Comparison between the quality and function of sperm after semen processing with two different methods

Aim: To compare the recovery rate of morphologically normal and chromatin condensed spermatozoa from native se-men samples using the SpermPrep~(TM) filtration columns and Percoll gradient centrifugation and to determine the influenceof the two processing techniques on fertilization and pregnancy rates in an IVF-ET program. Methods: Sixteen se-men samples obtained from patient's husband were included in this study. Each was divided into two aliquots. The firstaliquot was processed with SpermPrep~(TM) filtration columns and the second, Percoll gradient centrifugation. Smears weremade before and after semen processing with both methods for the evaluation of chromatin condensation (chromomycineCMA3) as well as morphology (strict criteria) of spermatozoa. One hundred and seventy oocytes were retrieved fromthe patients and the oocytes from each patient were subdivided into two sets : one set was inseminated using spermatozoaprocessed with SpermPrep~(TM) and the other inseminated after semen processing with Pe     

Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

AIM: To 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. METHODS: This 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. Results: Specimens 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. CONCLUSION: Semen 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.     

PureSperm离心法在宫腔内人工授精周期中优选精子的应用

目的 :探讨PureSperm离心法在宫腔内人工授精 (IUI)周期中优选精子的效果。　方法 :采用PureSperm单、双梯度离心法及Percoll双梯度离心法分别处理精液 ,比较 3种方法的处理效果 ,并将处理后的精子悬液施行夫精IUI。　结果 :单梯度PureSperm较双梯度的PureSperm及Percoll洗涤后精子密度显著增加 (P <0 .0 1) ,双梯度PureS perm及Percoll离心法两者差异无显著性 (P >0 .0 5 ) ,3种方法在洗涤后精子活率、畸形率及妊娠率比较差异均无显著性 (P >0 .0 5 )。洗涤后正常形态的精子数量明显增加 ,白细胞、上皮细胞及细胞碎片等非精子成分明显减少。　结论 :PureSperm离心法是一种高效、简便、安全的IUI精液处理方法。     

Morphometrical comparison of human spermatozoa obtained from semen and swim-up methodology

Because morphology is regularly established in semen smears, but not in swim-up spermatozoa, we were interested in comparing some morphological parameters of semen and swim-up spermatozoa to establish if the cells selected by the swim-up method were morphologically similar to those considered normal in semen. Normal human semen samples were divided into two aliquots. One of these aliquots was washed by centrifugation with B₂ medium and sperm smears were prepared with the resulting pellet as a control. The other aliquot was used to perform swim-up separation and the spermatozoa from the supernatant were used as experimental smears. Both groups were stained according to the triple stain technique and spontaneous acrosome reaction and viability were determined. Video microscopy and computer-assisted image processing of live and non-reacted sperm cells were used to establish morphometrical parameters of the sperm head in both populations. The following set of morphometrical parameters were considered: width, length, width/length ratio, acrosome area, head area, and acrosome area/head area ratio. An increase in head width, a decrease in head length and a subsequent increase of width/length ratio were found in swim-up cells compared with the control group. A slight increase in acrosome area/head area ratio was also observed in swim-up spermatozoa. Through the swim-up methodology we were able to select a subpopulation of oval shaped heads with spermatozoa having a bigger acrosome area in comparison to semen.     

Application of the hypo-osmotic swelling test to spermatozoa prepared by swim-up and discontinuous Percoll separation

The quality of spermatozoa prepared by washing and swim-up or by discontinuous Percoll centrifugation, was compared by applying the hypo-osmotic swelling (HOS) test to semen samples from 116 men of infertile couples. The HOS test performed on 95 normal semen samples showed that the percentage of swollen spermatozoa separated by both techniques was significantly higher than in the initial ejaculate ( p <0.001). The percentage of HOS-positive spermatozoa separated by the Percoll gradient technique was significantly higher than that separated by the swim-up technique ( p <0.001). On the contrary, in 21 abnormal semen samples, there was no significant difference in the percentage of spermatozoa which were positive in the HOS test between the Percoll gradient and the swim-up technique ( p =0.44). It is suggested that the Percoll gradient technique appears to be preferable to the swim-up technique when semen parameters are normal, but there is no significant difference between these two techniques in abnormal semen.     

Semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity

The objective of this study was to determine the effects of density gradient centrifugation on sperm cell DNA integrity and to correlate any detected DNA damage with semen analysis parameter. A total of 40 semen samples were collected from nonazoospermic men presenting for infertility evaluation at our department. Individual samples were divided into two parts: one part of the semen was washed and the remainder was prepared using the PureSperm density gradient centrifugation. Sperm DNA fragmentation as evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay, was monitored in the initially washed sample and in the different layers of the density gradient centrifugation. No significant correlations were observed between sperm DNA fragmentation, age of patient, concentration and motility. However, a significant correlation existed with strict spermatic morphology. Following density gradient centrifugation, the proportion of spermatozoa with DNA fragmentation decreased significantly when compared with whole semen. In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of DNA damage when compared with those remaining in the 70% and 50% layers. These results demonstrate that semen processing by the PureSperm gradient is useful in selecting sperm with higher double-strand DNA integrity.     

精液体外处理对精子核DNA链完整性影响的研究

目的 研究3种不同精子优选技术对精子运动参数及DNA链完整性的影响。方法 通过计算机辅助精液分析及彗星试验从精子运动参数及DNA链完整性两个方面评价精液体外处理对精子的影响。结果 上游法与Percoll密度梯度离心法相比,除前者精子回收率(15.499.39)%明显低于后者(42.807.17)%外,P<0.05,其他差别无显著性,PureSperm密度梯度离心法与Percoll密度梯度离心法比较,各运动参数差别无显著性(P>0.05)。精液经Percoll法及PureSperm法密度梯度离心和上游法处理后,总彗星细胞率较处理前降低(P<0.05)。结论 上游法、密度梯度离心法和Puresperm 均可以不同程度优化精液质量;对精子DNA链的损伤程度不同。     

Effects of antioxidants on human sperm preparationtechniques

The effect of two different sperm preparation techniques, Percoll gradient centrifugation and swim-up from a washed pellet were tested on the functional competence of the selected spermatozoa. Percoll gradient centrifugation brought about an improvement in sperm motility parameters such as curvilinear velocity and straight-line velocity, an increase in the rates of hyperactivation and the acrosome reaction and an increase in the percentage of motile spermatozoa after 24 h of incubation compared to the centrifugation, swim-up procedure. The effects of antioxidants such as dithiothreitol (DTT) or reduced glutathione (GSH), and reactive oxygen species-scavenging enzymes such as catalase or superoxide dismutase (SOD) during the stage of centrifugation before the swim-up procedure were also studied. Though all of these agents prevented the fall in sperm motility after 24 h incubation, only DTT and SOD improved the rates of both hyperactivation and the acrosome reaction. GSH also improved the acrosome reaction, whereas catalase was without significant effect on the rates of hyperactivation or the acrosome reaction. These results indicate that Percoll gradient centrifugation selects spermatozoa with better functional competence than does centrifugation swim-up. The damage caused by the centrifugation can be prevented by the addition of antioxidants, suggesting that the differences noted with the Percoll gradient method was due to better protection against peroxidative damage due to the centrifugation of unselected spermatozoa. However, the use of DTT is limited by virtue of the fact that this sulphydryl reducing agent leads to destabilization of the sperm chromatin. In contrast, GSH and SOD could have therapeutic potential.     

Influence of semen processing technique on human sperm DNA integrity

Objectives. To compare the effects of density-gradient centrifugation and swim-up technique on sperm DNA integrity.Methods. Semen samples (n = 22) were obtained from consecutive nonazoospermic men presenting for infertility evaluation. Individual samples were divided into three aliquots (whole semen, density-gradient centrifugation, and swim-up) for subsequent analysis of sperm motility and DNA integrity. Sperm DNA integrity was evaluated by flow cytometry analysis of acridine orange-treated spermatozoa and expressed as the percentage of spermatozoa demonstrating denatured DNA.Results. Mean sperm motility (±SEM) improved significantly after processing with two-layer density-gradient and swim-up compared with whole semen (65.6% ± 4.0% and 73.0% ± 3.0% versus 52.0% ± 3.6%, respectively, P <0.005), with no significant difference in motility between Percoll-treated and swim-up-treated spermatozoa. In contrast, the percentage of spermatozoa with denatured DNA was reduced significantly in swim-up-treated but not in Percoll-treated spermatozoa compared with whole semen (4.8% ± 1.2% and 13.6% ± 3.6% versus 10.1% ± 2.3%, respectively, P <0.0001).Conclusions. Although density-gradient centrifugation is comparable to swim-up technique in recovering spermatozoa with enhanced motility, spermatozoa recovered after swim-up possess higher DNA integrity. These data urge us to reexamine our current sperm processing techniques in order to minimize sperm DNA damage.     

Effect of freeze–thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality after freeze-thawing.     

Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rates in vitro

To determine which sperm movement characteristics are related to in vitro fertilization rates, semen and swim-up preparations used for in vitro fertilization in 108 patients were assessed using the Hamilton-Thorn HTM-2030 Motility Analyzer (HTMA) and other sperm tests. There were highly significant correlations between manual and HTMA results for sperm concentration (Spearman r = 0.881; P less than 0.001) and the percentage of motile spermatozoa (Spearman r = 0.580; P less than 0.001). The percentage of motile spermatozoa with average path velocities greater than 10 microns/s and greater than 20 microns/s, straight line and curvilinear velocity, linearity (straight line velocity vs curvilinear velocity), amplitude of lateral head displacement, and beat-cross frequency were significantly higher in the insemination medium after selection of motile spermatozoa by the swim-up technique than in the semen. Linearity (P less than 0.01), the percentage of morphologically normal spermatozoa (P less than 0.05) and straight line velocity (P less than 0.05) in semen, and the percentage of motile spermatozoa with average path velocities greater than 10 microns/s in both semen (P less than 0.05) and insemination medium (P less than 0.05) were significantly correlated with in vitro fertilization rate when examined by a nonparametric (Spearman) test. With logistic regression analysis of all data, only the diagnoses of male infertility and tubal disease, linearity in semen, and the percentage of motile spermatozoa with average path velocities between 10 and 20 microns/s in insemination medium were significantly related to in vitro fertilization rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of nuclear maturity in human spermatozoa obtained by sperm-preparation methods

Summary. Change in the nuclear maturity has been considered as an index of sperm quality. This is done by using aniline blue staining that stains spermatozoa with disturbances in the chromatin condensation. Therefore, this technique was used to evaluate the quality of sperm obtained by sperm preparation methods. In 14 semen samples from healthy donors with normal semen analysis and normal forms, the swim-up (SU), Percoll gradient centrifugation (PG), glass wool column filtration (GW) and sedimentationmigration (SM) were performed. GW and SM methods presented the lowest number of spermatozoa with alteration in the nuclear maturity (ANM), 11, 14% and 12, 42% compared to native semen (19%) ( P < 0.005 and P < 0.05 respectively). SU and PG did presented no significant difference compared to native semen. In tests using the four methods, approximately 60% of the ANM occurred in normal spermatozoa. In the cells with pathologic abnormalities and ANM, 74.5% corresponded to macrocephalic forms, as this abnormality correlated mainly with ANM. It is concluded that in a semen sample with a higher percentage of normal forms, approximately 19% will have an ANM. GW and SM methods obtain the lowest percentage of ANM. ANM presents itself in 98% of the macrocephalic forms and they are effectively isolated with the PG method. The practice of this simple technique may orientate towards the sperm preparation methods to be used in assisted reproduction.     

Comparison of centrifugation- and noncentrifugation-based techniques for recovery of motile human sperm in assisted reproduction

To compare standard density gradient centrifugation sperm preparation with a novel non-centrifugation-based dual-chamber capillary dish in efficiency for motile human sperm separation, approximately 3 mL fresh ejaculate specimens was obtained from 21 men (median age = 32 years. range 26-42 years) undergoing infertility evaluation. For each specimen, half of the sample was processed with a standard 45%/90% density gradient preparation (PureSperm. Nidacon International, Gothenburg, Sweden) followed by semen analysis. The other half was incubated in the Zech glass capillary dish (Astromedtec, Salzburg, Austria) consisting of 2 concentric wells overlaid by a U-ring and coverglass. After approximately 3 h, a 1-mL sample was taken from the central chamber and analyzed. Percentage motile sperm recovery, absolute (motile) cell number, and path velocities were compared for spermatozoa obtained from both methods. Both techniques reduced overall sperm concentration while enriching specimens with more motile spermatozoa. A trend towards higher % recovery of motile spermatozoa (p = .264) was observed with the Zech device, but at a cost of fewer absolute numbers of higher velocity cells (p = .004). The Zech device, therefore, localized a very small population of motile sperm without exposure to centrifugation stress, which has been considered potentially harmful to spermatozoa. This technique could theoretically improve efficiency by reducing time required to identify motile cells in in vitro fertilization where intracytoplasmic sperm injection is planned. However, refinements in incubation interval and suspension volumes are needed before this technique can be considered comparable to the density gradient method in recovering sperm for use in intrauterine insemination.     

Sperm evaluation in cryopreserved bovine semen recovered by two selection methods

Previous experiments have established that various semen manipulation techniques are able to increase the qualitative features of the spermatozoa used in different techniques of assisted reproduction, but practically no comparative data on frozen-thawed bovine semen have been found. The aim of this study was to compare the efficacy of two sperm selection methods: centrifugation on Percoll gradient and filtration through a Sephadex ion-exchange column, to improve the recovery of motile and morphologically normal spermatozoa, without inducing sperm damage, from cryopreserved bovine semen samples. Semen samples were thawed and centrifuged on a discontinuous Percoll gradient, or were filtered through a Sephadex G-15-120 column with the addition of ion exchangers. Sperm concentration, percentages of motile spermatozoa, acrosome integrity, superoxide dismutase activity and lipid peroxidation were evaluated in recovered samples and controls. The motility of spermatozoa obtained by Sephadex ion-exchange filtration (88.87 +/- 6.37%) and by Percoll gradient centrifugation (83.00 +/- 6.21%) were significantly greater than that of control samples (60.14 +/- 8.44%). Other results disclosed that both sperm selection methods significantly increased the percentage of intact acrosome and superoxide dismutase activity. In both cases, the number of recovered spermatozoa diminished significantly versus untreated samples. Although the number of recovered spermatozoa was low, these methods were effective to select viable sperm from cryopreserved bovine semen.     

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.     

A new CentriSwim procedure to increase the recovery of motile spermatozoa

A prospectively controlled in vitro study was performed to compare sperm concentration, sperm motility and progressive sperm motility recovered following the standard swim-up procedure and a new CentriSwim procedure. The CentriSwim procedure involves creating a centrifugal force to counteract the force of gravity during sperm swim-up procedure. Two aliquots of semen from 12 normozoospermic ejaculates and 12 laboratory-induced oligoasthenozoospermic specimens were diluted, centrifuged, and 1.0 ml of media layered over the sperm pellet. One aliquant was processed by standard swim-up technique. The other aliquant was processed by CentriSwim procedure involving centrifugation at 200 rpm on a 2-cm radius upward-directing arm, at an angle of 60 degrees for 10 min, creating roughly 0.8 g centrifugal force at room temperature (22-24 degrees C) to counteract the force of gravity. The numbers of spermatozoa recovered from the upper 0.5 ml of the medium following CentriSwim from the normozoospermic ejaculates and laboratory-induced oligoasthenozoospermic specimens were significantly higher than following standard swim-up procedure. No statistical differences in the recovery of percentage sperm motility and progressive sperm motility between the two techniques were observed. In conclusion, the CentriSwim procedure yields higher numbers of motile spermatozoa than the standard swim-up technique.     

优选技术对人精子染色体及超微结构的影响

目的 :了解优选技术对人精子染色体及超微结构的影响。　方法 :分别应用上游法、Percoll密度梯度离心法及双曲管优选法处理精液 ,观察优选前后精子染色体畸变率、精子性染色体比例及超微结构相对正常的精子百分率。　结果 :精子染色体畸变率、精子性染色体比例及超微结构相对正常的精子百分率在 3种优选方法优选前后均无显著改变 (P >0 .0 5 )。　结论 :3种优选方法不会增加精子超微结构的损害 ,对精子染色体没有产生影响     

Computer-assisted assessment of sperm morphology: comparison with conventional techniques

The aim of the present study was to compare conventional and computer-assisted morphology assessment of spermatozoa. Sixty-two semen samples from patients undergoing in vitro fertilization (IVF) and 40 samples from patients undergoing an intracytoplasmic sperm injection (ICSI) were studied using both techniques. The percentage of normal spermatozoa found was closely correlated between the techniques (r=0.788, p < 0.0001). The intra-operator variation was low for both techniques but the inter-operator variation was much higher with the conventional than with the computer-assisted method (coefficient of variation = 0.43 vs. 0.08, respectively, for conventional and computer-assisted assessments). The percentage of spermatozoa with normal morphology, as well as sperm motility, was significantly enhanced after PureSperm preparation, whatever the method used for assessment. In the IVF study, fertilization rate was poorly correlated with sperm morphology using both methods. However, combined with motility, morphology assessed with the computer allowed discrimination of two groups of patients with significantly different fertilization rates (30.5 +/- 5.4% vs. 63.1 +/- 5.4%, p < 0.0001). In contrast, the fertilization rate in ICSI was influenced neither by sperm morphology nor by motility. In conclusion, computer-assisted assessment of sperm morphology has a slightly better predictive value for ART than conventional assessment, but above all is much more reproducible, allowing standardization.     

Comparison of two sperm preparation techniques using automated sperm motion analysis: migration sedimentation versus swim-up

Migration sedimentation and spermatozoa swim-up techniques were used for obtaining spermatozoa from the semen samples of 39 infertile men. Concentration, percentage of motile sperm, velocity, linearity, and motility index of the sperm preparations obtained by both methods were compared using the CellSoft automated sperm motion analyzer. The mean velocity of the spermatozoa obtained after the migration sedimentation technique was significantly higher than that with swim-up technique. Since it is not necessary to centrifuge spermatozoa with the migration sedimentation technique, this method may be more desirable than other techniques using centrifugation.     

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay （SCSA） parameters, that is, DNA fragmentation index （%DFI） and high DNA stainability （%HDS）, were assessed on the flesh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation （DGC） was followed by swimup treatment in comparison with DGC alone （P 〈 0.01）. Although the %DFI increased in a time-dependent manner with incubation both at room temperature （RT） and at 37℃ in air, the %DFI after 24 h at RT was significantly lower than that at 37℃ （P 〈 0.05）. Incubation with 5% CO2 was effective in maintaining sperm motility （P 〈 0.01）; however, it induced further elevation of %DFI （P 〈 0.001）. Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.