Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis.

Previous studies suggest that sperm DNA fragmentation may be associated with aneuploidy. However, currently available tests have not made it possible to simultaneously perform DNA fragmentation and chromosomal analyses on the same sperm cell. The recently introduced sperm chromatin dispersion (SCD) test allows users to determine this relationship. Semen samples from 16 males, including 4 fertile donors, 7 normozoospermic, 3 teratozoospermic, 1 asthenozoospermic, and 1 oligoasthenoteratozoospermic, were processed for DNA fragmentation analysis by the SCD test using the Halosperm kit. Three-color fluorescence in situ hybridization (FISH) was performed on SCD-processed slides to determine aneuploidy for chromosomes X, Y, and 18. Spermatozoa with DNA fragmentation showed a 4.4 +/- 1.9-fold increase in diploidy rate and a 5.9 +/- 3.5-fold increase in disomy rate compared to spermatozoa without DNA fragmentation. The overall aneuploidy rate was 4.6 +/- 2.0-fold higher in sperm with fragmented DNA (Wilcoxon rank test: P < .001 in the 3 comparisons). A higher frequency of DNA fragmentation was found in sperm cells containing sex chromosome aneuploidies originated in both first and second meiotic divisions. The observed increase in aneuploidy rate in sperm with fragmented DNA may suggest that the occurrence of aneuploidy during sperm maturation may lead to sperm DNA fragmentation as part of a genomic screening mechanism developed to genetically inactivate sperm with a defective genomic makeup.

Evidence of modified nuclear protein matrix in human spermatozoa with fragmented deoxyribonucleic acid.

Human spermatozoa were processed for determination of DNA fragmentation with use of an in situ diffusion assay, so that those cells containing DNA fragmentation produce extensive peripheral dissemination of DNA fragments after lysis in an agarose microgel. Quantification of specific protein staining confirmed that sperm cells without DNA fragmentation had almost complete removal of nuclear matrix proteins, whereas spermatozoa with DNA fragmentation tended to retain residual nucleoskeletal protein in a collapsed and condensed state. This result suggests that a modified nuclear protein matrix associates with fragmented sperm DNA.

Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear damage level, evidenced by the sperm chromatin dispersion test.

The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile patients with varicocele and compared with those obtained in a group of 51 normozoospermic patients, 103 patients with abnormal standard semen parameters, and 22 fertile men. The spermatozoa were processed to discriminate different levels of DNA fragmentation using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. In this technique, after an acid incubation and subsequent lysis, those sperm cells without DNA fragmentation show big or medium-sized halos of dispersion of DNA loops from the central nuclear core. Otherwise, those spermatozoa containing fragmented DNA either show a small halo, exhibit no halo with solid staining of the core, or show no halo and irregular or faint stain of the remaining core. The latter, that is, degraded type, corresponds to a much higher level of DNA-nuclear damage. The varicocele patients showed 32.4% +/- 22.3% of spermatozoa with fragmented DNA, significantly different from the group of fertile subjects (12.6% +/- 5.0%). Nevertheless, this was not different from that of normozoospermic patients (31.3% +/- 16.6%) (P = .83) and with abnormal semen parameters (36.6% +/- 15.5%) (P = .31). No significant differences were found between the normozoospermic patients and the patients with abnormal semen parameters. Strikingly, the proportion of the degraded cells in the total of sperm cells with fragmented DNA was 1 out of 4.2 (23.9% +/- 12.9%) in the case of varicocele patients, whereas it was 1 out of 8.2 to 9.7 in the normozoospermic patients (11.1% +/- 9.9%) in the patients with abnormal sperm parameters (12.2% +/- 8.3%) and in the fertile group (10.3% +/- 7.2%). Thus, whereas no differences in the percentage of sperm cells with fragmented DNA were evident with respect to other infertile patients, individuals with varicocele exhibit a higher yield of sperm cells with the greatest nuclear DNA damage level in the population with fragmented DNA. This finding illustrates the value of assessing different patterns of DNA-nuclear damage within each sperm cell and the particular ability of the Halosperm kit to reveal them.

Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test.

OBJECTIVE: To improve the sperm chromatin dispersion (SCD) test and develop it as a simple kit (Halosperm kit) for the accurate determination of sperm DNA fragmentation using conventional bright-field microscopy. DESIGN: Method development, comparison, and validation. SETTING: Medical genetics laboratory, academic biology center, and reproductive medicine centers. PATIENT(S): Male infertility patients attending the Reproductive Medicine Center. A varicocele patient and a group of nine fertile subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): [1] The quality of chromatin staining in relaxed sperm nuclear halos and tail preservation; [2] SCD scoring reproducibility; [3] comparison with the sperm chromatin structure assay in 45 samples; [4] frequency of sperm with DNA fragmentation after incubation with increasing doses of the nitric oxide donor sodium nitroprusside and in sperm samples for 9 fertile men, 46 normozoospermic patients, 23 oligoasthenoteratozoospermic patients, and a subject with varicocele. RESULT(S): The sperm nuclei with DNA fragmentation, either spontaneous or induced, do not produce or show very small halos of DNA loop dispersion after sequential incubation in acid and lysis solution. The improved SCD protocol (Halosperm kit) results in better chromatin preservation, therefore highly contrasted halo images can be accurately assessed using conventional bright-field microscopy after Wright staining. Moreover, unlike in the original SCD procedure, the sperm tails are now preserved, making it possible to unequivocally discriminate sperm from other cell types. The chi2 test did not detect significant differences in the mean number of sperm cells with fragmented DNA as scored by four different observers. The intraobserver coefficient of variation for the estimated percentage of spermatozoa with fragmented DNA ranged from 6% to 12%. There was good correlation between the SCD and the sperm chromatin structure assay DNA fragmentation index (intraclass correlation coefficient R: 0.85; percent DNA fragmentation index mean difference: 2.16 significantly higher for SCD). Using the Halosperm kit, a dose-dependent increase in sperm DNA damage after sodium nitroprusside incubation was detected. The percentage of sperm cells with fragmented DNA in the fertile group was 16.3 +/- 6.0, in the normozoospermic group, 27.3 +/- 11.7, and in the oligoasthenoteratozoospermic group, 47.3 +/- 17.3. In the varicocele sample, an extremely high degree of nuclear disruption was detected in the population of sperm cells with fragmented DNA. CONCLUSION(S): The improved SCD test, developed as the Halosperm kit, is a simple, cost effective, rapid, reliable, and accurate procedure, for routinely assessing human sperm DNA fragmentation in the clinical andrology laboratory.

Critically short telomeres are associated with sperm DNA fragmentation.

The influence of critical telomeric attrition, a well-known trigger of apoptosis and cell arrest, on sperm DNA fragmentation was studied in late-generation knockout mice for Terc, the RNA component of telomerase, as a model of choice. Terc knockout mice had a sixfold mean increase in the percentage of sperm cells with fragmented DNA.

Halosperm is an easy, available, and cost-effective alternative for determining sperm DNA fragmentation.

The characteristics of Halosperm make this kit a reasonable alternative to allow basic and clinical research on sperm DNA fragmentation in any basic laboratory around the world.

Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains approximately 2.5-fold higher density of background DNA breaks with 3'-OH ends, and also approximately 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei.