Sperm DNA Fragmentation Values Research Articles

Inter-center variation in the efficiency of sperm DNA damage reduction following density gradient centrifugation

This was a prospective multicenter study aiming at comparing the efficiency of sperm selection by density gradient centrifugation (DGC) in reducing sperm DNA fragmentation (SDF) in different ART centers. The study was designed using 290 semen samples collected from 10 different ART centers performing artificial insemination, in vitro fertilization and blind assessment of SDF at the University facilities. The results showed that while there was a significant reduction in the SDF levels in sperm isolated from the gradient centrifuged pellet (DGC) compared to neat semen samples (NSS), there was also significant inter-center variability in the efficiency to reduce SDF values by DGC (78.5% to 29.2%). Surprisingly, for some patients, the level of SDF actually increased following sperm selection. The main conclusions derived from this study were that 1) isolation of sperm from the gradient pellet by DGC must be performed using validated, optimized protocols; 2) routine comparison of SDF values in NSS semen and in processed sperm after DGC or swim-up must be recommended as part of the internal quality control (QC) of ART laboratories to test the efficacy of sperm processing; and 3) SDF values in processed spermatozoa should be obtained to compare with the pregnancy rate when insemination or fertilization is about to be performed, otherwise, attempts to predict pregnancy outcome from SDF could be biased or are essentially meaningless.

Does centrifugation and semen processing with swim up at 37°C yield sperm with better DNA integrity compared to centrifugation and processing at room temperature?

AIM:To evaluate whether semen processing at 37°C yield sperm with better DNA integrity compared to centrifugation and processing at room temperature (RT) by swim-up method.SETTINGS:This study was done at tertiary care center attached to Reproductive Medicine Unit and Medical College.DESIGN:Prospective pilot study.PATIENTS:Normozoospermic men (n = 50) undergoing diagnostic semen analysis.MATERIALS AND METHODS:Normozoospermic samples (World Health Organization, 2010 criteria) after analysis was divided into two aliquots (0.5 mL each); one was processed at 37°C and the other at RT by swim-up method. DNA fragmentation of both samples post wash was calculated by acridine orange method.STATISTICAL ANALYSIS USED:The values of sperm DNA fragmentation were represented as mean and standard error (mean ± SEM) of the mean. Paired t-test was used for calculating the sperm DNA integrity difference between post wash at RT and 37°C.RESULTS:Statistically significant difference was not observed in post wash sperm DNA fragmentation values at 37°C compared to RT.CONCLUSION:Our data represents that there was no significant difference in sperm DNA fragmentation values of samples processed at 37°C and at RT. Hence, sperm processing at 37°C does not yield sperm with better DNA integrity compared to centrifugation and processing at RT.

Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from Opuntia ficus-indica

To analyze the effect of cryopreservation on sperm DNA fragmentation (SDF) in two cytometric sperm populations, PI(brighter) and PI(dimmer), and to test the effects of Opuntia ficus-indica (OFI) extracts, which contain antioxidants and flavanoids, and of resveratrol on cryopreservation of human semen. In vitro prospective study. Institutional study. Twenty-one normozoospermic men undergoing semen analysis for couple infertility. Cryopreservation using the routine method in the presence of OFI extracts or resveratrol. Measurement of SDF by TUNEL/PI flow cytometric method to evaluate sperm motility (by automated motion analysis, CASA system) and viability (by eosin/nigrosin staining) in the two populations of sperm PI(br) and PI(dim). Cryopreservation induced an increase of SDF only in the PI(br) sperm population. The increase was negatively dependent on the basal values of SDF in the same population. Addition of OFI extracts and resveratrol to the cryopreservation medium slightly but statistically significantly reduced SDF in the PI(br) population without affecting the deleterious effect of cryopreservation on sperm motion parameters or viability. The increase of SDF in the PI(br) population, which is unrelated to semen quality, suggests that caution must be taken in using cryopreserved semen, as morphologically normal and motile sperm may be damaged. The addition of substances with multifunctional properties such as OFI extracts to cryopreservation medium is only slightly effective in preventing the dramatic effects on SDF.

Is sperm DNA fragmentation a good marker for field AI bull fertility?1

This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of infertility of Holstein bulls. Cryopreserved sperm samples from 201 Holstein bulls provided by an AI center were used in the analyses of SDF at 0 (SDF_0) and 6 (SDF_6) h of incubation at 37°C. A refinement of the sperm chromatin dispersion test implemented in the Sperm-Halomax kit was employed to measure SDF. Records on routinely collected semen traits (volume, concentration, mass and individual motility evaluated in the fresh ejaculate, and individual motility in post-thawed semen straws) were provided by the AI center. Artificial insemination bull fertility was obtained from official field recording as successful or failed insemination. The results show that the average SDF was low (around 3.5%) at 0 and 6 h of incubation. A moderate effect of inbreeding depression was found. Estimated heritability for SDF traits were moderately high (0.41 and 0.29 for SDF_0 and SDF_6, respectively) and estimated repeatability of SDF measures in the same animal were high (0.73 and 0.70 for SDF_0 and SDF_6, respectively). An overall estimated service bull value (ESBV) obtained through statistical modeling that allowed for adjustment of systematic environmental effects not specific to a bull and of the female contribution to fertility, and the estimated genetic values (EGV) were obtained from field-recorded AI information. The ESBV and EGV were also obtained for all semen traits. Moderately large and negative Pearson correlation coefficients were observed between SDF traits and male fertility ranging from (-0.43 to -0.50; P <0.001). Results of stepwise regression analyses showed that SDF_6 had the largest partial r(2) (0.15 to 0.26) among all semen characteristics. Overall, the selected semen traits explained 25% and 31% of the observed variability in bull fertility measured as EGV and ESBV, respectively. When looking at the predictive ability of bull fertility categories, the results of discriminant and logistic regression analyses showed that low-fertility bulls (those in the 10th or lower percentile in the fertility distribution) can be accurately identified by using measures of SDF alone or in combination with sperm motility. Values of SDF around 7% to 10% could be used as indicators of low AI success.

Response

We thank the authors of the commentary “Flow Cytometry TUNEL Standardization for Assaying Sperm DNA Fragmentation,” for appreciating our study on terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay to detect sperm DNA fragmentation (Muratori et al, 2010a). Furthermore, we are glad an independent laboratory confirmed the drastic effects both on reproducibility provoked by the many existing variants of TUNEL assay and on the accuracy obtained by excluding M540 bodies from flow cytometric analyses of sperm. Reproducible and accurate measures are crucial to assess the reliability of a technique, and only with reliable techniques can the clinical value of sperm DNA fragmentation be speedily assessed (Muratori et al, 2010b). Unfortunately, most of the procedures to assess sperm DNA fragmentation are not standardized, and not all the flow cytometric assays take into account nonsperm elements, including M540 bodies, whereas a consensus on the fittest procedure(s) to detect sperm DNA fragmentation is sought.

Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate

Semen specimens (one ejaculate from each of 20 consenting study participants) were subjected to routine semen analysis, an in vitro sperm binding assay (HBA), and a sperm chromatin dispersion assay (HaloSperm), both before and after cryopreservation using cryoprotectant media supplemented with either egg yolk or soy lecithin. Comparing the equivalency of the two phospholipid cryopreservation supplements with regard to postthaw functional parameters demonstrated that there were no statistically significant differences between the two supplements for [1] recovery of motile sperm, [2] maintenance of sperm cell morphology, [3] maintenance of the ability of sperm to bind to hyaluronate in vitro, or [4] maintenance of sperm DNA integrity.

Comparing flow cytometry and fluorescence microscopy for analyzing human sperm DNA fragmentation by TUNEL labeling

Conflicting results are reported by recent studies comparing flow cytometry (FCM) and fluorescence microscopy (FM) for detecting sperm DNA fragmentation by TUNEL assay. Each of the two technologies has specific peculiarities and limitations, but whereas the limitations of FM observation are well known, the biases due to the inability of FCM to recognize morphologically analyzed cells are less explored. In particular, so far, FCM analysis of sperm DNA fragmentation have included in the analyses M540 bodies, round semen structures exhibiting FSC/SSC properties similar to sperm. Semen M540 bodies, altogether with the occurrence of two sperm populations with different nuclear staining, concur to an underestimation of values of sperm DNA fragmentation by FCM. However, even considering such bias, the observed discrepancies between the performance of FM and FCM are not fully explained. We discuss here the possible variables that may affect the results of each of the two technologies and the necessary efforts to be taken to address this issue.

Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study

To assess the value of sperm DNA fragmentation, measured by the sperm chromatin dispersion (SCD) test, in predicting fertilization rate, embryo quality, and pregnancy outcome. Prospective study. Four French infertility centers, from January to August 2005. Six hundred twenty-two couples participating in their first IVF or ICSI program. Analysis of DNA fragmentation by the sperm chromatin dispersion test in sperm samples used for IVF or ICSI. Correlations and associations between sperm parameters, sperm DNA integrity, and pregnancy outcomes. A statistically significant correlation was observed between sperm DNA fragmentation rate and the following sperm characteristics: sperm motility, morphology, and concentration. We found a statistically significant relationship between sperm DNA fragmentation rate and fertilization rate, and we were able to suggest a threshold sperm DNA fragmentation rate of 18%, above which fragmentation rate was predictive of fertilization rate. Regarding embryo quality, we observed a relationship between sperm DNA fragmentation and embryo quality. No significant relationship was found between sperm DNA fragmentation rate and clinical pregnancies or births. The results of this study confirm the utility of the sperm chromatin dispersion test for assessment of DNA fragmentation.

Significant decrease in sperm deoxyribonucleic acid fragmentation after varicocelectomy

To measure sperm DNA integrity values before and after varicocelectomy in patients with elevated preoperative levels of sperm DNA fragmentation. Retrospective. Private urology clinic. Eleven patients with grade 1, 2, or 3 varicocele. Varicocelectomy. Sperm DNA fragmentation values were assessed before and after varicocelectomy. Ninety percent of the patients showed a significant decrease in sperm DNA fragmentation levels. 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.