The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation, and copy number in normal and abnormal human ejaculates.

BACKGROUND: While it is common to clinically evaluate sperm nuclear DNA fragmentation, less attention has been given to sperm mitochondrial DNA. Recently, a digital PCR assay has allowed accurate estimation of the proportion of fragmented mtDNA molecules and relative copy number. OBJECTIVES: To determine the correlation of classical sperm parameters, average mtDNA copies per spermatozoon and the level of mtDNA fragmentation (SDF-mtDNA) to that of nuclear DNA fragmentation (SDF-nDNA), measured as the proportion of global, single-strand DNA (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs). To determine whether the level of nuclear and mitochondrial DNA fragmentation and/or copy number can differentiate normozoospermic from non-normozoospermic samples. MATERIALS AND METHODS: Ejaculates from 29 normozoospermic and 43 non-normozoospermic were evaluated. SDF was determined using the sperm chromatin dispersion assay. mtDNA copy number and SDF-mtDNA were analyzed using digital PCR assays. RESULTS: Relative mtDNA copy increased as sperm concentration or motility decreased, or abnormal morphology increased. Unlike SDF-mtDNA, mtDNA copy number was not correlated with SDF-nDNA. SDF-mtDNA increased as the concentration or proportion of non-vital sperm increased; the higher the mtDNA copy number, the lower the level of fragmentation. Non-normozoospermic samples showed double the level of SDF-nDNA compared to normozoospermic (median 25.00 vs. 13.67). mtDNA copy number per spermatozoon was 3× higher in non-normozoospermic ejaculates (median 16.06 vs. 4.99). Although logistic regression revealed SDF-Global and mtDNA copy number as independent risk factors for non-normozoospermia, when SDF-Global and mtDNA copy number were combined, ROC curve analysis resulted in an even stronger discriminatory ability for predicting the probability of non-normozoospermia (AUC = 0.85, 95% CI 0.76-0.94, p < 0.001). CONCLUSION: High-quality ejaculates show lower nuclear SDF and retain less mtDNA copies, with approximately half of them fragmented, so that the absolute number of non-fragmented mtDNA molecules per spermatozoon is extremely low.

Addendum to: The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation and copy number in normal and abnormal human ejaculates.

BACKGROUND: While the kinetics of human sperm nuclear DNA fragmentation (SDF-nDNA) following ejaculation have been described, the dynamics and relationships of mitochondrial DNA copy number per spermatozoon (mtDNAcn) and fragmentation (SDF-mtDNA) remain unexplored. OBJECTIVES: To compare post-ejaculatory kinetics of mtDNAcn, SDF-mtDNA and SDF-nDNA, global, single-strand DNA breaks (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs) in normozoospermic and non-normozoospermic samples. MATERIALS AND METHODS: 28 normozoospermic and 43 non-normozoospermic ejaculates were evaluated at 0, 6, 24 and 48 h of incubation in vitro. SDF-nDNA was determined by sperm chromatin dispersion (SCD) assays. mtDNAcn and SDF-mtDNA were analysed by dPCR. RESULTS: SDF-nDNA-global values increased as a consequence of quadratic SDF-SSBs and linear SDF-DSBs kinetics. Non-normozoospermic samples showed a slower SDF-global rate between 6-24 h, due to lesser SSBs production. Regarding SDF-DSBs, non-normozoospermic samples exhibited a faster initial increase rate, followed by a slower final increment. The mtDNAcn median value decreased linearly, being 3.2× higher in non-normozoospermics at all time points; mtDNAcn in both cohorts reduced to half of the baseline by 48 h. mtDNAcn was identified as a risk factor for discriminating non-normozoospermia, a finding that was further strengthen when combined with SDF-Global or SDF-DSBs values. SDF-mtDNA frequencies were identical, increasing over time correspondingly in both cohorts. The mtDNA fragmentation rate was initially fast, decreasing progressively with time for both cohorts; half of the initially unfragmented copies were fragmented after 48 h. Rates of mtDNAcn loss and SDF-mtDNA increase were only marginally correlated with the rates of nuclear fragmentation. CONCLUSION: mtDNA fragmentation and loss occur post ejaculation. Their dynamics are likely to be associated with different and/or uncoupled mechanisms to that which cause nuclear DNA fragmentation. Our results indicate that while mtDNA fragmentation is not influenced by the sperm quality, the number of copies of sperm mtDNAcn can potentially serve as a risk factor for predicting non-normozoospermia.

Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay.

Sperm DNA fragmentation (SDF) dynamic assays were piloted on 4 fresh ejaculates to examine the possible sperm toxicity of three common antibiotics, ciprofloxacin, doxycycline and ampicillin, incubated at a concentration estimated to be reached in semen in vivo, and 100×, for 24 h. SDF was assessed in terms of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs). Low and high concentrations of ciprofloxacin and high concentration of doxycycline significantly increased the SDF rate, due to sperm containing SSBs. Ampicillin did not affect SDF dynamics at any dose. Based on these results, the effect of antibiotics on the global-SDF dynamics was further examined in 21 ejaculates assessed at 0, 4 and 6 h. Ciprofloxacin increased the rate of SDF at the low concentration in 17 from 21 subjects; the high concentration resulted in a stronger effect in all individuals. A significant increase in the rate of SDF in 17 ejaculates was also noted when spermatozoa were incubated with the high concentration of doxycycline. The dynamic SDF assay is a rapid and sensitive tool to evidence sperm toxicity. Ciprofloxacin should be avoided when it is necessary to preserve sperm quality for reproductive purposes and as additive in semen diluents.

DNA fragmentation of human spermatozoa: Simple assessment of single- and double-strand DNA breaks and their respective dynamic behavioral response.

BACKGROUND: Procedures to detect sperm DNA fragmentation (SDF), like the sperm chromatin dispersion (SCD) test, determine the "global" SDF without discriminating between spermatozoa with single-strand DNA breaks only (SDF-SSBs) and those containing double-strand DNA breaks (SDF-DSBs). OBJECTIVES: (a) To validate a test to distinguish human spermatozoa with massive DSBs (DSB-SCD assay), (b) to study the baseline SDF-SSBs and SDF-DSBs, and (c) to assess their dynamics in vitro. MATERIALS AND METHODS: (a) SDF-DSBs were determined by visualization of diffused DNA fragments from spermatozoa lysed under non-denaturing conditions. This was validated by in vitro incubation with DNase I and the comet assay. (b) Baseline SDF-DSBs and SDF-SSBs were determined in ejaculates from 95 males. (c) Their dynamic appearance was studied in samples untreated or exposed to hyperthermia, acidic pH, nitric oxide released by sodium nitroprusside (SNP), and the metabolic energy inhibitors 2-deoxy-D-glucose and antimycin A. RESULTS: (a) DNase I and comet assay experiments confirmed that the assay successfully determined SDF-DSBs. (b) The higher the SDF of the semen sample, the higher the frequency of SSBs, whereas DSBs behaved independently. Abnormal samples showed higher SDF than normozoospermic, the difference being only significant for SDF-SSBs. (c) During the first hours of incubation, the linear rate of increase in SDF-SSBs was 3.7 X higher than that of SDF-DSBs. All hazardous agents accelerated the SDF rate when compared to untreated spermatozoa, primarily being associated with SDF-SSBs. SNP treatment was the most damaging, rapidly inducing spermatozoa with SSBs which progressively evolved to DSBs. Remarkably, this phenomenon was also evidenced after acute SNP exposure, revealing cryptic sperm damage. CONCLUSION: The DSBs-SCD is an easy complement for SDF assessment. The dynamic study of SSBs and DSBs may improve the evaluation of sperm quality in clinical settings, particularly "unmasking" the presence of non-specific cryptic sperm damage that might otherwise go undetected.