Background: Varicocele is a common condition involving the dilation of veins in the scrotum, often linked to male infertility and testicular dysfunction. This study aimed to elucidate the molecular effects of successful varicocele treatment on sperm proteomes following percutaneous sclero-embolization. Methods: High-resolution tandem mass spectrometry was performed for proteomic profiling of pooled sperm lysates from five patients exhibiting improved semen parameters before and after (3 and 6 months) varicocele sclero-embolization. Data were validated by Western blot analysis. Results: Seven proteins were found exclusively in varicocele patients before surgery—such as stathmin, IFT20, selenide, and ADAM21—linked to inflammation and oxidative stress. After sclero-embolization, 55 new proteins emerged, including antioxidant enzymes like selenoprotein P and GPX3. Thioredoxin (TXN) and peroxiredoxin (PRDX3) were upregulated, indicating restoration of key antioxidant pathways. Additionally, the downregulation of some histones and the autophagy-related protein ATG9A suggests a shift toward an improved chromatin organization and a healthier cellular environment post-treatment. Conclusions: Varicocele treatment that improves sperm quality and fertility parameters leads to significant proteome modulation. These changes include reduced oxidative stress and broadly restored sperm maturation. Despite the limited patient cohort analyzed, these preliminary findings provide valuable insights into how varicocele treatment might enhance male fertility and suggest potential biomarkers for improved male infertility treatment strategies.

Male accessory gland inflammation (MAGI) can impair male fertility through inflammation-driven oxidative stress and direct sperm damage; nutraceutical approaches may be useful when antibiotics are not indicated. Here, we evaluated a 3-month treatment with a Graminex™-based dietary supplement (Deprox-HP) in twenty MAGI patients integrating conventional semen analysis and oxidative stress assessment with sperm proteomics before and after therapy. After treatment, total and progressive sperm motility increased significantly, whereas sperm concentration and sperm morphology showed a non-significant upward trend. Sperm lipid peroxidation decreased markedly, while the antioxidant capacity showed a non-significant increase. Analysis of the sperm proteome demonstrated a clear PRE–POST clustering, consistent with treatment-associated remodeling. POST samples showed upregulation of proteins linked to sperm motility, redox homeostasis, mitochondrial metabolism and membrane remodeling. Two pregnancies occurred during the treatment period; in both cases, lipid peroxidation decreased along with an increase of morphologically typical spermatozoa, and sperm proteomics showed a concordant post-treatment shift enriched in flagellar and mitochondrial respiratory/redox compartments. Moreover, we found a selective enrichment POST treatment in these two patients of TEX50, a crucial protein involved in acrosome/head-stability during epididymal transit. Overall, Deprox-HP was associated with reduced oxidative membrane damage and a coordinated sperm proteomic shift consistent with improved motility.

Microfluidic platforms have emerged as critical technologies for exploring sperm chemotaxis, providing precise gradient control, and facilitating in-depth behavioral assessment. We designed a novel, user-friendly microfluidic device that is optimized for human sperm morphology and motility. The device was validated using two well-established sperm chemoattractants, progesterone and bourgeonal, demonstrating its reliability and reproducibility. Given the key role of olfactory receptors (ORs) in mediating sperm chemotaxis, the newly developed device was employed to identify additional receptors that may contribute to sperm behavior. Using the Atlas database, we identified OR2H1 as a candidate receptor. It is enriched in testis-derived cells, particularly in early and late spermatids, and it is broadly expressed across human spermatozoa. We demonstrated that OR2H1’s ligand, methional, a sulfur-containing aldehyde naturally found in vaginal fluid and biosynthesized by Lactococcus lactis, significantly enhances sperm migration and progressive motility. Methional stimulation also triggered increased intracellular calcium levels, indicating receptor activation. Computer-assisted sperm analysis revealed that methional treatment improved sperm linearity, straightness, and wobble without affecting the average velocity, suggesting enhanced directional movement. These findings provide evidence that methional promotes sperm chemotaxis via OR2H1 and highlight the potential role of the vaginal microbiome in influencing human fertility.

Human follicular development depends on coordinated communication between granulosa cells (GCs) and oocytes through endocrine cues, direct contacts, and extracellular vesicles (EVs). Exosomes are key EV mediators of intrafollicular signaling, but their cargo and functions in gonadotropin-stimulated GCs remain poorly defined. The human granulosa-like tumor cell line KGN was used to investigate exosome secretion and protein composition following human chorionic gonadotropin (hCG) stimulation. Exosomes were isolated by ultracentrifugation, characterized via nanoparticle tracking analysis (NTA), Scanning Electron Microscopy (SEM) and Western blotting, and analyzed using high-resolution mass spectrometry. Comparative proteomics integrating exosomal profiles with the whole secretome were performed, followed by bioinformatic analyses of protein networks, gene ontology, and pathway enrichment. hCG reshaped exosomal cargo, identifying 59 proteins enriched in exosomes, including Integrin α3 (ITGα3), Galectin-3-binding protein (LGALS3BP), tetraspanins (CD63, CD151), and proteasome subunits. Functional enrichment indicated roles in extracellular matrix remodeling, integrin signaling, proteostasis, and steroidogenesis. Comparison with the secretome revealed distinct protein distributions, supporting selective exosomal packaging. Western blot confirmed increased ITGα3 and LGALS3BP levels in exosomes upon hCG treatment. In conclusion, hCG modulates exosome cargo composition in granulosa cells, uncovering a novel mechanism of extracellular regulation.