Defective sperm zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization

Human Reproduction vol.15 no.3 pp.702 708, 2000 Defective sperm zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization D.Y.Liu 1 and H.W.G.Baker However, oocyte factors appear to be uncommon causes for complete failure of fertilization. Standard follicle stimulation University of Melbourne Department of Obstetrics and Gynaecology, Reproductive Biology Unit, Royal Women s Hospital treatments rarely produce uniformly abnormal or immature and Melbourne IVF, Victoria, Australia oocytes. 1 Although most of the patients with failure of fertilization To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, University of Melbourne, Royal in standard IVF can now be treated by intracytoplasmic Women s Hospital, Carlton, Victoria 3053, Australia sperm injection (ICSI, Van Steirteghem et al., 1993), diagnosing the causes of failure of fertilization in standard Sperm zona pellucida binding and penetration were IVF is important. Ideally, they should be detected before IVF assessed on the oocytes that failed to fertilize from couples is started. It is known that sperm zona pellucida interaction is with 3 oocytes treated by standard in-vitro fertilization important in human fertilization (Overstreet and Hembree, (IVF). There were four groups: fertilization rate 0% 1976; Yanagimachi, 1994). Tests for sperm zona pellucida (n 369), 1 25% (n 194), 26 50% (n 81) and binding and penetration have been developed and the results 51 95% (n 100). Of the couples with zero fertilization are highly correlated with fertilization rates in vitro (Burkman rate 70% had 5 spermatozoa bound per zona pellucida and et al., 1988; Liu et al., 1989b; Oehninger et al., 1989; Franken 42% had no spermatozoa penetrating the zona pellucida of et al., 1993; Liu and Baker 1994a). any oocyte. In contrast, in the 51 95% fertilization rate There are many reports on the relationships between sperm group, only 17% had 5 spermatozoa bound per zona factors and fertilization rates in vitro. However, there are pellucida and 6% had no spermatozoa penetrating the usually only small numbers of couples with failure of fertilizazona pellucida. There was a significantly higher frequency tion included in each study and it is difficult to determine of poor sperm morphology ( 5% normal) in couples with the frequency of various factors contributing to failure of zero fertilization rate (36%) than in the fertilization rate fertilization. In the present study, a large number of couples group 51 95% (7%). Incubation of oocytes from 68 couples with failure of fertilization were studied in order to analyse the with zero fertilization rate and low sperm zonae pellucidae frequency of the various abnormalities. Sperm zona pellucida binding with fertile donor spermatozoa resulted in normal binding and sperm zona pellucida penetration were assessed sperm zona pellucida binding and most zonae pellucidae on all oocytes that failed to fertilize. being penetrated. In conclusion, defective sperm zona pellucida interaction was the major cause for low fertilization rates in standard IVF. This was usually because of Materials and methods defects of the spermatozoa rather than defects of the Subjects oocytes. Sperm defects likely to cause failure of fertilization Couples (n 563) were included in the study on the basis that at should be diagnosed before commencing IVF and the least three mature oocytes were inseminated with husband s fresh patients directed to intracytoplasmic sperm injection. ejaculated spermatozoa and 25% of the oocytes fertilized. In Key words: IVF failure/spermatozoa/sperm zona interaction/ addition, 181 couples with 25% of oocytes fertilized but some zona pellucida which failed to fertilize were randomly selected and included as a positive control group. The couples were treated at the Royal Women s Hospital and Melbourne IVF programmes from June 1992 to December 1996. During this time only three patients were identified who had only immature oocytes collected that all failed to fertilize. Introduction Couples with sperm autoimmunity, fewer than three mature oocytes In standard in-vitro fertilization (IVF), complete failure of inseminated, oocytes inseminated with frozen husband or donor fertilization occurs in 10 15% of treatments. Although the spermatozoa or spermatozoa obtained surgically from the male genital causes may be unclear, many studies indicate that sperm tract were not included in the study. The 563 couples with 25% fertilization had IVF performed for: defects appear to be the major contributors (Mahadevan and unexplained infertility in 193 (34%), tubal occlusion in 120 (21%), Trounson, 1984; Jeulin et al., 1986; Kruger et al., 1988; Liu endometriosis in 72 (13%), male infertility in 156 (28%) and mixed et al., 1989a,b,c, Liu and Baker 1992a; Franken et al., 1993; male and female factors in 22 (4%). The reasons for IVF in the 181 Oehninger et al., 1997). Oocyte immaturity or abnormalities couples with 25% fertilization rate were unexplained infertility in can also contribute to failure of fertilization. Where the majority 48 (26%), tubal occlusion in 50 (28%), endometriosis in 42 (23%), of oocytes fertilize, the few that do not fertilize often have male factor in 34 (19%) and mixed male and female factors in defects (Bedford and Kim, 1993; Van Blerkom et al., 1994). seven (4%). 702 European Society of Human Reproduction and Embryology

Sperm zona interaction and IVF All oocytes that had failed to fertilize in vitro were examined for sperm zona pellucida binding and penetration as described below without knowledge of the results of IVF. Patients consented to use of the failed fertilized oocytes for research studies and the Royal Women s Hospital Research and Ethics Committees approved the study. IVF procedures The clinical and laboratory aspects of the IVF procedures are described elsewhere (Liu et al., 1988). The swim-up technique was used for selection of motile spermatozoa for insemination of the oocytes in the majority of couples. Gradient separation techniques were used for some semen samples with severe oligozoospermia or asthenozoospermia (Ng et al., 1992). The main purpose of using these alternative methods for selection of motile spermatozoa was to obtain a higher yield of motile spermatozoa for insemination of the oocytes. Usually 10 20 10 4 in 0.6 ml culture medium were used for insemination of one to three oocytes in each well of a four-well multidish (Nunclon, Kamstrup, Roskilde, Denmark). Higher concentrations of spermatozoa were used for insemination of oocytes when the percentage of normal sperm morphology was 10% or there was previous low fertilization ( 25%). The fertilization rate was the quotient of number of oocytes fertilized and the number of mature oocytes inseminated. Fertilized oocytes included those with two or more pronuclei observed 16 20 h after insemination. Other abnormal fertilizations such as the appearance of only one pronucleus or two pronuclei delayed until 48 h after insemination were not included. Of the 563 couples with 25% fertilization in standard IVF, 180 were subsequently treated by ICSI. The ICSI procedure is described in detail elsewhere (Liu et al., 1997). Average fertilization rates, as defined above, from one or more ICSI treatments were analysed. Sperm tests Semen analyses were performed before commencing IVF treatment by standard methods (World Health Organization, 1987). At the time of preparation of the insemination suspension, sperm concentration was determined using a haemocytometer and sperm progressive motility assessed by counting 100 spermatozoa using a phase contrast microscope. Spermatozoa in the insemination medium were obtained after embryos or unfertilized oocytes had been removed at 48 h after insemination. The insemination medium from all the culture dishes was pooled for the same couple. The spermatozoa were washed with 10 ml of 0.9% NaCl and the sperm pellet was resuspended in ~20 µl of 0.9% NaCl. The sperm suspension was smeared on a glass slide for staining of morphology. The density and evenness of spread of the spermatozoa were checked microscopically before the slides were left to dry. Assessment of sperm zona pellucida binding The oocytes which had failed to fertilize were pipetted (inner diameter 250 µm) gently several times to dislodge spermatozoa not tightly bound to the zona pellucida and any remaining cumulus cells. The number of spermatozoa tightly bound to the zona pellucida was counted with an inverted phase contrast microscope with magnification of 250. When there were 100 spermatozoa bound to one zona pellucida, it was impossible to count the number of spermatozoa accurately and the number was recorded as 100 for subsequent analysis (Liu et al., 1989a). Assessment of sperm zona pellucida penetration Because it was difficult to determine which spermatozoa were penetrating into the zona pellucida or perivitelline space (PVS) and which were bound to the surface of the zona pellucida, a technique was used to remove all the spermatozoa bound to the surface of the zona pellucida (Liu and Baker, 1994a,b). The oocytes were aspirated in and out of a pipette of inner diameter slightly smaller than the oocyte (inner diameter 120 µm) and the spermatozoa on the surface of the zona pellucida were sheared off. Only spermatozoa with their heads either partially or wholly embedded in the zona pellucida or in the PVS remained. These spermatozoa could not be removed by further repeated pipetting. After this pipetting procedure, spermatozoa with heads in the zona pellucida or PVS were easily seen and counted under the microscope. The accuracy of this technique was confirmed by histological examination of serial cross-sections of oocytes (Liu and Baker, 1994a). Binding of donor spermatozoa to the zona pellucida of oocytes from couples with failure of fertilization and low sperm zona pellucida binding To determine if low sperm zona pellucida binding was due to spermatozoa or oocyte defects in couples with complete failure of fertilization, oocytes from 68 couples with an average of 5 (mean 1.0, range 0 3.4) spermatozoa bound per zona pellucida were selected to test zona pellucida binding of spermatozoa from fertile donors. Usually four to five randomly selected oocytes were tested for each couple. The oocytes were incubated with swim-up spermatozoa from fresh or frozen donor semen at 72 86 h after the IVF insemination with husband s spermatozoa. Unfertilized oocytes used at 72 86 h have normal capacity for binding of spermatozoa. There was no difference in sperm zona pellucida penetration between fresh and frozen donor spermatozoa. Motile spermatozoa (1 2 10 6 ) were incubated with four to five oocytes in 1 ml human tubal fluid (HTF; Irvine Scientific, Irvine, CA, USA) medium at 37 C in5%co 2 in air for 2 h. The HTF medium was supplemented with 10% human serum (ICN Pharmaceuticals Inc., Costa Mesa, CA, USA) inactivated by heating at 56 C for 30 min and sterilized by filtration (0.22 µm). After incubation, the oocytes were washed with four changes of HTF with extensive aspiration in and out of a glass micropipette (inner diameter 250 µm) to dislodge loosely attached spermatozoa. The numbers of spermatozoa bound to and penetrating in the zona pellucida or PVS were assessed as described above. Statistical analysis The geometric mean of number of spermatozoa bound to all zonae pellucidae for each patient was calculated by natural log transforma- tion. The significance of differences between mean results of sperm tests for couples with zero (IVF rate 0) or some fertilization (IVF rate 0) was determined by t-tests. Correlations between sperm test results were examined by non-parametric (Spearman) test. Relation- ships between sperm test results and fertilization were examined by 703 Assessment of sperm morphology Morphology slides were stained with the Shorr method after the smears were fixed in 90% ethanol for 30 min (Jeulin et al., 1986; Liu et al., 1988). Sperm morphology was assessed following the World Health Organization (1987) criteria for the silhouette plus internal staining characteristics with the acrosomal region being clearly seen, regular in shape and occupying at least half of the sperm head. The percentage of spermatozoa with normal morphology was determined by assessing 200 spermatozoa from 10 individual fields under oil immersion with magnification of 1000 and bright-field illumination. These modified criteria are similar to the Kruger strict criteria (Kruger et al., 1988). Our previous study showed that 48 h incubation had no effect on sperm morphology and the similar results were obtained from the same sperm samples with or without 48 h incubation (Liu et al., 1988).

D.Y.Liu and H.W.G.Baker Table I. Mean SD and range (in brackets) of sperm tests and IVF results Variable Fertilization rate group 0% 1 25% 26 50% 50% No. of patients 369 194 81 100 No. of oocytes inseminated 8 5.2 (3 30) 11 5.5 (4 34) 11 6(3 35) 13 6.7 (3 33) No. of oocytes fertilized 0 1.5 0.7 (1 6) 4 1.9 (1 11) 9 4.9 (2 22) Fertilization rate (%) 0 16 5.8 (4 25) 37 7.5 (26 50) 73 11 (53 95) Spermatozoa inseminated ( 10 4 /ml) a 29 23 (1 160) 30 21 (3 130) 26 21 (1 95) 18 17 (6 105) % motility of spermatozoa inseminated 77 24 (5 100) 78 23 (5 100) 84 22 (5 100) 86 21 (5 100) No. spermatozoa bound per ZP a 9 17 (0 100) 12 18 (0 100) 26 29 (0 100) 36 32 (0 100) % of zona pellucida penetrated a 37 39 (0 100) 67 34 (0 100) 79 29 (0 100) 89 27 (0 100) % normal sperm morphology a 12 11 (0 60) 14 11 (0 56) 17 12 (0 52) 25 14 (0 56) a P 0.01, comparing fertilization rate (FR) 0 and 1 25% groups with FR 26 50% and 51 95% groups. ZP zona pellucida. logistic regression analysis using the SPIDA statistical package (Macquarie University, Sydney, Australia). unexpectedly low (but nevertheless significant) between sperm morphology and motility, and sperm zona pellucida binding and penetration. Results Sperm tests and IVF results Sperm zona pellucida binding and penetration, and in-vitro fertilization While the purpose of this study was mainly to determine The distributions of numbers of spermatozoa bound to the frequency of various sperm defects in couples with complete zona pellucida and proportion of zona pellucida penetrated for failure of fertilization, all couples with 25% fertilization different fertilization rate groups are shown in Figure 1 and rates were included and also a small number of randomly Figure 2. There were significantly higher proportions of couples selected couples with 25% fertilization rates. Mean sperm with low sperm zona pellucida binding (fewer than five test and IVF results are shown in Table I. There was a wide spermatozoa bound per zona pellucida) with zero or low range for the sperm test results and numbers of oocytes ( 25%) fertilization rates than with fertilization rates of inseminated. The number of spermatozoa inseminated was 25%. Over 70% of couples with zero fertilization rates had higher in couples with low or zero fertilization rates because 5 spermatozoa bound per zona pellucida. In contrast, only of the practice of increasing the number of spermatozoa for 17% of couples with fertilization rate 50% had 5 insemination in patients with severe sperm defects in the spermatozoa bound per zona pellucida. There was a signistandard IVF programme. The number of spermatozoa bound ficantly (P 0.001) higher proportion of patients (42%) with per zona pellucida, proportions of zona pellucida penetrated zero sperm zona pellucida penetration with zero fertilization and percentage normal sperm morphology were significantly rates than with higher fertilization rates. higher in couples with fertilization rates of 26 95% than those with 25% fertilization rate. Sperm morphology and in-vitro fertilization Correlations between sperm test results For couples with zero fertilization rates, over 35% had normal When all sperm test results from the 744 couples were sperm morphology 5%. In contrast only 7% of patients analysed by Spearman test, there were statistically significant with fertilization rate 51 95% had normal morphology 5% correlations between most of the sperm test results. In particuzero sperm zona pellucida penetration and low sperm zona (Figure 3). For 142 couples with zero fertilization rates, lar, sperm motility was significantly correlated with normal sperm morphology (r 0.323, P 0.001), the number of pellucida binding (fewer than five spermatozoa bound per zona spermatozoa bound per zona pellucida (r 0.144, P 0.001) pellucida), 37% had normal sperm morphology 5%. and the proportion of zona pellucida penetrated (r 0.202, P 0.001). Similarly, normal sperm morphology was significantly Logistic regression analysis correlated with number of spermatozoa bound per zona Since most patients (n 563) were selected because of low pellucida (r 0.172, P 0.001) and the proportion of zona fertilization rates ( 25%) their data were not included in the pellucida penetrated (r 0.176, P 0.001). The number of logistic regression analyses. Only data from the other randomly spermatozoa bound/zona pellucida was highly significantly selected couples (n 181) with fertilization rates 25% were correlated with the proportion of zonae pellucidae penetrated examined by logistic regression analysis (Table II). In this (r 0.744, P 0.001). It should be noted that there was a group of patients, both percentage normal sperm morphology high proportion of patients with normal semen analysis but poor and mean number of spermatozoa bound per zona pellucida sperm zona pellucida binding and penetration (see below), and were significantly related to fertilization rate (Table II). When this may explain why the Spearman correlation coefficient was the 103 couples with all oocytes having spermatozoa 704

Sperm zona interaction and IVF penetrating the zona pellucida were analysed, percentage normal sperm morphology was significantly related to fertilization rate (Table II). Binding and penetration of fertile donor spermatozoa to the zona pellucida of oocytes from couples with zero fertilization There were 68 couples who had complete failure of fertilization and fewer than five spermatozoa bound to each zona pellucida (mean SD, 1.0 1.1 per zona pellucida). Re-incubation of 310 oocytes from these couples (four to five oocytes per patient) with fertile donor spermatozoa resulted in large numbers (96 12, range 38 to 100 per zona pellucida) of spermatozoa binding to the zona pellucida and 94% (range 75 100%) of oocytes had one or more donor spermatozoa penetrating the zona pellucida. No couples had failure of penetration of all oocytes with donor spermatozoa. This result indicates that the low sperm zona pellucida binding during IVF was mainly because of a defect in the husband s spermatozoa and not a problem with the oocytes. Sperm zona pellucida binding and penetration in couples with zero fertilization but normal semen analysis results There were 310 couples with zero fertilization, who had pre-ivf semen analysis results from our laboratory. Overall, 52% (160) had normal results (sperm concentration 20 10 6 /ml, progressive motility 25%, and normal sperm morphology 15%). Of these, 75% (122) had low sperm zona pellucida binding (fewer than five spermatozoa bound per zona pellucida) and 47% (71) had no spermatozoa penetrating the zona pellucida of all the oocytes that failed to fertilize in vitro. ICSI results for couples with failure of fertilization in standard IVF Couples with either zero fertilization rate (n 113) or 25% fertilization rates (n 67) from one or more standard IVF treatment cycles were subsequently treated using ICSI. An average fertilization rate of 58% (range 0 100%) was achieved. Only five couples had zero fertilization with ICSI, and one of these had only a single oocyte injected. Figure 1. Frequency distribution of average number of spermatozoa bound per zona pellucida in fertilization rate groups (FR) 0%, 1 25%, 26 50% and 51 95%. There was a significantly higher proportion of low sperm zona pellucida binding ( 5/zona pellucida) in the group with FR 0% than in the other groups with FR 0% (P 0.001, χ 2 -test). Figure 2. Frequency distribution of percentages of zona pellucida penetrated in fertilization rate groups (FR) 0%, 1 25%, 26 50% and 51 95%. There was a significantly higher proportion of low zona pellucida penetration ( 20%) in the group with FR 0% than in the other groups with FR 0% (P 0.001, χ 2 -test). 705

D.Y.Liu and H.W.G.Baker Discussion The present study showed that 70% of couples with complete failure of fertilization in standard IVF had 5 spermatozoa bound per zona pellucida and 43% had no spermatozoa penetrating the zona pellucida. In contrast, 35% of patients had normal sperm morphology 5%. Thus defective sperm zona pellucida binding and penetration are the major causes of zero fertilization rates in vitro. The human zona pellucida plays a major role as a physiological barrier and selects morphologically normal spermatozoa. This study also further confirms that sperm abnormalities are the most frequent factors contributing to defective sperm zona pellucida binding and penetration for patients with all or most oocytes failing to fertilize in vitro. Failure of fertilization because of immaturity of all the oocytes collected for standard IVF is rare. In contrast a small proportion of immature oocytes in each stimulation cycle is quite common. An attempt was made to limit the possibility that fertilization failure may be directly related to oocyte immaturity by excluding all immature oocytes in the calculation Figure 3. Frequency distribution of percentage normal sperm morphology in fertilization rate groups (FR) 0%, 1 25%, 26 50% and 51 95%. There was a significantly higher proportion of poor sperm morphology (normal 0 5%) in the group with FR 0% than in the other groups with FR 0% (P 0.01, χ 2 -test). 706 of fertilization rates for each couple. Therefore couples with all immature oocytes or fewer than three mature oocytes inseminated were not included in the study. Although the zona pellucida of immature human oocytes allows normal binding and penetration of fertile donor spermatozoa, immature oocytes do not fertilize normally (Lopata and Leung, 1988). Over the time of this study, only three couples were seen with complete failure of fertilization with all oocytes inseminated being immature. They represent 1% of all couples with failure of fertilization and were not included in the study. To determine if oocyte abnormalities other than immaturity could contribute to defective sperm zona pellucida binding and penetration, oocytes with low zona pellucida binding and no penetration of husband s spermatozoa were re-incubated with spermatozoa from fertile men. Large numbers of spermatozoa bound to zona pellucida and one or more spermatozoa penetrated most of the zona pellucida. No couple had failure of penetration of all oocytes after re-insemination with fertile donor spermatozoa. This result further indicates that failure of sperm zona pellucida binding and penetration in couples with complete failure of fertilization is mainly due to abnormalities of the spermatozoa and not of the oocytes (Liu et al., 1989a; Liu and Baker 1994a). In the present study higher concentrations of donor spermatozoa were used (1 2 10 6 /ml) than previously (Liu et al., 1989b), or for IVF, to ensure that spermatozoa would be able to penetrate the zona pellucida during the 2 h incubation. Oocytes that failed to fertilize in vitro can be used for tests of sperm zona pellucida binding and penetration, and zona pellucida-induced acrosome reaction (Liu et al., 1989b; Liu and Baker 1996). When data from couples with fertilization rates ranging from 26 to 95% were analysed by logistic regression, normal sperm morphology was highly significantly related to fertilization rates. These results further suggest that sperm morphology is one of the most important sperm characteristics for fertilization in standard IVF (Jeulin et al., 1986; Kruger et al., 1988; Liu et al., 1988; Claassens et al., 1992; Liu and Baker 1992a,b,c; Donnelly et al., 1998; Duran et al., 1998). Couples with zero fertilization rates, low sperm zona pellucida binding and zero sperm zona pellucida penetration often had poor sperm morphology: 35% had normal sperm morphology 5%. On the other hand, IVF rates in the couples with all oocytes having spermatozoa penetrating the zona pellucida were correlated with normal sperm morphology. Previous studies showed that although the human zona pellucida is selective for binding spermatozoa with normal morphology, some spermatozoa with abnormal morphology are still capable of binding to and penetrating the zona pellucida (Liu and Baker 1992b,c; Garrett et al., 1997). The couples with normal sperm zona pellucida binding despite high proportions of spermatozoa with abnormal morphology may have low fertilization rates because of defects later in the fertilization process such as sperm fusion with the oolemma, nuclear decondensation or formation of the male pronucleus. Abnormal sperm morphology is highly correlated with sperm nuclear immaturity or abnormalities assessed with the acridine orange staining (Claassens et al., 1992; Liu and Baker, 1992c; Duran et al., 1998). However, there is no evidence to suggest that

Sperm zona interaction and IVF Table II. Significant variables related to fertilization rates by logistic regression analysis of results for patients with fertilization rates (FR) 26 95% Group Regression SE z P coefficient Patients with FR of 26 95% (n 181) No. of spermatozoa bound per zona pellucida 0.004 0.002 2 0.05 Normal sperm morphology (%) 0.024 0.003 8 0.001 Patients with FR of 26 95% and all oocytes having spermatozoa penetrating the zona pellucida (n 103) Normal sperm morphology (%) 0.024 0.004 6 0.001 the phenotype of individual spermatozoa reflects the genotype spermatozoa (Liu et al., 1989b; Liu and Baker, 1996). The it carries (Cummins and Jequier, 1994). oocytes can be stored in concentrated salt solution Because failure of fertilization in standard IVF is mainly (Yanagimachi et al., 1979; Liu et al., 1989b). Ethics due to defective sperm zona pellucida binding and penetration Committee approval and patient consent is required for using associated with sperm abnormalities, it is therefore expected the unfertilized oocytes for the tests. There should be no that the couples would have a higher fertilization rate with ICSI. difficulty in obtaining such permission, as there is no intention Many sperm defects, including motility disorders, abnormal to fertilize the oocytes and the material is to be discarded. We morphology, and inability of spermatozoa to bind or penetrate have been using such oocytes for sperm oocyte interaction the zona pellucida, are bypassed by ICSI. In this study, the tests for many years. Over 95% of patients are willing to average fertilization rate with ICSI was 58% in 180 couples donate their unfertilized oocytes for either clinical tests or with zero or very low ( 25%) fertilization rates in previous research. However, the number of human zonae pellucidae standard IVF. Pre-IVF assessment of sperm function is important available for routine tests of sperm function is limited, particu- to assign patients to standard IVF or ICSI. This should larly in small IVF clinics. In future, recombinant human zona avoid unexpected failure of fertilization by standard IVF. On pellucida 3 might be useful for routine tests of human sperm the other hand, couples with adequate sperm function may not function (Van Duin et al., 1994; Whitmarsh et al., 1996). need ICSI and should be treated with standard IVF. In summary, defective sperm zona pellucida binding and At the present time, most couples treated with ICSI have penetration are the major causes for zero or low fertilization obvious severe sperm defects such as oligozoospermia, rates with standard IVF. Failure of sperm zona pellucida asthenozoospermia, and teratozoospermia alone or in combina- binding and penetration is mainly due to abnormalities of the tion. These couples are easily identified by routine semen spermatozoa not the oocytes. Most severe sperm defects are analysis. However, in the present study, it was found that 52% obvious from standard semen analyses but pre-ivf assessment of couples (160 of 310) with zero fertilization rate had normal of the capacity of spermatozoa to bind to and penetrate the pre-ivf semen analysis results and 47% of them had no zona pellucida will provide useful information for diagnosis spermatozoa penetrating the zona pellucida of any of the and management of couples with idiopathic infertility with oocytes. It is suspected that many of these couples with normal normal standard semen analysis. semen analysis and failure of fertilization may have disordered zona pellucida-induced acrosome reaction (Liu and Baker, 1994b). Spermatozoa from these couples bind to the zona Acknowledgements pellucida but do not undergo the acrosome reaction and are We thank Mingli Liu for technical assistance and all the scientists in unable to penetrate the zona pellucida. Patients with this both Melbourne IVF and Royal Women s Hospital IVF laboratories condition have very low or zero fertilization rate with standard for collecting the oocytes and sperm samples for this study. The IVF but high fertilization and pregnancy rates with ICSI (Liu Royal Women s Hospital Research Committee supported this study. et al., 1997). Our preliminary results indicate that up to onethird of normozoopsermic infertile men may have disordered References zona pellucida-induced acrosome reaction (Liu and Baker, Bedford, J.M. and Kim, H.H. (1993) Sperm/egg binding patterns and oocyte unpublished data). In order to reduce the incidence of failure cytology in retrospective analysis of fertilization failure in vitro. Hum. of fertilization in standard IVF in unexplained infertile couples, Reprod., 8, 453 463. pre-ivf screening for this condition is highly recommended Burkman, L.J., Coddington, C.C., Franken, D.R. et al. (1988) The hemizona assay (HZA): development of a diagnostic test for the binding of human so that the couples can be assigned directly to ICSI instead of spermatozoa to the human hemizona pellucida to predict fertilization standard IVF (Liu and Baker, 1994b; Liu et al., 1997). potential. Fertil. Steril., 49, 688 697. In clinical IVF, usually 20 30% of oocytes fail to fertilize. Claassens, O.E., Menkveld, R., Franken, D.R. et al. (1992) The acridine These oocytes are valuable material for sperm zona pellucida orange test: determining the relationship between sperm morphology and fertilization in vitro. Hum. Reprod., 7, 242 247. interaction tests. The zona pellucida of unfertilized oocytes is Cummins, J.M. and Jequier, A.M. (1994) Treating male infertility needs more still capable of binding spermatozoa, inducing the acrosome clinical andrologist, not less. Hum. Reprod., 9, 1214 1219. reaction and being penetrated by the acrosome reaction Donnelly, E.T., Lewis, S.E.M., McNally, J.A. et al. (1998) In vitro fertilization 707

D.Y.Liu and H.W.G.Baker and pregnancy rates: the influence of sperm motility and morphology on implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, IVF outcome. Fertil. Steril., 70, 305 314. 1061 1066. Duran, E.H., Gurgan, T., Gunalp, S. et al. (1998) A logistic regression model Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill, J. including DNA status and morphology of spermatozoa for prediction of (eds), The Physiology of Reproduction, 2nd edition. Raven Press, New fertilization in vitro. Hum. Reprod., 13, 1235 1239. York, pp. 189 317. Franken, D.R., Kruger, T.F., Lombard, C. et al. (1993) The ability of the Yanagimachi, R., Lopata, A. and Odom, C.B. (1979) Retention of biological hemizoan assay to predict human fertilization in vitro in different consecutive characteristics of zona pellucida in highly concentrated salt solution: the IVF/GIFT cycles. Hum. Reprod., 8, 1240 1244. use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Garrett, C., Liu., D.Y. and Baker, H.W.G. (1997) Selectivity of the human Fertil. Steril., 31, 562 574. sperm zona pellucida binding process to sperm head morphometry. Fertil. Whitmarsh, A.J., Woolnough, M.J. and Moore, H.D.M. (1996) Biological Steril., 67, 362 371. activity of recombinant human ZP3 produced in vitro: potential for a sperm Jeulin, C., Feneux, D., Serres, C. et al. (1986) Sperm factors related to failure function test. Mol. Hum. Reprod., 2, 911 919. of human in vitro fertilization. J. Reprod. Fertil., 76, 735 744. World Health Organization (1987) WHO Laboratory Manual for Examination Kruger, T.F., Acosta, A.A., Simmons, K.F. et al. (1988) Predictive value of of Human Semen and Semen Cervical Mucus Interaction. Cambridge abnormal sperm morphology in in vitro fertilization. Fertil. Steril., 49, University Press, Cambridge, pp. 1 10. 112 117. Liu, D.Y. and Baker, H.W.G. (1992a) Tests of human sperm function and Received on June 24, 1999; accepted on November 15, 1999 fertilization in vitro. Fertil. Steril., 58, 465 483. Liu, D.Y. and Baker, H.W.G. (1992b) Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro. J. Fertil. Reprod., 94, 71 84. Liu, D.Y. and Baker, H.W.G. (1992c) Sperm nuclear chromatin normality: relationship with sperm morphology, sperm zona pellucida binding, and fertilization rates in vitro. Fertil. Steril., 58, 1178 1184. Liu, D.Y. and Baker, H.W.G. (1994a) A new test for the assessment of sperm zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro. Hum. Reprod., 9, 489 496. Liu, D.Y. and Baker, H.W.G. (1994b) Disordered acrosome reaction of sperm bound to the zona pellucida (zona pellucida): a newly discovered sperm defect causing infertility with reduced sperm zona pellucida penetration and reduced fertilization in vitro. Hum. Reprod., 9, 1694 1770. Liu D.Y. and Baker, H.W.G. (1996) A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona pellucida: lack of relationship with ionophore A23187-induced acrosome reaction. Hum. Reprod., 11, 551 557. Liu, D.Y., Du Plessis, Y.P., Nayudu, P.L. et al. (1988) The use of in vitro fertilization to evaluate putative tests of human sperm function. Fertil. Steril., 49, 272 277. Liu, D.Y., Lopata, A., Johnston, W.I.H. et al. (1989a) Human sperm zona binding, sperm characteristics and in-vitro fertilization. Hum. Reprod., 4, 696 701. Liu, D.Y., Clarke, G.N., Lopata, A. et al. (1989b) A zona pellucida binding test and in vitro fertilization. Fertil. Steril., 52, 281 287. Liu, D.Y., Bourne, H. and Baker, H.W.G. (1997) High fertilization and pregnancy rates after intracytoplasmic sperm injection in patients with disordered zona pellucida-induced acrosome reaction. Fertil. Steril., 67, 955 958. Lopata, A. and Leung, P.C. (1988) The fertilizability of human oocytes at different stages of meiotic maturation. In Jones, H.W. Jr, Schrader, C. (eds), In vitro Fertilization and Other Assisted Reproduction. New York Academy of Sciences, New York, pp. 324 336. Mahadevan, M.M. and Trounson, A.O. (1984) The influence of seminal characteristics on the success rate of human in vitro fertilization. Fertil. Steril., 42, 400 405. Ng, F.L.H., Liu, D.Y. and Baker, H.W.G. (1992) Comparison of Percoll, Mini- Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum. Reprod., 7, 261 266. Oehninger, S.C., Coddington, C.C. and Scott, R. (1989) Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil. Steril., 51, 665 669. Oehninger, S., Mahony, M. and Ozgur, K. (1997) Clinical significance of human sperm zona pellucida binding. Fertil. Steril., 67, 1121 1127. Overstreet, J.W. and Hembree, W.C. (1976) Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro. Fertil. Steril., 17, 815 831. Van Blerkom, J., Davis, P.W. and Merriam, J. (1994) A retrospective analysis of unfertilized and presumed parthenogenetically activated human oocytes demonstrates a high frequency of sperm penetration. Hum. Reprod., 9, 2381 2388. Van Duin, M., Polman, J.E.M., Ingeborg, TM et al. (1994) Recombinant human zona pellucida protein ZP3 produced by Chinese hamster ovary cells induces the human sperm acrosome reaction and promotes sperm egg fusion. Biol. Reprod., 51, 607 617. Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization and 708