DNA Fragmentation Rate Research Articles

P-782 The influence of microfluidic preparation of spermatozoa for ICSI (intracytoplasmic sperm injection) in an unselected IVF population on fertilization rate, embryo-quality and pregnancy rate

Abstract Study question Does the microfluidic technique as a new preparation option for sperm in ICSI during IVF have a better outcome in fertilization, blastocysts and pregnancy rate? Summary answer The patients that had the sperm preparation for ICSI with the microfluidic sperm selection had similar results as with the gradient centrifugation selection. What is known already Semen analysis is a poor predictor of the fertilization potential of spermatozoa and a male factor may contribute to poor outcomes of IVF procedure despite a normal semen analysis. Traditionally, sperm processing is accomplished by density gradient centrifugation or swim-up methods but this have been shown in some studies to increase reactive oxygen species and induce sperm DNA fragmentation. The microfluidic sperm selection (ZyMot- ICSI) is based on the selection of the spermatozoa with the lowest DNA fragmentation rate but studies do not prove better clinical outcomes after this method. Study design, size, duration This is a prospective randomized study carried out in a university level fertility clinic in Oradea, Romania from January 2022 to December 2022, including 239 patients in total. The patients that underwent IVF-ICSI procedures for various indications were randomized in two groups: the control group swim-up technique was used for sperm selection for ICSI during IVF and the study group with the ZyMot-ICSI (microfluidic sperm selection technique) was used. Participants/materials, setting, methods We conducted a prospective trial comparing 119 couples that were allocated to the classic swim-up technique (control group) and 120 couples that were allocated with the microfluidic technique was used (study group) in our university level clinic to go through IVF. Inclusion criteria were couples with known infertility requiring IVF and willing to participate in the study. Exclusion criteria were: severe oligoasthenospermia (concentration < 5x10^6 spermatozoa/ml or motility <10%), oocyte or sperm donation. Main results and the role of chance The statistical analysis showed that there is no significant difference between the fertilization rate (study vs control p = 0.37 with absolute difference -1.182, 95% CI (-7.2, 5.4)) or blastocyst rate (study vs control p = 0.88 with absolute difference -0.5 95% (-6.5, 6)) nor is there a difference in clinical pregnancy. Even if microfluidic preparation of spermatozoa did not seem to improve the results it may be utilized more broadly for standard IVF, intrauterine insemination and could also improve workflow, decrease intervention by laboratory personal and provide more consistent incubation conditions. Limitations, reasons for caution The population studied was inclusive with unselected IVF indications and did not attempt to isolate male factor infertility cases or patients with a history of elevated sperm DNA fragmentation. Wider implications of the findings Microfluidic sperm preparation performs similarly to density gradient centrifugation in sperm preparation for IVF in an unselected population. Trial registration number Not applicable

P-053 Sperm aneuploidy and DNA integrity pre- and post-chemotherapy in men with testicular cancer

Abstract Study question Are fragmentation levels (single-double stranded DNA breaks) or aneuploidy frequencies in sperm increased in patients with testicular cancer who recovered spermatogenesis after chemotherapy (CT)? Summary answer Similar levels of sperm DNA fragmentation and sperm aneuploidy frequency are found in men with testicular cancer pre- and post-chemotherapy. What is known already Testicular cancer is the most frequent solid tumor among young men. Therefore, the restoration of fertility and achievement of fatherhood in survivors have become important concerns. The integrity of genetic material in sperm is crucial since some paternal genes are essential for early embryo development. Although previous studies analyzed the effect of CT on sperm DNA integrity, they did not differentiate single vs. double-stranded DNA breaks. Few data are available on aneuploidy in sperm from testicular cancer survivors, so it remains to be elucidated whether cancer treatments may lead to an increased risk of aneuploidy syndromes in offspring. Study design, size, duration Prospective multicenter study including 31 males with a history of testicular cancer who requested semen cryopreservation between 2007-2021. All patients included underwent CT and recovered spermatogenesis once treatment ended. Post-CT samples were collected and seminal parameters, single-double stranded DNA breaks and the proportion of sperm aneuploidies were compared with one aliquot of the initial ejaculate. The project was approved by an Institutional Review Board and written informed consent for participation was obtained from each subject. Participants/materials, setting, methods Patient´s age prior CT was 31.23yrs (IC 95%: 29.29-33.17). Mean interval time between 1st and 2nd samples was 4.53yrs (IC 95%: 3.42-5.64). TUNEL quantified overall sperm DNA fragmentation by flow cytometry analyzing at least 20,000 cells, while γH2AX staining was applied for double DNA fragmentation assessment. Sperm aneuploidies were calculated by using fluorescence in situ hybridization (FISH) probes for chromosomes 13, 18, 21, X, Y. A paired t-test was used to compare variables pre- and post-CT. Main results and the role of chance Similar results were found when sperm quality parameters were compared. Mean sperm concentration pre-CT was 33.03M/mL (IC 95%: 23.98-42.07) and 36.41 M/mL (IC 95%: 27.85-44.96) in post-CT ejaculates (N.S.). A comparable rate of motile spermatozoa was observed in pre-CT vs. post-CT samples [(43.81% (IC 95%: 38.42-49.19) vs. 44.73% (IC 95%: 38.95-50.51), N.S.]. Regarding TUNEL assay results, the sperm DNA fragmentation rate was 21.65% (IC 95%: 17.66-25.63) in pre-CT samples and 23.00% (IC 95%: 19.90-26.61) in post-CT samples (N.S.). Furthermore, similar results were obtained when double-stranded fragmentation levels (H2AX) were compared between pre and post-CT [10.2% (IC 95%: 6.45-13.96) vs. 12.44% (IC 95%: 8.75-16.13) N.S.]. Comparable sperm disomic proportions were found in PRE and POST-CT samples: [(Cr.13 =0.02%, Cr.18 =0.01%, Cr.21=0.30%, Cr.XY=0.84%) vs. (Cr.13 =0%, Cr.18 =0%, Cr.21=0%, Cr.XY=0.33%), N.S]. Results were also evaluated according to the time elapsed between the PRE and POST -CT samples. No significant differences were found for seminal values (sperm concentration, p = 0.26; motility, p = 0.68), overall sperm DNA fragmentation (p = 0.68), double-strand DNA fragmentation (p = 0.90) and the frequency of sperm disomies (Cr. 13 p = 0.79, Cr.18 p = 0.46, Cr. 21 p = 0.85, Cr.XY p = 0.05). Limitations, reasons for caution The main limitations of the present study are that we only recruited patients who recovered spermatogenesis and that our data evaluated a long-term effect of CT. Wider implications of the findings All men wishing to have their own children after gonadotoxic treatment should receive adequate counseling about the side effects of the therapy. Based on our results on sperm DNA integrity and euploidy, patients who recovered spermatogenesis should not be advised to use samples that were preserved before undergoing CT. Trial registration number na

The effects of coenzyme Q10 and curcumin supplementation in freezing medium for human sperm cryopreservation

BackgroundDespite its routine and frequent application, cryopreservation of human sperm is far from the desired efficacy, as freezing and thawing impair motility, viability, acrosomal unity, and DNA integrity. ObjectivesIn this study, the authors aimed to investigate whether adding antioxidants, coenzyme Q10, and curcumin into the freezing medium provide better efficacy in the cryopreservation of human sperm. MethodsThe semen samples from 40 healthy men aged 18–45 were collected in sterile containers by masturbation. Samples within normal reference values for sperm concentration (≥15 million/mL) and motility (progressive motile ≥ 32% and total motility ≥ 40%) were included in the study. Semen samples were equally divided into five groups and evaluated; i) pre-freezing sperm suspension, ii) frozen-thawed control (Ctrl) without any supplementation in freezing medium, iii) frozen-thawed with curcumin supplementation of 0.25 mM (Cur), iv) frozen-thawed coenzyme Q10 supplementation of 25 µM (CoQ10) and v) frozen-thawed curcumin (0.25 mM) plus coenzyme Q10 (25 µM) supplementation (CurCoQ10) into the freezing medium. Liquid nitrogen vapour freezing and rapid thawing were performed in each group (ii-v). Sperm motility, viability, acrosome integrity, and DNA fragmentation rates were compared and ultrastructural evaluations by transmission electron microscopy were undertaken between the groups. Additionally, the total antioxidant capacity/total oxidant capacity values were measured. ResultsAccording to CASA results, progressive motility was significantly higher in the CoQ10 group (9.4 ± 7.6) when compared with the Ctrl (7.1 ± 6.3), Cur (6.4 ± 4.8) and CurCoQ10 (8.1 ± 7.7) groups (p < 0.05). Flow cytometry results showed no difference in the viability and acrosome integrity values after thawing, but DNA fragmentation was significantly increased in the curcumin-added groups (p < 0.05). Acrosomal changes and sub-acrosomal defects were seen in all groups after thawing at the ultrastructural level. Mitochondrial membrane structure was preserved in CoQ10 and CurCoQ10 groups. ConclusionsOur results suggested that sperm ultrastructural morphology and motility were better preserved in the CoQ10 group during cryopreservation. In curcumin groups, DNA fragmentation and head defects were increased.

Shaping the future of male reproductive health: fostering talent at the 14th Network of Young Researchers in Andrology meeting.

The 14th Network of Young Researchers in Andrology (NYRA) meeting was a 2-day conference held at the University of East Anglia in Norwich, UK, organized by the NYRA. The meeting brought together researchers and experts to discuss and exchange ideas on male infertility and spermatogenesis. The meeting covered a wide range of topics related to male germline research, including the impact of mutations in the male germline on future generations, the use of innovative sequencing tools for the study of male infertility, and the intricate germline epigenome. The impact of aging on spermatogenesis was also discussed, with a focus on the increased DNA fragmentation rates, changes in DNA methylation patterns, and longer telomeres associated with aging sperm. Additionally, progress on fertility preservation options for children undergoing gonadotoxic cancer treatments was presented. The meeting also featured workshops on leadership and career development strategies in science, providing a valuable opportunity for young researchers to learn from experts in the field and exchange ideas with their peers. Overall, the meeting provided a platform for researchers to discuss the latest developments in male germline research, highlighting the importance of empowering young researchers to tackle male reproductive health.

Sperm DNA fragmentation and apoptosis in the sperm of men with oligozoospermia are closely related to anti-ODF2 autoantibodies.

Around 15% of couples of childbearing age suffer from infertility; in 50% of these cases, the male factor is present. In this study, we investigated the association between anti-ODF2 autoantibody existence and the DNA fragmentation and apoptosis of sperm in oligozoospermia men. 35 fertile men and 57 oligozoospermia men are enrolled in this study as control and case groups, respectively. After the identification of ODF2 as a possible target of anti-sperm antibodies in sera of oligozoospermia men using two-dimensional gel electrophoresis followed by western blotting and mass spectrometry, the case group serums were screened for anti-ODF2 autoantibodies and divided into anti-ODF2 negative (N=24) and positive (N=33) subgroups to follow assays. The mRNA expression levels of ODF2, Caspases 3, 8, 9, BAX, and BCL-2 were evaluated via qRT-PCR in spermatozoa samples of mentioned groups. DNA fragmentation and apoptosis rate of spermatozoa in studied groups were assessed using an SDF kit and flow cytometry, respectively. Mass spectrometry showed that ODF2 is one of the anti-sperm antibodies targeted in oligozoospermia patients. 33 of 57 oligozoospermia men had anti-ODF2 autoantibody in their sera. An elevated expression of ODF2 mRNA was observed in spermatozoa of anti-ODF2+ patients compared to anti-ODF2- patients and controls. There was an increased expression level of Caspase 3, 8, 9, and BAX and decreased expression of BCL-2 in spermatozoa of anti-ODF2+ patients compared to anti-ODF2- patients and controls. Noticeable increases in DNA fragmentation and apoptosis rate of anti-ODF2+ patients' spermatozoa were observed compared to anti-ODF2- patients and healthy controls spermatozoa. A positive correlation was observed between ODF-2 expression and DNF fragmentation and apoptosis rate of anti-ODF2+ patients' spermatozoa. Our results revealed that ODF2 is one of the main spermatozoa structural proteins, which is one of the anti-sperm antibodies targets, and its dysregulated expression may result in an increased rate of sperm DNA fragmentation and apoptosis.

MP12-06 SOCIOECONOMIC DETERMINANTS AS A PREDICTOR OF HORMONE AND SEMEN PARAMETERS

You have accessJournal of UrologyCME1 Apr 2023MP12-06 SOCIOECONOMIC DETERMINANTS AS A PREDICTOR OF HORMONE AND SEMEN PARAMETERS Jordan Kassab and Larry Lipshultz Jordan KassabJordan Kassab More articles by this author and Larry LipshultzLarry Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003227.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Social determinants of health, including economic resources, lifestyle choices, and access to adequate healthcare, have been shown to predict certain health outcomes. While past studies have indicated varying fertility outcomes as a result of social determinants of health, little research has been done to look into how biological markers are affected. Thus, the aim of this study is to investigate whether hormone and semen quality parameters are influenced by socioeconomic determinants. METHODS: Using the Distressed Community Index (DCI), disparate communities were identified by ZIP code within a ten-mile radius of our university hospital. The DCI compiles seven metrics regarding income and vulnerability to generate a percentile score with lower scores indicating better economic well-being. We identified zip codes that fall within the highest quintile of scores and zip codes that fall within the lowest quintile of scores in order to provide a stratified patient cohort. Using these zip codes, 179 urology patients were included in our retrospective chart analysis noting DNA fragmentation rates, testosterone, PSA, and total sperm count. T-tests for each parameter were calculated using SPSS. RESULTS: The statistical analysis revealed that semen parameters greatly differed between the low and high DCI score patient cohorts. The low DCI cohort had less than half of the DNA fragmentation rate of the high DCI cohort (12.64% vs. 30.50%, p=.0245). The low DCI group had significantly higher testosterone levels (632.5 ng/dL vs. 353.7 ng/dL, p=.00001). Additionally, PSA levels varied significantly between the low and high DCI groups (0.685 ng/mL vs. 3.28 ng/mL, respectively, p=.0125). Lastly, the total sperm count varied greatly between the two groups: the low DCI group had an average of 125 million and the high DCI group had an average of 68.3 million (p=.0316). CONCLUSIONS: Our study has demonstrated that men residing within wealthier and less vulnerable areas had documented more normal DNA fragmentation rates, testosterone and PSA levels, and total sperm counts. This relationship emphasizes the need to prioritize efforts towards supporting urologic care in disadvantaged communities. Our findings establish the basis for further population based studies to address the impact of socioeconomic status as a factor of urologic health outcomes. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e135 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jordan Kassab More articles by this author Larry Lipshultz More articles by this author Expand All Advertisement PDF downloadLoading ...

Comparative Study of Sperm Selection Techniques for Pregnancy Rates in an Unselected IVF-ICSI Population.

(1) Background: Semen analysis is a poor predictor of the fertilization potential of spermatozoa and a male factor may contribute to poor outcomes of the IVF procedure, despite a normal semen analysis. The microfluidic sperm selection (ZyMot-ICSI) is based on the selection of the spermatozoa with the lowest DNA fragmentation rate, but studies do not prove better clinical outcomes after this method. (2) Methods: We conducted a retrospective trial comparing 119 couples that were allocated to the classic gradient centrifugation sperm method (control group), and 120 couples that were allocated with the microfluidic technique being used (study group) at our university-level clinic, to go through IVF. (3) Results: The statistical analysis showed that there is no significant difference between the fertilization rate (study vs. control p = 0.87), but regarding blastocyst rate (study vs. control p = 0.046) and clinical pregnancy (p = 0.049), there is quite a significant statistical difference. Microfluidic preparation of spermatozoa seems to improve the results and it may be utilized more broadly for ICSI, and could also improve the workflow in standard IVF, decrease intervention by laboratory personnel and provide more consistent incubation conditions. (4) Conclusions: The patients that had the sperm preparation for ICSI with the microfluidic sperm selection had slightly better results compared with the gradient centrifugation selection.

Comparison between density gradient centrifugation method, an extended version of the horizontal swim up method and the combination of both for sperm selection.

To compare the degree of efficiency between density gradient centrifugation (DGC) method and an extended horizontal swim-up (SU) method. A total of 97 couples undergoing in vitro fertilization were enrolled in the study. Semen samples were divided into three aliquots and treated using DGC, extended horizontal SU, and combined methods. DNA fragmentation and chromatin decondensation were detected in native semen samples and their three corresponding aliquots. The corresponding mature oocytes of each semen sample were divided into two sibling cultures. The first sibling culture was microinjected with semen pellets from DGC, and the second sibling culture was microinjected with semen pellets from the combination of both methods. Fertilization rate and embryonic development were assessed at day 3. DNA fragmentation and chromatin decondensation was significantly low in DGC and extended horizontal SU samples; however, the rates of DNA fragmentation and chromatin decondensation were significantly lower in extended horizontal SU samples than in DGC samples. The lowest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with both methods. The highest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with DGC. No significant difference was found in the fertilization rate or day 3 embryos between sibling cultures. The combination of DGC and the extended horizontal SU techniques is best for giving the lowest rates of sperm DNA fragmentation and chromatin decondensation.

Correlation analysis of sperm DNA fragmentation index with semen parameters and the effect of sperm DFI on outcomes of ART

Routine semen analysis provides limited information about a man’s male reproductive potential and can not always fully explain male infertility. Many male infertilities are caused by sperm DNA defects that routine semen quality analyses fail to detect. In this study, we analyzed the association of sperm DNA fragmentation index (DFI) with the semen routine, sperm morphology, in vitro fertilization embryo transfer (IVF-ET)/intracytoplasmic sperm injection (ICSI). Further, we explored the predictive value of sperm DFI in evaluating male fertility and the outcome of IVF-ET/ICSI. Data on sperm DFI, sperm routine, and sperm morphology were collected from 1462 males with infertility. According to DFI levels, there were 468 cases in group I (DFI ≤ 15%), 518 cases in group II (15% < DFI < 30%), and 476 cases in group III (DFI ≥ 30%). The correlations of sperm DFI with semen routine and malformation rate were analyzed. Seminal plasma malondialdehyde (MDA), and total antioxidant capacity (TAC) were assessed. Sperm DFI, semen routine, and sperm morphology were detected in male patients of 101 pairs of IVF-ET/ICSI infertile couples and subdivided into IVF-I group (DFI ≤ 15%), IVF-II group (15% < DFI < 30%), IVF-III group (DFI ≥ 30%), ICSI-I group (DFI ≤ 15%), ICSI-II group (15% < DFI < 30%) and ICSI-III group (DFI ≥ 30%) according to DFI value. The effect of sperm DFI on the outcome of IVF-ET/ICSI was analyzed. There were significant differences in sperm survival rate, sperm concentration, and PR% between groupIII and group II (P < 0.01). There were significant differences in sperm survival rate, sperm concentration and PR% between group III and group I (P < 0.01). There was no significant difference in semen volume, age, abstinence days, or percentage of normal sperm between the three groups (P > 0.05). DFI was positively correlated with MDA content ( P < 0.01) and negatively correlated with TAC (P < 0.01). Sperm DFI was negatively correlated with sperm survival rate, sperm concentration, and PR% (P < 0.01). There was no correlation with age, abstinence days, semen volume, or percentage of normal-form sperm (r = 0.16, 0.05, 0.04, -0.18, p > 0.05). Compared with IVF-I group, the sperm concentration and PR were decreased in IVF-III group. The sperm malformation rate was higher in IVF-III group than that in IVF-II group. Comparatively, the PR was decreased in ICSI-III group. The sperm malformation rate was higher in ICSI-III group than that of the ICSI-I group (P < 0.05). There were no statistically significant differences in fertilization rate, cleavage rate, embryo rate, and clinical pregnancy rate between IVF group or ICSI group, and between all subgroups (P > 0.05). Sperm DFI is negatively associated with sperm survival rate, sperm concentration, and PR%. Antioxidants can decrease the rate of DNA fragmentation. Sperm DFI has proven to be very valuable in the male fertility evaluation, but its significance as a predictor of pregnancy outcomes following assisted reproductive technology. (ART) requires further investigation.

The impact of varicocelectomy on sperm DNA fragmentation and pregnancy rate in subfertile men with normal semen parameters: A pilot study

The impact of varicocelectomy on sperm DNA fragmentation and pregnancy rate in subfertile men with normal semen parameters: A pilot study

Association between The SIRT1 and SIRT3 Levels and Gene Polymorphisms with Infertility in War Zones of Kermanshah Province, Iran: A Case-Control Study.

War toxin, mustard gas, alkylating agent results in male infertility via inducing reactive oxygen species (ROS) production and DNA mutagenesis. SIRT1 and SIRT3 are multifunctional enzymes that involve in the DNA repair, oxidative stress responses. This study aim is to assess the correlation between serum levels of SIRT1, SIRT3 and both rs3758391T>C and rs185277566C>G gene polymorphisms with infertility in the war zones of Kermanshah province, Iran. In this case-control study based on the semen analysis, samples were divided into two groups infertile (n=100) and fertile (n=100). High-performance liquid chromatography (HPLC) method was used to determine the malondialdehyde level, and also a sperm chromatin dispersion (SCD) test was used to evaluate the DNA fragmentation rate. Using the colorimetric assays, superoxide dismutase (SOD) activity was measured. SIRT1 and SIRT3 protein levels were determined by using ELISA. The genetic variants of SIRT1 rs3758391T>C, and SIRT3 rs185277566C>G, were detected by polymerase chain reaction-restriction fragment length (PCR-RFLP) technique. Malondialdehyde (MDA) level and the percentage of DNA fragmentation were higher in infertile samples, but serum levels of SIRT1 and SIRT3, and SOD activity was lower in infertile compared to fertile samples (P<0.001). The TC+CC genotypes and the C allele from SIRT1 rs3758391T>C polymorphism, and CG+GG genotypes and the G allele from SIRT3 rs185277566C>G polymorphism could increase risk of infertility (P<0.05). The results of this study suggest that war toxins through the impact on genotypes, decreasing levels of SIRT1 and SIRT3 and increasing levels of oxidative stress, lead to defects in the concentration, motility and morphology of sperms and thus, infertility in men.

Estrogenic and Non-Estrogenic Disruptor Effect of Zearalenone on Male Reproduction: A Review.

According to some estimates, at least 70% of feedstuffs and finished feeds are contaminated with one or more mycotoxins and, due to its significant prevalence, both animals and humans are highly likely to be exposed to these toxins. In addition to health risks, they also cause economic issues. From a healthcare point of view, zearalenone (ZEA) and its derivatives have been shown to exert many negative effects. Specifically, ZEA has hepatotoxicity, immunotoxicity, genotoxicity, carcinogenicity, intestinal toxicity, reproductive toxicity and endocrine disruption effects. Of these effects, male reproductive deterioration and processes that lead to this have been reviewed in this study. Papers are reviewed that demonstrate estrogenic effects of ZEA due to its analogy to estradiol and how these effects may influence male reproductive cells such as spermatozoa, Sertoli cells and Leydig cells. Data that employ epigenetic effects of ZEA are also discussed. We discuss literature data demonstrating that reactive oxygen species formation in ZEA-exposed cells plays a crucial role in diminished spermatogenesis; reduced sperm motility, viability and mitochondrial membrane potential; altered intracellular antioxidant enzyme activities; and increased rates of apoptosis and DNA fragmentation; thereby resulting in reduced pregnancy.

Gauging DNA degradation among common insect trap preservatives

AbstractGenetic methods for species identification are becoming increasingly popular and can accelerate insect monitoring. However, obtaining good DNA quality and quantity from insect traps remains a challenge for field studies. Ethylene glycol, propylene glycol, and Renner solution have been previously suggested as suitable preservatives for the collection of genetic material, but a systematic overview of their performance under compromising field conditions is lacking. Here we experimentally test whether and how different preservatives affect DNA quality under different conditions and evaluate how choice of preservative may affect metabarcoding and more demanding downstream applications (e.g., RADseq). For this, we used the house cricket, Acheta domesticus (L.) (Orthoptera: Gryllidae), and tested propylene glycol, ethylene glycol, and Renner solution for their ability to preserve DNA over 27 days in various dilutions and temperatures. DNA quality was measured as DNA fragmentation and success rates in PCR amplifying a COI fragment of 658, 313, or 157 bp. Undiluted propylene glycol and ethylene glycol always retained high molecular weight DNA at room temperature. No high molecular weight DNA was preserved at 37 °C or in any dilution. Nevertheless, the COI sequence could be amplified from samples at every condition. Renner solution did not preserve high molecular weight DNA and fragmentation increased over time at 37 °C until amplification was impossible. The results suggest that propylene glycol and ethylene glycol are suitable preservatives for collecting both genetic and morphological material, but dilution or high temperatures compromise their ability to preserve high molecular weight DNA. For genomic approaches requiring high DNA quality, additional preservatives may need to be tested.

Effects of in vitro repaglinide supplementation on improving sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men.

Human sperm motility and hyperactivation (HA) are induced by different factors such as intracellular calcium concentration. Repaglinide is an antidiabetic drug that, via the blocking of ATP-sensitive potassium channels (K-ATP channels), depolarization of the β-cell membrane, and opening of the voltage-gated calcium channels leads to an increase in intracellular calcium. The present study aimed to examine the effects of repaglinide on in vitro sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men. Semen samples were collected from two groups of normozoospermic donors and asthenozoospermic patients. The samples were washed free of seminal plasma and then treated with medium alone (control) or with 100 nM and 1µM concentrations of repaglinide. After 1 h of incubation, percent sperm motility and hyperactivation were assessed; after 2 h of incubation, sperm viability and DNA fragmentation rate were evaluated by the Eosin-Y and acridine orange staining, respectively. In both groups, repaglinide at a concentration of 100 nM and 1µM significantly improved percent sperm motility, hyperactivation, and vital sperms with normal DNA; in specimens from normozoospermic men, the 1µM concentration had a noticeable effect on progressive motility; in samples from asthenozoospermic men, the highest hyperactivation rate was seen at a concentration of 100 nM as compared with the 1µM concentration and controls (p<0.05). Our results suggest that repaglinide can improve sperm motility, hyperactivity, viability, and DNA integrity in both normozoospermic and asthenozoospermic men.

Possible Harmful Effects of Smoking Hookah on Sperm DNA Fragmentation Index and Protamine Genes Expression in Normozoospermic Men

In recent years, smoking water pipes or hookah has increased among adolescents in most countries. Although there is evidence in support of the negative effects of this type of smoking on human health, such as the increased risk of lung disease, little is known about the potential effects of hookah smoking on the male reproductive system, especially on the molecular aspects of sperm. This cross-sectional study examined sperm DNA fragmentation index, protamine 1 and 2 (PRM1 and PRM2) genes expression, and oxidant status in normozoospermic hookah smokers in comparison with non-smoker controls. Our results showed significantly higher rates of DNA fragmentation, protamine deficiency, and abnormal chromatin condensation in the spermatozoa of hookah smokers (P < .0001). Also, protamine gene expression showed a remarkable decrease in hookah smokers (1.55 ± 2.54 and 0.33 ± 0.54) compared to the controls (3.49 ± 5.41 and 1.22 ± 1.96), although the reduction was not statistically significant (P = .155 and P = .066, respectively). Moreover, a significantly higher level of semen MDA was observed in the case group compared to the controls (0.39 ± 1.04 vs 0.15 ± 0.21; P = .013). According to our study, although hookah smoking does not have a significant effect on sperm parameters, it may have deleterious effects on DNA integrity, oxidative status, and nuclear protein levels of spermatozoa.

Влияние длины молекул и природы среды на фрагментацию ДНК ультразвуком

Fragmentation of DNA by ultrasound is the most optimal method of artificial degradation of DNA molecules. It requires no reagents and makes it possible to obtain "pure" DNA samples for further use. However, despite an active usage of ultrasonic fragmentation in studies, a number of issues related to the applicability of mechanically degraded DNA for standard molecular biological operations remain poorly studied. This report presents data on the effect of the size of DNA molecules (on DNA amplicons) and the composition of aqueous solutions on the efficiency of DNA destruction by ultrasound. It has been shown that the rate of DNA fragmentation positively correlates with an increase in the length of molecules. In solutions with high ionic strength, the rate of fragmentation increases dramatically. The presence of organic solvents that do not affect hybridization slightly reduces the rate of ultrasonic destruction. With an increase in the length of DNA molecules, the influence of denaturing agents changes and leads to an increase in the rate of fragmentation. dsDNA molecules smaller than one persistent length (about 150 bp) are not fragmented.

Combinational Therapy of Acute Lymphoblastic Leukemia with Cyclophosphamide and BV6 Synergistically Induces Apoptosis in Leukemic Cells

Background: Acute lymphoblastic leukemia (ALL), a hematological malignancy, is associated with the accumulation of lymphoblasts in hematopoietic tissues and the peripheral blood (PB). A major obstacle in ALL treatment is the failure to undergo apoptosis, thus resulting in relapses. Cyclophosphamide is an alkylating agent widely used to treat various cancers. BV6 is another agent, under preclinical and clinical trials, which inhibits the function of inhibitors of apoptosis proteins (IAPs) in cells. According to previous studies, IAPs are highly overexpressed in ALL cells. Also, the intrinsic apoptosis pathway is reported to be dysfunctional in ALL cells, making BV6 a new promising option due to its potential to induce extrinsic cell death as well. Methods: In the current study, the inhibitory effect of the combinational treatment of cyclophosphamide and BV6 was studied on the cell growth of eleven ALL patients using an MTT test. Subsequently, the effect of the treatment was studied on TNF-α secretion as well. Finally, we measured the DNA fragmentation rates of treated cells as well. Results: The results indicated a synergistic effect between the drugs on the viability of cells. Also, the impact of the cotreatment was increased in a time-dependent manner. We also found that BV6, but not cyclophosphamide, induces TNF-α secretion, activating the extrinsic apoptosis pathway as well. Conclusion: BV6, by inducing intrinsic and extrinsic pathways, overcomes cyclophosphamide resistance. These results suggest the combinational use of BV6 and cyclophosphamide for the treatment of ALL patients. Inhibition of IAPs helps overcome the failure to undergo apoptosis and can improve the prognosis of ALL patients.

Effects of Jinkui Shenqi and Wuzi Yanzong pill on sperm motility and sperm DNA fragmentation rate in asthenospermia

This study was designed to compare the effects of Jinkui Shenqi and Wuzi Yanzong pill on sperm motility and sperm DNA fragmentation rate in patients with asthenospermia. 130 cases were randomly divided into an observation and control group (n=65). The control group was treated with the Wuzi Yanzong pill while the observation group with the Jinkui Shenqi pill. The sperm motility parameters rate (PR), semen concentration, sperm motility, DFI and α-glucosidase, fructose, seminal plasma zinc (Zn), acid phosphatase (ACP) in seminal plasma biochemistry and other indexes of were observed. The biochemical indexes of seminal plasma of α-glucosidase, fructose, Zn, ACP in two groups were significantly (p&lt;0.05) improved after treatment. Compared with the control group, the indexes of the observation group improved more obviously after treatment. Pearson correlation analysis of DFI and PR indexes in 130 patients before treatment showed that sperm DFI and PR percentage were negatively correlated in asthenospermia patients (r =-0.572, P&lt;0.05). There was no significant difference in DFI, semen concentration, PR, and sperm motility between the two groups before treatment. The DFI, semen concentration, PR and sperm viability of the two groups showed a tendency to improve after treatment, and the effect of the observation group was less significant than that of the control group (p&lt;0.05). Two groups of patients have completed treatment successfully, no adverse events occurred during treatment. Jinkui Shenqi pill can effectively treat asthenospermia, which can effectively improve the effect of sperm motility in patients. It has less adverse reactions, safe and reliable, and is worthy of promotion.

The effect of pentoxifylline and calcium ionophore treatment on sperm cell biology in oligoasthenoteratozoospermia samples.

The objective of this study was to assess the effects of pentoxifylline (PTX) and Ca2+ ionophore (CI) A12387 treatment on some biological characteristics of sperm cells in oligoasthenoteratozoospermia (OAT) patients. After processing, each sample was divided into four groups: 1, control; 2, exposed to 3.6 mM PTX; 3, exposed to 5 μm calcium ionophore (CI); and 4, exposed to both PTX and CI; 30 min at 37°C. Sperm motility was measured before and after preparation. Acrosome reaction (AR), status of sperm vacuoles, mitochondrial membrane potential (MMP) and DNA fragmentation were assessed using PSA-FITC staining, motile sperm organelle morphology examination (MSOME), JC-1 staining and sperm chromatin dispersion (CSD) test, respectively. Treatment with PTX and CI led to increased and decreased sperm motility, respectively (P < 0.05). Furthermore, vacuole status and rates of sperm DNA fragmentation were not significantly different among groups (P > 0.05). Moreover, the data showed that the rates of AR and disrupted MMP were significantly different between groups (P < 0.05). In conclusion, in vitro application of PTX not only did not have any adverse effects on sperm cell biology characteristics, but also can rectify the harmful effect of CI.

Using Dextran Instead of Egg Yolk in Extender for Cryopreservation of Spermatozoa of Dogs of Different Ages.

Egg yolk is a very common supplement of extenders aimed to protect sperm from cryoinjury, but due to their biological risks and difficulties with media standardization, there is a search for alternative. In addition, sperm cryoresistance can be affected by the initial decrease of their functional characteristics caused by age. The aim of this work was to evaluate the efficiency of using dextran (molecular weight 500 kDa) in the extenders instead of egg yolk for the cryopreservation of spermatozoa of dogs (Chinese Crested breed) of different ages. The obtained ejaculates were divided into three groups depending on the animal's age: 1-3, 4-6 and 7-10 years old. Sperm was cryopreserved by using 7% glycerol and 20% egg yolk, or 20% dextran. The cryoresistance of spermatozoa of the oldest age category was dramatically decreased, which was manifested in their morphology, motility, and DNA fragmentation rate. There were no differences between the cryoprotectant effect of the dextran-based extender on spermatozoa and the egg yolk-based extender in all age categories of dogs. However, given the benefits of dextran-containing media, its use for the cryopreservation of canine spermatozoa has potential benefits that need to be confirmed by sperm fertilization outcomes.