Human Sperm Motility Research Articles

Treatment of human sperm with GYY4137 increases sperm motility and resistance to oxidative stress.

Hydrogen sulfide (H2S) has been shown to play a significant role in oxidative stress across various tissues and cells; however, its role in sperm function remains poorly understood. This study aimed to investigate the protective effect of GYY4137, a slow-releasing H2S compound, on sperm damage induced by H2O2. We assessed the effects of GYY4137 on motility, viability, lipid peroxidation and caspase-3 activity in human spermatozoa in vitro following oxidative damage mediated by H2O2. Spermatozoa from 25 healthy men were selected using a density gradient centrifugation method and cultured in the presence or absence of 10 μM H2O2, followed by incubation with varying concentrations of GYY4137 (0.625-2.5 μM). After 24 h of incubation, sperm motility, viability, lipid peroxidation, and caspase-3 activity were evaluated. The results indicated that H2O2 adversely affected sperm parameters, reducing motility and viability, while increasing oxidative stress, as evidenced by elevated lipid peroxidation and caspase-3 activity. GYY4137 provided dose-dependent protection against H2O2-induced oxidative stress (OS). We concluded that supplementation with GYY4137 may offer antioxidant protection during in vitro sperm preparation for assisted reproductive technology.

The involvement of lysine glutarylation and mitochondria in spermatic movement deficiency induced by hexavalent chromium

Chromium (Cr) and hexavalent chromium Cr(VI) compounds are extensively utilized in various industrial processes, leading to significant environmental pollution. Animal studies have indicated that Cr(VI) can easily penetrate into male germ cells, thereby impairing male fertility. However, there is limited information on how Cr(VI) impacts human spermatozoa functions and the mechanisms involved. This study aims to elucidate the impact of Cr(VI) on human spermatozoa functions and the underlying mechanisms involved. We observed that K2CrO4 inhibited human spermatozoa motility in vitro, with the inhibition being positively correlated with both the concentration of K2CrO4 and the duration of exposure. Additionally, K2CrO4 was found to reduce lysine glutarylation (Kglu) levels and increase the accumulation of reactive oxygen species (ROS) within the cells, leading to mitochondrial depolarization and a subsequent decrease in adenosine triphosphate (ATP) content. Interestingly, sodium glutarate (Na-glu), a precursor of glutaryl-CoA, demonstrated the ability to partially ameliorate the detrimental effects of K2CrO4 on spermatozoa motility. These results suggest that the environmental pollutant Cr(VI) may impair human spermatozoa motility by disrupting lysine glutarylation and mitochondrial functions.

Decreased human sperm motility and vitality after fast gravity load changes in a parabolic flight

Little is known about the effects of low gravity on human gametes. The aim of this study was to analyze if fresh human sperm samples after fast gravity load changes suffered any detrimental effect in comparison to the splits maintained in Earth's gravity. Fifteen fresh samples from normozoospermic donors were analyzed. Statistically significant differences in vitality (69.7 ± 9.9 % vs 72.4 ± 9.7 %, [95 % CI: 0.002,0.07]); motile sperm concentration (23.7 ± 15.3 M/ml vs 31.5 ± 25.1 M/ml, [95 % CI: 1.03,14.65]); grade “a” sperm concentration (8.7 ± 6.5 M/ml vs 11.7 ± 9.9 M/ml, [95 % CI: 0.71,5.28]); percentage of progressive motility sperm (30 ± 12.9 % vs 36 ± 14.3 %, [95 % CI: 0.10,0.37]) and curvilinear velocity VCL: 45.7 ± 12.8 μm/s vs 47.7 ± 13.3 μm/s, [95 % CI: 0.79,3.22]) were observed. No statistical differences were observed in other sperm kinematic parameters, morphology, DNA fragmentation, apoptosis, and oxidative stress. In conclusion, even though it did not result in a total loss, heavy gravity load changes including microgravity causes a significant decrease in sperm vitality and motility suggesting that negative consequences would be even higher if the exposure were longer. The results obtained indicate that further research is really needed before Assisted Reproduction will be considered for the future human reproduction outside the Earth.

O-096 Human fallopian tube organoids provide a favorable environment for sperm motility

Abstract Study question Does a human fallopian tube (HFT) organoid model offer a favorable apical environment for human sperm survival and motility? Summary answer The apical compartment of a new model of human fallopian tube organoids provides after differentiation a favorable environment for sperm motility. What is known already Human fallopian tubes are the site of major events that are crucial for achieving an ongoing pregnancy, such as gamete survival and competence, fertilization steps, and preimplantation embryo development. In order to better understand the tubal physiology and tubal factors involved in these reproductive functions, and to improve still suboptimal in vitro conditions for gametes preparation and embryo culture during In Vitro Fertilization IVF, we sought to develop a human fallopian tube organoid model from isolated adult stem cells allowing spermatozoa coculture in the apical compartment. Study design, size, duration Over a two-year period, fallopian tube tissues were collected for organoid culture purposes from 10 “donor” patients undergoing bilateral salpingectomy by laparoscopy for definitive sterilization. After tissue digestion, adult stem cells from the isthmus and ampulla regions were separately seeded in 3D Matrigel and cultured with conventional growth factors for organoid culture and specific factors for differentiation of the female genital tract. Participants/materials, setting, methods HFT organoids were characterized by light microscopy, scanning and transmission electron microscopy, immunofluorescence and transcriptomic analysis. Following simultaneous organoid culture on specific inserts, spermatozoa from five donors were placed either in control media or in the apical compartment of colon or HFT organoids (isthmus and ampulla separately) for 96 hours. Vitality and motility were assessed at 0, 48, and 96 hours on 200 spermatozoa in each condition and in duplicate and compared using Wilcoxon test. Main results and the role of chance Specific fallopian tube differentiation of our model was confirmed by immunofluorescence, transcriptome analysis and electron microscopy observations that exhibited ciliary and secretory cells. We succeeded in releasing spermatozoa in the apical compartment of HFT organoids and in recovering them for sperm analysis. Sperm vitality values are similar in HFT organoids and in commercial sperm media. We demonstrated a superiority of HFT organoid apical compartment for sperm motility compared with other controls (colon organoids, organoid culture media, and conventional commercial sperm washing and fertilization media). At 48 hours of incubation, progressive sperm motility was higher in the apical compartment of HFT organoids (ampulla 30.6% ±17.3, isthmus 29.3% ±14.8) than in commercial fertilization media (15.3% ±14.6) (p < 0.05) and compared with all other conditions. At 96 hours progressive sperm motility was almost nil (<1%) in all conditions except for spermatozoa within HFT organoids (p < 0.05): 11.5% ±14.7 and 12.9% ±16.9 in ampulla and isthmus organoids, respectively. Limitations, reasons for caution This was an in vitro study in which conditions of organoid culture could not exactly mimic the in vivo environment of extracellular matrix and the vascularization of fallopian tubes. Wider implications of the findings This work opens up perspectives for better understanding of HFT physiology. For the first time, it highlights the possibility of developing HFT organoids for reproductive purposes. In the future, it could help us to improve gametes fertilizing abilities and culture conditions of embryos during human assisted reproductive technologies. Trial registration number not applicable

P-042 Enhancing human sperm motility using high-frequency ultrasound

Abstract Study question Is high-frequency ultrasound effective in the treatment of low-motility sperm for use in assisted reproduction? Summary answer Our study reveals that immotile and poorly motile sperm respond to ultrasound treatment and show up to 266% boost to motility parameters upon ultrasound exposure What is known already Sperm motility is central to both natural and assisted reproduction. This crucial importance has led to the utilization of chemical drugs such as pentoxifylline, theophylline, and other phosphodiesterase inhibitors to improve motility. However, these drugs are embryotoxic with a potentially harmful effect on sperm longevity. In a recent study, we showed that sperm cells at the population level showed a 32% boost to motility after 15 seconds of exposure to ultrasound. However, to translate these findings into clinical applications, it is crucial to identify which subset of sperm within a population is most affected by the treatment. Study design, size, duration We conducted single-cell study aimed at comprehensively assessing how different sperm motility grades are affected by ultrasound exposure. We utilized a droplet acoustofluidic system using which sperm cells were isolated in droplets and their responses to ultrasound were monitored. A total of 239 individual sperm from three biologically independent human samples were included in this study and parameters such as sperm motility, viability, DNA integrity, and mitochondrial membrane potential (MMP) upon ultrasound exposure were analysed. Participants/materials, setting, methods Fresh human semen samples were collected from healthy donors with the approval of the Monash University Human Research Ethics Committee. Three human donors contributed to a total of sixteen independent experiments performed in this work. The manual tracking plugin in ImageJ was used to measure sperm motility. The LIVE/DEAD™ Sperm Viability Kit (Invitrogen, USA) was used to evaluate sperm viability and JC-1 (Invitrogen, USA) was used to monitor the MMP. Main results and the role of chance We classified the sperm cells based on their pre-exposure straight-line velocity (VSL) into three categories of grade C (non-progressive, VSL < 5 µm s-1), grade B (slow progressive, 5 ≤ VSL< 25 µm s-1), and grade A (rapid progressive, 25 µm s-1 ≤ VSL) cells. Our single-cell analysis results show that 20 seconds of ultrasound exposure boosts the motility of grade C sperm up to 266% and as a result, 90% of total sperm are graded as progressively motile after exposure. Through ANOVA tests, we showed that non-progressive sperm cells show a boost to all of the motility parameters more than the other grades. In addition, ultrasound exposure renders 34% of live immotile sperm motile with 24% of the cells graded as progressive and the remaining 10% showed a twitching motility after exposure. We also showed that ultrasound exposure induces flagellum deformation to 16% of total live immotile sperm cells, providing opportunities to detect live sperm in testicular samples using this approach. Finally, we explored the non-invasiveness of the ultrasound platform and showed that there is no detrimental effect on sperm DNA integrity and viability post exposure. Limitations, reasons for caution A larger sample size could provide a higher statistical confidence to our study. Treatment of patient samples (testicular or asthenozoospermic) and evaluation of embryo safety must be included in future studies to evaluate the effectiveness of this method for clinical use. Wider implications of the findings Our acoustic mechanotherapy method could enhance sperm motility to potentially benefit assisted reproduction outcomes. It also enables new opportunities for detecting live sperm in testicular samples. Trial registration number not applicable

Exogenous Oxidative Stress in Human Spermatozoa Induces Opening of the Mitochondrial Permeability Transition Pore: Effect on Mitochondrial Function, Sperm Motility and Induction of Cell Death.

Oxidative stress (OS) and disrupted antioxidant defense mechanisms play a pivotal role in the etiology of male infertility. The alterations in reactive oxygen species (ROS) production and calcium (Ca2+) homeostasis are the main activators for the mitochondrial permeability transition pore (mPTP) opening. The mPTP opening is one of the main mechanisms involved in mitochondrial dysfunction in spermatozoa. This alteration in mitochondrial function adversely affects energy supply, sperm motility, and fertilizing capacity and contributes to the development of male infertility. In human spermatozoa, the mPTP opening has been associated with ionomycin-induced endogenous oxidative stress and peroxynitrite-induced nitrosative stress; however, the effect of exogenous oxidative stress on mPTP opening in sperm has not been evaluated. The aim of this study was to determine the effect of exogenous oxidative stress induced by hydrogen peroxide (H2O2) on mPTP opening, mitochondrial function, motility, and cell death markers in human spermatozoa. Human spermatozoa were incubated with 3 mmol/L of H2O2 for 60 min, and intracellular Ca2+ concentration, mPTP opening, mitochondrial membrane potential (ΔΨm), ATP levels, mitochondrial reactive oxygen species (mROS) production, phosphatidylserine (PS) externalization, DNA fragmentation, viability, and sperm motility were evaluated. H2O2-induced exogenous oxidative stress caused increased intracellular Ca2+, leading to subsequent mPTP opening and alteration of mitochondrial function, characterized by ΔΨm dissipation, decreased ATP levels, increased mROS production, and the subsequent alteration of sperm motility. Furthermore, H2O2-induced opening of mPTP was associated with the expression of apoptotic cell death markers including PS externalization and DNA fragmentation. These results highlight the role of exogenous oxidative stress in causing mitochondrial dysfunction, deterioration of sperm motility, and an increase in apoptotic cell death markers, including PS externalization and DNA fragmentation, through the mPTP opening. This study yielded new knowledge regarding the effects of this type of stress on mitochondrial function and specifically on mPTP opening, factors that can contribute to the development of male infertility, considering that the role of mPTP in mitochondrial dysfunction in human sperm is not completely elucidated. Therefore, these findings are relevant to understanding male infertility and may provide an in vitro model for further research aimed at improving human sperm quality.

Association between mitochondrial DNA genotype and sperm motility in humans

The relationship between genetic alterations in mitochondrial DNA (mtDNA) and progressive motility (PR) and rapid progressive motility (grade A) of ejaculated human spermatozoa remains unclear. In this study, we explored the association between human mtDNA genotype and sperm PR and grade A by analyzing mtDNA copy number, loci, haplogroup, rearrangement, deletions, and duplications and sperm motility parameters. Human sperm mtDNA copy number, loci and haplogroups were not associated with human sperm motility PR or A grade. However, the cumulative frequency of human sperm mtDNA rearrangements (including deletions and duplications) in participants with high PR and grade A ratio was higher than in participants with low PR and grade A ratio. Additional studies are needed to understand the relationship between mtDNA genotypes, including deletions and duplications, and human sperm motility.

Artificial activation of sperm motility in vitro.

The sperm activation method is a modern methodological approach that is used more and more often in practice. The number of studies focused on methods of artificial activation of human sperm motility are constantly increasing. Standard sperm selection methods can fail in some cases, among other things, because very young sperm are isolated that have not yet completed their development. In these cases, artificial stimulation of their movement can have a positive effect and greatly facilitate and faster the process of selecting suitable sperm. Methylxanthines are most often used as activating agents. However, opinions on the safety of using these substances on sperm are not uniform. The aim of the thesis is to present current knowledge about artificial activation of sperm motility for in vitro fertilization and subsequent embryonic development. Research of relevant literature in Web of Science, Scopus, PubMed/Medline databases. The literature analysis shows that this method is safe and effective in the selection of immotile spermatozoa. Scientific studies have been conducted to verify the safety and reliability of this method. The conclusion of these studies is the positive impact of this method of selection, especially in cases of sperm obtained from testicular tissue after method testicular sperm extraction. In these cases, the method of artificial sperm activation facilitated and accelerated the selection of sperm before intracytoplasmic sperm injection. Undamaged spermatozoa, which are immobile due to incomplete maturation, were activated.

Disposables used cumulatively in routine IVF procedures could display toxicity.

Is there a cumulative toxicity of disposables used in IVF procedures? A toxicity may be detected when consumables are used cumulatively, while no toxicity is detected when the same consumables are used and tested individually. Many components of items used in IVF laboratories may impair human embryonic development. Consequently, it is necessary to screen all reagents and materials which could be in contact with gametes and embryos. Toxicity tests, such as the mouse embryo assay and the human sperm motility assay (HSMA), are used by manufacturers as quality control tools to demonstrate the safety of their products. This evaluation is currently individually performed for each single consumable. However, during an IVF cycle, several devices are used sequentially, potentially creating a cumulative exposure to chemical contaminants, which could not be detected for individually tested consumables. The objective of this observational study conducted from March 2021 to October 2022 was to evaluate with the HSMA methodology if there was a cumulative toxicity when several disposables are sequentially used. Fourteen categories of consumables currently used in routine IVF procedures were studied, which included devices used for sperm and oocyte collection (cups, condoms, and oocyte aspiration needles), manipulation (flasks, tubes, tips, pipettes, embryo transfer catheters, syringes, and gloves), culture (dishes), and storage (straws). After obtaining patient consent, the surplus semen assessed as having normal parameters according to the World Health Organization 2010 criteria were used to perform the HSMAs. First, each consumable was tested individually. Then, associations of three, four, and five consumables, previously validated as non-toxic when tested individually, were analyzed. HSMAs were conducted three times to ensure reproducibility, with a defined toxicity threshold of a sperm motility index (SMI) below 0.85 in at least two of three tests. Thirty-six references of disposables were first individually tested across 53 lots. Forty-nine (92%) demonstrated compliance. However, four (8%) devices revealed toxicity: one lot of 1 ml syringes, two lots of sperm cups, and one lot of 25 cm2 flasks. These four references were excluded from the IVF routine procedures. A total of 48 combinations of consumables were assessed, involving 41 lots from 32 references that were previously individually tested. Among the evaluated combinations, 17 out of 48 (35%) associations exhibited toxicity with a SMI below 0.85 for two of the three tests (n = 8) or all the three tests (n = 9). Notably, three out of 17 (18%) of the three-consumable associations, five out of 16 (31%) of the four-consumable associations, and nine out of 15 (60%) of the five-consumable associations were found not compliant. The toxicity did not originate from a single consumable, because only consumables that were individually pre-validated as non-toxic were included in the combinations, but the toxicity had a cumulative origin. The risk of cumulative toxicity increased with the number of consumables included in the association (Cochran-Mantel-Haenszel statistic, P = 0.013). The high proportion of non-compliant combinations of disposables can be attributed directly to the extreme rigorous extraction conditions employed during the tests, which could deviate from the conditions encountered in routine clinical use. Also, the methodology employed in the HSMAs (e.g. toxicity extraction duration, sperm concentrations, and protein supplementation of the medium) can influence the sensitivity of the tests. This study highlights the significance of performing toxicity testing on devices before introducing them into clinical practice. Disposables should be tested individually to detect immediate toxicities and also in combination. Our results advocate rationalizing the number of consumables used in each IVF procedure and re-evaluating the use of glass consumables. This study received fundings from GCS Ramsay Santé pour l'Enseignement et la Recherche (Paris, France) and the Centre de Biologie Médicale BIOGROUP(Le Chesnay-Rocquencourt, France). The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the reported research. N/A.

In vitro effects of the combination of serotonin, selenium, zinc, and vitamins D and E supplementation on human sperm motility and reactive oxygen species production.

Infertility affects 15% of all couples worldwide and 50% of cases of infertility are solely due to male factors. A decrease in motility in the semen is considered one of the main factors that is directly related to infertility. The use of supplementation to improve the overall sperm quality has become increasingly popular worldwide. The purpose of this study was to evaluate whether sperm motility was affected by the combination of serotonin (5-HT), selenium (Se), zinc (Zn), and vitamins D, and E supplementation. Semen samples were incubated for 75 min at 37°C in medium containing varying concentrations of 5-HT, Se, Zn, vitamin D, and E. 5-HT (200 μM), Se (2 μg/ml), Zn (10 μg/ml), vitamin D (100 nM), and vitamin E (2 mmol) have also been shown to increase progressive sperm motility. Three different mixtures of supplements were also tested for their combined effects on sperm motility and reactive oxygen species (ROS) production. While the total motility in the control group was 71.96%, this was found to increase to 82.85% in the first mixture. In contrast the average ROS level was 8.97% in the control group and decreased to 4.23% in the first mixture. Inclusion of a supplement cocktail (5-HT, Se, Zn, vitamins D and E) in sperm processing and culture medium could create an overall improvement in sperm motility while decreasing ROS levels during the incubation period. These molecules may enhance the success of assisted reproduction techniques when present in sperm preparation medium.

Oxidative Stress and Acrosomal Status of Human Spermatozoa Subjected to Hydrophobic Carbon Soot Treatments.

The fourth industrial revolution extensively reshapes the reality we are living in by blurring the boundaries of physical, digital and biological worlds. A good example is the previously unthinkable incursion of nanoscale waste materials, such as soot, into the technologies for assisted reproduction. Although the rapeseed oil soot may efficiently enhance the progressive motility of human spermatozoa, it is yet unknown whether this material induces undesirable oxidative stress and premature acrosome reaction, endangering the sperm-oocyte fusion and blastocyst formation. In an attempt to clarify this issue, we reveal that the three-hour incubation of human semen mixed with three main types of soot does not cause oxidative stress and spontaneous acrosome reaction of the sperm. These unique findings are attributed to synchronous elimination and stabilization of the oxidants via hydrogen bonding to the acidic groups of the soot (i.e., C=O and/or C-O-C) and electron donation by its basic chemical sites (i.e., C-OH and/or COOH). Moreover, the soot nanoparticles are electrostatically attracted by discrete positively charged areas on the sperm head, increasing its negative charge and in some cases interfering the acrosome reaction. Such novel mechanistic insights emphasize the credibility of rapeseed oil soot to confidently shift from the purely diagnostic and therapeutic phases in reproductive medicine to research dealing with the effect of carbon nanomaterials on the embryo development and implantation.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. N/A. This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Improving human sperm motility via red and near-infrared laser irradiation: in-vitro study

Improved sperm motility is necessary for successful sperm passage through the female genital system, efficacious fertilization, and a greater probability of pregnancy. By stimulating the mitochondrial respiratory chain, low-level laser photobiomodulation has been shown to increase sperm motility and velocity. The respiratory chain in mitochondria is the primary site of action for cytochrome c oxidase because it can absorb light in the visible and infrared ranges. The present study aimed to investigate the effects of red laser 650 nm, near infrared laser (NIR) 980 nm, and combination of both on human spermatozoa motility and DNA integrity at different doses. An in-vitro controlled trial was performed in Al Zahraa university hospital laboratory using thirty fresh human semen specimens. Samples were exposed to red laser 650 nm, near infrared laser (NIR) 980 nm, and combination of both for various irradiation times. Sperm motility for the test and control aliquots was assessed as recommended in the manual of WHO-2021. Sperm chromatin integrity was evaluated using the Sperm Chromatin Structure Assay. Results revealed almost 70%, 80% and 100% increase in the total motility after 3 min of the 650-nm, 980-nm and the combined laser irradiation, respectively. Additionally, the Sperm Chromatin Dispersion assay was carried out on sperm heads utilizing human sperm DNA fragmentation, demonstrating that none of the three laser types had any discernible effects.Graphical abstract

HIF‐1α Affects Sperm Autophagy and Motility

Male infertility is frequently caused by decreased sperm motility. Sperm of low progressive motility showed abnormal energy metabolism and elevated autophagy rate, although the specific mechanisms remain elusive. Hypoxia‐inducible factor 1α (HIF‐1α) is a transcription factor activated by hypoxia and plays a crucial role in regulating biological oxygen homeostasis. Negative correlation between sperm motility and HIF‐1α expression and a potential association with the PI3K/Akt/mTOR signaling pathway were observed. To further explore the mechanisms by which HIF‐1α impacts sperm motility, normal human sperm were treated with cobalt chloride (CoCl2) or YC‐1. Sperm autophagy rate, ATP production, levels of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and HIF‐1α, p‐AKT, p‐mTOR, and LC3B were assessed. The results showed that CoCl2 or YC‐1 decreased sperm motility and increased sperm autophagy rate. The levels of intracellular ROS, HIF‐1α, p‐AKT, p‐mTOR, and LC3B were significantly increased, while the levels of sperm ATP and MMP reduced. These findings suggested that HIF‐1α might affect mitochondrial structure and energy generation, leading to increased autophagy rate and decreased motility in human sperm, with the PI3K/Akt/mTOR pathway potentially playing a role in this process by exerting an inhibitory effect on autophagy.

Oxidative Stress Induces Changes in Molecular Markers Associated with Ferroptosis in Human Spermatozoa.

Ferroptosis is a type of iron-dependent regulated cell death characterized by increased bioavailability of redox-active iron, loss of GPX4 antioxidant capacity, and oxidation of polyunsaturated fatty acid-containing phospholipids mediated by reactive oxygen species (ROS). The aim of this study was to evaluate the effect of oxidative stress induced by arachidonic acid (AA) on ferroptotic cell death in human spermatozoa. Spermatozoa from normozoospermic donors were exposed to AA (5, 25, and 50 µM) for 1 hour at 37 ℃, including an untreated control. Oxidative stress was confirmed by evaluation of cytosolic and mitochondrial ROS production, viability, mitochondrial membrane potential (ΔΨm) and motility. Subsequently, molecular markers of ferroptosis including iron content, levels of GPX4, SLC7A11, ACSL4, IREB2 and lipid peroxidation were evaluated. The analyses were carried out using either flow cytometry, a microplate reader or confocal laser microscopy. AA-induced oxidative stress showed increased cytosolic and mitochondrial ROS production accompanied by impairedΔΨm, viability and motility in human spermatozoa. These results were associated with biochemical and molecular markers related to ferroptotic cell death including an increase in iron content in the form of ferrous (Fe2+) ions, SLC7A11, ACSL4, IREB2, a decrease in the level of GPX4, and an increase in the level of lipid peroxidation compared to the untreated control. This study revealed that AA-induced oxidative stress induces cell death with biochemical characteristics of ferroptosis in human spermatozoa, demonstrating another mechanism of alteration of sperm function induced by oxidative stress and could establish new therapeutic objectives to prevent the decrease in sperm quality mediated by oxidative stress.

Potentials of electron microscopic examination in the diagnosis of genetically determined disorders related to morphology and motility of human spermatozoa

Despite infertility is known as a serious medical and social issue, commercial panels for the determination of genetically caused disorders of morphology and motility of spermatozoa are not available in the territory of the Eurasian Economic Union. Electron microscopic examination of spermatozoa (EMIS) represents a promising method that allows to visualize and analyze structural anomalies in spermatozoa at a level which is not accessible by other methods. The study included transmission electron microscopy (TEM) of spermatozoa. Native sperm was diluted and fixed with 2.5% glutaraldehyde. Ultrathin sections were obtained on an UltraCut III microtome. Analysis was performed at magnifications of x4000, x25000. Spermatozoa were analyzed on a JEM-1011 electron microscope at different magnifications to identify the general appearance, axonemal anomalies, chromatin of the nucleus, and mitochondria. EMIS serves as a tool for detailed analysis of the morphology of spermatozoa in patients with asthenozoospermia (AZS) and teratozoospermia (TZS), and their combination. The obtained data provide a basis for more accurate diagnosis and personalized treatment approaches, contributing to increased effectiveness in overcoming infertility.

The adenosine A2A receptor in human sperm: its role in sperm motility and association with in vitro fertilization outcomes.

The involvement of ATP and cAMP in sperm function has been extensively documented, but the understanding of the role of adenosine and adenosine receptors remains incomplete. This study aimed to examine the presence of adenosine A2A receptor (A2AR) and study the functional role of A2AR in human sperm. The presence and localization of A2AR in human sperm were examined by western blotting and immunofluorescence assays. The functional role of A2AR in sperm was assessed by incubating human sperm with an A2AR agonist (regadenoson) and an A2AR antagonist (SCH58261). The sperm level of A2AR was examined by western blotting in normozoospermic and asthenozoospermic men to evaluate the association of A2AR with sperm motility and in vitro fertilization (IVF) outcomes. A2AR with a molecular weight of 43 kDa was detected in the tail of human sperm. SCH58261 decreased the motility, penetration ability, intracellular Ca2+ concentration, and CatSper current of human sperm. Although regadenoson did not affect these sperm parameters, it alleviated the adverse effects of SCH58261 on these parameters. In addition, the mean level of A2AR in sperm from asthenozoospermic men was lower than that in sperm from normozoospermic men. The sperm level of A2AR was positively correlated with progressive motility. Furthermore, the fertilization rate during IVF was lower in men with decreased sperm level of A2AR than in men with normal sperm level of A2AR. These results indicate that A2AR is important for human sperm motility and is associated with IVF outcome.

Bioactivity Examination of Uncaria gambir (W.Hunter) Roxb on In Vitro Human Sperm Motility

Globally, 48 million couples experience infertility, where male infertility factors contribute to 50% of cases. Spermatozoa motility is a crucial parameter in assessing male fertility. Antioxidants act as the body's defence against excessive ROS and can be used as a treatment for male infertility. One of the local plants in Central Kalimantan that is potentially rich in antioxidants is Bajakah Kalalawit (Uncaria gambir (W.Hunter) Roxb. However, there is limited research on the bioactivity of Bajakah Kalalawit on spermatozoa in vitro. This study aims to determine the effect of ethanol extract of Uncaria gambir (W. Hunter) Roxb on spermatozoa motility in vitro. Bajakah Kalalawit stems were extracted using a 3x24-hour maceration technique with 96% ethanol. After obtaining the concentrated extract, the secondary metabolite compound content was analyzed through a phytochemical screening. The sample used was in vitro human spermatozoa that were washed and added to Bigger Whitten Whittingham medium, then incubated at 37°C for 1 hour with a 96% ethanol extract of Bajakah Kalalawit at doses of 50ng/ml, 100ng/ml, 500ng/ml, and 1000ng/ml, as well as a control group (Bigger, Whitten & Whittingham medium only). The results showed that the compounds contained in the ethanol extract of Uncaria gambir (W.Hunter) Roxb were terpenoids, flavonoids, phenolics, steroids, saponins, alkaloids, and tannins. Spermatozoa motility significantly increased in the treatment groups starting from doses of 50ng/ml, 100ng/ml, 500ng/ml, and 1000ng/ml compared to the control group. Ethanol extract of Uncaria gambir (W.Hunter) Roxb could increase spermatozoa motility in vitro and succeeded in improving reproductive technology.

#83 : Human Sperm Cryo-Survival Rate

Background and Aims: Sperm cryopreservation plays an important role in the suite of services available to fertility patients. The ability to preserve the motility and viability of human spermatozoa is a key component in delivering successful assisted reproductive technologies (ART) outcomes. Cryopreservation methods expose sperm to stress, resulting in cellular damage which consequently leads to a reduction in their survival rate. The aim of this study was to evaluate the average cryo-survival rate of semen samples frozen in a clinical setting, to help improve and manage patient expectations prior to accessing this service. Method: Pre and post sperm freeze data from patients who presented at the Genea Sydney clinic for sperm cryopreservation between 2017 and 2022 was analysed. The post-thaw survival rate for 1467 specimens was assessed by averaging the total post-thaw motility with total pre-freeze motility. Only ejaculated samples, frozen on-site, who had a raw motility calculated as well as a post-thaw motility analysis able to be performed were included in the study. Results: The range of post-thaw survival was 57% to 67% per annum over the six years of examined data, with an overall cryo-survival rate of 62%. Conclusions: The data shows a consistent recovery rate of post-thaw sperm motility over the time period analysed. Literature indicates average sperm cryo-survival rates range from 30% to 50%, and previous advice to patients has been to expect around 50% sperm survival. With this new data, Genea can revise its guidance to match the current average figure of around 60%. In addition, future internal freeze optimisation studies can use this figure as a benchmark to improve upon techniques and processes.

Low human sperm motility coexists with sperm nuclear DNA damage and oxidative stress in semen.

Low sperm motility, one of the common causes of male infertility, is associated with abnormal sperm quality. Currently, important sperm/semen biomarkers are sperm chromatin status and oxidation‒reduction potential (ORP) in semen. Because the association between sperm motility and these biomarkers is still not fully clarified, our study was designed to verify the distribution and risk of sperm DNA fragmentation (SDF) and oxidative stress in semen in asthenozoospermic men. This study was carried out on discharged sperm cells of asthenozoospermic men (isolated asthenozoospermia or coexisted with reduced sperm number and/or morphology), nonasthenozoospermic men (reduced total sperm count and/or sperm morphology) (experimental groups) and normozoospermic men (proven and presumed fertility) (control group). Basic semen analysis was evaluated according to the 6th edition of the World Health Organization manual guidelines. SDF was assessed using the sperm chromatin dispersion test, while static(s) ORP in semen was measured by means of a MiOXSYS analyser. The men from the asthenozoospermic group had lower basic semen parameters than those from the control and nonasthenozoospermic groups. In men with poor sperm motility SDF and sORP, prevalence and risk for>20% SDF (high level of DNA damage) and for>1.37 sORP (oxidative stress) were significantly higher than those of control and nonasthenozoospermic subjects. The risk for sperm DNA damage and oxidative stress in asthenozoospermic men was over 10-fold higher and almost 6-fold higher than those in control subjects and almost or over 3-fold higher than those in nonasthenozoospermic men. Poor human sperm motility coexisted with low basic sperm quality. Sperm DNA damage and oxidative stress in semen were much more frequent in asthenozoospermia. These abnormalities can decrease the sperm fertilizing capability under both natural and medically assisted reproduction conditions. Thus, in asthenozoospermia, the evaluation of sperm chromatin status and oxidation-reduction potential in semen is justified and inevitable, and the appropriate antioxidant therapy can be suggested.