University of Melbourne Department of Obstetrics and Gynaecology, Reproductive Services, Royal Womens Hospital and Melbourne IVF, Victoria 3035, Australia
1 To whom correspondence should be addressed at: e-mail: dyl{at}unimelb.edu.au
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: male infertility/spermZP binding/teratozoospermia/ZP-induced acrosome reaction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spermoocyte interaction, including spermZP binding, the ZP-induced acrosome reaction (AR), spermZP penetration and spermoolemma fusion are critical sequential steps for human fertilization (Yanagimachi, 1994; Wassarman, 1999
). Although both sperm and oocyte abnormalities can cause interruption of these stepsleading to failure of fertilizationin clinical IVF, sperm defects are the most frequent cause of defective spermZP interaction, where most (or all) of the oocytes from a patient fail to fertilize (Liu and Baker, 2000
).
Previous studies have shown that the human ZP selectively binds sperm with normal morphology; in particular, the shape and relative size of the acrosome region are important (Liu and Baker, 1992a; Garrett et al., 1997
). Morphologically abnormal sperm such as small round-headed sperm lacking the acrosome are not capable of binding to the ZP (von Bernhardi et al., 1990
; Bourne et al., 1995
), though this is a rare cause of male sterility. Most commonly, teratozoospermic semen contains a mixture of various types of abnormal morphology (Liu and Baker, 1992b
). Sperm with normal-shaped heads but with small or abnormally shaped acrosome regions are less capable of binding to the ZP (Liu and Baker, 1992a
,b; Ombelet et al., 1995
; Menkveld et al., 1996
; Garrett et al., 1997
). Therefore, the acrosome region is considered to be one of the most important features for the assessment of sperm morphology (Jeulin et al., 1986
; Liu and Baker, 1992a
,b; Ombelet et al., 1995
; Menkveld et al., 1996
).
In humans, sperm require a normal intact acrosome for binding to the ZP, and the physiological AR occurs on the surface of the ZP after tight binding to the ZP (Cross et al., 1988; Tesarik, 1989
; Yanagimachi, 1994
; Liu and Baker, 1996a
,b; Wassarman, 1999
). The human ZP is a very efficient inducer of the AR, and the ZP-induced AR is highly correlated with spermZP penetration in vitro (Cross et al., 1988
; Coddington et al., 1990
; Liu and Baker, 1994
, 1996a,b). Although disordered ZP-induced AR (DZPIAR) was discovered in patients with unexplained failure of fertilization with standard IVF (Liu and Baker, 1994
), more recently it has been found in prospective studies that 2530% of men with unexplained infertility, normal semen analysis and normal spermZP binding have low ZPIAR (Liu et al., 2001
; Liu and Baker, 2003
). In the present study, the frequency of defective spermZP binding and the ZP-induced AR in infertile men with isolated teratozoospermia was investigated.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human oocytes
Oocytes which showed no evidence of two pronuclei or cleavage at 4860 h after insemination in a clinical IVF programme were used for the ZP-induced AR tests. If the oocyte 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) that was slightly smaller than the oocyte diameter (Liu and Baker, 1994, 1996a). Most of the oocytes were obtained from patients with partial failure of fertilization in standard IVF, and more than 50% of these unfertilized oocytes had one or a few sperm penetrating the ZP from the IVF insemination. It has been shown previously that oocytes with less than 10 sperm penetrating the ZP have a similar ability for subsequent spermZP binding and ZP-induced AR as do those with no sperm penetration (Liu and Baker, 1996a
). Oocytes with more than 10 sperm penetrating the ZP were not used. Likewise, degenerate, activated or morphologically abnormal oocytes were not used for the spermZP interaction test. Oocytes were pooled from several patients and either used for the test on the same day or kept in the incubator and used within next 23 days.
All patients signed consent forms permitting use of their unfertilized oocytes or sperm samples for research. The study was approved by The Royal Womens Hospital Research and Ethics Committees.
Semen analysis
Semen samples were obtained by masturbation after 25 days abstinence. All sperm tests were performed after liquefaction of the semen within 1 h. Sperm concentration and motility in semen were determined using standard methods (World Health Organization, 1992).
Morphology of sperm in both semen and insemination medium (swim-up motile sperm) were assessed on smears prepared by washing of sperm with 10 ml 0.9% sodium chloride (World Health Organization, 1992). Morphology slides were stained with the Shorr method after the smears were fixed in 90% ethanol for 30 min (Jeulin et al., 1986; Liu and Baker, 1992b
). Washing sperm to remove seminal plasma or protein decreases background staining and produces clearer images of sperm. The percentage of normal sperm morphology was assessed according to strict (Tygerberg) 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 x1000 under bright-field illumination.
Sperm preparation
Motile sperm were selected by a swim-up technique as follows: 0.3 ml of semen was carefully added to the bottom of a test tube (12x75 mm) containing 0.7 ml human tubal fluid (HTF; Irvine Scientific, Irvine, CA, USA) supplemented with 10% heat-inactivated human serum (ICN Biomedicals, Irvine, CA, USA). Care was taken to avoid disturbing the interface between the 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 with serum supplemented HTF to a sperm concentration of 2x106/ml for the spermZP interaction test.
SpermZP binding
For the spermZP interaction tests, motile sperm (2x106) in 1 ml of medium were incubated with four oocytes for 2 h at 37°C in 5% CO2 in air. After incubation, the oocytes were transferred to phosphate-buffered saline (PBS), pH 7.4, containing 2 mg/ml bovine serum albumin (BSA) and washed by repeated aspiration with a glass pipette (inside diameter 250 µm) to dislodge sperm that were loosely adherent to the surface of the ZP. Sperm binding to all four oocytes was assessed. Under these experimental conditions, with the high concentration of sperm in the insemination medium (20-fold more than standard IVF insemination), the number of sperm bound tightly to the ZP was more than 100 per ZP with sperm from fertile or normospermic men. For this study, sperm samples with an average of no more than 40 sperm per ZP were considered to have poor spermZP binding, and no further testing was carried out (Liu and Baker, 2000
).
ZP-induced AR
For sperm samples without poor ZP-binding, 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) which was slightly smaller than the oocyte (Liu and Baker, 1994
, 1996a). This was carried out on a glass slide with
5 µl PBS containing 0.2% BSA, and the removed ZP-bound sperm were smeared over a limited area (
16 mm2), which was marked with a glass-pen on the back of the slides to help locate the sperm under the microscope. This pipetting procedure for removing sperm from the surface of ZP does not affect sperm motility, morphology and acrosome status (Liu and Baker, 1996a
).
The acrosome status of sperm removed from the ZP was determined with fluorescein-labelled Pisum sativum agglutinin (PSA; Sigma Co., St Louis, MO, USA), using a modification of a previously published method (Cross et al., 1986). Sperm smears were fixed in 95% ethanol for 30 min after drying in air and then stained in 25 µg/ml PSA in PBS for at least 2 h at 4°C. The slide was washed and mounted with distilled water and the percentage of AR determined by scoring 200 of the sperm removed from all four ZPs per test, using a fluorescence microscope with oil immersion at x400 magnification. When more than half a sperm head fluoresced brightly and uniformly, the acrosome was considered to be intact. Sperm with a fluorescing band at the equatorial segment or no staining of the head (a rare pattern) were considered to be acrosome-reacted. As only motile sperm are capable of binding to the ZP, sperm removed from the ZP were alive and motile at the time of ZP-binding, and thus no test of sperm viability was carried out.
Statistical analysis
The significance of correlations between spermZP binding and the ZP-induced AR and other sperm characteristics were examined using Spearman tests. Multiple regression analysis was also used to determine which of sperm characteristics was the most significantly related to spermZP binding or the ZP-induced AR. Sperm concentration was analysed after log transformation. Differences of the ZP-induced AR in men with low and high sperm concentrations were examined using non-parametric (Wilcoxon rank sum) tests.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Frequency of defective spermZP binding and low ZPIAR with teratozoospermia
In the present study, a threshold average number of sperm bound per ZP of 40 was used as a threshold for poor spermZP binding. A ZP-induced AR of
16% was used for classification of low ZPIAR according to previous reports (Liu and Baker, 1994
; Liu et al., 2001
). Among infertile men with isolated teratozoospermia, 31% (39/125) had poor spermZP binding; among the other 86 men, 48% (41/86) had low ZPIAR. Overall, only 36% (45/125) had normal ZP-binding and normal ZP-induced AR. In other words, 64% had either poor spermZP binding or low ZPIAR.
As shown above, the sperm count was significantly correlated with the ZP-induced AR, and men with sperm counts between 2060x106/ml had significantly lower mean (± SD) ZP-induced AR than those with sperm counts >60x106/ml (17 ± 15 versus 29 ± 22; Wilcoxon test, z = 3.218, P < 0.001). The frequency of low ZPIAR was 64% in men who had both serve teratozoospermia (normal morphology 5%) and low normal sperm counts (2060x106 /ml). In contrast, the frequency of low ZPIAR was 31% (13/42) in men with sperm counts >60x106/ml.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, only 31% of the severely teratozoospermic men had poor spermZP binding. This low proportion might be due to the high sperm concentration used for insemination (about 20-fold higher than used with standard IVF). Previously, the threshold of no more than 40 sperm bound per ZP with a sperm concentration of 2x106/ml was seen to be equivalent to no more than two sperm bound per ZP with a sperm concentration in the insemination medium of 1x105/ml (as used with oocytes in standard IVF; Liu and Baker, 2000). Others have also shown that increasing the number of sperm for insemination in teratozoospermic men will increase the number of sperm bound to the ZP (Oehninger et al., 1988
).
In the present study, it was found that 48% of teratozoospermic men without severely impaired spermZP binding had ZP-induced AR 16%, which is classified as low ZPIAR. This frequency of low ZPIAR with teratozoospermia is much higher than in men with unexplained infertility with normal semen analysis (
2530%; Liu et al., 2001
; Liu and Baker, 2003
). When the sperm count in semen was taken into account, over 64% of severe teratozoospermic men had low ZPIAR if they also had sperm counts between 20 and
60x106/ml (men with sperm counts <20x106/ml were excluded from this study). In contrast, with teratozoospermia and a sperm count >60x106/ml, the frequency of low ZPIAR was 31%, and this was similar to the value found in normozoospermic infertile men (Liu et al., 2001
; Liu and Baker, 2003
). Therefore, sperm count could be an important additional indicator for predicting which infertile men with isolated teratozoospermia are more likely to have impaired spermZP interaction. This finding may be very useful clinically as many IVF/ICSI clinics do not perform spermZP interaction tests routinely. The ZPIAR is highly correlated with spermZP penetration and fertilization rates in vitro in normozoospermic infertile men (Liu and Baker, 1996b
; Esterhuizen et al., 2001
; Liu and Baker, 2003
). The results of a previous study showed that normozoospermic infertile men with <10% ZPIAR had zero spermZP penetration, while in those men with <16% ZPIAR, fewer than 20% of the ZP were penetrated in vitro (Liu and Baker, 1996b
). Therefore, patients with both normal sperm morphology of
5% plus sperm count <60x106/ml should be recommended for ICSI because of the high frequency of low ZPIAR and the high risk of poor fertilization rates with standard IVF.
The finding of a significant correlation between ZP-induced AR and sperm concentration was consistent with previous studies in men with normal semen analysis and unexplained infertility (Liu et al., 2001; Liu and Baker, 2003
). This may suggest that men with sperm concentrations within the lower region of the conventional normal range (
60x106/ml) may have a degree of impaired spermatogenesis causing sperm dysfunction, particularly in the ability to interact with oocytes. However, further study is needed to determine the underlying cause of the high frequency of low ZPIAR with sperm counts in the range of 2060x106/ml. This may also indicate that a standard lower reference limit of 20x106/ml is too low.
Calcium ionophore A23187-induced AR may predict sperm fertilizing ability in vitro (Cummins et al., 1991; Yovich et al., 1994
), and a significant correlation was found between A23187-induced AR and IVF rate in patients with teratozoospermia (Liu and Baker, 1998
). However, in normospermic men, A23187-induced AR was not correlated with either the ZP-induced AR or spermZP penetration (Liu and Baker, 1996b
). Therefore, A23187-induced AR may not reflect the ability of sperm to undergo the physiological AR as induced by the ZP, and tests using A23187-induced AR will not predict ZPIAR. It has been reported (Calvo et al., 1989
) that follicular fluid induced-AR distinguished a subgroup of men with unexplained infertility who were not detected by routine semen analysis. Although others showed that progesterone-induced AR is predictive of fertility in men with unexplained infertility, it is not known whether progesterone-induced AR test results are related to ZPIAR (Krausz et al., 1996
).
In clinical IVF, about 2030% of inseminated oocytes fail to fertilize, and these are valuable for testing sperm function for other clinical patients before they commence ART treatment. Most oocytes that fail to fertilize in vitro can be used for testing ZP-induced AR; immature oocytes (germinal vesicle oocytes) may also be used in this test. However, degenerate, spontaneously activated and morphologically abnormal oocytes and those with more than 10 sperm penetrated into the ZP are not used. It is important to use a group of oocytes (optimally four) rather than a single oocyte for each test, due to variability in the quality of individual oocytes. Differences between ZP-induced AR from different batches of four oocytes or two sperm samples in the same men are <15% (Liu and Baker, 1996a; Liu et al., 2001
). However, in practical terms, routine testing for ZPIAR is difficult and is usually limited to a small proportion of patients because insufficient human oocytes are available. In future, the development of recombinant human ZP or other alternative tests that do not require native human ZP will have great potential for routine testing for the ZP-induced AR (van Duin et al., 1994
; Brewis et al., 1996
; Whitmarsh et al., 1996
; Dong et al., 2001
).
The results of the present study provide a more detailed explanation of why teratozoospermia is associated with infertility and poor results with standard IVF. The spermZP interaction tests using unfertilized oocytes from IVF may be useful for distinguishing severe and less severely infertile men with isolated teratozoospermia. One-third of severe teratozoospermic men may still have a chance to achieve fertilization either in vivo or in vitro with standard IVF, but two-thirds should be treated by ICSI from the outset.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baker, H.W.G. (2001) Male infertility. In DeGroot, L.J. and Jameson J. L. (eds), Endocrinology, 4th edn. W.B.Saunders Co., Philadelphia, PA, pp. 23082328.
Baker, H.W.G., Liu, D.Y., Bourne, H. and Lopata, A. (1993) Diagnosis of sperm defects in selecting patients for assisted fertilization. Hum. Reprod., 8, 17791780.[ISI][Medline]
Bourne, H., Liu, D.Y., Clarke, G.N. and Baker, H.W.G. (1995) Normal fertilization and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm. Fertil. Steril., 63, 13291332.[ISI][Medline]
Brewis, I.A., Clayton, R., Barratt, C.L.R., Hornby, D.P. and Moore, H.D.M. (1996) Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. Mol. Hum. Reprod., 2, 583589.[Abstract]
Calvo, L., Vantman, D., Banks, S.M., Tezon, J., Koukoulis, G.N., Dennison, L. and Sherins, R. (1989) Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil. Steril., 52, 10481054.[ISI][Medline]
Coddington, C.C., Fulgham, D.L., Alexander, N.J., Johnson, D., Herr, J.C. and Hodgen, G.D. (1990) Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody. Fertil. Steril., 54, 504508.[ISI][Medline]
Cohen, J., Edwards, R., Fehilly, C., Fishel, S., Hewitt, J., Purdy, J., Rowland, G., Steptoe, P. and Webster, J. (1985) In vitro fertilization: a treatment for male infertility. Fertil. Steril., 43, 422432.[ISI][Medline]
Cross, N.L., Morales, P., Overstreet, J.W. and Hanson, F.W. (1986) Two simple methods for detecting acrosome-reacted human sperm. Gamete Res., 15, 213226.[ISI]
Cross, N.L., Morales, P., Overstreet, J.W. and Hanson, F.W. (1988) Induction of acrosome reaction by the human ZP. Biol. Reprod., 38, 235244.[Abstract]
Cummins, J.M., Pember, S.M., Jequier, A.M., Yovich, J.L. and Hartmann, P.E. (1991) A test of the human sperm acrosome reaction following ionophore challenge (ARIC). Relationship to fertility and other seminal parameters. J. Androl., 12, 98103.
Dong, K.W., Chi, T.F., Juan, Y.W., Chen, C.W., Lin, Z., Xiang, X.Q., Mahony, M., Gibbons, W.E. and Oehninger, S. (2001) 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., 184, 835843.[CrossRef][ISI][Medline]
Esterhuizen, A.D., Franken, D.R., Lourens, J.D.H. and van Rooyen, L.H. (2001) Clinical importance of zona pellucida induced acrosome reaction (ZIAR): predictive value of the ZIAR test for in vitro fertilization. Hum. Reprod., 16, 138144.
Garrett, C., Liu, D.Y. and Baker, H.W.G. (1997) Selectivity of the human sperm-zona pellucida binding process to sperm head morphometry. Fertil. Steril., 67, 362371.[CrossRef][ISI][Medline]
Jeulin, C., Feneux, D., Serres, C., Jouannet, P., Guillet-Rosso, F., Belaosch-Allart, J., Frydman, R. and Testart, J. (1986) Sperm factors related to failure of human in vitro fertilisation. J. Reprod. Fertil., 76, 735744.[Abstract]
Krausz, C., Bonaccorsi, L., Maggio, P., Luconi, M., Criscuoli, L., Fuzzi, B., Pellegrini, S., Forti, G. and Baldi, E. (1996) Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization. Hum. Reprod., 11, 16611667.[Abstract]
Kruger, T.F., Acosta, A.A., Simmons, K.F., Swanson, R.J., Matta, J.F. and Oehninger, S. (1988) Predictive value of abnormal sperm morphology in in vitro fertilisation. Fertil. Steril., 49, 112117.[ISI][Medline]
Liu, D.Y. and Baker, H.W.G. (1992a) Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro. J. Fertil. Reprod., 94, 7184.
Liu, D.Y. and Baker, H.W.G. (1992b) Tests of human sperm function and fertilisation in vitro. Fertil. Steril., 58, 465483.[ISI][Medline]
Liu, D.Y. and Baker, H.W.G. (1994) 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., 9, 16941700.[Abstract]
Liu, D.Y. and Baker, H.W.G. (1996a) A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona pellucida: lack of relationship with ionophore A23187-induced acrosome reaction. Hum. Reprod., 11, 551557.[Abstract]
Liu, D.Y. and Baker, H.W.G. (1996b) 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., 66, 312315.[ISI][Medline]
Liu, D.Y. and Baker, H.W.G. (1998) Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen. Hum. Reprod., 13, 905910.[Abstract]
Liu, D.Y. and Baker, H.W.G. (2000) Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinic in vitro fertilization. Hum. Reprod., 15, 702708.
Liu, D.Y. and Baker, H.W.G. (2003) Disordered zona pellucida induced acrosome reaction and failure of in vitro fertilization in patients with unexplained infertility. Fertil. Steril., 79, 7480.[CrossRef][ISI][Medline]
Liu, D.Y., Clarke, G.N., Martic, M., Garrett, C. and Baker, H.W.G. (2001) Frequency of disordered zona pellucida (ZP)-induced acrosome reaction in infertile men with normal semen analysis and normal spermatozoa-ZP binding. Hum. Reprod., 16, 11851190.
Menkveld, R., Rhemrev, J.P., Franken, D.R., Vermeiden, J.P.W. and Kruger, T.F. (1996) Acrosomal morphology as a novel criterion for male fertility diagnosis: relation with acrosin activity, morphology (strict criteria), and fertilization in vitro. Fertil. Steril., 65, 637644.[ISI][Medline]
Oehninger S., Acosta, A.A., Morshedi, M., Veeck, L., Swanson, R.J., Simmons, K. and Rosenwaks, Z. (1988) Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities. Fertil. Steril., 50, 283287.[ISI][Medline]
Ombelet, W., Menkveld, R., Kruger, K.T. and Steeno, O. (1995) Sperm morphology assessment: historical review in relation to fertility. Hum. Reprod. Update, 1, 543557.[Abstract]
Tesarik, J. (1989) Appropriate time of the acrosome reaction is a major requirement for the fertilising spermatozoa. Hum. Reprod., 4, 957961.[Abstract]
van Duin, M., Polman, J.E.M., de Breet, I.T.M., Bunschoten, H., van Ginneken, K., Grootenhuis, A., Brindle, J. and Aitken, R.J. (1994) Recombinant human ZP protein ZP3 produced in Chinese hamster ovary cell induces the human sperm acrosome reaction and promotes sperm-egg fusion. Biol. Reprod., 50, 607617.[Abstract]
von Bernhardi, R., de Ioannes, A.E., Blanco, L.P., Herrera, E., Bustos-Obregon, E. and Vigil, P. (1990) Round-headed spermatozoa: a model to study the role of the acrosome in early events of gamete interaction. Andrologia, 22, 1220.[ISI][Medline]
Wassarman, P.M. (1999) Mammalian fertilization molecular aspects of gametes adhesion, exocytosis, and fusion. Cell, 96, 175183.[ISI][Medline]
Whitmarsh, A.J., Woolnough, M.J., Moore, H.D.M., Hornby, D.P. and Barratt, C.L.R. (1996) Biological activity of recombinant human ZP3 produced in vitro: potential for a sperm function test. Mol. Hum. Reprod., 2, 911919.[Abstract]
World Health Organization (1992) WHO Laboratory Manual for Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge University Press, Cambridge, pp. 120.
Yanagimachi, R. (1994) Mammalian fertilisation. In Knobil, E. and Neill, J. (eds), The Physiology of Reproduction, 2nd edn. Raven Press, New York, pp. 189317.
Yovich, J.M., Edirsinghe, W.R. and Yovich, J.L. (1994) Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study. Fertil. Steril., 61, 902910.[ISI][Medline]
Submitted on November 5, 2002; accepted on December 18, 2002.