Evaluation of Nanopore Sequencing on Polar Bodies for Routine Pre-Implantation Genetic Testing for Aneuploidy.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) using polar body (PB) biopsy offers a clinical benefit by reducing the number of embryo transfers and miscarriage rates but is currently not cost-efficient. Nanopore sequencing technology opens possibilities by providing cost-efficient and fast sequencing results with uncomplicated sample preparation work flows. METHODS: In this comparative experimental study, 102 pooled PB samples (99 passing QC) from 20 patients were analyzed for aneuploidy using nanopore sequencing technology and compared with array comparative genomic hybridization (aCGH) results generated as part of the clinical routine. Samples were sequenced on a Nanopore MinION machine. Whole-chromosome copy-numbers were called by custom bioinformatic analysis software. Automatically called results were compared to aCGH results. RESULTS: Overall, 96/99 samples were consistently detected as euploid or aneuploid in both methods (concordance = 97.0%, sensitivity = 0.957, specificity = 1.0, positive predictive value = 1.0, negative predictive value = 0.906). On the chromosomal level, concordance reached 98.7%. Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, and 97 monosomies in 70 aCGH samples.The whole nanopore work flow is feasible in under 5 h (for one sample) with a maximum time of 16 h (for 12 samples), enabling fresh PB-euploid embryo transfer. A material cost of US$ 165 (EUR 150)/sample possibly enables cost-efficient aneuploidy screening. CONCLUSIONS: This is the first study systematically comparing nanopore sequencing with standard methods for the detection of PB aneuploidy. High concordance rates confirmed the feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient work flow reveals the clinical utility of this technology, making it clinically attractive for PB PGT-A.

Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study.

The aim of this pilot study was to assess if array comparative genomic hybridization (aCGH), non-invasive preimplantation genetic screening (PGS) on blastocyst culture media is feasible. Therefore, aCGH analysis was carried out on 22 spent blastocyst culture media samples after polar body PGS because of advanced maternal age. All oocytes were fertilized by intracytoplasmic sperm injection and all embryos underwent assisted hatching. Concordance of polar body analysis and culture media genetic results was assessed. Thirteen out of 18 samples (72.2%) revealed general concordance of ploidy status (euploid or aneuploid). At least one chromosomal aberration was found concordant in 10 out of 15 embryos found to be aneuploid by both polar body and culture media analysis. Overall, 17 out of 35 (48.6%) single chromosomal aneuploidies were concordant between the culture media and polar body analysis. By analysing negative controls (oocytes with fertilization failure), notable maternal contamination was observed. Therefore, non-invasive PGS could serve as a second matrix after polar body or cleavage stage PGS; however, in euploid results, maternal contamination needs to be considered and results interpreted with caution.

University courses, eating problems and muscle dysmorphia: are there any associations?

BACKGROUND: Orthorexia and muscle dysmorphia are disorders affecting above all young adults whose prevalence and social impact are still unclear. We aimed to evaluate the prevalence of the traits of orthorexia and muscle dysmorphia among freshmen attending university courses focused on nutrition (Dietetics) and body care (Exercise and Sport Sciences). Students of Biology were considered as a control group. The prevalence of eating disorder (ED) traits were also evaluated. METHODS: All participants (n = 440; n = 53 Dietetics school, n = 200 Exercise and Sport Sciences school, n = 187 the Biology school) completed the following questionnaires: ORTO-15, Muscle-Dysmorphic-Disorder-Inventory, and Eating Attitudes Test-26. RESULTS: The prevalence of the traits of EDs, orthorexia, and muscle dysmorphia was 9.1%, 25.9%, and 5.9%, respectively. When compared to other students, those attending the Dietetics school showed a 2-fold higher risk of EDs and those from the Exercise and Sport Sciences school a 10-fold higher risk of muscle dysmorphia. The prevalence of orthorexia traits was high in all schools (35.9%, 22.5%, 26.5% in Dietetics, Biology, and Exercise and Sport Sciences schools, respectively). Overall, individuals with traits of any of these disorders were more frequently on diet or on supplement use. In a logistic regression model, attending the Dietetics school (OR = 2.71; 95% CI 1.14-6.48) was significantly associated with the ED traits, but not with the orthorexia traits (OR = 1.75; 95% CI 0.93-3.29), while attending the Exercise and Sport Sciences school was significantly associated with the muscle dysmorphia traits (OR = 5.15; 95% CI 1.44-18.4). Finally, when evaluating the relationships among the types of study programs as dependent variables and traits of these disturbances, the associations between the traits of ED (OR = 3.35; 95% CI 1.38-8.13) and matriculation at the school of Dietetics, and between the traits of muscle dysmorphia (OR = 4.32; 95% CI 1.16-16.1) and the choice of the Exercise and Sport Sciences school were confirmed. CONCLUSIONS: The choice of the university courses might be influenced by pre-existing disorders in eating behaviors, which were relatively frequent in the considered sample.

Impact of snacking pattern on overweight and obesity risk in a cohort of 11- to 13-year-old adolescents.

OBJECTIVES: The association between snacking habits and overweight in adolescents is unclear. We evaluated the relation between snacking patterns and overweight/obesity in a cohort of 11- to 13-year-old Italian adolescents. METHODS: The dietary habits of 400 randomly selected adolescents were evaluated; those with body mass index ≥ 85 th percentile were considered as overweight/obese. Participants were classified based on the percentage of caloric intake from snacks (<15%, 15%-20%, >20%), snacking frequency (1, 2, ≥ 3), and timing of consuming the most caloric snack (morning, afternoon, evening). RESULTS: A minority of participants (13/400, 3.3%) did not consume any snacks; 5/13 (38.5) of them were overweight/obese. Among snackers (387/400), overweight/obesity prevalence was 10.4%, 14.4%, 20.5%, respectively, in those consuming <15%, 15% to 10%, and >20% of their energy intake from snacks. In a Poisson regression model, the overweight/obesity relative risks (RRs) were 1.35 (95% confidence interval [CI] 0.58-3.15) and 2.32 (1.10-4.89) for 15% to 20% and >20% calories/day from snacks, respectively. Overweight/obesity prevalence (from 9.6% to 22.6%) was correlated with snacking frequency (RR 2.20, 95% CI 0.92-5.27, and RR 4.17, 95% CI 1.60-10.9, for 2 and ≥ 3 snacks per day, respectively). The most caloric snacks were consumed in the morning (180/387) and afternoon (179/387); 28.6% of the predominantly evening snackers (28/387) were overweight/obese (RR 3.12, 95% CI 1.17-8.34). CONCLUSIONS: Increased snacking calories, frequency, and evening snacking are independently associated with overweight/obesity in Italian middle-school adolescents.

Vive la Difference! The Effects of Natural and Conventional Wines on Blood Alcohol Concentrations: A Randomized, Triple-Blind, Controlled Study.

Different alcoholic beverages can have different effects on blood alcohol concentration (BAC) and neurotoxicity, even when equalized for alcohol content by volume. Anecdotal evidence suggested that natural wine is metabolized differently from conventional wines. This triple-blind study compared the BAC of 55 healthy male subjects after consuming the equivalent of 2 units of alcohol of a natural or conventional wine over 3 min in two separate sessions, one week apart. BAC was measured using a professional breathalyzer every 20 min after consumption for 2 h. The BAC curves in response to the two wines diverged significantly at twenty minutes (interval T20) and forty minutes (interval T40), and also at their maximum concentrations (peaks), with the natural wine inducing a lower BAC than the conventional wine [T20 = 0.40 versus 0.46 (p < 0.0002); T40 = 0.49 versus 0.53 (p < 0.0015); peak = 0.52 versus 0.56 (p < 0.0002)]. These differences are likely related to the development of different amino acids and antioxidants in the two wines during their production. This may in turn affect the kinetics of alcohol absorption and metabolism. Other contributing factors could include pesticide residues, differences in dry extract content, and the use of indigenous or selected yeasts. The study shows that with the same quantity and conditions of intake, natural wine has lower pharmacokinetic and metabolic effects than conventional wine, which can be assumed due to the different agronomic and oenological practices with which they are produced. It can therefore be hypothesized that the consumption of natural wine may have a different impact on human health from that of conventional wine.

Prevalence of obesity in Italian adolescents: does the use of different growth charts make the difference?

BACKGROUND: Since populations are becoming increasingly multi-ethnic, the use of local or international charts is a matter of debate. This study aimed to evaluate how the choice of cut-off thresholds affected prevalence of underweight (UW), overweight (OW), obesity (OB) in 1200 11-12-year Italian adolescents, and how their somatic growth depended on parental origin. METHODS: The height, weight and body mass index were expressed as standard deviation score (SDS) using Italian (ISPED-2006) and UK (UK-1990) charts. The classification of UW/OW/OB was computed with the IOTF international cut-offs, and thresholds were identified as centiles corresponding to BMI values of 18.5/25.0/30.0 kg/m2 at 18-year in ISPED-2006 or UK-1990 references. RESULTS: About 30% participants had non-Italian parents, above all from North-Africa and Romania. Referring to the UK-1990 charts, all groups showed negative mean SDS for height, and positive SDS for weight and BMI. Referring to the ISPED-2006 charts, all mean SDS were negative. Percentage of UW individuals was higher in accordance with ISPED-2006 than with UK-1990 charts, whereas percentages of OW/OB were higher with UK-1990 than ISPED-2006 charts. The results obtained using IOFT cut-offs were similar to UK-1990 cut-offs. These results were due to the different shape of age-dependent cut-off centiles. Independently by the parental origin, the percentages of adolescents classified as OW/OB were closer to the expected values using the ISPED-2006 then the UK-1990 cut-offs. The results suggested the use of the Italian references for adolescents with immigrant parents. CONCLUSION: The use of local charts seems more appropriate at least in Italian adolescents in the age range studied.

Random spray Retinex: a new Retinex implementation to investigate the local properties of the model.

In order to investigate the local filtering behavior of the Retinex model, we propose a new implementation in which paths are replaced by 2-D pixel sprays, hence the name "random spray Retinex." A peculiar feature of this implementation is the way its parameters can be controlled to perform spatial investigation. The parameters' tuning is accomplished by an unsupervised method based on quantitative measures. This procedure has been validated via user panel tests. Furthermore, the spray approach has faster performances than the path-wise one. Tests and results are presented and discussed.

Longitudinal study of quality of life in advanced cancer patients on home parenteral nutrition.

Since there is little knowledge regarding the quality of life (QoL) of cancer patients on home parenteral nutrition (HPN), we planned a prospective, longitudinal, double-center study to investigate the changes of QoL in these patients. One hundred and eleven adult cancer patients who were candidates for HPN following the indications of the European guidelines were consecutively enrolled. For QoL analysis, EORTC QLQ-C30 questionnaires were filled at the HPN start and after 1, 2, 3, and 4 months, and scores changes over time were analyzed according to the univariate mixed-effects linear model for repeated measures. Most patients had gastrointestinal cancers, were severely malnourished, and were in stage IV; two-thirds were still receiving oncologic treatments. Median weight loss over 3 months and body mass index were 11.7% and 20.7, respectively. Median survival was 4.7 (1-42) months; 67 and 34% of patients survived 3 and 6 months, respectively. Global QoL, physical functioning, role functioning, emotional functioning, appetite loss, and fatigue scores had a statistically significant trend over time (P < 0.001, P < 0.001, P = 0.007, P < 0.001, P = 0.004, P = 0.022, respectively). At the univariate analyses, the determinants significantly associated with changes in trend over time for physical, role, and emotional functioning were oncologic treatments (P < 0.001, P = 0.014, P = 0.040, respectively) and for appetite loss they were weight loss and Karnofsky performance status (P = 0.003, P = 0.023, respectively). Global QoL, physical, role, and emotional functioning improved during HPN even in advanced cancer patients on oncologic treatments.

Comparison of in vitro fertilization outcomes in ICSI cycles after human sperm preparation by density gradient centrifugation and direct micro swim-up without centrifugation.

OBJECTIVE: The aim of this study was to evaluate the efficacy of a non-expensive, easy and fast technique (direct micro swim-up) for sperm preparation in intracytoplasmic sperm injection (ICSI) treatments without the use of centrifuge. METHODS: We carried out a multicentric study in which a total of 140 ICSI-cycles were included. Sibling oocytes were divided into two groups according to semen preparation procedures: group A, discontinuous gradients (DG) (oocytes n=668), and group B, direct micro swim-up (MSU) (oocytes n=660). We analyzed differences in some key performance indicators. RESULTS: Fertilization rates were not statistically different between the DG and MSU groups (76.0% vs. 81.8%, respectively, p=0.248); while significant differences were found in blastulation rates per fertilized oocytes (41.7% vs. 58.5%, p=0.009), blastulation rates per D3 embryos (46.1% vs. 63.7%, p=0.045), and pregnancy rates (25.8% vs. 41.9%, p=0.045). The abortion rate was reduced in the MSU group as compared to DG, but not in a significant manner (12.9% vs. 29.4%, p=0.161). CONCLUSION: The MSU procedure has the advantage of reducing costs, time and mismatches, while ensuring comparable, and in some cases, better results than DG treatments. This technique can therefore be used as an alternative method to other conventional semen treatments.

Assay of diffusion of different botulinum neurotoxin type a formulations injected in the mouse leg.

Botulinum neurotoxin type-A (BoNT/A) is very effective in the therapy of a wide range of human syndromes characterized by hyperactivity of peripheral cholinergic nerve terminals. Little diffusion of this toxin from the site of injection is commonly observed, but even minor changes in this property would greatly affect the validity of the treatment. Different pharmacological formulations of BoNT/A are available, and they may have different diffusion characteristics due to protein complex size, product format, and pharmacological properties. Here we assessed the extent of diffusion of three commercial preparations of BoNT/A: Botox (Allergan), Dysport (Ipsen), and Xeomin (Merz Pharmaceuticals) using a novel and highly sensitive test based on neural cell adhesion molecule (N-CAM) expression in muscle. N-CAM is a membrane glycoprotein that accumulates on muscle fibers after denervation and is not expressed in untreated adult muscle. This allows fine monitoring of the functional diffusion of this toxin, and the sensitivity of this assay is emphasized by the use of the mouse model because of the small muscle dimensions. The results presented here indicate that there is no significant difference between Botox, Dysport, and Xeomin with respect to diffusion into adjacent muscles in the mouse leg.

Different mechanisms of inhibition of nerve terminals by botulinum and snake presynaptic neurotoxins.

The different mode of action on peripheral nerve terminals of the botulinum neurotoxins and of the snake presynaptic phospholipase A2 neurotoxins is reviewed here. These two groups of toxins are highly toxic because they are neurospecific and at the same time are enzymes that can modify many substrate molecules before being inactivated. The similarity of symptoms they cause in humans derives from the fact that both botulinum neurotoxins (seven serotypes named A-G) and snake presynaptic PLA2 neurotoxins block the nerve terminals and that peripheral cholinergic terminals are major targets. Given this general similarity of targets and clinical symptoms, the specific molecular and cellular mechanisms at the basis of their action are very different. This difference appears evident from the beginning of intoxication, i.e. neurotoxins binding to peripheral nerve terminals and proceeds with the different site of actions and molecular targets.

Neuromuscular paralysis and recovery in mice injected with botulinum neurotoxins A and C.

Botulinum neurotoxin type A (BoNT/A) is commonly used in human therapy. This treatment may induce immunoresistance and preliminary evaluation of other botulinum neurotoxin serotypes suggested botulinum neurotoxin type C (BoNT/C) to be a good alternative to BoNT/A. Here, we have further characterized the biological activities of BoNT/C using a variety of experimental approaches. Muscle paralysis and time of recovery of mouse hind limb injected with BoNT/A or BoNT/C were assayed with the Digit Abduction Scoring assay. The extent and duration of paralysis were similar with the two toxin serotypes. Extensor digitorum longus or tibialis anterior muscles were dissected at times of complete paralysis and of complete recovery. Muscle weight and force were significantly reduced in mice injected with BoNT/A and BoNT/C, and some atrophy persisted for a long time. In BoNT/C-treated junctions, nerve terminal sprouting was prominent, indicating that the capacity to extend the field of innervation is not hampered by BoNT/C. BoNT/C induced a marked decrease in the frequency of miniature endplate potentials and in the amplitude of endplate potentials. 3,4-diaminopyridine reversed the effect of BoNT/C by increasing the amplitude of synchronized endplate potentials. The present study shows an extensive similarity in the biological activities of BoNT/A and BoNT/C, further supporting the suggestion that BoNT/C is a valid alternative to BoNT/A.

Mathematical definition and analysis of the retinex algorithm.

We present a detailed mathematical analysis of the original Retinex algorithm due to Land and McCann [J. Opt. Soc. Am. 61, 1 (1071)]. To this end, we propose an analytic formula that describes the algorithm behavior. More than one Retinex version (e.g., with and without threshold) is examined. The behavior of Retinex varying the number of paths is predicted, and its recursive iterations are mathematically analyzed using the formula. The mathematical setting presented serves as a common ground for the various Retinex implementations. Its validity is confirmed by the tests on images that we have performed.