Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm

Transcription

1 Human Reproduction Vol.22, No.10 pp , 2007 Advance Access publication on July 26, 2007 doi: /humrep/dem245 Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm D.Y. Liu 1,4, M.L. Liu 1, G.N. Clarke 2 and H.W.G. Baker 1,3 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: þ ; Fax: þ ; dyl@unimelb.edu.au 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 ( ) 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 , 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 and 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. Keywords: hyperactivation; sperm zona pellucida binding; zona pellucida-induced acrosome reaction; IVF; ICSI Introduction 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 2632 # The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Sperm hyperactivation and zona-induced acrosome reaction 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. Materials and Methods 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 The patients were selected for the study when they had at least 2 x 10 6 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 þ b þ c) and progressive motility (a þ 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 1000 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 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 /ml for the ZP-binding test. Human oocytes Oocytes which showed no evidence of two pronuclei or cleavage at 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 mm) 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 ( in 1 ml medium) selected by PureSperm were incubated with four oocytes in 4-well culture plates (Nunc, Rosilde, Denmark) for 2 h at 378C in5%co 2 in air. After this 2 h incubation period, each group of four oocytes was transferred to phosphatebuffered 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 mm) 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 mm) slightly smaller than the oocyte (Liu and Baker, 1996a,b; Liu et al., 2001). This was performed on a glass slide with 5 ml PBS containing 0.2% BSA and dislodged sperm were smeared in a limited area (16 mm 2 ), 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 airdrying, sperm smears were fixed in 95% ethanol for 30 min and then 2633

3 Liu et al. stained using 25 mg/ml PSA in PBS for 2 h at 48C. The slides were washed and mounted with distilled water and 200 sperm per sample were counted with a fluorescence microscope using excitation wavelengths of nm and a magnification of 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 (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 378C. The Burkman (1991) criteria for HA were used as follows: VCL 100 mm/ s, LIN 65% and amplitude of lateral head displacement (ALH) 7.5 mm. The motility was defined as the percentage of sperm with VAP.7.5 mm/s. Because the sperm concentration was only /ml when the sperm were incubated in culture medium, the sperm concentration was adjusted to /ml by centrifugation at 600g for 5 min and resuspended in 100 ml of the same medium. A sample of 5 ml was placed in a Microcell (20 mm depth) for assessment of motility and velocities. For each sperm sample, an average of six (five to seven) fields with a total of 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 nonparametric analysis of variance. Table 1: Mean + SD and range of all sperm tests results in infertile men Tests n Mean + SD Range Semen (assessed by manual methods) Volume of ejaculate (ml) Sperm concentration (10 6 /ml) Total motility (%) Progressive motility (%) Live (%) Normal sperm morphology (%) Insemination medium (assessed by CASA, manual and sperm-oocyte interaction tests) Normal sperm morphology (%) No. sperm bound/zp a ZP-induced AR (%) b HA motility (%) VSL (mm/s) VCL (mm/s) VAP (mm/s) STR (%) LIN (%) BCF (Hz/s) ZP, zona pellucida; HA, hyperactivation; VSL, straight line velocity; VCL, curvilinear velocity; VAP, average path velocity; LIN, linearity; STR, straightness; AR, acrosome reaction; BCF, beat cross frequency; CASA, computer-assisted sperm analysis. a For the number of sperm bound/zp the median was 100 with quartiles 60 and 100; b ZP-induced AR was not tested in 12 men who had low sperm ZP binding and too few ZP-bound sperm. Results 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. 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 2634 Figure 1: Correlation between HA and the ZP-induced AR(A) Dot-plot of individual results (n ¼ 117, Spearman rho ¼ 0.626, P, 0.001). (B) Mean and SEM for various HA groups. The ZP-induced AR was significantly lower in all groups of men with HA,7% compared with all groups with HA 7% (all P, 0.001)

4 Sperm hyperactivation and zona-induced acrosome reaction 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 ¼ 60, Figure 2: Correlation between HA and number of sperm bound/zp in: (A) all 129 samples (a total of 67 dots plotted: 47 dots represent individual sample and 20 dots represent other 62 samples which had identical values, Spearman rho ¼ 0.019, n ¼ 129, P. 0.05); (B) the subgroup of 69 samples with 100 sperm bound/zp (a total of 58 dots plotted: 54 dots represent individual sample and 4 dots represent other 11 samples which had identical values, Spearman rho ¼ 0.112, n ¼ 69, P. 0.05). Table 2: Spearman (rho) correlation between semen analysis, velocities and HA and sperm ZP binding and the ZP-induced AR (NS: not significant) Tests No. sperm bound/zp ZP-induced AR (%) rho P-value rho P-value Semen (assessed by manual methods) Sperm concentration (10 6 /ml) NS 0.300,0.001 Live sperm (%) NS NS Total motility (%) NS NS Progressive motility (%) NS NS Normal sperm morphology (%) 0.346, NS Insemination medium (assessed by CASA) Normal sperm morphology (%) 0.446, NS VSL (mm/s) NS NS VAP (mm/s) NS NS VCL (mm/s) NS 0.208,0.05 HA motility (%) NS 0.626,0.001 STR (%) NS NS LIN (%) NS NS BCF (Hz) 0.202, ,0.01 mean + SD 9.0 þ 7.8%), sperm bound/zp (n ¼ 57, %) and,40 sperm bound/zp (n ¼ 12, %). 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). In multiple regression analysis models with all the variables included, only HA was significantly related to the ZP-induced AR (regression coefficient ¼ 0.668, SEM ¼ 0.088, P, ) 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 ¼ 31.14, SEM ¼ 6.862, P, ) and BCF (regression coefficient ¼ 1.534, SEM ¼ 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 ( versus %, 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 ¼ 0.964, P, ). Discussion This study is the first to show that HA of capacitated human sperm is significantly correlated with the ZP-induced AR of Table 3: Spearman (rho) correlation between HA and other sperm parameters obtained from routine semen analysis and CASA (n ¼ 129, NS for not significant) HA motility (%) rho P-value Semen (assessed by manual methods) Sperm concentration (10 6 /ml) ¼0.003 Live sperm (%) NS Total motility (%) ¼0.003 Progressive motility (%) 0.312,0.001 Normal sperm morphology (%) ¼0.001 Insemination medium (assessed by CASA) Normal sperm morphology (%) ¼0.002 VSL (mm/s) 0.429, VAP (mm/s) 0.696, VCL (mm/s) 0.491, STR (%) NS LIN (%) NS BCF (Hz) ,

5 Liu et al. Figure 3: Correlation between the percentage of HA sperm and normal sperm morphology in (A) semen (Spearman rho ¼ 0.320, n ¼ 129 P ¼ 0.001) and (B) insemination medium (Insem, B, Spearman rho ¼ 0.314, n ¼ 129, P ¼ 0.001) 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 2636 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 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

6 Sperm hyperactivation and zona-induced acrosome reaction 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 408C 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 ( 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. Acknowledgements 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 References Bastiaan H, Franken D. The influence of homogenous zona pellucida on human spermatozoa hyperactivation, acrosome reaction and zona binding. Andrologia 2007;39:7 11. Brewis IA, Clayton R, Barratt CLR, Hornby DP, Moore HDM. Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. Mol Hum Reprod 1996;2: Burkman LJ. Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm population. Arch Androl 1984;13: Burkman LJ. Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermatozoa using computerized analysis. Fertil Steril 1991;55: Chan PJ, Corselli JU, Patton WC, Jacobson JD, King A. Enhanced fertility after heat-induced hyperactivation. Fertil Steril 1998;69: Check JH, Bollendorf, Lee MA, Nazari A, Nowroozi K. Correlation of computerized semen analysis with successful fertilization of oocytes in an in vitro fertilization program. Arch Androl 1990;24: Coddington CC, Franken DR, Burkman LJ, Oosthuizen WT, Kruger T, Hodgen GD. Functional aspects of human sperm binding to the zona pellucida using the hemizona assay. J Androl 1991;12:1 8. Dong KW, Chi TF, Juan YW, Chen CW, Lin Z, Xiang XQ, Mahony M, Gibbons WE, Oehninger S. Characterization of the biologic activities of a recombinant human zona pellucida protein 3 expressed in human ovarian teratocarcinoma (PA-1) cells. Am J Obstet Gynecol 2001;184: Garrett C, Liu DY, Clarke GN, Rushford DD, Baker HWG. Automated semen analysis variables: zona pellucida preferred sperm morphometry and straight-line velocity are related to pregnancy rate in subfertile couples. Hum Reprod 2003;18: Green S, Fishel S. Morphology comparison of individually selected hyperactivated and non-hyperactivated human spermatozoa. Hum Reprod 1999;14: Hirano Y, Shibahara H, Obara H, Suzuki T, Takamizawa S, Yamaguchi C, Tsunoda H, Sato I. Relationship between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rate in vitro. J Assist Reprod Genet 2001;18: Ho HC, Suarez SS. Hyperactivation of mammalian spermatozoa: function and regulation. Reprod 2001;122: Hoodbhoy T, Dean J. Insights into the molecular basis of sperm-egg recognition in mammals. Reproduction 2004;127: Irvine DS, Macleod IC, Templeton AA, Masterton A, Taylor A. A prospective clinical study of the relationship between the computer-assisted assessment of human semen quality and the achievement of pregnancy in vivo. Hum Reprod 1994;9:

7 Liu et al. Johnston RC, Mbizvo MT, Summerbell D, Kovacs GT, Baker HWG. Relationship between stimulated hyperactivated motility of human spermatozoa and pregnancy rate in donor insemination: a preliminary report. Hum Reprod 1994;9: Katz DF, Drobnis EZ, Overstreet JW. Factors regulating mammalian sperm migration through the female reproductive tract and oocytes vestments. Gamete Res 1989;22: Kay VJ, Robertson L. Hyperactivated motility of human spermatozoa: a review of physiological function and application in assisted reproduction. Hum Reprod Update 1988;4: Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilisation. Fertil Steril 1988;49: Kulin S, Bastiaans BA, Hollanders HM, Janssen HJG, Goverde HJM. Human serum and follicular fluid stimulate hyperactivation of human spermatozoa after preincubation. Fertil Steril 1994;62: Larsen L, Scheike T, Jensen TK, Bonde JP, Ernst E, Hjollund NH. Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. Hum Reprod 2000;15: Liu DY, Clarke GN, Baker HWG. Relationship between sperm motility assessed with the Hamilton-Thorn Motility Analyzer and fertilization rates in vitro. J Androl 1991;12: Liu DY, Baker HWG. Tests of human sperm function and fertilisation in vitro. Fertil Steril 1992;58: Liu DY, Baker HWG. Disordered acrosome reaction of sperm bound to the ZP (ZP): a newly discovered sperm defect causing infertility with reduced sperm ZP penetration and reduced fertilisation in vitro. Hum Reprod 1994;9: Liu DY, Baker HWG. 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 1996a;11: Liu DY, Baker HWG. Relationship between the zona pellucida (ZP) and ionophore A23187 induced acrosome reaction and the ability of sperm to penetrate the ZP in men with normal sperm ZP binding. Fertil Steril 1996b;66: Liu DY, Clarke GN, Martic M, Garrett C, Baker HWG. Frequency of disordered zona pellucida (ZP)-induced acrosome reaction in infertile men with normal semen analysis and normal sperm ZP binding. Hum Reprod 2001;16: Liu DY, Baker HWG. Frequency of defective sperm ZP interaction in severely teratospermic infertile men. Hum Reprod 2003;18: Liu DY, Baker HWG. High frequency of defective sperm ZP interaction in oligozoospermic infertile men. Hum Reprod 2004;19: Liu DY, Garrett C, Baker HWG. Clinical application of sperm-oocyte interaction tests in in vitro fertilization-embryo transfer and intracytoplasmic sperm injection programs. Fertil Steril 2004;82: Martic M, Moses E, Adams T, Liu DY, Gook D, Garrett C, Dunlop ME, Baker HWG. Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line. Asian J Androl 2004;6:3 13. Mbizvo MT, Burkman LJ, Alexander NJ. Human follicular fluid stimulates hyperactivated motility in human sperm. Fertil Steril 1991;54: Mortimer ST, Anne M, Mortimer D. Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa. Hum Reprod 1998;13: Munire M, Shimizu Y, Sakata Y, Minaguchi R, Aso T. Impaired hyperactivation of human sperm in patients with infertility. J Med Dent Sci 2004;51: Pacey AA, Davies N, Ladbrook MB, Barratt CLR, Cook ID. The potential shortcomings of measuring hyperactivated motility by computer-aided sperm analysis when sperm motion is multiphasic. Hum Reprod Update 1997;3: Rankin TL, Coleman JS, Epifano O, Hoodbhoy T, Turner SG, Castle PE, Lee E, Gore-Langton R, Dean J. Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptor with human homologues. Dev Cell 2003;5: Robertson L, Wolf DP, Tash JS. Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm. Biol Reprod 1988;39: Shalgi R, Phillips DM. Motility of rat spermatozoa at the site of fertilization. Biol Reprod 1988;39: Shibahara H, Obara H, Avustawati, Hirano Y, Suzuki T, Ohno A, Takamizawa S, Suzuki M. Prediction of pregnancy by intrauterine insemination using CASA estimated and strict criteria in patients with male factor infertility. Int J Androl 2004;27: Stauss CR, Votta TJ, Suarez SS. Sperm motility hyperactivation facilitates penetration of the hamste zona pellucida. Biol Reprod 1995;53: Suarez SS, Katz DF, Overstreet JW. Movement characteristics and acrosome status of rabbit spermatozoa recovered at the site and time of fertilization. Biol Reprod 1983;29: Suarez SS, Katz DF, Meizel S. Changes in motility that accompany the acrosome reaction in hyperactivted hamster sperm. Gamete Res 1984;10: Suarez SS, Osman RA. Intiation of hyperactivated flagellar bending in mouse sperm within the female reproductive tract. Biol Reprod 1987;36: Suarez SS, Pacey AA. Sperm transportation in the female reproductive tract. Hum Reprod Update 2006;12: van Duin M, Polman JEM, de Breet ITM, Bunschoten H, van Ginneken K, Grootenhuis A, Brindle J, Aitken RJ. Recombinant human ZP protein ZP3 produced in Chinese hamster ovary cell induces the human sperm acrosome reaction and promotes sperm-egg fusion. Biol Reprod 1994;50: Wang C, Lee GS, Leung A, Surrey ES, Chan SY. Human sperm hyperactivtion and acrosome reaction and their relationship to human in vitro fertilization. Fertil Steril 1993;59: Whitmarsh AJ, Woolnough MJ, Moore HDM, Hornby DP, Barratt CLR. Biological activity of recombinant human ZP3 produced in vitro: potential for a sperm function test. Mol Hum Reprod 1996;2: World Health Organisation. WHO Laboratory Manual for Examination of human Semen and Semen-Cervical Mucus Interaction. Cambridge: Cambridge University Press, 1999, Yanagimachi R. In vitro acrosome reaction and capacitation of golden hamster spermatozoa by bovine follicular fluid and its fractions. J Exp Zool 1969;170: Yanagimachi R. The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil 1970;23: Yanagimachi R. Mammalian fertilisation. In: Knobil E (ed). The Physiology of Reproduction. 2nd edn. New York: Raven Press,1994, Yao Y, Ho P, Yeung WS. Effects of human follicular fluid on the capacitation and motility of human spermatozoa. Fertil Steril 2000;73: Zhu JJ, Barratt CLR, Cook ID. Effect of human cervical mucus on human sperm motion and hyperactivation in vitro. Hum Reprod 1992;7: Submitted on March 20, 2007; resubmitted on June 1, 2007; accepted on June 28,