Sperm DNA integrity: correlation with sperm plasma membrane integrity in semen evaluated for male infertility

Previous independent studies have indicated that abnormally low parameters of sperm DNA integrity and sperm membrane integrity correlate to reduced fertility due in part to implantation disorders. The purpose of our study was to evaluate the relationship between sperm plasma membrane functional integrity assessed by the hypo-osmotic swelling test (HOST) and sperm DNA integriy test assesed by DNA fragmentation index (DFI). Semen samples from 102 random patients were evaluated in terms of standard semen parameters and assessed by DFI and HOST. Both tests showed a significant correlation to standard semen parameters (p < .05). In addition, patients with abnormal HOST results had a higher likehood of a subnormal or abnormal DFI result (p < .001). Our results suggest that a common sublethal insult may manifest as abnormalities in both the nucleus and the plasma membrane that act at the implantation and/or subsequent levels of development rather than at the fertilization stage.     

Influence of pentoxifylline on sperm membrane functional integrity.

In epididymal mouse spermatozoa, the effects of dibutyryl cyclic adenosine monophosphate 1 mmol/L (dbcAMP), pentoxifylline 5 mmol/L (PX), and/or mastoparan 50 mumol/L (MT) were evaluated for the following parameters: percentage of motile cells and response to hypoosmotic shock (HOS). The gametes were incubated during 80 min (A) or 200 min (B) in Tyrode's medium, and the drugs were added during the last 20 min. In A, dbcAMP + PX (61.5 +/- 5.4%; n = 10) enhanced and MT decreased significantly the population of motile cells (13.4 +/- 5.4%; n = 6) (control: 47.6 +/- 3.9%; n = 11). In B, PX significantly increased this parameter and MT plus PX also exerted a significant detrimental effect. Responses to HOS dropped significantly in the presence of PX + MT in A or in B; in this latter condition a similar decrease was evoked by MT alone. A positive correlation between percentages of swollen and motile spermatozoa was detected in A or in B in samples incubated with PX (r = .58, n = 11 and r = .76, n = 10; p < .05, respectively). These results that support that, in mouse sperm tail, PX would preserve functional membrane integrity, a relevant condition for adequate motility.     

流式细胞术检测吸烟对精子质膜完整性的影响

目的:应用荧光染料SYBR-14/PI双色标记法进行流式细胞术检测不育患者精子质膜完整性,分析吸烟对精子质膜完整性的影响并探讨其临床意义。方法:收集202例男性精液标本,其中132例为本院就诊男性不育患者,分为大烟量组(n=68)与小烟量组(n=64),正常生育男性为正常对照组(n=70)。通过计算机辅助精液分析系统进行精液常规分析。精液标本经PBS洗涤处理后用荧光染料SYBR-14/碘化丙锭(PI)双染后上流式细胞仪分析,用SYBR-14+/PI-(绿)表示质膜完整精子(PMI);SYBR-14-/PI+(红)表示质膜破损而坏死的精子;SYBR-14+/PI+(绿,红)表示正处于由活到死过度状态的精子。检测精子质膜完整性,并对精子质膜完整性与部分精液参数做相关性分析。结果:大烟量组与小烟量组质膜破损的坏死精子(SYBR-14-/PI+%)、过渡态的精子(SYBR-14+/PI-%)与正常对照组存在显著差异(P<0.01或0.05)。大烟量组SYBR-14+/PI-%明显低于小烟量组,SYBR-14-/PI+%明显高于小烟量组。所有202例标本SYBR14+/PI-%与精子活动率呈显著正相关(r=0.938,P=0.000)。结论:应用流式细胞术SYBR-14/PI双染法能快速、准确地检测精子质膜完整性。吸烟会导致精子质膜完整性下降,引起精子活力下降,可能是导致男性不育的重要原因之一。     

不同获能时相对人精子膜完整性的影响

本研究用人精子低渗肿胀试验（ＨＯＳ－ｔｅｓｔ）分析了１２例正常育龄男子的精液标本。精子分别用常规洗涤法、上游法和加入钙离子载体（Ａ２３１８７）诱导获能等三种不同方法处理后，分别在液化后、上游１ｈ后、获能３ｈ、６ｈ、２４ｈ后测定精子的低渗肿胀率，结果显示经不同方法处理、在不同获能时相之后的精子，在２４ｈ之内其肿胀率保持在８７．８±１４．６％～７３．３±１１．９％之间，统计学分析表明各标本组肿胀率之间无明显差异。本研究提示在２４ｈ内精子膜的完整性不受获能时相及不同处理方法的影响。     

溶脲脲原体感染对男性不育患者精子质膜完整性的影响

目的:应用荧光染料SYBR-14/碘化丙锭(PI)双标法流式细胞术检测溶脲脲原体(Uu)感染对男性不育患者精子质膜完整性的影响。方法:收集63例男性精液标本,分为Uu感染组(n=32)和正常对照组(n=31)。通过计算机辅助精液分析系统进行精液常规分析,培养法进行Uu检测。精液标本经PBS洗涤处理后用荧光染料SYBR-14/PI双染后上流式细胞仪分析,用发绿色荧光精子百分率(SYBR-14+/PI-%)表示质膜完整精子的比例,检测精子质膜完整性。结果:Uu感染组精子质膜完整性[(45.14±10.69)%versus(72.68±9.91)%]和(a+b)级精子百分率[(23.29±8.81)%versus(46.32±9.54)%]均显著低于正常对照组(P<0.01),两组间精液量、pH值、精子浓度差异无显著性(P>0.05)。结论:Uu感染引起精子质膜完整性下降,可能是导致男性不育的重要原因之一。     

Detrimental effects of cryopreservation on the structural and functional integrity of the sperm membrane.

Many centers have been disappointed with the pregnancy rate following the insemination of cryopreserved-thawed sperm, despite the maintenance of an adequate motile density. The possibility exists that damage to the sperm membrane might occur despite preservation of other semen parameters. Simple measurements of structural integrity (viability) and functional integrity (hypoosmotic swelling test) were performed on thawed specimens. In each instance, both the viability and HOS scores were less than the critical 50% level. Specimens from three different commercial centers had very poor HOS and viability scores from two of the centers, and, though the scores were generally greater than or equal to 50% from the third center, this was achieved by eliminating 11 of 12 donors. Reducing the glycerol concentration from 12 to 7% and switching from Nunc vials to plastic embryo straws did not improve the poor sperm membrane tests. The possibility exists that if modification of the cryopreservation technique leads to improved HOS and viability scores, perhaps improved pregnancy results will be realized.     

流式细胞术检测精索静脉曲张患者精子质膜完整性的研究

目的:应用荧光染料SYBR-14/PI双色标记法进行流式细胞术检测精索静脉曲张患者精子质膜的完整性并探讨其临床意义。方法:随机收集120例男性精液标本,其中90例左侧精索静脉曲张患者分为3组:VC1组(精索静脉曲张Ⅰ度,n=30)、VC2组(精索静脉曲张Ⅱ度,n=30)和VC3组(精索静脉曲张Ⅲ度,n=30),正常生育男性为正常对照组(n=30)。通过计算机辅助精液分析系统进行精液常规分析。精液标本经PBS洗涤处理后用荧光染料SYBR-14/碘化丙锭(PI)双染后上流式细胞仪分析,用发绿色荧光精子百分率(SYBR-14+/PI-%)表示质膜完整精子(PMI)的比例,检测精子质膜完整性,并预见精子的受精能力。结果:精索静脉曲张各组患者与正常对照组之间SYBR-14+/PI-%、SYBR-14-/PI+%均存在统计学差异(P<0.01)。精索静脉曲张患者(VC1、VC2、VC3)代表精子质膜完整性指标(SYBR-14+/PI-%)的百分率分别是:[(54.85±3.78)%]、[(45.37±4.12)%]、[(35.14±4.91)%],均显著低于正常对照组[(70.79±6.71)%],其活力顺序为VC30.05)。结论:应用流式细胞术SYBR-14/PI双染法能快速、准确地检测精子质膜完整性,精索静脉曲张引起精子质膜完整性下降,可能是导致男性不育的重要原因之一。     

三种精子质膜完整性检测方法的比较

目的对6-羧基二乙酸荧光素／碘化丙啶（6-CFDA／PI）双荧光标记方法进行改良并应用于人精子质膜完整性的检测,并与单用伊红Y染色、低渗肿胀试验（HOS）两种检测方法进行比较。方法随机选择30例本所人类精子库志愿者精液标本（密度≥20×10^6／ml、活力（a＋b）≥50%）,分别采用单用伊红Y染色、HOS和6-CFDA／PI双荧光标记试验检测精子质膜完整性。结果30例精液标本伊红Y染色活精子比率、HOS肿胀精子数比率、6-CFDA／PI双荧光标记结果分别为（68．8±7．6）％、（68．7±7．8）％和（67．9±7．8）％,两两比较,无显著性差异（P均〉0．05）,但双荧光标记能显示死活精子的过渡状态。相关性分析结果显示,三种方法检测的活精子比率均与前向运动（a＋b）精子呈显著正相关。结论6-CFDA／PI双荧光标记检测方法与单用伊红染色、HOS两种常用检测精子质膜完整性方法具有一致性,同时能鉴定出精子过渡的中间状态。     

Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation

Cigarette smoking is a serious health problem of our society. It is known that cigarette smoke is a cell mutagen and carcinogen, and that it may affect adversely male fertility. The possible detrimental effects on sperm cells are of great interest but the data available to support this statement are somewhat elusive. To approach this problem we examined conventional semen parameters, plasma membrane translocation of phosphatidylserine (PS) (annexin V/6-CFDA cell staining) and sperm DNA integrity (comet assay) in a group of healthy man smoking cigarettes on a regular basis. The results of the study were compared with the results of the same tests in healthy non-smoking donors. Significant difference in standard sperm parameters between the two groups was not found. Intensive expression of PS on the sperm plasma membrane surface (assayed by annexin V positive staining) was detected in the smokers group. There is a significant increase of population of apoptotic spermatozoa in ejaculates of smokers. Albeit DNA damages (high frequencies of double- and single- stranded DNA breaks) in spermatozoa of smokers are increased compared with non-smokers, but this difference is not statistically significant. Sperm DNA integrity of healthy smokers remains in the normal range, but a clear negative trend is observed, especially in respect of disturbance of plasma membrane phospholipid asymmetry.     

膜联蛋白5对人精子膜及DNA完整性的影响

目的:研究膜联蛋白5(Annexin5)对人精子细胞膜及DNA完整性的影响。方法:①精子膜完整性测定:按精子浓度>20×106/ml;活率>60%选取标准收集精液标本53份,分为3组,实验组为47.5μl精液中加入2.5μl10-6mol/L的Annexin5;阴性对照组为47.5μl精液标本中加入2.5μl1mol/L的Tris-HCl(pH8.0,25℃);空白对照组为47.5μl精液标本中加入2.5μl0.01mol/L的PBS(pH7.4),作用20min后,通过精子低渗肿胀试验(HOS)检测精子膜的完整性。②DNA完整性测定:同方法①,3组实验标本作用20min后,每份标本加入2.5μl0.02mol/L的H2O2,作用60min,通过吖啶橙(AO)荧光染色检测精子核DNA的完整性。结果:An-nexin5处理20min后低渗肿胀精子百分率与空白对照组及阴性对照组比较均具有极显著差异[(66.17±12.02)%vs(58.13±13.08)%,P<0.01;(66.17±12.02)%vs(59.94±11.91)%,P<0.01];空白对照组与阴性对照组比较无显著性差异。加入H2O2后,Annexin5组的DNA碎片指数与空白对照组及阴性对照组比较均具有极显著差异[(6.39±1.07)%vs(11.16±1.16)%,P<0.01;(6.39±1.07)%vs(10.86±1.05)%,P<0.01],空白对照组与阴性对照组比较无显著性差异。结论:Annexin5蛋白能在体外提高低渗肿胀精子百分率,对精子膜的完整性具有保护作用,同时对HO作用引起的精子核DNA破坏起保护作用。     

Sperm toxicity evaluated by the overnight sperm survival test

A subnormal sperm stress test has also been associated with implantation failure despite apparently normal fertilization; however, this test is cumbersome and time-consuming. The overnight sperm survival test has been considered to possibly demonstrate lipid peroxidation abnormalities similar to the sperm stress test. The present study evaluated whether lower overnight survival scores were associated with lower pregnancy and implantation rates following in vitro fertilization-embryo transfer. The results showed no adverse effect of poor overnight survival test scores. Possibly, the overnight survival test, though similar in some respects to the sperm stress test, is not similar for properties of predicting embryo implantation defects. Corroboration that subnormal stress tests predicts implantation disorders is needed.     

Cryodamage to plasma membrane integrity in head and tail regions of human sperm

Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions ofindividual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocytepenetration rate. Methods: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a sin-gle test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. Results: After cryop-reservation, there was a marked decline in the percentage of spermatozoa with Type Ⅳ membrane integrity (head mem-brane intact/tail membrane intact), and a significant increase in those with Type Ⅰ (head membrane damaged/tail mem-brane damaged) and Type Ⅲ (head membrane damaged/tail membrane intact) membrane integrity (n = 50, P <0.01). The value of Type Ⅲ integrity had a wide range of variability, whereas Type Ⅱ (head membrane intact/tailmembrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type Ⅳin     

SYBR-14/PI双染法流式细胞术检测精子质膜完整性的研究

目的:探讨应用荧光染料SYBR-14/碘化丙啶(SYBR-14/PI)双色标记法进行流式细胞术检测精子质膜完整性的可行性及其临床意义。方法:收集208例男性精液标本,按WHO精液分析标准分为正常组(n=31)与异常组(n=177)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后经SYBR-14/PI双染后上流式细胞仪(FCM)分析,用发绿色荧光精子百分率(SYBR-14+/PI-%)表示质膜完整精子(PMI)的比例。结果:正常组与异常组SYBR-14+/PI-与SYBR-14-/PI+精子百分率均存在统计学差异(P均<0.05)。正常组精子SYBR-14+/PI-%为(55.66±20.64)%,显著高于异常组[(39.71±19.21)%,P=0.000]。208例标本中,SYBR-14+/PI-%与精子活动率呈显著正相关(r=0.408,P=0.000),与(a+b)级精子百分率呈显著正相关(r=0.398,P=0.000),与d级精子百分率呈显著负相关(r=-0.413,P=0.000);SYBR-14-/PI+%与精子活动率呈显著负相关(r=-0.380,P=0.000),与(a+b)级精子百分率呈显著负相关(r=-0.397,P=0.000),与d级精子百分率呈显著正相关(r=0.385,P=0.000);SYBR-14+/PI+%与精子活动率呈正相关(r=0.172,P=0.013),与(a+b)级精子百分率呈正相关(r=0.177,P=0.011),与d级精子百分率呈负相关(r=-0.164,P=0.018)。结论:应用流式细胞术SYBR-14/PI双染法检测精子质膜完整性具有可行性,可用于评估男性生育力。     

Expression of lectin receptors on the membrane surface of sperm of fertile and subfertile boars by flow cytometry

Studies suggest that carbohydrates are important in different stages of fertilization. Plasma membrane changes accompanying in vitro capacitation and acrosome reaction (AR), such as removal or appearance of specific glycoproteins, have been studied using lectins that bind specifically to carbohydrate residues. In specialized artificial insemination farms and semen production centers, identification of boars with decreased fertilization ability (subfertility) is a newborn necessity. This investigation is a sequential study to determine the kinetics of surface carbohydrates turnover during in vitro capacitation and AR in fertile and subfertile boar sperm. Flow cytometry determinations of the binding of three FITC-labeled lectins were assessed. WGA binding was significantly lower in fresh, capacitated, and acrosome-reacted sperm of subfertile boars than in fertile boars. Con-A binding was not significantly different in fresh sperm of fertile and subfertile boars. However. Con-A labeling in capacitated, and acrosome-reacted sperm differed significantly in both groups. UEA binding increased only in capacitated sperm of subfertile boars. These findings could be used as indicators of capacitation and AR and may also be a good indicator of sperm fertilizing ability in boars.     

Evaluation of human sperm membrane integrity using the water test and the hypoosmotic test

The objective of the study was to compare the water test and the hypoosmotic test (HOS) in the assessment of the human sperm membrane. A total of 686 semen samples from human male donors were subjected to water and HOS tests after routine semen evaluation. The mean percentage of swollen spermatozoa was 71.8 +/- 9.6% in the HOS test and 67.8 +/- 9.4% for the water test; these values were not statistically different. The correlation of coefficients between the water test and the HOS test was highly significant whether the values for the HOS test were higher or lower than 60% (P < 0.001). A poor correlation was obtained when the two tests were compared for sperm counts either higher or lower than 20 x 6 ml-1 and when the results for both tests were compared with the percentage of eosin-Y staining spermatozoa. A poor correlation was also obtained when the results of each test were compared with eosin-Y staining spermatozoa in normal and abnormal semen samples. The coefficient of regression between the two tests showed a high correlation (P < 0.001). In conclusion, even though a high correlation between the HOS test and water test was observed in this study, it is not possible to recommend assessment of sperm membrane integrity using the water test and the consequent replacement of the HOS test in routine practice. Further studies are necessary to establish the best test for sperm vitality.     

Correlation between hypo-osmotic swelling test and 'water test' to assess human sperm membrane integrity

Summary. Fifty-nine men who requested vasectomy and 43 infertile patients had a semen analysis performed prior to surgery or during evaluation. A hypo-osmotic swelling test (HOS) and a new 'Water test' were performed simultaneously, in order to assess correlation between these two procedures. Our results showed that values obtained with the 'Water test' were significantly higher than those obtained with the HOS test ( P < 0.001). These findings suggest that it is necessary to determine normal values for this new test before introducing it in the routine semen analysis.     

Effect of carboplatin on the functional integrity of the human sperm membrane in vitro

Although it is well known that carboplatin is a drug that binds directly to DNA, causing DNA-DNA and DNA-protein cross-links, which is the presumptive method for killing cells, the whole mechanism of action of carboplatin on spermatozoa is unclear. There are no published data in peer-reviewed journals focused on the interaction between carboplatin and cell membranes. Therefore, the purpose of this study was to investigate the minimal concentration of carboplatin that would affect the functional integrity of the human sperm membrane in an in vitro model. Human-ejaculated spermatozoa were obtained from 20 healthy normozoospermic donors. Solutions (SOL) of 0.5 mL of the semen samples and 0.5 mL NaCl (0.9%) containing increasing concentrations (7.5, 15, 30, and 60 ng) of carboplatin per 1 mL of SOL were prepared. Then, the hypoosmotic-swelling (HOS) test and the eosin test were performed on these samples and compared with the control (no carboplatin) group. Significant damage to the plasma membrane in the head region (eosin test positive) and in the tail region of spermatozoa, as assessed by the HOS test, was observed in concentrations of 30 and 60 ng carboplatin per 1 mL of SOL in comparison to the values evaluated in the control group. The results demonstrate that a minimal carboplatin concentration of 30 ng/mL causes significant damage to membrane integrity of spermatozoa in healthy volunteers.     

Current assessment of sperm DNA integrity

Conventional semen analysis is rapidly losing its place as the gold standard of diagnosis and the cornerstone of treating the infertile male in modern times. Recent technology allows scientists to analyze sperm fertilizing potential and subsequent embryonic growth by studying factors that have previously escaped traditional parameters. It has become increasingly evident that nuclear DNA arrangement is essential to the fertilizing potential of sperm. A vast array of tests are now available to examine the genetic makeup of individual spermatozoa, ranging the entire gamut from simple bench top assays performed routinely to complex flow cytometric assays requiring highly-skilled technologists. Future research to compare these new tests to those more commonly in use, correlating them with reproductive outcome promises to fill the current void in the field of male infertility, paring innovative diagnostic (and prognostic) technological standards to the already existing sophisticated assortment of successful treatment modalities.     

Various physical stress factors on rat sperm motility, integrity of acrosome, and plasma membrane

The objective of this study was to determine the effects of various physical interventions such as centrifugation regimes, Percoll gradient separation, and repeated pipetting on various viability parameters of epididymal sperm of Fischer 344 (F-344) and Sprague-Dawley (SD) rat strains. Three experiments were conducted. In experiment 1, sperm motility and acrosomal and membrane integrity were compared after exposing sperm samples to 200, 400, 600, and 800 x g centrifugal forces for 5, 10, or 15 minutes. In experiment 2, sperm motility and acrosomal and membrane integrity were compared after passing them through a Percoll separation using centrifugal forces of 600, 800, 1000, and 1200 x g for either 15 or 30 minutes. In experiment 3, the effect of repeated pipetting (2, 4, 6, 8, and 10 times) on motility and membrane integrity of rat sperm was compared with that on mouse, ram, bull, and boar sperm. The results revealed that both F-344 and SD rat sperm motility and membrane integrity were significantly affected by centrifugation (P < .05). The acrosomal integrity of SD rat sperm was affected after using 800 x g centrifugation force for 10 or 15 minutes (P < .05), whereas F-344 rat sperm acrosomal integrity was not affected by any centrifugation regimes (P > .05). Sperm from SD rats also had higher motility and membrane integrity loss than did sperm from F-344 rats after centrifugation and pipetting (P < .05). Percoll gradient separation did not cause significant motility loss or acrosomal damage to either F-344 or SD sperm (P > .05). Repeated pipetting had a dramatic adverse effect on both rat and mouse sperm motility (P < .05) as compared with sperm from bull, boar, and ram, which were not affected at all (P > .05). These data suggest that rat sperm have unique properties that need to be considered during centrifugation, Percoll gradient separation, and pipetting procedures.