摘要
研究背景：
据报道世界范围内约有15%-20%的育龄夫妇不能生育,且近半个世纪以来,由于环境、心理、社会等因素的影响,男性精子的数量和质量出现了明显下降的趋势,男女不育不孕的比例也由3：7上升到5：5,男性生殖健康正受到严重威胁。解脲支原体属于条件致病微生物,可黏附在泌尿生殖道的黏膜及上皮细胞上,导致泌尿生殖道感染,并可从多方面影响男性生育能力,如通过干扰精子发生、影响精子代谢、影响精卵结合及生殖道局部免疫等机制,但其致男性不育的具体机制目前还不完全清楚。并有部分研究显示解脲支原体对男性不育并无显著的影响。由于各个实验室的检测试剂和方法的不同,不同地区男性不育患者精液中解脲支原体检出率差异很大,可高达5%-58%,而正常生育者携带率为3%-31%。
解脲支原体对精液质量的影响同样在以往的研究中未达到共识,临床研究表明,解脲支原体感染患者存在精子数量降低,精子畸形率增加,液化时间延长及抗精子抗体阳性率上升等表现,而有些研究却得到了阴性结果。导致上述分歧可能与受试个体的数目、区域、社会背景有关,解脲支原体不同生物群致病能力不同,也可能是原因之一。解脲支原体按照基因型不同,共分为2个生物群：biovar I和biovar II,研究显示不同的生物群致病能力不同,biovar II可能与非淋菌性尿道炎、盆腔炎、宫颈炎、不良怀孕关系更加密切,解脲脲原体不同生物群与定植和感染的关系是目前该领域的研究热点。
活性氧主要由白细胞和精子产生并释放,生理状态下,对维系精子的正常功能起到了重要的作用,然而精液中活性氧水平异常增加,破坏氧化还原系统平衡,会造成精子膜结构的氧化损伤,精子活动力下降,影响精子获能和顶体反应的发生,并可攻击精子核DNA,造成精子核DNA的损伤如染色体交联、染色体缺失、碱基对修饰、单股和双股DNA断裂等,过多的活性氧还可诱导精子细胞凋亡,线粒体是介导细胞凋亡最重要的细胞器,高水平的活性氧可破坏线粒体膜,引起细胞色素c的释放,激发半胱氨酸蛋白酶的级联反应,触发细胞凋亡。精液中活性氧的来源众多,研究显示感染可导致精液中活性氧的异常增加,然而迄今只有少数报道涉及某一特殊感染对精液中活性氧生成的影响。
研究目的：
1.调查研究解脲支原体及其生物群在不育和可生育人群中的感染率。
2.研究不同生物群解脲支原体感染对精子常规参数,精子运动参数,精子形态以及精液白细胞的影响；探讨不同生物群解脲支原体感染与精液白细胞的关系及白细胞在解脲支原体感染影响精子参数中的作用。
3.进一步研究不同生物群解脲支原体感染对精液中活性氧产生,精子膜的脂质过氧化以及核DNA损伤的影响,并探讨活性氧产生与精液白细胞的关系。
研究方法：
1.解脲支原体及其生物群在不育和可生育男性人群中的比例
对2012年7至2013年5就诊于汕头大学医学院第一附属医院的223例不育男性患者和146例可生育男性人群进行研究,研究对象的选取均严格按照本文的筛选标准,采用传统培养法检测解脲支原体在男性不育人群和可生育人群中的感染率,并捡出感染阳性患者,使用实时荧光定量PCR法鉴定其生物群。
2.不同生物群解脲支原体感染对精子常规参数,精子运动参数和精液白细胞的影响及白细胞在其中的作用
采用手淫法收取精液,液化后,检测精子密度、活力、精子运动参数、白细胞、精子形态等参数,采用国产WLJY-9000型彩色精子自动分析系统分析,应用计算机精子形态分析软件结合人工判读进行分析,先后按照支原体感染种类和精液白细胞正常与否对所测标本的上述指标进行分组比较。
3.不同生物群解脲支原体感染对精液中活性氧产生,精子膜的脂质过氧化以及精子核DNA损伤的影响
以鲁米诺为探针,运用化学发光法检测精子活性氧(Reactive Oxygen Species, ROS)水平。分别采用硫代巴比妥酸法和黄嘌呤氧化酶法检测精液中丙二醛(Malondial-dehyde, MDA)和总超氧化物歧化酶(Total Superoxide Dismutase, T-SOD)的浓度。采用精子染色质结构分析检测精子核DNA完整性,采用单细胞凝胶电泳法检测精子核DNA断裂损伤
统计学处理
数据分析使用SPSS13.0统计软件。率的比较使用卡方检验；多组间的比较使用ANOVA analysis, LSD用于组间比较；两组数据的比较使用t检验,结果表示为均值士标准差。Levene's test被用作方差同源性检验,如方差不齐,使用Welch检验,组间比较采用Dunnett T3。P<0.05被认为具有统计学意义。
研究结果：
1.解脲支原体及其生物群在不育和可生育男性人群中的比例
(1)解脲支原体在不育和可生育男性人群中的比例
对223例我院就诊的不育男性和146例可生育人群精液中的解脲支原体感染率检测,结果显示虽然不育男性人群中的感染率为33.6%(75/223)较可育男性人群的感染率24.7%(36/146)有增高的趋势,但两者之间未具有统计学差异(χ2=3.379,P=0.066)。
(2)解脲支原体不同生物群在不育和可生育男性人群感染阳性标本中的检出率
使用实时荧光定量PCR法检测解脲支原体感染阳性标本两种生物群的检出率,结果显示biovar II群在不育男性感染阳性标本中的检出率42.7%(32/75)显著高于可生育人群阳性标本中的检出率22.2%(8/36),具有统计学差异(χ2=4.411,P=0.036)。
(3)解脲支原体不同感染类型在不育和可生育男性人群中的比例
在不育男性患者和可生育人群中分别有8例(10.7%)和3例(8.3%)被发现有混合感染(biovarⅠ和biovarⅡ同时存在),二者之间差异没有统计学意义(χ2=0.148,P=0.700)。
2.不同生物群解脲支原体感染对精子常规参数,精子运动参数以及精液白细胞的影响及白细胞在其中的作用
(1)不同生物群解脲支原体感染对精子常规参数,精子运动参数以及精液白细胞的影响
按照感染类别将标本划分为biovarⅠ, biovar Ⅱ,混合感染和未感染组共4组,比较分析了4组的精液常规参数和运动参数,结果显示精子总活力在4组之间存在统计学差异(P=0.001)。其他参数诸如精子浓度,前向运动力虽然感染组较非感染组有降低的趋势,但差异没有统计学意义(P>0.05)。组间比较结果显示biovarⅡ感染组的精子浓度(59.25±49.21)以及biovarⅡ(54.95±16.21)和混合感染组(51.26×20.88)的总活力与未感染组(77.03±×43.97；66.29±×16.14)之间具有统计学差异(P=0.038；P<0.001,P=0.005)。精子运动参数的分析结果显示biovarⅡ感染组的曲线速度(Mean Curvilinear Velocity, VCL)(P<0.001)、直线速度(Mean straight-line velocity, VSL)(P=0.002)、平均路径速度(Mean Average Path, VAP)(P=0.024)、直线性(Mean Percentage Lineality, LIN)(P=0.009)、摆动性(Swing, WOB)(P=0.004)以及混合感染组的VCL(P=0.017)、 VSL(P=0.007)、LIN(P=0.016)与未感染组相比差异具有统计学意义；biovar Ⅰ感染组与未感染组相比各参数均无统计学差异(P>0.05)。
同时我们发现biovar Ⅰ感染组71人中有12人精液白细胞增高,biovar Ⅱ感染组40人中有12人白细胞增高,混合感染组11人中有3人白细胞增高,未感染组258人中有25人白细胞增高。比较白细胞异常增加比率的结果显示四组之间存在统计学差异(χ2=14.842,P==0.002)。组间结果比较显示白细胞计数异常增加比例在biovar II感染组与未感染组之间具有统计学差异(χ2=13.136,P<0.001),而biovar I感染组和混合感染组与未感染组之间差异没有统计学意义(χ2=2.901,P=0.089；χ2=3.498,P=0.61),提示biovar II感染与精液中白细胞增加关系更加密切。
(2)精液白细胞异常增加对精子常规参数、运动参数以及精子形态的影响
白细胞异常组与白细胞正常组相比,精液常规参数中总活力、前向运动力和正常精子比率在两组之间差异具有统计学意义(P=0.008；P=0.005；P=-0.003)；精子运动参数中VCL (P=0.023)、VSL (P=0.010)、VAP (P=0.011)、WOB(P<0.001)在两组间具有统计学差异；精子形态异常率在两组间具有统计学差异(P--0.008),其中头部异常增加明显(P=0.005)。以上结果提示白细胞异常增加可以导致精子质量和运动能力的下降以及畸形精子的比率增加。
3.不同生物群解脲支原体感染对精液活性氧产生,精子膜的脂质过氧化以及精子核DNA损伤的影响
(1)不同生物群解脲支原体感染对精液氧化还原系统及脂质过氧化的影响
我们按感染分类先后比较了4组间的精液ROS、MDA和T-SOD水平,结果显示ROS水平在biovar II感染组(2.66±0.85)、混合感染组(2.82±±1.37)与未感染组(1.944±0.79)之间具有统计学差异(P<0.001,P=0.003)；而biovar I感染组(2.20±±0.94)与未感染组之间差异没有统计学意义(P=0.075)；MDA的检测结果显示biovar II感染组(1.25±0.61nmol/l)、混合感染组(1.30±0.61nmol/l)与未感染组(0.94±0.52nmol/l)之间具有统计学差异(P=0.004,P=0.040)；而biovar I感染组(1.05±±0.55)与未感染组之间差异没有统计学意义(P=0.226)；T-SOD检测结果显示biovar I (63.57±8.95), biovar II感染组(62.71±7.10),混合感染组(61.81±±7.62)虽然低于未感染(64.49±±7.23)组,4组之间差异没有统计学意义(P=0.556)。
(2)不同生物群解脲支原体感染对精子DNA氧化损伤的影响
使用精子染色质结构分析法对4组不同人群精子细胞染色质完整性进行分析比较,结果显示biovar Ⅱ感染组的DNA碎片指数(DNA Fragmentation Index, DFI%)(22.59±7.59).高DNA可染性(High DNA Stainable, HDS%)(10.58±3.74)与未感染组(18.32±7.62；8.06±4.23)相比具有统计学差异(P=0.003；P=0.001)；混合感染组DFI%(23.44±4.89)与未感染组之间具有统计学差异(P=0.032),而HDS%(9.73±3.37)在两组间差异无统计学意义(P=0.181); biovar I感染组DFI%(20.03±7.21), HDS%(8.92±3.60)与未感染组之间差异均无统计学意义(P=0.155；P=0.175)。使用单细胞凝胶电泳检测精子核DNA损伤,结果显示biovar M感染组(1.06±0.15)与未感染组(0.94±0.12)之间彗星尾距(Tail Moment, TM)具有统计学差异(P<0.001),而biovarⅠ感染组(0.96±0.11)和混合感染组(1.05±0.15)与未感染组之间差异没有统计学意义(P=0.257,P=0.224)。
结论：
1.解脲支原体在不育男性人群中的感染率虽然较可生育男性人群高,但二者之间并不具有统计学差异,而解脲支原体阳性标本中biovarⅡ生物群的检出率在两组间具有统计学差异,提示biovarⅡ群感染可能与男性不育密切相关。
2.按感染类别分组进行的精子参数比较研究显示只有总活力在4组之间存在统计学差异,组间比较发现biovarⅡ感染组的精子浓度及biovarⅡ和混合感染组的总活力与未感染组之间具有统计学差异。以上的结果提示biovarⅡ较biovarI对精子参数影响更加显著,主要体现在精子活力方面,比较精子运动参数的结果提示biovarⅡ感染组的VSL、VCL、VAP、LIN、WOB与未感染组之间具有统计学差异。主要涉及以下机制①解脲支原体的强粘附作用②感染致精液中活性氧增加,破坏精子膜结构及引起线粒体膜电位的改变,影响精子的能量供应,降低精子活动力。同时,我们发现白细胞计数异常增加比例在biovarⅡ感染组与未感染组之间具有统计学差异。以往研究显示白细胞是精液中活性氧的一个重要来源,结合上述结果,推测biovarⅡ群感染所致的精液白细胞异常增加并产生过多的活性氧可能是导致精子活力下降的主要原因之一。
3.为了证实白细胞增加对精液质量的影响,我们把精液标本分为白细胞正常组和白细胞异常组,分别比较了两组的精子常规参数,运动参数以及精子形态。结果显示精子常规参数中总活力、前向运动力和正常精子比率在两组之间具有统计学差异。精子运动参数VCL、VSL、VAP、WOB白细胞异常组明显低于白细胞正常组,白细胞异常组精子畸形率明显增加,并以头部畸形为主,提示精液中白细胞的异常增加可能导致精子质量的显著下降。
4.进而我们研究比较了不同生物群解脲支原体对ROS产生的影响,结果显示biovar II感染组和混合感染组的ROS水平与未感染组之间具有统计学差异。解脲支原体感染引起的活性氧增加主要有两个来源,一方面,解脲支原体对精子最有毒害的代谢产物是H2O2和NH3。H2O2本身对精子是有害的,并且可以转化成OH基自由基,而后者对细胞膜和DNA具有强毒性。除此之外脲原体磷脂酶A和C可以改变细胞膜的脂质结构,从而导致其完整性的损失并增加其渗透性。另一方面,解脲支原体(主要是biovar II群)感染可引起精液中白细胞异常增加,并产生过多的ROS。我们的结果提示biovar II群感染引起的精液白细胞异常增加可能是活性氧过量产生的最主要原因。MDA的检测结果也验证了此观点,biovar II和混合感染组的MDA水平显著增加,表明ROS所致的精子膜脂质过氧化增加,流动性下降,精子活动能力降低。然而此次研究中精液T-SOD水平未在感染组和未感染组之间发现有统计学差异。
5.精子DNA染色质结构分析实验中的DFI%, HDS%以及单细胞凝胶电泳中的TM值在biovar II感染组与未感染组之间具有统计学差异,提示biovar II群感染可引起精液白细胞增加,释放过量ROS,破坏精子DNA的完整性,导致DNA的断裂损伤增加,最终影响男性的生育能力,并可能导致孕后的不良后果。
创新之处：
1.揭示了不同生物群解脲支原体在不育和可生育男性人群中的分布,biovarⅡ群在男性不育人群感染阳性标本中的检出率显著高于可生育人群。
2.比较研究了不同生物群解脲支原体对精子质量和精液白细胞的影响,提出biovarⅡ群对精液质量的影响更加明显,尤其是精子总活力,同时biovarⅡ群感染可引起精液中白细胞的明显增加。
3.从活性氧产生的角度,比较研究了不同生物群解脲支原体对ROS产生,精子膜脂质过氧化以及精子核DNA断裂损伤的影响,提出biovarⅡ群可增加精液中ROS水平,引起精子膜脂质过氧化以及精子核DNA损伤,其机制可能与精液中白细胞增加有关。
Background
It is reported about15%-20%couples in the worldwide suffering from the infertile problem at the child-bearing age. Due to the influence of environment, psychological and social factors, the male sperm quality and quantity showed obvious downward trend, and the ratio of male and female infertility have changed from3:7to5:5. Ureaplasma Urealyticum (U.urealyticum), belonging to the conditions of pathogenic microorganism, can adhere to genitourinary tract and cause urinary tract infections. It can also affect male fertility in several ways, such as interfering with spermatogenesis, affecting sperm metabolism and sperm-oocyte binding, as well as triggering immune mechanisms in the genital tract. But the mechanism of male infertility caused by the U.urealyticum infection is not fully clear. Meanwhile some studies have shown that no significant effects of U.urealyticum were seen on male infertility. Because various reagent and method were used in the laboratories, the prevalences of U.urealyticum differed from each other. It varied from5%to58%in the infertile male seminal fluids and3%to31%in the fertile male seminal fluids. It still remains controversy about the influence of U.urealyticum on semen quality. Some studies showed that UU infections may alter various characteristics of semen, such as sperm motility, density, pH and morphology, and that antibiotic treatment can lead to the improvement of semen quality. While other studies show no influence of U.urealyticum infection on semen quality. On the basis of genotypic characteristics, U. urealyticum is divided into two biovars-biovar I (ureaplasma parvum, parvo) composed of serovars1,3,6and14, and biovar II (ureaplasma urealyticum, T960) composed of serovars2,4,5,7,8,9,10,11,12and13. Infection by biovar II is more closely involved with several inflammatory pathologies, such as nongonococcal urethritis (NGU), prostatitis and pelvic inflammatory disease than is biovar I. There are two main sources for production of free radicals in the semen:leukocytes and sperm. The same reactive oxygen species (ROS) that under physiological conditions are an inseparable element of the fertilization process, as well as being important regulatory factors in the control of spermatogenesis efficiency, may under pathological conditions (ROS excess) be responsible for structural, metabolic, and functional disorders of the male germ cells. ROS can damage the sperm membrane which in turn reduces the sperm's motility and ability to fuse with the oocyte. It can attack the integrity of DNA in the sperm nucleus by causing base modifications, DNA strand breaks, and chromatin cross-linking. High levels of ROS may destroy the mitochondrial membrane, the most important organelle mediating cell apoptosis, release cytochrome c, trigger the cysteine protease cascade and induce cell apoptosis at last.
Objective:
1. Investigate the prevalence of U. urealyticum and its different biovars in the infertile and fertile men.
2. Reveal the impacts of two biovars of U. urealyticum infection on sperm parameters, movement parameters and morphous of sperm cells. Investigate the relationship between U. urealyticum infection and seminal leukocytes as well as the role of seminal leukocytes in the progress of U. urealyticum infection in semen.
3. Study the effects of two biovars of U. urealyticum on ROS generation in semen as well as the lipid peroxidation of sperm membrane and DNA damage. Discuss the relationship among U. urealyticum infection, ROS generation and leukucytes elevation in semen.
Methods:
1. The prevalence of U. urealyticum and different biovars in the infertile and fertile men.
From July2012to February2014,223patients with infertility and146fertile whose wives had non-assisted pregnancies in the past and were clinically asymptomatic were selected from the Infertility Center, the First Affiliated Hospital of Shantou University Medical College (SUMC), China. All the subjects were screened strictly by the exclusion criteria for participants. Traditional culture method was used to detect U. urealyticum infection in the semen of infertile male and fertile male. Then the biovar species were identified by real-time PCR with U. urealyticum positive specimen.
2. The impacts of two biovars of U. urealyticum infection on sperm parameter and the role of leukocytes in the progress
Semen samples were obtained by masturbation. After semen production, the samples were liquefied at37℃for about30minutes. Routine semen analysis, including volume, pH, spermatozoa count, motility, morphology and leukocyte counts was carried out according to WHO guidelines by using a WLJY-9000TYPE WEILI Color Sperm Analysis System. The computer sperm morphology analysis software combined with artificial judgement was used to analysis.
3. The effects of different biovars of U. urealyticum on ROS generation, lipid peroxidation of sperm membrane and DNA damage
ROS production was measured using luminol as the probe by the chemiluminescence method. Semen malondial-dehyde content in semen was measured by the sulfo-barbitone acid method; total superoxide dismutase in semen was measured by the xanthine oxidase method. Sperm nuclear DNA damage was assessed of by sperm chromatin structure assay (SCSA) and single-cell gel electrophoresis (SCGE).
Statistical Analysis:
Data was analyzed by SPSS version13.0for Windows. Differences in proportions were compared using chi-square test. For more than two groups, one-way analysis of variance (ANOVA) was used. After ANOVA, a LSD test was applied to analyze the difference between two groups. The Levene's test was used for homogeneity of the variance analysis, and in the case of non-homogeneity, a Welch test was used. Statistical significance was set at P<0.05. Results were expressed as mean±SD.
Results:
1. The prevalence of U. urealyticum and different biovars in the infertile and fertile men.
The prevalence of U. urealyticum-positive in semen specimens from infertile men (75/223,33.6%) tended to be higher than that in the fertile men (36/146,24.7%), but it did not reach statistical significance (χ2=3.379, P=0.066). Among U. urealyticum-positive semen, biovar II was isolated from32/75(42.7%) infertile cases and8/36(22.2%) fertile cases (χ2=4.411, P=0.036). Eight (10.7%) infertile and three (8.3%) fertile men harbored mixed pathogens, but no significant difference existed between two groups (χ2=0.148, P=0.700).
2. The impacts of two biovars of U. urealyticum infection on sperm parameter and the role of leukocytes in the progress
(1) The impacts of two biovars of U. urealyticum infection on sperm parameters, motion parameters and leukocyte counts in semen.
In order to identify the effects of biovar I and biovar II on semen, we compared semen parameters between infected and uninfected groups. Among the biovar I, biovar II, mixed infection and uninfected groups, only the total motility showed significant difference between infected and uninfected individuals (P=0.001). Although other parameters including spermatozoa concentration and progressive motility in the positive groups tended to be lower than those in the uninfected group, the differences were statistically insignificant (P>0.05). The spermatozoa concentration (59.25±49.21) in the biovar II group, and the total motility in the biovar II (54.95±16.21) and mixed infection (51.26±20.88) groups were significantly lower than the uninfected group separately (P=0.038; P<0.001, P=0.005). When compared with uninfected group, the sperm motion parameters showed significant decreases of VCL(P<0.001), VSL(P=0.002), VAP(P=0.024), LIN(P=0.009), WOB(P=0.004) in Biovar II and VCL(P=0.017), VSL(P=0.007), LIN(P=0.016) in mixed infection group, but there was no difference between the biovar I and uninfected groups (P>0.05). A total of12/71subjects in biovar I,12/40subjects in biovar II,3/11subjects in mixed infection and25/258subjects in uninfected group were found Leukocytes elevated (≥106/ml). There were significant differences among the four groups(χ2=14.822, P=0.002).When compared with uninfected group separately, the leukocytes in biovar II increased significantly (χ2=13.136, P<0.001), but the leukocytes showed no significant difference in the biovar I and mixed infection groups (χ=2.901,P=0.089;χ2=3.498, P=0.61). The results indicated that biovar II infection had more close relationship with increased leukocyts in semen.
(2) The impacts of leukocytes on sperm parameters, motion parameters and sperm morphology.
Subsequently we compared the semen parameters between the normal leukocytes group and abnormal leukocytes group. The results showed that the semen parameters including total motility, progressive motility and normal forms in the abnormal leukocytes group decreased significantly compared with normal leukocytes group (P=0.008; P=0.005; P=0.003). The sperm motion parameters including VCL (P=0.023)、VSL (P=0.010)、VAP (P=0.011)、WOB (P<0.001) were showed significant lower in the abnormal leukocytes group than abnormal leukocytes group. The ratio of sperm abnormality in the abnormal leukocytes group increased significantly compared with the normal leukocytes group (P=0.008),in which the head abnormal increased significantly (P=0.005). The above results suggested increased leukocytes can lead to a decline in semen quality and increased rates of sperm abnormality.
3. The effects of different biovars of U. urealyticum on ROS generation, lipid peroxidation of sperm membrane and DNA damage
(1) The effects of different biovars of U. urealyticum on ROS generation and lipid peroxidation.
To further explore the effect of abnormal increase of leukocyte caused by the biovar II on semen redox system, we compared the ROS, MDA and T-SOD levels in the semen of four groups(including71cases in biovar Ⅰ,40cases in biovar II,11cases in mixed infection and80cases in uninfected group). The results showed ROS levels (using the log-transformation) were significantly higher in biovar II (2.66±0.85) and mixed infection (2.82±1.37) groups than that in controls (1.94±0.79)(P<0.001, P=0.003; respectively), but no significant difference was observed between the biovar I group (2.20±0.94) and uninfected group(P=0.075). MDA in the biovar II (1.25±0.61nmol/l) and mixed infection (1.30±0.61nmol/l) groups were approximately35%higher than that in the negative group (0.92±0.12nmol/l)(P=0.004,P=0.040), but no significant difference was observed between the biovar I group (1.05±0.55) and uninfected group (P=0.226). Although T-SOD levels in biovar I (63.57±8.95), biovar II infection (62.71±7.10), mixed infections group (61.81±7.62) showed lower than the uninfected group(64.49±7.23), the differences among the four groups were not statistically significant (P=0.556).
(2) The effects of different biovars of U. urealyticum on sperm DNA damage.
Data of SCSA indicated there were significant increases of DFI%and HDS%in the biovar II (18.32±7.62;8.06±4.23) and DFI%in mixed infection groups (23.44±4.89) compared to the negative group (18.32±7.62;8.06±4.23)(P=0.003; P=0.001, P=0.032respectively), but no significant difference was observed between the biovar I group (20.03±7.21,8.92±3.60) and uninfected group (P=0.155; P=0.175). SCGE analysis, in terms of tail moments, showed higher motility in the biovar II (1.06±0.15) than in the uninfected group (0.94±0.12)(P<0.001), but no significant difference was observed between the biovar I group (0.96±0.11), mixed infection groups (1.05±0.15) and uninfected group (P=0.257, P=0.224).
Conclusion:
1. The infection rate by U. urealyticum showed a tendency to be higher in the infertile group than in the fertile group, but it did not reach statistical significance. However, stratification into different biovar infections revealed that the prevalence of biovar II in infertile men was significantly higher than that in fertile men. These results demonstrated the biovar II infection was more frequent in infertile men, suggesting the role in male infertility.
2. In this study, the semen volume, pH, spermatozoa count, motility, and morphology with the presence of biovar I and biovar II were compared. Only the total motility showed a significantly decrease between infected and uninfected individuals. Spermatozoa concentration in the biovar II group, total motility in the biovar II and mixed infection groups were significantly reduced (P<0.05).The above results suggested that compared with biovar I, biovar II had more significant impact on sperm parameters, expecially in sperm motility. The results of sperm movement parameters also showed that VSL, VCL, VAP, LIN and WOB in biovar II were significantly lower than the uninfected group. At the same time, leukocyte counts in the biovar II were significantly higher than that in the negative group, but no significant difference was observed between the biovar I group, mix infection group and uninfected group. The mechanisms of sperm motility decrease include U. urealyticum's strong ability of adhesion to sperm; ROS increase which can attack and induce lipid peroxidation, which disrupts the integrity of the plasmamembrane and impairs sperm motility. In addition, ROS can change the mitochondrial membrane potential, which may reduce sperm energy supply and eventually slow sperm movement. Combined with the results of semen parameters change and leukocytes elevation, increased ROS seemed to play a more important role in this process, because the significant reduction in sperm motility and increased leukocytes occurred in biovar II group at the same time, and leukocytes have been considered to be an important source of ROS in semen.
3. To verify the effect of leukocytes elevation on the spermatozoa quality, we compared the semen parameters, sperm motion parameters and sperm morphology between the normal leukocytes group and abnormal leukocytes group. The results showed that the semen parameters in the abnormal leukocytes group including total motility, progressive motility and normal forms, the sperm motion parameters including VCL, VSL, VAP and WOB decreased significantly compared with normal leukocytes group. Meanwhile the ratio of sperm abnormality (especially the head abnormality) in the abnormal leukocytes group increased significantly compared with the normal leukocytes group. All these data suggested that excessive leukocytes may result in the decrease of spermatozoa quality.
4. Further, we explored the effect of abnormal increase of leukocyte caused by the biovar II infection on semen redox system. We compared the ROS, MDA and T-SOD levels in the semen of four groups. The results showed ROS levels (using the log-transformation) were significantly higher in biovar II and mixed infection groups than that in uninfected group, but no significant difference was observed between the biovar I and uninfected group. The data indicated that the biovar II infection may increase the leukocyte counts in the semen and subsequently produce excessive ROS. Two mechanisms were involved in the excessive ROS generation caused by U urealyticum infection. First, the most toxic agents for spermatozoa are the metabolic products of U urealyticum, which include H2O2and ammonia (NH3). Although H2O2is itself harmful to sperm, it is also a source of hydroxide anion (OH), which is a highly toxic radical for cell membranes. Furthermore, Ureaplasma phospholipases A and C may influence changes in the lipid composition of the cell membranes of male gametes, leading to loss of integrity and increased permeability. Second, U urealyticum can induce ROS generation through increasing leukocyte concentration in semen. Our results suggested that leukocytes elevation may be more responsible for the ROS increase. MDA results also supported this view, and indicated biovar II was more likely to cause sperm membrane lipid peroxidation. But results of T-SOD showed no significant difference between infected and non-infected group.
5. Our study showed the biovar II and mixed infection groups displayed elevated DFI%and HDS%values in the SCSA assay. The data of significant increase compared to the negative group strongly suggested DNA damage caused by biovar II infection may damage sperm DNA integrity and interfere with male fertility.
Innovation points:
1. Investigated the prevalence of U. urealyticum and its different biovars in the infertile and fertile men, demonstrated the biovar II infection was more frequent in infertile men.
2. Studied the impacts of two biovars of U. urealyticum infection on sperm parameter and the role of leukocytes in the progress, found spermatozoa concentration and total motility in biovar II and mix infection groups were lower significantly than in the U. urealyticum negative group; cases with leukocytes increasing in biovar II were significantly higher.
3. Explored the effects of two biovars of U. urealyticum on semen redox system(including ROS, MDA and T-SOD) as well as sperm DNA damage, suggested that leukocyte-induced ROS elevation owing to the biovar II infection may be more responsible for spermatozoa membrane and DNA damage.
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