Monoclonal antibody against human sperm acrosome inhibits sperm penetration of zona-free hamster eggs

Human spermatozoa were exposed to a monoclonal antibody (C11H), which recognizes sperm acrosin. The antibody was presented to the sperm during capacitation and/or insemination, and its effect on penetration was tested using zona-free hamster eggs. An inhibitory effect on penetration was observed when the antibody was present during insemination but not when it was included only in the capacitation medium. As judged by immunofluorescence microscopy, most of the sperm bound to the egg surface were devoid of acrosomal staining. Some of the bound sperm were stained at their equatorial segments. Sperm that had penetrated the ooplasm did not exhibit immunofluorescence.     

Association of human sperm nuclear decondensation and in vitro penetration ability

Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in vitro spontaneous nuclear decondensation of human sperm and its fertilizing ability was studied in infertile male patients. The ability of sperm to fertilize an egg using the discontinuous two-layer Percoll method was significantly correlated to the percentage of decondensed swollen head (r = 0.43; P less than 0.005). The fertilizing ability of sperm processed with Test-Yolk buffer was correlated with the percentage of sperm at the fully decondensed stalk stage (r = 0.51; P less than 0.05). There were insignificant correlations for the whole-wash centrifugation, cryopreserved-thawed and swim-up methods. Samples of sperm that were positive (greater than 0% fertilization) in the sperm penetration assay had a higher percentage of decondensed sperm heads (66.7% vs. 20.6%) after Percoll wash or whole-wash centrifugation (60.5% vs. 44.3%) treatments compared with samples with no fertilization. Treatments that included Test-Yolk resulted in high percentages of decondensed swollen heads. The results suggest a positive association between sperm nuclear decondensation and the fertilizing ability of sperm, and affirm the importance of nuclear decondensation to the study of fertilization events.     

Morphological factors influencing the penetration of human sperm into cervical mucus in vitro

The efficiency of cervical mucus in filtering out single, multiple and associated abnormalities of human spermatozoa was determined. Twenty semen samples which gave a normal in vitro cervical mucus penetration test (CMPT) were analysed before and after migration using a detailed classification system (13 categories). The % of normal forms was significantly increased in cervical mucus (59.5 vs 33.2%), whereas the % of sperm with single, multiple or associated abnormalities of the midpiece or of the flagellum were found to decrease significantly in cervical mucus. Sperm with single or multiple abnormalities confined to the head migrated similarly to normal forms. The decrease in amorphous and elongated tapering sperm was explained by their more frequent association with other defects of the midpiece and/or of the flagellum.     

Correlation between sperm penetration into the human zona pellucida and in vitro fertilization rates

Summary.  Sperm penetration into the zona pellucida of unfertilized oocytes, and its correlation with in vitro fertilization rates of the sibling oocytes, were assessed. This was performed in order to evaluate the prediction rate of the sperm penetration test into the zona pellucida. Unfertilized oocytes ( n =1872) from 371 cycles were pipetted through a microcapillary, and the remaining sperm cells penetrating the zona pellucida were counted. The mean (±SD) number of spermatozoa that penetrated the zona pellucida of unfertilized oocytes was 12.9±16.37. A significant correlation was found between the fertilization rate and the mean number of spermatozoa that penetrated into the zona pellucida of the unfertilized sibling oocytes (r = 0.48; P < 0.001), or the percent of unpenetrated zonae pellucidae in a cohort (r= —0.43; P < 0.001). However, a distinct variation in the number of spermatozoa that penetrated into the zona pellucida was detected. A step-wise regression analysis proved the number of spermatozoa penetrating the zona pellucida to be more predictive for fertilization rates than the variable of percent of unpenetrated zonae pellucidae. The results imply that although there is interdependence between penetration into the zona pellucida and fertilization rate, the predictive value of sperm penetration test for prognosis and future management after the first in vitro fertilization attempt, is limited.     

TEST-egg yolk buffer storage increases the capacity of human sperm to penetrate hamster eggs in vitro

A total of 85 semen samples from infertility clinic patients were examined to study the effect of storage at 4 degrees C in TES-Tris (TEST)-egg yolk buffer for 24 h on the penetrating capacity of sperm in the zona-free hamster egg penetration assay (HEPA). The mean sperm penetration rate and the fertilization index increased significantly after storage in TEST-egg yolk buffer. Only five out of the 85 samples (5.9%) failed to show any improvement in sperm penetration rate after cold storage. The sperm penetration rate before cold storage showed no significant correlations with routine semen characteristics, semen ATP concentration or the functional integrity of sperm membranes as measured by the hypo-osmotic swelling technique. Significant but low correlations were observed between sperm penetration rate after cold storage and the following semen parameters: sperm count, % motility, total number of motile sperm, % normal sperm morphology, total number of normal sperm, semen ATP concentration and sperm penetration rate before cold storage. Partial correlation analysis revealed that the positive correlation between semen ATP concentration and sperm penetration rate after cold storage was not a direct relationship but was due to the correlation with sperm count. The combination of sperm penetration rate before cold storage, sperm count and % normal sperm morphology accounted for 26.2% of the variation in sperm penetration rate after cold storage by stepwise multiple regression analysis, while sperm penetration rate before cold storage alone explained 13.5% of the variation. The results indicate that TEST-egg yolk buffer treatment can enhance sperm penetration rate in vitro and may be useful in the treatment of impaired sperm fertility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of white blood cells on the in vitro penetration of zona-free hamster eggs by human spermatozoa

The presence of white blood cells in semen has been associated with male infertility. Previous studies indicate that pyospermia occurs in conjunction with decreases in sperm motility, number of normal sperm forms, and penetration rates in the zona-free hamster egg sperm penetration assay. We have evaluated the relationship of seminal white blood cells and sperm function, as reflected in the zona-free hamster egg penetration assay, and have investigated the possible mode of action of the white cells. Egg penetration rates decreased when white blood cells from fertile or potentially fertile donors were added to their sperm suspensions prior to preincubation and at insemination in the in vitro assay. Zona-free hamster egg penetration assay results were also inhibited when the supernatant from white blood cells incubated in Biggers, Whitten, and Whittingham (BWW) medium overnight were introduced to sperm-oocyte suspensions at insemination. Conversely, egg penetration rates were enhanced in samples from hypofertile individuals when white blood cell concentrations in the semen or WBC/sperm ratios were reduced, either by physical removal or as a result of antibiotic therapy. The physical presence of leukocytes, and possibly, the extracellular release of lysosomal enzymes may be responsible for the inhibitory effects in vitro. Although the mechanism(s) by which white blood cells interfere with the fertilizing capacity of spermatozoa are not clear, it is quite obvious that their presence in the in vitro environment is undesirable and can mask an individual's actual fertilizing potential.     

Sperm penetration through the human zona pellucida as a predictor of in vitro fertilization

The aim of this study was to determine the predictive value of sperm penetration into the perivitelline space of human cadaveric oocytes on in vitro fertilization outcome. Forty-two patients with tubal infertility undergoing ovarian stimulation with gonadotropin for in vitro fertilization and embryo transfer participated in the study. The number of spermatozoa bound to the human zona pellucida, the percentage of cadaveric oocytes with one or more spermatozoa in the perivitelline space, and the in vitro fertilization outcome were evaluated. Spermatozoa from 37 of 42 patients were able to penetrate the perivitelline space of cadaveric oocytes as well as to fertilize human oocytes in vitro. In three individuals, no penetration of the perivitelline space of cadaveric oocytes was observed and no in vitro fertilization was detected. Only two patients were able to fertilize the couple's oocytes without penetration of the cadaveric oocytes. Based on these results the specificity and the sensitivity of the assay to predict in vitro fertilization was 100% and 94.1%, respectively. Accordingly, these results suggest that sperm-zona penetration is a useful bioassay to predict male fertility potential in IVF outcome.     

Relationship between the human sperm hypo-osmotic swelling test and sperm penetration assay

The hypo-osmotic swelling (HOS) test has been proposed as a useful assay in the diagnosis of the infertile male. A good correlation between the HOS test and the sperm penetration assay (SPA) in fertile and normal semen samples was initially found, but subsequently, no significant correlation was demonstrated with fertile and infertile patients. To validate the potential clinical usefulness of the HOS test, we evaluated 92 ejaculates using the HOS test, SPA, and traditional semen parameters. The methodology originally described by Jeyendran et al (1984) was used for the HOS test. The SPA was performed by the original procedure using an 18-hour preincubation period, and for 28 ejaculates, a modified procedure using TEST-yolk buffer was performed. Values of 60% or more for the HOS and 1% or more for the SPA were considered positive, and less than 60% for HOS and 0% for SPA were considered negative when the standard SPA was performed. For the TEST-yolk buffered SPA, values of 20% or more were considered positive. The sensitivity of the HOS test was 87%, but the specificity was 36%. The association of the two tests over and above that expected by chance (Kappa) was only 0.23. Using logistic regression, both sperm count (P less than 0.001) and morphology (P less than 0.025) were significant predictors of the SPA classification, but the HOS test did not improve the predictive results (P greater than 0.50).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of human sperm hyperactivated motility and its relationship with the zona-free hamster oocyte sperm penetration assay

The authors studied hyperactivated motility of human spermatozoa as a method of evaluating capacitation by examining its relationship to results of zona-free hamster oocyte sperm penetration assays (SPA) of semen samples from 50 men attending the infertility clinic. Hyperactivated motility was assessed in the seminal plasma and after swim-up preparation of spermatozoa at 1, 3, and 24 hours of incubation in capacitation media using a computer-assisted semen analysis system equipped with a hyperactivation module. Hyperactivated motility reached a peak at 1 hour and plateaued at 3 hours. The percentage of spermatozoa in seminal plasma with star-spin hyperactivated motility was significantly lower in the group showing no penetration in the SPA. The hyperactivated motility characteristics did not differ in the groups with positive or negative penetration. Correlation analysis failed to show any significant relationship between the hyperactivated motility parameters and SPA score. When the hyperactivated motility characteristics were compared in samples with normal and abnormal semen analyses, the total percentage of spermatozoa with hyperactivated motility and the percentage with star-spin at 3 hours were significantly lower in the group with abnormal semen analysis. The data indicate that lower hyperactivated motility of spermatozoa was found in patients with a score of zero for SPA and in patients with abnormal semen analysis. It was concluded that although no direct correlations were found between the results of SPA and hyperactivated motility, evaluating hyperactivated motility may still be useful as an early indicator of capacitation abnormalities of human spermatozoa not measured by SPA.     

Technique to examine human sperm chromosomes using zona-free hamster eggs

In experiments with repeated semen samples from two donors, zona-free hamster eggs were used for activation of human spermatozoa to metaphase. When mean number of spermatozoa per penetrated egg was low (1.0-3.3) after insemination in vitro, further incubation resulted in activation of spermatozoa to metaphase. High sperm/egg ratios, 4.2 and 7.5, yielded no sperm chromosomes. The preincubation and insemination intervals necessary for low sperm/egg ratios were individual.     

Effect of caffeine on human sperm penetration into zona-free hamster ova

The effect of caffeine on spermatozoal ability to penetrate zona-free hamster ova was examined on fresh and frozen-thawed semen samples. The mean motility of 10 fresh semen samples incubated with caffeine significantly increased from 29% to 35%. Sperm penetration into zona-free hamster ova did not differ between the control group and the specimens to which caffeine was added. The same effect of caffeine on sperm motility and hamster ova penetration was noted in the frozen-thawed sperm samples. Motility was enhanced by 21%, but hamster ova penetration did not significantly change. The increase in sperm motility caused by caffeine does not change the fertilizing ability of fresh and frozen-thawed human sperm.     

Human sperm penetration in different media

Sperm penetration into bovine cervical mucus and hen egg white using capillary tube penetration was investigated to verify the suitability of the capillary tube penetration test with hen egg white as a test of human sperm function. Semen samples from 50 consecutive patients were used for penetration tests and spermatozoa of a further 10 semen samples were penetrated into bovine cervical mucus and hen egg white for special motility assessment by computer-assisted motility analysis. Penetration tests revealed the well-known different ability of spermatozoa to penetrate into cervical mucus and a different penetration of spermatozoa into egg white for two nearly equal groups (n = 24 and n = 26, respectively). One group showed penetration comparable with cervical mucus and one group a very fast penetration up to the limit of the scale of measurement. Motility assessment of spermatozoa that penetrated into cervical mucus and egg white revealed significant differences in straight-line velocity, linearity and lateral head displacement. The number of spermatozoa selected actively during the penetration procedure was significantly higher in cervical mucus than in hen egg white. Spermatozoa selected by bovine cervical mucus and hen egg white exhibited a different motility pattern. There was significantly better linearity and less lateral head displacement in egg white than in cervical mucus. Sperm penetration into hen egg white appeared to be influenced by different sources of egg white.     

Abnormal sperm morphology and other semen parameters related to the outcome of the hamster oocyte human sperm penetration assay

A new method for evaluation of sperm morphology using strict criteria is currently used in the andrology laboratory at the Eastern Virginia Medical School. A prospective study was designed to evaluate the following semen parameters in samples of all patients over a set period of time: sperm concentration and motility, and normal sperm morphology. These factors were correlated with results of the hamster zona-free oocyte/human sperm penetration assay (SPA). One hundred patients with a sperm concentration ranging from 2 to 219 X 10(6)/ml, a motile sperm fraction ranging from 6.9 to 87%, and normal sperm morphology ranging from 1 to 39%, were evaluated. The statistical analysis system general linear model was used to judge the influence of the different variables. There was a statistically significant relationship between the per cent of sperm with normal morphology and penetration rate in the SPA (P = 0.001). Outcome of the SPA was also correlated with in vitro fertilization, retrospectively, in 84 patients. Thirty-eight patients had an SPA less than 10%, with no fertilization in vitro in 13 patients (33.3%) and fertilization in 25 (66.7%). Forty-five had an SPA greater than 10% with fertilization in 37 (82.2%) and no fertilization in eight (17.8%) patients.     

Correlation of the bovine cervical mucus penetration test with human sperm characterisitics in 1,406 ejaculates

The bovine cervical mucus penetration test (BCMPT) was performed to determine its usefulness in screening the ability of sperm to successfully penetrate mucus in vitro. Ejaculates were obtained by masturbation from patients attending an infertility clinic. Routine semen analysis was performed using a microcomputerized multiple-exposure photography system. The BCMPT was performed. Overall, the average penetration of the mucus was 38 +/- 0.46 mm. Of the 1,406 ejaculates analyzed, 244 (17%) displayed a negative result (0-20 mm), 291 (21%) a questionable result (21-30 mm), and 871 (62%) a positive result (>30 mm). A highly significant (p < .001) correlation between mucus penetration distance and sperm MD (r = 0.541), MI (r = 0.484), count (r = 0.475), motility (r = 0.448), velocity (r = 0.400) and morphology (r = 0.369) was observed. Overall, the finding of an abnormal semen parameter resulted in a 34 +/- 5% accurate prediction of a negative or questionable BCMPT (<30 mm), while a normal semen parameter resulted in a 90 +/- 4% accurate prediction of a positive BCMPT (>30 mm). Sperm MD showed the strongest positive predictive value (98%), while morphology showed the greatest negative predictive value (50%). Of the 1,406 samples, 25 +/- 2% of the samples with normal semen parameters displayed a negative BCMPT. Conversely, 6 +/- 2% of samples with abnormal parameters showed a positive BCMPT. The BCMPT successfully identifies a significant subpopulation of patients as having an inadequate penetration of mucus with otherwise normal semen characteristics.     

己酮可可碱对精子活力的体外改善作用

目的体外分析己酮可可碱(Pentoxifylline，PF)对精子活力的改善作用，探讨PF最佳作用浓度和时间，为实施人工授精奠定基础。方法本实验就PF的不同浓度(0．6mmol／L、3．0mmol／L)和不同时间(30min，60min)，恒温下与70例弱精子的体外孵育，并与对照比较，观察精子活力特性的改善情况。结果PF的不同浓度及不同时间对精子活力均有改善作用，而用PF的0．6mmol／L浓度、孵育30min，对体外弱精子的活力有显著增强作用。结论PF对体外弱精子的活力有改善作用，药物浓度和作用时间直接影响孵育效果。     

Effect of cyclosporine on human sperm motility in vitro

Cyclosporine affects motility and viability of human sperm when incubated together in vitro. Sperm motility was almost reduced to nil following 10 min of incubation with cyclosporine at a concentration of 1 mg/mL. However, 200 micrograms/mL of the drug has no effect on motility and viability when tested for up to 60 min under standard laboratory conditions. Cyclosporine effect on sperm was both dose and time dependent. Sperm sensitivity and susceptibility to cyclosporine even to lower doses increased significantly following withdrawal of bovine serum albumin from the incubating medium. Compared to untreated controls, lactate dehydrogenase was estimated higher by more than 2 to 4 times in the sperm-free incubating media, suggesting an altered membrane porosity in the affected spermatozoa.     

rhGM-CSF体外对人精子运动参数的影响

目的观察体外添加不同浓度的重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)对人精子运动参数的影响,探讨其在精子运动中的作用机制。方法健康生育男性和弱精子症患者各10例手淫取精,经简易上游优化处理后的精子与不同浓度rhGM-CSF溶液孵育10min、30min、60min后,采用计算机辅助的精液分析系统检测精子各项运动参数的变化。结果对精液运动参数正常的标本,1ng/ml和10ng/ml的rhGM-CSF溶液能显著提高精子的活率、前向性运动百分率,在10ng/ml时平均直线运动速度也有显著提高。对弱精子症患者精液标本,1ng/ml和10ng/ml的rhGM-CSF溶液能显著提高精子的前向运动百分率和直线运动速度,10ng/ml的rhGM-CSF对精子活率和平均路径速度也有显著性提高。两类精子的曲线运动速度均无显著性变化。结论1ng/ml～10ng/ml浓度范围的rhGM-CSF体外能显著改善精子的运动功能。