The renin angiotensin system (RAS) appears to influence male fertility at multiple levels. In this work, we analyzed the relationship between the RAS and DNA integrity. Fifty male volunteers were divided into two groups (25 each): control (DNA fragmentation ≤20%) and pathological (DNA fragmentation >20%) cases. Activities of five peptidases controlling RAS were measured fluorometrically: prolyl endopeptidase (which converts angiotensin [A] I and A II to A 1-7), neutral endopeptidase (NEP/CD10: A I to A 1-7), aminopeptidase N (APN/CD13: A III to A IV), aminopeptidase A (A II to A III) and aminopeptidase B (A III to A IV). Angiotensin-converting enzyme (A I to A II), APN/CD13 and NEP/CD10 were also assessed by semiquantitative cytometry and quantitative flow cytometry assays, as were the receptors of all RAS components: A II receptor type 1 (AT1R), A II receptor type 2 (AT2R), A IV receptor (AT4R or insulin-regulated aminopeptidase [IRAP]), (pro)renin receptor (PRR) and A 1-7 receptor or Mas receptor (MasR) None of the enzymes that regulate levels of RAS components, except for APN/CD13 (decrease in fragmented cells), showed significant differences between both groups. Micrographs of RAS receptors revealed no significant differences in immunolabeling patterns between normozoospermic and fragmented cells. Labeling of AT1R (94.3% normozoospermic vs 84.1% fragmented), AT4R (96.2% vs 95.3%) and MasR (97.4% vs 87.2%) was similar between the groups. AT2R (87.4% normozoospermic vs 63.1% fragmented) and PRR (96.4% vs 48.2%) were higher in non-fragmented spermatozoa. These findings suggest that fragmented DNA spermatozoa have a lower capacity to respond to bioactive RAS peptides.
Angiotensins ; DNA Fragmentation ; Humans ; Insulin ; Male ; Renin-Angiotensin System/physiology* ; Spermatozoa

ObjectiveTo investigate the effects of resveratrol in the cryopreservation medium on the quality and function of post-thaw sperm.

METHODSSemen samples were obtained from 50 normozoospermic and 50 oligoasthenozoospermic men, liquefied and then cryopreserved in the glycerol-egg yolk-citrate (GEYC) medium with or without 30 μmol/L resveratrol. Sperm motility, viability and acrosome reaction (AR) were examined before and after thawing. Sperm lipid peroxidation and the level of reactive oxygen species (ROS) were measured using commercial malondialdehyde (MDA) and the ROS assay kit. Sperm mitochondrial membrane potential (MMP) and DNA damage were determined by Rhodamine 123 staining and TUNEL.

RESULTSThe percentage of progressively motile sperm (PMS), total sperm motility, sperm viability, MMP and AR were significantly decreased (P <0.05) while the levels of sperm ROS, MDA and DNA fragmentation index (DFI) remarkably increased in both the normozoospermia and oligoasthenozoospermia groups after cryopreservation as compared with those in the fresh ejaculate (P <0.05). In comparison with the non-resveratrol control, the post-thaw sperm cryopreserved with 30 μmol/L resveratrol showed markedly higher PMS (［32.7 ± 4.8］ vs ［43.1 ± 6.3］ %, P <0.05), total motility (［44.8 ± 6.9］ vs ［56.9 ± 7.4］ %, P <0.05), viability (［52.3 ± 6.1］ vs ［67.5 ± 5.6］ %, P <0.05), MMP (［56.5 ± 7.0］ vs ［63.4 ± 7.5］ %, P <0.05) and AR (［16.6 ± 3.8］ vs ［26.3 ± 4.7］ %, P <0.05) but lower ROS, MDA and DFI (all P <0.05) in the normozoospermia group, and so did the post-thaw sperm in the oligoasthenozoospermia group, with a particularly lower DFI (［28.5 ± 4.8］ vs ［36.3 ± 5.7］%, P <0.01).

CONCLUSIONSResveratrol in the cryopreservation medium can improve the quality and function of post-thaw human sperm by reducing cryopreservation-induced sperm injury and the level of ROS.

Acrosome ; drug effects ; Animals ; Antioxidants ; pharmacology ; Cryopreservation ; methods ; DNA Fragmentation ; Humans ; Lipid Peroxidation ; Male ; Malondialdehyde ; Membrane Potential, Mitochondrial ; Reactive Oxygen Species ; analysis ; Resveratrol ; pharmacology ; Semen Analysis ; Semen Preservation ; adverse effects ; Sperm Motility ; drug effects ; Spermatozoa ; drug effects ; physiology

ObjectiveTo explore Mycoplasma genitalium (MG) infection in the urogenital tract of infertile men and its influence on semen quality.

METHODSSemen samples were collected from 352 infertile males in the Center of Reproductive Medicine of Nanjing General Hospital from March to July 2015. MG infection was detected by real-time fluorescence simultaneous amplification and testing and semen analyses were conducted according to the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed) on the semen pH value, semen volume, total sperm count, sperm concentration, total sperm motility, percentages of progressively motile sperm (PMS) and immotile sperm (IMS), and sperm DNA fragmentation index (DFI). The data obtained were subjected to statistical analysis by t-test and non-parametric test (Wilcoxon test).

RESULTSMG infection was found in 3.4% (12/352) of the infertile patients. Compared with the MG-positive cases, the MG-negative ones showed a significantly higher semen volume (［2.85 ± 0.14］ vs ［3.84 ± 0.12］ ml, P = 0.008) and percentage of PMS (［15.86±1.72］ vs ［60.95 ± 5.63］ %, P = 0.032) but a lower DFI (［30.73 ±2.24］ vs ［20.71 ± 1.55］%, P = 0.014). However, no statistically significant differences were observed between the two groups in the semen pH value (7.38 ±0.02 vs 7.39 ± 0.01, P = 0.774), sperm concentration (［52.96 ± 15.78］ vs ［60.05 ± 4.29］×10⁶/ml, P = 0.683), sperm count (［154.15 ± 46.37］ vs ［221.56 ± 15.43］×106, P = 0.236), total sperm motility (［29.04 ± 3.11］ vs ［33.52 ± 1.51］ %, P = 0.626), or percentage of IMS (［23.57 ± 0.99］ vs ［62.34 ± 1.69］ %, P = 0.691).

CONCLUSIONSUrogenital MG infection is common in infertile males and potentially affects the semen quality, especially sperm vitality of the patient.

DNA Fragmentation ; Humans ; Infertility, Male ; microbiology ; physiopathology ; Male ; Male Urogenital Diseases ; microbiology ; Mycoplasma Infections ; complications ; Mycoplasma genitalium ; Semen ; Semen Analysis ; Sperm Count ; Sperm Motility ; Spermatozoa ; physiology

Objective: Sperm DNA fragmentation (SDF) is widely used to predict male infertility and the methods of detecting SDF are varied. This study aimed to compare two methods of SDF detection and investigate the correlation between SDF and sperm quality. METHODS: Using sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCD), we detected SDF in 108 semen samples collected in the Center of Reproduction and Genetics of Suzhou Municipal Hospital. We compared the results of the two methods and analyzed the correlations of SDF routine semen parameters, sperm morphology and the age of the patients. RESULTS: A significant consistency was found in the SDF index (DFI) between the two methods (P<0.01). The DFI was correlated negatively with sperm motility, the percentage of progressively motile sperm, and that of morphologically normal sperm (P <0.01), but positively with the teratozoospermia index (P <0.01 in SCSA and P <0.05 in SCD). The DFI measured by SCSA showed a significantly positive correlation with the patients' age (P <0.01), but not that obtained by SCD. CONCLUSIONS The results of both SCSA and SCD play an important role in predicting sperm quality. As a clinical index, the DFI has a predictive value for male infertility. However, the results of different detecting methods vary widely, which calls for further studies on their standardization.
Chromatin ; genetics ; physiology ; DNA Fragmentation ; Humans ; Infertility, Male ; diagnosis ; Male ; Semen ; physiology ; Semen Analysis ; Sperm Motility ; Spermatozoa ; physiology ; ultrastructure

Objective: To study the correlation of the gene expressions of Chk1 and Chk2 with sperm concentration and motility. METHODS: According to sperm concentration and motility (percentage of progressively motile sperm), we divided 80 semen samples into four groups of equal number: normal control, oligozoospermia (OS), asthenospermia (AS), and oligoasthenozoospermia (OAS). We detected the sperm DNA fragmentation index (DFI) and viability and determined the expressions of Chk1 and Chk2 in the sperm by RT-PCR and Western blot. RESULTS: Statistically significant differences were not found in sperm DFI among the control, OS, AS, and OAS groups (21.24±6.93, 19.67±7.64, 21.52±6.92, and 19.28±11.55, P>0.05), but observed in sperm concentration, progressive motility, and viability between the DFI >30% and DFI ≤30% groups (P<0.01). Compared with the normal control, sperm viability was remarkably decreased in the OS, AS, and OAS groups (［83.48±9.87］% vs ［63.86±9.16］%, ［50.45±16.99］%, and ［39.21±15.74］%, P<0.05). RT-PCR showed remarkable differences among the control, OS, AS, and OAS groups in the relative expression level of Chk1 mRNA (0.73±0.22, 0.62±0.14, 1.03±0.39, and 0.92±0.071, P<0.01), which was correlated positively with sperm concentration (b = 80.661, P<0.01) but negatively with sperm motility (b = －19.275, P < 0.01), as well as in that of Chk2 mRNA (0.66±0.30, 0.27±0.09, 0.59±0.19, and 0.42 ± 0.11, P<0.01), which was correlated negatively with sperm concentration (b = －90.809, P<0.01) but positively with sperm motility (b = 27.507, P <0.01). The relative expression levels of the Chk1 protein were significantly different among the four groups (0.63±0.05, 0.42±0.03, 1.13±0.08, and 0.87±0.07, P<0.01), which was correlated positively with sperm concentration (b = 55.74, P<0.01) but negatively with sperm motility (b =－22.649, P<0.01), and so were those of the Chk2 protein (1.23±0.36, 0.37±0.16, 0.87±0.08, and 0.68±0.12, P<0.01), which was correlated negatively with sperm concentration (b =－53.001, P<0.01) but positively with sperm motility (b = 16.676, P < 0.01). CONCLUSIONS Chk1 and Chk2 are significantly expressed in human sperm. In case of sperm DNA damage, up-regulated Chk1 expression may enhance sperm apoptosis and lead to asthenospermia, while increased Chk2 expression may inhibit spermatogenesis and result in oligospermia.
Apoptosis ; Asthenozoospermia ; genetics ; Checkpoint Kinase 1 ; genetics ; metabolism ; Checkpoint Kinase 2 ; genetics ; metabolism ; DNA Damage ; DNA Fragmentation ; Gene Expression ; Humans ; Male ; Oligospermia ; genetics ; Semen Analysis ; Sperm Count ; Sperm Motility ; genetics ; Spermatozoa ; physiology

Objective: To clarify the roles of yam polysaccharide (YPS) in improving sperm viability and protecting sperm DNA integrity in vitro and provide a new approach to the treatment of oligoasthenozoospermia. METHODS: We collected samples by masturbation from 36 normal fertile males aged 27－39 years. Each sample was divided into six groups: blank control or treated with normal saline, vitamin C solution, and YPS solution at low (0.25 mg/ml), medium (1.0 mg/ml) or high concentration (5.0 mg/ml). Using eosin-Y staining, sperm hypotonic swelling (HOS) and sperm chromatin diffusion (SCD) test, we observed the effects of different concentrations of YPS on sperm viability, membrane integrity and nuclear DNA. RESULTS: After 24 and 48 hours of treatment, sperm viability was markedly reduced in the vitamin C (［28.5 ± 3.1］ and ［6.5 ± 1.2］%), low-YPS (［31.3 ± 3.5］ and ［6.5 ± 2.2］%), medium-YPS (［37.1 ± 3.5］ and ［9.5 ± 2.8］%) and high-YPS groups (［38.3 ± 3.3］ and ［9.0 ± 3.2］%) as compared with the blank control (［17.3 ± 2.1］ and ［3.2 ± 1.3］%) (P <0.01) and normal saline groups (［13.4 ± 4.1］ and ［3.1 ± 2.0］%) (P <0.01), and it was significantly higher in the medium- and high-YPS than in the vitamin C group (P <0.05 and P <0.01). The rate of sperm DNA fragmentation was remarkably decreased at 48 hours in the vitamin C (［30.5 ± 3.1］%), low-YPS (［29.4 ± 2.6］%), medium-YPS (［28.5 ± 2.3］%) and high-YPS groups (［27.9 ± 1.9］%) in comparison with the blank control (［41.7 ± 2.2］%) (P <0.01) and normal saline groups (［42.1 ± 3.3］%), markedly lower in the medium- and high-YPS than in the blank control, normal saline and vitamin C groups (P <0.05 or P <0.01), but with no statistically significant difference between the low-YPS and vitamin C groups (P >0.05). CONCLUSIONS Yam polysaccharide can improve sperm viability and protect sperm DNA integrity in vitro.
Adult ; Ascorbic Acid ; pharmacology ; DNA ; drug effects ; DNA Fragmentation ; Dioscorea ; chemistry ; Humans ; Male ; Polysaccharides ; pharmacology ; Semen Analysis ; Sperm Motility ; Spermatozoa ; drug effects ; physiology ; Vitamins ; pharmacology

Toxoplasma gondii penetrates all kinds of nucleated eukaryotic cells but modulates host cells differently for its intracellular survival. In a previous study, we found out that serine protease inhibitors B3 and B4 (SERPIN B3/B4 because of their very high homology) were significantly induced in THP-1-derived macrophages infected with T. gondii through activation of STAT6. In this study, to evaluate the effects of the induced SERPIN B3/B4 on the apoptosis of T. gondii-infected THP-1 cells, we designed and tested various small interfering (si-) RNAs of SERPIN B3 or B4 in staurosporine-induced apoptosis of THP-1 cells. Anti-apoptotic characteristics of THP-1 cells after infection with T. gondii disappeared when SERPIN B3/B4 were knock-downed with gene specific si-RNAs transfected into THP-1 cells as detected by the cleaved caspase 3, poly-ADP ribose polymerase and DNA fragmentation. This anti-apoptotic effect was confirmed in SERPIN B3/B4 overexpressed HeLa cells. We also investigated whether inhibition of STAT6 affects the function of SERPIN B3/B4, and vice versa. Inhibition of SERPIN B3/B4 did not influence STAT6 expression but SERPIN B3/B4 expression was inhibited by STAT6 si-RNA transfection, which confirmed that SERPIN B3/B4 was induced under the control of STAT6 activation. These results suggest that T. gondii induces SERPIN B3/B4 expression via STAT6 activation to inhibit the apoptosis of infected THP-1 cells for longer survival of the intracellular parasites themselves.
Animals ; Antigens, Neoplasm/genetics/*metabolism ; *Apoptosis ; Cell Line ; DNA Fragmentation ; Humans ; Macrophages/*cytology/metabolism ; Mice ; Mice, Inbred BALB C ; STAT6 Transcription Factor/genetics/*metabolism ; Serpins/genetics/*metabolism ; Toxoplasma/genetics/*physiology ; Toxoplasmosis/genetics/*metabolism/parasitology/*physiopathology

BACKGROUNDVibrio vulnificus (Vv) is an estuarine bacterium that can cause primary septicemia as well as serious wound infections. However, little is known about the mechanisms by which Vv infects dendritic cells (DCs) and its effects on cytoskeleton. In this study, we aimed to investigate the invasion, internalization, and the organelles damage of the cultured dendritic cells (a DC 2.4 strain) during Vv infection.

METHODSThe study model was the cultured DCs infected by a Vv 1.758 strain. Electron microscopy was used to observe the localization of bacteria at the different time points of infection, cell morphology, and the process of organelles changes. The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined under a fluorescence microscope.

RESULTSThe Vv were pinocytosised into the DC cells through double-sides, and localized at 1 - 2 mm of the inner side membrane. It took 1.3, 1.9, and 3.4 hours to reach the infection ratio of 25%, 50%, and 75%, respectively. Using electron microscopy, the DCs had been observed to have developed chromatin aggregation within 4.0 hours, and significant cytoskeleton structure disruption was noted within 6.0 hours.

CONCLUSIONThe high lethality of Vv infection may be associated with the direct disruption of the DCs cytoskeleton structure.

Animals ; Apoptosis ; physiology ; Cells, Cultured ; Cytoskeleton ; metabolism ; ultrastructure ; DNA Fragmentation ; Dendritic Cells ; metabolism ; microbiology ; ultrastructure ; Mice ; Microscopy, Electron ; Microscopy, Electron, Transmission ; Vibrio Infections ; metabolism ; Vibrio vulnificus ; pathogenicity

OBJECTIVETo investigate the effect of high ligation of the spermatic vein (HLSV) on DNA fragmentation in varicocele (VC) patients.

METHODSThirty-four VC patients underwent HLSV. Sperm motion indexes and the results of papanicolaou staining and DNA fragmentation detection were analyzed before and 3 months after the operation according to the WHO guidelines.

RESULTSCompared with pre-operation, HLSV achieved a significant increase in the percentage of morphologically normal sperm (P < 0.01), and remarkable decreases in DNA fragmentation, sperm deformity index (SDI) and multiple anomalies index (MAI) (P < 0.01). The patients also showed significant increases in sperm concentration and the percentages of grade b sperm (P < 0.05) and grade a and a + b sperm (P < 0.01) after the operation. The post-operative percentages of sperm DNA fragmentation in those with grades I - III VC were markedly lower (P < 0.01), but showed no significant difference from that in those with subclinical VC (P > 0.05). The percentage of big-halo sperm was significantly increased (P < 0.01), while those of the medium-, small- and non-halo sperm remarkably decreased (P < 0.01) after HLSV.

CONCLUSIONHLSV can effectively improve the sperm quality of VC patients.

Adolescent ; Adult ; DNA Fragmentation ; Humans ; Ligation ; methods ; Male ; Sperm Count ; Sperm Motility ; Spermatozoa ; physiology ; Varicocele ; genetics ; surgery ; Veins ; surgery ; Young Adult

OBJECTIVETo study the correlation between sperm DNA fragmentation index (DFI), age of male, various parameters of sperm, rates of fertilization, high quality embryo and pregnancy and implantation rates.

METHODSOne hundred and eleven infertile couples were selected randomly, and DFI was tested by flow cytometry for the sperm used for IVF. The patients were divided into different groups according to the DFI scores. The results of each group were analyzed.

RESULTSThe IVF normal fertilization was significantly lower in couples with sperm DFI over 10% (60.5%) than that in couples with DFI below 10% (70.1%) (P<0.05). Significantly positive correlation was found between DFI and the age of male (r=0.624, P<0.05). DFI was also significantly negatively correlated with the percentage of linearly progressive sperm (r=-0.360, P<0.05). There was no significant correlation between the rates of high quality cleaved embryos, pregnancy and implantation rate and sperm DFI.

CONCLUSIONDFI scores are increased with male's age, and it can influence the sperm motility. DFI=10% can be considered as a critical point which can be used to estimate the clinical fertility rate of IVF. But it could not provide relative information about the rates of high quality embryos and pregnancy for infertile couples undergoing IVF procedure.

Adult ; Aging ; genetics ; physiology ; DNA Fragmentation ; Embryo Implantation ; genetics ; Female ; Fertilization in Vitro ; Humans ; Male ; Pregnancy ; Regression Analysis ; Spermatozoa ; metabolism ; physiology