Search Results (21)

Sperm preparation is a critical step in assisted reproduction, aiming to isolate spermatozoa with optimal characteristics and high fertilizing potential. Traditional sperm selection methods involve centrifugation, which may cause sperm damage. Microfluidic sperm sorting (MSS) offers an alternative approach, mimicking the female reproductive tract environment, avoiding centrifugation, and reducing manipulation and processing time. This study aims to compare the performance of MSS and Swim-up (SU) in 26 normozoospermic, 31 hyperviscous normozoospermic, 15 oligozoospermic, and 9 asthenozoospermic subjects. Semen samples were collected from male subjects undergoing routine semen analysis at Careggi University Hospital, Florence. Sperm selection was carried out using both SU and MSS. The parameters assessed included sperm motility, viability, concentration, kinematics, DNA fragmentation (sDF), chromatin compaction, and oxidative status. Both SU and MSS improved sperm characteristics compared to unselected samples. MSS isolated high-quality spermatozoa with lower sDF and higher chromatin compaction than SU, not only in normozoospermic samples but also in samples with semen defects like hyperviscosity, low concentration and/or motility, and high sDF. In conclusion, the use of microfluidics may enhance the chances of successful fertilization and improve reproductive outcomes, especially for individuals with compromised semen quality where conventional methods may fail. Full article

30 percent of infertile men are diagnosed with idiopathic infertility. This study aimed to assess oxidative stress in the semen of 77 patients with idiopathic infertility by measuring F₂-isoprostane (F₂-IsoP), resolvin D1 (RvD1) levels, and semen parameters. The presence and localization of 8-IsoProstaglandin F_2α were determined using immunofluorescence. No significant correlations were observed for F₂-IsoP and RvD1 levels with the semen variables. Based on F₂-IsoP levels, individuals were classified into two groups: Group 1 (F₂-IsoPs ≤ 29.96 ng/mL, 51%) and Group 2 (F₂-IsoPs > 29.96 ng/mL, 49%). In comparison to Group 1, Group 2 showed significantly higher F₂-IsoP levels (13.33 ng/mL vs. 44.80 ng/mL; p < 0.05), a lower progressive motility percentage (30% vs. 25%; p < 0.05), and increased RvD1 levels (36.09% vs. 44.94%). Immunofluorescence analysis revealed a different localization of 8-IsoProstaglandin F_2α in the ejaculated sperm of Group 1 compared to that observed in Group 2. A weak signal was detected in the sperm tail (Group 1, 79.1% vs. Group 2, 36.9; p < 0.01). In spermatozoa of Group 2 patients, a strong signal in the acrosome, midpiece, and tail was highlighted. These findings suggest the need to test oxidative stress during routine semen analysis in patients with idiopathic infertility to improve diagnosis and treatment. Full article

Background: 16S rRNA analysis has been used in various diseases to identify pathogenic bacteria. In particular, pathogens that are difficult to cultivate or previously unknown can be detected with great certainty. In chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), a distinction between bacterial and non-bacterial genesis is essential with regard to categorization and therapy. The objective of this study is to investigate the value of 16S rRNA gene sequence analysis in the routine management of patients with CP/CPPS especially after failure to detect a pathogen in conventional culture and polymerase chain reaction for sexually transmitted diseases (STI-PCR). Methods: In total, 228 patients with CP/CPPS were prospectively enrolled and received a comprehensive andrological work-up. Microbial analysis consisted of standard bacterial cultures and the detection of sexually transmitted pathogens by PCR using urine specimens from a 2-glass test and semen analysis. 16S rRNA gene sequence analysis was performed in patients with urine and semen of patients without bacterial pathogens in microbiological culture and STI-PCR. Results: In 184 of 199 (92%) patients with negative ejaculate culture and negative STI-PCR, no pathogen could be detected by 16S rRNA analysis and in the case of a positive result, the analysis only showed non-pathogenic bacteria of the normal flora. There was no statistical association between the 16S rRNA analysis and the inflammatory markers or the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) scores. Conclusions: At least in our study cohort, the 16S rRNA analysis provided no additional benefit following microbiological culture and STI-PCR in the categorization of patients with CP/CPPS. Full article

Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections in all genders worldwide. Its association with male infertility is deeply investigated, although there are conflicting data on the role of the virus in the impairment of semen quality and reduced reproductive outcomes. In this study, we considered 335 semen samples of males (age: 37.63 ± 6.02 years) belonging to infertile couples who did not conceive a pregnancy after 12 months of unprotected intercourse. Residual semen samples, after routine sperm analysis, were used to amplify and type viral DNA. Positive or negative HPV semen samples were compared. In total, 42.51% (139/327) were positive for at least one HPV genotype, and in 54.68% (76/139), positivity was due to a high-risk (HR) genotype. The most prevalent was HPV-16 (16.55%) followed by HPV-52 (10.07%) and HPV-51 (7.91%). Overall, no significant differences emerged in terms of sperm concentration, sperm motility, and morphology between the two groups. However, a considerable reduction in sperm motility was found in the presence of HPV-51 or HPV-52. These data point to the importance of HPV screening in semen analysis to evaluate patients that might have a higher risk of infertility according to the type of HPV genotype. Full article

Artificial insemination (AI) plays a critical role in livestock reproduction, with semen quality being essential. In swine, AI primarily uses cool-stored semen adhering to industry standards assessed through routine analysis, yet fertility inconsistencies highlight the need for enhanced semen evaluation. Over 10-day storage at 17 °C, boar semen samples were analyzed for motility, morphology, sperm membrane integrity, apoptosis, and oxidative stress indicators. Additionally, machine learning tools were employed to explore the potential of Raman and near-infrared (NIR) spectroscopy in enhancing semen sample evaluation. Sperm motility and morphology gradually decreased during storage, with distinct groups categorized as “Good” or “Poor” survival semen according to motility on Day 7 of storage. Initially similar on Day 0 of semen collection, “Poor” samples revealed significantly lower total motility (21.69 ± 4.64% vs. 80.19 ± 1.42%), progressive motility (4.74 ± 1.71% vs. 39.73 ± 2.57%), and normal morphology (66.43 ± 2.60% vs. 87.91 ± 1.92%) than their “Good” counterparts by Day 7, using a computer-assisted sperm analyzer. Furthermore, “Poor” samples had higher levels of apoptotic cells, membrane damage, and intracellular reactive oxygen species on Day 0. Conversely, “Good” samples maintained higher total antioxidant capacity. Raman spectroscopy outperformed NIR, providing distinctive spectral profiles aligned with semen biochemical changes and enabling the prediction of semen survival during storage. Overall, the spectral profiles coupled with machine learning tools might assist in enhancing semen evaluation and prognosis. Full article

Male factors may be present in up to 50–70% of infertile couples and the prevalence of male infertility accounts for 20–30% of infertility cases. Understanding the mechanisms and causes behind male infertility remains a challenge, but new diagnostic tools such as DNA fragmentation might aid in cases where the routine semen analysis is insufficient. DNA fragmentation, which refers to damages or breaks of the genetic material of the spermatozoa, is considered one of the main causes of male infertility due to impaired functional capability of sperm. The aim of the present narrative review is to investigate and enlighten the potential correlation between DNA fragmentation and male infertility parameters such as the seminal profile and the reproductive outcomes. Comprehensive research in PubMed/Medline and Scopus databases was conducted and 28 studies were included in the present review. Fourteen studies provided data regarding the impact of DNA fragmentation and seminal parameters and showed a correlation of significantly lower sperm count, lower concentration, motility, and abnormal morphology with an increased DNA fragmentation index (DFI). Similarly, 15 studies provided data regarding the impact of DFI on reproductive outcomes. Two studies showed higher aneuploidy rates with higher DFI values, and seven studies showed significantly lower pregnancy rates and live birth rates with higher DFI values. Ultimately, the studies included in this review highlight, collectively, the importance of measuring sperm DFI in the assessment of male infertility. Further studies are needed to explore the effectiveness of interventions aiming to reduce DFI levels. Full article

Sperm oxidative stress has been extensively associated to male infertility. However, tests to detect this parameter have not been yet introduced in clinical practice and no definitive data are present on the extent of oxidative stress in male infertility. In this study, we used a novel and reliable flow cytometric method to reveal sperm ROS production in subfertile patients (n = 131) and in healthy donors (n = 31). Oxidative stress was higher in subfertile patients (14.22 [10.21–22.08]%) than in healthy donors (9.75 [8.00–14.90]% (p < 0.01)), but no correlation was found with age, semen quality or sDF. We also failed to detect an increase in sperm ROS production with semen viscosity or leukocytospermia, but a sharp impact of semen bacteria was evident (with bacteria: 31.61 [14.08–46.78]% vs. without bacteria: 14.20 [10.12–22.00]%, p < 0.01). Finally, after establishing a threshold as the 95th percentile in healthy donors, we found that 29% of subfertile patients exceeded this threshold. The percentage decreased to 25.56% when we excluded subjects with bacteriospermia and increased to 60.87% when only these patients were considered. In conclusion, 29% of subfertile patients showed an excessive sperm ROS production. Surprisingly, this parameter appears to be independent from routine semen analysis and even sDF determination, promising to provide additional information on male infertility. Full article

Sperm DNA fragmentation (sDF) is a DNA damage able to predict natural conception. Thus, many laboratories added tests for the detection of sDF as an adjunct to routine semen analysis with specific indications. However, some points related to sDF are still open. The available tests are very different each from other, and a direct comparison, in terms of the prediction of reproductive outcomes, is mandatory. The proposed mechanisms responsible for sDF generation have not yielded treatments for men with high levels of sDF that have gained the general consent in clinical practice, thus requiring further research. Another relevant point is the biological meaning to attribute to sDF and, thus, what we can expect from tests detecting sDF for the diagnosis of male infertility. SDF can represent the “tip of iceberg” of a more extended and undetected sperm abnormality somehow impacting upon reproduction. Investigating the nature of such a sperm abnormality might provide novel insights into the link between sDF and reproduction. Finally, several studies reported an impact of native sDF on assisted reproduction technique outcomes. However, to fertilise the oocyte, selected spermatozoa are used where sDF, if present, associates with highly motile spermatozoa, which is the opposite situation to native semen, where most sDF associates with non-viable spermatozoa. Studies comparing the impact of sDF, as assessed in both native and selected spermatozoa, are needed. Full article

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze–thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze–thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze–thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3–8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at −196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all r_s ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial r_s ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (r_s = 0.366, p = 0.014) and necrosis (r_s = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial r_s ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (r_s = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze–thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood. Full article

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler^® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/μL in the minimum extraction volume (40 μL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R², and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event. Full article

Effective wild cat conservation programs with assisted reproductive technologies are being developed in different parts of the world. The flat-headed cat, fishing cat, and Asiatic golden cat are three species among nine wild Felidae in Thailand that are in need of urgent conservation efforts. Here, we assessed routine sperm characteristics and we report the detection of protein biomarkers related to the fertilization process, IZUMO1 and the CRISP family, and apoptotic markers, active or cleaved caspase-3, in semen samples collected from these wild cats. IZUMO1 was located in the equatorial segment of the sperm head, which is the region involved in gamete interaction. The highest levels of IZUMO1 were found in both the sperm pellet and the seminal plasma of the flat-headed cat, as determined by immunoblotting. CRISP2, a sperm–egg fusion assisting protein, and CRISP3 were found in both the sperm pellet and the seminal plasma, and the highest levels were observed in the fishing cat. Positive correlations between certain semen parameters and IZUMO1, CRISP2, and CRISP3 expression were also demonstrated. Cleaved caspase-3 was found in all sperm samples in all three species and was associated with an increase in DNA fragmentation and a decrease in certain semen characteristics such as motility, viability, and intact acrosomes. Our results suggest that the analysis of IZUMO1, the CRISP family, and cleaved caspase-3, along with the routine sperm characteristics, may allow for better success in breeding management in wild Felidae, particularly in the flat-headed cat and the fishing cat. Full article

Background: The aim of this case–control study is to investigate possible associations between GSTM1 polymorphism and redox potential with sperm parameters. Methods: The study group consisted of sperm samples from 51 infertile men according to the WHO guidelines. The control group included 39 samples from men with normal seminal parameters. DNA was extracted and genotyped for the detection of the GSTM1 polymorphism. An evaluation of the static redox potential (sORP) using the MiOXSYS^TM system was conducted. Results: The frequency of the GSTM1-null genotype was higher in infertile male individuals (60.78%) than in the controls (41.03%) and was associated with a 2.228-fold increased risk for male infertility. Fertile controls carrying the GSTM1-null genotype presented a lower percentage of typical sperm morphology and lower slow progressive motility. An excess of redox potential was observed in infertile males compared to fertile ones. In the control group higher sORP values had a positive correlation with immotility percentage and a negative correlation regarding total motility. In the study group sORP values had a negative correlation with total count, concentration, and slow progressive motility. Conclusions: The present study highlights that GSTM1 polymorphism and redox potential affect both fertile and in fertile males. Moreover, redox potential levels could be used as an additional indicator along with the routine semen analysis for a comprehensive screening between infertile and fertile men. Full article

Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR^®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry. Full article

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure. Full article

Semen cryobanks are critical for preserving autochthonous and rare breeds. Since sperm cryopreservation has been optimized for commercial breeds, non-commercial ones (often endangered) must be characterized to ensure the germplasm’s viability. This study reports an investigation of the “Asturiana de la Montaña” breed (AM), a valuable Spanish autochthonous cattle breed adapted to the mountainous Atlantic environment. The survey included cryopreserved semen doses from 40 bulls stored at the Principado de Asturias Germplasm Bank. Data were obtained from the routine fresh semen analysis, CASA (motility), and flow cytometry analyses of fresh and post-thawing semen, and the 56-day non-return-rate (NRR) in heifers and cows (all results as 1st and 3rd quartiles). Fresh samples (artificial vagina) were within the normal range for cattle (4–6 mL, 5–10 × 10⁹/mL; mass motility 5). Post-thawing results showed motility below typical for commercial breeds (total motility 26–43%, progressive 14–28%), with higher values for viability (47–62%). Insemination results showed a good performance for this breed (NRR: 47–56%; higher for heifers). Sperm volume increased with age, with little or no effects on sperm quality. Few associations were found between post-thawing quality or freezability and NRR, LIN being the variable more strongly associated (positively). The AM semen bank shows a good prospect for preserving and disseminating the genetics of this breed. This survey indicates that dedicated research is needed to adapt freezing protocols to this breed, optimizing post-thawing results. Full article