用顶体反应释放的顶体酶活性评价顶体反应功能的研究

目的:研究用顶体反应释放的顶体酶活性评价人类精子顶体反应功能。方法:10例健康生育男子精液,采用Percoll密度梯度分离法优选精子,用FITC-PSA荧光染色法分析顶体反应(acrosome reaction,AR),顶体酶活性采用分光光度比色法测定。结果:A23187和孕酮诱导的精子AR释放的顶体酶活性和顶体反应率均明显增加(P<0.05)。随A23187浓度增加,AR释放的顶体酶活性和顶体反应率均明显增加(P<0.01)。A23187诱导AR释放的顶体酶活性与相应顶体反应率呈明显正相关关系(r=0.950,P<0.001)。随孕酮浓度增加,AR释放的顶体酶活性和顶体反应率均明显增加(P<0.05)。孕酮诱导的AR释放的顶体酶活性与相应顶体反应率亦呈明显正相关关系(r=0.921,P<0.001)。结论:孕酮可诱导人精子顶体反应,用顶体反应释放的顶体酶活性可评价人精子顶体反应功能。     

人精子顶体反应检测临床应用价值探讨

[目的]检测Ca2 载体A23187诱导精子顶体反应(AR)率,探讨其临床应用价值。[方法]对51例患者检测A23187诱导精子AR率,分析其与精液常规分析、宫腔内人工授精(IUI)、体外受精(IVF)、单精子卵泡浆内注射(ICSI)结果的关系。[结果]①精液常规分析对诱导AR率的相关性不显著;②诱导AR率低于18.1%理论值时,IVF受精率理论值为0;③3次IUI失败者AR率较受精正常者降低(P<0.05)。④不明原因不育者精子AR率与IVF受精率、卵裂率关系呈正相关(r=0.89,P<0.05),与ICSI受精率、卵裂率无显著关系;用ICSI治疗,其受精率、妊娠率高于IVF治疗组。⑤IVF受精不良病例AR率低,用ICSI治疗后受精率、优质胚胎率升高(P<0.05)。[结论]Ca2 载体A23187诱导顶体反应能预测精子的受精能力,有助于男性不育的临床诊断与治疗。     

精子顶体反应率与精液相关参数之间关系的研究

目的研究精子顶体反应率与精液相关参数之间的关系,探讨精子顶体反应对男性不育的影响。方法收集2012年10月至2013年4月间在广东省妇幼保健院生殖中心就诊的180例男性患者的精液标本,采用钙离子载体A23187诱导精子发生顶体反应(AR),按照顶体反应值,分AR>3%、AR≤3%两组,对AR与各项精液参数进行相关性分析。结果两组间精子活动力、精液量、精子密度、精子总数无显著差异(P>0.05),而精子正常形态率与精浆抗精子抗体(antisperm antibody,AsAb)差异显著(P<0.05)。结论精子顶体反应与精子质量有关,是相对独立的男性不育因素,是男性生育能力评估的重要参考指标。     

优选和冻融对精子顶体反应及顶体反应释放的顶体酶活性影响

目的:研究优选和冻融对人精子顶体反应(acrosome reaction,AR)和AR释放的顶体酶活性影响。方法:10例健康生育男子精液,用Percoll密度梯度分离法和上游法优选精子;用3种冷冻保护剂进行冷冻;用孕酮诱导AR;采用FITC-PSA荧光染色法分析AR;采用分光光度比色法测定顶体酶活性。结果:Percoll法和上游法优选后,顶体反应率和AR释放的顶体酶活性均较优选前显著升高(P<0·05),但Percoll法和上游法优选组间比较差异不显著(P>0·05)。精子冻融后甘油组和GYC组顶体反应率和AR释放的顶体酶活性均下降,与冷冻前比较差异均有显著性(P<0·01),GYCG组与冻融前组比较,差异不显著(P>0·05)。结论:优选后顶体反应率及AR释放的顶体酶活性增加,GYCG对精子顶体反应和AR释放的顶体酶活性有保护作用。     

局部注射甲氨喋呤配伍中药治疗输卵管妊娠的临床观察

目的:寻找一种能提高生育力,减少并发症,无毒副反应,简便、无损伤、药物用量小的输卵管妊娠的药物治疗方案。方法:对86例未破裂或符合条件的流产型输卵管妊娠病人,按治疗方案不同分两组进行对照研究,A组彩色阴道超声引导局部注射MTX治疗,B组彩色阴道超声引导局部注射MTX+中药联合治疗。结果:A、B两组治愈率分别为96.8%、98.1%,无显著性差异(P>0.005);A、B两组的生殖状态(宫内妊娠和足月活产)85.7%、97.8%,有显著性差异(P<0.005);A、B两组的并发症(再次输卵管妊娠)分别为7.2%、2.2%,有显著性差异(P<0.005);血绒毛膜促性腺激素(β-HCG)B组均比A组下降明显(P<0.005);病灶包块缩小或消失天数B组均比A组明显缩短(P<0.005);输卵管通畅率B组明显高于A组(P<0.05)。结论:彩色阴道超声引导局部注射MTX+中药联合治疗未破裂或符合保守治疗的流产型输卵管妊娠具有方法简便、疗效高、无毒副反应、保持优良的生殖状态和减少并发症等优点,值得临床推广应用。     

精索静脉曲张对精子形态的影响

目的:探讨精索静脉曲张对精子形态的影响。方法:分析71例已有生育男性和417例男性不育患者精液样本,其中不育患者分为精索静脉曲张组Ⅰ°(130例)、Ⅱ°(64例)、Ⅲ°(88例)和无精索静脉曲张组(135例)4组。采用精子形态检测系统下人工修正方法进行精子形态分析。结果:精索静脉曲张Ⅱ°和Ⅲ°组正常形态精子百分率均显著低于生育组(P<0.05,P<0.001),但两组之间差异没有显著性(P>0.05)。精索静脉曲张Ⅰ°组正常形态精子百分率与生育组之间差异无显著性(P>0.05)。精索静脉曲张不育组正常形态精子百分率显著低于生育组和无精索静脉曲张组(P<0.005,P<0.05),梨形头精子、尾部/中部缺陷精子百分率显著高于生育组(P<0.05,P<0.005),大头精子、颈部/中段缺陷精子和尾部缺陷精子百分率显著高于无精索静脉曲张组(P<0.05),而其它畸形精子百分率显著低于无精索静脉曲张组(P<0.005)。结论:精索静脉曲张能够影响精子形态,Ⅱ°和Ⅲ°能导致正常形态精子百分率下降。     

米非司酮在异位妊娠药物治疗中的作用及孕酮在其监测中的意义探讨

目的:探讨不同剂量米非司酮和甲氨喋呤(MTX)联用治疗异位妊娠的效果以及血清孕酮(P)检测在其治疗过程中的价值。方法:选择适合药物治疗的异位妊娠患者79例随机分为3组,A组25例:MTX40mg/m2单次肌肉注射;B组34例:MTX用法同A组加米非司酮150mg口服;C组20例:MTX用法同A组加米非司酮300mg口服。治疗过程中监测B超、血清β-HCG和孕酮(P)。结果:3组治疗成功率、平均住院日等方面均无显著性差异(P>0.05),检测血清β-HCG和孕酮发现孕酮(P)<5ng/ml所需时间明显短于平均住院日(P<0.05),血清β-HCG下降正常所需时间与孕酮(P)<1.5ng/ml所需时间相比明显延长,差异有显著性意义(P<0.05)。结论:米非司酮治疗异位妊娠作用不肯定,一定范围内剂量大小无明显意义,而孕酮在其安全性监测方面有临床价值,值得推广应用。     

二硫化碳吸入染毒对雄性大鼠生殖功能及子代影响的研究

目的探讨二硫化碳对雄性大鼠生殖功能及子代的影响。方法选用健康雄性Wistar大鼠40只,随机分为4组,以不同浓度二硫化碳(0、50、250、1250mgm3)静式吸入染毒,共10周。染毒结束前1周随机选对照、低、高浓度组各5只按经典致畸试验与健康雌鼠1∶2合笼交配,观察雌鼠受孕率、计流产数、吸收胎、活胎总数、每窝平均活胎数及称胎鼠体重,检查胎鼠外观及内脏、骨骼畸形,测量身长、尾长、腹围、肛殖距等;染毒结束后,称染毒大鼠体重及各脏器重、计算系数,测量睾丸横径,计附睾尾精子总数、精子活动率及分级、精子畸形率等。结果实验组雌鼠受孕率均低于对照组,但差异无显著性(P>0.05);高浓度组胎鼠各生长发育指标明显低于对照组(P<0.05或P<0.01);染毒大鼠体重、各脏器系数均低于对照组,但只有高浓度组大脑脏器系数与对照组比较差异有显著性(P<0.05);实验组睾丸脏器系数有随染毒剂量增加而降低的趋势,但只有高浓度组与对照组比较有差异(P<0.05);附睾重随染毒剂量增加而减轻,中、高浓度组与对照组比较差异分别有显著性(P<0.05)和极显著性(P<0.01),附睾尾精子总数及精子活动率染毒组均低于对照组,中、高浓度组与对照组比较差异分别有显著性(P<0.05)和极显著性(P<0.01),活动精子分级以原地运动为主。结论二硫化碳染毒对雄性大鼠生殖功能及子代有一定影响,可导致雌鼠受孕率降低、流产率升高,高浓度组胎鼠生长发育迟缓和畸形率增高,可能与雄性大鼠精子质、量下降有关。     

氟对雄性大鼠下丘脑-垂体-性腺轴内分泌干扰作用的实验研究

目的探讨氟对雄性大鼠下丘脑-垂体-性腺轴生殖内分泌干扰作用。方法选用4～5周龄雄性Wister大鼠36只,体重60～70g。随机分为Ⅰ高氟组(水氟100mg/L)、Ⅱ低氟组(水氟30mg/l)、Ⅲ对照组(纯净水)。8周后处死动物,做精子质量分析,并用放免法测定血清下丘脑、垂体、性腺三个层面生殖激素水平。结果与对照组比较,高氟组动物体重、睾丸及附睾重量均明显下降(P<0.05),低氟组动物体重、睾丸及附睾重量均明显升高(P<0.05);高氟组、低氟组动物右侧睾丸、附睾脏器系数均呈现下降趋势,但差别无统计学意义(P>0.05)。高氟组和低氟组与对照组比较精子密度、精子活率明显下降(P<0.05);高氟组亦明显低于低氟组(P<0.05)。与对照组比较,高氟组精子畸形率明显升高(P<0.05);低氟组精子畸形率有上升趋势,但差异无统计学意义(P>0.05)。对畸形精子进行分析,发现三组动物精子畸形类型有所不同,高氟组体部及混合畸形比例明显升高(P<0.05)。畸形指数高氟组为1.56,低氟组为1.42。大鼠血清促性腺激素释放激素(GnRH)浓度均值高氟组、低氟组均明显高于对照组(P<0.05);高氟组亦明显高于低氟组(P<0.05)。卵泡刺激素(FSH)浓度均值高氟组、低氟组均明显高于对照组(P<0.05);高氟组亦明显高于低氟组(P<0.05)。间质细胞刺激素(ICSH)浓度均值高氟组、低氟组分别低于对照组,但差异无显著性(P>0.05)。ICSH/FSH比值高氟组、低氟组均明显低于对照组(P<0.05);高氟组亦明显低于低氟组(P<0.05)。血清睾酮(T)均值高氟组明显低于对照组(P<0.05);亦明显低于低氟组(P<0.05)。血清雌二醇(E2)高氟组、低氟组均高于对照组(P<0.05);高氟组亦明显高于低氟组(P<0.05)。T/ICSH比值高氟组、低氟组均明显低于对照组(P<0.05);高氟组亦低于低氟组(P<0.05)。结论氟对雄性大鼠生殖系统的影响作用部位可能不在下丘脑;氟可能影响垂体分泌ICSH功能;T/ICSH更能反映睾丸间质细胞功能的早期变化。氟能影响下丘脑-垂体-性腺轴各层面血清生殖激素水平,因而具有生殖内分泌干扰作用。     

卵子激活剂A23187对睾丸精子卵胞浆内单精子显微注射后妊娠结局的影响

目的 评估卵子激活剂在临床中能否改善睾丸取精患者行卵胞浆内单精子显微注射(ICSI)后的妊娠结局。方法 本研究选取2014年1月至2020年4月期间在西北妇女儿童医院生殖中心睾丸取精术(TESA)后行ICSI的374对不孕夫妇作为研究对象,按照ICSI后是否采用卵子激活剂A23187行人工卵子激活术(AOA)随机分为卵子激活组(n=47)和常规对照组(n=327)。以女方年龄、促排卵方案、移植的胚胎类型、胚胎质量、胚胎数量作为匹配条件,按照1∶3进行倾向评分匹配后,对两组的实验室指标和妊娠结局进行比较。结果 匹配前,卵子激活组女方年龄较小,成熟卵子数较多,差异有统计学意义(t值分别为2.72、-2.56,P<0.05);匹配后,两组间基线资料均无显著性差异(P>0.05);匹配前,两组间在受精率、优质胚胎率、可用胚胎率和囊胚形成率之间无显著性差异(P>0.05);匹配后,卵子激活组的可用胚胎率低于对照组(χ²=47.68,P<0.05);采用Logistic回归模型评估卵子激活处理对临床妊娠率的独立作用,结果发现匹配后未调整模型、调整模型均...     

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin-fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3-8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.     

Effects of Genistein Isoflavone (4',5',7-Trihydroxyisoflavone) and Dexamethasone on Functional Characteristics of Spermatozoa

Caudal epididymal spermatozoa were used to study the influence of genistein isoflavone and dexamethasone (dxm) on the functional characteristics of spermatozoa. The effects of genistein alone and in combination with dxm on sperm motility, sperm morphology, spontaneous acrosome reaction (AcR), and ionophore A23187-induced AcR were investigated. The FITC-PSA/Hoechst 33258 staining procedure was used to assess sperm cell viability and AcR status and thus to differentiate between true AcR and acrosome degeneration. The overall results indicated that (1) lower doses of genistein alone, or in combination with dxm, did not significantly influence sperm motility or sperm morphology; (2) ionophore A23187 induced AcR in rat spermatozoa; (3) there appeared to be no direct correlation between sperm motility and AcR, (4) higher doses of genistein, alone or in combination with dxm, significantly interfered with percentage sperm motility and caused significant detachment of sperm heads but did not cause morphological defects; and (5) higher doses of genistein caused significant decrease in sperm acrosome reactivity with long duration of exposure. In view of the fact that sperm capacitation and AcR are physiological prerequisites for successful fertilization of oocytes, the findings suggest that chronic exposure of spermatozoa to high doses of genistein could be associated with infertility problems through suppression/inhibition of AcR and sperm motility. Dexamethasone did not appear to influence the effect of genistein on the functionality of postspermatogenic spermatozoa.     

A MODIFIED ACROSOME INDUCTION TEST

Many types of acrosome induction tests require special equipment and reagents that are not available to most clinicians; thus, simpler tests seem desirable. A modified acrosome induction test has been developed that uses basic reagents and a light microscope, which are available in most office settings. A hypoosmotic swelling test and a double stain (Bismark brown and rose Bengal) were combined to evaluate the viable acrosome reaction (AR) among 74 infertile men and 42 control men. The study included 34 infertile males without varicoceles, 20 with nonrepaired varicoceles and 20 with repaired varicoceles. On each test day, a specimen from a fertile donor was run as a control. The spontaneous acrosome reaction was recorded in semen before and after capacitation. The final % viable acrosome reaction equaled the capacitated value minus the spontaneous value for whole semen. The mean % viable AR among the control specimens was 16% with no values less than 10%. This mean value for controls was significantly greater than the mean % viable AR in each patient group. There were no overlaps in the 95% confidence intervals. When the study group was stratified according to normal acrosome induction tests or &gt;10% viable AR, 30 patients had a normal test and 44 had abnormal tests. Six patients with varicoceles and an abnormal acrosome induction test had a varicocelectomy, and 2 (33%) converted their acrosome induction test to normal after at least 6 months of follow-up. Nine patients had in vitro fertilization (IVF), 3 had a poor result, and all had an abnormal acrosome induction test. Six had a good result with IVF and all 6 had a normal acrosome induction test. Thus, the acrosome induction test described in this report may be performed in any office laboratory to detect subtle male factor problems. The results may be helpful for planning IVF, intracytoplasmic sperm injection, or varicocele surgery for infertile men.     

SPERM-IMMOBILIZING ANTIBODIES BLOCK CAPACITATION IN HUMAN SPERMATOZOA

Sperm-immobilizing antibodies block human fertilization by interfering with the acrosome reaction (AR). To clarify the mechanism of blockage of AR by sperm-immobilizing antibodies, the authors examined their effects on the increase of intracellular free Ca 2+ concentration induced by follicular fluids (Ca 2+ influx) in spermatozoa and on their capacitation. Sperm-immobilizing antibodies did not suppress Ca 2+ influx induced by follicular fluid, but they inhibited capacitation of human spermatozoa. Namely &#106 %AR (%AR after addition of an AR inducer - %AR before treatment) induced by progesterone was significantly ( p <. 0001) lower when spermatozoa were incubated in human tubal fluid medium cotaining antibody-positive serum (1.2%), compared to that when incubated in control medium (19.2%). Furthermore, the proportion of both spermatozoa that became capacitated and ones that had become capacitated decreased significantly ( p <. 0001) after 2, 4, and 6 h of incubation in medium containing antisperm antibody-positive serum, compared to those of spermatozoa incubated in control medium. In conclusion, sperm-immobilizing antibodies may be closely related to their blockage of capacitation.     

The in vitro effect of levonorgestrel on the acrosome reaction of human spermatozoa from fertile men

The objective of the study was to evaluate the effect of levonorgestrel (LNG) on the occurrence of acrosome reaction (AR) of capacitated spermatozoa from fertile men. A total of 20 semen samples from four fertile men were evaluated. The spermatozoa were separated by swim-up, and subsequently incubated for 20 h under capacitating conditions. Capacitated spermatozoa were exposed to three different concentrations of LNG (200, 400 and 800 ng/mL), follicular fluid (20% v/v), and ethanol or human tubal fluid medium (HTF) as a control. The AR rate and the ratio of live to dead spermatozoa were assessed after 15 and 30 min of incubation at 37 degrees C and 5% CO(2). The different treatments were compared with follicular fluid and HTF medium as positive and negative controls. The main results showed that the AR rate after 15 min of exposure was not affected by LNG and was significantly higher with follicular fluid than with all the other treatments. At 30 min of exposure, the three LNG concentrations induced a greater rate of AR than the HTF and a trend of higher AR rate with greater concentration was observed. Follicular fluid induced a significantly higher rate of AR than the other treatments. In conclusion, the addition of LNG in vitro to capacitated human spermatozoa is associated with a dose-dependent increased rate of AR, but such increase was not as great that induced by follicular fluid.     

Lifestyle and demographic factors associated with human semen quality and sperm function

In this study, we sought to investigate the effect of lifestyle and demographic factors on classic and functional semen parameters. Three hundred and twenty-eight subjects who underwent semen analysis were recruited. Routine SA, sperm vitality, acrosome reaction (AR) assay and sperm DNA fragmentation index (DFI) were analyzed. Demographic and lifestyle information, including (1) BMI, (2) current smoking and alcohol drinking frequency, (3) sleep habits, (4) daily fluid intake, (5) weekly meat intake, (6) sports frequency, (7) trouser cell phone use, (8) age, and (9) abstinence time, were collected. Generalized additive models were used to analyze the possible non-linear association. The results showed that total sperm count (TSC) was significantly associated with age (P = 0.001), abstinence time (P = 0.001) and daily coffee intake (P = 0.044). Semen volume was significantly associated with age (P < 0.001) and daily coffee intake (P < 0.001). Sperm concentration was significantly associated with abstinence time (P = 0.011) and average sleep duration (P = 0.010). Sperm motility was significantly associated with age (P = 0.002) and daily juice intake (P = 0.001). Total motile sperm count was significantly associated with age (P = 0.003) and abstinence time (P = 0.009). DFI was significantly associated with age (P = 0.002), irregular sleeping habit (P = 0.008) and abstinence time (P = 0.032). The percentage of AR sperm was significantly associated with daily juice intake (P = 0.013). In conclusion, DFI and TSC were the most sensitive semen parameters for demographic and lifestyle features, whereas age had more influence on semen parameters than other demographic and lifestyle features.

Abbreviations: BMI: body mass index; SA: semen analysis; AR: acrosome reaction; DFI: DNA fragmentation index; GAM: generalized additive model; TSC: total sperm count; TMC: total motile sperm count; IUI: intrauterine insemination; SCSA: sperm chromatin structure assay; SD: standard deviation; IQR: interquartile range; CBAVD: congenital bilateral absence of vas deferens; NEQAS: national external quality assessment service; HTF: human tubal fluid; HSA: human serum albumin.     

受精调控机制的研究进展

人类和哺乳动物的受精机制是近年来的研究热点.通过生物化学、转基因和基因敲除等研究得出的受精机制主要分为4个部分：①精子获能;②精子结合至卵子透明带;③精子与透明带结合诱发顶体反应（AR）;④精子与卵子的结合和膜融合.笔者拟就近期研究阐明的精子获能、AR及精、卵融合的新信息、新观点,进行综述如下.     

Boar semen can tolerate rapid cooling rates prior to freezing

Two experiments were performed in the present study that demonstrated that boar spermatozoa are capable of surviving rapid cooling rates within a range of 15-5 °C before freezing. Boar ejaculates diluted in Beltsville thawing solution (BTS) (1:1, v/v) were held at 17-20 °C and shipped over a 24-h time period from two AI centres to a cryobiology laboratory, where they were pooled (Experiment 1) or cryopreserved individually (Experiment 2) using a standard 0.5-mL straw freezing protocol. The effects of cooling before freezing were assessed after thawing through the objective evaluation of sperm motility and flow cytometric analysis of membrane integrity, acrosomal status, changes in membrane lipid architecture monitored by merocyanine and annexin V binding and intracellular production of reactive oxygen species. In Experiment 1 (six replicates), two semen pools (five ejaculates per pool) were cooled from 15 to 5 °C at rates of 0.08, 0.13, 0.40 and 1.50 °C min(-1). These cooling rates did not result in any significant differences (P>0.05) in any of the post-thaw sperm assessments, even in thawed samples incubated under capacitation conditions. In Experiment 2, three individual ejaculates from 16 boars were slowly (0.08 °C min(-1)) or rapidly (1.5 °C min(-1)) cooled before freezing. A consistent interboar variability (P<0.01) was detected, which was independent of the cooling rate used. Cooling rate only significantly influenced (P<0.05) sperm assessments in four of 16 boars, which exhibited slightly higher percentages of motile cells and intact plasma and acrosomal membranes in the samples that had been cooled slowly. These findings demonstrate that boar spermatozoa undergoing cryopreservation can withstand rapid cooling rates before freezing.