抗精子多肽抗体的制备和对人精子功能影响的实验研究
|
摘要
受精过程包括精子成熟、附着、结合、顶体反应、穿卵、融合、皮质反应、双方配子的带反应和核聚变等过程，而抗精子抗体可通过降低精子速度、减少顶体反应率、抑制精卵结合等途径来影响精子受精功能，导致免疫性不孕。利用精子抗原来制作避孕疫苗，已成为免疫避孕的研究热点。
精子蛋白SP17位于精子顶体部位，与精子顶体跨膜蛋白Cyritestin一样，均在精-卵透明带结合、精-卵膜结合和融合过程中发挥重要作用。本研究利用已经获得的含SP17和Cyritestin表位的人工合成35肽（P3肽）和抗P3肽兔血清，探讨抗P3抗体与人精子的结合和对受精过程的影响及其程度，评价人工合成的P3肽对人精子功能的影响，旨在对其生物学特性进行深入研究。
实验分两部分进行。第一部分运用试剂盒，将P3多肽通过EDC与KLH载体蛋白结合，免疫BALB/C小鼠，收获血清经过柱纯化，获得抗P3抗体，用FITC直接标记，通过流式细胞仪检测抗体与精子结合的阳性率，即抗体与人精子的结合率，用荧光显微镜观察抗体与人精子的结合部位，并且采用免疫组化方法，以抗Cyritestin单抗为阳性对照，观察含抗P3抗体的兔血清与人精子的结合部位。第二部分将人精子与含抗体血清共同孵育，利用CASA系统检测人精子活动力，用FITC-PSA法及考马斯亮蓝法检测顶体反应率，通过SPA法检测穿卵率，评价抗体对人精子功能的影响及其程度。
抗体经纯化、标记后与精子表面的结合率可达97.7%，抗体血清与人精子孵育后，用羊抗人IgG检测阳性率从49.2%降至36.4%，荧光显微镜见精子头颈部着色，部分见赤道板或顶端荧光增强，免疫组化染色见精子头部着深褐色，与阳性对照一致。抗体血清与人精子孵育后运动速度、各项运动精子百分率及其他各项指标均显著下降，并且与抗体浓度变化有一定关系，
精子顶体反应率及穿卵率也显著下降。
结论：P3肽在精子头部表面有表达，特异性抗P3抗体可与其高度结合；抗P3抗体可显著影响精子的活动力、顶体反应和穿卵率，可干扰精子受精功能，因此P3肽对人精子受精功能有影响，具有一定的开发前景。
There are many events in fertilization such as maturation of egg and sperm, attachment, binding, acrosomal reaction, penetration, fusion, cortical reaction, zona reaction and nuclear fusion of both gamete. It is well known that anti-sperm antibody could impair fertility in couples by resulting in immunological infertility through damaging sperm motility, reducing acrosome reaction, inhibiting sperm penetration zona pellucida, and so on. Many special sperm antigens have been utilized to prepare contraception vaccine for controlling fertilization, which is focused in immunological contraception research.
Sperm protein Sp17 is one of sperm antigens on acrosomal cap, and can bind specially with ovum zona pellucida. Cyritestin, a cross-membrane protein of acrosome, as Sp17 can also effect on sperm penetration. Both Sp17 and Cyritestin play important role in process of sperm-zona pellucida or sperm-member of egg binding and fusion. This study utilized P3 multiple peptides which was synthetic polypeptide containing both epitopes of Sp17 and Cyritestin and anti-P3 multiple peptides serum of rat. Our study is aimed to investigate the biological property of anti-P3 antibody, and its efficiency against sperm-egg conjugation and anti-fertilization.
This research is divided into two parts. In part one, we conjugated the P3 peptide and carrier protein KLH with EDC, and immunized BALB/C mouse with the purified compound P3-KLH. Serum were collected, anti-P3 antibody was purified using immobilized diaminodipropylamine column. The purified anti-P3 antibody was then labeled with FITC, and used to detect the sperm's positive percent by flow cytometry. Then we confirmed the binding site of antibody
against P3 peptide by immunofluorescence and immunocytochemistry techniques separately.
In part two, after swim-up collection, the motile sperm were incubated with antiserum against P3 peptide and control serum. Parameters of sperm motility, including sperm head amplitude, linearity, straight and curvilinear velocity, motility percentage, were evaluated and quantified using Computer-aided semen analysis system. Acrosomal reaction rate was determined by FITC-PSA fluorescence stain and Coomassie's blue stain. The penetrate capacity was assessed by hamaster zona-free egg penetration assay.
Brown reaction appeared on the sperm head surface by pre-incubating with antibody serum and also with the positive control. The positive rate of sperm conjugating with FITC-anti-P3 antibody is 97.7%. The conjugating positive rate of FITC-goat-anti-human IgG with sperm was reduced from 49.2% to 36.4% by acquired antiserum. After incubation with acquired antibody serum, all the parameters of sperm quality (motility) were decreased significantly, including sperm velocity, percent motility, ALH, LIN, the rate of AR, the rate of SPA.
Our results suggested that: on the surface of sperm head, there is an epitope as P3 peptide, and anti-P3 antibody could conjugate with sperm specially and excellently; the anti-P3 antibody serum could reduce the motility, acrosome reaction and penetrate capacity of sperm and disturb sperm's fertilization ability. So, the potential practicability of synthetic P3 peptide needs to be further determined.
精子蛋白SP17位于精子顶体部位，与精子顶体跨膜蛋白Cyritestin一样，均在精-卵透明带结合、精-卵膜结合和融合过程中发挥重要作用。本研究利用已经获得的含SP17和Cyritestin表位的人工合成35肽（P3肽）和抗P3肽兔血清，探讨抗P3抗体与人精子的结合和对受精过程的影响及其程度，评价人工合成的P3肽对人精子功能的影响，旨在对其生物学特性进行深入研究。
实验分两部分进行。第一部分运用试剂盒，将P3多肽通过EDC与KLH载体蛋白结合，免疫BALB/C小鼠，收获血清经过柱纯化，获得抗P3抗体，用FITC直接标记，通过流式细胞仪检测抗体与精子结合的阳性率，即抗体与人精子的结合率，用荧光显微镜观察抗体与人精子的结合部位，并且采用免疫组化方法，以抗Cyritestin单抗为阳性对照，观察含抗P3抗体的兔血清与人精子的结合部位。第二部分将人精子与含抗体血清共同孵育，利用CASA系统检测人精子活动力，用FITC-PSA法及考马斯亮蓝法检测顶体反应率，通过SPA法检测穿卵率，评价抗体对人精子功能的影响及其程度。
抗体经纯化、标记后与精子表面的结合率可达97.7%，抗体血清与人精子孵育后，用羊抗人IgG检测阳性率从49.2%降至36.4%，荧光显微镜见精子头颈部着色，部分见赤道板或顶端荧光增强，免疫组化染色见精子头部着深褐色，与阳性对照一致。抗体血清与人精子孵育后运动速度、各项运动精子百分率及其他各项指标均显著下降，并且与抗体浓度变化有一定关系，
精子顶体反应率及穿卵率也显著下降。
结论：P3肽在精子头部表面有表达，特异性抗P3抗体可与其高度结合；抗P3抗体可显著影响精子的活动力、顶体反应和穿卵率，可干扰精子受精功能，因此P3肽对人精子受精功能有影响，具有一定的开发前景。
There are many events in fertilization such as maturation of egg and sperm, attachment, binding, acrosomal reaction, penetration, fusion, cortical reaction, zona reaction and nuclear fusion of both gamete. It is well known that anti-sperm antibody could impair fertility in couples by resulting in immunological infertility through damaging sperm motility, reducing acrosome reaction, inhibiting sperm penetration zona pellucida, and so on. Many special sperm antigens have been utilized to prepare contraception vaccine for controlling fertilization, which is focused in immunological contraception research.
Sperm protein Sp17 is one of sperm antigens on acrosomal cap, and can bind specially with ovum zona pellucida. Cyritestin, a cross-membrane protein of acrosome, as Sp17 can also effect on sperm penetration. Both Sp17 and Cyritestin play important role in process of sperm-zona pellucida or sperm-member of egg binding and fusion. This study utilized P3 multiple peptides which was synthetic polypeptide containing both epitopes of Sp17 and Cyritestin and anti-P3 multiple peptides serum of rat. Our study is aimed to investigate the biological property of anti-P3 antibody, and its efficiency against sperm-egg conjugation and anti-fertilization.
This research is divided into two parts. In part one, we conjugated the P3 peptide and carrier protein KLH with EDC, and immunized BALB/C mouse with the purified compound P3-KLH. Serum were collected, anti-P3 antibody was purified using immobilized diaminodipropylamine column. The purified anti-P3 antibody was then labeled with FITC, and used to detect the sperm's positive percent by flow cytometry. Then we confirmed the binding site of antibody
against P3 peptide by immunofluorescence and immunocytochemistry techniques separately.
In part two, after swim-up collection, the motile sperm were incubated with antiserum against P3 peptide and control serum. Parameters of sperm motility, including sperm head amplitude, linearity, straight and curvilinear velocity, motility percentage, were evaluated and quantified using Computer-aided semen analysis system. Acrosomal reaction rate was determined by FITC-PSA fluorescence stain and Coomassie's blue stain. The penetrate capacity was assessed by hamaster zona-free egg penetration assay.
Brown reaction appeared on the sperm head surface by pre-incubating with antibody serum and also with the positive control. The positive rate of sperm conjugating with FITC-anti-P3 antibody is 97.7%. The conjugating positive rate of FITC-goat-anti-human IgG with sperm was reduced from 49.2% to 36.4% by acquired antiserum. After incubation with acquired antibody serum, all the parameters of sperm quality (motility) were decreased significantly, including sperm velocity, percent motility, ALH, LIN, the rate of AR, the rate of SPA.
Our results suggested that: on the surface of sperm head, there is an epitope as P3 peptide, and anti-P3 antibody could conjugate with sperm specially and excellently; the anti-P3 antibody serum could reduce the motility, acrosome reaction and penetrate capacity of sperm and disturb sperm's fertilization ability. So, the potential practicability of synthetic P3 peptide needs to be further determined.
引文
1. Hiroi M：[Elucidation of the mechanism of fertilization and clinical application of assisted reproductive technology] Nippon- Sanka- Fujinka- Gakkai-Zasshi 1996，48(8): 578-90
2. Shibahara H，Shigeta M，Inoue M，Hasegawa A，Koyama K，Alexander NJ，Isojima S：Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse. Hum-Reprod 1996，11(12): 2595-9
3. Yao YQ，Yeung WS，Ho PC： The factors affecting sperm binding to the zona pellucida in the hemizona binding assay.Hum-Reprod 1996，11(7): 1516-9
4. Moller CC, Bleil JD, Kinloch RA, et al：Structural and functional relationships between mouse and hamster zona pellucida glycoproteins. Dev Biol 1990,137:276-286
5. Wen Y, Richardson RT, O'Rand M：Processing of the sperm protein Sp17 during the acrosome reaction and characterization as a calmodulin binding protein。Dev Biol 1999,206:113-122.
6. Lum L, Blobel CP：Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin. Dev Biol 1997, 191:131-145.
7. Waters SI,White JM：Biochemical and molecular characterization of bovine fertilin alpha and beta (ADAM 1 and ADAM 2): A candidate sperm-egg binding/fusion complex. Biol Reprod 1997,56: 1245-1254
8. Harlow E，Lane D：Antibodies: A laboratory Manual. Cold Spring Harbor Laboratory. 1988 Harbor, NY, 56-100
9. Male D, Champion B, Cooker A：Advanced Immunology. J.B.Lippincott Co. Gower Medical Publishing, 1987, London: 8.1-8.8
Williams A，Ibrahin I：A new mechanism involving cyclic tautomers for the
10. reaction with nucleophilies of the water-soluble peptide coupling reagent 1-Ethyl-3-(3-dimethylaminopropyl) carbodimide-HCl (EDC). J.A.C.S. 1981,103: 7090-7095.
11. Bronson R, Cooper G,Hjort T,Ing R, Jones WR,Wang SX, et al：Anti-sperm antibodies, detected by agglutination, immobilization, microcytotoxicity and immunobead-binding assays. J Reprod Immuno 1985，8:279-99
12. Hinting A，Vermeulen L，Comhaire F：The indirect mixed antiglobulin reaction test using a commercially available kit for the detection of antisperm antibodies in serum. Fertil-Steril 1988，49(6): 1039-44.
13. Ke RW，Dockter ME，Majumdar G，Buster JE，Carson SA：Flow cytometry provides rapid and highly accurate detection of antisperm antibodies. Fertil-Steril 1995, 63(4): 902-6
14. Hass GG,Cunnngham ME：Identification of antibody-lade sperm by cytofluorometry. Fertil steril 1984, 42:606-13
15. Haas GG，D'Cruz OJ，DeBault LE：Comparison of the indirect immunobead, radiolabeled, and immunofluorescence assays for immunoglobulin G serum antibodies to human sperm. Fertil-Steril 1991, 55(2): 377-88
16. Nikolaeva MN，Verbitskii MS，Sukhikh GT： [The use of flow cytofluoro- metry for determining antisperm antibodies by sperm immobilization and sperm toxicity methods]. Biull-Eksp-Biol-Med 1992, 114(10): 398-9
17. Raymond WK，Michael ed，G.Majumdar：Flow cytometry provides rapid and highly accurate detection of antisperm antibodies. Fertil Steril 1995, 63(1): 902-6
18. Simon CN, Julian NR, Lan LS, David HB：Detecion of antisperm antibodies in semial plasma by flow cytometry: comparison with indirect immunobead binding test. Fertil Steril 1997, 68(6):1114-9
19. 蔡文琴 王伯云主编：实用免疫细胞化学与核酸分子杂交技术。成都，四川科技出版社，1994。
20. Tsai CC，et al：Antibodies affecting sperm motion。Fertile Steril 1987，47（3）：460
21. 黄天华，高晓平，漆著，李丹，尹丽林：人精子染色体研究-一种稳定的人精子染色体制备技术。遗传与疾病1987，4（3）：374-376
22. Biggers JD， Whitten WK， Whittingham DG：The culture of mouse embryos in vitro. In: J.C. Daniel, Editor, Methods in mammalian embryology, Freeman, San Francisco, 1971，86ˉ116
23. Makler: a new chamber for rapid sperm scount sand smotility estimation. Fertil Steril 1978，130：313
24. WHO: WHO laboratory manual for the examination of human semen and semencervical mucus interaction. Singapoire published by press concern.1992, 15-55
25. Kohn FM，Mack SR，Schill WB，Zaneveld LJ：Detection of human sperm acrosome reaction: comparison between methods using double staining, Pisum sativum agglutinin, concanavalin A and transmission electron microscopy. Hum-Reprod 1997，12(4): 714-21
26. 黄宇烽，许瑞吉主编：男科诊断学。上海：第二军医大学出版社，1999。
27. Mahadevan M，Trounson AO： Relationship of fine structure of sperm head to fertility of frozen human semen. Fertil-Steril 1984, 41(2): 287-93
28. 高晓萍，李丹，黄明孔：固定、低渗同步法制备受精卵标本。癌变.畸变.突变 1994，6（5）：16-18
29. 陈欣洁，孙筱放：人精子体外获能最佳时间的选择。生殖与避孕 1998，18（6）：367-8
30. Dunphy bc，Kay R，Barratt cr，etａl：Quality control during the conventional analysis of semen：an essential exercise. J-Androl 1989，10：378
31. Neuwinger J，Behere hm，Nieschlag E：External quality control in the andrology laboratory：an experimental multicenter trail. Fertil Steril 1990，54：308
Krause W：The significance of computer-assisted semen analysis (CASA) for
32. diagnosis in andrology and fertility prognosis. Int J Androl 1995，18：32ˉ35
33. Hirano Y，Shibahara H，Obara H，Suzuki T：Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro. J-Assist-Reprod-Genet 2001，18(4): 213-8
34. Joshi N， Kodwany G， Balaiah D， Parikh M， Parikih F：The importance of computer-assisted semen analysis and sperm function testing in an IVF program. Int J Fertil Men Stud 1996，41: 46ˉ52
35. Amann RP：Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989，10：89ˉ98
36. Barratt CLR，Tomlinson MJ，Cook ID：Prognostic significance of computerized motility analysis for in vivo fertility. Fertil Steril 1993，60：520ˉ525
37. Macleod IC，Irvine DS：The predictive value of computer-assisted semen analysis in the context of a donor insemination programme. Hum Reprod 1995，10：580ˉ586
38. Mortimer ST, Swan MA：Kinematics of capacitating human spermatozoa analysed at 60 Hz. Hum Reprod 1995，10（4）：873-879
39. Eilish TD，Sheena EM，James AM，William T：In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertil Steril 1998, 70（2）： 305-314
40. Green S， Fishel S， Hall J， Hunter A，Flemming S， Hobson G：Computer image sperm selection as a novel approach to subzonal insemination in the sperm. Hum Reprod 1995，10：1430ˉ1434
41. D'Cruz OJ，Haas GG，Wang BL，DeBault LE：Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm. J-Immunol 1991，146(2): 611-20
Coonrod SA, Weschusin ME, Naz RK：monoclonal antibody to human fertilization antigen (FA-1) inhibits bovine fertilization in vitro application
42. in immunocontraception. Biol Reprod 1994,51:14
43. Check JH，Adelson HG， Bollendorf A：Effect of antisperm antibodies on computerized semen analysis. Arch Androl 1991，27：61ˉ63
44. 罗丽兰主编，生殖免疫学。北京，人民卫生出版社。1998, 10：28-30。
45. Lansford B，Haas GG， DeBault LE， Wolf DP：Effect of sperm-associated antibodies on the acrosomal status of human sperm. J Androl 1990，11(6)：532ˉ538
46. Sukcharoen N，Keith J， Irvine DS， Aitken RJ：Definition of the optimal criteria for identifying hyperactivated human spermatozoa at 25Hz using in vitro fertilization as a functional end point. Hum Reprod 1995, 10：2928ˉ2937
47. 袁玉英、石其贤：精子功能的评价。生殖与避孕1998，18（1）：58-61
48. Yanagimachi R，Yanagimachi H，Rogers BJ：The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol-Reprod 1976，15(4)：471-6
49. 杨戎、魏莎莉、程法馨：去透明带地鼠卵穿透试验与人工授精受孕率关系的作用研究。中国实用妇科与产科杂志1998，14（2）：93-95
50. Haas GG，Sokoloski JE，Wolf DP：The interfering effect of human IgG antisperm antibodies on human sperm penetration of zona-free hamster eggs. Am-J-Reprod-Immunol 1980，1(1): 40-3
51. Tsukui S，Noda Y，Yano J，Fukuda A，Mori T：Inhibition of sperm penetration through human zona pellucida by antisperm antibodies. Fertil-Steril 1986，46(1): 92-6
52. Abdel-Latif A,Mathur S, Rust PF,et al：Cytotoxic sperm antibodies inhibit sperm penetration of zona-free hamaster eggs. Fertil Steril 1986,45(4): 542
53. Bronson RA,Cooper GW,Rosenfeld GL：Ability of antibody-bound human sperm to penetrate zona-free hamaster eggs. Fertil Steril 1983,40(1): 91
2. Shibahara H，Shigeta M，Inoue M，Hasegawa A，Koyama K，Alexander NJ，Isojima S：Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse. Hum-Reprod 1996，11(12): 2595-9
3. Yao YQ，Yeung WS，Ho PC： The factors affecting sperm binding to the zona pellucida in the hemizona binding assay.Hum-Reprod 1996，11(7): 1516-9
4. Moller CC, Bleil JD, Kinloch RA, et al：Structural and functional relationships between mouse and hamster zona pellucida glycoproteins. Dev Biol 1990,137:276-286
5. Wen Y, Richardson RT, O'Rand M：Processing of the sperm protein Sp17 during the acrosome reaction and characterization as a calmodulin binding protein。Dev Biol 1999,206:113-122.
6. Lum L, Blobel CP：Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin. Dev Biol 1997, 191:131-145.
7. Waters SI,White JM：Biochemical and molecular characterization of bovine fertilin alpha and beta (ADAM 1 and ADAM 2): A candidate sperm-egg binding/fusion complex. Biol Reprod 1997,56: 1245-1254
8. Harlow E，Lane D：Antibodies: A laboratory Manual. Cold Spring Harbor Laboratory. 1988 Harbor, NY, 56-100
9. Male D, Champion B, Cooker A：Advanced Immunology. J.B.Lippincott Co. Gower Medical Publishing, 1987, London: 8.1-8.8
Williams A，Ibrahin I：A new mechanism involving cyclic tautomers for the
10. reaction with nucleophilies of the water-soluble peptide coupling reagent 1-Ethyl-3-(3-dimethylaminopropyl) carbodimide-HCl (EDC). J.A.C.S. 1981,103: 7090-7095.
11. Bronson R, Cooper G,Hjort T,Ing R, Jones WR,Wang SX, et al：Anti-sperm antibodies, detected by agglutination, immobilization, microcytotoxicity and immunobead-binding assays. J Reprod Immuno 1985，8:279-99
12. Hinting A，Vermeulen L，Comhaire F：The indirect mixed antiglobulin reaction test using a commercially available kit for the detection of antisperm antibodies in serum. Fertil-Steril 1988，49(6): 1039-44.
13. Ke RW，Dockter ME，Majumdar G，Buster JE，Carson SA：Flow cytometry provides rapid and highly accurate detection of antisperm antibodies. Fertil-Steril 1995, 63(4): 902-6
14. Hass GG,Cunnngham ME：Identification of antibody-lade sperm by cytofluorometry. Fertil steril 1984, 42:606-13
15. Haas GG，D'Cruz OJ，DeBault LE：Comparison of the indirect immunobead, radiolabeled, and immunofluorescence assays for immunoglobulin G serum antibodies to human sperm. Fertil-Steril 1991, 55(2): 377-88
16. Nikolaeva MN，Verbitskii MS，Sukhikh GT： [The use of flow cytofluoro- metry for determining antisperm antibodies by sperm immobilization and sperm toxicity methods]. Biull-Eksp-Biol-Med 1992, 114(10): 398-9
17. Raymond WK，Michael ed，G.Majumdar：Flow cytometry provides rapid and highly accurate detection of antisperm antibodies. Fertil Steril 1995, 63(1): 902-6
18. Simon CN, Julian NR, Lan LS, David HB：Detecion of antisperm antibodies in semial plasma by flow cytometry: comparison with indirect immunobead binding test. Fertil Steril 1997, 68(6):1114-9
19. 蔡文琴 王伯云主编：实用免疫细胞化学与核酸分子杂交技术。成都，四川科技出版社，1994。
20. Tsai CC，et al：Antibodies affecting sperm motion。Fertile Steril 1987，47（3）：460
21. 黄天华，高晓平，漆著，李丹，尹丽林：人精子染色体研究-一种稳定的人精子染色体制备技术。遗传与疾病1987，4（3）：374-376
22. Biggers JD， Whitten WK， Whittingham DG：The culture of mouse embryos in vitro. In: J.C. Daniel, Editor, Methods in mammalian embryology, Freeman, San Francisco, 1971，86ˉ116
23. Makler: a new chamber for rapid sperm scount sand smotility estimation. Fertil Steril 1978，130：313
24. WHO: WHO laboratory manual for the examination of human semen and semencervical mucus interaction. Singapoire published by press concern.1992, 15-55
25. Kohn FM，Mack SR，Schill WB，Zaneveld LJ：Detection of human sperm acrosome reaction: comparison between methods using double staining, Pisum sativum agglutinin, concanavalin A and transmission electron microscopy. Hum-Reprod 1997，12(4): 714-21
26. 黄宇烽，许瑞吉主编：男科诊断学。上海：第二军医大学出版社，1999。
27. Mahadevan M，Trounson AO： Relationship of fine structure of sperm head to fertility of frozen human semen. Fertil-Steril 1984, 41(2): 287-93
28. 高晓萍，李丹，黄明孔：固定、低渗同步法制备受精卵标本。癌变.畸变.突变 1994，6（5）：16-18
29. 陈欣洁，孙筱放：人精子体外获能最佳时间的选择。生殖与避孕 1998，18（6）：367-8
30. Dunphy bc，Kay R，Barratt cr，etａl：Quality control during the conventional analysis of semen：an essential exercise. J-Androl 1989，10：378
31. Neuwinger J，Behere hm，Nieschlag E：External quality control in the andrology laboratory：an experimental multicenter trail. Fertil Steril 1990，54：308
Krause W：The significance of computer-assisted semen analysis (CASA) for
32. diagnosis in andrology and fertility prognosis. Int J Androl 1995，18：32ˉ35
33. Hirano Y，Shibahara H，Obara H，Suzuki T：Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro. J-Assist-Reprod-Genet 2001，18(4): 213-8
34. Joshi N， Kodwany G， Balaiah D， Parikh M， Parikih F：The importance of computer-assisted semen analysis and sperm function testing in an IVF program. Int J Fertil Men Stud 1996，41: 46ˉ52
35. Amann RP：Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989，10：89ˉ98
36. Barratt CLR，Tomlinson MJ，Cook ID：Prognostic significance of computerized motility analysis for in vivo fertility. Fertil Steril 1993，60：520ˉ525
37. Macleod IC，Irvine DS：The predictive value of computer-assisted semen analysis in the context of a donor insemination programme. Hum Reprod 1995，10：580ˉ586
38. Mortimer ST, Swan MA：Kinematics of capacitating human spermatozoa analysed at 60 Hz. Hum Reprod 1995，10（4）：873-879
39. Eilish TD，Sheena EM，James AM，William T：In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertil Steril 1998, 70（2）： 305-314
40. Green S， Fishel S， Hall J， Hunter A，Flemming S， Hobson G：Computer image sperm selection as a novel approach to subzonal insemination in the sperm. Hum Reprod 1995，10：1430ˉ1434
41. D'Cruz OJ，Haas GG，Wang BL，DeBault LE：Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm. J-Immunol 1991，146(2): 611-20
Coonrod SA, Weschusin ME, Naz RK：monoclonal antibody to human fertilization antigen (FA-1) inhibits bovine fertilization in vitro application
42. in immunocontraception. Biol Reprod 1994,51:14
43. Check JH，Adelson HG， Bollendorf A：Effect of antisperm antibodies on computerized semen analysis. Arch Androl 1991，27：61ˉ63
44. 罗丽兰主编，生殖免疫学。北京，人民卫生出版社。1998, 10：28-30。
45. Lansford B，Haas GG， DeBault LE， Wolf DP：Effect of sperm-associated antibodies on the acrosomal status of human sperm. J Androl 1990，11(6)：532ˉ538
46. Sukcharoen N，Keith J， Irvine DS， Aitken RJ：Definition of the optimal criteria for identifying hyperactivated human spermatozoa at 25Hz using in vitro fertilization as a functional end point. Hum Reprod 1995, 10：2928ˉ2937
47. 袁玉英、石其贤：精子功能的评价。生殖与避孕1998，18（1）：58-61
48. Yanagimachi R，Yanagimachi H，Rogers BJ：The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol-Reprod 1976，15(4)：471-6
49. 杨戎、魏莎莉、程法馨：去透明带地鼠卵穿透试验与人工授精受孕率关系的作用研究。中国实用妇科与产科杂志1998，14（2）：93-95
50. Haas GG，Sokoloski JE，Wolf DP：The interfering effect of human IgG antisperm antibodies on human sperm penetration of zona-free hamster eggs. Am-J-Reprod-Immunol 1980，1(1): 40-3
51. Tsukui S，Noda Y，Yano J，Fukuda A，Mori T：Inhibition of sperm penetration through human zona pellucida by antisperm antibodies. Fertil-Steril 1986，46(1): 92-6
52. Abdel-Latif A,Mathur S, Rust PF,et al：Cytotoxic sperm antibodies inhibit sperm penetration of zona-free hamaster eggs. Fertil Steril 1986,45(4): 542
53. Bronson RA,Cooper GW,Rosenfeld GL：Ability of antibody-bound human sperm to penetrate zona-free hamaster eggs. Fertil Steril 1983,40(1): 91