不育症患者精子肌酸激酶活性及其同工酶测定

背景:作为胞浆中的一种生化标志物,人精子中肌酸激酶活性增高与精浆残余量和异常功能精子比例二者有关。它即是成熟精子的标志,又与受精能力相关。目的:观察精子肌酸激酶活性及精子M-肌酸激酶、B-肌酸激酶同工酶的相对含量在健康生育男性和精子健康男性精子中的差异,评价其在男性精子健康临床诊断中的意义。设计:病例-对照观察。单位:四川省计划生育科学研究所生化室。对象:于1999-01/2000-10选择四川省计划生育科学研究所专科门诊收治男性精子健康患者94例,已排除女方不孕原因和无精子症,平均年龄31岁。精子计数>2×1010L-1的精子健康男性80例,为精子正常组,精子计数<2×1010L-1的精子健康男性患者14例,为少精组。同期精液常规检查完全健康且已有后代的健康男性供精者27例,为健康对照组。方法:所有对象禁欲3～5d,用手淫法采集精液,经37℃液化后进行精液常规检查。酶动力学分光光度法测定精子肌酸激酶总活性,琼脂糖电泳凝胶光密度扫描测定肌酸激酶的同工酶相对含量。主要观察指标:精子计数,精子存活率,运动精子百分比,精子肌酸激酶总活性和M-肌酸激酶及B-肌酸激酶同工酶的相对含量。结果:纳入患者94例和健康对照者27例,均进入结果分析。①少精组的精子计数,精子存活率和运动精子百分比(Ⅱ Ⅲ,WHO分类法)均显著低于健康对照组,精子正常组除精子计数外,其他指标也显著低于健康对照组健康对照组、精子正常组、少精组精子计数分别为(6.05±0.81)×1010L-1,(7.76±1.37)×1010L-1,(1.46±0.19)×1010L-1,P<0.01;健康对照组、精子正常组、少精组精子存活率分别为85.1%,56.8%,58.2%,P<0.01;健康对照组、精子正常组、少精组运动精子百分比(Ⅱ Ⅲ)分别为62.9%,34.6%,29.5%,P<0.01]。②少精组肌酸激酶活性显著高于健康对照组(少精组、健康对照组每1×108精子中肌酸激酶活性分别为(9.000±6.117),(1.933±0.943)kat,P<0.05),精子正常组每1×108精子中肌酸激酶活性为(2.800±0.862)kat,虽略高于健康对照组,但两者比较差异无显著性(P>0.05)。③精子正常组和少精组M-肌酸激酶同工酶含量都显著减少(健康对照组、精子正常组、少精组分别为30.5%,19.0%,18.0%,P<0.05),提示两组患者的精子分化程度与健康对照组精子分化程度可能有明显不同。结论:精子肌酸激酶活性测定对少精子症的诊断和病因分析具有一定的意义。精子M-肌酸激酶同工酶相对含量在不同精子健康男性群体中的分布规律及其在男性精子健康诊断中的作用有待更深入的研究。

Determination of sperm creatine kinase and its isozyme in infertile patients

BACKGROUND: As a biochemical marker of cytoplasm, the increased activity of creatine kinase (CK) in human spermatozoa is correlated with both the residual cytoplasma and the ratio of sperm with abnormal func tion. It is a marker of mature sperm and associated with the potential of in semination. OBJECTIVE: To investigate the differences of CK activity in sperm and the relative contents of sperm CK-MM and CK-BB isoenzymes between normal fertile males and infertile males, and evaluate its significance in clinical diagnosis of male infertility. DESIGN: Case-control observation. SETTING: Laboratory of Biochemistry, Family Planning Research Insti tute of Sichuan Province. PARTICIPANTS: Ninety-four male infertile patients between January 1999 and October 2000 were selected from the Department of Family Planning Sciences of Sichuan Province, who had no aspermatism with their wives proved to be fertile. The average age of subjects were 31 years. Eighty subjects with the sperm count ＞ 2×1010 L-1 were taken as normal sperm group and 14 subjects with the sperm counts ＜ 2×1010 L-1 were considered as oligospermia group. Semen obtained from 27 healthy males who were normal in routine examinations and with children was taken as the healthy control group.METHODS: Semen sample collected by masturbation after abstinence of 3 to 5 days was incubated at 37 ℃ for liquefication and routinely analyzed.Total activity of CK in sperm was determined by using a kinetic spectrophotometry and the relative contents of CK isozyme was determined by agarose gel electrophoresis followed by density scanning of CK isozyme.MAIN OUTCOME MEASURES: Sperm counts, percentages of viability and motility of sperm, total CK activity and the relative contents of CK-MM and CK-BB isozyme in spermatozoa.RESULTS: A total of 94 enrolled patients and 27 normal controls were involved in the analysis of results. ①Sperm counts, percentage of viability and motility in oligospermia group ( Ⅱ + Ⅲ, WHO method) were obviously lower than those in the healthy control group, and those in the normal sperm group, except the sperm counts, were remarkably lower than the healthy control group the sperm counts in healthy control group, normal sperm group and oligospermia group was (6.05 ±0.81 )×1010 L-1, (7.76±1.37)×1010 L-1 and (1.46±0.19)×1010 L-1 respectively (P ＜ 0.01). The survival rate in healthy control group, normal sperm group and oligospermia group was 85.1%,56.8% and 58.2%, P ＜ 0.01, and the sperm motility was 62.9% ,34.6% and 29.5% respectively, P ＜ 0.01].②Total activity of sperm CK in oligospermia group was significantly higher than the healthy control group respectively (9.000±6.117) and (1.933 ±0.943) kat/108 sperm,P＜ 0.05],although that in the normal sperm group (2.800±0.862) kat/108 sperm was a little higher than the healthy control group, while there were no significant difference between the two groups (P ＞ 0.05).③The relative contents of sperm CK-MM isozyme in the normal sperm group and oligospermia group were obviously decreased (that in the healthy control group, normal sperm group and oligospermia group was 30.5%, 19.0% and 18.0% respectively,P ＜ 0.05), which implied a remarkable difference in sperm differentiation between healthy control group and the latter two groups.CONCLUSION: The determination of sperm CK is meaningful for the diagnosis and pathogenesis of oligospermia. More work should be done on the distribution of sperm CK-MM isozyme in different infertile population as well as its importance in the diagnosis of infertility.