Daniel R. Franken

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.

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.

Normal sperm morphology and chromatin packaging: Comparison between aniline blue and chromomycin A3 staining

The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra‐ and inter‐assay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra‐ and inter‐assay assessments were <12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r=0.40 (P=0.001) and r=0.33 (P=0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (<4% normal forms) when compared with normozoospermic men (>14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.

Sperm morphology: assessing the agreement between the manual method (strict criteria) and the sperm morphology analyzer IVOS * †

OBJECTIVES To correlate the percentage normal morphology reported by different observers and a computerized method (IVOS; Hamilton-Thorne Research, Beverly, MA) on a slide-by-slide basis using strict criteria: [1] Experienced observer (T.F.K.) versus experienced observer (R.M.), [2] experienced observer (T.F.K.) versus sperm morphology analyzer (IVOS), and [3] repeatability of normal and abnormal cells (IVOS). DESIGN SETTING, PATIENTS: Slides from 30 different patients from the Tygerberg IVF program were selected randomly. Microscopic fields and sperm cells were chosen randomly and percent normal morphology was recorded (objectives 1 and 2). The same slides were used and a cell-by-cell repeatability was done as outlined (objective 3). RESULTS Experiment 1 (objective 1): there was no significant bias between T.F.K. and R.M. The limits of agreement were 8.6% and -7.3%. The SDs were not significantly different (P = 0.1283). The Spearman correlation coefficient between readers was 0.83. Experiment 2 (objective 2): the same findings were reported but the limits of agreement were 12.1% and -15.5%. The Spearman correlation coefficient was 0.85. The limits of agreement was tighter below 20% normal forms (+8.4 and -6.6). Experiment 3 (objective 3) (repeatability): 255 cells were analyzed three times in succession. Estimating pairwise agreement, the kappa statistic for the pairs are 0.85, 0.80, and 0.85, respectively, which compares favorably with the second canonical moment of 0.8329 (kappa = 0.83). DISCUSSION The computers ability to classify normal morphology per slide is promising. Below 20% normal forms, the limit of agreement is tight. Because of the 6% higher reading compared with the manual method, different thresholds possibly will be developed to identify subfertile from fertile patients. The computer gives excellent repeatability of normal and abnormal cells. Based on results obtained, this system can be of clinical value both in IVF units and andrology laboratories but more clinical data is required in this field.

Clinical significance of human sperm-zona pellucida binding

OBJECTIVE To assess the relationship between sperm morphology and motion parameters and sperm-zona pellucida (ZP) binding capacity under hemizona assay (HZA) conditions and to determine the discriminatory power of the HZA for the prediction of in vitro sperm fertilizing ability. DESIGN Prospectively designed study. SETTING Academic tertiary centers. PATIENT(S) One hundred ninety-six couples undergoing IVF therapy participated in this study. INTERVENTION(S) Hemizona assay and IVF results were determined for each couple. MAIN OUTCOME MEASURE(S) Computerized sperm motion analysis, sperm morphology (strict) criteria), and HZA results were correlated with fertilization outcome. RESULT(S) Among sperm parameters from the original ejaculates, morphology was the best predictor of sperm-ZP binding ability; hyperactivated motility was the best predictor of HZA results after swim-up separation of the motile sperm fractions. The HZA index provided the highest discriminatory power for fertilization success/failure, with an overall accuracy of 86%. CONCLUSION(S) Sperm morphology and hyperactivated motility showed a high correlation with the capacity of sperm to achieve tight binding to the ZP. The excellent positive and negative predictive values of the HZA for fertilization outcome provide additional support for the use of this functional bioassay in the decision-making process within the assisted reproduction setting.

Localization of Tyrosine Phosphorylated Proteins in Human Sperm and Relation to Capacitation and Zona Pellucida Binding

Abstract Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.

The hemizona assay: Its role in identifying male factor infertility in assisted reproduction

OBJECTIVES To identify male factor infertility among a group of patients in an assisted reproductive program (phase 1) and to evaluate the hemizona assay (HZA) in the diagnosis and prognosis of such a program (phase 2). DESIGN The IVF performance of normal gametes in the Tygerberg program were critically evaluated. Female patients were classified as pure tubal factor infertility, having a normal FSH:LH ratio on day 3 of the menstrual cycle. All participating women produced three or more preovulatory oocytes at retrieval and were inseminated with sperm considered normal by all present diagnostic criteria. The total and normal fertilization rate thresholds were defined in that group. Using those thresholds, couples tested for sperm binding in the HZA (n = 48) were used and divided into two groups according to their fertilization rates, namely group 1, low fertilization (< 55%) and group 2, normal fertilization (> 55%). SETTING University-based tertiary care center. PATIENTS Ninety-nine couples (589 oocytes) with pure tubal factor infertility and normal male factor were used in phase 1. Forty-eight couples with normal and abnormal male factors that had both HZA performed and IVF treatment were included in phase 2. RESULTS Investigation of the performance of normal gametes in 99 couples (589 oocytes) revealed the total fertilization rate (total number of oocytes fertilized/total number of oocytes inseminated) was (mean +/- SD) 88.6% +/- 16.8% and the normal fertilization rate (total number of oocytes with normal fertilization/total number of oocytes inseminated) was 81.3% +/- 22%. The minimum total fertilization rate that can be considered normal in the Tygerberg program using mean--2 SD is therefore 55% and for normal fertilization rate is 37%. The group with low fertilization rate (< 55%) showed a mean hemizona index (HZI) significantly lower; nevertheless, the distribution overlapping indicates a low discriminating power of the HZA. A sensitivity of 75% and a specificity of 75% were found; the positive and negative predictive values were 81% and 68%, respectively. CONCLUSIONS The results indicated the HZA and HZI contribute important information and can serve in conjunction with other semen characteristics as useful tools during the diagnosis of the male factor in assisted reproduction.

Defective sperm decondensation: a cause for fertilization failure

Summary. The study aimed to evaluate the role of chromatin packaging (CMA3 staining), sperm morphology during sperm‐zona binding, sperm decondensation and the presence of polar bodies in oocytes that failed in vitro fertilization (IVF). The percentage CMA3 staining categorized the data into three groups,  < 44%, n = 10;  ≥ 44–59%, n = 10; and ≥60%, n = 29. Morphology groups were ≤ 4% (n = 11);  > 4–14% (n = 19); and >14% (n = 19). One hundred and seventy‐two oocytes that failed IVF were evaluated for sperm‐zona binding, ooplasma penetration and sperm decondensation. Odds ratio analyses indicated that being in the ≥60% CMA3 staining group resulted in a 15.6 fold increase in the risk of decondensation failure, relative to CMA3 staining of <44%. For morphology, there was a 2.17 fold decrease in the risk of fertilization failure in the morphology group with >4–14% normal cells, while it increased 2.45 fold for the morphology group with ≤4% normal cells. Using CMA3 fluorescence to discriminate, 51% of the oocytes in the group with elevated CMA3 fluorescence had no sperm in the ooplasma compared to 32% and 16% penetration failure in the CMA3 staining groups ≥44–59% and <44%, respectively. Sperm chromatin packaging quality and sperm morphology assessments are useful clinical indicators of human fertilization failure. Immunofluorescence techniques could be used to provide a clear diagnosis of failed fertilization.

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa.

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 μm² vs. 18.8 ± 1.4 μm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Human preovulatory oocytes have a higher sperm-binding ability than immature oocytes under hemizona assay conditions: evidence supporting the concept of "zona maturation".

OBJECTIVE The purpose of this study was to investigate the sperm-binding potential of human oocytes at different stages of nuclear maturation under hemizona assay (HZA) conditions. DESIGN This was a prospective study designed in a blinded fashion. SETTING Academic research environment approved by the Institutional Review Board. PATIENTS Surplus oocytes, donated by patients undergoing in vitro fertilization therapy after gonadotropin stimulation, were analyzed. Semen from a fertile donor was used in all assays. INTERVENTIONS Five groups of oocytes were considered: (1) immature, prophase I; (2) metaphase I; (3) metaphase II; (4) inseminated, unfertilized metaphase II; and (5) immature, prophase I oocytes matured in vitro to metaphase II. Oocytes were stored in salt solution (pH 7.2) and microbisected before assay. MAIN OUTCOME MEASURE(S) Tight binding of sperm to the zona pellucida under HZA conditions was evaluated after 4 hours of gametes coincubation. RESULTS Metaphase II oocytes (groups 3 and 4) had significantly higher binding than other groups (P = 0.0001). The mean value of the difference between the two halves (hemizona) was not significant, thus showing a small intra-assay variation for all maturational stages. CONCLUSIONS Full meiotic competence of human oocytes is associated with an increased zona pellucida-binding potential.