Comparison of the effects on penetration capacity of human spermatozoa between using Ham's F-10 medium and a medium based on the composition of human tubal fluid

The penetration ability of human spermatozoa on zona-free hamster ovum when using Ham's F-10 (HF-10) medium was compared with that when using a medium based on the composition of human tubal fluid. Forty semen specimens were each divided into two aliquots; one aliquot was washed with and incubated in HF-10 medium and the other in HTF medium. The penetration rate of sperm with motility between 30 to 60 percent was significantly increased when HTF medium was used (67.9 +/- 6.9%) compared with the rate when HF-10 medium was used (49.2 +/- 7.3%) (P less than 0.05). This study shows that the ZFHO penetration ability of human spermatozoa with poor motility can be improved by using HTF medium as compared with the use of HF-10 medium.     

Adenosine 5''-Triphosphate (ATP) Concentration and Acrosin Activity in Human Spermatozoa. Their Relation with the Sperm Penetration Assay (SPA)

Fertility of men depends on the quality of semen. The aim of the present paper is to determine both the acrosin activity by radioimmunoassay and ATP concentration by bioluminescence in human spermatozoa, and evaluate these results in those samples with normal or low sperm penetration according to SPA test. Ejaculates obtained from 42 untreated men, were studied one hour after the obtention. These materials were divided into two groups:20 human semen samples with "in vitro" potentiality to penetrate zone-free hamster ova, between 15% to 98% and 22 human semen with SPA test between 0% to 14%. When we compare the group with normal penetration response vs that group with low or absent penetration ones, a significant decrease of ATP and acrosin concentrations was observed (P less than 0.001). Nevertheless no significant difference was observed in relation with percentage of motility, volume (ml), sperm concentration (10(6)/ml), percent of quick progressive spermatozoa and number of gametes capable of migrating into the medium layer (10(6)), between the group with low or absent penetration test against that one with normal zona-free hamster egg test.     

Effect of Urea and Detergents on the Ability of Human Spermatozoa to Penetrate Zona-Free Hamster Oocytes

Washing of human spermatozoa with either BWW alone or with the same buffer containing 0.1 M urea, 0.005% SDS or 0.001% NP40 affected the penetration ability of the gametes into zona-free hamster oocytes to various degrees. In contrast, human spermatozoa washed with BWW buffer containing 0.3 M urea had an increased ability to fuse with the heterologous oocytes when compared to controls capacitated with BWW/BSA. Moreover, the presence or absence of BSA in the insemination medium did not further modify this enhanced penetration pattern. The BWW, BWW/0.1 M urea and BWW/SDS treatments apparently mimicked some of the in vitro capacitation properties of albumin-containing media; the BWW/0.3 M urea treatment overpowered the capacitation and acrosomal reaction extent obtained with BWW+BSA. In all samples the motility of the spermatozoa washed with BWW buffer alone or containing various additives (but no albumin) was significantly decreased if compared to the motility of semen samples washed with albumin containing media. However, each sperm sample behaved differently when exposed to a given buffer.     

Comparative study of the kinetics of the acrosome reaction and survival of human spermatozoa in various media

The in-vitro kinetics of the acrosome reaction and the survival of human spermatozoa were studied under different capacitating conditions. Human preovulatory follicular fluid (FF), isotonic BWW (N-BWW) and hypertonic BWW (H-BWW) were tested. Motile sperm selected by migration in these media were examined after 1, 3, 5 and 22 h of incubation under 5% CO2. The kinetics of the reaction in the population of live, morphologically normal sperm was dependent on both the culture medium and time of incubation. In the first hour, the mean percentage of acrosome-reacted sperm in H-BWW and FF was significantly greater than in N-BWW. The proportion of reacted cells increased significantly after 3 h in N-BWW (P = 0.001), after 5 h in FF (P = 0.03) and after 22 h in H-BWW (P = 0.01). A significant decrease in sperm viability was registered at 3 and 22 h of incubation (P less than 0.002) in all media. These results demonstrate that both H-BWW and FF stimulate the acrosome reaction while survival is optimal in the latter.     

Cytochalasin D inhibits penetration of hamster eggs by guinea pig and human spermatozoa

Fertilization experiments using zona-free hamster eggs and spermatozoa from both guinea pig and human were conducted in the presence of cytochalasin D to evaluate the possible role of actin filaments in fertilization processes. When the actin filament inhibitor, cytochalasin D, was added to fertilization media at concentrations of 10 to 30 microM, penetration of eggs was significantly inhibited. Preincubation of the eggs with cytochalasin D and washing prior to addition of spermatozoa had no effect on penetration as quantitated by the number of swollen heads in the egg cytoplasm. However, spermatozoa preincubated with cytochalasin D and subsequently washed prior to egg addition showed reduced penetration of the same magnitude as when spermatozoa and eggs were coincubated with cytochalasin D. Both the percentage of zona-free eggs showing decondensed sperm heads and the penetration indices (total decondensed spermatozoa/total eggs) were significantly affected when spermatozoa were exposed to cytochalasin D. The DMSO vehicle used to dissolve cytochalasin D had little effect on the number of decondensed heads. When the concentration of cytochalasin D was increased (DMSO remaining constant) in human sperm experiments, percent penetration decreased and progressively fewer decondensed spermatozoa were recorded, indicating dose-responsiveness to cytochalasin D. Motility parameters of human spermatozoa were not altered at any of the concentrations of cytochalasin D tested. Neither guinea pig sperm motility nor acrosome reaction was altered significantly by cytochalasin D or the DMSO vehicle. These experiments suggest that cytochalasin D may be an inhibitor of some fertilization processes such as sperm penetration or sperm head decondensation.     

Comparison of the penetration ability of human spermatozoa into bovine cervical mucus and zona-free hamster eggs

In vitro bovine cervical mucus (BCM) penetration tests, sperm penetration assays (SPA) using zona-free hamster eggs, and routine semen analyses were performed on a total of 136 freshly collected semen samples from men who were seen at an infertility clinic. The correlations between bovine cervical mucus penetration and other semen parameters were the percent motile spermatozoa (r = 0.48), progressive motility grade (r = 0.44), sperm count (X 10(6)/ml) (r = 0.47), the percent normal morphology (r = 0.32) and the percent eggs penetrated (r = 0.46) (P less than 0.0001 for each correlation coefficient). When known fertile (n = 32) and infertile (n = 18) groups were tested, positive mucus penetration was associated 75% correctly and positive egg penetration was associated 90% correctly to clinical status. The mucus test had no false-negative results and the SPA had no false-positive results in these groups. It appears, then, that the mucus test and sperm penetration assay, although contributing different elements of data to an infertility evaluation, are both useful adjuncts to a semen analysis.     

The influence of medium composition, osmolarity and albumin content on the acrosome reaction and fertilizing capacity of human spermatozoa: development of an improved zona-free hamster egg penetration test

The influence of medium composition, osmolarity and albumin concentration on the ability of human spermatozoa to undergo the acrosome reaction and penetrate zona-free hamster ova has been investigated. Raising the osmolarity but not the albumin concentration of the media was found to significantly increase the proportion of spermatozoa exhibiting an acrosome reaction and penetrating hamster ova, without influencing motility. There was, however, no correlation between the size of the acrosome reacted population and penetration rates between samples, suggesting that the zona-free hamster egg penetration test is more than just a measure of the availability of acrosome reacted cells. As a result of this study, a revised protocol for the hamster egg assay is described which is shorter and considerably more sensitive than conventional procedures.     

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.     

Teratospermia in domestic cats compromises penetration of zona-free hamster ova and cat zonae pellucidae

The ability of spermatozoa to bind and penetrate zona-free hamster ova and the zonae pellucidae of domestic cat oocytes in vitro was compared between normospermic (greater than 60% structurally normal spermatozoa per ejaculate) and teratospermic (less than 40% normal spermatozoa per ejaculate) domestic cats. The effects of culture media (Biggers, Whitten, Whittingham [BWW] versus modified Krebs Ringer bicarbonate [mKRB]) and simple dilution (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU) on penetration also were examined. High percentages of structurally normal spermatozoa were bound to zona-pellucida-free hamster ova regardless of the morphological forms in the inseminant. Mean percent normal spermatozoa bound to ova in DR, NS, and SU sperm aliquots from teratospermic male cats were not different (P greater than 0.05) from similarly treated normospermic aliquots. However, the percent penetration of hamster ova by normospermic ejaculates (10.5%) was superior (P less than 0.05) to that of teratospermic ejaculates (2.8%). Although swim-up processing improved percent sperm motility, progressive motility, and normal morphology in teratospermic ejaculates (P less than 0.05), no difference was observed in ovum penetration among the DR-treated, NS-treated, and SU-treated spermatozoa (P greater than 0.05). Culture medium had no effect on sperm binding in the hamster assay, but ovum penetration rate by spermatozoa in the normospermic ejaculates was enhanced (P less than 0.05) using mKRB (13.5%) when compared with BWW (7.6%) medium. Spermatozoa from teratospermic cats were capable of binding and penetrating cat zonae; however, sperm-zona interaction (defined as percent of oocytes with spermatozoa binding to or penetrating into the zona) was different (P less than 0.05) between normospermic (65.3%) and teratospermic (24.2%) cats.(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.     

Acridine orange fluorescence as male fertility test

Acridine orange fluorescence, semen analysis, and zona-free hamster egg penetration tests were performed on 100 consecutive semen samples. No significant correlation was detected between sperm motility and red fluorescence. However, abnormal sperm morphology correlated significantly with red fluorescence (p less than or equal to 0.001) and fluorescence index (p less than or equal to 0.001). Presence of a high percentage of red fluorescing sperm did not prevent their penetration of zona-free hamster egg, indicating that the presence of abnormal DNA had no effect on the sperm fertility. The significance of acridine orange fluorescence male fertility test requires further elucidation.     

A prospective study of the relationship between semen quality and fertility in cases of unexplained infertility

The male partners of 68 couples exhibiting 5.1 +/- 0.3 (SEM) years of unexplained infertility were assessed using the conventional criteria of semen quality, the movement characteristics of the spermatozoa and the outcome of the zona-free hamster egg penetration test. After a follow-up period of 2.3 +/- 0.06 (SEM) years, 25 (37%) of these patients were found to have initiated a pregnancy, thereby permitting an analysis of those aspects of semen quality which most accurately predicted their subsequent fertility. A multivariate discriminant analysis revealed that the conventional semen profile, per se, was not of significant value in discriminating the incidence of pregnancy. However, significant discrimination (P = 0.0173) was obtained when the postcapacitation movement characteristics of the spermatozoa were incorporated into the analysis. The accuracy of this prognosis was further increased if either the duration of infertility or the outcome of the zona-free hamster egg penetration test was taken into consideration. Overall classifications of fertility were then 76.3% and 76.5% accurate, respectively. These results suggest that in vitro assessments of human sperm function are of significant value in evaluating male fertility.     

Diagnostic value of sperm function tests and routine semen analyses in fertile and infertile men

The results of routine semen analyses, the zona-free hamster oocyte penetration test, the hypoosmotic swelling test, and semen adenosine triphosphate levels were studied in 66 fertile and 130 infertile men. Multivariate discriminant analysis demonstrated that routine semen parameters including semen volume, sperm count, percent sperm motility, and percent normal spermatozoa in combination could predict the fertility of these patients with 70.4% accuracy. Of the three sperm function tests evaluated, the zona-free hamster oocyte penetration test and the hypoosmotic swelling test were selected by the multivariate discriminant analysis as variables capable of providing significant information on the fertility status of the patients. However, the addition of the results of these two tests to the routine semen analysis did not significantly improve the predictability of fertility. The overall correct prediction rate was 77.6% after incorporation of the results of these two sperm function tests. In this group of subjects, the presently available sperm function tests did not predict the fertility status of a patient with a high degree of accuracy.     

Adenosine triphosphate levels in human spermatozoa

ATP levels in human spermatozoa have been positively correlated with good motility. This has given rise to the impression that good ATP levels per se are related to good motility, i.e., the higher the ATP level, the better the motility and fertilizing potential. There was no direct correlation between motility percentage, forward progression, viability percentage, and ATP levels (when expressed per 1 million spermatozoa) in the general population. This finding was not unexpected since other factors, such as defects of the microtubules and viscosity of the semen, could affect the motility in some patients. However, when semen from individual patients was assessed, the motility percent, viability, and ATP concentration decreased by comparable levels over a 4-h period postejaculation. Semen samples with normal counts of spermatozoa (greater than 20 X 10(6)/ml) had higher levels of ATP than samples from patients with oligozoospermia. Spermatozoa from patients whose semen contained less than 0.60 X 10(-2) nmol ATP/1 million spermatozoa showed a rapid drop in motility over a 4-h period compared with semen samples where the motility remained above 10% motile over this period, the latter samples having ATP levels averaging 3.30 X 10(-2) nmol/1 million spermatozoa.     

Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa

A poor penetration rate of glycerol-treated, cryopreserved human spermatozoa as compared to untreated fresh control, was observed in the zona-free hamster oocyte test. Similarly, glycerol treatment of freshly ejaculated spermatozoa depressed the penetration rate unless the culture medium also contained glycerol. Immediately after thawing, glycerol-treated, cryopreserved spermatozoa possessed adequate progressive motility, but their incubation in glycerol-free culture medium caused a severe reduction in motility. Even if the same number of progressively motile, cryopreserved, glycerol-treated spermatozoa as unfrozen spermatozoa were added to the eggs, a much lower penetration rate was obtained by the treated spermatozoa. It is concluded that spermatozoa develop a glycerol dependence and that removal of glycerol from the surrounding medium, as most likely occurs when spermatozoa pass through the cervix, reduces both the motility and the ability of spermatozoa to become capacitated and fuse with oocytes. Thus, glycerol is not an optimal cryopreservative agent. Further, the decreased oocyte penetration rate of glycerol-treated, cryopreserved spermatozoa is due to other factors besides the decrease in sperm motility.     

Acrosome-reacting capacity of frozen-thawed human semen: relation to hamster ova penetration

Interspecies egg penetration requires completion of capacitation and acrosome reaction of the spermatozoa. The ability of frozen-thawed human semen to accomplish this reaction was studied, and its relation to the zona-free hamster egg penetration was investigated. The percentage of reacted cells in the population of live spermatozoa estimated by the triple-staining technique was found to be greater in frozen than in fresh samples. However, the egg-penetrating ability of cryopreserved semen remained significantly low. Individual differences in the ability to undergo the acrosome reaction and its relation to penetration are discussed.     

Cryosurvival of human spermatozoa frozen in eight different buffer systems

The present study was conducted to ascertain optimal cryoconditions for human spermatozoa by comparing the relative cryoprotective efficiency of eight buffer systems and assessing various cryovials and thaw rates under two freeze rates. Spermatozoa that were cryopreserved in one of four zwitterion buffers (TES-Tris-citrate-egg yolk-glycerol; TES-Tris-citrate-I) maintained higher progressive motility at 0, 1, 2, and 4 hours post-thaw as compared to cells frozen in glycerol only, citrate-egg yolk-glycerol and TES-Tris-citrate-egg yolk without glycerol (TES-Tris-citrate-III; P less than 0.01). Freezing in TES-Tris-citrate-I also resulted in spermatozoa that penetrated the furthest distance through cervical mucus and possessed the highest percent live spermatozoa when compared to other cryoprotective media. Spermatozoa were analyzed for their ability to penetrate zona-free hamster ova and no difference was found between buffers when the assay was corrected for progressive motility. After removal of seminal plasma/buffers and incubation for 2 hours in BWW, TES-Tris-citrate-II and TES-Tris-citrate-milk showed the greatest sperm longevity (P less than 0.05). Pooled semen was extended in TES-Tris-citrate-I and frozen in straws or ampoules in static N2 vapor or in pellets on dry ice. Thaw bath temperatures ranged from 0 to 37 C. Post-thaw progressive motility and cervical mucus penetration were similar in all treatment groups. In conclusion, the present results indicate the use of TES-Tris-citrate-I for cryopreservation of human spermatozoa. With this optimal cryoprotective buffer, the containers and thaw rates used have little effect on human sperm cryosurvival.     

Penetration of sperm from teratospermic men into zona-free hamster eggs

The ability of sperm samples from male partners of infertile couples, with isolated teratospermia (ITS), to penetrate zona-free hamster eggs was examined. The ITS patients had a significantly lower proportion of normal spermatozoa than did a control group of men of proven fertility (23.6% and 47.8%, respectively; P less than 0.001), while the mean total sperm count and the % motility did not differ between the two groups. The mean hamster egg penetration rate of sperm from ITS patients was 2.64% compared to 31.1% in the control group. A significant negative correlation was found in the ITS group between the proportion of a) abnormal forms and the ability to penetrate zona-free hamster eggs, and b) pyriform-shaped sperm and the penetration rate. Of the sperm parameters which were examined, only the morphology could be correlated with the rate of penetration.     

Relationship of acrosin activity to sperm function tests

Summary.  Acrosin activity of spermatozoa from infertile patients and fertile volunteers was measured. Acrosin activity of spermatozoa from asthenozoospermic patients and patients with unexplained infertility was lower than that from fertile volunteers. We utilize the zona-free hamster egg sperm penetration test to select candidates for conventional in vitro fertilization among unexplained infertile patients. The zona-free hamster egg sperm penetration test, however, requires several hours and special equipment which are not used in the clinical setting. It is preferable that other sperm function tests or a combination of them can replace this test. Thus three distinct tests of sperm function, namely, acrosin activity, Penetrak® test, hypo-osmotic swelling test, were compared with the hamster test, using spermatozoa from patients with unexplained infertility.
A combination of the Penetrak® test and measurement of acrosin activity could predict the results of the zona-free hamster egg sperm penetration test with 88.2% accuracy. Thus, the hamster test should be done when either Penetrak® test or measurement of acrosin activity showed abnormal values.     

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.