Search Results (1,152)

Myocardial infarction (MI) and its sequelae continue to be the leading cause of mortality globally. Following MI, a series of structural pathophysiological changes occur in the myocardium, including sympathetic remodeling. Tyrosine hydroxylase (TH), protein gene product 9.5 (PGP9.5), and synaptophysin (SYN) are recognized as key markers of sympathetic nerves. However, the expression patterns of these biomarkers during sympathetic remodeling, particularly their temporal profiles, remain insufficiently characterized. A cohort of 60 healthy adult male C57BL/6 mice was randomly divided into a control group (n = 12) and four MI groups with postoperative intervals of 2, 5, 7, and 10 days (n = 12/group). MI was induced via permanent ligation of the left anterior descending coronary artery (LAD). Cardiac tissues were subjected to histological analyses (HE and Masson’s trichrome staining), immunohistochemical profiling, and quantitative reverse-transcriptase PCR (qRT-PCR) (TH, PGP9.5, and SYN). Immunohistochemical staining revealed that TH-, PGP9.5-, and SYN-immunopositive sympathetic nerves were present in the epicardium, myocardial interstitium, and the periphery of small blood vessels in normal mice. Normal cardiomyocytes were negative for TH but exhibited focal expression of PGP9.5 and SYN. In the myocardial infarction tissue, TH-positive staining indicated sympathetic nerve proliferation in the epicardium, myocardial infarction border zone, and infarct zone, with peak expression occurring at 7 days post-MI. In contrast to TH, PGP9.5 exhibited prominent immunoreactivity, specifically localized to the infarct core and peri-infarct zone cardiomyocytes, while SYN was primarily located in fibroblast-like cells within the same region. qRT-PCR analyses revealed that the time-dependent trends of TH, PGP9.5, and SYN mRNAs exhibited similarities, peaking between 5 and 7 days post-MI. TH demonstrates higher specificity than PGP9.5 and SYN in sympathetic nerve identification, solidifying its role as the optimal biomarker for post-MI sympathetic remodeling. The ectopic expression of PGP9.5 and SYN in non-neuronal cells within myocardial infarction tissue remains speculative and requires further mechanistic studies for validation. Full article

Background/Objectives: Exogenous ketone supplements elevate circulating ketones without carbohydrate restriction, but their long-term hepatic safety remains unclear. This study evaluated the formulation-dependent impact of chronic ketone supplementation on liver histopathology, inflammatory signaling, and systemic biomarkers in rats. Methods: Male Sprague-Dawley rats were orally administered 1,3-butanediol (BD), medium-chain triglycerides (MCTs), ketone ester (KE), ketone electrolytes/salts (KSs), or a ketone salt–MCT combination (KSMCT) for 4 weeks. In a separate arm, animals received standard diet (SD), or SD supplemented with low-dose KE (LKE) or high-dose KE (HKE), for 83 days. Liver structure was assessed by hematoxylin and eosin staining with quantification of red blood cell density and lipid accumulation. Inflammatory and metabolic responses were evaluated by TNF-α and arginase immunohistochemistry. Serum biochemistry included glucose, proteins, electrolytes, and liver and kidney function markers. Results: BD and KE induced macrovesicular steatosis, vascular congestion, and elevated TNF-α and arginase expression, consistent with hepatic stress. MCT caused moderate hepatocellular ballooning and lipid deposition, whereas KS preserved near-normal hepatic morphology. KSMCT produced intermediate effects, reducing lipid accumulation and TNF-α compared with MCT or KE alone. KE supplementation caused dose-dependent reductions in globulin and elevations in creatinine, while HKE reduced sodium and glucose levels. Conclusions: Chronic hepatic responses to exogenous ketones are highly formulation dependent. KS demonstrated the most favorable safety profile under the tested conditions, maintaining normal hepatic structure, while BD and KE elicited adverse changes. Formulation choice is critical for the safe long-term use of exogenous ketones. Full article

Background/Objectives: Hemp (Cannabis sativa L. subsp. sativa) is a plant within the Cannabis sativa species and utilized for several applications, including antioxidation, antihypertension, and anti-inflammation. To our knowledge, no prior study has assessed the acute and sub-chronic oral safety of hemp leaf oil in Sprague-Dawley rats under Thailand-compliant THC levels. This study investigates the acute and sub-chronic effects of Hemp leaf oil (HLO) on the heart, liver, and kidneys of male and female Sprague-Dawley rats. Methods: Six-week-old male and female Sprague-Dawley rats were administered HLO (1.5 mL/kg) intragastrically, either as a single dose or a repeat dose over 28 days. Results: No changes in body or organ weights were observed following acute and sub-chronic HLO administration in sex-matched groups. Moreover, blood pressure and heart rate remained comparable across groups after acute and sub-chronic HLO treatment. Both acute and sub-chronic administration of HLO did not influence electrolyte balance, liver enzymes, total protein, albumin, blood urea nitrogen, or creatinine levels. Hematoxylin and eosin staining revealed the normal morphology of the heart, liver, and kidneys in rats subjected to HLO, during both acute and sub-chronic treatment. Conclusions: In conclusion, our data suggested that both acute and sub-chronic administration of HLO at 1.5 mL/kg could be safe for the vital organs. These findings support the potential use of HLO in therapeutic applications, particularly in scenarios when the safety of essential organs is at stake. Full article

Objective: The high expression of prostate-specific membrane antigen (PSMA) associated with neovascularization in non-prostatic malignant tumors and metastatic lesions has been documented in many studies. By taking advantage of the absence of PSMA-related background activity in brain tissue, in recent years, PSMA has been used for the imaging of glial tumors, especially for postoperative follow-up. The aim of this prospective study was to investigate the diagnostic value of ⁶⁸Ga-PSMA-11 PET/CT by comparing ⁶⁸Ga-PSMA-11 PET/CT, ¹⁸F-FDG PET/CT, and MRI findings in patients with brain metastases (BM). Materials and Method: In this prospective study, 27 cases, 11 female and 16 male, with a mean age of 59.48 ± 12.21 years, were included. Patients diagnosed with BM on ¹⁸F-FDG PET/CT or CT/MRI at initial diagnosis or in the follow-up period were included in the study. PET findings of BM lesions obtained from ¹⁸F-FDG and ⁶⁸Ga-PSMA-11 PET/CT imaging, demographic characteristics, histopathological data of the primary foci, and other clinical features were evaluated together. Results: Twenty-four (89%) patients were included in the study for restaging, two (7%) patients for local recurrence assessment, and one (4%) patient for local recurrence and suspicion of additional lesions. The indications for ¹⁸F-FDG PET/CT were breast carcinoma for 37% (n:10), followed by lung carcinoma for 26% (n:7), colorectal adenocarcinoma for 14% (n:4), squamous cell larynx carcinoma for 7% (n:2), gastric signet ring cell carcinoma for 4% (n:1), pancreatic NET3 for 4% (n:1), thyroid papillary carcinoma for 4% (n:1), and malignant melanoma for 4% (n:1). In total, 26/27 included patients had PSMA-positive brain metastases but only one patient had PSMA-negative brain metastases with ⁶⁸Ga-PSMA-11 PET/CT imaging. This patient was followed with a diagnosis of primary larynx squamous carcinoma and had a mass suspected of brain metastases. Further tests and an MRI revealed that the lesion in this patient was a hemorrhagic metastasis. The smallest metastatic focus on ⁶⁸Ga-PSMA-11 PET/CT imaging was 0.22 cm, also confirmed by MRI (range: 0.22–2.81 cm). The mean ± SD SUVmax of the BM lesions was 17.9 ± 8.6 and 6.8 ± 5.2 on ¹⁸F-FDG PET/CT and ⁶⁸Ga-PSMA-11 PET/CT imaging, respectively. Metastatic foci that could not be detected by ¹⁸F-FDG PET/CT imaging were successfully detected with ⁶⁸Ga-PSMA-11 PET/CT imaging in 11 cases (42%). The distribution and number of metastatic lesions observed on cranial MRI and ⁶⁸Ga-PSMA-11 PET/CT were compatible with each other for all patients. Immunohistochemical staining was performed in the primary tumor of 10 (38%) cases, and positive IHC staining with PSMA was detected in 5 patients. In addition, positive IHC staining with PSMA was detected in all of the four surgically excised brain metastatic tumor foci. Conclusions: In this study,⁶⁸Ga-PSMA-11 PET/CT appears to be superior to ¹⁸F-FDG in detecting BM from various tumors, largely due to its high expression associated with neovascularization and the absence of PSMA expression in normal brain parenchyma. This lack of physiological uptake in healthy brain tissue provides excellent tumor-to-background contrast, further supporting the advantage of ⁶⁸Ga-PSMA-11 over ¹⁸F-FDG for BM imaging. However, larger studies are required to confirm these findings, particularly through comparisons across tumor types and histopathological subgroups, integrating PSMA immunohistochemistry (IHC) scores with ⁶⁸Ga-PSMA-11 uptake levels. Beyond its diagnostic potential, our results may also inform PSMA-targeted therapeutic strategies, offering new perspectives for the management of patients with brain metastases from diverse primary tumors. Full article

This study investigated the structural features of adult duck liver and compared pathological alterations induced by duck Tembusu virus (DTMUV, strain XZ-2012) and lipopolysaccharide (LPS). Histological techniques (HE, reticular fiber, and trichrome staining) revealed normal duck liver exhibited reddish-brown coloration with indistinct lobule boundaries and no prominent bile ducts. Kupffer cell distribution was mapped via jugular ink injection. DTMUV infection caused liver swelling, congestion, and yellowish discoloration. Histopathology showed lymphocyte infiltration around central veins and portal areas, increased reticular fibers, thickened basement membranes, hepatocyte vacuolation, and erythrocyte accumulation in sinusoids. In contrast, LPS exposure led to mild hepatic enlargement without vacuolar degeneration but with marked perivascular lymphocyte aggregation and reticular fiber proliferation. Both treatments elevated Kupffer cell numbers. These findings demonstrate distinct liver injury patterns: DTMUV induces direct hepatocellular damage with inflammatory responses, while LPS triggers intense immune cell recruitment without significant hepatocyte degeneration. The study provides insights into avian viral versus bacterial pathogenesis and liver defense mechanisms, offering a foundation for further research into waterfowl infectious diseases. Full article

Background/Objectives: Gastric cancer (GC), a prevalent global malignancy and a leading cause of cancer-related mortality, has a poorly understood prognosis related to TRIP13 expression. TRIP13 has a recognized part in driving tumor progression across different cancer types, yet its precise role in GC remains beyond our full comprehension. Our study aimed to explore TRIP13’s prognostic value and function in GC patients. Methods: We extensively explored TRIP13’s influence on GC prognosis, functionality, and immune response by examining various cancer-related databases like UALCAN, GEPIA, GEO, and TIMER. Immunohistochemistry (IHC) staining was also conducted to assess the link between TRIM13 and GC patient survival. Results: TRIP13 expression levels were found to be significantly elevated in GC tissues compared to normal tissues through analysis of mRNA data from multiple public databases. IHC analysis exposed elevated TRIP13 protein levels in GC tissues and connected it with tumor depth. Prognostic evaluation demonstrated that GC patients exhibiting heightened TRIP13 expression endured a diminished overall survival rate. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that genes related to TRIP13 are involved in processes such as the cell cycle and DNA repair. Additionally, TRIP13 expression was found to correlate with ferroptosis-related genes and may play a role in regulating ferroptosis. Immune cell infiltration analysis demonstrated that TRIP13 expression is negatively correlated with the infiltration of CD4⁺ T cells, CD8⁺ T cells, and B cells. Conclusions: TRIP13 emerges as a candidate independent prognostic indicator and a promising intervention point for GC treatment. Full article

Background/Objectives: Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. Inflammation and metabolic dysregulation, particularly those related to obesity, have emerged as critical contributors to CRC progression. Interleukin-6 (IL-6) and retinol-binding protein 4 (RBP4), an adipokine involved in metabolic regulation, may be key mediators of these processes. This study aimed to evaluate the expression levels of IL-6 and RBP4 in CRC tissues and their associations with clinicopathological features and overall survival. Furthermore, in silico analyses were performed to explore the molecular networks and signaling pathways related to both biomarkers. Methods: Immunohistochemical staining of IL-6 and RBP4 was conducted in 118 CRC and matched adjacent normal tissues. Expression levels were assessed using the H-score system and correlated with clinical parameters. Survival analysis was performed using Kaplan–Meier curves. In silico analyses were based on RNA-seq data from TCGA and included pathway enrichment, gene co-expression, and protein–protein interaction networks. Results: IL-6 and RBP4 expression were significantly elevated in tumor tissue compared to adjacent normal mucosa. High IL-6 expression correlated with age and obesity measures, while RBP4 expression showed significant associations with pT stage, lymph node involvement, TNM stage, and obesity-related parameters. Kaplan–Meier analyses indicated shorter overall survival in patients with high IL-6 or RBP4 expression. In silico analysis confirmed upregulation of IL6 and RBP4 in CRC and highlighted immune-related pathways for IL-6 and developmental signaling for RBP4. Conclusions: Elevated expression of IL-6 and RBP4 in CRC tissue is associated with adverse clinical features and reduced survival, underscoring their potential role as prognostic biomarkers. These findings support the involvement of inflammation and metabolic dysfunction in CRC progression and suggest IL-6 and RBP4 as candidates for future targeted therapeutic approaches. Full article

Background/Objectives: Chronic endometritis (CE) is a subclinical inflammation of the endometrium that affects female fertility. Although awareness of its impact on reproductive outcomes has increased significantly, clinical management—especially the diagnostic value of hysteroscopy and the effectiveness of perioperative antibiotic prophylaxis in improving fertility—remains unclear. Methods: This retrospective analysis involved 136 infertile women (30–44 years) who underwent diagnostic hysteroscopy between 2022 and 2023 at the Military Institute of Medicine in Warsaw. Women with intrauterine pathologies or other infertility factors were excluded. Hysteroscopic indicators of chronic endometritis (CE) included micropolyps and endometrial hyperemia. Endometrial biopsies were stained with CD138 and CE was diagnosed based on ≥5 plasma cells per 10 high-power fields. A single oral dose of azithromycin was administered post-procedure and pregnancy outcomes were assessed 12 months later. Results: CE was histologically confirmed in 29.2% of patients. The presence of micropolyps demonstrated a strong correlation with CE (p < 0.0001), although CE was also found in 21% of patients with normal hysteroscopic findings. While CE status did not significantly influence pregnancy rates, patients who received azithromycin exhibited a significantly higher conception rate (53% vs. 21%, p = 0.022). Additionally, secondary infertility was associated with higher reproductive success compared to primary infertility (54% vs. 24%, p = 0.022). Conclusions: Micropolyps are a specific hysteroscopic marker of CE. However, histologic inflammation markers may be present even in the absence of abnormal hysteroscopic findings. Furthermore, the routine use of antibiotic prophylaxis is associated with improved reproductive outcomes. Full article

Background and Objective: The color normalization of breast cancer immunohistochemistry (IHC)-stained images helps change the color distribution of undesirable IHC-stained images to be more interpretable for the pathologists. This will affect the Allred score that the pathologists use to estimate the drug quantity for treating breast cancer patients. Methods: A new color normalization technique based on sparse stain separation and self-sparse fuzzy clustering is proposed. Results: The quaternion structural similarity was used to measure the quality of the normalization algorithm. Our technique has a structural similarity score lower than other techniques, and the color distribution similarity is closer to the target. We applied automated and unsupervised nuclei classification with Automatic Color Deconvolution (ACD) to test the color features extracted from normalized images. Conclusions: The classification result from our unsupervised nuclei classification with ACD is similar to other normalization methods, but it offers an easier perception to the pathologists. Full article

Objectives: Tobacco has been associated with the development of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC). This study aimed to evaluate the in vitro and in vivo changes caused by carcinogen 4-nitroquinoline 1-oxide (4NQO), simulating smoking conditions. Materials and Methods: In the in vitro study, normal keratinocytes were exposed to 1.3 µM and 2.6 µM concentrations of 4NQO to induce dysplastic transformation (H-DISP) and malignant transformation (H-SCC), respectively. The cells were collected and subjected to hematoxylin and eosin (H&E) staining and immunocytochemistry with Ki-67. For the in vivo study, female C57BL/6J mice were divided into a pure control (PC) group and experimental groups exposed to 50 µg/mL (NQ) and 100 µg/mL (CM) of 4NQO in autoclaved drinking water. Each group was euthanized after 8, 12, 16, and 20 weeks of exposure. The tongues were collected, processed, stained with H&E, and analyzed using conventional light microscopy. Results: In vitro, significant morphological changes were observed in the H-DISP and H-SCC groups, with a cell proliferation index exceeding 30% in the H-DISP group. In vivo, the CM group showed greater progression to severe dysplasia/carcinoma within a shorter treatment period compared to the NQ group. Conclusions: We established critical doses and exposure durations for 4NQO, both in vitro and in vivo, to induce cellular changes and the formation of OL and OSCC, providing a standardized model for studies related to oral carcinogenesis. Full article

Yin Yang 1 (YY1) is a multifunctional transcription factor implicated in gene regulation, cell proliferation, and survival. While its role in breast cancer (BC) has been explored, its prognostic significance remains controversial. In this study, we evaluated nuclear YY1 expression in 276 BC tissue samples using immunohistochemistry (IHC), tissue microarrays (TMAs), and digital pathology (DP). Nuclear staining was quantified using Aperio ImageScope software, focusing on tumor regions to avoid confounding from stromal or non-tumor tissues. This selective and standardized approach enabled precise quantification of YY1 expression. Our results show elevated median YY1 expression in tumor vs. normal matched tissues (p < 0.001). The optimal cutoff for medium-intensity nuclear YY1 expression in tumor areas for overall survival (OS) was established by a receiver operating characteristic (ROC) curve (AUC = 0.718, 95% CI: 0.587–0.849, p = 0.008). In contrast, ROC curves showed no prognostic impact (AUC and p-value) for YY1 quantification in whole spots (tumor + normal). As a categorical variable, high YY1 expression was correlated with more aggressive BC features, including tumor size > 3 cm (57.7% vs. 44.2% p = 0.037), the triple-negative breast cancer (TNBC) molecular subtype (27.3% vs. 13.9% p = 0.026), and advanced prognostic stage (III) (31.8% vs. 16.7% p = 0.003), while as a continuous variable, YY1 was associated with higher histological (p = 0.003) and nuclear grades (p = 0.022). High YY1 expression was significantly associated with a reduced OS of BC patients, as shown by Kaplan–Meier curves (HR = 2.227, p = 0.002). Since YY1 was significantly enriched in TNBC, we evaluated its prognostic resolution in this subgroup. But, probably due to the small number of patients within this subset, our results were not statistically significant (HR = 1.317, 95% CI: 0.510–3.405, p = 0.566). Next, we performed multivariate Cox regression, confirming YY1 as an independent prognostic factor for overall survival (HR = 1.927, 95% CI: 1.144–3.247, p = 0.014). In order to improve prognostic value, we constructed a mathematical model derived from the multivariate Cox regression results, including YYI, AJCC prognostic stage (STA), and axillary lymph node dissection (ALN), with the following equation: h(t) = h₀(t) × exp (0.695 × YY1 + 1.103 × STA − 0.503 × ALN). ROC analysis of this model showed a better AUC of 0.915, similar sensitivity (83.3%), and much higher specificity (92%). Bioinformatic analysis of public datasets supported these findings in BC, showing YY1 overexpression in multiple cancer types and its association with poor outcomes in BC. These results suggest that YY1 may play a role in tumor progression and serve as a valuable prognostic biomarker in BC. DP combined with molecular data enhanced biomarker accuracy, supporting clinical applications of YY1 in routine diagnostics and personalized therapy. Additionally, developing a combined score based on the modeling of multiple prognostic factors significantly enhanced survival predictions, representing a practical tool for risk stratification and the guidance of therapeutic decisions. Full article

Background and Objectives: The Drosophila retinal determination network comprises the transcription factor Teashirt (Tsh) and the transcription co-regulator C-terminal Binding Protein (CtBP), both of which are essential for normal adult eye development. Both Tsh and CtBP show a pattern of co-expression in the proliferating cells anterior to the morphogenetic furrow that demarcates the boundary between the anteriorly placed proliferating eye precursor cells and the posteriorly placed differentiating photoreceptor cells in the larval eye-precursor tissue, the eye–antennal disc. The disc ultimately develops into the adult compound eyes, antenna, and other head structures. Both Tsh and CtBP were found to interact genetically during ectopic eye formation in Drosophila, and both were present in molecular complexes purified from gut and cultured cells. However, it remained unknown whether Tsh and CtBP molecules could interact in the eye–antennal discs and elicit an effect on eye development. The present study answers these questions. Methods: 5′ GFP-tagging of the tsh gene in the Drosophila genome and 5′ FLAG-tagging of the ctbp gene were accomplished by the CRISPR-Cas9 and BAC recombineering methods, respectively, to produce GFP-Tsh- and FLAG-CtBP-fused proteins in specific transgenic Drosophila strains. Verification of these proteins’ expression in the larval eye–antennal discs was performed by immunohistological staining and confocal microscopy. Genetic screening was performed to establish functional interaction between Tsh and CtBP during eye development. Scanning Electron Microscopy was performed to image the adult eye structure. Co-immunoprecipitation and GST pulldown assays were performed to show that Tsh and CtBP interact in the cells of the third instar eye–antennal discs. Results: This study reveals that Tsh and CtBP interact genetically and physically in the Drosophila third instar larval eye–antennal disc to regulate adult eye development. This interaction is likely to limit the population of the eye precursor cells in the larval eye disc of Drosophila. Conclusions: The relative abundance of Tsh and CtBP in the third instar larval eye–antennal disc can dictate the outcome of their interaction on the Drosophila eye formation. Full article

This study aimed to evaluate how acidic and alkaline staining solutions affect the optical properties (mean color change, ΔE*), geometric characteristics (surface roughness, Ra), and bacterial adhesion of zirconia Ceramill Zolid PS computer-aided design/computer-aided manufacture (CAD/CAM) material after 21 days of immersion. Ninety-six zirconia CAD/CAM Ceramill Zolid multilayer PS specimens were prepared and allocated to eight groups based on the pH values of the immersion solutions; the acidic solutions included Mirinda Citrus, CodeRed, yerba mate tea, Saudi coffee, and Nescafe (A–E), and the alkaline solutions included artificial saliva, DZRT (tobacco-free nicotine pouches), and smokeless tobacco (F–H). The specimens were immersed for 21 days at 37 °C, with the solutions replaced every 12 h to ensure consistency. Color changes were measured using a VITA Easyshade V spectrophotometer, and Ra was evaluated via white-light interferometric microscopy. The bacterial adhesion of Streptococcus mutans was quantified by counting colony-forming units (CFUs, CFU/mm²). Statistical analyses included the Shapiro–Wilk test for normality, one-way ANOVA with Tukey’s HSD post hoc test for group comparisons, and paired t-tests, with significance set at <0.05. The recorded pH values of the staining materials ranged from acidic (Mirinda Citrus: 3.23) to alkaline (smokeless tobacco: 8.54). Smokeless tobacco caused the most unacceptable mean color change (ΔE* = 6.84), followed by DZRT (ΔE* = 6.46), whereas artificial saliva produced the least discoloration (ΔE* = 2.15), with statistically significant differences among the solutions (p < 0.001). The Ra measurements varied significantly (p < 0.001), with Nescafe demonstrating the lowest value (0.486 µm) and DZRT the highest (0.748 µm). S. mutans adhesion was the highest for CodeRed (546.75 CFU) and the lowest for smokeless tobacco (283.92 CFU), demonstrating significant variation across groups (ANOVA, p < 0.001). The acidic and alkaline solutions significantly altered the optical properties, Ra, and bacterial adhesion of zirconia Ceramill Zolid PS CAD/CAM, with acidic solutions leading to higher bacterial adhesion. Full article

Accurate and automated segmentation of white blood cells (WBCs) in whole slide images (WSIs) is a critical step in computational pathology. This study presents a comprehensive evaluation and enhancement of the StarDist algorithm, leveraging its star-convex polygonal modeling to improve segmentation precision in complex WSI datasets. Our pipeline integrates tailored preprocessing, expert annotations from QuPath, and adaptive learning strategies for model training. Comparative analysis with U-Net and Mask R-CNN demonstrates StarDist’s superiority across multiple performance metrics, including Dice coefficient (0.89), precision (0.99), and IoU (0.95). Visual evaluations further highlight its robustness in handling overlapping cells and staining inconsistencies. The study establishes StarDist as a reliable tool for digital pathology, with potential integration into clinical decision-support systems. In addition to Dice and IoU, metrics such as Aggregated Jaccard Index and Boundary F1-Score are gaining popularity for biomedical segmentation. Preprocessing techniques like Macenko stain normalization and adaptive histogram equalization can further improve generalizability. QuPath, an open-source digital pathology platform, was utilized to perform accurate WBC annotations prior to training and evaluation. Full article

Introduction: Sperm morphology is a fundamental parameter in the evaluation of male infertility, offering critical insights into reproductive health. However, traditional manual assessments under microscopy are limited by operator dependency and subjective interpretation caused by biological variation. To overcome these limitations, there is a need for accurate and fully automated classification systems. Objectives: This study aims to develop a two-stage, fully automated sperm morphology classification framework that can accurately identify a wide spectrum of abnormalities. The framework is designed to reduce subjectivity, minimize misclassification between visually similar categories, and provide more reliable diagnostic support in reproductive healthcare. Methods: A novel two-stage deep learning-based framework is proposed utilizing images from three staining-specific versions of a comprehensive 18-class dataset. In the first stage, sperm images are categorized into two principal groups: (1) head and neck region abnormalities, and (2) normal morphology together with tail-related abnormalities. In the second stage, a customized ensemble model—integrating four distinct deep learning architectures, including DeepMind’s NFNet-F4 and vision transformer (ViT) variants—is employed for detailed abnormality classification. Unlike conventional majority voting, a structured multi-stage voting strategy is introduced to enhance decision reliability. Results: The proposed framework consistently outperforms single-model baselines, achieving accuracies of 69.43%, 71.34%, and 68.41% across the three staining protocols. These results correspond to a statistically significant 4.38% improvement over prior approaches in the literature. Moreover, the two-stage system substantially reduces misclassification among visually similar categories, demonstrating enhanced ability to detect subtle morphological variations. Conclusions: The proposed two-stage, ensemble-based framework provides a robust and accurate solution for automated sperm morphology classification. By combining hierarchical classification with structured decision fusion, the method advances beyond traditional and single-model approaches, offering a reliable and scalable tool for clinical decision-making in male fertility assessment. Full article