Background: In Vietnam, the proportion infertile couples was about 7-10% of reproductive couples. Assisted reproductive technologies appeared and expanded rapidly. In 1970, sperm preparation techniques were found out, artificial insemination became widespread. Sperm preparation stage is very important in assisted reproductive techniques and increasingly improved. Objectives: To compare the efficiency of two sperm preparation techniques: swim \ufffd?up and discontinuous density gradient. Subjects and method: This was a cross-sectional described study included 30 normal semen samples were chosen based on WHO 1999 criteria. We examined sperm vitality, concentration, motility, morphology before and after applied these techniques. Results: Both procedures resulted in a significant higher percentage of vitality, motility, morphology compared with the original semen sample (p<0.01), but the concentration reduced approximately 5 times from whole semen to sediment when swim \ufffd?up was used and 3 times with density gradient, the difference was statically significant (p< 0.01). Conclusion: Normal semen should be prepared by these method. But in oligozoospermic man, to obtain a higher concentration of sperm, semen should be prepared by density gradient method.
Spermatozoa ; Centrifugation ; Density Gradient ;

No abstract available.
Centrifugation, Density Gradient* ; Humans* ; Spermatozoa*

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*

There are several different methods of purifying Treponema pallidum(TP) from rabbit testicular tissue. Among them, we compared the use of differential centrifugation, which has been most widely used, to Percoll density gradient centrifugatian, a newly applied method, in purifying TP from rabbit testicular tissue by checking the protein concentration of the TP suspension, hemagglutination assay using sheep erythrocytes sensitized by TP, IgM-TP-enzyme-linked immunosorhent, assay(IgM-TP-ELISA) and eJect,ron microscopic observation. The protein concent,ration af TP antigen suspension (2x10(8)TP/ml) purified by Percoll density gradient centrifugation (lower band) was the lowest (129.0pg/ml) when compared to those purified by differential centrifugation (324.0pg/ml) and Percoll density gradient centrifugatian (upper band) (560.2pg/ml). Sheep erythrocytes sensitized by TP purified by Percoll density gradient centrifugation(lower band) showed the same resiilts as those using a commercii1 TPHA kit when tested with positive and negative control sera. The sensitivity and specificity of the IgM-TP-ELISA were 88.5%(23/26') and 86.4%(19/2Z) respectively using TP as an antigen purified by differential centrifugation. The rates were 96.29% (25/26) and 95.5%(2l/22) using TP purified by Percoll density gradient centrifugation. As shown by the electron microscopy, T. pizllida purified by clifferential centritugation and Percoll density gradient centrifugatiori were structurally unaltered, and Percoll-purified TP contained much less tissue debris than TP prepared by differ ential centrifugation. Therefor e, Percoll density gradient centrifugation is considered to be a better method of purifying TP from rabbit testicular tissue when compared to differential centrifugatian, as a matter of fact, Perrol1 density gradient centrifugation has been applieci successfully in the study of the physiology, recombinant DNA techniques, and antigenic structure of TP and to the preparation of the antigen for the FTA-ARS and TP-ELISA
Centrifugation ; Centrifugation, Density Gradient ; DNA, Recombinant ; Erythrocytes ; Hemagglutination ; Microscopy, Electron ; Physiology ; Sensitivity and Specificity ; Sheep ; Treponema pallidum* ; Treponema*

Separation of Langerhans cells in epidermis of 16 healthy Korean individuals were performcd. Separation of Langerhans cells by density gradient centrifugation on a colloidal sillica(percoll) polyvinilpyrrolidone gradient. And autologous, allogeneic mixed skin cell leukocyte culture reaction was done with each fractionatcd cpidermal cell suspensions. Also lymphocytes, epidermal cells was cultured in media alone, respectively. The results was quantitated by the incorporation of H-thymidine by p-liquid scintillation counter. The densities of I angerhans cells within the epidermal cells, fraction-2 was most higher concentration (22.0+2.8%) and fraction-5 was most lower concentration (3.4+ l.9%). 2. In the comparison of the results of Langehans cells enriched and depleted population in autologous mixed skin cell leukocyte culture reaction, the former was higher than the latter on lymphocyte stimulatory capacity. There was significant differences(p<0.005) And also same as result in allogeneic mixed skin cell leukocyte culture reaction. 3. Langerhans cells enriched fraction in this study was more lymphocyte stimulatory capacity than depleted fraction in allogeneic mixed skin cell leukocyte culture(p<0.01~0.05). Ailogeneic mixed skin cell leukocyte culture reaction was more lymphocyte stimulatory capacity than the autologous(p<0.005~0.05).
Centrifugation, Density Gradient* ; Colloids* ; Epidermis ; Humans* ; Langerhans Cells* ; Leukocytes* ; Lymphocytes ; Scintillation Counting ; Silicon Dioxide* ; Skin* ; Suspensions

OBJECTIVETo separate high-quality sperm by PureSperm centrifugation applied to intrauterine insemination (IUI) cycles.

METHODSWe compared the separate results after washing the semen with one-layer and two-layer PureSperm gradient centrifugation methods and two-layer Percoll gradient centrifugation method, and used the recovered high-quality sperm for IUI.

RESULTSThe density of the sperm washed with one-layer PureSperm centrifugation method was significantly higher than that washed with two-layer Percoll and two-layer PureSperm centrifugation methods(P < 0.01), but there were no differences in all the results between the use of two-layer Percoll and two-layer PureSperm(P > 0.05). No significant differences in the motility, teratozoospermia and IUI results were found when the three methods were used for sperm preparation(P > 0.05). The percentage of morphologically normal sperm was markedly increased, and the non-sperm components such as leucocytes, epithelial cells and cellular fragments were significantly reduced after washed by the two methods.

CONCLUSIONPureSperm centrifugation is a safe, efficient and easy method for separating high-quality sperm on intrauterine insemination cycles.

Cell Separation ; methods ; Centrifugation, Density Gradient ; Female ; Humans ; Insemination, Artificial ; Male ; Spermatozoa ; cytology

OBJECTIVETo discuss the clinical significance of determining immature germ cells (IGC) in human semen.

METHODSDiscontinuous Percoll gradients technique was employed to separate different cells and May-Grunwald-Giemsa staining and fluorescein isothiocyanate (FTTC)-Mab-CD45 was adopted to identify IGCs and leukcocytes in semen. The IGCs in 30 semen samples were determined including 10 fertile and 20 infertile cases.

RESULTSIGCs concentrated in gradient fractions with 30% to 45% Percoll and leukocytes concentrated in 50%-55% Percoll fractions. The concentration of IGCs was (0.70 +/- 0.40) x 10(6) ml in the fertile group and (1.28 +/- 0.70) x 10(6)/ml in the infertile group(P < 0.05). There was no statistical correlation between the IGC concentration and the sperm density, vitality and normal morphology(P > 0.05).

CONCLUSIONThe use of the discontinuous Percoll gradient method can reach the best separation of IGCs in the ejaculate and it is possible to be used as a clinical index to reflect semen quality.

Cell Separation ; Centrifugation, Density Gradient ; Humans ; Male ; Semen ; cytology ; Spermatozoa ; cytology

OBJECTIVETo study the effect of Percoll selection technique on normal morphology and acrosin activity of human spermatoza.

METHODSThe sperm morphology and sperm acrosin activity were analyzed by automated sperm morphology analyzer(ASMA) and spectrocolorimetry.

RESULTSThe normal morphology sperm rate and acrosin activity were significantly increased after Percoll selection technique (P < 0.001).

CONCLUSIONPercoll selection technique could affect normal morphology sperm ratio and acrosin activity.

Acrosin ; metabolism ; Adult ; Centrifugation, Density Gradient ; Fertilization in Vitro ; Humans ; Male ; Spermatozoa ; cytology ; enzymology

AIMTo 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate.

METHODSThis prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels.

RESULTSSpecimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10(6) vs. 17.6 x 10(6)), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate.

CONCLUSIONSemen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.

Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Cryopreservation ; methods ; Humans ; Male ; Prospective Studies ; Sperm Motility ; Spermatozoa

OBJECTIVESTo develop a simple and effective method by which spermatids can be isolated from mouse testis.

METHODSCombination of enzymatic digestion was used to prepare suspension of spermatogenic cells from adult mouse testis, and then a modified discontinuous Percoll gradient (15%, 22%, 30%, 40%, 50%, 60%) centrifugation method was introduced to isolate spermatids from the cellular suspension. The content of spermatids in each isolated fraction by Percoll method was determined by morphology (Wright-Giemsa staining) and flow cytometry analysis, and the viability of spermatogenic cells was assessed using Eosin Y exclusion test.

RESULTSMore than 97% of the testicular cells remained their viability after enzymatic digestion. After Percoll centrifuged, six fractions were formed. In each isolated fraction, the 22% fraction contained mostly spermatids(mean 86.7%) and cell viability was more than 85.5%. While in the 30% fraction, immature spermatogenic cells were present, and more than 92% of the cells remained their viability.

CONCLUSIONSA large of relatively purified spermatids can be isolated from mouse testis by enzymatic digestion combined discontinuous Percoll gradient centrifugation method.

Animals ; Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Male ; Mice ; Spermatids ; cytology ; Testis ; cytology