Hum. Reprod. Advance Access originally published online on July 26, 2007
Human Reproduction 2007 22(10):2632-2638; doi:10.1093/humrep/dem245
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Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm
1 Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women’s Hospital, 132 Grattan Street, Carlton 3053, Australia 2 Andrology Laboratory, Royal Women’s Hospital, 132 Grattan Street, Carlton 3053, Australia 3 Reproductive Services and Melbourne IVF, Royal Women’s Hospital, 132 Grattan Street, Carlton 3053, Australia
4 Correspondence address. Tel: +61-3-9344-2042; Fax: +61-3-9347-1761; E-mail: dyl{at}unimelb.edu.au
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Abstract |
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BACKGROUND: The aim of this study was to determine the relationship between human sperm hyperactivation (HA), sperm–zona pellucida (ZP) binding and the ZP-induced acrosome reaction (AR) of ZP-bound sperm in vitro.
METHODS: Sperm samples from 129 infertile men were studied. Motile sperm (2 x 106) selected by Pure sperm were incubated with four oocytes in 1 ml human tubal fluid supplemented with 10% human serum. After 2-h incubation, the number of sperm bound to the ZP and the AR of ZP-bound sperm were examined. Velocities and HA of sperm in insemination medium were assessed by Hamilton-Thorn Sperm Analyzer.
RESULTS: The HA was highly correlated with the ZP-induced AR in all the subjects (rho = 0.626, P < 0.001). In the 69 men with 
100 sperm bound/ZP, allowing accurate counts, HA was not significantly correlated with sperm–ZP binding. Men with <7% HA sperm were more likely to have very low ZP-induced AR. Only normal sperm morphology was significantly correlated with sperm–ZP binding (rho = 0.346 and 0.446 in semen and insemination medium, respectively, both P < 0.001). Sperm motility and velocities were significantly correlated with sperm morphology but not with either sperm–ZP binding or the ZP-induced AR.
CONCLUSIONS: The correlation of HA with the ZP-induced AR of ZP-bound sperm suggests a mechanistic link between HA and the physiological AR in humans. Assessment of HA of capacitated sperm in vitro may be a useful clinical test for male infertility associated with defective ZP-induced AR that does not require the use of human oocytes.
Key words: hyperactivation/sperm–zona pellucida binding/zona pellucida-induced acrosome reaction/IVF/ICSI
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Introduction |
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During the process of human fertilization, sperm must be capable of binding to the zona pellucida (ZP), undergoing the acrosome reaction (AR) on the surface of the ZP, penetrating the ZP and fusing with oolemma (Yanagimachi, 1994
). However, human sperm, like many other mammalian sperm are not ready to fertilize oocytes immediately after ejaculation. Sperm need to swim out from seminal plasma and undergo capacitation either in vivo in the female reproductive tract or in vitro in conditioned culture medium to obtain the capacity to interact with oocytes (Shalgi and Phillips, 1988; Yanagimachi, 1994). During sperm capacitation both in vivo and in vitro, there are numerous changes in the sperm plasma membrane involving biochemical and molecular events, including changes in the pattern of sperm motility known as hyperactivation (HA). The HA was first reported by Yanagimachi (1969) who found that HA of hamster sperm played a critical role in ZP penetration during the process of fertilization. Since then numerous studies on sperm HA both in vivo and in vitro in various mammalian species, including humans, have been reported in the literature (see the reviews by Yanagimachi, 1994; Pacey et al., 1997; Kay and Robertson, 1998; Ho and Suarez, 2001). In general, HA can occur in vivo during transportation of sperm in the female reproductive tract (mostly in the ampulla of the oviduct) and also in conditioned media during in vitro culture, and it may facilitate sperm progression towards the oocyte and penetration of its vestments (Yanagimachi, 1994; Ho and Suarez, 2001; Suarez and Pacey, 2006).
HA was observed in over 80% of capacitated sperm in some mammalian species such as hamster (Yanagimachi, 1970), mouse (Suarez and Osman, 1987) and rabbit (Suarez et al., 1983). In contrast, in humans, HA was observed in 
20% of capacitated sperm from fertile men (Burkman, 1984; Robertson et al., 1988). It was previously reported that human seminal plasma inhibited the HA in vitro (Mortimer et al., 1998) whereas both human follicular fluid and cervical mucus stimulated the HA in vitro (Mbizvo et al., 1991; Zhu et al., 1992; Kulin et al., 1994; Yao et al., 2000). In the hamster, there is a relationship between HA and sperm–ZP penetration (Yanagimachi, 1994) and inhibition of HA of capacitated and acrosome reacted sperm bound to the ZP blocked sperm–ZP penetration (Stauss et al., 1995). Furthermore, HA of hamster sperm was also highly correlated with the proportion of sperm undergoing the AR (Suarez et al., 1984). In human sperm, a preliminary study with very few sperm samples tested showed that there was weak correlation between HA and sperm–ZP binding capacity in the hemizona binding assay (Coddington et al., 1991). However, so far it is still unclear if HA can be used as a useful biomarker for the sperm fertilizing ability. Today, computer-assisted sperm analysis (CASA) systems such as the Hamilton-Thorne Sperm Analyser can be used to measure the HA of sperm after culture in vitro. The aim of this study was to determine the relationship between HA of capacitated sperm and sperm–ZP binding and the ZP-induced AR of ZP-bound sperm in vitro.
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Materials and Methods |
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Samples and semen analysis
Semen samples were obtained by masturbation after 2–5 days abstinence from 129 infertile men who attended our infertility clinics in both the Royal Women’s Hospital and Melbourne IVF between January and December 2006. The patients were selected for the study when they had at least 2 x 106 motile sperm recovered by colloidal silica gradient (PureSperm, Nidacon International AB, Molndal, Sweden) centrifugation. Routine semen analysis was performed after liquefaction of the semen within 1 h according to World Health Organization (WHO) manual (World Health Organization, 1999
). Both total motility (WHO criteria a + b + c) and progressive motility (a + b) were assessed manually by counting 200 sperm. Viability of sperm was assessed by Eosin Y exclusion.
Morphology of sperm in both semen and PureSperm preparation was assessed on smears prepared by washing of sperm with 10 ml 0.9% sodium chloride. Washing sperm to remove seminal plasma or protein in medium decreases background staining and produces clearer images of sperm (Liu and Baker, 1992). Morphology slides were stained with the Shorr method after the smears were fixed in 90% ethanol for 30 min (World Health Organization, 1999). The percentage of normal sperm morphology was assessed according to strict criteria (Kruger et al., 1988). For each sperm sample, 200 spermatozoa were scored from at least 10 individual fields using oil immersion with magnification of 1000x under bright-field illumination.
Sperm preparation
Motile sperm were selected using the PureSperm with two layers of 1 ml 40% and 1 ml 80% PureSperm. About 0.5–1 ml of semen or concentrated sperm suspension by centrifugation (only for oligozoospermic semen) was gently added to the top layer of PureSperm and centrifuged at 600g for 15 min. The pellet of motile sperm obtained from PureSperm preparation was washed once with 1 ml of human tubal fluid (HTF) supplemented with 10% inactivated human serum (MP Biomedicals, Irvine, CA, USA). Then the washed sperm pellet was resuspended in serum-supplemented HTF to a sperm concentration of 2 x 106/ml for the ZP-binding test.
Human oocytes
Oocytes which showed no evidence of two pronuclei or cleavage at 48–60 h after insemination in routine IVF, or after injection in ICSI, or immature (germinal vesicle or metaphase I) oocytes not injected in ICSI were used for the sperm–ZP binding test. Our previous study confirmed that the ZP of immature oocytes was similar to the ZP of unfertilized mature oocytes for binding sperm and induction of the AR (Liu and Baker, 1996a,b). If the oocytes had sperm bound to the ZP from the IVF insemination, these were removed by aspiration using a fine glass pipette with an inner diameter (120 µm) slightly smaller than the oocyte diameter (Liu and Baker, 2004). Oocytes with >10 sperm penetrating the ZP, or degenerate, activated or morphologically abnormal oocytes were not used. Oocytes were pooled from several patients and used for the test on the same day or kept in the incubator and used within the next 3 days.
All patients signed consent forms permitting use of their gametes (unfertilized or immature oocytes and sperm samples) for research. The Royal Women Hospital Research and Ethics Committees approved the study.
Sperm–ZP binding
Motile sperm (2 x 106 in 1 ml medium) selected by PureSperm were incubated with four oocytes in 4-well culture plates (Nunc, Rosilde, Denmark) for 2 h at 37°C in 5% CO2 in air. After this 2 h incubation period, 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). The oocytes were then flushed several times to dislodge loosely adherent sperm using a fine 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 phase contrast microscope and the average number of sperm bound per ZP was used as endpoint. Oocytes with 
100 sperm bound were recorded as 100 since it is impossible to count the number accurately. Under these experimental conditions, <40 sperm bound/ZP is considered to be low binding (Liu et al., 2004). In this study, the sperm–ZP binding test was designed to detect low sperm–ZP binding and it is possible small differences in numbers of sperm bound/ZP in men with normal ZP-binding above 100/ZP may have been obscured.
Assessment of the AR of sperm bound to the ZP
After counting the number of sperm bound to the ZP, all sperm bound to 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 (Liu and Baker, 1996a,b; Liu et al., 2001). This was performed on a glass slide with 5 µl PBS containing 0.2% BSA and dislodged 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 for assessment of the AR. For sperm samples with <40 sperm bound/ZP for all the four oocytes, the ZP-induced AR was not determined because there were insufficient sperm for accurate assessment. Our previous study confirmed that this pipetting procedure for removing sperm from the surface of ZP had no effect on sperm motility, morphology and acrosome status (Liu and Baker, 1996a).
The sperm bound to the ZP were removed and smeared on a glass slide as described above. The AR of dislodged ZP-bound sperm was assessed using Pisum sativum agglutinin (PSA) conjugated with fluorescein isothiocyanate (Sigma Chemical Company, St Louise, MO, USA) as described previously (Liu and Baker, 1996a). After air-drying, sperm smears were fixed in 95% ethanol for 30 min and then stained using 25 µg/ml PSA 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 400x. 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 (a rare pattern) were considered acrosome reacted.
Assessment of sperm velocity and HA
After 2 h incubation of motile sperm in medium (separate aliquot not exposed to oocytes), sperm motility, velocity (VSL, straight line velocity; VCL, curvilinear velocity and VAP, average path velocity) and other movement characteristics (LIN, linearity; STR, straightness) were measured by Hamilton-Thorn Motility Analyzer (IVOS 10, 60 Hz, Hamilton-Thorn Research, Danvers, MA, USA) at 37°C. The Burkman (1991) criteria for HA were used as follows: VCL 100 µm/s, LIN 65% and amplitude of lateral head displacement (ALH) 7.5 µm. The motility was defined as the percentage of sperm with VAP >7.5 µm/s. Because the sperm concentration was only 2 x 106/ml when the sperm were incubated in culture medium, the sperm concentration was adjusted to 20 x 106/ml by centrifugation at 600g for 5 min and resuspended in 100 µl of the same medium. A sample of 5 µl was placed in a Microcell (20 µm depth) for assessment of motility and velocities. For each sperm sample, an average of six (five to seven) fields with a total of 300–400 sperm was assessed.
Statistical analysis
Correlations between the percentage of HA sperm and the sperm–ZP binding, the ZP-induced AR and other sperm characteristics were analysed by the non-parametric (Spearman) test. Linear regression analysis was also performed after normalizing transformations (cube root for semen volume and concentration, logarithmic for HA and ZP-induced AR) to determine which sperm test results were independently significantly related to sperm–ZP binding or the ZP-induced AR. The significance of differences for the ZP-induced AR between patients with various percentages of HA sperm were examined by non-parametric analysis of variance.
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Results |
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There were wide ranges for all the sperm tests results obtained from the 129 infertile men (Table 1). For example, the number of sperm bound per ZP ranged from 1 to >100, ZP-induced AR from 1 to 84% and HA from 0 to 38%. The average of HA was 8% and ZP-induced AR of ZP bound sperm was 19%. Sixty men with >100 sperm bound/ZP had a mean HA of 9.0% with a range of 0–38% and the actual numbers of sperm bound/ZP were not counted for these 60 samples as it was impossible to count the number accurately.
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The percentage of HA after 2 h incubation was strongly correlated with the ZP-induced AR but not with sperm–ZP binding (Figs 1 and 2, Table 2). Importantly, men with <7% HA had significantly lower ZP-induced AR than those with
7% HA. Using ZP-induced AR <16% for diagnosis of defective ZP-induced AR (DZPIAR, Liu et al., 2004), a cut-off value of <7% HA for prediction of DZPIAR gives a sensitivity (true positive) rate of 75% and a specificity of 75% (false positive 25%). Lack of significant relationship between HA and sperm–ZP binding was also further confirmed by analysis of the data from 69 men (with an actual average number of sperm bound/ZP assessed) after excluding the 60 men with >100 sperm bound/ZP (Fig. 2). There was also no significant difference (P > 0.05) in percentage HA sperm between three groups of men with >100 sperm bound/ZP (n = 60, mean ± SD 9.0 + 7.8%), 40–100 sperm bound/ZP (n = 57, 7.3 ± 6.0%) and <40 sperm bound/ZP (n = 12, 7.5 ± 6.1%).
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Sperm concentration in semen and VCL in the insemination medium were also significantly correlated with the ZP-induced AR but to a lesser degree than HA (Table 2). Beat cross frequency (BCF) was negatively correlated with the ZP-induced AR. Only normal sperm morphology in both semen and insemination medium and BCF were significantly correlated with sperm–ZP binding (Table 2). All the other variables including VSL, VAP, STR and LIN were not significantly correlated with either sperm–ZP binding or the ZP-induced AR. Most semen analysis parameters such as sperm concentration, total motility, progressive motility and normal morphology were significantly correlated with HA (Table 3, Fig. 3). Also as expected most CASA variables such as velocities (VSL, VCL and VAP) and movement characteristics (BCF) were highly correlated with the HA since HA sperm were defined based on the values of VCL, LIN and ALH (Table 3).
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In multiple regression analysis models with all the variables included, only HA was significantly related to the ZP-induced AR (regression coefficient = 0.668, SEM = 0.088, P < 0.0001) and the other variables which were significant on their own did not add to the model. Similarly, only sperm normal morphology in insemination medium (regression coefficient = 31.14, SEM = 6.862, P < 0.0001) and BCF (regression coefficient = 1.534, SEM = 0.682, P < 0.05) were significantly related to sperm–ZP binding.
To confirm that the results of HA assessed by CASA were consistent and reproducible, duplicate tests were performed in 25 sperm samples from different men. Similar results were obtained from the duplicate tests (7.32 ± 6.6 versus 7.24 ± 6.1%, P > 0.05) with mean difference 0.1% and SD 1.3%. Spearman’s test showed a high correlation between the two tests results (rho = 0.964, P < 0.0001).
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Discussion |
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This study is the first to show that HA of capacitated human sperm is significantly correlated with the ZP-induced AR of ZP-bound sperm in vitro, suggesting that the subpopulation of HA motile sperm may be more likely to acrosome react after binding to the ZP. It has previously been reported in the hamster that HA of capacitated sperm was highly correlated with both the AR (Suarez et al., 1984
) and sperm–ZP penetration in vitro (Yanagimachi, 1994). It is believed that the HA motile sperm signifies the final stage, or completion, of capacitation (Katz et al., 1989) and thus it is logical that HA motile sperm would be more responsive to initiation of the AR triggered by the ZP after sperm binding. As calcium influx plays a central role for both HA and the AR, it is most likely they share the same early phase of signal transduction during sperm capacitation. Both HA motility and the ZP-induced AR will ultimately enhance the ability of sperm to penetrate the ZP and finally to fertilize oocytes. Therefore, it is likely that sperm penetration of the ZP requires both HA motility and the ZP-induced AR.
In this study, all sperm samples were from infertile men and thus there was a relatively high proportion of men with low ZP-induced AR which was associated with low HA. Thus, men with <7% HA sperm after 2 h pre-incubation were likely to have very low ZP-induced AR which results in reduced sperm–ZP penetration. When we used HA <7% as the cut-off value for prediction of men with low ZP-induced AR (<16%), the true positive proportion was 75% and false positive was 25%. While the clinical usefulness of this level of prediction is still limited in comparison with routine semen analysis results, the HA could provide additional information for evaluating sperm function. Most importantly further confirmatory studies could indicate whether HA could be an alternative test for prediction of defective ZP-induced AR without using human oocytes. It is known that the ZP-induced AR is highly correlated with sperm–ZP penetration (Liu and Baker, 1996b). Disordered ZP-induced AR causes severe infertility and failure of fertilization with standard IVF in infertile men with normal routine semen analysis and normal sperm–ZP binding (Liu and Baker, 1994; Liu et al., 2001). Sperm samples with either defective sperm–ZP binding, or normal sperm–ZP binding but defective ZP-induced AR, are associated with reduced sperm–ZP penetration leading to failure of fertilization in vitro and infertility in vivo (Liu and Baker, 2003,2004; Liu et al., 2004). The frequency of defective sperm–ZP binding was 10% in infertile men with normal semen and up to 25% in those with severe teratozoospermic semen (abnormal morphology 5%, Liu et al., 2004). However, defective ZP-induced AR with normal ZP-binding is about twice as frequent as defective sperm–ZP binding in the same groups of infertile men (Liu and Baker, 2003,2004; Liu et al., 2004). It is important to diagnose patients with defective ZP-induced AR before commencement of assisted reproduction treatment since sperm from these men will have a reduced ability to penetrate the ZP and the patients will be at high risk of low or zero fertilization rates if they are treated by standard IVF. Patients with this condition should be treated by ICSI.
At present, defective ZP-induced AR can only be diagnosed by performing sperm–ZP interaction tests using human oocytes, e.g. those that failed to fertilize during clinical IVF/ICSI. Unfortunately, routine testing of sperm–ZP interaction is difficult because there are a very limited number of unfertilized oocytes available from clinical IVF/ISCI. Alternative testing without using human oocytes for assessment of human sperm function is more practical for routine use. Currently, there are no other agents which can replace the human ZP for induction of the human AR. There was no correlation between either calcium ionophore A23187
[GenBank]
or progesterone-induced AR and the human ZP-induced AR (Liu and Baker, 1996a; Liu et al., 2004). While recombinant human ZP3 (rhZP3) has been produced by several groups, it has little biological activity when compared with native human ZP for binding sperm or inducing the AR (van Duin, et al., 1994; Brewis, et al., 1996; Whitmarsh, et al., 1996; Dong, 2001; Martic et al., 2004). Even when rhZP1, 2 and 3 were co-expressed in a human kidney cell line to produce recombinant proteins glycosylated in a human pattern, the recombinant proteins still had no activity to bind human sperm or induce the human AR in vitro (Martic et al., 2004). Similarly, transgenic mice with ZP2 and ZP3 replaced with the human ZP proteins have oocytes that still bind mouse sperm and fertilize, but do not bind human sperm (Rankin et al., 2003; Hoodbhoy and Dean, 2004). Therefore, there is currently no substitute for human ZP. Results of the present study showed that HA of capacitated sperm was strongly correlated with the ZP-induced AR. Therefore, assessment of HA of capacitated sperm by CASA may be a useful alternative test for prediction of the ZP-induced AR. Today, CASA systems have been widely used in many large Andrology laboratories and thus routine assessment of HA of capacitated sperm can be easily performed. While the predictive value of HA for the ZP-induced AR is limited, particularly in men with HA 7%, those with low (<7%) HA usually have low ZP-induced AR.
In the literature, it has been reported that sperm velocities (VSL, VCL and VAP) or some of the movement characteristics assessed by CASA, together with normal sperm morphology, were significantly related to fertilization rate in IVF, natural conception or pregnancy with intrauterine insemination in subfertile men (Check et al., 1990; Liu et al., 1991; Irvine et al., 1994; Hirano et al., 2001; Larsen et al., 2000; Garrett et al., 2003; Shibahara et al., 2003). However, there are not many studies on the relationship between HA and the sperm fertilizing ability. Wang et al. (1993) reported that HA of sperm after 6 h incubation was significantly correlated with fertilization rate in IVF. Burkman (1984) showed that infertile men had a significantly lower proportion of sperm developing HA than sperm from fertile men, and a similar finding was also reported by Munire et al. (2004). In donor insemination with frozen semen, it was reported that the proportion of sperm with HA stimulated by pentoxifylline was significantly related to the pregnancy rate (Johnston et al., 1994). Enhancement of HA by exposing sperm to 40°C significantly increased human sperm ZP-free hamster egg penetration (Chan et al., 1998). A preliminary report by Coddington et al. (1991) showed that HA of sperm in culture medium was weakly correlated with sperm–ZP binding assessed by hemizona binding assay. However, our study found no significant correlation between HA of capacitated sperm and sperm–ZP binding. It is likely that only motility or velocity but not HA is necessary for sperm to bind to the ZP. Green and Fishel (1999) also observed that the subpopulation of HA sperm had significantly better morphology than that of non-HA sperm. Most recently, Bastiaan and Franken (2006) reported that solubilized human ZP significantly stimulated sperm HA in vitro, and the level of the solubilized ZP-stimulated HA was highly correlated with the proportion of sperm with normal morphology (strict criteria). The present study with a large number of samples from infertile men also showed that the percentage of HA sperm of capacitated sperm was significantly correlated with normal morphology of sperm in semen and insemination medium, and also with sperm concentration and total and progressive motility.
In this study, HA was not significantly correlated with sperm–ZP binding, suggesting that HA is unlikely to be essential for sperm to bind to the ZP. However, the design of the sperm–ZP binding test, to make it sensitive for detecting low binding, may have obscured a small effect on sperm–ZP binding because a large proportion of the subjects did not have the average number of sperm bound/ZP determined accurately: 60 had >100 sperm bound to all four ZP. However, we consider this unlikely because HA was not significantly correlated with sperm–ZP binding when the data from the other 69 men (with <100 sperm bound/ZP) were analysed. Furthermore, there were no statistically significant differences in average HA between men with excellent binding (>100 sperm bound/ZP), intermediate binding (40–100 sperm bound/ZP) and low binding (<40 sperm bound/ZP).
In conclusion, HA of capacitated sperm is highly correlated with the ZP-induced AR of ZP-bound sperm, suggesting that the subpopulation of motile sperm capable of HA may be those which can also undergo the ZP-induced AR. Both HA and the AR play an important role in sperm–ZP penetration during fertilization. For clinical application, the assessment of HA of capacitated sperm by CASA may be a useful alternative routine test for the prediction of the ZP-induced AR without the need to use human ZP.
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Acknowledgements |
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The authors thank all the scientists in both Royal Women’s Hospital and Melbourne IVF Laboratories for collecting the oocytes and scientists in the Andrology Laboratory for sperm samples. This study was supported by the National Health and Medical Research Council with a grant number 400069.
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Submitted on March 20, 2007; resubmitted on June 1, 2007; accepted on June 28, 2007.
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