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*

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

OBJECTIVETo compare the effect of continuous and discontinuous density gradient centrifugation for purification of human pancreatic islets with COBE 2991 cell processor.

METHODSHuman pancreases were obtained from brain-dead donors and stored in cold UW solution. The connective tissues were removed from the pancreases, and the pancreatic ducts were perfused with a cold enzyme (Liberase). The islets were then separated by gentle mechanical dissociation and purified with discontinuous (10 pancreases) or continuous (8 pancreases) gradients of HCA-Ficoll in COBE 2991 cell processor. Samples were collected in duplicate for determination of the quantity of islets, islet equivalents (IEQ), and the purity.

RESULTSThe weights of the pancreases before and after connective tissue removal and pancreas duct perfusion, and the quantity of islets obtained (including islets quantity of different diameters and total IEQ) after dissociation were not significantly different. Continuous gradient of HCA-Ficoll, compared with discontinuous gradient, resulted in significantly greater final islet quantity (55,000 IEQ vs 206,000 IEQ, P=0.000) and islet purity (58.0%-/+8.0% vs 33.5%-/+10.3%, P=0.000) and also greater number of islets with a diameter lager than 200 microm (P<0.01).

CONCLUSIONContinuous density gradient centrifugation can be more effective than discontinuous gradient in islet purification.

Cell Count ; Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Humans ; Islets of Langerhans ; cytology ; Organ Size

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

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