Relationships between the age of 25,445 men attending infertility clinics and sperm chromatin structure assay (SCSA®) defined sperm DNA and chromatin integrity.

OBJECTIVE: To determine relationships between age of men with potential male factor infertility and sperm chromatin structure assay (SCSA) measures of sperm DNA fragmentation (SDF) and high DNA stainable sperm (HDS), and to compare these data with those obtained from healthy donor men without reproductive issues. DESIGN: Retrospective study. SETTING: Infertility clinics and diagnostic laboratory. PATIENTS: A total of 25,445 men attending infertility clinics. Donors were 87 men working at Lawrence Livermore National Laboratory. INTERVENTION: None. MAIN OUTCOME MEASURES: SCSA measures (% DNA fragmentation index (DFI), X DFI, SD DFI, and %HDS) of men aged 21-80 years. RESULTS: In the study population, advancing paternal age was associated with increased sperm DNA fragmentation (SDF) scored as increased percentage of sperm in semen ejaculates with measurable DNA strand breaks (%DFI). The slope of increase in %DFI prior to age 41.6 years was 0.39, which increased after age 41.6 to more than double at a slope of 0.86. These changes in DNA/chromatin in more than 25,000 aging men attending infertility clinics are similar to those seen over the same age span (20-80 years) in 87 nonpatient, healthy men without reproductive issues. For the age group 20-50 years, there was no major significant difference in %DFI between patients and donor men. According to a logistic regression model, the estimated probability is that, for example, a 40-year-old and a 50-year-old man have a 20% and 40% chance, respectively, to have a pathological DFI ≥25% by age factor alone. The condensation of sperm chromatin in patients increased with age in a linear fashion, from a mean of 12.2 %HDS at age 20-25 to a mean of 7.9 %HDS at age 60-65. Patients had a greater %HDS than donors across all ages. CONCLUSIONS: The great heterogeneity of both DFI and HDS values at a specific age prevents the automatic translation of age into an index of DNA fragmentation. However, it reinforces the idea that both DFI and HDS evaluation can play a role in detecting potential male infertility in cases that are not resolved by routine testing and in cases of multiple miscarriages. DFI and HDS data can help clinicians to predict a man's fertility potential, to consider corrective therapeutic approaches, as well as to assess the risk to the offspring's health.

Luminal fluid of epididymis and vas deferens contributes to sperm chromatin fragmentation.

STUDY QUESTION: Do the luminal fluids of the epididymis and the vas deferens contribute to sperm chromatin fragmentation (SCF) in mice? SUMMARY ANSWER: The luminal fluids of both organs are required for activating SCF in mice, but the vas deferens luminal fluid does this more efficiently than that of the epididymis. WHAT IS KNOWN ALREADY: Mice sperm have the ability to degrade their DNA in an apoptotic-like fashion when treated with divalent cations in a process termed SCF. SCF has two steps: the induction of reversible double-strand DNA breaks at the nuclear matrix attachment sites, followed by the irreversible degradation of DNA by nuclease. Single stranded DNA breaks accompany SCF. STUDY DESIGN, SIZE, DURATION: Luminal fluids from two reproductive organs of the mouse (B6D2F1 strain), the epididymis and vas deferens, were extracted and tested for SCF activation with divalent cations using four different combinations of the sperm and the surrounding luminal fluids: (i) in situ--sperm were kept in their luminal fluid and activated directly; (ii) reconstituted--sperm were centrifuged and resuspended in their luminal fluid before SCF activation; (iii) mixed--sperm were centrifuged and resuspended in the luminal fluid of the other organ; (iv) no luminal fluid--sperm were centrifuged and reconstituted in buffer. All four experiments were performed without (controls) and with divalent cations (resulting in SCF). For each experimental condition, two different mice were used and the analyses averaged. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA damage by SCF was analyzed by three different methods, the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) analysis and field inversion gel electrophoresis. MAIN RESULTS AND THE ROLE OF CHANCE: In all three assays that we used, the vas deferens luminal fluid was much more efficient in stimulating SCF in the sperm from either source than that of the epididymis (P < 0.0001). Vas deferens sperm were capable of initiating lower levels of SCF in the absence of luminal fluid (P < 0.0001). LIMITATIONS, REASONS FOR CAUTION: Analyses were performed in only one species, the mouse, but we used three separate assays in our analysis. WIDER IMPLICATIONS OF THE FINDINGS: The data suggest that the luminal fluid of the male reproductive tract interacts with sperm during their transit providing a mechanism to degrade the DNA. We hypothesize that this is part of an apoptotic-like mechanism that allows the reproductive tract to eliminate defective sperm. The SCF model also allowed us to identify differences in the types of DNA lesions that the three tests can identify, providing important background information for the use of these tests clinically.

Boar fertility and sperm chromatin structure status: a retrospective report.

Little information exists about boar sperm chromatin quality and fertility within a commercial setting. The objective of this report is to provide information about boar sperm chromatin integrity and its relationship to fertility. The sperm chromatin structure assay (SCSA) was used retrospectively to characterize sperm from 18 sexually mature boars having fertility information. Boar fertility was defined by farrow rate (FR) and average total number of pigs born (ANB) per litter of gilts and sows mated to individual boars. Fertility data was compiled for 1867 matings across the 18 boars. The SCSA uses flow cytometry to evaluate the structural integrity of sperm nuclear DNA. The SCSA parameters measured in this retrospective analysis were the percentage DNA fragmentation index (%DFI) and standard deviation of the DNA fragmentation index (SD DFI). The %DFI and SD DFI showed the following significant negative correlations with FR and ANB; %DFI vs FR, r = -0.55, P < .01; SD DFI vs FR, r = -0.67, P < .002; %DFI vs ANB, r = -0.54, P < .01; and SD DFI vs ANB, r = -0.54, P < .02. Although more information is required to better understand the relationship between DFI and boar fertility, this report suggests that the SCSA assay may be an important assay for identification of boars having potential for lowered fertility.

Significant decrease in sperm deoxyribonucleic acid fragmentation after varicocelectomy.

OBJECTIVE: To measure sperm DNA integrity values before and after varicocelectomy in patients with elevated preoperative levels of sperm DNA fragmentation. DESIGN: Retrospective. SETTING: Private urology clinic. PATIENT(S): Eleven patients with grade 1, 2, or 3 varicocele. INTERVENTION(S): Varicocelectomy. MAIN OUTCOME MEASURE(S): Sperm DNA fragmentation values were assessed before and after varicocelectomy. RESULTS(S): Ninety percent of the patients showed a significant decrease in sperm DNA fragmentation levels. CONCLUSIONS(S): Although this study was small, 10 of the 11 patients with varicocele showed a significant decrease in sperm DNA fragmentation after varicocele repair. Elevated sperm DNA fragmentation has been shown to have a significant negative effect on pregnancy outcome with use of in vivo, IUI, routine IVF, and to a lesser extent intracytoplasmic sperm injection fertilization; therefore pregnancy outcome may improve after varicocelectomy.

Analysis of sperm DNA fragmentation using flow cytometry and other techniques.

The goal of a rapid, precise and objective sperm measure of sperm DNA fragmentation, in situ, was realised a quarter century ago by the pioneer development of the sperm chromatin structure assay (SCSA). Since then, measurements on many thousands of sperm samples from a variety of animals and humans exposed to reproductive toxicants and having different diseases or infertility pathologies have solidly established the utility of sperm DNA fragmentation in experimental and clinical settings. New methods have been developed that measure sperm DNA fragmentation by different means such as the enzymatic labelling of broken DNA strands (Tunel assay by light microscopy or flow cytometry) and the light microscope observations of DNA fragments in the Comet assay and the negative loops of sperm chromatin in the Halo sperm assay. These assays have proven useful in the areas of livestock and captive wildlife reproduction efficiency, toxicology and more recently in the human infertility clinic. A significant problem is the lack of quality control and standards of threshold values between laboratories. At this time, only the SCSA has been deemed sufficiently rigorous in methodology and established clinical thresholds for utility in the human infertility clinic.

Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay.

OBJECTIVE: To investigate how moderate and/or high levels of DNA fragmentation (DFI), as measured by the sperm chromatin structure assay (SCSA), affect either IVF or IVF with intracytoplasmic sperm injection (ICSI) fertilization, cleavage, blastulation, implantation, and pregnancy. DESIGN: Retrospective clinical study. SETTING: Academic human reproduction laboratory. PATIENT(S): Eighty-nine couples undergoing IVF with conventional fertilization or ICSI. INTERVENTION(S): Sperm chromatin structure assay testing (SCSA) of semen aliquot taken from ejaculate used for assisted reproductive technology (ART). MAIN OUTCOME MEASURE(S): Related DFI to conventional semen parameters and cycle-specific outcomes after ART. RESULT(S): No patients achieved clinical pregnancy if SCSA values exceeded the DFI (27%, P<.01), moderate DFI (15%, P<.01), or high DFI (15%, P<.05) thresholds. Dividing the DFI sperm population into moderate-fragmentation and high-fragmentation categories did not improve the prognostic value of the SCSA. No coefficient of determination (r(2)) between SCSA parameters and conventional parameters exceeded 0.29. CONCLUSION(S): Sperm chromatin structure assay identified thresholds for negative pregnancy outcome after ART not identified using conventional semen parameters. This is the first study analyzing the clinical value of sperm DFI to [1] include a large number of ART patients (n = 89), [2] perform SCSA analysis on a semen aliquot from the ejaculate used for ART, and [3] examine how the extent (moderate and high DFI) of DFI influenced ART outcomes.

Chemopreventive effects of dietary mustard oil on colon tumor development.

Fatty acid composition of dietary fat is one of the detrimental factors in colon cancer development. Fats containing omega 6-polyunsaturated fatty acids (e.g. corn oil) enhance and omega 3-polyunsaturated fatty acids (e.g. fish oil) reduce chemically-induced colon cancer in animal studies. The purpose of this study is to investigate the effects of dietary mustard oil (containing omega 3-polyunsaturated fatty acid) on azoxymethane-induced colon cancer in rats and compare with corn and fish oil treated groups. Colon tumor incidence and multiplicity were found to be 90, 75, and 50% and 1.7, 0.8, and 0.4 tumors/rat in corn, fish and mustard oil treated groups respectively. Omega-3 polyunsaturated fatty acid levels were highest in serum and colon microsomal fractions of the fish oil group followed by the mustard oil group. Corn oil group had the highest level of omega 6-polyunsaturated fatty acid levels in serum and colon microsomal fractions. The results indicate that dietary mustard oil is more effective in preventing colon cancer in rats than dietary fish oil.