The effects of follicular fluid from patients with different indications for IVF treatment on the binding of human spermatozoa to the zona pellucida

This study compared the effects of human follicular fluid (hFF) from women with endometriosis, tubal factor and male factor on the zona binding capacity of human spermatozoa. Samples of hFF were collected from 30 patients, 10 patients for each of the indications of infertility, at the time of oocyte retrieval in an in-vitro fertilization/embryo transfer programme. The hemizona binding assay (HZA) was used to assess the effect of these hFF on the zona binding potential of human spermatozoa. The mean numbers of spermatozoa bound to the zona pellucida after treating the spermatozoa with hFF from endometriosis, tubal factor and male factor were 90.5 +/- 20.9, 108.9 +/- 22.3 and 101.2 +/- 13.4 respectively. These were significantly lower than their corresponding controls, the spermatozoa of which were incubated with Earle's balanced salt solution (endometriosis 238.7 +/- 34.7; tubal factor 210.8 +/- 41.6; male factor 205.4 +/- 26.3; P <0.002). The hemizona binding index (HZI) was similar between male factor samples (52.0 +/- 6.7) and tubal factor samples (53.8 +/- 4.2). Spermatozoa incubated with hFF from endometriosis patients (36.0 +/- 4.1) had an HZI that was significantly lower than those treated with hFF from tubal factor patients (P <0.01). Probably due to small sample size, the differences in HZI between endometriosis samples and male factor samples did not reach statistical significance (P = 0.076). These data suggest that there was a stronger sperm-zona binding inhibitory effect of hFF from patients with endometriosis than from those without the disease.      

Effect of antisperm antibodies present in human follicular fluid upon the acrosome reaction and sperm-zona pellucida interaction

PROBLEM: To determine the ability of IgGs isolated from follicular fluids (hFFIgGs) to induce the acrosome reaction (AR) in human spermatozoa and to inhibit sperm-zona pellucida (ZP) interaction. METHOD OF STUDY: Incubation of capacitated spermatozoa with hFFIgGs (n = 40) and assessment of their effect on the AR or hemizona (HZ) assay in a condition that allows sperm-ZP interaction, avoiding acrosomal exocytosis. RESULTS: hFFIgGs from different women varied in their ability of inducing the AR. Those hFFIgGs with the highest AR-inducing capacity evoked the exocytotic response in most of the different sperm donors tested [high Induction Frequency (IF)]. Some of these antibodies were also able of inhibiting sperm binding to ZP [low HZ Index (HZI)]. A significant correlation was found between the IF and the HZI for each hFFIgG. CONCLUSIONS: Human follicular fluid contains antibodies capable of inducing the AR and inhibiting sperm-ZP binding, suggesting that they could be directed towards ZP receptors. hFFIgGs would constitute a tool for the identification of sperm entities involved in fertilization.     

Anti-ZP3 Antibodies Binding to the Human Zona Pellucida: Effect of Oocyte-Storage Conditions

PROBLEM: The zona pellucida protein 3 (ZP3) is a zona pellucida (ZP) glycoprotein crucially involved in fertilization. ZP3 plays a major role in sperm binding and induction of the acrosome reaction. In different species, ZP3 proteins differ in their primary structure as derived from cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertilization outcome under in vitro conditions. METHOD: In these studies, we used antisera generated against synthetic ZP3 peptides to compare antibody binding patterns to ZP with sperm-ZP binding capacity under different HZA conditions. RESULTS: Analysis of antibody binding to hemizonae derived from metaphase II human oocytes that were used either after refrigeration at 4°C or stored in a hyperosmotic salt solution revealed a strong reaction with human ZP3. However, treatment of human oocytes using a protocol to freeze embryos with the addition of 1,2 propanediol drastically reduced binding of ZP3 antibodies to the hemizonae. Nevertheless, no significant difference of sperm binding occurred under HZA conditions when oocytes were refrigerated, salt-stored, or frozen with 1,2 propanediol. CONCLUSIONS: Our results indicate that the ZP3 protein backbone might be altered by 1,2 propanediol-treatment while the glycoprotein-receptor remains intact. We conclude that antisera against ZP3 peptides can be used as markers for the ZP3 protein backbone in human oocytes and might be useful tools for the evaluation of ZP3 protein integrity.     

Immunology: Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse

The blocking effects of complement-dependent sperm immobilizingantibodies in the sera of infertile women and monoclonal antispermantibodies against humans and mice on fertilization were investigated.The hemizona assay (HZA) and sperm penetration assay (SPA) wereused to study the inhibitory effects of sera from 22 infertilepatients positive for sperm immobilizing antibodies. Use ofthese tests allowed us to differentiate whether the antibodyblocked sperm—zona pellucida tight binding and/or spermpenetration into the ooplasm. The zona pellucida penetrationassay (ZPA) was also used to study the effects of four monoclonalantibodies (mAbs) on human sperm penetration into the zona pellucida.Seven mAbs against murine spermatozoa were tested for theirinhibitory effects on in-vitro fertilization (IVF) and HZA inmice. Of 22 patient sera with sperm immobilizing antibodies,21 (95.5%) inhibited HZA attachment and penetration, whereasthis did not occur in any of 13 patient sera without these antibodies.However, 19 of 22 (86.4%) patient sera with sperm immobilizingantibodies and eight of 13 (61.5%) patient sera without theseantibodies inhibited the SPA. Two (2C6, 1G12) of four mAbs againsthuman spermatozoa showed strong inhibitory effects in all theassays (HZA, ZPA and SPA). One mAb (3B10) did not inhibit HZAbut blocked ZPA and SPA. Another mAb (H6-3C4) seemed to haveno inhibitory effects on fertilization. Two (Vx 5 and Vx 8)of seven mAbs against murine spermatozoa inhibited IVF in micebut did not block mouse HZA. These findings suggest that antispermantibodies block fertilization at specific stages. Some of themmay inhibit sperm capacitation and thus prevent all processesof fertilization that follow. Some other antibodies may notaffect capacitation and sperm binding to zona pellucida butinhibit the acrosome reaction, followed by the blocking of spermpenetration through zona pellucida and ooplasm.     

Glycosidic residues involved in human sperm-zona pellucida binding in vitro

Glycosidic residues of the mammalian zona pellucida (ZP) are known to be involved in sperm binding, suggesting the presence of complementary carbohydrate binding sites on spermatozoa. However, in previous studies, in which sperm suspensions were incubated with monosaccharides, no inhibitory effect was observed. Results of studies in which sperm were treated shortly after swim-up suggest that the use of non-capacitated cells may explain the apparently conflicting results. In the present report, we studied the effect of preincubation of capacitated spermatozoa with different monosaccharides on their ability to bind to ZP. After 5 h under capacitating conditions, spermatozoa were incubated in medium with or without a monosaccharide, resuspended in fresh medium and used for hemizona (HZ) binding assay. When ZH were incubated with spermatozoa treated with N-acetyl-D- glucosamine, D-mannose, D-fucose, L-fucose or D-galactose, a significant decrease in the number of spermatozoa bound was observed (level of inhibition: 62, 58, 82, 68 and 48% respectively) while treatment of spermatozoa with D-glucose produced no inhibition. Sugar treatment neither altered sperm motility nor the rate of acrosome reaction. These results suggest that N-acetylglucosamine, mannose, fucose and galactose residues are involved in human sperm-zona pellucida binding in vitro.      

The effect of seminal plasma on human sperm-zona pellucida binding

The aim of this study was to determine the effect of differentconcentrations of seminal plasma (SP) in insemination mediumon human sperm-zona pellucida (ZP) binding.The effects of freshSP [50%(n = 10) and 1% (n = 9)],frozen-thawed versus fresh SP[5% (n = 10)] and frozenthawed SP [5% (n = 10) and 1% (n = 9)]on sperm-ZP binding were examined using hemizona assays. Thevalidity of the hemizona assay as performed in this study wasalso examined. Relative sperm binding percentages were determinedfor respective hemizonae, and the hemizona index was calculatedfor each experimental group and compared statistically. The50% native SP inhibited sperm-ZP binding by -70% (P = 0.0051),while 1% native SP enhanced sperm-ZP binding by –350%(P =0.0051). Significantly more spermatozoa (mean ± SD,172.00± 54.12) bound to zonae in the presence of 5%frozen-thawedSP than with 5% SP that had not been frozen (mean ±SD,127.00 ± 69.18; P = 0.0431). The 1 and 5% frozen-thawedSP stimulated sperm-ZP binding by -400 and -250% respectively(P = 0.0077 and 0.0051 respectively). It is concluded that SPconcentrations found in insemination media during assisted reproductivetechniques do not inhibit but in fact enhance sperm-ZP binding.     

Species specificity of human and murine anti-ZP3 synthetic peptide antisera and use of the antibodies for localization and identification of ZP3 or ZPC domains of functional significance

The mammalian zona pellucida has an important function in the fertilization process. The zona pellucida protein 3 (ZP3 or ZPC) is the ligand for primary sperm binding and induces the acrosome reaction. In various species, ZP3 primary structures are highly conserved as revealed by cDNA cloning. The objective of these studies was to localize ZP3 protein using antisera generated against defined synthetic peptides that are specific for mouse or for human ZP3. Immunohistochemistry and transmission electron microscopy were applied to murine and human ovary sections. Immunochemical studies were performed in hemizonae pellucidae from microbisected human oocytes. Using the competitive hemizona assay and various anti-ZP3 antibodies, we further intended to identify human ZP3 epitopes of functional significance. Our results showed that antiserum AS ZP3-9 (mouse specific) detected mouse ZP3 protein in mouse oocytes and in immunoblots, whereas AS ZP3-14 (human specific) detected human ZP3 protein in human ovary sections, native hemizonae pellucidae and in immunoblots. ZP3 material was also detected in cumulus cells by immunohistochemistry. Ultrastructural studies showed an equal distribution of ZP3 throughout the zona pellucida. The human competitive hemizona assay revealed that none of the anti-ZP3 synthetic peptide antisera affected sperm binding suggesting that those epitopes are not involved in primary sperm binding. Anti-porcine ZP3 beta protein antibodies (polyclonal) blocked human sperm-zona pellucida binding. In summary, these anti-ZP3 synthetic peptide antibodies specifically reacted with intact ZP3 protein (murine and human) but did not inhibit human sperm-zona pellucida binding; anti-ZP3 antibodies can therefore be used as biomarkers for ZP3 localization and function.     

Andrology: The ability of the hemizona assay to predict human fertilization in different and consecutive in-vitro fertilization cycles

The objective of this prospective study was to examine the abilityof the hemizona assay (HZA) to predict fertilization outcomeof mature, pre-ovulatory oocytes under in-vitro fertilization(IVF) conditions. Since a large number of patients were evaluatedover a long period, the power of the HZA to prognosticate fertilizationresults in the same and subsequent (consecutive) IVF cyclesof those same patients was assessed. For IVF, only metaphaseII oocytes were used. For the HZA, both fresh oocytes donatedby patients at the time of IVF and oocytes recovered from surgicallyremoved ovarian tissue (and salt-stored) were used, and bisectedby micromanipulation techniques. Matching hemizonae were co-incubatedeither with spermatozoa from the patient (test) or from a fertileman (control) for 4 h. The number of spermatozoa tightly boundto the zona was counted. Patients (n = 112) were divided intotwo groups based on HZA results (expressed as HZA index or HZI):HZI 30% (n = 72) and <30% (n = 40). The patients with HZI<30% had significantly lower fertilization rates in boththe HZA—IVF cycle and in subsequent cycles compared topatients with HZI 30% (P < 0.03). Linear discriminant analysisindicated the HZA to have a sensitivity of 84%, and positiveand negative predictive values of 85 and 70% respectively, forprediction of fertilization outcome in a total of 233 cycles.It was concluded that the HZA is a good predictor of fertilizationrate in vitro, and can be used in the IVF setting to supplyadditional clinical information in malefactor patients.     

The effect of pentoxifylline on sperm movement characteristics and zona pellucida binding potential of teratozoospermic men

This study investigates the effect of pentoxifylline on spermmovement and zona pellucida binding. Spermatozoa from nine teratozoospermicpatients (10.2 ± 6% normal forms) were included in thestudy. Test samples were diluted with a 4 mM solution of pentoxifylline,and control samples with Ham's F-10 culture medium only. Zonapellucida binding potential was measured under hemizona assayconditions (HZA). Sperm motility was evaluated at the start(0 h postswim-up) and end (4 h) of the HZA, under test-tubeconditions and under HZA conditions (50µ droplet underoil). Motilityparameters tested included the curvilinear velocity(VCL), straight line velocity (VSL) and linearity (LIN). Comparedto the controls, pentoxifylline-treated samples showed an immediatestimulation of sperm motility, under test-tube conditions, witha significant elevation of VCL at 0 h incubation (102.77 ±14.4 versus control value 84.60 ± 10.6 µm/s; P< 0.005), which however was reversed after 4 h incubation(73.16 ± 4.6 versus control value 85.47 ± 12.8µm/s; P < 0.005). A decline in sperm motility from0 to 4 h incubation was noted for all the pentoxifylline-treatedsamples, under both test tube conditions (VCL: 102.77 ±14.4 versus 73.16 ± 4.6 µm/s, P <<;<<0.005; VSL: 27.2 ± 10 versus 10.66 ± 2.2 µm/P <<; 0.005; LIN: 23.65 ± 7.1 versus 11.86 ±1.8%, P<<; 0.005) and HZA conditions (VCL: 100.04 ±13.1 versus 76.00 ± 7 µm/s, P <<; 0.005;VSL: 26.40 ± 8.7 versus 9.14 ± 4.5 µm/s,P <<; 0.005; LIN: 26.2 ± 12 versus 11.05 ±4.3%, P <<; 0.005). Pentoxifylline also showed no effecton the zona binding capacity of the sperm population, sinceno significant difference in zona binding capacity was seenbetween control and test samples. In conclusion, the effectof pentoxifylline on teratozoospermic spermatozoa is short-lived,and despite its stimulatory potential pentoxifylline shows nobeneficial use in enhancing zona pellucida binding of teratozoospermicspermatozoa.     

Tyrosine phosphorylation of a 95 kDa protein and induction of the acrosome reaction in human spermatozoa by recombinant human zona pellucida glycoprotein 3

Protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated and capacitated human spermatozoa was investigated in response to recombinant human zona pellucida glycoprotein (rhZP3) produced by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA. rhZP3-containing medium promoted the AR in a high proportion of capacitated spermatozoa (48.6 +/- 3.2%; P < 0.01) compared with control (no rhZP3) samples (14.8 +/- 2.1%). However, rhZP3-containing medium did not cause increased acrosomal exocytosis in non-capacitated spermatozoa (16.8 +/- 3.0%). Induction of the AR was associated with increased tyrosine phosphorylation of a 95 +/- 5 kDa epitope only in capacitated spermatozoa. A dose-dependent increase in the protein phosphorylation of a 95 kDa epitope in response to rhZP3 was detected by [gamma-32P]- ATP labelling of detergent-solubilized sperm proteins. When spermatozoa were co-incubated with monoclonal antibody 97.25 (mAb 97.25) recognizing a 95 kDa tyrosine kinase epitope, there was no rhZP3 induction of tyrosine phosphorylation of the 95 kDa protein. Such co- incubation also markedly inhibited the AR (23.9 +/- 3.1%). These results support the model that initial interaction of the fertilizing spermatozoon with ZP3 involves the tyrosine phosphorylation of a 95 kDa tyrosine kinase protein and that this requires capacitation.      

Immunoreactivity and in-vitro effect on human sperm-egg binding of antibodies against peptides corresponding to bonnet monkey zona pellucida-3 glycoprotein

The following synthetic peptides were made as immunogens for development of a zona-based contraceptive vaccine: P1, KQPFWLLQGGASRAETSVQPVLVE [amino acids (aa) 23-45 with an additional K at the N-terminus]; P2, FSEEKLVFSLRLMEENC (aa 164-179 with an additional C at the C-terminus and T170 replaced by V); and P3, CSFSKSSNSWFPVEGPADICQCC (aa 300-322). The aa are numbered on the basis of bonnet monkey ZP3 precursor protein. Antibodies against an additional peptide P4, KGDCGTPSHSRRQPHVVSQWSRSA (aa 324-347), significantly inhibits human sperm-oocyte binding. In addition, antibodies against cocktail of peptide-diphtheria toxoid conjugates also significantly inhibit the binding of spermatozoa to zona pellucida in a hemizona assay. These results will further help in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to ZP3.      

Evidence for the participation of beta-hexosaminidase in human sperm-zona pellucida interaction in vitro

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.     

Low proportions of sperm can bind to the zona pellucida of human oocytes

BACKGROUND: Sperm binding to the zona pellucida (ZP) is required for human fertilization. Under experimental conditions not limited by ZP binding sites, the cumulative numbers of sperm binding tightly to the ZP will asymptote with time to the total number of sperm in the insemination medium capable of binding. METHODS: Numbers of ZP-bound sperm were counted after groups of 10 oocytes were incubated with 2x10(4) motile sperm in 20 micro l droplets. The time-course of sperm binding was measured in three consecutive 2 h incubation periods using fresh oocytes for each period (n = 12). Using the kinetic theory of gases to model sperm-oocyte collision rates, the time-course results were extrapolated to give the total proportion of motile sperm capable of binding to the ZP. ZP binding of sperm after 4 h incubation was studied in 20 fertile and 20 normozoospermic subfertile men. RESULTS: The percentage of motile sperm capable of binding was for fertile men: mean 14% (range 8-25) and for the subfertile: 4.3% (range 0.1-13, P < 0.001). Sperm morphology correlated with the proportion of ZP-bound sperm. CONCLUSIONS: More than 75% of motile sperm from fertile men have no ability to bind to the ZP. This finding has important implications for improvement of semen analysis.     

Fertilization and early embryology: The factors affecting sperm binding to the zona pellucida in the hemizona binding assay

Sperm binding to the zona pellucida is a prerequisite for fertilization.The hemizona binding assay (HZA) is commonly used to evaluatethe zona-binding capacity of spermatozoa. The present studyreports three factors that affect HZA. They were the base mediumused, the protein source and the size of pipette used for removingloosely bound spermatozoa during HZA. The number of spermatozoabound on the hemizona was compared between (1) Earle's balancedsalt solution (EBSS) and Ham's F-10, and (2) between human serumand bovine serum albumin (BSA). Results indicated that EBSSand human serum both significantly increase the number of boundspermatozoa when compared to Ham's F-10 (P < 0.0001, pairedt-test) and BSA (P < 0.05, paired t-test) respectively. Pipettesof different diameters were used to study the effect of sizein removing loosely bound spermatozoa on hemizona. Data showedthat the diameter of the pipette should be 200 mm, in ordernot to remove bound spermatozoa excessively. These results emphasizethe importance of standardization of the protocol of the hemizonaassay worldwide to be able to compare results between differentlaboratories.     

A new combined in-vitro test model for the identification of substances affecting essential sperm functions [published erratum appears in Hum Reprod 1997 Nov;12(11):2580]

Binding of mammalian spermatozoa to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. It has been postulated that xenobiotics that are released in the environment as well as exposure to pharmaceutical medications may be associated with reproductive problems in men and wildlife. Examining physiological and non-physiological effects of particular compounds on sperm functions requires high quality in-vitro test systems. We established a reliable combined in-vitro test system with bovine gametes and evaluated if aliquots of pooled post-thaw spermatozoa are suitable for examining essential sperm functions. Using cryopreserved semen, the PSA-FITC/Hoechst 33258 staining procedure was applicable to evaluate the acrosomal status and cell viability. In the bovine hemizona assay, hemizona indices revealed no differences between cryopreserved and fresh semen. Treatment of post-thaw bovine spermatozoa with progesterone (1 microM or bovine follicular fluid (20%) induced the acrosome reaction from 12% (untreated spermatozoa) to 25% (P < 0.001) and to 22% [corrected] (P < 0.01), respectively. Incubation of both compounds (1 microM progesterone and 20% follicular fluid) raised the percentage of acrosome-reacted spermatozoa to 30% (P < 0001). Our results demonstrate that cryopreserved semen can be integrated into an in-vitro screening model for reproductive toxicology testing. Pooled, cryopreserved bovine spermatozoa will thus permit reproducible experiments for clinical and basic science purposes and may also be applicable for the human system.      

Angiotensin II in human seminal fluid

The renin-angiotensin system (RAS) and angiotensin II are important in sperm function and male fertility. Angiotensin II type I (AT1) receptors have been identified in developing and ejaculated human spermatozoa, and angiotensin can stimulate sperm motility, the acrosome reaction and binding to the zona pellucida. However, there is little information on the availability of the hormone to spermatozoa during the reproductive process. Seminal plasma and blood plasma obtained from normal and subfertile subjects was extracted, and angiotensin content was analysed by radioimmunoassay. Values obtained for blood angiotensin II were within the normal range at 16.0 +/- 3.1 pg/ml (mean +/- SEM). Values for seminal plasma were usually 3-5 fold higher, at 51.6 +/- 9.3 pg/ml (n = 34, P < 0.0001). High performance liquid chromatography analysis showed that approximately 80% of the immunoreactive angiotensin was attributable to angiotensin II itself. However, seminal plasma angiotensin II concentrations were not correlated with blood angiotensin II, sperm concentration or sperm motility. The results show that immunoreactive angiotensin from a source other than the circulation is available to spermatozoa in human ejaculates. The results are consistent with the concept that angiotensin II has an important role in male fertility.     

Fertilization and early embryology: Female age does not affect the capacity of human zona pellucida to bind spermatozoa

The purpose of this study was to evaluate the effect of femaleage on the capacity of the zona pellucida to bind spermatozoa.A total of 1008 unfertilized oocytes obtained from 210 women(aged 21–43 years) participating in the in-vitro fertilizationprogramme were tested using a hemizona assay. Spermatozoa takenfrom a cryopreserved pool of fertile donor specimens servedas a control in the hemizona assay, and were used to assessthe ability of the zona pellucida to bind spermatozoa. The mean± SD number of spermatozoa attached to the hemizona was107 ± 42. The binding capacity of different oocytes fromthe same cohort varied substantially (coefficient of variation= 28%). Age was not found to be correlated with the number ofspermatozoa bound to the zona pellucida (r = -0.02; P > 0.1).It was concluded that female age has no role in the abilityof the human zona pellucida to bind spermatozoa. Key words:female age/spermatozoa/zona binding.     

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.     

The influence of human follicular fluid on the acrosome reaction, fertilizing capacity and proteinase activity of human spermatozoa

The present study was designed to assess physiological and enzymatic changes in human spermatozoa following incubation in human follicular fluid (HFF). Initially, it was determined that infertility patients (n = 29) scored dramatically higher on the hamster egg penetration test (HEPT) when spermatozoa were incubated with HFF (22.9 +/- 4.4%) rather than the standard swim-up alone (4.6 +/- 1.1%). To further evaluate this effect, in experiment I, spermatozoa were obtained from proven fertile individuals and evaluated following exposure to three different HFF samples as well as control treatments (medium, cul de sac fluid and heparin). There were no significant differences in HEPT scores following sperm incubation in the three different follicular fluids although incubation in all the fluids significantly (P less than 0.01) enhanced sperm penetration (%PEN) when compared to the standard swim-up and other control treatments. The absence of an effect of cul de sac fluid on spermatozoa indicates that not all body fluids contain factors which stimulate sperm fertilizing capacity. The effect of HFF was demonstrated in a infertile patient population as well as in donors of proven fertility. In experiment II, the effect of HFF on the acrosome reaction (%AR), sperm fertilizing capacity and changes in sperm proteolytic enzymes were determined. There was no significant difference in the %AR between freshly ejaculated (7.9 +/- 3.1) and medium incubated (9.4 +/- 1.6) spermatozoa; however, in both of these treatments the %AR was less (P less than 0.01) than for spermatozoa treated with HFF (45.6 +/- 4.7). The %PEN following incubation of spermatozoa in HFF (52.2 +/- 6.8) was greatly increased (P +/- 0.01) compared to the standard swim-up (19 +/- 3.9).     

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona spellucida: lack of relationship with ionophore A23187-induced acrosome reaction

Acrosome reactions induced by the calcium ionophore A23187 [GenBank] andzona pellucid a (ZP) were studied. Sperm samples were obtainedfrom fertile men or men with normal semen analysis and normalsperm-ZP binding. Oocytes were obtained, with the consent ofthe patients, after the failure of fertilization in vitro. Motilespermatozoa selected by a swim-up technique were incubated with10 µM A23187 [GenBank] for 1 h, four oocytes for 2 h or solubilizedZP (4 ZP/µl) for 2 h. Spermatozoa bound to the ZP weredislodged and collected in a small volume of phosphate-bufferedsaline by aspirating the oocytes with a glass pipette with aninner diameter (120 µm) slightly smaller than the diameterof the oocyte. The acrosome status of the spermatozoa was determinedusing fluorescein-labelled Pisum sativum agglutinin. The proportionof spermatozoa undergoing the acrosome reaction on the ZP at2 h varied over a wide range (5–99%), but the agreementbetween results for the same semen sample exposed to differentgroups of oocytes was good: the standard deviations of the differencesbeing 9%. Pre-incubation of spermatozoa for 2 h did not increasethe ZP-induced acrosome reaction. Re-incubation of ZP with thesame sperm suspension for 2 h after removing ZP-bound spermatozoafrom the first 2 h incubation produced a significantly lowerZP-induced acrosome reaction in the second incubation (22 ±16%) than in the first incubation (30 ± 14%; P < 0.001,n = 20). There was no significant difference in the ZP-inducedacrosome reaction with oocytes with ZP which had or had notbeen penetrated by spermatozoa during the in-vitro fertilizationinsemination. Pre-incubation of spermatozoa with solubilizedZP blocked sperm-ZP binding. However, the acrosome reactioninduced by solubilized ZP (4 ZP/µl) was significantlylower than the acrosome reaction induced by intact ZP (10 ±5 and 30 ± 13% respectively, n = 11, P < 0.001), butthere was a high correlation (Spearman r = 0.822, P < 0.01)between the results. On the other hand, although the averageof the acrosome reaction was similar for A23187 [GenBank] (42%) and forZP (43%), there was no significant correlation between the resultsfor the two stimuli (n = 60). In conclusion, a useful methodfor assessing the ZP-induced acrosome reaction has been developedusing oocytes which failed to fertilize in vitro. The lack ofa relationship between the results of the chemical (A23187 [GenBank] )and physiological (ZP) stimali for the acrosome reaction inthe same subjects questions the biological basis of using A23187 [GenBank] for tests of sperm function. Solubilized human ZP in a concentrationthat blocks sperm-ZP binding is a less efficient inducer ofthe acrosome reaction than is intact ZP. It is possible thatthe three-dimensional structure of the ZP is important for inductionof the acrosome reaction or that spermatozoa which bind to theZP are more likely to acrosome react Assessment of the physiologicalacrosome reaction for diagnosis of sperm defects which interferewith the fertilization process should be concentrated on thespermatozoa which are capable of binding to the ZP.