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

D.Y. Liu1 and H.W.G. Baker

University of Melbourne Department of Obstetrics and Gynaecology, Reproductive Biology Unit, Royal Women's Hospital and Melbourne IVF, Victoria, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sperm–zona pellucida binding and penetration were assessed on the oocytes that failed to fertilize from couples with >=3 oocytes treated by standard in-vitro fertilization (IVF). There were four groups: fertilization rate 0% (n = 369), 1–25% (n = 194), 26–50% (n = 81) and 51–95% (n = 100). Of the couples with zero fertilization rate 70% had <=5 spermatozoa bound per zona pellucida and 42% had no spermatozoa penetrating the zona pellucida of any oocyte. In contrast, in the 51–95% fertilization rate group, only 17% had <= 5 spermatozoa bound per zona pellucida and 6% had no spermatozoa penetrating the zona pellucida. There was a significantly higher frequency of poor sperm morphology (<= 5% normal) in couples with zero fertilization rate (36%) than in the fertilization rate group 51–95% (7%). Incubation of oocytes from 68 couples with zero fertilization rate and low sperm–zonae pellucidae binding with fertile donor spermatozoa resulted in normal sperm–zona pellucida binding and most zonae pellucidae 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 defects of the spermatozoa rather than defects of the oocytes. Sperm defects likely to cause failure of fertilization should be diagnosed before commencing IVF and the patients directed to intracytoplasmic sperm injection.

Key words: IVF failure/spermatozoa/sperm–zona interaction/zona pellucida


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In standard in-vitro fertilization (IVF), complete failure of fertilization occurs in 10–15% of treatments. Although the causes may be unclear, many studies indicate that sperm defects appear to be the major contributors (Mahadevan and Trounson, 1984Go; Jeulin et al., 1986Go; Kruger et al., 1988Go; Liu et al., 1989aGo,bGo,c, Liu and Baker 1992aGo; Franken et al., 1993Go; Oehninger et al., 1997Go). Oocyte immaturity or abnormalities can also contribute to failure of fertilization. Where the majority of oocytes fertilize, the few that do not fertilize often have defects (Bedford and Kim, 1993Go; Van Blerkom et al., 1994Go). However, oocyte factors appear to be uncommon causes for complete failure of fertilization. Standard follicle stimulation treatments rarely produce uniformly abnormal or immature oocytes.

Although most of the patients with failure of fertilization in standard IVF can now be treated by intracytoplasmic sperm injection (ICSI, Van Steirteghem et al., 1993), diagnosing the causes of failure of fertilization in standard IVF is important. Ideally, they should be detected before IVF is started. It is known that sperm–zona pellucida interaction is important in human fertilization (Overstreet and Hembree, 1976Go; Yanagimachi, 1994Go). Tests for sperm–zona pellucida binding and penetration have been developed and the results are highly correlated with fertilization rates in vitro (Burkman et al., 1988Go; Liu et al., 1989bGo; Oehninger et al., 1989Go; Franken et al., 1993Go; Liu and Baker 1994aGo).

There are many reports on the relationships between sperm factors and fertilization rates in vitro. However, there are usually only small numbers of couples with failure of fertilization included in each study and it is difficult to determine the frequency of various factors contributing to failure of fertilization. In the present study, a large number of couples with failure of fertilization were studied in order to analyse the frequency of the various abnormalities. Sperm–zona pellucida binding and sperm–zona pellucida penetration were assessed on all oocytes that failed to fertilize.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Couples (n = 563) were included in the study on the basis that at least three mature oocytes were inseminated with husband's fresh ejaculated spermatozoa and <25% of the oocytes fertilized. In addition, 181 couples with >25% of oocytes fertilized but some 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. Couples with sperm autoimmunity, fewer than three mature oocytes inseminated, oocytes inseminated with frozen husband or donor spermatozoa or spermatozoa obtained surgically from the male genital tract were not included in the study.

The 563 couples with <=25% fertilization had IVF performed for: unexplained infertility in 193 (34%), tubal occlusion in 120 (21%), endometriosis in 72 (13%), male infertility in 156 (28%) and mixed male and female factors in 22 (4%). The reasons for IVF in the 181 couples with >25% fertilization rate were unexplained infertility in 48 (26%), tubal occlusion in 50 (28%), endometriosis in 42 (23%), male factor in 34 (19%) and mixed male and female factors in seven (4%).

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., 1988Go). 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., 1992Go). 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 10–20x104 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., 1997Go). 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, 1987Go). 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 morphology
Morphology slides were stained with the Shorr method after the smears were fixed in 90% ethanol for 30 min (Jeulin et al., 1986Go; Liu et al., 1988Go). 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 x1000 and bright-field illumination. These modified criteria are similar to the Kruger `strict' criteria (Kruger et al., 1988Go). 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., 1988Go).

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 x250. 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., 1989aGo).

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, 1994aGo,bGo). 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, 1994aGo).

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 (1–2x106) were incubated with four to five oocytes in 1 ml human tubal fluid (HTF; Irvine Scientific, Irvine, CA, USA) medium at 37°C in 5% CO2 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 transformation. 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. Relationships between sperm test results and fertilization were examined by logistic regression analysis using the SPIDA statistical package (Macquarie University, Sydney, Australia).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sperm tests and IVF results
While the purpose of this study was mainly to determine frequency of various sperm defects in couples with complete failure of fertilization, all couples with <=25% fertilization rates were included and also a small number of randomly selected couples with >25% fertilization rates. Mean sperm test and IVF results are shown in Table IGo. There was a wide range for the sperm test results and numbers of oocytes inseminated. The number of spermatozoa inseminated was higher in couples with low or zero fertilization rates because of the practice of increasing the number of spermatozoa for insemination in patients with severe sperm defects in the standard IVF programme. The number of spermatozoa bound per zona pellucida, proportions of zona pellucida penetrated and percentage normal sperm morphology were significantly higher in couples with fertilization rates of 26–95% than those with <25% fertilization rate.


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Table I. Mean ± SD and range (in brackets) of sperm tests and IVF results
 
Correlations between sperm test results
When all sperm test results from the 744 couples were analysed by Spearman test, there were statistically significant correlations between most of the sperm test results. In particular, sperm motility was significantly correlated with normal sperm morphology (r = 0.323, P < 0.001), the number of spermatozoa bound per zona pellucida (r = 0.144, P < 0.001) and the proportion of zona pellucida penetrated (r = 0.202, P < 0.001). Similarly, normal sperm morphology was significantly correlated with number of spermatozoa bound per zona pellucida (r = 0.172, P < 0.001) and the proportion of zona pellucida penetrated (r = 0.176, P < 0.001). The number of spermatozoa bound/zona pellucida was highly significantly correlated with the proportion of zonae pellucidae penetrated (r = 0.744, P < 0.001). It should be noted that there was a high proportion of patients with normal semen analysis but poor sperm–zona pellucida binding and penetration (see below), and this may explain why the Spearman correlation coefficient was unexpectedly low (but nevertheless significant) between sperm morphology and motility, and sperm–zona pellucida binding and penetration.

Sperm–zona pellucida binding and penetration, and in-vitro fertilization
The distributions of numbers of spermatozoa bound to the zona pellucida and proportion of zona pellucida penetrated for different fertilization rate groups are shown in Figure 1Go and Figure 2Go. There were significantly higher proportions of couples with low sperm–zona pellucida binding (fewer than five spermatozoa bound per zona pellucida) with zero or low (<25%) fertilization rates than with fertilization rates of >25%. Over 70% of couples with zero fertilization rates had <=5 spermatozoa bound per zona pellucida. In contrast, only 17% of couples with fertilization rate >50% had <=5 spermatozoa bound per zona pellucida. There was a significantly (P < 0.001) higher proportion of patients (42%) with zero sperm–zona pellucida penetration with zero fertilization rates than with higher fertilization rates.



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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, {chi}2-test).

 


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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, {chi}2-test).

 
Sperm morphology and in-vitro fertilization
For couples with zero fertilization rates, over 35% had normal sperm morphology <=5%. In contrast only 7% of patients with fertilization rate 51–95% had normal morphology <=5% (Figure 3Go). For 142 couples with zero fertilization rates, zero sperm–zona pellucida penetration and low sperm–zona pellucida binding (fewer than five spermatozoa bound per zona pellucida), 37% had normal sperm morphology <=5%.



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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, {chi}2-test).

 
Logistic regression analysis
Since most patients (n = 563) were selected because of low fertilization rates (<=25%) their data were not included in the logistic regression analyses. Only data from the other randomly selected couples (n = 181) with fertilization rates >25% were examined by logistic regression analysis (Table IIGo). In this group of patients, both percentage normal sperm morphology and mean number of spermatozoa bound per zona pellucida were significantly related to fertilization rate (Table IIGo). When the 103 couples with all oocytes having spermatozoa penetrating the zona pellucida were analysed, percentage normal sperm morphology was significantly related to fertilization rate (Table IIGo).


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Table II. Significant variables related to fertilization rates by logistic regression analysis of results for patients with fertilization rates (FR) 26–95%
 
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. Over- all, 52% (160) had normal results (sperm concentration >=20x106/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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 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, 1988Go). 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., 1989aGo; Liu and Baker 1994aGo). In the present study higher concentrations of donor spermatozoa were used (1–2x106/ml) than previously (Liu et al., 1989bGo), 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., 1989bGo; Liu and Baker 1996Go).

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., 1986Go; Kruger et al., 1988Go; Liu et al., 1988Go; Claassens et al., 1992Go; Liu and Baker 1992aGo,bGo,cGo; Donnelly et al., 1998Go; Duran et al., 1998Go). 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 1992bGo,cGo; Garrett et al., 1997Go). 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., 1992Go; Liu and Baker, 1992cGo; Duran et al., 1998Go). However, there is no evidence to suggest that the phenotype of individual spermatozoa reflects the genotype it carries (Cummins and Jequier, 1994Go).

Because failure of fertilization in standard IVF is mainly due to defective sperm–zona pellucida binding and penetration associated with sperm abnormalities, it is therefore expected that the couples would have a higher fertilization rate with ICSI. Many sperm defects, including motility disorders, abnormal morphology, and inability of spermatozoa to bind or penetrate the zona pellucida, are bypassed by ICSI. In this study, the average fertilization rate with ICSI was 58% in 180 couples with zero or very low (<=25%) fertilization rates in previous standard IVF. Pre-IVF assessment of sperm function is important to assign patients to standard IVF or ICSI. This should avoid unexpected failure of fertilization by standard IVF. On the other hand, couples with adequate sperm function may not need ICSI and should be treated with standard IVF.

At the present time, most couples treated with ICSI have obvious severe sperm defects such as oligozoospermia, asthenozoospermia, and teratozoospermia alone or in combination. These couples are easily identified by routine semen analysis. However, in the present study, it was found that 52% of couples (160 of 310) with zero fertilization rate had normal pre-IVF semen analysis results and 47% of them had no spermatozoa penetrating the zona pellucida of any of the oocytes. It is suspected that many of these couples with normal semen analysis and failure of fertilization may have disordered zona pellucida-induced acrosome reaction (Liu and Baker, 1994bGo). Spermatozoa from these couples bind to the zona pellucida but do not undergo the acrosome reaction and are unable to penetrate the zona pellucida. Patients with this condition have very low or zero fertilization rate with standard IVF but high fertilization and pregnancy rates with ICSI (Liu et al., 1997Go). Our preliminary results indicate that up to one-third of normozoopsermic infertile men may have disordered zona pellucida-induced acrosome reaction (Liu and Baker, unpublished data). In order to reduce the incidence of failure of fertilization in standard IVF in unexplained infertile couples, pre-IVF screening for this condition is highly recommended so that the couples can be assigned directly to ICSI instead of standard IVF (Liu and Baker, 1994bGo; Liu et al., 1997Go).

In clinical IVF, usually 20–30% of oocytes fail to fertilize. These oocytes are valuable material for sperm–zona pellucida interaction tests. The zona pellucida of unfertilized oocytes is still capable of binding spermatozoa, inducing the acrosome reaction and being penetrated by the acrosome reaction spermatozoa (Liu et al., 1989bGo; Liu and Baker, 1996Go). The oocytes can be stored in concentrated salt solution (Yanagimachi et al., 1979Go; Liu et al., 1989bGo). Ethics Committee approval and patient consent is required for using the unfertilized oocytes for the tests. There should be no difficulty in obtaining such permission, as there is no intention to fertilize the oocytes and the material is to be discarded. We have been using such oocytes for sperm–oocyte interaction tests for many years. Over 95% of patients are willing to donate their unfertilized oocytes for either clinical tests or research. However, the number of human zonae pellucidae available for routine tests of sperm function is limited, particularly in small IVF clinics. In future, recombinant human zona pellucida 3 might be useful for routine tests of human sperm function (Van Duin et al., 1994Go; Whitmarsh et al., 1996Go).

In summary, defective sperm–zona pellucida binding and penetration are the major causes for zero or low fertilization rates with standard IVF. Failure of sperm–zona pellucida binding and penetration is mainly due to abnormalities of the spermatozoa not the oocytes. Most severe sperm defects are obvious from standard semen analyses but pre-IVF assessment of the capacity of spermatozoa to bind to and penetrate the zona pellucida will provide useful information for diagnosis and management of couples with idiopathic infertility with normal standard semen analysis.


    Acknowledgments
 
We thank Mingli Liu for technical assistance and all the scientists in both Melbourne IVF and Royal Women's Hospital IVF laboratories for collecting the oocytes and sperm samples for this study. The Royal Women's Hospital Research Committee supported this study.


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Carlton, Victoria 3053, Australia Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 24, 1999; accepted on November 15, 1999.