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Evaluation on Sperm Acrosome Integrity of Infertile Men with Varicocele
Journal of Reproduction & Contraception
http://www.RandC.cn
2007 Mar; 18(1):19-24
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Evaluation on Sperm Acrosome Integrity of Infertile Men with Varicocele P. Tzvetkova1, Wei-jie ZHU2, Jing LI3, D. Tzvetkov4 1. Institute of Experimental Morphology and Anthropology with Museum, Bulgarian Academy of Science, Sofia, Bulgaria 2. Center for Reproductive Immunology Research, Jinan University, Guangzhou 510632, China 3. Medical College, Jinan University, Guangzhou 510632, China 4. Department of Urology, Medical University, Sofia, Bulgaria
Objective To determine the sperm acrosome integrity of samples from infertile men with varicocele. Methods Forty-nine infertile men with varicocele were divided into three groups according to the grade of varicocele. Group A (grade I), B (grade II), and C (grade III) consisted of 15, 18, and 16 cases, respectively. Besides, 15 semen samples from normospermic donors were used as the control. The acrosome integrity of sperm was examined with fluorescein-labeled Pisum sativum agglutinin. Acrosomal ultrastructure was observed with transmission electron microscopy. Results In three varicocele groups, most samples had high sperm abnormal morphology rates. There were significant differences in acrosome integrity rates between each varicocele group and the control (P<0.01). Group C had the lowest acrosome integrity rate among the three groups. Ultrastructural observation showed that acrosome malformations revealed acrosomal membranes defects, swelling, hypoplasia, and dissolution of the matrix. Conclusions Infertile men with varicocele had low level of acrosome integrity. Severe varicocele for infertile men might be associated with severe acrosomal defects. Evaluating sperm acrosome should aid the understanding of the sperm structural state and benefit the treatment for infertile men. Key words: varicocele; acrosome; sperm; infertility Corresponding authors: P. Tzvetkova; Tel: +359-2439000; E-mail:[email protected] Wei-jie ZHU; Tel: +86-20-85225718; E-mail:[email protected] 19
Male factor infertility has an impact on approximately 50% of infertile couples. A number of aetiologies have been identified, in which varicocele has been widely considered as a common cause of male infertility. At present, varicoceles are reported in 35%-40% of men with primary infertility, and in up to 80% of men with secondary infertility[1-5]. A varicocele is a pathologic phenomenon caused by vascular lesions of the pampiniform plexus[4]. The role of varicocele in male infertility is associated with an impairment of testicular and epididymal functions[3-6]. Many studies indicate that infertile men with varicocele have poor semen quality, including decreased sperm motility, count, and normal morphology[3-8]. However, the mechanism of the fertility disturbance induced by varicocele, especially on aspects of sperm structure and function, still needs to be investigated. In the present study, the sperm acrosome integrity rates of samples from infertile men with varicocele were examined. In addition, acrosomal ultrastructure was observed with transmission electron microscopy.
Materials & Methods Patients Forty-nine men with varicocele who visited the infertility clinic were included in the present study. The patients age was 32.3 ± 5.6 years, and infertility duration was 5.4 ± 3.2 years. Varicoceles were graded according to the criteria described by the World Health Organization (WHO) manual[9]: graded I, the varicocele could only be palpated after the Valsalva maneuver in the upright position; graded II, the varicocele was easily palpable but not visible; and graded III, the varicocele was easily visible. Based on their grades, 49 patients were divided into three groups: group A ( grade I, n=15 ), group B ( grade II, n=18 ), and group C ( grade III, n=16 ). Besides, 15 semen samples from normospermic donors were used as the control. Semen samples Semen samples were obtained by masturbation after 3-7 d of sexual abstinence. Semen routine examination was performed according to the WHO laboratory manual[10]. Sperm morphology was assessed after slide staining with the modified Papanicolaoustaining method and using strict criteria. Special attention was given to the acrosome morphology. Sperm acrosome integrity test The acrosome integrity of sperm was examined with fluorescein-labeled Pisum sativum agglutinin (PSA; Sigma Chemical Company, USA) as described[11, 12]. Briefly, sperm smears were fixed in 95% ethanol for 30 min after air drying and then stained in 25 µg/ml PSA for 2 h. The slide was washed and mounted with distilled water. The acrosome status was determined with a fluorescence microscope and oil immersion at a magnification of × 400. 20
Transmission electron microscopy Sperm from group C (n=5) were prepared for observation of transmission electron microscopy (TEM) using a routine method as previously described[13]. Ultrastructural changes of sperm were observed with transmission electron microscope (PHILIPS TELNAI-10). Statistical analysis The results were expressed as mean ± standard deviation. Difference between two groups was evaluated by t-test. A difference with P<0.05 was considered significant.
Results Of the 49 patients studied, 12 in group B and 15 in group C had bilateral varicoceles, and all cases in group A had unilateral varicoceles. Sperm parameters for three varicocele groups and the control are shown Table 1. Sperm motility, count, and normal morphology in three varicocele groups were significantly lower compared with the control (P<0.01). Among the three groups, the lowest percentage of morphologically normal sperm was group C. Samples in group C had high abnormal morphology rates, ranged from 92% to 100%. Under the fluorescence microscope, sperm acrosome was clearly demonstrated. Sperm with normal acrosomes showed that more than half a sperm head were brightly and uniformly fluorescing (Figure 1). Abnormal acrosomes showed many defective types (Figure 1), including irregular acrosome cap, irregular distribution of fluorescence, small acrosome, large acrosome, vacuoles in the acrosomeal region. The acrosome integrity rates for all groups are shown in Table 1. There were significant differences in acrosome integrity rates between each varicocele group and the control (P<0.01). Group C had the lowest acrosome integrity rate among the three groups. Under TEM, a few acrosomes had normal features, which was full acrosomal coverage of the anterior part of the nucleus. However, sperm heads with abnormal shapes were frequently seen. Vacuolization or condensed granules of chromatin aggregation was frequently observed in the heads, which often had poorly defined indistinct, unclear, or fuzzy membranes (Figures 2, 3). The acrosome malformations revealed acrosomal hypoplasia, rarefaction, swelling, and dissolution of the matrix. Table 1 Sperm parameters from infertile men with varicocele (x- ±s) Group A B
Motility(%) 61.0±9.2 52.5±11.2*
C Control
36.8±7.4* 68.5±5.3
Count (×106/ml) 80.5±14.9* 69.4±19.4* 39.1±12.7* 147.2±33.5
Normal morphology (%) 12.2±5.3* 9.7±4.4* 3.8±2.8* 60.4±8.6
Intact acrosome (%) 52.5±6.2* 38.6±10.2* 23.3±6.9* 86.4±4.5
*:P<0.01, compared with the control 21
Figure 1
Sperm with normal acrosome (△) and abnormal acrosome (
) (PSA stain, original
magnification×1 000)
Figure 2 Sperm with swollen acrosome (TEM, original magnification, ×20 000)
Figure 3 Sperm with poorly defined indistinct and fuzzy membranes (TEM, original magnification, ×20 000)
Discussion The acrosome of human sperm is a relatively inconspicuous, cap-like structure that consists of outer acrosome membrane, matrix, and inner acrosome membrane, and covers the anterior three quarters of the nucleus[14, 15]. To achieve successful fertilization in natural condition or in vitro, the acrosome must be induced to undergo the acrosome reaction. Many studies have suggested that human sperm bind to the zona pellucida with the acrosome intact and the acrosome reaction occurs on the zona pellucida. Furthermore, only acrosome-reacted sperm can penetrate into the zona pellucida and bind to and fuse with the oolemma[11, 16-18]. Therefore, the acrosome normality is the basis for the normal physiological fertilization. In the present study, abnormalities of the acrosome were a common finding in sperm samples from varicocele patients. All three varicocele groups had high abnormal rates. Because of 22
the important role of the acrosome in the fertilization, defective acrosomes will influence the acrosome reaction, which may lead to reduced fertility. It indicated that acrosome defects might contribute the infertile state associated with varicocele in some such patients. The acrosome develops from the Golgi complex during early stages of spermatid differentiation. Most acrosomal defects are associated with the abnormal function of the Golgi complex, which causes developmental disturbances and errors of the acrosome[14, 15]. From our observation, a few samples, especially from group C, unintact acrosomes were in a great proportion. These results indicated that severe varicocele for infertile men might be associated with severe acrosomal defects. According to the pathogenetic mechanism of the acrosome, acrosome malformations seem to be difficult to treat efficiently. No improvement in the acrosome reaction was noted post-varicocelectomy[4, 19]. With the development of assisted reproductive techniques(ART), intracytoplasmic sperm injection (ICSI) has provided substantial fertilization rates in cases of dramatically diminished sperm functions[20]. By using ICSI, all barriers to sperm penetration of the oocyte are bypassed, and the intact acrosome and acrosome reaction are not necessary since the sperm is injected directly into the ooplasm. Therefore, it suggested that varicocele men with poor acrosomes should be considered to select ICSI to treat their infertility in order to improve the fertilization efficacy. In the present study, varicocele patients demonstrated low sperm motilities, low sperm counts, and low normal sperm morphology rates, which were in agreement with previous reports[2-8, 21, 22]. Obviously, affected sperm parameters, especially high abnormal sperm morphology, were related to reduced fertility[17, 23, 24]. On the other hand, we noticed that some sperm with “normal” shaped head had abnormal acrosmes. Besides, acrosome malformations did not show special types by using both light and electron microscopy in this study. Because acrosome defects are associated with spermatogenesis, the reasons or mechanisms need further investigation. In conclusion, our study revealed that infertile men with varicocele had abnormal acrosome integrity, and some acrosome defects could not be showed by routine light microscopy. Because the acrosome can not be clearly visualized in live and unstained human sperm due to its relatively thin and translucency, special techniques such as PSA method should be used to determine its morphology or integrity. Combined with routine semen analysis, evaluating sperm acrosome should aid the understanding of the sperm structural state and also provide information relevant to the treatment of infertile men with varicocele.
References 1. Pasqualotto FF, Lucon AM, Hallak J, et al. Induction of spermatogenesis in azoospermic men after varicocele repair. Hum Reprod, 2003, 18(1):108-12. 2. Benoff S, Gilbert BR. Varicocele and male infertility: Part I, Preface. Hum Reprod Update, 2001, 7(1):47-51. 23
3. Jarow JP. Effects of varicocele on male infertility. Hum Reprod Update, 2001, 7(1):59-64. 4. Naughton CK, Nangia AK, Agarwal A. Varicocele and male infertility: Part II, Pathophusiology of varicoceles in male infertility. Hum Reprod Update, 2001, 7(5):473-81. 5. Hauser R, Paz G, Botchan A, et al. Varicocele and male infertility: Part II, Effect on sperm functions. Hum Reprod Update, 2001, 7(5):482-5. 6. Cockett ATK, Takihara H, Cosentino MJ. The varicocele. Fertil Steril, 1984, 41(1):5-11 7. Ismail MT, Sedor J, Hirsch I. Are sperm motion parameters influenced by varicocele ligation? Fertil Steril, 1999, 71(5):886-90. 8. Liang WB, Zhu WJ, Su ZX. Observation on sperm penetration rate for infertile men with varicocele. J Reprod Contracep, 2002, 13(1):10-5. 9. WHO. Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Cambridge: Cambridge University Press, 1993. 10. WHO. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge: Cambridge University Press, 1999. 11. Liu DY, Baker HWG. Acrosome atatus and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro. Hum Reprod, 1994, 9(4):673-9. 12. Tang FX, Zhu WJ, Li J. Effect of cryopreservation on changes in human sperm acrosome integrity and ultrastructure. Chinese J Birth Health & Heredity (in Chinese), 2004, 12(3):105-7. 13. Zhu WJ, Li J. Evaluation on the morphology and membrane integrity of immotile human sperm. J Reprod Contracep, 2006, 17(2):80-4. 14. Zamboni L. The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril, 1987, 48(5):711-34. 15. Bartoov B, Eltes F, Weissenberg R, et al. Morphological characterization of abnormal human spermatozoa using transmission electron microscopy. Arch Androl, 1980, 5(5):305-22. 16. El-Ghobashy AA, West CR. The human sperm head: a key for successful fertilization. Hum Reprod, 2003, 24(2):232-8. 17. Liu DY, Baker HWG. Tests of human sperm function and fertilization in vitro. Fertil Steril, 1992, 58(3):46583. 18. Garrett C, Liu DY, Baker HWG. Selectivity of the human sperm-zona pellucida binding process to sperm head morphometry. Fertil Steril, 1997, 67(2):362-71. 19. Benoff S, Barcia M, Hurley I, et al. Classification of male factor infertility relevant to in vitro fertilization insemination strategies using mannose ligands, acrosome status and anticytoskeletal antibodies. Hum Reprod, 1996, 11(2):1 905-18. 20. Palermo G, Cohen J, Rosenwaks Z. Intracytoplasmic sperm injection: a powerful tool to overcome fertilization failure. Fertil Steril, 1996, 65(5):899-908. 21. Newton R, Schinfeld J, Schiff I. The effect of varococelectomy on sperm count, motility, and conception rate. Fertil Steril, 1980, 34(3):250-4. 22. Marks JL, McMahon R, Lipshultz L. Predictive parameters of successful varicocele repair. J Urol, 1986, 136 (3):609-12. 23. Donnelly ET, Lewis AEM, McNally JA, et al. In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertil Steril, 1998, 70(2):305-14. 24. Baccetti B, Capitani S, Collodel G, et al. Recent advances in human sperm pathology. Contraception, 2002, 65(4): 283-7. (Received on December 28, 2006)
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http://www.RandC.cn
2007 Mar; 18(1):19-24
[email protected]
Evaluation on Sperm Acrosome Integrity of Infertile Men with Varicocele P. Tzvetkova1, Wei-jie ZHU2, Jing LI3, D. Tzvetkov4 1. Institute of Experimental Morphology and Anthropology with Museum, Bulgarian Academy of Science, Sofia, Bulgaria 2. Center for Reproductive Immunology Research, Jinan University, Guangzhou 510632, China 3. Medical College, Jinan University, Guangzhou 510632, China 4. Department of Urology, Medical University, Sofia, Bulgaria
Objective To determine the sperm acrosome integrity of samples from infertile men with varicocele. Methods Forty-nine infertile men with varicocele were divided into three groups according to the grade of varicocele. Group A (grade I), B (grade II), and C (grade III) consisted of 15, 18, and 16 cases, respectively. Besides, 15 semen samples from normospermic donors were used as the control. The acrosome integrity of sperm was examined with fluorescein-labeled Pisum sativum agglutinin. Acrosomal ultrastructure was observed with transmission electron microscopy. Results In three varicocele groups, most samples had high sperm abnormal morphology rates. There were significant differences in acrosome integrity rates between each varicocele group and the control (P<0.01). Group C had the lowest acrosome integrity rate among the three groups. Ultrastructural observation showed that acrosome malformations revealed acrosomal membranes defects, swelling, hypoplasia, and dissolution of the matrix. Conclusions Infertile men with varicocele had low level of acrosome integrity. Severe varicocele for infertile men might be associated with severe acrosomal defects. Evaluating sperm acrosome should aid the understanding of the sperm structural state and benefit the treatment for infertile men. Key words: varicocele; acrosome; sperm; infertility Corresponding authors: P. Tzvetkova; Tel: +359-2439000; E-mail:[email protected] Wei-jie ZHU; Tel: +86-20-85225718; E-mail:[email protected] 19
Male factor infertility has an impact on approximately 50% of infertile couples. A number of aetiologies have been identified, in which varicocele has been widely considered as a common cause of male infertility. At present, varicoceles are reported in 35%-40% of men with primary infertility, and in up to 80% of men with secondary infertility[1-5]. A varicocele is a pathologic phenomenon caused by vascular lesions of the pampiniform plexus[4]. The role of varicocele in male infertility is associated with an impairment of testicular and epididymal functions[3-6]. Many studies indicate that infertile men with varicocele have poor semen quality, including decreased sperm motility, count, and normal morphology[3-8]. However, the mechanism of the fertility disturbance induced by varicocele, especially on aspects of sperm structure and function, still needs to be investigated. In the present study, the sperm acrosome integrity rates of samples from infertile men with varicocele were examined. In addition, acrosomal ultrastructure was observed with transmission electron microscopy.
Materials & Methods Patients Forty-nine men with varicocele who visited the infertility clinic were included in the present study. The patients age was 32.3 ± 5.6 years, and infertility duration was 5.4 ± 3.2 years. Varicoceles were graded according to the criteria described by the World Health Organization (WHO) manual[9]: graded I, the varicocele could only be palpated after the Valsalva maneuver in the upright position; graded II, the varicocele was easily palpable but not visible; and graded III, the varicocele was easily visible. Based on their grades, 49 patients were divided into three groups: group A ( grade I, n=15 ), group B ( grade II, n=18 ), and group C ( grade III, n=16 ). Besides, 15 semen samples from normospermic donors were used as the control. Semen samples Semen samples were obtained by masturbation after 3-7 d of sexual abstinence. Semen routine examination was performed according to the WHO laboratory manual[10]. Sperm morphology was assessed after slide staining with the modified Papanicolaoustaining method and using strict criteria. Special attention was given to the acrosome morphology. Sperm acrosome integrity test The acrosome integrity of sperm was examined with fluorescein-labeled Pisum sativum agglutinin (PSA; Sigma Chemical Company, USA) as described[11, 12]. Briefly, sperm smears were fixed in 95% ethanol for 30 min after air drying and then stained in 25 µg/ml PSA for 2 h. The slide was washed and mounted with distilled water. The acrosome status was determined with a fluorescence microscope and oil immersion at a magnification of × 400. 20
Transmission electron microscopy Sperm from group C (n=5) were prepared for observation of transmission electron microscopy (TEM) using a routine method as previously described[13]. Ultrastructural changes of sperm were observed with transmission electron microscope (PHILIPS TELNAI-10). Statistical analysis The results were expressed as mean ± standard deviation. Difference between two groups was evaluated by t-test. A difference with P<0.05 was considered significant.
Results Of the 49 patients studied, 12 in group B and 15 in group C had bilateral varicoceles, and all cases in group A had unilateral varicoceles. Sperm parameters for three varicocele groups and the control are shown Table 1. Sperm motility, count, and normal morphology in three varicocele groups were significantly lower compared with the control (P<0.01). Among the three groups, the lowest percentage of morphologically normal sperm was group C. Samples in group C had high abnormal morphology rates, ranged from 92% to 100%. Under the fluorescence microscope, sperm acrosome was clearly demonstrated. Sperm with normal acrosomes showed that more than half a sperm head were brightly and uniformly fluorescing (Figure 1). Abnormal acrosomes showed many defective types (Figure 1), including irregular acrosome cap, irregular distribution of fluorescence, small acrosome, large acrosome, vacuoles in the acrosomeal region. The acrosome integrity rates for all groups are shown in Table 1. There were significant differences in acrosome integrity rates between each varicocele group and the control (P<0.01). Group C had the lowest acrosome integrity rate among the three groups. Under TEM, a few acrosomes had normal features, which was full acrosomal coverage of the anterior part of the nucleus. However, sperm heads with abnormal shapes were frequently seen. Vacuolization or condensed granules of chromatin aggregation was frequently observed in the heads, which often had poorly defined indistinct, unclear, or fuzzy membranes (Figures 2, 3). The acrosome malformations revealed acrosomal hypoplasia, rarefaction, swelling, and dissolution of the matrix. Table 1 Sperm parameters from infertile men with varicocele (x- ±s) Group A B
Motility(%) 61.0±9.2 52.5±11.2*
C Control
36.8±7.4* 68.5±5.3
Count (×106/ml) 80.5±14.9* 69.4±19.4* 39.1±12.7* 147.2±33.5
Normal morphology (%) 12.2±5.3* 9.7±4.4* 3.8±2.8* 60.4±8.6
Intact acrosome (%) 52.5±6.2* 38.6±10.2* 23.3±6.9* 86.4±4.5
*:P<0.01, compared with the control 21
Figure 1
Sperm with normal acrosome (△) and abnormal acrosome (
) (PSA stain, original
magnification×1 000)
Figure 2 Sperm with swollen acrosome (TEM, original magnification, ×20 000)
Figure 3 Sperm with poorly defined indistinct and fuzzy membranes (TEM, original magnification, ×20 000)
Discussion The acrosome of human sperm is a relatively inconspicuous, cap-like structure that consists of outer acrosome membrane, matrix, and inner acrosome membrane, and covers the anterior three quarters of the nucleus[14, 15]. To achieve successful fertilization in natural condition or in vitro, the acrosome must be induced to undergo the acrosome reaction. Many studies have suggested that human sperm bind to the zona pellucida with the acrosome intact and the acrosome reaction occurs on the zona pellucida. Furthermore, only acrosome-reacted sperm can penetrate into the zona pellucida and bind to and fuse with the oolemma[11, 16-18]. Therefore, the acrosome normality is the basis for the normal physiological fertilization. In the present study, abnormalities of the acrosome were a common finding in sperm samples from varicocele patients. All three varicocele groups had high abnormal rates. Because of 22
the important role of the acrosome in the fertilization, defective acrosomes will influence the acrosome reaction, which may lead to reduced fertility. It indicated that acrosome defects might contribute the infertile state associated with varicocele in some such patients. The acrosome develops from the Golgi complex during early stages of spermatid differentiation. Most acrosomal defects are associated with the abnormal function of the Golgi complex, which causes developmental disturbances and errors of the acrosome[14, 15]. From our observation, a few samples, especially from group C, unintact acrosomes were in a great proportion. These results indicated that severe varicocele for infertile men might be associated with severe acrosomal defects. According to the pathogenetic mechanism of the acrosome, acrosome malformations seem to be difficult to treat efficiently. No improvement in the acrosome reaction was noted post-varicocelectomy[4, 19]. With the development of assisted reproductive techniques(ART), intracytoplasmic sperm injection (ICSI) has provided substantial fertilization rates in cases of dramatically diminished sperm functions[20]. By using ICSI, all barriers to sperm penetration of the oocyte are bypassed, and the intact acrosome and acrosome reaction are not necessary since the sperm is injected directly into the ooplasm. Therefore, it suggested that varicocele men with poor acrosomes should be considered to select ICSI to treat their infertility in order to improve the fertilization efficacy. In the present study, varicocele patients demonstrated low sperm motilities, low sperm counts, and low normal sperm morphology rates, which were in agreement with previous reports[2-8, 21, 22]. Obviously, affected sperm parameters, especially high abnormal sperm morphology, were related to reduced fertility[17, 23, 24]. On the other hand, we noticed that some sperm with “normal” shaped head had abnormal acrosmes. Besides, acrosome malformations did not show special types by using both light and electron microscopy in this study. Because acrosome defects are associated with spermatogenesis, the reasons or mechanisms need further investigation. In conclusion, our study revealed that infertile men with varicocele had abnormal acrosome integrity, and some acrosome defects could not be showed by routine light microscopy. Because the acrosome can not be clearly visualized in live and unstained human sperm due to its relatively thin and translucency, special techniques such as PSA method should be used to determine its morphology or integrity. Combined with routine semen analysis, evaluating sperm acrosome should aid the understanding of the sperm structural state and also provide information relevant to the treatment of infertile men with varicocele.
References 1. Pasqualotto FF, Lucon AM, Hallak J, et al. Induction of spermatogenesis in azoospermic men after varicocele repair. Hum Reprod, 2003, 18(1):108-12. 2. Benoff S, Gilbert BR. Varicocele and male infertility: Part I, Preface. Hum Reprod Update, 2001, 7(1):47-51. 23
3. Jarow JP. Effects of varicocele on male infertility. Hum Reprod Update, 2001, 7(1):59-64. 4. Naughton CK, Nangia AK, Agarwal A. Varicocele and male infertility: Part II, Pathophusiology of varicoceles in male infertility. Hum Reprod Update, 2001, 7(5):473-81. 5. Hauser R, Paz G, Botchan A, et al. Varicocele and male infertility: Part II, Effect on sperm functions. Hum Reprod Update, 2001, 7(5):482-5. 6. Cockett ATK, Takihara H, Cosentino MJ. The varicocele. Fertil Steril, 1984, 41(1):5-11 7. Ismail MT, Sedor J, Hirsch I. Are sperm motion parameters influenced by varicocele ligation? Fertil Steril, 1999, 71(5):886-90. 8. Liang WB, Zhu WJ, Su ZX. Observation on sperm penetration rate for infertile men with varicocele. J Reprod Contracep, 2002, 13(1):10-5. 9. WHO. Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Cambridge: Cambridge University Press, 1993. 10. WHO. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge: Cambridge University Press, 1999. 11. Liu DY, Baker HWG. Acrosome atatus and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro. Hum Reprod, 1994, 9(4):673-9. 12. Tang FX, Zhu WJ, Li J. Effect of cryopreservation on changes in human sperm acrosome integrity and ultrastructure. Chinese J Birth Health & Heredity (in Chinese), 2004, 12(3):105-7. 13. Zhu WJ, Li J. Evaluation on the morphology and membrane integrity of immotile human sperm. J Reprod Contracep, 2006, 17(2):80-4. 14. Zamboni L. The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril, 1987, 48(5):711-34. 15. Bartoov B, Eltes F, Weissenberg R, et al. Morphological characterization of abnormal human spermatozoa using transmission electron microscopy. Arch Androl, 1980, 5(5):305-22. 16. El-Ghobashy AA, West CR. The human sperm head: a key for successful fertilization. Hum Reprod, 2003, 24(2):232-8. 17. Liu DY, Baker HWG. Tests of human sperm function and fertilization in vitro. Fertil Steril, 1992, 58(3):46583. 18. Garrett C, Liu DY, Baker HWG. Selectivity of the human sperm-zona pellucida binding process to sperm head morphometry. Fertil Steril, 1997, 67(2):362-71. 19. Benoff S, Barcia M, Hurley I, et al. Classification of male factor infertility relevant to in vitro fertilization insemination strategies using mannose ligands, acrosome status and anticytoskeletal antibodies. Hum Reprod, 1996, 11(2):1 905-18. 20. Palermo G, Cohen J, Rosenwaks Z. Intracytoplasmic sperm injection: a powerful tool to overcome fertilization failure. Fertil Steril, 1996, 65(5):899-908. 21. Newton R, Schinfeld J, Schiff I. The effect of varococelectomy on sperm count, motility, and conception rate. Fertil Steril, 1980, 34(3):250-4. 22. Marks JL, McMahon R, Lipshultz L. Predictive parameters of successful varicocele repair. J Urol, 1986, 136 (3):609-12. 23. Donnelly ET, Lewis AEM, McNally JA, et al. In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertil Steril, 1998, 70(2):305-14. 24. Baccetti B, Capitani S, Collodel G, et al. Recent advances in human sperm pathology. Contraception, 2002, 65(4): 283-7. (Received on December 28, 2006)
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