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.

Percutaneous repair of Achilles tendon ruptures with Tenolig: quantitative analysis of postural control and gait pattern.

BACKGROUND: Surgical approach in Achilles tendon's rupture involved during the last years has becoming safer and less invasive as possible. Lots of study investigate the outcomes of the mini-invasive technique with Tenolig proving its good results, but never in the long-term. OBJECTIVES: Our study want to emphasize the effectiveness of this treatment exploring the postural and gait patterns in a 24-month follow up. METHOD: Patients did self-training exercises without specific supervision, instead of a particular postoperative rehabilitation protocol. We compared 21 patients to a control group of 19 health subjects using a clinical examination, a podobarometric and an optokinetic analysis. RESULTS: Data shows no differences in time-distance parameters, despite a reduction of propulsion phase data, confirmed also by kinetic analysis. Podobarometric results show only a decrease in the anterior pressure of the injured limb (p=0.09). In standing an increase of anterior-posterior oscillation of the COP (center of pressure) (p=0.03). CONCLUSIONS: The results underline the long-term outcome effectiveness of the technique but some functional alterations remain. This could be the reason of the weakness, which always affected the patients. Reduction of the triceps elongation and restoration of strength during the propulsion phase should be the key points in postoperative physiotherapy.

A novel application of nylon membranes to the luminescent determination of benzo[a]pyrene at ultra trace levels in water samples.

Very simple and highly sensitive methods are presented for the determination of benzo[a]pyrene, one of the most carcinogenic polycyclic aromatic hydrocarbons (PAHs). The approaches are based on solid-phase extraction of the analyte on a nylon membrane via a syringe procedure, and its fluorescent or phosphorescent determination on the solid surface. While the native fluorescence of benzo[a]pyrene retained on a nylon surface is measured directly, room-temperature phosphorescence is induced by spotting a few microlitres of thallium(I) nitrate solution on the surface (heavy-atom effect). An enhancement of the phosphorescence signal was corroborated when the measurements were carried under a nitrogen atmosphere. The analytical figures of merit obtained under the best experimental conditions demonstrate the capability of detecting benzo[a]pyrene at a sub-parts-per-trillion (sub-ng L(-1)) level. The potential interference from other common PAHs and also from different metal ions was studied. The feasibility of determining benzo[a]pyrene in real samples was successfully evaluated through the analysis of spiked tap, underground and mineral water samples of different origins. Recoveries obtained from spiked river waters were successfully compared with those provided by a reference method, through rigorous statistical analysis.

Bioactivation and toxicity in vitro of HCFC-123 and HCFC-141b: role of cytochrome P450.

The bioactivation and cytotoxicity in vitro of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) and 1,1-dichloro-1-fluoroethane (HCFC-141b), two replacements for some ozone-depleting chlorofluorocarbons (CFC), were investigated in rat liver microsomes and isolated rat hepatocytes. Both compounds were activated by cytochrome P450 to reactive metabolites, as indicated by: (i) the depletion of exogenous and cellular glutathione, (ii) the increased LDH release from hepatocytes, (iii) the loss of microsomal P450 content and activities, and (iv) the formation of free radical species observed in the presence of the two compounds. Moreover, the formation of two stable metabolites and an increased production of conjugated dienes, a marker of lipid peroxidation, were observed for both HCFC-123 and HCFC-141b. The biotransformation of both compounds by pyridine- and phenobarbital-induced rat liver microsomes and the inhibition of LDH release by 4-methylpyrazole and troleandomycin indicate that P450 2E1, 2B and, possibly, also 3A are the isoforms involved in the bioactivation and toxicity of HCFC-123 and HCFC-141b in the rat.

Bioactivation to free radicals and cytotoxicity of 1,1-dichloro-1-fluoroethane (HCFC-141b).

1. The in vitro bioactivation by rat liver microsomes and the cytotoxicity in rat hepatocytes of 1,1-dichloro-1-fluoroethane (HCFC-141b), a replacement for some ozone depleting chlorofluorocarbons (CFC), have been investigated. 2. Anaerobic incubations of liver microsomes from pyridine-induced rats with HCFC-141b in the presence of the spin-trapping agent N-t-butyl-alpha-phenylnitrone (PBN) resulted in the formation of a typical ESR radical signal. 3. In the presence of HCFC-141b, a dose-dependent formation of conjugated dienes was observed that was partially inhibited by PBN, glutathione (GSH) and vitamin C. Moreover, HCFC-141b increased the release of lactate dehydrogenase (LDH) and the depletion of cellular glutathione in isolated rat hepatocytes under both normoxic and hypoxic conditions. 4. HCFC-141b-dependent cytotoxicity was completely prevented by PBN under both conditions and it was partially prevented under normoxic conditions by the broad-spectrum P450 inhibitor metyrapone, the P4502E1 specific inhibitor 4-methylpyrazole and the P4503A-specific inhibitor troleandomycin. Interestingly, HCFC-141b-dependent glutathione depletion was not prevented by PBN, metyrapone, 4-methylpyrazole or troleandomycin, whereas two glutathione depletors, 2,6-dimethyl-2,5-heptadien-4-one (phorone) and diethylmaleate, partially prevented LDH release. 5. The present results indicate that HCFC-141b is reductively metabolized in vitro to free radical intermediates by P450, in particular by the CYP2E1 and, to a lower extent, CYP3A isoforms, leading to peroxidative membrane damage and glutathione-independent cytotoxicity.

Bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) in isolated rat hepatocytes.

The bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), a replacement for some ozone-depleting chlorofluorocarbons, were investigated using freshly isolated hepatocytes from non-induced male rats. A time- and concentration-dependent increase in the leakage of lactate dehydrogenase and a concentration-dependent loss of total cellular glutathione were observed in cells incubated with 1, 5 and 10 mM HCFC-123 under normoxic or hypoxic (about 4% O2) conditions. Lactate dehydrogenase leakage was completely prevented by pretreating the cell suspension with the free radical trapper N-t-butyl-alpha-phenylnitrone. The aspecific cytochrome P450 (P450) inhibitor, metyrapone, totally prevented the lactate dehydrogenase leakage from hepatocytes, while two isoform-specific P450 inhibitors, 4-methylpyrazole and troleandomycin (a P450 2E1 and a P450 3A inhibitor, respectively), provided a partial protection against HCFC-123 cytotoxicity. Interestingly, pretreatment of cells with glutathione depletors, such as phorone and diethylmaleate, did not enhance the HCFC-123-dependent lactate dehydrogenase leakage. Two stable metabolites of HCFC-123, 1-chloro-2,2,2-trifluoroethane and 1-chloro-2,2-difluoroethene, were detected by gas chromatography/mass spectrometry analysis of the head space of the hepatocyte incubations carried out under hypoxic and, although at a lower level, also normoxic conditions, indicating that reductive metabolism of HCFC-123 by hepatocytes had occurred. The results overall indicate that HCFC-123 is cytotoxic to rat hepatocytes under both normoxic and hypoxic conditions, due to its bioactivation to reactive metabolites, probably free radicals, and that P450 2E1 and, to a lower extent, P450 3A, are involved in the process.