Sperm Chromatin Structure Assay Test Research Articles

Establishing the normal range of sperm DNA fragmentation index (% DFI) for rhesus macaques

The Sperm Chromatin Structure Assay (SCSA) is a robust test with high repeatability and precision. It is a clinically accepted assay that defines risk for infertility in men by measuring the degree of DNA fragmentation (% DFI) in sperm. The objective of this study was to adapt and validate the SCSA for rhesus macaques (Macaca mulatta) and establish a range for % DFI in fertile males. Sperm samples from two different males were used to produce a % DFI validation curve before establishing a range using additional samples from n = 11 males. Sperm labeled with acridine orange were analyzed by flow cytometry to measure green fluorescence (native or intact DNA) and red fluorescence (fragmented DNA). Data were exported to FlowJo software to determine the % DFI for each sample. DNA fragmentation ranged from 0.1 to 2.4% DFI, with a mean ± SD = 1.1 ± 0.7% DFI (validation curve optimized to R2 > 0.95). In conclusion, we were able to successfully validate the SCSA in our institution and establish the first normal range for sperm DNA fragmentation in rhesus macaques. Our study provides a quantitative baseline for future evaluations to assess macaque fertility through the SCSA test.

8 Effects of magnetic-activated cell sorting on human sperm motility and DNA fragmentation index

Selection of spermatozoa before their use for assisted reproductive techniques is an important step in therapy of human infertility. The DNA fragmentation index of sperm plays a major role in pregnancy rates following IVF and intracytoplasmic sperm injection (ICSI). Sperm analyses and standard sperm selection methods in many cases do not eliminate a sufficient proportion of sperm with apoptosis and DNA fragmentation. Magnetic-activated cell sorting (MACS) is a selection method that eliminates apoptotic spermatozoa based on the presence of externalized phosphatidylserine residues. The aim of our study was to evaluate the effect of MACS on human sperm motility and DNA fragmentation index (DFI) in a patient population. The participants were 63 male patients of an IVF clinic, 34 to 45 years old, with 3 years of primary infertility due to male factor. Semen analysis was performed according to the World Health Organization guidelines (2010) and revealed oligoasthenoteratozoospermia in 63 patients. The DFI of fresh semen samples was evaluated using the sperm chromatin structure assay (SCSA) test and revealed DFI 32.4±5.9%. The SCSA test was done on a flow cytometer CyFlow Space (Sysmex-Partec; Evenson 2016 Anim. Reprod. Sci. 169, 56-75; https://doi.org/10.1016/j.anireprosci.2016.01.017). Sperm motility was studied on Hamilton Thorne IVOS. For MACS, we used the MACS® ART Annexin V system (Miltenyi Biotec). The semen sample was diluted to a concentration 10×106 spermatozoa mL−1. After double-density gradient centrifugation, the pellet was resuspended in 100µL of MACS Art Annexin V reagent and added MACS Art Binding Buffer (BB) to 500µL. The sample was gently mixed and incubated for 15min at room temperature. After rinsing the column with BB, the sperm-bead suspension was added on top with BB and, immediately after that, the annexin V-negative and annexin V-positive fractions were obtained (MiniMACS; Miltenyi Biotec). Data were evaluated by ANOVA Student’s t-test. Fresh semen samples collected from the patients had an average sperm concentration of 29.7±5.7×106 mL−1, motility of 32.7±5.9%, and DFI of 32.4±5.9%. Motility of spermatozoa after MACS for the annexin-negative fraction was 47.2±6.3% and for the annexin-positive fraction was 3.5±2.3% (P<0.003). Similarly, the annexin-negative spermatozoa had a lower DFI (10.5±3.8%) rate than did the annexin-positive fraction (67.8±5.9%; P<0.003). The MACS technique allowed a significant reduction of DNA fragmentation levels (from 32.4% for the original sample to 10.5% for the annexin-negative; P<0.01). The separation of a distinct population of nonapoptotic spermatozoa with intact membranes may optimize outcomes from IVF and ICSI procedures. Magnetic-activated cell sorting of human sperm using annexin-V microbeads results in selection of a population with enhanced motility and reduced DFI rates.

TUNEL assay: Establishing a sperm DNA fragmentation cut-off value for Egyptian infertile men.

Male factor infertility is responsible for half of all infertility cases. Conventional semen analysis is inadequate to evaluate male fertility. Sperm DNA fragmentation (SDF) test can be done by: direct methods such as Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) and Comet assay, or indirect like Sperm Chromatin Structure Assay (SCSA) and Sperm Chromatin Dispersion (SCD). TUNEL assay measures both single- and double-strand breaks and is technically less demanding, while SCSA tests for the susceptibility for nuclear DNA denaturation and samples should be sent to the reference lab. Studies showed that a single cut-off value does not fit all. Therefore, this study aimed at establishing a cut-off value to discriminate between fertile and infertile Egyptian men. We enrolled 354 infertile men and 40 proven fertile volunteers.TUNEL assay was performed using Apo-Direct kit and bench top flow cytometer.The calculated SDF cut-off value was 20.3% with a sensitivity of 96.6% and specificity of 87.5%, and the overall accuracy of the test was 95.7%. Sperm DNA fragmentation Test using TUNEL assay is valuable tool for male infertility evaluation, and it assists in offering the best treatment options based on it's results.

Evaluation of sperm DNA fragmentation using multiple methods: a comparison of their predictive power for male infertility.

ObjectiveThe usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay.MethodsIn this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group).ResultsThe alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r =0.70 and r =0.68, respectively; p<0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r =0.77, p<0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r =0.59, r =0.57, and r =0.72, respectively; p<0.001).ConclusionThe TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.

Effect of long-term storage in Safe Cell+ extender on boar sperm DNA integrity and other key sperm parameters

BackgroundThere is some controversy about the extent of changes in different sperm cell features in stored boar semen, especially regarding the potential role of the DNA fragmentation assay for assessment of sperm fertilizing ability. The aim of this study was to assess the effect of time of storage and the dynamic changes in sperm cell characteristics in normospermic boar semen stored in long-term extender, in order to determine the susceptibility to damage of particular structures of spermatozoa during cooling and storage at 17 °C for 240 h post collection. The study included five ejaculates from each of seven boars of the Polish Large White breed (n = 35 ejaculates). The sperm characteristics were assessed using a flow cytometer and a computer assisted sperm analyzer on samples at 0, 48, 96, 168 and 240 h post collection.ResultsThe sperm chromatin structure assay (SCSA) showed a significant abrupt increase (P < 0.01) in the DNA fragmentation index (%DFI) after 48 h of semen storage with only subtle changes thereafter, not exceeding 5% on average after 240 h of storage. The use of a combination of SYBR-14/PI stains did not reveal any significant changes in the percentage of live sperm cells up to 168 h of semen storage. A significant (P < 0.01) decrease in the percentage of live spermatozoa with intact acrosomes was observed after prolonged semen storage (168 h). A significant and progressive decrease in sperm motility was recorded during the whole period of semen storage.ConclusionsStorage of boar semen extended in long-term diluent at 17 °C for 48 h initially induced a decrease in the integrity of sperm DNA. This suggests that the structure of boar sperm DNA is susceptible to damage, especially during semen extension and at the beginning of sperm storage. These findings support the opinion that the SCSA test has only a low potential for routine assessment of boar semen preserved in the liquid state and for assessment of sperm quality changes during 10 days of semen preservation. Remarkably, the integrity of acrosomes and plasma membranes remained nearly unchanged for 7 days.

Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls?

BackgroundIn humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs.MethodsForty Nellore bulls were divided into three age groups: 1.8 to 2 years – young bulls; 3.5 to 7 years – adult bulls; and 8 to 14.3 years – aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA).ResultsWe report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05).ConclusionThese results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity.

Chromomycin A3 staining, sperm chromatin structure assay and hyaluronic acid binding assay as predictors for assisted reproductive outcome

Functional sperm tests such as the sperm chromatin structure assay (SCSA), chromomycin A3 staining (CMA(3)) and hyaluronic acid binding assay (HBA) have been suggested as predictive tests of fertility in vitro. This study aimed to define the clinical role of these functional parameters in assisted reproduction in a prospective cohort study. Conventional sperm diagnosis (motility, morphology and concentration) as well as SCSA, CMA(3) and HBA tests were performed on 205 semen samples [74 IVF, 94 ICSI and 37 combined IVF/intracytoplasmic sperm injection (ICSI)]. Main outcome parameters were fertilization rate, clinical pregnancy rate and take-home baby rate. The study showed that each of the three functional sperm tests was related to one or more conventional and one or more functional sperm tests, indicating that spermatozoa from patients with abnormal conventional semen parameters have a higher likelihood for multiple functional abnormalities. Only SCSA and CMA(3) staining were shown to have a limited predictive value when IVF or combined IVF/ICSI was applied. The proposed threshold value of <or=15% for predicting good fertilization rates and obtaining a pregnancy in IVF could only be confirmed for percent HDS (high DNA stainability in SCSA). ICSI outcome was not influenced by any of the conventional or functional sperm parameters.

DNA Status on Thawed Semen from Fighting Bull: A Comparison Between the SCD and the SCSA Tests

The assessment of sperm chromatin status is compulsory in a complete spermiogram. Here we applied the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test to assess the chromatin status of three fighting bulls. Cryopreserved semen (two straws/bull) were analysed by duplicate after thawing and after 6 h at 37 degrees C with and without oxidative stress (1 mm FE(2+)). Results (SCD: percentage of spermatozoa with halo; SCSA: SD-DFI, %DFI and HDS) were analysed for differences between bulls and treatments, sensitivity and specificity (receiver operating characteristic curves) and repeatability (repeatability coefficients as 2SD of duplicate differences).%DFI for the three bulls was below 2% at 0 h, indicating no risk for fertility according to previous reports. It increased slightly for two of the bulls after FE(2+) treatment (%DFI < 5%) and more pronouncedly for the other bull (C, %DFI approximately 10%), which merits further investigation. SCD rendered higher percentage of halos for bull C, but could not discriminate between samples with and without oxidizing treatment (AUC: 0.52). SCSA (%DFI) showed a high discriminating ability between treatments (AUC: 0.96). The repeatability coefficient was also higher for SCD (5.9) than for %DFI (1.8), indicating lower repeatability for SCD. Overall, %DFI might be the most useful parameter for assessing sperm chromatin on fighting bull. SCD might yield different information than SCSA, hence further research is warranted.

The sperm chromatin structure assay (SCSA) as prognostic factor in IVF/ICSI program

In this study 60 couples undergoing intracytoplasmic sperm injection (ICSI) procedures were enrolled. All men were classified into two groups regarding to the DNA fractionation index (DFI) threshold value: group I < 15% and group II =or> 15%. In group I, median DFI was 4%, normal pre-implantation embryo development was observed and eleven pregnancies were achieved. In group II, median DFI was 23% and normal pre-implantation embryo development was also observed, but only two pregnancies were achieved. Our results suggest that the patients included in the assisted reproductive techniques (ART) should be diagnosed with the SCSA test and the DFI may be related to the outcome of fertilization process as well as to the number of transferred embryos and pregnancy.

Sperm DNA damage in men from infertile couples

To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay (SCSA) were carried out. Ninety-seven men (28% of the whole study group) had a DNA fragmentation index (DFI)> 20%, and 43 men (12%) had a DFI>30%. In the group of men with abnormal semen parameters (n = 224), 35% had a DFI>20%, and 16% had a DFI>30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters (n=126). Men with low sperm motility and abnormal morphology had significantly higher odds ratios (ORs) for having a DFI>20% (4.0 for motility and 1.9 for morphology) and DFI>30% (6.2 for motility and 2.8 for morphology) compared with men with normal sperm motility and morphology. In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique (ART).

Comparison of Chromatin Assays for DNA Fragmentation Evaluation in Human Sperm

Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT-mediated-dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r > .866; P < .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (< or =15%, >15%-<30%, and > or =30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P < .05) under SCSA (22.0 +/- 1.6 vs 11.8 +/- 1.4), TUNEL (19.5 +/- 1.3 vs 11.1 +/- 0.9) and SCD (20.4 +/- 1.3 vs 10.8 +/- 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P > .05) between infertile men (31.3 +/- 2.4) and donors (32.7 +/- 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.

EVALUATION OF SPERM NUCLEAR MORPHOLOGY IN SPECIMENS WITH ABNORMAL SPERM CHROMATIN STRUCTURAL ASSAYS

Two recent tests have claimed to identify the subfertile male even when other semen parameters were normal: the sperm chromatin structure assay (SCSA) and abnormal sperm nuclear morphology using much higher magnification. The present study attempted to determine if having a high (> 30%) DNA fragmentation index (DFI), thus resulting in an abnormal SCSA test, is associated with a greater likelihood of sperm with abnormal nuclei. Four males with high DFI scores (57.6%, 65.4%, 31.0%, and 35.3%) had their nuclei evaluated by a complex microscope set-up that magnifies the sperm at least 6000x. The corresponding % of normal nuclei was 0%, 20.0%, 23.7% and 40.0%. The mean and median % of normal nuclei was 20.9+/-16.43 and 21.8, respectively. More studies of similarly matched refractory in vitro fertilization cases, where males have normal DFI scores, are needed to determine if having a high DFI index is associated with a lower percentage of normal nuclei.

Comparison of the Halosperm ® test kit with the Sperm Chromatin Structure Assay (SCSA ®) infertility test in relation to patient diagnosis and prognosis

The Halosperm test kit, the latest of the sperm DNA fragmentation tests, is considered by Fernández et al. to be a suitable replacement for the Sperm Chromatin Structure Assay (SCSA) test. Although the test is ingenious and interesting, the data lack the statistical rigor of the SCSA test.

Sperm DNA damage in neat and density gradient fractions of patient and donor semen samples by different chromatin evaluation assays

Interest in evaluating chromatin quality has increased since DNA damage in sperm from infertile men has been associated with infertility and poor embryo quality. We compared the neat semen and density gradient isolated sperm fractions for DNA damage with different assays. Fresh semen samples from 54 patients undergoing IVF with a wide range of semen quality and 7 fertile donors were collected under IRB approval and evaluated for sperm DNA damage. Fresh ejaculates with 2–3 days of sexual abstinence were collected by masturbation and evaluated according to WHO criteria (1999). Briefly, an aliquot of neat semen was frozen for sperm chromatin structure assay (SCSA). The remaining sample was subjected to a discontinuous ISolate density gradient (Irvine) at 300 × g for 15 minutes. The supernatant, 35% and 90% fractions were washed in mHTF (Irvine) at 300 × g for 10 minutes. The sperm from the neat sample, supernatant, 35% and 90% fractions were evaluated for DNA damage using TdT-mediated-dUTP nick end labeling (TUNEL), sperm chromatin dispersion (SCD) test and acridine orange staining technique (AOT). All the assays were performed following the established protocols. The SCSA evaluated more than 5000 sperm and the results are expressed as percent DNA fragmentation index (DFI). A total of 500 sperm from neat and gradient fractions were evaluated in each assay under a fluorescent microscope. The data was analyzed with ANOVA, repeated measures analysis and Pearson correlation tests. Sperm DNA damage in neat semen under SCSA, TUNEL and SCD test correlated (r=0.86: P>0.05) but no correlation was found for AOT. Differences were observed for DNA damage within each assay for sperm in neat, supernatant, 35% and 90% fractions. The results for sperm DNA damage were different (p<0.05) between patients and donors under each assay except for AOT, which showed significantly (p<0.05) higher DNA damage compared to SCSA, TUNEL and SCD. The AOT showed extreme variations for sperm DNA damage within gradient fractions for the same individual and the problems of indistinct colors, rapid fading and heterogeneous staining of the slides were encountered. The DNA damage was significantly (p<0.05) lower in patient and donor sperm isolated from the 90% pellets under TUNEL and SCD test. The values in the figure are the average percent damage ± SEM. These data demonstrate that the SCSA, TUNEL and SCD test show similar predictive values for the assessment of sperm DNA damage. The AOT shows high DNA damage in sperm and the variations in results make AOT of questionable clinical value. Density gradient centrifugation significantly improves the chromatin quality of sperm in the 90% pellet. Lastly, the data demonstrate a lower sperm DNA damage in donors compared to infertile patients.

Nurture vs nature: how can we optimize sperm quality?

Nurture vs nature: how can we optimize sperm quality?

Sperm chromatin structure assay (SCSA) parameters provide additional prognostic information to conventional semen analysis for couples trying to conceive

Sperm chromatin structure assay (SCSA) parameters provide additional prognostic information to conventional semen analysis for couples trying to conceive