精子DNA损伤与不明原因复发性流产的关系

目的:探讨不明原因复发性流产与男方精子DNA损伤的关系。方法:精子DNA断裂损伤检测采用精子染色质扩散试验(sperm chromatin dispersion,SCD),以DNA断裂指数(DNA fragmentation index,DFI)表示。分别测定不明原因复发性流产(试验组,56例)男方与无流产史(对照组,31例)男方精子的DFI。结果:试验组DFI(11.0%～56.9%)高于30%的有21例(37.5%),对照组DFI(10.0%～36.8%)高于30%的有8例(25.8%)。复发性流产组的男方精子DFI均数比对照组精子DFI高(29.4%vs 25.5%),且差异显著(P<0.05)。结论:不明原因复发性流产可能与精子DNA损伤存在一定关系。     

Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions

The aim of this study was to examine the possible relationship between sperm DNA integrity and chromatin packaging evaluated by cytochemical assays, traditional sperm parameters and recurrent spontaneous abortion (RSA) of unknown origin. In this cohort study, 40 couples with a history of RSA and 40 couples with proven fertility were considered as case and control groups respectively. The semen samples of all husbands were analysed for sperm parameters and also sperm chromatin and DNA integrity assessed using cytochemical tests including aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), acridine orange (AOT) and nuclear chromatin stability assay. Among different sperm parameters, only slow motility was significantly different between the two groups. In sperm chromatin evaluations, there were significant differences between the two groups in all of the tests. In addition, the majority of semen samples in RSA patients exhibited upper percentages of abnormal spermatozoa than the cut-off values regarding different cytochemical assays. Our study showed that in the cases of RSA, slow motility had a significant reduction in comparison with controls and also spermatozoa of men from RSA group had less chromatin condensation and poorer DNA integrity than spermatozoa that obtained from fertile men with no history of RSA.     

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。     

Sperm DNA fragmentation,recurrent implantation failure and recurrent miscarriage

Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.     

Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss

Previous studies have indicated that sperm quality may be related to unexplained recurrent pregnancy loss. This study evaluated the degree of sperm DNA fragmentation using the TUNEL assay on sperm from 24 couples with unexplained recurrent pregnancy loss (RPL) compared to sperm from 2 control groups: donors of known fertility and unscreened men from the general population. The percentage of sperm staining positive for DNA fragmentation was increased (p < .001) in the RPL group (38 +/- 4.2) compared to the donor (11.9 +/- 1.0) or general population (22 +/- 2.0) control groups. In the RPL group, no correlation was observed between semen quality parameters and the TUNEL data. These data indicate that some RPL patients have a significant increase of sperm DNA fragmentation, which may be causative of pregnancy loss in some patients.     

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）.     

Sperm ubiquitination and DNA fragmentation in men with occupational exposure and varicocele

Assessment of sperm ubiquitination and DNA fragmentation as sperm functional markers are proposed to complement routine semen analysis. This study focuses on the evaluation of these markers in infertile men with varicocele or exposed to occupational background. The results were compared with normozoospermic men. Semen parameters in both groups were lower than those in the control group. Ubiquitination median, as a marker for functionality of the ubiquitin–proteasome system, was also lower in both groups. The ubiquitination median showed a significant positive correlation with motility in both groups, while it showed only a negative correlation with sperm morphology in the varicocele group. DNA fragmentation showed a significant correlation with semen parameters, in total varicocele and also total exposure groups. In conclusion, significant difference of sperm ubiquitination between normal and study groups further validates that sperm ubiquitination as a potential molecular marker for sperm evaluation in addition to routine semen analysis in clinical laboratories.     

精子DNA碎片与复发性流产相关性的系统评价

目的系统评价精子DNA碎片与复发性流产之间的相关性。方法以"Sperm DNA integrity","The sperm DNA integrity","sperm DNA fragmentation index","sperm DNA fragmentation","SDF","DFI","Habitual Abortion","Habitual Abortions","Miscarriage,Recurrent","Recurrent Miscarriage","Recurrent Miscarriages","Abortion,Recurrent","Recurrent Abortion","Recurrent Abortions","Recurrent Early Pregnancy Loss","recurrent spontaneous abortion"为主题词及自由词检索PubMed、The Cochrane Library、Embase、Web of science,检索时间为各数据库建库至2019年7月10日,对最终纳入的非随机对照研究根据纽卡斯尔-渥太华评分(NOS)进行质量评价,运用系统评价方法阐明两者之间的相关性。结果经过仔细的搜索和排除后,最后纳入28篇非随机对照研究。结果显示:应用Acridine orange test(AO)、Comet assay(Comet)、Sperm Chromatin Structure assay(SCSA)、Sperm Chromatin Dispersion(SCD)、The terminal deoxyuridine nick labeling assay(TUNEL)方法测定精子DNA碎片,复发性流产与精子DNA碎片存在明显的相关性。结论复发性流产患者配偶精子DNA碎片指数是高于配偶无复发性流产史的正常生育男性。     

Analysis of sperm chromosomal aneuploidy and DNA integrity in infertile couples with unexplained recurrent miscarriage

Objective: To evaluate sperm chromosome aneuploidy and DNA fragmentation in infertile couples with unexplained recurrent miscarriage (URM).Methods: Multi-color fluorescence in situ hybridization (FISH) protocol was performed with the probes specific for the chromosomes 13, 18, 21, X and Y in the control group (n= 5) and the infertile patients with URM (n=12). The level of DNA fragmentation was determined by Sperm Chromatin Dispersion (SCD) test in the patients with URM (n=48) and the fertile men (control group, n=32).Results: The patients with URM had total numerical abnormality rate of 3.91% (n=12) for the chromosomes 13, 18 and 21 and 1.98% for the chromosomes X and Y respectively, which significantly higher than that of the control group (1.29% and 0.61%, n = 5, P<0.01). A proportion of total sperm DNA fragmentation was detected in infertile patients with URM (2.1±10.3%) (n = 48), which significantly higher than the control group(2.1±5.2%, P<0.01).Conclusions: Spermatozoa from couples with URM contained high rates of DNA fragmentation and chromosomal aneuploidy. Screening for sperm chromosomal aneuploidy and sperm DNA fragmentation may provide the useful information for investigating male unexplained infertility.     

Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations

We herein summarise the evidence concerning the impact of sperm DNA fragmentation in various clinical infertility scenarios and the advances on sperm DNA fragmentation tests. The collected evidence was used to formulate 41 recommendations. Of these, 13 recommendations concern technical aspects of sperm DNA fragmentation testing, including pre-analytical information, clinical thresholds and interpretation of results. The remaining 28 recommendations relate to indications for sperm DNA fragmentation testing and clinical management. Clinical scenarios like varicocele, unexplained infertility, idiopathic infertility, recurrent pregnancy loss, intrauterine insemination, in vitro fertilisation/intracytoplasmic sperm injection, fertility counselling for men with infertility risk factors and sperm cryopreservation have been contemplated. The bulk evidence supporting the recommendations has increased in recent years, but it is still of moderate to low quality. This guideline provides clinicians with advice on best practices in sperm DNA fragmentation testing. Also, recommendations are provided on possible management strategies to overcome infertility related to sperm DNA fragmentation, based on the best available evidence. Lastly, we identified gaps in knowledge and opportunities for research and elaborated a list of recommendations to stimulate further investigation.     

Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables

OBJECTIVE

To present the first UK data on sperm DNA fragmentation levels in subfertile men and fertile controls, the correlation with semen variables, and to assess the effect on the outcome of intracytoplasmic sperm injection (ICSI).

PATIENTS, SUBJECTS AND METHODS

In all, 56 subfertile men undergoing ICSI (28 with positive and 28 with a negative outcome for paternity) and 10 control fertile semen donors were recruited. The sperm DNA fragmentation index (DFI) was assessed on raw pre‐preparation samples using the sperm chromatin structure assay. A mean of 5212 sperm were analysed per sample and DFI data are presented by fertility status, ICSI outcome and correlated with semen variables (assessed using World Health Organisation criteria).

RESULTS

Total DFI was significantly higher in subfertile men than in fertile controls (mean and median of 22.8% and 17.0% vs 8.4% and 5.0%; P < 0.001), as was the proportion of both moderate DFI (16.4% and 13.0% vs 6.4% and 4.0%; P = 0.001) and high DFI (6.2% and 6.1 vs 2.0% and 1.0%; P = 0.01). This difference remained significant when the control men were compared only with the subfertile men with successful paternity. There was no significant difference in DFI in the subfertile men when analysed by ICSI outcome (mean and median of 24.5% and 17.0% vs 22.3% and 21.0% for successful and unsuccessful cycles, respectively; P = 0.94). There was a positive statistically significant correlation (r = 0.37; P = 0.02) between the DFI and sperm morphology.

CONCLUSIONS

This study confirms a relationship between male subfertility and sperm DFI; we discuss the correct role for genetic testing of sperm in the evaluation of subfertile men. Although DNA fragmentation data might help to decide a suitable treatment, once it is decided to proceed with ICSI, DFI levels have no effect on the outcome.     

Elevated sperm DNA fragmentation does not predict recurrent implantation failure

In this study, we sought to determine whether sperm DNA fragmentation (DFI%) and high DNA stainability (HDS%) evaluated by sperm chromatin structure assay (SCSA) predict recurrent implantation failure (RIF) or pregnancy rate. A retrospective study was performed of consecutive cycles of ICSI treatment from 2009 to 2018. A total of 386 couples that underwent 1,216 frozen embryo transfer (FET) cycles were analysed. Mean female and male age was 34 ± 3.6 years and 37.3 ± 6.6 years, respectively, and a median total motile sperm count (TMSC) was 43.5 [9.9–105.5] million. Overall median DFI% and HDS% was 12 [7.1–18.9] and 9.6 [6.5–14.4] respectively. On multivariable analysis, DFI% and HDS% were not associated with RIF (DFI%: OR = 1.01, 95% CI: 0.98–1.04, p = .414; HDS%: OR = 0.97, 95% CI: 0.94–1.01, p = .107) or IVF success, defined as clinical pregnancy (DFI%: OR = 1.00, 95% CI: 0.99–1.01, p = .641; HDS%: OR = 1.01, 95% CI: 0.99–1.02, p = .565). We found that neither DFI% or HDS%, as assessed by SCSA, were predictive of RIF or pregnancy rate. This finding suggests that sperm DNA fragmentation does not predict RIF or pregnancy rate.     

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human‐mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation.     

Sperm nuclear histone H2B： correlation with sperm DNA denaturation and DNA stainability

Aim： To examine the relationship between sperm DNA damage and sperm nuclear histone （H2B） staining. Methods： We evaluated sperm samples from 14 consecutive asthenoteratozoospermic infertile men and six consecutive fertile controls. Sperm nuclear histone （H2B） staining and sperm chromatin integrity （assessed by sperm chromatin structure assay and expressed using the percentage of （i） DNA fragmentation index [%DFI] and （ii） high DNA stainability [%HDS）]） were evaluated. Results： Histone H2B immunocytochemistry demonstrated two nuclear staining patterns： （i） focal punctate staining; and （ii） diffuse staining. Infertile men had a higher mean percentage of spermatozoa exhibiting diffuse H2B staining than did fertile men （7.7%± 4.6% vs. 1.6% ±1.2%, respectively, P 〈 0.01）. We observed significant relationships between the proportion of spermatozoa with diffuse nuclear histone staining and both sperm %DFI （r = 0.63, P 〈 0.01） and sperm %HDS （r = 0.63, P 〈 0.01）. Conclusion： The data demonstrate that infertile men have a higher proportion of spermatozoa with diffuse histone H2B than do fertile men and suggest that sperm DNA damage might, at least in part, be due to abnormally high histone H2B levels.     

Sperm nuclear DNA fragmentation and its association with semen quality in Greek men

Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non‐normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non‐normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility.     

Reciprocal balanced translocation: infertility and recurrent spontaneous abortions in a family

In this case report we present a family with infertile, azoospermic but otherwise apparently healthy males with history of recurrent spontaneous abortions (RSA) in females. Karyotype of the infertile man revealed a reciprocal balanced translocation t(8; 13) with breakpoints at 8q22 and 13p11.2. The reported reciprocal balanced translocation is associated with azoospermia. The same translocation is probably the cause of RSA in females of the family.     

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility – idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.     

不明原因复发性流产女性FMR1基因CGG重复序列多态性研究

目的 探讨不明原因复发性流产女性脆性X智力障碍1(FMR1)基因CGG重复序列多态性分布特点.方法 收集2018年1月1日至2019年12月31日在石家庄市第四医院复发性流产门诊就诊的具有不明原因复发性流产史的340例患者为研究组;同期在石家庄市第四医院妇科门诊就诊的340例年龄匹配的无流产史女性为对照组.抽取外周血,...     

Oxidative stress status and sperm DNA fragmentation in fertile and infertile men

Evidence suggests that disturbing the balance between reactive oxygen species levels and antioxidant contents in seminal plasma leads to oxidative stress resulting in male infertility. This study was carried out to identifying clinical significance of seminal oxidative stress and sperm DNA fragmentation in treatment strategies of male infertility in southwest Iran. Sperm parameters, lipid peroxidation and activity of antioxidant enzymes were assessed in fertile (n = 105) and infertile (n = 112) men. Malondialdehyde (MDA) levels in seminal plasma were found to be higher significantly (p < .001) in patients. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in seminal plasma were significantly (p < .001) lower in infertile men. Significant negative correlations were observed between MDA levels and sperm motility and normal morphology. Spermatozoa with fragmented DNA were higher (p < .001) in infertile men and significantly correlated with MDA levels and SOD and GPx activities. MDA of 4.2 nmol/ml, SOD of 4.89 U/ml and GPx of 329.6 mU/ml were optimum cut‐off limits to discriminate infertile patients from fertile men. The results show the leading role of oxidative stress in aetiology of male infertility in southwest Iran and indicate that evaluation of seminal antioxidant status and DNA integrity can be helpful in men attending infertility clinics during fertility assessment.     

Sperm DNA fragmentation in donors and normozoospermic patients attending for a first spermiogram: Static and dynamic assessment

Static assessment of sperm DNA Fragmentation (SDF at the time of ejaculation or sperm thawing when cryopreserved) and the dynamic assessment of SDF (SDF assessed after T2 hr, T6 hr and T24 hr of sperm thawing) were used to establish cut‐off values associated with sperm donors when compared with closely related normozoospermic patients. Cryopreserved samples from donors revealed SDF levels two times lower in comparison with the patients. Donor sperm DNA exhibited a 2.5 times higher longevity when compared with the patients. Static values of SDF after thawing of approximately 11% identify the donors with a 71% of sensitivity and 84% specificity. With respect to the dynamic assessment, SDF increases of 2.3 per hr during the first 2 hr of incubation identify the donors with 70% of sensitivity and 66% of specificity. Creating the Rate of Combined Damage (RCD) defined as the product of SDF‐T₀ by the increase in the damage registered during the first 2 hr of incubation (r‐SDF‐T_0–2), an index of RCD = 22.2 units has an identification capacity of donors with a 78% sensitivity and 77% specificity. Such cut‐off values could be used to characterise donors with high chromatin resistance to damage when meeting the above‐established criteria.