in catchments and (iii) isotopic analysis of river biota to verify whether isotopic variations were indicative of anthropic activities in the watershed, the associated alteration of water quality, and the consequent impact on snail abundance and diversity. Variation in the δ15N of epilithon within and between rivers reflected localised and diffuse N inputs from inorganic and organic sources. Negative epilithon δ15N values (<0‰) indicated inorganic pollution from agriculture. Values between 4‰ and 8‰ and those above 8‰ respectively indicated moderate organic pollution from urban areas, and high organic pollution, mostly from waste waters. The diversity and abundance of snails decreased with increasing water pollution. While their isotopic variations reflected between-river differences, they failed to indicate within-river variations in anthropogenic N inputs, since the proportion of epilithon in their diet varied along the rivers. Concluding, epilithon was a reliable indicator of anthropogenic N sources across a wide range of nutrient concentrations and anthropogenic inputs, and the proposed approach allowed us to determine the nature of nitrogen pollutants, their sources, location and dispersion along rivers embedded in complex human landscapes.

Clinical and dermoscopic features of genital pigmented Bowen disease.

Pigmented Bowen disease (pBD) is an uncommon variant of squamous cell carcinoma in situ. Sometimes it can show clinical and dermoscopic features that are seen in other pigmented lesions of the skin and mucosa, making the diagnosis difficult. We report six cases of pBD occurring on the anogenital area, and discuss the importance of dermoscopy for improving the diagnostic accuracy in pBD.

A participação da família na reabilitação psicossocial do sujeito emsofrimento psíquico / Family involvement in psychosocial rehabilitation of individuals under psychological suffering

Objetivo: conhecer a participação da família na reabilitação psicossocial do sujeito em sofrimento psíquico. Métodos: trata-se de uma pesquisa qualitativa, exploratória e descritiva, realizada em 2012. Os sujeitos do estudo foram seis familiares de usuários de um Centro de Atenção Psicossocial (CAPS). Foi utilizada a análise temática conforme Minayo, que tem como foco encontrar núcleos de sentido na comunicação apresentada pelo material estudado. Resultados: emergiram desse estudo dois eixos temáticos: A inserção do sujeito em sofrimento psíquico na sociedade sob a ótica da família e A participação da família na reabilitação psicossocial do sujeito em sofrimento psíquico. Conclusão: há um certo isolamento do usuário no serviço substitutivo. É importante que os profissionais de saúde invistam na autonomia dos sujeitos, contribuindo para o conhecimento do usuário e sua família acerca dos seus direitos e da proposta da reforma psiquiátrica. Colaborando com o aumento do compromisso da família neste processo.

Objective: to now the family's participation in psychosocial rehabilitation of the individual underpsychological distress. Methods: this is a qualitative, exploratory and descriptive study, held in2012. The study subjects were relatives of individuals with psychological distress, users of aPsychosocial Care Center (CAPS). The thematic analysis according to Minayo was used, whose focusis to find nuclei of meaning in the communication presented by material studied. Results: this studyemerged the themes: The insertion of the subject in psychological distress in society from theperspective of the family and the family's participation in psychosocial rehabilitation of the subject in psychological distress. Conclusions: there’s a certain isolation used in substitute service. It isunderstood that it is important for health professionals bet on education, increasing the user'sknowledge and his family about their rights and psychiatric reform proposal. This may collaboratewith the commitment of the family in this process.

Patients with Duchenne and Becker muscular dystrophies are not more asymmetrical than healthy controls on timed performance of upper limb tasks.

This study aimed to investigate possible asymmetries and relationships between performance of dominant and non-dominant upper limbs (UL) in patients with Duchenne and Becker muscular dystrophies (DMD/BMD), to compare UL performance of patients and healthy subjects and to investigate the relationship between timed performance of UL and age, motor function and muscle strength in DMD/BMD patients. Sixteen patients with DMD and 3 with BMD were evaluated with Jebsen-Taylor Test (timed performance), Vignos scale and Dimension 3 of Motor Function Measure (motor function), and Medical Research Council scale (muscle strength) on a single session. ANOVA showed no asymmetry between dominant and non-dominant UL, except in the writing subtest, in patients and in healthy controls. There were relationships between dominant and non-dominant UL performances. Correlations between timed performance, motor function and muscle strength were found, but age was not correlated with these variables. These findings may reduce the assessment time, prevent fatigue and provide more accurate clinical reasoning involving UL in DMD/BMD treatment.

Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome.

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. METHODS: We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. RESULTS: We found extensive mutational and copy-number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. CONCLUSION: This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success.

Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data.

BACKGROUND: Exome or whole-genome deep sequencing of tumor DNA along with paired normal DNA can potentially provide a detailed picture of the somatic mutations that characterize the tumor. However, analysis of such sequence data can be complicated by the presence of normal cells in the tumor specimen, by intratumor heterogeneity, and by the sheer size of the raw data. In particular, determination of copy number variations from exome sequencing data alone has proven difficult; thus, single nucleotide polymorphism (SNP) arrays have often been used for this task. Recently, algorithms to estimate absolute, but not allele-specific, copy number profiles from tumor sequencing data have been described. MATERIALS AND METHODS: We developed Sequenza, a software package that uses paired tumor-normal DNA sequencing data to estimate tumor cellularity and ploidy, and to calculate allele-specific copy number profiles and mutation profiles. We applied Sequenza, as well as two previously published algorithms, to exome sequence data from 30 tumors from The Cancer Genome Atlas. We assessed the performance of these algorithms by comparing their results with those generated using matched SNP arrays and processed by the allele-specific copy number analysis of tumors (ASCAT) algorithm. RESULTS: Comparison between Sequenza/exome and SNP/ASCAT revealed strong correlation in cellularity (Pearson's r = 0.90) and ploidy estimates (r = 0.42, or r = 0.94 after manual inspecting alternative solutions). This performance was noticeably superior to previously published algorithms. In addition, in artificial data simulating normal-tumor admixtures, Sequenza detected the correct ploidy in samples with tumor content as low as 30%. CONCLUSIONS: The agreement between Sequenza and SNP array-based copy number profiles suggests that exome sequencing alone is sufficient not only for identifying small scale mutations but also for estimating cellularity and inferring DNA copy number aberrations.

Vision, light and aging: a literature overview on older-age workers.

BACKGROUND: In many western countries individuals will need to continue their professional careers beyond the current retirement age. This requires adaptation of the working conditions to compensate for age related visual changes. OBJECTIVE: The aim of this paper is to compile and structure knowledge concerning age related changes in visual and non-visual functions among older-age workers and to describe in what way these changes relate to light and work performance. METHOD: An overview of the literature was performed in PubMed and EMBASE concerning visual changes among elderly people, light, visual ergonomics and consequences at work. RESULTS: Visual conditions and lighting design have an impact on work performance in those over age 65 even if there are few studies available. Natural age related changes in the eyes or ocular diseases can result in reduced visual function and performance. Moreover, evidence of the importance of light and dark rhythms for circadian regulation is mounting; there are indications that the older-age population might need specific attention related to this issue. Finally, visual deteriorations might also, secondarily, induce strained postures and musculoskeletal symptoms, pain and injury. CONCLUSION: Age-related changes in the eyes and also ocular diseases among older-age people have an impact on well-being and work performance, and therefore call for reconsideration of their working conditions. Knowledge about how visual functions, light and ocular diseases is needed for work design and preventive actions.

A miniature temperature high germanium doped PCF interferometer sensor.

We report in this paper a high thermal sensitivity (78 pm/°C) modal interferometer using a very short Photonic Crystal Fiber stub with a shaped Germanium doped core. The Photonic Crystal Fiber is spliced between two standard fibers. The splice regions allow the excitation of the core and cladding modes in the PCF and perform an interferometric interaction of such modes. The device is proposed for sensitive temperature measurements in transmission, as well as in reflection operation mode with the same high temperature sensitivity.

Spheroidal Fabry-Perot microcavities in optical fibers for high-sensitivity sensing.

All-optical-fiber Fabry-Perot interferometers (FPIs) with microcavities of different shapes were investigated. It was found that the size and shape of the cavity plays an important role on the performance of these interferometers. To corroborate the analysis, FPIs with spheroidal cavities were fabricated by splicing a photonic crystal fiber (PCF) with large voids and a conventional single mode fiber (SMF), using an ad hoc splicing program. It was found that the strain sensitivity of FPIs with spheroidal cavities can be controlled through the dimensions of the spheroid. For example, a FPI whose cavity had a size of ~10x60 µm exhibited strain sensitivity of ~10.3 pm/µÎµ and fringe contrast of ~38 dB. Such strain sensitivity is ~10 times larger than that of the popular fiber Bragg gratings (~1.2 pm/µÎµ) and higher than that of most low-finesse FPIs. The thermal sensitivity of our FPIs is extremely low (~1pm/°C) due to the air cavities. Thus, a number of temperature-independent ultra-sensitive microscopic sensors can be devised with the interferometers here proposed since many parameters can be converted to strain. To this end, simple vibration sensors are demonstrated.

Prenatal stress induces intrauterine growth restriction and programmes glucose intolerance and feeding behaviour disturbances in the aged rat.

There is growing evidence that prenatal adversities could be implicated in foetal programming of adult chronic diseases. Since maternal stress is known to disturb the foetal glucocorticoid environment, we examined the consequences of prenatal stress on foetal growth, on glucose-insulin metabolism and on feeding behaviour in the aged male rat. In foetuses at term, maternal stress reduced body, adrenal and pancreas weight as well as plasma corticosterone and glucose levels. In aged male rats (24 months of age), prenatal stress induced hyperglycaemia and glucose intolerance and decreased basal leptin levels. Moreover, after a fasting period, they showed an increased food intake. These data suggest that maternal stress induces a long-lasting disturbance in feeding behaviour and dysfunctions related to type 2 diabetes mellitus. This programming could be linked to the early restricted foetal growth and to the adverse glucocorticoid environment in utero.