Effects of long and short ejaculatory abstinence on sperm parameters: a meta-analysis of randomized-controlled trials.

Purpose: This study aimed to investigate the effects of ejaculatory abstinence on sperm parameters. Methods: This analysis was registered in PROSPERO (CRD42023472124). We performed a search on PubMed using the following text terms: (("sperm parameters" OR "sperm analysis" [Mesh]) AND ("sperm DNA fragmentation" OR "DNA fragmentation" [Mesh]) AND ("sexual abstinence" [Mesh] OR "abstinence")) and an advanced search in Scopus using the terms ("sperm parameters" OR "sperm parameters" OR "DNA fragmentation") AND ("abstinence"). The sperm parameters that were investigated were sperm volume, total sperm motility, progressive sperm motility, sperm concentration, sperm morphology, and sperm DNA fragmentation (SDF). A two-day cut-off as a "short" or "long" abstinence period has been defined. Results: Thirteen studies published between 2013 and 2022 were included in this meta-analysis. A total of 2,315 patients, ranging from 6 to 836 from each cohort, were enrolled in the study. We showed that longer abstinence time was associated with greater sperm concentration (mean difference [MD]: 8.19; p <0.01), sperm volume (MD: 0.96; p <0.01), and higher SDF (MD: 3.46; p <0.01), but lower progressive sperm motility (MD: -1.83; p <0.01). Otherwise, no statistically significant difference was observed in patients with longer vs. shorter abstinence times regarding total sperm motility (MD: -1.83; p = 0.06). Meta-regression analysis showed that days of abstinence were positively and linearly related to sperm concentration (slope: 3.74; p <0.01) and SDF (slope: 0.65; p = 0.044). Conclusions: According to our data, short ejaculatory abstinence is associated with better sperm quality. Indeed, a higher percentage of progressive sperm motility and lower levels of SDF have been reported in a short abstinence cohort. In contrast, the long abstinence group reported a higher sperm concentration. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023472124.

Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.

Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

Antifungal activity of human antimicrobial peptides targeting apoptosis in Candida auris.

Introduction. Innovative antifungal therapies are of crucial importance to combat the potentially life-threatening infections linked to the multidrug-resistant fungal pathogen Candida auris. Induction of regulated cell death, apoptosis, could provide an outline for future therapeutics. Human antimicrobial peptides (AMPs), well-known antifungal compounds, have shown the ability to induce apoptosis in pathogenic fungi.Hypothesis/Gap Statement . Although it is known that AMPs possess antifungal activity against C. auris, their ability to induce apoptosis requires further investigations.Aim. This study evaluated the effects of AMPs on the induction of apoptosis in C. auris.Methods. Human neutrophil peptide-1 (HNP-1), human ß-Defensins-3 (hBD-3) and human salivary histatin 5 (His 5) were assessed against two clinical C. auris isolates. Apoptosis hallmarks were examined using FITC-Annexin V/PI double labelling assay and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick-end labelling (TUNEL) to detect phosphatidylserine externalization and DNA fragmentation, respectively. Then, several intracellular triggers were studied using JC-10 staining, spectrophotometric assay and 2',7'-dichlorofluorescin diacetate staining to measure the mitochondrial membrane potential, cytochrome-c release and reactive oxygen species (ROS) production, respectively.Results and conclusion. FITC-Annexin V/PI staining and TUNEL analysis revealed that exposure of C. auris cells to HNP-1 and hBD-3 triggered both early and late apoptosis, while His 5 caused significant necrosis. Furthermore, HNP-1 and hBD-3 induced significant mitochondrial membrane depolarization, which resulted in substantial cytochrome c release. In contrast to His 5, which showed minimal mitochondrial depolarization and no cytochrome c release. At last, all peptides significantly increased ROS production, which is related to both types of cell death. Therefore, these peptides represent promising and effective antifungal agents for treating invasive infections caused by multidrug-resistant C. auris.

Genotyping of rams based on melatonin receptor 1A gene polymorphisms: a tool in sire selection?

Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.

Development of a thermotaxis and rheotaxis microfluidic device for motile spermatozoa sorting.

Male infertility is a pervasive global reproductive challenge, primarily attributed to a decline in semen quality. Addressing this concern, there has been a growing focus on spermatozoa sorting in assisted reproductive technology. This study introduces a groundbreaking development in the form of a thermotaxis and rheotaxis microfluidic (TRMC) device designed for efficient motile spermatozoa sorting within a short 15-min timeframe. The TRMC device mimics the natural sperm sorting mechanism of the oviduct, selecting spermatozoa with superior motility and DNA integrity. The experimental outcomes demonstrate a remarkable enhancement in the percentage of progressive spermatozoa following sorting, soaring from 3.90% to an impressive 96.11% when subjected to a temperature decrease from 38 °C to 35 °C. Notably, sperm motility exhibited a substantial 69% improvement. The TRMC device exhibited a commendable recovery rate of 60.93%, surpassing current clinical requirements. Furthermore, the sorted spermatozoa displayed a notable reduction in the DNA fragmentation index to 6.94%, signifying a substantial 90% enhancement in DNA integrity. This remarkable advancement positions the TRMC device as highly suitable for applications in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), offering a promising solution to male infertility challenges.

Increased scalability and sequencing quality of an epigenetic age prediction assay.

Epigenetic ageing in a human context, has been used to better understand the relationship between age and factors such as lifestyle and genetics. In an ecological setting, it has been used to predict the age of individual animals for wildlife management. Despite the importance of epigenetic ageing in a range of research fields, the assays to measure epigenetic ageing are either expensive on a large scale or complex. In this study, we aimed to improve the efficiency and sequencing quality of an existing epigenetic ageing assay for the Australian Lungfish (Neoceratodus forsteri). We used an enzyme-based alternative to bisulfite conversion to reduce DNA fragmentation and evaluated its performance relative to bisulfite conversion. We found the sequencing quality to be 12% higher with the enzymatic alternative compared to bisulfite treatment (p-value < 0.01). This new enzymatic based approach, although currently double the cost of bisulfite treatment can increases the throughput and sequencing quality. We envisage this assay setup being adopted increasingly as the scope and scale of epigenetic ageing research continues to grow.

Assessing DNA Degradation through Differential Amplification Efficiency of Total Human and Human Male DNA in a Forensic qPCR Assay.

The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of degradation. The possibility of deriving information on DNA degradation was evaluated in a forensic qPCR assay not specifically designed to detect DNA fragmentation, the Plexor HY (Promega), by calculating the ratio between the estimated concentrations of autosomal (99 bp) and Y-chromosomal (133 bp) targets ("[Auto]/[Y]"). The [Auto]/[Y] ratio measured in 57 formalin-fixed, paraffin-embedded samples was compared to a quality score (QS) calculated for corresponding STR profiles using quantitative data (allele peak height). A statistically significant inverse correlation was observed between [Auto]/[Y] and QS (R = -0.65, p < 0.001). The [Auto]/[Y] values were highly correlated (R = 0.75, p < 0.001) with the "[Auto]/[D]" values obtained using the PowerQuant (Promega) assay, expressly designed to detect DNA degradation through simultaneous quantification of a short (Auto) and a long (D) autosomal target. These results indicate that it is possible to estimate DNA degradation in male samples through Plexor HY data and suggest an alternative strategy for laboratories lacking the equipment required for the assessment of DNA integrity through dedicated qPCR assays.

Cell based and In vivo systematic evaluation of some Egyptian plant extracts targeting breast cancer.

The prevalence of breast cancer as a significant public health concern necessitates continued exploration of natural resources for novel anti-cancer agents is crucial. MATERIAL AND METHODS: Anticancer activity of plant extracts on monolayer breast cancer cell line (MCF7) with lower levels of toxicity towards normal (RPE1) underwent further assessment using a three-dimensional model (3D). The extract's effects were investigated through multiple assays including apoptosis induction using quantifying cleaved cytokeratin-18 (CK18) and DNA fragmentation. Additionally, the expression of Bcl-2 and Bax was quantitative using real-time PCR. The median lethal dose (LD50) was determined by the acute oral toxicity, while biomarkers associated with tumorigenesis, metastasis, and cell death were quantified by ELISA. RESULTS: Limoniastrum monopetalum and Bauhinia variegata exhibited the most potent antitumor efficacy among the investigated extracts. They demonstrated potent cytotoxicity against MCF7 with no significant effect on hTERT RPE-1, with an IC50 of 100 µM. The extract demonstrated effectiveness in killing cancer cells within 3D tumor-like structures, induced apoptosis through caspase-3 activation and cleavage of cytokeratin-18, up-regulated the tumor suppressor p53, down-regulated the anti-apoptotic Bcl-2 gene, and caused DNA fragmentation. Acute oral toxicity studies in mice indicated low toxicity, and in a syngeneic mouse tumor model, the extract significantly inhibited tumor growth, suggesting its potential for further development. CONCLUSION: Limoniastrum monopetalum and Bauhinia variegata exhibited the most potent antitumor efficacy among the investigated extracts.

Mitochondrial genome fragmentation is correlated with increased rates of molecular evolution.

While mitochondrial genome content and organization is quite diverse across all Eukaryotes, most bilaterian animal mitochondrial genomes (mitogenomes) exhibit highly conserved gene content and organisation, with genes typically encoded on a single circular chromosome. However, many species of parasitic lice (Insecta: Phthiraptera) are among the notable exceptions, having mitogenomes fragmented into multiple circular chromosomes. To better understand the process of mitogenome fragmentation, we conducted a large-scale genomic study of a major group of lice, Amblycera, with extensive taxon sampling. Analyses of the evolution of mitogenome structure across a phylogenomic tree of 90 samples from 53 genera revealed evidence for multiple independent origins of mitogenome fragmentation, some inferred to have occurred less than five million years ago. We leveraged these many independent origins of fragmentation to compare the rates of DNA substitution and gene rearrangement, specifically contrasting branches with fragmented and non-fragmented mitogenomes. We found that lineages with fragmented mitochondrial genomes had significantly higher rates of mitochondrial sequence evolution. In addition, lineages with fragmented mitochondrial genomes were more likely to have mitogenome gene rearrangements than those with single-chromosome mitochondrial genomes. By combining phylogenomics and mitochondrial genomics we provide a detailed portrait of mitogenome evolution across this group of insects with a remarkably unstable mitogenome structure, identifying processes of molecular evolution that are correlated with mitogenome fragmentation.

Phase angle at bioelectric impedance analysis is associated with detrimental sperm quality in idiopathic male infertility: a preliminary clinical study.

Background: In 2020, 38% of adults were affected by obesity, while infertility globally affected 1 in 6 people at some stage of their lives.Body mass index (BMI) provides an easy but occasionally inaccurate estimation of body composition. To achieve a more precise assessment, bioelectric impedance analysis serves as a validated tool that administers electrical energy through surface electrodes. Phase angle as a function of the relationship between tissues resistance and reactance, is a trustworthy predictor of body composition and cell membrane integrity. Objectives: We aim to assess whether there is an association between phase angle and seminal parameters, as well as sperm DNA fragmentation percentage. Design: Semen samples of 520 idiopathic infertile patients were analyzed according to 2021 World Health Organization guidelines and evaluated for sperm DNA fragmentation rate. Each participants underwent bioelectric impedance analysis. Results: Median age was 40 years old, median BMI was 26.3 kg/m2, median phase angle was 6.2°. In the logistic regression analysis adjusted for age and total intracorporeal water, phase angle (continuous) was significantly associated with oligozoospermia (odds ratio [OR]:0.4; p<0.01) and sperm morphology (OR: 0.65; p=0.05) and slightly with sperm DNA fragmentation (OR: 0.98; p=0.07). In subgroup analysis, the logistic regression analysis adjusted for the mentioned parameters showed that a phase angle between 6.2 and 7 (°) (OR: 0.63; p=0.02) and >7 (°) (OR: 0.12; p<0.01) were associated with a reduced risk of oligozoospermia compared to values <6.2 (°). Similarly, a phase angle between 6.2 and 7 (°) (OR: 0.57; p< 0.01 and OR: 0.58; p= 0.01) and PA > 7 (°) (OR: 0.12; p= 0.03 and OR: 0.21; p< 0.01) were associated with a reduced risk of lower sperm concentration and lower total sperm count, respectively, compared to a phase angle < 6.2 (°). Conclusion: Our study suggests a negative association between phase angle and detrimental sperm parameters in male idiopathic infertility.

Impact of Escherichia coli, Candida non-albicans, and Trichomonas vaginalis on Semen Chemical and Functional Parameters: an In-Vitro Study.

BACKGROUND: Infertility is a worldwide medical issue in which infection is recognized to play a major role. Pathogens trigger various mechanisms that impact fertility, either directly by affecting the physiological indices of semen or indirectly by disrupting the process of spermatogenesis. In the current work, the effect of in-vitro cultivation of Escherichia coli (E. coli), Candida non-albicans (C. non-albicans), and Trichomonas vaginalis (T. vaginalis) (as the most frequently reported sexually transmitted infections) was assessed on the physiological functions of the spermatozoa and the chemical characteristics of the seminal fluid. METHOD: The semen samples were exposed to cultures of E. coli, C. non-albicans, and T. vaginalis. The study analyzed the changes in motility, agglutination, viability, DNA fragmentation index (DFI%), seminal pH, and biochemical parameters at 1/2, 1, 1.5, 2, 2.5, 3.5 and 4 hours. RESULTS: Incubation of the semen samples with E. coli resulted in a progressive increase in agglutination, pH, and nitrite. The seminal glucose and the sperm motility, on the other hand, were reduced. The sperm vitality and seminal protein remained unaffected. C. non-albicans induced three forms of agglutination (head-to-head, tail-to-tail, and head-to-tail), lowered pH values and decreased the sperm motility, but did not alter the seminal protein, glucose, nitrite, nor the spermatozoa viability at the different tested time intervals. T. vaginalis resulted in increased seminal protein, and reduced glucose, pH, and motility. It also induced minimal agglutination and caused unchanged nitrite and sperm viability. The DFI% was increased in all pathogens with the C. non-albicans showing the highest DNA fragmentation index. CONCLUSION: Urogenital infection with E. coli, C. non-albicans, or T. vaginalis is assumed to affect the quality of semen through DNA fragmentation, agglutination and altered seminal chemical microenvironment.

Reproductive outcomes in patients with high levels of sperm DNA fragmentation using testicular sperm for intracytoplasmic injection: a retrospective analysis.

This study aims to compare the embryological and clinical parameters of intracytoplasmic sperm injection (ICSI) cycles using testicular versus ejaculated sperm in male patients with elevated sperm DNA fragmentation (SDF). A total of 73 ICSI cycles were examined in couples where the male partner exhibited high levels of SDF. ICSI was performed using either ejaculated or testicular sperm. The primary outcomes were rates of blastocyst formation, high-quality embryo development, and clinical pregnancy. The DNA fragmentation index (DFI) for testicular sperm (16.81 ± 17.51) was significantly lower than that of ejaculated sperm (56.96 ± 17.56). While the blastocyst formation rate was significantly higher in the testicular sperm group compared to the ejaculated sperm group, no statistically significant differences were noted in fertilization rate (72.15% vs. 77.23%), rate of high-quality embryo formation (47.17% vs. 46.53%), clinical pregnancy (50% vs. 56.52%), Cumulative pregnancy (70.2% vs. 55.6%), or live birth rate (43.75% vs.43.48%). Testicular spermatozoa have no additional advantage over ejaculated spermatozoa except for blastocyst quality in patients with high SDF, the use of testicular spermatozoa for the first ICSI cycle in male infertility patients with high SDF should be undertaken after much consideration at present.

Correlation between standard sperm parameters and sperm DNA fragmentation from 11,339 samples.

Conventional semen parameters have long been considered fundamental in male fertility analyses. However, doubts have been raised regarding the clinical utility of the assessment of spermatozoa (sperm) DNA damage. In this retrospective study, we investigated the potential correlation between conventional semen parameters and semen DNA fragmentation (SDF) assessed as sperm DNA damage, in 11,339 semen samples collected between January 2019 and June 2022. We observed significant negative correlations between the DNA fragmentation index (DFI) and sperm viability (correlation coefficient [r] = -0.514) as well as progressive sperm motility (r = -0.512, p < 0.05). Samples were categorized into three groups according to DFI levels (Groups A, B, and C: ≤15%, 15 < DFI ≤30%, and >30%, respectively). Furthermore, the percentage of semen samples with normal sperm conventional parameters in Groups A, B, and C was 76.7% (4369/5697), 61.4% (2351/3827), and 39.7% (721/1815), respectively. Moreover, according to the reference values of conventional sperm parameters, the samples were divided into Groups F, G, and H with all normal, only one abnormal, and > two abnormal parameters, respectively. In addition, the proportions of samples with abnormal DFI values (>30) in Groups F, G, and H were 9.7% (721/7441), 23.1% (618/2676), and 39.0% (476/1222), respectively. Multivariate logistic regression models demonstrated that sperm vitality, progressive sperm motility, normal sperm form, total sperm count, semen volume, age, and some sperm kinematics collectively improved the area under the receiver operating characteristic curve (AUROC) to 0.861, surpassing the predictive value of a single predictor of pathologically damaged sperm DNA. Our study suggests that samples with abnormal sperm parameters may have a higher likelihood of high DNA fragmentation. Furthermore, certain semen parameters could be potential indicators of sperm DNA fragmentation, aiding sperm selection in assisted reproductive procedures.

Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Sperm Chromatin Dispersion Test Detects Sperm DNA Fragmentation Mainly Associated with Unviable Spermatozoa and Underestimates the Values with Respect to TUNEL Assay.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

Zinc oxide nanoparticles preserve the quality and fertility potential of rooster sperm during the cryopreservation process.

Sperm cryopreservation is one of the main methods for preserving rooster sperm for artificial insemination (AI) in commercial flocks. Yet, rooster sperm is extremely susceptible to reactive oxygen species (ROS) produced during the freezing process. Oxidative stress could be prevented by using nanoparticles containing antioxidants. The present study was conducted to investigate the effect of zinc oxide nanoparticles (ZnONP) in rooster semen freezing extender on quality parameters and fertility potential. For this aim, semen samples were collected and diluted in Lake extenders as follows: control: Lake without ZnONP, ZnO100: Lake with 100-µg zinc oxide (ZnO), ZnONP50: Lake with 50-µg ZnONP, ZnONP100: Lake with 100-µg ZnONP and ZnONP200: Lake with 200-µg ZnONP. After freezing and thawing, sperm motility, viability, membrane integrity, morphology, mitochondrial activity, acrosome integrity, DNA fragmentation, lipid peroxidation and ROS, as well as fertility and hatchability were assessed. According to the current results, higher rates of motility, membrane integrity, mitochondrial activity, acrosome integrity and live cells were detected in the ZnO100, ZnONP50 and ZnONP100 groups compared to other groups (p ≤ .05). Yet, the percentage of dead cells, DNA fragmentation, lipid peroxidation and ROS levels were lower in the mentioned groups (p ≤ .05). Furthermore, a higher percentage of fertility was observed in the ZnO100 and ZnONP100 groups than in the control group (p ≤ .05). In conclusion, the use of 100-µg ZnO and 50- to 100-µg ZnONP represents a valuable and safe additive material that could be used to improve the quality and fertility potential of rooster sperm under cryopreservation conditions.

Elevated sperm DNA fragmentation is correlated with an increased chromosomal aneuploidy rate of miscarried conceptus in women of advanced age undergoing fresh embryo transfer cycle.

Background: Male sperm DNA fragmentation (SDF) may be associated with assisted reproductive technology (ART) outcomes, but the impact of SDF on the occurrence of aneuploid-related miscarriage remains controversial. Methods: Genome-wide single-nucleotide polymorphism-based chromosomal microarray analysis was performed on 495 miscarried chorionic villus samples undergone IVF/ICSI treatment from the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University. SDF was assessed using sperm chromatin structure assay. Patients were divided into four groups according to embryo transfer cycle type and maternal age, and the correlation between SDF and chromosome aberration was analyzed. A receiver operating characteristic (ROC) curve was utilized to find the optimal threshold. Results: Total chromosomal aneuploidy rate was 54.95%, and trisomy was the most common abnormality (71.32%). The chromosomally abnormal group had higher SDF than the normal group (11.42% [6.82%, 16.54%] vs. 12.95% [9.61%, 20.58%], P = 0.032). After grouping, elevated SDF was significantly correlated with an increasing chromosome aneuploidy rate only in women of advanced age who underwent fresh embryo transfer (adjusted odds ratio:1.14 [1.00-1.29], adjusted-P = 0.045). The receiver operating characteristic curve showed that SDF can predict the occurrence of chromosomal abnormality of miscarried conceptus in this group ((area under the curve = 0.76 [0.60-0.91], P = 0.005), and 8.5% was the optimum threshold. When SDF was ≥ 8.5%, the risk of such patients increased by 5.76 times (adjusted odds ratio: 6.76 [1.20-37.99], adjusted-P = 0.030). Conclusion: For women of advanced maternal age undergoing fresh embryo transfer, older oocytes fertilized using sperm with high SDF in IVF/ICSI treatment might increase the risk of chromosomal abnormality in miscarried conceptus.