Swim-down separation of progressively motile sperm from poor quality human semen by the modified funnel procedure

The simultaneous swimming up and down from identical semen specimens suggested that the separation efficiency of progressively motile sperm by the swim-down method was superior to that of the swim-up method. The swimming down was performed in a small plastic funnel, the bottom of which was plugged with a 2.5 ml disposable syringe. In this funnel, 2.0 ml of 70% Percoll was poured and then the sperm resuspension was overlaid. After 1 h, progressively motile sperm penetrating into the lower part of Percoll layer (1.6 ml) was collected in the syringe. The technical strategies to yield higher density of progressively motile sperm were found to be centrifugal concentration of the sperm from whole ejaculate prior to the swimming down, and subsequent re-centrifugation of the separated sperm after the swimming down. Oligo-asthenozoospermic semen (22 +/- 4.4 x 10(6) ml1, 5.5 +/- 4.4% motility, n = 8) was processed by the present method, yielding sperm qualities of 32 +/- 19 x 10(6) ml1, 74 +/- 14% in the final preparations. Overall improvement in fertility index (sperm density ml1 x motility % x 10(8)) reached 27-folds on average.     

Washing and concentration of human semen by Percoll density gradients and its application to AIH

Human sperm for use in artificial insemination with husband's semen (AIH) were prepared by two methods: the monolayer Percoll method and the discontinuous Percoll density gradient of four steps. The former focused mainly on the oligozoospermic semen concentration, with the sperm concentration and motility being improved 4.29 +/- 3.7 times and 1.92 +/- 1.1 times, respectively (n = 41). The latter was used principally for the selective isolation of forward motile sperm, with the sperm motility being increased from 42.3 +/- 29.5% to 76.3 +/- 11.0% after centrifugation. The clinical application of these procedures resulted in 35 (the monolayer Percoll method) and 10 (the discontinuous Percoll density gradient) successful deliveries with greater enhancement of sperm qualities. The number of females was predominate over the males in the delivered cases: 31:34 (100:45.1) females to males.     

重组人睾丸精子结合蛋白对人精子运动参数的影响

目的:探讨重组人睾丸精子结合蛋白(TSBP)对体外培养人精子运动参数的影响。方法:将22例生育男性的精液经Percoll密度梯度离心后,分别与0.01mg/ml及0.1mg/ml的重组His6-TSBP在体外共同孵育1h或3h,同时设立对照组,Western印迹检测重组His6-TSBP与精子膜的结合情况,计算机辅助精液分析(CASA)系统测定重组His6-TSBP对精子运动参数的影响。将12例弱精子症患者的精液按同样方法处理,检测重组His6-TSBP对弱精子症患者精子运动参数的影响。结果:0.1mg/ml重组His6-TSBP与生育男性精子作用1h可以提高体外培养精子的前向运动百分率(a+b级精子百分率),培养3h后前向运动百分率和活率均有所提高,差异具有显著性(P<0.05);0.01mg/ml重组His6-TSBP对检测各指标均无显著性影响。0.1mg/ml重组His6-TSBP与弱精子症患者精子作用3h可以提高精子前向运动百分率(P<0.05),但对活率无显著影响。结论:0.1mg/ml的重组His6-TSBP在体外可以提高生育男性精子的前向运动百分率和活率及弱精子症患者精子的前向运动百分率。     

A simplified technique for freezing human sperm for AIH: cryosyringe/floating platform of liquid nitrogen vapor.

A cryosyringe and floating platform were developed to simplify the procedures for human sperm cryopreservation without sacrificing post-thaw sperm quality. The cryosyringe was developed as a vessel for cryopreservation and as a device for post-thaw insemination. A platform-like polystyrene foam board 3-cm in height was floated on the surface of liquid nitrogen in a polystyrene foam freezing container. The cryosyringe containing sperm/KS-II medium mixture was placed on it for 5 min, then it was dropped into the liquid nitrogen. Freezing velocity was -27 degrees C/min. Poor quality semen specimens (40 +/- 14 x 10(6)/ml, 18 +/- 2.6% motility, n = 9) were concentrated (87 +/- 46 x 10(6)/nl, 29 +/- 9.1%) previously and then cryopreserved in the cryosyringe or in a commercial tuberculin syringe under the conditions established in the present study. The post-thaw sperm motility of the specimens in the cryosyringe (18 +/- 7.4%) was significantly superior (p less than 0.05) to that in the commercial tuberculin syringe (13 +/- 5.3% motility).     

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.     

Separation of cryopreserved human semen using Sephadex columns, washing, or Percoll gradients.

The following methods were evaluated for their ability to separate motile cryopreserved sperm from semen after thawing: single washing, Percoll separation followed by a single washing, and Sephadex column separation. For Sephadex separation, washing, and Percoll separation, percent recovery of motile sperm was 65%, 76%, and 28%, and motility was 81%, 39%, and 60%, respectively. Percoll separation and washing were the best methods for removing seminal constituents, but sperm velocity and linearity were lower after Percoll separation and washing than after Sephadex separation. During 3 hours of incubation, there was an additional decrease in the motility, viability (exclusion of supravital dye), velocity, linearity, and intact acrosomes of Percoll-separated sperm, indicating that Percoll separation may not be suitable for cryopreserved sperm. Motile, washed sperm also had lower velocities and higher spontaneous acrosome reactions than Sephadex-separated sperm, but velocity and linearity were maintained during incubation. When semen was separated with Sephadex followed by washing, motility was well maintained (84%). The Sephadex method is a promising technique for selecting and concentrating motile cryopreserved sperm.     

Relationship between age and semen parameters in men with normal sperm concentration: analysis of 6022 semen samples

This study evaluates retrospectively the relationship between age and semen parameters among men with normal sperm concentration. It was based on computerized data and performed in an Academic Fertility and IVF Unit. Six thousand and twenty-two semen samples with sperm concentrations of >or=20 x 10(6) ml(-1) were examined according to WHO criteria and analysed in relation to patients' age. For each age group, mean values +/- SD of semen volume, sperm concentration, percentage of motile spermatozoa, normal morphology, acrosome index, total sperm count/ejaculate, total motile sperm count/ejaculate and sexual abstinence duration were examined. A peak semen volume of 3.51 +/- 1.76 ml(-1) was observed at age >or=30 to <35 years and a lowest volume of 2.21 +/- 1.23 ml(-1) was observed at age >or=55 years (P<0.05). Sperm motility was found to be inversely related to age with peak motility of 44.39 +/- 20.69% at age <25 years and lowest motility of 24.76 +/- 18.27% at age >or=55 years (P<0.05). A reduction of 54% was observed for total motile sperm, between values of 103.34 +/- 107 x 10(6) at age >or=30 to <35 years and 46.68 +/- 53.73 x 10(6) (P<0.05) at age >55 years. A statistically significant and inverse relationship was observed between semen volume, sperm quality and patient age, in spite of prolonged sexual abstinence duration. Top sperm parameters were observed at age >or=30 to <35 years, while the most significant reduction in sperm parameters occurred after the age of 55 years.     

Bias to routine semen analysis by uncontrolled changes in laboratory environment—detection by long-term sampling of monthly means for quality control

To detect systematic bias in the results of routine semen analysis over time, monthly means of semen parameters determined by the recommended WHO methods were computed. The analysis was based on a total sample size of 1784 ejaculates and included 18 months of observation. In addition to slight changes of morphology estimates caused by a change of laboratory staff, a major bias in the measurement of sperm motility could be detected. This observation triggered a search for changes in protocols not previously given the required attention. It revealed that the newly introduced use of polypropylene syringes with a mounted needle for accurate measurement of seminal volume impaired sperm motility. More detailed investigation by computerized sperm motion analysis in 10 semen samples treated simultaneously in different ways revealed that predominantly it was the needle which caused the drop in proportion of motile sperm (glass cylinder: 50.3 +/- 4.1% vs. syringe + needle: 26.6 +/- 5.3%; mean +/- SEM) and not the contact with the plastic material alone (syringe alone: 43.4 +/- 4.8%). Other motion parameters such as curvilinear velocity (36.0 +/- 1.6 microns/sec), linearity (78.5 +/- 8.4%) and lateral head displacement (3.8 +/- 0.9) were not influenced by the different methods of handling. The results indicate that long-term sampling of monthly means may serve as part of a quality control scheme in semen analysis.     

Quantitative assessment of the serum-induced stimulation of human sperm motility

In previous studies, sperm populations with high motility and fertilizing potential have been isolated using Percoll gradients and/or addition of serum or caffeine, but no objective measurements of the motility parameters affected have been presented. In addition, the types of sperm populations and the incidence of positive or negative responses to these treatments have never been documented. In this paper, the proportion of samples responding positively or negatively to these treatments and the type of motility stimulation observed was measured using the CellSoft computer-assisted digital image analysis system. Semen, obtained from the Infertility Clinic, was washed on discontinuous Percoll gradients. Sperm motility parameters were measured in the original semen and after washing through a Percoll gradient in the presence or absence of serum or caffeine. Percoll washing permitted a good recovery of essentially pure sperm. The Percoll treatment had little effect on the percentage of motile sperm but caused a decrease in the linearity-related parameters. A high proportion of the washed sperm samples supplemented with serum showed an increase in at least one of the motility parameters when compared to sperm populations in the original semen (68% of cases) or after Percoll washing (96% of cases). These effects were maintained for several hours. The stimulation of motility was concentration-dependent, with an optimum around 25-30% serum. Caffeine (4 mM) increased linear velocity but lowered linearity. In contrast to caffeine, which stimulated sperm motility for less than 1 h, the effects of human serum lasted for more than 16 h.     

Techniques for selection of normal-chromatin sperm preparations.

Various sperm preparation techniques, swim-up and Percoll gradient, and the newly developed Wang's tube system, were evaluated for their ability to recover normal-chromatin sperm. Twenty human semen samples, collected by masturbation, were studied simultaneously with the three methods. Analysis by Acridine orange fluorescence test was performed on all samples. Pretreated semen contains 58 +/- 22% green sperm (fertile/normal). Treatment with Wang's tube system resulted in 99 +/- 1.0% green sperm; Percoll gradient, 78 +/- 11%; and swim-up technique, 72 +/- 15%. It would appear that Wang's tube system yields a high-quality sperm preparation with enough concentration, very active forward progression, and greatly improved sperm morphology, while containing normal-chromatin, double-stranded DNA.     

Mecobalamin Promotes Mouse Sperm Maturation/Mecobalamin verbessert die Reifung von Mäusespermatozoen

The effect of Mecobalamin (alpha-(5,6-dimethyl benzimidazolyl)-Co-methyl-cobamide: Me-B 12) on sperm production in the oligozoospermic mice experimentally induced by the treatment with adriamycin (0.3 mg/kg, three times a week for 5 weeks) was evaluated quantitatively by means of equilibrium sedimentation in Percoll. After centrifugation, the distribution profile of the sperm showed two peaks, i.e. the first peak near the bottom consisting of mature sperm with good motility and the second peak containing immature and/or immotile sperm. By oral administration of Me B 12 (1.0 mg/kg/day) to the oligozoospermic mice for 10 weeks, the sperm count, sperm motility, motile sperm count, diameter of seminiferous tubules and the percentage of good motile sperm with higher apparent density were increased as compared with those of the control. These results suggest that Me-B 12 enhanced the testicular function, resulting in an increased output of mature sperm.     

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.     

Are conventional sperm morphology and motility assessments of predictive value in subfertile men?

Sperm morphology and motility are believed to be important prognostic factors for fertility. Results of a group of 67 men investigated for primary infertility who had mean sperm concentrations greater than 5 million per ml and who later produced pregnancies, were compared with those of 67 matched controls who remained infertile. All female partners were potentially fertile. The groups were matched for other known prognostic factors for fertility, namely, wife's age, the duration of infertility, sperm concentration and varicocele size. There were no significant differences between the two groups overall in the (mean +/- SEM)% of sperm with normal morphology (58.3 +/- 2.1; 58.5 +/- 2.2), or motility (40.6 +/- 1.8; 37.0 +/- 2.0). However, among oligospermic men from the two groups, sperm motility was significantly higher (P less than 0.05) in the subsequently fertile group (43.1 +/- 2.6%) than in the persistently infertile group (33.3 +/- 3.7). These results indicate that sperm morphology as currently assessed may not be important in predicting fertility in subfertile men with a mean sperm concentration over 5 million/ml and the % sperm motility may only be a relevant predictor in oligospermic men.     

Effects of semen characteristics on IUI combined with mild ovarian stimulation

To determine the influence of sperm parameters inseminated on the outcome of intrauterine insemination (IUI) in patients undergoing ovarian stimulation with clomiphen citrat (CC) or human menopausal gonadotropin (HMG) therapy, a retrospective review was performed for 2 years on data from the IUI program. 190 couples underwent a total of 268 IUI cycles in which CC or HMG was used for ovulation induction. The initial sperm concentration (mil/ml), motility (percent), preprocessing total motile sperm (TMS) count (million), fast motile sperm (percent) and postprocessing sperm concentration (mil/ml), motility (percent), TMS count, fast motile sperm (percent), sperm morphology, hypoosmotic swelling (HOS) scores, semen leuocytes, and bacteria were analyzed. 268 inseminations were followed by a pregnancy rate of 12% and couple pregnancy rate of 17%. On multivariable logistic regression analysis, total motile sperm (TMS) count, percent motility, and percent of fast motile sperm were independent prognostic factors of fertility. The impact of the preprocessing and postprocessing sperm parameters on pregnancy outcome after IUI was evaluated. There was a trend toward an increasing percent of conception with increasing TMS count, motility, and percent of fast motile sperm. The TMS count, motility and percent of fast motile sperm independently predict success with IUI. Patients with original sperm motility > or = 30% had a higher cumulative pregnancy rate (74%) than patient with motility < 30% (p < 0.005). Pregnancy rate increased 4 times with motility of > or = 30%.     

Increase in progressive motility and improved morphology of human spermatozoa following their migration through Percoll gradients

Human spermatozoa were separated on the basis of their motility in a discontinuous Percoll-gradient made up in tissue culture medium containing 10% (v/v) human serum (TCMS). Portions of ejaculates were placed on top of the gradients. After 3 h at 37 degrees C the bottom 1.5 ml was collected and the sperm washed free of the Percoll solution by centrifugation at 240 X g after dilution in TCMS. In this way the spermatozoa were separated from seminal fluid by means of the swimming rate of the sperm. When semen samples from normal men were used, total recovery of sperm after separated on a Percoll gradient was 21 +/- 2.3%. The progressive motility index increased by a factor of 15 +/- 1 when comparing separated samples with the same unseparated ejaculate, and the frequency of sperm with normal morphology increased from 60 to 85%. The improvements in these semen samples was attributable to the Percoll separation as the washing procedure itself was without effect. Using this method sperm of relatively unifirm motility and morphology can be collected. These may then be used for further biochemical and physiological studies. Improved sperm quality was also obtained when samples from patients with abnormal semen profiles were separated in this way, although the degree of improvement was much more variable than that obtained with semen from normal fertile men. This indicates that this method can be used in clinical practice in selected cases for the preparation of sperm for insemination or for in-vitro fertilization.     

Tadalafil as an in vitro sperm motility stimulant

Tadalafil (Cialis) is a known oral selective phosphodiesterase-5 inhibitor used widely in the management of erectile dysfunction. To assess its ability on human sperm motility in vitro, 70 asthenozoospermic semen specimens delivered by masturbation were investigated. Semen samples were divided equally into four tubes, one as a control and to the others tadalafil dissolved solution was added in vitro in three different concentrations (4.0, 1.0, 0.5 mg ml(-1) respectively). The tubes were incubated and were followed up for sperm motility per cent changes for 0.5, 1, 2, 3 h. It was found that the concentration used played an important role in the degree of sperm enhancement. Specimens treated with 4 mg ml(-1) tadalafil solution demonstrated a significant decrease in sperm motility compared with the controls. Specimens treated with 1.0 mg ml(-1) solution demonstrated significant increase in sperm progressive forward motility. Specimens treated with 0.5 mg ml(-1) solution demonstrated significant increases in sperm motility but lower than that of 1 mg ml(-1) concentration. It is concluded that in vitro use of tadalafil solution in special concentration has a significant stimulatory effect on asthenozoospermic sperm motility.     

Effect of sperm preparation techniques by density gradient on intra-individual variation of sperm motility

The purpose of this study was to examine the effect of sperm preparation by density gradient on the intra-individual variation in sperm motility. Patients presenting for density gradient (DG) sperm preparation were analyzed retrospectively. Patients who had more than one preparation were included. The variation within each patient was studied using the coefficient of variation (CV = standard deviation/mean x 100). Density gradient preparation resulted in a reduction in the CV of sperm motility (CV motility before DG: 19.8 +/- 15.82% vs. CV motility after DG: 15.9 +/- 17.97%, p < 0.001). However, CV of sperm concentration (44.2 +/- 26.51%) and CV progressively motile sperm (49.2 +/- 28.48%) remained very high after DG. This variability should be reflected in counseling patients undergoing intrauterine insemination.     

The motility of epididymal or testicular spermatozoa does not directly affect IVF/ICSI pregnancy outcomes

Our objective was to determine whether the presence of motility in surgically obtained sperm samples improves fertilization and pregnancy rates for patients undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). This was a retrospective study in a hospital-based infertility center. Sixty-seven couples with a diagnosis of azoospermia or severe oligozoospermia who had undergone a sperm retrieval procedure in conjunction with 100 IVF/ICSI cycles from 1995 to 2004 were evaluated. The impact of sperm motility on fertilization and clinical pregnancy rates was determined. The motile and nonmotile sperm groups differed in the number of mature oocytes retrieved (10.7 +/- 5.8 vs 13.4 +/- 6.0), but fertilization (56.7% vs 59.1%) and embryo cryopreservation rates (35.9% vs 39.3%) were statistically similar. Clinical pregnancy rates did not differ between the motile (38.5%) and nonmotile (31.2%) groups, nor did they differ between obstructive and nonobstructive patients (35.3% vs 26.7%). There was also no statistical difference in pregnancy rates between testicular and epididymal aspiration (35.3% vs 26.7%), although epididymal sperm were significantly more likely to be motile than testicular sperm (100% vs 39.3%, P < .0001). Epididymal aspiration is more likely to produce motile sperm than testicular sperm retrieval. The use of motile sperm from epididymal or testicular samples, however, does not appear to enhance fertilization or clinical pregnancy rates.     

Concentrations of calmodulin in sperm in relation to their motility in fertile euspermic and infertile asthenozoospermic men

The relationship between calmodulin and sperm motility was assessed in euspermic and asthenozoospermic men using radioimmunoassay and time-lapse photography, respectively. There was a significant decrease in the % sperm motility, mean sperm velocity, motility index and % of progressively motile sperm in the asthenozoospermic group when compared with euspermic men. The former also exhibited a higher % of sperm with erratic or circular motility. Calmodulin concentration in sperm from the asthenozoospermic men was 4.8 +/- 1.4 micrograms/mg protein compared with 12.6 +/- 2.3 in euspermic men (P less than 0.0005). The differences observed in sperm motility characteristics between the two groups may, thus, be due to the observed differences in the concentration of calmodulin.     

Value of the hamster oocyte test and computerised measurements of sperm motility in predicting if four or more viable embryos will be obtained in an IVF cycle

The experimental group consisted of men from 81 couples waiting for in vitro fertilization (IVF), about half of whom had sperm dysfunction defined by a negative post-coital test. A diagnostic semen sample was subjected to a hamster oocyte penetration test (HOPT) after stimulation of the acrosome reaction with A23187 +/- pentoxifylline and to computerized sperm motility measurements (CASA) as well as conventional semen analysis according to the WHO protocol. Logistic regression was used to identify parameters that predicted the probability of achieving four or more viable embryos at IVF among the 65 couples from whom four or more oocytes were collected. The number of oocytes available and whether the woman had previously been pregnant (ever pregnant) were important factors but once these had been taken into account a number of sperm parameters had additional predictive power. The most useful of these were the percentage sperm static (CASA) or the percent sperm progressively motile (conventional semen analysis) in the Percoll preparation. A model incorporating the number of oocytes collected, ever pregnant and percentage sperm static achieved 85% correct prediction of outcome in the experimental dataset but only 62% correct prediction in an independent set of 280 IVF cycles. The percentage of hamster oocytes penetrated was a significant predictor but had no advantage over simple motility measurements. The results illustrate the difficulty of basing a prognosis for achieving satisfactory fertilization in IVF on the properties of spermatozoa.