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.     

Human oviductal cells and their conditioned medium maintain the motility and hyperactivation of human spermatozoa in vitro

The effects of the co-incubation of human oviductal cells withhuman spermatozoa on the general motility pattern and hyperactivationof spermatozoa in vitro were studied using computer-assistedsperm analysis. Co-incubation preserved all the sperm motilityparameters, with the exception of the percentage of hyperactivation(HA), beat cross frequency (BCF), and the percentage of transitionalhyperactivated (THA) spermatozoa, when compared with the initialmotility pattern of the spermatozoa. The HA and THA decreased,and BCF increased after co-incubation for 3 h; these levelsthen remained stable up to 5 h. The control spermatozoa showeda continuous significant change after 5 h incubation. The oviductalcell-conditioned medium maintained all the motility parametersof spermatozoa even after 5 h incubation. These data suggestedthat human oviductal cells could maintain the motility of spermatozoain vitro. Similar effects were also observed when conditionedmedium was used to treat subnormal spermatozoa.     

Apoptosis and necrosis in human ejaculated spermatozoa

BACKGROUND: Features of both apoptosis and necrosis have been reported in ejaculated human spermatozoa. This study examines the relative contribution of these two modes of cell death to the demise of these terminally differentiated cells. METHODS: Sperm fractions were prepared from aliquots of semen samples from young normozoospermic donors by simple washing from seminal plasma, by discontinuous density gradient centrifugation or by swim-up technique. They were subsequently incubated in vitro at 37 degrees C for 24 h. Sperm motility, viability, and the presence of two apoptotic markers, phosphatidylserine externalization (annexin-V binding) and DNA fragmentation (TUNEL), were examined before incubation and again after 4 and 24 h of incubation. RESULTS: The swim-up technique was the most efficient in terms of the recovery of viable, motile and non-apoptotic spermatozoa, followed by density gradient centrifugation and finally simple washing. No changes in the parameters tested were observed after 4 h of incubation, but a significant decrease in sperm motility and viability was detected after 24 h irrespective of the sperm preparation technique employed. However, these changes were not accompanied by any increase in the incidence of spermatozoa showing markers of an active apoptotic process. CONCLUSIONS: Healthy human ejaculated spermatozoa appear incapable of initiating apoptosis, at least under in vitro conditions.     

Comparative analysis of motility characteristics of Percoll-selected spermatozoa populations from fresh and cryopreserved semen

The proportion and quality of motility of spermatozoa in normozoospermicejaculates were assessed using computerassisted semen analysis.The ejaculate was split and the motility re-assessed followingseparation on a Percoll gradient with or without cryomediumand cryopreservation. Cryopreservation caused a significantdecrease in the proportion of motile spermatozoa and in theirvelocity and amplitude of lateral head displacement. The initialdecrease in the proportion of motile spermatozoa was found tobe in part an effect of the cryomedium. The use of Percoll gradientseparation did not initially change these effects but after4 h incubation differences in velocity and amplitude of lateralhead displacement between samples were no longer evident. Percoll-selected,cryopreserved spermatozoa had both a stable proportion of motilespermatozoa and a stable velocity for at least 48 h, whereasin fresh spermatozoa populations, similarly separated usingPercoll, the proportion of motile spermatozoa had decreasedby 24 h and the velocity was lower at 48 h. Percoll preparationis an effective method for the selection of motile spermatozoafrom cryopreserved semen which, after a short incubation, havesimilar motility characteristics to fresh spermatozoa.     

Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples.

Two methods of density gradient centrifugation, Percoll (P) and mini-Percoll (MP), were compared with the swim-up technique for preparing spermatozoa from each of 40 abnormal semen samples. P and MP produced similar results with a mean recovery of spermatozoa with progressive motility which was significantly higher (18-19%) than that achieved with swim-up (5%). However, the swim-up method resulted in the recovery of spermatozoa with a higher mean motility (89 versus 58%), velocity (69 versus 56 microns/s), percentage with normal morphology (22 versus 16%) and intact acrosomes (61 versus 36%) than P and MP. The mean amplitude of lateral head displacement was the only characteristic of spermatozoa in semen which correlated with the recoveries of motile spermatozoa. Combining MP and swim-up methods for 10 samples produced a higher recovery (11 versus 6.9%) of spermatozoa with significantly better mean motility (94 versus 87%) than did swim-up alone. Although P and MP resulted in greater yields of motile spermatozoa than the swim-up preparation, the latter procedure selected higher proportions of spermatozoa with improved characteristics (velocity, intact acrosomes and normal morphology) which correlate with fertilization rates in vitro. It is concluded that P and MP are not superior to swim-up. However, sequential MP and swim-up preparation improves yields of high quality spermatozoa from some abnormal semen samples and therefore has potential for improving fertilization rates.     

Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime

While washing of human sperm cells by centrifugation and resuspensionis a procedure in widespread use, there have been indicationsthat this procedure per se may be harmful to the cells. Theobjective of this study was to investigate this question. Tothis end, a method for the clean separation of motile humanspermatozoa from seminal plasma in the absence of centrifugationwas developed, using a modified swim-up procedure, in whichliquefied semen was mixed with an equal volume of 30 mg/ml dextranin medium, and the mixture overlaid with medium containing 5mg/ml bovine serum albumin, forming two discreet layers withstable interface. The percentage of motile cells in a givensample was consistently >80% immediately after recovery.Damage to the cells was assessed by loss of motile cells duringincubation up to 96 h post-recovery. Comparison of aliquotsof spermatozoa obtained by the dextran swim-up procedure showedthat the aliquot subjected to centrifugation had 4 ±3% motile cells after 48 h, while the untreated aliquot had52 ± 12%. The aliquots showed no difference 1 h postrecovery.Similar results were obtained with spermatozoa that had beencentrifuged in seminal plasma and resuspended in fresh plasma,then recovered by dextran swim-up. The delayed onset of motilityloss in the centrifuged samples implies that this treatmentinduces sublethal damage in the cells. Comparison of the standardswim-up and Percoll gradient methods for sperm recovery, bothof which involve centrifugation steps, showed decline in motilityof the samples similar to that seen with dextran swim-up ofcentrifuged cells. We conclude that centrifugation per se inducessublethal damage in human spermatozoa, independently of treatmentmethod, and suggest that recovery methods for human spermatozoawhich avoid centrifugation might partially alleviate the damageincurred by these cells during cryopreservation.     

The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization

Silane-coated silica particles (PureSperm) were evaluated as an alternative to Percoll for gradient separation of spermatozoa, for use in assisted reproduction. Recovery of motile and morphologically normal spermatozoa after using a four-layer Percoll and a two- and four-layer PureSperm gradient respectively was recorded. In-vitro fertilization (IVF) results after using PureSperm for the sperm preparation were also evaluated. No difference in sperm recovery or sperm motility was found when comparing the use of Percoll and the four-layer gradient of PureSperm. When using a two-layer PureSperm gradient, motility was significantly decreased (P < 0.05) compared to Percoll. Normal sperm morphology increased from 8-17.2% after using Percoll and to 12.7% and 11.4% after using a four-layer and a two-layer PureSperm gradient respectively. All gradient preparations showed a significant decrease in the teratozoospermia index compared to the ejaculate (P < 0.01). No significant differences in IVF results regarding fertilization and pregnancy rates were found when PureSperm or the swim-up technique were used for the sperm preparation. PureSperm seems to be an acceptable alternative to Percoll but although the percentage of sperm recovery was higher after PureSperm we still recommend the swim-up technique to be the first choice, as a higher percentage of progressive motile spermatozoa is obtained without using other chemicals than IVF culture medium.     

In-vitro effects of anti-sperm antibodies on human sperm movement

The present study was designed to investigate whether autoantibodlesto external domains of the sperm plasma membrane affect themovement of normal motile spermatozoa. Eight sera and 20 semInalplasma samples containing high levels of anti-sperm antibodiesas well as antibodies eluted from the sperm fraction of 19 autounmuneejaculates were incubated with donor's motile spermatozoa, obtainedby swim-up migration in Tyrode's solution. Sperm movement wasanalysed using is exposure microphotography when >70% ofthe spermatozoa were coated with antibodies (after 30–90mi of incubation). At least 50 tracks of progressively motilespermatozoa were analysed in order to obtain the mean valuesof the amplitude of lateral head displacement (ALH) and thevelocity of progression (VSL). Serum antibodies and sperm elutedantibodies had quite consistent but opposite effects on spermmovement; serum antibodies Increased ALH and decreased VSL whereaseluted antibodies decreased ALH and increased VSL. Seminal antibodiesdid not affect these two parameters significantly. Furthermore,seminal antibodies and sperm eluted antibodies obtained fromthe same ejaculates had distinct effects on ALH and/or VSL.This diversity was apparently not linked to antibody isotypeor localization on the sperm membrane; it might be due to differencesin the composition of the extracellular media. These resultssuggest a dynamic effect of anti-sperm antibodies on sperm movement,a possibility that merits further investigation.     

Co-incubation of human spermatozoa with Chlamydia trachomatis serovar E causes premature sperm death

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54x10(6) EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5x10(6) per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems.     

Phosphatidylinositol 3-kinase inhibition enhances human sperm motility

BACKGROUND: The number of spermatozoa with forward motility after capacitation procedures represents the limiting factor for application of IVF versus intracytoplasmatic sperm injection (ICSI) procedure in cases of oligoasthenozoospermia. The possibility of increasing this number may thus be of help to the patient. A complex array of signalling pathways is involved in the regulation of sperm motility and recent data pointed out an important role for kinase/phosphatase-regulated phosphorylation of proteins. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K), a lipid and protein kinase involved in the regulation of several biological aspects of somatic cells, on human sperm motility by using the specific PI3K inhibitor LY294002. METHODS AND RESULTS: We demonstrated that in-vitro incubation of swim-up selected or unselected human spermatozoa with LY294002 determined an increase of percentage forward motility in all the treated samples. The effect was dose-dependent with an EC(50) of 1.09 +/- 0.54 micromol/l. LY294002 also increased sperm movement characteristics and hyperactivation as evaluated by computer-assisted motion analyser. The compound was also able to overcome the detrimental effect of hydrogen peroxide and lithium chloride on sperm motility. CONCLUSIONS: Our results suggest a negative role for PI3K in the development and maintenance of sperm motility and suggest a possible use of PI3K inhibitors to enhance motility in cases of asthenozoospermia.     

Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing

The purpose of this study was to examine if selecting a sperm population with improved motion characteristics before freezing reduces the deleterious effects of cryopreservation. Semen specimens from 15 normal donors were divided into two equal aliquots. The first aliquot received no treatment (control), and the second was processed by swim-up from a washed sperm preparation to select a sperm population with better motility and motion characteristics (swim-up). Both aliquots were cryopreserved by the liquid nitrogen vapour method. Percentage motility and motion characteristics were evaluated by computer-assisted semen analysis. Acrosome integrity as well as spontaneous and calcium ionophore-induced acrosome reactions before freezing and after thawing were assessed by fluorescein isothiocyanate conjugated peanut agglutinin combined with a supra vital dye (Hoechst-33258). Swim-up processing enabled selection of a sperm population with better motion characteristics, percentage motility and viability before freezing (P < 0.001), but with no difference in percentage of acrosome-intact spermatozoa (P = 0.63). After thawing, the swim-up specimens exhibited faster velocity and progression than untreated specimens (P < 0.001). They also had higher percentages of spermatozoa with intact acrosomes and spermatozoa able to undergo acrosome reaction in response to calcium ionophore (P < 0.05). Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality.     

Endeometriosis: The influence of peritoneal fluid from patients with minimal stage or treated endometriosis on sperm motility parameters using computer-assisted semen analysis

The aim of this study was to determine the influence of peritonealfluid from patients with minimal stage or treated endometriosison sperm motility parameters. Peritoneal fluid aspirated atdiagnostic laparoscopy for unexplained infertility from womenduring the luteal phase of the menstrual cycle (days 20–23)was incubated for 5 h with fresh semen samples obtained frommen of recently proven fertility. Spermatozoa were preparedby a swim-up technique from unprocessed semen. Using computer-assistedsemen analysis (Hamilton-Thorn Research, MA, USA), sperm motilityand motion parameters were observed at 0, 120, 180 and 300 min.Compared with spermatozoa incubated in Earle's balanced saltsolution/human serum albumin, the percentage motility, percentageprogressive motility and progressive velocity of spermatozoaincubated in peritoneal fluid from patients without visibleendometriosis were significantly higher (P< 0.05). Maximaleffect was observed at 3 h and maintained until 5 h. We concludethat in an in-vitro study, in contrast to peritoneal fluid frompatients with minimal stage endometriosis, peritoneal fluidfrom patients with unexplained infertility and no visible endometriosiscan improve sperm motility when compared with culture medium.     

Andrology: Percoll semen preparation enhances human oocyte fertilization in male-factor infertility as shown by a randomized cross-over study

The aim of this study was to compare two methods of semen preparation:multiple tube swim-up and Percoll separation, using a randomizedcross-over clinical study, in which sperm parameters, oocytefertilization rates, embryo quality and cell stage were analysed.Overall, there was no difference between the two preparationmethods in the normozoospermic cycles. In the male-factor cycles,Percoll extracted a higher total number of spermatozoa (P =0.02), increased the concentration of motile spermatozoa (P= 0.02), increased the total number of motile spermatozoa persample (P = 0.02), and enhanced the recovery rate of motilespermatozoa (P = 0.04) compared to swim-up. There was a significantimprovement in fertilization rates (P = 0.0006), in the percentageof embryos over 2-cell stage on day of transfer (P = 0.004),and in the number of replaced embryos per transfer (P = 0.01)in the Percoll as compared to swim-up cycles. There was no significantdifference in embryo quality. We conclude, therefore, that inadvanced reproductive procedures where sperm dysfunction exists,semen preparation with Percoll should replace the swim-up technique.     

IVF treatment of moderate male factor infertility: a comparison of mini-Percoll, partial zona dissection and sub-zonal sperm insertion techniques

In this study we examined various techniques of in-vitro fertilization(IVF) for treating couples in whom the male had subnormal semenparameters. We compared two sperm preparation methods (mini-Percolland conventional swim-up) for efficiency of recovery after preparationand for fertilization rates after IVF, and compared the suitabilityof partial zona dissection (PZD) and sub-zonal sperm insertion(SUZI) to patients with different types of male factor infertility.The mini-Percoll technique allowed the recovery of significantlymore motile spermatozoa from the same semen sample comparedto the swim-up method. More oocytes were fertilized after spermatozoawere prepared by the mini-Percoll technique. An increased numberof spermatozoa recovered from an ejaculate led to an improvementin the quality of spermatozoa in the insemination droplet. Subsequently,when using the PZD technique, the fertilization rate increasedwhen there was a higher number of spermatozoa in the patient'sejaculate. When comparing the two micromanipulation techniques,SUZI provided patients with oligoasthenzoo-spermia (i.e. <10 x 10⁶ spermatozoa/ml and 40% motility) with a higher chanceof obtaining 2-pronculeate eggs.     

Optimizing sensitivity of the human sperm motility assay for embryo toxicity testing

The human sperm motility assay was used as a measure of quality control in the IVF laboratory. The effects of albumin supplementation and incubation time on the sensitivity of the human sperm motility assay were investigated. The assay was also compared with mouse embryo development. The human sperm motility assay and mouse embryo development assays were performed on 25 items commonly used in IVF laboratories. Sperm motility assay was conducted after 2, 4, 6, 8, 24 and 48 h incubation intervals under standard embryology conditions. A calculated sperm motility index value <0.75 was used to indicate sperm toxicity. It was found that optimum sensitivity (P < 0.01) of the human sperm motility assay was attained in the absence of albumin after 4, 8 and 48 h incubation periods. Items identified to be sperm toxic within 8 h by the human sperm motility assay were considered to be of clinical significance due to the close concordance of these results with mouse embryo development.     

Pentoxifylline induces hyperactivation and acrosome reaction in spermatozoa of golden hamsters: changes in motility kinematics

Pentoxifylline (PF) is used to enhance motility of spermatozoa from infertile human subjects. We have previously shown that 0.45 mM PF improved capacitation of spermatozoa and fertilization of oocytes in vitro in hamsters. The present study was carried out to assess PF- induced changes in motility kinematics of hamster spermatozoa by a computer-aided sperm analyser (CASA) and determine the timing of onset of hyperactivation (HA) and acrosome reaction (AR) in PF-treated spermatozoa. Motility kinematics were analysed by CASA for 0-8 h in the absence or presence of 0.45 mM PF in Tyrode's medium supplemented with lactate, pyruvate and polyvinyl alcohol (TLP-PVA) or in TLP-PVA with bovine serum albumin (TALP-PVA). Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Both in TALP-PVA and TLP-PVA, PF markedly increased SMI, especially the quality of motility (P < 0.02) by 2-3 h which was sustained up to 6 h. The motility kinematic data of PF-treated spermatozoa in TALP-PVA showed that average path velocity, curvilinear velocity and amplitude of lateral head displacement significantly (P < 0.05) increased as early as 2 h, with the expected decrease in straightness (STR) and linearity (LIN). Similar changes were also observed with PF-treated spermatozoa in TLP-PVA. Moreover, the percentage of hyperactivated spermatozoa in PF-treated samples was significantly (P < 0.001) higher than the untreated control at 2 h. To determine whether PF could induce AR, independent of bovine serum albumin, quantitative AR was assessed by observing the presence or absence of acrosomal cap on viable spermatozoa. PF significantly (P < 0.001) increased the percentage of AR as early as 2 h, reaching maximum at 4 h both in TALP-PVA (P < 0.05) and in TLP-PVA (P < 0.001). These results show that, in hamsters, PF induces early onset (by 2 h) of HA and AR and increases the proportion of spermatozoa undergoing physiological maturation.      

Hyper-osmotic condition enhances protein tyrosine phosphorylation and zona pellucida binding capacity of human sperm

BACKGROUND: The aim of this study was to determine the effect of culture medium osmolality, in the range known to occur in the male and female reproductive tracts, on human sperm tyrosine phosphorylation and sperm-zona pellucida (ZP) interaction in vitro. METHODS: Motile sperm (2x10(6)), selected by swim-up from semen of normozoospermic men with normal sperm-ZP binding, were incubated with or without four oocytes in 1 ml human tubal fluid (HTF) medium with different osmolalities (150, 200, 280, 350, 400 mOsm/kg) adjusted by variation of the NaCl concentration. After 2 h incubation, the number of sperm bound to the four ZP was examined, sperm motility and velocities were assessed by Hamilton-Thorn Motility Analyzer (IVOS 10) and sperm tyrosine phosphorylation was assessed by both western immunoblotting and immunofluorescence with an anti-phosphotyrosine monoclonal antibody (PY20). The effect of hyper-osmolality (400 mOsm/kg) on the ZP-induced acrosome reaction (AR) was also determined. RESULTS: Incubation of human sperm in hyper-osmotic medium significantly increased tyrosine phosphorylation and the number of sperm bound to the ZP. In contrast, hypo-osmotic medium significantly decreased both tyrosine phosphorylation and sperm-ZP binding. Medium with high osmolality (400 mOsm/kg) significantly reduced the ZP-induced AR. Both hypo- and hyper-osmotic media significantly decreased average sperm percentage progressive motility and velocities. CONCLUSION: Incubation of human sperm in hyper-osmotic media was associated with significantly increased tyrosine phosphorylation and ZP-binding ability but severely reduced the ZP-induced AR.     

Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound

Spermatozoa-zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0. 14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0. 32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.     

Selection and characterization of human acrosome reacted spermatozoa

In order to study the characteristics of human acrosome reactedspermatozoa, we developed a method to select them. Spermatozoa,obtained from 20 fertile volunteers, were selected by usingGB24 antibody fixed on magnetic immunobeads, after acrosomereaction induced by 50% follicular fluid. Bead-bound spermatozoawere then detached using sheep anti-mouse IGG F(ab')2 antibody.This method allowed recovery of 170 ± 48x10³ spermatozoa(n = 20), free of GB24 antibody, as assessed by incubation withFITC-rabbit anti-mouse antibody. The percentage of acrosomereacted spermatozoa in the selected population was 88 ±3% versus 32 ± 6% in the whole sperm population. Concerningsperm morphology, the percentage of head abnormalities was lowered(15 ± 3% versus 20 ± 3%). The motility of selectedspermatozoa was dramatically reduced (7 ± 3% versus 53± 7% in the whole population) despite no difference inviability (84 ± 3% versus 80 ± 4%). However, theviability after an 18 h incubation was very low (1 ±0.5% versus 46 ± 5%). These results show that acrosomereaction occurs in the most morphologically normal spermatozoaand is followed by a loss in motility and a decrease in longevity.