Nonobstructive azoospermia (NOA), the most severe form of male infertility, frequently arises from genetic defects that disrupt spermatogenesis. In this study, a novel hemizygous missense variant (NM_001184767.2 [c.G1069A; p.V357I]) is identified in the X-linked BEN domain-containing 2 (BEND2) gene of a patient with NOA characterized by spermatocyte maturation arrest. Whole-exome sequencing and Sanger validation confirmed that this rare variant is absent in fertile controls and that no pathogenic variants were detected in established NOA genes. Computational analysis predicted potential structural alterations via AlphaFold modeling, leading to the hypothesis that the ability of BEND2 to recognize genomic targets may be compromised. The patient’s phenotype phenocopies the meiotic arrest observed in Bend2-knockout mice. Expression profiling confirmed predominant BEND2 transcription in human and mouse testes, peaking in early spermatocytes and coinciding with meiotic initiation, with reduced transcript levels detected in the proband’s peripheral blood compared with those in an obstructive azoospermia control. This study reports a pathogenic BEND2 variant associated with NOA with spermatocyte arrest, highlighting its critical role in human meiosis and expanding the genetic etiology of male infertility.

Biallelic variants in fibrous sheath-interacting protein 2 (FSIP2) gene are a known cause of multiple morphological abnormalities of the sperm flagella (MMAF). This study aimed to identify novel FSIP2 variants and evaluate their impact on sperm ultrastructure and intracytoplasmic sperm injection (ICSI) outcomes. Whole-exome sequencing (WES) was employed to screen a cohort of 92 MMAF patients, with candidate variants validated via Sanger sequencing and third-generation sequencing. We identified one homozygous variant in a proband from a consanguineous family and two pairs of compound heterozygous variants in two unrelated, non-consanguineous families. Routine semen analysis demonstrated markedly reduced motility across all probands. Detailed morphological and ultrastructural assessments using Papanicolaou staining, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) demonstrated that approximately 80.0% of spermatozoa exhibited pathological elongation of the mitochondrial sheath in the midpiece. Furthermore, 50.0%–70.0% of spermatozoa displayed fibrous sheath dysplasia or loss in the principal piece. Immunofluorescence assays and Western blotting confirmed that FSIP2 protein localization was disrupted, and the expression of key axonemal assembly factors was dysregulated. Notably, successful pregnancies were achieved via ICSI in the partners of two probands. This study expands the mutational spectrum of FSIP2 in both consanguineous and non-consanguineous populations. Ultrastructural abnormalities, such as mitochondrial sheath elongation and fibrous sheath disassembly, highlight FSIP2’s critical role in flagellar assembly. Clinical results further support ICSI as an effective therapeutic intervention for affected individuals.

Low-intensity pulsed ultrasound (LIPUS) has emerged as a non-invasive therapeutic approach with regenerative potential in various tissues. This study explores its effects on testicular steroidogenesis in male rats. Twenty-four 9-month-old rats were allocated into three groups: control, LIPUS, and LIPUS-M. The LIPUS group underwent pulsed ultrasound therapy targeting the testes twice weekly for 4 weeks, whereas the LIPUS-M group continued with maintenance therapy administered once every 2 weeks for 6 months. At the end of the experiment (16 months), blood and testicular tissues were collected for analysis. Our results showed that LIPUS therapy significantly elevated serum testosterone level and increased the number of Leydig cells (LCs), as indicated by steroidogenic acute regulatory protein (STAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) immunofluorescence. Collagen IV staining showed increased interstitial vascularization, and senescence-associated β-galactosidase (SA-β-Gal) staining suggested reduced cellular senescence. Although the LIPUS-M group also showed increased LC counts and angiogenesis, serum testosterone levels were not elevated. These findings demonstrate that LIPUS can activate LCs and enhance endogenous testosterone production, supporting its potential as a noninvasive regenerative approach for hypogonadism.

Human sperm cryopreservation is essential for assisted reproductive technology; however, the freeze–thaw process reduces motility and increases DNA fragmentation. Rho-associated coiled-coil kinase (ROCK) inhibitors improve post-thaw survival in several cell types, yet their application to human sperm remains underexplored. We evaluated whether ROCK inhibition reduces DNA fragmentation during cryopreservation by liquid nitrogen vapor slow freezing or vitrification. Each semen sample was divided into four aliquots: 2 were cryopreserved via liquid nitrogen vapor slow freezing with or without ROCK inhibitor and 2 underwent vitrification with or without ROCK inhibitor. After 1 week of cryostorage, samples were thawed at the room temperature, and DNA fragmentation was quantified using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. DNA fragmentation decreased with ROCK inhibitor versus control after liquid nitrogen vapor slow freezing (mean ± standard deviation [s.d.]: 10.47% ± 6.77% vs 13.54% ± 7.0%; P = 0.002) and after vitrification (mean ± s.d.: 12.51% ± 5.84% vs 16.5% ± 10.77%; P = 0.006). ROCK inhibition thus reduces post-thaw sperm DNA fragmentation with both cryopreservation methods, particularly with liquid nitrogen vapor slow freezing, supporting its potential to improve sperm storage for assisted reproductive technology.

The aim of this study was to explore the predictors of International Society of Urological Pathology (ISUP) Grade Group (GG) upgrading in nonclinically significant prostate cancer (non-csPCa) biopsy specimens guided by transrectal ultrasound (TRUS) following radical prostatectomy (RP). A retrospective analysis was conducted on clinical data from 231 patients who underwent TRUS-guided biopsy and RP between January 2018 and December 2023. Univariate and multivariate logistic regression analyses were employed to identify independent predictors of postoperative ISUP GG upgrading. The diagnostic efficiency of these predictors was evaluated using receiver operating characteristic (ROC) curves. Additionally, ROC analysis was used to compare the predictive performance of different positive target (PT) imaging methods across prostate volume (PV) subgroups. The study included 111 (48.1%) patients in the GG concordance group and 120 (51.9%) patients in the GG upgrading group. Univariate analysis revealed that PV, prostate-specific antigen density, Prostate Imaging Reporting and Data System version 2.1 (PI-RADS v2.1), magnetic resonance imaging (MRI) PT, contrast-enhanced ultrasound (CEUS) PT, and clinical T stage significantly influenced GG upgrading (all P < 0.05). Multivariate logistic regression revealed PV (OR = 0.974; 95% CI: 0.955–0.994; P = 0.010), MRI PT (OR = 3.902; 95% CI: 1.507–10.102; P = 0.005), and CEUS PT (OR = 2.280; 95% CI: 1.073–4.845; P = 0.032) as independent predictors. The prediction model demonstrated the highest diagnostic performance (area under the receiver operating characteristic curve [AUC] = 0.738, P < 0.001). MRI PT showed superior predictive efficacy compared with CEUS PT in patients with a PV <30.13 ml. In conclusion, PV, CEUS PT, and MRI PT were identified as independent predictors of postoperative ISUP GG upgrading. The predictive model exhibited the best overall performance, and compared with CEUS PT, MRI PT demonstrated greater predictive accuracy for smaller prostates.