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）.

Novel association between sperm deformity index and oxidative stress-induced DNA damage in infertile male patients

Aim:To investigate the impact of abnormal sperm morphology using the sperm deformity index (SDI) on reactive oxygen species (ROS) production and its correlation with sperm DNA damage.Methods:Semen samples were collected from men undergoing infertility screening (n=7) and healthy donors (n=6).Mature spermatozoa were isolated and incubated with 5 mmol/L β-nicotinamide adenine dinucleotide phosphate (NADPH) for up to 24 h to induce ROS.Sperm morphology was evaluated using strict Tygerberg's criteria and the SDI.ROS levels and DNA damage were assessed using chemiluminescence and terminal deoxynucleotidyl transferase-mediated fluorescein- dUTP nick end labeling (TUNEL) assays,respectively.Results:SDI values (median [interquartiles]) were higher in patients than donors (2 [1.8,2.1] vs.1.53 [1.52,1.58],P=0.008).Aliquots treated with NADPH showed higher ROS levels (1.22 [0.30,1.87] vs.0.39 [0.10,0.57],P=0.03) and higher incidence of DNA damage than those not treated (10 [4.69,24.85] vs.3.85 [2.58,5.10],P=0.008).Higher DNA damage was also seen following 24 h of incubation in patients compared to donors.SDI correlated with the percentage increase in sperm DNA damage following incubation for 24 h in samples treated with NADPH (r=0.7,P=0.008) and controls (r=0.58,P=0.04). Conclusion:SDI may be a useful tool in identifying potential infertile males with abnormal prevalence of oxidative stress (OS)-induced DNA damage.NADPH plays a role in ROS-mediated sperm DNA damage,which appears to be more evident in infertile patients with semen samples containing a high incidence of morphologically abnormal spermatozoa.

Comparative study of the effects of three semen preparation media on semen analysis,DNA damage and protamine deficiency,and the correlation between DNA integrity and sperm parameters

Semen samples collected from 28 male partners of infertile couples were divided into three equal aliquots and prepared with three selected media,such as PureSperm （Nidacon,Gothenburg,Sweden）,Sil-Select Plus^TM （Fertipro,Beemem,Belgium） and SpermGrad^TM（Vitrolife,Gothenburg,Sweden）. The differences in mean percentages of semen parameters were assessed by repeated measures analysis. Correlations of sperm DNA damage,as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay,and of protamine deficiency,as measured by chromomycin A3 （CMA3） staining with sperm parameters,were determined by Pearson＇s correlation. After preparation with all three media,sperm concentrations decreased （P〈0.05） while percentages of sperm with normal morphology increased （P〈0.05）. Percentages of sperm motility,rapid motility and progressive motile concentration （PMC） increased （P〈0.05） for each ofthese parameters,PureSperm preparation gave the best results （P〈0.05）. The percentage of DNA damage decreased in the PureSperm and Sil-Select Plus preparations （17.9% and 31.3%,respectively,P〈0.05） and increased in the SpermGrad preparation （56.3%,P〈0.05）. Protamine deficiency also decreased in all three kinds of media,59.3%,47.7% and 40.3% for PureSperm,Sil-Select Plus and SpermGrad preparations,respectively （P〈0.05）. The percentage of DNA-damaged sperm was negatively correlated with the percentages of sperm motility,rapid motility and PMC,but was positively correlated with static motility （P〈0.05）. This comparative study and correlation analysis revealed that PureSperm preparation yielded sperm with the best motility and the lowest percentage of protamine deficiency. The Sil-Select Plus preparation yielded sperm with the lowest amount of DNA damage. The SpermGrad preparation had a high percentage of sperm with normal morphology,but also had the highest percentage of sperm with DNA damage. Sperm DNA damage was correlated with percentages of sperm motility,rapid motility,static motility and PMC.

Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice

This study was carried out to determine if exposure to hot environmental temperatures had a direct, detrimental effect on sperm quality. For this the effect of whole-body heat exposure on epididymal spermatozoa of laboratory mice was investigated. C57BL/6 mice （n = 7） were housed in a microclimate chamber at 37℃-38℃ for 8 h per day for three consecutive days, while control mice （n = 7） were kept at 23℃-24℃. Cauda epididymal spermatozoa were obtained 16 h after the last heat treatment. The results showed that sperm numbers were similar in the two groups （P = 0.23）, but after heat treatment, a significant reduction in the percentage of motile sperm was present （P 〈 0.0001）. Membrane changes of the spermatozoa were investigated by staining with phycoerythrin （PE）- conjugated Annexin V, which detects exteriorization of phosphotidylserine from the inner to the outer leaflet of the sperm plasma membrane, and 7-aminoactinomycin D （7-AAD）, which binds to the sperm nucleus when the plasma membrane is damaged. The percentage of spermatozoa showing positive staining with Annexin V-PE or 7-AAD or both, was significantly higher （P 〈 0.05） in heat-exposed mice compared with controls. These results show that whole-body heat exposure to 37℃-38℃ induces membrane changes in the epididymal spermatozoa of mice, which may lead to apoptosis.

Aberrant protamination in sperm correlates to anomalous nuclear and cytoplasmic architectures in infertile males with sperm dysmorphology

Aberrant sperm protamination is linked to sperm dysmorphology and nuclear chromatin condensation.Yet,its effects on sperm cytoplasmic maturation remain largely unexplored.The relationships of protamines,sperm morphology,DNA damage,and cytoplasmic remodeling were illustrated in this study to provide fresh perspectives on the mechanisms of male infertility.A total of 205 infertile males were allocated into 5 groups according to the percentage of spermatozoa exhibiting abnormal morphology within their samples.Sperm concentration,motility,abnormal sperm morphology,cytoplasmic droplets(CDs),and excess residual cytoplasm(ERC)were analyzed according to the World Health Organization manual(2010).Sperm nuclear vacuoles(NVs)were determined by propidium iodide(PI)staining.Sperm protamine expressions(P1 and P2)were detected by western blot.DNA damage was measured by acridine orange test(AOT)to calculate the proportion of sperm with single-strand DNA breaks(SSBs).Our data showed that sperm concentration and motility in infertile males significantly decreased with the severity of abnormal sperm morphology(both P<0.01).P1 level,P1/P2 ratio,and SSB rate increased with the severity of sperm dysmorphology,whilst the P2 level decreased(all P<O.01).NVs,CDs,and ERC were more common in males with sperm dysmorphology and positively correlated with the SSB rate(all P<O.01).The relationships between the SSB rate and the P1/P2 ratio were also significant(P<0.01).Aberrant protamination may cause sperm dysmorphology and compromise male fertility by impairing sperm's nucleus and cytoplasm maturation,with the P1/P2 ratio potentially serving as a valuable indicator of sperm quality and male fertility.

A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome

Sperm DNA damage is prevalent among infertile men and is known to influence natural reproduction. However, the impact of sperm DNA damage on assisted reproduction outcomes remains controversial. Here, we conducted a meta-analysis of studies on sperm DNA damage （assessed by SCSA, TUNEL, SCD, or Comet assay） and clinical pregnancy after IVF and/or ICSI treatment from MEDLINE, EMBASE, and PUBMED database searches for this analysis. We identified 41 articles （with a total of 56 studies） including 16 IVF studies, 24 ICSI studies, and 16 mixed （IVF ＋ ICSI） studies. These studies measured DNA damage （by one of four assays： 23 SCSA, 18 TUNEL, 8 SCD, and 7 Comet） and included a total of 8068 treatment cycles （3734 IVF, 2282 ICSI, and 2052 mixed IVF ＋ ICSI）. The combined OR of 1.68 （95% Ch 1.49-1.89; P 〈 0.0001） indicates that sperm DNA damage affects clinical pregnancy following IVF and/or ICSI treatment. In addition, the combined OR estimates of IVF （16 estimates, OR = 1.65; 95% CI： 1.34-2.04; P 〈 0.0001）, ICSI （24 estimates, OR = 1.31; 95% Ch 1.08-1.59; P = 0.0068）, and mixed IVF ＋ ICSI studies （16 estimates, OR = 2.37; 95% Ch 1.89-2.97; P〈 0.0001） were also statistically significant. There is sufficient evidence in the existing literature suggesting that sperm DNA damage has a negative effect on clinical pregnancy following IVF and/or ICSI treatment.

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.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling （18% w/v raffinose, RS-C） were compared with spermatozoa vitrified using 0.25 M sucrose （SV） or 18% w/v raffinose （RV）. The motility, vitality, and DNA damage （TUNEL assay） of fresh control （FC） spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes （n = 267） were randomly assigned into three groups for insemination： RV （n = 102）, RS-C （n = 86）, and FC （n = 79）. The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility （P 〈 0.01） and vitality （P 〈 0.05） were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose （15 - 1.8% [SV] vs 26 - 2.8% [RV] and 27 - 1.2% [RS-C]; P 〈 0.01）. Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa （P = 0.0053）. This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.

Supplementation with L-Carnitine Rescues Sperm Epigenetic Changes in Asthenospermic Male Semen with Altered Acetyl-L-Carnitine Levels

Objective:To investigate the relationship between the concentration of L-carnitine in semen and sperm parameters and investigate the epigenetic profile in sperm cell after L-carnitine usage.Methods:From February 2017 to February 2018,46 semen samples from asthenospermic males and 41 semen samples from healthy donors were acquired.Motility parameters were assessed using computer-assisted sperm analysis(CASA,n=78)and the DNA fragmentation index(DFI)was evaluated through flow cytometry(n=86),%DFI=%cells outside main population.Other oxidative stress markers,such as reactive oxygen species(ROS)levels(n=86)and the mitochondria DNA copy numbers,were detected(n=78).The concentration of L-carnitine and acetyl-L-carnitine was detected(n=82),and methylation was analyzed(n=30).After that,we collected 13 fresh semen samples from asthenospermic males and 23 fresh semen samples from healthy donors.These samples were used in a freeze-thaw model that was used to determine whether adding L-carnitine could change sperm progressive motility(n=23),apoptosis index(n=9),and methylation analysis(n=7).In total,we have done 13 asthenospermia samples for Western blot,and except for the poor Western result,we analyzed 6 samples for H3K9ac detection,7 samples for H3K9m3 and H3K27m3 detection,and immunofluorescence(n=3).Finally,we had recruited 30 volunteers,and they were given oral administration of L-carnitine for 3 months and then collected semen samples at different time points for methylation analysis.Results:The concentration of acetyl-L-carnitine is negatively correlated with the%DFI value(r^2=0.1090;P=0.0026),and the concentration of acetyl-L-carnitine is positively correlated with sperm forward motility(r^2=0.0543;P=0.0458)and ROS(r^2=0.1854;P<0.0001),and the acetyl-L-carnitine level is negatively correlated with%DFI in asthenospermia(r^2=0.1701;P=0.0066),and the level of acetyl-L-carnitine in asthenospermic semen is significantly lower than the normal group(P=0.0419).In addition,this study indicates that adding L-carnitine significantly improved sperm motility(P=0.0325)and reduced sperm apoptosis(P=0.0032).Importantly,Western blotting(P=0.0429)and immunofluorescence staining results showed that the addition of L-carnitine reduced H3K9Me3 methylation level in sperm,respectively.Furthermore,semen samples from asthenospermic patients had reduced methylation levels in a specific region(16^thP=0.0003;17^thP=0.0016)of the brain-derived neurotrophic factor(BDNF)promoter.The 16^th methylation decreased with age(r^2=0.1564;P=0.0306),and the 17^th methylation was decreased after treatment with L-carnitine for 28 days(P=0.0341).Conclusion:L-carnitine can reduce the%DFI and also affect the methylation of the histone modification marker in sperm as a possible epigenetic regulator.

The paternal genome and the health of the assistect reproductive technology child

As a number of children born by assisted reproductive technology （ART） are increasing each year across the developed world, the health of such offspring is a matter of public concern. Does the integrity of the paternal genome impact on offspring health？ In societal terms, as birth rates fall, and the Western population become unsustainable, do the benefits outweigh the costs of creating and providing for this ART conceived subpopulation？ There are little data to date to answer these questions. The long-term health of such children has largely been ignored, and success measured only by early （prebirth） outcomes such as embryo quality or pregnancy. However, there are powerful paradigms such as ageing and smoking that give vital clues as to the potential impact of unhealthy spermatozoa on disease risk, mental and physical health, fertility and mortality of these offspring.