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Dermatitis , Neoplasias Cutáneas , Humanos , Microscopía Confocal , Piel , Coloración y EtiquetadoRESUMEN
The treatment of choice for malignant eyelid tumors is surgical excision. If this is not feasible or undesirable, a number of alternative treatments are available. Possible systemic preparations are vismodegib and sonidegib for basal cell carcinoma as well as cetuximab and cemiplimab for squamous cell carcinoma. Cryodestruction is possible for superficial tumors. In situ findings can be treated with the local preparations imiquimod or 5fluorouracil and with photodynamic therapy. An interdisciplinary cooperation with dermatologists is advisable.
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Carcinoma Basocelular , Carcinoma de Células Escamosas , Neoplasias de los Párpados , Fotoquimioterapia , Neoplasias Cutáneas , Neoplasias de los Párpados/terapia , Humanos , Neoplasias Cutáneas/terapiaRESUMEN
BACKGROUND: Ex vivo confocal laser scanning microscopy (CLSM) is a novel diagnostic tool for the fast examination of native tissue. However, CLSM produces black/white/green images, depending on the refraction indices of the tissue structures, complemented by nuclear fluorescence staining, which the vast majority of Mohs surgeons and dermatopathologists are not trained to interpret. Digital staining is applicable to ex vivo CLSM investigations to simulate the images of conventional slides stained with haematoxylin and eosin (H&E). OBJECTIVES: The aim of our study was to evaluate in detail the appearance of human skin structures using digitally stained ex vivo CLSM images and compare the results to that of conventional H&E slides of the same specimen. METHODS: After providing informed consent, 26 patients donated their Burow's triangles (healthy skin) that resulted from plastic reconstruction after the R0 excision of skin tumours. After being investigated by ex vivo CLSM, including automated digital staining (VivaScope 2500M-4G, MAVIG GmbH), the specimens were fixed in formalin, embedded in paraffin and stained with H&E. RESULTS: Almost all skin structures in the digitally stained ex vivo CLSM images morphologically resembled the structures in the histopathological images acquired from H&E slides. Due to the high refraction index of melanin, the hair shafts appeared bright pink, and the melanocytes and melanophages were poorly imaged, resulting in a strong pink appearance that vastly differed from the appearance of conventional H&E-stained histopathology. CONCLUSIONS: Digital staining of ex vivo CLSM images is an easy and highly useful tool to facilitate the interpretation of black-field images generated by confocal laser scanning microscopy for dermatopathologists and Mohs surgeons who are familiar with H&E staining. Unlike the pigmented structures, the cutaneous and subcutaneous structures had excellent visualization with only minimal differences from their appearance on H&E slides.
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Neoplasias Cutáneas , Humanos , Melanocitos , Microscopía Confocal , Piel/diagnóstico por imagen , Neoplasias Cutáneas/diagnóstico por imagen , Coloración y EtiquetadoRESUMEN
BACKGROUND: Classic galactosemia is a rare inborn error of carbohydrate metabolism, caused by a severe deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). A galactose-restricted diet has proven to be very effective to treat the neonatal life-threatening manifestations and has been the cornerstone of treatment for this severe disease. However, burdensome complications occur despite a lifelong diet. For rare diseases, a patient disease specific registry is fundamental to monitor the lifespan pathology and to evaluate the safety and efficacy of potential therapies. In 2014, the international Galactosemias Network (GalNet) developed a web-based patient registry for this disease, the GalNet Registry. The aim was to delineate the natural history of classic galactosemia based on a large dataset of patients. METHODS: Observational data derived from 15 countries and 32 centers including 509 patients were acquired between December 2014 and July 2018. RESULTS: Most affected patients experienced neonatal manifestations (79.8%) and despite following a diet developed brain impairments (85.0%), primary ovarian insufficiency (79.7%) and a diminished bone mineral density (26.5%). Newborn screening, age at onset of dietary treatment, strictness of the galactose-restricted diet, p.Gln188Arg mutation and GALT enzyme activity influenced the clinical picture. Detection by newborn screening and commencement of diet in the first week of life were associated with a more favorable outcome. A homozygous p.Gln188Arg mutation, GALT enzyme activity of ≤ 1% and strict galactose restriction were associated with a less favorable outcome. CONCLUSION: This study describes the natural history of classic galactosemia based on the hitherto largest data set.
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Galactosemias/patología , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , Adolescente , Adulto , Estudios de Cohortes , Femenino , Galactosemias/genética , Homocigoto , Humanos , Recién Nacido , Masculino , Mutación/genética , Tamizaje Neonatal , Sistema de Registros , Estudios Retrospectivos , Adulto JovenRESUMEN
In dermato-oncological treatment there are many gonadotoxic interventions. Alkylating and hormonally active substances as well as gonadal irradiation, in particular, are known to have a fertilization-limiting effect in men. Since 2017 certified skin cancer centers in Germany therefore have the task to implement counselling on preservation of fertility. This is supported by the S2k guidelines on preservation of fertility in oncological treatment. Because recommendation of the various interventions from the dermato-oncological guidelines are dependent on the stage, the authors advocate at least the question "Is the desire to have children of interest to you?" when patients reach the appropriate stage. Fertility protection of men via cryopreservation of ejaculates or testicular tissue is then a simple and safe option. The procedure is standardized and usually available. In addition, the possibility of cryopreservation of testicular tissue from prepubertal male children and infants is now available via the new Androprotect project. If signs of hypogonadism occur during therapy, a treatment can be considered by weighing up the effects of testosterone but in this case it is important to take the anabolic and immunomodulating effects into account.
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Andrología , Neoplasias , Preservación de Semen , Criopreservación , Preservación de la Fertilidad , Alemania , Humanos , Masculino , Oncología Médica , TestículoRESUMEN
To study apoptosis as a functional pathway in mature spermatozoa and apoptosis correlated to the acrosome reaction via the intracellular calcium concentration, semen samples from 27 healthy human donors were treated with inducers of apoptosis (betulinic acid, thapsigargin), inducers of the acrosome reaction (thapsigargin, calcium ionophore) or hydrogen peroxide to produce reactive oxygen species with and without prior incubation with a calcium chelator. Computer-assisted sperm analysis, flow cytometry, and transmission electron microscopy were performed to analyze changes in the acrosomal status and in apoptotic features. Betulinic acid, thapsigargin, and the calcium ionophore treatment resulted in an increased number of sperm cells with caspase 9 and caspase 3 activation, disrupted mitochondrial membrane potential, and a reacted acrosome. Sperm motility was decreased in all cases. Transmission electron analyses showed ultra-morphological changes, such as membrane integrity, membrane blebbing, the formation of head vacuoles, defects of the nuclear envelope, nuclear fragmentation, and the acrosome reaction. Acrosome reaction and apoptotic features decreased due to the reduction in intracellular calcium by the calcium chelator NP-EGTA, AM. Therefore, apoptotic cell death in acrosome-reacted sperm cells mediated by high intracellular calcium levels is possible.
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Apoptosis , Espermatozoides/fisiología , Reacción Acrosómica/fisiología , Apoptosis/efectos de los fármacos , Calcio , Citometría de Flujo , Humanos , Masculino , Potencial de la Membrana Mitocondrial , Estrés Oxidativo , Triterpenos Pentacíclicos , Motilidad Espermática , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructura , Tapsigargina/farmacología , Triterpenos/farmacología , Ácido BetulínicoRESUMEN
BACKGROUND: Long-chain fatty acid oxidation disorders (LC-FAOD) lead to accumulation of high concentrations of potentially toxic fatty acid intermediates. Newborn screening and early intervention have reduced mortality, but most patients continue to experience frequent hospitalizations and significant morbidity despite treatment. The deficient energy state can cause serious liver, muscle, and heart disease, and may be associated with an increased risk of sudden death. Triheptanoin is a medium odd-chain fatty acid. Anaplerotic metabolites of triheptanoin have the potential to replace deficient tricarboxylic acid (TCA) cycle intermediates, resulting in net glucose production as a novel energy source for the treatment of LC-FAOD. STUDY DESIGN: A single-arm, open-label, multicenter Phase 2 safety and efficacy study evaluated patients with severe LC-FAOD evidenced by ongoing related musculoskeletal, cardiac, and/or hepatic events despite treatment. After a four-week run-in on current regimen, investigational triheptanoin (UX007) was titrated to a target dose of 25-35% of total daily caloric intake. Patients were evaluated on several age/condition-eligible endpoints, including submaximal exercise tests to assess muscle function/endurance (12-minute walk test; 12MWT) and exercise tolerance (cycle ergometry), and health related quality of life (HR-QoL). Results through 24weeks of treatment are presented; total study duration is 78weeks. RESULTS: Twenty-nine patients (0.8 to 58years) were enrolled; most qualified based on severe musculoskeletal disease. Twenty-five patients (86%) completed the 24-week treatment period. At Week 18, eligible patients (n=8) demonstrated a 28% increase (LS mean=+181.9 meters; p=0.087) from baseline (673.4meters) in 12MWT distance. At Week 24, eligible patients (n=7) showed a 60% increase in watts generated (LS mean=+409.3W; p=0.149) over baseline (744.6W) for the exercise tolerance test. Improvements in exercise tests were supported by significant improvements from baseline in the adult (n=5) self-reported SF-12v2 physical component summary score (LS mean=+8.9; p<0.001). No difference from baseline was seen in pediatric parent-reported (n=5) scores (SF-10) at Week 24. Eighteen patients (62%) had treatment-related adverse events, predominantly gastrointestinal (55%), mild-to-moderate in severity, similar to that seen with prior treatment with medium chain triglyceride (MCT) oil. One patient experienced a treatment-related serious adverse event of gastroenteritis. One patient discontinued from study due to diarrhea of moderate severity; the majority of patients (25/29; 86%) elected to continue treatment in the extension period. CONCLUSIONS: In patients with severe LC-FAOD, UX007 interim study results demonstrated improved exercise endurance and tolerance, and were associated with positive changes in self-reported HR-QoL.
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Ácidos Grasos/toxicidad , Errores Innatos del Metabolismo Lipídico/tratamiento farmacológico , Resistencia Física/efectos de los fármacos , Triglicéridos/administración & dosificación , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Errores Innatos del Metabolismo Lipídico/metabolismo , Errores Innatos del Metabolismo Lipídico/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Calidad de Vida , Resultado del Tratamiento , Triglicéridos/farmacología , Prueba de Paso , Adulto JovenRESUMEN
During pregnancy, a successful and safe therapeutic management of patients is possible to lower the burden of disease. Often topical therapy in combination with intensive basic skin care is sufficient. Drug therapies may also be used, most often for systemic diseases such as autoimmune diseases or psoriasis. An early change in therapy is also key during planned pregnancies so that treatments can be switched, adjusted, reduced or closely monitored. Another point to consider is to keep drug dosing as low as possible (without occlusion in local therapy) or short termed (with the exception of autoimmune or malignant diseases). An interdisciplinary collaboration between obstetrics and gynecology/rheumatology/internal medicine/dermatology as well as pharmacologists is of utmost importance.
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Antibacterianos/administración & dosificación , Antiinflamatorios/administración & dosificación , Antineoplásicos/administración & dosificación , Fármacos Dermatológicos/administración & dosificación , Complicaciones del Embarazo/tratamiento farmacológico , Enfermedades de la Piel/tratamiento farmacológico , Administración Cutánea , Administración Tópica , Monitoreo de Drogas/métodos , Medicina Basada en la Evidencia , Femenino , Humanos , Embarazo , Complicaciones del Embarazo/diagnóstico , Enfermedades de la Piel/diagnóstico , Resultado del TratamientoRESUMEN
High amounts of nicotinamide phosphoribosyltransferase (NAMPT) were found in human seminal plasma. This enzyme influences energy metabolism and apoptosis and is essential for the regulation of cellular nicotinamide adenine dinucleotide (NAD)+ levels in somatic cells. NAD+ is required as a co-substrate for dehydrogenases, which are potentially important for spermatogenesis. The functional significance of intra- and extracellular NAMPT in human reproduction, however, has not been defined yet. The objectives of the study were therefore to determine NAMPT protein expression in human spermatozoa and testes, the secretion of NAMPT by spermatozoa depending on their maturation stage and the impact of NAMPT enzymatic function on sperm viability, motility, fertilisation capacity and induction of apoptosis. Firstly, we detected NAMPT protein in different cell types of human testes. NAMPT protein was also detected in spermatozoa, with significantly higher amounts in immature than in mature ejaculated spermatozoa. Additionally, NAD+ levels were significantly higher in immature than in mature spermatozoa. Secondly, NAMPT was released into the supernatant of human spermatozoa, with significantly higher NAMPT levels in supernatant of immature spermatozoa compared with mature cells. Finally, the specific inhibition of the enzyme by FK866 did not influence motility, capacitation or apoptosis signalling. In summary, NAMPT is produced in human spermatozoa in a maturation-dependent manner.
Asunto(s)
Nicotinamida Fosforribosiltransferasa/metabolismo , Maduración del Esperma/fisiología , Espermatozoides/metabolismo , Acrilamidas/farmacología , Inhibidores Enzimáticos/farmacología , Fertilidad/efectos de los fármacos , Fertilidad/fisiología , Humanos , Masculino , Piperidinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Capacitación Espermática/efectos de los fármacos , Capacitación Espermática/fisiología , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
BACKGROUND: Enzyme replacement therapy (ERT) for infantile-onset Pompe disease has been commercially available for almost 10 years. We report the experience of its use in a cohort treated at three specialist lysosomal treatment centres in the UK. METHODS: A retrospective case-note review was performed, with additional data being gathered from two national audits on all such patients treated with ERT. The impact on the outcome of various characteristics, measured just prior to the initiation of ERT (baseline), was evaluated using logistic regression. RESULTS: Thirty-three patients were identified; 13/29 (45%) were cross-reactive immunological material (CRIM) negative, and nine were immunomodulated. At baseline assessment, 79% were in heart failure, 66% had failure to thrive and 70% had radiological signs of focal pulmonary collapse. The overall survival rate was 60%, ventilation-free survival was 40% and 30% of patients were ambulatory. Median follow-up of survivors was 4 years, 1.5 months (range 6 months to 13.5 years). As with previous studies, the CRIM status impacted on all outcome measures. However, in this cohort, baseline failure to thrive was related to death and lack of ambulation, and left ventricular dilatation was a risk factor for non-ventilator-free survival. CONCLUSION: The outcome of treated patients remains heterogeneous despite attempts at immunomodulation. Failure to thrive at baseline and left ventricular dilation appear to be associated with poorer outcomes.
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Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/mortalidad , Cardiomiopatías/metabolismo , Cardiomiopatías/mortalidad , Reacciones Cruzadas , Terapia de Reemplazo Enzimático/métodos , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Humanos , Lactante , Lisosomas/metabolismo , Masculino , Estudios Retrospectivos , Tasa de Supervivencia , Reino Unido , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/mortalidadRESUMEN
Dermatologists administer a broad spectrum of systemic medications. However, our current knowledge of potential risks to male fertility is still limited, particularly with the new emerging therapies in dermato-oncology. Individual differences in susceptibility and a history of andrological disorders influence prognostic values. For fertility protection, a thoughtful selection of medication and/or sperm cryopreservation remain the best options.
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Fármacos Dermatológicos/efectos adversos , Fármacos Dermatológicos/uso terapéutico , Infertilidad Masculina/inducido químicamente , Infertilidad Masculina/prevención & control , Preservación de Semen/métodos , Enfermedades de la Piel/tratamiento farmacológico , Humanos , Infertilidad Masculina/diagnóstico , Masculino , Enfermedades de la Piel/complicaciones , Resultado del TratamientoRESUMEN
Dilated cardiomyopathy is a rare complication in propionic acidaemia (PA). Underlying pathophysiological mechanisms are poorly understood. We present a child of Pakistani consanguineous parents, diagnosed with late-onset PA at 18months of age. He presented a mild phenotype, showed no severe further decompensations, normal growth and psychomotor development on a low protein diet and carnitine supplementation. At 15years, a mildly dilated left ventricle was noticed. At 17years he presented after a 2-3month history of lethargy and weight loss with severe decompensated dilated cardiomyopathy. He was stabilised on inotropic support and continuous haemofiltration; a Berlin Heart biventricular assist device was implanted. He received d,l-hydroxybutyrate 200mg/kg/day, riboflavin and thiamine 200mg/day each and coenzyme Q10 (CoQ10). Myocardial biopsy showed endocardial fibrosis, enlarged mitochondria, with atypical cristae and slightly low respiratory chain (RC) complex IV activity relative to citrate synthase (0.012, reference range 0.014-0.034). Myocardial CoQ10 was markedly decreased (224pmol/mg, reference range 942-2738), with a marginally decreased white blood cell level (34pmol/mg reference range 37-133). The dose of CoQ10 was increased from 1.5 to 25mg/kg/day. Cardiomyopathy slowly improved allowing removal of the external mechanical cardiac support after 67days. We demonstrate for the first time low myocardial CoQ10 in cardiomyopathy in PA, highlighting secondary mitochondrial impairment as a relevant causative mechanism. According to these findings, a high-dose CoQ10 supplementation could be a potential adjuvant therapeutic to be considered in PA-related cardiomyopathy.
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Cardiomiopatías/complicaciones , Mitocondrias/química , Miocardio/patología , Acidemia Propiónica/tratamiento farmacológico , Acidemia Propiónica/fisiopatología , Ubiquinona/análogos & derivados , Vitaminas/uso terapéutico , Adolescente , Biopsia , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/patología , Humanos , Lactante , Masculino , Resultado del Tratamiento , Ubiquinona/análisis , Ubiquinona/uso terapéuticoRESUMEN
BACKGROUND: Panobinostat, a pan-deacetylase inhibitor, overcomes imatinib resistance in preclinical models of gastrointestinal stromal tumours (GIST). Here we determined the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT) of panobinostat in combination with imatinib (IM) for treatment of patients with refractory GIST. METHODS: Following a 7-day run-in phase of IM (400 mg per day), escalating doses of panobinostat were added following a '3 plus 3' design. Twelve heavily pretreated GIST patients were enrolled in two dose levels. RESULTS: Most common adverse events were thrombocytopenia, anaemia, fatigue, creatinine elevation, nausea, emesis and diarrhoea. Twenty micrograms of panobinostat and 400 mg IM were declared the MTD. Pharmacologically active concentrations of panobinostat and IM were achieved as evidenced by histone H3 acetylation in blood mononuclear cells in vivo and inhibition of the IM-resistant KIT (D816) mutation in vitro. In FDG-PET-CT scans after IM run-in and following 3 weeks panobinostat treatment, 1 out of 11 evaluable patients showed a metabolic partial response, 7 patients were metabolically stable and 3 patients progressed. Longest treatment duration was 17 weeks (median 6). CONCLUSION: Panobinostat and IM can be administered at doses achieving target inhibition in vivo. Further clinical exploration of patients with treatment-refractory GIST is warranted. Correlative studies in this trial may help to optimise dosing schedules in GIST.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Gastrointestinales/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Benzamidas/administración & dosificación , Benzamidas/efectos adversos , Femenino , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/efectos adversos , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/efectos adversos , Mesilato de Imatinib , Indoles/administración & dosificación , Indoles/efectos adversos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Panobinostat , Piperazinas/administración & dosificación , Piperazinas/efectos adversos , Pirimidinas/administración & dosificación , Pirimidinas/efectos adversosRESUMEN
BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype--genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.
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Encefalopatías Metabólicas Innatas/genética , Creatina/deficiencia , Creatina/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/deficiencia , Adulto , Niño , Creatina/genética , Genes Ligados a X , Pruebas Genéticas , Genotipo , Humanos , Masculino , Fenotipo , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , Estudios RetrospectivosRESUMEN
Inhibin B is an important serum marker of spermatogenesis, whereas sensitivity and predicting power for the spermatogenic situation at several ages are under debate. We performed a retrospective analysis of data from 2448 men who attended our University-based male infertility clinic to evaluate inhibin B in relation to age and semen sample qualities in comparison with FSH. Moreover, the range of inhibin B in 82 nonobstructive azoospermic patients was correlated with the sperm retrieval in testicular sperm extraction procedures. Inhibin B correlated with FSH (Spearman rank correlation (R)=-0.50; P<0.00001). Inhibin B and inhibin B/FSH ratio (IFR) showed an inverse U-shaped dependence on age, whereas FSH showed a U-shaped dependence on age (optimum 20-40 years). However, in men with normal spermiograms inhibin B concentrations did not differ between age groups. Their levels of inhibin B amounted to 130.5, 54.5-247âng/l (median, 10th-90th precentile), and of IFR to 38.3, 12.5-104.8 (median, 10th-90th percentile), which might be taken as the reference range. Using the 10th percentile of IFR, correct classification in normal or pathological semen groups was achieved in 99.1%. The percentage of aniline blue-negative spermatozoa, i.e. mature spermatozoa with protamines, did not correlate with FSH (P>0.05) but with inhibin B (R=0.15, P<0.001). The probability of retrieving testicular spermatozoa decreased with declining inhibin B: <20âng/l sperm could never be found. Our results from a large group of men with a wide spectrum of semen qualities allow estimating reference values for inhibin B and IFR. Inhibin B and especially the IFR are more sensitive markers of male infertility than FSH alone.
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Envejecimiento/sangre , Hormona Folículo Estimulante Humana/sangre , Infertilidad Masculina/diagnóstico , Inhibinas/sangre , Análisis de Semen , Adolescente , Adulto , Factores de Edad , Anciano , Envejecimiento/patología , Biomarcadores/sangre , Biopsia , Humanos , Infertilidad Masculina/sangre , Infertilidad Masculina/patología , Modelos Lineales , Masculino , Persona de Mediana Edad , Análisis Multivariante , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Análisis de Semen/métodos , Recuento de Espermatozoides , Motilidad Espermática , Recuperación de la Esperma , Adulto JovenRESUMEN
The inclusion of apoptotic spermatozoa during assisted reproductive techniques (ART) may be one reason for suboptimal success rates. The aim of our study was to evaluate the potential of routine semen preparation to eliminate spermatozoa with activated apoptosis signalling. Semen samples from 20 infertility patients scheduled for ART procedures were investigated. Following density gradient centrifugation (DGC) and swim-up, aliquots were taken from each sample to analyse motility, Caspase-3 activation (CP3) and integrity of the mitochondrial membrane potential (MMP) using flow cytometry. Aliquots from the neat semen served as controls. Semen samples of patients contained 53.8 +/- 17.7% spermatozoa with disrupted MMP and 51.8 +/- 14.9% with active CP3. Preparation by DGC and swim-up resulted in improvement of progressive motility (+43.5%) and reduction of spermatozoa with disrupted MMP (-34.3%) and activated CP3 (-25.7%, P < 0.01). Minimal reduction of spermatozoa with disrupted MMP and active CP3 was 6.0% and 0.7%, maximum reduction was 65.5% (disrupted MMP) and 49.3% (CP3). Semen samples of subfertile patients contain high levels of spermatozoa with activated apoptosis signalling. Although there was a reduction in the majority of the samples, profound interindividual differences in the separation effect demand further development of innovative molecular-based separation methods to deplete apoptotic spermatozoa.
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Apoptosis , Infertilidad Masculina/terapia , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Caspasa 3/metabolismo , Separación Celular/métodos , Centrifugación por Gradiente de Densidad , Femenino , Citometría de Flujo , Humanos , Masculino , Potencial de la Membrana Mitocondrial , Embarazo , Resultado del Embarazo , Técnicas Reproductivas AsistidasRESUMEN
Nitric oxide (NO) is known to be involved in multiple signal transduction pathways of male germ cells, including sperm capacitation. In somatic cells, NO production was found to be part of apoptosis signalling. The aim of our study was to further clarify the role of NO in spermatozoa by investigation of NO synthase activity with regard to sperm maturity and sperm apoptosis signalling. Semen specimens from 19 healthy donors were subjected to density gradient centrifugation to separate the predominantly mature and immature sperm fraction. NO synthase activity was evaluated using diaminofluoresceine-2-diacetate by FACS. Apoptosis signalling was monitored by flowcytometric analyses of caspase-3 (CP3) and integrity of the transmembrane mitochondrial potential (TMP). TUNEL assay was used to detect DNA fragmentations. Maturity of human spermatozoa was associated with increased NO synthase activity and inactivated apoptosis signalling (lower levels of disrupted TMP, active CP3 and DNA fragmentations, P < 0.05). Activation of apoptosis signalling was significantly negatively correlated to NO production, indicating a rather anti-apoptotic effect of NO. This might underline the recently proposed role of NO in physiological sperm signal transduction, e.g. during capacitation.