Sperm DNA Fragmentation Assay Research Articles

#227 : Is Sperm DNA Fragmentation Assay a Useful Predictor in ART Outcomes?

Background and Aims: The Sperm DNA Fragmentation (SDF) assay has been a promising tool for evaluation of male fertility in assisted reproductive technology (ART). Our primary aim is to assess the impact of SDF levels (<15% and ≥15%) on the laboratory and clinical outcomes; and secondary aim to evaluate those outcomes among 3 ART subgroups namely IVF–ICSI Split, IVF and ICSI cycles. Method: This retrospective cohort study was conducted on 1148 males who presented along with their partners for infertility management at the PIVET Medical Centre between 2013 and 2022 and had their SDF assay performed by Halosperm. They undertook 1600 ART cycles with outcomes divided into 2 groups based on SDF levels <15% and ≥15%. Categorical data were analyzed by Chi-squared or Fisher’s exact test. Results: The overall rates of fertilization, blastocyst development, good–quality blastocyst, clinical pregnancy, miscarriage and livebirth rates were comparable between the <15% and ≥15% SDF groups. Nevertheless, when comparing these outcomes according to female age groups, the clinical and livebirth rates were significantly higher in women <35 years in the SDF <15% (44.1%, p=0.04; 38.6%, p<0.01). Among the 3 ART modalities, the fertilization rates were optimised in both SDF groups by ICSI (75.5%, p<0.0001; 74.1%, p<0.0001), however ICSI also has the lowest blastocyst rate in both SDF groups (p<0.0001 and p<0.001). The IVF-ICSI Split modality generated the highest rate of good-quality blastocysts in both SDF groups <15% (44.9%, p<0.0001) and ≥15% (50.0%, p<0.001). Although the clinical pregnancies were not highest in the IVF modality, it carried the lowest miscarriage rate resulting in the highest live birth rate in SDF <15% group where the women were aged <40 years. Conclusion: The SDF assay is predictive of clinical and livebirth rates for women aged <35 years. IVF is an effective modality among women aged <40 years where the SDF level is <15%.

#108 : Methods of Sperm DNA Fragmentation (SDF) Testing and Interpretation: Results from the Global Andrology Forum Survey

Background and Aims: Sperm DNA fragmentation (SDF) is caused by apoptosis, oxidative stress or incomplete chromatin maturation and can result in male infertility and poor reproductive outcomes. Despite growing trends, there are no specific recommendations on specific diagnostic tests used, assay cut-offs or abstinence periods prior to SDF testing. Therefore, we conducted a global survey to evaluate the approaches used to diagnose SDF, towards a consensus in the field of SDF research and clinical management. Method: The survey collected cross-sectional global data from 55 countries following the CHERRIES protocol. 436 respondents included clinicians, researchers and reproductive biologists from academic, public and private health sectors, all of whom utilize SDF testing in their routine patient care and practice (Fig. 1). Results: Survey results showed that TUNEL (28.6%), SCSA (24.1%), SCD (19.1%) and Comet (5.3%) were most frequently used to diagnose SD (Fig. 2A). Assays were chosen primarily due to their availability (70.0%), cost (44.05%) and accuracy (35.48%). Published evidence on assays (25.71%), training of staff (25.48%), cost to patients (21.67%), assay time (18.81%), ease of interpretation (18.33%) and performance (17.38%) were taken lesser into consideration when selecting SDF diagnostic tests (Fig. 2B). Assay cut-offs of “30% or higher” (33.7%) were most frequently used to diagnose SDF (Fig. 2C). Finally, an abstinence period of 3-5 days was mostly recommended (53.6%; Fig. 2D). The results of this survey provide valuable data on SDF assays used in clinical practice as well as factors affecting assay choices such as availability of resources, personnel and complexity in assay processing. As decreased abstinence period is associated with lower SDF, testing is recommended with an abstinence period of less than five days. We observed that there was a lack of standard cut-off values, and every laboratory should establish its own reference values based on predictive values for fertility outcomes. Conclusion: We present new data associated with SDF testing in real-world practice, on a global scale. The trends and conclusions derived from the study are recommended for inclusion into the 6[Formula: see text] Edition WHO Manual, towards optimized patient care.

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.

Recurrent pregnancy loss: a male crucial factor-A systematic review and meta-analysis.

Recurrent pregnancy loss (RPL), defined as two or more failed clinical pregnancies, affects 1%-3% of couples trying to conceive. Nowadays up to 50% of cases remain idiopathic. In this context, paternal factors evaluation is still very limited. The aim is to address the topic of the male factor in RPL with a broad approach, analyzing collectively data on sperm DNA fragmentation (SDF) and semen parameters. We systematically searched in Pubmed/MEDLINE and Google Scholar from inception to February 2023. A protocol has been registered on PROSPERO (ID number CRD42022278616). PRISMA guidelines were followed. Pooled results from 20 studies revealed a higher DNA fragmentation rate in the RPL group compared to controls (mean difference [MD] 9.21, 95% CI 5.58-12.85, p<0.00001, I2 98%). Age, body mass index (BMI), smoking, and alcohol intake were not associated with DNA fragmentation. Subgroup analysis by different SDF assays (TUNEL and COMET at a neutral pH vs. indirect assessment with other assays) and ethnicity did not highlight different results (p = 0.25 and 0.44). Results pooled from 25 studies showed a significant difference comparing RPL and control groups regarding ejaculation volume (MD -0.24, 95% CI -0.43; -0.06, p 0.01, I2 66%), total sperm number (MD -10.03, 95% CI -14.65; -5.41, p<0.0001, I2 76%), total sperm motility (MD -11.20, 95% CI -16.15; -6.25, p<0.0001, I2 96%), progressive sperm motility (MD -7.34, 95% CI -10.87; -3.80, p<0.0001, I2 97%), and normal sperm morphology (MD -5.99, 95% CI -9.08; -2.90, p 0.0001, I2 98%). A sub-analysis revealed that Asian and Africans, but not white-European RPL men had lower progressive sperm motility compared to controls. In conclusion, current review and meta-analysis findings suggested that SDF and some specific semen parameters were associated with RPL in a multi-ethnic evaluation. This effort opens future direction on a growing awareness of, first, how the male factor plays a key role and, second, how appropriate would be to establish a direct dialogue between the gynecologist and the urologist. We performed a systematic review and meta-analysis on the male component of RPL. We found that sperm DNA fragmentation and some specific sperm parameters are significantly associated with RPL.

DSC2 suppresses the growth of gastric cancer through the inhibition of nuclear translocation of γ-catenin and PTEN/PI3K/AKT signaling pathway.

Globally, gastric cancer (GC) is still a major leading cause of cancer-associated deaths. Downregulated desmocollin2 (DSC2) is considered to be closely related to tumor progression. However, the underlying mechanisms of DSC2 in GC progression require further exploration. We initially constructed different GC cells based on DSC2 contents, established the mouse tumor xenografts, and subsequently performed clonal formation, MTT, Caspase-3 activity, and sperm DNA fragmentation assays to detect the functions of DSC2 in GC growth. Subsequently, we performed western blot, Co-IP, and immunofluorescence assays to investigate the underlying mechanisms through pretreatment with PI3K inhibitor, LY294002, and its activator, recombinant human insulin-like growth factor I (IGF1). DSC2 could significantly inhibit the viability of GC cells at both in vitro and in vivo levels. The underlying mechanism may be that DSC2 binds the γ-catenin to decrease its nuclear level, thereby downregulating the anti-apoptotic factor BCL-2 expression and upregulating the pro-apoptotic factor P53 expression, which adjusts the PTEN/PI3K/AKT signaling pathway to promote the cancer cell apoptosis. Our finding suggests that DSC2 might be a potential therapeutic target for the treatment of cancers, most especially GC.

The Sperm DNA Fragmentation Assay with SDF Level Less Than 15% Provides a Useful Prediction for Clinical Pregnancy and Live Birth for Women Aged under 40 Years.

This retrospective cohort study was conducted on 1148 males who presented along with their partners for infertility management at the PIVET Medical Centre between 2013 and 2022 and had a sperm DNA fragmentation (SDF) assay performed by Halosperm, thereafter participating in 1600 assisted reproductive technology (ART) cycles utilising one of three modalities, namely, IVF-Only, ICSI-Only or IVF-ICSI Split cycles. The outcomes from the ART cycles were then analysed as two groups based on SDF levels <15% and ≥15%. The study showed the unadjusted fertilization rates were not different between the groups, neither across the four female age ranges. However, when the fertilization rates were adjusted for the mature oocytes (metaphase-II oocytes), there was a highly significant difference in fertilization rates in favour of the group with SDF levels < 15% where the women were in the younger age grouping of <35 years (78.4% vs. 73.0%; p < 0.0001). Overall, there was no difference in the rates of blastocyst development nor clinical pregnancy rates between the two SDF groups, but there was a significantly higher pregnancy rate for the younger women (<35 years) with the group of SDF level < 15% (44.1% vs. 37.4%; p = 0.04). Similarly, there was no difference in the miscarriage rates overall with respect to SDF groups, and no clear picture could be deciphered among the women's age groups. With respect to cumulative live births, this reflected the pregnancy rates with no overall difference between the two SDF groups, but there was a significantly higher cumulative live birth rate for women <35 years where the SDF level was <15% (38.6% vs. 28.6%; p < 0.01). Among the three modalities, the highest cumulative live birth rate occurred within the group with SDF level < 15%, being highest with the IVF mode, particularly for women aged <40 years (43.0% vs. 37.7% for IVF-ICSI Split and 27.9% for ICSI; p = 0.0002), noting that the IVF case numbers were disproportionately low.

Does folate deficiency alter sperm parameters in infertile males?

Introduction: There is not enough evidence regarding the influence of folate deficiency in the etiology of idiopathic male infertility. Objectives: The objective of the current study was to examine whether there is an association between serum levels of folate and sperm parameters among infertile males. Patients and Methods: This cross-sectional study was undertaken on a sample of infertile males referring to an infertility treatment clinic in Isfahan province, Iran. Semen parameters, comprising sperm concentration, progressive/total motility, morphology, semen leukocyte, agglutination, and vitality were assessed based on standard protocols. The sperm DNA fragmentation assay (SDFA) and sperm chromatin maturity assay (SCMA) were also assessed by the sperm chromatin dispersion and aniline-blue tests respectively. Results: In general, 70 infertile males including 46 with sufficient serum folate level and 24 with insufficient serum folate level were examined. Subjects with sufficient serum folate level had a significantly higher mean of sperm concentration, vitality, progressive motility, total motility, SDFA, and SCMA (P&lt;0.05). Higher serum folate level was also associated with lower risk of abnormal sperm agglutination (OR: 0.09, 95% CI: 0.01-0.65), and morphology (OR: 0.12, 95% CI: (0.02-0.60) after adjustment for potential confounders. Conclusion: The present study found a significant association between serum folate level and infertility among Iranian infertile males. However, prospective large-scale population-based research is required to confirm the finding.

Ceratonia siliqua L improved cryodamage of asthenozoospermic specimens: An experimental study.

Sperm freezing is an important procedure in assisted reproductive technology. Freezing results in physical and chemical changes in the sperm. Ceratonia siliqua L (C.siliqua) is a tree that has antioxidant properties. This study aimed to investigate the effect of different concentrations of C.siliqua in a freezing medium on semen parameters, and some biochemical parameters in asthenozoospermic specimens. Forty asthenozoospermic specimens (semen specimens with motility 32%) were obtained from men aged between 20-40 yr according to the World Health Organization criteria. Each sample was divided into 6 groups: I) fresh, II) control, III) 5, IV) 10, V) 20, and VI) 30 µg/ml C.siliqua extract were added to a freezing medium respectively. Then sperm parameters, malondialdehyde, total antioxidant capacity, reactive oxygen species, and sperm DNA assay were evaluated using related protocols after thawing. Data analysis shows that sperm parameter, and total antioxidant capacity level increased at a concentration of 20 µg/ml of C.siliqua extract compared to the other concentrations of C.siliqua extract after cryopreservation and thawing (p 0.001). Also, the sperm DNA fragmentation assay, reactive oxygen species, and malondialdehyde levels were significantly reduced by adding 20 µg/ml of C.siliqua extract to the sperm freezing medium compared to the other treated groups after cryopreservation (p 0.001). C.siliqua extract significantly improved sperm parameters after cryopreservation and thawing in asthenozoospermic specimens, and the greatest impact was observed at the 20 µg/ml C.siliqua L extract concentration (p 0.001).

Technical Aspects and Clinical Limitations of Sperm DNA Fragmentation Testing in Male Infertility: A Global Survey, Current Guidelines, and Expert Recommendations.

Sperm DNA fragmentation (SDF) is a functional sperm abnormality that can impact reproductive potential, for which four assays have been described in the recently published sixth edition of the WHO laboratory manual for the examination and processing of human semen. The purpose of this study was to examine the global practices related to the use of SDF assays and investigate the barriers and limitations that clinicians face in incorporating these tests into their practice. Clinicians managing male infertility were invited to complete an online survey on practices related to SDF diagnostic and treatment approaches. Their responses related to the technical aspects of SDF testing, current professional society guidelines, and the literature were used to generate expert recommendations via the Delphi method. Finally, challenges related to SDF that the clinicians encounter in their daily practice were captured. The survey was completed by 436 reproductive clinicians. Overall, terminal deoxynucleotidyl transferase deoxyuridine triphosphate Nick-End Labeling (TUNEL) is the most commonly used assay chosen by 28.6%, followed by the sperm chromatin structure assay (24.1%), and the sperm chromatin dispersion (19.1%). The choice of the assay was largely influenced by availability (70% of respondents). A threshold of 30% was the most selected cut-off value for elevated SDF by 33.7% of clinicians. Of respondents, 53.6% recommend SDF testing after 3 to 5 days of abstinence. Although 75.3% believe SDF testing can provide an explanation for many unknown causes of infertility, the main limiting factors selected by respondents are a lack of professional society guideline recommendations (62.7%) and an absence of globally accepted references for SDF interpretation (50.3%). This study represents the largest global survey on the technical aspects of SDF testing as well as the barriers encountered by clinicians. Unified global recommendations regarding clinician implementation and standard laboratory interpretation of SDF testing are crucial.

Association between Seminal Oxidation-Reduction Potential and Sperm DNA Fragmentation-A Meta-Analysis.

Seminal oxidative stress and sperm DNA damage are potential etiologies of male factor infertility. The present study aims to evaluate the relationship between oxidation-reduction potential (ORP), a measure of oxidative stress, and sperm DNA fragmentation (SDF) by conducting a systematic review and meta-analysis of relevant clinical data. A literature search was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The COVIDENCE tool was used to screen and identify studies evaluating seminal ORP and SDF. Studies (n = 7) that measured seminal ORP and SDF of 3491 semen samples were included in the analysis. The fixed-effects model revealed a significant pooled correlation coefficient (r = 0.24; p < 0.001) between seminal ORP and SDF. Furthermore, subgroup analyses indicated that the pooled correlation coefficient between ORP and sperm chromatin dispersion (SCD) assay was less than other SDF assays (0.23 vs. 0.29). There was a moderate level of heterogeneity (I2 = 42.27%) among the studies, indicating a lack of publication bias. This is the first meta-analysis to reveal a positive correlation between seminal ORP and SDF. Furthermore, this study indicates the role of oxidative stress in the development of sperm DNA damage and thus warrants prospectively exploring the clinical value of these sperm function tests.

P-029 Sperm DNA fragmentation measured by novel sperm chromatin dispersion-based assay is associated with semen parameter and embryo quality after IVF

Abstract Study question To evaluate the association between sperm DNA fragmentation measured by two new SCD-based assay and sperm parameters as well as ART outcomes. Summary answer Sperm DNA fragmentation assessed by novel sperm DNA fragmentation assay is significantly negative correlated with semen parameters and embryo quality. What is known already Sperm DNA fragmentation (SDF) is one of the major defects in sperm chromatin. This may lead to male infertility, abnormal embryological development, and pregnancy loss. To date, SDF testing has emerged as a means of diagnosis in some medical institutions as an additional assessment for predicting fecundity and guiding treatment regimen. Assisted reproductive technology (ART) such as IVF and ICSI have played a pivotal role in the treatment of infertility over two decades. However, a parts of patients with high level of SDF underwent ART may still have poor clinical outcome. Study design, size, duration A total of 200 participants was included in this prospective cohort study. Data was collected from the reproductive medicine center from June 2020 through December 2021. Participants/materials, setting, methods This study included 200 infertile couples attending for assisted reproductive treatment. Semen sample collected on the day of fertilization was analyzed for SDF by using LensHooke® R10 (R10) and LensHooke® R11 (R11) kits. Semen analysis parameters, embryologic outcome, and clinical outcome after ART treatment were compared between groups with low-level SDF and high-level SDF. Main results and the role of chance The SDF assessed by R10 was negatively correlated with sperm total motility (Spearman's ρ=-0.24,P=0.0007). The SDF assessed by R11 was negatively correlated with sperm concentration (Spearman's ρ=-0.26, P = 0.0002), total motility (Spearman's ρ=-0.45, P &amp;lt; 0.0001), progressive motility (Spearman's ρ=-0.29, P &amp;lt; 0.0001), and normal morphology (Spearman's ρ=-0.43, P &amp;lt; 0.0001). Moreover, SDF obtained by the R10 in asthenozoospermic (26.9% ± 19.5%, p &amp;lt; 0.05) was significantly higher than that in normozoospermic samples (14.8% ± 8.9%). SDF obtained by the R11 in oligozoospermic (21.1% ± 17.9%,p&amp;lt;0.01), asthenozoospermic (25.3% ± 19.4%, p &amp;lt; 0.001), teratozoospermic (17% ± 13.2%, p &amp;lt; 0.01), and oligoasthenoteratozoospermic samples (35.6% ± 24.5%, p &amp;lt; 0.0001) were significantly higher than that in normozoospermic samples (7.9% ± 4.2%). In addition, there was significant difference in good embryo rate post IVF between the groups of low SDF (&amp;lt; 14%) and high SDF (≥ 14%), as measured by R11. Limitations, reasons for caution To focus on male factor-related infertility, the female partner with an age above 38 years old was not enrolled, however, other female factors were not precluded from this study. Wider implications of the findings Sperm DNA fragmentation has proven to be a valuable biomarker in male fertility evaluation, while its significance as a predictor of clinical outcomes following ART requires further investigation. Trial registration number CS2-20012

Relationship Among Traditional Semen Parameters, Sperm DNA Fragmentation, and Unexplained Recurrent Miscarriage: A Systematic Review and Meta-Analysis.

Several studies have explored the relationship among traditional semen parameters, sperm DNA fragmentation (SDF), and unexplained recurrent miscarriage (RM); however, the findings remain controversial. Hence, we conducted a meta-analysis to explore the relationship among traditional semen parameters, SDF, and unexplained RM. Multiple databases, including PubMed, Google Scholar, MEDLINE, Embase, Cochrane Library, Web of Science, and China National Knowledge Infrastructure (CNKI), were searched to identify relevant publications. From the eligible publications, data were extracted independently by two researchers. A total of 280 publications were identified using the search strategy. According to the inclusion/exclusion criteria, 19 publications were eligible. A total of 1182 couples with unexplained RM and 1231 couples without RM were included in this meta-analysis to assess the relationship among traditional semen parameters, SDF, and unexplained RM. Our results showed that couples with unexplained RM had significantly increased levels of SDF and significantly decreased levels of total motility and progressive motility compared with couples without RM, although significant differences were not observed in the semen volume, sperm concentration, and total sperm count between couples with and without RM. The SDF assay may be considered for inclusion in evaluations of couples with unexplained RM.

Comparison between SPATA18 and P53 Gene Expressions in The Sperm Cells Obtained from Normospermic and Asthenospermic Samples: A Case-Control Study.

Background:Improving sperm motility results in increasing the success of a treatment cycle. Recently, sperm RNA has been used for diagnostic purposes such as whole seminal fluid, sperm analysis, and sperm quality test in patients undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). SPATA18-P53 pathway is considered an essential pathway related to sperm mitochondria, which controls mitochondrial quality by eliminating its oxidative proteins. Oxidative stress may decrease sperm motility and affect sperm quality negatively due to an increase in P53 expression. SPATA18 protein is found in satellite fibers related to outer dense fibers in the middle piece of sperm. The downregulation of SPATA18 in the asthenospermia group can represent this gene’s critical function in sperm motility and fertility. The present study aimed to assess the relationship between SPATA18 and P53 gene expression in sperm cells obtained from normospermia and asthenospermia.Materials and Methods:In this case-control study, the quantitative real-time polymerase chain reaction (RT-PCR) technique was used to measure the SPATA18 and P53 gene expression level in sperm samples collected from 21 patients and 63 healthy individuals. Further, the sperm DNA fragmentation assay (SDFA) kit was applied to determine the relative apoptosis level in cells and evaluate the biochemical information related to the patients’ sperm samples. Furthermore, all the participants completed the consent form, and the ethics committee confirmed the study.Results:Based on the results, the P53 and SPATA18 gene expression levels in most of the samples, in which motility was less than 40%, increased and decreased (P≤0.001), respectively.Conclusion:The SPATA18 and P53 gene expression levels increased and decreased in the asthenospermic patients, respectively, compared to the control group. Thus, the P53 and SPATA18 expression levels can be used as an appropriate marker for diagnosing sperm motility in males.

Assessment of the Impact Induced by Different Incubation Time, Storage Time, Storage Medium and Thawing Methods on Sperm DNA Fragmentation Assay: A Before-After Study.

The sperm DNA fragmentation has been considered an important index in the field of male infertility. Our study aims to evaluate the impact of different factors, including incubation time, storage time, storage medium and method of thawing, on DNA fragmentation of semen samples. This study was designed as a before-after study in five experiments. Experiment 1 was conducted to assess the effect of storage time in liquid nitrogen on 15 semen samples. In experiment 2, DNA fragmentation was performed on 10 semen samples with different incubation times before freezing. In experiments 3, 4, two different storage media and thawing methods were applied respectively in two separate groups, each containing 30 samples and the DNA fragmentation index (DFI) was measured using the sperm chromatin structure assay method. Data were analysed using Stata version 11. There was a significant increase in sperm DNA fragmentation of samples stored in liquid nitrogen for 1 month. This increase occurred in the first 2 weeks. Furthermore, our results showed a significant increase in the DFI after 120 min of incubation at room temperature (RT) and also thawing in RT separately. It is better to use fresh samples to measure DNA fragmentation up to 2 h after ejaculation to achieve more accurate results. Furthermore, if sperm freezing is inevitable, the use of a water bath (37°C) to thaw will be the most appropriate option, as it can lead to less DNA damage.

Sperm DNA Fragmentation in Reproductive Medicine: A Review.

Approximately 15% of the world's couples suffer from infertility during their reproductive period of which the male factor is responsible for 50% of cases. Male factor infertility is multifactorial in origin, and sperm DNA fragmentation (SDF) has also been linked to male infertility including idiopathic male infertility. Some degree of controlled DNA nicking is essential for adequate DNA compaction, but excessive SDF is usually associated with reduced male fertility potential, reduced fertilisation, poor embryo quality, recurrent pregnancy loss and poor assisted reproductive techniques (ARTs) outcomes. Although semen analysis remains the gold standard for diagnosis of male factor infertility worldwide, its limitations motivated the search and the development of complementary tests of sperm function and integrity. SDF assay is an emerging diagnostic tool in infertile men, and several indications for SDF testing in infertile couples have also been proposed. The use of SDF in routine male infertility assessment is, however, still controversial. Furthermore, both direct and indirect SDF tests are now available. Hence, the present review was conducted to summarise the recent evidence of SDF, underlying mechanisms, clinical indications, diagnostic tests, as well as the role of SDF in male factor infertility, pregnancy and ART outcomes.

Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay.

Sperm DNA fragmentation (SDF) dynamic assays were piloted on 4 fresh ejaculates to examine the possible sperm toxicity of three common antibiotics, ciprofloxacin, doxycycline and ampicillin, incubated at a concentration estimated to be reached in semen in vivo, and 100×, for 24h. SDF was assessed in terms of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs). Low and high concentrations of ciprofloxacin and high concentration of doxycycline significantly increased the SDF rate, due to sperm containing SSBs. Ampicillin did not affect SDF dynamics at any dose. Based on these results, the effect of antibiotics on the global-SDF dynamics was further examined in 21 ejaculates assessed at 0, 4 and 6h. Ciprofloxacin increased the rate of SDF at the low concentration in 17 from 21subjects; the high concentration resulted in a stronger effect in all individuals. A significant increase in the rate of SDF in 17 ejaculates was also noted when spermatozoa were incubated with the high concentration of doxycycline. The dynamic SDF assay is a rapid and sensitive tool to evidence sperm toxicity. Ciprofloxacin should be avoided when it is necessary to preserve sperm quality for reproductive purposes and as additive in semen diluents.

P–004 Effect of varicocelectomy on sperm DNA fragmentation rates in infertile men with clinical varicocele: a systematic review and meta-analysis

Abstract Study question Does varicocelectomy improve sperm DNA quality in men with infertility and clinically detected varicoceles? Summary answer Varicocelectomy reduces sperm DNA fragmentation (SDF) rates in infertile men with clinical varicocele. What is known already Varicocele has been linked to male infertility through various non-mutually exclusive mechanisms, including an increase in reactive oxygen species (ROS) production that may lead to sperm DNA damage. Damage to sperm DNA may result in longer time-to-pregnancy, unexplained infertility, recurrent pregnancy loss, and failed intrauterine insemination or in vitro fertilization/intracytoplasmic sperm injection. Therefore, interventions aimed at decreasing SDF rates, including varicocele repair, have been explored to improve fertility and pregnancy outcomes potentially, either by natural conception or using medically assisted reproduction. Study design, size, duration Systematic review and meta-analysis Participants/materials, setting, methods We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Our systematic search included PubMed/Medline, EMBASE, Scielo, and Google Scholar to identify all relevant studies written in English and published from inception until October 2020. Inclusion criteria were studies comparing SDF rates before and after varicocelectomy in infertile men with clinical varicocele. Articles were included if the following SDF assays were utilized: SCSA, TUNEL, SCD test, or alkaline Comet. Main results and the role of chance Thirteen studies fulfilled the inclusion criteria and were selected for the analysis. The estimated weighted mean difference of SDF rates after varicocelectomy was –6.58% (13 studies, 95% CI –8.33%, –4.84%; I2=90% p &amp;lt; 0.0001). Subgroup analysis revealed a significant decrease in SDF rates using SCSA (eight studies, WMD –6.80%, 95% CI –9.31%, –4.28%; I2=89%, p &amp;lt; 0.0001), and TUNEL (three studies, WMD –4.86%, 95% CI –7.38%, –2.34%; I2=89%, p &amp;lt; 0.0001). The test for subgroup difference revealed that pooled results were conservative using the above SDF assays. Comet and SCD tests were used in only one study each; thus, a meta-analysis was not applicable. The studies were further categorized by the surgical technique (microsurgical versus non-microsurgical). This subgroup analysis showed a significant decrease in SDF rates using microsurgical technique (10 studies, WMD –6.70%, 95% CI –9.04%, –4.37%; I2=91%, p &amp;lt; 0.0001). After varicocelectomy, SDF rates were also decreased when non-microsurgical approaches were used, albeit the effect was not statistically significant (2 studies, WMD –6.84%, 95% CI –10.05%, 1.38%; I2=86%) (Figure 3). The heterogeneity was not materially affected by performing analyses by the above subgroups, suggesting that the SDF assay and surgical technique do not explain the inconsistency in the treatment effect across primary studies. Limitations, reasons for caution There were no randomized controlled trials comparing varicocelectomy to placebo for alleviating SDF levels. Heterogeneity was high, which may be explained by the low number of included studies. Pregnancy data are not available in most studies, thus the impact of reduced SDF after varicocelectomy on pregnancy rates unclear. Wider implications of the findings: Our study indicates a positive association between varicocelectomy and reduced postoperative SDF rates in men with clinical varicocele and infertility, independentetly of the assays used to measure SDF. These findings may help counsel and manage infertile men with varicocele and high SDF levels. Trial registration number Not applicable

P–103 Novel sperm preparation techniques compared with conventional preparation method

Abstract Study question Which sperm preparation technique separate the best quality sperm? Summary answer Microfluidic method improved the sperm parameters and decreased sperm DNA damage. What is known already About 40% of infertility issues are due to male factor. One of the known causes of male infertility is associated with low sperm parameters and high level DNA fragmentation. Sperm preparation techniques in ICSI procedures is used in order to obtain the best-quality sperm. Study design, size, duration The present study was designed to compare Microfluidic, Zeta potential, Magnetic Activated Cell Sorting (MACS) and Swim-up methods for sperm preparation and the effect of these methods on semen parameters and sperm DNA integrity in infertile men (n = 25) with a mean age of 38. Participants/materials, setting, methods In this study, each sample was divided into 4 groups, one part for preparing by Microfluidic method, one of them for preparing by Swim -up method, the other one was prepared by MACS and the last one was prepared by zeta potential. Then sperm count, viability, motility and morphology were assessed according to WHO 2010. DNA damage were assessed by Sperm DNA Fragmentation assay and sperm chromatin packaging assessed by CMA3 staining test Main results and the role of chance Sperm parameters including viability, motility, and morphology in the Microfluidic method were significantly improved and sperm DNA damage were significantly lower than three other methods (P-value &amp;lt;0.05). The sperm parameters and sperm DNA damage after preparation by MACS and Zeta potential methods were not significantly different however in the Swim-up method sperm parameters were lower than three other methods (P-value &amp;lt;0.05). Limitations, reasons for caution The fertilization and pregnancy rate of the resulting embryos are not available. Wider implications of the findings: Our results showed that Microfluidic can be an effective way to improve sperm quality of infertile male compared to conventional preparation methods. We also found instead of the MACS method, we can use the Zeta potential method according to their costs, for sperm preparation during ART cycle. Trial registration number *

Investigation of molecular cryopreservation, fertility potential and microRNA-mediated apoptosis in Oligoasthenoteratozoospermia men

Investigation of the cryo-injury mechanism can provide novel insight into cryopreservation. The objective of this study is to assess the effect of cryopreservation on fertility potential, motility, oxidative stress (OS), DNA fragmentation, microRNAs (miRNAs), and apoptotic target genes in the infertile men compared to the fertile men. All 40 samples were divided into two leading groups of fresh and cryopreserved sperms. Each main group was subdivided into three groups including, Normozoospermia, and Mild, and Severe Oligoasthenoteratozoospermia (OAT). In all collected samples the following were assessed: microRNA-34c (miR-34c) and miR-184, P53 and Caspase9 using Quantitative real-time polymerase chain reaction (RT-PCR), malondialdehyde (MDA), Superoxide dismutase (SOD) using imaging multi-mode reader, and DNA fragmentation using Sperm DNA Fragmentation Assay Test (SDFA). Within the studied groups, immotile spermatozoa were increased due to cryopreservation. We observed an increasing levels of SOD, MDA, and DNA fragmentation. Also, cryopreservation was associated with decreasing the expression of P53, mir-43c, and miR-184 while capase9 was showed enhancing expression after freeze-thawing of sperm cells. During cryopreservation, sperm fertility and motility were influenced via apoptosis cascade-mediated mitochondrial dysfunctions such as caspase9. Also, we found that miR-34c, miR184, and P53 could impact fertility potential. In Addition, there was a meaningful correlations between microRNAs and motility post freeze-thawing process in Severe Oligoasthenoteratozoospermia men.

In vivo acridine orange human spermatozoa staining-A new perspective for RNA detection and spermatozoa morphology evaluation.

Significant increases in male infertility and the still unresolved questions on the compatibility and interpretation of current methods in infertility diagnostics call for new protocols. Morphology, genome damage, RNA content and quantity are currently in practice as the major parameters in evaluation of sperm quality. However, results of various methods are not always in mutual concordance. In this study, in vivo acridine orange (AO) staining, which is presently in application in the estimation of genome damage in reticulocytes, was adjusted for spermatozoa staining. Ten men suffering from oligoasthenoteratozoospermia (OAT) and 10 healthy fertile men were analysed using in vivo AO staining. Microscopic analysis was performed by fluorescent and confocal fluorescent microscopy. Our results show that this method preserves spermatozoa membranes, which enables new insight into spermatozoa genome damage, RNA content in residual cytoplasm, damage of neck area with mitochondrion and tail pathology. The introduced method explains the difference between results of sperm DNA fragmentation assay and the globally used AO staining and opens new options for the development of automated systems. In conclusion, the results of our study offer (a) an innovative approach to the analysis of spermatozoa pathology, (b) enable localization and quantification of RNA in residual cytoplasm, (c) a significant contribution to research of aetiology of infertility in men, (d) open new perspectives for the automatization of sperm quality estimation and (e) improve the personalized approach in the selection of in vitro fertilization protocols.