Search Results (171)

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

Background/Objectives: Low semen quality and male infertility are critical global health issues. Emerging research highlights that nutritional factors could play a significant role in determining reproductive outcomes. Understanding and optimizing these dietary influences, including the role of polyphenols, is crucial for developing targeted strategies to improve male fertility. We aimed to explore the relationship between the intake of different classes of polyphenols and semen quality indicators in a cohort of healthy young males. Methods: This is a secondary analysis involving 106 male individuals, aged 18–35 years, from the FERTINUTS trial. Dietary intake was assessed using 3-day dietary records, and semen quality parameters were analyzed. Multivariable linear regression analysis was employed to evaluate the associations between dietary polyphenol consumption and semen quality indicators. Results: Our findings revealed both positive and negative associations between polyphenol consumption and sperm morphology parameters. A higher intake of total polyphenols was associated with a lower percentage of abnormalities in sperm heads but a higher rate of abnormalities in the principal piece. Similar results were observed for lignan and flavonoid intake. Additionally, a higher intake of flavonoids was also associated with a greater percentage of normal sperm forms. In contrast, a higher dietary intake of stilbenes was associated with a higher percentage of combined abnormalities. Conclusions: Higher intake of polyphenols, particularly flavonoids and lignans, was associated with improved sperm head morphology but also with increased tail abnormalities, although no associations with motility or vitality were observed. These results suggest that specific polyphenol classes may have both beneficial and adverse effects on sperm structure, warranting consideration of compound type and dosage in dietary recommendations. Further studies are needed to determine whether these morphological changes impact fertilization outcomes and reproductive potential. Full article

Under tropical conditions, seasonal variations may also influence the sperm characteristics of Bos indicus. The objective was to evaluate the motility of thawed sperm of bulls from the Peruvian tropics throughout the year. Over 24 months, 129 ejaculates were evaluated based on semen quality and subjected to slow horizontal freezing in 0.5 mL straws. After thawing, the individual, seasonal period, and season effect on motility and kinetic parameters were analyzed using a Sperm Class Analyzer^® (Microptic S.L.U., Barcelona, Spain). There was an individual effect on volume, motility, fresh concentration, and kinetic parameters when thawed. In the dry period, higher straight-line velocity (VSL) (p < 0.05) and beat cross frequency (BCF) were found than in the rainy period (p < 0.01). In summer and autumn, there was greater total motility, fast, circular routes, curvilinear velocity, average path velocity, VSL, amplitude of lateral displacement of the head, and BCF (p < 0.01). Greater volume and motility were found in winter and spring, but in summer and autumn, greater speed and vigor of movement were obtained in thawed sperm. The variation in annual climate patterns influences the seminal quality of bulls, and its effect needs to be assessed to propose adaptation strategies to climate change in tropical areas. Full article

The aim was to evaluate the effectiveness of semen cryopreservation and oocyte vitrification in roe deer as a potential method of gamete preservation for endangered deer species. Sperm were isolated from the cauda epididymis of fourteen bucks (n = 14). The motility measure (CASA) and morphology of fresh semen (FS) and frozen–thawed semen (TS) were compared. A hyaluronic binding assay was used to distinguish between mature FS spermatozoa expressing hyaluronan receptors and immature FS lacking these receptors, and the mitochondrial membrane potential (MMP) in TS was determined (flow cytometry). A Sperm–Hyaluronan Binding Assay (HBA) showed a viability rate of 61.9% in FS and 78.2% in TS. Oocytes received from eight does (n = 8) underwent a viability test and vitrification, and fresh oocytes from the other eight does (n = 8) were fertilized with TS and embryos were cultured until the blastocyst stage. The number of isolated oocytes, cumulus–oocyte complexes (COCs), cleaved embryos, and expanded blastocysts was evaluated. Higher percentages of morphological factors (acrosome, head, midpiece, and tail shape) were observed in FS compared to TS, whereas the motility and progressive movement were greater in TS (p ≤ 0.001). The viability was 50.5% and MMP was 40.6% in TS. A total of 311 oocytes were collected and from 150 COCs and 125 blastocysts developed. The viability of thawed oocytes after vitrification was 81%. The viability of vitrified oocytes and cryopreserved sperm confirmed the effectiveness of freezing protocols and highlights the potential for their implementation in other deer species. Full article

The development of biometric techniques in domestic animals has greatly advanced scientific practices in wildlife research. The association between seminal characteristics and body and testicular biometry enables the selection of suitable breeders, though appropriate measurement techniques are required. The present study assessed differences among conventional methods and formulas for estimating testicular parameters. Testicular length, width, and thickness were measured using three methods in 13 adult male domestic cats. Testicular area, volume, and weight were estimated, from which the gonadosomatic index (GSI) was calculated. Sperm were collected using an alpha-2 adrenergic agonist and urethral catheterization, and characterized in terms of volume, vigor, total motility, progressive motility, concentration, plasma membrane integrity, and morphology. The three methods were consistent in terms of testicular area, volume, weight, and GSI. Moderate positive correlations were observed for testicular weight (r = 0.61, p < 0.05) and GSI (r = 0.58, p < 0.05). Testicular parameters showed strong positive correlations among each other (r > 0.80, p < 0.05). We observed a moderate positive correlation between head length and progressive motility (r = 0.65, p < 0.05). In conclusion, all testicular measurement and estimation techniques showed comparable performance. Therefore, testicular biometry is useful for selecting breeding males in feline conservation programs, wherein larger body biometrics are related to improved seminal and reproductive parameters. Full article

Oxidative stress plays a critical role in regulating sperm function, yet species-specific antioxidant mechanisms remain poorly understood. This study compared hydrogen peroxide (H₂O₂) tolerance in horse and human sperm and investigated the roles of catalase and glutathione peroxidase (GPx) in horses. A H₂O₂ dose–response assay (0–2000 µM) showed that horse sperm were significantly more resistant to oxidative damage, with an IC₅₀ for progressive motility over 14-fold higher than that of human sperm (391.6 µM vs. 27.3 µM). Horse sperm also accumulated more intracellular H₂O₂ without loss of motility or viability. DNA damage assays (Halo and SCSA) revealed H₂O₂-induced fragmentation in human but not horse sperm. Enzyme inhibition experiments in horse sperm using 3-amino-1,2,4-triazole (catalase inhibitor) and (1S,3R)-RSL3 (GPx inhibitor) at 250 µM H₂O₂ showed that catalase inhibition severely impaired motility and increased intracellular H₂O₂ > 100-fold, while GPx inhibition had a milder effect (~5-fold increase). Immunocytochemistry localized catalase to the sperm head, particularly the post-acrosomal region, challenging the notion that sperm lack peroxisomes. The dependence of horse sperm on oxidative phosphorylation may drive the need for enhanced antioxidant defenses. These findings reveal species-specific oxidative stress adaptations and highlight catalase as a key antioxidant in equine sperm. Full article

The study investigated the sperm motility and morphometry of pre-freeze and post-thaw boar epididymal semen cooled at increasing holding times at 18 °C. A total of 50 testes of heterogeneous boars were collected (5 testes/day) from the local abattoir and transported to the laboratory at 5 °C within 30 min after slaughter. Semen was retrieved from the caudal part of the epididymis using the slicing float-up method, diluted with Beltsville Thawing Solution extender, pooled in a 50 mL centrifuge tube/5 testes/day, and cooled at 18 °C. Following each holding time (0, 3, 6, 9, 12, 24, and 48 h), the cooled semen sample was re-suspended with Fraction A extender and stored at 5 °C for an additional 45 min. A cooled resuspended semen sample was then diluted with Fraction B extender, loaded into 0.25 mL straws, and frozen using liquid nitrogen vapour. Thawing was accomplished by immersing the semen straws in warm (37 °C) water for 1 min and the samples were evaluated for sperm motility and morphometry traits using the computer-assisted sperm analyzer system. The data were analyzed using variance analysis. Descriptive statistics were used to assess sperm morphometry, establishing the minimum and maximum values. Boar epididymal sperm survived for up to 48 h when held at 18 °C. Furthermore, the highest post-thawed sperm motility rates were observed in semen frozen after 3 h of holding time, with a sperm total motility of 85.9%, a progressive motility of 60.3%, and a rapid motility of 33.2%, as compared to other holding times (p < 0.05). The acceptable ranges for pre-freeze and post-thawed sperm morphology were head length (8.4–9.1 µm), width (4.4–4.8 µm), area (29.9–38.2 µm²), perimeter (20.1–23.7 µm), midpiece width (1.1–2.8 µm), and sperm shape, were consistent regardless of the holding time. A holding time of 3 h enhances the cryoresistance of sperm cooled at 18 °C. Therefore, these findings suggest that boar epididymal sperm can be effectively conserved and can maintain fertilization capability when cooled for 3 h at 18 °C before freezing. Full article

Purpose: Non-obstructive azoospermia (NOA) presents major challenges in assisted reproductive technology (ART) due to the extremely low number of viable sperm within testicular tissue. In Micro-TESE procedures, embryologists manually search for sperm under DIC microscopy—a slow, labor-intensive process. We aim to streamline this process with an efficient computational detection tool. Methods: We present SD-CLIP (Sperm Detection using Classical Image Processing), a lightweight, real-time algorithm that simulates sperm structure detection from unstained DIC images. The model first identifies convex sperm head candidates based on shape and width using edge gradients, then confirms the presence of a tail via principal component analysis (PCA) of pixel clusters. Results: Compared to the MB-LBP + AKAZE method, SD-CLIP improved processing speed by 4× and achieved a 3.8× higher posterior probability ratio, making detected sperm candidates significantly more reliable. Evaluation was performed on both human Micro-TESE and mouse testis images, demonstrating robustness in low-sperm environments. Conclusions: SD-CLIP simulates a domain-specific image interpretation model that identifies sperm morphology with high specificity. It requires minimal computational resources, supports real-time integration, and could be extended to automated sperm extraction systems. This tool has clinical value for accelerating Micro-TESE and increasing success rates in ART for NOA patients. Full article

Phospholipase C zeta (PLCζ) has emerged as a pivotal sperm-specific factor responsible for triggering oocyte activation, a process essential for successful fertilization and early embryogenesis. A narrative review was conducted to examine the molecular architecture and biochemical features of PLCζ, with particular emphasis on how its distinctive structural domains facilitate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) and the induction of calcium (Ca²⁺) oscillations in the oocyte. Notably, PLCζ exhibits unique sensitivity to basal Ca²⁺ levels and the capacity to sustain repetitive intracellular Ca²⁺ transients that drive meiotic progression and block polyspermy. Clinically, PLCζ deficiency—whether caused by genetic mutations, reduced expression, or improper localization—represents a unifying explanation for certain forms of male infertility, including total fertilization failure (TFF) following intracytoplasmic sperm injection (ICSI). Globozoospermia is a prime example; this condition is characterized by round-headed sperm devoid of acrosomes and exhibiting significantly reduced or absent PLCζ and often results in fertilization failure. Diagnostic methods such as immunofluorescence, Western blotting, and the mouse oocyte-activation test collectively support the identification and characterization of PLCζ-related defects, while genetic testing for mutations in the PLCZ1 gene has proven valuable for identifying hereditary causes of sperm-borne oocyte-activation deficiency (OAD). Therapeutic approaches range from assisted oocyte activation (AOA) with calcium ionophores to emerging interventions that introduce functional PLCζ protein or mRNA directly into the oocyte. These advancements demonstrate the rapid translation of foundational discoveries into clinically actionable interventions. Future investigations are poised to refine diagnostic assays, standardize measurement protocols, and explore the potential of gene therapy or CRISPR/Cas9-mediated correction for heritable PLCζ abnormalities. By addressing both the molecular basis and translational applications of PLCζ, recent findings underscore its indispensable role in fertility care and lay out a path toward further innovation in assisted reproductive technologies. Full article

To perform accurate computer vision quality assessments of sperm used within reproductive medicine, a clear separation of each sperm component from the background is critical. This study systematically evaluates and compares the performance of Mask R-CNN, YOLOv8, YOLO11, and U-Net in multi-part sperm segmentation, focusing on the head, acrosome, nucleus, neck, and tail. This study conducts a quantitative analysis using a dataset of live, unstained human sperm, employing multiple metrics, including IoU, Dice, Precision, Recall, and F1 Score. The results indicate that Mask R-CNN outperforms other models in segmenting smaller and more regular structures (head, nucleus, and acrosome). In particular, it achieves a slightly higher IoU than YOLOv8 for the nucleus and surpasses YOLO11 for the acrosome, highlighting its robustness. For the neck, YOLOv8 performs comparably to or slightly better than Mask R-CNN, suggesting that single-stage models can rival two-stage models under certain conditions. For the morphologically complex tail, U-Net achieves the highest IoU, demonstrating the advantage of global perception and multi-scale feature extraction. These findings provide insights into model selection for sperm segmentation tasks, facilitating the optimization of segmentation architectures and advancing applications in assisted reproduction and biological image analysis. Full article

Sperm cryopreservation is helpful for maintaining the genetic diversity of fish species. This study was aimed at developing efficient methods to cryopreserve the sperm of three fish species, including koi carp (Cyprinus carpio L.), Ya fish (Schizothorax prenanti), and Glyptosternum maculatum. Firstly, based on the analysis of sperm viability, the cryomedium, dilution ratio, volume, and cooling procedure were assessed and optimized in koi carp. The results showed that the highest sperm viability was up to 63.23 ± 1.36% after a 14-day cryopreservation using the optimal method, briefly, sperm frozen with a volume of 50 μL (Vol.sperm:Vol.cryomedium = 1:9) of cryomedium containing 10% DMSO and 3% sucrose in D17 through ultrarapid cooling. Secondly, both the mitochondrial membrane potential and the DNA fragmentation index of sperm were examined and found to be significantly damaged after the cryopreservation. Intriguingly, the fertilization rate of sperm after 14-day cryopreservation is up to 63.03 ± 1.36% and the elongation of cryopreservation time (210 days) just slightly affected the fertilization rate (55.09 ± 4.70%) in koi carp. Thirdly, the optimal cryopreservation method was applied to cryopreserve Glyptosternum maculatum sperm; the cell viability was 45.39 ± 4.70%. And then this method, after a minor modification (3% sucrose of cryomedium replaced with 3% SMP) was adopted to cryopreserve Ya fish sperm, the cell viability was up to 70.45 ± 2.23%. Lastly, the ultrastructure and morphology of sperm was observed by SEM, and it was found that the cryopreservation prominently caused sperm head swelling and tail shortening in three fish species. In conclusion, this study established effective methods for cryopreserving sperm in three fish species and elaborated the injuries on sperm caused by cryopreservation. And the findings facilitate developing more protocols with practical value to cryopreserve sperm in different fish species. Full article

Type 1 diabetes (T1D) is a chronic metabolic disease defined by sustained hyperglycemia, leading to oxidative stress (OS) and systemic complications, including male subfertility. This study investigates the potential impact of T1D-induced OS on microtubule (MTs) dynamics and microtubule-associated proteins (MAPs) in the testis and spermatozoa (SPZ). Using a streptozotocin-induced T1D rat model, we examined the expression and localization of key MAPs, including Microtubule Affinity-Regulating Kinase 4 (MARK4), Microtubule-Associated Protein 1A (MAP1A), Dynein Light Chain LC8-Type 1 (DYNLL1), Prolyl Endopeptidase (PREP), and Radial Spoke Head 6 Homolog A (RSPH6A), alongside sperm functional parameters. Our findings showed that T1D significantly impaired the expression and distribution of these proteins, which may affect MTs organization and be associated with cytoskeletal disorganization, and impaired germ cell differentiation. Moreover, T1D rats exhibited reduced sperm count, viability, and motility, accompanied by increased DNA fragmentation and chromatin defects. Elevated levels of 4-hydroxy-2-nonenal (4-HNE), a marker of OS, were detected in SPZ, particularly in the acrosome and flagellum, correlating with mitochondrial dysfunction and ATP depletion. Additionally, decreased intracellular Ca²⁺ levels, downregulation of Cation Channel of Sperm (CATSPER) and Voltage-Dependent Anion Channel 3 (VDAC3), and altered tubulin acetylation, possibly due to imbalanced Alpha-Tubulin N-Acetyltransferase 1 (ATAT1) and Histone Deacetylase 6 (HDAC6) expression, were also associated with impaired sperm motility. The combined data suggest that T1D-induced OS is linked to disrupted MTs dynamics, which may contribute to testicular dysfunction and reduced sperm quality, potentially affecting male fertility. A better understanding of these associations may support the development of therapeutic strategies to mitigate the reproductive consequences of T1D and improve male fertility outcomes. Full article

The testis-specific serine kinases (TSSKs) are post-meiotically expressed in testicular germ cells. Their testis-specific expression, together with their putative role in phosphorylation pathways, suggests that TSSKs have relevant roles in spermiogenesis, sperm function, or both. Independent Tssk3 and Tssk6 knockout mice, as well as the double Tssk1/Tssk2 KO males, are sterile. However, the double KO results are silent regarding the individual roles of TSSK1 and TSSK2. The aim of this study was to develop independent mutant mouse models of Tssk1 and Tssk2, using CRISPR/Cas9, to evaluate their independent roles in reproduction. Male heterozygous pups were used to establish one Tssk1 and two independent Tssk2 mutant lines. Natural mating mutant Tssk1 and Tssk2 homozygous males but not females were found to be sterile. Additionally, homozygous males have lower sperm numbers and decreased motility, and were infertile in vitro. Anti-TSSK2 antibodies were validated against Tssk2 mutants and used in Western blot and immunofluorescence experiments. TSSK2 is localized to the sperm head; importantly, it is present in the testes and sperm from Tssk1 mutant mice, confirming individual mutation. Our results indicate that both TSSK1 and TSSK2 are individually essential for male reproduction and support both kinases as suitable nonhormonal male contraceptive targets. Full article

Male infertility is often linked to sperm quality issues; however, the mechanisms behind these alterations remain unclear in certain contexts. This study investigates the impact of anomalous Rho GTPase activation—a process triggered by bacterial toxins—on human sperm structure and function. Human spermatozoa were exposed in vitro to a Rho GTPase activator derived from Escherichia coli under both capacitating and non-capacitating conditions. The results showed increased RhoA GTPase activity in non-capacitating conditions, without affecting viability or mitochondrial membrane potential. However, progressive motility decreased across both conditions, while non-progressive motility and acrosome reaction rates increased. Additionally, intracellular calcium levels rose exclusively in non-capacitating conditions. Structural analysis revealed an increase in abnormal sperm morphology, particularly vacuoles in the sperm head. These findings highlight that anomalous Rho GTPase activation disrupts essential processes like motility and capacitation, which are crucial for successful fertilization. This study provides novel insights into how bacterial infections may induce sperm damage, proposing that Rho GTPase activity could serve as a biomarker for evaluating sperm quality in cases of infertility linked to urogenital infections. Understanding these mechanisms may improve diagnostic and therapeutic approaches for male infertility associated with bacterial pathogens. Human spermatozoa were exposed in vitro to a Rho GTPase activator derived from Escherichia coli under both capacitating and non-capacitating conditions. Full article

Penaeus monodon (black tiger shrimp) is one of the important shrimp species in aquaculture. Cryopreserving its sperm not only provides technical support for breeding but also effectively prevents the decline of genetic resources, promoting the sustainable development of its aquaculture industry. This study screened different types of diluents, cryoprotectants, and concentrations and explored equilibration time, cooling protocols, and thawing conditions, ultimately determining the optimal cryopreservation protocol for P. monodon sperm. The results showed that the optimal cryopreservation protocol involved using natural seawater as the diluent with 10% dimethyl sulfoxide (DMSO) as the cryoprotectant, in which the sperm suspension and cryoprotectant were mixed at a 1:1 (v/v) ratio and equilibrated at 4 °C for 30 min. Subsequently, cooling was performed using a programmable controlled-rate freezer: the temperature was reduced to −20 °C at −5 °C/min and held for 5 min; then cooled to −80 °C at −10 °C/min and held for 5 min; finally, the temperature was reduced to −180 °C at −20 °C/min. After cooling, the sperm samples were transferred to liquid nitrogen for long-term storage. The results demonstrated that thawing in a 37 °C water bath achieved the highest sperm motility compared to conditions at 27 °C, 32 °C, 42 °C, and 60 °C. After 15 days of liquid nitrogen storage, the sperm survival rate was 53.33 ± 9.18%. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed that the sperm structure was intact before freezing, with a rounded head, a distinct acrosomal spike anterior to the head, a concentrated nucleus in the head, dense chromatin, and a smooth cell membrane surface. However, after freezing and thawing, the acrosomal spikes of some sperm were fractured, and the membrane structure was damaged. Enzyme activity analysis showed that during liquid nitrogen storage from 0 to 15 days, the enzyme activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) in sperm gradually increased with significant differences observed compared to day 0 (p < 0.05). The activity of malondialdehyde (MDA) showed a gradual increase at 0, 5, and 10 days, but then decreased at day 15. The enzyme activity of catalase (CAT) showed no significant changes from 0 to 10 days (p > 0.05) but significantly increased on day 15 (p < 0.05). The activity of total superoxide dismutase (T-SOD) showed no significant changes from 0 to 5 days (p > 0.05) but significantly increased from days 10 to 15 (p < 0.05). These findings provide valuable insights into the cryopreservation of P. monodon sperm and will guide the optimization of cryoprotectant combinations and freezing protocols aimed at improving sperm survival rates. Full article