整合素配体玻连蛋白在人精子的表达及其与受精的关系

目的 :进一步研究整合素配体玻连蛋白 (Vn)在人精子的表达及其与受精的关系。方法 :选用 1 4例生育力正常的成年男性及 8例精液常规分析正常的不明原因不育男性患者的精液标本 ,液化后提取上游精子。精子体外获能后用兔抗人 Vn多克隆抗体及羊抗兔 Ig G-FITC行免疫染色。然后用流式细胞仪计数 Vn表达阳性精子百分数。部分获能精子同时与去透明带金黄地鼠卵行异种体外受精 (SPA)以检测其受精力 ,比较两组获能精子表面Vn表达阳性精子百分率及受精率差异并分析受精率与 Vn表达阳性的获能精子百分数之间的相关性。结果 :生育组与不育组获能精子 Vn表达水平分别为 2 1 .2 4± 1 1 .70 %与3.6 4± 3.2 7% ,不育组明显低于生育组 (P<0 .0 5)。生育组受精率大于 1 0 % ,不育组受精率小于 1 0 % ,符合划分生育力正常与异常的标准。所有标本 Vn表达阳性的获能精子百分率与精子受精率间具有相关性 (r=0 .476 )。结论 :人获能精子表面存在一定水平的整合素配体玻连蛋白表达 ;Vn参与受精过程 ;Vn表达异常可能与一些不明原因的不育有关     

男性不育症患者外周血白细胞雄激素受体的改变及意义

目的：探讨男性不育与外周血白细胞雄激素受体表达的关系。方法：根据精液分析及睾丸活检病理检查结果,将67例男性不育症患者分成少精子症组(n=21)、弱精子症组(n=15)、阻塞性无精子症组(n=14)和非阻塞性无精子症组(n=17),采用放射配体结合分析法检测不育症患者外周血白细胞雄激素受体(AR),同时采用放射免疫法检测血清睾酮(T)和雌二醇(E_2)水平,并以22例正常生育男性为对照。结果：外周血白细胞AR含量少精子症组、弱精子症组、阻塞性无精子症组与对照组比,均无统计学差异(P＞0．05)；非阻塞性无精子症组(782±98)与对照组(913±104)相比,差异有统计学意义(P＜0．01)。男性不育症各组与对照组相比,E_2和T水平差异均无统计学意义(P＞0．05)；而男性不育症患者精子密度与白细胞AR含量呈正相关(r=0．233,P=0．010)。结论：一部分男子不育的发生、发展过程可能与AR表达下调有密切关系。     

整合素亚基α_5、β_1在人精子的表达及其与受精的关系

目的 :研究精子表面整合素亚基 α5、β1的表达及其与精子功能状态和受精力的关系 ;整合素亚基 α5、β1的表达与不明原因不育症的关系。方法 :1 3例生育力正常男性的精液标本 ,9例临床诊断为不明原因不育症患者的精液标本。以精子穿透去透明带的金黄地鼠卵行体外受精试验 (SPA)检测精子受精力 ;对新鲜、获能和孕酮诱导顶体反应后精子行间接免疫荧光染色 ,流式细胞仪检测精子表面整合素亚基α5、β1表达阳性的精子百分率。以三色法染色 ,观察精子顶体反应的发生率。结果 :流式细胞仪检测显示正常组新鲜精子表面α5亚基的表达 (1 3 .3± 5 .4% )与对照组 (1 0 .7± 6 .4% )无显著差异 (P>0 .0 5 )。获能组精子表面 α5亚基的表达率 (6 5 .7± 1 7.6 % )比新鲜组和对照组显著增高 (P<0 .0 5 )。孕酮诱导顶体反应后 ,α5亚基的表达率 (6 5 .3± 1 8.9% )比新鲜组和对照组显著增高 (P<0 .0 5 ) ,但与获能精子组相比无显著差异 (P>0 .0 5 )。β1亚基的表达率分别为新鲜组1 2 .9± 7.4% ,获能组 1 5 .9± 7.9% ,诱导顶体反应组 1 6 .9± 6 .2 % ,与对照组 (分别为 :9.3± 3 .4% ,1 7.3± 7.8% ,1 2 .2± 8.7% )比较均无显著差异 (P>0 .0 5 )。获能精子表面α5亚基的阳性表达率与受精率有一定的线性相关 (r=0 .     

不育男性精浆左旋肉碱浓度与精子活力和浓度的关系

目的:探讨精浆左旋肉碱测定在男性不育症诊疗中的临床意义。方法:按照第5版《世界卫生组织人类精液检查与处理实验室手册》的参考值,将不育男性按照精液常规分析结果分为精子活力正常组(前向运动精子百分率≥32%)(n=283)和弱精子症组(前向运动精子百分率32%)(n=892)。通过比色法检测精浆中的游离左旋肉碱含量,分析左旋肉碱浓度与精子活力、精液浓度的相关性。通过受试者操作特征分析曲线(receiver operating characteristic curve,ROC curve)确定左旋肉碱浓度的阈值,以阈值为分界点,将弱精子症组患者分为高于左旋肉碱阈值组和低于左旋肉碱阈值组,分析左旋肉碱与精子活力和精子浓度的相关性。结果:弱精子症组的精浆左旋肉碱浓度(384.14±188.81μmol/L)显著低于精子活力正常组(434.04±171.77μmol/L,P0.05)。精子活力正常组和弱精子症组精浆中的左旋肉碱含量与前向运动精子百分率和精子浓度的相关性极低或不相关(r0.2)。肉毒碱检测的ROC曲线下面积(AUC)为0.592,阈值为380.9,低于左旋肉碱阈值弱精子症组与前向运动精子百分率有较弱的正相关关系(r=0.329,P=0.000)。结论:对于拟行辅助生育的不育男性患者,精浆中左旋肉碱浓度作为精浆生化的指标之一,可能对弱精子症患者有一定的临床参考意义。     

生育与不育男性生育力指数的比较

目的:评估生育力指数(FI)在判断男性生育能力中的作用。方法:对不育组(n=124)和生育组(n=62)进行精液常规检查,并计算FI[FI=精子密度(106/ml)×精子活动力×精子正常形态率]。结果:生育组FI为13.23(24.16),高于不育组的5.69(10.62)(t=5.657,P=0.001)。生育组FI(P2.5,P97.5)的范围为2.06-56.85。结论:FI较单个精液参数更能客观反映男性生育能力,当FI<2.0时男性生育概率将下降。     

精子双链DNA与习惯性流产相关性研究

目的:探讨精子双链DNA(dsDNA)与习惯性流产(RA)的相关性。方法:109例其妻子发生不明原因的自然流产≥3次的男性(研究组),平均年龄28.7±0.1岁;60例为妻子正常生育的健康男性志愿者(对照组),平均年龄26.7±0.2岁。采用吖啶橙染色法在倒置荧光显微镜下计算精子双链DNA百分率。结果:研究组中双链DNA≥66%有87例(79.82%);55±10%有6例(5.50%);45±10%有12例(11.00%);35±10%有2例(1.83%);<25%有2例(1.83%);对照组:60例,均为双链DNA≥66%者;经χ2检验研究组精子双链DNA百分率较对照组明显降低,具统计学差异(P<0.01);经Pearson相关系数分析精子双链DNA百分率与习惯性流产发生次数呈明显负相关性(r=-0.510,P<0.01)。结论:精子双链DNA降低可能与配偶习惯性流产的发生具有相关性。     

精子质膜磷脂酰丝氨酸外翻与线粒体膜电位的关系

目的:探讨精子早期凋亡过程中质膜磷脂酰丝氨酸(PS)外翻与线粒体膜电位丧失的相关性。方法:42例精液标本用AnnexinV/PI和Rh123/PI双染法染色后,用流式细胞术(FCM)分析。结果:AnnexinV/PI染色法中,与正常生育组比,不育组活精子和死精子百分率均存在统计学差异(P=0.001,P=0.003),凋亡精子百分率无统计学差异。Rh123/PI染色法中,与正常组相比,线粒体膜电位正常精子百分率和死精子百分率均存在统计学差异(P=0.000,P=0.001),线粒体膜电位丧失精子百分率差异无显著性(P=0.120)。两组中凋亡精子百分率与线粒体膜电位丧失精子百分率间均呈正相关。结论:精子存在质膜PS外翻和线粒体膜功能紊乱,且两者间存在相关性。     

来曲唑对EM大鼠P450arom mRNA及COX-2mRNA表达的影响

目的:探讨来曲唑对大鼠EM模型子宫内膜异位病灶中P450arom mRNA、COX-2 mRNA表达的影响及其治疗EM的作用机制。方法:参照Jones法用自体移植建立EM大鼠模型,随机将建模成功的大鼠分为来曲唑组及盐水对照组,比较治疗前后EM大鼠异位病灶体积的变化。用RT-PCR法检测各组大鼠异位病灶中P450arom mRNA及COX-2 mRNA的表达。结果:来曲唑组异位病灶体积明显缩小或萎缩,与对照组差异有统计学意义(P<0.05)。来曲唑治疗后P450arom mRNA、COX-2 mRNA在异位内膜中表达降低,与对照组比较均有统计学差异(P<0.05),且两者变化有相关性(r=0.918,P=0.001)。结论:来曲唑对大鼠异位病灶体积的影响明显,短时间内就有明显效果,可能通过降调P450arom mRNA、COX-2 mRNA在异位内膜组织中的表达起作用。     

解脲脲原体感染与精子DNA完整性的相关性的研究

目的:探讨不育男性解脲脲原体(Uu)感染与精子核DNA完整性及精液各参数的相关性。方法:95例不育症患者的精液用Uu培养液进行解脲脲原体的培养,按WHO的标准,用计算机辅助精液分析系统(CASA)进行精液各参数的分析,然后通过精子低渗肿胀实验,吖啶橙(AO)荧光染色检测精子膜的完整性和精子核DNA的完整性。结果:Uu阳性40例,Uu阴性55例,Uu阳性组与阴性组比较,精子核DNA完整性与精子的密度、a+b级精子百分率以及精子的肿胀率都有显著性差异(P<0.01,P<0.05)。同时Uu阳性组与阴性组的精子核DNA完整性与精子的肿胀率存在显著的正相关性(r=0.438,P=0.014;r=0.438,P=0.01)。结论:Uu感染能够降低精子膜的完整性以及精子核DNA的完整性,这可能是Uu致男性不育的又一因素。     

性表达上调雌性大鼠乳腺组织P450arom的表达

目的:研究性表达对成年雌性大鼠乳腺组织芳香化酶(P450arom)表达的影响。方法:12只成年雌性SD大鼠随机分为禁欲组和性表达组。光镜下观察各组乳腺组织结构,采用放射免疫法检测外周血及乳腺组织中雌激素水平,采用免疫组织化学技术和Western blotting技术检测乳腺组织P450arom表达变化。结果:与性表达组比较,禁欲60d后,成年雌性大鼠乳腺组织雌激素水平下降(P<0.05),同时乳腺组织P450arom表达也下调(P<0.01)。各组乳腺组织结构光镜下未见明显异常。结论:性表达可能通过上调乳腺组织P450arom表达引起乳腺组织雌激素水平升高。     

Testing the fertilizing ability of motile spermatozoa separated by Percoll in patients with abnormal sperm analysis or sperm penetration

The Percoll density-gradient technique was used to select motile sperm from men who exhibited abnormal semen analysis or unexplained infertility. The fertilizing ability of the spermatozoa was evaluated by the hamster ova sperm penetration assay (SPA). The current work is a novel approach in the usage of Percoll in an attempt to improve the quality of the sperm of nonfertile patients and to examine its results by SPA. The method succeeded in improving the penetration score of the fertile controls by 15% to 30%. However, no improvement of the low penetration score of the nonfertile patients was demonstrable. The difficulties inherent in using the Percoll density gradient technique are discussed.     

Sperm acrosin activity and fluorescence microscopic assessment of proacrosin/acrosin in ejaculates of infertile and fertile men.

OBJECTIVE: To compare biochemically active with immunoreactive sperm acrosin in fertile and infertile men. SETTING: This study was conducted in a tertiary care center, the Andrology Clinic, Department of Internal Medicine, University of L'Aquila. PATIENTS: We evaluated the males in 40 infertile couples with no recognized cause of female infertility and 20 fertile men. INTERVENTIONS: Ejaculates were collected under standardized conditions of abstinence. MAIN OUTCOME MEASURES: Total sperm acrosin activity was measured on a spectrophotometer in washed sperm stored at -80 degrees C for 1 to 6 days. The percent of spermatozoa immunostained by an antiserum against proacrosin/acrosin by indirect immunofluorescence (IFL) was determined on methanol fixed sperm smears. RESULTS: Biochemically active acrosin was correlated to immunoreactive acrosin (P = 0.0028), and both were inversely correlated to the percent of spermatozoa with an abnormal head (P = 0.00024 for acrosin activity and P = 0.0013 for IFL). Biochemically active and immunoreactive acrosin were lower in infertile compared with fertile men (P = 0.0012 and P = 0.0009, respectively). Sixty-eight percent of ejaculates with an acrosin activity lower than the limit value observed in fertile men showed a normal sperm morphology and a normal immunoreactivity for acrosin. CONCLUSIONS: A low sperm acrosin activity in teratospermic ejaculates is because of a lack or a defect of the immunogenic and functional domains of the protein. A low sperm acrosin in infertile men with normal semen parameters results from a possible functional defect of the enzyme that is immunohistochemically detected in spermatozoa.     

Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization.

OBJECTIVES: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). DESIGN: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. PATIENTS, PARTICIPANTS: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. RESULTS: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71%. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. CONCLUSIONS: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm.     

Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men

OBJECTIVE: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters. DESIGN: Prospective, observational study. SETTING: University infertility clinic.Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy. Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF. MAIN OUTCOME MEASURE(S): Sperm DD and DF in fertile and infertile men. RESULT(S): The mean (+/-SE) rates of DD and DF were significantly higher in infertile subjects compared to fertile controls, respectively: 25.4 +/- 3.0 vs. 10.2 +/- 2.3 (P=.028) and 27.6 +/- 2.5 vs. 13.3 +/- 2.5% (P=.016). DF and DD correlated strongly (r = 0.71, P<.0001). Also, DD and DF correlated negatively with standard semen parameters (concentration, motility, and morphology), the strongest correlation being with sperm motility. CONCLUSION(S): The strong correlation between sperm DD and DF, and the higher levels of sperm DNA damage in infertile compared with fertile men, indicate that male infertility is associated with poor sperm DNA integrity. Although infertile men may father children with assisted conception, fertilization with DNA-damaged spermatozoa may increase the risk of genetic disease in the offspring.     

The relationship between the human sperm hypoosmotic swelling test, routine semen analysis, and the human sperm zona-free hamster ovum penetration assay

The functional integrity of sperm membranes of 270 semen samples collected from fertile men and the male partners in couples with infertile marriages was assessed by the hypoosmotic swelling test and the results correlated with routine semen analysis and the human sperm zona-free hamster ovum penetration assay. Semen samples with abnormal semen parameters had lower values of percentage of swollen sperm after hypoosmotic treatment in comparison with those with normal semen parameters. A weak positive correlation was observed between sperm swelling and sperm morphologic features (r = 0.32, P less than 0.05) and between sperm swelling and sperm motility (r = 0.22, P less than 0.05). Insignificant correlation was observed between sperm swelling and in vitro sperm fertilizing capacity, as assessed by the zona-free hamster ovum penetration assay. The results indicate that the sperm swelling test and the zona-free hamster ovum penetration assay are evaluating different functional qualities of sperm that are apparently not associated with each other.     

Relationship between Fertilizing Ability of Ejaculated Human Spermatozoa and Its Chromatin Heterogeneity

OBJECTIVE: The aim of this study was to examine the relationship between the fertilizing ability of ejaculated human sperm and its chromatin heterogeneity. METHOD: We used the D-AO staining method (acridine orange epifluorescence accompanied by diamide, a thiol oxidizing agent) to analyze the sperm chromatin structure of infertile patients with IVF-ET treatment. SDS-PAGE was performed to analyze the sperm nuclear proteins collected from patients with immature sperm (stained red with D-AO staining) and proven fertile men. RESULTS: 1. It was suggested that D-AO staining allowed the immature sperm to be divided into two groups. One was immature sperm, which had disturbance of S-S formation in the epididymides, and the other was sperm that which had an abnormal exchange process of nuclear proteins in the testes. 2. The stainability after D-AO staining showed no correlation with the findings of semen analysis. 3. The fertilization rate of IVF-ET was significantly correlated to the percentage of green sperm staining with D-AO staining. 4. In the pregnant group after IVF-ET, it was noticed that sperm of the green type with D-AO staining was increased in comparison with the non-pregnancy group. 5. The fertilization rate in the group of the sperm stained red with D-AO staining was increased to 73.5% by ICSI. 6. Definite differences were noticed between the protein components of the patients with immature sperm and those of the proven fertile men by analysing with SDS-PAGE. CONCLUSION: D-AO staining was an efficient method for evaluating the fertilizing ability of human ejaculated sperm, and to determine an appropriate ART tool such as ICSI.     

Levels of cholesterol and phospholipids in freshly ejaculated sperm and Percoll-gradient-pelletted sperm from fertile and unexplained infertile men

Cholesterol and phospholipid levels were determined in individual sperm samples obtained from 20 fertile and 20 unexplained infertile men. The determination was performed on both washed freshly ejaculated sperm and Percoll-gradient-pelletted sperm. Although sperm cholesterol levels in unexplained infertile patients were significantly lower, i.e., 10.6 +/- 1.3 (mean +/- SD) nmol/10(7) freshly ejaculated sperm and 5.4 +/- 0.7 nmol/10(7) Percoll-gradient-pelletted sperm as compared with 19.9 +/- 1.9 nmol/10(7) and 12.6 +/- 1.5 nmol/10(7) for corresponding sperm populations in fertile donors. Motility parameters measured in 10 sperm samples of the two groups of fertile and unexplained infertile men revealed increases in the amplitude of lateral head displacement and decreases in percent of straightness in sperm tracks from unexplained infertile men.     

Cytotoxic sperm antibodies inhibit sperm penetration of zona-free hamster eggs

Zona-free hamster egg sperm penetration assay was used to study the effects of cytotoxic sperm antibodies on egg penetration by the sperm of fertile and infertile men. Twenty-nine fertile and 9 infertile men did not have significant cytotoxic sperm antibodies in their serum and seminal plasma; 7 infertile men were positive for these antibodies in serum and seminal plasma. Two others were positive in sera, and 14 were positive in seminal plasma. Sperm from 18 of 23 (78%) infertile men with sperm antibodies had poor egg penetration (less than or equal to 20%) compared with only 6 of 38 (16%) nonautoimmune men (P less than 0.0001). Sperm from nonautoimmune fertile men were coated with seminal plasma and serum of autoimmune men and serum of isoimmune women, resulting in a significant decrease in hamster egg penetration. Sixteen of 21 (76%) seminal plasma samples with cytotoxic sperm antibodies reduced the control sperm penetration of hamster eggs by greater than or equal to 50%. Coating of sperm from fertile men with serum and seminal plasma samples from non-sperm-immune fertile and infertile subjects did not alter their penetration of hamster eggs. Coating of sperm from autoimmune men with cytotoxic antibody-positive autologous seminal plasma samples resulted in a significant decrease of egg penetration. The inhibitory effect of antibody-positive seminal plasma samples on egg penetration by control sperm was abrogated when the samples were preabsorbed with sperm. It is concluded that cytotoxic sperm antibodies, especially those in seminal plasma, inhibit hamster egg penetration by autologous and control sperm. This may explain in part the incidence of infertility associated with sperm antibodies.