Sperm Reactive Oxygen Species Research Articles

P-041 Impact of cigarette smoking on level of sperm reactive oxygen species in normozoospermic men

Abstract Study question Does cigarette smoking influence the levels of sperm reactive oxygen species (ROS) in men with normal semen routine parameters? Summary answer Cigarettes impact the sperm ROS of normozoospermic smokers, whose sperm ROS level is elevated when they smoke equal to or more than 10 cigarettes daily. What is known already Increasing evidence indicates that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated ROS levels in semen. Cigarette smoking leads to an imbalance between ROS and antioxidants in the semen of smokers, which may negatively affect semen quality and male fertility. However, few studies concern whether cigarette smoking would influence the sperm ROS levels in normozoospermic men. Study design, size, duration Data of semen analysis from January 2021 to December 2023 were involved. Men with normal semen routine parameters, including sperm concentration, total sperm number, sperm progressive motility, total motility and normal forms of sperm morphology, were included in the study. Sperm ROS levels of normozoospermic men were retrospectively analyzed. Men with varicocele and leucocytospermia were excluded. Participants/materials, setting, methods Sperm count and motility were analyzed by the computer-assisted sperm analysis (CASA) system, while sperm morphology was examined after the Diff-Quik rapid staining procedure. Sperm ROS was measured by flow cytometry using the probe DCFH-DA. Sperm ROS levels of smokers and non-smokers were compared by the Mann-Whitney U test. The Kruskal-Wallis H test was used for the comparison of sperm ROS levels among the control group and smoking subgroups. Main results and the role of chance A total of 2946 subjects were involved in the present study. The included subjects were divided into smokers (n = 730) and non-smokers (n = 2216) according to the clinical records. And the included smokers were subsequently divided into three subgroups according to the daily cigarettes taken, which were group A (1∼9 cigarettes/day, n = 184), group B (1∼19 cigarettes/day, n = 315), and group C (>20 cigarettes/day, n = 231). No statistically significant differences in age or BMI were found between smokers and non-smokers with normozoospermia. The sperm ROS level of the smoker was higher than that of the non-smoker [14.53% (14.84%) vs. 13.07% (12.09%), p = 0.001]. Compared to the control group (non-smoker), the sperm ROS levels of group B [13.07% (12.09%) vs. 15.44% (14.28%), p = 0.002] and group C [13.07% (12.09%) vs. 14.80% (14.31%)., p = 0.042] were significantly elevated. ROS levels in the control group and group A were comparable [13.07% (12.09%) vs. 13.46% (15.74%)., p = 0.391]. Limitations, reasons for caution It is a single-center study and the above results were from a retrospective analysis. Wider implications of the findings The above results indicate that cigarette smoking, although it would not deteriorate semen routine parameters, may increase the ROS level in sperm. Elevated ROS in sperm may lead to an imbalance of redox in sperm, which may contribute to male infertility. Trial registration number not applicable

P-099 The development of a novel sperm washing medium, enriched with serotonin as the active constituent, with the objective of enhancing sperm motility and viability

Abstract Study question Is it possible to develop a novel product that increases the success rate of treating male infertility through assisted reproductive techniques? Summary answer Sperm washing media, aiming to improve sperm quality, enhance sperm motility, and generally support male reproductive health in the field of assisted reproductive techniques. What is known already Infertility impacts 15% of all couples globally, with 50% of infertility cases attributed solely to male factors. Reduced semen motility is identified as a primary factor directly associated with infertility. The utilization of supplementation to enhance overall sperm quality has gained increasing popularity worldwide. The sperm preparation process in Assisted Reproductive Technology (ART) is crucial for successful fertilization, with sperm washing mediums playing a critical role in enhancing efficiency. Sperm-washing media is a specialized solution utilized in assisted reproductive technologies such as intrauterine insemination and in vitro fertilization to remove the seminal fluid and debris for sperm to undergo capacitation. Study design, size, duration In this study, 200 normozoospermic men were included, and their semen samples were analyzed over an 18-month period following the guidelines outlined by the World Health Organization (2010) to assess the efficacy of serotonin, selenium, zinc, L-Carnitine, and Coenzyme Q10 supplementation on sperm motility, viability, ROS and DNA damage. Participants/materials, setting, methods Sperm samples obtained from 200 volunteer men undergoing infertility assessments were subdivided into three samples.The sperm samples underwent preparation using the swim-up technique. The study involved the examination of samples in three distinct washing media: (1) Origio, (2) Irvine Scientific, (3) a prototype (serotonin, selenium, zinc, L-carnitine, and Coenzyme Q10). Each medium was analyzed for motility, apoptosis, reactive oxygen species (ROS), and DNA damage. Main results and the role of chance It is found that mean basal total sperm counts are 38 million. After sperm washing, mean total sperm counts of Origio®, Irvine® and Prototype are 15 million respectively (p > 0.05). As total progressive motility is % 40 before washing, it is increased to 62%,56% and 64% after washing with Origio®, Irvine® and Prototype, respectively (p > 0.05). Annexin V staining is used for detection of apoptosis in sperm samples. On average, early apoptosis over the total sperm count(TSC) per subgroups are 34.67% for Prototype, 33.17% for Origio® and 30.96% for Irvine®, whereas late apoptosis ratios are found significantly lower in Prototype compared to Origio and Irvine; 8.7%, 28.26% and 27.74%, respectively. Ratio of necrosis over TSC per subgroups is 3.15% for Prototype, 13.49% for Origio® and 18.78% for Irvine® whereas viability ratios are as 53.48%, 25.08%, 22.52%, respectively.(p < 0.05). ROS levels in semen after washing are significantly lower for Prototype (5.9%, 10.29%, and 18.22%, Prototype, Origio® and Irvine®, respectively) (p < 0.05). Limitations, reasons for caution Semen samples from males aged 28 to 45, who are unable to conceive, do not smoke, and have not undergone any surgical interventions, are collected from patients after a sexual abstinence period of 3-4 days. Wider implications of the findings We propose that our serotonin-activated washing media for sperm preparation can serve as a suitable choice for selecting high-quality sperm in assisted reproductive technology programs. These molecules may enhance the success of assisted reproduction techniques when present in sperm preparation media. Trial registration number SerotoneX

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.

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.

Pyrroloquinoline Quinone Improves Ram Sperm Quality through Its Antioxidative Ability during Storage at 4 °C.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm's high ability of fertilization.

Epigenetic transgenerational effects of PM2.5 collected from southern Taiwan on sperm functions and DNA methylation in mouse offspring

During respiration, particulate matter with a diameter of 2.5 µm or less (PM2.5) suspended in the atmosphere enters the terminal alveoli and blood. PM2.5 particles can attach to toxic substances, resulting in health problems. Limited information is available regarding the effects of prenatal exposure to water-soluble PM2.5 (WS-PM2.5) and water-insoluble PM2.5 (WI-PM2.5) on male reproduction. In addition, whether exposure to these particles has transgenerational effects remains unknown. We investigated whether prenatal exposure to WS-PM2.5 and WI-PM2.5 disrupts sperm function in generations F1, F2, and F3 of male mice. Pregnant BALB/c mice were treated using intratracheal instillation on gestation days 7, 11, and 15 with 10 mg of a water extract or insoluble PM2.5. On postnatal day 105, epididymal sperm count, motility, morphology, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, the sperm chromatin DNA fragmentation index (DFI), and testicular DNA methyltransferase (Dnmt) levels were evaluated in all generations. Whole-genome bisulfite sequencing was used to analyze the DNA methylation status of generation F3. According to the results, exposure to WS-PM2.5 affected sperm morphology, ROS production, and mean DFI in generation F1; ROS production and mean DFI in generation F2; and sperm morphology and MMP in generation F3. Similarly, exposure to WI-PM2.5 affected sperm morphology, ROS production, mean DFI, %DFI, and Dnmt1 expression in generation F1; sperm morphology, MMP, and ROS production in generation F2; and sperm morphology, ROS, and %DFI in generation F3. Two hypermethylated genes, PRR16 and TJP2, were observed in the WS-PM2.5 and WI-PM2.5 groups, two hypomethylated genes, NFATC1 and APOA5, were observed in the WS-PM2.5 group, and two hypomethylated genes, ZFP945 and GSE1, were observed in the WI-PM2.5 group. Hence, prenatal exposure to PM2.5 resulted in transgenerational epigenetic effects, which may explain certain phenotypic changes in male reproduction.

Antioxidant Effects of Myo-Inositol Improve the Function and Fertility of Cryopreserved Boar Semen.

During cryopreservation, sperm undergoes structural and molecular changes such as ice crystal formation, DNA fragmentation, and reactive oxygen species (ROS) production, leading to decreased sperm quality after thawing. Antioxidants play a crucial role in preventing these damages, both in vivo and in vitro. One potent antioxidant is myo-inositol, known for its protective effects on sperm against ROS. This study aimed to investigate the protective effect of myo-inositol on cryopreserved boar semen. The semen was diluted, cooled, and cryopreserved using a BF5 extender. It was then divided into five groups: control and different concentrations of myo-inositol (0.5, 1, 1.5, and 2 mg/mL). The post-thaw evaluation included assessments of motility, viability, acrosome integrity, mitochondrial membrane potential (MMP), caspase activity, gene expression, ROS levels, apoptosis, and IVF with treated semen. Results showed that myo-inositol at 0.5 mg/mL improved motility, acrosome integrity, and fertilization ability. It also reduced the expression of pro-apoptotic genes and increased SMCP expression. Lower concentrations also demonstrated improved viability and reduced apoptosis and ROS levels. In conclusion, myo-inositol treatment during cryopreservation improved sperm quality, reduced apoptosis and ROS levels, and enhanced fertility rates in boar semen.

P-033 Impact of the sperm reactive oxygen species on Intrauterine Insemination outcome

Abstract Study question Does sperm reactive oxygen species values affect the pregnancy rate after Intrauterine Insemination (IUI)? Summary answer Sperm reactive oxygen species values affect pregnancy rate and can be used as a predictive marker for IUI outcome. What is known already Oxidative stress resulting from excessive production of reactive oxygen species (ROS) can profoundly affect the sperm and subsequent functional sperm integrity. Diagnostics to measure semen oxidative stress have been added to the 6th edition of the WHO laboratory manual for examining and processing of human semen, under advanced examinations. Although a semen analysis has classically been used as the gold standard for determining a man’s fertility, basic sperm analysis alone cannot accurately predict infertility since between 6 and 27 % of men with normal semen parameters are infertile. Determination of oxidative stress in diagnosing infertility may contribute to better outcomes. Study design, size, duration This study was based on semen analyses of 50 male patients whose samples were used for IUI treatment between November 2022 and January 2023. The oxidation-reduction potential was measured using novel galvanostat-based technology, the MiOXSYS System. Briefly, 30 μL of liquefied semen at room temperature were applied to the MiOXSYS sensor. Both absolute ORP (mV) and normalized sORP values ( mV/10 6 sperm/ml) based on sperm concentration were calculated. Participants/materials, setting, methods Sperm analysis were performed according to the WHO laboratory manual sperm criteria (Sixth edition 2021). All participants followed a period of 3–5 days of sexual abstinence and had a diagnosis of unexplained infertility (normozoospermia). Ovarian stimulation for IUI cycle was initiated on the 5th day of menstruation by using recombinant gonadotropins. Clinical pregnancy was determined based on the Beta-hCG values. For statistical comparison between group values the Mann-Whitney U test was used. Main results and the role of chance According to the guidelines of the World Health Organization, all participants included in the study had normal BMI (21.85±3.80 kg/m2). The average age of the female patients included in the study was 33.9 ± 3.1 years while the average age of the male patients was 37.8 ± 4.8 years. Out of the 50 patients, 32% (N = 16) got pregnant. For the study propose sperm ORP and sORP values were compared between patients based on the pregnancy outcome. Statistical analysis showed a significant difference in ORP values. Patients with a positive pregnancy outcome after IUI had a lower ORP value compared to patients where IUI was not successful [25.65 [6.7-52.6] mV vs. 40.2 [6.3-109.8] mV, respectively, p = 0.02]. Additionally, normalized values according to the sperm concentration (sORP) were also significantly different between those groups [ 0.37(0.17-1.44) mV/10 6 sperm/ml vs. 0.88(0.13-20.81) mV/10 6 sperm/ml, respectively p = 0.006]. Due to the above-mentioned significant difference logistic regression analysis were performed, and the obtained results confirm that sperm ORP (p = 0.02) as well sORP (p = 0.016) significantly affects the pregnancy rate after the IUI procedure. Limitations, reasons for caution The limitation of the presented study was a relatively small patient cohort. Additionally, pregnancy was determined based on the beta hCG values, therefore future research should link ORP values and live birth rate as well. Wider implications of the findings In the present study suggests ORP as an additional parameter with diagnostic value to identify the best therapeutic approach concerning IUI in couples that show minimal or no impairments of the WHO semen parameter. Trial registration number not applicable

P-085 Reactive molecules drive sperm capacitation via protein posttranslational modifications

Abstract Study question Are S-sulfenylation and S-nitrosylation, two essential posttranslational modifications (PTMs), increased during sperm capacitation? Which proteins are subjected to S-sulfenylation? Summary answer S-sulfenylation and S-nitrosylation increase during sperm capacitation. S-sulfenylation affects certain proteins, including A-kinase anchor protein 13, Fibrilline-1, and Spermatogenesis-associated protein 7. What is known already There is a significant increase in reactive oxygen species (ROS) and reactive nitrogen species (RNS) production within sperm capacitation. In contrast to tyrosine phosphorylation, a well-established mark of sperm capacitation, ROS, and RNS lead to PTMs themselves and modify thiol groups of cysteines. Despite these facts, the dynamics of ROS/RNS-derived PTMs during sperm capacitation remain unknown. Therefore, we consider both S-nitrosylation and S-sulfenylation of sperm proteins as novel modifications associated with the capacitation. Study design, size, duration C57BL6 mouse sperm were isolated from the caudae epididymae of 12-20-week-old males (n = 5). The study compared non-capacitated and capacitated spermatozoa using proteomic profiling. Participants/materials, setting, methods Sperm proteins were extracted into RIPA buffer. The protein lysate was incubated with 5mM dimedone, a hapten that specifically labels S-sulfenylated proteins, and immunogenically detected with an anti-dimedone antibody (Merk, Germany). Alternatively, S-nitrosylated proteins were detected by the anti-nitrosocysteine antibody (Merk, Germany). S-sulfenylated and S-nitrosylated proteins were visualized by western blotting. S-sulfenylated proteins were simultaneously identified by mass spectrometry. Main results and the role of chance We demonstrated an increase in S-sulfenylation and S-nitrosylation along with sperm capacitation. In addition, both S-sulfenylation and S-nitrosylation are positively correlated with tyrosine phosphorylation. Using mass spectrometry, we identified S-sulfenylated proteins, mostly belonging to the cytoskeleton: A-kinase anchor protein 13, Fibrilline-1, Spermatogenesis-associated protein 7, and Rab-like protein 2A. Limitations, reasons for caution The limitation of this study is the instability of PTMs. ROS/RNS-derived PTMs hypothetically arise and cease during protein sample preparation. The experiment was limited to one animal model. Further experiments should be performed on other mammalian models. Wider implications of the findings Studying novel sperm PTMs and their mechanism of action may improve the diagnosis of male infertility. Further, it could bring a novel approach to modulate the fertilizing ability of sperm used for in vitro fertilization. Trial registration number 260 536

The influence and mechanistic action of sperm DNA fragmentation index on the outcomes of assisted reproduction technology.

We investigated the influence of DNA fragmentation index (DFI) on in vitro fertilization (IVF), embryo transfer (ET), and intracytoplasmic sperm injection (ICSI). We analyzed the semen parameters of 61 cycles in infertile couples undergoing IVF-ET and ICSI and determined DFI by sperm chromatin dispersion testing. Based on DFI, the patients were differentiated into a control group (DFI < 25%, n = 35) and a test group (DFI ≥ 25%, n = 26). Flow cytometry and immunofluorescence were used to investigate the extent of sperm reactive oxygen species (ROS) and apoptosis. We also investigated the effect of DFI on pregnancy outcomes of IVF-ET/ICSI. DFI was negatively related to sperm motility and positively correlated with ROS and apoptosis (P < 0.05). Abnormally elevated DFI reduced the rate of transplantable, high-quality embryos, implantation, clinical pregnancy, delivery, and live birth after IVF-ET, and increased the chance of early abortion per transfer cycle (P < 0.05). However, there was no significant correlation between DFI and fertilization rate, cleavage rate, transplantable rate, high-quality embryo rate, implantation rate, clinical pregnancy rate, early abortion rate, delivery rate and live birth rate when assisted by ICSI (P > 0.05). Sperm DNA integrity is crucial for fertilization and the development of healthy offspring. ROS may increase the level of DFI by inducing apoptosis in sperm.

Obesity-induced oxidative stress and mitochondrial dysfunction negatively affect sperm quality.

Obesity is a systemic metabolic disease that can induce male infertility or subfertility through oxidative stress. The aim of this study was to determine how obesity impairs sperm mitochondrial structural integrity and function, and reduces sperm quality in both overweight/obese men and mice on a high-fat diet (HFD). Mice fed the HFD demonstrated higher body weight and increased abdominal fat content than those fed the control diet. Such effects accompanied the decline in antioxidant enzymes, such as glutathione peroxidase (GPX) and catalase and superoxide dismutase (SOD) in testicular and epidydimal tissues. Moreover, malondialdehyde (MDA) content significantly increased in sera. Mature sperm in HFD mice demonstrated higher oxidative stress, including increased mitochondrial reactive oxygen species (ROS) levels and decreased protein expression of GPX1, which may impair mitochondrial structural integrity and reduce mitochondrial membrane potential (MMP) and ATP production. Moreover, cyclic AMPK phosphorylation status increased, whereas sperm motility declined in the HFD mice. Clinical studies demonstrated that being overweight/obese reduced SOD enzyme activity in the seminal plasma and increased ROS in sperm, accompanied by lower MMP and low-quality sperm. Furthermore, ATP content in the sperm was negatively correlated with increases in the BMI of all clinical subjects. In conclusion, our results suggest that excessive fat intake had similar disruptive effects on sperm mitochondrial structure and function, as well as oxidative stress levels in humans and mice, which in turn induced lower sperm motility. This agreement strengthens the notion that fat-induced increases in ROS and impaired mitochondrial function contribute to male subfertility.

Double-edged sword: effects of human sperm reactive oxygen species on embryo development in IVF cycles

BackgroundThe exact role of sperm reactive oxygen species (ROS) in early embryo development has yet to be fully identified, and most of existing research did not differentiate female infertility factors, ignoring the importance of oocyte quality in embryo development and the large differences in oocyte quality in women with infertility of different etiologies. And there has been no relevant report on whether different types of sperm ROS have distinct effects on embryo development. This study aimed to study the impact of selected sperm ROS, namely, sperm mitochondrial ROS (mROS) and hydrogen peroxide, on human embryo development after conventional in vitro fertilization (IVF) cycles in patients with normo-ovulatory infertility vs. anovulatory infertility.MethodsThis was a prospective investigation including 393 couples underwent IVF cycles, among whom 90 patients had anovulatory infertility and 303 patients had normo-ovulatory infertility in a public university-affiliated in vitro fertilization center. Sperm mROS and hydrogen peroxide testing were performed by flow cytometry and analyzed for their relationship with embryo development indices on days 1–6 after IVF. Multivariate logistic regression analysis was used to control for female potential confounders. The nonlinear effects of sperm ROS on embryo development were analyzed by the Restricted cubic spline (RCS) method.Results1. Multivariate linear logistic regression analysis showed that high proportion of mROS positive sperm improved the 2PN rate (OR = 1.325, 95% CI: 1.103–1.595), day 3 embryo utilization rate (OR = 1.362, 95% CI: 1.151–1.614) and good-quality day 3 embryo rate (OR = 1.391, 95% CI: 1.089–1.783) in patients with anovulatory infertility. High percentage of sperm mROS and hydrogen peroxide had adverse effects on cleavage-stage embryo and blastocyst development in patients with normo-ovulatory infertility. 2. For patients with polycystic ovarian syndrome (PCOS) anovulatory infertility, there were significant distinct effects on embryo development indices between sperm mROS and hydrogen peroxide, and the increased rate of sperm mROS improved the good-quality day 3 embryo rate (OR = 1.435, 95% CI: 1.045–1.981); however, high percentage of sperm hydrogen peroxide reduced the blastocyst utilization rate (OR = 0.555, 95% CI: 0.353–0.864) and the good-quality blastocyst rate (OR = 0.461, 95% CI: 0.292–0.718). 3. Multivariate RCS analysis revealed that sperm ROS had a nonlinear (such as a parabolic curve) effect on embryo development in patients with anovulatory infertility (P < 0.05), and either greatly increased or greatly decreased affected cleavage-stage embryo and blastocyst development. The effects of sperm ROS in patients with normo-ovulatory infertility were both linear and nonlinear.ConclusionsThese findings indicate that contrary effects of sperm mROS on embryo development depending on whether patients treated with IVF cycles had normal ovulation. Regardless of whether the patients ovulated normally, increased sperm hydrogen peroxide rate damaged blastocyst development. It is necessary to evaluate male sperm ROS levels and the female ovulatory state to determine an individualized intervention plan before starting cycles, as this may be beneficial for infertile couples.

Effects of capacitating media and incubation time on collared peccary sperm quality, acrosome reaction, and binding ability

Context Several assisted reproduction techniques have been proposed for collared peccaries due to the interest in its conservation. However, there is little information about the requirements for sperm capacitation, an initial step in in vitro fertilisation. Aims We aimed to determine the optimal conditions for collared peccary sperm capacitation by comparing Tyrode’s albumin lactate pyruvate (TALP) or the same media plus heparin, caffeine, or their combination at different exposure times (1, 3, and 6 h). Methods The samples were evaluated for kinetic parameters, membrane functionality and integrity, mitochondrial activity, morphology, DNA integrity, reactive oxygen species (ROS) expression levels, capacitation, and sperm binding ability using swine oocytes. Key results Samples incubated with caffeine or heparin had a higher percentage of capacitated spermatozoa. The maximum percentage of capacitation was achieved after 3 h of incubation with either agent. Moreover, spermatozoa subjected to heparin capacitation showed better motility than when subjected to caffeine, and lower ROS expression levels after 1 h. No differences were observed among incubation times for the binding ability. Conclusions In summary, collared peccary spermatozoa can be capacitated with caffeine or heparin; however, heparin better maintains sperm motility and ROS expression levels. The co-incubation of gametes in a medium with heparin for 3 h could be efficient for in vitro fertilisation in collared peccaries. Implications This result will contribute to the development of assisted reproduction techniques for conservation and productivity of collared peccaries.

Cellular responses and microRNA profiling in bovine spermatozoa under heat shock.

Elevated temperatures disturbed sperm physiology. Bovine sperm cells exposed to heat shock led to diminished mitochondrial activity, fertilizing ability, increased oxidative stress and caspase activity concomitant with a delay in embryonic developmental kinetics and modulation of sperm-borne microRNAsmiRNAs. Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41°C for 4 h. Heat shock reduced sperm motility after 3-4 h at 41°C while mitochondrial activity was reduced by 38.5 and 41°C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified >300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41°C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm.

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n=5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n=5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.

Mitochondrial metabolism determines the functional status of human sperm and correlates with semen parameters

The diagnosis of male infertility is based essentially on the patient’s medical history and a standard semen analysis. However, the latter rarely provides information on the causes of a possible infertility, emphasizing the need to extend the analysis of the sperm function. Mitochondrial function has been associated with sperm function and dysfunction, the latter primarily through the production of excessive amounts of reactive oxygen species (ROS). We hypothesized that analysis of sperm mitochondrial metabolism together with sperm ROS production could be an additional tool to improve routine semen analysis, after appropriate validations. To test our hypothesis, we performed several experiments using a non-routine method (high-resolution respirometry, HRR) to access mitochondrial function. First, we investigated whether mitochondrial function is related to human sperm motility and morphology. When mitochondrial metabolism was challenged, sperm motility decreased significantly. Additionally, morphological abnormalities in the sperm mid-piece and mitochondria were associated with global sperm defects evaluated by routine methods. Subsequently, sperm mitochondrial function was assessed by HRR. Respiratory control ratio (RCR) was determined and evaluated in the context of classical sperm analysis. In parallel, sperm hydrogen peroxide (H2O2) production and seminal plasma (SP) antioxidant capacity were measured. The percentage of sperm with progressive motility correlated positively with RCR, SP antioxidant capacity, and negatively with the concentration of extracellular H2O2 production ([H2O2]). The percentage of normal sperm morphology correlated positively with RCR and negatively with [H2O2]. Sperm morphology did not correlate with seminal plasma antioxidant capacity. Furthermore, Receiver Operating Characteristic curves were used for the first time to test the diagnostic ability of RCR, [H2O2], and SP antioxidant capacity as binary classifiers. An RCR cut off value of 3.2 was established with a sensitivity of 73% and a specificity of 61%, using reference values considered normal or abnormal in routine semen analysis. The cut off value for [H2O2] was 0.2 μM/106 sperm (sensitivity = 65%, specificity = 60%). There were no reference values for SP antioxidant capacity that distinguished between abnormal and normal sperm samples. We conclude that sperm mitochondrial function indices in combination with [H2O2] may be useful tools to complement the routine semen analysis.

P-037 A fluorescent probe for Reactive Oxygen Species (ROS) detection identifies spermatozoa with a better reporductive performance

Abstract Study question Could Reactive Oxygen Species (ROS) detected in human spermatozoa represent a predictive marker of fertilization ability? Summary answer ROS detected by CellROX® Orange probe is related to a better sperm quality and function, indicating the sensitivity of the probe to identify physiological ROS. What is known already Oxidative stress (OS), defined as an unbalance between ROS production and antioxidant defenses, is one of the causes of male infertility. A small amount of ROS is necessary for the physiological sperm function, however high ROS levels could impair fertility potential inducing damages at membrane, protein and DNA levels. Previous studies performed by using different methods and probes for OS evaluation in semen or in spermatozoa highlighted the negative role of ROS on sperm functions. However, such studies were not conclusive because of the small number of included subjects and of high variability in the cohorts. Study design, size, duration Observational study conducted on 73 male partners of infertile couples attended consecutively to the Andrology Laboratory of Careggi University Hospital of Florence from September to December 2021. Analyses were performed on both washed and Swim-up selected spermatozoa. Participants/materials, setting, methods After routine semen analysis, washed and Swim-up selected spermatozoa were incubated with CellRox®Orange (a fluorescent probe able to reveal OS in viable cells), at the concentration of 1 µM for 30 minutes at 37°C, 5%CO2, and revealed by flow cytometry. Sperm DNA fragmentation (by TUNEL/PI method), sperm kinematic parameters and hyperactivated motility (by C.A.S.A. system) were also assessed. In some samples a double staining with CellRox®Orange and Annexin V (which stains phosphatidylserine externalization) was performed. Main results and the role of chance We found that the percentage of spermatozoa positive to CellRox® Orange is positively correlated with routine seminal parameters. Although this result appears in contrast with most studies in literature reporting negative correlations between OS and semen parameters, it can be explained by the fact that the probe is specific for viable spermatozoa characterized by a better motility and morphology. Considering the importance of distinguishing between positive and negative ROS in spermatozoa, we further investigated the significance of ROS detected by this probe. To understand whether the probe marks spermatozoa with a better quality, CellRox® Orange positivity was evaluated in Swim-up selected spermatozoa finding significantly higher levels of CellRox® Orange positivity respect to unselected samples. Furthermore, the fact that most of the CellRox® Orange positive spermatozoa were negative for Annexin V (which reveals cells with early signs of apoptosis) is a further confirmation of the good quality of CellRox® Orange positive spermatozoa. Another evidence is represented by the finding that we observed a negative correlation between OS detected by CellRox® Orange and sperm DNA fragmentation, although several studies have shown a positive relationship between these two parameters (when OS is measured by different probes). Limitations, reasons for caution Up to now, the study involved a limited number of subjects. Further experiments should be performed to increase the number of subjects in order to confirm the results. Wider implications of the findings Results of this study together with those previously published evidence that different OS is detected depending on which probe is used. CellRox® Orange seems to be sensible for physiological ROS detection, therefore, it could be employed in future studies aimed to evaluate the association between OS and assisted reproduction outcomes. Trial registration number not applicable

P-430 Sperm genomic integrity, telomere dynamics and gene expression in recurrent implantation failure

Abstract Study question Does sperm affect the implantation and early embryogenesis? Summary answer Deranged sperm genomic integrity, limited repair mechanisms, aberrant gene expression have the potential to be transmitted to developing embryo and affect implantation and embryonic development. What is known already The terminally differentiated, transcriptionally quiscent sperm cells are vulnerable to a host of factors which might affect its function. The spermatozoa with truncated repair mechanisms have the potential to fertilize the oocyte, but eventually result in high rates of pre and post implantation losses. Oxidative stress, deranged genomic integrity, aberrant gene expression in the sperm cell has the potential to affect fertilisation potential, implantation and embryonic viability. This has been seen to be correlated with decreased fertilisation, clevage rates and blastocyst development and thus impaired implantation rates. Study design, size, duration A case control study of 75 men from infertile couples who had recurrent implantation failure (RIF) in IVF cycles and 75 fertile controls at AIIMS, New Delhi, India. Study duration was 2 years. Participants/materials, setting, methods Semen samples from men from couples who had RIF were analyzed as per WHO 2010. Sperm reactive oxygen species (ROS) and DNA fragmentation index (DFI) was assessed by chemiluminiscence and sperm chromatin structure assay (SCSA) respectively. Relative sperm telomere length was evaluated from sperm DNA by q-PCR analysis. The expression of genes pertinent for early embryonic development was done by qPCR. The relative quantification was done after normalizing with GAPDH and β-actin by 2-ΔΔCt method. Main results and the role of chance Seminal ROS levels (RLU/sec/million sperm) were seen to be significantly higher [57.75 (10.1-1186.9)] in cases with respect to controls [16.7(1.15-53.9)] (p &amp;lt; 0.001***). The mean DFI of men undergoing ART was significantly higher (37.7 ± 5.7) vs 23.2 ± 4.6%; p &amp;lt; 0.001***) in cases as compared to controls. ROS and DFI levels correlated negatively with sperm concentration and progressive motility (p &amp;lt; 0.001***). We analyzed the expression of FOXG1, SOX3, STAT4, RPS6, RBM9, RPL10A, RPS17, RPL29, WNT5A, HSP90, TOMM7, EIF5A genes. The expression of SOX3, RBM9, WNT5A, HSP90, TOMM7, and EIF5A showed a significant difference from control levels. The relative sperm telomere length was found to be significantly lower in RIF patients as compared to controls (p &amp;lt; 0.001***). Limitations, reasons for caution The current study was a case control study and lacked randomization and also is limited by low sample size. There is a lack of stratification in the enrollment of participants due to difference in specific risk of occurrence and varied clinical history. Wider implications of the findings The analysis of spermatozoal gene expression is important for understanding the sperm differentiation, fertilization and early embryonic events. Correlation with oxidative stress, genomic integrity and telomere length may help in regulationof gene expression. It may help in establishing sperm gene expression as a potential biomarker. Trial registration number Not applicable

Cyanidin-3-Ο-glucoside supplementation in cryopreservation medium improves human sperm quality.

Currently, the cryopreservation of human spermatozoa must overcome the adverse effects of excessive oxidation. In this study, we aimed to evaluate the effect of supplementation of cryopreservation medium with cyanidin-3-Ο-glucoside (C3G) on sperm quality. Semen samples were obtained from men with normozoospermia according to WHO criteria (n=39). The sperm parameter values were compared after cryopreservation in medium supplemented with and without C3G.Compared with the control group (without additive), low doses (50 μM and 100 μM) of C3G improved sperm viability and motility and decreased the reactive oxygen species (ROS) of spermatozoa, while high doses (200 μM) of C3G did not obviously enhance sperm quality. The amount of DNA fragmentation index (DFI) and high DNA stainability (HDS) after freezing were higher in the control group than in the C3G supplementation groups. Low-concentration C3G supplementation (50μM) was negatively correlated with sperm ROS levels (r= -0.2, p= 0.03). Collectively, our findings suggest that C3G could be an efficient semen cryoprotectant that ameliorates oxidative stress in human sperm during cryopreservation.

Synergistic effects of myo-inositol and melatonin on cryopreservation of goat spermatozoa.

Overproduction of reactive oxygen species (ROS) during sperm cryopreservation has a detrimental effect on sperm parameters. Therefore, the use of antioxidants in the sperm freezing extender can reduce ROS destructive effects. In this study, we investigated whether co-supplementation of melatonin and myo-inositol into the semen extender can improve the post-cryopreservation quality of goat spermatozoa. After the freeze-thawing process, sperm motility, viability, plasma membrane and acrosome intact morphology were improved in the combined myo-inositol and melatonin group compared to both individual and the control groups (p<.05). In addition, the mean of sperm ROS, DNA damage and lipid peroxidation were reduced in co-supplementation of myo-inositol and melatonin compared to their individual counterparts (p<.05). Therefore, the synergistic effects of myo-inositol and melatonin on the cryopreserved spermatozoa are highly likely mediated through the reduction in important factors involved in the sperm lipid peroxidation. Finally, we used the cryopreserved spermatozoa for in vitro production of embryos. Results showed that combined group of myo-inositol and melatonin improved the cleavage rate compared to both individual and control groups, although blastocyst rate was improved using both individual and combined groups. In conclusion, co-supplementation of melatonin and myo-inositol is a promising approach for the improvement of goat sperm cryopreservation.