Lani J. Burkman

The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential * † ‡

The authors present their initial results with the hemizona assay (HZA), which was developed to predict the fertilizing potential of spermatozoa. The HZA uses the matching halves of a human zona pellucida from a nonfertilizable and nonliving oocyte, providing an internal control on zona-to-zona variability. Maximal binding of human sperm to the hemizona usually occurred after 4 to 5hours of coincubation. Sperm from fertile men exhibited significantly higher binding capacity to hemizonae compared with sperm from men who had fertilization failure during in vitro fertilization (IVF) treatment. The HZA index is calculated as follows: (bound sperm from subfertile male) ÷ (bound sperm from fertile male)×100. These findings demonstrate that the HZA may be a useful diagnostic tool in male infertility evaluations.

DISCRIMINATION BETWEEN NONHYPERACTIVATED AND CLASSICAL HYPERACTIVATED MOTILITY PATTERNS IN HUMAN SPERMATOZOA USING COMPUTERIZED ANALYSIS

The objective identification of human hyperactivated (HA) motility has been controversial. The present study defines new criteria for automatic sorting of all HA patterns that are consistent with the classical descriptions. Sperm from fertile men were prepared in Hams F-10 medium. Based on the authors previously published slow-motion method, 342 sperm were selected that represented the non-HA, circling high-curvature, thrashing, star, and helical patterns. Automatic sorting for HA was achieved with the use of three criteria: linearity less than or equal to 65 and velocity greater than or equal to 100 microns/s and head displacement greater than or equal to 7.5 microns. Additional criteria were determined for identifying each HA class separately. Whether using the new or old method, the incidence of HA is significantly associated with multiple fertilization endpoints.

Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome

The hemizona assay (HZA) was used in a prospective, blinded study to assess the relationship between tight sperm binding in the HZA and sperm fertilizing ability in in vitro fertilization (IVF). In each controlled assay, the authors compared sperm binding of proven fertile men with that of patients undergoing IVF. Human oocytes stored in a salt solution were used in the study, and binding results were correlated with the fertilization rate of preovulatory oocytes during IVF. Patients with poor fertilization rates in IVF had significantly lower binding than those cases with successful fertilization (7.3 +/- 1.4 versus 62.1 +/- 10.9, respectively; mean +/- standard error, P less than 0.02). Based on current standards, the HZA was able to predict fertilization accurately in 26 of 28 cases (sensitivity of 83%, specificity of 95%, positive predictive value of 83%). The authors conclude that the HZA is a valuable tool for evaluating dysfunctional sperm-zona pellucida binding, with good predictive value for fertilization in vitro.

The hemizona assay (HZA): A predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment

The hemizona assay (HZA) was developed to assess human sperm fertilizing potential. This blinded study investigated the relationship between sperm binding to the hemizona and in vitro fertilization (IVF) success (36 patients). Nonliving human oocytes were recovered from excised ovaries and stored. Each zona pellucida was cut into equal hemispheres by micromanipulation. For the HZA, one droplet exposed a hemizona to abnormal spermatozoa, while the control droplet contained the matching hemizona and spermatozoa from normal semen. After 4 hr, the number of tightly bound spermatozoa was counted. Binding to the hemizona was significantly higher for those having IVF success (mean of 36.1±7, versus 10.4±4 from the failure group;P<0.05). Fewer sperm from the failure group had a strictly normal morphology (3,2 versus 12.7%;P<0.05, Kruger method). Tight zona binding was significantly correlated with the percentage motile sperm, percentage normal morphology, and seminal sperm concentration. These results enhanced our confidence that the HZA is diagnostic for identification of patients at high risk of failing to achieve fertilization in vitro.

Human follicular fluid stimulates hyperactivated motility in human sperm

Since human follicular fluid (FF) is known to enhance the acrosome reaction and capacitation, we investigated whether hyperactivated motility is stimulated by FF. Follicular fluid-treated sperm exhibited a threefold increase in hyperactivation compared with the controls. The use of fetal cord serum in the medium, instead of bovine serum albumin, supported the same high levels of hyperactivation, although the peak occurred at 3 hours rather than 5 hours of capacitation. When sperm were treated with a steroid-rich fraction of the FF, hyperactivation was stimulated to the same degree as with whole FF. In contrast, no stimulation occurred when sperm were treated with a FF fraction stripped of steroids. The FF enhancement of hyperactivation in vitro could augment the fertilizing capacity of subfertile sperm samples, providing also a glimpse of possible in vivo events as sperm traverse the FF-laden cumulus oophorus.

Male factor evaluation in in vitro fertilization: Norfolk experience

Thirty-three patients from the in vitro fertilization (IVF) program at Norfolk are critically reviewed. A battery of tests was designed and an endocrine investigation was carried out on these patients. The fertilization rate for preovulatory oocytes was lower than in the normal male population (39.6% versus 88.6%). When total concentration of sperm with rapidly progressive motility was less than 6 X 10(5), to fertilize several eggs together the fertilization rate was zero. No fertilization was obtained when the number of sperm with rapidly progressive motility recovered after the separation was less than 1.5 X 10(6). The hamster zona-free oocyte penetration test correlated well with the human IVF system. The other parameters investigated did not show good correlation. When fertilization was achieved, the results of the IVF procedure in the series reviewed rendered a 30.8% pregnancy rate per transfer in 26 transfers. Fifty percent of the pregnancies were normal (either ongoing or delivered). Thirty-seven percent were preclinical miscarriages, and 12.5% were clinical abortions. In the abnormal male population, higher concentrations of sperm per egg should be used for insemination for achievement of optimum fertilization rates. Once fertilization is obtained, the results do not differ substantially from the IVF population at large.

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution

Summary. The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.

Defining the valid hemizona assay: accounting for binding variability within zonae pellucidae and within semen samples from fertile males

OBJECTIVE To achieve a better understanding of the variability in sperm and oocyte binding capacities will optimize use of the hemizona assay (HZA) as a predictor of sperm function. DESIGN Limitations of the HZA were more clearly delineated by current studies: (1) variability of sperm binding capacity of men over a 90-day interval; (2) variability of sperm binding using different oocytes; and (3) lower limits of the number of sperm bound from the fertile control in two laboratories. PATIENTS Semen was obtained from proven fertile men and one subfertile individual. MAIN OUTCOME MEASURE The number of sperm tightly bound to the hemizona were measured and compared. RESULTS In the initial study, 6 fertile control men exhibited a similar degree of variability in zona binding when studied over a 90-day interval. Average sperm binding for individuals ranged from 68 to 127. Second, 3 of the 15 simultaneous assays showed very low numbers of sperm bound, indicating that 20% of the zonae had poor binding. Third, from 18 men who had 0% fertilization in an in vitro fertilization system using mature oocytes, evaluation of their sperm by HZA was performed. The sperm bound poorly and the 95% confidence interval was 20 sperm bound. Thus, the fertile controls should bind greater than 20 sperm to distinguish them from the infertile group in the HZA system resulting in a valid assay. CONCLUSIONS With these guidelines, applications of the HZA may be made with greater reassurance of a valid bioassay of sperm fertilizing potential.