Successful treatment of severe MSUD in Bckdhb-/- mice with neonatal AAV gene therapy.

Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb-/- mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha-/- mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb-/- mice at 1014 vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb-/- mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.

Nontargeted urine metabolomic analysis of acute intermittent porphyria reveals novel interactions between bile acids and heme metabolism: New promising biomarkers for the long-term management of patients.

Acute intermittent porphyria is an inherited error of heme synthesis. The underlying pathophysiology, involving mainly hepatic heme synthesis, is poorly understood despite its occurrence, and the severity of acute porphyria attack is still difficult to control. A better understanding of the interactions between heme synthesis and global metabolism would improve the management of AIP patients. An untargeted metabolomic analysis was performed on the urine of 114 patients with overt AIP and asymptomatic carriers using liquid chromatography coupled to high-resolution mass spectrometry. The collected data were analyzed by combining univariate and multivariate analyses. A total of 239 metabolites were annotated in urine samples by matching chromatographic and mass spectral characteristics with those from our chemical library. Twenty-six metabolites, including porphyrin precursors, intermediates of tryptophan or glycine metabolism and, unexpectedly, bile acids, showed significant concentration differences between the phenotypic groups. Dysregulation of bile acid metabolism was confirmed by targeted quantitative analysis, which revealed an imbalance in favor of hydrophobic bile acids associated with changes in conjugation, which was more pronounced in the severe phenotype. Using a random forest model, the cholic acid/chenodeoxycholic acid ratio enables the differential classification of severe patients from other patients with a diagnostic accuracy of 84%. The analysis of urine samples revealed significant modifications in the metabolome of AIP patients. Alteration in bile acids provides new insights into the pathophysiology of chronic complications, such as primary liver cancer, while also providing new biomarker candidates for predicting the most severe phenotypes.

Roux-en-Y gastric bypass induces hepatic transcriptomic signatures and plasma metabolite changes indicative of improved cholesterol homeostasis.

BACKGROUND & AIMS: Roux-en-Y gastric bypass (RYGB), the most effective surgical procedure for weight loss, decreases obesity and ameliorates comorbidities, such as non-alcoholic fatty liver (NAFLD) and cardiovascular (CVD) diseases. Cholesterol is a major CVD risk factor and modulator of NAFLD development, and the liver tightly controls its metabolism. How RYGB surgery modulates systemic and hepatic cholesterol metabolism is still unclear. METHODS: We studied the hepatic transcriptome of 26 patients with obesity but not diabetes before and 1 year after undergoing RYGB. In parallel, we measured quantitative changes in plasma cholesterol metabolites and bile acids (BAs). RESULTS: RYGB surgery improved systemic cholesterol metabolism and increased plasma total and primary BA levels. Transcriptomic analysis revealed specific alterations in the liver after RYGB, with the downregulation of a module of genes implicated in inflammation and the upregulation of three modules, one associated with BA metabolism. A dedicated analysis of hepatic genes related to cholesterol homeostasis pointed towards increased biliary cholesterol elimination after RYGB, associated with enhancement of the alternate, but not the classical, BA synthesis pathway. In parallel, alterations in the expression of genes involved in cholesterol uptake and intracellular trafficking indicate improved hepatic free cholesterol handling. Finally, RYGB decreased plasma markers of cholesterol synthesis, which correlated with an improvement in liver disease status after surgery. CONCLUSIONS: Our results identify specific regulatory effects of RYGB on inflammation and cholesterol metabolism. RYGB alters the hepatic transcriptome signature, likely improving liver cholesterol homeostasis. These gene regulatory effects are reflected by systemic post-surgery changes of cholesterol-related metabolites, corroborating the beneficial effects of RYGB on both hepatic and systemic cholesterol homeostasis. IMPACT AND IMPLICATIONS: Roux-en-Y gastric bypass (RYGB) is a widely used bariatric surgery procedure with proven efficacy in body weight management, combatting cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD). RYGB exerts many beneficial metabolic effects, by lowering plasma cholesterol and improving atherogenic dyslipidemia. Using a cohort of patients undergoing RYGB, studied before and 1 year after surgery, we analyzed how RYGB modulates hepatic and systemic cholesterol and bile acid metabolism. The results of our study provide important insights on the regulation of cholesterol homeostasis after RYGB and open avenues that could guide future monitoring and treatment strategies targeting CVD and NAFLD in obesity.

Combining metabolomics and machine learning models as a tool to distinguish non-classic 21-hydroxylase deficiency from polycystic ovary syndrome without adrenocorticotropic hormone testing.

STUDY QUESTION: Can a combination of metabolomic signature and machine learning (ML) models distinguish nonclassic 21-hydroxylase deficiency (NC21OHD) from polycystic ovary syndrome (PCOS) without adrenocorticotrophic hormone (ACTH) testing? SUMMARY ANSWER: A single sampling methodology may be an alternative to the dynamic ACTH test in order to exclude the diagnosis of NC21OHD in the presence of a clinical hyperandrogenic presentation at any time of the menstrual cycle. WHAT IS KNOWN ALREADY: The clinical presentation of patients with NC21OHD is similar with that for other disorders of androgen excess. Currently, cosyntropin stimulation remains the gold standard diagnosis of NC21OHD. STUDY DESIGN, SIZE, DURATION: The study was designed using a bicentric recruitment: an internal training set included 19 women with NC21OHD and 19 controls used for developing the model; a test set included 17 NC21OHD, 72 controls and 266 PCOS patients used to evaluate the performance of the diagnostic strategy thanks to an ML approach. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fifteen steroid species were measured in serum by liquid chromatography-mass spectrometry (LC-MS/MS). This set of 15 steroids (defined as 'steroidome') used to map the steroid biosynthesis pathway was the input for our models. MAIN RESULTS AND THE ROLE OF CHANCE: From a single sample, modeling involving metabolic pathway mapping by profiling 15 circulating steroids allowed us to identify perfectly NC21OHD from a confounding PCOS population. The constructed model using baseline LC-MS/MS-acquired steroid fingerprinting successfully excluded all 17 NC21OHDs (sensitivity and specificity of 100%) from 266 PCOS from an external testing cohort of originally 549 women, without the use of ACTH testing. Blood sampling timing during the menstrual cycle phase did not impact the efficiency of our model. LIMITATIONS, REASONS FOR CAUTION: The main limitations were the use of a restricted and fully prospective cohort as well as an analytical issue, as not all laboratories are equipped with mass spectrometers able to routinely measure this panel of 15 steroids. Moreover, the robustness of our model needs to be established with a larger prospective study for definitive validation in clinical practice. WIDER IMPLICATIONS OF THE FINDINGS: This tool makes it possible to propose a new semiology for the management of hyperandrogenism. The model presents better diagnostic performances compared to the current reference strategy. The management of patients may be facilitated by limiting the use of ACTH tests. Finally, the modeling process allows a classification of steroid contributions to rationalize the biomarker approach and highlight some underlying pathophysiological mechanisms. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by 'Agence Française de Lutte contre le dopage' and DIM Région Ile de France. This study was supported by the French institutional PHRC 2010-AOR10032 funding source and APHP. All authors declare no competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

A Machine Learning Approach for the Prediction of Testicular Sperm Extraction in Nonobstructive Azoospermia: Algorithm Development and Validation Study.

BACKGROUND: Testicular sperm extraction (TESE) is an essential therapeutic tool for the management of male infertility. However, it is an invasive procedure with a success rate up to 50%. To date, no model based on clinical and laboratory parameters is sufficiently powerful to accurately predict the success of sperm retrieval in TESE. OBJECTIVE: The aim of this study is to compare a wide range of predictive models under similar conditions for TESE outcomes in patients with nonobstructive azoospermia (NOA) to identify the correct mathematical approach to apply, most appropriate study size, and relevance of the input biomarkers. METHODS: We analyzed 201 patients who underwent TESE at Tenon Hospital (Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris), distributed in a retrospective training cohort of 175 patients (January 2012 to April 2021) and a prospective testing cohort (May 2021 to December 2021) of 26 patients. Preoperative data (according to the French standard exploration of male infertility, 16 variables) including urogenital history, hormonal data, genetic data, and TESE outcomes (representing the target variable) were collected. A TESE was considered positive if we obtained sufficient spermatozoa for intracytoplasmic sperm injection. After preprocessing the raw data, 8 machine learning (ML) models were trained and optimized on the retrospective training cohort data set: The hyperparameter tuning was performed by random search. Finally, the prospective testing cohort data set was used for the model evaluation. The metrics used to evaluate and compare the models were the following: sensitivity, specificity, area under the receiver operating characteristic curve (AUC-ROC), and accuracy. The importance of each variable in the model was assessed using the permutation feature importance technique, and the optimal number of patients to include in the study was assessed using the learning curve. RESULTS: The ensemble models, based on decision trees, showed the best performance, especially the random forest model, which yielded the following results: AUC=0.90, sensitivity=100%, and specificity=69.2%. Furthermore, a study size of 120 patients seemed sufficient to properly exploit the preoperative data in the modeling process, since increasing the number of patients beyond 120 during model training did not bring any performance improvement. Furthermore, inhibin B and a history of varicoceles exhibited the highest predictive capacity. CONCLUSIONS: An ML algorithm based on an appropriate approach can predict successful sperm retrieval in men with NOA undergoing TESE, with promising performance. However, although this study is consistent with the first step of this process, a subsequent formal prospective multicentric validation study should be undertaken before any clinical applications. As future work, we consider the use of recent and clinically relevant data sets (including seminal plasma biomarkers, especially noncoding RNAs, as markers of residual spermatogenesis in NOA patients) to improve our results even more.

Adrenal adaptation in potassium-depleted men: role of progesterone?

BACKGROUND: In rodents, the stimulation of adrenal progesterone is necessary for renal adaptation under potassium depletion. Here, we sought to determine the role of progesterone in adrenal adaptation in potassium-depleted healthy human volunteers and compared our findings with data collected in patients with Gitelman syndrome (GS), a salt-losing tubulopathy. METHODS: Twelve healthy young men were given a potassium-depleted diet for 7 days at a tertiary referral medical centre (NCT02297048). We measured by liquid chromatography coupled to tandem mass spectroscopy plasma steroid concentrations at Days 0 and 7 before and 30 min after treatment with tetracosactide. We compared these data with data collected in 10 GS patients submitted to tetracosactide test. RESULTS: The potassium-depleted diet decreased plasma potassium in healthy subjects by 0.3 ± 0.1 mmol/L, decreased plasma aldosterone concentration by 50% (P = 0.0332) and increased plasma 17-hydroxypregnenolone concentration by 45% (P = 0.0232) without affecting other steroids. CYP17 activity, as assessed by 17-hydroxypregnenolone/pregnenolone ratio, increased by 60% (P = 0.0389). As compared with healthy subjects, GS patients had 3-fold higher plasma concentrations of aldosterone, 11-deoxycortisol (+30%) and delta 4-androstenedione (+14%). Their post-tetracosactide progesterone concentration was 2-fold higher than that of healthy subjects and better correlated to plasma potassium than to plasma renin. CONCLUSION: The increase in 17-hydroxypregnenolone concentration after mild potassium depletion in otherwise healthy human subjects suggests that 17 hydroxylation of pregnenolone prevents the increase in progesterone observed in potassium-depleted mice. The unexpected over-response of non-mineralocorticoid steroids to tetracosactide in GS subjects suggests that the adrenal system not only adapts to sodium depletion but may also respond to hypokalaemia.

CYP46A1 gene therapy deciphers the role of brain cholesterol metabolism in Huntington's disease.

Dysfunctions in brain cholesterol homeostasis have been extensively related to brain disorders. The main pathway for brain cholesterol elimination is its hydroxylation into 24S-hydroxycholesterol by the cholesterol 24-hydrolase, CYP46A1. Increasing evidence suggests that CYP46A1 has a role in the pathogenesis and progression of neurodegenerative disorders, and that increasing its levels in the brain is neuroprotective. However, the mechanisms underlying this neuroprotection remain to be fully understood. Huntington's disease is a fatal autosomal dominant neurodegenerative disease caused by an abnormal CAG expansion in huntingtin's gene. Among the multiple cellular and molecular dysfunctions caused by this mutation, altered brain cholesterol homeostasis has been described in patients and animal models as a critical event in Huntington's disease. Here, we demonstrate that a gene therapy approach based on the delivery of CYP46A1, the rate-limiting enzyme for cholesterol degradation in the brain, has a long-lasting neuroprotective effect in Huntington's disease and counteracts multiple detrimental effects of the mutated huntingtin. In zQ175 Huntington's disease knock-in mice, CYP46A1 prevented neuronal dysfunctions and restored cholesterol homeostasis. These events were associated to a specific striatal transcriptomic signature that compensates for multiple mHTT-induced dysfunctions. We thus explored the mechanisms for these compensations and showed an improvement of synaptic activity and connectivity along with the stimulation of the proteasome and autophagy machineries, which participate to the clearance of mutant huntingtin (mHTT) aggregates. Furthermore, BDNF vesicle axonal transport and TrkB endosome trafficking were restored in a cellular model of Huntington's disease. These results highlight the large-scale beneficial effect of restoring cholesterol homeostasis in neurodegenerative diseases and give new opportunities for developing innovative disease-modifying strategies in Huntington's disease.

Biallelic pathogenic variants in the lanosterol synthase gene LSS involved in the cholesterol biosynthesis cause alopecia with intellectual disability, a rare recessive neuroectodermal syndrome.

PURPOSE: Lanosterol synthase (LSS) gene was initially described in families with extensive congenital cataracts. Recently, a study has highlighted LSS associated with hypotrichosis simplex. We expanded the phenotypic spectrum of LSS to a recessive neuroectodermal syndrome formerly named alopecia with mental retardation (APMR) syndrome. It is a rare autosomal recessive condition characterized by hypotrichosis and intellectual disability (ID) or developmental delay (DD), frequently associated with early-onset epilepsy and other dermatological features. METHODS: Through a multicenter international collaborative study, we identified LSS pathogenic variants in APMR individuals either by exome sequencing or LSS Sanger sequencing. Splicing defects were assessed by transcript analysis and minigene assay. RESULTS: We reported ten APMR individuals from six unrelated families with biallelic variants in LSS. We additionally identified one affected individual with a single rare variant in LSS and an allelic imbalance suggesting a second event. Among the identified variants, two were truncating, seven were missense, and two were splicing variants. Quantification of cholesterol and its precursors did not reveal noticeable imbalance. CONCLUSION: In the cholesterol biosynthesis pathway, lanosterol synthase leads to the cyclization of (S)-2,3-oxidosqualene into lanosterol. Our data suggest LSS as a major gene causing a rare recessive neuroectodermal syndrome.

Restoring brain cholesterol turnover improves autophagy and has therapeutic potential in mouse models of spinocerebellar ataxia.

Spinocerebellar ataxias (SCAs) are devastating neurodegenerative disorders for which no curative or preventive therapies are available. Deregulation of brain cholesterol metabolism and impaired brain cholesterol turnover have been associated with several neurodegenerative diseases. SCA3 or Machado-Joseph disease (MJD) is the most prevalent ataxia worldwide. We show that cholesterol 24-hydroxylase (CYP46A1), the key enzyme allowing efflux of brain cholesterol and activating brain cholesterol turnover, is decreased in cerebellar extracts from SCA3 patients and SCA3 mice. We investigated whether reinstating CYP46A1 expression would improve the disease phenotype of SCA3 mouse models. We show that administration of adeno-associated viral vectors encoding CYP46A1 to a lentiviral-based SCA3 mouse model reduces mutant ataxin-3 accumulation, which is a hallmark of SCA3, and preserves neuronal markers. In a transgenic SCA3 model with a severe motor phenotype we confirm that cerebellar delivery of AAVrh10-CYP46A1 is strongly neuroprotective in adult mice with established pathology. CYP46A1 significantly decreases ataxin-3 protein aggregation, alleviates motor impairments and improves SCA3-associated neuropathology. In particular, improvement in Purkinje cell number and reduction of cerebellar atrophy are observed in AAVrh10-CYP46A1-treated mice. Conversely, we show that knocking-down CYP46A1 in normal mouse brain impairs cholesterol metabolism, induces motor deficits and produces strong neurodegeneration with impairment of the endosomal-lysosomal pathway, a phenotype closely resembling that of SCA3. Remarkably, we demonstrate for the first time both in vitro, in a SCA3 cellular model, and in vivo, in mouse brain, that CYP46A1 activates autophagy, which is impaired in SCA3, leading to decreased mutant ataxin-3 deposition. More broadly, we show that the beneficial effect of CYP46A1 is also observed with mutant ataxin-2 aggregates. Altogether, our results confirm a pivotal role for CYP46A1 and brain cholesterol metabolism in neuronal function, pointing to a key contribution of the neuronal cholesterol pathway in mechanisms mediating clearance of aggregate-prone proteins. This study identifies CYP46A1 as a relevant therapeutic target not only for SCA3 but also for other SCAs.

Membrane re-arrangements and rippled phase stabilisation by the cell penetrating peptide penetratin.

Cell penetrating peptides are promising vectors for molecular drug delivery in eukaryotic cells. Despite of their discovery 20years ago, the mechanisms of peptide membrane crossing are still controversial. The different suggested penetration mechanisms reflect the high sequence and structural diversity of cell penetrating peptides. The fundamental step for peptide penetration into the cytosol is the crossing of the membrane lipid barrier at the level of the plasma membrane or the endosomes. Therefore, the study of the peptide-lipid interaction is the key for peptide penetration mechanisms understanding. In order to study the changes in lipid organisation induced by the cell penetrating peptide penetratin, several experiments by three different physicochemical approaches were performed. X-ray diffraction data shows that penetratin is able to induce membrane phase separation and lipid rearrangements observed by inter-lipid distances. These changes are accompanied by a temperature stable behaviour of some of the induced membrane domains. The membrane environment fluorescent probe laurdan showed that, in DMPC and DMPC/DMPG membranes, the peptide induces de-packing of lipids. Calorimetric analyses show that penetratin favours the gel phase to gel-like rippled phase transition. Overall, the data suggest both, that the rippled phase is a heterogeneous structure formed by gel-like and fluid-like coexisting components, and that the penetratin-induced membrane heterogeneity could be important for membrane destabilisation during cell penetration.

CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease.

Huntington's disease is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (Exp-HTT) leading to degeneration of striatal neurons. Altered brain cholesterol homeostasis has been implicated in Huntington's disease, with increased accumulation of cholesterol in striatal neurons yet reduced levels of cholesterol metabolic precursors. To elucidate these two seemingly opposing dysregulations, we investigated the expression of cholesterol 24-hydroxylase (CYP46A1), the neuronal-specific and rate-limiting enzyme for cholesterol conversion to 24S-hydroxycholesterol (24S-OHC). CYP46A1 protein levels were decreased in the putamen, but not cerebral cortex samples, of post-mortem Huntington's disease patients when compared to controls. Cyp46A1 mRNA and CYP46A1 protein levels were also decreased in the striatum of the R6/2 Huntington's disease mouse model and in SThdhQ111 cell lines. In vivo, in a wild-type context, knocking down CYP46A1 expression in the striatum, via an adeno-associated virus-mediated delivery of selective shCYP46A1, reproduced the Huntington's disease phenotype, with spontaneous striatal neuron degeneration and motor deficits, as assessed by rotarod. In vitro, CYP46A1 restoration protected SThdhQ111 and Exp-HTT-expressing striatal neurons in culture from cell death. In the R6/2 Huntington's disease mouse model, adeno-associated virus-mediated delivery of CYP46A1 into the striatum decreased neuronal atrophy, decreased the number, intensity level and size of Exp-HTT aggregates and improved motor deficits, as assessed by rotarod and clasping behavioural tests. Adeno-associated virus-CYP46A1 infection in R6/2 mice also restored levels of cholesterol and lanosterol and increased levels of desmosterol. In vitro, lanosterol and desmosterol were found to protect striatal neurons expressing Exp-HTT from death. We conclude that restoring CYP46A1 activity in the striatum promises a new therapeutic approach in Huntington's disease.

Antenatal manifestations of inborn errors of metabolism: biological diagnosis.

Inborn errors of metabolism (IEMs) that present with abnormal imaging findings in the second half of pregnancy are mainly lysosomal storage disorders (LSDs), cholesterol synthesis disorders (CSDs), glycogen storage disorder type IV (GSD IV), peroxisomal disorders, mitochondrial fatty acid oxidation defects (FAODs), organic acidurias, aminoacidopathies, congenital disorders of glycosylation (CDGs), and transaldolase deficiency. Their biological investigation requires fetal material. The supernatant of amniotic fluid (AF) is useful for the analysis of mucopolysaccharides, oligosaccharides, sialic acid, lysosphingolipids and some enzyme activities for LSDs, 7- and 8-dehydrocholesterol, desmosterol and lathosterol for CSDs, acylcarnitines for FAODs, organic acids for organic acidurias, and polyols for transaldolase deficiency. Cultured AF or fetal cells allow the measurement of enzyme activities for most IEMs, whole-cell assays, or metabolite measurements. The cultured cells or tissue samples taken after fetal death can be used for metabolic profiling, enzyme activities, and DNA extraction. Fetal blood can also be helpful. The identification of vacuolated cells orients toward an LSD, and plasma is useful for diagnosing peroxisomal disorders, FAODs, CSDs, some LSDs, and possibly CDGs and aminoacidopathies. We investigated AF of 1700 pregnancies after exclusion of frequent etiologies of nonimmune hydrops fetalis and identified 108 fetuses affected with LSDs (6.3 %), 29 of them with mucopolysaccharidosis type VII (MPS VII), and six with GSD IV (0.3 %). In the AF of 873 pregnancies, investigated because of intrauterine growth restriction and/or abnormal genitalia, we diagnosed 32 fetuses affected with Smith-Lemli-Opitz syndrome (3.7 %).

Severe X-linked chondrodysplasia punctata in nine new female fetuses.

OBJECTIVES: Conradi-Hünermann-Happle [X-linked dominant chondrodysplasia punctata 2 (CDPX2)] syndrome is a rare X-linked dominant skeletal dysplasia usually lethal in men while affected women show wide clinical heterogeneity. Different EBP mutations have been reported. Severe female cases have rarely been reported, with only six antenatal presentations. METHODS: To better characterize the phenotype in female fetuses, we included nine antenatally diagnosed cases of women with EBP mutations. All cases were de novo except for two fetuses with an affected mother and one case of germinal mosaicism. RESULTS: The mean age at diagnosis was 22 weeks of gestation. The ultrasound features mainly included bone abnormalities: shortening (8/9 cases) and bowing of the long bones (5/9), punctuate epiphysis (7/9) and an irregular aspect of the spine (5/9). Postnatal X-rays and examination showed ichthyosis (8/9) and epiphyseal stippling (9/9), with frequent asymmetric short and bowed long bones. The X-inactivation pattern of the familial case revealed skewed X-inactivation in the mildly symptomatic mother and random X-inactivation in the severe fetal case. Differently affected skin samples of the same fetus revealed different patterns of X-inactivation. CONCLUSION: Prenatal detection of asymmetric shortening and bowing of the long bones and cartilage stippling should raise the possibility of CPDX2 in female fetuses, especially because the majority of such cases involve de novo mutations.

Exosomes participate in the alteration of muscle homeostasis during lipid-induced insulin resistance in mice.

AIMS/HYPOTHESIS: Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route. METHODS: Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA). RESULTS: HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues. CONCLUSIONS/INTERPRETATION: Exosomes act as 'paracrine-like' signals and modify muscle homeostasis during high-fat diets.

Machine learning approach to assess the association between anthropometric, metabolic, and nutritional status and semen parameters.

Many lifestyle factors, such as nutritional imbalance leading to obesity, metabolic disorders, and nutritional deficiency, have been identified as potential risk factors for male infertility. The aim of this study was to evaluate the relationship between semen parameters and anthropometric, metabolic and nutritional parameters. Relationship was first assessed individually, then after the application of a previously constructed and validated machine learning score that allows their combination. Anthropometric, metabolic, antioxidant, micronutrient, and sperm parameters from 75 men suffering from idiopathic infertility from four infertility centers in France (Jean-Verdier ART Center Hospital, Bondy; North Hospital ART Center, Saint-Étienne; Navarre Polyclinic ART Center, Pau; and Cochin Hospital ART Center, Paris) between September 2009 and December 2013 were collected. After assessing standard correlation analysis, a previously built machine learning model, providing a score ranging from 0 (the poorest) to 1 (the most favorable), was calculated for each man in the study cohort. This machine learning model, which separates infertile/fertile men with unexplained infertility on the basis of their bioclinical signature, provides a more holistic evaluation of the influence of the considered markers (anthropometric, metabolic, and oxidative status). We observed a significant correlation of some anthropometric, metabolic, and nutritional disorders with some sperm characteristics. Moreover, an unfavorable machine learning score was associated with a high level of sperm DNA fragmentation. Favorable anthropometric, metabolic, and oxidative patterns, which may reflect an appropriate lifestyle, appear to positively impact overall health, in particular reproductive function. This study, consistent with previous publications, suggests that beyond semen quality parameters, in an essential assessment of male fertility, other key factors should be taken into account. In this regard, the application of emerging artificial intelligence techniques may provide a unique opportunity to integrate all these parameters and deliver personalized care.

Effect of Stool Sampling on a Routine Clinical Method for the Quantification of Six Short Chain Fatty Acids in Stool Using Gas Chromatography-Mass Spectrometry.

Short chain fatty acids (SCFAs) are primarily produced in the caecum and proximal colon via the bacterial fermentation of undigested carbohydrates that have avoided digestion in the small intestine. Increasing evidence supports the critical role that SCFAs play in health and homeostasis. Microbial SCFAs, namely butyric acid, serve as a principal energy source for colonocytes, and their production is essential for gut integrity. A direct link between SCFAs and some human pathological conditions, such as inflammatory bowel disease, irritable bowel syndrome, diarrhea, and cancer, has been proposed. The direct measurement of SCFAs in feces provides a non-invasive approach to demonstrating connections between SCFAs, microbiota, and metabolic diseases to estimate their potential applicability as meaningful biomarkers of intestinal health. This study aimed to adapt a robust analytical method (liquid-liquid extraction, followed by isobutyl chloroformate derivatization and GC-MS analysis), with comparable performances to methods from the literature, and to use this tool to tackle the question of pre-analytical conditions, namely stool processing. We focused on the methodology of managing stool samples before the analysis (fresh stool or dilution in either ethanol/methanol, lyophilized stool, or RNAlater®), as this is a significant issue to consider for standardizing results between clinical laboratories. The objective was to standardize methods for future applications as diagnostic tools. In this paper, we propose a validated GC-MS method for SCFA quantification in stool samples, including pre- and post-analytical comparison studies that could be easily used for clinical laboratory purposes. Our results show that using lyophilization as a stool-processing method would be the best method to achieve this goal.

[Artificial intelligence: to a better predictive strategy for testicular sperm extraction outcome in azoospermia]. / Intelligence artificielle : vers une meilleure stratégie de prédiction du résultat de la biopsie testiculaire dans un contexte d'azoospermie.

Azoospermia, defined as the absence of sperm in the semen, is found in 10-15 % of infertile patients. Two-thirds of these cases are caused by impaired spermatogenesis, known as non-obstructive azoospermia (NOA). In this context, surgical sperm extraction using testicular sperm extraction (TESE) is the best option and can be offered to patients as part of fertility preservation, or to benefit from in vitro fertilization. The aim of the preoperative assessment is to identify the cause of NOA and evaluate the status of spermatogenesis. Its capacity to predict TESE success remains limited. As a result, no objective and reliable criteria are currently available to guide professionals on the chances of success and enable them to correctly assess the benefit-risk balance of this procedure. Artificial intelligence (AI), a field of research that has been rapidly expanding in recent years, has the potential to revolutionize medicine by making it more predictive and personalized. The aim of this review is to introduce AI and its key concepts, and then to examine the current state of research into predicting the success of TESE.

Modulation of Brain Cholesterol Metabolism through CYP46A1 Overexpression for Rett Syndrome.

Rett syndrome (RTT) is a rare neurodevelopmental disorder caused by mutation in the X-linked gene methyl-CpG-binding protein 2 (Mecp2), a ubiquitously expressed transcriptional regulator. RTT results in mental retardation and developmental regression that affects approximately 1 in 10,000 females. Currently, there is no curative treatment for RTT. Thus, it is crucial to develop new therapeutic approaches for children suffering from RTT. Several studies suggested that RTT is linked with defects in cholesterol homeostasis, but for the first time, therapeutic evaluation is carried out by modulating this pathway. Moreover, AAV-based CYP46A1 overexpression, the enzyme involved in cholesterol pathway, has been demonstrated to be efficient in several neurodegenerative diseases. Based on these data, we strongly believe that CYP46A1 could be a relevant therapeutic target for RTT. Herein, we evaluated the effects of intravenous AAVPHP.eB-hCYP46A1-HA delivery in male and female Mecp2-deficient mice. The applied AAVPHP.eB-hCYP46A1 transduced essential neurons of the central nervous system (CNS). CYP46A1 overexpression alleviates behavioral alterations in both male and female Mecp2 knockout mice and extends the lifespan in Mecp2-deficient males. Several parameters related to cholesterol pathway are improved and correction of mitochondrial activity is demonstrated in treated mice, which highlighted the clear therapeutic benefit of CYP46A1 through the neuroprotection effect. IV delivery of AAVPHP.eB-CYP46A1 is perfectly well tolerated with no inflammation observed in the CNS of the treated mice. Altogether, our results strongly suggest that CYP46A1 is a relevant target and overexpression could alleviate the phenotype of Rett patients.

Dose-response evaluation of intravenous gene therapy in a symptomatic mouse model of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare, autosomal recessive neurodegenerative disease caused by deficient activity of the lysosomal enzyme arylsulfatase A (ARSA), resulting in sulfatide accumulation and subsequent demyelination and neuronal damage within the central and peripheral nervous systems. Three clinical forms of MLD have been described, based on age at symptom onset. The most frequent and severe forms have an early onset, with the disease progressing rapidly toward severe motor and cognitive regression and ultimately premature death. There are currently no approved therapies for most of these early-onset patients once symptoms are present. Thus, it is crucial to develop new approaches to treat symptomatic patients. Here, we proposed a gene therapy approach based on the intravenous delivery of AAVPHP.eB encoding ARSA. MLD mice were treated at 6 months for a dose-response study and at 9 months to assess late-treatment efficacy. Therapeutic efficacy was evaluated 3 or 6 months after injection. We demonstrated a broad transduction in the central nervous system, a complete correction of sulfatide storage, and a significant improvement in neuroinflammation at low dose and late treatment. Taken together, this work establishes a strong rationale for proposing a phase I/II clinical trial in MLD patients.

Plasma 21-deoxycortisone: a sensitive additive tool in 21-hydroxylase deficiency in newborns.

OBJECTIVE, DESIGN, AND METHODS: Although 17-hydroxyprogesterone (17OHP) has historically been the steroid assayed in the diagnosis of congenital adrenal 21-hydroxylase deficiency (CAH-21D), its C11-hydroxylated metabolite, 21-deoxycortisol (21DF), which is strictly of adrenal origin, is assayed in parallel in this pathology. This steroid (21DF) is oxidized by 11beta-hydroxysteroid dehydrogenase type 2 into 21-deoxycortisone (21DE). In the context of CAH-21D confirmation testing, confounding factors (such as intensive care unit admission, stress, prematurity, early sampling, and variations of sex development) can interfere with the interpretation of the gold-standard biomarkers (17OHP and 21DF). Since its tissue concentrations are especially high in the placenta, we hypothesized that 21DE quantification in the neonatal periods could be an interesting biomarker in addition to 17OHP and 21DF. To verify this hypothesis, we developed a new mass spectrometry-based assay for 21DE in serum and applied it to newborns screened for CAH-21D. RESULTS: In newborns with CAH-21D, the mean serum levels of 21DE reached 17.56 ng/mL (ranging from 8.58 ng/mL to 23.20 ng/mL), and the mean 21DE:21DF ratio was 4.99. In contrast, in newborns without CAH-21D, the 21DE serum levels were low and not statistically different from the analytical 21DE limit of quantification (0.01 ng/mL). CONCLUSION: Basal serum 21DE appears to be a novel sensitive and specific biomarker of CAH-21D in newborns.