DNA Fragmentation Rate Research Articles

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.

Cellular and Molecular Events that Occur in the Oocyte during Prolonged Ovarian Storage in Sheep.

Simple SummaryEstablishing efficient in vitro embryo production (IVP) protocols in sheep usually requires prolonged transportation of post-mortem ovaries since adult animals are often slaughtered in abattoirs far from laboratories. In this study, different analyses were carried out to investigate important cellular and molecular aspects of hypoxic injury on excised ovaries over time in order to understand the factors jeopardizing the development of competent oocytes during prolonged transport times. We observed that, when ovaries were stored for more than 7 h, the quality and developmental potential of oocytes and cumulus cells were greatly reduced. Moreover, the use of medium TCM199 over saline solution also had deleterious effects. Beyond transport time, strategies aimed at reducing these damages may improve oocyte quality and developmental competence.For the past two decades, there has been a growing interest in the application of in vitro embryo production (IVP) in small ruminants such as sheep. To improve efficiency, a large number abattoir-derived ovaries must be used, and long distances from the laboratory are usually inevitable when adult animals are used. In that scenario, prolonged sheep ovary transportation may negatively affect oocyte developmental competence. Here, we evaluated the effect of ovary storage time (3, 5, 7, 9, 11 and 13 h) and the medium in which they were transported (TCM199 and saline solution) on oocyte quality. Thus, live/dead status, early apoptosis, DNA fragmentation, reduced glutathione (GSH) and reactive oxygen species (ROS) content, caspase-3 activity, mitochondrial membrane potential and distribution, and relative abundance of mRNA transcript levels were assessed in oocytes. After in vitro maturation (IVM), cumulus cell viability and quality, meiotic and fertilization competence, embryo rates and blastocyst quality were also evaluated. The results revealed that, after 7 h of storage, oocyte quality and developmental potential were significantly impaired since higher rates of dead oocytes and DNA fragmentation and lower rates of viable, matured and fertilized oocytes were observed. The percentage of cleavage, blastocyst rates and cumulus cell parameters (viability, active mitochondria and GSH/ROS ratio) were also decreased. Moreover, the preservation of ovaries in medium TCM199 had a detrimental effect on cumulus cells and oocyte competence. In conclusion, ovary transport times up to 5 h in saline solution are the most adequate storage conditions to maintain oocyte quality as well as developmental capacity in sheep. A strategy to rescue the poor developmental potential of stored oocytes will be necessary for successful production of high-quality embryos when longer ovarian preservation times are necessary.

Dynamic assessment of human sperm DNA damage III: the effect of sperm freezing techniques.

Among the currently available strategies for sperm freezing, vitrification may be considered as the leading alternative to conventional cryopreservation. Nevertheless, a direct comparison of both techniques with respect to the iatrogenic sperm DNA damage has not been performed yet. As such, this study was focused to assess the static and dynamic behavior of human sperm DNA damage following thawing of cryopreserved or vitrified spermatozoa. Semen samples were obtained from fifty donors with a normal spermiogram, and divided into four aliquots. The first aliquot represented the neat sample. In the second aliquot the seminal plasma was discarded, and the resulting sperm pellet was resuspended in PBS. The third fraction was used for slow freezing and the fourth fraction was subjected to vitrification. Each set of samples was incubated at 37°C for 24h and sperm DNA damage (SDF) was assessed using the chromatin-dispersion test following 0h, 2h, 4h and 24h of incubation. When comparing the rate of DNA fragmentation (r-SDF) at 2h, significant differences were observed between the PBS group, cryopreserved (p .000) or vitrified semen (p .015). Furthermore, the sperm longevity comparison using Kaplan-Meier survival curves revealed significant differences between cryopreservation and vitrification (p .000). Our data suggest that exposure of spermatozoa to low temperatures, independently of the chosen freezing protocol, leads to a higher susceptibility of sperm DNA towards damage. This damage is nevertheless lower following vitrification in comparison to traditional cryopreservation. As vitrification leads to a smaller proportion of spermatozoa with DNA damage, we may recommend its use in reproductive techniques which rely on a longer sperm survival, such as artificial insemination.

Our experience of application of low-level laser therapy in red spectrum in male idiopathic secretory infertility

Introduction. Due to insufficient efficacy of medical therapy of idiopathic secretory male infertility there is a need to develop and apply more resultative methods of treatment.
 The aim of our study was to assess the efficacy of low-level laser therapy (LLLT) in red spectrum on sperm parameters, sperm DNA fragmentation (SDNAF) and MAR-test rate in men with idiopathic infertility.
 Patients and methods. 85 men with idiopathic infertility were examined, 50 of them (first group) were treated with LLLT and 35 (second group) didnt have course of LLLT. Average age of patients of the first and second groups was 31.4 1.3 and 32.3 1.4 years. LLLT was performed in red spectrum on a Rubin-C device (Russia). 10 laser therapy procedures were carried out every two days with active luminescence for 10 minutes of both testicles. Male hormone panel, semen analysis, MAR-test, SDNAF were assessed before and after the treatment.
 Results. LLLT in patients of the first group resulted in significant increase in ejaculate volume, sperm viability, number of morphologically normal sperm forms (by an average of 11, 9 and 23% respectively), and active mobile sperm forms by an average of 14 and 19% one and two months after the therapy respectively. In 12 patients of the first group with initially increased SDNAF its level became normal after the treatment. Significant decrease of MAR-test we searched out in patients of the first group with its initial level less than 30%. As a result of the treatment, pregnancies developed in 10 (20%) out of 50 couples in the first group.
 Conclusions. LLLT in red spectrum leads to significant improvement of sperm quality in patients with idiopathic infertility, normalizes sperm DNA-fragmentation level and decreases MAR-test level.

Effect of ejaculatory abstinence period on sperm DNA fragmentation and pregnancy outcome of intrauterine insemination cycles: A prospective randomized study.

To evaluate the effect of the ejaculatory abstinence period on sperm DNA fragmentation and pregnancy rates in IUI cycles. One hundred and twenty couples with unexplained infertility were prospectively randomized into two groups on the second day of their cycle. In group A, patients had 1-day ejaculatory abstinence period; in group B patients had 3-day ejaculatory abstinence period. Each patient was stimulated with gonadotropins, and IUI was performed. The primary outcome measure was clinical pregnancy and sperm DNA fragmentation rate. The pregnancy rate was 17.3% and 18.5% in group A and group B, respectively. No significant difference was found among the groups (p = 0.803). The sperm DNA fragmentation rate for group A (20.71 ± 11.01) and group B (23.78 ± 12.64) were almost similar (p = 0.187). The sperm DNA fragmentation rate was 24.89 ± 12.89 in pregnant couples and 21.71 ± 11.69 in non-pregnant couples (p = 0.288). Even after a short abstinence period of 1day, compared to the standard abstinence period of 3days, pregnancy rates are similar among unexplained infertility couples undergoing ovarian stimulation and IUI. The study was registered at ClinicalTrials.gov with ID NCT04361292. Date of registration: 27 April 2020. The study was registered retrospectively.

Diagnostic performance of discontinuous density gradient centrifugation for estimating human semen quality.

Semen analysis plays an important role in the diagnosis of male infertility. However, many studies have demonstrated that the current methods of semen analysis are inefficient for assessing male fertility. To test whether prior discontinuous density gradient centrifugation (DDCG) improves the performance of semen analysis in diagnosing male infertility. Infertile men and fertile men were recruited from the clinic. Pre- and post-DDGC values for the semen parameters of sperm concentration, total sperm number, percent total motility, percent progressive motility, percent normal sperm morphology, and sperm DNA fragmentation rate were compared. A total of 528 men (252 infertile men and 276 fertile men) were enrolled in the present study. After DDGC, sensitivity was significantly increased for sperm concentration, total sperm number, and sperm morphology (P<.01); specificity was significantly increased for progressive motility and sperm morphology (P<.01); and diagnostic accuracy was significantly improved for all of these parameters (area under the curve (AUC): P<.01). Total motility and sperm DNA fragmentation rate exhibited no obvious change in sensitivity, specificity or accuracy after DDGC (all P>.01). For the combination of all these semen parameters, diagnostic accuracy improved significantly after DDGC (AUC: P<.01). In a multiple regression analysis, only sperm morphology and sperm DNA fragmentation rate had P values less than 0.05 before DDGC, whereas all parameters except total sperm number contributed to the equation after DDGC. DDGC is a mature, standardized procedure for clinical commonly used to optimize spermatozoa. The diagnostic accuracy of semen analysis was significantly improved after DDGC, which indicated that assessing "functional spermatozoa" might be a more suitable method for semen analysis than the WHO 2010 criteria. Assessing semen parameters after DDGC might improve their diagnostic accuracy for male infertility.

Aneuploidy Rate, DNA Fragmentation, Tubulin and Centrin Content in Different Groups of Male Infertility

In this study, we aimed to compare DNA fragmentation ratios, centrosomal protein concentrations (centrin and tubulin) and numerical chromosomal abnormalities between four groups including asthenozoospermia, teratozoospermia, oligoasthenozoospermia and normozoospermia patients, and to emphasize the necessity of investigating genetic and protein content of the spermatozoa also in patients with a semen analysis within normal reference range. Evaluation of semen analysis of the patients was performed based on WHO 2010 criteria. As a result of the statistical analysis, it was observed that the sperm DNA fragmentation rate in the normozoospermia group (6.50 ± 3.46%) was significantly lower than those in the teratozoospermia (19.69 ± 9.86%) and oligoasthenoteratozoospermia group (32.47± 14.13%) (p<0.001). For centrin and tubulin proteins, highest concentrations were observed in normozospermia group (p<0.05). When incidence of numerical abnormalities were compared between four groups, it was found that there were statistically significant differences between groups in terms of nullisomies X, Y and 13, and 21 (p<0.05). In conclusion, we suggest that abnormalities in semen parameters seem to be associated with chromosomal abnormalities; and sperm aneuploidy rate is also at a considerable level even in normozoospermic patients. Keywords: DNA fragmentation, teratozoospermia, centrin, tubulin DOI: 10.7176/JMPB/68-04 Publication date: September 30 th 2020

The redox function of apurinic/apyrimidinic endonuclease 1 as key modulator in photodynamic therapy

Photodynamic therapy (PDT) is an anticancer modality depicting an induced oxidative stress as the mechanism of action that ultimately culminates in cell death. The apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a key protein promoting bad prognostic in several cancer types. APE1/Ref-1 is able to regulate cell response to oxidative stress by two basic protein activities, including a reduction-oxidation-function and a DNA repair-function. Therefore, the efficacy of anticancer therapies is negatively affected by APE1-overexpression. Thus, here it was evaluated the potential of APE1-chemical inhibitors as sensitizers for PDT in two different cancer cell lines (A549 and HeLa cells). Both functions of APE1 were addressed using E3330 (redox-function) and CRT0044876 (DNA repair-function) molecules. A detailed cytotoxicity screening (cell viability, cell cycle kinetics, mitochondrial perturbation, and cell death) indicated HeLa cells as extremely sensitive (~ 3.5×) to the combination of PDT with E3330 when compared to A549 cells. The treatment using PDT with E3330 induced downregulation of APE1 as detected by Western Blot. The APE1's downregulation correlated to an increase of DNA fragmentation (17% and 66% in A549 and HeLa cells, respectively) and cell death rate (total: 24% and 74% in A549 and HeLa cells, respectively) characterized by annexin V and 7-AAD markers as well as a considerable difference in superoxide detected in mitochondria (29% and 78% in A549 and HeLa cells, respectively). This study definitively detected an increase in PDT efficacy when APE1's redox function is dysregulated by E3330.

Mitigating the Effects of Oxidative Sperm DNA Damage.

Sperm DNA damage is correlated with reduced embryo development and increased miscarriage risk, reducing successful conception. Given its links with oxidative stress, antioxidants have been investigated as a potential treatment, yet results are conflicting. Importantly, individual antioxidants are not identical in composition, and some compounds may be more effective than others. We investigated the use of the polyphenol-rich, high-antioxidant-capacity fruit acai as a treatment for elevated sperm DNA fragmentation (>16%), measured by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Following ≥ 74 days of treatment, we observed a significant decrease in sperm DNA fragmentation (−17.0% ± 2.5%) to 11.9 ± 1.7% (0–37%), with a 68.6% success rate (defined as post-treatment TUNEL < 16%). Post-treatment decreases in DNA fragmentation and success rates were not significantly impacted by low motility and/or concentration, or exceptionally high (> 25%) TUNEL. Treatment significantly reduced concentration in men with normal semen parameters, but 88% remained normal. Overall, successful treatment was not associated with age, semen parameters or TUNEL result at baseline. However, body mass index was significantly higher in nonresponders at baseline. This study provides evidence of a low-cost, effective treatment for elevated sperm DNA damage using acai.

In Vivo and In Vitro Evaluation of Bull Semen Processed with Zinc (Zn) Nanoparticles.

Defective sperms cause fertilization failure under both in vivo and in vitro conditions. Therefore, providing optimal conditions during semen storage is a prerequisite for maintaining viability. The current study investigated bull semen quality in vitro and in vivo when zinc (Zn) nanoparticles were used as antioxidant during semen processing and cryopreservation. In total, 32 ejaculates were collected from four Holstein bulls. All ejaculates were pooled and diluted with Bioxcell-extender containing 0 (control group), 10-6, 10-5, 10-4, 10-3, and 10-2M of Zn nanoparticles. Several physical and biochemical sperm parameters were determined after freeze-thawing process. In vitro embryo development rate and pregnancy rate were monitored after in vitro fertilization or artificial insemination using semen treated with Zn nanoparticles. Plasma membrane integrity was improved (P < 0.05) in bull semen treated with 10-6M (69.3%), and 10-2 (62.4%) of Zn nanoparticles compared to untreated group (51.3%). In addition, proportions of live spermatozoa with active mitochondria were increased (P < 0.05) in semen supplemented with Zn nanoparticles at concentration of 10-6M (67.3%), and 10-2 (85.3%) compared to control group (49.8%). Moreover, the level of MDA was lower (P < 0.05) in semen with Zn nanoparticles at 10-6M (2.97mol/mL) and 10-2 (2.7mol/mL) concentrations than control semen samples (3.77mol/mL). However, sperm total and progressive motility, sperm viability, DNA fragmentation, and pregnancy rate were not affected by treatment of semen with Zn nanoparticles. On the other hand, supplementation of in vitro maturation media with 10-6M Zn nanoparticles has increased blastocyst rate (P < 0.05) compared to other experimental groups, while addition of Zn nanoparticles-treated sperm during in vitro fertilization did not affect embryo development rate. In conclusion, supplementation of Zn nanoparticles to semen has improved its quality without affecting embryo development rate in vitro. However, in vitro embryo development rate was increased when Zn nanoparticles were supplemented to IVM media. This support the notion of Zn nanoparticles beneficial action on improving bovine gametes quality without affecting pregnancy rate.

Modified permeable cryoprotectant-free vitrification method for three or fewer ejaculated spermatozoa from cryptozoospermic men and 7-year follow-up study of 14 children born from this method.

What technique can be used to successfully cryopreserve three or fewer ejaculated spermatozoa from cryptozoospermic men and is the physical and cognitive development of children born after this technique normal? The modified cryopreservation method for three or fewer human spermatozoa from cryptozoospermic men showed a recovery rate above 95% and a survival rate just under 90%, and the physical and cognitive abilities of the children born after ICSI were comparable to those born after natural conception. Clinical outcomes of ICSI using cryptozoospermic men's ejaculated spermatozoa are considered to be inferior to that using testicular spermatozoa from microsurgical testicular sperm extraction (Micro-TESE), possibly because the DNA fragmentation rate is higher in ejaculated spermatozoa than in testicular spermatozoa from Micro-TESE. Evaluation of the efficiency of cryopreservation of three or fewer spermatozoa was conducted retrospectively at St. Mother Clinic. The physical and cognitive development of children born after this method was studied between 2011 and 2018. This study included 28 cryptozoospermic men who had three or fewer morphologically normal and motile spermatozoa in their ejaculate after centrifugation and who preferred using cryopreserved spermatozoa to Micro-TESE. Control subjects were 31 cryptozoospermic patients using fresh spermatozoa from their ejaculates and 20 non-obstructive azoospermic patients with fewer than 10 spermatozoa obtained by TESE and vitrified. Clinical outcomes among three groups, vitrified spermatozoa from the ejaculate, fresh spermatozoa from the ejaculate and vitrified spermatozoa from the testis, were statistically analysed. For the 7-year follow up study of the 14 children born after ICSI using the ejaculated vitrified spermatozoa, the Japanese government-issued Boshi Kenko Techo (Mother-Child Handbook) and Kinder Infant Development Scale (KIDS scale) were used to determine whether their physical and cognitive development was comparable to that of naturally conceived children. Recovery and survival rates were 97.8% (510/521) and 87.1% (444/510) for vitrified spermatozoa from the ejaculate and 92.7% (152/164) and 60.5% (92/152) for vitrified spermatozoa from the testis. Clinical pregnancies (%), miscarriages (%) and live birth rates (%), respectively, among the three groups were as follows: vitrified spermatozoa from the ejaculate: 15(25.0), 2(13.3), 13(21.7); fresh spermatozoa from the ejaculate: 26(24.3), 5(19.2), 20(18.7); and vitrified spermatozoa from the testis: 3(16.7), 0(0.0), 3(16.7). Among the groups, there were no statistically significant differences except for the sperm survival rate and the oocyte fertilisation rate, which were lower for vitrified spermatozoa from the testis compared with vitrified spermatozoa from the ejaculate. The 7-year follow-up study showed that the physical and cognitive development of 14 children born after ICSI using vitrified ejaculated spermatozoa from the ejaculate was comparable to that of naturally conceived children. The maximum number of spermatozoa to which this method can be applied successfully is about 10. When the number of aspirated spermatozoa is over 10, some of them change direction after colliding with each other inside the aspiration pipette and reach the mineral oil, and once this happens, they cannot be expelled out of the pipette. Even though we did not find evidence of DNA fragmentation, further studies with larger participant numbers and longer time periods are necessary. This technique is very useful for the cryopreservation of very small numbers of testicular spermatozoa (fewer than 10) in order to avoid or reduce Micro-TESE interventions. No external funding was received to undertake this study. There are no competing interests. N/A.

RS-1 enhanced the efficiency of somatic cell reprogramming via activation of RAD51 in somatic cell nuclear transfer

Background & Aim Reprogramming efficiency of somatic cell nuclei is very low in both somatic cell nuclear transfer (SCNT) and iPSCs technologies. Recently, it was reported that the genetic instability and DNA damage arise prior to transcriptional activity during reprogramming. Subsequently, in the absence of mitotic nuclear remodeling, DNA replication is delayed and errors are exacerbated (Egli D et al., 2017). Methods, Results & Conclusion Here, in order to find a regulator of DNA damage during reprogramming events, we observed the effect of RAD51 stimulatory compound (RS-1) supplements in DNA repair process of mouse SCNT embryos in the early embryogenesis and compared with in vitro fertilization (IVF)-derived embryos. First, Rad51 mRNA expression in both embryos of SCNT and IVF groups was observed at 1-cell, 2-cell, and 4-cell stages. In IVF group, relative Rad51 mRNA expression was markedly decreased from 1-cell to 2-cell stages and then restored in 4 cell stage. However, in SCNT group, its expression decreased from 1-cell to 2-cell stages at a similar rate with IVF group, but not recovered at 4-cell stage. Based on these results, we have observed DNA damage detected by rH2AX and DNA repair of double strand breaks by Rad51 expressions. The rH2AX expression was highly localized in the nucleus of IVF embryo when compared with SCNT groups until 10 h after reconstruction of embryos. However Rad51 was not expressed in the nucleus until 10 h after SCNT moreover, at 12h after SCNT, rH2AX showed highly dotted in nuclear in both IVF and SCNT+RS-1 group. A large number of embryos were observed Rad51 expression only in IVF and SCNT+RS-1 groups. In this study, we found different expression of some cell cycle-related genes among IVF, SCNT and SCNT+RS-1 groups by RNA-sequencing. The SCNT+RS-1 group significantly enhanced blastocyst formation rate of the cloned embryos when compared to those of the RS-1-untreated group, and reduced the rate of DNA fragmentation. Furthermore SCNT+RS-1 group improved full term clone mice rates and significantly increased the derivation efficiency of pluripotent stem cells from cloned embryos (SCNT-PSCs). Based on these results, we suggest that DNA damage and Rad51-mediated DNA repair during DNA replication could complement each other and its regulation has plays an important role in somatic cell nuclei reprogramming process.

Sperm Parameters before and after Swim-Up of a Second Ejaculate after a Short Period of Abstinence.

Background: Recent studies have supported the beneficial effects of a short abstinence period on sperm parameters. The aim of this study was to assess sperm motility, morphology and DNA fragmentation before and after swim-up of a second ejaculate obtained after a short abstinence period in normozoospermic men and oligo-astheno-teratozoospermic (OAT) patients. Material and methods: Semen analyses and swim-up preparations of two consecutive semen samples (collected within 1 h) were carried out in 30 normozoospermic and 35 OAT patients enrolled in an assisted reproductive technique (ART) program. Results: Compared to the first ejaculate, the second sample showed a higher percentage of spermatozoa with normal form (p < 0.01) and lower percentage of spermatozoa with DNA fragmentation (p < 0.01) in normozoospermic men, whereas a higher percentage of spermatozoa with progressive motility (p < 0.001) and normal morphology (p < 0.0001) was found in OAT patients. Swim-up separation showed a lower DNA fragmentation rate (p < 0.05) in the second ejaculate in normozoospermic men, whereas the second ejaculate of OAT patents showed an increase in normally-shaped spermatozoa (p < 0.01) and lower percentage of spermatozoa with fragmented DNA (p < 0.001) compared to the first one. Conclusions: Swim-up separation of a second ejaculate collected within 1 h might be suggested for ART procedures, especially in OAT patients.

Cyclooxygenase 1 (COX1) as an indicator of sperm quality in humans.

Sperm quality is important for in vitro fertilisation and embryo transfer and intracytoplasmic sperm injection in the treatment of human infertility. The purpose of this study was to screen for biomarkers that could evaluate sperm quality. We analysed semen samples in 172 fertile males; multivariate logistic regression analysis showed that the levels of COX1 (17.5ng/ml) in seminal plasma may represent a useful biomarker for sperm quality (area under the curve: 0.745; sensitivity: 0.808; specificity: 0.722). Analysis indicated that the values of parameters related to sperm quality changed significantly (p<.05) between COX1 level≥17.5ng/ml group and COX1 level<17.5ng/ml group. Further analysis of two consecutive semen samples (1-hr interval) from 48 subjects revealed that the first semen samples (COX1 levels≥17.5ng/ml) had a higher sperm concentration and a larger proportion of spermatozoa showing progressive motility, a lower rate of sperm DNA fragmentation and a lower proportion of spermatozoa undergoing the acrosome reaction spontaneously (p<.05); identical results were observed for the second semen samples. These data indicated that COX1 could be used as an indicator for sperm status and may be helpful for identifying better quality spermatozoa for artificial insemination.

Seasonal variations in sperm DNA fragmentation and pregnancy rates obtained after artificial insemination with cooled-stored stallion sperm throughout the breeding season (spring and summer)

The aim of this study was to assess seasonal variations during different periods of the breeding season (spring and summer) on stallion sperm DNA fragmentation and in vivo fertility associated with cooled-stored semen samples. Ejaculates were collected from eleven stallions and assessed for sperm motility (assessed by computer-assisted sperm analysis) and plasma membrane integrity (evaluated under fluorescence microscopy). Sperm DNA fragmentation (evaluated by the Sperm Chromatin Dispersion test) was assessed in cooled-stored semen at 5 °C for up to 24 h. Artificial insemination was performed throughout the breeding season. Mares were inseminated with cooled-stored semen (up to 24 h) every other day until ovulation. Pregnancy rates per cycle were determined detecting the embryonic vesicle by ultrasonography fifteen days after ovulation. Values (mean ± SD) for progressive sperm motility were significantly higher (P < 0.05) in spring (53.57 ± 9.97%) in comparison to summer (41.37 ± 10.81%). No significant differences in plasma membrane integrity were found between seasons (P > 0.05). Sperm DNA fragmentation was significantly lower (P < 0.01) in spring in comparison to summer after 0h (4.81 ± 1.87% vs. 8.77 ± 5.78%), 6h (9.00 ± 3.19% vs. 18.73 ± 8.22%) and 24h (14.6 ± 4.13% vs. 30.14 ± 9.85%) of cooled-storage. Pregnancy rates per cycle were also significantly higher (P < 0.01) in spring (50%) in comparison to summer (37%). There was a moderate negative relationship between positive pregnancies and sperm with fragmented DNA (r = - 0.619; P < 0.001). Semen samples associated with moderate fertility levels (Pregnancy rate < 50%) showed a higher percentage of sperm with fragmented DNA compared to samples obtaining higher fertility levels. In conclusion, seasonal variations were found during the breeding season, obtaining lower sperm DNA fragmentation and higher pregnancy rates in spring. Additionally, samples with the highest proportion of sperm with fragmented DNA showed the lowest fertility levels throughout the breeding season.

The Effects of Calligonum Extract and Low-Intensity Ultrasound on Motility, Viability, and DNA Fragmentation of Human Frozen-Thawed Semen Samples.

BackgroundThe study aimed to evaluate the impact of Calligonum extract and US radiation on sperm parameters of cryopreserved human semen samples.Materials and MethodsIn this experimental study, twenty-five semen specimens were obtained from healthy semen donors and incubated in human tubal fluid (HTF) medium supplemented with 10% human serum albumin (HSA) for 45 minutes. Samples were treated with Calligonum extract (10 μg/ml) alone (CGM group) and US radiation (LIPUS- exposed group) alone or a combination of both treatments (CGM+LIPUS). The US group received US stimulation (in both continuous and pulsed wave modes) at a frequency of 1 MHZ and intensity of 200 mW/cm2 for 200 seconds. Sperm morphology was assessed by Diff-Quik staining. The DNA fragmentation was evaluated the Halo sperm kit. Sperm parameters was analyzed by a computer-assisted semen analysis system. Reactive oxygen species (ROS) was assessed by flow cytometry.ResultsThe results showed that the treatment with Calligonum extract significantly (P<0.05) increased the progressive motility of spermatozoa in the CGM group as compared with the control group. The application of low-intensity US significantly (P<0.05) decreased the motility and viability of spermatozoa in the US group when compared with the control group. Our findings also indicated that the use of both low-intensity US in continuous mode and Calligonum extract slightly increased progressive motility; however, such an increase was not statistically significant. The rate of DNA fragmentation was considerably higher (P<0.05) in control and LIPUS-exposed groups than the other groups.ConclusionTreatment of spermatozoa with Calligonum extract slightly improved the sperm parameters due to its antioxidant activity, on the other hand, according to our results, US radiation did not improve sperm parameters which may be due to interference with the motility of sperm, as well as its physical effects on spermatozoa.

Specific tail swelling pattern in hypo-osmotic solution as a predictor of DNA fragmentation status in human spermatozoa.

ObjectiveThe aim of this study was to investigate DNA fragmentation status in human spermatozoa according to specific tail swelling patterns determined via hypo-osmotic swelling test (HOST).MethodsFrozen semen samples from 21 healthy donors were thawed and prepared by the swim-up technique for use in intracytoplasmic sperm injection. The semen samples were treated for 5 minutes as part of the HOST procedure and then underwent the sperm chromatin dispersion test using a Halosperm kit. DNA fragmentation status (large halo, medium halo, small halo, no halo, or degraded) and the specific tail swelling pattern (“a”–“g”) were assessed at the level of a single spermatozoon. A total of 42,000 spermatozoa were analyzed, and the percentage of spermatozoa without DNA fragmentation (as evidenced by a large or medium halo) was assessed according to the specific tail swelling patterns observed.ResultsThe HOST examinations showed that >93% of spermatozoa across all types displayed no DNA fragmentation. The percentage of spermatozoa without DNA fragmentation was 100% in type “d”, 98.67% in type “g”, and 98.17% in type “f” spermatozoa.ConclusionWe found that the type “d” spermatozoa displayed no DNA fragmentation, but the other types of spermatozoa also displayed very low rates of DNA fragmentation. This result may be associated with the processing of the spermatozoa by density gradient centrifugation and the swim-up technique.

Effect of cryopreservation on sperm DNA fragmentation and apoptosis rates in the testicular tissue of domestic cats

The objectives of the present study were to evaluate the damage caused by cryopreservation on sperm DNA and estimate the percentage of cell apoptosis in tissue after thawing. Testicles of cats were sectioned into of 0.3 cm3 and 0.5 cm3 fragments and evaluated for DNA damage using acridine orange and semi-quantitatively through histo-morphological and immunohistochemical methods (caspase-3). Other fragments were placed in cryotubes with diluent containing either 3% glycerol or 3% propanediol, and were cryopreserved. Evaluation using acridine orange indicated there was a difference with use of propanediol and glycerol on DNA damage in 0.5 cm3fragments, with the latter being more effective than the former for cryopreservation. Results from histomorphological evaluations indicated there was a greater cell integrity among germ cells that were not cryopreserved, based on criteria assessed (detachment of cells from basal membrane, retraction of seminiferous tubule epithelium, visibility of the spermatogonia nucleoli and nuclear spermatogonia condensation), for both sizes of fragments. The values for these variables decreased after cryopreservation, with there being no differences as a result of size of fragment stored or between cryoprotectants used (P > 0.05). The staining for caspase-3 differed for the cytoplasm, nuclei and germ cells. Assessment of these staining patterns indicated the fresh fragments had an amount of cell damage and there was a similar amount of damage detected in cryopreserved fragments. This finding indicated that there was considerable efficacy in preserving the tissue fragments with use of the freezing protocols that were evaluated in this study.

Influence of in vitro capacitation time on structural and functional human sperm parameters.

A cascade of dramatic physiological events is linked to the sperm acrosome reaction and binding to the oocyte's zona pellucida during human sperm capacitation. However, structural and functional sperm changes during capacitation currently remain poorly defined. Here, we performed a multibiomarker approach based on the utilization of sperm concentration, motility, viability, morphology, acrosome reaction, tyrosine phosphorylation, DNA fragmentation, and lectin-binding sites to analyze the impact caused by swim-up selection times (uncapacitated, 1 h capacitated, and 4 h capacitated) on sperm function and structure in normozoospermic samples. We found that a 4 h swim-up capacitation increased sperm quality, because a large number of cells with normal morphology and lower DNA fragmentation rates were recovered. Furthermore, the long-term capacitation induced a higher percentage of cells with tyrosine phosphorylation of the principal piece as well as a redistribution of lectin-binding sites. Overall, the multivariate biomarkers analyzed showed a less variable distribution on spermatozoa recovered after 4 h capacitation than that with the shorter capacitation time. These findings stress the importance of capacitation time as a relevant factor in sperm quality with potential biological reproductive implications both for basic research and in assisted reproduction techniques.

MSOME and IMSI: reasonable rationale, selective clinical value

Purpose: Motile sperm organelle morphology examination (MSOME) is a real-time high magnification motile sperm examination applied prior to intracytoplasmic morphologically selected sperm injection (IMSI) - intracytoplasmic injection of the selected spermatozoon. Over the past decade, there is an important shift concerning MSOME, focusing on the shape of the sperm head and presence of vacuoles, resulting with different selection criteria. The authors investigated the relevance of sperm vacuoles phenomenon. From a clinical perspective they focused on prospective trials aiming to distinguish scenarios in which IMSI may improve clinical outcome compared with intracytoplasmic sperm injection (ICSI). Materials and Methods: The authors performed this review based on a primary literature search of publications, which as of November 1, 2017, were listed in PubMed under the search phrase &lt;motile-sperm organelle morphology examination&gt;, &lt;MSOME&gt;, &lt;intra-cytoplasmic morphologically selected sperm injection&gt;, &lt;IMSI&gt;, and &lt;sperm vacuoles&gt;. The references of these manuscripts were further reviewed when considered relevant to the subject. Results: Inadequate chromatin compaction leads to higher rate of DNA fragmentation and aneuploidies, which may be followed by the appearance of large vacuoles. Distinguishing between large and small vacuoles remains a challenge. IMSI may expand blastocyst formation in cases of previous embryos developmental arrest and may be used in cases of isolated teratospermia and repetitive ICSI cycles failures. Conclusions: Solid evidence supports the association between non-adequate sperm DNA compaction and DNA defects, resulting with abnormal vacuoles. Current prospective studies emphasize ICSI as first treatment for male factor during assisted reproductive technology (ART), while the role of IMSI in selective clinical scenarios warrants further large randomized controlled studies.