High Levels Of Sperm DNA Fragmentation Research Articles

Reproductive outcomes in patients with high levels of sperm DNA fragmentation using testicular sperm for intracytoplasmic injection: a retrospective analysis

This study aims to compare the embryological and clinical parameters of intracytoplasmic sperm injection (ICSI) cycles using testicular versus ejaculated sperm in male patients with elevated sperm DNA fragmentation (SDF). A total of 73 ICSI cycles were examined in couples where the male partner exhibited high levels of SDF. ICSI was performed using either ejaculated or testicular sperm. The primary outcomes were rates of blastocyst formation, high-quality embryo development, and clinical pregnancy. The DNA fragmentation index (DFI) for testicular sperm (16.81 ± 17.51) was significantly lower than that of ejaculated sperm (56.96 ± 17.56). While the blastocyst formation rate was significantly higher in the testicular sperm group compared to the ejaculated sperm group, no statistically significant differences were noted in fertilization rate (72.15% vs. 77.23%), rate of high-quality embryo formation (47.17% vs. 46.53%), clinical pregnancy (50% vs. 56.52%), Cumulative pregnancy (70.2% vs. 55.6%), or live birth rate (43.75% vs.43.48%). Testicular spermatozoa have no additional advantage over ejaculated spermatozoa except for blastocyst quality in patients with high SDF, the use of testicular spermatozoa for the first ICSI cycle in male infertility patients with high SDF should be undertaken after much consideration at present.

Outcomes comparison of testicular versus ejaculated sperm for intracytoplasmic sperm injection in infertile men with high DNA fragmentation: updated systematic review and meta-analysis.

The testicular sperm instead of ejaculated sperm for intracytoplasmic sperm injection (ICSI) in infertile men with high sperm DNA fragmentation (SDF) is a controversial topic. This updated systematic review and meta-analysis aims to evaluate whether couples with high level of SDF will benefit more from intracytoplasmic sperm injection with testicular sperm (Testi-ICSI) as compared to intracytoplasmic sperm injection with ejaculated sperm (Ejac-ICSI). A systematic search was conducted according to PRISMA guidelines, using PubMed, Embase, Web of Science and the Cochrane Central Register of Controlled Trials (CENTRAL), encompassing studies from the earliest record until May 2022. We included studies analyzing comparative pregnancy outcomes of testicular versus ejaculated sperm for ICSI in infertile men with high DNA fragmentation. The risks of bias and certainty of evidence were assessed using the Risk Of Bias In Non-randomized Studies of Interventions (ROBINS-I) and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework, respectively. Eleven studies were included. Meta-analysis showed that SDF levels revealed a significant difference association [odds ratio (OR) =-25.81; 95% confidence interval (CI): -34.82, -16.81; I2=94%; P<0.00001] between testicular and ejaculated sperm. Compared with Ejac-ICSI, a non-significant tendency was observed for fertilization rates (FRs) in the Testi-ICSI group (OR =0.87; 95% CI: 0.67, 1.12; I2=81%; P=0.28). However, there was significant difference pointing to better outcomes for Testi-ICSI in clinical pregnancy rates (CPRs) (OR =2.36; 95% CI: 1.71, 3.24; I2=0%; P<0.00001), live birth rates (LBRs) (OR =3.10; 95% CI: 2.13, 4.51; I2=4%; P<0.00001) and miscarriage rates (MRs) (OR =0.28; 95% CI: 0.13, 0.60; I2=0%; P=0.001). Results of this updated meta-analysis reveal that SDF rates are lower in testicular sperm than in ejaculated sperm and that Testi-ICSI is correlated with better clinical outcomes, including higher CPRs, higher LBRs, and lower MRs in infertile males with high SDF levels. Nevertheless, with the overall low to moderate quality of the studies, further well-designed controlled studies are required.

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.

DNA Integrity in Absolute Teratozoospermia Patients and its Impact on Assisted Reproductive Technology Outcome

Numerous studies showed that Sperm DNA Fragmentation (SDF) is inversely correlated to semen parameters and that high SDF is a major contributor to failure of conception. In the present study, the severity of Sperm DNA Fragmentation in absolute teratoozoospermia patients and its impact on ICSI outcome compared to males with normal semen was examined. A prospective study was performed including 36 absolute teratozoospermia and 30 controls. The mean of DNA Fragmentation Index (DFI) using Sperm Chromatin Dispersion (SCD) test in the absolute teratozoospermia was 42.9 ± 13.2, compared to 24.59 ± 6.9 for the controls. The percentage of patients with high DFI (&gt;30) was higher in the absolute teratozoospermia group 78% than in the control group 30%. Moderate DFI (15-30%) was more common in controls 60% than in absolute teratozoospermia 22%. Normal DFI (≤ 15%) was completely absent in absolute teratozoospermia. The pregnancy rate following Intracytoplasmic Sperm Injection (ICSI) in the control group was 66.6% and 50% in the absolute teratozoospermia. The miscarriage rate was 26% in the control group compared to 53% in the absolute teratozoospermia. Live birth was significantly lower in absolute teratozoospermia compared to fertile men; 19% and 52% respectively. High levels of SDF are predominant in absolute teratozoospermia patients consequently affecting the outcome of ICSI.

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-096 Clinical outcome of ICSI, PICSI and testicular spermatozoa-ICSI procedures in overcoming in patients with high level of sperm DNA fragmentation

Abstract Study question To compare the clinical effectiveness of ICSI, PICSI and testicular spermatozoa-ICSI (T-ICSI) in patients with abnormal level of sperm DNA fragmentation (SDF). Summary answer Clinical effect of T-ICSI procedure exceeds the effect of PICSI and ICSI in male infertility with high level of SDF. What is known already The effectiveness of ART in male infertility depends on sperm chromatin structure integrity. High level of SDF may be reason of IVF/ICSI procedure failures. Several studies have demonstrated impaired clinical outcomes of ICSI in patients with abnormal level of SDF. SDF has a positive correlation with pregnancy loss. It has been shown the benefit of using of testicular spermatozoa in ICSI in high level of SDF. PICSI is a method of the selection of sperm that may reduce the risk of reproductive losses. The data on the comparison of the effectiveness of these methods in high SDF are limited. Study design, size, duration Data was collected from 2019 to 2021 years. The couples with male factor of infertility were enrolled in the study. All the patients had abnormal level of SDF (30-45%). The age of women was less than 35 year. The couples were randomly selected and divided in 3 groups according the ART method: I (n = 25) ICSI; II (n = 22) PICSI; III (n = 20) T-ICSI procedure. Participants/materials, setting, methods The trial involved 67 couples. The mean patients age was 33,3±6,2 y (24-46), the women age - 29,1±2,3 (25-33). All of the men were non-smokers, had idiopathic pathospermia, high level of SDF and underwent unsuccessful courses of empirical therapy in anamnesis. SDF level was measured by TUNEL method. 52 (77,6%) couples had unsuccessful attempts of ICSI including pregnancy loss. The fertilization, blastocyst formation (Gr A/B), clinical pregnancy and live birth rates were measured. Main results and the role of chance It was not significant difference in SDF level in the groups: I group - 37,3±3,1%; II group - 37,2±3,5%; III group - 37,8±3,2% (III). The fertilization rate was comparable in the groups I, II and III - 65,4%; 66,6%; 63,3% respectively. Blastocyst formation (good quality embryos) rate was 41,2%; 46,1%; 56,1%(p &amp;lt; 0001) in I, II and III groups respectively. Clinical pregnancy and life birth rates were 36,0% [9/25]; 40,9%[9/22] (p &amp;lt; 0,05); 45,0%[9/20] (p &amp;lt; 0001) and 20,0% [5/25]; 27,2%[6/22]; 35%[7/20] in ICSI, PICSI, T-ICSI groups respectively. It was significant difference in miscarriage rate 16%, 13,7% (p &amp;lt; 005), 10% (p &amp;lt; 0001) in I, II и III groups respectively. Limitations, reasons for caution T-ICSI might compensate for ART failure in high level of SDF cases. Testicular sperm retrival is an invasive intervention, and its benefit must be carefully justified in each cases. Further studies are needed to determine whether infertility with sperm DNA damage can be overcomed by less invasive methods than T-ICSI. Wider implications of the findings There was no significant difference in fertilization rate in ICSI, PICSI and T-ICSI. T-ICSI demonstrated the best blastocyst formation rate and clinical outcome in trial. According results of our investigation PICSI might increase pregnancy rates and reduce pregnancy losses when compared with ICSI in infertility in high SDF. Trial registration number not applicable

O-134 Predictive value of seminal oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) analysis for reproductive outcomes of intracytoplasmic sperm injection (ICSI) cycles

Abstract Study question Do seminal oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) predict ICSI cycle outcomes? Summary answer Both SDF and seminal ORP have strong prognostic value in predicting good fertilization (≥80%), blastocyst development (≥60%), clinical pregnancy and live birth. What is known already Seminal oxidative stress (OS) has been found to play an important role in various aetiologies of male infertility and is induced by the imbalance between reactive oxygen species (ROS) and antioxidants. OS is a major cause of sperm DNA fragmentation (SDF). It is well established that high levels of seminal OS and SDF are negatively associated with ART outcomes. Previous studies have associated high levels of SDF with an increase risk of miscarriage and a decrease in live birth rate. Study design, size, duration This prospective study included 144 couples who had a fresh autologous ICSI cycle between June 2018 and December 2020. There was no restriction to patients with severe male factor infertility. All cryopreservation and third party assisted reproductive techniques were excluded. Participants/materials, setting, methods This multi-centre study was performed at three ART Clinics in Cape Town, South Africa. All couples included in the study had fresh autologous ICSI cycles with freshly ejaculated semen. Seminal ORP testing was performed using the MiOXSYS system and SDF by means of a fluorescence microscopic TUNEL assay on freshly ejaculated semen samples that were used for ICSI. The ART outcomes evaluated were fertilization rate, blastocyst development rate, clinical pregnancy rate and live birth rate. Main results and the role of chance The study shows that seminal ORP significantly negatively correlates with fertilization rate (r=-0.267; P = 0.0012), blastocyst development rate (r=-0.389; P &amp;lt; 0.0001), pregnancy (r=-0.296; P = 0.0003) and live birth (r=-0.353; P &amp;lt; 0.0001). ROC curve analysis shows significant predictive power for ORP for fertilization (≥80%; AUC=0.652; P = 0.0012), blastocyst development rate (≥60%; AUC=0.762; P &amp;lt; 0.0001), pregnancy (AUC=0.677; P = 0.0002) and live birth (AUC=0.729; P &amp;lt; 0.0001). Comparable results were obtained for SDF. An average cut-off value for ORP of 0.55 mV/106 sperm/mL was calculated. The AUCs between the SDF and ORP results for all reproductive outcome parameters did not differ. For all reproductive outcome parameters, normal sperm morphology shows the lowest predictive power. ORP with male age as confounding factor had significant effects on odds ratios (OR) of fertilization (OR = 0.1446; CI: 0.048-0.430; P = 0.0001) and blastocyst development (OR = 0.2468; CI: 0.095-0.635; P = 0.0029), while it was not significant for pregnancy and life birth. Multivariate logistic regression with fertilization, blastocyst development, pregnancy and live birth as dependent parameters and high/low seminal ORP, primary and secondary infertility, unexplained infertility, polycystic ovaries, endometriosis, tubal factor myomectomy, ovarian insufficiency and advanced maternal age as independent variables showed overall model fits (P &amp;lt; 0.0002) with a significant (P &amp;lt; 0.0084) influence of ORP on all reproductive outcome parameters. Limitations, reasons for caution Since only ICSI patients were included in this study, this might have contributed to the fact the cut-off value of ORP is lower than the published cut-off. A further limitation is the use of a fluorescence microscopic TUNEL assay which drastically limited the number of sperm evaluated. Wider implications of the findings This is the first study showing predictive value of seminal ORP for reproductive outcome including pregnancy and live birth. The method is easy and much quicker than current methods for the determination of SDF. Hence, it can be used a point of care method. Trial registration number Not applicable

Beyond conventional sperm parameters: the role of sperm DNA fragmentation in male infertility.

Infertility is a condition that widely affects couples all over the world. In this regard, sperm DNA fragmentation can lead to harmful reproductive consequences, including male infertility and poor outcomes after assisted reproductive techniques. The investigation of sperm DNA fragmentation in male infertility diagnostics has constantly increased over time, becoming more common in clinical practice with the recent publication of several guidelines regarding its testing. This narrative review aims to provide a comprehensive overview of the pathogenesis and causes of sperm DNA fragmentation, as well as the assays which are commonly performed for testing. Moreover, we discuss the most recently published evidence regarding the use of sperm DNA fragmentation testing in clinical practice, highlighting the implications of high sperm DNA fragmentation rate on human reproduction, and the therapeutic approaches for the clinical management of infertile patients. Our review confirms a significant harmful impact of sperm DNA fragmentation on reproduction and points out several interventions which can be applied in clinics to reduce sperm DNA fragmentation and improve reproductive outcomes. Sperm DNA fragmentation has been shown to adversely impact male fertility potential. As high sperm DNA fragmentation levels have been associated with poor reproductive outcomes, its testing may significantly help clinicians in defining the best therapeutic strategy for infertile patients.

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.

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–043 Six years’ retrospective statistical study (2013 – 2018) investigating the impact of Sperm DNA fragmentation on sperm analysis parameters

Abstract Study question What is the relationship between Sperm DNA fragmentation (SDF) levels and sperm analysis (Spermocytogramme) parameters results? Summary answer SDF level of patients with pathological spermocytogramme presents negative correlations to total spermatozoa mobility, vitality and concentration, and positive correlation to sperm morphology defects. What is known already The relationship between SDF and Sperm analysis parameters and especially sperm morphology needs to be more studied since few studies over the last years were focused on this relationship. However, abnormalities in these two parameters are considered as the most important biological indicators of male infertility. The pathogenesis of Teratozoospermia (&amp;lt;4% morphologically normal sperm cells according to WHO 2010) is continuously increasing over the last decade according to several studies. In addition, SDF is also increasing over the years because of several factors such as pollution, stress and lifestyle changing. Study design, size, duration Retrospective study including 331 infertile patients undergoing SDF-index testing with Spermocytogramme from January 2013 – December 2018. Patients divided into two groups: 143 patients with normal-Spermocytogramme and 188 patients with pathological-Spermocytogramme. Each group includes patients with abnormal SDF levels (&amp;gt;30%). Statistical analyzes were performed using SPSS22.0 for Windows-software. Kolmogorov–Smirnov-test for normality analysis and comparisons by Student-t-test or Mann–Whitney U-test, as appropriate. Pearson/Spearman’ tests for correlations were used as appropriate, P-value&amp;lt;0.05 was considered as significant. Participants/materials, setting, methods 143 patients with normal Spermocytogramme (2.8% abnormal-SDF) vs 188 patients with pathological Spermocytogramme (10.6% abnormal-SDF). WHO–2010 instructions for sperm-analysis were used through Makler®-counting-chamber (Sefi-Medical Instrument Ltd) for sperm-concentration and motility-determination using Sperm-class-analyzer-software (CASA-system (Microptic®)) to detect sperm abnormalities. Normozoospermia was determined when sperm progressive-motility is ≥ 32%, sperm-concentration ≥15x106/mL, and sperm-morphology ≥4%. “Diff-Quick” staining-method for the coloration of the fixed-sperm-slides was used for Sperm-morphology analysis. GoldCyto Sperm®Kit (Goldcyto Biotech corp.) was used to analyze SDF. Main results and the role of chance SDF is significantly higher in pathological spermocytogramme’ patients than in normal spermocytogramme’ patients (17.02 ± 11.88 vs 12.16 ± 9.58 respectively). In patients with pathological spermocytogramme, SDF is negatively correlated to Progressive sperm motility (r= –0.137; p = 0.042), Total sperm motility (r= –0.153; p = 0.036), vitality (r=–0.140; p = 0.048) and concentration (r=–0.195; p = 0.007). In the other hand, SDF presented positive correlation with teratozoospermia and especially with sperm midpiece defects (r = 0.171; p = 0.02). However, SDF did not present any correlation with age, testosterone levels and total ejaculated sperm volume. However the latter was positively correlated to spermatozoa midpiece and head defects (r = 0.156; p = 0.034; r = 0.203; p = 0.006, respectively). These results are in accordance with García-Ferreyra et al. (2014) who found that men with abnormal spermatozoa morphology showed high levels of DNA fragmentation, Sá et al. (2015) who confirmed that semen with lower concentration, motility and morphology have higher levels of SDF and showed that sperm head staining patterns are correlated with the degree of SDF. In addition, recently the study of Jakubik-Uljaszstudy et al. (2020) could confirms our results when it concluded that detailed sperm structural defects coexist with abnormal nuclear sperm DNA dispersion and that men with teratozoospermia may have a higher risk for sperm DNA damage. Limitations, reasons for caution Our study is a retrospective statistical investigation that included patients attending to the laboratory for fertility diagnosis after a period of infertility. Meta-analyzes studies in addition to more prospective-randomized-controlled-trials with couples undergoing assisted-reproductive-treatments and in comparison with fertile men are needed to confirm the relationship between SDF and spermocytogramme defects. Wider implications of the findings: These results should interest andrologists, reproductive science fundamentalists and embryologists who want to improve the investigations on the origin of infertility especially when it comes from male side. Trial registration number Not applicable

Molecular Analysis of DPY19L2, PICK1 and SPATA16 in Italian Unrelated Globozoospermic Men.

This study aims to evaluate genetic contribution and sperm DNA fragmentation (SDF) in a cohort of 18 unrelated globozoospermic Italian men (Group G). Semen samples were assessed according to the WHO 2010 Laboratory Manual and compared with 31 fertile controls. We focused our genetic analysis on the exons of the main globozoospermia-associated genes, performing qualitative PCR to assess deletion of DPY19L2 and sequencing to detect mutations of SPATA16 and PICK1. SDF was evaluated using the TUNEL assay. In Group G, 10 patients had a complete form of globozoospermia, whereas 8 patients had a partial form. Molecular analysis revealed deletion of DPY19L2 in six of the patients, all of them with complete globozoospermia, while no mutations were found in the examined exons of PICK1 and SPATA16. TUNEL analysis showed a higher SDF% in Group G. Our findings confirm DPY19L2 defects as the most frequent genetic alteration in Italian patients contributing to globozoospermic phenotypes. Furthermore, spermatozoa with acrosomal defects could also display high levels of SDF as a possible consequence of abnormally remodeled chromatin. The possible effect on offspring of chromatin structure abnormalities and altered DNA integrity should be carefully evaluated by clinicians, especially regarding the feasibility and safety of artificial reproductive techniques, which represent the only treatment that allows these patients to conceive.

The Negative Impact of Varicocele on Basic Semen Parameters, Sperm Nuclear DNA Dispersion and Oxidation-Reduction Potential in Semen.

Since varicocele is so common in infertile men, this study intends to analyse the relationships between varicocele and conventional semen characteristics, sperm nuclear DNA dispersion and oxidation-reduction potential (ORP) in semen. Varicocele-positive and varicocele-negative infertile men (study groups) showed significantly lower standard sperm parameters and higher sperm DNA fragmentation (SDF) and ORP in semen than healthy volunteers and subjects with proven fertility (control groups). A lower proportion of low SDF levels (0–15% SDF) and higher incidence of high SDF levels (>30% SDF), as well as a higher prevalence of high ORP values (>1.37 mV/106 sperm/mL), were found in the study groups vs. the control groups. Moreover, infertile men had significantly lower odds ratios (ORs) for low SDF levels and significantly higher ORs for high SDF levels and high ORP. SDF and ORP were negatively correlated with sperm number, morphology, motility and vitality. Furthermore, a significant positive correlation was found between SDF and ORP. The obtained results suggest that disorders of spermatogenesis may occur in varicocele-related infertility. These abnormalities are manifested not only by reduced standard semen parameters but also by decreased sperm DNA integrity and simultaneously increased oxidative stress in semen.

Magnetic-Activated Cell Sorting (MACS): A Useful Sperm-Selection Technique in Cases of High Levels of Sperm DNA Fragmentation

Magnetic-activated cell sorting (MACS) can be used to separate apoptotic sperm with high proportions of fragmented DNA from the rest, thus improving the overall quality of the seminal sample. Therefore, the aim of this retrospective study was to investigate the efficiency of the MACS technique to increase reproductive outcomes in patients with high levels of sperm DNA fragmentation (SDF) undergoing intracytoplasmic sperm-injection (ICSI) cycles. In this study, we analyzed a total of 724 assisted-reproduction-technique (ART) cycles that were divided into two groups: the study group (n = 366) in which the MACS selection technique was performed after density-gradient centrifugation (DGC), and the control group (n = 358) in which only DGC was used for sperm selection. Reproductive outcomes were analyzed in both groups according to three different ART procedures: preimplantation genetic testing for aneuploidy (PGT-A), and autologous and oocyte-donation cycles. The MACS group showed significantly lower miscarriage rates in autologous ICSI cycles, higher pregnancy rates in oocyte-donation cycles, and a significant increase in live-birth rates in both autologous and oocyte-donation cycles. Overall, these results suggested that the MACS technique can be effectively used to eliminate sperm with high SDF levels, and therefore may help to improve reproductive outcomes in couples undergoing ART.

Relationship between sperm morphology and sperm DNA dispersion.

BackgroundThe pathogenesis of teratozoospermia (<4% morphologically normal sperm cells) and the relationship between sperm morphological abnormalities and abnormal sperm nuclear DNA fragmentation, which are considered indicators of male fertility, have not been elucidated. Our research was designed to determine the prevalence of different sperm DNA fragmentation (SDF) levels in men with teratozoospermia and to establish a discriminating threshold value for SDF in assessing sperm morphology.MethodsBasic semen characteristics and detailed sperm morphological analysis (head, neck, midpiece, and tail defects and excess residual cytoplasm) (WHO, 2010), and the nuclear sperm DNA dispersion test were performed on semen samples obtained from 523 men with teratozoospermia (n=296) and those without teratozoospermia (n=227).ResultsSubjects with abnormal sperm morphology had not only lower results for standard sperm characteristics, including detailed sperm morphological abnormalities, but also a higher proportion of sperm cells with SDF vs. men with normal sperm morphology. Moreover, significantly fewer subjects with low SDF levels (≤15%), more subjects with high SDF levels (>30%) and a higher odds ratio (OR) for having high SDF levels were found in the group of men with teratozoospermia vs. men without teratozoospermia. However, the receiving operating characteristic (ROC) curve analysis indicated that a SDF >18% was a significant negative predictive value to distinguish between men with normal sperm morphology or men with abnormal sperm morphology. The optimal area under the ROC curve (AUC) was 0.746. In the group of men with teratozoospermia, a higher incidence of men with >18% SDF and a higher OR for having >18% SDF were observed. SDF negatively correlated with sperm number, morphologically normal sperm cells, sperm motility and sperm vitality but positively correlated with the teratozoospermia index (TZI) and detailed sperm morphological abnormalities.ConclusionsThe obtained findings demonstrated that: (I) detailed sperm structural defects coexist with abnormal nuclear sperm DNA dispersion, (II) men with teratozoospermia may have a higher risk for sperm DNA damage, (III) the calculated optimal SDF value of 18% measured by the DNA sperm dispersion test is the best criterion to predict normal and abnormal sperm morphology.

Impact of sperm DNA fragmentation on ICSI outcome and incidence of apoptosis of human pre-implantation embryos obtained from in vitro matured MII oocytes

BackgroundSperm DNA integrity and oocyte quality significantly affect embryo development and survival. The current study evaluated embryo development and quality, as well as the expression level of apoptosis-related genes and microRNAs in embryo derived from in vitro matured MII oocytes according to sperm DNA fragmentation (SDF) level. MethodsThe semen and immature oocytes were collected from 50 ICSI cycles with any recognizable female factor infertility. After ovarian stimulation, germinal vesicle stage (GV) oocytes were collected and incubated in in vitro maturation (IVM) medium for 24 h. Next, reactive oxygen species (ROS) level of media culture was determined. Using by sperm chromatin dispersion (SCD) test, the SDF levels of processed semen were assessed and categorized into SDF ≤ 30% and SDF>30%. Seventy two hours after intracytoplasmic injection, the embryo development and quality score were recorded in the groups I (GV-MII + SDF≤ 30%) and II (GV-MII + SDF> 30%). Also, the apoptosis incidence of embryos at morula stage was evaluated at molecular and cellular levels by quantitative real time PCR and TUNEL staining, respectively. ResultsCleavage rate did not differ between two groups. The quality score of embryos obtained from IVM matured oocytes and high level of SDF was significantly lower than that of low level of SDF (P < 0.05). The embryos from group II had a significant reduction of the expression of BCL-2 compared to group I (P < 0.05). Also, they showed an increase in relative transcription of pro-apoptotic microRNAs; miR 15a and miR 16-1 versus group I (P < 0.05). A rise of TUNEL positive blastomers of embryo was observed at group II versus group I, but it did not reach to significantly level. ConclusionThe IVM oocytes, probably, did not suffice to recover the high level of paternal genomic damage and inhibition of apoptosis pathway beginning.

What should be done for men with sperm DNA fragmentation?

In an age when a small quantity of sperm can lead to pregnancy through in vitro fertilization or intracytoplasmic sperm injection, selecting healthy sperm is important. Sperm DNA fragmentation (SDF) is known to be higher in infertile men. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and the alkaline comet test are SDF tests that directly measure DNA damage and have shown closer correlations with assisted reproduction results than indirect tools such as the sperm chromatin structure assay or the sperm chromatic dispersion test. It is difficult; however, to endorse a single test as the best test overall; instead, it is best to select a testing method based on each patient's clinical condition and goals. In a couple struggling with infertility, if the male partner has a high level of SDF, he should aim to decrease SDF through lifestyle modifications, antioxidant treatment, and ensuring an appropriate duration of abstinence, and physicians need to treat the underlying diseases of such patients. If sperm DNA damage continues despite the patient's and physician's efforts, other methods, such as micromanipulation-based sperm selection or testicular sperm extraction, should be used to select healthy sperm with nuclear DNA integrity.

Sperm DNA fragmentation (SDF) index on day of transvaginal oocyte retrieval (TVOR) does not correlate with the incidence of segmental aneuploidy in blastocysts analyzed with targeted next generation sequencing (NGS)

Sperm DNA fragmentation (SDF) is more common in infertile men and has been found to be associated with recurrent miscarriage in some studies1. SDF testing evaluates the extent of DNA strand breaks in a given specimen and has been proposed as an adjunct to routine semen analysis. Given that high levels of SDF indicate a greater number of DNA strand breaks in sperm, some have proposed that the association between high SDF and poor reproductive performance may reflect segmental aneuploidy that has not traditionally been detectable with preimplantation genetic screening (PGS)2. However, the increased resolution of NGS allows for improved identification of segmental imbalances and may be useful in addressing this question3. To evaluate the association between segmental aneuploidy (duplications and deletions) and sperm DNA fragmentation levels among trophectoderm biopsy samples evaluated with targeted NGS. This was a subgroup analysis of a prospective cohort study of females 35-40 years of age undergoing IVF with ICSI and PGS between December 2014 and May 2017. Routine clinical care was performed through TVOR, at which time a 0.5mL semen aliquot was isolated from the fresh specimen. SDF was evaluated with the Sperm Chromatin Structure Assay (SCSA®). Beginning in July 2016, PGS samples were evaluated using NGS and segmental aneuploid calls were included in clinical reports (in addition to whole chromosome aneuploidy). Reports indicated the % of fragmented sperm DNA (DFI). Patients were divided into 2 groups: (<15% and >15%) and incidence of segmental aneuploidy was compared between the groups in 2 ways: 1) any segmental aneuploidy (among all embryos, including those with an additional whole chromosome aneuploidy) and 2) segmental aneuploidy only (no other whole chromosome aneuploidy). A total of 169 cases were included in the analysis. DFI <15% was found in 123 specimens, while DFI >15% was found in 45 specimens. The median DFI for the entire study population was 11 (interquartile range: 8 to 15). Men in the 15% DFI group were older (40.6 vs. 38.0, p<0.01), but there was no difference in the age of the female partner (37.9 vs. 37.4, p = 0.71). There was no difference in the rate of conversion to blastocyst. The incidence of any segmental aneuploidy and only segmental aneuploidy was no different between the groups. The incidence of whole chromosome aneuploidy was also no different (Table 1). Specimens with elevated DNA Fragmentation used for ICSI do not result in an increased incidence of segmental aneuploidy in trophectoderm biopsy samples evaluated by NGS. These results suggest three possible rationales: 1) DNA damage in the male may be at least partially repaired by oocyte-derived mechanisms, 2) small deletions and duplications may be below the diagnostic threshold of current NGS technologies, or 3) SDF testing, which relies on average levels among pooled sperm, may be limited in its ability to prognosticate embryonic segmental aneuploidy.Tabled 1Table 1DFI ≤15% (N=126)DFI >15% (N=43)P ValueDFI %9 (7-12)20 (16-27)<0.001Female Age (years)37.4 ± 1.637.9 ± 1.90.71Male Age (Years)38 ± 5.940.6 ± 5.9<0.01Oocytes Retrieved13.2 ± 8.111.5 ± 9.00.18Blastulation Rate48.2% (525/1089)50.8% (159/313)0.45Whole Chromosome Aneuploidy (per embryo)34.7% (182/585)35.8% (57/159)0.86Any segmental aneuploidy (including in embryos with whole chromosome aneuploidy also)10.7% (56/525)11.9% (19/159)0.75Only segmental aneuploidy present (no other whole chromosome aneuploidy)5.3% (28/525)5% (8/159)0.99 Open table in a new tab

Reproductive outcomes of testicular versus ejaculated sperm for intracytoplasmic sperm injection among men with high levels of DNA fragmentation in semen: systematic review and meta-analysis

To compare sperm DNA fragmentation (SDF) levels between testicular and ejaculated sperm and to evaluate outcomes of intracytoplasmic sperm injection (ICSI) with the use of testicular (Testi-ICSI) versus ejaculated (Ejac-ICSI) sperm in nonazoospermic men with high SDF. Systematic review and meta-analysis. Not applicable. Normo- and oligozoospermic men with high levels of SDF in semen subjected to Testi-ICSI or Ejac-ICSI. Summary mean difference (MD) and odds ratio (OR) were calculated with the use of an inverse variance model and fixed- or random-effects models, respectively. Primary outcomes were SDF levels, clinical pregnancy rates (CPRs), and live birth rates (LBRs). Secondary outcomes were fertilization and miscarriage rates. Five studies involving 143 patients provided paired SDF rates for testicular and ejaculated sperm, revealing lower SDF in testicular sperm (MD -24.58%). Four studies involving 507 cycles and 3,840 oocytes reported clinical outcomes of Testi-ICSI and Ejac-ICSI. Fertilization rates were not different between sperm sources, but a trend to lower rates was observed with Testi-ICSI. CPRs were higher for Testi-ICSI than for Ejac-ICSI, as were LBRs, whereas miscarriage rates were reduced with Testi-ICSI. Testicular sperm have lower levels of SDF than ejaculated sperm, with Testi-ICSI for high post-testicular SDF men improving reproductive outcomes compared with Ejac-ICSI. Infertile couples may benefit from Testi-ICSI if male partners have confirmed high SDF in the ejaculate.

Recurrent pregnancy loss: current perspectives.

Recurrent pregnancy loss is an important reproductive health issue, affecting 2%–5% of couples. Common established causes include uterine anomalies, antiphospholipid syndrome, hormonal and metabolic disorders, and cytogenetic abnormalities. Other etiologies have been proposed but are still considered controversial, such as chronic endometritis, inherited thrombophilias, luteal phase deficiency, and high sperm DNA fragmentation levels. Over the years, evidence-based treatments such as surgical correction of uterine anomalies or aspirin and heparin for antiphospholipid syndrome have improved the outcomes for couples with recurrent pregnancy loss. However, almost half of the cases remain unexplained and are empirically treated using progesterone supplementation, anticoagulation, and/or immunomodulatory treatments. Regardless of the cause, the long-term prognosis of couples with recurrent pregnancy loss is good, and most eventually achieve a healthy live birth. However, multiple pregnancy losses can have a significant psychological toll on affected couples, and many efforts are being made to improve treatments and decrease the time needed to achieve a successful pregnancy. This article reviews the established and controversial etiologies, and the recommended therapeutic strategies, with a special focus on unexplained recurrent pregnancy losses and the empiric treatments used nowadays. It also discusses the current role of preimplantation genetic testing in the management of recurrent pregnancy loss.