Sperm DNA Integrity Testing Research Articles

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.

Correlation analysis of age and MTHFR C677T polymorphism with sperm motility and sperm DNA integrity.

Several factors, including age and polymorphisms in genes such as MTHFR, affect sperm quality. However, the relationship between MTHFR C677T polymorphism and infertility remains unclear. Hence, the purpose of this study was to investigate the effects of age and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on the forward progressive sperm motility and sperm DNA integrity. For this purpose, a total of 845 men were recruited from the hospital's infertility clinic for semen analysis, sperm DNA integrity testing, and MTHFR C677T genotyping. The participants were grouped by age (<30 years, 30-35 years, >35 years) to determine the relationship between age and the progressive motility of sperms (PR%) and sperm DNA integrity. The effect of MTHFR C677T polymorphism on sperm motility and sperm DNA integrity was also analyzed in all the age groups. Results showed that PR% and DNA fragmentation index (DFI) differed significantly among the groups (P<0.001). The correlation analysis showed that age was positively correlated with DFI and negatively correlated with PR%. DFI correlated negatively with PR% (P <0.01). When the age groups were classified based on the C677T genotype, we observed that PR% and DFI did not differ significantly between different genotypes within the same age range (P>0.05). The conclusion was that age exerted a negative effect on sperm motility and sperm DNA integrity. Age correlated positively with DFI and negatively with PR%. MTHFR C677T polymorphism did not affect forward motile sperm count and sperm DNA integrity. Our observations will be useful for fertility guidance and MTHFR genotype interpretation in the clinic for couples of childbearing age.

High-throughput sperm DNA analysis at the single-cell and population levels.

Clinical semen quality assessment is critical to the treatment of infertility. Sperm DNA integrity testing provides critical information that can steer treatment and influence outcomes and offspring health. Flow cytometry is the gold standard approach to assess DNA integrity, but it is not commonly applied at the clinical level. The sperm chromatin dispersion (SCD) assay provides a simpler and cheaper alternative. However, SCD is low-throughput and non-quantitative - sperm assessment is serial, manual and suffers inter- and intra-observer variations. Here, an automated SCD analysis method is presented that enables quantitative sperm DNA quality assessment at the single-cell and population levels. Levering automated optical microscopy and a chromatin diffusion-based analysis, a sample of thousands of sperm that would otherwise require 5 hours is assessed in under 10 minutes - a clinically viable workflow. The sperm DNA diffusion coefficient (DDNA) measurement correlates (R2 = 0.96) with DNA fragmentation index (DFI) from the cytometry-based sperm chromatin structure assay (SCSA). The automated measurement of population-level sperm DNA fragmentation (% sDF) prevents inter-observer variations and shows a good agreement with the SCSA % DFI (R2 = 0.98). This automated approach standardizes and accelerates SCD-based sperm DNA analysis, enabling the clinical application of sperm DNA integrity assessment.

Technical Evaluation of Commercial Sperm DFI Quality Control Products in SCSA Testing.

With the increase in population and the advancement of medicine, people even more hope that their offspring will be healthier. DNA fragmentation rate is currently one of the more common indicators for evaluating sperm fertility and predicting the outcome of pregnancy. In order to evaluate the technical performance and application value of commercial sperm DFI (DNA fragmentation index) quality control products in the flow cytometry sperm chromatin structure analysis (SCSA), this paper uses flow cytometry to test commercial sperm DFI quality control products (Celula) and laboratory routine self-made DFI quality control products. In this paper, the quality control of new commercial sperm DFI and laboratory-made quality control were compared and tested for 30 consecutive days. And this paper monitors the response of commercial quality control products to the interference of key reagent parameters. This paper compares the stability of the test results of two quality control products and their sensitivity to the interference of key performance parameters of the detection reagent. Experimental results show that commercial sperm DFI quality control products can simulate sperm DNA damage to achieve accurate detection of DNA integrity. The stability of commercial sperm DFI quality control (CV = 2.47%) is better than that of laboratory-made quality control (CV = 11.22%). The new commercial sperm DFI quality control product can sensitively detect changes in the concentration of acidified solution and staining solution at the same time. It can effectively control the quality of detection reagents and experimental procedures. The new commercial sperm DFI quality control product can effectively control sperm DNA integrity testing. It can be used as an external quality control product for quality control of test results to ensure that more accurate test results are provided to the clinic.

Sperm DNA fragmentation and male fertility: a retrospective study of 5114 men attending a reproductive center.

The sperm DNA fragmentation index (DFI) was quantitatively measured and its relationship with age, semen quality, and infertility conditions was investigated. Semen routine test and sperm DFI were performed in 2760 infertile male and 2354 male whose spouse experienced at least one unexplained miscarriage to analyze the correlation between sperm DNA damage, semen routine parameters, and age. Sperm DFI was significantly lower from patients whose wife experienced unexplained miscarriage compared to infertility males (p = 0.000). An inverse correlation between sperm DFI and sperm progressive motility was observed (rs = - 0.465, p = 0.000) and sperm DFI was positively correlated with age (rs = 0.255, p = 0.000). However, the correlation between sperm DFI and sperm concentration, semen volume, total sperm count, and motile sperm count were not proved. Sperm DFI is an important indicator for evaluating the quality of semen. Sperm DNA integrity testing is preferentially recommended to those who have decreased sperm progressive motility, especially older men. An integrative analysis of sperm DFI, sperm progressive motility, age, and infertility conditions can provide a more comprehensive assessment of male fertility.

Sperm DNA Integrity and Male Fertility in Farm Animals: A Review.

The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.

Effect of whey protein supplementation on sperm quality and fertility in male mice

Protein supplements are a billion-dollar industry and the intake of these supplements is increasing, especially among young men. However, little is known about whether consumption of these products affects the reproductive health. The aim of this study was to assess the effect of whey protein supplementation on the sperm quality and reproductive health of male mice. A total of 48 male NMRI mice were fed with either plain tap water or a high dose of whey protein (Whey100, BodyLab) supplemented in the drinking water for 3 months. Mice was individually housed with two female mice for five days and reproductive parameters were assessed. DNA fragmentation index (DFI) was assessed at 0 h and 4 h of in vitro incubation using a sperm DNA integrity test (SDI®-test). No significant differences were detected between the groups in the epididymal sperm count, sperm motility, DFI, oxidation-reduction potential (ORP), serum testosterone, body and seminal vesicles weights, relative testis and epididymal weights, testicular morphology, number of impregnated females, or litter size. No correlation was found between ORP and DFI. These results suggest that the highest recommended human dose of whey protein supplementation do not significantly impair the sperm quality and fertility in male mice.

Interaction between serum levels of Anti-Mullerian Hormone and the degree of sperm DNA fragmentation measured by sperm chromatin structure assay can be a predictor for the outcome of standard in vitro fertilization.

Serum levels of Anti-Mullerian Hormone (AMH) have been shown to be biomarker for prediction of the quantitative aspects of ovarian reserve. On the male side, sperm chromatin structure assay (SCSA) DNA fragmentation index (DFI) has been demonstrated to be an important predictor of outcomes in standard IVF procedures but to less degree in intracytoplasmic sperm injection procedures (ICSI). The purpose of this study was to investigate whether the combination of female AMH serum levels and sperm DFI adds to prediction of the outcome of assisted reproduction. A total of 352 couples was included (ICSI-148: IVF-204) A venous blood sample was drawn for AMH analysis before IVF/ICSI treatment. DFI was measured in the ejaculate used for assisted reproduction. Regression models for the following odds ratio calculations were constructed: for obtaining at least one Good Quality Embryo; for live birth in all procedures; for pregnancy in procedures where embryo transfer was performed; for miscarriage. For DFI increase by 10 percentage points (not increased DFI as reference) odds ratio for Good Quality Embryo was statistically significantly lower when AMH was at lower quartile (AMH <12 pmol/L; OR = 0.29, 95% CI: 0.14–0.59,) but not when AMH was at upper quartile (AMH ≥ 36 pmol/L; OR = 0.95, 95% CI: 0.43–2.13,). The marginal effect of an increase in DFI by 10 percentage points was statistically significant only when AMH < 25.2 pmol/L. Similar results were obtained as considers live birth following standard IVF. No interactions were seen for standard IVF in relation to the risk of miscarriage and for any of the outcomes when ICSI was used as method of treatment. We conclude that the impact of high DFI on the outcome of standard IVF is most pronounced if the female partner has relatively low AMH levels. This finding may help in defining the role of sperm DNA integrity testing in management of infertile couples. It may also explain some of the heterogeneity in results of studies focusing on predictive value of DFI measurements in assisted reproduction.

Effect of nordihydroguaiaretic acid on spermatogenesis and fertility in rats.

Nordihydroguaiaretic acid (NDGA) is a naturally occurring lignan with potent antioxidant activity. Currently, it is in clinical trials as anticancer agent. As there is no earlier report on the effect of NDGA on spermatogenesis and fertility, this study was designed to investigate this aspect. Administration of NDGA to rats for 60days produced degenerative changes in testis but had no effect on sperm DNA integrity test and androgen receptor expression. Ultrastructural studies revealed loss of integrity of cells in seminiferous tubules, vacuolation and presence of apoptotic bodies. Derangement of the outer dense fibres was noted in some sperm flagella. Acrosome formation appears to be normal. About 13.7% of epididymal spermatozoa had deformations like short tail or rounded head. This may explain the lower fertility index in NDGA-treated group. No external deformations in newborns were noted. In conclusion, NDGA may have adverse effects on spermatogenesis.

Sperm DNA integrity testing-a valuable clinical tool.

For long, spermatozoa were only considered as vectors carrying the paternal genetic material to the oocyte. However, over the recent years, the important contribution of the spermatozoa to embryogenesis has become better understood. It is now evident that sperm cells are highly differentiated, polarized and specialized, containing the paternal constituents required for fertilization and normal embryonic development.

Sperm DNA integrity testing: a valuable addition to the tool box of infertility clinicians.

ART (assisted reproductive technologies) clinicians worldwide should take note of the recent article by Agarwal et al . (1), which provides both a pertinent and timely recommendation; the combined evaluation of oocyte quality and sperm DNA integrity represents a critical step towards achieving reproductive success, whether via natural conception or following ART.

Finding the fit: sperm DNA integrity testing for male infertility.

Complementing the medical history and physical examination, the semen analysis has been an essential laboratory test for the evaluation of male fertility for over 50 years. However, the concept that fertility is defined by threshold values of semen parameters is fundamentally flawed (1). At best, the semen analysis suggests that the probability of achieving fertility is lower than normal (2). In addition, the definition of “normal” semen parameters is constantly challenged: witness the five editions of WHO manuals in which fertile semen parameters have been redefined over 36 years (3). Lastly, wide intra-individual variation in semen quality (2), and seasonal (4) and geographic variations (5) further complicate the potential of the semen analysis to predict fertility. So, something better is needed to help us determine male fertility potential.

A few more fragments: putting sperm DNA integrity testing into clinical practice.

While routine semen analysis has been the cornerstone of the male fertility evaluation, we know that it is far from a perfect predictor of fertility, and there will be some couples who struggle to obtain a live birth despite the male having a normal semen analysis. Indeed, the World Health Organization semen analysis normal reference ranges were based on samples from fertile men (1). The LIFE study, a prospective observational cohort study of 501 couples, which looked at the relationship between routine semen parameters and time to pregnancy, found that on multivariate analysis, none of the routine semen parameters (volume, concentration, motility or morphology) were associated with time to pregnancy. Only male age and female body mass index (BMI) were associated with the time to pregnancy (2). From both this data and others, we know that the standard semen analysis is not a perfect predictor for male infertility (3). These limitations have led to the investigation of sperm DNA fragmentation as an adjunctive test to guide the management of the sub-fertile couple.

Pros and cons of sperm DNA fragmentation testing: weighing the evidence.

Conventional semen parameters are poor predictors of reproductive outcomes, and do not reliably discriminate between fertile and infertile men (1,2). A growing body of evidence suggests that markers of sperm DNA integrity may help differentiate fertile from infertile men, but the clinical value of sperm DNA fragmentation testing remains a matter of debate (3). Although guidelines and best practice statements published by the American Urological Association (AUA), European Association of Urology (EAU) and the American Society of Reproductive Medicine (ASRM) all acknowledge the potential contribution of sperm DNA fragmentation to male factor infertility, they do not recommend the routine use of sperm DNA integrity tests in the evaluation and treatment of infertile couples (4-6).

Relationship between phospholipase C-zeta, semen parameters, and chromatin status

ABSTRACTThe need for additional tests to complement basic sperm analysis in clinics is well appreciated. In this regard, a number of tests such as sperm DNA integrity test as a tool in diagnosis and treatment of infertility are suggested. But recent studies have focused on main sperm factors involved in oocyte activation such as phospholipase C-zeta (PLCζ) that initiate intracellular Ca2+ signaling and embryogenesis. Therefore, this study aimed to investigate the relationship between PLCζ, basic semen parameters, sperm DNA fragmentation (SDF), and protamine deficiency in men with normal (n=32) and abnormal (n=23) semen parameters. Unlike SDF and protamine deficiency, as negative factors related to fertility, the mean value of PLCζ as positive factor related to infertility was significantly lower in men with abnormal semen parameters compared to men with normal semen parameters. Significant correlations were also observed between sperm concentration, motility, and abnormal morphology with the percentage of PLCζ positive spermatozoa. In addition, logistic regression analysis revealed that sperm morphology is more predictive than sperm motility and concentration for PLCζ presence. In addition, a statistically significant negative relationship was observed between the percentage of PLCζ positive spermatozoa and SDF. These findings suggested during ICSI, selection of sperm based on morphology has a profound effect on its ability to induce oocyte activation based on the likelihood of PLCζ expression. Therefore, assessment of PLCζ as an index for fertilization potential of a semen sample in men with severe teratozoospermia may define individuals who are candidates for artificial oocyte activation (AOA) and may avoid failed fertilization post ICSI.

Sperm DNA Integrity Test and Assisted Reproductive Technology (Art) Outcome

Summary The objective of the study was to investigate the influence of sperm DNA fragmentation index (DFI) by DNA integrity test on pregnancy outcome and pregnancy loss after assisted reproductive technology (ART) procedure: autologous intracytoplasmic sperm injection (ICSI), donation eggs ICSI, and intrauterine insemination (IUI). We investigated men from 531 couples undergoing autologous ICSI procedure (n=416), from couples undergoing donation eggs procedure (n=39) and IUI (n=76). We performed the following interventions: semen analysis, DNA integrity test, embryo scoring by Gardner and Schoolcraft grading system (1999). The study showed no statistically significant differences between the group regarding pregnancy rate (χ2=0.55; p&gt;0.05; OR=1.25, 95% Cl 1.23-1.46; p&gt;0.05). However, with increased levels of DFI, the number of pregnancy losses became higher (including biochemical pregnancies and spontaneous abortions) at OR=5.65 (95% Cl 4.32-7.11; p=0.05). We examined the percentage of grade I blastocysts (by Gardner and Schoolcraft, 1999) before donation eggs embryo transfer and found a statistically significant correlation with both the DFI (χ2=7.80; p&lt;0.05) and sperm morphology (χ2=6.14; p&lt;0.05). Analysis of the relationship between DFI and IUI output (clinical pregnancy, miscarriage) revealed significant correlations in both directions: between DFI and pregnancy rate after IUI (χ2=6.29; p&lt;0.05) and between the DFI and pregnancy development after IUI (χ2=6.87; p&lt;0.05). The three group categories (autologous, heterologous ICSI procedures and IUI) studied showed that sperm samples with DFI&gt;27% were associated with increased riskofearlypregnancyloss. Men with infertility should undergo DNA fragmentation assay in addition to the standard semen analysis. When DFI exceeds 27%, ICSI should be a method of choice, even in cases where the conventional parameters of semen analysis tests are normal.

PP-002 - Sperm DNA integrity test and ART outcome

PP-002 - Sperm DNA integrity test and ART outcome

Study of Evaluation of Causes of Male Infertility at a Tertiary Care Hospital

ABSTRACTIntroductionWorldwide 580 million males experience infertility at some point of time in their life; of these, 372 million reside in low- and middle-income countries. Male subfertility is one of the most rapidly growing fields in medicine, with dramatic advances and treatment. If a male infertility factor is present, it is almost always defined by the finding of an abnormal semen analysis, although other male factors may play a role even when the semen analysis is normal.AimsTo classify the causes of male infertility at pre-testicular, testicular, and post-testicular levels.Materials and methodsThis study was conducted at the Department of Obstetrics and Gynecology in collaboration with the Department of Radio Diagnosis, Institute of Postgraduate Medical Education and Research, Kolkata.ResultsPre-testicular cause was seen in 7% of patients. Erectile dysfunction is the commonest. In testicular causes, varicocele is the most common (29.6%), followed by idiopathic (25.9%) and trauma in 14.8%, and other causes, such as torsion, infective orchitis, and cryptorchism show a common incidence of 7.4%. In post-testicular, varity in 51.8% is due to ejaculatory duct obstruction and accessory gland dysfunction, and acquired hernia surgeries have a common incidence of 14.81%. The least common cause is epididymal asthenozoospermia.ConclusionIn male infertility evaluation history and clinical examination, semen analysis is the common tool for all groups of patients. Pre-testicular male infertile is mainly due to consequences of primary or secondary dysfunction of endocrine or exocrine glands influencing the male reproductive axis. Testicular group of male infertility are due to varity of genetic, congenital, and acquired insults to the proper testicular function; post-testicular cause of infertility is due to pathology in the pathway of sperm passage; this is influenced by congenital and acquired defects and diseases respectively. The idiopathic group of patients needs further evaluation by use of advanced seminal tests like immunological tests, semen culture, special staining of the spermatozoa, sperm DNA integrity tests, appropriate genetic evaluation and. Evaluation of ultrastructural abnormalities of spermatozoa, for detection of defects in outer dense fibers, microtubules, mitochondria, connecting piece, and acrosome.How to cite this articleUmashankar KM, Mukherjee J, Banerjee SN, Cristy R, Seal BN, Drakshyani CL. Study of Evaluation of Causes of Male Infertility at a Tertiary Care Hospital. J South Asian Feder Obst Gynae 2016;8(2):113-122.

Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage.

Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

Increased DNA Fragmentation in Patients with Infertility in Dakar (Senegal)

The presence of DNA fragmentation in semen is associated with poor results in ART (Assisted Re-productive Technology). This has led to the introduction of sperm DNA integrity testing in the clinical assessment of male infertility. The TUNEL assay is one of the most commonly used tests to measure DNA fragmentation in research studies, but it is now being optimized for routine clinical use. So, the objectives of our study were to install the TUNEL assay in our laboratory and to use it to assess the degree of DNA fragmentation in our patients dealing with couple infertility. Forty seven (47) and 25 samples from patients followed up for infertility and known fertile men were analyzed between August 2013 and July 2015 respectively. The sperm samples were evaluated according to the World Health Organization Guidelines. Sperm DNA damage was assessed by the TUNEL assay, with a standardized protocol that we adapted for reading using fluorescence microscopy instead of flow cytometry. The Sperm DNA Fragmentation Index was significantly higher in patients with infertility compared to the controls (22.2% ± 5.6% vs. 16.7% ± 0.7%) with a p < 0.05. This Index was also high in patients with varicocele compared to the control group (22.8% ± 9.2 vs. 16.7% ± 0.7%) (p < 0.01). After establishing the ROC curve (Receiver Operating Characteristics curve), we found a 15% threshold of DNA fragmentation, separating the patients with infertility from the control group. In our study, a 15% threshold positivity of DNA fragmentation using the TUNEL assay was highly associated with infertility. The results indicate that high level DNA fragmentation is associated with infertility.