Andrology: Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro

It is believed that during the process of human fertilization,acrosome-intact spermatozoa bind to the surface of the zonapellucida which triggers the acrosome reaction and the enzymesreleased facilitate sperm penetration through the zona pellucida.We describe here reduced frequency of the acrosome reactionon the zona pellucida as a cause of infertility in 10 coupleswith long durations of infertility (average 6 years) and low(<15%, n= 3) or zero (n= 7) fertilization rates in vitro.Sperm concentration, motility, velocity (Hamilton-Thorn), morphologyand DNA normality were within the normal range in all the patients.Electron microscopy of spermatozoa did not reveal any specificultrastructural defects. All couples were negative for antispermantibodies by immunobead tests. Oocytes from other patientswhich failed to fertilize in in-vitro fertilization and normaldonor spermatozoa were used as controls for sperm-zona pellucidabinding and penetration experiments. Acrosome status of spermatozoabound to the zona pellucida was assessed with a fluorescentlectin and electron microscopy. The mean number of spermatozoabound to the zona pellucida was not significantly differentbetween patients and controls. However, the acrosome reactionof spermatozoa bound to the zona pellucida after 2 h incubationwas significantly lower (P< 0.001) in the patients (mean5%, range 0–16) than in the controls (mean 68%, range44–96). No zona pellucida (out of 40) was penetrated bypatient spermatozoa whereas most (39/40) zonae were penetratedby control spermatozoa (average 27 spermatozoa/four zonae pellucidae).The spontaneous acrosome reaction of spermatozoa in inseminationmedium was not different between patients (4%) and controls(3%), the acrosome reaction induced by calcium ionophore waslow (21 and 43% respectively) in six of the eight patients examined.In conclusion, these patients have spermatozoa with a disorderof the zona pellucida-induced acrosome reaction that resultsin failure of sperm-zona pellucida penetration and explainstheir infertility.     

Zona-opening of hamster oocytes: a comparative study using macro- and micro-manipulation methods

The present study compared a new macro-manipulation techniquefor zona-opening of hamster oocytes with existing micro-manipulationtechniques. In experiment I the zona pellucida of hamster oocyteswas partially opened with a 32 gauge steel needle (macro-manipulation)and these oocytes were then co-incubated with human spermatozoaat 36.5°C in 5% CO₂ in air for 3.5 h. Zona-free and zona-intactoocytes similarly inseminated served as controls. Of 113 oocytes,30 (26%) lysed following zona-opening with the macro technique.The sperm penetration rates of zona-opened and zona-free oocyteswere 37% (31/83) and 60% (49/81) respectively (P<0.01), with12 and 28% oocytes respectively showing polyspermia (P<0.05).No sperm penetration occurred in zona-intact oocytes. In experimentII the zona pellucida of hamster oocytes was partially openedby macro-or micro-manipulation techniques before the oocyteswere coincubated with human spermatozoa as in experiment I.Nine of 156 (6%) macro-manipulated and 11 of 133 (8%) micro-manipulatedoocytes lysed following zona-opening. Of 147 remaining macro-manipulatedand 122 remaining micro-manipulated oocytes, 80 (55%) and 79(64%) respectively were penetrated (P>0.05), with 30 and37% respectively showing polyspermia (P>0.05). These resultsdemonstrate that macro-manipulation results in similar penetrationand polyspermia rates as compared to conventional micro-manipulation.     

A Mono-Percoll separation technique improves sperm recovery of normal and male factor specimens when compared with the swim-up technique

The purpose of this study was to determine the effects of asimplified 80% Mono-Percoll sperm separation procedure on bothnormal and male factor semen samples compared with the standardswim-up technique. The parameters examined include sperm concentration,motility and morphology, total motile functional spermatozoaand percentage recovery. Normal patients demonstrated enhancedsperm parameters with the Mono-Percoll compared with the swim-uptechnique for concentration (67x10⁶ versus 42x10⁶/ml, P <0.001), motility (66 versus 59%, P < 0.001), morphology (56versus 49%, P < 0.005) and percentage recovery (60 versus42%, P < 0.005). Male factor patients showed enhanced spermparameters with the Mono-Percoll procedure compared with theswim-up technique for motility (53 versus 42%, P < 0.05)and percentage recovery (54 versus 29%, P < 0.005), withno significant difference in concentration and morphology. Insummary, the Mono-Percoll sperm recovery procedure is significantlybetter than the swim-up technique for male factor patients andpatients with normal sperm parameters.     

The effect of smoking and varicocele on human sperm acrosin activity and acrosome reaction

Smoking and varicocele are frequent findings in the medicalhistory and physical examination of patients attending and rologicaloutpatient departments. However, data about their influenceon human semen parameters, such as sperm concentration and motility,are contradictory. Therefore, the purpose of this study wasto examine sperm function (acrosin activity and induction ofthe acrosome reaction) in smokers (n = 130) and varicocele patients(n = 30)compared with normal fertile donors (n = 20). The acrosomereaction was detected by triple staining after 3 h ofincubationat 37°C, followed by treatment with 0.1%dimethyl sulphoxide(spontaneous acrosome reaction) and 10 µM calcium ionophoreA23187 (induced acrosome reaction) for 1 h at 37°C. Acrosinactivity was measured by gelatinolysis. The diameters aroundthe sperm heads after gelatinolysis and the percentages of spermatozoashowinghalo formations were evaluated. The inducibility of theacrosome reaction was significantly lower in semen samples fromsmokers than in those from the fertile group (7.1 ±3.2versus 11.2 ± 4.0%, P < 0.01), whereas no statisticallysignificant difference was demonstrated in spermatozoa frompatients with varicocele (9.3 ± 4.3%). Both the percentagesof spermatozoa with halo formation (53.3 ±20.0 versus76.6 ± 13.6%, P < 0.05) and the halo diameters (16.1± 6.6 versus 31.0 ± 14.5 urn, P < 0.001) weresignificantly lower in the varicocele group than in thesamplesfrom fertile men. These data suggest that smoking and varicoceleaffect sperm function, and that the standard semen parametersalone are insufficient to evaluate the influence of both factorson human male fertility.     

Andrology: An auto-controlled study in in-vitro fertilization reveals the benefit of Percoll centrifugation to swim-up in the preparation of poor-quality semen

The efficiency of spermatozoa prepared by swim-up or by Percollcentrifugation was assessed in an in-vitro fertilization programmeon 71 semen samples of a well-defined quality [total numberof type A (WHO criteria) motile spermatozoa]: category I (n= 21) with > 100 x 10⁶, II (n = 31) with 15–100 x 10⁶,III (n = 11) with 5–15 x 10⁶ and IV (n = 8) with <5 x 10⁶ type A motile spermatozoa. Oocytes were inseminated4 h after oocyte retrieval, alternately with spermatozoa derivedfrom swim-up and Percoll preparation. Both selection proceduresresulted in a significantly higher (P < 0.001) percentagemotility as compared to fresh semen. For low-quality samples(III and IV), however, swim-up was more effective in selectinghighly motile (P = 0.004) and morphologically normal spermatozoa(P < 0.05). For high-quality samples, this difference mighthave been masked by introducing a swim-up step to remove Percollparticles. Regardless of the initial sperm quality, the meanfertilization rate was significantly higher (P = 0.003) whenPercoll-treated spermatozoa were used for insemination (51.3versus 37.8%). For semen of groups I and II, no difference infertilization capacity was observed according to the sperm preparationmethod. Despite the lower percentage motility and normal morphologyfor the Percoll compared to the swim-up treatment in groupsIII and IV, fertilizing capacity was significantly (P < 0.001)in favour of this selection method (65.3 versus 26.5% in groupIII, 47.6 versus 11.6% in group IV). Based on these results,it may be concluded that a subgroup of patients exhibiting poorsemen quality can benefit from Percoll semen preparation interms of improved fertilizing capacity.     

Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization

Human and bovine cervical mucus penetration tests (n = 57) wereperformed preceding IVF to test their prognostic value as spermfunction tests for IVF. This evaluation also induded resultsfrom conventional semen analysis and from a computerized spermanalysis system. The bovine cervical mucus penetration testwas shown to be at least as valuable as the human cervical mucuspenetration test in evaluating sperm function. The migrationdistance of the vanguard sperm (P < 0.001) and the spermdensity at a fixed migration distance in the mucus column (P< 0.05) correlated most closely with the IVF results. A clearparallelism with the out come of the ‘swim up’ techniquewas also found. Of the sperm parameters examined, only spermmotility In the ejaculate (P < 0.05) correlated significantlywith the results of IVF. It is concluded that the outcome ofa bovine cervical mucus penetration test depends on the samesperm functions as re quired for IVF. Therefore, this test maybe of predictive value in an IVF programme.     

Deterioration of sperm quality in young healthy Belgian men

We have retrospectively analysed the sperm characteristics of416 consecutive healthy young men who presented themselves inthe past 19 years as candidate sperm donors. Ejaculate volumeincreased slightly (P = 0.067), and average sperm concentrationdecreased (P = 0.035) by 12.4xlO6ml over the observation period,so that sperm count per ejaculate remained unchanged (P = 0.91).In contrast, sperm morphology (r = –0.23, P < 0.0001),rapid progressive motility (r = –0.42, P < 0.0001)and total motility (r = –0.33, P < 0.0001) presentedan important and time-related decrease. When a quadratic modelwas used rather than a linear one to analyse the data on rapidprogressive motility, there appeared to have been no furtherdecline since 1990. The average proportion of spermatozoa withnormal morphology decreased from 39.2% in the period 1977–1980to 26.6% in 1990–1995 (P < 0.0001), and the mean percentageof spermatozoa with rapid progressive motility decreased from52.7 to 31.7% (P < 0.0001). The percentage of candidate donorswith sperm characteristics below the 5th percentile cut-offvalue of a normal fertile population increased from 13 to 54%during the observation period (P < 0.0001). Since the techniqueof semen analysis has remained essentially unchanged in-so-faras has been practically possible, as has the method of recruitmentof candidate sperm donors, the observed deterioration of spermcharacteristics is considered to reflect degeneration of spermproduction among men aged between 20 and 40 years.     

Semen parameters and sperm morphology in men in unexplained recurrent spontaneous abortion, before and during a 3 year follow-up period

To investigate the role of the ‘male factor’ inthe patho-genesis of recurrent spontaneous abortion (RSA), especiallysperm morphology abnormalities, 120 previously selected coupleswith unexplained RSA were studied for sperm parameters retrospectivelyand prospectively. The patients were subdivided into three subgroups,depending on their reproductive outcome during the 3 years offollow-up study: (i) 48 RSA couples who achieved a successfulpregnancy; (ii) 39 RSA couples who experienced further abortions;and (iii) 33 RSA couples who experienced infertility duringthe follow-up period. A semen analysis was performed twice atthe time of inclusion in the study, and twice again during the3 year follow-up period. No significant differences in semenparameters were observed between the RSA males and fertile controls.Instead, significant differences were observed between the groupof RSA couples who experienced infertility during the follow-upand the other two groups (RSA couples who achieved successfulpregnancy and RSA couples who experienced miscarriages and nolive birth during the follow-up) for sperm concentration (P< 0.01 and P < 0.01 respectively), sperm motility (P <0.01 and P < 0.01 respectively) and sperm morphology abnormalities(P < 0.01 and P < 0.01 respectively). Sperm morphologyabnormalities do not seem to be involved in determining RSA;instead, they are an aetiological factor in determining infertilityin patients, along with the other semen parameters, in the RSAcouple‘s subsequent reproductive life. Semen analysisis an important test in the clinical management of RSA couples.     

Fertilization and early embryology: Human follicular fluid inhibits the binding of human spermatozoa to zona pellucidain vitro

The effect of human follicular fluid on human zona pellucidabinding of spermatozoa was investigated using the hemizona bindingassay (HZA). This effect was compared to that of progesterone,a known component of human follicular fluid. Exposure of spermatozoato 25% pooled human follicular fluid for 1 h significantly reducedthe number of spermatozoa bound to zona pellucida when comparedto those without human follicular fluid treatment (149.1 ±30.7 versus 177.1 ± 33.8, P 0.01). The same phenomenonwas observed after 3 h of treatment The corresponding numbersof bound spermatozoa were 140.4 ± 19.1 and 200.2 ±23.4 (P 0.0001). Progesterone (1.0µg/ml) stimulated thezona pellucida-binding capacity of spermatozoa significantlyunder the same conditions (P 0.01). The numbers of bound spermatozoaafter 1 and 3 h progesterone treatment were 235.5 ± 44.7(control, 168.1 ± 32.9) and 204.3 ± 27.4 (control,162.3 ± 20.1) respectively. HZA comparing the effectsof human follicular fluid and progesterone at concentrationsequivalent to those found in human follicular fluid using matchinghemizonae confirmed the inhibitory effect of human follicularfluid on sperm binding to zona pellucida (80.4 ± 28.4versus 149.8 ± 35.2, P 0.05). This inhibitory effectwas also found in another eight individual human follicularfluid samples. Both human follicular fluid and progesteronedid not affect the motility and viability of the treated spermatozoawhen compared to the controls with the same incubation period.Although more spermatozoa underwent the acrosome reaction after1 and 3 h of human follicular fluid treatment than in the control,the extent was comparable to those after progesterone treatmentThese results suggested that human follicular fluid inhibitedthe zona pellucida-binding capacity of spermatozoa in vitro.This inhibitory effect of human follicular fluid was not mediatedby progesterone, and did not result from the effects of humanfollicular fluid on sperm motility, viability and acrosome reaction.     

Fertilization and early embryology: Does zona pellucida thickness influence the fertilization rate?

In human in-vitro fertilization (IVF), the cumulus oophorusis routinely removed to assess fertilization and hence the thicknessof the zona pellucida is measurable. This study aimed to measurethe thickness of the zona pellucida and to assess its influenceon fertilization rate in IVF programmes. The zona pellucidathickness varies from 10 to 31 µm with a mean of 17.5µm. One-way analysis of variance revealed that in IVFtrials performed with normal semen, the zona pellucida of fertilizedoocytes (16.6 ± 3.2 µm) was significantly thinnerthan the zona pellucida of unfertilized oocytes (18.9 ±4.0 µm; P < 0.001). As measured on micro-injected oocytes,the zona pellucida thickness did not change between ovulationand 16–20 h after fertilization. Zona pellucida thicknesswas not related to ooplasm diameter. In conclusion, zona pellucidathickness appears to be an additional factor that should betaken into account when interpreting the fertilization rate.Zona pellucida thickness influences sperm penetration, evenwhen the spermatozoa are considered normal. From a clinicalpoint of view, a thick zona pellucida (22 µm) could bean indicator for the use of micro-injection procedures.     

Sperm selection by PD-10 sephadex columns: comparison with SpermPrep filtration and Percoll centrifugation

The efficacy of a disposable, prepacked column (PD-10) containingSephadex G-25, to select motile spermatozoa, was compared withanother column for sperm filtration (SpermPrep) and centrifugationthrough Percoll gradients. Aliquots of washed sperm suspensionswere processed by the three techniques. The number of motilecells and the proportion of total spermatozoa selected was similarfor all methods. Recovery of spermatozoa showing optimal movementwas 145.9 ± 30% (mean ± SEM) with PD-10 columnsand 131.9 ± 32% with Percoll, both significantly higherthan SpermPrep (71.9 ± 11%; P < 0.05). The straightline velocity of motile cells was lower in samples processedby SpermPrep (29.3 ± 2 µm/s) compared to both PD-10(34.7 ± 1 µm/s) and Percoll (34.9 ± 2 µm/s;P = 0.07). When whole semen was processed, total sperm recoverywith PD-10 was 61.7 ± 8% versus 47.7 ± 7% withPercoll (P < 0.001). Percoll centrifugation improved thepercentage of morphologically normal spermatozoa more than PD-10.Similar proportions of motile spermatozoa and cells with optimalmotility were obtained by both methods. We conclude that PD-10filtration columns can be used to prepare semen in the laboratoryas a practical alternative to other methods.     

Andrology: Co-culture with Vero cell monolayer maintains the motility of asthenozoospermic semen samples

The clinical effectiveness of co-culture with Vero (Green monkeykidney) cell monolayer in maintaining the motility and viabilityof fresh asthenozoospermic semen (18 samples) and frozen–thawedsemen with poor motility (motility fraction <50%) (15 samples)in a 24-h period was evaluated. Co-culture with Vero cell monolayerin human tubal fluid (HTF) medium for 24 h resulted in a statisticallybetter maintenance of motility percentage (P < 0.005), meanamplitude of lateral head displacement (ALH) (P < 0.005),and mean track speed (VCL) (P < 0.05) than culture in HTFmedium alone. However, these motility parameters (motility percentage,ALH, VCL) declined soon after removal of spermatozoa from themonolayer. Co-culture with Vero cell monolayer also maintainedthe viability percentage of these sperm samples (52% of theoriginal value) after the 24-h period compared with culturein HTF medium alone (22% of the original) (52% versus 22%, P< 0.05).It is concluded that Vero cell monolayer is effectivein the maintenance of motility and viability of asthenozoospermicsemen or frozen–thawed semen with poor motility.This co-culturesystem may be beneficial in enhancing the in-vitro performanceof asthenozoospermic semen samples in the practice of assistedreproductive technology.However, its safety needs further evaluation.     

Fertilization and early embryology: Prolonged sperm-oocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate

A reduced time interval of oocyte exposure to spermatozoa wasinvestigated to assess whether it could enhance oocyte developmentand improve embryo viability, especially in cases of male factorinfertility. A total of 167 patients were included in a prospectiverandomized study. They were randomly allocated to two majorstudy groups, A (n = 85) and B (control group; n = 82). Theoocytes from group A patients were exposed to spermatozoa foronly 1 h; those from group B were exposed for 16 h. The twostudy groups were then subdivided according to semen qualityfor further analysis of the results. Significantly higher percentageswere obtained in group A than in group B in terms of the fertilizationrate (74 versus 68%, P < 0.025), cleavage rate (53 versus41%, P < 0.005), pregnancy rate (27 versus 12%, P < 0.05)and implantation rate (11 versus 6%, P < 0.05). In addition,an increased fertilization rate was achieved in oocytes exposedto male factor spermatozoa for only 1 h compared with the conventionalincubation period (78 versus 65%, P < 0.01). Advanced cellularstages (55 versus 41%, P < 0.02) and higher implantationrates (13 versus 4%, P < 0.05) were attained in the subgroupwhose oocytes were exposed to normal spermatozoa for 1 h comparedwith the male factor spermatozoa with the standard culture interval.The higher fertilization rates, enhanced embryo developmentand viability achieved in group A indicate that prolonged exposureof oocytes to high concentrations of spermatozoa is detrimental,decreasing sperm-oocyte interaction and subsequent embryo implantation,particularly in male factor patients.     

Andrology: Density adjustment of software settings minimizes bias in automated sperm motility estimation

To minimize overestimation of motility, it is recommended thatfresh semen be diluted with seminal plasma prior to automatedanalysis. However, for glycerolated or cryo-preserved sementhis is impractical, and alternative methods are needed to minimizeautomated motility bias. In the present study, the proportionof motile spermatozoa was determined in fresh, diluted and cryopreservedsemen (n = 25 ejaculates) using visual and automated methods.The effect of software settings on motility was investigatedby assessing samples at a range of modified settings. At standardsettings, automated motility was biased in fresh semen (+7.2%)after dilution with cryopreservative (–2.9%) and aftercryopreservation (–7.8%) (P < 0.0001 versus visual).Automated motility was inversely related to the minimum numberof frames for motility sampling (P < 0.0001), with mean estimatesof 41.0, 46.1, 52.0 and 58.2% generated at settings of 8, 4,2 and 1 frame(s) respectively (n = 15 fresh, diluted and cryopreservedsamples). Based on an arbitrary ordinal scale, a method wasdeveloped whereby motility sampling was adjusted prior to analysisaccording to sperm density. Analysis of an independent set ofsemen samples with density-adjusted software settings reducedbias in automated estimates (n = 30) before and after freezing(P < 0.0001). In addition, bias was no longer related tosperm density. In conclusion, modification of software settingsis an effective alternative to dilution to minimize bias inautomated motility estimates in fresh, diluted and cryopreservedhuman semen.     

Relationships between motility parameters, morphology and acrosomal status of human spermatozoa

A total of 130 semen samples were examined for motility (bycomputer-assisted sperm analysis), morphology and acrosomalstatus. A high positive correlation was found between percentagesof normal forms and progressive motility in the whole semen(r = 0.539, P < 0.0001) as well as in the Percoll fraction(r = 0.702, P < 0.0001). Among the specific abnormalities,acrosome defects were most highly correlated with progressivemotility (r = –0.492, P < 0.0001, in the Percoll fraction).The percentage of total spontaneously acrosome-reacted spermatozoain the Percoll fraction was negatively correlated with the progressivemotility (r = –0.499, P < 0.0001) and with the percentageof normal forms (r = –0.430, P < 0.0001). Surprisingly,the percentage of total spontaneously acrosome-reacted spermatozoawas poorly linked with head abnormalities but displayed significantpositive correlations with the percentages of bent tails (r= 0359, P < 0.0001) and of coiled tails (r = 0371, P <0.0001). These data suggest that sperm defects are often linkedtogether, reflecting spermiogenesis and/or epididymal dysfunctions.     

The possible meaning of transferrin and its soluble receptors in seminal plasma as markers of the seminiferous epithelium

Transferrin (Tf) and soluble transferrin receptors (S-Tf-R)were measured by enzyme immunoassay in seminal plasma of 130semen samples. The mean concentration of S-Tf-R in cases withnormozoospermia was 10.4 IU/ml (95% confidence interval: 9.5–11.3)and it was significantly lower in patients with oligozoospermia(6.6, 95% CI: 5.8–7.5, P < 0.001), asthenozoospermia(8.5, 95% CI: 5.5–10.7, P < 0.05), azoospermia of primarytesticular origin (7.9, 95% CI: 6.1–9.6, P < 0.05)and post-vasectomy samples (5.9, 95% CI: 5.4–6.9, P<0.001). The concentration of S-Tf-R in post-vasectomy sampleswas lower than that in patients with azoospermia of primarytesticular origin (P< 0.05; positive likelihood ratio= 7at value of 8.3 IU/ml). S-Tf-R was positively correlated withmotile sperm concentration (r= 0.50, P< 0.0001), percentagemotility (r= 0.38, P< 0.001), percentage of normal forms(r = 0.43, P < 0.001), sperm linear velocity (r= 0.42, P<0.001), and ATP concentration (r= 0.67, P< 0.0001). Folliclestimulating hormone (FSH) was found to be negatively correlatedwith the concentrations of both Tf (r= -0.31, P< 0.05) andof S-Tf-R (r= -0.45, P< 0.01). The mean concentration ofTf in seminal plasma was 50.4 µg/ml (35.9–67.2)in samples with normozoospermia (n= 22), and the concentrationwas significantly lower in patients with oligozoospermia (P<0.05), azoospermia of testicular origin (P < 0.001), andpost-vasectomy samples (P< 0.001). Seminal Tf was correlatedwith motile sperm concentration (r = 0.36, P< 0.001), percentageof motile spermatozoa (r= 0.25, P < 0.05), linear velocity(r= 0.24, P< 0.05) and ATP concentration (r= 0.44, P<0.001). The concentration of Tf was positively correlated withthat of S-Tf-R both in cases with spermatozoa present (r= 0.66,P < 0.001), and in cases with azoospermia of testicular origin(r = 0.51, P < 0.05) but not in vasectomy cases. It is concludedthat S-Tf-R in seminal plasma is a marker of spermatogenesisand may give information on the presence or absence of spermatogeneticcells in cases with azoospermia. Further investigations areneeded to assess its usefulness for clinical practice.     

Andrology: The use of hemizona assay in the evaluation of the optimal sperm preparation technique

The objective of the study was to evaluate the benefit of differentsperm preparation methods by using the hemizona assay. A totalof 58 men admitted to the male infertility clinic for evaluationwere tested by routine semen analysis and hemizona assay. Fivedifferent techniques (swim-up, TEST-yolk buffer, Percoll, pentoxifyllineand progesterone) were used for preparation of sperm suspensions.The effect of these treatments on the sperm-binding capacityusing the hemizona assay was assessed. The routine swim-up preparationwas used as the reference method. Of the four preparation methods,only the TEST-yolk buffer and pentoxifylline exhibited an overallstatistically significant improvement in sperm-binding capacityin comparison with the swim-up preparation method (P = 0.01and 0.001 respectively). Following preparation with Percolland progesterone there was no change in the mean value of bindingcapacity, compared with swim-up. However, examination of theeffect of the four treatments on each specimen individuallyyielded a diversity in the response, e.g. having the capabilityto enhance, damage or be ineffective in sperm binding capacity.The results support the conclusion that in-vitro sperm preparationmethods can affect sperm binding to the zona pellucida. Sincethere is a diversity in the response of sperm samples to differenttreatments, the hemizona assay can be used in selecting theoptimal sperm preparation method prior to its use for assistedreproductive techniques. This is advocated mainly for the ‘malefactor’ group.     

Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen

Peroxidative damage induced by reactive oxygen species (ROS)has been proposed as one of the major causes of defective spermfunction. The ROS detected in semen reflect an imbalance betweenROS generation and degradation. The objective of the presentstudy was to investigate the relationship between the oxidativeand anti-oxidative potential in semen of infertile patientsand healthy donors. Specimens were obtained from 28 patientsand 18 healthy donors (controls). A conventional spermiogram,measurement of luminol-chemiluminescence (CL) in washed semen,and high performance liquid chromatography determination ofascorbic acid and urate concentrations in seminal plasma wereperformed. Oligozoospermic patients exhibited higher CL signalsthan controls (P < 0.001). Normozoospermic patients showedlower ascorbic acid (mean ± SE: 491 ± 46 µM,P < 0.04) and urate concentrations (320 ± 22 µM,P < 0.009) than controls (612 ± 35 and 426 ±26 µM respectively). Seminal plasma ascorbic acid wasnegatively correlated with the CL signals (P < 0.0006) andpositively correlated with the percentage of spermatozoa withnormal morphology (P < 0.006). This is the first report ofa correlation between the anti-oxidant ascorbic acid in seminalplasma and ROS generation in human semen. Furthermore, the reducedascorbic acid/urate concentrations found in semen of normozoospermicpatients might be indicative of a reduced anti-oxidative protection.     

The influence of sera, follicular fluids and seminal plasma on human sperm-zona pellucida binding

The purpose of this study was to assess the influence of male, female and fetal cord sera, follicular fluid, and seminal plasma on human sperm-zona pellucida binding, using the hemizona assay. Steroids, gonadotrophins, growth hormone and prolactin concentrations in follicular fluid and sera were also analysed. The influence of follicular fluid (10 or 50%, v/v) and sera (10%) on sperm-zona pellucida binding was investigated by supplementing the sperm processing medium as well as the sperm-hemizona incubation medium. Different seminal plasma concentrations (1 or 10%) were added to the sperm-hemizona incubation medium. Supplementation with 10% day 3 donor serum was used as a control throughout experimentation. Although supplementation with male sera and fetal cord serum exerted a stimulatory effect (36 and 90% respectively; P < 0.029) on sperm-zona pellucida binding, hemizona indices obtained with addition of male sera, fetal cord serum and sera obtained from sub-fertile in-vitro fertilization (IVF) patients on day 12 of their menstrual cycle did not differ significantly (P > 0.05). Final progesterone concentrations in sperm-zona pellucida incubation media (10% follicular fluid supplementation), which ranged from 0.788 to 3.85 microg/ml, enhanced sperm binding to the zonae by >100% (P < 0.02). The utilization of follicular fluid (10%) as a natural physiological stimulus to enhance sperm-zona pellucida binding in an IVF setting is recommended. The presence of seminal plasma in the spermzona pellucida incubation media showed no beneficial effect on the binding ability of sperm, and can be viewed as an unfavourable substance in the proximity of the oocyte.