DNA Fragmentation Rate Research Articles

Effect of paternal health on pregnancy loss-A review of current evidence.

Pregnancy loss has multifactorial causes, and the maternal risk factors are the most investigated. Therefore, this review investigates the current literature regarding the effect of paternal health on pregnancy loss. This review is conducted according to the PRISMA guidelines. The electronic databases PubMed and Medline were the primary sources of information. The online tool covidence.org was used for the screening process. The Newcastle-Ottawa Scale was used for assessment of risk of bias across the non-RCT (Randomized Controlled Trials) included studies. Six cohort studies and one randomised clinical trial were included for assessment in this review. Especially three large retrospective studies reported that circulatory paternal health issue, increasing metabolic syndrome diagnoses and paternal age was significantly associated with a higher risk of pregnancy loss. Lower pregnancy loss was also found in couples with diabetes in the man compared to couples without diabetes. One study suggests a connection between varicocelectomy and improved sperm DNA fragmentation and lower abortion rate. This review confirms that paternal age, somatic health and particularly health regarding cardiovascular and metabolic disease are associated positively with risks of pregnancy loss. However, further research may lead to evidence, which are more conclusive.

MP21-18 IT'S NOT ABOUT TIME: DOES ABSTINENCE ALTER SEMINAL OXIDATIVE STRESS OR DNA FRAGMENTATION RATES?

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation I (MP21)1 Sep 2021MP21-18 IT'S NOT ABOUT TIME: DOES ABSTINENCE ALTER SEMINAL OXIDATIVE STRESS OR DNA FRAGMENTATION RATES? Kevin Campbell, John Sullivan, Oscar Velazquez, and Larry Lipshultz Kevin CampbellKevin Campbell More articles by this author , John SullivanJohn Sullivan More articles by this author , Oscar VelazquezOscar Velazquez More articles by this author , and Larry LipshultzLarry Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002006.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Seminal oxidative stress and DNA fragmentation have been associated with male infertility, poor embryo development, and recurrent miscarriage. Oxidative stress can be measured using the static oxidation-reduction potential (ORP) as well as chemiluminescence assays for reactive oxygen species (ROS). Studies have independently shown ORP and ROS values to be respectively increased in semen samples of abnormal quality compared to those of normal samples. Additionally, semen parameters have been shown to change reflecting the number of days of abstinence (DOA) prior to a patient providing a semen sample. Since many fertility patients travel a long distance or are not counseled about the number of days abstinence before collecting, we designed this study to evaluate the relationship between number of days of abstinence, oxidative stress, DNA fragmentation, and age of patients. METHODS: Semen samples from a high-volume men’s fertility clinic were retrospectively evaluated for DOA, DNA fragmentation, and oxidative stress quantified by ROS or ORP. DOA samples were then stratified by DNA damage percentage and ROS/ORP for association. RESULTS: A total of 857 semen samples were identified by those with patient DOA, including 818 with ROS values, 199 with ORP, and 857 with DNA damage testing. DOA showed no significant correlation when stratified by ROS (R2=0.003, p=0.4523), ORP (R2=0.001, p 0.595), or DNA damage (R2=0.001, p=0.461). CONCLUSIONS: A patient’s DOA prior to providing a semen sample does not correlate with DNA damage percentage or oxidative stress measured by ORP or ROS. Therefore, these more analytically complicated tests may be done at any visit, regardless of the day’s abstinence. Consequently, these results can be ready for the patient’s subsequent visit, at which time a DOA controlled sample can be obtained for better determination of abstinence-related sperm density and motility. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e353-e353 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kevin Campbell More articles by this author John Sullivan More articles by this author Oscar Velazquez More articles by this author Larry Lipshultz More articles by this author Expand All Advertisement Loading ...

Effects of Selenium Supplementation on Sperm Parameters and DNA-Fragmentation Rate in Patients with Chronic Autoimmune Thyroiditis.

Background: Selenium (Se) is an essential component of selenoenzymes, which have catalytic and antioxidant functions. A low Se status has been reported in patients with chronic autoimmune thyroiditis (AT) who benefit from Se supplementation. The role of Se in male reproduction is still a matter of debate. Although Se and selenoenzymes ensure sperm viability and protect against increased oxidative stress, only a few studies have assessed the effects of the administration of Se alone on sperm parameters, providing contrasting results. Aim: The aim of this study was to assess the effects of oral Se supplementation on conventional sperm parameters and DNA fragmentation (SDF) in patients with AT of reproductive age with normal thyroid function. Patients and Methods: Only patients with AT and normal thyroid function were selected for this study. All included patients underwent oral Se supplementation at the dose of 83 µg once daily (Syrel®, IBSA) for six months. Sperm conventional parameters, SDF, and thyroid function were assessed before and at the end of the treatment. Results: Twenty AT patients with normal weight were enrolled. After Se supplementation, they showed a higher sperm concentration, a higher percentage of sperm with progressive motility, and a higher percentage with normal morphology. They also had lower semen leukocyte concentration, and a lower percentage of spermatozoa with DNA fragmentation compared with pre-treatment values. Free-thyroxine serum levels increased significantly, whereas free triiodothyronine showed an upward trend. The thyroid-stimulating hormone did not change significantly. Conclusion: Se supplementation may represent a possible non-hormonal therapeutic choice for the treatment of male infertility, although further studies are needed to confirm this evidence. The possible thyroid hormone dependency of these findings needs to be clarified.

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 < 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 < 0.0001), and TUNEL (three studies, WMD –4.86%, 95% CI –7.38%, –2.34%; I2=89%, p < 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 < 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–019 Sperm parameter and ICSI / IVF outcomes after sperm selection using microfluidic sperm separator and density gradient centrifugation with swim-up in split semen sample

Abstract Study question To analyze whether microfluidic sperm selection (MSS) by ZyMōt™ improves sperm DNA fragmentation rate and embryonic development compared to density gradient centrifugation with swim-up (DGCS). Summary answer MSS by ZyMōt™ selects sperm for clinical use with less DNA damage significantly compared to DGCS. What is known already Conventional sperm preparation methods, such as density gradient centrifugation and the swim-up method utilize centrifugation during processing, may damage the sperm. MSS may allow for improved selection of normal sperm compared with conventional sperm preparation as it yields sperm with a lower DNA fragmentation rate. However, there are few clinical studies by sibling oocytes study compared to DGCS. Study design, size, duration This prospective study was performed between March 2020 and May 2020 at a reproductive center. All patients involved gave written consent, and institutional review board approval was granted. A total of 575 metaphase II oocytes were collected from 49 cycles. Wife’s age was 34.7 ± 3.9 years old. Raw sperm concentration and motile sperm concentration was 63.1 ± 78.7M/mL, and 41.6 ± 67.7M/mL, respectively. Participants/materials, setting, methods Patients who performed ART for the first or second time were divided into two groups according to MSS and DGCS. Sperm DNA fragmentation rate (SDFR) and motile sperm concentration were compered between MSS and DGCS. SDFR was measured by sperm chromatin structure assay (SCSA) using a flow cytometer. Sibling oocytes were randomized into MSS-IVF, DGCS-IVF, MSS-ICSI, and DGCS-ICSI. Rate of two pronuclear (2PN) oocytes, blastocysts development, and good-quality blastocysts were compared between each group. Main results and the role of chance SDFR was 13.5 ± 11.8% for raw semen. SDFR was significantly lower after MSS (3.6 ± 4.1%) than that for raw semen and after DGCS (17.4 ± 14.8%) (P < 0.01). Motile sperm concentration after MSS (19.0 ± 28.3M/mL) was significantly higher after than after DGCS (15.4 ± 15.3M/mL) (P < 0.01). The number of IVF performed was 145 for MSS and 132 for DGCS. IVF results (MSS vs DGCS) were 2PN rate (73.1% vs 72.0%), blastocysts development rate (65.3% vs 55.4%), and good quality blastocysts rate (43.2% vs 34.9%). The number of ICSI performed was 149 for MSS and 149 for DGCS. ICSI results (MSS vs DGCS) were 2PN rate (77.9% vs 79.2%), blastocysts development rate (68.8% vs 65.8%), and good quality blastocysts rate (35.8% vs 30.6%). No significant difference was observed between MSS and DGCS for each parameter both IVF and ICSI. Limitations, reasons for caution The participants were limited to those who collected semen of 2mL or more and motile sperm concentration of above 1M/mL, because semen sample needed to be divided to MSS and DGCS. Wider implications of the findings: This is the first study to conducted in sibling oosytes study with MSS and DGCS, in both IVF and ICSI. MSS is effective in collecting sperm with less DNA damage compared to DGCS. Motile sperm concentration after using MSS is sufficient to perform IVF as well as DGCS. Trial registration number Not applicable

P–037 Influence of sperm selection by microfluidic device on outcomes of in vitro fertilization cycles

Abstract Study question Does sperm selection by a microfluidic sperm-sorting device improve laboratory outcomes of intracytoplasmic sperm injection cycles? Summary answer The ZyMōtTMdevice seems a good alternative to the conventional swim-up technique, possibly increasing progressive sperm motility post processing and blastulation rate. What is known already It is well-known that high levels of sperm DNA fragmentation negatively impact not only early embryonic development but also embryo genome activation. Microfluidic technologies for processing sperm samples selection such as the ZyMōtTM device have been developed to sort sperm with lower rates of sperm DNA fragmentation in order to improve in vitro fertilization (IVF) outcomes. Moreover, it offers several advantages: working with very small volume samples, high sensitivity and low reaction times, automatic sample treatment and analysis, among others. However, the real benefits on the clinical and laboratory outcomes of IVF cycles is yet to be determined. Study design, size, duration We performed a retrospective study including 126 seminal samples processed between May 2019 and December 2020. These samples belong to 63 infertile couples that underwent 2 consecutive IVF cycles using the swim-up technique in the first cycle (n = 63) and the ZyMōtTM device in the second (n = 63). The oocytes of the female partners were recovered and inseminated, and the embryonic culture was carried out until the blastocyst stage, when the quality of these embryos was analyzed. Participants/materials, setting, methods: A total of 118 seminal samples from 59 couples were included. Four couples were excluded due to insufficient seminal volume / concentration (2) or absence of mature oocytes recovered (2). Laboratory outcomes were evaluated including semen parameters post processing, as well as fertilization, cleavage, blastulation (blastocyst among cleavage stage) and top-quality blastocyst rates. Continuous variables were analyzed by paired Student’s t-test or Wilcoxon signed-rank test, as appropriate. Differences were considered significant when p-value < 0.05. Main results and the role of chance We did not find any difference in the percentage of non-progressive sperm motility [0 (0–33%) vs. 0 (0–10%); p = 0.063] and immotile sperm [0 (0–70%) vs. 0 (0–80%); p = 0.095] post processing when comparing the swim-up technique with the ZyMōtTM device. Likewise, no significant differences were detected regarding fertilization [83% (0–100%) vs. 89% (0–100%); p = 0.104] and cleavage [100% (0–100%) vs. 100% (80–100%); p = 0.217] rates in both groups. Nevertheless, there was a significant difference in the percentage of progressive sperm motility [100% (20–100%) vs. 100% (10–100%); p = 0.012] post processing, as well as blastulation [37% ± 0.32 vs. 50% ± 0.3; p = 0.031] and top-quality blastocyst [13.5% (0–100%) vs. 33% (0–100%); p = 0.009] rates, favoring the ZyMōtTM device. Limitations, reasons for caution The retrospective design and the small sample size make the study prone to bias. Furthermore, we do not have data on sperm DNA fragmentation pre and post processing. Clinical outcomes were not evaluated due to insufficient statistical power, since embryo transfer has not yet been accomplished in several patients. Wider implications of the findings: Besides the ZyMōtTM device has been indicated as a new tool to potentially improve laboratory and clinical IVF outcomes, it offers the advantage of being safe, fast, easy-to-use and less influenced by human errors. Further well-designed prospective studies are needed to prove its cost-effectiveness. Trial registration number Not applicable

Effects of L-carnitine combined with pancreatic kininogenase on thioredoxin 2, thioredoxin reductase 1, and sperm quality in patients with oligoasthenospermia.

BackgroundTo study the effects of L-carnitine (LC) combined with pancreatic kininogenase on thioredoxin 2 (Trx 2), thioredoxin reductase 1 (TrxR 1), and sperm quality in patients with oligoasthenospermia.MethodsA total of 300 male infertility patients with oligoasthenospermia who were treated in the andrology clinic of our hospital from December 2019 to December 2020 were randomly divided into an LC group and combined treatment group, and 50 males with normal semen were selected as a control group. The computer-assisted semen analysis system (CASA) was used to detect the total number, vitality, and forward motility of the sperm before and after treatment, and sperm morphology was detected by the Diff-Quik method of the sperm staining kit. Sperm chromatin dispersion (SCD) method was used to detect sperm DNA fragments, and Western-blot was used to detect the protein expression of Trx 2 and TrxR 1.ResultsThere were no significant differences in sperm density, motility rate, forward motile sperm rate, and DNA fragmentation rate in oligoasthenospermia patients before treatment (P>0.05). However, after 1 month of treatment, the sperm density, motility rate, and forward motile sperm rate were all higher than before treatment (P<0.05), while the DNA fragmentation rate was lower than before treatment. At the same time, each index of semen in the combination group was higher than that in the LC group (P<0.05), and the total effective rate in the combination group was significantly higher than in the LC group (P<0.01). The expression of Trx2 protein in oligoasthenospermia patients was significantly increased (P<0.05), while the expression of TrxR1 protein was significantly decreased (P<0.05). After 3 months of treatment, the expression of Trx2 protein was significantly decreased (P<0.05), while the expression of TrxR1 protein was significantly increased (P<0.05).ConclusionsThe results suggest Trx 2 and TrxR 1 may be candidate protein markers for oligoasthenospermia. LC combined with pancreatic kininogenase in the treatment of male oligoasthenospermia can effectively promote sperm maturation, enhance sperm motility, and improve semen quality, which has high application value.

Impact of Paternal Genome with a High DNA Fragmentation Index (&gt;60%) on Early Embryonic Development

: Objectives: The objective of this study is to propose thresholds of the sperm DNA fragmentation rate (IFA≤30% IFA31%-60% IFA&gt;60%), in order to assess the clinical effects of the paternal genome on intra cytoplasmic sperm injection parameters, in particular the effect of the latter on early embryonic development. Materials and Methods: The procedure is a retrospective study, which involved 101 patients enrolled in an ICSI program with their partners. The index of spermatic DNA fragmentation rate was measured using the Sperm Chromatin Dispersion assay. Results: There is a negative correlation between high levels of the spermatic DNA fragmentation index and spermiological characteristics: Concentration P=0.002 and mobility P=0.0001. For ICSI results, there are different observations on the existence of a correlation between the spermatic DNA fragmentation index and fertility rate. On the other hand, the rate of sperm DNA fragmentation does not seem to influence early embryonic development, and even couples whose partners have a high fragmentation index manage to obtain the best quality embryos (P=0.002). We observe a decrease in the rate of implantation with an increase in the rate of alteration of the sperm genome, but this remains insignificant P &gt; 0.05. Conclusion: ICSI remains the only alternative for men with a high rate of sperm DNA fragmentation. Moreover, the operator seems to influence the results more than is suggested. This does not exclude the paternal effect which may influence the quality of the concepltus later on. Keywords: DNA Fragmentation Index, ICSI, Fertilization Rate, Embryos Quality.

IT TAKES TWO TO TANGO: COMPARING SPERM PREPARATION TECHNIQUES AND THE EFFECT ON EMBRYO GRADING

Male factor infertility accounts for 40-50% of infertility among couples seeking assisted reproductive technology (ART). Research on a new parameter of sperm quality, known as DNA fragmentation, suggests that fragmentation rates >27% may hinder embryo development and increase rates of miscarraige, while lower fragmentation rates are associated with improved outcomes. Recently, microfluidic sperm sorting as a sperm selection method has shown to decrease DNA fragmentation rates by positively selecting healthy sperm, compared to the standard method of density gradient centrifugation.

Design, Synthesis, In silico and In Vitro Anticancer Activity of Novel Bis‐Furanyl‐Chalcone Derivatives Linked through Alkyl Spacers

AbstractA novel series of bis(furan‐based chalcone) derivatives linked to aliphatic linkers, with the furan units at the A‐ or B‐rings, were synthesized and evaluated as anticancer agents. Chalcones 5 a–c in which the furan ring designed to be a B‐ring were obtained from the Knoevenagel condensation reactions of the appropriate bis(acetyl) compounds 3 a–c with two equivalents of furan‐2‐aldehyde. Likewise, the condensation of bis(aldehydes) 7 a–c with two equivalents of 2‐acetylfuran afforded the corresponding chalcones 9 a–c, in which the furan rings represent the A‐rings of the chalcones, in excellent yields. The synthesized compounds have been fully characterized by 1H‐NMR, 13C‐NMR, and elemental analysis. The in vitro anticancer activity of the prepared compounds was tested against A549, HCT116, HePG2, PC3, A431 and BJ1 cell lines using MTT assay, gene expression analysis of skin and lung cancer related genes, comet assay, and DNA fragmentation assy. Compounds 9 a and 9 c were found to be the most promising, with IC50 (24.9 and 13.7 μg/ml, respectively) against A549 compared with doxorubicin (IC50, 28.3 μg/ml) and IC50 (26.1 and 14.4 μg/ml, respectively) against A431 in comparison to doxorubicin (IC50, 24.9 μg/ml). Comapred with neagtive control cell lines, the gene expression levels of hBD‐2 and hBD‐3 genes were decreased singnificantly (P&lt;0.05) in positive control and skin cancer cell lines (A431) treated with compounds 9 a and 9 c. The expression levels of FOSB and IL‐8 genes were increased singnificantly (P&lt;0.05) in positive control and lung cancer cell lines (A549 and A541) treated with compounds 9 a and 9 c, respectively, comapred with neagtive control cell lines. The DNA damage was increased significantly (P&lt;0.001) in treated skin and lung cell lines samples compared with negative control. The negative control skin and lung cancer cell lines showed a significant decrease (P&lt; 0.05) in DNA fragmentation rate compared with positive control. On the other hand, the DNA fragmentation rates were increased significantly (P&lt;0.001) in treated skin and lung cell lines samples compared with negative control. In summary, the synthesized compounds 9 a and 9 c showed a significant anticancer activity against lung and skin cancer cell lines compared with known chemotherapeutic drug (doxorubicin).

Predictive Significance of Sperm DNA Fragmentation Testing in Early Pregnancy Loss in Infertile Couples Undergoing Intracytoplasmic Sperm Injection.

ObjectiveThis study aimed to determine the role of sperm DNA fragmentation as a marker that could predict early pregnancy loss (EPL), either independently or in combination with another marker or markers, after intracytoplasmic sperm injection (ICSI) cycles.MethodsThis prospective descriptive cohort study retrieved data from 162 couples who underwent their first ICSI cycles at the Center for Reproductive Endocrinology and Infertility of Hue University Hospital in Vietnam from May 2018 to December 2019. General characteristics, semen parameters, sperm DNA fragmentation index (DFI), fertilization, embryo development, pregnancy rates, and EPL were assessed. The receiver-operating characteristic (ROC) method was performed to identify the threshold of DFI in EPL. Multivariate analysis was used to demonstrate the relationship between the sperm DNA fragmentation level and EPL.ResultsOf 162 ICSI cycles, 23 (14.2%) involved EPL. There was no significant difference between the sperm DNA fragmentation rate and the overall rate of pregnancy loss, although the negative pregnancy outcome group had a median DFI that was higher than that of the positive pregnancy outcome group (20% vs 17.8%). The ROC analysis indicated that a sperm DNA fragmentation rate of 16.6% was the priority cut-off that could be used to distinguish EPL with a sensitivity of 73.9% (95% confidence interval [CI], 67.15–80.67) and specificity of 47.48% (95% CI, 39.79–55.17). The multivariate analysis confirmed that in female factors such as age, body mass index (BMI), and sperm DNA fragmentation level affected the EPL rate. However, a combination of the sperm DNA fragmentation level and female age or female BMI could not sufficiently predict EPL.ConclusionEPL can be affected by multiple factors including sperm DNA fragmentation; however, there is no sufficient evidence indicating that sperm DNA fragmentation, both as a single marker and combined with other markers, is a good predictor of EPL.

Study of Sperm Dna Fragmentation Index in Patients from Rural Areas of Wardha Region

Background: Sperm DNA fragmentation test is an advanced test for proper sperm function with respectto semen analysis, as semen analysis is used for motility and morphology assessment, it cannot explainsperm at molecular level and thus SDF test has been introduced to evaluate male fertility. The integrity ofgenetic material in sperm is crucial for successful fertilization &amp; normal embryo development as this willlead to proper transmission of genetic information. Patients having varicocele, recurrent failure of ARTtecniques and improper lifestyle (radiation, smoking, alcohol, BMI, ageing, etc) are considered for spermDNA fragmentation test. SDF test is useful in selecting patients for the ART procedures i.e. IVF/ICSI/IUIand highest pregnancy and live birth outcomes.Objectives:· To predict male infertility with normal semen parameters &amp; its diagnostic values.· To investigate the clinical factors related to sperm DNA fragmentation in male with infertility.· To investigate the rate of sperm DNA fragmentation in male related to improper fertilization, embryodevelopment and recurrent pregnancy loss.Methods: The participants (n=40) with male fertility problems will be included in randomised control trial.All 40 participants will be randomly selected to evaluate their sperm DNA integrity from their respectivesemen samples using sperm chromatin dispersion test.Discussion: Efficiency of the Sperm DNA Fragmentation test is assessed by performing sperm chromatindispersion test on respective processed semen sample using Halosperm G2 test kit.

Spermatic profile of captive giant anteaters (Myrmecophaga tridactyla): Knowing more to preserve better

The giant anteater (Myrmecophaga tridactyla) is being threatened by natural habitat destruction and fragmentation, illegal hunting and road kills. In this context, the generation of basic information on the reproductive parameters of this species is vital, aiming to improve reproductive management via, amongst others, assisted reproductive technologies. This study aimed to describe the morphological and functional features of semen collected from captive giant anteaters. Electroejaculation was performed in 13 animals housed in zoos located in São Paulo state, Brazil. Semen samples were collected from 13 animals in 16 procedures. Samples were evaluated for volume, motility, vigor, pH, concentration, sperm morphology, and functional tests. The following mean values were obtained: volume 1.28 ± 0.27 mL; motility 28.3 ± 6.2%; vigor 2.4 ± 0.25; concentration 129.4 ± 36.1 × 106 sperm/mL; pH 7.4 ± 0.2. Total acrosome, head, midpiece, and tail sperm abnormalities were 3.2 ± 0.8%, 25.4 ± 3.6%, 20.7 ± 3.2%, and 14.7 ± 2.6%, respectively. Intact acrosome was found in 83.7 ± 3.1% and intact membrane in 81.1 ± 4.0% of all samples collected. Mitochondrial activity was 66.4 ± 6.0% (Class I), 18.7 ± 2.9% (Class II), 8.0 ± 2.0% (Class III), 3.9 ± 1.0% (Class IV), and 3.0 ± 0.9% (Class V). Sperm DNA fragmentation rate was 13.2 ± 3.7%. These results indicated that electroejaculation is a feasible method for semen collection in giant anteaters, allowing a more detailed description of the semen in this species.

The impact of varicocelectomy on sperm DNA fragmentation and pregnancy rate in subfertile men with normal semen parameters: A pilot study

ABSTRACT Objective: To evaluate the outcome of microscopic subinguinal varicocelectomy on sperm DNA fragmentation (SDF) and pregnancy rate in men with normal semen parameters. Patients and methods: A pilot study that included male patients with a minimum of a 1-year history of male subfertility, normal semen parameters, a high percentage of SDF, and clinically palpable varicoceles. Microscopic subinguinal varicocelectomy was carried out for 45 patients (study group), while 40 patients had no intervention (control group). Semen analysis and SDF were measured before and at 6 months after the varicocelectomy. The pregnancy rate was assessed at the 6- and 12-month follow-ups. Results: Between July 2014 and January 2019, 85 subfertile men were included in the study and completed 12 months of follow-up. The two groups were comparable in terms of their age, body mass index, infertility duration, infertility type, varicoceles laterality, and varicoceles grade (P values = 0.84, 0.34. 0.35, 1, 0.39, and 0.46, respectively). At 6 months after varicocelectomy, the mean SDF was reduced in both groups, and this reduction was statistically higher in the varicocelectomy group (P < 0.001). After 1-year, spontaneous pregnancy was achieved in 62% of the patients in the varicocelectomy group compared to 30% in the control group (P = 0.009). Conclusion: Varicocelectomy has a positive impact on SDF and spontaneous pregnancy in infertile men with clinically palpable varicoceles and normal semen parameters. Abbreviations: BMI: body mass index; DFI: DNA fragmentation Index; SDF: sperm DNA fragmentation

Healthy ageing and spermatogenesis.

Delayed family planning and increased parental age increase the risk for infertility and impaired offspring health. While the impact of ageing on oogenesis is well studied, this is less understood on spermatogenesis. Assessing ageing effects on the male germline presents a challenge in differentiating between the effects of ageing-associated morbidities, infertility and 'pure' ageing. However, understanding the impact of ageing on male germ cells requires the separation of age from other factors. In this review, we therefore discuss the current knowledge on healthy ageing and spermatogenesis. Male ageing has been previously associated with declining sperm parameters, disrupted hormone secretion and increased time-to-pregnancy, among others. However, recent data show that healthy ageing does not deteriorate testicular function in terms of hormone production and spermatogenic output. In addition, intrinsic, age-dependent, highly specific processes occur in ageing germ cells that are clearly distinct from somatic ageing. Changes in spermatogonial stem cell populations indicate compensation for stem cell exhaustion. Alterations in the stem cell niche and molecular ageing signatures in sperm can be observed in ageing fertile men. DNA fragmentation rates as well as changes in DNA methylation patterns and increased telomere length are hallmarks of ageing sperm. Taken together, we propose a putative link between the re-activation of quiescent Adark spermatogonia and molecular changes in aged sperm descending from these activated spermatogonia. We suggest a baseline of 'pure' age effects in male germ cells which can be used for subsequent studies in which the impact of infertility or co-morbidities will be studied.

Application of leukocyte subsets and sperm DNA fragment rate in infertile men with asymptomatic infection of genital tract.

About 15% of male infertility is due to genital tract infection and inflammation, some of them have no clinical symptoms, but manifested as leukocytospermia (LCS). Leukopenia will lead to functional impairment of male sperm and integrity damage of sperm morphology. A large amount of reactive oxygen species (ROS) produced by leukocytes can damage sperm nuclear DNA. The aim of this study was to investigate the correlation between leukocyte subsets and sperm DNA fragmentation rate in semen of infertile men with asymptomatic infection of genital tract. One hundred and eight cases of infertile men were enrolled, who were admitted to our hospital from May 2016 to September 2018, and all had genital tract infections. After routine sperm analysis, realtime PCR was performed for detecting the infection of chlamydia trachoma (CT) and Ureaplasma urealyticum (UU). Furthermore, total leukocyte count in semen was evaluated by detection of CD45 molecules using immunocytochemistry. Flow cytometry was used for subset analysis, monocyte/macrophages were evaluated by CD14, and activated macrophages were evaluated by HLA-DR antigen. Sperm DNA fragmentation index (DFI) were evaluated by SCD method and 8-hydroxydeoxyguanosine (8-OHdG) expression were evaluated by chromatin diffusion method and TUNEL method; the correlation analysis was conducted between semen leukocyte subsets, sperm DNA fragment rate and conventional semen parameters. There was a significant correlation among the concentrations of cells expressing HLA-DR antigen and those expressing CD14 (P<0.01), but the concentrations of differential leukocyte subsets all had no significant correlation with sperm DFI, the percentage of 8-OHdG-expressing cells and routine semen parameters. The percentage of 8-OHdG-expressing sperm was positively correlated with the percentage of sperm fragments (r=0.42, P<0.01), and negatively correlated with sperm concentration (r=-0.32, P<0.01). After adjustment for possible confounders including age, abstinence time and smoking, the percentage of 8-OHdG-expressing sperms independently associated with sperm concentration (β=-0.30; P=0.006) and DFI (β=0.180, P=0.06). Among infertile men with genital tract infection, the sperm DFI is associated with decreased semen quality and not the concentration of differential leukocyte subsets.

Microfluidic sperm selection yields higher sperm quality compared to conventional method in ICSI program: A pilot study

ABSTRACT An appropriate preparation technique, should be capable of isolating highquality spermatozoa for intracytoplasmic sperm injection (ICSI). The aim was to assess sperm quality parameters, DNA integrity, embryo development, and clinical outcomes using a practical and accessible Microfluidic Sperm Sorting (MSS) technique. A total of 95 ICSI cases performed using sperm samples were prepared with our MSS (group 1) or by Direct Swim Up (DSU; control) method (group 2). Both sperm quality parameters and sperm DNA fragmentation (SDF) were compared between the groups. DNA fragmentation was assessed using Sperm Chromatin Dispersion (SCD) test and fine morphology was assessed using Motile Sperm Organelle Morphology Examination (MSOME). Embryo development and clinical outcomes were compared between the groups. In the MSS group, progressive motility and the fraction of Class I sperm morphology sperm were significantly higher compared to DSU group (P < 0.01 and P < 0.001, respectively). Moreover, the rates of DNA fragmentation and immotile spermatozoa were significantly lower in MSS when compared to DSU group (P < 0.001). Also, higher rates of high-quality embryo formation (P < 0.001), implantation (P = 0.04) and pregnancy (P = 0.05) were achieved in the MSS compared to DSU groups. The MSS technique proved to be a noninvasive, disposable, easy to use, and inexpensive method for separation of high-quality spermatozoa. Both laboratory parameters and clinical outcomes were improved with application of MSS for neat sperm collection in ICSI. Abbreviations ICSI: Intracytoplasmic Sperm Injection; MSS: Microfluidic Sperm Sorting; Sperm DNA Fragmentation (SDF); SCD: Sperm Chromatin Dispersion; MSOME: Motile Sperm Organelle Morphology Examination; DGC: Density Gradient Centrifugation; DSU: Direct Swim Up; ROS: Reactive Oxygen Species; ART: Assisted Reproducetive Technology

Sperm DNA Fragmentation Has a Negative Impact on Cumulative Live Birth Rate of IVF-ET/ICSI in Patients with Oligozoospermia

The relationship between sperm DNA fragmentation and in vitro fertilization (IVF) outcome was uncertain today. In the past, researchers studying the pregnancy outcome of IVF, mainly base on fresh embryo transplantation cycle, recently, cumulative live birth rate was consider as a new indicator of IVF successful rate which is used to evaluate the pregnancy outcome by fresh or thawed transferred embryos until live birth or use up all embryos in one oocyte collection cycle, is more comprehensive. The purpose of this research is to investigate the relationship between sperm DNA fragmentation and cumulative live birth rate. 1019 couples who were assisted by IVF in our center from Jan 2012 to Dec 2016 were studied according to inclusive criteria, and divided into four groups by DNA fragmentation index (DFI), which were determined by sperm chrome structure assay (SCSA), then the fertilization rate, No. of Embryo available and cumulative live birth rate between the groups were analyzed. Cumulative live birth curve was plotted to study the time to reach live birth, and Cox regression model was used in order to find out the factors affecting pregnancy outcome. Study results showed no significant difference in cumulative live birth rate between the four groups, but DFI ≥40% group were significantly lower than the other three groups. In the cumulative live birth curve figure, except the high DFI group, the curve of the other three groups is relatively close. Cox regression model suggest that DFI was not the main factor affecting pregnancy outcome. Further studies in these patients with oligozoospermia showed that when DFI increase, a decrease trend was appeared in cumulative live birth rate, and spend more time to reach live birth studied by the live birth curve. Cox model showed that DFI was the impact factor of pregnancy outcome in oligozoospermia patients. In conclusion, abnormal sperm DNA fragmentation have a negative impact on cumulative live birth rate in patients with oligozoospermia, but further research was still needed.

IUI: Optimizing results, minimizing complications

Intrauterine insemination is a procedure in Assisted Reproductive Techniques (ART) where processed and concentrated motile sperms are placed directly into the uterine cavity. Although NICE [2013] recommends In Vitro Fertilisation over intrauterine insemination after 2 years of unprotected intercourse, Intrauterine Insemination (IUI) appears to be a low cost and effective option before proceeding for In Vitro Fertilisation (IVF). Minimal requirement for IUI is patency of at least one fallopian tube. Adequate number of motile sperms should be present in pre wash specimen of semen. At total sperm count 3–5 million, pregnancy rate is less than 1% per cycle. In unexplained infertility there may be undetected subtle functional defects in follicular development, maturation and ovulation like luteinised unruptured follicle, luteal phase defect. Ovarian stimulation increases the number of dominant follicles and improves their quality resulting in improvement in pregnancy rate. Single IUI has benefits of fewer visits, less cost and stress. Evidence suggests no difference in clinical pregnancy rate between single versus double IUI. WHO recommends ejaculatory abstinence of 2–7 days before semen collection (WHO, 2010), for diagnostics and semen preparation. Shorter time interval between processing and insemination leads to less sperm chromatin decondensation & sperm DNA fragmentation and higher Pregnancy Rate. The luteinizing hormone surge can be detected almost 36 hours before ovulation in serum and 24 hours before ovulation in urine. The optimal time interval between human chorionic gonadotropin (hCG) injection and IUI seems to be between 12 and 36 hours; 24 hours after leutinising hormone (LH) Surge. Nature of sperms is the best guide for choice of technique. “Swim up” techniques are recommended in cases of normozoospermia and “Density Gradient” should be chosen in cases of any pathology of semen. The pregnancy rate per cycle is highest in the first three treatment cycles. Most couples show acceptable cumulative ongoing pregnancy rates after six cycles of IUI with ovulation induction. In vitro fertilization should be considered after 3–6 cycles taking all factors into consideration.

Toxicity evaluation of Eleutherine plicata Herb. extracts and possible cell death mechanism.

Eleutherine plicata has been shown to be a promising medicinal plant, and its activity has been associated with naphthoquinones. The present study aimed at evaluating the cytotoxicity, genotoxicity, and oral toxicity of the ethanol extract (EEEp), dichloromethane fraction (FDMEp) of E. plicata, and isoeleutherin. For the cytotoxicity evaluation, the viability test (MTT) was used. Genotoxicity was accessed through the Comet assay (alkaline version), acute and subacute oral toxicities were also evaluated. The antioxidant capacity of the samples in the wells where the cells were treated with E. plicata was evaluated. Furthermore, the participation of caspase-8 in the possible mechanism of action of isoeleutherin, eleutherin, and eleutherol was also investigated through a docking study. FDMEp and isoeleutherin were cytotoxic, with higher rates of DNA fragmentation observed for FDMEp and isoeleutherin, and all samples displayed higher antioxidant potential than the control. In the acute oral toxicity test, EEEp, FDMEp, and isoeleutherin did not cause significant clinical changes. In the subacute toxicity assay, EEEp and FDMEp also did not cause clinical, hematological, or biochemical changes. The three compounds bound similarly to caspase-8. Despite the results of cytotoxicity, in vitro studies demonstrated that the use of EEEp appears to be safe and cell death may involve its binding to caspase-8.