Nuclear DNA Integrity Research Articles

Moment of induction and duration of experimental varicocele in rats: effects on semen quality.

Varicocele is a condition known to cause damage to seminal parameters and sperm function. Furthermore, it has been hypothesized that the varicocele effect on fertility is time-dependent; however, little is known about the consequences of its establishment time on reproductive organs and/or sperm function. This study aimed to evaluate the effect of the duration of experimental varicocele on reproductive organs, sperm parameters, and sperm function. Varicocele induction surgeries were performed in Wistar rats aged 40 or 100 days old. At 160-day-old, analyses were performed, including biometry of reproductive organs (prostate, seminal vesicles, epididymis, and testis), sperm parameters (vitality, morphology, and motility), and sperm function tests (nuclear DNA integrity, acrosome integrity, and mitochondrial activity). The analysis of the biometry of reproductive organs showed no differences between distinct ages in which varicocele was induced. The total abnormal sperm morphology was bigger in animals with varicocele induced to 100 days old than in animals with varicocele induced to 40 days old. Regarding nuclear DNA integrity, animals of varicocele induced to 100 days old showed worse results compared to animals of varicocele induced to 40 days old. Other parameters analyzed showed no differences between varicocele groups. In this study conducted on rats, we conclude that varicocele adversely affects sperm, particularly its function. However, we did not observe a negative progressive effect on sperm.

The Effects on Post-Thaw Sperm Quality and Nuclear DNA Integrity of Supplementation of Low-Density Lipoprotein to Freezing Extender in the Mouse

Mice are an important research tool for genetic and molecular biology, allowing researchers to explore a variety of human illness models. Egg yolk is a common component of semen extenders for domestic animals and low-density lipoproteins (LDL) from egg yolk have some cryoprotective properties. This study aimed to investigate sperm quality characteristics and nuclear DNA integrity after post-thawing in an extender (18% raffinose + 3% skim milk) supplemented with different concentrations of LDL (2.5%, 5.0%, 7.5%, or 10%) in mice. 18% Raffinose+3% skim milk extender was used as a control group without LDL. CD-1 mice were used in the study, and semen was collected from the cauda epididymis and diluted with the extender. The straws were then frozen and thawed to evaluate progressive motility, viability, plasma membrane (HOST), acrosome, and nuclear DNA integrity parameters. Fresh sperm had the highest progressive motility, viability, plasma membrane integrity, and longevity (endurance) of progressive motility for 4 h in HTF solution. The greatest spermatologic results, including nuclear DNA integrity, were determined in fresh sperm (p

The Impact of PSR™ (Plant Small RNA Technology), Tea Extract, and Its Principal Components on Mitochondrial Function and Antioxidant Properties in Skin Cells

Objective: This study explored the impact of a black tea extract obtained through (plant small RNA) PSRTM technology, characterized by its abundance of small molecules, particularly citric acid—an antioxidant and tricarboxylic acid (TCA) cycle contributor—on mitochondrial health. The primary focus was to assess whether this extract could counteract reactive oxygen species (ROS)-induced mitochondrial alterations associated with aging, which lead to impaired mitochondrial function, reduced ATP production, and increased ROS generation. Methods: The PSRTM extraction method was employed to obtain a high content of polyphenols and small molecules, particularly citric acid. Results: In comparison with a conventional extract, the PSRTM extract demonstrated significant enhancements in aconitase activity, an ROS-sensitive enzyme in the TCA cycle, as well as basal respiration and ATP synthesis in fibroblast cells and skin biopsies. Moreover, the PSRTM extract effectively reduced ROS production by safeguarding this critical enzyme within the Krebs cycle and displayed superior capabilities in scavenging free radicals when exposed to UV-induced stress. When administered post-UV exposure, the PSRTM extract protected nuclear DNA by reducing the formation of cyclobutane pyrimidine dimers (CPDs) and promoting DNA repair mechanisms. Furthermore, the extract exhibited beneficial effects on the extracellular matrix, characterized by a reduction in matrix metalloprotease 1 (MMP1) and an increase in fibrillin 1 expression. Conclusions: These findings collectively suggest that the PSRTM extract holds promising antiaging potential, potentially functioning as a mitochondrial nutrient/protector due to its multifaceted benefits on mitochondrial function, nuclear DNA integrity, and the extracellular matrix.

The Relationship between Body Mass Index, Metal Elements, and Antioxidant Capacity of Semen on The Human Sperm Chromatin.

Sperm chromatin abnormalities are defects in nuclear maturation and DNA integrity. These defects originated from defective spermatogenesis due to a lack of DNA repair during chromatin remodeling. Changes in semen elements can cause damage to chromatin. There is little information about the relationship between changes in trace metal elements and total antioxidant capacity (TAC) with sperm chromatin damage. The present study was conducted to determine the relationship between Selenium (Se), Iron (Fe), Zinc (Zn), Copper (Cu) and the TAC of semen with the status of human sperm chromatin. In this experimental study, semen samples (n=30) were collected from healthy men referred to Kermanshah Motazadi Hospital and stored in liquid nitrogen; after thawing and centrifugation, sperm were separated. The atomic absorption method was used to measure the concentration of metal elements. The TAC was evaluated using the ferric-reducing antioxidant capacity of the plasma method. Furthermore, the integrity of sperm chromatin was measured using the sperm DNA fragmentation (SDF) method. The status of sperm chromatin had a non-significant correlation with body mass index (BMI, P=0.25, r=0.21) and a non-significant negative correlation with sperm count (P=0.71, r=-0.71) and motility (P=0.75, r=0.61). In addition, there was no significant relationship between sperm chromatin and the TAC of semen (P=0.92, r=0.01). Additionally, there was no significant correlation between Se, Zn, or Cu concentration (P>0.05) and Fe concentration, which had a partially positive relationship with the chromatin state of sperm (P=0.24, r=0.20). The trace metal elements in the seminal fluid did not play a significant role in the status of sperm chromatin.

Sperm telomere length is associated with sperm nuclear DNA integrity and mitochondrial DNA abnormalities among healthy male college students in Chongqing, China.

Is sperm telomere length (STL) associated with sperm nuclear DNA damage and mitochondrial DNA abnormalities? Sperm telomere length is related to sperm nuclear DNA integrity and mitochondrial DNA abnormalities in healthy young college students. Many studies have revealed the correlations between sperm genetic alterations in both the nucleus and mitochondria and sperm functionality, however, the possible associations between the telomere, an important component of chromosome, and conventional indicators of mitochondrial DNA and nuclear DNA changes have not been investigated. A prospective cohort study, Male Reproductive Health in Chongqing College Students (MARHCS), was conducted from June 2013 to June 2015. We pooled data collected from the follow-up study in 2014 and a total of 444 participants were included. STL was measured by quantitative (Q)-PCR. Sperm nuclear DNA integrity was determined using sperm chromatin structure assay (SCSA) and comet assay. Mitochondrial DNA damage was assessed by mitochondrial DNA copy number (mtDNAcn) evaluated with Q-PCR, and mtDNA integrity was determined with long PCR. The univariable-linear regression analysis revealed that STL was significantly positively correlated with markers of sperm nuclear DNA damage including the DNA fragmentation index (DFI) and comet parameters (the percentage of DNA in the tail, tail length, comet length, and tail moment). Additionally, STL was also significantly positively correlated with mtDNAcn and significantly negatively correlated with mtDNA integrity. After adjustment for potential confounders, these relationships remained appreciable. Moreover, we investigated potential effects of biometric factors, including age, parental age at conception, and BMI on STL and found that STL was increased with paternal age at conception. A mechanistic explanation of the correlation between STL, sperm nuclear DNA integrity, and mtDNA abnormalities cannot be provided with a cross-sectional study design, so well-designed longitudinal studies are still necessary. In addition, a single semen samples were provided and were not all obtained at the same time point, which may increase the intraindividual bias in this study. The findings extend the literature including assessment of mitochondrial dysfunction, sperm nuclear DNA damage, and telomere length and provide new insights into the relevance of STL in male reproduction. This work was supported by the National Natural Science Foundation of China (No. 82073590), the National Natural Science Foundation of China (No. 81903363), the National Natural Science Foundation of China (No. 82130097), and the National Key R&D Program of China (2022YFC2702900). The authors declare no conflicts of interest. N/A.

An Overview of Adding Rho-Associated Coiled-Coil Kinase and Knockout Serum Replacement with Trehalose to a Low Glycerol Tris-Based Semen Extender

It is known that livestock animal semen is very sensitive to cold shock during freezing processes, and this sensitivity directly affects post-thaw sperm qualities which are progressive motility, mitochondrial membrane potential, sperm nuclear DNA integrity and in vitro spermatological parameters such as plasma membrane and acrosome integrity, and sperm fertility. In addition, with the sudden decrease in the total antioxidant level of the semen after thawing, the sperm cells are insufficient to tolerate their damage. Consequently, significant losses occur in sperm fertility. For this reason, researches on freezing the semen of livestock animals include semen processing; cryopreservation/cryogenic damage – thawing methods - sperm extenders, added antioxidants, the mechanisms of action and metabolic pathways of these antioxidants and physiological and metabolic parameters such as sperm fertility. It has been explained that low dose glycerol (trehalose added to increase the cryoprotectant effect) added to the extender in the freezing of livestock animal semen, knockout serum replacement (KSR) and Rho-associated coiled-coil kinase (ROCK), which are antioxidant additives, can increase the in vitro quality parameters of frozen thawed semen.

Unravelling joint cytotoxicity of ibuprofen and oxytetracycline on Chlamydomonas reinhardtii using a programmed cell death-related biomarkers panel

Pharmaceutical active compounds (PhACs) are emerging contaminants that pose a growing concern due to their ubiquitous presence and harmful impact on aquatic ecosystems. Among PhACs, the anti-inflammatory ibuprofen (IBU) and the antibiotic oxytetracycline (OTC) are two of the most used compounds whose presence has been reported in different aquatic environments worldwide. However, there is still scarce information about the cellular and molecular alterations provoked by IBU and OTC on aquatic photosynthetic microorganisms as microalgae, even more if we refer to their potential combined toxicity. To test the cyto- and genotoxicity provoked by IBU, OTC and their binary combination on Chlamydomonas reinhardtii, a flow cytometric panel was performed after 24 h of single and co-exposure to both contaminants. Assayed parameters were cell vitality, metabolic activity, intracellular ROS levels, and other programmed cell death (PCD)-related biomarkers as cytoplasmic and mitochondrial membrane potentials and caspase-like and endonuclease activities. In addition, a nuclear DNA fragmentation analysis by comet assay was carried out.For most of the parameters analysed (vitality, metabolic activity, cytoplasmic and mitochondrial membrane potentials, and DNA fragmentation) the most severe damages were observed in the cultures exposed to the binary mixture (IBU+OTC), showing a joint cyto- and genotoxicity effect. Both PhACs and their mixture caused a remarkable decrease in cell proliferation and metabolic activity and markedly increased intracellular ROS levels, parallel to a noticeable depolarization of cytoplasmic and mitochondrial membranes. Moreover, a strong increase in both caspase and endonuclease activities as well as a PCD-related loss of nuclear DNA integrity was observed in all treatments. Results analysis showed that the PhACs caused cell death on this non-target organism, involving mitochondrial membrane depolarization, enhanced ROS production and activation of PCD process. Thus, PCD should be an applicable toxicological target for unraveling the harmful effects of co-exposure to PhACs in aquatic organisms as microalgae.

Opioid replacement therapy with methadone or buprenorphine effects on male mice reproduction.

Opioid use disorders are commonly treated by long-acting agonist opioids including methadone and buprenorphine which could affect various aspects of male reproduction especially spermatogenesis. We aimed to determine whether detoxification with methadone or buprenorphine was associated with reproductive disorders in male mice. We orally induced morphine dependence in NMRI male mice, and then performed detoxification programs using either methadone or buprenorphine. Testis architecture and sperm parameters including sperm nuclear DNA integrity, mitochondrial activity, oxidative stress in seminal plasma, and routine sperm parameters were assessed to find the involved mechanisms. The number of Leydig cells and the thickness of germinal epithelium reduced following morphine use and increased differently after detoxification with methadone or buprenorphine. Morphine dependence and detoxification with methadone and buprenorphine had different effects on sperm parameters. Morphine altered chromatin integrity, mitochondrial activity, and oxidative stress in sperm. Detoxification with methadone improved mitochondrial activity but worsened chromatin integrity, whereas detoxification with buprenorphine improved neither chromatin integrity nor mitochondrial activity. Seminal plasma oxidative stress was higher in the treated groups compared to control groups but was comparable among treatment groups. Our study revealed that long-term morphine use followed by detoxification with methadone or buprenorphine impairs testis structure and sperm parameters. Detoxification from morphine use with methadone and buprenorphine led to different preclinical outcomes in semen quality parameters, including chromatin integrity. Therefore, clinical detoxification protocols should be performed more cautiously, considering the desire of the individuals to reproduce.

Selecting motile, non-apoptotic and induced spermatozoa for capacitation without centrifuging by MACS-Up method.

In newly improved MACS-Up method, magnetic field has been applied to separate non-apoptotic spermatozoa directly from the neat semen. The spermatozoa during passing through a viscous layer, located on the neat semen, contacted with progesterone and induced for the capacitation. Then, a clean population of non-apoptotic, and capacitated spermatozoa were selected in the pure culture media. Selected spermatozoa may be useful for use in ART. The 80semen samples from normozoospermic individuals were divided separately into 4 attempts. Semen analysis, SCSA (sperm chromatin structure assay), FLICA (fluorescein-labelled inhibitors of caspase) methods, immunoassay of phosphorylation of tyrosine residues of sperm proteins, nuclear DNA integrity, caspase 3 activity and sperm capacitation rate were all performed for evaluation of sperm parameters respectively. To examine all aspects, the MACS-Up method compared with DGC (density gradient centrifuging) and MACS-DGC methods separately. This method can isolate non-apoptotic spermatozoa directly from the neat semen, which has similar performance compared to the MACS-DGC method. Movement and passing spermatozoa through the viscous layer, and contact with progesterone, significantly induced spermatozoa for capacitation compared with the control group. Also, the MACS-Up in comparison with routine DGC method could select spermatozoa with significantly higher total and progressive motility, DNA integrity, induced sperm population for capacitation and normal morphology. MACS-Up can be developed as an effective, short-time, and ease of performing method and used practically to select functional spermatozoa as novel sperm selection procedure. However, for clinical use of MACS-Up, all clinical aspects of this method should be considered and evaluated.

Nucleus Near-Infrared (nNIR) Irradiation of Single A549 Cells Induces DNA Damage and Activates EGFR Leading to Mitochondrial Fission.

There has been great interest in identifying the biological substrate for light-cell interaction and their relations to cancer treatment. In this study, a near-infrared (NIR) laser is focused into the nucleus (nNIR) or cytoplasm (cNIR) of a single living cell by a high numerical aperture condenser to dissect the novel role of cell nucleus in mediating NIR effects on mitochondrial dynamics of A549 non-small cell lung cancer cells. Our analysis showed that nNIR, but not cNIR, triggered mitochondrial fission in 10 min. In contrast, the fission/fusion balance of mitochondria directly exposed to cNIR does not change. While the same phenomenon is also triggered by single molecular interactions between epidermal growth factor (EGF) and its receptor EGFR, pharmacological studies with cetuximab, PD153035, and caffeine suggest EGF signaling crosstalk to DNA damaging response to mediate rapid mitochondrial fission as a result of nNIR irradiation. These results suggest that nuclear DNA integrity is a novel biological target for cellular response to NIR.

The relevance of mitochondrial DNA variants fluctuation during reprogramming and neuronal differentiation of human iPSCs.

SummaryThe generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.

The effect of micronutrient supplementation on spermatozoa DNA integrity in subfertile men and subsequent pregnancy rate

Background Male infertility has been related to an increased sperm DNA fragmentation index (DFI). Nutritional factors may improve sperm nuclear DNA integrity and thus pregnancy rates. Objective: To evaluate the effect of micronutrient supplementation on sperm DNA integrity in subfertile men and subsequent pregnancy rates. Methods In this retrospective comparative study 339 subfertile males were included on whom a sperm chromatin dispersion test (SCD) was performed as a method to detect DNA fragmentation, as well as an initial semen analysis. Of all, n = 162 received a nutritional management program for three months, consisting of two daily capsules of a standardized combined micronutrient formulation together with a guidance to diet modification and to lifestyle changes (study group). Each capsule contained L-carnitine, L-arginine, coenzyme Q10, zinc, vitamin E, folic acid, glutathione, and selenium. The control group consisted of those patients who did not receive the active treatment (n = 177), yet were instructed to engage in a healthy lifestyle, including a modification of their regular diet. The SCD test was repeated for both groups after three months. As part of the routine follow up, pregnancy rate was assessed six months after the second SCD test. Males with complete follow up and healthy female partners (aged 18 to 40 years) where included. Results Data of men with an initial mean DFI of >15% were analyzed first (n = 81;46 study and 35 control patients). After three months, both groups displayed a significant decrease of mean DFI values; however, the mean percent difference was higher in the study group (10.46 ± 1.20 % vs. 5.29 ± 0.57 %; p < .001). Then, the entire population was considered (n = 339). After three months, only the study group displayed a significant decrease of mean DFI initial values (10.48 ± 7.76 % to 6.51 ± 4.61%; p < .001); and the percent difference was higher in the study group (3.97 ± 0.28 % vs. 0.91 ± 0.28 %; p < .001). At six months follow-up, pregnancy rate was higher in the study group (27.78% vs. 15.25%, p = .002). Conclusion Both regimes significantly reduced sperm DNA fragmentation among subfertile men with a DFI >15%; however, when any baseline DFI value was considered, only micronutrient supplementation achieved a better result on DFI and thus pregnancy rate was higher.

Can leukocytospermia predict prostate cancer via its effects on mitochondrial DNA?

Leukocytospermia was previously reported to affect sperm quality by the production of reactive oxygen species (ROS) leading to oxidative stress (OS). In turn, OS decreases sperm functional integrity, increases sperm DNA damage and ultimately alters fertility status. To elucidate the impact of leukocytospermia on sperm nuclear DNA integrity and mitochondrial DNA (mtDNA) structure, we conducted a study including 67 samples from infertile patients with low level of leucocytes (Group 1: n=20) and with leukocytospermia (Group 2: n=47). In addition to standard sperm parameters' assessment, we measured the levels of inflammation biomarkers [interleukin-6 (IL-6) and interleukin-8 (IL-8)] and evaluated the oxidative status [malondialdehyde (MDA) and enzymatic and non-enzymatic antioxidants]. In addition, we evaluated the level of sperm nuclear DNA fragmentation and analysed mitochondrial DNA (mtDNA) of sperm cells by sequencing of 5 genes [cytochrome oxidase I (COXI), cytochrome oxidase II (COXII), cytochrome oxidase III (COXIII), adenosine triphosphate synthase 6 (ATPase 6) and adenosine triphosphate synthase 8 (ATPase 8)]. As expected, patients with leukocytospermia had significantly higher MDA levels (32.56±24.30 nmole/ml) than patients without leukocytospermia (17.59±9.60nmole/ml) (p<.018). Also, sperm DNA fragmentation index (DFI) was significantly higher in Group 2 (33.05±18.14%) as compared to Group 1 (14.19±9.50%) (p<.001). The sequencing of mtDNA revealed a high number of substitutions in Group 2 (n=102) compared to Group 1 (n=5). These substitutions were observed mainly in COXI. Among COXI substitutions found in Group 2, twelve changes were previously described in patients with prostate cancer and six of them were shown associated with this pathology. These findings suggest that leukocytospermia may predispose to the manifestation of prostate cancer through modification of mitochondrial DNA and this may be promoted by OS.

The Toolbox of Methods for Multidirectional Characterization of Dietary Nucleic Acids; Verification for Raw and Processed Food Products

Currently, the nutritional value of food is associated mainly with components such as proteins, carbohydrates, and lipids. However, another important macromolecules present in many foods are dietary nucleic acids (dietNA), i.e., DNA as well as both coding and non-coding RNAs. In the context of food chemistry and nutrition, dietNA are nowadays vastly neglected. In consequence, there are no dedicated methodologies to characterize dietNA. In this study, using raw or processed meat and plant products as model foodstuffs, we developed a toolbox of methods borrowed from other fields (histology, toxicology, molecular biology) that enable the initial characterization of dietNA as a necessary step on the way to systematic evaluation of their nutritional role. The proposed set of methods embraces (i) paraffin embedding of food samples and their staining to visualize the distribution and variety of dietNA in situ; (ii) comet assay to assess integrity of nuclear DNA with possible detection of DNA damage; (iii) dietNA isolation with and without RNAse digestion to determine the content of both DNA and RNA; (iv) electrophoretic separation of isolates to profile dietNA fragments. Such a combined methodological approach revealed clear differences between dietNA derived from raw and processed food products. We believe that the presented set of methods will encourage the broader research on dietNA to understand their role as a nutritionally relevant food component.

Nuclear localization of BCR and cortactin indicates their potential role in regulation of actin branching in nucleus.

To study cellular localization of full-length breakpoint cluster region (BCR), Pleckstrin homology domain of BCR and cortactin and determine whether they can coexist in cell nucleus. HEK293T cell line was transfected with pECFP-BCR, pEGFP-PH and pmTagRFP-N1-CTTN using polyethyleneimine. Live cells were imaged in cell culture dishes with glass coverslip attached to the bottom with Leica SP8 STED 3D confocal microscope in the environmental chamber. Obtained images were processed and analyzed with Fiji software. We identified colocalization of full-length BCR and cortactin in nucleus of cell undergoing terminal phase of cell division. We did not observe nuclear localization of cortactin in non-dividing cell. Both Pleckstrin homology domain and full-length BCR exhibited cytoplasmic as well as nuclear localization. Colocalization of BCR with cortactin in cell nucleus indicates their potential role in regulation of actin network allowing for the maintenance of nuclear architecture and DNA integrity.

Mitochondrial DNA lesions and copy number are strain dependent in endurance-trained mice.

In this pilot work, we selected two inbred strains that respond well to endurance training (ET) (FVB/NJ, and SJL/J strains), and two strains that respond poorly (BALB/cByJ and NZW/LacJ), to determine the effect of a standardized ET treadmill program on mitochondrial and nuclear DNA (nucDNA) integrity, and mitochondrial DNA (mtDNA) copy number. DNA was isolated from plantaris muscles (n = 37) and a gene‐specific quantitative PCR‐based assay was used to measure DNA lesions and mtDNA copy number. Mean mtDNA lesions were not different within strains in the sedentary or exercise‐trained states. However, mtDNA lesions were significantly higher in trained low‐responding NZW/LacJ mice (0.24 ± 0.06 mtDNA lesions/10 Kb) compared to high‐responding strains (mtDNA lesions/10 Kb: FVB/NJ = 0.11 ± 0.01, p = .049; SJL/J = 0.04 ± 0.02; p = .003). ET did not alter mean mtDNA copy numbers for any strain, although both sedentary and trained FVB/NJ mice had significantly higher mtDNA copies (99,890 ± 4,884 mtDNA copies) compared to low‐responding strains (mtDNA copies: BALB/cByJ = 69,744 ± 4,675; NZW/LacJ = 65,687 ± 5,180; p < .001). ET did not change nucDNA lesions for any strain, however, SJL/J had the lowest mean nucDNA lesions (3.5 ± 0.14 nucDNA lesions/6.5 Kb) compared to all other strains (nucDNA lesions/6.5 Kb: FVB/NJ = 4.4 ± 0.11; BALB/cByJ = 4.7 ± 0.09; NZW/LacJ = 4.4 ± 0.11; p < .0001). Our results demonstrate strain differences in plantaris muscle mtDNA lesions in ET mice and, independent of condition, differences in mean mtDNA copy and nucDNA lesions between strains.

One day is better than four days of ejaculatory abstinence for sperm function.

SummaryAccording to the World Health Organization guidelines, ejaculatory abstinence (EA) of 2–7 days is recommended for semen analysis. This study aimed to determine how seminal quality may be affected by two EA periods from the same man. Seminal samples from 65 men were evaluated by conventional semen analysis and qualitative characteristics after 1 and 4 days of EA (two samples/man). The semen was qualitatively analyzed by examining oxidative activity (intracellular and seminal plasma), sperm function (acrosome integrity, mitochondrial activity, and nuclear DNA integrity), and epididymal function. As expected, samples collected after 1 day of EA showed a decrease in volume and sperm total number compared to samples collected after 4 days of EA. The sperm motility of the samples collected after 1 day of EA was better compared to samples collected after 4 days of EA. Oxidative activity measured was lower after 1 day of EA compared with those measured after 4 days of EA. With regards to sperm function, samples collected after 1 day of EA showed an increase in acrosome integrity, mitochondrial activity, and nuclear DNA integrity compared with samples collected after 4 days of EA. Epididymal function showed no difference between the two-time points. Although samples collected after 4 days of EA showed better results for sperm quantity, samples collected after 1 day of EA showed better qualitative results, including motility, oxidative activity, and sperm function. Thus, it can be concluded that sperm storage at the epididymal tail may make spermatozoa more susceptible to oxidative damage.Lay summaryAccording to the World Health Organization guidelines, stopping ejaculation for 2 to 7 days is recommended before sperm collection for semen analysis. However, the evidence that supports these recommendations is limited. Our study aimed to compare how sperm quality was affected in samples collected after stopping ejaculation for 1 day and 4 days (two samples per man) in a total of 65 men. Although sample collection after stopping ejaculation for 4 days showed better semen quantity (volume and sperm concentration), sample collection after stopping ejaculation for 1 day showed better sperm motility and function. If not ejaculated, sperm are stored in the epididymis tail located in the scrotum beside the testicles and our study suggests that longer sperm storage may damage sperm quality. The results from this study may be used to inform guidance for sperm collection for use in assisted reproduction techniques, and lead to an improvement in both fertilization and implantation rates.

In vitro neuroprotective effects of farnesene sesquiterpene on alzheimer’s disease model of differentiated neuroblastoma cell line

Objective To investigate neuroprotective properties of the farnesene sesquiterpene on the experimental Alzheimer’s disease model in vitro. Methods Human neuroblastoma cell line (SHSY-5Y) was differentiated into neuron-like cells by using retinoic acid to constitute the in vitro Alzheimer’s Disease model. β-amyloid 1-42 protein was applied to the transformed cells for 24 and 48 hours in a wide dose ranges (3.125-200 μM) to establish AD cytotoxicity. Then, farnesene was applied to cell cultures in a wide spectrum dose interval (1.625-100 μg/ml) to investigate neuroprotective effect against β-amyloid for 24 and 48 hours. 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release tests were executed to determine cytotoxicity in the Alzheimer model. Nuclear DNA integrity of cells was examined under the fluorescent microscope using the Hoechst 33258 staining method. Furthermore, acetylcholinesterase (AChE) activity, total antioxidant capacity (TAC) and total oxidative status (TOS) levels were analyzed to understand the protection mechanism of the farnesene application on the cell culture model. Finally, flow cytometry analysis was used to find out the cell death mechanism after beta-amyloid and farnesene application to the cell culture. Results Cell viability tests revealed significant neuroprotection against β-amyloid toxicity in both 24 and 48 hours and the Hoechst 33258 fluorescence staining method showed a significant decrease in necrotic deaths after farnesene application in the cell cultures. Finally, flow cytometry analysis put forth that farnesene could decrease necrotic cell death up to 3-fold resulted from beta-amyloid exposure. Conclusion According to the investigations, farnesene can potentially be a safe, anti-necrotic and neuroprotective agents against Alzheimer’s disease.

Vitamin C and ubiquinone have the same ability in reducing the spermatozoa DNA fragmentation index in infertile men at Doctor Soetomo General Hospital, Surabaya, Indonesia

Background: Infertility is one of the most common health problems in the world. Malefactors contribute to 50% of cases and about 15-30% of infertile men are categorized as unexplained infertility. There have been numerous studies investigating the role of spermatozoa DNA fragmentation in male infertility. Spermatozoa nuclear DNA integrity has been suggested as a better predictor of male infertility and oxidative stress has been known related to it. This study was conducted to determine the oral supplementation of Vitamin C and Ubiquinone in reducing the spermatozoa DNA fragmentation index (DFI) among infertile men. Methods: This was an experimental clinical trial with a pre-test and post-test group design. Thirty infertile men were randomized double-blindingly into 3 groups: Vitamin C and Ubiquinone; Vitamin C and Placebo; Ubiquinone and Placebo groups, each treatment was given for 35 days. The DFI was evaluated by Sperm Chromatin Dispersion (SCD) test, before and after treatment. Data were analyzed using SPSS version 21 for Windows.Results: The average age of respondents was 32.6 years old, followed by 4.6 years of infertility duration in 5.8 years of marriage. Most of the respondents had a normal Body Mass Index (BMI) (46.7%), no smoking history (75.0%), no history of heat (90.0%) or chemical (73.3%) exposure, and diagnosed with primary male infertility (90.0%). Vitamin C group showed a significant reduction in the spermatozoa DFI (95% CI, p&lt;0.05), while the other two groups showed no differences in the spermatozoa DFIs (95% CI, p&gt;0.05). Comparison analysis showed no differences in the reduction of the spermatozoa DFIs (95% CI, p&gt;0.05) among these three groups. Conclusion: The recent findings suggest that oral supplementation of vitamin C can reduce the spermatozoa DFI, while ubiquinone and the combination of vitamin C and ubiquinone could not reduce the spermatozoa DFI.

Obesity and ageing: Two sides of the same coin.

Conditions and comorbidities of obesity mirror those of ageing and age-related diseases. Obesity and ageing share a similar spectrum of phenotypes such as compromised genomic integrity, impaired mitochondrial function, accumulation of intracellular macromolecules, weakened immunity, shifts in tissue and body composition, and enhanced systemic inflammation. Moreover, it has been shown that obesity reduces life expectancy by 5.8 years in men and 7.1 years in women after the age of 40. Shorter life expectancy could be because obesity holistically accelerates ageing at multiple levels. Besides jeopardizing nuclear DNA and mitochondrial DNA integrity, obesity modifies the DNA methylation pattern, which is associated with epigenetic ageing in different tissues. Additionally, other signs of ageing are seen in individuals with obesity including telomere shortening, systemic inflammation, and functional declines. This review aims to show how obesity and ageing are "two sides of the same coin" through discussing how obesity predisposes an individual to age-related conditions, illness, and disease. We will further demonstrate how the mechanisms that perpetuate the early-onset of chronic diseases in obesity parallel those of ageing.