The human embryonic genome is karyotypically complex, with chromosomally abnormal cells preferentially located away from the developing fetus.

STUDY QUESTION: Are chromosome abnormalities detected at Day 3 post-fertilization predominantly retained in structures of the blastocyst other than the inner cell mass (ICM), where chromosomally normal cells are preferentially retained? SUMMARY ANSWER: In human embryos, aneuploid cells are sequestered away from the ICM, partly to the trophectoderm (TE) but more significantly to the blastocoel fluid within the blastocoel cavity (Bc) and to peripheral cells (PCs) surrounding the blastocyst during Day 3 to Day 5 progression. WHAT IS KNOWN ALREADY: A commonly held dogma in all diploid eukaryotes is that two gametes, each with 'n' chromosomes (23 in humans), fuse to form a '2n' zygote (46 in humans); a state that remains in perpetuity for all somatic cell divisions. Human embryos, however, display high levels of chromosomal aneuploidy in early stages that reportedly declines from Day 3 (cleavage stage) to Day 5 (blastocyst) post-fertilization. While this observation may be partly because of aneuploid embryonic arrest before blastulation, it could also be due to embryo 'normalization' to a euploid state during blastulation. If and how this normalization occurs requires further investigation. STUDY DESIGN, SIZE, DURATION: A total of 964 cleavage-stage (Day 3) embryos underwent single-cell biopsy and diagnosis for chromosome constitution. All were maintained in culture, assessing blastulation rate, both for those assessed euploid and aneuploid. Pregnancy rate was assessed for those determined euploid, blastulated and subsequently transferred. For those determined aneuploid and blastulated (174 embryos), ICM (all 174 embryos), TE (all 174), Bc (47 embryos) and PC (38 embryos) were analyzed for chromosome constitution. Specifically, concordance with the original Day 3 diagnosis and determination if any 'normalized' to euploid karyotypes within all four structures was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients (144 couples) were undergoing routine preimplantation genetic testing for aneuploidy in three IVF clinical settings. Cleavage-stage biopsy preceded chromosome analysis by next-generation sequencing. All patients provided informed consent. Additional molecular testing was carried out on blastocyst embryos and was analyzed for up to four embryonic structures (ICM, TE, Bc and PC). MAIN RESULTS AND THE ROLE OF CHANCE: Of 463/964 embryos (48%) diagnosed as euploid at Day 3, 70% blastulated (leading to a 59% pregnancy rate) and 30% degenerated. Conversely, of the 501 (52%) diagnosed as aneuploid, 65% degenerated and 35% (174) blastulated, a highly significant difference (P < 0.0001). Of the 174 that blastulated, the ratio of '(semi)concordant-aneuploid' versus 'normalized-euploid' versus 'other-aneuploid' embryos was, respectively, 39%/57%/3% in the ICM; 49%/48%/3% in the TE; 78%/21%/0% in the PC; and 83%/10%/5% in the Bc. The TE karyotype therefore has a positive predictive value of 86.7% in determining that of the ICM, albeit with marginally higher aneuploid rates of abnormalities (P = .071). Levels of abnormality in Bc/PC were significantly higher (P < 0.0001) versus the ploidy of the ICM and TE and nearly all chromosome abnormalities were (at least partially) concordant with Day 3 diagnoses. LIMITATIONS, REASONS FOR CAUTION: The results only pertain to human IVF embryos so extrapolation to the in vivo situation and to other species is not certain. We acknowledge (rather than lineage-specific survival, as we suggest here) the possibility of other mechanisms, such as lineage-specific movement of cells, during blastulation. Ethical considerations, however, make investigating this mechanism difficult on human embryos. WIDER IMPLICATIONS OF THE FINDINGS: Mosaic human cleavage-stage embryos can differentiate into a euploid ICM where euploid cell populations predominate. Sequestering of aneuploid cells/nuclei to structures no longer involved in fetal development has important implications for preimplantation and prenatal genetic testing. These results also challenge previous fundamental understandings of mitotic fidelity in early human development and indicate a complex and fluid nature of the human embryonic genome. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Organon Pharmaceuticals and Merck Serono by grants to W.G.K. W.G.K. is also an employee of AdvaGenix, who could, potentially, indirectly benefit financially from publication of this manuscript. R.C.M. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133747. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.K.G. provides paid consultancy services for Care Fertility. TRIAL REGISTRATION NUMBER: : N/A.

Tubulointerstitial nephritis and uveitis in Northern Ireland.

OBJECTIVES: This paper looks at patients with a diagnosis of tubulointerstitial nephritis and uveitis (TINU) presenting to the Northern Ireland regional adult and paediatric uveitis service in the Belfast Health and Social Care Trust. The demographic distribution, treatment required and the visual and renal outcomes of these patients are documented. METHODS: Data were collected retrospectively on 24 patients with TINU using the Northern Ireland Electronic Care Record, central pathology records alongside the adult and paediatric uveitis databases from 2011 to 2021. Patients were categorised into two groups using the Mandeville classification system. Standard Uveitis Nomenclature (SUN) was used to classify the uveitis. RESULTS: The population prevalence is at least 12.6 cases per million based on a population of 1.9 million. Nineteen of 24 cases were definite TINU and five of 24 probable. Seventeen out of 24 had biopsy-positive TIN, all of which met all of the Mandeville clinical diagnostic features required for a definite diagnosis. All but one presented with acute bilateral anterior uveitis. The paediatric cases ranged from age 12 to 18 at age of onset with a mean age of 14. Of the 18 adult onset cases, the age ranged from 20 to 76 years. The mean age of onset for the adult cases was 53 years. Of these patients 71% were female; 42% required second-line immunosuppression for ocular disease. Visual acuity was maintained. Follow-up time ranged from 3 months to 16 years. No patient developed long-term renal impairment. CONCLUSIONS: TINU is a cause of uveitis in both the paediatric and adult populations. In Northern Ireland average age with TINU was older than much of the published literature. Long-term immunosuppression for uveitis may be required as ongoing ocular, rather than renal inflammation seemed to require treatment.

Analysis of bovine blastocysts indicates ovarian stimulation does not induce chromosome errors, nor discordance between inner-cell mass and trophectoderm lineages.

Contemporary systems for oocyte retrieval and culture of both cattle and human embryos are suboptimal with respect to pregnancy outcomes following transfer. In humans, chromosome abnormalities are the leading cause of early pregnancy loss in assisted reproduction. Consequently, pre-implantation genetic testing for aneuploidy (PGT-A) is widespread and there is considerable interest in its application to identify suitable cattle IVP embryos for transfer. Here we report on the nature and extent of chromosomal abnormalities following transvaginal follicular aspiration (OPU) and IVP in cattle. Nine sexually mature Holstein heifers underwent nine sequential cycles of OPU-IVP (six non-stimulated and three stimulated cycles), generating 459 blastocysts from 783 oocytes. We adopted a SNP-array approach normally employed in genomic evaluations but reanalysed (Turner et al., 2019; Theriogenology125: 249) to detect levels of meiotic aneuploidy. Specifically, we asked whether ovarian stimulation increased the level of aneuploidy in either trophectoderm (TE) or inner-cell mass (ICM) lineages of blastocysts generated from OPU-IVP cycles. The proportion of Day 8 blastocysts of inseminated was greater (P < 0.001) for stimulated than non-stimulated cycles (0.712 ± 0.0288 vs. 0.466 ± 0.0360), but the overall proportion aneuploidy was similar for both groups (0.241 ± 0.0231). Most abnormalities consisted of meiotic trisomies. Twenty in vivo derived blastocysts recovered from the same donors were all euploid, thus indicating that 24 h of maturation is primarily responsible for aneuploidy induction. Chromosomal errors in OPU-IVP blastocysts decreased (P < 0.001) proportionately as stage/grade improved (from 0.373 for expanded Grade 2 to 0.128 for hatching Grade 1 blastocysts). Importantly, there was a high degree of concordance in the incidence of aneuploidy between TE and ICM lineages. Proportionately, 0.94 were "perfectly concordant" (i.e. identical result in both); 0.01 were imperfectly concordant (differing abnormalities detected); 0.05 were discordant; of which 0.03 detected a potentially lethal TE abnormality (false positives), leaving only 0.02 false negatives. These data support the use of TE biopsies for PGT-A in embryos undergoing genomic evaluation in cattle breeding. Finally, we report chromosome-specific errors and a high degree of variability in the incidence of aneuploidy between donors, suggesting a genetic contribution that merits further investigation.

The complex phenotype of spinocerebellar ataxia type 48 in eight unrelated Italian families.

BACKGROUND AND PURPOSE: Heterozygous mutations in the STUB1 gene have recently been associated with an autosomal dominant form of spinocerebellar ataxia (SCA) associated with cerebellar cognitive-affective syndrome (CCAS), named SCA48. METHODS: Molecular screening was performed in a cohort of 235 unrelated patients with adult-onset, autosomal dominant (17) or sporadic (218) cerebellar ataxia, negative for pathological trinucleotide expansions in the common SCAs, FRDA and FXTAS loci, by using targeted multigene panels or whole-exome sequencing. Bioinformatics analyses, detailed neurological phenotyping and family segregation studies corroborated the pathogenicity of the novel STUB1 mutations. Clinico-diagnostic findings were reviewed to define the phenotypic spectrum. RESULTS: Eight heterozygous STUB1 mutations were identified, six of which were novel in 11 patients from eight index families, giving an estimated overall frequency of 3.4% (8/235) for SCA48 in our study cohort, rising to 23.5% (4/17) when considering only familial cases. All our SCA48 patients had cerebellar ataxia and dysarthria associated with cerebellar atrophy on brain magnetic resonance imaging; of note, many cases were also associated with parkinsonism, chorea and dystonia. CCAS also occurred frequently, whereas definite signs of pyramidal tract dysfunction and peripheral nervous system involvement were absent. One SCA48 patient presented with hypogonadism, associated with other autoimmune endocrine dysfunctions. CONCLUSIONS: Our results support SCA48 as a significant cause of adult-onset SCA. Besides CCAS, our SCA48 patients often showed movement disorders and other clinical manifestations previously described in SCAR16, linked to biallelic variants in the same gene, thus suggesting a continuous clinical spectrum and significant overlap amongst recessive and dominantly inherited mutations in STUB1.

ERIGO: a possible strategy to treat orthostatic hypotension in progressive supranuclear palsy? A feasibility study.

Progressive supranuclear palsy (PSP) is a neurodegenerative disease of the central nervous system, presenting with different clinical phenotypes, all involving the extrapyramidal system. Orthostatic hypotension (OH) is a common symptom of cardiovascular autonomic dysfunction. OH is defined as a fall in systolic blood pressure of at least 20 mmHg and/or a fall in diastolic blood pressure of at least 10 mmHg on standing or head-up tilt. In this pilot study, we tested the feasibility and efficacy of the ERIGO® device in managing OH non-responsive to conventional treatments in a sample of patients with PSP. OH was chosen as the primary outcome, as the symptom is a serious complication in neurodegenerative disorders, challenging the rehabilitation treatment. Six patients received intensive training using ERIGO®, a robot- assisted tilt table with an integrated leg movement system that allows progressive verticalization of the patient, and application of functional electrical stimulation. In all the participants, OH improved after the training with the device, suggesting that robotic verticalization may be a feasible and effective tool in improving blood pressure stability in patients with PSP. Further studies in larger samples, also including patients with other neurodegenerative disorders associated with OH, are needed to confirm these promising results.

Prevalence and phenotype of the c.1529C>T SPG7 variant in adult-onset cerebellar ataxia in Italy.

BACKGROUND AND PURPOSE: Hereditary ataxias are heterogeneous groups of neurodegenerative disorders, characterized by cerebellar syndromes associated with dysarthria, oculomotor and corticospinal signs, neuropathy and cognitive impairment. Recent reports have suggested mutations in the SPG7 gene, causing the most common form of autosomal recessive spastic paraplegia (MIM#607259), as a main cause of ataxias. The majority of described patients were homozygotes or compound heterozygotes for the c.1529C>T (p.Ala510Val) change. We screened a cohort of 895 Italian patients with ataxia for p.Ala510Val in order to define the prevalence and genotype-phenotype correlation of this variant. METHODS: We set up a rapid assay for c.1529C>T using restriction enzyme analysis after polymerase chain reaction amplification. We confirmed the diagnosis with Sanger sequencing. RESULTS: We identified eight homozygotes and 13 compound heterozygotes, including two novel variants affecting splicing. Mutated patients showed a pure cerebellar ataxia at onset, evolving in mild spastic ataxia (alternatively) associated with dysarthria (~80% of patients), urinary urgency (~30%) and pyramidal signs (~70%). Comparing homozygotes and compound heterozygotes, we noted a difference in age at onset and Scale for the Assessment and Rating of Ataxia score between the two groups, supporting an earlier and more severe phenotype in compound heterozygotes versus homozygotes. CONCLUSIONS: The SPG7 c.1529C>T (p.Ala510Val) mutants accounted for 2.3% of cerebellar ataxia cases in Italy, suggesting that this variant should be considered as a priority test in the presence of late-onset pure ataxia. Moreover, the heterozygous/homozygous genotype appeared to predict the onset of clinical manifestation and disease progression.

Dietary Patterns and Retinal Vessel Caliber in the Irish Nun Eye Study.

BACKGROUND: Retinal vessel abnormalities are associated with cardiovascular disease risk. Widening of retinal venules is associated with increased risk of stroke while narrowing of retinal arterioles independently predicts incident hypertension, coronary heart disease and diabetes. Dietary factors are known to play an important role in cardiovascular health. However, few studies have examined the association between dietary patterns (DPs) and retinal microvascular health. OBJECTIVE: To examine the association between 'a posteriori'-derived DPs and retinal vascular caliber (RVC) in older women with a restricted lifestyle. METHODS: This was a cross-sectional study of 1233 participants (mean age: 76.3 years) from the Irish Nun Eye Study (INES). Computer-assisted software was used to measure RVC from digital eye images using standardized protocols. Dietary intake was assessed using a food frequency questionnaire (FFQ). DP analysis was performed using principal component analysis from completed FFQs. Regression models were used to assess associations between DPs and retinal vessel diameters, adjusting for age, body mass index, refraction, hypertension, diabetes mellitus, ischemic heart disease, cerebrovascular accident and fellow eye RVC. RESULTS: Two DPs were identified: a 'healthy' pattern with high factor loadings for fruit, vegetables, wholegrains and oily fish and an 'unhealthy' pattern with high factor loadings for sugar and sweets, chips, high fat dairy products and French fries. Adjusted linear regression analysis revealed that those who adhered most closely to the unhealthy DP had wider central retinal venular equivalent (CRVE) (p=0.03) and narrower central retinal arteriolar equivalent (CRAE) (p=0.01) compared to the least unhealthy DP. No independent relationship was observed between the healthy DP and RVC. CONCLUSION: In this cohort of older women with a restricted lifestyle, an unhealthy DP was independently associated with an unfavorable retinal profile, namely a widening of retinal venules and narrowing of retinal arterioles.

Severe 5,10-methylenetetrahydrofolate reductase deficiency: a rare, treatable cause of complicated hereditary spastic paraplegia.

BACKGROUND AND PURPOSE: Juvenile- or adult-onset forms of severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency manifesting as complicated hereditary spastic paraplegia have rarely been described. METHODS: Two siblings with mental retardation developed a progressive spastic paraparesis in their late teens. Their diagnostic assessment included extensive neurophysiologic, neuroimaging and metabolic studies. RESULTS: Brain magnetic resonance imaging showed occipital white matter alterations, and electromyography documented a mixed polyneuropathy. Severe hyperhomocisteinemia (>150 µmol/L) associated with the characteristic amino acid profile suggested a diagnosis of severe MTHFR deficiency, confirmed by MTHFR direct sequencing. Treatment with betaine and vitamins benefitted patients' symptoms and diagnostic features. CONCLUSIONS: Severe MTHFR deficiency can be a rare, treatable cause of autosomal recessive complicated hereditary spastic paraplegia. Its screening should be part of the diagnostic flowchart for these disorders.

Speckle variance OCT for depth resolved assessment of the viability of bovine embryos.

The morphology of embryos produced by in vitro fertilization (IVF) is commonly used to estimate their viability. However, imaging by standard microscopy is subjective and unable to assess the embryo on a cellular scale after compaction. Optical coherence tomography is an imaging technique that can produce a depth-resolved profile of a sample and can be coupled with speckle variance (SV) to detect motion on a micron scale. In this study, day 7 post-IVF bovine embryos were observed either short-term (10 minutes) or long-term (over 18 hours) and analyzed by swept source OCT and SV to resolve their depth profile and characterize micron-scale movements potentially associated with viability. The percentage of en face images showing movement at any given time was calculated as a method to detect the vital status of the embryo. This method could be used to measure the levels of damage sustained by an embryo, for example after cryopreservation, in a rapid and non-invasive way.

Action on diabetic macular oedema: achieving optimal patient management in treating visual impairment due to diabetic eye disease.

This paper identifies best practice recommendations for managing diabetes and sight-threatening diabetic eye disease. The authors provide an update for ophthalmologists and allied healthcare professionals on key aspects of diabetes management, supported by a review of the pertinent literature, and recommend practice principles for optimal patient management in treating visual impairment due to diabetic eye disease. In people with diabetes, early optimal glycaemic control reduces the long-term risk of both microvascular and macrovascular complications. The authors propose more can and should be done to maximise metabolic control, promote appropriate behavioural modifications and encourage timely treatment intensification when indicated to ameliorate diabetes-related complications. All people with diabetes should be screened for sight-threatening diabetic retinopathy promptly and regularly. It is shown that attitudes towards treatment adherence in diabetic macular oedema appear to mirror patients' views and health behaviours towards the management of their own diabetes. Awareness of diabetic macular oedema remains low among people with diabetes, who need access to education early in their disease about how to manage their diabetes to delay progression and possibly avoid eye-related complications. Ophthalmologists and allied healthcare professionals play a vital role in multidisciplinary diabetes management and establishment of dedicated diabetic macular oedema clinics is proposed. A broader understanding of the role of the diabetes specialist nurse may strengthen the case for comprehensive integrated care in ophthalmic practice. The recommendations are based on round table presentations and discussions held in London, UK, September 2016.

The loss of CCR6+ and CD161+ CD4+ T-cell homeostasis contributes to disease progression in SIV-infected rhesus macaques.

Although previous studies have shown that CD4+ T cells expressing CCR6 and CD161 are depleted from blood during HIV infection, the mechanisms underlying their loss remain unclear. In this study, we investigated how the homeostasis of CCR6+ and CD161+ CD4+ T cells contributes to SIV disease progression and the mechanisms responsible for their loss from circulation. By comparing SIV infection in rhesus macaques (RMs) and natural host sooty mangabeys (SMs), we found that the loss of CCR6+ and CD161+ CD4+ T cells from circulation is a distinguishing feature of progressive SIV infection in RMs. Furthermore, while viral infection critically contributes to the loss of CD161+CCR6-CD4+ T cells, a redistribution of CCR6+CD161- and CCR6+CD161+CD4+ T cells from the blood to the rectal mucosa is a chief mechanism for their loss during SIV infection. Finally, we provide evidence that the accumulation of CCR6+CD4+ T cells in the mucosa is damaging to the host by demonstrating their reduction from this site following initiation of antiretroviral therapy in SIV-infected RMs and their lack of accumulation in SIV-infected SMs. These data emphasize the importance of maintaining CCR6+ and CD161+ CD4+ T-cell homeostasis, particularly in the mucosa, to prevent disease progression during pathogenic HIV/SIV infection.

Myotonic dystrophy type 1: role of CCG, CTC and CGG interruptions within DMPK alleles in the pathogenesis and molecular diagnosis.

Myotonic dystrophy type 1 (DM1) is a multisystem neuromuscular disease caused by a CTG triplet expansion in the 3'-untranslated region (3'-UTR) of DMPK gene. This CTG array is usually uninterrupted in both healthy and DM1 patients, but recent studies identified pathological variant expansions containing unstable CCG, CTC and CGG interruptions with a prevalence of 3-5% of cases. In this review, we will describe the clinical, molecular and genetic issues related to the occurrence of variant expansions associated with DM1. Indeed, the identification of these complex DMPK alleles leads to practical consequences in DM1 genetic counseling and testing, because these exams can give false negative results. Moreover, DM1 patients carrying interrupted alleles can manifest either additional atypical neurological symptoms or, conversely, mild, late-onset forms. Therefore, the prognosis of the disease in these patients is difficult to determine because of the great uncertainty about the genotype-phenotype correlations. We will discuss the putative effects of the variant DM1 alleles on the pathogenic disease mechanisms, including mitotic and meiotic repeats instability and splicing alteration typical of DM1 tissues. Interruptions within the DMPK expanded alleles could also interfere with the chromatin structure, the transcriptional activity of the DM1 locus and the interaction with RNA CUG-binding proteins.

DJ-1 modulates mitochondrial response to oxidative stress: clues from a novel diagnosis of PARK7.

DJ-1 mutations are associated to early-onset Parkinson's disease and accounts for about 1-2% of the genetic forms. The protein is involved in many biological processes and its role in mitochondrial regulation is gaining great interest, even if its function in mitochondria is still unclear. We describe a 47-year-old woman affected by a multisystem disorder characterized by progressive, early-onset parkinsonism plus distal spinal amyotrophy, cataracts and sensory-neural deafness associated with a novel homozygous c.461C>A [p.T154K] mutation in DJ-1. Patient's cultured fibroblasts showed low ATP synthesis, high ROS levels and reduced amount of some subunits of mitochondrial complex I; biomarkers of oxidative stress also resulted abnormal in patient's blood. The clinical pattern of multisystem involvement and the biochemical findings in our patient highlight the role for DJ-1 in modulating mitochondrial response against oxidative stress.

Validation of plasma microRNAs as biomarkers for myotonic dystrophy type 1.

Non-invasive and simple to measure biomarkers are still an unmet need for myotonic dystrophy type 1 (DM1). Indeed, muscle biopsies can be extremely informative, but their invasive nature limits their application. Extracellular microRNAs are emerging humoral biomarkers and preliminary studies identified a group of miRNAs that are deregulated in the plasma or serum of small groups of DM1 patients. Here we adopted very stringent selection and normalization criteria to validate or disprove these miRNAs in 103 DM1 patients and 111 matched controls. We confirmed that 8 miRNAs out of 12 were significantly deregulated in DM1 patients: miR-1, miR-27b, miR-133a, miR-133b, miR-206, miR-140-3p, miR-454 and miR-574. The levels of these miRNAs, alone or in combination, discriminated DM1 from controls significantly, and correlated with both skeletal muscle strength and creatine kinase values. Interestingly, miR-133b levels were significantly higher in DM1 female patients. Finally, the identified miRNAs were also deregulated in the plasma of a small group (n = 30) of DM2 patients. In conclusion, this study proposes that miRNAs might be useful as DM1 humoral biomarkers.

STAT3 Activation in Circulating Monocytes Contributes to Neovascular Age-Related Macular Degeneration.

Infiltrating macrophages are critically involved in pathogenic angiogenesis such as neovascular agerelated macular degeneration (nAMD). Macrophages originate from circulating monocytes and three subtypes of monocyte exist in humans: classical (CD14(+)CD16(-)), non-classical (CD14(-)CD16(+)) and intermediate (CD14(+)CD16(+)) monocytes. The aim of this study was to investigate the role of circulating monocyte in neovascular age-related macular degeneration (nAMD). Flow cytometry analysis showed that the intermediate monocytes from nAMD patients expressed higher levels of CX3CR1 and HLA-DR compared to those from controls. Monocytes from nAMD patients expressed higher levels of phosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3), and produced higher amount of VEGF. In the mouse model of choroidal neovascularization (CNV), pSTAT3 expression was increased in the retina and RPE/choroid, and 49.24% of infiltrating macrophages express pSTAT3. Genetic deletion of the Suppressor of Cytokine Signalling 3 (SOCS3) in myeloid cells in the LysM-Cre(+/-):SOCS3(fl/fl) mice resulted in spontaneous STAT3 activation and accelerated CNV formation. Inhibition of STAT3 activation using a small peptide LLL12 suppressed laserinduced CNV. Our results suggest that monocytes, in particular the intermediate subset of monocytes are activated in nAMD patients. STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD.