Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization （WHO） manual-based semen parameter＇s concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay （SCSA）, a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection （ICSI）.

Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF

The aim of this study was to investigate whether the sperm chromatin structure assay （SCSA） results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitrofertilization （IVF）. A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index （DFI） subgroups and high DNA stainability （HDS） subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.

Mixed secondary chromatin structure revealed by modeling radiation-induced DNA fragment length distribution

Spatial chromatin structure plays fundamental roles in many vital biological processes including DNA replication, transcription,damage and repair. However, the current understanding of the secondary structure of chromatin formed by local nucleosomenucleosome interactions remains controversial, especially for the existence and conformation of 30 nm structure. Since chromatin structure influences the fragment length distribution(FLD) of ionizing radiation-induced DNA strand breaks, a 3D chromatin model fitting FLD patterns can help to distinguish different models of chromatin structure. Here, we developed a novel "30-C" model combining 30 nm chromatin structure models with Hi-C data, which measured the spatial contact frequency between different loci in the genome. We first reconstructed the 3D coordinates of the 25 kb bins from Hi-C heatmaps. Within the25 kb bins, lower level chromatin structures supported by recent studies were filled. Simulated FLD patterns based on the 30-C model were compared to published FLD patterns induced by heavy ion radiation to validate the models. Importantly, the 30-C model predicted that the most probable chromatin fiber structure for human interphase fibroblasts in vivo was 45% zig-zag 30 nm fibers and 55% 10 nm fibers.

Targeting the chromatin structural changes of antitumor immunity

Epigenomic imbalance drives abnormal transcriptional processes,promoting the onset and progression of cancer.Although defective gene regulation generally affects carcinogenesis and tumor suppression networks,tumor immunogenicity and immune cells involved in antitumor responses may also be affected by epigenomic changes,which may have significant implications for the development and application of epigenetic therapy,cancer immunotherapy,and their combinations.Herein,we focus on the impact of epigenetic regulation on tumor immune cell function and the role of key abnormal epigenetic processes,DNA methylation,histone post-translational modification,and chromatin structure in tumor immunogenicity,and introduce these epigenetic research methods.We emphasize the value of small-molecule inhibitors of epigenetic modulators in enhancing antitumor immune responses and discuss the challenges of developing treatment plans that combine epigenetic therapy and immuno-therapy through the complex interaction between cancer epigenetics and cancer immunology.

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）.

Mechanisms and clinical correlates of sperm D damage

Among the different DNA anomalies that can be present in the male gamete, DNA fragmentation is the most frequent, particularly in infertile subjects. There is now consistent evidence that a sperm containing fragmented DNA can be alive, motile, morphologically normal and able to fertilize an oocyte. There is also evidence that the oocyte is able to repair DNA damage; however, the extent of this repair depends on the type of DNA damage present in the sperm, as well as on the quality of the oocyte. Thus, it is important to understand the possible consequences of sperm DNA fragmentation （SDF） for embryo development, implantation, pregnancy outcome and the health of progeny conceived, both naturally and by assisted reproductive technology （ART）. At present, data on the consequences of SDF for reproduction are scarce and, in many ways, inconsistent. The differences in study conclusions might result from the different methods used to detect SDF, the study design and the inclusion criteria. Consequently, it is difficult to decide whether SDF testing should be carried out in fertility assessment and ART. It is clear that there is an urgent need for the standardisation of the methods and for additional clinical studies on the impact of SDF on ART outcomes.

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay （SCSA） parameters, that is, DNA fragmentation index （%DFI） and high DNA stainability （%HDS）, were assessed on the flesh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation （DGC） was followed by swimup treatment in comparison with DGC alone （P 〈 0.01）. Although the %DFI increased in a time-dependent manner with incubation both at room temperature （RT） and at 37℃ in air, the %DFI after 24 h at RT was significantly lower than that at 37℃ （P 〈 0.05）. Incubation with 5% CO2 was effective in maintaining sperm motility （P 〈 0.01）; however, it induced further elevation of %DFI （P 〈 0.001）. Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.

The impact of male overweight on semen quality and outcome of assisted reproduction

It is well-documented that male overweight and obesity causes endocrine disorders that might diminish the male reproductive capacity; however, reports have been conflicting regarding the influence of male body mass index （BMI） on semen quality and the outcome of assisted reproductive technology （ART）. The aim of this study was to investigate whether increased male BMI affects sperm quality and the outcome of assisted reproduction in couples with an overweight or obese man and a non-obese partner. Data was prospectively collected from 612 infertile couples undergoing ART at a Danish fertility center. Self-reported information on paternal height and weight were recorded and BMI was calculated. The men were divided into four BMI categories： underweight BMI 〈 20 kgm^-2, normal BMI 20-24.9 kg m^-2, overweight BMI 25-29.9 kgm^-2 and obese BMI 〉 30 kgm^-2. Conventional semen analysis was performed according to the World Health Organization guideline and sperm DNA integrity was analyzed by the Sperm Chromatin Structure Assay （SCSA）. No statistically significant effect of male BMI was seen on conventional semen parameters （sperm concentration, total sperm count, seminal volume and motility） or on SCSA-results. Furthermore, the outcome of ART regarding fertilization rate, number of good quality embryos （GQE）, implantation and pregnancy outcome was not influenced by the increasing male BMIo

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.

Effect of in ovo folic acid injection on hepatic IGF2 expression and embryo growth of broilers

Background： Insulin-like factor 2（IGF2） plays an important role in embryonic growth process by modulating intermediary metabolism and cell proliferation. Folic acid is involved in one carbon metabolism and contributes to DNA methylation which is related to gene expression. The purpose of this study was to explore whether folic acid could regulate IGF2 expression via epigenetic mechanism and further promote embryonic growth of new-hatched broilers.Methods： In the present study, 360 fertile eggs were selected and randomly assigned to four treatments. On11 embryonic day of incubation（E11）, 0, 50, 100 and 150 μg folic acid were injected into eggs respectively.After hatched, growth performance of broilers were calculated. Hepatic IGF2 expression, methylation level and chromatin structure of promoter region were analyzed.Results： Results have showed that IGF2 expression was up-regulated in 150 μg folic acid group（P 〈 0.05） and other two dose of folic acid did not affect gene expression（P 〉 0.05）. Meanwhile, methylation level of IGF2 promoter were lower in 100 and 150 μg groups, which was consistent with lower expression of DNA methyltransferase1（DNMT1）（P 〈 0.05）. What＇s more, chromatin looseness of IGF2 promoter was higher in 150 μg group than control group（P 〈 0.05）. Further, birth weight（BW）, liver and bursa index of new-hatched chickens in 150 μg folic acid group were higher than the other groups（P 〈 0.05）. There were positive correlations between hepatic IGF2 expression and BW and organs index（P 〈 0.05）.Conclusion： In conclusion, our data have demonstrated that 150 μg folic acid injection on E11 could up-regulate IGF2 expression by modulating DNA hypomethylation and improving chromatin accessibility in the gene promoter region,and ulteriorly facilitate embryonic growth and organ development of broilers.

Relationship between apoptotic markers in semen from fertile men and demographic, hormonal and seminal characteristics

Apoptosis in the testis has two putative roles during normal spermatogenesis; limitation of the germ cell population to numbers that can be supported by the Sertoli cells, and, possibly, selective depletion of meiotic and postmeiotic abnormal germ cells. We investigated the demographic and biological correlates of the pro-apoptotic marker Fas and the anti-apoptotic marker Bcl-xL in sperm cells of fertile men. Six hundred and four men from Greenland, Poland and Ukraine were consecutively enrolled during their pregnant wife＇s antenatal visits. Semen analysis was performed as recommended by the World Health Organization. Immunofluorescence coupled to flow cytometry was utilized for detection of apoptotic markers in the sperm cell. DNA damage was assessed by flow cytometry using both the sperm chromatin structure assay （SCSA） and the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） assay. The percentage of Fas-positive sperm cells was higher in men with high total sperm count （P〈O.01）, more motile sperms （P=-O.04） and fewer sperm head defects （P=-O.05）. These associations were consistent within and across study regions. Furthermore, testosterone, follicle-stimulating hormone （FSH） and sexual hormone-binding globulin （SHBG） were significantly negatively correlated with Fas within and across regions as well. The data indicated no association between the anti-apoptotic Bcl-xL marker and semen or personal characteristics. The finding of Fas-positive sperm cells associated with better semen quality in a cohort of spouses of pregnant women seems different from previous data obtained in infertile men and warrants further investigation to clarify the biological significance of sperm apoptotic markers.

Illuminating the structure and dynamics of chromatin by fluorescence labeling

BACKGROUND： Visualization of chromosomal loci location and dynamics is crucial for understanding many fundamental intra-nuclear processes such as DNA transcription, replication, and repair. OBJECTIVE： Here, we will describe the development of fluorescence labeling methods for chromatin imaging, including traditional as well as emerging chromatin labeling techniques in both fixed and live cells. We will also discuss current issues and provide a perspective on future developments and applications of the chromatin labeling technology. METHODS： A systematic literature search was performed using the PubMed. Studies published over the past 50 years were considered for review. More than 100 articles were cited in this review. RESULTS： Taking into account sensitivity, specificity, and spatiotemporal resolution, fluorescence labeling and imaging has been the most prevalent approach for chromatin visualization. Among all the fluorescent labeling tools, the adoption ofgenome editing tools, such as TALE and CRISPR, have great potential for the labeling and imaging of chromatin. CONCLUSION： Although a number of chromatin labeling techniques are available for both fixed and live cells, much more effort is still clearly required to develop fluorescence labeling methods capable of targeting arbitrary sequences non-intrusively to allow long-term, multiplexing, and high-throughput imaging of genomic loci and chromatin structures. The emerging technological advances will outline a next-generation effort toward the comprehensive delineation of chromatin at single-cell level with single-molecule resolution.

Effect of BNT162b2 mRNA COVID-19 vaccine on sperm morphokinetics and DNA integrity: A prospective observational study in Japan

Objective:To assess whether the coronavirus disease 2019(COVID-19)mRNA vaccine affects sperm morphokinetics using a computer-assisted semen analyzer and other semen parameters using a sperm chromatin structure assay.Methods:Healthy male volunteers in two Japanese clinics between May 2021 and December 2021 were prospectively analyzed.Participants donated sperm twice,two days apart,in the following phases:before vaccination,2 weeks after the first vaccine dose,and 2,4,and 12 weeks after the second dose.Basic sperm parameters,sperm motility characteristics,and the percentage of DNA-damaged sperm were compared among the different phases.Results:Ninety-six semen samples from ten volunteers,who were vaccinated with the BNT162b2 mRNA vaccine,were evaluated.There were no significant differences between any phases in basic semen findings and parameters of the sperm chromatin structure assays.Regarding sperm motion characteristics,the average linear velocity,beat-cross frequency,and sperm motility index significantly decreased after the second vaccine dose(P=0.018,P=0.003,and P=0.027,respectively),with no significant differences between any two phases by post-hoc pairwise comparisons.Conclusions:After COVID-19 mRNA vaccination,while sperm motion characteristics might fluctuate,no apparent deterioration of basic sperm parameters or sperm DNA integrity was observed.Given the adverse effects of COVID-19 on sperm,our findings suggest that there might be no reason to refrain from vaccination for healthy individuals.

Mechanism of chromatin remodeling revealed by the Snf2-nucleosome structure

Subject Code:C05With the support from the National Natural Science Foundation of China,a collaborative study by the research teams led by Chen Zhucheng(陈柱成)and Li Xueming(李雪明)at the School of Life Sciences,Tsinghua University,recently reported their work,titled“Mechanism of chromatin remodeling revealed

Structure and regulation of the chromatin remodeller ISWI

Subject Code:C05With the support by the National Natural Science Foundation of China,the research team led by Dr.Chen Zhucheng(陈柱成)at the School of Life Science,Tsinghua University,Beijing,recently reported their work,titled'Structure and regulation of the chromatin remodeller ISWI',in Nature(2016,540:466—469).Chromatin is the life blueprint of eukaryotes.Chromatin remodellers utilize the energy of ATP hydrolysis to move,destabilize,eject,or restructure nucleosomes,building and rebuilding the blueprint

Chromatin Remodeling in Stem Cell Maintenance in Arabidopsis thaliana

Pluripotent stem cells are able to both self-renew and generate undifferentiated cells for the formation of new tissues and organs. In higher plants, stem cells found in the shoot apical meristem （SAM） and the root apical meristem （RAM） are origins of organogenesis occurring post-embryonically. It is important to understand how the regulation of stem cell fate is coordinated to enable the meristem to constantly generate different types of lateral organs. Much knowledge has accumulated on specific transcription factors controlling SAM and RAM activity. Here, we review recent evidences for a role of chromatin remodeling in the maintenance of stable expression states of transcription factor genes and the control of stem cell activity in Arabidopsis.

Exploring CTCF and cohesin related chromatin architecture at HOXA gene cluster in primary human fibroblasts

Spatial expression patterns of homeobox （HOX） genes delineate positional identity of primary fibroblasts from different topo- graphic sites. The molecular mechanism underlying the establishing or maintaining of HOX gene expression pattern remains an attractive developmental issue to be addressed. Our previous work suggested a critical role of CTCF/cobesin-mediated high- er-order chromatin structure in RA-induced HOXA activation in human teratocarcinoma NT2/D1 cells. This study investigated the recruitment of CTCF and cohesin, and the higher-order chromatin structure of the HOXA locus in fetal lung and adult foreskin fibroblasts, which display complementary HOXA gene expression patterns. Chromatin contacts between the CTCF-binding sites were observed with lower frequency in human foreskin fibroblasts. This observation is consistent with the lower level of cohesin recruitment and 5＇ HOXA gene expression in the same cells. We also showed that CTCF-binding site A56 （CBSA56） related chromatin structures exhibit the most notable changes in between the two types of cell, and hence may stand for one of the key CTCF-binding sites for cell-type specific chromatin structure organization. Together, these results im- ply that CTCF/cohesin coordinates HOXA cluster higher-order chromatin structure and expression during development, and provide insight into the relationship between cell-type specific chromatin organization and the spatial collinearity.

Noncoding RNA-chromatin association:Functions and mechanisms

Pervasive transcription of the mammalian genome produces hundreds of thousands of noncoding RNAs(ncRNAs).Numerous studies have suggested that some of these ncRNAs regulate multiple cellular processes and play important roles in physiological and pathological processes.Notably,a large subset of ncRNAs is enriched on chromatin and participates in regulating gene expression and the dynamics of chromatin structure and status.In this review,we summarize recent advances in the functional study of chromatin-associated ncRNAs and mechanistic insights into how these ncRNAs associate with chromatin.We also discuss the potential future challenges which still need to be overcome in this field.

DNA cytosine methylation in plant development

Cytosine bases of the nuclear genome in higher plants are often extensively methylated.Cytosine methylation has been implicated in the silencing of both transposable elements （TEs） and endogenous genes,and loss of methylation may have severe functional consequences.The recent methylation profiling of the entire Arabidopsis genome has provided novel insights into the extent and pattern of cytosine methylation and its relationships with gene activity.In addition,the fresh studies also revealed the more dynamic nature of this epigenetic modification across plant development than previously believed.Cytosine methylation of gene promoter regions usually inhibits transcription,but methylation in coding regions （gene-body methylation） does not generally affect gene expression.Active demethylation （though probably act synergistically with passive loss of methylation） of promoters by the 5-methyl cytosine DNA glycosylase or DEMETER （DME） is required for the uni-parental expression of imprinting genes in endosperm,which is essential for seed viability.The opinion that cytosine methylation is indispensible for normal plant development has been reinforced by using single or combinations of diverse loss-of-function mutants for DNA methyltransferases,DNA glycosylases,components involved in siRNA biogenesis and chromatin remodeling factors.Patterns of cytosine methylation in plants are usually faithfully maintained across organismal generations by the concerted action of epigenetic inheritance and progressive correction of strayed patterns.However,some variant methylation patterns may escape from being corrected and hence produce novel epialleles in the affected somatic cells.This,coupled with the unique property of plants to produce germline cells late during development,may enable the newly acquired epialleles to be inherited to future generations,which if visible to selection may contribute to adaptation and evolution.

Histone Acetyltransferase AtGCN5/HAG1 Is a Versatile Regulator of Developmental and Inducible Gene Expression in Arabidopsis

Histone acetylation/deacetylation is a dynamic process and plays an important role in gene regulation. Histone acetylation homeostasis is regulated by antagonist actions of histone acetyltransferases （HAT） and deacetylases （HDAC）. Plant genome encodes multiple HATs and HDACs. The Arabidopsis HAT gene AtGCNS/HAGlplays an essential role in many plant development processes, such as meristem function, cell differentiation, leaf and floral organogenesis, and responses to environmental conditions such as light and cold, indicating an important role of this HAT in the regulation of both long-term developmental switches and short-term inducible gene expression. AtGCN5 targets to a large number of promoters and is required for acetylation of several histone H3 lysine residues. Recruitment of AtGCN5 to target promoters is likely to be mediated by direct or indirect interaction with DNA-binding transcription factors and/or by interaction with acetylated histone lysine residues on the targets. Interplay between AtGCN5 and other HATand HDAC is demonstrated to control specific regulatory pathways. Analysis of the role of AtGCN5 in light-inducible gene expression suggests a function of AtGCN5 in preparing chromatin commitment for priming inducible gene activation in plants.