An atlas of cell types in the mouse epididymis and vas deferens

Following testicular spermatogenesis, mammalian sperm continue to mature in a long epithelial tube known as the epididymis, which plays key roles in remodeling sperm protein, lipid, and RNA composition. To understand the roles for the epididymis in reproductive biology, we generated a single-cell atlas of the murine epididymis and vas deferens. We recovered key epithelial cell types including principal cells, clear cells, and basal cells, along with associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Moreover, our data illuminate extensive regional specialization of principal cell populations across the length of the epididymis. In addition to region-specific specialization of principal cells, we find evidence for functionally specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Our dataset extends on existing knowledge of epididymal biology, and provides a wealth of information on potential regulatory and signaling factors that bear future investigation

The impact of pre- and post-image processing techniques on deep learning frameworks: A comprehensive review for digital pathology image analysis

Recently, deep learning frameworks have rapidly become the main methodology for analyzing medical images. Due to their powerful learning ability and advantages in dealing with complex patterns, deep learning algorithms are ideal for image analysis challenges, particularly in the field of digital pathology. The variety of image analysis tasks in the context of deep learning includes classification (e.g., healthy vs. cancerous tissue), detection (e.g., lymphocytes and mitosis counting), and segmentation (e.g., nuclei and glands segmentation). The majority of recent machine learning methods in digital pathology have a pre- and/or post-processing stage which is integrated with a deep neural network. These stages, based on traditional image processing methods, are employed to make the subsequent classification, detection, or segmentation problem easier to solve. Several studies have shown how the integration of pre- and post-processing methods within a deep learning pipeline can further increase the model's performance when compared to the network by itself. The aim of this review is to provide an overview on the types of methods that are used within deep learning frameworks either to optimally prepare the input (pre-processing) or to improve the results of the network output (post-processing), focusing on digital pathology image analysis. Many of the techniques presented here, especially the post-processing methods, are not limited to digital pathology but can be extended to almost any image analysis field

The impact of pre- and post-image processing techniques on deep learning frameworks: A comprehensive review for digital pathology image analysis.

Recently, deep learning frameworks have rapidly become the main methodology for analyzing medical images. Due to their powerful learning ability and advantages in dealing with complex patterns, deep learning algorithms are ideal for image analysis challenges, particularly in the field of digital pathology. The variety of image analysis tasks in the context of deep learning includes classification (e.g., healthy vs. cancerous tissue), detection (e.g., lymphocytes and mitosis counting), and segmentation (e.g., nuclei and glands segmentation). The majority of recent machine learning methods in digital pathology have a pre- and/or post-processing stage which is integrated with a deep neural network. These stages, based on traditional image processing methods, are employed to make the subsequent classification, detection, or segmentation problem easier to solve. Several studies have shown how the integration of pre- and post-processing methods within a deep learning pipeline can further increase the model's performance when compared to the network by itself. The aim of this review is to provide an overview on the types of methods that are used within deep learning frameworks either to optimally prepare the input (pre-processing) or to improve the results of the network output (post-processing), focusing on digital pathology image analysis. Many of the techniques presented here, especially the post-processing methods, are not limited to digital pathology but can be extended to almost any image analysis field

New insights into androgen-dependent Wolffian duct development

In mammals, male and female fetal reproductive tracts are initially indistinguishable with Wolffian ducts (WD) and Mullerian ducts (MD) present in both sexes. Androgens play a vital role in masculinisation of the fetus, including WD rescue and development in males; however, the mechanisms that underlie this process are unknown. The aims of the current study therefore were to investigate the timing and mechanisms involved in androgen mediated WD development. The present study exposed pregnant rats to the androgen receptor antagonist, flutamide (50 or 100 mgkg⁻¹), Di(n-butyl) phthalate (DBP; 500 mgkg⁻¹) which reduces testicular testosterone production, and/or exogenous testosterone during specific time windows in fetal life in order to establish the key time windows for androgenregulated WD development and the possible mechanisms involved.These studies confirmed the vital role for androgens in WD development and highlighted their critical involvement in establishing the early patterning of WD development between el5.5-17.5, prior to any sign of morphological differentiation. At this stage, androgens receptors (AR) are only expressed in the stroma, not in the epithelium, thus androgens must regulate WD differentiation via stroma-epithelial interactions. Contrastingly, high levels of androgens were not required during morphological differentiation of the WD between el 9.5- 21.5 (when coiling occurs). Impaired androgen action during the correct window of development disrupted WD development as evidenced by reduced coiling of the future epididymis. This was likely due to a demonstrated reduction in cell proliferation in both stromal and epithelial compartments, impaired stromal differentiation, reduced epithelial cell height and ultimately epithelial degradation. These stromal abnormalities were noted prior to observing any obvious abnormalities in the epithelium, further highlighting regulation of the epithelium by the stroma. The mechanisms involved in this impaired WD development include interruption to the basement membrane and extracellular matrix, as evidenced by altered expression of some intermediate filaments. These were similar to the mechanisms noted in the regressing female WD but impaired androgen action did not induce apoptosis in the male WD, which was observed in the regressing WD in females. Maternal exposure to testosterone during gestation was able to rescue the female WD and even induce some degree of morphological differentiation, although this was to a lesser degree than that noted in the normal male

Investigation of intra-tumour heterogeneity to identify texture features to characterise and quantify neoplastic lesions on imaging

The aim of this work was to further our knowledge of using imaging data to discover image derived biomarkers and other information about the imaged tumour. Using scans obtained from multiple centres to discover and validate the models has advanced earlier research and provided a platform for further larger centre prospective studies. This work consists of two major studies which are describe separately: STUDY 1: NSCLC Purpose The aim of this multi-center study was to discover and validate radiomics classifiers as image-derived biomarkers for risk stratification of non-small-cell lung cancer (NSCLC). Patients and methods Pre-therapy PET scans from 358 Stage I–III NSCLC patients scheduled for radical radiotherapy/chemoradiotherapy acquired between October 2008 and December 2013 were included in this seven-institution study. Using a semiautomatic threshold method to segment the primary tumors, radiomics predictive classifiers were derived from a training set of 133 scans using TexLAB v2. Least absolute shrinkage and selection operator (LASSO) regression analysis allowed data dimension reduction and radiomics feature vector (FV) discovery. Multivariable analysis was performed to establish the relationship between FV, stage and overall survival (OS). Performance of the optimal FV was tested in an independent validation set of 204 patients, and a further independent set of 21 (TESTI) patients. Results Of 358 patients, 249 died within the follow-up period [median 22 (range 0–85) months]. From each primary tumor, 665 three-dimensional radiomics features from each of seven gray levels were extracted. The most predictive feature vector discovered (FVX) was independent of known prognostic factors, such as stage and tumor volume, and of interest to multi-center studies, invariant to the type of PET/CT manufacturer. Using the median cut-off, FVX predicted a 14-month survival difference in the validation cohort (N = 204, p = 0.00465; HR = 1.61, 95% CI 1.16–2.24). In the TESTI cohort, a smaller cohort that presented with unusually poor survival of stage I cancers, FVX correctly indicated a lack of survival difference (N = 21, p = 0.501). In contrast to the radiomics classifier, clinically routine PET variables including SUVmax, SUVmean and SUVpeak lacked any prognostic information. Conclusion PET-based radiomics classifiers derived from routine pre-treatment imaging possess intrinsic prognostic information for risk stratification of NSCLC patients to radiotherapy/chemo-radiotherapy. STUDY 2: Ovarian Cancer Purpose The 5-year survival of epithelial ovarian cancer is approximately 35-40%, prompting the need to develop additional methods such as biomarkers for personalised treatment. Patient and Methods 657 texture features were extracted from the CT scans of 364 untreated EOC patients. A 4-texture feature ‘Radiomic Prognostic Vector (RPV)’ was developed using machine learning methods on the training set. Results The RPV was able to identify the 5% of patients with the worst prognosis, significantly improving established prognostic methods and was further validated in two independent, multi-centre cohorts. In addition, the genetic, transcriptomic and proteomic analysis from two independent datasets demonstrated that stromal and DNA damage response pathways are activated in RPV-stratified tumours. Conclusion RPV could be used to guide personalised therapy of EOC. Overall, the two large datasets of different imaging modalities have increased our knowledge of texture analysis, improving the models currently available and provided us with more areas with which to implement these tools in the clinical setting.Open Acces

Development of Synchrotron Based Imaging Tools for Benign Prostatic Hyperplasia Using an Induced Canine Model

Benign prostatic hyperplasia (BPH) is a disease that develops spontaneously in men and dogs, and the dog is an accepted model to study BPH in men. As the gland can also be the site of a malignant cancer, definitive diagnosis relies on histological evaluation of prostate tissue following an invasive biopsy. The use of a new imaging technique, synchrotron-based phase contrast computed tomography (PC-CT) for excised prostates shows great detail of the internal structure of the gland, comparable to that of low power histology. Considering this fact, our main objective was to induce BPH in dogs using a combination of hormones dihydrotestosterone (DHT) and estradiol and verify if PC-CT imaging of in situ prostate glands of live dogs allowed for improved non-invasive imaging details compared to conventional medical imaging modalities and early diagnosis of BPH. Two studies were conducted to achieve the objectives. The first study involved the induction of BPH using intact male dogs with DHT and estradiol. The control group (n=3) received triolein carrier and the BPH induction group (n=3) received the hormone combination of DHT and estradiol (dissolved in triolein) injections three times/week for 35 weeks. Parameters that were assessed to diagnosis BPH were in vivo prostate volumes calculated using computed tomography (CT) images, volume of excised prostates determined using water displacement, 3D measurements of the excised gland, semen analysis, digital rectal exam (DRE), hormone analysis (estradiol, testosterone, DHT and canine prostate specific esterase CPSE), morphometric analysis and histological assessment of the tissue slides. The results showed that the gland volumes calculated both in vivo and ex vivo and the 3D measurements from BPH induction group were numerically higher than the control group. Sperm count decreased for all dogs but was reduced to zero or almost zero in the BPH induction group. The CPSE hormone analysis indicated that 1 dog from the treatment and 1 dog from the control group had BPH according to the manufacture’s defined threshold value. DHT hormone levels were higher for the induction group than controls throughout the entire study. This consequently affected the endogenous testosterone and estradiol, which were both decreased due to the negative feedback in the hypothalamic-pituitary-gonadal (HPG) axis. For DRE, prostates from all dogs were found to be enlarged in size by the end of the study and the histological diagnosis revealed that all dogs have a certain degree of BPH. The second study involved the imaging of all six dogs with conventional non-invasive modalities (magnetic resonance imaging, MRI; CT; positron emission tomography, PET-CT; ultrasound, US; radiographs) plus the innovative PC-CT technique at the Canadian Light Source (CLS). None of these techniques resulted in images with the same level of fine detail as that obtained with previous PC-CT imaging of excised canine prostates. Comparisons among images from the various modalities determined that the best modality for the visualization of the internal structures of the prostate gland such as capsule, parenchyma, septa, lobe, urethra and cysts was MRI (T2), followed by US and CT. PC-CT images were comparable with PET-CT, allowing the visualization of the lobes and urethra filled with tracer. In conclusion, all dogs developed BPH, either spontaneously (control group) or following induction (treatment group). Also, images of the in situ prostate gland of dogs were acquired for the first time at the CLS with the PC-CT technique. Although the quality and resolution was not as expected in comparison with PC-CT images of excised canine prostates, this technique shows promise and with additional study and development has the potential to become a useful diagnostic methodology

Characterisation of androgen receptor function in the male reproductive system through conditional gene targeting

Androgen receptor (AR) signalling is essential for the development and function of the male reproductive system. Conditional gene ablation using the Cre-loxP system has previously assisted in the elucidation of the role of AR in different cell types. The aim of this study was to examine the effects of the ablation of AR in previously untargeted cell types, with the hypothesis that this will have significant and novel effects on reproductive development and function that have not been previously documented by current models of androgen disruption. In these studies, three Cre recombinase lines were empirically validated for action in the male reproductive system, before being used to ablate AR and the phenotypes of the resulting lines were characterised. Endothelial-specific receptor tyrosine kinase (Tie2)-Cre was shown to target the vascular and endothelial cells of the testis, and used to ablate AR in these cells. The testes of the resulting Tie2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. Aquaporin 2 (Aqp2)-Cre was shown to target the post-meiotic germ cells of the testis, and was used to ablate AR in these cells. The testes of the resulting Aqp2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. It was concluded that the Ar gene was dispensable in the endothelial cells and post-meiotic germ cells of the testis for normal spermatogenesis. Forkhead box protein G1 (FoxG1)-Cre was shown to target the caput epididymal epithelium and pituitary, and used to ablate AR in these cells. d100 FoxG1-ARKO mice had a severe testicular phenotype, with sloughing of the seminiferous epithelium, atrophy of some seminiferous tubules and distension of the rete testis with spermatozoa. Despite the severe testis phenotype, ablation in the testis was incomplete and restricted to a small percentage of Leydig cells, with no ablation in Sertoli cells. Ablation of AR in the embryonic pituitary did not cause adult serum testosterone or LH concentrations to change, nor did it cause changes in other pituitary hormone transcripts. Mosaic ablation of AR in the caput epididymal epithelium was shown to impair epididymal development, with failure of initial segment (segment I) development and a significant decrease in epithelial cell height and lumen diameter in the remaining proximal caput epididymis (segment II). Dysfunction of the caput epididymis resulted in the failure of spermatozoa to transit the efferent ducts into the epididymis correctly: instead they were found to stall in the efferent ducts and produce a block. The testicular phenotype could be explained as the result of fluid backpressure effects resulting from the efferent duct block. Consequently, low concentration of spermatozoa in the cauda epididymis resulted in infertility in the FoxG1-ARKO, which represents a new model of obstructive azoospermia

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated

Embryonic Stem Cells

Embryonic stem cells are one of the key building blocks of the emerging multidisciplinary field of regenerative medicine, and discoveries and new technology related to embryonic stem cells are being made at an ever increasing rate. This book provides a snapshot of some of the research occurring across a wide range of areas related to embryonic stem cells, including new methods, tools and technologies; new understandings about the molecular biology and pluripotency of these cells; as well as new uses for and sources of embryonic stem cells. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines