Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function*

Transcription

1 FERTILITY AND STERILITY Copyright 1996 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function* Sergio Oehninger, M_D.t:J: Elvira Hinsch, Ph.D. Susanne Pfisterer, M.D. Lucinda L. Veeck, D.Sci.t Paul Kolm, Ph.D.t Wolf-Bernhard Schill, M.D. Gary D. Hodgen, Ph.D.t Klaus-Dieter Hinsch, M.D.II The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia, and Zentrum fur Dermatologie und Andrologie, Justus-Liebig-Universitat Giessen, Giessen, Germany Objective: To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. Design: Prospectively designed, blinded, internally controlled study. Setting: Tertiary care academic center. Patients: Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. Interventions: Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3-6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3-7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-anti peroxidase method and diaminobenzidine as color reagent. Results: A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3-6- treated hemizonae than patients with sperm factor. Conclusions: These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting. Fertil Steril 1996;65: Key Words: ZP3, antiserum, oocyte, IVF Disorders of fertilization observed during IVF therapy may be due to subtle or more severe gamete Received March 6, 1995; revised and accepted August 23, * Supported in part by funds from the Bundesministerium fur Forschung und Technologie, Germany and by an institutional grant from Eastern Virginia Medical School, Norfolk, Virginia. t Department of Obstetrics and Gynecology and Office of Biostatistics, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School. Zentrum fur Dermatologie und Andrologie, Justus-Liebig Universitiit Giessen. II Reprint requests: Klaus-Dieter Hinsch, M.D., Zentrum fur Dermatologie und Andrologie, Justus-Liebig-Universitiit Giessen, Gaffky-Str 14, Giessen, Germany (FAX: ). abnormalities also responsible for pregnancy failure during natural reproduction (1,2). Among these disorders, total or partial loss of fertilization capacity particularly are disturbing for patients and their physicians. Contemporary methods of oocyte micromanipulation, particularly intracytoplasmic sperm injection, currently are being used successfully to assist fertilization in some of these couples (3). Nevertheless, the basic underlying gamete defects responsible for failed fertilization are very poorly understood in the majority of cases. In couples with repeated cycles offertilization failure, sperm and/or oocyte defects are suspected (4). We have observed a high frequency of severe sperm Oehninger et al. ZP3 antiserum and oocyte defects 139

2 abnormalities in some of these patients. The most common male gamete defects associated with fertilization failure are poor morphology and several dysfunctional capacities, including motility abnormalities, acrosome reaction deficiencies, and an abnormal ability to interact with the zona pellucida (ZP) and/ or the oolemma (1, 2, 5). The hemizona assay, an internally-controlled, homologous test that evaluates tight binding of sperm to the ZP, has been instrumental in the identification of some of these dysfunctions (1, 2, 6-8). Oocyte abnormalities are present in ~25% of cycles with failed fertilization (4). A degenerative appearance, the presence of refractile bodies, and a vacuolated ooplasm are morphological features often observed in oocytes that fail to fertilize (4, 9, 10). Defects of cytoplasmic factors of the oocyte, mitochondria-cell respiratory function, and the nuclearchromosomal apparatus probably also are involved as causative factors (11). A rather high, yet variable rate of chromosomal disorders has been reported in human unfertilized oocytes (12). The role of the ZP, a structure responsible for the initial specific spermoocyte recognition event, has not been evaluated comprehensively in patients with failed fertilization. So far, studies have been hindered by the difficulty in obtaining a specific tool to examine the human ZP structure and function. Full-length complementary DNA clones of human ZP protein 3 (ZP3) have been isolated and the genomic loci of human ZP3 and ZP2 have been well characterized (13). Tight binding of sperm to the egg is thought to be supported by ZP3 and complementary sperm-binding proteins present in the sperm plasma membrane. ZP3 also triggers the acrosome reaction, whereas ZP2 seems to function as a second ligand to maintain the binding of acrosome-reacted spermatozoa to the ZP (14-16). Recently, Hinsch et a1. (17) have generated antisera against synthetic ZP3 peptides and identified ZP3 protein in various mammalian oocytes. They demonstrated that one of the antisera, antiserum 6 (AS ZP3-6), reacted strongly with human ZP3 protein. This antiserum was generated against a synthetic peptide conserved throughout species (conserved ZP3 epitope) (17). In the present study, we used AS ZP3-6 as a novel tool to examine ZP integrity and function in patients who demonstrated total or partial fertilization failure during IVF treatment. Patients MATERIALS AND METHODS A total of 37 patients undergoing IVF treatment during Norfolk series 53 and 54 (January through June 1994) were evaluated prospectively. They were included in the study based upon the following criteria: a minimum of two mature, preovulatory oocytes (metaphase II) obtained at the time of egg retrieval and partial or total failed fertilization of preovulatory oocytes using standard culture and insemination conditions. Failed fertilization was diagnosed when absence of pronuclear formation was demonstrated 20 hours postinsemination (4). Oocytes with delayed fertilization or polyspermia were not included in the study. The etiology of infertility per individual couple was diagnosed as follows: male infertility (n = 17), tubal factor (n = 12), endometriosis (n = 4), and unexplained (n = 4). The females' mean ages were 33.4 ± 3.6 years (range 28 to 42 years). Male factor infertility was diagnosed when semen abnormalities were demonstrated following guidelines established at Norfolk, i.e., sperm concentration < 20 X ml (oligozoospermia), progressive motility < 30% (evaluated by computerized semen analyzer, asthenozoospermia), and/or <4% normal morphology (assessed by strict criteria, teratozoospermia) in the original ejaculate (5, 18, 19). Ovarian stimulation for the purpose of multifollicular development was performed using two standard protocols (20): patients :5 38 years of age or with a basal day 3 serum FSH level :5 15 mivlml (RIA; Leeco Diagnostics, Inc., Southfield, MI; conversion factor to SI unit, 1.00) (n = 33 patients) were stimulated using a luteal suppression protocol with leuprolide acetate (Lupron; TAP Pharmaceuticals, Abbott Park, IL) and a gonadotropin combination of FSH (Metrodin; Serono Laboratories, Randolph, MA) and hmg (Pergonal; Serono) following established guidelines; patients > 39 years of age or with a basal day 3 serum FSH level ~ 16 mivlml (n = 3 patients) were stimulated with gonadotropins alone (pure FSH treatment) following protocols publish- ;. ed elsewhere (20). Human chorionic gonadotropin (10,000 IV 1M) (Steris Laboratory, Phoenix, AZ) was administered when at least two follicles were > 16 mm in diameter. Transvaginal follicular aspiration was performed 34 hours later. Gamete processing, cell culture techniques, and ET procedures were as previously described (9, 10). Oocyte maturity was classified as described by Veeck (21). Oocyte Storage and Microbisection Preovulatory oocytes showing absence of pronuclear formation (failed fertilization) were kept in culture medium for an additional 24-hour period and then stored in a hyperosmotic salt solution (consisting of 1.5 M MgCI 2, 0.1% polyvinylpyrrolidone, 140 Oehninger et ai. ZP3 antiserum and oocyte defects Fertility and Sterility

3 and 40 mm HEPES buffer, ph 7.4) at 4 C for up to 30 days. This storage method is known to preserve the biochemical, physical, and biologic properties of the ZP (1, 2, 22). Before each assay, individual 00- cytes were removed from salt storage and rinsed in medium (Ham's F-10; GIBCO Laboratory, Grand Island, NY) 5 to 10 times. Narishige micromanipulators (Narishige, Tokyo, Japan) were mounted on a phase-contrast microscope (Nikon Diaphot, Garden City, NY) and used to microbisect the oocytes into matching hemizonae as described previously (1, 2,6). Immunochemical Detection of ZP3 in Human Hemizonae Hemizonae from the unfertilized oocytes were mounted immediately to polylysine-coated eightwell glass slides (one hemizona per well). Hemizonae were air dried overnight and stored exsiccated at room temperature for several days. For immunochemical studies, hemizonae were treated with AS ZP3-6 (test) or a control antibody (AS ZP3-7) (17). AS ZP3-6 was raised against a synthetic peptide derived from amino acid sequences that are highly conserved in the structure of ZP3 from different mammalian species (and that therefore recognizes human ZP3, as demonstrated earlier); AS ZP3-7 was generated against a synthetic peptide specific for mouse ZP3 (negative control) (17). Specific binding was visualized with anti-rabbit IgG antibodies (Dako, Hamburg, Germany), the peroxidase-antiperoxidase method (PAP, Dako), and 3,3'-diaminobenzidine (DAB; Sigma, Deisenhofen, Germany) as color substrate, as described previously (17). Slides containing the hemizonae were mounted with ACCD (Baxter Diagnostics, Inc., McGraw Park, IL) and a coverslip and results of immunostaining were evaluated and photographed using a Zeiss Axioskop microscope (Zeiss, Oberkochen, Germany). Staining intensity was scored from 0 (no staining) to 10 (most intense staining) blindly by two independent observers and the results were averaged (17). Experimental Design and Statistical Analysis From each microbisected oocyte, a hemizona was treated with AS ZP3-6 (test) while its matching half was treated with AS ZP3-7 (negative control). The use of the two matching hemizona halves allowed us to perform an internally controlled study on a single egg, thereby eliminating concerns about intra-assay (intraegg) variability (1, 2, 6). A total of 104 oocytes (1 to 8 eggs per patient) were analyzed. Studies were performed in three different experiments; at each time, and in addition to the patients' oocytes, hemi- zonae derived from noninseminated oocytes from healthy egg donors (volunteers participating in the oocyte donation program that donated eggs for research under approval from the Institutional Review Board of Eastern Virginia Medical School) were used as positive controls. Two oocytes from fertile donors were examined in each of the three experiments. Staining scores with AS ZP3-6 ranged from 5 to 9; intensity of staining was used as a (positive) control to score the hemizonae from the patients' study group (exposed to AS ZP3-6 and AS ZP3-7). Patients were divided into three gamete factors based upon clinical and IVF data as follows: "sperm factor" group, consisting of patients with a diagnosis of male factor based upon the original semen evaluation and sperm characteristics at the time of IVF; "oocyte factor" group, based upon the identification of morphological oocyte abnormalities at the time of IVF (these included excessive egg cytoplasm granularity, multiple vacuoles and/or presence of refractile bodies, excluding oocytes diagnosed as degenerative) (9, 10); and an "unknown factor" group, which included those couples with a normal semen assessment and no demonstrable oocyte abnormalities. For statistical calculations the following parameters were considered: "gamete factor" (dividing patients as depicted above into three groups based upon the clinical diagnosis of sperm, oocyte, or unknown factors), "fertilization factor" (dividing patients into two groups, i.e., total or partial failure of fertilization), and "antiserum factor" (i.e., antiserum staining results (scoring) of test hemizonae versus control hemizonae). The data were analyzed by a mixed-model analysis of variance (ANOVA) with gamete factor, fertilization factor, and antiserum factor treated as fixed effects and subjects within antiserum factor (n = 37 different couples) as a random effect. Comparisons were performed between the sperm factor and oocyte factor groups; the unknown factor (possibly comprising a mixture of subtle sperm and/or oocyte defects and truly unknown factors) was analyzed separately. The influence of female's age and basal serum FSH levels on hemizonae staining results was analyzed by including these factors as covariates of oocyte staining scores. Results are presented as mean::!::: SD. P levels < 0.05 were considered significant. RESULTS Of the 37 couples, 10 had total failure of fertilization (0 fertilized oocytes of 52 inseminated oocytes or 0% fertilization rate), whereas 27 had partial failed fertilization (170 fertilized oocytes of 296 inseminated oocytes, with a fertilization rate ranging from Oehninger et a1. ZP3 antiserum and oocyte defects 141

4 Table 1 Antiserum Staining Scores for Test (AS ZP3-6) and Control (AS ZP3-7) Hemizonae No. of Failed Antiserum hemizonae Gamete fertilization factor pairs factor factor (hemizonae) Score* studied Oocyte Total Test 3.2 ± Oocyte Total Control 1.6 ± Sperm Total Test 3.68 ± Sperm Total Control 1.44 ± Oocyte Partial Test 3.25 ± Oocyte Partial Control 1.87 ± Sperm Partial Test 5.11 ± Sperm Partial Control 1.29 ± Unknown Partial Test 3.65 ± Unknown Partial Control 1.41 ± * Values are means ± SD. 20% to 85%). Within the group with total failed fertilization there were eight couples with a sperm factor (characterized by an abnormal sperm concentration, progressive motility or morphology, alone or in combination) and two couples with an oocyte factor (showing a combination of egg abnormalities, which included the presence of cytoplasmic vacuoles, excessively dark and granular cytoplasm, refractile bodies, or a degenerative appearance). Within the group with partial failed fertilization there were 7 couples with a sperm factor, 5 couples with an oocyte factor, and 15 couples with unknown factor (no demonstrable gamete abnormalities). Table 1 depicts mean hemizonae staining scores for AS ZP3-6 (test) and AS ZP3-7 (control) for patients divided according to gamete factor and fertilization factor. Gamete factors include oocyte, sperm, or unknown factors; failed fertilization factors include groups with either total failure or partial failure of fertilization. The means ± SD are presented for the antiserum factor (i.e., hemizonae exposed to test or control antiserum). Figure 1 shows the reactivity of AS ZP3-6 test and AS ZP3-7 (internal, negative control) for matching hemizonae pairs. Panel A presents a couple with sperm factor: Al shows the test hemizona and A2 shows the control, matching hemizona (scores of 8 and 2, respectively). Panel B depicts a couple with oocyte factor: Bl shows the test hemizona and B2 shows the control, matching hemizona (scores of 2 and 1, respectively). Panel C displays an oocyte from a fertile woman used as a positive control (oocyte donor): Cl shows the test hemizona and C2 shows the control hemizona (scores of 9 and 3, respectively). Table 2 presents the results of the three-way ANOVA (interactions of gamete by fertilization by antiserum factors). The effect of the antiserum factor alone (test hemizonae versus control hemizonae) was significant (P = ). This indicated that the test hemizonae mean was significantly greater than the control hemizonae mean when combined over the gamete or fertilization factors. The effect of gamete and fertilization factors alone was not significant. More importantly, the gamete by antiserum interaction also was significant (P = ), indicating a significant effect of hemizonae staining score (test versus control) depending upon the source of gamete factor (sperm or oocyte factor groups). Other interactions between factors were not significant. Table 3 presents the predicted means ± SD from the ANOVA in which hemizonae immunostaining results were modeled as a function of the gamete and antiserum factors only. The significant interaction is apparent. For the sperm factor, there was a significant difference between the test versus control hemizonae (P < ). In the oocyte factor, scores for test versus control hemizonae were not significantly different (P = 0.14). Additionally, the interaction between gamete factors (sperm versus oocyte) showed a significant difference for test hemizonae (P = ) but not for control hemizonae (P = 0.47). It must be emphasized that within individual patients in which multiple oocytes could be examined we observed either a similar, homogeneous score for all oocytes or marked score variations producing a spectrum of staining intensity. The mean for unknown factor test hemizonae was intermediate to the means for sperm factor and oocyte factor (Table 1), suggesting a combined contributing effect of both gamete factor groups. Three of 15 couples within this unknown factor group showed very low antiserum staining scores in test, similar to those observed in the oocyte factor group (data not shown). Female's age and basal serum FSH levels did not significantly influence antiserum staining scores (P = 0.3) and, consequently, had little impact on the predicted test hemizonae and control hemizonae means (data not i shown). DISCUSSION This study demonstrates the feasibility of using AS ZP3-6 as a novel tool for the evaluation of ZP integrity and function in human unfertilized oocytes. For the first time, anomalies of human ZP3 have been identified using a specific ZP3 antiserum. Furthermore, powerful statistical correlations between clinical and laboratory analyses of the female and male patients' gametes and ZP3 anomalies detected by immunostaining suggest that AS ZP3-6 can be used as a biomarker for oocyte defects that are responsible for fertilization disorders within the assisted reproduction setting. 142 Oehninger et al. ZP3 antiserum and oocyte defects Fertility and Sterility

5 Figure 1 Immunoreactivity of AS ZP3-6 (test) and AS ZP3-7 (internal, negative control) for matching hemizonae pairs for selected couples. See text for explanation. Antiserum ZP3-6 is an antiserum generated against a synthetic ZP3 peptide conserved throughout species (specific for ZP3) that reacts strongly with human ZP3 protein both in isolated oocytes and in ovary sections (17). The antiserum recognizes the protein backbone ofzp3, the primary sperm receptor on the ZP and the physiological inducer of the acrosome reaction (14-17). Because of the pivotal role of this glycoprotein in the process of fertilization, we speculated that, in some patients with a deficient fertilization capacity under IVF conditions, a ZP abnormality (structural or functional abnormality of ZP3) could be demonstrated. We therefore sought to investigate this hypothesis using the hemizona assay model. The micro bisection of the oocyte into matching hemizonae provides a Oehninger et al. ZP3 antiserum and oocyte defects 143

6 Table 2 Analysis of Variance Comparing Sperm Factor Versus Oocyte Factor F Degrees of Source Statistic freedom Probability Gamete factor , Fertilization factor , Antiserum factor , * Gamete factor , X fertilization factor Gamete factor X antiserum , * factor Fertilization factor X , antiserum factor Gamete factor X , fertilization factor X antiserum factor * Statistically significant. unique means to assess ZP biologic properties in an internally controlled assay (1,2). We have reported previously that human metaphase II oocytes that were used fresh, refrigerated at 4 C, or stored in a hyperosmotic salt solution revealed a strong and similar reactivity with AS ZP3-6 (23). Furthermore, we have shown that the protein backbone of ZP3 recognized by this antiserum is not influenced by prior contact of the oocyte with sperm (similar immunostaining was observed in noninseminated and inseminated oocytes) (23). Here, we have extended the use of AS ZP3-6 to the clinical arena and we delineate its potential use as a diagnostic tool. Until the advent ofivf technology, mature (preovulatory) oocytes were virtually impossible to obtain. We acknowledge that these 00- cytes were obtained after ovarian hyperstimulation, failed to fertilize after in vitro insemination, and had been under in vitro culture conditions for >48 hours after aspiration from the ovary, and that this represents a weakness of these analyses. Nevertheless, all oocytes used in the studies were mature (metaphase II) at the time of aspiration and were treated in an identical fashion thereafter (21). Moreover, the study is strengthened by the concomitant use of a negative control (AS ZP3-7, which specifically recognizes mouse ZP3) internally during each assay and positive controls performed during the different experiments (oocytes from fertile women that showed very strong immunoreactivity to AS ZP3-6). Our studies demonstrated that the group of patients defined as oocyte factor (based upon morphological abnormalities of the female gamete observed at the time of oocyte identification and during 48 hours of in vitro incubation) had very low immunostaining scores for AS ZP3-6. Conversely, patients defined as sperm factor, who presumably had achieved low fertilization rates due to dysfunctional male gametes, had immunostaining scores closely resembling those observed in the oocytes of fertile women (positive controls). This suggests that fertilization failure (total or partial) in these male factor cases was due to sperm deficiencies in the absence of any oocyte morphological abnormalities and with no demonstrable ZP3 immunostaining deficiency. Therefore, our studies show that a group of patients presenting morphological (cytoplasmic) oocyte defects have ZP deficiencies as well. They are characterized by an abnormal integrity and/or function of the protein backbone of ZP3. Whether these defects are directly responsible for a deficient gamete interaction manifested at the level of sperm-zona binding or sperm-zona penetration leading to fertilization failure remains to be elucidated. We recently have reported that a successful sperm-zona binding capacity still may be present in cases in which the protein backbone is immunologically abnormal (i.e., in oocytes cryopreserved using 1,2 propanediol) (23). This might indicate that changes of the ZP3 protein backbone do not influence the functional integrity of the glycoprotein receptor that initiates primary sperm binding to the ZP (23). This in turn may suggest the possibility of a deficient induction of the acrosome reaction and/or an abnormal sperm-zona penetration as causative factors leading to fertilization failure. Poor AS ZP3-6 immunostaining was observed in some patients with no demonstrable gamete defects (unknown group). This underscores the need for improved assays directed toward the identification of more subtle oocyte abnormalities and also suggests that some cases with poor fertilization results with otherwise normal gametes may be associated with alterations of the ZP. It is interesting to note that, within any given cohort of oocytes from a single patient, we could either observe all oocytes showing i very similar, homogenous immunostaining score or very dissimilar scores, revealing an immunostaining spectrum ranging from high intensity to almost no Table 3 Predicted Values for Gamete Factor by Antiserum Factor From the ANOVA Model* Gamete factor Oocyte Sperm P Hemizonae test 2.96 ± ± * Values are means ± SD. Antiserum factor Hemizonae control 1.80 ± ± P < Oehninger et al. ZP3 antiserum and oocyte defects Fertility and Sterility

7 staining. This suggests that, in some patients, not all preovulatory oocytes from the same cohort have the same ZP structure and/or function and may explain different degrees of fertilization success for individual oocytes. Whether ZP3 abnormalities detected by AS ZP3-6 are intrinsic or stimulation derived (all patients were stimulated with hmg with or without a GnRH agonist) remains to be resolved. Sperm binding capacity to the ZP has been shown to be affected by different types of ovarian stimulation regimens (24). We detected no relationship between female's age or basal serum FSH levels (fundamental determinants of ovarian response to hyperstimulation in terms of number of oocytes and pregnancy outcome during IVF treatment) and immunostaining scores (20, 25). A larger number of patients, including poor responders and those with polycystic ovarian syndrome, needs to be evaluated to determine whether any parameter(s) of the controlled ovarian hyperstimulation process can be predictive of ZP abnormalities. We conclude that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF therapy. This newly developed biomarker of oocyte integrity and/or function may help in the decision-making process in the IVF and oocyte micromanipulation for assisted fertilization setting. Acknowledgments. The authors gratefully acknowledge Ms. Pauline M. Clynes and Ms. Dara Willett-Leary of The Jones Institute for Reproductive Medicine for critical editorial contribution and preparation of this manuscript, respectively. REFERENCES 1. Oehninger S. Diagnostic significance of sperm-zona interaction. In: Smith S, editor. Reproductive medicine review. Vol. 1. 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