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PubMed2026

Combined effects of urine exposure and cryopreservation on sperm quality: an in vitro study of retrograde ejaculation.

Sperm quality influences fertility and offspring health through both genomic inheritance and epigenetic inheritance. Thus, for use in clinical-assisted reproductive technology (ART), spermatozoa must have optimal genomic and epigenetic structures. In patients with retrograde ejaculation, spermatozoa are usually recovered from urine and then cryopreserved for ART. However, the effects of urine exposure and subsequent freeze-thaw cycles on sperm quality remain unclear. This is particularly true for epigenetic changes and their underlying mechanisms. In this study, we examined how different durations of urine exposure (10 min and 40 min) followed by freeze-thaw cycles affected sperm motility, DNA integrity, and methylation levels of imprinting genes (H19-imprinted maternally expressed transcript [ H19 ], mesoderm-specific transcript [ MEST ], and the transposable element Alu [ Alu ]). As the duration of urine exposure increased, sperm motility (median [interquartile range]) decreased from 48.0% (39.0%-52.5%) to 1.0% (1.0%-5.0%), the DNA fragmentation index (DFI; median [interquartile range]) increased from 12.0% (9.3%-19.9%) to 23.5% (13.9%-33.9%), the MEST methylation level (mean ± standard deviation [s.d.]) increased from 3.8% ± 1.5% to 11.5 ± 1.2%, and the H19 methylation level (mean ± s.d.) decreased from 86.9% ± 0.9% to 82.1% ± 0.5%. The freeze-thaw process further reduced sperm motility, while the DFI and methylation levels of MEST and H19 did not significantly change. The Alu methylation level remained stable. These findings demonstrate that urine exposure affects sperm motility, DNA integrity, and methylation levels of some imprinting genes. These effects intensify over time. In contrast, the freeze-thaw process impacts only sperm motility. In clinical practice, minimizing exposure to urine might improve sperm quality.

Humans↗

PubMed2026

Aquablation/AquaBeam Waterjet Therapy for Benign Prostatic Hyperplasia: Three-Year Functional and Ejaculatory Outcomes in a Multicenter Real-World Italian Cohort.

OBJECTIVE: To evaluate the 3-year functional outcomes of Aquablation/AquaBeam waterjet therapy for bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH), with particular attention to urinary symptoms, quality of life, continence, ejaculatory function, and complications. METHODS: We performed a retrospective analysis of a prospectively maintained multicenter database including 218 consecutive men who underwent Aquablation/AquaBeam for symptomatic BPH between January 2019 and January 2022 at three referral centers. Functional outcomes assessed preoperatively and during follow-up included International Prostate Symptom Score (IPSS), IPSS quality-of-life item (IPSS-QoL), maximum urinary flow rate (Qmax), post-void residual urine (PVR), continence, and ejaculatory function. Median follow-up was 36 months. Ejaculatory preservation was evaluated only in patients with preserved antegrade ejaculation at baseline. RESULTS: Median age was 61 years (IQR 57-66), median prostate-specific antigen (PSA) was 2.52 ng/mL (IQR 0.40-21.60), and median prostate volume was 55 mL (IQR 40-73). Median operative time was 56 min, while median catheterization time and length of hospital stay were both 48 h. Functional improvements were evident from the 3-month follow-up and remained stable through 36 months. At 36 months, median IPSS improved to 5 and median Qmax to 18 mL/s. Median PSA changed modestly from 2.52 ng/mL at baseline to 2.75 ng/mL at 36 months. No cases of de novo urinary incontinence were observed throughout follow-up. Among patients with preserved antegrade ejaculation at baseline, 87% maintained antegrade ejaculation at 36 months. Most postoperative complications were minor (Clavien-Dindo grade I-II, 20.2%). One grade III rectal injury occurred (0.5%). Median hemoglobin decreased from 15.2 g/dL preoperatively to 14.0 g/dL before hospital discharge, and two patients required blood transfusion. During the 36-month follow-up, no surgical retreatment was required, whereas 11 patients (5.0%) received temporary medical retreatment. CONCLUSION: In this multicenter real-world cohort, Aquablation/AquaBeam was safe and effective over 3 years, providing durable improvement in lower urinary tract symptoms and quality of life while preserving continence and antegrade ejaculation in most patients. These findings support Aquablation/AquaBeam as a valuable minimally invasive surgical option for selected men with BPH, particularly those who prioritize preservation of ejaculatory function. Longer-term and comparative studies are warranted. TRIAL REGISTRATION: 48281.

Humans↗

PubMed2025

Silent cells? Potential for context-dependent gene expression in mature sperm.

Sperm are traditionally viewed as transcriptionally and translationally silent cells. However, observations that components of the cellular machinery of gene expression are maintained in ejaculated sperm are increasingly cited as challenges to this fundamental assumption. Here, we critically evaluate these arguments and present three lines of evidence from both model and non-model systems that collectively raise the question of whether ejaculated sperm may be capable of active gene expression. First, and critical for arguments surrounding the possibility of differential gene expression, we review recent evidence that spermatozoa may retain the capacity to transcribe and translate their genomes. Second, we highlight how sperm cells can exhibit differential transcript quantities across different post-ejaculation environments. Third, we ask whether the accumulating evidence of remarkable phenotypic plasticity in post-ejaculatory sperm phenotypes could be mechanistically underpinned by changes in sperm gene expression. While these lines of evidence are indirect and do not definitively show transcription of sperm genomes, we highlight how emerging technologies may enable us to test this hypothesis explicitly. Our review advocates for progress in this field and highlights several important evolutionary, ecological and practical implications that will probably transcend disciplines to the clinical and applied reproductive sectors.

Male↗

PubMed2026

Age-related differences in semen quality in Holstein-Friesian bulls: a paired within-bull comparison of early and mature reproductive stages.

Genomic selection has changed dairy cattle breeding by increasing the use of young bulls for artificial insemination and shortening the reproductive lifespan of sires. Under these conditions, semen quality at the beginning of commercial use has become an important practical issue. Semen samples from 39 fertile Holstein-Friesian bulls used for commercial AI were collected between 2013 and 2016, during the introduction of genomic selection in Poland. This paired within-bull study compared semen collected from the same bulls at an early reproductive stage (13-20 months; young bulls, YB) and at full maturity (5-6 years; mature bulls, MB). The evaluation included conventional ejaculate traits, CASA-derived motility and kinematic descriptors, mtDNA copy number, and mitochondrial content per sperm cell. Importantly, all ejaculates met the quality requirements for commercial insemination. Ejaculate volume, sperm concentration, mitochondrial DNA copy number, and mitochondrial content did not differ significantly between age groups. The CASA-derived sperm movement profile, in contrast, differed with age. Semen from young bulls showed a higher proportion of progressively motile spermatozoa, whereas semen from mature bulls showed higher velocity-related parameters, including VSL, VCL, and STR. These findings indicate that bull age mainly affected sperm movement characteristics rather than semen output or mitochondrial content. Overall, the results support the use of young bulls in artificial insemination programs and show that age-related differences in semen quality are expressed mainly through changes in the post-thaw sperm motility and kinematic profile.

Animals↗

PubMed2026

Protamine gene expression is associated with sperm motility in rams: An integrative experimental and gene network analysis.

Protamine 1 (PRM1) and protamine 2 (PRM2) are essential regulators of sperm chromatin condensation and genome integrity, and their dysregulation has been associated with impaired male fertility. However, their role in rams remains insufficiently characterized. This study investigated the relationship between protamine gene expression and semen quality in rams and explored their potential upstream regulatory mechanisms using gene regulatory network (GRN) analysis. Fifteen ejaculates from five rams were analyzed. Based on total sperm motility using computer-assisted sperm analysis (CASA), ejaculates were classified into a high-motility group (n = 8) and a low-motility group (n = 7). PRM1 and PRM2 expression levels were quantified by RT-qPCR. Following normality confirmation (p > 0.05), parametric tests were applied using the ejaculate as the biological experimental unit. Samples with reduced motility showed significantly lower expression of both protamines (p < 0.01). Moreover, progressive sperm motility was strongly correlated with both PRM1 (r = 0.71, p = 0.019) and PRM2 (r = 0.69, p = 0.03) transcript levels. Cross-species GRN inference using scGeneRAI and a reference human spermatogenesis dataset identified several hypothesis-generating candidate transcription factors, including HMGB4, HMGB1, H2AFZ, NKX6-1, and SMC3, consistently supported across multiple bootstrap resampling runs. These findings demonstrate a strong association between protamine expression and sperm motility in rams. While the identified candidate regulators provide a valuable framework for future species-specific validation, they also highlight promising candidate molecular biomarkers of male fertility in livestock.

Gene regulatory networks↗

PubMed2026

Deoxyribonucleic acid methylation abnormalities at imprinted loci in oligospermic and azoospermic men.

OBJECTIVE: To assess deoxyribonucleic acid (DNA) methylation at imprinted and repetitive genomic regions in ejaculated and testicular sperm from men with oligospermia, azoospermia, and those undergoing vasectomy reversal (VR), compared with fertile controls. DESIGN: Observational case-control study. SUBJECTS: Samples were obtained from 68 men, including 29 infertile men (18 oligospermic [5-15 million/mL], 11 severely oligospermic [<5 million/mL]) and 20 fertile controls with confirmed natural conceptions. Testicular tissue was collected from 11 azoospermic men (7 obstructive azoospermia [OA], 4 nonobstructive azoospermia [NOA]) and 8 men with prior paternity undergoing VR. EXPOSURE: Sperm DNA methylation at four imprinted genes (H19, GTL2, MEST, and LIT1) and one repetitive element (LINE1). MAIN OUTCOME MEASURES: Methylation levels at CpG sites determined by bisulfite pyrosequencing. RESULTS: The H19 was significantly hypomethylated only in severe oligospermia compared with fertile controls, whereas other groups showed nonsignificant trends with overlapping distributions. In contrast, MEST was significantly hypermethylated in oligospermic, azoospermic, and VR groups compared with fertile controls. No significant differences were observed for GTL2, LIT1, or LINE1. CONCLUSION: The DNA methylation abnormalities in sperm are locus-specific and vary across infertility phenotypes. MEST alterations were consistent across groups, whereas H19 changes were limited to severe oligospermia. Similar methylation patterns in testicular sperm from azoospermic and VR groups suggest that epigenetic alterations may reflect the testicular environment or obstruction, or differences between testicular and ejaculated sperm.

Humans↗

PubMed2026

Sperm subpopulations differing in mitochondrial abundance show divergent nuclear allele frequencies.

Mammalian ejaculates contain heterogeneous sperm subpopulations that differ in subcellular architecture and developmental history, despite appearing morphologically uniform. The extent to which this cellular heterogeneity reflects underlying nuclear genomic structure within a sire remains largely unexplored. Mitochondrial architecture in sperm is established during spermatogenesis, with final assembly and organization occurring during spermiogenesis under nuclear genomic control, positioning variation in mitochondrial abundance and organization as a potential phenomic indicator of within-sire allelic segregation. Here, we tested whether sperm subpopulations defined by differing mitochondrial abundance exhibit systematic differences in nuclear allele representation. Boar sperm were resolved into low and high mitochondrial subpopulations using fluorescence-activated cell sorting based on MitoTracker™ Green fluorescence while excluding debris, doublets, and non-viable cells. Epifluorescence microscopy confirmed that high MitoTracker™ Green fluorescence sperm possessed longer mitochondrial sheaths, validating a structural distinction between subpopulations. Whole-genome sequencing of paired mitochondrial subpopulations from three boars was performed, and allelic ratio distortion was evaluated relative to heterozygous baseline populations. Analyses across heterozygous loci genome-wide identified candidate allele frequency shifts between mitochondrial-defined subpopulations, suggesting non-random segregation of alleles within ejaculates. Using a minimum sequencing depth of 30 reads in both sorted fractions, 182 candidate SNPs were identified with evidence of allele-frequency differences between mitochondrial fluorescence-defined subpopulations. These findings suggest that sperm mitochondrial abundance can potentially serve as an indirect, high-throughput marker of nuclear genomic heterogeneity within sires. This proof-of-concept framework establishes a foundation for future studies integrating sperm phenotyping, genome-wide allele-frequency analysis and functional validation to better characterize gamete-level heterogeneity.

Male↗

PubMed2026

Long-term outcomes of surgical correction of ventral penile curvature in children: Patient-reported measures, surgical results, and decisional regret.

INTRODUCTION: There is a dearth of data on long-term outcomes, including patient-reported outcomes (PROMs), decisional regret, and complication rates for surgical correction of isolated ventral penile curvature in childhood. PATIENTS AND METHODS: Twenty-six children treated for ventral curvature between 1993 and 2008 were identified; 24 met inclusion criteria (isolated ventral curvature without hypospadias or need for urethral reconstruction). Surgical correction consisted of degloving alone or degloving with Nesbit-like dorsal plication when residual curvature >20° persisted after degloving. PROMs were collected via pre-mailed validated questionnaires after puberty: Danish Prostatic Symptom Score (DAN-PSS) for LUTS, Erection Hardness Score (EHS) for erectile function, Penile Perception Score (PPS) for cosmetic perception, and items assessing decisional regret and perceived appropriateness of surgical timing. RESULTS: Curvature was corrected intraoperatively in all 24 patients. Twelve underwent degloving alone and 12 required additional dorsal plication. During long-term follow-up (median 14.2 years), one patient (4%) underwent re-operation for residual curvature, and three (13%) underwent cosmetic revisions; two (8%) underwent cystoscopy for flow concerns. 71% returned PROMs at a median age of 16.2 years. LUTS were uncommon, with low bother scores. Erectile function was favorable: 87% (13/15) reported EHS 4 and 93% (14/15) reported ejaculation. Cosmetic outcomes were favorable, with PPS dissatisfaction rates comparable to controls. Two patients reported dissatisfaction with overall appearance, and one with residual subjective curvature. No patient expressed decisional regret, and 88% felt timing of surgery was appropriate. CONCLUSION: Early surgical correction of isolated ventral penile curvature using degloving with or without dorsal plication provided durable anatomical correction and favorable long-term functional and cosmetic outcomes. These findings support early intervention as an effective approach with sustained patient-perceived benefits.

Humans↗

PubMed2026

Spermidine and melatonin ameliorate heat stress-induced decline in sheep semen quality.

Heat stress impairs reproductive performance in sheep through endocrine disruption and oxidative stress. This study evaluated the protective effects of spermidine (SPD) and melatonin (MT) supplementation on semen quality in Dorper rams during summer. Twenty-four rams were randomly assigned to a control group, an SPD group (5 mg/kg, dietary supplementation), or an MT group (60 mg, subcutaneous implantation) and treated for 60 days. The temperature-humidity index (THI) was monitored throughout the experimental period. Compared with the control group, MT significantly reduced serum cortisol concentration on day 30 (P < 0.05), whereas no significant differences were observed at the other sampling time points. Serum testosterone, spermidine, and melatonin concentrations remained unchanged throughout the study (P > 0.05). SPD supplementation significantly increased ejaculate volume on day 35 and sperm motility on day 42 (P < 0.05), whereas MT did not significantly affect these parameters. Neither treatment reduced the overall sperm abnormality rate. However, both SPD and MT significantly decreased the proportion of acephalic and decaudated sperm on day 56 (P < 0.05). Neither treatment significantly affected pregnancy rate, delivery rate, or the expression of PMFBP1 and SUN5 proteins in semen (P > 0.05). Regarding oxidative stress, MT significantly downregulated CAT protein expression (P < 0.05), whereas SPD significantly reduced MDA content and SOD1 protein expression (P < 0.05); MT showed similar but non-significant trends for these two markers (P > 0.05). Collectively, these findings demonstrate that SPD and MT exert distinct protective effects against heat stress, with SPD improving selected semen quality traits and both treatments reducing sperm head-tail separation, although these benefits did not translate into improved reproductive performance.

Animals↗

PubMed2026

Mitochondrial uncoupler BAM15 attenuates cryopreservation-induced damage in human sperm by stabilizing mitochondrial homeostasis†.

Human sperm cryopreservation is essential for sperm banking and assisted reproduction, yet freeze-thaw stress promotes oxidative injury that reduces motility and damages the acrosome and nuclear DNA. Here, we tested whether the mitochondrial uncoupler BAM15 improves post-thaw human sperm quality and examined mechanisms linked to mitochondrial homeostasis. Ejaculates were cryopreserved using a standard protocol supplemented with graded concentrations of BAM15. After thawing, total and progressive motility and viability were assessed. Flow cytometry quantified the DNA fragmentation index and the proportion of high DNA stainability cells. Mitochondrial membrane potential, intracellular reactive oxygen species, and lipid peroxidation were measured to evaluate mitochondrial function and oxidative status. Ultrastructural preservation of the acrosome, plasma membrane, midpiece mitochondria, and flagellar axoneme was examined by transmission electron microscopy. Compared with untreated controls, BAM15 increased total and progressive motility and improved viability. BAM15 reduced DNA fragmentation and decreased high DNA stainability, indicating enhanced genomic integrity. Consistently, BAM15 improved mitochondrial membrane potential while suppressing intracellular reactive oxygen species and lipid peroxidation, supporting attenuation of freeze-thaw oxidative damage. Transmission electron microscopy further revealed more continuous acrosomal and plasma membranes, fewer swollen or vacuolated midpiece mitochondria, and improved preservation of axonemal architecture. Collectively, these findings identify BAM15 as a promising cryopreservation supplement that stabilizes mitochondrial homeostasis and improves the functional and structural quality of human sperm after thawing.

Humans↗