Density gradient centrifugation was performed to isolate motile sperm. Samples were incubated with different concentrations (0, 1, 10, 100, and 1,000 ng/mL) of RA. The non-exposed group (0 ng/mL) was defined as the control group. Samples were analyzed for motility at different time points (0, 60, 150, 240, and 300 minutes) and for vitality and oxidation reduction potential (ORP) (at 0, 240, and 300 minutes). Sperm motility was assessed manually and motion kinetic parameters were recorded by computer aided semen analysis.RESULTS: RA at any tested concentration significantly increased sperm motility compared to the control in a time-dependent manner with a maximum increase after 240 minutes. Motion kinetic parameters were not comparable. For sperm vitality, supplementation with RA significantly maintained survival at higher levels, while non-treated sperm gradually died. These higher levels of vitality were maintained with rising RA concentrations of up to 1,000 ng/mL. A non-significant trend of increased ORP was observed in all study groups.CONCLUSIONS: RA increases sperm motility and maintains vitality at any concentration tested. Therefore, RA might be utilized to improve sperm quality in asthenozoospermic specimens. However, further investigation is ongoing to evaluate the effect of RA on other sperm parameters.]]>
Centrifugation, Density Gradient ; Humans ; In Vitro Techniques ; Methylphenidate ; Oxidation-Reduction ; Oxidative Stress ; Semen ; Semen Analysis ; Sperm Motility ; Spermatozoa ; Tissue Donors ; World Health Organization

OBJECTIVE: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. METHODS: Thirty men with a total motile spermatozoa count >80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. RESULTS: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p<0.05 for each when compared with 6.9% in raw semen). The DNA fragmentation level was significantly reduced after DGC using PureSperm, PureCeption, and SpermGrad media (6.0%, 6.5%, and 4.9%, respectively; p<0.05 for each when compared with 11.2% in raw semen), but not after DGC using Sidney media (8.5%, p>0.05). HA binding ability did not change after DGC using any of the four media. CONCLUSION: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.
Centrifugation, Density Gradient ; DNA Fragmentation ; DNA ; Humans ; Hyaluronic Acid ; Male ; Semen ; Spermatozoa

OBJECTIVE: The aim of this study was to investigate DNA fragmentation status in human spermatozoa according to specific tail swelling patterns determined via hypo-osmotic swelling test (HOST).METHODS: Frozen semen samples from 21 healthy donors were thawed and prepared by the swim-up technique for use in intracytoplasmic sperm injection. The semen samples were treated for 5 minutes as part of the HOST procedure and then underwent the sperm chromatin dispersion test using a Halosperm kit. DNA fragmentation status (large halo, medium halo, small halo, no halo, or degraded) and the specific tail swelling pattern (“a”–“g”) were assessed at the level of a single spermatozoon. A total of 42,000 spermatozoa were analyzed, and the percentage of spermatozoa without DNA fragmentation (as evidenced by a large or medium halo) was assessed according to the specific tail swelling patterns observed.RESULTS: The HOST examinations showed that >93% of spermatozoa across all types displayed no DNA fragmentation. The percentage of spermatozoa without DNA fragmentation was 100% in type “d”, 98.67% in type “g”, and 98.17% in type “f” spermatozoa.CONCLUSION: We found that the type “d” spermatozoa displayed no DNA fragmentation, but the other types of spermatozoa also displayed very low rates of DNA fragmentation. This result may be associated with the processing of the spermatozoa by density gradient centrifugation and the swim-up technique.
Centrifugation, Density Gradient ; Chromatin ; DNA Fragmentation ; DNA ; Humans ; Infertility ; Semen ; Semen Preservation ; Sperm Head ; Sperm Injections, Intracytoplasmic ; Spermatozoa ; Tail ; Tissue Donors

OBJECTIVE: The aim of this study was to compare the efficacy of swim-up and density gradient centrifugation (DGC) for reducing the amount of sperm with fragmented DNA, sex chromosome aneuploidy, and abnormal chromatin structure. METHODS: Semen samples were obtained from 18 healthy male partners who attended infertility clinics for infertility investigations and were processed with swim-up and DGC. The percentages of sperm cells with fragmented DNA measured by the sperm chromatin dispersion test, normal sex chromosomes assessed by fluorescence in situ hybridization, and abnormal chromatin structure identified by toluidine blue staining were examined. RESULTS: The percentage of sperm cells with fragmented DNA was significantly lower in the swim-up fraction (9.7%, p=0.001) than in the unprocessed fraction (27.0%), but not in the DGC fraction (27.8%, p=0.098). The percentage of sperm cells with normal X or Y chromosomes was comparable in the three fractions. The percentage of sperm cells with abnormal chromatin structure significantly decreased after DGC (from 15.7% to 10.3%, p=0.002). The swim-up method also tended to reduce the percentage of sperm cells with abnormal chromatin structure, but the difference was not significant (from 15.7% to 11.6%, p=0.316). CONCLUSION: The swim-up method is superior for enriching genetically competent sperm.
Aneuploidy* ; Centrifugation, Density Gradient* ; Chromatin ; DNA Fragmentation* ; DNA* ; Fluorescence ; Humans ; In Situ Hybridization ; Infertility ; Male ; Methods ; Semen ; Sex Chromosomes* ; Spermatozoa* ; Tolonium Chloride ; Y Chromosome

OBJECTIVE: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. METHODS: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. RESULTS: The sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). CONCLUSION: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.
Apoptosis ; Centrifugation* ; Centrifugation, Density Gradient ; Chromatin* ; DNA Fragmentation ; DNA* ; Methods ; Product Packaging* ; Semen ; Semen Analysis ; Sperm Motility ; Spermatozoa*

OBJECTIVETo investigate the influence of different methods of semen preservation and processing on sperm DNA integrity.

METHODSWe collected semen samples from 100 normozoospermic male volunteers and, following homogeneous mixing, preserved them by means of snap freezing, slow freezing, or at the room temperature for 4 and 24 hours. Meanwhile we processed the semen by washing, swim-up, and density gradient centrifugation (DGC). Then we obtained the sperm DNA fragmentation index (DFI) by sperm chromatin dispersion test and measured total sperm motility and DFI after cultured for 24 hours following processing.

RESULTSThe sperm DFIs after 4 hours of preservation by snap freezing, slow freezing, and at the room temperature were (27.3 ± 6.4)%, (26.9 ± 6.1)%, and (24.7 ± 6.8)%, respectively, and that after preserved at the room temperature for 24 hours was (35.6 ± 9.0)%, with statistically significant differences between the first three and the 24-hour room temperature preservation groups (P < 0.05) but not among the former three groups (P > 0.05). The sperm DFI was significantly higher in the samples processed by washing ([13.7 ± 2.0]%) than in those processed by swim-up ([9.1 ± 1.3]%) and DGC ([8.0 ± 2.5]%) (P < 0.05), and it was the lowest in the DGC group after 24-hour culture ([11.5 ± 4.2]%) as compared with the other groups (P < 0.05).

CONCLUSIONSperm DNA integrity is influenced by different semen preservation conditions and processing methods.

Centrifugation, Density Gradient ; DNA Fragmentation ; Humans ; Male ; Semen ; Semen Analysis ; Semen Preservation ; methods ; Sperm Motility ; Spermatozoa ; cytology

OBJECTIVE: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. METHODS: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). RESULTS: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. CONCLUSION: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.
Centrifugation, Density Gradient ; Embryonic Structures ; Female ; Fertilization ; Fertilization in Vitro ; Humans ; Infertility ; Oocytes ; Pregnancy Rate ; Reproductive Techniques ; Reproductive Techniques, Assisted ; Retrospective Studies ; Semen ; Spermatozoa

To improve a fast and high-quality isolation method for culturing the primary cardiomyocyte and fibroblast in vitro, the neonatal Wistar rats were decapitated accordingly and left ventricles were isolated under the sterile condition. The ventricles were chopped and digested in the enzyme solution containing 0.5 mg/mL type II collagenase. During this process, the digesting time, frequency and stirring speed, centrifuging frequency and speed were strictly controlled. The cardiomyocytes were separated from the cardiac fibroblast by using the Percoll density gradient centrifugation. The cell viability was tested by staining with 0.2% trypan blue. The purity of cardiomyocytes and fibroblasts were determined by immunoflourescent staining with anti-cTnI, anti-Vimentin and anti-α-SMA antibodies. The results indicated that with this protocol, the viability and purity of cardiomyocytes were 92% and 95%. The automobile pulse of the adhered cardiomyocyte was visible. For fibroblasts, the cell viability and purity were 96% and 94%. Our results demonstrate that this advanced isolation method is reproducible, and can simultaneously produce high-quality primary cardiomyocytes and fibroblasts for the future study.
Animals ; Cell Separation ; methods ; Cell Survival ; Centrifugation, Density Gradient ; Fibroblasts ; Heart Ventricles ; cytology ; Myocytes, Cardiac ; Povidone ; Rats ; Rats, Wistar ; Silicon Dioxide

OBJECTIVETo investigate the influence of the time interval from the end of semen processing to artificial intrauterine in semination with husband's sperm (AIH-IUI) on the rate of clinical pregnancy.

METHODSThis study involved 191 AIH-IUI cycles with the same ovulation induction protocol. After Percoll density gradient centrifugation, we divided the sperm into four groups based on the incubation time: 0-19, 20-39, 40-59, and 60-80 min, and again into another four groups according to the total progressively motile sperm count (TPMC): (0-9), (10-20), (21-30), and > 30 x 10(6). We analyzed the correlation of the clinical pregnancy rate with the time interval from the end of sperm processing to AIH-IUI and with other influencing factors, such as maternal age, infertility duration, and semen quality.

RESULTSThe rate of clinical pregnancy was significantly higher in the 20-39 min group (18.3%) than in the 0-19, 40-59, and 60-80 min groups (12.7, 11.4 and 9.1%) (all P < 0.05). The (10-20) x 10(6) group achieved a remarkably higher pregnancy rate (16.7%) than the (0-9), (21-30), and > 30 x 10(6) groups (0, 11.4, and 8.3%) (all P < 0.05). Logistic multivariate analysis showed that the rate of clinical pregnancy was decreased with the increased age of the women (OR 0.89, 95% CI 0.83-0.94) but significantly elevated in the 20-39 min group (OR 2.11, 95% CI 1.34-3.13) and of (10-20) x 10(6) group (OR 2.06, 95% CI 1.32-3.46).

CONCLUSIONThe time interval from the end of sperm processing to AIH-IUI is a most significant factor influencing the rate of clinical pregnancy of AIH-IUI.

Centrifugation, Density Gradient ; Female ; Humans ; Infertility ; therapy ; Insemination, Artificial, Homologous ; statistics & numerical data ; Male ; Pregnancy ; Pregnancy Rate ; Semen ; Semen Analysis ; Sperm Count ; Spermatozoa ; Time Factors

OBJECTIVETo explore the expression patterns of the chemokine CXCL12 and its receptor CXCR4 in human sperm.

METHODSWe collected semen samples from 10 fertile men, performed density gradient centrifugation, and then determined the expressions of both CXCL12 and CXCR4 in the sperm by RT-PCR and immunofluorescence staining.

RESULTSRT-PCR revealed the mRNA expressions of CXCL12 (0.641 +/- 0.180) and CXCR4 (0.464 +/- 0.100) in the sperm. However, only CXCR4 rather than CXCL12 was expressed at the protein level, and the positive staining for CXCR4 was observed mainly in the posterior part of the acrosome.

CONCLUSIONCXCL12 and CXCR4 are involved as important molecules in regulating the function of human sperm.

Acrosome ; metabolism ; Centrifugation, Density Gradient ; Chemokine CXCL12 ; metabolism ; Humans ; Male ; Receptors, CXCR4 ; metabolism ; Signal Transduction ; Spermatozoa ; metabolism