Protective Effect of Raphanus sativus Seed Extract on Damage Induced by In Vitro Incubation and Cryopreservation of Human Spermatozoa.

Sperm selection by swim-up in terms of deoxyribonucleic acid fragmentation as measured by the sperm chromatin dispersion test is altered in heavy smokers

Processing of semen can result in increased sperm DNA fragmentation

Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate

Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection

Novel technologies for selecting the best sperm for in vitro fertilization and intracytoplasmic sperm injection

Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study

Study question Is the sperm chromatin dispersion (SCD) test reliable for predicting reproductive technology (ART) treatment outcomes? Summary answer Assessment of DNA fragmentation using the SCD test failed to predict pregnancy or live births rates. What is known already The detrimental effect of high DNA fragmentation on semen quality, fertility, and poor ART outcomes has been well-studied in recent years. Still, it remains contentious. Several techniques can detect sperm DNA fragmentation, such as TUNEL, comet assay, and SCSA. Although these techniques have been implemented in many andrology laboratories, they remain complex, time-consuming, and expensive. On the other hand, the SCD test indirectly estimates the DNA fragmentation level by manually quantifying the number of halos representing nuclear dispersion after sperm lysis and acid denaturation. While the SCD test is commonly used, it remains subjected to bias and high variability. Study design, size, duration Systematic review and meta-analysis. A PRISMA-guided literature search in the English language was performed in January 2021 using PubMed/MEDLINE, Scopus, and Google scholar. We looked for rates of fertilization, cleavage rate embryos, implantation, clinical pregnancy, miscarriage, and live birth. MedCalc software using the random effects model was used, Sensitivity analysis was conducted to examine heterogeneity and the robustness of the results. Participants/materials, setting, methods After duplicate removal, 179 abstracts were assessed for eligibility, and 55 full-text articles were screened. Forty-one articles were included in the qualitative syntheses, and 16 were included in the quantitative synthesis, representing 6989 participants. Main results and the role of chance No significant difference was identified between the group with high SDF vs. low SDF regarding fertilization (SMD −0.334, 95% CI −0.695, 0.027, p = 0.07), clinical pregnancy (OR 0.901, 95% CI 0.719, 1.130, p = 0.367), miscarriage (OR 0.901, 95% CI 0.719, 1.130, p = 0.367) and live birth (OR 1.105, 95% CI 0.627, 1.946, p = 0.730) rates. The rates of cleavage stage embryos (SMD -1.153, 95% CI -2.113, −0.194, p = 0.019) and implantation (OR 0.472, 95% CI 0.303, 0.737, p = 0.001) were significantly lower in the group with high SDF vs. low SDF. Limitations, reasons for caution A high index of heterogeneity and publication bias was reported regarding fertilization (I2 = 93.23%, Egger’s test: intercept -5.29866, 95% CI -10.41, −0.19, p = 0.02) and cleavage-stage embryos(I2 = 98.49%, Egger’s test: intercept -14.02395, 95% CI -27.47, −0.57, p = 0.02) Wider implications of the findings Assessment of DNA fragmentation using the SCD test failed to predict pregnancy or live births. As the goal of ART is live birth, clinicians should consider the limited yield of this assay to predict the likelihood of live birth. Trial registration number NA

The determination of reproductive safety in men during and after cancer treatment

Sperm chromatin dispersion (SCD) test identifies more spermatozoa with DNA damage than the TUNEL assay in men with unexplained infertility

Study question In men undergoing fertility treatment, is microfluidic device a superior processing option compared to Density Gradient Centrifugation (DGC) in terms of DNA Fragmentation Index (DFI)? Summary answer Microfluidic sperm sorting devices appear to provide sperm populations with reduced DNA fragmentation compared to DGC. What is known already DFI has been proposed as an additional predictor of male fertility, as elevated sperm DNA fragmentation has been associated with poor clinical outcomes in Assisted Reproductive Technology (ART). So far, numerous studies have investigated the potential adverse effects of DGC on sperm motility, viability and DNA integrity. Microfluidic devices (MFD) address this challenge by sorting a smaller population of progressively motile and morphologically normal spermatozoa with the required DNA integrity. At the moment, there is no consensus on the most appropriate sperm processing method for optimizing outcomes. Study design, size, duration This blinded, controlled observational clinical study included 34 men undergoing ART and was conducted at Embryolab Fertility Clinic, Thessaloniki, Greece, from June 2023 to December 2024. Each semen sample was evaluated for DFI in its unprocessed state, after DGC and after processing with MFD. DFI was assessed in 500 spermatozoa per sample, using the same sperm chromatin dispersion test. The evaluations were performed by three experienced embryologists, and were blinded to the processing method. Participants/materials, setting, methods Sperm samples were collected via masturbation and were allowed to liquefy at room temperature. Basic semen analysis was conducted according to World Health Organization (WHO) guidelines. The mean age was: 41.5±8.07. DFI was assessed and interpreted as the percentage of spermatozoa with fragmented DNA. Exclusion criteria included retrograde ejaculation, surgically retrieved sperm, severe oligo-astheno-teratospermia, and azoospermia. The cut off limit for DFI was 30%. Main results and the role of chance The statistical software R was utilized for data analysis (age and by-subject variability were accounted for). A p-value below 0.05 was taken to indicate that the difference observed reached statistical significance. Both processing methods demonstrated statistically significant differences compared to the DFI of unprocessed semen samples. The mean DFI of the raw samples was found to be 20.88±15.39%. The DFI of sperm processed using a microfluidic device was 8.29±7.42% (p < 0.05), while the DFI of sperm processed with DGC was 43.09±23.77% (p < 0.05). The difference in DFI between the two processing methods (MFD versus DGC) was 34.79±24.9% (p < 0.05). Subsequently, the samples were divided into two groups based on their raw sample DFI: Group A with normal DFI (24 samples) and Group B with abnormal DFI (10 samples). Using the microfluidic device revealed a statistically significant decrease of DFI in both groups (Group A, p < 0.05 and Group B, p < 0.05) while a similar statistically significant increase in DFI was observed with DGC (Group A, p < 0.05 and Group B, p = 0.0025). Comparing the two sperm processing methods (MFD and DGC) in both groups (A and B) the difference in DFI was 28.71±22.34% in Group A and 49.4±24.33% in Group B. Limitations, reasons for caution The study was performed on a small sample size and did not include clinical outcomes. Wider implications of the findings The outcome of the study indicates that the use of the MFD yields spermatozoa with lower DFI, compared to DGC, improving overall patient prognosis by counterbalancing paternal factor. This finding was consistent to all samples, independent of the DFI in the unprocessed samples. Trial registration number No

Comparación entre el test de fragmentación de ADN espermático mediante la técnica de SCD y el índice de vitalidad medida con el test de naranja de acridina

Koala (Phascolarctos cinereus) sperm nuclei show a tendency to swell after cryopreservation, but it is uncertain whether this phenomenon is associated with DNA fragmentation. In this study, we validated a modified version of the sperm chromatin dispersion test (SCDt) for use with koala spermatozoa, which is the first use of the test for a marsupial. Cryopreserved spermatozoa (multiple straws) from a single koala were used to explore the relationship between sperm morphology, viability, chromatin dispersion, and DNA fragmentation. A SCDt prototype kit (Sperm Halomax) was specifically developed for koala spermatozoa with the use of a lysing solution that did not contain dithiothreitol. DNA fragmentation of lysed and nonlysed spermatozoa was examined in microgel slides and validated by means of in situ nick translation (ISNT). The SCDt was then applied to the analysis of extended and frozen-thawed semen samples of 3 different koalas. Spermatozoa were classified into 3 distinct koala sperm morphotypes (KSMs) after the SCDt: 1) KSM-1, rod-shaped cells with no halo of DNA; 2) KSM-2, rounded nuclei with various degrees of halo formation about a dense chromatin core; and 3) KSM-3, rod-shaped or rounded nuclei consisting of an inner chromatin core but with large dispersed halos of stellar chromatin. Although ISNT after the SCDt did not label KSM-1, both KSM-2 and KSM-3 stained positively for DNA fragmentation. ISNT was not able to differentiate between KSM-2 and KSM-3. Although application of the SCDt to the spermatozoa of another 3 koalas showed no difference in the percentage of the 3 sperm morphotypes found between extended and frozen-thawed semen, thawed spermatozoa incubated at 35 degrees C for 2 hours showed an increase in the incidence of KSM-3 and a corresponding decrease in KSM-2. We propose that KSM-1 and KSM-2 represent nuclei that show either no, or only limited, sperm DNA fragmentation, respectively. It is likely that the halos formed around KSM-2 are from DNA that is damaged as part of the normal processing of the spermatozoa and is a consequence of the lack of cysteine residues and associated stabilizing disulfide bonds in marsupial sperm DNA. "True" sperm DNA damage is most likely associated with KSM-3, which shows a massive dispersion of chromatin similar to that described in other species. A model of koala sperm chromatin structure is proposed to explain the behavior of the sperm nuclei after the SCDt. Further studies are required to determine whether DNA damage found in KSM-2 is indicative of single-stranded DNA breakage associated with an inherent lack of cysteine residues in marsupial sperm chromatin. Conversely, it will also be important to establish whether KSM-3 is caused by an increased incidence of double-stranded DNA breakage and whether this abnormality is correlated with impaired fertility as it is in other species.

Effect of exogenous β-nicotinamide adenine dinucleotide phosphate reduced (NADPH) on Sperm DNA integrity

There are extremely limited studies that have investigated DNA fragmentation in amphibians and even less information exists pertaining to DNA fragmentation in the spermatozoa. A DNA fragmentation assay applied and validated for amphibian spermatozoa would not only provide the first evidence of this phenomenon in a new major taxon, but also prove a valuable tool for developing improvements in ART used in the captive propagation of threatened and endangered species. Consequently, the fundamental aim of this project was the development and validation of the sperm chromatin dispersion (SCD) test for amphibian spermatozoa with the purpose of investigating the dynamic behaviour of amphibian sperm DNA in response to cryopreservation. The SCD test was first applied to African clawed frog (Xenopus laevis) as an amphibian sperm model using a species-specific modified lysing solution for protein depletion. Sperm DNA fragmentation (SDF) was assessed immediately following activation of sperm motility (T0) and again after one hour and 24 hours of incubation in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. Levels of SDF revealed by the SCD test were highly correlated with results produced using in situ nick hybridisation with DNA-specific molecular probes (ISNT) and the double comet assay (r = 0.9613). The alkaline step of the double-comet assay revealed the extensive presence of structural single-stranded DNA breaks (SSB) and provided grounds for the suggestion that alkali labile sites (ALS) are a constitutive feature of African clawed frog sperm chromatin. SDF is a dynamic process and has been shown to increase with incubation at room temperature as well as following cryopreservation. However, the mechanisms of DNA cryoinjury and the resulting effect on the potential reproductive output of ART remain open to interpretation. Consequently, dynamic changes to the basal level of DNA fragmentation were examined in fresh African clawed frog spermatozoa in comparison with the post-thaw integrity of cryopreserved (-80 °C) and frozen (-20 °C) sperm DNA. SDF was examined initially at T0 and over incubation of up to 60 minutes. The difference in SDF was only marginal for fresh, frozen (-20 °C) and cryopreserved (-80 °C) spermatozoa examined at the onset of the experiment immediately upon thawing, however, a significant increase (p = 0.004) in SDF, albeit of a low magnitude, was observed in activated cryopreserved (-80 °C) spermatozoa following incubation. Although African clawed frog spermatozoa appeared to be highly resistant to cryopreservation-induced DNA damage, cryopreserved-thawed spermatozoa yielded a significantly lower (p = 0.0065) fertilisation rate than fresh spermatozoa. In order to provide further insight into cryoinjury of amphibian sperm chromatin, the dynamic loss of DNA integrity in fresh and cryopreserved cane toad (Bufo marinus) spermatozoa was correlated with changes in sperm viability and motility. Analysis of SDF was conducted at T0 immediately following activation and/or thawing and again following T3 and T6 hours of incubation at room temperature. Sperm motility was activated by decreasing the osmolality of the sperm extender to < 50 mOsmol kg-1 with dH20. Sperm viability was assessed using a dual emission technique that incorporated the nucleic stains SYBR-14 and propidium iodide. Spermatozoa were classified as either live (green fluorescence) or dead (red fluorescence). Dead spermatozoa were further classified into two categories; dead-compact, where spermatozoa retained a normal elongated, rod-like morphology and dead-swollen, where spermatozoa appeared enlarged with less defined morphology. Assessment of SDF at the population level in fresh spermatozoa, as well as the dynamics of sperm chromatin longevity, revealed that cane toad sperm DNA was highly stable throughout the duration of the incubation period. Results demonstrated a surprisingly poor recovery of both motility and viability in cane toad spermatozoa following freeze-thawing and a significant increase in the proportion of spermatozoa showing nuclear swelling (p < 0.0001). The incidence of dead-swollen spermatozoa observed in the cane toad was interpreted to represent the transition from ASM-1 to ASM-2 described in African clawed frog spermatozoa and lent support to the hypothesis that ALS may exist in the sperm chromatin of both amphibians.

Study question Does sperm DNA integrity affect clinical outcomes of ICSI? Summary answer Use of high DNA integrity sperm selected by microfluidics sperm sorting results in lower miscarriage rates in the patients of 39-years old and more. What is known already High sperm DNA damage is associated with decreased normal fertilization, embryo development and pregnancy rates, and an increased miscarriage rate. On the other hand, oocytes from older women have decreased pregnancy rate, and increased miscarriage rate because of possibility of low ability to repair sperm with DNA fragmentation, and dramatical increases of aneuploidy as women age. A microfluidic sperm selection chamber (MSS, ZyMōt™; DxNow) is a device designed to collect sperm with higher chromatin integrity than density gradient centrifugation (DGC). Study design, size, duration Sperm analysis was performed by sperm chromatin dispersion (SCD) test and comet assay in the same sample of 15 cases between October 2020 and February 2021. ICSI outcomes by DGC and MSS were compared with blastocyst development, and pregnancy rates in vitrified-thawed single blastocyst transfers cycle for 518 cases between August 2018 and May 2021. Participants/materials, setting, methods SCD test was optimized as a rapid procedure, with sperm showing a halo deemed normal, and those without a halo abnormal. Comet assay results were analyzed using CometScore 2.0, with comparison of %Tail DNA. ICSI outcomes were analyzed using multiple logistics regressions of male and female ages. Main results and the role of chance We found a positive correlation between male age and sperm DNA fragmentation rates in raw semen using SCD test (r = 0.70) and Comet assay (r = 0.42). Higher DNA integrity sperm could select using MSS than DGC. In this study with ICSI outcomes, 170 of 318 (53.5%) blastocyst transfers resulted in pregnancy, and 49 (28.8%) subsequently miscarried. The data were classified according to less than or more than 39 years old of male age detected by multiple logistics regressions. In patients with ≥39 years of male age, the female age was significantly higher and blastocyst and pregnancy rates were significantly lower, and the miscarriage rate was significantly higher than &amp;lt;39 years of male age. Since sperm DNA fragmentation increased in accordance with male age, we compared MSS and DGC in the patients with male age ≥39 years. There was no significant difference in blastocyst, pregnancy, and miscarriage rates in female age &amp;lt;39 years. While in ≥ 39 years of female age, blastocyst and pregnancy rates in MSS were not significantly different from DGC, but the miscarriage rate in MSS was significantly lower than in DGC (27.3 vs. 57.1%). Limitations, reasons for caution The sample size for each study was small. Analysis of sperm DNA fragmentation and samples in ICSI outcomes were not the same. The retrospective nature of ICSI outcomes in this study does not allow controlling of unknown confounders. Wider implications of the findings Sperm DNA fragmentation depended on male age affected fertility outcomes. However, when male age is higher, masking the effect of male age by female age. In this study, we found out the improvement of ICSI outcome by using high DNA integrity sperm selected by MSS in both ≥39 years. Trial registration number Not applicable