Original contributionsIndividuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay
Abstract
Eight monthly semen samples from 45 men not known to be exposed to industrial toxicants were measured by the flow cytometric sperm chromatin structure assay (SCSA). This assay determines susceptibility of sperm DNA to in situ, acid-induced denaturation and is quantitated by the metachromatic shift of acridine orange fluorescence from green (native DNA) to red (denatured DNA). The observed green versus red fluorescence scattergram (cytogram) patterns were generally unique between donors and homogeneous within a donor over time. Within a donor, the cytogram patterns were the same whether intact sperm cells or detached nuclei were measured. For some individuals the cytogram patterns differed for some months and then returned to the original pattern. Intraclass correlations for mean and standard deviation of alpha t [αt = red/(red + green) fluorescence] were higher (.67 to .90) than any classically measured semen variables, suggesting that SCSA results within an individual were more consistent than other measures. Furthermore, average within-donor CV of αt parameters expressed as a percent of any given individual's means was around 10%, which is significantly lower than those derived from common semen measures. The SCSA is an objective, technically sound, biologically stable, sensitive, and feasible measure of semen quality.
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Cited by (256)
Melatonin or L-arginine in semen extender mitigate reductions in quality of frozen-thawed sperm from heat-stressed rams
2022, Animal Reproduction ScienceOur objective was to determine effects of melatonin or L-arginine on quality of frozen-thawed sperm from heat-stressed (HS) rams. Ten Dorset rams were randomly allocated to either scrotal neck insulation for 3.5 d or whole-body heating (28 °C and 30–34% RH for 8 h/d for 4 consecutive days). Semen was collected before HS then once weekly for 1–5 wk, extended (Steridyl CSS One Step ®), and divided into 5 aliquots: control (no additive) or 0.5- or 1-mM of melatonin or L-arginine. For total and progressive motility (CASA), there were effects of group (P = 0.023 and P = 0.008, respectively); for morphological abnormalities (eosin-nigrosin), effects of group (P = 0.01) and a group*week interaction (P = 0.03); and for acrosome integrity (FITC-PSA), effects of group (P = 0.046) and week (P = 0.001). All 4 treatments improved motility (~5–10% points), whereas 1 mM of either compound optimized abnormalities and acrosomal integrity (~7% and 12% points, respectively). For superoxide dismutase and catalase, there were effects of week (P = 0.01 and P = 0.045, respectively), with 1 mM of either additive yielding best results. For DNA fragmentation index (DFI%), there was an effect of week (P = 0.01), and a group*week interaction (P = 0.05), with all 4 treatments reducing DFI%. For total ROS%, there was an effect of week (P = 0.044) and a group*week interaction (P = 0.037), with 1 mM melatonin or L-arginine being best. The hypothesis that melatonin or L-arginine improve quality of frozen-thawed sperm from HS rams was supported; 1 mM of either gave best results, except 0.5 mM minimized DFI%.
Reliability of the sperm chromatin dispersion assay to evaluate sperm deoxyribonucleic acid damage in men with infertility
2022, Fertility and SterilityTo investigate the intraindividual agreement of the sperm chromatin dispersion (SCD) assay results to assess sperm DNA fragmentation (SDF) in men with infertility.
Diagnostic test reliability study.
Andrology laboratories.
A total of 219 men with infertility.
Sperm DNA fragmentation assessment in two ejaculates of the same subjects within a 3-month interval, using the SCD assay performed and analyzed by the same observers under similar testing conditions.
Cohen’s κ statistics to assess the degree of agreement between the pairs of results after converting the nominal SCD values into categorical data, that is, normal (<20%), intermediate (21%–29%), and high (≥30%) SDF rates. We also assessed the pairs of results using reliability measures for numerical variables (intraclass correlation coefficient for consistency using the two-way mixed-effects model and Bland–Altman plots).
The degree of agreement in classifying patients according to normal and pathological SDF classes was overall substantial (κ = 0.632; 95% confidence interval [CI], 0.546–0.718).
A total of 76.7% of individuals were classified under the same class using paired ejaculates. The agreement rate was highest (approximately 80%) in ejaculates initially classified as either normal or high and lowest (approximately 60%) among those with intermediate SDF levels. The frequency of intermediate SDF ejaculates downgraded to normal or upgrade to high SDF classes in the second test was similar (approximately 20%). The intraclass correlation coefficient was 0.856 (95% CI, 0.812–0.887), and the mean difference between the pairs of observations was 0.80% (95% CI, −0.72 to 2.23), indicating no systematic difference between paired observations.
Our study indicates a substantial intraindividual agreement of paired SCD assay results to classify men with infertility into three SDF categories: normal, intermediate, and high. The reliability of the SCD assay was adequate and exceeded 0.80 using two ejaculates analyzed within a 3-month interval under similar conditions. Although this evidence overall supports a single SCD test for patient classification using predefined SDF thresholds, particularly when the first test shows normal or high SDF levels, one in four men will have discordant values in paired ejaculates. Clinicians should be judicious when using SDF test results in decision-making.
Confiabilidad del ensayo de dispersión de cromatina espermática para evaluar el daño del ácido desoxiribonucléico en hombres con infertilidad.
Investigar la concordancia de los resultados en el mismo individuo del ensayo de dispersión de la cromatina espermática (DCE) para evaluar la fragmentación de ADN espermática (FDE) en hombres con infertilidad.
Estudio de confiabilidad de una prueba diagnóstica.
Laboratorios de andrología.
Un total de 219 hombres con infertilidad.
Evaluación de la fragmentación de ADN espermática en dos eyaculados del mismo sujeto en un intervalo de 3 meses, usando el ensayo DCE, llevado a cabo y analizado por los mismos observadores bajo condicione de examen similares.
Análisis estadístico K de Cohen para evaluar el grado de concordancia entre los pares de resultados, después de convertir los valores nominales de DCE en datos categóricos, así: normal (<20%), intermedio (21%-29%) y alto (>/= 30%) de tasas de FDE. Nosotros también evaluamos los pares de resultados usando medidas de confiabilidad para variables numéricas (coeficiente de correlación intraclase para consistencia, usando el modelo de efecto-mixto de doble vía y los plots de Bland-Altman).
El grado de concordancia en la clasificación de pacientes de acuerdo a las clases normal y patológica de FDS fue en general sustancial ( k =0.632; intervalo de confianza del 95% (CI), 0.546-0.718).
Un total de 76.7% de los individuos fueron clasificados en la misma clase usando eyaculados pareados. La tasa de concordancia más alta (aproximadamente 80%) en eyaculados clasificados inicialmente como normales o alto y más bajo (aproximadamente 60%) entre aquellos con niveles intermedios de FDE. La frecuencia de eyaculados con FDE intermedia degradados a categoría normal o ascendidos a categoría alta de FDE en la segunda prueba, fue similar (aproximadamente 20%) El coeficiente de correlación intra-clase fue 0.856 (IC 95%, 0.812-0.887) y la diferencia media entre observaciones pareadas fue 0.80% (IC 95%, -0.72 a 2.23), indicando que no hubo una diferencia sistemática entre observaciones pareadas.
Nuestro estudio indica una concordancia sustancial intra-individuo en los resultados de los ensayos pareados de DCE para clasificar los hombres con infertilidad en tres categorías de FDE: normal, intermedia y alta. La confiabilidad del ensayo de DCE fue adecuada y excedió 0.80 usando dos eyaculados analizados en un intervalo de 3 meses bajo condiciones similares. A pesar de que esta evidencia soporta el uso de un ensayo único de DCE para clasificar los pacientes usando umbrales predefinidos de FDE, particularmente cuando el primer examen muestra resultados normales o altos, uno en cuatro hombres tendrá valores discordantes en eyaculados pareados. Los clínicos deberían tener un juicio adecuado cuando usen los resultados de FDE en el proceso de toma de decisiones.
Age-related changes in human conventional semen parameters and sperm chromatin structure assay-defined sperm DNA/chromatin integrity
2021, Reproductive BioMedicine OnlineWhat are the correlations between male age, traditional semen parameters, sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) in a sufficiently large sample size?
Retrospective cohort study of 18,441 semen samples, with data divided into seven age groups according to male age: ≤25, 26–30, 31–35, 36–40, 41–45, 46–50 and ≥51 years.
Age was negatively correlated with semen volume, total sperm count, motility and HDS, and positively correlated with sperm concentration and DFI (P < 0.001). After 35 years of age, semen volume and total sperm count began to decline. After 30 years of age, motility and HDS decreased consistently. Sperm concentration and DFI increased from 26–30 years of age. DFI was negatively correlated with sperm concentration, total sperm count, motility and normal morphology (P < 0.001) and positively correlated with semen volume and HDS (P < 0.001). HDS was negatively correlated with all parameters (P < 0.001) except semen volume (r = –0.013, P = 0.074) and DFI (r = 0.124, P < 0.001). Patients aged ≥40 years had higher DFI than those aged <40 years in the entire cohort, in the abnormal semen parameters cohort, and in the normal semen parameters cohort (OR 2.145, 2.042, 1.948, respectively, P < 0.001). The ≥40 years age group had a lower HDS than the <40 years age group in the entire cohort and abnormal semen parameters cohort (OR 0.719, 0.677, respectively, P < 0.001).
Ageing is a negative effector of sperm quantity and quality, and routine sperm parameters have weak but significant correlations with sperm DNA/chromatin integrity.
Relationships between the age of 25,445 men attending infertility clinics and sperm chromatin structure assay (SCSA®) defined sperm DNA and chromatin integrity
2020, Fertility and SterilityTo 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.
Retrospective study.
Infertility clinics and diagnostic laboratory.
A total of 25,445 men attending infertility clinics. Donors were 87 men working at Lawrence Livermore National Laboratory.
None.
SCSA measures (% DNA fragmentation index (DFI), X DFI, SD DFI, and %HDS) of men aged 21−80 years.
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.
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.
Relacion entre la edad de 25,445 hombres que asisten a clínicas de infertilidad y el ensayo de estructura de cromatina espermática (SCSA®) definieron la integridad del ADN y cromatina espermática.
determinar las relaciones entre la edad de los hombres con posible esterilidad por factor masculino y los valores del ensayo de estructura de la cromatina espermática (SCSA) en cuanto a fragmentación del ADN espermático (SDF) y ADN altamente teñible (HDS), y comparar estos datos con los obtenidos de donantes hombres sanos sin problemas reproductivos.
Estudio retrospectivo.
Clínicas de infertilidad y laboratorio de diagnóstico.
Un total de 25,445 hombres que fueron a clínicas de infertilidad. Los donantes fueron 87 hombres que trabajaban en el Laboratorio Nacional Lawrence Livermore.
ninguna.
valores del SCSA (% de índice de fragmentación de ADN (DFI), X DFI, SD DFI y % HDS) en hombres de 21 a 80 años.
En la población de estudio, el avance de la edad paterna se asoció con un aumento de la fragmentación del ADN espermático (SDF) calificado como un mayor porcentaje de espermatozoides en el semen eyaculado con roturas medibles de la cadena de ADN (% DFI). La pendiente de aumento en % DFI antes de la edad de 41,6 años fue de 0,39, que aumentó después de la edad de 41,6 a más del doble con una pendiente de 0,86. Estos cambios en el ADN / cromatina en más de 25,000 hombres de edad avanzada que asisten a clínicas de infertilidad son similares a los observados en la misma edad (20 a 80 años) en 87 hombres sanos, no pacientes y sin problemas reproductivos. Para el grupo de edad 20 a 50 años, no hubo una diferencia significativa importante en el % de DFI entre pacientes y hombres donantes. Según un modelo de regresión logística, la capacidad de probabilidad estimada es que, por ejemplo, un hombre de 40 y 50 años tiene una probabilidad del 20% y 40%, respectivamente, de tener un DFI R25% patológico por factor de edad solo. La condensación de la cromatina espermática en pacientes aumentó con la edad de forma lineal, de una media de HDS del 12.2% a los 20 - 25 años a una media de HDS del 7.9% a los 60 – 65 años. Los pacientes tenían un HDS mayor que los donantes en todas las edades.
la gran heterogeneidad de los valores de DFI y HDS a una edad específica impide la traducción automática de la edad a un índice de fragmentación del ADN. Sin embargo, refuerza la idea de que tanto la evaluación DFI como HDS pueden desempeñar un papel en la detección de infertilidad masculina potencial en casos que no se resuelven mediante pruebas de rutina y en casos de abortos espontáneos múltiples. Los datos de DFI y HDS pueden ayudar a los médicos a predecir el potencial de fertilidad de un hombre, a considerar enfoques terapéuticos correctivos, así como a evaluar el riesgo para la salud de la descendencia.
Responses to genotoxicity in mouse testicular germ cells and epididymal spermatozoa are affected by increased age
2019, Toxicology LettersThe increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage.
Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay.
Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage.
There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately.
Association between sperm DNA fragmentation and idiopathic recurrent pregnancy loss: a systematic review and meta-analysis
2019, Reproductive BioMedicine OnlineSperm DNA fragmentation (sDF) has emerged as a valuable tool for evaluating male fertility, yet the relationship between DNA fragmentation in the male gamete and idiopathic recurrent pregnancy loss (RPL) remains a topic of ongoing debate. Hence, a meta-analysis was conducted of 12 prospective and 2 retrospective studies involving 530 men with a history of RPL who underwent sDF testing compared with 639 fertile control participants. The main outcome measures were sDF measured by comet assay, TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL), sperm chromatin dispersion (SCD) or sperm chromatin structure assay. Overall, couples with a history of idiopathic RPL demonstrated higher levels of sDF than fertile couples (average mean difference 11.98, P < 0.001). Subgroup analysis demonstrated a similar average mean difference between the RPL and control groups using SCD compared with TUNEL, while mean paternal age and mean sperm motility in the RPL groups tested by meta-regression demonstrated no significant effect on the mean differences in sDF (P > 0.10). These results support the diagnostic value of sDF over standard semen analysis, as well as a possible paternally derived genetic origin of unexplained RPL. Further prospective studies are required to further assess the predictive utility of sDF for assessing couples with unexplained RPL.