Degree Of Sperm DNA Fragmentation Research Articles

Expression of DDSR1 Long Non-Coding RNA and Genes Involved in the DNA Damage Response in Sperm with DNA Fragmentation.

The molecular mechanism responsible for sperm DNA fragmentation is not fully understood. Therefore, identifying genes related to the response to DNA damage is an important area of research. Recently, the role of long non-coding RNAs (LncRNAs), especially DNA damage-sensitive RNA1 (DDSR1) in male infertility has been highlighted. In this research, a protein-protein interaction network (PPIN) was constructed using the STRING database, and functional classification was conducted using webgestalt servers. Subsequently, a group of 40 males with a high degree of sperm DNA fragmentation (DFI ≥ 25%) was compared to a control group of 20 healthy males with a normal sperm DNA fragmentation rate (DFI < 25%). To assess gene expression, real-time polymerase chain reaction (PCR) analysis was performed on DNA samples obtained from both healthy and infertile males. Our findings revealed that infertile men with an abnormal DFI index showed significantly lower expression levels of the long noncoding RNA DDSR1, as well as the genes BRCA1, MRE11A, RAD51, and NBN, compared to the control group. Pathway analysis of the network proteins using Reactome indicated involvement in crucial cellular processes such as the cell cycle, DNA repair, meiosis, reproduction, and extension of telomeres. In conclusion, the downregulation of LncRNA and genes associated with the DNA damage response in males with an abnormal DFI suggests that these factors may contribute to the development of sperm DNA fragmentation and could potentially serve as diagnostic markers for further investigation in therapeutic interventions in the future.

Антиоксидантная терапия в коррекции ДНК-фрагментации сперматозоидов.

Introduction. The progressive deterioration of reproductive function, observed throughout the world in recent decades, is an extremely alarming trend, while the incidence of male infertility is increasing. The universal pathogenetic mechanism of spermatogenesis disorders, which underlies almost all forms of male infertility, is oxidative stress of sperm. It has various manifestations, the essence of which, as a rule, boils down to the formation of mitochondrial dysfunction and fragmentation of sperm DNA. It has been found that consuming antioxidants and microelements helps increase the chances of conception for subfertile couples, and also reduces the risk of reproductive losses. Of greatest and justifiable interest are medicinal complexes designed to reduce the level of oxidative stress and sperm DNA fragmentation. The purpose of the study was to evaluate the effect of antioxidant therapy on the degree of sperm DNA fragmentation in men suffering from infertility. Materials and methods. The study involved 42 men aged 25 to 37 years with an established diagnosis of male infertility who were treated at the Center for Family Health and Reproduction. The patients were divided into 2 groups, comparable according to the main criteria assessed. Patients in the control group (12 people, 29%) received standard antioxidant therapy. Patients of the main group (30 people, 71%) received the BESTFertil-DHA® complex. The course of treatment for patients in both groups was 3 months. Control examinations were carried out at 1.5 and 3 months from the start of therapy. Spermogram data and assessment of the degree of DNA fragmentation in sperm using the SCD (Sperm chromatin dispersion test) method were used as the main criterion for assessing the effectiveness of therapy. Results. After three months of taking BESTFertil-DHA®, in 16 patients (53%), the degree of sperm DNA fragmentation decreased to less than 15%. Against this background, 5 couples became pregnant naturally, and 4 couples became pregnant as a result of the in vitro fertilization procedure. The average level of sperm DNA fragmentation was less than 13% (p &lt; 0.05). A decrease in the level of DNA fragmentation was noted in 53% of patients after 1.5 months of taking BESTFertil-DHA®, which is especially important when preparing patients for assisted reproductive technology programs. In patients taking BESTFertil-DHA®, the total progressive motility of spermatozoa «A+B» increased by 28%, and the concentration of spermatozoa in 1 ml of ejaculate – by 61%. In the control group, the degree of sperm DNA fragmentation did not undergo statistically significant changes during treatment. Conclusion. BESTFertil-DHA® statistically significantly helps to reduce the degree of DNA fragmentation in sperm, increase the number of actively motile forms of sperm and reduce the degree of teratozoospermia.

Evaluation of telomere length, reactive oxygen species, and apoptosis in spermatozoa of patients with oligospermia.

50% of cases of infertility are caused by male factor, which acquired or congenital problems may bring on. Male infertility can be caused by oligospermia and asthenozoospermia, which are common. Since the same mutations that cause azoospermia in some people also cause oligozoospermia in others, oligozoospermia may be thought of as a less severe form of azoospermia. Studies have demonstrated telomere length, catalase activity, super oxide dismutase (SOD), and DNA fragmentation can be influential factors for male infertility. The amount of apoptosis, oxidative stress factors, telomere length, and DNA fragmentation were some aspects of healthy sperm that we chose to look into in this study and compare to oligospermia individuals. Oligospermia patients (n = 24) and fertile men (n = 27) semen samples were collected, and the apoptosis rate of sperms in both groups was analyzed (Flow cytometry). Also, gene expression of apoptotic and antiapoptotic markers and telomere length were examined (real-time polymerase chain reaction). The sperm DNA fragmentation kit was used to determine DNA fragmentation and to evaluate catalase and SOD activity; the specific kits and methods were utilized. Higher expression levels of caspase3 (p = .0042), caspase8 (p = .0145), caspase9 (p = .0275), and BAX (p = .0202) mRNA were observed in patients who had oligospermia. In contrast, lower mRNA expression of BCL-2 (p = .0009) was detected in this group. In addition, telomere length was decreased in the oligospermia group (p < .0001) compared to the health group. Moreover, the frequency of apoptosis is induced in patients (p = .0026). The catalase activity is low (p = .0008), but the SOD activity is high (p = .0015) in the patient group. As a result of our findings, we may list the sperm cell apoptosis rate, telomere length, the degree of sperm DNA fragmentation, and lastly, the measurement of significant and efficient oxidative stress markers like SOD and catalase in semen plasma among the principal diagnostic characteristics for oligospermia. Future studies will be better able to treat oligospermia by showing whether these indicators are rising or falling.

The impact of asymptomatic and mild COVID-19 on sperm characteristics

Background: Although the pandemic of the new coronavirus infection (COVID-19) is characterized by mostly mild and asymptomatic cases (more than 80%), it appears important to assess the potential risks for the male reproductive system, in particular, for the parameters of the sperm. Aim: To study the effect of a mild and asymptomatic course of the new coronavirus infection (COVID-19) on the male sperm's parameters. Methods: The study included 397 patients who applied for a spermogram. All the patients underwent a spermogram study according to the WHO protocol, with the additionally measured degree of sperm DNA fragmentation, acrosine activity, fructose level, zinc level, citric acid level, neutral-glucosidase activity. A test for sperm binding to hyaluronic acid (HBA test) was performed, and the serum antibodies (IgM and IgG) against SARS-CoV-2 were analyzed. The study was conducted twice with an interval of 35 months. Results: At the first examination, normozoospermia was found in 33.5% (n=133) of patients, pathospermia in 66.5% (n=264). All the patients had no antibodies against SARS-CoV-2 (COVID-19). When the analysis was repeated after 35 months, antibodies (IgG) against SARS-CoV-2 were detected in 144 patients: in 14.6% (n=21) the coronavirus infection was mild, and in 85.4% (n=123) it was asymptomatic. Those patients who received some type of a treatment (andrologic, anticovid, and/or other) were excluded from the further study. The subsequent analysis of the data from the untreated patients (131 patients without antibodies against SARS-CoV-2 and 93 patients with antibodies (IgG) against SARS-CoV-2) was performed using the Student's paired test. For all the parameters of the standard spermogram according to the WHO protocol, the found changes after the mild and asymptomatic form of COVID-19 were not statistically significant. Also, the changes in the degree of fragmentation of sperm DNA, the enzymatic activity of acrosine, and other estimated parameters of the sperm (levels of fructose, citric acid, zinc, and the activity of neutral-glucosidase) were not statistically significant. The results of the test for the spermatozoa's interaction with hyaluronic acid (HBA test) worsened and had statistically significant differences after the coronavirus infection in both the mild form (p=0.006) and asymptomatic form (p=0.001). There were no statistically significant changes in the sperm parameters (including those from the HBA test) in the patients who had not had the new coronavirus infection. Conclusions: No statistically significant data were obtained on the effect of mild and asymptomatic forms of the course of the new coronavirus infection on the parameters of the standard spermogram, on the degree of fragmentation of spermatozoa's DNA and the enzymatic activity of acrosin. Mild and asymptomatic forms of the new coronavirus infection course can negatively affect the interaction of spermatozoa with hyaluronic acid (HBA test), that indicates the impairment of the spermatozoa binding to the zona pellucida. One should pay attention to the HBA test when examining the sperm of patients who have suffered the new coronavirus infection.

Does sperm DNA fragmentation affect clinical outcomes during vitrified-warmed single-blastocyst transfer cycles? A retrospective analysis of 2034 vitrified-warmed single-blastocyst transfer cycles.

Sperm DNA is essential in embryo development. The sperm DNA fragmentation index (DFI), which reflects the degree of sperm DNA fragmentation (SDF), is a crucial biomarker in evaluating the sperm quality. However, whether SDF influences the clinical outcomes after in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) remains controversial. This study aimed to investigate the relationship between sperm DNA SDF and clinical outcomes of vitrified-warmed single-blastocyst transfer cycles. A total of 2034 vitrified-warmed single-blastocyst transfer cycles (536 from ICSI and 1498 from IVF) were included in this analysis. According to the sperm DFI, all cycles were divided into two groups (DFI < 27.3% group and DFI ≥ 27.3% group). The Mann-Whitney and chi-squared tests were used to compare patient characteristics and clinical outcomes between the two groups. Furthermore, logistic regression analysis was performed to analyze the association between SDF and clinical outcomes. The chi-squared test showed no differences in positive human chorionic gonadotropin (HCG) rate, clinical pregnancy rate, miscarriage rates, and live birth rate between the two groups. Logistic regression analysis indicated that SDF was not a prognostic predictor of positive HCG, clinical pregnancy, miscarriage, and live birth. SDF was not associated with clinical outcomes either in ICSI or IVF cycles during vitrified-warmed single-blastocyst transfer cycles.

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

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

Sperm DNA fragmentation and total antiradical activity of sperm in men with exaggerated anamnesis

Various intracellular and extracellular stimuli can promote excessive accumulation of reactive oxygen species and lead to detrimental effects on sperm function, in particular, damage to sperm DNA, thus often resulting into infertility. The aim of the study was to assess the degree of sperm DNA fragmentation by flow cytometry and to study total antiradical activity of sperm in men with infertility and chronic prostatitis using spectrophotometric technique. A total of 75 men were examined. The comparison group consisted of 45 men with chronic prostatitis beyond exacerbation. The observation group included 30 men with chronic prostatitis in remission state. We have found that in the subjects during the period of acute inflammation, pathological fragmentation of sperm DNA was revealed, being statistically significant (p = 0.004). It was registered 1.7 times more often than in the examined men during the remission of chronic prostatitis. Increased fragmentation of the sperm DNA was noted in 50% of men from comparison group. In the patients from observation group, the degree of sperm DNA fragmentation over reference ranges was observed in 80% of the subjects. In men diagnosed with chronic prostatitis at the exacerbation stage, there is a statistically significant (p 0.05) relationship between sperm DNA fragmentation and development of acute inflammatory process. It was shown that in the subjects from observation group, the mean value of antiradical sperm activity did not show statistically significant differences (p = 0.378) compared with results found in men from the comparison group. The presented results suggest a higher degree of sperm DNA fragmentation in the examined patients with chronic prostatitis at the stage of decompensation, which may point to excessively increased production of free radicals during the acute inflammatory process. Obviously, exacerbation of chronic prostatitis contributes to imbalance in the redox homeostatic system in men, thus creating prerequisites for excess of reactive oxygen species and significant fragmentation of sperm DNA. The identified changes, i.e., an increased sperm DNA fragmentation levels and a deficiency of total antiradical activity of sperm in men with chronic urogenital diseases, may play a pathogenetic role in occurrence and further development of infertility. The degree of sperm DNA fragmentation and markers of redox processes may serve as informative activity indices of the inflammatory process.

105 Increasing fertility of interspecific hybrid males using biotechnological approaches

The results of AI depend on many factors, with the quality of semen being one of the most important. Not all male hybrids can meet the requirements for semen quality, because they often have reduced fertility following cryopreservation. Thus, it is necessary to improve semen processing before use in AI. The aim of the study was to evaluate the effectiveness of using the “swim-up” flotation method to improve sperm quality of hybrid males of the Ovis genus. Semen from interspecific hybrid rams (1/4 Argali×3/4 Romanov, n=15; 1/8 Argali×7/8 Romanov, n=15) was freshly obtained, frozen–thawed, and processed by the swim-up method. Evaluation of sperm motility was determined using computer-assisted semen analysis. Statistical analysis was performed using SPSS vs.15.0 (ANOVA and t-test; SPSS Inc.). Semen was collected during the breeding season (October–December) via artificial vagina. Assessment of acrosome integrity was determined using differential staining with a Diachem diff-quick kit (NPF ABRIS+). The degree of sperm DNA fragmentation was determined using the acridine-orange test. The sperm freezing/thawing cycle was accompanied by sperm damage and an increase in the proportion of immobile sperm from 10 to 58%, with non-progressive movement increasing from 9 to 19.3%. The number of spermatozoa with abnormal morphology doubled, and the DNA fragmentation index increased from 16 to 26%. Use of the swim-up procedure allowed us to sort progressively motile spermatozoa. The content of progressively motile spermatozoa in the samples obtained from the supernatant was 86%, which was 2.3 times higher than in frozen–thawed sperm (P≤0,01). The obtained results show the effective use of the swim-up procedure to determine the quality of semen in hybrid rams. These studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.

Fragmentation of sperm DNA: clinical significance, reasons, methods of evaluation and correction

A review of the main causes of male infertility in the aspect of the relationship with the degree of sperm DNA fragmentation is presented. Information is provided on the main methods for assessing sperm DNA fragmentation and its effect on male fertility. The effect of oxidative stress on the integrity of sperm DNA structure, the reparative capabilities of antioxidant therapy, and the effect of varicocele on male fertility are described.

Interaction between serum levels of Anti-Mullerian Hormone and the degree of sperm DNA fragmentation measured by sperm chromatin structure assay can be a predictor for the outcome of standard in vitro fertilization.

Serum levels of Anti-Mullerian Hormone (AMH) have been shown to be biomarker for prediction of the quantitative aspects of ovarian reserve. On the male side, sperm chromatin structure assay (SCSA) DNA fragmentation index (DFI) has been demonstrated to be an important predictor of outcomes in standard IVF procedures but to less degree in intracytoplasmic sperm injection procedures (ICSI). The purpose of this study was to investigate whether the combination of female AMH serum levels and sperm DFI adds to prediction of the outcome of assisted reproduction. A total of 352 couples was included (ICSI-148: IVF-204) A venous blood sample was drawn for AMH analysis before IVF/ICSI treatment. DFI was measured in the ejaculate used for assisted reproduction. Regression models for the following odds ratio calculations were constructed: for obtaining at least one Good Quality Embryo; for live birth in all procedures; for pregnancy in procedures where embryo transfer was performed; for miscarriage. For DFI increase by 10 percentage points (not increased DFI as reference) odds ratio for Good Quality Embryo was statistically significantly lower when AMH was at lower quartile (AMH <12 pmol/L; OR = 0.29, 95% CI: 0.14–0.59,) but not when AMH was at upper quartile (AMH ≥ 36 pmol/L; OR = 0.95, 95% CI: 0.43–2.13,). The marginal effect of an increase in DFI by 10 percentage points was statistically significant only when AMH < 25.2 pmol/L. Similar results were obtained as considers live birth following standard IVF. No interactions were seen for standard IVF in relation to the risk of miscarriage and for any of the outcomes when ICSI was used as method of treatment. We conclude that the impact of high DFI on the outcome of standard IVF is most pronounced if the female partner has relatively low AMH levels. This finding may help in defining the role of sperm DNA integrity testing in management of infertile couples. It may also explain some of the heterogeneity in results of studies focusing on predictive value of DFI measurements in assisted reproduction.

Effect of endocrine disrupting chemicals bisphenol A and triclosan on semen quality

Aim. To establish a relationship between the concentration of endocrine disruptors (bisphenol A, triclosan) in testicular tissues or seminal fluid and semen quality in men and experimental animals.&#x0D; Methods. 53 samples of seminal fluid were investigated from men with normo- and pathozoospermia. In seminal fluid the level of bisphenol A and triclosan were determined by gas chromatography-mass spectrometry. The spermiological study was carried out according to the World Health Organization guidelines (2010). The experimental part of the study was performed on adult male lab Wistar rats (n=28). The amount of spermatozoa per 1 ml of sperm was determined with the calculation of their motility and atypical forms. The concentration of bisphenol A and triclosan in the testicles was measured by gas chromatography-mass spectrometry.&#x0D; Results. Bisphenol A and triclosan were detected in 100 and 84.9% of the ejaculate samples with a median concentration of 0.15 (0.06–0.31) ng/ml and 0.13 (0.05–0.22) ng/ml, respectively. With the Spearman's rank correlation coefficient, statistically significant correlation was found between the concentration of endocrine disruptors and ejaculate parameters: between bisphenol A and the total count of spermatozoa, their concentration, the proportion of progressively motile spermatozoa, the proportion of spermatozoa with normal morphology and degree of sperm DNA fragmentation. Triclosan concentration correlated with the proportion of spermatozoa with normal morphology and the degree of sperm DNA fragmentation. The experimental part of the study demonstrated that bisphenol A reduced the total number of spermatozoa with an increase in the proportion of atypical forms of spermatozoa and a violation of the proportion of fertile spermatozoa compared to the group of intact animals. Triclosan reduced the proportion of progressively motile spermatozoa.&#x0D; Conclusion. Bisphenol A and triclosan have a negative effect on sperm quality in men and experimental animals.

Evaluation of sperm DNA fragmentation index, Zinc concentration and seminal parameters from infertile men with varicocele.

The aim of this study was to investigate the effect of varicocele on DNA fragmentation index (DFI), zinc concentration and seminal parameters in infertile patients. In this prospective study, 179 men with at least 1-year history of infertility and varicocele were examined for semen quality at Hanoi Medical University Hospital (HMUH), Hanoi, Vietnam. In addition, an inverse correlation between zinc concentration and the degree of sperm DNA fragmentation in patients with clinical varicocele was found. The difference in mean values of sperm DNA fragmentation index in patients with various grades of varicoceles can be neglected, whereas most patients with varicocele of grades II and III had DFI >30%. Varicocele is associated with high levels of DNA damage in spermatozoa and reduced zinc levels that correlate with different grades of disease. Therefore, DNA fragmentation index and zinc concentration can be used as essential additional diagnostic test for patients with clinical varicocele. A study should be conducted to evaluate the benefits of zinc supplementation to improve seminal parameters in patients with varicocele.

Environmental Exposure to Endocrine Disruptor of Bisphenol A and Semen Quality of Men

Background: The reproductive health disorders in men are one of the urgent problems of international medicine. The prevalence of idiopathic male infertility has the highest rate. Oxidative stress, genetic factor, and endocrine disruptors are considered to be the most probable causes for the idiopathic male infertility. In this regard, studying the effect of endocrine disruptors, in particular bisphenol A on male reproductive health, becomes actual and relevant. Aims: The aim of the study was to reveal the relationship between the concentration level of bisphenol A (BPA) in seminal fluid and semen quality in men with normo- or pathozoospermia, as well as between the concentration level of bisphenol A and the level of total testosterone and estradiol in plasma. Materials and methods: 53 samples of seminal fluid of men with normo- or pathoospermia were studied. In seminal fluid the concentration of bisphenol A was determined by gas chromatography with mass spectrometry (GC-MS). The spermiological analysis was performed according to the WHO recommendations (2010) including the evaluation of sperm count/concentration, motility and morphology, and DNA fragmentation index. In addition, the concentration of total testosterone and estradiol in plasma was determined. The results were statistically processed using the Mann–Whitney U test, the correlation analysis, the paired regression method, and the ROC curves to determine the cut-off point for BPA in the seminal fluid. The results were considered statistically significant at p0.05. Results: In 100% of the ejaculate samples BPA with a median concentration of 0.15 (0.06–0.31) ng/ml was detected. Using the Spearman rank correlation coefficient, statistically significant correlations between the concentration of BPA and the total count (r=-0.330, p=0.016), concentration (r=-0.309; p=0.024), the proportion of progressively motile spermatozoa (r=-0.575; p=0.001), the proportion of spermatozoa with normal morphology (r=-0.397, p=0.003), the degree of sperm DNA fragmentation (r=0.349, p=0.025), and the concentration of total testosterone (r =-0.616; p0.001) were registered. A statistically significant inverse linear relationship (r=-0.406, p=0.003) and (r =-0.364, p=0.048) was determined by a paired linear regression between the BPA concentration in the seminal fluid and the proportion of progressively motile spermatozoa, and the total testosterone level respectively. To assess the risk of pathozoospermia, the threshold value of seminal BPA concentration was determined using the analysis of ROC-curves; the cut-off point was 0.1025 ng/ml. Conclusions: BPA in the seminal fluid influences negatively on the quality of the sperm and suppress the level of total testosterone in plasma.

Topology of chromosome centromeres in human sperm nuclei with high levels of DNA damage.

Several studies have shown that the ‘poor’ sperm DNA quality appears to be an important factor affecting male reproductive ability. In the case of sperm cells from males with the correct somatic karyotype but with deficient spermatogenesis, resulting in a high degree of sperm DNA fragmentation, we observed changes in the preferential topology of the chromosome 7, 9, 15, 18, X and Y centromeres. The changes occurred in radial localization and may have been directly linked to the sperm chromatin damage. This conclusion is mainly based on a comparison of FISH signals that were observed simultaneously in the TUNEL-positive and TUNEL-negative sperm cells. The analyzed cells originated from the same ejaculated sample and FISH was performed on the same slides, after in situ TUNEL reaction. Based on the observed changes and previous data, it appears that the sperm nucleus architecture can be disrupted by a variety of factors and has a negative influence on spermatogenesis at the same time. Often, these factors coexist (e.g. chromosomal translocations, aneuploidies, a higher DNA fragmentation, abnormal seminology), but no direct correlations between the factors were observed.

Sperm DNA oxidative damage and DNA adducts

The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers’ percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm.

Ultrastructural features of human sperm cells cryopreserved by different methods

Cryopreservation of human spermatozoa has been recognized as a key strategy for management of male fertility. Nevertheless, current protocols of sperm freezing are neither optimal nor standardized between different labs (1). In this study we compare the ultrastructural features of human normospermic sperm samples (according to WHO parameters 2010) from 5 different freezing techniques in order to identify the best methods of cryopreservation. After informed con- sent, 21 normospermic patients (from the Medically Assisted Procreation PMA Center of the Fondazione IRCCS Policlinico San Matteo in Pavia) were recruited and both traditional and improved analysis of sperm quality were applied, in order to define critical steps of cryopreservation. Cryopreservation of human spermatozoa has been related to decreased motility associated with impaired velocity and viability of sperm pre-freeze and post-thaw. For all applied methods there was a significant reduction of progressive and total motility (P) as a result of freezing. To investigate ultrastructural details, 5 additional cryopreserved samples by the best two freezing methods were analyzed with elec- tron microscopy (TEM). Preliminary data showed the minimal differences between the protocols, with a large number of queues detached and large quantities of cyto- plasmic debris after of the first protocol. Spermatozoa appear to be better preserved in the second analyzed method, despite both procedures induced deteriorations at ultrastructural level (2). Other non-routine analysis will be performed to determine whether the cooling time to +4°C may affect the procedure; Comet Assay (to assess the degree of sperm DNA fragmentation) (3) and flow cytometry (to study light scat- ters patterns and membrane integrity) (4) will be applied.

Evaluation of sperm DNA integrity and its effect on embryo development using time-lapse microscopy

To examine whether there are any correlation between the degree of sperm DNA fragmentation as measured with Sperm Chromatin Structure assay (SCSA) and early embryo development assessed by time-lapse microscopy.

Incipient post‐zygotic barrier in a model system of ecological speciation with gene flow

The role of post-zygotic isolation in nonallopatric ecological speciation is still mostly unknown and information on the nature and strength of these barriers in well-known speciation models is essential for a deeper understanding of such processes. The Galician ecotypes of the marine snail Littorina saxatilis represent one of the best studied cases of nonallopatric ecological speciation. Here, we test the existence of incipient post-zygotic isolation by comparing the fertility of male hybrids with that of both pure forms [ridged and banded (RB) and smooth and unbanded (SU) ecotypes]. We analysed the degree of sperm DNA fragmentation (SDF) of individuals morphologically classified as RB, SU and hybrids, sampled from two locations. SDF analyses were chosen to study sperm quality because, in other animal species, SDF rates correlate with important parameters for speciation research, such as fertilization and abortion rates and viability of adult progeny. In the present work, hybrids showed significantly higher SDF rates than RB and SU males in one location and significantly higher variances in both locations. These results suggest the existence of an incipient post-zygotic barrier, the strength of which may vary across the Galician shore, and highlight the potential of SDF analyses for speciation research.

Is intracytoplasmic morphologically selected sperm injection (IMSI) beneficial in the first ART cycle? A multicentric randomized controlled trial

Intracytoplasmic morphologically selected sperm injection (IMSI), by selecting spermatozoa at high magnification improves the outcome of intracytoplasmic sperm injection (ICSI) mainly after several failures. However, only few monocentric randomized studies are available and they do not analyse results as a function of sperm characteristics. In 255 couples attempting their first assisted reproductive technology (ART) attempt for male infertility (motile sperm count <1×10⁶ after sperm selection, but at least 3×10⁶ spermatozoa per ejaculate to allow a detailed analysis of sperm characteristics), a prospective randomized trial was performed to compare the clinical outcomes of IMSI and ICSI and to evaluate the influence of sperm characteristics on these outcomes. IMSI did not provide any significant improvement in the clinical outcomes compared with ICSI neither for implantation (24% vs. 23%), nor clinical pregnancy (31% vs. 33%) nor live birth rates (27% vs. 30%). Moreover, the results of IMSI were similar to the ICSI ones whatever the degree of sperm DNA fragmentation, nuclear immaturity and sperm morphology. These results show that IMSI instead of ICSI has no advantage in the first ART attempts. However, this does not rule out IMSI completely and more randomized trials must be performed especially regarding patients carrying severe teratozoospermia, or high sperm DNA fragmentation levels or having previous ICSI failures.

Sperm DNA fragmentation in couples with unexplained recurrent spontaneous abortions

The aim of the present study was to evaluate the degree of sperm DNA fragmentation in couples with idiopathic recurrent spontaneous abortion (RSA) and in those with no history of infertility or abortion. In this cohort study, 30 couples with RSA and 30 fertile couples as control group completed the demographic data questionnaires, and their semen samples were analysed according to World Health Organization (WHO) standards (September 2009-March 2010) for evaluation of sperm DNA fragmentation, using sperm chromatin dispersion (SCD) technique. The percentage of morphologically normal sperm was significantly lower in RSA patients compared with control group (51.50 ± 11.60 versus 58.00 ± 9.05, P = 0.019), but not in other parameters. Additionally, the level of abnormal DNA fragmentation in the RSA group was significantly higher than in the control group (43.3% versus 16.7%, P = 0.024). Our results indicated a negative correlation between the number of sperm with progressive motility and DNA fragmentation (r = -0.613; P < 0.001). The sperm from men with a history of RSA had a higher incidence of DNA fragmentation and poor motility than those of the control group, indicating a possible relationship between idiopathic RSA and DNA fragmentation.