Assessment Of Sperm DNA Fragmentation Research Articles

Sperm DNA Fragmentation: Causes, Effects and Relevance in IVF Outcomes

Sperm DNA fragmentation, characterized by damaged or broken DNA strands within sperm cells, has been recognized as an important factor in male infertility and poor ART outcome. This lecture aims to provide an overview of the causes, effects, and relevance of sperm DNA fragmentation in in vitro fertilization (IVF) outcomes and look into the latest evidences from the literatures. Causes: Sperm DNA fragmentation can result from various factors, including oxidative stress, advanced paternal age, lifestyle factors such as smoking and excessive alcohol consumption, environmental exposures, genotoxic agents, and underlying genetic abnormalities. These factors can induce DNA damage during sperm development, during the transit of sperm through the male reproductive tract or during the storage of the sperm in epididymis. Relevance in IVF Outcomes: The assessment of sperm DNA fragmentation has emerged as a valuable tool in evaluating male infertility and predicting IVF success. Several methods, including the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL), comet assay, and other commercial detection kits, are available to measure sperm DNA fragmentation. Previous small cohort studies have demonstrated that higher levels of sperm DNA fragmentation are associated with lower pregnancy rates, decreased embryo quality, and an increased risk of pregnancy loss in IVF cycles. Therefore, incorporating sperm DNA fragmentation analysis into the routine assessment of male infertility can aid in optimizing treatment strategies and improving IVF outcomes. Recent evidences: Latest large clinical studies showed a limited association to IVF outcome. The DFI remains controversial as a predictive tool. Nonetheless, sperm DNA fragmentation is still believed to be a significant factor affecting male fertility and IVF outcomes. Further research is warranted to explore the mechanisms underlying DNA fragmentation, develop standardized assessment methods, and explore potential interventions to mitigate the adverse effects of sperm DNA damage on IVF success.

Sperm chromatin structure assay (SCSA®) and flow cytometry-assisted TUNEL assay provide a concordant assessment of sperm DNA fragmentation as a function of age in a large cohort of approximately 10,000 patients

BackgroundSperm DNA integrity is increasingly seen as a critical characteristic determining reproductive success, both in natural reproduction and in assisted reproductive technologies (ART). Despite this awareness, sperm DNA and nuclear integrity tests are still not part of routine examinations for either infertile men or fertile men wishing to assess their reproductive capacity. This is not due to the unavailability of DNA and sperm nuclear integrity tests. On the contrary, several relevant but distinct tests are available and have been used in many clinical trials, which has led to conflicting results and confusion. The reasons for this are mainly the lack of standardization between different clinics and between the tests themselves. In addition, the small number of samples analyzed in these trials has often weakened the value of the analyses performed. In the present work, we used a large cohort of semen samples, covering a wide age range, which were simultaneously evaluated for sperm DNA fragmentation (SDF) using two of the most frequently used SDF assays, namely the TUNEL assay and the sperm chromatin structure assay (SCSA®). At the same time, as standard seminal parameters (sperm motility, sperm morphology, sperm count) were available for these samples, correlations between age, SDF and conventional seminal parameters were analyzed.ResultsWe show that the SCSA® and TUNEL assessments of SDF produce concordant data. However, the SDF assessed by TUNEL is systematically lower than that assessed by SCSA®. Regardless of the test used, the SDF increases steadily during aging, while the HDS parameter (High DNA stainability assessed via SCSA®) remains unchanged. In the cohort analyzed, conventional sperm parameters do not seem to discriminate with aging. Only sperm volume and motility were significantly lower in the oldest age group analyzed [50–59 years of age].ConclusionsIn the large cohort analyzed, SDF is an age-dependent parameter, increasing linearly with aging. The SCSA® assessment of SDF and the flow cytometry-assisted TUNEL assessment are well correlated, although TUNEL is less sensitive than SCSA®. This difference in sensitivity should be taken into account in the final assessment of the true level of fragmentation of the sperm nucleus of a given sample. The classical sperm parameters (motility, morphology, sperm count) do not change dramatically with age, making them inadequate to assess the fertility potential of an individual.

Sperm DNA Fragmentation after Cryopreservation and Sperm Selection Has No Implications for Clinical Pregnancies and Live Births after Intrauterine Insemination with Donor Sperm.

Intrauterine insemination with donor sperm (IUI-D) requires multiple in vitro manipulations such as sperm selection and cryopreservation during which spermatozoa may be exposed to oxidative stress (OS) and other insults that may produce potential damage including sperm DNA fragmentation (SDF). High levels of SDF, referring to damage or breaks in the genetic material of sperm cells, are linked to an increased risk of reproductive failure. This retrospective, observational study set out to evaluate whether SDF assessment could predict clinical outcome in an IUI-D program, where sperm donors are selected on strict conventional semen parameters. A total of 18 donors and 106 recipients were matched for IUI-D. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. We counted 78 live births (18.2% of cycles), while 20 pregnancies ended in miscarriage (4.7% of cycles), 1 in extra-uterine pregnancy and 1 in stillbirth. Female age significantly influenced clinical pregnancy and miscarriage rates. SDF increased after cryopreservation (26.3 ± 14.5%; p < 0.001) and more so after post-thaw density gradient (34.9 ± 22.1%; p = 0.04) without affecting clinical pregnancy (OR [95% CI] 1.01 [0.99; 1.02]; p = 0.27), live birth (1.00 [0.99; 1.02]; p = 0.72) and miscarriage rates (1.02 [1.00; 1.05]; p = 0.08). The implications of our findings extend to a better selection of sperm donors and a better sperm preparation technique tailored to the donor semen's properties in order to maximize the chances of a favorable treatment outcome.

Paternal Age Matters: Association with Sperm Criteria's- Spermatozoa DNA Integrity and Methylation Profile.

Advanced age has been reported to negatively affect sperm parameters and spermatozoa DNA integrity. A decline in sperm criteria was also associated with altered epigenetic marks such as DNA methylation with a potential downstream impact on in vitro fertilization success and clinical outcomes. The aim of the present retrospective study was to clarify the association between advanced paternal age (APA) and sperm parameters, DNA integrity and DNA methylation profile. A total of 671 patients consulting for infertility underwent sperm analysis, sperm DNA integrity assessment and methylation level measurement. The principal finding was that individuals over 40 years of age exhibit a significant increase in DNA fragmentation levels compared to the younger group (15% versus 9%, respectively, p = 0.04). However, there was no significant difference in DNA decondensation and sperm parameters in association with APA. In addition, a drop in the global methylation level was also found in men over 40 years (6% in the young group versus 2% in the old group, p = 0.03). As a conclusion, men over 40 years are at higher risk of elevated sperm DNA fragmentation and lower methylation level. Based on these observations, it is recommended that the assessment of sperm DNA fragmentation should be taken into consideration particularly after the age of 40. Our findings support the idea that paternal age is a crucial factor that should not be neglected during fertility evaluation and treatment since it is associated with epigenetics changes in sperm. Although the underlying mechanism remains to be clarified, we believe that environmental and professional exposure factors are likely involved in the process.

P-059 Sperm DNA fragmentation (SDF) after cryopreservation and sperm selection: implications for clinical pregnancies and live births after intrauterine insemination with donor sperm

Abstract Study question Can SDF assessment pre-cryopreservation, post-thaw and after post-thaw sperm selection predict clinical outcome in a heterologous intra-uterine donor insemination program (IUI)? Summary answer SDF increased post-cryopreservation in donor sperm and after post-thaw density gradient without affecting clinical pregnancy, live birth and miscarriage rates. What is known already Sperm cryopreservation is effective for preservation of male fertility and facilitates the storage of donor semen, while infectious disease screening can be completed and confirmed negative. Sperm viability and motility are the most vulnerable parameters during the freeze-thaw process. Inherent reactive oxygen species may induce SDF via oxidative stress. Controversial results are reported regarding induction of SDF after cryopreservation due to differences in cryo tolerance, methods of cryopreservation and methods used to evaluate SDF. Sperm selection post-thaw is indispensable to select a normal motile fraction for insemination. Again, results are controversial regarding influence on SDF levels after sperm selection post-thaw. Study design, size, duration This was a prospective, observational study. Between March 2015 and March 2019, eighteen potential sperm donors were screened via a three step plan – semen assessment, medical assessment and serological blood tests. In accordance with the Belgian legislation (2007), a sperm donor was matched to obtain a maximum of 6 pregnancies in acceptor women/couples, allowing for more than one offspring/woman or couple. A total of 106 acceptors were matched for heterologous intra-uterine insemination. Participants/materials, setting, methods Semen samples were collected after 2-7 days abstinence and standard semen parameters analyzed within 60 mins after ejaculation. Within the same time frame SDF test was performed using TUNEL assay both before and after cryopreservation and after a two-step discontinuous density gradient centrifugation post-thaw. Samples were cryopreserved by the conventional slow freezing method using sperm freeze solution. A yield of ≥ 2 M progressive spermatozoa post-thaw after selection was sufficient to contemplate IUI. Main results and the role of chance Mean female age at first cycle was 33.9 ± 4.1 years and donor age 28.5 ± 5.6 years. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. Excluding three ongoing pregnancies, we counted 75 live births (17.6% of cycles or 77.3% of pregnancies), while 21 pregnancies ended in miscarriage (4.9% of cycles or 21.6% of pregnancies) and 1 resulted in stillbirth. Progressive motility (59.3 ± 12.5%) decreased significantly after cryopreservation (30.0 ± 13.2%; p &amp;lt; 0.001), but increased post-thaw after density gradient centrifugation (61.6 ± 16.0%; p &amp;lt; 0.001). On the contrary, SDF (12.0 ± 5.9%) increased post-cryopreservation (26.3 ± 14.5%; p &amp;lt; 0.001) and further increased after sperm selection (34.9 ± 22.1%; p = 0.04). Using multilevel mixed-effects logistic regression (Odds ratio [95%CI]), we found that female age significantly influenced clinical pregnancy (0.911 [0.847-0.981]; p = 0.01), live birth (0.894 [0.834-0.959]; p = 0.002) and miscarriage rates (1.180 [1.033-1.347]; p = 0.015). There was no significant age-adjusted effect of SDF post-thaw after density gradient selection on clinical pregnancy (1.007 [0.994-1.021]; p = 0.30), live birth (1.001 [0.988-1.014]; p = 0.84) or miscarriage (1.021 [0.997-1.046]; p = 0.08). Median time-to-live birth was 4 cycles. Analyses of time-to-live birth with mixed-effects Cox models revealed no significant association with progressive motility; SDF before or after cryopreservation and after sperm selection post-thaw. Limitations, reasons for caution Results concern a small donor population, with high normal semen parameters and should therefore be extrapolated cautiously to subfertile men with normal and subnormal semen parameters. Wider implications of the findings In a donor IUI program with strict selection on conventional sperm parameters, differences in SDF before and after cryopreservation and gradient centrifugation do not change IUI outcomes, although cryopreservation and centrifugation increase SDF. Findings could be useful for the optimization of sperm freezing and selection in different assisted reproductive procedures. Trial registration number not applicable

Novel sperm chromatin dispersion test with artificial intelligence-aided halo evaluation: A comparison study with existing modalities.

Sperm chromatin dispersion test is a common and inexpensive technique to assess sperm DNA fragmentation, but its subjectivity in assessing a small number of spermatozoa is a disadvantage. To study the efficacy of a new sperm chromatin dispersion test kit (R10) combined with an artificial intelligence-aided halo-evaluation platform (X12) and compare the results to those of existing sperm DNA fragmentation testing methods. Semen samples from normozoospermic donors (n=10) and infertile men with abnormal semen parameters (n=10) were enrolled. DNA fragmentation indices were examined by multiple assays, including R10, Halosperm G2 (G2), sperm chromatin structure assay, and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling. In R10 assay, the DNA fragmentation indices were obtained both manually (manual R10) and by X12 (AI-R10). The obtained DNA fragmentation indices were analyzed by agreement analyses. The DNA fragmentation indices obtained by manual R10 and those obtained by AI-R10 showed a strong significant correlation (r=0.97, p<0.001) and agreement. The number of spermatozoa evaluated by AI-R10 was 2078 (680-5831). The DNA fragmentation indices obtained by manual R10 and AI-R10 both correlated with those of G2 (r=0.90, p<0.001; r=0.88, p<0.001). Between the AI-R10 and G2 results, Passing-Bablok regression showed no systematic or proportional difference, and Bland-Altman plots revealed overall agreement and a mean bias of 6.3% with an SD of 6.9% (95% limit of agreement: -7.2% to 19.9%). AI-R10 and sperm chromatin structure assays showed systematic differences with a mean bias of -1.9%, while AI-R10 and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling revealed proportional differences with a mean bias of -10.7%. The novel sperm chromatin dispersion kit and artificial intelligence-aided platform demonstrated significant correlation and agreement with existing sperm chromatin dispersion methods by assessing greater number of spermatozoa. This technique has the potential to provide a rapid and accurate assessment of sperm DNA fragmentation without technical expertise or flow cytometry.

Sperm DNA fragmentation testing in clinical management of reproductive medicine.

Approximately 8%-12% of couples worldwide face infertility, with infertility of individuals assigned male at birth (AMAB) contributing to at least 50% of cases. Conventional semen analysis commonly used to detect sperm abnormalities is insufficient, as 30% of AMAB patients experiencing infertility show normal results in this test. From a genetic perspective, the assessment of sperm DNA fragmentation (SDF) is important as a parameter of sperm quality. In this narrative study, we review and discuss pathophysiological causes, DNA repair mechanisms, and management of high SDF. We then summarize literature exploring the association between SDF and reproductive outcomes. Recent systematic reviews and meta-analyses have revealed a significant association between high SDF in AMAB individuals and adverse reproductive outcomes including embryo development, natural conception, intrauterine insemination, and invitro fertilization. However, the association with live birth rates and pregnancy rates following intracytoplasmic injection remains inconclusive. The disparities among quantitative assays, inconsistent reference range values, absent high-quality prospective clinical trials, and clinical heterogeneity in AMAB patients with elevated SDF represent the main limitations affecting SDF testing. The evaluation and management of SDF plays an important role in a subset of AMAB infertility, but widespread integration into clinical guidelines will require future high-quality clinical trials and assay standardization.

Male Infertility Coexists with Decreased Sperm Genomic Integrity and Oxidative Stress in Semen Irrespective of Leukocytospermia.

Our research was designed to verify the relationship between male infertility, basic semen characteristics (with respect to detailed sperm morphology), sperm DNA fragmentation (SDF), oxidation-reduction potential in semen (ORP), and leukocytospermia. The obtained results showed that infertile groups (with or without leukocytospermia) had significantly lower basic semen characteristics and higher SDF, raw ORP, and static ORP (sORP) than fertile controls. The thresholds of 13% SDF (AUC = 0.733) and 1.40 sORP (AUC = 0.857) were predictive values for discriminating infertile from fertile men. In infertile groups, a higher prevalence and risk for >13% SDF and >1.40 sORP were revealed. Unexpectedly, leukocytospermic subjects had lower sORP, prevalence, and risk for >1.40 sORP than leukocytospermic-negative men. These groups did not differ in SDF and raw ORP. Both SDF and sORP negatively correlated with basic semen parameters but positively correlated with sperm head and midpiece defects. sORP positively correlated with sperm tail defects, immature sperm cells with excess residual cytoplasm, and SDF. In turn, raw ORP negatively correlated with sperm count but positively correlated with SDF and sORP. These findings indicate that (1) there is a relationship between male infertility, SDF, and OS in semen; (2) in infertile men, there is a clinically significant risk of SDF and OS irrespective of leukocytospermia; and (3) the assessment of SDF and oxidative stress should be independent of leukocytospermia.

Sperm DNA integrity and male infertility: a narrative review and guide for the reproductive physicians.

Background and ObjectiveConventional semen analysis (SA) remains an essential tool in the initial male fertility evaluation and subsequent follow-up. However, it neither provides information about the functional status of spermatozoa nor addresses disorders such as idiopathic or unexplained infertility (UI). Recently, assessment of sperm DNA fragmentation (SDF) has been proposed as an extended sperm test that may help overcome these inherent limitations of basic SA. In this review, we aim to: (I) discuss the pathophysiological aspects of SDF, including natural repair mechanisms, causes, and impact on reproductive outcomes; (II) explain different assessment tools of SDF, and describe potential therapeutic options to manage infertile men with high SDF; and (III) analyse the strengths, weaknesses, opportunities and threats (SWOT) of current research on the topic.MethodsThis review was constructed from original studies, systematic reviews and meta-analyses that were published over the years up until August 2021, related to the various aspects of SDF.Key Content and FindingsDifferent mechanisms lead to high SDF, including defective chromatin packaging, apoptosis, and seminal oxidative stress. The relevance of sperm DNA integrity to male fertility/infertility has been supported by the frequent observation of high levels of SDF in infertile men, and in association with risk factors for infertility. Additionally, high SDF levels have been inversely correlated with the outcomes of natural pregnancy and assisted reproduction. Terminal deoxynucleotidyl transferase dUTP nick end labelling, sperm chromatin structure assay, sperm chromatin dispersion, and Comet assay are four commonly used assays for measurement of SDF. Addressing lifestyle risks and underlying conditions, antioxidants, hormonal therapy, and advanced sperm selection techniques have all been proposed as potential therapeutic options to lower SDF.ConclusionsThe sum of literature provides evidence of detrimental effects of high SDF on both natural and assisted fertility outcomes. Standardization of the techniques used for assessment of SDF and their incorporation into the work up of infertile couples may have significant implications on the future management of a selected category of infertile men with high SDF.

P-056 The reproductive toxicity of alginate coated silver nano-rod on sperm

Abstract Study question What is the toxic effects of Au@Ag core-shell NR on sperm function? Summary answer Although sperm count and morphology did not differ significantly, changing the shape and envelope of the silver nanostructure did not reduce its toxicity. What is known already Spermatogenesis is a developmental process of germ cell reproduction that depends on hormonal or dynamic interplays between sertoli cells and germ cells. Silver is the most common noble metal used in the synthesis of nanoscale materials, particularly in room sprays, cosmetics, toys, clinical catheters, and wound dressings. The increasing usability of silver nanoparticles (Ag NP) has increased human exposure. Many studies have demonstrated that Ag nanoparticles, depending on their shape, size, and concentration, have cytotoxic effects that reduce cell viability. The toxicity of silver Nano-Rod by deformation and coating with alginate has been studied. Study design, size, duration Forty-eight male mice were divided into five groups (two treatments, two shams, and one control). The treated groups received the Ag Nano-Rod at a dose of 1.5 mg/kg/day intraperitoneally for fourteen and thirty-five days. The sham groups were treated with identical amounts and days, and the control group received no material. Finally, the cauda epididymis of the animals was removed. Participants/materials, setting, methods In the present study, Ag Nano-Rod were characterized by HR-TEM, FT-IR, UV-visible, XRD and ICP-MS. Sperm analysis, including count, motility, viability, and morphology, was studied according to the criteria of WHO. A phase-contrast microscope was used to count the spermatozoa and assess their motility. Viability and morphology were examined by eosin-nigrosin and papanicolaou staining, respectively. TUNEL assay and gene expression for apoptotic and autophagic markers were also performed. Main results and the role of chance High magnification images TEM were obtained. According to this, the Ag nanostructure was rod-shaped and the average size of the particles was 50nm. The UV-visible confirms the formation of the synthesized nanosilver (rod shape). The change in nanostructure coating from CTAB to alginate was studied by FTIR analysis, and XRD proved the presence of silver in the composition. The results of sperm analysis showed that there was no significant decrease in sperm count and morphology but the parameters of motility and viability of the nanosilver groups showed a significant decline compared to the control group (P &amp;lt; 0.05). Assessment of sperm DNA fragmentation by the TUNEL assay showed that the nanosilver groups had significantly higher DNA fragmentation and the real-time PCR results also confirmed this. Expression of autophagic and apoptotic markers indicates activation of the apoptosis pathway compared to the autophagy pathway. The expression of LC3 and Beclin-1 (markers that initiate and continue the autophagy) was reduced compared to the sham and control groups. Also, examination of apoptotic pathway genes showed a decrease in Bcl-2 expression and an increase in Bax and caspase-3 expression. Limitations, reasons for caution This study requires further research, including protein expression in these two pathways. Wider implications of the findings This study investigated the acute (14 days) and chronic (35 days) effects of silver nanorod on reproductive toxicity, which can lead to significant changes in sperm quality. This reduction in quality was observed mainly in the chronic phase and severely affected sperm motility. Trial registration number Not Applicable

P-033 A third of men with normospermia attending for initial fertility assessment could have DNA damage

Abstract Study question What is the incidence of sperm DNA damage in men with normal semen analyses attending for initial fertility investigation? Summary answer Half of men (54%) attending for infertilty investigation had a normal semen analysis of which a third (32%) of these had abnormal sperm DNA. What is known already Conventional semen analysis assesses sperm concentration, motility, and morphology but with a high degree of biological variability. Around 25% of men are given a diagnosis of unexplained infertility by normal semen analysis. The World Health Organisation (WHO) stated in 2021 that ‘clinically there is a growing awareness that chromosomal anomalies and gene mutations underlie a diverse spectrum of male infertility,’ so now recommends that sperm DNA is determined as an extended analysis. Sperm DNA damage occurs more often in infertile men and DNA damage is associated with recurrent pregnancy loss and decreased live birth rates following fertility treatment. Study design, size, duration A prospective cohort study of men attending a secondary fertility clinic were offered a standard semen analysis plus sperm DNA fragmentation using SpermComet technology. UK IRAS ethical approval was obtained. Men attending were given a patient information leaflet at their first appointment. The study duration was 6 months. In total, 142 men were recruited out of a possible maximum of 409 attendees. Participants/materials, setting, methods Participants included men who had been referred as a couple to a secondary level infertility clinic for an initial assessment. Information leaflets were either posted to them prior to their first appointment or given to them at the initial nurse-led consultation. Those who subsequently contacted our research team re-attended for a sperm analysis and an aliquot was cryopreserved and sent in weekly batches to Examen (Northern Ireland) for SpermComet DNA fragmentation assessment. Main results and the role of chance Between May and November 2021, there were 409 attendees of whom 142 consented to the study. Sperm DNA quality for the participants was assessed and reported as average, low and high DNA damage as a percentage compared with clinical thresholds used by Examen from their sperm bank of 63 fertile sperm donors who had recently achieved clinical pregnancies. Of the 142 recruited, 77 men (54%) attending for initial investigations had normospermia assessed against the WHO criteria, so were initially classified as unexplained. Of these, 25 (32%) had abnormal sperm DNA values. The average comet score was 39.7% +/-1.3, low comet score was 37.8% +/- 4.4 and high comet score was 17.0% +/-2.0. These values were all significantly outside ( p &amp;lt; 0.001) of the fertile parameters from 63 fertile donors used by Examen (fertile range: average &amp;lt;26%%, low &amp;gt;74% and high &amp;lt;4%). Our results suggest that up to a third of men reported to have normospermia will have DNA damaged sperm. This is important to consider before labelling a couple as 'unexplained'. Knowing the male's sperm DNA fragmentation assessment would allow for further discussion and exploration of lifestyle and dietary advice. Limitations, reasons for caution During the study period, not all attendees agreed to participate as it required an additional hospital visit. In this single site study, men have not yet been followed up for future fertility outcome such as natural conception and need or success of assisted conception. Wider implications of the findings Sperm DNA damage was found in a third of men who would previously have been classified as fertile and offered no further investigations or treatment to improve their sperm quality. Thus, this tool could be a useful adjunct to semen analysis to guide these couples’ future treatment pathways. Trial registration number not applicable

Apoptotic M540 bodies present in human semen interfere with flow cytometry-assisted assessment of sperm DNA fragmentation and oxidation

BackgroundThe use of flow cytometry (FC) to evaluate sperm DNA fragmentation via deoxynucleotidyl transferase terminal fluorescein dUTP nick-end labeling (TUNEL) has shown inconsistencies compared with conventional fluorescent microscopic analyses. It has been hypothesized that the observed discrepancies could be attributed to the presence of apoptotic bodies that can be labeled with merocyanine 540, the so-called M540 bodies. In order to verify this hypothesis and determine the accuracy of our in-house FC-assisted evaluation of spermatozoa parameters, we used FC to evaluate both the fragmentation of sperm DNA using the TUNEL assay and the oxidation of sperm DNA using the 8-OHdG assay on semen samples with or without M540 bodies.ResultsWe show that the presence of M540 bodies lead to underestimation of both the level of sperm DNA fragmentation and sperm DNA oxidation when using FC assisted detection systems. We also observed that this situation is particularly pertinent in semen samples classified as abnormal with respect to the routine WHO semen evaluation as they appear to contain more M540 bodies than normal samples.ConclusionsWe conclude that M540 bodies interfere with both FC-conducted assays designed to evaluate sperm nuclear/DNA integrity. Exclusion of these contaminants in unprepared semen samples should be performed in order to correctly appreciate the true level of sperm DNA/nuclear damage which is known to be a critical male factor for reproductive success.

Reliability of the sperm chromatin dispersion assay to evaluate sperm deoxyribonucleic acid damage in men with infertility

To 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.

DNA fragmentation of human spermatozoa: Simple assessment of single- and double-strand DNA breaks and their respective dynamic behavioral response.

Procedures to detect sperm DNA fragmentation (SDF), like the sperm chromatin dispersion (SCD) test, determine the "global" SDF without discriminating between spermatozoa with single-strand DNA breaks only (SDF-SSBs) and those containing double-strand DNA breaks (SDF-DSBs). (a) To validate a test to distinguish human spermatozoa with massive DSBs (DSB-SCD assay), (b) to study the baseline SDF-SSBs and SDF-DSBs, and (c) to assess their dynamics in vitro. (a) SDF-DSBs were determined by visualization of diffused DNA fragments from spermatozoa lysed under non-denaturing conditions. This was validated by in vitro incubation with DNase I and the comet assay. (b) Baseline SDF-DSBs and SDF-SSBs were determined in ejaculates from 95 males. (c) Their dynamic appearance was studied in samples untreated or exposed to hyperthermia, acidic pH, nitric oxide released by sodium nitroprusside (SNP), and the metabolic energy inhibitors 2-deoxy-D-glucose and antimycin A. (a) DNase I and comet assay experiments confirmed that the assay successfully determined SDF-DSBs. (b) The higher the SDF of the semen sample, the higher the frequency of SSBs, whereas DSBs behaved independently. Abnormal samples showed higher SDF than normozoospermic, the difference being only significant for SDF-SSBs. (c) During the first hours of incubation, the linear rate of increase in SDF-SSBs was 3.7 X higher than that of SDF-DSBs. All hazardous agents accelerated the SDF rate when compared to untreated spermatozoa, primarily being associated with SDF-SSBs. SNP treatment was the most damaging, rapidly inducing spermatozoa with SSBs which progressively evolved to DSBs. Remarkably, this phenomenon was also evidenced after acute SNP exposure, revealing cryptic sperm damage. The DSBs-SCD is an easy complement for SDF assessment. The dynamic study of SSBs and DSBs may improve the evaluation of sperm quality in clinical settings, particularly "unmasking" the presence of non-specific cryptic sperm damage that might otherwise go undetected.

Comparison of different mathematical models to assess seasonal variations in the longevity of DNA integrity of cooled‐stored stallion sperm

Dynamic assessment of sperm DNA fragmentation (SDF) has shown to give fuller understanding of stallion semen quality; however, there have been limited attempts to use this parameter to investigate seasonal changes in productive functions. The aims of this study were to: (a) establish a reliable mathematical model to describe the longevity of cooled-stored sperm DNA integrity; (b) to examine the effect of seasonal variations on SDF. Ejaculates were cooled to 5°C, and SDF was analysed after 0, 6 and 24hr of storage. The coefficient of determination (R2 ) was calculated after fine-tuning linear (LIN), exponential (EXP) and second order polynomial (POL) models. R2 was significantly higher (p<.001) for POL than for LIN and EXP. The rate of DNA degradation was calculated using the slopes of POL equations. After assessing the rate of change of the POL functions, significant differences between the acceleration of DNA fragmentation were found (p<.01) among seasons, being higher for winter and summer than spring and autumn. In conclusion, DNA analysis of stallion sperm fits better to a second order polynomial mathematical model, being spring the best season to collect and process cooled stallion semen in order to maintain the DNA integrity of the stallion sperm.

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

Sperm DNA Fragmentation: A New Guideline for Clinicians.

Sperm DNA integrity is crucial for fertilization and development of healthy offspring. The spermatozoon undergoes extensive molecular remodeling of its nucleus during later phases of spermatogenesis, which imparts compaction and protects the genetic content. Testicular (defective maturation and abortive apoptosis) and post-testicular (oxidative stress) mechanisms are implicated in the etiology of sperm DNA fragmentation (SDF), which affects both natural and assisted reproduction. Several clinical and environmental factors are known to negatively impact sperm DNA integrity. An increasing number of reports emphasizes the direct relationship between sperm DNA damage and male infertility. Currently, several assays are available to assess sperm DNA damage, however, routine assessment of SDF in clinical practice is not recommended by professional organizations. This article provides an overview of SDF types, origin and comparative analysis of various SDF assays while primarily focusing on the clinical indications of SDF testing. Importantly, we report four clinical cases where SDF testing had played a significant role in improving fertility outcome. In light of these clinical case reports and recent scientific evidence, this review provides expert recommendations on SDF testing and examines the advantages and drawbacks of the clinical utility of SDF testing using Strength-Weaknesses-Opportunities-Threats (SWOT) analysis.

Utility and Predictive Value of Human Standard Semen Parameters and Sperm DNA Dispersion for Fertility Potential

Because the assessment of sperm DNA fragmentation (SDF) plays a key role in male fertility, our study was designed to find the relationships between SDF and standard semen parameters. The receiver operating characteristic (ROC) curve showed that 18% SDF is a prognostic parameter for discriminating between men with normal and abnormal standard semen parameters (n = 667). Men with > 18% SDF had significantly lower quality semen, a higher prevalence of abnormal semen characteristics, and a higher odds ratio for abnormal semen parameters compared to men with ≤ 18% SDF. An ROC analysis provided predictive values for age and semen parameters to distinguish between men with SDF > 18% and men with ≤ 18% SDF. SDF was positively correlated with male age and teratozoospermia index but negatively with sperm concentration, total number of spermatozoa, sperm morphology, progressive motility, and vitality. Our study shows that 18% SDF has a predictive value for distinguishing between men with normal and abnormal semen characteristics. Men with >18% SDF have a higher risk for abnormal semen parameters, while age and obtained semen parameters have a predictive value for SDF. There is a relationship between SDF and conventional sperm characteristics, and thus, SDF can be incorporated into male fertility assessment.

Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size.

The sperm chromatin structure assay is a method for assessment of sperm DNA fragmentation, a parameter reported to be negatively related to field fertility in several mammal species. This method calculates a DNA fragmentation index (DFI) whose high values indicate abnormal chromatin structure. In this study, running from March 2010 until June 2017, the aim was to assess sperm DFI in stored liquid extended semen from two different pig breeds, Norwegian Landrace (NL; n=693) and Norwegian Duroc (ND; n=655), and to evaluate the influence on total number of piglets born (TNB). There was a significantly higher median DFI (p<0.0001) in ejaculates from the 478 ND boars compared to the 452 NL boars. Data from 19,496 NL litters and 3,877 ND litters of the same boars were retrieved. For either breed, sow herd (p<0.0001), parity (p<0.05) and DFI (p<0.05) showed significant effects on TNB. The DFI was negatively correlated to TNB in both breeds. The boars with the 5% lowest TNB had a least square means DFI of 3.05% and 2.24% in NL and ND, respectively, compared to 1.67% and 1.23% for the boars with the 5% highest TNB (p<0.01). The DFI and the motility of the same semen samples were negatively correlated (p<0.0001), and the high and low TNB groups showed significant differences in motility. However, this difference could not be used for practical prediction of TNB group (92.1% vs. 89.7%; p=0.0038 and 92.3% vs. 89.5%; p=0.018; NL and ND, respectively). In conclusion, our results indicate that sperm DNA integrity in semen with good motility and morphology may be an additional prediction parameter for fertility in pigs.

A treatment algorithm for couples with unexplained infertility based on sperm chromatin assessment

ObjectiveTo design a reproductive treatment algorithm based on the sperm DNA fragmentation (SDF) for couples with unexplained infertility following a poor intrauterine insemination (IUI) outcome.DesignCouples that failed IUI with no apparent reproductive issue in both partners were allocated to diverse reproductive treatments on the basis of SDF.SettingReproductive medical center in an academic setting.Patient(s)Over 4 years, couples with an unexpected poor IUI outcome and no apparent female or male partner reproductive issues were recruited.Intervention(s)IUI, IVF, and ICSI were performed in the standard fashion following sperm SDF assays.Main outcomes measure(s)Fertilization rate, implantation rate, pregnancy characteristics, and delivery rates.Result(s)A total of 354 couples with unexplained infertility and normal semen parameters underwent 1133 IUI cycles. Clinical pregnancy rate (CPR) with IUI at our center in an age-matched cohort is 23.9% while the study cohort had 1.8%. Following SDF assessment, couples with failed IUI attempts but normal SDF (SCSA 9.8 ± 4.6%; TUNEL 11.8 ± 6.2%) underwent IVF with a CPR of 12.7%; those with abnormal SDF underwent ICSI with ejaculated spermatozoa, resulting in a CPR of 18.7%. This group included couples with normal SDF that had failed IVF. Couples with abnormal SDF that failed ICSI with ejaculated spermatozoa achieved a CPR of 31.0% with surgically retrieved spermatozoa.Conclusion(s)Couples with unexplained infertility that present with unexpectedly poor IUI outcomes can be funneled into a treatment algorithm guided by the integrity of the sperm genome for higher chances of pregnancy using an alternate method of insemination.