Sperm Damage Research Articles

Correlation of IGF2 levels with sperm quality, inflammation, and DNA damage in infertile patients.

Insulin-like growth factor 2 (IGF2) is a critical endocrine mediator implicated in male reproductive physiology. To investigate the correlation between IGF2 protein levels and various aspects of male infertility, specifically focusing on sperm quality, inflammation, and DNA damage, a cohort of 320 male participants was recruited from the Women's Hospital, Zhejiang University School of Medicine (Hangzhou, China) between 1st January 2024 and 1st March 2024. The relationship between IGF2 protein concentrations and sperm parameters was assessed, and Spearman correlation and linear regression analysis were employed to evaluate the independent associations between IGF2 protein levels and risk factors for infertility. Enzyme-linked immunosorbent assay (ELISA) was used to measure IGF2 protein levels in seminal plasma, alongside markers of inflammation (tumor necrosis factor-alpha [TNF-α] and interleukin-1β [IL-1β]). The relationship between seminal plasma IGF2 protein levels and DNA damage marker phosphorylated histone H2AX (γ-H2AX) was also explored. Our findings reveal that IGF2 protein expression decreased notably in patients with asthenospermia and teratospermia. Correlation analysis revealed nuanced associations between IGF2 protein levels and specific sperm parameters, and low IGF2 protein concentrations correlated with increased inflammation and DNA damage in sperm. The observed correlations between IGF2 protein levels and specific sperm parameters, along with its connection to inflammation and DNA damage, underscore the importance of IGF2 in the broader context of male reproductive health. These findings lay the groundwork for future research and potential therapeutic interventions targeting IGF2-related pathways to enhance male fertility.

Effects of nano-berberine and berberine loaded on green synthesized selenium nanoparticles on cryopreservation and in vitro fertilization of goat sperm

After cryopreservation, reactive oxygen species (ROS) can damage sperm. Antioxidants are the primary defense against oxidative damage. Berberine is a bioactive alkaloid found in Berberis vulgaris, Curcuma longa, and Ergon grape, and is a potent antioxidant. Due to the negative effects of free radicals in oxidative stress processes, antioxidant chemicals are required to protect sperm. However, berberine has low bioavailability, making it less effective. Loading techniques on nanoparticles and nanotechnology can help overcome this limitation. Selenium nanoparticles were synthesized with barberry extract, and berberine was loaded on them. Berberine nanoparticles were then synthesized using anti-solvent precipitation with a syringe pump technique. The synthesis of nanoparticles was confirmed by EDX, UV-visible, FE-SEM, Zeta-Potential, and FTIR tests. In this experiment, we aim to investigate the impact of nano-berberine and berberine loaded on Se-NPs on goat sperm parameters after freeze-thawing. We assessed the generation of reactive oxygen species (ROS), in vitro fertility, and the subsequent embryo development of zygote with treated sperm after determining the optimal concentration of various chemicals on sperm parameters. The study found that all treatments had significant differences from the control group in terms of motility, viability, DNA and membrane integrity, ROS level, lipid peroxidation, in vitro fertility ability, and the capacity to develop inseminated oocytes (p < 0.05). The most significant outcomes were observed with berberine loaded on Se-NPs and the combination of selenium nanoparticles with berberine nanoparticles.

Effect of natural astaxanthin on sperm quality and mitochondrial function of breeder rooster semen cryopreservation

Cryopreservation causes higher reactive oxygen species (ROS) concentrations, leading to oxidative stress and lipid peroxidation damage sperm, and using antioxidants can improve semen quality after freeze-thaw. Natural astaxanthin (ASTA) can be inserted into cell membranes and its antioxidant properties are stronger than other antioxidants. We aimed to investigate the effects of ASTA supplementation in the Beltsville Poultry Semen Extender (BPSE) on post-thaw rooster semen quality and to explore the potential mechanism of rooster semen quality change. The qualifying semen ejaculates collected from 30 adult male Jinghong No.1 laying hen breeder roosters (65wk old) were pooled, divided into four aliquots, and diluted with BPSE having different levels of ASTA (0, 0.5, 1, or 2μg/mL). Treated semen was cryopreserved and kept in liquid nitrogen. The entire experiment was replicated three times independently. Sperm viability, motility, curvilinear velocity, amplitude of lateral head displacement, straightness, plasma membrane integrity, and acrosome integrity were observed highest (P < 0.05) with 1μg/mL ASTA at freeze-thawing. Higher (P < 0.05) antioxidant enzyme (CAT-like, SOD) activities and free radical (·OH, O2.-) scavenging ability, less ROS and malondialdehyde (MDA) concentrations were recorded with the addition of appropriate concentrations of ASTA compared to control. In addition, the levels of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP), and lactate dehydrogenase (LDH) in the 1μg/mL ASTA group improved compared to the control group, and decreased the amount of AIF protein level but increased the Bcl-2 protein level (P < 0:05). Collectively, these results demonstrate that adding ASTA in the BPSE promoted rooster freeze-thaw sperm quality, which may be related to reducing ROS levels, protecting the antioxidant defense system, preventing lipid peroxidation, improving mitochondrial structural and functional integrity, and inhibiting sperm apoptosis.

Nme8 is essential for protection against chemotherapy drug cisplatin-induced male reproductive toxicity in mice.

Cisplatin (CP), a chemotherapy drug commonly used in cancers treatment, causes serious reproductive toxicity. With younger cancer patients and increasing survival rates, it is important to preserve their reproductive capacity. NME8 is highly expressed in testis and contains thioredoxin and NDPK domains, suggesting it may be a target against the CP-induced reproductive toxicity. We deleted exons 6-7 of the Nme8 in mice based on human mutation sites and observed impaired transcript splicing. In mice, Nme8 was not essential for spermatogenesis, possibly due to functional compensation by its paralog, Nme5. Nme8 expression was elevated and translocated to the nucleus in response to two weeks of CP treatment. Under CP treatment, Nme8 deficiency further impaired antioxidant capacity, induced lipid peroxidation and increased ROS level, and failed to activate autophagy, resulting in aggravated DNA damage in testes and sperm. Consequently, the proliferation and differentiation of spermatogonia and the meiosis of spermatocyte were almost completely halted, and sperm motility was impaired. Our research indicates that NME8 protects against CP-induced testis and sperm damage. This may provide new insights into the physiological functions of the Nme family and potential targets for preserving fertility in young male cancer patients.

Association of mixed exposure to microplastics with sperm dysfunction: a multi-site study in China

Microplastics are environmental pollutants detected in various human organs and tissues. These particles originate from multiple sources including the degradation of larger plastic items and the intentional inclusion in consumer goods. Potential risks for human health resulting from microplastics exposure have also been reported. However, the distribution in the male reproductive system and its effect remains largely unknown. This study aims to investigate the presence of multiple microplastics in human semen and urine and their association with sperm quality in a multi-site study across China. We conducted a cross-sectional study involving 113 male participants from three regions in China. Semen and urine samples were collected and analysed using Raman microscopy to detect eight types of microplastics: polystyrene (PS), polypropylene (PP), polycarbonate (PC), polyethylene (PE), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), and acrylonitrile butadiene styrene (ABS). Semen quality parameters, including total sperm count, concentration, motility, and morphology, were assessed. Statistical analyses, including single and multi-variable models, were used to evaluate the relationship between microplastic exposure and semen quality, with a focus on PTFE, after adjusting confounding factors of age, body mass index (BMI), smoking, alcohol drinking, and sites. Microplastics were detected in all semen and urine samples, with participants typically exposed to 3-5 different types. The detection rates of PS, PP and PE were the highest. Notably, PTFE exposure was significantly associated with decreased semen quality. Participants exposed to PTFE showed reductions in total sperm count [188.90±163.71 vs. 207.67±132.36 million, p=0.091], sperm concentration [52.13±47.47 vs. 58.32±37.26 million/mL, p=0.041], and progressive motility [40.29%±19.06 vs. 34.11%±17.02, p=0.083]. The multi-linear regression analysis indicated that each additional type of microplastic exposure was associated with a significant decrease in total sperm number [β=-15.4 (95% CI:-25.6,-5.2)], sperm concentration [β=-7.2 (95% CI:-12.4,-2.0)], and progressive motility [β=-8.3 (95% CI:-13.5,-3.1)]. Latent category analysis further refined these groups by types of microplastic exposure, highlighting specific types more strongly associated with decreased semen quality (OR=3.5, 95% CI: 1.8, 6.9, p<0.001). The nomogram can be used to assess the risk of sperm damage by combining the type of microplastic exposure in urine with age and BMI. Our findings highlight the potential reproductive health risks posed by microplastic contamination, particularly PTFE, a non-stick pan coating material, and raise concerns about the potential of urine testing as an indicator of male reproductive microplastic exposure. Future research is warranted to further elucidate the mechanisms underlying the adverse effects of microplastics on male fertility and cross-generational effects. This study was funded by the Clinical Research Project of Shanghai Municipal Commission of Health and Family planning (20224Y0085), Open Fund Project of Guangdong Academy of Medical Sciences (YKY-KF202202), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-064), Shanghai Clinical Research Center for Gynecological Diseases (22MC1940200), Shanghai Urogenital System Diseases Research Centre (2022ZZ01012), Key Discipline Construction Project (2023-2025) of Three-Year Initiative Plan for Strengthening Public Health System Construction in Shanghai (GWVI-11.1-35, GWVI-11.2-YQ29) and Shanghai Frontiers Science Research Base of Reproduction and Development.

Progesterone promotes CXCl2-dependent vaginal neutrophil killing by activating cervical resident macrophage-neutrophil crosstalk.

Vaginal infections in women of reproductive age represent a clinical dilemma with significant socioeconomic implications. The current understanding of mucosal immunity failure during early pathogenic invasions that allows the pathogen to grow and thrive is far from complete. Neutrophils infiltrate most tissues following circadian patterns as part of normal repair, regulation of microbiota, or immune surveillance and become more numerous after infection. Neutrophils are responsible for maintaining vaginal immunity. Specific to the vagina, neutrophils continuously infiltrate at high levels, although during ovulation, they retreat to avoid sperm damage and permit reproduction. Here we show that, after ovulation, progesterone promotes resident vaginal macrophage-neutrophil crosstalk by upregulating Yolk sac early fetal organs (FOLR2+) macrophage CXCl2 expression, in a TNFA-patrolling monocyte-derived macrophage-mediated (CX3CR1hiMHCIIhi-mediated) manner, to activate the neutrophils' capacity to eliminate sex-transmitted and opportunistic microorganisms. Indeed, progesterone plays an essential role in conciliating the balance between the commensal microbiota, sperm, and the threat of pathogens because progesterone not only promotes a flurry of neutrophils but also increases neutrophilic fury to restore immunity after ovulation to thwart pathogenic invasion after intercourse. Therefore, modest progesterone dysregulations could lead to a suboptimal neutrophilic response, resulting in insufficient mucosal defense and recurrent unresolved infections.

Hormone-Driven Temperature Optimization for Elevated Reproduction in Goldfish (Carassius auratus) under Laboratory Conditions.

This study investigates the efficacy of hormone-induced artificial reproduction in goldfish (Carassius auratus) under controlled temperatures. Ovaprim injections significantly enhanced ovulation and sperm production compared to controls. Medium temperature (22 °C) produced the highest ovulation rates, fastest ovulation timing, and optimal sperm quality (motility and morphology) compared to high (28 °C) and low (16 °C) temperature groups. The low-temperature group exhibited reduced sperm motility duration and higher rates of sperm and larvae damage. The sperm volume of the high-temperature group was higher, but their post-injection survival rates were lower. Furthermore, the lowest spawning rate and low egg quality were noted in the high temperature. Cryopreservation using extender E4 (15% DMSO) exhibited superior post-thaw sperm motility and achieved higher fertilization rates. Fertilization rates, embryo development, and larval survival were all highest at the medium temperature. Larvae hatched from fresh sperm at medium temperature exhibited faster growth and fewer deformities. These findings suggest that hormone stimulation coupled with a medium temperature regimen is critical for successful artificial reproduction in goldfish. Cryopreservation with extender E4 holds promise for sperm banking; however, further optimization is necessary to improve fertilization success with thawed sperm. Future research could explore the influence of temperature on sperm physiology and refine cryopreservation protocols to enhance fertilization rates.

Integrated cytological and transcriptomic analyses provide new insights into restoration of pollen viability in synthetic allotetraploid Brassica carinata.

Upregulation of genes involved in DNA damage repair and sperm cell differentiation leads to restoration of pollen viability in synthetic allotetraploid B. carinata after chromosome doubling. Apart from the well-known contribution of polyploidy to crop improvement, polyploids can also be induced for other purposes, such as to restore the viability of sterile hybrids. The mechanism related to viability transition between the sterile allodiploid and the fertile allotetraploid after chromosome doubling are not well understood. Here, we synthesised allodiploid B. carinata (2n = 2x = 17) and allotetraploid B. carinata (2n = 4x = 34) as models to investigate the cytological and transcriptomic differences during pollen development. The results showed that after chromosome doubling, the recovery of pollen viability in allotetraploid was mainly reflected in the stabilisation of microtubule spindle morphology, normal meiotic chromosome behaviour, and normal microspore development. Interestingly, the deposition and degradation of synthetic anther tapetum were not affected by polyploidy. Transcription analysis showed that the expression of genes related to DNA repair (DMC1, RAD51, RAD17, SPO11-2), cell cycle differentiation (CYCA1;2, CYCA2;3) and ubiquitination proteasome pathway (UBC4, PIRH2, CDC53) were positively up-regulated during pollen development of synthetic allotetraploid B. carinata. In summary, these results provide some refreshing updates about the ploidy-related restoration of pollen viability in newly synthesised allotetraploid B. carinata.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG+GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

The Association Between Oxidative Stress and Sperm Parameters in Patients with Acromegaly.

Spermatozoa are susceptible to oxidative radicals when antioxidant defenses are inadequate. The extent to which oxidative radicals contribute to sperm damage in patients with acromegaly remains unclear. This study aimed to investigate and elucidate this relationship. The overall status of oxidants and antioxidants in both seminal plasma and serum of patients with acromegaly compared to a control group of healthy individuals was investigated. In addition, sperm parameters, including important measures such as growth hormone and insulin-like growth factor-1 concentrations. Twenty-two patients with acromegaly with controlled disease and 14 healthy controls were included. The total oxidant status was significantly higher in the semen samples of the patients with acromegaly. A negative correlation was found between sperm total oxidant status and total sperm count and sperm concentration. Similarly, a negative correlation was found between the total sperm count and the sperm oxidative stress index. In individuals diagnosed with acromegaly, there was a statistically significant increase in sperm growth hormone levels. Conversely, the level of insulin-like growth factor 1 was significantly increased in the sperm of the control group, which consisted of healthy individuals. The correlation analysis revealed a significant relationship between venous total oxidant status and growth hormone levels in semen. The elevated levels of reactive oxygen radicals in individuals with acromegaly suggest a possible link between oxidative stress and its effects on semen quality.

Impact of lifestyle on male fertility - review

Introduction and aim of study: The World Health Organization defines infertility as the disease of reproductive system defined by the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse. It is estimated that 30% of infertility is due to the male factor, and 25% to the coexistence of the male and female factors. The purpose of this review study was to provide an overview of lifestyle risk factors for male infertility and their impact on the organism. Materials and methods: The authors conducted an extensive review of articles available in PubMed, Google Scholar, UpToDate, Science Direct, and Cochrane databases. The keywords infertility, lifestyle, testosterone, men were the basis of the review. Results: Leading an unhealthy lifestyle can significantly impact male fertility. The function of the reproductive system can be affected by inadequate body weight, as well as leading a sedentary lifestyle, using stimulants or anabolic steroids. They not only affect the endocrine system by disrupting steroidogenesis and spermatogenesis but may also directly damage sperm. This leads to impaired semen quality and increases the risk of recurrent pregnancy loss and poor pregnancy outcomes. Conclusion: Lifestyle significantly affects male fertility. A balanced diet, adequate physical activity and avoidance of stimulants can all have a beneficial effect.

The Role of Selected Elements in Oxidative Stress Protection: Key to Healthy Fertility and Reproduction.

Oxidative stress and its relationship to fertility and reproduction is a topic of interest in medicine, especially in the context of the effects of trace elements and micronutrients. Oxidative stress occurs when there is an excess of free radicals in the body, which can lead to cell and tissue damage. Free radicals are reactive oxygen species (ROS) that can be formed as a result of normal metabolic processes, as well as under the influence of external factors such as environmental pollution, UV radiation, and diet. Oxidative stress has a significant impact on fertility. In men, it can lead to DNA damage in sperm, which can result in reduced semen quality, reduced sperm motility and increased numbers of defective sperm, and free radical damage to sperm cell membranes causing a reduction in the number of available sperm. In women, oxidative stress can affect the quality of female reproductive cells, which can lead to problems with their maturation and with embryo implantation in the uterus and can also affect ovarian function and disrupt hormonal regulation of the menstrual cycle. A proper balance of trace elements and micronutrients is key to protecting against oxidative stress and maintaining reproductive health. Supplementation with appropriate elements such as zinc, selenium, copper, manganese, chromium, and iron can help reduce oxidative stress and improve fertility. This work discusses the effects of selected elements on oxidative stress parameters specifically in terms of fertility and reproduction.

Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus)

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

The influence of rutin and chlorogenic acid on oxidative stress and in vivo fertility: Evaluation of the quality and antioxidant status of post-thaw semen from Azari water buffalo bulls.

The vulnerability of buffalo sperm to cryoinjury necessitates the improvement of sperm cryo-resistance as a critical strategy for the widespread use of assisted reproductive technologies in buffalo. The main aim of the present study was to evaluate the effects of different concentrations of rutin and chlorogenic acid (CGA) on buffalo semen quality, antioxidant activity and fertility during cryopreservation. The semen was collected and pooled from the 3 buffaloes using an artificial vagina (18 ejaculations). The pooled sperm were divided into nine different groups: control (Tris-based extender); 0.4, 0.6, 0.8 and 1mM rutin (rutin+Tris-based extender); and 50, 100, 150 and 200µM CGG (CGA+Tris-based extender). Sperm kinematics, viability, hypo-osmotic swelling test, mitochondrial activity, antioxidant activities and malondialdehyde (MDA) concentration of frozen and thawed buffalo sperm were evaluated. In addition, 48 buffalo were finally inseminated, and pregnancy was rectally determined 1 month after insemination. Compared to the control group, adding R-0.4, R-0.6, CGA-100 and CGA-150 can improve total and progressive motility, motility characteristics, viability, PMF and DNA damage in buffalo sperm. In addition, the results showed that R-0.4, R-0.6, CGA-50, CGA-100 and CGA-150 increased total antioxidant capacity, catalase, glutathione peroxidase and glutathione activities and decreased MDA levels compared to the control group. Furthermore, it has been shown that adding 150µM CGA and 0.6mM rutin to an extender can increase in vivo fertility compared to the control group. In conclusion, adding rutin and CGA to the extender improves membrane stability and in vivo fertility of buffalo sperm by reducing oxidative stress.

Association between semen microbiome disorder and sperm DNA damage.

DNA fragmentation index (DFI), a new biomarker to diagnose male infertility, is closely associated with poor reproductive outcomes. Previous research reported that seminal microbiome correlated with sperm DNA integrity, suggesting that the microbiome may be one of the causes of DNA damage in sperm. However, it has not been elucidated how the microbiota exerts their effects. Here, we used a combination of 16S rRNA sequencing and untargeted metabolomics techniques to investigate the role of microbiota in high sperm DNA fragmentation index (HDFI). We report that increased specific microbial profiles contribute to high sperm DNA fragmentation, thus implicating the seminal microbiome as a new therapeutic target for HDFI patients. Additionally, we found that the amount of Lactobacillus species was altered: Lactobacillus iners was enriched in HDFI patients, shedding light on the potential influence of L. iners on male reproductive health. Finally, we also identified enrichment of the acetyl-CoA fermentation to butanoate II and purine nucleobase degradation I in the high sperm DNA fragmentation samples, suggesting that butanoate may be the target metabolite of sperm DNA damage. These findings provide valuable insights into the complex interplay between microbiota and sperm quality in HDFI patients, laying the foundation for further research and potential clinical interventions.IMPORTANCEThe DNA fragmentation index (DFI) is a measure of sperm DNA fragmentation. Because high sperm DNA fragmentation index (HDFI) has been strongly associated with adverse reproductive outcomes, this has been linked to the seminal microbiome. Because the number of current treatments for HDFI is limited and most of them have no clear efficacy, it is critical to understand how semen microbiome exerts their effects on sperm DNA. Here, we evaluated the semen microbiome and its metabolites in patients with high and low sperm DNA fragmentation. We found that increased specific microbial profiles contribute to high sperm DNA fragmentation. In particular, Lactobacillus iners was uniquely correlated with high sperm DNA fragmentation. Additionally, butanoate may be the target metabolite produced by the microbiome to damage sperm DNA. Our findings support the interaction between semen microbiome and sperm DNA damage and suggest that seminal microbiome should be a new therapeutic target for HDFI patients.

Cucumeropsis mannii seed oil mitigates bisphenol-A-induced sperm and hormonal damages in F1-generation of F0-exposed male rats: An in-vivo and in-silico analysis

Exposure to endocrine-disrupting chemicals such as bisphenol A (BPA) has been linked to the sharp drop in male fertility observed worldwide. This study evaluates the protective benefits of Cucumeropsis mannii seed oil (CMSO) against BPA-induced transgenerational sperm indices and reproductive hormone disruptions in rats. Twenty-four F0-male rats (aged 4–5 weeks) were randomly assigned to six groups (n = 4 each) after CMSO characterization for flavonoids. Group A received 1 ml of olive oil. Group B received 100 mg/kg body weight (BW) of BPA whereas Group C had a BW of 7.5 ml/kg CMSO. Groups D, E, and F were administered 100 mg/kg_BW of BPA in addition to 7.5, 5.0, and 2.5 ml/kg_BW of CMSO, respectively. After two weeks of treatment, four mature female rats were introduced to each group and allowed to stay together with male rats for seven days to confirm mating. Thereafter, the pregnant female rats were separated from the male according to their groups and allowed to give birth after 21 days of gestation period. At six weeks, the F1-male rats were isolated from each group and sacrificed for biochemical analysis. In-silico, CMSO flavonoids were assessed for drug-likeness and oral absorbability, followed by molecular docking to study their androgen receptor targeting mechanisms. Results showed that total flavonoids were 17.3652 ± 8.85 g/100 g. In-vivo, BPA significantly (p < 0.005) dysregulated sperm indices and male fertility hormones in F1 rats, but co-administration with CMSO restored these dysregulations in F1 rats. In-silico, the druglike molecules exhibited stronger binding affinities (−6.2 to −8.5 kcal/mol) for the androgen receptor than BPA (−6.1 kcal/mol) within the binding pockets. We postulated that CMSO mitigates transgenerational sperm indices and male fertility hormone disruption in F1 male offspring following F0 male exposure to BPA perhaps by utilizing androgen receptor signaling.

A novel murine model mimicking male genital Neisseria species infection using Neisseria musculi†.

With ~78 million cases yearly, the sexually transmitted bacterium Neisseria gonorrhoeae is an urgent threat to global public health due to continued emergence of antimicrobial resistance. In the male reproductive tract, untreated infections may cause permanent damage, poor sperm quality, and subsequently subfertility. Currently, few animal models exist for N. gonorrhoeae infection, which has strict human tropism, and available models have limited translatability to human disease. The absence of appropriate models inhibits the development of vital new diagnostics and treatments. However, the discovery of Neisseria musculi, a mouse oral cavity bacterium, offers much promise. This bacterium has already been used to develop an oral Neisseria infection model, but the feasibility of establishing urogenital gonococcal models is unexplored. We inoculated mice via the intrapenile route with N. musculi. We assessed bacterial burden throughout the male reproductive tract, the systemic and tissue-specific immune response 2-weeks postinfection, and the effect of infection on sperm health. Neisseria musculi was found in penis (2/5) and vas deferens (3/5) tissues. Infection altered immune cell counts: CD19+ (spleen, lymph node, penis), F4/80+ (spleen, lymph node, epididymus), and Gr1+ (penis) compared with noninfected mice. This culminated in sperm from infected mice having poor viability, motility, and morphology. We hypothesize that in the absence of testis infection, infection and inflammation in other reproductive is sufficient to damage sperm quality. Many results herein are consistent with outcomes of gonorrhoea infection, indicating the potential of this model as a tool for enhancing the understanding of Neisseria infections of the human male reproductive tract.

O-306 Sperm DNA methylome changes in testicular cancer patients following chemotherapy treatment

Abstract Study question Does chemotherapy (CT) treatment for testicular cancer induce sperm methylome changes? Summary answer The analysis of 27 pre- and post-CT samples from testicular cancer survivors identified 65 differentially methylated regions. What is known already Research has shown that cancer treatment can cause DNA damage and aneuploidy in sperm, even years after the treatment. However, there is limited information available on the impact of CT on the sperm epigenome, and most of the information has been obtained from rodent models. Two recent studies, one involving one patient and the other involving three, have investigated this topic in humans. Both studies have suggested that cancer survivors may experience changes in sperm’s DNA methylation following oncological treatment. Study design, size, duration This is a multicenter prospective study. We included 27 patients [31.23 years (CI 95% 29.29-33.17)] diagnosed with testicular cancer who cryopreserved semen before CT (2009-2020). After receiving oncological treatment, these patients recovered their spermatogenesis. Patients who agreed to participate in this IRB-approved study, donated a second semen sample after their recovery, which was also frozen. Participants/materials, setting, methods Semen samples were thawed; sperm DNA was isolated and subsequently hybridized using the Human Infinitum DNA Methylation 850K EPIC BeadChip array platform (Illumina). A paired differential methylation analysis was performed between pre-CT and post-CT samples from the same patient, fitting a linear model of the methylated positions and regions. Minfi and DMRcate R packages were used to find differentially methylated positions (DMPs) and regions (DMRs). Subsequently, the functional impact of involved genes was evaluated. Main results and the role of chance DNA methylation data were available for the 54 sperm samples (27 collected before CT and 27 after CT treatment). Results were controlled by age and time elapsed between the cryopreservation of pre-CT and post-CT samples, which was 4.01 years (CI 95%: 3.42-5.64). After filtering for initial QC, the assessment was done on 445.000 CpGs per sample. The analysis revealed that a total of 228 CpG positions showed a tendency towards statistical significance. DMRcate identified 65 DMRs (40 hypomethylated and 25 hypermethylated) that reached a smoothed FDR p-value &amp;lt; 0.05 when compared post-CT samples to pre-CT within the same patient. We observed that 61 genes were located within DMRs, being TBC1D20, EN1, HOXB1 and SCNN1A the ones within the most significant DMRs (min-smoothed-FDR &amp;lt; 0.001). The functional enrichment analysis of putatively affected genes revealed that the biological processes more affected were: Regulation of macromolecule biosynthetic process, hormone receptor binding, signaling pathways regulating pluripotency of stem cells and pathways in cancer. Limitations, reasons for caution Epigenetics may be affected during cryopreservation and thawing. To ensure comparability, we also froze the samples after undergoing the CT process. Due to the high-throughput nature of the study and the limited access to samples, we only achieved marginal significance. Therefore, our results should be further validated. Wider implications of the findings This is the largest study suggesting distinct sperm DNA methylation signatures in testicular cancer survivors. Our findings emphasize the importance of counseling cancer patients in pretreatment sample collection and may explain the reported increase in birth defects after treatment. Trial registration number NOT APPLICABLE

P-050 Microfluidic chip-sorted spermatozoa selection yields embryo euploidy rate and reproductive outcomes similar to density-gradient centrifugation

Abstract Study question Does microfluidic chip-sorted spermatozoa selection (MSS) improve good quality blastocyst (GQB) and embryo euploidy rates compared to density gradient centrifugation (DGC)? Summary answer The use of MSS before intracytoplasmatic sperm injection (ICSI) does not improve good quality blastocist and euploidy rates comparing to DGC. What is known already The integrity of sperm DNA plays a crucial role in embryonic development. Conventional sperm selection methods, such as DGC, may potentially damage sperm DNA due to oxidative stress generated during centrifugation. One of the recent advances in sperm selection techniques is the microfluidic chamber, which, requiring less processing time and eliminating the need for centrifugation, allows for the selection of sperm with better mobility, morphology, and reduced DNA damage. Consequently, improved GQB and euploid embryo rates, as well as enhanced reproductive outcomes, are expected. Study design, size, duration Retrospective study of 1739 blastocysts from 404 couples undergoing 544 autologous cycles of ICSI/PGT-A (preimplantation genetic testing for aneuploidies) from January 2020 to December 2022. Those patients with sperm concentration &amp;lt;5 × 106/mL or motility &amp;lt;10%, altered karyotype, women aged &amp;gt; 43 years, uterine anomalies and thrombophilia were excluded. Besides comparing euploidy rates and blastocyst formation, reproductive outcomes were analyzed and compared. Participants/materials, setting, methods According to the sperm selection technique, two groups were established. One group underwent MSS (ZyMōt ICSI® or ZyMōt Multi®), resulting in 295 blastocysts from 96 cycles. It was compared with a control group (DGC) including 1444 blastocysts from 448 ICSI/PGT-A cycles. PGT-A was performed in day 5/6 using Next Generation Sequencing (NGS) technology. For a 95% confidence interval, statistical significance was considered at p &amp;lt; 0.05. Chi-square analysis was used for the statistical evaluation. Main results and the role of chance Both groups were comparable in terms of demographic data, including age, sperm analysis parameters, and cycle characteristics. There were no significant differences between the DGC and MSS groups in the women’s age (mean ± SD) (37.9 ± 3.9 vs. 38 ± 3.9 years, p = 0.99) or men’s age (38.4 ± 7.1 vs. 39.8 ± 5.9 years, p = 0.11). No significant differences were found between the DGC and MSS groups in terms of GQB rate (49.3% vs. 50.7%, p = 0.55), euploidy rate (35.8% vs. 32.2%, p = 0.23), fertilization rate (69.5% vs. 69.5%, p = 0.99), clinical pregnancy rate per transfer (63% vs. 76%, p = 0.13), clinical miscarriage (9% vs. 21%, p = 0.33), and live birth rate per transfer (44% vs. 37%, p = 0.42). The results also showed a higher number of euploid embryos per initiated cycle in the DGC group (1.154) compared to the MSS group (0.989). Despite this difference, the results were not statistically significant (p &amp;gt; 0.05). Limitations, reasons for caution The limitations of this study include the potential impact of selection bias, as it is a retrospective study in which groups were not randomly assigned. Additionally, the low number of blastocysts in the MSS group and the potential bias of DNA fragmentation, which was not evaluated in the study. Wider implications of the findings Both methods demonstrated similar effectiveness. While MSS could potentially be more effective in samples with high DNA fragmentation and they feature a user-friendly application, its major limitation lies in its challenging application with samples having a low concentration of motile sperm. Specific semen samples may need different sperm selection methods. Trial registration number Not Applicable

Skoochies and its component substances induced testicular damage and impaired sperm function via increased generation of reactive oxygen species and impairment of the glutathione system in rats

ObjectiveTo study examined the effect of skoochies, an illicit cocktail drink, on testicular and sperm function in male rats. DesignTwenty-five adult male Wistar rats were assigned randomly into five groups (n = 5), as follows: Normal saline (NS group), skoochies (SK group), cannabis sativa (CS group), codeine (CO group), and tramadol (TM group). The cocktail (skoochies) used for this study was formulated using the following composition: codeine (5mg/kg), tramadol (20mg/kg) and cannabis extract (2mg/kg). These doses are as previously reported. Administration was done once daily for twenty-eight (28) days. ResultsSkoochies increased reactive oxygen species generation and impaired the antioxidant system resulting in inflammation that eventually damage the testicular tissue. Skoochies causes oxido-inflammatory injury to the testicular tissues resulting in impaired testicular functions. This was evident by the distorted cytoarchitecture, reduced sperm count, motility and impaired testicular DNA integrity. ConclusionThus, our results infer that Skoochies induced the impairment of testicular and sperm function through the increased generation of reactive oxygen species and impairment of the glutathione system.