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The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples.

ABSTRACT: 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.

SUBMITTER: Petersen CG

PROVIDER: S-EPMC6210622 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples.

Petersen Claudia G CG Mauri Ana L AL Vagnini Laura D LD Renzi Adriana A Petersen Bruna B Mattila Mariana M Comar Vanessa V Ricci Juliana J Dieamant Felipe F Oliveira Joao Batista A JBA Baruffi Ricardo L R RLR Franco Jose G JG

JBRA assisted reproduction 20181101 4

<h4>Objective</h4>This study aimed to evaluate the effects of male age on sperm DNA damage.<h4>Methods</h4>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 A<sub>3</sub>), abnormal mitochondrial membrane potential (MMP/MitoTracker Green), and apoptosis (annexin V) were recorded. Fo ...[more]

PMID: 30106542

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Project description:Background and objectives: Male infertility is a global health dilemma and Follicle-Stimulating Hormone (FSH) administration has shown promising results. Several studies showed that infertile men with normal semen parameters have low levels of DNA damage while infertile men with abnormal semen parameters have more damage at the DNA level. Sperm DNA damage may affect the reproductive outcome and has been associated with failure in the achievement of competent embryos and pregnancy fulfillment. The aim of this study was to evaluate whether the administration of recombinant FSH (Gonal-f® PEN 900 IU) could improve sperm DNA fragmentation in men with infertility. The secondary endpoints of this study were to evaluate the FSH effects on sperm parameters and hormonal assets. Methods: A longitudinal, prospective, multicenter, open-label clinical trial was carried out. Infertile couples were recruited from six Italian Reproductive Medical Centers and 115 infertile men were enrolled for this study. All participants were treated with subcutaneous injections of Gonal-f® 150 IU every other day, within a 3 month-time frame. The semen samples were examined in accordance to the 2010 World Health Organization criteria. Sperm DNA Fragmentation (DFI) was determined by fluorescence microscopy using terminal deoxynucleotidyl transferase-mediated d-UTP Nick-end Labeling (TUNEL) assay. Statistical analysis was performed using both the t-test for paired samples and the Wilcoxon signed-rank test. Results: FSH administration improved DFI in 67% of patients, with an average decrease of 35.4% compared to the baseline. This improvement is more evident in men with basal DFI lower than 17% and in those with FSH basal levels between 2.16 and 4.27 IU/L. In addition, FSH enhanced the gonadal function, increasing the hormones AMH and Inhibin B and semen parameters. Limitation of these results are represented by the absence of a placebo group and of FSHR genotype stratification sub-analysis. Conclusion: Recombinant FSH 150 IU is well tolerated and effective in eliciting a significant DFI reduction as well as in improving gonadal function.Trial registrationEUDRACT Number 2010-020196-23. Registred 14 April 2011.

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.

Dataset Information

The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples.

Publications

The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples.

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