Microbiological Evaluation and Sperm DNA Fragmentation in Semen Samples of Patients Undergoing Fertility Investigation.
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SUBMITTER: Pagliuca C
PROVIDER: S-EPMC8145398 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Research questionTo examine associations between sperm mitochondrial DNA copy number (mtDNAcn), sperm mitochondrial DNA deletions (mtDNAdel), semen parameters and clinical infertility in an IVF setting.DesignA total of 125 sperm samples were collected from men undergoing assisted reproductive procedures in an IVF clinic in Western Massachusetts, USA. Sperm mtDNAcn and mtDNAdel were measured by probe-based quantitative polymerase chain reaction. Semen parameters, clinical diagnoses of infertility, and infertility based on consecutive semen parameters, were fitted with mtDNAcn and mtDNAdel in linear models. The utility of sperm mtDNAcn and mtDNAdel to predict infertility was assessed by receiver operating characteristic curves.ResultsAdjusting for relevant covariates, both sperm mtDNAcn and mtDNAdel were associated with lower sperm concentration, count, motility and morphology (P ≤ 0.03). Sperm mtDNAcn and mtDNAdel were also associated with increased risks of clinical infertility based on current and consecutive semen samples. Sperm mtDNAcn had high predictive accuracy for consecutive diagnoses of clinical infertility (C-statistic: 0.91), whereas sperm mtDNAdel had moderate predictive accuracy (C-statistic: 0.75).ConclusionsSperm mtDNAcn is a measure of consecutive abnormal semen parameters and has promise as a diagnostic test.
| S-EPMC6339832 | biostudies-literature
Project description:Many studies have now confirmed that sperm DNA fragmentation (SDF) is associated with a poorer outcome of some forms of assisted reproduction technology. For this reason, SDF is an important parameter to evaluate in male fertility assessment. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled to flow cytometry is one of the most promising methods for SDF quantification. Several kits for the detection of DNA fragmentation are currently available on the market and all are recommended as equally appropriate to quantify SDF. In this work we compared for the first time the efficacy of two different types of TUNEL kits for SDF quantification: one using an indirect antibody-based labeling system (BrdUTP/fluorescein-anti-BrdUTP) and another using a direct labeling system (fluorescein-dUTP). We demonstrated that TUNEL indirect labeling system largely underestimates SDF when compared with the direct labeling, the differences ranging from 19.2% to 85.3% (p<0.05, n = 22). We observed that these differences were most pronounced among dead spermatozoa where indirect labeling stained 40.1% [23.6%, 58.2%] and the direct system 65.7% [36.5%, 90.9%] (n = 10, p<0.05). Interestingly, we found that both systems stained the living spermatozoa with the same efficiency. We showed that the differences are due to the steric hindrance of the antibody during its binding to the BrdUTP. Indeed, after sperm DNA decondensation, the percentages of TUNEL positivity increased significantly from 46.3% [31.8%, 61.7%] to 97.5% [96.1%, 98.8%] (p<0.05, n = 5). Our results are important for future use of TUNEL in clinical practice. Laboratories relying on the use of an antibody-based system heavily underestimate SDF, most particularly in infertile patients with reduced sperm motility. As a consequence, the kit using BrdUTP/fluorescein-anti-BrdUTP should not be recommended as a method to assay DNA damage in semen. This study represents one further step in the standardization of TUNEL among laboratories.
| S-EPMC5546573 | biostudies-literature
Project description:OBJECTIVE:This study aimed to evaluate the effects of male age on sperm DNA damage. METHODS:This cross-sectional study included semen samples collected from 2,178 men seen at an infertility clinic. For DNA integrity analysis, the proportions of spermatozoa showing DNA fragmentation (TUNEL assay), abnormal chromatin packaging/underprotamination (chromomycin A3), abnormal mitochondrial membrane potential (MMP/MitoTracker Green), and apoptosis (annexin V) were recorded. For group comparisons, enrolled subjects were divided into three groups based on their ages: ?35 years; 36-44 years; and ?45 years. The associations between age and sperm parameters were assessed using Spearman's rank correlation coefficient. RESULTS:Although aging did not affect sperm apoptosis (p>.05), sperm DNA fragmentation and MMP deteriorated significantly with age (p<.05). Chromatin packaging/protamination improved significantly with age (p<.05). CONCLUSION:Sperm DNA fragmentation worsened with age and was apparently associated with mitochondrial damage. The age-related increase in sperm DNA damage suggests that delaying childbearing, not only in women but also in men, might jeopardize a couple's reproductive capacity. The increase seen in chromatin packaging might represent a protective feature for DNA. However, additional studies must be performed to confirm the results concerning chromatin packaging/protamination.
| S-EPMC6210622 | biostudies-literature
Project description:Although thousands of genetically modified mouse strains have been cryopreserved by sperm freezing, the likelihood of cryorecovery success cannot be accurately predicted using conventional sperm parameters. The objective of the present study was to assess the extent to which measurement of a sperm DNA fragmentation index (DFI) can predict sperm quality and fertility after cryopreservation. Using a modified TUNEL assay, we measured and correlated the DFI of frozen-thawed sperm from 83 unique mutant mouse strains with sperm count, motility and morphology. We observed a linear inverse correlation between sperm DFI and sperm morphology and motility. Further, sperm DFI was significantly higher from males with low sperm counts compared to males with normal sperm counts (P?<?0.0001). Additionally, we found that viable embryos derived using sperm from males with high DFI (62.7?±?7.2% for IVF and 73.3?±?8.1% for ICSI) failed to litter after embryo transfer compared to embryos from males with low DFI (20.4?±?7.9% for IVF and 28.1?±?10.7 for ICSI). This study reveals that measurement of DFI provides a simple, informative and reliable measure of sperm quality and can accurately predict male mouse fertility.
| S-EPMC7052244 | biostudies-literature
Project description:PURPOSE: To investigate the effects of male aging on sperm quality and sperm DNA fragmentation. METHODS: The ejaculates of 320 unselected men attending a fertility clinic and, as a control, 84 normozoospermic men without any history of ART were analyzed according to WHO guidelines. Sperm DNA fragmentation was measured by flow cytometry after staining with propidiumiodide. RESULTS: The patients were divided into four groups: <30 years, 30-35 years, 36-39 years and >or=40 years. Sperm motility decreased with increasing age whereas sperm concentration, morphology, and DNA fragmentation fluctuated throughout the four groups both among patients and among controls. However, we could not detect any significant correlation between male age and conventional semen parameters or sperm DNA fragmentation, respectively, neither in the patients' group nor among the controls. This also applies to a classification of patients and controls into only two age groups with a cut-off point at 35 years. CONCLUSIONS: Our findings suggest that neither the routinely assessed semen parameters nor the amount of spermatozoa with fragmented DNA are affected by male age.
| S-EPMC2649333 | biostudies-other
Project description:Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2'-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.
| S-EPMC4461587 | biostudies-literature
Project description:BackgroundIt has been identified that incidence of infertility was about 20% among couples worldwide, about 50% caused by male elements. However, conventional semen laboratory detections could not handle clinical needs, which led to more comprehensive parameters for male fertility evaluation. We aimed to investigate the clinical relationship of age-linked changes and the sperm chromatin structure assay (SCSA) sperm DNA fragmentation index (DFI), and routine semen characteristics among subfertile Chinese males.Methods1790 clinical semen specimens were enrolled from February 2018 to October 2019. Clinical and laboratory data including routine semen analyses, sperm DFI, and sperm morphology were collected and showed age-related alterations in semen parameters.ResultsOur results, displayed an increase in sperm DFI with age, were demonstrated in three age-groups, particularly within the ≥35-year cohort. There were positive and inverse correlations of sperm DFI with abnormal semen characteristics and with normal morphological parameters, respectively. Furthermore, age, sperm morphology, concentration, and progressive motility, immotile sperm percentage, semen volume, sperm survival, and high acridine orange DNA stainability (indicating immature forms) were found to be independent risk factors affecting sperm DNA integrity. Likewise, men aged ≥35 years had a higher sperm DFI than did normozoospermic men in the overall cohort. Routine semen characteristics, sperm DFI, and morphology tended to alter with age.ConclusionsThe SCSA sperm DFI showed the greatest clinical application in the assessment of male fertility in this study, which should help infertility clinics decide on reproductive options for the treatment of older infertile couples.
| S-EPMC7755769 | biostudies-literature
Project description:ObjectiveTo examine the factors associated with increased deoxyribonucleic acid fragmentation index (DFI), evaluate the pregnancy outcomes of men with increased DFI, and compare three independent DFI assays.DesignSecondary analysis.SettingNine US-based fertility centers.PatientsInfertile men (N = 147) with sperm concentration ≤15 × 106/mL, motility ≤40%, or normal morphology ≤4% were enrolled. The female partners were ovulatory, ≤40 years old, and had documented tubal patency.InterventionsAt a baseline visit, the men provided a semen sample. The couples attempted conception without assistance for 3 months and with ovarian stimulation and intrauterine insemination in the subsequent 3 months.Main outcome measuresThe DFI was analyzed using the sperm chromatin structure assay (SCSA) with increased DFI defined as >30%. The predictors of increased DFI were determined by a multivariable linear regression model. The pregnancy outcomes were compared using the χ2 test. The independent DFI assays (SCSA, deoxynucleotidyl transferase-mediated dUTP nick end labeling, and Comet) were compared with Pearson and Spearman correlations.ResultsThe 19% of men with increased DFI were older (36.0 vs. 33.0 years) and had lower total sperm motility (38.2% ± 20.5% vs. 45.2% ± 15.6%). Increased male age was found to be a significant predictor of DFI (0.75, 95% confidence interval [0.06, 1.45]). Increased DFI was not associated with conception or live birth. There was a modest correlation of the deoxynucleotidyl transferase-mediated dUTP nick end labeling assay with the SCSA (r = 0.34) and Comet assay (r = 0.19).ConclusionsOlder age was associated with increased DFI among infertile men. The DFI assays were only weakly correlated, indicating a standard definition of DFI is needed to truly interrogate how sperm deoxyribonucleic acid fragmentation impacts male fertility.
| S-EPMC8441563 | biostudies-literature
Project description:The main goal of semen processing in Assisted Reproductive Techniques (ART) is to select sperm with good viability and, at the same time, remove Reactive Oxygen Species (ROS) sources (such as leukocytes) and reduce the percentage of morphologically abnormal sperm for fertility treatment. We performed a comparative analysis on sperm DNA fragmentation after Density Gradient Centrifugation (DGC) using products sold by two competing companies. Our results showed comparable DNA Fragmentation Index (DFI) after treatment with both DGC products. However, in both cases, a comparable number of samples do not benefit from the treatment. Interestingly, increasing evidences indicated that male age has a negative impact on sperm DNA fragmentation, but the mechanisms underlying age-dependent patterns of sperm decline have not yet been fully understood. Thus, we performed a comparative analysis of DFI before and after treatment with DGC products in age-stratified sample populations. Our results showed a worsening of the baseline DFI in the eldest group and the benefits of DGC on sperm DNA were compromised. In conclusion, our work consolidates the current evidences suggesting that both paternal and maternal aging, critically affects reproductive success.
| S-EPMC6535069 | biostudies-literature
Project description:The evaluation of morpho-functional sperm characteristics alone is not enough to explain infertility or to predict the outcome of Assisted Reproductive Technologies (ART): more sensitive diagnostic tools are needed in clinical practice. The aim of the present study was to analyze Sperm DNA Fragmentation (SDF) and sperm-borne miR-34c-5p and miR-449b-5p levels in men of couples undergoing ART, in order to investigate any correlations with fertilization rate, embryo quality and development. Male partners (n = 106) were recruited. Semen analysis, SDF evaluation and molecular profiling analysis of miR-34c-5p and miR-449b-5p (in 38 subjects) were performed. Sperm DNA Fragmentation evaluation- a positive correlation between SDF post sperm selection and the percentage of low-quality embryos and a negative correlation with viable embryo were found. SDF > 2.9% increased the risk of obtaining a non-viable embryo by almost 4-fold. Sperm miRNAs profile-we found an association with both miRNAs and sperm concentration, while miR-449b-5p is positively associated with SDF. Moreover, the two miRNAs are positively correlated. Higher levels of miR-34c-5p compared to miR-449b-5p increases by 14-fold the probability of obtaining viable embryos. This study shows that SDF, sperm miR-34c-5p, and miR-449b-5p have a promising role as biomarkers of semen quality and ART outcome.
| S-EPMC9864861 | biostudies-literature