Background Patients with incident venous thromboembolism (VTE) have a high risk of recurrence. Recent studies have shown that plasma levels of human epididymis protein 4 (HE4) are associated with increased risk of future incident VTE. Objective To investigate whether plasma HE4 levels are associated with risk of recurrent VTE. Methods Patients with first-lifetime VTE ( n = 896) derived from the Trøndelag Health Study (HUNT3; 2006-2019) were followed from the VTE date. Symptomatic and objectively confirmed VTE recurrences were recorded during the study period. Plasma HE4 levels were measured by Somascan at the time of inclusion in the source cohort (HUNT3, prior to incident VTE). Cox regression models were used to estimate hazard ratios (HRs) for VTE recurrence across quartiles of plasma HE4 levels adjusted for age, sex, and body mass index. Subgroup analyses by type of incident VTE were also performed. Results During follow-up, 116 patients developed recurrent VTE. We found no significant association between quartiles of plasma HE4 levels and absolute or relative risk of recurrent VTE. The HR for recurrence in individuals with elevated plasma HE4 levels (Q4) versus low HE4 levels (Q1) was HR: 0.82 (95% confidence interval [CI]: 0.46-1.49). Similarly, no association was found between plasma HE4 levels and recurrent VTE in any subgroup analyses (index deep vein thrombosis, pulmonary embolism, provoked VTE, or unprovoked VTE). Conclusion Our results suggest that inherent plasma HE4 levels are not useful as a risk biomarker for recurrent VTE.

Sperm present a unique paradox: heavily compacted chromatin silences transcription, and loss of cytoplasm during spermiation eliminates the conventional translational apparatus, yet sperm require ∼10 days of protein-demanding epididymal maturation to acquire motility and fertilization competence. How sperm produce the necessary proteins has remained enigmatic. Here, we show that mammalian sperm sustain protein synthesis through a biphasic translational program. In testicular sperm, a residual cytoplasmic droplet retains the full complement of translational machinery, including ribosomes and intact tRNAs, and supports nascent protein synthesis. As sperm enter the epididymis, this droplet is progressively fragmented, and protein synthesis shifts to the midpiece, where mitochondrial translation machinery becomes active. Proteomic and functional analyses indicate that both systems contribute to sperm maturation. Inhibiting cytoplasmic translation in testicular sperm or mitochondrial translation in epididymal sperm abolishes motility. These findings reveal how cytoplasm-free sperm overcome transcriptional silence to complete maturation and support male fertility.

1. This study aimed to evaluate the bioactivation of methyl-parathion (Me-Pa) and the expression and activity of mouse cytochrome P450 (Cyp) isoforms involved, in the cauda epididymis-vas deferens (CE-VD) and testis, and in the liver for comparison.2. Microsomes were obtained from adult mice treated and non-treated with phenobarbital (PB), and the bioactivation of Me-Pa was indirectly evaluated by in vitro acetylcholinesterase inhibition. The protein content was evaluated by immunoblotting, and the enzymatic activities by resorufin ether-O-dealkylation and 16α- and 6β-testosterone hydroxylation. Me-Pa bioactivation was observed in the testis at a lower rate than in the liver and was detected in the CE-VD only after PB induction.3. The protein content and enzymatic activities associated with the Cyps evaluated were observed in all tissues in different proportions, depending on the enzyme. In all cases, protein content and enzymatic activities in the liver were higher than in reproductive tissues, except for Cyp1a2, for which constitutive expression was not observed in the CE-VD, but its associated enzymatic activity was higher compared to the testis and liver.4. These findings suggest that mouse reproductive tissues are metabolically active in biotransforming xenobiotics, such as Me-Pa, contributing to its toxicity.

Human epididymis protein 4 (HE4; WFDC2) has emerged as a profibrotic biomarker associated with adverse outcomes across multiple diseases. However, its prognostic value in patients with established coronary artery disease (CAD) remains insufficiently characterized and was addressed in the present study. We prospectively studied 395 patients with angiographically confirmed CAD. Circulating HE4 levels were measured at baseline and analyzed both as log2-transformed values and by using a data-derived cut-off of 8.0 ng/mL, derived from Youden's index. The primary endpoints were major adverse cardiovascular events (MACE) and all-cause mortality during a 10-year follow-up. Multivariable Cox regression models were adjusted for traditional risk factors, including age, body mass index, hypertension, type 2 diabetes mellitus, LDL cholesterol, HDL cholesterol, and the presence of significant coronary artery disease (Model 1), and additionally for renal function, C-reactive protein, fibrinogen, and N-terminal pro-B-type natriuretic peptide (Model 2). Higher HE4 levels, analyzed as a log2-transformed continuous variable, indicated a significantly increased risk of MACE and all-cause mortality across all adjustment models, with HRs of 1.2-1.4 per doubling of HE4. Also, the categorical HE4 cut-off was significantly associated with all endpoints across all adjustment levels, and the addition of HE4 to the fully adjusted baseline model improved overall risk reclassification across all endpoints. In conclusion, HE4 is significantly associated with long-term cardiovascular outcomes and all-cause mortality in patients with established CAD and may provide prognostic information beyond inflammatory and cardiorenal markers.

CLASP2, a microtubule plus-end tracking protein, exhibits changes in phosphorylation as well as localization along the microtubule in response to insulin stimulation. We report that CLASP2 is expressed in insulin target tissues such as skeletal muscle and adipose tissue and that CLASP2 protein abundance is decreased in epididymal fat of insulin resistant mice. Using a commercially available proline-directed phospho-antibody together with site-directed mutagenesis and quantitative proteomics, we establish that mouse CLASP2 (Uniprot ID Q8BRT1) undergoes insulin-stimulated phosphorylation at serines 884/893/1004/1005/1021 in 3T3-L1 adipocytes. Treatment of cells with the ERK inhibitor PD98059 inhibits the insulin-stimulated phosphorylation of CLASP2 at the proline-directed serines 893/1004/1005/1021. In vitro incubation of CLASP2 with ERK2 increases CLASP2 phosphorylation, whereas phosphorylation of a Ser884/893/1004/1005/1021Ala CLASP2 penta-mutant is decreased, indicating ERK2 is capable of directly phosphorylating CLASP2. When expressed in 3T3-L1 adipocytes, the CLASP2 penta-mutant presents significantly diminished insulin-stimulated microtubule plus-end trailing versus wildtype CLASP2. The lack of proline-directed, insulin-stimulated CLASP2 phosphorylation results in reduced insulin-induced microtubule stabilization. We introduce insulin-stimulated, proline-directed CLASP2 phosphorylation under control of the ERK pathway as a contributing factor for insulin-induced microtubule stabilization.

Psychological stress is a risk factor for male infertility, and adolescence is a critical period of vulnerability to stress. However, the effects of psychological stress on spermatogenesis in adolescent males remain unclear. To investigate this, we used a restraint stress model in adolescent Kunming mice, where animals were restrained for 2 and 4 h per day from postnatal day 28 to 56. Restraint stress resulted in a decreased sperm concentration and serum testosterone levels, together with increased sperm abnormalities, synaptonemal complex protein3 (SYCP3)-positive germ cells in the epididymis, and serum corticosterone levels in adult mice. Additionally, restraint stress altered seminiferous tubule histoarchitecture and the distribution of spermatogenic stages. Restraint-stressed mice exhibited increased proportions of zygotene- and pachytene-stage spermatocytes and a reduced proportion of diplotene-stage spermatocytes. Further analysis showed altered γH2AX and ataxia-telangiectasia and rad3-related (ATR) localization patterns together with increased frequencies of chromosomal synapsis abnormalities and abnormal chiasma formation, which were accompanied by increased spermatocyte apoptosis. The mRNA expression levels of key meiotic genes, including SPO11 initiator of meiotic double stranded breaks (SPO11), DNA meiotic recombinase 1 (DMC1), synaptonemal complex protein1 (SYCP1) and SYCP3, were reduced in stressed mice. In addition, restraint stress increased germ cell apoptosis and reduced litter size in adulthood. In conclusion, adolescent exposure to psychological stressors such as restraint stress is associated with impaired spermatogenesis and reduced reproductive capacity in adult male mice.

The aim of this study was to characterize the proteome of sperm and seminal plasma (SP) from rams with high semen freezability (HF) and low semen freezability (LF). Eleven ejaculates were collected from 12 rams, at 3-day intervals, and cryopreserved (132 semen samples). For proteomic analysis, sperm and SP samples were pooled separately for each ram. Pools 1 and 2 included ejaculates obtained on odd-numbered and even-numbered collection days, respectively. This generated 24 sperm and 24 SP pools. Sperm and SP proteins were analyzed by mass spectrometry, and computational biology platforms, used for calculation of differentially abundant proteins (DAPs; p < 0.05) in HF (n = 5) and LF (n = 7) rams. Total sperm motility decreased from 91.87% ± 0.63% in fresh to 65.65% ± 2.58% in frozen-thawed semen of HF rams; and decreased from 89.47% ± 1.15% to 50.05% ± 2.33% in LF rams. Reduction in total sperm motility after cryopreservation was lower in HF (26.22% ± 2.39%) compared to LF rams (37.70% ± 2.23%; p = 0.006). Sperm progressive motility decreased less in HF rams (from 87.20% ± 0.50% to 58.12% ± 2.82%) than in LF rams (from 85.54% ± 1.41% to 44.00 ± 2.13; p = 0.006). Rams with the HF phenotype had better plasma membrane (39.00% ± 2.79% vs. 15.14% ± 0.92%) and acrosome integrity (37.54% ± 2.72% vs. 15.40% ± 1.92%; p < 0.001) than LF rams. Sperm motility was similar in fresh semen of HF and LF rams. There were 1599 and 1677 sperm proteins identified in HF and LF rams, with 73 and 76 DAPs, respectively. SP had 1127 proteins, with 25 and 45 DAPs in HF and LF rams. This is the greatest coverage ever published about the ram SP proteome. Sperm proteins with greater abundance in HF rams were extracellular and immune-related proteins, antiproteases, chaperones and structural regulators, contributing to cytoskeletal integrity, apoptotic resistance and maintenance of proteostasis. Sperm proteins with greater abundance in LF rams were mainly mitochondrial subunits, axonemal components, and bioenergetic enzymes, indicating greater reliance on oxidative processes. Major components of HF SP participate in sperm protection, stabilization of membrane lipids, calcium fluxes, epididymal maturation and ATP availability. The major LF SP proteome was associated with cellular stress, inflammatory responses, oxidative damage and ATP production. Thus, the nature of sperm and SP proteome atlas potentially defines ram sperm cryoresilience.

Topiramate is a widely prescribed antiepileptic drug with a multimodal mechanism of action. However, its effects on male reproductive function remain poorly characterized. This study was conducted to investigate the impact of this drug on testicular and epididymal function in adult male Wistar rats. Animals were treated orally for 21 days with topiramate at 40 or 160 mg/kg. Serum testosterone levels, testicular histomorphology, spermatogenic indices, sperm parameters, and epididymal duct contractility were subsequently evaluated. The treatment did not alter serum testosterone levels at either dose. However, both doses reduced Sertoli cell and round spermatid numbers, increased primary spermatocyte counts, and decreased meiotic and overall spermatogenic yields, whereas only the higher dose (160 mg/kg) reduced Sertoli cell efficiency and daily sperm production. The lower dose (40 mg/kg) increased sperm morphological abnormalities, whereas the higher dose reduced total and progressive sperm motility and induced both head and tail defects. Additionally, treatment with topiramate at 160 mg/kg prolonged sperm transit time in the cauda epididymis and reduced contractile responses of the distal cauda epididymal duct to KCl, noradrenaline, and carbachol, suggesting impaired epididymal transport and potential ejaculatory dysfunction. Altogether, these findings demonstrate that topiramate adversely affects male reproductive function. Given its widespread use in individuals of reproductive age, these results underscore the need for translational studies in humans to assess potential reproductive risks associated with this therapy.

Prenatal exposure to the environmental endocrine disruptor di-n-butyl phthalate (DBP) can impair male reproductive development, but the upstream regulation of oxidative injury remains incompletely defined. In male C57BL/6 offspring assessed at postnatal day 35, prenatal DBP exposure was associated with testicular histopathology, lower testis weight and serum testosterone, impaired spermatogenic and epididymal sperm parameters, oxidative stress, and apoptosis. Testicular RNA sequencing identified Phf10, which encodes a PBAF chromatin-remodeling subunit, as a DBP-responsive candidate. Tissue analyses showed increased PHF10 with preferential enrichment in SOX9-positive Sertoli cells. In TM4 Sertoli cells, DBP increased PHF10, reactive oxygen species (ROS), and mitochondrial-apoptosis markers; Phf10 knockdown attenuated these responses, whereas PHF10 overexpression enhanced them. N-acetylcysteine reduced DBP-induced ROS and apoptosis without significantly altering DBP-induced PHF10 expression, supporting PHF10 as an upstream contributor to ROS-associated injury under the tested conditions. PHF10 ChIP-based analyses identified candidate redox-related loci associated with PHF10 occupancy and DBP-responsive expression. Promoter-reporter and mRNA-decay experiments supported transcriptional activation of Phf10 rather than increased mRNA stability, and pharmacologic PPARα inhibition attenuated DBP-associated PHF10 induction and downstream injury markers. Collectively, these findings support a PPARα-associated PHF10/redox/apoptosis mechanism in DBP-induced testicular injury, while direct promoter regulation, in-vivo cell-specific causality, dose relevance, and human applicability remain to be established.

Round spermatid elongation represents a pivotal morphological transformation essential for functional sperm formation. The present study investigated how perfluorooctanoic acid (PFOA) disrupts this process. Eight-week-old male ICR mice (n = 42, 14 per group) received daily oral gavage of PFOA at doses of 0 (Ctrl group), 1 (P1 group) or 10 (P10 group) mg/kg for 35 days. PFOA exposure dose-dependently reduced epididymal sperm count and elevated the ration of round spermatid to elongated spermatid. Moreover, TPPP2, RhoC and ATG4, several proteins specifically expressed in elongated spermatids, were downregulated. Ultrastructure of the chromatoid body (CB)-a specialized structure in round spermatids participating in spermatid elongation-was impaired, indicating that PFOA inhibits round spermatid elongation. Mechanistically, PFOA disrupted the ubiquitin-proteasome system (UPS) by downregulating testicular E2-conjugating enzyme, E3 ubiquitin ligases, testis-specific α4 subunit PSMA8 and two spermatoproteasome activators. Concurrently, chromatin remodeling was disrupted, as evidenced by reduced replacement of histone variants and attenuated post-translational modifications (PTMs)-particularly acetylation and methylation of histone H3 and H4. Consequently, PFOA decreased protamines (PRMs) levels and impaired disulfide bond formation, thereby inhibiting the histone-to-protamine transition (HPT) and chromatin condensation. Collectively, our data demonstrate that PFOA suppresses round spermatid elongation by impeding ubiquitin-proteasome-mediated chromatin remodeling.

Post-Orgasmic Illness Syndrome (POIS) is a rare con-dition characterized by flu-like symptoms after ejacula-tion, lasting several days and impairing quality of life. A 62-year-old man developed POIS after percutaneous ep-ididymal sperm aspiration, with symptoms lasting seven days and worsening over six years. Multiple treatments offered minimal relief until combined therapy with tam-sulosin, ebastine, escitalopram, and montelukast achieved complete resolution and restored sexual function. Immune hypersensitivity to seminal or epididymal antigens is a leading hypothesis. The success of α-blockers, antihista-mines, and SSRIs highlights the interplay between im-mune, autonomic, and psychological mechanisms. POIS is a complex, underrecognized disorder. Multimodal therapy addressing both immune and neuropsychological pathways may yield optimal outcomes. Greater awareness and stan-dardized diagnostic and treatment criteria are needed to improve management and research.