不育夫妇血清抗精子抗体和抗心磷脂抗体的表达及意义
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摘要
目的:近年来探讨不孕不育的免疫学因素正成为生殖医学研究的一个热点,在众多免疫因素中抗精子抗体(Atisperm Atibody,AsAb)和抗心磷脂抗体(Aticardiolipin Atibody,ACA)越来越引起人们的重视。精子对男性自身而言为一种隐蔽抗原,精子对女性而言是一种异己抗原,但正常情况下在生理保护机制作用下,并没有抗精子抗体的产生,当生理保护机制被破坏后即会导致机体产生抗精子抗体。抗心磷脂抗体是抗磷脂抗体的一种,正常情况下,带负电荷的心磷脂位于细胞膜脂质双层的内层,不被免疫系统识别,一旦暴露,心磷脂抗原即刺激机体产生一种自身免疫性抗体即抗心磷脂抗体。本实验通过检测不育夫妇的血清抗精子抗体三种亚型IgG、IgM、IgA和抗心磷脂抗体三种亚型IgG、IgM、IgA的表达,探讨抗精子抗体和抗心磷脂抗体在不孕不育中的作用。
方法:1研究对象:选取2008年5月至2008年10月在河北医科大学第三医院泌尿外科门诊和白求恩国际和平医院男科门诊就诊的不育夫妇50对作为实验组(男性年龄25～31岁、平均26.70士1.67,女性年龄23～32岁、平均26.22士3.05)均为首次检测血清抗精子抗体和抗心磷脂抗体者。入选条件:结婚1年以上、夫妇性生活正常、未采用任何避孕措施而无孕育史的夫妇。男方无遗传性疾病家族史,无慢性疾病,泌尿外科检查未发现睾丸、附睾及输精管异常。女方无遗传性疾病家族史,无慢性疾病,卵泡发育正常,有正常的排卵周期,妇科检查阴道、子宫、输卵管及卵巢未发现异常。正常对照组:体格检查健康的有正常生育史的育龄非孕状态夫妇40对(男性年龄24～31岁、平均26.35士1.85;女性年龄23～33岁、平均26.63士3.47),泌尿外科及妇科检查无异常。
2标本采集:采取清晨空腹静脉血3ml,不抗凝,分离血清后保存于-20℃冰箱中待测。所有入选男性均在禁欲5-7天后,采取精液进行精液分析。
3检测方法:用酶联免疫吸附测定法(ELISA)检测血清中抗精子抗体和抗心磷脂抗体的表达。酶联免疫吸附测定法(ELISA)检测试剂盒购于深圳市安群生物工程有限公司,按照试剂盒说明进行实验操作并对结果进行判定。
4统计学分析:所得抗精子抗体和抗心磷脂抗体的数据采用计数资料表示,采用Fisher确切概率法分析。精液分析结果以x±s表示,采用成组t检验法分析;所有数据均在微机上用SAS8.1统计软件包进行统计分析。以P<0.05作为差异有统计学意义的指标。
结果:1抗精子抗体及抗心磷脂抗体结果:实验组男性抗精子抗体阳性率与对照组相比差异有统计学意义(30 % vs 5%,P<0.01)见Fig1及Table3。实验组女性抗精子抗体阳性率与对照组相比差异有统计学意义(34% vs 12.5%,P<0.05)见Fig1及Table5。实验组男性抗心磷脂抗体阳性率与对照组相比差异无统计学意义(6% vs 5%,P>0.05)见Fig1及Table4。实验组女性抗心磷脂抗体阳性率与对照组相比差异有统计学意义(32% vs 10%,P<0.05)见Fig1及Table6。2精液分析结果:实验组男性中抗精子抗体阳性且抗心磷脂抗体阴性者与对照组中抗精子抗体阴性且抗心磷脂抗体阴性者相比,精液量、PH值、精子密度及b级精子差异无统计学意义(P>0.05);实验组较对照组精子活率、a级精子低,直线速度和曲线速度慢,具有统计学意义(P<0.01)见Table7。
结论:1抗精子抗体和抗心磷脂抗体是导致不孕不育的重要免疫因素,抗精子抗体可影响男性和女性的生育能力,而抗心磷脂抗体主要损害女性的生育功能。2抗精子抗体可通过降低精液质量而导致不孕不育。
Objective:In recent years many investigations are focus on the immunological factors of infertility and investigators pay much attention to antisperm antibody(AsAb) and anticardiolipin antibody(ACA) among those factors.Antisperm antibody is a hidden antigen for men and is a foreign antigen for women,but normally there is no AsAb ,because of some physiological protective mechanisms.If these physiological protective mechanisms are destroied AsAb will be produced.ACA is one of anti-phospholipid antibodies,it has negative charge ,normally ACA locates in the inner layer of double-layer lipid cellular membrane and is not be detected by immune system .If cuorin is exposed ACA will be produced.This study is designed to detecte the expression of three subtypes (IgG、IgM、IgA ) of AsAb and ACA and to analyze the function of AsAb and ACA in infertility.
Method: 1 Object:50 infertile couples were choosed from out-patient department(OPD) of urology of the thrid hospital of He Bei Medical University and OPD of andrology of Bai Qiuen international peace hospital during may 2008 to october 2008 as experimental group(the age of men 25～31,mean 26.7士1.67;the age of women 23～32,mean 26.22士3.05).All of these couples did not detect AsAb and ACA before.Election qualification: They married over one year,have normal sexual life and do not use any contraception but they are infertile .All the men do not have genetic disease family history,have no chronic disease and have normal testicle、epididymis and deferent canal. All the women do not have genetic disease family history,have no chronic disease and follicular development are normal,they have normal ovulatory cycle、vagina、uterus、ovarian duct and ovaries.Normal control group:40 healthy couples(the age of men 24~31,mean 26.4士1.85;the age of women 23~33,mean 26.63士3.47 ) who have normal childbearing history but are not pregnant when they are detected,the urinarysurgery and gynecologic examinations are normal.
2 Sample collection: Taking 3ml venous blood in the morning but donot anticoagulate, abstracting serum and then put the serum in -20℃refrigerator.Collecting and analyzing semen after sexual abstinence for 5-7days.
3 Detection Method: Choosing ELISA to detect AsAb and ACA in serum.we buy ELISA kit form Shenzhen Anqun Biotechnology Company limited and follow the kit instruction when doing experiment and assessing results.
4 Statistic Analysis: The result of AsAb and ACA are represented by numeration data and the Data are analyzed through x2 or fisher’s exact test. The results of semen parameter are represented by x±s and the Data are analyzed through tort′test. All the data are analyzed in computerized SAS8.1software. For each of these analyses, two-tailed probability values <0.05 are designed as significant.
Result: 1 The result of AsAb and ACA:The positive rate of AsAb in male infertile group is higher than control group and the difference is significant(30 % vs 5%,P<0.01)see Fig1 andTable3. The positive rate of AsAb in female infertile group is higher than control group and the difference is significant(34% vs 12.5%,P<0.05)see Fig1 and Table5.The difference of positive rate of ACA between infertile male group and control group is no significant(6% vs 5%,P>0.05)see Fig1 and Table4. The positive rate of ACA in female infertile group is higher than control group and the difference is significant(32% vs 10%,P<0.05)see Fig1 and Table6.
2.Semen analysis:When comparing semen parameter between AsAb positive but ACA negative infertile men in the experimental group and AsAb negative and ACA negative fertile men in the control group,there is no difference in capacity of semen、PH、density of sperm and sperm in B stag(eP>0.05).But the motility rate of sperm、sperm in A stage、rectilinearity、linear velocity and curve velocity in control group are hingher than experimental group and the differences are significant(P<0.01) see Table7.
Conclusion: 1 AsAb and ACA are imporant immunological factors that can cause infertility, AsAb can damage male and female potentia generandi but ACA only decrease female potentia generandi .2 AsAb can impact the quality of semen and then result in infertility.
方法:1研究对象:选取2008年5月至2008年10月在河北医科大学第三医院泌尿外科门诊和白求恩国际和平医院男科门诊就诊的不育夫妇50对作为实验组(男性年龄25～31岁、平均26.70士1.67,女性年龄23～32岁、平均26.22士3.05)均为首次检测血清抗精子抗体和抗心磷脂抗体者。入选条件:结婚1年以上、夫妇性生活正常、未采用任何避孕措施而无孕育史的夫妇。男方无遗传性疾病家族史,无慢性疾病,泌尿外科检查未发现睾丸、附睾及输精管异常。女方无遗传性疾病家族史,无慢性疾病,卵泡发育正常,有正常的排卵周期,妇科检查阴道、子宫、输卵管及卵巢未发现异常。正常对照组:体格检查健康的有正常生育史的育龄非孕状态夫妇40对(男性年龄24～31岁、平均26.35士1.85;女性年龄23～33岁、平均26.63士3.47),泌尿外科及妇科检查无异常。
2标本采集:采取清晨空腹静脉血3ml,不抗凝,分离血清后保存于-20℃冰箱中待测。所有入选男性均在禁欲5-7天后,采取精液进行精液分析。
3检测方法:用酶联免疫吸附测定法(ELISA)检测血清中抗精子抗体和抗心磷脂抗体的表达。酶联免疫吸附测定法(ELISA)检测试剂盒购于深圳市安群生物工程有限公司,按照试剂盒说明进行实验操作并对结果进行判定。
4统计学分析:所得抗精子抗体和抗心磷脂抗体的数据采用计数资料表示,采用Fisher确切概率法分析。精液分析结果以x±s表示,采用成组t检验法分析;所有数据均在微机上用SAS8.1统计软件包进行统计分析。以P<0.05作为差异有统计学意义的指标。
结果:1抗精子抗体及抗心磷脂抗体结果:实验组男性抗精子抗体阳性率与对照组相比差异有统计学意义(30 % vs 5%,P<0.01)见Fig1及Table3。实验组女性抗精子抗体阳性率与对照组相比差异有统计学意义(34% vs 12.5%,P<0.05)见Fig1及Table5。实验组男性抗心磷脂抗体阳性率与对照组相比差异无统计学意义(6% vs 5%,P>0.05)见Fig1及Table4。实验组女性抗心磷脂抗体阳性率与对照组相比差异有统计学意义(32% vs 10%,P<0.05)见Fig1及Table6。2精液分析结果:实验组男性中抗精子抗体阳性且抗心磷脂抗体阴性者与对照组中抗精子抗体阴性且抗心磷脂抗体阴性者相比,精液量、PH值、精子密度及b级精子差异无统计学意义(P>0.05);实验组较对照组精子活率、a级精子低,直线速度和曲线速度慢,具有统计学意义(P<0.01)见Table7。
结论:1抗精子抗体和抗心磷脂抗体是导致不孕不育的重要免疫因素,抗精子抗体可影响男性和女性的生育能力,而抗心磷脂抗体主要损害女性的生育功能。2抗精子抗体可通过降低精液质量而导致不孕不育。
Objective:In recent years many investigations are focus on the immunological factors of infertility and investigators pay much attention to antisperm antibody(AsAb) and anticardiolipin antibody(ACA) among those factors.Antisperm antibody is a hidden antigen for men and is a foreign antigen for women,but normally there is no AsAb ,because of some physiological protective mechanisms.If these physiological protective mechanisms are destroied AsAb will be produced.ACA is one of anti-phospholipid antibodies,it has negative charge ,normally ACA locates in the inner layer of double-layer lipid cellular membrane and is not be detected by immune system .If cuorin is exposed ACA will be produced.This study is designed to detecte the expression of three subtypes (IgG、IgM、IgA ) of AsAb and ACA and to analyze the function of AsAb and ACA in infertility.
Method: 1 Object:50 infertile couples were choosed from out-patient department(OPD) of urology of the thrid hospital of He Bei Medical University and OPD of andrology of Bai Qiuen international peace hospital during may 2008 to october 2008 as experimental group(the age of men 25～31,mean 26.7士1.67;the age of women 23～32,mean 26.22士3.05).All of these couples did not detect AsAb and ACA before.Election qualification: They married over one year,have normal sexual life and do not use any contraception but they are infertile .All the men do not have genetic disease family history,have no chronic disease and have normal testicle、epididymis and deferent canal. All the women do not have genetic disease family history,have no chronic disease and follicular development are normal,they have normal ovulatory cycle、vagina、uterus、ovarian duct and ovaries.Normal control group:40 healthy couples(the age of men 24~31,mean 26.4士1.85;the age of women 23~33,mean 26.63士3.47 ) who have normal childbearing history but are not pregnant when they are detected,the urinarysurgery and gynecologic examinations are normal.
2 Sample collection: Taking 3ml venous blood in the morning but donot anticoagulate, abstracting serum and then put the serum in -20℃refrigerator.Collecting and analyzing semen after sexual abstinence for 5-7days.
3 Detection Method: Choosing ELISA to detect AsAb and ACA in serum.we buy ELISA kit form Shenzhen Anqun Biotechnology Company limited and follow the kit instruction when doing experiment and assessing results.
4 Statistic Analysis: The result of AsAb and ACA are represented by numeration data and the Data are analyzed through x2 or fisher’s exact test. The results of semen parameter are represented by x±s and the Data are analyzed through tort′test. All the data are analyzed in computerized SAS8.1software. For each of these analyses, two-tailed probability values <0.05 are designed as significant.
Result: 1 The result of AsAb and ACA:The positive rate of AsAb in male infertile group is higher than control group and the difference is significant(30 % vs 5%,P<0.01)see Fig1 andTable3. The positive rate of AsAb in female infertile group is higher than control group and the difference is significant(34% vs 12.5%,P<0.05)see Fig1 and Table5.The difference of positive rate of ACA between infertile male group and control group is no significant(6% vs 5%,P>0.05)see Fig1 and Table4. The positive rate of ACA in female infertile group is higher than control group and the difference is significant(32% vs 10%,P<0.05)see Fig1 and Table6.
2.Semen analysis:When comparing semen parameter between AsAb positive but ACA negative infertile men in the experimental group and AsAb negative and ACA negative fertile men in the control group,there is no difference in capacity of semen、PH、density of sperm and sperm in B stag(eP>0.05).But the motility rate of sperm、sperm in A stage、rectilinearity、linear velocity and curve velocity in control group are hingher than experimental group and the differences are significant(P<0.01) see Table7.
Conclusion: 1 AsAb and ACA are imporant immunological factors that can cause infertility, AsAb can damage male and female potentia generandi but ACA only decrease female potentia generandi .2 AsAb can impact the quality of semen and then result in infertility.
引文
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1郭应禄,胡礼泉,主编.男科学.第一版.北京:人民卫生出版社,2004:191-213
2范海涛,郭航.不同睾丸活检术前后血清抗精子抗体的检测.中华男科学杂志,2005,11:862-863
3 Chavez Badiola A,Drakeley AJ,Finney V. Necrospermia, antisperm antibodies, and vasectomy. Fertility and Sterility,2008,89(4):723.e5-732.e7
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1郭应禄,胡礼泉,主编.男科学.第一版.北京:人民卫生出版社,2004:191-213
2范海涛,郭航.不同睾丸活检术前后血清抗精子抗体的检测.中华男科学杂志,2005,11:862-863
3 Chavez Badiola A,Drakeley AJ,Finney V. Necrospermia, antisperm antibodies, and vasectomy. Fertility and Sterility,2008,89(4):723.e5-732.e7
4薛宁,崔明玉.精索静脉曲张不育症的病理观察及免疫组化研究.中国计划生育学杂志,2006,8:482-484
5刘子明,郑新民,杨志伟等.单侧睾丸扭转引发对侧睾丸生精损伤免疫机制的实验研究.中华泌尿外科杂志,2006,27(1):59-62
6张群锋,罗志刚.衣原体感染不孕不育患者血清中自身抗体的检测.现代生物医学进展,2008,8(6):1096-1097
7 Dimitrova D,Kalaydjiev S,Mendizova A. Circulating antibodies to human spermatozoa in patients with ulcerative colitis. Fertility and Sterility,2005,84(5):1533-1535
8 Yasuoka H,Kuwana M.Autoantibody response against a novel testicular antigen protein highly expressed in testis (PHET) in SSc patients. Autoimmunity Reviews ,2007,6(4):228–231
9 Donatella Paoli, Barbara Gilio, Emanuela Piroli et al. Testicular tumors as a possible cause of antisperm autoimmune response.Fertil Steril,2009,91(2):414-419
10 Oliva A,Spira A,Multigner L.Contribution of environmental factors to the risk of male infertility.Hum Reprod,2001,16 (8): 1768-1776
11马丽娜,李凤云,冯锡才.不孕不育与支原体、衣原体感染和抗精子抗体的关系.实用全科医学,2008,6(5):463-464
12曹桂范,王红,苏禄辉.女性慢性生殖道炎症患者血清抗精子抗体的检测分析.中国优生与遗传杂志,2005,13(1):113-114
13邓超干,沈彦珍,林小丹.女性不孕与支原体、衣原体感染和抗精子抗体的相关性.中国优生与遗传杂志, 2005,13(10):122-130
14王铮,黄国香,胡边等.不孕不育患者抗精子抗体的临床研究.中国优生与遗传学杂志,2006,14(8):97-98
15陈志刚.不孕不育患者1200例血清抗精子抗体分析.临床和实验医学杂志,2007,6(10):37-38