[The miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance].

OBJECTIVE: To study the miR-184 level in the seminal plasma exosome of male infertility patients and its clinical significance. METHODS: Between 2015 and 2019, we collected 285 seminal plasma samples from 97 azoospermia (AS) and 96 asthenospermia (AZS) patients and 92 age-matched normal fertile controls in Jiangsu Provincial Hospital of Traditional Chinese Medicine, General Hospital of Eastern Theater Command and the First Hospital Affiliated to Wenzhou Medical University, identified the isolated seminal plasma exosomes by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot, and detected the miR-184 level in the seminal plasma exosomes by quantitative real-time PCR (qRT-PCR). We determined the clinical value of the miR-184 level and its correlation with semen parameters by multiple statistics, predicted the target genes and involved pathways of miR-184 by bioinformatic algorithms, and analyzed their relationship with male infertility. RESULTS: NTA, TEM and Western blot exhibited plenty of exosomes in the seminal plasma of the patients. The results of qRT-PCR showed that the miR-184 level in the seminal plasma exosome was dramatically decreased in the AS patients compared with that in the normal fertile controls (0.227 ［0.092, 0.790］ vs 0.650 ［0.408, 1.061］, P < 0.01), but increased in AZS males in comparison with that in the control group (1.176 ［0.661, 1.946］ vs 0.650 ［0.408, 1.061］, P < 0.01). The areas under the ROC curve (AUC) for differentiating the AS and AZS patients from the controls were 0.866 (95% CI: 0.815－0.916) and 0.724 (95% CI: 0.653－0.795), respectively, and that for differentiating the AS from the AZS group was 0.964 (95% CI: 0.943－0.985). The miR-184 level in the seminal plasma exosome of the AZS patients was correlated positively with the sperm count (r = 0.243, P = 0.017) but negatively with the percentage of progressively motile sperm (r = －0.407, P = 0.006). Bioinformatics analysis indicated that the downstream target genes of miR-184 were significantly enriched in the protein regulatory pathways closely related to male reproduction and spermatogenesis. CONCLUSIONS: The miR-184 level in the seminal plasma exosome of infertility patients is significantly different from that of normal fertile males, which may serve as a potential auxiliary marker for the diagnosis of and participate in the development and progression of male infertility.

[A260G and A386G single nucleotide polymorphisms of the DAZL gene are not correlated with male infertility in the Chinese population of Zhejiang Province].

OBJECTIVE: To assess the association of the A260G and A386G single nucleotide polymorphisms (SNP) of the DAZL gene with male infertility in the Chinese population of Zhejiang Province. METHODS: We collected the peripheral blood samples from 317 idiopathic infertile males with azoospermia or oligozoospermia and 246 normal fertile men, and genotyped the polymorphic loci of the A260G and A386G polymorphisms of the DAZL gene using the SNaPshot technique. RESULTS: The DAZL gene A260G was found genetically polymorphic in the Chinese population of Zhejiang Province, with the gene frequencies and their distribution consistent to the Hardy-Weinberg equilibrium. The frequencies of the AA, AG and GG genotypes of the A260G polymorphism were 92.3%, 7.3%, and 0.4% respectively in the normal controls and 94.3%, 5.7%, and 0% in the infertile patients, with no statistically significant differences between the two groups (P = 0.43, OR = 0.78, 95% CI 0.413-1.46). Heterozygosis (AG) of A386G was found in 1 of the control males but not in the infertile patients, while homozygosis (GG) of A386G was not observed in either group (P = 0.259, OR = 0.698, 59% CI: 0.374-1.306). CONCLUSION: A260G and A386G SNPs of the DAZL gene are not associated with spermatogenic failure and neither represents a molecular marker for the genetic diagnosis of male infertility in the Chinese population of Zhejiang Province.

[Sperm DNA damage and sperm-nucleoprotein transition correlate to acrosin activity and seminal parameters].

OBJECTIVE: To investigate the correlation of sperm DNA damage and sperm-nucleoprotein transition with acrosin activity and seminal parameters. METHODS: We collected 535 semen samples, assessed sperm DNA damage by sperm chromatin dispersion test, and analyzed the correlation of sperm DNA damage and sperm-nucleoprotein transition with acrosin activity and seminal parameters according to the WHO criteria. RESULTS: Statistically significant differences were observed in sperm DNA damage among sperm-nucleoprotein transition, acrosin activity, sperm concentration and the percentage of grade a + b sperm (P < 0.01). Sperm DNA damage was positively correlated with age, sperm-nucleoprotein transition, sperm concentration and the percentage of grade d sperm (P < 0.01 or P < 0.05), but negatively correlated with acrosin activity (P < 0.001). Stepwise linear regression analysis demonstrated that age, sperm concentration, the percentage of grade d sperm, sperm-nucleoprotein transition and acrosin activity were independent variables related to the DNA fragmentation index (DFI). The abnormality rates of sperm-nucleoprotein transition, acrosin activity, sperm concentration and graded a + b sperm were significantly higher in the sperm DNA damage group (DFI > or = 30%) than in the normal control (DFI < 30%) (P < 0.01). CONCLUSION: Sperm DNA damage is closely related with sperm-nucleoprotein transition, acrosin activity and seminal parameters, which may become another important independent parameter for the evaluation of sperm quality.

The CFTR polymorphisms poly-T, TG-repeats and M470V in Chinese males with congenital bilateral absence of the vas deferens.

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have also been frequently identified in patients with CBAVD. However, the distribution of the CFTR polymorphisms M470V, poly-T, TG-repeats and F508del mutation in the Chinese CBAVD population with presumed low cystic fibrosis (CF) frequency remains to be evaluated. Samples obtained from 109 Chinese infertile males with CBAVD and 104 normal controls were analyzed for the presence of CFTR (TG)m(T)n, M470V and F508del by PCR amplification followed by direct sequencing. Our study showed that the F508del mutation was not found in our patients. The 5T mutation was present with high frequency in Chinese CBAVD patients and IVS8-5T linked to either 12 or 13 TG repeats was highly prevalent among CBAVD patients (97.22% of 72 cases and 96.91% of 97 alleles with IVS8-5T). Moreover, a statistically significant relationship between TG12-5T-V470 haplotype and CBAVD was detected. This study indicated that the CFTR polymorphisms poly-T, TG-repeats and M470V might affect the process of CBAVD in the Chinese population.

[Inhibitory effect of siRNA on bcr-abl gene expression in K562 cell line].

To explore a new way to treat CML, inhibitory effect of small interfering RNA (SiRNA) on bcr-abl fusion gene expression of K562 cell line was studied. SiRNA for bcr-abl gene was designed and transfected into K562 cells, bcr-abl gene expression was tested by RT-PCR. The results showed that bcr-abl gene expression was inhibited by using siRNA in dose-dependent manner and reduced to 19.9% and 26.6% of the control at 24 and 48 hours after transfection with 0.2 micro g siRNA respectively. K562 cells proliferation was suppressed finally, but bcr-abl gene expression restored at 72 hours. In conclusion, anti-bcr-abl siRNA can effectively inhibit bcr-abl gene expression of K562 cell line.