Sperm DNA Damage Research Articles

A Review Diagnosing Male Infertility: Beyond Conventional Semen Analysis

Infertility, defined as the inability of couples to conceive after one year of consistent, unprotected sexual contact, is a widespread concern affecting a significant portion of the population. Notably, between 14 to 17% of couples may encounter infertility at some point during their reproductive journey. This review delves into the realm of diagnosing male infertility beyond conventional semen analysis. Conventional semen analysis, which is a globally accepted tool for male infertility, primarily focuses on assessing sperm count, motility, viability, and morphology, leaving certain aspects of sperm functionality unexplored. These limitations necessitate the integration of advanced semen analysis techniques to provide a more comprehensive evaluation. Advanced semen analysis encompasses a range of sophisticated tests that probe deeper into the intricacies of male fertility. These tests include assessing sperm penetration capabilities, acrosomal discharge, and spermatozoa Reactive Oxygen Species (ROS) levels. Moreover, advanced semen analysis encompasses tests like sperm chromatin structure assay, DNA damage assessment, sperm proteomics, sperm metabolomics, and high sperm magnification microscopy, all of which shed light on various facets of sperm function and quality. While advanced semen analysis techniques offer a more comprehensive understanding of male infertility, their uptake and utilization in clinical practice have been limited. This review aims to elucidate the roles, merits, and drawbacks of both conventional and advanced semen analysis in diagnosing male infertility. This review sheds light on the strengths and weaknesses of each approach, it promotes a more nuanced approach to male infertility diagnosis, enhancing the prospects of successful conception for infertility challenged couples.

Lipid mixtures (from a liposome kit) and melatonin improve post-thawed Angora goat sperm parameters

Semen freezing and storing has been widely used in reproductive biotechnology, being applied to certain males of livestock breeds or animal species with economic value such as the Angora goat. The development of a semen extender with the cryoprotective agents can prevent the deterioration of sperm parameters after thawing. This study aimed to investigate lipid mixtures (from a liposome kit, Lps) and melatonin (Mel) at different doses to prevent the deterioration of sperm parameters and to provide the cryoprotective effects on sperm DNA. The Angora goat ejaculates were collected and pooled. They were divided into seven equal volumes, and each of them was diluted with the extenders of the experimental groups with additives (Lps 321.99 μg/mL, Lps 841.33 μg/mL, Mel 0.25 mM, Mel 1 mM, Lps 321.99 μg/mL + Mel 1 mM, Lps 841.33 μg/mL + Mel 0.25 mM) and no additives (control group). After the freeze-thawing process, motility, viability, acrosome integrity, DNA double-strand breaks, and abnormal DNA integrity were assessed for different extender groups. It was determined that the use of Lps alone at low dose or the combination of Lps and Mel had significant cryoprotective effects on motility, viability, acrosome integrity, and DNA damage in Angora goat sperm. This study will help us to understand the effects of Lps and Mel used alone or in combination at different doses and which doses give the optimum spermatological parameter rates following the freeze-thawing process, and hence it will shed light on further studies.

DNA damage, obesity and obesity-related health complications: what are new data telling us?

Obesity is associated with increased DNA damage, which may in turn contribute to the development of obesity-related complications. DNA damage can also affect adipocyte biology, resulting in increased adiposity. Carefully managed weight loss programs can reverse this process. This article surveys new data that support these contentions. Whole exome sequencing analyses have identified rare variants linked to high BMI and adiposity. Two of the identified genes are linked to DNA damage and DNA repair, suggesting that DNA damage itself may play a role in the cause of obesity. It has also been recognized that obesity increases DNA damage in breast tissue of carriers of BRCA mutations and rates of tumour formation in BRCA1+ mice, indicating effect of obesity on cancer development in high-risk populations. In addition, obesity promotes cancer cell chemoresistance by decreasing fatty acid oxidation involved in cellular DNA damage response, leading to apoptotic cellular death. Obesity is also associated with a reduced capacity of oocytes to repair sperm DNA damage, leading to lower in-vitro fertilization rates in women with obesity. DNA damage and cellular responses to DNA damage can be both the result and the cause of obesity and can strongly influence the development and treatment of obesity-associated diseases.

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.

Transcriptomic and proteomic features of a mouse model of sperm DNA damage induced by benzo(a)pyrene

This study replicated a mouse model of sperm DNA damage induced by benzo(a)pyrene (BaP), and the transcriptomic and proteomic features of the model were examined to clarify the pathways related to BaP-induced damage to sperm DNA. Male mice in the BaP group were subjected to BaP at a dosage of 100 mg/kg/d or an equivalent quantity of saline solution in the control group for 60 days. Subsequently, the DNA fragmentation index (DFI) in sperm was assessed using a sperm chromatin structure assay (SCSA). RNA-seq and data-independent acquisition (DIA) were used to identify the mRNA and protein expression patterns in the testis. The sperm DFI significantly increased in the BaP group. Compared to the control group, the BaP group exhibited differential expression of 240 genes (referred to as DEGs) and 616 proteins (referred to as DEPs). These molecules included Aldh1a1, Cyb5r3, Fads1, Oxsm, Rcn3, and Prss45. Pathways in cancer, the PI3K-Akt signaling pathway, metabolic pathways, and the MAPK signaling pathway were the primary areas where these genes showed enrichment. BaP can damage the DNA of sperm and affect metabolism, the PI3K-Akt pathway, and pathways associated with cancer signaling.

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Increased Sperm DNA Damage in Sprague-Dawley Rats Exposed to Dextromethorphan as an Antitussive

Introduction: Male reproductive functions have been observed to be negatively impacted by the antitussive dextromethorphan (DM). It was also discovered that DM has an impact on micronutrients (such as zinc, calcium, and selenium) which are crucial for both capacitation and the acrosome reaction, which can result in infertility. Using Sprague-Dawley rats as models, we investigated the effects of quercetin and rutin on DM-induced toxicity in males in relation to apoptotic protein markers. Methodology: For a period of 16 weeks, 80 male rats, weighing 150 ± 30 g, were utilized and split into four groups. Twenty animals were employed in each group. For a period of 16 weeks, Group B received 20 mg/kg, Group C received 40 mg/kg, and Group D received 80 mg/kg of DM. Group A acted as the control group and received 1 ml of distilled water (DW). Five randomly chosen animals from each group were put to sleep at the end of the DM treatment period, and their testes were taken out to measure DNA fragmentation using comet assay. To ascertain the pace of recovery, the animals were then split into three groups, E–G, and kept for a total of 16 weeks. Group G received 1 ml of DW, Group F was given rutin (25 mg/kg), and Group E was given quercetin (50 mg/kg). Results: Comparing DM-treated groups to control revealed a significant dose-dependent decrease in DNA fragmentation. When DM-treated and recovery-alone groups were contrasted with rutin and quercetin groups, an improvement in DNA was seen. Conclusion: Significant improvements in the parameters were observed when rutin and quercetin supplements were used, which may lessen the harmful effects of DM and enhance male fertility.

Effect of Testosterone and Antioxidant-Loaded Nanoliposomes on the Performance of Normozoospermic Samples: A Case-Control Study

Introduction: Antioxidants reduce the harmful effects of reactive oxygen species (ROS) in sperm. Additionally, nanoliposomes can be used as an efficient new drug delivery system that mitigates the adverse effects of drugs. This study aimed to investigate the use of nanoliposomes for delivering drugs such as resveratrol, catalase, resveratrol-catalase, and testosterone to normozoospermic samples to minimize the side effects of ROS. Methods: This study took place at Yazd Reproductive Sciences Institute in Iran from October 2018 to December 2019. It involved 450 samples divided into three groups. Each of the 15 normozoospermic sperm samples collected from men was divided into 10 parts and analyzed in three groups: free drugs, nanoliposomes, and a control group. The study analyzed sperm parameters, PRM2 (Protamine 2) and HSP70 (heat shock proteins) expression, as well as sperm acrosome reaction and DNA fragmentation before and after freezing. Results: Nanoliposomes improved sperm motility and viability before freezing (P&lt;0.05). Free resveratrol-catalase reduced motility before freezing (P&lt;0.05). PRM2 and HSP70 expression were lower in the blank and testosterone nanoliposome groups (P&lt;0.0001). After freezing, nanocarriers and free testosterone improved sperm motility (P&lt;0.05). Resveratrol and testosterone-loaded nanoliposomes increased sperm viability (P&lt;0.01). Nanoliposomes caused less sperm DNA damage (P&gt;0.05). Free catalase reduced PRM2 and HSP70 expression (P&lt;0.0001). Conclusion: Nanoliposomes can be used as an efficient drug delivery system to decrease ROS production or their side effects. Additionally, during freezing, they improve sperm viability and reduce DNA damage.

Evaluation of Sperm DNA Fragmentation Using Halosperm Technique after the Freezing-Thawing Process in Men: A Study on the Validation of the SCD Protocol.

DNA fragmentation index (DFI) enhances routine semen analysis by providing valuable insights into male reproductive potential. Utilizing Halosperm test, a sperm chromatin dispersion (SCD) assay based on induced condensation. The purpose of this study was to assess sperm DNA damage both before and after freezing. By following the specified kit instructions, an attempt was made to validate the SCD test protocol, with a particular emphasis on the implications of sperm freezing on its DNA integrity. In total, 380 fresh human semen samples from normozoospermic patients were frozen at -20°C for 10 days, using SCD cryopreservation reagent. Routine semen analysis and DNA fragmentation index (DFI) were determined for each sample before freezing and after thawing. Semen morphology and sperm DFI were compared before and after freezing/thawing process. There was a significant decrease in sperm normal morphology after thawing (9.31±2.42% vs. 7.1±1.53%, p<0.05, respectively). The sperm head, midpiece, and tail defect rate increased after freezing at -20°C. Moreover, DFI was significantly higher after thawing compared to before freezing (20.71±1.61% before freezing vs. 29.1±0.21% after thawing with p<0.001). Cryoconservation of semen samples at -20°C for 10 days using SCD cryopreservation reagent seems to damage sperm morphology, resulting in a reduction in sperm DNA integrity. The measurement of DFI on a fresh sample remains the most reliable technique for obtaining accurate results.

Effects of intratesticular injection of hypertonic mannitol and saline on the quality of donkey sperm, indicators of oxidative stress and testicular tissue pathology

ObjectivesThe aim of the present study was to examine donkey sperm quality after intratesticular injection of hypertonic mannitol (HM) and saline (HS).MethodsRandomly assigned to five treatment groups were 15 adult male donkeys: (1) Control group (no treatment), (2) Surgery group (surgical castration for testosterone control), (3) NS group (normal saline intratesticular injection), (4) HS group (hypertonic saline), and (5) HM group. We injected 20 mL per testicle. We took 5 mL blood from all donkeys before injection. Castration was performed under general anesthesia 60 days later. Samples included blood and testicular tissue. Total motility (TM), progressive motility (PM), movementy features, DNA damage, morphology, viability, and plasma membrane functionality were evaluated. Hormone analyses, histomorphometric studies and oxidative stress indices including total antioxidant capacity (TAC), glutathione peroxidase (GPx), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and NADP+/NADPH were evaluated. Apoptosis, pyroptosis-related Bax, Caspase-1, GSDMD, and Bcl-2 expression were also assessed.ResultsIn HS and HM groups, testosterone, epididymal sperm count, motility, viability, and plasma membrane functionality dropped while sperm DNA damage increased. HS and HM groups had significantly lower histomorphometric parameters, TAC, GPx, SOD, GSH, and Bcl-2 gene expression. MDA, NADP+/NADPH, Bax, Caspase-1, and GSDMD gene expression were substantially higher in the HS and HM groups than in the control group.ConclusionsToxic effects of hypertonic saline and mannitol on reproductive parameters were seen following, hence, they might be considered as a good chemical sterilizing treatment in donkeys.

Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. The study is funded by the Iran University of Medical Sciences and no competing interest exists. N/A.

(203) Impact of Smoking on Sperm Chromatin Integrity

Abstract Introduction Semen analysis is an essential component of infertility evaluation, but it may still fail to detect subtle sperm defects present in patients with male factor infertility. Although estimates vary, approximately 15% of patients with male factor infertility have normal spermiograms It is necessary to identify diagnostic measures for male infertility that are easy to perform, inexpensive, and provide an accurate diagnosis. Tests that assess sperm quality should not only identify the ability of spermatozoa to reach the oocyte but also their ability to fertilize the oocyte and activate embryo growth. The estimation of sperm DNA damage fills the above need in the diagnosis of male infertility Abnormalities in the male genome characterized by damaged sperm DNA may be indicative for male subfertility regardless of routine semen parameters, and these parameters do not reveal sperm DNA defects Men with normal semen analysis may also have a high degree of DNA fragmentation, which can be a major cause of undiagnosed/unexplained infertility. Sperm DNA fragmentation may result from aberrant chromatin packaging during spermatogenesis, defective apoptosis before ejaculation or excessive production of reactive oxygen species (ROS) in the ejaculate. Objective To study the impact of cigarette smoking on sperm chromatin integrity in infertile patients. Methods The present study included eighty infertile male patients recruited from the Andrology Outpatient Clinic at Al-Azhar University Hospitals The patients were divided into two groups: - Group I: forty nonsmoker infertile males. (Control Group). Group II: forty cigarette smoker infertile males. (Study Group) Results The current study revealed that smoking increases the abnormality of sperm chromatin integrity in sperms of smokers. This is evident by the positive correlation of increased head stainability of sperms in smokers compared to nonsmokers. The value of partially stained sperm heads using aniline blue is calculated from the total staining score formula, this score was found to increase significantly among smokers in comparison to nonsmokers. In the present study, the increased severity of smoking (smoking index), was not correlated with the degree of sperm head stainability. This mean that, smoking insult on sperm chromatin integrity is crucial regardless the duration or the amount of the consumed cigarettes Conclusions There is a significant increase of sperm chromatin damage in smoker infertile patients than nonsmoker infertile patients and these damages may affect the quality of the ejaculated spermatozoa and decrease their fertility potential. Not only semen parameters but also sperm chromatin condensation is affected significantly by smoking. Aniline blue staining is an effective and inexpensive tool for assessment of the fertilization potentiality of infertile patients. A significant correlation was found between the percentage of spermatozoa with normal sperm chromatin condensation and normal morphology. Cigarette smoking may have a negative impact on sperm quality and DNA integrity. Therefore, it would be advisable for men to abstain from smoking to avoid decreased fertility. Disclosure No.

Role of DNase Activity in Human Sperm DNA Fragmentation.

In this clinical era of intracytoplasmic sperm injection (ICSI), where a single spermatozoon is chosen for fertilization, the diagnostic functionality of the classical parameters typically associated with fertilization, such as sperm concentration, sperm motility, acrosome integrity, and mitochondria, is perhaps becoming less critical. In contrast, the contribution of sperm DNA quality to our understanding of the impact of male fertility within the context of ICSI is gaining increasing interest and importance. Even with respect to natural conception, high levels of sperm DNA fragmentation (SDF) in the ejaculate can adversely affect reproductive outcomes. However, the precise origin of SDF pathology in sperm cells is often ambiguous and most likely to be multifactorial. Hence, the genetic makeup of an individual, unbalanced REDOX processes, enzymatic activity, environmental and lifestyle factors, and even damage during sperm handling in the laboratory all operate in a unique and often synergistic manner to produce or induce sperm DNA damage. Surprisingly, the contribution of active enzymes as potential agents of SDF has received much less attention and, therefore, is likely to be underrated. This review highlights the roles of different enzymes related to the degradation of sperm DNA as possible effectors of DNA molecules in spermatozoa.

Isolated teratozoospermia: revisiting its relevance in male infertility: a narrative review.

Basic semen analysis is the first step in the evaluation of male infertility. It includes an assessment of sperm morphology which is believed to reflect on overall spermatogenesis and sperm function. Teratozoospermia, defined as abnormal sperm morphology, is frequently present in association with severe oligoasthenozoospermia, but isolated teratozoospermia (in the presence of normal counts and motility) is a poorly understood clinical entity with conflicting implications in terms of fertility potential and treatment strategies. The following paper aims to: (I) discuss the classification of sperm morphology, causes, and molecular mechanism of teratozoospermia; (II) analyze the clinical significance and potential treatment options of isolated teratozoospermia as a cause of male infertility and a predictor of fertility outcome; and (III) provide a SWOT (strengths, weaknesses, opportunities, and threats) analysis based on the existing literature on this topic. A comprehensive search from database inception to 25 April 2023 was conducted in PubMed for relevant papers relating to isolated teratozoospermia in male infertility. Finally, seven systematic reviews/reviews/meta-analyses and 81 original articles were synthesized into the current narrative review. Classification of sperm morphology has evolved significantly since the first edition of the World Health Organization (WHO) Manual of Human Semen Analysis. Kruger's strict criteria are the most used classification and have been shown to correlate with fertility outcomes. There are many causes of teratozoospermia including genetic and environmental factors and physical conditions like varicocele. Teratozoospermia correlates with sperm DNA damage, elevated oxidative stress, low antioxidant function, and apoptotic alterations, which can result in impaired spermatozoa function and lower pregnancy rates. However, the clinical correlation between teratozoospermia and assisted reproductive technology (ART) outcome shows conflicting data with recent meta-analyses suggesting that isolated teratozoospermia was not associated with poor fertility outcomes from ART and that intrauterine insemination (IUI) can be an effective option even in the presence of teratozoospermia. There is very limited data on effective therapeutic options to treat idiopathic isolated teratozoospermia. The opportunity for future research is huge to fill the gap in the medical literature on this topic. Contemporary literature on isolated teratozoospermia shows conflicting results in terms of its actual clinical implication in male infertility and the utility of available treatment options. Further research is warranted on this clinical entity to improve sperm function and future paternity.

Characterization of sperm quality in captive-bred barramundi (Lates calcarifer): Effect on spawning performance and paternal contribution in mass-spawning events

Barramundi (Asian sea bass, Lates calcarifer) is a species with a mass-spawning reproductive strategy whereby adults synchronously release their gametes into the water column. In captivity, this reproductive strategy usually results in uneven paternal contributions to each cohort of progeny, creating skews in family size that hinder the effectiveness of selective breeding programs. As sperm quality influences fertilization success and early larval development, we investigated the relationship between sperm quality and spawning performance. Accordingly, we assessed three established breeding cohorts. Sperm samples (n = 22) were collected by cannulation. Sperm motility was assessed using CASA, and sperm viability and DNA integrity were evaluated using dual-fluorescence stains by flow cytometry. Broodstock were induced to spawn across two consecutive nights, and offspring were collected at 2.5 h and 12 h post-fertilization (hpf) and 24 h and 48 h post-hatching (hph). Offspring were assessed for morphological abnormalities, and key morphological parameters were recorded. Offspring collected at 2.5 hpf and 24 hph were genotyped to determine their parentage and examine the relationship between sperm quality of individual males and offspring survival. Results highlighted that male condition factor (K) and sperm quality were highly variable within each breeding cohort. Males with a lower condition factor showed lower sperm motility and velocity. In contrast, males with a higher condition factor showed higher sperm motility and velocity but also higher levels of sperm DNA damage. While all males were capable of fertilization, males with a lower condition factor had greater paternal contribution. In this study, a reduction in skewed paternity was also found on Night 2 of spawning compared to Night 1. Moreover, males from Tank B, which had the lowest level of sperm DNA damage, had the most even paternal contribution. Conversely, highly skewed maternal contributions were reported across all spawning events. The results of this study suggest that additional complex dynamics and further unmeasured variables, such as spawning behavior, social hierarchy, and the spawning induction procedure, may also skew family sizes. Therefore, further development of artificial reproductive technology is recommended to gain greater control over individual contribution and overcome current breeding bottlenecks.

Assessment of the impact of direct in vitro PFAS treatment on mouse spermatozoa.

Poly- and per-fluoroalkyl substances (PFAS) are synthetic environmentally persistent chemicals. Despite the phaseout of specific PFAS, their inherent stability has resulted in ubiquitous and enduring environmental contamination. PFAS bioaccumulation has been reported globally with omnipresence in most populations wherein they have been associated with a range of negative health effects, including strong associations with increased instances of testicular cancer and reductions in overall semen quality. To elucidate the biological basis of such effects, we employed an acute in vitro exposure model in which the spermatozoa of adult male mice were exposed to a cocktail of PFAS chemicals at environmentally relevant concentrations. We hypothesized that direct PFAS treatment of spermatozoa would induce reactive oxygen species generation and compromise the functional profile and DNA integrity of exposed cells. Despite this, post-exposure functional testing revealed that short-term PFAS exposure (3 h) did not elicit a cytotoxic effect, nor did it overtly influence the functional profile, capacitation rate, or the in vitro fertilization ability of spermatozoa. PFAS treatment of spermatozoa did, however, result in a significant delay in the developmental progression of the day 4 pre-implantation embryos produced in vitro. This developmental delay could not be attributed to a loss of sperm DNA integrity, DNA damage, or elevated levels of intracellular reactive oxygen species. When considered together, the results presented here raise the intriguing prospect that spermatozoa exposed to a short-term PFAS exposure period potentially harbor an alternate stress signal that is delivered to the embryo upon fertilization. PFAS are synthetic chemicals widely used in non-stick cookware, food packaging, and firefighting foam. Such extensive use has led to concerning levels of environmental contamination and reports of associations with a spectrum of negative health outcomes, including testicular cancer and reduced semen quality. To investigate the effects of PFAS on male reproduction, we incubated mouse sperm in a cocktail of nine PFAS at environmentally relevant concentrations before checking for a range of functional outcomes. This treatment strategy was not toxic to the sperm; it did not kill them or reduce their motility, nor did it affect their fertilization capacity. However, we did observe developmental delays among pre-implantation embryos created using PFAS-treated sperm. Such findings raise the intriguing prospect that PFAS-exposed sperm harbor a form of stress signal that they deliver to the embryo upon fertilization.

Making immotile sperm motile using high-frequency ultrasound.

Sperm motility is a natural selection with a crucial role in both natural and assisted reproduction. Common methods for increasing sperm motility are by using chemicals that cause embryotoxicity, and the multistep washing requirements of these methods lead to sperm DNA damage. We propose a rapid and noninvasive mechanotherapy approach for increasing the motility of human sperm cells by using ultrasound operating at 800 mW and 40 MHz. Single-cell analysis of sperm cells, facilitated by droplet microfluidics, shows that exposure to ultrasound leads to up to 266% boost to motility parameters of relatively immotile sperm, and as a result, 72% of these immotile sperm are graded as progressive after exposure, with a swimming velocity greater than 5 micrometer per second. These promising results offer a rapid and noninvasive clinical method for improving the motility of sperm cells in the most challenging assisted reproduction cases to replace intracytoplasmic sperm injection (ICSI) with less invasive treatments and to improve assisted reproduction outcomes.

(304) Effects of Wireless Radiation Exposure on Genetic Variants in Human Sperm: A Preliminary Prospective Study

Abstract Introduction In the present era, the extensive use of modern cell phones has raised the health risks associated with the radiofrequency-electromagnetic radiation (RF-EMR) they emit, which the human body can absorb. RF-EMR exposure can potentially harm the process of spermatogenesis by increasing oxidative stress, elevating mitochondrial reactive oxygen species (ROS), and triggering the formation of free radicals that leads to sperm DNA damage. Our previous research revealed decreased sperm motility and viability in semen samples exposed to RF-EMR through WiFi calling. Objective To investigate the effects of WiFi radiation on genetic variants in human sperm using current-generation smartphones. Methods Six healthy, normozoospermic men aged 25-35 provided semen samples for this study. A current generation smartphone was used in WiFi calling mode, specifically through Whatsapp voice call, to serve as the source of RF-EMR. The call duration was six hours. Semen samples were analyzed based on the 2010 WHO guidelines before and after the radiation exposure. The genomics facility at our university extracted DNA from semen samples of each participant before and after RF-EMR exposure to perform whole exome sequencing with alignment to the reference genome. After sequencing, we compared genetic variants for each participant before and after RF-EMR exposure. Results After six hours of exposure, a significant decrease in sperm progressive motility was observed (p&amp;lt;0.05). Following exposure, the median number of exonic, nonsynonymous variants absent before exposure increased to 4 (IQR 3). Across the four successfully sequenced samples, we found 20 new, exonic, nonsynonymous variants after RF-EMR exposure. Significantly, male reproductive tissues expressed 12 variants, five associated with ciliary function and two related to mitochondrial function (see Table 1). Gene Chromosome Variant Location Function SPDYE11 Chr7 A &amp;gt; C Spermatids • Acetylcholine receptor activity • Cell cycle regulation SPDYE17 Chr7 A &amp;gt; C Spermatids • Protein kinase activity • Cell cycle regulation PRB2 Chr12 T &amp;gt; G salivary gland • Cell cycle regulation GGT2 Chr22 G &amp;gt; A Spermatids • Cell cycle regulation RGPD3 Chr2 C &amp;gt; T Spermatids • Ciliary function EPPK1 Chr8 G &amp;gt; A Spermatids, spermatocytes • Epithelial structure GOLGA6L4 Chr15 G &amp;gt; A Spermatids, spermatocytes • Ciliary function TCP11X2 ChrX T &amp;gt; C Spermatids • Acrosome reaction • Cilia function • Spermatid development HRNR Chr1 A &amp;gt; G Mitochondria • Metal ion binding POUF1 Chr13 &amp;gt; GCC Mitochondria • Structure &amp; function of spermatozoa NPIPB13 Chr16 T &amp;gt; A Spermatids • Ciliary function • Sperm motility NBPF11 Chr1 A &amp;gt; C Spermatids, Sertoli cells, Leydig cells • Cell cycle regulation • Microtubule development ANKRD20A1 Chr9 T &amp;gt; A Spermatocytes • Ciliary function Table 1. Candidate genes with variants after six hours of phone radiation exposure Conclusions Based on our pilot study, we observed a decrease in sperm progressive motility and an increase in potentially related genetic variants associated with sperm motility following six hours of exposure to RF-EMR. These preliminary results highlight the need for further research to investigate the effects of RF-EMR exposure on semen parameters and to determine the clinical significance of these genetic variants. Disclosure No.

Embryo development is impaired by sperm mitochondrial-derived ROS

BackgroundBasal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development.ResultsWe first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation.ConclusionCollectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.

AMELIORATING POST-THAWED SEMEN OF BUFFALO BULLS USING A MILK-BASED EXTENDER SUPPLEMENTED WITH RESVERATROL

The objective of the current study was to investigate the influence of adding resveratrol (Res) to a milk-based extender on some post-thawed semen quality of Iraqi buffalo bulls for different preservation periods (cooling at 5˚C, 48 h, 1, and 2 months post-cryopreservation; PC). Four bulls, 3.5 to 4 years old, were used from 11th November 2020 to 1st June 2021. Semen samples were collected, pooled, and divided into four groups using different Res concentrations (0, 100, 150, and 200 μM) added to the milk extender. The Res-150 μM group exhibited higher (P≤0.01) plasma membrane, acrosome integrity percentages, and total antioxidants concentration (TAC) compared to the other groups. Sperm abnormalities and DNA damage percentages, as well as malondialdehyde (MDA) concentration, were lesser (P≤0.01) in the Res-150 μM group. Finally, Res-150 μM added to a milk-based extender increased the percentage of post-thawed live sperm and TAC in seminal plasma and minimized abnormal sperm percentage, DNA damage percentage, and MDA concentration in the seminal plasma of Iraqi buffalo bulls.