Poor Sperm Chromatin Condensation Is Associated with Cryopreservation-Induced DNA Fragmentation and Cell Death in Human Spermatozoa.

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze-thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze-thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze-thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3-8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at -196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all rs ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial rs ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (rs = 0.366, p = 0.014) and necrosis (rs = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial rs ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (rs = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze-thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood.

Revalidation of DNA Fragmentation Analyses for Human Sperm-Measurement Principles, Comparative Standards, Calibration Curve, Required Sensitivity, and Eligibility Criteria for Test Sperm.

(1) Background: Double-strand breaks (DSBs) in a single nucleus are usually measured using the sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, and comet assay (CA). Mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFGE) and angle-modulated two- dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) were developed to observe DNA fragmentation in separated motile sperm. (2) Methods: Comparative standards, calibration curves, required sensitivity levels, and eligibility criteria for test sperm were set up to validate the measurement principles of these tests. (3) Results: The conventional methods overlooked the interference of nucleoproteins in their measurements. In-gel proteolysis improves the measurement accuracies of 1D- and 2D-SCPFGE. Naked DNA is suitable for comparative standards and test specimens. Moreover, several dysfunctions that might induce DNA damage are observed in the separated motile sperm. Overall, the discussion highlights the need to revisit the conventional univariable analyses based on the SCSA, SCD test, and CA. (4) Conclusions: Human infertility is a complex syndrome, and the aim of quality control in intracytoplasmic sperm injection is to identify the underlying dysfunctions remaining in the separated motile sperm that render them ineligible for injection. Multivariable analyses with special consideration to confounding factors are necessary in future cohort studies.

DNA fragmentation of lymphocytes and sperm cells induced by nickel released from orthodontic archwires: A preliminary study.

Orthodontic brackets and archwires placed intraorally are subject to corrosion, leading to the release of cytotoxic metal ions. The aim of this study was to determine whether the use of orthodontic NiTi archwires increases systemic Ni levels and cause alterations on the DNA of cells unrelated to the oral environment such as lymphocytes and sperm cells. Human urine, semen and blood samples were collected before (baseline) sham placement of orthodontic archwires and 15 and 30 days after placement. Lymphocytes and sperm cells cells were evaluated by comet assay. Ni concentration levels in urine increased significantly between baseline and 15 days (p<0.01) and 15 and 30 days of exposure (p<0.01). Progressive decrease in sperm viability and motility was observed between the sampling periods. Lymphocytes and sperm cells showed DNA fragmentation. The increase in systemic concentration of nickel induced structural damage in the DNA of lymphocytes and human sperm cells.

Nucleic Acid Target Sensing Using a Vibrating Sharp-Tip Capillary and Digital Droplet Loop-Mediated Isothermal Amplification (ddLAMP).

Nucleic acid tests are key tools for the detection and diagnosis of many diseases. In many cases, the amplification of the nucleic acids is required to reach a detectable level. To make nucleic acid amplification tests more accessible to a point-of-care (POC) setting, isothermal amplification can be performed with a simple heating source. Although these tests are being performed in bulk reactions, the quantification is not as accurate as it would be with digital amplification. Here, we introduce the use of the vibrating sharp-tip capillary for a simple and portable system for tunable on-demand droplet generation. Because of the large range of droplet sizes possible and the tunability of the vibrating sharp-tip capillary, a high dynamic range (~2 to 6000 copies/µL) digital droplet loop-mediated isothermal amplification (ddLAMP) system has been developed. It was also noted that by changing the type of capillary on the vibrating sharp-tip capillary, the same mechanism can be used for simple and portable DNA fragmentation. With the incorporation of these elements, the present work paves the way for achieving digital nucleic acid tests in a POC setting with limited resources.

Preparation, characterization and antioxidant and anticancerous potential of Quercetin loaded ß-glucan particles derived from mushroom and yeast.

ß-glucans are polysaccharides found in the cell walls of various fungi, bacteria and cereals. ß-glucan have been found to show various kinds of anti-inflammatory, antimicrobial, antidiabetic antioxidant and anticancerous activities. In the present study, we have isolated ß-glucan from the baker's yeast Saccharomyces cerevisiae and white button mushroom Agaricus bisporus and tested their antioxidant potential and anticancerous activity against prostate cancer cell line PC3. Particles were characterized with zeta sizer and further with FTIR that confirmed that the isolated particles are ß-glucan and alginate sealing made slow and sustained release of the Quercetin from the ß-glucan particles. Morphological analysis of the hollow and Quercetin loaded ß-glucan was performed with the SEM analysis and stability was analyzed with TGA and DSC analysis that showed the higher stability of the alginate sealed particles. Assessments of the antioxidant potential showed that Quercetin loaded particles were having higher antioxidant activity than hollow ß-glucan particles. Cell viability of the PC3 cells was examined with MTT assay and it was found that Quercetin loaded alginate sealed Agaricus bisporus derived ß-glucan particles were having lowest IC50. Further ROS generation was found to increase in a dose dependent manner. Apoptosis detection was carried out with Propidium iodide and AO/EtBr staining dye which showed significant death in the cells treated with higher concentration of the particles. Study showed that particles derived from both of the sources were having efficient anticancer activity and showing a dose dependent increase in cell death in PC3 cells upon treatment.

Impact of cryoprotectant-free sperm vitrification in pulled-glass capillary on sperm parameters and DNA integrity: A lab trial study.

Background: Vitrification is a recently introduced yet widely applied assisted reproduction technique. So far, the effects of the chemicals and devices in vitrification on sperm motility and DNA integrity are still unclear. Objective: This study aimed to examine sperm quality, as determined by semen analysis and sperm DNA integrity when vitrified with or without cryoprotectant agents (CPAs) using pulled-glass capillaries. Materials and Methods: Between February and June 2020, 50 infertile men from the Hue Center for Reproductive Endocrinology and Infertility, Hue University of Medicine and Pharmacy, Vietnam, were enrolled. Sperm samples, prepared using the swim-up technique, were divided into 2 groups: vitrification with CPAs (group 1) and without CPAs (group 2). Vitrified sperm samples were preserved in 10 µL pulled-glass capillaries. Motility, sperm membrane integrity, and the DNA fragmentation index were tested. Results: Sperm motility in vitrified media with CPAs (54.4 ± 11%) was statistically higher than in media without CPAs (51.14 ± 10.6%, p < 0.05). CPAs did not affect sperm membrane integrity or large halo ratio (71.34 ± 8.47 vs. 70.38 ± 8.11 and 50.84 ± 18.92 vs. 51.98 ± 19.44, respectively). Group 2 exhibited a lower DNA fragmentation index than group 1 after vitrification (14.2 ± 8.47 vs. 12.60 ± 9.03, p = 0.021). Conclusion: Using a pulled-glass capillary for sperm vitrification, the presence of CPAs in the vitrification medium resulted in higher progressive motility and lower DNA fragmentation index than the medium without CPAs.

One out of two idiopathic infertile men has pathologic sperm DNA fragmentation values: Potential implications for clinical practice.

OBJECTIVES: To investigate the distribution of sperm DNA fragmentation (SDF) values and their association with clinical and seminal parameters in idiopathic infertile men. DESIGN, PATIENTS, MEASUREMENTS: Data from 3224 primary infertile men (belonging to couples having failed to conceive a pregnancy within 12 months) who underwent a thorough diagnostic work-up were analysed. A SDF value ≥ 30% (according to Sperm Chromatin Structure Assay) was considered pathologic. We excluded: (1) men with genetic abnormalities; (2) men with history of cryptorchidism; (3) men with biochemical hypogonadism; (4) men with clinical varicocele; and (5) men with other possible known aetiological factors. Descriptive statistics and logistic regression analyses were used to describe the whole cohort. RESULTS: Of all, 792 (23%) men with at least one abnormal WHO semen parameter but without any identified aetiologic factor for infertility, were considered as idiopathic infertile men. Of 792, 418 (52.7%) men had SDF ≥30%. Men with pathologic SDF were older (p = .02), had higher Follicle-stimulating hormone (FSH) (p = .04) but lower total testosterone (p = .03) values than those with SDF <30%. The homoeostatic model assessment index for insulin resistance (HOMA-IR) was higher in men with SDF ≥30% (p = .01). Idiopathic infertile men with SDF ≥30% presented with lower sperm concentration (p < .001) and lower progressive sperm motility (p < .01) than those with SDF < 30%. Logistic regression analysis revealed that older age (OR: 1.1, p = .02) and higher HOMA-IR score (OR: 1.8, p = .03) were associated with SDF ≥ 30%, after accounting for FSH and sperm concentration values. CONCLUSIONS: Approximately half of infertile men categorized as idiopathic had pathologic SDF values. Idiopathic infertile men with pathologic SDF showed worse clinical, hormonal and semen parameters than those with normal SDF values. These results suggest that including SDF testing could be clinically relevant over the real-life management work-up of infertile men.

Correlation of Sperm DNA Fragmentation Index With Semen Parameters, Lifestyle and Clinical Pregnancy Outcome After Intracytoplasmic Injection.

Objective: This study aimed to measure the correlation of sperm DNA fragmentation with semen parameters, lifestyle, and fertility outcomes after intracytoplasmic injection (ICSI). Materials and methods: The partners who were candidates for ICSI with a history of one In vitro fertilization (IVF) failure or male factor were recruited in the study. Semen parameters including sperm count, motility, and morphology as well as DNA fragmentation index (DFI) (that were divided into 2 groups as high (>15%), and low (≤15%) fragmentation scales) were evaluated either. The correlation of DFI with semen parameters, lifestyle, and clinical pregnancy after ICSI were compared between groups. Results: In 120 included couples, 59 men (49.2%) had DFIs ≤ 15% and 61 (50.8%) cases had DFIs >15%. In the group with higher DFI, abnormal morphology (p=0.010) was higher whereas, progressive motility (p=0.001), total motility (p<0.001), and total count (p<0.001) of sperm were significantly lower. In addition, the DFI was significantly higher in the subgroup of male infertility (0.012). Logistic regression showed that a lower risk of DFI>15% was associated with higher values of progressive motility (OR=0.97, p=0.001), total motility (OR=0.96, p=<0.001), count (OR=0.96, p=<0.001) and even clinical pregnancy (OR=0.27, p=0.011). However, a history of testicular surgery was associated with a higher risk of DFI>15% (OR=3.37, p=0.046). Although no correlation was found between male age and lifestyle components with DFI, the number of embryos was lower in DFI≥15% (p<0.001). Conclusion: DFI provide a clinically important measurement of sperm quality and have an impact on IVF outcomes; however, lifestyle components may not correlate with DFI.

What sperm parameters effect blastocyst formation and quality during ICSI with severe male infertility.

Sperm-derived genetic material contributes half of the genome to the embryo, hence it's crucial to investigate which sperm parameter influences blastocyst formation in the intracytoplasmic sperm injection (ICSI) cycles with severe male infertility. The retrospective study analyzed 296 ICSI cycles with severe oligoasthenoteratozoospermia (OAT) and 99 ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A). Following the correlation analysis, data stratifications were performed in the OAT ICSI subgroup. The results showed that the matching blastocyst in the OAT ICSI cycles had inferior sperm parameters. DFI and sperm morphology had an influence on the blastocyst formation rate and the high-quality blastocysts formation rate on Day6, but no significant effect on the blastocyst development on Day 5. The high-quality blastocysts formation rate and ratio of high-quality blastocyst on Day 6 were demonstrably better in the subgroup of the teratozoospermic morphology when DFI was within the normal range. In the case of the normal sperm morphology, no statistically significant difference was found in blastocyst development, although there were numerical differences within different DFI subgroups. It was concluded that the blastocyst quality and development declined with the decreased sperm qualities.

Bacteriospermia-Related Male Infertility: A Case Report on Diagnostic and Therapeutic Approaches.

Male infertility is significantly affected by bacteriospermia, defined by the presence of bacteria in semen. This case report aims to address the effects of bacteriospermia and its correlation with semen characteristics, sperm DNA fragmentation (SDF), and reproductive outcomes. The 33-year-old male was diagnosed with bacteriospermia caused by two gram-negative bacteria: Escherichia coli and Klebsiella pneumoniae. It was found that sperm parameters such as mobility, number, and morphology were compromised in sperm, indicating poor fertility. In addition, SDF analysis has revealed a high DNA fragmentation index (DFI), emphasizing the detrimental effects of bacteriospermia on the integrity of sperm. Antibiotic therapy and antioxidant supplements have been used as therapeutic measures to reduce the impact of bacterial infections and DNA damage caused by oxidative stress (OS). Follow-up assessments showed significant improvements in the integrity of the sperm DNA and the resolution of microbial colonization, which ultimately led to successful in vitro fertilization (IVF) and embryo transfer (ET), leading to a positive pregnancy outcome.

Sperm DNA fragmentation and infertility: a narrative review.

PURPOSE: The purpose of this narrative review is to provide a practical understanding of sperm DNA fragmentation (SDF) in the management of male infertility. METHODS: A search for systematic reviews and meta-analyses (SRMA) on SDF between April 1st, 2018 and April 1st, 2023 was performed using PubMed and articles were selected as per their relevance to the topic. Guidelines from major societies were also reviewed. Three clinical cases are reported and discussed. RESULTS: The search initially identified 80 articles. We selected 13 SRMAs based on their relevance to the topic. Of the 13 SRMAs, 7 evaluated the effect of SDF on assisted reproductive technology (ART) outcomes and recurrent pregnancy loss, 3 studied the effect of varicocele repair on SDF, and 3 evaluated the role of SDF involving lifestyle and environmental health factors including body mass index and male factor treatment strategies. CONCLUSION: Evidence suggests that increased SDF has a negative impact on natural pregnancy and ART outcomes. SDF testing may be particularly important in the infertility evaluation of men with varicoceles, idiopathic or unexplained infertility, recurrent pregnancy loss, or previous ART failure. Further studies are needed on SDF testing and the implications it can have on male factor infertility and pregnancy outcomes as well as its implementation in the setting of ART.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences.

BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.

Effects of long and short ejaculatory abstinence on sperm parameters: a meta-analysis of randomized-controlled trials.

Purpose: This study aimed to investigate the effects of ejaculatory abstinence on sperm parameters. Methods: This analysis was registered in PROSPERO (CRD42023472124). We performed a search on PubMed using the following text terms: (("sperm parameters" OR "sperm analysis" [Mesh]) AND ("sperm DNA fragmentation" OR "DNA fragmentation" [Mesh]) AND ("sexual abstinence" [Mesh] OR "abstinence")) and an advanced search in Scopus using the terms ("sperm parameters" OR "sperm parameters" OR "DNA fragmentation") AND ("abstinence"). The sperm parameters that were investigated were sperm volume, total sperm motility, progressive sperm motility, sperm concentration, sperm morphology, and sperm DNA fragmentation (SDF). A two-day cut-off as a "short" or "long" abstinence period has been defined. Results: Thirteen studies published between 2013 and 2022 were included in this meta-analysis. A total of 2,315 patients, ranging from 6 to 836 from each cohort, were enrolled in the study. We showed that longer abstinence time was associated with greater sperm concentration (mean difference [MD]: 8.19; p <0.01), sperm volume (MD: 0.96; p <0.01), and higher SDF (MD: 3.46; p <0.01), but lower progressive sperm motility (MD: -1.83; p <0.01). Otherwise, no statistically significant difference was observed in patients with longer vs. shorter abstinence times regarding total sperm motility (MD: -1.83; p = 0.06). Meta-regression analysis showed that days of abstinence were positively and linearly related to sperm concentration (slope: 3.74; p <0.01) and SDF (slope: 0.65; p = 0.044). Conclusions: According to our data, short ejaculatory abstinence is associated with better sperm quality. Indeed, a higher percentage of progressive sperm motility and lower levels of SDF have been reported in a short abstinence cohort. In contrast, the long abstinence group reported a higher sperm concentration. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023472124.

Effect of Postbiotics Derived from Lactobacillus rhamnosus PB01 (DSM 14870) on Sperm Quality: A Prospective In Vitro Study.

Vaginally administered postbiotics derived from Lactobacillus were recently demonstrated to be effective in alleviating bacterial vaginosis and increasing pregnancy rates. However, their potential effect on sperm quality has not been well investigated. This controlled in vitro study aimed to assess the dose- and time-dependent effects of postbiotics derived from Lactobacillus rhamnosus PB01 (DSM 14870) on sperm quality parameters. The experiment was conducted in vitro to eliminate potential confounding factors from the female reproductive tract and vaginal microbiota. Sperm samples from 18 healthy donors were subjected to analysis using Computer-Aided Sperm Analysis (CASA) in various concentrations of postbiotics and control mediums at baseline, 60 min, and 90 min of incubation. Results indicated that lower postbiotic concentration (PB5) did not adversely affect sperm motility, kinematic parameters, sperm DNA fragmentation, and normal morphology at any time. However, concentrations exceeding 15% demonstrated a reduction in progressively motile sperm and a negative correlation with non-progressively motile sperm at all time points. These findings underscore the importance of balancing postbiotic dosage to preserve sperm motility while realizing the postbiotics' vaginal health benefits. Further research is warranted to understand the underlying mechanisms and refine practical applications in reproductive health.

Development and Performance Evaluation of a New Test Kit for Quantifying the Degree of DNA Fragmentation.

BACKGROUND: PCR-based genetic testing of agricultural products and foods is widely used for detecting various analytical targets such as genetically modified organisms and food allergens. However, it is difficult to obtain accurate genetic testing results from processed foods because DNA is fragmented by heat and pressure during food processing. Thus we previously developed an analytical method to quantitatively evaluate the degree of DNA fragmentation for the purpose of quality control of genetic testing for processed foods. OBJECTIVE: Our previous analytical method requires four PCR primer sets, resulting in high reagent costs and heavy analytical workloads. Therefore, we attempted to develop an easy-to-use test kit for quantifying the degree of DNA fragmentation and to evaluate its analytical performance. METHODS: To simplify the analysis procedure, we used only two primer sets. In addition, no-fragmentation control templates were prepared to obtain stable measurement results. The precision of the simplified analysis was evaluated through blind tests among laboratories. RESULTS: It was confirmed that plant species and extracted DNA concentrations had little effect on analysis with the newly developed test kit. In addition, the analytical values indicating the degree of DNA fragmentation exhibited small variability among laboratories. CONCLUSION: We confirmed the high practicality of the developed test kit. Because DNA fragmentation in cells is a universal phenomenon, we anticipate that the test kit will be used not only for quality control of genetic testing but also for food testing, medical diagnostics and other applications in a range of fields. HIGHLIGHTS: The newly developed test kit enables quantitative evaluation of the degree of DNA fragmentation in a simple manner.

An alternative approach of TUNEL assay to specifically characterize DNA fragmentation in cell model systems.

DNA damage is one of the most important effects induced by chemical agents. We report a comparative analysis of DNA fragmentation on three different cell lines using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, generally applied to detect apoptosis. Our approach combines cytogenetic techniques and investigation in detached cellular structures, recovered from the culture medium with the aim to compare the DNA fragmentation of three different cell line even beyond the cells adherent to substrate. Consequently, we detect any fragmentation points on single chromosomes, whole nuclei and other cellular structures. Cells were exposed to resveratrol (RSV) and doxorubicin (Doxo), in single and combined treatments. Control and treated astrocytes showed DNA damage in condensed nuclei and detached structures. Caco-2 cells showed fragmented DNA only after Doxo-treatment, while controls showed fragmented chromosomes, indicating DNA damage in replicating cells. MDA-MB-231 cells showed nuclear condensation and DNA fragmentation above all after RSV-treatment and related to detached structures. This model proved to perform a grading of genomic instability (GI). Astrocytes show a hybrid level of GI. Caco-2 cells showed fragmented metaphase chromosomes, proving that the DNA damage was transmitted to the daughter cells probably due to an absence of DNA repair mechanisms. Instead, MDA-MB-231 cells showed few or no fragmented metaphase, suggesting a probable activation of DNA repair mechanisms. By applying this alternative approach of TUNEL test, we obtained data that can more specifically characterize DNA fragmentation for a suitable application in various fields.

Shugan Tongluo Qiangjing recipe protects against varicocele of EVC rats through modulating sperm DNA damage, telomere expression and oxidative stress.

Varicocele (VC) refers to expansion and tortuosity of spreading venous plexus in spermatic cord due to poor blood flow. This study aimed to investigate effects of Shugan Tongluo Qiangjing recipe (SGTL) on sperm DNA damage and oxidative stress in experimental VC (EVC) rats. EVC model was established by partial ligation of left renal vein. Spermatic vein diameter, testicular weight, sperm DNA fragmentation index (DFI) were evaluated. Telomere reverse transcriptase (TERT) expression, telomere gene transcription, and testicular tissue morphology were determined·H2O2, catalase, SOD, T-AOC were measured with colorimetry. SGTL significantly decreased spermatic vein diameter (P=0.000) and increased testicular weight (P=0.013) of rats compared those of EVC rats. SGTL maintained testicular tissue morphology in EVC rats. SGTL markedly reduced sperm DFI value in sperm of rats compared to EVC rats (P=0.000). SGTL significantly enhanced TERT expression and telomere gene transcription (P=0.028) in testis of rats compared to EVC rats. SGTL reduced H2O2 levels (P=0.001) and promoted CAT activity (P=0.016), SOD activity (P=0.049), and T-AOC activity (P=0.047) of rats, compared to EVC rats. In conclusion, SGTL could reduce pathogenic process of EVC by reducing sperm DNA damage and regulating telomere length in EVC rats, which may be related to oxidative stress regulation.

Prospective reproductive outcomes according to sperm parameters, including DNA fragmentation, in recurrent pregnancy loss.

RESEARCH QUESTION: Are the prospective reproductive outcomes in couples experiencing recurrent pregnancy loss (RPL) related to the sperm DNA fragmentation index (DFI), as measured by sperm chromatin structure assay, sperm morphology and sperm concentration at referral? DESIGN: This prospective cohort study included 95 couples seen between 1 April 2018 and 1 December 2019 at the tertiary Copenhagen RPL Unit, Copenhagen University Hospital, Rigshospitalet and Hvidovre Hospital, Denmark. The couples had experienced three or more unexplained consecutive pregnancy losses or two late pregnancy losses (>12 weeks gestation). Follow-up was 12-31 months. RESULTS: Eighty-one of 95 (85.3%) couples achieved pregnancy after referral. In the first pregnancy after referral, 46 (56.8%) couples achieved a live birth, and 35 (43.2%) couples experienced another pregnancy loss. There was no significant difference in baseline DFI between couples that experienced pregnancy loss [median 11.7, interquartile range (IQR) 9.1-17.3] and couples that achieved a live birth (median 12.5, IQR 9.3-16.5; P = 0.971). Improving sperm morphology increased the odds of a live birth after referral (adjusted OR 1.26, 95% CI 1.05-1.52; P = 0.014). DFI and sperm concentration were not associated with the outcome of the first pregnancy after referral. Overall, 35.9% of the men had DFI ≥15 at inclusion. Couples that failed to achieve pregnancy had a higher median DFI of 17.7 (IQR 7.7-27.2) compared with the rest of the cohort (median 12.0, IQR 9.3-16.5; P = 0.041). CONCLUSIONS: At referral, sperm DFI, morphology and concentration cannot be used to identify RPL couples at risk of another pregnancy loss. Increased baseline DFI was associated with difficulty achieving another pregnancy, and improving sperm morphology was associated with increased odds of a live birth.

Exogenous Oxidative Stress in Human Spermatozoa Induces Opening of the Mitochondrial Permeability Transition Pore: Effect on Mitochondrial Function, Sperm Motility and Induction of Cell Death.

Oxidative stress (OS) and disrupted antioxidant defense mechanisms play a pivotal role in the etiology of male infertility. The alterations in reactive oxygen species (ROS) production and calcium (Ca2+) homeostasis are the main activators for the mitochondrial permeability transition pore (mPTP) opening. The mPTP opening is one of the main mechanisms involved in mitochondrial dysfunction in spermatozoa. This alteration in mitochondrial function adversely affects energy supply, sperm motility, and fertilizing capacity and contributes to the development of male infertility. In human spermatozoa, the mPTP opening has been associated with ionomycin-induced endogenous oxidative stress and peroxynitrite-induced nitrosative stress; however, the effect of exogenous oxidative stress on mPTP opening in sperm has not been evaluated. The aim of this study was to determine the effect of exogenous oxidative stress induced by hydrogen peroxide (H2O2) on mPTP opening, mitochondrial function, motility, and cell death markers in human spermatozoa. Human spermatozoa were incubated with 3 mmol/L of H2O2 for 60 min, and intracellular Ca2+ concentration, mPTP opening, mitochondrial membrane potential (ΔΨm), ATP levels, mitochondrial reactive oxygen species (mROS) production, phosphatidylserine (PS) externalization, DNA fragmentation, viability, and sperm motility were evaluated. H2O2-induced exogenous oxidative stress caused increased intracellular Ca2+, leading to subsequent mPTP opening and alteration of mitochondrial function, characterized by ΔΨm dissipation, decreased ATP levels, increased mROS production, and the subsequent alteration of sperm motility. Furthermore, H2O2-induced opening of mPTP was associated with the expression of apoptotic cell death markers including PS externalization and DNA fragmentation. These results highlight the role of exogenous oxidative stress in causing mitochondrial dysfunction, deterioration of sperm motility, and an increase in apoptotic cell death markers, including PS externalization and DNA fragmentation, through the mPTP opening. This study yielded new knowledge regarding the effects of this type of stress on mitochondrial function and specifically on mPTP opening, factors that can contribute to the development of male infertility, considering that the role of mPTP in mitochondrial dysfunction in human sperm is not completely elucidated. Therefore, these findings are relevant to understanding male infertility and may provide an in vitro model for further research aimed at improving human sperm quality.

The Association of Sperm DNA Fragmentation With Serum Lipid Profile Among Males With Abnormal Semen Parameters: An Analytical Cross-Sectional Study.

BACKGROUND: Through the ages, infertility, affecting 8% to 12% of couples worldwide, has been a perturbing clinical problem. Approximately 40% to 50% of all infertility cases are due to 'male factor' infertility. Semen analysis is crucial in routinely evaluating idiopathic male infertility. Studies support the idea that semen parameters are associated with serum lipids and sperm DNA fragmentation (SDF). Therefore, it is possible to evaluate male infertility by serum lipid levels, especially before assisted reproduction technology, and modify it by bringing about lifestyle modifications. This study aimed to measure the correlation of SDF with levels of total cholesterol (TC), triglycerides (TG), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) among males with abnormal semen parameters. METHODS: A cross-sectional analytical study was conducted in the infertility clinic of a tertiary care hospital. A total of 106 infertile males with abnormal semen analysis as per the WHO criteria (2010) were enrolled in the study. After routine semen analysis, SDF was studied using the comet assay. The serum fasting lipid profile was analyzed using the spectrophotometric kit in the autoanalyzer. The relationship of SDF with serum lipid profile parameters was analyzed. RESULTS: Out of 106 infertile men, 52% (n = 55) had severe SDF. A modest positive correlation was observed between SDF (percentage of DNA in comet tail) and serum lipid values (serum TG, serum LDL, and serum VLDL). CONCLUSIONS: Our study is novel in its research on the correlation between SDF and serum lipid values. Based on the findings of our study, it can be concluded that a significant level of SDF was observed in men with high levels of serum TG, LDL, and VLDL. This provokes a potential relationship between sperm DNA integrity and serum lipid profile, which warrants further research.