Computer-assisted sperm analysis parameters in young fertile sperm donors and relationship with age

Sperm parameter values have been shown to decline with age, according to conventional sperm analysis. However, the effect of age on sperm kinematic parameters has been rarely studied, especially in young fertile men. Here, we studied Computer-Assisted Sperm Analysis (CASA) parameters in a large cohort of men with proven fertility, in order to determine if there is a decline with age in this young fertile population. This retrospective analysis of CASA parameters was conducted on all donors included in the sperm donor programme in the Assisted Reproductive Techniques (ART) Centre of the University Hospital of Nantes between 2006 and 2009. Sperm concentration, motility, and kinetic parameters were recorded by a HTM-Ceros system and compared in 3 groups of sperm donors according to their age:?<35 years, 36-40 years, and 41-44 years. A total of 362 ejaculates from 138 donors were analyzed. Values for ALH, VCL, LIN, and STR significantly decreased with age. Sperm concentration, motile sperm proportion, and other kinetic parameters did not differ significantly among the groups. The use of CASA allowed the identification of ALH, VCL, LIN, and STR age-related decrease in young men with proven fertility.     

积水输卵管液对精子运动及形态学的影响

目的:通过体外研究积水输卵管液对精子运动及形态学的影响,探讨输卵管积水引起不孕的原因,并为单侧输卵管积水(对侧输卵管通畅)患者的处理提供依据。方法:优化处理后的高质量的精子体外与输卵管积水患者的输卵管液(积水组n=18)及正常输卵管液(对照组n=18)孵育2h、7h和24h后,精子分析仪(CASA)测量精子的各项运动参数,应用精子Diff-Quick涂片染色法分析精子形态。结果:①培养2h、7h和24h后,各时间点积水组精子的前向性运动百分率、平均曲线运动速度(VCL)、平均直线运动速度(VSL)、平均路径运动速度(VAP)、快速直线运动精子活率(MOT)、直线性(LIN)、摆动性(WOB)及前向性(STR)各项精子运动参数均低于对照组(P<0.05),且输卵管积水加快精子的运动速度参数(VCL、VSL、VAP)随时间延长而降低的速度(P<0.05)。②培养2h、7h和24h后,积水组与对照组精子的正常形态率对比无统计学差异,且不随时间延长而改变(P>0.05)。结论:输卵管积水在体外能降低精子的前向性运动百分率、精子运动速度参数(VSL、VCL、VAP)及精子运动方式参数(MOT、LIN、WOB、STR)各项运动指标,并提示输卵管积水不仅干扰了精子活动力,并可能对精子的获能产生不良影响,推测体内输卵管积水对精子产生一定的损害作用,从而影响受孕,但对精子正常形态精子率未有明显影响。     

辛硫磷对大鼠精子生成量和精子运动能力的影响

为探讨有机磷杀虫剂辛硫的雄性生殖毒性,运用动物实验研究了不同剂量辛硫磷径口染毒大鼠６０天对精子生成量和精子运动能力的影响。采用计算机辅助精子分析仪分析精了轨迹。结果显示：辛硫磷２４．５和７３．５ｍｇ／ｋｂＢＷ染毒组,大鼠睾丸精子生成量低于对照组。     

S型氯代甘油醇对大鼠精子运动和超活化抑制作用研究

目的研究S型氯代甘油醇(SACH)对大鼠精子运动和超活化的影响和可能机制。方法 20只成熟雄性SD大鼠随机分为4组,分别给予0、2.5、5.0和10mg/kg BW SACH,连续灌胃染毒52天。取附睾尾精子,获能条件下培养5h,以计算机辅助精子分析系统检测精子运动和超活化运动,同时检测精子特异的3-磷酸甘油醛脱氢酶(GAPDS)活性、三磷酸腺苷(ATP)和环磷酸腺苷(cAMP)水平,观察己酮可可碱(PTF)对SACH的拮抗作用。结果 SACH染毒组大鼠精子曲线运动速率(VCL)、平均路径速率(VAP)、直线运动速率(VSL)和精子头侧摆幅度(ALH)与对照组相比受到显著抑制(P0.01),运动直线性(LIN)显著增强(P0.01)。SACH使VCL≥400μm/s或LIN≤20%的比例显著减少(P0.01和P0.05),表明精子超活化受抑制。SACH显著抑制GAPDS活性,ATP和cAMP水平也随染毒剂量增加呈下降趋势。PTF可改善精子cAMP水平,部分缓解精子运动和超活化的抑制程度。结论 SACH抑制大鼠精子GAPDS活性导致ATP和cAMP水平下降,可能是影响精子运动和超活化的重要机制。     

精子核成熟度与精子形态的关系

目的:研究精子核成熟度与精子形态关系,探讨精子形态异常机制。方法:收集55例精液样本,其中生育组19例,畸形精子症组36例。应用CASA分析精子密度、活力,伊红染色检测精子活率,联苯胺染色分析精液白细胞浓度,改良巴氏染色分析精子形态,苯胺蓝染色法评价精子核成熟度。结果:畸形精子症组苯胺蓝染色阳性率显著高于生育组(P<0.05)。头部异常、颈部异常、尾部异常、无定型、其它畸形精子组苯胺蓝染色阳性率显著高于形态正常精子组苯胺蓝染色阳性率(P<0.05)。结论:精子核成熟异常可导致精子形态异常。     

Morphometric analysis of sperm used for IVP by three different separation methods with spatial light interference microscopy

ABSTRACT

The goal of this study was to characterize sperm populations resulting from three different methods of sperm selection used for bovine in vitro fertilization. We compared sperm selection with discontinuous Percoll gradients, Swim-Up, and electro-channel. Spatial light interference microscopy (SLIM) was used to evaluate the morphology of the spermatozoa and computer-assisted semen analysis (CASA) was used to evaluate the motility behavior of the sperm. Using these two technologies, we analyzed morphometric parameters and the kinetic (motility) patterns of frozen-thawed Holstein bull spermatozoa after sperm selection. For the first time, we have shown that these methods used to select viable spermatozoa for in vitro fertilization (IVF) result in very different sperm subpopulations. Almost every parameter evaluated resulted in statistical differences between treatment groups. One novel observation was that the dry mass of the sperm head is heavier in spermatozoa selected with the electro-channel than in sperm selected by the other methods. These results show the potential of SLIM microscopy in reproductive biology.

Abbreviations: SLIM: spatial light interference microscopy; CASA: computer aided sperm analysis; IVF: in vitro fertilization; BSA: bovine serum albumin; QPI: quantitative phase imaging; IVEP: in vitro embryo production; IACUC: institutional animal care and use committee; CSS: Certified Semen Services; AI: artificial insemination; TALP: Tyrode’s Albumin Lactate Pyruvate; MEC: medium for electro-channel; PDMS: polydimethylsiloxane; EC: electro-channel; TM, %: total motility; PM, %: progressive motility; RM, %: percentage of rapid sperm motility; VAP, μm/s: average path velocity; VSL, μm/s: straight-line velocity; VCL, μm/s: curvilinear velocity; ALH, μm: amplitude of lateral head displacement; BCF, Hz: beat cross frequency; STR, %: straightness; LIN, %: and linearity; GLS: generalized least squares; ANOVA: analysis of variance; LSD: Least Significant Difference; SPSS: Statistical Package for the Social Sciences; PCA: principal components analysis     

精液白细胞与精子运动参数关系的研究

目的:探讨精液白细胞与精子运动参数关系。方法:234例不育患者,采用精子自动分析系统分析精子运动参数,采用联苯胺法分析精液白细胞。结果:白细胞精子症组精子运动参数VCL、VSL、VAP均显著低于非白细胞精子症组,两组比较差异有显著性(P<0.001,P<0.001,P<0.001);白细胞精子症组MAD、BCF显著高于非细胞精子症,两组比较差异亦有显著性(P<0.001,P<0.001)。精液白细胞与VCL、VSL、VAP均呈显著负相关(r=-0.368,P<0.001;r=-0.362,P<0.001;r=-0.359,P<0.001),与MAD、BCF均呈显著正相关(r=0.134;P<0.05,r=0.210,P<0.001)。结论:精液白细胞可影响精子运动参数。     

New method to estimate the possibility of natural pregnancy using computer-assisted sperm analysis

The World Health Organization (WHO) criteria, which include percent motility and sperm concentration, are the only criteria for evaluating sperm quality and conception ability. However, these criteria are insufficient to evaluate the possibility of natural pregnancy. Thus, an index that can directly evaluate the possibility of a natural pregnancy is necessary. A new sperm energy theory without approximation was developed to assess the possibility of natural pregnancy based on mechanical sperm energy. Sperm motility parameters were measured using computer-assisted sperm analysis (CASA) in 129 ejaculated semen samples from 50 men in couples diagnosed with infertility, in which no abnormalities were found in women (sterile group), and 157 ejaculated semen samples from 57 men who had already fathered children in natural pregnancies (control group). A total of 129 subjects were selected from the control group and classified as the fertile group in order of the sample measurement date. The sperm energy index (SEI) and mean sperm energy index (MEI) were accurately obtained according to the methods described by the new sperm energy theory. SEI reflects total mechanical energy of the sperm in a visual field during CASA measurements. MEI reflects the mean mechanical energy of one sperm in a measurement field. All subjects with (MEI)/(SEI)?>?2 were assigned to the sterile group. The larger the SEI, the higher was the probability of predicting fertile subjects. The probability of predicting fertile subjects was approximately 60% with a SEI of?>?0.5, 70% with a SEI of?>?1, 80% with a SEI of?>?3, and 90% with a SEI of?>?6 in cases where (MEI)/(SEI) is?<?2. The data support the view that this novel method can be used to estimate the possibility of a natural pregnancy.     

精子DNA碎片率对男性生育力评估的预测价值

目的：探讨精子DNA碎片率（DFI）对男性生育力评估方面的预测价值。方法：选择2016年1月-2018年7月于上海市第十人民医院生殖医学中心就诊的460例男性不育患者纳入观察组,同期107例正常生育男性纳入对照组。计算机辅助精液分析系统（CASA）检测精液常规,精子染色质结构分析检测精子DFI。将精液常规结果分为精液正常、少精子、弱精子和少弱精4类。比较观察组和对照组的精液常规构成比和精子DFI;比较不同年龄（＜30岁,30～39岁,≥40岁）患者精子DFI水平的变化趋势及不同水平精子DFI（≤15%,15%～30%,≥30%）患者的体外受精（IVF）/胞浆内单精子注射（ICSI）的生殖结局。结果：观察组中精液正常、少精子、弱精子、少弱精所占比列分别为22.17%、13.26%、52.62%和11.95%,对照组分别为60.96%、34.59%、1.75%和2.70%。与对照组相比,观察组精液正常比例下降,少精子、弱精子和少弱精比例增加,差异有统计学意义（P＜0.05）。与对照组相比,观察组精子DFI≤15%所占比例下降,DFI≥30%比例增加,差异有统计学意义（P＜0.05）。随着年龄增加,精子DFI≤15%患者所占比例下降,精子DFI（15%～30%）与精子DFI≥30%患者所占比例增加,3组间精子DFI的分布比例差异有统计学意义（P＜0.05）。精子DFI≥30%来源的IVF或ICSI胚胎,其早期流产率显著升高（31%和25%,均P＜0.05）,但受精率、卵裂率、生化妊娠率、临床妊娠率、着床率等指标差异均无统计学意义（P＞0.05）。结论：不育男性的精子DNA碎片率更高,并且随年龄的增加而增加。DFI≥30%的精子受精的胚胎早期流产率较高,影响辅助生殖技术的妊娠结局,因此DFI对不育男性的生育力评估有实用参考价值。     

Evaluation of the Sperm Motility Analyzer System (SMAS) for the Assessment of Sperm Quality in Infertile Men

Eighty-one fresh semen samples were analyzed to compare the sperm parameters obtained using the new Sperm Motility Analysis System (SMAS; version 1.0, Kaga Electronics, Tokyo, Japan) with the CellSoft^TM Series 3000 (CRYO Resources, New York, USA) computer-assisted semen analysis (CASA) system and conventional manual semen analysis, based on WHO guidelines.. Significant correlations of sperm concentration (p<0.0001) and sperm motility (p<0.0001) were observed between SMAS and manual semen analysis estimates. There were also significant correlations of sperm concentration (p=0.0003) and sperm motility (p<0.0001) between SMAS and CellSoft estimates. Significant correlations for motility-related parameters were demonstrated in sperm velocity (p<0.0001), and linearity (linear velocity (VSL) divided by curvilinear velocity (VCL)×100) (p<0.0001), amplitude of lateral head displacement (ALH) (p<0.0001), and beat/cross frequency (BCF) (p=0.0127), between SMAS and CellSoft estimates. In this study, we showed the usefulness of the new SMAS, which has high reliability in estimating sperm concentration, sperm motility, velocity and linearity compared with CellSoft. SMAS can be a promising alternative, providing cost-effective semen analysis with the utility of the CASA system.     

Profiling of sperm proteins and association of sperm PDC-109 with bull fertility

Abstract

The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n?=?12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n?=?3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1–5.6, 37%) and basic (pI; 7.9–10.0, 27%) proteins and their pattern of expression varied significantly (p?<?0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4?kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p?<?0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.     

Study on sperm function in men long after cessation of gossypol treatment

Sperm function was assessed in 19 men 3-10 years after cessation of gossypol treatment and 2-9 years after recovery of normal sperm density. Nineteen normal fertile men of similar age served as the controls. The zona-free hamster egg-sperm penetration assay (SPA) revealed a highly significant difference (P less than 0.01) in the penetration rates between the treated and the control groups. Hormone assays indicated that there were no significant differences in circulating levels of luteinizing hormone (LH) and testosterone (T) between the two groups, but the follicle stimulating hormone (FSH) level of the treated group was significantly higher than that of the controls (P less than 0.05). The results showed that sperm function in the treated group was lower than that in the control, which may be a result of persistent gossypol-mediated damage to the testes.     

Thiol oxidation by nitrosative stress: Cellular localization in human spermatozoa

Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS^® system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility.

Abbreviations: DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO^?: peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation ANOVA: analysis of variance     

精子DNA碎片与精液各参数的相关性分析

目的探讨男性不育患者精子DNA碎片与精液各参数的相关性,研究精子DNA完整性在评估男性生育力方面的应用价值。方法回顾性分析2014年1月至2015年11月就诊于邢台不孕不育专科医院生殖中心男科的不育患者265例,采集精液标本,通过计算机辅助行常规精液分析,采用瑞-吉染色镜检观察精子形态及通过精子染色体扩散试验(SCD法)行精子DNA碎片率检测,对精子DNA碎片率与年龄、精液各参数(精子浓度、前向运动精子、正常精子形态)进行分组比较及相关分析。结果不同年龄段精子DNA碎片组间比较差异有统计学意义(P0.05);不同精子DNA碎片化指数(DNA fragmentation index,DFI)、精子浓度组间比较差异无统计学意义(P0.05),前向活动精子、正常形态精子组间比较差异有统计学意义(P0.05);年龄与精子DNA碎片存在正相关关系,前向运动精子率、正常形态精子率均与精子DNA碎片存在负相关关系。结论年龄是影响精子DNA损伤的因素,精子DNA损伤可能是导致精子活力和正常形态精子的影响因素。     

SQA—V全自动精子质量分析仪与计算机辅助精液分析系统结果比较

目的评估SQA—V全自动精子质量分析仪与计算机辅助精液分析系统（CASA）分析的精液各项主要参数的可比性。方法 对50份精液标本采用SQA-V全自动精子质量分析仪与CASA同时进行检测。结果在检测精子密度,精子活率,A级精子活力百分率和A＋B级精子活力百分率的结果上,SQA-V全自动精子质量分析仪与CASA的结果差异无统计学意义（P〉0．05）。结论SQA—V全自动精子质量分析仪与CASA在各项主要参数上具有较好的一致性。     

精子因素对反复自然流产的影响

目的 分析精子因素对反复自然流产的影响.方法 病例组:21例不明原因反复自然流产患者丈夫,对照组:20名已生育男子,采用精子染色质结构分析方法 分析精子DNA完整性,参照WHO标准分析精液质量,选用X、Y和18号染色体探针进行荧光原位杂交(FISH)检测精子的非整倍体性.结果 病例组正常形态率(10.8±2.8)%低于对照组,DNA碎片指数(DNA fragmentation index,DFI)值(21.7±6.7)%、18染色体非整倍体率(0.23±0.09)%、性染色体非整倍体率(0.80±0.14)%均明显高于对照组,差异均具有统计学意义(P＜0.01).结论 精子形态、DNA完整性和染色体非整倍体率是反复自然流产的相关性因素.     

精浆抗精子抗体对精子形态及运动特征的影响

目的:探讨精浆抗精子抗体(AsAb)对精子形态和运动特征的影响。方法:检测251例男性不育患者精液样本,采用酶联免疫法进行AsAb检测,计算机自动分析精子运动特征,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:251例男性不育患者中,AsAb阳性率为17·13%。AsAb阳性组正常形态精子百分率与阴性组无显著性差异(P>0·05),大头精子和锥形头精子百分率显著高于AsAb阴性组(P<0·05,P<0·005)。精浆AsAb阳性组a+b级精子百分率,精子运动参数MAD、LIN、WOB均明显低于阴性组,两组比较差异有显著性(P<0·05,P<0·01)。结论:精浆AsAb不影响正常形态精子百分率,但能使锥形头精子百分率显著升高;使a+b级精子百分率、精子运动参数MAD、LIN、WOB明显降低。     

Adding bovine seminal plasma prior to freezing improves post-thaw bull sperm kinematics but decreases mitochondrial activity

Variations in fertility between bulls with comparable sperm quality could be due to differences in their seminal plasma (SP). The aim of this study was to investigate the effect of adding bovine SP from bulls of known fertility to SP-free sperm samples. After removal of SP by Single Layer Centrifugation, resuspended sperm pellets were treated with SP from high or low fertility bulls at 0% (control), 1%, or 5% before freezing. Sperm quality was evaluated after thawing. Data were analyzed using Proc MIXED, SAS®. Bovine SP at 1% or 5% SP1 and SP5, respectively, decreased average path velocity, curvilinear velocity, and amplitude of lateral head displacement whereas wobble and linearity were increased. In addition, the proportion of spermatozoa with high mitochondrial membrane potential (MMP) was lowest for treatment with SP5 compared to SP1 and control. The proportion of SP did not affect other parameters of sperm quality. Thus, adding 5% bovine SP produced a favorable effect on some sperm velocity parameters but had an unfavorable effect on MMP. There were no differences in effect between SP from high and low fertility bulls.

Abbreviations: AI: artificial insemination; BCF: beat cross frequency; CASA: computer-assisted sperm analysis; IVF: in vitro fertilization; MMP: mitochondrial membrane potential; SLC: single layer centrifugation; SP: seminal plasma     

The Expression of Mannose-Ligand Receptor is Correlated with Sperm Morphology

The objective of the present study was to evaluate the association between the expression of sperm mannose-ligand receptors and sperm morphology. Sperm samples were obtained from 45 men, 30 fertile sperm donors and 15 infertile men. Sperm concentration, motility and morphology were evaluated and then incubated with control medium (Ham's F-10 + 1% HSA) for 4 h. Expression of mannose-ligand receptors was evaluated by mannosylated-BSA-FITC (subdivided into 3 patterns: I, for uncapacitated sperm; II, for capacitated; and III, for acrosome-reacted sperm). The mean ( &#45 SE) frequencies of sperm cells of the total sperm population that expressed patterns I, II, and III were 88 &#45 2.1%, 7 &#45 1.6%, and 5 &#45 0.8%, respectively, for fertile men, and 90 &#45 2.1%, 7 &#45 1.3%, and 3 &#45 0.5%, respectively, for infertile men. The rate of pattern III expression of mannose-ligand receptors was significantly higher in the fertile group compared to the infertile patients ( p <. 01). A poor but significant correlation was observed between the rate of pattern III and the percentage of normal-forms sperm cell in the ejaculate ( r =. 35, p =. 018). Fertile sperm samples express more advanced patterns of mannose-ligand receptors compared to infertile men. This phenomenon is related to the morphology of human sperm cell in the ejaculate more than to any other basic sperm characteristics.     

输精管吻合术后精子超微结构观察

目的:观察输精管吻合术后精子超微结构的变化。方法:采用光学显微镜、扫描和透射电子显微镜对20例接受输精管吻合术者和10例正常生育男性精子的形态和超微结构改变进行观察、统计、分析。结果:①光镜和扫描电镜分析显示吻合术后精液中正常形态精子显著减低;②两组精子尾畸在光镜下有明显差异,在扫描电镜下无差异;③两组精子头畸在扫描电镜下有显著性差异;④在透射电镜下吻合术后精子多数均属于复合型精子结构异常,大部分均有多个部位和多个细胞器结构异常。结论:吻合术后精子有多种类型和复合型的异常改变,这些改变与不育男子精子的改变相似。提示:输精管结扎术后精子形态学的改变,是使吻合术后生育能力降低的主要原因之一。