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1.
Surg Endosc ; 36(3): 2208-2216, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34973079

RESUMO

BACKGROUND: Postoperative reflux can compromise anastomotic healing after Ivor-Lewis oesophagectomy (ILE). We report on Pre-emptive Active Reflux Drainage (PARD) using a new double-lumen open-pore film drain (dOFD) with negative pressure to protect the anastomosis. METHODS: To prepare a dOFD, the gastric channel of a triluminal tube (Freka®Trelumina, Fresenius) is coated with a double-layered open-pore film (Suprasorb®CNP drainage film, Lohmann & Rauscher) over 25 cm. The ventilation channel is blocked. The filmcoated segment is placed in the stomach and the intestinal feeding tube in the duodenum. Negative pressure is applied with an electronic vacuum pump (- 125 mmHg, continuous suction) to the gastric channel. Depending on the findings in the endoscopic control, PARD will either be continued or terminated. RESULTS: PARD was used in 24 patients with ILE and started intraoperatively. Healing was observed in all the anastomoses. The median duration of PARD was 8 days (range 4-21). In 10 of 24 patients (40%) there were issues with anastomotic healing which we defined as "at-risk anastomosis". No additional endoscopic procedures or surgical revisions to the anastomoses were required. CONCLUSIONS: PARD with dOFD contributes to the protection of anastomosis after ILE. Negative pressure applied to the dOFD (a nasogastric tube) enables enteral nutrition to be delivered simultaneously with permanent evacuation and decompression.


Assuntos
Esofagectomia , Refluxo Gastroesofágico , Anastomose Cirúrgica , Drenagem/métodos , Nutrição Enteral/métodos , Esofagectomia/métodos , Refluxo Gastroesofágico/prevenção & controle , Humanos , Intubação Gastrointestinal/métodos
2.
Mol Hum Reprod ; 25(6): 283-294, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30892608

RESUMO

Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of prepubertal boys or in treatment of male adults suffering from meiotic arrest. Prior to therapeutic application, in vitro propagation of rare hSSCs is mandatory. As the published data points to epigenetic alterations in long-term cell culture of spermatogonia (SPG), an initial characterisation of their DNA methylation state is important. Testicular biopsies from five adult normogonadotropic patients were converted into aggregate-free cell suspensions. FGFR3-positive (FGFR3+) SPG, resembling a very early stem cell state, were labelled with magnetic beads and isolated in addition to unlabelled SPG (FGFR3-). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19 and MEG3), maternally imprinted (KCNQ1OT1, PEG3, and SNRPN), pluripotency (POU5F1/OCT4 and NANOG), and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, and L1TD1) genes on either single cells or pools of 10 cells. Both spermatogonial subpopulations exhibited a methylation pattern largely equivalent to sperm, with hypomethylation of hSSC marker and maternally imprinted genes and hypermethylation of pluripotency and paternally imprinted genes. Interestingly, we detected fine differences between the two spermatogonial subpopulations, which were reflected by an inverse methylation pattern of imprinted genes, i.e. decreasing methylation in hypomethylated genes and increasing methylation in hypermethylated genes, from FGFR3+ through FGFR3- SPG to sperm. Limitations of this study are due to it not being performed on a genome-wide level and being based on previously published regulatory gene regions. However, the concordance of DNA methylation between SPG and sperm implies that hSSC regulation and germ cell differentiation do not occur at the DNA methylation level.


Assuntos
Metilação de DNA/fisiologia , Espermatogônias/metabolismo , Alelos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Metilação de DNA/genética , Epigênese Genética/genética , Humanos , Masculino , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Células-Tronco/metabolismo
3.
Mol Hum Reprod ; 24(3): 123-134, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29304256

RESUMO

STUDY QUESTION: Is it possible to induce in vitro reorganization of primary human testis cells from testicular sperm extraction (TESE) biopsies, maintain their long-term cultivation in a 2D system and identify cellular compositions? SUMMARY ANSWER: In vitro reorganization of primary human testis cells from TESE biopsies and their long-term cultivation on uncoated cell culture dishes is feasible and the cellular compositions can be uncovered through gene expression and microscopic analyses. WHAT IS KNOWN ALREADY: It has been shown in the rodent model that mixtures of testicular cell types are able to reassemble into clusters when cultivated on different kinds of surfaces or three-dimensional matrices. Two recent publications demonstrated the ability of primary human testicular cells to assemble into testicular organoids and their cultivation for a period of 3-4 weeks. STUDY DESIGN SIZE, DURATION: Primary human testis cells from TESE biopsies from 16 patients were reorganized in vitro and the clusters were cultivated long term on uncoated cell culture dishes, providing a solid ground for in vitro spermatogenesis. Gene expression analysis as well as fluorescence/transmission electron microscopy (TEM) were employed to uncover the cellular composition of the clusters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsies from adult, normogonadotropic patients displaying full spermatogenesis (n = 11), hypospermatogenesis (n = 2), predominantly full spermatogenesis with some hypospermatogenic tubules (n = 1), meiotic arrest (n = 1) or mixed atrophy (n = 1) were enzymatically digested and dispersed cells were cultivated on 96-well plates or chamber dishes as aggregate-free cell suspensions. Time-lapse imaging of cluster formation was performed over a period of 48 h. For receptor tyrosine kinase inhibition of cluster formation, cells were treated twice with K252a within 2-3 days. Immunofluorescence staining and confocal microscopy was carried out on clusters after 1-3 weeks of cultivation to identify the presence of Sertoli cells (SC) (SOX9), peritubular myoid cells (SMA), Leydig cells (LC) (STAR), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Single clusters from four patients and a pool of eight larger clusters from another patient were manually picked and subjected to quantitative real-time PCR to evaluate the presence of SC (SOX9, AR), LC (INSL3, STAR, HSD3B1), peritubular myoid cells (ACTA2), fibroblasts (FSP1), endothelial cells (CD34), macrophages (CD68), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Finally, an ultrastructural investigation was conducted based on TEM of clusters from six different patients, among them 3-month cultivated large clusters from two patients. MAIN RESULTS AND THE ROLE OF CHANCE: Quantitative PCR-based analysis of single-picked testicular cell clusters identified SC, peritubular myoid cells, endothelial cells, fibroblasts, macrophages, spermatids and LC after 1, 2 or 3 weeks or 3 months of cultivation. Immunofluorescence positivity for SC and peritubular myoid cells corroborated the presence of these two kinds of testis niche cells. In addition, round as well as elongated spermatids were frequently encountered in 1 and 2 weeks old clusters. Transmission electron microscopical classification confirmed all these cell types together with a few spermatogonia. Macrophages were found to be of the proinflammatory M1 subtype, as revealed by CD68+/CD163-/IL6+ expression. Time-lapse imaging uncovered the specific dynamics of cluster fusion and enlargement, which could be prevented by addition of protein kinase inhibitor K252a. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Cell composition of the clusters varied based on the spermatogenic state of the TESE patient. Although spermatids could be observed with all applied methods, spermatogonia were only detected by TEM in single cases. Hence, a direct maintenance of these germ cell types by our system in its current state cannot be postulated. Moreover, putative dedifferentiation and malignant degeneration of cells in long-term cluster cultivation needs to be investigated in the future. WIDER IMPLICATIONS OF THE FINDINGS: This work demonstrates that the reorganization of testicular cells can be achieved with TESE biopsies obtained from men enroled in a standard clinical assisted reproduction program. The formed clusters can be cultivated for at least 3 months and are composed, to a large extent, of the most important somatic cell types that are essential to support spermatogenesis. These findings may provide the cellular basis for advances in human in vitro spermatogenesis and/or the possibility for propagation of spermatogonia within a natural stem cell niche-like environment. STUDY FUNDING AND COMPETING INTERESTS: The project was funded by a DFG grant to K.v.K. (KO 4769/2-1). The authors declare they have no conflicts of interest.


Assuntos
Espermatogônias/metabolismo , Testículo/metabolismo , Biópsia , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Humanos , Masculino , Reação em Cadeia da Polimerase , Protaminas/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Espermatogônias/citologia , Espermatozoides/citologia , Espermatozoides/fisiologia , Testículo/citologia
7.
Hum Reprod ; 28(3): 590-8, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23303554

RESUMO

STUDY QUESTION: What are the reference values for delineating altered somatic cell gene expression from transcript enrichment/dilution in gene expression studies of human spermatogenesis? SUMMARY ANSWER: We have designed a crosstable and rule-of-thumb values for different stages of spermatogenic impairment that define the reference cut-off values for altered gene expression in Sertoli and Leydig cells in the context of impaired spermatogenesis. WHAT IS KNOWN ALREADY: Morphometrical studies have shown that on the cellular level, impaired spermatogenesis results in a relative enrichment of somatic cell types. However, until now it is not known how this affects transcript levels in gene expression studies. STUDY DESIGN, SIZE DURATION: In this study, 31 testis biopsies from men with different stages of spermatogenic impairment (full spermatogenesis, hypospermatogenesis, meiotic arrest, spermatogonial presence, Sertoli cell-only syndrome, complete tubular atrophy) were used to define reference ratios of somatic transcript enrichment/dilution. The reference ratios were validated on an independent test set of 28 samples and on gene expression data from men with Y-chromosomal microdeletions. PARTICIPANTS/MATERIAL, SETTING, METHODS: High-quality microarray data were filtered with respect to Sertoli- and Leydig-cell-specific genes. General reference enrichment/dilution factors for these two cell types for all combinations of spermatogenic impairment were calculated using robust permutation statistics. To validate the specificity of the filtered transcripts, we calculated ratios for an independent test set of spermatogenic impairment and for transcriptional data from men with Y-chromosomal microdeletions, and checked the functional enrichment (gene ontology) and cellular localization of the corresponding proteins in a histological database and assessed their correlation with testicular size. MAIN RESULTS AND THE ROLE OF CHANCE: Filtering of Sertoli- and Leydig-cell-specific genes resulted in a set of 54 and 332 transcripts, respectively. These were used in defining robust reference dilution/enrichment factors of somatic transcripts for all spermatogenic levels and were compiled in a reference crosstable. Validation on an independent test set showed ratios within 0.5 units of our reference crosstable. Analysis of the resulting transcripts with respect to functional enrichment for Sertoli- and Leydig-cell-specific functions and protein expression, as obtained from an immunohistochemical database, indicated filtering of data sets highly enriched for Sertoli and Leydig cell function. The dilution/enrichment ratios differed significantly when transcripts were interrogated in samples with Y-chromosomal microdeletions, pointing to an overall decreased expression of somatic markers in a genetically altered background. LIMITATIONS, REASONS FOR CAUTION: The defined reference ratios might apply with some restrictions in samples that display very heterogeneous histology (e.g. Sertoli cell only with a significant proportion of spermatogenic foci) or when spermatogenic impairment is a consequence of an altered genetic background. WIDER IMPLICATIONS OF THE FINDINGS: The reference dilution/enrichment values for somatic testicular transcripts as defined in this study are to be seen as cut-off values for discriminating between simple transcript dilution/enrichment as a consequence of an altered germ cell composition and actual transcriptional regulation. Future studies dealing with transcriptional changes in testicular somatic cells in a background of altered germ cell quantities should consider these correction factors in order to avoid the description of transcriptional changes that are based simply on shifts in somatic cellular quantities. STUDY FUNDING/COMPETING INTEREST(S): Financial support was from grant Sp721/1-3 of the German Research Foundation. There are no competing interests to be declared.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Infertilidade Masculina/metabolismo , Espermatogênese , Testículo/metabolismo , Transcrição Gênica , Adulto , Inteligência Artificial , Biomarcadores/metabolismo , Biópsia , Deleção Cromossômica , Cromossomos Humanos Y/metabolismo , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Infertilidade Masculina/etiologia , Infertilidade Masculina/patologia , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , RNA Mensageiro/metabolismo , Síndrome de Células de Sertoli/metabolismo , Síndrome de Células de Sertoli/patologia , Síndrome de Células de Sertoli/fisiopatologia , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Aberrações dos Cromossomos Sexuais , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/metabolismo , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/patologia , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/fisiopatologia , Testículo/patologia
8.
Chirurgie (Heidelb) ; 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38085297

RESUMO

INTRODUCTION: Early postoperative reflux (PR) can compromise anastomotic healing after Ivor Lewis esophagectomy (ILE) and poses a risk for aspiration. Anastomotic insufficiency is the most threatening surgical complication. We present the protective method of pre-emptive active reflux drainage (PARD) with simultaneous enteral feeding. We report our experience with this new safety concept in esophageal surgery in a cohort of 43 patients. MATERIALS AND METHODS: For PARD we use a double lumen open porous film drainage (dOFD). To create the dOFD, the gastric tube of a Trelumina probe (Freka®Trelumina, Fresenius) is coated with a double-layered open-pore drainage film (Suprasorb®CNP drainage film, Lohmann & Rauscher) over a length of 25 cm. The dOFD is endoscopically inserted into the tubular stomach intraoperatively after completion of the anastomosis. Continuous negative pressure is applied with an electronic pump (-125 mm Hg). The PR is continuously aspirated completely and the stomach and anastomotic region are decompressed. At the same time, nutrition is delivered via an integrated intestinal tube. Depending on the results of the endoscopic control after 5 days, PARD is either continued or terminated. RESULTS: During the observation period (2017-2023), PARD was used in all patients (n = 43) with ILE. The healing rate under PARD was 100% and healing was observed in all anastomoses. No additional endoscopic procedures or surgical revisions of the anastomoses were required. The median duration of PARD was 8 days (range 4-21). We observed problems in the healing of the anastomosis in 20 of 43 patients (47%) for whom we defined endoscopic criteria for at-risk anastomosis. CONCLUSIONS: Our results suggest that PARD has a strong protective effect on anastomotic healing and may reduce the risk of anastomotic insufficiency. The integrated feeding tube of the dOFD allows early postoperative enteral feeding while simultaneously applying negative pressure. PARD appears to prevent the negative consequences of impaired anastomotic healing.

9.
Chirurgie (Heidelb) ; 94(12): 1022-1033, 2023 Dec.
Artigo em Alemão | MEDLINE | ID: mdl-37882839

RESUMO

INTRODUCTION: Early postoperative reflux (PR) can compromise anastomotic healing after Ivor Lewis esophagectomy (ILE) and poses a risk for aspiration. Anastomotic insufficiency is the most threatening surgical complication. We present the protective method of pre-emptive active reflux drainage (PARD) with simultaneous enteral feeding. We report our experience with this new safety concept in esophageal surgery in a cohort of 43 patients. MATERIALS AND METHODS: For PARD we use a double lumen open porous film drainage (dOFD). To create the dOFD, the gastric tube of a Trelumina probe (Freka®Trelumina, Fresenius) is coated with a double-layered open-pore drainage film (Suprasorb®CNP drainage film, Lohmann & Rauscher) over a length of 25 cm. The dOFD is endoscopically inserted into the tubular stomach intraoperatively after completion of the anastomosis. Continuous negative pressure is applied with an electronic pump (-125 mm Hg). The PR is continuously aspirated completely and the stomach and anastomotic region are decompressed. At the same time, nutrition is delivered via an integrated intestinal tube. Depending on the results of the endoscopic control after 5 days, PARD is either continued or terminated. RESULTS: During the observation period (2017-2023), PARD was used in all patients (n = 43) with ILE. The healing rate under PARD was 100% and healing was observed in all anastomoses. No additional endoscopic procedures or surgical revisions of the anastomoses were required. The median duration of PARD was 8 days (range 4-21). We observed problems in the healing of the anastomosis in 20 of 43 patients (47%) for whom we defined endoscopic criteria for at-risk anastomosis. CONCLUSIONS: Our results suggest that PARD has a strong protective effect on anastomotic healing and may reduce the risk of anastomotic insufficiency. The integrated feeding tube of the dOFD allows early postoperative enteral feeding while simultaneously applying negative pressure. PARD appears to prevent the negative consequences of impaired anastomotic healing.


Assuntos
Esofagectomia , Refluxo Gastroesofágico , Humanos , Esofagectomia/efeitos adversos , Endoscopia , Drenagem/efeitos adversos , Drenagem/métodos , Refluxo Gastroesofágico/etiologia , Refluxo Gastroesofágico/prevenção & controle , Refluxo Gastroesofágico/cirurgia
10.
Histochem Cell Biol ; 138(5): 759-72, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22777346

RESUMO

Human spermatogonia (Spg) and their fetal precursors express fibroblast growth factor receptor 3 (FGFR3). To further elucidate the role of FGFR3 in the control of Spg self-renewal, proliferation, and/or differentiation, and to narrow down the FGFR3-positive cell type(s) in the normal adult human testis, tissue sections and whole mount preparations of seminiferous tubules were analyzed combining immunofluorescence and confocal fluorescence microscopy. FGFR3 protein was chiefly observed in cellular membranes and cytoplasmic vesicles of a subpopulation of type A Spg, which comprised the chromatin rarefaction zone-containing type A(dark). Cytoplasmic expression of FGFR3 and nuclear expression of proliferation-associated antigen KI-67 were mutually exclusive. Similarly, FGFR3-positive Spg were negative for Doublesex and Mab-3 related transcription factor 1 (DMRT1). By contrast, undifferentiated embryonic cell transcription factor 1 (UTF1) and survival time-associated PHD finger in ovarian cancer 1 protein (SPOC1) were co-expressed in the nuclei of FGFR3-positive Spg. Whole mounted seminiferous tubules illustrated the clonogenic arrangement of the FGFR3/UTF1 double-positive Spg, which mainly occurred as pairs or quadruplets and, different from the KIT-positive Spg, showed no overlap with KI-67 labeled clusters. Taken together, in the adult human testis, FGFR3 expression is a feature of small clones of rarely dividing type A Spg which resemble "undifferentiated" Spg, including the spermatogonial stem cells.


Assuntos
Divisão Celular , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/biossíntese , Espermatogônias/metabolismo , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Imunofluorescência , Humanos , Antígeno Ki-67/biossíntese , Masculino , Microscopia Confocal , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas c-kit/análise , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/análise , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Espermatogênese/fisiologia , Testículo/citologia , Testículo/metabolismo , Transativadores/biossíntese , Fatores de Transcrição/análise , Fatores de Transcrição/biossíntese
11.
Hum Reprod ; 27(11): 3233-48, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22926843

RESUMO

BACKGROUND: Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS: We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS: Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS: Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapies.


Assuntos
Criptorquidismo/metabolismo , Regulação da Expressão Gênica , Infertilidade Masculina/metabolismo , Proteoma/metabolismo , Espermatogênese , Testículo/metabolismo , Adulto , Motivos de Aminoácidos , Animais , Criança , Criptorquidismo/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Infertilidade Masculina/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Proteoma/química , Proteoma/genética , RNA Mensageiro/metabolismo , Ratos , Especificidade da Espécie , Testículo/crescimento & desenvolvimento , Testículo/patologia
12.
Reproduction ; 143(1): 45-57, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22046055

RESUMO

It is unclear whether the distinct nuclear morphologies of human A(dark) (Ad) and A(pale) (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear 'vacuolar spaces' representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex- and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing.


Assuntos
Espermatogônias/citologia , Espermatogônias/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Espermatogênese , Espermatogônias/classificação , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
13.
AIDS Res Ther ; 9(1): 2, 2012 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-22260499

RESUMO

Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted.

14.
Mol Hum Reprod ; 17(11): 669-78, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21593214

RESUMO

Human spermatozoal RNA features special characteristics such as a significantly reduced quantity within spermatozoa compared with somatic cells is described as being devoid of ribosomal RNAs and is difficult to isolate due to a massive excess of genomic DNA in the lysates. Using a novel two-round column-based protocol for human ejaculates delivering highly purified spermatozoal RNA, we uncovered a heterogeneous, but specific banding pattern in microelectrophoresis with 28S ribosomal RNA being indicative for the amount of round cell contamination. Ejaculates with different round cell quantities and density-purified spermatozoa revealed that 18S rRNA but not 28S rRNA is inherent to a pure spermatozoal fraction. Transmission electron microscopy showed monoribosomes and polyribosomes in spermatozoal cytoplasm, while immunohistochemical results suggest the presence of proteins from small and large ribosomal subunits in retained spermatozoal cytoplasm irrespective of 28S rRNA absence.


Assuntos
RNA Ribossômico 18S/química , RNA Ribossômico 28S/química , Ribossomos/metabolismo , Espermatozoides/química , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , RNA Ribossômico 28S/genética
15.
Hum Genet ; 128(4): 421-31, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20668881

RESUMO

Y-chromosomal microdeletions (YCMD) are the major genetic cause of male infertility. To date, it is not known which global changes are induced by the presence of AZFc or AZFb+c deletions in the human testicular transcriptome. We investigated this question by microarray analysis in which we had to eliminate the 'germ cell effect', i.e., the dominating effect of germ cell transcripts due to the quantitative difference in germ cell composition in samples with/without YCMD. This problem was tackled by selecting 26 samples from an initial cohort of 34 samples by their homogeneity in respect to cellular composition as obtained from gene expression clustering. This way, the 'germ cell effect' was minimized, and a distinct 'deletion effect' became more apparent. Several hundred genes are influenced by YCMD as shown on the three different phenotypes hypospermatogenesis, meiotic arrest, and Sertoli-cell only syndrome. We validated on an independent cohort of samples five genes by quantitative real-time PCR that are expressed in germ cells or the somatic compartment and which are exclusively altered by the presence of YCMD. We conclude that the deletion of Y-chromosomal genes has a significant effect on spermatogenesis by modulating the transcriptional network of the germ cell and somatic compartment.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Y/genética , Perfilação da Expressão Gênica , Testículo/metabolismo , Biópsia , Análise por Conglomerados , Estudos de Coortes , Loci Gênicos , Células Germinativas/metabolismo , Células Germinativas/patologia , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Meiose/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plasma Seminal/genética , Síndrome de Células de Sertoli/genética , Espermatogênese/genética , Testículo/patologia
16.
Hum Reprod ; 25(5): 1104-12, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20208059

RESUMO

BACKGROUND: A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type. METHODS: The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets. RESULTS: Highly significant differences in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast growth factor receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-1 (CTAG1A/B), undifferentiated embryonic cell transcription factor 1 (UTF1) and synaptosomal-associated protein, 91 kDa homolog (SNAP91) were shown to represent specific biomarkers of human spermatogonia. CONCLUSIONS: These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation.


Assuntos
Espermatogônias/citologia , Espermatogônias/metabolismo , Testículo/citologia , Testículo/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Desmogleína 2/genética , Desmogleína 2/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imuno-Histoquímica , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento de Interação de Proteínas , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/embriologia
18.
World J Urol ; 28(5): 631-5, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20043168

RESUMO

INTRODUCTION: Enhancer of Zeste 2 (EZH2) is an epigenetic transcriptional repressor involved in cell cycle control and cell fate decisions. Since these processes play key roles during intact spermatogenesis, deregulation of EZH2 expression may contribute to the development and progression of benign and malignant testicular diseases. The objective of this study was to investigate the expression profile of EZH2 in testicular germ cell tumors (TGCT) and spermatogenic defects. MATERIAL AND METHODS: Real-time RT-PCR was applied to quantify the m-RNA expression of EZH2 in 64 seminomas 36 non-seminomas, 4 carcinomas in situ (CIS), 40 samples harboring impaired spermatogenesis and 24 normal testicular reference biopsies. RESULTS: EZH2 was expressed in 99% of TGCT samples and in all biopsies with intact spermatogenesis. Its expression levels were highest in normal testicular tissue, and continuously decreased with malignant transformation to CIS and further progression to invasive TGCT (P < 0.001). EZH2 tumor levels were not related to the histological TGCT subtype or clinical tumor stage. Comparison of distinct stages of spermatogenic failure revealed an inverse association of EZH2 levels to the severity of the spermatogenic defect (P < 0.001). CONCLUSION: Our data strongly suggest that in TGCT EZH2 does not exert its often assumed oncogenic properties during malignant transformation and progression. High EZH2 levels in normal testicular tissue and the inverse association of its expression levels with the severity of spermatogenic failure point to its potential value as a molecular marker for spermatogenic defects and may indicate an important physiological role of EZH2 during intact spermatogenesis.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/fisiologia , Neoplasias Embrionárias de Células Germinativas/metabolismo , Espermatogênese/fisiologia , Neoplasias Testiculares/metabolismo , Fatores de Transcrição/metabolismo , Biópsia , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Masculino , Neoplasias Embrionárias de Células Germinativas/patologia , Complexo Repressor Polycomb 2 , Estudos Retrospectivos , Seminoma/metabolismo , Seminoma/patologia , Índice de Gravidade de Doença , Neoplasias Testiculares/patologia , Testículo/metabolismo , Testículo/patologia
19.
Innov Surg Sci ; 5(1-2): 67-73, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33506096

RESUMO

OBJECTIVES: We describe the first application of intrauterine negative-pressure therapy (IU-NPT) for an early rupture of a uterine suture after a third caesarean section with consecutive peritonitis and sepsis. Because all four quadrants were affected by peritonitis, a laparotomy was performed on the 15th day after caesarean section. Abdominal negative-pressure wound therapy (A-NPWT) of the open abdomen was initiated. During the planned relaparotomy, a suture defect of the anterior uterine wall was identified and sutured. In the second relaparotomy, the suture appeared once more insufficient. CASE PRESENTATION: For subsequent IU-NPT, we used an open-pore film drainage (OFD) consisting of a drainage tube wrapped in the double-layered film. The OFD was inserted into the uterine cavity via the uterine defect and IU-NPT was established together with A-NPT. With the next relaparotomy, local inflammation and peritonitis had been resolved completely. IU-NPT was continued transvaginally, the uterine defect was sutured, and the abdomen was closed. Vaginal IU-NPT was also discontinued after another eight days. CONCLUSIONS: By using IU-NPT, local infection control of the septic focus was achieved. The infectious uterine secretions were completely evacuated and no longer discharged into the abdominal cavity. As a result of the applied suction, the uterine cavity collapsed around the inlaid OFD. The total duration of IU-NPT was 11 days. The uterine defect was completely closed, and a hysterectomy was avoided. The patient was discharged four days after the end of IU-NPT. IU-NPT follows the same principles as those described for endoscopic negative-pressure wound therapy of the gastrointestinal tract.

20.
Endosc Int Open ; 6(7): E865-E871, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29978007

RESUMO

BACKGROUND AND STUDY AIMS: Endoscopic negative pressure therapy (ENPT) has been developed to treat gastrointestinal leakages. Up to now, ENPT has usually been performed with open-pore polyurethane foam drains (OPD). A big disadvantage of the OPDs is their large diameter. We have developed a new, small-bore open-pore film drainage (OFD). Herein we report our first experience in a case series of 16 patients. PATIENTS AND METHODS: OFD is constructed with a drainage tube and a very thin double-layered open-pore drainage film (Suprasorb CNP, Drainage Film, Lohmann & Rauscher International, Germany). The distal end of the tube is wrapped with only one layer of film. OFD is placed into the gastrointestinal leakage site with common endoscopic techniques. The tube is connected to an electronic vacuum device and continuous negative pressure of -125 mmHg applied. RESULTS: From 2013 to 2016, 16 patients were treated with the new OFD device. In 10 patients, transmural intestinal defects (4 esophageal, 4 rectum/colon, 1 duodenal, 1 pancreatic cyst) were closed with ENPT in median time of 12 days (range 3 - 34 days). Five of the 10 patients were treated solely with OFD devices. In five patients ENPT started with ODP and changed to OFD when the cavity was shrunken to a channel with a small opening. In four patients postoperative gastric reflux was eliminated for 5 to 16 days. CONCLUSIONS: Small-bore OFD opens up promising new treatment options within ENPT. OFD can be used in endoscopic closure management of intestinal leakages in the upper and lower gastrointestinal tract. Gastric reflux can be eliminated in an active manner. OFD can be inserted nasally. OFD may be an adequate substitute for OPD, especially when placement of the larger OPD is difficult.

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