Dynamics, ultrastructure and gene expression of human in vitro organized testis cells from testicular sperm extraction biopsies.

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.

Trends in practice and outcomes from 2011 to 2015 for surgical aortic valve replacement: an update from the German Aortic Valve Registry on 42 776 patients.

OBJECTIVES: Surgical aortic valve replacement (sAVR) is coming under close scrutiny with the recent upswing in the use of less invasive approaches. The aim of this analysis was to identify current trends in patient selection, procedural characteristics and outcomes after sAVR in Germany. METHODS: We analysed data from 42 776 patients included in the German Aortic Valve Registry who underwent sAVR with and without coronary artery bypass surgery (CABG) between 2011 and 2015. Baseline, procedural and short-term outcome parameters were analysed. RESULTS: Of all registered patients, 26 618 (62.2%) underwent isolated sAVR and 16 158 (37.8%) sAVR + CABG. The median age was 72 years, and the median Society of Thoracic Surgeons Predicted Risk of Mortality (STS PROM) was 2.3%. From 2011 to 2015, there was a decline in STS PROM (2.4-2.2%, P < 0.001) and a decline in risk factors, such as pulmonary hypertension (9.1-3.2%, P < 0.001), occlusive arterial disease (19.6-17.7%, P = 0.003), mitral regurgitation ≥2° (10.6-7.6%, P < 0.001) and New York Heart Association Class III/IV (65.3-59.2%, P < 0.001). In-hospital mortality was 2.3%, 1.3% had disabling stroke, 0.4% residual aortic regurgitation ≥2°, and the incidence of new-onset pacemaker/implantable cardioverter-defibrillator implantation was 3.9%. There was an increase in the use of biological valves in patients <65 years (50.1-65.7%, P < 0.001), and the proportion of rapid deployment valves increased significantly (1.5-8.4%, P < 0.001) over the investigated time period. CONCLUSIONS: Both isolated sAVR as well as sAVR + CABG resulted in excellent in-hospital outcomes based on >42 000 patients treated between 2011 and 2015. The implementation of alternative treatment strategies has resulted in palpable changes in patient and device selection.

German CABG score: a specific risk model for patients undergoing isolated coronary artery bypass grafting.

BACKGROUND: A specific risk model concerning mortality of patients undergoing isolated coronary artery bypass grafting (CABG) is developed based on the national quality benchmarking mandatory by law in Germany. METHODS: On the basis of the national data pool from 2004, a risk score model for patients undergoing isolated CABG was developed and finally adjusted with the data of 43,145 patients of the year 2008. Modeling was performed by logistic regression analysis. This risk model was validated with the 2007 data pool which comprised 45,569 patients. RESULTS: Observed in-hospital mortality after isolated CABG procedures was 3.0% in 2008. Hosmer-Lemeshow test p value was 0.189 and area under receiver operating characteristic curve was 0.826. Applying the German CABG score for 2007 resulted in an observed-to-expected mortality ratio of 1.01. CONCLUSION: The German CABG score for in-hospital mortality is a risk score with proven validity for isolated CABG, developed by means of the patient population in Germany. It can be used for the assessment of patient risk groups and for interhospital benchmarking. We encourage other researchers to apply and validate this score in comparable health care systems.

German Aortic Valve Score: a new scoring system for prediction of mortality related to aortic valve procedures in adults.

OBJECTIVES: The aim of the study was to establish a scoring system to predict mortality in aortic valve procedures in adults [German Aortic Valve Score (German AV Score)] based upon the comprehensive data pool mandatory by law in Germany. METHODS: In 2008, 11 794 cases were documented who had either open aortic valve surgery or transcatheter aortic valve implantation (TAVI). In-hospital mortality was chosen as a binary outcome measure. Potential risk factors were identified on the basis of published scoring systems and clinical knowledge. First, each of these risk factors was tested in an univariate manner by Fisher's exact test for significant influence on mortality. Then, a multiple logistic regression model with backward and forward selection was used. Calibration was ascertained by the Hosmer-Lemeshow method. In order to define the quality of discrimination, the area under the receiver operating characteristic (ROC) curve was calculated. RESULTS: In 11 147 of 11 794 cases (94.5%), a complete data set was available. In-hospital mortality was 3.7% for all patients, 3.4% in the surgical group (95% confidence interval 3.0-3.7%, n = 10 574) and 10.6% in the TAVI group (95% confidence interval 8.2-13.5%, n = 573). Based on multiple logistic regression, 15 risk factors with an influence on mortality were identified. Among them, age, body mass index and left ventricular function were categorized in three (body mass index, left ventricular dysfunction) or 6 subgroups (age). The Hosmer-Lemeshow method corroborated a valid concordance of predicted and observed mortality in 10 different risk groups. The area under the ROC curve with a value of 0.808 affirmed the quality of discrimination of the established scoring model. CONCLUSIONS: It is well known that a predictive model works best in the setting where it was developed; therefore, the German AV Score fits well to the patient population in Germany. It was designed for fair and reliable outcome evaluation. It allows comparison of predicted and observed mortality for conventional aortic valve surgery and transcatheter aortic valve implantation in low-, moderate- and high-risk groups. Thus, it enables primarily a risk-adjusted benchmark of outcome and fosters the efforts for continuous improvement of quality in aortic valve procedures.

Angiotensin-converting enzyme insertion/deletion polymorphism and retinal artery occlusion.

PURPOSE: An insertion/deletion (I/D) polymorphism of the gene for angiotensin-converting enzyme (ACE) is associated with higher ACE plasma levels and activity. This enzyme is known to play an important role in blood pressure regulation and the ACE I/D gene polymorphism has been suggested as a risk factor for atherosclerotic vascular diseases. The purpose of the present study was to investigate a hypothesized association between the ACE I/D polymorphism and retinal artery occlusion (RAO). METHODS: A total of 159 patients with RAO and 304 control subjects were enrolled in the present retrospective case-control study. ACE I/D genotypes were determined by polymerase chain reaction. RESULTS: Allelic frequencies and genotype distribution of the ACE I/D polymorphism did not significantly differ between patients and control subjects (ACE DD 25.8% versus 28.0%; p = 0.36). A logistic regression analysis predicted the presence of RAO by arterial hypertension and current smoking status, but not by ACE I/D genotypes. CONCLUSION: Our data suggest that the ACE I/D polymorphism is not a major risk factor for RAO.