These results suggest that, with high sensitivity and representativity, cfs-mRNA could be non-invasive biomarkers for identifying the presence of germ cells or complete obstruction in azoospermia.

Cell-free seminal mRNA (cfs-mRNA) contains testis-specific transcripts from bilateral testes. This study determined the presence of DEAD box polypeptide 4 (DDX4) in cfs-mRNA to identify and characterize the incidence of Sertoli cell-only (SCO) syndrome in men with non-obstructive azoospermia (NOA). DDX4 cfs-mRNA was determined in 315 men with NOA, and compared with testicular samples obtained by microdissection from 19 NOA patients. Karyotype and azoospermia factor microdeletion analysis were performed, and clinical features were evaluated. Negative DDX4 cfs-mRNA suggestive of SCO was found in 13.7% of NOA patients, with a similar incidence in NOA men with known genetic causes and those without known genetic causes. DDX4 cfs-mRNA was absent in 44% of SCO cases diagnosed by testicular histopathology, but present in all patients presenting with maturation arrest or hypospermatogenesis. Furthermore, 84.2% of NOA men with DDX4 cfs-positive mRNA had a DDX4-positive testicular sample. In NOA men without genetic causes, SCO patients discriminated by negative DDX4 cfs-mRNA showed different clinical features when compared with non-SCO cases. These results suggest that the evaluation of DDX4 cfs-mRNA is more accurate than testicular histopathology in discriminating SCO, and also permits the identification of a specific group of NOA men with distinct clinical features.

A group of 20 cows and heifers experienced poor conception rates and probable ovarian dysfunction after being artificially inseminated. When first examined, some showed signs of vulvovaginitis, with pustular, ulcerative lesions consistent with a herpesvirus infection. They had had no contact with bulls during the current breeding season. Bovine herpesvirus type 5 (BHV-5) was isolated from samples of frozen semen from the batch that had been used for the artificial insemination programme. BHV-5 was also isolated from the semen of a second apparently healthy bull during routine screening of its semen.

BACKGROUND Sperm DNA integrity is crucial for normal fertilization, implantation, and embryo development. Several assays are available to assess sperm DNA fragmentation but are limited by high price, complicated processes and low accuracy. Additionally, the evaluation parameter cannot accurately show the degree of sperm DNA damage. METHODS We developed a secondary amplification detection system based on terminal deoxynucleotidyl transferase (TdT) and endonuclease IV (Endo IV), which could efficiently detect the number of 3'-OH. We used this detection system to detect the amount of 3'-OH in DNA single strand at standard concentration. We then interrupted the double strand of genomic DNA through ultrasound and enzyme digestion, and then used the detection system. Finally, we used this method to detect the number of breakpoints of human sperm DNA and calculated the mean number of breakpoints of sperm DNA. RESULTS We successfully detected the number of 3'-OH in DNA single strand at standard concentration and created the standard curve. The linear range for the increase rate of fluorescence intensity over the concentration of substrate DNA was from 0.1 nM to 15 nM. The detection method was successfully verified on λ DNA and human sperm DNA. CONCLUSION This method, which involves direct detection of actual DNA fragmentation, can measure the specific degree of sperm DNA fragmentation. It also has advantages, such as short time-consumption, simple operation, high analytical sensitivity, and low requirement for instrumentation, which makes it conducive to clinical application.

Aim: To investigate the regulation of microRNA-124 ­(miRNA-124) on NF-κB pathway from H₂O₂-induced apoptosis and oxidative stress in human lens epithelial cells (hLEC). Methods: The MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to detect hLEC ­viability. HLECs were divided into Blank, H₂O₂, mimics (miRNA-124 mimics) + H₂O₂, NC+ H₂O₂, pyrrolidine dithiocarbamate (PDTC; NF-κB signaling pathway inhibitor) + H₂O₂, and inhibitors (miRNA-124 inhibitors) + PDTC + H₂O₂ groups. Quantitative real-time polymerase chain reaction and Western blot were employed to detect mRNA and protein expressions, Dichloro-dihydro-fluorescein diacetate to measure reactive oxygen species (ROS) production, and AnnexinV-FITC/PI staining to determine cell apoptosis. The mitochondrial membrane potential (MMP) was detected by fluorescence probe JC-1. Results: The H₂O₂-induced hLEC showed reductions in cell viability with decreased miRNA-124 but increased p-p65 in a dose-/time-dependent manner. Furthermore, ROS production, malondialdehyde content, Bax and Caspase-3 expressions, and cell apoptosis were elevated in H₂O_2-induced hLEC, whereas the activities of superoxide dismutase and glutathione peroxidase, Bcl-2 expression, MMP, as well as the mitochondrial energy metabolism genes were reduced. Additionally, miRNA-124 mimics and PDTC both decreased the p-p65 and reversed the cytotoxicity in H₂O₂-induced hLEC. Conclusion: MiRNA-124 prevents H₂O₂-induced oxidative stress and apoptosis in hLEC through suppressing the activation of the NF-κB pathway.