Zona-free Hamster Egg Penetration Test Research Articles

Treatment of infertile patients by intrauterine insemination of washed spermatozoa.

Thirty-five couples with a variety of infertility problems were treated by a sperm-washing and intrauterine-insemination method. The husband's semen was washed with a modified Biggers, Whitten, and Whittingham's medium to obtain a seminal plasma-free, highly concentrated sperm suspension for intrauterine insemination. Couples receiving these inseminations were evaluated by semen analysis, zona-free hamster egg penetration test (EPT), post-coital test (PCT), antisperm antibody test (ASAT), and other appropriate infertility tests to determine the possible cause of infertility. The pregnancy rate as a group was 17%, while the spontaneous rate was found to be only 7%. Patients with a poor PCT result had a good success rate (25%) especially if the male's fertilizing ability appeared normal in the EPT (50% success). This treatment did not help men with poor EPT results or reduced sperm concentration (5% and 0% success). By excluding unsuccessful patients attempting less than three months, the calculated conception rate for this treatment is 33%. No infection or other serious complications were reported. It is concluded that this method is an uncomplicated and effective treatment for certain infertility patients. The patients most likely to be assisted by this method can be identified by using the EPT, PCT, and ASAT, and these patients should be treated for a minimum of three cycles.

Immunogenicity and contraceptive potential of recombinant human sperm associated antigen (SPAG9)

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm–egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm–egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.

Characterization of immune response in mice to plasmid DNA encoding human sperm associated antigen 9 (SPAG9)

To examine the immunogenicity of the plasmid DNA encoding human sperm associated antigen 9 (hSPAG9), the cDNA corresponding to hSPAG9 was cloned in mammalian expression vector pcDNA 3.1 down stream of cytomegalovirus promoter. Immunization of female BALB/cJ mice with pcDNA–hSPAG9 plasmid DNA in saline by intramuscular (i.m.), by adsorbing onto gold microcarriers (delivered by gene gun) and by recombinant hSPAG9 (r-hSPAG9) protein generated antibody response against Escherichia coli expressed r-hSPAG9 protein and native SPAG9 in human sperm. Although mice immunized with r-hSPAG9 protein exhibited highest antibodies titres ( P < 0.001), the difference in the antibody titres seen by the two modes of plasmid DNA delivery were not significant ( P > 0.05). A dominant IgG1 isotype response was observed in mice immunized with pcDNA–hSPAG9 plasmid DNA delivered by gene gun as compared to a mixed IgG1–IgG2a isotype response in mice immunized with r-hSPAG9 protein and pcDNA–hSPAG9 plasmid DNA delivered by i.m. Further, antibodies generated by pcDNA–hSPAG9 plasmid DNA localized acrosomal compartment of human sperm and inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. These studies for the first time, demonstrate the feasibility of generating an immune response to sperm specific hSPAG9 protein by DNA vaccine and that antibodies thus generated recognize native SPAG9 in human sperm.

A monoclonal antibody to human SP-10 inhibits in vitro the binding of human sperm to hamster oolemma but not to human Zona pellucida.

SP-10 is a sperm intra-acrosomal protein, specific to the testis, that is believed to play an important role in egg-sperm binding. While the molecular characterization of the SP-10 protein has been clarified, little is yet known of its functional role in fertilization. We therefore established a monoclonal antibody (mAb pep-SP10) against a peptide (pep-SP10) that included the most hydrophilic portion of human SP-10 between the 135th and 149th amino acids. Human SP-10 was found to be localized in the equatorial region of acrosome-reacted sperm by immunofluorescent staining using our mAb pep-SP10. Monoclonal Ab pep-SP10 inhibited sperm-oolemma binding in the zona-free hamster egg penetration test, but it did not inhibit sperm-zona binding in the hemizona assay. Furthermore, we demonstrated that the oolemmal ligands of human SP-10 did not include beta(1) integrins, the most promising candidates for oocyte ligands involved in sperm-oolemma binding, based on the findings of a human sperm-cultured cell binding assay using F9 mouse embryonal carcinoma cells and F9-transformed cells lacking beta(1) integrins. In conclusion, our present data suggest that human SP-10, expressed on the equatorial region of acrosome-reacted sperm, indeed mediates sperm-oolemma binding in a beta(1) integrin-independent manner, but not sperm-zona binding.

A sialoglycoprotein, gp20, of the human capacitated sperm surface is a homologue of the leukocyte CD52 antigen: analysis of the effect of anti-CD52 monoclonal antibody (CAMPATH-1) on capacitated spermatozoa.

In this study we performed N-terminal sequence analysis of gp20, a 20 kDa sialoglycoprotein on the human sperm surface previously identified by radiolabelling of the sialic acid residues of sperm surface. We found 100% identity with the N-terminus of CD52, an antigen expressed on almost all human leukocytes. We also show that, like CD52, gp20 behaves as a glycosylphosphatidylinositol (GPI)-anchored protein and that anti-gp20 antiserum reacts with an antigen on leukocytes of the same molecular weight as CD52. Using CAMPATH-1, the monoclonal antibody against CD52, in fluorescent staining of capacitated spermatozoa, Western blot analysis and the zona-free hamster egg penetration test, we found that the effect of this antibody was different from that of our anti-gp20. Western blot analysis revealed a well-defined 20 kDa band with anti-gp20, whereas a 14-20 kDa band was detected with CAMPATH-1. Anti-gp20 stained the equatorial region of the sperm head, whereas CAMPATH-1 stained the tail in immunofluorescence analysis of capacitated spermatozoa. A dose-dependent inhibitory effect was seen with CAMPATH-1, similar to that previously detected with anti-gp20, in a zona-free hamster egg penetration test. However, with CAMPATH-1 agglutination of motile spermatozoa was detected, and this was not present with anti-gp20. This suggests that the epitopes recognized by the two antibodies are different.

The predictive value of the zona-free hamster egg penetration test in relation to in-vitro fertilization at various insemination concentrations.

The aim of the study was to evaluate the predictive value of the zona-free hamster egg penetration test (ZHEPT) for success in in-vitro fertilization (IVF) at various insemination concentrations ranging between 0.1 and >0.6 x 10(6)/ml. The ZHEPT was assessed using sperm samples from 87 couples undergoing IVF treatment. A similar test was simultaneously performed on the same semen sample following ionophore induction of the acrosome reaction (ZHEPTii test). Both the tests were poorly correlated with the fertilization rate of IVF at all the insemination concentrations except at >0.6 x 10(6)/ml, when there was good correlation between the ZHEPTii test and the fertilization rate. Following exclusion of two cases with an oocyte problem, further statistical analysis revealed that both the ZHEPT and ZHEPTii tests were poorly correlated with fertilization rate in IVF in this treatment group. This study suggests that the ZHEPT (with and without ionophore induction of the acrosome reaction) has a poor predictive value for the success of fertilization in IVF treatment at any insemination concentration.

Improvement of Motility and Fertilization Potential of Postthaw Human Sperm Using Glutamine

The effectiveness of three amino acids, glutamine, proline, and histidine, and one amino acid-related compound, betaine, in preserving human sperm diluted v/v in a basal medium (BM) containing 14% glycerol during the freeze–thaw (FT) process was studied. At 80 mMin BM, only glutamine improved the 5- to 60-min postthaw total and progressive motilities and velocity. The presence of glutamine at 80 mMis not sufficient to achieve lower concentrations of the toxic agent glycerol in FT medium. Glutamine may therefore have a mechanism of protection on human spermatozoa that is independent from that of glycerol. The zona-free hamster egg penetration test showed that the percentage of eggs penetrated was significantly greater when spermatozoa were frozen–thawed with 80 mMglutamine in BM. Consequently, the presence of glutamine at 80 mMin a glycerol–FT medium maintains human sperm motility and fertilizing ability during the FT process.

Long-term follow-up of couples after hamster egg penetration testing

To determine the clinical usefulness of the zona-free hamster egg penetration test as a long-term prognostic indicator for future pregnancy. Division of Reproductive Endocrinology and Infertility at the Washington University Medical Center. All couples (n = 148) who had a hamster egg penetration assay performed between March 1, 1985 and December 31, 1986 were identified and followed with direct or telephone contact up to 68 months after the initial assay. The monthly fecundity rates using life table analysis and the 5-year incidence of pregnancy were categorized by the percentage of hamster eggs penetrated and by history of previous urologic surgery. There were no significant differences in the rate nor incidence of pregnancy in couples with hamster egg penetration scores of 0%, > 0% and < or = 10%, > 10% and < or = 20%, or > 20%. Although men with previous urologic surgery tended to have lower scores, there was no significant difference in the 5-year incidence of pregnancy. The hamster egg penetration score is not predictive of incidence of pregnancy nor time to conception.

Computer-assisted assessment of human sperm morphology: usefulness in predicting fertilizing capacity of human spermatozoa

The usefulness of sperm morphology to predict the outcome of human sperm fertilizing capacity was examined. Semen samples from 50 male patients attending the infertility clinic of a tertiary referral institution were studied. Sperm morphology was classified both by visual assessment and computer-assisted image analysis. In addition, morphometric analysis of the spermatozoa was measured by the morphologizer. Multivariate discriminant analysis was used to evaluate the usefulness of these morphology parameters for predicting the outcome of the zona-free hamster oocyte sperm penetration assay. The manually derived percent of spermatozoa with normal and small head were selected to be of discriminating value in predicting the outcome of the zona-free hamster oocyte penetration test. The accuracy of correctly classifying the outcome of zona-free hamster oocyte penetration test by these two parameters in combination was 84%, whereas assessment of sperm morphology with morphometric analysis by the morphologizer selected a total of eight variables, which together predicted sperm fertilizing capacity with 74% accuracy. Addition of the morphologizer-derived parameters to those derived manually did not significantly improve the predictive value. We conclude that the results of the zona-free hamster egg penetration test could be predicted using manual assessment of sperm morphology and computer-assisted morphometric analysis did not add further information.

Treatment of spermagglutination with proteolytic enzymes, II. Sperm function after enzymatic disagglutination

Antibody-mediated spermagglutination is responsible for infertility in some couples and fertilization in vitro can also be impaired by these antibodies. Having previously demonstrated the possibility of enzymatic disagglutination in such situations, the functional potential of disagglutinated spermatozoa has now been assessed. Chymotrypsin (500 U/ml) and papain (50 U/ml) resulted in impairment of oocyte penetration in the zona-free hamster egg penetration test. Trypsin (500 U/ml), while having no effect on egg penetration of normal spermatozoa, significantly improved oocyte penetration of spermatozoa which had been previously incubated with spermagglutinating antibody-positive sera. Sperm-mucus interaction was not improved, however, by trypsin treatment of agglutinated spermatozoa. This technique may be of value in conjunction with in-vitro fertilization in situations where spermagglutination exists, and also possibly with intra-uterine insemination if improved fertilizing ability can be confirmed in vitro.

The effects of lymphokines and monokines on human sperm fertilizing ability in the zona-free hamster egg penetration test

Antisperm cell-mediated immunity has been associated with infertility in men and women. The purpose of this study was to determine whether the products of activated lymphocytes and macrophages (Iymphokines and monokines) affect the fertilizing ability of human spermatozoa in the zona-free hamster egg penetration test. Of a panel of six different purified cytokines tested, the Iymphokine 'Y-interieron and the mono kine tumor necrosis factor significantly affected the ability of human sperm to penetrate hamster eggs. An inhibitory effect was observed when motile sperm were harvested for use in the assay by a swim-up technique after overnight incubation in low amounts of these cytokines and also when the factors were added to the cultures at the time sperm were introduced to the eggs. These data contribute further evidence that soluble factors produced by activated lymphocytes and macrophages in reproductive tissues could be significant mediators of immunologic infertility. (AM J OSSTET GVNECOL 1989;160:1154-9.)

The effects of lymphokines and monokines on human sperm fertilizing ability in the zona-free hamster egg penetration test

Antisperm cell-mediated immunity has been associated with infertility in men and women. The purpose of this study was to determine whether the products of activated lymphocytes and macrophages (lymphokines and monokines) affect the fertilizing ability of human spermatozoa in the zona-free hamster egg penetration test. Of a panel of six different purified cytokines tested, the lymphokine -y-interferon and the monokine tumor necrosis factor significantly affected the ability of human sperm to penetrate hamster eggs. An inhibitory effect was observed when motile sperm were harvested for use in the assay by a swim-up technique after overnight incubation in low amounts of these cytokines and also when the factors were added to the cultures at the time sperm were introduced to the eggs. These data contribute further evidence that soluble factors produced by activated lymphocytes and macrophages in reproductive tissues could be significant mediators of immunologic infertility. (Am J Obstet Gynecol 1989;160:1154-9.)

Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test

The acrosomal status of human sperm during preparation for the process of zona-free hamster egg penetration test (ZFHEPT) was determined. The incidence of acrosome reaction (AR), as assessed by triple-stain technique, was significantly increased after 24 hours of incubation at 4 degrees C in TES-Tris (TEST)-yolk buffer, but the absolute values were relatively low (20% or less). Sperm from fertile donors and infertile patients with normal or abnormal semen analysis displayed similar capacity to undergo the AR in vitro. Although a positive correlation was found between the incidence of AR and the score of ZFHEPT, a remarkable individual variation was noted. The incidence of AR in freely swimming human sperm does not accurately reflect the fertilizing ability of the sperm.

Effects of relaxin on the fertilization capacity of human spermatozoa

Relaxin, a polypeptide hormone found in human seminal plasma, exerted favorable effects on the function of spermatozoa. This study was designed to investigate the effect of this hormone on the fertilization capacity of human spermatoxoa from 10 oligospermic men (group A), 11 asthenospermic men (group B), 10 normospermic infertile men (group C), and 10 men with verified fertility (group D). We compared the effects of purified porcine relaxin at different concentrations (0, 50, and 150 ng/ml). Parameters include motility, hypoosmolar swelling test, and zona-free hamster egg penetration test. In group A and group B, relaxin increased the results of the hamster egg penetration test and motility. In group C, results of the hamster egg penetration test and motility improved only at the concentration of 50 ng/ml. In group D, relaxin increased the results of the hamster egg penetration test only at a concentration of 150 ng/ml. Results of the hypoosmolar swelling test were not consistently altered in any study group. Relaxin appears to enhance the fertilization capacity and motility of human spermatozoa in oligospermic and asthenospermic subjects.

Heterologous penetration of zona-free hamster ova by ejaculated domestic cat spermatozoa

Hypertonic high-salt Biggers, Whitten, and Whittingham medium was compared with either a complex culture medium (synthetic tubal fluid) or a variant of Tyrode's medium for capacitating donor human spermatozoa to be used in the zona-free hamster egg penetration test. Five sperm preparations showed a slightly higher penetration rate in synthetic tubal fluid than in high-salt Biggers, Whitten, and Whittingham medium, five showed equivalent results, and only two had slightly lower penetration rates. However, in modified Tyrode's medium only one sample showed a lower penetration, while six showed comparable results and five showed higher rates of penetration. This last group included two samples that had demonstrated zero penetration in high-salt Biggers, Whitten, and Whittingham medium. Although no overall significant differences between media in the two series of experiments could be discerned with paired t tests, in view of the lower incidence of false negative results in modified Tyrode's medium and the hamster egg penetration test.

Application of a Poisson-gamma model to study the influence of gamete concentration on sperm-oocyte fusion in the zona-free hamster egg penetration test.

A Poisson-gamma distribution model has been developed for studying sperm-oocyte interaction in the zona-free hamster egg penetration test. With the aid of this model we have analysed the relationship between the penetration results achieved with semen of normal fertile men and the concentration of spermatozoa and oocytes used in the assay. From this analysis we have drawn up reference tables estimating the number of spermatozoa and oocytes which must be present in the assay for the result to represent a significant (P less than 0.05) decline in sperm function relative to the normal fertile population. Since there is, as yet, no fixed protocol for the penetration assay, these tables have been constructed to fit a range of methodologies varying with respect to the nature of the preincubation phase and including (a) a 7-h preincubation in Medium BWW, (b) a 3-h preincubation in hyperosmotic Medium BWW or (c) a 3-h preincubation in the presence of A23187.

Clinical evaluation of the zona‐free hamster egg penetration test in the management of the infertile couple. Prospective and retrospective studies

Clinical evaluation of the zona‐free hamster egg penetration test in the management of the infertile couple. Prospective and retrospective studies

GROUP DISCUSSION

GROUP DISCUSSION

The use of strontium-substituted media for capacitating human spermatozoa: an improved sperm preparation method for the zona-free hamster egg penetration test

An improved procedure for human sperm capacitation based upon calcium deprivation using a modified Tyrode's medium is described. Replacement of CaCl2 by SrCl2 significantly increased the penetration of zona-free hamster eggs after 20 hours of sperm preincubation, compared with parallel sperm aliquots incubated in normal medium. The effect of an extra 4 hours' sperm preincubation produced no significant shift in fertilizing ability. Addition of 0.1 mM ethyleneglycol-bis-(beta-amino-ethyl ether)-N,N,N',N'- tetraacetic acid (EGTA) to the strontium-substituted medium resulted in a further significant increase in the penetration rate as well as a significant increase in polyspermy. The average increase in the penetration capacity (penetration rate X polyspermy) of the strontium/EGTA-preincubated spermatozoa was approximately tenfold, with no significant shift as a result of an extra 4 hours' preincubation.

Elaboration of a new culture medium for physiological studies on human sperm motility and capacitation.

The formulation of a new medium based upon published data on human tubal fluid and blood plasma is described. Sperm motility was well maintained for periods of up to 6 h in this 'synthetic tubal fluid' (STF), and movement characteristics (velocity of progression and amplitude of lateral head displacement) were quantitatively and qualitatively similar to values previously reported for other complex media. STF also supported human sperm capacitation and the spontaneous acrosome reaction as determined using the zona-free hamster egg penetration test. Spermatozoa pre-incubated in STF containing blood plasma levels of taurine (86 microM) for 3 h penetrated significantly more oocytes than parallel sperm populations pre-incubate in STF lacking taurine (P less than 0.001). This difference was no longer significant after 5 h of pre-incubation. These findings indicate a possible role for taurine in human sperm capacitation, and demonstrate the potential value of STF for performing more physiological invitro studies on human sperm function.