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Hum. Reprod. Advance Access originally published online on April 23, 2007
Human Reproduction 2007 22(7):1878-1884; doi:10.1093/humrep/dem087
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Comparison of the frequency of defective sperm–zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen

De Yi Liu1,4, Ming Li Liu1, Claire Garrett1 and H.W. Gordon Baker1,2,3

1 Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, 132, Grattan Street, Carlton, Victoria 3053, Australia 2 Reproductive Services, Royal Women's Hospital, 132, Grattan Street, Carlton, Victoria 3053, Australia 3 Melbourne IVF, 320 Victoria Parade, East Melbourne 3002, Australia

4 Correspondence address. Tel: +61-3-9344-2042; Fax: +61-3-9347-1761; E-mail: dyl{at}unimelb.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The aim of this study was to compare the frequency of defective sperm–zona pellucida (ZP) binding (DSZPB) and defective ZP-induced acrosome reaction (DZPIAR) in subfertile men (i.e. male partners of infertile couples) with normal and abnormal semen analyses.

METHODS: A total of 1030 subfertile men with normal semen analysis (n = 255), oligozoospermia (count < 20 x 106/ml, n = 136), severe teratozoospermia (strict normal morphology ≤ 5%, n = 294) and mild-moderate teratozoospermia (morphology 6–14%, n = 345) were studied. Unfertilized oocytes from clinical in vitro fertilization/intracytoplasmic sperm injection were used for sperm–ZP interaction tests. After 2 h incubation of motile sperm with four oocytes, sperm tightly bound to the ZP, and the AR of ZP-bound sperm (ZPIAR) were assessed. An average of < 40 sperm bound/ZP and < 16% ZPIAR were used for diagnosis of DSZPB or DZPIAR.

RESULTS: For the groups of men with normal semen or mild-moderate teratozoospermia, severe teratozoospermia and oligozoospermia, the frequencies of DSZPB were: 13, 21, 29 and 28%, respectively, and in those normal SZPB, DZPIAR were 27, 36, 56 and 68%, respectively. Overall DSZPB and ZPIAR were 36, 49, 68 and 77% for the four groups, respectively. The highest frequencies of defective sperm–ZP interaction were in the oligozoospermia and severe teratozoospermia groups. In the normal and teratozoospermia groups, subjects with a relatively low sperm concentration (20–60 x 106/ml) had a significantly higher frequency of DZPIAR.

CONCLUSION: Defective sperm–ZP interaction is a major mechanism of male infertility. DZPIAR is more frequent than DSZPB in subfertile men with either normal or abnormal semen, suggesting that sequential sperm–ZP interaction tests are essential to detect these sperm defects.

Key words: male infertility/semen analysis/sperm–zona pellucida interaction


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Statistical analysis
 Results
 Discussion
 Acknowledgements
 References
 
Infertility has a major public health impact as it affects ~10–15% of couples of reproductive age worldwide. Although assisted reproductive technology (ART) including conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are now effective procedures for management of human infertility not treatable by other methods, the precise cause of infertility for many of the couples undergoing ART is unknown. This is particularly true where the female has no absolute barrier to fertility and the male has semen analyses that are normal or mildly to moderately abnormal. Because the cause of infertility for many of couples seen in the ART clinics is unclear, it is often difficult to decide which of the ART treatments, conventional IVF or ICSI, will offer the couple with the best outcomes with minimum intervention and cost.

For ICSI, a single sperm is selected and injected directly into cytoplasm of each oocyte and therefore certain sperm functions are not required for fertilization. In contrast, in conventional IVF all sperm functions particularly sperm binding to the zona pellucida (ZP) and penetration of the ZP are essential for achieving fertilization (Liu and Baker 1992Go; Yanagimachi, 1994Go; Wassarman 1999Go). Thus, as a general rule, infertile couples with sperm defects are treated by ICSI and those without sperm defects are treated by conventional IVF. Therefore, it is very important to identify sperm defects that would impair fertilization with conventional IVF before commencing ART treatment. Currently in most ART clinics, assignment of patients for treatment with IVF or ICSI is mainly on the basis of conventional semen analysis results. However, semen analysis only determines sperm count, motility, viability and morphology, which do not accurately reflect many other sperm functions such as sperm–ZP binding (SZPB) and penetration (Overstreet et al., 1980Go; Oehninger et al., 1989Go, 1997Go, 2000Go; Liu and Baker, 1992Go, 1994Go; Mackenna et al., 1993Go; Liu et al., 2004Go, Sifer et al., 2005Go).

Before the introduction of ICSI, couples with normal or mild-moderate abnormalities of the semen analysis were treated by standard IVF but the frequency of low fertilization rate (<30%) was very high ranging from 15 to 30% of the patients undergoing IVF treatments. We showed that defective SZPB (DSZPB) and penetration were major contributors for failure of fertilization in IVF (Liu and Baker, 2000Go). Although some patients with low fertilization rates and defective sperm–ZP interaction had obvious semen abnormalities including oligozoospermia and teratozoospermia, many of the patients had normal semen analyses. Therefore defective sperm–ZP interaction can occur with both normal and abnormal semen. We previously reported particularly high frequencies of defective sperm–ZP interaction with both oligozoospermia and severe teratozoospermia (Liu and Baker 2003Go, 2004Go). However, the frequency of DSZPB in subfertile men with normal semen analysis and mild-moderate teratozoospermia has not been reported before.

In this study, we have analyzsed all patients (n = 1030) who had sperm–ZP interaction tests performed over the past 10 years, including 75 oligozoospermic and 125 severe teratozoospermic men reported previously (Liu and Baker, 2003Go, 2004Go), to compare the frequencies of defects of SZPB and ZP-induced acrosome reaction (ZPIAR) between subfertile men with normal semen and three categories of abnormal semen analysis results (oligozoospermia, mild-moderate teratozoospermia and severe teratozoospermia).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Statistical analysis
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
The subjects were 1030 men who are likely to be subfertile, being the male partners of couples being investigated for infertility at the Melbourne IVF Reproductive Services clinics between February 1995 and March 2006. All couples had tried for natural conception for at least 12 months without success. Semen samples were obtained by masturbation after 2–5 days abstinence. Sperm count and motility were performed after liquefaction within 1 h of collection of semen according to the World Health Organization manual (1992). Percent normal sperm morphology was assessed by scoring 200 sperm according to strict criteria on Shorr stained smears under oil immersion with magnification 1000 and bright field illumination (Kruger et al., 1988Go; World Health Organization, 1992). Morphology of motile sperm selected by swim-up was also assessed.

Reproducibility of the SZPB and ZPIAR within subjects between different ejaculates and with different batches of unfertilized oocytes was studied in 176 men who had two sperm–ZP interaction tests performed within 2–30 weeks.

Patients were divided into four groups based on the result of the semen analysis performed on the same sample as was tested for sperm–ZP interaction: normal semen (n = 255, sperm count ≥ 20 x 106/ml, motility ≥ 30%, normal morphology ≥ 15%), mild-moderate teratozoospermia (n = 345, normal morphology 6–14% and sperm count ≥ 20 x 106/ml), severe teratozoospermia (n = 294, normal sperm morphology ≤ 5% and sperm count ≥ 20 x 106/ml) and oligozoospermia (n = 136, sperm count < 20 x 106/ml). In the oligozoospermic group, only those samples with at least 1 x 106 motile sperm recovered after swim-up were included.

All patients signed consent forms permitting use of their gametes (unfertilized oocytes and sperm samples) for research. The Royal Women Hospital Research and Ethics Committees approved the project.

Human oocytes
Oocytes for the ZP-interaction tests were obtained from the clinical IVF/ICSI program. These failed to fertilize and showed no evidence of pronuclei or cleavage up to 60 h after insemination or were immature oocytes (germinal vesicle to metaphase I [MI] stages) not used for ICSI. Oocytes with any remaining cumulus and corona cells or sperm bound to the ZP from the IVF insemination had these removed by repeated aspiration of the oocyte using a fine glass pipette with an inner diameter (120 µm) slightly smaller than the oocyte diameter (Liu and Baker, 1994Go, 1996aGo,bGo). Degenerate, activated or morphologically abnormal oocytes as well as oocytes with > 10 sperm penetrating the ZP were not used for the test. All the oocytes were obtained on day 3 of insemination and the oocytes were pooled from several patients and used for the test on the same day or kept in the 5% CO2 incubator and used within the next 3 days.

Sperm preparation
Motile sperm were selected by swim-up technique as follows: 0.3–0.5 ml of semen (or sperm pellet for oligozoospermic semen) was carefully added to the bottom of a test tube (12 x 75 mm) containing 0.7 ml human tubal fluid (HTF) supplemented with 10% human serum (ICN Pharmaceuticals, Costa Mesa, CA, USA). Care was taken to avoid bubbles and not disturb the interface between semen and the medium. After incubation for 1 h, 0.5 ml of the top layer of the medium containing motile sperm was aspirated. The motile sperm suspension was then centrifuged at 1000 g for 5 min, the supernatant removed and the sperm pellet washed again with 1 ml fresh HTF by centrifugation at 1000 g for 5 min. The washed sperm pellet was resuspended in serum supplemented HTF to a motile sperm concentration of 20 x 106/ml for subsequent experiments.

Design of sequential sperm–ZP interaction tests
In this study, we used a sequential sperm–ZP interaction test to screen for two sperm defects: (i) DSZPB and (ii) defective ZPIAR (DZPIAR) in men with normal SZPB (Fig. 1). A relatively high concentration (2 x 106/ml) of motile sperm was incubated with a group of four oocytes for each test. This enabled counting of at least 200 ZP-bound sperm recovered from the four oocytes after assessment of SZPB.


Figure 1
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  		Figure 1: Diagram showing the sequential sperm–ZP interaction tests, as described in detail in the Materials and Methods section.

 
Sperm–ZP binding
As illustrated in Fig. 1, motile sperm (2 x 106/1 ml or 1 x 106/0.5 ml medium for some oligozoospermic samples) selected by swim-up were incubated with four randomly selected oocytes in 4-well culture plates (Nunc, Rosilde, Denmark) for 2 h at 37°C in 5% CO2 in air. After 2 h incubation, each group of four oocytes was transferred to phosphate-buffered saline (PBS) containing 2 mg/ml bovine serum albumin (BSA, Commonwealth Serum Laboratory, Parkville, Victoria, Australia), and then the oocytes were flushed several times to dislodge loosely adherent sperm using a pipette approximately twice the diameter of the oocyte (250 µm) in three separate wells containing 0.5 ml PBS with 0.2% BSA. The number of sperm bound to each of four oocytes was counted using an inverted contrast microscope, and average number of sperm bound per ZP was used as the endpoint. Oocytes with > 100 sperm bound/ZP was recorded as 100 since it was difficult to accurately count sperm bound above this number.

Assessment of acrosome status of sperm bound to the ZP
For sperm samples with normal ZP binding (average ≥ 40 sperm/ZP), all sperm bound to the surface of the four ZPs were removed by repeated vigorous aspiration with a narrow gauge pipette with an inner diameter (~120 µm) slightly smaller than the oocyte (Fig. 1, Liu and Baker, 1994Go, 1996bGo). This was performed on a glass slide with ~5 µl PBS containing 0.2% BSA, and the removed ZP-bound sperm were smeared in a limited area (~16 mm2), which was marked on the back of the slides with a glass pen to help find the sperm under the microscope. This pipetting procedure for removing sperm from the surface of ZP does not affect sperm motility, morphology or acrosome status (Liu and Baker, 1996bGo).

The acrosomes of the ZP-bound sperm were assessed using Pisum sativum agglutinin conjugated with FITC (PSA-FITC, Sigma Chemical Company, St Louise, MO, USA) as described previously (Cross et al., 1986Go; Liu and Baker 1996bGo). Sperm smears were fixed in 95% ethanol for 30 min after air-drying and then stained using 25 µg/ml PSA-FITC in PBS for 2 h at 4°C. The slides were washed and mounted with distilled water and 200 sperm per sample were counted with a fluorescence microscope using excitation wavelengths of 450–490 nm and a magnification of x 400. When more than half the head of a sperm was brightly and uniformly fluorescing, the acrosome was considered intact. Sperm with a fluorescing band at the equatorial segment or without fluorescence in acrosome region were considered reacted.

The ZPIAR results for two samples (one from the normal group and another one from the mild-moderate group) were not obtained due to accidental damage to the smear during the process of PSA staining.

Thresholds for DSZPB and DZPIAR
A threshold of < 40 sperm bound/ZP was used as cut-off for classification of DSZPB. This threshold is approximately equivalent to < 2 sperm bound/ZP with standard IVF insemination (Liu and Baker, 2003Go, 2004Go). The ZPIAR < 16% was used for classification of DZPIAR according to our previous report that men with < 16% ZPIAR would have very low (<30%) sperm–ZP penetration and fertilization rates with standard IVF (Liu and Baker, 1996aGo). Our previous study of normal fertile men (n = 111), whose partners were 16–32 weeks pregnant, showed the normal range of the ZPIAR averaged 48% with a range of 20–98% (Liu et al., 2003Go).


   Statistical analysis
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
Results
 Discussion
 Acknowledgements
 References
 
Correlations between results of SZPB, ZPIAR and other sperm characteristics were examined by Spearman's tests. Differences in frequency of DSZPB and DZPIAR between the semen groups was examined by {chi}2-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
Discussion
 Acknowledgements
 References
 
Reproducibility of sperm binding and the ZPIAR between ejaculates within the same men
All results for the two semen samples from the 176 men were highly correlated (Table 1), particularly sperm concentration, motility, morphology and SZPB. However, live sperm and the ZPIAR results showed a less significant correlation between two semen samples. The mean and standard deviation (SD) of the difference between two results were 13% (SD 20%) for SZPB and 8% (SD 9%) for the ZP induced AR. When the cut-off values used for diagnosis of DSZPB and DZPIAR were applied, the proportion of discrepant results (one normal and one abnormal) were 7% (12 of 176) for SZPB and 15% (21 of 126) for ZPIAR.


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  		Table 1: Correlations (Spearman's {rho} and P-value) between sperm tests results on two semen samples from the same 176 men

 
Semen analysis and SZPB and the ZPIAR
There was a wide range of sperm test, SZPB and the ZPIAR results in each of the four groups (Table 2). Since the four groups of patients were divided according to semen analysis results, all semen analysis results except live sperm were significantly different.


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  		Table 2: Mean ± SD (range) for all sperm tests results in 1030 subfertile men with normal semen analysis, mild-moderate teratozoospermia, severe teratozoospermia and oligozoospermia

 
Correlations between semen analysis results and SZPB and ZPIAR
In the normal semen analysis group, total motility, progressive motility and normal morphology in semen were significantly correlated with SZPB. Sperm concentration was significantly correlated with the ZPIAR. The other semen analysis results were not significantly correlated with either SZPB or ZPIAR (Table 3).


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  		Table 3: Correlations between results of semen analysis and SZPB, and the ZPIAR in the four groups of subfertile men with normal semen analysis, mild-moderate teratozoospermia, severe teratozoospermia and oligozoospermia (Spearman's {rho} and P-value, NS for not significant)

 
In the mild-moderate teratozoospermia group, sperm concentration was weakly correlated with the ZPIAR. All the other semen parameters were not correlated with either SZPB or the ZPIAR (Table 3).

In the severe teratozoospermia group, total and progressive motility were significantly correlated with SZPB. In contrast, sperm concentration and, to a lesser extent, sperm morphology in semen and in an insemination medium were significantly correlated with ZPIAR (Table 3).

In the oligozoospermia group, total and progressive motility in semen were significantly correlated with the SZPB. Sperm morphology in semen and insemination medium were significantly, though weakly, correlated with the ZP induced AR (Table 3).

Frequency of defective sperm–ZP interaction in subfertile men with normal and abnormal semen
The SZPB and the ZPIAR results in the four semen groups are shown in Fig. 2. For the four groups, normal semen, mild-moderate teratozoospermia, severe teratozoospermia and oligozoospermia, the frequency of DSZPB was 13, 21, 29 and 28%, respectively. The frequency of DSZPB was significantly higher in the abnormal semen analysis groups. In men with normal SZPB, the frequency of DZPIAR was 27, 36, 56 and 68% in the same groups and the difference between the normal and each abnormal semen group was statistically significant. In all men with either normal or abnormal semen, the frequency of DZPIAR was significantly higher than the frequency of DSZPB (Fig. 2A).


Figure 2
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  		Figure 2: Comparison of the frequency of DSZPB and DZPIAR (A) or a combination of both DSZPB and DZPIAR (B) between men with normal, mild-moderate teratozoospermic (M terato), severe teratozoospermic (S terato) and oligozoospermic (Oligo) semen.

 
The overall frequencies of defective sperm–ZP interaction (DSZPB and DZPIAR both combined) were 36, 49, 68 and 77% (Fig. 2B). The severe teratozoospermia and oligozoospermia groups had a higher frequency of defective sperm–ZP interaction than the other groups. Although the normal semen and both teratozoospermia groups had by definition normal sperm concentrations ≥ 20 x 106/ml, sperm concentration was still highly significantly associated with ZPIAR. Men with sperm concentrations 20–60 x 106/ml had a higher frequency of DZPIAR than those with sperm concentrations greater than 60 x 106/ml (Fig. 3).


Figure 3
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  		Figure 3: Comparison of the frequency of DZPIAR in men with normal semen or in teratozoospermic (mild-moderate and severe) groups with sperm concentrations 20–60 x 106/ml or > 60 x 106/ml.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
Acknowledgements
 References
 
These results confirm there is a high frequency of defective sperm–ZP interaction in subfertile men. Low SZPB was found in 13, 21, 29 and 28% in men with normal, mild-moderate teratozoospermia, severe teratozoospermia and oligozoospermia. Although subfertile men with oligozoospermia and severe teratozoospermia had significantly higher frequencies of DSZPB, overall 72% of subfertile men with either normal or abnormal semen had normal SZPB results. However, in the men with normal SZPB, DZPIAR was found in 27, 36, 56 and 68% in the four groups, respectively, when a cut-off value of < 16% ZPIAR was used for diagnosis of DZPIAR. When sperm from normal fertile men were tested with the same ZP-interaction test, ZPIAR was an average of 48% with a wide range from 20 to 98% (Liu et al., 2003Go). The frequency of DZPIAR was about two to three times higher than the DSZPB in each group. Therefore, it is important to use sequential sperm–ZP interaction tests for detection of both DSZPB and DZPIAR. In contrast with other previously reported sperm–ZP interaction tests such as hemizona assay, SZPB ratio and the solubilized ZPIAR tests (Liu and Baker, 1992Go, 1994Go; Oehninger et al., 1997Go, 2000Go; Esterhuizen et al., 2001Go; Bastiaan et al., 2003Go), the sequential sperm–ZP interaction tests described in this study can detect two sperm defects, DSZPB and DZPIAR from the same test. Alternatively, assessment of sperm–ZP penetration would indicate either DSZPB or DZPIAR. However, most of the unfertilized oocytes obtained from clinical IVF/ICSI are not suitable for sperm–ZP penetration tests since many of the oocytes from IVF have sperm penetrating the ZP. Although these oocytes can be used to test for SZPB and the ZPIAR, they are not useful for sperm–ZP penetration tests (Liu and Baker, 1996bGo).

In total, 36% of subfertile men with normal semen analysis had defective sperm–ZP interactions, which may be the real cause of their infertility. This result further indicates that conventional semen analysis is limited for predication of the sperm fertilizing ability. Without sperm–ZP interaction tests, over one-third of subfertile men with normal semen analysis will not be diagnosed. In most clinical ART services, as these men have normal semen analysis, the couples are treated by IVF in the initial cycle. If IVF fails then they will be treated with ICSI. This means patients are put at risk of having low or complete failure of fertilization in IVF and may waste one cycle with the attendant costs both financially and emotionally. This could be minimized by screening for defective sperm–ZP interaction before commencing the ART treatment. Patients identified with DSZPB or DZPIAR regardless of semen analysis results should be assigned to ICSI in which a sperm will be injected into the cytoplasm to bypass the ZP. Thus, the sperm–ZP interaction tests should be useful in diagnosis of the cause of the infertility and management by ART.

In the severe teratozoospermia and oligozoospermia groups, only 28 and 29% had DSZPB but 56 and 68% had DZPIAR. The overall, frequency of defective ZP interaction was 68 and 77% for these two groups. This is consistent with the high incidence of low or zero fertilization rates seen when patients with severe teratozoospermia and oligozoospermia were treated with conventional IVF (Baker et al., 1993Go). Thus, in the clinical situation, such patients do not need tests for sperm–ZP interaction because they have such a high frequency of defective sperm–ZP interaction and semen analysis results are sufficient for making the clinical decision to assign these patients directly to ICSI. The small proportions of patients in these groups (23 and 32%) who have normal sperm–ZP interactions could be distinguished by performing sperm–ZP interaction tests if they particularly wished to have treatment with IVF rather than ICSI.

Although all infertile patients irrespective of sperm defects now could be treated by ICSI, most clinics do not do this because (i) the ICSI procedure is more invasive and costly than IVF, (ii) ICSI damages a small proportion of oocytes, (iii) immature oocytes are not used for ICSI but these might mature and fertilize in standard IVF, (iv) there is evidence for less embryo utilization (embryos/oocytes) with ICSI than IVF (Bhattacharya et al., 2001Go) and (v) patients may prefer to have treatment with IVF than ICSI or want to know why they need to be treated by ICSI. Therefore, at the moment, IVF should still be the first option for most patients with normal or mild-moderate abnormalities of semen analysis and ICSI is mainly for couples with severe sperm defects. Sperm–ZP interaction tests can be useful to identify otherwise hidden sperm defects that are unable to be predicted by routine semen analysis (Overstreet et al., 1980; Mackenna et al., 1993Go, Oehninger et al., 1997Go, 2000Go; Liu et al., 2004Go; Sifer et al., 2005Go). In the past decade, several similar sperm–ZP interaction tests have been developed and evaluated extensively, such as SZPB ratio test, hemizona assay and solubilized ZPIAR; all these tests are valid for prediction of sperm fertilizing ability in vitro and useful for diagnosis and management of male infertility in clinical ART (Oehninger et al., 1989Go, 1997Go, 2000Go; Liu and Baker 1992Go, 1994Go; Esterhuizen et al., 2001Go; Bastiaan et al., 2003Go). Most recently, Arslan et al. (2006) reported that the hemizona assay was highly predictive of pregnancy outcomes in couples with male factor and unexplained infertility undergoing controlled ovarian hyperstimulation with intrauterine insemination.

As all the oocytes used for the tests were obtained from unfertilized oocytes or immature (GV or MI) from clinical IVF/ICSI, the quality of individual oocytes may be variable for capacity of binding of sperm or induction of the AR of ZP-bound sperm. However, our previous study showed that similar results of ZPIAR were obtained when the same sperm was tested using two different batches of oocytes (Liu and Baker, 1996bGo). In this study, 179 men had two sperm–ZP interaction tests performed with different ejaculates within 2–30 weeks. There was a high correlation between the two results of all sperm tests including SZPB and the ZPIAR. However there was an upward trend in the latter results of the second test because we selected subjects with low results in the first test for repeat sperm–ZP interaction tests and this would be expected as a result of regression to the mean (Baker and Kovacs, 1985Go). Variability of tests using the cut-off values resulted in 7 and 15% of men with different results in the two tests for diagnosis of DSZPB or DSZPIAR. Mixed results for DZPIAR were about two times higher than for DSZPB. This may suggest that (i) activity of the ZP of unfertilized oocytes for induction of the AR may be more variable between individual batches of oocytes and/or (ii) ZPIAR is more variable between ejaculates. Although variability of results of SZPB is much less than for routine semen analysis results, the tests are not highly reproducible, and therefore, for clinical application, a second test should be performed to confirm the results in men with either low SZPB or low ZPIAR in one test.

Another important factor related to the ZPIAR was sperm concentration in semen. The oligozoospermia group had the highest frequency of DZPIAR. Even in men in the normal semen and teratozoospermia groups who by definition had sperm concentrations > 20 x 106/ml, sperm concentration was significantly correlated with ZPIAR. Men with relatively lower (≤60 x 106/ml) sperm concentrations had DZPIAR more often than those with relative higher (> 60 x 106/ml) sperm concentrations. It is likely that DZPIAR originates from abnormal spermatogenesis and this requires further study.

For all the groups studied except the mild-moderate teratozoospermic group, both sperm motility and morphology were highly correlated with SZPB. Also sperm morphology was weakly correlated with the ZPIAR. These results may further suggest that overall sperm quality revealed by routine semen analysis may relate to the frequency of defective sperm–ZP interaction. However, routine semen analysis results cannot accurately predict the ability of sperm–ZP interaction.

At the present, human oocytes are essential for sperm–ZP interaction tests since binding of sperm to the ZP is highly species-specific and no animal ZP or other substitute is available. Oocytes which failed to fertilize in clinical IVF/ICSI are the major source, but the number of oocytes available is very limited and therefore routine testing for all patients is not possible. Although recombinant human ZP3 has been produced by several independent groups, it is not consistently as active as native human ZP for binding sperm or inducing the AR (van Duin et al., 1994Go; Brewis et al, 1996Go; Whitmarsh et al, 1996Go; Dong, 2001Go). We expressed rhZP1, 2 and 3 alone or in combination in a human kidney cell line to produce recombinant proteins glycosylated in a human pattern, but none bound human sperm or induced the human AR in vitro (Martic et al., 2004Go). Furthermore, transgenic mice with ZP2 and 3 replaced with the human ZP proteins have oocytes that still bind mouse sperm and fertilize, but human sperm are unable to bind to the ZP of these oocytes (Rankin et al., 2003Go; Hoodbhoy et al., 2005). Therefore, there is currently no substitute for human ZP and human oocytes that failed to fertilize in clinical IVF remain a valuable resource for studying human sperm–ZP interaction (Liu et al., 2004Go).

Our recent studies showed that assessment of the proportions of sperm exposing actin on the surface of the head or becoming tyrosine phosphorylated during in vitro culture correlate with SZPB capacity (Liu et al., 2005; 2006). However, these tests cannot predict ZPIAR. During human fertilization, only motile and acrosome intact sperm bind to the ZP and then the AR occurs on the surface of ZP, induced by the ZP proteins (Cross et al., 1988Go; Tesarik, 1989Go). However, most studies of the human AR in the literature involve model systems (membrane preparations and permeabilized sperm) and other stimuli (progesterone and calcium ionophore), which may not reflect the physiological human ZPIAR. For example, there is no relationship between ZPIAR and either the calcium ionophore-induced AR or the spontaneous AR occurring with incubation, whereas there is close relationship between ZPIAR and the AR induced with solubilized human ZP (Cross et al., 1988Go; Liu and Baker, 1996aGo,bGo). Because the ZPIAR is not correlated with the calcium ionophore A23187 [GenBank] -induced AR, tests for A23187 [GenBank] -induced AR cannot predict DZPIAR. Therefore, at the moment, intact or solubilized human ZP is essential for testing the physiological AR for predicting the sperm fertilizing ability (Liu and Baker, 1996aGo,bGo; Esterhuizen et al., 2001Go; Bastiaan et al., 2003Go; Liu et al., 2004Go). In future, development of alternative tests for prediction of sperm–ZP interaction that do not require human oocytes will be a major advance for routine assessment of human semen.

In conclusion, the frequency of defective sperm–ZP interaction was significantly higher in subfertile men with abnormal semen than in those with normal semen, being highest with oligozoospermia and severe teratozoospermia. Defective sperm–ZP interaction is the major mechanism of infertility due to sperm abnormalities. DZPIAR is more frequent than DSZPB in men with normal and abnormal semen. The use of sperm–ZP interaction tests would improve both diagnosis of the cause of infertility and the management of infertility by ART. The results of this study explain why so many infertile couples with mild-moderate teratozoospermia require treatment by ICSI rather than IVF.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Acknowledgements
References
 
The authors thank scientists in both Royal Women's Hospital and Melbourne IVF Laboratories for collecting the oocytes and scientists in the Andrology Laboratory, Department of Pathology for sperm samples. This study was supported by the National Health and Medical Research Council with a grant number 400069.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 11, 2006; resubmitted on March 4, 2007; accepted on March 6, 2007.


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