OBJECTIVE:There is a rising demand for assisted reproductive medicine, including sperm, oocyte and embryo donation. Besides medical and legal considerations, genetic testing, including carrier screening for multiple autosomal and X-linked recessive disorders plays an essential role in evaluating hereditary risk among donors and therefore exclude them from the donation process. METHODS:A retrospective study was conducted on oocyte donors from a private clinic of assisted reproduction who underwent genetic testing between June 2014 and September 2023. Pre and post-test procedures were performed at the private clinic while karyotyping and carrier screening for Cystic Fibrosis, Fragile X syndrome and Spinal Muscular Atrophy were performed at the Genetic Unit of Faculty of Medicine, University of Porto. RESULTS:Among 581 donors, 81 women were excluded from the donation process since 5/563 had an alteration in karyotype, 57/581 were carriers of a Cystic Fibrosis Transmembrane conductance Regulator pathogenic variant or had a 5T allele, 11/394 had Survival of Motor Neuron 1 deletion and 8/426 had an intermediate or premutation allele in Fragile X Messenger Ribonucleoprotein gene. While recommendations from fertility societies advocate for comprehensive screening, opinions differ on the mandatory implementation of expanded carrier screening. CONCLUSIONS:In conclusion, the genetic tests and the pre and post-test counseling is imperative to optimize reproductive outcomes in the oocyte donation process.

Autosomal recessive keratitis-ichthyosis-deafness syndrome (KIDAR MIM #242150) is a very rare disorder caused by pathogenic loss-of-function variants in the AP1B1 gene. So far, nine patients have been reported in the literature and more clinical descriptions are essential to further delineate the phenotype of KIDAR. Here we report a new patient with KIDAR and compare the clinical findings with those from the other published cases with molecular confirmation. We describe a 14-year-old male born to non-consanguineous parents with unremarkable family history. The patient had fetal ascites, neonatal pancreatic insufficiency with consequent failure to thrive, feeding difficulties, recurrent infections and sepsis. The skin examination was remarkable for an ichthyosis with conspicuous palmoplantar keratoderma, sparse and brittle hair with alopecia on the vertex and slight bilateral ectropion. He had short stature, thin build, frontal bossing, small teeth and prominent abdomen. Additional features were congenital profound bilateral sensorineural deafness, photosensitivity and photophobia. Mild global developmental delay was noted. Persistent mild anemia, neutropenia, thrombocytopenia, and low serum copper, ceruloplasmin and growth hormone were also present. Brain magnetic resonance imaging (MRI) showed cerebral atrophy and thin corpus callosum. Genetic testing revealed a homozygous deletion in the AP1B1 gene, possibly including the same exons as a previously reported deletion. Comparing the phenotypes of all reported individuals, they are highly concordant and major features are enteropathy with feeding difficulties, failure to thrive, ichthyosis, palmoplantar keratoderma, sensorineural deafness and sparse and brittle hair. Here we report other features present in more than one patient that could be part of the phenotypic spectrum and suggest copy number variation analysis to be performed alongside sequencing of the AP1B1 gene in case of suspicion.

Treatable targets that hinder heart failure development following myocardial infarction remain limited. Through an unbiased transcriptional regulation study for ischemic heart disease, we identified the protein disrupted in schizophrenia 1 (DISC1), which has been almost solely characterized in the brain. Here, we show that loss of DISC1 is a major driver of heart disease and ischemic damage. Silencing of DISC1 sensitizes human cardiomyocyte cell lines to hypoxia, whereas DISC1 overexpression is cardioprotective. Mechanistically, we provide evidence that reduced DISC1 protein levels interrupt multiple signaling processes vital for cardiomyocyte survival whereas overexpression of DISC1 enhances pro survival signalling. The present study has implications for understanding scenarios where cardiomyocytes are unable to increase the levels of DISC1 during ischemia, e.g., in individuals with DISC1 genetic defects. Indeed, patients with severe mental illness display 20 years earlier mortality, with IHD as the greatest cause of death. The present study can thus shed light on this long-term enigma in epidemiology. Furthermore, the promising data showing activation of pro survival signalling processes with DISC1 overexpression opens new directions towards potential treatment strategies of patients suffering from ischemic heart disease.

Strong evidence has suggested an important role of telomeres in meiosis, fertilization, and embryo development. To determine if sperm telomere length (STL) in sperm purified by differential gradient centrifugation followed by swim-up (selected STL) is correlated with sperm quality and clinical outcomes. Relative selected STL was assessed by quantitative polymerase chain reaction (Q-PCR) in 78 consecutive assisted reproductive technology (ART) treatments during 2017. Statistical analyses were performed in the totality of patients, and in normozoospermic and non-normozoospermic patients. These included correlations between selected STL and sperm quality parameters, embryological parameters (multivariable linear regression), and clinical parameters (multivariable logistic regression). No significant correlations were found between selected STL and sperm quality in the total population. However, selected STL was significantly correlated with total sperm count (r = 0.361; P = 0.039) and sperm DNA fragmentation-post-acrosomal region pattern (r = − 0.464; P = 0.030) in normozoospermic patients. No relation was observed between selected STL and clinical outcomes in any clinical group. As the correlations observed in normozoospermic patients were not representative of the whole heterogeneous population, differences in the sperm characteristics of the study population may lead to discrepant results when evaluating the association of STL with sperm quality. Since the total population selected STL was not related with sperm quality and with clinical outcomes, results do not support the use of selected STL measurement to evaluate the reproductive potential of the male patient or to predict the success rates of ART treatments.

Background 12q14 microdeletion syndrome is characterized by low birth weight and failure to thrive, proportionate short stature and developmental delay. The opposite syndrome (microduplication) has not yet been characterized. Our main objective is the recognition of a new clinical entity - 12q14 microduplication syndrome. - as well as confirming the role of HMGA2 gene in growth regulation. Case presentation Array Comparative Genomic Hybridization (CGH), Karyotype, Fluorescence in situ Hybridization, Quantitative-PCR analysis and Whole exome sequencing (WES) were performed in a girl presenting overgrowth and obesity. Array CGH identified a 1.5 Mb 12q14.3 microduplication involving HMGA2, GRIP1, IRAK3, MSRB3 and TMBIM4 genes. Karyotype and FISH showed that duplication was a de novo insertion of 12q14.3 region on chromosome 9p resulting in an interstitial microduplication. Q-PCR confirmed the duplication only in the proband. WES revealed no pathogenic variants. Conclusions Phenotypic comparison with patients with 12q14 microdeletion syndrome showed a reciprocal presentation, suggesting a phenotypically recognizable 12q14 microduplication syndrome as well as confirming the role of HMGA2 gene in growth regulation. It is also indicative that other genes, such as IRAK3 and MSRB3 might have of role in weight gain and obesity.

Matrix metalloproteinases (MMP) are important for cardiac remodeling. Recently, microRNA (miR)-451a has been found to inhibit the expression of both MMP-2 and MMP-9 in human malignancies, but its role in cardiomyocytes has not been explored. We hypothesized that miR-451a modulates MMP-2 and MMP-9 levels in human cardiomyocytes. The role of miR-451a on regulation of MMP-2 and MMP-9 was evaluated in two separate pathological models using Cor.4U human inducible pluripotent stem cell-derived cardiomyocytes (hiPS-CMs): 1) endothelin-1 (ET-1), and 2) 48-h hypoxia (1% O2). Both models were transfected with synthetic miR-451a mimics or scramble control. Expression of both mRNA and miR was determined by quantitative real-time polymerase chain reaction and protein activity by (MMP-2/9) activity assay. Bioinformatic analyses were performed using Targetscan 7.1 and STRING 10.5. hiPS-CMs stimulated by hypoxia increased both MMP-2 and MMP-9 expression levels compared with normoxia (P < 0.05), whereas ET-1 stimulation only increased the MMP-9 level compared with vehicle controls (P < 0.05). miR-451a mimics prevented the increase of MMP-2 and MMP-9 expression in both models. Protein activity of MMP-2 and MMP-9 was confirmed to be lower following treatment with miR-451a mimic compared with scramble-controls. Six of 28 predicted gene transcripts of miR-451a were linked to MMP-2 and MMP-9; Macrophage migration inhibitory factor (MIF) was the only predicted target of miR-451a that was increased by ET-1 and hypoxia and reduced following miR-451a mimic transfection. miR-451a prevent the increase of MMP-2 and MMP-9 in human cardiomyocytes during pathological stress. The modulation by miR-451a on MMP-2 and MMP-9 is caused by MIF.

Objectives: Hypertension is a worldwide public health problem and the Renin Angiotensin System (RAS) has been a focus of researchers in the area of hypertension and to identify the components involved in the onset of hypertension and the involvement of angiotensin converting enzyme (ACE) in this problem, RAS has been the object of many studies. ACE converts angiotensin I into angiotensin II, a potent vasoconstrictor. Studies suggest the association of ACE isoforms, especially the 90 kDa isoform, with hypertension. The objective of this study was to investigate the association between ACE isoforms and the presence of arterial hypertension in the second phase of the prospective study (MONICA Project) in the population of Vitória - ES, Brazil. Methods: The demographic, clinical and biochemical parameters of 220 individuals were evaluated. Urine samples were concentrated and ACE isoforms were identified by the Western Blotting technique. Results: The groups were classified as Group 1 (presence of isoforms with 65, 90 and 190 KDa); Group 2 (65 and 90 kDa isoforms) and Group 3 (65 and 190 kDa isoforms). The results showed a high prevalence of the 90 kDa isoform with a higher incidence of hypertension (higher in Group 2) after five years of segment. The groups containing 90 kDa isoform showed higher systolic and diastolic pressure profiles in the second phase and the frequency of normotensives expressing the isoforms with 65 and 190 kDa was higher than that the hypertensive ones. Loss of isoform expression at 190 kDa increases the chances of developing hypertension as well as family history of hypertension. Conclusion: The results suggest the 90-kDa N-domain isoform as a possible biological marker of hypertension, which confirms the results obtained in the first phase of the study.

OBJECTIVESDuring open-heart surgery, the myocardium experiences ischaemia-reperfusion injury. A single bout of moderate, 30-min exercise induces preconditioning and protects the heart from ischaemia-reperfusion injury in rats, but this has never been investigated in humans. We aimed to investigate whether 1 bout of moderate exercise 24 h prior to surgery protects against mitochondrial and cardiac damage.METHODSPatients scheduled for elective coronary artery bypass were eligible for this pilot study. Twenty were included and randomized to the treadmill exercise group (the EX group, n = 10) 24 h preoperatively or to standard presurgical procedures (control n = 10). Right atrial (RA) and left ventricular (LV) biopsies were collected immediately before and as long as possible after aortic cross-clamping to assess the primary outcome of mitochondrial respiration by respirometry, in addition to reactive oxygen species production by fluorometry and apoptotic transcripts. Cardiac troponin T and creatine kinase myocardial brain were measured in plasma at arrival, before surgery and 6 and 24 h postoperatively.RESULTSMitochondrial respiration was lower in the EX group after surgery in the LV (Complex I -22%, P < 0.05 and maximal -23%, P < 0.05) and the right atrium (Complex I -25%, P < 0.05). Transcript level of the apoptosis-related marker caspase 3 was increased 1.5-fold in the LV prior to surgery in the EX group when compared with the control group, P < 0.05. Cardiac troponin T was 45% higher in the EX group than in the control group 6 h postoperatively (P = 0.03), although not significant when corrected for aortic cross-clamping time.CONCLUSIONSResults indicate that exercise did not precondition the heart against surgery-related damage. Exercise may render the myocardium and mitochondria more vulnerable to perioperative damage.Clinical trials registration numberNCT00218985 (https://clinicaltrials.gov/ct2/show/NCT00218985).

The study aimed to describe the ultrastructure of two human mature oocyte intracytoplasmic dysmorphisms, the bull-eye inclusion and the granular vacuole, with evaluation of clinical outcomes after intracytoplasmic sperm injection (ICSI) treatment.We retrospectively evaluated 4099 consecutive ICSI cycles during the period 2003-2013. Three groups were compared: controls, those with a bulls-eye inclusion, and those with granular vacuoles. Oocyte dysmorphisms were evaluated by transmission electron microscopy and in situ fluorescence hybridization (FISH). Detailed data on demographic and stimulation characteristics, as well as on embryological, clinical, and newborn outcomes, are fully presented.The bull-eye inclusion is a prominent smooth round structure containing trapped vesicles, being surrounded by lipid droplets. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when transferred embryos were exclusively derived from dysmorphism oocytes. The granular vacuole is delimited by a discontinuous double membrane and contains lipid droplets and vesicles. As FISH analysis revealed the presence of chromosomes, they probably represent pyknotic nuclei. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when at least one transferred embryo was derived from dimorphic oocytes.Poor clinical outcomes were observed with transfer of embryos derived from dysmorphism oocytes, although without causing gestation or newborn problems. The bull-eye inclusion and granular vacuoles may thus be new prognostic factors for clinical outcomes.

Extracellular vesicles (EVs) are major conveyors of biological information, mediating local and systemic cell-to-cell communication under physiological and pathological conditions. These endogenous vesicles have been recognized as prominent drug delivery vehicles of several therapeutic cargoes, including doxorubicin (dox), presenting major advantages over the classical approaches. Although dox is one of the most effective anti-tumour agents in the clinical practice, its use is very often hindered by its consequent dramatic cardiotoxicity. Despite significant advances witnessed in the past few years, more comprehensive studies, supporting the therapeutic efficacy of EVs, with decreased side effects, are still scarce. The main objective of this study was to evaluate the role of the gap junction protein connexin43 (Cx43) in mediating the release of EV content into tumour cells. Moreover, we investigated whether Cx43 improves the efficiency of dox-based anti-tumour treatment, with a concomitant decrease of cardiotoxicity. In the present report, we demonstrate that the presence of Cx43 in EVs increases the release of luciferin from EVs into tumour cells in vitro and in vivo. In addition, using cell-based approaches and a subcutaneous mouse tumour model, we show that the anti-tumour effect of dox incorporated into EVs is similar to the administration of the free drug, regardless the presence of Cx43. Strikingly, we demonstrate that the presence of Cx43 in dox-loaded EVs reduces the cardiotoxicity of the drug. Altogether, these results bring new insights into the concrete potential of EVs as therapeutic vehicles and open new avenues toward the development of strategies that help to reduce unwanted side effects.

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication.

Catecholamines (CA) play an important role in cardiovascular (CDV) disease risk. Namely, noradrenaline (NA) levels positively correlate whereas adrenaline (AD) levels negatively correlate with obesity and/or CDV disease. Western diets, which are tipically rich in Ω-6 fatty acids (FAs) and deficient in Ω-3 FAs, may contribute to the development of obesity, type 2 diabetes and/or coronary artery disease. Taking this into consideration and the fact that our group has already described that saturated FAs affect catecholamine handling by adrenal chromaffin cells, this work aimed to investigate the effect of unsaturated FAs upon catecholamine handling in the same model. Our results showed that chronic exposure to unsaturated FAs differently modulated CA cellular content and release, regardless of both FA series and number of carbon atoms. Namely, the Ω-6 arachidonic and linoleic acids, based on their effect on CA release and cellular content, seemed to impair NA and AD vesicular transport, whereas γ-linolenic acid selectively impaired AD synthesis and release. Within the Ω-9 FAs, oleic acid was devoid of effect, and elaidic acid behaved similarly to γ-linolenic acid. Eicosapentaenoic and docosahexaenoic acids (Ω-3 series) impaired the synthesis and release of both NA and AD. These results deserve attention and future development, namely, in what concerns the mechanisms involved and correlative effects in vivo.

Janete Quelhas-Santos, Maria Paula Serrao, Isabel Soares-Silva, Catia Fernandes-Cerqueira, Liliana Simoes-Silva, Maria Joao Pinho, Fernando Remiao, Benedita Sampaio-Maia, Gary V. Desir, and Manuel Pestana Nephrology Research and Development Unit, Faculty of Medicine, University of Porto, Porto, Portugal; Nephrology and Infectious Diseases Research and Development Group, Instituto Nacional de Engenharia Biomedica-(I3S); Faculdade de Medicina da Universidade do Porto, Department of Pharmacology and Therapeutics, Porto, Portugal; Centro de Quimica da Universidade do Porto/Servico de Toxicologia, Faculdade de Farmacia, University of Porto, Porto, Portugal; Faculty of Dental Medicine, University of Porto, Porto, Portugal; Department of Medicine, Veterans Affairs Connecticut Healthcree System, Yale University, New Haven, Connecticut; Faculdade de Medicina da Universidade do Porto, Department of Renal, Urological, and Infectious Diseases, Porto, Portugal; and Department of Nephrology, Sao Joao Hospital Center, Entidade Publica Empresarial, Porto, Portugal

Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency.

Essential hypertension is associated with alterations of sodium homeostasis, which promote abnormal accumulation of water in the intravascular compartment leading to blood pressure elevations. Salt‐inducible kinase 1 (SIK1) belongs to the AMPK family and a single nucleotide polymorphism within the human SIK1 gene is associated with hypertension [lower SIK1 activity is associated with higher blood pressure (HBP)]. SIK1 network mediates sodium‐dependent signals that modulate the activity of the Na+,K+‐ATPase. We aimed to study the role of SIK1 in cardiovascular and renal homeostasis using SIK1 knockout (KO) mice. No differences in BP were observed between sik1‐/‐ and sik1+/+ mice under 0.3% NaCl diet; however, the sik1‐/‐ mice challenged with an acute or chronic 8% NaCl diet showed increased SBP. We found that under an 8% NaCl diet, Na+, K+ and Cl‐ excretion was significantly lower in sik1‐/‐ mice compared with wild type mice, despite similar plasma aldosterone levels. The impaired ability of sik1‐/‐ to eliminate Na+ under 8% NaCl diet was accompanied by hyperkalemia. Signs of fibrosis were observed in sik1‐/‐ mice kidneys (increased glomerular and interstitial collagen deposition) vs. wild type mice. At the molecular level, this phenotype was accompanied by an increased in collagen α1 and α4 and MMP9 mRNA, and a reduction in E‐cadherin and nephrin mRNA levels. These results indicate that the presence of SIK1 is necessary to prevent the development of hypertension.Grant Funding Source: Supported by FCT (PIC/IC/83204/2007).

MicroRNAs (miRNAs) are regulators of gene expression and play a key role in the pathophysiology of various disease processes, namely cardiovascular disease. This study aimed at the identification of the miRNA expression pattern of peripheral blood mononuclear cells derived from hypertensive subjects (HT) and non-hypertensive subjects with cardiac disease (CD) using a genome-wide approach. Furthermore, we explored the potential of orthogonal partial least squares-discrimination analysis (OPLS-DA) to analyze miRNA expression data. The homogeneity of the population was assessed with Principal Component Analysis (PCA) and verified by the clear separation of the three groups on the basis of the miRNA expression levels. Moreover, local PCA of HT group indicates the splitting of this group in two distinct subgroups. This distinction correlates with two subject’s characteristics (smoking habits and history of myocardial infarction) that are closest to the CD group. OPLS-DA analysis confirms the separation of three groups and identified the most important miRNA to discrimination between groups. Among the most influential miRNA to the positive differentiation of HT from CD and control, several of them are relevant to endothelial dysfunction. On the other hand, miRNAs associated with angiogenesis positively differentiate CD from HT and control. Mir-186, a regulator of myogenin, is the most important miRNA to discriminate HT from CD. These results provide evidence of altered miRNA profile in hypertensives and cardiac disease and demonstrate that PCA and OPLS-DA are very useful and robust tools to analyze how different miRNAs contribute to the separations of groups and to find interesting patterns within the multidimensional data.