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The aim of this study, based on a prospective analysis, was to evaluate the accuracy of the TEST-yolk buffer enhanced hamster egg penetration test (HEPT) and acrosin activity in discriminating fertile from infertile males as compared with the conventional semen profile. The incidence of spontaneous pregnancies was monitored during a follow-up period ranging between 18-24 months in 62 couples without untreatable female infertility. The diagnostic accuracy of the HEPT was significantly higher than that of the conventional semen profile, as evaluated with fitted ROC curves. The best discriminatory ability was exhibited by a penetration index (penetrations/oocyte) of 2 as cut-off value. In our series it gave a positive predictive value of 77.8% and a negative predictive value of 92.3%. Acrosin activity did not show a discriminating power significantly higher than the conventional semen profile, but an acrosin activity <20 microIU/10(6) spermatozoa was found only among patients who did not achieve a pregnancy. In conclusion, this prospective analysis indicates that the TEST-yolk buffer enhanced HEPT constitutes a significant improvement over the conventional semen profile in discriminating fertile from infertile males. Acrosin activity could provide a useful adjunctive test because of the high negative predictive value of its poor results.

Objective: To determine whether antisperm antibodies can interfere with the induction of the acrosome reaction (AR) by the zona pellucida (ZP) and whether this interference also can occur in the absence of an inhibitory effect on ZP binding., Design: Prospective in vitro study., Setting: A tertiary care center, the Andrologic Clinic, University of L'Aquila., Patient(s): Sera from 12 infertile patients with high titers of circulating antibodies directed against the sperm head were studied., Intervention: None, Main Outcome Measure(s): The effect of antisperm antibodies on ZP binding was evaluated by matching antibody-exposed and nonexposed donor sperm suspensions labeled with fluorescein or rhodamine, respectively, and incubated with the same salt-stored human ZPs. The effect of antibodies on ZP-induced AR was determined by challenging antibody-exposed and nonexposed donor sperm suspensions with human ZPs disaggregated with acidic NaH2PO4. Acrosomal status was evaluated using fluorescein-labeled Pisum sativum agglutinin and supravital stain Hoechst 33258. In some selected cases, the effect of antisperm antibodies on the acrosomal status of sperm bound to intact ZP also was evaluated using transmission electron microscopy., Result(s): Five of 12 sera exhibited an inhibitory effect on ZP binding. An inhibition of AR induction by disaggregated ZPs (ranging from 64% to 98%) was produced by all 5 sera with an inhibitory effect on ZP binding and by 2 of 7 sera without an inhibitory effect on ZP binding. The different effects of antisperm antibodies on AR induction by disaggregated ZP were confirmed by comparing with ultrastructural evaluations on the acrosomal status of sperm bound to intact ZP., Conclusion(s): Antisperm antibodies can interfere with the induction of AR by ZP. This inhibition can occur even in the absence of an inhibitory effect on ZP binding. Neither effect may occur.

Problem: This study was performed to evaluate the occurrence as well as the level of the interference of sperm-associated antibodies on fertilization process., Method: Motile sperm suspensions from 28 infertile patients with high degree of autoimmunization against the sperm head were tested with the zona pellucida (ZP) binding test and with the sperm penetration assay (SPA) enhanced with TEST-yolk buffer. Both tests were also performed using donor sperm exposed and non-exposed to the patients' circulating sperm antibodies., Results: A low ZP-binding was exhibited by sperm from 50% of patients with normal semen profile. All normozoospermic patients with low ZP-binding showed circulating sperm-antibodies with inhibitory effect on ZP-binding, while no patient with normal ZP-binding showed circulating sperm-antibodies with inhibitory effect. No normozoospermic patient exhibited a negative SPA result, and only in 16% of cases the penetration index was slightly less than 2 (the lowest value exhibited by fertile controls). Circulating antisperm-antibodies did not significantly affect the hamster egg penetration., Conclusion: Even in the presence of high degree of autoimmunization against the sperm head, sperm fusion with oolemma is not impaired after sperm preincubation with TEST-yolk buffer, while an impairment of the ZP-binding is demonstrable in half cases, when non-immunologic factors are excluded. A substantial role in this interference is likely exerted by IgG antibodies transuded from the blood into the genital tract.

Objective: To determine the relationship between acrosome reactions and hamster egg penetration after ionophore challenge in nonteratozoospermic semen samples., Setting: A tertiary care center, the Andrologic Clinic, Department of Internal Medicine, University of L'Aquila., Patients: Twenty-five nonteratozoospermic patients with infertile marriages and nine fertile men., Interventions: The hamster egg penetration assay and the acrosome reaction assessment were performed on capacitated sperm suspensions in basal conditions and after ionophore challenge with ionomycin., Main Outcome Measure: The relationship between the acrosome reactions and the hamster egg penetration was analyzed., Results: Although in basal conditions the spontaneous acrosome reaction rate was not correlated with the hamster egg penetration, after ionophore challenge both the induced acrosome reaction rate and the acrosome reaction increase (induced minus spontaneous acrosome reaction rate) correlated with the hamster egg penetration. The highest correlation was observed between the induced acrosome reaction rate and the penetration index. Considering a penetration index = 2 as the threshold of a good response of the hamster egg penetration assay to the ionophore challenge, significantly lower acrosome reaction rates and increases were associated with poor hamster egg penetration. However, different penetration indexes occurred at the same level of induced acrosome reaction rate., Conclusions: The capability of sperm to react after ionophore challenge reflects to some extent the capability of the acrosome-reacted sperm to undergo the changes of the plasma membrane that are necessary for sperm-oocyte fusion. The simple evaluation of the acrosome reactions may represent a useful complement rather than a substitute for the hamster egg penetration assay in monitoring the responses of human sperm to the ionophore challenge. An impaired inducibility of the acrosome reactions may account for poor hamster egg penetrations exhibited by nonteratozoospermic semen samples.

Objective: To determine if a low acrosin activity in otherwise normal ejaculate from infertile patients is associated with an impairment of sperm functions involved in the hamster egg penetration., Setting: A tertiary care center, the Andrology Clinic, Department of Internal Medicine, University of L'Aquila., Patients: Nine infertile patients with low acrosin activity in otherwise normal ejaculate (including normal immunoreactivity for acrosin) were studied; nine fertile men served as a control group. The two groups were homogeneous for seminal parameters., Interventions: The hamster egg penetration assay and the acrosome reaction rate assessment were performed on capacitated sperm suspensions in basal conditions and after ionophore challenge., Main Outcome Measures: The penetration rate, the penetration index, and the acrosome reaction rate were measured and compared between the groups., Results: No penetration was achieved in seven patients and a low penetration was achieved in two cases. The difference with the control group was significant. The ionophore challenge was associated to penetration of hamster eggs in seven of nine patients, but the penetration index was significantly lower than the controls. Acrosin activity was correlated to hamster egg penetration. Both spontaneous and induced acrosome reaction rate were similar in the two groups., Conclusions: A low sperm acrosin activity in otherwise normal ejaculate is associated with an impaired hamster egg penetration. This impairment does not seem to be due to altered dynamics of acrosome reaction.

Problem: Following the demonstration that antisperm antibodies do not affect the spontaneous acrosome reactions (AR) of human sperm used for the hamster egg penetration assay (HEPA), we evaluated the effect of the ionophore challenge on HEPA and AR of antibody-coated sperm., Method: Motile sperm suspensions from donors were exposed to serum and seminal plasma samples with sperm-head-directed antibodies, washed, capacitated, and challenged with ionomycin., Results: The proportion of acrosome-reacted sperm, assessed by fluoresceinated Pisum sativum agglutinin and supravital stain Hoechst 33258, was significantly higher in the antibody-exposed sperm than in the controls (47.0 +/- 13.2% vs. 39.3 +/- 12.1, respectively; P < 0.05). However, the hamster egg penetration rate and index were not significantly different between antibody-exposed sperm and controls (98.0 +/- 3.7% vs. 85.9 +/- 16.2% and 6.64 +/- 4.1 vs. 6.81 +/- 4.8, respectively). Only one of eight test samples with antisperm antibodies, where the highest increase of AR rate compared with controls was observed (55.0% vs. 35.6%) produced a substantial increase of penetrations per oocyte (7.4 vs. 4.1)., Conclusion: The ionophore challenge enabled us to recognize a subtle effect of anti-sperm antibodies on the dynamics of acrosomal loss, the clinical relevance of which needs to be elucidated.

Objective: To determine if the overcoming of the cervical mucus barrier removes the interference of sperm-bound antibodies with fertility., Design: Prospective case series., Settings: University-based intrauterine insemination (IUI) homologous program., Patients: Nineteen patients with all spermatozoa in the ejaculate coated by antisperm antibodies. As control group, 86 consecutive patients without antisperm antibodies, treated for oligoasthenozoospermia or mucus hostility., Interventions: Intrauterine inseminations (at least 3 attempts per couple)., Main Outcome Measures: The outcome of IUIs, demographic, and seminal parameters were compared between the two groups., Results: No pregnancy occurred in the couples with male immunological infertility, treated by 110 IUIs. Twenty-three pregnancies occurred in 22 (25.6%) of the control group couples who were treated by 411 IUIs. In the group of patients without antisperm antibodies, we demonstrated that the pregnancy rate (PR)/couple in oligoasthenozoospermia without teratozoospermia was similar to that achieved in normozoospermia (35% versus 38.9%), whereas it was significantly affected by teratozoospermia (3.6%). Only three patients with antisperm antibodies had teratozoospermia. Comparing the PR per couple and per cycle between the two groups of patients (with and without antisperm antibodies), excluding the patients with teratozoospermia, significant differences resulted (P less than 0.005 and P less than 0.005, respectively). The motile sperm count was not significantly different between the two groups, which also resulted to be homogeneous for demographic data. Moreover, the motile sperm count was not different between the patients with and without antisperm antibodies, who had successful IUI., Conclusions: The analysis of this trial suggests that the failure of IUI in the treatment of male immunological infertility is imputable to antisperm antibodies when they involve all spermatozoa, regardless of semen quality.