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For decades, clinicians have developed medications and therapies to alleviate the symptoms of Parkinson's disease, but no treatment currently can slow or even stop the progression of this localized neurodegeneration. Fortunately, sparked by the genetic revolution, stem cell reprogramming research and the advancing capabilities of personalization in medicine enable forward-thinking to unprecedented patient-specific modeling and cell therapies for Parkinson's disease using induced pluripotent stem cells (iPSCs). In addition to modeling Parkinson's disease more accurately than chemically-induced animal models, patient-specific stem cell lines can be created, elucidating the effects of genetic susceptibility and sub-populations' differing responses to in vitro treatments. Sourcing cell therapy with iPSC lines provides ethical advantages because these stem cell lines do not require the sacrifice of human zygotes and genetically-specific drug trails can be tested in vitro without lasting damage to patients. In hopes of finally slowing the progression of Parkinson's disease or re-establishing function, iPSC lines can ultimately be corrected with gene therapy and used as cell sources for neural transplantation for Parkinson's disease. With relatively localized neural degeneration, similar to spinal column injury, Parkinson's disease presents a better candidacy for cell therapy when compared to other diffuse degeneration found in Alzheimer's or Huntington's Disease. Neurosurgical implantation of pluripotent cells poses the risk of an innate immune response and tumorigenesis. Precautions, therefore, must be taken to ensure cell line quality before transplantation. While cell quality can be quantified using a number of assays, a yielding a high percentage of therapeutically relevant dopaminergic neurons, minimal de novo genetic mutations, and standard chromosomal structure is of the utmost importance. Current techniques focus on iPSCs because they can be matched with donors using human leukocyte antigens, thereby reducing the severity and risk of immune rejection. In August of 2018, researchers in Kyoto, Japan embarked on the first human clinical trial using iPSC cell therapy transplantation for patients with moderate Parkinson's disease. Transplantation of many cell sources has already proven to reduce Parkinson's disease symptoms in mouse and primate models. Here we discuss the history and implications for cell therapy for Parkinson's disease, as well as the necessary safety standards needed for using iPSC transplantation to slow or halt the progression of Parkinson's disease.
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Generation of midbrain dopaminergic (mDA) neurons from human pluripotent stem cells provides a platform for inquiry into basic and translational studies of Parkinson's disease (PD). However, heterogeneity in differentiation in vitro makes it difficult to identify mDA neurons in culture or in vivo following transplantation. Here, we report the generation of a human embryonic stem cell (hESC) line with a tyrosine hydroxylase (TH)-RFP (red fluorescent protein) reporter. We validated that RFP faithfully mimicked TH expression during differentiation. Use of this TH-RFP reporter cell line enabled purification of mDA-like neurons from heterogeneous cultures with subsequent characterization of neuron transcriptional and epigenetic programs (global binding profiles of H3K27ac, H3K4me1, and 5-hydroxymethylcytosine [5hmC]) at four different stages of development. We anticipate that the tools and data described here will contribute to the development of mDA neurons for applications in disease modeling and/or drug screening and cell replacement therapies for PD.
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Separación Celular/métodos , Neuronas Dopaminérgicas/metabolismo , Técnicas de Sustitución del Gen , Genes Reporteros , Mesencéfalo/citología , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Secuencia de Bases , Diferenciación Celular , Línea Celular , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Análisis de Secuencia de ARN , Transcripción Genética , Tirosina 3-Monooxigenasa/metabolismo , Proteína Fluorescente RojaRESUMEN
: Data suggest that myoblasts from various sources, including bone marrow, skeletal muscle, and adipose tissue, can restore muscle function in patients with urinary incontinence. Animal data have indicated that these progenitor cells exert mostly a paracrine effect on the native tissues rather than cell regeneration. Limited knowledge is available on the in vivo effect of human stem cells or muscle progenitors on injured muscles. We examined in vivo integration of smooth muscle progenitor cells (pSMCs) derived from human pluripotent stem cells (hPSCs). pSMCs were derived from a human embryonic stem cell line (H9-ESCs) and two induced pluripotent stem cell (iPSC) lines. pSMCs were injected periurethrally into urethral injury rat models (2 × 106 cells per rat) or intramuscularly into severe combined immunodeficiency mice. Histologic and quantitative image analysis revealed that the urethras in pSMC-treated rats contained abundant elastic fibers and thicker muscle layers compared with the control rats. Western blot confirmed increased elastin/collagen III content in the urethra and bladder of the H9-pSMC-treated rats compared with controls. iPSC-pSMC treatment also showed similar trends in elastin and collagen III. Human elastin gene expression was not detectable in rodent tissues, suggesting that the extracellular matrix synthesis resulted from the native rodent tissues rather than from the implanted human cells. Immunofluorescence staining and in vivo bioluminescence imaging confirmed long-term engraftment of pSMCs into the host urethra and the persistence of the smooth muscle phenotype. Taken together, the data suggest that hPSC-derived pSMCs facilitate restoration of urethral sphincter function by direct smooth muscle cell regeneration and by inducing native tissue elastin/collagen III remodeling. SIGNIFICANCE: The present study provides evidence that a pure population of human smooth muscle progenitor cells (pSMCs) derived from human pluripotent stem cells (hPSCs) (human embryonic stem cells and patient induced pluripotent stem cells) restores urethral sphincter function by two mechanisms: modulation of extracellular matrix protein metabolism in vivo and pSMC proliferation and differentiation into smooth muscle cells to regenerate the muscle layer in the lower urinary tract. These findings on the in vivo effects of human pSMCs should aid in optimizing regenerative therapies using human myoblasts.
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Miocitos del Músculo Liso/citología , Células Madre Pluripotentes/citología , Células Madre/citología , Uretra/lesiones , Uretra/patología , Animales , Western Blotting , Supervivencia Celular , Rastreo Celular , Colágeno Tipo III/metabolismo , Elastina/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Mediciones Luminiscentes , Ratones SCID , Miocitos del Músculo Liso/trasplante , Ratas Desnudas , Incontinencia Urinaria de Esfuerzo/terapiaRESUMEN
There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 10(6) cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function.
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Mioblastos/trasplante , Miocitos del Músculo Liso/citología , Células Madre Pluripotentes/citología , Trasplante de Células Madre , Incontinencia Urinaria de Esfuerzo/terapia , Animales , Células Cultivadas , Femenino , Humanos , Mioblastos/citología , Células Madre Pluripotentes/trasplante , RatasRESUMEN
Despite the potential of stem cell-derived neural transplants for treating intractable neurological diseases, the global effects of a transplant's electrical activity on host circuitry have never been measured directly, preventing the systematic optimization of such therapies. Here, we overcome this problem by combining optogenetics, stem cell biology, and neuroimaging to directly map stem cell-driven neural circuit formation in vivo. We engineered human induced pluripotent stem cells (iPSCs) to express channelrhodopsin-2 and transplanted resulting neurons to striatum of rats. To non-invasively visualize the function of newly formed circuits, we performed high-field functional magnetic resonance imaging (fMRI) during selective stimulation of transplanted cells. fMRI successfully detected local and remote neural activity, enabling the global graft-host neural circuit function to be assessed. These results demonstrate the potential of a novel neuroimaging-based platform that can be used to identify how a graft's electrical activity influences the brain network in vivo.
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Cuerpo Estriado/fisiología , Xenoinjertos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Animales , Encéfalo/fisiología , Mapeo Encefálico , Cuerpo Estriado/cirugía , Células Madre Embrionarias/fisiología , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Optogenética , RatasRESUMEN
OBJECTIVE: To study the relationship between semen quality and current health status in a cohort of men evaluated for infertility. DESIGN: Cross-sectional study. SETTING: Fertility clinic. PATIENT(S): Nine thousand three hundred eighty-seven men evaluated for infertility between 1994 and 2011. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Charlson comorbidity index, medical diagnoses by organ system. RESULT(S): At the time of evaluation, 9,387 men with a mean age of 38 years had semen data available. Of these men, 44% had at least one medical diagnosis unrelated to infertility. When stratifying the cohort by the Charlson comorbidity index (CCI), differences in all measured semen parameters were identified. Men with a higher CCI had lower semen volume, concentration, motility, total sperm count, and morphology scores. In addition, men with diseases of the endocrine, circulatory, genitourinary, and skin diseases all showed significantly higher rates of semen abnormalities. Upon closer examination of diseases of the circulatory system, men with hypertensive disease, peripheral vascular and cerebrovascular disease, and nonischemic heart disease all displayed higher rates of semen abnormalities. CONCLUSION(S): The current report identified a relationship between medical comorbidites and male semen production. Although genetics help guide a man's sperm production, his current condition and health play an important role.
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Enfermedades Cardiovasculares/epidemiología , Enfermedades del Sistema Endocrino/epidemiología , Infertilidad Masculina/epidemiología , Infertilidad Masculina/patología , Enfermedades Urogenitales Masculinas/epidemiología , Análisis de Semen/estadística & datos numéricos , Enfermedades de la Piel/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , California/epidemiología , Estudios de Cohortes , Comorbilidad , Estudios Transversales , Estado de Salud , Humanos , Incidencia , Masculino , Pronóstico , Factores de Riesgo , Estadística como Asunto , Adulto JovenRESUMEN
Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1â hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.
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Desarrollo Embrionario/genética , Mitosis/genética , Estrés Oxidativo/genética , Espermatozoides/metabolismo , Aneuploidia , Blastocisto/citología , Blastocisto/metabolismo , Daño del ADN/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Oocitos/citología , Oocitos/metabolismo , Espermatozoides/patologíaRESUMEN
Although 10-15 % of couples are infertile, little is known of the diverse, underlying pathologies in men and women with poor germ cell production; furthermore, for those with few or no high-quality germ cells, there are few options available for treatment. Thus, over the last decade, concerted efforts have been aimed at developing a biological system to probe the fundamentals of human egg and sperm production via pluripotent stem cell cells with the hopes of informing clinical decisions and ultimately providing alternative methods for therapy which may include developing a source of germ cells ultimately for reproductive purposes.
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Diferenciación Celular/genética , Células Madre Embrionarias/citología , Células Germinativas/citología , Células Madre Pluripotentes Inducidas/citología , Femenino , Humanos , Masculino , Biología Molecular/métodos , Óvulo/citología , Espermatozoides/citologíaRESUMEN
Systematically tunable in vitro platforms are invaluable in gaining insight to stem cell-microenvironment interactions in three-dimensional cultures. Utilizing recombinant protein technology, we independently tune hydrogel properties to systematically isolate the effects of matrix crosslinking density on cardiomyocyte differentiation, maturation, and function. We show that contracting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) remain viable within four engineered elastin-like hydrogels of varying crosslinking densities with elastic moduli ranging from 0.45 to 2.4 kPa. Cardiomyocyte phenotype and function was maintained within hESC embryoid bodies for up to 2 weeks. Interestingly, increased crosslinking density was shown to transiently suspend spontaneous contractility. While encapsulated cells began spontaneous contractions at day 1 in hydrogels of the lowest crosslinking density, onset of contraction was increasingly delayed at higher crosslinking densities up to 6 days. However, once spontaneous contraction was restored, the rate of contraction was similar within all materials (71 ± 8 beats/min). Additionally, all groups successfully responded to electrical pacing at both 1 and 2 Hz. This study demonstrates that encapsulated hESC-CMs respond to 3D matrix crosslinking density within elastin-like hydrogels and stresses the importance of investigating temporal cellular responses in 3D cultures.
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Retroviral overexpression of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) generates induced pluripotent stem cells (iPSCs). However, the integration of foreign DNA could induce genomic dysregulation. Cell-permeant proteins (CPPs) could overcome this limitation. To date, this approach has proved exceedingly inefficient. We discovered a striking difference in the pattern of gene expression induced by viral versus CPP-based delivery of the reprogramming factors, suggesting that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In gain- and loss-of-function studies, we find that the toll-like receptor 3 (TLR3) pathway enables efficient induction of pluripotency by viral or mmRNA approaches. Stimulation of TLR3 causes rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming. Activation of inflammatory pathways are required for efficient nuclear reprogramming in the induction of pluripotency.
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Péptidos de Penetración Celular/metabolismo , Reprogramación Celular , Inmunidad Innata , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Línea Celular , Fibroblastos/metabolismo , Humanos , Inflamación/metabolismo , Factor 4 Similar a Kruppel , FN-kappa B/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Retroviridae/metabolismo , Receptor Toll-Like 3/metabolismoRESUMEN
Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (SNCA). We observed that the iPSCs readily differentiated into functional neurons. Importantly, the PD-affected line exhibited disease-related phenotypes in culture: accumulation of α-synuclein, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide induced oxidative stress. These findings show that the dominantly-acting PD mutation is intrinsically capable of perturbing normal cell function in culture and confirm that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain.
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Neuronas Dopaminérgicas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Estrés Oxidativo , Enfermedad de Parkinson/patología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Adulto , Animales , Muerte Celular , Neuronas Dopaminérgicas/patología , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , Fenotipo , Embarazo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , alfa-Sinucleína/químicaRESUMEN
An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.
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Anticuerpos Monoclonales/metabolismo , Células Madre Pluripotentes/citología , Polisacáridos/metabolismo , Antígenos Embrionarios Específico de Estadio/metabolismo , Teratoma/patología , Animales , Biomarcadores , Diferenciación Celular , Células Cultivadas , Biología Computacional , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Análisis por Micromatrices , Células Madre Pluripotentes/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Teratoma/metabolismoRESUMEN
Using donated human embryos for scientific research raises ethical questions about the donation process. We describe a two-stage consent process designed to help couples make informed decisions about embryo disposition. This consent methodology minimizes conflict of interest, respects patient choice, and provides a much-needed resource to patients and the research community.
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Destinación del Embrión , Investigación con Células Madre , Destinación del Embrión/ética , Investigaciones con Embriones/ética , Humanos , Consentimiento Informado , Métodos , Investigación con Células Madre/éticaRESUMEN
Studies of Parkinson's disease (PD) have been hindered by lack of access to affected human dopaminergic (DA) neurons. Here, we report generation of induced pluripotent stem cells that carry the p.G2019S mutation (G2019S-iPSCs) in the Leucine-Rich Repeat Kinase-2 (LRRK2) gene, the most common PD-related mutation, and their differentiation into DA neurons. The high penetrance of the LRRK2 mutation and its clinical resemblance to sporadic PD suggest that these cells could provide a valuable platform for disease analysis and drug development. We found that DA neurons derived from G2019S-iPSCs showed increased expression of key oxidative stress-response genes and α-synuclein protein. The mutant neurons were also more sensitive to caspase-3 activation and cell death caused by exposure to stress agents, such as hydrogen peroxide, MG-132, and 6-hydroxydopamine, than control DA neurons. This enhanced stress sensitivity is consistent with existing understanding of early PD phenotypes and represents a potential therapeutic target.
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Dopamina/metabolismo , Células Madre Pluripotentes Inducidas/patología , Mutación/genética , Neuronas/patología , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/genética , Amidas/farmacología , Sustitución de Aminoácidos/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Leupeptinas/farmacología , Mesencéfalo/patología , Ratones , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Oxidopamina/farmacología , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
INTRODUCTION: The age-specific prevalence of polycystic ovaries (PCO), as defined by the Rotterdam criteria, among normal ovulatory women, has not yet been reported. It is also uncertain whether these women differ from their peers in the hormonal or metabolic profile. METHODS: A total of 262 ovulatory Caucasian women aged 25-45 yr, enrolled in a community-based ovarian aging study (OVA), underwent transvaginal ultrasound assessment of ovarian volume and antral follicle count (AFC) in the early follicular phase and were categorized as to whether they met the Rotterdam definition of PCO by AFC (≥12 in one ovary) and/or by volume (>10 cm(3) for one ovary). The effect of age on prevalence of PCO was assessed. Serum hormones and metabolic measures were compared between women meeting each element of the Rotterdam criterion and those without PCO using age-adjusted linear regressions. RESULTS: The prevalence of PCO by AFC was 32% and decreased with age. Those with PCO by AFC had lower FSH; higher anti-Müllerian hormone, estrone, dehydroepiandrostenedione sulfate, and free androgen index; and slightly higher total testosterone than those without PCO. However, slightly higher body mass index and waist circumference were the only metabolic differences. Women with PCO by volume had higher anti-Müllerian hormone and free androgen index but did not differ in any other hormonal or metabolic parameter. DISCUSSION: PCO is a common, age-dependent finding among ovulatory women. These women lack the metabolic abnormalities seen in PCO syndrome. Isolated PCO in an ovulatory woman is not an indication for metabolic evaluation.
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Quistes Ováricos/epidemiología , Ovario/diagnóstico por imagen , Adulto , Factores de Edad , Andrógenos/sangre , Hormona Antimülleriana/sangre , Índice de Masa Corporal , Ensayo de Inmunoadsorción Enzimática , Estrona/sangre , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Persona de Mediana Edad , Quistes Ováricos/sangre , Quistes Ováricos/diagnóstico por imagen , Prevalencia , Análisis de Regresión , UltrasonografíaRESUMEN
Developments in genomic medicine will likely explain much of what is now considered idiopathic male infertility. Indeed, our understanding of the genetic defects that cause infertility is no longer confined to chromosomal aneuploidies (e.g., Klinefelter syndrome) and single-gene defects (cystic fibrosis and congenital absence of the vas deferens). The past decade has seen that isolated Y-chromosomal loci can influence spermatogenesis (AZF regions) and that the human X chromosome is likely to be an important source of spermatogenesis genes. More recently, the finding that faulty recombination occurs in male infertility has large implications not only for the cause of the infertility but also for the use of affected gametes. Indeed, as our understanding of genetic infertility matures, so too will the importance and complexity of genetic counseling and testing for patients who use assisted reproduction.
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Infertilidad Masculina/genética , Aneuploidia , Cromosomas/genética , Cromosomas/fisiología , Cromosomas Humanos X/genética , Cromosomas Humanos Y/genética , Reparación del ADN , Femenino , Asesoramiento Genético , Humanos , Masculino , Mutación/genética , Mutación/fisiología , Recombinación Genética , Translocación GenéticaRESUMEN
Spermatocytic seminomas are solid tumors found solely in the testis of predominantly elderly individuals. We investigated these tumors using a genome-wide analysis for structural and numerical chromosomal changes through conventional karyotyping, spectral karyotyping, and array comparative genomic hybridization using a 32 K genomic tiling-path resolution BAC platform (confirmed by in situ hybridization). Our panel of five spermatocytic seminomas showed a specific pattern of chromosomal imbalances, mainly numerical in nature (range, 3-24 per tumor). Gain of chromosome 9 was the only consistent anomaly, which in one case also involved amplification of the 9p21.3-pter region. Parallel chromosome level expression profiling as well as microarray expression analyses (Affymetrix U133 plus 2.0) was also done. Unsupervised cluster analysis showed that a profile containing transcriptional data on 373 genes (difference of > or = 3.0-fold) is suitable for distinguishing these tumors from seminomas/dysgerminomas. The diagnostic markers SSX2-4 and POU5F1 (OCT3/OCT4), previously identified by us, were among the top discriminatory genes, thereby validating the experimental set-up. In addition, novel discriminatory markers suitable for diagnostic purposes were identified, including Deleted in Azospermia (DAZ). Although the seminomas/dysgerminomas were characterized by expression of stem cell-specific genes (e.g., POU5F1, PROM1/CD133, and ZFP42), spermatocytic seminomas expressed multiple cancer testis antigens, including TSP50 and CTCFL (BORIS), as well as genes known to be expressed specifically during prophase meiosis I (TCFL5, CLGN, and LDHc). This is consistent with different cells of origin, the primordial germ cell and primary spermatocyte, respectively. Based on the region of amplification defined on 9p and the associated expression plus confirmatory immunohistochemistry, DMRT1 (a male-specific transcriptional regulator) was identified as a likely candidate gene for involvement in the development of spermatocytic seminomas.