Localisation and quantification of alkali‐labile sites in human spermatozoa by DNA breakage detection–fluorescence in situ hybridisation

The localisation and quantification of constitutive alkali‐labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD–FISH) and alkaline single‐cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD–FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility.     

精子DNA碎片指数和精子畸形率对ICSI临床结局的影响

目的:通过评估ICSI治疗前的精子畸形率(SMR)和精子DNA碎片指数(DFI),探讨精子DFI和SMR对卵胞浆内单精子注射(ICSI)助孕结局的影响。方法:共入组79对因少弱精子症实施第一周期ICSI治疗的不孕夫妇,在进入治疗周期前3⁶个月,评价精子浓度、前向运动精子百分率、SMR及DFI。主要观察SMR和DFI与ICSI结局参数的关系。结果:79例少弱精子症患者DFI正常51例,异常28例,异常组的DFI值明显升高(14.18%vs 41.47%);巧合的是,SMR正常组同样为51例,异常组28例,异常组的SMR值亦明显升高(87.88%vs98.46%)。按DFI正常(DFI≤25%)与异常(DFI>25%)分组,或按SMR正常(≤96%)与异常(>96%)分组,组间的双方年龄、女方BMI、获卵数、移植胚胎数等基本情况差异无统计学意义。DFI正常和异常组间,SMR正常和异常组间的受精卵子数、可移植胚胎数、早期流产率无显著差异;异常组生化妊娠率(43.5%vs 61.5%)和临床妊娠率(39.1%vs 56.4%)降低,但差异无统计学意义(P=0.19及0.10)。精子DFI与SMR呈显著正相关(r=0.231,P<0.05)。结论:精子DFI增高(>25%),与按严格标准检测的SMR增高(>96%)男性行ICSI治疗,生化妊娠率和临床妊娠率降低,但与正常者比较未发现有统计学差异,可能与样本量小有关,有必要深入研究。     

DNA fragmentation assessment by flow cytometry and Sperm-Bos-Halomax (bright-field microscopy and fluorescence microscopy) in bull sperm

The aim of this study was to find the relationship between fertility (as 90-day non-return rates) and DNA fragmentation assessed by two techniques [sperm chromatin structure assay (SCSA) and Sperm-Bos-Halomax (SBH)]. Furthermore, other quality parameters were achieved (motility, morphological abnormalities, cytoplasmic droplets, viability, capacitation and acrosomal and mitochondrial status) and their correlations with fertility were analysed. Bulls were divided into three fertility groups: high [non-return rate (NRR) >or= 80], medium (80 < NRR >or= 70) and low (70 < NRR > 40). The results of this study indicate that there is a good correlation between fertility and different parameters of sperm quality (SBH and SCSA parameters, % of spermatozoa with head, neck and total abnormalities, and % of spermatozoa with proximal cytoplasmic droplets) and differences between fertility groups were observed in some of them (SBH and SCSA parameters and % of spermatozoa with head, neck and total abnormalities). In this sense, SBH parameters rendered good correlations with fertility (r = -0.42 using bright light microscope and r = -0.47 with fluorescence). Also, standard deviation of DNA fragmentation index (SD-DFI) and DFIh (cells with High DNA fragmentation index) showed good correlations with fertility (r = -0.41 and r = -0.29). No correlations were observed between SCSA and SBH parameters. A multiple regression shows that four parameters (% of proximal cytoplasmic droplets, % of intact acrosomes in total population, SD-DFI and percentage of fragmented DNA detected by bright light microscope) present a good predictive value of the fertility of sperm samples (r(2) = 0.34, p < 0.001).     

Human sperm DNA integrity in normal and abnormal semen samples and its correlation with sperm characteristics

Reports indicate an increase in the incidence of DNA fragmentation in male factor infertility and its role in the outcome of assisted reproductive techniques (ART). However, reports are conflicting between the relationships of sperm DNA integrity with conventional semen parameters. We examined the relationship between conventional sperm parameters and DNA integrity using acridine orange (AO) test. The study included 373 patients and 28 fertile volunteers. DNA normality was compared with semen parameters between the patient and donor populations. Significant correlations were noted between DNA normality and sperm concentration ( r  = 0.18, P  = 0.000), motility ( r =  0.21, P  = 0.0001), rapid motility (0.19, P  = 0.000), normal morphology by World Health Organization ( r =  0.15, P  = 0.019) and head defects ( r =  −0.15, P  = 0.023). A significant difference was noted in AO levels between donors and patients with asthenozoospermia ( P  = 0.002) and oligoasthenozoospermia ( P  = 0.001). A significant difference in DNA integrity was noted in samples having <30% and >30% normal morphology. A wide range of % DNA normality was observed in the patient group. Sperm assessment for DNA status using AO is reliable and shows good correlation with sperm count, motility and morphology. Assessment of sperm DNA status with AO staining may be helpful prior to ART.     

Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation

Varicocele has been associated with reduced male reproductive potential. With the advances in biomolecular techniques, it has been possible to better understand the mechanisms involved in testicular damage provoked by varicocele. Current evidence suggests the central role of reactive oxygen species (ROS) and the resultant oxidative stress (OS) in the pathogenesis of varicocele-associated male subfertility although the mechanisms have not yet been fully described and it is likely to be multifactorial. Excessive ROS is associated with sperm DNA fragmentation, which may mediate the clinical manifestation of poor sperm function and fertilization outcome related to varicocele. Testing of ROS/OS and DNA fragmentation has the potential to provide additional diagnostic and prognostic information compared to conventional semen analysis and may guide therapeutic management strategies in individual patient.     

Impact of cigarette‐smoking on sperm DNA methylation and its effect on sperm parameters

DNA methylation is an epigenetic modification of the genome. The purpose of this study was to determine the influence of cigarette‐smoking on sperm DNA methylation from a genomewide survey of sperm samples and to ascertain its effect on sperm parameters. Twenty‐eight sperm DNA samples (from 14 fertile smokers as a case study and 14 proven fertile nonsmokers as controls) were subjected to Infinium 450K BeadChip arrays to identify the changes in the DNA methylation level between the two groups. Then, deep bisulphite sequencing was used to validate five CpGs on 78 samples. The results from the Infinium 450K found that only 11 CpGs showed a significant difference in DNA methylation between the case and the control groups. Five CpGs of the eleven (cg00648582, cg0932376, cg19169023, cg23841288 and cg27391564) underwent deep bisulphite sequencing where cg00648582, related to the PGAM5 gene, and the cg23841288 CpGs, related to the PTPRN2 gene amplicons, showed a significant increase in their DNA methylation level in more than one CpG in the case group. In contrast, a significant decrease was found at cg19169023 and at its various neighbouring CpGs in the TYRO3 gene‐related amplicons. Furthermore, this study demonstrated a significant correlation between the variation in sperm DNA methylation level and standard sperm parameters in the case group.     

Relationship between sperm quality and chromatin condensation measured by sperm DNA fluorescence using flow cytometry

DNA flow cytometry of sperm from 100 randomly chosen men undergoing fertility investigation revealed a general association between reduced sperm quality, as judged by conventional parameters, and the appearance of sperm with lower degrees of chromatin condensation in the ejaculate as measured by DNA fluorescence intensity. Chromatin hypocondensation, as measured by increased fluorescence, was manifested to different degrees in different samples. In many cases of more extreme sperm pathology, such as oligoasthenoteratozoospermia (OAT), the whole population of spermatozoa appeared to be affected. Significant numbers of hypercondensed spermatozoa were present in both normozoospermic men and men with different degrees of disturbance in sperm quality. All of the different parameters of sperm quality could be correlated significantly with certain of the flow parameters, although not one in particular could be used to predict deviations from the normal flow profile. In several asthenoteratozoospermic men and a small proportion of men with OAT, the DNA profiles were normal, implying that in these cases the disturbance may not be so fundamental. The presence of leucocytes in the ejaculate was associated with a general increase in the preponderance of hypocondensed subpopulations of spermatozoa in men with OAT as well as in normozoospermic subjects, emphasizing the effect of inflammatory conditions in the reproductive tract on sperm quality.     

Influence of extended incubation time on Human sperm chromatin condensation,sperm DNA strand breaks and their effect on fertilisation rate

The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double‐strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double‐strand breaks (r = ?0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates.     

Aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity

The purpose of this study was to evaluate the aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity. A total of 108 males (65 males suffering from reduced fecundity as cases and 43 proven fertile males as a control) were included in the study. Thirty samples were subjected to 450K arrays as a screening phase, and then, three CpG sites located in the following genes: TYRO3, CGβ and FAM189A1 were selected to validate on 78 samples using deep bisulphite sequencing. A significant difference in the methylation level was found between cases and controls at all CpGs in TYRO3 gene‐related amplicon (CpG1, p ≤ .003, CpG2, p ≤ .0001, CpG3, p ≤ .003 and CpG4, p ≤ .030) and CpG1 in CGβ gene‐related amplicon (p ≤ .0001). Besides, a significant difference was found at two CpGs (CpG1, p ≤ .004 and CpG2, p ≤ .002) tested in the FAM189A1 gene‐related amplicon. A significant correlation was found between the methylation level at CpG1 in the FAM189A1 gene and the different types of sperm motility. In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was showed. In addition, a relationship between variations in the methylation level of these CpGs and sperm motility has been observed.     

吸烟对精子染色质结构完整性影响的探讨

目的:探讨吸烟对精子染色质结构完整性的影响。方法:784例男性不育患者从病例库中选取,根据吸烟与否及每日吸烟支数(≤10、11~19、≥20)和吸烟年限(≤5、6~9、≥10)进行分组,比较各组患者精液常规检测参数及精子染色质结构受损和精子核成熟情况。精子染色质结构完整性采用流式细胞仪检测,DNA损伤程度和不成熟精子指数分别以DNA损伤指数(DFI)和高DNA着色性(HDS)来表示。结果:吸烟≥20支/日和吸烟年限≥10年的患者组精液量、前向运动精子比例低于其他组而精子畸形率高于其他组(P<0.05);吸烟组男性的DFI和HDS值均升高(P<0.05);HDS与前向运动精子比例呈负相关(r=-0.18,P<0.05),DFI与HDS均与精子畸形率呈正相关(r=0.31与r=0.39,P均<0.05)。结论:日吸烟量≥20支或吸烟年限≥10年对男性的精液量、前向运动精子比例和精子畸形率都有影响,吸烟影响男性精子DNA完整性和精子核成熟。     

Effects of microsurgical varicocelectomy on semen analysis and sperm function tests in patients with different grades of varicocele: Role of sperm functional tests in evaluation of treatments outcome

It seems that varicocele play a role in male infertility, as such, their prevalence increases from 15% in the normal population to 80% in secondary infertility subjects. Varicoceles may have negative effects on semen quality. Our goal was to assess the effects of microsurgical varicocelectomy on semen analysis and sperm functional tests in men with different grades of varicoceles. Thirty infertile men with different grades of varicoceles (grades 1 to 3) were enrolled in our study. Semen quality was assessed by semen analysis according to the WHO guideline (WHO, 1999) and four different sperm functional tests (aniline blue, toluidine blue, chromomycin A3 and TUNEL test) were carried out before and 3 months after microsurgical varicocelectomy (M‐varicocelectomy). When considered all three grades together, we showed that M‐varicocelectomy had statistically significant effects on all four types of sperm functional tests (p value<0.05). It also had positive effects on conventional semen parameters, although the effects were not statistically significant for some parameters (for example sperm count). When analysed separately (based on varicocele grades) the surgery, although caused improvements in semen quality, but may have more statistically significant effects on patients with varicocele of higher grade. In addition, in varicocele of lower grade (for example grade 2), sperm function test may be a better predictor of surgical success than the conventional semen analysis. Thus, we show that not only M‐varicocelectomy has significant positive effect on semen quality but also if sperm functional tests become more affordable in the future, because they yield more precise results, their use in daily practice may increase significantly in patients with varicoceles.     

Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage

Abnormal imprinted genes methylation in spermatozoa has been shown to be associated with subfertility. However, the relationship between sperm DNA damage and specific imprinted genes methylation remains unclear. In this study, DNA methylation levels were determined at seven imprinted genes loci (H19, INS‐IGF2, KCNQ1, MEG3, MEST, PEG3 and SNRPN) in 66 semen samples using the MSRE‐qPCR method. The semen samples were divided into two groups according to the threshold value (25%) of DNA fragmentation index (DFI). We found that the mean methylation level at IGF2 (cg17037101) in the group with DFI ≥ 25% was lower than that in the group with DFI < 25% (13.7 ± 3% vs. 31.5 ± 5.3%, p = 0.0053). However, the methylation levels of other CpGs did not differ from the imprinted genes. Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2 (cg17037101) methylation level was negatively correlated with sperm DFI (r = ?0.448, p = 0.0038), and the KCNQ1 (cg24932449) methylation level was positively correlated with sperm DFI (r = 0.354, p = 0.0273). Our results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.     

微流控芯片精子分选法对精子常规参数及DNA完整性的影响

目的:研究微流控芯片精子优选技术对精子常规参数及DNA完整性的影响。方法:自行设计制作微流控芯片,利用芯片处理技术和上游法对40例精液标本进行精子优选,通过计算机辅助精液分析系统及染色质扩散试验从精子常规参数及DNA完整性两个方面评价精液体外处理对精子的影响。结果:精液经微流控芯片法和上游法处理后,精子活力、精子正常形态率以及精子尾部肿胀率均有显著提高(P<0.01),精子的DNA损伤率明显降低(P<0.01)。微流控芯片法与上游法相比,优选后前者精子DNA损伤率明显低于后者[(8.4±5.8)%vs(16.4±9.2)%,P<0.01],而其他参数差异无显著性。结论:微流控芯片技术在精子优选中能获得精子DNA损伤程度小的高质量精子。     

反复自然流产与精子DNA完整性的相关性研究

目的 探讨反复自然流产与精子DNA完整性的相关性。方法 按纳入标准将研究对象分成反复自然流产患者丈夫组（n=58）和正常生育男性对照组（n=50），采用吖啶橙实验（AOT）对研究对象精子DNA完整性进行检测，并对研究对象精液进行常规分析。砖果两组研究对象年龄、吸烟史、饮酒史无统计学差异（P〉0．05）；反复自然流产患者丈夫组精液的精子密度、精子活力显著低于正常生育对照组（P〈O．05）；反复自然流产患者丈夫组精子DNA的完整性低于正常生育对照组，差异有统计学意义（P〈O．05）。砖论精子DNA完整性损伤是反复自然流产的可能原因，精子DNA完整性损伤导致反复自然流产的致病机理尚需进一步研究。     

Aneuploidy and DNA fragmentation in morphologically abnormal sperm

In light of the relative success of ICSI in the treatment of male infertility, much importance has been made to the selection of morphologically viable sperm. However, correlation between specific sperm morphology and chromosomal abnormalities is still relatively limited and less is known about the connection between sperm morphology and DNA integrity. Sperm obtained from isolated teratozoospermic men ( n  = 10) and control men ( n  = 9) were analysed using FISH (for chromosomes 13, 18, 21, X and Y) and TUNEL assays to determine the level of aneuploidy and DNA fragmentation. Sperm morphology was evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. Sperm from teratozoospermic men, compared with fertile men, had higher rates of total chromosomal abnormality ( p  < 0.05), total aneuploidy ( p  < 0.01) and chromosome 13 disomy ( p  < 0.01). Associations between particular types of sperm morphology and chromosomal abnormalities were observed in both control (tapered heads) and teratozoospermic (amorphous heads and tail abnormalities) samples. Levels of DNA fragmented sperm were higher in teratozoospermic men than in the control men (60.28 ± 21.40% vs. 32.40 ± 17.20%, p  < 0.05) and positively correlated to sperm with bent necks in control samples and round heads in teratozoospermic samples ( p  < 0.05). Sperm of isolated teratozoospermic men have higher rates of chromosomal abnormalities and DNA fragmentation than that of the fertile controls. Specific abnormal sperm morphology can be correlated to chromosomal abnormalities and level of DNA fragmentation in sperm.     

DNA methylation level of spermatozoa from subfertile and proven fertile and its relation to standard sperm parameters

The epigenetic mechanism plays an important role in spermatogenesis such as DNA methylation where this episode is represented by either switching genes on or off. Twenty‐eight samples (14 case and 14 controls) were subjected to Infinium 450K BeadChip arrays to identify genomic regions that differ in sperm DNA methylation patterns in the subfertile compared to the proven fertile group. Then two CpGs were validated by deep bisulphite sequencing on 82 sperm samples. The results screening study revealed eight CpGs were significantly different in their sperm DNA methylation levels between cases and control group. The results of the validation study for the two CpGs (cg19779893 and cg19406113) showed that a significant variation in the methylation level at 2 CpGs of 3 CpGs related to cg19779893 site amplicon in cases compared to the controls. Moreover, six CpGs related to the cg19406113 site amplicon showed significant differences in sperm DNA methylation between the cases and the control group. Furthermore, there was a significant decrease in the sperm parameters in the cases compared to the control group. This study found two CpGs altered in their sperm DNA methylation levels. In addition, a strong association was found between changes in the sperm DNA methylation levels in these CpGs sites and sperm parameters.     

Fluorescence in situ hybridisation sperm examination is significantly impaired in all categories of male infertility

To study the outcome of FISH sperm examination in cases with sperm pathology and outline the potential correlation with certain chromosomal defects. A retrospective study of prospectively collected data was performed in IAKENTRO, Infertility Treatment Center. Rates of abnormal FISH semen examination were compared between male infertility patients and fertile controls. Detection of abnormal FISH semen examination as well as each chromosomal abnormality detected was correlated with each sperm deficiency (asthenozoospermia, oligozoospermia and teratozoospermia) in a univariate regression model. There were 72 male partners included, of which 52 male infertility patients and 20 controls. The rate of abnormal sperm FISH examination was significantly higher in patients’ group (55.8% vs. 15.0% for controls, p = .002). Asthenozoospermia, oligozoospermia and teratozoospermia were significantly correlated with detection of abnormal FISH examination (p = .004, p = .01 and p < .001 respectively). Teratospermia was significantly correlated with increased aneuploidy rate for chromosome 17 (p = .005), chromosome X (p = .05) and Y (p = .03). FISH examination reveals pathology in a significant proportion of patients with sperm defects and should be recommended to achieve early detection of chromosomal defects that may postpone favourable reproductive outcome.     

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.     

Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, the comet assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay.     

Sperm DNA damage in men from infertile couples

Aim： To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. Methods： A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay （SCSA） were carried out. Results： Ninety-seven men （28% of the whole study group） had a DNA fragmentation index （DFI） 〉 20%, and 43 men （12%） had a DFI 〉 30%. In the group of men with abnormal semen parameters （n = 224）, 35% had a DFI 〉 20%, and 16% had a DFI 〉 30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters （n = 126）. Men with low sperm motility and abnormal morphology had significantly higher odds ratios （ORs） for having a DFI 〉 20% （4.0 for motility and 1.9 for morphology） and DFI 〉 30% （6.2 for motility and 2.8 for morphology） compared with men with normal sperm motility and morphology. Conclusion： In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique （ART）.