Effects of dibutyryl cyclic guanosine monophosphate on human spermatozoal motility and penetration of zona-free hamster oocytes

The possible effects of N2,2'-O-dibutyryl 3'-5'-cyclic guanosine monophosphate (dbcGMP), imidazole (IM) or their combination on human spermatozoal motility and penetration of zona-free hamster oocytes were investigated. Motility of human spermatozoa was assessed at 0, 0.5, 2, 3, 5 and 8 h over an 8-h incubation period with various concentrations of dbcGMP, IM or dbcGMP plus IM. Both percentage motile spermatozoa and progression of human spermatozoa decreased with increasing concentrations of dbcGMP (12-96 mM) and IM (10-80 mM). This inhibitory effect on motility was more pronounced when dbcGMP was added with IM. For assessment of human spermatozoal penetration into zona-free hamster oocytes, spermatozoa were treated for 30 min with either dbcGMP, IM or both after 5 h of capacitation pre-incubation at 37 degrees C. Alternatively, the compounds were only added during the capacitation preincubation period. At concentrations of dbcGMP less than or equal to 24 mM, neither of the treatments had a significant effect on the penetration rate of zona-free hamster oocytes in comparison with the controls. However, a higher concentration of dbcGMP (48 mM) inhibited the penetration rate. A similar trend was observed when dbcGMP plus IM were added. IM alone had no significant effect on the penetration rate at the concentrations tested (10-40 mM). Our data indicated an inhibitory effect of dbcGMP on both human spermatozoal motility and penetration into zona-free hamster oocytes.     

Variable kinematics of capacitating human spermatozoa

Human spermatozoa were prepared by swim-up fromsemen into in-vitrofertilization (IVF) culture medium, and their movement recordedby videomicrography using NTSC video to give 60 images/s onfreeze-frame playback. Trajectoriesin which spermatozoa appearedto switch fromone type of motility pattern to another (fromnon-hyperactivatedto transitional or star-spin hyperactivated),were reconstructed manually and the movement characteristicsdetermined for short consecutive sequences along eachtrack.The spermatozoa were able to switch between allof these motilitypatterns, apparently at random. The seobservations illustratedthat hyperactivated motility is areversible state in human spermatozoa,with phase changesfrom hyperactivated to non-hyperactivatedmotility patternsalong trajectories. It was not necessary forspermatozoato pass through the transitional hyperactivated motilityphasewhen switching from non-hyperactivated to starspinhyperactivatedmotility or vice versa.     

Antisperm antibodies induce polyspermy by promoting adherence of human sperm to zona-free hamster eggs

Certain antisperm antibodies (ASAs) in the sera of infertilemen or women promote the penetration of zona-free hamster eggsby spermatozoa. We have shown previously that this enhancementof penetration occurs through mechanisms other than an alterationin the acrosomal structure of antibody-labelled spermatozoa.In the present study, small limited populations of antibody-labelledand antibody-free spermatozoa from fertile donors were observedserially by phase contrast, epifluorescence and scanning electronmicroscopy following their adherence to the oolemma of zona-freehamster eggs. The adherence of ASA-labelled spermatozoa to thezona-free hamster egg was markedly increased and occurred earlierwhen compared with antibody-free spermatozoa from the same ejaculate.The likelihood of entry of an antibody-labelled spermatozooninto the ooplasm, however, once adherent to the oolemma wasnot different to that of an antibody-free spermatozoa, and theuhrastructural steps of incorporation of antibody-labelled humanspermatozoa were also similar to those observed during fertilizationof hamster eggs by spermatozoa of several other species. Theseobservations indicate that ASAs promote polyspermic fertilizationof zona-free hamster eggs primarily through then-enhancementof sperm-oolemmal binding.     

Glass wool column filtration of human semen: relation to swim-up procedure and outcome of IVF

The number and viability of spermatozoa recovered by glass woolcolumn filtration and a swim-up procedure were compared usingdifferent types of ejaculates, such as normal, asthenozoospermicand very viscous oligozoospermic semen. The filtration procedureresulted in significantly (P < 0.01) higher recovery of viablespermatozoa than the swim-up procedure from all types of ejaculatesstudied. Further, the spermatozoa from 50 (78.1%) of the 64ejaculates filtered through glass wool column fertilized atleast one intact hwnan egg in an in-vitro fertilization (IVF)procedure. it is concluded that glass wool column filtrationis superior to the swim-up procedure since it yields a higherrecovery of viable spermatozoa that are potentially fertile.Therefore, the glass wool column filtration procedure used toprepare spermatozoa may be of benefit for IVF, intra-uterineinsemination, in-vitro fertilization and GIFT (gamete intra-Fallopiantransfer), especially in cases of poor quality semen.     

Production of Mouse Monoclonal Antibodies Directed Against the Oolemma of Human and Hamster Oocytes by Intra-splenic Injection of Oocytes

PROBLEM: To develop an additional approach for the study of oolemmal surface moieties involved in gamete interactions, we decided to obtain monoclonal antibodies by intrasplenic injection of human and hamster oocytes in Balb/c mice. METHOD: Two Balb/c males were injected three times intrasplenically at 15-day intervals with approximately 40 zona-free hamster and 3–5 zona-free human oocytes. After the third injection, spleen cells were fused and hybridomas developed. We used a novel screening system based upon the use of sections of frozen human and hamster eggs, tested by means of indirect immunofluorescence. The antibodies that we produced were evaluated for their ability to interfere with the zona-free hamster eggs penetration by human spermatozoa. The B2B5 antibody was also developed as ascitic fluid and further characterized. RESULTS: Seven antibodies reactive with hamster oocytes were produced. Six of them also reacted with human oolemmas. The binding was confined to the oolemma, and no staining of the zona nor the cytoplasm was present. One of these antibodies reduced the penetration of zona-free hamster eggs by human spermatozoa. This antibody, B2B5, an IgM kappa, was confirmed to interact with the oolemma by means of indirect immunofluorescence of fresh eggs and Covasphere binding. B2B5 did not react with other human or hamster tissues except capacitated human spermatozoa. The reactivity with the oolemma of hamster oocytes was not lost after egg penetration by human sperm. CONCLUSIONS: Intrasplenic immunization using zona-free human and hamster oocytes allows the production of anti-oolemma antibodies. A system of screening based upon the use of sections of frozen eggs also allows an easy and quick scoring of many supernatants. B2B5 monoclonal anti-oolemma antibody deserves further studies in that is able to interfere with fertilization and its antigen appears to be confined to the gametes surface.     

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.     

Analysis of human sperm-derived pronuclei by three-colour fluorescent in-situ hybridization

We describe a modification of the human sperm-zona-free hamster egg fusion method that permits the study of aneuploidy in sperm-derived pronuclei by multicolour fluorescent in-situ hybridization (FISH). Zona- free hamster eggs and human spermatozoa were fused and cultured for 15 h in the presence of colcemid (1 microg/ml of medium) to obtain hamster oocytes arrested at metaphase II and human spermatozoa at the pronuclear stage. By applying a whole human genomic DNA probe we confirmed that 100% of pronuclei tested (372/372) were of human origin. One-colour fluorescent in-situ hybridization using a centromeric 18 probe was applied to 919 pronuclei with different dithiothreitol (DTT) pretreatments: 50 mM (10 min) or 25 mM (20 and 25 min). The highest hybridization efficiency was obtained with treatment with 25 mM DTT for 20 min (90.3%). Sex chromosome aneuploidy was analysed by three-colour FISH in a total of 2596 pronuclei from a normal donor. Hybridization efficiency was 98.6%. The disomy rates for X, Y and XY chromosomes (0.11, 0.04 and 0.08% respectively) were similar to data reported for sperm nuclei by three-colour FISH and to those obtained in sperm chromosomes. These results suggest that selection of potentially fertile spermatozoa (spermatozoa able to fertilize zona-free hamster eggs and produce a pronucleus) does not imply chromosomal selection.      

Diagnostic accuracy of computer-assisted sperm motion analysis

One retrospective and two prospective studies were conducted among 218 couples treated with in-vitro fertilization (IVF) to establish the reproducibility and diagnostic accuracy of computer-assisted sperm analysis (CASA) with swim-up spermatozoa for the prediction of the fertilization rate of oocytes in vitro. Based on the results of a preliminary retrospective analysis in 49 patients, the 'curvilinear velocity' (VCL) was chosen as the most distinctive motion parameter of sperm function and the median was used to represent the entire sperm population. The number of inseminated motile spermatozoa was then adjusted to median VCL during two subsequent prospective studies with clinical IVF. Whereas in the first prospective study (90 couples) the threshold values of VCL with regard to the number of spermatozoa inseminated were based on the results of the preliminary retrospective study (49 couples), in the second prospective study (79 couples) the settings were based on the results of the first prospective study. The reproducibility of CASA was tested by analysing the motion characteristics of spermatozoa at different intervals after termination of swim-up, by repeated analysis of the same video-recording of the incubated spermatozoa by different observers, and by the repeated video- recording of the freshly prepared sperm samples and analysis of both video-recordings by the same observer. Under these conditions the frequency of disagreement between two measurements varied between 2.0 and 8.2%. In both prospective studies the sensitivity of CASA for the prediction of fertilization was high (74.0%), whereas the specificity was low (40.0%). In contrast to successful fertilization, unsuccessful fertilization of oocytes in vitro could not be predicted reliably with CASA. However, the pregnancy rate per cycle among patients with predicted low fertilization rates was significantly lower (5.3%) than in couples with high predicted fertilization rates (24.3%, P < 0.001). Therefore, CASA of washed spermatozoa may still help to identify couples who would benefit more from intracytoplasmic sperm injection (ICSI) than from IVF. A definite threshold level could not be identified for any of the motion parameters to distinguish the motion characteristics of fertilizing and non-fertilizing spermatozoa. Using various algorithms for hyperactivated motility, the percentage of hyperactivated spermatozoa was significantly higher among the successfully fertilizing patients than among the nonfertilizing group. However, the absolute number of hyperactivated spermatozoa added to the oocytes was higher in non-fertilizing couples. Therefore, the lack of fertilization in some patients may be caused by a generalized defect in sperm function rather than by insufficient hyperactivation.      

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.     

Correlation between intracellular cAMP content, kinematic parameters and hyperactivation of human spermatozoa after incubation with pentoxifylline

Various compounds have been used in the attempt to improve sperm motility, including pentoxifylline (PF), a methylxanthine derivative. It has been postulated that PF, being a phosphodiesterase inhibitor, increases sperm kinematic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. However, it has not been clarified whether the biological effects of PF on sperm motility correlate with its ability to increase intracellular cAMP levels. To examine this relationship, the kinematic parameters, hyperactivation, and intracellular cAMP content were evaluated in motile spermatozoa, obtained by discontinuous Percoll gradient and swim- up from 21 normozoospermic semen samples, incubated without and with PF for 0, 1, 2, and 4 h. PF increased beat cross frequency after 1 and 2 h of incubation, curvilinear velocity and lateral head displacement (ALH) after 4 h, and hyperactivation after 1, 2, and 4 h, and decreased linearity (LIN) after 1 h of incubation. The intracellular cAMP content of spermatozoa incubated with PF increased at all time-points examined. Both intracellular cAMP content and increase in hyperactivation in response to PF decreased with the length of incubation. In the absence of PF, cAMP content was unchanged and was correlated significantly only with ALH and the percentage of spermatozoa with hyperactivated motility. Following incubation with PF, cAMP content correlated with hyperactivation and all sperm kinematic parameters, with the exception of LIN and straightness. These findings suggest that the beneficial effects of PF on sperm kinematic parameters and hyperactivation are related to its ability to increase intracellular cAMP content.      

Andrology: Nuclear decondensation of sperm head and failure at in-vitro fertilization: an ultrastructural study

The problem of unexplained male infertility was investigatedby electron microscopic study of spermatozoa from 51 males.The subjects were subdivided as follows: group A (n = 25) normalfertile males (controls), group B (n = 13) successful in-vitrofertilization (IVF) cases (fertilization rate >50%), groupC (n = 13) failed IVF cases. All subjects included in groupsB and C had a 6–12 year history of childlessness and IVFwas employed when other methods of assisted reproduction failed.The study of spermatozoa in fertile males (controls) was carriedout to establish baseline ultrastructural abnormalities. Inall 51 cases, an average of 330 (280–800) sperm headsand 660 (330–1190) sperm tails were studied. Decondensationof nuclear chromatin was observed in 70 ± 15% (mean ±SD) of spermatozoa in failed IVF cases, 16 ± 5% in successfulIVF cases and 7 ± 3% in controls. These results werefound to be statistically significant (P > 0.001). The meanvalue for motility of spermatozoa in all three groups was withinaccepted limits of normality. It is concluded that decondensationof nuclear chromatin seen by electron microscopy is one of themost important causes of male infertility. It is advocated thatelectron microscopic examination of semen should be carriedout in all cases of longstanding, unexplained male infertilitybefore embarking upon IVF programmes.     

Successful in-vitro fertilization pregnancy with spermatozoa from a patient with Kartagener's syndrome: case report

This paper reports on the successful treatment by in-vitro fertilization (IVF) of a couple in whom the male partner had Kartagener's syndrome. His spermatozoa were severely asthenozoospermic with deficient dynein arms and disordered microtubular configuration. On computer-assisted sperm analysis (CASA) motile spermatozoa displayed straight non-progressive motility with minimal amplitude of lateral head displacement and none were hyperactivated. This is the first case report in which spermatozoa with axonemal disruption in a man with immotile cilia syndrome (ICS) have been shown to be able to penetrate the zona pellucida and fertilize oocytes. IVF may be a suitable treatment for certain variants of ICS.     

Human epididymal secreted protein CD52 on ejaculated spermatozoa: correlations with semen characteristics and the effect of its antibody

CD52 is a glycosylphosphatidylinositol-anchored glycoprotein secreted by the epididymis where it is incorporated into sperm membranes. The antigen is common to spermatozoa and lymphocytes, and its role is not known. Quantitative analysis using immunostaining with the monoclonal antibody CAMPATH-1G and flow cytometry indicated positive signals from approximately 80% of viable, washed ejaculated spermatozoa. Staining intensity increased after capacitation overnight, and decreased after short incubation with high density lipoprotein. After incubation of Percoll-washed spermatozoa with CAMPATH-1G, motility was reduced from 83 to 74% after 5 min and from 73 to 52% after 3.5 h. Swimming velocities were reduced by approximately 30% and linearity by 15% in 5 min, but no further decreases were observed over 3.5 h. After 20 min incubation with the antibody, cell viability was decreased by 10 and 20% in freshly washed and capacitated spermatozoa respectively. Comparison of fertile and infertile patients revealed no difference in the percentages of immunostained spermatozoa or in staining intensity and there were no differences in sperm labelling between normozoospermia and teratozoospermia. Compared with normozoospermia, percentages of stained spermatozoa were lower by 20 and 27% in asthenozoospermia and oligozoospermia respectively, whereas staining intensity in asthenozoospermia was less than half that in oligozoospermia. The correlation of percentages of stained spermatozoa with percentages of motile cells suggests the involvement of epididymal CD52 in the maturation of sperm function.      

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation

Oxygen radical generation is known to be detrimental to sperm function, especially motility, through the lipid peroxidation of the membranes. Generation of reactive oxygen species can be induced by leukocyte contamination, sperm centrifugation and the presence of abnormal spermatozoa with excess residual cytoplasm. This study aims to evaluate the effect on sperm motility of incubation in an antioxidant-containing solution, during liquefaction and centrifugation. Thirty semen samples were each divided into two equal parts: one mixed with Tyrode's solution, the other with a salt solution containing antioxidants (Sperm- Fit; Ellios Bio-Media, Paris, France). All the procedures were identical in the two groups. The ratio of leukocytes to spermatozoa was significantly correlated with the motility after liquefaction and after a 24 h incubation in routine in-vitro fertilization (IVF) medium and with the number of motile spermatozoa recovered after Percoll preparation. Moreover, when this ratio was > or = 0.2, all motility parameters were lowered. Incubation with Sperm-Fit allowed a higher percentage of motility after Percoll preparation when the ratio was > or = 0.2 (48 +/- 5% versus 41 +/- 6% for Sperm-Fit and Tyrode's solution respectively; P < 0.05) and a greater number of motile spermatozoa recovered after Percoll preparation, whatever the ratio (3.2 +/- 1.0 x 10(6) versus 2.4 +/- 0.7 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio > or = 0.2; 18.1 +/- 3.4 x 10(6) versus 14.4 +/- 2.9 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio < 0.2; P < 0.05). These results show that incubation with antioxidants during liquefaction and centrifugation increases recovery of motile spermatozoa.      

Case report: high fertilization rate in conventional in-vitro fertilization utilizing spermatozoa from an oligozoospermic subject presenting microdeletions of the Y chromosome long arm

A case is reported in which a high fertilization rate was achieved by conventional in-vitro fertilization (IVF), using spermatozoa from an oligozoospermic man carrying a microdeletion of the long arm of the Y chromosome. The patient presented with idiopathic infertility of 10 years duration; the fertility status of his wife was completely normal. After IVF, five out of eight oocytes retrieved showed normal fertilization and four showed normal embryo cleavage. Four embryos were transferred; however, pregnancy did not result. These results demonstrate that spermatozoa from oligozoospermic patients carrying a Yq microdeletion are fully competent in achieving capacitation, acrosome reaction and fertilizing ability during IVF. Therefore, although definitive conclusions cannot be made from a single case report, we suggest that Yq microdeletion analysis should be considered in oligozoospermic patients undergoing conventional IVF.      

Importance of the use of short and long sperm pre-incubation periods in assessing the fertilizing capacity of human spermatozoa by hamster test

The fertilizing capacity of semen samples of seven fertile menwas studied in vitro using zona-free hamster ova (hamster test).Spermatozoa of each sample were pre-incubated 4 or 24 h beforeova were inseminated for 3 h. An extended insemination for anadditional 3 h was also performed after the shorter pre-incubation.The overall ovum penetration rate changed from 0 to 100% (mean52%) after short (4 h) sperm pre-incubation and from 10 to 100%(mean 55%) after long (24 h) pre-incubation. Taking into accountthe combination of short and long pre-incubation periods, alldonors reached a penetration rate of 67 to 100%. The resultsindicate that both short and long pre-incubation periods mustbe employed when the hamster test is used diagnostically toassess the fertilizing capacity of human spermatozoa.     

Effect of human cervical mucus on human sperm motion and hyperactivation in vitro.

The aim of our experiment was to examine the effect of exposure to human cervical mucus on quantitative sperm motility with specific reference to hyperactivated sperm motility. Human spermatozoa were allowed to penetrate cervical mucus for 20 min before swimming into Earle's balanced salt solution tissue culture medium for 25 min. The sperm motion characteristics were compared to those which had been obtained from a direct swim-up for 45 min. Spermatozoa treated with mucus were more 'active' than the control group. Multivariate statistical analysis indicated that cervical mucus promotes hyperactivated motility and that sperm sub-populations exposed to cervical mucus are very heterogeneous, as indicated by the numbers and motility characteristics of spermatozoa.     

Sperm Antibodies and Human Fertilization

ABSTRACT: Previous investigations using zona-free hamster oocytes and salt-stored human zonae pellucidae, and retrospective analysis of routine human in vitro fertilization (IVF) results have suggested that sperm antibodies can impair the fertilizing capacity of human spermatozoa. The results of our investigations using viable human oocytes confirm that human antispermatozoal antibodies can inhibit fertilization by human spermatozoa. In the future we plan to examine the mechanism of fertilization inhibition, including characterization of the sperm antigens involved. These studies may facilitate the development of iromunocontraceptive vaccines and of new treatments for immunological infertility.     

Andrology: Effects of platelet activating factor on human sperm function in vitro

The direct effects of platelet activating factor (PAF) and thespecific PAF receptor antagonist, CV-3988, on the fertilizingability of human spermatozoa were investigated. PAF (10^–7–10^–11M) increased the human sperm penetration rates in a sperm penetrationassay at all doses >10^–11 M. In contrast, treatmentof the spermatozoa with 10^–5 CV-3988 caused a significantdecrease in human sperm penetration of zona-free hamster oocytesand adversely affected sperm motility after 24 h of incubation.This suppression was reversed by the addition of PAF. The acrosomereaction was also enhanced by PAF treatment of spermatozoa butthis effect was not observed in calcium-free medium. While 10^–5M CV-3988 decreased the acrosome reaction, the inhibition wasalso reversed by the addition of PAF. These results suggestthat PAF may have a direct role in the fertilizing capacityof human spermatozoa. These findings also suggest that PAF mayhave a clinical application in an in-vitro fertilization programme.