Zona-free Hamster Oocyte Penetration Test Research Articles

Adhesion molecules and matrix proteins on human spermatozoa.

Ejaculated spermatozoa and spermatogenic cells express alpha- and beta-chains of beta 1, 3 and 4 integrins as well as their ligands fibronectin and laminin in an extended intra- and interindividual variation and in different patterns of location. The mRNA transcripts of these molecules were detectable by nested polymerase chain reaction in the spermatozoa. The conclusion of a functional competence of these adhesion molecules (AM) was supported by their relation to the results of the zona-free hamster oocyte penetration (HOP) test, the in vitro fertilization of human oocytes and cell attachment assays. AM labelling was influenced by the disintegration of the sperm plasma membrane, especially in seminal plasma, by progesterone, human follicular fluid and microorganisms, but was barely modified by sperm cryopreservation. Despite substantial advances in the knowledge about sperm adhesion molecules, many questions remain to be answered.

Platelet activating factor improves the in vitro penetration of zona free hamster eggs by buffalo ( Bubalus bubalis) spermatozoa

Twelve buffalo bulls of Murrah breed, selected on the basis of their conception rates, were classified into low-, moderate- and high-fertility groups. Frozen semen was thawed and treated with 200 μM platelet activating factor (PAF) for 15 min at 37 °C and 5% CO 2. In both treated and control (no PAF) semen samples (five replicates per bull), the following were assessed: motility, acrosome reaction (AR) evaluation (for 10 replicates of each bull), and zona-free hamster oocyte penetration test—to determine aspects of fertilization in vitro, viz., sperm attached per ovum (SA/O), fertilization percent (FP), fertilization index (FI), and polyspermic ova (PO). There was an effect of group ( P < 0.01) on all parameters; all except motility were increased by PAF treatment. However, the group X treatment interaction was not significant for any parameter. The overall mean values of motility, AR, SA/O, FP, FI, and PO, for controls, treated spermatozoa and (net change) were: 42.89 ± 0.85, 36.65 ± 0.85, (−6.24); 28.94 ± 0.46, 61.44 ± 0.58, (32.50); 126 ± 2, 145 ± 2, (19); 74.21 ± 1.59, 89.11 ± 1.18, (14.90); 0.79 ± 0.02, 1.10 ± 0.03, (0.31) and 5.22 ± 1.22, 21.69 ± 1.88, (16.47)%, respectively. In conclusion, PAF significantly increased the AR and other aspects of fertilization, despite a small reduction in motility.

Effect of two ram sperm capacitating media on acrosome reaction and zona-free hamster oocyte penetration test

An in vitro zona-free hamster oocyte penetration test was utilized in 24 trials in order to evaluate the capacitation media used for ram sperm. A pool of fresh semen was collected from three crossed breed rams. Two semen drops were washed by centrifugation and incubated in high ionic strength treatment (HIS) or in a defined medium with HEPES, on heat ewe serum and heparin. After the incubation to promote capacitation, simplified triple-stain technique was used to evaluate the spontaneous acrosome reaction of the capacitated sperm. Superovulation in 96 golden hamsters was induced by PMSG and hCG. The oocytes were treated with hyaluronidase and trypsin to remove, respectively, the cumulus cells and the zona pellucida. Oocytes and capacitated sperm were incubated during 3 hours for further penetration. Then, oocytes were fixed and stained, being evaluated under phase contrast microscope. No significant statistical difference (p >; 0.05) was found between media, concerning the penetration rate of the capacitated sperm and between number of sperm viable with acrosome reaction after the capacitation treatment using two different media. It was concluded that both media utilized were effective in capacitating ram sperm.

Adhesion molecules of spermatozoa mediate likely sperm-oocyte interactions

Ejaculated spermatozoa and spermatogenic cells express α- and β-chains of β1-, β3- and β4-integrins as well as their ligands laminin and fibronectin. These adhesion molecules (AM) showed an extended intra- and interindividual variation and different patterns of location. The mRNA transcripts of the AM were detectable by nested polymerase chain reaction in the spermatozoa. The conclusion of a functional competence of the AM was supported by the correlation of the AM-expression on the spermatozoal surface after the acrosome reaction with three function tests: (i) the zona-free hamster oocyte penetration (HOP) test, (ii) the in vitro fertilization of human oocytes and (iii) the cell attachment assays. AM labelling was modified by progesterone, human follicular fluid, disintegration of the sperm plasma membrane in seminal plasma, and micro organisms. In contrast, the sperm cryopreservation did barely influence the labelling of spermatozoal AM.

Effects of human oviductal cell coculture on various functional parameters of human spermatozoa

Objective: To investigate the effect of human oviductal cells on various sperm functions in vitro. Design: Controlled experimental laboratory study. Setting: University gynecology unit. Patient(s): Women undergoing tubal ligation or hysterectomy and men who were visiting our subfertility clinics. Intervention(s): Coculture of oviductal cells with human spermatozoa in vitro; sperm functions were determined after coculture. Main Outcome Measure(s): Capacitation, acrosome reaction, zona binding, and oocyte fusion. Result(s): Oviductal cells and conditioned medium induced more spermatozoa to capacitate than did control medium. Although there was no difference in the spontaneous acrosome reaction between any of the groups, the coculture group had a lower percentage of acrosome-reacted spermatozoa after calcium ionophore challenge than did the control and conditioned medium groups. Coculture and conditioned medium treatment reduced the number of spermatozoa bound to the zona pellucida. The penetration rate and penetration index of the control spermatozoa in the zona-free hamster oocyte penetration test were significantly higher than that of the cocultured or conditioned medium–treated spermatozoa. Conclusion(s): Human oviductal cells promoted capacitation, stabilized the acrosome, and suppressed binding to the zona pellucida and fusion with the oocyte in vitro.

The role of sperm creatine kinase in the assessment of male fertility

The lack of reliable methods to assess sperm fertilizing potential has been a long-standing problem for infertile couples and for their physicians. The most widely used tests, the measurements of sperm concentrations, motility, velocity and morphology in the ejaculate, are of limited utility. Indeed, following intrauterine insemination, a treatment that compensates for low motile sperm concentrations, there were no significant differences found in semen parameters among those who did or did not achieve pregnancies. Other available assays probing for selected sperm functions, such as membrane integrity, acrosome enzyme activity, bovine cervical mucus penetration test, zona-free hamster oocyte penetration test and sperm binding to various carbohydrates,10–13have all failed thus far to consistently predict male fertility. It became increasingly obvious that there was a need to identify cellular markers of sperm quality and fertilizing potential.

* - The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration

To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

Comparative study on the use of human follicular fluid or egg yolk medium to enhance the performance of human sperm in the zona-free hamster oocyte penetration assay

A prospective study was conducted on 61 unselected semen samples from infertile patients to compare the efficacy of human follicular fluid (hFF) or TEST-egg yolk medium treatment in enhancing sperm performance in the zona-free hamster oocyte penetration (HOP) test. Both hFF and TEST-egg yolk enhanced penetration scores significantly in the HOP test in comparison with the control swim-up sperm preparation. The TEST-egg yolk medium treatment, however, exhibited the highest penetration scores, and there were higher percentages of preparations passing the 10 or 20% penetration rate cut-off point with sperm exposed to the TEST-egg yolk medium than those exposed to hFF. These results indicate that modification of the original HOP-test protocol by incorporation of TEST-egg yolk medium gives a greatly increased penetration rate when compared to the addition of hFF in such a system.

Standard sperm morphology as a predictor of male fertility potential.

There are many clinicians who believe that standard sperm morphological criteria as determined by the World Health Organization (WHO) may be one of the most reliable indices of male fertility potential. Many previous studies of sperm morphology have lacked meticulous correction of all female infertility factors in forming their conclusions. Other studies may have compared morphological analysis to either the zona-free hamster oocyte penetration test or fertilization rates with in vitro fertilization/embryo transfer (IVF-ET). The former may not be a valid "gold standard" with which to compare, and the latter might not truly reflect the in vivo situation in view of the markedly different type of sperm-oocyte interaction. These data failed to find a strong correlation of abnormal sperm morphology (as determined by WHO criteria) to identify the subnormal male. When motile densities were normal (greater than 10 x 10(6)/ml), 87% of the female partners of men with normal morphology achieved pregnancies within 6 months compared to 82% with subnormal morphology. Even subnormal motile densities and morphology did not predict the subfertile male. These data strongly suggest that the normals established by WHO for morphology are inadequate to distinguish normal from subnormal. Perhaps increasing the percentage of abnormal sperm morphology to be considered subfertile might prove more useful, or perhaps using another type of morphological assessment might be more effective.

Cyclic-adenosine monophosphate (cAMP) is elevated during capacitation of bovine sperm by heparin or oviduct fluid

Objective: To investigate the effect of human oviductal cells on various sperm functions in vitro.Design: Controlled experimental laboratory study.Setting: University gynecology unit.Patient(s): Women undergoing tubal ligation or hysterectomy and men who were visiting our subfertility clinics.Intervention(s): Coculture of oviductal cells with human spermatozoa in vitro; sperm functions were determined after coculture.Main Outcome Measure(s): Capacitation, acrosome reaction, zona binding, and oocyte fusion.Result(s): Oviductal cells and conditioned medium induced more spermatozoa to capacitate than did control medium. Although there was no difference in the spontaneous acrosome reaction between any of the groups, the coculture group had a lower percentage of acrosome-reacted spermatozoa after calcium ionophore challenge than did the control and conditioned medium groups. Coculture and conditioned medium treatment reduced the number of spermatozoa bound to the zona pellucida. The penetration rate and penetration index of the control spermatozoa in the zona-free hamster oocyte penetration test were significantly higher than that of the cocultured or conditioned medium–treated spermatozoa.Conclusion(s): Human oviductal cells promoted capacitation, stabilized the acrosome, and suppressed binding to the zona pellucida and fusion with the oocyte in vitro.

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.

Relative ability of modified versions of the hamster oocyte penetration test, incorporating hyperosmotic medium or the ionophore A23187, to predict IVF outcome.

This study was designed to assess the relationship between IVF outcome and the results obtained with two modified versions of the zona-free hamster oocyte penetration test in which the spermatozoa were pre-incubated with either hyperosmotic medium or the divalent cation ionophore A23187. When the former system was used, a poor correlation with IVF outcome was observed. Samples screened prior to IVF exhibited a 60% false negative rate (failed penetration test, successful IVF), while for those assessed concurrently with IVF, the equivalent figure was 85.7%. Addition of A23187 optimized the penetration system giving higher levels of sperm--oocyte fusion and a more accurate prediction of the capacity of the spermatozoa to fertilize human ova in vitro. With this system the false negative rate was 4.3% for screened samples and 0% for those assessed simultaneously with IVF. These results suggest that the A23187-enhanced system may be of value as a screening criterion for IVF.

GROUP DISCUSSION

GROUP DISCUSSION

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

WHO protocol for the zona‐free hamster oocyte penetration test

International Journal of AndrologyVolume 9, Issue s6 p. 197-199 Free Access WHO protocol for the zona-free hamster oocyte penetration test First published: October 1986 https://doi.org/10.1111/j.1365-2605.1986.tb00953.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume9, Issues6October 1986Pages 197-199 ReferencesRelatedInformation

Application of Poisson distribution theory to the zona-free hamster oocyte penetration test to assess sperm function of men with asthenozoospermia.

Assessments of the penetrating potential of human spermatozoa were carried out using the zona-free hamster oocyte penetration test on 4 groups of subjects exhibiting normal fertility, idiopathic asthenozoospermia(less than 40% motility), asthenozoospermia associated with varicocele and oligoaesthenozoospermia (less than 20 X 10(6) spermatozoa/ml and less than 40% motility). When the Poisson model was used to correct the results of the in-vitro penetration experiments for differences in motile sperm concentration, significant differences were apparent between the normal fertile controls and all 3 categories of asthenozoospermic patient. Furthermore, the penetrating ability of the motile spermatozoa from patients presenting with a varicocele or oligoaesthenozoospermia was significantly less than that for the group in which asthenozoospermia was the only detectable defect. These results emphasize the practical significance of the Poisson model in the analysis of male fertility and demonstrate that the asthenozoospermic condition is associated with a significant reduction in the fertilizing potential of the motile spermatozoa.

Diagnostic value of the zona-free hamster oocyte penetration test and sperm movement characteristics in oligozoospermia.

A group of 27 oligozoospermic patients were followed up for 4.2 +/- 1.1 years in a prospective study designed to determine which aspects of semen quality are of value in the diagnosis of fertility. The semen analysis included the zona-free hamster oocyte penetration test, the assessment of sperm movement characteristics by time exposure photomicrography and a conventional semen profile. During the follow up period, 7 patients (25.9%) initiated a pregnancy. The identity of these fertile subjects could not have been ascertained by any single criterion of sperm movement or any feature of the conventional semen profile. The zona-free hamster egg penetration rates were also of little value in this respect since 4 of the 7 fertile patients scored 0% in this assay. Using a multivariate discriminant analysis, however, a combination of 7 criteria was identified, including hamster oocyte penetration and elements of both sperm movement and the semen profile, which could predict the fertility of these patients with 85.2% accuracy.